Purpose: To compare the effects of different cell inoculation strategies on cellular attachment and viability as well as on osteogenic differentiation capacity and subsequent bone graft functionality after implantation of cellularized scaffolds in a mandibular defect rabbit model. Materials and Methods: Bone marrow stromal cells (BMSCs) were inoculated into the scaffolds of beta-tricalcium phosphate/chitosan (β-TCP/CS) either once or multiple times and their efficiency assessed based on cell viability, initial seeding efficiency, alkaline phosphatase activity in cell lysates, mineralization activity in cryosections of cell-seeded scaffolds by von Kossa staining, and osteogenic differentiation on reverse transcriptase polymerase chain reaction. Results: Compared with one-time inoculation of BMSCs, multiple inoculations led to higher cell viability, increased proliferation, more homogeneous distribution in the scaffold, better in vitro osteogenic differentiation, and improved in vivo bone repair in a rabbit model of mandibular defect. Conclusion: Multiple inoculations have many advantages over the one-time inoculation method and should be further researched for bone tissue engineering. Schlagwörter: bone marrow stromal cells, inoculation, seed cells