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Bone Morphogenetic Proteins: From Basic Science to Clinical Applications
A. H. Reddi, PhD
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A.H. Reddi, PhD Research Building 1, Room 2000, Center for Tissue Regeneration and Repair, Department of Orthopaedic Surgery, University of California-Davis School of Medicine, 4635 Second Avenue, Sacramento, CA 95817. E-mail address: ahreddi@ucdavis.edu
In support of the research or preparation of this manuscript, the author received grants or outside funding from Lawrence Ellison Endowed Chair. The author did not receive payments or other benefits or a commitment or agreement to provide such benefits from a commercial entity. No commercial entity paid or directed, or agreed to pay or direct, any benefits to any research fund, foundation, educational institution, or other charitable or nonprofit organization with which the author is affiliated or associated.
The role of bone morphogenetic proteins (BMPs) in bone formation during development and in fracture-healing is now well established. In experimental animals, BMPs elicit bone formation in ectopic sites and healing of critical-sized segmental bone defects. Many of the studies on the capacity of BMPs to elicit the healing of segmental bone defects have been carried out in orthopaedic research laboratories and are familiar to orthopaedic surgeons.

The Journal of Bone & Joint Surgery.  2001; 83:S1-S6 
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However, until recently little was known about the cellular and molecular mechanisms by which BMPs elicit bone formation. In a series of stunning studies over the last several years, molecular cell biologists working intensively in several laboratories have elucidated some of these mechanisms. When BMPs bind to their cell surface receptors on mesenchymal cell, a BMP signaling cascade is activated. Signals are sent via specific proteins to the cell nucleus. This results in the expression of genes that lead to the synthesis of macromolecules involved in cartilage and bone formation, and the mesenchymal cell becomes a chondrocyte or an osteoblast.
The development of knowledge in this area of BMP signal transduction during the last several years has been phenomenal and has provided a substantial amount of new information that is clear-cut, specific, and useful. Some of this new information may be of clinical relevance because it suggests potential therapeutic approaches to enhance or suppress new bone formation. Several of the studies on the mechanisms of BMP signal transduction presented at the International Conference on Bone Morphogenetic Proteins (held in Lake Tahoe, California, June 7 through 11, 2000) have been included in this supplement. In each article, the authors have included in the introductory section a lucid summary of the development of knowledge in a particular area.
In the general introduction that follows, the role of bone matrix as a repository of BMPs is described.
 
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+Fig. 1:BMP (bone morphogenetic protein) receptors and signaling cascades. BMPs are dimeric ligands with a cysteine knot in each monomer fold. Each monomer has two ß sheets (represented as two pointed fingers). In the functional dimer, the fingers are oriented in opposite directions. BMPs interact with both type-I and type-II BMP receptors. BMPR-II phosphorylates the GS domain of BMPR-I. The interaction between type-I and type-II receptors forms the signal-transducing complex. BMP type-I receptor kinase complex phosphorylates the trimeric receptor-regulated signaling substrate Smad 1, 5, or 8. This phosphorylation of Smads 1, 5, and 8 by type-I receptor kinase is inhibited and modulated by inhibitory Smads 6 and 7. Phosphorylated Smad 1 or 5 interacts with Smad 4 (common functional partner) and enters the nucleus to activate the transcriptional machinery for early BMP-response genes. The actions of signaling Smads are terminated by the degradation of the ubiquinated Smads by proteasomes. The bioavailability of BMP for interaction with BMP receptors is determined by BMP binding to extracellular matrix components such as heparan sulfate and collagen IV. In addition, BMP antagonists such as noggin, chordin, and DAN bind with high affinity to BMP and prevent its interaction with BMP receptors. Thus, there is a very intricate regulation of the biological actions of BMPs by BMP antagonists and extracellular matrix. Further interaction of soluble BMPs with extracellular matrix components renders them insoluble and matrix-bound and restricts their mobility and promotes contact-mediated short-range actions that are critical in bone. (Adapted, with permission, from Reddi AH. Role of morphogenetic proteins in skeletal tissue engineering and regeneration. Nat Biotechnol . 1998;16:247.)

 
Anchor for JumpAnchor for JumpTable I:  Bone Morphogenetic Protein Family* in Humans and Chromosome Location
BMP SubfamilyGeneric NameBMP DesignationChromosome Location
BMP 2/4BMP-2ABMP-220
BMP-2BBMP-414
BMP-3OsteogeninBMP-3  4
Growth/differentiation factor-10 (GDF-10)BMP-3B10
OP-1/BMP-7BMP-5BMP-5  6
Vegetal related-1 (Vgr-1)BMP-6  6
Osteogenic protein-1 (OP-1)BMP-720
Osteogenic protein-2 (OP-2)BMP-8-
Osteogenic protein-3 (OP-3)BMP-8B-
OthersBMP-9BMP-9-
BMP-10BMP-10-
Growth/differentiation factor-11 (GDF-11)BMP-11-
CDMPCartilage-derived morphogenetic protein-1 (CDMP-1) growth/differentiation factor-5 (GDF-5)BMP-1420
Cartilage-derived morphogenetic protein-2 (CDMP-2) growth/differentiation factor-6 (GDF-6)BMP-13-
Cartilage-derived morphogenetic protein-3 (CDMP-3) growth/differentiation factor-7 (GDF-7)BMP-12-
OthersBMP-15BMP-15-
BMP-16BMP-16-
*BMP-1 is not a BMP family member with seven canonical cysteines. It is a procollagen-C proteinase related to Drosophila tolloid, and it may play a role in modulating BMP actions by the proteolysis of BMP antagonists/binding proteins, such as noggin, chordin, and gremlin.
Demineralized bone matrix is the initial biomaterial for the isolation, purification, and molecular cloning of BMPs. Over a century ago, Nicholas Senn 43 of Rush Medical College in Chicago demonstrated that decalcified bone could be used in the treatment of osteomyelitis. Lacroix 27 hypothesized the role of an osteogenic inducer called osteogenin in bone. Urist 47 made the key discovery that demineralized, lyophilized rabbit bone induced new bone formation in intramuscular sites. Reddi and Huggins 34 induced endochondral bone morphogenesis by the subcutaneous implantation of particles of demineralized bone matrix, and they subsequently described the multiple steps in bone morphogenesis.
Bone morphogenesis is a sequential cascade with three key phases: chemotaxis and mitosis of mesenchymal cells, differentiation of the mesenchymal cells initially into cartilage, and replacement of the cartilage by bone 35 . The sequential cascade begins with the binding of plasma fibronectin 50 to the implanted demineralized matrix, which facilitates mesenchymal cell attachment and maximal proliferation on day 3. Chondroblast differentiation is evident on day 5. Maximal chondrogenesis is observed on days 7-8. On day 9, hypertrophy of cartilage is observed, with concomitant mineralization of the cartilage matrix. Angiogenesis and vascular invasion is a prerequisite for osteoblast differentiation and is maximal on days 10-11. The newly formed endochondral bone is remodeled and is the site of hematopoiesis 37,38 . The sequential bone morphogenesis in response to demineralized bone matrix mimics the early stages of skeletal morphogenesis in limb buds in embryos and fracture-healing in adults, recapitulating embryonic bone development and morphogenesis. Thus, it is possible to isolate the key signals for bone morphogenesis from demineralized bone matrix.
In general, a requirement for the purification of an osteoinductive BMP is a satisfactory bioassay. The demineralized bone matrix is insoluble. Dissociative extractants such as 4M guanidine hydrochloride have been used to extract 2 to 3% of the proteins. Once separated, neither the solubilized proteins in the extract nor the insoluble collagenous residue were osteoinductive in an ectopic site in a twenty-eight-day-old rat. However, combining the extract with the collagenous matrix residue rendered the recombined matrix osteoinductive 41,42 . Thus, there was collaboration between a soluble signal in the extract and the insoluble extracellular matrix substratum. This finding in our laboratory made it possible for us to design a satisfactory bioassay in which extracted proteins were recombined with the insoluble collagenous residue, and the reconstituted material was then implanted in an ectopic site. The development of this bioassay made it possible to isolate and purify the BMPs. The partial amino-acid sequence of osteogenin (BMP-3) was determined 31 . The amino-acid sequence data permitted the design of cognate oligonucleotide probes, allowing molecular cloning of the BMPs. The bioassay facilitated the molecular cloning and recombinant expression of the BMPs by biotechnology organizations. At last count, there were more than fifteen BMPs ( Table I ). This list does not include BMP-1, as it is an enzyme, procollagen C-proteinase, involved in proteolytic processing of mature collagen.
Bone morphogenetic proteins are dimeric molecules with two polypeptide chains held together by a single disulfide bond 33,48 . Each monomeric chain is biosynthesized as a polypeptide chain of more than 400 amino acids with the precursor, including the pro-region and the mature BMP. Certain subtilisin-like proteases 1,12,13 are involved in processing of pro-BMPs to mature BMPs of approximately 120 to 140 amino acids with canonical seven cysteine residues, one of which is involved in a critical interchain disulfide bond. The other six cysteines in each monomer are involved in three intrachain disulfide bonds. The crystal structure of BMP-7 has been determined by x-ray crystallography and highlights the characteristic cysteine knot of members of the BMP family 20 .
As noted above, in the bone morphogenetic cascade, cartilage differentiation, hypertrophy, and cell death are followed by bone formation. In this regard, all BMPs are cartilage morphogenetic proteins since cartilage is formed first. It is noteworthy that extracts of bovine articular cartilage contain distinct cartilage-derived morphogenetic proteins (CDMPs), also known as growth/differentiation factors (GDFs) 5 through 7. CDMPs/GDFs are members of the BMP family 9,45,46 .
Recombinant human BMP-4 (BMP-2B) and purified BMP-3 stimulate chondrogenesis in limb bud mesodermal cells 10 . Thus, BMPs may play a role in early chondrogenesis. In addition, BMPs have a profound role in the maintenance of the articular cartilage phenotype 2,3 . It is well known that in monolayer cultures chondrocytes progressively undergo dedifferentiation and lose their cartilage-specific type-II collagen matrix, which is replaced by type-I collagen. However, in explant cultures of articular cartilage, matrix encases the chondrocytes and the dedifferentiation is prevented and/or delayed. In serum-free chemically-defined medium, recombinant BMP-4 and BMP-7 stimulate proteoglycan synthesis in bovine and porcine articular cartilage explants 2,3,29,32 . This observation has prompted the investigation of their potential utility in cartilage regeneration and repair 7 .
Pleiotropy is the property of a single gene or gene product, such as protein, to have a multiplicity of different biological actions 34 . Since bone morphogenetic proteins govern the three key steps in the bone-induction cascade (chemotaxis and mitosis, differentiation into cartilage, and then replacement by bone), BMPs are true pleiotropic morphogens. In addition, BMPs regulate hematopoiesis, stimulate extracellular matrix synthesis, and influence cell survival maintenance and cell death (apoptosis). Recombinant BMP-4 stimulated chemotaxis of human peripheral blood monocytes at femtomolar concentrations 17 . The mitogenic action of BMP-4 on mesenchymal cells is at picomolar concentration. At a slightly higher concentration, BMP-4 initiates in vitro chondrogenesis 10 . It is important to emphasize that these concentrations are in solution in vitro , and in vivo BMPs are bound to extracellular matrix components such as collagens I and IV, heparan sulfate, heparin, and the bone mineral hydroxyapatite. Thus, when BMPs are bound to extracellular matrix, the local concentration is difficult to determine and may offer an optimal molecular conformation for biological actions. This aspect of the molecular cell biology of morphogens has considerable physiological significance for local contact-mediated short-range actions in bone formation by osteoblasts and remodeling by osteoclasts. BMPs are pleiotropic morphogens that act at concentration-dependent thresholds that are critical in local cellular environments and are dependent on context and microenvironment.
The use of genetic approaches including homologous recombination (gene knockouts) or members of the BMP family has revealed actions beyond bone. BMP-2 null mice have defects in heart development 57 . Induction of mesoderm is impaired in BMP-4 null mice 53 . Overexpression of BMP-6 in transgenic mice under the control of Keratin 10 promoter leads to psoriasis, implicating BMP-6 in skin development 19,30,49 . BMP-7 is critical for eye and kidney development. Thus, BMPs have actions beyond bone and perhaps they should be called body morphogenetic proteins to signify their wide-ranging role.
The biological actions of BMPs are mediated via specific BMP receptors ( Fig. 1 ). BMP receptors are of two types, I and II, and are serine/threonine protein kinases. These kinases are enzymes that phosphorylate proteins called Smads and activate them. The activated Smads are then translocated to the nucleus, where they participate in the transcriptional regulation of the expression of genes involved in cartilage and bone formation. There are eight different Smads ( Fig. 1 ). Smads 1, 5, and 8 are substrates for BMP receptors. Smads 2 and 3 are substrates for TGF-ß and activin receptors. Phosphorylation of Smad 1, 5, or 8 activates it to interact with common functional partner Smad 4, and this heteromeric complex enters the nucleus to activate or turn on BMP-responsive genes. There are two inhibitory Smads, 6 and 7, that normally reside in the nucleus and act as a relay to inhibit or turn off BMP type-I receptor kinase-mediated phosphorylation of Smads 1, 5, and 8. Thus, there is an intricate homeostatic regulation of the BMP receptor-activated turning on of genes and their turning off by Smads 6 and 7 through the inhibition of type-I BMP receptor kinase phosphorylation.
The term Smad originated as follows: In 1997, studies of the decapentaplegic (dpp) signaling pathway in Drosophila identified a gene named Mad (mothers against decapentaplegic) which, when mutated, abolished dpp signaling 22 . A similar gene, named Sma , was identified in Caenorhabditis elegans , and both Mad and Sma were implicated in signal transduction pathways activated by BMPs. When related genes with diverse names were rapidly identified in vertebrates, it became apparent that a unified system of nomenclature was needed for these vertebrate homologues. It was therefore proposed that Smad (merging the terms Sma and Mad ), be used to define the cytoplasmic proteins activated by BMPs 18 . Subsequent studies divided Smad proteins into three groups: receptor-activated Smads (R-Smads); inhibitory Smads (I-Smads), and the co-activator Smad 4, which binds to activated R-Smads.
Recent work has also identified BMP-specific antagonists (such as noggin 58 and chordin) and members of the DAN family (such as gremlin 24 ). These antagonists bind to the BMPs with the same affinity as BMP receptors do. Local irradiation also inhibits BMP-induced bone formation 51,52 . An overexpression or dysregulation of BMP pathways may lead to heterotopic bone formation or fibrodysplasia ossificans progressiva (FOP). BMPs have been implicated in FOP 26,44 . The pioneering work of Sakou has implicated BMPs in ossification of the posterior longitudinal ligament of the spine in Japanese patients 21,56 . The BMP-specific antagonists such as noggin or chordin might be used therapeutically in clinical conditions with pathologically excessive bone formation.
The osteoinductive capacity of BMPs has been demonstrated in preclinical models, and the efficacy of BMPs for the treatment of orthopaedic patients is now being evaluated in clinical trials 4-6,11,14-16,40,55 . The clinical applications of recombinant BMP-2 and BMP-7 (also known as osteogenic protein-1 [OP-1]) are being studied most extensively. Friedlaender et al. carried out a clinical trial of the efficacy of OP-1 in the treatment of tibial nonunions, and an article describing their results is included in part 2 of this supplement.
At the end of the session on "Clinical Applications of the BMPs in Orthopaedic Surgery" at this conference, a discussion followed that focused on unresolved issues related to the use of the BMPs in the treatment of problem fractures and nonunions. The question was raised, "What should be the design of clinical trials to demonstrate the efficacy of the BMPs in the treatment of problem fractures and nonunions?" Several participants addressed this question. Their thoughts and some guidelines for the design of clinical trials are presented in the Commentary section at the end of this issue. Concerns were expressed as to whether carrier and delivery systems with requisite properties, which adequately immobilize the growth factor and release it with desirable kinetics, will be used in the treatment of orthopaedic patients.
It was suggested that further progress in the clinical application of the BMPs will depend upon the development of carriers with ideal release kinetics for the delivery of the BMPs. In the section entitled "Delivery Systems for the BMPs" in part 2 of this supplement, articles are included that describe the preparation of new carriers 23,29,40 with defined BMP-release kinetics. These carriers may yield even better results in experimental models of the osteoinductive effects of the BMPs and in clinical trials.
Note: I wish to thank Mrs. Rita Rowlands for her truly outstanding help in the preparation of this manuscript and the references. This work was supported by the Lawrence J. Ellison Chair in Musculoskeletal Molecular Biology.
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+Fig. 1:BMP (bone morphogenetic protein) receptors and signaling cascades. BMPs are dimeric ligands with a cysteine knot in each monomer fold. Each monomer has two ß sheets (represented as two pointed fingers). In the functional dimer, the fingers are oriented in opposite directions. BMPs interact with both type-I and type-II BMP receptors. BMPR-II phosphorylates the GS domain of BMPR-I. The interaction between type-I and type-II receptors forms the signal-transducing complex. BMP type-I receptor kinase complex phosphorylates the trimeric receptor-regulated signaling substrate Smad 1, 5, or 8. This phosphorylation of Smads 1, 5, and 8 by type-I receptor kinase is inhibited and modulated by inhibitory Smads 6 and 7. Phosphorylated Smad 1 or 5 interacts with Smad 4 (common functional partner) and enters the nucleus to activate the transcriptional machinery for early BMP-response genes. The actions of signaling Smads are terminated by the degradation of the ubiquinated Smads by proteasomes. The bioavailability of BMP for interaction with BMP receptors is determined by BMP binding to extracellular matrix components such as heparan sulfate and collagen IV. In addition, BMP antagonists such as noggin, chordin, and DAN bind with high affinity to BMP and prevent its interaction with BMP receptors. Thus, there is a very intricate regulation of the biological actions of BMPs by BMP antagonists and extracellular matrix. Further interaction of soluble BMPs with extracellular matrix components renders them insoluble and matrix-bound and restricts their mobility and promotes contact-mediated short-range actions that are critical in bone. (Adapted, with permission, from Reddi AH. Role of morphogenetic proteins in skeletal tissue engineering and regeneration. Nat Biotechnol . 1998;16:247.)

Anchor for JumpAnchor for JumpTable I:  Bone Morphogenetic Protein Family* in Humans and Chromosome Location
BMP SubfamilyGeneric NameBMP DesignationChromosome Location
BMP 2/4BMP-2ABMP-220
BMP-2BBMP-414
BMP-3OsteogeninBMP-3  4
Growth/differentiation factor-10 (GDF-10)BMP-3B10
OP-1/BMP-7BMP-5BMP-5  6
Vegetal related-1 (Vgr-1)BMP-6  6
Osteogenic protein-1 (OP-1)BMP-720
Osteogenic protein-2 (OP-2)BMP-8-
Osteogenic protein-3 (OP-3)BMP-8B-
OthersBMP-9BMP-9-
BMP-10BMP-10-
Growth/differentiation factor-11 (GDF-11)BMP-11-
CDMPCartilage-derived morphogenetic protein-1 (CDMP-1) growth/differentiation factor-5 (GDF-5)BMP-1420
Cartilage-derived morphogenetic protein-2 (CDMP-2) growth/differentiation factor-6 (GDF-6)BMP-13-
Cartilage-derived morphogenetic protein-3 (CDMP-3) growth/differentiation factor-7 (GDF-7)BMP-12-
OthersBMP-15BMP-15-
BMP-16BMP-16-
*BMP-1 is not a BMP family member with seven canonical cysteines. It is a procollagen-C proteinase related to Drosophila tolloid, and it may play a role in modulating BMP actions by the proteolysis of BMP antagonists/binding proteins, such as noggin, chordin, and gremlin.
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