We purified a factor that induces bone formation >300,000-fold from guanidinium chloride extracts of demineralized bone. Fifty nanograms of highly purified protein was active in an in vivo cartilage and bone-formation assay. The activity resided in a single gel band, corresponding to a molecular mass of %30 kDa, which yielded proteins of 30, 18, and 16 kDa on reduction. The partial amino acid sequence obtained from these proteins confirmed our identification of specific factors that induce new bone formation in vivo.Bone is a complex tissue that undergoes constant remodeling in response to changing physical demands. The signals that control resorption and formation, whether from humoral or localized growth and differentiation factors, extracellular matrix, or other presently unknown controls, require much further study. One approach to studying bone development is use of in vivo ectopic bone formation-the best characterized model of which is induction by demineralized bone implanted intramuscularly or subcutaneously. During this sequence of events (i) mesenchymal cells are seen to migrate into the implant, proliferate after several days, and condense in regions. (ii) Chondroblasts, believed to be derived from the early-appearing mesenchymal cells, form a cartilaginous template in the area of presumptive bone. (iii) At 10-14 days, the cartilage hypertrophies, and the cartilage extracellular matrix is vascularized by hematopoietic and endothelial cells. (iv) The cartilage is gradually removed and replaced by bone, and at the end of 21 days an ossicle of bone, complete with marrow, has been formed. This response is localized to the implant itself. The morphological but not temporal developmental sequence is the same as seen in embryonic endochondral bone formation and adult fracture repair (1-3).This induction of the natural sequence of bone formation immediately suggested potential application for human therapeutics and for developmental studies. Thus began the search for a factor, or factors, named bone morphogenetic protein (BMP) by Urist (1), that could induce bone formation. BMP was characterized as an activity tightly bound to the matrix of demineralized bone and extractable by denaturing solvents (4). Implantation of protein itself was sufficient to induce bone, but reconstitution of the factor with a collagenous matrix (5, 6) or synthetic matrices (7, 8) enhanced sensitivity of the assay. Although purification and characterization have been hampered by the cumbersome in vivo assay, numerous reports have described osteoinductive factors (8-10). Additionally, many other growth factors, namely fibroblastic growth factor, platelet-derived growth factor, transforming growth factors 81 and /82, insulin-like growth factors I and II, and bone-derived growth factor, have been implicated in bone development by their presence in bone and their effect on cartilage and bone cells in vitro, although no direct osteoinductive role has yet been identified in vivo (for reviews, see refs. 11 and 12). We used the rat ...
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