Mechanism of mechanical stress-induced osteoblast differentiation and osteogenesis

机械应力（MS）是骨形成最有效的诱导因子之一。然而，细胞接受并将信号转导成成骨的机制尚不清楚。先前的研究表明，MS在体内和体外均可引起成骨细胞和骨细胞中许多基因表达水平的变化。然而，这些变化都不是骨细胞所特有的。此外，尚不清楚哪种类型的细胞导致ms诱导的OBs增加。因此，本研究的目的是确定哪些细胞分化为OBs，并检查成骨细胞特异性基因的表达如何变化。为了评估这些问题，我们在培养中选择了小鼠颅骨缝合。颅骨缝合线在添加10%胎牛血清、青霉素G和链霉素的BGJb培养基中，37℃、5% CO_2空气中培养。使用Elgiloy正畸金属丝制成的螺旋弹簧连续施加拉伸应力。组织学研究显示，从形态学和碱性磷酸酶染色判断，OB分化大约在6小时开始。类骨形成向缝线中心延伸，在12小时清晰地检测到，此后一直持续。新合成的类骨散在区域在48小时内钙化。RT-PCR结果显示，BMP-4基因表达量在6小时升高，24小时达到2倍。原位杂交显示BMP-4基因在preOBs中表达增加，并在3小时后在preOBs旁边的纺锤形成纤维细胞中被诱导表达。这些事件发生在骨形成部位的前部，向缝合线的中心移动。Cbfal是一种ob特异性转录因子，在先前诱导BMP-4表达的preOBs中，Cbfal在3小时时被诱导表达。这些变化序列随着时间的推移向缝线中心推进，这与OBs的分化和随后的成骨一致。这些观察结果清楚地表明，成纤维细胞可以通过MS向OBs分化，而BMP-4作为一种自分泌和旁分泌因子在这一过程中起着关键作用。为了进一步鉴定受拉伸胁迫诱导的OB分化基因，采用改良的RNA任意引物(RAP)-PCR技术分析了受拉伸胁迫显著影响的OB分化基因，共分离了20多个克隆，证实这些基因的表达在ms作用下发生了显著变化，其中一些克隆与已知的粘附分子和离子通道等基因高度同源。四联素和适应素就是这样的例子。大多数其他基因与数据库中任何已报道的基因都不同源，因此似乎是迄今尚未确定的新基因。这些基因的特征和功能分析目前正在进行中。少

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 … More 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. Less