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

采用截骨渐进牵张的方法延长57只大鼠的骨组织，以BMP-2、-4、-6、-7和GDF-5为探针，采用原位杂交和Northern印迹分析的方法研究了机械张应力对骨形态发生蛋白基因表达的影响。股骨截骨后有7d的滞后期，然后用小型外固定器以0.25 mm/12h的速度逐渐牵张21d。随着牵张的开始，骨痂被拉长，并最终分离成近端和远端的片段，在中间形成了一个纤维带。骨形态发生蛋白-2和骨形态发生蛋白-4的表达在这一阶段被显著诱导。它们的信号在软骨细胞和成骨细胞及其前体细胞中广泛检测到，这些细胞在纤维间带承受机械拉伸应力。BMP-6和GDF-5mRNAs仅在纤维带两端软骨内成骨的软骨细胞中表达。随着牵张的推进，软骨从两端逐渐吸收，膜内骨化直接形成新骨。随着牵张时间的延长，BMP-6和GDF-5的表达逐渐减弱，而BMP-2和BMP-4的表达一直维持在较高水平。牵张完成后，纤维带间融合，延长的节段巩固。骨形态发生蛋白-2、骨形态发生蛋白-4、骨形态发生蛋白-6和骨形态发生因子-5在该阶段均无表达。在整个实验过程中都没有检测到BMP-7的信号。这些数据表明，破坏成骨过程中良好且不间断的骨形成归功于机械张应力促进了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.