We have purified and characterized active recombinant human bone morphogenetic protein (BMP) 2A. Implantation of the recombinant protein in rats showed that a single BMP can induce bone formation in vivo. A doseresponse and time-course study using the rat ectopic bone formation assay revealed that implantation of 0.5-115 ,ug of partially purified recombinant human BMP-2A resulted in cartilage by day 7 and bone formation by day 14. The time at which bone formation occurred was dependent on the amount of BMP-2A implanted; at high doses bone formation could be observed at 5 days. The cartilage-and bone-inductive activity of the recombinant BMP-2A is histologically indistinguishable from that of bone extracts. Thus, recombinant BMP-2A has therapeutic potential to promote de novo bone formation in humans.The therapeutic potential for bone formation induced by demineralized bone or its extracts has long been recognized (1-4), but the definition of the factor(s) responsible has remained elusive. We previously described the molecular cloning of the genes for bone morphogenetic protein (BMP) 1, 2A, 2B, and 3, using peptide sequence information from a group of proteins purified from such an extract (5, 6). Each of these proteins was implicated in cartilage and bone formation by preliminary experiments which demonstrated in vivo cartilage induction at 7 days (5) in the rat ectopic bone-formation system (7). We now describe the purification and characterization of recombinant human BMP-2A, produced by a Chinese hamster ovary (CHO) cell line, and its activity in ectopic bone formation. METHODS 2220The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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 ...

Using a novel purification scheme, we have characterized BMP as a 30kD fraction. After reduction, which destroys biological activity, this fraction was shown to be comprised of individual polypeptides with molecular weights of 30kD, 18kD and 16kD. Molecular cloning of these polypeptides resulted in the identification of 4 previously undescribed genes (BMP-1, BMP-2A, BMP-2B, and BMP-3) each of which is capable of directing de novo cartilage formation in vivo. While BMP-1 appears to be unrelated to other known growth factors, the derived amino acid sequence of BMP-2A, 2B, and 3 indicate that they are new members of the TGFb gene family. BMP-1, 2A and 2B are expressed in rat embryos during morphogenesis and can be localized by in situ hybridization to developing limb buds. BMP-3 localizes to neural ectoderm and later on in development to newly forming periosteum. Comparisons to other members of the TGFb family suggest that these newly identified BMPs are involved in pattern formation during early skeletal development.

Transposon mutagenesis of the bacterium Myxococcus xanthus with the transposon Tn5 revealed a special class of bacterial mutants that transduced the transposon through culture supernatant fluids. Virus-like particles copurified with transducing activity. Transposon tagging for detecting these virus-like particles may be generally useful in isolating endogenous viral agents capable of transferring genetic information between cells.