Oxidized alginate-gelatin (ADA-GEL)/silk fibroin/Cu-Ag doped mesoporous bioactive glass nanoparticle-based hydrogels for potential wound care treatments

The present work focuses on developing 5% w/v oxidized alginate (alginate di aldehyde, ADA)-7.5% w/v gelatin (GEL) hydrogels incorporating 0.25% w/v silk fibroin (SF) and loaded with 0.3% w/v Cu-Ag doped mesoporous bioactive glass nanoparticles (Cu-Ag MBGNs). The microstructural, mechanical, and biological properties of the composite hydrogels were characterized in detail. The porous microstructure of the developed ADA-GEL based hydrogels was confirmed by scanning electron microscopy, while the presence of Cu-Ag MBGNs in the synthesized hydrogels was determined using energy dispersive x-ray spectroscopy. The incorporation of 0.3% w/v Cu-Ag MBGNs reduced the mechanical properties of the synthesized hydrogels, as investigated using micro-tensile testing. The synthesized ADA-GEL loaded with 0.25% w/v SF and 0.3% w/v Cu-Ag MBGNs showed a potent antibacterial effect against Escherichia coli and Staphylococcus aureus. Cellular studies using the NIH3T3-E1 fibroblast cell line confirmed that ADA-GEL films incorporated with 0.3% w/v Cu-Ag MBGNs exhibited promising cellular viability as compared to pure ADA-GEL (determined by WST-8 assay). The addition of SF improved the biocompatibility, degradation rate, moisturizing effects, and stretchability of the developed hydrogels, as determined in vitro. Such multimaterial hydrogels can stimulate angiogenesis and exhibit desirable antibacterial properties. Therefore further (in vivo) tests are justified to assess the hydrogels’ potential for wound dressing and skin tissue healing applications.


Introduction
Skin is the first defense line of the human body against injuries, infectious diseases, and dehydration.Further, it is responsible for sensory detection [1], thermoregulation, and protection against mechanical and thermal trauma [2,3].Nevertheless, inefficient skin healing is prone to burns and bruises due to poor circulation, excessive swelling, poor nutrition, abnormal bacterial response, and trauma.The frequency of diseases caused by infectious wounds has raised recently [4,5]; thus, various anti-infectious wound treatments are suggested for clinicians and patients.For instance, dehydration can be inhibited to maintain a humid wound environment by absorbing and retaining a large volume of fluid while at the same time avoiding maceration of the peri-wound skin, thus providing moisturizing effects.Enhancing angiogenesis and collagen synthesis can also provide protection against secondary infections and promote tissue repair [2,6].
The type and location of the wound, the patient's condition, and medical resources can all affect the acceptable rate of complications that can occur due to wound closure delays [7].Delays in wound closure can cause infection, scarring, and tissue damage [8,9].The severity and incidence of these problems vary on factors such as wound type (surgical, traumatic, and chronic wounds have differing tolerable rates of complications) [10][11][12].Controlled environments and sterile techniques are expected to reduce complications in surgical wounds, but wounds in certain areas of the body, such as the face or hands, may have lower acceptable complications due to their aesthetic and functional importance [13,14].Patients with underlying medical conditions, such as diabetes or immunodeficiency, usually have a higher risk of complications [3,15].In general, the goal of any wound treatment is to mitigate complications to an acceptable level by achieving suitable in-vitro and invivo properties of the wound dressing which should be then validated by successful clinical trials.The proposed solution should not only contribute positively to the common qualitative (and quantitative) markers (like enhanced healing rate, better mechanical strength, non-infectious environment, proangiogenic behavior) of soft tissue repair, but should also exhibit specific properties related to a particular wound type.For instance, hydrogels for skin regeneration should possess high water retention capability for treating burn wounds, anti-microbial properties for abrasive wounds, etc.The proposed biomaterial approach, in general, should be capable of addressing all the combined qualitative requirements in the wound healing process, acting as a barrier against secondary infections, eliciting tissue regeneration, assuaging wound-associated pain, and fostering high-quality wound healing.Additionally, hydrogels should possess elasticity, exhibit non-antigenic properties, and enable the effective management of wound exudate [16][17][18].
Wound care and hydrogels as wound dressings have changed greatly over the years due to advancements in biomaterials technology and medical research [19][20][21].From woven cotton structures to synthetic bioengineered materials and natural tissue replacements, wound care treatments have improved and become more specialized [22][23][24].For instance, moisture-retentive hydrogels are designed to create and maintain a moist environment around the wound [25].This promotes wound healing by stimulating cell migration, angiogenesis, and granulation tissue formation [26].Hydrogels, hydrocolloids, foams, and films as moisture-retentive biomaterials can be used to treat ulcers, burns, and surgical wounds [15,27].Recently, impregnated wound hydrogels include the addition of antimicrobials, silver, iodine, or other drugs to fight infection or promote healing [28][29][30].Biomaterials science and biotechnology research continue to advance hydrogels to improve wound care outcomes [31][32][33][34].In this regard, research is being carried out continuously worldwide to develop hydrogels that can meet the complex requirements involved in wound healing, in particular tackling the delay in wound closure, usually linked to reduced angiogenesis, and the high potential of infections.
In the context of wound treatment methods, hydrogels are promising candidates as their threedimensional (3D) hydrophilic network [35][36][37] can be conveniently exploited to absorb from 10% to 20% (an arbitrarily minimum range) of their comparable water content until the system reaches an equilibrium point [38,39].
Alginate is a long unbranched copolymer made up of (1-4)-linked -D-mannuronic acid (M) and -L-guluronic acid (G) monomers and is being used in bioprinting due to its biocompatibility and quick ionic gelation by divalent cations [5,40].Gelatin (GEL) is a biodegradable and biocompatible protein polymer produced by denaturing collagen, which can be used in biomedical applications such as tissue engineering, hydrogel fabrication, wound dressing, gene therapy, and drug delivery [41,42].GEL can be combined with alginate to improve the degradation behavior and printability of alginate by optimizing the viscosity and processability of the hydrogel.The covalent cross-linking of GEL with alginate dialdehyde (ADA) (oxidized alginate) has been investigated to develop ADA-GEL hydrogels [42], which can aid the migration, growth, and proliferation of cells.Additionally, the soft and rubbery nature of ADA-GEL provides a moist environment at the wound site, which can decrease pain and increase leucocyte phagocytic activity and enzymatic activity of injured cells [43].
Silk fibroin (SF) is a natural protein that can be incorporated into ADA-GEL hydrogels to further improve their biocompatibility, chemical stability and mechanical integrity [44].SF is obtained from silk cocoons (Bombyx mori) [45] and can be functionalized and crosslinked [44].SF-based hydrogels are highly flowable, which leads to self-healing and electrically conductive hydrogels [46].Moreover SF provides a moisturizing effect to the wound site due to the amino acid content present in it [47][48][49][50].
Bioactive glasses (BGs) can facilitate the formation of a bond between implants or scaffolds and host tissues due to their surface bioreactivity in contact with the biological environment [51].Mesoporous bioactive glass nanoparticles (MBGNs) are being increasingly investigated as suitable inorganic materials to promote vascularization, bone formation, and tissue integration [52][53][54].MBGNs can be doped with various metallic ions to enhance their therapeutic effect.For instance, Aqib et al [55] and Bari et al [56] doped Ag and Cu in MBGNs and reported that incorporating Cu and Ag into MBGNs led to antibacterial and angiogenic effects, respectively.Application of MBGNs in wound healing approaches has been also reported [57,58].
The present study aims to develop a new strategy for designing SF containing ADA-GEL hydrogels loaded with Cu-Ag MBGNs in order to address unmet requirements in wound dressing technologies.We propose that wound care treatments may benefit from ADA, GEL, SF, and Cu-Ag doped MBGNs due to the potential synergistic effect of these components.The ADA-GEL hydrogel should ensure that the body's natural healing processes are not inhibited.The acceptable swelling and degradation properties of ADA-GEL hydrogel will provide a moist environment, ultimately aiding in cell migration and tissue regeneration, followed by enhanced healing rate.The controlled release of the antibacterial ions Cu and Ag from MBGNs can effectively prevent or treat infections.Moreover ADA-GEL hydrogels loaded with Cu-Ag MBGNs are hypothesized to induce angiogenesis (due to the local release of Cu), in addition to exhibiting antibacterial properties.The combination of 0.25% w/v SF and ADA-GEL hydrogels loaded with Cu-Ag MBGNs is being reported here for the first time to the best of the authors' knowledge.The present characterization of the new multimaterial hydrogels by in vitro studies should provide preliminary but promising evidence that the developed hydrogels have potential to reduce complications in wound healing by promoting angiogenesis and antibacterial effects.Favorable in vitro data will justify further research, including in vivo studies, to evaluate the efficacy and safety of the hydrogels in reducing complications in various wound healing scenarios.

Synthesis of 5% w/v ADA-7.5% w/v GEL films
ADA was synthesized by the controlled oxidation of sodium alginate (Na-Alg) in equal volume ratios of ethanol and water following previously published protocols [40,59].Briefly, 1.605 g of sodium metaperiodate and 5 g of Na-Alg were dissolved in 25 ml D.W. and 25 ml ethanol, respectively, to obtain a homogeneous solution.The periodate solution was then added dropwise to the Na-Alg dispersion with continuous stirring under dark conditions at room temperature.After 6 h, the reaction was quenched by mixing 5 ml of ethylene glycol (EG) to the suspension under continuous stirring for 30 min.Afterwards, the obtained suspension was dialyzed against deionized water using a dialysis membrane with a molecular weight cutoff (MWCO) between 6000 and 8000 kDa for 4-7 d (deionized water was changed after every 24 h) until the suspension was periodate free.0.5 ml aliquot of dialysate was added to 0.5 ml of 1% w/v solution of Ag (NO 3 ) 2 to confirm the absence of periodate in the suspension.In this case, if no precipitates are formed, it confirms that the dialysate is periodate free.Finally, the ADA solution was freeze-dried for 3 d (i.e.lyophilized).
ADA and GEL of different compositions were prepared in equal volumes in PBS following a previously published protocol [60].Briefly, 5% w/v of frozen ADA solution and 7.5% w/v of GEL were added separately to PBS and continuously stirred at 40 • C until homogenous solutions were formed.Both solutions were then passed through a 0.45 µm sterile filter to remove undissolved particles.Filtered solutions were then mixed under continuous stirring to facilitate cross-linking between ADA and GEL and eventually form 5% w/v ADA-7.5% w/v GEL hydrogels.Figure 1 illustrates all the steps involved in synthesizing 5% w/v ADA-7.5% w/v GEL hydrogels.The optimization of hydrogel composition involved a systematic 'trialand-error' approach to achieve the ideal combination of 5% w/v ADA and 7.5% w/v GEL.The focus was on attaining optimal viscosity and ensuring highquality hydrogel formation to impart structural integrity.This was crucial to maintain the stability of the resulting film, preventing it from breaking or flowing easily.The viscosity of the hydrogel precursor solution played a pivotal role in ensuring the uniformity of the film, which is especially significant in applications such as wound dressings and transdermal drug delivery patches.The hydrogel synthesized from 5% w/v ADA and 7.5% w/v GEL yielded the best results in terms of stability (data not shown here), which was in agreement with previous studies [42,60].The 5% w/v ADA −7.5% w/v GEL hydrogel was thus utilized in the present study.Ajisawa [61] proposed a mechanism for the complete dissolution of silk fibroin (SF) using a ternary solvent.Following that technique, SF solution was prepared by dissolving it in a 1:2:8 molar ratio of calcium chloride: ethanol: water at a temperature of 55 • C for 4 h under continuous stirring, respectively.5 g of SF was dissolved in 150 ml solvent.Figure 2 shows the schematic illustration of the preparation of the SF solution.
A low-temperature sol-gel method, namely the modified Stöber process, was used to prepare Cu-Ag MBGNs.Cu-Ag MBGNs were synthesized following the method specified by Bano et al [52].First, 0.56 g CTAB was dissolved in 26 ml of distilled water under stirring for 30 min at 40 • C. 8 ml ethyl acetate was then slowly added to the solution.Next, 0.3 ml of ammonium hydroxide (32 vol.%) was diluted in 26 ml of distilled water, then added to the solution to maintain the pH at ∼9.5.Then, 6 ml of TEOS was added dropwise into the solution under continuous stirring.Later, 0.1925 g of copper nitrate, 0.0275 g AgNO 3 , and 2.53 g Ca(NO 3 ) 2 were added into the solution (for the preparation of 1 mol.% Ag and 7 mol.% Cu MBGNs) followed by magnetic stirring for 30 min.The nominal composition was 70 SiO 2 -22 CaO-1 Ag 2 O-7CuO in mol.%.The solution was left overnight for the reaction to occur.Afterwards, the suspension was centrifuged at ∼8000 rpm for 10 min to precipitate nanoparticles from the solution.The precipitated particles were washed with water and ethanol three times.Ultimately, the particles were dried in an oven at 70 • C for 12 h, followed by calcination at 700 • C for 5 h to obtain Ag-Cu MBGNs.
In order to prepare the ADA-GEL composite films, 0.25% w/v SF solution and 0.05% w/v, 0.1% w/v, 0.2% w/v, and 0.3% w/v Cu-Ag MBGNs were added to 5% w/v ADA-7.5% w/v GEL separately, under continuous stirring, before cross-linking the ADA-GEL hydrogel.The 5% w/v ADA-7.5% w/v GEL solution incorporating 0.25% w/v SF and different concentrations of Cu-Ag MBGNs was left to stir for 1 h to allow SF to react to form a homogeneous solution.The hydrogel composite blend was then cast into petri dishes.An equal volume of 0.1 M calcium chloride solution was added to the cast hydrogel in the petri-dishes.The films were freeze dried and stored for future use. Figure 3 shows an schematic illustration of the process followed to fabricate 5% w/v ADA-7.5% w/v GEL/0.25% w/v SF/0.3% w/v Cu-Ag MBGNs films.

Characterization of the hydrogels 2.3.1. Morphological analysis
The microstructural features of the produced hydrogel films were examined using scanning electron microscopy (SEM, LEO 435VP, Carl Zeiss™ AG, Jena, Germany) at an accelerating voltage of 10 kV.The hydrogel films were coated with gold (approximately 10 nm) by sputtering (Q150/S, Quorum Technologies™, Lewes, UK) to make them conductive and to reduce charging during SEM imaging.SEM micrographs at different magnifications were captured to examine the morphology of hydrogels.

Compositional analysis
A SEM equipped with energy-dispersive x-ray spectroscopy (EDX) capability (LEO 435VP, Carl Zeiss™ AG) was used to investigate the hydrogel films qualitatively.Further, the changes in the chemical bonds of the synthesized hydrogels were investigated via Fourier transform infrared spectroscopy (FTIR).Additionally, a background scan was conducted with 128 runs.The chemical structure of the hydrogels was analyzed with 128 transmittance scans with 4 cm −1  resolution in Happ-Genzel apodization and in a wavenumber range between 400 and 4000 cm −1 .The FTIR spectra were smoothed by 15 points to minimize the noise.

Micromechanical testing
The micromechanical characterization of hydrogel films was carried out using a tensile testing rig (Linkam TST350, UK) equipped with a 200 N load cell, according to the ASTM D882 standard.The dimensions of the samples were 26 mm × 7 mm × 0.5 mm, and the samples were stretched at a rate of 83.35 µm s −1 .The tests were performed on three different hydrogel films (5% w/v ADA-7.5% w/v GEL, 5% w/v ADA-7.5% w/v/ 0.25% w/v SF, 5% w/v ADA-7.5% w/v GEL/0.25% w/v SF/0.3% w/v Cu-Ag MBGNs).

Surface area and pore size analysis
The Brunauer-Emmett-Teller (BET) surface area and pore-type of hydrogels were measured by adsorption isotherm using a surface area analyzer (GOLD APP, V-Sorb 2800, China) through N 2 adsorption at 77 K. Prior to analysis, the hydrogel was degassed at 130 • C for 3 h.

Swelling/deswelling behavior of hydrogels
Swelling/deswelling tests were carried out on hydrogels under sterile conditions.After preparing hydrogel films, they were left to dry until they completely shrunk/deswelled.The initial weight of five different samples of each composition of hydrogels was measured, and then these samples were immersed in water for different periods, i.e. 15 min, 30 min, 1 h, 2 h, 3 h, 3.5 h, 4 h, 4.5 h, 5 h, 5.5 h, and 6 h.The wet weight of the gel was determined by first removing any excess surface water by placing it on a filter paper for a few minutes in air.Then, the hydrogels were weighed, and the swollen hydrogels were freeze-dried and weighed again.Finally, the swelling ratio percentages of the samples were calculated by equation (1).Each experiment was repeated 7 times (n = 7) where, %SR is the swelling ratio percent, W d is the weight of freeze dried hydrogels, and W s is the weight of swollen hydrogels.

Ion release studies (ICP-OES)
To investigate the Cu and Ag ion release from the prepared hydrogels loaded with 0.3% w/v Cu-Ag MBGNs, films were cut into 1 × 1 × 1 cm 3 cubes.The experiment was performed in triplicate (n = 3).

In vitro degradation studies
The degradation rate of the synthesized films was monitored by immersing 200 mg of the film (cut into 1 cm × 1 cm × 1 cm dimensions) in 15 ml DPBS.
Initially, the films were weighed before incubation and later on the films were incubated in an orbital shaking incubator at 37 Next, the samples were sterilized under UV light for 45 min.Afterwards, hydrogel films were placed on the agar plates and incubated at 37 • C for 24 h.After 24 h, the cultured agar plates were photographed, and the images were analyzed using ImageJ® software for inhibition zone measurements (each test was carried out in triplicate).
The turbidity test was also performed to quantify the antibacterial effect of the hydrogels.A sterile wooden tip was used to scratch bacteria from a frozen sample, and then the scratched bacteria cells were poured into LB media to grow E. coli and S. aureus bacteria in separate glass test tubes (Pyrex, U.K.).The medium contained a bacterial concentration of 0.015 ± 0.002 (OD 600 ) after 24 h of incubation (the same initial concentration was used for both types of bacteria) and was ready to use.In the test tubes, solutions containing 5% w/v ADA-7.5% w/v GEL (control) and 5% w/v ADA-7.5% w/v GEL/0.25% w/v SF/0.3% w/v Cu-Ag MBGNs were introduced and incubated for 24 h.Afterwards, with absorbance at 600 nm, an optical density (OD) measurement was done (OD 600 ).The OD 600 medium was used as a standard for each test.

Colony formation of bacteria (CFU)
To evaluate the antimicrobial effect, the spread plate method was used.The spread plate method is typically used to count the number of colonies on an agar plate.E. coli and S. aureus strains were cultured in nutrient broth.In order to investigate the antimicrobial activity of hydrogels against E. coli and S. aureus, 1 ml of culture broth was inoculated in 9 ml of nutrient broth.5% w/v ADA-7.5% w/v GEL/0.25% w/v SF/0.3% w/v Cu-Ag MBGNs and 5% w/v ADA-7.5% w/v GEL hydrogels were placed in the solution inoculated with E. coli and S. aureus.Afterward, the samples were incubated at 37 • C for 48 h.Later on, samples were centrifuged at 11 000 rpm for 10 min, and 1 ml of supernatant was inoculated with a bacterial culture of E. coli and S. aureus and were then incubated for 24 h at 37 • C.After that, serial dilution (n = 17) was performed, 50 µl of aliquots were plated on nutrient agar plates and plates were incubated at 37 • C for 24 h.After incubation, the number of colonies was counted and converted to CFU/ml [62].

Chorioallantoic membrane (CAM) assay studies
The CAM assay is a biological characterization technique that is used to test the angiogenic (formation of new blood vessels) potential of scaffolds, hydrogels, and membranes [63].For this purpose, day 6 fertilized eggs were obtained from the 'Jadeed Hatcheries Private Limited, Islamabad, Pakistan.'Day 6 fertilized eggs were initially cleaned with 30% ethanol solution and then placed in the incubator at 37 • C and 55%-65% relative humidity.The digital egg incubator (HHD 435) was switched on for 24 h before placing the eggs in order to achieve the required temperature and humidity.The eggs were taken out from the egg incubator on day 7 (implantation day) one by one inside the biosafety cabinet and cleaned with 70% ethanol.Later on, a small square (1 cm 2 ) was made on the top of the eggshell with a sterilized saw blade.The sterilized hydrogel sample was implanted inside the egg, and the egg was closed with parafilm and adhesive tape.The implanted eggs were marked with a unique number and placed inside the incubator.This process was repeated for all 30 eggs.Each type of sample was implanted in 5 eggs and then placed in the incubator for seven more days.Samples were retrieved on day 14, and the eggs were sacrificed.Prior to sacrificing, a picture of those eggs was taken, in which the embryo was at the developing stage.Three of the best samples from each group were compared with the control (5% w/v ADA-7.5% w/v GEL) in order to investigate the angiogenic potential of 5% w/v ADA-7.5% w/v GEL/0.25% w/v SF/0.3% w/v Cu-Ag MBGNs hydrogels.

Cell culture
Fibroblasts (NIH3T3-E1) derived from mouse NIH/Swiss embryo were seeded in DMEM (Sigma-Aldrich, St Louis, MO) containing 10% heatinactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin antibiotics (Sigma-Aldrich, St Louis, MO) in a humidified incubator at 37 • C and 5% CO 2 .Before starting the experiment, all films were sterilized under gamma irradiation for 24 h.For cell studies, squared films (1 cm × 1 cm) loaded with different concentrations (0.05% w/v-0.3%w/v) of Cu-Ag MBGNs were prepared in a similar manner as those used for swelling studies, and placed in a 24-well tissue culture plate to determine the effect of the samples on fibroblast cell lines.Cells with a density of 5 × 10 4 cells ml −1 of medium in each well were seeded on the control plate (without films).After 5 h of seeding, 1% v/v WST-8 into the well plates containing the samples was added, and the well plates were incubated for 3 h.10 µl of the culture sample was taken from each well after incubation and transferred to a 96 well plate to evaluate the cellular viability of films by measuring absorbance with a microplate reader and following the WST-8 protocol.
For biological testing, different Cu-Ag MBGN compositions (ranging from 0.05% w/v to 0.3% w/v) were tested.Copper has been studied for its pro-angiogenic properties, promoting blood vessel formation.Cu-Ag MBGNs are designed to enhance angiogenesis and using higher concentrations may be aimed at maximizing this effect.Angiogenesis is a critical process in wound healing, tissue regeneration, and other biological functions.By testing different concentrations, the intention was to identify whether the material has the potential to be effective in scenarios where a robust angiogenic response is desired.The different concentrations were thus used to assess whether Cu-Ag MBGNs can elicit a strong angiogenic response.Meanwhile it is also important to confirm biological responses that maximize the therapeutic benefits while minimizing any potential harm.So, it was important to verify that our selected fabricated films (5% w/v ADA-7.5% w/v GEL/0.25% w/v SF/0.3% w/v Cu-Ag MBGNs hydrogels) would not adversely impact angiogenic potential (CAM assay), cellular viability and, instead, enhance biocompatibility.Thus, cell-culture and CAM studies were conducted using different Cu-Ag MBGNs concentrations.
Each experiment was performed in pentaplicate under sterile conditions, and then the mean values along with the standard deviation were reported.In order to assess the statistical differences between the groups analysis of variance (ANOVA) was conducted.

Morphological analysis
SEM images of the hydrogels indicate an interconnected porous microstructure (figure 4(A)).This interconnected pore structure, which changes as a function of time during hydrogel degradation, is favorable for wound healing because it facilitates the transmission of nutrients and cell waste products in wound defects, as well as enabling proliferation and communication of cells.Figure 4(B) shows that SF was successfully incorporated into 5% w/v ADA-7.5% w/v GEL hydrogels through the physical crosslinking method.Furthermore, the inherent porous nature of the composite hydrogels was maintained.A dense packing of fibers within the pores is visible in figure 4(B), indicating that SF has consolidated and dispersed homogeneously within the porous ADA-GEL microstructure.In addition, it is evident from SEM images that 5% w/v ADA-7.5% w/v GEL and 5% w/v ADA-7.5% w/v GEL/0.25% w/v SF hydrogels were successfully formed with suitable porosity in the range from ∼12 µm to nanometers [46].All hydrogels exhibited a porous structure with varying pore diameters.The mechanical properties of hydrogels are influenced by their porosity.The pore size of the 5% w/v ADA-7.5% w/v GEL/0.25% w/v SF/0.3% w/v Cu-Ag MBGNs hydrogel (figure 4(C)) was lower than the pore size of the 5% w/v ADA-7.5% w/v GEL, and 5% w/v ADA-7.5% w/v GEL/0.25% w/v SF hydrogels (figures 4(A) and (B)), implying that the former has a denser structure and pore sizes have changed from ∼12 µm to nanometers.As a result, we conclude that the current porous morphology, which includes interconnected pore sizes at the micro and nanometer scales, contributes significantly to the hydrogels' mechanical properties [64].Figure 4(C) shows that 0.25% w/v SF and 0.3% w/v Cu-Ag MBGNs were successfully incorporated in 5% w/v ADA-7.5% w/v GEL hydrogel through physical crosslinking.Again, the inherent pore nature of the hydrogels was maintained.Both SF and Cu-Ag MBGNs were well dispersed in the 5% w/v ADA-7.5% w/v GEL matrix.
In general, the porous microstructure of a biomaterial should imitate the natural extracellular matrix, stimulate cell growth and tissue development, provide more surface area for cells to adhere to, and improve nutrient and waste exchange due to increased fluid flow.Cell attachment and function require careful regulation of pore size, dispersion, and porosity.Moreover, a regulated pore size decrease can also help cells connect by providing more surface area for them to adhere to.In this study, the incorporation of Cu-Ag MBGNs and SF caused a regulated pore size by occupying space within the hydrogel network, creating a dense packing structure with smaller pores well-distributed in the matrix, which may enhance protein attachment and cellular adhesion [65].
Cu-Ag MBGNs include nanostructured features, as shown in figure 4(D).The elemental map of a 0.3% w/v Cu-Ag MBGNs particles (figure 5) reveals the elemental composition of Cu-Ag MBGNs.Elements present in MBGNs such as Si, Ca, Cu, Ag, were detected by EDX, confirming the incorporation of 0.3% w/v Cu-Ag MBGNs in the 5% w/v ADA-7.5% w/v GEL matrix.It is evident from figure 5 that the MBGNs (Si (green), O (blue), C (orange), Ag (yellow), Cu (red), Ca (light yellow)) are distributed uniformly throughout the matrix.Here, it is noteworthy that Cu and Ag are elements required for their angiogenic and antibacterial potential while Si, Ca are the main constituents of the bioactive glass.Na appears due to the Na-Alg which is a component of the hydrogel matrix.

FTIR analysis
FTIR is a valuable analytical technique due to its ability to provide crucial information about the molecular composition and structure of biological samples.The 5% w/v ADA-7.5% w/v GEL FTIR spectrum (figure 6) exhibits peaks at 1651 cm −1 , 1564 cm −1 (aliphatic C-H bending vibrations), 1265 cm −1 (stretching vibration of C-N bond,) and 1125 cm −1 (asymmetric C-O-C stretching of ADA) [66].The spectrum exhibits n(C=N) absorption bands at 1651 cm −1 and 1564 cm −1, indicating the formation of Schiff 's base through the crosslinking between the amino groups of lysine or hydroxylysine of GEL and the free aldehyde groups of ADA [66,67].The peaks of SF are amide I at 1655 cm −1 (C=O stretching), amide II at 1598 cm −1 (N-H deformation and C-N stretching), and amide III at 1255 cm −1 (N-H deformation and C-N stretching) [68,69].The amide II and amide III peaks from SF did not appear in the FTIR pattern of 5% w/v ADA-7.5% w/v GEL/0.25% w/v SF hydrogels.Additionally, the region of the O-H and N-H bonds presented essential changes [67,69].The band around 3200 cm −1 -3400 cm −1 is Figure 6.FTIR spectra for 5% w/v ADA-7.5% w/v GEL, 5% w/v ADA-7.5% w/v GEL/0.25% w/v SF, 5% w/v ADA-7.5% w/v GEL/0.25% w/v SF/0.3% w/v Cu-Ag MBGNs hydrogels showing the characteristic peaks explained in the text.
attributed to the H-O-H bending vibration of surface hydroxyl groups and absorbed water molecules and amide III [66].With the addition of 0.25% w/v SF in the 5% w/v ADA-7.5% w/v GEL hydrogels, the intensity of the peak decreased and the broadness increased, which can be attributed to the formation of inter-and intramolecular hydrogen bonds, which became broader.There is also a slight peak shifting of amide I and amide II in 5% w/v ADA-7.5% w/v GEL/0.25% w/v SF hydrogels.The shift of the amides is characteristic for the β-sheet structure of SF within the hydrogel and could be attributed to the hydrogen-bonded from the intermolecular hydrogen bonds between the carbonyl groups of the SF and the hydrogen donating groups of 5% w/v ADA-7.5% w/v GEL [70].A new absorbance peak that appeared at ∼2927 cm −1 is attributed to new hydrogen bonds and free amide groups (on the backbone of gelatin) [70].This could be due to the hydrogen bonding between the amide groups of SF and hydroxyl groups of Cu-Ag MBGNs [52].The Cu-Ag MBGNs spectrum exhibited characteristic peaks at 1144 cm −1 (Si-O stretching vibration), 698 cm −1 and 1081 cm −1 (Si-O) [52,71].In the spectra of composite hydrogels (ADA-GEL/SF/Cu-Ag MBGNs), the C-O-C stretching peak at 1125 cm −1 from 5% w/v ADA-7.5% w/v GEL overlaps with the Si-O stretching peak at 1015 cm −1 from Cu-Ag MBGNs (resulting in the peak broadness).The peak at 1125 cm −1 is shifted to lower wavenumbers, and the peak intensity increases as Cu-Ag MBGNs were added.Hence, especially in the spectrum of 5% w/v ADA-7.5% w/v GEL/0.25% w/v SF/0.3% w/v Cu-Ag MBGNs, the overlapped peak at 1125 cm −1 is broader than the same peak of the 5% w/v ADA-7.5% w/v GEL and 5% w/v ADA-7.5% w/v GEL/0.25% w/v SF samples.The peak at 698 cm −1 is ascribed to the Cu-O bond [72,73], which is attributed to the Cu-Ag MBGNs.The cross-linking between 5% w/v ADA and 7.5% w/v GEL contributes to the acceptable mechanical strength of the hydrogels.

Equilibrium swelling/deswelling behavior
Swelling behavior of the hydrogels reflects the ability of the materials to retain and diffuse water, which is an important property of hydrogel-based materials for wound healing applications.
Figure 7 shows the variation of the SR of 5% w/v ADA-7.5% w/v GEL, 5% w/v ADA-7.5% w/v GEL/0.25% w/v SF and 5% w/v ADA-7.5% w/v GEL/0.25% w/v SF/0.3% w/v Cu-Ag MBGNs hydrogels over time.Generally, a strong gain of weight can be observed, with an increase of > 25% after 3 h for the 5% w/v ADA-7.5% w/v GEL films.Furthermore, the addition of SF leads to a decrease in the swelling behavior (i.e.< 25%).This fact can be attributed to the diffusion of water inside the network, which is likely reduced by electrostatic and hydrophobic interactions that occur between the protein chains (SF) and the 5% w/v ADA-7.5% w/v GEL molecules.The addition of 0.3% w/v Cu-Ag MBGNs led to a further reduction in swelling behavior compared to the 5% w/v ADA-7.5% w/v GEL.The observed differences in water uptake ability may be due to strong interactions between 5% w/v ADA-7.5% w/v GEL, 0.25% w/v SF and 0.3% w/v Cu-Ag MBGNs.Moreover, a decrease in pore size of hydrogel films due to the addition of 0.3% w/v Cu-Ag MBGNs could lead to a reduced water uptake.The addition of 0.3% w/v Cu-Ag MBGNs to the hydrogel films might fill up the hydrogel porosity, thus providing less space for absorbing water.Increased swelling behavior of 5% w/v ADA-7.5% w/v GEL hydrogels affects negatively the mechanical strength of the material; thus a controlled hydrogel swelling is required and should be accomplished for successful biomedical applications.Thus, 5% w/v ADA-7.5% w/v GEL/0.25% w/v SF/0.3% w/v Cu-Ag MBGNs absorbed less water than 5% w/v ADA-7.5% w/v GEL hydrogels [64].

Mechanical properties
Most previous studies have reported the mechanical behavior of ADA-GEL hydrogels (e.g.scaffolds and films) under compression and indentation load (see for example [66,69,74,75]), whereas less than a handful of articles have studied the tensile behavior of 5% w/v ADA-7.5% w/v GEL hydrogels (see for example [74]).The tensile strength of ADA-GEL hydrogels has been reported to range between 0.1 and 10 MPa depending on processing routes, ADA-GEL composition, and testing conditions [75], whereas the elastic modulus and elongation at break under tensile stress for 5% w/v ADA-7.5% w/v GEL/0.25% w/v SF and 5% w/v ADA-7.5% w/v GEL/0.25% w/v SF/0.3% w/v Cu-Ag MBGNs hydrogels have yet to be investigated.Table 1 reports the mechanical properties of 5% w/v ADA-7.5% w/v GEL, 5% w/v ADA-7.5% w/v GEL/0.25% w/v SF and 5% w/v ADA-7.5% w/v GEL/0.25% w/v SF/0.3% w/v Cu-Ag MBGNs hydrogels, indicating a significant variation in the elastic modulus and elongation at break with Cu-Ag MBGNs additions.Unexpectedly, the elastic modulus was lower for 5% w/v ADA-7.5% w/v GEL/0.25% w/v SF and 5% w/v ADA-7.5% w/v GEL/0.25% w/v SF/0.3% w/v Cu-Ag MBGNs than for 5% w/v ADA-7.5% w/v GEL.However, it has been shown that in some cases incorporating second phases in hydrogels can reduce the elastic modulus [76] in agreement with the present results.Wound healing requires a hydrogel that is not excessively stiff but supports the wound site, conforms to the skin, and resists mechanical stress without tearing [77].The modulus should not be too high because stiffness might be uncomfortable for the patient and could impair tissue growth and movement.Thus, in the present study, the decreasing trend in elastic modulus with 0.25% w/v SF and 0.3% w/v Cu-Ag MBGN addition seems to be favorable for wound healing applications since a lower elastic modulus means that the hydrogel is more flexible and can better conform to the shape of the wound.

Samples
Elastic modulus (kPa) % Elongation 5% w/v ADA-7.5% w/v GEL 96 ± 16 48 ± 3 5% w/v ADA-7.5% w/v GEL/0.25% w/v SF 21 ± 4 58 ± 14 5% w/v ADA-7.5% w/v GEL/0.25% w/v SF/0.3% w/v Cu-Ag MBGNs 18 ± 8 24 ± 9 ADA-7.5% w/v GEL/0.25% w/v SF/0.3% w/v Cu-Ag MBGNs).One of the reasons for this behavior may be the interaction between 0.25% w/v SF and 0.3% w/v Cu-Ag MBGNs with the ADA-GEL matrix, affecting the crosslinking between 5% w/v ADA and 7.5% w/v GEL [15].Since the 0.25% w/v SF and 0.3% w/v Cu-Ag MBGNs were incorporated before the cross-linking of ADA-GEL, the additions likely result in the hindrance of the effective cross-linking, consequently leading to a reduced number of overall cross-links.This reduction in the number of crosslinks in 5% w/v ADA-7.5% w/v GEL/0.25% w/v SF and 5% w/v ADA-7.5% w/v GEL/0.25% w/v SF/0.3% w/v Cu-Ag MBGNs may result in reduced elastic modulus and ultimate tensile strength of the system [78].Additionally, the reduced crosslinking between 5% w/v ADA and 7.5% w/v GEL due to 0.25% w/v SF and 0.3% w/v Cu-Ag MBGNs additions should also detrimentally affect the stretchability of the produced hydrogels, as evidenced by the reduced elongation at break (table 1).

ICP analysis
Ion-release of the synthesized 5% w/v ADA-7.5% w/v GEL hydrogels loaded with 0.3% w/v Cu-Ag MBGNs was tracked under dynamic conditions in DMEM at 37 • C for 7 d.This release of Cu ions is considered to be beneficial for the angiogenic properties of bioactive glasses [56,73].Figure 8(B) shows the release profile of Ag ions.Almost a constant release of Ag ions was observed in the first week of incubation.Ag ions released from 0.3% w/v Cu-Ag MBGNs samples were within the concentration range of 0.04 µg ml −1 -1.2 µg ml −1 which has been proven to induce significant antibacterial activity against Gram-positive and Gramnegative bacterial strains [52].A burst release of both ions (Cu, Ag) was observed from 0.3% w/v Cu-Ag MBGNs loaded in the 5% w/v ADA-7.5% w/v GEL hydrogel, which might be due to the concentration gradient between the particles and the physiological solution.The release of Ag ions is particularly beneficial for antibacterial effects.Thus, it was concluded that the co-substitution of Cu and Ag ions in MBGNs can provide multiple therapeutic effects, specifically an angiogenic effect [79] due to the release of Cu ions, and an antibacterial effect due to the release of Ag ions (in addition to the fact that Cu ions are also antibacterial).The antibacterial and angiogenic potential of the synthesized 5% w/v ADA-7.5% w/v GEL/0.25% w/v SF/0.3% w/v Cu-Ag MBGNs hydrogel is in good agreement with the ion-release results.

In vitro degradation studies
In vitro degradation analysis was used to evaluate the degradation rate of ADA-GEL-based films with time in a physiological environment using DPBS.Figure 9 shows that initially pure ADA-GEL exhibited a faster degradation rate than the composite films.Degradation values of 50% and 100% for the 5% w/v ADA-7.5% w/v GEL film were calculated at the 3rd and the 7th day of incubation, respectively.The degradation rate of 5% w/v ADA-7.5% w/v GEL/0.25% w/v SF films was 37.5%, at the 3rd day, and it was 87%, and 92.5% at the 7th and 15th days, respectively.The 5% w/v ADA-7.5% w/v GEL/0.25% w/v SF film did not show 100% degradation which might be due to the strong hydrophobic interactions of β-sheets present in the protein structure.5% w/v ADA-7.5% w/v GEL/0.25% w/v SF/0.3% w/v Cu-Ag MBGNs showed 25%, 62%, and 70% degradation at the 3rd, 7th, and 15th day of incubation, respectively.It is clear that 5% w/v ADA-7.5% w/v GEL/0.25% w/v SF/0.3% w/v Cu-Ag MBGNs films showed the lowest degradation rate, likely due to the incorporation of MBGNs, an inorganic material.The other reason for the relative low degradation of the composite hydrogel might be the strong crosslinking (reduced swelling rate) between the 5% w/v ADA-7.5% w/v GEL, 0.25% w/v SF, and 0.3% w/v Cu-Ag MBGNs.The hydrogel is biodegradable, so that in wound healing applications, it would gradually break down as the wound heals.This feature reduces the need for frequent dressing changes, minimizing trauma to the wound and improving patient comfort.Moreover the hydrogel composition can be tailored to specific wound types and patient needs.In fact different ratios of the components could be used to tailor the properties of the composite hydrogel for application in chronic wounds, burns, and surgical incisions.

Surface area and pore size analysis
BET adsorption data was used to determine the surface area of the synthesized hydrogel.Figure 10 shows the convex shaped (towards x-axis) type-III isotherm which indicates the unimpeded multilayer formation of N 2 molecules over the hydrogel surface.This type of behavior indicates the micro-porosity in the test sample.The surface area of the final hydrogel was 3.3 m 2 g −1 .SEM images (figure 4) of the final hydrogel display that the 0.25% w/v SF and 0.3% w/v Cu-Ag MBGNs have covered the surface of the ADA-GEL matrix and infiltrated the pores, which could be the reason for the reduced surface area of the composite hydrogel.

Antibacterial studies
Antibacterial tests were performed against S. aureus and E. coli bacteria.The antibacterial activity of the 5% w/v ADA-7.5% w/v GEL and 5% w/v ADA-7.5% w/v GEL/0.25% w/v SF/0.3% w/v Cu-Ag MBGNs was investigated against E. coli and S. aureus using the disc diffusion and turbidity tests.Figure 11(A) shows the antibacterial effect of 5% w/v ADA-7.5% w/v GEL and 5% w/v ADA-7.5% w/v GEL/0.25% w/v SF/0.3% w/v Cu-Ag MBGNs by the disc diffusion method.The samples containing 0.3% w/v Cu-Ag MBGNs formed a clear inhibition zone, measuring 10 mm against E. coli and 8 mm against S. aureus, confirming the antibacterial activity of the synthesized composite hydrogels.Figures 11(B) and (C) show the results of the turbidity test by measuring OD 600 via UV-vis spectrophotometer, indicating the quantitative growth of E. coli and S. aureus against 5% w/v ADA-7.5% w/v GEL and 5% w/v ADA-7.5% w/v GEL/0.25% w/v SF/0.3% w/v Cu-Ag MBGNs hydrogels.The OD 600 of the 5% w/v ADA-7.5% w/v GEL was 0.015 ± 0.002, while the OD 600 of 5% w/v ADA-7.5% w/v GEL/0.25% w/v SF/0.3% w/v Cu-Ag MBGNs was 0.009 ± 0.003 after 24 h of incubation.It can be hypothesized that after 6 h incubation, the Ag ions start to be released in a significant concentration, preventing E. coli and S. aureus bacteria growth.Following 24 h of incubation, it was observed that the release of Ag ions was sufficient to stop both bacteria cells from growing adequately.It was also revealed that the pure ADA-GEL samples allow both strains to survive on their surfaces.As a result, the 0.3% w/v Cu-Ag MBGNs incorporated in the 5% w/v ADA-7.5% w/v GEL/0.25% w/v SF hydrogels significantly inhibited the growth of both strain cells.It has been reported in the literature that the release of Ag ions from MBGNs is highly effective against bacteria [55], as Ag ions can rupture the walls of bacterial cells, allowing ions to enter the cells and interfere with DNA replication, eventually resulting in cell death.Furthermore, the antibacterial activity of the hydrogels is also enhanced by the presence of Cu, which is also an antibacterial element, as stated previously.The antibacterial analysis confirmed thus the bactericidal potency of the present hydrogels justifying future in-vivo studies, for example considering infected wound models.

Colony forming units (CFU)
We investigated the antimicrobial effect of 0.3% w/v Cu-Ag MBGNs loaded 5% w/v ADA-7.5% w/v GEL/0.25% w/v SF films.Bacterial counts (CFU/ml) were evaluated from the cultured plates via colony counting (table 2).It was anticipated that the release of Cu and Ag ions from hydrogels would cause a strong antibacterial effect.In the present study, we observed complete inhibition of S. aureus growth on all the incubated plates.However, partial inhibition was observed against E. coli.

CAM assay
A CAM assay study was performed to study the potential of Cu-Ag MBGNs in supporting new blood vessel formation.The results show that 3 chicks out of the original five fertilized eggs per group survived (figure 12).The first sample contained 0.05% w/v Cu-Ag MBGNs and the concentration of the Cu-Ag MBGNs increased in the other samples.As shown in figure 12(A), the blue circle and the blue square on the 0.1% w/v Cu-Ag MBGNs and 0.2% w/v Cu-Ag MBGNs samples showed the position of the hydrogel on the CAM.The difference in the angiogenesis performance could be attributed to variation of Cu and   12 confirmed that the maximum number of blood vessels was observed in the 0.3% Cu-Ag MBGNs loaded in 5% w/v ADA-7.5% w/v GEL/0.25% w/v SF samples, which is in agreement with Zhao' study [80].Since Cu is known for its angiogenic effect, Cu ions have been used to enhance angiogenesis due to their ability to stabilize the expression of hypoxia-inducible factor (HIF-1α), enhancing the formation of blood vessels [80,81].Moreover, the release of Cu ions can stimulate the transforming growth factor-β (TGF-β), which is vital for the growth of new blood vessels.It was also observed that all samples containing MBGNs showed angiogenic potential (figure 12(A)).Overall, it was found that angiogenesis increases with increasing MBGN concentration.The maximum angiogenic potential was observed in the sample incorporating 0.3% w/v Cu-Ag MBGNs compared to the other samples.Statistically significant differences were noted between hydrogel samples containing 0.05% w/v and 0.1% w/v Cu-Ag MBGNs.Statistically significant differences were also observed between the 0.05% w/v Cu-Ag MBGNs, 0.2% w/v Cu-Ag MBGNs, and 0.3% w/v Cu-Ag MBGNs (figure 12(B)).It is hypothesized that Cu ions can trigger the vascular endothelial growth factor receptor, followed by the release of vascular endothelial growth factor, fibroblast growth factor, and angiopoietins, leading to the migration of endothelial cells.Cu ions may contribute to the formation of sprouts from existing blood vessels, a critical step in the angiogenic process.So, the enhanced angiogenic potential triggered by Cu ion release may lead to an enhanced healing rate.

Cell biology studies
Cell viability must be tested to ensure that the hydrogel does not hinder the natural healing process and supports cell growth and tissue repair.The cellular viability of the control (medium), 5% w/v ADA-7.5% w/v GEL film, 5% w/v ADA-7.5% w/v GEL/0.25% w/v SF, and 5% w/v ADA-7.5% w/v GEL/0.25% w/v SF/0.3% w/v Cu-Ag MBGNs films incorporated with different concentrations of Cu-Ag MBGNs was significantly increased as compared to the control sample (figure 13).NIH3T3 fibroblasts were adhered to 5% w/v ADA-7.5% w/v GEL films after an initial incubation period of 12 h, while after 24 h, the cells started to spread throughout the 5% w/v ADA-7.5% w/v GEL films' surface.It was observed that as the cell culture time increased the number of cells (cell density) increased, which resulted in the increase of cellular viability.Initially, up to 3 d, 5% w/v ADA-7.5% w/v GEL films incorporated with 0.25% w/v SF, and 0.05% w/v, 0.1% w/v, 0.2% w/v, and 0.3% w/v of Cu-Ag MBGNs exhibited significantly reduced cell viability (<45% compared to the control), which could be due to the higher concentration of released Cu, Si, and Ca ions, leading to a pH increase.Cell density at day 5 was almost twice as compared to day 1.The cell viability of 5% w/v ADA-7.5% w/v GEL films incorporated with 0.05% w/v, and 0.1% w/v of Cu-Ag MBGNs was higher than that obtained from the other concentrations (0.2% w/v, and 0.3% w/v), at 7th day.This result could be due to the controlled and sustained release of Cu and Ag ions for these concentrations of Cu-Ag MBGNs .Hydrogel porosity affects cellular attachment and function, while the reduction in pore size associated with the incorporation of Cu-Ag doped MBGNs into hydrogels might reduce the overall surface area accessible to cells.This may seem counterintuitive, however the pro-angiogenic properties based on the controlled release of Cu ions can outweigh the potential drawbacks of pore size reduction.The controlled release of these ions within the hydrogel matrix can create a favorable microenvironment for angiogenesis, supporting the formation of functional blood vessels, and ultimately modulating the cellular response and promoting specific aspects of wound healing [73].These films contain a greater amount of pores and sites for cell attachment, resulting in contact inhibition delay.Nonetheless, the relative cell viability in the sample groups was still higher until the 7th day, demonstrating the non-cytotoxicity of the Cu-Ag MBGNs loaded hydrogels.
The cell viability of all groups increased with the concentration of the dissolution products up to seven days, although there was no significant difference between the concentrations.The results showed that the cytotoxicity of (0.05% w/v-0.3%w/v) Cu-Ag MBGNs was related to the concentration of Cu and Ag ions.It has been reported that Cu-Ag MBGNs could exert viability effects at a relatively low concentration (0.05% w/v, 0.1% w/v), while a relatively high concentration could cause toxicity toward healthy cells or bacteria (as the cellular viability of the ADA-GEL films incorporated with Cu-Ag MBGNs at concentrations of 0.2% w/v, 0.3% w/v is lesser than for concentrations of 0.05% w/v, and 0.1% w/v).
Therefore, the release of Cu and Ag ions (e.g. both the concentration and the release profile) needs to be adjusted depending on the required applications.In conclusion, our results suggest that the developed 5% w/v ADA-7.5% w/v GEL films containing Cu-Ag MBGNs stimulate significantly cellular proliferation without cytotoxicity, under the conditions of the present experiments, and can be considered an appropriate candidate for wound healing applications.

Conclusions
This study successfully synthesized 5% w/v ADA-7.5% w/v GEL, 5% w/v ADA-7.5% w/v GEL/0.25% w/v SF, and 5% w/v ADA-7.5% w/v GEL/0.5% w/v SF/0.3% w/v Cu-Ag MBGNs hydrogels and characterized their physicochemical and biological properties.The incorporation of SF and Cu-Ag MBGNs into the ADA-GEL matrix resulted in hydrogels with tailored mechanical, morphological, and biological properties which are required for wound healing applications.SEM images confirmed that SF and Cu-Ag MBGNs were successfully incorporated into the matrix without compromising significantly the inherent porous nature of 5% w/v ADA-7.5% w/v GEL.FTIR analysis provided insight into the reaction chemistry between Cu-Ag MBGNs, SF, and ADA-GEL, confirming the successful crosslinking between ADA-GEL and 0.25% w/v SF and the presence of Cu-Ag MBGNs in the hydrogels.Composite hydrogels exhibited lower mechanical property than the neat 5% w/v ADA-7.5% w/v GEL hydrogels, which is desirable for applications in wound healing.As expected, addition of 0.3% w/v Cu-Ag MBGNs imparted significant antibacterial activity against E. coli and S. aureus, while the controlled release of Cu and Ag ions further enhanced the antibacterial efficacy.Overall, the encouraging in vitro results suggest that the developed hydrogels hold promise as an acceptable solution for the development of wound dressings with improved properties.The biomaterials developed exhibit flexibility in terms of structural, physical, and chemical properties in the range needed for wound healing applications in addition to be able to improve healing through angiogenesis and antimicrobial properties.In future work, in-vivo studies should be performed in detail to evaluate the clinical potential of these hydrogels in different wound healing scenarios.

Figure 2 .
Figure 2. Process flow for the synthesis of SF solution.

Figure 5 .
Figure 5. Elemental map (EDX results) of Cu-Ag MBGNs showing the uniform distribution of Cu, Si, Ag, and Ca present in MBGNs.

Figure 8 (
A) shows the release of Cu ions from the hydrogels indicating a rapid release in the first 7 d.The cumulative release of Cu ions showed a linear behavior until 6 d of incubation.

Figure 12 .
Figure 12. (A) Evaluation of the angiogenic potential of the 5% w/v ADA-7.5% w/v GEL hydrogels via CAM assay studies.Angiogenic potential was observed at day 14 and the blue circle and blue square show the position of the hydrogel sample on the CAM, (B) quantification of angiogenesis induced by hydrogel samples retrieved from the eggs at day 14 by Image J software.Significant difference of p < 0.05 was observed between 0.05% w/v Cu-Ag MBGNs and 0.1% w/v Cu-Ag MBGNs, 0.05% w/v Cu-Ag MBGNs and 0.2% w/v Cu-Ag MBGNs and 0.05% w/v Cu-Ag MBGNs and 0.3% w/v Cu-Ag MBGNs samples.(Data represent the mean ± standard deviation).Results were statistically analyzed with control group by ANOVA with statistical difference of * p < 0.05, * p < 0.005, and ns means non significant difference.

Figure 13 .
Figure 13.Cellular viability of control (tissue culture plate) and 5% w/v ADA-7.5% w/v GEL films against fibroblast (NIH3T3) cells measured by WST-8 up to 7 d of culture.(Data represent the mean ± standard deviation).Results were statistically analyzed with control group by ANOVA with statistical difference of * p < 0.05 and ns means non significant difference.
• C in a physiological environment (pH = 7.4) for different time intervals (0 d, 1 d, 3 d, 5 d, 7 d, 9 d, 11 d, 13 d, and 15 d).At each time interval, films were taken out of DPBS, put into the tissue paper to soak up the excess DPBS, and then the weight was recorded.The degradation rate was calculated using equation (2): w/v Cu-Ag MBGNs.First, 15 ml of nutrient agar was poured into petri dishes, and then a volume of 15 µl of overnight cultured bacterial strains, i.e.Escherichia coli (E.coli) and Staphylococcus aureus (S. aureus), having an optical density of 0.015 (OD 600 ) was spread over the plates to form a uniform bacterial lawn.