Degradation, bioactivity and cytotoxicity evaluation of polyhydroxyalkanoate (PHA) reinforced with nano-calcium phosphate (nCaP) and chitosan for bone regeneration

Polyhydroxyalkanoates (PHA) exhibit tremendous potential for bone tissue regeneration, owing to their biocompatibility and biodegradability. To address limitations in supporting bone growth, researchers have employed a strategic approach of reinforcing PHA with calcium phosphate (CaP), leading to transformative advancements. This comprehensive study investigates the bioactivity properties of composites prepared using biodegradable PHA reinforced with nano-CaP and chitosan (CH), which serve as natural carriers for growth factors and demonstrate antimicrobial properties. Various in vitro methods, including Tris-HCL degradation, simulated body fluids (SBF), and cytotoxicity tests, were employed to evaluate the performance of the composites. PHA served as the matrix, while nano-CaP (3-15wt%) was incorporated as a reinforcement along with a constant 10wt% of CH. The results revealed slower and steady degradation rates for both PHA and PHA/n-CaP/CH composites, as evidenced by water uptake and mass change profiles. SBF testing, confirmed by scanning electron microscopy-energy-dispersive X-ray spectroscopy (SEM-EDX) analysis, demonstrated the formation of an apatite layer on the composites' surface within three days, indicating excellent bioactivity potential of nano-CaP. Furthermore, the sustained apatite layer formation after 28 days strongly indicated the composites' effectiveness in promoting bone integration in vivo. Moreover, the composites maintained a neutral pH of Tris-HCl degradation and SBF media, closely resembling the physiological environment (pH 7.40). Cytotoxicity evaluations using the Alamar Blue assay confirmed the non-toxicity of the composites to osteoblast cells, accompanied by enhanced cell proliferation and viabilities exceeding 100%. Additionally, the osteogenic differentiation of human fetal osteoblasts assessed via alkaline phosphatase activity testing further emphasized the potential of PHA and PHA/n-CaP/CH composites as promising materials for bone regeneration applications. Collectively, these findings highlight the remarkable prospects of PHA-based composites in advancing bone tissue engineering and regeneration therapies.