In Vitro Biodegradation and Biocompatibility of Bacterial Nanocellulose–Chitosan-Based Hydrogel Scaffolds for Bone Tissue Engineering

Abstract

Bacterial nanocellulose (BNC) has many advantageous physicochemical characteristics, including high mechanical strength, high porosity, excellent water adsorption, and biocompatibility, making it a promising option for a wide range of biomedical applications. However, the limited biodegradability of BNC within the human body could reduce its utility in this field. In the present study, we investigated the in vitro biodegradability of a BNC composite of bacterial nanocellulose–chitosan–alginate–gelatin (BNC–CS–AG–GT). This BNC–CS–AG–GT hydrogel scaffold was shown to be gradually degraded during immersion in simulated body fluid (SBF) with the addition of lysozyme. Furthermore, the compressive strength of the BNC–CS–AG–GT hydrogel slowly decreased in correlation with incubation time: by 8 weeks of incubation in SBF, the compressive strength was reduced from ∼68 to ∼25 MPa, coupled with a 54% weight reduction. In cell culture, the BNC–CS–AG–GT scaffold was noncytotoxic. Cultivation of osteogenic MC3T3-E1 cells in osteogenic medium within a BNC–CS–AG–GT hydrogel for 4 weeks showed that the BNC–CS–AG–GT hydrogel supports cell adhesion and cell proliferation and promotes alkaline phosphatase (ALP) activity and mineralization in vitro. Moreover, BNC–CS–AG–GT exhibited strong antibacterial properties. The favorable biodegradability, mechanical properties, biocompatibility, and antibacterial activity of the BNC–CS–AG–GT hydrogel scaffold indicate that it has potential as a promising candidate for applications in bone tissue engineering. However, although these findings suggest that BNC–CS–AG–GT hydrogels have osteogenic potential in vitro, future additional studies in vivo and extended osteogenic differentiation assays are required to confirm the efficacy of BNC–CS–AG–GT scaffolds under physiological load conditions.

1. Introduction

Bone tissue engineering attempts to integrate scaffolds and cells in an optimal environment in order to promote bone repair and regeneration. Appropriate scaffolds provide a three-dimensional (3D) structure that mimics the extracellular matrix and should enable cells to attach, proliferate, and differentiate in bone formation processes. Scaffolds should also possess specific functional characteristics that are important for applications in bone tissue engineering, including biocompatibility, biodegradability, bioresorbability, mechanical properties, and antibacterial properties, , together with properties that promote osteoconduction, osteoinduction, and osteointegration. Biodegradability in particular is considered to be a critical factor for bone tissue engineering applications where a temporary structural support for the regeneration of native tissue is needed. Controlled degradation of these biomaterials is essential to ensure that the scaffold breaks down over time, coinciding with the growth and maturation of the regenerated tissue.

Bacterial nanocellulose (BNC) is an interesting biomaterial that can be obtained from microbial fermentation and is produced by bacteria such as Acetobacter, Komagataeibacter, and Agrobacterium. , Structurally, BNC is a linear polysaccharide composed of glucose–glucose units connected by β-1,4-glycosidic bonds. BNC has gained attention for its unique properties, including high purity, high water content, and its nanofibrillar structure, making it a potential candidate for various biomedical applications, including tissue engineering. − Nonetheless, like other forms of cellulose, BNC cannot be biodegraded by enzymatic degradation processes within the human body. To date, there is a lack of comprehensive published data on the hydrolytic cleavage of BNC. Thus, this limited understanding of the biodegradation of BNC composites has emerged as a crucial constraint, which prevents its utilization as a scaffold material in some biomedical applications.

Chitosan (CS) is a natural polysaccharide derived from chitin, which is commonly found in the exoskeletons of crustaceans, such as shrimp and crabs. CS has been investigated for various biomedical applications due to its biocompatibility and biodegradability. In the human body, CS can be enzymatically degraded by enzymes such as lysozyme, which is naturally present in bodily fluids like saliva, tears, and mucus. Lysozyme catalyzes the hydrolysis of the β-(1,4)-glycosidic bonds in CS, breaking it down into smaller oligosaccharides and eventually into monomers such as glucosamine and N-acetyl-glucosamine. The final product of biodegradation is nontoxic to the cells. −

Alginate (AG) is a natural polysaccharide derived from brown seaweed, and it is composed of two monomers: β-d-mannuronic acid and α-l-guluronic acid bonded by 1-4 glycosidic linkage. , AG is known for its biocompatibility, biodegradability, and ability to form hydrogels under mild conditions, making it suitable for various applications in the field of tissue engineering and regenerative medicine. ,

Gelatin (GT) is a protein derived from the partial hydrolysis of collagen, which is a structural protein found in the connective tissues of animals. GT is widely used in the food industry, pharmaceuticals, and various biomedical applications, including drug delivery and tissue engineering. GT is commonly used in tissue engineering due to its biodegradability and ability to support cell adhesion and proliferation. , In addition, GT can be degraded by lysozyme. −

In this study, BNC, CS, AG, and GT were selected because of their complementary roles in scaffold design: BNC provides nanoscale structural reinforcement, mimicking ECM fibrils; CS contributes to improved biodegradability and provides intrinsic antimicrobial activity; AG enables ionic cross-linking for hydrogel formation and dimensional stability; and GT improves cell adhesion and protein release. Their excellent biocompatibility and biodegradability, structural similarity to native ECM, well-established safety profiles, clinical availability, and proven synergistic interactions provide enhanced mechanical stability and stimulate cellular responses. These polymers have established safety profiles, good printability, and reproducible cross-linking compatibility (via Ca²⁺ with AG), making them suitable for bone tissue engineering applications. − While other materials like tannic acid or hyaluronic acid are promising, for the present study, this combination of BNC, CS, AG, and GT was chosen to achieve controlled biodegradation, favorable biological responses, and more feasible scaffold fabrication.

The objective of the present study was to investigate the in vitro biodegradation of BNC–CS-based hydrogel scaffolds using simulated body fluid (SBF) supplemented with lysozyme. BNC and BNC–CS are produced biosynthetically from Acetobacter xylinum. , BNC and BNC–CS were composited with AG and GT to increase biodegradation rates and biocompatibility. After in vitro biodegradation in fluids SBF supplemented with lysozyme, the resulting soluble degradation products were analyzed for total protein and sugar content by Bradford protein assay and high-performance liquid chromatography (HPLC), respectively. During degradation, the morphology, chemical structure, and mechanical properties of the hydrogel scaffolds were monitored. Additionally, the hydrogel scaffolds were evaluated for cytotoxicity, in vitro biocompatibility, and antimicrobial properties.

2. Materials and Methods

2.1. Preparation of Hydrogel

2.1.1. Preparation of Bacterial Nanocellulose and Bacterial Nanocellulose–Chitosan

BNC and BNC–CS were prepared by following the optimal procedures described previously. , A. xylinum (AGR60) (supplied by Pramote Thamarat from the Institute of Research and Development of Food Product, Kasetsart University, Bangkok, Thailand) was used for BNC and BNC–CS biosynthesis, and the culture medium consisted of coconut water from mature coconuts (Burapha City Bang Wua Fresh Market, Chachoengsao, Thailand), supplemented with 5% (w/v) sucrose, 0.50% (w/v) ammonium sulfate, and 1% (v/v) acetic acid. For BNC–CS biosynthesis, CS (MW 30,000 and DAC 85%, purchased from Seafresh Chitosan Company Limited, Thailand) was added at 0.75% (w/v) to the culture medium. To start the biosynthesis, the culture of A. xylinum (AGR60) at 5% (v/v) was added to the culture medium and incubated at 30 °C for 7 days. Then BNC and BNC–CS were harvested, washed with DI water, treated with 1% (w/v) sodium hydroxide to eliminate bacterial cells, and rinsed until reaching a pH of 7.0. The purified BNC and BNC–CS were subsequently homogenized at 20,000 rpm for 30 min to form a slurry, centrifuged to remove excess water, and stored at 4 °C before use. Schematic diagrams for BNC–CS production and the proposed mechanism of BNC–CS biosynthesis from A. xylinum are shown in Figure S1.

2.1.2. Preparation of Alginate–Gelatin

GT powder of 15% (w/v) (300 bloom and type A, purchased from Sigma-Aldrich, USA) was dissolved in 1× phosphate buffered saline (PBS) at 50 °C with constant stirring until complete dissolution. Then AG powder at 2% (w/v) (purchased from Sigma-Aldrich, USA) was added to the GT solution. The mixture of AG and GT (AG–GT) was thoroughly stirred at 50 °C until a uniform, gel-like solution was achieved.

2.1.3. Fabrication of Bacterial Nanocellulose–Alginate–Gelatin and Bacterial Nanocellulose–Chitosan–Alginate–Gelatin Scaffolds

The 3D hydrogel scaffolds of BNC–AG–GT and BNC–CS–AG–GT were fabricated by blending BNC or BNC–CS slurry with AG–GT solution at weight ratios of 80:20 BNC or BNC–CS/AG–GT. The mixtures were stirred at 30 °C for 24 h, cast into 24-well plates, and refrigerated at 4 °C for 24 h before cross-linking in a 1% (w/v) calcium chloride (CaCl₂) aqueous solution at room temperature for 1 h. Subsequently, the hydrogels of BNC–AG–GT and BNC–CS–AG–GT were rinsed with distilled water to eliminate excess chlorides and stored in distilled water at 4 °C before use. The schematic diagram for the fabrication process of the hydrogel scaffolds is shown in Figure S2A.

2.2. In Vitro Study of Biodegradability

The hydrogel scaffolds were cut into a cylinder shape (diameter 5 mm and thickness 5 mm) by using a biopsy punch (size diameter 5 mm) and sterilized at 121 °C for 15 min. The SBF solution was prepared by dissolving chemical reagents, NaCl (0.3319 g), Na₂HPO₄ (0.0071 g), NaHCO₃ (0.1134 g), Na₂SO₄ (0.0035 g), KCl (0.1862 g), CaCl₂ (0.0138 g), and MgCl₂·6H₂O (0.0152 g) (Sigma-Aldrich, USA) in 50 mL final volume deionized water and the pH was adjusted to 7.4 using 1 M HCl. The biodegradation of the hydrogel scaffolds was studied in SBF solution with 0.5 mg/mL lysozyme , (from human neutrophils, Sigma-Aldrich, Cat. L8402, Lot 0000294498) with a reported purity of ≥95% (confirmed by SDS–PAGE, Certificate of Analysis provided by the manufacturer, Table S1), and the hydrogel scaffolds were incubated at 37 °C for 8 weeks. The summarized procedure for in vitro biodegradation of the hydrogel is shown in Figure S2B. The soluble (solution) and nonsoluble (hydrogel) from in vitro biodegradation were further analyzed and characterized as shown in Figure S2C.

2.3. Protein and Sugar Concentration Analysis

2.3.1. Protein Concentration Analysis

The solutions from the in vitro biodegradation were separated by centrifugation. The supernatants were analyzed for the concentration of protein by a Bradford protein assay (Bio-Rad, Hercules, CA). The standard calibration curve of bovine serum albumin (BSA, Thermo Fisher Scientific, USA) was prepared by adding 1 mg/mL BSA stock solution into an Eppendorf tube and then filling with PBS to make up a volume of 1 mL. Supernatants and a standard solution of the calibration curve were introduced into the 96-well plate (10 μL). Each 96-well plate was added with 250 μL of Bradford’s reagent for the detection of protein content. Afterward, the 96-well plate was incubated in a dark place at room temperature for 10 min. Finally, the produced color was analyzed by a spectrophotometer (MultiskanGo, Thermo Fisher Scientific, USA) at 595 nm. The protein concentration of supernatants from in vitro biodegradation was calculated by using the standard curve.

2.3.2. Sugar Concentration Analysis

The solutions from in vitro biodegradation were separated by centrifugation and filtered through a syringe filter (filter, 0.2 μm). The supernatants were analyzed by using HPLC (ALLTECH ELSD 2000ES). The supernatants (10 μL) were then injected onto a Rezex RPM-Monosaccharide Pb²⁺ (300 × 7.8 mm, particle size 8 μm) column (Phenomenex, USA) maintained at 75 °C and eluted with a 100% water mobile phase at a flow rate of 0.6 mL/min.

2.4. Characterizations of Biodegradable Hydrogel Scaffolds

2.4.1. Swelling Degree of Scaffolds

The hydrogels were prepared in a cylindrical form (5 mm diameter × 5 mm height) using a biopsy punch. The samples were immersed in SBF (pH 7.4) at 37 °C for 3 h. After incubation, excess solution on the surface was gently removed using lint-free tissue (Kimwipe), and the swollen weight (W _w) was recorded. The swelling degree (%) was calculated according to eq

$$\mathrm{s}\mathrm{w}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{d}\mathrm{e}\mathrm{g}\mathrm{r}\mathrm{e}\mathrm{e}(\%)=\left(\frac{W_{\mathrm{w}}-W_0}{W_0}\right)\times 100$$ 1

where W ₀ is the initial weight of the hydrogel and W _w is the wet weight of the sample after immersion.

2.4.2. Weight Loss of Scaffolds

The hydrogel scaffolds were removed from the SBF solution, rinsed with distilled water, dried, and weighed. The remaining weights of the hydrogel scaffolds were measured. The weight loss (%) of the hydrogel scaffold was calculated by using eq

$$\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\mathrm{l}\mathrm{o}\mathrm{s}\mathrm{s}(\%)=\left(\frac{W_{\mathrm{i}}-W_t}{W_{\mathrm{i}}}\right)\times 100$$ 2

where W _i represents the initial weight of hydrogels and W _t represents the remaining weight of hydrogel scaffolds at time t.

2.4.3. Morphology Observation of Scaffolds

The morphology of hydrogel scaffolds was observed by using a scanning electron microscope (SEM), JEOL, JSM-IT-500HR (Tokyo, Japan). The specimens were freeze-dried by critical point drying and sputtered with gold. The accelerating voltage was adjusted to 5–10 kV.

2.4.4. Pore Size Analysis

The pore sizes observed from SEM images were determined manually using ImageJ software (version 1.54k). The diameter of each pore was an average length of 6 straight lines across the pore. Three images were used for determination of each scaffold. The average pore diameter was reported as an average value determined from 150 pores.

2.4.5. Fourier Transform Infrared Spectrometer Analysis

Fourier transform infrared (FTIR) spectra of the samples were obtained using a Spectrum One FTIR Spectrometer (Perkin Elmen, USA). The FTIR spectra were recorded in the wavenumber range from 4000 to 400 cm^–1 at the resolution of 4 cm^–1.

2.4.6. Mechanical Test

The hydrogel scaffolds formed in vitro biodegradation were placed under compressive load using a universal testing machine (INSTRON, MA, USA) at a crosshead speed of 0.5 mm/min up to failure or until the sample reached a 50% reduction in height. The test was performed by following the procedure in ASTM F2027. The reported values were the average values determined from five specimens.

2.5. Cytotoxicity Evaluation of Scaffolds

The cytotoxicity of hydrogel scaffolds was assessed following ISO10993-5 and ISO10993-12. , The L929 cell cultures were utilized in Dulbecco’s modified eagle medium (DMEM, Thermo Fisher Scientific, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) served as the culture medium. The hydrogel scaffolds were sterilized and immersed in DMEM medium at 37 °C for 24 h. The extracted medium was used for culturing L929 cells. Negative and positive controls were established using DMEM medium with and without 10% (v/v) DMSO, respectively. L929 cells were seeded in 96-well plates at a density of 104 cells per well and incubated at 37 °C under 5% CO₂ for 24 h. The medium in each well was replaced with extracted medium, negative control, or positive control. The plates were then incubated at 37 °C under 5% CO₂ for an additional 24 h. Cell viability was determined using PrestoBlue (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. Briefly, the cell culture medium was replaced with 100 μL of medium containing 10% (v/v) PrestoBlue in DMEM medium, and the samples were shielded with aluminum foil to prevent light exposure. After incubating at 37 °C for 1 h, the absorbance values at wavelengths of 570 and 600 nm were measured using a spectrophotometer (MultiskanGo, Thermo Fisher Scientific, USA).

2.6. Cytotoxicity Evaluation of Biodegradation Products from Scaffolds

The cytotoxicity of the soluble biodegradation products resulting from the in vitro biodegradation of hydrogel scaffolds that degraded by lysozyme for 8 weeks was assessed using an adaption of the testing method described in the previous reports. − The L929 cell cultures were utilized in DMEM (Thermo Fisher Scientific, USA) supplemented with 10% FBS (Gibco, USA) served as the culture medium. The soluble biodegradation products were diluted to concentrations of 25% (v/v), 50% (v/v), and 75% (v/v) in DMEM, while a 100% (undiluted) biodegradation product was also tested. Negative and positive controls were established using DMEM medium with and without 10% (v/v) DMSO, respectively. L929 cells were seeded in 96-well plates at a density of ≈10⁴ cells per well and incubated at 37 °C under 5% CO₂ for 24 h. The medium was then replaced with the biodegradation product dilutions, undiluted product, negative control, or positive control, and the plates were incubated for another 24 h at 37 °C under 5% CO₂. The viability of the cell was determined using PrestoBlue (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. Briefly, the cell culture medium was replaced with 100 μL of medium containing 10% (v/v) PrestoBlue in DMEM medium, and the samples were shielded with aluminum foil to prevent light exposure. After incubating at 37 °C for 1 h, absorbance at 570 and 600 nm wavelengths was measured using a spectrophotometer (MultiskanGo, Thermo Fisher Scientific, USA).

2.7. In Vitro Biocompatibility

2.7.1. Cell Culture Preparation

MC3T3-E1 cells were procured from Biomedia, Thailand. These cells were cultured in alpha-modification of Eagle’s medium (α-MEM, Gibco, USA) supplemented with 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin (Gibco, USA) within a 75 cm cell culture flask (CORNING, USA) and maintained at 37 °C under 5% CO₂ for 24 h. The culture medium was refreshed every 3–4 days. Subculturing was carried out when cells reached 80% confluence using TrypLE Express (Gibco, USA).

2.7.2. Cell Encapsulation

Cell encapsulation within hydrogel scaffolds was prepared according to optimized protocols detailed in previous studies. BNC–AG–GT and BNC–CS–AG–GT solutions were sterilized via autoclaving at 121 °C for 15 min. A suspension containing 100 μL of MC3T3-E1 cells was combined with 900 μL of either BNC–AG–GT or BNC–CS–AG–GT solution (seeding density of 5 × 10⁵ cells per 1 mL hydrogel). For AG–GT hydrogel serves as a control to compare the effects of BNC and BNC–CS on cell proliferation and cell activity. This cell-encapsulated hydrogel was then transferred into a 24-well plate and cross-linked using a 1% (w/v) CaCl₂ solution for 1 h under aseptic conditions. Afterward, the cell-encapsulated hydrogel constructs were rinsed with PBS to eliminate excess chloride and cultured in osteogenic medium [α-MEM supplemented with 10% (v/v) FBS, 1% (v/v) penicillin/streptomycin, 10 mM β-glycerol phosphate (Sigma-Aldrich, USA), and 50 μM ascorbic acid (Sigma-Aldrich, USA)] within 24-well plates (CORNING, USA), maintaining them at 37 °C under 5% CO₂ for a period of 28 days. The culture medium was refreshed every 3–4 days.

2.7.3. Cell Proliferation Assay

The MC3T3-E1 cell-encapsulated hydrogel was cultured in medium for a period of 28 days. The MC3T3-E1 cell-encapsulated hydrogel was evaluated for cell proliferation at 3, 7, 14, and 28 days using 1 mL culture medium containing 10% (v/v) PrestoBlue under the PrestoBlue assay.

2.7.4. Live/Dead Cell Viability

The viability of MC3T3-E1 cell-encapsulated hydrogel was evaluated based on a live/dead viability/cytotoxicity kit (Thermo Fisher Scientific, UK) following the manufacturer’s guidelines.

2.7.5. Cell Morphology Observation

The morphology of MC3T3-E1 cell-encapsulated hydrogel scaffolds was observed at 7, 14, and 28 days via scanning electron microscopy (SEM). After the removal of the culture medium, the MC3T3-E1 cell-encapsulated hydrogel scaffolds were washed with PBS and fixed using 2.5% glutaraldehyde. The fixed cells were dehydrated in serial dilutions of ethanol and dried in an automated Leica EM-CPD300 critical point dryer (Leica Microsystems, Austria). Then, the fixed cell-encapsulated hydrogel scaffolds were coated with gold using sputtering and observed by SEM using a JEOL JSM-IT-500HR instrument (JEOL, Tokyo, Japan).

2.7.6. Alkaline Phosphatase Activity Assay

The MC3T3-E1 cells encapsulated within the hydrogel scaffolds were cultured for 28 days. After removal of the culture medium, the cells were rinsed with PBS and then lysed using SDS lysis buffer (containing 150 mM NaCl, 15 mM sodium citrate, and 0.02% SDS) for a duration of 30 min. Subsequently, 10 μL of the cell lysate was combined with 100 μL of the p-nitrophenyl phosphate liquid substrate (Sigma-Aldrich, USA), and the mixture was incubated at 37 °C for 15 min. The reaction was halted by the addition of 0.02 N NaOH, and the enzyme activity was assessed by measuring the absorbance at 405 nm. The protein content was determined using a Bradford protein assay (Bio-Rad, Hercules, CA).

2.7.7. Alkaline Phosphatase Staining

The MC3T3-E1 cells encapsulated within hydrogel scaffolds were cultured in a culture medium for 28 days. After the removal of the culture medium, the encapsulated MC3T3-E1 cells were fixed using 4% formaldehyde in PBS for 30 min, followed by rinsing with DI water three times. The fixed cells were then immersed in 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP-NBT, obtained from Sigma-Aldrich, USA) to assess alkaline phosphatase (ALP) activity and were observed under a microscope (IX81, Olympus).

2.7.8. Alizarin Red Staining and Quantitative Analysis

The MC3T3-E1 cell-encapsulated hydrogel scaffolds were fixed by using 4% formaldehyde in PBS for 30 min, followed by rinsing with DI water three times. The fixed cells were stained with 2% (w/v) Alizarin Red Staining (ARS) (Sigma, USA) for 60 min at a pH of 4.2, followed by multiple washes with PBS. The Alizarin Red-stained scaffolds were observed under a microscope (IX81, Olympus) and then eluted and quantified by using a 10% (w/v) cetylpyridinium solution with absorbance measured at 557 nm.

2.8. Antibacterial Activity Assays

Antimicrobial characteristics of scaffolds against Staphylococcus aureus and Escherichia coli were assayed by applying the modified JIS Z 2801 method. The stocks of S. aureus and E. coli cells were prepared and incubated at 37 °C for 16–20 h. The sterilized scaffolds cut into a cylinder shape were subjected to an autoclave at a temperature of 121 °C for 15 min. The bacterial cell suspension of 1 mL with the initial cell concentration ranging from 1.5–4 × 10⁶ CFU/mL was dropped into nutrient broth medium on scaffold surfaces and incubated at 37 °C for 24 h. After that, the samples underwent energetic shaking within 10 mL of pH 7.4 PBS by using a speed of 200 rpm and were maintained there for 1 min. Then the plate count method was used to determine the number of viable bacterial cells in a sample. The reduction (%) of bacteria cells was calculated by using the following eq

$$\mathrm{r}\mathrm{e}\mathrm{d}\mathrm{u}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{o}\mathrm{f}\mathrm{b}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{e}\mathrm{r}\mathrm{i}\mathrm{a}\mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{s}(\%)=\left(\frac{B-A}{B}\right)\times 100\%$$ 3

where A represents the number of viable bacteria after the treatment for 24 h (CFU/mL) and B represents the number of viable bacteria before the treatment (CFU/mL).

2.9. Statistical Analysis

The statistical analysis was conducted using GraphPad Prism 10.1.0 software. The results are expressed as mean ± standard deviation. Group differences were analyzed by using one-way or two-way ANOVA, followed by Tukey’s honest significant difference test for multiple comparisons. A p-value <0.05 was determined as statistically significant.

3. Results and Discussion

3.1. Biosynthesis

Schematic illustrations of BNC–CS production and a proposed mechanism of BNC–CS biosynthesis by A. xylinum are shown in Figure S1 (in Supporting Information). The culture medium for A. xylinum consisted of sucrose and CS. It is possible that A. xylinum secretes sucrase or invertase enzymes, which catalyze the hydrolysis of sucrose into glucose and fructose. Additionally, specific enzymes are released by A. xylinum that are capable of degrading CS into Glc and GlcNAc. As can be seen in Figure S3 (in Supporting Information), HPLC chromatograms of medium culture show a decrease in the peak assigned to sucrose and an increase in glucose, fructose, Glc, and GlcNAc peaks from day 1 to day 3. During biosynthesis by A. xylinum, it has been suggested that glucose, fructose, and GlcNAc could be absorbed and reacted with UTP (uridine-5-triphosphate) to form uridine-5′-diphosphate. Formation of BNC is catalyzed by cellulose synthase, and these cellulose chains are excreted through pores in the cell walls of A. xylinum. The biochemical pathways involved are regulated by the bacterial cellulose synthesis ABCD (bcsABCD) operon. − The bcsABCD operon encodes expression of several important components of the bacterial cellulose synthase complex, including the c-di-GMP-binding subunit and catalytic subunit, which are important for biosynthesis and production of cellulose. , As can be seen in HPLC chromatograms of medium culture on day 7, peaks corresponding to glucose, fructose, and GlcNAc were decreased, indicating that these sugars were consumed during the biosynthesis of cellulose. Sugar standard peaks from HPLC analysis are shown in Figure S4 in the Supporting Information. Previously, it has been suggested that BNC–CS could be formed via hydrogen bonding between the amino/hydroxyl groups of CS and the hydroxyl groups of BNC. ,

3.2. In Vitro Biodegradability

Biodegradation of the hydrogel scaffolds in vitro was examined by incubating the hydrogel scaffolds in SBF solution with and without lysozyme at 37 °C for 8 weeks. The addition of lysozyme in SBF is intended to mimic physiological conditions. Macroscopic figures of the hydrogel scaffolds during the biodegradation process are shown in Figure . BNC, BNC–CS, BNC–AG–GT, BNC–CS–AG–GT, and AG–GT hydrogel scaffolds in SBF solution without and with lysozyme are shown in Figure A,B, respectively. These results clearly show that BNC was highly stable in SBF in systems with and without lysozyme: no degradation of BNC in SBF was observed during the 8 week incubations. In contrast, the AG−GT hydrogel scaffold was significantly degraded in SBF solution, and this degradation was enhanced in the presence of lysozyme. Moreover, AG–GT scaffolds became transparent, broke into fragments, and were observed to be completely degraded after immersion for 6 weeks in SBF with lysozyme.

1.

Morphology (A,B) and weight loss (C,D) of scaffolds during in vitro biodegradation in SBF solution without lysozyme (A,C) and with lysozyme (B,D).

In addition, the degree of swelling of all scaffolds was assessed in SBF solution (without lysozyme) to evaluate the water uptake capacity during immersion (Figure S7). The degree of swelling remained below 2% for all formulations over 7 days of incubation, indicating that the hydrogels were already highly hydrated during the preparation phase and exhibited minimal additional water absorption. Consistently, no visible swelling was observed in the macroscopic images of the scaffolds during prolonged immersion (Figure A,B). These findings are in agreement with previous reports that BNC-based and AG-containing hydrogels contain a high intrinsic water content after preparation , and thus show negligible additional swelling when placed in aqueous environments. Furthermore, this suggests that the weight loss reported in the following degradation experiments can be mainly attributed to polymer dissolution or enzymatic activity, rather than swelling-induced effects. ,,

Based on weight loss (%) measurements, AG–GT showed the most dramatic degradation with 100% weight loss upon incubation in a system with lysozyme for 6 weeks. The AG–GT hydrogel scaffold contains AG and GT, both of which are biodegradable in the SBF solution. On the other hand, lysozyme can cleave the β-(1 → 4) glycosidic bonds present in the polysaccharide chains of CS , and polypeptide chains of GT. ,− , The degradation rate of GT by lysozyme may vary depending on specific factors, such as concentration.

According to data obtained from weight loss measurements, BNC was not degraded in SBF either with or without the addition of lysozyme. In addition, although BNC–CS was not degraded upon incubation in SBF alone, slow degradation of BNC–CS was detected in the presence of lysozyme. For the other scaffolds, the level of weight loss progressively increased from 0 to 8 weeks in SBF with or without lysozyme. Upon incubation in SBF solution with lysozyme, the tested hydrogels displayed different susceptibilities to degradation, with AG–GT > BNC–CS–AG–GT > BNC–AG–GT > BNC–CS, respectively. In support of this, after 8 weeks of incubation, weight losses of 100%, 53.7%, 45.2%, and 13.3% were recorded for AG–GT, BNC–CS–AG–GT, BNC–AG–GT, and BNC–CS, respectively. The SBF solution could react with calcium in the cross-linked AG hydrogel, leading to the formation of calcium phosphate (Ca₃(PO₄)₂) and resulting in degradation of the calcium AG polymer. Lysozyme may stimulate degradation of CS-based biomaterials by cleaving 1,4-beta-glycosidic bonds in their polysaccharide units. , To ensure traceability and reproducibility of the enzyme used in the degradation tests, the lysozyme used in the present study was a high-purity-grade enzyme (≥95% pure by SDS–PAGE, Sigma-Aldrich, Cat. L8402) with verified activity, as shown in Table S1 (Supporting Information). To the best of our knowledge, this is the first report to demonstrate that the integration of CS in a BNC network yields a BNC–CS scaffold that is susceptible to degradation by lysozyme. Previously, a physically cross-linked CS gel with tunable mechanical properties was applied to CS scaffold in order to regulate its biodegradability, stability, and physical properties.

The functional groups present on CS can interact with BNC, affecting both the structural integrity and the biodegradability of the resulting BNC–CS scaffold. In particular, CS has amino (−NH₂) and hydroxyl (−OH) functional groups that are able to form hydrogen bonds and ionic interactions with the hydroxyl and acetal groups of BNC. These interactions can enhance the mechanical stability of the scaffolds, while retaining enough flexibility for biomedical use. , Additionally, because CS is enzymatically degradable by lysozyme, its integration into the BNC matrix enhances the biodegradability of the scaffold, making it more appropriate for tissue engineering. The addition of CS also improves the water absorption capacity of the scaffold, promoting cellular adhesion and proliferation.

Note that although a BNC–CS–GT control group without AG could have allowed for a clearer interpretation of the individual contribution of GT to scaffold degradation, it was not included in the present study. This is because AG was used in the present study for cross-linking to form a stable 3D hydrogel scaffold from homogenized BNC or BNC–CS fibers. In the presence of calcium chloride, carboxylate groups in sodium AG can interact with calcium ions, which replace sodium ions and form strong ionic cross-links between adjacent AG chains. This form of mediated ionic cross-linking is a crucial step that creates a stable network; without the use of AG for cross-linking, the scaffolds cannot be firmly fabricated. In addition, the observed weight loss in AG-containing scaffolds may result not only from lysozyme activity but also from the intrinsic susceptibility of AG to ion-exchange effects and partial hydrolytic degradation in SBF (pH 7.4). Namely, divalent Ca²⁺ cross-links can be gradually displaced by monovalent ions (e.g., Na⁺, K⁺, and Mg²⁺), leading to weakening of the hydrogel network and accelerating mass loss over time, − which can influence scaffold degradation.

In order to provide more supporting data with respect to scaffold biodegradability and stability, the morphology, chemical structure, mechanical properties, and degradation products from incubation of BNC, BNC–CS, and other BNC composite scaffolds in SBF with or without lysozyme were further investigated, as shown in Sections 3.3.1, 3.3.2, 3.3.3, and 3.4, respectively.

3.3. Characterizations

3.3.1. Morphology

SEM images of the morphology of the outer surface and internal parts of the hydrogel scaffolds, before and after incubation in SBF solution without and with lysozyme, are shown in Figures and , respectively. Pore sizes of the hydrogel scaffolds before and after 8 weeks of incubation were determined using ImageJ software, as shown in Table . The pore sizes of BNC and BNC–CS scaffolds were in the range of 262–308 μm for the outer surface and 273–328 μm for the internal part, while the pore sizes of BNC–AG–GT and BNC–CS–AG–GT scaffolds were in the range of 55–60 μm for the outer surface and 326–358 μm for the internal part. The morphology of the BNC scaffold was unchanged during the 8 week incubation, consistent with the fact that the BNC scaffold was highly stable and did not degrade in SBF with or without lysozyme. The BNC–CS hydrogel in SBF solution without lysozyme was also very stable and showed no morphological changes over 8 weeks in SBF. However, biodegradation of the BNC–CS hydrogel was observed in SBF solution supplemented with lysozyme: in agreement with this, the BNC–CS scaffold pore size in the outer surface and internal part became significantly larger while fibers became smaller. On the other hand, BNC–AG–GT and BNC–CS–AG–GT hydrogel scaffolds were biodegradable in SBF solutions either with or without lysozyme, and the pore size of the outer surface and internal hydrogel scaffolds was found to be significantly larger after 8 weeks incubation. The morphology changes and increased pore sizes observed for the BNC–CS and BNC–CS–AG–GT hydrogel scaffolds are consistent with the enhanced biodegradability of these hydrogel scaffolds under the action of lysozyme in SBF solution.

2.

SEM images of scaffolds during in vitro biodegradation in SBF solution without lysozyme: outer surface (A) and internal part (B).

3.

SEM images of scaffolds during in vitro biodegradation in SBF solution with lysozyme: outer surface (A) and internal part (B).

1. Pore Size of Scaffolds During In Vitro Biodegradation in SBF Solution without the Lysozyme.

pore size of hydrogels (μm)			BNC	BNC–CS	BNC–AG–GT	BNC–CS–AG–GT
without lysozyme	surface	0 week	266.05 ± 28.34	262.47 ± 34.37	59.48 ± 17.44	57.35 ± 14.76
		8 weeks	270.67 ± 33.92	258.55 ± 26.74	103.61 ± 26.41	96.72 ± 31.29
	internal	0 week	274.54 ± 37.59	285.37 ± 37.52	348.85 ± 36.89	338.15 ± 34.65
		8 weeks	288.94 ± 56.43	281.69 ± 42.79	417.48 ± 24.03	414.42 ± 38.55
with lysozyme	surface	0 week	262.84 ± 29.22	308.11 ± 28.73	56.02 ± 15.28	55.36 ± 18.44
		8 weeks	269.32 ± 31.86	397.49 ± 47.58	116.74 ± 34.42	427.53 ± 56.38
	internal	0 week	272.87 ± 53.94	327.56 ± 38.76	326.27 ± 31.22	358.36 ± 42.71
		8 weeks	269.31 ± 48.21	446.82 ± 36.95	379.83 ± 23.53	463.01 ± 29.33

3.3.2. Fourier Transform Infrared Spectrophotometric Analysis

Functional groups present on the investigated hydrogel scaffolds were characterized by FTIR in wave numbers ranging from 4000 to 400 cm^–1, as shown in Figures and . FTIR spectra of hydrogel scaffolds before the in vitro biodegradation test (week 0) in SBF without and with lysozyme are shown in Figures A and A. For BNC, the broad peak at 3393 and 3402 cm^–1 was attributed to the stretching frequency of −OH groups. The band at 1654 cm^–1 indicates H–O–H bending by absorbed water, while the band at 1007 cm^–1 represents C–O–C stretching. For the BNC–CS hydrogel scaffold, characteristic peaks from CS were observed at 1372 and 1383 cm^–1. This observation is in line with findings previously described for the biosynthesis of bacterial cellulose-CS. The AG–GT hydrogel scaffolds show characteristic peaks from both AG and GT. Specifically, signature peaks from AG were observed at 3256–3373 cm^–1 for –OH stretching and at 824–821 cm^–1 for the COO– groups in mannuronidic acid residues. GT peaks were also evident, including those at 1604–1628 cm^–1 for amide I, 1503–1509 cm^–1 for amide II, and 1178–1172 cm^–1 for amide III. For BNC–AG–GT hydrogel scaffolds, the broad peak observed at 3275–3284 cm^–1 was attributed to the presence of hydrogen bonds. Characteristic peaks corresponding to GT and AG bands were identified at 1621–1627 cm^–1, 1513–1523 cm^–1, 1181–1186 cm^–1, and 832–814 cm^–1. In the case of BNC–CS–AG–GT hydrogel scaffolds, a characteristic CS peak was observed at 1341–1352 cm^–1, along with bands attributed to –OH stretching at 3268–3296 cm^–1. Additionally, characteristic peaks associated with GT and AG bands were detected at 1603–1632 cm^–1, 1501–1514 cm^–1, 1174–1182 cm^–1, and 822–819 cm^–1. The functional peaks identified by FTIR for all of the hydrogel scaffolds are similar to those previously reported. FTIR spectra of hydrogel scaffolds following 8 weeks of incubation in SBF without and with lysozyme are shown in Figures B and B, respectively. Samples of BNC and BNC–CS in an SBF solution without lysozyme have similar peak patterns before and after the biodegradability test. Meanwhile, for BNC–CS, incubation in SBF solution with lysozyme for 8 weeks resulted in a decrease in the peaks for some functional groups. In addition to a reduction in intensity, small shifts were observed in the characteristic bands of the scaffolds, such as the amide I region (from 1628 to 1615 cm^–1) and the carboxylate group (COO^–) around 820–824 cm^–1. These shifts suggest a possible hydrolysis or rearrangement of hydrogen bonding due to enzymatic degradation.

4.

FTIR of scaffolds after the incubation period of 0 day (A) and 8 weeks (B) in SBF solution without lysozyme.

5.

FTIR of scaffolds after the incubation period of 0 day (A) and 8 weeks (B) in SBF solution with lysozyme.

The intensities of some functional peaks for BNC–AG–GT, BNC–CS–AG–GT, and AG–GT hydrogel scaffolds also decreased after incubation in SBF solution without and with lysozyme for 8 weeks. This observed decrease in FTIR peak intensity and peak shifts may indicate chemical degradation or disruption of molecular interactions in the hydrogel network.

3.3.3. Mechanical Property

The effect of biodegradation on the mechanical properties of the hydrogel scaffolds was also determined. Specifically, the compressive strength of the hydrogel scaffolds was investigated following incubation in SBF with and without lysozyme (Figure ). BNC was found to have a high compressive strength (∼185 MPa), and no significant change in compressive strength was observed following the degradation test with/without lysozyme for 8 weeks. BNC–CS had an even more outstanding compressive strength (214–218 MPa), which was relatively higher than that of BNC. For the biosynthesis of BNC, the addition of CS in the culture medium could have resulted in improved mechanical properties of the BNC–CS composite. ,, However, the mechanical properties of BNC–CS deteriorated with the incubation time in SBF solution supplemented with lysozyme. The compressive strength of BNC–CS was significantly reduced to 193 MPa after incubation in SBF for 8 weeks with lysozyme.

6.

Compressive strength of scaffolds during in vitro biodegradation in SBF solution without lysozyme (A) and with lysozyme (B). The data are represented as mean ± standard deviation, n = 3, a significant difference at *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

BNC–AG–GT and BNC–CS–AG–GT scaffolds had compressive strengths of ∼52 MPa and ∼68 MPa, respectively, which were much higher than that of AG-GT (<10 MPa). The compressive strengths of BNC–AG–GT, BNC–CS–AG–GT, and AG–GT significantly decreased with time during degradation in SBF solution with and without lysozyme for 8 weeks to ∼25 MPa, ∼28 MPa, and ∼0 MPa (complete degradation), respectively. A larger reduction in compressive strength was observed following incubation in SBF with lysozyme, most likely due to the degradation of CS and GT by lysozyme. AG–GT was shown to have very low compressive strength and poor stability, suggesting it may not be suitable for use as a scaffold in bone tissue engineering.

Notably, the compressive strengths observed for native BNC and BNC–CS hydrogels in the present study (185–218 MPa) were markedly higher than those published for hydrated BNC-based hydrogels, which are typically reported to be in the range of 11–40 kPa. − This difference may arise from the dense nanofibrillar network of our biosynthesized hydrogels. Both BNC–AG–GT and BNC–CS–AG–GT scaffolds exhibited superior compressive strengths compared to many other reported hydrogel scaffolds, suggesting that they possess sufficient stiffness, and that they could be promising candidates to support osteoblast adhesion, proliferation, differentiation, and ALP activity in tissue engineering, which are essential for bone tissue engineering applications. ,

3.4. Products Detectable from Degradation of Hydrogel Scaffolds

Protein and sugars were the main soluble products detected following the biodegradation of the hydrogel scaffolds in SBF solution supplemented with lysozyme. Total protein and sugar contents following incubation of the hydrogel scaffolds in SBF solution supplemented with lysozyme at 37 °C for 8 weeks were analyzed.

3.4.1. Total Protein Concentration

Total protein concentrations present in solution following in vitro biodegradation in SBF without/with lysozyme are shown in Figure A,B. As can be seen, AG–GT had a much higher protein concentration in solution than the other scaffolds due to the high protein content present in the AG–GT scaffold, which contains a significant amount of GT. In addition, it was observed that protein concentrations increased during degradation of the AG–GT hydrogel scaffolds in the presence of lysozyme (Figure B), as GT could also be degraded by lysozyme. − Moreover, at weeks 6 and 8, the protein concentration of the AG–GT hydrogel scaffolds remained constant, consistent with the complete degradation of the AG–GT scaffolds. These observations are in agreement with weight loss results obtained for the hydrogel scaffolds (Figure D).

7.

Protein concentration in the solution during in vitro biodegradation in SBF solution without lysozyme (A) and with lysozyme (B); glucose concentration in the solution during in vitro biodegradation in SBF solution with lysozyme of BNC–CS and BNC–CS–AG–GT scaffolds (C). The data are represented as mean ± standard deviation, n = 3, a significant difference at *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

3.4.2. Sugar Concentration

Sugar concentrations in solution following in vitro biodegradation of BNC, BNC–CS, BNC–AG–GT, and BNC–CS–AG–GT scaffolds were analyzed by HPLC. HPLC chromatograms for sugar peaks are shown in Figure S5 (see the Supporting Information). Analysis of results for BNC–CS and BNC–CS–AG–GT hydrogel scaffolds shows the appearance of glucose peaks resulting from biodegradation at 4 and 8 weeks. However, no sugar peaks were observed for BNC and BNC–AG–GT hydrogel scaffolds or for hydrogels incubated without lysozyme. Measured glucose concentrations following the biodegradation of BNC–CS and BNC–CS–AG–GT in SBF with lysozyme are shown in Figure C. BNC–CS and BNC–CS–AG–GT hydrogel scaffolds were degraded by incubation in SBF with lysozyme, and the presence of glucose is an expected breakdown product. However, lysozyme is unable to degrade cellulose fibril networks in BNC and BNC–AG–GT. These results indicate that biosynthesis of BNC–CS by A. xylinum may result in the formation of fibril networks linked between CS and cellulose since lysozyme would then be able to cut the glycoside bonds between the polysaccharide units in this type of copolymer. BNC–CS hydrogels might be randomly degraded by lysozyme, starting from CS linked via cellulose chains on the surface. The degradation of CS by lysozyme would result in cleavage of β-(1,4)-glycosidic bonds into glucosamine, N-acetyl-glucosamine, small oligosaccharides, and glucose. , In the present study, it was shown that BNC–CS could be gradually degraded into glucosamine, N-acetyl-glucosamine, and glucose during in vitro biodegradation in SBF solution with lysozyme.

The concentration of glucose was calculated using a calibration curve created from a glucose standard (Figure S6 in the Supporting Information). The concentration of glucose gradually increased with degradation time from 2 to 8 weeks. BNC–CS–AG–GT showed a higher glucose concentration than BNC–CS. AG–GT could be degraded in SBF solution in the presence of lysozyme, possibly because it possesses a more porous structure and surface area that is accessible to lysozyme for hydrolysis of glycosidic bonds. In agreement with this, considerably greater degradation was seen for BNC–CS–AG–GT than BNC–CS.

3.5. Cytotoxicity Assay

3.5.1. Cytotoxicity of Hydrogel Scaffolds

An extracted medium of hydrogel scaffolds was used for culturing L929 cells for 24 h. Cell viability was determined according to ISO10993-5 and ISO10993-12 guidelines for cytotoxicity testing. , Treatments that resulted in a cell viability greater than 70% were considered to be noncytotoxic, as specified in the standard. As shown in Figure A, control (P) treatment of L929 cells with a positive control cytotoxic reagent (DMSO) resulted in very low remaining cell viability, confirming the cytotoxic nature of this positive control. On the other hand, treatment of cells with cell growth medium, a noncytotoxic negative control (N), resulted in retention of 100% cell viability, providing a reference point for defining normal cellular proliferation. Culturing cells in an extracted medium of BNC hydrogel resulted in a cell viability of 83.75%, which is above the standard cytotoxicity threshold. Thus, BNC hydrogels can be considered to be noncytotoxic. Use of an extracted medium of BNC–CS or BNC–AG–GT gave relatively higher cell viabilities of 87.41% and 92.06% respectively. Similarly, using an extracted medium of BNC–CS–AG–GT yielded a cell viability of 94.27%. Based on our results, incorporation of CS, AG, and GT into the BNC–CS–AG–GT scaffold may promote cell adhesion and subsequent cell proliferation because of their adequate physicochemical properties and biological activities, while all hydrogel samples can be considered to be noncytotoxic.

8.

Cytotoxicity of hydrogel scaffolds (A) and cytotoxicity of soluble biodegradation products of hydrogel that degraded by lysozyme for 8 weeks were mixed with culture medium at 25% (B), 50% (C), 75% (D), and 100% (E) concentration. The data are represented as mean ± standard deviation, n = 3, a significant difference at *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

3.5.2. Cytotoxicity of Biodegradation Products from Hydrogel Scaffolds

Degradation of hydrogel scaffolds by incubation in SBF with lysozyme for 8 weeks resulted in the formation of specific biodegradation products. The in vitro cytotoxicity of these biodegradation products was evaluated using an adaptation of testing methods described in previous reports. − That is, L929 cells were cultured in the presence of different concentrations of degradation products mixed with culture medium at ratios of 25%, 50%, 75%, and 100% (v/v) for 24 h. At a concentration of 25% (Figure B), the degradation products from all hydrogel scaffolds were noncytotoxic, with more than 70% cell viability. Treatment of cells with biodegradation products from BNC, BNC–CS, BNC–AG–GT, and BNC–CS–AG–GT resulted in a cell viability of 78.26%, 84.14%, 93.71%, and 96.43%, respectively. Slightly reduced viabilities were obtained upon treatment of cells with a 50% ratio of degradation products in the culture medium (Figure C). The highest cell viability (93.73%) was obtained upon treatment of cells with biodegradation products from BNC–CS–AG–GT. However, considerably lower cell viabilities (68.14%, 76.09%, 81.62%, and 84.59%) were obtained upon increasing the ratio of biodegradation products from BNC, BNC–CS, BNC–AG–GT, and BNC–CS–AG–GT to 75% (Figure D). Low cell viabilities were obtained in the presence of biodegradation products at a 100% concentration (Figure E). Taken together, these results suggest that biodegradation products from BNC, BNC–CS, BNC–AG–GT, and BNC–CS–AG–GT are not cytotoxic. However, the use of biodegradation products in the cell culture medium at a ratio of more than 50% can affect cell viability due to the associated reduction in available nutrients. Moreover, our results suggest that addition of biocompatible polymers, such as CS, AG and GT into the BNC–CS–AG–GT hydrogel scaffold could improve cell viability, since the actual degradation products, such as protein, glucosamine, N-acetyl-glucosamine, small oligosaccharides, and glucose, should have positive effects on cell viability.

3.6. In Vitro Biocompatibility

3.6.1. Cell Proliferation and Attachment

BNC–AG–GT and BNC–CS–AG–GT hydrogel scaffolds were evaluated for biocompatibility with osteoblast tissue in vitro. For this purpose, MC3T3-E1 cells were encapsulated within the hydrogel matrix, as shown in Figure . Cell proliferation of MC3T3-E1 in the hydrogel scaffolds is shown in Figure A. Based on these results, MC3T3-E1 cell proliferation was observed to be significantly higher in the presence of both BNC–AG–GT and BNC–CS–AG–GT hydrogel scaffolds vs controls. Cross-sectional observation of cell viability based on fluorometric detection in hydrogel scaffolds is shown in Figure B, and the morphology of cell adhesion in hydrogel scaffolds from SEM observations is shown in Figure C.

9.

In vitro biocompatibility of MC3T3 cells in BNC–AG–GT and BNC–CS–AG–GT hydrogel scaffolds. Cell proliferation in hydrogel scaffolds at 3–28 days (A). Live/Dead assay was used to assess cell viability in hydrogel scaffolds at 7, 14, and 28 days (B). SEM images indicating cell attachment, growth, and proliferation in BNC–AG–GT and BNC–CS–AG–GT hydrogel scaffolds for 7, 14, and 28 days (C). The data are represented as mean ± standard deviation, n = 3, a significant difference at *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

According to the SEM images in Figure C, good cell distribution, high cell proliferation, and good attachment of live cells were observed for all studied hydrogel scaffolds, which suggested adequate mass transport throughout the BNC–AG–GT and BNC–CS–AG–GT scaffolds. Moreover, MC3T3-E1 cells encapsulated in BNC–CS–AG–GT scaffolds exhibited the highest cell proliferation compared to those encapsulated in BNC–AG–GT hydrogel scaffolds or controls. Previously, a scaffold composed of AG/bacterial cellulose nanocrystals–CS–GT (Alg/BCNs–CS–GT) fabricated via internal gelation and layer-by-layer electrostatic assembly of polyelectrolytes was shown to have excellent cyto-compatibility, and promoted cell adhesion and proliferation in MG63 and MC3T3-E1 cells. Similarly, BNC hydrogels were reported with excellent biocompatibility, and facilitated effective cell attachment and proliferation in MC3T3-E1 osteoblasts, which are crucial for bone regeneration. MC3T3-E1 cells showed proliferative activity in the presence of BNC and GT-modified BNC scaffolds, indicating their suitability for bone tissue engineering due to their biocompatibility. In the present study, a BNC–CS–AG–GT hydrogel scaffold was shown to preferentially promote cell viability and higher levels of cell proliferation in MC3T3-E1 cells compared to either BNC–AG–GT scaffolds or controls.

Degradation analysis revealed a gradual and controlled loss of weight in BNC-based hydrogels, indicating that this scaffold maintains its structural integrity over a time frame compatible with early bone regeneration phases. This is particularly relevant since a gradual degradation rate allows for progressive replacement of the scaffold by new tissue. Moreover, cytocompatibility results demonstrated that all of the tested hydrogel formulations promoted high cell viability (>80%) and good cellular adhesion, confirming the noncytotoxic nature of the degradation products. These findings are consistent with previous reports for BNC-based composites, which were shown to exhibit excellent biocompatibility in vitro. − Together, our results provide strong evidence that the developed hydrogels possess favorable degradation profiles and bioactivities, making them promising candidates for bone tissue engineering scaffolds.

3.6.2. Alkaline Phosphatase Activity and Mineralization

MC3T3-E1 cells encapsulated in the hydrogel scaffold were evaluated by ALP staining and ALP activity measurements, where ALP is an enzyme marker of early osteoblast cells. In Figure A,B, results from examination at 7 and 14 days demonstrate that ALP expression and activity were significantly higher in MC3T3-E1 cells encapsulated in BNC–CS–AG–GT scaffolds than in those encapsulated in BNC–AG–GT scaffolds. Based on observations for 7–28 days, ALP activity in MC3T3-E1 cells encapsulated in a BNC–CS–AG–GT hydrogel scaffold was highest on day 14, and significantly decreased on day 28. In addition, MC3T3-E1 cell encapsulated-hydrogels were evaluated by ARS staining to investigate the mineralization capacity of MC3T3-E1, which occurs during the late stages of osteoblast differentiation. In this process, the extracellular matrix is gradually mineralized as Ca deposits during the formation of mineralized bone nodules. ARS activity was monitored in MC3T3-E1 cells encapsulated in hydrogel scaffolds for 7–28 days. As can be seen in Figure C,D, relative ARS activity of MC3T3-E1 cells encapsulated in the BNC–CS–AG–GT hydrogel scaffold was significantly higher than in the presence of either the BNC–AG–GT scaffold or control. At the same time, quantitative analysis of calcium deposition showed an increase in calcium deposits in the hydrogels from days 7, 14, and 28, respectively. Based on evaluation of ALP activity, ARS activity and mineralization in MC3T3-E1 cells, the BNC–CS–AG–GT hydrogel scaffold significantly improved the expression and function of bone mineralization compared to the BNC–AG–GT scaffold or control. Previously, incorporation of GT in a CS hydrogel was shown to have promise in promoting osteogenic activity, as evidenced by ALP activity and mineralization assays. A recent report on MC3T3-E1 osteoblasts showed enhanced differentiation in the presence of a BNC/hydroxyapatite composite hydrogel, which also promoted ALP activity, mineralization, and bone healing in vitro.

10.

(A) ALP staining of MC3T3-E1 cells in hydrogels for 7, 14, and 28 days. (B) Relative ALP activity of MC3T3-E1 cells in hydrogels for 7–28 days. (C) ARS staining of MC3T3-E1 cells in hydrogels for 7–28 days for quantitative analysis of calcium deposition. (D) Relative ARS activity analysis of MC3T3-E1 cells in hydrogels for 7–28 days. The data are represented as mean ± standard deviation, n = 3, a significant difference at *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

A previous study of CS-based scaffolds indicates that CS could have significant effects on bone development by promoting the adherence of calcium ions, an important constituent for hydroxyapatite nucleation. In addition, the positively charged calcium ions can interact with negatively charged glycosaminoglycans and proteins that play a significant role in the bone mineralization process. , Moreover, CS-derived materials have been reported to enhance osteoblast proliferation, differentiation, and ALP activity, all important factors for the regeneration of bone tissue. ,

The enhanced osteogenic responses observed in the presence of BNC–CS–AG–GT scaffolds, as demonstrated by higher ALP activity and mineralization compared to BNC–AG–GT scaffolds (Figure ), could be partly due to their superior mechanical properties. Increased compressive strength has been reported to positively regulate osteoblast adhesion, proliferation, and differentiation through mechano-transduction pathways, leading to upregulation of ALP expression and enhanced calcium deposition. − This suggests that the improved compressive strength measured for the BNC–CS–AG–GT scaffolds could provide favorable mechanical cues for osteogenic differentiation, contributing to their superior ALP activity and promoting mineralization.

3.7. Antibacterial Testing

The antibacterial properties of the tested hydrogel scaffolds were evaluated against E. coli, representing Gram-negative bacteria, and S. aureus, representing Gram-positive bacteria. The antibacterial efficacy of the hydrogel scaffolds was quantitatively estimated using the colony-forming unit (CFU) method (cell numbers are shown in Supporting Information and Tables S2 and S3). The effects of the hydrogels on the reduction of bacterial cell growth are shown in Figure . As can be seen, both BNC–CS and BNC–CS–AG–GT hydrogel scaffolds have strong antimicrobial effects, resulting in 100% (BNC–CS) and 100% (BNC–CS–AG–GT) reductions in the growth of E. coli and a 100% (BNC–CS) and 89.16% (BNC–CS–AG–GT) reduction in the growth of S. aureus. In contrast, BNC and BNC–AG–GT did not inhibit the growth of either E. coli or S. aureus. Specifically, negative growth reduction percentages were obtained for BNC and BNC–AG–GT, with −12.04% and −14.29% against E. coli and −6.47% and −6.32% against S. aureus, respectively, indicating a lack of antibacterial activity. Therefore, the integration of CS into the scaffold could play an important role in the antimicrobial activities of BNC–CS and BNC–CS–AG–GT hydrogels. CS has been reported to have potent antimicrobial characteristics due to its ability to disrupt bacterial cell membranes. The BNC–CS–AG–GT composite showed significant antimicrobial activity; however, its antibacterial properties were relatively lower against S. aureus compared to E. coli. This could be due to intrinsic structural differences between Gram-negative (E. coli) and Gram-positive (S. aureus) bacteria, which may determine their susceptibility to the composite hydrogels.

11.

Antibacterial testing, reduction of bacterial cells on hydrogel scaffold surface (A), and images of agar plates of E. coli and S. aureus colonies at 24 h (B).

Previously, it was reported that a BNC-quaternized CS hydrogel exhibited excellent antibacterial activity against E. coli and S. aureus, with killing rates of 80.8% and 81.3%, respectively, making it a promising wound healing material. Similarly, a cellulose nanofiber embedded CS/tannin hydrogel showed exceptional antibacterial activity (>99.9%) even against drug-resistant bacteria, making it effective for infected wound healing. In addition, the antibacterial properties of BNC–CS scaffolds were enhanced by incorporating nanosilver, resulting in 59% and 44% improvements in antibacterial activity against S. aureus and E. coli, respectively. Two antibacterial mechanisms have been proposed for CS. In the first model, positively charged CS has been hypothesized to interact with the negatively charged surface of bacterial cells, thereby enhancing membrane permeability and inhibiting bacterial cell growth. In the second model, binding of positively charged CS with DNA could suppress the production of mRNA in bacterial cells. In general, antibacterial properties are an important feature of hydrogels that ensure their safety for biomedical applications and also prolong the shelf life of hydrogel products made from biomaterials.

The distinct biological and degradation responses observed in the present study can be attributed to the synergistic roles of the individual scaffold components: CS enhances mechanical strength and provides antimicrobial activity; GT supports cellular attachment and accelerates degradation; AG maintains dimensional stability through ionic cross-linking; and BNC reinforces the fibrous architecture of the hydrogel, mimicking native ECM. This combination results in scaffolds with improved bioactivity and controlled degradation compared to single-polymer hydrogels.

Overall, BNC–CS–AG–GT hydrogels displayed favorable degradation behavior, good mechanical stability, high cyto-compatibility, strong osteogenic potential (as indicated by ALP activity and mineralization), and inherent antimicrobial properties. However, the osteogenic assessments in the present study are limited because the ALP and mineralization assays were conducted in vitro and thus can only provide indirect evidence for bone-forming capacity. Therefore, further in vivo studies under mechanical loading are needed for a comprehensive evaluation of the effects of the hydrogel scaffolds.

4. Conclusion

In the present work, BNC, BNC–CS, BNC–AG–GT, and BNC–CS–AG–GT were developed as 3D-scaffolds and were investigated for in vitro biodegradation and biocompatibility for bone tissue engineering. The in vitro biodegradation of the tested hydrogel scaffolds was performed in SBF with/without lysozyme. During in vitro biodegradation without lysozyme for 8 weeks, morphology changes and degradation were observed only for BNC–AG–GT and BNC–CS–AG–GT; however, during in vitro biodegradation with lysozyme, morphology changes and degradation were observed for BNC–AG–GT, BNC–CS–AG–GT, and BNC–CS. In SBF solution supplemented with lysozyme, degradation products of the hydrogel scaffolds exhibited an increase in protein concentration over incubation time, while a gradual increase in glucose concentration was also observed following biodegradation of BNC–CS and BNC–CS–AG–GT. In addition, the compressive strength of BNC–CS, BNC–AG–GT, BNC–CS–AG–GT, and AG–GT significantly decreased with time during degradation in SBF with lysozyme. After 8 weeks in SBF solution supplemented with lysozyme, the compressive strength of BNC–CS–AG–GT was reduced from approximately 68 to 25 MPa, with a weight loss of ∼54%. It is well established that BNC is not enzymatically degradable in the body. However, incorporation of CS in BNC through biosynthetic processes in A. xylinum produces a native BNC–CS that can be degraded by lysozyme, a naturally occurring enzyme in the body. Moreover, by incorporating a substance like AG–GT, the degradation rate is increased because lysozyme helps break the hydrogel down into smaller particles. However, no degradation was observed for BNC alone in SBF with or without lysozyme. According to cytotoxicity measurements against L929 cells for 24 h, degradation products from all tested hydrogel scaffolds were nontoxic. Biocompatibility studies using the osteogenic cell line MC3T3-E1 encapsulated in BNC–AG–GT or BNC–CS–AG–GT hydrogel scaffolds demonstrated that these scaffolds strongly support cellular activities such as increased cell proliferation and cell adhesion while promoting a significant increase in ALP activity and calcium deposition. In addition, BNC–CS and BNC–CS–AG–GT hydrogel scaffolds showed strong antimicrobial properties against both Gram-positive and Gram-negative bacteria. BNC–CS–AG–GT hydrogel scaffolds induced the greatest enhancement in cell proliferation, ALP activity, ARS activity, and mineralization in MC3T3-E1 cells: better than those associated with BNC–AG–GT scaffolds. Based on this work, it is possible that they could be designed to control the degradation rate during bone development. Consequently, BNC–CS–AG–GT hydrogel scaffolds appear to be more suitable than BNC–AG–GT, and they could be promising candidates for applications in bone tissue engineering. The experimental data shown here suggest that the tested BNC–CS–AG–GT hydrogel scaffold has desirable biodegradability and biocompatibility properties. Nevertheless, these conclusions are based solely on in vitro findings. Therefore, further in vivo studies and osteogenic differentiation experiments under physiological loading conditions are required to confirm their long-term effectiveness in bone regeneration.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c03747.

• Additional experimental details, materials, and methods, including schematic illustrations of BNC–CS production, schematic illustrations of the fabrication process, HPLC chromatograms of medium culture, HPLC chromatograms of sugar standard, HPLC chromatograms of soluble degradable products of hydrogel scaffolds in SBF with lysozyme, calibration curve of glucose standard, swelling degree (%) of hydrogel during 7 days of incubation in SBF, and certificate of analysis (COA) of lysozyme used in the biodegradation experiments and number of living bacterial cells (CFU/mL) at 0 and 24 h contact time intervals with hydrogel scaffold against E. coli and S. aureus (PDF)

The manuscript was written through the contributions of all authors. All authors have given approval to the final version of the manuscript. P.P.P.: Conceptualization, data curation, formal analysis, investigation, methodology, software, and writingoriginal draft. S.Y.: Conceptualization, funding acquisition, investigation, methodology, resources, validation, and visualization. M.P.: Conceptualization, funding acquisition, investigation, methodology, project administration, resources, supervision, validation, visualization, writingreview and editing, and funding acquisition.

The authors declare no competing financial interest.