Influence of skeletal muscle on embryonic long bone development.

Abstract

Skeletal muscle and its mechanical activity have been proposed as important extrinsic influences on bone development. Previous anatomical studies have identified several aspects of long bone growth and morphology sensitive to muscle activity, but how these relationships are mediated at a cellular and molecular level remains unknown. The present studies tested the hypothesis that functioning skeletal muscle stimulates long bone growth and refines skeletal shape by influencing distinct populations of skeletal cells and expression patterns of specific matrix genes and paracrine regulatory factors. Chick embryo long bone development was studied after inducing muscular atrophy and functional paralysis by surgical denervation or systemic neuromuscular blockade. Consistently reduced longitudinal growth was observed in experimental limbs, and severe reductions in cartilage volume growth at the bone ends eventually resulted in altered bone proportions. By contrast, osteoblast-mediated appositional growth of the diaphysis was minimally affected. Growth reduction was associated with reductions of over 30% in chondrocyte proliferation in the growth plates and epiphyses and reductions of over 50% in levels of cartilage-specific type II collagen mRNA in whole bones, but with only relatively minor changes in hypertrophic chondrocyte size and no reduction in bone-specific type I collagen mRNA. These findings suggest that skeletal muscle modulates growth in part through specific effects on cartilage matrix production and production of new chondrocytes. No differences in mRNA levels for a panel of regulatory factors, including transforming growth factor $\beta$3 and its type II receptor, basic fibroblast growth factor and its type 3 receptor, and bone morphogenetic protein-4, were observed in whole bone samples, suggesting that global changes in their expression do not mediate muscle-induced stimulation of bone growth. Morphologic refinements, including asymmetric arrangement of diaphyseal bone trabeculae and development of specialized protuberances serving in muscle attachment, occurred in normal embryos and were associated with local variations in the expression of type I and type II collagen by osteoblasts and chondrocytes, respectively. These specializations of both structure and collagen expression were not seen in the muscular-deficient limb. Localized regulation of matrix genes thus represents one strategy by which cells may refine skeletal shape during development.