**Part B: Improvement of the Optical Properties of Cellulose Nanocrystals Reinforced Thermoplastic Starch Bio-Composite Films by Ex Situ Incorporation of Green Silver Nanoparticles from** *Chaetomorpha linum*

**Nour Houda M'sakni 1,2,\*,† and Taghreed Alsufyani 1,\*,†**


**Abstract:** The study was used in the context of realigning novel low-cost materials for their better and improved optical properties. Emphasis was placed on the bio-nanocomposite approach for producing cellulose/starch/silver nanoparticle films. These polymeric films were produced using the solution casting technique followed by the thermal evaporation process. The structural model of the bio-composite films (CS:CL-CNC7:3–50%) was developed from our previous study. Subsequently, in order to improve the optical properties of bio-composite films, bio-nanocomposites were prepared by incorporating silver nanoparticles (AgNPs) ex situ at various concentrations (5–50% *w*/*w*). Characterization was conducted using UV-Visible (UV-Vis), Fourier Transform Infrared (FTIR), Scanning Electron Microscope (SEM) and Transmission Electron Microscope (TEM) to understand the structure–property relationships. The FTIR analysis indicated a reduction in the number of waves associated with the OH functional groups by adding AgNPs due to the formation of new hydrogen bonds between the bio-composite matrix and the CL-WE-AgNPs. Based on mathematical equations, the optical bandgap energy, the energy of Urbach, the edge of absorption (Ed), and the carbon clusters (N) were estimated for CS:CL-CNC and CS:CL-CNC-AgNPs (5–50%) nanocomposite films. Furthermore, the optical bandgap values were shifted to the lower photon energy from 3.12 to 2.58 eV by increasing the AgNPs content, which indicates the semi-conductor effect on the composite system. The decrease in Urbach's energy is the result of a decrease in the disorder of the biopolymer matrix and/or attributed to an increase in crystalline size. In addition, the cluster carbon number increased from 121.56 to 177.75, respectively, from bio-composite to bio-nanocomposite with 50% AgNPs. This is due to the presence of a strong H-binding interaction between the bio-composite matrix and the AgNPs molecules. The results revealed that the incorporation of 20% AgNPs into the CS:CL-CNC7:3–50% bio-composite film could be the best candidate composition for all optical properties. It can be used for potential applications in the area of food packaging as well as successfully on opto-electronic devices.

**Keywords:** green macroalga; red sea; *Chaetomorpha linum*; cellulose nanocrystals; starch; green silver nanoparticles; bio-nanocomposite films; optical properties

#### **1. Introduction**

The development of the bio-composites was based on the environmental knowledge that was generated over time. The remaining natural starch is a vital biomaterial for making environmentally friendly materials. In order to improve the properties of these biomaterials, a new area was created, including the development of bio-nanocomposites [1]. This is a continuous polymer phase in which loads of at least one size on a nanoscale are dispersed.

**Citation:** M'sakni, N.H.; Alsufyani, T. Part B: Improvement of the Optical Properties of Cellulose Nanocrystals Reinforced Thermoplastic Starch Bio-Composite Films by Ex Situ Incorporation of Green Silver Nanoparticles from *Chaetomorpha linum*. *Polymers* **2023**, *15*, 2148. https://doi.org/10.3390/ polym15092148

Academic Editor: Marcelo Antunes

Received: 25 March 2023 Revised: 26 April 2023 Accepted: 28 April 2023 Published: 30 April 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

At this scale, the surface effects become predominant with volume effects in order to obtain original properties different from massive objects. The synthesized nanocomposites have demonstrated their versatility as a catalyst in the esterification response for the production of bio-diesel, a very good potential substitute for Gram-positive antimicrobial activity, as well as Gram-negative micro-organisms and an effective material for energy storage applications [2]. In this regard, nanostructures and nanocomposites, due to their small size, good catalytic activity, high surface area and outstanding selectivity, can play an important role in the near future [3]. Nanofillers, by their specific properties and the multitude of forms they can take, produce functional materials of interest in a number of areas such as electronics, medicine, cosmetics, optical physics and packaging [1].

Polymer matrices used for the production of CNC-reinforced composites could be divided into two parts: biodegradable and non-biodegradable polymers [4]. For example, natural polymers including by-products of cellulose, starch, natural rubber, and biopolymers such as polyhydroxyalkanoate (PHA), polylactic acid (PLA), polycaprolactone (PCL), etc., were widely used as biodegradable polymers to prepare bio-nanocomposites reinforced with cellulose nanocrystals [4,5]. Therefore, in our recent studies [6], we sought to reinforce the bio-composite film, with the introduction of a green *C. linum* silver nanoparticle in order to improve the optical properties of the biofilm. The study provided not only the optical property but also the characterization that led to the development of an optoelectronic bio-nanocomposite.

Due to remarkable physical and chemical properties, noble metallic nanoparticles play their role in various domains such as biological markers [7], treatment of cancer tumors [8], fluorescence [9], improving the efficiency of solar panels [10] or the signal in Raman spectroscopy [11]. Moreover, through enhanced optical response, these nanoparticles are indirectly used to characterize the physical properties of other particles such as catalysis [12]. However, integrating metallic nanoparticles in the polymer matrix is a fundamental and critical industrial challenge. In fact, their load properties, their nature and their presence in the matrix considerably alter the mechanical, thermal, electrical or optical properties, as well as contributing to simplifying and reducing the cost of the transformed material. In recent years, silicon nanowires decorated with silver nanoparticles have also been identified as semi-conductor and antibacterial unidimensional synthetic nanomaterials [13]. AgNPs with diverse properties such as catalytic activity [14], Raman diffusion [15], good conductivity [16], anti-microbial [17] and optical activity [18] have generated considerable interest in the area of nanotechnology [19]. AgNPs can be synthesized using physical, biological and chemical approaches [20]. However, physical–chemical techniques are highly productive in designing well-defined nanoparticles, but they have certain limitations, such as the use of hazardous chemicals, high costs, time-consuming processes, and the generation of impurities [21]. Green synthesis has gained more interest and actively increased progress in the fields of science and industry due to its ecological, cost-effective and non-hazardous nature [22]. To synthesize AgNPs, a metal precursor, reducing agents, and a non-toxic stabilizing/capping agent were required [23,24]. However, the presence of biomolecules of natural active agents in plants plays a significant role in reducing and stabilizing nanoparticles [24,25]. The phytochemical substances involved in reducing and capping nanoparticles are terpenoids, flavonoids, phenols, alkaloids, polysaccharides, proteins, enzymes, amino acids, etc. [26]. In addition, other active agents were reported such as linalool, quinol, methyl chavicol, eugenol, chlorophyll, caffeine, theophylline, ascorbic acids, and so on [27,28]. Several methods have been developed for incorporating nanoparticles into a polymer matrix in two ways, which are known as ex situ and in situ [29].

Each organic semiconductor has its own energy levels that basically depend on the molecular structure. Thus, the electronic bandgap is the energy difference between the lowest unoccupied molecular orbital (LUMO) and highest occupied molecular orbital (HOMO) level [30]. This difference in energy reveals the bandgap that electrons can only penetrate by external excitation. In general, it is 1 to 3 eV for organic semiconductors and

zero for conductors with overlapping valence and a conductive band. When it is high (>6 eV), the material is insulating, since it does not transfer electrons [31].

The objective of this study was to Integrate ex situ AgNPs as an electron donor reinforcement at the bandwidth of the bio-composite developed in our recent study [6] in order to develop new low-bandgap bio-nanocomposites for an optoelectronic application. The main requirement was to reduce the HOMO energy level as well as the bandgap of the polymers in order to increase the open circuit voltage of organic photovoltaic solar cells.

#### **2. Experimental Work**

#### *2.1. Materials*

Macroalgae thalli belonging to the order Cladophorales, *Chaetomorpha linum* were used as raw material to prepare AgNPs. Green algae were collected on the southwest coast of the Red Sea in Jeddah KSA (coordinates 21◦37 41 N and 39◦6 11 E). A CS:CL-CNC7:3–50% bio-composite (Table 1) was developed by reinforcing the thermoplastic starch matrix with cellulose nanocrystals (CL-CNCs) derived from *C. linum* algae biomass, according to the treatment detailed in our previous studies [6]. Silver nitrate-AgNO3 (99.8%) was purchased from VWR, PROLABO.

**Table 1.** Composition of the cellulose nanocrystals, starch, glycerol, and CL-WE-AgNPs used in the investigation for the development of bio-composite and, bio-nanocomposites films.


#### 2.1.1. Colloidal Silver Nanoparticle (AgNP) Synthesis

The hydrosoluble polymers were obtained from 2 g of dry *C. linum* powder (CL-R) by extraction for 2 h in hot deionized water (1:40 w:w, 80 ◦C) with mechanical stirring. The mixture was filtered through a cloth and then centrifuged (5000 rpm) for 20 min to produce the CL-W fraction, as illustrated in Figure 1a. The filtrate was precipitated with the addition of 95% ethanol (40:60 v:v) to remove the remaining salts as well as low-molecular-mass polymers (proteins and polysaccharides) [32]. After decanting for 12 h (4 ◦C), the insoluble material was centrifuged out (5000 rpm) for 30 min to obtain the CL-WE fraction and recovered in deionized water. This fraction was used as a reducing and stabilizing agent for AgNPs synthesis [32]. To synthesize the colloidal AgNPs, 10 mL of seaweed extract was added to 90 mL of a 1 mM aqueous solution of silver nitrate (AgNO3) with constant stirring. The pH was adjusted to 9 by using 1 M NaOH solution to promote the reduction of Ag+ ions at ambient temperature. Within hours, the color changed from yellow to dark brown, which confirmed the formation of AgNPs [33]. The synthesized material was labeled CL-WE-AgNPs.

**Figure 1.** Schematic diagram of (**a**) elaboration of CL-WE-AgNPs from *C. linum*, and (**b**) elaboration of bio-nanocomposites.

#### 2.1.2. Development of Optical Bio-Nanocomposite Films

To improve the performance of the bio-composite packaging, which was produced with the best formulation made for the biodegradable film (containing cellulose nanocrystals and thermoplastic starch), CL-WE-AgNPs were added in order to enhance the optical properties of bio-composite films.

Firstly, five solutions were prepared using the CS:CL-CNC7:3–50% bio-composite, and they were mixed successively with different compositions of CL-WE-AgNPs by weight with 190 mL of distilled water, stirred for five minutes and sonicated for 15 min. Then, the solutions were heated in a water bath at 85 ◦C for 30 min. The solution was moved into a Petri dish and stored in the oven for 24 h at 45 ◦C for drying. The films were peeled and retained in a desiccator (48 h) to control moisture (Figure 1b). The composition of the materials was fixed in cellulose nanocrystals, starch, and glycerol, which vary only with the percentage by weight of AgNPs, as shown in Table 1. Consequently, the percentage by weight of CL-WE-AgNPs was derived from the total quantity of mixture added. The resulting biofilms are named: CS:CL-CNC7:3-AgNPs (5%), CS:CL-CNC7:3-AgNPs (10%), CS:CL-CNC7:3-AgNPs (20%), CS:CL-CNC7:3-AgNPs (40%), and CS:CL-CNC7:3-AgNPs (50%) for the bio-nanocomposite films and CS: CL-CNC7:3–50% for the controlic biocomposite film.

#### *2.2. Characterization Methods*

#### 2.2.1. UV-Visible Analysis

For optical properties, the UV-Vis-NIR (JASCO; V670) (JASCO; V670, Easton, Portland, OR, USA) spectrophotometer was used to study the optical transmittance (T) and absorbance (A) of films prepared over the wavelength range of 190 to 900 nm (Figure 8a,b).

Determining the bandgap value of both amorphous materials and polymers is crucial for their applications. The most popular technique to stimulate the bandgap is a measurement of the optical absorption coefficient. This coefficient was determined by mean absorbance (A), and Equation (1) was followed [34]:

$$\alpha = \frac{2.303\,\mathrm{A}}{\mathrm{d}(\mathrm{cm})} \tag{1}$$

where d = 0.02 cm, and the film thickness was determined by a Vernier caliper. The functions of the absorption coefficient with incident photon energy for bio-composite films CS:CL-CNC7:3–50% and for bio-nanocomposite films CS:CL-CNC7:3-AgNP (5–50%) are shown, respectively, in Figure 9a,b.

The energy bandgap (Eg) deviation of all the prepared films was determined by intercepting the plotted linear portion (α hν) <sup>2</sup> versus hν, as shown in Figure 10a, pursuing Tauc's method (Equation (2)) [35,36]:

$$\alpha \mathbf{h} \mathbf{v} = \mathbf{B} (\mathbf{h} \mathbf{v} - \mathbf{E} \mathbf{g})^{\mathbf{n}} \tag{2}$$

where B is the width parameter of the absorption edge, hν is the incident photon energy calculated from hν (eV) = 1240/λ (nm), and n is the factor that takes 3/2 or 1/2 for direct transitions and 2 or 3 for indirect transitions relaying on the forbidden or allowed transition, respectively.

For determining the band tail that refers to the width of localized states, the absorption coefficient α(v) near the band edge as exponential dependence of photon energy (hv) was determined from the Urbach relationship (Equation (3)) [37,38]:

$$\mathfrak{\alpha}\left(\mathbf{h}\mathbf{v}\right) = \mathfrak{\alpha}\_0 \mathbf{e}^{\left(\frac{\mathbf{h}\mathbf{v}}{\mathbb{E}\_0}\right)}\tag{3}$$

where α<sup>0</sup> is known as the constant and Ee denotes the band tail (Urbach tail), referring to the localized state's width (Figure 10b).

The number of carbon atoms (N) in a cluster is calculated from the optical energy bandgap (Eg) using the following relation (Equation (4)) [39,40]:

$$\text{Eg} = \text{34.4} / \sqrt{\text{N}} \tag{4}$$

#### 2.2.2. FTIR Analysis

Several stages involved in the development of bio-nanocomposite films were studied by FTIR (Thermo spectrophotometer, Nicolet IR 200, Madison, WI, USA). FTIR spectra were recorded between 4000 and 400 cm−<sup>1</sup> and compared with data already reported to distinguish the signal in a specific manner.

#### 2.2.3. SEM and TEM Analyses

The JEOL model JEM-2000FX (Tokyo, Japan) instrument operated at an accelerating voltage of 200 Kilo voltage used to determine SEM (scanning electron microscope), EDX (energy-dispersive X-ray spectroscopy), and TEM (transmissions electron microscopy) measurement.

SEM and EDX images were taken for the characterization of the morphology, and the microstructures of all the materials were obtained at different stages of the bio-nanocomposite film development process.

In order to better clarify the morphology and size of the particles, TEM analysis was applied. A few drops of sonicated powdered sample were prepared and placed on a carbon-coated copper grid and air-dried for 1 h. The CL-WE-AgNP sample was selected for TEM analysis.

#### **3. Results and Discussion**

### *3.1. Impact of AgNPs Density on Optical Responses of Bio-Nanocomposite Films*

3.1.1. Synthesis of Colloid Silver Nanoparticles (AgNPs)

AgNPs (5, 10, 20, 40 and, 50%) were ex situ incorporated in (CS:CL-CNC7:3–50%) biofilm employing the matrix containing CL-CNC from *C. linum* (3 g), CS (7 g) and 50% glycerol as plasticizer agent to improve the optical properties of our previous biofilm [6]. The reduction of AgNPs was carried out by UV-Visible, SEM-EDX, TEM, and FTIR.

The reduction of the Ag+ ion into AgNPs was analyzed by color change (Figure 2a–d). Before adding the solution (AgNO3, 1 mM), the supernatant of the *C. linum* extract precipitated in ethanol (CL-WE) was pale white–yellow (Figure 2b); it turned to a yellow–brown color after 30 min of contact (Figure 2c) and then brownish at the end of the reaction with the ions (after 48 h of contact) (Figure 2d).

These results were confirmed by UV-Visible characterization spectrophotometry, which is a technique that proved to be very useful for the rapid analysis of colloidal solutions of AgNPs. This helps to determine whether the synthesis process was terminated by the formation of nanoparticles. The formation and stability of the reduced silver nanoparticles in the colloidal solution were monitored by UV-Vis spectrophotometric analysis [41]. The UV-Vis spectra showed a maximum absorbance at 415 nm that increased with the incubation time of silver nitrate with *C. linum* extract (Figure 2a) [42]. The presence of an absorbance peak at approximately 415 nm makes clear the formation of AgNPs in the solution, which is due to surface plasmon resonance (SPR) electrons present on the nanoparticle surface. The intensity of the SPR band increased with reaction time (30 min, 2 h, 6 h, 12 h, 24 and 48 h) and showed a maximum absorbance at 432 nm after 48 h (Figure 2a), indicating the synthesis of the AgNPs [42]. It is reported earlier that the absorbance at about 430 nm for silver is a feature of these noble metallic particles [43,44]. These results confirmed that 48 h time is the longest synthesis time at the present temperature and pH. It was observed that with an increase in the contact time between CL-WE and AgNO3, the absorption peak shifted to a higher wavelength (from 415 to 432 nm), indicating an increase in the size of the AgNPs synthesized extract. These results are similar to those reported in the literature [41,42,45].

**Figure 2.** (**a**) UV-Vis absorption spectrum of silver nanoparticles CL-WE-AgNPs synthesized using aqueous dialyzed extract of fresh *C. linum* (CL-WE) as a function of reaction time, (**b**) beginning of the reaction (pale white–yellow), (**c**) after 30 mn (yellow–brown color) and (**d**) after 48 h (brownish color) at 40 ◦C, and pH = 9.

#### 3.1.2. Morphological Analysis of Synthesized AgNP and Its By-Products

A scanning electron microscope with an energy-dispersive X-ray spectrometer (SEM-EDX) was used to determine the silver concentration of the nanoparticles (Figure 3a–c). However, the EDX analysis of the aqueous extract of *C. linum* (CL-W) (Figure 3a) showed a high percentage of chlorine and salt, while after precipitation with ethanol (Figure 3b), the percentage of chlorine was decreased, so we note the presence of sulfur specific for glucosamines, which confirms the role of ethanol purification. After the reduction of the silver ions with the aqueous extract (CL-WE) (Figure 3c) which performs both reducing and stabilizing effects, a new distinct peak was detected at 2.983 keV in the CL-WE-AgNPs. Prasad, Kambala [46] have shown that AgNPs generally exhibit an absorption peak in the region of 3 keV due to the phenomenon of surface plasma resonance. The appearance of this peak (Figure 3c) confirmed the presence of elemental silver in the nanoparticles thus produced (CL-WE-AgNPs) with a silver concentration of around 38.41 ± 1.06%, which was detected after an incubation of 48 h. The morphology of the CL-WE-AgNPs shows that several co-components appear alongside silver: in particular, iron, magnesium, zinc, cadmium, chlorine, sulfate ... This is due to the reduction of silver by the aqueous *C. linum* extract, which was accompanied by the reduction of those trace metals and led to the formation of co-nanoparticles as a trace. Despite the low metal content (zinc and cadmium ≤ 1 ppm, iron ≤ 2 ppm, and magnesium ≤ 5 ppm per mass) that coexist with silver (originally VWR Chemicals commercial silver nitrate), ethanol precipitates of aqueous extract have been able to reduce these metals and obtain co-nanocomposites alongside nanocomposite silver synthesis. This explains the effect of ethanol precipitation of the plant extract that characterizes our method of green synthesis of AgNPs, which promotes the increase in the reducing properties of the plant extract following the purification of ethanol.

**Figure 3.** SEM and EDX image of silver nanoparticles synthesized steps: (**a**) CL-W, (**b**) CL-WE, (**c**) CL-WE-AgNPs.

Figure 4 represents the TEM image of CL-WE-AgNPs (48 h) determining that the AgNPs are well defined and are spherical with slight agglomeration. The presence of agglomeration could be due to the drying effects produced during sample preparation [47,48]. The aggregation behavior of AgNPs is mostly affected by pH and electrolyte concentration [49], while the presence of biomolecules can improve particle stability due to the biomolecular coronary effect [49]. In our work, we show that a degree of detected aggregation can be attributed to the ethanol precipitation effect of water extract (CL-W) that removes the salt that coexists with biomolecules. As shown, the size of nanoparticles increases with increasing concentration of AgNO3. The Ag size range thus detected varied from 20 to 30 nm with an average of 21.4 nm. In comparison to the previous research, it was concluded that AgNPs with 20 nm size have a plasmon resonance band around 430 nm (violet absorption) and are brown [50].

#### 3.1.3. FTIR Analysis of Synthesized AgNPs and Its By-Products

The biomolecules present in the root extract of *C. linum* played an active role in reducing Ag+ to AgNPs, as confirmed by the FTIR analysis. Figure 5a,b represent the FTIR spectrum of *C. linum* extract before and after ethanol precipitation. The FTIR spectra of dried *C. linum* extract before purification by ethanol (CL-W) (Figure 5a) have shown many peaks at 614 cm−1, 645 cm−1, 706 cm−1, 839 cm−1, 863 cm−1, 924 cm−1, 1004 cm−1, 1047 cm−1, 1095 cm<sup>−</sup>1, 1224 cm−1, 1412 cm−1, 1538 cm−1, 1645 cm−1, 2297 cm−1, 2842 cm−1, 2910 cm−1, 2938 cm<sup>−</sup>1, 3279 cm−<sup>1</sup> and 3338 cm<sup>−</sup>1. However, the FTIR spectra of dried *C. linum* extract after purification with ethanol precipitation (CL-WE) have shown disparate peaks at 614 cm−1, 645 cm−1, 924 cm−1, and 2297 cm−<sup>1</sup> specifying aliphatic bromo compound, aliphatic-chloro compound, nucleic acid (other phosphate-containing compounds) [51,52] and nitrile compounds [53], respectively. Meanwhile, the decrease in the peak at 1539 cm−<sup>1</sup> is of amide II [54], explaining the effect of precipitation in ethanol which eliminates the salt compounds and minimizes the presence of free protein and increases the level of polysaccharides in the solution [32]. The appearance of the strong peak at 1220 cm−<sup>1</sup> is

specific to the asymmetric stretching vibration of sulfate groups commonly available in *C. linum* in the form of sulfated polysaccharides [55], which are used for the stabilization of AgNPs [56]. However, the corresponding bands observed at 1545 cm−<sup>1</sup> and 1643 cm−<sup>1</sup> are assigned successively to the amide II from proteins and the stretching vibration of the (NH) C=O group. After the reduction of AgNO3, the shift of the bands from 1538 cm−<sup>1</sup> and 1524 cm−<sup>1</sup> is attributed to the involvement of the secondary amines in the reduction process and the binding of the (NH) C=O group with nanoparticles. Since a member of the (NH) C=O group within the cage of cyclic peptides is involved in stabilizing the nanoparticles, the shift of the (NH) C=O band is quite small. Thus, the peptides play a major role for the reduction of Ag<sup>+</sup> to AgNPs. New bands in CL-WE-AgNPs at 1133 cm−<sup>1</sup> and 1467 cm−<sup>1</sup> (Figure 5b) may be attributed as vibrations of the glycosidic C-O bond (C-O-C) stretches from carbohydrates as well as to C=C-C, aromatic ring stretch, and the aromatic compound.

#### *3.2. Characterization of Bio-Nanocomposite Films* 3.2.1. Morphological Analysis

To analyze the surface morphology of the films produced, and to show the distribution of AgNPs in the bio-nanocomposites (Starch/Cellulose/AgNPs) thus formed, a scanning electron microscope with an energy-dispersive X-ray spectrometer (SEM-EDX) was used. Figure 6a–e provides specific information about the structure and changes in the optical properties of films resulting from the addition of AgNPs. The SEM image of the film without AgNPs (CS:CL-CNC7:3–50%) in one of our recent studies [6] shows a porous surface with rough tufts. In contrast, the SEM images of the films with AgNPs (CS:CL-CNC7:3-AgNP5–50%) (Figure 6a–e) show more or less smooth surfaces with the emergence of some small aggregates corresponding to CL-WE-AgNPs in the form of white markings merged into nanoclusters [57,58]. The SEM images (Figure 6c) show a better distribution of CL-WE-AgNPs in CS/CL-CNC7:3–50% films with a percentage of 20% of AgNPs, indicating sufficient interfacial interaction with the matrix of the CS/CL-CNC mixture and the CL-WE-AgNPs [59].

**Figure 5.** FTIR analysis of (**a**) *C. linum* water extract before and after purification with ethanol precipitation and (**b**) colloidal silver nanoparticle CL-WE-AgNPs synthesis.

#### 3.2.2. FTIR Analysis

After the preparation of the bio-nanocomposites by the modification of CS:CL-CNC7:3– 50% films, by the incorporation of different levels of CL-WE-AgNPs (5–50%), FTIR measurements (Figure 7, Table S1) were performed to verify the formation of chemical bonds between the functionalized CL-WE-AgNPs and the matrix of CS:CL-CNC7:3–50%, which was carried on from our previous article for the current study [6]. The shape of the curves showed that the interactions of the matrix chains increase with the velocity of the nanoparticles (Figure 7). For all the matrices (CS:CL-CNC7:3–50%-AgNPs), new bands have appeared (1642 cm−<sup>1</sup> and 1787 cm−1) showing a better interaction in the order of CL-WE-AgNPs rate: 5%, 10%, 40%, 50%, to 20%. Although the bio-nanocomposite containing 20% CL-WE-AgNPs seems best in terms of composition, it suggests a good intermolecular interaction between the different compositions of the matrix.

**Figure 6.** SEM and EDX analysis of

bio-nanocomposite

 films

CS:CL-CNC7:3-AgNPs

 (5–50%).

**Figure 7.** FTIR analysis of development bio-nanocomposite films (400–1800 cm<sup>−</sup>1).

The band appeared at 1643 cm−<sup>1</sup> specifying the groups of water released from CL-CNC, as we stated in one of our recent studies [6]. The absorbance increases as CL-WE-AgNPs are added, indicating that water absorption is inversely proportional to the proportion of hydrophilic CL-WE-AgNPs [60–62]. However, the band around 1787 cm−<sup>1</sup> is attributed to the elongational vibrations of the C=O bond which occur due to the presence of hemicellulose residues from the CL-WE-AgNPs matrix on cellulose chains [60,63].

The number of waves associated with OH functional groups at 3321 cm−<sup>1</sup> was reduced due to the formation of new hydrogen bonds between the bio-composite matrix and the CL-WE-AgNPs [64]. A similar result was reported by previous research, which is explained in terms of weakened hydrogen bonding due to electron delocalization [65,66].

#### *3.3. Optical Properties of the Synthesized Bio-Nanocomposites Films: The Ability to Protect Films against UV*

The absorbance of the bio-composite films of CS:CL-CNC:7:3–50% (control) [6] and with 5 to 50% of CL-WE-AgNPs was incorporated over the wavelength range of 190 to 900 nm, as shown in Figure 8a. As we stated in one of our recent studies [6], the starch/cellulose/glycerol-based bio-composite film exhibited UV absorbance at 273 nm but was not present in visible regions. In contrast, CS-CL-CNC-AgNPs (5–50%) films showed strong absorbance bands in the UV range around 212–300 nm (Figure 8a), which was due to the presence of AgNPs in the matrix. This explains that these bio-nanocomposites could be used in different fields of application. This is due to efficient UV absorbers, mainly for UV-C rays (100–280 nm), but also for UV-B rays (280–315 nm). He also noted that the intensity of the absorption peaks increases with the increase in the AgNPs content in CS-CL-CNC-AgNPs films (5–50% by weight), explaining a higher UV absorption ability for CS-CL-CNC-AgNPs 50% film compared to other matrices. Figure 8b revealed a strong absorption peak between 336 and 600 nm with a maximum absorbance of about 420–434 nm,

which is the typical plasmon resonance band of AgNPs [33,67]. The peak indicates that the Ag<sup>+</sup> ions in the solution (AgNO3, 1 mM) were successfully reduced to AgNPs.

**Figure 8.** (**a**) UV-visible absorption spectra of bio-composite formulation with the integration of CL-WE-AgNPs (10%, 20%, and 50%), and (**b**) Optical absorbance spectra of development bionanocomposite films.

The absorption coefficient (α) of the bioplastic film (CS-CL-CNC7:3–50%) is calculated according to Equation (1) and illustrated in (Figure 9a,b). The α-values of the film (Figure 9a) are high and draw a broad spectrum in the spectral region > 1.55 eV, which recommends the film for specific applications in the field of solar cells. Two absorption peaks also appeared at hν ≈ 2.1 eV and 4.6 eV in the visible and UV spectra range, respectively. These peaks are attributed to the π–π\* transition between bonding and molecular orbital antibonding [68,69].

**Figure 9.** Optical absorption coefficient as a function of photon energy of (**a**) CS-CL-CNC7:3–50% bio-composite film [6] and (**b**) CS-CL-CNC-AgNPs (5–50%) development bio-nanocomposite films at ambient temperature.

The band structure of solid materials can be identified by studying the optical absorption spectrum [70,71]. Optical absorption studies on polymer blend films without and

containing various concentrations of CL-WE-AgNPs (5–50%) are presented in Figure 9b. It was carried out to determine the optical constants: namely the position of the edge of the fundamental band and the optical bandgap (Table 2).

**Table 2.** Values of absorption edge (Ed), bandgap (Eg), band tail (Ee), and carbon cluster number N of CS-CL-CNC7:3–50% bio-composite film [6], and CS-CL-CNC-AgNPs (5–50%) development bio-nanocomposite films, at ambient temperature.


Figure 9b demonstrated the optical absorption coefficients versus the photon energy of the biofilm mixture of CL-NCN and starch (CS-CL-CNC7:3–50%) and the bionanocomposites films (CS-CL-CNC-AgNPs (5–50%). In particular, the optical absorption coefficients of the CS-CL-CNC7:3–50% film decrease as the AgNP content increases (Table 2). This reduction in Ed is attributed to the increase in the number of charge carriers by the structural modifications of the polymer matrix, which is due to molecular interactions between polymeric chains and CL-WE-AgNP [72]. They are also related to the changes in the number of electrons and the holes in the conductive and valence bands [73]. Specifically, the optical absorption edge shift explains the electronic coupling between CL-WE-AgNPs and CS-CL-CNC7:3–50% [72,73].

The optical bandgap energy (Eg) is the most interesting parameter for integrated optical optoelectronic and photovoltaic devices [74]. Therefore, by extrapolating the linear region to the abscissa, we obtain the energy of the optical bandgap of the amorphous material (Figure 10a). Table 2 shows that the energy value of the bandgap of the CS-CL-CNC7:3–50% film without CL-WE-AgNPs is 3.12 eV and decreases with increasing AgNPs content to 2.58 eV for the CS:CL-CNC-AgNPs 50% nanocomposite film. In this case, the bandgap energy is lower (<3 eV), indicating that it is a semi-conductor [75]. In particular, the reduction of Eg of the polymer matrix of the different films is attributed to the fact that the content of CL-WE-AgNPs is responsible for modifying the degree of disorder of the polymer as well as its structure. As a result, these changes reflect the localized states in the bandgap, which are responsible for decreasing the bandgap energy of the polymer [39,76]. Thus, the reduction in the bandgap is due to the addition of CL-WE-AgNPs in the CS:CL-CNC-7:3–50% matrix, which was carried on from our previous article for the current study [6]. This is due to the increased carrier–carrier interaction in the valence and conduction bands and subsequently the displacement of the valence and conduction band. However, the decrease in Eg reflects the formation of charge transfer complexes (CTCs) as trap levels between the bands of the HOMO, which is mainly carried by the silver metal and is an MO type Ag-d(π), and the LUMO, which is carried by the polymer matrix and it is an MO type (π\*) [77]. This highlights the good miscibility between the CL-WE-AgNPs and the polymer matrix [39].

**Figure 10.** (**a**) Relationship between (αhν) <sup>2</sup> verses photon energy (hν), (**b**) Absorption coefficient Ln α versus photon energy for CS-CL-CNC7:3–50% bio-composite film [6], and CS-CL-CNC-AgNPs (5–50%) development bio-nanocomposite films at ambient temperature.

The Urbach curve is shown in Figure 10b, and the logarithm of the absorption coefficient (α) is plotted as a function of the photon energy (hv). Table 2 groups the estimated values of the tail of the samples (Ee) which were obtained by the inverse of the slope of the linear part of these curves (Equation (3)). The absorption edge called the Urbach energy, and it depends on induced disorder, static disorder, temperature, thermal vibrations in the lattice, strong ionic bonds and on average photon energies [78]. In our study, the temperature and the thickness of films were fixed, respectively, at ambient temperature and 0.02 cm. It is apparent in Figure 10b that the incorporation of CL-WE-AgNPs significantly reduces the absorption edge and decreases the Ee values in the films (Table 2) [79]. Indeed, the values of Ee decrease from 6.89 to 3.62 eV with the increase in the concentration of CL-WE-AgNPs from 0 to 50% by weight. This decrease in the Urbach energy is the result of the decreased disorder of the biopolymers matrix, and/or this was attributed to an increase in crystalline size [78]. Results were explained by several studies, showing that AgNPs [39] and other nanoparticles such as carbon nanotubes [79] lead to a redistribution of states from the band to the tail and thus promote a large number of tail-to-tail transitions [39,79]. Other studies showed that the width of the Urbach tail decreased by moving from thicker film to finer film, which meant from order to disorder [80]. However, the addition of CL-WE-AgNPs costs a charge transfer complex and reduces both Eg and Ee. At the same time, nanocomposite films achieve a good transparency of about 20%, and these values are suitable for certain applications. The correlation between Urbach energy and film thickness should be investigated.

The carbon atom number in clusters of CS-CL-CNC7:3–50% films and different bionanocomposite films were calculated (Equation (4)) and are regrouped in Table 2. The value of N for a bio-composite film without CL-WE-AgNPs is around 121.56, which increases to 177.78 in CS:CL-CNC-AgNPs 50% bio-nanocomposites. The increase in N value is due to the conjugation in monomer units of CS:CL-CNC7:3–50% matrix post embedding of AgNPs. Meanwhile, the band tail and the number of carbon clusters in the samples increased with increasing AgNPs contents [39].

The increase might be due to the breaking of electrons in the C–H bonds due to the liberation of hydrogen [81]. During irradiations, there is the release of gases from the polymeric material. These released gases such as H2, H, CO, and CO2 can be led to the carbonaceous clusters (being rich charge carriers) in the polymer matrix. This carbonaceous cluster impacts the various physical properties of the polymeric material. Hence, the increase may be due to the carbon network and bonding of polymer–metal, and it ensures the conductivity of polymers [40]. Another study also shows that the value of the optical bandgap Eg shows a decreasing trend with the fluence of the irradiated ions and with two kinds of ions (Si8+ and Ne6+) [82]. In addition, the number of carbon atoms per conjugation length increases with the increase in the ion dose built into the bio-composite matrix [82]. The formation of these clusters in polymer films with ion irradiation has been investigated extensively [82,83], and it is explained by the carbon clusters, which are supposed to be carriers in electrical conductivity, being formed along the latent pathways of energy ions in polymers.

#### **4. Conclusions**

The improvement of the optical properties of the bio-composite film was obtained by integrating ex situ variable percentages of CL-WE-AgNPs (5–50%) synthesized from *C. linum* by green means. SEM/EDX confirmed a uniform dispersion of CL-WE-AgNPs in the bio-composite matrix with small agglomerations. A fundamental study of the optical properties of films was carried out to determine the absorption of UV with the presence of AgNPs in composites. However, a decrease in optical bandgap, edge absorption, and Urbach energy was observed compared to the CS:CL-CNC7:3–50% film developed in our recent study. The bandgap of the bio-composites decreases from 3.12 to 2.58 eV with the increase in the AgNPs content to 50%. In this case, the bandgap energy is lower (<3 eV), indicating that it is an improved semi-conductor. The decrease in Urbach energy results from a decrease in the matrix of biopolymers and/or an increase in crystalline size. Furthermore, the cluster carbon number increased, respectively, from 121.56 to 177.75 from bio-composite to bio-nanocomposite with 50% AgNPs. This is due to the presence of a strong H-binding interaction between the bio-composite matrix and the AgNP molecules. Consequently, the incorporation of AgNPs into the bio-composite film CS/CNC costs a load transfer complex and reduces Eg and Ee. At the same time, nanocomposite films attain a good transparency of about 20%. It could be applied to potential applications in the field of food packaging and can be used successfully on opto-electronic devices. Due to the promising properties of bio-composites, several new ones are emerging, and the assessment of their risks requires an individual approach of each nanomaterial. As a result of concerns about the safe use of bio-nanocomposites, further research into their mechanical properties and biological activity is required to provide a clear response regarding how and what nanomaterials can be a viable alternative to applications in many different areas.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/polym15092148/s1, Table S1: Assignment of bands found in FTIR spectra of different isolated water extract, silver nanoparticles and bio-nanocomposite.

**Author Contributions:** Both authors contributed equally to this study, including experiment performance, data analyses, discussion, writing, and reviewing. All authors have read and agreed to the published version of the manuscript.

**Funding:** The publication was funded by the Deanship of Scientific Research, Taif University for funding this work.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** The researchers would like to acknowledge Deanship of Scientific Research, Taif University for funding this work.

**Conflicts of Interest:** The authors declare that they have no conflict of interest.

#### **References**


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## *Article* **Physico-Mechanical, Thermal, Morphological, and Aging Characteristics of Green Hybrid Composites Prepared from Wool-Sisal and Wool-Palf with Natural Rubber**

**Seiko Jose 1,2, Puthenpurackal Shajimon Shanumon 3, Annmi Paul 3, Jessen Mathew <sup>3</sup> and Sabu Thomas 1,3,\***


**Abstract:** In the reported study, two composites, namely sisal-wool hybrid composite (SWHC) and pineapple leaf fibre(PALF)-wool hybrid composite (PWHC) were prepared by mixing natural rubber with equal quantities of wool with sisal/PALF in a two-roll mixing mill. The mixture was subjected to curing at 150 ◦C inside a 2 mm thick mold, according to the curing time provided by the MDR. The physico-mechanical properties of the composite *viz*., the tensile strength, elongation, modulus, areal density, relative density, and hardness were determined and compared in addition to the solvent diffusion and thermal degradation properties. The hybrid composite samples were subjected to accelerated aging, owing to temperature, UV radiation, and soil burial tests. The cross-sectional images of the composites were compared with a scanning electron microscopic analysis at different magnifications. A Fourier transform infrared spectroscopic analysis was conducted on the hybrid composite to determine the possible chemical interaction of the fibres with the natural rubber matrix.

**Keywords:** aging; coarse wool fibre; hybrid composites; natural rubber; PALF; sisal fibre

### **1. Introduction**

Green composites are expected to be the next generation of sustainable composite materials, and both academia and industry are interested in them [1]. These materials are made from natural resources that are renewable, recyclable, and biodegradable. Green composites are typically made by combining natural resins with plant and animal fibres. Natural fibres are demonstrating that they are a more ecologically friendly, cost-effective, and a lighter alternative to synthetic fibres [2]. Bio-resins, which are derived from protein, starch, and vegetable oils, have been created as an alternative to petroleum-based polymers. Compared to synthetic fibres, natural plant-based fibres have a number of clear advantages, such as a reasonable price, good mechanical properties, thermal and acoustic insulation, and can degrade naturally. The short natural fibre reinforced rubber composites have been found to possess a good dimensional stability and high green strength [3].

Sisal is a commercially valuable fibre, extracted from *Agave sisalana* leaves. It is primarily utilized in the production of carpets, insulating panels, and maritime ropes and is commercially grown in Brazil, Tanzania, Kenya, and Madagascar. It has tremendous tensile strength and is quite robust. Many research attempts have been reported for the use of sisal fibre in composites [4]. Pineapple is one of the most popular fruits extensively grown in Costa Rica, the Philippines, Brazil, Thailand, China, and India. The pineapple leaf fibre (PALF) is extracted from the leftover leaves of the pineapple plant. Of all of the natural fibres derived from plant leaves, PALF has the largest proportion of cellulose content and the lowest microfibrillar angle, which results in an exceptionally good tensile strength. PALF isused for a variety of purposes, such as the creation of textiles [5], paper [6], and

**Citation:** Jose, S.; Shanumon, P.S.; Paul, A.; Mathew, J.; Thomas, S. Physico-Mechanical, Thermal, Morphological, and Aging Characteristics of Green Hybrid Composites Prepared from Wool-Sisal and Wool-Palf with Natural Rubber. *Polymers* **2022**, *14*, 4882. https://doi.org/10.3390/ polym14224882

Academic Editor: Raffaella Striani

Received: 20 October 2022 Accepted: 10 November 2022 Published: 12 November 2022

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

composites [7]. Both PALF and sisal fibres are extracted by a mechanical extraction process known as decortication.

Wool is a protein fibre obtained from sheep. Based on the fibre fineness, it is categorized as fine, medium coarse and very coarse (kempy). The fine and medium coarse wool is employed in the apparel and carpet industries, respectively. The very coarse wool fibre has medullation, as result it is highly brittle and does notfind applications in the above said industries. Currently, they are used in decentralized sectors, *viz*., quilt industries, handmade felt, and so forth [8]. Few studies have been reported about the use of coarse wool in composites [9–11].Natural rubber (NR) is one of the unavoidable polymers in the world. It is extracted as a resin from the *Hevea brasiliensis* tree and further dried into sheets or blocks. The plastic qualities of the natural rubber are transformed into elastic through vulcanization, ultimately resulting in the hardness and resilience of NR. The NR is a highly preferred matrix for the composite researchers.

Several recent studies have been reported, regarding the fabrication of PALF and sisal fibre reinforced natural rubber composite [12,13]. Sisal fibre is considered as an important reinforcement, due to the presence of excess cellulose components which make them less susceptible to moisture [14]. The physical and mechanical characteristics of the hybrid composites are determined by factors, such as type of fibre, aspect ratio, orientation, length, and interfacial adherence to the matrix [15].

The objective of our work is to give a value addition to the highly coarse wool, which has no other purpose at the moment. In our previous work, we employed coarse wool fibre as reinforcement in the rubber matrix and subsequently made few prototypes. However, we realized the need of improving the mechanical properties of the wool- NR composite, without compromising the "natural touch". Thus, it is decided to mix coarse wool with appropriate plant fibre and to prepare the hybrid composites with better mechanical properties. As per the author's knowledge, the hybrid composite of wool with other plant fibres in the natural rubber matrix, has never been reported. Hybrid composites are often prepared to conceal the flaws of one or more component fibres. Many research attempts have been reported on the hybrid composites of natural fibres [16,17]. In this study, two hybrid composites (wool + sisal + rubber) and (wool + PALF + rubber) were prepared by mixing equal quantities of wool with sisal/PALF in a rubber matrix. The morphological, thermal, physico-mechanical properties, and accelerated aging of these hybrid composites were analyzed and compared.

#### **2. Materials and Methods**

The natural rubber of grade ISNR-5 (Indian Standard Natural Rubber) was sourced from M/S Malankara Plantations, Thodupuzha, Kerala, India. The coarse wool fibre (Patawadi sheep breed) (bundle strength—13.67 g/tex, fibre diameter—44 microns), sisal fibre (bundle strength—30.9 g/tex, fineness—30 tex, density—1.45 g/cm3) was supplied by ICAR- Central Sheep and Wool Research Institute, Avikanagar, Rajasthan, India, and ICAR-Sisal Research Station, Odisha, India, respectively. The PALF was extracted from remnant pineapple leaves after cultivation, using a decorticator at Mahatma Gandhi University, Kottayam, Kerala, India. (bundle strength—38.5 g/tex, fineness—3.5 tex, density—1.43 g/cm3). The chemicals used in the rubber vulcanization *viz*., sulphur (sp. gravity 2.05), zinc oxide (sp. gravity 5.55), stearic acid (sp. gravity 0.85), Wingstay L (antioxidant), and CBS (N-cyclohexylbenzothiazylsulphenamide), were purchased from Sameera Chemicals, Kottayam, Kerala, India.

#### *2.1. Preparation of the SWHC and PWHC*

The wool, sisal, and PALF fibre were chopped in to 1.5 cm length. The NR was adequately masticated in a two-roll mixing mill (300 × 500 cm) for two minutes. The masticated rubber, the vulcanizing agents, and the fibreswere combined, as mentioned in Table 1. Just before adding sulphur, the wool fibre was added to the NR polymer matrix, along with the sisal fibre for the SWHC and withthe PALF for the PWHC. Care was taken

to preserve the compound flow direction so that the majority of the fibres followed the same flow path. In order to ensure an equal distribution of thefibres in the polymer matrix, samples were milled for 10 min [18].


**Table 1.** List of rubber compounding ingredients [19,20].

phr indicates parts per hundred of rubber.

Using a Moving Die Rheometer (Rheometer MDR 2000, Alpha Technology), the curing properties of the SWHC and the PWHCwere studied, in accordance with the ASTM D5289 method at 150 ◦C. The compositeswerevulcanized at 150 ◦C inside a 2 mm thick mold, according to the curing time provided by the MDR at 100 bar pressure (5 min for curing for both composites (see t90 vales in Table 2). Following thevulcanization, the hybrid composite samples were removed from the mold and cooled. The samples were pre-conditioned at 25 ◦C and 65% RH before further analysis. For each set of composites, fivereplicas were prepared. The cure rate index, which is a measurement of difference between t90 (optimum cure time)and ts2 (insipient scorch time), was calculated using the formula [21]

$$\text{Cure rate index} = 100 / (t90 - t \text{s} \mathbf{2}) \tag{1}$$

**Table 2.** Torque and cure time values of the SWHC and PWHC samples.


#### *2.2. Analysis of the Physico-Mechanical Properties of the Composites*

A universal testing machine (Tinius Olsen H50KT) was employed for the determination of the tensile and tear strength of the SWHC and PWHC. The samples were analyzed, in accordance with the ASTM D412 and ASTM D624 standards, respectively. Three samples were tested for each composite and the average result was calculated. The moisture content of the hybrid composites was determined, in accordance with ASTM D2495-07. The hardness of the SWHC and PWHC was assessed with the aid of a Shore-A hardness tester (Presto), following the ASTM D-2240 guidelines. The areal density of the composites was calculated, using the following formula.

$$Area\ density = \frac{Weight\ of\ the\ composite\ \left(\text{g}\right)}{Area\ of\ the\ composite\ \left(cm^2\right)} \times 10000\tag{2}$$

The relative density of the SWHC and PWHC in water was calculated according to ASTM D792, using the equation below.

$$\text{Relative density} = \left(\frac{\text{Weight in air}}{\text{Weight in air} - \text{Weight in water}}\right) \times \text{Density of water} \tag{3}$$

The SWHC and PWHC were analyzed for their solvent diffusion properties using water and toluene. Three replicas from each composite were cut in a round disc shape (2 cm diameter).Prior to dipping in the solvent, both specimens underwent preconditioning (25 ◦C and 65% RH) and were weighed. The hybrid composite samples were dipped in their respective solvents and removed from the solvents in predefined intervals. The specimens were gently hand pressed in between a blotting paper, to remove the surplus solvent and weighed. The procedure was repeated until a swelling equilibrium was reached [18].The mole% solvent uptake of the composite samples was calculated using the Equation (4). Qt represents the solvent's mole% uptake at a certain time t. Further, to investigate the diffusion properties of the SWHC and PWHC with water and toluene, a graph of Qt vs. √<sup>t</sup> was generated.

$$\text{Qt} = \left(\frac{\left(\text{Mass of solvent absorbed by the composite} / \text{Molar mass of the solvent}\right)}{\text{Initial mass of the composite}}\right) \times 100\tag{4}$$

The crosslink density, associated with the composites immersed in toluene is calculated using the following sets of equations below [18]

$$\mathbf{U} = 1/2M\mathbf{c} \tag{5}$$

$$Mc = -\frac{\rho\_{\text{p}} \text{ Vs } \phi \text{\AA}^{\text{1}} \text{\AA}}{\left(\ln\left[1-\phi\right]+\phi+\text{\mathcal{K}}\phi^{2}\right)}\tag{6}$$

$$\Phi = \frac{\frac{\text{W1}}{\rho\_{\text{p}}}}{\left(\frac{\text{W1}}{\rho\_{\text{p}}}\right) + \left(\frac{\text{W2}}{\rho\_{\text{s}}}\right)}\tag{7}$$

$$\chi = \text{ } \pounds + \frac{\text{Vs}}{\text{RT}} \left( \delta p - \delta \text{s} \right)^2 \tag{8}$$

The crosslinking density of the material is given by Equation (5). Equation (6) is used to compute the molar masses between the crosslinks, or "*Mc*". Equation (7) is used to compute "φ", which is the volume fraction of rubber at the swelling equilibrium. "ρp" stands for the polymer density, "ρs" for the solvent density, and "Vs" for the molar volume of each solvent. Equation (8) can be used to calculate "χ" which is the interaction parameter between the polymer and the solvent. Equation (6) would be used to derive "Mc", using the values of "φ" and "χ", as determined by Equations (7) and (8), respectively. In Equation (4), "β" "*δs*", and "*δp*" stand in for the lattice constant (zero for polymers), the solubility parameter of the solvent, and the solubility parameter of the polymer, respectively. "R" is the universal gas constant and "T" is the temperature. For all testing, five replicas were made and the average value was taken.

#### *2.3. FTIR, SEM, TGA, and the Aging Analysis of the Composites*

With a Perkin Elmer Spectrum-2 spectrometer, the FT-IR spectra of theSWHC and PWHC were recorded across a range of 4000 cm−<sup>1</sup> to 400 cm−1, using the attenuated total reflection (ATR). The spectrum was obtained after 24 consecutive scans. The JEOL-JSM-6390 scanning electron microscope was used to examine the surface morphological properties of the hybrid composites. The samples were sputter coated with gold-palladium to prevent electron beams from having any charge effects during the examination. The images were captured at various magnifications at a 20 kV accelerating voltage. The

thermogravimetric analysis was carried out, using TA instruments (SDT Q600) in an inert atmosphere at temperatures ranging from 25 ◦C to 700 ◦C. The heating rate, 10 ◦C/min and a DTA sensitivity of 0.001 ◦C were maintained throughout the analysis. The composite samples were subjected to accelerated aging to temperature (ASTMD 573-04), UV, and biodegradation as per the standard methods reported in our previous studies [22].

#### **3. Results and Discussion**

#### *3.1. Cure Characteristics*

Figure 1 shows the cure characteristics of the SWHC and PWHC. It is apparent from the figure that both composite mixtures followed almost the same pattern in the MDR curve. It can be clearly understood from the graph that the time required for the initiation of the crosslinking is nearly the same for the SWHC and PWHC. There is an initial decrease in torque that was noted in both the SWHC and PWHC, because of the softening of the rubber polymer matrix, when subjected to heat. When the crosslinking was initiated, with respect to time, the torque increased to a maximum, where the crosslinking was the highest and then showed a slight reduction, and then became almost constant. The corresponding torque and cure time values are shown in Table 2.

**Figure 1.** Cure characteristics of the SWHC and PWHC.

In fibre reinforced rubber composites, the maximum torque is an indication of the extent of the crosslinking and the stiffness, while the minimum torque indicates the fibre content present in it [23]. The maximum torque for the PWHC (48.68) is marginally higher than that for the SWHC (38.36). The value of ts2 (insipient scorch time) is same for both composites, which indicate that the vulcanization in both composites begins at the same time and the similar value of t90 shows that the vulcanization proceeds to a completion at the similar time, for the composites.

Regardless of the same values for t90 and ts2 in both the SWHC and PWHC, the maximum torque for the PWHC was found to be high. This indicates that the crosslinking associated with the vulcanization was increased by the addition of PALF in the wool-NR matrix, in comparison to the addition of the sisal fibre. The cure rate index is almost same for both composites, which indicates that the rate of curing is almost the same for the SWHC and PWHC [24].Since the rubber content is lower in the NR hybrid composite, the cure curve declined after reaching t90. This might be due to the over curing of the composites. Similar observations have been reported elsewhere [25].

#### *3.2. Tensile and Tear Properties*

The stress–strain curve of the composite samples is depicted in Figure 2. The curves indicate that the PWHCs could withstand more stress in comparison with SWHC, meanwhile the latter possess a higher elongation. The corresponding tensile strength data is shown in the Table 3.

**Figure 2.** Stress–strain curve of the SWHC and PWHC.


It is evident from Table 3 that the tensile strength for the PWHC (11.14 MPa) is almost double thanthat for the SWHC (6.09 MPa). The higher tensile strength of the PWHC, in comparison with the SWHC may be due to the following reasons. (i) The higher tensile strength of the PALF (38.5 g/tex) than sisal fibre (30.9 g/tex), (ii) the dense packing of the fibre, the lack of voids and a better interfacial adhesion of the PALF in the rubber matrix, as observed from the SEM images, (iii) the better transfer of stress between the NR and PALF, compared to that between the NR and sisal fibre, as indicated by the tear analysis (Table 3). Thus, Young's modulus associated with the PWHC is higher than that of the SWHC. The lower value of Young's modulus and the elongation (%) indicates that the PWHC has a much better resistance to elastic deformation, compared to the SWHC.

The tear strength of a polymer indicates the ability of the polymer to withstand tearing or cracking, when they are subjected to an external force. The tear strength of the PWHC (85.0 N/mm) is significantly higher than that for the SWHC (45.9 N/mm). This is because the transfer of stress in the PALF incorporated composite, is better than that in the sisal fibre incorporated composite. The lower tear strength is also an indication of a poor interaction between the fibre and the polymer matrix [26]. The SWHC possessed a higher elongation at break (5.42%) than the PWHC (4.49%).

#### *3.3. Moisture Absorption and the Hardness Properties*

In comparison with the synthetic fibre composites, the moisture content of the SWHC and the PWHC was found to be in a higher range (Table 4). The higher moisture content of the developed hybrid composites may be due to the high inclusion (100 phr) of hydrophilic natural fibres. In comparison with the SWHC, the PWHC has a marginally lower moisture content (5.90%), perhaps due to the fact that sisal fibres absorb more moisture than the PALF [27]. In the context of composites, the high moisture content of the natural fibre is a major concern to researchers. Sisal and PALF are lignocellulosic, and the presence of hemicelluloses causes a high moisture uptake. The presence of moisture in the natural fibre reinforced composites causes a weaker interaction between the fibres and the matrix [28].


SWHC 6.57 91.30 2707.84 1.09

**Table 4.** Moisture content and hardness of the PWHC and SWHC.

Rubber is a soft polymer. The inclusion of natural fibres, such as wool, sisal, and PALF, in large quantity significantly increase the hardness of the composites. A good network formation of a natural fibre inside of the soft rubber polymer matrix may be the reason behind it, as a result, the Shore A hardness increased. The hardness of the PWHC was 91.56 and that of the SWHC was 91.3. Though there exists a considerable difference in the mechanical properties, hardness (91), areal density (2700 g/m2), and relative densities (1.11 g/cm3) of both, the PWHC and SWHC were found to be almost the same. It can be seen from Table 4 that the relative density of both hybrid composites is almost same.

#### *3.4. FTIR Analysis*

Figure 3a displays the FTIR spectra of sisal, PALF, and wool fibre. The sharp peak for wool fibre, seen at 1640 cm−1, is due to the amide I group present in the wool protein, while the peak at 3272 cm−<sup>1</sup> denotes the amide N-H stretching vibration. The bending vibration of the C-N-H bond corresponds to the peaks at 1520 cm−<sup>1</sup> [29–31]. The FTIR spectra shows similar peaks for both the PALF and sisal, as theyare lignocellulosic fibres. In case of the sisal fibre and PALF, the broad peak at 3332 cm−<sup>1</sup> corresponds to the -OH stretching vibrations from cellulose while the symmetric and asymmetric stretching of the CH2 groups is indicated by the peak at 2886 cm−<sup>1</sup> [32]. The peaks at 1732 cm−<sup>1</sup> can be attributed to the stretching vibration of C=O groups in hemicellulose and the peaks in between 1627–1606 cm<sup>−</sup>1, indicate the aromatic C=C stretching vibrations in lignin [33,34]. It can also be observed that the intensity of the peak at 1606 cm−<sup>1</sup> is higher for the sisal fibre, compared to PALF, indicating that the lignin content is higher for the sisal. The C-O/ C-C group stretching is indicated by the peaks at 1017 cm−<sup>1</sup> [35].

**Figure 3.** FTIR spectrum of (**a**) sisal fibre, PALF and wool fibre (**b**) vulcanized rubber, PWHC and SWHC.

It can be clearly observed, from the Figure 3b, that the SWHC, PWHC, and the vulcanized rubber shows almost the same peaks. The peaks observed at 2920 cm−<sup>1</sup> and 2848 cm−<sup>1</sup> indicate the symmetrical stretching of the -CH3 bonds and -CH2- bonds, respectively. The characteristic in-plane bending of the amide II group in the wool protein is denoted by the peak at 1536 cm−1. The sharp peaks observed at 1452 cm−<sup>1</sup> and 1370 cm−<sup>1</sup> indicate the deformation of the -CH3 bonds, while the out of plane bending for the C=C-H group is shown by the peak at 830 cm−<sup>1</sup> [36]. All of these peaks are characteristic of the vulcanized rubber sample. It is observed from the spectra that the peaks corresponding to the wool, PALF, and sisal are not prominent and are masked by the natural rubber. No shifts in the peaks, even after the addition of the fibres, indicates that there is no chemical interaction between the fibres and the polymer matrix, leading to the conclusion that the interaction may be physical, involving van der Waals forces or hydrogen bonds.

#### *3.5. SEM Analysis*

Figure 4a–d shows the cross-sectional scanning electron microscopic images of the SWHC and Figure 4e–h shows that of the PWHC. It is visible from the images that in both the composites, a good network of fibres is present throughout the polymer matrix. It can also be seen from the images that the wool fibre (with scales) is distributed evenly with the sisal fibre (without scales) in the SWHC and with PALF (without scales) in the PWHC. Most of the fibres are in a uniform direction inside the rubber matrix. The absence of large voids in the composites indicates that the hybrid composites are properly prepared without the entrapment of air inside them. However; while comparing the SEM images (Figure 4d,h), which is of the cross sectional images of the SWHC and PWHC, respectively, it is found that the number of voids are comparatively higher in the SWHC than in the PWHC. This indicates a good adhesion between the PALF and NR matrix. The fibre pull-out is visible from the matrix in Figure 4b,f, and the images suggest that in the PWHC (Figure 4h), the voids formed at the root of the pulled-out fibres, are relatively small when compared to that in the SWHC. The higher interfacial adhesion between the natural fibres and the polymer matrix keeps the fibres intact with the matrix, which can also be the reason for the higher tensile strength, shown by the PWHC (11.14 MPa), compared to the SWHC (6.09 MPa) [37]. It has been reported that the formation of large voids is an indication of the poor interfacial adhesion between the fibres and the matrix [13].It is inferred from the SEM analysis that the failure mechanism in these composites was the fibre pull-out, the fibre fracture, and the interfacial debonding.

Figure 5a shows the TG curve of the SWHC and PWHC. Due to the similarity in the chemical nature and composition, both composites followed almost the same pattern. As discussed regarding the physical properties of the composites, the composites have a moisture content of 6–7%. The minor weight loss at 110 ◦C, may be due to the removal of moisture from the composites. The TGA shows a major weight reduction between 250 ◦C and 400 ◦C and both composites showed a weight loss of 84.14%, until a constant weight was reached at a temperature of 442.5 ◦C.

The thermal degradation of the composites may be explained, based on the degradation of the individual components. In the case of the wool fibre, the thermal degradation takes place in three steps [38]. The first stage of the degradation takes place between 100 ◦C and 135 ◦C and is attributed to the loss of moisture content [39]. During the second step, a maximum weight loss occurred between 218 ◦C and 390 ◦C, due to the breakdown of the microfibril-matrix structure and the disulfide linkages [38]. In the third step, various peptide bonds present in the wool were broken at around 390–500 ◦C. Above 500 ◦C, the char oxidation reactions dominated [31,40].

**Figure 4.** SEM images of the SWHC (**a**–**d**) and the PWHC (**e**–**h**) at various magnifications.

#### *3.6. Thermogravimetric Analysis*

Being lignocellulosic in nature, in both the sisal and PALF, after the removal of moisture at 110 ◦C, the second weight loss corresponds to hemicellulose's degradation that starts at about 190 ◦C. Further, cellulose starts degrading from 290 ◦C up to 360 ◦C. The lignin degradation starts at about 280 ◦C and continues even above 500 ◦C [32]. For the vulcanized rubber, the degradation begins at about 200 ◦C and is completed at about 475 ◦C, where the maximum weight loss is obtained at 358 ◦C, which may be attributed to the oxidation of the rubber [41]. It is also inferred from the data that the incorporation of wool, sisal, and PALF, slightly increases the thermal stability of the vulcanized rubber.

**Figure 5.** (**a**) TGA and (**b**) DTG curve of the SWHC and PWHC.

The degradation process has demonstrated one corresponding weight-loss peak in the DTG curves, as shown in Figure 5b, which corresponds to a single turn in the TG curves and was caused by thermal scissions of the C-C chain bonds in the natural rubber matrix [42].The DTG curves show that at 361.67 ◦C, both composites show an equal rate of weight loss (0.8373%/◦C), with respect to the temperature. The results indicate that the SWHC and PWHC possess a similar range of magnitude when considering their thermal stability, which may be due to the fact that both sisal fibre and PALF are plant fibres that havean almost similar chemical structure and properties.

#### *3.7. Solvent Diffusion*

The uptake of toluene and water by the SWHC and PWHC, via the diffusion, was analyzed and plotted between Qt (mole% uptake of solvent) and √t (min). The process of diffusion is a parameter for the kinetics and is related to the nature of the polymer, the nature of the fillers added, its free volume, the extent of crosslinking, etc. [18]. It is apparent from Figure 6a that the rate of diffusion and the quantity of the toluene absorption is higher in the SWHC, in comparison with the PWHC. The low absorption and diffusion of toluene in the PHWC may be due to the dense packing of PALF inside the rubber matrix, which restrict the diffusion of the aromatic solvent [43]. This is also supported by the SEM images, which showed a higher packing density, a better adhesion, and less void contents in the PWHC than the SWHC. At the time of saturation, the SWHC showed a weight gain of 184.65%, while it was 95.85% for the PWHC.

**Figure 6.** Diffusion curve of the SWHC and PWHC in (**a**) toluene (**b**) water.

Interestingly, it can be also seen from Figure 6b, that the mole% uptake of water does not show much difference for both the SWHC and PWHC, although the SWHC graph shows a marginally higher uptake of water. Both composites showed similar rates of diffusion up to the saturation. The water may enter the composite though the small cracks and pores and generates diffusion pathways. Both the sisal fibre and PALF are hygroscopic and allow the diffusion of water through them whilst, the matrix is hydrophobic. At the time of saturation, the SWHC gained 14.99% and the PWHC gained 14.39% weight, respectively. These high water absorption properties, though common in natural fibre-reinforced composites, are not in an appreciable quality for the composites.

It can also be observed from Table 5 that both composites possess a similar crosslink density. The crosslink density was defined as the density of chains or segments that connect two infinite sections of the polymer network [44]. The value of "Mc", which is the molar masses between the crosslinks, is so high that it can be considered as an indication of the greater crosslinking of the networks present in the composites [44].

**Composite Mc (g/mol) Crosslink Density (g**·**mol/cc)** SWHC 62,840.43 7.96 <sup>×</sup> <sup>10</sup>−<sup>6</sup> PWHC 65,247.92 7.66 <sup>×</sup>10−<sup>6</sup>

**Table 5.** Crosslink density and Mc of the SWHC and PWHC.

#### *3.8. Accelerated Thermal and UV Aging*

The composites of the NR are susceptible to degradation by heat, UV radiation, ozone, humidity, etc. [45]. The PWHC and SWHC (Figure 7) were subjected to the accelerated thermal and UV degradation and the change in their mechanical properties were analyzed. In large chain macromolecules with complicated crosslinked structures, the application of heat, as well as radiation, can cause scissions, not only to the main chain, but also to the side chains which may lead to a loss of weight and the emission of gases with low molecular weights. As a result, the exposure to heat/radiation can cause changes to the chemical structure of the composites, such as the chain scission, crosslink formation, and breakage [46].It can be observed from Figure 8 that there is a slight increase in the tensile strength and Young's modulus after the thermal aging for both the SWHC and PWHC. This increase might be due to the formation of new crosslinks when the vulcanized NR is subjected to heating [47]. When exposed to prolonged UV radiation, the tensile strength increased for both the SWHC and PWHC, although there was a significant reduction in Young's modulus of the material. The trend shown by the un-aged, thermal aged, and UV aged samples for both the PWHC and SWHC, was similar, such that there is an increase in their tensile strengths. In the case of their stress % (elongation at break %), the two composites showed a different trend, such as in the SWHC, the stress % demonstrates an increase after the UV aging, with respect to the un-aged samples, where the stress % for the PWHC remains stagnant. Similarly, Young's modulus for both the PWHC and SWHC showed a similar trend, such as an increase in the modulus after thermal aging and a decrease in the modulus after UV aging.

**Figure 7.** Images of the SWHC (**green**) and the PWHC (**yellow**). Note: Pigments were added during the composite preparation for identification.

**Figure 8.** *Cont*.

**Figure 8.** Mechanical properties of the SWHC and PWHC after the thermal and UV aging.

#### *3.9. Biodegradation*

Biodegradation is a process in which a compound decomposes due to the enzymes or chemicals secreted by bacteria or fungi present in soil. Both the SWHC and PWHC were subjected to accelerated biodegradation for 60 days through a soil burial test, as mentioned earlier. Once the stated period was over, the reduction in weight for the SWHC was found to be 2.43%, while it was 2.34% for the PWHC. It can be seen from Table 6 that both composites showed an almost equal loss of weight. It appears that the biological decomposition occurred at a very slow rate. This may be due to the following reason. (1) The vulcanized rubber, as it is poorly biodegradable, due to the presence of high crosslinking. (2) Though wool, sisal, and PALF are natural fibres that degrade over time, the presence of lignin in the PALF and the sisal mask and protects the cellulose and hemicellulose from a rapid degradation by the microorganisms, due to the presence of the aromatic and crosslinked structure of the lignin [13]. (3) The extensive packing of fibres in the matrix can slow down the degradation process, since the tight network prevents the excessive growth of microorganisms [48].Above all, these facts and the presence of high amounts of natural fibres in the developed composites resulted in an increase in the absorption of moisture which eventually led to the growth of microorganisms that caused the mass degradation of the composite.

**Table 6.** Weight reduction of the SWHC and PWHC after 60 days of soil burial test.


Note: The values in the parenthesis indicate the standard deviation.

#### **4. Conclusions**

Hybrid green composites, SWHC (sisal fibre + coarse wool fibre + NR) and PWHC (PALF + coarse wool fibre + NR) were fabricated and compared for their morphological, physical, mechanical, and aging properties. In comparison with the SWHC, the PWHC showed a higher tensile strength and modulus, a higher tear strength, a low moisture absorption and low mole% uptake (diffusion) of toluene and water. The PHWC showed a higher torque during the cure analysis. The results obtained from the FTIR spectraprovided no valid evidence for any chemical interaction between the polymer matrix and the natural fibres in both the SWHC and PWHC. An analysis of the SEM images showed that the PWHC had a better packing of fibres, thereby an increased interfacial adhesion between the fibres and the polymer matrix. The thermal degradation characteristics remained as constant. Both the hybrid composites showed a slow pace of degradation while subjected to the soil burial test. It is concluded that the newly developed hybrid composites can be regarded as good substitutes for non-biodegradable composites and can be considered as potential material for packing and household applications.

**Author Contributions:** Conceptualization, S.J.; Methodology, J.M.; Validation, S.T.; Formal analysis, J.M., P.S.S., A.P. and S.J.; Investigation, S.J., J.M. and S.T.; writing, P.S.S., A.P. and S.J.; Original draft preparation, P.S.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


### *Article* **PLA-Based Hybrid Biocomposites: Effects of Fiber Type, Fiber Content, and Annealing on Thermal and Mechanical Properties**

**Supitcha Yaisun 1,2 and Tatiya Trongsatitkul 1,2,3,\***


**Abstract:** In this study, we utilized a hybridization approach for two different fibers to overcome the drawbacks of single-fiber-reinforced PLA composites. Coir fiber and bamboo leaf fiber were used as reinforcing natural fibers as their properties complement one another. Additionally, we combined thermal annealing with hybridization techniques to further improve the overall properties of the composites. The results showed that the hybridization of BF: CF with a ratio of 1:2 gave PLA-based hybrid composites optimal mechanical and thermal properties. Furthermore, the improvement in the thermal stability of hybrid composites, attributable to an increase in crystallinity, was a result of thermal annealing. The improvement in HDT in annealed 1BF:2CF hybrid composite was about 13.76% higher than that of the neat PLA. Annealing of the composites led to increased crystallinity, which was confirmed using differential scanning calorimetry (DSC). The synergistic effect of hybridization and annealing, leading to the improvement in the thermal properties, opened up the possibilities for the use of PLA-based composites. In this study, we demonstrated that a combined technique can be utilized as a strategy for improving the properties of 100% biocomposites and help overcome some limitations of the use of PLA in many applications.

**Keywords:** polylactic acid; coir fiber; bamboo leaf fiber; hybrid composites; annealing; biocomposite

#### **1. Introduction**

Biopolymers are presently viewed as promising substitutes for traditional petroleumbased polymers, as the latter have contributed to environmental issues related to pollution, greenhouse gas emissions, and the depletion of fossil fuel reserves. Growing environmental awareness and sustainability concerns among consumers have driven industries to search for alternative, more environmentally friendly materials [1]. Polylactic acid or PLA stands out as one of the extensively studied and commonly used biopolymers, garnering significant interest for traditional uses like packaging materials, fiber production, and more recently, in composite materials for diverse practical and mechanical applications. PLA occupies a central role in the eco-friendly polymer market and emerges as a highly promising choice for future advancements [2–4]. However, the use of PLA has been limited by its thermal properties. It has a low heat-softening temperature and low thermal stability as compared with petroleum-based polymers polyethylene (HDPE), polypropylene (PP), and polystyrene (PS).

To overcome these limitations while maintaining a status of being 100% biodegradable, natural fibers used as reinforcement in PLA-based biocomposites have been investigated to improve PLA performance [5]. An increasing tendency has been observed in favor of incorporating natural fibers as a reinforcement in polymer composites. This inclination is driven by their adaptability during processing, well-defined strength characteristics,

**Citation:** Yaisun, S.; Trongsatitkul, T. PLA-Based Hybrid Biocomposites: Effects of Fiber Type, Fiber Content, and Annealing on Thermal and Mechanical Properties. *Polymers* **2023**, *15*, 4106. https://doi.org/10.3390/ polym15204106

Academic Editor: Raffaella Striani

Received: 30 June 2023 Revised: 7 October 2023 Accepted: 9 October 2023 Published: 16 October 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

ready availability, biodegradable nature, cost-effectiveness (in terms of volume), and environmentally friendly attributes. Polymer composites incorporating natural fibers present numerous notable benefits compared with traditional synthetic alternatives, including their biodegradability, environmentally friendly characteristics, affordability, ready availability, low weight, and more. These natural fiber-reinforced polymer composites are receiving increasing recognition and wider acceptance across various applications, such as food packaging, interiors of automobiles, railway coaches, and airplanes, as well as storage solutions and construction.

Recently, coir fibers (CFs) have stood out as excellent choices due to the fruit's remarkable versatility, serving a purpose as a food and contributing to the production of a diverse array of industrial products. The extensive cultivation of coir crops results in substantial biomass accumulation, often leading to its disposal in landfills or inappropriate sites, giving rise to significant social and environmental challenges. On the other hand, coir fiber-reinforced polylactic acid (PLA) biocomposites have gained substantial research attention for their use in various applications [6–10]. Coir fibers possess low cellulose (36–43%) and hemicellulose (0.2%), a high lignin content (41–45%), and a high microfibrillar angle (30–45◦, which results in their relatively low tensile strength and modulus as well as the highest elongation at the break among other typical natural fibers [11–13], as shown in Table 1. Because coir fibers possess a significant amount of lignin, they exhibit durability, resistance to weather, a degree of waterproofing, and the potential for chemical modification. Additionally, these fibers can be stretched beyond their elastic limit without breaking, showcasing a high elongation at the break [14]. Many studies in the literature contain comprehensive analyses of the structural, morphological, mechanical, and thermal characteristics of coir fibers [15–20]. Bamboo leaf fiber (BF) is one of the most abundantly available waste materials. BF has a shorter growing time than its culm. Studies on the use of BF as a reinforcement material have not yet been widespread. Up to now, studies have indicated that adding fibers into PLA matrices can improve the performance of PLA composites. The selection of plant fibers determines the end properties of composites [21].

Incorporating natural fibers into polymer composites can be challenging due to certain inherent characteristics that have potential downsides. These characteristics include limited protection against microbial attacks, inadequate resistance to moisture, poor adhesion in the fiber–matrix surface, and a tendency to form aggregates during processing. These limitations can be addressed by alterations to the surface of fibers; this is achievable using chemical techniques like mercerization [22], dewaxing, acetylation, chemical grafting, bleaching, delignification, and salinization [23].

Because each fiber possesses unique advantages and drawbacks, reinforcing a given polymer with a combination of two or more fiber types may help to overcome the drawbacks of each fiber and, subsequently, improve the properties of the composite overall. This technique is known as "hybrid composite" and has recently attracted significant attention from researchers [9,24,25].

To further improve the mechanical properties and service temperatures of PLA-based composites, we used thermal annealing together with hybridization in this study. Thermal annealing can be carried out by subjecting specimens to a high temperature, above its cold-crystallization temperature, and then using a slow cooling rate to induce the formation of a crystallized structure that enhances the thermal properties of the material, as well as the mechanical properties [26–28]. Thus, the service temperature and mechanical performance of the PLA-based composite are expected to improve.

Many researchers try to improve natural fiber composite properties by carrying out chemical treatment of the natural fiber [29]. However, because most of the chemical treatment techniques use strong acidic or basic chemicals, they are inherently harmful to the environment. Therefore, we focused on developing a 100% eco-friendly material in this work. We strategically combined fiber hybridization and annealing to overcome the drawbacks of PLA composites. In this study, first, we investigated the effect of fiber types (coir and bamboo leaf fibers), fiber loading (5, 10, and 15 wt%), and thermal annealing

on morphology, tensile properties, thermal properties, crystallinity, and heat distortion temperature (HDT) of a PLA-based single-fiber composite. Then, we combined the fiber in various ratios to create PLA-based hybrid composites. The fiber loading of 10 wt% was kept constant. The ratio of coir fiber and bamboo leaf fiber was varied. The BF: CF ratios were 1:1, 1:2, and 2:1. The fibers were incorporated into the PLA matrix using twin screw extrusion, and the test specimens were prepared with compression molding. To enhance the crystallization of PLA, annealing of PLA-based hybrid composites was performed at 120 ◦C for 30 min [30]. With these combined techniques, an improvement in the overall properties of composites could be expected.


**Table 1.** The chemical, mechanical, and physical properties of natural fibers [31].

#### **2. Materials and Methods**

#### *2.1. Materials*

Polylactic acid (PLA) (grade LX175, Purac Ltd., Ban Chang, Thailand) used was at an extrusion grade with a density of 1.24 g/cm3 and a melt flow index of 3 g/10 min. Coir (*Cocos nucifera* L., Thailand) and bamboo leaf (*Bambusa ventricosa McClure*, Thailand) were purchased from local farmers in Nakhon Ratchasima, Thailand.

#### 2.1.1. Fiber Preparation

The dried bamboo leaf and coir were crushed into fine fibers with shorter lengths using a wood crusher machine (CT, CGR-20, Chareon Tut Co., Ltd., Samutprakarn, Thailand) for 1 h. The fibers with a diameter in the range of 45–106 μm were obtained. The obtained fibers were given code names as bamboo leaf fiber (BF) and coir fiber (CF).

#### 2.1.2. Preparation of PLA-Based Composites

A list of samples used in this study is shown in Table 2. The composites were prepared with the melt mixing technique using a co-rotating twin screw extruder (Brabender, DSE 35/17D, Brabender GmbH & Co. KG, Duisburg, Germany). The fibers and PLA were dried in a hot air oven at 80 ◦C for 4 h before use. Immediately after drying, the PLA and fiber underwent melt mixing in the twin screw extruder at the screw speed of 20 rpm and melting temperature of 170 ◦C. The compound pellets were then compression molded at 170 ◦C for 10 min to form test specimens (See Figure 1). To investigate the effect of annealing on the composite's properties, samples were annealed at 120 ◦C for 30 min in a hot air oven (Despatch, LAC series, Despatch Industries, Inc., Lakeville, MN, USA) before being left cool at room temperature.


**Table 2.** Composition of PLA-based composites.

**Figure 1.** Photograph of PLA, PLA composite, and PLA-based hybrid composite specimens.

#### *2.2. Characterization and Test*

#### 2.2.1. Tensile Test

The tensile test of PLA and its composite was carried out according to ASTM D638 [32]. Five dog bone-shape specimens with a gauge length of 50 mm were tested at room temperature (~25 ◦C) using a universal testing machine (UTM, INSTRON/5565, Instron Co., Ltd., Norwood, MA, USA). The test was performed using a 5 kN load cell at 5 mm/min crosshead speed. The reported value is an average value from five replications. The error bars shown in the graph represent the standard deviation value.

#### 2.2.2. Morphological Study

The tensile fractured surfaces of PLA and its composites were used in the investigation of the composites' morphological structure. The fractured surface was used as it can reveal information on the distribution and dispersion of the reinforcing agents and the adhesion between the fibers and matrix as well as the failure mode (brittle or ductile fracture). The fractured surfaces were then sputtered coated with gold for 3 min before being examined using a scanning electron microscope, SEM (JEOL, model JSM6400, JEOL Ltd., Tokyo, Japan), at 5–10 kV.

#### 2.2.3. Differential Scanning Calorimetry (DSC)

The crystallization and melting behaviors of PLA and PLA-based hybrid composites were determined using differential scanning calorimetry (DSC: Mettler Toledo STARe SW 8.1, Mettler-Toledo International Inc., Greifensee, Switzerland). A sample was heated from 25 to 200 ◦C with a heating rate of 10 ◦C/min (first heating scan). After keeping the sample at 200 ◦C for 1 min, it was cooled to 25 ◦C. Finally, it was heated again to 200 ◦C (second heating scan). The degree of crystallinity (*Xc*) of the neat PLA, PLA composites, and PLA hybrid composites was calculated using Equation (1) [33].

$$X\_{\rm c} = \frac{\Delta H\_{\rm m} - \Delta H\_{\rm c\varepsilon}}{\omega \Delta H\_{\rm m}^{o}} \times 100\% \tag{1}$$

where Δ*Hm* is the heat of melting and Δ*Hcc* is determined by integrating the areas (J/g) under the peaks. Δ*H<sup>o</sup> <sup>m</sup>* is a reference value and represents the heat of melting if the polymer were 100% crystallinity (93.7 J/g for PLA) [34] and *ω* is the weight fraction of the PLA in the composites.

#### *2.3. Heat Deflection Temperature (HDT)*

An HDT/VICAT manual heat deflection tester (model HDV1, Atlas Electric Devices Co., Chicago, IL, USA) was used to measure the heat deflection temperature (HDT) of PLA and its composites. The test was carried out using a load of 0.455 MPa, as specified by ASTM D648 [35].

#### **3. Results and Discussion**

#### *3.1. Effect of Fiber Type and Content on Tensile Properties of PLA-Based Composites*

The tensile properties including modulus tensile strength, and elongation at break of PLA and PLA single-fiber composites at various fiber loading are depicted in Figure 2. Generally, neat PLA showed better tensile strength and elongation at break than those of the PLA composites. This finding is in agreement with those reported by others [36,37]. The neat PLA possessed the highest tensile strength and elongation at break of 51.85 MPa and 8.13%, respectively. The presence of natural fiber in PLA composite increased the tensile modulus. The maximum tensile moduli were obtained from the composites containing 20 wt% fiber loading. Improvements in tensile moduli in the PLA composites over the neat PLA were 41.18% and 40.20% for 20BF/PLA and 20CF/PLA, respectively.

Increasing the fiber loading resulted in an increase in the tensile moduli of PLA composites, whereas the tensile strength and elongation at break of PLA composites were decreased. The results suggested that with the presence of the natural fiber, the PLA-based composite became more brittle. These results were expected, and they indicated a poor adhesion between the natural fiber and the PLA matrix. Similar results were reported by other researchers who suggested that these findings were associated with a low strength of adhesion among the fibers within composite materials. The decrease in tensile strength with the increasing fiber content may also be due to an agglomeration of fibers in the PLA matrix [36–38]. The poor adhesion among the composite components stemmed from the low hydrophilicity and polarity of PLA as compared with those of the plant fibers, which possess polar hydroxyl groups on their surfaces. The PLA composites reinforced with plant fibers tend to display inadequate interfacial adhesion, consequently leading to ineffective stress transfer from the matrix to the fibers [39–41]. A poor adhesion between the fibers and the PLA matrix could be seen in the results of the morphological study reported in the next section. In addition, it was known that adding rigid particles into a polymer matrix generally resulted in an increased stiffness of the polymer.

As compared with the same fiber content, the presence of BF and CF in the PLA matrix gave the same moduli value, which increased with the fiber loading. Therefore, the type of fiber was an insignificant parameter in increasing the composite stiffness. In other words, the fiber content played a dominant role in controlling the composite moduli. On the other hand, the type of fiber seemed to have a significant effect on the tensile strength and elongation at break. The BF/PLA composites showed slightly higher tensile strength, while the CF/PLA composites presented higher elongation at break. These findings could plausibly be explained by the nature of the fiber (see Table 1). The coir fiber possessed a low tensile strength and high stretching ability as compared with the other fibers. Therefore, a composite of such fiber would sustain the same characteristics as its

component [11–14]. Additionally, one could speculate that combining the two fibers would yield the best characteristic of both fibers. To investigate the effect of fiber hybridization, we considered using 10 wt% fiber for further study. Although the PLA composite containing 20 wt% of fiber possessed the highest modulus, because of the diminishing tensile strength and elongation at break, it may not be optimal for further improvement in the composite properties. PLA composites with 10 wt% fiber content possessed overall optimal properties and offered the possibility for greater improvement as well as the opportunity to understand the different effects of different fibers in hybridization on the composite performance. Therefore, hybrid composites with different ratios of BF: CF were prepared with a constant fiber loading of 10 wt%.

**Figure 2.** Tensile properties of PLA and PLA composites with varied fiber content 5–20 wt% (**a**) tensile modulus, (**b**) tensile strength, and (**c**) elongation at break.

#### *3.2. Effect of Fiber Ratio and Annealing on Tensile Properties of PLA-Based Composites*

Hybrid composites with a constant fiber loading of 10 wt% were prepared with different BF: CF ratios (1:1, 1:2, and 2:1). As can be seen in Figure 3, similar to the results found for single-fiber composites, the modulus of the hybrid composites was dependent only on the fiber loading. Different BF: CF fiber ratios gave insignificantly different modulus as well as tensile strength. Among the hybrid composites, 1BF: 2CF showed the highest tensile properties including tensile modulus, tensile strength, and elongation at break. It was interesting to note that elongation at break of the composites seemed to improve with the CF fiber content. As the ratio of the reinforced fiber changed, the decrease in CF content in the composite resulted in a decrease in elongation at break. This finding that CF was beneficial in improving elongation at break of single-fiber-reinforced PLA remained true even in the hybrid composite, where two different fibers were combined.

Annealing was the strategy we used in combination with fiber hybridization. The increases in modulus and tensile strength were expected as annealing generally increased and improved crystallinity and crystal growth. The crystalline phase in a semicrystalline polymer is known to be the main contributor to the hardening and strengthening of the polymer. As expected, further increases in modulus and tensile strength were obtained after thermal treatment/annealing of the PLA hybrid composites. However, elongation at break of all annealed samples suffered, as shown in Figure 3. These could also be explained by the increase in crystallinity in post-annealing samples. Higher crystalline materials resulted in harder material, whereas the portion of amorphous regions providing the elasticity of the sample was reduced. The presence of an increased crystal portion restricted the chain movement in the samples, resulting in lower extensibility. The DSC results reported in the next section confirm this speculation. It should be noted that the hybrid composite containing a high content of coir fiber showed a lower degree of elongation at break dimension after annealing. The decrease in elongation at break was about 7% and 10% in 10CF and 1:2 BF: CF composites, respectively, and about 30% for the other composites. This result indicated the strong dependency of elongation at break on fiber type even after annealing, where crystallinity should be dominant. To our knowledge, this particular point has not been reported elsewhere and may be worth investigating in depth in future studies.

#### *3.3. Morphology of PLA-Based Composites*

SEM micrographs of the tensile fractured surface of PLA and its composites are shown in Figure 4. The surface of neat PLA was smooth, all the composites were present with voids. Figure 4b–g shows the void due to fiber pull-out and poor adhesion between the fibers and matrix [42]. The void size tends to increase with increasing fiber content. Composites with CF showed more voids with greater sizes than those with BF. These voids generate weak zones where the load-bearing capacity tends to drop, leading to a reduction in tensile strength and elongation at break of PLA composites. When comparing BF composites and CF composites, the fractured surface investigation revealed that CF composites were more ductile as compared with the BF composites. The yielding feature of the PLA matrix on the fractured surface of CF composites was plausibly due to the ability of CF to elongate to a greater extent than BF [14]. Thus, when the extensional force was applied, the CF held the composites together, impeding a brittle failure and allowing a greater extension length before braking. This finding agreed well with the tensile results shown in Figure 2. The results also suggested that CF may have a better adhesion between fibers and the PLA matrix; otherwise, fracture surface yield could not occur.

(**c**)

**Figure 3.** Tensile properties of PLA, PLA composites, and PLA hybrid composites with and without thermal treatment (annealing at 120 ◦C for 30 min): (**a**) tensile modulus, (**b**) tensile strength, and (**c**) elongation at break.

**Figure 4.** SEM micrographs of the tension-fractured surfaces of (**a**) neat PLA, (**b**) 5BF/PLA, (**c**) 10BF/PLA, (**d**) 20BF/PLA, (**e**) 5CF/PLA, (**f**) 10CF/PLA, and (**g**) 20 CF/PLA.

#### *3.4. Thermal and Crystallinity of PLA-Based Composites*

Differential scanning calorimetry (DSC) was used to investigate the effect of fiber type, fiber content, and thermal treatment in promoting the crystallinity of the PLA matrix. The

DSC data including crystallinity (*Xc*) calculated using Equation (1), melting temperature (Tm), and cold crystallization temperature (Tcc) from the first heating scan are presented in Figure 5 and Table 3. It can be seen that the Tg of PLA was 61.18 ◦C. The Tg slightly decreased with the addition of fiber into the PLA matrix. The TCC was observed in all non-annealed samples, indicating that PLA molecules were unable to crystallize during the cooling phase. It is well-known that PLA's crystal formation is naturally low and requires significant encouragement to induce crystallization [43]. An increase in crystallinity in PLA results in an improvement in several properties such as tensile and thermal properties [44]. Tcc of PLA composites increased with the presence of fiber. This indicated a greater amount of the non-crystalline phase in the composite as compared with that of the neat PLA. The result agreed well with the crystallinity (*XC*). This was plausibly due to the incorporated fiber restricting the mobility of PLA chains together with the fast-cooling conditions during compression molding [45]. However, the Tg and Tcc peaks disappeared from the curve after thermal annealing. The disappearance of the peaks signified the growth of crystals in PLA (crystal perfection). This phenomenon was attributed to the rearrangement of PLA molecules upon high temperature and slow cooling. The molecules had sufficient time to slowly crystallize.

**Figure 5.** DSC thermograms of PLA, PLA composites, PLA hybrid composites (**a**) before and (**b**) after heat treatment.


**Table 3.** Transition temperatures obtained from the DSC first heating scan of PLA and its composites, with and without thermal treatment.

The *XC* of the neat, non-annealed PLA was 6.1%, which showed the tendency to decrease with increasing fiber content. The nucleating ability of both fillers was insufficient to obtain the dominant crystallinity of the PLA-based composites. The existence of both fibers hindered the mobility of PLA chains, leading to the poor rearrangement of PLA molecules and thus, lower crystallinity. The higher results for *XC* of post-annealed samples were due to sufficient time and energy for the PLA molecules to rearrange and overcome the hindrance of the fiber to crystallization during the annealing process.

#### *3.5. Heat Deflection Temperature (HDT)*

The heat deflection temperature (HDT) is commonly used to determine the maximum service temperature of a material. PLA possesses a low service temperature, which limits its use in various applications. For semicrystalline polymers such as PLA, HDT is strongly dependent on crystallinity. The poor HDT of PLA is partly due to its naturally low crystallinity. Figure 6 shows the HDT results of PLA and its composites. The HDT of neat PLA was about 53.33 ◦C, which was rather low for various applications such as automotive parts and packaging. With the presence of natural fiber in the PLA matrix, the HDT increased to 55.67 and 59.33 ◦C for the 10BF/PLA and 10CF/PLA composites, respectively. The increase in HDT in this case was due to the higher stiffness (moduli) of the single-fiber composites. As discussed previously, the presence of fiber in the composites reduced the crystallinity slightly; therefore, the increase in HDT in the composite was not from the crystallinity.

**Figure 6.** HDT data for PLA, PLA composites, and PLA hybrid composites before and after heat treatment.

In the case of hybrid composites created by adding BF: CF in different ratios (1BF:2CF, 1BF:1CF, and 2BF:1CF) into the PLA matrix, the HDT value of 1BF:2CF was the highest among those hybrid composites. The HDT value of 2BF:1CF was 57.33 ◦C, which was a

7.5% improvement as compared with the neat PLA. The increases in HDT for PLA hybrid composites were also due to the increase in stiffness with the presence of fiber and not because of crystallinity. Stiffness is defined as the ability of a material to resist deformation under load. On the other hand, HDT is a measurement of the material while temperature is increased. Therefore, the modulus of PLA composites and PLA hybrid composites were also examined for an analysis of HDT. Referring to Figures 2a and 3a, it was shown that the modulus of PLA composites and PLA hybrid composites were higher than that of the neat PLA.

Annealing of the PLA composites gave rise to HDT in all samples. This was because annealing increased both the crystallinity and modulus. The increase in HDT value of the post-annealing samples might be due to the increase in crystallinity and consequently, the modulus/stiffness. The high-temperature exposure and slow cooling process of PLA composites induce the formation of a crystallized structure that enhances thermal properties. The maximum HDT obtained in this work was 61.7 ◦C in an annealed 10CF sample, which was an 8.3 ◦C increase over the non-annealed neat PLA. Other researchers also reported an increase in HDT over the same temperature range. A further increase in HDT up to 120 ◦C could be achieved using nucleating agents together with thermal treatment manipulation [46].

#### **4. Conclusions**

In summary, we illustrated the possibility of improving PLA properties using combined techniques of hybridization and thermal treatment. The hybrid composites created using two different fibers can offer beneficial properties of the individual fibers used. Selecting a proper pair of fibers is critical to obtaining properties that complement one another. In this work, the PLA composite reinforced with coir fiber offers better elongation at break and HDT than BF composites, while bamboo leaf fiber offers better tensile strength than CF composites. The fiber content played an important role in dominating the mechanical properties of the final composite, specifically the stiffness (moduli). The PLA composites consisting of 10 wt% of fiber possessed the most balanced properties in terms of tensile modulus, tensile strength, and elongation at break. Hybridization of the BF: CF with the ratio of 1:2 gave the most desirable properties. Thermal treatment or annealing further improved the mechanical and thermal properties of hybrid composites. These improvements are attributed to the increased degree of crystallinity brought about by the exposure to high temperatures and slow cooling. However, the result of tensile properties showed lower tensile strength, as compared with the neat PLA, due to poor adhesion between fiber and matrix. Further studies can be performed to investigate several strategies to improve the adhesion between fibers and the PLA matrix. It can be expected that when the adhesion between fibers and the matrix is improved, the properties of PLA-based hybrid composites will be greatly improved, which will consequently open more possibilities for the utilization of PLA in various applications.

**Author Contributions:** Conceptualization, T.T. and S.Y.; methodology, T.T. and S.Y.; validation, T.T. and S.Y.; formal analysis, S.Y.; investigation, S.Y.; writing—original draft preparation, S.Y.; writing—review and editing, T.T. and S.Y.; visualization T.T. and S.Y.; supervision, T.T.; project administration, T.T.; funding acquisition, T.T. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was funded by Suranaree University of Technology (SUT) and the Center of Excellence on Petrochemical and Materials Technology (PETROMAT) High Performance and Smart Materials (HPSMs) program, 2016.

**Institutional Review Board Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available upon request from the corresponding author.

**Acknowledgments:** The authors thank the Suranaree University of Technology for providing equipment and facilities.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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### *Review* **Polymer-Based Hydrogel Loaded with Honey in Drug Delivery System for Wound Healing Applications**

**Siti Nor Najihah Yasin, Zulfahmi Said \*, Nadia Halib, Zulaiha A Rahman and Noor Izzati Mokhzani**

Department of Basic Sciences and Oral Biology, Faculty of Dentistry, Universiti Sains Islam Malaysia, Tower B, Persiaran MPAJ, Jalan Pandan Utama, Pandan Indah, Kuala Lumpur 55100, Malaysia; sitinajihah.yasin@gmail.com (S.N.N.Y.); nadia.halib@usim.edu.my (N.H.); zulaiha@usim.edu.my (Z.A.R.); n.izzati9802@raudah.usim.edu.my (N.I.M.)

**\*** Correspondence: zulfahmi@usim.edu.my

**Abstract:** Excellent wound dressings should have crucial components, including high porosity, nontoxicity, high water absorption, and the ability to retain a humid environment in the wound area and facilitate wound healing. Unfortunately, current wound dressings hamper the healing process, with poor antibacterial, anti-inflammatory, and antioxidant activity, frequent dressing changes, low biodegradability, and poor mechanical properties. Hydrogels are crosslinked polymer chains with three-dimensional (3D) networks that have been applicable as wound dressings. They could retain a humid environment on the wound site, provide a protective barrier against pathogenic infections, and provide pain relief. Hydrogel can be obtained from natural, synthetic, or hybrid polymers. Honey is a natural substance that has demonstrated several therapeutic efficacies, including antiinflammatory, antibacterial, and antioxidant activity, which makes it beneficial for wound treatment. Honey-based hydrogel wound dressings demonstrated excellent characteristics, including good biodegradability and biocompatibility, stimulated cell proliferation and reepithelization, inhibited bacterial growth, and accelerated wound healing. This review aimed to demonstrate the potential of honey-based hydrogel in wound healing applications and complement the studies accessible regarding implementing honey-based hydrogel dressing for wound healing.

**Keywords:** wound healing; wound dressings; hydrogel; honey; natural polymer; synthetic polymer

#### **1. Introduction**

Wounds are typically defined as damage to the skin as well as to epidermal- or dermallayer structures. They can be categorized as acute or chronic wounds depending on their duration and the nature of the healing process [1]. Wound healing is a dynamic and sophisticated tissue regeneration process that repairs the damaged skin and other soft tissues locally or systematically. It involves four temporal stages: hemostasis, inflammation, proliferation, and remodeling [2].

Wound dressings play a significant role in offering the optimum conditions for wound healing and protecting the wounds from further damage and infection. Conventional dressings, including gauzes, plasters, and bandages, are used as primary and secondary dressings for protection against microbial infections [3]. However, these dressings absorb a high amount of moisture on the wound, and can dry and adhere to the wound surface and cause pain when removed [4]. Additionally, some of these dressings might not have antimicrobial, antioxidant, and other bioactive components [5]. Therefore, it is essential to design appropriate dressings that can be easily detached and do not cause any harm to the surface of wounds during dressing replacement [6]. Additionally, it should offer excellent antimicrobial activity, excellent mechanical properties, be able to deliver bioactive agents [7], provide a physical protective barrier, promote the deposition of the extracellular matrix (ECM), and maintain an optimal environment on the wound site, as well as promote the process of wound healing [8].

**Citation:** Yasin, S.N.N.; Said, Z.; Halib, N.; Rahman, Z.A.; Mokhzani, N.I. Polymer-Based Hydrogel Loaded with Honey in Drug Delivery System for Wound Healing Applications. *Polymers* **2023**, *15*, 3085. https://doi.org/10.3390/ polym15143085

Academic Editors: Raffaella Striani and Marcel Popa

Received: 29 March 2023 Revised: 1 May 2023 Accepted: 18 May 2023 Published: 18 July 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Hydrogels are three-dimensional (3D) networks made up of hydrophilic polymers formed through hydrogen or covalent crosslinking using the process of a physical or chemical reaction [9]. Hydrogels are commonly employed as wound dressings and have been proven effective, mainly for treating wounds and skin ulcers [10]. These 3D polymer networks can absorb a tremendous amount of liquid, offer a humid environment, excellent biocompatibility [11], biodegradability, and adhesion that can efficiently stimulate cell proliferation and facilitate the process of wound healing as well as improve the stage of wound repair [5]. In addition, the hydrophilic groups in the polymeric chains cause the hydrogel dressings to retain water, with a higher water content ensuring a good porosity, softness, and elasticity [12] and a cooling effect, thus minimizing pains upon removal [13].

Hydrogels can be prepared through natural polymers, including alginate, chitosan, hyaluronic acid, cellulose, starch, gelatin, etc., or synthetic polymers such as polyvinyl alcohol (PVA), polyacrylamide, polyethylene glycol (PEG), etc., or a combination of both polymers [14]. These combinations, which are known as hybrid polymers, could enhance their individual physicochemical, mechanical, and biological properties and promote healing [13]. Some examples of this combination include PVA/chitosan [15], PVA/starch [16], polyacrylamide/chitosan [17], etc. Hydrogel-based wound dressings are well recommended for their healing-promoting properties, which speed up the proliferation and epithelialization processes [8]. Therefore, they are acceptable as first aid for wound care [18]. Excellent wound dressings that are prepared from the polymers as mentioned earlier can be further enhanced by incorporating nature-based bioactive agents [19].

Honey has been applied for centuries as a treatment for infected wounds to accelerate the process of wound healing. Honey is gaining considerable attention as a regenerative agent to treat ulcers, bed sores, skin infections, wounds, and burns. It offers antimicrobial, anti-inflammatory, and antioxidant activity and maintains a moist wound environment that acts as a protective barrier to prevent infection [20]. It also demonstrates the ability to recover the development of new tissue to heal the wound through epithelization. Honey rapidly clears wounds when applied topically to promote the deep healing of wounds with infection [21]. The lower pH of honey (pH 3.5–4.0) could inhibit protease activity, which increases oxygen release from hemoglobin, and promotes macrophage and fibroblast activity on a wound site. In contrast, hydrogen peroxide sterilizes the wound and stimulates vascular endothelial growth factor (VEGF) production [22].

The application of honey-based hydrogel wound dressings has been practiced in the biomedical field. Some studies have demonstrated that honey-based hydrogel dressings can increase water absorption and swellability, support epithelization, stimulate cell proliferation, inhibit bacteria growth, and accelerate wound healing [23–25]. This review aimed to focus on the potential of using honey incorporated into hydrogel patch formulation as a promising approach for wound healing applications and highlight the honey-based hydrogel's properties in treating wound infection.

#### **2. The Phase of Wound Healing**

The process of wound healing is dynamic and sophisticated, consisting of four overlapping phases: hemostasis, inflammation, proliferation, and remodeling [26,27] (Figure 1). Hemostasis begins with blood coagulation, including the formation of fibrin clots, degranulation, platelet accumulation, and release of growth factors. As a result, fibroblasts, macrophages, and endothelial cells become involved in wound healing [28].

In the inflammatory stage, neutrophils protect the wound against pathogenic infections and cleanse the wound from cellular debris to create a favorable condition for rapid healing [29]. Exudates are responsible for inflammation symptoms, such as redness, warmth, erythema, and swelling of the damaged skin. New epithelial cells infiltrate the wound environment to replace the dead cells due to damaged skin [19]. The severity of the damage determines the duration of the hemostasis and inflammatory stages [30].

In the proliferative stage, cell proliferation and connective tissue formation occur. Next, ECM components, such as hyaluronic acid and glycosaminoglycan, contribute to the

production of granulation tissue to replace the primary clot development. This stage can last several days to a few weeks following injury [31]. The final stage is a remodeling or maturation phase that starts around weeks and lasts up to months. Finally, the surface of the damaged tissue is fully recovered with fibroblast cells with a scar formation [32,33].

**Figure 1.** The phases of wound healing. Adapted from Ref. [33].

#### **3. Classification of Wound Dressings**

Acute or chronic wounds need proper treatment to evade any shortcomings that may arise throughout the healing process. Conventional, bioactive, and interactive dressings and skin substitutes are applied to treat wounds [19] (Figure 2). Conventional wound dressings, or passive dressings, protect wounds against external substances, infection, and damage. Additionally, these dressings function to control blood, cover and absorb wounds, and cushion the damage. Examples of conventional dressings are gauze, plasters, and bandages. However, some limitations of these dressings are that they do not provide a moist environment to the wound bed [34] and must be changed frequently during the healing process, which may cause more skin damage [35].

Bioactive wound dressings are designed to provide bioactive compounds. To improve the therapeutic efficiency of these dressings, they might be incorporated with antimicrobial agents, growth factors, nutrients, nanoparticles, vitamins, plant extracts, and other natural biomaterials to the wound site to promote the healing process [36]. Some formulationbased bioactive wound dressing examples include sponges, foams, wafers, hydrogels, films, membranes, and nanofibers [37]. In addition, bioactive wound dressings have properties that include non-toxicity, excellent biocompatibility, and biodegradability [19].


**Figure 2.** Classification of wound dressings. Adapted from Ref. [19].

Interactive dressings are applied directly to the wound site, removing debris, providing a moist environment, and preventing infections [38]. Examples of these dressings include sprays, films, foams, nanofibers, and sponges. In addition, they are favorable for reepithelization due to excellent oxygen concentration and pH control [39].

Skin substitutes are wound dressings that are developed to restore damaged skin. They are made up of epidermal and dermal layers that are formed by fibroblasts and keratinocytes on collagen matrices. The primary mechanism of these dressings is to secrete and stimulate growth factors through which epithelization is achieved [3]. Autografts, acellular xenografts, and allografts are some forms of skin substitute wound dressings. The advantages of these wound dressings include minimizing scar formation, providing pain relief, and accelerating healing. However, some limitations of these dressings are possible disease transmission, long preparation time, poor keratinocyte attachment, difficulty handling, etc. [40].

#### **4. Polymer-Based Hydrogels for Wound Healing**

The polymer-based hydrogel can be employed in biomedical applications for wound healing, drug delivery, and tissue engineering [41]. They can be classified as natural, synthetic, and hybrid (combination of natural and synthetic) polymers (Figure 3). Excellent polymer-based hydrogel wound dressings should have appropriate features, including good biocompatibility and biodegradability, meaning the hydrogels could fully degrade after a duration [5,11,42]. Additionally, it should have adequate adhesion and excellent mechanical properties to ensure it can adhere to and cover the wounds entirely to prevent microbial infection [43]. In addition, the hydrogels should provide and maintain a humid environment at the wound site for cell migration and proliferation [11,44]. Therefore, dressing selection needs careful consideration before promoting the healing process [45].

**Figure 3.** Types of polymers.

#### *4.1. Natural Polymer*

Natural polymers have been applied over the centuries as the primary bioactive substance in biomedical fields [45]. These polymers are naturally synthesized and extracted from organisms and plants. Natural polymers including chitosan, collagen, starch, cellulose, sodium alginate, agarose, gelatin, and hyaluronic acid are some examples that are broadly utilized in synthesizing hydrogel wound dressings [14,46].

The interest in utilizing natural polymers as a hydrogel includes for their biodegradability, biocompatibility, non-toxicity, and low immunogenicity, and the structures are similar to that of ECM [47]. They may also produce by-products that are well tolerated by living organisms without triggering toxic reactions when subjected to enzymatic degradation [48]. However, they have some limitations, such as pathogenic contamination, uncontrollable degradation rate, complex modification, and low mechanical properties, which may restrain tissue regeneration application [49].

#### *4.2. Synthetic Polymer*

Synthetic polymers are beneficial in a few properties over natural polymers, such as endless forms, tunable properties, non-toxicity, and established structures [45]. Synthetic polymers are typically designed to mimic the structures of natural polymers, with minor modifications to enhance desired properties. These polymers contribute to forming a controlled 3D network with a high molecular weight, new functional groups, and charged groups. Some examples of synthetic polymers include PVA, PEG, polyurethane (PU), polyvinyl pyrrolidone (PVP), and polyacrylamide [5]. In addition, the derivations of cellulose, acrylic acid polymers, and vinyl polymers are some of the most commonly used synthetic polymers [46]. The limitations of these polymers are that they have insufficient cell adhesion sites, require chemical modifications to improve cell adhesion [50], are impermeable to drugs and proteins, and have poor mechanical stability [51].

#### *4.3. Hybrid-Based Polymers*

Hybrid-based polymers are developed through the combination of at least two or more polymer-based natural and synthetic polymers. Each polymer holds specific physicochemical and biological properties in a blending [52]. Several researchers have investigated if hybrid hydrogels can be widely used to overcome the limitations of both polymer types [13], as they possess the advantages of both natural and synthetic polymers in terms of their physicochemical, mechanical, and biological activities [53]. Additionally, these hybrid hydrogels could offer excellent flexibility, biocompatibility, biodegradability, and high absorption capacity and promote wound healing [54]. Some examples of hybrid polymers include PVA/sodium alginate [55], PEG/chitosan [56], PVP/keratin [57], poly(N-isopropyl acrylamide)/cellulose [58], chitosan/poly(N-vinyl-2-pyrrolidone)/polyacrylic acids [59], etc.

#### **5. Physicochemical Properties and Composition of Honey**

Honey is a raw substance with a sophisticated chemical composition and woundhealing properties. In addition, it has a broad range of physicochemical properties and compositions dependent upon its botanical and geographical areas [60].

The physicochemical composition of honey includes acidity, pH, moisture, ash content, hydroxymethylfurfural (HMF), sugar content, and enzyme activity. The chemical composition of honey encompasses various constituents that contribute to its biological activities. These constituents include proteins, organic acids, enzymes, phenols, flavonoids, vitamins, etc. These components play a role in honey's beneficial effects and potential wound-healing properties [61].

A previous study has investigated the nutritional index of honey from diverse botanical areas. They found that honey's moisture content ranges from 27–31% of honey. The ash levels were 0.15–0.9%, while the protein content was 0.2–0.8%. Additionally, the sugar content for glucose, fructose, and sucrose was 29–31%, 45–48%, and 2–4%, respectively. The HMF value also should not be more than 60 mg/kg. Its pH levels are between 3.24 to 6.1. Honey contains several active compounds, including flavonoids, organic acids, phenolic acid, vitamins, and enzymes, that may improve wound healing [62].

#### **6. Biological Activity of Honey in Wound Healing**

Honey has been well-known for wound treatment since ancient times. The healing properties of honey are related to its antioxidant, anti-inflammatory, and antimicrobial activities, and its capabilities of maintaining a moist wound environment, protecting the wound, and preventing pathogenic infection [20]. The immunological activity of honey is also crucial in wound healing as it has pro- and anti-inflammatory properties [63]. In addition, honey has antimicrobial properties and has the potential to counter wound infections and function as a physical barrier to the wound area, as well as promote wound healing [21]. The antimicrobial properties found in honey play a significant role in the body's response to tissue damage [64].

Honey may aid in regenerating damaged tissues and wound healing as it contains a high sugar content, reactive oxygen species generation, and anti-inflammatory properties [65]. Additionally, honey can sterilize wound infection, stimulate the growth of tissues and re-epithelization, and reduce scar formation. These factors contribute to the four phases of wound healing, as stated above [66]. Honey demonstrates diverse effects in each stage of the wound-healing process [67]. During the inflammatory stage, honey inhibits bacterial placement, lowers pH, increases antioxidant action, increases peroxide generation, and releases pro-inflammatory cytokines [68]. It then promotes epithelization and proliferation while decreasing edema and exudate in the wound during the proliferative stage. Next, during the remodeling stage, honey helps to recover the wound and prevent scar formation [32].

Additionally, hydrogen peroxide (H2O2) production on glucose is another characteristic of honey that causes antimicrobial action. This compound catalyze by glucose oxidation of glucose which lead to the production of gluconic acid and hydrogen peroxide [68]. The

formation of gluconic acid contributes to a decrease in pH levels, while hydrogen peroxide enhances the antimicrobial properties of honey. This cascade of events, which includes pH reduction to levels between 3.5–4.0, is crucial for initiating the tissue repair process [68,69]. Furthermore, H2O2-dependent honey may stimulate the synthesis of vascular endothelial growth factor (VEGF) and sterilize the wound site [22]. In addition to glucose oxidase, bees create invertase, which enhances the osmotic potential of honey by breaking down sucrose into fructose and glucose (Figure 4) [69]. The production of H2O2 also tends to be toxic to the cellular tissue when it is too saturated. However, it can be countered with an antioxidant compound inside the honey [70].

**Figure 4.** The fundamental principle of honey in wound healing. Adapted from Ref. [69].

The antioxidant properties are also other medicinal properties of honey that have been studied. The antioxidant action in honey is enhanced by the presence of phenolic compounds [66]. Plants create various secondary metabolites in response to environmental stresses and oxidative damage. These compounds are transferred to honey through nectar. The phenolic compounds are divided into two categories, which are phenolic acids and flavonoids. Free radicals are scavenged by phenolic acids and flavonoids, which reduce tissue damage and inflammation. In biomedical fields, honey has been employed to treat wounds, burns, and inflammation, and has a synergistic effect when combined with antimicrobial agents [22,71]. Previous research discovered that the relative positions of OH groups in the aromatic ring affect the antioxidant effect of phenolic acids and are also found to be the most potent antioxidant among all phenolic acid compounds [72].

The current therapeutic applications used in wound management are beneficial for inhibiting bacterial infection and promoting healing [73]. Using natural products with antimicrobial properties in biomedical research has garnered considerable attention in modern medicine [71]. The excellent properties of natural products, including honey, curcumin, and aloe vera, are the most prominent arguments for applying natural products in treating wounds [74]. Honey has been a topical treatment since ancient times and has been officially recognized as a medical device in conventional medicine, and can be combined with silver dressings or other formulations [56,63]. Table 1 shows the comparison between honey and some bioactive substances that are commonly utilized in wound healing applications.


**Table 1.** Comparison between honey and other bioactive substances for wound healing applications.

#### **7. Application of Honey-Based Hydrogel for Wound Healing**

Hydrogels combined with honey have multiple benefits and are considered ideal wound dressings to promote healing [20,78,79] (Figure 5). Hydrogels are 3D structures crosslinked with hydrophilic characteristics that can hold abundant volumes of water and other liquids [11,80]. Thus, it is applicable for wound healing due to its high porosity, excessive water content, ability to release therapeutic agents, excellent biocompatibility, biodegradability, and it can accelerate the wound healing process [5,19,81]. Furthermore, honey has been traditionally utilized as a wound dressing to accelerate and enhance the process of wound healing. Therefore, incorporating honey into the hydrogel could be effective for wound healing [20,82].

**Figure 5.** Schematic of honey-based hydrogel for wound healing. Adapted from Ref. [82].

Chopra et al. prepared a natural chitosan and PVA to formulate a hydrogel film incorporating honey for wound healing treatment and evaluated their physicochemical and mechanical properties. The findings showed that the thickness and weight of the films were between 0.041 ± 0.006 to 0.055 ± 0.004 mm, and 0.425 ± 0.02 to 0.480 ± 0.04 g, respectively. The folding endurance ranged from 350 ± 15 to 445 ± 7. The folding endurance values increase as the chitosan concentration increases from F1–F5 (0.25–2%). The formulation of batch F5 (2% of chitosan) gives a smooth surface and homogenous form with little porosity, exhibiting excellent structural integrity. Additionally, the hydrogel's moisture content increases as the chitosan concentration increases. Swelling analysis indicates that increasing the chitosan concentration may increase the water's swelling ratio. The F5 showed the highest swelling ratio, with 300% after 24 h. For mechanical characteristics, the results showed a value from 4.74 ± 0.83 to 38.36 ± 5.39 N for tensile strength, and 30.58 ± 3.64 to 33.51 ± 2.47 mm for elongation at the break, respectively. A strong interaction and network between the polymers could enhance the mechanical characteristics of the hydrogel film. In addition, an antimicrobial study against *Staphylococcus aureus* (*S. aureus*) demonstrated that a honey-based hydrogel film exhibited antimicrobial activity with excellent bacteriostatic ability. The F4 showed an excellent antimicrobial effect, with the diameter of the inhibition zone being 5.01 ± 0.32 mm [83].

Additionally, a study by Gopal et al. incorporated Kelulut honey and Tualang honey into cellulose/PEG hydrogels to treat wound infections. The finding showed that the honey hydrogels showed excellent antimicrobial activity compared to the control hydrogels. Tualang honey hydrogels exhibited the highest zone of inhibition for negative *Escherichia coli* (*E. coli*), and *S. aureus*, which could be influenced by the highly acidic component with pH 3.55–4.0 which may inhibit both bacteria. For *E. coli*, the Kelulut honey hydrogels showed slightly higher inhibition zones than the Tualang honey hydrogels. Meanwhile, for *S. aureus*, the Tualang honey hydrogels exhibited higher inhibition zones than the Kelulut honey hydrogels. In vitro cell viability testing indicated that both honey-based hydrogels recorded the maximum cell viability (90%) compared to control hydrogels without the incorporation of honey, which recorded the minimum viability [23].

Lo et al. conducted a study that formulated cellulose/poly(lactic-co-glycolic acid) (PLGA) patches incorporated with Kelulut honey for aphthous stomatitis treatment. The ATR–FTIR study was utilized to analyze the morphology of the patches. In vitro cell viability analysis indicated that the Kelulut honey patch stimulated an incre in cell viability percentage by more than 90% compared to the control, which can promote angiogenesis by supporting tissue regeneration and skin re-epithelization. Additionally, the PLGA polymer released the honey into the extracellular matrix and rapidly closed the wound gap. In vivo analysis also demonstrated that the honey patches could inhibit the growth of *E*. *coli* in the first 2 h, followed by the inhibition of *S. aureus* in the next 2 h [84].

Zekry et al. investigated the PVA/honey hydrogel for wound healing. They preparedManuka honey (MH)/pomegranate peel powder (PPP)/PVA (10%/1%/12%), MH/PPP/PVA (20%, 2%, 10.5%), MH/PPP/PVA (25%/2.5%/9.7%), MH/PPP/BV/PVA (25%/2.5%/0.01%/9.7%), and LH/PPP/BV/PVA (25%/2.5%/0.01%/9.7%). Scanning electron microscopy (SEM) was used to analyze the morphological structures of all formulations. The in vitro release study displayed that the honey was released over 24 h with a low adhesion to the wound site, stimulating cell proliferation and re-epithelization. Additionally, in vivo analysis of the wound healing activity indicated that all treated groups achieved complete healing on day 10, compared to the PVA control group (day 13) and no treatment groups (day 14) which demonstrated slowed healing processes. Moreover, at day 3 and 5, the commercial Medihoney® group showed a higher percentage of wound closure compared to the PVA control and no treatment groups. Additionally, the honey hydrogel inhibited 90–98% of the *S. aureus* and *E. coli* growth, which showed good antimicrobial activity compared to controls [85].

Samraj et al. studied a combination of Kelulut honey with curcumin in the nanofibrous composite hydrogel membrane to treat wound healing. The findings showed that the impregnation of curcumin and honey promotes healing by stimulating cell migration and promoting recovery through anti-inflammatory properties. In addition, impregnating honey with curcumin promotes new cell regeneration and prevents scar formation. In vitro and in vivo rat models showed improved recovery and no cytotoxicity compared to control groups without treatment. Furthermore, antioxidant and antimicrobial studies demonstrated that the activity of wound healing with the hydrogel membrane was significantly higher than curcumin and honey alone. Therefore, incorporating honey into composite hydrogel membranes may assist in wound healing [86].

A previous study by Noori et al. developed a nanocomposite hydrogel using PVA/chitosan /honey/montmorillonite (PCMH). SEM and XRD were employed to perform the morphological analysis of the hydrogel film. Additionally, swelling tests were performed at 37 ◦C, and the results demonstrated that the swelling increased as the temperature increased. Furthermore, the 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide (MTT) analysis revealed that the PCMH hydrogel had a higher cell viability above 75% after 24 h, indicating no cytotoxicity. For pure chitosan, it was shown that the cell viability was more than the control group. This indicated that the pure chitosan itself could stimulate cell proliferation. An in vitro study against *S. aureus* has shown that PCMH hydrogel showed a more significant antibacterial value of higher than 99%, which demonstrated that it can restrict the growth of bacteria. Additionally, wound healing activity was evaluated in rats through in vivo analysis, and the results showed that PCMH hydrogel reduced the wound area more significantly than the control group and showed better wound healing ability, a rapid rate of honey release, restricted bacterial growth, and reduced the length of the wound healing process through cell reepithelization and proliferation. These results indicate that honey-based hydrogels could be applied as a wound-healing treatment [24].

The studies in vitro and in vivo performed by El-Kased have incorporated Egyptian honey (25, 50, and 75%) into chitosan/polyacrylic acid hydrogels for treating burn-wound healing. The findings showed that all hydrogel formulations exhibited a rapid swelling behavior due to their porous structure, providing a large surface area for rapid solvent uptake. Additionally, the swelling index was found to be inversely proportional to the honey concentration, indicating that an increase in honey concentration results in a decrease in the hydrogel's swelling percentage. This factor may be affected by the polymer's viscosity, which can impact the swelling process. In vitro release studies revealed that the release of honey from the hydrogel depended upon the honey concentration. Among all formulations, hydrogels with the lowest concentration of honey (25%) showed superior sustained release with 70% of release over 3 h. In vitro antimicrobial analysis showed that 75% of honey incorporated into hydrogels showed the highest healing rate as it stimulated cell re-epithelization and excellent antimicrobial activity compared to pure honey and commercial products [87].

Yang et al. developed nanofibrous silk fibroin and polyethylene oxide (PEO) with various concentrations of Manuka honey (10%, 30%, 50%, and 70% *w*/*v*) using an electrospinning technique. The FTIR was used to study the structural behavior of the fibrous matrices. The findings showed that the honey-based hydrogel dressings exhibited an-

timicrobial activity against *E. coli*, *S. aureus*, *P. aeruginosa*, and MRSA, in which the results revealed that the non-honey dressing was approximately zero, but antimicrobial activity improved to around a 50%, 28%, 57% and 40% inhibition of *E. coli*, *S. aureus*, *P. aeruginosa*, and MRSA, respectively, for the 70% *w*/*v* honey hydrogel over 24 h. Furthermore, in vitro biocompatibility analysis showed that hydrogel containing honey had a higher viability than the control. However, the increasing concentration of honey did not change the cell viability, demonstrating that the incorporation of honey does not negatively affect the excellent biocompatibility of the hydrogel. Additionally, in vivo analysis in a rat dorsal wound model showed that the wounds treated with 70% honey hydrogel wholly recovered, whereas both the control group and commercial Aquacel®Ag wound dressing group had only slight reductions in wound size [88].

Another study by Tavakoli and Tang fabricated a polyvinyl alcohol/Manuka honey hybrid hydrogel wound dressing with borax as a crosslinking agent. Hydrogels prepared with 1% borax demonstrated adequate biocompatibility, a sustained release of honey in the ulcer bed, and no burst release of antibiotics. The addition of borax also increased the mechanical durability of the honey/PVA hybrid and prevented hydrogel degradation during the swelling process. This thin layer of hydrophilic gel may improve the wound-healing process and reduce the risk of contamination. The results demonstrated that the honey showed good antibacterial activity against *S. aureus* in all samples, especially in the samples with a 1% crosslinking agent. The results showed that the PVA/borax/honey hybrid hydrogel demonstrated the greatest swellability and stability and had excellent antimicrobial activity, and indicated that PVA/honey hydrogel produced the best characteristics for applying to wound dressing [25].

Durai and Sizing fabricated chitosan hydrogel films impregnated with 8% Manuka honey to treat wounds. The results revealed that honey increased the folding endurance, with the honey hydrogel films surviving a mean of 289 folds compared to 143 folds for the non-honey films. This result demonstrates a greater flexibility of the honey hydrogel film due to the hygroscopic effect of honey. Additionally, honey reduced the swelling ratios of the hydrogel films and inhibited the growth of *S. aureus* and *E. coli*. In an in vivo analysis of a rat dorsal wound model, the honey hydrogel showed an increased wound gap compared with control groups of non-honey and ointment. The honey hydrogel and non-honey hydrogel showed closures of 94% and 78% after 12 days of treatment, compared with the ointment-treated group and the non-treated control whose wounds showed closures of 86% and 64%, respectively [89].

Zohdi et al. developed a hydrogel dressing incorporating Gelam honey into the polyvinyl pyrrolidone (PVP)/protein-free agar/polyethylene glycol (PEG) hydrogel with a 6%, 8%, 10%, and 15% concentration of honey. The finding showed that the honey hydrogel and the control group had good uniformity and transparency with a 3–4 mm thickness. Additionally, the pH of the honey hydrogel was slightly acidic, with a value of pH 4.3, while the control group had a pH of 5.3. This slight acidity in the hydrogel may be due to the natural acidic properties of honey, which typically has a pH ranging from 3.2 to 4.5. For swelling analysis, the honey hydrogel demonstrated a high capability in absorbing fluid compared to the control group. The in vivo analysis in rats revealed that the honey hydrogel dressing stimulated wound closure and promoted the process of reepithelization better than the control group. Furthermore, the histopathological analysis showed that the honey hydrogel attenuated the inflammatory response on day 7, earlier than the control group. Moreover, honey hydrogel facilitates the growth of granulation tissue and blood capillary and collagen synthesis, which is effected by the generation of hydrogen peroxide by honey [90].

Khoo et al. compared a Tualang honey wound dressing and hydrofiber silver-treated wound dressing. The results demonstrated that the Tualang honey dressing had more flexibility, less adherence, easily peeled, and caused less fluid accumulation in the wound site. Furthermore, according to an in vivo study, using Tualang honey for dressing burn wounds resulted in significantly greater wound contraction than applying hydrofiber silver dressing. Furthermore, on day 6, the wound area became smaller and showed increasing cell epithelization. Additionally, the Tualang honey -treated wound dressing showed a lower bacterial growth of *Pseudomonas aeruginosa*-inoculated wounds and excellent antibacterial activity [91].

#### **8. Cell Migration and Proliferation on Honey-Based Wound Dressings**

The scratch- or wound-healing assay is a cost-effective and straightforward experimental method for investigating cell migration [92]. The assay involves growing a cell monolayer in a multiwell assay plate, creating a "wound" or scratch, and then capturing images at regular intervals to measure and quantify cell migration [93] as shown in Figure 6. Scratch assays are commonly employed to study the molecular mechanisms that influence cell migration and to identify therapeutic compounds that can modulate cell migration for potential treatments. Therefore, it is crucial to develop reliable methods for quantifying and comparing migration rates of different scratch assays to advance biomedical research [94]. The wound closure percentage was calculated using the following formula:

$$\% \text{ Volume Classure} = \frac{\text{A}\_0 - \text{A}\_{\text{T}}}{\text{A}\_0} \times 100\%$$

where A0 is the wound area measured after scratching, and AT is the area of the wound measured at a predetermined time.

**Figure 6.** Illustration of in vitro wound healing assay. (**A**) Fibroblast cells form a confluent monolayer. (**B**) In vitro "wound" was created by a straight line scratch across the fibroblast monolayer [94].

There are limited studies on utilizing cell-culture applications to perform cellular migration upon honey-based dermal wound dressings, as there are broad studies that have carried out the application of honey dressing in animals to study the effectiveness of honey in wound healing. However, several studies utilize pure honey (with a dilution factor) for wound healing analysis.

For instance, Chaudhary et al. studied the cell migration assay under 0.1% of Manuka honey and 0.1% Jamun honey on primary fibroblast cells from a neuron differentiation medium (NDM) and a decalcified bone matrix (DBM) skin. The results showed that both kinds of honey could stimulate cell proliferation against fibroblast cells over 24 h. However, DBM cells with Manuka honey and Jamun honey migrated faster than NDM cells at 24 h [95].

Ranzato et al. performed a scratch-wound assay on the fibroblast cells using 0.1% *v*/*v* Manuka, buckwheat, acacia honey, and platelet lysate (PL). The finding showed that the cells exposed to buckwheat and acacia honey showed a higher rate of wound closure at 24 h compared to controls, while Manuka honey showed a lower effect against fibroblast cells [96].

The study by Ebadi and Fazeli performed a wound healing analysis on human dermal fibroblasts using honey and an ethanol extract of propolis (EEP). The finding showed that 100 μg/mL and 200 μg/mL concentrations of EEP demonstrated the highest percentages

of wound closure compared to the control and DMSO control. After 48 h, the wound healed entirely at the 100 μg/mL and 200 μg/mL concentrations. For the honey analysis, the 25 μg/mL to 200 μg/mL concentrations showed a slight increase in the percentage of wound closure, while for the 100 μg/mL and 200 μg/mL concentrations, the wound healed after 48 h, faster than both control groups. The EEP and honey concentrations of 100 μg/mL and 200 μg/mL showed remarkable wound closure after 24 and 48 h compared to both control groups [97].

An MTT assay also can be performed to assess cell proliferation. A study by Lau et al. performed a cell proliferation assay under different concentrations of Tualang honey on human periodontal ligament fibroblast cells (HPDLF). The finding showed that 0.02% Tualang honey concentration stimulated a higher proliferation rate than the control. However, at a higher concentration of Tualang honey (5%), the cells became rounded and floating, indicating that a higher honey concentration could inhibit cell proliferation [98].

Additionally, Shamloo et al. studied the cell proliferation and biocompatibility of human fibroblast cells using various concentrations (0, 5, 10, and 20%) of a chitosan/honey hydrogel. The finding demonstrated that a 10% concentration of chitosan/honey hydrogel stimulated the highest cell proliferation compared to other hydrogels. It also demonstrated that the addition of honey into hydrogel could offer maximum nutrients for cells, which may increase cell proliferation, as well as cell viability [99].

A study by Sarhan et al. analyzed the cell proliferation of human fibroblast cells when using various types of honey hydrogel (0, 25, 50, 75, and 100% extraction). The findings showed that 100% honey extraction stimulated the highest cell proliferation (>90%) compared to other hydrogels and the positive control, commercial Aquacel®Ag. In this study, the Aquacel®Ag showed cytotoxic signs with a cell viability of 9% [100].

#### **9. Toxicological Information of Honey-Based Wound Dressings**

It is essential to consider the toxicological aspects associated with honey-based wound dressings [101]. Among many types of toxicological analyses, the MTT assay is a widely utilized method to evaluate cell viability and cytotoxicity in vitro, which makes it suitable for toxicological analysis in wound-healing applications [102]. Table 2 shows in vitro MTT assays related to applying honey-based wound dressings.


**Table 2.** In vitro MTT assay of honey-based hydrogel wound dressings.


#### **Table 2.** *Cont.*

#### **10. Regulatory Information of Honey-Based Wound Dressing**

Honey-based wound dressings are classified as medical devices. They are regulated by various regulatory agencies worldwide [66], including the US Food and Drug Administration (FDA) [105], European Medicines Agency (EMA) [106], National Medical Products Administration (NMPA) [107], Therapeutic Goods Administration (TGA) [108], Health Sciences Authority (HSA) [109], and Medical Device Authority (MDA) [110]. It should be noted that the regulatory requirements for honey-based wound dressings may vary depending on the country and region in which they are commercialized [66].

The importance of providing the regulation information before they can be sold in a country is to assure the quality, efficacy, and safety of products that are used for wound care [111]. Regulatory bodies set standards and guidelines for manufacturing, labeling, and marketing wound-care products, including honey-based wound dressings, to ensure that they meet specific criteria and do not harm patients [112]. By adhering to these regulations, manufacturers can ensure that their products are effective and safe for use, and healthcare providers and patients can have confidence in their products [113]. Additionally, regulatory information can help healthcare providers. Patients make informed decisions about wound care products based on their specific needs and circumstances [111,114]. Table 3 describes the regulatory requirements for honey-based wound dressings based on the country.

**Table 3.** Description of the regulatory body in different countries.



#### **Table 3.** *Cont.*

#### **11. Patent Information on Honey-Based Wound Dressings**

Patent information in wound dressing refers to the documentation of a novel invention or discovery related to wound dressings, registered with the appropriate government agency for exclusive rights of use and distribution by the inventor or assignee for a certain period [115]. This information can include detailed descriptions of the wound dressing composition, manufacturing methods, potential applications, and any relevant testing or clinical trial results [116].

The importance of patent information in wound dressing lies in the potential value it can offer to researchers, manufacturers, and clinicians involved in wound care. By studying patented wound dressings, researchers can gain insights into new materials and technologies that may improve the efficacy and safety of wound healing [117]. In addition, manufacturers can use this information to develop and market innovative wound dressings that offer unique benefits to patients. Moreover, clinicians can stay informed about new wound dressing options that may help their patients heal faster and with fewer complications [118]. Overall, patent information in wound dressing is an essential resource for anyone involved in wound-care research, development, and clinical practice, providing insights into new technologies and innovations that may help improve patient outcomes and advance the field of wound healing [117–119]. Table 4 describes patent information for honey-based wound dressings.

**Table 4.** Patent information of honey-based wound dressings.


#### **Table 4.** *Cont.*


#### **12. Commercialized Product of Honey-Based Wound Dressings**

Commercializing a honey-based wound dressing involves bringing the product to market and selling it to healthcare providers, medical facilities, and end-users [129]. Table 5 shows some recent honey-based wound dressings commercialized in the market. These commercialized honey-based wound dressings effectively manage and treat various wounds, including burns, diabetic ulcers, surgical wounds, pressure ulcers, etc. [25,130]. They are also known for reducing inflammation and promoting faster healing compared to traditional wound dressings. However, they should be used under the guidance of a healthcare professional before independent application of these dressings [1,131].


**Table 5.** Product commercialization of honey-based wound dressings.

#### **13. Conclusions and Future Perspectives**

Wound healing is a sophisticated process that involves the replacement of damaged tissue layers and cellular structures. Numerous approaches have focused on wound-care management, including developing new therapeutic approaches and technologies for wound management. Hydrogel wound dressings have gained attention among researchers due to their rapid wound healing properties and their ability to offer a moist environment, good biodegradability, and protection against bacterial infections. Improving the physicochemical, mechanical, and biological properties, and the wound-healing ability of hydrogel materials, is the primary goal when developing hydrogels, mainly achieved through blending natural and synthetic polymers with the addition of other bioactive substances, such as honey, which is beneficial for wound healing. The addition of honey during in vivo and in vitro studies into formulated hydrogel wound dressings has been found to prevent bacterial infections, enhance their absorption capacity, and accelerate wound healing, due to its anti-inflammatory, antimicrobial, and antioxidant activities. Moreover, the blending of polymers could be enhanced by incorporating other additives, such as cross-linkers, to enhance their mechanical properties, flexibility, biocompatibility, biodegradability, high absorption, etc. Although there are extensive in vivo and in vitro analyses that have shown efficacy in wound healing, its implementation in clinical fields still needs to be managed to ensure the safety and effectiveness of polymer-based hydrogel formulations in human applications.

**Author Contributions:** Conceptualization, S.N.N.Y., Z.S., N.H., Z.A.R. and N.I.M.; writing—original draft preparation, S.N.N.Y., Z.S. and N.I.M.; writing—review and editing, S.N.N.Y., Z.S., N.H., Z.A.R. and N.I.M.; supervision, Z.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Ministry of Higher Education (MoHE) of Malaysia under the Fundamental Research Grant Scheme (FRGS) (FRGS/1/2020/STG05/USIM/03/1).

**Institutional Review Board Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors thank the Universiti Sains Islam Malaysia (USIM) and the Ministry of Higher Education (MoHE) of Malaysia.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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### *Review* **Polymeric Materials Obtained by Extrusion and Injection Molding from Lignocellulosic Agroindustrial Biomass**

**Ada Pacheco 1, Arian Evangelista-Osorio 1, Katherine Gabriela Muchaypiña-Flores 1, Luis Alejandro Marzano-Barreda 1, Perla Paredes-Concepción 2, Heidy Palacin-Baldeón 1, Maicon Sérgio Nascimento Dos Santos 3, Marcus Vinícius Tres 3, Giovani Leone Zabot <sup>3</sup> and Luis Olivera-Montenegro 1,2,\***

	- La Molina 15024, Peru; pparedes@usil.edu.pe

**Abstract:** This review presents the advances in polymeric materials achieved by extrusion and injection molding from lignocellulosic agroindustrial biomass. Biomass, which is derived from agricultural and industrial waste, is a renewable and abundant feedstock that contains mainly cellulose, hemicellulose, and lignin. To improve the properties and functions of polymeric materials, cellulose is subjected to a variety of modifications. The most common modifications are surface modification, grafting, chemical procedures, and molecule chemical grafting. Injection molding and extrusion technologies are crucial in shaping and manufacturing polymer composites, with precise control over the process and material selection. Furthermore, injection molding involves four phases: plasticization, injection, cooling, and ejection, with a focus on energy efficiency. Fundamental aspects of an injection molding machine, such as the motor, hopper, heating units, nozzle, and clamping unit, are discussed. Extrusion technology, commonly used as a preliminary step to injection molding, presents challenges regarding fiber reinforcement and stress accumulation, while ligninbased polymeric materials are challenging due to their hydrophobicity. The diverse applications of these biodegradable materials include automotive industries, construction, food packaging, and various consumer goods. Polymeric materials are positioned to offer even bigger contributions to sustainable and eco-friendly solutions in the future, as research and development continues.

**Keywords:** agroindustrial wastes; biomaterials; cellulose; lignocellulosic biomass

#### **1. Introduction**

The growing awareness of environmental challenges and the search for sustainable solutions have led to a critical evaluation of the way natural resources and waste are managed [1,2]. The continuous growth of the global population and the increasing demand for food and energy have made the effective management of agricultural and food waste a fundamental area of concern [3,4]. On an annual basis, a considerable quantity of agroindustrial wastes, arising from the production of food and crops, amasses on a global scale. This accumulation has adverse repercussions not only for the environment, but also for the global economy [5]. These residues not only represent a loss of valuable resources, but also cause increasing emissions of greenhouse gases, thus contributing to climate change [6]. Furthermore, at various stages across the food supply chain, from production to consumption, there is a disconcerting level of food loss and waste [7,8]. The food industry

**Citation:** Pacheco, A.; Evangelista-Osorio, A.; Muchaypiña-Flores, K.G.; Marzano-Barreda, L.A.; Paredes-Concepción, P.; Palacin-Baldeón, H.; Dos Santos, M.S.N.; Tres, M.V.; Zabot, G.L.; Olivera-Montenegro, L. Polymeric Materials Obtained by Extrusion and Injection Molding from Lignocellulosic Agroindustrial Biomass. *Polymers* **2023**, *15*, 4046. https://doi.org/10.3390/ polym15204046

Academic Editor: Raffaella Striani

Received: 21 August 2023 Revised: 3 October 2023 Accepted: 3 October 2023 Published: 10 October 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

has embarked on a concerted endeavor to curtail food loss and waste, embracing strategies that champion the reevaluation of food waste. In this process, the concept of the circular economy has assumed a central role, advocating for the conversion of waste into valuable resources [9,10].

These waste materials, primarily composed of lignocellulosic biomass, can be efficiently converted into biopolymers [11,12]. Biomass, mainly comprising cellulose, hemicellulose, and lignin, necessitates pretreatment to reduce its refractory nature and enhance accessibility within its structure [13]. There are four types of pretreatment methods: physical (milling, extrusion, sonication, microwave, ultrasound, ozonolysis, and pyrolysis), chemical (alkali, dilute acid, ionic liquid, organic solvent, and oxidative delignification), physicochemical (CO2 explosion, steam explosion, hydrothermal, liquid hot water, and ammonia fiber explosion), and biological [14–16]. The characteristics of the feedstock, energy requirements, cost, and product recovery should be considered when choosing the pretreatment method [17].

Extrusion and injection molding are widely used manufacturing technologies in the plastics industry [18]. By leveraging this technology, biomass can be transformed into highquality polymeric materials with desirable properties. Extrusion involves the continuous melting, mixing, and shaping of the biopolymers, while injection molding enables the precise and efficient formation of complex shapes through the injection of molten materials into molds [19].

The use of lignocellulosic agroindustrial biomass for polymeric materials offers numerous advantages, such as reduced dependence on polymers based on non-renewable fossil fuels, thereby promoting sustainability, and reducing the environmental impact [20]. In addition, this approach contributes to efficient waste management, reducing the burden on landfills [21].

Reinforced polymers, also called composites, are the union of two materials, a matrix, and a reinforcement, characterized by one being lightweight and the other strong [22]. The matrix can be polymeric, ceramic, or metallic, while the reinforcement can be fibers, particles, or laminates [23]. A challenge in the formation of composites is the coupling of the hydrophilic interfaces in the reinforcement and the hydrophobic interfaces in the polymeric matrix [24]. Fiber reinforcement is mainly composed of lignocellulosic mass. Agroindustrial wastes are increasingly used due to their low cost, biodegradability, improved properties, and composite quality [24].

This review delves into the latest innovations and research trends in the use of lignocellulosic biomass from agroindustrial wastes, with the purpose of developing polymeric materials using extrusion and injection molding technologies. Additionally, it provides an overview of agroindustrial biomass, its properties, pretreatment methods, and extrusion and injection molding processes. The review also underscores the wide-ranging industrial applications of these materials and outlines potential future developments.

#### **2. Sources and Components of Lignocellulosic Agroindustrial Biomass**

Lignocellulosic agroindustrial biomass, a highly renewable and cost-effective natural resource, is derived from agricultural residues (husks, bagasse, seeds, roots, leaves, stems, seed pods, and straw), food processing waste (peels, skin, shells, oil cakes, and egg waste), and forestry by-products [9,25]. The primary components of biomass are cellulose, hemicellulose, and lignin. The components can vary based on factors such as the type of biomass, location, climate, and harvesting season [12].

#### *2.1. Cellulose*

Cellulose (C6H10O5)n is the most abundant renewable natural polymer in nature. In general, lignocellulose-based biomaterials have a large proportion of total cellulose content, highly interlaced by a significant amount of covalent bonds with high rigidity to form extremely strong and resilient components [26]. Generally, cellulose from algae is approximately 70 wt% and cellulose from plant-based materials ranges from 40 to

60 wt% [27]. Nonetheless, some studies indicate that the cellulose content in some plants, such as hop stems, can reach 70 wt%, which allows the substance to be widely used for biopolymer production and applications [28]. Spontaneously, cellulose molecules form large agglomerates that aggregate into microfibrils, which are constituents commonly called crystalline and amorphous zones [29]. The structure of the multiple components that comprise the complex are presented in Figure 1. The structure of cellulolytic chains is made up of cellulose microfibrils, intimately intertwined in complexes based on lignin and hemicellulose.

**Figure 1.** Matrix with cellulose macrofibrils and microfibrils intimately intertwined by the matrix of lignin and hemicellulose.

One of the main assertions about cellulose, and its stocked reserve of highly renewable and widely investigated organic constituents, is directed at the ease of obtaining the biopolymer. Expressive amounts of cellulose are easily verified in a series of plant species, marine algae, marine animals, bacteria, and vegetable residual biomass, which represent up to 50 wt% of the total weight of the biowaste [30]. Additionally, cellulose promotes high resistance in the plant cell wall, mainly due to the large number of glucose monomeric units covalently linked through β-1,4 glycosidic bonds [31].

Furthermore, other characteristics of the cellulose complex give rise to the recalcitrant characteristic of lignocellulose-based materials, such as the high crystalline performance of the matrix, a significant degree of polymerization (up to 10,000 units), and the presence of an intricate network of hydroxyl groups associated with intramolecular hydrogen bonds in cellulose [32]. The glucose-rich aggregate of hydroxyl compounds forms intertwined hydrogen bonds that provide resilience to the molecular structure and connect with neighboring particles to form a network of microfibrils. The hundreds of bonds that involve intermolecular and intramolecular hydrogen molecules and the intertwining of crystalline and non-crystalline zones are intimately responsible for the two-phase structure of cellulose, in which the regions of high crystallinity or cellulose nanocrystals (CNC) stand out [33]. Conversely, more susceptible molecular chains are called amorphous zones, which are easily degraded to obtain a highly soluble and reactive amorphous material. This performance promotes a drastic decline in solubility in liquid contents and an increase in resistance to molecular chain disfigurement by the action of water [34]. Additionally, cellulolytic chains

include β-D-glucopyranose elements interconnected via β-(1,4)-glycosidic bonds. Cellulose holds up to 1400 D-glucose units directly disposed to structure microfibrils units, which are broadly grouped to configure cellulose fibrils, which are structured under a highly rigid and vigorous matrix, rich in cellulose and hemicellulose [35].

The diversity of applications of cellulose complexes is closely associated with a range of matrix dominances, such as low density, biodegradability, significant porosity, and improved physical and mechanical mechanisms [36]. Cellulose is easily obtained from natural sources, which corroborates its high accessibility, cost effectiveness, applicability, reduced or minimal toxicity, and biocompatibility [37]. The total cellulose content and the arrangement of the crystalline zones are dependent on the plant species and the lignocellulose content, which is directly associated with the resistance potential of the biomaterial and the difficulty of breaking the complex by the action of hydrolysis [38]. Furthermore, there is a diversity in the secondary structures derived from cellulose, or crystal arrangements, such as cellulose I, cellulose II, cellulose III, and cellulose IV [39]. Cellulose I is associated with natural cellulose, easily found in nature. Cellulose II and cellulose III are by-products of the original cellulose, generally obtained through the regeneration of cellulose I. Finally, cellulose IV is obtained from cellulose III using procedures involving high temperatures and glycerol. The different crystal arrangements vary in terms of the characteristic attributes, such as hydrophilicity, mechanical potential, and stability performance [40].

Recently, cellulose-based exploration has been promoted due to a series of benefits, such as cost effectiveness, efficiency, physical and mechanical properties, the low degree of the environmental impact, exuberance, and capacity for nanoscale structure, among others [41]. A variety of technological innovations have been widely explored for the isolation of cellulose from lignocellulosic waste. The high interest has broken sustainability boundaries under the concept of biorefineries, because there is a wide spectrum of applications for cellulose-rich biomaterials or secondary bioproducts. Among the main industrial complexes that instigate research associated with cellulose are the food industry [42], textile industry [43], energy production [44], building and engineering industry [45], biomedicine [46], pharmaceuticals industry [47], adsorption [48], and wastewater treatment [49], among others.

#### Nanocellulose

Nanocellulose is a biopolymer originating from cellulose and occurring at the nanoscale, obtained mainly from marine and land plants, animals, and bacteria in four primary forms: CNC, cellulose nanofibers or nanofibrillated cellulose (NFC), microfibrillated cellulose (MFC), and microbial or bacterial nanocellulose (BNC) [50]. Nanocellulose is characterized as highly resistant fibers, with a diameter of less than 100 nm and a density of up to 1.6 g/cm3. A high abundance of hydroxyl functional groups can be easily adapted to express high performance [51]. Nanocellulose provides a highly modifiable surface, significant mechanical strength, high hydrophilicity, and biocompatibility [52]. During the hydrolytic process, the amorphous zone of the cellulose fibers is cleaved to form an extremely strong and crystalline nanoscale structure with a rod-like arrangement [53]. Most commonly seen, CNC features lengths of up to 100–300 nm and up to 5–50 nm in diameter, with a rich hydrogen bonding matrix, allowing for high voltage transfer. An NFC is commonly synthesized using chemical pretreatments and homogenization is carried out in high-pressure conditions [54]. NFCs constitute nanoscale fibrils, with a width between 2 and 60 nm, and are established from the agglomeration of cellulose chains, generated by hydrogen bonds, and comprise crystalline and amorphous zones, easily synthesized from the discharge of fibrils from microfiber bundles under strategies of mechanical fibrillation [55]. Furthermore, BNC consists of the application of microorganisms as primary sources of biopolymers, mainly due to the rapid microbial growth and high availability of the product. The literature indicates two dominant procedures to produce BNC based on microbial agents: static culture and agitated culture. Static culture refers to the accumulation of BNC forming a thick and whitish layer or cuticle. Agitated culture spontaneously

produces cellulose in the culture medium, forming irregular agglomerates or suspended fibers [54].

CNCs are nanoparticles abundantly rich in fragments of the cellulose chain, rigorously ordered in a crystalline structure of up to 100 nm. CNCs indicate high thermal stability, in addition to a higher surface area and crystallinity compared to primitive cellulose [41]. NFCs are frequently produced by many mechanical procedures, such as milling/refining, high-pressure homogenization, ultrasound-assisted treatment, microwave, steam explosion, and microfluidization, and by a series of chemical processes, such as TEMPO oxidation, persulfate oxidation ammonium, carboxymethylation, and cationization [40].

The direct alteration of the surface of the cellulose nanoparticles allows access to the biopolymer for a variety of purposes (Figure 2). Modifications based on hydroxyl groups allow the improvement of the biomaterial and intensify its potential use. Chemical reactions involving oxidation and acetylation processes or the addition of functional materials, polymers, and functional groups on the surface of the nanogranules allow the surface properties of the nanocellulose to be improved and associate with different non-polar matrices or change its affinity with certain polar and non-polar molecules [56]. The use of nanocellulose has aroused extensive industrial interest and has shed light on a variety of operations, such as the paper industry [57], packaging [58], cosmetics [59], the pharmaceuticals industry [60], medicine [61], biomedicine [62], paints and coating [63], hydrogel synthesis [64], and filtrations [65]. Nanocellulose has two basic disadvantages, namely a high number of hydroxyl compounds, which causes strong and resistant interactions by hydrogen molecules between two bundles of nanofibrils, and high hydrophilicity, which does not allow its application for a variety of industrial purposes, such as coating paper or composites, for example, without inducing a prominent surface modification to degrade the number of hydroxyl interactions and to stimulate compatibility with several other matrices [66].

**Figure 2.** Main advantages and current applications of cellulose-based biomaterials and cellulose primary configurations.

#### *2.2. Lignin*

Lignin is one of the most exuberant organic materials in nature and its content range is 15–30% in plants. However, these concentrations are variable depending on the type of biomass, plant characteristics, plant growth environment, and constitution of the cellulose wall [67]. Moreover, lignin is the only renewable aromatic polymer in nature [68]. Approximately 50 to 70 million tons of lignin are produced worldwide [69]. This panorama is directly associated with the widespread use of lignin as a source for the production of biofuels, since about 60 billion gallons of biofuels should be produced annually. Approximately 0.75 billion tons of biomass rich in lignin is required, indicating that the conversion of plant biomass will result in at least 0.225 billion tons of lignin as a by-product [70]. In the plant spectrum, lignin encompasses the free space between the cellulose and hemicellulose bands, establishing a highly resistant and rigid structure, whose purpose is to act in the performance of water and nutrient transport in the stems of plants [68]. The lignin matter is closely associated with the mechanical properties of the plant cell wall and the mechanical resistance provided by the biopolymer is significantly superior to the resistance provided by the cellulose content [71].

The inflexibility of lignin is highly influenced by the aromatic chains in the compounds in signapyl alcohol, *p*-coumaryl alcohol, and coniferyl alcohol. Furthermore, plant species with high lignin production have large amounts of lignin-synthesizing enzymes, such as phenylalanine ammonia lyase (PAL), caffeic acid *O*-methyltransferase (COMT), 4-coumarate coenzyme A ligase 3 (4CL3), cinnamyl alcohol dehydrogenase 2/7 (CAD2/7), cinnamoyl-CoA reductase 20 (CCR20), and cinnamate 4-hydroxylase (C4H) [72]. Lignin is also composed of three hydroxycinnamic alcohols, cetearyl alcohol, and mustard alcohol via ether associations, C-C chains, among others [33]. There is a significant diversity in distinct, highly polar chemical groups allocated in the structural complex of lignin, such as methoxyl, hydroxyl, carbonyl, and carboxyl, granting lignin high resistance to the action of enzymes, chemical solvents, or water hydrolysis [20].

Lignin acts as a carrier of fundamental materials, such as water and nutritional substances, and as a component of structural support for plant organs, arranging the matrix that also composes cellulose and hemicellulose in the complex [73]. The lignin content in the plant may vary with the species and the morphological organ, since there is a diversity in the scientific investigations that have indicated different concentrations of lignin in different organs of the same plant [74–76]. The high accessibility and ease of obtaining lignin from natural sources is key to a wide range of industrial applications, from adsorbent materials to biofuels and power generation [77]. The sustainable footprint of lignin provides the basis for the synthesis of biomaterials that convert the uncontrolled use of chemical resources to the production of electricity [78]. The spectrum of direct applications of lignin includes the engineering industry [79], biomedicine and biotechnology [80], medicine [81], biopesticides and biofertilizers [82], wastewater treatment [83], biofuels [84], adsorbents [85], carbon fibers [86], adhesives [87], dispersants [88], anti-UV filters [89], and the pharmaceuticals industry [90].

#### **3. Modification and Characterization of Cellulose**

Cellulose is widely obtained from lignocellulose-rich materials, bacteria, marine animals, and algae [91]. With the intensification of sustainable assertions in recent years, the exploration of polymers of natural origin has gained attention, which directly reflects the exploration of technological strategies and processes that involve the modification of these materials to enhance performance. The structural modification of the cellulose surface aims to reduce the high hydrophilicity of biomaterials, as well as to intensify the rupture of the long chain of hydroxyl groups that sustain the material. To improve treatment performance, it offers appropriate cost effectiveness and generates bioproducts in an environmentally friendly context. Pretreatments involving cellulose materials can be of enzymatic origin or TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl). These procedures aim to increase the reactivity of cellulose, especially in the transfiguration of hydroxyl groups into carboxylate groups [54]. The subtopics described below provide a better understanding of the processes involved in configuration changes in cellulose-based biomaterials.

#### *3.1. Surface Modification*

The surface arrangement of nanocellulose can be easily configured through the continuous action of surfactants rich in highly hydrophobic and hydrophilic groups, or the adsorptive process based on polyelectrolytes [66]. There is a diversity of surfactants, such as fluorosurfactants adherent to the cellulose structure, cationic surfactants, and polyelectrolyte compounds, adapting the hydrophobic potential and improving specific properties [54]. Alterations in the hydroxyl groups that form the surface structure of nanocellulose are appropriate to enhance the spectrum of action of these biopolymers, especially in association with other materials to configure the structural properties of nanocellulose and improve the field of affinity with highly polar and/or non-polar matrices [56]. The modification of the surface structure using the adsorption method is segmented into two main classifications: the polyelectrolyte method and specific groups aimed at the adsorption of some points. The polyelectrolyte method has high potential as it involves different polyelectrolytes with opposite charges and specific nanoparticles to adapt the desired properties to the nanoparticles, with ease of modification through the adsorption of the nanoparticles and CNFs [92].

#### *3.2. Grafting*

Graft polymerization is a cellulose modification strategy whose purpose is to stimulate highly resistant covalent bonds to generate a branched copolymer, without affecting the primary characteristics of the biomaterial [37]. The grafting procedure drastically reduces the interaction between solutes and unattractive aggregates with the cellulolytic surface, providing groups suitable for designing electrostatic repulsion from the membrane surface or enhancing hydrophilicity to enhance water-surface interactivity [93]. The grade of the grafted polymer directly affects the properties of the natural fiber, mainly the mechanical characteristics, elasticity, potential absorption, ion exchange competence, propensity for rupture of the resistant structure with extreme conditions of temperature and abrasion, and resistance [94].

Generally, the grafting procedure involves different mechanisms of action: (i) "grafting into" a step directly related to reactions between the functional groups of different polymers; (ii) "grafting from" refers to a polymer with functional groups that enhance the polymerization of vinylic monomers, in which the highly reactive sites belonging to the main chain are stimulated by chemical treatments or irradiation; and (iii) "grafting through" which implies the (co)polymerization of macromonomers [95]. Modification of the surface of cellulose by polymerization provides for the alteration of specific physical and chemical properties that may suit the desired purpose [96].

The effect of the grafting of cellulose in polylactide was evaluated after the synthesis of a series of cellulose ester–graft–polylactide (CeEs-g-PLA) copolymers. A series of CeEs-g-PLA copolymers was synthesized using one-pot reactions involving acylation and ring-opening polymerization. With the increasing degree of acyl group substitution, the copolymers presented enhanced thermal stability and thermoplasticity due to the intermolecular interactions between the acyl groups and polylactide sidechains. Therefore, the feed content of the acyl agent has a significant influence on the structural characteristics of the graft copolymer, because the acylation proceeds predominantly at the hydroxy groups in the cellulose backbone and, then, the PLA chains are grafted onto the remaining unreacted hydroxy groups [97].

Green biofilms with antimicrobial activity were developed from PLA and cyclic Nhalamine 1-chloro-2,2,5,5-tetramethyl-4-imidazolidinone (MC) grafted microcrystalline cellulose (g-MCC) fibers. The grafting percentage was 10.24%. The grafting improved the compatibility between g-MCC and PLA, leading to an excellent dispersion of g-MCC in the film matrix, and a superior transparency of the g-MCC/PLA compared to that of the MCC/PLA films. The enhanced compatibility of the g-MCC/PLA films produced better mechanical properties, including the mechanical strength, elongation at break and initial modulus than those of both the MCC/PLA and MC/PLA composites. The oxidative

chlorine of g-MCC/PLA was highly stable compared to that of MC/PLA films, providing long-term antimicrobial activity [98].

Incorporating the surface-grafted cellulose nanocrystals (CNCs) with enantiomeric polylactide (PLLA or PDLA) was presented as an effective and sustainable way to modify PLLA. The CNCs with identical content and length of PLLA and PDLA were prepared and blended with PLLA. The rheological properties of PLLA/CNC-g-D are improved, indicating that the stereocomplexation can improve the interfacial strength as compared with the conventional van der Waals force in PLLA/CNC-g-L. The matrix crystallizes at a higher rate in PLLA/CNC-g-L than PLLA/CNC-g-D. PLLA/CNC-g-L15 reached its half crystallinity in 8.26 min, while a longer period of 13.41 min was required for PLLA/CNCg-D15. The formation of low content sc-PLA at the interface may restrict the diffusion of PLLA, but contribute less to generate crystalline nuclei, which synergistically leads to the retarded crystallization kinetics in PLLA/CNC-g-D [99].

#### *3.3. Chemical Procedures*

Chemical-based modification procedures involve changes in the basic properties of cellulose, such as the hydrophilic or hydrophobic potential, elasticity, water absorption, adsorptive or ion exchange performance, and resistance to adversity. The dominant basic chemical modification strategies for cellulose are esterification, etherification, halogenations, oxidation, and treatment with alkaline compounds [54]. Changes in the nanocellulose complex significantly increase the degradability and biocompatibility of the biopolymer with other biomaterials [53]. Furthermore, considering the low cost–benefit and process efficiency, Table 1 indicates the main segments and pretreatments for modifying the pulp structure, from specific chemical methods to mechanical base changes. The procedures increase the cellulolytic reactivity and enhance the conversion of compounds into desired functional groups to adapt to promising characteristics and properties.

#### *3.4. Other Treatments*

Furthermore, the diversity of viable alternatives has been applied to biowaste treatment. These strategies concentrate techniques of mechanical and/or thermal and chemical activities to alter the physicochemical properties of the feedstocks. Among the mechanical and physical methods, the drying method and the milling strategy are valid alternatives that have been widely explored. The drying method is extremely necessary for preparing the raw material before applying other pretreatment strategies, especially for eliminating moisture from the material, which improves process efficiency and requires lower temperature and calorific value [100]. Cellulose drying conditions directly influence its dissolution and some studies have led to a parameterization of conditions to optimize the cellulose dissolution process [95]. The drying process can be conducted by oven drying and/or freeze drying, hot pressing, and supercritical drying with CO2. Furthermore, the drying procedure or wetting/drying cycles, called hornification, provide higher dimensional stability and less material degradation through increases in molecular packing. This procedure can be controlled, for example, by the time and/or number of cycles and drying requirements [101].

Among the mechanical methods, strategies aimed at reducing the particle size and increasing the contact area between the solid matrix and the solvent are widely applied. The milling strategy involves the effectiveness of the mechanical and thermal effects to redesign the fiber matrix and provide a wide spectrum of applications for the biomaterials, based on the adjustment of high pressure, collision, and absorption, in addition to a significant increase in temperature [102]. Additionally, the milling procedure is an extremely efficient strategy for modifying the crystalline structure of cellulose, as it enables the optimization of cellulose hydrolysis, interrupting the crystallinity (cellulose I) of native cellulose through increased contact with acid by cellulose [103].

According to physiochemical methods, they are the most common alternatives, mainly due to the modifications in the properties of the material, as well as the increase in inter-

molecular interactions. These methods involve steam explosion, wet oxidation, liquid hot water (LHW), and microwave-assisted and ultrasound-assisted extractions, and have been widely explored due to the high rupturing of the lignocellulose complex and minimization of the crystallinity of the cellulose. Accordingly, the steam explosion process is an environmentally viable strategy to modify cellulose fibers through the intensification of fibrillation, providing the synthesis of nanofibers [104]. Furthermore, the steam explosion procedure promotes the rupture of lignocellulosic biomass components by steam heating, shear forces, and hydrolysis of glycosidic bonds by the organic acid formed during the process. The steam explosion procedure facilitates the rupture of lignocellulosic structures, promoting the modification of the physical properties of the material (specific surface area, water retention capacity, color, etc.) and increasing the rate of enzymatic hydrolysis of the cellulose components [105].

Wet oxidation is an interesting alternative applied to the functionalization of cellulose because the process results in products with different structures and properties depending on the substrate, reagents, reaction parameters, and medium. The strategy provides new, high-performance materials based on cellulose, with the possibility of a variety of applications [106]. The oxidation process involves changing the performance of nanofibrils, facilitating their dissolution in water. This scenario results in a high degree of cellulose processing, without requiring the use of chemical products [107]. Furthermore, pretreatment with liquid hot water (LHW) is an interesting strategy, since it does not involve the addition of chemicals and has moderate process operating conditions. The procedure involves the application of water associated with an increase in temperature, drastically reducing the pH of the medium releasing carboxylic acids and intensifying the rupture of the structural matrix of the lignocellulosic biomass. Consequently, there is a significant increase in the accessible surface area, intensifying the action of the enzymes and the fermentation process [108].

Furthermore, microwave-assisted technology is a promising technique applied to lignocellulose-rich structure modification processes and extraction procedures. The alternative applies microwaves to significantly increase the temperature of the medium, reducing the reaction time, improving the process efficiency, and establishing uniform operating conditions, such as fast heating speed, uniform heating, and no temperature gradient occurrence [109]. In the hydrolysis processes, the microwave-assisted treatment significantly promotes the transformation of cellulose into C6 molecules with high selectivity. High-temperature conditions act positively on hydrolysis performance, since the microwave-assisted process allows superior operating conditions compared to conventional hydrothermal systems. It was pointed out that high temperatures promoted an intensification of the association at the molecular level between the microwaves and cellulose (through the primary alcohol groups, –CH2OH groups), redirecting the energy to the surrounding molecular structure to initiate the cleavage of polysaccharide chains [110].

Ultrasound-assisted technology has been indicated as an efficient strategy in the extraction and rupture processes of the lignocellulosic complex. The energy intensity of the process increases the mass transfer of the biomass components to the extraction solution and, under established conditions, causes the acoustic cavitation process, in which the waves produced by the equipment propagate in expansion and compression cycles. Large amounts of microbubbles are formed and collide with strong motion. The friction between the microbubbles releases a significant amount of energy in the configuration of shock waves, which come into contact with the material rich in lignocellulose and promote its disintegration, facilitating the extraction and modification processes [111]. The hydrodynamic forces produced lead to the defibrillation of the biomass, which may be pure cellulose, microcrystalline cellulose, or other components of interest. The direct rupture of the biomass promotes the formation of filament aggregates with different sizes. The performance of the process depends directly on the characteristics of the material, since the ultrasonic bath acts on the crystalline structure of cellulose in different ways, based on

the type of biomass, operating conditions, concentration of lignocellulose, and degree of crystallinity [112].

On the other hand, the use of organic solvents is still one of the main alternatives adopted as a pretreatment. The replacement of the primary hydroxyl groups in cellulose by other molecules results in the intensification of the diversity of chemical reactions, in addition to contributing to an increase in grafting efficiency and the performance of functional groups for structural modifications. The main chemical reaction alternatives applied for the structural modification of cellulose are esterification, oxidation/amidation, and silanization. Esterification is generally carried out by an acylation process with carboxylic acid anhydride and 4-dimethylaminopyridine or strong acid as a catalyst. The oxidation process involves distinct C6 hydroxyl groups under moderate aqueous conditions; in addition to modifying the biopolymers and causing strong bonds at one end and adapting them with specific functional groups at the other to adapt to the matrix [113]. The application of silane is widely carried out, since the process intensifies the interfacial interaction between the hydroxyl groups of cellulose. The silanol agent is produced and can react with the hydroxyl groups of cellulose or condense on cellulose surfaces since they have the same reactive groups (-OH). Furthermore, thermal treatments can allow condensation between the OH groups of hydrolyzed silanes and cellulose, assuming chemical modification [114]. Nevertheless, these materials are highly harmful and their recovery after the extraction procedure requires additional steps, which results in higher process complexity and increased cost–benefit [115]. The continued use of solvents in pretreatment procedures is still inevitable, mainly due to the high proportion of solid dissolution, mass and heat transfer, viscosity reduction, and effectiveness in the separation and purification operation [116].

**Table 1.** Current advantages and limitations to the main cellulose-based modification processes.


#### **4. Modification and Characterization of Lignin**

Lignin is the second most abundant biopolymer in nature, with a highly resilient structure and strong antioxidant activity. The molecular design of lignin indicates a significant concentration of functional groups, with easy alteration of properties based on chemical modification procedures [121]. The concentration and design of the lignin matrix varies depending on the type of biomass and lignocellulose content [122]. The golden age of exploring highly sustainable energy sources based on the use of materials rich in lignocellulose comes from strategies for a diversity of applications, such as the mass production of biofuels and other biochemical products to satisfy energy demand. Some essential plant materials from widely cultivated crops, such as sugar cane, corn, and sorghum, are promising for processes involving the synthesis of first-generation biofuels and chemical products of interest [33]. Under biorefinery concepts, the type of biomass is also strongly influenced by local characteristics, such as agricultural management, climate performance, and raw material availability. Since the bioeconomy approach has emerged as a strategy faithfully associated with the valorization of residues of plant origin, the requirement for natural biopolymers has fueled interest in technological alternatives and methods associated with the modification of lignin [123].

Considering that the structure of lignin is rich in a diversity of active groups, lignin can react chemically from different aspects, such as halogenation, nitration, phenylation, graft copolymerization, alkylation, dealkylation, sulfomethylation, acylation, ammonization, esterification, and hydrogenolysis. Furthermore, lignin has satisfactory compatibility with other biopolymers or natural fibers due to its hydrophilic nature, which establishes the application of lignin polar groups as agents to increase compatibility with essentially hydrophobic polymers. Furthermore, cross-linking with other polymers is desirable from the application of their hydroxyl groups to give rise to new materials, such as aromatic chemicals and bio-based polymeric materials [124].

One of the main methods of modifying lignin consists of the esterification of the biopolymer in reactions involving carboxylic acids, anhydrides, and acid chlorides. In this case, the modification of lignin by esterification reaction causes significant changes in its properties, such as better UV absorption, altered thermal stability, higher compatibility with the matrix, improved mechanical properties, better dimensional stability, improved hydrophobicity, and higher resistance to microbial decomposition [125]. Other surface modification strategies, such as conductive polymer coating, gold spray coating, and metal oxide coating, have received attention, due to the tunable physicochemical properties that have a wide range of uses, such as energy storage, sensors, and adsorption propensity [126]. Other methods involve physical modification techniques, which do not involve reactions between the functional groups present in lignin, but explore physical strategies that promote new and distinct properties of the modified material. Among these techniques, the application of gamma irradiation, sorption of metal ions, and plasma treatment are excellent exemplifications. These alternatives cause strong variations in the morphology of lignin, ease the rupture of the rigid matrix, and cause alterations in the surface characteristics of the material.

#### **5. Manufacturing Technology**

#### *5.1. Extrusion Technology*

An extrusion machine can be a single or twin-screw machine. A twin-screw extruder offers better efficiency results by reducing the melting and mixing time. Three important aspects related to extrusion technology are polymer melting, solids transport, and melt flow, which are controlled by computer models. These extrusion models are limited to pure polymers, so when making a composite there are difficulties in the fluidity of the reinforcing material [122]. However, a model called global GSEM has recently been developed for the extrusion of reinforced polymers in single-screw extruders with flood and starvation feeding, where starvation feeding has advantages to melting, less agglomeration, and better compound mixing [127].

Extrusion is a technology that is generally used as a preliminary step to injection molding. In extrusion, the matrix and the reinforcement are mixed to form granules, which are then laminated with injection technology [23,128].

There is research using extrusion as a pre-injection stage using vegetable-based materials. Mainly when producing pellets, this is the case in the study of thermoplastic starch and polylactic acid with tannins to delay biodegradation [129], to evaluate compatibilizers between polylactic acid and thermoplastic starch [130], with residues of soy, polyvinyl alcohol, and starch [131], or the use of bagasse cassava with polylactic acid for the production of tubes for seedlings [132].

#### *5.2. Injection Molding (IM)*

The injection molding process has four relevant phases: plasticization, injection, cooling, and ejection [133]. During the first phase, the material is inserted into the barrel through a hopper and is melted using a rotating screw and internal heating units. Once the material is melted, it continues to the next phase, where the material is injected into the mold at a set speed and pressure parameters. For this, the screw is shifted to the front to avoid pressure variation and backward movement of the material in the barrel or deformation of the material. After this, the molded part goes to the cooling phase where the pressure and temperature decrease. This phase ends when the material solidifies. Finally, in the ejection, the part is removed by opening the mold [134–136]. IM technology demands high-energy consumption during processing [137]. The cooling phase is the most time-consuming stage in the cycle, taking between 50% to 80% of the cycle, so it is the stage that consumes the most energy [138]. As a result, there are more and more studies on improving energy efficiency at different stages of the process [139–142].

#### Parts of an Injection Molding Machine

The fundamental aspects to consider for IM are the machine specifications and the material to be used. Optimizing these aspects can ensure the reduction of defects and the quality of the final products [143]. An injection molding machine has a motor. It can be an AC motor or a hydraulic motor, with the hydraulic motor being the most used due to its excellent characteristics, such as less force required to start the movement and less overload on the rotating screw [134]. Then, it has a hopper to receive and store the material until it passes into the barrel to be melted, with the help of the heating units and the rotary screw. As the pellets are moved forward by the screw, they gradually melt, and are entirely molten by the time they reach the front of the barrel. In this part, there are temperature control sensors for the resistors. To complete the injection unit parts, we have the non-return check valve and the nozzle that contacts the mold and through which the material is injected. In the clamping unit, there are the fixed platen and the mobile platen that hold the mold [22,144].

Additionally, water is involved in the injection process. The plastic, which has the consistency of warm honey, is too viscous to flow through the narrow vents. To speed up the plastic's solidification, coolant, typically water, flows through channels inside the mold just beneath the surface of the interior. After the injected part solidifies, the mold opens. As the mold opens, the volume increases without introducing air, which creates tremendous suction that holds the mold together [145]. The extrusion and injection molding process described is illustrated in the following Figure 3.

**Figure 3.** Extrusion and injection molding to produce composite-based parts.

#### *5.3. Materials*

The materials used for IM can be thermoplastic or thermosetting. Some of the polymers used are PA 6, PC, PE-HD, PE-LD, PP, and PS, although there is an extensive variety [146]. Currently, due to the growing interest in biodegradable compounds, petroleum-derived polymers are being replaced by biopolymers obtained naturally or synthetically, such as PLA, TPS, PGA, PHB, PLLA, etc. To select the most suitable polymer for IM, it is important to consider some of the relevant inherent properties, such as strength, flexibility, toughness, thermoresistance, and cost [147].

#### *5.4. Polymer Composites: Issues, Challenges, and Progress*

#### 5.4.1. Cellulose and Hemicellulose Used in Injection Molding

Cellulose and hemicellulose in injection molding are generally used as reinforcing materials in bonding to a matrix polymer. The mechanical, thermal, and morphological properties of injection molded reinforced composites are the focus of research and discussion, since these properties are parameters to evaluate the improvements that the addition of lignocellulose to the polymer matrix can provide. The parameters of reinforced polymers are mainly linked to the pretreatment of the fiber, the percentage of the filler to be used, the dispersion of the fibers in the matrix, the technology used, the fiber length, and the properties of the matrices [148].

The compatibility between the matrix and the reinforcement represents a challenge due to the hydrophilic behavior of the filler and the hydrophobic behavior of the matrix, resulting in fiber agglomeration [149]. For this reason, coupling agents that act both in the matrix and the filler are currently used to improve the adhesion, heat resistance, and mechanical properties of the composite. The most used coupling agents are epoxy and maleic groups and glycidyl methacrylate because of their favorable compatibility [150–152]. One of the agents most widely used as a compatibilizer is maleic anhydride grafted polypropylene (MAPP), as it provides good adhesion when a polypropylene matrix is used [153,154]. The correct adhesion between the fiber and the matrix will integrate the fiber-dependent strength and modulus and the matrix-dependent thermal stability.

For the formation of parts by extrusion and IM using biomass as reinforcement, it is important to consider the processing temperature. Lignocellulose has two zones where its main components are lost, between 200–250 ◦C where amorphous cellulose and hemicellulose are degraded, and between 360–540 ◦C where lignin is degraded [150,155–157]. This parameter can affect the tensile strength and stiffness of the obtained product [158]. Organoleptic characteristics, such as odor and color, are also affected by high temperatures, even though cellulose has a high thermal resistance. Hemicellulose, on the other hand, decomposes producing an inappropriate odor, however, this can be minimized with odor attenuators [159]. The color of the molded part can undergo variations depending on the reinforcement material used, such as turning a dark brown color due to the Maillard reaction [160].

In injection molding and extrusion, the reinforcement material and the matrix material influence the rheological, mechanical, and thermal characteristics. Overfilling can reduce the contact between the reinforcement surfaces and the matrix due to the lack of available contact surfaces in the matrix, which will affect the mechanical properties by preventing energy absorption and enhancement of the matrix polymer [161]. Regarding the rheological properties, the increase in filler material does not significantly affect the viscosity or melt temperature [153], but it can generate an increase in pressure, which can cause clogging of the nozzle during injection and generate defective parts [162]. In some cases, the coupling agent has been shown to reduce viscosity by providing lubrication, which may reduce the pressure required during injection [163]. In the study on a composite reinforced with coffee husk flour, they evidenced fractures in the rough surface due to the increase in filler [160]. Increased filler and poor adhesion can affect the toughness of the composite and promote brittleness, as evidenced in tests conducted between linseed meal and PLA. However, this can be significantly reduced with the use of linseed derivatives, such as oil, which serve as a plasticizer during extrusion [164].

Extrusion is commonly used as a previous step to IM, used to make the blend of the reinforced composite. Hence, it is very important to try to optimize the parameters during this process. A failure related to the extrusion of fiber-reinforced polymers is breakage due to stress accumulation in the fibers. This is mainly due to the control of parameters through the extruder flow [165]. The size of the fibers and the shearing can also affect the mechanical properties since the adhesion between the compounds is reduced [151,166]. It reduces the surface interaction between the filler and the matrix, as well as overfilling, promoting agglomeration and a reduction of Young's modulus [167]. The tensile modulus will increase as the fiber length increases [158]. One technique that has shown promising results in the processing of polymer composites before injection is solid-state extrusion (SSE), as it favors fiber distribution and dispersion [149].

#### 5.4.2. Lignin-Based Polymeric Materials

Due to its hydrophobicity and rigidity, lignin is of direct use, however, it requires hard work for its integration with a polymer matrix [168]; in addition to being incompatible with various aliphatic polyesters, such as PLA and PLC, impairing its mechanical properties [169]. In the evaluation of the addition of unmodified lignin extracted from tobacco in HDPE, it was found that the injection molding parameters are not affected and the dispersion using a single-screw extruder is adequate; however, the increase in lignin decreases the resistance to traction [170]. A coupling agent in lignin-reinforced composites, such as maleic anhydride grafting, can improve the tensile strength and ethylenebutylacrylate glycidylmethacrylate terpolymer (EBGMA) impact resistance. The combination of both can offer better results in terms of both characteristics [171]. More recent studies have seen advances in composites by extrusion and injection with hybrid components (pp/lignin/linen) using MAPP to ensure adhesion, obtaining improvements in stiffness and strength [172].

Regarding advances in extrusion and injection technologies, biobased polyethylene and kraft lignin processed using reactive extrusion with dicumyl peroxide (DCP) offer suitable results in terms of the mechanical properties and dispersion in lignin, thus being an effective and sustainable alternative [173]. Kraft lignin can also be used as a biocoupling agent when modified by phenolation or glyoxalation, giving similar results to those obtained with maleic anhydride grafting concerning the mechanical properties [174].

#### **6. Applications**

In recent years, the number of biodegradable materials from different agroindustrial wastes and by-products has increased because of the need to replace the use of conventional petroleum-based plastics. In this context, developing biodegradable plastic (natural polymers or biopolymers) is necessary to avoid recycling and environmental pollution issues. It also has several advantages, such as renewability and biodegradability, and can be part of sustainable consumption that minimally affects the environment [175]. This agroindustrial biomass may be directly incorporated into polymer matrices, reinforcing filler composites [175], or used as the source of particular compounds to modify the polymer materials [176].

Corn, wheat, rice, soybean straw, sugarcane bagasse, orange waste, coffee industry by-products (coffee husk, spent coffee grounds) [177], avocado seed flour [175], banana and pineapple wastes, cornhusk, malt bagasse, and a diversity of residues are used in polymer matrices (polyolefins, low-density polyethylene, polyhydroxybutyrate, high-density polyethylene, and polypropylene). They are used for the manufacturing of natural fiber composites (NFCs), mainly to promote mechanical reinforcement and thermal or acoustic insulation [178], since they have thermal conductivity similar to these materials. They have already been implemented in the automotive, aerospace, and defense industries, where innovations are being made [179]. It uses trays prepared by thermopressing in a compression molding machine to fabricate biodegradable trays for semi-rigid packaging [180].

In agricultural, agroindustrial wastes, such as corn and wheat-waste flour, sunflower seed husks, rice husks, yerba mate waste, and cellulose paper, are used in the development of biodegradable and compostable pots for seedling growth containers molded from the obtained thermocompressed sheets using a mold with the specified dimensions [181]. In civil construction, studies have been developed on the application of vegetable fibers as reinforcement in cement-based composites and particleboards for building construction and infrastructure, for applications as ceilings and as structural components [182].

Applications of polymeric materials from agroindustrial biomass include household goods, sports equipment, musical instruments, toys, office supplies, flexible cards, and within the automotive industry in the form of pellets by injection molding and extrusion [67,158]. Regarding the food industry, it has been used extensively for the formulation of food packaging and containers, such as trays, plates, bags, cups, and lids. In the food services sector, it has been used to produce spoons, forks, knives, and drinking straws, as shown in Table 2.


**Table 2.** Applications of compounds.


**Table 2.** *Cont.*

The use of PLA in combination with cassava bagasse accelerates biodegradation faster, its use as seedling tubes increases the phosphorus content of the soil [132], cassava bagasse also increases the tensile strength, the modulus of elasticity, and lowers the water absorption capacity [201]. Besides, their use as lignocellulosic nanofibers, in combination with cassava starch, they obtain good intermolecular interaction and barrier properties, which can be applied in food packaging [202]. Moreover, cassava could also be used as a matrix. One study used cassava starch with glycerol and water, this mixture in combination with acerola improves the elongation at break, but reduces the mechanical properties and elasticity due to its high sugar content, while the mixture with added grape residues, improves the mechanical properties and elasticity [197]. Furthermore, grape pomace extract as an antimicrobial additive in bactericidal isotactic polypropylene shows low water vapor permeability [203]. PLA composites in combination with wheat straw were also developed, demonstrating rapid crystallization for a shorter molding time, and increased flexural modulus, and water permeability for packaging [200]. In addition, the use of ultrafine wheat fiber, blended with PHBV, improves the water vapor transfer rate, favoring

its use in fresh produce packaging [204]. On the other hand, lignocellulosic nanofibers developed from wheat straw, blended with PLA and Ecoflex®, resulted in high water vapor permeability and antioxidant capacity for lettuce packaging [205]. Other research, using raw wheat bran composites and PBS, showed a noticeable impact on the rate of decomposition in an accelerated ageing environment [206]. PLA with mango by-products (20% tegument) achieves good mechanical properties, such as an elastic modulus up to 38% by IM, fiber provides higher stiffness, applicable for rigid packaging [187]. Mango seed and its use as a flour, mixed with glycerol, increases the mechanical and barrier properties, and has good antioxidant capacity [207,208]. In addition, the development of bioPP/mango peel flour for wood product applications and compatibilized with an itaconic acid copolymer, results in increased Shore D hardness, tensile strength, and an increased fracture toughness of 29.69% [185]. PLA with coffee grounds (SCG) by IM and compatible with oligomers, possesses high thermal stability, tensile strength, and elongation at a break of 39.6%, due to its lipid content. These were applicable for utensils and tableware [183]. Another study also incorporated the use of PLA and SCG through the blown extrusion process to produce biocomposite films, showing that the elongation at break increases with increasing SCG, while the tensile strength and hardness decrease [199]. This is because oil extraction from SCG increases the flexibility in films [208], and its incorporation also increases the content of antioxidants and microbial activity [209]. The use of coffee waste continues to increase, such as the production of coffee capsules based on coffee silverskin (tegument covering the endosperm) with PHBV copolymers by IM, resulting in a low breaking strength but an increase in the elastic modulus and crystallinity [192]. Food packaging was also made from coffee husks, HDPE, and ABS, as a result of the increased tensile modulus and tensile strength [195].

Yerba mate waste at 20% by weight, blended with PP and HDPE, showed good modulus and tensile strength, viable for wood composites [188]. Likewise, the use of yerba mate residue with PLA increases the flexibility and preserves the antioxidant properties, applicable in films [210]. Bean waste can also be applied in film making, its use at 30% by weight, in combination with PBSA/PHBV, increases the modulus of elasticity and decreases the tensile strength, applicable in the production of films [166]. Banana fiber and Mater-Bi® were used in the creation of biodegradable bags, presenting greater strength and flexibility due to the fibers. In addition, the ripening of bananas is delayed by 1 to 2 weeks [189]. On the other hand, banana fibers with PVA increase the tensile strength and minimum water absorption for film making [211]. Similarly, banana fiber (especially canary fiber) has a higher tensile strength and modulus of elasticity compared to other fibers, such as sisal, jute, flax, cotton, and coconut [212].

Walnut shells were combined with PP by means of IM for panel, board, and plywood production, demonstrating that using PP and MAPP as a bonding precursor provides stiffness and thermal stability [184]. In the production of wood-based panels, walnut shells could be added up to 20% in order to fulfil their mechanical properties [213]. In addition, it has been shown that nut shells are very fragile in combination with PLA, so alkaline treatments are used [214] or plasticizers, for example, epoxidized oils are used [215]. Another study used PLA with durian skin fiber and additionally epoxidized palm oil, which resulted in improved processability and energy reduction, applied in biodegradable packaging [198]. Moreover, sheep wool fibers were used as reinforcement for PLA plasticized with maleinized linseed oil, resulting in poor tensile properties, but an increase in the modulus of elasticity and the elongation at break [193]. The increased use of wool fibers does not generate good adhesion in the polymer matrix, which decreases the tensile forces [216]. As a solution, silane treatment generates good compatibility and adhesion to wool fibers, increasing the mechanical properties [217]. Recycled cotton fiber waste has been used with bio-PET by IM and showed poor mechanical properties, such as tensile strength, due to different polarities; however, they have a high modulus of elasticity and hardness, applicable for rigid packaging [191]. On the other hand, films with cotton and elastane residues through dissolution and regeneration, obtain high transparency

and good tensile strength, applicable for packaging materials [218]. Another study used Carbocal® (sugar-beet residue) with LLDPE, resulting in stiffer composites; the thermal resistance and modulus of elasticity increased by 150% with the use of 50% Carbocal® [194]. The use of sugar beet with PVA increases the mechanical properties and water resistance for the formation of biodegradable films [219].

#### **7. Concluding Remarks and Future Trends**

Advances in polymeric materials derived from agroindustrial biomass using extrusion and injection molding techniques present a promising avenue for sustainable material development. Biomass offers renewable, eco-friendly feedstock for biomaterial production, reducing the environmental impact and providing cost savings. The modification of cellulose, achieved through surface modification, grafting, chemical procedures, and molecule chemical grafting, improves the properties and versatility of these materials. However, more studies are needed to optimize the modification and processing techniques, improve material compatibility, and explore new applications. Extrusion technology melts, mixes, and homogenizes the composites to form granules. Twin-screw extrusion is suggested as it provides greater efficiency in the process, followed by the use of injection molding technology to obtain the desired shape. The parameters in extrusion and injection molding should be optimized, as they will depend on its shear and composition. The fiber size is of vital importance because it can reduce the mechanical properties, such as Young's modulus of elasticity, which is why solid-state extrusion is recommended, as it favors a better distribution of the fibers. In addition, to overcome the differences in polarity between the matrix and the reinforcements, coupling agents are used to improve the adhesion, mechanical properties, and thermal stability of the reinforcements. Continued advancements in this field will contribute to the transition towards more eco-friendly and resource-efficient material solutions. Extrusion and injection molding techniques offer remarkable advantages as a result of their application in multiple industrial sectors, such as the automotive industry, textiles, pharmaceuticals, the biomedical industry, various packaging applications, and many others.

According to this review, it is evident that the trend is for biodegradable materials applied to the area of agriculture and food. The most common biodegradable polymer used is polylactic acid. Depending on the application and rigidity of the material, extrusion or injection is preferable. For example, more flexible materials, such as bags, are better for extrusion and more rigid materials, such as utensils or tableware, are preferable.

**Author Contributions:** Conceptualization, L.O.-M. and L.A.M.-B.; methodology, G.L.Z.; Project administration, L.O.-M.; investigation, A.P., A.E.-O., K.G.M.-F., L.A.M.-B., H.P.-B., P.P.-C., M.S.N.D.S., M.V.T., G.L.Z. and L.O.-M.; resources, G.L.Z., H.P.-B. and K.G.M.-F.; writing—original draft preparation, K.G.M.-F., L.A.M.-B., H.P.-B., P.P.-C., M.S.N.D.S., M.V.T., G.L.Z. and L.O.-M.; writing—review and editing, L.O.-M. and G.L.Z.; visualization, L.O.-M. and H.P.-B.; supervision, L.O.-M., P.P.-C., L.A.M.-B. and G.L.Z.; Data curation, A.P. and A.E.-O. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors acknowledge the scientific and technical support from the Pilot Plant of Agroindustrial Engineering at the University San Ignacio de Loyola and the Integrated Laboratory of Agroindustrial Processes Engineering (LAPE) at the Federal University of Santa Maria.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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### *Review* **Polymeric Scaffolds Used in Dental Pulp Regeneration by Tissue Engineering Approach**

**Vinna K. Sugiaman 1,\*, Jeffrey 2, Silvia Naliani 3, Natallia Pranata 1, Rudy Djuanda <sup>4</sup> and Rosalina Intan Saputri 5,6**


**Abstract:** Currently, the challenge in dentistry is to revitalize dental pulp by utilizing tissue engineering technology; thus, a biomaterial is needed to facilitate the process. One of the three essential elements in tissue engineering technology is a scaffold. A scaffold acts as a three-dimensional (3D) framework that provides structural and biological support and creates a good environment for cell activation, communication between cells, and inducing cell organization. Therefore, the selection of a scaffold represents a challenge in regenerative endodontics. A scaffold must be safe, biodegradable, and biocompatible, with low immunogenicity, and must be able to support cell growth. Moreover, it must be supported by adequate scaffold characteristics, which include the level of porosity, pore size, and interconnectivity; these factors ultimately play an essential role in cell behavior and tissue formation. The use of natural or synthetic polymer scaffolds with excellent mechanical properties, such as small pore size and a high surface-to-volume ratio, as a matrix in dental tissue engineering has recently received a lot of attention because it shows great potential with good biological characteristics for cell regeneration. This review describes the latest developments regarding the usage of natural or synthetic scaffold polymers that have the ideal biomaterial properties to facilitate tissue regeneration when combined with stem cells and growth factors in revitalizing dental pulp tissue. The utilization of polymer scaffolds in tissue engineering can help the pulp tissue regeneration process.

**Keywords:** biocompatible; biodegradable; polymers; scaffolds; tissue engineering

### **1. Introduction**

Pulpal pathosis is one of the most common oral diseases due to persistent stimulation from trauma, dental caries, or iatrogenic causes. Dental caries occur because of bacterial infection on the tooth surface, which consists of enamel and dentin. Untreated dental caries trigger an inflammation response in the dental pulp, and chronic inflammation in the pulp tissue leads to permanent healthy tissue loss [1,2].

The current pulpal pathosis treatments are root canal treatment and pulp revascularization [2]. Root canal treatment is the treatment of choice in dentistry, which is effective for severe pulpal pathosis conditions. This treatment has a high success rate, but the tooth loses pulp tissue as a result. Thus, despite the treatment's benefits, the treated tooth becomes nonvital, which increases the risk of fracture and a decrease in the pulp defense mechanism and sensory function [2,3].

**Citation:** Sugiaman, V.K.; Jeffrey; Naliani, S.; Pranata, N.; Djuanda, R.; Saputri, R.I. Polymeric Scaffolds Used in Dental Pulp Regeneration by Tissue Engineering Approach. *Polymers* **2023**, *15*, 1082. https:// doi.org/10.3390/polym15051082

Academic Editor: Raffaella Striani

Received: 8 January 2023 Revised: 16 February 2023 Accepted: 18 February 2023 Published: 21 February 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Therefore, regenerative endodontic treatment to restore normal pulp functioning via complex dentin–pulp regeneration has recently been developed. The treatment aims to replace the pathological or nonvital pulp tissue with new healthy tissue [2,4].

Regenerative tissue engineering technology is improving rapidly. In pulp tissue regeneration, three important aspects have been developed for their utilization in the technique: stem cells, growth factors, and biomaterials/scaffolds [2,5]. Stem cells represent one of the key elements in tissue engineering technology. Stem cells are unspecialized cells that have the ability to regenerate, proliferate, and differentiate into specific cells [6,7]. After an injury, these cells play a role in healing via tissue regeneration [2,8].

A growth factor or morphogen is a protein or signaling molecule that bonds to specific membrane cell receptors which control and coordinate all cellular functions, such as cell signaling, cell proliferation, and matrix synthesis [6,9]. Growth factors play an important role in increasing the regenerative effect and control function of stem cells. Examples of growth factors that play a role in the signaling process of dentin and pulp regeneration are bone morphogenic proteins (BMP) such as BMP-2, BMP-4, BMP-7, and transforming growth factor β-1(TGF-β1) [4,10,11].

A scaffold or biomaterial is a framework or structure that provides a three-dimensional (3D) growth space for cells and regulates cell function and metabolism. The scaffold creates a microenvironment that promotes cells' regenerative capacities and multipotentialities. These conditions promote tissue regeneration. Recently, many natural or synthetic scaffold materials have been used for pulp regeneration [2,12]. Bioactive scaffolds stimulate the proliferation and differentiation of stem cells into odontoblast-like cells to regenerate pulp tissue [13,14]. Therefore, the role of scaffolds in tissue regeneration is important, becoming the mediator that facilitates the transfer of stem cells and/or growth factors at the location of the local receptor [15].

Each component in tissue engineering has a different effect in supporting the pulp regenerative process, but a combination of these three components gives the best results [2,4]. Dental tissue engineering is expected to provide tooth vitality, with pulp tissue similar to that of a normal tooth. Therefore, it is important to guide cell interactions with extracellular matrices, which is accomplished by using scaffolds and cell culture techniques [15].

This review will describe the latest developments regarding the usage of natural or synthetic scaffold polymers that have the ideal biomaterial properties to facilitate tissue regeneration when combined with stem cells and growth factors to revitalize dental pulp tissue. The utilization of polymer scaffolds in tissue engineering can help the pulp tissue regeneration process. This article is the first to discuss the various types of scaffolds with their various advantages and disadvantages that can be utilized in regenerating dental pulp tissue.

#### **2. The Dental Pulp**

Dental pulp is a loose connective tissue that occupies the root canal and is surrounded by dentin. Dental pulp consists of blood vessels, nerves, and odontoblasts, which line the predentine layer in the pulp tissue. Thus, pulp plays a role in providing nutrition, vitality, and pathogen detection through its sensory function as an infection response. Pulp tissue has sensitivity and immunoprotective attributes that maintain pulp homeostasis, facilitate its regenerative ability, and form reactionary dentin [2,16–18].

Histologically, dental pulp consists of several zones: the dentinoblastic zone, the cell-free zone, the cell-rich zone, and the pulp core. The primary cells of the pulp layer are odontoblasts, fibroblasts, macrophages, undifferentiated ecto-mesenchymal cells, and other immunocompetent cells [19,20]. The dentinoblastic zone functionally forms the pulp– dentin complex. This zone is the first line of reparative dentine formation and provides protective responses toward external stimulation, whereas the pulp core is rich in nerves and blood vessels which provide the pulp with nutrition and sensory functioning [2,19].

Therefore, the loss of pulp tissue causes a loss of vitality and sensitivity in the tooth and leads to uncontrolled infections in the surrounding tissues. This condition needs complex treatment, such as root canal treatment, which renders the tooth nonvital and brittle, which influences the patient's quality of life [17,18].

#### **3. Dental Pulp Regeneration**

Pulp regeneration is a healing process regarding the injured or lost parts of the dental pulp and results in the re-establishment of its complete biological function [2,21]. Ideal pulp regeneration should generate pulp structure and function as similar as possible to healthy tissue. This regeneration involves the regeneration of the dentin–pulp complex, blood vessels, and nerves, which reach a favorable level of reconstruction through the angiogenesis and neurogenesis processes. Other than that, it also involves the rehabilitation of pulp physiological functioning, represented by sensation, nutrition, and immunological defense [2,6].

Illustrated by the formation of connective tissue, with cell density and an architecture similar to that of healthy pulp, successful pulp regeneration consists of nerves and blood vessels able to secrete new dentin as healthy pulp at a controlled rate. Vascular tissue plays a role in providing nutrition, oxygen, cell immunity, and the recruitment and circulation of cells, which maintains the tissue's vitality and viability, while the nerves are fundamental to cell regulation, which manages the regeneration process and provides defense mechanisms and tissue repair [6,22].

Regenerated blood vessels should be connected to the periapical bone tissue, which surrounds the tooth; therefore, it can receive regular blood flow and transport nutrition for regenerating the tissue or dentin. Other than that, the regenerating tissue should be innervated, with the tooth maintaining heat/cold and pain sensations [17,23]. Therefore, vascular and nerve supply should be maintained through the apical foramen, which is one of the aims of the pulp regenerative process.

In the regeneration process, stem cells proliferate and differentiate into endothelial cells for angiogenesis/vasculogenesis and move into odontoblasts to carry out the dentin reparative process. At the beginning of the process, angiogenic signals, such as fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and transforming growth factor β (TGF-β), are released by endothelial cells, injured pulp cells, and the extracellular matrix (ECM), which causes stem cell migration and stimulates neo-angiogenesis [24,25].

#### **4. Endodontic Regeneration**

Infected dental pulp needs root canal treatment (RCT), which is a conservative but effective treatment. Traditionally, in this treatment, the pulp tissue is removed and replaced by synthetic obturation materials, such as paste or gutta-percha [13,17]. RCT aims to remove the space for potential microbiome reinfection and create a healing environment by mechanical or chemical disinfection, which is continued by inert material closure [2,26]. The treatment has a high success rate in dentistry, with 97% of one million teeth able to retain functionality for around 8 years [13,17].

Teeth that receive RCT experience severe defects regarding hard tissue, devitalized pulp from denervation, and avascularity. This leads to an increased risk of fracture, the disruption of the pulpal defense mechanisms, and a loss of physiological functions, such as nutrition and sensation [2,17,27]. In order to prevent these side effects, an effective treatment strategy is needed for the revitalization of the pulp. The emergence of tissue engineering technology and regenerative treatments provides the possibility of developing regenerative endodontic treatments [17].

RCT causes the tooth to be nonvital and susceptible to structural changes [28]; the challenge in modern dentistry is to maintain pulp vitality. Thus, an interdisciplinary approach to regenerative treatments has developed, which utilizes living cells to heal, replace, and restore damaged human tissues and organs to reach their normal level of functioning. One of these treatments is stem cell engineering, which has the potential to be the future of regenerative treatment [29,30].

Dental tissue regeneration can be obtained by the regeneration of each part of a tooth's structure, which consists of enamel, dentin, pulp, alveolar bone, cementum, and periodontal ligament or by regenerating the whole tooth structurally and functionally [15,31]. Regenerative endodontics is one of the endodontic treatments that focus on replacing the damaged pulp tissue through tissue regeneration to restore tooth vitality, leading to an increase in patient quality of life. Regenerative tissue should have healthy pulp properties, such as the ability of the dentin-deposition process, reinnervation, and vascularization [17,26].

#### **5. Tissue Engineering**

Tissue engineering technology is an interdisciplinary science that implements the biological principles of regenerative treatment techniques, with a focus on repairing and restoring the biological function of cells, tissues, and organs that have been injured by internal or external factors [6,32]. Tissue engineering technology aims to contribute to the restoration of damaged tissue function and structure by utilizing stem cell interactions, scaffolds/biomaterials, and growth factors. The proper combination of these three elements enables the manipulation of the biomimetic microenvironment containing the vascular system, which normally maintains nutrition supply, waste disposal, inflammatory response, and pulp regeneration [2,6,33]. In tissue engineering, angiogenesis has an important role in nutrition supply and the potential recruitment of stem cells [4,34].

In tissue engineering technology, pulp regeneration might be achieved via the utilization of three key elements: (i) stem cells, (ii) scaffolds, and (iii) signaling molecules such as growth factors. Firstly, the pulp regeneration process might be achieved through stem cell isolation and in vitro manipulation. After this, the cells are cultured in the scaffold and combined with the growth factor, which is then all transplanted into the root canal [35–37].

Every individual element has a different impact on pulp regeneration, but with all elements supporting each other, this might provide a favorable result. The proper combination of these three elements provides a micro-biomimetic environment, influencing the overall accomplishment of pulp regeneration. This result might be achieved by the formation of a fully functional vascular system, thus providing adequate nutrition supply, waste disposal, and inflammation response, leading to satisfactory pulp regeneration [2].

#### *5.1. Dental Stem Cells*

Mesenchymal stem cells (MSCs) are a type of stem cell that is suitable for regenerative treatment because of its high proliferation and multipotential ability [29,38]. According to the minimal criteria of the International Society for Cellular Therapy, MSCs are marked with positive (CD29, CD44, CD73, CD90, CD105, and Stro-1) and with negative hematopoietic markers (CD14, CD34, and CD45) [13,39].

MSCs can be isolated from different locations in the oral and maxillofacial regions, such as from dental pulp stem cells (DPSCs) and the stem cells exfoliated from human deciduous teeth (SHED) and can be isolated from healthy pulp tissue. These cells could be differentiated in vitro into adipocytes, odontoblasts, osteoblasts, and chondroblasts, which form dentin or pulp tissue after in vivo transplantation [13,29]. Other cells, such as dental follicle progenitor stem cells (DFPCs), periodontal ligament stem cells (PDLSCs), and stem cells from apical papilla (SCAPs), can be differentiated in vitro into adipocytes, odontoblasts, cementoblast-like cells, and connective tissue [5,13,29,40].

Each type of stem cell has different properties: SHED and SCAP have higher proliferation activity compared to DPSC, although all stem cells possess the potential to regenerate dentin and pulp [5,13].

#### *5.2. Growth Factors*

Signaling molecules, such as stem cell factor (SCF), stromal-cell-derived factor (SDF-1α), platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), and granulocyte colony-stimulating factor (G-CSF), can be used for pulp tissue regeneration [17]. Several growth factors, such as SDF-1α, bFGF, and PDGF, are chemotaxis molecules and

correlate to blood vessels, nerves, and dentin in the pulp regeneration process. PDGF and VEGF contribute to vasculogenesis/angiogenesis, while NGF contributes to the growth and survival of the nerves; BMP-7 contributes to the differentiation and mineralization of odontoblasts [36,37]. Growth factors play a role in the restoration of stimulation of a structure and the physiology of tissue function in damaged tissue [2].

#### *5.3. Scaffolds*

A scaffold is a three-dimensional frame microenvironment that facilitates attachment, cellular infiltration, differentiation, proliferation, and stem cell metabolism with the aid of growth factors. The frame has to provide support for nutrition and oxygen diffusion in the regeneration process and should have biodegradable properties because it will be replaced by the new tissue [4,6,41].

Different types of developed scaffold materials or models have certain levels of flexibility and degradability [6]. Currently, natural or synthetic scaffolds have started to be commonly used in pulp tissue regeneration [2]. The scaffolds that have been used are tissue extracts, such as blood clots, platelet-rich fibrin (PRF), platelet-rich plasma (PRP), tricalcium phosphate ceramic, hydroxyapatite calcium, and mineral trioxide aggregate and synthetic polymers such as polylactic-co-glycolic acid, polylactic acid, and biopolymers such as collagen, hydrogel, hyaluronan, and chitosan [4].

Blood clots represent one type of scaffold that has natural properties from which natural substances such as collagen, chitosan, fibrin, hyaluronic acid, gelatin, alginate, and peptide-based scaffolds can be derived. These scaffolds have been studied as scaffolds for pulp regeneration because of their biocompatibility, biomimetic properties, availability, cost-effectiveness, and ease of conversion (into hydrogel) [13,42].

Other than natural scaffolds, there have been several synthetic polymers developed, such as polyglycolic acid (PGA), poly(d,l-lactide-coglycolide) (PLGA), polylactic acid (PLA), poly(l-lactic) acid (PLLA), and polycaprolactone (PCL), and inorganic calcium phosphates, such as hydroxyapatite (HA) or beta-tricalcium phosphate (β TCP), as well as a combination of silica glass and phosphate. Synthetic scaffolds have been studied considerably as scaffolds that have the potential for tooth regeneration because of their nontoxicity, biodegradability, and ease with which to manipulate properties, including mechanical rigidity and degradation rate [2,15,42].

In contrast to natural scaffolds, synthetic scaffolds can be prepared in unlimited numbers because they are produced in a controlled environment according to a desirable shape. This condition allows for the obtainment of the scaffold in accordance with cell differentiation properties, certain pore characteristics, and certain mechanical, chemical, and degradation rate properties according to the desired application [15,43,44].

This polymer is a biomaterial that is commonly used to form scaffolds with characteristics that are related to differentiation in their composition, structure, and macromolecule arrangement [15]. In recent studies, scaffolds have shown the potential to be bioactive carriers and have recapitulated the interaction between stem cells, progenitor cells, microphysiological environments, and extracellular matrices [13]. In regenerative endodontic treatment, polymer scaffold usage could provide physiological environments to increase the biological performance in the pulp regeneration process. This process consists of revascularization and revitalization processes. This scaffold influences cell migration, viability, discharge, proliferation, recruitment, and degradability [45].

Although scaffolds have huge potential, there are challenges that need to be overcome, such as integrating the scaffold with complicated morphologies without damaging the surrounding tissues. For tooth regeneration, scaffolds require several general characteristics, such as being easy to manipulate, having bioactive and biodegradable properties, having adequate porosity and physical and mechanical strength, having low immunogenicity, and being able to support vascularization [15,43].

Other criteria, such as having an adequate shape, size, and pore volume, are important for the penetration and diffusion of growth factors, nutrition, and waste discharge between the cells [13,15]. Therefore, a scaffold's criteria and design create favorable microenvironments that are important as a foundation to then perform tissue engineering technology processes. This microenvironment supports the organization of cell functioning regarding self-renewal and differentiation, supporting cell and growth factor transportation, creating an environment for cell activities, and promoting communication between cells, which leads to tissue regeneration [2,13,46]. These scaffold characteristics represent important keys to the process of tissue regeneration because they play vital roles in defining cell behavior and tissue formation [13].

To confirm the success of the cell growth and differentiation processes in tissue engineering, scaffold materials must be able to interact with host tissues and provide an ideal environment for tissue growth [29,46]. The ideal scaffold for pulp regeneration should fulfill three criteria: biocompatibility, adequate rigidness to withstand mastication force, and tight sealing with dentin to prevent micro-organism infiltration [29,44]. Other than that, the degradation process of a scaffold is usually one of the factors that plays a role in treatment failure [47]. The rate of scaffold degradation should be complementary to the rate of new tissue formation and should not produce harmful waste side products [15,48,49]. Utilization of the use of scaffolds in tissue engineering technology must fulfill several characteristics which can be seen in Figure 1.

**Figure 1.** Scaffold for Tissue Engineering.

#### 5.3.1. Scaffolds Made of Natural Polymers

One of the tissue engineering triad elements in regenerative endodontics is scaffolds, which work as biological and structural support for cell growth and differentiation. Proper scaffold selection is a challenge in the dentin–pulp regeneration process [50]. Cells' migration, proliferation, and differentiation correlate with the choice of a scaffold's physical properties, such as appropriate viscoelasticity to mimic the real pulp tissue [51]. The application of scaffolds for dental pulp regeneration should be able to mimic the microenvironment in the root canal and provide mechanical support [52,53].

The application of 3D bioprinting technology to scaffold-making can precisely mimic external and internal morphologies. The 3D scaffold has moderate porosity, which allows nutrition and oxygen infiltration, leading to the occurrence of metabolic activities [53]. The application of scaffolds via the injection process is recommended because it can adapt well to the shape of the pulp chamber and root canal so that cell and matrix interaction can occur efficiently [50].

To date, scaffolds are classified as natural and synthetic scaffolds based on the material source and biomaterial properties used [54]. Scaffolds for tissue regeneration using natural or synthetic materials are continually being developed [55]. Natural scaffolds come from the host or natural materials. Examples of host scaffolds are blood clots, autologous platelet concentrates, and decellularized extracellular matrices [54]. Examples of natural material scaffolds are collagen, alginate, chitosan, hyaluronic acid, and fibrin [50,51,53,54]. Natural material scaffolds have the advantage of cell recognition and adhesion from molecular signaling, although the application of this type of scaffold has the limitation of product variation, risk of pathogen transmission, poor mechanical properties, and immunological responses to foreign objects [52]. The shape of the scaffold can be a porous sponge, a solid block, a sheet, or a hydrogel [56].

Collagen is a scaffold material that has the closest viscoelasticity to real pulp tissue [51]. The combination of natural materials, such as collagen and the host's blood clot, show predictable patterns for tissue formation and mineralization in human dental structures when compared to collagen or blood clots individually. The application of one type of scaffold, such as a blood clot, does not provide stable results for the tissue regeneration process [57]. Instability and unpredictable clinical results from the blood clot are the consequences of unregulated stem cells in the pulp chambers, including the difficulties of bleed formation and hemostasis [52].

When compared to blood clots, platelet-rich plasma (PRP) and platelet-rich fibrin (PRF) provided lower increases in dental root length and less effectivity in root development [58]. PRP from the host's blood contains high platelet, growth factor, and cytokine concentrations, which increase the ability of wound healing and stem cell recruitment from the pulp and increase SCAP proliferation. While PRF contains plentiful growth factors, which can stimulate cell differentiation as well as cell adhesion and migration [59]. The advantages of materials with rich platelet concentrations, such as PRF or PRP, are the increases in the level of angiogenesis and revascularization, which is fundamental to accomplishing endodontic regeneration therapy [56]. Hydrogel-based collagen could mimic the interaction between cells and extracellular matrices in vivo and organize cell growth, which is used for tissue engineering [60].

Polymer materials, such as gelatin and fibrin, are commonly used as natural scaffolds. Gelatin is a biopolymer protein that comes from collagen hydrolysis, which facilitates the proliferation and differentiation of odontoblasts in dental pulp stem cells (DPSCs) [50]. Gelatin is a partial hydrolysate from animals. When compared to gelatin, hydrogel gelatin has better biocompatibility because of its low immunogenicity properties [50,53]. A gelatinbased matrix showed better endodontic therapy results when compared to fibrin-based matrix groups after 12 weeks follow-up in mini-pig immature dental models [50].

Other studies into fibrin-based scaffolds in hydrogel showed that this material was compatible with dental pulp regeneration by supporting pulp-like tissue formation [61]. Fibrin is a natural protein polymer that forms part of blood clot formation. Hydrogelbased fibrin can stimulate pulp-like tissue formation with an odontoblast layer in the root canal system [50]. The advantages of these materials are good cytocompatibility, physical kinetic degradation, and nontoxic degradation products, and they are also easy to inject into the pulp canal. Other natural materials, such as alginate, chitosan, collagen, and hyaluronic acid, or synthetic materials, such as polyethylene glycol, poly (D,L) lactic acid, and fibrin-based bio-ink for 3D printing, were added to increase the structural and functional properties of fibrin scaffolds [61].

Alginate is a natural polymer from algae, which has good biocompatibility properties, is cost-effective, has low cytotoxicity, and has an optimal structure for nutrition exchange [45,52,53]. Alginate hydrogels were formed by crosslinking polysaccharide and divalent cations to form an ion bridge in water-insoluble tissue [52]. Alginate hydrogels are able to arrange themselves in accordance with mechanical properties, such as rigidity and stress relaxation, to regulate stem cell activity [45]. Alginate has proper mechanical properties but can be applied in the form of hydrogel injection or bone porosity, which enables the natural structure to be loaded with growth factor [56] The macroporosity of alginate scaffolds enables the exchange between nutrition and metabolism waste. However, scaffolds that consist of only alginate have a limited role in endodontic regenerative therapy; therefore, its combination with other materials, such as bioactive polymers, is needed [52].

Hyaluronic acid (HA) is a biopolymer that can be modified and processed for biomedical applications, and it can be combined with other materials to increase its favorable

properties [60]. HA in dental pulp was found to decrease dental development in the odontogenesis process [52]. When applied to exposed pulp, HA can stimulate the production of reparative dentin. HA can be applied in 3D-sponge form to create a proper environment for blood vessel proliferation and stem cell differentiation [56]. HA is formed by d-glucuronic acid and N-acetyl-D-glucosamine and is commonly available in the form of liquid injection [45]. HA degradation products include pro-angiogenic growth factors, which represent the revascularization elements of dental regeneration tissue, although HA has the disadvantages of poor mechanical properties and can cause hypersensitivity reactions [52].

Chitosan is a widely used natural scaffold [62]. Chitosan is a cation polymer from chitin [55] Chitosan has good biocompatibility, biodegradation, and other favorable biological properties, such as being antimicrobial, fungistatic, and noncarcinogenic, with hemostatic and protein fusion abilities, as well as being able to stimulate cell adhesion, proliferation, and differentiation [55,62]. However, the application of chitosan is difficult because of the complex gelation and degradation process due to unusual polycationics and a highly crystalline structure, which limits the application of this type of scaffold to the form of a natural injection [52]. The hydrogel form of chitosan can be injected into the dental pulp chamber [62]. Chitosan can be applied as an individual scaffold or in combination with polymers or other biomaterials to produce a large number of matrices for tissue engineering purposes. The addition of chitosan scaffolds into the blood for endodontic regeneration procedures can stimulate the formation of new soft tissue (as proven by histological regeneration) without the formation of mineralized tissue around the pulp canal wall [55]. Additional photo-biomodulation therapy could increase in vitro stem cell survival, proliferation, and migration from the root papilla [62].

When comparing several natural scaffolds, other studies have shown that human teeth can be applied as scaffolds for periodontal ligament and pulp regeneration [26]. Scaffolds from natural materials have higher biocompatibility and bioactivity properties when compared to synthetic scaffolds, whereas synthetic scaffolds have higher controlled degradation levels and mechanical properties [63]. The application of scaffolds that are not limited to the use of only one material, i.e., those that can be combined, can provide better endodontic regeneration therapy.

#### 5.3.2. Scaffolds Made of Synthetic Polymers

The implantation of 3D scaffolds in the appropriate living cells that secrete their own extracellular matrix (ECM) can provide an acceptable environment. The adequate porosity and permeability of a polymeric scaffold are essential for guiding and supporting the cultured cells' ability to produce tissue. Synthesizing synthetic biodegradable polymers is challenging in tissue engineering applications [64,65].

The progenitor/stem cells should then be able to attach, travel through, proliferate, and organize themselves spatially in 3D space and differentiate into odontogenic, vasculogenic, and neurogenic lineages with the support of an adequate scaffold for dentin–pulp regeneration. Furthermore, the biocompatibility of the material is critical to avoid any negative reactions from the host tissue. Biodegradability that can be adjusted to match the rate of regeneration is critical for facilitating constructive remodeling. As a result of scaffold deterioration, a series of tissue responses occur, comprising the targeted tissue replacement of the scaffold, vascularization, differentiation, spatial structure, and cellular infiltration [66–68].

Metals, ceramics, and polymers are examples of materials that can be used to make scaffolds. Both dental and bone implants are frequently made of metallic alloys. When it comes to bone tissue engineering, ceramics with strong osteoconductivity have been used, although metals and ceramics have substantial disadvantages because metals do not biodegrade and do not serve as a matrix that mimics biological processes for the proliferation of cells and tissue creation. Additionally, due to brittleness, ceramics are difficult to convert into highly porous structures and have a limited capacity for biodegradation. In contrast, polymers can be molecularly designed to have increased biodegradability and excellent processing flexibility. Therefore, for tissue engineering, polymers are the most common type of scaffolding material [31,68–70].

Biological recognition represents one potential benefit of naturally generated polymers, which may help to stabilize cell adherence and ensure proper function. The synthetic polymers used as scaffolding materials have been spurred on by the challenges associated with natural polymeric materials, such as their complex purification, structural composition, pathogen transmission, and immunogenicity. When compared to naturally occurring extracellular matrix (ECM) proteins, synthetic polymers offer better processing flexibility and no immunological issues. Functionalized scaffolds that combine the benefits of synthetic and natural polymeric materials can be made by adding bioactive molecules to synthetic polymers [69–71].

The advantages of synthetic polymers include nontoxicity, biodegradability, and the ability to precisely manipulate their physicochemical characteristics, such as degradation rate, structural rigidity, microstructure, and porosity [72–74]. Natural polymers are mostly broken down by enzymes, but synthetic polymers are typically broken down by simple hydrolysis. However, because of the relative acidity of the hydrolytically destroyed byproducts, synthetic polymers might cause localized pH reductions and a chronic or acute inflammatory host response [74–76].

Tissue engineering frequently uses poly (-hydroxy acids), such as poly (lactic acid), poly (l-lactic acid), poly (glycolic acid), polyethylene glycol, and their copolymers poly [(lactic acid)-co-(glycolic acid)] (PLGA) and poly-epsilon caprolactone (PCL), which appears to be the most synthetic polymeric material. These polymers have an established track record and have been approved by the FDA for specific human applications (e.g., sutures). Two of the synthetic polymer scaffolds that have been suggested for dental tissue engineering are PGA and PLA, which are biodegradable polyesters that can be produced from a range of renewable sources. When compared to PGA, PLA, which is an aliphatic polyester, is more hydrophobic [66,69,74,76–78].

The synthetic scaffold known as PGA, which has been used for cell transplantation, breaks down when the cells secrete an ECM. Several cell types, including cellular origins of dental pulp, pulpal fibroblasts, and ex vivo human pulp tissue cells, have been shown to be able to adhere and develop on PGA scaffolds. The copolymers of PGA and PLA that are sown with dental pulp progenitor cells have been shown in rabbit and mouse xenograft models to produce pulp-like tissue [66,69,74,75].

Since structural strength is vital in many applications, PLLA, an extremely strong polymer, has been used in several of them. Nanofibrous scaffolds have been created from it that resemble the structure of genuine collagen (a crucial element of ECM). It has been shown that nanofiber PLLA scaffolds promote cell attachment and differentiation. Previous studies demonstrated how PLLA scaffolds could stimulate the development of endothelial cells from dental pulp cells and odontoblasts [66,69,75]. This was demonstrated by utilizing PLGA as a scaffold from which dentin-like tissue could emerge and in which pulp-like tissue could be repaired over the course of 3 to 4 months. A 50:50 blend of PLGA degrades after around 8 weeks. PCL, a slowly disintegrating polymer, has been utilized in bone tissue engineering projects either by itself or in conjunction with hydroxyapatite [75].

A different type of polymer, polyethylene glycol, is utilized in tissue engineering techniques, such as pulp regeneration. Dental pulp progenitor cells have been transformed to create 3D-tissue constructs while being linked to electrospun polyethylene glycol scaffolds. These artificial polymer scaffolds have also been utilized to convey a range of substances, including anti-inflammatory drugs, growth hormones, and sticky proteins. Such scaffolds could not only support cell growth and proliferation but could also reduce pulpitis and aid in pulpal healing. Synthetic polymer scaffolds have better handling characteristics and a more straightforward manufacturing process, which improves their potential for endodontic regeneration. They do, nevertheless, differ significantly from the natural dental

pulp extracellular environment. As a result, ECM-based natural scaffolds that are closer to the microenvironment have been developed [66,74,79,80].

Planting human exfoliated deciduous teeth stem cells (SHED) on dentin disks with PLA resulted in the structure of odontoblast-like cells, new dentin, and vascularized pulplike tissue. A study by Huang et al. illustrated that when implanted in vivo into an empty root canal area, the stem cell constructions made from the apical papilla (SCAPs) and L-lactide, poly-D, and glycoside were able to create soft tissue that resembles pulp, with the continual addition of new dentin to the surface. However, synthetic polymers have the potential to cause an immediate or long-lasting inflammatory response. Additionally, the locally decreased pH brought on by the hydrolytically degraded metabolites may impair its clinical use [66,75].

Several methods have been used to construct 3D scaffolds from poly (hydroxy acids). The inability of the poly (a-hydroxy acids) chains to allow functional groups, however, restricts the incorporation of biologically active moieties onto the scaffolding surface. In order to increase the functioning of these polymers and broaden their usage, significant efforts have been made in this direction; creating copolymers out of a-hydroxy acids with additional monomers that have functional pendant groups, including amino and carboxyl groups, is one technique. In one study, ring-opening polymerization was used to copolymerize (RS)-b-benzyl malate and L-lactide; then, the benzyl groups were removed to create (RS)-b-malic acid) poly (L-lactide) with connected carbonyl compounds [69,81,82].

In order to copolymerize this with L-lactide, benzyloxymethyl methyl glycolide and benzyloxymethyl glycolide are required, which have preserved hydroxyl groups. The matching hydroxylated PLLA copolymers were produced when the benzyloxymethyl groups were unprotected. Comparable carboxylic acid functionalized copolymers can be created using succinic anhydride [69,83].

The researchers created a poly [(L-lactic acid)-co-(L-lysine)] containing a useful lysine residue that they further linked to the RGD peptide. Even though the development of functional groups in random copolymers by lactide/glycolide copolymerization with additional monomers can be successful, this procedure frequently affects the physical characteristics of the starting homopolymers, such as crystallinity and mechanical strength. Numerous block and graft copolymers based on poly(a-hydroxy acid) have been developed and made as a result of this [69,84].

Polymer PEG, or poly (ethylene glycol), is the component that is most frequently used in (a-hydroxy acids). PL(G)A/PEG diblock, triblock, and multiblock copolymers could be made by the ring-opening of PEG and certain catalysts and the presence of glycolide/lactide polymers. However, the hydroxyl or carboxyl (functional groups) in the block copolymers containing PEG are only present at the end of each PEG segment, and the content in these block copolymers is very low, further restricting chemical alterations. Numerous block and graft copolymers made without PEG have been described [69,85].

Amphiphilic poly [hydroxyalkyl (meth) acrylate)] is a variety of biodegradable polymer. Copolymers of -graft-poly (L-lactic acid) (PHAA-gPLLA) with hanging hydroxyl groups were employed to successfully produce 3D-nanofibrous scaffolds. The further functionalization of these copolymers can result in biomimetic scaffolds that are more hydrophilic, degrade more quickly, and have uses in tissue engineering [69,86].

The fabrication of highly porous poly (α-hydroxy acid) scaffolds can be used for tissue engineering based on star-shaped functional poly(ε-caprolactone). The functional groups were added to PCL chains using similar methods. Examples of these methods include the copolymerization of ε-caprolactone and a-chloro-ε-caprolactone to produce functionalized PCL copolymers, and the subsequent addition of carboxyl, pendant hydroxyl, and epoxide groups via atom transfer radical addition. In order to produce the pendant hydroxyl groups in the PCL copolymers, ε-caprolactone was copolymerized with another monomer, 5-ethyleneketal-ε-caprolactone, and the resulting molecule was subsequently deacetylated to convert the ketone groups into hydroxyl groups [69,87].

However, these deprotection processes (as well as the synthesis of these functional comonomers) are typically challenging and time-consuming. Aside from poly (3-hydroxybutyrate), polyurethanes, polycarbonate, poly (ortho ester), poly (propylene fumarate), and polyphosphazenes, other synthetic biodegradable polymers have also been used as scaffolding biomaterials. Comparatively, there are many fewer reports of the functionalization of these biomaterials (a-hydroxy acids), which include the creation of functionalized PC using synthetic methods [69,87,88].

Pendant amino groups were added to PC chains after polymerizing the cyclic carbonate monomer (2-oxo-[1,3]-dioxan-5-yl) carbamic acid benzyl ester and disposing of the protective benzyloxy carbonyl groups. The pendant amino groups' further functionalization was shown using RGD peptide grafting; synthetic efficiency should be considered, given the number of steps in this reaction cycle [69,89].

The five distinctive structural characteristics of these PAs are as follows: (1) an extended alkyl tail that contributes to the molecule's amphiphilic characteristic; (2) maintenance of the structure by possessing four consecutive cysteine residues that create disulfide bonds; (3) a flexible hydrophilic head group due to the three glycine residues in the linker region, which separates the hard cross-linked region; (4) phosphorylated serine residues that interact strongly with calcium ions to encourage mineralization; and (5) an effective RGD peptide [69].

The high electrostatic interaction between molecules causes the PAs to self-assemble into nanofibrous networks when the pH is changed or when divalent ions are added, as evidenced by this study. Additionally, the hydrophilic peptide signals can be displayed in a specific way on the surfaces of the produced nanostructures due to the molecule's amphiphilic characteristics. However, the creation of sufficient mechanical three-dimensional structures from these PAs must be addressed, as is true for several other hydrogel materials. Proteinase-sensitive motifs represent an inventive technique to make biomaterials react to cells [69,90].

As cell-ingrowth frameworks for tissue formation, Hubbell et al. presented a valuable example of how to build synthetic PEG-based hydrogels. The functionalization molecules for PEG chains in hydrogel networks, which also include pendant oligo peptides (RGDSP) for cell attachment, are matrix metalloproteinase (MMP)-sensitive peptides. The material's reaction to the MMPs secreted by cells is controlled by the MMP-sensitive binding agent. This hydrogel, with a PEG foundation, functions as a biomaterial and reacts to cells. The authors also showed that these gels could promote bone regeneration and are efficient delivery systems for recombinant human bone morphogenetic protein-2 (rhBMP-2) [49,69].

Many of the requirements for the dental pulp tissue engineering approach may be accommodated by self-assembling, adaptable, and customizable peptides. Due to the peptide chains' natural amino acid makeup, they can produce biodegradable products. The potential for uniform cell encapsulation, the rapid transport of nutrients and metabolites, and the characteristics of peptide hydrogel systems are affected by their viscoelastic properties, which are comparable to the properties of collagenous tissues such as dental pulp [66,91].

The term "bioceramic scaffolds" refers to a group of materials, including glass ceramics, bioactive glasses, and calcium/phosphate compounds. Calcium phosphate-based (CaP) ceramics are the biomaterials that are utilized most frequently. Due to their characteristics of osteoclast genesis, nontoxicity, antigenicity, osteoinduction, bone bonding, and similarity to mineralized tissues, CaP scaffolds, such as -TCP or HA, have been extensively explored for bone regeneration. Three-dimensional CaP porous granules have demonstrated their potential in the engineering of dental tissue by providing excellent 3D-substrate characteristics for hDPSC growth and odontogenic differentiation. Pure TCP scaffolds are doped with SiO2 and ZnO to increase their mechanical stability and capacity for cellular proliferation. Glass ceramics made of SiO2 Na2OCaOP2O5 are bioactive and offer ideal crystallization conditions. The osteoblastic activity of the substance is increased by the release of dissolving products, such CaP [15,75].

Ceramic scaffolds can be altered to control the dissolving rate, provide the appropriate permeability, and control certain surface properties to promote cellular activity. The mechanical rigidity of the scaffold is influenced by variations in pore size and volume. Glass ceramics made of magnesium can increase mechanical strength and provide a high rate of bioactivity. Excellent hDPSC attachment, proliferation, and differentiation have been demonstrated by niobium-doped fluorapatite glass ceramics [75,92].

The several disadvantages of bioceramics include a longer creation time, the lack of an organic phase, nonhomogeneous particle size and form, huge grains, difficulty to shape, brittleness, slow degradation, and high density. Bioceramics are fragile and have little mechanical strength when individually utilized. This drawback can be remedied by combining them with polymer scaffolds [75,92]. Comparison of various types of scaffolds for tissue engineering can be seen in Table 1.

**Table 1.** Comparison of various types of scaffolds for tissue engineering.



**Table 1.** *Cont.*

Tissue engineering technology requires a scaffold as a porous structure that can assist in tissue regeneration. In addition to various scaffold properties with various advantages needed to provide mechanical support in the regeneration process, tortuosity is also an important parameter in developing the permeability of 3D scaffolds to be used in tissue engineering technology. This affects the occurrence of cell attachment, proliferation, differentiation, and cell migration in the process of tissue regeneration [101,102].

Research on tissue engineering technology has not been widely carried out in humans, so this study cannot discuss how far its success has been when applied to living tissue. Therefore, the application of various types of polymer scaffolds needs to be developed further.

#### **6. Conclusions**

Various types of scaffolds, both natural and synthetic, can be used to regenerate dental pulp by utilizing tissue engineering technology. Scaffolds made from natural materials have advantages in cell recognition and molecular signal adhesion, while synthetic scaffolds can be made in unlimited quantities. However, a better effect might be realized if the two types of scaffolds are combined to obtain good mechanical properties so that they can support pulp regeneration properly. In the future, it is hoped that more extensive research can be carried out on various types of scaffolds so that not only polymer-based scaffolds are described for the regeneration of dental pulp tissue.

**Funding:** This research was funded by Maranatha Christian University.

**Institutional Review Board Statement:** Not applicable.

**Data Availability Statement:** Data sharing not available.

**Acknowledgments:** The authors would like to thank the Faculty of Dentistry, Maranatha Christian University and the Faculty of Dentistry, Jenderal Achmad Yani University.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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### *Review* **Polymers Use as Mulch Films in Agriculture—A Review of History, Problems and Current Trends**

**Zinnia Mansoor 1,2, Fideline Tchuenbou-Magaia 3, Marek Kowalczuk 4, Grazyna Adamus 4, Georgina Manning 1, Mattia Parati 1, Iza Radecka 1,\* and Habib Khan 1,\***


**Abstract:** The application of mulch films for preserving soil moisture and preventing weed growth has been a part of agricultural practice for decades. Different materials have been used as mulch films, but polyethylene plastic has been considered most effective due to its excellent mechanical strength, low cost and ability to act as a barrier for sunlight and water. However, its use carries a risk of plastic pollution and health hazards, hence new laws have been passed to replace it completely with other materials over the next few years. Research to find out about new biodegradable polymers for this purpose has gained impetus in the past few years, driven by regulations and the United Nations Organization's Sustainable Development Goals. The primary requisite for these polymers is biodegradability under natural climatic conditions without the production of any toxic residual compounds. Therefore, biodegradable polymers developed from fossil fuels, microorganisms, animals and plants are viable options for using as mulching material. However, the solution is not as simple since each polymer has different mechanical properties and a compromise has to be made in terms of strength, cost and biodegradability of the polymer for its use as mulch film. This review discusses the history of mulching materials, the gradual evolution in the choice of materials, the process of biodegradation of mulch films, the regulations passed regarding material to be used, types of polymers that can be explored as potential mulch films and the future prospects in the area.

**Keywords:** mulch films; biodegradability; biopolymers; SDGs; plastic pollution

#### **1. Introduction**

The technique of mulching has been a part of agricultural practice for a long time. In simple terms, mulches are defined as materials that are applied directly onto the surface of soil for various purposes such as the protection of seedlings and young shoots through insulation, reduction of evaporation, control of weed growth and prevention of soil erosion [1]. They specifically protect delicate crop species from unfavourable abiotic and biotic stresses that may occur as a result of extreme weather conditions, insects, pests and weeds. Therefore, mulches are commonly used in agriculture to prevent loss of crop yield [2].

The history of using mulching to enhance crop production dates back to around 500 BCE, as shown in Figure 1. It is from that age that the first documented proof of the use of organic matter as a mulch film has been obtained [3]. The material used gradually changed to stones, pebbles and volcanic ash in the 1600s, although these were mostly used in arid regions. In the 1800s the Parisian market gardeners found that the use of straw as mulching material for strawberry production was beneficial. Thus, over a span of hundreds of years, different naturally available materials were tried and used for mulching

**Citation:** Mansoor, Z.; Tchuenbou-Magaia, F.; Kowalczuk, M.; Adamus, G.; Manning, G.; Parati, M.; Radecka, I.; Khan, H. Polymers Use as Mulch Films in Agriculture—A Review of History, Problems and Current Trends. *Polymers* **2022**, *14*, 5062. https://doi.org/10.3390/ polym14235062

Academic Editor: Raffaella Striani

Received: 19 October 2022 Accepted: 17 November 2022 Published: 22 November 2022

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

depending upon climatic conditions in different parts of the world [4]. As science advanced in the 20th century, mulching was also revolutionized. Paper sheets were introduced as mulch films in the 1920s, followed by the commercialization of plastic, specifically polyethylene films for mulching in the late 1950s. Plastic mulching gained popularity and proved to be very effective. However, the negative impact of plastic became evident within three decades and by the early 1980s photo-degradable and oxo-degradable plastics were introduced as an alternative to polyethylene based films. It soon became apparent that these polymers did not degrade in field conditions and generated microplastics [5]. Research was accelerated in this area and in 2006 the first biodegradable plastic mulch film was introduced in the market commercially. Following this, a number of biodegradable mulch films were manufactured by companies throughout the world. It was not until 2021, however, that the Food and Agriculture Organization, which is a part of the United Nations Organization, gave its recommendation to replace conventional and non-biodegradable polymers with bio-based and biodegradable materials for mulching practices [6].

**Figure 1.** The history and development of different mulching materials.

Various anthropogenic activities over the years have led to the production of greenhouse gases, which, in turn, have resulted in climate change. The emission and accumulation of these gases in the atmosphere has led to global warming, i.e., an increase in average temperatures and fluctuations that contribute to extreme weather conditions, as well as alterations in precipitation patterns throughout the world. Such changes in agricultural regions have an adverse impact on crop production, thus creating problems when providing adequate food supply for an increasing population in an efficient and sustainable manner [7]. The limited availability of arable land, depletion of water sources for irrigation, soil erosion, overexploitation of natural resources, pollution of ecosystems and climate change are some of the factors that restrict food crop production and yields [8].

In 2015, under the banner of the United Nations Organization, the international community developed a series of Sustainable Development Goals, known as SDGs, which include ensuring access to food for all, increasing agricultural productivity and achieving zero hunger by 2030 [9]. These goals not only imply the provision of food to achieve zero hunger on a global scale, but also aim to enhance agricultural productivity in a sustainable, manageable and efficient manner. The primary focus is to increase yields of food crops by adopting farming practices that are environmentally friendly and ecologically viable [10]. Mulching is already commonly used all over the world as a strategy to improve crop yield and prevent losses. The global mulch market is estimated at USD 3.5 billion in the year 2020, and is projected to amount to USD 5.1 billion by 2027 [11]. Considering the expansion of the market, it is pertinent to check that the practice is in line with the UN SDGs, specifically in terms of responsible production and consumption (Figure 2). Currently, the main material used for mulch films is polyethylene plastic, which is raising concerns. These mulch films do not degrade naturally and need to be removed from fields after harvesting. They cannot be recycled, and the debris left in soil contributes to soil pollution. A lack of disposal options for used plastic films adds to land and water pollution [12]. Therefore, there is an urgent need to explore other alternative polymers that may be used to replace plastic for mulching purposes [13].

**Figure 2.** The need for mulching as an agricultural practice and the materials available for use as mulch films.

Biodegradable polymers may be one solution, and have the potential to replace plastics for many applications. However, these polymers have certain limitations which are restricting their use on a commercial scale for the purpose of mulching. Factors that need to be considered before a polymer can be used include not only its physical and chemical properties but also the source and method of production. These polymers have different mechanical properties, which, in turn, affect their biodegradation in the environment [14]. Moreover, legislation and laws passed over the past few years have defined and strict criteria regarding biodegradation, chemical composition, deployment and toxicity for materials that may be used as a mulch film [15].

This review discusses the practice of mulching in agriculture, its benefits, the materials used as mulch films, and the alternate options that can be explored for sustainable agricultural practices. It also includes an overview of the biodegradation process that occurs in the soil for the breakdown of mulching materials, factors affecting this process, and a list of

different commercially available mulches, highlighting the direction of further research in this area.

#### **2. The Benefits of Mulch Films**

The increasing food demand of a growing world population has driven the need to increase agricultural crop yields. Protected cultivation is one way to help optimize the yield of crops. This involves controlling the microclimate around the growing plant to protect it from harsh climatic conditions [16]. Mulching is based on the principle of protected cultivation and involves the application of a protective ground cover made of different materials that may be organic or synthetic, to improve the growth and yield of agricultural crops. The word mulch is derived from the German word 'molsch', which means 'easy to decay' [3].

The use of mulch films in agriculture has several benefits when compared to nonmulched crop production. These may be categorized as improvements in the soil microenvironment or economic advantages (Figure 3a). Soil moisture content is an important factor that affects the growth of plants. Winds, high temperature, adverse climatic conditions and particularly weed infestation can contribute to the reduction of soil moisture. Mulches have been reported to increase the percolation and water retention capacity of soil, to the extent that the use of mulching material can reduce the irrigation requirement of crop plants. This is attributed to their water retention ability, which reduces the runoff from the soil profile [17,18]. This can indirectly contribute to salinity mitigation, as well. Various studies have demonstrated that the application of mulch films reduces the impact of salt toxicity on plants and helps in soil reclamation [19]. Mulches are also associated with the protection of soil from wind and water erosion, as well as the reduction of the compaction of soil. It has been observed that crop growth is negatively affected due to erosion and soil compaction in the absence of mulch films (Figure 3b). Mulches protect the soil by breaking the speed of water, especially in slopes or hilly areas, thereby increasing the infiltration rate. Similarly, the presence of covering material in the form of mulch films prevents soil erosion by winds [20]. Mulch materials also reduce the impact of weathering and the beating action of heavy rain, and the weight of feet and tyres of heavy machinery, helping to overcome the problem of compaction in soil [21].

**Figure 3.** A comparison of the effect of mulch films on plant growth and soil—(**a**) in the presence of mulch films and (**b**) in the absence of mulch films.

It is important to control the temperature of soil, since temperature fluctuations adversely affect the development of roots [20]. Since the material used for mulching covers the soil surface entirely, it helps to maintain the optimal soil temperature required for plant growth. Studies have shown that the application of mulch films keeps the soil temperature warmer on chilly days, and cooler during hot spells [17,22]. Mulches also contribute to the soil nutrient content, since these are broken down or degraded by soil microorganisms into simpler compounds that become a part of the soil itself [23]. Weed control is one of the key benefits that mulching provides. When mulch is spread on the surface of the soil, it acts as a barrier to the passage of sunlight, which reduces germination of weeds, especially small-seeded weed species. This phenomenon has been observed and widely used in nurseries as well as agricultural fields [24].

The presence of heavy metals in soil affects the growth of plants. Mulches can be used as a remediation strategy for the removal of these heavy metals from soil. Organic and plant-based mulch material forms complexes with heavy metals and converts them into a form that renders them non-toxic for plants [25]. Mulches can also be used as control release composite films, which are embedded with fertilizers, herbicides or pesticides. This strategy allows the gradual and slow release of these materials from the mulch films, ensuring that these are constantly available for use by the plant instead of being run off after the first application [26,27].

Generally, mulching decreases the stress level on plants, leading to better growth. This leads to enhanced crop yield and improved product quality. The crop may also be harvested earlier and tends to be more profitable [28]. All these facts confer economic advantages to the use of mulch films.

#### **3. Types of Mulch Films**

The use of mulch films dates back to ancient civilizations. The earliest mulch material comprised by-products from the agricultural and forestry industries, and included the trimmings of trees and shrubs, animal waste, stubble and residues of crop plants [20]. With time the material was modified, and now mulches can be categorized as organic or inorganic mulches. Organic mulches are made from materials found in nature and are usually broken down into simpler compounds by soil microorganisms. Inorganic mulches, on the other hand, are made of synthetic material that does not decompose easily [29]. The different types of materials used for mulching and their application in the field are shown in Figure 4.

**Figure 4.** Application of different mulch films in the field—(**A**): Compost, (**B**): Straw, (**C**): Bark, (**D**): Newspaper, (**E**): Woodchips, (**F**): Sawdust, (**G**): Plastic, (**H**): Black plastic, (**I**): LDPE. Adapted from [30].

The type of mulch used is governed by many factors, including the plant species, soil type and characteristics, the cost and availability of mulching material, as well as the regulations and law governing the region [31]. For instance, for vineyards or fruit orchards, the mulch film is generally thick and has a lifespan of years until it becomes

ineffective. For vegetable fields, on the other hand, thinner mulch films are used that last one growing season only [32]. Similarly, the effect of each mulching material is a combination of various factors including the amount of material applied, the carbon and nitrogen ratio of the material, its thickness, colour and other physical attributes, and the amount of toxic substances present [33]. Each material has certain advantages and disadvantages regarding its use for mulching, as summarized in Table 1 [34–72].


**Table 1.** Comparison of the sources, advantages and disadvantages of organic and synthetic mulch films.

#### *3.1. Organic Mulches*

Organic mulches mainly comprise animal or plant residues such as compost, manures, straws, husks, saw dust, grass and paper clippings, and wood/bark chips (Figure 4A–F). The application of compost and manure is an age-old practice in many regions of the world. The use of compost is as a way of recycling waste. It is cheap and readily available. Studies have exhibited that repeated use of compost for mulching over a number of years can increase the organic content of the soil and improve plant yield [34]. However, the use of compost as mulch has the added risk of phytotoxicity due to high nitrogen content. If applied near the stalks it absorbs moisture that promotes diseases and pests, leading to low crop yields [35]. Depending on the source of compost, it may contain heavy metals that accumulate in the soil as well as plants. While the presence of these heavy metals may not lead to toxicity in plants, it renders the crop unfit for human consumption. Compost is therefore not preferred anymore as a mulching material [36].

Other organic materials that can be used include straw and husks. These materials have a long life span when used for mulching and are effective for vegetables grown in winter months [37]. They are inexpensive, readily available and field trials have exhibited that they are good at preventing water loss via evaporation [38]. Both husk and straw mulches have the potential to increase crop yield and can significantly lower water losses from a well-irrigated system [39]. However, they often contaminate the soil with weed seeds and in addition harbour pests such as termites, snails, slugs and earwigs, leading to losses in crop yield, reducing their use for mulching [40].

Sawdust is readily available and has exhibited potential as a mulching material specifically for acid loving plants. It is very efficient in minimizing water runoff and soil erosion [41]. Studies have shown that it is not efficient in controlling the growth of weeds and also tends to harden over time, preventing water from reaching the deeper layers of the soil [42]. Another disadvantage is the low nitrogen content of sawdust, which means that it

uses up nitrogen from the soil as it decomposes. This has been seen in field trials where the use of sawdust mulch reduced the number of flowers produced and had an adverse impact on plant growth [43].

Grass clippings are very effective as a mulching material when applied as an appropriately thick layer. If the layer is too thick, it prevents air from penetrating, resulting in rotten and odorous clippings. If the layer is thin, the grass clippings decompose quickly, and need to be replenished frequently to maintain efficacy of the mulching material [44]. Fresh grass clippings also have the potential to develop their own root systems and offer growth competition to the crop plants. The use of these has also been shown to increase soil temperature and affect plant growth negatively in hot climates, limiting their application as mulching material [45].

Paper mulches are made up of cellulose fibres which can be decomposed naturally by most soil-borne microorganisms. Sheets of paper, mainly newspapers with black ink, can be used as mulch, as a part of a recycling strategy. The colour of paper used for mulching has an impact on the control of weed growth. Black paper mulching has shown promising results for decreasing the growth of weeds in lettuce and cantaloupe farming [46]. However, the main problem is that due to their light weight, these clippings are easily blown away by the wind. When applied as sheets, they tear apart easily when damp or wet, and are penetrated by weeds. Paper mulches degrade quickly so they cannot be used for long-term cultivation. These disadvantages limit the use of paper as a mulching material [40,47].

Wood and bark chips are preferred as mulching materials since these are very effective in retaining moisture in the soil. They also allow proper aeration and contribute to the organic content of the soil. Pine bark makes an attractive, usually dark-coloured mulch and may be used for ornamental plants. However, bark chips and pine needles have been reported to reduce the soil pH upon application and cause phytotoxicity [48]. The decomposition of woody material results in the release of phenolic acids which contribute to acidification of the soil, affecting plant growth. While this may be beneficial for crops that require an acidic pH for growth, it limits the large-scale use of woody material for mulching [49].

The main factors that limit the use of organic mulches are that they may not be available in adequate amounts throughout the year, have an inconsistent quality and are extremely labour intensive [50].

#### *3.2. Synthetic Mulches*

Inorganic or synthetic mulches are made up of materials such as glass, rubber sheets and plastic [51]. Glass mulches are usually made from chips of bottled glass as a method of recycling parts that cannot be separated from organic matter and other debris. These are exquisitely used to aesthetically modify a landscape while functioning as a mulch film [1]. However, the use of glass mulch films has become limited over the years because of their low efficiency in controlling weed growth. The ability of glass to reflect light reduces the sunlight that can reach the soil and has an impact on the soil temperature [52]. Moreover, the incorporation of non-recyclable glass into soil disturbs the physico-chemical characteristics of the soil, which subsequently impairs plant growth [53].

Rubber mulches are frequently made by shredding worn out tyres as a strategy of recycling. These shredded tyres include very fine particulate matter that can be inhaled, ingested or taken up transdermally, increasing health risks. The use of rubber mulches is also associated with the hazard of ignition since rubber catches fire quickly and is very difficult to extinguish [54]. Moreover, it has also been reported that rubber mulches are less effective in controlling weed growth compared to organic mulches [55]. In fact, high levels of zinc incorporated in tyres during manufacturing is released in the soil from the mulch and leads to zinc toxicity in plants, which adversely affects their growth [56].

Plastic mulch films (Figure 4G–I) have been used extensively since the 1950s and their global market has shown a continuous growth with a worth of USD 4.1 billion a year [57]. These are very effective in controlling weed growth and over time they have increased

global grain and cash crop yields by 15% and 40%, respectively [58]. Polyethylene plastic is the most popular and frequently used inorganic mulch throughout the world because of its efficacy and low price [59]. It is a low-density plastic that is highly resistant to weathering due to its chemical structure, which comprises saturated hydrocarbon chains [60]. These mulch films have low cost, low frequency of replacement, versatility and strength, and are therefore very effective in the field [61].

However, there are several problems associated with the use of plastic mulch films, including laborious and costly removal from the fields after use, lack of sustainable endof-life management options and the addition of plastic waste to the environment [62]. At the end of the growing season, the mulch film is often contaminated with plant as well as soil debris which restricts its recycling. Therefore, these are categorized as single-use plastic films and are usually landfilled or incinerated, contributing significantly to plastic pollution [63]. Complete removal of polyethylene mulch films is often not possible from fields, which has led to considerable amounts of macro and microplastic debris in soils as shown in Figure 5 [54]. This accumulation of plastic residue in soil has a negative impact on crop production by affecting the plant growth-promoting bacteria (PGPB), damaging soil structure, retarding root growth and development, and altering the carbon concentration of the soil [65]. In some countries the lack of disposal options means that farmers simply stockpile these mulch films after use, which subsequently leads to the dispersion of the mulch fragments into the environment by water and wind erosion, causing pollution [66].

**Figure 5.** The debris of black plastic mulch films—(**a**): piece of plastic mulch film, (**b**): microplastics collected from soil after removal of mulch film. Adapted from [67].

Most countries in the world are now opting for sustainable practices and have banned single-use plastics to overcome plastic waste generation and pollution. This has necessitated the need to look for sustainable and eco-friendly alternatives that can be used as mulch films [68]. As a solution, pro-oxidant additive containing (PAC) plastics have been formulated. These are chemically similar to low density polyethylene plastics but contain a pro-oxidant additive that increases oxidation and degradation of the polymer in the presence of light [69]. This photo-oxidation reduces the molar mass and adds oxygenated groups that make the polymer more prone to breakdown by microorganisms under aerobic environments. These plastics are also referred to as 'oxo-degradable' plastics. An example is Oxo-PP or oxo-degradable polypropylene [70]. PAC plastic mulches have been reported to be as efficient as conventional plastic mulches in the field for controlling the growth of weeds. They also have no adverse impact on soil health [71].

However, these mulch films have low strength and can break down prematurely while being spread onto the field. This fragility and their lightweight nature makes the application very difficult. Moreover, the absence of UV light reduces degradation of these plastics once they are incorporated into the soil. Even if there is breakdown in the soil, it is never fully biodegraded. The mulch pieces persist in soil and watersheds, and form micro plastics that have the ability to adsorb toxic pesticides, insecticides and herbicides, and carry them into the food chain [3]. Research reports suggest that while these mulch films are more expensive than polyethylene plastic films, they contribute equally to the generation of micro plastics that pollute the soil [72]. Many countries in the European Union, including France, have completely banned the use of PAC plastics, making it imperative to explore more sustainable, economical and environment friendly alternatives [66].

#### **4. Current Trends in Mulching Material**

In recent years a lot of research has been carried out to produce mulch material that is both sustainable and eco-friendly. The material for a mulch film should ideally be bio-based and very prone to biodegradation [73]. To replace plastic, it should have similar mechanical properties including a high tensile strength and high percentage elongation. The European Union has set up the product standards for biodegradable mulch films that are to be used in the horticulture and agriculture sectors. The first and only standard (EN 17033) has specified values that must be met, in terms of biodegradability, deployment, chemical composition and eco-toxicity, for a material to be used as a mulch film [15]. The generally acceptable standard for biodegradability is that the material should completely breakdown to carbon dioxide, water, methane, inorganic compounds and biomass within a year of application without producing any visible, toxic residue [74].

The use of biodegradable material imparts the main advantage of being tilled into soil once used, so that it can be degraded by the action of soil microbiota, reducing the costs of labour and disposal [75]. To replace conventional polyethylene mulch, the material should have the ability to maintain the optimum microclimate for plant growth and degrade completely without producing any harmful toxic products. In terms of efficacy, it should possess high tensile strength, good water retention capacity and allow for easy application. The film should be readily available, especially in the cropping season, and be inexpensive so that farmers can use it. Any material that is to be used as mulch should meet the minimum design requirements according to EN17033 and ISO 23517:2021 [76,77]. The main attributes required in a material to be used as mulch film are shown in Figure 6.

**Figure 6.** Properties of the ideal mulch film materials.

#### **5. Biodegradable Polymers for Mulch Films**

The term biodegradation refers to the breakdown of macromolecules by the action of microorganisms. Molecular level studies have shown that it is a two-step process [78]. In the first step fragmentation occurs in which the high molecular weight polymer chain is broken down into smaller units that may be oligomeric units with polar chain ends or monomers with specific properties. Fragmentation may occur through hydrolysis, which

may or may not be enzyme-mediated, oxidation or other chemical reactions depending on the environment as well as the chemical structure of the polymers. In the second step, oligomers and monomers formed in the first step are mineralized by microorganisms to produce carbon dioxide, methane, water, and biomass. The products formed may vary slightly depending upon the microorganisms involved and the aerobic/anaerobic nature of the process [79]. The schematic diagram of the process of degradation is shown in Figure 7.

**Figure 7.** The two steps of biodegradation of mulch films.

Polymers may constitute aliphatic chains or aromatic groups. Generally aliphatic polymers are easily hydrolysable while aromatic polymers require harsh conditions for breakdown, such as extremely acidic environments at high temperatures. Most biodegradable polymers are therefore aliphatic, but some aliphatic-aromatic polymers that have a limited number of aromatic units may also be included in this category [80].

Many factors affect the biodegradation of a polymer, including environmental conditions and the characteristics of the polymer, as shown in Figure 8. Environmental conditions may further be categorized as abiotic or biotic factors [50]. Abiotic factors include temperature, moisture, pH, and the presence of UV radiation. It has been reported in numerous studies that the temperature of the soil has a significant effect on the initiation of the biodegradation process, and lower temperatures decrease the rate at which the polymer is broken down [81]. Soil moisture is important and may become a limiting factor when fragmentation proceeds through hydrolysis. It is less likely to slow down degradation during irrigation periods, but has a marked effect if the moisture content drops too low or increases to the extent of making the soil anoxic [74,82]. Soil pH has a marked effect on the metabolism of microorganisms which, in turn, has an impact on their ability to degrade mulch films. It has been observed that generally soils with a neutral pH show maximum biodegradation, although depending on the type of microorganisms present there may be exceptions [83]. Ultraviolet radiation has a direct impact on the breakdown of mulch films. Experiments have shown that UV radiation speeds up biodegradation two-fold [84]. Biotic factors that affect biodegradation include the type of microorganisms present, the enzymes produced by them, and their ability to colonize the surface of the mulch films to initiate biodegradation. Climate and soil characteristics define the type of microbial communities present indigenously. Whilst it is mostly bacteria that are involved in biodegradation, the presence of fungi may accelerate the process in some cases [85].

**Figure 8.** The factors that affect biodegradation of mulch films.

It is interesting to note that the biodegradability of a polymer is not linked to its source but is dependent on its physiochemical properties such as molar mass, cross-linking, functional groups, crystallinity, flexibility and the presence of co-polymers, additives or cross-linkers [86]. As a general rule, an increase in the molar mass leads to a decrease in degradation. This is also linked to the solubility of the polymer, since high molecular weight compounds have low solubility which makes them unfavourable for microbial attack [87]. Similarly, highly cross-linked polymers have slower degradation since the close mesh-like structure makes the polymer inaccessible to both microbes and water molecules. The polymer needs to be mechanically broken down into smaller pieces before biodegradation can occur [88]. The type of bonds and functional groups present in the polymer are directly linked to its degradation. The presence of hydrophobic and non-polar functional groups makes a polymer less prone to biodegradation [89]. The breakdown of a mulch film is dependent on the type of polymer present. The biodegradation mechanism becomes more complex if the film is made of more than one type of polymer. These copolymer mulches or blends may be degraded at a slower or at times greater rate than single polymer mulches. For instance, it has been observed that PBAT blends degrade more slowly compared to PBAT mulches, whilst PLA blends show better degradation than mulches made of PLA alone [90,91]. Sometimes additives are included in polymer blends to increase the solubility or flexibility of the product. The presence of additives, specifically in co-polymers, may contribute to a reduction in its biodegradation. Depending on the type of additive, it may form cross-links with the polymer upon exposure to solar radiation. This cross-linking reduces accessibility to microbial enzymes and water, which leads to a decline in the degradation rate [92]. The degree of crystallinity of a polymer is one of the main factors that affect the biodegradation phenomenon. The more crystalline a polymer is, the denser the packing of molecules, which slows down the rate at which it can be broken down [93]. Although flexibility of a polymer seems more linked to its mechanical properties, it is surprising that it also has an impact on the rate of biodegradation. It has been observed that polymers with increased flexibility show enhanced biodegradation [94].

Biodegradable polymers may be derived from fossil fuels or renewable resources, or even a blend of both (Figure 9). They usually contain ester, amide and ether functional groups. Considering the already limited fossil fuel supplies, renewable resource-based materials or bio-based polymers are preferred over conventional petroleum based products due to easy availability and superior degradability [95].

**Figure 9.** A comparison of the diversity of plastics and bio-based polymers. Abbreviations include: PBAT—Polybutyrate Adipate Terephthalate, PCL—Polycaprolactone, PE—Polyethylene, PET— Polyethylene Terephthalate, PP—Polypropylene, PHA—Polyhydroxyalkanoate, PLA—Poly(Lactic Acid), PBS—Poly(Butylene Succinate), PBSA—Poly(Butylene Succinate Adipate). Adapted and modified from [96].

#### *5.1. Fossil Fuel Based Biodegradable Polymers Used for Mulching*

Biodegradable material made from synthetic polymers or derived from fossil fuels that have been used as mulch films includes polyurethanes such as polybutylene adipate terephthalate or PBAT, poly ε-caprolactone or PCL, and polybutylene succinate PBS [97]. The chemical structures of fossil fuel based biodegradable polymers is shown in Figure 10.

**Figure 10.** The chemical structures of fossil fuel based biodegradable polymers [98–100].

#### 5.1.1. Polybutylene Adipate Terephthalate

Polybutylene adipate terephthalate (PBAT) is a derivative of common petrochemicals and is considered to be fossil fuel based. It is a co-polymer of 1,4-butanediol, adipic acid and terephthalic acid. Therefore, it is an example of an aromatic–aliphatic polymer, with properties that are partially attributed to the aromatic group and partially to the aliphatic chain. It has a higher elongation break than most other polymers and is flexible [101]. PBAT also has good stretchability, impact resistance, extensibility and heat resistance, properties that are desirable for a material to be used as mulch film. The butylene adipate group imparts good soil biodegradability, making it a potential alternative to plastic mulches [102]. However, it is both expensive and highly sensitive to UV radiation. In field applications, PBAT undergoes severe crosslinking, which makes it brittle and reduces its efficacy, so it is generally recommended for short season crops only [103]. To overcome these limitations, blends of PBAT have been formulated with PLA, PPC, and/or starch. These co-polymers have exhibited increased durability and less brittleness. Additionally, they have a considerably lower cost of manufacturing since the blend is less expensive than the pure PBAT film. Now, many countries are producing PBAT-based mulch films commercially and their use is reported to have beneficial effects [104]. Blends of PBAT also have the property of functioning as controlled release systems via mulch films when fertilizers, fungicides, or herbicides are embedded into these films. The slow release of these compounds increases their efficiency and enhances crop production [2,24].

The degradability of these films may not be same in all types of soil. Studies have shown that the type of microorganisms present in the soil and the composition of soil structure influenced the extent of biodegradability of PBAT-based mulch films in field experiments [105], while more than 90% degradation of these blends under aerobic soil, within two years of application, has been documented. However, the persistence of fragments not converted to carbon dioxide or organic carbon within this time is not accounted for. There is a possibility that these micro-fragments and other chemical constituents will accumulate over time after repeated applications in the same field [106]. The degradation of PBAT under soil conditions results in the production of adipic acid and terephthalic acid, both of which are categorized as slight to moderate environmental toxins [107,108]. The presence of these compounds in the form of micro plastics has been detected in soils where PBAT-based mulch films were applied. Additionally, it was noticed that the presence of these micro plastics reduced the electrical conductivity and nitrate content in soil, which decreased the availability of water-soluble nutrients [109]. Studies have been carried out to compare the effect of mulch residues left in the soil. The results of these studies show that compared to LDPE plastic, PBAT mulch residues have a negative and harmful impact on soil bacterial community and plant growth. Further work and research need to be undertaken to ensure that PBAT-based mulch films are not hazardous to the environment, as well as the plants and microbial community of soil [110]. Since it is a petroleum-based polymer, long term use and availability of PBAT remains questionable [111].

#### 5.1.2. Poly ε-Caprolactone

Poly ε-caprolactone (PCL) is an aliphatic, synthetic, and thermoplastic polyester derived from petrochemical feedstocks. It is produced by ring-opening polymerization of εcaprolactone, which is obtained from crude oil. It has ester linkages and can be easily broken down by microorganisms that produce the enzymes lipase or esterase [112]. The chemical structure of PCL imparts it flexibility, low melting temperature and variable viscosity. It can be moulded easily, making it a potential candidate for mulching material [113]. However, PCL mulches have poor impact, weak tear strength behaviour and there are reports on film extrusion for these mulches. It is biodegradable, but the degradation rate is slow. Therefore, it is often mixed with starch to form blends with enhanced biodegradability. The application of these blends in the field has exhibited promising results. It has been observed that these films show better degradation compared to polyethylene mulches and have a positive impact on root growth and density, which is an important indicator of plant

growth [114]. Trials have also demonstrated that PCL-starch based mulch films can be degraded in most soil types and are effective in conserving soil moisture under various environmental conditions [74]. Many PCL-starch based blends are commercially available and being used as mulch films in many countries as an alternative to plastic mulches [99].

However, results of the field trials have indicated that these mulch films degrade in a shorter time if certain fungi and actinomycetes are present in the soil. In some conditions, these films degrade within a short span of 60 days, reducing their practical value and importance in agricultural practices [115]. With this in mind, this problem and the fact that the source of this polymer is non-renewable, concerns have been raised over its sustainability. Therefore, better alternatives are being searched for use in the agricultural sector [116].

#### 5.1.3. Poly Butylene Succinate

Poly Butylene Succinate (PBS) is a thermoplastic polyester which has physico-chemical properties such as conventional non-biodegradable plastic. It is made up of 1,4-butanediol and succinic acid [117]. PBS is a synthetic, petroleum-based polymer with good thermal stability and desirable mechanical properties. Its melting point is higher than other synthetic polymers but lower compared to natural polymers, so it can be melted in a shorter time and blended with other materials to develop films [118].

PBS is synthesized through various processes including co-polycondensation and reactive and physical blending, all of which impart different physical characteristics to the product formed. The higher the degree of crystallinity in the polymer, the lower its potential to be degraded by enzymes or microorganisms [119]. To overcome this problem, amorphous domains are added to the polymer by making blends with other materials. These blends have adequate strength and are available commercially as mulch films [120]. Interestingly, these PBS-based mulch films function as controlled release systems and have shown efficacy when embedded with different beneficial chemical compounds such as fertilizers or herbicides [121].

PBS and its blends are broken down by microorganisms that produce esterases. It has been found that the degradation rate of a PBS-based mulch film is influenced by the presence of certain fungi in the soil, as well as the availability of nitrogen and carbon sources to these microorganisms [122]. This may be linked to the fact that microorganisms do not produce esterases under conditions of nitrogen limitation. Similarly, the availability of carbon source plays a role in the regulation of esterase production. Therefore, soil characteristics and the type of indigenous microorganisms present are decisive factors in biodegradation of PBS-based mulch films [86]. It is important to understand that while the formulation of blends seems to be the most plausible solution to altering the properties of the polymer, PBS-based blends are often immiscible, resulting in phase separation which leads to poor mechanical properties. An alternative approach is the use of cross linkers or compatibilizer compounds in the blend, which enhance the phase mixing properties. However, the addition of these compounds compromises the biodegradability of the end-product [123]. It has been found that, compared to laboratory conditions, the biodegradation of PBS and its blends under field conditions takes longer. Low degradation of PBS-based mulches, with less than 3% degradation in more than 100 days, has been reported [124,125]. The price of PBS compared to petrochemical-based plastics is higher due to the cost of production. The large-scale production and application of PBS blends as mulch films is limited because of slow degradation, high cost and the fact that the polymer is derived from fossil fuels which are non-renewable [126].

#### *5.2. Bio-Based Polymers Used for Mulching*

Bio-based polymers are defined as materials formed naturally by living organisms over many growth cycles. These include lipids, proteins, and carbohydrates. Lipids are hydrocarbons, chemically, and may exist as esters, acid polyesters or free acids. They include hydrogenated fats, oils, fatty acids, and waxes [127]. Although lipids can undergo biodegradation, they are hydrophobic in nature and cannot be used as mulch films. The preparation and handling of films made of pure lipids is also very difficult, limiting their uses [128].

Proteins may be produced by microbial cells or extracted from plants and animals. These can be easily degraded in the environment by indigenous bacteria and fungi. Films made from soy protein, zein protein, wheat gluten, collagen and gelatine have been tried for mulching applications but have not been successful. The main problem associated with the use of protein-based mulches is their high-water sensitivity, which reduces the efficiency of these films [14].

The most abundant among these polymers are carbohydrates; mainly polysaccharides. These may be produced by the bacteria or exist naturally in plants and animals. Naturally occurring polysaccharides include chitin, alginate, starch, and cellulose (Figure 11). Chemically, all of these are made up of monosaccharides that are linked together by glycosidic bonds, but the presence of various functional groups and charges impart versatility [99]. The key advantages associated with the use of these materials for mulching are enhanced biodegradability, non-toxicity, availability and low cost [127]. Bacteria can utilize carbohydrates to synthesize polymers. Two such polymers are polylactic acid and polyhydroxyalkanoates, which are chemically polyesters that are synthesized from a carbohydrate base [129]. The chemical structures of PLA and PHAs are shown in Figure 11.

**Figure 11.** The chemical structures of bio-based biodegradable polymers (PLA\* is a synthetically produced bio-based polymer, whilst PHA, Chitin, Alginate, Starch and Cellulose are naturally produced bio-based polymers) [98–100].

#### 5.2.1. Polylactic Acid

Polylactic acid (PLA) was discovered in the 1930s and is produced by lactic acid forming microorganisms. It can be manufactured using renewable resources as the substrate for the microorganisms, such as corn, sugar beet starch and other agricultural products [129]. As suggested by the name, PLA is made from monomers of lactide which are synthesized

from lactic acid. Commercially available PLA is usually a co-polymer of poly L-lactic acid and poly D-lactic acid. The monomers are produced by bacteria but the polymer is made through a synthetic process usually involving ring opening polymerization and poly-condensation. The ratio of these two polymers influences the physical properties of PLA, including its melting temperature and crystallinity, as well as molecular weight [130]. Although the large-scale production of PLA is relatively inexpensive, the total cost of the polymer is high when compared to commercially available plastics [131]. PLA has good thermoplasticity, biocompatibility and processability which allows for a wide range of applications. However, as a mulch film, brittleness is its only drawback which reduces its efficiency [132].

To counter the problems of high cost and brittleness, blends of PLA are made with other polymers. A common blend is to mix PLA with starch, which has been shown to enhance the mechanical properties of PLA. Formulation of PLA-starch blend also reduces the cost of the mulch films, making it more economical for use [133]. Studies have shown that PLA blends as mulch films are effective, biodegradable and have good mechanical properties as well as higher water holding capacity [132,134]. Additionally, PLA-based mulch films can act as controlled release systems which allow for the release of chemical compounds embedded in the film over time, to enhance plant growth while preventing leaching of these compounds. Several studies have shown their efficacy as controlled release systems when PLA-based mulch films are embedded with herbicides [2,135,136].

However, the formation of these blends is challenging and difficult. The noncompatibility of composites is an area that is still under research. It has been found that formation of unstable bubbles in the film may occur after blending, leading to wrinkles and tears in the sheet. Similarly, electrostatic attraction may result in adhesion between these films, reducing the efficacy in agricultural applications [99,137]. To overcome this problem, plasticizers are added to the blends. Plasticizers are compounds used as additives, which are added to polymers to make them softer and more pliable. However, the addition of some plasticizers to these blends is not permitted due to the stringent requirements of manufacturing materials allowed for used in agriculture. These plasticizers are not biodegradable and are released into the environment once the mulch is applied and degraded [138]. Another problem related to the use of PLA based mulch films is the commercial production of PLA using genetically modified organisms. In many European countries, as well as the USA, there are strict restrictions on the use of GMOs for manufacturing agricultural products. For example, the National Organic Program rule in the USA states that any synthetic biodegradable mulch must be produced without involvement of any GMOs, restricting the use of such products on a large-scale [139].

#### 5.2.2. Polyhydroxy Alkanoates

Polyhydroxy alkanoates (PHA) are a class of aliphatic polyesters that are produced by many bacterial species as distinct granules. Discovered in 1925, the polyesters are synthesized intracellularly and used as storage polymers for carbon sources in many prokaryotes [140]. PHA granules differ in their content and chain arrangement depending on the microorganism used for production. PHAs are polyesters of hydroxyalkanoates where the number of monomeric units may vary from as little as 600 to as many as 35,000. The presence of different functional groups on the monomeric units is used as a classification system for types of PHAs [141]. The carbon source utilized for production, constituents of the media, fermentation process and conditions, as well as the downstream processes used for purification are other factors that have an effect on the structure and composition of the polymer [142–144]. More than 150 different PHAs are known to date, based on the combination of the monomeric units that make up the chain. The most common and well-known PHAs are poly 3-hydroxybutyrate(PHB) and poly 3-hydroxybutyrate-co-3 hydroxyvalerate (PHBV). Both are short chain polymers and available commercially [145].

Generally, the mechanical properties of PHAs do not support their use as mulch films. However, blending different PHAs together or with other polymers improves their mechanical strength. The main feature of PHAs that imparts an advantage is their ability to function as controlled release systems for agricultural chemicals. Studies have shown that PHB blends are particularly effective as mulch films for the slow and controlled release of pesticides and fungicides [146,147]. Blends of PHB with PLA have desirable mechanical properties such as polyethylene mulch films [91]. Biodegradation of PHB blends has been studied in a wide range of conditions including a controlled laboratory environment as well as natural habitats including different types of soil, river water, activated sludge, and compost. PHB is known to degrade rapidly in both aerobic and anaerobic environments, and thus can be disposed of easily without any negative impact on the environment [148,149].

The desirable characteristics such as structural variability, raw material availability, and biodegradability of PHB polymers make them an ideal candidate for replacing plastic as mulch films. Yet, despite the microbial source and versatility, the large-scale production of PHAs is restricted by the high cost of the manufacturing process [14]. Different production strategies, including the use of waste material as a carbon source, have been employed to decrease the cost of production. While these studies have shown positive results, the extraction of PHAs from bacterial cells still contributes significantly to the higher cost of the process. Extensive chemical extraction methods cause degradation of the polymer during the purification process. Therefore, efficient biological methods for extraction of PHAs are being investigated to decrease the cost of the production and to ensure that the useful properties of the polymer are not lost [150].

#### 5.2.3. Chitin

Chitin is found naturally in the cell walls of fungi and yeast, and in the exoskeleton of arthropods. The most abundant crystalline form of the polymer is α-chitin, which may be obtained from seafood waste in large quantities [151]. It is made up of D-glucosamine and N-acetyl-D-glucosamine residues which are linked together. The deacetylation of chitin forms chitosan. Chitosan readily dissolves in dilute acids but remains insoluble in water. It can also be moulded to form films. These two features make it a good choice for use as a material from which to make mulch films [152]. Chitosan is the second largest and abundant biological polysaccharide found in nature. However, films of pure chitosan may be brittle due to low fluidity of the linear chain. Plasticizers such as glycerol may be added to chitosan to enhance its flexibility and improve fluidity [153].

When used for mulching, chitosan has exhibited the potential to control weed growth much better compared to some herbicides [154]. It can also function effectively as a controlled release system and chitosan mulch films may be loaded with fungicides to protect plants against fungal diseases [155]. Chitosan is also considered to promote plant growth by contributing to soil nutrients, and many studies have shown that it enhances the growth rate, number of flowers and quality of vegetables when used as a mulch for various plant species [156–158]. However, it has been observed that the use of chitosan-based mulch affects soil temperature, increasing it during the day and lowering it at night, which has an adverse impact on plant growth [159]. Moreover, the production of chitosan from chitin is an expensive process. The interference in soil temperature and high cost mean that chitin and chitosan are usually not preferred as a mulching material [14,152].

#### 5.2.4. Alginate

Alginate is an aliphatic, water-soluble polymer found in the cell wall of brown algae. It is chemically made up of β-d-mannuronic acid (monomer M), and α-l-guluronic acid (Monomer G). The sequential arrangement and proportions of these two monomers in the alginate chain impart its different properties [160]. It can form three-dimensional mesh networks in the presence of cations, where the cation acts as a cross-linker that joins the alginate chains from the G residues. This property has been exploited for the production of gels or films from alginate, mainly with calcium ions, for the controlled release of active agents [161]. Alginate mulches have been produced as a solution of sodium alginate which

is sprayed on the soil, where it forms cross-linking with naturally occurring calcium ions. Once the water has evaporated, a thin layer of the polymer appears on the soil surface that functions as a mulch film [162]. Field trials have shown that these alginate mulches improve plant growth and have a positive impact on the population of soil microorganisms including fungi and actinomycetes. It also lowers soil temperature, making it a good option for use in hot climatic zones [163]. Alginate is known to act as a biostimulant and promotes plant growth, specifically through better root development and fruit quality, and it also improves the plant's ability to tolerate salt stress. Therefore, the use of alginate mulches may contribute in many ways to enhanced plant growth [164–166]. Moreover, alginate is non-toxic and biodegradable, and has good water holding capacity [167].

However, the main problem with the use of alginate-based mulch films is their dependency of synthesis on the presence of calcium ions in the soil, since the films cannot be produced in the absence of these ions. Furthermore, the stiff structure of sodium alginate and the rigidity of the resulting cross-linked film leads to rips and tears in the mulch film, which paves way for the growth of weeds [14]. A method to overcome this problem is the formation of blends including polyglycerol or hydroxyethylcellulose. While these blends have shown potential to be used as mulch films, the cost of production increases considerably, reducing the overall efficacy of the product [168].

#### 5.2.5. Starch

Starch is the main storage polysaccharide found in plants and is made up of the chains of amylose and amylopectin. Amylose is linear and consists of glucose units linked by α-1, 4 glycosidic linkages, while amylopectin has branches that stem out of the main chain. The main chain is made up of glucose units joined by α-1, 4 glycosidic linkages while the branch points have α-1, 6 glycosidic linkages [169]. Starch is found in the form of granules in seeds, roots, and tubers. Depending upon the plant source, starch may consist of amylose, amylopectin and other compounds such as lipids, proteins and phosphate or ester groups in minor quantities. The proportion of amylose and amylopectin influences the properties of starch. High amylose starches produce stiffer gels and stronger films, while high amylopectin starches produce softer gels which are more stable over longer periods of time. The proportion of these components is the main factor that helps to choose the source of starch and its applicability [170]. Commercially, starch is extracted from corn, wheat, rice, sorghum and potato depending on the geographical regions and local availability [171].

Starch is one of the most abundant and cheap polymers that can be used to replace plastic. It can be easily processed without any stringent requirements and can be produced commercially using already installed plastic processing machinery/equipment [172]. Considering these attributes and the fact that it can be used easily to form a film through the process of gelatinization, it is a good choice for use as a mulching material [173]. However, it has poor water resistance or high hydrophilicity which leads to enhanced degradation. In fact, it has been observed that more than 33% of the starch mulch film degrades within 55 days of application as a mulching material [64]. Starch films also have low elastic strength and are brittle, which means that they often tear apart while being spread on the field [68]. One way to overcome these problems is to modify or blend starch with other materials such as glycol, chitosan, and PLA. Films synthesized in this manner have exhibited better elastic strength but are expensive to produce and are often sensitive to high humidity. Moreover, the mechanical properties of these films are still not comparable to plastic mulch films, limiting their potential use in agriculture [174,175].

#### 5.2.6. Cellulose

Cellulose is the main structural component of plant cell walls and is the most abundant polysaccharide in nature. It is a linear homopolymer made up of D-glucose units joined through β- 1, 4 glycosidic bonds [176]. Cellulose is found in plants and can also be produced by a number of microorganisms including common soil-borne bacteria. Naturally occurring cellulose is found in two crystalline or allomorphic forms, Iα and Iβ, depending upon the

network of hydrogen bonds formed between the hydroxyl groups of cellulose chains. Iα is predominantly found in plants, while Iβ is the form of cellulose produced by bacteria [177]. These allomorphs vary in their solubility because of the hydrogen bonding pattern. Bacterial cellulose is preferred over plant cellulose due to relative abundance and ease of production and extraction [178]. Additionally, bacterial cellulose has good biodegradability, purity, water holding capacity, transparency, flexibility, and greater mechanical strength, making it an ideal material to replace plastic for mulch films [179]. Field trials have demonstrated that mulch films made of bacterial cellulose better retain soil moisture and are effective in providing a suitable microclimate for plant growth. Furthermore, these films can be modified as composite films to release nutrients by the addition of fertilizers [180,181].

The main challenge for the production of cellulose on a commercial scale is the higher cost of this process. Scientists are investigating the use of cheap waste material as a substrate for the bacteria to produce cellulose to decrease the cost of production [182,183]. Other aspects to reduce production costs include developing new strategies for agitated and static culturing and designing new, cost-effective fermentation vessels [184].

A comparison of the advantages and disadvantages of biodegradable polymers and their degradation times is given in Table 2 [103–184] and Figure 12, respectively [185–189].


#### **6. Commercially Available Biodegradable Mulches—Current Trends and Challenges**

Despite the challenges and problems related to production of biodegradable mulch films, consistent research efforts over a number of years have resulted in the development of some commercial mulching materials. All these mulch films are polysaccharide-based and are manufactured by blending two or more polymers [99].

The first biodegradable mulch film used in agriculture was Mater-Bi®, which was produced by Novamont, an Italian company. The mulch film is made of a blend of poly ε-caprolactone and thermoplastic starch and possesses reasonable biodegradability and mechanical strength [82]. Its application in the field has demonstrated that the material can be used effectively for mulching purposes [190]. Other commercially available mulch films include Biomax® TPS, Biopar®, Bionelle, Biosafe™, Ingeo®, and WeedGuardPlus [65,191,192]. These films are manufactured by various companies in different countries and are being used in farming practices (Table 3).

**Table 3.** Some commercially available mulch films and the crops grown using them.


Results obtained from field experiments have indicated that biodegradable mulch films are as effective as polyethylene mulch in controlling weed infestation and promoting plant growth. However, the increasing demand for food and cash crops has led to a continuous rise in the global consumption of plastic mulch film. Although there is a wide range of biodegradable material available on the market, the quantity is still not sufficient to completely replace non-degradable plastic mulch films [202]. The main challenge associated with the large-scale production of biodegradable mulch films is the economic viability of the production process. The profit and economic feasibility analysis of biodegradable mulches indicates that if these are to be used as alternatives to plastic mulches, governments need to provide subsidies to promote their use through extensive marketing [203–205].

#### **7. Future Prospects of Biodegradable Mulch Films**

Despite extensive research in the development of biodegradable mulch films commercially, the large-scale use of these materials is still limited. The biodegradable mulching material needs to be cost effective and easily accessible to the farmers for complete replacement of polyethylene in the field. Commercially available biodegradable mulch films are not preferred by most farmers because these are expensive, difficult to manage and require specialized equipment for application [206]. This means that although some alternatives to plastic mulches are available, they are not being used. Governments need to play a part in subsidising these mulch films and raising awareness about their benefits over plastic mulch films. For example, some regional authorities in Spain are giving incentives to farmers who use biodegradable mulch films to promote their use [204]. Such policies need to be adopted globally to encourage the use of biodegradable materials.

There is a need to develop polymers suitable for mulching in different climatic zones, with a wide range of temperatures and soil types, so that these can be utilized for the production of different crops [207]. It is currently a challenge to develop a material that fits the criteria of mulching products with good physical characteristics, durability, and

biodegradability. In this context, while there are standard testing methods for determination of mechanical strength, biodegradation analysis is still raising questions on many platforms. The biodegradability testing methods have several shortcomings, since these are mostly carried out in laboratory conditions and rely on indirect testing methods without taking soil characteristics into account. It is important to understand that the biodegradation process in the field is affected by many factors and the polymer may not exhibit the same rate of breakdown in lab and field conditions [208]. Even if the polymer degrades in soil, the longterm effects of any residual matter left after biodegradation of these polymers is yet to be investigated. Therefore, there is a dire need to develop testing methods that can estimate the biodegradation of the polymer in field conditions and allow for a close-to-reality simulation of the possible residual effects [139].

Among all polymers that may be used, cellulose offers potential for further research since the only problem associated with it is the cost of production. New processing methodologies such as 3D printing and electrospinning are also being considered for production of mulch material and can be used for lowering the cost of cellulose [209]. However, work in this area is still limited to laboratory trials, and field application still needs to be carried out to commercialize these products for further use [86].

#### **8. Conclusions**

This review provides a detailed account of the history of mulching, the concerns about the use of plastic mulch films and their impact on the environment. It also discusses the alternative biodegradable polymers with the potential to be used for mulching, including those produced naturally by microorganisms, animals and plants as well as some derived from fossil fuels, alongside their advantages and limitations. Most of the biodegradable polymers have characteristics that are a compromise in terms of parameters that need to be met for agricultural use in terms of maintaining the optimum microclimate for plant growth for the whole growth cycle and sufficient high tensile strength. For example, materials that exhibit good biodegradation often lack mechanical strength, and vice versa. The formation of blends or use of co-polymers appears to be a promising solution but presents a limitation of increasing the cost of production significantly. The future outlook for the development of biodegradable mulch films is nonetheless favourable. The cost of production can be reduced by adopting waste valorisation strategies wherever possible, and advanced scientific techniques can be used to improve the quality of the polymer produced so that it can meet the required standards. The discussion reflects the potential for research and development in the design of biodegradable mulch films. Further research in all domains of production, design, characterization, biodegradation analysis and commercialization needs to be carried out to encourage the use of biodegradable polymers for mulching to promote sustainable practices.

**Author Contributions:** This review article was written by Z.M., H.K. and I.R. were the main people involved in the planning of this review. M.K., G.A., M.P., G.M., F.T.-M. worked on the interpretation of the data. All authors were involved in the final editing of this article. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the University of Wolverhampton Research Investment Fund (RIF4) and the Commonwealth Scholarship Commission (CSC) UK (reference PKCS-2021-645).

**Institutional Review Board Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** Many thanks to the University of Wolverhampton, School of Sciences for their financial support and the library at the University of Wolverhampton for provision of necessary sources. We gratefully acknowledge support through Commonwealth Scholarship PKCS-2021-645.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


### *Article* **Preparation and Characterization of Acrylonitrile Butadiene Rubber Reinforced with Bio-Hydroxyapatite from Fish Scale**

**Namthip Bureewong 1,2, Preeyaporn Injorhor 1,2, Saifa Krasaekun 1,2, Pawida Munchan 1,2, Oatsaraphan Waengdongbang 1,2, Jatuporn Wittayakun 3, Chaiwat Ruksakulpiwat 1,2,\* and Yupaporn Ruksakulpiwat 1,2,\***


**Abstract:** This work aims to enhance the mechanical properties, oil resistance, and thermal properties of acrylonitrile butadiene rubber (NBR) by using the *Nile tilapia* fish scales as a filler and using bis(triethoxysilylpropyl)tetrasulfide (TESPT) as a coupling agent (CA). The prepared fish scale particles (FSp) are B-type hydroxyapatite and the particle shape is rod-like. The filled NBR with FSp at 10 phr increased tensile strength up to 180% (4.56 ± 0.48 MPa), reduced oil absorption up to 155%, and increased the decomposition temperature up to 4 ◦C, relative to the unfilled NBR. The addition of CA into filled NBR with FSp at 10 phr increased tensile strength up to 123% (5.62 ± 0.42 MPa) and percentage of elongation at break up to 122% relative to the filled NBR with FSp at 10 phr. This work demonstrated that the prepared FSp from the *Nile tilapia* fish scales can be used as a reinforcement filler to enhance the NBR properties for use in many high-performance applications.

**Keywords:** acrylonitrile butadiene rubber; fish scale; bis(triethoxysilylpropyl)tetrasulfide; composite; compatibilizer

#### **1. Introduction**

*Nile tilapia* is a freshwater fish that finds public favor in consumption, which makes it popular in aquaculture. About 140,000 tons of *Nile tilapia* fish were produced in Thailand, and 23% of those were processed into fillets. That left about 2% of the *Nile tilapia* fish scales as waste [1–3]. Generally, fish scales are considered discarded waste from the food processing industry and are often disposed of in landfills, which pollute both soil and water resources [4,5]. Therefore, the development of fish scales into functional materials is interesting because it could reduce the impact of environmental pollution and increase their value. Normally, the fish scales are rich in hydroxyapatite, collagen, polysaccharide, and chitin, which contain the mineral elements magnesium, calcium, fluoride, and phosphorus [6,7]. Recently, fish scales have been used to produce various products, such as fertilizer and fillers for plastic and rubber industrials. Additionally, it also has a wide use in tissue engineering for biomedical applications [3,4]. Hydroxyapatite from fish scales has gained the attention of many researchers. Majhool et al. [8] have prepared hydroxyapatite from tilapia fish scales. They expected that it can be used as a potential filler in polymers. Similar to Prasad et al. [9], they have used hydroxyapatite from fish scales as a filler in polylactic acid (PLA) composites for use as fixation devices. Their work revealed that hydroxyapatite can be improved the wettability and thermal stability of PLA/hydroxyapatite composites.

**Citation:** Bureewong, N.; Injorhor, P.; Krasaekun, S.; Munchan, P.; Waengdongbang, O.; Wittayakun, J.; Ruksakulpiwat, C.; Ruksakulpiwat, Y. Preparation and Characterization of Acrylonitrile Butadiene Rubber Reinforced with Bio-Hydroxyapatite from Fish Scale. *Polymers* **2023**, *15*, 729. https://doi.org/10.3390/ polym15030729

Academic Editor: Raffaella Striani

Received: 29 November 2022 Revised: 25 January 2023 Accepted: 28 January 2023 Published: 31 January 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Acrylonitrile butadiene rubber (NBR) is a synthetic rubber that has excellent resistance to solvents and oils due to the presence of polar cyanide groups in the NBR backbone. In addition, NBR is convenient to use in various industrial applications because of its moderate cost, processability, and heat resistance. The majority of NBR applications are used in petroleum and automobile industrials, such as fuel hoses, gaskets, oil seals, radiator hoses, v-belts, etc. [10,11]. However, NBR has drawbacks in tensile strength, flexibility and flame resistance, etc., that restrict its potential to be used in many applications requiring high performance [10–15]. Several researchers have recently been fabricating elastic composites, especially NBR-based nanocomposites, not only to improve the mechanical properties of NBR but also to enhance the resistance to solvents, oils, and heat [13,16,17].

However, although the addition of nanofillers to NBR has several advantages, as mentioned, it also has some disadvantages, such as the ability to agglomerate due to the filler–filler interactions, which limits the potential of the filler to improve the performance of NBR vulcanizate. Therefore, the addition of coupling agents could be an alternative way to reduce these problems because it would reduce the filler–filler interactions and enhance the filler–rubber interactions, which would improve the overall polymer composite properties [18,19]. Normally, bis(triethoxysilylpropyl)tetrasulfide (TESPT) is typically the coupling agent (CA) that is widely used in the rubber industry due to its low cost, availability, and the simplicity of the process [20]. TESPT is a bifunctional compound which is composed of two functionally active end groups. It can act as a bridge between filler and rubber via chemical linkages in the sulfur vulcanization, so enhancing of the rubber–filler interaction occurred [21].

In this work, alkali heat treatment was used to prepare *Nile tilapia* fish scales as fish scale particles (FSp). The method was based on previous research [22]. The particle size of FSp was expected to decrease with increasing treatment time and can be used as a filler in NBR. The characteristics of FSp were characterized using an X-ray diffractometer (XRD), an energy dispersive X-ray spectrometer (EDS), Fourier transform infrared spectroscopy (FTIR), a nitrogen adsorption–desorption analyzer, and a field emission scanning electron microscope (FE-SEM). The effects on the cure characteristics, mechanical properties, morphological properties, oil resistance, and thermal properties of the filled NBR with FSp at different contents (0, 5, and 10 phr) were investigated. Furthermore, the effects of CA on NBR samples with optimal FSp content were compared. NBR composites filled with FSp were expected to provide the oil resistance properties. This is a novelty of the work.

#### **2. Materials and Methods**

#### *2.1. Materials*

*Nile tilapia* fresh fish scales of approximately 3 kg were collected from the local market in Nakhon Ratchasima, Thailand. Acrylonitrile butadiene rubber (NBR) with the trademark NANCAR® 3345 was supplied by NANTEX Industry Co., Ltd. (Kaohsiung, Taiwan). Bis(triethoxysilylpropyl)tetrasulfide (TESPT) with the product ID KBE-846 was supplied by Shin-Etsu Chemical Co., Ltd. (Tokyo, Japan). Hydrochloric acid (HCl) with AR grade and 37% purity was supplied by RCI Labscan Co., Ltd. (Bangkok, Thailand). Sodium hydroxide (NaOH) with RPE grade and 99% purity was supplied by Carlo Erba (Milan, Italy). Stearic acid (SA), zinc oxide (ZnO), Di(benzothiazol-2-yl)disulfide (MBTS), N-Cyclohexylbenzothiazole-2-sulfenamide (CBS), and sulfur (S) were supported by Chemical Innovation Co., Ltd. (Bangkok, Thailand).

#### *2.2. Preparation of Fish Scale Particles (FSp) from Fresh Fish Scales*

The method for preparing FSp was adapted from Kongsri et al. and Injorhor et al. [22,23]. The collected fresh fish scales were washed with tap water several times to remove dirt, and dried using a hot-air oven at 60 ◦C for 12 h to obtain the dried fish scales. The protein and fat on the surface of the dried fish scales were removed using HCl solution at 0.1 M under stirring for 2 h at room temperature. Then, the removed fish scales were filtered, washed with DI water until they reached pH = 7, and dried at 60 ◦C using a hot-air oven. The prior

removed fish scales were alkali heat treated with 50% (*w*/*v*) of NaOH solution at 70 ◦C for 7 h. Afterwards, the slurry of fish scales was filtered, washed with DI water until it reached pH = 7, and dried using a hot-air oven at 60 ◦C. The FSp were then obtained.

#### *2.3. Characterizations of FSp*

The crystalline phase compositions and diffraction lines of FSp were analyzed by an X-ray diffractometer (XRD, D2 PHASER, Bruker, Billerica, MA, USA) with Cu Kα radiation source operated at 30 kV and current 10 mA. The 2θ range carried out was between 10 and 60 degrees.

The elemental compositions of FSp were analyzed by an energy dispersive X-ray spectrometer (EDS, EDAX Genesis 2000, AMETEK, Inc., Berwyn, PA, USA) in a scanning electron microscope (SEM-EDS, JSM-6010LV, JEOL, Tokyo, Japan).

The functional groups of FSp were analyzed by Fourier transform infrared spectroscopy (FTIR, TENSOR 27, Bruker, Billerica, MA, USA). The sample was mixed with potassium bromide (KBr) using agate mortar and pressed into a disk to obtain the test specimen with a smooth surface for transmittance measurement. The wavenumber range of 4000 to 400 cm−<sup>1</sup> with resolution of 4 cm−<sup>1</sup> and number of scans of 64 were used to collect data.

The characteristics of FSp in terms of BET surface area, total pore volume and particle size were determined from nitrogen adsorption–desorption analysis, which was performed on BELSORP-mini II (MicrotracBEL, Osaka, Japan). The sample was degassed at 160 ◦C for 6 h before analysis. The microstructure of FSp was acquired using a field emission scanning electron microscope (FE-SEM, AURIGA, Carl Zeiss, Oberkochen, Germany) at 3 kV. The samples were sputtered with gold for 3 min at 10 mA beforehand.

#### *2.4. Preparation of NBR/FSp and NBR/FSp/CA Composites*

The NBR/FSp and NBR/FSp/CA composites were prepared by compounding using an internal mixer at 70 ◦C with roller speed at 40 rpm and vulcanizing using a compression molding machine at 160 ◦C with an optimal cure time of each compound that was determined using a moving die rheometer (MDR). First, the NBR was masticated for 4 min, followed by the addition of different FSp contents (0, 5, and 10 phr), CA, and vulcanizing agent for 2 min. Then, the accelerators and activators were added separately for 1 min. The sample codes and compositions of NBR/FSp and NBR/FSp/CA composites are listed in Table 1.


**Table 1.** Sample codes and compositions of NBR/FSp and NBR/FSp/CA composites.

\* phr = part per hundred of rubber.

#### *2.5. Characterizations of NBR/FSp and NBR/FSp/CA Composites*

The cure characteristics, such as minimum torque, maximum torque, scorch time, and optimal cure time of NBR/FSp and NBR/FSp/CA composites, were determined using an MDR (M-2000AN, GOTECH, Taichung, Taiwan) according to ASTM D2084 with a temperature at 160 ◦C. The cure rate index of NBR/FSp and NBR/FSp/CA composites were calculated by following the equation:

$$\text{Cure rate index} = 100 / (\text{optimal curve time} - \text{second time}) \tag{1}$$

The modulus at 100% elongation (M100), modulus at 300% elongation (M300), tensile strength, and percentage of elongation at break of NBR/FSp and NBR/FSp/CA composites were measured according to ASTM D412 using a universal testing machine (UTM, Model:5565, INSTRON, Norwood, MA, USA) with a load cell of 5 kN and crosshead speed of 500 mm/min.

The hardness of NBR/FSp and NBR/FSp/CA composites were measured according to ASTM D2240 using a hardness tester (HPE II, Bareiss, Stouffville, ON, Canada) with the Shore A test method.

The secondary electron images of NBR/FSp and NBR/FSp/CA composites were acquired using a field emission scanning electron microscope (FE-SEM, AURIGA, Carl Zeiss, Oberkochen, Germany). The tensile fracture surfaces of NBR/FSp and NBR/FSp/CA composites were coated with gold before the SEM observation.

The oil resistance in terms of change in mass percentage of NBR/FSp and NBR/FSp/CA composites were performed according to ASTM D471 by immersing the standard specimens in toluene for 22 h. The equation that was used to calculate the change in mass percentage of NBR/FSp and NBR/FSp/CA composites is:

$$
\Delta \text{M (\%)} = [(\text{M}\_2 - \text{M}\_1)/\text{M}\_1] \times 100\tag{2}
$$

where ΔM is the change in mass (%), M1 is the initial mass of the specimen in air (g), and M2 is the mass of specimen in air after immersion (g).

The thermogravimetric analyzer (TGA, TGA/DSC 1, METTLER TOLEDO, Greifensee, Switzerland) was used to analyze the thermal decompositions of NBR/FSp and NBR/FSp/CA composites. The specimens were placed in an alumina pan and heated from room temperature up to 650 ◦C under nitrogen with a heating rate of 10 ◦C/min and a gas flow rate of 20 mL/min.

#### **3. Results and Discussion**

#### *3.1. Characterizations of FSp*

The XRD pattern of FSp is represented in Figure 1. The sample consisted entirely of the hydroxyapatite phase with defined peaks following the Crystallography Open Database (COD 9001345) and similar to the standard of JCDPS 00-009-0432 [24] without other phases. The characteristic peaks of the FSp are similar to the synthetic hydroxyapatite of Pon-On et al. [25] and the fish scale nano-hydroxyapatite of Kongsri et al. [23]. It was confirmed that the obtained FSp are a type of hydroxyapatite, which is a bio-material.

**Figure 1.** XRD pattern of FSp compared with COD 9001345.

The elemental compositions and EDS spectra of FSp are shown in Figure 2. It confirms that the major constituents are calcium (Ca), phosphorous (P), and oxygen (O). Moreover,

the typical presence of sodium (Na) and magnesium (Mg) are significant factors in bone and tooth growth [26]. However, the Ca/P of FSp is 1.86, which is higher than the stoichiometric ratio of 1.67 for stoichiometric hydroxyapatite because of the presence of carbonate (CO3 <sup>2</sup>−) ions that substitute phosphate (PO4 <sup>3</sup>−) in the hydroxyapatite structure (B-type hydroxyapatite) [25].

The functional groups of FSp are shown in Figure 3. The broad band around 3500 cm−<sup>1</sup> corresponds to the OH− stretching vibration of adsorbed water. The bands at 1471 and 1415 cm−<sup>1</sup> correspond to the asymmetric stretching vibration of the CO3 <sup>2</sup><sup>−</sup> band, and a band at 873 cm−<sup>1</sup> corresponds to the bending vibration of CO3 <sup>2</sup><sup>−</sup> [23,27]. The presence of CO3 <sup>2</sup><sup>−</sup> bands indicated that some PO4 <sup>3</sup><sup>−</sup> groups were replaced with CO3 <sup>2</sup><sup>−</sup> groups. These results confirmed the FSp are B-type hydroxyapatite, which corresponds to the EDS result and with Kongsri et al. [23]. In addition, the strong band at 1043 cm−<sup>1</sup> corresponds to the stretching vibration of PO4 <sup>3</sup>−, and the sharp bands at 601 and 563 cm−<sup>1</sup> correspond to the degenerate bending vibrations of PO4 <sup>3</sup><sup>−</sup> in a hydroxyapatite structure.

**Figure 3.** FTIR spectrum of FSp.

The adsorption–desorption isotherm of FSp is represented in Figure 4 and the information on FSp analysis in terms of BET surface area and total pore volume are listed in Table 2. The isotherm shape shows unrestricted monolayer–multilayer adsorption up to high P/P0 without the final saturation plateau. Therefore, the isotherm of FSp is fitted to the second (II) type of the IUPAC classification given by nonporous adsorbents [28,29]. The BET surface area of FSp shows a higher value as compared to the extracted hydroxyapatite from carp fish that was reported by Muhammad et al. [30]. In addition, the BET surface area of FSp shows a higher value than the commercial hydroxyapatite in these reports [23,30]. For use as filler in composite materials, the high surface area of FSp has an advantage in terms of greater interactions with the matrix. It was expected to be a bio-filler for improving NBR composites.

**Figure 4.** Physisorption isotherm of FSp.

**Table 2.** Information on FSp analysis.


The microstructure of FSp is shown in Figure 5. The FE-SEM images show the FSp in a rod-like shape with some agglomerates of FSp due to the static force between the FSp. However, the particle size of the FSp is in the range of the nanoscale.

**Figure 5.** FE-SEM images of FSp at (**a**) ×50,000 and (**b**) ×100,000 magnification.

#### *3.2. Characterizations of NBR/FSp and NBR/FSp/CA Composites*

The cure characteristics such as minimum torque, maximum torque, scorch time, optimal cure time, and cure rate index of NBR/FSp and NBR/FSp/CA composites are listed in Table 3. Normally, the minimum torque is related to the viscosity of the rubber compound, while the maximum torque is related to the rigidity of the rubber vulcanizate. In the case of the addition of filler to rubber, these properties are also related to the nature of the filler. In addition, the scorch time is the time with no crosslinks in the rubber compound, which is an important parameter for the safe processing of rubber in molds. The optimal cure time is another important parameter that determines the time required to produce the rubber products [15,31–35]. The value of minimum torque of NBR-5FSp tends to increase as compared to unfilled NBR because the dispersion of FSp at 5 phr reduces the mobility of the macromolecular chains of NBR, which causes the viscosity of the compound to increase [15,32,34]. Meanwhile, the value of minimum torque of NBR-10FSp decreases as compared to unfilled NBR and NBR-5FSp because the addition of FSp at this amount has agglomerated in the NBR matrix. The values of maximum torque of the filled NBR with FSp are higher than unfilled NBR because the stiffness of the FSp increases the rigidity of composite vulcanizates. The values of scorch time and optimal cure time of filled NBR with FSp decrease as compared to unfilled NBR because the mineral content of FSp acts as an activator for composite vulcanizates [4,34]. Meanwhile, the NBR-10FSp slightly decreases the values of scorch time and optimal cure time as compared to NBR-5FSp because the system of filled NBR with increasing FSp content becomes more heated from the filler friction that affects the increased degree of curing. These results resemble the cure characteristics of filled rubbers with prepared hydroxyapatite that were reported by Nihmath and Ramesan [15,34]. Therefore, the use of FSp as a filler in NBR is an alternative idea for producing NBR composite because it can reduce the optimal cure time of NBR to obtain the product in a shorter time. However, the NBR-10FSp-CA shows the value of minimum torque decreasing as compared to filled NBR because the CA acts as a lubricant, which causes the viscosity of the compound to decrease. Moreover, the value of the optimal cure time of NBR-10FSp-CA tends to increase as compared to filled NBR because the CA coats on the surface of FSp, which reduces their activator activity, which causes the optimal cure time of the composite vulcanizate to increase.


**Table 3.** Cure characteristics of NBR/FSp and NBR/FSp/CA composites.

Figure 6 represents the mechanical properties of NBR/FSp and NBR/FSp/CA composites, and Table 4 shows the values of the mechanical properties of NBR/FSp and NBR/FSp/CA composites in terms of M100, M300, tensile strength, percentage of elongation at break, and hardness. It is well known that the characteristics and dispersion of filler are directly related to the properties of the polymer composite. Additionally, the area under the stress–strain curves is related to the toughness of the polymer. In general, the mechanical properties depend on the nature of the filler, dispersion, and the interaction between the filler and polymer matrix [4,15,34,36]. The modulus and tensile strength of filled NBR with FSp show increased values as compared to unfilled NBR because of the interactions between FSp and the NBR matrix, which improve the fracture resistance of NBR composites. However, the percentage of elongation at the break of filled NBR with FSp shows decreased values as compared to unfilled NBR because the addition of FSp restricts the molecular motions of the NBR matrix, which reduces the elasticity of NBR. When

compared to the filled NBR with FSp at 5 and 10 phr, the NBR-10FSp shows higher values of tensile strength and percentage of elongation at break that resemble the mechanical properties reported by Akbay et al. [4]. According to this research, the tensile strength and percentage of elongation at break of the rubber were increased by increasing the fish scale content, because the calcium oxide (CaO) content of fish scale acts as a vulcanizate activator that improved the mechanical strength and flexibility of the rubber composite. Meanwhile, the hardness values of filled NBR increase with increasing FSp content because the stiffness of FSp improves the resistance to indentation of NBR composite, which resembles the hardness results in these reports [15,34]. Moreover, the NBR-10FSp-CA shows increased values of tensile strength and percentage of elongation at break as compared to filled NBR because the CA enhances the chemical interaction between FSp and the NBR matrix, which assists the stress-transfer of NBR composite but has no effect on the modulus value. Figure 7a represents the schematic of NBR/FSp/CA interactions. The first step is CA hydrolysis, which generates the silanol groups (Si-OH) on the side chains of CA. The second step is that the Si-OH of CA and the Ca of FSp generate an ionic reaction together. In addition, the sulfur (S) of the CA and the carbon (C) on the NBR chains undergo a crosslinking reaction. Figure 7b represents a sketch of the interface interactions between FSp, CA, and NBR chains. These interactions are similar to these reports [19,37,38]. All results show that the modulus, tensile strength, and hardness of NBR are improved by adding the FSp with increasing contents. In addition, the tensile strength, percentage of elongation at break, and hardness of NBR composites are also improved by using a CA to obtain superior values.

**Figure 6.** Mechanical properties of NBR/FSp and NBR/FSp/CA composites showing (**a**) Stress–strain curves and (**b**) Tensile strength and percentage of elongation at break.



**Figure 7.** (**a**) Schematic representation of NBR/FSp/CA interactions and (**b**) Sketch of the interface interaction between FSp, CA, and NBR chains.

Figure 8 presents the FE-SEM images of tensile fracture surfaces of the NBR/FSp and NBR/FSp/CA composites. Generally, the morphological properties of the polymer composite are necessary to report in order to understand the dispersion, compatibility, and characteristics of the filler after the mixing and forming processes [13,32,39]. The unfilled NBR shows the smooth fracture surface without the particles on the surface (Figure 8a). On the other hand, the filled NBR with FSp shows the dispersion of FSp with some agglomerates on the rough fracture surface (Figure 8b,c). These characteristics tend to increase with increasing FSp content in NBR (Figure 8c). In general, the dispersion of the filler should be uniform with no agglomerates in a polymer composite. Nevertheless, the mechanical properties in terms of modulus, tensile strength, and hardness of filled NBR increase with increasing FSp content. This indicated that the FSp are effective reinforcement fillers for improving the mechanical properties of NBR, although the FSp exhibit non-uniform dispersion and have some agglomeration in the NBR matrix. Additionally, the roughness on the fracture surface indicates the resistance to fracture of the polymer composite due to the good mechanical interlocking between filler and matrix [32]. That corresponds to the results of tensile strength and percentage of elongation at break of filled NBR, which increase with increasing FSp content. In the case of the addition of CA, the NBR-10FSp-CA shows the image of fracture surface with similar characteristics as compared to the NBR-10FSp. However, the FSp on the fracture surface of NBR-10FSp-CA tend to be more embedded in the NBR matrix than the FSp on the fracture surface of NBR-10FSp (Figure 8d), which affects the increased tensile strength and the percentage of elongation at break of the NBR-10FSp-CA. This indicated that the CA increases the reinforcement efficiency of the FSp and NBR matrix.

**Figure 8.** FE-SEM images of (**a**) Unfilled NBR, (**b**) NBR-5FSp, (**c**) NBR-10FSp, and (**d**) NBR-10FSp-CA composites.

Figure 9 depicts the oil resistance of NBR/FSp and NBR/FSp/CA composites in terms of change in mass percentage. The oil resistance is an important parameter for polymer products that are used in petroleum applications. In general, the oil resistance depends on the interactions between solvent and polymer, crosslink density, and crystallinity of polymer [15,34]. The oil resistance of NBR is enhanced by adding the FSp, because the intermolecular forces between FSp and NBR increase the energy required to separate the NBR molecules for the penetration of oil molecules, which affects the decreased swelling percentages of filled NBR with FSp. In addition, this result also depends on the content of FSp added, which shows a decrease in the swelling percentage with increasing FSp content in NBR. Meanwhile, the value of the swelling percentage of NBR-10FSp-CA is no different when compared to NBR-10FSp, which indicates that the CA has no effect on this property. The values of the swelling percentage of unfilled NBR, NBR-5FSp, NBR-10FSp, NBR-10FSp-CA are 93.91%, 64.33%, 60.73%, and 58.41%, respectively. Therefore, although it is well known that NBR is a synthetic rubber that has excellent resistance to solvents and oils, the filled NBR with increasing FSp content increases the oil resistance efficiency.

**Figure 9.** Oil resistance of NBR/FSp and NBR/FSp/CA composites.

The TGA thermograms of NBR/FSp and NBR/FSp/CA composites are presented in Figure 10, and the thermal properties in terms of the initial degradation temperature (Tonset), temperature at maximum weight loss level (Tmax), final degradation temperature (Tendset), and residue percentages of NBR/FSp and NBR/FSp/CA composites are listed in Table 5, according to the TGA thermograms of NBR composites, which can divide the stages of weight loss into three stages: 35–350, 350–500, and 500–650 ◦C. The first stage shows a slightly decreased weight loss percentage in all samples, which is about 3% due to

the volatile water. Meanwhile, the decomposition of organic compounds from filler and polymer is the reason for the maximum weight loss in all samples that is shown in the second stage. However, the values of Tonset of NBR tend to increase with increasing FSp content, and the results of Tmax and Tendset also show this tendency. The reason for these results is that the decomposition temperature of FSp is higher than the NBR matrix, so the FSp act as a heat barrier during the thermal decomposition process of NBR composites, which resembles the thermal properties reported in these reports [13,40,41]. According to these studies, the thermal stability of the polymer was increased by increasing the prepared hydroxyapatite content. Additionally, due to the incomplete decomposition of organic compounds in the second stage, all samples exhibit a slightly decreased weight loss percentage in the third stage. At a temperature of 650 ◦C, the values of residue percentage of NBR increased with increasing FSp content due to the remaining inorganic compounds from FSp in NBR composites. Nevertheless, the NBR-10FSp and NBR-10FSp-CA show similar results in their thermal properties. Therefore, the thermal properties of NBR are increased by adding FSp with increasing content, but the CA has no effect on these results.

**Figure 10.** TGA thermograms of NBR/FSp and NBR/FSp/CA composites.


**Table 5.** Thermal properties of NBR/FSp and NBR/FSp/CA composites.

#### **4. Conclusions**

FSp were successfully obtained from *Nile tilapia* fish scales biowaste and used to prepared NBR/FSp composites. The obtained FSp are B-type hydroxyapatite with a rodlike shape. The FSp were the effective reinforcement filler in the NBR matrix because it enhanced the tensile strength, oil resistance, and thermal properties of NBR. Moreover, the scorch time and optimal cure time of NBR also reduced with increasing FSp content, resulting in a shorter time to obtain the NBR product. The addition of CA gave the best properties, because the CA enhanced the tensile strength and percentage of elongation at break of NBR-10FSp. The obtained NBR composites filled with FSp are expected to be used

in sealing gadgets that can resist oil. In the future, the NBR composites will compare with other bio-fillers.

**Author Contributions:** Conceptualization, Y.R. and C.R.; methodology, N.B., Y.R. and C.R.; validation, J.W., Y.R. and C.R.; formal analysis, N.B., P.I., S.K., P.M. and O.W.; investigation, N.B., P.I., S.K., P.M. and O.W.; resources, Y.R. and C.R.; data curation, N.B., P.I., S.K., P.M. and O.W.; writing—original draft preparation, N.B. and P.I.; writing—review and editing, N.B., J.W., Y.R. and C.R.; visualization, Y.R. and C.R.; supervision, Y.R. and C.R.; project administration, Y.R. and C.R.; funding acquisition, Y.R. and C.R. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Thailand Science Research and Innovation (TSRI), Grant No. 160344.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors are grateful to Suranaree University of Technology (SUT), to Thailand Science Research and Innovation (TSRI), to the National Science, Research and Innovation Fund (NSRF), to the Research Center for Biocomposite Materials for Medical Industry and Agricultural and Food Industry for the financial support, and to the Chemical Innovation Co., Ltd. for supporting the chemicals.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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### *Review* **Role of Polymers in Microfluidic Devices**

**Laila A. Damiati 1,\*, Marwa El-Yaagoubi 2, Safa A. Damiati 3, Rimantas Kodzius 4,5, Farshid Sefat 6,7 and Samar Damiati 8,\***

	- <sup>5</sup> Faculty of Medicine, Vilnius University, 03101 Vilnius, Lithuania
	- <sup>6</sup> Interdisciplinary Research Centre in Polymer Science & Technology (Polymer IRC), University of Bradford, Bradford BD7 1DP, UK
	- <sup>7</sup> Department of Biomedical and Electronics Engineering, School of Engineering, University of Bradford, Bradford, BD7 1DP, UK
	- <sup>8</sup> Department of Chemistry, College of Sciences, University of Sharjah, Sharjah 27272, United Arab Emirates
	- **\*** Correspondence: ladamiati@uj.edu.sa (L.A.D.); sdamiati@sharjah.ac.ae (S.D.)

**Abstract:** Polymers are sustainable and renewable materials that are in high demand due to their excellent properties. Natural and synthetic polymers with high flexibility, good biocompatibility, good degradation rate, and stiffness are widely used for various applications, such as tissue engineering, drug delivery, and microfluidic chip fabrication. Indeed, recent advances in microfluidic technology allow the fabrication of polymeric matrix to construct microfluidic scaffolds for tissue engineering and to set up a well-controlled microenvironment for manipulating fluids and particles. In this review, polymers as materials for the fabrication of microfluidic chips have been highlighted. Successful models exploiting polymers in microfluidic devices to generate uniform particles as drug vehicles or artificial cells have been also discussed. Additionally, using polymers as bioink for 3D printing or as a matrix to functionalize the sensing surface in microfluidic devices has also been mentioned. The rapid progress made in the combination of polymers and microfluidics presents a low-cost, reproducible, and scalable approach for a promising future in the manufacturing of biomimetic scaffolds for tissue engineering.

**Keywords:** polymers; microfluidics; lab-on-chip; biomedical engineering; drug carrier; artificial cell; 3D bioprinting

#### **1. Introduction**

Polymeric biomaterials have been used to provide artificial matrices that can mimic the biological cell. This requires appropriate biophysical and biochemical properties, such as certain topography, stiffness, signaling, and growth factors [1]. Polymers are commonly used in tissue engineering scaffolds and wound dressing due to their ability to enhance cellular regeneration. Further, drugs are encapsulated in the polymeric particles to generate drug vehicles that can improve drug uptake into the disease sites and bioavailability. However, the physical and self-assembled properties of polymers, such as charges, composition, biodegradation, shape, size, and surface chemistry, play a dominant role in determining polymer behavior within biological environments [2]. Further, these interactions are directed by the physicochemical properties of the polymers in micro or nanostructures. The developed polymeric models are able to navigate the body, infect and transform cells, or repair damaged cells. The incorporation of cells into polymeric matrix can be performed by cell implantation into readily prepared polymer matrix. This strategy has a significant drawback, namely the lack of good integration between cells and polymer matrix. Another

**Citation:** Damiati, L.A.; El-Yaagoubi, M.; Damiati, S.A.; Kodzius, R.; Sefat, F.; Damiati, S. Role of Polymers in Microfluidic Devices. *Polymers* **2022**, *14*, 5132. https://doi.org/10.3390/ polym14235132

Academic Editor: Raffaella Striani

Received: 12 October 2022 Accepted: 22 November 2022 Published: 25 November 2022

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

alternative strategy relies on the fabrication the polymer matrix with encapsulated cells, which allows development of complex cellular microenvironments. New techniques, such as microfluidics, 3D printing, and electrospinning, enable direct cell integration into the matrix to mimic the matrix of desired tissue [1].

Microfluidics technology, also known as lab-on-chip technology, has been used as a platform for biomedical engineering applications [3]. Generally, a microfluidic chip is network of microchannels incorporated into the microenvironment by several holes throughout the chip. Microfluidics allow the integration of biological and chemical processes on a single platform. These microdevices allow controlling of the flow behavior of small volumes of fluids in micro-chambers in the range of tens to hundreds of micrometers. Microfluidics are widely used to synthesize polymeric particles for various applications involving drug carrier vehicles, as well as bioarchitecture models mimicking cell-like structures or extracellular matrix (ECM). Furthermore, recent studies have demonstrated the possibility of using microfluidic chips as an artificial cell chassis. Depending on the application, glass or polymer can be used to manufacture microfluidic devices and several parameters should be taken into consideration in the fabrication of a microfluidic chip, such as the compatibility of constructed materials with various solvents and channel geometries [4–7]. Polymer-based chips are usually selected due to their cost efficiency, suitable optical transparency, elasticity, and appropriate mechanical and chemical properties [8]. Several polymers, such as polydimethylsiloxane (PDMS) and poly(methyl methacrylate) (PMMA), are employed to fabricate microfluidic devices [3]. However, polymers have some limitations regarding their properties, including operation temperature range limitations, higher autofluorescence, and the limited availability of surface modification techniques [9]. The fabrication of polymer microfluidic devices is relatively simple, and hazardous etching reagents are not needed to create the polymer microstructure [10].

It is common to use natural polymers, such as polysaccharides and bacterial polyesters, to generate polymer-based therapeutics, while it is common to use synthetic polymers as building blocks for microfluidic devices. Figure 1 shows different applications of utilizing polymers and microfluidics. Biodegradable and bioreducible polymers that are used for polymeric drug/gene delivery systems are rapidly emerging in pharmaceutical fields. Combining therapeutic agents with polymers can improve their safety and efficacy by controlling the rate, time, and preferentially delivers the therapeutic agents to the target site in the body [11]. Combining microfluidic devices and polymers presents unique advantages for the development of efficient carriers of a wide range of drugs and genetic materials (e.g., polymersomes). Microfluidic technology enables the production of highly stable, uniform, monodispersed particles with higher encapsulation efficiency [5]. Furthermore, many studies showed the possibility of using polymer-based bioinks in 3D printing for applications in tissue engineering and regenerative medicine. There are several natural (e.g., alginate, collagen, agarose) and synthetic (e.g., Pluronic and poly(ethylene glycol) (PEG)) polymeric biomaterials that are used as bioinks for 3D printing based on their ability to support cell growth, mechanical properties, and printability. Combining of cells, biomedical polymers and biosignals is the basic requirement to develop 3D tissues or organ structures [12]. The fabrication of vessel-like microfluidic channels is an example of organ fabrication and thick tissue. Besides supporting the mechanical integrity, the printed 3D microfluidic network enables fluid transport. The microfluidic architecture allows media transport, including nutrients, oxygen, water, and removal of the waste in the same manner [13]. Recent developments in droplet microfluidics allowed the creation of versatile vesicles with a structure that resembles the biological membrane. These artificial cell-like structures with well-defined size enable the implementation of various biological reactions within a compartment separated by a membrane that mimics a natural cell membrane [3]. In this perspective, this review deals with polymers used to either fabricate microfluidic devices or create functional particle/matrix models using polymers and microfluidic chips. The review first provides an overview of the different types of polymers. Then, it highlights some of the recent advances in the design of microfluidics and polymers for various

biomedical engineering applications, including drug delivery, 3D bioprinting, and artificial cell-like structures.

**Figure 1.** Utilizing of microfluidic chips and polymers for various applications (Created with Biorender.com, accessed on 12 October 2022).

#### **2. Polymers Used in Microfluidic Devices**

A single polymer unit may be composed of hundreds or millions of monomers. They have one of the four basic polymer structures: linear, branched, cross-linked, or networked (Figure 2). The two types of polymers are natural and synthetic. Natural polymers can be extracted from biological systems such as plants, algae, microorganisms, and animals, which have a similar ECM structure to native tissues. Synthetic polymers are similar to natural polymers, but they are much cheaper, can be produced at large scale, and have long shelf life compared with natural polymers. As such, generally, they present good cellular attachment, which improves the cellular behaviors and prevents immunological reactions [1,14].

**Figure 2.** From monomers to polymers. Linear, branched, cross-linked, and networked structures in polymers (Created with Biorender.com, accessed on 12 October 2022).

Choosing the right material is the first and most critical step in designing a successful microfluidic device. A wide range of constraints and requirements dictates the selection of the material for a specific component. The design of the device, the compatibility of the material with the chemicals, as well as the applied temperature and pressure are crucial considerations in material selection. Additionally, the final application of the device is an essential consideration. For example, devices intended for in vivo applications in tissue engineering must be nontoxic, exhibit a slow and predictable degradation rate, have nontoxic and safe degradation products, and potentially capable of mimicking certain physical and chemical properties of the native ECM or of supporting other agents that play such roles. The architecture of the device can also influence the choice of materials. For example, in devices that contain microfluidic systems, the materials have to be mechanically robust but have a controlled degree of flexibility [15,16]. The material also has to be compatible with microfabrication techniques, easily processed in mild conditions, and cheap to manufacture, among others [17]. Polymers are classified into two major groups: biodegradable and biostable polymers. These two types of polymers are commonly used as scaffolds or bioactive coatings in biomedical applications [8,15]. The next section focuses on these two classes of polymers for the manufacture of microfluidic devices and their biomedical applications.

#### *2.1. Biodegradable Polymers*

Sustainable polymers from various renewable resources can be directly obtained from biomass (proteins and polysaccharides), or through chemical modifications of natural polymers [18]. However, there are many sources of natural and synthetic biodegradable polymers. Natural polymers are derived from natural raw materials and available in large quantities while synthetic polymers are synthesized by the chemical polymerization of bio-monomers.

#### 2.1.1. Natural Biodegradable Polymers

In this section, we distinguish between natural polymers, which are produced outside the human body (xenobiotic polymers), and proteins, which are native to the human body, such as the ECM proteins. The use of natural biopolymers in microfluidics provides many advantages, such as surface chemistry biocompatibility and having the same mechanical properties of the native proteins of interest [16,19].

Natural polymers, such as chitosan, alginate, and gelatin, are also biologically derived and biodegradable polymers. They are used in the manufacturing of biodevices that are intended to interact with the biological systems of the human body. The crosslinking ability of these natural polymers, which is induced by physical and chemical stimuli, makes them ideal for the preparation of microgels for microfluidic devices. The two natural biopolymers, alginate and gelatin, were used as substrates to make two types of hydrogelbased microfluidics. Subsequently, the fabricated hydrogel microchannels can be used as platforms to provide 3D cell culture environments for mammalian cells: fibroblasts and vascular endothelial cells. The developed enclosed microchannel models are simple and reproducible and do not require complicated operations [16,19].

One class of natural biomaterials that is a good candidate for microfluidic devices is silk fibroin (SF) [20]. SF protein, originally found in the silkworm *Bombyx mori*, is a highmolecular-weight protein that primarily consists of hydrophobic residues. This protein is approved by Food and Drug Administration (FDA) for many medical applications, such as drug delivery and tissue engineering. SF can be easily processed to form hydrogels, films, and nanofiber mats under mild conditions [21]. In recent years, SF has also been used to fabricate microfluidic devices due to its excellent biocompatibility, robust mechanical properties, and slow proteolytic degradation rate [16,20]. The solubility and mechanical properties of SF materials are linked to its secondary structure. Whereas self-assembled β-sheet structures are responsible for the mechanical stability and water insolubility of SF, the amorphous regions, including random coil, α helix, and β turn structures, contribute

to the elasticity and solubility of the biomaterial. Thus, SF-based microfluidic fabrication strategies allow the rapid and scalable production of devices without the need for harsh processing conditions that require cytotoxic reagents. Mao et al. used SF and chitosan to construct porous SF–CS scaffolds with predefined microfluidic channels. The generated model showed structural properties suitable for seeding and growth of hepatic cells. Mass transport and uniform cell distribution within the 3D scaffold were successfully achieved [19].

In addition to all the above-mentioned advantages of natural polymers, the inclusion of natural ECM proteins into microfluidic devices allows the reproduction native cell– biomaterial interactions in vitro [22]. The use of ECM proteins is crucial in controlling cell function overall via other physicochemical mechanisms such as specific cell binding domain sequences. Proteins, such as fibronectin, vitronectin, and collagen I, contain the amino acid sequence of arginine–glycine–aspartic acid (RGD), which supports the adhesion of cells and to control stem cell differentiation. For example, Arik et al. reported the fabrication of a collagen-I-based membrane incorporated in an organ-on-chip device [23]. The membrane demonstrated permeability, as well as the adhesion of both endothelial and epithelial cells. Moreover, they characterized the degradation and remodeling of the basement membrane by a protease. Natural proteins offer an environment that more closely mimics that of the body and more realistically mimics the cell–ECM interactions, which are crucial for tissue engineering. However, these biomaterials have a complex structural composition that prevents complete control over their composition and other factors, such as molecular weight, immune response, degradation, and mechanical properties. As an alternative, scientists have focused on the development and use of synthetic polymers, which have more tunable properties [22].

#### 2.1.2. Synthetic Biodegradable Polymers

Synthetic polymers were proposed as ideal candidates for the fabrication of biodegradable microstructures, including microfluidic biomaterials [16,24]. Poly(glycolic acid) (PGA), poly(lactic acid) (PLA), and their copolymer poly(lactic acid-co-glycolic acid) (PLGA), belong to the linear aliphatic polyesters family [25] (Figure 3). This polymer family is one of the most widely used in tissue engineering and drug delivery [25,26]. These polymers have several advantages, such as low cost, ease of processing, and well-characterized biological behavior. These polymers (PLA, PGA, and PLGAs) are among the few synthetic polymers approved by the U.S. FDA for certain human clinical applications [26]. These polymers degrade through the hydrolysis of the ester bonds [27]. Although PGA and PLA belong to the same family, they also display distinct properties. For instance, because of its very hydrophilic nature, PGA rapidly degrades in aqueous solutions. However, PGA and PLA show the same behavior in vivo: they lose mechanical integrity in a period between two and four weeks [28]. Conversely, PLA contains a methyl group, which renders the chains more hydrophobic and hence reduces the affinity to water, and displays a slower hydrolysis rate (months to years) [28]. This class of biodegradable polymers is suitable for microfluidics because of the wide range of tunable properties [27]. They can be modulated by adjusting the lactide-to-glycolide ratio. The physical properties of the copolymer PLGA are defined by the properties of both pure PGA and PLA [25]. The presence of PLA makes it more hydrophobic than PGA. Hence, lactide-rich PLGA copolymers are less hydrophilic and more slowly degrade. Additionally, PLA exhibits relatively a high glass transition temperature (Tg = 50–80 ◦C) and melting point (Tm = 173–178 ◦C). PLGA blends of various copolymer ratios exhibit a reduced phase transition temperature (Tg, PLGA75/25 = 54 ◦C) and melting point (Tm, PLGA75/25 = 80 ◦C) [29]. Poly(caprolactone) (PCL) is another example of an aliphatic polyester used in microfluidics. PCL demonstrates advantageous properties for replica molding strategies, such as a low melting point (Tm = 57 ◦C) and low glasstransition temperature (Tg = −62 ◦C) [30]. PCL can be degraded by micro-organisms as well as by the hydrolysis of its ester linkage in physiological conditions [31]. However, PCL materials have a substantially slower biodegradation rate than PLA and PGA, making it suitable for the use in long-term implantable systems. Biodegradable cell-support scaffolds play an important role in the growth of engineered tissue and the delivery of biologically active agents. Therefore, the concept of biodegradable microfluidic devices formed by various biodegradable polymers has attracted considerable research attention. For example, microstructured PLGA films were used to construct a high-resolution and high-precision 3D device. The developed device allows diffusion distance reduction in cell-seeded scaffolds with convective transport [32]. PLA microchannels have been widely generated by 3D printing. Kadimisetty et al. developed a microfluidic immunoarray using PLA and a 3D printer. The fabricated device was low cost and could sensitively detect prostate cancer biomarker proteins [33].

**Figure 3.** Synthetic bio-degradable polymer structures.

Poly(1,3-diamino-2-hydroxypropane-co-polyol sebacate) (APS) is another biodegradable elastomeric polymer used to construct microfluidic scaffolds. The simple microchannel network design exhibited a very low degradation rate while retaining the elastomeric properties required for tissue scaffold applications [24].

#### *2.2. Biostable Polymers*

PDMS is a mineral–organic polymer structurally composed of silane-oxygen backbones covered with alkyl groups. Depending on the size of the monomer chain, noncross-linked PDMS may be almost liquid (low amount of n monomer) or semi-solid (high amount of n monomer) [34]. The high level of viscoelasticity displayed by the polymer chain is due to the siloxane bonds in the polymer structure. After cross-linking with a curing agent, PDMS becomes a hydrophobic elastomer [34]. One of the main reasons for the success of PDMS in microfluidics is the ease of PDMS device fabrication, which also allows mass production. Among many other methods, PDMS microchips can be fabricated through microscale molding processes [35]. For example, a silicon wafer with patterns can be used as a mold master. Prepolymer PDMS is poured into the mold master. Then, cured PDMS is peeled off from the master to be pasted on a flat plate, i.e., PMMA, glass, etc. [34]. The flat support should be drilled in advance to provide access ports for the introduction of reagents and samples. PDMS can precisely replicate structures down to the submicron size [36]. Due to the favorable optical properties of PDMS (almost no absorbance in the visible wavelength range), fluorescent dyes are widely used for the detection and quantification of molecules in most biochemical analyses. In addition, PDMS is transparent, biocompatible, nontoxic, and displays high gas permittivity, so has been traditionally used as a biomaterial in catheters, insulation for pacemakers, and ear and nose implants [10].

The combination of its elastic properties, easy processability, and the other properties mentioned above make PDMS an ideal candidate for use in microfluidic devices for biomedical and cell applications.

Many studies have been performed to further examine the compatibility of PDMS with both microfluidic technology and biomedical applications [37]. In terms of microfluidic technology, the effects of the structure and surface of PDMS in widely used microfluidics methods, such as spin coating and chemical immersion, on different liquid chemicals have been studied. Successful spin-coating of PDMS depends on the crosslinking ratio; increased amounts of crosslinker agent in the formulation decrease film thickness. Additionally, whereas chemical immersion (solvents such as alcohol, toluene, acetone, etc.) does not result in major changes in the surface hydrophilicity of PDMS, macrotexture distortion and destructions are observed with strong acids (hydrofluoric, nitric, sulfuric, and hydrofluoric acids) and bases (potassium hydroxide). For biomedical applications, the effect of oxygen plasma and sterilization and the exposure to tissue culture media was also explored. Oxygen plasma exposure increases PDMS surface hydrophilicity, whereas a following exposure to air leads to hydrophobic recovery. UV and alcohol sterilization do not affect the PDMS surface microtexture, element concentration, hydrophilicity, or mechanical properties. Finally, immersion in tissue culture media increases the surface concentration of oxygen relative to silicon [38].

Despite all these advantages, the use of PDMS is limited due to challenges encountered in microfluidics. For example, incomplete curing of PDMS leaves uncrosslinked oligomers within the material, which can leach out and contaminate the culture medium. Other problems, such as incompatibility with some organic solvents, water evaporation, channel deformation, and adsorption of biomolecules onto channel walls, present severe limitations to the use of PDMS for microfluidics applications [39].

#### Thermoplastics

Thermoplastics are plastic polymer materials that have emerged as a commercially viable material. Their use has recently increased, being widely applied to fabricate microfluidics platforms for biomedical applications. The most commonly used thermoplastics are PMMA, polycarbonate (PC), polystyrene (PS), polyvinyl chloride (PVC), Cyclo-olefincopolymer (COC), and Cyclo-olefinpolymer (COP) [39,40] (Figure 4).

**Figure 4.** Most used thermostable polymers structures for microfluidic chips.

Because of their linear structure, their thermoplastic rigidity resists temperature and pressure changes. The properties of the most common thermoplastics used for chips fabrication are summarized in Table 1. Thermoplastic-based materials have good physical and chemical characteristics, such as high chemical and mechanical stability; low water-

absorption capacity; acid/base resistivity; and are suitable for mass production at low cost. In term of fabrication, thermoplastics can be softened after exposure to heat at their transition temperature (Tg), making them processable around this temperature. During cooling, the softened polymer hardens, and it takes the shape of the container or mold, without any chemical change. They can be reshaped multiple times by reheating, which is important for the molding and microfluidics fabrication process [41].


**Table 1.** Properties of the most used biocompatible thermoplastics in the microfluidic field.

One of the first properties to consider in cell biology is biocompatibility. According to Table 1, most of the thermoplastics are biocompatible. However, for long-term applications, some of the materials can be problematic. For example, polycarbonates can be experience surface erosion during in vivo applications. In addition, bisphenol A (BPA), which is hazardous in food contact situations, might be released during hydrolysis.

PVC can release toxic gases during manufacturing, and nylon is a heat-sensitive material. Resistance to solvents is also a main criterion that must be considered for microdevice fabrication and biomedical applications (sterility). PS is widely used in molecular and cell biology studies due to its biocompatibility and its high resistivity to alcohols, polar solvents, and alkalis [50]. PMMA is affected by ethanol, isopropyl alcohol, acetone, and other important solvents used in microfabrication and sterilization [51]. When working with cell cultures, low water absorption is beneficial because the cells consume more oxygen from water, which can be limited by the absorption of water onto the polymer surface.

The optical properties of the selected material (e.g., transparency and autofluorescence) are crucial. Consequently, PMMA, polyethylene terephthalate (PET), and polypropylene (PP) are less suitable for applications that require further reactions inside the microfluidic devices under a microscope. Additionally, PC displays high autofluorescence, so PC is difficult to use when working with fluorescently labelled cells or materials. In contrast, PS has high transparency, and the surface of PS is suitable for long-term cell studies [41]. Table 2 highlights some studies that used polymers as a chassis or to functionalize sensing surface.


**Table 2.** Some studies using polymers for microfluidics devices for various biological applications.

#### **3. Polymers as Drug Carriers**

Generally, a drug is any bioactive molecule, including medicine, small molecules, and proteins, e.g., growth factors and nucleic acids [63]. Different polymers have been used in drug delivery approaches: i. a drug can be directly incorporated onto scaffolds throughout the casting process [64], ii. bulk hydrogels [65,66], iii. drug reversibly and covalently conjugated to the matrix [67], iv. micro- or nanodrug particles spread on the

surface [68–70]. However, all of these methods have advantages and disadvantages in terms of drug stability [63]. When manufacturing new drug delivery system, different factors should be taken into consideration for instance cost, efficacy, and properties differences. Advances in manufacturing techniques may produce more complex drug carrier designs to allow specific drug release targeted to a particular disease [71]. Using a microfluidic platform approach can allow generation of drug carriers that can meet the sophisticated requirements of biomedical applications [72] (Figure 5).

**Figure 5.** A scheme of different approaches of using polymer for drug delivery. Microfluidics control synthesis of various drug delivery systems. Subsequently, microfluidic chips can be used for cell culture and drug toxicity screening (Created with Biorender.com, accessed on 12 October 2022).

Drug delivery devices have potential to be used for various clinical applications, such as tissue regeneration, diabetes, oncology, and infectious diseases. Moradikhah et al. used a cross-junction microfluidic device to prepare alendronate-loaded chitosan nanoparticles. They showed that this system substantially enhanced the osteogenic differentiation of human adipose MSCs, so can be a suitable component of bone tissue engineering scaffolds [73]. Mora-Boza et al. illustrated that their fabricated hMSC-laden microcarriers based on in situ ionotropic gelation of water-soluble chitosan in a microfluidic device using antioxidant glycerlphytate and tripolyphosphate maintained cell viability over time and increased the secretion of paracrine factor [74]. An example of oral delivery drug was examined by Jaradat et al.; insulin was encapsulated into various PLGA nanoparticles prepared by the microfluidic technique. They found that the mucopenetrating heparin sulfate-conjugated PLGA nanoparticles enhance insulin permeability in a triple-cultured intestinal model compared with unmodified and free insulin nanoparticles [75]. Another model developed by Damiati et al. used PLGA to generate indomethacin-loaded PLGA microparticles employing a 3D flow-focusing microfluidic chip. This model not only successfully incorporates indomethacin, which is a poorly water-soluble drug and nonsteroidal anti-inflammatory drug, but the authors also developed an artificial neural network as in silico tool to predict size microparticles [76,77].

An example of using polymers in drug delivery in cancer is biodegradable polymeric nanocapsules. Oxaliplatin, irinotecan, and 5-fluorouracil chemotherapy drugs were encapsulated and carried on a coaxial glass capillary microfluidic device, which the potential for targeting tumors as the drug release could be controlled [78]. Hong et al. reported that the synthesized amphiphilic tri-chain tricarballylic acid-poly (ε-caprolactone) methoxypolyethylene glycol (Tri-CL-mPEG) and enzyme-targeted tetra-chain pentaerythritol-

poly (ε-caprolactone)-polypeptide (PET-CL-P) using microfluidics continuous granulation technology improved the bioavailability and antitumor effects of curcumin in a mouse model [79]. A recent review by Salari et al. provides a comprehensive assessment of studies in the field of polymer-based drug delivery for anti-cancer therapy. In their study, 71 papers were investigated, and they conclude that the polymeric nanoparticles have influential roles in cancer treatment comparing to the conventional chemotherapy. Polymeric nanoparticles were able to reduce the cytotoxicity following chemotherapy drug administration, enhance therapeutic agents solubility, and inhibit tumor growth rate [80].

As bacterial infections are posing a major threat to human health, in addition to increasing antibiotic resistance, new methods for bacterial detection are necessary to reduce disease spread. Recently, advances in antibiotic treatment have focused on the targeted delivery of antibiotics, as well as antibiotics alternatives, such as antimicrobial polymers, peptides, nucleic acids, and bacteriophages [81]. Borro et al. reported that by using polymyxin B-aliginate-Ca2+ microgels prepared by 3D printing, the microfluidic mixer affected the charge contrast and composition of the microgel formation and the interaction with bacteria-mimicking liposomes at different ionic strengths [82]. Additionally, a P-based nanoparticles delivery system was used as therapy against bacterial biofilm infections. Huang et al. used PLGA-based nanoformations combined with carbon quantum dots (CQDs) using a microfluidic flow-focusing pattern to load different types of antibiotics, e.g., azithromycin and tobramycin. They found that the azithromycin-loaded CQD–PLGA hybrid nanoparticles showed synergistic chemo-photothermally antibiofilm effects against *Pseudomonas aeruginosa* [83]. Norries et al. illustrated that the hydrogel developed from the poly(2-hydroxyethyl methacrylate) (PHEMA) and coated with ciprofoxacin antibiotic reduced the biofilm production of *Pseudomonas aeruginosa* [84].

#### **4. Polymers as Bioink for 3D Printing**

Three-dimensional (3D) printing is a development technique that has been used during the last decade to produce microfluidic devices. It has many advantages such as low cost, enabling the easy design of complex 3D structures and rapid prototyping. However, 3D printing has some limitations regarding the size of the microchannels and some final steps that are related to the laborious fabrication [85].

Different methods can be used to produce printed porous materials: i. curing a porous monolithic polymer sheet into the chosen pattern with photolithography, ii. screen-printing silica gel particles with gypsum, and iii. dispensing silica gel particles with polyvinyl acetate binder using a 3D printer. All three approaches can be successfully used in microfluidics [86].

Hydrophilic and hydrophobic polymers can be used to generate 3D-printed microfluidic droplets to prevent water-in-oil or oil-in-water droplets from sticking to the interior device surfaces. Warr et al. investigated two different approaches to avoid this issue: First, different resins were tested to evaluate their suitability for droplet formation and material properties. They found that the hexanediol diacrylate/lauryl acrylate resin forms the best hydrophobic solid polymer that prevents aqueous droplets from attaching to the device wall. Second, they formed a fully 3D microfluidic annular channel-in-channel geometry that forms droplets that do not contact channel walls. As such, this geometrical approach can be used with hydrophilic reins [87].

Distler et al. found that 3D-printed hydrogel is more electroactive and cytocompatible and enhances cell adhesion and proliferation compared with a 2D flat hydrogel. This kind of hydrogel formulation has shown promise in in vitro studies, cell therapy, and assisted tissue engineering electrical stimulation [88]. Wright et al. used a hydrogel composed of calcium crosslinked alginate (polypyrrole–alginate composite) as bioink for tissue engineering. They found that PC12 neural cells adhere and proliferate slightly better than alginate scaffold alone [89]. A compensation between metallic and polymer materials was used to fabricate a novel complex 3D structure. A soft polymer was cast and cured into a 3D-printed thin-shelled metallic mold, followed by metallic mold etching in an acidic solvent, which did not affect the soft polymer. This approach provided various polymeric complex structures [90].

At present, organ failure is a worldwide issue, and allograft organ transplantations are seriously limited due to donor organ shortages, immune rejection, and ethical conflicts, so finding an alternative solution is crucial [91–93]. Several polymers have been used for bioartificial organ manufacturing with different types of cells, e.g., stem cells, various growth factors, and vascular and neural networks. However, 3D bioprinting technology is a challenging engineering approach. Cooperation is required between different fields, such as biomaterials, biology, medicine, physics, chemistry, bioinformatics, and engineering, to fulfill all the requirements from the molecular to organ levels. Further, 3D bioprinting of polymers needs to meet several basic requirements to be applied in clinical applications. These requirements include biocompatibility, biostability, good mechanical properties, bioprintability, biodegradability, suturable with host vascular and nerves, permeability for nutrients and gases, and sterilizability [93].

#### **5. Polymers as Artificial Cells or Organs**

Numerous researchers have been trying to reduce the gap between the structures that can be designed and produced in the laboratory and those found in biology. Biological cells provide multiple functions, such as synthesizing proteins and lipids, storing genetic materials, storing and harvesting energy, etc. [92,94] (Figure 6). Additionally, homogeneous cells organize into specific tissues, whereas heterogeneous cells aggregate into an organ with specific physiological functions [93]. As such, creating an artificial cell or organ that has the same compartmentalized, multifunctional architecture is a challenging task. Two fundamental approaches have been considered for artificial cell constriction: top-down and bottom-up approaches. The top-down approach starts from living organisms, moving down the genome to the lowest number of genes that are essential for maintaining cell viability and functionality. The bottom-up approach starts from scratch by using biological and nonbiological molecules to build up a "living" artificial organelle or cell [95].

**Figure 6.** Architecture of typical biological cell and artificial cell. Left: Eukaryotic cell containing different types of organelles. Right: artificial "synthase" cell that mimic that structure of biological cell (Created with Biorender.com, accessed on 12 October 2022).

Several researchers have tried to mimic the natural cell or tissue function and reduce the gap between normal and artificial cells. In studies involving artificial cells, microfluidics provides a powerful tool to produce a large number of compartments with different size ranges [96,97]. For instance, a circular design PDMS microfluidic compartmentalized co-culture platform was developed by Park et al. In the fabricated model, neurons and oligodendrocytes are co-cultured in two separate compartments connected by arrays of

shallow axon-guiding microfluidic channels. The chip design offers physical and fluidic isolation between the soma and the axon/glia compartments [98]. In an attempt to mimic the structure of biological cells, alginate was used as a biomaterial in artificial systems, and four types of glass microfluidics with flow-focusing or co-flowing droplet generators were used to produce alginate droplets. The generated alginate microgels exhibited various architectures, including individual monodisperse or polydisperse beads, small clusters, and multicompartment systems [99]. For cell culture, microfluidic systems are mainly fabricated with silicon, PDMS, and borosilicate. These materials have been used to test the mammalian embryos within microfluidic systems [100]. Moreover, many microfluidic devices have been reported to enhance cell growth, differentiation, and micro-environmental changes in various perfusion system [101–104]. In 3D cellular environment, combining PDMS and hydrogel into hybrid device has been used to produce 3D-ECM of aligned for endothelial cell cultures [105]. A study by Leclerc et al. illustrated that the culture of fetal human hepatocytes (FHHs) microfluidic bioreactors is promising for liver tissue engineering. They found that the albumin production by FHHS was four times higher than in static culture which can be influenced to the potentiality of fetal liver cells maturation [106].

These fabricated models show the use of a variety of polymers as distinctive biomaterials and the ease of using microfluidic platforms, which can be used to construct simple mimics of cellular environments or cellular architectures, and thus offer a promising approach for synthesizing bioarchitectures. Table 3 summarizes some studies used polymers and microfluidics in applications described in this review.


**Table 3.** Summary of some studies used polymers and microfluidics for several applications include drug delivery, 3D printing, and artificial cells.


#### **Table 3.** *Cont.*

#### **6. Conclusions**

The combination of natural/synthetic polymers and new biofabrication techniques, such as microfluidics, offers promising approach for tissue engineering scaffolds. Polymers and microfluidics enable rapid prototyping, reliability, as well as easy and low-cost manufacturing in research laboratories and for commercialization. Currently, there are many polymer-based drug delivery systems approved by FDA that are available on the market. Further, for commercial mass production, thermoplastics are used to develop standard microfluidic devices. However, despite scientific progress in biofabrication technologies, we are still in the early stages of the development of microfluidic technology for tissue engineering applications. There are serious obstacles to be overcome in producing a functional, complex, and large-scale system.

**Author Contributions:** Conceptualization, L.A.D., M.E.-Y. and S.D.; writing—original draft preparation, L.A.D., M.E.-Y. and S.D.; review and editing, S.A.D., R.K. and F.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Article* **The Effect of Dual-Modification by Heat-Moisture Treatment and Octenylsuccinylation on Physicochemical and Pasting Properties of Arrowroot Starch**

**Herlina Marta 1,\*, Ari Rismawati 1, Giffary Pramafisi Soeherman 2, Yana Cahyana 1, Mohamad Djali 1, Tri Yuliana <sup>1</sup> and Dewi Sondari <sup>3</sup>**


**Abstract:** Starch is widely applied in various industrial sectors, including the food industry. Starch is used as a thickener, stabilizer, or emulsifier. However, arrowroot starch generally has weaknesses, such as unstable under heating and acidic conditions, which are generally applied to processing in the food industry. Modifications were applied to improve the characteristics of native arrowroot starch. In this study, arrowroot starch was modified by heat-moisture treatment (HMT), octenylsuccinylation (OSA), and dual modification between OSA and HMT in a different sequence—-namely, HMT followed by OSA, and OSA followed by HMT. This study aims to determine the effect of different modification methods on the physicochemical and functional properties of native arrowroot starch. The result shows that both single HMT and dual modification caused damage to native starch granules, such as the formation of cracks and roughness. For single OSA treatment, especially, there is no significant change in granule morphology after modification. All modification treatments did not change the crystalline type of starch but reduced the RC of native starch. Both single HMT and dual modifications (HMT-OSA, OSA-HMT) increased pasting temperature and setback, but, conversely, decreased the peak and the breakdown viscosity of native starch, whereas single OSA had the opposite trend compared with the other modifications. HMT played a greater role in increasing the thermal stability and the retrogradation ability of arrowroot starch. Both single modifications (HMT and OSA) increased the hardness and gumminess of native starch, and the opposite was true for the dual modifications. HMT had a greater effect on color characteristics, where the lightness and whiteness index of native arrowroot starch decreased. Single OSA modification increased swelling volume higher than dual modification. Both single HMT and dual modifications increased water absorption capacity and decreased the oil absorption capacity of native arrowroot starch.

**Keywords:** arrowroot starch; heat-moisture treatment; octenylsuccinilation; dual-modification; physicochemical properties; functional properties

#### **1. Introduction**

Arrowroot (*Maranta arundinacea* L.) is a tuber plant with a rhizome root, which is elongated like an arrow. Arrowroot tubers have been considered inferior commodities and have not been utilized optimally. Arrowroot tuber is one of the commodity sources of carbohydrates, which can be processed into starch or flour. Starch can be utilized in many ways, such as a food thickener, stabilizer, and emulsifier [1]. Starches that are commonly found in the market are corn starch (maize) and cassava starch (tapioca). However, there

**Citation:** Marta, H.; Rismawati, A.; Soeherman, G.P.; Cahyana, Y.; Djali, M.; Yuliana, T.; Sondari, D. The Effect of Dual-Modification by Heat-Moisture Treatment and Octenylsuccinylation on Physicochemical and Pasting Properties of Arrowroot Starch. *Polymers* **2023**, *15*, 3215. https:// doi.org/10.3390/polym15153215

Academic Editor: Raffaella Striani

Received: 12 June 2023 Revised: 24 July 2023 Accepted: 25 July 2023 Published: 28 July 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

are still many potential plants that are potential sources of starch, and one of them is arrowroot tubers.

Native arrowroot starch, however, as with starches from other sources, has several limitations, such as low thermal stability and susceptibility to acidic conditions, which are generally used in processing in the food industry [2]. To improve the characteristics of native arrowroot starch, modifications were applied. One modification method that can be used to enhance thermal stability is the heat-moisture treatment (HMT) method [3,4]. However, several studies have shown that HMT increases starch syneresis [5,6]. Dual modification technology is an alternative to overcome the weakness of HMT starch by combining HMT modification treatment with other modification treatments, such as chemical modification. This study combined both modified treatments using heat-moisture treatment and octenylsuccinilation using octenyl-succinic anhydride (OSA). Modification of OSA has advantages because it can increase the hydrophobicity of starch [7,8]. Thus, OSA-modified starch can be applied as an emulsifier or used as a fat replacer in high-fat products, resulting in decreased fat products [8–10].

Information regarding the modification of arrowroot starch modified by two methods, HMT and OSA, is still limited. This current study aimed to determine the physicochemical and pasting properties of native and modified starches for both single and dual modifications using HMT and OSA in the reverse sequence. HMT and OSA-modified starch is expected to be an ingredient in the manufacture of a functional food because each single modification treatment has advantages from a health perspective. For example, HMTmodified starch has very slowly digested starch, making it suitable for diabetics [11], while single OSA modification can produce starch that can replace the role of fat and could thus be used to produce low-fat products [8].

#### **2. Materials and Methods**

#### *2.1. Materials*

Arrowroot tubers (*Maranta arundinacea* L.) cultivated in Pangandaran, West Java, Indonesia were used as the raw material for starch extraction. All chemicals used for the modification process were 2-octen-1-ylsuccinic anhydride (OSA) (Sigma-Aldrich, St. Louis, MO, USA), hydrochloric acid, sodium hydroxide, ethanol 95%, acetic acid, isopropanol, AgNO3, and phenolphthalein, with specification analytical grade.

#### *2.2. Arrowroot Starch Isolation*

Arrowroot starch isolation method was followed according to Marta et al. [11] with a slight modification. The tubers were peeled and cut into small pieces, and were then further soaked in water with a tuber/water ratio of 1:5. The tubers were crushed to form a pulp using a blender (Sharp EM-121-BK, Chiba, Japan) for 2 min. The resulting slurry was squeezed using a muslin cloth. The slurry was allowed to precipitate for 18 h and decanted to separate starch and supernatant. The resulting starch was washed and centrifugated using SL 16 Centrifuge, Thermo Scientific, Waltham, MA, USA at 5000 rpm for 2–3 min to precipitate the starch. Starch washing and centrifugation were repeated 3 times in order to obtain clean starch. The resulting starch was dried in a drying oven at 50 ◦C for 24 h, and was then dried and sieved using a 100-mesh sieve.

#### *2.3. Heat-Moisture Treated (HMT) Starch Preparation*

Preparation of HMT-starch refers to Marta et al. [12]. The moisture content of arrowroot starch was adjusted to 30% (±2%) by adding distilled water, and it was then equilibrated at 4 ◦C for 24 h in the refrigerator. The starch was then transferred to a tightly-closed Teflon and heated at 100 ◦C for 8 h. The modified starch was dried in a drying oven at 50 ◦C for 24 h, and was then ground and sieved using a 100-mesh sieve.

#### *2.4. Octenylsuccinilated (OSA) Starch Preparation*

Preparation of OSA-starch refers to Marta et al. [13] with a slight modification. The starch sample was first made into a 30% (*w*/*w*) starch suspension by dissolving the starch in distilled water. The pH of the starch suspension was then adjusted to pH 8 using 1 M NaOH before the addition of 3% OSA solution (*w*/*w*). The esterification was then carried out by stirring the solution for 3 h. During the modification process, the pH of the suspension was adjusted and maintained in the range of 8.0–8.5 using 1 M NaOH and 1 M HCl. After the reaction, the starch suspension was adjusted to pH 6.5 using 1 M HCl. Starch was centrifuged at 5000 rpm for 2–3 min. The precipitated starch was washed using distilled water and then centrifuged again, and this process was repeated 2–3 times. The starch was then dried in a drying oven at 50 ◦C for 24 h. The dried modified starch was ground and sieved (no. 100 mesh sieve).

#### *2.5. Dual Modification by HMT Followed by OSA (HMT-OSA)*

The native starch of arrowroot was modified by HMT (as mentioned in Section 2.3) and followed by OSA modification, as mentioned in Section 2.4, and the obtained starch was named HMT-OSA starch.

#### *2.6. Dual Modification by OSA Followed by HMT (OSA-HMT)*

The native starch of arrowroot was modified by OSA (as mentioned in Section 2.4) and then followed by HMT modification, as mentioned in Section 2.3, and the obtained starch was named OSA-HMT starch.

#### *2.7. Granule Morphology Observation Using Scanning Electron Microscopy (SEM)*

The granular morphology of native and modified arrowroot starch was determined using scanning electron microscopy (SEM), a JEOL JSM-6360 LA at 15 kV (JEOL, Tokyo, Janpan). Mounted starch samples were coated with gold/palladium at 8–10 mA for 10–15 min under low pressure (less than 10 tors). Representative digital images of native and modified starch granules were obtained at 5000 and 10,000 magnifications.

#### *2.8. Starch Crystallinity Using X-ray Diffractometer (XRD)*

The crystallinity of native and modified arrowroot starch was measured using an X'Pert PRO series PW3040/60 X-ray diffractometer (Malvern Panalytical, Malvern, UK) that operated using Cu-K alpha radiation with a wavelength of 1.540 nm as an X-ray source at 40 kV and 30 mA. The diffraction angle (2θ) scanning was from 3.0◦ to 50.0◦ with a scanning rate time of 2.9 s. OriginLab Program was used to calculate the relative crystallinity of the starch samples.

#### *2.9. Thermal Properties Determination Using Differential Scanning Calorimetry (DSC)*

Thermal properties of starch samples were measured using DSC-Q100, TA Instruments (New Castle, DE, USA). The parameters observed were onset temperature (To), peak temperature (Tp), conclusion temperature (Tc), and enthalpy of gelatinization (ΔH). Starch was made into a slurry, with the ratio of water and starch being 3:1. The slurry was then hermetically sealed using a DuPont encapsulation press before weighing. The starch samples were heated at a rate of 5 ◦C/min from 20 to 100 ◦C.

#### *2.10. Pasting Properties Determination Using Rapid Visco Analyzer (RVA)*

The pasting properties of arrowroot starch were determined using an RVA Starch-Master 2, Parten Instruments. A total of 3 g of starch samples were added with 25 mL of distilled water in the RVA canister tube, and stirred in an RVA canister at 960 rpm for 10 s. RVA was set with a temperature profile, initially held at 50 ◦C for 1 min; the heating was from 50 to 95 ◦C for 3.7 min; the temperature was held at 95 ◦C for 2.5 min; and cooling was then achieved at 50 ◦C in 3.8 min and then kept at 50 ◦C for 2 min. The gel was then maintained at 50 ◦C for 2 min with constant paddle rotational speed (160 rpm) used

throughout the analysis, and the total analysis time was 12 min. The pasting properties included the following parameters: pasting temperature (PT), peak viscosity (PV), hold viscosity (HV), final viscosity (FV), breakdown (BD), and setback (SB).

#### *2.11. Texture Properties Evaluation Using Texture Analyzer (TA)*

The texture properties of starch gels were evaluated on a TA-XT express enhanced Stable Micro System (Surrey, UK). Exponent Lite Express software was used to collect and calculate the data obtained. The gelatinized starch in the canister after the RVA measurement was poured into cylindrical plastic tubes (20 mm diameter, 40 mm deep) and then kept at 4 ◦C for 24 h to form a solid gel. Each gel sample in the tube was penetrated with a cylindrical probe (P36/R) at a speed of 5 mm/s to a distance of 10 mm for two penetration cycles. The texture profile curves were used to calculate hardness, adhesiveness, springiness, cohesiveness, and gumminess.

#### *2.12. Functional Properties*

Swelling volume and solubility of arrowroot starch were measured referring to Marta et al. [13]. A total of 0.35 g (db) of starch sample was mixed with 10 mL distilled water and put into a centrifuge tube. It was then mixed using a vortex mixer for 20 s, and the sample was then heated in a water bath at 92.5 ◦C for 30 min and stirred regularly. The starch sample was cooled for 1 min in ice water and centrifuged at 3500 rpm for 15 min, and then the supernatant was separated and the volume was measured. Swelling volume was calculated using the equation:

$$\text{Swelling volume} \,(\text{mL/g}) = \frac{\text{total volume} - \text{supermatant volume}}{\text{weight of sample (db)}} \tag{1}$$

After being separated, the supernatant was dried in a drying oven for 24 h. Solubility was calculated using the equation:

$$\text{Solubility } (\%) = \frac{\text{weight of dried segment}}{\text{weight of sample } (\text{db})} \times 100\% \tag{2}$$

Water absorption capacity (WAC) and oil absorption capacity (OAC) were measured referring to Marta et al. [13]. One gram (db) of the starch sample was added with 10 mL of distilled water or oil into a centrifuge tube, and then mixed using a vortex for 20 s. Samples were stored at room temperature for 1 h and then centrifuged at 3500 rpm for 30 min. The volume of the supernatant (water or oil) was measured and separated. WAC and OAC are calculated using the following equation:

$$\text{WAC (g/g)} = \frac{\text{volume of water absorbed}}{\text{weight sample (db)}} \tag{3}$$

$$\text{OAC} \left( \text{g/g} \right) = \frac{\text{volume of oil absorbed}}{\text{weight sample (db)}} \tag{4}$$

Freeze-thaw stability or syneresis was determined by a previous method [12] with a slight modification. An aqueous starch suspension (5%) was prepared and heated at 95 ◦C for 30 min with constant light stirring, then cooled to room temperature in an ice water bath. A total of 20 g of aliquots of the paste was then taken and put into a centrifuge tube, and a freeze-thaw cycle was then carried out with storage at 4 ◦C for 24 h, frozen at −15 ◦C for 48 h, and then thawed at 25 ◦C for 3 h. The samples were then centrifuged at 3500 rpm for 15 min. The supernatant was separated from the gel. The syneresis was calculated using the following equation:

$$\text{Symmetris} \left( \% \right) = \frac{\text{weight of separated water}}{\text{weight of starch paste}} \times 100\% \tag{5}$$

#### *2.13. Color Analysis Using CM-5 Spectrophotometer*

The starch color was measured using a CM-5 Spectrophotometer (Konica Minolta Co., Osaka, Japan) with SpectraMagic NX version 3.0 Software. The samples were placed in a glass cell and above the light source. After that, measurements were taken at room temperature. The parameters measured were CIE-lab namely, the L\*, a\*, and b\* values in each sample. L\* value indicates the lightness (whiteness/darkness), representing dark (0) to light (100); a\* value indicates the degree of red-green color ((+) redness/(−) greenness); and b\* value indicates the degree of the yellow-blue color ((+) yellowness/(−) blueness). The whiteness index for native and modified starches was determined using the following equation [14]:

$$\text{Whiteness Index} = 100 \, - \sqrt{(100 \, - \, \text{L}^\*)^2 + \text{a}^{\*2} + \text{b}^{\*2}} \tag{6}$$

Total color differences between starch samples were calculated using the following equation [15]:

$$
\Delta \mathbf{E} = \sqrt{\left(\mathbf{L}^\* - \mathbf{L}\_\mathbf{n}^\*\right)^2 + \left(\mathbf{a}^\* - \mathbf{a}\_\mathbf{n}^\*\right)^2 + \left(\mathbf{b}^\* - \mathbf{b}\_\mathbf{n}^\*\right)^2} \tag{7}
$$

L\*, a\*, and b\* were parameters for modified starch, and L∗ n, a<sup>∗</sup> n, and b<sup>∗</sup> <sup>n</sup> were parameters for native starch.

#### *2.14. Statistical Analysis*

Data are displayed as the mean ± SD. Experiments were all performed in triplicate. One-way analysis of variance (ANOVA) was used to analyze all of the data, and the Duncan test was used to compare the sample mean at a significance level of 5% (*p* < 0.05). The statistical software program IBM SPSS Statistics version 25 was used to examine all of the data.

#### **3. Results**

#### *3.1. Granule Morphology*

The granule morphology of native and modified arrowroot starches is presented in Figure 1. The granules of arrowroot starch are spherical and ellipsoid to oval-shaped. There was no damage on the surface of starch granules after OSA treatment. In HMTmodified starch, in both single and dual modifications (HMT, HMT-OSA, and OSA-HMT), the damage occurred on the surface of the granules, where the surface became rougher due to the formation of fine cracks.

**Figure 1.** Granule morphology of native and modified arrowroot starches with 5000× and 10,000× magnification. OSA = octenyl-succinic anhydride treatment; HMT = heat-moisture treatment; HMT-OSA = HMT followed by OSA treatment; OSA-HMT = OSA followed by HMT treatment.

#### *3.2. Starch Crystallinity*

The crystallinity properties of native and modified arrowroot starch are presented in Figure 2. According to the diffractograms, both native and modified arrowroot starch possessed an A-type crystalline pattern, which was indicated by several diffraction peaks at 15◦, 17◦, 18◦, 23◦, and 26◦ (2θ), showing that all of the treatments did not alter the crystalline pattern of the arrowroot starches. However, in terms of relative crystallinity (RC), the modified starches have lower RC compared to the native starch. The native arrowroot starch has an RC value of 30.96%, while OSA, HMT, HMT-OSA, and OSA-HMT starches have 30.25%, 28.26%, 24.88%, and 26.94% RC values, respectively. Additionally, HMT-modified starch in both single and dual modifications (HMT, HMT-OSA, and OSA-HMT) has a greater effect on RC than single OSA modification.

**Figure 2.** X-ray diffractograms of native and modified arrowroot starches. OSA = octenyl-succinic anhydride treatment; HMT = heat-moisture treatment; HMT-OSA = HMT followed by OSA treatment; OSA-HMT = OSA followed by HMT treatment.

#### *3.3. Thermal Properties*

DSC is used for thermal analysis in order to determine the transition of starch crystallinity caused by heating with the following parameters: (1) To, or temperature onset, is the temperature at which gelatinization begins, and is also defined as the melting temperature of the weakest crystals in starch granules; (2) Tp, or peak temperature, represents the the endothermic peak on the DSC thermogram; (3) Tc, or conclusion temperature, is the final temperature at which the sample is wholly gelatinized, and is sometimes known as the crystalline melting temperature (high-perfection crystalline); and (4) ΔH or enthalpy (J/g) is calculated based on the DSC endotherm, expressing the energy required to break the double helix structure during starch gelatinization [12,16].

The thermal properties of native and modified arrowroot starches are presented in Table 1. All modified starch has a lower To than native starch, except for HMT starch. Tp native starch decreased after modification from 50.46 ◦C to 39.83–44.36 ◦C. Both single methods (OSA and HMT) decreased Tc, but, conversely, both dual modification methods increased the Tc of native starch. The temperature range (Tc–To) of modified starches was higher than native starch, except for HMT-starch, whereas the enthalpy of modified starch decreased significantly from 1117.08 (J/g) to 936.44–647.30 (J/g).


**Table 1.** Thermal properties of native and modified arrowroot starches.

Means marked with different letters are significantly different (*p* < 0.05). OSA = octenyl-succinic anhydride treatment; HMT = heat-moisture treatment; HMT-OSA = HMT followed by OSA treatment; OSA-HMT = OSA followed by HMT treatment.

#### *3.4. Pasting Properties*

The viscoamylograph and pasting properties parameters of native and modified arrowroot starches are presented in Figure 3 and Table 2, respectively. Both single HMT and dual modifications (HMT-OSA, OSA-HMT) increased PT and SB of native starch, but, conversely, decreased PV and BD viscosity of native starch, whereas single OSA has the opposite trend compared with the other modification treatments.

**Figure 3.** Viscoamylograph of native and modified arrowroot starches. OSA = octenyl-succinic anhydride treatment; HMT = heat-moisture treatment; HMT-OSA = HMT followed by OSA treatment; OSA-HMT = OSA followed by HMT treatment.



Means marked with different letters are significantly different (*p* < 0.05). OSA = octenyl-succinic anhydride treatment; HMT = heat-moisture treatment; HMT-OSA = HMT followed by OSA treatment; OSA-HMT = OSA followed by HMT treatment.

#### *3.5. Texture Properties*

Texture properties parameters of native and modified arrowroot starches are presented in Table 3. Both single modifications, OSA and HMT alone, increased the gel hardness of native arrowroot starch, and the converse effect was seen for the dual modifications. All modified starches have higher adhesiveness and lower springiness and cohesiveness than native starch. Both dual modifications, HMT-OSA and OSA-HMT, decreased the gumminess of native arrowroot starch from 208.95 to 29.21–59.60.


**Table 3.** Texture profile of native and modified arrowroot starches.

Means marked with different letters are significantly different (*p* < 0.05). OSA = octenyl-succinic anhydride treatment; HMT = heat-moisture treatment; HMT-OSA = HMT followed by OSA treatment; OSA-HMT = OSA followed by HMT treatment.

#### *3.6. Functional Properties*

OSA modification, for both single OSA and dual HMT-OSA, significantly increased the SV of native starch, but we saw a converse trend for OSA-HMT. All modified starch has lower solubility and higher syneresis than native starch (Table 4). HMT starch, for both single HMT and dual modifications (HMT-OSA, OSA-HMT), increased the water absorption capacity (WAC) and decreased the oil absorption capacity (OAC) of native starch. All modification treatments increased the syneresis of native starch.


**Table 4.** Functional properties of native and modified arrowroot starches.

Means marked with different letters are significantly different (*p* < 0.05). OSA = octenyl-succinic anhydride treatment; HMT = heat-moisture treatment; HMT-OSA = HMT followed by OSA treatment; OSA-HMT = OSA followed by HMT treatment.

WAC describes the amount of water available for gelatinization [12]. WAC of native and modified arrowroot starches ranges from 0.83–1.60 g/g (db). The WAC of native arrowroot starch is 1.13 g/g db, which is smaller than in the previous study, where it was 1.81 g/g db [17]. Meanwhile, the ability of starch to absorb oil is called OAC, which could also represent the emulsifying properties of the starch [18]. The OAC of native and modified arrowroot starches range from 2.15–2.33 g/g db. All of the modification methods, except for OSA, decreased the OAC of native starch from 2.33 g/g db to 2.15–2.21 g/g db.

#### *3.7. Color Characteristics*

The color parameters of native and modified arrowroot starches are L\*, a\*, and b\*, whiteness index, and ΔE (Table 5). HMT modification of both single HMT and dual modifications (HMT-OSA and OSA-HMT) significantly decreased the L\* value of native arrowroot starch. Single OSA did not alter the a\* value, whereas HMT modification of both single and dual had a different effect on the a\* value of native arrowroot starch. Native and OSA-modified arrowroot starches have a negative value of a\*, which indicates that the

color tends towards greenness, whereas all HMT modifications have a positive value of a\*, which indicates that the color tends towards redness. All modified starch has a lower whiteness index than native starch, except for OSA starch. OSA starch has the highest whiteness index among all of the samples. ΔE is a value indicating the total color difference between a modified starch and the native starch. The ΔE of modified starch ranged from 0.64 to 1.61. The higher the ΔE, the greater the color differences between modified and native starches, whereas the HMT starch has the highest ΔE among other modified starches. The color images of native and modified arrowroot starches which were captured from Spectrophotometer CM-5 are presented in Figure 4.


**Table 5.** Color parameters of native and modified arrowroot starches.

Means marked with different letters are significantly different (*p* < 0.05). OSA = octenyl-succinic anhydride treatment; HMT = heat-moisture treatment; HMT-OSA = HMT followed by OSA treatment; OSA-HMT = OSA followed by HMT treatment.

**Figure 4.** The color of arrowroot starch (**a**) native, (**b**) OSA, (**c**) HMT, (**d**) HMT-OSA, and (**e**) OSA-HMT. The color images were captured from Spectrophotometer CM-5.

#### **4. Discussion**

#### *4.1. Granule Morphology*

Native arrowroot starch has a round, oval shape with a granular surface that is slightly textured without cracks, and this is in line with some of the previous studies [1,19]. The morphology of OSA starch granules did not show significant changes compared to native starch granules. This result was consistent with the study of OSA-modified Japonica rice starch [20] and OSA-modified sago starch [13], whereas the HMT caused damage to the starch granules, with cracks forming and the surface of the granules becoming rougher. Some of the previous studies also reported granule surface deformation after HMT [5,6,12], and this is due to the thermal strength, which changes the morphology of the arrowroot starch [11]. Both dual-modified starch granules (OSA-HMT and HMT-OSA) showed similar surface characteristics to the HMT starch. The surface of the granules became rougher, and there were also indentations. This indicated that thermal treatment significantly dominates the alteration of granule morphology of dual-modified arrowroot starch.

#### *4.2. Crystallinity*

X-ray Diffraction (XRD) is a method for assessing and quantifying long-range crystalline order in starch [21]. Figure 2 shows that native and modified starches have a similar crystallinity pattern, i.e., an A-type crystalline pattern, as indicated by several diffraction peaks at 2θ 15◦, 17◦, 18◦, 23◦, and 26◦ [22], which indicated that all of the modification treatments did not significantly affect the crystalline type of native arrowroot starch. Several studies have reported that native arrowroot starch has an A-type crystalline pattern [1,23]. However, this is not in agreement with another study by Nogueira et al. [24], which reported that arrowroot starch has a C-type crystalline pattern, indicating a mixture of polymorphs types A and B. The HMT showed no change in the crystalline pattern, similar to the study of Marta et al. [11] on banana starch, and a similar trend has been seen on OSA-sago starch [13].

Relative crystallinity (RC) was calculated based on the ratio of the diffraction peak area (crystalline area) to the total diffraction area [25]. Arrowroot native starch has an RC of 30.96%, which was in range with the other studies (52.84% [26] and 28.8–30.2%) [27]. All modification treatments, however, decreased the RC of native starch—-that is, from 30.96% for native arrowroot starch to 30.25% for OSA-treated starch, 28.26% for HMT-treated starch, 284.88% for HMT-OSA, and 26.94% for OSA-HMT. Several studies have reported that HMT decreased the RC, such as in sago starch [13], banana starch [11,13]; mango kernel starch [28], breadfruit starch [6], rice, cassava, and pinhão starches [29]. Decreased RC in hydrothermally modified starches, both for single HMT and dual modifications (HMT, HMT-OSA, and OSA-HMT), can be associated with changes in the crystalline phase (amylopectin) of starch, where dehydration and double helix movements can disrupt starch crystallinities and change the crystal orientation of the semi-crystalline fraction to the amorphous phase [30–32] or possibly partial gelatinization [4,33]. The RC of OSA starches, for both single and dual modified starches (OSA, OSA-HMT, and HMT-OSA), was lower than its native counterpart, which was in line with some of the previous studies [13,34,35]. These results indicated that OSA esterification occurs in the amorphous region of starch granules and slightly changes the starch crystal structure.

#### *4.3. Thermal Properties*

Esterification using OSA on arrowroot starch causes a decrease in To, Tp, and ΔH compared to native starch, which is in agreement with some of the previous studies [36,37]. The introduction of OSA molecules changes the degree of hydrogen bonding, which tends to weaken the interactions between the starch macromolecules, allowing the granules to swell and melt at lower temperatures [7,38], whereas heat-moisture treatment could weaken the interaction between amylose-amylose, amylose-amylopectin, and amylose-lipid, which leads to imperfect crystal formation. This phenomenon could cause a decrease of Tc and ΔH in HMT-treated starch [39]. Some previous studies have reported that HMT-modified starch showed higher gelatinization parameters (To, Tp, Tc) than native starch [12,30,40,41], whereas in this study, To and Tp of HMT starch were not significantly different to native starch. Both dual-modified starch HMT-OSA and OSA-HMT starches tend to have thermal characteristics that are almost similar, where the To, Tp, Tc, and Tc–To are not significantly different from each other, whereas ΔH of OSA-HMT starch is significantly higher than HMT-OSA starch.

#### *4.4. Pasting Properties*

The increase in PT and the decrease in PV on HMT starch, both for single HMT and dual modifications (HMT-OSA and OSA-HMT), could be due to the partial breakage of the ordered chain structure and the rearranging of the broken molecules, facilitated by the high temperature (100 ◦C) and limited moisture content (30%) during HMT. As a result, the forces of the intra-granular bonds would be augmented, and the linkages between starch chains would be strengthened. The HMT-treated starch samples needed greater heat for structural breakdown and paste production, resulting in a lower paste viscosity [4]. On the other hand, an increase in PV was observed in single OSA-treated starch. According to Bajaj et al. [8], substituting bulky octenyl groups could decrease the inter- and intramolecular bond between starch granules, resulting in limited incorporation of water into starch molecules. When OSA groups are substituted, the starch granule is destroyed, which enhances swelling and raises PV. The hydrophobic properties of OSA also increased the viscosity of starch. This characteristic was discovered advantageous for making mayonnaise with improved emulsion stability [8].

The significant difference in PV between HMT-OSA and OSA-HMT-treated starch is because HMT facilitated this OSA particle to attack more of the –OH group in starch. According to Park et al. [42], HMT before cross-linking could increase the phosphate content of the starch, showing that the HMT facilitates the incorporation of the cross-linking agent to react more with starch granules. This occurred in the octenyl group as well. The OSA modification increased the BD, which was in line with some of the previous studies [8,43], whereas the other modifications significantly decreased the BD of the native starches. The lower BD indicated the higher thermal stability of starch. All HMT modifications, both single and dual modifications, have a higher SB than native and single OSA starch, which indicates that HMT increases the ability of starch to retrograde.

From the industrial point of view, the modification of arrowroot starch could give arrowroot more value, as the starch could be used more in the food industry. HMT and dual-modified starches (OSA-HMT and HMT-OSA starcher) have a higher thermal stability than native starch, as shown by the lower breakdown viscosity of the starches. Starch with good thermal stability could be used as a thickening agent for food products that need to be sterilized, e.g., sauce, paste, etc. [39], whereas OSA starch has a lower ability to retrograde, which indicates that the starch may be used as an ingredient in baby food and baked goods (as it can inhibit staling on bread).

#### *4.5. Texture Properties*

Texture parameters in starch have an important role because starch can be a texturizer agent, such as a thickening and gelling agent. TPA (texture profile analysis) is used to observe texture parameters, such as hardness, adhesiveness, springiness, cohesiveness, and gumminess [44]. Gel hardness or hardness is related to the strength of the gel network, and changes in hardness are related to the effect of swelling granules and amylose content [44,45], and hardness increases with increasing amylose content. The hardness of HMT and OSA-modified starches were higher than native starch, whereas the amylose content of native starch was lower than both HMT and OSA starch. The amylose content for native, HMT, and OSA starches are 29.15%db, 36.01%db, and 33.44%db, respectively. The gel network structure depends on the quantity and intensity of hydrogen bonds formed between the amylose chains [46]. Conversely, the hardness of dual-modified starch (OSA-HMT and HMT-OSA starch) decreased very sharply compared to native starch, and it was not in line with the amylose content of the dual-modified starches, where the hardness decreased with increasing amylose content. The amylose content of HMT-OSA and OSA-HMT starches was 35.32%db and 36.36%db, respectively. This indicated that hardness is not only affected by amylose content. The adhesiveness of native starch was higher than all of the modified starches, which is in line with the other studies [47,48]. All modification treatments did not significantly affect the springiness of starch gel, but they decreased the cohesiveness, which may be due to the degradation of starch molecules, resulting in a weaker starch network structure [49]. In terms of gumminess, dual-modified starches tend to have lower values compared to both native and single-modified starch. The low level of elasticity in the dual-modified starch is influenced by its low hardness and low cohesive strength compared to single-modified starch (OSA and HMT).

#### *4.6. Functional Properties*

Swelling volume (SV) measures the hydration capacity of starch molecules due to the presence of water trapped in granules [50]. SV is related to amylose leaching or the solubility of starch [51]. Solubility indicates the amount of amylose leaching, which dissociates and diffuses out of the starch granules during the gelatinization process, resulting in starch swelling [10].

The SV of HMT starch decreased when compared to native starch, which is in line with other studies [2,12,52]. Furthermore, the decrease might be due to the rearrangement of starch molecules, increased intramolecular forces [52], and amylose-amylose, amylopectinamylose, and amylopectin-amylopectin interactions, which become stronger where the

starch granules become more rigid [11]. Among other modified starches, OSA starch has the highest SV. Furthermore, Park et al. [51] reported that the SV of OSA arrowroot starch was significantly higher than its native starch. This increase in swelling strength is associated with an increase in the ability of water to percolate into starch granules [53]. HMT-OSA-modified starch had a higher SV than OSA-HMT starch. It was presumed that the HMT process resulted in cracks and porous starch granules. When the OSA treatment was applied, the presence of succinate groups could weaken the internal bonds in the starch granules and increase the percolation of water so that the SV of HMT-OSA starch became higher [53,54].

All modified starches have lower solubility than native starch, and this is caused by amylose leaching during the starch gelatinization process. Amylose is in the crystalline region and is relatively small in size and linear shape, making it easier to leach out from the starch granules [25]. However, another study [24] reported that HMT on corn starch increased its solubility, which was influenced by the treatment time: the longer the treatment time, the more solubility [2].

Among the modified starches, OSA starch has the lowest WAC (0.83 g/g db). The reduced WAC on OSA starch was due to the presence of a hydrophobic substituent group from OSA that replaces the hydroxyl group on starch granules, which causes an increase in the hydrophobicity of starch [55], whereas HMT starch increased WAC (1.35 g/g db), which is in agreement with some of the previous studies [51,56]. The increased WAC of HMT starch was caused by the breaking of hydrogen bonds in the crystalline and amorphous regions, resulting in the expansion of the amorphous areas, which increased the hydrophilic properties of the starch [18,57]. The presence of pores on the surface of starch granules can also increase WAC because it will be easier for water to diffuse into the granules [12]. The increased WAC in dual-modified starch (HMT-OSA and OSA-HMT) was significantly influenced by the second modification treatment applied because HMT-OSA starch showed a lower WAC than OSA-HMT starch. OSA treatment after HMT was suspected to reduce starch hydrophilicity and increase its hydrophobicity due to the presence of OSA groups, which decreased WAC.

The esterification process with OSA increased starch's hydrophobicity, thereby increasing the OAC [7]. On the other hand, the OAC is related to the degree of substitution (DS), where the oil absorption capacity increases with the degree of substitution of OSA [58].

Freeze-thaw stability was determined as syneresis (%). Syneresis releases water from gel or starch paste during cooling, storage, and freeze-thawing [59]. High syneresis indicates low stability at low-temperature storage [12]. Native arrowroot starch has the lowest syneresis among other starches, and this is in agreement with some of the previous studies on arrowroot starch [19,60]. All modifications applied in this study tend to increase syneresis. However, when compared with modified starches, OSA starch has higher stability, which indicates that OSA starch is more stable at low-temperature storage conditions. A previous study reported that the modification of starch into succinate derivatives (OSA starch) can improve the freeze-thaw stability (FTS) of corn and amaranth starch [50]. This is associated with a steric effect on the OSA group, which can prevent starch chain alignment when stored at low temperatures [7], whereas in this study, OSA modification cannot improve the FTS of native arrowroot starch. Increased syneresis in HMT starch might be due to the resulting random interactions reducing the water-holding capacity of the starch gel [61]. The intensity of syneresis depends on various factors, such as the composition of the amylose fraction, the length of the amylopectin chains, and the degree of polymerization of amylose and amylopectin [57].

HMT and OSA-HMT decrease SV and SOL and increase the WAC of native starch, which indicates that HMT and OSA-HMT starches can be applied in pasta and noodle formulations. Marta et al. [62] have reported that HMT banana starch can be used as an ingredient in noodle production. All modified starches have higher sineresis, so they cannot be applied in frozen foods.

#### *4.7. Color Analysis*

Color is a characteristic that has an important role in determining the quality and the level of consumer acceptance in selecting a product. In flour or starch products, consumers generally prefer products that are white or bright in color. To determine the color of both native and modified arrowroot starch objectively, color analysis was performed. Color testing of products, mainly arrowroot starch, can be carried out using a stand-alone, topport color measuring instrument, which was the Spectrophotometer CM-5 in this research. The color of each starch sample was then interpreted by referring to the CIELAB systems through the L\*. a\*, and b\* values [63].

The highest lightness (L\*) was shown by OSA starch (96.39), while HMT starch showed the lowest lightness (94.96). There was a significant difference in lightness, especially for HMT-modified starch. This is presumably because thermal treatment can cause the degradation of color pigments in starch. The redness (a\*) in arrowroot starch with a negative value is indicated by both native and OSA starches, which indicated a tendency to be green in color [64]. Meanwhile, the positive values for HMT starches, both single HMT and dual modified starches (HMT-OSA and OSA-HMT starches), indicated that these starches tended to be red. The color shift to red was caused by the application of thermal treatment. The level of yellowness (b\*) in all arrowroot starch (native and modified) has a positive value, indicating that all starch leads to a yellow color. The highest b\* value was shown by HMT starch (4.07), while the lowest was indicated by OSA starch (2.13). This means that HMT starch is more yellow than OSA starch (Figure 3).

The whiteness index (WI) is based on a scale of 0–100, with the highest value described as the highest level of lightness. The WI value is positively correlated with the L\* value; the higher the WI, the higher the L\* value. HMT-modified starch, for both the single HMT and dual-modified starches, had low WI. The HMT modification process that was carried out reduced the lightness level of starch. Total color difference (ΔE) is a parameter used to assess how much change or difference can be seen in the Lab\* value results in ingredients or food after specific treatments [65]. Dual modification, especially OSA-HMT starch, has the smallest ΔE value, which indicates that the color produced between native and OSA-HMT starch is not much different, whereas HMT starch showed the highest ΔE value, in which there was a significant difference/change in starch color compared to native starch.

#### **5. Conclusions**

All modification treatments, both for the single and dual modifications, had significant effects on granule morphology, crystallinity, thermal, pasting, and functional properties, texture, and color characteristics of native arrowroot starch. The granules of arrowroot starch are spherical and ellipsoid to oval shaped. There was no damage on the surface of the starch granules after OSA. In HMT-modified starch, both for single and dual modifications (HMT, HMT-OSA, and OSA-HMT), the damage occurred on the surface of the granules. Native and modified starches have a similar crystallinity pattern, which was an A-type crystalline pattern. All modification treatments decreased the RC of native starch, where HMT has a greater effect on RC than OSA. Both single HMT and dual modification (HMT-OSA, OSA-HMT) increased PT and SB, and, conversely, decreased PV and BD viscosity of native starch. All HMT treatments, both for single and dual modifications, can improve the thermal stability of native starch, especially the HMT single treatment, whereas OSA single treatment has the opposite trend compared with the other modification treatments. OSA starch has a firm texture characteristic, which is indicated by the high value of hardness and gumminess. Both single OSA treatment and HMT-OSA significantly increased the SV of native starch, but, conversely, trend for the OSA-HMT. All modified starches have lower solubility and higher syneresis than native starch. Both single HMT modification and dual modifications (HMT-OSA, OSA-HMT) increase WAC and decrease the OAC of native starch. HMT starch for both single and dual modifications have low L\* and WI values, while OSA starch has the brightest color among other starches. Based on pasting and functional properties, both HMT and OSA-HMT starch can be applied in pasta and

noodle formulation and also as a thickening agent, whereas OSA starch can be used as an ingredient in baby food and bakeries because it has a low ability to retrograde.

**Author Contributions:** Conceptualization, H.M. and M.D.; methodology, H.M. and D.S.; software, A.R.; validation, H.M, Y.C. and M.D.; formal analysis, A.R. and G.P.S.; investigation, H.M. and T.Y.; data curation, D.S., A.R. and G.P.S.; writing—original draft preparation, H.M., A.R. and G.P.S.; writing—review and editing, Y.C., M.D. and T.Y.; visualization, A.R. and G.P.S.; supervision, H.M., Y.C. and M.D.; funding acquisition, H.M. and Y.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Grant of Ministry of Education, Culture, Research, and Technology (MoECRT), Republic of Indonesia [grant number: 2393/UN6.3.1/PT.00/2022] and The APC was funded by Universitas Padjadjaran.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors acknowledge the facilities, scientific and technical support from Advanced Characterization Laboratories Cibinong—Integrated Laboratory of Bioproduct, National Research and Innovation Agency through E-Layanan Sains, Badan Riset dan Inovasi Nasional.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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## *Article* **Toward a Circular Bioeconomy: Development of Pineapple Stem Starch Composite as a Plastic-Sheet Substitute for Single-Use Applications**

**Chanaporn Thongphang 1, Atitiya Namphonsane 1, Sombat Thanawan 2, Chin Hua Chia 3, Rungtiwa Wongsagonsup 4,5, Siwaporn Meejoo Smith <sup>1</sup> and Taweechai Amornsakchai 1,\***


**Abstract:** Plastic waste poses a significant challenge for the environment, particularly smaller plastic products that are often difficult to recycle or collect. In this study, we developed a fully biodegradable composite material from pineapple field waste that is suitable for small-sized plastic products that are difficult to recycle, such as bread clips. We utilized starch from waste pineapple stems, which is high in amylose content, as the matrix, and added glycerol and calcium carbonate as the plasticizer and filler, respectively, to improve the material's moldability and hardness. We varied the amounts of glycerol (20–50% by weight) and calcium carbonate (0–30 wt.%) to produce composite samples with a wide range of mechanical properties. The tensile moduli were in the range of 45–1100 MPa, with tensile strengths of 2–17 MPa and an elongation at break of 10–50%. The resulting materials exhibited good water resistance and had lower water absorption (~30–60%) than other types of starch-based materials. Soil burial tests showed that the material completely disintegrated into particles smaller than 1 mm within 14 days. We also created a bread clip prototype to test the material's ability to hold a filled bag tightly. The obtained results demonstrate the potential of using pineapple stem starch as a sustainable alternative to petroleum-based and biobased synthetic materials in small-sized plastic products while promoting a circular bioeconomy.

**Keywords:** biodegradable plastic; starch; circular economy; pineapple; tensile strength

#### **1. Introduction**

In recent years, there has been growing concern about the environmental impact of plastics, which are known to persist in the environment and harm living organisms [1]. To address this issue, a variety of solutions have been proposed, including the use of biodegradable polymers in single-use applications and outright bans on plastic use in some countries [2]. Biodegradable polymers are available in a variety of forms, including fully biobased polylactic acid (PLA), partially biobased polybutylene succinate (PBS), fully synthetic polybutylene adipate terephthalate (PBAT), and natural polymers such as starch, polyhydroxyalkanoates (PHAs), and polyhydroxybutyrate (PHB). However, not all biodegradable polymers are created equal, and it is important to understand the specific properties and limitations of each type. For example, polylactic acid (PLA), polybutylene

**Citation:** Thongphang, C.; Namphonsane, A.; Thanawan, S.; Chia, C.H.; Wongsagonsup, R.; Smith, S.M.; Amornsakchai, T. Toward a Circular Bioeconomy: Development of Pineapple Stem Starch Composite as a Plastic-Sheet Substitute for Single-Use Applications. *Polymers* **2023**, *15*, 2388. https://doi.org/ 10.3390/polym15102388

Academic Editor: Raffaella Striani

Received: 26 April 2023 Revised: 15 May 2023 Accepted: 17 May 2023 Published: 19 May 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

adipate terephthalate (PBAT), and polybutylene succinate (PBS) are widely used biodegradable polymers, but they do not easily biodegrade in natural environments. Instead, they require specific conditions, such as controlled humidity and temperature, which are typically only found in industrial composting facilities [3,4]. Therefore, it is still crucial to ensure that these biodegradable materials are properly disposed of and collected in such facilities to ensure their complete degradation. On the other hand, starch-based materials, polyhydroxyalkanoates (PHAs), and polyhydroxybutyrates (PHBs) are fully biodegradable in natural environments and may be a more appropriate choice for certain applications where collection and recycling are not easy or economical, such as small and lightweight objects or products. These materials can biodegrade completely without requiring specialized industrial composting facilities and may, therefore, offer a more practical and environmentally friendly solution for certain types of waste.

Considering the availability, cost of production, and other environmental impacts, starch may be the material of choice to make bioplastics and, indeed, there are many reviews available [5–7]. However, unmodified starch has poor properties, especially low mechanical strength and poor water resistance. Thus, it requires modification or blending with other polymers to make it more useful [8–11]. Unfortunately, most starches are used for human consumption. Using them for materials would certainly interrupt food supply chains and limit access to food for vulnerable groups, making it unsustainable. Researchers have investigated various nonconventional or nonfood starches for use in material applications [12–18]. However, the availability of these alternative starch sources is relatively limited. As a result, much of the research on starch-based materials has focused on modifying traditional food-grade starch to improve its properties. One of the main challenges of using starch as a material is its poor water resistance and low mechanical strength. To address these limitations, researchers have developed a range of modification methods. Initially, modification was achieved through a single method, but the resulting products still had limitations. Over time, researchers developed more advanced modification techniques, such as dual modifications [19–22] or combining starch with various fillers [23–25]. This not only adds complexity to the process but also produces products with higher intensity in terms of material and energy, leading to a higher carbon footprint. Therefore, finding starch that does not require modification or requires very little modification would make it more sustainable.

Recently, our group started investigating pineapple stem starch (PSS) as a promising material for a variety of applications. This is because the PSS has a relatively high amylose content and can be obtained from pineapple field waste, which is abundantly available in Thailand [26–28]. We reported that the PSS film exhibits good properties, such as high water resistance, low water absorption, and good mechanical strength, while still being readily biodegradable [29]. As a result, the PSS film has been proposed for use in single-use or disposable applications.

This study aims to extend the potential applications of PSS by developing a biodegradable composite suitable for single-use purposes. The high amylose content of PSS is expected to confer good water-resistant properties to the composite. The matrix material used for the composite was unmodified and raw PSS. The mechanical properties of the material were enhanced by adding glycerol and calcium carbonate as modifiers. By exploring the range of mechanical properties achievable through these modifications, we can identify suitable applications for our composite material. This knowledge is critical for developing sustainable alternatives to conventional plastics and reducing the environmental impact of single-use products.

Glycerol was chosen as the primary plasticizer in this study due to its widespread use and effectiveness in previous research [5,30,31] and its availability as a byproduct from the biodiesel industry [32–34]. Similarly, calcium carbonate was chosen as the primary filler due to its wide use in plastic manufacturing and its availability from natural sources, such as the Earth's crust [35,36], or byproducts from food industries, such as eggshells and seashells [37,38]. Notably, the use of PSS, a nonconventional food-grade starch, in

this research ensures that it does not pose a threat to food security. This approach not only makes use of waste or byproducts but also supports the principles of a circular economy and sustainability. The use of renewable resources such as PSS, glycerol, and calcium carbonate in the development of biodegradable composites can significantly reduce reliance on nonrenewable resources and mitigate the environmental impact associated with conventional plastics.

#### **2. Materials and Methods**

#### *2.1. Raw Material and Chemicals*

Pineapple stem waste, a byproduct of a proprietary bromelain extraction process, was obtained from Hong Mao Biochemicals Co., Ltd., (Rayong, Thailand). In general, the process involves crushing peeled pineapple stems to disrupt the cell structure and extract liquid by centrifugation [28]. The remaining solid material was dried under the sun for a few days and further ground into a powder using a grinder. The stem powder was collected by sieving (80 mesh) to separate the coarse fibers, cell wall, and other solid contaminants, which constitute about 56% of the whole mass. The powder was used as obtained without further washing. The extractive constituents make up about 15% of the dry powder. The characteristics of the powder are similar to that obtained by the wet milling process reported previously [27]. Commercial-grade glycerol was obtained from local stores and the uncoated-grade calcium carbonate was produced by Surint Omya Chemicals (Kok Toom, Thailand) Co., Ltd., (Lopburi, Thailand).

#### *2.2. Preparation of Starch Paste and Composites*

A starch paste was prepared by mixing a predetermined amount of PSS, water, and glycerol in a glass beaker. The amount of glycerol was varied at 20, 30, 40, and 50% based on the weight of the PSS. For all formulations, the amount of water was fixed at the same weight as PSS. The mixture was left for at least 60 min at ambient condition before being gelatinized in a household microwave (Toshiba, model ER-G33SC(S), Toshiba Thailand Co., Ltd., Nonthaburi, Thailand) set at 50% of maximum power (1100 W) for 90 s. The gelatinized PSS was left to cool down to room temperature. A predetermined amount of calcium carbonate (0, 20, and 30% wt. of PSS) was then added into the paste on a laboratory 2-roll mill until a homogeneous mixture was obtained. The mixing time was approximately 15 min. After mixing, the mixture was sheeted out to a thickness of approximately 1 mm. The sheet was then dried in a hot-air oven at 80 ◦C for 6 h. The samples were then left in an ambient environment to gain equilibrium moisture content at least 7 days before any measurements were made. The sample code is represented as GXCaY, where X and Y are the amounts of glycerol and calcium carbonate, respectively.

#### *2.3. Characterization of PSS Composites*

#### 2.3.1. Fourier-Transform Infrared Spectroscopy (FTIR)

The FTIR spectra of the materials were recorded with a spectrophotometer (Frontier, Perkin Elmer, Waltham, MA, USA) in the attenuated total reflection (ATR) mode using a diamond crystal. The measurements were performed at room temperature over a range of 4000 to 400 cm−<sup>1</sup> with 16 scans and a resolution of 4 cm<sup>−</sup>1.

#### 2.3.2. X-ray Diffraction (XRD)

The X-ray diffraction patterns of the materials were obtained from a benchtop X-ray powder diffractometer (D2 Phaser, Bruker AXS GmbH, Karlsruhe, Germany) using an X-ray wavelength of 1.54 Å with a step scan of 15 s/point over the 2θ of 5–40 degrees. The percentage of crystallinity of each PSS composite sample was determined using the following equation:

$$\text{Crystallinity (\%)}=A\_{\text{c}}/(A\_{\text{c}}+A\_{\text{a}}) \times 100\tag{1}$$

where *A*c = the area of crystalline region and *A*a = the area of amorphous region. The peaks belonging to calcium carbonate were excluded from the calculation.

#### 2.3.3. Mechanical Properties

Tensile test: Specimens were punched out from a 1 mm sheet with a cutter (ISO 37 type 2 dumbbell die). Tensile tests were carried out on a universal testing machine (Instron 5569, Instron, High Wycombe, UK) according to ISO 527-3 with a long-travel, contact-type extensometer. A crosshead speed of 50 mm/min was used. The secant modulus at 1% and the tensile strength and elongation at break were obtained as average values from five specimens.

Hardness and density: The hardness of the material was determined according to the durometer method or Shore hardness of ISO 7619-1 (Zwick Model 7206.07, Zwick, Ulm, Germany). The density of the material was determined following Archimedes' principle with a density kit on a laboratory balance (XS105, Mettler Toledo, Greifensee, Switzerland) according to method A of ISO 1183. The specimen was weighed in air and then weighed when immersed in distilled water using a sinker and wire to hold the specimen completely submerged. The measurement was carried out at 25 ◦C and the density was calculated using the below equation. The water density for the calculation was set at 1.00 g/cm3.

$$\text{Density} = \mathcal{W}\_{\text{air}} / (\mathcal{W}\_{\text{air}} - \mathcal{W}\_{\text{water}}) \tag{2}$$

where

*W*air = weight of the sample in air. *W*water = weight of the sample in water.

#### 2.3.4. Morphology

The fractured surfaces of the specimens obtained from the tensile tests were observed with a scanning electron microscope (SEM) (JSM-IT500, JEOL, Tokyo, Japan). The samples were coated with platinum before the observation.

#### 2.3.5. Water Solubility and Absorption

A piece of specimen was immersed in distilled water for 24 h and its weights (wet and dried) were monitored. The amount of water absorbed was determined following ISO 62 at 25 ◦C. The water resistance was determined qualitatively by observing the uptake of water by the sheet samples. The water solubility and absorption of the sheets were determined from the following equations:

$$\text{Water solubility} = ((w\_{\text{i}} - w\_{\text{fd}})/w\_{\text{i}}) \times 100\tag{3}$$

$$\text{Water absorption} = ((w\_{\text{f}} - w\_{\text{i}})/w\_{\text{i}}) \times 100\tag{4}$$

where

*w*fd is the weight of the dried PSS sheet after being immersed in distilled water. *w*<sup>f</sup> is the weight of the wet PSS sheet after being immersed in distilled water. *w*<sup>i</sup> is the initial weight of the PSS sheet.

#### 2.3.6. Soil Burial Test

This test can be used to determine the biodegradability of starch-based materials by microorganisms [39]. The test was slightly modified from a protocol reported previously [39]. Specimens of size 4.0 × 4.0 cm<sup>2</sup> were cut and put in envelopes made from a high-density polyethylene net for easy recovery. The envelopes were buried at the edge of a garden of the department building, about 10 cm beneath the surface. The pH of the soil was measured to be 7.5. The area was under the shade of trees and was watered every week. No attempt was made to regulate the moisture content and temperature of the area to obtain a natural environment. The envelopes were taken out for the observation of samples after different periods of time. The state of biodegradation was evaluated visually.

#### *2.4. Statistical Analysis*

Statistical analysis was performed using analysis of variance (ANOVA) with the Data Analysis tool in the Microsoft Excel (Office16) program. The *t*-test method, with two-sample assuming unequal variances, was performed to analyze differences among the means at a confidence level of 95%.

#### **3. Results and Discussion**

#### *3.1. Fourier-Transform Infrared Spectroscopy (FTIR)*

Figure 1 displays the FTIR spectra of the PSS composites containing different amounts of glycerol and calcium carbonate. In the controlled system without calcium carbonate, the amount of glycerol did not significantly affect the FTIR spectra of the composites. The FTIR spectra of the PSS with only glycerol were very similar to that of other starches that have been well understood and documented [40,41]. The peak positions and their corresponding functional group vibrations are listed in Table 1. When calcium carbonate was added, obvious changes were noticed at two positions (shaded area in Figure 1) belonging to calcium carbonate, i.e., the asymmetric stretching peak at 1416 cm−<sup>1</sup> and out-of-plane bending at 876 cm−<sup>1</sup> [42]. The intensity of these peaks increased on increasing the amount of calcium carbonate.

**Figure 1.** FTIR spectra of PSS composites containing different amounts of glycerol and calcium carbonate: (**a**) no calcium carbonate, (**b**) 20% calcium carbonate, and (**c**) 30% calcium carbonate.


**Table 1.** FTIR peak positions and corresponding functional group vibrations.

#### *3.2. X-ray Diffraction (XRD)*

Figure 2 displays the XRD patterns of the PSS composites containing different amounts of glycerol and calcium carbonate. All PSS composites have a certain degree of crystallinity.

The crystalline structure is different from that of the original PSS, which has an A-type structure, similar to other types of starches as shown previously [27]. The appearance is similar to that of PSS film prepared by solution casting [29] and that of ozonated cassava starch films [11]. These crystalline peaks are attributed to the spontaneous recrystallization of amylose molecules during film drying [43,44] or retrogradation. It was stated that retrograded starch is always B-type regardless of the starch type [11,45]. The crystallinity index decreased on increasing the amount of glycerol added. In systems containing calcium carbonate, five new peaks appeared, which were characteristics of calcium carbonate. The patterns were simply a combination of the PSS matrix and calcium carbonate. The diffraction intensity of PSS dropped significantly but still displayed crystallinity. The drop in intensity is due to the lesser amount of PSS within the measurement volume of XRD. Again, for each set of calcium carbonate contents, the crystallinity index decreased on increasing the amount of glycerol. It is worth noting that most reported thermoplastic starches do not exhibit such a distinct crystalline structure as is observed here [20,25,46]. This is presumably due to the high amylose content of PSS as it is the component that undergoes rapid reordering to form double helices and crystallites [47,48].

**Figure 2.** X-ray diffraction patterns of PSS composites containing different amounts of glycerol and calcium carbonate: (**a**) no calcium carbonate, (**b**) 20% calcium carbonate, and (**c**) 30% calcium carbonate. The number on each pattern indicates the crystallinity of the starch matrix. \* indicate calcium carbonate diffraction peaks.

#### *3.3. Mechanical Properties*

Figure 3 displays the stress–strain curves of the PSS composites containing different amounts of glycerol and calcium carbonate. For the system without calcium carbonate (Ca0), that with 20% glycerol displayed a sharp rise in stress as it was extended, and the stress reached a maximum point and then dropped slightly and broke at about 5% strain. When the glycerol content increased to 30, 40, and 50%, the stress dropped significantly, but the material could still be extended to a strain of about 50%. The maximum stress decreased with increasing glycerol content. When 20% calcium carbonate was added, the stress dropped from that without calcium carbonate but still broke at a strain of about 5%. On increasing the glycerol content to 30% and beyond, a similar pattern was observed, i.e., the stress dropped sharply and further decreased with increasing glycerol content and failure strains increased to about 40–50%. For the last set with 30% calcium carbonate, a very different pattern of behavior was seen. The composite with a glycerol content of 20% displayed a sharp rise in stress and then leveled off and failed at much a greater strain

of about 25%. On increasing the glycerol content to 30, 40, and 50%, the stress dropped in steps while the failure strain also increased in steps, reaching a value of about 45%. Average values for the moduli, tensile strength, and elongation at break are shown in Figure 4. The range of moduli obtained was about 45–1120 MPa, and the tensile strength was about 3.0–17.4 MPa. It should be noted that the modulus and tensile strength of the PSS sheet with glycerol content of 20 wt.% were much greater than that of other starches with similar glycerol content, which were about 95.0–529.0 MPa and 5.7–12.0 MPa, respectively [49,50].

**Figure 3.** Stress–strain curves of PSS composites containing different amounts of glycerol and calcium carbonate: (**a**) no calcium carbonate, (**b**) 20% calcium carbonate, and (**c**) 30% calcium carbonate.

Figure 5 displays the hardness of the PSS composites containing different amounts of glycerol and calcium carbonate. At the lowest glycerol content of 20%, the hardness of the composite was about 90–95 Shore A, and the hardness decreased with increasing glycerol content. The lowest hardness obtained was about 63 Shore A for PSS with 50% glycerol without calcium carbonate. For each glycerol content, the hardness increased with increasing calcium carbonate content.

**Figure 4.** Modulus, tensile strength, and elongation at break of PSS composites containing different amounts of glycerol and calcium carbonate. Different letters on each bar indicate statistically significant differences in the means.

The densities of the PSS composites containing different amounts of glycerol and calcium carbonate are shown in Figure 5b. A trend similar to that for hardness was observed here. For each set of calcium carbonate contents, the density decreased with

increasing glycerol content, and for each glycerol content, the density increased with increasing calcium carbonate. These results are to be expected as calcium carbonate has a density of 2.65 g/cm<sup>3</sup> [35] while that of thermoplastic starch with 35 wt.% glycerol is about 1.4 g/cm3 [51]. By assuming these values, the observed densities of the PSS composite sheets fit well with the calculated values.

**Figure 5.** Hardness (**a**) and density (**b**) of PSS composites containing different amounts of glycerol and calcium carbonate. Different letters on each bar indicate statistically significant differences in the means.

#### *3.4. Morphology*

Figures 6–8 display the scanning electron micrographs of tensile-fractured specimens of G20Ca0, G30Ca0, G40Ca0, and G50Ca0. It is apparent that all specimens contained numerous voids. Presumably these voids occurred because of syneresis process in which water is expelled from the starch network due to retrogradation [52] and then evaporates away in the drying stage. The presence of voids agrees well with the decrease in density with increasing glycerol content observed in Figure 5b. This observation can further support the decrease in tensile modulus and tensile strength of the films with increasing glycerol content. For specimens without calcium carbonate (Figure 6), the fracture surface displayed a very rough morphology. It seems that as the amount of glycerol increases, the size of the voids increases. With calcium carbonate added, the fracture surface showed brittle behavior and no other feature was seen.

**Figure 6.** SEM micrographs of tensile-fractured surface of (**a**) G20Ca0, (**b**) G30Ca0, (**c**) G40Ca0, and (**d**) G50Ca0 specimens (scale bar = 100 μm).

**Figure 7.** SEM micrographs of the tensile-fractured surface of (**a**) G20Ca20, (**b**) G30Ca20, (**c**) G40Ca20, (**d**) G50Ca20 specimens (scale bar = 100 μm).

**Figure 8.** SEM micrographs of the tensile-fractured surface of (**a**) G20Ca30, (**b**) G30Ca30, (**c**) G40Ca30, (**d**) G50Ca30 (scale bar = 100 μm).

#### *3.5. Water Solubility and Absorption*

Figure 9 displays the water solubility of different PSS composite sheets. Solubility increases with increasing glycerol content indicating more material is leached out. Since both starch and glycerol are water soluble, it follows that the leached material could be both. With the addition of calcium carbonate, the water solubility decreased but still increased with increasing glycerol content. This is to be expected as calcium carbonate is not water soluble. Considering that glycerol molecules are small and readily soluble in water and the water solubility is close to but less than the amount of glycerol added, it is likely that some glycerol could still be trapped inside the composites.

**Figure 9.** Water solubility of PSS composites containing different amounts of glycerol and calcium carbonate.

Figure 10 displays the water absorption of the PSS composite sheets. For all composites, it is seen that the water absorption increased with increasing immersion time and then leveled off after a certain period of time. The points where the water absorption starts to level off seem to change with the calcium carbonate content, i.e., it increased with increasing calcium carbonate content. In addition, the maximum water absorption for each set of calcium carbonate content depends on the glycerol content. For specimens without calcium carbonate, maximum water absorption decreased with increasing glycerol content. These data should not be treated as evidence for actual lower water absorption since glycerol leaching did occur as will be shown in the next section, and more discussion will follow.

**Figure 10.** Water absorption of PSS composites containing different amounts of glycerol and calcium carbonate of 0 (**a**), 20 (**b**) and 30 (**c**) wt.%.

For composites with calcium carbonate, Figure 10b,c, the maximum water absorption decreased, and similar trend was seen that maximum water absorption decreased with increasing glycerol content, but the change was not regularly spaced as in the system without calcium carbonate. This could suggest complex interactions within the structure of the composites. To clearly understand the behavior, further work would be needed, and this will be reported in future correspondence.

#### *3.6. Soil Burial Test*

Figure 11 displays photographs of some selected composite samples before and after the burial test. All composite samples clearly deteriorated in the burial test but at different rates. It appears that, after 7 days, the composites became moldy but still maintained their original shape. After 15 days, composites with little or no CaCO3 were broken into small pieces, while those with a higher content largely retained their shape. After 30 days, all composites completely disintegrated, and nothing could be recovered.

**Figure 11.** Photographs of some PSS composite specimens before and after burial in the soil for different periods of time.

#### **4. Discussion and Potential Applications**

It has been shown that composites with a wide range of properties can be prepared from pineapple stem waste, an abundant agricultural waste in Thailand and many other countries. A wide range of properties were obtained from the inherent property of PSS, which has a high amylose content, due to its ability to accept a large amount of plasticizer and filler. The material is completely biodegradable in a natural environment. Thus, properties can be adjusted or tailored to suit different applications. It can be used to replace synthetic plastics in applications where collection for recycling is difficult or not economical and the plastic is likely to leak into the environment, such as bread clips, cotton buds, or golf tees. Compostable versions of these types of products are being offered, such as cardboard bread clips [53]. To demonstrate potential applications of the PSS composite sheet, simple bread clips were cut from a sheet using a manual punching tool, and their photographs are shown in Figure 12. The bread clip was chosen as an example as its function is just to carry

some information (related to the product in the packaging) and allow re-closure of a bag with very little stress on the clip. Some composite formulations were found to be too hard and broke during punching, while some gave a good cut. The clips were found to be able to close a sample plastic bag nicely as shown in Figure 12. The clip in the figure was cut from G40Ca0 (without calcium carbonate).

**Figure 12.** Photographs of simple bread clips made from PSS composites compared with a commercial clip (**left**) and its use for closing a plastic bag (**right**).

In summary, our study demonstrated the potential of using pineapple stem starch, glycerol, and calcium carbonate for the production of biodegradable composites. The utilization of waste, byproducts, and renewable sources for these materials offers numerous advantages, including a reduction in the energy required for raw material production, lower carbon dioxide emissions, and a reduced need for land and water resources, compared to the use of edible starch. Furthermore, the use of pineapple stem starch, which is not conventionally used as food, does not pose a threat to food security, and edible starches can be reserved for food production. We have also shown that a range of composite formulations can be achieved with varying mechanical and water-resistant properties, making them suitable for various applications. For instance, our proposed application of using the composite as bread clips is a practical example of how it can be used to replace single-use plastics in everyday items that are too small for people to collect for recycling. Moreover, the observed range of properties could serve as a starting point for future research, where other sustainable materials can be incorporated to obtain specific properties tailored to various applications. Overall, our findings hold great potential for advancing sustainable materials and circular economy, reducing plastic waste, and mitigating the environmental impact of plastic production and disposal. With the increasing concern over climate change and resource depletion, our study offers a promising solution toward a more sustainable future.

#### **5. Conclusions**

Biodegradable plastic sheets with a wide range of mechanical properties were successfully developed from PSS. The high amylose content allows a sufficient degree of crystallinity to impart a good starting point. The use of simple chemicals, such as glycerol as plasticizer and calcium carbonate as reinforcement, provides an opportunity to alter the mechanical properties to suit various applications. The material is specifically useful for applications where strength is not so critical and collection back for recycling is difficult. Since it is starch-based, the material is readily biodegradable within a short period of time and should leave no microplastics and other contaminants behind. In addition, the rate at which the composite deteriorates can be controlled via the filler or other additive contents. **Author Contributions:** Conceptualization, T.A. and C.H.C.; methodology, C.T., A.N. and T.A.; validation, and data curation, C.T. and A.N.; writing—original draft preparation, T.A.; writing—review and editing, T.A., S.T., C.H.C., R.W. and S.M.S.; funding acquisition, T.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Mahidol University (Basic Research Fund: fiscal year 2022; grant No. BRF1-046/2565).

**Institutional Review Board Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** We thank Mahidol University Frontier Research Facility (MU-FRF) for instrument support and the MU-FRF scientists, Nawapol Udpuay, Chawalit Takoon, and Suwilai Chaveanghong, for their kind assistance in the operation of SEM.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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