Growth factors in the initial stage of bone formation, analysis of their expression in chondrocytes from epiphyseal cartilage of rat costochondral junction

Abstract

Introduction. In endochondral ossification septoclasts and osteoclasts (also called chondroclasts) release growth factors deposited in non-calcified and calcified zones of the growth plate. They stimulate, within the metaphysis, initial stages of the bone formation. We have recently reported quantitation of several growth factors in calcified cartilage from calf costochondral junction. Data from the analogous human cartilage could possibly help to choose efficient combinations of growth factors for clinical applications, but the amount of the calcified cartilage needed for analysis of numerous growth factors would be difficult to collect. The estimation of growth factors expression in endochondral chondrocytes may, indirectly, indicate which of them play a leading role in the stimulation of osteoprogenitor cells in metaphysis. To test this hypothesis, we used rat chondrocytes to evaluate mRNA levels of several growth factors. Materials and methods. Chondrocytes were isolated from proliferative and hypertrophic zones of the epiphyseal cartilage forming costochondral junctions of inbred Lewis rats. The total RNA was isolated from chondrocytes and the level of mRNA for bone morphogenetic proteins 1-7 (BMP-1-7), vascular endothelial growth factor A (VEGF-A), basic fibroblast growth factor (bFGF), growth/differentiation factor 5 (GDF-5), NEL-like protein 1 (NELL-1), transforming growth factor beta 1 (TGF-β1), mesencephalic astrocyte-derived neurotrophic factor (MANF), connective tissue growth factor (CTGF), osteoclast-stimulating factor 1 (OSTF-1) and insulin-like growth factor 1 (IGF-1) was evaluated using real-time PCR method. Results. All studied factors were expressed. The highest level of mRNA was detected for CTGF, MANF, VEGF-A and TGF-β1. Expression was also quite high for BMP-1, BMP-2, BMP-5, BMP-6, BMP-7, IGF-1, GDF-5 and OSTF-1. Very low level of mRNA was detected for BMP-3, BMP-4 and NELL-1. Conclusions. Chondrocytes from the proliferative and hypertrophic zones of the growth plate produce factors involved in the cartilage metabolism and bone formation. The determination of these growth factors in humans could help to choose their optimal composition necessary for stimulation of bone formation in clinical practice. In rat the best stimulation of bone formation would presumably be achieved with a mixture of BMP-2, BMP-5, BMP-6 and BMP-7.