Propolis-functionalized acellular fish skin scaffolds as biotechnological platforms for antimicrobial activity and regenerative wound healing

Abstract

Background

Biotechnological advances in tissue engineering increasingly exploit naturally derived scaffolds as bioactive delivery systems. Acellular fish skin (AFS) provides a collagen-rich extracellular matrix favorable for tissue regeneration but lacks intrinsic antimicrobial activity. Propolis, a polyphenol-rich natural resin, exhibits antimicrobial, antioxidant, and immunomodulatory properties, yet its direct application is limited by burst release and local cytotoxicity. In this study, we aimed to develop and characterize a propolis-functionalized AFS scaffold as a multifunctional biomaterial capable of combining sustained antimicrobial activity with regenerative support for wound-healing applications.

Results

Lipid-preserved Tilapia-derived AFS was biofunctionalized with ethanolic propolis extract (EPE) through a vacuum-assisted impregnation process to obtain an EPE-AFS hybrid scaffold. The biotechnological modification preserved the collagenous architecture and porosity of the matrix, enhanced surface hydrophilicity (contact angle reduced from 91° ± 2° to 67° ± 3°), and maintained mechanical integrity (~ 13 MPa). Spectroscopic and microscopic analyses (FTIR, FE-SEM) confirmed successful deposition of polyphenolic components. The release profile demonstrated sustained polyphenol diffusion over 96 h (> 90% cumulative release). Functionally, cellular assays confirmed enhanced fibroblast adhesion and migration on propolis-functionalized scaffolds. Gene expression analysis revealed transcriptional upregulation of extracellular matrix remodeling and angiogenesis-associated markers (COL1A1, COL3A1, VEGF, TGF-β1), alongside downregulation of inflammation-related transcripts (IL-6, MMP-9). Antibacterial evaluation based on turbidity measurements indicated effective growth inhibition against Staphylococcus aureus and Escherichia coli.

Conclusions

This study demonstrates a biotechnological strategy for functionalizing natural collagen scaffolds with propolis-derived polyphenols, yielding a dual-action biomaterial that combines sustained antibacterial growth suppression with enhanced regenerative-associated signaling in vitro. The EPE–AFS construct represents a promising nature-derived platform for further preclinical investigation in wound-healing applications. While these findings reveal a pro-regenerative and anti-inflammatory transcriptional profile in fibroblasts, additional validation—including endothelial and immune cell functional assays and in vivo wound models—is required to confirm angiogenic, immunomodulatory, and therapeutic efficacy under physiologically relevant conditions.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12896-026-01155-9.

Background

Skin wound healing is a dynamic and tightly regulated process that can be profoundly disrupted by infection and excessive inflammation, particularly in chronic wounds. Breach of the skin barrier—due to trauma, burns, or pathological conditions—facilitates microbial invasion, delays tissue repair, and increases the risk of complications and morbidity [1]. Chronic non-healing wounds affect approximately 1–2% of the population in developed countries, representing a significant and growing clinical burden [2, 3]. A major contributing factor is the persistence of biofilm-forming microorganisms within the wound bed, which sustain inflammatory signaling and impair normal healing cascades [4]. This establishes a self-perpetuating cycle of infection and delayed regeneration, underscoring the need for therapeutic strategies that concurrently address microbial burden and tissue repair.

Conventional wound dressings remain limited in their ability to meet these multifactorial demands. Passive materials such as foams and hydrocolloids primarily maintain moisture balance but lack active antimicrobial or regenerative functions. In contrast, antimicrobial dressings (e.g., silver-based systems) reduce microbial load but do not provide biological cues for tissue repair and may induce cytotoxic effects in host cells [5–7]. These limitations have driven the development of multifunctional wound dressings capable of modulating the wound microenvironment. An optimal advanced dressing should not only maintain hydration and gas exchange but also prevent infection while actively promoting granulation, angiogenesis, and re-epithelialization [8]. Achieving this requires the integration of structural scaffolds with bioactive agents that regulate inflammation and microbial activity. In this context, natural biomaterials are particularly advantageous due to their extracellular matrix (ECM)-mimetic architecture, intrinsic biocompatibility, and chemical versatility [9].

Acellular fish skin (AFS) has recently emerged as a promising regenerative scaffold, as it preserves native ECM ultrastructure while removing immunogenic cellular components. This results in a collagen-rich matrix that supports cell adhesion, migration, and neovascularization [10]. Among available sources, tilapia skin is especially attractive due to its high type I collagen content and well-organized fibrillar architecture, which can be retained following decellularization [11, 12]. Preclinical studies using tilapia-derived acellular dermal matrices have demonstrated accelerated re-epithelialization, enhanced granulation tissue formation, increased angiogenesis, and improved collagen remodeling in full-thickness wound models [12, 13]. Importantly, scalable processing and sterilization approaches have been developed that preserve biocompatibility while supporting translational feasibility, and in vivo evaluations confirm a favorable safety profile [11, 14]. In addition, tilapia skin is widely available as a food-industry byproduct, offering cost-effective and batch-consistent sourcing compared with mammalian-derived matrices [10]. Collectively, these attributes position AFS as a robust ECM-based platform for regenerative wound therapies, particularly when combined with complementary bioactive functionalities.

Propolis, a resinous natural product derived from honeybees, represents a compelling bioactive component for addressing infection and inflammation in wounds. It is rich in polyphenolic compounds, including flavonoids and phenolic acids such as caffeic acid phenethyl ester, which confer broad-spectrum antimicrobial activity against bacteria, fungi, and viruses [15, 16]. Mechanistically, propolis disrupts microbial membranes, inhibits key enzymatic processes, and interferes with biofilm formation [4]. Beyond antimicrobial effects, propolis exhibits well-documented antioxidant and anti-inflammatory properties, largely attributed to its polyphenol content [6, 17]. Through reactive oxygen species (ROS) scavenging and modulation of inflammatory cytokines, propolis may help attenuate the chronic inflammatory state characteristic of non-healing wounds [18]. Furthermore, accumulating evidence indicates that propolis can enhance wound healing by promoting fibroblast proliferation, angiogenesis, and ECM remodeling, while reducing healing time and scar formation [19]. However, direct application of propolis extracts is limited by rapid dilution, uncontrolled release, and potential cytotoxicity at high local concentrations, necessitating controlled delivery strategies.

Integrating AFS with propolis offers a rational approach to developing a multifunctional wound dressing that combines structural and biochemical therapeutic modalities. Incorporation of propolis into biomaterial scaffolds enables sustained release of its bioactive constituents, thereby maintaining prolonged antimicrobial and immunomodulatory activity at the wound site while avoiding burst-associated toxicity [20]. For example, propolis-loaded electrospun nanofibers have demonstrated controlled release profiles with effective antibacterial activity and improved wound healing outcomes in vivo [21]. In the context of AFS, the porous collagen-rich ECM can serve as a biological reservoir that localizes and stabilizes propolis-derived polyphenols, facilitating sustained delivery while preserving scaffold integrity. Concurrently, the antimicrobial and anti-inflammatory actions of propolis can modulate the wound microenvironment, reducing infection and inflammatory stress, thereby enhancing the regenerative performance of the ECM scaffold. This reciprocal interaction supports a synergistic therapeutic paradigm, wherein AFS provides structural and biological guidance, and propolis actively conditions the healing milieu.

Based on this rationale, we developed a propolis-functionalized AFS scaffold as a bioactive wound dressing that integrates infection control with regenerative support. To the best of our knowledge, this represents the first incorporation of plant-derived propolis into a decellularized fish dermal matrix for advanced wound care applications. The present study systematically characterizes the physicochemical properties of the ethanolic propolis extract (EPE)-coated AFS scaffold, evaluates its polyphenol release kinetics, and assesses its biological performance in vitro. We hypothesize that this composite scaffold can achieve sustained polyphenol delivery with potent antibacterial activity while preserving the intrinsic regenerative cues of the fish-derived ECM. Such a dual-function system directly targets the coupled challenges of infection and impaired healing in chronic wounds, offering a promising strategy for next-generation biomaterial-based wound therapy [22].

Methods

Preparation of AFS

Fresh Tilapia skin was obtained from a licensed seafood processing facility and processed within 6 h of harvest. After rinsing with PBS (pH 7.4), scales and subcutaneous fat were removed aseptically. The tissue was immersed in hypertonic saline solution (3× PBS containing 1 M NaCl) at 4 °C for 2 h under gentle agitation (50 rpm). Subsequently, samples were incubated in 0.5% (v/v) Triton X-100 in PBS at 4 °C for 24–36 h. The samples were washed in PBS (3×, 15 min each), then treated with nuclease solution (50 U/mL DNase I, 10 U/mL RNase A in 50 mM Tris-HCl, 10 mM MgCl₂, pH 7.5) for 2–4 h at room temperature. If DNA content exceeded the decellularization benchmark (< 50 ng/mg dry tissue), tissues were exposed to 0.05–0.1% SDS in PBS at 4 °C for 1–2 h, followed by five sequential PBS washes. To prevent lipid oxidation, an antioxidant rinse with PBS supplemented with 0.01% (w/v) α-tocopherol or 0.1 mM ascorbic acid was applied for 15 min. Sterilization was achieved by immersion in 70% ethanol at 4 °C for 2 min, followed by air drying in a Class II biosafety cabinet. Scaffolds were vacuum-sealed under nitrogen and stored at − 20 °C.

Preparation and characterization of EPE

Raw propolis was obtained from a certified apicultural supplier (Urmia, Iran) and stored at 4 °C until use. Propolis powder was extracted in 70% ethanol (30% w/v) at 20–22 °C for 48 h with shaking (100 rpm). Extracts were filtered using Whatman No.1 filter paper and 0.45 μm PTFE membranes, and stored at 4 °C in the dark.

Total phenolic content was quantified by the Folin–Ciocalteu assay at 765 nm using a UV-Vis spectrophotometer (Lambda 25, PerkinElmer, Fremont, CA, USA), expressed as mg gallic acid equivalents (GAE)/mL. HPLC analysis was conducted on an Agilent 1260 Infinity system (Agilent Technologies, Santa Clara, CA, USA) using a C18 column (Phenomenex, Torrance, CA, USA) with detection of CAPE, chrysin, and galangin against standards.

EPE loading and scaffold functionalization

AFS scaffolds were cut into 1 cm² pieces and equilibrated in sterile PBS for 5 min prior to coating. EPE (100 µL/cm²) was applied dropwise under vacuum-assisted impregnation using a laboratory desiccator at 300–500 mbar. This loading was selected because it reproducibly wetted and impregnated the scaffold surface without visible pooling, enabling uniform coating for characterization. At this dose, the scaffold provided a quantifiable polyphenol payload (≈ 8–9 mg GAE released per cm² over 120 h) with sustained release, while maintaining cytocompatibility in HFF-1 cultures. The scaffolds were subsequently air-dried in a biosafety cabinet, followed by overnight vacuum drying.

Residual double-stranded DNA content was determined using the PicoGreen^® assay and quantified on a fluorescence plate reader (Synergy H1, BioTek, Winooski, VT, USA). Surface morphology was examined by field emission scanning electron microscopy (FE-SEM; MIRA3, TESCAN, Brno, Czech Republic) after sputter-coating the samples with gold using a Q150R ES system (Quorum Technologies, Lewes, UK). Chemical functional groups were characterized by Fourier-transform infrared spectroscopy (FTIR) in attenuated total reflectance (ATR) mode on a Nicolet iS50 spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Surface wettability was determined by measuring static water contact angles with a DSA25 goniometer (Krüss GmbH, Hamburg, Germany). Mechanical properties were assessed using an Instron 5565 universal testing machine (Instron, Norwood, MA, USA) equipped with a 100 N load cell. Rectangular specimens (10 mm × 40 mm) with a gauge length of 20 mm were tested at a crosshead speed of 5 mm/min, and stress–strain curves were generated and analyzed with Bluehill software (Instron, Norwood, MA, USA).

In vitro release of EPE phenolics

To evaluate the release kinetics of polyphenols from EPE–coated scaffolds, samples (1 cm², n = 3 per time point) were immersed in 2 mL PBS, pH 7.4, and incubated at 37 °C under constant orbital shaking (60 rpm). At predetermined intervals (3, 6, 12, 24, 48, 72, 96, and 120 h), the entire supernatant was collected and replaced with fresh PBS of equal volume to maintain sink conditions. Total phenolic content in the collected supernatants was quantified by Folin–Ciocalteu assay: 200 µL of sample was mixed with 1 mL of 10% Folin–Ciocalteu reagent (Sigma-Aldrich) and incubated for 5 min, followed by the addition of 800 µL 7.5% sodium carbonate solution. After incubation for 30 min at room temperature in the dark, absorbance was measured at 765 nm using a UV-Vis spectrophotometer (Lambda 25, PerkinElmer, Fremont, CA, USA). Concentrations were calculated against a gallic acid standard curve (0–200 µg/mL; R² > 0.99) and expressed as mg GAE.

Cell culture

Human foreskin fibroblasts (HFF-1, ATCC^® SCRC-1041™, Manassas, VA, USA) were expanded in Dulbecco’s Modified Eagle Medium (DMEM, high glucose; Gibco, Invitrogen, Paisley, UK) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco) and 1% penicillin–streptomycin solution (100 U/mL penicillin, 100 µg/mL streptomycin; Gibco). Cells were maintained in a humidified incubator at 37 °C with 5% CO₂ and 95% relative humidity. Cells were subcultured at 70–80% confluence using 0.05% trypsin–EDTA (Gibco) and passages 5–10 were used for all experiments.

Cytocompatibility (MTT assay)

Cytocompatibility of the scaffolds was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich). HFF-1 cells were seeded into 96-well tissue culture plates (Corning, USA) at a density of 1 × 10⁴ cells/well in 200 µL of complete DMEM and allowed to adhere overnight. Sterilized scaffolds (1 cm²) were placed in experimental wells, while untreated cells served as controls. After 1, 3, and 5 days, 20 µL of MTT solution (5 mg/mL in PBS) was added per well and incubated at 37 °C for 4 h. Formazan crystals were solubilized with 150 µL dimethyl sulfoxide (DMSO, Sigma-Aldrich), and absorbance was recorded at 570 nm with a reference at 630 nm using the Synergy H1 microplate reader (BioTek, Winooski, VT, USA). Results were expressed as percentage cell viability relative to control.

Quantification of scaffold-associated cell number (PicoGreen dsDNA assay)

Scaffold-associated cell number was quantified using the Quant-iT™ PicoGreen^® dsDNA assay (Invitrogen, Thermo Fisher Scientific, USA). HFF-1 fibroblasts were seeded onto sterilized scaffolds at a density of 1 × 10⁵ cells/cm² and cultured under standard conditions for 3 and 5 days. At each time point, samples were gently washed with PBS to remove non-adherent cells and transferred to microcentrifuge tubes. Cells were lysed using a Triton X-100–based lysis buffer, and scaffolds were mechanically agitated to ensure complete DNA release. Lysates were collected, and dsDNA content was quantified according to the manufacturer’s protocol using PicoGreen reagent. Fluorescence was measured using a Synergy H1 microplate reader (BioTek) with excitation/emission wavelengths of 480/520 nm. DNA concentration was determined from a λ-DNA standard curve and expressed as total dsDNA per scaffold. Measurements were performed in triplicate (n = 3).

Cell adhesion and morphology

For SEM, HFF-1 fibroblasts were seeded on scaffolds (1 × 10⁵ cells/cm²) and cultured for 3 and 5 days. Samples were washed with PBS and fixed in 2.5% glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) for 2 h at 4 °C, followed by post-fixation in 1% osmium tetroxide for 1 h. Sequential dehydration was performed in graded ethanol (30–100%), and scaffolds were dried at the critical point (CPD 030, Bal-Tec, Liechtenstein). Surfaces were sputter-coated with gold (Q150R ES, Quorum Technologies, Lewes, UK) and imaged using FE-SEM (MIRA3, TESCAN, Brno, Czech Republic) at an accelerating voltage of 15 kV.

Scratch wound assay

To assess cell migration, HFF-1 cells were seeded into 6-well plates at 2 × 10⁵ cells/well and grown to near confluence. A uniform scratch was created in the monolayer using a sterile 200 µL pipette tip, and the wells were gently washed twice with PBS to remove detached cells. Fresh serum-reduced medium (DMEM containing 1% FBS) was added with or without scaffolds. Wound closure was monitored at 0, 24, and 48 h using an inverted phase-contrast microscope (Eclipse Ti-U, Nikon, Tokyo, Japan). Images were analyzed using ImageJ software (NIH, Bethesda, MD, USA) to calculate the percentage of wound closure according to the formula:

  

where A₀ is the initial wound area and At is the wound area at time t.

Quantitative real-time PCR (qPCR)

Total RNA was extracted from HFF-1 fibroblasts cultured on different scaffolds using the RNeasy Mini Kit (Qiagen, Hilden, Germany) with on-column DNase digestion to eliminate genomic DNA contamination. RNA concentration and purity were assessed spectrophotometrically (NanoDrop 2000, Thermo Fisher Scientific, USA), and samples with A260/A280 between 1.8 and 2.0 were used. Reverse transcription was performed with 1 µg RNA using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) following the manufacturer’s protocol. qPCR reactions were carried out in 20 µL volumes containing 10 µL PowerUp™ SYBR™ Green Master Mix (Thermo Fisher Scientific), 0.4 µM forward and reverse primers, and 2 µL diluted cDNA template. Amplifications were performed on a QuantStudio™ 5 Real-Time PCR System (Thermo Fisher Scientific) under the following conditions: initial denaturation at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Melt curve analysis confirmed specificity of amplification. Relative expression levels were calculated using the 2^–ΔΔCt method with GAPDH as the internal reference gene.

Antibacterial efficacy assay

The antibacterial activity of EPE-AFS was assessed against Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922) using a turbidity assay. Overnight cultures in tryptic soy broth (TSB; BD Biosciences, USA) were adjusted to ~ 1 × 10⁶ CFU/mL (OD600 ≈ 0.1). Treatments included uncoated AFS, EPE-AFS (100 µL/cm²), soluble EPE (phenolic content matched to scaffold), growth control (GC), and gentamicin (10 µg/mL) as a positive control. Sterilized scaffolds (1 cm²) were placed in 96-well plates with bacterial suspensions (200 µL/well) and incubated at 37 °C for up to 24 h.

Optical density at 600 nm (OD600) was recorded at 0, 3, 6, 12, and 24 h (Synergy H1, BioTek, USA). Group-matched bacteria-free blanks (scaffold or EPE in sterile TSB) were subtracted to correct for color and scattering. Growth inhibition (%) was calculated relative to GC:

Statistical analysis

All experiments were performed in triplicate unless otherwise stated, and data are presented as mean ± standard deviation (SD). Statistical analyses were carried out using GraphPad Prism (v9.0). Normality of data distribution was confirmed prior to analysis. Comparisons between two groups were conducted using unpaired Welch’s t-tests, while multiple-group or time-course data were analyzed by one- or two-way ANOVA with Tukey’s post-hoc test. A p-value < 0.05 was considered statistically significant.

Results

AFS ECM characterization by histology and biochemical assays

Histological evaluation of NFS and AFS by H&E and Masson’s trichrome staining revealed clear differences in tissue morphology and cellular content (Fig. 1A). H&E staining of NFS showed dense collagen bundles with abundant cellular components, whereas AFS exhibited complete absence of visible nuclei together with preservation of the overall ECM framework. Masson’s trichrome staining showed intense collagen-associated blue staining in NFS and retained collagen staining in AFS, although with lower density and altered fiber organization after decellularization.

Fig. 1.

Histological evaluation and biochemical composition of NFS and AFS scaffolds. (A) Representative histological images of NFS and AFS stained with H&E (top) and Masson’s trichrome (bottom). Scale bar = 100 μm. (B–D) Quantitative biochemical analysis. (B) Hydroxyproline assay showing collagen retention in AFS with a modest reduction compared with NFS (*p < 0.05). (C) GAG content measured by DMMB assay showing partial GAG loss after decellularization (*p < 0.05). (D) DNA quantification by PicoGreen demonstrating marked removal of cellular material after decellularization. Data are presented as mean ± SD (n = 3). Statistical comparisons were performed using Welch’s t-tests

Biochemical assays further quantified ECM preservation and cellular removal (Fig. 1B–D). Hydroxyproline content decreased from 6.25 ± 0.54 to 5.20 ± 0.27 µg·mg⁻¹ dry tissue, corresponding to a 16.8% reduction. GAG content decreased from 18.3 ± 2.1 to 13.1 ± 1.7 µg·mg⁻¹ dry tissue, corresponding to an approximately 28.4% reduction. DNA content, measured by PicoGreen, decreased from 416.7 ± 35.1 to 11.7 ± 3.5 ng·mg⁻¹ dry tissue, representing an approximately 97.2% reduction.

Morphological and physicochemical characterization of EPE-coated AFS

FE-SEM analysis

FE-SEM analysis showed that uncoated AFS possessed a highly porous fibrillar architecture. Surface images revealed a continuous collagen fiber network with large interconnected voids (Fig. 2A). Cross-sectional images showed a multilayered lamellar structure with visible interlamellar spaces and preserved depth-wise organization (Fig. 2B). Following EPE coating, the overall fibrous structure remained detectable, but the fiber surfaces displayed a roughened, particulate-like layer (Fig. 2C). Some pore openings appeared partially narrowed or bridged by deposited material, although most pores remained open. Cross-sectional SEM of EPE-coated AFS showed preservation of the layered architecture, together with localized material deposition along interlamellar regions and more irregular lamellar contours (Fig. 2D).

Fig. 2.

Multimodal characterization of AFS scaffolds and the effect of EPE coating. (A) Surface (en-face) FE-SEM image of uncoated AFS showing a highly porous, interconnected collagen fibrillar network with open voids. (B) Cross-sectional (through-thickness) FE-SEM image of uncoated AFS revealing the native lamellar/stratified collagen organization. (C) Surface (en-face) FE-SEM image of EPE-coated AFS demonstrating deposition of a roughened particulate layer on collagen fibrils and partial surface pore coverage. (D) Cross-sectional (through-thickness) FE-SEM image of EPE-coated AFS showing preservation of the layered lamellar architecture after coating, with localized infilling along interlamellar spaces. (E) FTIR spectra of propolis (blue), AFS (orange), and AFS–propolis (green). (F) Surface wettability of AFS and EPE-coated scaffolds. (G) Stress–strain profiles of native fish skin, AFS, and EPE-coated AFS

FTIR analysis

FTIR spectroscopy was used to compare the chemical signatures of uncoated AFS, EPE, and EPE-coated AFS (Fig. 2E). The spectrum of uncoated AFS showed characteristic collagen-associated bands, including amide I at approximately 1640 cm⁻¹, amide II at approximately 1540 cm⁻¹, and amide III at approximately 1235 cm⁻¹, together with a broad band near 3300 cm⁻¹. The EPE spectrum exhibited a broad O–H stretching band in the 3350–3300 cm⁻¹ region, aromatic C = C bands around 1600–1500 cm⁻¹, and C–O-related bands near 1260 and 1030 cm⁻¹. The spectrum of EPE-coated AFS contained features attributable to both AFS and EPE, including retained amide I and II bands and increased broadening in the 3200–3500 cm⁻¹ region, together with shoulders in the 1260–1020 cm⁻¹ region.

Surface wettability

Static water contact angle measurements showed that uncoated AFS had a contact angle of 91.2 ± 1.4°, whereas EPE-coated AFS showed a significantly reduced contact angle of 66.7 ± 1.5° (Fig. 2F), indicating increased surface hydrophilicity after coating.

Tensile mechanical properties

Uniaxial tensile testing demonstrated distinct mechanical responses for NFS, AFS, and EPE-coated AFS (Fig. 2G). NFS exhibited the highest tensile strength, reaching approximately 17.8 MPa, with failure strain exceeding 30%. After decellularization, AFS showed a reduced ultimate tensile strength of approximately 12.6 MPa and a failure strain of approximately 17%. EPE-coated AFS displayed a peak stress of approximately 13.4 MPa and a slightly lower failure strain of approximately 15%.

In vitro EPE release profile

The release behavior of propolis-derived phenolic constituents from EPE-coated AFS was quantified by the Folin–Ciocalteu assay and expressed as GAE over 120 h (Fig. 3A). At 3 h, the scaffold released 0.82 ± 0.09 mg GAE, corresponding to 11.0% of the soluble EPE reference measured at the same time point (7.43 ± 0.26 mg GAE). Cumulative release increased to 22.5% at 6 h, 34.4% at 12 h, and 50.5% at 24 h. Release then continued progressively, reaching 68.4% at 48 h, 81.5% at 72 h, and 90.6% at 96 h. At 120 h, cumulative release reached 8.34 ± 0.31 mg GAE, corresponding to approximately 93.1% of the soluble EPE reference at the same time point (8.96 ± 0.02 mg GAE).

Fig. 3.

Cumulative release kinetics of propolis phenolics and in vitro cytocompatibility of AFS scaffolds. (A) Cumulative release of total phenolic content, expressed as GAE, from EPE-coated AFS scaffolds versus soluble EPE control in PBS at 37 °C over 120 h. Data are mean ± SD (n = 3). (B) Fibroblast metabolic activity assessed by MTT assay at days 1, 3, and 5 across control, uncoated AFS, EPE solution, and EPE-coated AFS scaffolds. Data are mean ± SD (n = 3); **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. control. (C) Quantification of scaffold-associated cell number by PicoGreen dsDNA assay at days 3 and 5. Data are mean ± SD (n = 3); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. control

In vitro cytocompatibility

MTT assay

The MTT assay showed a time-dependent increase in fibroblast metabolic activity in all groups (Fig. 3B). Cells cultured on uncoated AFS exhibited significantly higher metabolic activity than the no-scaffold control by day 5. EPE-coated AFS further increased cell viability, showing approximately 15% higher activity than uncoated AFS at day 5. Cells treated with free EPE solution showed the highest viability at each time point, with approximately 8% higher MTT absorbance than the EPE-coated AFS group at day 5. All groups maintained viability above the 70% threshold for non-cytotoxicity according to ISO 10993-5.

Scaffold-associated cell number by PicoGreen dsDNA assay

PicoGreen dsDNA quantification was performed at days 3 and 5 to assess scaffold-associated cellular accumulation (Fig. 3C). At day 3, the EPE-coated AFS group exhibited lower dsDNA content than uncoated AFS. By day 5, dsDNA content increased in both scaffold groups, and EPE-coated AFS exceeded uncoated AFS, indicating greater scaffold-associated cellular accumulation at the later time point.

Fibroblast adhesion and surface colonization

FE-SEM was used to evaluate fibroblast adhesion and matrix deposition on control, uncoated AFS, and EPE-coated AFS at days 3 and 5 post-seeding (Fig. 4). At day 3, the control and uncoated AFS groups showed extensive cellular spreading and interconnected fibrillar surface features. In contrast, EPE-coated AFS showed reduced early surface coverage with sparse fibrillar deposition. By day 5, the control and uncoated AFS surfaces were largely covered by dense cellular and matrix layers. The EPE-coated AFS group also showed increased surface coverage, improved cell spreading, and more continuous matrix deposition relative to day 3.

Fig. 4.

FE-SEM evaluation of fibroblast adhesion and ECM deposition on scaffolds at days 3 and 5 post-seeding. Representative FE-SEM images showing fibroblast morphology and surface-associated matrix deposition on control, uncoated AFS, and EPE-coated AFS at day 3 and day 5 after seeding. Images illustrate temporal changes in cell attachment, spreading, and ECM coverage across the different scaffold conditions

Scratch wound assay

Scratch wound assays were performed using HFF-1 fibroblasts cultured under four conditions: control, uncoated AFS, EPE solution, and EPE-coated AFS (Fig. 5). At 0 h, all groups showed comparable initial wound widths. At 24 h, both EPE-treated groups exhibited significantly greater wound closure than control (p < 0.05), while uncoated AFS showed an intermediate response. Soluble EPE produced the greatest early closure (p < 0.01 vs. control). EPE-coated AFS also significantly enhanced wound closure relative to control (p < 0.01). At 48 h, soluble EPE showed the greatest closure (p < 0.001 vs. control), followed by EPE-coated AFS (p < 0.001 vs. control) and uncoated AFS (p < 0.01 vs. control), whereas the control group showed the slowest closure.

Fig. 5.

Effect of propolis delivery and AFS scaffolds on fibroblast wound closure in vitro. Representative phase-contrast micrographs of scratch wound assays performed in HFF-1 human dermal fibroblasts cultured under four conditions (control, uncoated AFS, EPE solution, and EPE-coated AFS) at 0, 24, and 48 h (left). Red dashed lines indicate the initial wound boundaries at 0 h and are carried forward to subsequent time points for visual comparison. Quantitative analysis of wound closure is shown on the right. Data are presented as mean ± SD (n = 3 independent experiments). Statistical significance was assessed by two-way ANOVA followed by Tukey’s multiple-comparison post hoc test (*p < 0.05, **p < 0.01, ***p < 0.001). Scale bar: 200 μm

Transcriptional responses of fibroblasts analyzed by qRT-PCR

Quantitative RT-PCR analysis was performed in HFF-1 fibroblasts cultured under control, uncoated AFS, EPE solution, and EPE-coated AFS conditions (Fig. 6). Uncoated AFS increased the expression of TGF-β1, COL1A1, FN1, MMP-2, and MMP-9 relative to control. Propolis-treated groups showed increased expression of VEGF-A and Ki67 relative to untreated and uncoated scaffold groups. Direct comparison between delivery modes showed that VEGF-A and Ki67 expression were higher in the EPE solution group than in the EPE-coated AFS group (p < 0.05 for both genes). For ECM-associated genes, TGF-β1 and COL1A1 did not differ significantly between EPE solution and EPE-coated AFS at the measured endpoint, whereas FN1 expression was modestly but significantly lower in the EPE-coated AFS group.

Fig. 6.

Transcriptional profiling of fibroblasts cultured with uncoated AFS, EPE solution, and EPE-coated AFS. Quantitative RT-PCR analysis of HFF-1 fibroblasts under four culture conditions (control, uncoated AFS, EPE solution, and EPE-coated AFS). Panels show genes related to proliferation/angiogenesis (VEGF-A, Ki67), ECM synthesis/remodeling (TGF-β1, COL1A1, FN1), matrix remodeling (MMP-2, MMP-9), pro-inflammatory cytokines (IL-1β, TNF-α, IL-6), inflammation-associated mediators (COX-2, iNOS, NF-κB p65), and anti-inflammatory/antioxidant response (IL-10, Nrf2, HO-1). Data are presented as fold change relative to untreated control (mean ± SD, n = 3 independent experiments). Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple-comparison post hoc test (*p < 0.05, **p < 0.01, ***p < 0.001)

Pro-inflammatory cytokines (IL-1β, TNF-α, IL-6) and inflammation-associated mediators (COX-2, iNOS, NF-κB p65) were reduced in the propolis-treated groups relative to control. When the two propolis groups were compared directly, some inflammatory markers, including IL-1β and NF-κB p65, were slightly higher in EPE-coated AFS than in EPE solution, although both remained below control levels. MMP-9 expression was lower in EPE-coated AFS than in EPE solution. Propolis treatment also increased expression of IL-10, Nrf2, and HO-1. Among these, IL-10 expression was higher in the EPE solution group than in the EPE-coated AFS group.

Antibacterial growth inhibition assessed by turbidity analysis

Antibacterial activity was evaluated against S. aureus and E. coli using blank-corrected OD₆₀₀ measurements after 24 h incubation (Fig. 7). For S. aureus, EPE-coated AFS reduced blank-corrected turbidity to 0.207 ± 0.025, corresponding to 75.1 ± 3.3% inhibition relative to the growth control (0.833 ± 0.025). The phenolic-equivalent soluble EPE condition produced 76.0 ± 3.6% inhibition (0.200 ± 0.030). Uncoated AFS showed only minor inhibition of approximately 5.5 ± 2.1%. Gentamicin produced ≥ 97% inhibition.

Fig. 7.

Antibacterial evaluation of EPE-coated AFS against S. aureus and E. coli. Bacterial growth was assessed by turbidity (OD₆₀₀) after 24 h incubation with the indicated treatments. The soluble EPE group was matched to the scaffold phenolic payload, and gentamicin (10 µg/mL) was used as a positive control. Antibacterial activity was determined using blank-corrected OD₆₀₀ measurements. Data are presented as mean ± SD (n = 3). Statistical analysis was performed as indicated in the figure; ***p < 0.001 and ****p < 0.0001 vs. growth control

For E. coli, EPE-coated AFS achieved 71.2 ± 2.7% inhibition with an OD₆₀₀ value of 0.260 ± 0.020, while the soluble EPE condition achieved 69.0 ± 2.6% inhibition with an OD₆₀₀ of 0.280 ± 0.020. Uncoated AFS showed approximately 6% inhibition (0.850 ± 0.010), and gentamicin again produced ≥ 97% inhibition. The soluble EPE condition was matched to the scaffold’s phenolic payload, and no significant difference in OD₆₀₀ was detected between soluble EPE and EPE-coated AFS at the 24 h endpoint.

Discussion

This study presents a multifunctional ECM-based platform that integrates a structurally preserved AFS scaffold with a propolis-derived bioactive coating, enabling coordinated regulation of regeneration and antimicrobial activity. The data support a model in which the collagen-rich scaffold provides a permissive microenvironment for cellular integration, while the propolis coating introduces sustained biochemical cues that modulate inflammation, oxidative stress, and tissue remodeling.

Decellularization is central to scaffold performance, requiring effective removal of immunogenic components while preserving ECM architecture. The observed structural and compositional features are consistent with detergent–enzymatic protocols that preferentially eliminate nucleic acids and soluble proteoglycans while retaining fibrillar collagen [23, 24]. While collagen preservation maintains mechanical integrity and cell–matrix signaling, the reduction in GAGs reflects a known trade-off, as GAG depletion may impair growth factor retention and matrix hydration [25]. Within this context, the introduction of a bioactive coating represents a rational strategy to compensate for biochemical losses without disrupting structural integrity.

The AFS scaffold retained a porous, lamellar architecture characteristic of fish dermis, which is favorable for cell infiltration and nutrient transport. Importantly, EPE coating preserved this hierarchical structure while introducing surface roughness and localized pore modulation. Such topographical modification is known to influence protein adsorption and integrin-mediated adhesion, potentially enhancing interfacial bioactivity without compromising bulk permeability [26–28]. This balance between structural preservation and surface functionalization is critical for maintaining scaffold performance in regenerative applications.

FTIR analysis indicates that propolis components associate with the scaffold through noncovalent interactions, including hydrogen bonding and hydrophobic interactions, without altering the collagen backbone [29–31]. This interaction mode is advantageous, as it preserves ECM conformation while enabling reversible binding and controlled release of polyphenolic compounds. Polyphenol–protein affinity is well documented in biomaterials systems and is directly linked to sustained release behavior in natural extract-functionalized scaffolds.

Surface wettability plays a pivotal role in early biological responses. The shift toward moderate hydrophilicity following EPE coating falls within the optimal range for protein adsorption and integrin engagement. Polyphenol-induced increases in surface polarity, driven by hydroxyl-rich functional groups, have been widely associated with improved cell–material interactions and enhanced cytocompatibility [32, 33]. This physicochemical modification complements the biochemical activity of propolis, reinforcing the functional relevance of the coating at the biointerface.

Mechanical characterization further supports scaffold suitability. Although decellularization reduces tensile strength due to disruption of native crosslinking and GAG loss, the scaffold retains mechanical properties within ranges relevant for dermal substitutes [34, 35]. The partial recovery of tensile strength after EPE coating is consistent with phenolic-mediated interactions that enhance intermolecular cohesion within collagen networks [36]. Such reinforcement, coupled with maintained structural integrity, supports handling stability and potential translational applicability.

A defining feature of the EPE–AFS system is its sustained release profile. The release kinetics are consistent with diffusion-controlled transport coupled with reversible polyphenol–matrix interactions. This behavior aligns with prior studies demonstrating prolonged release of polyphenols from ECM-like substrates [20, 21]. Importantly, the absence of an excessive burst release suggests controlled delivery that may reduce cytotoxicity associated with high local concentrations. Sustained release is particularly advantageous in chronic wound environments, where prolonged modulation of inflammation and microbial activity is required.

Biologically, the AFS scaffold provides a favorable substrate for fibroblast activity, consistent with the established role of collagen matrices in supporting dermal cell adhesion and proliferation [10]. Propolis functionalization further enhances this response, likely through antioxidant and immunomodulatory effects mediated by polyphenolic constituents such as CAPE, chrysin, and galangin [37, 38]. These compounds are known to regulate oxidative stress and inflammatory signaling, thereby promoting a pro-regenerative microenvironment. Scaffold-mediated delivery enables sustained exposure to these bioactives, which may be critical for maintaining therapeutic activity over time.

The temporal modulation of cell–material interactions observed in this system highlights the importance of delivery kinetics. The initial attenuation of cell adhesion followed by enhanced cellular accumulation suggests that polyphenol coatings can regulate early cell–surface interactions while supporting subsequent proliferation. Such delayed yet sustained cellular engagement may be beneficial in wound healing, where excessive early fibroblast activity can contribute to fibrosis. Temporal control of bioactive presentation is therefore an important design parameter in regenerative biomaterials.

Fibroblast migration, a key determinant of wound closure, was enhanced in the presence of propolis, consistent with reported effects of polyphenols on cytoskeletal dynamics and matrix remodeling pathways, including FAK and MMP signaling [10, 37]. The collagen-rich scaffold further supports directional cell migration by providing a structurally organized substrate. The combined influence of biochemical and structural cues enables coordinated regulation of cellular motility and matrix deposition.

At the transcriptional level, fibroblast responses reflect the combined influence of ECM-derived signals, propolis bioactivity, and delivery mode. Distinct gene expression patterns observed between soluble and scaffold-mediated propolis exposure suggest that cellular signaling is sensitive to temporal and spatial presentation of bioactive compounds. Such delivery-dependent modulation is consistent with the kinetics-driven nature of ligand–receptor interactions and downstream signaling pathways.

The anti-inflammatory effects of propolis are supported by reduced expression of NF-κB–associated mediators and pro-inflammatory cytokines, consistent with extensive evidence of polyphenol-mediated NF-κB inhibition [39, 40]. Concurrent upregulation of antioxidant pathways, including Nrf2 signaling, indicates activation of cytoprotective responses. This coordinated modulation of inflammatory and oxidative pathways is particularly relevant in chronic wounds, where persistent inflammation and oxidative stress impede healing.

Matrix remodeling dynamics are also influenced by propolis treatment. Regulation of MMP expression suggests a controlled balance between ECM degradation and synthesis, which is essential for effective tissue repair. Excessive proteolysis is a hallmark of non-healing wounds, and its modulation represents a critical therapeutic target. The observed effects are consistent with previous reports linking propolis to MMP regulation during wound healing [41].

The antibacterial activity of the EPE–AFS scaffold demonstrates effective growth suppression against both Gram-positive and Gram-negative bacteria. This broad-spectrum activity reflects the multi-target mechanisms of propolis-derived polyphenols, including membrane disruption, enzyme inhibition, and oxidative stress induction [42]. Notably, scaffold-mediated delivery achieves antibacterial efficacy comparable to soluble propolis at the evaluated time point while providing sustained release. This sustained exposure is particularly relevant for preventing biofilm formation and maintaining antimicrobial activity in wound environments.

Methodologically, the use of blank-corrected turbidity measurements ensures reliable quantification of antibacterial activity in chromophoric and matrix-containing systems [43–45]. While turbidity assays do not distinguish between bacteriostatic and bactericidal effects, they provide an appropriate first-line evaluation for release-based biomaterials. Further studies incorporating CFU enumeration and biofilm models will be necessary to fully characterize antimicrobial mechanisms and efficacy.

Conclusion

This study presents a biotechnological strategy for functionalizing AFS scaffolds with propolis-derived polyphenols to generate a bioactive ECM platform integrating antibacterial activity with regeneration-associated cellular responses in vitro. Vacuum-assisted impregnation enabled uniform, non-destructive coating while preserving collagen architecture, porosity, and mechanical integrity, and supported sustained release of phenolic constituents over multiple days. The EPE–AFS construct maintained fibroblast viability, enhanced migration and ECM-related gene expression, upregulated angiogenesis-associated signaling (VEGF), and attenuated inflammation- and matrix-remodeling–related mediators (IL-6, MMP-9). Concurrently, the scaffold demonstrated broad-spectrum antibacterial growth inhibition against both Gram-positive and Gram-negative bacteria under a sustained-release profile.

Collectively, these findings support EPE–AFS as a multifunctional, in vitro–validated biomaterial that combines localized antibacterial exposure with a pro-regenerative cellular microenvironment. The antibacterial effect was attributable to propolis functionalization, while the preserved collagen matrix provided structural support for cell attachment and migration. This dual functionality highlights the potential of ECM-based delivery systems to couple infection control with regenerative support within a single platform.

This work is intentionally limited to an in vitro proof-of-concept. Accordingly, in vivo validation is required to assess wound healing efficacy, host response, scaffold degradation, and antibacterial performance under physiologically relevant conditions. Future studies will focus on standardized full-thickness wound models, including infected settings, with evaluation of wound closure, tissue regeneration, angiogenesis, inflammatory response, and bacterial burden. Complementary mechanistic studies and translation-oriented optimization will be necessary to define the therapeutic potential of sustained polyphenol delivery within ECM scaffolds for advanced wound care.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

SB and SM were responsible for the methodology, investigation, and preparation of the original draft. NZ and AAS performed the data curation, project administration and formal analysis. MD and YP supervised the project, managed the overall workflow, and contributed to the writing, review, and editing of the manuscript.

Funding

No funding.

Data availability

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

Declarations

Ethics approval and consent to participate

This study was approved by the Ethics Committee of Semnan University of Medical Sciences, Semnan, Iran (Approval code: IR.SEMUMS.REC.1401.333). Informed consent to participate was not applicable because this study did not involve human participants, human tissue, or identifiable human data. This study did not involve human participants, human tissue, or identifiable human data; therefore, ethical approval and informed consent were not required. All experiments were conducted using commercially available human fibroblast cell lines (HFF-1, ATCC^® SCRC-1041™), animal-derived acellular tilapia skin obtained from a licensed seafood supplier, and standard laboratory bacterial strains (Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922). All procedures involving cell culture, bacterial handling, scaffold sterilization, and in vitro biological testing were performed in accordance with the institutional biosafety guidelines and standard laboratory biosafety practices of Urmia University of Medical Sciences.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.