*2.2. Surface Functionalization of SSD/Collagen Peptide-P(3HB-co-4HB) Scaffolds*

Surface functionalization of P(3HB-*co*-4HB) was carried out by salt leaching and solvent casting technique followed by freeze-drying method. Briefly, P(3HB-*co*-4HB) copolymer was dissolved in chloroform (5.5% w/v) and sodium bicarbonate (NaHCO3) particles sieved with known mesh sizes (200 μm) were added as porogen with mass ratio of salt:polymer at 6:1. The resulting polymer matrix was washed with deionized water to leach out the porogens. The scaffolds were freeze-dried for 24 h and later vacuum-dried for 48 h (BINDER GmbH, Tuttlingen, Germany) to remove any remaining solvent.

There were two types of scaffolds prepared using the various functionalization combination methods by incorporating different concentration of collagen peptide (2.5 wt.%, 5 wt.%, 7.5 wt.%, 10 wt.%, 12.5 wt.%) and 0.04% (w/v) of SSD. Collagen peptide powder from Tilapia fish skin with high purity (95%) and molecular weight of less than 3000 Da was used (Hainan Zhongxin Chemical Co. Ltd., Haikou, China).

The SSD coat/collagen peptide coat-P(3HB-*co*-4HB) scaffold (SCCC) was prepared by coating different concentration of collagen peptide in the silver (I) sulfadiazine (Sigma Aldrich) dispersed in hydrochloric acid solution (1.0 mM, pH 3.0).

The preparation of SSD blend/collagen peptide coat-P(3HB-*co*-4HB) scaffold (SBCC) was prepared with SSD added into the dissolved P(3HB-*co*-4HB) with NaHCO3 porogen and then solvent cast, as mentioned above.

Cross-linking was carried out using GA vapor-phase technique where the scaffolds were placed in an airtight desiccator containing 25% aqueous GA solution heated to 100 ◦C. Subsequently, the samples were washed for 24 h to remove GA, and then dried in vacuum for 24 h [8,25]. The scaffolds will be known as SCCC and SBCC from here on.

#### *2.3. Characterization of Scaffolds*

The functional group present in the scaffolds fabricated were determined and analyzed using FTIR-ATR spectrophotometer (Model RX1, PerkinElmer, Buckinghamshire, UK). The spectra of the samples were obtained in the range of wave number between 650 cm−<sup>1</sup> and 4000 cm−1. The spectrum of the FTIR was recorded in transmittance mode as function of wave number and the results were computed after 4 automated scans [24]. The surface

morphology of the scaffolds coated with gold were mounted on aluminium stump and was observed using scanning electron microscopy (SEM) (Leo Supra 50 VP Field Mission SEM, Carl-Ziess SMT, Oberkochen, Germany). The scaffolds were cut into 1 cm × 1 cm. The dry weight before immersion (*mo*) was used as the initial weight of the scaffolds. The scaffolds were immersed in distilled water for 24 h. In order to obtain the wet weights (*mf*), the immersed scaffolds were removed from the solution, gently wiped with absorbent paper and air-died for 15 s before weighing. Water uptake was calculated using the formula below:

$$Water\,\,uptake=\left(m\_0-m\_f\right)/\,m\_0\times100\%\tag{1}$$

The contact angle of the fabricated scaffolds was conducted by using sessile drop method (KSV CM200 Contact Angle) to determine their wettability properties. The scaffolds were cut into 1 cm × 1 cm pieces. The scaffolds were placed on the instrument and the droplet of water was then deposited on the polymer surface by a specialized microsyringe. The water droplet was observed from the computer screen and the contact angle was calculated. The porosity of the scaffolds was calculated using Image Analyser Software (Olympus Co. Ltd., Tokyo, Japan). The values of 100 different spots were analyzed and averaged [8].

#### *2.4. Antimicrobial Activity*

Four bacterial strains, which include *Bacillus licheniformis*, *Staphylococcus aureus* ATCC 12600, *Escherichia coli* ATCC 11303 and *Pseudomonas aeruginosa* ATCC 17588, were used. Briefly, the tested bacterial suspensions (1.5 × 106 CFU/mL) were transferred in sterilized nutrient broth. Then, 20 <sup>μ</sup>L of the bacteria suspension (7.5 × <sup>10</sup><sup>5</sup> CFU/mL) was added to each antimicrobial coated porous scaffold. The incubation is done under suitable conditions for varied time intervals (0, 6, 12 and 24 h). In every 6 h interval, the scaffold with bacteria adhesion was dissolved in 10 mL of distilled water and vortexed. After that, 100 μL of the bacterial suspension was spread on nutrient agar to observe the colonization of bacteria. The percentage of dead cells is calculated relatively to the growth control by determining the number of living cells (CFU/mL) of each scaffold using the agar plate count method. The percentages of inhibition were calculated using following Equation:

$$\text{C\%} = (\text{Co} - \text{Ce}) \; / \; \text{Co} \times 100\text{\%} \tag{2}$$

where *C*% is percentage of inhibition, *Ce* is CFU after incubation period and *Co* is initial CFU before incubation period.

#### *2.5. Biocompatibility and Cell Proliferation Evaluation*

Mouse fibroblast cell culture (L929, ATCC) was cultured in cell culture flasks containing Modified Eagle Medium (MEM) supplemented with 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, 1 mM of sodium pyruvate, 1000 U/mL penicillin-streptomycin and 10% (*v/v*) of bovine calf serum, which were incubated at 37 ◦C in 5% (*v/v*) CO2 for 2–3 days. The various scaffolds fabricated and its positive control (P(3HB-*co*-4HB) without collagen were cut in size (6 mm in diameter) fitting the 96-well flat bottom culture plate and sterilized under UV cross-linker (Spectrolinker™, XL-1000 UV Cross-linker, Westbury, NY, USA) at 1200 μJ/cm<sup>2</sup> for 30 min [8,9]. The scaffolds were then placed in the 96-well flat bottom culture plate. Suspension of the mouse fibroblast cell lines (L929) [2.5 × 104 cells/mL] were directly cultivated onto the scaffolds and film. The seeded scaffolds and film were incubated in a 5% (*v/v*) CO2 incubator at 37 ◦C for 96 h. The cells viability and proliferation were assayed with MTS[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4 sulfophenyl)-2H-tetrazolium/PMS (phenazinmethosulfate). MTS and PMS solution were used to evaluate the biocompatibility of the fabricated of scaffolds. Standard curve was plot based on the cell density from the range of 1 × 103 to 5 × <sup>10</sup><sup>5</sup> cells/mL. The media was used as the positive control and scaffolds without any incorporation of collagen peptide

were used as negative control. The absorbance values were plotted against the counted cell numbers, and thus a standard curve was established [9].

#### *2.6. Statistical Analysis*

The qualitative results were presented as means and standard deviation (s.d). The qualitative data were analyzed using ANOVA and Tukey's HSD test with SPSS 20.0 software. All *p* values < 0.05 were considered significant.

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

#### *3.1. Biosynthesis of P(3HB-co-4HB) via Batch Fermentation*

The biosynthesis of P(3HB-*co*-4HB) copolymer was carried out using *Cupriavidus malaysianesis* USMAA1020 transformant, which possessed an excess copy of the phaC gene. This cultivation regulated 4HB molar fraction to achieve 95 mol% of P(3HB-*co*-4HB) with PHA content of 78 wt.% and its concentration at 17.3 g/L in 20 L bioreactor, the mixed substrates of 1,6-hexanediol and 1,4-butanediol at 1:5 ratio. The high 4HB monomers are favored for implantable medical products. This was in agreement with the previous study [23], the 1,6-hexanediol and 1,4-butanediol were utilized as carbon sources as 4-hydroxybutyryl-CoA was initially formed and converted to 4-hydroxybutyrate. The copolymer was recovered by the chloroform extraction method and subjected to physical properties. Based on the results obtained, as summarized Table 2, the average molecular weight (MW) of the polymer was 585 kDa while the polydispersity index was in the range 3.2. Besides, the tensile strength of the polymer was recorded at about 23 MPa with the elongation at break around 611%.

#### **Table 2.** Physical and mechanical properties of P(3HB-*co*-4HB).


Values are mean ± SD of three replicates; <sup>a</sup> Determined using Gotech Al-3000 tensile Machine; <sup>b</sup> Calculated from GPC analysis, Mn: number-average molecular weight; Mw: weight average molecular weight; Mw/Mn: polydispersity index.

#### *3.2. Fabrication of SBCC and SCCC Scaffolds*

In this study a three-dimensional, porous scaffold was successfully engineered with the use of a combination of techniques, namely particle leaching and freeze-drying. Figure 1 shows a schematic of the fabrication of porous antimicrobial SSD-P(3HB-*co*-4HB)-collagen peptide scaffolds termed as SBCC and SCCC scaffolds. The system contained two phases in developing a highly porous, well interconnected pore structure of the scaffold. The first phase involved the particle leaching using NaHCO3 (200 μm), followed by the freezedrying technique. The combination of methods has shown many advantages over other methods as it is easier to control pore structures. This will produce porous scaffolds with open surface pores and interconnected bulk pores which will facilitate cell seeding and homogeneous cell distribution and promote tissue regeneration [26,27]. Despite the homogenous pores' structures, the surface properties of these polymers are hydrophobic which will possibly inhibit the infiltration of cell suspension into the scaffolds preventing smooth cell seeding.

Therefore, it is crucial to modify the surface characteristic from hydrophobic to hydrophilic to facilitate cell seeding. In this case, the surface of the porous P(3HB-*co*-4HB) scaffolds was coated with hydrophilic collagen peptide to increase the hydrophilicity of the surface, thus improving cell interaction [28–30]. In this study, apart from the surface modification of P(3HB-*co*-4HB) scaffold with collagen layer, incorporation of antimicrobial agent, SSD was carried out. This was executed by introducing the SSD either through blending (SBCC) or coating of the scaffolds (SCCC). Fabrication of scaffolds that release

the antimicrobial agents or respond to infections is crucial in developing biomaterials in tissue engineering [31–33].

**Figure 1.** Schematic of fabrication of SBCC and SCCC scaffolds using a combination of salt leaching modification and freeze-drying technique with collagen peptide coating and cross-linking with glutaraldehyde.

> Besides, surface morphology is crucial in developing biomaterials as this determines the cell-matrix interface interactions. As seen in Figure 2, the SEM micrographs reveal the formation of the three-dimensional interconnected porous structure of SBCC and SCCC scaffolds. Interestingly, the pore sizes observed using SEM were much smaller than the range of porogen sizes (NaHCO3) used to create them. This could be attributed to the combination of techniques used, mainly freeze-drying. Hence, combining salt leaching with freeze-drying may enhance pore interconnectivity and assist the formation of homogenous pores ranging from 100 to 200 μm [34]. However, SCCC scaffolds exhibited rougher appearance with less interconnection and possessed numerous macropores as compared to SBCC. Basically, the porous-based connectivity surface is favored to enhance the ECM architecture and provide a larger space to induce cell-material interactions [35–39]. Additionally, both the scaffolds created similar morphology with generally amorphous pores with smooth edge.

> The fabricated scaffolds differed in terms of their construction. SSD coated onto the porous SCCC scaffolds showed solubility in aqueous medium in contrast to the impregnated collagen in SBCC. Hence, the collagen peptide coated P(3HB-*co*-4HB) porous scaffold were then cross-linked via GA vapor phase. The dissolution analysis on scaffolds of cross-linking and uncross-linking scaffolds were shown in Figure 3. The percentage of dissolutions was significantly higher for uncross-linked SCCC scaffolds with the amount retained only between 15% and 45%. On the contrary, the crosslinked SSD/collagen peptide-coated P(3HB-*co*-4HB) scaffold exhibited collagen retain percentage from up to 80%

to 90%. This demonstrates that cross-linking with GA enhanced the scaffolds resistance to dissolution. After GA vapor cross-linking, the membranes became visibly yellowish and shrunk dimensionally. The aldimine linkages (CH=N) between the free amine groups of protein and GA attributes to the color change, whereas the covalent bond formed between the aldehyde groups of GA caused shrinkage [40,41]. The aldimine linkage was a reflection of Schiff base reaction, whereby the carbon in the aldehyde group of GA was attacked by nucleophilic nitrogen in the amino group of collagen peptides, and hence replaced the oxygen in the aldehyde group and eliminated water molecule [30]. Nonetheless, GA cross-linking with vapor phase methodology showed low or no detectable cytotoxic effects [25,42]. As described by Teixera et al. (2021), this will enable a sustainable approach in achieving green methodology and the lowest environment impact possible at all stages of fabrication for biomedical application [43].

**Figure 2.** Micrograph of porous structure of (**a**) control-P(3HB-*co*-4HB), (b) SCCC 2.5 wt.%, (**c**) SCCC 5 wt.%, (**d**) SCCC 7.5 wt.%, (**e**) SCCC 10 wt.%, (**f**) SCCC 12.5 wt.%, (**g**) SBCC 2.5 wt.%, (**h**) SBCC 5 wt.%, (**i**) SBCC 7.5 wt.%, (**j**) SBCC 10 wt.% and (**k**) SBCC 12 wt.%.

**Figure 3.** The percentage of collagen retained on cross-linked SCCC and uncross-linked SCCC scaffold. Data represent means ± SD (*n* = 3). Mean data accompanied by different alphabets as of cross-linked SCCC scaffolds (a–b); and uncross-linked SCCC (A–D) indicates significant difference within each respective group (Tukey's HSD test, *p* < 0.05).

#### *3.3. Functional Group Identification Using FTIR Analysis*

FTIR analysis shown in Figure 4 was carried as an evidential analysis to determine and analyze the characteristic bands that correlate to functional groups of the fabricated scaffolds. The FTIR spectrum for collagen (a) showed symmetric and unsymmetric stretching of the primary amine (NH2) bands at 3275 cm−<sup>1</sup> and 3150 cm<sup>−</sup>1, respectively. The hydroxyl (OH) from carboxylic acid portion also is expected to be overlapped with the symmetric amine at 3275 cm−1. The moderate peaks at 2937 cm−<sup>1</sup> represent CH3 (bend) and CH2 (stretch) of the alkanes' substructure. A strong band at 1633 cm−<sup>1</sup> represents (C=O) from the amide moiety. Another strong peak can be seen at 1531 cm−<sup>1</sup> and represents NH2 bending [44–48]. The peaks of the (C=C) bands of the aromatic portion also can be clearly observed between the peaks of 1531 cm−<sup>1</sup> to 1449 cm−1. In addition, a moderate peak at 920.89 cm−<sup>1</sup> would represent a C-H (out-of-plane) band from the aromatics.

In the case of P(3HB-*co*-4HB) polymer (b), moderate peaks at 2963 cm−<sup>1</sup> and 2899 cm<sup>1</sup> represent CH3 (bend), CH2 (stretch) and CH of the alkanes' substructure. A strong band at 1633 cm−<sup>1</sup> represents (C=O) and another strong band at 1161 cm−<sup>1</sup> exhibits the (C-O) band [8,49].

Comparatively, the FTIR spectra of SBCC (c) and SCCC (d) are rather comparable to each other as they exhibit all the expected bands and peaks of the designated collagen, P(3HB-*co*-4HB) polymer and pure SSD. The major characteristic absorption peaks in both FTIR spectra of SBCC (c) and SCCC (d) ca. 3283, 3150, 2900, 1719, 1630, 1540, 1450 and 1164 cm−1. The absorption peak at 3283 and 3150 cm−<sup>1</sup> are assigned to NH2 symmetric and asymmetric stretching, respectively. A distinctive peak at 2900 cm−<sup>1</sup> represents CH3 (bend) and CH2 (stretch) of the alkanes' substructure. Whilst the strong peak at 1719 cm−<sup>1</sup> represents (C=O) peak. The absorption peak at 1630 cm−<sup>1</sup> corresponds to NH2 bending. The peaks at 1540 cm<sup>−</sup>1, 1450 cm−<sup>1</sup> belong to the peaks of the (C=C) bands of the aromatic portion. The peaks of asymmetric stretching vibration of (SO2) group cannot be resolved in these spectra as the band of (C-O) can be dominantly seen in this fingerprint region at 1164 cm−<sup>1</sup> [50].

**Figure 4.** FTIR spectra of scaffolds (**a**) collagen, (**b**) P(3HB-*co*-4HB), (**c**) SBCC, and (**d**) SCCC. Arrows I, II, III indicate amide I, amide II and amide III, respectively.

It was observed that the prominent characteristic peaks of SBCC (c) and SCCC (d) with a few bands shift in comparison to each other with the dominant characteristics are from the P(3HB-*co*-4HB) polymer (b) which are indicative of the reservation of the chemical aspect of these blends production. It can be concluded that the SSD did not engage with its active groups in any chemical interaction with any of the components of SBCC (c) and SCCC (d) built up. From the FTIR spectra of the two, there is also no evidence of electrostatic interaction nor chemical reaction have taken place between all the materials that made up the blend due to very little shift of all the vibrational wavenumbers (i.e., less than 5 cm<sup>−</sup>1) throughout the major bands of interest.

### *3.4. Porosity Analysis*

The pores in scaffolds are imperative as they provide an ideal framework for cells to bind, proliferate and form extracellular matrix. As such, here the porosity was determined with six different collagen concentrations of the scaffolds. The fabricated scaffolds exhibited a gradual drop of the pore size from 145 to 53 μm with increasing collagen concentrations (Figure 5). Similarly, the porosity of the SBCC declined by 50% from the control scaffold. This decrease in porosity could have been due to the larger collagen layer deposits on the surface of scaffolds [51–55]. Based on various studies, pore sizes above 100 μm are ideal for cell infiltration and migration. Interestingly, 10 wt.% scaffolds resulted in a desirable pore size despite the higher concentration of collagen peptide. In developing biomaterial, pore structures of scaffolds play a crucial role in facilitating cell seeding, cell penetration and distribution in the scaffolds. Thus, the adhesion of cells and formation of new tissues and organs occurs [56–58]. It is emphasized that an ideal scaffold depends on biomaterial source, fabrication technique and the pore geometry. As such, it is vital to develop a scaffold with specific porosity properties for potential application in tissue engineering and regenerative medicine [59,60].

**Figure 5.** Porosity analysis of SSD coated/collagen coated P(3HB-*co*-4HB); SCCC and SSD blend/collagen coated P(3HB*co*-4HB); SBCC scaffolds. Data represent means ± SD (*n* = 3). Mean data accompanied by different alphabets as of SCCC scaffolds (a–d) and SBCC scaffolds (A–D) indicates significant difference within each respective group (Tukey's HSD test, *p* < 0.05).

#### *3.5. Hydrophilicity of Fabricated Scaffolds*

The hydrophilicity of the SCCC and SBCC scaffolds was determined using water contact angle analysis (Table 3). The graph clearly showed a decline in the contact angle as the concentration of collagen peptide increases, thus indicating the increase of hydrophilicity. Ideally, a contact angle of less than 90◦ indicates that the surface is wet-prone, hence being categorized as a hydrophilic surface [61–63]. Whole wetting was observed with the water droplet becoming a flat puddle with 0◦ contact angle on SBCC and SCCC with 10 wt.% and 12.5 wt.% collagen peptides. Additionally, the collagen peptide coating enhanced the surface wettability of sample scaffolds. The significant hydrophilicity enhancing effect of collagen peptide could be associated with the amino groups in collagen [64,65].

The wettability analysis of different sample collagen concentrations is demonstrated in Figure 6. A steady rise of water uptake percentage with the increment of collagen peptide concentrations can be observed. Water uptake ability elucidates the hydrophilicity of fabricated scaffolds which will increase the efficiency of absorption of essential supplements required for cell attachment. Overall, the collagen peptide coated P(3HB-*co*-4HB) scaffold absorbed a larger amount of water, exceeding 100% (*v/v*) of the total volume of the scaffold even at the low concentrations of collagen peptide (2.5 wt.%). As anticipated, the results pointed out that the hydrophilicity of both SCCC and SBCC scaffolds have similar water uptake ability. The water uptake ability properties of scaffolds are crucial in order to enhance the proliferation of a cell. The optimal design of a scaffold strongly depends on both materials and the surface treatment in modulating cell seeding and proliferation [60,66].
