Effect of nanocomposite chitosan/hydroxyapatite pH-induced hydrogels on the osteogenic differentiation of spheroids from adipose stem cells

Chitosan is gaining scientific recognition as a hydrogel in bone tissue engineering (BTE) due to its ability to support osteoblast attachment and proliferation. However, its low mechanical strength and lack of structural integrity limit its application. Nanometric hydroxyapatite (HA) is used as a filler to enhance the mechanical properties and osteoinductivity of hydrogels. In this study, chitosan-based hydrogels were systematically compared by adding 10 %, 20 %, and 30 % HA to evaluate their impact on chemical-physical properties and cellular behavior. Mechanical reinforcement of HA was evaluated by rheological and mechanical tests, with results showing a marked increase in stiffness and mechanical strength as HA concentration increased. Specifically, the Young's modulus and the compression strength increased from 26.8 kPa for chitosan alone to 63.8 kPa and with values reaching 183 kPa for the 30 wt% HA sample. Swelling tests revealed a decrease in water absorption with higher HA concentrations, while weight loss measurements showed that the addition of HA improved hydrogel stability. Biological analysis demonstrated that stem cells maintained viability, with osteopontin expression observed after 14 days of culture, indicating successful differentiation toward osteoblasts. This study highlights the significant potential of HA-enhanced chitosan hydrogels for BTE applications, with improved mechanical properties and osteoinductive capabilities.