LONG BONE FRACTURE HEALING: MECHANISMS OF ENDOCHONDRAL OSSIFICATION

Abstract

Introduction: Our previous work has shown that angiogenesis occurs within the cartilaginous callus during long bone fracture healing1. Our aim in this study was to investigate the mechanisms involved in endochondral ossification within callus tissue during the secondary stages of fracture healing.

Methods: In this study, immunohistochemical techniques were used to localise the following proteins within the fracture callus at different times following injury. The angiogenic factors vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) were localised; bFGF is also involved in matrix remodelling and cell proliferation. In addition, urokinase plasminogen activator and its receptor (uPA and uPAr respectively), a proteolytic enzyme involved in matrix remodelling, was immunolocalised. The model used for the study was a standardised midtibial osteotomy performed on New Zealand white rabbits.

Results: Results showed that from early time points, VEGF and bFGF were detected in pericellular locations around the chondrocytes. However, VEGF was only detected around the chondrocytes in close proximity to infiltrating vessels whereas homogenous localisation of bFGF was seen throughout the cartilage. Urokinase was localised throughout the cartilage callus as well as within vascular cavities and its receptor was detected on the chondrocyte cell surface from early time points.

Discussion: The immunolocalisation of VEGF around large hypertrophic chondrocytes in close proximity to infiltrating vessels suggests that this growth factor plays a role in chondrocyte hypertrophy and death, similar to its role in the growth plate. In contrast, bFGF was strongly detected throughout cartilage from early time points, suggesting that it may also be involved with cartilage proliferation during callus formation and subsequent matrix remodelling. The localisation of urokinase around chondrocytes and within vascular cavities suggests that this enzyme plays an important role in matrix remodelling and the vascularisation of the cartilage. In conclusion, our work suggests that the following sequence of events takes place within the cartilaginous callus. Firstly, bFGF is involved in the rapid proliferation of chondrocytes in the early stages of cartilage formation following fracture. Chondrocytes then express high levels of urokinase and its receptor which is actively involved in the degradation of cartilage matrix and formation of vascular spaces which leads to angiogenesis, VEGF expression, chondrocyte hypertrophy and eventual death.