Incorporation of biphasic calcium phosphate microparticles in injectable thermoresponsive hydrogel modulates bone cell proliferation and differentiation

•Bone cells in hydrogel composite showed enhanced cell proliferation and osteogenic differentiation.•Subcutaneously implanted bone cells in hydrogel composite showed ectopic bone formation in nude mice.•The hydrogel composite can serve as an injectable material for osteoblast delivery in orthopedic applications. To provide osteoblast cells with a three-dimensional environment closer to bone matrix, an engineered construct mimicking bone components have been designed and evaluated. A biocompatible injectable thermo-responsive hydrogel, hyaluronic acid-g-chitosan-g-poly (N-isopropylacrylamide) (HA-CPN), was used as a biomimetic organic gel matrix while embedded biphasic calcium phosphate (BCP) ceramic micoparticles were used to replace mineralized matrix. The physicochemical properties of HA-CPN/BCP hydrogel composite were characterized by X-ray diffraction, thermogravimeric analysis, differential scanning calorimetric, and Fourier transform infrared spectroscopy. Human fetal osteoblast cells were cultured in vitro in HA-CPN and HA-CPN/BCP scaffold to study cell viability, proliferation, gene expression, and maintenance of their osteoblastic differential potentials. Overall, HA-CPN/BCP can serve as a better injectable carrier for bone cells than HA-CPN with enhanced proliferation rate and alkaline phosphatase activity from biochemical assays, improved calcium deposition and mineralization of extracellular matrix by histological examinations, and elevated gene expression of all essential osteoblastic genes from quantitative real-time PCR, to provide a cell/scaffold construct with higher mechanical strength and elasticity. From in vivo nude mice subcutaneous implantation experiments, the injected cell mass can form ectopic bone tissue in HA-CPN/BCP hydrogel composite.