The aim of the present study was to evaluate new bone formation around the periosteum within the space formed by a polytetrafluoroethylene tube on the surface of rat calvarial bone. The experimental model consisted of 40 rats divided into eight groups (five rats per group). Each group was divided into the experimental group and the control groups, and it divided on 1, 2, 6 and 8 weeks group respectively. For the experimental groups, a polytetrafluoroethylene tube was placed under a raised periosteal flap, and for the control groups, the periosteum was removed and a teflon tube was placed under the skin on the calvarial bone. All surgical sites were then sutured. We examined new bone formation using micro-computed tomography, histology by staining with hematoxylin and eosin, and immunochemistry by antibodies specific to bone morphogenetic protein-2 (BMP-2) and runt-related gene-2 (Runx2). The experimental and control groups were compared at 1, 2, 6, and 8 weeks after surgery. The histological analysis clearly demonstrated that specimens from the experimental groups showed some bone formation at 2, 6, and 8 weeks after surgery. In contrast, the control groups demonstrated new bone only at 6 and 8 weeks after surgery. New bone was produced only from the calvarial bone side; it was not observed from the periosteum side. Micro-computed tomography scan revealed that bone mineral content (BMC), bone mineral density (BMC), and bone volume/tissue volume ratio (BV/TV) all increased in a time-dependent manner in the experimental groups. In the control groups, the bone volume/tissue volume ratio, bone mineral content, and bone mineral density increased slightly from 6 to 8 weeks after surgery. In all experimental and control groups, BMP-2 positive and Runx2-positive cells appeared on the new bone. In conclusion, these results suggest that the periosteum plays a role in promoting new bone formation.

The objective of this study was to investigate the osteogenic potential of human dental follicle cells (hDFC) in vivo. hDFC were isolated from dental follicle tissue by enzymatic digestion and cultured in growth medium (GM). hDFC were grown in a three-dimensional (3D) culture using gelatin sponges in osteogenic induction medium (OIM) or GM. The cells were transplanted onto calvaria bone of immunodeficient rats (n = 3). Hematoxylin and eosin (H-E) staining and immunohistochemistry were performed, and bone formation was analyzed with micro-computed tomography (micro-CT). H-E staining showed newly formed bone after transplantation of hDFC grown in a 3D culture in OIM. Immunohistochemistry showed BMP-2, Runx2, and Osterix expression in the hDFC transplantation site during the early stage of bone formation. Moreover, micro-CT showed that the transplanted hDFC that were 3D-cultured in OIM promoted good bone quality and bone volume compared to the other two groups. Thus, human dental follicles may be a potentially useful cell source for regenerative therapy.