Involvement of BMP-2, TGF-β2 and TGF-β3 Signaling in Initial and Early Stages of Heterotopic Ossification in a rat Experimental Model

This study focused on the localization and expression of bone morphogenetic protein 2 (BMP-2) and different isoforms of transforming growth factor beta (TGF-beta(1), TGF-beta(2) and TGF-beta(3)) in the initial and early stages of heterotopic ossification (HO) employing an animal model mimicking the situation after total hip arthroplasty (THA). Bone growth was induced in rats using beta-tricalcium phosphate implants immersed either in osteoinductive rhBMP-2 solution or in saline and implanted at the site where the HO is usually expected to develop after THA. Implants were removed at 3 or 21 days after the operation and handled according to stereology principles. mRNA expression and protein staining of growth factors in different types of tissues was determined by in situ hybridization and immunohistochemistry, respectively. After three days, TGF-beta(3) content in the undifferentiated mesenchymal-like cells in the rhBMP-2 treated implants was, as assessed by immunohistochemistry, 49.6% higher compared to the saline treated group (p=0.024). This was also supported by in situ hybridization of mRNA of TGF-beta(3), which showed stronger expression in rhBMP-2 treated group. Immunohistochemical investigation showed that after 21 days the connective tissue in the rhBMP-2 treated implants contained more TGF-beta(1),TGF-beta(2) and TGF-beta(3), compared to BMP-2 and osteoblasts contained significantly (27.2%) more TGF-beta(3) compared to TGF-beta(1) (p=0.045). In the formed HO the proportion of the TGF-beta(2) and TGF-beta(3), producing bone tissue was increased by 32.1% and 47.8% respectively, compared to the TGF-beta(1) producing bone tissue (p=0.007 and p=0.006) and although this difference was not so clear at mRNA level, this suggests that TGF-beta(2) and TGF-beta(3) signaling seem to play an important role during initial and early stages of HO formation.