Mechanism of mechanical stress-induced osteoblast differentiation and osteogenesis

• Project/Area Number: 10470388
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Functional basic dentistry
• Research Institution: Niigata University
• Principal Investigator: KAWASHIMA Hiroyuki Faculty of Dentistry, Niigata University Professor, 歯学部, 教授 (40169719)
• Co-Investigator(Kenkyū-buntansha): YOSHIZAWA Tatsuya Faculty of Dentistry, Niigata University Assistant, 歯学部, 助手 (40313530) ; ISHIBASHI Osamu Faculty of Dentistry, Niigata University Assistant, 歯学部, 助手 (70293214) ; IKEGAME Mika Faculty of Dentistry, Niigata University Assistant, 歯学部, 助手 (70282986)
• Project Period (FY): 1998 – 1999
• Project Status: Completed (Fiscal Year 1999)
• Budget Amount: ¥12,800,000 (Direct Cost: ¥12,800,000); Fiscal Year 1999: ¥2,800,000 (Direct Cost: ¥2,800,000); Fiscal Year 1998: ¥10,000,000 (Direct Cost: ¥10,000,000)
• Keywords: Mechanical stress / osteogenesis / osteoblast differentiation / BMP-4 / gene cloning

Research Abstract

Mechanical stress (MS) is one of the most potent inducer of bone formation. The mechanism by which cells receive and transduce the signal into osteogenesis, however, remains unknown. Previous studies have demonstrated that MS causes changes in expression levels of many genes in osteoblasts (OBs) and osteocytes both in vivo and in vitro. However, none of these changes are specific to bone cells. Moreover it is not clear which type of cells contributed to the increased OBs induced by MS.The purpose of this study, therefore was to identify which cells differentiate into OBs and to examine how the expression of genes that are specific to osteogenic cells does change. To assess these problems, we have chosen mice calvarial suture in culture. Calvarial sutures were incubated in BGJb medium supplemented with 10% FBS, penicillin G and streptornycin at 37℃ and 5% CO_2 in air. Tensile stress was continuously applied using a helical spring made of the Elgiloy orthodontic wire. Histological study revealed that OB differentiation seems to begin about 6 hrs under the tensile judging by morphological appearance and alkaline phosphatase staining. Osteoid formation was extended toward the center of the suture which was clearly detected at 12 hr and continued thereafter. Scattered areas of newly synthesized osteoid were calcified by 48 hr. RT-PCR demonstrated that BMP-4 gene expression was increased at 6 hr and reached 2-fold by 24 hr. In situ hybridization revealed that BMP-4 gene expression increased in preOBs and was induced in spindle-shaped fibroblastic cells next to the preOBs at 3 hr. These events took place at the front of bone formation site which moved toward the center of the suture. Expression of Cbfal, an OB-specific transcription factor, was induced in the preOBs in which BMP-4 expression was previously induced at 3 hr. These sequences of changes advanced with time toward the center of the suture which was consistent with the differentiation of OBs and subsequent osteogenesis. These observations clearly indicate that fibroblasts can differentiate into OBs by MS, and that BMP-4, as an autocrine and paracrine factor, play a critical role in this process. To further identify genes involved in the tensile stress induced OB differentiation genes whose expression are markedly affected by the tensile stress were analyzed by a modified differential display using RNA Arbitrary Primed (RAP)-PCR Over twenty clones were isolated and confirmed that expression of these genes were dramatically changed by MS.Among these several clones were highly homologous to known genes such as adhesion molecule and ion channel. Tetranectine and adaptin are such examples. Most of other genes are not homologous to any reported genes in the data bank and thus appeared to be new genes so far unidentified. Characterization and functional analysis of these genes are currently in progress.