In vitro transformation of mesenchymal cells derived from embryonic muscle into cartilage in response to extracellular matrix components of bone.

Abstract

Subcutaneous implantation of demineralized diaphyseal bone matrix into rats induces cartilage and bone formation in vivo. When minced skeletal muscle is cultured on hemicylinders of demineralized bone in vitro, mesenchymal cells are transformed into chondrocytes. In the present investigation, the potential of extracellular matrix components of bone to trigger cartilage differentiation in vitro was examined. Extraction of bone hemicylinders with 6 M guanidine HCl resulted in the absence of chondrogenesis in vitro and endochondral bone formation in vivo. Biologically inactive hemicylinders of bone were then reconstituted with the guanidine extract and also with partially purified components extracted from bone matrix and bioassayed. Reconstitution completely restored the ability to elicit chondrogenesis in vitro and endochondral bone differentiation in vivo. Reconstitution of the whole guanidine extract on Millipore filters coated with gels of tendon collagen (type I) and subsequent culture with minced skeletal muscle also resulted in cartilage induction in vitro. These observations show that the extracellular matrix of bone is a repository of factors that govern local cartilage and bone differentiation.Extracellular matrix is necessary for adhesion, proliferation, and subsequent differentiation of cells (1-3). The extracellular matrix of bone (bone matrix) has been shown to elicit local differentiation of cartilage and bone when implanted subcutaneously in rats (4-6). A similar developmental sequence can be achieved in part, in vitro, by culturing skeletal muscle on a substratum of demineralized bone; under these conditions, muscle-derived cells form hyaline cartilage (7-10). The precise mechanisms underlying these phenotypic transformations are not well understood; however, surface charge and geometry of the matrix have been shown to be important in this regard (11,12). When components of bone matrix were dissociatively extracted and reconstituted with the inactive residual collagenous matrix, full bone induction activity was restored (13). The putative transforming proteins, with an apparent molecular mass of <50,000 daltons, appear to be related in several species of mammals (14) and have been shown to stimulate fibroblast proliferation in vitro (15).To gain further insights into the mechanism(s) of cell transformation in vitro, we have examined the action of extracellular components of bone matrix on embryonic skeletal muscle in vitro. The results reveal that fractions that elicit osteogenesis in vivo are also competent to transform mesenchymal cells derived from muscle into chondrocytes in vitro. MATERIALS AND METHODSTissue Preparation and Cultivation. The source tissue consisted of embryonic skeletal muscle excised from the thighs of 19-to 20-day fetal rats. Thigh muscle was aseptically isolated into an aliquot of ice-cold complete culture medium as described (16). During the isolation procedure, contaminant dermal, vascular, and nervous tissues were mechanically removed. Cultures consisted of aliqu...

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Isolation of osteogenin, an extracellular matrix-associated, bone-inductive protein, by heparin affinity chromatography.

“…This advance provided a method for assaying soluble components for their ability to induce endochondral bone differentiation in vivo and permitted their further purification. The putative differentiation factors have a molecular mass of <50 kDa (6), appear to be homologous in several species of mammals (7), stimulate fibroblast proliferation (8), and transform mesenchymal cells derived from muscle into chondrocytes in vitro (9). Aggregates of the bone morphogenetic protein and other proteins from bovine demineralized matrix induce formation of new bone (10).…”