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PubMed1983

Analysis of cartilage differentiation from skeletal muscle grown on bone matrix. III. Environmental regulation of glycosaminoglycan and proteoglycan synthesis.

The ability of numerous nutritional and topographic factors to influence differentiation of embryonic mesenchyme has given rise to several theories which attempt to explain the development of muscle and cartilage from these similar-appearing cells. Some theories are challenged by the observation that a substratum of demineralized bone is capable of supporting the transformation of skeletal muscle into cartilage in vitro and that the potential to form cartilage still resides within cloned myoblasts and fibroblasts of skeletal muscle. In the present study, culture media CMRL-1066, minimal essential medium (MEM), and F-12 provide varied nutritional environments and are tested for their ability to support the morphological and biochemical transformation of skeletal muscle into cartilage. Morphologically, CMRL-1066 reproducibly supports hyaline cartilage formation, whereas MEM does so in only one out of three explants onto demineralized bone, and F-12 is incapable of supporting formation of a hyaline matrix. Biochemically, each medium is sufficient to elicit synthesis of cartilage-like patterns of sulfated glycosaminoglycans and proteoglycan monomer. Synthesis of hyaluronic acid (HA) initially increases in explants grown in CMRL-1066, but decreases prior to chondrogenesis. MEM elicits a similar increase in HA synthesis, but the subsequent decrease is not as rapid. In F-12, synthesis remains depressed throughout the experiment. The data show that increases in HA synthesis occur concurrent with the appearance of fibroblast-like cells, which normally precede chondroblasts. Decreases in HA synthesis correlate well with the onset of chondrogenesis. Explants grown in CMRL-1066 reproducibly from cartilage and synthesize the greatest amounts of proteoglycan aggregate. Those grown in MEM form cartilage infrequently, synthesize reduced amounts of proteoglycan aggregate-like material, and contain greater amounts of HA, of low molecular weight. The data demonstrate that chondrogenesis can be subtly regulated by environmental factors, and such factors regulate both the morphological and biochemical expression of the phenotype through HA synthesis.

Animals↗
PubMed1994

Localization of the mRNA for bone matrix proteins during fracture healing as determined by in situ hybridization.

The expression of the mRNAs for osteonectin (ON), osteopontin (OPN), osteocalcin (OC), and matrix Gla protein (MGP) was studied by in situ hybridization during the healing process of an experimental fracture in adult rat femora. At day 1 postoperatively, ON mRNA was detected in the proliferating periosteum. At day 3, ON, OPN, and OC mRNAs were detected in woven bone. From day 5, MGP and ON mRNAs were detected in the immature chondrocytes. From day 7, ON, OPN, and OC mRNAs were detected in the osteoblastic cells in newly formed endosteal trabecular bone. OPN mRNA was also detected in some of the osteocytes in trabecular bone. From day 14, OPN and MGP mRNAs were detected in newly formed periosteal hypertrophic chondrocytes, and the ON, OPN, and OC mRNAs were detected in osteoblastic cells in newly formed periosteal trabecular bone. Although the cell types that expressed each mRNA in fractured bones were similar to those in embryonic bones, the time course of these mRNA expression in fractured bones was different from that in embryonic bones. We considered that this system is useful to investigate the phenotypic change in osteogenic and chondrogenic lineage cells that appears during fracture healing at the molecular level.

Animals↗
PubMed1993

Surface adhesion and attachment factors in bone morphogenetic protein-induced chondrogenesis in vitro.

Bone morphogenetic protein (BMP) induces cartilage and bone development in embryonic and post-fetal life. Bone morphogenetic protein-induced chondrogenesis in outgrowths of muscle connective tissues on various furrowed and unfurrowed fibronectin-coated substrata was observed by biochemical and histologic methods. The substrata consisted of cellulose acetate membranes, atelocollagen, denatured autoclaved demineralized bone matrix, bone matrix deactivated by extraction of BMP with guanidine hydrochloride (GuHCl), and aggregates of insoluble BMP and associated noncollagenous proteins (BMP/NCP) prepared from bovine bone. The furrows, which were mechanically cut into the substrata, were intended to increase surface area and provide extra spaces for cell proliferation under compression. The extent to which extracellular attachments were required for induced cartilage development was reflected in the quantity of cartilage formed when BMP/NCP, either in insoluble or in water-soluble form, was introduced was greatest on GuHCl-extracted bone matrix. On cellulose acetate and atelocollagen, BMP-induced cartilage development was relatively scanty. A substratum of bone matrix, denatured by autoclaving at a minimum of 125 degrees, permitted cell-to-cell adhesion but not cell-to-substratum attachments; the end product was loose fibrous connective tissue only. In contrast, cartilage development occurred on surfaces of undissolved particles of BMP/NCP. Water-soluble human BMP induced development of masses of amorphous cartilage. Even after it was extracted with GuHCl, rat bone matrix may have retained trace amounts of endogenous BMP. Thus, when the requirements of cells for cell-to-cell adhesion and cell-to-substratum attachment, including mechanical factors such as furrows to enlarge surface area, were met, cartilage development was a manifestation of temporal, spatial, and BMP distribution patterns. In situ hybridization and immunofluorescent microscopy with the aid of antirecombinant BMP antibiotics may provide new information about morphogenesis.

Animals↗
PubMed1991

Changes in the gene expression of collagens, fibronectin, integrin and proteoglycans during matrix-induced bone morphogenesis.

Subcutaneous implantation of demineralized bone matrix in rat results in the local cartilage and bone development. This in vivo model of bone formation was used to examine the expression patterns of cartilage and bone specific extracellular matrix genes. The steady state levels of mRNA in implants for cartilage specific type II collagen, type IX collagen, proteoglycan link protein and cartilage proteoglycan core protein (aggrecan) were increased during chondrogenesis and cartilage hypertrophy. Fibronectin mRNA levels were high during mesenchymal cell migration, attachment and chondrogenesis. Integrin (beta 1 chain) mRNA was expressed throughout the endochondral bone development. Type I collagen mRNA levels in implants increased as early as day 3, reached its peak during osteogenesis. These gene markers will be useful in the study of the mechanism of action of bone morphogenetic proteins present in the demineralized bone matrix.

Animals↗