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To investigate the osteoconductive effect of a chitosan scaffold in a rat skull defect model. Previous publications have demonstrated the osteoinductive properties as scaffold materials with growth factors; however, whether chitosan alone has osteoconductive ability is unclear. This study used cross-linked chitosan scaffolds for in vivo evaluation of scaffold-supported bone regeneration in rat calvarial defects using histopathological analysis and examination of alkaline phosphatase (ALP), calcium, phosphorus, and calcitonin serum levels.Scaffolds were made of cross-linked chitosan. After the defect was filled with the scaffold, the periosteum was carefully repositioned and sutured to stabilize the scaffold. The effects of the scaffold on wound repair were examined microscopically. Morphological radiographic and histopathological analyses of wound repair ratios were performed at 3 and 4 weeks after the defects were made.Using the cross-linked chitosan biomaterial of the wounds. The amount of regenerated bone measured was significantly greater in the chitosan-treated group than in the control group. The ALP level in the chitosan group at 4 weeks was higher than at baseline and at the 4-week follow-up in the control group (P < 0.05).The results show that cross-linked chitosan has an osteoconductive effect on bone regeneration in vivo.