Changes in proteoglycan types during matrix-induced cartilage and bone development

Subcutaneous implantation of demineralized bone matrix powder in rats induces migration of host cells into the site and results in a sequential chondrogenic-osteogenic development. The proteoglycans synthesized in these subcutaneous plaques at different stages during this sequence were labeled in vivo with [³⁵S]sulfate and then isolated using associative and dissociative extractions followed by density gradient centrifugation. The predominant proteoglycan synthesized in Day 7 plaques, when cartilage formation is maximal, has similar elution profiles on Sepharose 2B to proteoglycans isolated from rat femoral head cartilage. The proteoglycans from both of these cartilages contain 95% chondroitin 4-sulfate, very little keratan sulfate, and form aggregates. However, the chondroitin sulfate chains from the Day 7 plaque proteoglycans are somewhat larger. On Day 9, prior to osteogenesis, the hypertrophied cartilage matrix undergoes extensive calcification. At this time, there is a decline in synthesis of cartilage-type proteoglycan and the appearance of a smaller size proteoglycan which increases further on Days 11 to 14, when the plaques contain primarily osteogenic cells. The smaller bone-type proteoglycan has larger chondroitin sulfate chains than in the cartilage proteoglycans. Autoradiography of ³⁵S-labeled plaques provided evidence that osteoblasts and osteocytes are the site of synthesis of bone-type proteoglycan. A small proteoglycan with very similar characteristics is also synthesized on Day 4 by pre-chondrogenic plaques. An associative extraction procedure employing 0.5 M guanidine.HCl and protease inhibitors was developed to extract proteoglycans in aggregated form from the plaques. The effectiveness of this procedure for extracting proteoglycans from the femoral head and xiphosternum cartilages was also studied.