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

在57只大鼠中，采用断骨和逐渐牵张的方法实现了骨延长，并采用BMP-2、-4、-6、-7和GDF-5探针，采用原位杂交和northern blot分析了机械张力应力对骨形态发生蛋白（BMPs）基因表达的影响。股骨截骨后有7天的滞后期，直到使用小型外固定架以0.25 mm/12 h的速度逐渐牵张21天。当牵张开始时，愈伤组织被拉长，最终分离成近端和远端节段，在中间形成纤维间带。在此阶段显著诱导BMP-2和BMP-4 mRNA的表达。它们的信号广泛存在于软骨细胞和成骨细胞及其在纤维带间维持机械张力应力的前体细胞中。BMP-6和GDF-5 mrna仅在纤维带间两端的软骨细胞中检测到，这是软骨内成骨发生的地方。随着牵张的进展，软骨从两端逐渐被吸收，新骨通过膜内骨化直接形成。在这一阶段，BMP-6和GDF-5 mRNA的信号下降，而BMP-2和-4 mRNA的信号在持续分心的情况下保持在较高水平。牵张完成后，纤维带间融合，延长节段巩固。BMP-2、-4、6和GDF-5在此阶段均无表达。在整个实验过程中从未检测到BMP-7的信号。这些数据表明，在破坏成骨过程中，良好且不间断的骨形成是由于机械拉伸应力增强了BMP-2和BMP-4基因的表达。丰富的BMP-2和bmp -4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.