Characterization of a novel biomaterial obtained by culturing Ganoderma sessile on a substrate composed of hydroxyapatite nanoparticles and alginate with potential for bone tissue regeneration

Oral diseases represent a significant public health challenge, particularly periodontal and peri-implant diseases that result in considerable bone loss and diminished quality of life. To address these issues, tissue engineering techniques such as guided bone regeneration (GBR) use barrier membranes to promote healing of bone defects. This study introduces a novel GBR membrane that employs Ganoderma sessile mycelium as a microstructural director of the biomaterial, yielding a mycelium-alginate-nanohydroxyapatite (My‐ALG‐HA) composite. The mycelium colonizes the ALG‐HA substrate, forming a porous trabecular bone-like network. Water contact angle assays indicated an anisotropic interaction behavior, and tensile testing confirmed the material ductility. Adding mycelium‐derived molecules increases the biomaterial’s hydrophobicity and improves its resistance to degradation under simulated physiological conditions. Additionally, an indirect crystal-violet assay using biomaterial dispersions showed osteoblast-like cell (MC3T3‐E1) cytocompatibility, highlighting its potential as an advanced solution for bone tissue regeneration in regenerative medicine. This approach combines the unique hierarchical structure and hydrophobic properties of Ganoderma mycelium alongside the cytocompatibility and hydrophilicity of the ALG‐HA system to improve structural integrity and biological functionality. These results establish a preliminary physicochemical basis for further optimization of wet‐state stability, suture retention and indirect cell-material interactions.

One of the major challenges in periodontology is the regeneration of periodontal defects, which requires the restoration of lost alveolar bone tissue (Namanloo et al., 2022). Successful repair depends on preventing the rapid migration of non-osteogenic cells into the wound site while facilitating the recruitment and repopulation of osteoblast cells to the lesion for bone formation (Bădărău-Șuster et al., 2025). Guided bone regeneration (GBR) represents one of the most promising tissue-engineering strategies to address this challenge. In GBR, barrier membranes function as physical filters that promote bone defect healing by excluding soft-tissue ingrowth. Depending on their degradation profile, these membranes can be classified as non-resorbable or resorbable (Duarte et al., 2025). Non-absorbable membranes, such as polytetrafluoroethylene (PTFE) and titanium, are inert and provide excellent mechanical properties but require surgical removal. In contrast, absorbable membranes, like collagen, offer better biocompatibility but often face challenges related to rapid degradation rate and low mechanical strength (Angjelova et al., 2024). Therefore, there is a need for new biomaterials that can overcome the biomechanical limitations of current options, including managing chewing forces, addressing limited blood supply to tooth and implant surfaces, and controlling microbial contamination (Wang et al., 2023).

In the search for improved biomaterials, scientists are increasingly drawing inspiration from natural systems. In this context, fungal-derived biomaterials display notable properties attributed to their mycelium (D’Elía et al., 2023). The structural framework of these novel materials consists of fungal mycelium growing onto a substrate to form an interconnected network. In this system, the mycelium functions as a natural bio-adhesive, binding the substrate particles together through hyphal extension. The resulting composite exhibits a highly porous scaffold architecture (Mohseni et al., 2023), a key feature for bone tissue regeneration. Fungal mycelium also has natural hydrophobicity and antibacterial properties (Rojas-Osnaya et al., 2024), offering a promising solution to the increasing problem of bacterial drug resistance (Jin et al., 2024).

Recently, promising results have been obtained for using fungal mycelia, and their derivates, in tissue engineering applications (Antinori et al., 2021; Ruggeri et al., 2023; Wu, 2014). Narayanan et al. produced nanofibrillar scaffolds from Aspergillus sp. mycelium that exhibited biocompatibility with human keratinocytes and potential for tissue regeneration (Narayanan et al., 2020). Additionally, Ganoderma lucidum mycelium-derived fibers demonstrated biocompatibility with fibroblasts while promoting cell proliferation and collagen production (Wu, 2014). With respect to bone tissue applications, it has been demonstrated that α-chitin, a key structural component of the fungal cell wall, promotes the deposition of hydroxyapatite and facilitates keratinocyte migration to wound sites, both critical processes for effective healing (Antinori et al., 2021; Ruggeri et al., 2023).

Several fungal species have been reported to produce bioactive compounds with potential applications in biomedicine. A notable example is the genus Ganoderma, which is known for its diverse bioactive metabolites (Pan et al., 2013; Ryu et al., 2020; Sułkowska-Ziaja et al., 2023; Paterson, 2006). In particular, Ling Zhi-8 (LZ-8), a protein derived from G. lucidum acts as a potent immune modulator by enhancing cytokine expression, making it a promising candidate for osteoporosis treatment and prevention (Yang et al., 2020). Interestingly, the bioactive components of Ganoderma work synergistically and their medicinal efficacy reduces when isolated (Ferreira et al., 2015). Thus, utilizing complete mycelium as a biomaterial represents an effective strategy for exploit its full therapeutic potential.

In this work, we developed a novel biomaterial by culturing Ganoderma sessile mycelium on an potentially osteogenic substrate composed of alginate (ALG) and hydroxyapatite nanoparticles (HA) (D’Elía et al., 2013; D’Elía et al., 2015). The objective of this study is to improve the structural and biological properties of ALG-HA membranes previously developed by our research group (D’Elía et al., 2020) through the incorporation of a fibrous mycelium-based framework. The resulting mycelium–alginate–nanohydroxyapatite (My-ALG-HA) composite was designed to provide improved functionality for applications in guided bone regeneration (GBR) by better mimicking the native bone microstructure, increasing porosity, and modulating hydrophobic–hydrophilic properties. We characterized component retention by XRD and FTIR, examined structure and surface properties by electron microscopy and water contact angle measurements, and evaluated tensile behavior and PBS stability in the My-ALG-1.1HA lead composition. We also performed a preliminary indirect assessment of MC3T3-E1 cell response to dispersions of the different HA concentrations. The study was designed as an initial physicochemical characterization, laying the basis for future investigations into osteogenic induction and clinical GBR performance.

Synthesis of My‐ALG‐HA biomaterials

Despite the extensive research conducted on G. lucidum, the mycelium of G. sessile was chosen for the production of this biomaterial due to its genetic and phylogenetic relationship with G. lucidum and its genetic and phenotypic similarity (Galappaththi et al., 2022; Viceconte et al., 2021). Additionally, G. sessile is a well-known species that has been extensively studied by this research team (Postemsky et al., 2017). The mycelium of G. sessile was inoculated on substrates formed by ALG-HA hydrogels with in vitro osteogenic potential (D’Elía et al., 2020), to obtain biomaterials with superior biological and mechanical characteristics.

The substrate hydrogel was synthesized using a methodology developed previously (D’Elía et al., 2020), with modifications. Briefly, 2% w/v alginic acid was prepared in ddH 2 O and kept under 350 rpm stirring at 60 °C for 30 min. As a source of nutrients necessary for the mycelium’s growth, malt extract, yeast extract and sucrose were incorporated by sequence, and stirred in the same conditions until their complete solubilization, to obtain final concentrations of 2% w/v, 0.2% w/v and 1% w/v respectively. A volume of 8 mL of this substrate solution was added to a 60 mm diameter Petri dish and dried at 50 °C for 24 h. After drying, the membrane formed was carefully detached and immersed in 15 mL of crosslinking solution composed of 110 mM CaCl 2 and customized hydroxyapatite nanoparticles (HA) (D’Elía et al., 2013) solutions in ddH 2 O in different concentrations: 0, 1.1, 4.4% and 8.8% w/v. The substrates were stirred at 150 rpm, at room temperature (RT) for 4 h, to allow Ca 2+ -mediated ionic crosslinking of alginate chains in the presence of HA and then, they were sterilized by autoclave at 120° C for 20 min.

Under a laminar flow hood, a 4 mm 2 portion of G. sessile mycelium, strain E47 (CERZOS-UNS-CONICET, Bahía Blanca, Argentina), was inoculated on the substrate, and incubated at 25 °C in the darkness for 12 days. Then, mycelium-substrates were frozen at −30 °C for 72 h and dehydrated for 24 h in a freeze-dryer (Rificor L-A-B4), under a pressure of 0.038 mmHg for 24 h. Finally, they were autoclaved at 120 °C for 20 min, to inactivate and sterilize the mycelium. The resulting biomaterial exhibit two asymmetric surfaces: a mycelium-rich side that corresponds to the surface initially inoculated with the fungal mycelium, and an alginate-rich side (ALG-rich side) that corresponds to the side in contact with the substrate during biomaterial formation. Final biomaterials were named My-ALG, My-ALG-1.1HA, My-ALG-4.4HA, and My-ALG-8.8 HA. Biomaterials without HA served as control of the HA effect. The impact of varying HA concentrations in the mycelium colonization of the substrate over time was assessed through photographic documentation. The final My-ALG-HA biomaterial macrostructure aspect was studied by measuring the coverage area using ImageJ software before and after freeze-drying and autoclaving.

To corroborate the complete mycelium inactivation in the final biomaterials, a 4 mm 2 portion of autoclaved G. sessile E47 mycelium was re-cultivated in MYSA culture medium (2% w/v malt extract, 0.2% w/v yeast extract and 1% w/v sucrose, 2% agar, pH = 6), and incubated at 25 °C in the darkness for 60 days. The absence of mycelium colonization of the substrate was considered as a non-viable mycelium.

Physicochemical analysis

X-ray Powder Diffraction (XRD) measurements were conducted using a Philips PW 1710 diffractometer equipped with a Cu Kα radiation source (λ = 1.5418 nm) and a graphite monochromator, operating at 45 kV, 30 mA, and 25 °C.

Also, previously dried and grounded biomaterials were homogenized with 1% w/w KBr (FTIR grade, 99%, Sigma-Aldrich, ref. n. 221,864). The mixture was further dried at 60 °C for 48 h and placed in a micro-sample cup. The characterization was performed using a Fourier transform infrared spectroscopy (FTIR, NICOLET Nexus 470) with an AVATAR Smart Diffuse Reflectance accessory. The measurements were done at 2 cm -1 resolution and 64 scans min -1. The KBr spectrum was collected and subtracted from the samples’ spectra.

Structural analysis and surface characterization

Transmission electron microscopy (TEM)

The cellular ultrastructure was studied to evaluate the effects of autoclaving (120 °C, 20 min) on the hyphae structure. A sample of biomaterial was immersed in a 0.067 M phosphate-buffered saline (PBS, pH = 7.2) solution, with glutaraldehyde at 2.5%. Subsequently, it was washed three times with PBS. For fixation, 2% OsO 4 was added and immersed for 1 h, then washed four times with ddH 2 O for 15 min. Dehydration was carried out using a gradient of ethanol/distilled water: 30, 50% v/v for 15 min and 70% v/v overnight. Ethanol reached 96%, and dehydration continued with 50% v/v acetone/ethanol for 15 min until reaching 100% acetone for 1 h. Then the samples were infiltrated with Spurr resin/acetone at RT: 1:3, 1:1 v/v (1 h each) and 3:1, v/v overnight. The samples continued in pure resin for a day. The polymerization of the resin was carried out at 70 °C overnight. Ultrathin sections were obtained with an ultramicrotome (LKB, 2088), deposited on 300 mesh copper grids and contrasted with uranyl acetate (1 min) and lead citrate (5 min). Transition electron microscopy (TEM) measurements were carried out on a JEOL 100CXII microscope with a nominal resolution of 0.6 nm, operated with an acceleration voltage of 80 kV, and magnification ranges of ×80,000–×100,000.

Field emission scanning electron microscopy (FE-SEM)

The biomaterial was cut and mounted on stubs with double-sided tape. Gold or carbon was then evaporated using an Argon plasma metal evaporator (91,000 Model 3, Pelco). Biomaterial was dispersed on an aluminium conductive adhesive tape (3M®) and metallized with gold or carbon in a sputter coater. The observation was performed in a variable pressure Scanning Electron Microscope (SEM) (LEO EVO 40XVP-M) equipped with SE, SEVP and BSE detectors and connected to a microanalysis system, operated at 7.0 kV with a resolution (WD) of 2.1 nm. Images of representative areas were acquired at different magnifications (25000x and 30000x). The associated energy-dispersive X-ray spectroscopy (EDS) (Oxford X-max 50) provided qualitative information about surface elemental composition.

Water contact angle assay

The wettability of the two asymmetric surfaces of the biomaterial, the mycelium-rich side and the ALG-rich side, was evaluated by water contact angle measurements. Measurements were performed on samples before and after freeze-drying and autoclaving to assess the effect of processing on surface wettability. The water contact angle between the biomaterial surface and a water drop was measured at RT using a tailored system described by Belén and col. (Belén et al., 2022). Briefly, the system consists of placing biomaterial disks in sample holders aligned with a digital microscope. Subsequently, a tube was placed above the sample holder to form the drop. The other end of the tube connects to a syringe attached to a syringe pump, Thermometric 612 syringe pump 2, forming reproducible drops in all measurements. A water droplet of 2.0 μL was deposited over the disk to acquire the image taken within 3 s of liquid deposition by using the digital microscope (Andonstar digital microscope). All images were processed using ImageJ software (Schneider et al., 2012) and the contact angle measurements were carried out with an accuracy of ±1 using the Drop Snake and LB-ADSA available module (Stalder et al., 2006). The reported contact angle values are based on six to sixteen repeats.

Mechanical and stability properties

The My-ALG-1.1HA was preselected as the lead candidate, and all mechanical and stability assessments were performed exclusively on this material.

Tensile properties

The biomaterial was sliced into strips 5 cm long and 1 cm wide. Using an Instron Universal Testing Machine (Model 3369) equipped with a 1 kN load cell, the Young’s modulus (E), maximum tensile stress (σ max), maximum tensile strain (ε max), yield stress (σ y), and yield strain (ε y) were measured longitudinally. These tests were conducted at a speed of testing of 1 mm/min and an initial separation gap of 0.5 cm. The maximum tensile stress (σ max) was calculated by dividing the maximum load needed to fracture the biomaterial by its cross-sectional area. Each experiment was performed on six specimens.

Gravimetric assay

A portion of 28 cm 2 of biomaterial was placed in a pre-weighed Petri dish and 5 mL of sterile 1X PBS was added to each dish to cover the biomaterials samples. The dishes were then sealed with cling film and incubated in a thermostatic bath at 37 °C for 24 h. After this period, the PBS was removed, and the biomaterials were oven-dried at 50 °C for 24 h. Each dried sample was then weighed. Data was accurately recorded, and the samples were once again submerged in 5 mL of sterile PBS. This process was repeated to achieve the following submerging times: 1, 2, 4, 7 and 14 days, and it was conducted in quadruplicate. The degradation profile was represented as a plot of weight loss (Equation 1) over time (Tampieri et al., 2014):

W 0 and W t, are initial weight and weight at the specific time point, respectively.

Preliminary indirect cell-response assay: osteoblast cells viability and morphology

As a first approach, osteoblast cell viability was assessed by crystal violet assay and cell morphology was examined by optical microscopy. MC3T3-E1 cells were cultured in flasks in Dulbecco′s Modified Eagle′s Medium (DMEM; Sigma-Aldrich St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P-S) in an incubator with a humidified atmosphere of 5% CO 2 in air at 37 °C. The medium was changed every other day. Upon 80% confluence, cells were detached with a minimum amount of trypsin, which was inactivated with complete medium after 5 min. The cells were then re-cultured or used for the experiments. The biomaterial samples were washed two times with ddH 2 O for 15 min and dried at RT under sterile conditions. Then, they were mortared for 10 min in order to obtain powders and sterilized by autoclaving at 120 °C for 20 min. Afterwards, each biomaterial powder was immersed in DMEM 10% FBS; 1% P-S, in order to obtain a final biomaterial concentration of 1 mg/mL, and incubated at 37 °C in a water bath for 24 h while shaking at 30 rpm. MC3T3-E1 cells at passage 6 were seeded into 96-well plates at a density of 7,800 cells/cm 2 and cultured to let attach for 24 h. The next day, cell culture media was aspirated and cells were washed with 1X PBS. Then, attached cells were treated with 250 µL of 1 mg/mL biomaterial powder dispersions in complete medium, while medium without biomaterial was used as control. After 48 h of cell culture, each well was washed with PBS. For the crystal violet stain, a protocol described by Principe and col (Principe et al., 2023). was followed. Briefly, the cells attached to the bottom of the plate were fixed with 4% paraformaldehyde for 7 min, stained with 0.1% crystal violet solution for 10 min at RT and quantified by adding 0.2% Triton x-100. Finally, the absorbance at 590 nm was measured using a microplate reader (Biotec Synergy-HT). For cell viability evaluation, the average absorbance of the control wells, which received no treatment, was set to 100%, and the percentage of cell growth in each well was calculated relative to the control (% of control).

Statistical analysis

All experiments were performed in triplicate or sextuplicate, and the results are expressed as mean ± standard deviation. Statistical analysis was performed using Excel 2016 (Microsoft Corporation; USA). A one-way ANOVA was performed after confirming normality and homogeneity of variance. Student’s t-test was used to calculate the p-value and p < 0.05 was considered significant.

Mycelium growth and final My-ALG-HA biomaterial obtaining

To develop final My-ALG-HA biomaterials, G. sessile mycelium was inoculated and cultured on various substrates composed of ALG cross-linked by CaCl 2, and different concentrations of HA: 0, 1.1, 4.4% and 8.8% w/v. Photographic documentation revealed that the temporal progression of mycelium growth was comparable across the different substrates, with full substrate colonization observed by day 12 under all tested conditions (Figure 1). These findings suggest that the varying HA concentrations incorporated into the substrates do not significantly impact mycelium growth and colonization.

A biomaterial intended for biomedical applications must be both sterile and inactive. Consequently, it was necessary to inactivate the mycelium cells in the final My-ALG-HA biomaterial, which was achieved by autoclaving at 120 °C for 20 min. After completing the procedure for obtaining the final My-ALG-1.1HA biomaterial, it was observed that the macrostructural characteristics were preserved, with only a 21.70% reduction in the coverage area (Figure 2).

The inactivation was assessed by re-incubating a fragment of the treated biomaterial on MYSA culture media. The re-growth assay showed that, following these treatments, the mycelium exhibited no detectable biological activity after 60 days of culturing (Supplementary Figure S1).

Physicochemical analysis

X-ray diffraction (XRD) spectra showed no significant differences among biomaterials containing different amounts of HA (My-ALG-1.1HA, My-ALG-4.4HA and My-ALG-8.8HA) (Figure 3A and Supplementary Figure S2). The crystalline phases of the My-ALG-1.1HA lead composition were compared with pure synthetic HA nanoparticles, and its XRD pattern showed diffraction peaks (211) (130), and (213), matching those of synthetic HA (Figure 3B). Hydroxyapatite is a substrate key component and, therefore, of the final biomaterial. Additionally, the spectrum showed diffraction peaks at 24.18° and 37.35°, associated with the crystalline planes (0022) and (1032) of calcium oxalate dihydrate crystals, respectively (sample R050242.1 from the RRUFF dataset; Lafuente et al., 2015) (Figure 3A). The formation of calcium oxalate by fungi serves as a calcium reservoir, modulating phosphate availability in their environment. Specifically, Ganoderma mycelium produces calcium oxalate crystals when cultured in a Ca 2+ -enriched medium. In this regard, weddellite is the predominant form, characterized by a dehydrated tetragonal atomic arrangement (Guggiari et al., 2011). Interestingly, calcium oxalate crystals serve as common binding sites for bone proteins, such as osteopontin (Thurgood et al., 2012). This protein plays several critical roles in bone physiology, including promoting osteoblast adhesion and migration (Chen et al., 1993). Consequently, the presence of calcium oxalates in this biomaterial may facilitate the adhesion of bone proteins and act as nucleation centers for the formation of additional hydroxyapatite nanocrystals in situ. Thus, this potential interaction should be further explored.

The amorphous background observed in the My-ALG-HA biomaterials spectrum can be attributed to the amorphous components of the mycelium, which was also evident in the My-ALG spectrum (Figure 3C).

The Fourier transform infrared spectroscopy (FTIR) technique was used to analyze the composition, chemical interaction between the different components of the biomaterial (Figure 4; Table 1). The FTIR spectra of My-ALG-HA and the control without mycelium, ALG-1.1HA, show several common absorption bands. The broad band at 3700–3080 cm -1 corresponds to O–H stretching from hydrogen bonded hydroxyl groups, which may arise from mannuronic/guluronic acid units in ALG, hydroxyl rich polysaccharides (β glucans/chitin-containing matrix) in the mycelium, and, most importantly, a broad contribution from adsorbed water. The band at 2930–2900 cm -1, assigned to C–H stretching, is characteristic of the uronic acid backbone of ALG, but it also overlaps with lipids, proteins, and cell wall carbohydrates from the mycelium. In the 1660–1580 cm -1 region, the band related to amide I (C=O) is attributed to protein bound polysaccharides or glycoproteins from the mycelium, although it may also include contributions from adsorbed water, and it is present in all samples (Figure 4A).

Two characteristic bands of the ALG-1.1HA control material are observed at 1620 cm -1 and 1419 cm -1, both assigned to carboxylate (COO −) stretching of mannuronic/guluronic acid units in ALG. These bands are present in ALG-1.1HA and remain detectable in My-ALG-HA samples, although their intensity may vary, In the fingerprint region, the bands at 1087 cm -1 and 1032 cm -1 are associated with mannuronic and guluronic pyranose rings and phosphate vibrations of HA; they are also present in the control but are attenuated in the mycelium-containing samples (Figure 4B).

A key difference between mycelium containing samples and the control, ALG-1.1HA, is the band at 1320 cm -1, assigned to O–H bending and related to the chitin/glucan rich fungal cell wall. This band is absent in the ALG-1.1HA, confirming the contribution of mycelial components. In addition, the bands around 1069 cm -1 and in the 1043–1026 cm -1 region are associated with the carbohydrate fingerprint of polysaccharides from the mycelium, and their appearance or increased intensity in My-ALG-HA further indicates that a proportion of mycelium components, including proteins, glucans, galactans, heterogalactans, and galactoglucomannans, remained structurally stable following inactivation and sterilization treatments. Notably, these molecules are widely recognized as the principal bioactive constituents of Ganoderma, where their synergistic interactions may enhance a broad spectrum of biological activities (Ferreira et al., 2015).

Compared with ALG-HA, the mycelium containing samples show some bands that are attenuated, especially in the 1087 cm -1 and 1032 cm -1 regions, and absent at 1320 cm -1 region, that could indicate interactions between mycelium and the alginate/HA network. The increase in HA nanoparticle content at the tested concentrations did not appear to alter molecular interactions, consistent with previous observations in similar biomaterials. On the other hand, retention of the main ALG bands in the My-ALG and My-ALG-HA samples indicates that the principal functional groups were largely preserved during autoclaving. This is also particularly evident because of the presence of the main ALG-HA bands, located at 1620 cm -1 and 1419 cm -1, of our previously developed biomaterial, which was sterilized by UV irradiation (D’Elía et al., 2020). However, this does not exclude chain scission; further studies, such as rheological measurements, would be required to support such conclusions.

Structural analysis and surface characterization

TEM analysis of transversal hyphal sections revealed that, compared with the non-autoclaved biomaterial (Figure 5A), the autoclaved material (Figure 5B) exhibited a marked loss of structural integrity. This result is consistent with the re-growth assay (Supplementary Figure S1), indicating that the mycelium is no longer active. However, the thickness of the hyphal cell wall showed no significant differences before and after autoclaving, measuring 76.63 nm ± 15.19 nm and 77.14 nm ± 34.67 nm, respectively (Supplementary Figure S4A). These results suggest that the cell walls were preserved in the final sterile and inactivated My-ALG-HA biomaterial.

Electron-dense particles morphologically compatible with the HA nanoparticles used during synthesis were observed localized externally to the hyphae (Figure 5C). Their morphology remained consistent with that of the particles originally used in the biomaterial synthesis (Supplementary Figures S4C, D). and SEM-EDS independently showed co-localized calcium and phosphorus in irregular mineral deposits (Figures 6H–J). However, while TEM morphology alone cannot definitively distinguish HA from other electron-dense components, the particles are confidently described as presumed HA nanoparticles based on the available evidence previously described. Biomaterial My-ALG-8.8HA was selected for TEM because its higher HA content ensures clear visualization of particle localization and morphology.

Scanning electron microscopy (SEM) analysis was employed to investigate the microstructural organization of the composite biomaterial. The resulting micrographs revealed a well-defined and hierarchical architecture, in which the mycelium plays a central role in guiding structural formation. When the mycelium colonized the ALG-HA substrate, it led to the formation of a porous trabecular bone-like network with a hierarchical structure (Figure 6; Supplementary Figure S5). The resultant network structure is interesting as interconnected pores are essential for vital cell functions such as nutrient and oxygen supply and waste removal (Percival et al., 2024).

As expected, no mineral deposits were observed in the My-ALG biomaterial (Figure 6A). On the other hand, two distinct types of mineral structures were identified in My-ALG-1.1HA, My-ALG-4.4HA and My-ALG-8.8HA (Figures 6B–F). The first type consists of irregular-shaped structures, which could be attributed to HA (Figure 6E). The second type of minerals observed consists of tetragonal-shaped formations, likely corresponding to the “envelope-shaped” morphology typical of calcium oxalate dihydrate crystals (Laube et al., 2014) (Figure 6F). The presence of both hydroxyapatite and calcium oxalate dihydrate crystals was previously confirmed by XRD.

To determine the elemental composition of the mineral structures observed, scanning electron microscopy-energy dispersive X-ray analysis (SEM-EDS) was employed. The co-localization of calcium and phosphate atoms in the irregular-shaped minerals could be attributed to hydroxyapatite (HA) nanoparticles, as indicated by the arrows in Figures 6H–J. In contrast, the tetragonal-shaped mineral structures exhibit a notable absence of phosphorus (Figure 6I), while calcium is present, along with a higher concentration of oxygen atoms, as indicated by the squares in Figures 6I,J. Calcium and oxygen elements are characteristic of calcium oxalate crystals.

Surface wettability, defined by hydrophobicity or hydrophilicity, is crucial for interactions between the biomaterial and its biological environment. This property influences ion interactions, protein adsorption and cell adhesion (Guo et al., 2016). The wettability of a surface is often expressed in terms of contact angle (CA), the angle at which a liquid-vapor interface meets the solid surface in an air environment. A water CA greater than 65° indicates hydrophobic surfaces, while angles less than 65° suggest hydrophilic surfaces (Vogler, 1998). Wettability assays showed that My-ALG-HA biomaterial has different water CA depending on the side tested (Figure 7).

The side where the mycelium was initially inoculated, named “mycelium-rich side”, is hydrophobic showing water CA of 143.10° ± 5.18° (Figure 7A) and 147.77° ± 9.10° (Figures 7A,C,E) before and after freeze-drying and autoclaving, respectively. This low wettability is expected due to the presence of hydrophobic mannoproteins and hydrophobins, in the outermost layer of the fungal cell wall. In other research, Antinori and col. found that Ganoderma mycelium exhibits a highly hydrophobic nature with water contact angles greater than 120° (Antinori et al., 2021). In this sense, the micrometric roughness of the fibrous mycelium-based biomaterial strongly contributes to the hydrophobic character (Barylyak et al., 2024). However, at the “ALG-rich side”, the biomaterial exhibits water CA of 36.54° ± 5.38° and 38.37° ± 1.72°, before and after freeze-dry and autoclaving respectively (Figures 7B,D,E). This hydrophilicity could be due to the higher amount of alginate molecules, compared to mycelium, exposed on the surface. Water contact angle assays demonstrated the anisotropic water-interaction behavior of the biomaterial.

Hydrophobic surfaces could be advantageous due to bacterial adhesion inhibition (Song et al., 2015). Moreover, hydrophobic regions can impart mechanical robustness and stability to the biomaterial structure (Gregurec et al., 2016) while hydrophilic regions facilitate cell-biomaterial interactions and nutrients and waste exchange (Zhou et al., 2023). Thus, combining hydrophobic and hydrophilic sides within an anisotropic biomaterial could create a more biomimetic microenvironment that better regulates cell behavior and tissue regeneration.

Mechanical and stability properties

The macroscopic analysis of the My-ALG-1.1HA membrane demonstrates an integrated structure that is both facile to handle and flexible (Figure 8A). Additionally, superficial variations in color and texture between the mycelium-rich side and the ALG-rich side are clearly evident at the macroscopic scale (Figures 8B,C). This observation is consistent with findings from previous section about anisotropy in surface water affinity.

The mechanical strength of biomaterials must match the requirements of the specific regenerated tissue. For instance, GBR membranes require adequate mechanical support to facilitate bone growth and withstand physiological loads. The design must consider the mechanical properties to ensure the scaffold can support tissue regeneration effectively. Stress-strain curves obtained from the tensile test showed that My-ALG-1.1HA lead composition has distinct stages: elastic region, yielding and a significant plastic region (Figure 8D).

The mechanical strength parameters, elastic modulus and maximum tensile stress were, 25.28 ± 1.14 MPa and 0.32 ± 0.07 MPa respectively, were comparable to other mycelium-based biomaterials (Antinori et al., 2021; Elsacker et al., 2022). Interestingly, the maximum tensile strain was 22.20% ± 5.83% (Table 2), similar to other Ganoderma mycelium biomaterial (Antinori et al., 2021), but higher than mineralized mycelium biomaterials (D’Elía et al., 2020; Elsacker et al., 2020). Recent investigations have demonstrated that the tensile strength of oral mucosa tissues varies significantly, ranging from 0.83 MPa to 4.75 MPa, depending on the specific anatomical site. Interestingly, although these values exceed the tensile strength of the My-ALG-1.1HA biomaterial, the Young’s modulus of My-ALG-1.1HA biomaterial is in the range of oral mucosal tissues, 2.22 MPa–54.72 MPa (Jung et al., 2020). This similarity to oral tissues may make the biomaterial more useful for GBR, because matching the mechanical behavior of native tissues is important for good integration and function. Comparisons with oral mucosa or other mycelium-based materials provide valuable contextual insights; however, due to differences in thickness, hydration, test geometry, and loading rate, these comparisons should be interpreted as complementary rather than indicating direct equivalence.

In our previous work (D’Elía et al., 2020), we proved the HA contribution to the brittleness of ALG-HA membranes. In this opportunity, the effect of the same amount of HA in M-ALG-1.1HA seems to be masked by the mycelium presence, obtaining a more ductile biomaterial. The constant stress plateau observed in the stress-strain curve of My-ALG-1.1HA after the yield point shows the material’s ability to absorb significant energy before failure. This is a consequence of a material’s crystal structure rearrangement, which increases its toughness. In this sense, ductile biomaterials would be advantageous due to their capability to sustain large strains before fracture, which could help absorb energy and distribute loads in orthopedic applications (Barber et al., 2021).

Studying the degradation rate of biomaterials is essential, as it must be synchronized with the bone healing process to ensure optimal outcomes in bone tissue regeneration. Figure 8F shows the weight loss of My-ALG-1.1HA biomaterial over time in PBS. The weight loss of the biomaterial significantly increased over the first week after immersion. On days 1, 2, and 3, the degradation rates were 49.7% ± 0.6%, 59.7% ± 1.2%, and 64.1% ± 0.8%, respectively. Following day 7 of immersion, the weight started to rise steadily, reaching 67.4% ± 0.6% by day 7% and 70.9% ± 0.6% by day 16. Compared to previous findings (D’Elía et al., 2020), the degradation of My-ALG-1.1HA biomaterial was significantly slower than that of ALG-1.1HA membranes, with half of the biomaterial degrading approximately 380% more slowly. It indicates that incorporating mycelium molecules into this novel biomaterial enhances its hydrophobicity, increasing its resistance to degradation in a simulated physiological environment. Further improvements in the synthesis and composition can be done to extend stability include increasing or homogenizing ionic crosslink density, adding a secondary covalent/photo-crosslinked network, or forming alginate–chitosan polyelectrolyte complexes (Yu et al., 2023; Zhu et al., 2024).

Because contact angle and tensile behavior were not measured for the 4.4% and 8.8% HA compositions, no HA concentration-dependent conclusion can be drawn for these outcomes.

Preliminary indirect cell-response assay

The cytocompatibility assay was performed by culturing MC3T3-E1 osteoblast-like cells with dispersions of My-ALG, My-ALG-1.1HA, My-ALG-4.4HA, and My-ALG-8.8HA biomaterial powders in complete medium. Cellular morphology was evaluated via optical microscopy after 48 h of incubation. The results indicated that cell size, shape, and spreading were preserved in the presence of the biomaterials, with no observable alterations compared to the untreated control group (Figures 9A,B). The crystal violet assay, which indirectly quantifies adherent cells, shows that under the tested conditions there was no statistically significant reduction relative to the untreated control in the number of cells remaining attached (Figure 9C; Supplementary Table S1). These findings indicate that, under the conditions of this indirect assay, neither the mycelium nor the HA at the selected concentrations (1.1%, 4.4%, and 8.8%) exert any apparent adverse effects. These results provide an initial basis for additional studies to assess osteogenic potential, cell interactions on the intact anisotropic surfaces, alkaline phosphatase (ALP) activity, osteogenic-marker expression, and mineralization.

This study establishes the feasibility of growing Ganoderma sessile on Ca 2+ -crosslinked ALG–HA substrates to obtain an anisotropic biomaterial composed by mycelium, alginate, hydroxyapatite composite. The mycelium’s successful colonization of the substrate resulted in a mineralized network development characterized by a hierarchical fibrillar structure. In bone tissue environment, this structure is important because the interconnected pores are needed for nutrients and oxygen supply, as well as the removal of waste. Characterization through XRD, FTIR and electron microscopy supported retention of the principal organic and mineral components. In the My-ALG-1.1HA lead composition, the two surfaces displayed different wettability and tensile testing showed ductile behavior. The biomaterial maintains favorable osteoblast-like cell morphology and cytocompatibility. The novelty of the work lies in using intact Ganoderma mycelium as a structure-directing biological framework within an ALG–HA composite. The presence of Ganoderma mycelium active biomolecules highlights its promising therapeutic potential; however, further studies are required to fully characterize these compounds and confirm their biological efficacy. Future work should assess direct osteoblast adhesion, viability, differentiation and mineralization. Moreover, integrating advanced analytical techniques with targeted biological evaluations will be key to elucidating the therapeutic properties of the My–ALG–HA biomaterial. Overall, these findings emphasize the potential of mycelium-based composites to advance bone tissue regeneration and open new avenues for innovation in the field.