MATRIX METALLOPROTEINASE 9 (MMP 9) INDUCES THE FORMATION OF CARTILAGE CANALS IN THE CHONDRO-EPIPHYSIS OF THE NEONATAL RABBIT: A CHORIOALLANTOIC MEMBRANE (CAM) CULTURE STUDY.

Abstract

Introduction: Chondro-epiphyseal cartilage is generally resistant to vascular invasion. At the time of formation of the secondary ossification center in skeletal ‘long’ bones, the anti-angiogenic nature of cartilage is altered in favor of angiogenesis and vascular invasion takes place. We studied the control of this angiogenic ‘switch’ by experimentally investigating two factors which might influence vascular invasion. MMP 9 is a 92Kda gelatinase which degrades collagen types IV, V and X and gelatin (denatured collagen). It has been implicated in the control of endochondral ossification at the growth plate and has been shown to modulate endothelial cell morphogenesis. Basic Fibro-blast Growth Factor (b-FGF) is a cytokine with well established angiogenic capability and has also been implicated in the development of the growth plate. We investigated whether MMP-9 caused an effect on the development of the vasculature of the chondro-epiphysis of neo-natal rabbits and compared this to the effects of b-FGF.

Materials and Methods: The CAM Culture consists of placing a small tissue explant onto the the chorioallantoic membrane of 10 day-old chick embryos and continuing culture for a further 10 days. CAM derived vessels will invade the tissue, unless anti-angiogenic factors are present. Hence, CAM culture is used as an assay system for angiogenesis and factors that will influence it. We utilized the CAM culture model to investigate vascular in-growth into explants of femoral and humeral heads from 4 day old postnatal rabbits to test the influence of MMP-9 and b-FGF. A small nylon membrane, pre-soaked in a solution containing the factor, was placed on to a tangential cut across the perichondrium. The explant was then cultured on the CAM for 3–10 days.

Results: In control epiphyses, the in-growth of CAM derived blood vessels was rare and invasion of cartilage canals through the perichondrium seldom occurred, thus confirming the anti-angiogenic nature of epiphyseal cartilage. The initial presence of MMP 9 caused a tremendous increase in the de novo vascular invasion. MMP 9 treated epiphyses contained numerous large cartilage canals. In b-FGF treated epiphyses, a greater level of vascular in-growth was seen compared with controls, but this was not as marked as with MMP 9.

Our findings indicate that b-FGF and perhaps, more interestingly, MMP-9 are implicated in the activation of the angiogenic ‘switch’ at the chondroepiphysis leading to vascular invasion. The fact that MMP-9 can act as a stimulator to angiogenesis is a novel finding. The mechanism of action remains unclear although it is possible that it is involved in the deactivation of inhibitors of vasculogenesis or the activation of angiogenic factors, or both.