Abstract
Bio-composites play a crucial role in tissue repair by supporting cell growth and promoting regeneration. Hydroxyapatite (HA), the primary mineral component of human bone and teeth, is widely used in bone tissue engineering due to its excellent biocompatibility and osteoconductivity. Bovine-derived hydroxyapatite (BHA) provides a cost-effective and efficient source for producing bone substitutes. Chitosan, a natural polymer known for its biodegradability, non-toxicity, biocompatibility, and antimicrobial properties, is another promising biomaterial for biomedical applications. Therefore, this study aimed to develop a BHA–chitosan biocomposite using lyophilization and to evaluate its physicochemical properties, biocompatibility, and potential for in vivo bone regeneration.
Bovine-derived hydroxyapatite (BHA) was produced through a successful defatting and deproteinizing procedure. The BHA cubes were milled into a nano-sized powder using a ball mill and then mixed with chitosan (CS) at a chitosan/nano-BHA ratio of 40/25 g to prepare the bio-composite (CS-BHA). The mixture was dissolved in 1% acetic acid to create a gel, which was subsequently freeze-dried at -40°C and lyophilized to form a porous scaffold (CS-BHA). The scaffold was characterized using various analytical techniques to assess its physicochemical properties. The in-vitro biocompatibility of the bio-composite was evaluated using human osteosarcoma cells. In-vivo bone regeneration was evaluated by implanting the CS-BHA scaffold in a critical-sized defect (5 mm) in the rat cranium.
Fourier-transform infrared spectroscopy (FTIR) confirmed the characteristic vibrational peaks of hydroxyapatite and chitosan. Scanning electron microscopy (SEM) showed the scaffold had an interconnected porous network. Energy-dispersive X-ray (EDX) analysis revealed high levels of organic phases, while the inorganic phases were mainly composed of calcium and phosphorus. Live/dead staining, cell proliferation, and alkaline phosphatase (ALP) assays demonstrated that the bio-composite was non-toxic, supporting cell adhesion, proliferation, and differentiation. Micro-CT, histological, and immunohistochemical analyses at 8 weeks demonstrated enhanced bone formation and density, with evidence of woven bone, blood vessel formation, and active migration of osteoblasts and osteoclasts near the implantation site.
The bovine-derived hydroxyapatite–chitosan (CS–BHA) biocomposite developed in this study exhibited excellent physicochemical properties essential for osteoconductivity, supporting cell adhesion, proliferation, and differentiation in vitro. Moreover, in vivo studies confirmed the scaffold’s ability to promote bone formation by enhancing vascularization and cellular activity. These findings indicate that the CS–BHA biocomposite is a promising candidate for future bone tissue engineering and regenerative medicine applications.