Tissue Engineered Bone Formation Using Chitosan/Tricalcium Phosphate Sponges

Background: Chitosan is a biodegradable natural polymer that has been shown to improve wound healing. This study aimed to develop chitosan/tricalcium phosphate (TCP) sponges as tissue engineering scaffolds for bone formation by three-dimensional osteoblast culture. Methods: The sponges were prepared by freeze-drying and cross-linking a mixture of chitosan solution with TCP. Fetal rat calvarial osteoblastic cells were isolated, cultured, and seeded into the sponges. The cell-sponge constructs were cultured for 56 days. Cell proliferation, alkaline phosphatase (ALPase) activity, and calcium deposition in the cell-sponge constructs were measured at 1, 7, 14, 28, and 56 days. Histologic examination was performed with light microscopy and scanning electron microscopy. Results: Chitosan/TCP sponges supported the proliferation of osteoblastic cells as well as their differentiation as indicated by high ALPase activities and deposition of mineralized matrices by the cells. Light and scanning electron microscopic examination indicated that seeded osteoblastic cells were well attached to sponge matrices and proliferated in a multi-layer fashion. Small bone-like spicules were observed on the sponge matrix at 14 days. Seeded cells appeared to be embedded in the newly formed tissue matrix, which is characteristic of the osteoblast differentiation and their progression into osteocytic cells. The amount of mineralized tissue formed in the sponge at 56 days was significant. Conclusions: These results suggest that the chitosan/TCP sponge is a feasible tool as a scaffolding material to grow osteoblast in a three-dimensional structure for transplantation into a site for bone regeneration.