*Article* **Chitosan Reinforced with Kenaf Nanocrystalline Cellulose as an Effective Carrier for the Delivery of Platelet Lysate in the Acceleration of Wound Healing**

**Payal Bhatnagar <sup>1</sup> , Jia Xian Law 2,\* and Shiow-Fern Ng 1,\***

> <sup>1</sup> Centre for Drug Delivery Technology, Faculty of Pharmacy, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, Kuala Lumpur 50300, Malaysia; payalbhatnagar2020@gmail.com

<sup>2</sup> Centre for Tissue Engineering & Regenerative Medicine, 12th Floor, Clinical Block, UKM Medical Centre, Jalan Yaa'cob Latif, Cheras, Kuala Lumpur 56000, Malaysia

**\*** Correspondence: lawjx@ppukm.ukm.edu.my (J.X.L.); nsfern@ukm.edu.my (S.-F.N.); Tel.: +60-3-9145-7677 (J.X.L.); +60-3-9289-7977 (S.-F.N.); Fax: +60-3-9289-7060 (S.-F.N.)

**Abstract:** The clinical use of platelet lysate (PL) in the treatment of wounds is limited by its rapid degradation by proteases at the tissue site. This research aims to develop a chitosan (CS) and kenaf nanocrystalline cellulose (NCC) hydrogel composite, which intend to stabilize PL and control its release onto the wound site for prolonged action. NCC was synthesized from raw kenaf bast fibers and incorporated into the CS hydrogel. The physicochemical properties, in vitro cytocompatibility, cell proliferation, wound scratch assay, PL release, and CS stabilizing effect of the hydrogel composites were analyzed. The study of swelling ratio (>1000%) and moisture loss (60–90%) showed the excellent water retention capacity of the CS-NCC-PL hydrogels as compared with the commercial product. In vitro release PL study (flux = 0.165 mg/cm2/h) indicated that NCC act as a nanofiller and provided the sustained release of PL compared with the CS hydrogel alone. The CS also showed the protective effect of growth factor (GF) present in PL, thereby promoting fast wound healing via the formulation. The CS-NCC hydrogels also augmented fibroblast proliferation in vitro and enhanced wound closures over 72 h. This study provides a new insight on CS with renewable source kenaf NCC as a nanofiller as a potential autologous PL wound therapy.

**Keywords:** chitosan; kenaf; nanocrystalline cellulose; platelet lysate; wound healing

#### **1. Introduction**

In the last decade, biopolymers have attracted substantial attention mainly because of their renewability, biocompatibility, biodegradability, and abundance. Nanocellulose, a biopolymer derived from plants, is a promising biomaterial in skin grafts, implants, tissue engineering, and wound healing. Nanocellulose are classified into nanocrystalline cellulose (NCC) and cellulose nanofibrils (CNF). NCC consists of amorphous and highly ordered crystalline cellulose structures. NCC exhibits overwhelming performance as a nanomaterial because of its dimensions 5–100 nm in diameter with lengths up to hundreds of nanometers (100–600 nm). NCC can be obtained from inexpensive biomass renewable resources, such as wood, shrubs, and herbs. Plant-derived NCC is highly beneficial for wound dressing applications because of its high capability to absorb liquids and form translucent films [1]. These properties are crucial for non-healing and chronic wounds, where exudates need to be managed adequately. In addition, the translucency of NCC allows the wound development to be evaluated without needing to remove the dressing. Agricultural waste is a valuable source of advanced biomaterials. Nanocellulose may be obtained from agro-waste and further used as reinforcement in biopolymers. Kenaf (*Hibiscus cannabinus* L.) is an important industrial crop with high economic and ecological importance in many tropical countries, including Malaysia. Kenaf plant components that

**Citation:** Bhatnagar, P.; Law, J.X.; Ng, S.-F. Chitosan Reinforced with Kenaf Nanocrystalline Cellulose as an Effective Carrier for the Delivery of Platelet Lysate in the Acceleration of Wound Healing. *Polymers* **2021**, *13*, 4392. https://doi.org/10.3390/ polym13244392

Academic Editors: Bramasta Nugraha and Faisal Raza

Received: 22 October 2021 Accepted: 30 November 2021 Published: 15 December 2021

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**Copyright:** © 2021 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 of industrial importance include bast and core fibers. Bast fibers are isolated from the outer layer of plant fibers (30% of dry weight), whereas the core refers to the inner fiber layer (70% of dry weight). Bast fibers have been extensively investigated for their excellent mechanical properties because of their high length-to-diameter ratio and superior crystallinity, allowing these fibers to be applied as a reinforcing agent with other natural or synthetic polymers for biomedical applications [2]. NCC isolated from kenaf waste has been extended to various scientific research for their special characteristics, such as sustainability, affordability, and well-established mechanical, thermal, and electrical properties, which attracted scientists to develop new promising materials with unique values [3]. Currently, the use of kenaf-derived nanocelluloses is limited to the packaging and paper industries. The use of kenaf-based NCC as wound dressing has yet to be researched and explored.

Platelet lysate (PL) derived from platelet-rich plasma (PRP) is an autologous source of therapeutic proteins that is extensively researched in hard-to-heal wounds and tissue regeneration [4–6]. PL is a rich source of growth factors (GFs), including epidermal growth factor (EGF), insulin-like growth factor (IGF), transforming growth factor beta (TGF-β), platelet-derived growth factor (PDGF), which are crucial in regulating cell proliferation, migration, and differentiation by binding to specific transmembrane receptors on target cells. EGF secreted during the hemostasis of inflammation by platelets exhibits chemotactic effect on keratinocytes by promoting re-epithelialization [7,8]. TGF-β has three isoforms (i.e., TGF-β; 1–3). TGF-β1 is highly produced during wound healing by platelets, neutrophils, macrophages, and fibroblasts and is responsible for the synthesis of extracellular matrix components. IGF is discharged by platelets at the beginning of hemostasis. It entices leukocytes and participates in cell inflammation and proliferation. Thus, IGF serves a regulatory function in fibroblast proliferation in tissues. The PL serves as the cellular induction of normal wound healing responses and PL therapy has clinical significance regardless of the wound etiology. Hence, PL applications have gained considerable popularity in tissue regeneration and wound healing. GFs are released quickly from PL; thus, their activity and clinical efficacy are easily lost [9]. As a result, hydrogels or carriers suitable for PL are highly sought after by researchers.

Chitosan (CS) is a polysaccharide which chemically exist as (1→4)-2 amino-2-deoxy-β-D-glucan; originated from chitin, a key constituent of crustacean outer skeletons commonly used as an antimicrobial agent for preventing and treating infections owing to its natural antimicrobial property [10]. CS shows unique hemostatic properties and promotes the infiltration and migration of neutrophils and macrophages in early stages of wound healing, making it a suitable biopolymer for wound dressing [11]. CS exists in the form of 3D hydrogel polymer networks that can absorb and retain a large quantity of moisture on account of the abundance of hydrophilic groups [12]. CS dressings provoke minimal adverse reactions with little or almost absent fibrous encapsulation and provide protection against bacterial infections [13]. Recently, CS and modified CS have been deemed suitable for the delivery of PRP or PL to wounds [6–15]. Rossi et al. demonstrated via PDGF evaluation that CS dressings maintain the platelet GF in unaltered active form [14]. CS fibers also tightly bind with major plasma proteins and at specific platelet surface proteins. The combination of CS with PL shows the sustained release of GFs and increases glycoprotein IIIa expression in platelets [16].

Despite the significant therapeutic potential of PL in wound repair, PL generally suffers clinical limitations, such as short half-life, instability, degradation by protease at the tissue site, and toxicity at high systemic dose [17]. An efficient delivery system has been sought to stabilize PL for its sustained release in wound healing. The applicability of NCC as a potential cell culture scaffold has been previously reported because it provides the desired 3D environments for the growth and differentiation of skin cells [18,19]. A successful PL delivery system from hydrogels could be developed by mimicking tissue regeneration in terms of endogenous release profiles of PL. This research aimed to develop a CS hydrogel reinforced with NCC and loaded with PL as a controlled-release vehicle for wound healing. CS with a 2% *w*/*v* was employed as a PL stabilizing agent and cell growth-promoting polymer and added with NCC 0.4% *w*/*v* to provide a nanocellulose network that can facilitate the loading and delivery of PL in wound healing.

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

#### *2.1. Materials*

Kenaf raw bast fiber was provided by the National Kenaf and Tobacco Board (Lembaga Kenaf dan Tembakau Negara, LKTN), Malaysia. Low-molecular-weight (LMW, analytical grade) CS (molecular weight of 50–190 kDa, 75–85% degree of deacetylation) was purchased from Sigma Aldrich (Dublin, Ireland), glacial acetic acid (GAA; 100%) was purchased from R.M Chemicals Pvt. Ltd. (Chandigarh, India), PL, cell culture media, and primary skin cells were received from Centre For Tissue Engineering and Regenerative Medicine (CTERM), UKM. Enzyme-linked immunosorbent assay (ELISA) kit was obtained from Insphiro Technology (Selangor, Malaysia). Alamar Blue was obtained from Invitrogen (Waltham, MA, USA). Dulbecco's phosphate-buffered saline (DPBS) and fetal bovine serum (FBS) were purchased from Biowest (Riverside, MO, USA). Collagenase type I was obtained from Worthington (Columbus, OH, USA), Dulbecco's modified Eagle's medium (F12: DMEM; 1:1), antibacterial–antimycotic, Glutamax, and 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid (HEPES) were procured from Gibco (Grand Island, NY, USA). All chemicals, such as sodium hydroxide (NaOH), sodium chlorite (NaClO2), and sulfuric acid (H2SO4), were analytical grade (Dublin, Ireland).

#### *2.2. Preparation of Nanocrystalline Cellulose from Kenaf Bast Fibers*

NCC was extracted from raw kenaf bast fibers adapted from previous method with slight modifications [20]. Briefly, the retting of raw bast fibers was carried out by soaking it in distilled water overnight and filtered several times with subsequent drying at 60 ◦C in the oven. The dried fibers were ground and sieved to obtain finer fibers. Thereafter, pulverization was carried out by treating the fibers with 5% (*w*/*v*) NaOH at 80 ◦C for 2 h, and this step was carried out thrice to accomplish delignification. The fibers were then thoroughly washed and filtered using distilled water to remove residual chemicals. Subsequently, the alkali-treated fibers were bleached using a combination of acetic acid and sodium chlorite to remove residual lignin and hemicellulose. The bleached pulp was filtered and washed numerous times after a series of bleaching procedures until the pH reached 7. The bleaching treatment was repeated five times at 80 ◦C for 3 h to obtain white fibers and thereafter stored in water swollen state. Finally, acid hydrolysis was commenced by immersing 4% (*w*/*w*) bleached kenaf bast fibers into 65% (*v*/*v*) sulfuric acid at a temperature of 45 ◦C for 30 min. The fiber suspension was constantly homogenized using a magnetic stirrer. Then, the fiber suspension was diluted by adding distilled water and allowed to cool. Afterwards, the acid was removed by centrifuging the fiber suspension at 12,000× *g* rpm for 30 min, and this process was repeated five times for complete removal of residual acid. Then, the fiber pellet was dispersed in distilled water and poured into a dialysis bag (MWCO 11 KDa; cellulose acetate) under slow stirring until complete neutralization. Subsequently, the suspension of tiny fibers was ultrasonicated for 5 min at a frequency of 20 kHz with an amplitude of 80%. This process was carried out under an ice box to prevent overheating. The resulting thick suspension containing the NCC was stored at 4 ◦C and freeze-dried for further characterization and hydrogel preparation.

#### *2.3. PL Processing and Quantification of GFs*

The PL was processed aseptically by freeze-thawing for one cycle and subsequently centrifuged at 5000× *g* rpm at 4 ◦C for 20 min. The resulting pellet containing cell debris was discarded and the processed PL was kept at −80 ◦C with the addition of 40 IU of LMW heparin as an anticoagulant until further use.

The concentrations of key GFs (EGF and TGF) were determined using ELISA based on Sandwich-ELISA using 96-well plates in accordance with the manufacturer's instructions. Optical density (OD) were measured under 450 nm wavelength using a microplate reader Bio-Rad (Berkeley, CA, USA).

#### *2.4. Preparation of CS Hydrogel and CS-NCC Composite Hydrogels*

The porous CS hydrogels were prepared as previously described method with slight modifications [21]. In brief, 2% (*w*/*v*) CS solution was prepared by dissolving fine CS powder in 1% GAA, and pH was adjusted to 7.0 using 1 M NaOH. Subsequently, the solution was stirred on a magnetic stirrer for 1 h at room temperature. Afterward, CS solution was transferred into 24-well tissue-culture plates and frozen at −20 ◦C for 24 h, followed by freeze drying (Labconco, Topeka, KS, USA, Model no.117; weight-24 kg) at −80 ◦C for 4 days to ensure complete drying.

CS hydrogels containing PL (CS-PL) were made by mixing 1 mL of PL/gram of CS hydrogel (2% *w*/*v*) on a magnetic stirrer for 30 min at 20 ◦C. Then, the mixture was transferred to a 24-well tissue-culture plate for lyophilization at −80 ◦C for 4 days.

As for the CS-NCC composite hydrogel, NCC was dispersed in deionized water (DI) to produce a 0.4% (*w*/*v*) concentration [22]. Then, the slurry of NCC was added to the 2% (*w*/*v*) solution of CS. The resulting dispersion was ultra-sonicated at an amplitude of 40% for 5 min to homogenize NCC in CS solution and then allowed to mix for 1 h on a magnetic stirrer in a closed reactor. Then, acetic acid (1%) was added to solubilize the CS and the mixture was mechanically stirred for the next 5 h to obtain a complete dissolution of CS. Consequently, hydrogels were formed through the neutralization of the viscous suspension of CS by pouring it on a petri plate containing 1 M NaOH for 1 h. The prepared hydrogels were washed with distilled water until a neutral pH was obtained. Finally, the composite hydrogel of CS-NCC without using any specific cross-linkers were prepared and subsequently freeze-dried for further characterization. The CS-NCC-PL gel was prepared by thorough mixing of the CS-NCC gel with PL (1 mL/g). The CS-NCC gel was stored in the refrigerator at 4 ◦C. Upon use, PL (stored at −80 ◦C) was thawed to room temperature prior and added into the CS-NCC gel via simple mixing. The unused CS-NCC-PL hydrogel was stored at −20 ◦C but thawed to room temperature before tests.

#### *2.5. Characterization of NCC and Hydrogels*

#### 2.5.1. Chemical Composition, Fiber Yield, and Zeta Potential

Chemical composition of NCC was determined in accordance with TAPPI standard methods T 222 (acid-insoluble lignin in wood and pulp) [23]. The fiber yield of NCC was calculated in terms of percentage (%) of the initial weight of bleached fibers after hydrolysis. The suspension of the fiber obtained after dialysis treatment was freeze-dried and compared with the initial weight of fiber. The final weight of NCC (*Mf*) and the initial weight of fiber (*Mi*) were measured to calculate the yield using Equation (1) [24]. Fiber yield was calculated using the following equation.

$$\text{Yield (\%)}=\text{(Mf/Mi)} \times 100\tag{1}$$

The zeta-potential of NCC was determined using Zetasizer Nano-ZS (Malvern Instruments Ltd., Malvern, UK) to identify the electrical charges of NCC. The NCC samples were tested after acid hydrolysis of the fiber suspension.

#### 2.5.2. X-ray Diffraction Characterization (XRD)

XRD patterns of bleached and NCC from kenaf bast fibers were performed on X-ray diffractometer (Empyrean PANalytical, Marvin, UK) to examine the changes in crystallinity before and after chemical treatment. The diffraction intensity of Cu Kα radiation (λ = 0.1542 nm; 40 kV and 40 mA) was measured in a 2θ range between 5◦ and 70◦ with scan rate of 0.5◦ per min. Peak analysis was performed using Diffrac.EvaV4.0 software. Meanwhile, the crystallinity index of fibers was calculated as previously described method [25] using the following Equation (2).

$$\text{CrI} \text{ (\%)}=\text{[I}\_{002} - \text{I}\_{\text{amorphous}}/\text{I}\_{002}] \times 100\tag{2}$$

where CrI is the crystallinity index is the maximum peak intensity at the (002) plane (around 22.5◦ for native cellulose) and Iamorph is the minimum intensity of the amorphous portion taken at 2θ = 18◦

#### 2.5.3. Scanning Electron Microscopy (SEM)

A SEM instrument (Carl Zeiss Merlin Compact-Germany, Oberkochen, Germany) was used to observe the surface morphology of NCC. The acceleration voltage was set up in the range of 5 to 20 kV, and dried samples were sputter-coated with gold to avoid the charging effect during SEM observations. The fiber diameter was measured by using Smart TIFF image viewer software.

2.5.4. Fourier Transform Infrared Spectroscopy-Attenuated Total Reflectance (FTIR-ATR) Spectroscopy

FTIR-ATR spectroscopy (Spectrum 100; Perkin Elmer, Walthman, MA, USA) was performed on raw fibers, bleached fibers, NCC, CS, PL, CS-NCC, CS-PL, and CS-NCC-PL hydrogels.

Infrared spectra of CS hydrogel control, PL, NCC, CS hydrogel-reinforced PL, and NCC were determined between 4000 and 650 cm−<sup>1</sup> using FTIR-ATR spectrometer (Spectrum 100; Perkin Elmer, Walthman, MA, USA). The spectra were acquired using 32 scans and a 4 cm−<sup>1</sup> resolution.

#### 2.5.5. Swelling Behavior of Hydrogels

The swelling behavior of freeze-dried samples of CS, CS-NCC, CS-NCC-PL, CS-PL hydrogel, and Intrasite™ gel as positive control were tested in DI at 37 ◦C for 3, 6, and 24 h. The dried samples were weighed and placed in 20 mL of DI, and the hydrogels were allowed to reach their swelling equilibrium. The weights of the hydrogels (W2) were measured at 3, 6, and 24 h of duration. All formulations were run in triplicates, and average values were presented. The swelling ratio was calculated using the following Equation (3).

$$\text{Swelling ratio (\%)} = (\text{W2} - \text{W1}) / (\text{W1}) \times 100 \tag{3}$$

where W1 is the weight before swelling and W2 is the weight after swelling

#### 2.5.6. Moisture Loss Study

The moisture retention capacity of the hydrogels was evaluated using the desiccant method of Standard Test Method for Water Vapour Transmission of Materials (ASTM E96/E96M-16, 2015) [26]. Hydrogel samples (0.5 g, triplicates) were spread in thin layers in a 2 cm-diameter crucible. The samples were then placed into airtight containers lined with a bed of silica gel as a desiccant. After 24 h, the crucibles containing the samples were re-weighed and percentage of moisture loss was calculated using the following Equation (4).

$$\text{Moisture Loss (\%)} = \text{(W2/W1)} \times 100\tag{4}$$

where W1 is the weight of the crucible before drying and W2 is the weight of the crucible after drying. Moisture loss was the water loss from the exposed surface of hydrogels over 24 h. The test was conducted on the CS, CS-NCC, CS-PL, CS-NCC-PL hydrogels. Intrasite™ gel served as the positive control.

#### 2.5.7. In Vitro Protein Release Assessment

The in vitro release profile of PL was investigated on two hydrogel preparations: CS-PL and CS-NCC-PL. Protein release was studied using Franz diffusion cell (Permegear

Inc., Hellertown, PA, USA) in triplicates. All samples were prepared by mixing 5 mg/g of PL in hydrogel, and the proteins were incorporated into the hydrogel overnight at −10 ◦C. The release study was conducted using a cellulose acetate membrane with a mesh size of 0.45 µm. DI at pH 7.0 was used as the receptor medium. The receptor chamber was filled with DI until the mark of the sampling port was reached. The cellulose acetate membrane was then placed on top of the receptor chamber opening (0.7855 cm<sup>2</sup> ). The receptor chamber was maintained at 37 ± 1 ◦C in a circulating water bath. The hydrogel weighing 1 g was placed on top of the cellulose acetate membrane, and all of the compartments were held together with a clamp. Samples (1 mL) were withdrawn at predetermined time intervals at 3, 6, and 24 h. The receptor medium was replaced with an equal volume of DI to maintain the sink conditions. The released proteins in the media were added with a few drops of Bradford reagent and assayed using a UV-Vis spectrophotometer (Shimadzu, Kyoto, Japan) at 280 nm. DI served as a blank. The absorbance of each sample was determined and recorded. The concentration of unknown proteins was then calculated by plotting the calibration curve. The cumulative amount of proteins that permeated out of the membrane was calculated and plotted against time (h). This experiment was repeated in triplicates for the in vitro release and characterization of therapeutic proteins present in PL that were incorporated in the hydrogel. A validated UV spectrophotometric method was used to quantify the PL loaded in the hydrogel systems at 280 nm to determine the concentration of proteins released at 3, 6, and 24 h.

#### *2.6. Cytocompatibility Studies*

#### 2.6.1. Isolation of Human Dermal Fibroblast (HDF)

This study was approved by the Universiti Kebangsaan Malaysia Research Ethics Committee (UKM PPI/111/8/JEP-2021-052). Redundant abdominoplasty skin tissue samples were received from the patient with written informed consent and processed within 48 h with the ISO protocol followed at CTERM. The skin was completely rinsed in DPBS, cut into small pieces (1–2 cm<sup>2</sup> ), and immersed overnight in 10 mL of serum-free Epilife medium containing 25.3 mg of Dispase at 2–8 ◦C to isolate the epidermis from the dermis layers. On the next day, the epidermis layer was separated from the dermis layer. Thereafter, the dermis was chopped into smaller pieces and digested with 0.6% collagenase type I (Worthington, Columbus, OH, USA) for 4–8 h in an incubator shaker maintained at 37 ◦C. The cell suspension was centrifuged at 5000× *g* rpm for 5 min at 37 ◦C, and the cell pellet was rinsed with DPBS after trypsinization with TE-EDTA (0.05%). Finally, the cell pellet was re-suspended in F12: DMEM (1:1) supplemented with 10% FBS, 1% antibacterial–antimycotic, 1% Glutamax, and 2% HEPES. Cells were cultured at 37 ◦C in 5% CO<sup>2</sup> with the medium changed every 2–3 days.

#### 2.6.2. Cell Culture

HDFs were used to study the cytocompatibility of the hydrogels. Cells with the passage of P1-3 were cultured in F12: DMEM supplemented with 10% FBS at 37 ◦C in an incubator supplemented with 5% CO2. PL (10%) was used alone, as well as in all hydrogel formulations. The culture medium was replenished every 2–3 days and subsequently trypsinized once more than 90% confluence was achieved and transferred to T75 flasks for further assay.

#### 2.6.3. Cell Viability

The cytotoxic effect of the fabricated hydrogels (CS, CS-NCC, and CS-NCC-PL) on skin cells was investigated. Cell viability assay was performed indirectly using primary HDFs (CTERM, UKM Medical Centre, Kuala Lumpur, Malaysia) cultured in DMEM by utilizing Alamar Blue. All cells were maintained at 37 ◦C in humidified 5% CO<sup>2</sup> atmosphere. In this study, all hydrogel samples were washed with sterile DPBS and then incubated in a culture medium for 24 h to obtain the leachates (membrane sterilized 0.22 µm) from the hydrogel. HDFs with passage number P2 were seeded onto a 96-well plate at a density

of 10<sup>4</sup> cells/well and incubated in fresh media for 24 h at 37 ◦C to ensure cell attachment and proliferation. Leachates from the hydrogels were sterilized by membrane filtration (0.45 µm). Afterward, the culture medium was replaced with leachates of the hydrogels of volume 200 µL in each well. The microplate was then incubated for 24, 48, and 72 h, and 10% of Alamar Blue was added at the end of each time points and incubated for 4 h in the dark. Finally, OD was measured at 570 nm using a microplate reader (BioTek PowerWave XS, Winooski, VT, USA). The results were calculated as percentage cell viability relative to the control group (cells without hydrogel treatment) from Equation (5).

$$\text{Cell viability (\%)} = \text{[OD of treated/OD of control]} \times 100\tag{5}$$

## 2.6.4. Cell Proliferation

All hydrogel samples were placed in a micro well plate and washed with sterile DPBS prior to analysis. Thereafter, culture media were added and then incubated for 24 h to retrieve the extract from the hydrogels indirectly as previously mentioned in cell viability assay. HDFs (1 <sup>×</sup> <sup>10</sup><sup>4</sup> ) were seeded in a 96-well plate for 24 h and subsequently treated with sterile hydrogel extracts. DMEM was used as control and incubated for 24, 48, and 72 h. Alamar Blue cell proliferation assay was carried out as per the manufacturer's protocol in accordance with previous work [27]. Alamar Blue (10%) was added into each well containing the control and treatment groups and incubated at 37 ◦C for 4 h in the dark. Absorbance was recorded using a spectrophotometer (BioTek, PowerWave XS, USA) at 570 and 600 nm. The percent reduction in Alamar Blue was calculated from following [Equation (6)].

Percent reduction (%) = [(εOX) λ2Aλ1 − (εOX) λ1Aλ2]/[(εRED) λ1A'λ2 − (εRED) λ2A'λ1] × 100 (6)

where (εOX) λ2 = 117,216, (εOX) λ1 = 80,586, (εRED) λ1 = 155,677, (εRED) λ2 = 14,652. Aλ1 and Aλ2 = Observed absorbance reading for the test well at 570 nm and 600 nm, respectively. A'λ1 and A'λ2 = Observed absorbance reading for control well at 570 and 600 nm, respectively.

#### 2.6.5. Scratch Wound Assay

The in vitro wound scratch assay is an economic and fast method to predict the wound healing ability of compounds by assessing their migration rate on the skin cells. In this work, the migration rate of the HDFs was calculated via scratch assay to ensure that the hydrogel formulations do not interfere in wound healing. The HDFs were seeded in a 12 well plate (Greiner Bio-One, Kremstest, Austria) and then incubated at 37 ◦C in humidified 5% CO<sup>2</sup> atmosphere until 100% confluence. The spent culture was discarded, and a scratch was made at the middle of each well on a cell monolayer using a sterile 10 µL pipette tip. Afterward, the cells were rinsed with DPBS by slight swirling the microplate, and different hydrogel extracts were subsequently added to the scratched cells. Cells without treatment served as the control group. Wound closure was observed through live imaging by acquiring images every 60 min for 72 h at three spots per well using a Nikon A1R-A1 CLSM. Cell migration rate was calculated for 24 h. The tissue-culture plate inclosing cells was fixed inside the Chamlide Incubator System (Live Cell Instrument, Seoul, Korea) at 37 ◦C and 5% CO2. The images were analyzed by NIS Elements AR 3.1 (Nikon). The migration rate of the cells was calculated from following [Equation (7)].

Cell migration rate = (measurement at 0 h − measurement at 24 h)/24 h (7)
