In-vitro and in-vivo wound healing studies of Ag@TiO2NRs/GG hydrogel film for skin tissue regeneration

One million cases of skin wounds, either closed or open wounds, necessitate wound treatments to improve the quality of life. In this study, gellan gum biopolymer (Ag@TiO2NRs/GG) hydrogel film with Ag loaded TiO2 nanorods was fabricated for wound healing dressing. The wound healing performance of Ag@TiO2WR/GG hydrogel was tested in vitro and in vivo to investigate its ability to regenerate skin tissue. FTIR, XRD, and SEM were used to examine the physical and chemical properties of prepared Ag@TiO2NRs/GG hydrogel film, as well as pure Ag and Ag@TiO2NRs. The FTIR spectra revealed the functional groups of Ag, TiO2NRs, GG, and their interactions. The hydrogel film was in an amorphous form, according to XRD analysis, due to the helical structure of GG and the presence of Ag and TiO2NRs in distinct phases. The SEM image shows agglomeration of Ag particles and elongated TiO2 nanorods, indicating that Ag@TiO2NRs were successfully incorporated onto GG hydrogel film. Human skin fibroblast cells (CRL2522) were used to study the in vitro wound healing of Ag@TiO2NRs/GG hydrogel film for cell viability and proliferation. After 72 h, ∼98,022 cells well−1 were counted, indicating that the Ag@TiO2NRs/GG was biocompatible and non-toxic. In vivo wound healing on Sprague Dawley rats revealed 100% wound healing after 14 days of treatment with Ag@TiO2NRs/GG hydrogel film. On a treated skin wound, ultrasound images revealed a thicker epidermis, clear dermis, and subcutis layer, indicating a positive correlation between wound healing and skin tissue regeneration.


Introduction
The skin is the largest organ in the human body and serves as a barrier against environmental hazards, mechanical stresses, and infections. Their structure, which includes the epidermis, dermis, and subcutis, is essential to their function [1,2]. A wound is the disruption of the integrity or malfunction of skin tissue, which prevents the skin from performing its functions. As a result, wound management, including repair and treatment, has emerged as a global medical issue that must be addressed. It entails a series of highly controlled and complex reactions that are triggered by tissue destruction to restore the functionality and integrity of the damaged tissues [3,4]. Around 2 million people in Europe alone receive treatment for acute or chronic wounds. Chronic wounds have a severe impact, frequently acting as a complication of some chronic diseases such as diabetes [5,6]. This trend is likely to worsen as a result of the impact of chronic diseases and an ageing skin fibroblast cells (CRL2522) and Sprague Dawley rats, respectively. The skin regeneration of a wound treated with Ag@TiO 2 NRs/GG hydrogel was studied utilising a real-time high resolution 20 MHz ultrasound DermaLab Combo. The novelty of this study was the fabrication of synthesised TiO 2 NRs loaded with Ag incorporated GG hydrogel film used for wound dressing with good biocompatibility and high antibacterial properties. Moreover, the present study was performed with both in vitro and in vivo wound healing experimental for skin tissue regeneration. Furthermore, in vivo toxicity of the Ag@TiO 2 /GG hydrogel film was carried out which indicates that the sample is non-toxic, and it is potentially safe to use.

Preparation of argentum particles (Ag)
The Ag particles were produced through chemical reduction method using NaBH 4 as the reducing agent. A beaker containing 100 ml of 0.1 mM NaBH 4 as a reducing agent was placed in an ice bath for 10 min to prevent degradation. Using a dropper, 10 ml of 1.0 mM AgNO 3 was slowly added to the NaBH 4 solution while stirring. One second for each drop of AgNO 3 solution until finished and the stirrer was turned off. The solution's colour changed. The formation of nanoparticles was indicated by the yellow colour appearance. The solution is dried in an oven at 80°C for 48 h to produce Ag particles powder for characterisation.
Preparation of argentum loaded titanium dioxide nanorods (Ag@TiO 2 NRs) A modified hydrothermal approach was utilised to synthesise titanium dioxide nanorods (TiO 2 NRs) using commercial titanium dioxide (TiO 2 ) powder as a precursor [28]. A 100 ml aqueous solution of 10 M NaOH was mixed with 1 g of commercial TiO 2 before being agitated with a magnetic stirrer for 30 min. After that, the suspension solution was put into a Teflon-lined autoclave reactor for hydrothermal treatment in the furnace at 150°C for 24 h. When the reaction is complete, the resulting white precipitate is washed with deionised water and 0.1 M HCl. The solution was filtered and washed with deionised ethanol and water. The white precipitate was then dried at 40˚C for 24 h before being calcined at 500˚C for 2 h. For the preparation of Ag-loaded TiO 2 NRs, 1 g of prepared TiO 2 NRs was mixed with 100 ml of 0.1 mM NaBH 4 to form a suspension. Using a dropper, 10 ml of 1.0 mM AgNO 3 was added gradually to the suspension while stirring. To obtain the Ag@TiO 2 NRs sample, the suspension was centrifuged, and the precipitate was washed 3 times with deionised water and dried at 80°C for 48 h. Before characterisation, the powder was calcined for 2 h at 500°C.
Preparation of argentum loaded titanium dioxide nanorods incorporated gellan gum (Ag@TiONRs/GG) hydrogel film The suspension was created by dispersing 0.01 g of Ag@TiO2NRs in 100 ml of a solution made up of 1 g GG, 0.5 g glycerol, 5 ml of CaCl2 (0.1 M), and distilled water. A bath-style ultrasonic cleaner (FS 140H, Ultrasonic Cleaner, Fisher Scientific, Pittsburg, PA, USA) was used to sonicate the suspension for 30 min After that, the mixture was stirred for two hours at 70°C. To create a hydrogel film, the suspension was transferred to a petri dish and dried for 24 h at 50°C. The hydrogel film was removed from the casting dish and treated for at least 48 h in a humidity chamber with the temperature set to 25°C and 50% RH before the subsequent test. The optimization research was carried out to ascertain the ideal material concentration needed to produce a good biofilm [29].

Characterisation
A Perkin Elmer Spectrum 100 FT-IR spectrophotometer equipped with a PIKE Miracle ATR accessory was used to obtain ATR-FTIR spectra between 4000 and 400 cm −1 wavelength number (single-bounce beam path, 45°i ncidence angle, 16 scans, 4 cm −1 resolution). At room temperature, Rigaku Miniflex (II) x-ray diffractometer was used to conduct an XRD examination from 10°to 80°of 2θ. A JOEL JSM 6360 lA electron microscope was used to take image for scanning electron microscopy (SEM) coupled with energy dispersive x-ray (EDX). The samples were coated with gold using Auto Fine Coater (JFC-1600) prior to the SEM, EDX mapping and elemental analysis. Nitrogen adsorption-desorption isotherms were measured at 77 K using ASAP2000 adsorption apparatus from Micromeritics for surface area, pore volume and pore size determination. The samples were degassed at 100°C for 4 h under vacuum before analysis.

Antibacterial study
An antibacterial experiment was performed using both gram-positive (Staphylococcus aureus) and gramnegative (Escherichia coli) microorganisms. Agar from the Muller-Hinton, MH, DifcoTM standard growing medium was sterilised in an autoclave for 15 min at 120°C. In order to ensure that the bacteria were in stable and uncontaminated conditions, they were subcultured in MH agar and incubated aerobically at 37°C for 24 h before the bacterial injection. Using a simple optical density measurement on a Biomerieux Densicheck Plus Spectrophotometer at 600 nm, bacterial concentrations were determined. The bacterial suspensions used in this study were altered to achieve turbidities at 0.5 McFarland standards. Gram positive, Staphylococcus aureus and gram negative, Escherichia coli inoculants were distributed evenly in sterile petri plates with MH agar. With a sterile cotton swab, all microorganisms were rubbed onto the agar plates' surface. Penicillin, employed as a control sample, pure GG, and the Ag@TiO 2 NRs/GG hydrogel samples were all gently placed into the agar. Triplicate plates containing samples and agar inoculated with bacteria were incubated at 37°C for 24 h. After that, the observations were made on the clear zone of each plate.

Cell viability and proliferation
The hydrogel film samples were put in a 96-well plate (Nunc, Germany) and sterilised under UV light for 20 min in a biological safety cabinet after being bathed in 70% alcohol for 5 min. Before CRL2522 cells were seeded into the wells holding the samples, the hydrogel film was soaked in culture medium (EMEM alone) for 24 h. The supernatant was then collected, and the hydrogel film was then incubated at 37°C in a humidified atmosphere of 5% CO 2 for 24, 48, and 72 h. The absence of hydrogel samples in the EMEM culture medium served as the experiment's negative control. Using a calcein-AM staining process (Life Technologies, USA), the viability of cells in contact with hydrogel film samples and negative control sample over 24 h, 48 h, and 72 h of incubation time was observed using light microscope (Olympus TH4-200) equipped with a fluorescence filter (Olympus U-RFL-T UV with blue light excitation). CellTiter 96 ® aqueous one solution test (Promega, USA) with tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium, inner salts; MTS(a)] and electron-coupling reagent (phenazineethosulfate) was used to measure the proliferation of normal human skin cell. Except for the negative control, the media in each well containing the prepared samples were replaced with new media and incubated for 3 h before to the addition of the assay solution (20 μl in each well). Then, 100 μl of the inoculants were added to fresh wells, and microplate reader (Multiskan Ascent 96/384, USA) was used to measure the absorbance at 490 nm. The conversion of absorbance values to cell number was done using calibration curves of CRL2522 cells in 96-well plates under the same conditions.
In-vivo wound healing study Experiment protocol The effect of Ag@TiO 2 NRs/GG hydrogel on wound healing was assessed using a female Sprague Dawley rat as an animal model. Rats weighing 200-250 ± 24.82 g of body weight and aged 6-8 weeks wered divide into 2 groups contain three rats of each. The experiment was carried out in accordance with industry standards. The animal ethics committee (AEC) of Universiti Malaysia Terengganu approved the experimental design for animal use with the approved number of JKEPHT/2022/49. All the animals were kept in hygienic metabolic cages, and they were given a balanced commercial diet as well as water. Every effort was taken to minimise animal suffering and to demonstrate consistent results for the operations and treatments the number of animals used is minimal.
We hereby confirm that the study is reported in accordance with ARRIVE guidelines (https:// arriveguidelines.org).

Full-thickness excision wound model
The rat was sedated with ether anaesthesia prior to wound induction. A hair clipper was used to shave the hairs from the dorsal side. The area was then sterilised with 70% v/v ethanol. Then, with an 8.00 mm diameter sterile biopsy punch, a full thickness excision wound that extends through subcutaneous tissue at the back of the rat was created. The Ag@TiO 2 NRs/GG hydrogel film was applied to the rat after the wound had been created and the hydrogel film was covered with adhesive tape to keep it in place. The wound was left untreated in the control group for comparison study.

Pro-healing parameters
The wound area was measured at 3, 7, and 14 days of treatment using J image software. The wound healing efficiency (WH) was then calculated using the following formula [30]: For in vivo biocompatibility and toxicity study, the rats were observed at least once during the first 30 min after the Ag@TiO 2 NRs/GG hydrogel film was applied on wound. Then the rats were monitor occasionally during the 24 h with particular attention during the first 4 h. Further monitoring was carried out daily for the next 14 days. If the rats died, the Ag@TiO 2 NRs/GG hydrogel film was referred to as toxic otherwise, the hydrogel film is safe and non-toxic.

Results and discussion
The photo images of the synthesis Ag, Ag@TiO 2 NRs, and Ag@TiO 2 NRs/GG hydrogel films are shown in figure 1, and their chemical nature was investigated using ATR-FTIR analysis. The spectra in figure 2 show a broad vibration band at around 3200 cm −1 , which is assigned to the characteristic sketching of the O-H bond, indicating the presence of absorbed water in all studied samples. An additional absorption peak at approximately 663 cm −1 was observed for the Ag sample (figure 2(a), which was attributed to the Ag nanoparticle [31]. However, the peaks shifted to 770 cm −1 for the Ag@TiO 2 NRs sample ( figure 2(b)) as a result of the chemical interaction between Ag and TiO 2 and the metal oxide bonding of the Ti-O stretching vibration [32]. The weak absorption peaks of metal oxide stretching vibration in the Ag@TiO 2 NRs/GG hydrogel film (figure 2(c) revealed that the metal oxide bonding and Ag were shielded from oxidation by the polysaccharide [33]. Another characteristic peak in the Ag sample at 1303 cm −1 is attributed to the sketching vibrations of O-H, as described by other researchers [34]. The peak ascribed to O-H vibrations changed to 1622 cm −1 and 1629 cm −1 for Ag@TiO 2 NRs and Ag@TiO 2 NRs/GG hydrogel film samples, respectively. The shifting and broadening of the measured peaks indicate that Ag@TiO 2 NRs are present within the matrix material [33]. The vibration peak appeared at 1379 cm −1 in Ag@TiO 2 NRs and Ag@TiO 2 NRs/GG hydrogel samples, stating the interaction between Ag and TiO 2 NRs. Another peak at 1209 cm −1 in the Ag@TiO 2 NRs/GG hydrogel film was caused by the interaction of the Ag@TiO 2 NRs filler with the GG matrix [35].   [36]. The data were compared to JCPDS file no. 87-0720, which was in good accordance with the standard values. Using the Debye-Scherrer formula, the highest intensity diffraction peak at 38.5°indexed as the (111) plane was used to identify crystalline Ag nanoparticles [36]. Ag nanoparticles with an average crystallite size of 25.0 nm were discovered. The Ag@TiO 2 NRs was successfully synthesised in this study because all peaks belonging to the Ag FCC structure were observed at 38. As reported by other researchers, a prominent peak at ∼25°is exclusive to the anatase TiO 2 (101) crystallographic plane [37,38]. The presence of sharp peaks was attributed to the crystalline structure of the Ag@TiO 2 NRs sample. Due to the strong interactions between Ag@TiO 2 NRs and GG, the crystalline region of Ag@TiO 2 NRs is reduced in Ag@TiO 2 NRs/GG blends, and the appearance of a broad peak proves the amorphous morphology of structure, which is in line with previous findings [39,40]. The GG double helical structure also led to the amorphous condition of the Ag@TiO 2 NRs/GG hydrogel sample because the molecules are randomly arranged and not evenly packed, which gives them flexibility and elasticity [41]. GG is defined as a short-range order of repeating units that is incapable of ordered arrangement [42]. The presence of the peaks assigned to Ag FCC and anatase TiO 2 in figure 3(c) verified the presence of Ag@TiO 2 NRs as a final product in the GG hydrogel film as a separated phase.
The SEM images in figure 4 show the morphology of pure Ag, Ag@TiO 2 NRs, and Ag@TiO 2 NRs/GG hydrogel films. For the Ag sample, the particle is texturally globular with some evidence of aggregation in a few sections. This is due to the use of manual sampling. The particles are nearly homogeneous, with sizes ranging from 20 to 40 nm ( figure 4(a). The SEM micrograph in figure 4(b) shows the presence of agglomerated Ag and elongated rod-like particles attributed to TiO 2 in the Ag@TiO 2 NRs sample. The calcination process used to prepare Ag@TiO 2 NRs resulted in Ag agglomeration and bundles of agglomerated TiO 2 NRs. It is well  understood that the heating process promotes particle agglomeration, and that calcination at high temperatures causes more nanorods to form in bundles [43]. In this study, it was determined that the diameter of elongated rod-like particles is approximately 85 nm, and that their length is in the hundreds. In figure 4(c), elongated Ag@TiO 2 WRs were observed on the surface of hydrogel, indicating that Ag@TiO 2 NRs had been successfully incorporated onto the GG hydrogel film.
The Ag@TiO 2 NRs was homogeneously distributed on the film surface as the typical elements of Ag, Ti, and O were detected using energy dispersive of x-ray (EDX) mapping as shown in figure 5. The weight percentage of Ag, Ti and O were estimated to be 9.71 wt%, 52.07 wt%, and 35.08 wt%, respectively.
The Ag@TiO 2 NRs also possessed a large specific surface area of ∼116.42 m 2 /g, due to their elongated nanostructures. Large surface area is important to induce the generation of radical oxygen species (ROS) [44]. The typical nitrogen adsorption-desorption isotherms of the Ag@TiO 2 NRs sample is shown in figure 6. The  isotherm displays a type IV isotherm represent mono-and multilayer adsorption plus capillary condensation in mesoporous materials. While the hysteresis in adsorption and desorption isotherm is assigned to type H3 loop suggesting the presence of slit-shaped pores due to the aggregation plate-like particles [45]. The pore volume and pore size of Ag@TiO 2 NRs was found to be 0.4664 cm 3 g −1 and 14.34 nm, respectively.
In vitro wound healing performance of Ag@TiO 2 NRs/GG hydrogel film was evaluated for cell viability on human skin fibroblast cells (CRL2522). The choice of this cell because the fibroblasts are the main cell type involved in the regulation of extracellular matrix (ECM) protein production [46]. They also play a critical role in supporting key processes in normal wound healing such as breaking down the fibrin clot, creating new ECM and collagen structures to support the other cells associated with effective wound healing, as well as skin regeneration [47]. Figure 7 depicts fluorescence images of cells taken at various time intervals after culture with prepared Ag@TiO 2 NRs/GG hydrogel and tissue culture polystyrene plate (TCPP) as a control sample for comparison. After 24 to 72 h of incubation, the cell was extremely healthy and grew well on both samples. Within 72 h, the cell increased gradually for both samples; however, the Ag@TiO 2 NRs/GG hydrogel film was discovered to be the most advantageous in promoting fibroblast cell proliferation.
The cell viability observed is approximately equal to the proliferation quantified using the CellTiter 96 ® aqueous one solution assay on the samples. The findings revealed that the Ag@TiO 2 NRs/GG hydrogel film significantly increased fibroblast cell proliferation compared to the control sample (figure 8). After 72 h of exposure to the Ag@TiO 2 NRs/GG hydrogel film, the number of cells was discovered to be ∼98,022 cells well −1 , compared to ∼80,622 cells well −1 for the TCPP control sample. The results show that Ag@TiO 2 NRs/GG hydrogel film outperforms TCPP in terms of cell adhesion and growth. The high cell viability and proliferation observed in this study suggest that Ag and TiO 2 NRs were both preferred for cell interaction and were non-toxic. The elongated shape of Ag@TiO 2 NRs on GG hydrogel film improved cell adhesion, which aided cell proliferation [48,49]. Researchers have widely reported TiO 2 's beneficial effect on cellular functions such as cell adhesion and proliferation [50,51]. In addition, it is commonly known that TiO 2 nanomaterials, including TiO 2 nanorods, are biologically inert and nontoxic to both human and animal cells [52,53]. Researchers have also reported that the toxicity of Ag nanoparticles is negligible [54,55]. Previously, in vivo inhalation toxicity has been conducted on eight-week-old Sprague-Dawley rats for 28 days and found no different changes in body weight, haematology, or biochemical values relative to the Ag nanoparticles dose [56]. Kim et al (2008) measured the oral toxicity of various doses of 60 nm Ag nanoparticles to Sprague-Dawley rats for 28 days. After the testing period, there were minute, but different, changes in bodyweight between male and female rats. Furthermore, neither the micronucleate polychromatic erythrocytes, nor the ratio of polychromatic erythrocytes to total erythrocytes differed between the rats exposed to Ag nanoparticles and the control rats. They suggested that the Ag nanoparticles in fact did not induce genetic toxicity in rats [57].
Moreover, the results of the study revealed that the Ag@TiO 2 NRs/GG hydrogel film was non-toxic and completely biocompatible. During the study, there were no instances of death of the rats indicating no appreciable toxicity to organs. A previous study also indicated that there were no signs of pathological changes when the rats were treated with Ag/TiO 2 hydrogel film [32].
Sprague Dawley rats with wounds treated with Ag@TiO 2 NRs/GG hydrogel film were used in the in vivo wound healing experiment. An examination was also conducted on a control sample in which the wound was cleaned with distilled water. On days 3, 7, and 14 after treatment, wound healing was observed, as depicted by the photo image in figure 9.
On the third day, the wounds treated with Ag@TiO 2 NRs/GG hydrogel film appeared to be dried and darker. On day 14, wound healing was 100% for wounds treated with Ag@TiO 2 NRs/GG hydrogel film, as shown in figure 10. On the other hand, wound contraction rates of 77.40% were calculated for the control sample. Previously, wound contraction was 81.44% and 53.95% on the seventh day for treatments with Ag@TiO 2 NRs/GG hydrogel film and control sample, respectively. On the third day, the wound contraction was 51.62% and 42.86% for each treatment.  Lower wound contraction for the control sample is due to the inflammatory period of wounds. According to the previous research, the improper shift of macrophages from the M1 (proinflammatory) to M2 (antiinflammatory and pro healing) phenotype is the fundamental cause of inflammation [58]. Proinflammatory substances are continually secreted by macrophages with the M1 phenotype, which causes significant tissue damage. The TiO 2 in hydrogel sample could help in the change from the M1 to the M2 phenotype which will release the polyamine interleukin (IL)-10 that has anti-inflammatory and tissue-repair properties to promote the healing of wounds [58]. Additionally, more in-depth mechanistic studies have demonstrated that Ag nanoparticle can activate the complement system in wounds by increasing the expression of anti-inflammatory cytokines [59]. The hydrogel film also could up-regulate the expression of transforming growth factor beta 1 (TGF)-β1 and vascular endothelial growth factor (VEGF), and down-regulate the level of IL-6 and tumor necrosis factor alpha (TNF-α), indicating that hydrogel film could effectively stimulate fibroblast growth and angiogenesis, inhibit inflammation [60]. High expression of TGF-β1 can increase immunoregulatory activity and promote the proliferation of fibroblasts, which is consistent with the increase of fibroblasts [61]. Moreover, VEGF can induce inflammatory cells to enter the injury site, stimulate endothelial cell proliferation and migration to improve vascular permeability, promote angiogenesis, improve wound epithelialization and  collagen deposition [62]. Thus, the synergy effects of Ag and TiO 2 in GG hydrogel film can stimulate the skin wound healing and tissue regeneration.
The findings indicate that the Ag@TiO 2 NRs/GG hydrogel film has excellent wound healing properties. This is likely due to the antibacterial activity of light-irradiated reactive oxygen species (ROS) generated by GG hydrogel containing Ag@TiO 2 NRs, which played a crucial role in wound healing [32]. TiO 2 is a photocatalytically active material to produce reactive ROS via the redox reaction of electron and hole pairs with oxygen and water, respectively. The ROS produced could hasten wound healing by increasing the expression of vascular endothelial and induced fibroblast growth factors [63,64]. ROS could also regulate blood vessel formation (angiogenesis) and optimise blood flow into the wound-healing area [65]. TiO 2 dissociated into titanium ions, which were then released into the wound. This ion release inhibited microbial proliferation and, as a result, aided in the acceleration of the wound healing process [66]. While the presence of Ag in the hydrogel film killed more than 90% of the major contributors to wound infections, such as Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, and Escherichia coli [67,68]. Moreover, these major contributors frequently form biofilms in infected wounds, making it more difficult to treat wound infections [69]. However, researchers found that both Gram positive and Gram-negative microorganisms were almost equally able to be inhibited by Ag from forming biofilms [70]. Ag treatment inhibited biofilm formation by methicillin-resistant Staphylococcus aureus and methicillin-resistant Staphylococcus epidermidis [71]. Staphylococcus epidermidis was treated with Ag, which inhibited biofilm formation by more than 95% [72]. Thereby, the synergy of Ag's antibacterial and anti-biofilm formation combined with TiO 2 NR's excellent photocatalytic properties for ROS generation supports the good performance of Ag@TiO 2 NRs/GG hydrogel film to accelerate wound healing for skin regeneration. Table 1 provides a summary of the antibacterial activity of pure GG, Ag@TiO 2 NRs/GG hydrogel film, and penicillin as a control sample. Pure GG showed no antibacterial effect against any of the tested microorganisms. While, the exhibition zone using Ag@TiO 2 NRs/GG hydrogel film were 17 ± 0.14, and 16 ± 0.10 mm against Staphylococcus aureus, and Escherichia coli, respectively which is better than the control sample (penicillin). Figure 11 depicts an ultrasound image of a treated skin wound with Ag@TiO 2 NRs/GG hydrogel film. The image shows the epidermis layer at the bottom, followed by the dermis and subcutis sections. The dermis layer was distinguished by varying intensities (different colours) present on the wound, whereas the subcutis layer was distinguished by low intensity areas due to the homogeneous composition. At day 14, the wound treated with Ag@TiO 2 NRs/GG hydrogel film showed the formation of a thicker epidermis layer and a clear dermis line. Various colour intensities demonstrated in the dermis layer of the rats wound were improved progressively directed towards the complete epithelialisation of skin. This result demonstrated that the dermis and epidermis layers of the skin were well-developed. Subcutis areas were also growing and well distributed, containing Figure 11. Ultrasound images of wound contraction on skin Sprague Dawley rats after treated with Ag@TiO 2 NRs/GG hydrogel film on day 3, 7 and 14. Table 1. Inhibition zone of pure GG, Ag@TiO 2 NRs/GG hydrogel film and penicillin against gram-positive and gram-negative bacteria.
homogeneous structures such as fat, water, and blood. Comparative ultrasound images of the skin on days 3 and 7 are also depicted in figure 11.

Conclusion
The solvent-casting method for skin tissue regeneration was used to create an Ag@TiO 2 NRs/GG hydrogel film wound dressing. The physical and chemical properties of the hydrogel film were investigated using FTIR, XRD, and SEM. The presence of agglomeration of Ag and elongated TiO 2 NRs on the surface of GG hydrogel film was seen in SEM images, showing that the Ag@TiO 2 NRs was successfully dispersed in GG film. The combination of Ag and TiO 2 NRs promotes cell viability and proliferation. When wounds on rats are treated with Ag@TiO 2 NRs/GG hydrogel film, they heal completely by day 14. An ultrasound image of the treated skin shows the formation of larger epidermis, dermis, and subcutis layers, indicating excellent skin tissue regeneration.