For this experimental study, 18 male laboratory mice of the BALB/c strain aged 8-10 weeks were used. The animals were obtained from the Experimental Medical Research Centre of Birjand University of Medical Sciences, Birjand, Iran. Ethical standards for work with laboratory animals were applied throughout the work. The study protocol was approved by the ethics committee of Birjand University of Medical Sciences (IR.BUMS.REC.1400.364). The mice were housed in clean individual cages with free access to food and water. A 12-hour light-dark cycle was maintained, and the temperature was set at 22-23 degrees Celsius.

Initially, 18 healthy male BALB/c mice were kept in an appropriate environment. To induce a skin wound, the animals were anesthetized by intraperitoneal injection of a combination of ketamine and xylazine (100:10 mg/kg). The hair on the dorsal neck was shaved, and a full-thickness skin wound, encompassing the dermis and hypodermis, was created with a scalpel under sterile conditions and in accordance with surgical principles. The mice were divided into experimental and control groups, each containing 9 mice. A one-centimeter incision was made on the back of all mice and subsequently sutured. In the experimental group, a single dose of 0.05 ml (equivalent to 1150 μg) of hyaluronic acid (REVOFIL Plus, Korea) was injected on both sides of the wound immediately after suturing. The control group was kept without the injection of any substances at the site of the suture. Finally, photographs were taken from the wound site along with length measurements. On days 7, 14, and 21, three mice from each group were anesthetized, and skin samples were collected for histological evaluation.

Tissue samples were fixed in a 10% formaldehyde solution, dehydrated with ascending concentrations of alcohol, and cleared in xylene during tissue processing stages. Subsequently, they were embedded and blocked in paraffin, and 5 μm sections were cut using a microtome. Finally, the sections were stained with hematoxylin and eosin (H&E) and subjected to the trichrome Mason technique to evaluate collagen synthesis and arrangement. After staining, slides were prepared for interpretation, and ultimately, 12 slides from each group were randomly examined by a pathologist to assess tissue changes, which included evaluating the number of fibroblasts, collagen density, epithelial thickness, and granulation tissue extension. The findings were then reported accordingly. Slides were imaged using a Euromex-CMEX-10 camera at magnifications of 10, 40, and 100. For histological analysis, the images were processed with Image J analysis software.

Quantitative parameters, including the number of fibroblasts, collagen fiber thickness, epithelialization, and the area of granulation, were assessed using Image J software. The quantitative data were expressed as mean ± standard deviation (SD). One-way repeated-measure analysis of variance, Bonferroni test, and independent samples t-test were applied for data analysis. Statistical analysis was performed using SPSS version 26. A statistical significance level of 0.05 or lower was considered significant.

The results of this study revealed that on days 7 and 14 following the injection of artificial hyaluronic acid, the fibroblast count in the experimental group was significantly higher than in the control group (Table 1) (Fig. 1). However, by day 21 post-injection, the mean fibroblast count did not differ significantly between the two groups.

The results of this study showed a significantly higher collagen fiber density in the intervention group compared to the control group on days 14 and 21 post-injection. Furthermore, the collagen fibers in the experimental group demonstrated improved organization and arrangement at the wound site in the experimental group on both days 14 and 21 than in the control group (Table 1) (Fig. 1).

The results of this study indicated no significant difference in area of granulation tissue on day 7 after injection. However, on days 14 and 21 after injection, the granulation tissue area in the experimental group was significantly greater (Table 1) (Fig. 1).

The results of this study showed no significant difference in epithelial thickness between the two groups on days 7 and 14 post-injection. However, on day 21 post-injection, the epithelial thickness in the experimental group was significantly greater than that in the control group (Table 1) (Fig. 1). Furthermore, microscopic examination revealed a higher number of hair follicles at the wound site in the experimental group compared to the control group on days 14 and 21.

In our study, the number of fibroblasts was significantly higher in the experimental group compared to the control group on days 7 and 14 after injection. This finding aligns with previous research demonstrating beneficial effect of hyaluronic acid on fibroblast proliferation. For instance, Hameed et al. investigated the role of hyaluronic acid in the progression of wound healing; their study reported fibroblast growth factor levels in the tissue increased locally at day 21.^[15] In a systematic review conducted by Price et al., the effects of hyaluronic acid on skin regeneration and wound healing were examined. The study concluded that HA, due to its long-chain molecular structure, stimulates fibroblast proliferation both in vivo and in vitro conditions. ^[16] Recent studies have also confirmed the beneficial effect of hyaluronic acid on fibroblast phenotype and proliferation.^{[17, 18]}

For instance, research has shown that HA facilitates transforming growth factor-β1 (TGF-β1)-dependent fibroblast proliferation, highlighting its role in wound healing processes.^{[19, 20]} Additionally, in vitro studies have demonstrated that the addition of HA to human dermal fibroblast cultures increases proliferation and synthesis of collagen type I (COL1A1), indicating its potential in enhancing skin regeneration.^[21]

A recent study published in Scientific Reports examined the combined effects of green tea and HA gel on fibroblast activation and wound healing. The research demonstrated that HA plays a significant role in tissue repair mechanisms by enhancing fibroblast proliferation and migration. The study concluded that the synergistic application of green tea and HA gel can effectively promote wound healing, highlighting HA’s involvement in tissue repair processes.^[22]

These findings underscore the multifaceted role of HA in modulating fibroblast activity, thereby contributing to improved wound healing and tissue regeneration.

On the other hand, some findings align with existing literature indicating that HA plays a crucial role in ECM remodeling and collagen fiber organization. For instance, studies have shown that HA can modulate the deposition of fibronectin and collagen, essential components of the ECM, during myofibroblast formation.^[14]

Furthermore, the study demonstrated an increase in the synthesis of collagen type I as a result of fibroblast proliferation.^[17] Existing literature highlights a notable impediment in the accumulation of a disorganized collagen matrix within fetal wounds, resulting in a distinctive scarless healing process. This favorable outcome is attributed to the heightened and prolonged presence of HA in fetal wounds compared to adult wounds. Consequently, interventions aimed at sustaining a matrix rich in HA represent promising strategies for achieving scarless repair and regenerative healing.^[23]

Our study further demonstrated that direct injection of HA in the experimental group resulted in a significant increase in collagen fiber density and improved organization compared to the control group.

Our findings demonstrated a significant increase in the granulation tissue area in the experimental group on days 14 and 21 post-injection. Similarly, Hameed et al. reported that the application of HA exosomes in wound models stimulated fibroblasts to produce higher levels of angiogenic growth factors, such as VEGF, thereby enhancing wound healing.^[15] Additionally, the hygroscopic properties of HA were found to play a crucial role in maintaining hydration and supporting the structural integrity of the extracellular matrix (ECM) during the wound healing process. This support, in turn, enhanced cell migration, adhesion, and proliferation within the granulation tissue.^[24] Moreover, evidence indicates that HA possesses the ability to modulate the inflammatory response by inhibiting inflammatory proteases, thereby promoting improved tissue granulation and stabilization.^[25]

The results of our study revealed a significantly greater epithelial thickness in the experimental group compared to the control group on day 21 post-injection. Similarly, Kamdem et al., in their investigation of using HA dressings for second-degree burns in children, demonstrated that epithelialization occurred in 96.3% of the burn wounds studied within an average period of 14 days after the proce- dure.^[26] Additionally, Leite et al. demonstrated the efficacy of 0.2% hyaluronic acid in improving skin abrasions in rats. Their study revealed that on days 7 and 14, the HA group had a higher rate of epithelialization compared to other groups, and with all lesions achieving complete re-epithelialization.^[25]

Our research findings showed that the experimental group had a significantly greater number of hair follicles on days 14 and 21 post-injection. In a study by Kim et al. on the impact of hyaluronic acid microsponges on minoxidil absorption, it was demonstrated that hyaluronic acid promotes the proliferation, migration, and accumulation of dermal papilla cells, thereby contributing to hair growth. The topical application of hyaluronic acid in mice with chemically induced alopecia led to a reduction of hair loss.^[27] Furthermore, the application of hyaluronic acid was found to stimulate hair growth around the wounds.^[15]