Heterotopic ossification induced by Achilles tenotomy via endochondral bone formation: Expression of bone and cartilage related genes

Animal model for heterotopic ossification (HO) induced by Achilles tenotomy in rats has been used in the literature. However, the molecular mechanism remains unclear. Here, we studied bone and cartilage related genes and their possible roles in this model. Thirty rats underwent bilateral midpoint Achilles tenotomy through a posterior approach under aseptic condition. At 3, 5 and 10 weeks post-operation, X-ray and histological examinations were carried out to investigate the formation of HO. At different phases of HO formation, mRNA levels of transforming growth factor (TGF)-β1, TGF-β2, TGF-β3, bone morphogenetic proteins (BMP)-2, BMP-4, BMP-7, hypoxia inducible factor (HIF)-1α, Sox9, Runx2, vascular endothelial growth factor (VEGF), aggrecan and collagen type I, II and X were evaluated by real-time RT-PCR. Protein levels of TGF-β1, TGF-β2, TGF-β3, BMP-2, BMP-4, BMP-7, HIF-1α, Sox9 and Runx2 were also examined by immunohistochemical staining. During the chondrogenic phase, the expressions of HIF-1α and Sox9 were significantly up-regulated. Runx2 expression was significantly up-regulated during osteogenic phase, while HIF-1α and Sox9 expression was significant decreased. TGF-β1 mRNA levels were rather constant, and the mRNA levels of TGF-β2, TGF-β3 and BMPs were changed throughout HO formation. The presences of the proteins of HIF-1α, Sox9, Runx2, TGF-βs and BMPs within the HO tissues were confirmed by immunohistochemical staining. Our study indicates that HO induced by Achilles tenotomy is by endochondral bone formation, and HIF-1α activation plays an important role during chondrogenesis in this model. Furthermore, the model provides a new experimental system to study endochondral ossification.