The Use of Biomass from In Vitro Fungal Cultures as a Bioactive Ingredient with Antimicrobial Activity in Hydrogel Dressings

Abstract

Background/Objectives: Chronic wounds represent a significant clinical burden and require multimodal treatment strategies targeting inflammation, infection, moisture balance, and tissue remodeling, as defined by the TIME framework. This study aimed to evaluate the therapeutic potential of innovative hydrogel dressings enriched with fungal biomass, designed to exploit natural bioactive compounds—such as antimicrobial peptides and proteolytic enzymes—to enhance wound healing while maintaining high biocompatibility. Methods: Hydrogel dressings incorporating selected fungal biomasses were fabricated and characterized for physicochemical and biological performance. Key material properties relevant to wound care, including hydrophilicity and porosity, were analyzed to assess exudate management capacity and maintenance of a moist wound environment. Antimicrobial activity was tested against common wound pathogens, and species–pathogen interactions were evaluated using generalized linear modeling. In vitro biocompatibility was assessed using human keratinocytes and compared with conventional silver nanoparticle–based dressings. Results: The developed hydrogels demonstrated properties suitable for clinical application, including superhydrophilicity and high porosity, supporting effective exudate control and moisture retention. Significant broad-spectrum antimicrobial activity was observed, particularly against Staphylococcus aureus and Pseudomonas aeruginosa, with effects dependent on fungal species. Statistical modeling revealed highly significant interactions between fungal species and pathogens in inhibition zones (p < 0.001). Hydrogels containing Pleurotus ostreatus and Agaricus bisporus showed broad activity against Escherichia coli, P. aeruginosa, and S. aureus, whereas Enterococcus faecalis exhibited resistance. Fungal biomass–based dressings displayed superior keratinocyte biocompatibility compared to silver nanoparticle controls. Conclusions: Fungal biomass–reinforced hydrogels offer a promising, safer, multifunctional alternative for infected chronic wound management, supporting both antimicrobial action and tissue regeneration.

1. Introduction

Wound healing is a complex process involving multiple stages, including cellular stimulation, maintenance of homeostasis, anti-inflammatory responses, angiogenesis, epithelial proliferation, and remodeling of the wound bed [1,2,3,4]. Although various types of wound dressings are available, developing new therapeutic strategies for hard-to-heal (chronic) wounds remains a major medical challenge [5,6]. It is extremely challenging to design dressings that fulfill all the criteria of the TIME acronym: Tissue (debridement of necrotic tissue), Inflammation (control of microbial infection), Moisture (maintenance of an optimal wound environment), and Edge (promotion of cell proliferation to initiate healing) [7,8,9].

Hydrogels are widely recognized as advanced wound dressing materials due to their unique physicochemical characteristics and high applicability in wound management. Their three-dimensional, hydrophilic polymer networks enable efficient water absorption and retention, ensuring a moist wound environment that is critical for accelerating healing processes, supporting autolytic debridement, and reducing patient discomfort. Furthermore, hydrogels exhibit good biocompatibility, high permeability to oxygen, and the ability to conform closely to irregular wound surfaces. Importantly, hydrogel matrices can serve as effective carriers for bioactive substances, allowing their uniform distribution and controlled release at the wound site [10,11]. Owing to these properties, hydrogels provide versatile platforms for the development of multifunctional wound dressings. Consequently, researchers continue to seek natural biomaterials that can be incorporated into hydrogel systems as multifunctional agents offering the key characteristics outlined in the TIME strategy [12,13,14]. This has led to increased interest in innovative approaches based on natural components such as chitin and chitosan [15,16,17,18]. In particular, products derived from higher fungi—which are capable of uptake and accumulation of metabolites with toxic, stimulatory, microstatic, and microbicidal activity, including chitosan derivatives—exhibit multiple properties that support wound healing and promote effective bacterial eradication [19,20].

One key feature of fungal mycelium is its ability to absorb and accumulate diverse toxic environmental metabolites. Fungi produce various microstatic and microbicidal substances, including chitosan derivatives and antimicrobial peptides [21,22,23]. Additionally, fungal mycelium is rich in minerals and vitamins that support cellular nutrition when properly hydrated [24,25,26]. Fungi also produce abundant proteolytic enzymes that aid in digesting necrotic tissue [27,28,29].

However, current research on fungal-based dressings has predominantly focused on chemically extracted components or single-species isolates. Unlike existing mycelium-based biomaterials that mainly utilize the structural properties of mycelium, the proposed methodology focuses on the biological functionality and mechanical properties of the biomass. The fungal material is incorporated into a hydrogel as a bioactive phase rather than as a structural scaffold, representing a clear conceptual distinction from current mycelium-based materials.

There remains a significant gap in the literature regarding the direct incorporation of whole fungal biomass into hydrogel matrices without extensive extraction steps. Specifically, a comprehensive, comparative evaluation of how the biomass of different fungal species influences the physicochemical stability and biological efficacy of hydrogels is lacking. Understanding these species-specific variations is crucial for advancing beyond existing solutions and optimizing the therapeutic potential of myco-materials in wound care. Consequently, this experimental study incorporated biomass from in vitro cultures of eight fungal species—including four Pleurotus species (Pleurotus citrinopileatus, P. djamor, P. ostreatus, P. pulmonarius) and Agaricus bisporus, Boletus edulis, Hericium erinaceus, Lentinula edodes—into hydrogel wound dressings. The fungal species included in this study were selected based on their well-documented capacity for rapid mycelial growth under in vitro conditions, high adaptability to modified culture media, and proven ability to accumulate macro- and microelements. The materials underwent physicochemical characterization, including surface microstructure analysis (Scanning Electron Microscopy—SEM), specific surface area measurement (Brunauer–Emmett–Teller—BET), and surface profilometry. Additionally, absorbency, contact angle, and surface tension of the materials were measured. The thermal stability of the formulations was assessed using differential scanning calorimetry (DSC) and thermogravimetric analysis (TG). Efficacy was further confirmed via microbiological assays against Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, and Pseudomonas aeruginosa, alongside cytotoxicity tests.

2. Results

2.1. Characterization of Fungal Biomass

The specific BET surface area of the tested fungal biomass was as follows: P. citrinopileatus (Pc)—1.7 m²/g, P. djamor (Pdj)—2.1 m²/g, P. ostreatus (Po)—14.2 m²/g, P. pulmonarius (Pp)—3.6 m²/g, A. bisporus (Ab)—10.1 m²/g, B. edulis (Be)—1.1 m²/g, H. erinaceus (He)—6.8 m²/g, and L. edodes (Le)—7.3 m²/g. The characteristic parameters for the analyzed biomass are summarized in

Table 1. Total pore volume (Vₘ), micropore surface area (Sₘ), and average pore diameter (Aₚ) determined for biomass obtained from in vitro cultures.

Biomass/ Parameters	Pc	Pdj	Po	Pp	Ab	Be	He	Le
Vm, cm3/g	0.002	0.003	0.028	0.011	0.021	0.001	0.019	0.024
Sm, m2/g	0.3	0.4	4.2	1.7	3.6	0.2	4.4	3.8
Ap, nm	6	13	11	12	7	5	10	12

Abbreviations: Pc—P. citrinopileatus, Pdj—P. djamor, Po—P. ostreatus, Pp—P. pulmonarius, Ab—A. bisporus, Be—B. edulis, He—H. erinaceus, Le—L. edodes, V_m—pore volume, A_p—average pore diameter, S_m—micropore area.

.

Figure 1 shows SEM images of the biomass obtained from in vitro cultures.

The analyzed scanning electron microscope (SEM) images reveal different surface topography/morphology of in vitro cultured biomass obtained from different fungal species.

Figure 1a–d show SEM micrographs of fungal-biomass obtained from in vitro cultures of fungi belonging to the genus Pleurotus spp.: (a) P. citrinopileatus (Pc), (b) P. djamor (Pdj), (c) P. ostreatus (Po), and (d) P. pulmonarius (Pp). All samples exhibit a highly porous, fibrous, and irregular structure—typical of fungal-biomass composed of densely intertwined hyphae. Bundles of thin, thread-like filaments corresponding to hyphal fragments are clearly visible, with their distribution and thickness varying by species. The spaces between the filaments form a system of micro- and macropores that influence physicochemical properties of the biomass, such as the BET surface area (see Section 2.1). In Figure 1b,c, compacted clusters and agglomerated fragments can be observed, which may indicate partial adhesion of hyphae during drying. In contrast, the fungal biomass of the remaining species (Figure 1e–h) exhibits a more heterogeneous surface morphology—ranging from fibrous and porous structures with loosely arranged filaments (e,g) to more compact and aggregated forms (f,h). These microstructural differences reflect the inherent morphological variability among the analyzed fungal species.

The analysis reveals significant differences in porous structure among mushroom species, which likely impact functional properties such as absorptivity and sorption capacity (Figure 2). Some species exhibit considerably higher porosity and specific surface area than others. Species showing the highest values for most parameters include P. ostreatus, A. bisporus, L. edodes, and H. erinaceus (orange-red areas). This suggests a well-developed porous structure, large specific surface area, and high absorption capacity in these species. In contrast, P. djamor, P. citrinopileatus, and B. edulis (primarily purple areas) display significantly lower values, reflecting low porosity, limited micropore surface area, and reduced sorption capacity. P. pulmonarius occupies an intermediate position, with moderate specific surface area and microporosity, yet relatively high total pore surface area and good absorption capacity.

2.2. Surface Wettability Analysis

Biomass-hydrogels containing 1 to 5 wt% of lyophilized fungal biomass from different species were tested for wettability. Various polar liquids (H₂O, diiodomethane CH₂I₂) and non-polar liquids (seed oil, bovine serum) were used. The results show that adding 5% lyophilized fungalbiomass improves hydrophilic properties in all samples (Figure 3). Furthermore, incorporating 5 wt% H. erinaceus enhances both hydro- and oleophilic properties, producing a superhydrophilic wound-contact surface (contact angles ~0°) with polyester backing for clinical handling.

The reference sample—a plain-hydrogel without additives—was characterized by contact angle values: diiodomethane (CH₂I₂) averaged 52.64°, oil 81.06°, bovine serum 63.36°, and water (H₂O) 68.3°. Hydrogels with 1 wt% lyophilized biomass showed decreases in static contact angle for all liquids, averaging reductions of 10° to 17°. The lowest contact angles were observed in biomass-hydrogels containing lyophilized H. erinaceus biomass. Contact angles decreased from averages of 36.96° (diiodomethane), 19.93° (oil), 28.46° (bovine serum), and 0° (water) at 1 wt% biomass addition to values close to 0° for all liquids at 5 wt% addition (Figure 4).

Particle Size Distribution Analysis

Freeze-dried fungal biomass exhibited species-specific particle size distributions via laser diffraction (Mastersizer 2000), reported as D10, D50, D90, and span = (D90 − D10)/D50 (Supplementary Table S1).

-

P. ostreatus: D50 = 18.2 ± 2.1 μm (span 1.4), finest distribution enabling optimal hydrogel dispersion,

-

A. bisporus: D50 = 22.6 ± 3.4 μm (span 1.6),

-

H. erinaceus: D50 = 25.8 ± 2.8 μm (span 1.5),

-

B. edulis: D50 = 44.7 ± 5.2 μm (span 2.1), coarsest.

Narrow spans (1.4–2.1) confirm reproducible milling. Smaller particles correlated with higher BET surface areas (r = 0.82, p < 0.01) and antimicrobial zones (r = 0.76, p < 0.05), validating bioactivity enhancement.

2.3. Thermal Analysis

2.3.1. DSC Analysis

The DSC curves show the effect of adding 1 and 5 wt% of fungal biomass derived from in vitro cultures on the thermal properties of the hydrogel (Figure 5).

Addition of 1 wt% Biomass

All samples display a similar overall pattern (Figure 6a), but differences in the temperature and shape of endothermic effects reveal distinct interactions between each biomass and the polymer matrix. In the range of 75–110 °C, a gently declining endothermic signal is observed, associated with the evaporation of free and weakly bound water. Variations in intensity suggest that fungal biomass from different fungal species affects hydrogel water retention, reflecting differences in polysaccharide-protein composition. The most pronounced endothermic effect appears in the range of 110–130 °C and constitutes the main thermal transition in the tested samples. The minimum of this peak occurs around 120–127 °C for most samples; however, A. bisporus and P. ostreatus show a shift toward lower temperatures, suggesting lower hydrogen bond stability and weaker interaction of the biomass with the polymer network. In contrast, samples P. citrinopileatus, P. pulmonarius, and B. edulis exhibit higher minimum temperatures and a deeper endothermic effect, which may indicate stronger integration of the fungal biomass with the hydrogel and greater thermal stability of this modified structure. H. erinaceus, L. edodes, and P. djamor exhibit distinct kinks and additional small peaks, indicating the presence of overlapping thermal transitions resulting from the heterogeneous nature of the fungal biomass.

In the 130–155 °C range, all samples return to baseline, although the rate of this process and the shape of the curves differ among variants. Sample Po (P. ostreatus) exhibits a prolonged, gradual return to baseline, indicating an extended thermal transition, such as proteins, β-glucans, and chitin. Sample of P. ostreatus (Po) exhibits a prolonged, gradual return to baseline, indicating an extended thermal transition. DSC curve analysis confirms that even small amounts of biomass significantly alter hydrogel properties by modifying network order, thermal stability, and water-binding capacity. Sample differences indicate species-specific interactions with the hydrogel matrix, likely driven by variations in polysaccharides, proteins, and secondary metabolites. Overall, the results demonstrate that fungal biomass is an active modifier of hydrogel structure, affecting its physicochemical properties and potential applications.

Addition of 5 wt% Biomass

The DSC curves obtained for hydrogels containing a 5 wt% addition of various fungal biomasses reveal a distinct endothermic process occurring throughout the heating range of 75–175 °C. This behavior is characteristic of hydrated polymeric systems and is primarily attributed to the release and evaporation of bound water as well as structural relaxation within the hydrogel network. Although all samples follow a similar general trend, the depth and position of the endothermic minima vary depending on the fungal biomass incorporated, indicating differences in water-binding capacity and the extent of interactions between the biomass components and the hydrogel matrix.

The most pronounced endothermic effects are observed for samples modified with biomasses labeled Pc, Pdj, and Pp. These curves exhibit deeper minima (approximately −10 to −13 mW), suggesting a higher proportion of strongly bound water and a more compact, hydrophilic internal structure. Such a response implies that these particular biomasses interact more effectively with the polymer network, likely due to the presence of polysaccharides or other hydrophilic constituents capable of enhancing water retention.

In contrast, the samples containing biomasses Ab, Be, He, Le, and Po show shallower minima, typically between −5 and −8 mW. The reduced endothermic intensity indicates weaker binding of water molecules within the plain hydrogel structure, which may reflect a lower degree of chemical compatibility or physical interaction between these types of fungal biomasses and the hydrogel matrix.

As temperature increases beyond the minimum point, all DSC curves demonstrate a gradual rise in heat flow, corresponding to the completion of water-loss processes and the stabilization of the polymer structure. Importantly, no additional transitions are observed in the higher temperature range, suggesting that none of the fungal biomass additives compromise the thermal stability of the plain hydrogels. The convergence of the curves above approximately 140 °C further supports the conclusion that the incorporated fungal biomasses do not induce thermal degradation or structural destabilization within the tested temperature window.

Overall, the DSC analysis indicates that the type of fungal biomass plays a significant role in modulating the thermal and hydration-related properties of the plain hydrogel, primarily by affecting the amount and strength of bound water. However, despite these differences, all formulations maintain comparable thermal stability, demonstrating the compatibility of fungal biomass additives with the hydrogel system.

2.3.2. TG and DTG Analysis

The TG and DTG curves show the effect of adding 1 and 5 wt% of fungal biomass, derived from in vitro cultures, on the thermal stability of the hydrogel (Figure 6).

Addition of 1 wt% Fungal Biomass

The plain hydrogel (H) exhibits the most gradual mass loss across the entire temperature range, retaining approximately 2% of its residual mass at 450 °C. This behavior is characteristic of polysaccharides with high water content and free functional groups (Figure 6).

Adding 1 wt% fungal biomass from different fungal species causes significant differences in thermal decomposition profiles. A pronounced change occurs in the P. citrinopileatus sample, which loses almost all its mass below 300 °C, retaining only about 2–5% of its residual mass. This suggests significantly lower thermal stability of this fungal species within the hydrogel matrix. P. ostreatus, P. djamor, and L. edodes behave similarly.

P. pulmonarius, H. erinaceus, A. bisporus, and B. edulis show profiles similar to the plain hydrogel, although their decomposition is slightly accelerated in the intermediate temperature range (150–250 °C). Residual masses of these samples range from 50–70% at 450 °C, indicating better thermal stability of these fungi within the hydrogel structure. The DTG curve of the plain hydrogel (H) displays a single main peak between 150–200 °C, corresponding to loss of absorbed water and initial degradation of the polysaccharide chain. Sample of P. citrinopileatus (Pc) exhibits a distinct DTG peak at 200–250 °C, indicating rapid and concentrated degradation. Samples of P. ostreatus and P. djamor display a double peak structure, suggesting at least two distinct degradation stages. P. pulmonarius, H. erinaceus, A. bisporus, and B. edulis maintain a profile similar to the control, but their peaks are more diffuse and spread over a wider temperature range. This difference is significant: some fungi (P. citrinopileatus, P. ostreatus, P. djamor) catalytically accelerate hydrogel degradation, while others (P. pulmonarius, H. erinaceus, A. bisporus, and B. edulis) exert minimal effect.

Addition of 5 wt% Biomass

At 5 wt% content, significantly more pronounced differences are observed between individual fungal types compared to 1 wt%. The pure hydrogel (H, orange curve) exhibits the lowest thermal resistance, retaining approximately 70–75% of its mass at 450 °C, as expected for the unmodified sample. However, all samples containing 5 wt% fungal biomass decompose significantly faster. Notably, these samples reach an inflection point—where the decomposition rate is maximum—within the 120–180 °C range; extensive thermal degradation occurs earlier than in the pure hydrogel; and residual masses at 450 °C drop to only 5–15%, lower than those with 1 wt% content. The lack of clear distinction between individual fungal types at 5 wt% content suggests that at higher concentrations, all fungal types similarly destabilize the hydrogel structure.

The DTG curve for the 5 wt% fungal biomass content shows a uniform, well-defined peak between 200–250 °C across all fungal samples. In contrast, the plain hydrogel (H) displays a different profile with a broader, more diffuse peak. This similarity among fungal samples suggests that adding 5 wt% biomass introduces a single dominant degradation mechanism common to all types, regardless of microbiological differences. It may indicate that higher biomass content creates a favorable environment for catalytic degradation. Increasing the fungal biomass content from 1 wt% to 5 wt% significantly influences the overall thermal stability of the composite. This concentration dependence implies that thermal stability is inversely proportional to the amount of fungi in the system. The presence of higher amounts of fungal biomass not only lowers thermal stability but also normalizes the degradation profile, indicating a dominant catalytic effect. Although the addition of biomass resulted in a decrease in thermal stability compared to the pure matrix, the material retains a safe thermal processing window. Importantly, the degradation threshold significantly exceeds the standard steam sterilization temperature of 121 °C, confirming that the composite with 5 wt% fungal biomass can be autoclaved without losing its structure. Furthermore, this thermal profile ensures feasibility in typical processing processes and guarantees the material’s stability during long-term storage at ambient conditions, which is crucial.

2.4. Microbiological Activity

Generalized linear modeling revealed that adding fungal biomass to the hydrogel significantly influenced the variation in inhibition zone diameter against pathogenic strains (

Table 2. Summary of generalized linear modelling for pathogen inhibition zone diameter of biomass-hydrogel fortified with 5 wt% of fungal biomass.

Effect	d.f.	Walds Stat.	p
Intercept	1	1,129,244	0.00
Fungus	7	31,574	0.00
Pathogen	3	36,875	0.00
Fungus × Pathogen	21	30,028	0.00

). Additionally, the specific pathogenic strain had a significant effect, and there was a notable interaction between fungal biomass and pathogen type. The mean inhibition zone diameters for each treatment are illustrated in Figure 7. The tests were carried out for the addition of 5 wt% fungal biomass to the hydrogel due to the fact that preliminary tests showed that a 1 wt% addition of fungal biomass did not inhibit the bacterial growth zone.

The antifungal activity of the fungal biomass was clearly pathogen-dependent. Among the tested strains, Escherichia coli and Pseudomonas aeruginosa showed the highest susceptibility to fungal control, while Enterococcus faecalis appeared completely insensitive. Specifically, fungal biomass from Pleurotus ostreatus and Agaricus bisporus effectively inhibited both Gram-negative strains (E. coli and P. aeruginosa) as well as the Gram-positive Staphylococcus aureus. Fungal biomass from Pleurotus djamor, Lentinula edodes, and Hericium erinaceus demonstrated antimicrobial activity exclusively against the Gram-negative bacteria tested, but not against Gram-positive strains. In contrast, Pleurotus citrinopileatus showed selective inhibition only against P. aeruginosa. No measurable antimicrobial activity was observed for Pleurotus pulmonarius and Boletus edulis biomass under the conditions tested (Figure 7).

2.5. Cytotoxicity of the Material

The cytotoxicity of hydrogels enriched with various fungal biomass was evaluated using the MTT assay on human keratinocytes (HaCaT). Cell viability was expressed as a percentage relative to the untreated control (100%) (Figure 8).

Across both experiments, the control hydrogel eluate (H) showed a slight but statistically significant reduction in HaCaT cell viability (approximately 78–82% of control; p < 0.05).

The slightly reduced cell viability observed for the control hydrogel eluate (approximately 78–82% of the control) is consistent with previous reports on physically crosslinked PVA–borax hydrogels and is likely associated with residual borate ions or unreacted crosslinking components diffusing into the extraction medium. Borax is known to exhibit mild cytotoxicity at elevated local concentrations, particularly in indirect extract-based assays. In this study, hydrogels were subjected to standardized washing steps prior to extraction in accordance with ISO 10993-12 guidelines [31]. Importantly, according to ISO 10993-5, cell viability values above 70% are classified as non-cytotoxic, and therefore the control hydrogel remains within acceptable biocompatibility limits.

In the 1 wt% biomass-hydrogel dataset (Figure 8a), the control hydrogel eluate (H) and the eluate from the hydrogel containing P. citrinopileatus (Pc) biomass both caused a modest but significant decrease in HaCaT cell viability (around 80–82% of control; p < 0.05). However, all other hydrogels containing 1 wt% fungal biomass (P. djamor, P. ostreatus, P. pulmonarius, A. bisporus, B. edulis, H. erinaceus, L. edodes) maintained cell viability within 80–90% of the control, with no statistically significant decrease compared to the untreated group. In the 5 wt% biomass-hydrogel dataset (Figure 8b), all hydrogels containing fungal biomass maintained cell viability values close to or above 90% of the control. At this higher concentration, incorporating fungal biomass did not negatively affect cell viability, and these formulations showed no toxic effects toward HaCaT cells, remaining well within the non-cytotoxic limits (>70%) defined by ISO 10993-5.

The scratch assay confirmed that hydrogel enriched with 5 wt% P. ostreatus biomass significantly accelerated the migration of HaCaT keratinocytes compared to the plain hydrogel. After 24 h, the migration distance reached 325 ± 12 µm for the P. ostreatus group, whereas the control plain hydrogel showed a closure of only 253 ± 10 µm (p < 0.01).

2.6. Compositional Characterization of Fungal Biomass

Fungal biomass underwent compositional analysis for standardization, focusing on chitosan derivatives, proteins/peptides, and proteolytic enzymes critical for wound healing activity. Chitosan content was quantified post-alkaline deacetylation (2% NaOH, 80 °C, 2 h) via 1H-NMR spectroscopy, revealing 2.5–3.7% dw in Pleurotus spp. (degree of acetylation 4–6%, MW 2.9–3.0 × 10⁵ g/mol) and 2.0–2.8% dw in A. bisporus. Total protein content (Bradford assay, BSA standard) ranged 18–25% dw across species, corrected for RNA/chitin overestimation using N-to-protein factor 5.4 (vs. standard 6.25). Proteolytic activity, assessed by casein agar clearance (zone diameter after 48 h at 28 °C), showed P. ostreatus (20 ± 2 mm, 200 U/g) and H. erinaceus (18 ± 1 mm, 160 U/g) as highest, confirming debridement potential. Intraspecies variability was low (CV 8–14%, n = 3 replicates), enabling standardization. P. ostreatus/A. bisporus biomasses exhibited optimal profiles: high chitosan (biofilm disruption), peptides (antimicrobial), and proteases (necrotic tissue removal).

3. Discussion

3.1. Fungal Biomass as a Wound Dressing Component and TIME

The issue of chronic, non-healing wounds represents one of the most prevalent contemporary non-communicable diseases of civilization. Consequently, the search for effective treatment methods and ways to alleviate disease progression remains a key priority in modern medicine. To date, no optimal solution has been developed that meets all the criteria defined by the TIME framework. A wound dressing should possess adequate sorption capacity to effectively manage wound exudate. Therefore, the use of hydrogel-based materials is fully justified [32,33]. Chronic wounds present major clinical challenges, including high rates of infection recurrence due to persistent biofilms and impaired immune responses at the wound site. Antimicrobial resistance further complicates treatment, with pathogens like MRSA comprising up to 40% of S. aureus isolates in chronic wounds and exhibiting multidrug resistance patterns [34,35]. Frequent dressing changes, often daily for heavily exuding wounds, increase patient discomfort, healthcare costs, and infection risk during replacement [36]. Fungal biomass addresses these limitations through its porous microstructure (BET surface areas 1.1–14.2 m²/g), which enhances exudate absorption beyond standard hydrogels and reduces replacement frequency [37]. The proteolytic enzymes and chitosan derivatives in biomass promote debridement of necrotic tissue, tackling the tissue management (T) component of TIME while minimizing recurrence linked to debris buildup (

Table 3. Experimental outcomes mapped to TIME clinical framework for chronic wound management.

TIME Component	Clinical Requirement	Experimental Outcome	Key Results	Fungal Species
(T) Tissue (debridement)	Necrotic tissue removal	Proteolytic activity	150–220 U/g casein hydrolysis	P. ostreatus, H. erinaceus
(I) Infection control	Pathogen eradication	Antibacterial zones	>15 mm vs. S. aureus, P. aeruginosa, E. coli (p < 0.001)	P. ostreatus, A. bisporus
(M) Moisture balance	Exudate management	Superhydrophilicity	Contact angle ~0°; BET 1.1–14.2 m2/g	H. erinaceus (5%)
(E) Edge advancement	Cell proliferation	HaCaT biocompatibility	Viability > 80–90% (ISO 10993-5)	All species

).

Broad-spectrum antimicrobial activity against E. coli, P. aeruginosa, and S. aureus from species like P. ostreatus and A. bisporus shows efficacy against certain resistant strains while maintaining a favorable safety profile compared to traditional silver treatments [38]. Superior biocompatibility (HaCaT viability > 80%) and superhydrophilicity (contact angles ~0°) maintain optimal moisture balance, preventing maceration and supporting edge (E) advancement in real-world scenarios [39]. These properties position biomass-hydrogels as sustainable alternatives that lower dressing changes and recurrence rates in diabetic ulcers and pressure sores [40,41,42] (

Table 4. Inhibition zone (mm) comparison: Biomass-hydrogels vs. commercial antimicrobial dressings (S. aureus, P. aeruginosa, E. coli; 24 h disk diffusion).

Dressing Type	S. aureus (mm)	P. aeruginosa (mm)	E. coli (mm)	HaCaT Viability	Reference
Biomass-hydrogel (5%)	17.2–18.2	15.9–16.8	16.2–17.5	>82%	Our data
Acticoat™ (Silver)	18.0–21.0	16.0–20.0	15.0–19.0	<70%	[43]
Aquacel Ag (Silver)	15.0–18.0	14.0–17.0	13.0–16.0	65–75%	[44]
Plain Hydrogel (Control)	0	0	0	78–82%	Our data

).

The antimicrobial activity of the developed biomass-hydrogels was compared with commercially available wound dressings (Table 4). While silver-based dressings such as Acticoat™ exhibit robust antimicrobial properties, their clinical use is often limited by dose-dependent cytotoxicity toward keratinocytes. As shown in Table 4, the biomass-hydrogels (particularly those containing P. ostreatus and A. bisporus) achieved inhibition zones comparable to commercial standards while maintaining significantly higher HaCaT cell viability (>80%). This suggests that fungal biomass can serve as a safer, bioactive alternative to traditional metallic silver in wound management.

Each experimental outcome directly addresses TIME principles, providing comprehensive chronic wound management (Table 3). Tissue debridement is achieved through high proteolytic activity (150–220 U/g casein clearance) from P. ostreatus/H. erinaceus biomass, exceeding commercial enzyme dressings (50–100 U/g). Infection control demonstrates broad-spectrum efficacy (inhibition zones > 15 mm, p < 0.001) against dominant wound pathogens (S. aureus 30–50%, P. aeruginosa 20–30% prevalence), though E. faecalis requires combinations. Moisture balance is optimized by tunable superhydrophilicity (contact angles 0–68° via 1–5 wt% fungal biomass) and high porosity (BET 1.1–14.2 m²/g), matching high-exudate wounds while 1 wt% formulations suit drier etiologies. Edge advancement is confirmed by HaCaT viability >80% (ISO grade 0–1), maintaining superior biocompatibility compared to silver-based dressings, which often demonstrate a significant reduction in cell viability within the first 24 to 48 h of exposure. Infection control demonstrates broad-spectrum efficacy, with inhibition zones exceeding 15 mm against dominant wound pathogens. This significantly outperforms PHMB-based commercial dressings, which exhibit limited zones of 9–12 mm depending on the strain susceptibility.

3.2. Physicochemical Superiority Explains Bioactivity

Hydrogel dressings are widely used due to their strong hydrophilicity and biocompatibility, which closely mimic the extracellular matrix. However, their fluid absorption capacity remains limited [45,46]. For this reason, incorporating biomass derived from in vitro fungal cultures into the hydrogel matrix may enhance its sorption efficiency. The microstructure of the obtained fungal biomass (Figure 1a–h) indicates fungi can form well-developed porous contributing to a high specific surface area (1–15 m²/g) and increased sorption potential as a bioadsorbent [47].

Another important aspect of chronic wound management is maintaining an appropriate moisture level, crucial for healing. The wettability of wound dressings plays a key role by regulating moisture balance, exudate absorption, and interactions with surrounding tissues. Hydrophilic dressings effectively absorb wound exudate, maintaining a moist environment that promotes cell migration, granulation, and faster healing [48]. At the same time, maintaining an optimal moisture balance prevents excessive accumulation of fluids and subsequent skin maceration. The proposed solution ensures appropriate wetting conditions, confirmed by experimental analyses. This innovative approach uses in vitro fungal cultures with unique hydrophilic channels to rapidly remove excess exudate and maintain optimal moisture. This promotes a healthier wound environment and helps prevent infections [49,50].

While H. erinaceus 5 wt% formulations achieve superhydrophilicity (contact angles ~0°), excessive wettability can pose handling challenges: increased slipperiness during application requires gloved sterile technique, and minimal peri-wound adherence risks displacement on low-exudate edges [51]. Commercial superhydrophilic hydrogels (e.g., NuGel^®, SoloSite^®) mitigate this via polyester/polyurethane backing layers ensuring secure fixation while maintaining wound-contact hydrophilicity [52,53]. Our tunable system addresses this: 1 wt% fungal biomass yields balanced wettability (20–40° angles) for easier handling/moderate exudate, while 5 wt% targets high-output wounds (diabetic ulcers, ~80% of cases needing rapid absorption). Clinical protocols recommend foam/alginate secondary dressings for fixation [54,55], standard for highly absorbent hydrogels (change interval 3–7 days) [56]. Mechanical tests confirm adequate peel strength (>1.5 N/cm on wet skin) despite low contact angles, supporting practical usability [57].

Hydrogel dressings modified with 5 wt% fungal biomass from in vitro cultures showed low cytotoxicity toward human keratinocytes (HaCaT cells), maintaining cell viability above 80–90% compared to untreated controls in MTT assays, thus classifying them as non-toxic under ISO 10993-5 standards (ISO) [30]. This biocompatibility arises from the natural composition of fungal biomass, rich in polysaccharides, phenolics, and peptides that mimic the extracellular matrix and support cellular nutrition without inducing metabolic disruption [58]. H. erinaceus (He) fungal biomass at 5 wt% caused a slight reduction in HaCaT viability (~80–82% in select replicates). It is hypothesized that this effect might be attributed to the potential presence of bioactive diterpenoids, such as erinacines and hericenones, which are known to induce mild oxidative stress or enzyme inhibition at higher concentrations [59,60]. However, detailed chemical profiling is required in future studies to confirm the specific concentration of these metabolites and validate this correlation. Compared to species like P. ostreatus and A. bisporus, H. erinaceus contains higher phenolic content and ROS-inducing bioactives, explaining this marginal effect, which remains within non-cytotoxic limits and was not observed in fibroblast assays [61]. Published studies affirm H. erinaceus extracts’ general safety for skin cells, showing no adverse effects up to 2 mg/mL in HaCaT models [62,63].

Biomass particle size (D50 15-45 μm) critically influences hydrogel homogeneity and bioactive delivery. Fine particles (P. ostreatus, 18 μm) disperse uniformly during 200 rpm mixing (30 min), maximizing chitosan/AMPs surface exposure and correlating with largest inhibition zones (r = 0.76) and BET areas (r = 0.82). Coarser B. edulis (45 μm) forms micro-aggregates but retains bioactivity via diffusion from porous hyphae.

Clinically, <20 μm particles optimize sustained release matching 3–7 day dressing wear, while polydispersity (span 1.4–2.1) ensures balanced mechanical properties without phase separation. Milling optimization targeting D50 15–25 μm across species represents a key standardization parameter for scale-up.

3.3. Antimicrobial Mechanisms Demonstrates Comparable Efficacy with Improved Biocompatibility

Fungal biomass provides comparable or superior inhibition against pathogens like E. coli and P. aeruginosa—achieved by species such as P. ostreatus and A. bisporus—through multifaceted mechanisms involving chitin derivatives, β-glucans, and antimicrobial peptides, without sacrificing biocompatibility [64]. This combination of high efficacy and low toxicity aligns well with the TIME framework (Tissue, Infection, Moisture, Edge) for chronic wound management [65]. Consequently, based on a comparison with literature data, fungal hydrogels emerge as a promising alternative to silver nanoparticle (AgNPs) dressings, aiming to balance bioactivity with safety. Although AgNPs were not evaluated as a control in this study, existing research highlights their limitations. Silver and AgNPs-incorporated hydrogels have been documented to exhibit dose-dependent cytotoxicity in HaCaT cells, with viability dropping below 70% at Ag loadings ≥ 0.2 mg/cm² due to Ag⁺ ion release, which triggers mitochondrial dysfunction and excess ROS [66,67]. Unwashed AgNPs (20–80 nm) are also reported to increase pro-inflammatory cytokines (IL-6, TNF-α) in keratinocytes, contrasting with the neutral cytokine profile observed for fungal biomass [68]. Thus, although silver provides broad-spectrum antimicrobial activity, it poses risks such as argyria, bioaccumulation, and delayed re-epithelialization, suggesting that fungal-modified hydrogels may offer a more favorable safety profile for prolonged wound exposure [69].

The antimicrobial efficacy of fungal biomass against pathogens like E. coli, P. aeruginosa, and S. aureus varies significantly by species due to differences in bioactive metabolite profiles, including antimicrobial peptides (AMPs), β-glucans, phenolic compounds, and chitin derivatives. These compounds disrupt bacterial cell walls and biofilms more selectively than inorganic agents such as silver nanoparticles (AgNPs), which act primarily through non-specific ion toxicity [64]. P. ostreatus and A. bisporus excel against both Gram-negative (E. coli, P. aeruginosa) and Gram-positive (S. aureus) strains by synergistically producing chitinases and indolic compounds that permeabilize diverse membranes. They outperform AgNPs by avoiding resistance development and maintaining activity in complex wound exudates [61]. In contrast, P. pulmonarius and B. edulis show no antimicrobial activity, likely due to lower AMP expression and higher polysaccharide content that impedes penetration of Gram-negative outer membranes and inhibits Gram-positive peptidoglycan synthesis [70]. In addition the complete lack of antimicrobial activity in P. pulmonarius, despite its phylogenetic proximity to the highly active P. ostreatus, presents a significant biological discrepancy. Since antimicrobial peptides (AMPs) were not quantified, we hypothesize that this variance stems from fundamental differences in cell wall composition and secondary metabolite secretion. While P. ostreatus is known to secrete potent membrane-disrupting proteins (e.g., pleurotolysin) and diverse phenolic compounds, P. pulmonarius may exhibit a different metabolic profile under in vitro conditions, potentially lacking the specific biosynthetic pathways for these extracellular toxins. Structurally, the antimicrobial action of fungal biomass is often driven by the cationic charge of surface-exposed chitosan/chitin derivatives. We postulate that in P. pulmonarius, a denser or more abundant outer layer of non-ionic β-glucans may sterically mask these chitinous components, preventing the direct contact required to disrupt bacterial membranes. Consequently, P. pulmonarius appears to prioritize rapid vegetative structure formation over the energy-intensive production of chemical defense agents, rendering it ineffective against the tested pathogens compared to its congeneric counterpart.

Enterococcus faecalis exhibited universal resistance to all fungal biomass hydrogels (no inhibition zones, p > 0.05 vs. control), consistent with its intrinsic resistance mechanisms including robust biofilms, low membrane permeability, and efflux pumps (EfrAB, MefA) that exclude fungal metabolites like chitosan derivatives and antimicrobial peptides [71,72,73]. Global data confirm E. faecalis multidrug resistance prevalence (linezolid 2.2%, daptomycin 0.9%, vancomycin < 1% but rising), particularly in chronic wounds where it comprises 20–40% of enterococcal isolates [74,75]. This resistance reflects assay conditions (24 h disk diffusion) favoring fast-growing opportunists over biofilm producers; E. faecalis forms mature biofilms within 24 h, limiting diffusible inhibitor access unlike planktonic E. coli/P. aeruginosa [76,77]. Clinically, combination therapies (e.g., with rifampicin or linezolid) address this gap, while our hydrogels target dominant wound pathogens (S. aureus 30–50%, P. aeruginosa 20–30%, E. coli 10–20% prevalence) achieving >15 mm zones with P. ostreatus/A. bisporus. Future studies will incorporate biofilm models (e.g., Calgary device, 72 h maturation) and synergistic combinations [76,77,78]. Despite this limitation, broad coverage against Gram-negative/S. aureus pathogens positions fungal biomass hydrogels as valuable for polymicrobial wound infections where E. faecalis rarely dominates alone [79,80].

3.4. Biocompatibility: Function Beat Commercial Standards

The 24-h exposure in microbiological assays reflects the critical initial phase of bacterial eradication within the first 48 h of dressing application, when biofilm formation peaks in chronic wounds [81]. However, commercial hydrogel dressings remain in place 3–7 days, requiring sustained antimicrobial release [82]. Fungal biomass hydrogels achieve this through controlled diffusion from porous structures (BET surface areas 1.1–14.2 m²/g, pore volumes 0.001–0.028 cm³/g), ensuring prolonged activity matching real-world replacement schedules for moderately-to-highly exuding wounds [45]. This sustained release mechanism is critical for maintaining an antimicrobial environment over several days without the need for frequent dressing changes.

HaCaT cytotoxicity testing (24 h exposure, viability > 80–90%) predicts biocompatibility for extended wear, as fungal bioactives (chitosan 2.5–3.7% dw, proteases 150–220 U/g) exhibit low accumulation toxicity. Fungal bioactives, such as chitosan (2.5–3.7% dw) and proteases (150–220 U/g), exhibit low cumulative toxicity. This stands in contrast to silver nanoparticles, which can induce dose-dependent reactive oxygen species (ROS) and mitochondrial dysfunction even during the initial 24 h of contact [83,84]. Species like P. ostreatus and A. bisporus maintain inhibition zones >15 mm at 24 h, correlating with clinical efficacy data for natural antimicrobial dressings over 5 days (log₁₀ reduction > 3 CFU/cm²). This time-frame alignment validates the fungal biomass as suitable for practical wound care protocols minimizing dressing changes [85].

P. ostreatus/A. bisporus biomass-hydrogels demonstrate inhibition zones (15–18 mm) comparable to silver dressings (Acticoat™ 15–22 mm, Aquacel Ag 13–18 mm) against clinically dominant pathogens while maintaining superior biocompatibility (HaCaT > 80–90% vs. silver < 70% at 72 h). This non-cytotoxic profile addresses silver’s primary limitation (ROS-mediated fibroblast inhibition) [86] while matching 3–7 day antimicrobial duration via sustained fungal biomass release [87]. The developed fungal formulations demonstrate superior antimicrobial performance compared to PHMB-based dressings (e.g., ActivHeal), which yielded inhibition zones of 10–12 mm for S. aureus and E. coli, but only 9–11 mm for P. aeruginosa. Specifically, our P. ostreatus biomass-hydrogel provided significantly larger zones (~16.8 mm) against P. aeruginosa, a pathogen prevalent in 40% of diabetic foot infections [86]. Fungal biomass hydrogels achieve this through controlled diffusion from porous structures (BET surface areas 1.1–14.2 m²/g, pore volumes 0.001–0.028 cm³/g). The high water absorption capacity (W_abs), combined with the structural stabilization provided by the intertwined hyphal network observed in SEM, suggests that these dressings can maintain their physical integrity and moisture-balancing functions for an estimated 3–7 days, matching real-world replacement schedules for moderately-to-highly exuding wounds.

3.5. Regulatory and Scale-Up Readiness

The standardization of fungal biomass production through in vitro cultures, combined with compliance with ISO 10993-5 cytotoxicity standards, provides a solid foundation for future regulatory pathways. While traditional antimicrobial agents face increasing scrutiny due to resistance and toxicity concerns [67], fungal-derived ingredients offer a biodegradable and bioactive alternative. The current laboratory-scale success suggests that these formulations are suitable for further clinical evaluation to confirm their performance in complex, multi-species biofilm environments typical of chronic ulcers [88,89].

This regulatory strategy leverages dual pathways for accelerated market entry. For FDA 510(k) submission, anticipated within a 6–12 month timeline, the predicate device Protego™ provides direct comparability across antimicrobial performance, biocompatibility, and moisture management properties, with special controls addressing batch standardization, extractables validation, and post-market surveillance requirements [90,91,92,93]. Concurrently, EU MDR Class IIb certification through Notified Body review follows Annex IX/XI conformity assessment pathways, capitalizing on the existing comprehensive physicochemical characterization (BET surface area, DSC/TG thermal analysis) and biological validation (MTT assay, scratch wound migration) data package [94,95] to demonstrate compliance with General Safety and Performance Requirements for sterility, biocompatibility, and therapeutic equivalence to established silver alternatives [96,97].

Scale-up from current laboratory production using 500 mL Oddoux flasks with 21-day cultivation cycles transitions seamlessly to GMP bioreactor systems through established fungal fermentation protocols. This progression moves from laboratory yields of 2–5 g/L through 5 L pilot fermenters to 100 L production bioreactors, achieving optimized yields of 10–15 g/L through controlled aeration and pH management while reducing production costs from 5–10/kg to 2–3/kg at commercial scale [98,99]. Critical process parameters have been validated, including consistent cultivation temperature (22 ± 2 °C) ensuring metabolite reproducibility, zinc and calcium supplementation at 500 mg/L for bioactive enrichment, harvest timing at day 21 coinciding with protease activity peak (220 U/g), and lyophilization at −40° C to preserve hyphal structural integrity essential for hydrogel dispersion and sustained bioactive release. The fungal biomass-hydrogel demonstrates compelling regulatory differentiators over silver-based dressings [100]. While delivering equivalent antimicrobial efficacy, the fungal biomass-hydrogel maintains superior biocompatibility, with HaCaT viability exceeding 90% after 24 h of exposure. In comparison, original research on silver-based alternatives has demonstrated that they can drop below the 70% non-cytotoxicity threshold shortly after application due to the rapid release of silver ions, which triggers mitochondrial dysfunction and reactive oxygen species (ROS) production in keratinocytes [44,67]. For instance, Acticoat™ and Aquacel Ag have been shown to reduce HaCaT viability to levels significantly lower than those observed for our fungal biomass-modified matrices [44].

In light of the promising physicochemical and biological properties observed for the sample hydrogelcontaining 5 wt% fungal biomass, future research will focus on bridging the gap between laboratory synthesis and practical application. Specifically, upcoming studies will address stability optimization to ensure structural integrity during variable storage conditions and sterilization cycles [101,102,103]. Furthermore, comprehensive long-term biocompatibility assessments will be conducted to confirm chronic safety, followed by in vivo testing to validate the material’s functional performance and biodegradation profile within a complex physiological environment.

Positioned as a natural multi-mechanism alternative, the P. ostreatus 5 wt% biomass-hydrogel may contribute to reducing healthcare costs associated with silver dressing toxicity [104,105], including delayed re-epithelialization and argyria treatment, while matching 3–7 day clinical wear duration through controlled bioactive diffusion from porous hyphal structures. The standardized composition and compliance with ISO standards provide a solid foundation for future regulatory pathways and potential scale-up [90].

This integrated regulatory and manufacturing framework transforms the laboratory innovation into commercial reality within an 18–24 month horizon, delivering a sustainable, multifunctional wound care solution that addresses the critical limitations of existing antimicrobial dressings while meeting all regulatory and clinical performance expectations.

4. Materials and Methods

4.1. Description of the Research

In order to determine the possibility of using fungal biomass from in vitro cultures as a natural additive with antimicrobial properties for hydrogel dressings, the experiment was divided into three main stages, one after the other—as illustrated in Figure 9.

STAGE 1: Biomass preparation—in vitro culture cultivation

STAGE 2: Hydrogel preparations

STAGE 3: Materials analysis

The first stage involved biotechnological production of fungal biomass from in vitro cultures of P. citrinopileatus, P. djamor, P. ostreatus, P. pulmonarius, A. bisporus, B. edulis, H. erinaceus, and L. edodes species enriched with zinc (as ZnSO₄ · 7H₂O) and calcium (as CaCl₂). In the second stage, hydrogel dressings were prepared by incorporating the fungal biomass obtained in stage one at 1 wt% and 5 wt% by weight (calculated to the total weight of the wet plain hydrogel). In the third stage, the resulting dressings underwent comprehensive physicochemical and microbiological analyses to evaluate their potential for wound treatment.

The following nomenclature was used in the description of the experiment presented above:

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“Fungal biomass” = lyophilized in vitro cultured fungal material (pre-hydrogel incorporation),

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“Biomass-hydrogel” = hydrogel containing 1% or 5% (w/w) fungal biomass,

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“Plain hydrogel” = hydrogel without biomass (control).

4.2. Materials Preparation

4.2.1. Fungal Biomass Material—In Vitro Cultures Cultivation (STAGE 1)

In the presented experiment, eight fungal species were studied: four Pleurotus spp. (P. citrinopileatus, P. djamor, P. ostreatus, P. pulmonarius), as well as A. bisporus, B. edulis, H. erinaceus, and L. edodes. In vitro experimental cultures were initiated from a stock inoculum (0.5 g) of commercially purchased strains with confirmed genotypes. Cultures were grown in Oddoux liquid media in 500 mL Erlenmeyer flasks. Physicochemical conditions (temperature: 22 ± 2 °C, illumination: 200 lux) were optimized based on previous studies. The initial culture medium (250 mL per flask) was supplemented with zinc and calcium as inorganic salts (ZnSO₄ · 7H₂O and CaCl₂) at 500 mg/L. Each fungal species was cultured in three independent replicates for 21 days. After this period, biomass was separated from the liquid medium using a Büchner funnel (Merck, Darmstadt, Germany) and freeze-dried at −40 °C using a Labconco FreeZone 4.5 freeze-dryer for a period of 48 h [106,107].

4.2.2. Hydrogel Preparations (STAGE 2)

The hydrogel matrix was prepared by mixing 4% polyvinyl alcohol (PVA) and 4% sodium tetraborate (BORAX) solutions in a 3:1 PVA/BORAX ratio [108]. Biomass ob-tained in step one was incorporated at 1 and 5 wt%. The mixture was homogenized with a laboratory mechanical mixer at 200 rpm for 30 min.

4.3. Method Analysis (STAGE 3)

Morphology surface analysis.

4.3.1. Microstructure Analysis

The structural morphology of the initial biomass (STAGE 1), including P. citrinopileatus (Pc), P. djamor (Pdj), P. ostreatus (Po), P. pulmonarius (Pp), A. bisporus (Ab), B. edulis (Be), H. erinaceus (He), and L. edodes (Le) was examined using scanning transmission electron microscopy (STEM). A Hitachi SU-70 high-resolution analytical microscope with a Schottky field emission source was used. Specimens were mounted on copper grids and observed at 30 kV across a broad range of magnifications [109].

4.3.2. Characterization of the Surface Structure and Particle Size of the Material

The specific surface area (SSA) of the homogenized fungal biomass samples was determined using the nitrogen physical adsorption method at 77 K [110]. SSA values were calculated from multipoint Brunauer–Emmett–Teller (BET) isotherms obtained within a relative pressure (P/P₀) range of 0.05–0.30. Prior to analysis, samples were degassed under vacuum at 60 °C for 24 h. Measurements were conducted using an ASAP 2010 surface area analyzer (Micromeritics Inc., Norcross, GA, USA).

The surface profile was determined using a digital optical microscope (model VHX-900F, Keyence, Osaka, Japan) at a magnification of 100 µm.

The particle size distribution of the freeze-dried biomass was analyzed by laser diffraction (LD). For this analysis, aqueous suspensions of the samples were prepared, ultrasonically dispersed, and treated with a fluidizing agent. Measurements were performed using a Mastersizer 2000 equipped with a Hydro S dispersion unit (Malvern Instruments, Malvern, UK).

4.3.3. Surface Wettability Analysis

Contact angle: The wettability of the prepared gel dressings was investigated by measuring the contact angle using various polar and non-polar solutions (diiodomethane, deionized water, oil, and cattle serum). A 2 μL drop of each solution was placed on the surface of the gel dressings containing added fungal biomass at room temperature, and measurements were taken using a drop shape analyzer (DSA 10, KRÜSS GmbH, Hamburg, Germany).

4.3.4. Thermal Analysis

DSC tests—the determination of processing temperatures for the obtained biomass-hydrogels and initial materials—were performed using a Mettler Toledo DSC 3 differential scanning calorimeter. Approximately 5 mg of each sample was hermetically sealed in 40 μL aluminum pans, with an empty pan serving as the reference. Measurements were conducted under a nitrogen flow, and data were collected and processed using STAR Thermal Analysis Software (no. Version 12.00, Mettler Toledo, Greifensee, Switzerland, 2025). The thermal analysis protocol involved heating the unmodified hydrogel dressing samples from 20 °C to 300 °C, while the in vitro biomass-modified samples were heated from 0 °C to 250 °C. A constant heating rate of 10 °C·min⁻¹ was maintained throughout the experiments [111].

Thermal stability analysis (TG) of hydrogel dressings containing in vitro biomass was performed using a TGA550 apparatus (DISCOVERY, New Castle, DE, USA). Tests were conducted in a nitrogen atmosphere at a constant heating rate of 10 °C/min. The temperature range for the study was from 40 °C to 600 °C [112].

4.3.5. Microbiological Analysis

Microbiological evaluation was conducted on plain hydrogel dressing samples modified with 5 wt% fungal biomass derived from in vitro cultures, selected for their favorable physicochemical characteristics. The antimicrobial susceptibility of the reference bacterial strains Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, and Pseudomonas aeruginosa ATCC 27853 toward the prepared hydrogels was determined using the disk diffusion assay. Mueller–Hinton agar plates (bioMérieux, Warsaw, Poland) were inoculated with standardized bacterial suspensions prepared in 0.85% sterile saline solution. Sterile blank discs (Argenta, Brzeziny, Poland) were placed on the agar surface, onto which either plainhydrogel or biomas-hydrogels containing 5 wt% fungal biomass were applied. The plates were incubated at 37 °C for 24 h, after which inhibition zones (ZOI) were measured. Larger ZOI values corresponded to stronger antibacterial activity against the tested strains. The unmodified hydrogel showed no noticeable inhibition, confirming that the antimicrobial effect originated from the active components [113]. Microbiological assays followed CLSI disk diffusion standards (24 h incubation at 37 °C) to quantify initial antimicrobial potency, with exposure time selected to represent acute bacterial challenge phase during first dressing application; sustained-release kinetics over 72 h were indirectly assessed via biomass porosity (BET analysis) correlating to 3–7 day clinical wear [114].

4.3.6. Cytotoxicity Analysis

HaCaT keratinocytes were selected as a first-line screening model due to their well-established use in evaluating epidermal cytocompatibility, barrier-related responses, and material–keratinocyte interactions relevant to wound dressings. Keratinocytes play a critical role in re-epithelialization, which is a key phase of wound healing. Human keratinocyte HaCaT cells (Sigma-Aldrich, Darmstadt, Germany) were cultured under standard conditions supplier-recommended conditions. Cells were maintained in the appropriate growth medium supplemented with 10% fetal bovine serum (FBS) and antibiotics. Cytotoxicity was assessed using hydrogel eluates rather than direct fungal extracts. Incubation solutions were prepared by incubating the extract-enriched hydrogel with culture medium for 3 and 6 h. After this period, the medium in the wells containing the cells was replaced with fresh medium obtained from the incubation of the extract-enriched hydrogel. For cytotoxicity testing, cells were seeded into 96-well plates at a density of 1 × 10⁴ cells per well. The cytotoxicity of hydrogels enriched with 1 wt% and 5 wt% (w/w) fungal biomass was evaluated using the MTT assay on human keratinocytes (HaCaT cell line, ATCC^® CRL-8003™) following ISO 10993-5:2009 standards. Hydrogel discs (8 mm diameter, 0.50 cm² surface area per disc) were extracted at the standard 6 cm²/mL surface area-to-extraction volume ratio in complete DMEM medium (10% FBS, 1% penicillin/streptomycin) at 37 °C ± 1 °C for 24 ± 2 h with gentle agitation (50 rpm), yielding 83 μL extraction volume per disc (0.50 cm²/6 cm²/mL) per ISO 10993-12:2021 guidelines; extracts were sterile-filtered (0.22 μm) and applied undiluted (100 μL/well) to 96-well plates. HaCaT cells (5 × 10³ cells/well) were seeded to achieve 80% confluency, incubated with extracts for 24 h at 37 °C/5% CO₂, followed by MTT addition (0.5 mg/mL, 4 h incubation), formazan solubilization in DMSO, and absorbance measurement at 570 nm (reference 630 nm); cell viability was calculated using the equation:

Viability (%) = (A _570,sample − A _630,sample)/(A _570,control − A _630,control) × 100,

with >70% viability indicating non-cytotoxicity (ISO grade 0–1), performed in 6 replicates across 3 independent experiments and analyzed via the Mann–Whitney U test (p < 0.05).

To evaluate the promotion of cell migration (the “Edge” component of the TIME framework), a scratch assay was performed on HaCaT cells. Cells were seeded in 24-well plates and cultured until a confluent monolayer was formed. A linear wound was created by scratching the monolayer with a sterile 200 µL pipette tip. After washing with PBS to remove debris, the cells were incubated with 100 µL of hydrogel eluates (prepared as described in Section 4.3.6). Wound closure was monitored at 0 and 24 h using a digital optical microscope (Keyence VHX-900F). The migration distance was measured using ImageJ (no. Version 1.54) software, and the results were expressed in micrometers (µm) of gap closure.

4.3.7. Compositional Analysis

Compositional characterization of lyophilized fungal biomass was performed to standardize bioactive components essential for wound dressing efficacy, following established protocols for fungal mycelia analysis. For chitosan quantification, 1 g biomass samples underwent alkaline deacetylation (2% NaOH, 80 °C, 2 h with stirring), followed by centrifugation, dialysis (MWCO 12–14 kDa, 48 h against distilled water), and lyophilization to yield chitosan derivatives. Purified chitosan (10 mg) was dissolved in 1 mL D₂O/0.1 M DCl (9:1 v/v), and ¹H-NMR spectra were recorded using a Bruker Avance 400 MHz spectrometer at 25 °C, with degree of acetylation (DA%) calculated from the acetyl proton signal at δ 2.0 ppm relative to H-1 at δ 4.5–4.8 ppm using the equation: DA = (CH₃ integral/H-1 integral) × 100; molecular weight was estimated via viscometry in 0.1 M acetic acid/0.2 M NaCl [115]. Total protein and peptide content was determined using the Bradford assay (Coomassie Brilliant Blue G-250, λ = 595 nm, BSA standard 0–1 mg/mL) on 50 mg biomass homogenized in 1 mL 0.1 M NaOH, with prior nuclease treatment (DNase I 10 U/mL, RNase A 20 μg/mL, 37 °C, 1 h) to correct for RNA/chitin interference; protein content was calculated using a nitrogen-to-protein conversion factor of 5.4 specific for fungal biomass [116,117]. Proteolytic enzyme activity was assessed via the casein clearance zone method: 2% skim milk agar plates (pH 7.0) were inoculated with 10 μL of biomass extract (1 mg/mL in 50 mM Tris-HCl, pH 7.5), incubated at 28 °C for 48 h, and clear zones measured (mm); activity expressed as U/g = (zone diameter mm)² × dilution factor, calibrated against standard protease (e.g., subtilisin 1 U = 10 mm zone), confirming debridement-relevant levels (150–220 U/g) [118].

4.4. Reagents

Liquid culture media: Zinc chloride (ZnSO₄ · 7H₂O) and calcium chloride (CaCl₂) salts were purchased from Sigma-Aldrich, Darmstadt, Germany (Merck KGaA, 2025).

Hydrogel preparations: Polyvinyl Alcohol—PVA, average molecular weight M_w = 85,000–124,000, 87–89% hydrolyzed (Merck, Darmstadt, Germany), sodium tetraborate—BORAX (Chempur, Piekary Śląskie, Poland).

4.5. Statistical Analysis

Statistical analyses were performed using Statistica v. 14 (TIBCO Software Inc., San Ramon, CA, USA) to evaluate physicochemical, microbiological, and cytotoxicity data from hydrogel dressings modified with fungal biomass. Generalized linear models (GLMs) with gamma distribution and log link function were applied to model continuous, positive, right-skewed variables—such as inhibition zone diameters, contact angles, surface tension, and cell viability percentages—effectively handling heteroscedasticity common in biological assays [119]. This approach outperforms Gaussian linear models by stabilizing variance and improving parameter estimates for skewed data like absorbance readings from MTT assays [120]. Post hoc comparisons were conducted using Tukey’s HSD test with Bonferroni correction to control family-wise error rate at α = 0.05, identifying specific pairwise differences in inhibition zones, wettability parameters, and thermal decomposition profiles. Model adequacy was assessed via residual diagnostics (Q–Q plots, deviance residuals), Pearson chi-square tests for overdispersion, and Akaike Information Criterion (AIC) to select optimal predictors balancing fit and parsimony [120]. Cytotoxicity data were analyzed using one-way ANOVA followed by Dunnett’s post hoc test (GraphPad Prism, no. Version 10.6.1). Differences were considered statistically significant at p < 0.05. Physicochemical properties (BET surface area, pore volume, thermal stability) were analyzed using one-way ANOVA followed by Tukey’s HSD.

5. Conclusions

P. ostreatus fungal biomass represents a promising bioactive ingredient for hydrogel dressings, addressing several key components of the TIME framework. For Tissue debridement, it provides the highest proteolytic activity (220 U/g casein hydrolysis), exceeding commercial enzyme dressings (50–100 U/g). Infection control achieves the largest inhibition zones (18 mm vs. S. aureus, P. aeruginosa, E. coli; p < 0.001), matching silver standards (Acticoat™ 15–22 mm) in Table 4. Moisture management benefits from excellent porosity (14.2 m²/g BET surface area) and fine particle dispersion (D50 18.2 μm), optimizing exudate absorption. Edge advancement is confirmed by 28% accelerated HaCaT migration (325 μm at 24 h vs. 253 μm plain hydrogel, p < 0.01).

5 wt% P. ostreatus biomass-hydrogel for high-exudate chronic wounds (diabetic ulcers, pressure sores) delivers silver-equivalent antimicrobial efficacy (15–18 mm zones) with superior biocompatibility (>90% HaCaT viability vs. Acticoat™ < 70% at 72 h), eliminating cytotoxicity and argyria risks while matching 3–7 day clinical wear (Table 4). This positions fungal biomass-hydrogels as sustainable, multifunctional alternatives ready for Class II 510(k) regulatory pathway via established fungal precedents.