Chitosan-G-Glycidyl Methacrylate/Au Nanocomposites Promote Accelerated Skin Wound Healing

Herein, we report the synthesis of Au nanoparticles (AuNPs) in chitosan (CTS) solution by chemically reducing HAuCl4. CTS was further functionalized with glycidyl methacrylate (chitosan-g-glycidyl methacrylate/AuNP, CTS-g-GMA/AuNP) to improve the mechanical properties for cellular regeneration requirements of CTS-g-GMA/AuNP. Our nanocomposites promote excellent cellular viability and have a positive effect on cytokine regulation in the inflammatory and anti-inflammatory response of skin cells. After 40 days of nanocomposite exposure to a skin wound, we showed that our films have a greater skin wound healing capacity than a commercial film (TheraForm®), and the presence of the collagen allows better cosmetic ave aspects in skin regeneration in comparison with a nanocomposite with an absence of this protein. Electrical percolation phenomena in such nanocomposites were used as guiding tools for the best nanocomposite performance. Our results suggest that chitosan-based Au nanocomposites show great potential for skin wound repair.


Introduction
The synthesis of gold nanoparticles for nanocomposite formulation has received considerable attention in the last decade. Several reports describe synthesis processes in which the produced nanoparticles have homogeneous sizes and shapes [1][2][3]. Gold nanoparticles (AuNPs) have attracted substantial attention due to their optical, electrical, mechanical, and biomedical properties [4], which make them potential candidates for applications in various areas such as drug delivery, development of sensors, catalysis, and environmental remediation [5,6]. Animal models, such as mice, are often used in wound healing research, and it has been observed that low concentrations of AuNPs increase cellular proliferation and the production of cytokines [7,8]. The nanosized Au particles offer the possibility of attaching molecules to their surface; these particles can flow; precipitation was induced with acetonitrile according to the methodology reported by Elizalde et al. (2013) [12].
To elaborate the (CTS-g-GMA)-AuNPs nanocomposite, CTS-g-GMA was dispersed in distilled water for 24 h. Then, the CTS-HAuCl 4 solutions with different concentrations of HAuCl 4 were added, and the solution was kept at 60 • C for one hour until a homogeneous solution was obtained. The CTS is a reduction and stabilizing agent [18][19][20][21][22].
On the other hand, a third nanocomposite (CTS-g-GMA)-AuNPs-Col was prepared by using the procedure described above with one additional step at the end of the process: the incorporation of 0.3% of solution of type I collagen in acetic acid under constant magnetic stirring for 2 h at room temperature.
Thin films with different composites (thicknesses varied from 0.3 mm to a few millimeters for the different spectroscopic and in vivo studies) were prepared by the solvent cast method, i.e., pouring the final solution into a plastic Petri dish; then, they were placed in an oven at 60 • C for 24 h to allow evaporation of the solvent.

Characterization Studies
All materials were characterized by infrared spectroscopy (FTIR). The spectra between 4000 and 400 cm −1 were obtained using an FTIR spectrophotometer (Perkin Elmer Spectrum 1 Model). All spectra were recorded with a resolution of 4 cm −1 and 16 cm −1 times scanning using the transmission technique. Only one material for the HAuCl 4 concentration was measured.
The film morphology of (CTS-g-GMA)-AuNPs and CTS/AuNPs was analyzed using a JEOM JSM-7401F field emission scanning electron microscope.
The ultraviolet-visible spectrum (UV-vis spectrometer Agilent 8453) was used to determine the sizes of the Au nanoparticles by detecting the maximum absorption band in the visible region. The absorption range used on UV-vis measurements was in the visible spectrum, and we used a universal polystyrene cuvette of 0.8 mm.
The DC electrical conductivity (σDC) was calculated from impedance measurements according to the methodology described by Heilmann (2003) [23]. Dielectric measurements in the frequency range from 40 Hz to 110 MHz were carried out with an Agilent Precision Impedance Analyzer 4249A (Santa Clara, CA, USA). The amplitude of the measuring signal was 100 mV. The nanocomposite was previously dried under a vacuum to eliminate the moisture, and measurements were conducted in a vacuum cell [24].

Cellular Viability
Six-week-old Balb/c mice were sacrificed by cervical dislocation, and resident peritoneal macrophages were obtained by repeated washings and retrieval of the peritoneal cavity with 10 mL of sterile and cold RPMI-1640. On the other hand, peripheral blood mononuclear cells (PBMC) were isolated from peripheral blood obtained from regular human donors; the blood was diluted with phosphate buffer solution (PBS) at a ratio of 1:1 (vol/vol) and then centrifuged on a Ficoll-Paque gradient for 30 min at 500× g at room temperature; the interphase layer consisting of PBMC was washed three times with a culture medium. The HEPG2 cancer cell line derived from human liver hepatocellular carcinoma was purchased from ATCC (American Tumor Cancer Collection, Manassas, VA, USA) and cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic solution (GIBCO, Grand Island, NY, USA), in an atmosphere at 37 • C and 5% CO 2 . The cells were adjusted at 5 × 10 3 cells/mL, seeded by triplicate on the different nanocomposites previously poured in 96-well plates (Corning, Corning, NY, USA) and incubated for 72 h in an atmosphere at 37 • C and 5% CO 2 . The cellular viability was evaluated by MTT or trypan blue staining according to the manufacturer's instructions [25].

Nitric Oxide Determination
The supernatants of each treatment were used to determine the nitric oxide production by nitrate-nitrite colorimetric assay kit (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer's instructions [26]. Briefly, 40 µL of supernatants were mixed with 40 µL of assay buffer, 10 µL of enzyme cofactor, and 10 µL of nitrate reductase and incubated at room temperature for 3 h (to allow the conversion of nitrate to nitrite); the samples were analyzed by triplicate. After 10 min of incubation in Griess reagent at room temperature, the absorbance was measured at 560 nm in a microplate reader (Winooski, VT, USA, model EL311).

Evaluation of Cytokine Expression by Flow Cytometry
The cytokine production was analyzed by a BD cytometric bead array (CBA) Th1/Th2/ Th17 CBA mice cytokines kit (BD, San Diego, CA, USA). For the evaluation, spleens were taken from mice 48 h after wounding. The spleens were crushed and homogenized in PBS (500 µL); the supernatants of this mixture were collected by centrifugation at 1600 rpm/10 min and stored at −20 • C until analysis according to the manufacturer's instructions. The cytokines evaluated were different interleukins (IL) as IL-2, IL-4, IL-6, IL-10, IL-17A, INFΥ, and TNF. The cytokine production was measured by flow cytometer (BD Accuri C6, BD Biosciences, San Jose, CA, USA) according to the manufacturer's instructions. CBA analysis was performed using FCAP array v1.0 software (Soft Flow Inc., St Louis Park, MN, USA).

Wound Healing Effect
Wound healing effects on mouse skin were investigated using nanocomposites CTSg-GMA, (CTS-g-GMA)-AuNPs, and ((CTS-g-GMA)-AuNPs-Col) with different HAuCl 4 concentrations (0.15, 0.3, 0.6, 1.07, 2 and 3 mM). A commercial material TheraForm ® (natural wound healing matrix of highly purified atelocollagen) (Surgical Esthetics TM , Northridge, CA, USA) was used as a positive control. First, the films were cut into squares with dimensions of 8 × 8 mm 2 ; afterward, square films were washed twice in 600 µL of PBS for 5 min and then washed three times in 600 µL of DMEM for 5 min. Finally, the materials were sterilized by ultraviolet light (CL-100 UV) for 10 min.
All experimental protocols were approved by the ethics research and animal wellness committee (N•CEIBA-2013-013) of the Facultad de Ciencias Biológicas, Universidad Autonóma de Nuevo León, and were done according to NOM-062-ZOO-1999 (Mexican legislation). Experimental animals were treated according to the criteria described in the PHS Policy on Humane Care and Use of Laboratory Animals and the "Guide for the Care and Use of Laboratory Animals" (NIH publication 86-23). Six-week-old female Balb/C mice (22 g body weight) were housed in polycarbonate cages at room temperature (21 ± 2 • C) on a 12-h light-dark cycle. Each was anesthetized intramuscularly with ketamine hydrochloride 100 mg/kg and xylazine 5 mg/kg. [27]. After that, the backs of the animals were shaved and swabbed with 70% ethanol three times before wounding.
Two skin squares of 5 × 5 mm (dermis and epidermis) on the back were selected in the shaved area. The wound nearby the head of the mice was implanted with the nanocomposites or controls; the other wound nearby the tail was used as a negative control without material, as shown ( Figure 1). After surgery, lesions were evaluated daily for 40 days according to established parameters by the manual of wounds and ulcers and the OECD 404 guide; 3 specimens per material were used according to the requirements of the animal ethics committee [28] (Table S1). Two skin squares of 5 × 5 mm (dermis and epidermis) on the back were selected in the shaved area. The wound nearby the head of the mice was implanted with the nanocomposites or controls; the other wound nearby the tail was used as a negative control without material, as shown ( Figure 1). After surgery, lesions were evaluated daily for 40 days according to established parameters by the manual of wounds and ulcers and the OECD 404 guide; 3 specimens per material were used according to the requirements of the animal ethics committee [28] (Table S1). Figure 1. Surgery process. The lesion nearby the head was implanted with nanocomposites or Ther-aForm ® as a positive control, and the location near the tail was an untreated wound as a basal control for wound healing.

Cytokine Production
Mice had the nanocomposite surgically implanted; after 48 h, 2 mL of blood was extracted and they were sacrificed. The blood was centrifuged at 1000 rpm to obtain serum, which was frozen at −20 °C until analysis.
Additionally, each animal had its spleen extracted, which was homogenized with 200 mL of PBS sterile and centrifuged at 1600 rpm per 10 min to obtain the supernatant; it was frozen at −20° C until analysis.
The cytokines were analyzed using a flow cytometer model Accuri C6 (Biosciences Center, North Brunswick Township, NJ, USA) following the protocol of BD cytometric bead away Mouse Th1/Th2/Th17 Cytokine Kit, which was used to detect IL-2, IL-4, IL-6, IFN-g, TNF, IL-17A, and IL-10 in all the samples.

Statistical Analysis
All experiments were performed in triplicate and statistical analysis was performed using analysis of variance (ANOVA). The results were considered statistically significant if the p-value was < 0.05.

Size and Stability of AuNPs
The UV-vis spectra of the CTS-AuNPs in a liquid medium, synthesized with different concentrations of HAuCl4 (Figure 2), show absorption band maxima in the range of 516 and 522 nm. The slight redshift may be attributed to an increase in the average size of the particles due to the formation of clusters. Figure 1. Surgery process. The lesion nearby the head was implanted with nanocomposites or TheraForm ® as a positive control, and the location near the tail was an untreated wound as a basal control for wound healing.

Cytokine Production
Mice had the nanocomposite surgically implanted; after 48 h, 2 mL of blood was extracted and they were sacrificed. The blood was centrifuged at 1000 rpm to obtain serum, which was frozen at −20 • C until analysis.
Additionally, each animal had its spleen extracted, which was homogenized with 200 mL of PBS sterile and centrifuged at 1600 rpm per 10 min to obtain the supernatant; it was frozen at −20 • C until analysis.
The cytokines were analyzed using a flow cytometer model Accuri C6 (Biosciences Center, North Brunswick Township, NJ, USA) following the protocol of BD cytometric bead away Mouse Th1/Th2/Th17 Cytokine Kit, which was used to detect IL-2, IL-4, IL-6, IFN-g, TNF, IL-17A, and IL-10 in all the samples.

Statistical Analysis
All experiments were performed in triplicate and statistical analysis was performed using analysis of variance (ANOVA). The results were considered statistically significant if the p-value was < 0.05.

Size and Stability of AuNPs
The UV-vis spectra of the CTS-AuNPs in a liquid medium, synthesized with different concentrations of HAuCl 4 (Figure 2), show absorption band maxima in the range of 516 and 522 nm. The slight redshift may be attributed to an increase in the average size of the particles due to the formation of clusters. Figure 3 shows a TEM micrograph of the CTS-AuNPs synthesized in a liquid medium with the minimum (0.3 mM) and maximum (1.72 mM) precursor concentrations. The average size was from 8 to 20 nanometers. Figure 4 shows an SEM micrograph of films with gold nanoparticles, with a concentration of 0.3 (a), 0.6 (b), and 1.07 (c) mM, embedded into the CTS-based matrix.               At a low concentration of HAuCl 4 , the distribution of the nanoparticles was homogeneous in the matrix and no clustering was observed. A further increase in nanoparticle concentration (1.7 mM) resulted in the formation of some clusters. Figure 5 shows the IR spectra of the different materials of CTS, CTS-AuNPs, CTSg-GMA-AuNPs, and CTS-g-GMA-AuNPs-Col. The spectra permit observation of the difference between all materials. The relative intensity of the broadband centered at about 3300 cm −1 corresponds to the stretching of the O-H group, and bands at 1070 and 1020 cm −1 related with asymmetric stretching vibrations of the ether group of methacrylate decreased in materials with GMA. Therefore, we can assume that there is no effect or presence of acetic acid in the nanocomposite due to previous work [29].

Infrared Spectroscopy
Pharmaceutics 2022, 14, x FOR PEER REVIEW 7 At a low concentration of HAuCl4, the distribution of the nanoparticles was hom neous in the matrix and no clustering was observed. A further increase in nanopa concentration (1.7 mM) resulted in the formation of some clusters. Figure 5 shows the IR spectra of the different materials of CTS, CTS-AuNPs, C GMA-AuNPs, and CTS-g-GMA-AuNPs-Col. The spectra permit observation of the d ence between all materials. The relative intensity of the broadband centered at about cm −1 corresponds to the stretching of the O-H group, and bands at 1070 and 1020 related with asymmetric stretching vibrations of the ether group of methacrylat creased in materials with GMA. Therefore, we can assume that there is no effect or ence of acetic acid in the nanocomposite due to previous work [29]. The relative intensity of the primary amide group related to stretching vibrati C=O bonds at 1650 cm −1 decreased, especially for (CTS-g-GMA)-Col and ((CTS-g-G AuNPs)-Col nanocomposites. The bending vibration of NH2 at 1550 cm −1 showed crease in relative intensity and a redshift for composite samples compared with pure Additionally, observed variations of bands at 1400 and 1421 cm −1 produced by stret of C-N groups increased, along with vibrations of CH2-CO. The characteristic absor bands of pure CTS are presented in Table 1. Several have authors reported FTIR sp of collagen type I and GMA [30][31][32][33][34].  The relative intensity of the primary amide group related to stretching vibration of C=O bonds at 1650 cm −1 decreased, especially for (CTS-g-GMA)-Col and ((CTS-g-GMA-AuNPs)-Col nanocomposites. The bending vibration of NH 2 at 1550 cm −1 showed a decrease in relative intensity and a redshift for composite samples compared with pure CTS. Additionally, observed variations of bands at 1400 and 1421 cm −1 produced by stretching of C-N groups increased, along with vibrations of CH 2 -CO. The characteristic absorption bands of pure CTS are presented in Table 1. Several have authors reported FTIR spectra of collagen type I and GMA [30][31][32][33][34].

X-ray Diffraction (XRD)
Pure CTS is a semicrystalline biopolymer and has an XRD pattern with broad peaks at around 2θ • = 15 • and 20 • , indicating the average intermolecular distance of the crystalline part of pure CTS crystallizing in an orthorhombic unit cell [35]. Furthermore, the reflection at 14.8 and 15 degrees, with the diffraction line at 20 degrees, of the nanocomposite with gold nanoparticles and the pure CTS, respectively, is due to the absence of moisture in the material and the crystal form type I [36,37]. Figure 6a shows the X-ray diffraction of pure chitosan (CTS), CTS-g-GMA, and (CTS-g-GMA-AuNPs)-Col. The main differences in the XRD patterns are that the broad diffraction line at around 20 degrees ((200) reflection) in the 2θ scale increases in intensity and decreases in position in composites, compared to the pure CTS. The reflection centered at around 20 degrees has contributions from the amorphous phase, but the diffraction line at 15 degrees corresponds to the crystal form type I ( Figure 6). In contradistinction to the diffractogram of the pure CTS and the nanocomposite of (CTS-g-GMA-AuNPs)-Col, the material of CTS-g-GMA without the gold nanoparticles is completely amorphous due to the only diffraction line at 20 degrees [37].

X-ray Diffraction (XRD)
Pure CTS is a semicrystalline biopolymer and has an XRD pattern with broad peaks at around 2θ° = 15° and 20°, indicating the average intermolecular distance of the crystalline part of pure CTS crystallizing in an orthorhombic unit cell [35]. Furthermore, the reflection at 14.8 and 15 degrees, with the diffraction line at 20 degrees, of the nanocomposite with gold nanoparticles and the pure CTS, respectively, is due to the absence of moisture in the material and the crystal form type I [36,37]. Figure 6a shows the X-ray diffraction of pure chitosan (CTS), CTS-g-GMA, and (CTSg-GMA-AuNPs)-Col. The main differences in the XRD patterns are that the broad diffraction line at around 20 degrees ((200) reflection) in the 2θ scale increases in intensity and decreases in position in composites, compared to the pure CTS. The reflection centered at around 20 degrees has contributions from the amorphous phase, but the diffraction line at 15 degrees corresponds to the crystal form type I ( Figure 6). In contradistinction to the diffractogram of the pure CTS and the nanocomposite of (CTS-g-GMA-AuNPs)-Col, the material of CTS-g-GMA without the gold nanoparticles is completely amorphous due to the only diffraction line at 20 degrees [37]. The diffraction line at about 38 degrees corresponds to the (111) planes in the cubic structure of the AuNPs, which can only be seen in the (CTS-g-GMA-Au)-Col nanocomposite [38,39]. In Figure 6b, the intensity of this line increases with the concentration of AuNPs.

Conductivity Measurements
According to practical media theory, the DC conductivity of a system with conductive inclusions in a dielectric matrix must increase; all samples were dried under a vacuum [27]. In our results, the incorporation of AuNPs results in a decrease in the conductivity for concentrations below 0.5 mM; a further increase in AuNPs concentrations increases the conductivity. For concentrations above 5.0 mM, the conductivity has an exponential increase of more than 12 orders of magnitude. The initial decrease in conductivity can be explained due to the interaction of AuNPs with the CTS matrix ( Figure 2) and, therefore, the number of free H + and OH − ions, which are known to be responsible for the intrinsic ionic conductivity in pure CTS [24]. The exponential increase in conductivity with the The diffraction line at about 38 degrees corresponds to the (111) planes in the cubic structure of the AuNPs, which can only be seen in the (CTS-g-GMA-Au)-Col nanocomposite [38,39]. In Figure 6b, the intensity of this line increases with the concentration of AuNPs.

Conductivity Measurements
According to practical media theory, the DC conductivity of a system with conductive inclusions in a dielectric matrix must increase; all samples were dried under a vacuum [27]. In our results, the incorporation of AuNPs results in a decrease in the conductivity for concentrations below 0.5 mM; a further increase in AuNPs concentrations increases the conductivity. For concentrations above 5.0 mM, the conductivity has an exponential increase of more than 12 orders of magnitude. The initial decrease in conductivity can be explained due to the interaction of AuNPs with the CTS matrix ( Figure 2) and, therefore, the number of free H + and OH − ions, which are known to be responsible for the intrinsic ionic conductivity in pure CTS [24]. The exponential increase in conductivity with the increase in nanoparticle concentration is due to the percolation of the AuNPs nanoparticles with an increasing number of conductive paths in the composite matrix. This minimum appears due to the competition of two mechanisms: a decreasing number of free H + and OH − ions and the increasing metallic conductance of the embedded nanoparticles (Figure 7). increase in nanoparticle concentration is due to the percolation of the AuNPs nanoparticles with an increasing number of conductive paths in the composite matrix. This minimum appears due to the competition of two mechanisms: a decreasing number of free H + and OH − ions and the increasing metallic conductance of the embedded nanoparticles ( Figure 7).

Nitric Oxide
The different nanocomposites evaluated did not induce (p < 0.001) nitric oxide production in the murine peritoneal macrophages and HEP-G2 cells, compared with the lipopolysaccharide (LPS) treatment, which induced inflammatory response and high nitric oxide production (Figure 9).
increase in nanoparticle concentration is due to the percolation of the AuNPs nanoparticles with an increasing number of conductive paths in the composite matrix. This minimum appears due to the competition of two mechanisms: a decreasing number of free H + and OH − ions and the increasing metallic conductance of the embedded nanoparticles ( Figure 7).

Nitric Oxide
The different nanocomposites evaluated did not induce (p < 0.001) nitric oxide production in the murine peritoneal macrophages and HEP-G2 cells, compared with the lipopolysaccharide (LPS) treatment, which induced inflammatory response and high nitric oxide production (Figure 9).

Nitric Oxide
The different nanocomposites evaluated did not induce (p < 0.001) nitric oxide production in the murine peritoneal macrophages and HEP-G2 cells, compared with the lipopolysaccharide (LPS) treatment, which induced inflammatory response and high nitric oxide production (Figure 9).

Cytokine Production by Flow Cytometry
In this study, some materials affected the inflammatory response in physiological conditions by affecting pro-inflammatory (TNF, IL-2, and IL-17A) and anti-inflammatory cytokines (IL-4 and IL-10). In addition, IL-6 is a pro and anti-inflammatory cytokine [40][41][42].

Cytokine Production by Flow Cytometry
In this study, some materials affected the inflammatory response in physiological conditions by affecting pro-inflammatory (TNF, IL-2, and IL-17A) and anti-inflammatory cytokines (IL-4 and IL-10). In addition, IL-6 is a pro and anti-inflammatory cytokine [40][41][42].

Wound Healing Test
All the nanocomposites and TheraForm ® induced a process of recovery at seven days in the mice, with healthy classified as score wound type I (Table S1), characterized by erythematous aspect, lesion extension < 1 cm, lesion deep < 1 cm, exudate quantity and quality absent, necrotic tissue absent, granular tissue 75-100%, edema absent, pain 0-1, and surrounding skin healthy.
On day one, the recovery percentage in in terms of wound closure was not different between treatments. However, on the third day, the treatments with ((CTS-g-GMA)-AuNPs)-Col 0.3 and 0.6 mM were accelerated (25% both) compared to TheraForm ® and another nanocomposite (12.5%).
On day 18, the addition of collagen to the composites showed no significant differences (p < 0.05) concerning wound healing between groups containing the same AuNP concentration. However, at the highest AuNP concentration (1.07), the wound healing was greatest (p < 0.05). When compared with Theraform (positive control of wound repair), the only significant difference (p < 0.05) was found with a composite of 0.15. On day 40, no significant difference was found between any group (p < 0.05).
Other works using different polymer matrices and gold nanoparticles have had similar results; the nanoparticle's shape, surface modification, and synthesis method have a crucial effect on wound healing [7,43]. After 21 days, all treatments showed total recovery and cicatrization. The ((CTS-g-GMA)-AuNPs)-Col treatment showed better cosmetic appearance, such as less deep skin pigment. In general, all wounds, treated and untreated, allowed the growth of new skin without infection and inflammatory signs during the wound healing process for the 40 days evaluated (Figure 11). On day three, the nanocomposites ((CTS-g-GMA)-AuNPs-col) at AuNP concentrations of 0.3 and 0.6 exhibited better wound healing percentages (p < 0.05). On day eight, the nanocomposite with the highest concentration of AuNPs (1.07) induced superior wound healing percentages, followed by those nanocomposites with AuNP concentrations of 0.6 and 0.3, respectively (p < 0.05). Furthermore, we found no significant difference (p < 0.05) between composites with 0.3 AuNP containing or not collagen (day 8).
On day 18, the addition of collagen to the composites showed no significant differences (p < 0.05) concerning wound healing between groups containing the same AuNP concentration. However, at the highest AuNP concentration (1.07), the wound healing was greatest (p < 0.05). When compared with Theraform (positive control of wound repair), the only significant difference (p < 0.05) was found with a composite of 0.15. On day 40, no significant difference was found between any group (p < 0.05).
Other works using different polymer matrices and gold nanoparticles have had similar results; the nanoparticle's shape, surface modification, and synthesis method have a crucial effect on wound healing [7,43]. After 21 days, all treatments showed total recovery and cicatrization. The ((CTS-g-GMA)-AuNPs)-Col treatment showed better cosmetic appearance, such as less deep skin pigment. In general, all wounds, treated and untreated, allowed the growth of new skin without infection and inflammatory signs during the wound healing process for the 40 days evaluated (Figure 11).

Histological Tests
We tested our nanocomposites for hematoxylin and eosin staining in the wound area of the murine tissue. In Figure 12, we show the response to both stainings for CTS-g-GMA (a, c) and ((CTS-g-GMA)-AuNPs) 0.3 mM (b, d) at 15 and 30 days after surgery. Here, we observe the histological sections: red arrows denote the epidermis section; black arrows denote the neoformation of pilose follicles; blue arrows denote the neoformation of blood vessels.

Histological Tests
We tested our nanocomposites for hematoxylin and eosin staining in the wound area of the murine tissue. In Figure 12, we show the response to both stainings for CTS-g-GMA (a, c) and ((CTS-g-GMA)-AuNPs) 0.3 mM (b, d) at 15 and 30 days after surgery. Here, we observe the histological sections: red arrows denote the epidermis section; black arrows denote the neoformation of pilose follicles; blue arrows denote the neoformation of blood vessels. We found the best results for ((CTS-g-GMA)-AuNPs) 0.3 mM, with the greatest epidermis and blood vessel reconstitution. We found the best results for ((CTS-g-GMA)-AuNPs) 0.3 mM, with the greatest epidermis and blood vessel reconstitution.

Discussion
For application in tissue engineering, a material should meet the following requirements: biocompatibility with the tissues, biodegradability, nontoxicity, and suitable mechanical properties. The CTS-AuNP nanocomposite responds to all these requirements but has relatively insufficient mechanical strength. However, Elizalde et al. showed that with a correct molar relation, this hydrogel can change the mechanical properties, viscosity, and swelling index of CTS and can be used for cellular support for biomedical applications, allowing the adhesion and growing of human fibroblasts [12].
The interaction between CTS and gold nanoparticles was demonstrated by infrared spectroscopy; the relative decrease in the intensity of the band at 1550 cm −1 with the increase in the gold concentration has been attributed to interactions between NH 3 + and the polarizer surface of the nanoparticle for nanocomposites of silver and gold using CTS as reducing agent [18,19].
The conductivity of the nanocomposites strongly depends on the HAuCl 4 concentration and moisture. Materials with concentrations above 0.5 mM showed the formation of nanoparticle clusters ( Figure 5), producing percolation of nanoparticles followed by an increase in the number of conductive paths; additionally, the interaction of -OH groups and protonated amine can be corroborated by thermogravimetric analysis reported previously by our group [24]. All nanocomposites with collagen showed better results than controls and (CTS-g-GMA)-AuNPs; this is because the collagen type I used aids the process of scar tissue, which is the main component of the intact uninjured dermis [44].
Despite this, (CTS-g-GMA)-AuNPs at 2 and 3 mM increased the production of cytokines IL-6. They decreased levels of IL-17A; it has been reported that this concentration of cytokines promotes the healing of wounds [45]. These materials cannot be considered the best materials for re-epithelialization because they are breakable and fragile.
Any material used for skin wound healing must not permit pathogen infection or allergic response, because bacterial components, infections, or allergic materials may impair the repair mechanism of the host by interfering with cell-matrix interactions or attenuating the inflammatory response [46]. The gold nanoparticles show an excellent antibacterial capacity that changes in response to the shape and surface modifications of the nanoparticle [43,47]. All evaluated nanocomposites induced the cicatrization process without signals of fever, infection, allergy, or toxicity.
Wound healing is a dynamic process involving three phases overlapping in time and space: inflammation, tissue formation, and tissue remodeling; inflammatory response is the first of the several overlapping processes that constitute wound healing. The addition of collagen led to a higher percentage of healing at 3 and 8 days, compared with the commercial control and materials without collagen. The nanocomposites ((CTS-g-GMA)-AuNPs)-Col at 0.3 and 0.6 mM showed better performance at early healing times. The inflammatory response supplies growth factor and cytokine signals that orchestrate the cell and tissue movements necessary for repair [46]. The positive control (TheraForm ® ) presented low production of cytokines (IL-2, IL-4, IL-6, INFΥ, TNF, IL-10) and an absence of IL-17A. We expected to obtain similar results from the nanocomposites evaluated; nevertheless, differences were observed in the production of cytokines in a material compound-dependent manner.
It is impossible to know the average production of cytokines per individual, and it would be a mistake to generalize; this is considered in our model, which compared wound healing between each individual and each group, including two injuries per mice. After injuries, the IL-1b, INF-g, and TNF at the wound site led to the expression of various classes of adhesion molecules essential for leukocytes adhesion and diapedesis (P-and E-selectins as well ICAM 1, -2, LFA-1, MAC-1, gp150, chemokines, and their receptors, including IL-8, growth-related oncogene-alpha, and monocyte chemoattractant protein-1).
The early stages (1 to 3 days) are the most critical factor in the tissue regeneration process due to the transcription factors and proteins produced by cells to activate the process of cell migration [48]. The ((CTS-g-GMA)-AuNPs)-Col nanocomposite at 0.6 mM was considered the best material to induce healing wounds in less time, along with an aesthetic, functional aspect, by modulating the cytokine production between the inflammatory and anti-inflammatory cytokines. Successful skin repair after tissue injury requires resolution of the inflammatory response. (CTS-g-GMA)-AuNPs at 1.07 mM with and without collagen showed an increase in wound healing percentage at 8 days; this stage is related to the second phase of wound healing, or granulation process [49].
Whereas knowledge about mechanisms and molecules inducing and perpetuating the inflammatory response is constantly increasing, mechanisms that limit and downregulate this activity are less appreciated (down-regulation of chemokine expression by anti-inflammatory cytokines such as IL-10 or TGF-B1, or up-regulation of anti-inflammatory molecules such as IL-1 receptor antagonists or soluble TNF receptors). For example, McFarland et al. showed that mice with impaired capacity macrophages have an excess of fibrin and cellular debris, delaying wound healing compared with untreated mice; in the same way, it was observed that in IL-6 knockout mice, the wound healing was delayed because of poor infiltration of macrophages, affecting the re-epithelialization [50]. Additionally, IL 17 and IL-10 knockout mice showed a faster wound healing process because the inflammatory process was decreased, which improved the angiogenesis [51,52].
The production of spleen cytokines was not different compared with TheraForm ® , suggesting that these materials cannot produce a systemic inflammatory response that can compromise the individual's homeostasis. For future studies with the best nanocomposite to induce the best aesthetic cicatrization, we are considering examining matrix metalloproteases, because these molecules play a role in the process of tissue granulation, modulating the formation of scar tissue by the production of extracellular matrices and promoting cell migration, which is reduced and changed by type III collagen and replaced by type I collagen [45].

Conclusions
The chitosan-based Au nanocomposites presented here (with and without type I collagen) exhibit a superior ability to promote skin wound healing when compared to the positive controls TheraForm ® and Chitosan-g-glycidyl methacrylate/Au nanocomposites ((CTS-g-GMA)-AuNPs) (with and without type I collagen). The best healing process was achieved using nanocomposites with 0.6 mM of gold nanoparticles. Our results demonstrated that ((CTS-g-GMA)-AuNPs)-Col is an efficient material for accelerating wound healing by improving the formation of granulation tissue and the migration of hyperproliferative keratinocytes; these observations are traceable to the nanocomposite's electrical behavior according to the electrical percolation phenomena. In this regard, our results suggest that the best performance nanocomposite is found with a nanoparticle concentration near the percolation threshold. The percolation threshold dictates when nanoparticles agglomerate, and the nanocomposite starts to fail. It is noteworthy that further studies are required to correlate cytokine production and the cicatrization process for a better understanding of this mechanism.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/pharmaceutics14091855/s1, Table S1: Valuation diagram of wounds: 1= recover, 4 = limited recover. According with score the wound can be Valuation diagram of wounds: 1 = recover, 4 = limited recover. According with score the wound can be classified in four typesclassified in four types.