Bacterial biosurfactant-reinforced chitooligosaccharide/polyvinyl alcohol hydrogels accelerate methicillin-resistant Staphylococcus aureus-infected wound healing by attenuating its virulence factors
Geum-Jae Jeong, Dong-Joo Park, Ju-Hong Kang, Se-Chang Kim, Yu-Jin Ahn, Kyung-Jin Cho, Fazlurrahman Khan, Won-Kyo Jung, Young-Mog Kim
TL;DR
A new hydrogel made with a bacterial biosurfactant helps heal MRSA-infected wounds by reducing the bacteria's harmful effects and promoting tissue repair.
Contribution
A novel hydrogel combining a biosurfactant from Bacillus rugosus with chitooligosaccharide and polyvinyl alcohol is developed for MRSA-infected wound healing.
Findings
The hydrogel significantly enhanced cell proliferation, migration, and extracellular matrix deposition in wound healing.
It exhibited strong antibacterial, antibiofilm, and anti-virulence effects against MRSA.
In vivo tests showed accelerated wound healing and reduced MRSA burden with the hydrogel.
Abstract
Methicillin-resistant Staphylococcus aureus (MRSA) causes widespread infections and poses serious public health concerns. Its high level of resistance to multiple antibiotics has garnered growing interest in identifying and applying novel antibacterial compounds derived from natural sources. In this study, we purified a biosurfactant (BS) from Bacillus rugosus HH2 to develop a natural antibacterial agent. This agent was then reinforced with chitooligosaccharide (COS) and polyvinyl alcohol (PVA) to create a hydrogel that promoted healing in MRSA-infected wounds. The COS/PVA/BS hydrogel was readily fabricated via the freeze-thaw method and demonstrated excellent mechanical strength, biological activity, and biocompatibility. In vitro assays confirmed that the hydrogel significantly enhanced the proliferation, migration, angiogenesis, and extracellular matrix deposition of fibroblasts,…
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Taxonomy
TopicsWound Healing and Treatments · Bacterial biofilms and quorum sensing · Nanocomposite Films for Food Packaging
Introduction
1
Staphylococcus aureus is one of the principal pathogens responsible for infection-related mortality worldwide, accounting for approximately 1 million deaths annually [1]. It is widely recognized as a significant pathogen in community settings, where it frequently causes mild-to-moderate skin infections [2]. The severity of S. aureus infection can be attributed to a range of virulence factors and mechanisms. These include biofilm formation, which facilitates tissue colonization [3], immune evasion strategies, which enable the bacterium to bypass innate host defenses [4], and various toxic proteins that contribute to host cell and tissue degradation [5]. Methicillin-resistant S. aureus (MRSA) is a highly problematic multidrug-resistant strain that severely limits treatment options and poses a significant public health threat [1]. Therefore, alternative therapeutic strategies are urgently required to treat antibiotic-resistant infections. Novel antimicrobial agents, derived from natural sources, have emerged as promising alternatives [6].
Biosurfactants (BS), which are microbial products with surface-active properties, have garnered significant attention owing to their antimicrobial effects [7]. BS derived from Bacillus rugosus HH2 exhibits potent antibacterial activity against S. aureus, particularly MRSA [8]. B. rugosus HH2 BS has surfactin as the major component and has demonstrated a minimum inhibitory concentration (MIC) of 128–256 µg/ml against methicillin-sensitive S. aureus (MSSA) and MRSA. Transcriptomic analysis revealed that BS downregulates the genes involved in quorum sensing (QS) and bacterial attachment in MRSA, offering molecular insights into its potential for infection control. In addition, in silico docking suggested that BS has a high affinity for essential MRSA resistance-related proteins such as PBP2a and DNA gyrase. These results indicate that BS is a promising naturally derived antibacterial compound capable of addressing MRSA-related antibiotic resistance. However, natural antimicrobial agents often face challenges in clinical application, including low bioavailability and stability. To address these limitations, drug delivery systems that ensure the targeted and sustained release of natural antimicrobials at the site of infection should be utilized.
Hydrogels interact physicochemically with loaded drugs, protecting unstable drugs from degradation and enabling spatial and temporal control of drug release [9]. These multifunctional characteristics have been widely investigated in the design of hydrogel-based therapeutic platforms with enhanced clinical applicability [10]. Recent advances have revealed microenvironment-responsive hydrogel systems that release antibacterial agents in response to infection-specific cues, thereby accelerating chronic wound healing [11]. Given these properties, hydrogel-based drug delivery systems offer therapeutic benefits and are widely used in clinical applications. Chitosan (CS) is a hydrogel biomaterial with excellent physicochemical properties, including biocompatibility, biodegradability, antimicrobial, and antibiofilm activity [12,13]. However, unmodified CS has poor solubility under neutral conditions and low mechanical strength, limiting its applicability in biological environments [14]. To overcome these limitations, modifying the degree of deacetylation and polymerization of CS or converting it into low-molecular-weight chitooligosaccharides (COS) could enhance its potential for biomedical applications [15]. COS exert strong antimicrobial effects by neutralizing the negative charge on bacterial surfaces and altering membrane permeability [16]. Additionally, COS stimulates the expression of transforming growth factor-β (TGF-β), which promotes cell proliferation and migration, thereby facilitating rapid re-epithelialization and wound healing [17]. Polyvinyl alcohol (PVA) is one of the most commonly used hydrogel materials owing to its advantageous properties such as nontoxicity, biocompatibility, thermal stability, hydrophilicity, and ease of processing [18]. PVA can enhance mechanical properties by forming a double network with natural polymers through uncomplicated physical crosslinking processes, such as freeze-thaw cycles, eliminating the need for chemical crosslinkers [12].
Recent advances in wound healing biomaterials have emphasized the importance of multifunctionality, combining antibacterial and antibiofilm activities with the ability to promote angiogenesis, regulate inflammation, and remodel the extracellular matrix (ECM). This study presents the first bacterial BS-reinforced COS/PVA hydrogel dressing designed to accelerate the healing of MRSA-infected wounds. The dual-network structure of COS and PVA provided a hydrogel with excellent mechanical properties, whereas the reinforcement of BS with COS/PVA conferred antibacterial and antibiofilm properties. Furthermore, the hydrogel promoted fibroblast, keratinocyte, and endothelial cell responses, including proliferation, migration, ECM deposition, and tube formation in vitro. The COS/PVA/BS hydrogel effectively inhibited key virulence factors associated with MRSA wound infections, including hemolysin, lipase, and staphyloxanthin, as well as factors involved in biofilm formation at both the phenotypic and genotypic levels. Finally, an in vivo MRSA-infected wound model confirmed the hydrogel’s antibacterial properties, immunomodulation, and efficacy in promoting neovascularization and skin regeneration. Overall, this study introduces a multifunctional hydrogel with antibacterial, antibiofilm, anti-virulence, angiogenic, and immunomodulatory effects. These findings underscore the translational potential of the COS/PVA/BS hydrogels as novel therapeutic platforms for treating MRSA-infected wounds.
Materials and methods
2
Materials
2.1
Chloroform, Congo Red, COS (M_n_ = 5,000, >90% deacetylated), crystal violet solution, disodium hydrogen phosphate, dimethyl sulfoxide (DMSO), ethanol, hydrochloric acid, isopropyl alcohol, methanol, p-nitrophenyl palmitate, PVA (M_w_ = 89,000–98,000, >99% hydrolyzed), sodium carbonate, sodium deoxycholate, sodium hydroxide, and Triton X-100 were purchased from Sigma-Aldrich (MO, USA). Tryptic soy agar (TSA) and tryptic soy broth (TSB) were acquired from Difco (MI, USA). Endothelial Cell Growth Kit-BBE, Escherichia coli (25922), MSSA (6538), and vascular cell basal medium (VCBM) were purchased from the American Type Culture Collection (ATCC；VA, USA). 4′,6-diamidino-2-phenylindole (DAPI), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), bovine serum albumin (BSA), Dulbecco’s minimum Eagle’s medium (DMEM), fetal bovine serum (FBS), fluorescein diacetate (FDA), formalin, paraffin, phosphate-buffered saline (PBS), penicillin, propidium iodide (PI), streptomycin, SYTO 9, and trypsin (250 U/mg) were purchased from Thermo Fisher Scientific (MA, USA). Formaldehyde and glutaraldehyde were purchased from Wako Chemicals (Osaka, Japan). Gum arabic was obtained from Daejung Chemicals (Seoul, South Korea). Sheep blood was obtained from MB Cells (Seoul, South Korea). Tween 20 was purchased from BioBasic (Markham, Canada). MRSA (40510) was purchased from the Korean Culture Center of Microorganisms (KCCM) (Seoul, South Korea). Pseudomonas aeruginosa (1637) was obtained from the Korean Collection for Type Cultures (Jeongeup, South Korea). Analytical grade solvents were used in this study.
Extraction and partial purification of BS
2.2
Biosurfactants (BS) was extracted and partially purified as described in our previous studies [8,19]. B. rugosus HH2 was grown in TSB broth at 37 °C with shaking at 150 rpm for 120 h. The culture broth was centrifuged at 12,000 rpm for 20 min at 4 °C, and then filtered (0.2 µm) to obtain a supernatant free of cellular debris. Lipid precipitation was induced by adjusting the pH of the supernatant to 2 with 6 N hydrochloric acid, followed by overnight incubation at 4 °C. The lipid portion was recovered through high-speed centrifugation (12,000 rpm, 20 min, and 4 °C) and resuspended in deionized water. After adjusting the solution to pH 7 with 1 M sodium hydroxide, a chloroform-methanol mixture (2:1, v/v) was slowly added until the final solvent ratio of chloroform:methanol:water was 8:4:3. Following prolonged extraction, the upper organic layer was gently collected and the solvent was subsequently evaporated under reduced pressure using a rotary evaporator (Hei-VAP Expert, Heidolph, Schwabach, Germany). The final product was washed with deionized water and freeze-dried to obtain a BS concentration of 0.1035 g/l.
The anti-S. aureus activity of BS against MSSA and MRSA was evaluated by determining the MIC, minimum biofilm inhibitory concentration (MBIC), growth kinetics, time-kill kinetics, and using confocal laser scanning microscopy (CLSM). Detailed procedures are provided in the Materials and Methods section of the Supplementary Material.
Preparation of hydrogel
2.3
Four types of hydrogels were developed using the freeze-thaw method (Table S1). First, 10 wt% PVA and 2 wt% COS solutions were prepared in distilled water under mechanical stirring at 110 °C and room temperature for 2 h, respectively. To prepare COS/PVA/BSI, the PVA and COS solutions were mixed in a 5:5 ratio, which is the optimal ratio for gel formation that maximizes COS utilization (Fig. S1). Subsequently, 0.05 wt% BS was added, and the mixture was stirred for 2 h. After complete dissolution, the solution was poured into a mold, then stored at −18 °C for 18 h, followed by thawing at room temperature for 6 h. This freeze-thaw process was performed three times to induce physical crosslinking of the hydrogel. Other types of hydrogels were prepared using the same process but with different components. The obtained hydrogels were preserved at −4 °C or subjected to lyophilization for subsequent analyses.
Physicochemical characterization of hydrogels
2.4
Field-emission scanning electron microscopy
2.4.1
The microstructures of the hydrogels were analyzed using field-emission scanning electron microscopy (FE-SEM; JSM-IT800SHL, JEOL, Tokyo, Japan). The freeze-dried samples were vertically sectioned and sputter-coated with gold. Observations were conducted at an accelerating voltage of 3 kV, and images were acquired at a magnification of 250×. The pore diameters of the hydrogels were analyzed using the ImageJ software based on the FE-SEM images.
Fourier-transform infrared spectroscopy
2.4.2
To identify the functional groups of the pure material and hydrogels, Fourier-transform infrared spectroscopy (FTIR) was performed in the attenuated total reflectance mode using an FT-4100 spectrometer (Jasco, Tokyo, Japan), generating spectra in the 650–4000 cm^−1^ range.
X-ray diffraction
2.4.3
The phase structure and crystalline types of the hydrogels were characterized using X-ray diffraction (XRD) patterns obtained with an UltimaIV X-ray diffractometer (Rigaku, Tokyo, Japan) at a diffraction angle (2θ) range of 10°–50° with a scanning rate of 2°/min.
Determination of hydrogel swelling
2.4.4
To study the swelling behavior of the hydrogel, freeze-dried samples were placed in PBS (pH 7.4) and incubated at 37 °C. At specific time points, the samples were removed, gently blotted to remove the surface moisture, and weighed again. The swelling ratio was determined using the formula:
where Wt and W0 denote the hydrogel’s weight at a specific time point and its initial dry weight, respectively.
Determination of hydrogel degradation
2.4.5
To investigate the degradation behavior of the hydrogel, freeze-dried hydrogels were submerged in PBS (pH 7.4) and maintained at 37 °C. At specific time intervals, the hydrogels were removed, rinsed with deionized water, and freeze-dried. The degradation rate was calculated using the following formula:
where Wt and W0 denote the weight of the hydrogel at a specific time point and its initial dry weight, respectively.
Determination of hydrogel water retention
2.4.6
To evaluate the water retention behavior of the hydrogels, lyophilized hydrogels were immersed in PBS (pH 7.4) and maintained at 37 °C until the swelling equilibrium was achieved. At defined time intervals, the swollen hydrogels kept at 37 °C in an open flask were weighed to monitor changes. The water retention rate was calculated using the following formula:
where Wt and W0 denote the weight of the hydrogel at a specific time point and the initial weight of the swollen hydrogel, respectively.
Thermogravimetric analysis
2.4.7
The thermal properties of the hydrogels were investigated via thermogravimetric analysis (TGA) using a simultaneous thermal analyzer (SDT650, Waters, MA, USA). The analysis was conducted with a nitrogen purge and a temperature ramp of 10 °C/min, spanning a temperature interval of 50–650 °C.
Release profile
2.4.8
To evaluate the release of BS from the hydrogels, COS/PVA/BSI and COS/PVA/BSII were immersed in PBS adjusted to pH 7.4, and incubated at 37 °C with shaking at 90 rpm. Aliquots of the supernatant were collected at predetermined intervals, and an equivalent volume of fresh PBS was added to the original solution. The collected samples were analyzed spectrophotometrically at 230 nm using a microplate reader (Epoch 2, BioTek, VT, USA) [8]. The BS content in each sample was quantified using a standard concentration curve (Fig. S2). Time-dependent cumulative release data from the BS were used to develop release profiles.
Rheological analysis
2.4.9
The rheological properties of the hydrogels were analyzed at room temperature using a Discovery HR-2 Hybrid Rheometer (TA Instruments, DE, USA) with 8 mm parallel plate geometry and a 1 mm gap [20]. Strain scanning was performed to determine the viscoelastic behavior of the hydrogels by measuring the storage modulus (Gʹ) and loss modulus (Gʹʹ) at a constant frequency of 10 rad/s over a strain range of 0.01–1,000%. Frequency scanning was conducted to evaluate the dependence of Gʹ and Gʹʹ on frequency, with measurements taken over the range of 0.1–100 rad/s under a constant strain of 0.1%.
Biocompatibility of hydrogels
2.5
Cell culture
2.5.1
Human dermal fibroblasts (HDF) and human keratinocytes (HaCaT) were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin, whereas human umbilical vein endothelial cells (HUVEC) were cultured in VCBM using an Endothelial Cell Growth Kit-BBE. Cells were seeded at 5 × 10^4^ cells/ml, and the cultures were incubated at 37 °C under humidified conditions with 5% CO_2_. To prepare hydrogel extracts for in vitro analysis, the sterilized hydrogels were soaked in culture medium and maintained at 37 °C for 24 h.
Cell viability assay
2.5.2
Cell viability was determined using a standard MTT assay [21]. The cells were seeded in 48-well plates and treated with the hydrogel extracts for 24 h. After gently removing the medium, MTT reagent was added to each well, and the cells were incubated at 37 °C for 4 h. Subsequently, the supernatant was removed, and DMSO was added to solubilize the formazan crystals. The optical density (OD) at 540 nm was determined using a microplate reader. Cell viability was calculated using the following formula:
where ODsample, ODcontrol and ODblank represent the OD values of the sample, blank and negative control, respectively.
Live/dead staining was performed to evaluate cell viability further. Following 24 h incubation with the hydrogel extracts, the culture medium was gently aspirated, and the cells were stained with FDA and PI for 5 min. After washing with PBS, the cells were observed under a fluorescence microscope (Axio Observer A1, Zeiss, Jena, Germany).
Hemolysis
2.5.3
The hemocompatibility of the hydrogels was assessed using a hemolysis assay based on a previously described method [12]. A 10-fold dilution of fresh sheep blood was prepared to create the blood dilution. Each hydrogel sample was introduced into a prediluted blood suspension, followed by incubation at 37 °C for 1 h. Following incubation, the blood samples were centrifuged at 1,000 rpm for 10 min at 4 °C to separate the supernatant, and OD was then recorded at 540 nm using a microplate reader. Triton X-100 and PBS served as positive and negative controls, respectively. The hemolysis rate was calculated using the following formula:
where ODsample, ODpositive and ODnegative represent the OD values of the sample, positive control and negative control, respectively.
In vitro cellular functional assays
2.6
Cell migration assay
2.6.1
Cell migration was evaluated using a scratch assay. Cells were seeded in 12-well plates and incubated until they reached confluency. A wound scratch model was then created by scraping the cell monolayer with a sterile 200 µ pipette tip, followed by washing with PBS to remove debris. After treatment with the hydrogel extracts, cell migration was observed using an optical microscope, and images were captured after 24 h. The migration area was quantified using the ImageJ software.
In vitro tube formation assay
2.6.2
The 50 µL of Matrigel Basement Membrane Matrix (BD Biosciences, CA, USA) was added in 96-well plate and formed the basal membrane matrix at 37 °C. HUVEC were seeded with different hydrogel extracts and incubated for 6 h. Tube formation images were captured using an optical microscope. The ImageJ software was used to analyze the tube lengths and number of meshes.
Immunofluorescence staining
2.6.3
Immunofluorescence staining was performed to demonstrate ECM expression in HDF. The samples were then rinsed with PBS and fixed with 4% paraformaldehyde for 10 min at room temperature. After permeabilization with 0.1% Triton X-100 in PBS for 5 min, non-specific binding was blocked with 3% BSA in PBS containing 0.05% Tween 20 (PBS-T) for 1 h at room temperature. The following primary antibodies type I collagen (COLI) and α-smooth muscle actin (α-SMA) diluted in blocking solution were applied and incubated overnight at 4 °C. The cells were then washed thrice with PBS and incubated with goat anti-rabbit IgG Alexa Fluor 488 and goat anti-mouse IgG Alexa Fluor 562 diluted in blocking solution for 2 h in the dark. Nuclei were counterstained with DAPI for 10 min and images were obtained using a CLSM (LSM 900, Zeiss, Jena, Germany).
Sirius Red assay
2.6.4
HDF were fixed with 70% ice-cold ethanol for the Sirius Red assay. Deposited collagen was stained by incubating with 0.1% Sirius Red solution in saturated picric acid for 1 h, followed by washing with 0.01 N hydrochloric acid to remove residual unbound dye. The matrix was dissolved by gentle agitation in a 0.1 N sodium hydroxide solution to decompose the collagen dye complex, and OD was measured at 540 nm using a microplate reader.
In vitro anti-S. aureus activities of hydrogels
2.7
Bacteriostatic effect
2.7.1
The bacteriostatic effects of the hydrogels were confirmed by monitoring the growth curves of MSSA and MRSA after hydrogel treatment [22]. MSSA ATCC 6538 and MRSA KCCM 40510 were incubated in TSB at 37 °C with agitation at 150 rpm for 10 h. The cultures were diluted in TSB to adjust the cell density to 10^5^ colony-forming units (CFU)/ml. The hydrogel (Ф12 mm × 2 mm) was immersed in 5 ml diluted culture. The mixture was incubated at 37 °C and 150 rpm for 8 h. Aliquots (100 µl) of the bacterial culture were taken at 2 h intervals up to 8 h. The growth rate was determined by measuring OD at 600 nm using a microplate reader. The control group was cultured in a medium without the hydrogel. The bacteriostatic effect of the hydrogel was also evaluated against other clinically relevant pathogens such as E. coli and P. aeruginosa.
Bactericidal effect
2.7.2
The bactericidal effects of the hydrogels were evaluated by determining the CFU of MSSA and MRSA after hydrogel treatment [23]. MSSA ATCC 6538 and MRSA KCCM 40510 suspensions were prepared by diluting the cultures to 10^7^ CFU/ml in PBS. Subsequently, 5 ml diluted culture was treated with the hydrogel (Ф12 mm × 2 mm) and incubated at 37 °C with shaking at 150 rpm for 8 h. A 100 µl sample was collected, serially diluted in PBS, inoculated onto TSA plates, and cultured at 37 °C for 24 h. After incubation, the CFUs were quantified and presented as log CFU/ml. The control group was cultured in a medium without the hydrogel. The bactericidal effect of the hydrogel was also evaluated against other clinically relevant pathogens such as E. coli and P. aeruginosa.
FE-SEM
2.7.3
The bacterial morphology after hydrogel treatment was observed using FE-SEM [24]. Hydrogels (Ф12 mm × 2 mm) were incubated with 10^5^ CFU/ml MSSA ATCC 6538 and MRSA KCCM 40510 suspensions at 37 °C for 24 h. After incubation, the bacterial cultures were collected and plated onto 0.2 µm pore-sized nylon filters. The bacteria were fixed for 2 h at 4 °C with 2.5% glutaraldehyde and 2% formaldehyde, and then rinsed three times with PBS. The samples were then dehydrated using a graded ethanol series (30%, 50%, 70%, 80%, 95% and 100%) before critical-point drying. Following fixation and dehydration, the bacterial samples were observed using FE-SEM.
Antibiofilm effect
2.7.4
The antibiofilm effects of the hydrogels were evaluated using a crystal violet microtiter plate assay with slight modifications [25]. MSSA ATCC 6538 and MRSA KCCM 40510 cultures were grown overnight in TSB and diluted to approximately 10^6^ CFU/ml. Aliquots (200 µl) of the diluted bacterial suspension were inoculated into 96-well microplates and incubated at 37 °C for 24 h under static conditions to allow biofilm formation. After incubation, 200 µl the hydrogel extract was added to each well, while wells containing bacterial cultures with fresh medium served as the control group. The plates were further incubated at 37 °C for 8 h to allow the hydrogel extract to interact with the preformed biofilms. Subsequently, the supernatant was carefully removed, and the wells were gently washed with PBS to remove any remaining planktonic cells. The attached biofilms were stained with a 0.1% crystal violet solution for 20 min. The retained dye was solubilized in 95% ethanol, and the biofilm biomass was quantified by measuring OD at 570 nm using a microplate reader.
CLSM analysis was performed to further evaluate antibiofilm effects. Biofilm cells treated with hydrogel extract were stained with SYTO 9 and PI for 15 min. After washing with PBS, biofilm cells were observed under a CLSM.
Anti-virulence effect
2.7.5
The anti-virulence effects of the hydrogels on S. aureus were evaluated by analyzing their potential to reduce hemolysis, extracellular enzyme (lipase) output, staphyloxanthin pigment production, and slime layer development.
The inhibition of hemolysis was examined using sheep blood, following previously reported methods [26]. A 10-fold dilution of sheep blood was prepared in PBS. Cultures of MSSA ATCC 6538 and MRSA KCCM 40510 were prepared at a concentration of 10^5^ CFU/ml in TSB and exposed to the hydrogel (Ф12 mm × 2 mm), followed by incubation at 37 °C with shaking at 150 rpm for 24 h. After incubation, a 300 µl aliquot of bacterial culture was combined with 1 ml diluted sheep blood solution and incubated at 37 °C with agitation at 150 rpm for 1 h. The supernatant was carefully collected, and OD was recorded at 540 nm using a microplate reader. Cell cultures without the hydrogel served as controls.
To quantify the effect of the hydrogels on extracellular lipase production, a previously described method was followed [27]. MSSA ATCC 6538 and MRSA KCCM 40510 (10^5^ CFU/ml) cultures were exposed to hydrogel (Ф12 mm × 2 mm) and cultured at 37 °C with continuous shaking at 150 rpm for 24 h. The cell cultures were then centrifuged at 10,000 rpm for 10 min at 4 °C, and the supernatant was collected. A volume of 0.1 mL of the supernatant was combined with 0.9 mL of a substrate buffer composed of 10% (v/v) buffer A (3 mg/ml p-nitrophenyl palmitate dissolved in isopropyl alcohol) and 90% (v/v) buffer B (1 mg/mL gum arabic and 2 mg/mL sodium deoxycholate in 50 mm disodium hydrogen phosphate). The mixture was then incubated at 40 °C for 30 min in the dark. Afterward, 0.1 ml of 1 M sodium carbonate was added to stop the reaction. Following centrifugation at 10,000 rpm for 10 min at 4 °C, the supernatant was collected and analyzed spectrophotometrically at 405 nm using a microplate reader. The control group consisted of cultures without the hydrogel.
The ability of hydrogels to reduce staphyloxanthin production was assessed according to the method outlined in a previous study [28]. MSSA ATCC 6538 and MRSA KCCM 40510 cultures (10^5^ CFU/ml) were grown in TSB and incubated with the hydrogel (Ф12 mm × 2 mm) at 37 °C and 150 rpm for 24 h. After incubation, the cell cultures were centrifuged at 10,000 rpm for 10 min at 4 °C to collect the bacterial cells, which were then washed twice with PBS. The bacterial cells were suspended in methanol and stirred at 40 °C for 2 h to extract staphyloxanthin pigment. Following methanol extraction, the mixture was centrifuged at 10,000 rpm for 10 min at 4 °C to separate the components, and the resulting supernatant was obtained. OD was measured at 450 nm using a microplate reader. Cultures without hydrogels served as controls.
The ability of hydrogels to attenuate slime formation by S. aureus was assessed using the Congo Red method as previously described [29]. Hydrogel samples (Ф12 mm × 2 mm) were added to bacterial suspensions of MSSA ATCC 6538 and MRSA KCCM 40510 (10^5^ CFU/ml), and the mixtures were kept at 37 °C and 150 rpm for 24 h. Subsequently, the cell cultures were plated on TSA plates containing 0.8 g/l Congo Red and incubated at 37 °C for 24 h before imaging. Cell cultures without the hydrogel served as controls.
Gene expression changes induced by hydrogels
2.7.6
The expression of virulence- and resistance-related genes in MSSA and MRSA following hydrogel treatment was analyzed using real-time polymerase chain reaction (RT-PCR) [27]. Cultures of MSSA ATCC 6538 and MRSA KCCM 40510, prepared to 10^6^ CFU/ml, were exposed to hydrogels (Ф12 mm × 2 mm) and incubated at 37 °C with continuous shaking at 150 rpm for 24 h. Following incubation, cell pellets were obtained through centrifugation at 10,000 rpm for 10 min at 4 °C. Total mRNA was isolated using RiboEx reagent (GeneAll Biotechnology, Seoul, South Korea) following the manufacturer’s instructions, and its concentration and purity were assessed using a Nabi UV/Vis Nano Spectrophotometer (MicroDigital, Seongnam, South Korea). cDNA was synthesized using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, CA, USA) following the manufacturer’s instructions. The transcription levels of virulence-related genes (agrA, RNAIII, hla and saeR), resistance-related gene (mecA), and housekeeping genes (16S rRNA) in MSSA and MRSA strains were evaluated using gene-specific primers (Table S2). RT-PCR was performed using SYBR Green Universal Master Mix (Applied Biosystems, CA, USA) on a QuantStudio Real-Time PCR System (Applied Biosystems, CA, USA). The relative gene expression in MSSA and MRSA was evaluated using the 2^−ΔΔCT^ method [30].
In vivo wound healing in MRSA-infected wounds
2.8
All animal experiments were performed in accordance with the ethical principles established by the National Regulations of the Republic of Korea and complied with the National Research Council’s Guide for the Care and Use of Laboratory Animals. This study was approved by the Institutional Animal Care and Use Committee of Pukyong National University (PKNUIACUC-2025-02). Male ICR mice (6 weeks old and 20–25 g) were housed under standard laboratory conditions (12 h light/dark cycle, 25 °C, and 50%–70% humidity) with ad libitum access to food and water. The mice were randomly assigned to four experimental groups to evaluate the therapeutic efficacy of the hydrogels. Following anesthesia, the dorsal region of each mouse was shaved, and full-thickness wounds (5 mm in diameter) were surgically created using a sterile biopsy punch. The wounds were infected under aseptic conditions with 20 µl MRSA KCCM 40510 suspension (10^7^ CFU/ml) to establish an MRSA-infected wound model. Each hydrogel formulation was applied to the wound, followed by occlusive coverage with Tegaderm film (3M Healthcare, Seoul, South Korea). The control group did not receive hydrogel treatment. Wound healing progression was monitored by digital imaging at predefined time points (0, 3, 7, 10 and 14 d). The wound area was measured using the ImageJ software, and the wound healing rate was calculated using the following formula:
where A0 and Ad represent the initial wound areas on Day 0, 3, 7, 10 and 14 after hydrogel treatment, respectively. The in vivo antibacterial efficacy was evaluated by collecting tissue samples on Day 3 and 7, followed by dilution with PBS.
Histological analysis
2.9
Wound tissues were collected at predetermined time points, immersed in 10% formalin for 24 h for fixation, and embedded in paraffin. The embedded tissues were sectioned into 5 µm slices with a microtome, then deparaffinized and rehydrated through a graded ethanol series (70%, 80%, 95% and 100%). Hematoxylin and eosin (H&E) staining was performed to assess the general tissue morphology, and Masson’s trichrome (MT) staining was used to evaluate collagen deposition and tissue remodeling. The stained sections were analyzed using MoticEasyScan Infinity (Motic Hong Kong Limited, Hong Kong, China). Additionally, for safety evaluation, the lung, kidney, liver, spleen, heart and stomach tissues were fixed, sectioned, and stained with H&E for microscopic examination of histomorphological changes.
In vivo immunofluorescence analysis was performed using paraffin-embedded wound sections that were deparaffinized and rehydrated, followed by antigen retrieval. The sections were then rinsed with PBS-T, permeabilized with 0.1% Triton X-100 for 10 min, and blocked with 5% BSA to reduce nonspecific binding. Primary antibodies against cluster of differentiation 31 (CD31), vascular endothelial growth factor (VEGF), arginase-1 (Arg1), and inducible nitric oxide synthase (iNOS) were incubated overnight at 4 °C. After washing, goat anti-rabbit IgG Alexa Fluor 488 was added for 2 h, and the nuclei were counterstained with DAPI. Fluorescent images were obtained using a CLSM.
Statistical analysis
2.10
For statistical evaluation, a one-way analysis of variance with Dunnett’s post-hoc test was performed using the GraphPad Prism software (version 10.4.1). Results are presented as mean ± standard deviation. Statistical significance was defined as follows: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 indicate significant differences versus the control. ^#^P < 0.05, ^##^P < 0.01, ^###^P < 0.001, and ^####^P < 0.0001 indicate significant differences among PVA, COS/PVA, and COS/PVA/BSI versus COS/PVA/BSII. ns indicates no significant difference.
Results and discussion
3
Fabrication and characterization of biosurfactant reinforced-hydrogels
3.1
Controlling bacterial infections at wound sites is crucial for accelerating wound healing. In the present study, a BS with anti-MRSA activity was purified from B. rugosus HH2 to develop therapeutic agents targeting MRSA infections. The structure of B. rugosus HH2 BS was elucidated using ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry, FTIR, and nuclear magnetic resonance spectroscopy, which identified it as a surfactin homologue (Fig. 1A) [8]. The antibacterial properties of B. rugosus HH2 BS were then systematically assessed against S. aureus. The BS displayed a MIC of 128 µg/ml against both MSSA and MRSA (Fig. 1B). In addition, the MBICs were 32 µg/ml for MSSA and 16 µg/mL for MRSA, indicating that biofilm development was effectively suppressed at sub-MIC levels (Fig. 1C). The growth kinetics further revealed bacteriostatic effects below the MIC (Fig. S3A and S3B). At 1/4× MIC, exponential growth was delayed by approximately 8 h in MSSA and 4 h in MRSA. Time-kill assays demonstrated bactericidal activity, with cell counts reduced by approximately 3.8 log CFU/ml for MSSA and 3.2 log CFU/ml for MRSA after 24 h of treatment at 2× MIC (Fig. S3C and S3D). CLSM provided additional mechanistic evidence (Fig. S3E). Untreated cells showed intact membranes with intense green fluorescence, whereas cells exposed to 2× MIC BS exhibited a pronounced increase in red fluorescence, consistent with membrane disruption. Collectively, these results demonstrated that B. rugosus HH2 BS exerts both bacteriostatic and bactericidal effects against MSSA and MRSA, underscoring its potential as a broad-spectrum antibacterial agent.Fig. 1. Fabrication and visual characterization of the hydrogels. (A) Structure of BS derived from Bacillus rugosus HH2; (B) Antibacterial and (C) antibiofilm activity of BS derived from B. rugosus HH2 against MSSA and MRSA (n = 3). (D) Hydrogen bonding interactions between the hydroxyl groups of COS and PVA in the COS/PVA/BS hydrogel. (E) Hydrogen bonding interactions between the amine groups of COS and the carboxyl groups of BS in the COS/PVA/BS hydrogel. (F) Photographs of PVA, COS/PVA, COS/PVA/BSI, and COS/PVA/BSII hydrogels prepared in a vial through three freeze-thaw cycles. (G) Representative morphology of the developed hydrogels. The black scale bar represents 10 mm. (H) Representative FE-SEM image of the developed hydrogels. The yellow scale bar represents 100 µm. COS/PVA/BS hydrogel with (I) adhesive and (J) stretchable properties.Fig 1 dummy alt text
A BS-reinforced COS/PVA hydrogel was developed to facilitate the delivery of BS to wound sites, while improving the gel-forming properties of the polysaccharides. Crosslinking between COS and PVA results in the formation of a dual-network hydrogel with excellent biological and mechanical properties. PVA crystallized through freeze-thaw cycles, creating a primary physical crosslinking network [18], whereas COS and PVA formed a secondary physical crosslinking network via hydrogen bonding (Fig. 1D) [31]. To provide an antibacterial function, the COS/PVA hydrogel was reinforced with BS during the hydrogel formulation process before the gelation phase. Hydrogen bonding between the carboxyl group of the BS cyclic peptide and the amino groups of the COS side chains facilitated the formation of a hydrogel network (Fig. 1E) [32]. As shown in Fig. 1F and 1G, the precursor solutions of PVA, COS/PVA, COS/PVA/BSI, and COS/PVA/BSII successfully formed hydrogels after three freeze-thaw cycles. During this process, hydrogen bonding between COS and PVA contributes to the creation of a three-dimensional structure within the interpenetrating polymer network [33]. FE-SEM images revealed interconnected porous structures within the PVA, COS/PVA, COS/PVA/BSI, and COS/PVA/BSII hydrogels (Fig. 1H). The BS reinforcement did not alter the pore morphology. The average pore size of the COS/PVA hydrogel (92.17 ± 8.49 µm) was significantly smaller than that of the PVA hydrogel (70.76 ± 8.76 µm) (P < 0.0001), likely because of strong hydrogen bonding between COS and PVA (Fig. S4). The average pore sizes of COS/PVA/BSI and COS/PVA/BSII hydrogels were 63.02 ± 9.79 µm and 62.49 ± 10.66 µm, respectively, showing significant differences compared to the COS/PVA hydrogel (P < 0.05) (Fig. S4C, D, and E). These findings suggest that BS reinforcement increases the crosslinking density of hydrogels. Fig. 1I and 1J show that the COS/PVA/BS hydrogels exhibited adhesive and stretchable properties, indicating their potential to maintain close contact with the human skin and effectively accommodate everyday movements. Additionally, the COS/PVA/BS hydrogel exhibited strong adhesion to various organs, highlighting its broad applicability (Fig. S5).
The FTIR results revealed the chemical structures of the BS and various hydrogels (Fig. 2A). The spectrum of B. rugosus HH2 BS exhibited characteristic absorption bands at 3276, 1640 and 1536 cm^−1^, corresponding to N-H stretching, C=O stretching of amide groups, and C-N stretching vibrations, respectively [8]. The COS/PVA hydrogel exhibited a weakened peak at 1640 cm^−1^, associated with C=O stretching compared to that of COS alone, indicating intermolecular hydrogen bonding between COS and PVA [31]. Furthermore, the FTIR spectrum of the COS/PVA hydrogel showed no new peaks compared with those of COS and PVA, suggesting that COS and PVA formed a physically entangled network structure without chemical interactions [31]. Notable peaks corresponding to N-H stretching appeared in the 3282–3267 cm^−1^ range. Specifically, the 3282 cm^−1^ peak of COS/PVA shifted to 3274 cm^−1^ in COS/PVA/BSI and 3267 cm^−1^ in COS/PVA/BSII, indicating hydrogen bond formation between the carboxyl groups of BS and the amine groups of COS [32,34]. Meanwhile, the absorption bands at 1640, 1536 and 1086 cm^−1^ in the COS/PVA/BS hydrogels intensified with increasing BS content, which is consistent with the characteristic peaks of BS. These results support the hypothesis that a dual-network structure was formed through hydrogen bonding in the COS/PVA/BS hydrogels.Fig. 2. Physicochemical characterization of the hydrogels. (A) FTIR spectra of BS, COS, PVA, COS/PVA, COS/PVA/BSI, and COS/PVA/BSII hydrogel. (B) XRD patterns of COS, PVA, COS/PVA, COS/PVA/BSI, and COS/PVA/BSII hydrogel. (C) Swelling ratio of PVA, COS/PVA, COS/PVA/BSI, and COS/PVA/BSII hydrogel (n = 3). (D) Representative images of the developed hydrogels before and after swelling. The black scale bar represents 10 mm. (E) Degradation of PVA, COS/PVA, COS/PVA/BSI, and COS/PVA/BSII hydrogel (n = 3). (F) Water retention rate of PVA, COS/PVA, COS/PVA/BSI and COS/PVA/BSII hydrogel (n = 3). (G) TGA curve of PVA, COS/PVA, COS/PVA/BSI, and COS/PVA/BSII hydrogel. (H) Cumulative release of COS/PVA/BSI and COS/PVA/BSII hydrogel in PBS (pH 7.4) (n = 3). Strain sweep curve of (I) PVA, (J) COS/PVA, (K) COS/PVA/BSI, and (L) COS/PVA/BSII (n = 3).Fig 2 dummy alt text
The XRD patterns were used to confirm the chemical structure of the hydrogels (Fig. 2B). COS displayed 3f characteristic peaks at 23.7°, 37.5° and 43.7°, corresponding to crystalline phases, whereas the other regions indicated a dominant amorphous phase [35]. PVA exhibited semi-crystalline diffraction peaks at 19.5°, 37.5° and 43.8°, which were attributed to both intramolecular and intermolecular hydrogen bonding among the PVA chains. These broad regions reflect the amorphous nature of the hydrogels, confirming their semi-crystalline structure [36]. In the COS/PVA hydrogel, the intensities of the COS peak at 23.7° and the PVA peak at 19.5° decreased, likely because of reduced crystallinity resulting from hydrogen-bonding interactions between COS and PVA, indicating the formation of a porous hydrogel network [37]. The COS/PVA/BS hydrogel was consistent with that of COS/PVA but showed reduced intensities of the characteristic peaks (19.5°, 37.5° and 43.8°), suggesting reduced crystallinity of the COS/PVA/BS hydrogel because of hydrogen bonding interactions between COS and BS, confirming the successful reinforcement of BS in the COS/PVA hydrogel [38].
An ideal wound dressing should rapidly absorb excess wound exudates and prevent secondary infections caused by delayed exudate management. The swelling behavior of the hydrogels was evaluated at 37 °C (simulated body temperature) in PBS (pH 7.4) (Fig. 2C and 2D). All the hydrogels exhibited rapid swelling within the first 3 h and reached equilibrium shortly thereafter. The swelling ratios after 48 h for PVA, COS/PVA, COS/PVA/BSI and COS/PVA/BSII hydrogels were measured at 375.25% ± 10.58%, 1450.79% ± 10.37%, 1374.01% ± 56.05% and 1312.60% ± 9.75%, respectively. The COS/PVA hydrogel exhibited greater swelling than the PVA hydrogel, which agrees with the observation that the swelling capacity of the PVA hydrogel increased with the incorporation of higher amounts of natural polymers (COS) [39]. The swelling ratio of the COS/PVA/BS hydrogel was lower than that of the COS/PVA hydrogel, probably because of the increased crosslinking density and reduced pore size of the BS-reinforced hydrogel, which impeded water diffusion and consequently decreased the swelling ratio [40]. Furthermore, this finding is consistent with those of previous studies, suggesting that hydrogen-bonding interactions between drugs and polymers within hydrogels can compact the hydrogel structure, thereby reducing its water absorption capacity [41].
Degradation is a critical factor to consider when using biomaterials for medical applications, as it determines the duration for which wound dressings can effectively perform their functions while remaining on the skin. The degradation behavior of the hydrogels was evaluated in PBS adjusted to pH 7.4 at 37 °C (Fig. 2E). All hydrogels exhibited rapid degradation within the first 3 d. By Day 21, the degradation rates of PVA, COS/PVA, COS/PVA/BSI, and COS/PVA/BSII hydrogels were 20.07% ± 2.03%, 41.15% ± 1.13%, 42.63% ± 1.56% and 44.68% ± 2.10%, respectively. The COS/PVA hydrogel showed greater degradation than the PVA hydrogel, which was consistent with the observation that the COS/PVA hydrogel degrades faster than PVA alone [39]. For the COS/PVA/BS hydrogels, the degradation rate increased at higher BS concentrations, probably because of BS release from the hydrogels. These findings aligned with previous studies demonstrating that higher drug concentrations added to PVA/CS hydrogels resulted in faster degradation rates [42].
The water retention capacity of wound dressings is crucial for providing a moist environment that promotes wound healing by dissolving necrotic tissue and preventing scab formation. The water retention ability of the hydrogels was assessed at 37 °C in PBS (pH 7.4) (Fig. 2F). All the hydrogels showed similar trends in water retention over time. After 24 h, the water retention ratios for PVA, COS/PVA, COS/PVA/BSI, and COS/PVA/BSII hydrogels were 9.78% ± 2.12%, 15.30% ± 2.80%, 14.36% ± 1.40% and 12.79% ± 0.87%, respectively. Throughout the 24 h period, the water retention ratio of the COS/PVA hydrogel was higher than that of PVA alone, indicating that crosslinking between COS and PVA enhanced water retention. These results align with earlier reports indicating improved water retention upon the incorporation of CS into PVA [43]. However, no differences in water retention were observed in the COS/PVA/BS hydrogels after BS reinforcement.
In summary, the COS/PVA/BS hydrogels effectively absorbed wound exudates and exhibited excellent moisture retention properties. These features are likely to enhance the wound healing process by maintaining an optimal moist environment [44]. Additionally, the remarkable degradation stability of the hydrogel ensured its structural integrity and provided sustained protection at the wound site [45].
TGA is a critical method to assess the ability of biomaterials to retain their stability and functionality under extreme environmental conditions. The thermal stabilities of the hydrogels were evaluated using TGA (Fig. 2G). The PVA hydrogel exhibited a significant mass loss of 91.3% between 270 and 500 °C, which can be attributed to the decomposition of -CH_2_ and -CH-OH groups in the PVA [43]. The COS/PVA, COS/PVA/BSI, and COS/PVA/BSII hydrogels underwent weight loss during two distinct degradation stages. For the COS/PVA hydrogel, the first stage (250–300 °C) demonstrated a 26.4% mass loss, likely because of polymer chain decomposition [38], while the second stage (350–500 °C) exhibited an 83.4% mass loss, possibly related to unstable residues and exothermic decomposition [46]. Moreover, the incorporation of COS into PVA generally enhanced the thermal stability, which is consistent with previous findings where CS integration into PVA reduced thermal degradation [47]. In the COS/PVA/BSI and COS/PVA/BSII hydrogels, the first stage showed mass losses of 28.7% and 33.8%, respectively, whereas the second stage showed losses of 84.7% and 85.9%, respectively. The COS/PVA/BS hydrogel exhibited a slight increase in thermal degradation at higher BS concentrations, which is consistent with the trend observed for the CS/PVA hydrogel, where the addition of insulin resulted in increased thermal degradation compared to that of the CS/PVA hydrogel [48]. In conclusion, incorporating COS into PVA enhanced its thermal stability, whereas reinforcement with BS resulted in a slight increase in weight loss.
These findings underscore the significance of BS release from COS/PVA hydrogels in the treatment of MRSA infections. The COS/PVA/BS hydrogels demonstrated a time-dependent release of BS in PBS adjusted to physiological conditions (37 °C, pH 7.4) (Fig. 2H). Both COS/PVA/BSI and COS/PVA/BSII hydrogels exhibited a rapid initial release of BS within the first 12 h, reaching concentrations of 45.14 ± 5.05 µg/mL and 85.14 ± 4.04 µg/ml, respectively. This release pattern is consistent with the drug release behavior reported for CS/PVA hydrogels [12]. At the same time point, the COS/PVA/BSII hydrogels exhibited a higher release rate and greater cumulative drug release than the COS/PVA/BSI hydrogels. Following this burst phase, both COS/PVA/BSI and COS/PVA/BSII hydrogels maintained a sustained and gradual BS release, likely because of the mobility of the polymer chains within the COS/PVA hydrogel network [49], suggesting that the COS/PVA/BS hydrogels offer a therapeutic advantage for infectious wound healing by preventing MRSA colonization during the initial burst-release phase.
Structural stability and deformability of wound dressings are crucial for their practical clinical applications. The rheological properties of the hydrogels were assessed using the strain (Fig. 2I-2L) and frequency (Fig. S6-S6D) sweep measurements. The strain sweep results revealed that all hydrogels exhibited a higher Gʹ than Gʹʹ within the linear viscoelastic region, indicating the formation of stable crosslinked networks with semi-solid properties [50]. As strain increased, all hydrogels exhibited reversible elastic deformation, with the critical strain at which Gʹ fell below Gʹʹ indicating network disruption observed at 6.33% for PVA, 15.90% for COS/PVA, and 25.19% for COS/PVA/BSII. The addition of COS into PVA or reinforcement with BS improved the mechanical performance, likely because of increased hydrogen bonding within the hydrogel structure [51]. Similarly, the frequency sweep results confirmed that Gʹ exceeded Gʹʹ for all hydrogels, further verifying stable gel formation [52]. The COS/PVA/BS hydrogel exhibited consistent gel-like behavior across all frequency ranges, with no notable differences in Gʹ and Gʹʹ associated with BS concentration. The rheological analysis indicated that the COS/PVA/BS hydrogel exhibited excellent structural stability and mechanical performance.
Biocompatibility of hydrogels
3.2
To ensure patient safety, materials used for wound dressings must be biologically compatible. Given the significant trade-off between antimicrobial efficacy and cytotoxicity in the developed formulations, a thorough investigation of cytocompatibility is critical [53]. The cytotoxicity of the hydrogel was evaluated using live/dead staining assays and MTT analysis with HDF, HaCaT and HUVEC as model cells (Fig. 3A-3D). In the live/dead staining assay, most cells were predominantly alive and evenly distributed, indicating that the hydrogel exhibited minimal toxicity toward HDF, HaCaT, and HUVEC. MTT analysis revealed that the cell viability for all cell types exceeded 90%, surpassing the International Organization for Standardization (ISO) 10993-5 threshold for nontoxicity of 70% [54].Fig. 3. Biocompatibility of the hydrogels. CLSM images of (A) HDF, (B) HaCaT and (C) HUVEC cells following treatment with PVA, COS/PVA, COS/PVA/BSI and COS/PVA/BSII. The yellow scale bar represents 200 µm; (D) Cell viability of HDF, HaCaT and HUVEC cells following treatment with PVA, COS/PVA, COS/PVA/BSI and COS/PVA/BSII (n = 3); (E) Hemolysis rate and optical images of sheep blood cells following treatment with PVA, COS/PVA, COS/PVA/BSI and COS/PVA/BSII (n = 3).Fig 3 dummy alt text
As wound dressings come into direct contact with blood, exhibition of non-hemolytic activity or minimal induction of hemolysis by them is crucial. The hemolytic activity of the hydrogels was assessed using sheep blood cells (Fig. 3E). The supernatants of all the hydrogel samples appeared transparent, indicating no red blood cell destruction compared to the positive control. The hemolysis rates for all hydrogel samples were below 5%, which is well below the safety threshold specified by ISO 10993-4 [55].
According to our previous study, B. rugosus HH2 BS maintained the viability of HDF and HaCaT above 70% and showed negligible phytotoxic effects in a Raphanus sativus model at concentrations that exerted anti-MRSA effects [8]. These results indicated that the hydrogel exhibited excellent cytocompatibility and blood compatibility, while maintaining its antibacterial efficacy, underscoring its potential as an antimicrobial wound dressing for tissue engineering applications.
In vitro evaluation of wound healing-related cellular responses to hydrogels
3.3
Wound healing involves the coordinated activity of multiple cell types, and biomaterials play a crucial role in accelerating this process by promoting cell migration [56]. A cell scratch assay was performed to evaluate the effects of the hydrogels on the migration of HDF, HaCaT, and HUVEC (Fig. 4A--4F). Although the PVA hydrogel showed no significant impact on the migration of these cell types compared with the control group (P > 0.05), the COS/PVA hydrogel significantly enhanced HUVEC migration (P < 0.001). In addition, the reinforcement of BS in the COS/PVA hydrogel boosted the migration of all cell types. Notably, the COS/PVA/BSII hydrogel outperformed the COS/PVA/BSI hydrogel, showing significantly greater migration enhancement (P < 0.0001 for HDF, P < 0.01 for HaCaT, P < 0.05 for HUVEC). These findings underscore the contribution of the BS to the ability of the hydrogels to promote cell migration. Yan et al. reported that surfactin A stimulated the migration of HaCaT, HDF, and HUVEC [57]. Western blot analysis revealed that surfactin A activates the ERK1/2, JNK, and NF-κB p65 signaling pathways, driving HaCaT cell migration. In conclusion, the COS/PVA/BS hydrogel is a promising wound dressing candidate that effectively promotes the migration of HDF, HaCaT, and HUVEC via BS release.Fig. 4In vitro evaluation of wound healing-related cellular responses to hydrogels. (A-F) Scratch assay images and quantitative statistics showing the wound closure ratio of (A and D) HDF, (B and E) HaCaT, and (C and F) HUVEC following treatment with PVA, COS/PVA, COS/PVA/BSI, and COS/PVA/BSII (n = 3); (G) Image for tube formation of HUVEC following treatment with PVA, COS/PVA, COS/PVA/BSI and COS/PVA/BSII; (H-J) Quantification through counting the number of (H) nodes, (I) junctions, and (J) total capillary length (n = 3). The yellow scale bar represents 200 µm; (K) Immunofluorescence images of HDF stained with COLI (green) and α-SMA (red) following treatment with PVA, COS/PVA, COS/PVA/BSI and COS/PVA/BSII. The white scale bar represents 50 µm; (L-M) Quantitative analysis of (L) COLI and (M) α-SMA fluorescence intensity (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 versus control. ns means no significance versus control. #P < 0.05, ##P < 0.01, and ####P < 0.0001 for PVA, COS/PVA, and COS/PVA/BSI versus COS/PVA/BSII.Fig 4 dummy alt text
The tube formation assay was conducted to assess the angiogenic potential of the hydrogels using HUVEC seeded on Matrigel for 6 h (Fig. 4G). In line with the best practices, images were analyzed for branching complexity (nodes and junctions) and network extent (total length). Quantitative analysis using the ImageJ Angiogenesis Analyzer revealed that the COS/PVA and BS-reinforced hydrogels significantly promoted capillary-like network formation compared to the control and PVA groups. The number of nodes (branching points) and junctions progressively increased across groups, with the COS/PVA/BSII hydrogel exhibiting the highest values (Fig. 4H and 4I). Moreover, the total capillary length was markedly extended in both the COS/PVA/BSI and COS/PVA/BSII groups (Fig. 4J), indicating that BS acted as the primary angiogenic driver. Surfactin has been reported to upregulate HIF-1α and VEGF, promote cell migration, and regulate macrophage-related signaling pathways during wound healing [57]. These mechanisms are likely responsible for the higher node/junction density and longer network length observed in the hydrogels reinforced with BS.
To further examine ECM remodeling and fibroblast activation, HDF was treated with hydrogel extracts and analyzed using immunofluorescence staining for COLI and α-SMA (Fig. 4K) [58]. The fluorescence intensity was measured in ImageJ as marker/DAPI ratio. TGF-β, used as a positive control, significantly increased the levels of both COLI and α-SMA, confirming its well-known role in Smad-dependent fibroblast-to-myofibroblast transition [59]. Conversely, the hydrogel-treated groups showed much lower α-SMA expression levels, indicating minimal fibrotic activation. Among the hydrogels, TGF-β was 256.25% ± 27.19% for COLI expression, increasing incrementally from PVA (93.75% ± 19.84%) to COS/PVA (109.38% ± 9.62%), then to COS/PVA/BSI (136.25% ± 8.45%), and finally to COS/PVA/BSII (143.13% ± 6.58%). This suggests that COS contributed to baseline collagen synthesis, while adding BS further enhanced ECM deposition (Fig. 4L). However, α-SMA levels remained similar to the control (Fig. 4M), indicating that the hydrogels supported matrix assembly without causing excessive fibroblast contractility or fibrosis.
Consistently, Sirius Red staining of HDF further supported these findings (Fig. S7A). Cells treated with TGF-β showed the strongest red staining, indicating high collagen deposition [60], while the control, PVA, and COS/PVA groups exhibited minimal collagen accumulation. Conversely, BS-reinforced hydrogels (COS/PVA/BSI and COS/PVA/BSII) showed progressively stronger staining, consistent with quantitative measurements indicating significant increases in the BS-reinforced groups (Fig. S7B). This ranking aligns with typical TGF-β/Smad2/3-driven collagen synthesis in fibroblasts (positive control) and also reflects PVA’s relative bio-inertness; modest increases in COS- and BS-containing eluates likely represent low-level release of bioactives that support ECM assembly without reaching the profibrotic levels induced by TGF-β.
Taken together, these results show that BS-reinforced hydrogels enable controlled fibroblast activation necessary for ECM remodeling and wound healing, while avoiding pathological fibrosis. This balanced matrix response is beneficial for skin tissue regeneration because it promotes collagen deposition and tissue integration without causing scar-like contractile remodeling.
In vitro antibacterial and antibiofilm activities of hydrogels
3.4
Hydrogels, which have the potential to combine drug delivery, tissue regeneration, and antibacterial properties, hold significant promise for the management and control of wound-related infections. However, before assessing the in vivo applicability of these materials, evaluating their antibacterial efficacy in vitro, is crucial. The bacteriostatic effect of the hydrogels was evaluated by monitoring the growth curves of MSSA and MRSA over an 8 h period following hydrogel treatment (Fig. 5A and 5B). The OD_600_ values of the PVA and COS/PVA hydrogels were similar to those of the control group, indicating that these hydrogels did not inhibit MSSA or MRSA proliferation. Throughout the incubation period, the COS/PVA/BSI hydrogel group exhibited slower bacterial growth compared to the PVA and COS/PVA hydrogel groups, with MSSA and MRSA growth being inhibited by 50.60% ± 2.56% and 47.84% ± 2.92%, respectively, after 8 h of incubation. Moreover, in the COS/PVA/BSII hydrogel group, no noticeable increase in OD_600_ values was observed, and MSSA and MRSA growth were inhibited by 86.47% ± 0.17% and 83.93% ± 2.51%, respectively, at the 8 h time point. The more vigorous bacteriostatic activity observed with COS/PVA/BS correlated with its higher loading, which provided a cumulative release of approximately 85.14 µg/ml BS, sufficient to prevent MSSA and MRSA colonization. Therefore, 0.1 wt% BS reinforcement in COS/PVA/BSII was identified as the optimal loading concentration for hydrogel fabrication to achieve effective antibacterial activity.Fig. 5In vitro antibacterial and antibiofilm activity of the hydrogels against MSSA and MRSA. Growth curves of (A) MSSA ATCC 6538 and (B) MRSA KCCM 40510 cells following treatment with PVA, COS/PVA, COS/PVA/BSI and COS/PVA/BSII (n = 3); CFU measurement of (C) MSSA ATCC 6538 and (D) MRSA KCCM 40510 cells following treatment with PVA, COS/PVA, COS/PVA/BSI,; and COS/PVA/BSII (n = 3); (E) Proposed model illustrating the synergistic antibacterial effect of the COS/PVA/BS hydrogel against MRSA; (F) FE-SEM images of MSSA ATCC 6538 and MRSA KCCM 40510 cells following treatment with PVA, COS/PVA, COS/PVA/BSI and COS/PVA/BSII. The green scale bar represents 2 µm. Green-colored areas represent intact cells, while red-colored areas indicate the leakage of intracellular components caused by cell membrane damage at 15,000× magnification; Effects of PVA, COS/PVA, COS/PVA/BSI and COS/PVA/BSII treatment on (G) MSSA and (H) MRSA biofilm inhibition (n = 3); (I) Images of residual biofilms stained with crystal violet after treatment with PVA, COS/PVA, COS/PVA/BSI and COS/PVA/BSII; (J) Fluorescence images of MSSA and MRSA biofilms following treatment with PVA, COS/PVA, COS/PVA/BSI and COS/PVA/BSII. Images show merged channels of SYTO 9 (green) and PI (red) staining. The yellow scale bar represents 100 µm. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 versus control. ns means no significance versus control. #P < 0.05, ##P < 0.01, ###P < 0.001, and ####P < 0.0001 for PVA, COS/PVA, and COS/PVA/BSI versus COS/PVA/BSII.Fig 5 dummy alt text
The bactericidal effect of the hydrogels was evaluated by measuring the CFU of MSSA and MRSA cultures over an 8 h period following hydrogel treatment (Fig. 5C and 5D). The PVA hydrogel did not significantly reduce MSSA and MRSA counts (P > 0.05), whereas the COS/PVA hydrogel significantly reduced MSSA and MRSA counts by 2.17 ± 0.08 log CFU/mL and 2.13 ± 0.11 log CFU/ml, respectively (P < 0.0001). The incorporation of COS into PVA enhanced its bactericidal effect against MSSA and MRSA, likely because the positively charged amine groups in COS interacted with the negatively charged bacterial cell membranes [61]. This interaction compromises membrane integrity, resulting in the efflux of cellular contents and eventually triggering bacterial cell lysis [62]. Furthermore, treatment with the COS/PVA/BSI hydrogel significantly reduced MSSA and MRSA CFU counts by 3.74 ± 0.04 log CFU/ml and 3.29 ± 0.09 log CFU/ml, respectively (P < 0.0001), demonstrating a greater reduction than that observed with the COS/PVA hydrogel. Notably, an even more pronounced reduction was observed with the COS/PVA/BSII hydrogel (P < 0.0001), which showed a statistically significant difference compared with COS/PVA/BSI (P < 0.01 MSSA and P < 0.05 for MRSA). These results suggest that, similar to the bacteriostatic behavior of the COS/PVA/BS hydrogels, the increased bactericidal activity can be attributed to the enhanced BS content within the COS/PVA matrix. In summary, the bactericidal effect of the COS/PVA/BS hydrogel significantly increased with the incorporation of COS into PVA. This was further enhanced by the reinforcement of BS in the COS/PVA hydrogel. This effect was likely owing to the synergistic mechanism of COS degradation and BS release as the COS/PVA/BS hydrogel decomposed in the bacterial microenvironment (Fig. 5E). The FE-SEM analysis confirmed the antibacterial activity of the hydrogels against MSSA and MRSA (Fig. 5F). In the untreated and PVA hydrogel groups, MSSA and MRSA cells exhibited intact cell membranes and well-preserved spherical morphologies. In the COS/PVA hydrogel-treated group, a slight reduction in cell density was observed, accompanied by partial damage to the cell membrane, leading to the release of intracellular components. Moreover, treatment with the COS/PVA/BS hydrogel resulted in a pronounced decrease in cell density and extensive damage to the cell membrane compared with the COS/PVA hydrogel group. These results suggest that COS and BS in the COS/PVA/BS hydrogel disrupted the cell membrane structure of S. aureus, leading to the efflux of internal cellular components and resulting in vigorous antibacterial activity. This finding is consistent with earlier studies [8,61,62].
In addition to their activity against S. aureus, the antibacterial performance of the hydrogels was further assessed against other clinically relevant pathogens such as P. aeruginosa and E. coli. In the bacteriostatic assay, after 8 h of incubation, the COS/PVA/BSI and COS/PVA/BSII hydrogel groups showed reduced bacterial growth compared to the control against P. aeruginosa. In contrast, for E. coli, all hydrogel groups except PVA exhibited significant growth inhibition (Fig. S8A and S8B). Regarding bactericidal activity, the COS/PVA, COS/PVA/BSI and COS/PVA/BSII hydrogels significantly decreased the viable counts, with the most pronounced reduction observed for the COS/PVA/BSII hydrogel (Figs. S8C and S8D). These findings indicate that COS/PVA/BS hydrogels are also capable of inhibiting clinically critical Gram-negative pathogens, although their efficacy is not as strong as that observed against MRSA.
If wounds infected with pathogenic microorganisms are not thoroughly treated, these microorganisms can rapidly form biofilms within 24 h, which subsequently integrate into the wound microenvironment and delay the healing process [[62], [63], [64]]. The antibiofilm activity of the hydrogels against MSSA and MRSA was evaluated by crystal violet quantification (Fig. 5G-5I) and CLSM analysis (Fig. 5J). PVA hydrogel did not affect biofilm formation (P > 0.05), whereas the COS/PVA hydrogel reduced MSSA and MRSA biofilm formation by 23.78% ± 8.69% and 20.88% ± 2.63%, respectively. Notably, BS reinforcement led to even greater reductions in both strains in a concentration-dependent manner. CLSM analysis further supported these findings; biofilm cells treated with the PVA hydrogel exhibited strong green fluorescence, similar to that of the control, whereas those treated with the COS/PVA hydrogel displayed reduced green signal density. Moreover, the COS/PVA/BS hydrogels showed further green fluorescence suppression, accompanied by the presence of red fluorescent signals, indicative of damaged cells. These results closely parallel the antibacterial effects of the hydrogels. The antibiofilm effect of COS is attributed to the polycationic nature of its protonated amino groups, which electrostatically interact with the negatively charged biofilm components [59]. In contrast, BS has been reported to downregulate QS-related genes such as agrA and RNAIII, thereby impairing S. aureus biofilm formation [8].
In conclusion, these findings highlight the effectiveness of the COS/PVA/BS hydrogel, which exhibited bacteriostatic, bactericidal, and antibiofilm properties. Bacteriostatic activity prevents MRSA colonization at wound sites, thereby mitigating the risk of chronic wound development. This bactericidal effect inhibits bacterial infiltration into the wound area and promotes faster healing of infectious wounds. Significantly, the antibiofilm activity further suppressed the establishment of MRSA biofilms, which are critical contributors to delayed wound healing and therapeutic relapse. Notably, the COS/PVA/BS hydrogel is a promising alternative to conventional antibiotics because it releases BS, a naturally derived compound, thereby addressing both biofilm-associated infections and antibiotic treatment failures.
Anti-virulence effects of hydrogels against MRSA wound infection-associated virulence factors
3.5
Building on its in vitro antibacterial activity, the hydrogel was evaluated for its ability to attenuate the virulence factors of MSSA and MRSA.
Wound sites are prone to colonization by hemolytic S. aureus [65]. S. aureus produces four types of hemolysins, with α-hemolysin identified as a key factor in the initial formation of skin lesions [66]. Additionally, α-hemolysin plays a crucial role as a virulence factor, enabling S. aureus to cause localized pyogenic skin infections and sepsis [67]. The effects of the hydrogel on the hemolytic activities of MSSA and MRSA are shown in Fig. 6A and 6B. The PVA hydrogel did not significantly affect the hemolytic activity of MSSA or MRSA (P > 0.05). However, the COS/PVA hydrogel demonstrated significant anti-hemolytic activity against both MSSA and MRSA (P < 0.05). Furthermore, an increase in BS content correlated with a reduction in hemolytic activity. Notably, the COS/PVA/BSII hydrogel caused a substantial decrease in hemolytic activity, with reductions of 21.63% ± 0.08% for MSSA and 38.48% ± 1.33% for MRSA (P < 0.0001).Fig. 6. Effect of the hydrogels on virulence factors and gene expression in MSSA and MRSA. Hemolytic activity of (A) MSSA ATCC 6538 and (B) MRSA KCCM 40510 following treatment with PVA, COS/PVA, COS/PVA/BSI and COS/PVA/BSII (n = 3); Extracellular lipase production from (C) MSSA ATCC 6538 and (D) MRSA KCCM 40510 following treatment with PVA, COS/PVA, COS/PVA/BSI and COS/PVA/BSII (n = 3); Staphyloxanthin production from (E) MSSA ATCC 6538 and (F) MRSA KCCM 40510 following treatment with PVA, COS/PVA, COS/PVA/BSI and COS/PVA/BSII (n = 3); (G) Images of MSSA ATCC 6538 and MRSA KCCM 40510 cell pellets following treatment with the developed hydrogels. The black scale bar represents 5 mm; (H) Images of slime production by MSSA ATCC 6538 and MRSA KCCM 40510 following treatment with the developed hydrogels. The white scale bar represents 5 mm; Relative transcriptional profiles of (I) MSSA ATCC 6538 and (J) MRSA KCCM 40510 following treatment with the developed hydrogels (n = 3). Relative gene expression levels indicate transcriptional differences between treated and untreated cells, as determined by RT-PCR with 16S rRNA as the housekeeping gene; (K) Proposed mechanism of MRSA-infected wound healing acceleration by COS/PVA/BS hydrogel based on transcriptional changes. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 versus control. ns means no significance versus control. ^#^P < 0.05, ^##^P < 0.01, ^###^P < 0.001, and ^####^P < 0.0001 for PVA, COS/PVA and COS/PVA/BSI versus COS/PVA/BSII.Fig 6 dummy alt text
S. aureus produces lipase, which impairs the host’s immune defense by inhibiting granulocyte chemotaxis and reducing phagocytosis [68]. Lipase is a critical virulence factor that facilitates the breakdown of host tissues and promotes deep-seated wound infections by S. aureus [69]. Recent studies highlighted that S. aureus-derived lipases are critically involved in the development of drug resistance and surgical site infections [70]. The hydrogel exhibited a notable capacity to suppress lipase production in both MSSA and MRSA (Fig. 6C and 6D). A progressive reduction in extracellular lipase production was observed with the incorporation of COS into PVA, which was further enhanced by BS in the COS/PVA hydrogel. Notably, the COS/PVA/BSII hydrogel significantly inhibited lipase production by 66.41% and 52.08% in MSSA and MRSA isolates, respectively.
Staphyloxanthin is a golden pigment produced by S. aureus that contributes to its membrane integrity and antioxidant properties [71]. This pigment acts as a stress resistance factor by neutralizing the reactive oxygen species (ROS) generated by the host immune system, including O_2_, H_2_O_2_, and HOCl [72]. Consequently, staphyloxanthin-deficient mutants, although capable of normal growth, are rapidly killed by host neutrophil-derived ROS, leading to reduced abscess formation [73]. Recent studies have demonstrated that staphyloxanthin promotes S. aureus survival by protecting against oxidative stress and neutrophil phagocytosis, thereby impairing wound healing in a mouse model [74]. Accordingly, the effect of the hydrogel on staphyloxanthin production was investigated (Fig. 6E and 6F). All hydrogels suppress staphyloxanthin production by MSSA. However, for MRSA, only the hydrogels reinforced with BS significantly inhibited staphyloxanthin production (P < 0.01). Additionally, whereas the MSSA and MRSA pellets treated with the PVA and COS/PVA hydrogels exhibited a yellow color similar to that of the control group, those treated with the BS-reinforced hydrogels appeared white (Fig. 6G).
Slime, which comprises extracellular polymeric substances, plays a key role in the structure of S. aureus biofilms [75]. S. aureus can form biofilms at wound sites within 24 h, integrate into the wound environment, and contribute to chronic wound infections [76]. In chronic wounds, biofilms facilitate genetic exchange within polymicrobial communities, promoting antimicrobial resistance and causing symptoms such as necrosis, pain, excessive exudate, and malodor [77]. Accordingly, evaluating the effect of hydrogels on S. aureus slime production is essential for assessing their biofilm-forming capabilities (Fig. 6H). Colonies treated with PVA and COS/PVA hydrogels displayed a dark, reddish color comparable to that of the control group, suggesting continued slime production. In contrast, colonies treated with BS-reinforced hydrogels exhibited a bright red color, indicating complete slime production inhibition.
These results demonstrate that the COS/PVA/BS hydrogel effectively inhibits key virulence factors associated with wound infections. This study presents the first documented evidence of the ability of hydrogels to attenuate MRSA-associated virulence factors, including hemolysin, lipase, staphyloxanthin, and slime production. Additionally, RT-PCR analysis was performed to assess alterations in gene expression in MSSA and MRSA after exposure to hydrogel, providing insights into the mechanisms responsible for suppressing wound-associated virulence factors.
To elucidate the molecular mechanisms underlying the efficacy of the hydrogel, its effects on the expression of virulence- and resistance-related genes in MSSA and MRSA were examined (Fig. 6I and 6J). The PVA and COS/PVA hydrogels exhibited no significant effects or caused slight upregulation of virulence-related genes (agrA, RNAIII, hla, and saeR) in MSSA. Conversely, the BS-reinforced hydrogels significantly downregulated the expression of these virulence genes in MSSA (P < 0.05), with the most pronounced suppression observed for agrA (14.2-fold for COS/PVA/BSI and 9.3-fold for COS/PVA/BSII). In MRSA, the PVA hydrogel had no significant effect on any virulence genes (P > 0.05), whereas the COS/PVA hydrogel selectively and significantly downregulated agrA expression (P < 0.05). Notably, the COS/PVA/BSII hydrogel demonstrated a robust suppression of MRSA virulence genes, reducing the agrA, RNAIII, hla, and saeR expression levels by 4.4-, 2.8-, 14.6-, 2.1- and 4.8-fold, respectively. COS/PVA/BSII significantly downregulated the expression of the resistance-related gene mecA by 4.9-fold, representing the only hydrogel formulation that exhibited a significant inhibitory effect (P < 0.05).
The downregulation of agrA, RNAIII, hla, and saeR by the COS/PVA/BSII hydrogel offers insights into the underlying mechanism. The QS system, a crucial cell signaling mechanism in S. aureus, is fundamentally involved in modulating the production of virulence factors and biofilm development [78]. The accessory gene regulator locus, which encodes the QS system in S. aureus, comprises the response regulator AgrA and regulatory RNAIII [79]. Staphyloxanthin biosynthesis, regulated by agr, occurs independently of RNAIII [80]. RNAIII, a key regulatory RNA transcribed from the agr QS locus, orchestrates the expression of multiple virulence-associated proteins, including lipase, exoprotease, and α-hemolysin [80,81]. The virulence regulator saeR directly modulates hla transcription, which contributes to biofilm formation and α-hemolysin production [28]. These findings indicate that the COS/PVA/BSII hydrogel suppresses various virulence factors and biofilm formation by negatively regulating QS and virulence-related genes. Moreover, mecA downregulation by COS/PVA/BSII provided insights into the mechanisms of MRSA resistance. In MRSA, the mecA gene encodes PBP2a, a key factor in resistance to β-lactam antibiotics [78]. In summary, the COS/PVA/BSII hydrogel effectively inhibited key virulence factors associated with wound infections by downregulating QS-related and virulence regulator genes, while also exhibiting the potential to suppress MRSA resistance through mecA downregulation, thereby promoting the healing of MRSA-infected wounds (Fig. 6K).
In vivo efficacy of MRSA-infected wound treatment
3.6
A full-thickness MRSA-infected wound model was established in mice to evaluate the therapeutic effects of hydrogel [82]. Wound healing was monitored for 14 d after treatment with each hydrogel (Fig. 7A).Fig. 7COS/PVA/BS hydrogel promoted the healing of MRSA-infected full-thickness wounds in mice. (A) Scheme of the MRSA-infected wound healing experiment; (B) Images of infected wounds following treatment with PVA, COS/PVA, COS/PVA/BSI and COS/PVA/BSII at Day 0, 3, 7, 10, and 14. Each scale division on the ruler represents 1 mm; (C) Diagrams of the dynamic wound healing process in PVA, COS/PVA, COS/PVA/BSI and COS/PVA/BSII treatment groups at Day 0, 3, 7, 10 and 14; (D) Wound area of PVA, COS/PVA, COS/PVA/BSI and COS/PVA/BSII treatment groups at Day 0, 3, 7, 10 and 14 (n = 6); (E) Quantitative analysis of MRSA colonies in infected wound tissues following treatment with PVA, COS/PVA, COS/PVA/BSI, and COS/PVA/BSII at Day 3 and 7 (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 versus control. ns means no significance versus control. ^#^P < 0.05, ^##^P < 0.01, ^###^P < 0.001, and ^####^P < 0.0001 for PVA, COS/PVA and COS/PVA/BSI versus COS/PVA/BSII.Fig 7 dummy alt text
As shown in Fig. 7B, the representative wound images revealed significant variations in the rate of wound contraction across the treatment groups. The wounds treated with the PVA hydrogel exhibited a closure rate comparable to that of the control group over 14 d, with minimal initial contraction. The COS/PVA group demonstrated a moderate healing response with noticeable wound contractions emerging after 10 d. In contrast, both BS-reinforced hydrogels (COS/PVA/BSI and COS/PVA/BSII) markedly accelerated wound healing within the first 3 d. Notably, the COS/PVA/BSII group achieved near-complete epithelial coverage by 14 d. These visual observations are supported by a schematic diagram (Fig. 7C). The wound area was quantitatively analyzed (Fig. 7D). All hydrogel-treated groups showed a progressive decrease in wound area over time. On Day 3, the wound area in the COS/PVA/BSII group was 58.16% ± 11.63%, significantly smaller than that of the control (89.10% ± 5.87%), PVA (89.32% ± 5.47%), COS/PVA (87.05% ± 6.68%), and COS/PVA/BSI (73.51% ± 5.40%) groups. By 14 d, wounds treated with COS/PVA/BSII hydrogel had nearly completely healed, with a remaining wound area of only 2.87% ± 1.27%, compared to 9.21% ± 2.16% (control), 9.42% ± 1.21% (PVA), 5.15% ± 1.20% (COS/PVA), and 5.23% ± 3.02% (COS/PVA/BSI). Notably, the COS/PVA hydrogel group showed statistically significant healing after only 10 d, emphasizing the critical role of BS in promoting early-stage wound healing.
The in vivo antibacterial efficacy of the hydrogels was evaluated by isolating MRSA from tissues collected on Day 3 and 7 after hydrogel treatment and measuring the CFU (Fig. 7E). On Day 3, all hydrogels significantly reduced the MRSA levels in the wound tissues (P < 0.0001). Notably, incorporating COS into PVA and further enhancing the COS/PVA ratio with BS resulted in a greater reduction. Among them, the COS/PVA/BSII hydrogel demonstrated the most potent antibacterial effect, reducing MRSA by 1.94 ± 0.33 log CFU/ml, a statistically significant reduction compared to COS/PVA/BSI (P < 0.001). On Day 7, all hydrogels, except for PVA alone, significantly lowered the MRSA burden in wound tissues (P < 0.001), with the COS/PVA/BSII hydrogel exhibiting the highest bactericidal effect, achieving a reduction of 0.80 ± 0.13 log CFU/ml. Overall, the in vivo antibacterial efficacy of the hydrogels followed a trend similar to their in vitro antibacterial activities. These results indicated that the COS/PVA/BSII hydrogel could effectively control MRSA infection in vivo, contributing to the healing of infected wounds.
This enhanced wound closure can be attributed to the synergistic bioactivity of COS and BS, both of which have previously demonstrated potential for wound healing [8,36]. Collectively, these results indicate that the COS/PVA/BSII hydrogel not only provides a moist and protective environment conducive to wound healing but also effectively suppresses MRSA colonization in vivo, making it a promising therapeutic option for treating MRSA-infected wounds.
To comprehensively evaluate tissue regeneration, histological analysis of the wound tissue collected on Day 14 was performed using H&E and MT staining [83]. H&E staining revealed marked histological differences among the hydrogel-treated groups (Fig. 8A). The wounds in the control group exhibited discontinuous epidermis, dense infiltration of inflammatory cells, and immature dermal tissue, indicating delayed tissue remodeling. In contrast, the COS/PVA/BSII group displayed a well-organized granulation tissue, a fully regenerated epithelial layer, and reduced immune cell infiltration, indicating advanced wound healing and structural restoration. MT staining demonstrated that collagen fibers were sparse and loosely arranged in the PVA and COS/PVA groups (Fig. 8B). By contrast, the COS/PVA/BSI group promoted the formation of denser and more aligned collagen fibers. In contrast, the COS/PVA/BSII group exhibited well-organized collagen distribution throughout the dermis. These findings indicate active ECM remodeling and tissue regeneration. The epithelial gap was measured to quantitatively assess epithelial healing (Fig. 8C). The PVA group exhibited the largest residual gap between wound edges (1,522.36 ± 67.44 µm), followed by the COS/PVA group (1,296.75 ± 67.17 µm). Both COS/PVA/BSI (1,034.55 ± 21.41 µm) and COS/PVA/BSII (780.49 ± 86.02 µm) significantly reduced the epithelial gap, with the COS/PVA/BSII group showing a significantly reduced epithelial gap, indicating superior epithelial regeneration. The re-epithelialization rate was calculated to evaluate the extent of newly formed epidermis at the wound site (Fig. 8D). The COS/PVA/BSII group exhibited the highest re-epithelialization percentage (81.80% ± 1.13%), significantly surpassing that of the COS/PVA/BSI (77.80% ± 1.75%), COS/PVA (70.71% ± 1.96%), PVA (65.56% ± 2.62%), and control (42.34% ± 2.83%) groups. These results confirmed the superior ability of the COS/PVA/BSII hydrogel to promote rapid and complete epithelial barrier restoration. Finally, collagen deposition was quantified in MT-stained tissue sections (Fig. 8E). The COS/PVA/BSII group exhibited the highest collagen content (45.54% ± 1.76%), significantly greater than that of the COS/PVA/BSI (40.16% ± 2.31%), COS/PVA (29.39% ± 2.52%), PVA (26.60% ± 0.91%), and control (21.73% ± 2.85%) groups. In addition, H&E staining of major organs, including the lung, kidney, liver, spleen, heart, and stomach (Fig. S9), revealed no histopathological abnormalities such as necrosis or cytoarchitectural disruption in any of the treatment groups. These results indicated that the COS/PVA/BSII hydrogel was well tolerated in vivo and did not induce systemic toxicity or metabolic dysfunction, thereby confirming its favorable safety profile for treating MRSA-infected wounds.Fig. 8. Histological analysis of MRSA-infected wound tissue following hydrogel treatment. Representative (A) H&E and (B) MT staining images of MRSA-infected wound tissues on Day 14 following treatment with PVA, COS/PVA, COS/PVA/BSI and COS/PVA/BSII. The black and orange scale bars represent 1 mm and 200 µm, respectively; Analysis of (C) epithelial gap, (D) re-epithelialization, and (E) collagen deposition in wound tissues on Day 14 (n = 6). **P < 0.01 and ****P < 0.0001 versus control. ns means no significance versus control. ^#^P < 0.05, ^##^P < 0.01, ^###^P < 0.001, and ^####^P < 0.0001 for PVA, COS/PVA and COS/PVA/BSI versus COS/PVA/BSII.Fig 8 dummy alt text
Immunofluorescence staining for CD31 and VEGF was conducted to assess angiogenesis within the wound tissues on Day 7. As shown in Fig. 9A and 9E, CD31 expression was barely detectable in the control group but gradually increased in the PVA and COS/PVA groups. This finding is supported by previous studies demonstrating that functionalized CS and PVA-based hydrogels promote endothelial cell activation and neovascularization in infected wound models [84,85]. Notably, the BS-reinforced hydrogels (COS/PVA/BSI and COS/PVA/BSII) exhibited markedly stronger CD31-positive signals, indicating enhanced endothelial cell recruitment and the formation of well-defined microvascular structures. Similarly, VEGF, a pivotal growth factor driving endothelial proliferation and neovessel formation, displayed a comparable expression pattern (Fig. 9B and 9F). The BS-reinforced hydrogels showed significantly higher VEGF expression levels than the other groups, suggesting that the presence of BS synergistically promoted angiogenic signaling. These findings demonstrate that BS-reinforced COS/PVA hydrogels create a favorable microenvironment that enhances endothelial cell attachment, migration, and vascular sprouting. Furthermore, the in vivo angiogenic improvement observed aligns with the in vitro tube formation results, where BS-reinforced hydrogels significantly increased the number of nodes, junctions, and overall capillary length. Collectively, these findings indicate that reinforcing BS into COS/PVA hydrogels promotes angiogenesis by upregulating CD31 and VEGF markers, thereby supporting vascular remodeling and tissue regeneration during the infected wound healing process.Fig. 9. Characterization of angiogenesis and macrophage polarization on MRSA-infected the wound tissue. (A-D)Immunofluorescence images showing (A) CD31, (B) VEGF, (C) iNOS, and (D) Arg1 expression in wound tissue at Day 7 following treatment with PVA, COS/PVA, COS/PVA/BSI and COS/PVA/BSII; (E-H) Quantitative analysis of (E) CD31, (F) VEGF, (G) iNOS, and (H) Arg1 fluorescence intensity following treatment with PVA, COS/PVA, COS/PVA/BSI and COS/PVA/BSII. (n = 6). **P < 0.01 and ****P < 0.0001 versus control. ns means no significance versus control. ^#^P < 0.05, ^##^P < 0.01, and ^####^P < 0.0001 for PVA, COS/PVA and COS/PVA/BSI versus COS/PVA/BSII.Fig 9 dummy alt text
During wound healing, macrophages dynamically shift from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype, a transition essential for inflammation resolution and tissue repair. However, in chronic pathological conditions such as MRSA-infected wounds, persistent activation of M1 macrophages and impaired transition toward the M2 phenotype lead to excessive inflammation and delayed processes of re-epithelialization, granulation, and vascularization. Therefore, advanced wound dressings capable of promoting angiogenesis while modulating macrophage polarization are highly desirable for effective treatment of infected wounds [86,87]. Immunofluorescence staining was conducted to evaluate macrophage polarization within the wound sites on day 7. The expression of iNOS, a marker of pro-inflammatory M1 macrophages, showed no significant difference among the control, PVA, and COS/PVA groups but was notably downregulated in the BS-reinforced hydrogels (Fig. 9C and 9G). In contrast, Arg1, a representative M2 macrophage marker associated with anti-inflammatory and regenerative functions, exhibited a slight increase in the COS/PVA group compared to the control and was further upregulated by the concentration-dependent presence of BS in the COS/PVA/BSI and COS/PVA/BSII groups (Fig. 9D and 9H). DAPI staining confirmed uniform nuclear distribution across the groups, and the merged images clearly highlighted opposing patterns of iNOS downregulation and Arg1 upregulation in the COS/PVA/BSI and COS/PVA/BSII groups. These results suggest that the reinforcement of BS into COS/PVA hydrogels effectively suppresses the excessive pro-inflammatory M1 response while enhancing M2 macrophage polarization, thereby promoting an anti-inflammatory and pro-regenerative wound environment. This immunomodulatory balance aligns with previous studies reporting that biomaterials capable of regulating macrophage phenotypes can attenuate inflammation and accelerate tissue regeneration in infected wounds [88]. Taken together, the in vivo data demonstrate that the COS/PVA/BSI and COS/PVA/BSII hydrogels achieve immunomodulation by shifting macrophage polarization, which synergistically contributes to improved vascularization and tissue regeneration in chronic wound settings.
Overall, these results demonstrate that the COS/PVA/BSII hydrogel effectively promotes full-thickness wound healing by accelerating re-epithelialization, enhancing ECM deposition, and restoring the skin architecture. These therapeutic effects were likely attributable to the synergistic action of COS and BS in stimulating the proliferation of fibroblasts, keratinocytes, and vascular endothelial cells, while simultaneously maintaining potent antibacterial activity.
Conclusion
4
In this study, a double-network COS/PVA/BS hydrogel was developed, and its wound-healing properties in MRSA-related wound infections were evaluated. The COS/PVA/BS hydrogel was fabricated through a simple and efficient process, with bacteria-derived BS offering the advantage of a high production yield. The hydrogels exhibited excellent physicochemical characteristics as well as cytocompatibility with HDF, HaCaT, and HUVEC, and demonstrated hemocompatibility. Furthermore, in vitro assays demonstrated enhanced cell proliferation, migration, angiogenesis, and ECM synthesis. In addition, it showed potent antibacterial and antibiofilm activities against both MSSA and MRSA strains. Anti-virulence assessments revealed that the hydrogel effectively suppressed the key virulence factors associated with MRSA wound infections. RT-PCR analysis further elucidated the molecular mechanisms, showing that the COS/PVA/BSII hydrogel downregulated QS and virulence-related genes (agrA, RNAIII, hla and saeR), as well as an antibiotic resistance-associated gene (mecA). In a mouse model study, the hydrogel demonstrated a significant enhancement in the regeneration of full-thickness wounds infected with MRSA. This was achieved by effectively reducing the bacterial load, promoting re-epithelialization, enhancing collagen deposition and vascularization, and encouraging favorable macrophage polarization. Collectively, these factors contributed to restoring the overall integrity of the skin architecture. In summary, the COS/PVA/BS hydrogel exhibits excellent material and biological properties, highlighting its potential as a naturally derived therapeutic platform for treating wounds infected with antibiotic-resistant bacteria.
Conflicts of interest
The authors declare that there is no conflicts of interest.
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