A number of anabolic osteogenic factors have been investigated; however, effects of those on human skeletons remain uncertain yet. Parathyroid hormone (PTH) is a major regulator in calcium metabolism, can exert anabolic effects on net bone formation in clinical trials and its various effects have been reported in vivo and in vitro. Continuous infusion of PTH decreases bone mass whereas intermittent injection of PTH increases bone mass. These diverse effects, especially PTH's anabolic effect, have not been clearly reproduced in an in vitro system and the mechanism of these PTH effects in bone is still controversial. The purpose of this study was to establish a culture model to mimic anabolic effect of PTH and elucidate its mechanism. In primary culture system of rat bone marrow, cells were treated with PTH at different times for various periods and mineralized-nodule (MN) formation was evaluated at day 14. Osteogenic markers, such as osteopontin, osteocalcin, and bone sialoprotein were also evaluated. Continuous incubation in higher than 10 nM PTH completely inhibited the MN formation. In contrast, when the cells were treated with 100 nM PTH at an early stage (day 4), MN formation was stimulated. Furthermore, pulse incubation with PTH at the early stage stimulated ALP activity, the mRNA expression for bone matrix proteins whereas this PTH treatment did not affect 3[H] thymidine incorporation or DNA content. These results indicate that the target cells for the PTH anabolic effects are the immature cells of osteoblastic lineage and the PTH stimulates the osteoblastic cell differentiation without influencing the cellular proliferation, consequently increasing the mineralized tissue formation. These findings could partly explain the mechanism whereby PTH exerts the anabolic effect in bone and suggest that PTH could be an applicable agent for bone development in restoration of bone defect, fracture healing and cell-based tissue engineering.

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Tissue engineering is in the process of making the shift from bench to bed. In a common strategy of tissue engineering, stem cells were sorted from primary cultured cells by cell surface markers, and then differentiated into the suitable cells for tissue regeneration using differentiation-inducing agents. The more simple strategy is the better in clinical application. This study showed that human dental pulp cells (hDPCs) cultured primarily under ordinary serum- supplemented condition without cell sorting and osteogenic differentiation induction possessed the capability to generate bone tissue in vivo. The alkaline phosphatase activity of hDPCs increased during in vitro cell culture, and the expression of osteocalcin was detected in the primary outgrowth culture of hDPCs. The hDPCs generated ectopic bone tissues on the border of the porous hydroxyapatite scaffold at 12 weeks after implantation in all 10 cases. This ectopic bone formation by hDPCs was observed regardless of the developing stage of tooth as a cell source, the type of culture medium, serum concentration and implantation site. We did not use cell sorting and osteogenic differentiation-inducing agents throughout this study.
These results lead to set up a simple strategy of bone tissue engineering like not previous.

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Sonic hedgehog (Shh) is a cytokine which has important roles in cell differentiation and is believed to interact with other signaling molecules including bone morphogenetic proteins (BMPs). Given that tooth development involves reciprocal epithelial-mesenchymal interactions and cytodifferentiation processes, we asked whether Shh regulates odontoblast differentiation along with BMPs. Shh mRNA was produced by inner dental epithelium; the expression was not constant but varied with development and cytodifferentiation of ameloblasts along the cusp-to-cervix axis. Shh protein (SHH), but not RNA, was present in the underlying differentiating preodontoblasts, probably resulting from gradual diffusion from the epithelial layers. Patched-1, one of hedgehog receptors, was expressed in dental mesenchyme, reflecting paracrine loops of action. Interestingly, BMPs were also expressed in both preameloblasts and preodontoblasts, indicating functional interactions between the two signaling molecules during their differentiation. A new culture system has been established and the process of odontoblast differentiation were analyzed. SHH upregulated the expression levels of odontoblast differentiation markers including Alp and Osteopontin. The stimulatory effect of SHH on ALPase activity was synergistically enhanced by BMP2 or BMP4. Taken together, our data provide clear evidence that SHH is synthesized by dental epithelial layers, reaches the underlying dental mesenchyme, and appears to act as a paracrine factor which regulates odontoblast differentiation in concerted fashion with BMPs.

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Induced pluripotent stem (iPS) cells have a developmental potential similar to that of ES cells. There is a possibility to be able to use the mouse iPS cells as in vitro embryotoxicity test according to the cell differentiation level. We examined the possibility that the mouse iPS cells could be used instead of ES-D3 cells with the Embryonic Stem Cell Test (EST). The differentiation level of the mouse iPS cells in the same protocol as the EST was tested.
A pulse was observed in ES-D3 cells at a rate of 82%. However, only 52% of iPS cells showed a pulse, with a low rate of contraction. There was a marked difference in iPS cells at the beginning of cardiac muscle contraction. The iPS cell is expected to be applied to embryotoxicity, as well as ES cells. However, research on iPS cells began only three years ago, and optimal differentiation techniques for iPS cells must be developed in near future.

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The final goal of periodontal therapy is to produce periodontal regeneration. Gingival fibroblast (GF) plays an important role for periodontal regeneration by synthesizing growth factors such as basic fibroblast growth factor (bFGF) and insulin-like growth factor-I (IGF-I). Growth factor induces the recruitment and formation of new fibroblast, followed by cell proliferation and cell cycle progression by expressions of cdk and cyclin proteins. In the previous study, it has been demonstrated that bFGF induced regeneration of periodontium. However, it has not much been shown the effect of IGF-I on periodontal tissue. Thus, in this study, we investigated the effect of IGF-I on cell growth in human GF (hGF) compared with bFGF. Cells were cultured to semi-confluent in DMEM containing 10% FBS, and stimulated by 10 ng/ml of IGF-I or bFGF after arrest by DMEM containing 0.5% FBS for 24 h, and then cell proliferation, cell cycle, expressions of mRNA and protein controlling cell cycle were analyzed. In this study, we demonstrated that IGF-I increased cell growth in hGF through cell cycle progression, followed by expression of phospho-ser780-Rb regulated by cdks -1, -2, -4, -6 and cyclins -A, -B1, -D1, -E in the same manner as that of bFGF. Thus, IGF-I may be applied for periodontal tissue regeneration.

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In order to elucidate the involvement of adipocytokine action in bone formation, we demonstrated the expression of adipocytokine receptors, leptin receptor (LepR) and adiponectin receptors (AdipoR1 and AdipoR2), in cultured pro-osteoblastic cells during osteoblastic differentiation.
The mRNA expression levels of osteocalcin and Runx2 were increased significantly by culture with differential medium. Both adiponectin receptors, AdipoR1 and AdipoR2, were also increased significantly during osteoblastic differentiation, though there were no significant changes in the leptin receptor, LepR, level. This indicated that adiponectin rather than leptin may be involved in osteoblastic differentiation.
Circulating adiponectin includes both a full-length type and a globular type. We also investigated the effect of globular adiponectin (gAN) on the induction of osteoblastic properties. gAN significantly suppressed the induced gene expression of osteocalcin and adiponectin receptors.
Our results indicate that the expressions of adiponectin receptors may be involved in osteoblastic differentiation without involvement of the serum globular type of adiponectin.

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