Advanced Biomaterials in Tissue Engineering Based on Antibacterial and Antibiofilm Nanocomposites for Multidrug-Resistant Wound Pathogens

Abstract

The treatment of chronic wounds is critically challenged by resilient biofilms formed by multidrug-resistant (MDR) pathogens, which are largely impervious to conventional antibiotics. This study addresses this challenge by engineering a multifunctional hydrogel dressing that synergistically integrates bacterial cellulose (BC), MXene nanosheets, and hydroxyapatite (HAp). Plays a critical dual role in the composite, significantly boosting bioactivity while also stabilizing the MXene to prevent oxidation and maintain its antibacterial properties. As a result, the BC/MXene/HAp composite achieved a near-total (98%) eradication of viable cells in mature biofilms of challenging MDR pathogens, including MRSA and FQRPA. Such activity is driven by a dual-mechanism that effectively disrupts both the initial attachment of bacteria and the structural scaffold of established biofilms. Complementing this efficacy, in vitro assays with NIH-3T3 fibroblasts confirm that the composite supports excellent cell adhesion, proliferation, and metabolic activity, highlighting its high biocompatibility. This work demonstrates a nonantibiotic strategy to combat biofilms through a synergistically designed biomaterial. Given its robust performance and multifunctionality, this platform is a promising candidate for the development of advanced therapeutic dressings in regenerative wound care.

1. Introduction

The increasing prevalence of multidrug-resistant pathogens and biofilm-associated infections represents a major challenge for effective wound healing and significantly contributes to the global burden of antibiotic resistance. , Conventional wound dressings incorporating antiseptics or antibiotics are often associated with cytotoxic effects and the emergence of microbial resistance, highlighting the urgent need for alternative antimicrobial strategies.

One promising approach involves the functional modification of biomaterials with inorganic bioactive components, enabling not only antimicrobial activity but also the initiation of controlled host tissue responses that support and accelerate wound repair. In this context, bacterial cellulose (BC) has attracted considerable attention as a wound dressing material due to its ultrafine three-dimensional nanofibrous network, high water-holding capacity, and excellent biocompatibility. − However, pristine BC primarily acts as a passive physical barrier and lacks intrinsic antimicrobial and bioactive functions, necessitating the development of advanced multifunctional platforms capable of simultaneously suppressing biofilm formation and promoting tissue regeneration.

MXenes, a class of two-dimensional (2D) nanomaterials belonging to the family of transition-metal carbides, nitrides, and carbonitrides, have emerged as promising candidates for antimicrobial and antibiofilm applications. Growing evidence indicates that MXenes and MXene-based composites effectively inhibit biofilm formation and may actively contribute to biofilm disruption. In parallel, the incorporation of hydroxyapatite (HAp), a calcium phosphate mineral with a Ca/P ratio of 1.67, is known to accelerate wound healing by modulating inflammatory cell infiltration and stimulating fibroblast proliferation and macrophage migration. −

To the best of our knowledge, no reports in the biomaterials literature have described a ternary BC/HAp/MXene composite system. Nevertheless, numerous studies have demonstrated the individual and hybrid performance of BC-, MXene-, and HAp-based materials, providing strong experimental evidence of their intrinsic antimicrobial properties and their ability to stimulate skin and bone tissue regeneration. While BC-MXene composites have been explored for conductive scaffolds in tissue engineering, they often lack hemostatic efficacy and regenerative signaling crucial for dynamic wound environments. Similarly, MXene-HAp systems developed for bone repair emphasize osteoconductivity and structural rigidity but are ill-suited for soft tissue applications due to limited fluid absorption and passive biological responses. , Notably, although HAp can mitigate MXene cytotoxicity and support immunomodulation under external stimuli (e.g., magnetic fields), such programmable regulation has not been adapted to wound dressings. Moreover, neither binary system fully addresses the triad of infected wound management: rapid exudate control, potent antibiofilm action, and active transition from infection suppression to tissue regeneration.

In this study, we integrate MXene and HAp into a BC scaffold to develop a nonantibiotic wound dressing that synergistically combines MXene’s potent antibiofilm activity with HAp’s ability to enhance biocompatibility and stability. The resulting BC/MXene + HAp as a nonantibiotic wound dressing exhibiting antiseptic, antibiofilm, and antioxidant properties, addressing two critical limitations of current antimicrobial dressings.

2. Materials and Methods

2.1. Synthesis of BC/MXene, BC/HAp and BC/MXene + HAp Composites

The bacterial cellulose producer Komagataeibacter xylinus C-3 was cultured under static conditions in Hestrin–Schramm (HS) medium (2% glucose, 0.5% yeast extract, 0.5% peptone, 0.27% Na₂HPO₄, and 1.5% citric acid) − for 6–7 days at 29–30 °C. , The resulting BC films were washed with deionized water until a pH of 6.8–7.2 was achieved, then treated with 1% NaOH solution at 35 °C for 24 h to remove impurities, including producer endotoxins. After rinsing, the films were sterilized via autoclaving and used for subsequent experiments. ,

MXenes (Ti₃C₂T _x ) were synthesized from a MAX-phase precursor (Ti₃AlC₂, Carbon-Ukraine Ltd.) via selective aluminum etching. Briefly, 1 g of Ti₃AlC₂ was gradually added to 20 mL of 32% HCl solution containing 1.6 g LiF and incubated at 42 °C for 48 h under continuous stirring. The product was centrifuged at 3500 rpm and repeatedly washed with deionized water until a pH of 5.0–5.5 was achieved. Delamination of Ti₃C₂T _x flakes was performed via ultrasonication (5 min, 900 W). The suspension was centrifuged at 5000 rpm for 5 min to separate exfoliated nanosheets from residual sediment. , The concentration of Ti₃C₂T _x in the colloidal solution was determined by vacuum filtration through a PVDF membrane (0.45 μm), followed by drying at 110 °C for 12 h, yielding a final concentration of 11.5 mg/mL. ,

HAp was synthesized from eggshells through a calcination-precipitation method. First, 4 g of eggshells were calcined at 950 °C for 2 h to produce calcium oxide (CaO). Subsequently, 2 g of the resulting CaO was reacted with 40 mL of 6% phosphoric acid (H₃PO₄) solution under constant stirring for 1 h. The mixture was then homogenized via ultrasonication (32 kHz, 1 h) in an ice bath to ensure complete reaction and particle dispersion.

30.6 mg of HAp was gradually added to 4 mL (11.5 mg/mL) of freshly prepared Ti₃C₂T _x colloidal solution. The mixture was magnetically stirred for 2 h, followed by centrifugation at 4000 rpm for 10 min. The resulting precipitate was vacuum-dried at 100 °C for 12 h To fabricate the composites, bacterial cellulose films of uniform mass and thickness were employed. The BC matrix was functionalized through ex situ incorporation of three distinct solutions: (1) a freshly prepared Ti₃C₂T _x MXene colloidal suspension (5 wt %), (2) an aqueous hydroxyapatite solution (0.05 wt %), and (3) a preformed MXene + HAp composite dispersion (0.1 wt %). All reported mass fractions were calculated relative to the dry weight of BC. The BC films were immersed in each respective solution and incubated at 37 °C for 12 h under orbital shaking (140 rpm) to ensure homogeneous infiltration. The loading efficiency of each component into the BC matrix was quantified gravimetrically. Post incubation, residual solutions were subjected to vacuum filtration, and the retained solids were dried at 110 °C for 12 h. The mass of incorporated materials was determined by subtracting the filtered residue mass from the initial mass of components in the solution. This protocol yielded final composite compositions of 4 wt % Ti₃C₂T _x , 0.03 wt % HAp, and 0.08 wt % MXene + HAp, calculated relative to the dry weight of BC, demonstrating precise control over component distribution within the BC scaffold.

2.2. Structure and Composition Analysis

The surface morphology of the hydrogels was characterized using a field-emission scanning electron microscope (FE-SEM, JEOL JSM-7600F). Elemental composition and spatial distribution were analyzed via energy-dispersive X-ray spectroscopy (EDS) equipped with an X-Max 50 mm² silicon drift detector. To ensure representative data, elemental quantification was performed by averaging results from five distinct regions across the sample, enabling reliable assessment of the uniformity and integration of HAp within the BC and MXene matrix.

The crystal structure of the composites was analyzed using X-ray diffraction (XRD) with a PANalytical Empyrean X-ray diffractometer (Malvern Panalytical, The Netherlands). The XRD analysis was conducted in a θ −2θ configuration using a Cu Kα radiation source (λ = 0.154 nm, 2.2 kW ceramic anode). The X-ray source was operated at 40 kV and 40 mA, utilizing a divergent slit of 1° and an antiscatter slit of 1°. A PIXcel3D matrix detector with 256 channels was used for signal collection. The θ −2θ scan was performed over a range of 20°–100° with a step size of 0.015° and a scan rate of 2°/min. The resulting crystallographic data were further utilized for SAED pattern identification.

The advanced microstructure of the BC-based composites was investigated using transmission electron microscopy (TEM) on a JEOL JEM-ARM200CF operated at 200 kV. The electron-transparent sample was prepared via focused ion beam (FIB) milling using a Zeiss Auriga 60 FIB-SEM system, with final polishing performed at 5 kV and 240 pA to minimize surface damage. Elemental distribution was analyzed by energy-dispersive X-ray spectroscopy using a large-solid-angle (up to 0.98 sr) JEOL JED-2300T CENTURIO SDD detector (100 mm² active area), enabling high-sensitivity mapping of Ti, C, O, and F.

X-ray photoelectron spectroscopy (XPS) analysis of the BC/MXene + HAp composite was performed using a PREVAC EA15 hemispherical electron energy analyzer equipped with a 2D multichannel plate detector and an Al Kα X-ray source (1486.6 eV, PREVAC dual-anode XR-40B) under an ultrahigh vacuum of 1.2 × 10^–7 Pa. Survey spectra were acquired with a pass energy of 200 eV and a step size of 0.9 eV, while high-resolution spectra were collected at a pass energy of 100 eV and a step size of 0.05 eV to enhance spectral detail. Binding energy calibration was referenced to the adventitious carbon. Spectral deconvolution was carried out using CASA XPS software, employing a mixed Gaussian (70%)/Lorentzian (30%) line shape for peak fitting and a Shirley background correction to accurately determine the chemical states within the composite.

2.3. Biomedical Properties

2.3.1. Bacteriological Study of Burn Wounds

The samples of burn wound exudate were transported in Cary-Blair transport medium (Copan, Italy) while maintaining the required temperature range (2–8 °C). The delivery time to the laboratory did not exceed 7 h, with the maximum allowable interval being 24 h from the time of sample collection. As part of the study, the following were evaluated: (a) the degree of microbial contamination (CFU/g of tissue) using quantitative culture on blood agar (HiMedia, India) and MacConkey agar (BioRad, USA); (b) the presence of polymicrobial associations through microscopy of Gram-stained smears and analysis of colony morphology. Species identification of microorganisms and determination of their antibiotic susceptibility were performed using the automated VITEK 2 Compact system (bioMérieux, France).

2.3.2. Study of Antibiofilm Activity

The antibiofilm activity of BC, BC/HAp, BC/MXene, and BC/MXene + HAp against methicillin-resistant Staphylococcus aureus (MRSA) and fluoroquinolone-resistant Pseudomonas aeruginosa (FQRPA) was evaluated using the method [Stepanović, S., et al.]. with modifications.

The test materials were placed into wells of a 24-well plate (Costar) and incubated with 2 mL of bacterial suspension (10⁵ CFU/mL) in tryptic soy broth (TSB, HiMedia) containing 2% glucose. BC was used as a positive control. Incubation was carried out at 37 °C for 24 h under static conditions. After removing the supernatant, the materials were washed three times with phosphate-buffered saline (PBS).

Cells adhered to the material surface were stained with a 0.1% crystal violet solution (Sigma-Aldrich). After removing the dye, the materials were rinsed with distilled water and dried at 30 °C. The biofilm-associated dye was extracted using 95% ethanol. Optical density (OD) was measured at 570 nm using a BioTek Synergy H1 microplate spectrophotometer. Biofilm formation was assessed based on the following criteria: Negative: OD < 0.1; Weakly positive: 0.1 ≤ OD < 1; Positive: OD ≥ 1.

2.3.3. Study of Antibiofilm Activity Using the LCWB Model

In this study, the antibiofilm activity of materials was assessed using a modified lubbock chronic wound biofilm (LCWB) model, developed by San et al. The experimental design is illustrated in Figure . A dual-species biofilm was established by mixing 10 μL of S. aureus (MRSA, methicillin-resistant strain) and P. aeruginosa (FQRPA, fluoroquinolone-resistant) suspensions at a concentration of 10⁶ CFU/mL. The mixture was inoculated into polystyrene tubes containing Brucella broth (BB) supplemented with 0.1% bacteriological agar, 50% porcine plasma, 5% erythrocytes and 2% fetal bovine serum (FBS). Sterile pipet tips were placed in the medium to serve as a substrate for microbial adhesion and biofilm growth. Incubation was performed in an orbital shaker at 37 °C for 48 h. After incubation, the mature biofilm was harvested from the pipet tips, washed with sterile phosphate-buffered saline (PBS, pH 7.4), and transferred to an artificial “wound bed” composed of a two-layer agarized medium (Bolton broth with 1.5% agar and 1% gelatin).

4.

Lubbock chronic wound biofilm model.

To evaluate the antibiofilm effects of BC, MXene, HAp, and MXene + HAp, the materials were introduced during biofilm maturation. The tubes were incubated for 48 h at 37 °C. After incubation, the biofilm was mechanically disrupted via vortexing (1 min, 200 rpm), ultrasonication (3 min, 40 kHz, 100 W power), secondary vortexing (1 min). Serial dilutions of the suspension were plated on selective media: Mannitol Salt Agar (MSA) for S. aureus and Cetrimide Agar (CET) for P. aeruginosa. Plates were incubated for 24 h at 37 °C. Antibiofilm activity was expressed as the percentage reduction in CFU/mg of biofilm compared to the control.

The methodology of Kucera et al. was used to analyze the impact of composites (BC, BC/MXene, BC/HAp, BC/MXene + HAp) on mature biofilm. The wound bed with biofilm was covered with the test materials and incubated for 48 h at 37 °C. Post-treatment, the biofilm was disrupted as described above. Serial dilutions (10¹–10⁶) were plated on MSA and CET, followed by colony counting.

2.3.4. Study of Cytotoxicity and In Vitro Biocompatibility

The cytotoxicity of materials was investigated using mouse fibroblast NIH-3T3 cells obtained from the Biomedical Engineering Laboratory of Aachen University of Applied Sciences (Germany). Cells were cultured in modified DMEM medium (Gibco) supplemented with 10% fetal bovine serum (FBS, HyClone), 1% l-glutamine (PanEco), 2 mg/L lactalbumin hydrolysate (Sigma-Aldrich), and antibiotics (100 IU/mL penicillin, 100 μg/mL streptomycin). Incubation was performed at 37 °C in a 5% CO₂ atmosphere. The medium was replaced every 4 days. Cells were grown to 75–85% confluency, then dissociated with 0.25% trypsin–EDTA solution (PanEco) and resuspended at a concentration of 10⁴ cells/mL.

Material biocompatibility was evaluated according to ISO 10993-5:2023 , by assessing the following parameters: viability, adhesion, morphology, and cell proliferation.

Cell viability was determined after 72 and 120 h of incubation with materials. Cells were stained with 0.4% trypan blue solution (Sigma-Aldrich) at a 1:1 ratio. Viable (unstained) cells were counted using a Bio-Rad TC20 automated cell counter.

Cells were seeded in 96-well plates (5 × 10⁴ cells/well) and incubated for 24 h. Material samples (1 cm² area) were placed in wells and incubated for 24, 48, and 72 h. After material removal, 100 μL of MTT solution (0.5 mg/mL in medium) was added and incubated for 2 h. Formazan crystals were dissolved in 100 μL of lysis buffer (10% SDS, 40% DMSO, 50% isopropanol). Optical density was measured at 570 nm using a BioTek PowerWave 340 microplate spectrophotometer.

Adhesion properties, morphology, and proliferation of fibroblasts incubated with materials were evaluated using a Zeiss AxioVert 25 inverted microscope. Fluorescence images were acquired on days 3 and 5 of cultivation. Samples were prefixed with 4% formaldehyde solution, then stained with DHR-123 fluorescent dye (emitting green fluorescence in metabolically active cells). For actin filament staining (green), NIH3T3 cells were treated with Alexa Fluor 488 phalloidin (1:20, Invitrogen, USA). Alexa Fluor 488 phalloidin-labeled fluorophores were excited at 488 nm using a 505–530 nm bandpass filter.

The data are presented as mean ± standard deviation (SD) (n = 5). Statistical significance was determined using one-way analysis of variance (ANOVA, p < 0.05) in GraphPad Prism 10 software.

2.4. Tensile Testing of Composites

Tensile mechanical properties, including elastic modulus (Young’s modulus, E), tensile strength, and elongation at break, were determined via uniaxial tensile testing according to ASTM D638-V. Rectangular specimens with a nominal gauge length (l) of 20 mm were used. The cross-sectional area (S) of each specimen was calculated from its measured width (a) and thickness (b) prior to testing (S = a × b).

The elastic modulus (E) was calculated from the initial linear elastic region of the stress–strain curve using the formula

$$E=\frac{F/S}{\mathrm{\Delta }l/l}=\frac{Fl}{S\mathrm{\Delta }l}$$

where F is the applied normal tensile load (N), S is the original cross-sectional area (mm²), l is the original gauge length (20 mm), Δl is the change in length (elongation, mm) corresponding to the load F within the elastic deformation regime.

2.5. Statistical Analysis

All quantitative data are presented as mean ± standard deviation (SD). Biofilm formation assays (n = 3), CFU reduction assays (n = 3), MTT cytocompatibility assays (n = 5), and mechanical tensile tests (n = 5) were analyzed using one-way ANOVA followed by Tukey’s post hoc test. For the MTT assay measured at multiple time points, a two-way ANOVA (factors: treatment × time) was applied. Differences were considered statistically significant at p < 0.05 and highly significant at p < 0.01.

3. Results and Discussion

3.1. Characterization of the BC/MXene + HAp Nanocomposite

Representative scanning electron microscopy micrographs (Figure A,B) and corresponding EDS analysis (Table ) elucidate the distinct morphology and phase distribution within the BC-MXene hydrogels (See also Figure S1). The BC/MXene + HAp composite (Figure B) exhibits a particulate morphology characterized by bright, irregularly shaped hydroxyapatite agglomerates (1–5 μm diameter) embedded within a continuous matrix. EDS point analysis confirms the HAp phase via high Ca, P, and O signals (Ca/P atomic ratio ≈1.35, indicating calcium deficiency) and identifies a titanium-rich MXene phase (Ti₃C₂T _x ) by high Ti content and associated F. Elemental mapping reveals MXene flakes dispersed within the matrix and decorating HAp particle surfaces. The matrix itself, inferred to consist of bacterial cellulose, is overlaid with a sodium aluminosilicate phase (high Si, Na, Al). , In contrast, the BC/MXene composite (Figure A) presenting a more uniform, monolithic matrix with visible microcracking. EDS confirms the absence of Ca and P, while elemental maps and point analysis show homogeneous colocalization of titanium (MXene) and silicon within the matrix, forming a titanium silicate composite embedded in the BC network. The matrix here is identified as a sodium silicate (high Si, Na), lacking the aluminum found in the HAp-containing counterpart. This analysis validates the targeted compositional differences: the ternary composite combines distinct HAp particles with MXene in aluminosilicate/BC matrix, while the binary composite forms a homogeneous MXene-silicate/BC structure.

1.

Morphological and structural characterization of BC-based composites. (A,B) Scanning electron microscopy (SEM) images showing the surface morphology of (A) BC/MXene and (B) BC/MXene + HAp composites. (C) X-ray diffraction (XRD) patterns of pure bacterial cellulose (BC), BC/MXene, and BC/MXene + HAp. (D–G) Transmission electron microscopy (TEM) and elemental analysis of the BC/MXene + HAp composite: (D) FIB cross-section showcasing the internal lamellar structure and interface, accompanied by an EDS map; (E) detailed TEM image of HAp nanoparticles within the composite matrix; (F) corresponding selected area electron diffraction (SAED) pattern; and (G) BF-TEM image revealing the dispersion of HAp nanoparticles with the corresponding EDS elemental map highlighting the distribution of key elements.

1. EDS Analysis Results Corresponding to Figure A,B.

element	O	F	Na	Al	Si	P	Ca	Ti	Fe
BC/MXene (wt %)	49.89	0.1	1.62	0.0	21.97	0.49	0.3	19.44	6.18
BC/MXene + HAp (wt %)	42.93	0.0	3.6	0.41	5.89	12.59	23.68	3.62	4.14
pure HAp clusters (wt %)	40.05	0.0	0.28	0.09	0.25	20.91	35.99	0.32	2.11

A chemical composition of the composites was verified further by means of high-resolution X-ray photoelectron spectroscopy (HR-XPS). Obtained results elucidate the chemical states and interfacial interactions in BC/MXene and BC/MXene + HAp composites (Figure S2, Table ).

2. XPS Surface Composition and Line Fitting of Main Peaks.

composite/line	BC/MXene	binding energy [eV]	BC/MXene + HAp	binding energy [eV]
C 1s	C–O–H	284.3	C–O–H	283.1
	C–C	284.8	C–C	284.9
	CO/COOH	286.0	CO/COOH	288.6
O 1s	C–O–H	529.9	C–O–H	531.2
	O–C–O	530.5	O–C–O	532.9
	C(O)O	528.0	C(O)O	529.3
	P–O		P–O	532.0
Ti 2p	Ti4+	456.2	Ti4+	459.1
	Ti2+	454.8	Ti2+	458.3
	Ti–C	453.7	Ti–C	456.9
	Ti–O	457.2	Ti–O	460.0

The C 1s spectrum of BC/MXene (Figure S2A) show characteristic peaks at 284.8 eV (C–C, MXene sp²/sp³ carbon, , at 284.3 eV (C–OH, hydroxyl groups), and at 286 eV (CO/COOH). For BC/MXene + HAp (Figure S2D), the persistence of these peaks with minor intensity shifts suggests preserved MXene carbon states but modulated surface interactions with HAp.

The O 1s spectrum of BC/MXene (Figure S2C) shows contributions from C–OH (529.9 eV), CO (528.0 eV), and O–C–O (530.5 eV). In BC/MXene + HAp (Figure S2E), a distinct feature emerges near 532.0 eV, attributed to phosphate oxygen (P–O) from HAp, confirming its integration. Intensity changes at 529.2 eV imply enhanced hydroxyl interactions.

The deconvoluted Ti 2p spectra for both BC/MXene and BC/MXene + HAp (Figure S2C,F) reveal four primary components: the Ti–C bond of the MXene carbide backbone (453.7 eV) along with Ti²⁺ (∼454.8 eV), Ti⁴⁺ (∼456.2 eV), and Ti–O (∼457.2 eV) species. Appearance of the Ti–O peak indicates formation of surface titanium oxides and/or terminal groups (−OH, –O). The uniform positive shift of approximately 3.0–3.5 eV across the entire Ti 2p line in the BC/MXene + HAp composite indicates a systemic change in the local electrostatic environment at the MXene surface, consistent with interfacial dipole formation or work function alteration upon interaction with HAp. This interpretation is supported by a slight increase in spin–orbit splitting from 5.3 to 5.8 eV, suggesting enhanced polarization. Critically, HAp appears to modulate the oxidation process rather than prevent it. The reduced intensity of the Ti–C signal suggests partial oxidation, but the interfacial interactions via Ti–O–P coordination stabilize the resulting titanium species. The acquired FTIR spectra (Figure S3) confirm the successful integration of components within the composite, with spectrum for BC/MXene + HAp exhibiting a prominent carbonyl stretch at 1736 cm^–1 and a broad O–H stretch near 3254 cm^–1, characteristic of HAp, alongside peaks indicative of the BC matrix and MXene, while the deep trough at 2345 cm^–1 in spectrum for BC/MXene suggests potential CO₂ adsorption on MXene surfaces. This stabilization mechanism, complemented by FTIR evidence of successful chemical integration, allows the composite to maintain functional, redox-active sites, which is anticipated to contribute to the sustained antibiofilm activity of the material in aqueous environments.

The X-ray diffraction analysis confirms the phase composition and reveals structural interactions within the composites (Figure C). The pattern for pure BC (red) displays the characteristic diffraction profile of highly crystalline cellulose I with primary peaks at 2θ = 14.3°, 16.6°, and 22.5°, corresponding to the (1̅10) (110), and (200) planes, respectively. The sharpness of these peaks indicates a well-ordered structure. The BC/MXene composite (blue) pattern shows the persistence of the BC crystalline structure. However, the BC peaks exhibit slight broadening and a measurable shift to higher angles (e.g., the (200) peak), suggesting a decrease in crystallite size and a contraction of the BC unit cell due to interfacial stress induced by the incorporated MXene. The presence of MXene is indicated by low-intensity, broad peaks at ∼10.4° (002) and ∼35.7° (111), consistent with a low mass fraction (∼5 wt %) and a partially disordered or exfoliated structure. The weak intensity of these features precludes precise quantitative analysis of the MXene phase. Finally, the pattern for the BC/MXene + HAp ternary composite (green) is dominated by numerous sharp, well-defined diffraction peaks that are exclusively assigned to a highly crystalline hydroxyapatite (HAp) phase. Key HAp reflections (JCPDS #09-0432) are observed at 2θ = 25.9° (002), 31.8° (211), 32.9° (300), and 34.0° (202). The remarkable intensity of these peaks indicates that HAp is the major crystalline component in the composite. The underlying broad features of the BC matrix remain detectable but are significantly masked by the strong HAp signal.

TEM confirms interfacial bonding of the BC/MXene + HAp hydrogel, revealing its hierarchical structure. Composite cross-section prepared by focused ion beam (FIB) displaying BC/MXene lamellar structure with embedded HAp particles, about 200–500 nm in size (Figure D). The accompanying EDS map highlights the spatial distribution of calcium (green) localized at HAp regions and carbon (red) dominating the BC-MXene matrix.

High-resolution transmission electron microscopy (HRTEM) images (Figure E,F) resolve the crystalline lattice fringes of HAp nanoparticles (50–200 nm diameter), composed of smaller nanocrystals (10–20 nm). The corresponding SAED patterns, indexed along the [1100] zone axis of hexagonal HAp (ICSD 98-026-1063), validate the phase purity and preferred crystallographic orientation. Overall composite architecture: HAp nanoparticles anchored at the BC/MXene interface (Figure S4). It demonstrates successful integration, with distinct phase segregation evidenced by inverse Ca/C EDS signals.

High-resolution bright-field transmission electron microscopy (BF-HRTEM) images combined with EDS elemental mapping of a BC/MXene + HAp nanocomposite reveal the introduction of HAp into layered matrix of BC and Ti₃C₂T _x MXene (Figure G). Matrix exhibits a characteristic lamellar morphology with visible delamination gaps. Embedded within a void between the layers is an ovoid nanoparticle of hydroxyapatite, approximately 45–55 nm in length. Corresponding EDS maps (Figure G) display the spatial distribution of key elements: Titanium (Ti K, yellow) is uniformly distributed throughout the MXene-rich matrix but is absent in the HAp particle region; Carbon (C K, red) and Oxygen (O K, cyan) signals are widespread, originating from both the BC and MXene components, with a notable depletion in the particle area. The Fluorine (F K, green) signal is detected only in the matrix, confirming the presence of -F surface terminations typical of HF-etched MXene and further verifying that the particle is a distinct, fluorine-free phase. The lack of Ti and F in the embedded particle proves that HAp was successfully integrated as a separate phase.

3.2. Microbial Colonization and Emergence of Multidrug Resistance in Burn Wounds

A serious and pressing issue for all surgical hospitals, including burn units, is healthcare-associated infections caused by opportunistic multidrug-resistant microorganisms that frequently form biofilms. In this context, one of the primary objectives of the present study was to identify potentially dangerous microbial key markers.

A dynamic analysis of 60 microbiological samples from 20 patients (aged 15–60 years) with burns covering 5–20% total body surface area (II–III degree) was performed. The investigation assessed microbial colonization levels, detected biofilms, and identified pathogens.

A positive culture result, indicating wound infection, was detected in 76% of initial samples collected between days 3 and 5 postadmission (Figure ). Among these positive cultures, monomicrobial infections accounted for 78% of cases, while polymicrobial infections were observed in 22%. The most prevalent microbial isolates were P. aeruginosa (39.2%) and Acinetobacter baumannii (18.2%), followed by equal proportions of Klebsiella pneumoniae (12.1%) and S. aureus (12.1%). Less frequent isolates included Enterococcus faecalis (6.1%), Staphylococcus saprophyticus (6.1%), Staphylococcus epidermidis (3.1%), and Proteus vulgaris (3.1%). Notably, polymicrobial associations were detected as early as the first culture collection (days 3–5 of hospitalization), and by the end of the observation period, 75% of burn wounds demonstrated mixed microbial infections.

2.

Temporal progression of microbial infection complexity and antimicrobial resistance in burn wound infections over time postadmission.

Alarmingly, 32% of all positive cultures exhibited resistance to six or more antibiotics, indicating the development of multidrug resistance. Of particular concern was the observed doubling in the incidence of pan-resistance (resistance to all first-line and reserve antibiotics in the antibiogram panel) compared to initial culture results. By the fourth sampling series (day 24), 80% of the five isolated pathogens demonstrated pan-resistance to antimicrobial agents. This escalation occurred despite patients receiving comprehensive in-hospital treatment for a full month, including systemic antibiotic therapy combined with local wound care featuring antiseptic solutions and antibacterial ointment dressings.

Thus, the identified potentially hazardous key markers include: nonfermenting Gram-negative bacteria (P. aeruginosa and A. baumannii), antibiotic-resistant flora, and microbial biofilms, most frequently formed by P. aeruginosa and S. aureus. These findings align with published data on burn wound microbiota, where opportunistic pathogens, particularly the ESKAPE group (Enterococcus faecium, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa, and Enterobacter spp.) dominate. These pathogens are notorious for: high prevalence in healthcare settings, tendency to form polymicrobial associations, and multidrug resistance. ,

Given this evidence, methicillin-resistant S. aureus (MRSA) and fluoroquinolone-resistant P. aeruginosa (FQRPA) were selected as target microorganisms for subsequent antibiofilm activity testing of the studied materials. These indicator strains embody three critical characteristics: they are typical hospital-acquired contaminants, possess a high biofilm-forming capacity, and are clinically relevant MDR pathogens.

3.3. Estimation of Antibiofilm Activity

Current methods for diagnosing and testing antimicrobial activity against biofilms remain insufficiently effective. Conventional antimicrobial susceptibility tests, which involve exposing planktonic (suspended) cells to antibacterial agents, fail to reliably predict the efficacy of these agents against biofilm-embedded pathogens. Consequently, this study employed a modified biofilm formation assay in microtiter plates during the initial phase. Biofilms were cultivated on discs fabricated from experimental materials, which were then tested using a standardized 24-well plate setup. The biofilm formation on BC discs proved reproducible and straightforward. Crystal violet staining was used to quantify adherent biofilms. Data revealed that after 24 h, BC surfaces supported well-developed single- and dual-species biofilms. In contrast, biofilm formation on the tested composite materials was either minimal or absent in some cases (Figure ).

3.

Biofilm assay. (A)Visualization of biofilm formation on the surface of tested materials in a microtiter plate; (B)optical density of biofilms measured at 570 nm; (C)biofilm morphology (n = 3).

The reinforcing agents incorporated into BC differentially influenced the biofilm-forming capacity of target bacteria. A weak S. aureus biofilm was observed on the BC/MXene matrix, while BC/HAp and BC/MXene + HAp hydrogels showed no biofilm formation. P. aeruginosa exhibited lower sensitivity to the active components of the composite membranes: biofilm staining on HAp-containing discs was comparable to that on pure BC. BC/MXene and BC/MXene + HAp scaffolds demonstrated antibiofilm effects, though these were less pronounced against P. aeruginosa than against S. aureus.

Quantitative analysis (Figure C) showed that BC/MXene and BC/MXene + HAp significantly reduced biofilm biomass compared to pure BC (ANOVA, p < 0.05). For S. aureus, BC/MXene and BC/HAp decreased biofilm by 61.5% and 84.6%, while BC/MXene + HAp achieved the strongest inhibition (94%, Tukey, p < 0.01). For P. aeruginosa, BC/MXene showed the greatest reduction (47.6%), whereas BC/MXene + HAp and BC/HAp produced smaller decreases (28.6% and 9.5%, p < 0.05). In mixed-species biofilms, BC/HAp and BC/MXene + HAp reduced biomass by 75.0% and 84.7%, with the ternary composite again demonstrating the highest efficacy (p < 0.01).

To date, three primary biocidal mechanisms of MXene have been elucidated: (1) the “nanoknife” effect, involving physical disruption of bacterial cell walls through mechanical cutting; (2) reactive oxygen species (ROS) generation, inducing oxidative stress in microorganisms; and (3) nanothermal activity, which inactivates bacteria via localized heat generation under light exposure. MXene demonstrate antimicrobial efficacy against standard pathogenic models, including Escherichia coli, S. aureus, Bacillus subtilis, P. aeruginosa, and K. pneumoniae. − Notably, multiple studies report higher activity of MXene against Gram-positive bacteria compared to Gram-negative species. This disparity is widely attributed to structural differences in bacterial cell walls: Gram-negative bacteria (e.g., P. aeruginosa) possess an outer lipid membrane that may shield against direct MXene-induced damage, whereas Gram-positive bacteria (e.g., S. aureus), despite their thicker peptidoglycan layer, lack this protective barrier, rendering them more susceptible to MXene interactions.

Additional factors influencing antimicrobial activity include the surface charge of both composite materials and target cells. Oppositely charged composites and bacteria promote nanoparticle adsorption onto cell membranes. Upon interaction with MXene-containing materials, membrane integrity is compromised, leading to leakage of cellular components. MXene penetrate the cell through these breaches, where they further induce ROS production, including superoxide anions (O₂ ^–), hydrogen peroxide (H₂O₂), and hydroxyl radicals (−OH). Oxidative stress is increasingly recognized as a universal antibacterial mechanism of metal-, metal oxide-, and carbon-based nanomaterials.

A similar phenomenon was observed for dual-species biofilms: a statistically significant (p < 0.05) reduction in staining intensity compared to the control (pure BC) was detected on discs containing MXene. The maximum reduction in biofilm growth (80%) relative to the BC-only samples was demonstrated by the BC/MXene + HAp composite, indicating that HAp enhances the antibacterial efficacy of MXene. This enhancement may be attributed to the mitigation of MXene oxidation, which typically occurs in aqueous and biological environments. We hypothesize that two mechanisms contribute to this effect: (i) the interaction between free OH^– groups and the terminal T _x groups of MXene might form a protective surface layer; (ii) hydroxyapatite-derived OH^– groups at the MXene surface could reduce Ti cations mobility, thereby decreasing interactions with oxygen molecules.

HAp may also enhance the hydrophilicity of Ti₃C₂T _x MXene surfaces, thereby reducing bacterial adhesion, which is a critical factor in antimicrobial efficacy. This property is particularly vital for medical applications, as preventing bacterial colonization and biofilm formation is essential for minimizing infection risks.

Unlike Gram-negative bacteria, the cell walls of Gram-positive bacteria exhibit higher hydrophobicity due to their thick peptidoglycan layer, which hinders their adsorption onto hydrophilic surfaces. This may explain the reduced S. aureus biofilm volume observed on the BC/MXene + HAp composite and the predominance of rod-shaped P. aeruginosa cells in mixed microbial communities isolated from this material.

The absence of S. aureus biofilm on HAp-containing discs warrants specific attention. Most staphylococci, including S. aureus, possess the Bap protein (biofilm-associated protein), which facilitates bacterial attachment and intercellular adhesion. However, Bap activity is calcium-dependent: binding to Ca²⁺ ions render the protein incapable of mediating adhesion and biofilm formation. One plausible hypothesis is that Ca²⁺ ions released from the BC/HAp discs might diffuse into the nutrient medium and interact with the biofilm-associated protein (Bap), potentially impairing its adhesion-promoting function. If validated, this mechanism could explain the absence of S. aureus biofilms on HAp-containing discs. However, other factors (e.g., HAp-induced changes in surface hydrophilicity or ion-mediated disruption of fibrin networks) may also contribute.

Biofilms typically form on wound surfaces; however, in some cases, biofilms such as those produced by P. aeruginosa can penetrate deep into skin layers, evading detection by conventional diagnostic methods like wound swabs. As previously noted, developing effective antimicrobial and antibiofilm therapies requires appropriate microbiological models. While most current methodologies rely on planktonic microbial cultures, selecting an experimental platform for biofilm studies must balance two priorities: (1) mimicking in vivo tissue conditions where biofilms persist, and (2) adhering to ethical and humane principles by avoiding live animal models. A notable example of this dual approach is the Lubbock Chronic Wound Biofilm model, an in vitro system that replicates chronic wound environments. The LCWB model has gained traction due to its biological realism, cost-effectiveness, and ease of implementation, with a rapidly growing body of optimized studies validating its utility. This model provides a functional platform for evaluating antimicrobial and antibiofilm treatments under conditions that closely approximate in vivo scenarios.

This model comprises blood plasma, erythrocytes, and a mixed culture of S. aureus and P. aeruginosa, forming a coagulated dual-species biofilm system embedded within a fibrin network, which is a structure typical of in vivo wound environments. The system is characterized by rapid maturation of multispecies biofilms. The study was conducted following the experimental workflow outlined schematically in Figure .

S. aureus synthesizes coagulase, which generates an insoluble fibrin network that serves as a scaffold for wound biofilms. This network enables bacterial adhesion, not only for staphylococci but also for subsequent colonization by other species, forming polymicrobial biofilms. This mechanism represents a key factor explaining the frequent involvement of staphylococci in wound infections. They create a biogenic framework that facilitates multispecies biofilm development.

Biofilm formation occurs in sequential stages: (1) adhesion of microbial cells to the substrate, (2) colonization, and (3) maturation. Adhesion initiates the development of a three-dimensional structure that binds bacteria together, accurately replicating the spatial microbial colonization observed in chronic wounds. Consequently, the primary objective of this study was to evaluate the antibiofilm activity of the tested materials against both S. aureus and P. aeruginosa biofilms at two critical phases: initial formation and mature biofilm.

For the early stage inhibition assay, individual material components: BC, HAp, MXene, and MXene + HAp were introduced into a medium inoculated with a mixed bacterial culture. In the control group, biofilms were formed under the same conditions, but without the addition of the tested components (No treatment). The number of viable cells and LCWB weight for each species in the resulting biofilms was quantified (Figure ).

5.

Antibiofouling performance of membranes. (A)percentage reduction of CFU/mg of Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (FQRPA) and (B)mass of LCWB biofilms in forming and mature LCWB biofilms in the presence of BC, MXene, HAp, and MXene + HAp composite; (C)photographic images of Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (FQRPA) colonies.

According to the results of the study, treatment with BC did not lead to a reduction in bacterial cells. In contrast, in the presence of MXene, the growth of S. aureus decreased by 52%, and that of P. aeruginosa by 30%. When only HAp was added to the mixed biofilm, even fewer cells of S. aureus and P. aeruginosa were isolated: only 25% and 37%, respectively, compared to untreated suspensions. HAp, containing Ca²⁺, inhibits fibrin polymerization, thereby reducing ability of S. aureus to coagulate plasma and form an effective, complex fiber network. Fibrin creates a critical matrix during wound healing by serving as a substrate for cell attachment, proliferation, and extracellular material formation. Crucially, Ca²⁺ binding to the γ-chain of fibrinogen is fundamental for modulating fibrin polymerization. This promotes the subsequent aggregation of fibrin protofibrils into thick fibers. ,

Consistent with these findings, the results of this study demonstrate that HAp disrupts mature biofilms by degrading the fibrin scaffold that holds them together. This is corroborated by data showing that the mass of biofilms formed in HAp-containing media is, on average, four times smaller than in control samples (Table S1). When the MXene + HAp composite is introduced into the culture medium, biofilm growth is reduced by 93% and 84% for S. aureus and P. aeruginosa, respectively.

All reductions in CFU for MXene, HAp, and MXene + HAp were statistically significant compared to the untreated control (ANOVA, p < 0.05).

The mature biofilm collected had a firm-mucoid consistency and a macroscopic structure characterized by a central cavity (formed by the pipet tip), a surface biofilm region at the medium/air interface, and a lower region at the tip of the nozzle. Overall, no significant changes in microscopic morphology were observed during biofilm formation prior to transfer to the artificial wound bed. After applying BC/MXene patches to the mature biofilm, robust microbial inhibition was demonstrated, confirming a primary effect against S. aureus (67% reduction). Antibiofilm activity of MXene was also observed against P. aeruginosa (59% reduction) in a dual-species biofilm compared to control samples (pure BC). The greatest inhibitory effect was noted in the BC/MXene + HAp composite coating. In this experimental sample, CFU/mg reductions reached 98% for S. aureus and 86% for P. aeruginosa.

Prior studies suggest MXene can suppress bacterial growth through physical membrane disruption (“nanoknife” effect) and intracellular oxidative stress, , however the relative contributions of these mechanisms in our composite system require further validation. It is also known that in aqueous environments and in the presence of oxygen, individual MXene layers can aggregate, reducing their damaging effects. Immobilizing MXene within BC fibrils prevents this delamination, and HAp acts similarly to stabilize MXene. Furthermore, the HAp component directly mitigates MXene oxidation by forming stabilizing Ti–O–P bonds at their interface, which shield the MXene nanosheets from ROS. Such protective effect is supported by two main mechanisms: (1) OH^– ions from HAp form a protective layer by reacting with T _x groups (e.g., –O, –OH, or –F), thereby preventing oxidation; (2) hydroxide ions (OH^–) at terminal sites of MXene weaken the internal electric field of the composite, reducing the rate of Ti⁴⁺ and O₂ ^– interactions. Together, these mechanisms suppress oxidation and inhibit the synthesis of TiO₂ ²⁷.

It is also important to note that microorganisms can secrete extracellular polymeric substances (EPS) exopolysaccharides on their cell surfaces, which play a critical role in bionano interface interactions. EPS forms the matrix of the biofilm, in which bacterial cells are embedded. This matrix, primarily composed of proteins and polysaccharides, exhibits a high affinity for nanomaterials. This property may promote heteroagglomeration between MXene and bacterial cells. It is hypothesized that such physical bionano interface interactions also contribute to the toxicity of MXene. The “nanoknife” effect is the primary reason for the mechanical disruption of bacterial membranes.

Based on the obtained results, the tested materials can be ranked in the following order of antibiofilm activity: BC/MXene + HAp → BC/MXene → BC/HAp. In the LCWB model, these materials interfere with bacterial clustering during dual-species biofilm formation and can disaggregate and disperse microbial sessile cells in mature biofilms. Thus, during the initial phase of microbial colonization, the most active hydrogel (BC/MXene + HAp) delays infection progression. In mature biofilms, it disrupts bacterial clusters embedded in EPS, leading to the release and subsequent death of planktonic cells. Therefore, BC/MXene + HAp can be considered an effective nonantibiotic agent against resistant microorganisms isolated from chronic wounds.

3.4. Study of Biocompatibility and Cytotoxicity of Composite Materials Based on BC, BC/MXene, BC/HAp, and BC/MXene + HAp

The cytotoxicity of materials BC, BC/HAp, and BC/MXene + HAp was evaluated using the NIH-3T3 murine fibroblast cell line. All data obtained from the analysis were normalized to control cells.

On day 3 of the experiment, no significant differences were observed between the control groups and the groups treated with BC, BC/HAp, or BC/MXene + HAp. Cells formed a loose monolayer on the bottom of 12-well plates, with a notable number of mitotic cells present. Fibroblasts exhibited polygonal shapes, including elongated, spindle-shaped, and stellate morphologies. Their cytoplasm displayed small projections, and nuclei in most cells contained 2–3 nucleoli. Based on structural organization, the cells were classified as young fibroblasts. However, in the BC/MXene group, cell fragmentation was observed, indicating compromised adhesion.

Thus, the morphology of mouse fibroblasts on day 3 of cultivation reflected their functional characteristics: proliferative capacity and interaction with the microenvironment. Fibroblasts play a critical role in tissue repair, with their migration and proliferation essential for wound healing.

In both test and control cultures, localized areas contained cells with pyknotic nuclei and vacuolated cytoplasm. To objectively quantify dead cells and total cell counts, trypan blue exclusion assays were performed across experimental and control series (Table ).

3. Fibroblast Cell Counts after 3 and 5 Days of Exposure to Materials (*n = 7, *p ≤ 0.05).

	day 3		day 5
group	total cells	dead cells	total cells	dead cells
control	1.98 × 104	1.88 × 103	3.21 × 104	4.84 × 103
		9.5%		15.1%
BC	1.53 × 104	1.5 × 103	1.92 × 104	2.84 × 103
		9.8%		14.8%
BC/MXene	2.8 × 104	4.78 × 103	3.05 × 104	5.97 × 103
		17.1%		19.6%
BC/HAp	2.05 × 104	2.11 × 103	2.03 × 104	2.94 × 103
		10.3%		14.5%
BC/MXene + HAp	3.68 × 104	4.49 × 103	4.01 × 104	6.77 × 103
		12.2%		16.9%

On day 3, the percentage of dead cells in the BC, BC/HAp, and BC/MXene + HAp groups were 9.8%, 10.3%, and 12.2%, respectively, comparable to the control group (9.5%). Dead cell counts in both control and experimental cultures (treated with BC, BC/MXene, BC/HAp, and BC/MXene + HAp) remained within normal ranges for primary cultures. The highest percentage of dead cells was observed in the BC/MXene group (17.1%), representing a 7.6% increase compared to the control. The inclusion of HAp in the BC/MXene + HAp composite reduced MXene toxicity by 4.9%.

As reported in this study, the cytotoxicity of MXene arises from ROS generation. Upon contact with water, physiological fluids, or air, MXene surfaces produce free radicals, including superoxide radicals (O₂ ^–) and peroxides (H₂O₂). Hassan et al. described two mechanisms by which HAp mitigates MXene oxidation: (1) free OH^– groups from hydroxyapatite bind to terminal T _x groups (e.g., –O, –OH), forming a barrier against oxidation; (2) weakening of internal electric field: The presence of OH^– groups at MXene terminal sites slows Ti cation migration, reducing the chance of Ti⁴⁺ and O₂ ^– interactions, thereby suppressing TiO₂ synthesis.

By day 5, a significant increase in cell density was observed in both control and experimental cultures. No differences in cell arrangement or cytological features were noted between experimental groups (BC, BC/HAp, BC/MXene + HAp) and the control. Cells predominantly retained their phenotype as mature fibroblasts (spindle-shaped morphology, compact nuclei). The percentage of dead cells did not exceed 19.6%, consistent with norms for primary cultures. Cell density was highest in the central region of the wells, where cells formed compact clusters. Layered cell overlap was observed in localized areas. Mature fibroblasts dominated the well bottoms, exhibiting spindle-shaped morphology and small, dense nuclei. The number of dividing cells decreased, while cells with pyknotic nuclei (apoptotic markers) and cytoplasmic vacuolization/disintegration increased. Dead cell percentages were 15.1% (control) and 14.8%, 19.6%, 14.5%, and 16.9% for BC, BC/MXene, BC/HAp, and BC/MXene + HAp, respectively all within acceptable thresholds for primary cultures.

Thus, the cell growth patterns, spatial organization, total cell counts, and proportion of structurally compromised and dead cells collectively indicate that fibroblast cultures were in a stable growth phase by day 5 of cultivation. The presence of test materials in the nutrient medium exhibited no cytotoxic effects on the cells.

The percentage of dead cells in experimental cultures did not exceed 19.6%. These results demonstrate the stability of NIH-3T3 murine fibroblast cultures maintained for 5 days with composite materials, as well as the absence of cytotoxic disruption to cellular structural organization. Their sustained growth and proliferative activity confirm the biocompatibility of the materials.

Morphological analysis revealed a pronounced affinity of cells for the BC, BC/HAp, and BC/MXene + HAp composites compared to BC/MXene. The observed differences in adhesion are likely attributable to the structural properties of the composites. For instance, the porous architecture of BC and BC/HAp increases surface area, facilitating efficient cell attachment and proliferation. As demonstrated in the study by Chen et al., the highly ordered nanoporous structure of bacterial cellulose provides an optimal matrix for cell adhesion and growth in cultured systems.

The observed differences in cell adhesion can likely be attributed to structural variations among the materials. For example, the porous architecture of the materials promotes cell attachment and proliferation by increasing the surface area available for interaction. The highly developed porous structure of BC allows cells to adhere and thrive on its surface, ensuring robust adhesion and growth. A critical factor in enhancing cell–material interactions is the formation of adhesion foci-specialized protein complexes anchored by integrins. These transmembrane receptors mediate communication between the ECM and the cytoskeleton, regulating cellular migration and proliferation mechanisms. The incorporation of hydroxyapatite into the composite enhances the adhesive properties by facilitating Ca²⁺ ion binding to integrins, which activates ligand-dependent signaling pathways. , Thus, HAp not only mitigates MXene cytotoxicity but also modulates its biocompatibility at the molecular level. , The adhesive and proliferative properties of the cells were evaluated using fluorescence microscopy (Figure A).

6.

Viability and metabolic activity of NIH-3T3 murine fibroblast cells cultured with BC, BC/MXene, BC/HAp, and BC/MXene–HAp composites. (A)Fluorescent live/dead staining images showing cell morphology and viability after 24 h culture. (B)Quantification of cellular metabolic activity via MTT assay (absorbance at 570 nm) normalized to control (BC).

The cells retained their typical morphology and exhibited strong adhesion across all experimental groups. Visual analysis revealed no significant differences in the structure or morphology of cells cultured with BC, BC/HAp, and BC/MXene + HAp hydrogels. Metabolic activity was confirmed by intense green fluorescence, characteristic of viable cells.

Proliferative activity in the control group, observed on day 3, remained consistent throughout the experiment. Fluorescence microscopy showed that cells in the BC and BC/HAp groups proliferated 23% faster than those in the BC/MXene + HAp group by day 5 of cultivation. The incorporation of HAp into the BC/MXene composite not only improved the biocompatibility but also enhanced NIH-3T3 cell proliferation, indicating a synergistic effect between the components.

The colorimetric MTT assay was used to assess the metabolic activity of cells cultured with the materials, as well as their cytotoxicity (loss of viable cells) or cytostatic activity (shift from proliferation to quiescence). The reduction of the tetrazolium dye depends primarily on NAD­(P)­H-dependent oxidoreductase enzymes in the cell cytosol. Thus, MTT reduction correlates with cellular metabolic activity due to NADPH flux. Cells with low metabolic activity reduce minimal MTT, whereas rapidly dividing cells exhibit high MTT reduction. The MTT assay results are presented in Figure B.

The MTT assay showed that BC, BC/HAp, and BC/MXene + HAp did not induce cytotoxic effects at either 72 or 120 h (two-way ANOVA, p > 0.05). The modest reduction in metabolic activity observed for BC/MXene + HAp remained statistically insignificant (p > 0.05).

The metabolic activity of cells in the control group and those cultured with BC and BC/HAp samples increased at 72 and 120 h throughout the cultivation period. In the case of the BC/MXene + HAp experimental sample, a slight reduction in metabolic activity was observed in adherent cells on day 3 (≤19%) and day 5 (≤25%) of cultivation. Despite this, the BC/MXene + HAp material exhibited low cytotoxicity. The metabolic activity values correlated with the survival outcomes of cells cultured with the samples, indicating no toxic effects from the materials, as the cells retained their normal metabolic activity (full functionality).

3.5. Study of Mechanical Properties of Composite Materials Based on BC, BC/MXene, BC/HAp, and BC/MXene + HAp

The tensile testing results (Table ) reveal a complex interplay between the nanofillers (HAp, MXene) and the bacterial cellulose matrix, significantly altering the mechanical behavior. Pure BC exhibits a baseline tensile strength of 0.74 MPa, an elastic modulus of 1.63 MPa, and moderate ductility (45% elongation). Incorporation of hydroxyapatite into BC yields a slight but notable improvement, increasing tensile strength to 0.80 MPa and elongation to 52%, while maintaining a comparable modulus (1.54 MPa). This suggests effective load transfer and a reinforcing effect from the well-integrated HAp nanoparticles, enhancing both strength and toughness.

4. Mechanical Properties of BC-Based Composites under Tensile Loading .

sample	tensile load [N]	tensile strength [MPa]	elastic modulus [MPa]	elongation [%]
BC	13.5	0.74	1.63	45
BC/HAp	22.6	0.80	1.54	52
BC/MXene	15.1	0.20	0.25	85
BC/MXene + HAp	7.0	0.36	0.70	50

^a(n = 5).

Mechanical testing revealed statistically significant differences between groups (ANOVA, p < 0.05). BC/MXene exhibited a pronounced reduction in tensile strength and modulus compared to pure BC (p < 0.01). BC/HAp showed a modest but significant increase in elongation (p < 0.05). The BC/MXene + HAp composite displayed intermediate performance, demonstrating partial recovery of mechanical strength relative to BC/MXene (p < 0.05).

In stark contrast, the addition of MXene alone (BC/MXene system) drastically compromises stiffness and strength, reducing tensile strength to 0.20 MPa and modulus to a mere 0.25 MPa, despite inducing high ductility (85% elongation). This significant weakening is attributed to poor interfacial adhesion and/or MXene agglomeration disrupting the cohesive BC network. The ternary composite (BC/MXene + HAp) demonstrates an intermediate but compromised performance: its tensile strength (0.36 MPa) and modulus (0.70 MPa) are substantially lower than pure BC or BC/HAp, though improved compared to BC/MXene, while elongation (50%) reduces between BC and BC/MXene. This indicates that while synergistic interactions between HAp and MXene may provide partial reinforcement relative to BC/MXene, the overall mechanical integrity (strength and stiffness) is significantly reduced compared to BC or BC/HAp, likely due to phase incompatibility, uneven dispersion, or the dominant negative influence of the MXene on the load-bearing capacity of composite system.

4. Conclusions

The present study aimed to develop a potent nonantibiotic system for treating chronic wound infections, which are increasingly resistant to conventional antibiotics due to biofilm formation. To deconvolute the individual and synergistic contributions of each component, tests were performed on a sequences of material combinations, including pure bacterial cellulose (BC), binary composites (BC/MXene and BC/HAp), and the final ternary composite (BC/MXene + HAp).

The composite forms a hierarchical structure where HAp nanoparticles integrate at the BC-MXene interface, as verified by electron microscopy and elemental mapping. This integration is stabilized by chemical Ti–O–P bonds, which preserve MXene from oxidation and maintain its bioactivity. The exceptional antibiofilm activity of the composite, reducing MRSA and FQRPA viability by up to 98% and 86%, may arise from synergistic interactions between MXene and HAp. Importantly, the inclusion of HAp simultaneously resolved key limitations of the binary system by significantly improving biocompatibility and restoring mechanical strength.

Overall, the synergistic interplay between the components yields a multifunctional material with potent antimicrobial action and high bioactivity, presenting a promising next-generation platform for managing MDR-infected chronic wounds. Future work will focus on validating its efficacy in in vivo wound healing models, assessing long-term stability, and exploring its photothermal functionality to fully reveal its clinical potential.

No data was used for the research described in the article.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c11015.

• This material includes SEM images detailing the surface morphology of the composite, HR-XPS spectra, FTIR spectra, HRTEM images and a table detailing the LCWB weight and bacterial load (PDF)

A.B.T.: Conceptualization, Methodology, Investigation, Formal analysis, Writingoriginal draft, Visualization. V.B.: Conceptualization, Methodology, Investigation, Formal analysis, Writingoriginal draft, Writingreview and editing, Visualization. I.S.S.: Resources, Supervision, Funding acquisition, Writingreview and editing. I.A.: Investigation, Data curation. F.I.M.: Investigation, Validation. D.H.S.: Investigation, Resources. M.S.: Investigation, Formal analysis. K.R.: Investigation, Formal analysis. M.Č.: Investigation, Formal analysis. A.D.P.: Conceptualization, Project administration, Supervision, Funding acquisition, Writingreview and editing.

The authors declare no competing financial interest.