Tissue engineering has been rapidly developed in oral and maxillofacial reconstruction. Biocompatible scaffold from chemically composites seeded with stem cells is essential and several growth factors for bone formation and angiogenesis are also required. To overcome limited activity of new bone formation with scaffolds, several biomechanical stimulation methods on cells have been made to grow cells in scaffold. Several bioreactors have been developed for real tissue growth in culture laboratory. In addition to biological stimulants like BMP, growth factors and exogenous drugs, biomechanical stimulation technique has also been known as an effective method in cell differentiation. We developed our own bioreactor with tensile mechanical strains. Then we tested with it for detection of suitable biomechanical effect on the cell differentiation and proliferation. And we also compared the results with the effect of low intensity pulsed ultrasound (LIPUS). Mechanical strain group showed more rapid reaction with cell differentiation and proliferation than non-mechanical strain group. Mechanical strain groups stimulated with 0.5∼0.7Hz for 6 hours and 8 hours showed more active cell differentiation than the group with 0.5∼0.7Hz for 2.5 hours tensile strain stimulation. Group of LIPUS also showed more rapid reaction in cell differentiation and proliferation. LIPUS with 3MHz showed more cell reaction than the LIPUS group with 1MHz. Our results showed the positive effect on differentiation and proliferation of cell with mechanical tensile strain, LIPUS both.

Kisspeptin-10 (KP-10) has been reported to act as a tumor metastasis suppressor via its receptor, G protein-coupled receptor 54 (GRP54). The KP-10/GPR54/BMPs signaling pathway plays an important role in embryonic kidney development. However, its function in osteoblast differentiation is unknown. The aim of this study was to confirm the molecular mechanism for the action of KP-10 on osteoblast differentiation. Expression of the Bone morphogenetic protein-2 (BMP2) and osteogenic genes were determined by RT-PCR and real-time PCR analysis in C3H10T1/2 cells. Transient transfection assays were performed to confirm the effects of KP-10 on BMP2-Luc activity. BMP2 and phospho-Smad1/5/9 protein levels were determined by Western blot analysis. Alkaline phosphatase (ALP) staining experiment was performed to evaluate ALP activity. To further confirm the effect of KP-10-induced GPR54, we used GPR54 Knock out (KO) C3H10T1/2 cells. KP-10 significantly increased osteogenic gene such as Runx2, ALP and Dlx5 in C3H10T1/2 cells. The ALP staining levels were also increased by KP-10. Interestingly, BMP2 mRNA, protein expression and promoter activity were also increased by KP-10. However, KP-10-induced BMP2 expressions were not increased in GPR54 KO cells. These results suggest that KP-10 increases BMP2 expression through GPR54. Next, Western blot analysis shown Smad1/5/9 phosphorylation were enhanced in a time-dependent manner by KP-10 treatment. It is well known that BMP2 increased phosphorylation of Smad1/5/9 via BMP2 receptor. In addition, KP-10 increased NFATc4 mRNA levels and NFATc4 overexpression enhance BMP2 mRNA levels. To confirm the KP-10-induced BMP2 action, we used KP-10-treated medium in wild type cells and GPR54 KO cells. The osteogenic genes were not elevated by KP-10-treated medium (GPR54 KO cells) whereas increased expression levels by KP-10 medium (wild type cells). These data indicate that KP-10 induced osteoblast differentiation through NFATc4-mediated BMP2 signaling.