Objective To explore a method by using frozen autologous cranial bone combined with bone mesenchymal stem cells (BMSCs)-sheet to repair critical-size calvarial defects in rabbits.Methods BMSCs were cultured in vitro and induced into BMSCs-sheets by cell culture with Vitamin C.Twelve New Zealand rabbits were randomly divided into experimental group and control group (n=6); critical-size calvarial defect models in the two groups were established; the autogenous bone (diameter 15 mm) from the above critical-size calvarial defect rabbits was kept in-80 ℃ refrigerator.The rabbits in the experimental group were repaired by BMSCs-sheets and autologous cranial bone,while rabbits in control group were repaired only by autologous cranial bone.All rabbits were scaned by using competed tomography (CT) 12 weeks after transplantation and then sacrificed.Cranial defects were evaluated histologically and macroscopically.Results The rabbits in the experimental group showed a stronger potential in bone regeneration than those in the control group; the bone suture in the experimental group was better filled by dense bone,and had a harder texture as compared with those in the control group.CT scan showed high-density shadow in cranial defects of the experimental group,while low-density shadow was observed in CT scan of the control group.In experimental group,HE staining indicated partial healing in broken ends,formation of newly bone layer,dispersed bone cells,and nourishvessels; in contrast,the control group did not form a complete layer of bone.Conclusion Frozen autologous bone and cell-sheet combination is a promising strategy in improving the healing of large area bone defect.