Sulfated galactan (SG) derivatives, including degraded SG (DSG) and degraded SG supplemented with octanoyl ester (DSGO), were prepared using previously established methods [9, 13]. Briefly, initial SG was stirred in 0.1 M HCl (RCILabscan, Bangkok, Thailand; ratio 10:1) for 6 h at room temperature. The mixture was then neutralized to pH 8 and precipitated with 95% ethanol (RCILabscan, Bangkok, Thailand). The pellet was collected, re-suspended in distilled water, and dialyzed against distilled water in a dialysis bag for 24 h. DSG was obtained after freeze-drying overnight. DSGO was prepared by stirring DSG with pyridine (Merck, Darmstadt, Germany; ratio 12.5:1) for 6 h at room temperature. Afterward, octanoyl chloride (20 µL; Sigma-Aldrich, Merck, Darmstadt, Germany) was added and the mixture was stirred vigorously for an additional 24 h. The reaction mixture was then concentrated, and the remaining pyridine was removed by adding toluene (Merck, Darmstadt, Germany). The reaction mixture was subsequently dissolved in distilled water and precipitated with 95% ethanol, overnight. The pellet was collected, re-suspended in distilled water, and freeze-dried. DSG, with a molecular weight of 7.87 kDa, comprises complex structures of alternating 3-linked $\beta$-D-galactopyranose and 4-linked 3,6-anhydro-$\alpha$-L-galactopyranose or $\alpha$-L-galactopyranose-6-sulfate. DSGO, having a molecular weight of 152.79 kDa, consists of these complex DSG structures combined with octanoyl ester (as shown in Supplementary Figs. 1–4). The composition and structure of these derivatives were confirmed through gel permeation chromatography (GPC; Agilent Technologies, Santa Clara, CA, USA), Fourier-transform infrared spectroscopy (FTIR; Bruker, Ettlingen, Germany), and nuclear magnetic resonance (NMR; Bruker, Rheinstetten, Germany) analysis.

DSG and DSGO were incorporated into a simple ointment base as outlined in Table 1. The formulation consisted of 75% petroleum jelly (Phitsanuchemical, Phitsanulok, Thailand), 5% polyethylene sorbitol (Sigma-Aldrich, Merck, Darmstadt, Germany), and 19.5% water. Additionally, 0.5% of either DSG or DSGO was added as applicable. The components were thoroughly mixed using a homogenizer mixer (Homogenizer 270D JSR, WorldWide Trade, Pathum Thani, Thailand). The prepared ointments were stored at 4 °C until used.

Wistar rats 5 weeks of age and weighing 150–200 g were obtained from Nomura Siam International (Bangkok, Thailand) and maintained under standard laboratory conditions with unrestricted access to food and water. All experimental procedures adhered to the Guidelines for the Care and Use of Laboratory Animals at the University of Phayao. The study received approval from the Animal Ethics Committee at the University of Phayao Faculty of Medical Science, in line with the Ethics of Animal Experimentation by the National Research Council (Ethical Approval Number: 64 01 04 011). The rats were assigned to one of four groups, each comprising 6 animals: (1) Normal control, which received no ointment; (2) Ointment control, treated with a simple ointment; (3) DSG ointment, treated with an ointment containing 0.5% DSG; and (4) DSGO ointment, treated with an ointment containing 0.5% DSGO.

The rats in each group were anesthetized in the induction chamber using an isoflurane vaporizer set to a 5% flow rate, with the oxygen flow meter set to 1 liter per minute. To ensure proper anesthetization before beginning the procedure, the depth of anesthesia was verified by a firm toe pinch. A predetermined full-thickness skin area of 300 mm² was excised in the dorsal interscapular region [14, 15]. Post-surgery, the rats were left exposed to the open environment. The simple, DSG, and DSGO ointments were subsequently applied daily until complete healing was observed. Wound contraction and the epithelialization period were monitored throughout the healing process. On day 21, skin samples from the healed wounds of each group were collected for histopathological, immunohistochemical, and immunoblotting analyses. For skin sample collection, the rats were anesthetized in the induction chamber with the isoflurane vaporizer set to a 5% flow rate and the oxygen flow meter set at 1 liter per minute. The rats were then sacrificed by cervical dislocation. The skin at the wound site was removed and split into two pieces along the midline of the wound for further investigation.

After creating the excision wound, its margin was traced using transparent paper and the area was measured with graph paper. Wound contraction was measured daily until complete healing occurred and was expressed as a percentage of the healed wound area. Photographs of the wound were captured on days 0, 3, 7, 14, and 21. The measured surface area was then used to calculate the percentage of wound contraction, with the initial wound size of 300 mm² taken as 100%. The formula used for this calculation was as follows:

$$\%\text{wound contraction}=1-({\mathrm{WS}}_t/{\mathrm{WS}}_0)\times 100$$

wherein WS_t represents the wound size on a specific day, while WS₀ represents the wound size on day 0.

In the in vivo studies, wound size was measured on days 0, 3, 7, 14, and 21, with tissue samples being excised on day 21. Full-thickness wound tissue specimens from each group underwent a series of histological preparations; they were fixed in 10% neutral buffered formalin (RCILabscan, Bangkok, Thailand), dehydrated in graded ethanol (RCILabscan, Bangkok, Thailand), cleared with xylene (RCILabscan, Bangkok, Thailand), embedded in paraffin (Merck, Darmstadt, Germany), and sectioned at 5 µm thickness. Ten histological sections obtained from each paraffin-embedded skin sample were then stained with hematoxylin and eosin (H&E, 3 sections), and Masson’s trichrome (MT, 3 sections) (Bio-Optica, Milano, Italy) to highlight different tissue elements. The prepared slides were examined under a light microscope (Nikon Upright Microscope Eclipse Ni-U, Tokyo, Japan) equipped with a digital camera. The analysis was carried out in four histological fields per section. The thickness of skin, number of fibroblasts, blood vessels and collagen fibers in the dermis of the injured area were quantitatively assessed at a magnification of $\times$40. For counting fibroblasts and blood vessels, the sections were evaluated by three examiners to reduce bias, and the results were also confirmed with positive fibroblasts in $\alpha$-smooth muscle actin ($\alpha$-SMA) immunoreaction. The deposition of collagen fibers in the wound was quantitatively assessed by measuring the intensity of the blue color using ImageJ software version 1.32j (National Institutes of Health, Bethesda, MA, USA).

For the immunohistochemical assay, the expressions of $\alpha$-SMA, Ki67, vimentin, and E-cadherin were evaluated using specific primary antibodies for $\alpha$-SMA (cat no. ab124964; Abcam, Cambridge, UK), Ki-67 (cat no. ab15580; Abcam, Cambridge, UK), E-cadherin (cat no. ab231303; Abcam, Cambridge, UK) and vimentin (cat no. MA5-11883; Thermo Fisher Scientific, Waltham, MA, USA), all diluted at 1:300. The procedure began with sectioning formalin-fixed skin tissues at 5 µm thickness. These sections were then deparaffinized in xylene and rehydrated in a graded ethanol series. To block endogenous peroxidase activity, sections were treated with 3% hydrogen peroxide for 30 minutes, followed by immersion in 0.1 M citrate buffer (pH 6.0) at 98 °C for 20 minutes. After cooling, sections were blocked to prevent non-specific binding using 5% normal donkey serum (cat no. ab7475; Abcam, Cambridge, UK) for 1 hour at room temperature. Sections were incubated overnight at 4 °C with the primary antibodies, followed by incubation with secondary antibodies conjugated with horseradish peroxidase (HRP) (Abcam, Cambridge, UK), diluted at 1:500 for 1 hour at room temperature. The signal was developed using the HRP substrate chromogen (Vector Laboratories, Newark, CA, USA), and sections were counterstained with hematoxylin. They were then dehydrated in a graded ethanol solution, cleared with xylene, mounted, and covered for examination under a light microscope (Nikon Upright Microscope Eclipse Ni-U, Tokyo, Japan) equipped with a digital camera. Negative controls were prepared by omitting the primary antibodies and substituting them with blocking serum. The presence of an immunochemical positive signal was assessed under the microscope. The immunoreactive intensity of $\alpha$-SMA, Ki67, vimentin, and E-cadherin was quantitatively analyzed in four histological fields per section at a magnification of $\times$40 using ImageJ software version 1.32j (National Institutes of Health, Bethesda, MA, USA).

Collected tissue samples underwent protein extraction using lysis buffer containing a 100$\times$ protease inhibitor solution. Proteins were then separated using 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane (Merck, Darmstadt, Germany). The membrane was incubated overnight at 4 °C with specific primary antibodies for Collagen type 1 alpha 1 (Col1A1, cat no. A22089; ABclonal Science, Woburn, MA, USA), Ki67 (cat no. sc-23900; Santa Cruz Biotechnology, Dallas, CA, USA), E-cadherin (cat no. sc-21791; Santa Cruz Biotechnology, Dallas, CA, USA), Vimentin (cat no. sc-6260; Santa Cruz Biotechnology, Dallas, CA, USA), $\alpha$-SMA (cat no. sc-53015; Santa Cruz Biotechnology, Dallas, CA, USA), vascular endothelial growth factor (VEGF) (cat no. sc-7269; Santa Cruz Biotechnology, Dallas, CA, USA), Smad2/3 (cat no. 8685; Cell Signaling Technology, Danvers, MA, USA), Smad4 (cat no. 46535; Cell Signaling Technology, Danvers, MA, USA), phosphorylated Smad2/3 (cat no. 8828; Cell Signaling Technology, Danvers, MA, USA), phosphorylated Smad4 (cat no. AF8316; Affinity Biosciences, Minhang, Shanghai, China), and $\beta$-actin (cat. no. AF7018; Affinity Biosciences, Minhang, Shanghai, China), all diluted at 1:1000. This was followed by incubation with secondary antibodies conjugated with HRP, diluted at 1:2000 for 1 hour at room temperature. Immunoreactive proteins were detected using the Clarity™ Western ECL substrate (Bio-Rad Laboratories, Hercules, CA, USA). Relative protein expressions were quantified against an internal control, $\beta$-actin (Affinity Biosciences, Minhang, Shanghai, China) using the densitometry Scion Image 4.0 Software Package. Each analysis was performed in triplicate to ensure accuracy and reproducibility.

One-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test was employed to determine the differences between the treatment and control groups. If significant differences were identified, the data were further analyzed using the Krus-kal-Wallis H test, a nonparametric ANOVA, followed by the Mann-Whitney U-test. Statistical significance was defined as a p-value of less than 0.05. All statistical analyses were conducted using GraphPad Prism software version 5 (GraphPad, San Diego, San Diego, CA, USA).

The primary variables measured in the excision wound study were the rate of wound contraction and the period of epithelialization. Consequently, visible appearances and measurements of wound contraction are crucial parameters in the macroscopic evaluation of wound healing [16]. An effective wound healing agent should enhance the process of wound contraction (expressed as % of wound contraction) and reduce the time required for re-epithelialization [17]. In this study, the area of the healed wounds treated with DSG and DSGO ointments were measured on days 0, 3, 7, 14, and 21 post-surgeries. The results indicated that both DSG and DSGO ointments significantly increased the rate of contraction compared to the controls (normal and ointment controls). Notably, variable epithelial closure of the wound rim with both SG derivative ointments was observed between days 7 and 21 post-wound creation (Fig. 1). Macroscopic observations demonstrated that the healing process facilitated by the DSG and DSGO ointments was faster than that by the controls. Additionally, healing of the wound excision was quantified by calculating the percentage of wound contraction, as outlined in Table 2. The percentage of wound contraction in rats treated with DSG and DSGO ointments demonstrated a significant increase compared to controls. Notably, on day 14, the wound contraction percentages for the normal control, ointment control, DSG ointment, and DSGO ointment groups were 77.58 $\pm$ 4.98%, 78.92 $\pm$ 3.47%, 91.16 $\pm$ 4.62%, and 91.64 $\pm$ 2.98%, respectively. By day 21, these figures were 91.97 $\pm$ 0.39%, 90.02 $\pm$ 0.16%, 98.59 $\pm$ 0.69%, and 99.73 $\pm$ 0.45%, respectively. However, DSG and DSGO treatments also demonstrated effectiveness in wound healing comparable to that of 0.01% silver sulfadiazine, which served as the standard treatment (as shown in Supplementary Fig. 5). The enhanced wound contraction observed with DSG and DSGO ointments is likely due to their ability to stimulate the proliferation, migration, and differentiation of various cells toward the wound site [13], which in turn increases cell-cell and cell-extracellular matrix interactions and contractions [18]. However, comparisons between the SG derivatives (DSG and DSGO ointments) revealed no significant difference in wound contraction percentage (p $>$ 0.05), suggesting that both ointments are effectively healing excision wounds. This finding contrasts with previous in vitro studies indicating that DSGO has superior wound healing activity in fibroblast cultures [13], highlighting the complexity of translating in vitro results to animal models.

Fig. 1.

Degraded sulfated galactan (DSG) and degraded sulfated galactan supplemented with octanoyl ester (DSGO) ointments increased the rate of wound contraction in excision rats. Contraction of excision wounds in rats (n = 6 in each group), assessed on days 0, 3, 7, 14, and 21 post-creation, comparing normal control, ointment control, DSG ointment, and DSGO ointment.

At 21 days post-wounding, skin wounds treated with DSG and DSGO ointments exhibited better healing compared to controls. Histopathological examinations were performed on the wounded skin using H&E staining to assess key aspects of skin microstructure, such as epithelialization, cell proliferation, vascularization, and tissue granulation [19]. The histopathological findings for all groups displayed healed skin architecture characterized by re-epithelialization, restoration of connective tissue, and formation of new blood vessels (Fig. 2A). Notably, the skin from rats treated with DSG and DSGO ointments showed enhanced healing compared to those in the control groups, aligning with the results observed in wound contraction. Further evaluations included measuring the thickness of the skin, the extent of neovascularization and fibroblasts (Fig. 2B–D). Both DSG and DSGO ointments led to a significantly thicker epidermis compared to the controls. An increase in new blood vessel formation and fibroblasts in the dermis of rats treated with both DSG and DSGO ointments was observed, highlighting the angiogenic effectiveness and activation of fibroblasts by these compounds. This result aligns with previous studies demonstrating that ointments supplemented with extracts from Marantodes pumilum (Blume) Kuntze and Manilkara zapota L. enhanced wound healing in excision rats by promoting epithelialization, accelerating neo-collagen synthesis, and stimulating angiogenesis, as well as fibroblast proliferation and function, as observed in histological examinations [19, 20]. These findings underscore the capacity of DSG and DSGO ointments to not only support re-epithelialization and remodeling of connective tissue (fibers and cells) but also enhance neovascularization, thereby significantly improving the overall wound healing process [21].

Fig. 2.

Degraded sulfated galactan (DSG) and degraded sulfated galactan supplemented with octanoyl ester (DSGO) ointments improved the histopathological features of wounded skin in excision rats. Histopathological examination of skin wounds stained with hematoxylin and eosin (H&E), conducted on skin sections from the healed areas of wounds at day 21 across various treatment groups: normal control, ointment control, DSG ointment, and DSGO ointment (n = 6 in each group). Magnification: 40$\times$ (A). Black arrowheads indicate the stratum corneum of the epidermis; red arrowheads indicate the fibroblasts; arrows show the blood vessels in the dermis. Scale bar = 50 µm. Graphs display the thickness of the epidermis, stratum corneum (B), neovascularization in the dermis (C), and fibroblasts in the dermis (D) in different treatment groups. Data presented as mean $\pm$ SEM from three independent experiments. ^a denotes values significantly different from normal control, and ^b from ointment control (p 0.05).

After injury, damaged tissues release various cytokines that initiate the proliferation, migration, and differentiation of cells toward the wound area, a key process in tissue repair [19]. The proliferation phase, which includes cell migration and differentiation, is a critical hallmark of tissue repair and is associated with many biochemical events, particularly the formation of new blood vessels through the vascularization of endothelial cells. Keratinocytes proliferate to create new epithelial layers, while fibroblasts differentiate into myofibroblasts, contributing to wound contraction and size reduction [22]. Previous studies have shown that cellular signaling proteins such as Ki67, $\alpha$-SMA, E-cadherin, vimentin, and VEGF are crucial for cell proliferation, migration, and differentiation during the wound healing process [23, 24]. Ki67 protein, upregulated during all active phases of the cell cycle and absent in resting cells, serves as a reliable marker for cell proliferation [25]. Additionally, the transformation of activated fibroblasts into contractile myofibroblasts is marked by the expression of $\alpha$-SMA, an important indicator of myofibroblast formation and tissue repair [26]. Two additional regulated proteins, E-cadherin and vimentin, play critical roles in cell migration during wound healing. E-cadherin is a key component of adherens junctions and is essential for the collective directional migration of large epithelial sheets, facilitating coordinated cell movement necessary for wound closure [27]. Vimentin is crucial for fibroblast functions that are integral to the proliferation and remodeling phases of wound healing, supporting structural integrity and the function of cells during repair [28]. Typically, E-cadherin downregulation and vimentin upregulation are associated with a failure to heal [29, 30]. Angiogenesis, the formation of new blood vessels from pre-existing ones, is a vital component of the healing process. This biological mechanism restores blood flow to damaged tissues, providing the oxygen and nutrients necessary for the growth and proper function of repaired cells. VEGF is among the most potent proangiogenic growth factors in the skin. The presence and concentration of VEGF within a wound are crucial for promoting angiogenesis, thereby having a substantial impact on the rate and quality of wound healing [31].

Hence, we examined the effects of DSG and DSGO ointments on the protein expression of Ki67, $\alpha$-SMA, E-cadherin, vimentin, and VEGF in wounded rats. The results, depicted in Fig. 3, indicated that the expressions of Ki67, $\alpha$-SMA, and E-cadherin were significantly upregulated in both DSG and DSGO ointments compared to the normal control. However, changes in the expression of vimentin and VEGF were observed only in the DSGO ointment. Specifically, the expression levels of Ki67, $\alpha$-SMA, E-cadherin, vimentin, and VEGF in the DSG ointment were 1.40 $\pm$ 0.27, 1.25 $\pm$ 0.11, 1.68 $\pm$ 0.31, 0.98 $\pm$ 0.33, and 1.03 $\pm$ 0.23-fold that of the normal control, respectively. In contrast, expressions in the DSGO ointment were 2.59 $\pm$ 0.19, 1.25 $\pm$ 0.12, 3.01 $\pm$ 0.45, 1.67 $\pm$ 0.09, and 0.53 $\pm$ 0.21-fold that of the normal control, respectively. Interestingly, when comparing the two compound ointments, the DSGO ointment exhibited greater wound healing ability than the DSG ointment, which correlated with related protein expressions.

Fig. 3.

Degraded sulfated galactan (DSG) and degraded sulfated galactan supplemented with octanoyl ester (DSGO) ointments altered the expression of wound healing-related proteins in excision rats. Western blot analysis of protein expression related to skin healing in excision wounds of rats across different treatment groups (n = 6 in each group). Expressions of Ki67, $\alpha$-smooth muscle actin ($\alpha$-SMA), E-cadherin, vimentin, and vascular endothelial growth factor (VEGF) were detected in wound specimens collected at day 21 post-wound healing, relative to $\beta$-actin. Data are expressed as mean $\pm$ SEM from three independent experiments. ^a indicates values significantly different from normal control, ^b from ointment control, and ^c from DSG ointment (p 0.05).

Additionally, immunohistochemistry assays were performed to confirm the expression of Ki67, $\alpha$-SMA, E-cadherin, and vimentin in the wound tissue. The immunoreactions for Ki67, $\alpha$-SMA and E-cadherin were dramatically enhanced in the wound tissue treated with both DSG and DSGO ointments compared to the controls. However, there was no significant difference in the immunoreaction of vimentin between the groups (Fig. 4A,B). The unchanged level of vimentin in immunohistochemistry may indicate issues with the specificity and quality of antibodies, methodology, or experimental conditions [32]. The observed decrease in VEGF levels with DSGO ointment suggests that optimal protein expression levels may vary depending on the type of wound and the phases of healing. Inadequate levels of VEGF can lead to impaired healing, whereas excessively high levels might promote the formation of overabundant scar tissue [31]. These results underscore the beneficial effects of SG derivatives in wound healing processes, particularly through the altered expression of proteins associated with cell proliferation, migration, differentiation, and angiogenesis [33].

Fig. 4.

Immunohistochemical staining of wound healing-related proteins in skin of excision rats treated by degraded sulfated galactan (DSG) and degraded sulfated galactan supplemented with octanoyl ester (DSGO) ointments. (A) Immunohistochemical micrographs displaying dark brown staining on the skin of excision rats in various treatment groups (n = 6 in each group). Specimens were immunoassayed using antibodies against $\alpha$-smooth muscle actin ($\alpha$-SMA; myofibroblast marker), Ki67 (cell proliferation marker), E-cadherin, and vimentin (cell migration markers). Scale bar = 50 µm. (B) Graphs display the intensity of $\alpha$-SMA, Ki67, E-cadherin, and vimentin detected in wound specimens collected at day 21 post-wound healing using ImageJ software. Data are expressed as mean $\pm$ SEM from three independent experiments. ^a indicates values significantly different from normal control, ^b from ointment control, and ^c from DSG ointment (p 0.05).

Some bioactive compounds have proven highly effective in wound healing by enhancing wound closure through the stimulation and regulation of collagen biosynthesis within the wound area [34]. Collagen, a primary component of the extracellular matrix, plays a crucial role throughout the wound healing phases due to its regulatory functions [35, 36]. The process of wound healing significantly relies on the regulated synthesis and deposition of new collagen fibers [37]. The deposition of collagen in the healing tissue was assessed using MT staining. The results revealed that the tissues from the control groups (normal and ointment controls) exhibited loosely arranged thin collagen bundles in the dermis. In contrast, tissues treated with both DSG and DSGO ointments displayed thick collagen bundles that were densely arranged, as depicted in Fig. 5A. The intensity of MT staining, which indicated collagen bundle deposition, was further analyzed and is shown in Fig. 5B. This observation indicates that the ointments supplemented with SG derivatives promote wound healing by facilitating the deposition of collagen.

Fig. 5.

Degraded sulfated galactan (DSG) and degraded sulfated galactan supplemented with octanoyl ester (DSGO) ointments increased the collagen and Smad signaling in skin of excision rats. (A) Masson’s trichrome (MT) staining on skin sections from healed wound areas at day 21 in various treatment groups: normal control, ointment control, DSG ointment, and DSGO ointment (n = 6 in each group). Magnification: 40$\times$. Scale bar = 50 µm. (B) The graph displays the intensity of collagen deposition in the dermis by MT staining. (C) Western blot analysis demonstrating the expression levels of collagen (Col1A1) and Smad signaling proteins (Smad2/3, Smad4, phospho-Smad2/3, and phospho-Smad4) in excision wound samples at day 21 post-healing, relative to $\beta$-actin. Data are expressed as multiples of control and mean $\pm$ SEM from three independent experiments. ^a indicates values significantly different from normal control, ^b from ointment control, and ^c from DSG ointment (p 0.05).

Further analysis was conducted to determine the expression levels of Col1A1 and Smad proteins, which are critical regulators of collagen type I biosynthesis [38], in excised rats treated with DSG and DSGO ointments. According to the results depicted in Fig. 5C, there was a significant increase in the expression of Col1A1 and Smad proteins in the tissues of rats treated with both DSG and DSGO ointments compared to the normal control. Specifically, the expression levels of Col1A1, Smad2/3, phosphorylated Smad2/3, Smad4, and phosphorylated Smad4 in rats treated with DSG ointment were significantly elevated to 2.23 $\pm$ 0.21, 1.61 $\pm$ 0.37, 2.18 $\pm$ 0.30, 1.28 $\pm$ 0.18, and 2.34 $\pm$ 0.42-fold of normal control, respectively. Meanwhile, in rats treated with DSGO ointment, these levels were up-regulated to 4.65 $\pm$ 0.54, 1.47 $\pm$ 0.28, 2.38 $\pm$ 0.44, 1.61 $\pm$ 0.28, and 3.41 $\pm$ 0.55-fold of normal control, respectively. However, the increases in Col1A1 and Smad4 were not significant in the DSG ointment compared to the control ointment, suggesting that the formulated ointment itself may contribute to enhancing wound healing activity [14, 21]. Interestingly, the expression levels of Col1A1, phosphorylated Smad2/3, and phosphorylated Smad4 were significantly higher in the DSGO ointment compared to the DSG ointment. This indicates that the wound healing activity of both DSG and DSGO ointments enhances type I collagen synthesis, which is involved in the mediated phosphorylation of the Smad proteins signaling pathway [39].

Taken together, the results indicate that the wound healing process benefits from ointments supplemented with SG derivatives, particularly DSGO. This effect is observed as the provisional matrix, present immediately after wounding, is replaced by neo-formed connective tissue composed of small vessels, extracellular matrix, and fibroblastic cells. These cells become activated and differentiate into myofibroblasts, playing a crucial role in wound repair [33]. In addition, DSGO showed slightly better wound healing efficacy compared to DSG, suggesting that DSGO, which contains medium-chain fatty acids, enhances cellular uptake ability. This, in turn, activates mediators associated with fibroblast functions, potentially promoting wound healing [13, 39].