Cell loaded hydrogel containing Ag‐doped bioactive glass–ceramic nanoparticles as skin substitute: Antibacterial properties, immune response, and scarless cutaneous wound regeneration

Abstract An ideal tissue‐engineered dermal substitute should possess angiogenesis potential to promote wound healing, antibacterial activity to relieve the bacterial burden on skin, as well as sufficient porosity for air and moisture exchange. In light of this, a glass–ceramic (GC) has been incorporated into chitosan and gelatin electrospun nanofibers (240–360 nm), which MEFs were loaded on it for healing acceleration. The GC was doped with silver to improve the antibacterial activity. The bioactive nanofibrous scaffolds demonstrated antibacterial and superior antibiofilm activities against Gram‐negative and Gram‐positive bacteria. The nanofibrous scaffolds were biocompatible, hemocompatible, and promoted cell attachment and proliferation. Nanofibrous skin substitutes with or without Ag‐doped GC nanoparticles did not induce an inflammatory response and attenuated LPS‐induced interleukin‐6 release by dendritic cells. The rate of biodegradation of the nanocomposite was similar to the rate of skin regeneration under in vivo conditions. Histopathological evaluation of full‐thickness excisional wounds in BALB/c mice treated with mouse embryonic fibroblasts‐loaded nanofibrous scaffolds showed enhanced angiogenesis, and collagen synthesis as well as regeneration of the sebaceous glands and hair follicles in vivo.


| INTRODUCTION
Bacterial infection is a vicious, unaddressed problem in the healing of skin wounds. Prolonged bacterial infections are critical challenges in the management of chronic wounds. Bacterial biofilms play a significant role in persistent bacterial infections. [1][2][3] Skin substitutes are acellular or cellular tissue-engineered platforms used to restore the structure and function of the skin. 4 They are intended for providing temporary coverage or supporting permanent wound closure.
The use of skin substitutes decreases healing time, minimizes postoperative wound contraction, and enhances skin function. 5, 6 An ideal skin substitute should be biocompatible, biodegradable, minimally immunogenic, mechanically stable, and keep the wound interface moist while also containing antimicrobial activities. [7][8][9] Natural-based materials (e.g., chitosan and collagen) and synthetic biocompatible polymers (e.g., polyethylene oxide) have been explored to fabricate tissue-engineered skin scaffolds in various forms. 10,11 Fibrous and nanofibrous materials are important in bioengineering because they resemble the extracellular matrix (ECM), permeability, as well as large surface area. [12][13][14] Electrospinning is a common method for fabricating nanofibrillar structures due to its simplicity and relative ease of scaling up for industrial production. 15 Bioactive glasses and glass-ceramics (GC) are used as components of skin tissue engineering scaffolds to accelerate the healing process because of their angiogenic and anti-inflammatory characteristics. These biomaterials promote angiogenesis via their ionic dissolution products.
Bioactive glasses and GC increase the secretion of angiogenic growth factors from fibroblasts, such as vascular endothelial growth factor and basic fibroblast growth factor. [16][17][18][19] Trace elements such as Ca, P, Si, Cu, and Mg have also been reported to improve angiogenesis. The incorporation of Ag, Cu, or Zn improves antibacterial properties. It is possible to dope bioactive glasses and GC with these trace elements to augment these highly desirable properties. [19][20][21] In light of the potential angiogenic and wound healing properties of bioactive glasses and GC, the present study deals with the fabrication of a biocompatible skin substitute with improved antibacterial properties and low immunogenicity for skin regeneration. Silver ions were incorporated into the GC composition to endow the biomaterial with bactericidal and anti-biofilm activities. The nanofibrous scaffold was first evaluated for its physicochemical properties in vitro. An in vivo study was subsequently conducted to investigate the wound healing capability of the nanofibrous scaffold in excisional fullthickness wounds created in BALB/c mice with or without mouse embryonic fibroblasts (MEFs). Figure 1a shows the method used for synthesizing GC and Ag/GC via sol-gel reaction. Fourier transform-infrared (FT-IR) spectra of the pristine and Ag-doped GC powders are shown in Figure 2a. The peak observed at 1670 cm À1 was attributed to the vibrations of O H bonds. The peak at 621 cm À1 was attributed to the P O bonds. Two peaks at 926 and 1024 cm À1 were assigned to the Si O tension bonds, and the peak at 460 cm À1 was attributed to the Si O stretching mode. The data were suggestive of the formation of GC networks. 22 The small peak at 644 cm À1 and the change in peak intensity at 930 cm À1 in the Ag-doped powder was confirmative of Ag doping. Figure 2b shows the x-ray diffraction (XRD) pattern of the pristine GC powder and Ag-doped GC powder. According to The International

| Structural characterization of glass-ceramic powders
Center for Diffraction Data database, two crystalline phases were detected in each powder. The best overall matches were combeite (Na 6 Ca 3 Si 6 O 18 ; Code: 01-077-2189, predominant phase) and silicorhenanite (Na 2 Ca 4 (PO 4 ) 2 SiO 4 ; Code: 00-032-1053). The low-intensity noise from the XRD data indicates the co-existence of crystalline and amorphous phases in the structure. A slight peak shift toward higher angles and reduction of peak intensities were observed in the Ag-doped GC. [23][24][25] Field emission scanning electron microscopy (SEM) images of the pristine and Ag-doped GC powders are shown in Figure 2c. Both powders appeared as spherical nanoscopic agglomerates. The particle size range for the pristine GC powder was 60-180 nm with a mean size of 80 nm. Silver doping reduced the particle size range to 20-80 nm, with a mean particle size of 36 nm. Energy-dispersive x-ray analysis confirmed the presence of Si, Na, Ca, and P in both powder samples. Silver was additionally identified in the Ag-doped GC powder.

| Structural characterization of scaffolds
The nanofibrous scaffolds were fabricated via electrospinning ( Figure 1b). The presence of GC and Ag/GC within the nanofibrous scaffolds were evaluated to ensure the fabrication of the proper scaffolds for skin regeneration. Figure 2d shows the FT-IR spectra of the gelatin and chitosan (Ch)/ polyethylene oxide (PEO)/Gel and the Ag/GC-Ch/PEO/Gel electrospun scaffolds. The similarity of the two spectra suggests the presence of similar chemical bonds in the two nanofibrous scaffolds. The C O band at 1662 cm À1 was attributed to type I amide. The N H and C H bands at 1546 cm À1 were attributed to type II amide. The C N and N H bands at 1224 cm À1 corresponded to type III amide. The N H vibration at 3298 cm À1 was associated with amide groups. These peaks were indicative of the presence of gelatin and chitosan in the scaffolds. 26,27 The total porosity and pore distribution of the Ag/GC-Ch/PEO/Gel scaffold were investigated using mercury porosimetry. The results showed that the porosity distribution in this nanofibrous scaffold was in the range of 0.01-20 μm. The average pore size is 7 ± 2 μm (Figure 2e). Since pore size distribution for the Ag/GC-Ch/PEO/Gel scaffold was the same when evaluated by mercury porosimetry and SEM, the porosity of the other samples was estimated by SEM to be 44%, 39%, and 38% for the Ch/PEO/Gel, GC-Ch/ PEO/Gel and Ag/GC-Ch/Gel scaffolds, respectively. This can be related to the impairing effect of particles such as GCs on the structure of the polymeric scaffolds by serving as hard inclusions. 28,29 Thermal gravimetric analysis (TGA) measures the weight change of a sample as a function of temperature, while subjected to a controlled heating program. Figure 3b,c show TG and DTG thermograms of Ch/PEO/Gel, GC-Ch/PEO/Gel, and the Ag/GC-Ch/PEO/Gel F I G U R E 1 (a) GC and Ag/GC synthesis by the sol-gel method (b) GC-Ch/PEO/Gel and Ag/GC-Ch/PEO/Gel nanofibrous scaffold fabrication through electrospinning samples. The thermogram comparison of the samples exhibits that the thermal stability of GC-Ch/PEO/Gel is higher than the Ch/PEO/Gel. Surprisingly, it was observed that the thermal stability of Ag/GC-Ch/ PEO/Gel is lower than the Ch/PEO/Gel. This may be due to the nonoptimized amount of silver in the GC structure.
Brunauer-Emmett-Teller (BET) analysis was used to explain the physical adsorption of gas molecules on a solid surface and serves as the basis for an important analysis technique for the measurement of the specific surface area of materials. Figure 3d show the N 2 adsorption/desorption isotherms and the BET obtained data of Ag/GC-Ch/ PEO/Gel, GC-Ch/PEO/Gel, and Ch/PEO/Gel. The BET analysis showed that the specific surface area of Ag/GC-Ch/PEO/Gel was 12.396 m 2 /g compared to GC-Ch/PEO/Gel and Ch/PEO/Gel. This can be related to the presence of Ag nanoparticles in the GC composition. Moreover, the mean pore diameter of Ag/GC-Ch/PEO/Gel was 13.357 nm compared with Ch/PEO/Gel (Figure 3e).

| Antibacterial activities
The antibacterial properties of the nanofibrous scaffolds, with or without GC powder, were investigated using methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa. The most effective bacterial eradication was detected when the biofilms were exposed to Ag/GC-Ch/PEO/Gel (p < 0.001). Confocal laser scanning microscopy of 48 h old biofilms of MRSA (f) and P. aeruginosa (g). After incubation for 24 h, the bacteria were stained with fluorescein isothiocyanate and propidium iodide to evaluate morbidity (green fluorescence-live bacteria; red fluorescence-dead bacteria; f and g). Scale bars represent 25 μm nanofibrous scaffolds; Figure 4b,c). As shown in Figure 4d,E, suspensions of the Ag/GC-Ch/PEO/Gel scaffold had the highest biofilm eradication rate (89 ± 5%). The percentage of biofilm elimination by the Ag/GC-Ch/PEO/Gel scaffold increased from less than 20% to more than 80% (p < 0.001). Confocal laser scanning microscopy of live/dead-stained single-species bacteria biofilms showed dead MRSA biofilms ( Figure 4f) and P. aeruginosa biofilms ( Figure 4g) after exposure to the AG/GC-Ch/PEO/Gel scaffolds (red fluorescence). In contrast, biofilms derived from the control group and those exposed to the GC-Ch/PEO/Gel scaffolds were predominantly alive (green fluorescence).

| The effects of Ag-doped bioactive GC nanofibrous scaffolds on dendritic cells
To evaluate the immunogenicity of the nanofibrous scaffolds, bone marrow-derived dendritic cells were analyzed after the cells were exposed to the different scaffolds. Specifically, the levels of cell surface activation markers CD80 and CD86 were evaluated by cytofluorimetry. The expression of both CD80 and CD86 markers in (e) Percentage of hemolysis induced by different experimental scaffolds. The positive control was blood treated with water. Data are means ± standard deviations (n = 3). Groups connected by lines and labeled with asterisks are significantly different; significant differences were tested by one-way analysis of variance (ANOVA) and post hoc Tukey's multiple comparison tests (*p < 0.05; ***p < 0.001) dendritic cells that had been exposed to the Ch/PEO/Gel scaffold was largely unchanged. Conversely, both markers were upregulated in dendritic cells that had been exposed to the GC-Ch/PEO/Gel scaffold.
Dendritic cells that had been exposed to the Ag/GC-Ch/PEO/Gel scaffold showed no difference in the levels of CD80 and CD86 expression when compared to untreated dendritic cells (Figure 6a-c).
The release of pro-inflammatory cytokines interleukin-6 (IL-6) and IL-12 by the dendritic cells was also evaluated. Both IL-6 and IL-12 are produced by dendritic cells and macrophages in response to bacterial infections or stress conditions such as tissue injury. The cytokines promote a T helper type I-oriented response. 30,31 There was a release of IL-6 in the dendritic cell cultures that were exposed

| In vivo study
The wound healing efficacy of the Ag/GC-Ch/PEO/Gel nanofibrous scaffold, with or without MEFs, was evaluated using a BALB/c mouse excisional wound splinting model and followed histopathological analysis. As shown in Figure 7b,c, the size of all the wounds was approximately equal to the one on the third day. Wounds size treated with the Ag/GC-Ch/PEO/Gel nanofibrous scaffold, with or without MEFs, became considerably smaller in dimension than the control group after the third day. In comparison, wound size in the control wound area remained the same, with minimal change (9.12 ± 0.85 mm 2 ). On the seventh day after surgery, wound healing and skin regeneration in the Ag/GC-Ch/PEO/Gel and the Ag/GC-Ch/PEO/Gel containing MEFs improved significantly (6.27 ± 0.83 and 5.80 ± 0.45 mm 2 ) compared with the control group (9.12 ± 0.85 mm 2 ). Investigations on the 14th day revealed the same healing process in wounds that were treated by the nanofibrous scaffold; there was a lack of skin regeneration in the control group. Compared with other groups (4.03 ± 0.17 mm 2 ) and after 21 days, there was a significant decrease in wound area in those skin wounds that were treated with Ag/GC-Ch/PEO/Gel scaffolds containing MEFs (0.28 ± 0.04 mm 2 ). According to the macroscopic evaluation of wound healing (Figure 6b) as well as wound size (Figure 6c  Bone marrow-derived dendritic cells were exposed to the Ch/PEO/Gel, GC-Ch/PEO/Gel, Ag/GC-Ch/PEO/Gel scaffolds, or stimulated with lipopolysaccharide (LPS positive control). Culture supernatants were analyzed by enzyme-linked immunosorbent assay for IL-6 (d) and IL-12 p70 (e) production. (f) Inflammation was induced in BM-DCs by co-culturing cells with bacterial lipopolysaccharide in the presence of the different scaffolds or the medium. The IL-6 released in the supernatants was measured by ELISA. All the supernatants were assayed in duplicate. Data represent mean ± standard error of the mean. Results of a representative experiment (out of two) are shown. Significant differences were tested by one-way analysis of variance (ANOVA) and post hoc Tukey's multiple comparison tests (*p < 0.05; **p < 0.01; ***p < 0.001) The outermost skin layer (i.e., the epidermis) was not regenerated in the control group. Conversely, the epidermis was healed entirely in wounds that were treated with the Ag/GC-Ch/PEO/Gel nanofibrous scaffold. Regeneration of the epidermis was attributed to keratino- were only 9 ± 2 vessels in wounds derived from the control group.

| DISCUSSION
Regenerative medicine and tissue engineering are promising therapeutic approaches for repairing or replacing large skin wounds. An extensive array of biomaterials, semiconducting nanomaterials, bioactive glass, GC, and composite materials are available to develop skin substitutes. 34 Skin substitutes with angiogenic and antibacterial properties and the ability for tissue to regenerate are ideal alternatives to traditional dressings because they improve the wound healing process. 23,35 In the present work, GC-Ch/PEO/Gel nanofibrous scaffolds containing pristine GC or Ag-doped GC were synthesized. To enhance cellular interactions, 60-80 nm sol-gel derived GC or Ag/GC were incorporated into the electrospun scaffolds that had fibrils with diameters between 200 and 300 nm. The electrospinning setting employed produced Ag/GC-Ch/PEO/Gel scaffolds with a mean pore size of 7 ± 2 μm. A recent study reported that the optimal pore size for cellular infiltration and vessel formation in electrospun nonwoven scaffolds for bioresorbable vascular grafts is in the range of 5-20 μm. 36 X-ray diffraction showed that the GC powder had combeite as the predominant crystalline phase. This result was in agreement with the literature. 25,[37][38][39] The reason for the formation of this crystalline structure is that the temperature at which nitrates are completely removed is higher than the crystallization temperature of the glass. A lower degree of crystallinity was identified for Ag-GC, making the glassceramic more suitable for biological applications. Fourier transforminfrared spectroscopy confirmed that both the GC and Ag/GC powders possessed 45S5 bioglass characteristics. Energy-dispersive -ray analysis confirmed the presence of Ag in the Ag-doped GC powder. Infrared spectra of the scaffolds also identified the presence of gelatin (amide types I, II, III, and amide B), chitosan, PEO, and GC in the nanofibrous scaffold network.
Cell-scaffold interactions are significantly affected by the surface's wettability of the scaffolds, as this feature controls several key biological processes, including protein adsorption, cell attachment, and proliferation. 11,40 Furthermore, the contact angle is a proper assay to measure the surface moisture, which plays a vital role in wound healing. 41 The difference indicates that GC and Ag/GC containing The hemolysis assay is an essential blood compatibility test for determining a material's biocompatibility. Damaged RBCs release adenosine diphosphate, which increases platelet attraction and assembly to the material surface. This procedure may expedite the initiation of coagulation cascades and thrombosis, leading to disruption of the wound healing process. Accordingly, a desirable skin substitute should not harm the circulating RBCs at the wound site and should not compromise the activation of coagulation pathways. 50 The results indicate that all the experimental scaffolds examined, the GC-Ch/PEO/Gel scaffold, in particular, induced negligible hemolysis.
Biocompatible materials should be well-tolerated by the host. Consequently, they should be immunologically inert and should not induce inflammatory host immune responses. 51   until the gels were formed. The wet gels were dried at 120 C in an oven for 16 h to produce xerogels. The resultant powders were transferred to a furnace and sintered at 700 C to remove nitrates and stabilize the network. 54 Grinding of the sintered GC powder was performed using a ball mill to obtain nanoparticles (Figure 1a). Fourier transform-infrared spectroscopy was used to identify the functional groups of the prepared nanofibrous scaffolds. Briefly, 1 mg of each scaffold was mixed with 300 mg of KBr. The mixture was prepared as a pellet and analyzed over the range of 4000-400 cm À1 at a scanning speed of 2.60 Hz with a resolution of 4 cm À1 .

| Synthesis and structural characterization of GC and Ag/GC nanoparticles
Total porosity, average pore diameter, and pore size distribution of the Ag/GC-Ch/Gel scaffolds were evaluated using mercury porosimetry (PASCAL 140, Thermo-Finnigan LLC, San Jose, CA) using increasing pressures of 0.1-400 kPa. Pore size measurements were performed on the SEM micrographs of the prepared nanofibrous scaffolds using the ImageJ software.
The mechanical characteristics of the nanofibrous scaffolds were measured using SANTAM universal testing machine (STM-1 model).
The ends of the rectangular specimens were placed vertically on the tensile tester's two mechanical gripping components, leaving 50 mm gauge length for mechanical loading, and were pulled with a 5 mm/min rate.
Thermogravimetric analysis (TGA, L81A1750, Linseis) was employed to study the thermal stability of fabricated nanofibrous scaffolds. TGA analysis was recorded at 10 C/min in an N 2 atmosphere. N 2 adsorption-desorption isotherms were obtained on a Nova 2000 pore analyzer at 196 C under continuous adsorption condition.
Brunauer-Emmett-Teller (BET) analyses were utilized to determine the surface area, the pore size distribution and the pore volume.

| Contact angle measurement
The hydrophobic characteristics of each specimen were assessed using contact angle measurement by 2X lens and Protractor (AMCAP, VERSION 9.016). Nanofibrous scaffold samples were prepared into 12 Â 12 mm 2 square pieces and fixed on the assay plate. Afterward, a single drop with an approximate volume of 4 μl of distilled water was added to each sample at room temperature. Three different areas on each sample were measured, and the mean contact angle and standard deviation were calculated.

| Antibacterial evaluation
The bacterial death rate was evaluated using a modified AATCC-100 Test Method. The bacterial death rate was measured by providing

| Confocal laser scanning microscopy
Confocal laser scanning microscopy to assess the impact of the GCcontaining nanofibrous scaffold on the MRSA and P. aeruginosa biofilms. The biofilms were grown on glass coverslips as previously described. 56 In brief, 6-well microtiter plates were seeded with glass coverslips. Five milliliters of TSB with 2% glucose were added to each well. Three microliters of mid-exponential grown bacterial culture in TSB were added aseptically to the wells, followed by incubation at

| Cell viability and cell attachment measurement
For SEM examination of cell attachment, the prepared nanofibrous scaffolds were seeded with 1 Â 10 4 MEF for 3 days.

| Statistical analysis
Statistical analyses were performed using a one-way analysis of variance followed by post hoc Tukey's test. Data analyses were performed using GraphPad Prisma 9 software (San Diego, CA). A p-value below 0.05 was considered statistically significant.

| CONCLUSION
The present work demonstrated that mouse embryonic fibroblasts-loaded Ag/GC-Ch/PEO/Gel nanofibrous scaffolds enhanced the cutaneous wound healing process. Nanoscopical crystalline GC and Ag-doped GC powders were prepared. These bioactive powders were then used to fab-