Hyaluronic Acid Hydrogel Associated with Silver in the Treatment of Experimentally Induced Partial-Thickness Burn Wounds in Mice

Abstract

Burns are complex traumatic injuries that require specialized care. This study aimed to evaluate the therapeutic effects of a hyaluronic acid (HA) hydrogel associated with silver (HA+Ag) on partial-thickness burn wounds in mice, through macroscopic, morphometric, microscopic, and molecular analyses. Seventy-two BALB/c mice were divided into four groups: Control (saline), SSD (1.0% silver sulfadiazine cream), HA (1.0% hyaluronic acid hydrogel), and HA+Ag (1.0% hyaluronic acid hydrogel + 0.5% silver). At 7 days after injury (DAI), the HA+Ag group showed significantly greater wound contraction compared to the Control. Microscopic analysis revealed increased angiogenesis and fibroblast presence in the HA and HA+Ag groups, suggesting enhanced granulation tissue formation. No significant differences were observed in collagen fiber deposition. Gene expression analysis at 7 DAI showed higher levels of IL-1β, TNF-α, and IL-10 in the Control group, indicating a more intense inflammatory response in untreated animals. These differences were transient and were no longer observed at later time points. Therefore, the findings suggest that HA-based hydrogels, particularly when combined with silver, promote wound healing during the initial phase and represent a promising therapeutic alternative for partial-thickness burns.

Key words
Burns; Wound healing; Hyaluronic acid; Silver; Hydrogel

INTRODUCTION

Burns result from exposure to high-temperature substances, such as liquids and flames (86% of cases), electrical currents (4% of cases), and chemical substances, such as acids and alkalis (3% of cases) (Peck 2011, Schaefer & Szymanski 2023, Yakupu et al. 2022). Accurate classification of burns is crucial to guide initial treatment and early patient recovery, distinguishing between superficial-thickness (first-degree), partial-thickness (second-degree), and full-thickness (third-degree) burns (Jeschke et al. 2020, Warby & Maani 2023).

The World Health Organization (WHO) defines burns as a global public health problem, with approximately 180,000 annual deaths (WHO 2023). The incidence and mortality related to these injuries are directly linked to factors such as age, occupation, and socioeconomic circumstances. In addition to physical impacts, burn victims may suffer psychological consequences due to prolonged hospitalizations and stigmas, compromising their quality of life (Yakupu et al. 2022, Opriessnig et al. 2023).

Burn healing is a complex physiological process comprising three successive and overlapping phases: hemostasis/inflammation, proliferation, and remodeling (Mihai et al. 2018). Cytokines and growth factors play crucial roles in the inflammation phase and, consequently, in the regulation of healing. IL-1β and TNF-α are pro-inflammatory mediators that trigger inflammatory responses necessary for the removal of damaged tissue. On the other hand, IL-10 acts as an anti-inflammatory mediator, controlling excessive inflammatory responses and promoting the transition to the repair phase. TGF-β is essential in the formation of granulation tissue and the modulation of the extracellular matrix during healing. VEGF plays a crucial role in angiogenesis, stimulating the formation of new blood vessels, which is vital for providing nutrients and oxygen to injured tissues. The balanced regulation of these mediators is essential for effective healing (Rodrigues et al. 2019).

The relevance of preclinical studies not only supports the safety and efficacy of new burn treatments but also contributes to a more robust approach, directly reflecting on the quality and success of future burn treatments. Water-based dressings, especially hydrogels, stand out in this context, offering an effective protective barrier, a conducive environment for healing, autolytic debridement, stimulation of essential cellular components for the healing process, and pain relief. Thus, they constitute a promising and comprehensive alternative for the treatment of these complex wounds (Abdullahi et al. 2014, Dhaliwal & Lopez 2018, Chamkouri & Chamkouri 2021, Gospodarek et al. 2022).

Natural polymers, such as hyaluronic acid (HA), have been the subject of studies to develop new therapies for wound treatment. HA is a polysaccharide belonging to the glycosaminoglycan family and is biocompatible, biodegradable, hydrophilic, and non-immunogenic, naturally found in human body fluids (Dalmedico et al. 2016). Silver (Ag) is used for burn treatment due to its antimicrobial properties, being effective against a broad spectrum of gram-positive and gram-negative bacteria, encapsulated or not in biofilms (Wilkinson et al. 2011). Silver Sulfadiazine 1.0% (SSD) is the most widely used product in public burn treatment centers, acting by releasing Ag salts that confer its antimicrobial effect. However, this product can have adverse reactions, delaying re-epithelialization due to cytotoxicity to cells involved in healing (Oaks & Cindass 2023).

Hyaluronic acid (HA) is directly involved in the wound healing process. HA can present different molecular weights, with each size acting at distinct stages of healing. High molecular weight HA (HMW HA) during the inflammatory stage is degraded into low molecular weight HA (LMW HA) oligomers by hyaluronidase enzymes or by reactive oxygen species. HMW HA molecules have a structural function, forming porous networks that fill spaces in granulation tissue during the inflammatory phase, and also exhibit anti-angiogenic and anti-inflammatory properties. In contrast, LMW HA molecules (<1000 kDa) are pro-inflammatory and pro-angiogenic, promoting chemotaxis, cell proliferation, and consequently the expression of cytokines such as IL-1β, TNF-α, and IGF-1 (Dicker et al. 2014, Frenkel 2012, Graça et al. 2020, Kawano et al. 2021).

In vivo studies with murine models have shown the therapeutic potential of hydrogels containing HA and silver in wound healing. Hu et al. (2022) evaluated full-thickness skin and abdominal wall wounds in rats treated with dopamine-modified HA hydrogel, gelatin, and silver nanoparticles, observing stimulation of angiogenesis, granulation tissue formation, and accelerated repair. Taskan et al. (2021) tested an HA-based gel with antioxidants, coenzyme Q10, and vitamin E, observing increased fibroblast presence, reduced inflammation, and accelerated healing at 7 days.

Gonçalves et al. (2021) studied a formulation containing carboxymethyl chitosan (2.0%), HA (0.2%), and Ag (1.0%) in partial-thickness burns, with promising results during the inflammatory phase, including significant wound contraction, granulation tissue formation, inflammatory infiltration, and collagen deposition.

Contrary to previous studies, the present work evaluates the healing effects of a simple topical formulation combining hyaluronic acid (1.0%) with silver (0.5%), without chemical modification or the use of nanoparticles, applied to partial-thickness burns. Therapies that promote healing and inhibit microbial development are alternatives for wound treatment, especially those caused by burns. Thus, this study aims to evaluate the macroscopic, morphometric, microscopic aspects, and gene expression of inflammatory mediators in partial-thickness burn wounds induced in mice after treatment with HA+Ag hydrogel.

MATERIALS AND METHODS

Hydrogels

The hydrogels were formulated in the Experimental Pathology Laboratory of the Institute of Tropical Pathology and Public Health at the Federal University of Goiás. The hyaluronic acid used was of medium molecular weight (M_w 170,000–250,000 kDa; Sigma-Aldrich, USA). The formulations were weighed and prepared in sterile deionized water under laminar flow, with a final concentration of 1.0% for HA. For the HA+Ag formulation, a sterile silver nitrate solution (0.5%) was added. The hydrogels were prepared in sterile 1.5 mL Eppendorf tubes under aseptic conditions. The mixture was homogenized using a vortex, and hydrogel formation was visually confirmed through the inverted flask method. The formulations were stored at -8°C until use (Gonçalves et al. 2021, Wahid et al. 2017).

Animals

Seventy-two male BALB/c mice (Mus musculus), aged between 8 to 12 weeks and weighing approximately 30 g, from the animal facility of the Institute of Tropical Pathology and Public Health at the Federal University of Goiás, where the study was conducted, were used. Two animals were housed per polypropylene cage, separated by an acrylic divider, lined with wood shavings, and the cages were changed twice a week. The animals received autoclaved water and food ad libitum. Lighting, temperature, noise intensity, and relative air humidity were those of the general environment.

This study was approved by the Animal Ethics Committee of the Federal University of Goiás - Protocol No. 094/19. The animals were treated following the principles and guidelines of the National Council for the Control of Animal Experimentation, prioritizing their well-being (Concea 2018). Euthanasia of the animals was performed at the end of each experimental day (7, 14, and 21 days after burn induction - DAI). The animals were individually placed in a chamber with a flow of carbon dioxide (CO₂).

Experimental Groups

The animals were randomly distributed into 4 groups: Control: Saline; SSD: 1.0% Silver Sulfadiazine Cream; HA: 1.0% Hyaluronic Acid Hydrogel; and HA+Ag: 1.0% Hyaluronic Acid Hydrogel + 0.5% Silver. All groups were monitored for 7, 14, and 21 days after burn induction (DAI), with 6 mice used per experimental day.

Burn Induction and Debridement

On day 0, the animals were weighed and anesthetized by intraperitoneal administration of 10% ketamine and 2% xylazine, with 0.1 mL/10g of anesthetic solution. After anesthesia, the dorsal region of the animal was shaved, and the area to be burned was disinfected using sterile gauze soaked in 70% alcohol. To perform the lesions, the animal was placed inside a PVC plastic cylinder with a 1x1 cm2 opening and sealed ends. Then, a partial-thickness thermal injury was induced by immersing the region in boiling water at 95°C for 7 seconds (Bernardes et al. 2022, Cardoso et al. 2016, Carvalho et al. 2022, Fantinati et al. 2016).

On the second day, after lesion induction, the animals were weighed and anesthetized again, using the same protocol as for burn induction, and underwent surgical debridement (tangential excision), as indicated in the treatment of burns (ISBI Practice Guidelines Committee 2016). For this procedure, a scalpel and scissors were used to remove necrosis, and the skin was gently detached, preserving the subcutaneous muscle of the panniculus carnosus (R.C.S. Ribeiro, unpublished data).

The animals subjected to the lesion were treated daily with a uniform layer of the products, sufficient to cover the wound bed. The mice were evaluated (for the presence of pain and/or suffering) by a veterinarian and received analgesic medication: tramadol hydrochloride (Grünenthal do Brasil Farmacêutica Ltda.) diluted in the drinking water during the first seven days after the burn procedure. The diet remained ad libitum (Bernardes et al. 2022, Cardoso et al. 2016, Carvalho et al. 2022, Fantinati et al. 2016).

Evaluations

Macroscopic and Morphometric Analysis

On the established experimental days (7, 14, and 21), the presence of necrosis/scab was macroscopically analyzed, identified semi-quantitatively, following the following criteria: absent (score 0); discrete (score 1 – up to 25% of the affected area); moderate (score 2 – between 26 and 50% of the affected area); and accentuated (score 3 – above 50% of the affected area) (Fantinati et al. 2016).

For morphometric analysis of wound contraction, the lesions were photographed using a camera mounted on a tripod at a constant distance of 11 cm, recorded on day zero (burn induction) and at the end of the experiment (euthanasia). The burn area, in square centimeters, was delineated using the ImageJ v1.53 software (National Institutes of Health, USA). To determine the degree of wound contraction, the following mathematical equation adapted from Moraes et al. (2013):

$$\text{Percentage of wound closure (\%)}=[({\text{Area T}}^0-{\text{Area T}}^{\text{day of euthanasia}})/{\text{Area T}}^0]\times 100$$

Where: T⁰ = day of injury induction and T^{day of euthanasia} = days 7, 14 and 21 after injury induction.

Microscopic Analysis

For microscopic evaluation, wound fragments were fixed in 10% buffered formalin (pH 7.2); subsequently, this material was processed for paraffin embedding. The paraffin blocks were placed in a microtome (Leica RM2255), serial sections of the material (4 μm) were obtained, and placed on glass slides. The slides were stained using Hematoxylin and Eosin (H&E) and Picrosirius red (PS) techniques.

For the analysis of general pathological processes, the slides stained with H&E were evaluated using a binocular microscope (Leica DM750), coupled with a camera (Leica ICC50 HD) for image recording. The presence of the following parameters was analyzed: necrosis/scab, hemorrhage, fibrin, polymorphonuclear and mononuclear cell infiltrate, angiogenesis, fibroblasts, granulation tissue, and re-epithelialization, emphasizing that the entire extent of the slides was evaluated by the same evaluator. These parameters were identified semi-quantitatively, following the previously mentioned criteria and described by Fantinati et al. (2016).

For collagen quantification, the slides stained with Picrosirius red were evaluated under a binocular microscope (Zeiss Axiostar Plus) and recorded with a digital camera (Sony Alpha Nex-3). Collagen fibers were evaluated under polarized light, with the entire extent of the slides visualized. For this analysis, the ImageJ v1.53 software (National Institutes of Health, USA) was used, which generates results in pixels.

RNA Extraction

Samples stored in a freezer (-80°C) were subsequently thawed and homogenized in 1 mL of Trizol reagent (INVITROGEN, CARLSBAD, CA, USA), and RNA was obtained following the manufacturer’s protocol. The High-Capacity cDNA Reverse Transcription Kit (LIFE TECHNOLOGIES, 40 CARLSBAD, CA, USA) was used to reverse transcribe two micrograms of total mRNA.

Real-Time PCR for Cytokine Expression

PCR reactions were performed in duplicate containing 3.0 µL of LuminoCt ReadyMix 2x (Cat No. L6669, Sigma-Aldrich), 0.25 µL of primers with probe, 0.25 µL of water, and 4.0 µL of cDNA (5 ng/µL). The negative control used 4.0 µL of water instead of cDNA. The cycling conditions were as follows: 95°C for 10 minutes and 45 cycles of 95°C for 5 seconds and 60°C for 30 seconds. The endogenous (or reference) genes Gapdh (Mm99999915_g1) and 18s (Mm03928990_g1) were acquired from Thermo Fisher, while the endogenous genes Hprt (Mm.PT.39a.22214828) and Rplp0 (Mm.PT.58.43894205) were acquired from IDT DNA Technologies. Primers for the target genes were acquired from IDT DNA Technologies: Il-1β (Mm.PT.58.41616450); Tnf-α (Mm.PT.58.12575861); Il-10 (Mm.PT.58.13531087); Tgfβ (Mm.PT.58.11254750); and Vegf-α (Mm.PT.58.14200306). The relative gene expression values were obtained by analyzing the results in the StepOne™ and StepOnePlus™ Software v2.3 (Applied Biosystems).

Statistical Analysis

Statistical analysis was performed using the GraphPad Prism software v10.0. All variables were tested for normal distribution and homogeneous variance. For the analyses, the parametric ANOVA test and Tukey›s post-test, and the non-parametric Kruskal-Wallis test and Dunn›s post-test were used. Observed differences were considered significant when p<0.05.

RESULTS

Macroscopic and Morphometric Evaluations

Through macroscopic analysis, it was observed that at 7 DAI, all groups presented necrosis/scab. At 14 DAI, only one lesion in the Control group presented necrosis/scab, while the other groups did not show necrosis/scab formation, and this pattern remained at 21 DAI (Figure 1a).

Figure 1
a) Macroscopic aspects of partial-thickness burns induced in BALB/c mice after 7, 14, and 21 days of treatment. b) Degree of burn area contraction. Data are expressed as mean ± SD. Statistical test: ANOVA and Tukey’s post-test (*p<0.05). c) Microscopic aspects of partial-thickness burns induced in BALB/c mice after 7, 14, and 21 days of treatment. Staining: Hematoxylin and eosin (H&E). Magnification: 10x. Scale: 200 μm. 7 DAI – HA/HA+Ag (Angiogenesis); HA/HA+Ag (Fibroblasts). Control – Saline; SSD – 1.0% Silver Sulfadiazine Cream; HA – 1.0% Hyaluronic Acid Hydrogel; HA+Ag – 1.0% Hyaluronic Acid Hydrogel + 0.5% Silver.

At 7 DAI, burn wound contraction was significantly greater in the HA+Ag group compared to the Control group (p<0.05), while the other treatments did not interfere with burn contraction (Figure 1b).

Microscopic Evaluation

At 7 DAI, a higher presence of angiogenesis and fibroblasts was observed in the HA and HA+Ag groups compared to the SSD group (p<0.05) (Table I and Figure 1c).

Table I
Microscopic analysis of general pathological processes in experimentally induced partial-thickness burns in BALB/c mice. *DAI – days after burn induction; n – number of animals; PMN – Polymorphonuclear cells; MN – Mononuclear cells; Control – Saline; SSD – 1.0% Silver Sulfadiazine Cream; HA – 1.0% Hyaluronic Acid Hydrogel; HA+Ag – 1.0% Hyaluronic Acid Hydrogel + 0.5% Silver; min – minimum value; max – maximum value. For statistical analysis, changes were considered: 0 – absent; 1 – discrete; 2 – moderate; 3 – accentuated. Statistical test: Kruskal-Wallis and Dunn’s post-test (*p<0.05).

Pathological processes	DAI	Control (n=18) Median (min–max)	SSD (n=18) Median (min–max)	HA (n=18) Median (min–max)	HA+Ag (n=18) Median (min–max)	p	Dunn’s post-test
	7	3 (1-3)	3 (2-3)	3 (3-3)	3 (3-3)	0.2816
Necrosis/scab	14	0 (0-2)	0 (0-1)	0 (0-1)	0 (0-1)	0.8441
	21	0 (0-0)	0 (0-0)	0 (0-0)	0 (0-0)	1.000
	7	1.5 (1-3)	1 (1-2)	1 (1-2)	1 (1-2)	0.4355
Hemorrhage	14	0 (0-2)	1 (0-1)	0 (0-0)	0 (0-1)	0.1201
	21	0 (0-0)	0 (0-0)	0 (0-0)	0 (0-0)	1.000
	7	2 (1-3)	3 (1-3)	1 (1-3)	2 (1-3)	0.2103
Fibrin	14	0 (0-1)	0 (0-0)	0 (0-0)	0 (0-1)	0.5083
	21	0 (0-0)	0 (0-0)	0 (0-0)	0 (0-0)	1.000
	7	1 (1-2)	1 (1-2)	1 (1-1)	1 (1-1)	0.1623
PMN	14	0 (0-1)	0 (0-1)	0 (0-0)	0 (0-0)	0.589
	21	0 (0-0)	0 (0-0)	0 (0-0)	0 (0-0)	1.000
	7	3 (3-3)	3 (3-3)	3 (2-3)	3 (2-3)	0.2924
MN	14	2 (2-3)	2 (2-3)	2 (1-3)	2 (2-2)	0.5872
	21	1 (0-1)	1 (1-2)	1 (1-2)	1 (1-1)	0.3169
	7	2 (2-3)	2 (1-2)	3 (3-3)	3 (3-3)	0.0003*	HA > SSD; HA+Ag > SSD
Angiogenesis	14	2.5 (1-3)	2 (2-3)	2 (1-3)	2 (1-3)	0.7757
	21	1 (1-1)	1.5 (1-2)	2 (1-2)	2 (1-3)	0.0671
	7	2 (2-3)	2 (1-2)	3 (2-3)	3 (2-3)	0.0201*	HA > SSD; HA+Ag > SSD
Fibroblasts	14	2 (2-3)	2.5 (2-3)	2 (1-2)	2 (2-3)	0.1578
	21	1 (0-1)	1 (1-2)	1.5 (1-3)	1 (1-1)	0.0052
	7	2 (2-3)	2.5 (2-3)	3 (3-3)	3 (2-3)	0.1098
Granulation tissue	14	2 (1-3)	2 (2-3)	1.5 (0-2)	2 (1-2)	0.219
	7	1 (1-1)	1 (1-1)	1 (1-2)	1 (1-1)	0.1147
Re-epithelialization	14	3 (3-3)	3 (3-3)	3 (3-3)	3 (3-3)	1.000
	21	3 (3-3)	3 (3-3)	3 (3-3)	3 (3-3)	1.000

Collagen Quantification

There were no significant differences between the analyzed groups regarding collagen fiber deposition at 7, 14, and 21 DAI (Figure 2a and b).

Figure 2
a) Collagen fiber deposition in partial-thickness burns induced in mice after 7, 14, and 21 days of treatment. Staining: Picrosirius red. Magnification: 20x. Scale: 100 μm. b) Quantitative analysis of collagen fiber deposition in partial-thickness burns induced in mice after 7, 14, and 21 days of treatment. Data are expressed as mean ± SD. Statistical test: ANOVA and Tukey’s post-test (*p<0.05). Control – Saline; SSD – 1.0% Silver Sulfadiazine Cream; HA – 1.0% Hyaluronic Acid Hydrogel; HA+Ag – 1.0% Hyaluronic Acid Hydrogel + 0.5% Silver.

Real-Time PCR

The main findings of the macroscopic and microscopic analyses were concentrated at 7 DAI; therefore, the gene expressions of IL-1β, TNF-α, IL-10, TGF-β, and VEGF were evaluated during this experimental day. Higher gene expression of IL-1β and TNF-α was observed in the Control group compared to the SSD and HA+Ag groups (p<0.01) (p<0.05) (Figure 3a and b). Higher expression of IL-10 was also observed in the Control group compared to the SSD group (p<0.05) (Figure 3c).

Figure 3
Gene expression levels of IL-1β, TNF-α, IL-10, TGF-β, and VEGF in partial-thickness burns induced in BALB/c mice after 7 days of treatment. Statistical test: ANOVA and Tukey’s post-test (*p<0.05) (**p<0.01). Control – Saline; SSD – 1.0% Silver Sulfadiazine Cream; HA – 1.0% Hyaluronic Acid Hydrogel; HA+Ag – 1.0% Hyaluronic Acid Hydrogel + 0.5% Silver.

DISCUSSION

The present study evaluated the healing process of partial-thickness burns induced in mice after treatment with the HA+Ag hydrogel. It is important to note that the findings of the healing study refer to the animal’s response to the treatments performed during the 21-day experimental period.

HA is one of the main non-protein components of the extracellular matrix (ECM) of the skin, involved in the inflammatory response, cell migration, angiogenesis, and re-epithelialization, important in the phases of wound healing. Its intrinsic properties, such as biocompatibility, biodegradability, and hydrophilic nature, make it an alternative for the production of new treatments (Cortes et al. 2020, Ding et al. 2022).

At 7 DAI, it was observed that treatment with the hydrogels induced greater angiogenesis, presence of fibroblasts, and contraction, findings indicative of accelerated healing. Fibroblasts differentiate into myofibroblasts, which are cells found around the new ECM, joined together generating force, as they express α-actin, thus aiding in the wound contraction process. HA promotes fibroblast proliferation and chemotaxis and their subsequent differentiation into myofibroblasts in the injured area (Graça et al. 2020, Neuman et al. 2015, Baum & Arpey 2005, Balbino et al. 2005). Despite this finding, no significant increase in collagen fibers was observed after treatment with the hydrogels, which could be attributed to the possible increase in matrix metalloproteinases (MMPs), enzymes involved in the degradation of ECM components, such as collagen (Gill & Parks 2008, Raziyeva et al. 2021), however, it is important to note that this parameter was not analyzed in the present study.

HA has also been associated with the stimulation of angiogenesis, playing a role in cell mobility and the proliferation of endothelial cells lining blood vessels, through signaling pathways mediated by hyaluronan-mediated motility receptors (RHAMM), which act as receptors for HA. Studies indicate that the interaction between RHAMM and HA may play a role in the regulation of angiogenesis, contributing to processes such as endothelial cell migration and capillary formation (Graça et al. 2020, Messam et al. 2021, Sohr & Engeland 2008). The hyaluronic acid used in this study had a medium molecular weight (Mw 170,000–250,000 kDa). Different molecular weights are known to act at distinct stages of wound healing (Dicker et al. 2014, Kawano et al. 2021). In the present study, the HA and HA+Ag groups exhibited increased fibroblast presence and angiogenesis, important components in the formation of granulation tissue, as well as greater wound contraction compared with the control group. These findings suggest that the HA used in this study exerted its predominant effects during the early phase of the healing process.

A study conducted by Matsumoto et al. (2009) investigated the effect of HA-based spongy sheets and observed that after 7 days, the sheets promoted greater angiogenesis in excisional wounds induced in rats. Another study (Taskan et al. 2021) evaluated the effect of an HA-based gel in full-thickness wounds induced in rats and observed a higher number of fibroblasts and fewer inflammatory cells at 7 days. These results coincide with the findings of the present study.

Still at 7 DAI, the Control group showed higher expression of the inflammatory mediators IL-1β, TNF-α, and IL-10. Burns trigger an initial inflammatory response, and it is important to highlight the need for a balance in the production of these mediators (Hur et al. 2015). Despite the presence of pro-inflammatory mediators such as IL-1β and TNF-α, the presence of IL-10 indicates regulation and modulation of the inflammatory response. This effect was observed only in the Control group at 7 DAI; in the other treated groups, the evaluated genes were expressed but less significantly compared to the Control group. In the treated groups, the overall reduction in pro-inflammatory and anti-inflammatory cytokines suggests a balanced modulation of the inflammatory response. This balance may have prevented prolonged inflammation, which can delay tissue repair, while avoiding excessive suppression that could impair the early defense phase. A controlled inflammatory environment is known to favor a faster transition to the proliferative phase (Rodrigues et al. 2019).

The association of hyaluronic acid (HA) with silver offers advantages over formulations already available, such as silver sulfadiazine (SSD), which is considered the standard treatment in burn care centers. While SSD is effective in controlling microbial growth, its prolonged use can delay re-epithelialization due to cytotoxicity to keratinocytes and fibroblasts (Oaks & Cindass 2023). HA is one of the main components of the extracellular matrix, promoting hydration, cell migration, angiogenesis, and granulation tissue formation (Cortes et al. 2020, Dalmedico et al. 2016, Kawano et al. 2021). By associating silver with HA, the aim is to combine the broad-spectrum antimicrobial effect of silver with the healing properties of HA, keeping the wound moist, reducing healing time and minimizing cytotoxic effects, since the release of silver through polymeric hydrogels occurs more gradually (Gonçalves et al. 2021). In the present study, this combination promoted greater wound contraction, angiogenesis, and fibroblast presence already in the early phase of healing, with performance superior to the control, suggesting that the HA+Ag formulation may be promising, especially in cases where the risk of infection is high.

The association between hyaluronic acid (HA) and silver ions (Ag⁺) in the present study is presumed to occur through electrostatic interactions, primarily involving the negatively charged carboxylate groups of HA and the positively charged silver ions (Dalmedico et al. 2016, Dicker et al. 2014, Wilkinson et al. 2011). It is important to highlight that the hydrogel containing hyaluronic acid and silver used in this study was prepared by direct dispersion of the powdered components in sterile deionized water, following the approach described by Gonçalves et al. (2021), without the use of chemical cross-linking agents or purification steps. As no physicochemical analyses were performed to confirm the degree of complexation between the compounds, this represents a limitation of the study. However, the formulation exhibited appropriate consistency and adherence when applied to the wounds, and the biological responses observed suggest a potential synergistic effect between HA and Ag⁺. Future studies are needed to investigate the physicochemical properties and interaction mechanisms of this combination in more detail.

CONCLUSIONS

The hyaluronic acid-based hydrogels, with or without silver, promoted enhanced wound contraction, increased fibroblast presence, and angiogenesis at 7 days, indicating an acceleration of the early healing process. These effects were particularly evident in the HA+Ag group. Inflammatory mediator expression was higher in the control group, suggesting modulation of the inflammatory response in the treated groups. Therefore, the hydrogels demonstrated efficacy during the initial stage of partial-thickness burn healing and may represent a promising therapeutic alternative.

Acknowledgements

We would like to thank the Histotechnology, Morphometry, Innovation, and Experimental Pathology laboratories, coordinated by Ruy de Souza Lino Júnior, and the Anaerobes, Phenotyping, and Molecular Biology Laboratory, coordinated by Carla Afonso da Silva at the Institute of Tropical Pathology and Public Health of the Federal University of Goiás, where the hydrogels were prepared and histopathological analyses were performed. We would also like to thank the Laboratory of Cell Signaling, coordinated by Licio Augusto Velloso, located at the Obesity and Comorbidities Research Center of the University of Campinas, where molecular biology analyses were performed. This work was supported by the Coordination for the Improvement of Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) grant No. 88887.819590/2023-00.

• Data availability
  The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Edited by

• Handling editor
  Marcello Iacomini

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Publication Dates

• Publication in this collection
  03 Aug 2026
• Date of issue
  2026

History

• Received
  10 June 2025
• Accepted
  27 Dec 2025