Mechanism of mechanical stress-induced osteoblast differentiation and osteogenesis

机械应力是骨形成最有效的诱导剂之一。然而，细胞接收并将信号转化为骨生成的机制仍然未知。以往的研究表明，MS引起体内和体外成骨细胞（OBs）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特有的。此外，尚不清楚哪种类型的细胞导致MS诱导的OB增加。因此，本研究的目的是鉴定哪些细胞分化为OB，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，我们选择了小鼠颅骨缝在文化。将颅骨缝合线置于BGJb培养基中，加入10%FBS、青霉素G和链霉菌素，在37℃、5%CO_2空气中培养。使用Elgiloy正畸线制成的螺旋弹簧连续施加拉伸应力。组织学研究 关于我们 显示，通过形态学外观和碱性磷酸酶染色判断，OB分化似乎在拉伸下约6小时开始。类骨质形成向缝线中心延伸，在12小时时可清楚地检测到，此后继续。48小时后，新合成的类骨质的分散区域已发生钙化。RT-PCR显示，BMP-4基因表达在6小时时增加，24小时时达到2倍。原位杂交显示，BMP-4基因在前OB中表达增加，并在纺锤体中被诱导。3小时后，在前OB旁有成纤维细胞形成。这些事件发生在骨形成部位的前部，该部位向缝合线的中心移动。Cbfal，OB特异性转录因子的表达，诱导在preOBs中，BMP-4的表达先前在3 hr. These序列的变化，随着时间的推移，向中心的缝，这是一致的OB的分化和随后的成骨诱导。这些观察清楚地表明，成纤维细胞可以分化成OB的MS，和BMP-4，作为一种自分泌和旁分泌因子，在这个过程中发挥了关键作用。为了进一步鉴定与张应力诱导成骨细胞分化有关的基因，采用改良的差异显示技术，利用RNA任意引物（RAP）-PCR技术，对张应力显著影响成骨细胞分化的基因进行了分析。分离了20多个克隆，证实这些基因的表达受到了MS的显著影响。Tetranectine和adaptin就是这样的例子。其他基因大多数与数据库中已报道的基因不同源，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