Immunohistochemical analysis of bone bridge formation in the injured growth plate – a 9 month follow-up study

INTRODUCTION: Physeal bony bridges represent a major clincal problem as they impair the growth process, evoking limb lenght discrepancies. Furthermore, if they are partial or peripheral, they predispose to angular deformity, as the chondrocytes of the remaining parts of the growth plate continue the normal growth process. The factors involved in posttraumatic physeal bone bridge formation are still largely unknown. OBJECTIVES: The aim of this study was to investigate the role of several proteins involved in osteogenesis, i.e. osteoprotegerin (OPG), type X collagen (COLX) and bone morphogenic protein type 2 (BMP-2) that promote osteogenesis and RANK ligand (RANKL) and Dickkopf-1 (DKK1), both inhibitors of osteogenesis. Furthermore we investigated the expression of IL-6 to detect signs of inflammation during bone bridge formation. We hypothesize that the mentioned proteins could play a major role in bone bridge formation in the growth plate. METHODS: Thirty-six male Sprague–Dawley rats with a body weight of 140–160 g and four weeks of age were randomly assigned into nine experimental groups. These groups underwent surgery receiving an unilateral cylindrical defect of 1.2 mm in diameter into the distal growth plate of the left femur. The contralateral right femur of the animals served as control bone. At designated time points (1, 3, 7, 14 and 21 days as well as 1 month, 3 month, 6 month and 12 month) four animals were anasthetized and both femora were processed for immunohistochemical analysis of the above mentioned proteins. All animal experiments were conducted under animal ethical respect and were authorized by the Austrian Ministry of Science and Research (accreditation number BMWF-66.010/0113-II/10b/2009). RESULTS: Expression of OPG was significantly increased at the injury site in the first week post injury. As expected also IL-6 was significantly increased due to the trauma. Additionally to OPG and IL-6, 14 days post surgery, expression of COLX and BMP-2 was increased. From month 1 to month 3 after injury we found a significant increased expression of OPG, COLX and BMP-2. However, also expression of DKK-1 was increased. From month 6-9 also RANKL and COLX expression was up-regulated. CONCLUSION: From our resulst we conclude that bone bridge formation is an early event following injury and that OPG, COLX and BMP-2 are involved in this process. Furthermore we were able to confirm the clinical observation that the growth plate has the potential for remodeling by showing an up-regulated expression of the inhibitors of osteogenesis DKK1 and RANKL in a later stage of injury healing. The involvement of these two factors in bone bridge formation is a novel finding and has not been reported before.