Molecular mechanism of distraction osteogenesis : Effect of tension-stress on bone formation.

采用原位杂交和北方印迹技术，以骨形态发生蛋白（BMP-2、-4、-6、-7和GDF-5）为探针，研究了机械牵张对57只大鼠骨形态发生蛋白基因表达的影响。股骨截骨后有一个7天的延迟期，直到使用小型外固定器以0.25 mm/12 h的速率进行21天的逐渐牵引。随着牵引开始，骨痂被拉长，并最终分离成近端和远端段，在中间形成纤维中间区。BMP-2和BMP-4 mRNA的表达在此阶段被显著诱导。它们的信号被广泛检测到软骨细胞和成骨细胞及其前体细胞在纤维间区承受机械张力应力。BMP-6和GDF-5 mRNA仅在纤维间带两端的软骨形成细胞中检测到，其中发生软骨内骨化。随着牵引的推进，软骨从两端逐渐被吸收，新骨直接通过膜内骨化形成。BMP-6和GDF-5 mRNA的表达在此阶段有所下降，而BMP-2和-4 mRNA的表达则在牵张过程中一直维持在较高水平。牵张完成后，纤维间带融合，延长段巩固。BMP-2、BMP-4、BMP-6和GDF-5在此阶段均不表达。在整个实验过程中从未检测到BMP-7的信号。这些数据表明，良好的和不间断的骨形成过程中破坏成骨归因于BMP-2和BMP-4基因的表达增强的机械张力应力。BMP-2和BMP-4丰富的基因产物可通过旁分泌和自分泌机制诱导原位新骨形成。

Bone lengthening with osteotomy and gradual distraction was achieved in 57 rats and the effect of mechanical tension-stress on gene expression of bone morphogenetic proteins (BMPs) was investigated by in situ hybridization and northern blot analysis using probes of BMP-2, -4, -6, -7, and GDF-5. There was a lag phase for 7 days after femoral osteotomy until gradual distraction was carried out for 21 days at a rate of 0.25 mm/12 h using a small external fixator. As distraction was started, the callus were elongated and eventually separated into proximal and distal segments forming a fibrous interzone in the middle. Expression of BMP-2 and BMP-4 mRNA was dramatically induced at this stage. Their signals were detected widely among chondrogenic and osteogenic cells and their precursor cells sustaining mechanical tension-stress at the fibrous interzone. BMP-6 and GDF-5 mRNAs were detected exclusively in chondrogenic cells at the both ends of fibrous interzone, where endochondral ossification occurred. As distraction advanced, the cartilage was progressively resorbed from both ends and new bone was formed directly by intramembranous ossification. The signals of BMP-6 and GDF-5 mRNA declined by this stage, while those of BMP-2 and -4 were maintained in high level as long as distraction was continued. After completion of distraction, the fibrous interzone fused and the lengthened segment was consolidated. None of BMP-2, -4, 6, and GDF-5 was expressed at this stage. The signals of BMP-7 was never detected throughout the experiment. These data suggest that excellent and uninterrupted bone formation during distruction osteogenesis owes to enhanced expression of BMP-2 and BMP-4 genes by mechanical tension-stress. Abundant gene products of BMP-2 and -4 could induce in situ new bone formation by paracrine and autocrine mechanism.