Vascularized bone grafts have been widely used to replace bone lost by disease or trauma. While this approach has been generally successful, some problems remain. Autologous bone grafts are limited in supply and can occasionally cause severe surgical trauma. Recently, some studies were undertaken to evaluate the potential of using cultured cells and biomaterials to produce tissue-engineered bone.We created prefabricated vascularized bone grafts in the femur of rats by the combined use of a collagen sponge scaffold, osteoblasts differentiated from rat bone marrow, and a vascular bundle in a diffusion chamber. In the experimental group, the saphenous vessels of 8-week-old Fischer rats were exposed under general anesthesia and placed on slits cut into the diffusion chamber. Then, a collagen sponge scaffold seeded with osteoblasts was packed into the diffusion chamber. In the control group, the procedure was similar, without the placement of vessels. One, 3, 6, and 9 weeks after operation, the implants were harvested, and new bone formation was assessed histologically and biochemically.In the experimental group, histological analysis showed active bone formation in the diffusion chamber. Bone formation was confirmed by the presence of osteoblasts lining the surface of new bone. Substantial alkaline phosphatase activity in the composites was detected soon after the procedure. Osteocalcin, detected immediately after implantation, increased slightly later. In contrast, there was no significant bone formation in the control group.These results suggest that the combination of a collagen scaffold, osteoblasts, and a vascular bundle in the chamber may be a useful method for the prefabrication of vascularized bone grafts.