Development of Decellularized Fish Skin Scaffold Decorated with Biosynthesized Silver Nanoparticles for Accelerated Burn Wound Healing

In this study, decellularized fish skin (DFS) scaffold decorated with silver nanoparticles was prepared for accelerating burn wound healing. The silver nanoparticles (AgNPs) synthesized by the green and facile method using Aloe vera leaf at different incubating times were characterized by using X-ray Diffraction (XRD), Fourier Transform Infrared (FT-IR) Spectroscopy, and Ultraviolet-Visible Spectroscopy (UV-Vis spectroscopy). The different characterizations confirmed that the sizes of AgNPs prepared by incubating for 6 hours and 12 hours were 29.1 nm and 35.2 nm, respectively. After that, the different concentrations of the smallest AgNPs were used to dope the DFS scaffold to determine the cell viability. Additionally, an agar well diffusion method was used to screen for antimicrobial activity. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were used to correlate the concentration of AgNPs with its bactericidal effect which was seen from 50 μg/ml. Then, the toxicity with human cells was investigated using a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay with no significant cell viability from the concentration of 50 μg/ml to 200 μg/ml compared to the cocultured and commercial treatments.


Introduction
Globally, it is reported that 2,65,000 deaths occur each year from fres alone, and over 96% of fatal fre-related burns occur in developing countries including Nepal. In the context of Nepal, burn is the second most common injury in rural areas accounting for 5% of disabilities. Te leading cause of death of burn victims is due to infection [1]. In our country, with the lack of biobanks, burns are washed, treated, and covered in gauge bandages instead of the human or porcine skin [2]. Te bandage should be changed daily, otherwise the leaching problem can happen [3]. Te frequent changing of the bandages would expose the wound to bacteria and microbes, which leads to major disorder and impediment in wound repair. To overcome such problems, it is most necessary for an alternative system to heal burn wounds.
Tere has been intense investigation to fnd new natural biomaterials based on decellularized unbroken fsh skin scafold which can be used as the skin substitute in case of a severe burn. Te tilapia fsh skin has shown a high content of collagen types I and II proteins along with a great tension resistant property and enormous moisture content than human [4]. Moreover, such skin scafold also has been shown a less healing time and lessening in pain [5].
Te numerous nanoparticles were invented and used as a promoter of the wound healing process to improve the healing quality. Te nanoparticles, whose surface areas increase exponentially, are the key assets that determine their medical applications. Among diferent metal nanoparticles, silver (Ag), gold (Au), titanium (Ti), and zinc oxide (ZnO) are the most used and safe contender for wound dressing [6]. Among them, AgNPs are currently used nanoparticles, as therapeutic agents for burn wound healing due to their antimicrobial and anti-infammatory properties [7]. It is commonly used in the treatment of burns, wound infections, and diferent ulcers and also to improve infection prevention strategies [8]. Moreover, AgNPs possess features to modulate anti-infammatory cytokine release in order to promote the wound healing process and stimulate epidermal re-epithelialization through proliferation and relocation of keratinocytes [3,9]. Furthermore, it forms sulfuric bonds with either bacterial cell membrane proteins or thiol groups of various enzymes, especially those involved in the respiratory chain, and ultimately kills the cell. It can also interfere with deoxyribonucleic acid (DNA) with sulphur and phosphor bonds inhibiting the bacterial multiplication [6,9,10].
Te fsh skin is the envelope of the body that separates and protects the fsh from its environment. Te skin plays a relevant role in protection, communication, sensory perception, locomotion, respiration, ion regulation excretion, and thermal regulation [11]. Likewise, the fsh skin also plays a vital role in medical applications such as regenerating tissue in trauma, chronic wound-like burns, and diabetic wounds [12].
Recently, the fsh skin has been used broadly in the management of skin burns. Nile Oreochromis niloticus fsh skin which is commonly called tilapia has been used widely in medical applications [13]. Tis fsh is commonly found and cultivated in Tailand and Brazil. Tis fsh skin has the highest collagen content, i.e., collagen types I and III and Omega-3, which helps in the speedy recovery of burn [14,15]. Te Omega-3 content has been found to proliferate keratinocytes that enhance the wound healing process [16]. Collagens are the major proteins of extracellular matrix (ECM) which are the most abundant fbrous proteins in fsh. ECM is a three-dimensional network of extracellular macromolecules, such as collagen, enzymes, and glycoproteins, that provide structural and biochemical support of surrounding cells [17]. Similarly, Omega 3 helps in preventing extensive wound. Likewise, keratin fghts against diseasecausing microbes [18].
Researchers in Brazil created a sterilized tilapia skin wound dressing and carried out clinical trials for second and third-degree burns. So far, more than 56 patients have received this treatment [19]. Recently, tilapia collagen nanofbers were found to quickly and efectively promote the skin wound healing process and were also shown to promote cell adhesion, proliferation, and diferentiation in rats [20]. Moreover, another study carried out in vitro and in vivo experiments for wound healing evaluation and the results illustrated the promising application of marine collagen peptides from the tilapia skin [21].
In present days, the biosynthesis methods using plant extract are being immensely utilized in order to synthesize nanoparticles over the common chemical and physical methods [22]. Physical and chemical synthesis that uses speed, radiation, and chemicals as reducing agents requires high temperature, energy, and time to obtain nanoparticles [23]. Moreover, the toxic chemicals used in the chemical synthesis process likely generate harmful waste that might afect internal health if used as biomedical applications [24,25]. Likewise, chemically synthesized nanoparticles barely show any therapeutic properties [26]. On the other hand, the biosynthesized nanoparticles contain medicinally active phytochemicals from the plants that can be used as therapeutic agents [27,28]. In addition, plants have a complex network of antioxidant metabolites and enzymes that act together to protect cellular components from oxidative damage. Tis method is also eco-friendly, costefective, and follows an easy procedure comparatively with high yields [29,30]. Researchers have been exploring diferent resources available in the environment such as microorganisms, plants and plant extracts, diferent templates as virus's DNA, membranes, and diatoms to obtain the desired form of nanoparticles as per the feld of application.
Plant extracts ofer high-quality manipulation and control over crystal growth and their stabilization. In order to obtain nanoparticles with desired shape, size, and dispersity, the biosynthesis methods are performed using plant extracts [31]. Various plants such as Medicago sativa, Aloe vera leaves, Azadirachta indica leaves, Camellia sinensis, Capsicum annuum, Cinnamomum camphora leaves, Datura metel, Emblica ofcinalis fruit, Ocimum sanctum, and Geranium leaves have all been used to synthesize various metal nanoparticles [32,33]. When the metal core is capped with biological components, the therapeutic activity of these nanoparticles synthesized by plant-mediated synthesis is signifcantly improved [6].
Aloe vera extracts can be found in a wide range of medicinal and dermatological treatments. Many studies show that the Aloe vera herb may efectively heal burns, sunburns, infammatory skin diseases, and wounds when used topically [34]. Monosaccharides and polysaccharides, tannins, sterols, organic acids, enzymes, saponins, vitamins, and minerals are all found in Aloe vera [35]. Alpine, an anthraquinone heterosis, is the most active component in Aloe vera plant extract. Many researchers mentioned that Aloe vera inhibits the growth of some microorganisms, such as Streptococcus pyogenes and Shigella fexneri, against Gram-positive bacteria that cause food poisoning or diseases in humans and animals [36]. AgNPs synthesized with Aloe vera extract helps to incorporate benefciary components of Aloe vera in nanoparticles for the better healing process of burn wound [3].
Many illnesses in humans are associated with the accumulation of free radicals. Antioxidants are substances that supply electrons to damaged cells in order to prevent and stabilize the damage caused by free radicals [37]. It is generally understood that oxidation damages a variety of biological components which creates a variety of illnesses. Tus, several scientifc articles have been published which describe the close relationship between the oxidative damage and diferent illnesses caused by this, such as cancer, liver disease, Alzheimer's disease, aging, arthritis, infammation, diabetes, Parkinson's disease, atherosclerosis, and AIDS [38]. Hence, antioxidants have been used to treat a variety of illnesses in order to avoid oxidative damage.
To the best of our knowledge, there has not been any report on the composite scafold incorporating biosynthesized AgNPs on the fsh skin for the accelerated burn wound healing process. So, we are motivated to prepare a naturally obtained healing approach by incorporating biosynthesized AgNPs on the fsh skin.

Biosynthesis of AgNPs. AgNPs were biosynthesized from
Aloe vera extract using the hydrothermal method.

Preparation of Aloe vera Extract.
Te fresh leaves of an adult Aloe vera plant were selected and harvested. Te leaf base was cut and left for a few minutes to drain out the yellow resins. Ten, the leaves were washed with sterile water and cut into small pieces. After that, 50 g of leaves and 50 ml deionized water were boiled for 20 minutes and cooled at room temperature [3]. Te plant extract was fltered using Whatman flter paper and stored in the refrigerator at 4°C. Finally, the obtained plant extract was used as a reducing agent as well as capping/stabilizing agent in the synthesis of AgNPs. Te extracted Aloe vera which was used for the preparation of AgNPs from the hydrothermal method is shown in Figure S1 (supporting information).

Biosynthesis of AgNPs.
Te biosynthesized AgNPs were prepared using the hydrothermal method [3]. For this, 50 mg silver nitrate (AgNO 3 ) was added in 20 ml deionized water. It was then mixed with 20 ml of extract solution by vigorously stirring at room temperature for 30 minutes. Te mixture was then added to a sealed Tefon-lined vessel and incubated at 100°C for 6 hours and 12 hours. A grey precipitate was collected and purifed by redispersing with distilled water followed by centrifugation at 12,000 rpm for 15 minutes to obtain a pellet of AgNPs. Te centrifuging and redispersing processes were repeated for three times. Finally, AgNPs were obtained upon drying. Te reduction of clear AgNO 3 to brown color solution by Aloe vera extract incubated at 100°C for 6 h and 12 h and the obtained corresponding powder of AgNPs are shown in Figure S2 (supporting information).

Characterization of AgNPs.
FT-IR analysis was used to analyze the presence of diferent functional groups of synthesized AgNPs. Tis is the preferred method of infrared spectroscopy. In this instrument, the monochromator and the slits are replaced by an interferometer, usually of the Michelson type. Te diferent chemical structures produce diferent spectral fngerprints. Terefore, infrared spectroscopy is one of the most common and widely used spectroscopic techniques which can be used as a fngerprint for the purpose of comparing molecules.
XRD measurement of AgNPs synthesized by the Aloe vera leaf was carried out using Cu-Kα radiation source in the 2θ range of 20-80 at room temperature. Te primary use of this technique is the identifcation and characterization of compounds based on their difraction pattern. XRD relies on the dual wave/particle nature of X-rays to obtain information about the structure of crystalline materials. Tus, this was used to determine the phase variety and grain size of synthesized AgNPs. Te crystallite size of the synthesized AgNPs was determined by using Scherrer's equation as follows [45]: where D � average crystallite size, ß � line broadening in radians, λ � wavelength of X-ray, θ � Braggs angle, and k � constant. Te prepared AgNPs were further analyzed by UV-Vis spectroscopy for structural characterization. UV-Vis spectroscopic measurements were performed at room temperature using the UV-Vis spectrometer.

Antioxidant Assay.
Te antioxidant activity of the biosynthesized AgNPs was determined by the free radical scavenging assay, where the free radical used was 2, 2-International Journal of Biomaterials diphenyl-1-picrylhydrazyl (DPPH). Te free radical scavenging activity (RSA) was determined by monitoring the change in optical density (OD) of DPPH radical. For this assay, a stock solution of AgNPs, Aloe vera extract, and ascorbic acid was prepared using methanol. Ten, a serial dilution was carried out to obtain solutions of 200, 100, 50, and 25 μg/mL. A fresh DPPH solution of 0.2 mM was prepared by dissolving 7.88 mg of DPPH powder in 100 ml of methanol. Ten, a volume of 50 μl of each sample (25-200 μg/mL) was mixed with 150 μl of DPPH in a 96-well plate in triplicate. Te control was prepared by replacing the same volume of sample with absolute methanol. After 30 minutes of incubation at room temperature, the absorbance was measured at 517 nm using a Chromate ELISA reader. Te %RSA was calculated by using the following formula: (2)

Antibacterial Tests.
Te antibacterial activity was observed on Pseudomonas aeruginosa and Staphylococcus aureus by the agar well difusion test and broth dilution method. Te AgNPs were used as the test sample, while Aloe vera extract and distilled water were used as negative control, and Penicillin was used as positive control. Te susceptibility test was evaluated by the agar well difusion method on agar plates. Te agar plates were inoculated with bacterial strains under aseptic conditions, and the punched wells (6 mm diameter) were flled with test samples and incubated at 37°C for 24 hours. After incubation period, the diameter of the growth inhibition zones was measured. Te experiments were carried out in triplicates. Te MIC of the AgNPs was checked using 6 test tubes containing bacteria (S. aureus and P. aeruginosa) grown in a nutrient broth. Te 2 ml of bacteria containing broth was taken in all the tubes. Te MIC of AgNPs was tested through the serial twofold dilution's method. Te colloidal AgNPs solution with the concentrations from 12.5 μg/ml to 200 μg/ ml were taken (i.e., 12.5 μg/ml, 25 μg/ml, 50 μg/ml, 100 μg/ ml, and 200 μg/ml) and added at the test tubes and incubated for 8-10 hours. Te MIC was determined by the evaluation of turbidity of tubes with a constantly increasing concentration of antimicrobial agents. Te point at which no turbidity was observed was taken as MIC. Ten, MBC was measured after MIC determination. For this, AgNPs were pipetted onto nutrient agar plates and incubated at 37°C for 24 hours. Te MBC value was interpreted at the lowest concentration of colloidal AgNPs at which inoculated bacterial strains were completely killed.

Preparation of Decellularized Fish Skin Scafold.
Tilapia fsh were obtained from the Centre for Aquaculture-Agriculture Research and Production Pvt. Ltd (CAARP), Chitwan, Nepal. Te adult tilapia fsh, extraction skin tissue from tilapia fsh, and decellularized fsh skin scafold are shown in Figure S3 (supporting information). At frst, the fsh was descaled and the skin was peeled with the help of a sharp knife. Ten, the skin samples were washed two times with sterile phosphate bufer saline (PBS) containing 50 mm ascorbic acid and 500 ppm streptomycin to make it germfree. Tus, the prepared skin sample was used for further processing [46].
For decellularization, the skin sample was incubated in a PBS solution containing 0.5% Triton X and 0.02% sodium azide for 2 hours in 40 rpm at room temperature. Ten, the sample was washed two times with Hank's balanced salt solution (HBSS) for 10 minutes in 40 rpm at room temperature. Te sample was again treated with a decellularizing solution containing 0.5% sodium dodecyl sulphate (SDS) for 1 hour at the same rpm and temperature. Te resultant sample was washed with PBS [46].
Ten, the decellularized skin sample was incubated in a digestion containing 1 M Tris-HCl and 0.05 g/ml trypsin maintained at a pH of 8.5 for 2 hours at room temperature. Ten, the resultant decellularized skin sample was immersed in a prefreezing solution containing 7% dextran, 6% sucrose, 6% rafnose, and 1 mm EDTA in HBSS and stored at −20°C. Finally, these prepared samples were cut into diferent dimensions as required before being used for testing.

Histological Analysis.
Histological analysis was carried out to ensure the decellularization of fsh skin scafold. Te skin specimen was embedded in parafn blocks and sectioned with microtome. Sections of the fsh skin and decellularized fsh skin scafold were mounted on glass slides separately and stained with hematoxylin and eosin (H and E).
Te slides that were fxed using formaldehyde were dipped in distilled water. Tey were then transferred to the beaker containing haematoxyl in solution for 10 minutes. Tose were then transferred to the beaker under running tap water for 10 minutes. Tey were dipped in the acid alcohol (1 ml of HCl in 90 ml of 70% ethanol) unless they were converted to red. Te slides were then kept under the running tap water for 30 minutes. Tus, obtained slides were transferred to the beaker containing 80 ml of distilled water and were left for 10 minutes. Ten, they were dipped into a beaker containing eosin for 1 minute and were transferred to the beaker containing distilled water (80 ml) where those were only dipped. Te slides were then dipped in 70% ethanol followed by 95% and then absolute alcohol. Tus, resultant slides were then air-dried and dipped in the beaker containing xylene I for 10 minutes. Again, it was transferred to the beaker containing xylene II for 10 minutes. Finally, a drop of DPX was added on the top of the resultant slide and was covered gently with the cover slip so that there is no formation of bubbles.

In Vitro Degradation Rate Test.
Te DFS scafolds were cut into 2 cm × 2 cm dimensions. Te dry weights of the scafolds were taken. Ten, the scafolds were kept in 2 ml PBS solution (pH 7.4) under aseptic conditions in a sealed tube and incubated at 37°C for 1, 2, 3, 4, 5, 6, 7, 14, 21, and 28 days. After each time point, scafolds were taken out and dried and weighted.
Te degree of degradation of each scafold was defned as the weight loss percentage calculated by the following equation: where W 0 denotes initial weight of the dried scafold and W t denotes the weight of degraded scafold after respective time intervals.

Swelling Test.
For the swelling test, the dry weights of the scafolds were recorded before immersion. Ten, the scaffolds were immersed in PBS solution (pH 7.4) under aseptic conditions in a sealed tube and incubated at 37°C for 24 hours. Ten, the wet weights of the scafold were recorded. Te degree of the swelling capacity was calculated using the following equation: where W 0 represents the initial weight of the dried scafold before immersion and W t represents the weight of the scafold after immersion.

Moisture Content Test.
Te DFS scafolds were cut into 2 cm × 2 cm dimension. Te initial weights of the scafolds were recorded. Ten, the samples were kept in a desiccator containing calcium chloride (CaCl 2 ) until they reached the constant dry weight. Te amount of moisture contained in the scafold was defned as the weight loss of scafold upon drying in the desiccator.

Mechanical Test.
A folding endurance test was performed to test the mechanical strength of the DFS scafold. Six DFS scafolds were subjected to repeat folding at the same place until it breaks. Te folding endurance was given by the number of times the sample could be folded at the same place without breaking.

Permeability
Test. An apparatus was created by using two test tubes. Te scafold of 2 cm × 2 cm was wrapped between the test tubes. One test tube was flled with saltwater for higher concentration, and another was flled with distilled water for lower concentration. After 24 hours, levels of both the liquids were observed. Permeability of scafold was calculated by measuring the increased volume of salt solution. Te experiment was carried out in triplicates. Te amount of colloidal AgNPs absorbed into the DFS scafold was determined by comparing the absorbance of the AgNPs solution before and after doping. Te AgNPs' loading capacity of DFS scafold was calculated as the amount of the absorbed colloidal AgNPs per square millimeter of DFS scafold (μg/mm 2 ).

AgNPs' Release Rate Profle. DFS scafolds doped
with AgNPs were placed in 2 ml PBS solution and incubated at 37℃, and the absorbance values of PBS solution at 2, 4, and 24 hours were recorded at 450 nm Chromate ELISA reader. Te changes in the absorbance were observed to determine the rate of AgNPs released from the scafold. Te experiments were carried out in triplicates, and a sample without AgNPs was used as a control.

In Vitro Testing.
For the in-vitro testing, a cytocompatibility test must be performed. Te biocompatibility of biomaterials is mainly determined by its cytotoxicity and hemocompatibility. Te prepared samples should not cause any toxic reaction and immunological rejection in the body so that the biocompatibility test should be needed for those samples. Terefore, the individual materials of the fsh skin scafold and the nanoparticles doped fsh skin scafold should need the biocompatible test to ensure efective and safety uses for humans. Tis includes the cytotoxicity test, sensitization assay, hemocompatibility test, implantation test, irritation test, acute systemic toxicity, subchronic toxicity, genotoxicity, carcinogenesis bioassay, reproductive and developmental toxicity, pharmacokinetics, and preclinical safety test.
2.12.1. Cytotoxicity. Cytotoxicity assays are utilized during drug development before toxicological testing is performed. Tese assays are additionally used for controlling the quality of manufactured drug compounds. Tere are quantitative and qualitative methods of cytotoxicity testing. Te quantitative cytotoxicity assay mainly used is the MTT assay, and the qualitative assays used are the MEM elution method, the direct contact method, and the agar difusion method.
International Journal of Biomaterials 2.12.2. MTT Assay. Te cytotoxicity assay uses 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide dye, commonly referred to as MTT. MTT is a yellow-colored water-soluble compound which is split by mitochondrial succinate dehydrogenase, giving rise to the violet-colored formazan [47]. Te conversion of MTT to formazan only occurs in viable cells. Te formazans are insoluble in water but soluble in solvents such as dimethyl sulfoxide (DMSO) and isopropanol. After formazans are dissolved, the solution is taken for spectrophotometry. Te solution is kept in cuvette and kept in the spectrophotometer to fnd absorbance. Te absorbance of the solution is directly proportional to the concentration of the solution. If the solute in the solution is high, there will be a greater number of viable cells, whereas the lesser concentration, the number of viable cells will be less.
Here, the prepared scafold was placed in 3 diferent 96well plates. Fibroblast 3T3 cells were seeded in each well plate. Te plate was then incubated for 48 hours at 37°C, and 100 of 5 mg/ml MTT was added in the well plate. Te plate was again incubated for 4 hours, and 15% DMSO was added to it. Te absorbance reading in 96-well plates was carried out in chromate ELISA Reader supplemented with Chrome Manager Software at 595 nm.

FT-IR Analysis.
Te FT-IR analysis of AgNPs synthesized in 6 hours and 12 hours at 100°C is shown in Figure 1. As shown in the fgure, both graphs have similar absorbance bands within the same range. Here, strong absorbance bands were observed at 1609 cm −1 ,1524 cm −1 , 1383 cm −1 , and 1083 cm −1 which correspond to nitro compound, alkane, and amine, respectively [48]. Te band at 1609 cm −1 arises due to C-N and C-C stretching indicating the presence of proteins [49]. Te band around 1524 cm −1 and 1383 cm −1 corresponds to C�O stretching vibration (Amide I) and N-H bending vibrations (Amide II), and the band around 1296 cm −1 corresponds to the CH 2 wagging vibrations (Amide III) [50]. 1083 cm −1 corresponds to -C-O-of the ester group [51]. Te peaks observed in the range of 1000-450 cm −1 confrm the CH group [50].
Tus, the presence of biological molecules such as proteins confrmed the bio-fabrication of AgNPs. It also suggests that the proteins are responsible for the capping and stabilization of the synthesized AgNPs [52].
Te other medium to small peaks shows the presence of a halo compound and alcohol. Tis in total shows that Aloe vera successfully reduced AgNO 3 into AgNPs as well as capped the nanoparticle as most of the compound shown on the surface of the nanoparticle of Aloe vera.

XRD Analysis.
Te XRD patterns of AgNPs are prepared by using the two hydrothermal conditions, i.e., 100°C for 6 hours and 12 hours as shown in Figures 2(a) and 2(b), respectively. Te XRD analysis of synthesized AgNP extract from the Aloe vera leaf shows diferent peaks ranging from 30 0 to 80°. Te peaks are indexed with reference to the standard JCPDS card no. 04-783 for silver that are found to be 122, 111, 200, 222, and 311 around 32.5°, 38.3°, 50°, 60°, and 80°, respectively, corresponding to the cubic face of AgNPs [52,53]. Te peaks observed around 27°and 32°may be due to the leaf extract. Tese Braggs peaks might have resulted due to the capping agent, stabilizing the nanoparticles [54]. Te previous research works have showed that the highest peaks of green synthesis AgNPs were found between the ranges of 30-40 radians [52,55]. Te crystallite sizes of the AgNP particles prepared from 100°C for 6 hours and 12 hours are calculated using the Debye-Scherrer's equation which were found to be 29.1 nm and 35.2 nm, respectively [56]. Te XRD analysis data of the peak position, full-width-half-maximum (FWHM), size, and average size of both samples of AgNPs are shown in Table S1 (supporting fle).

UV-Vis Spectroscopy.
Te surface plasmon spectrum of synthesized AgNPs is shown in Figure 3. Te fgure illustrates that the AgNPs displayed a strong characteristic surface plasmon resonance band in the visible region, centered from 417 to 424 nm which is specifc for AgNPs. Similarly, the electronic transition of metallic silver appears in the range of around 300-330 nm. Besides these two peaks, there are not any other peaks, which indicate the absence of nanoparticle aggregation. Tus, UV-Vis spectroscopy further confrms the formation of silver nanoparticles [57,58].

Antioxidant Assay.
Free radical scavenging activity of the AgNPs was assessed by the DPPH assay. Te freshly prepared DPPH solution exhibited a deep purple color with maximum absorbance at 517 nm. Te disappearance of purple color on adding synthesized AgNPs might be due to the presence of antioxidants in the medium. Te diferent color during the antioxidant assay of biosynthesized AgNPs and Aloe vera extract compared with chemically synthesized AgNPs and ascorbic acid is shown in Figure S4 (supporting information).
Here, Figure 4 illustrates that the %RSA values are diferent between the values of Aloe vera extract and CAgNPs and between the values of GAgNPs and CAgNPs. Free radical scavenging activity of Aloe vera extract, AgNPs prepared from Aloe vera extract, and chemically prepared AgNPs on DPPH radical was found to increase in concentration, showing a maximum of 33%, 26%, and 17%, respectively, at 200 μg/ml. Te standard ascorbic acid, however, at this concentration exhibited 76% inhibition, but it cannot be used in a burn wound treatment. Te IC50 value of Aloe vera extract, AgNPs prepared from Aloe vera extract, and chemically prepared Aloe vera extract was found to be 307 μg/ml, 362 μg/ml, and 620 μg/ml, respectively, whereas the IC50 value of ascorbic acid was 7 μg/ml. Te experimentally obtained data of DPPH radical scavenging activity (%RSA) and IC50 value of Aloe vera extract, biosynthesized silver nanoparticles using Aloe vera leaf extract, chemically synthesized silver nanoparticles, and ascorbic acid (standard) are shown in Table S2 (supporting information). Tese obtained results are comparable with a scientifc article  International Journal of Biomaterials published by Barabadi et al., where the %RSA of green synthesized AgNPs from Cestrum nocturnum was found to be 29.5% [43]. Te previous result also demonstrates that the scavenging activity of Aloe vera extract is higher than GAgNPs and CAgNPs which might be due to the presence of antioxidant phenolic compounds, favonoids, ascorbic acid, β-carotene, and α-tocopherol in the Aloe vera extract [59]. In addition, the GAgNPs have greater scavenging activity than that of CAgNPs, further confrming that the antioxidant property of Aloe vera extract has made an important contribution in the increment of scavenging activity of AgNPs.
Burn wound on the skin is reported to be an oxidation process which generates free radicals from various cellular pathways [60]. Tus, the antioxidant GAgNPs would help in eradicating the free radicals from the wound and provide a healthy environment for the skin to heal. Figure 5 illustrates the digital image of the histological slide of the fsh skin before and after decellularization. Te histological study of slides under the microscope shows that the decellularization of the fsh skin with diferent decellularizing chemicals such as Triton-X, SDS retained the ECM components without any evidence of cellular and nuclear materials. Likewise, the H&E stain results of fsh skin tissue before and after decellularization confrmed the suitable maintenance of the ECM structure in DFS scafold. Moreover, the decellularization procedure removed the cells from the tissue and created more porous scafolds as shown in Figure 5. Tis facilitates the absorption of metal nanoparticles for the bioscafold development and application. Te porous structure of the scafold provides the large surface area for the cell attachment.

Histological Analysis.
Te decellularization procedure removes the cells as well as ensures the scafold is free from nuclear content as well as microbial contaminations. Te complete decellularization of the scafold was ensured by histological analysis of the scafold using the H&E staining technique. Te absence of nuclear content and the presence of an intact ECM structure only are confrmed by the absence of purple stain and the presence of pink stain. Te histological slide of the fsh skin sample showed the purple stain that indicates the presence of nucleus, whereas the histological slides of the DFS scafold showed the lesser presence of purple stain than pink stain, which indicates the decrease in nucleus content. Te DFS scafold showed a mesh-likereticular arrangement of fbers. Te absence of cells in the ECM matrix results in porosity which facilitates adsorption of AgNPs and provides a large surface area for cell attachment for better proliferation. Figure 6 shows the result of the contamination test. Tere were not any colonies of bacteria observed in the media even after 24 hours of incubation at 37°C which is an appropriate condition for bacterial colony formation. Tis proved that the DFS scafolds were free from microbial contaminations and can be safely used for in vitro experiments.

In Vitro Degradation Rate Analysis.
Te graph was plotted according to the data obtained from the experiment which is shown in the Figure 7. Te degradation rate of the DFS scafold was 19.4%, 29%, 33%, 40.3%, 43.4%, 47.9%, and 50% in the frst successive 7 days, respectively. Te degradation rate of DFS scafold was increased constantly by about 9% within 2 nd and 3 rd weeks. After the 3 rd week, slow degradation was perceived and about 70% of the scafold was degraded up to 28 days. Te experimental data for percentage weight loss of the DFS scafold during in vitro degradation at 37°C in PBS solution with a pH of 7.4 up to the 3 rd week are shown in Table S3 (supporting  information).
A degradation test is used to know the stability of the scafold during contact with the wound. An ideal scafold is supposed to degrade at a rate proportional to the healing rate of the wound. Burn wounds are usually healed within 14 to 21 days. If the scafold lasts up to 21 days, then it can be used as a treatment for burn wounds [61].
Te DFS scafold is ECM based scafold that consists of almost 80% collagen. Te degradation of the DFS scafold was similar to that of the collagen-based scafold [62]. Te error bars show sample standard deviation from triplicate measurements. Figure 8 shows the swelling ratio graph from the obtained results, and the corresponding data are shown in Table S4 (supporting  information). Also, the dried DFS scafold in desiccators after the swelling test is shown in Figure S5    International Journal of Biomaterials 2, 3, 12, and 24 hours, respectively. An ideal scafold is required to have a water absorption/water uptake capacity of 100-800% to prevent the buildup of a fuid and enhance the formation of new ECM [61]. Hydrophilicity and the microstructure of a scafold are the key determinants of a scafold's water uptake capacity [63]. Since the prepared DFS scafold contains an enormous amount of collagen fber, the scafold was easily wettable by polar solvents such as PBS. Collagen contains a large number of functional groups which are capable of binding water; therefore, they exhibit a high swelling ability. Likewise, the porous microstructure of collagen fber facilitates water uptake, making the scafold supportive for the wound healing process [62]. Te PBS solution having a pH of 7.4 that corresponds to the body's internal pH (e.g., blood) supports to examine the behavior of the material inside the   body. Te high swelling capacity of prepared DFS scafold verifed that it has a signifcant ability to uptake the excessive wound exudates. Te obtained result suggests that the DFS scafold possesses porous lamellar matrix spaces which increased the water containing capacity. Tus, the porous structure creates a suitable ambience for cell proliferation when used to heal the burn wound.

Swelling Analysis/Water Uptake Analysis.
3.9. Moisture Content. Figure 9 illustrates the moisture content of diferent samples. According to the graph shown in fgure, the DFS scafold possessed a high moisture content of 81.7 ± 3.6%. Te experiment was performed in triplets, and the result is presented as the mean ± standard deviation. Te data of initial and fnal weight of DFS scafold required to calculate moisture content percentage are given in Table S5 (supporting information). Te appropriate moisture content in the DFS scafold ensures the sufcient supply of moisture to the wound. A moist environment has been proven to enhance the wound healing process by promoting angiogenesis, cell adhesion, growth, migration, and collagen synthesis and facilitating re-epithelization and cessation of dead tissue and fbrin [64].

Permeability.
Te experimental setup of the permeability test is shown in Figure 10. It was observed that there was no increment of volume of liquid in all three samples. Te permeability test of the DFS scafold showed that the scafold is impermeable to the water and external environment. Correspondingly, the loaded AgNPs prevent infection and decrease bacterial load by their intrinsic antibacterial properties. Tis ensures that the scafold is the perfect barrier for the protection of burn wounds against external microbial contamination.

Mechanical Test.
Te mechanical test of the DFS scafold showed that the prepared scafold has high tensile strength, is highly fexible, and is easy to handle by clinicians. Te high tensile strength of the scafold means the Moisture content in (%) Figure 9: Moisture content of DFS scafold. Te moisture content of DFS scafold was found to be 81.7 ± 3.6%. Te graph is presented as the amount of moisture in % present per identical sample (N � 3). Te result is expressed as grams of water in 100 g of dry sample weight. Error bars are the standard deviation from triplicate measurements. mechanical integrity of the scafold is preserved to a proper level even after the chemical decellularization process. Te folding endurance tests demonstrate that the scafold does not break even after the repeated folding of the scafold more than 1500 times.

AgNPs' Loading Capacity of DFS Scafold.
Te doping of AgNPs on the DFS scafold depends upon the concentration of a solution of AgNPs and the area of the DFS scafold. Te doping of AgNPs adds antibacterial properties to the DFS scafold and improves the infammatory response during the initial phase of treatment. Te controlled release of AgNPs from the DFS scafold is essential to prevent infection due to opportunistic bacteria and guide the wound healing process. Te smallest size of AgNPs obtained from incubating 6 h was used to load in the fsh skin scafold.
Te loading of AgNPs on DFS at diferent concentrations is shown in Figure 11. Te fgure illustrates that the loading of AgNPs in 24 hours was 48.7 μg/mm 2 and 61.5 μg/mm 2 when doped in 150 μg/ml and 200 μg/ml, respectively. Te required data for calculation of AgNPs' loading capacity on DFS Scafold are shown in Table S6 (supporting information). Te results showed that the loading amount of AgNPs depends on the concentration of nanoparticles in the solution in which DFS scafold is dipped. Te porous structure of the DFS scafold might have facilitated the loading amount of nanoparticles in the scafold. Figure 12 demonstrates the absorbance taken at diferent time intervals of the prepared scafold. Te release rates of the scafold in 2, 4, and 24 hours were observed in a signifcant amount through the absorbance of the fuid at 430 nm which is shown in Figure 12. Te experimental data of the AgNPs' release profle from DFS scafold at diferent time intervals are shown in Table S7 (supporting information).

AgNPs' Release Rate Profle.
Te release rate of DFS scafold doped in 150 μg AgNPs is greater than that of DFS scafold doped in 200 μg AgNPs over the time period of 24 hours. Specifcally, most of the AgNPs were released in a burst in the frst 2 hours, with approx. 45 and 50.5% of the AgNPs being released from the DFS scafold. After the burst release phase, slow release was sustained for 24 hours. One potential reason for this behavior is that the collagens present on the DFS may serve as a steric barrier that retards the difusion of AgNPs from the scafolds [65]. Another potential reason for this behavior might be due to the stability of the biosynthesized AgNPs. Alternatively, the sustained release of AgNPs may result from the slow degradation of the collagen scafolds.
3.14. Antibacterial Assay. S. aureus, P. aeruginosa, E.coli, Enterobacter, A. baumannii, and K. pneumonia are reported to be the most prevalent bacterium in a burn wound [66]. Among these, the Gram-negative bacterium, S. aureus, is found to be more active during the wound healing process. So, antibacterial  tests were performed in P. aeruginosa, Gram-negative bacteria and S. aureus, and Gram-positive bacteria. For this, the AgNPs incubated for 6 h were used which was the smallest sized nanoparticle among the samples. Te antibacterial efect of AgNPs is infuenced by various factors such as shape, size, and colloidal state and surface charge. Reports suggest that the smaller the size of nanoparticles (<30 nm), more efective will be the antibacterial property [67]. For example, the biosynthesized AgNPs from Zataria multifora with an average size of 25.5 nm showed a MIC of 4 μg/ml against S. aureus that was lower than the commercial AgNPs [68]. Similarly, the biosynthesized AgNPs which reduced from polyphenol-rich plant extract, with an average size of 8.5 nm, exhibited the MIC of 1.25 μg/ml against P. aeruginosa. Likewise, photosynthesized nanosized Ag particles with an average diameter of 10-30 nm showed great antibacterial activity against A. baumannii with MIC and MBC of 62.5 and 250 μg/mL, respectively [69,70]. Meanwhile, among diferent shapes of nanoparticles, spherical nanoparticles were found to be more efective against bacteria. Smaller and spherical nanoparticles tend to interact with the bacterial cell wall, damage the lipid bilayer, and enter inside the cell resulting in cell death [71,72]. A study found that nanoparticles show higher antibacterial activity in colloidal form than in noncolloidal form. In a comparative study between cationic, anionic, neutral, and uncoated AgNPs, the cationic AgNPs showed the strongest antibacterial activity. Tis suggests that the surface charge also afects the activity of nanoparticles [73]. Figure 13 shows the schematic illustration to represent the proposed antibacterial mechanisms of silver nanoparticles.
In the previous fgure, AgNPs are attached to the bacterial cell wall, and we constantly infltrate it (a). Tis causes physical damage to the cell wall leading to leakage in the cellular content that leads to bacterial death. AgNPs penetrate inside the cell and interact with biomolecules such as ribosomes, nucleoid, plasmids, etc., (b), which causes an increase in reactive oxygen species (ROS). AgNPs also release Ag + ions, which also interact with biomolecules of the bacterial cell. Increased ROS leads to DNA damage, apoptosis, reduction in ATP generation, and lipid peroxidation, thus leading to bacterial death [74].
3.14.1. Susceptibility Test. Te disk difusion susceptibility test on ager petri plates exhibited a signifcant zone of inhibition with AgNPs solution which shown in Figure 14. Te mean diameter of the zone of inhibition was measured in millimeters (mm) and found to be 17.66 ± 1.5 mm for S. aureus and 8 ± 1 mm for P. aeruginosa, while penicillin (standard antibiotic) and Aloe vera extract did not show any antibacterial activity. Te experiment was performed in triplets, and the result is presented as mean ± standard deviation. Tus, this showed that the prepared AgNPs possessed antibacterial properties ( Figure 14).

MIC.
Te MIC of AgNPs was examined in P. aeruginosa and S. aureus bacteria by using the broth dilution method seen in Figure 15. Te MIC of AgNPs was indicated by the occurrence of a clear solution in the test tube. Te turbidity was seen from 50 μg/ml AgNPs in both bacteria. Tus, we can conclude that the MIC of AgNPs is 50 μg/ml. 3.14.3. MBC. Te MBC result of AgNPs against S. aureus and P. aeruginosa showed no bacterial growth upto 50 μg/ml of AgNPs which is shown in Figures 16(a) and 16(b), respectively. From this experiment, we can conclude that biosynthesized AgNPs have bactericidal efects as well.
3.15. MTT Assay/Cell Proliferation Assay. Te biocompatibility of DFS scafold and AgNPs were evaluated using the mouse fbroblast 3T3 (MF3T3) cell line. Te viability of mouse fbroblast 3T3 in the presence of test scafold was observed using the MTTassay for a period of 48 hours, and the results obtained are given in Figure 17. Te required data for calculation of cell viability % from OD value obtained from the MTTassay, ordinary two-way ANOVA analysis of the data obtained from the MTTassay, and ordinary two-way ANOVA multiple comparisons of the test samples are shown in Tables S8, S9, and S10, respectively (supporting information). Te data obtained from the ELISA Reader were analyzed using GraphPad Prism 9.2.0. Signifcant diferences between specimens were evaluated using two-way ANOVA.
Te values obtained from the analysis of the MTT assay indicated that the DFS scafold doped with diferent concentrations of AgNPs showed no signifcant diference from the control treatment (p < 0.05). However, the developed scafold helped in the proliferation of fbroblast cells. When the cells were seeded on the DFS scafold doped with AgNPs, MF3T3 cells attached and proliferated well, as shown by the increased metabolic activity over 48 hours in the MTT assay. In tissue engineering, one of the main requirements of a scafold is biocompatibility, which is the ability to support normal cellular activity without any toxic efects on the host tissue. Te developed scafold created a good ambience for the cell to proliferate without showing any toxicity to the cell.
AgNPs also showed the viability of MF3T3 cells, but the result was signifcantly less than the DFS scafold (p < 0.05). Tis result indicates that the silver nanoparticle has cell proliferation properties. Te cell proliferation of silver nanoparticles loaded with DFS scafold increases signifcantly. Te reason behind this might be due to the DFS scafold which provides a better environment for the cells to attach and proliferate than AgNPs alone. Furthermore, the viability of cells has been increased with an increase in the concentration of AgNPs. However, there is no signifcant increase in the viability of cells when the concentration of loaded silver nanoparticles is above 50 μg/ml in the DFS scafold. Tis shows that a minimum concentration of 50 μg/ ml of AgNPs will be enough for the cell to proliferate when loaded on the DFS scafold. Also, from the antimicrobial assay, it was seen that the concentration of 50 μg/ml of AgNPs was enough to inhibit bacterial growth. Tus, a concentration of 50 μg/ml AgNPs will be the appropriate concentration to be loaded on the scafold considering the relation between the release rate of nanoparticles and the area of the scafold.
For a wound to heal ideally, the healing agent must create a moisture environment, reduce infection, mimic the extracellular matrix feature, and reduce the wound scar [75].
During the infammatory phase which is the initial phase of the wound healing process, cells such as neutrophils and monocytes travel to the wound site to prevent probable   International Journal of Biomaterials 13 infection. Silver nanoparticles become more active in this phase, as it acts as an antibacterial medium and helps prevent potential bacterial growth and infection. AgNPs also activate macrophages and the immune cell [76]. Nanoparticles loaded with DFS scafold will act as a barrier to the outer environment. Te infammatory phase is followed by the proliferative phase where keratinocytes and fbroblasts are activated which assist in closure and restoration of the vascular network. In this phase, both AgNPs and DFS scafold will be active; DFS scafold creates a 3D structure for the fbroblast and keratinocytes to attach and proliferate. Also, AgNPs help in acceleration of fbroblast migration [76]. Similarly, AgNPs themselves have the wound healing property; bacterial synthesis of AgNPs using Bacillus cereus and Escherichia fergusonii showed accelerated formation of collagen and epithelization [77]. It is described in the literature that silver nanoparticles can modulate antiinfammatory cytokine release and promote rapid wound closure without increasing scarring. Tey can also promote epidermal re-epithelization by causing keratinocyte proliferation. Te moist environment of the DFS scafold helps in angiogenesis which is also a part of the proliferative phase. Te prepared scafold does not require any dressing due to its biodegradable property. Te scafold has been shown to degrade partially within 7 days, while the scafold takes 30 days to degrade fully. Considering the mentioned features, the present DFS scafold doped with AgNPs act as a promising biocompatible, wound healing agent. Tis concludes that the DFS scafold doped with AgNPs is a biocompatible antimicrobial scafold capable of enhancing the burn wound healing process.

Conclusions
A novel scafold with antibacterial activity was successfully prepared using decellularized fsh skin scafold and biosynthesized silver nanoparticles. Te FT-IR analysis of biosynthesized AgNPs concluded that some of the biological molecules of leaf are responsible for biotransformation of silver ions to AgNPs. Te XRD analysis elucidated that the crystallite size of biosynthesized AgNPs ranged between 29 nm and 35 nm. Te composite scafold contains a large amount of collagen type I that increases migration and proliferation which makes it an appropriate ECM-based decellularized scafold for tissue engineering and burn wound dressing. Moreover, the prominent characteristics of the composite scafold, such as encompassing high collagen content, appropriate antibacterial activity, a suitable microbial barrier, good fexibility, moisture content, swelling ratio, biodegradability, and biocompatibility with ease of formulation, make the proposed DFS scafold a suitable dressing material for burn wound healing. Te results obtained from the in vitro experiments met the expectations.

Data Availability
Te data used to support the fndings of this study are available within its supplementary materials.

Conflicts of Interest
Te authors declare that there are no conficts of interest. transforming our ideas into reality. First and foremost, they would like to convey their heartfelt gratitude to the College of Biomedical Engineering and Applied Sciences (CBEAS) for providing the required platform for us to carry out the project work. Tey are grateful to the Research Centre for Applied Sciences and Technology (RECAST) and Annapurna Research Centre for providing them with a laboratory to conduct this research. Tis work was supported by the College of Biomedical Engineering and Applied Sciences.

Supplementary Materials
XRD analysis data of peak position, full-width-halfmaximum (FWHM), calculation of size, and average size of both AgNPs samples; DPPH radical scavenging activity (% RSA) and IC50 value of Aloe vera extract, biosynthesized and chemically synthesized silver nanoparticles, and ascorbic acid (standard); percentage weight loss of the DFS scafold during in vitro degradation at 37°C in PBS solution, having pH 7.4; percentage of swelling ratio or water uptake capacity of DFS scafold at 1, 2, 3, 12, and 24 hours; percentage moisture content of DFS scafold; determination of nanoparticle loading capacity of DFS scafold by dipping in diferent concentrations of AgNPs solutions; the AgNP release profle from DFS scafold at diferent time intervals; ordinary two-way ANOVA analysis of the data obtained from the MTT assay; extracted Aloe vera which was used for the preparation of AgNPs in the hydrothermal method; samples of purifed AgNPs prepared in 6 h and 12 h; photographs of adult tilapia fsh, extraction of skin tissue from tilapia fsh, and decellularized skin scafold used in research; fgure of the antioxidant assay of biosynthesized AgNPs and Aloe vera extract compared with chemically synthesized AgNPs and ascorbic acid; samples of dried DFS scafold in desiccators after the swelling test. (Supplementary Materials)