Identification of novel genes regulating fracture healing and their functional analyzes

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To comprehensively evaluate gene expression in the early stage of fracture healing, we used a cDNA microarray with 2304 cDNA clones derived from an oligo-capped mouse embryo library. Closed middiaphyseal fractures were created in mouse tibiae and expression profiles were analyzed 3 days after fracture. The expression levels of six genes were up-regulated in comparison to those in unfractured bones, and 3 of 6 were identified as novel candidate genes involved in fracture repair. These are periostin, calumenin, and FHL-1, for which cloning has been already completed, but their function during bone formation remains to be elucidated. We also found that the expression of 11 genes was down-regulated. The up-regulation of the 6 genes was reconfirmed by semi-quantitative RT-PCR analysis. Furthermore, among the 6 genes, we analyzed the spatial and temporal expression of periostin by means of in situ hybridization and Northern blot analysis since it displayed the highest up-regulation ratio. A strong signal for periostin was detected in undifferentiated mesenchymal cells and immature osteoblastic cells in the periosteum between days 3 and 7, but the signal rapidly decreased by day 14. The temporal expression pattern exhibited a peak in expression on day 3, followed by a rapid decrease in expression on day 14. These findings suggest that periostin is a specific marker for preosteoblasts and plays an important role in callus formation during the early stage of fracture healing.