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Demineralized dentin matrix (DDM) is an osteoconductive and osteoinductive material that has been successfully used in sinus floor augmentation and alveolar ridge augmentation in clinical applications. It releases bone morphogenetic proteins (BMPs) and other growth factors, making DDM a suitable grafting material. However, the granular particle of DDM makes it difficult to anchor into the bone defect area. The aim of this study was to investigate the biological effects and osteoinductivity of the combination of DDM and Fibrin Glue (FG) at an optimal ratio on bone healing from a critical bone defect in an animal model. The mouse osteoblastic cell line (MC3T3-E1) was co-cultured with various ratios of DDM and FG to examine their effects on osteoblast proliferation and differentiation, as indicated by alkaline phosphatase (ALP) activity, osteocalcin (OC) production and mineralized nodules formation. The optimal ratio was then chosen for further study with a rabbit calvarial defective model, in which they were implanted with DDM or DDM-FG1 (1 g: 0.1 ml) and DDM-FG2 (1 g: 0.5 ml) compounds, or left blank for 2, 4, 8 and 12 weeks to investigate soft tissue and new bone regeneration. Micro-CT and histology analysis were used to evaluate the total grafting properties according to the different healing periods. The result from in vitro studies demonstrated that the ratio of 1:0.1 induced more ALP activity and mineralized nodules, while the ratio of 1: 0.5 (DDM-FG combined) induced more osteocalcin (OC) at specific time points. In the animal model, the 3D new bone volume in all DDM-FG treatment groups was significantly greater than that in the blank group at 2, 4, 8 and 12 weeks. Furthermore, the new bone volume was greater in DDM-FG2 when compared to the other groups during the early weeks of the healing period. In histological analysis, clusters of osteoblasts were formed adjacent to the DDM particles, and newly formed bone was observed in all groups, suggesting an osteoinductive property of DDM. Moreover, the greater new collagen synthesis observed at 4 weeks suggested that early bone healing was induced in the DDM-FG2 group. This study demonstrated that at an optimal ratio, the DDM-FG compound enhances osteogenic activities and bone regeneration., (© 2023. Springer Nature Limited.)