Protein extracts derived from bone can initiate the process that begins with cartilage formation and ends in de novo bone formation. The critical components of this extract, termed bone morphogenetic protein (BMP), that direct cartilage and bone formation as well as the constitutive elements supplied by the animal during this process have long remained unclear. Amino acid sequence has been derived from a highly purified preparation of BMP from bovine bone. Now, human complementary DNA clones corresponding to three polypeptides present in this BMP preparation have been isolated, and expression of the recombinant human proteins have been obtained. Each of the three (BMP-1, BMP-2A, and BMP-3) appears to be independently capable of inducing the formation of cartilage in vivo. Two of the encoded proteins (BMP-2A and BMP-3) are new members of the TGF-beta supergene family, while the third, BMP-1, appears to be a novel regulatory molecule.

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.

Adult bones have a notable regenerative capacity. Over 40 years ago, an intrinsic activity capable of initiating this reparative response was found to reside within bone itself, and the term bone morphogenetic protein (BMP) was coined to describe the molecules responsible for it. A family of BMP proteins was subsequently identified, but no individual BMP has been shown to be the initiator of the endogenous bone repair response. Here we demonstrate that BMP2 is a necessary component of the signaling cascade that governs fracture repair. Mice lacking the ability to produce BMP2 in their limb bones have spontaneous fractures that do not resolve with time. In fact, in bones lacking BMP2, the earliest steps of fracture healing seem to be blocked. Although other osteogenic stimuli are still present in the limb skeleton of BMP2-deficient mice, they cannot compensate for the absence of BMP2. Collectively, our results identify BMP2 as an endogenous mediator necessary for fracture repair.

Since the identification in 1988 of bone morphogenetic protein 2 (BMP2) as a potent inducer of bone and cartilage formation, BMP superfamily signalling has become one of the most heavily investigated topics in vertebrate skeletal biology. Whereas a large part of this research has focused on the roles of BMP2, BMP4 and BMP7 in the formation and repair of endochondral bone, a large number of BMP superfamily molecules have now been implicated in almost all aspects of bone, cartilage and joint biology. As modulating BMP signalling is currently a major therapeutic target, our rapidly expanding knowledge of how BMP superfamily signalling affects most tissue types of the skeletal system creates enormous potential to translate basic research findings into successful clinical therapies that improve bone mass or quality, ameliorate diseases of skeletal overgrowth, and repair damage to bone and joints. This Review examines the genetic evidence implicating BMP superfamily signalling in vertebrate bone and joint development, discusses a selection of human skeletal disorders associated with altered BMP signalling and summarizes the status of modulating the BMP pathway as a therapeutic target for skeletal trauma and disease.

Bone morphogenetic protein (BMP) family members, including BMP2, BMP4, and BMP7, are expressed throughout limb development. BMPs have been implicated in early limb patterning as well as in the process of skeletogenesis. However, due to complications associated with early embryonic lethality, particularly for Bmp2 and Bmp4, and with functional redundancy among BMP molecules, it has been difficult to decipher the specific roles of these BMP molecules during different stages of limb development. To circumvent these issues, we have constructed a series of mouse strains lacking one or more of these BMPs, using conditional alleles in the case of Bmp2 and Bmp4 to remove them specifically from the limb bud mesenchyme. Contrary to earlier suggestions, our results indicate that BMPs neither act as secondary signals downstream of Sonic Hedghog (SHH) in patterning the anteroposterior axis nor as signals from the interdigital mesenchyme in specifying digit identity. We do find that a threshold level of BMP signaling is required for the onset of chondrogenesis, and hence some chondrogenic condensations fail to form in limbs deficient in both BMP2 and BMP4. However, in the condensations that do form, subsequent chondrogenic differentiation proceeds normally even in the absence of BMP2 and BMP7 or BMP2 and BMP4. In contrast, we find that the loss of both BMP2 and BMP4 results in a severe impairment of osteogenesis.

Little is known about the regulatory signals involved in tendon and ligament formation, and this lack of understanding has hindered attempts to develop biologically based therapies for tendon and ligament repair. Here we report that growth and differentiation factors (GDFs) 5, 6, and 7, members of the TGF-␤ gene superfamily that are most related to the bone morphogenetic proteins, induce neotendon/ligament formation when implanted at ectopic sites in vivo. Analysis of tissue induced by GDF-5, 6, or 7, containing implants by currently available morphological and molecular criteria used to characterize tendon and ligament, adds further evidence to the idea that these GDFs act as signaling molecules during embryonic tendon/ligament formation. In addition, comparative in situ localizations of the GDF-5, 6, and 7 mRNAs suggest that these molecules are important regulatory components of synovial joint morphogenesis. ( J.

Characterization of the polypeptides present in bone-inductive protein extracts from bovine bone has led to the cloning of seven regulatory molecules, six of which are distantly related to transforming growth factor .8. The three human bone morphogenetic proteins (BMPs) we describe herein, BMP-5, BMP-6, and BMP-7, show extensive sequence similarity to BMP-2, a molecule that by itself is sufficient to induce de novo bone formation in vivo. The additive or synergistic contribution of these BMP-2-related molecules to the osteogenic activity associated with demineralized bone is strongly implicated by the presence of these growth factors in the most active fractions of highly purified bone extract.Protein extracts of demineralized bone contain an activity known as bone morphogenetic protein (BMP) (1, 2). We have extensively purified this bone-inductive activity from bovine bone (3) utilizing an in vivo assay system for ectopic cartilage and bone formation (4, 5). Initial peptide sequence information derived from this highly purified extract enabled the cloning of four polypeptides, BMP-1, BMP-2, BMP-3, and BMP-4. Due to the increasing size of the BMP family of molecules described in this paper, we refer to the molecule previously called BMP-2A simply as BMP-2 and rename BMP-2B as BMP-4. Preliminary studies with these recombinant (r) human (h) molecules indicated that they all had some cartilage-forming activity in the in vivo assay system (6). Subsequent analysis has demonstrated that rhBMP-2 by itself is sufficient to initiate the developmental cascade resulting in chondrogenesis and osteogenesis in vivo (7). Further biochemical characterization of the proteins present in active BMP preparations indicated that additional molecules related to BMP-2 were present. As part of an effort to clearly define the roles of all these molecules in cartilage and bone induction, we have attempted to identify cDNA clones corresponding to each protein in the bovine (b) BMP mixture.In the present study we report the isolation ofcDNA clones encoding human BMP-5, BMP-6, and BMP-7.* These proteins represent three members of the transforming growth factor p (TGF-f3) family of growth and differentiation factors and are most closely related to the bone-inductive molecule rhBMP-2. MATERIALS AND METHODSProtein Purification and Peptide Sequence Analysis. Bovine bone extract, which was purified as in ref. dialyzed, concentrated, and subjected to SDS/PAGE under nonreducing conditions. The 28-to 30-kDa region of the gel was excised; protein was electroeluted, reduced, alkylated, and submitted to SDS/PAGE. The resulting 14-to 20-kDa region was excised; protein was electroeluted, digested with trypsin, and sequenced as described (3).RNA Preparation. RNA was isolated from the human osteosarcoma cell line U-2 OS by a modification of the Nonidet P-40 lysis method (8). Bovine bone RNA was prepared from primary cultures of fetal long bone (9). Enrichment of polyadenylylated RNA was obtained by ohgo(dT)-cellulose chromatography.cDNA Library Con...

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