Matrix Metalloproteinase-9 Induces the Formation of Cartilage Canals in the Chondroepiphysis of the Neonatal Rabbit

Abstract

An investigation of matrix metalloproteinase-9 (MMP-9) and its influence on vascular invasion in the secondary ossification center at the chondroepiphysis of developing long bones was undertaken. The effect of MMP-9 was compared with that of basic fibroblast growth factor (b-FGF), a potent angiogenic factor, and we assessed the chorioallantoic membrane (CAM) culture as a model for angiogenesis in osteochondral tissue. Seventy-two femoral and seventy-two humeral heads of thirty-six four-day postnatal rabbits were dissected immediately after each animal was killed. Solutions of MMP-9, b-FGF, and phosphate-buffered saline solution were applied, and the femoral and humeral chondroepiphyseal explants were incubated for ten days in CAM culture. This was used as an in vivo model to investigate the growth of blood vessels into the femoral and humeral heads of the neonatal rabbit. The explants were harvested from the CAM culture and analyzed histologically. A three-day incubation was also performed to look for early signs of vascular ingrowth into the cartilage matrix. One hundred and twenty epiphyses from thirty rabbits were placed onto CAM culture successfully; of these, two were harvested at three days to assess early changes and 118 were harvested at ten days. Forty of the 118 cultures were still viable when harvested after ten days, giving a 33% yield. Both MMP-9 and b-FGF caused an increased vascular invasion into the chondroepiphysis. New blood vessels derived from the chorioallantoic membrane within cartilage canals were more numerous in MMP-9 treated epiphyses, and larger canals were more commonly seen when compared with a control group. These findings confirmed that b-FGF is angiogenic at the chondroepiphysis. Matrix metalloproteinase-9 appears to be implicated in vascular invasion and induces the formation of new cartilage canals at the chondroepiphysis. The CAM culture model was a useful model for investigating angiogenesis in osteochondral tissue. This study adds to the understanding of the complex biochemical interaction that occurs in cartilage when the advancing vasculature begins growing into the chondroepiphysis. A better knowledge of this angiogenic process will enable a better understanding of the pathological failure or disturbance of vasculogenesis, which results in dysplastic growth disorders and osteonecrosis.