This experimental study used a post-test-only control group design to evaluate the effects of DACC on inflammation and proliferation in diabetic ulcers in Wistar rats.

A total of 27 healthy male Wistar rats (Rattus norvegicus), aged 2–3 months and weighing 200–300 g, were obtained from the Central Laboratory of Food and Nutrition Studies, Gadjah Mada University. Using Federer’s formula, the rats were randomly assigned into three groups of nine, with an additional rat per group to anticipate potential mortality. The inclusion criteria required healthy male rats with normal activity and post-induction blood glucose levels of at least 250 mg/dL. Exclusion criteria included inactivity, illness, or failure to maintain hyperglycemia, while dropout criteria consisted of mortality or blood glucose levels below 250 mg/dL.

Hyperglycemia was induced via intraperitoneal injection of streptozotocin (45 mg/kg body weight) and nicotinamide (110 mg/kg body weight), dissolved in 0.01 M citrate buffer (pH 4.5). On day 4, blood glucose levels were measured from tail vein samples, and only rats with glucose levels ≥250 mg/dL were included in the study.

After confirmation of hyperglycemia, each rat was anesthetized with ketamine (80 mg/kg) and xylazine (10 mg/kg) administered intraperitoneally. A standardized 0.5 cm full-thickness wound was created on the shaved dorsal area using a biopsy punch under sterile conditions.

Following wound induction, each group received treatment according to its group assignment. The control group (C) received only a sterile gauze dressing, the saline-treated group (T1) received gauze soaked in 0.9% NaCl solution, and the DACC-treated group (T2) received a DACC-coated gauze dressing. Dressings were applied immediately after wound creation, secured with transparent adhesive film to prevent detachment, and replaced every 3 days for 14 days. Wounds were observed daily for general condition, dressing integrity, and signs of infection until the end of the study period.

The study used various laboratory instruments, including animal cages, feeding and hydration systems, sterile gloves, masks, glass slides, cover slips, microscopes, syringes, and micropipettes. Histological staining kits for hematoxylin-eosin (H&E) and Masson’s trichrome were used for tissue analysis. The primary materials included sterile gauze dressings, saline solution, and DACC-coated wound dressings.

The assessment of macrophage count was conducted on day 18 through histological analysis of H&E-stained tissue sections, examined under a light microscope at 400× magnification, with quantification performed in three different fields of view per sample. The inflammatory response was evaluated on day 7 by quantifying leukocyte infiltration in H&E-stained tissue, with microscopic examination performed at 400× magnification. IL-6 levels were determined using enzyme-linked immunosorbent assay, with normal serum levels considered to be below 4 pg/mL. Angiogenesis was assessed on day 18 through capillary density analysis in H&E-stained sections, observed at 400× magnification. Collagen deposition was analyzed on day 7 using Masson’s trichrome staining, with collagen fibers identified by their characteristic blue or greenish-blue coloration and quantified under 400× magnification. Fibroblast proliferation was evaluated on day 7 by counting spindle-shaped fibroblast cells in H&E-stained sections, also examined at 400× magnification, with three separate fields of view analyzed per sample. Histological evaluation was performed in a blinded manner by trained laboratory personnel at the Central Laboratory of Food and Nutrition Studies, Gadjah Mada University.

Statistical analysis was conducted using SPSS software (IBM Corp.). Normality testing was carried out using the Shapiro-Wilk test for small sample sizes (n<50), and quantitative data were expressed as mean±standard deviation. Comparisons between groups were analyzed using one-way analysis of variance (ANOVA) for variables with normally distributed data. For variables not meeting normality assumptions, such as angiogenesis (Shapiro-Wilk P<0.05), the Kruskal-Wallis non-parametric test was applied. Post hoc comparisons were performed using least significant difference (LSD) and Tamhane’s tests depending on homogeneity of variance. A P-value of less than 0.05 was considered statistically significant.

Ethical approval was obtained from the Commission of Medical Research Bioethics, Faculty of Medicine, Sultan Agung Islamic University (Ref. No. 425/X/2024/Komisi Bioetik). All procedures involving experimental animals were conducted in compliance with ethical guidelines for the humane treatment of laboratory animals.

The findings indicate no significant differences in macrophage count among the groups, suggesting that DACC did not significantly alter macrophage recruitment in diabetic ulcer healing. Elevated glucose levels in diabetic conditions can complicate the wound healing process, as diabetic wounds typically require 12–20 weeks to heal, compared to acute wounds, which heal within 3 weeks [10]. Macrophages are key regulators of the inflammatory phase in wound healing, particularly in diabetic ulcers, where chronic hyperglycemia sustains prolonged M1 macrophage activation, thereby delaying progression to the proliferative phase. In the present study, although the difference in macrophage count among the groups did not reach statistical significance (P=0.058), the DACC-treated group (T2) exhibited the lowest mean macrophage infiltration. This finding may indicate a trend toward reduced inflammation, potentially resulting from the antimicrobial activity of DACC. By binding microbial cell walls via hydrophobic interactions, DACC reduces the local microbial load without inducing resistance, thereby diminishing pathogen-associated molecular patterns that perpetuate macrophage-mediated inflammation [11].

The relatively short observation period of 14 days may have limited the detection of more pronounced shifts in macrophage dynamics, including phenotypic transitions from M1 to M2 subtypes. Additionally, dressing changes performed every 3 days, as opposed to more frequent interventions in other studies (e.g., Ronner et al., 2014 [12]), may have influenced bacterial colonization and subsequent immune activation. To further elucidate the immunomodulatory role of DACC, future research should incorporate longer follow-up periods and include phenotypic characterization of macrophages through immunohistochemical or molecular analyses to differentiate between pro-inflammatory and anti-inflammatory macrophage subsets.

The findings of this study indicate a significant difference in the mean leukocyte count, which reflects the degree of inflammation among the treatment groups. The highest mean leukocyte count was observed in the control group, which received only gauze and transparent adhesive film dressings, with a value of 18.933. In the T1 group, which received saline gauze and transparent film dressings, the mean leukocyte count was 11.663. Meanwhile, the T2 group which received DACC gauze and transparent film dressings exhibited the lowest mean leukocyte count, at 8.700. These findings suggest that DACC treatment effectively reduces the inflammatory response, likely by decreasing microbial load and modulating immune activity [4].

Post hoc LSD analysis revealed a significant reduction in IL-6 levels in the treatment groups compared to the control, suggesting that DACC administration effectively reduces inflammatory responses. IL-6 is a pro-inflammatory cytokine involved in chronic inflammatory conditions, including diabetic ulcers. Elevated IL-6 levels correlate with severe inflammation and delayed wound healing. Previous studies investigated the effects of DACC dressings on diabetic wound healing in rats. After 14 days of application, a significant reduction in IL-6 levels and an accelerated wound healing process were observed compared to the control. The 14-day duration of DACC treatment in this study allowed for monitoring of IL-6 reduction, which correlated with enhanced progression to the proliferative phase of wound healing [13]. The significant reduction in IL-6 levels observed in group T2, which received DACC treatment, compared to the control and T1 groups, suggests that DACC may exert a potent anti-inflammatory effect by lowering local cytokine expression.

This study demonstrated significant differences in angiogenesis among the groups (P=0.008) based on the Kruskal-Wallis test, with further details obtained through post hoc analysis. The control exhibited a mean angiogenesis level of 10.93±7.10. This group represents the natural wound healing process without intervention. The results in the control group demonstrated some level of angiogenesis, though at a lower rate than the treatment groups. This finding aligns with the literature, which states that wound healing in diabetic conditions tends to be slower due to chronic inflammation, vascular impairment, and oxidative stress, all of which hinder new blood vessel formation [14].

The mean angiogenesis level in group T1 was 12.78±6.83, higher than that of the control group. NaCl solution is known to have physiological effects that help cleanse the wound and create a more sterile environment. This result suggests that NaCl administration may enhance new blood vessel formation, although the difference compared to the control was not statistically significant (P=0.249). The potential mechanism of NaCl in promoting angiogenesis is related to its ability to reduce microbial load and create an environment conducive to endothelial cell activity. However, the lack of significant improvement compared to the control suggests that the effects of NaCl may be transient and insufficient to significantly accelerate wound healing in diabetic ulcers [15].

The DACC-treated T2 group exhibited a mean angiogenesis level of 9.22±10.72, which was lower than both the control and T1 groups. DACC is a physical antimicrobial agent that binds bacteria hydrophobically, reducing microbial burden without inducing antibiotic resistance [16]. However, the lower angiogenesis observed in this group suggests that while DACC effectively reduces infection, it may also alter the microenvironment of the wound, affecting endothelial cell migration and vessel formation.

No statistically significant differences were found in fibroblast count across the groups. However, clinically, fibroblast levels were highest in the T1 group (16.811), followed by the control group (16.044), and lowest in the T2 group (13.544). This numerical trend may suggest that saline treatment provides a more favorable microenvironment for fibroblast activity within the limited observation period. In contrast, the lower fibroblast count in the DACC-treated group may be related to DACC’s hydrophobic surface, which has been reported to minimize cellular adhesion. As fibroblasts play a key role in extracellular matrix production and tissue regeneration, the decreased presence observed in T2 may limit short-term reparative processes. Based on this, the relatively higher fibroblast presence in the saline and control groups may indirectly support the continued use of traditional wound care approaches such as simple cleansing and gauze dressing in early-stage diabetic wound management, especially when fibroblast stimulation is a clinical priority [17].

The lower fibroblast count in T2 (DACC-treated group) compared to the other groups contrasts with previous studies suggesting that DACC’s hydrophobic properties minimize cell adhesion [7]. The hydrophobic functionalization of DACC is believed to demonstrate antimicrobial properties by binding microorganisms through hydrophobic interactions. While DACC-coated dressings have shown promising results in infection prevention and wound healing, further investigation is needed to determine their long-term effects on fibroblast activity.

The results demonstrated that collagen levels were highest in group T2, followed by the control group (C), and lowest in group T1. These findings suggest that DACC application in diabetic ulcers promotes collagen synthesis, which is crucial for wound healing [18]. According to Alwi (2014), hyperglycemia in diabetes induces vascular abnormalities that affect skin and muscle tissue, increasing the risk of ulcer formation [4,19]. In diabetic ulcers, epidermal ulceration disrupts the extracellular matrix, contributing to loss of tissue integrity and a deficiency in type I collagen, which plays a key role in tissue regeneration [20].

This study has several limitations in its methodology. First, it focused solely on a single macrophage phenotype and did not include assessments of animal mobility, which may influence wound healing dynamics. Second, the 14-day observation period may be insufficient to fully capture the long-term effects of DACC, particularly on angiogenesis and fibroblast proliferation. Although no exogenous infection was induced, the potential for microbial contamination due to non-aseptic dressing procedures may better reflect real-world clinical conditions. However, the absence of a deliberately infected wound model limits the extrapolation of these findings to chronic or superinfected ulcers. Furthermore, the observed reduction in angiogenesis and fibroblast counts in the DACC-treated group despite increased collagen deposition deviates from established wound healing mechanisms and warrants further investigation. In the absence of mechanistic in vitro studies, such as endothelial (e.g., HUVEC) migration assays or fibroblast proliferation analyses, these results remain speculative. Lastly, the omission of wound closure rate, a gold standard indicator of healing progression, restricts the translational significance of the study. Future research should incorporate longer observation periods, infected wound models, quantitative wound area analysis, and targeted cellular assays to comprehensively evaluate the therapeutic efficacy of DACC on macrophage dynamics, inflammation, collagen deposition, angiogenesis, and fibroblast proliferation in diabetic ulcer healing.

This study demonstrates that DACC significantly reduces inflammation and enhances collagen deposition in diabetic ulcer healing. The DACC-treated group exhibited a notable reduction in IL-6 levels, macrophage count, and degree of inflammation, indicating its potential role in modulating inflammatory responses. Although angiogenesis and fibroblast proliferation were highest in the saline-treated group, the increased collagen deposition in the DACC-treated group suggests a beneficial effect on extracellular matrix formation and tissue remodeling. These findings highlight the potential of DACC as a promising wound care intervention, though further studies are needed to explore its long-term effects on angiogenesis and fibroblast activity in diabetic ulcer healing.