Development of New Chitosan-Based Complex with Bioactive Molecules for Regenerative Medicine

Abstract

Background/Objectives: The development of new materials incorporating bioactive molecules for tissue regeneration is a growing area of interest. The objective of this study was to develop a new complex specifically designed for bone and skin tissue engineering, combining chitosan, ascorbic acid-2-magnesium phosphate (ASAP), and β-tricalcium phosphate (β-TCP). Methods: Chitosan and the complexes chitosan/ASAP and chitosan/ASAP/β-TCP were prepared in membrane form, macerated to a particulate format, and then subjected to characterization through Fourier transform infrared (FTIR) spectroscopy, optical and scanning electron microscopy (SEM), zeta potential, thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC). Cell viability was evaluated through a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and with fluorescein diacetate (FDA) and propidium iodide (PI) staining in stem cells obtained from deciduous teeth. Statistical analyses were performed using analysis of variance (ANOVA), followed by Tukey’s test. Results: The FTIR results indicated the characteristic bands in the chitosan group and the complexation between chitosan, ASAP, and β-TCP. Microscopic characterization revealed a polydisperse distribution of micrometric particles. Zeta potential measurements demonstrated a reduction in surface charge upon the addition of ASAP and β-TCP to the chitosan matrix. TGA and DSC analyses further indicated complexation between the three components and the successful formation of a cross-linked structure in the chitosan matrix. Stem cells cultured with the particulate biomaterials demonstrated their biocompatibility. Statistical analysis revealed a significant increase in cell viability for the chitosan/ASAP and chitosan/ASAP/β-TCP groups compared to the chitosan control. Conclusions: Therefore, the chitosan/ASAP complex demonstrated potential for skin regeneration, while the chitosan/ASAP/β-TCP formulation showed promise as a biomaterial for bone regeneration due to the presence of β-tricalcium phosphate.

1. Introduction

The loss of tissue due to disease, trauma, or the natural aging process represents a significant challenge to human health. Regenerative medicine, which seeks to repair, regenerate, or replace damaged or diseased biological tissues, has emerged as a promising approach to treating these conditions [1].

Tissue engineering is a field of regenerative medicine that employs an interdisciplinary approach, integrating principles of engineering and biology, with the objective of developing functional replacements for damaged tissues [2]. The central triad for tissue engineering proposed by Langer and Vacanti is centered on cells, biomaterials, and growth factors. Biomaterials are of great importance in the field of tissue engineering due to their biocompatible properties, which permit them to replace or repair damaged tissues [3,4]. Biomaterials can be employed as scaffolds, serving as synthetic extracellular matrices to direct cell proliferation and differentiation, or as particulate materials, which can be utilized as wound fillers. The particles‘ utilization enables the controlled release of growth factors, drugs, or genes to specific areas within the biomaterial [5,6]. Materials of a particulate nature can be produced directly or after maceration.

The exploration of marine resources presents a dual opportunity: the potential to address environmental challenges and to advance the field of tissue engineering. The expansion of the global seafood industry has resulted in the generation of considerable waste, which frequently gives rise to environmental contamination and public health hazards [7]. The repurposing of these marine byproducts offers a promising avenue for the development of innovative and sustainable biomaterials for tissue engineering applications. This approach not only mitigates waste but also leverages the potential of marine organisms to create biocompatible and functional materials for regenerative medicine.

Chitin, the second-most abundant natural polymer on Earth, is primarily extracted from crustacean exoskeletons [8,9]. Composed of N-acetylglucosamine units, chitin undergoes deacetylation to form chitosan, a linear polysaccharide of glucosamine units linked by β-(1→4) glycosidic bonds. Structurally analogous to glycosaminoglycans, chitosan displays biocompatibility, biodegradability, and antimicrobial characteristics, rendering it a multifaceted material for drug and nutrient delivery, notably in the context of wound care [10,11,12]

Chitosan particles offer a promising solution for modern drug delivery, due to their favorable properties, including biodegradability, biocompatibility, stability, low toxicity, and ease of production. These nanoparticles have been utilized in a variety of fields, including parenteral and oral drug delivery, gene therapy, vaccine development, and others [13]. Given these advantageous properties, the incorporation of active molecules into chitosan-based systems has garnered significant interest.

One such bioactive molecule is vitamin C, which plays a pivotal role in numerous phases of wound healing. As a cofactor in collagen synthesis, vitamin C plays a pivotal role in bone and skin regeneration [14], rendering it a highly pertinent component for integration with chitosan-based systems. L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate (ASAP), a stabilized derivative of vitamin C [15,16], has demonstrated potential in this context. This bioactive molecule has been identified as a promising candidate for incorporation into biomaterials and has been investigated as a potential antioxidant in cosmetics. The literature reports the use of this compound at concentrations ranging from 0.001% to 3% [16]. The incorporation of ASAP into chitosan matrices has the potential to enhance the regenerative capacity of these biomaterials, particularly in the promotion of new tissue formation.

To enhance the efficacy of bone regeneration, this chitosan-based system can be augmented with bioceramic materials. Bioceramic materials are frequently employed in biomedical applications, particularly in the domain of bone tissue restoration [17]. Beta-tricalcium phosphate (β-TCP) is a biocompatible bioactive ceramic known for its osteoinductive and osteoconductive properties, making it a promising material for bone tissue engineering [18,19]. β-TCP is a versatile ceramic biomaterial frequently employed in dental implants, bone grafts, and other medical procedures aimed at restoring damaged or lost bone tissue [20]. The rapid degradation of β-tricalcium phosphate can be mitigated by complexing it with polymers such as chitosan, enabling a more controlled release of bioactive molecules [21].

This study developed, characterized, and tested chitosan-based particles complexed with ASAP and β-TCP. Several studies have been conducted to investigate the formulation of ascorbic acid-loaded chitosan [22,23,24,25,26]. However, to the best of our knowledge, no study has investigated the use of chitosan with the more stable ASAP and β-TCP. The chitosan-based complex with bioactive molecules developed in this study, derived from crustacean carapace, offers a promising approach for tissue regeneration, particularly in bone and skin applications.

2. Materials and Methods

2.1. Development of Chitosan Complexes

Chitosan (91.5% deacetylation, 75 mPas viscosity) was procured from Chitolytic (Toronto, ON, Canada) and dissolved in an aqueous solution of hydrochloric acid (0.9% HCl, Synth, Diadema, Brazil) at a temperature of 20 °C (Figure 1a).

To encapsulate L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (ASAP), the first complex, chitosan–ASAP, was employed in a 1:0.03 ratio with the active molecule code A8960 (Sigma-Aldrich, St. Luis, MO, USA), as illustrated in Figure 1b. The complexation process was carried out within a pH range of 6.3 to 6.5. This pH interval initiates the glucosamine macromolecule agglutination. Subsequently, a 0.9% aqueous solution of NaOH (Cromoline, Diadema, Brazil) was added dropwise to the solution until the pH stabilized at 7.2. This neutralization process followed the equation NaOH + HCl → NaCl + H₂O.

The chitosan, β-tricalcium phosphate (β-TCP), and ASAP complex (chitosan–β-TCP–ASAP in a 1:1:0.03 ratio) was prepared by diluting 1% β-TCP (product code 49963, Sigma-Aldrich, St. Luis, MO, USA) in 0.9% HCl and adding it slowly to the chitosan solution (Figure 1c) based on previous studies [27,28]. Subsequently, the ASAP was added in a gradual manner.

The coagulated materials from the three experimental groups were refrigerated for 24 h, and the pH of the solutions was confirmed to be 7.2 at room temperature. Subsequently, the gels formed were filtered and rinsed with Milli-Q water in order to eliminate the sodium chloride (Figure 1d).

The samples were transferred to a Petri dish and subjected to a freeze–thaw cycle every 3 h for 24 h. The macroscopic appearance after complexation, coagulation, and filtering was a pasty material (Figure 1e). Following this process, the materials were let out for 24 h at room temperature until completely dry. After 48 h, the appearance was of a membrane (Figure 1f). The particulate features of these complexes result from membrane maceration, rather than the direct development of a complex of particulate nature (Figure 1g). For biological tests in vitro, the biomaterials were submitted under ultraviolet light (UV) in a laminar hood for 3 h with agitation every 15 min (Figure 1h).

2.2. Characterization with Fourier Transform Infrared Spectroscopy (FTIR)

FTIR analysis was employed to identify the functional groups present in the samples of chitosan, the complexes chitosan/ASAP, and chitosan/ASAP/β-TCP. The spectral range used was between 4000 cm⁻¹ and 650 cm⁻¹. The outcomes were employed to generate a transmittance graph dependent on the wavenumber. The samples were applied directly to the diamond crystal, and the spectra were registered via attenuated total reflectance (ATR) [27]. The spectrophotometry studies were conducted using a Perkin Elmer spectrophotometer (Frontier model, São Paulo, Brazil).

2.3. Optical and Electron Microscopy Analises

To examine the morphology and size, the particles from the three experimental groups were evaluated using a Leica Dmi8 microscope (Leica Microsystems, Wetzlar, Germany), and scanning electron microscopy (SEM) was executed with the Hitachi Scanning Electron Microscope, model TM3000, Tokyo, Japan. Optical microphotographs were obtained using 10× and 20× objectives after the disposition of materials in glass microscope slides.

For evaluations via scanning electron microscopy, the particles were deposited on aluminum stubs with carbon conductive tape (Ted Pella, Inc., Redding, CA, USA). Subsequent to the SEM analysis, the particle size was determined through the utilization of the ImageJ software version number 1.53t (National Institutes of Health) on images of 50 and 200× magnification, with 100 measurements of the height and width of each experimental group of particles [29]. A magnification of 2000× was employed to visualize the surface of the particles.

2.4. Zeta Potential Measurements

The chitosan powder, both pure and complexed with bioactive compounds, was diluted in 1 mM NaCl to obtain a concentration of 1 mg/mL. The samples underwent ultrasound treatment for 10 min to disaggregate the particles. Next, approximately 1 mL of the sample was pipetted into Zetasizer DTS1061 cuvettes [30]. The zeta potential was ascertained by measuring the electrophoretic mobility of the particles using the Zetasizer nano ZS90 (Malvern Instruments, Worcestershine, UK) with dynamic light scattering. Measurements were repeated thrice, with a minimum of 12 executions for each measurement, and the results were presented as the mean ± standard deviation (SD).

2.5. Thermogravimetric Analysis (TGA) Experimental Procedures

The thermal decomposition behavior of the samples was analyzed via TGA. A total of approximately 15 mg of each sample was analyzed from 25 °C to 500 °C, with a heating rate of 10 °C/min. The analysis was conducted using a Q50 Thermoanalyzer (TA Instruments, New Castle, DE, USA) [31,32]. At the conclusion of the analysis, the residue was quantified at 500 °C. The weight changes were recorded with the instrument, and a weight change (%) by temperature chart was plotted.

2.6. Differential Scanning Calorimetry (DSC) Analysis

DSC analysis was conducted to examine the potential for phase transformations in the materials [33]. The assay was conducted in a Q20 (TA Instruments, New Castle, DE, USA) from 25 °C up to 200 °C using a heating rate of 10 °C/min, with samples of approximately 5 mg of the dried materials. Following the initial heating cycle, the samples were cooled at the same heating rate and subsequently reheated.

2.7. Mesenchymal Stem/Stromal Cell (MSC) Isolation, Cultivation, and Characterization

Biological tests were conducted on MSCs derived from the human pulp of deciduous teeth. The Human Research Ethics Committee of Universidade Federal do Rio Grande do Sul (UFRGS) approved this study through the Brazil Platform (Certificate of Presentation of Ethical Review—CAAE number 36403514.6.0000.5347).

Cell isolation and characterization followed procedures from prior laboratory studies [29,34]. Briefly, the stromal cells were isolated from dental pulp using the collagenase enzyme. They were cultured in Dulbecco’s Modified Eagle’s Medium with low glucose (DMEM, Sigma-Aldrich, St. Luis, MO, USA), supplemented with 2.5 g/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, Sigma-Aldrich code H4038, St Luis, MO, USA), 3.7 g/L sodium bicarbonate (Dinâmica Química Contemporânea, Indaiatuba, Brazil), 10% fetal bovine serum (FBS, Cultilab, Campinas, Brazil), 100 U/mL penicillin, and 100 μg/mL streptomycin (P/S, Sigma-Aldrich, Rehovot, Israel) in cell culture flasks (Kasvi, Guangzhou, China) and incubated at 37 °C and 5% CO₂ at a Thermo Electron Corporation incubator (Waltham, MA, USA). The cell culture media were refreshed every 3–4 days. When the cells reached confluence, cell passage was performed using a solution of trypsin-EDTA (Sigma-Aldrich, St. Luis, MO, USA) in phosphate-buffered saline (PBS) produced with monobasic potassium phosphate (Synth, Diadema, Brazil), sodium phosphate (Dinâmica Química Contemporânea, Indaiatuba, Brazil), sodium chloride (Neon, Suzano, Brazil), potassium chloride (CPQ, Diadema, Brazil) (0.25% for 3 to 5 min) to detach the cells from the culture flasks. The cells were then seeded at a density of 5000 cells per cm² of plastic.

The mesenchymal stromal cells were characterized through morphological analysis, in vitro cellular differentiation, and immunophenotypical analyses. The elongated and spindle-shaped fibroblast-like morphology of MSCs, alongside adherence to the culture plastic, was observed under an inverted optical microscope (Leica Microsystems, Wetzlar, Germany). Multidifferentiation assays with mesenchymal stem cells were performed using specific culture media and staining techniques. Osteogenic differentiation was induced by culturing cells in DMEM low-glucose culture media supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 1 μM dexamethasone (Sigma-Aldrich code D4902, St. Luis, MO, USA), 50 μM ASAP, and 15 mM β-glycerophosphate (Sigma-Aldrich code G9422, St. Luis, MO, USA) for 25 days, followed by Alizarin Red S (Sigma-Aldrich code A5553, Shanghai, China) staining. Adipogenic differentiation was achieved by culturing cells for 30 days in DMEM-supplemented media with 500 μM 3-isobutyl-1-methylxanthine (IBMX, Sigma-Aldrich code I7018, St. Luis, MO, USA), 1 μM dexamethasone, 1.74 μM insulin (Sigma-Aldrich code I6634, St. Luis, MO, USA), 50 μM indomethacin (Sigma-Aldrich code I7378, St. Luis, MO, USA), and 1 μM rosiglitazone (Sigma-Aldrich code R2408, St. Luis, MO, USA), followed by oil red O (Sigma-Aldrich code O0625, Bangalore, India) staining. Chondrogenic differentiation involved culturing in DMEM low-glucose media supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 0.1 μM dexamethasone, 50 μM ASAP, 0.01 mg/mL human recombinant insulin, 5.5 μg/mL human transferrin, 5 ng/mL sodium selenite (ITS, Sigma-Aldrich code I3146, St. Luis, MO, USA), and 10 ng/mL transforming growth factor beta 1 (ImmunoTools, Friesoythe, Germany) for 30 days, followed by Alcian Blue 8GX (Sigma-Aldrich code A5268, India) staining. An immunophenotypical assessment was performed using specific antibodies and flow cytometry prior to the tenth passage. Antibodies used included positive markers of mesenchymal stromal cells (CD29, CD44, CD73, CD90, CD105) and hematopoietic cell markers (CD14, CD34, CD45, CD184, HLA-DR, and STRO-1), with 7AAD used for cell viability assessment (Pharmingen, Becton Dickinson, San Jose, CA, USA). The analysis was conducted using FACSCanto™ II flow cytometer (BD Bioscience, San Jose, CA, USA) with FACSDiva™ software 6.0 version number (BD Biosciences, San Diego, CA, USA).

2.8. Cell Viability Assays

Post trypsinization, 10,000 cells per well were plated in 96-well culture plates. The particulate biomaterials were suspended in the culture medium and pipetted into the wells.

The particle concentrations utilized in this study were determined based on the findings of previous works. The chitosan group was administered 10 mg/mL of chitosan [35,36]. The chitosan/ASAP group received 10 mg/mL of chitosan and 0.3 mg/mL of ASAP. The concentration of 0.3 mg/mL of ASAP in the experimental groups had been previously established by pilot studies conducted by our research group [37]. Beta-TCP was utilized at a concentration of 10 mg/mL [38,39] in conjunction with 10 mg/mL chitosan and 0.3 mg/mL of ASAP, resulting in a chitosan/ASAP/β-TCP complex group that received 20.3 mg/mL.

The cells/samples were then incubated at 37 °C in a 5% CO₂ atmosphere. Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich code M2128, China) colorimetric assay. Cells were seeded directly onto culture plates and treated with particulate materials. The control group was treated with culture media alone to serve as a baseline for comparison. After 72 h, MTT (200 μL/well at 0.25 mg/mL in a 96-well plate) was added to each well and incubated for 3 h [29]. Next, the reagent was removed, and the formed crystals were dissolved with DMSO (Synth, Diadema, Brazil). The wells were agitated for ten minutes, and the absorbance was measured using a spectrophotometer (Multiskan Thermo Scientific, Shangai, China) at wavelengths of 570 and 630 nm. The results were calculated by subtracting the absorbance at 630 nm from the absorbance at 570 nm. Only viable cells are capable of reducing MTT and increasing the absorbance of the sample. The cell viability results were expressed as the mean ± SD of absorbance, determined by comparison with the control (untreated cells).

Additionally, the supernatant was removed from other wells, which were then washed with phosphate-saline buffer (PBS). Viable cells, stained green, were observed after being treated with fluorescein diacetate (FDA, 10 µg/mL, Sigma-Aldrich code F7378, St. Luis, MO, USA) and propidium iodide (PI, 5 µg/mL, Sigma-Aldrich code P4170, St. Luis, MO, USA) dissolved in PBS. Images were captured using a fluorescence microscope (Leica Dmi8, Leica Microsystems) [30].

2.9. Statistical Analysis

The MTT assay data were analyzed using ANOVA followed by Tukey’s post hoc test to compare the control group (cells cultivated in a culture plate) to the experimental groups (chitosan, chitosan/ASAP, and chitosan/ASAP/β-TCP). The control was deemed to be 100%, and the absorbance values of the experimental groups were calculated in relation to the control. The Bioestat 5.0 software program was used for all analyses, with a significant level of 5%.

3. Results

3.1. Fourier Transform Infrared Spectroscopy (FTIR)

The FTIR spectra of the samples are presented in Figure 2. The bands of pure chitosan in the region from 2886 to 2961 cm⁻¹ can be attributed to the symmetric stretching vibration of CH and CH₃ groups and the asymmetric stretching of CH₂. These observations are consistent with the presence of alkyl groups in structures such as chitin. In particular, the bands at 1380 and 1312 cm⁻¹ indicate CH₃ deformation and CH₂ agitation, respectively [40]. The distinct peaks at 1629 and 1662 cm⁻¹ are associated with the Amide I band [41]. This suggests the presence of two types of hydrogen bonds in a C=O group with the NH and OH groups of the chitin chains, thereby underscoring the intricate nature of the network of intramolecular interactions. Finally, bands ranging from 1027 to 1163 cm⁻¹ were identified. According to Fatima (2020), these bands are associated with the oxygen of the asymmetric bridge and the C-O stretching, which is a common feature of polysaccharides such as chitin [42].

With regard to the Chit/ASAP sample, the complexation of the materials is likely to overlap the stretching bands of chitosan with the characteristic bands of ASAP, rendering it impossible to identify the bands corresponding to this material. The 1408 cm⁻¹ band, which corresponds to the ionized form of ascorbic acid, was not detected. This indicates the existence of an interaction between ASAP and chitosan, resulting in the retention of ASAP in its non-ionized form [15].

FTIR analysis of the chitosan/ASAP/β-TCP complex revealed no bands corresponding to the amino group (around 1562 and 3300 cm⁻¹), indicating complete involvement in the complexation process [43]. The amide I band around 1640 cm⁻¹ due to C=O stretching vibrations was derived from residues that are not deacetylated in chitosan. The characteristic chitosan ring of CH vibrations was observed at 1327 cm⁻¹ [44]. The bands at 1092 and 1040 cm⁻¹ correspond to more than three components of the degenerated vibrational mode of asymmetrical P-O stretching in both the β-TCP and ASAP molecules.

Bands below 650 cm⁻¹, characteristic of β-TCP, were not detected due to the equipment’s detection limit.

The FTIR spectroscopy analysis provided valuable insights into the chemical interactions and structural characteristics of the complexes of chitosan with ASAP and β-TCP. These findings contribute to a better understanding of the complexation process and have implications for the design and development of chitosan-based biomaterials for various biomedical applications, including drug delivery and tissue engineering.

3.2. Microscopic Characterization

Optical microscopy revealed that both the complexed and uncomplexed chitosan particles exhibited irregular morphologies (Figure 3a).

Scanning electron microscopy (SEM) confirmed the presence of a heterogeneous particle size distribution, with particles observed at magnifications of 50× and 200× that were both smaller and larger than 100 μm (Figure 3b). At higher magnification (2000×), the irregular surface characteristics of the materials became apparent (Figure 3b).

In optical microscopy, the particles are scattered in a manner that allows the passage of light, thereby allowing their visualization. In scanning electron microscopy (SEM), the materials are grouped together, enabling the observation of particles of varying sizes.

The size analyses conducted using ImageJ following SEM at 50× and 200× magnification indicated a statistically similar distribution between the experimental groups (p = 0.3272), with a high degree of dispersion. The mean and standard deviation size of the chitosan particles was 136 ± 115 μm, while the mean size of the chitosan/ASAP particles was 146 ± 123 μm. The complex formed with β-TCP and ASAP exhibited a tendency toward larger particle sizes, with values of 163 ± 152 μm. The images obtained at 2000× magnification did not allow for the quantification of particle size, as not all of the microstructure was visible within the image. This impeded the exact observation of the edges of the material, which exceeded the dimensions of the photographic image, thereby preventing the accurate measurement. However, these images enabled the visualization of fine details of the surface of the samples.

The microscopic analysis indicates that the chitosan particles have a wide range of sizes and shapes, which suggest their polydisperse nature. These surface characteristics are the result of various factors, including polymer chain entanglement, aggregation, and interactions with the surrounding environment during particle formation.

3.3. Zeta Potential

The chitosan sample exhibited a zeta potential of 25.4 ± 1.4 mV. This value decreased significantly (p < 0.05) to 17.2 ± 0.3 mV in the chitosan/ASAP complex and further decreased (p < 0.05) to 14.9 ± 0.3 mV in the chitosan/ASAP/β-TCP complex (Figure 4). Figure S1 presents the zeta potential distribution graph generated by the Zetasizer equipment, while Table S1 presents the raw values of the zeta potential, along with the associated calculations, statistical analyses, and graphical representation.

The zeta potential values observed in this study indicate differences among the tested samples. The chitosan sample had the highest zeta potential value, indicating a higher surface charge density compared to the complexes containing ASAP and β-TCP. This higher zeta potential suggests stronger repulsive forces between chitosan particles, which may contribute to enhanced stability and dispersion in solutions. In contrast, the chitosan/ASAP sample exhibited a lower zeta potential, suggesting a decrease in surface charge density in comparison to chitosan alone. This decrease in zeta potential may be due to the inclusion of ASAP in the complex, which could introduce ions that neutralize the charge or modify the surface chemistry of the particles. Additionally, the chitosan/ASAP/β-TCP complex had the lowest zeta potential value. The incorporation of β-TCP into the sample resulted in a decrease in surface charge density compared to the chitosan/ASAP sample. This reduction in zeta potential may be attributed to the presence of β-TCP, which could shield or neutralize the surface charges of particles.

The zeta potential results indicate that the inclusion of the ASAP and β-TCP into chitosan particles significantly affects their surface charge characteristics. These findings have implications for the stability and performance of these composite materials in biomedical applications.

3.4. Thermogravimetric Analysis (TGA)

A weight loss by temperature chart for all samples is presented in Figure 5. The chitosan sample presented the highest weight loss for lower temperatures (25 to 100 °C), with an almost 19.2% loss and presenting greater weight loss velocity at 63.3 °C. All samples presented a prominent decomposition event between 200 and 320 °C, related to the thermal degradation of chitosan [45]. The chitosan sample presented 44.8% residue.

The higher mass loss in this temperature span observed for chitosan/ASAP samples further indicates the formation of a complex structure between the two components, which retains the same temperature degradation as pure chitosan. This sample presented the lowest residue content of all samples (33.6%). The chitosan/ASAP/β-TCP sample presented a smaller event between 165 and 205 °C, with less than 1.5% of mass loss, which could be related to water absorbed by β-TCP being vaporized. This more complex sample presented a higher residue content than the other two samples, with 62.3% residue, which can be attributed to the ceramic content added by the β-TCP.

3.5. Differential Scanning Calorimetry (DSC)

The thermograms for all samples are presented in Figure 6. No glass transition was observed for the materials, but all samples exhibited an endothermic event between 109 and 117 °C, which is indicative of the melting of chitosan. The lowest temperature at which this event was observed was in the chitosan sample. The addition of ASAP and β-TCP increased the melting temperature, further indicating the complexation between chitosan and ASAP.

The sample containing β-TCP presented a second endothermic event at 189 °C. This event could be related to the formation of a complex containing chitosan, ASAP and β-TCP, which presents an even higher melting temperature. However, this possibility requires further investigation. No peaks were observed during cooling or second heating, indicating that chitosan has undergone crosslinking during the initial heating process. This crosslinking prevents the material from melting after the linking process is complete, thereby imparting thermal stability to its structure.

3.6. Biological Test with Mesenchymal Stem/Stromal Cells (MSCs)

Prior to particle viability testing, the adherent cells were confirmed to have a characteristic mesenchymal stromal cell morphology. The cells displayed a fibroblast-like morphology and exhibited adherence and spreading on the culture plastic (Figure 7a). MSCs were successfully differentiated into multiple lineages (Figure 7a), according to previous studies by the research group [46,47]. Osteogenic differentiation was confirmed by the presence of Alizarin Red-positive mineralized bone matrix in red in the extracellular space, indicating the formation of osteocytes (Figure 7a). Adipogenic differentiation resulted in the formation of reddish lipid droplets oil red-stained, confirming their transformation into adipocytes (Figure 7a). In addition, MSCs demonstrated chondrogenic differentiation potential, as evidenced by the production of blue stained with Alcian blue glycosaminoglycans and a characteristic chondrocyte-like morphology (Figure 7a). Flow cytometry was used to immunophenotypically characterize the isolated MSCs. The results showed low expression (<1%) of the hematopoietic markers CD14, CD34, CD184, and CD45. Conversely, the cells showed high positive expression (>99%) for MSC markers CD29, CD90, CD105, CD44, and CD73. The mesenchymal stem/stromal cells obtained meet the criteria for defining multipotent mesenchymal stromal cells, as established by the International Society for Cellular Therapy and described by Dominici and colleagues (2006) [48].

The mitochondrial metabolism of stromal cells treated with the biomaterials was evaluated after 3 days using the MTT assay. There was no statistically significant difference in cell viability between the control group (cells were grown on a culture plate without any treatment) and the cells treated with chitosan particles. However, there was a significant increase in cell viability between the control group and both the chitosan/ASAP and chitosan/ASAP/β-TCP groups (p < 0.05). There was no significant difference between the complexed groups (Figure 7b). The data generated by the spectrophotometer from the MTT test, including the raw absorbance values, results of statistical calculations, the obtained graph, and photographs of the plate, are detailed in Table S2. Thus, the viability of cells treated with a concentration of 10 mg/mL chitosan particles was found to be similar to that of cells cultured in culture media without treatment. This indicates that chitosan alone did not significantly affect cell viability. Both the chitosan/ASAP and chitosan/ASAP/TCP complexes demonstrated enhanced cell viability in comparison to the control and chitosan alone, with statistically significant differences. This suggests that ASAP and β-TCP contribute to the positive enhancement of the complex’s biocompatibility.

Parallelly to the quantitative data obtained from the MTT assay, fluorescence microscopy demonstrated the presence of viable cells in all experimental groups, as indicated by the green fluorescence observed after staining with FDA (Figure 7c). No dead PI cells labeled red were detected.

Overall, the findings suggest that the chitosan complexes with ASAP and β-TCP have favorable biocompatibility and bioactive properties, which may be beneficial in tissue engineering and regenerative medicine.

4. Discussion

Chitosan has been studied and used in regenerative medicine and tissue regeneration [49,50,51]. From a surgical perspective, regenerative procedures are typically performed using autogenous grafts and collagen membranes. However, the use of autogenous biomaterials can cause greater patient discomfort due to the need for an additional surgical site. Additionally, collagen of animal origin may generate immunogenicity [52,53].

This study proposes using chitosan to develop a new biomaterial. This is because marine polymers pose less risk of immunogenicity compared to polymers from mammals [50,54]. Studies that improve the application of natural polymers for large-scale use and promote cell viability are relevant to tissue engineering.

The presence of primary amino groups in chitosan provides the possibility of modifications, such as N-acylation and N-allylation [55], as well as complexation with other molecules [56]. The coagulation process of chitosan occurs close to neutral pH, when the deprotonation of amine groups occurs, which bind to the complexed substance [57].

Injectable devices are becoming more commonly used to address skin defects and deficiencies associated with the aging process. Chitosan is a promising and innovative substance for this purpose, as its physical, chemical, and biological characteristics provide an excellent foundation for skin regeneration [43].

A search of the PubMed database for scientific papers containing chitosan and L-ascorbic acid 2-phosphate magnesium in the “Title/Abstract” search field did not yield any results. ASAP is an antioxidant derived from ascorbic acid (AA), but it is more stable than vitamin C, which allows for better preservation. On the other hand, 353 papers using chitosan and ascorbic acid were found in July 2024. Many of these papers were not related to the medical field. In some of these studies, AA was used as a reducing agent in the synthesis of various materials and not as a bioactive molecule. If the term “viability” was added to the search, the number of papers was reduced to 11. Among the studies focusing on the medical field with ascorbic acid incorporated in the material, an interesting work by Nasab and collaborators found that the viability of SC isolated from rats was maintained in chitosan/alginate membranes with or without AA [22]. It is possible that the use of ascorbic acid, which is less stable, contributed to the lack of a statistically significant difference in cell viability. In another paper, chitosan particles containing encapsulated AA were incorporated into electrospun polycaprolactone membranes with dexamethasone to enhance the osteogenic differentiation of stem cells [23]. In a study by Seddighian and coworkers, ascorbic acid also did not affect stromal cell viability. In chitosan and collagen scaffolds, the addition of ascorbic acid, dexamethasone, and 45S5 bioglass did not alter the viability of dermal fibroblasts [24]. The addition of 45S5 bioglass improved the mechanical properties and reduced the degradation rate of the composites. Lu and colleagues investigated the effect of modified chitosan oligosaccharides and ascorbic acid on the viability of L929 fibroblasts. AA at 0.1 and 0.5 mg/mL decreased cell viability by approximately 15%. Chitosan 4–thiobutylamidine conjugate–nitric oxide (chitosan–TBA-NO) with or without AA maintains cell viability similar to 100% [25]. L-ascorbic acid in chitosan with and without HCl (chitosan hydrochloride, CS-HCl) and with and without silicon tetraglycerolate (Si(OGly)4-3GlyOH) statistically decreased the viability of primary normal human dermal fibroblasts (NHDFs). Conversely, the addition of D-ascorbic acid to chitosan resulted in a decline in cell viability, though not to a statistically significant extent [26]. Therefore, it can be concluded that AA did not affect cell viability in the majority of the studies. The present study suggests that the use of ASAP, a more stable form of AA, may have contributed to the observed increase in viability. A previous study from our research group demonstrated that ASAP at a concentration of 300 μg/mL exhibited cytoprotective properties when in contact with zoledronate, an important drug used to treat bone diseases but with the side effect of osteonecrosis of the jaws [37].

With regard to β-TCP, a search was conducted on PubMed using the following keywords: “(chitosan [Title/Abstract]) AND (β-tricalcium phosphate [Title/Abstract]) AND (viability)”. This returned 14 results. However, none of these works described a complex formed with vitamins. All these studies with β-TCP aimed at bone regeneration, given its widespread use in this field due to its excellent biocompatibility and capacity to promote new bone growth. Furthermore, in all articles, scaffolds were produced and tested for their ability to support cell growth and/or differentiation. In the most recent of these papers, the authors produced a porous scaffold of hydroxyapatite from lobster, rather than β-TCP [58]. In the previous year, Boda and colleagues demonstrated the formation of new bone tissue in vivo using a β-tricalcium phosphate-modified aerogel containing polyvinyl alcohol/chitosan nano-spun scaffolds [59]. In 2022, Yoshida and colleagues conducted a comparative analysis of β-tricalcium phosphate, chitosan, and poly-caprolactone-based 3D melt-extruded composite scaffolds [60]. In the same year, another research team fabricated scaffolds comprising chitosan, polyvinyl alcohol (PVA), β-tricalcium phosphate, and cellulose nanocrystals [61]. Xu and colleagues constructed a bi-layered composite of chitosan/chitosan–β-tricalcium phosphate scaffold that could facilitate osteochondral defect repair [62]. A further study revealed that chitosan/poly(caprolactone)/nano β-TCP scaffolds promoted the enhanced osteogenic differentiation of stem cells [63]. In 2019, Matinfar, Mesgar, and Mohammadi conducted a study investigating the use of chitosan/carboxymethyl cellulose in conjunction with multiphasic calcium phosphate fibers [64]. Topsakal and colleagues developed electrospun polyurethane/chitosan/β-TCP scaffolds with enhanced amoxicillin loading and encapsulation efficiencies compared to previous research [65]. In a 2016 article, Muthukumar et al. produced biocompatible collagen, hydroxyapatite, chitosan, and beta-tricalcium phosphate scaffolds incorporated with ginseng compound K [66]. Additionally, in 2016, research demonstrated that stromal cells incorporated into beta-tricalcium phosphate/chitosan scaffolds enhance new bone formation and reconstruction in rabbits [67]. In order to create a non-toxic polyelectrolyte complex scaffold that mimics the structure of native osteochondral tissue, Algul D et al. fabricated multilayered β-TCP/chitosan–alginate polyelectrolyte complex scaffolds [68]. Lee and colleagues investigated the impact of varying β-TCP and chitosan concentrations in collagen membranes on cellular adhesion and mechanical stability. Their findings suggested that these modifications can effectively enhance the membrane’s biological and mechanical properties, offering a promising avenue for further research and development [69]. In another paper in 2012, Chen et al. devised a 3D-printed scaffold comprising polycaprolactone, chitosan, nanoclay, β-tricalcium phosphate, and anthracycline. This innovative construct offers mechanical support and facilitates localized sustained drug release, which may prove effective in eradicating residual cancer cells in the event of bone tumor resection [70]. The earliest work with chitosan and B-TCP studied the viability of the osteoblasts in calcium phosphate cement pastes with chitosan or with B-TCP, but not in those containing both substances [71].

In the present study, two chitosan-based complexes were introduced for investigation. The first was chitosan/ASAP, which combines chitosan with L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (ASAP). The second was chitosan/ASAP/β-TCP, which integrates β-tricalcium phosphate (β-TCP) to enhance bone regeneration potential. The primary objective was to develop materials for the regeneration of skin and bone tissue, utilizing the biocompatibility of chitosan and the bioactive properties of ASAP and β-TCP.

To characterize these complexes, Fourier transform infrared spectroscopy (FTIR) was employed to elucidate the nature of the chemical interactions, while optical and scanning electron microscopy (SEM) were utilized to ascertain the irregular morphology. Zeta potential measurements revealed alterations in the surface characteristics upon the addition of ASAP and β-TCP. Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) indicated that the chitosan/ASAP/β-TCP complex exhibited enhanced thermal stability and complexation.

With regard to stability, the chitosan/ASAP/β-TCP complex exhibited enhanced characteristics, as demonstrated by a range of analytical techniques. Fourier transform infrared spectroscopy confirmed the formation of a complex between chitosan, ASAP, and β-TCP, with the absence of characteristic amino group bands suggesting the presence of strong interactions that stabilize the composite. Zeta potential measurements indicated a reduction in surface charge, which suggests that particle repulsion was diminished and colloidal stability in the dispersed state was enhanced. Thermogravimetric analysis demonstrated that the composite exhibited increased thermal stability, as evidenced by a higher residue percentage due to the presence of β-TCP. The differential scanning calorimetry results indicated a higher melting temperature, thereby providing further evidence in support of the formation of a stable composite structure. These findings collectively indicate that the chitosan/ASAP/β-TCP complex possesses favorable stability properties, thereby establishing its potential for use in biomedical applications.

A viability test was employed to assess the biological compatibility of mesenchymal stem/stromal cells derived from dental pulp. The chitosan/ASAP and chitosan/ASAP/β-TCP complexes exhibited enhanced cell viability in comparison to chitosan alone, which is likely attributable to the ASAP’s capacity to facilitate cell proliferation and viability.

Therefore, besides the potential for bone regeneration, this study also suggests future investigations of the applicability of this novel complex for dermal biostimulation, as it combines materials with characteristics similar to existing biostimulators on the esthetics market [72], such as hydroxyapatite, which is similar to β-TCP. However, this material has the added benefit of chitosan being an antibacterial polymer [73,74] and containing a bioactive molecule (ASAP) that stimulates cell viability explored by the cosmetics industry [16].

5. Conclusions

The chitosan/ASAP and chitosan/ASAP/β-TCP complexes were developed and characterized. The analysis conducted, which combined FTIR spectroscopy, TGA, DSC, microscopic examination, and zeta potential measurements, provides valuable insights into the properties and potential applications of chitosan complexes with ASAP and β-TCP in biomedical contexts. The results indicate that chitosan complexes with ASAP and β-TCP possess favorable biocompatibility and bioactive properties, making them promising candidates for use in tissue engineering, regenerative medicine, and other biomedical fields. Future research directions include in vivo studies to validate the biocompatibility and efficacy of the developed chitosan complexes.