2.6.6. LIVE/DEAD® Cell Viability Assay

This assay was conducted to evaluate the functional status of the cells by identifying cytoplasmic esterase activity using the LIVE/DEAD™ Viability/Cytotoxicity kit for mammalian cells (Invitrogen). The kit comprises of calcein, which fluoresces green in living cells, and ethidium bromide, which fluoresces red in dead cells. In brief, the HDFs were plated

at the same seeding density as per cell viability and proliferation assay and maintained as above prior to treatment with samples. The cells were treated with calcein and ethidium bromide for 30 min as per the manufacturer's instructions. Later, the cells were rinsed with DBPS and observed using a Nikon A1R fluorescence microscope (Nikon, Tokyo, Japan).

#### 2.6.7. CS Stabilizing Effect on PL

A cell proliferation assay was carried out using protease degradation and heat treatment approaches to establish the protective effect of CS on PL.

#### Protease Degradation Test

Cell proliferation assay was conducted to determine the protective effect of CS against proteases on GF. In brief, all PL-loaded CS hydrogels and PL alone (10%) were subjected to 0.05% trypsin treatment at 37 ◦C for 1 h. Then, the HDFs (P2, 2 <sup>×</sup> <sup>10</sup><sup>4</sup> ) were incubated with trypsin-treated PL and CS hydrogel for 24 h followed by Alamar Blue addition to the cells and incubated for the next 4 h. Then, absorbance was recorded at 570 and 600 nm. Cell proliferation was calculated by percentage reduction in Resazurin. Cells without treatment served as control.

#### Heat Treatment Test

PL (10%) and CS hydrogels containing PL were exposed to 0, 25 ◦C, 37 ◦C, and 45 ◦C for 24 h to examine the stabilizing effect of CS on PL. Then, extracts of both heat-treated groups were added to the HDFs (P3, 2 <sup>×</sup> <sup>10</sup><sup>4</sup> ) as per above method, and cell proliferation was determined.

#### *2.7. Statistical Analysis*

Experiments were performed in triplicates and mean ± SD was reported. Data were analyzed using one-way analysis of variance and Tukey's multiple comparisons test, by using GraphPad Prism version 5.00 (GraphPad Software, La Jolla, CA, USA). The level of significance was set at *p* < 0.05.

#### **3. Results**

#### *3.1. Quantification of GFs in PL*

The concentrations of TGF-β and EGF from PL were measured using ELISA. ELISA was performed for standard and sample as per manual instructions, and concentrations were determined as 477.26 pg/mL and 150 ng/mL for EGF and TGF-β, respectively, from the calibration curves. The level of TGF-β was threefold higher than that of EGF, which is consistent with reported literature [28].

#### *3.2. Chemical Composition, Fiber Yield, Zeta Potential, and Crystallinity of NCC*

After various stages of chemical treatment on raw, alkali-treated, and bleached fibers, chemical composition of the fibers was determined as previously described by Tuerxun Duolikun (2018). The results are presented in Table 1. After a series of chemical treatment on the kenaf biomass fibers, cellulose content significantly increased from raw to bleached to 30% to 84%, whereas hemicellulose and lignin contents declined to 5% and 3%, respectively [29].

**Table 1.** Chemical composition of kenaf fibers after chemical treatments.


The fiber yield of the NCC after acid hydrolysis was 40% (of initial weight), which is consistent with the fiber yield of NCC from other plant sources, such as sisal (30%) and mengkuang leaves (28%) [30]. In general, fiber yield is dependent on pre-treatment methods and hydrolysis environment. The low fiber yield of NCC might be caused by sulfuric acid treatment during production, which caused the degradation and removal of amorphous and other non-cellulosic regions of the fibers that result in weight loss.

The zeta potential of the NCC suspension was recorded as (−10.9 ± 5.47 mV). The NCC becomes crystalline and possesses a negative charge on the surface of the cellulose chain. They become more stable through electrostatic repulsion among the negatively charged groups on the polymer chains [31]. The anionic NCC is found suitable to form composite hydrogels with cationic CS polymer. The abundance of hydroxyl groups in NCC cellulose is responsible for the negative charges that bonded with CS via the electrostatic interaction of protonation of NH<sup>2</sup> on CS and hydroxyl groups. Platelets also carry negative charges onto their surfaces due to the presence of sialic acid (N-acetyl-neuraminic acid) and amino acids such as glutamate and aspartate [32]. PL was loaded into NCC-reinforced CS hydrogel and associated by non-covalent bonding and subsequently stabilized by CS through protein bindings that promote fibrin gel formation and thus retained GF functionality for a long time [9].

Cellulose naturally comprises of crystalline and amorphous portions. The amorphous region (contains impurities of lignin, hemicellulose etc.) needs to be eliminated to make cellulose highly pure and crystalline with desirable properties. Therefore, chemical treatment including sulfuric acid hydrolysis was carried out to obtain an extremely crystalline and purified form of cellulose by hydrolyzing the amorphous region.

To investigate the crystallinity of the bleached fibers and effect of acid treatment on the resulting NCC, X-ray, diffractometry (XRD) was carried out. Figure 1 presents the obtained XRD patterns for kenaf bast fibers for bleached and acid treated fibers. The crystallinity index of both fibers were calculated using Diffrac.EvaV4.0 software. Diffraction peaks at 2θ value of 14.5◦ , 16.5◦ , and 22.5◦ at plane of 101, 10-1, and 002, respectively, were observed. From the diffraction pattern it could be noticed that cellulose was present in the form of cellulose [33,34]. Crystallinity index was calculated for the bleached fibers and NCC. For the kenaf biomass, the crystallinity index values of the bleached fibers and NCC were reported as 56% and 71.6%, respectively, in consistence with similar findings [35]. The higher crystallinity of NCC than the bleached fibers could be due to the removal of amorphous cellulosic region by acid hydrolysis. The XRD result suggested that the amorphous region of the bleached fibers degraded during extraction, whereas the crystalline region remained unaffected. The high crystallinity of NCC was related to the high tensile strength of the fibers. Therefore, the mechanical properties of the nanocomposite can be improved by using NCC as a reinforcing agent.

#### *3.3. Scanning Electron Microscopy*

Surface morphological analysis was performed on the bleached fibers and kenaf NCC. The SEM images are depicted in the Figure 2 at different magnifications. As shown in Figure 2a,c the bleached fiber bark structures were apparent. Meanwhile, NCC microphotographs in Figure 2b,d revealed that the surface of the NCC became smooth and the fibers appeared as a web-like network with diameters approximately 40–90 nm in consistent with similar SEM surface morphologies [24]. After a series of chemical treatments, impurities such as pectin, hemicellulose, and lignin were removed from the structure of the bleached fibers, as can be implied from the FTIR findings. The small diameter of NCC was because of acid hydrolysis, which significantly removed the amorphous portion from the crystalline part, which is in accordance with previous similar finding [34].

*Polymers* **2021**, *13*, x FOR PEER REVIEW 10 of 24

**Figure 1.** The XRD patterns of both NCC (Red) and (Bleached fibers (Black) from kenaf bast fiber. **Figure 1.** The XRD patterns of both NCC (Red) and (Bleached fibers (Black) from kenaf bast fiber.

**Figure 2.** Photographs of SEM images of (**a**) bleached fibers (**b**) NCC at 500X (**c**) bleached fibers (**d**) NCC at 20KX. **Figure 2.** Photographs of SEM images of (**a**) bleached fibers (**b**) NCC at 500X (**c**) bleached fibers (**d**) NCC at 20KX.

#### *3.4. FTIR-ATR Spectroscopic Analysis of NCC 3.4. FTIR-ATR Spectroscopic Analysis of NCC*

FTIR was carried out on raw fibers, bleached fibers, and NCC to determine their chemical composition after chemical and mechanical treatment. The results are shown in Figure 3a and Table 2. All spectra detected a broad and intense peak at 3300 cm−1 region which is attributed to the characteristic of polysaccharides hydroxyl bonds [36]. C–H symmetrical stretching and CH2 symmetrical stretching at 2900–2800 cm−1 revealed FTIR was carried out on raw fibers, bleached fibers, and NCC to determine their chemical composition after chemical and mechanical treatment. The results are shown in Figure 3a and Table 2. All spectra detected a broad and intense peak at 3300 cm−<sup>1</sup> region which is attributed to the characteristic of polysaccharides hydroxyl bonds [36]. C–H symmetrical stretching and CH2 symmetrical stretching at 2900–2800 cm−<sup>1</sup> revealed

polysaccharide, wax, and oil content of the fibers [36]. Another peak was observed in the

group present in lignin. The disappearance of this peak in bleached and NCC suggested the separation of lignin by chemical treatment [36].The vibration peak in the fibers at 1326– 1340 cm−1 was linked to bending of the C-H and C-O bonds in polysaccharide aromatic rings [36]. Absorption peak was shown in all samples at 1023–1030 cm−1, which were associated with C-O and C-N vibrations [36]. The absorption peak at 1634 cm−1 in NCC

was due to adsorbed water, which was suggestive of NCC [36].

polysaccharide, wax, and oil content of the fibers [36]. Another peak was observed in the spectra of the raw fibers at 1242 cm−<sup>1</sup> , which was associated with C-O stretching of the aryl group present in lignin. The disappearance of this peak in bleached and NCC suggested the separation of lignin by chemical treatment [36].The vibration peak in the fibers at 1326– 1340 cm−<sup>1</sup> was linked to bending of the C-H and C-O bonds in polysaccharide aromatic rings [36]. Absorption peak was shown in all samples at 1023–1030 cm−<sup>1</sup> , which were associated with C-O and C-N vibrations [36]. The absorption peak at 1634 cm−<sup>1</sup> in NCC was due to adsorbed water, which was suggestive of NCC [36]. *Polymers* **2021**, *13*, x FOR PEER REVIEW 12 of 24

**Figure 3.** FTIR analysis comparison of (**a**) NCC, bleached fiber and raw fiber (**b**) NCC, CS, PL, and CS-NCC-PL (**c**) CS-PL, CS and PL alone. **Figure 3.** FTIR analysis comparison of (**a**) NCC, bleached fiber and raw fiber (**b**) NCC, CS, PL, and CS-NCC-PL (**c**) CS-PL, CS and PL alone.

stretching and CH2

3345.74 3342.62 3322.16 O**–**H stretching 2909.88 2911.71 2909.90 C**–**H symmetrical

**Table 2.** FTIR characteristic peaks of Kenaf raw fibers, bleached fibers, and NCC.

**Bleached Fibers Peak** 

**Raw Fibers Peak (cm<sup>−</sup>1)** 


**Table 2.** FTIR characteristic peaks of Kenaf raw fibers, bleached fibers, and NCC.

FTIR was conducted to investigate the possible interaction of functional group between PL and CS hydrogel, as presented in Figure 3b. CS showed a broad band at 3500–3200 cm−<sup>1</sup> , which can be attributed to the O–H and N–H stretching vibrations of functional groups in hydrogen bonds. Characteristic absorption bands at 1634 cm−<sup>1</sup> corresponded to C=O stretching in the amide I vibration. The absorption band at 1540 cm−<sup>1</sup> appeared due to N–H bending in amide II vibration. The spectra at 1069 and 1024 cm−<sup>1</sup> corresponded to C–O stretching vibrations, which were characteristics of CS structure. Association of PL in CS hydrogel was confirmed by displacement of characteristic bands at 3219–3270 cm−<sup>1</sup> due to O-H stretching and 1634–1641 cm−<sup>1</sup> due to NH vibrations [37]. These modifications suggested a possible interaction between PL and CS.

FTIR spectra of NCC, CS, PL, and CS-NCC-PL are represented in Figure 3c. A characteristic peak of CS was observed at 3310 cm−<sup>1</sup> owing to the O-H group. CS-NCC was observed by shifting of the peak from 3337 cm−<sup>1</sup> to 3759 cm−<sup>1</sup> , which indicated possible overlapping of hydrogen bonds. PL with a characteristic peak at 3885 cm−<sup>1</sup> was due to O-H stretching that shifted slightly toward 3901 cm−<sup>1</sup> . Thus, CS incorporated with NCC and PL possessed two extra peaks, which corresponded to NCC and PL as compared with control CS hydrogel. These results indicate that NCC was prepared and successfully incorporated in CS hydrogel along with PL.

#### *3.5. Swelling Study*

Freeze drying allows the nucleation of ice crystals from solution and further growth along the lines of thermal gradients. Exclusion of the CS acetate salt from the ice crystal phase and subsequent ice removal by lyophilization generate a porous material [38]. Freeze drying is usually employed to produce porous CS hydrogels to assess their water swelling behavior effectively.

An ideal dressing must be capable of absorbing the wound exudates that could potentially cause bacterial infection at the wound bed. Swelling properties determines the moisture absorption capacity of a dressing, which is a crucial action for absorbing pus and exudates from weeping wounds. The swelling ratios over time of the freeze-dried hydrogels are shown in Figure 4. All hydrogels swelled at different rates, and equilibriums were achieved up to 1000 times of their initial weight within 24 h of water imbibition. During the initial swelling, water was absorbed via capillaries located in the internal structure of the hydrogels. As 3D networks, porous structures provide channels for the molecules to enter and escape. The hydrophilic groups (-OH/-COOH/-COO-) present in CS were bound with water molecules by hydrogen bonds that created a hydration layer. This phenomenon may explain why all hydrogels showed the fastest swelling at the beginning (<3 h). The addition of PL in the CS hydrogels reduced the swelling ratio (~500% reduction). The addition of PL may decrease availability of CS hydrophilic groups to bind with water. The swelling ratio also decreased further (~300% reduction) when NCC was

added into CS. This behavior could be ascribed to the fact that NCC lodges the free space volume in the CS polymeric matrix, thereby restricting the volume available for swelling and causing the formation of a rigid hydrogel structure that cannot be easily penetrated by water molecules. Hence, the water absorption decreased, which consequently decreased the swelling ratio [39]. However, the addition of PL to the CS-NCC hydrogel increased the swelling ratio owing to its large hydrophilic groups in molecular chains, which promoted the establishment of hydration layers [40]. The higher water holding capacities (>1000%) of the hydrogels indicate that the formulated hydrogels are suitable for medium to heavy suppurating wounds. This is a crucial property for a dressing in minimizing infection. At the same time, the hydrogels maintain a moist local microenvironment for tissue repair process to take place. *Polymers* **2021**, *13*, x FOR PEER REVIEW 14 of 24 are suitable for medium to heavy suppurating wounds. This is a crucial property for a dressing in minimizing infection. At the same time, the hydrogels maintain a moist local microenvironment for tissue repair process to take place.

**Figure 4.** Swelling ratio of Control (Intrasite Gel), CS hydrogel, CS-NCC hydrogel, CS-PL hydrogel and CS-NCC-PL hydrogel over 24 h. The error bars showed standard deviation based on triplicated samples at each time point. **Figure 4.** Swelling ratio of Control (Intrasite Gel), CS hydrogel, CS-NCC hydrogel, CS-PL hydrogel and CS-NCC-PL hydrogel over 24 h. The error bars showed standard deviation based on triplicated samples at each time point.

#### *3.6. Moisture Loss Study 3.6. Moisture Loss Study*

Moisture retention capacity is an important characteristic for a wound dressing. Hydrogels should provide a moist environment to the wound to accelerate healing [40]. Moisture loss from the different hydrogels was evaluated by employing the desiccation method. Moisture loss was expressed in percentage and calculated after 24 h of hydrogel drying. As shown in Figure 5, Intrasite™ hydrogel lost almost 80% of moisture after 24 h, followed by PL, which lost 90% of moisture. Meanwhile, the CS-PL hydrogel lost approximately 42% of moisture as compared with the CS hydrogel. The PL entrapped within the network of polymer and prevented moisture loss. Therefore, the hydrogels under investigation held moisture for a longer period as compared with the commercial hydrogel. The CS-NCC-PL hydrogel can maintain a moist environment on chronic wounds for an extended time. Moisture retention capacity is an important characteristic for a wound dressing. Hydrogels should provide a moist environment to the wound to accelerate healing [40]. Moisture loss from the different hydrogels was evaluated by employing the desiccation method. Moisture loss was expressed in percentage and calculated after 24 h of hydrogel drying. As shown in Figure 5, Intrasite™ hydrogel lost almost 80% of moisture after 24 h, followed by PL, which lost 90% of moisture. Meanwhile, the CS-PL hydrogel lost approximately 42% of moisture as compared with the CS hydrogel. The PL entrapped within the network of polymer and prevented moisture loss. Therefore, the hydrogels under investigation held moisture for a longer period as compared with the commercial hydrogel. The CS-NCC-PL hydrogel can maintain a moist environment on chronic wounds for an extended time.

**Figure 5.** Moisture loss profile of formulated hydrogels over 24 h (expressed in mean ±SD, *n* = 3). The asterisks (\*) represent significant difference (*p* < 0.05) compared to control. The hashtag (#) represent significant difference (*p* < 0.05) between the groups. **Figure 5.** Moisture loss profile of formulated hydrogels over 24 h (expressed in mean ±SD, *n* = 3). The asterisks (\*) represent significant difference (*p* < 0.05) compared to control. The hashtag (#) represent significant difference (*p* < 0.05) between the groups.

#### *3.7. In Vitro Protein Release Assay 3.7. In Vitro Protein Release Assay*

The cumulative release profiles of PL from the CS-PL and CS-NCC-PL hydrogels are represented in Figure 6. The PL released from the CS-PL hydrogel showed faster release at the first 3 h compared with that from CS-NCC. Thereafter, the PL release increased gradually (PL flux = 0.165 mg/cm2/h) and reached the maximum release of 5.22 ± 1.47 mg/cm2 at 24 h. During this time, proteins permeated from the inside matrix of the hydrogel via swelling and diffusion mechanism. In the CS-NCC-PL hydrogel, the rate of PL release was significantly decreased and much controlled throughout the test duration (PL flux = 0.075 mg/cm2/h). This phenomenon suggested that NCC might act as a nanofiller and provide sustained release because of its capacity to retain the proteins within the hydrogel matrix for a long period of time (maximum PL release of 3.00 ± 1.11 mg/cm2 at 24 h). For chronic wound healing, hydrogel comprises of platelet proteins, and a cocktail of various GFs is desired for tissue proliferation. The CS-NCC hydrogel composite is successful in controlling the PL release and is a promising vehicle for PL in The cumulative release profiles of PL from the CS-PL and CS-NCC-PL hydrogels are represented in Figure 6. The PL released from the CS-PL hydrogel showed faster release at the first 3 h compared with that from CS-NCC. Thereafter, the PL release increased gradually (PL flux = 0.165 mg/cm2/h) and reached the maximum release of 5.22 <sup>±</sup> 1.47 mg/cm<sup>2</sup> at 24 h. During this time, proteins permeated from the inside matrix of the hydrogel via swelling and diffusion mechanism. In the CS-NCC-PL hydrogel, the rate of PL release was significantly decreased and much controlled throughout the test duration (PL flux = 0.075 mg/cm2/h). This phenomenon suggested that NCC might act as a nanofiller and provide sustained release because of its capacity to retain the proteins within the hydrogel matrix for a long period of time (maximum PL release of 3.00 <sup>±</sup> 1.11 mg/cm<sup>2</sup> at 24 h). For chronic wound healing, hydrogel comprises of platelet proteins, and a cocktail of various GFs is desired for tissue proliferation. The CS-NCC hydrogel composite is successful in controlling the PL release and is a promising vehicle for PL in wound healing.

#### wound healing. 3.7.1. Cell Viability

The cytotoxic effect of hydrogel formulations on HDFs was tested by Alamar Blue assay. The results of cell viability are presented in Figure 7. This experiment revealed that the cell viability of the CS-PL and CS-NCC-PL hydrogels was higher than that of PL alone for 24, 48, and 72 h. This hike in cell viability of the PL-loaded hydrogels is probably due to the protective effect of CS-NCC on PL. As mentioned previously, CS can stabilize PL and protect it from degradation. Overall, the cytotoxic effect of hydrogels in this study with or without PL was negligible (*p* < 0.05, one-way ANOVA, Tukey's multiple comparison test). Therefore, the hydrogel formulations are non-toxic and safe to be applied onto wounds.

*Polymers* **2021**, *13*, x FOR PEER REVIEW 16 of 24

**Figure 6.** In vitro cumulative release profile of PL from CS-PL and CS-NCC-PL. (\*) means significant difference (*p* < 0.05) compared to CS-PL. **Figure 6.** In vitro cumulative release profile of PL from CS-PL and CS-NCC-PL. (\*) means significant difference (*p* < 0.05) compared to CS-PL. applied onto wounds.

**Figure 7.** Percentage cell viability of hydrogels on Human dermal fibroblast (HDF) at different time points using Alamar blue assay, *n* = 3, The asterisks (\*) represents statistically significant difference compared to control (*p* < 0.05) and hashtag (#) represent significant difference (*p* < 0.05) between **Figure 7.** Percentage cell viability of hydrogels on Human dermal fibroblast (HDF) at different time points using Alamar blue assay, *n* = 3, The asterisks (\*) represents statistically significant difference compared to control (*p* < 0.05) and hashtag (#) represent significant difference (*p* < 0.05) between groups.

#### 3.7.2. Cell Proliferation

groups.

groups.

**Figure 7.** Percentage cell viability of hydrogels on Human dermal fibroblast (HDF) at different time points using Alamar blue assay, *n* = 3, The asterisks (\*) represents statistically significant difference compared to control (*p* < 0.05) and hashtag (#) represent significant difference (*p* < 0.05) between Cell proliferation was analyzed through Alamar Blue assay to ascertain any change in cell proportion. In this study, CS-NCC-PL hydrogels were tested by treating HDFs (1 <sup>×</sup> <sup>10</sup><sup>4</sup> ) with sterile hydrogel leachates in the microplates for 24, 48, and 72 h. The results of cell proliferation are provided in Figure 8. PL alone showed low proliferation with an increasing trend over 3 days. The hydrogels enhanced the HDF proliferation from 24 h to 48 h, demonstrating the growth and proliferation of the HDFs. The PL-loaded hydrogels significantly augmented fibroblast proliferation (>100% viability) compared with the blank hydrogels or PL alone, indicating fast wound closure without causing any cell toxicity. Higher cell proliferation induced by combination of chitosan with PL is also in agreement with similar study where it showed that platelet-rich plasma with chitosaninduced growth factor enrichment can stimulate the growth of fibroblasts [41]. This dictates

the feasibility and safety of autologous PL in wound healing and complications in patients with intractable diseases such as diabetic ulcers and decubitus [41]. dictates the feasibility and safety of autologous PL in wound healing and complications in patients with intractable diseases such as diabetic ulcers and decubitus [41].

Cell proliferation was analyzed through Alamar Blue assay to ascertain any change in cell proportion. In this study, CS-NCC-PL hydrogels were tested by treating HDFs (1 × 104) with sterile hydrogel leachates in the microplates for 24, 48, and 72 h. The results of cell proliferation are provided in Figure 8. PL alone showed low proliferation with an increasing trend over 3 days. The hydrogels enhanced the HDF proliferation from 24 h to 48 h, demonstrating the growth and proliferation of the HDFs. The PL-loaded hydrogels significantly augmented fibroblast proliferation (>100% viability) compared with the blank hydrogels or PL alone, indicating fast wound closure without causing any cell toxicity. Higher cell proliferation induced by combination of chitosan with PL is also in agreement with similar study where it showed that platelet-rich plasma with chitosaninduced growth factor enrichment can stimulate the growth of fibroblasts [41]. This

*Polymers* **2021**, *13*, x FOR PEER REVIEW 17 of 24

3.7.2. Cell Proliferation

**Figure 8.** Cell proliferation study of hydrogels on human dermal fibroblast (HDF) at different time points using Alamar blue assay, *n* = 3, The asterisks (\*) represent statistically significant different (*p* < 0.05) compared to control, the hashtag (#) means statistically significant different (*p* < 0.05) between groups. **Figure 8.** Cell proliferation study of hydrogels on human dermal fibroblast (HDF) at different time points using Alamar blue assay, *n* = 3, The asterisks (\*) represent statistically significant different (*p* < 0.05) compared to control, the hashtag (#) means statistically significant different (*p* < 0.05) between groups.

#### *3.8. Wound Scratch Assay 3.8. Wound Scratch Assay*

Cell migration across the provisional gap was carried out to assess the healing potential of the hydrogels. The results in Figure 9 show the effect of hydrogels on the migration rate of the HDFs for 24 h. All hydrogel groups showed significant migration of cells as compared with the control group (*p* < 0.05, one-way ANOVA followed by Tukey's multiple comparison test). The migration rates of the HDFs after being treated with the CS-PL hydrogels was significantly higher than CS alone (*p* < 0.05). This indicates the PL stimulates the cell migration rate. The addition of NCC does not affect the migration rate as CS-NCC-PL and CS-PL group (*p* > 0.05). The wound area-time plot and micrographs (Figures 10 and 11) indicated that all wounds were nearly closed in less than 48 h despite of the differences in the migration rates of the control and treatment groups. The HDFs were managed to occupy the free space within a span of 48 h, indicating that the hydrogels efficiently healed the HDFs. Cell migration across the provisional gap was carried out to assess the healing potential of the hydrogels. The results in Figure 9 show the effect of hydrogels on the migration rate of the HDFs for 24 h. All hydrogel groups showed significant migration of cells as compared with the control group (*p* < 0.05, one-way ANOVA followed by Tukey's multiple comparison test). The migration rates of the HDFs after being treated with the CS-PL hydrogels was significantly higher than CS alone (*p* < 0.05). This indicates the PL stimulates the cell migration rate. The addition of NCC does not affect the migration rate as CS-NCC-PL and CS-PL group (*p* > 0.05). The wound area-time plot and micrographs (Figures 10 and 11) indicated that all wounds were nearly closed in less than 48 h despite of the differences in the migration rates of the control and treatment groups. The HDFs were managed to occupy the free space within a span of 48 h, indicating that the hydrogels efficiently healed the HDFs. *Polymers* **2021**, *13*, x FOR PEER REVIEW 18 of 24

**Figure 9.** Rate of cell migration of HDFs treated with different hydrogels; *n* = 3, \*—statistically significant different (*p* < 0.05) compared to CS and control. **Figure 9.** Rate of cell migration of HDFs treated with different hydrogels; *n* = 3, \*—statistically significant different (*p* < 0.05) compared to CS and control.

**Figure 10.** Wound area closure at different time points; *n* = 3.

significant different (*p* < 0.05) compared to CS and control.

**Figure 11.** Migration of HDFs treated with hydrogels for 24, 48, and 72 h. **Figure 11.** Migration of HDFs treated with hydrogels for 24, 48, and 72 h.

#### *3.9. LIVE/DEAD® Cell Viability Assay 3.9. LIVE/DEAD® Cell Viability Assay*

Cytotoxic effect of hydrogel formulations was further investigated using LIVE/DEAD™ Cell Viability Assay. This assay gives qualitative aspect of cell viability. The control and cells treated with the hydrogels lacked dead cells (stained red), as shown in Figure 12. Control and chitosan hydrogels with different combinations exhibited favorable cell viability after 24 h of incubation (captured in green color cells). In addition, after 48 h, cells continue to grow and become elongated for further maturation and differentiation. Additionally, these results comply with the results of Alamar Blue™ assay, suggesting the non-toxicity of the hydrogel after 24 and 72 h of treatment. Cell number and cell viability increased over the period of 72 h. Cytotoxic effect of hydrogel formulations was further investigated using LIVE/DEAD™ Cell Viability Assay. This assay gives qualitative aspect of cell viability. The control and cells treated with the hydrogels lacked dead cells (stained red), as shown in Figure 12. Control and chitosan hydrogels with different combinations exhibited favorable cell viability after 24 h of incubation (captured in green color cells). In addition, after 48 h, cells continue to grow and become elongated for further maturation and differentiation. Additionally, these results comply with the results of Alamar Blue™ assay, suggesting the non-toxicity of the hydrogel after 24 and 72 h of treatment. Cell number and cell viability increased over the period of 72 h.

**Figure 12.** (**a**) Live Dead cell viability Assay at 200X magnification, (**b**) Cell counting treated with different hydrogels at 24 h and 72 h (**c**) % cell viability compared to control. *n* = 3. The asterisks (\*)—statistically significant different (*p* < 0.05); ns—non-significant from control (untreated cells).

#### *3.10. CS Stabilizing Effect on PL* Cell proliferation assay was carried out to assess the protective effect of CS on GF

*3.10. CS Stabilizing Effect on PL* 

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Cell proliferation assay was carried out to assess the protective effect of CS on GF present in PL. CS along with NCC hydrogel with and without PL and PL alone groups were subjected to heat treatment and protease digestion. Afterward, the HDFs were subjected to these treatments for 24, 48, and 72 h. Results of these findings are provided in Figure 13a,b. All data show the significant protective effect of CS on PL for proteases and heat treatment. The PL loaded with CS or CS-NCC over 72 h had significantly higher proliferation than those unprotected PL. This protective effect of chitosan is supported with similar work carried out by Fisher et al., that proposed the mechanism where chitosan fibers tightly bind with major plasma proteins and a specific sub-set of platelet surface proteins which results in the acceleration of fibrin gel formation when platelet integrins contact with plasma proteins. This result indicates that CS can protect the GF present in PL, thereby promoting fast wound healing. present in PL. CS along with NCC hydrogel with and without PL and PL alone groups were subjected to heat treatment and protease digestion. Afterward, the HDFs were subjected to these treatments for 24, 48, and 72 h. Results of these findings are provided in Figure 13a,b. All data show the significant protective effect of CS on PL for proteases and heat treatment. The PL loaded with CS or CS-NCC over 72 h had significantly higher proliferation than those unprotected PL. This protective effect of chitosan is supported with similar work carried out by Fisher et al., that proposed the mechanism where chitosan fibers tightly bind with major plasma proteins and a specific sub-set of platelet surface proteins which results in the acceleration of fibrin gel formation when platelet integrins contact with plasma proteins. This result indicates that CS can protect the GF present in PL, thereby promoting fast wound healing.

**Figure 12.** (**a**) Live Dead cell viability Assay at 200X magnification, (**b**) Cell counting treated with different hydrogels at 24 h and 72 h (**c**) % cell viability compared to control. *n* = 3. The asterisks (\*) statistically significant different (*p* < 0.05); ns—non-significant from control (untreated cells).

**Figure 13.** Chitosan stability studies on GF present in PL by cell proliferation study using Alamar blue assay (**a**) Effect of proteases treatment (**b**) Effect of heat treatment, *n* = 3, The asterisks (\*) statistically significant different compared to PL alone (*p* < 0.05); The hashtag (#) means statistically significant different (*p* < 0.05) between groups. **Figure 13.** Chitosan stability studies on GF present in PL by cell proliferation study using Alamar blue assay (**a**) Effect of proteases treatment (**b**) Effect of heat treatment, *n* = 3, The asterisks (\*) statistically significant different compared to PL alone (*p* < 0.05); The hashtag (#) means statistically significant different (*p* < 0.05) between groups.

proteases at the tissue site. To address the challenges related to PL instability, we developed a CS hydrogel combined with kenaf-derived NCC. It can stabilize PL and control the release of PL onto the wound site for prolonged action. Kenaf biomass-derived NCC at 0.4% concentration acted as the nanofiller for CS hydrogel and controlled PL release at a slower rate (maximum PL release of 3.00 ± 1.11 mg/cm2 at 24 h) compared with the CS hydrogel alone. FTIR study confirmed the presence of NCCs and PL in the CS matrix. Swelling data revealed that NCC incorporation in the PL-loaded CS hydrogel possesses high swelling ratio and water retention capacity (>1000 times at less than 3 h), thereby benefiting the healing of high exudate wounds. In vitro study revealed that the hydrogels are non-toxic to host tissues (>100% HDF cell viability) and are able to close wounds at a faster rate compared with the controls. The protective effect of CS upon GFs in PL was also demonstrated via protease and heat treatment of the hydrogels. Thus, NCC-reinforced CS hydrogel emerged as a promising PL vehicle especially for autologous wound therapy for chronic wounds. This new hydrogel PL carrier may be extended to other autologous PRP therapies, such as rheumatoid arthritis, bone regeneration, low-

**Author Contributions:** Conceptualization and methodology, S.-F.N.; writing—original draft preparation, S.-F.N. and P.B.; review and editing, J.X.L. and S.-F.N. funding acquisition, S.-F.N. All

**Funding:** This research is financially supported by the Dana Impak Perdana (DIP) grant (DIP-2018-

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

grade musculoskeletal injuries, and dental applications.

028), Universiti Kebangsaan Malaysia (UKM).

**Informed Consent Statement:** Not applicable.

corresponding author.

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

authors have read and agreed to the published version of the manuscript.

**4. Conclusions** 

#### **4. Conclusions**

In general, GFs in PL suffer clinical limitations because of rapid degradation by proteases at the tissue site. To address the challenges related to PL instability, we developed a CS hydrogel combined with kenaf-derived NCC. It can stabilize PL and control the release of PL onto the wound site for prolonged action. Kenaf biomass-derived NCC at 0.4% concentration acted as the nanofiller for CS hydrogel and controlled PL release at a slower rate (maximum PL release of 3.00 <sup>±</sup> 1.11 mg/cm<sup>2</sup> at 24 h) compared with the CS hydrogel alone. FTIR study confirmed the presence of NCCs and PL in the CS matrix. Swelling data revealed that NCC incorporation in the PL-loaded CS hydrogel possesses high swelling ratio and water retention capacity (>1000 times at less than 3 h), thereby benefiting the healing of high exudate wounds. In vitro study revealed that the hydrogels are non-toxic to host tissues (>100% HDF cell viability) and are able to close wounds at a faster rate compared with the controls. The protective effect of CS upon GFs in PL was also demonstrated via protease and heat treatment of the hydrogels. Thus, NCC-reinforced CS hydrogel emerged as a promising PL vehicle especially for autologous wound therapy for chronic wounds. This new hydrogel PL carrier may be extended to other autologous PRP therapies, such as rheumatoid arthritis, bone regeneration, low-grade musculoskeletal injuries, and dental applications.

**Author Contributions:** Conceptualization and methodology, S.-F.N.; writing—original draft preparation, S.-F.N. and P.B.; review and editing, J.X.L. and S.-F.N. funding acquisition, S.-F.N. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research is financially supported by the Dana Impak Perdana (DIP) grant (DIP-2018- 028), Universiti Kebangsaan Malaysia (UKM).

**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:** Hydrogel formulation work and facilities was provided by the Faculty of Pharmacy, UKM and special thanks to Centre for Tissue Engineering and Regenerative Medicine (CTERM) for supporting the cell work.

**Conflicts of Interest:** The authors declare that they have no known competing financial interest or personal relationships that could have appeared to influence the work reported in this paper.
