Bone morphogenetic proteins (BMPs) are multifunctional growth factors that belong to the transforming growth factor beta superfamily. This literature review focuses on the molecular biology of BMPs, their mechanism of action, and subsequent applications. It also discusses uses of BMPs in the fields of dentistry and orthopedics, research on methods of delivering BMPs, and their role in tissue regeneration. BMP has positive effects on bone grafts, and their calculated and timely use with other growth factors can provide extraordinary results in fractured or nonhealing bones. Use of BMP introduces new applications in the field of implantology and bone grafting. This review touches on a few unknown facts about BMP and this ever-changing field of research to improve human life.
Bone, primarily an osseous tissue, is a relatively hard and lightweight composite material, formed mostly of calcium phosphate in the chemical arrangement termed calcium hydroxyapatite. Although bone is a rigid structure, it is dynamic as it is continuously being remodeled by the cells within it. This enables the turnover and replacement of the matrix in the interior of the bone throughout the life of a person.1,2
Three major cell types are found throughout the extracellular bone matrix: osteoblasts, osteocytes, and osteoclasts.2 Osteoblasts are mononucleate bone-forming cells that originate from mesenchymal stem cells and contribute to the production of new bone.1,2 They are responsible for producing and secreting major components of bone matrix, eg, type I collagen, osteocalcin, osteonectin, and alkaline phosphatase. Some osteoblasts remain active, synthesizing and regulating the deposition and mineralization of bone, while others become embedded in their own secretions and are trapped within the hard mineralized bone matrix.1,2 These cells form resting osteocytes, which do not multiply and are chiefly responsible for bone maintenance. The counterpart to the osteoblast is the osteoclast, a cell that is responsible for breaking down bone. Osteoclasts are giant multinucleated cells derived from hemopoietic stem cells in the bone marrow. These cells are involved in bone resorption and therefore remodeling.1,2
Bone Morphogenic Proteins
Bone morphogenetic proteins (BMPs) have roles in the processes of chemotaxis, mitogenesis and differentiation of mesenchymal stem cells, and promotion of angiogenesis.1 They induce bone formation through sequential multistep events that consist of the chemotaxis of progenitor cells; proliferation of mesenchymal cells; differentiation of cartilage; vascular invasion; and differentiation, mineralization, and remodeling of bone.3,4 Various BMPs are produced during the process of bone formation by spatially and temporally different profiles, and they have different roles during bone morphogenesis in vivo.4,5
Bone marrow stroma contains a population of multipotent stem cells that can differentiate into different cell lines, including fibroblasts, adipocytes, reticular cells, and osteogenic cells.6 Human bone marrow stromal cells can be induced to differentiate into cells expressing alkaline phosphatase activity, osteocalcin, and type I collagen.7 The complete induction of osteogenesis may require the effect of multiple local factors, acting in a coordinated fashion to control cell proliferation and generate cell differentiation through a multistep process.7
When bone is injured, such as by fracture, a local population of pluripotent progenitor cells is actived by different growth factors. The local cells are determined osteoprogenitors that live in the cambial layers of the periosteum, endosteum, and dura. Another class of cells, the inducible osteoprogenitor cells, such as pericytes, arrives at the injury site approximately 3–5 days after bone injury by transit in developing capillary sprouts.8,9 Pericytes may become osteoblasts following interactions with endogenous BMPs. According to Brighton and Hunt,10 a population of polymorphic mesenchymal cells can appear as early as 12 hours after fracture and become preosteoblasts. These cells possess multilineage potential and can convert to cartilage-forming chondrocytes or bone-forming osteoblasts, depending on the presence of environmental cues such as nutrient supply, BMP concentrations, growth factors, blood vessels, and mechanical stability.8,11
BMPs belong to a larger superfamily, known as the transforming growth factor beta (TGF-β), which includes multiple TGF-βs, activins, inhibins, Mullerian-inhibiting substance, and glial cell–derived neurotrophic factor.4 They are synthesized by osteoblasts as 400–500 amino acid peptides, each consisting of a leader sequence, a propeptide, and a mature osteoinductive domain at the carboxy-terminal.12 The mature domain of each BMP contains a region of 7 conserved cysteine amino acids, 6 of which are involved in forming a characteristic structural motif—a cysteine knot with 2 fingers, like double-stranded sheets.
The cysteine knot (as shown in Figure 1) affords significant stability to the mature protein. The molecules appears to be biologically active homodimers linked by a disulphide bond between the seventh conserved cysteine residues.12 Before secretion from the osteoblast, BMP molecules are cleaved between the propeptide and mature regions to release the active BMP dimer.12 There are 23 different members of TGF-β family, and their functions are described in brief in the Table.
BMPs bind and initiate a cell signal through a transmembrane receptor complex. Investigators have distinguished 2 basic types of BMP receptors: type I and type II. They are classified according to their molecular weights, the presence of a glycine/serine-rich domain (located on the type-I receptor), and the ability to bind a particular ligand.8,25 Binding of the BMP to at least one type-I and one type-II receptor is required to activate the BMP signal.33,34
Three type-I receptors, Alk2, Alk3 (BRIa), and Alk6 (BRIb), and 3 type-II receptors, BRII, ActRII, and ActRIIB have been identified.25,33 There is also an alternative splice variant of BRII, which lacks most of the C-terminal tail.35,36 The structure of the receptors37 reveals a typical pattern of 4 disulfide bridges that seem to occur in all the known receptor chains of the TGF-β superfamily. Additional disulfide bridges and the positions of a few amino acid residues seem to be characteristic for either the type I or the type II receptor proteins.37 In general, the type-I receptors are the high-affinity binding receptors, whereas the type- II receptors are low-affinity receptors.25,38 One exception is growth developing factor-9, which binds BRII with high affinity and ActRII with low affinity.39 The type- II receptors are continuously active (autophosphorylating) and function upstream of the type-I receptors but cannot independently initiate cell signals.8,40 The type–II receptors activate the type-I receptors.36
BMP receptor binding proteins
Very few proteins have been identified that bind to the BMP receptors. Most important of them is the SMAD family. The receptor-regulated Smads can bind to all type-I receptors and are involved in the Smad signaling cascade.18,43 XIAP is another BMP receptor binding protein that binds TAB1 and therefore may be the link to the TAB1/TAK1 pathway. XIAP belongs to the family of inhibitors of apoptosis. Another BMP receptor binding protein is BRAM1, which is identified by a yeast 2-hybrid screen and binds to Alk3 but not Alk2.41 BRAM1, localized in the cytoplasm is shown to inhibit the latent membrane protein 1 and tumor necrosis factor–α-mediated NF-κ B activation, but not c-jun-N terminal kinase (JNK) activation.44 The FK506 binding protein has also been shown to bind to Alk3 at the GS domain being released upon receptor activation.45
Eight different Smad proteins have been identified in mammals and are classified into 3 subgroups: receptor-regulated Smads (R-Smads), common-partner Smads (co-Smads), and inhibitory Smads (I-Smads).46 Smad proteins possess conserved N-terminal (MH1) and C-terminal (MH2) domains separated by less conserved threonine-, serine-, and proline-linking regions.8,47–49 Smads have L3 and SSXS motifs, which are used for binding to BMPs and phosphorylation, respectively. R-Smads transiently and directly interact with activated type-I receptors and become phosphorylated at SSXS motifs. R-Smads then form heteromeric complexes with co-Smads and Smad4s to translocate into the nucleus, where they activate transcription of various target genes.46 Smad1, Smad5, and presumably Smad8 are thought to act as specific R-Smads for BMPs. Human Smadl is activated and directly phosphorylated on a serine residue by type-I BMP receptors.50 After activation, Smadl associates with Smad4 as a hetero-oligomer that rapidly accumulates in the nucleus of the cell and may play a role in bone formation.48 Smad1 and Smad5 have been well-characterized and implicated in BMP-2 and BMP-4 signaling in mammals. Smad8 is closely related to Smad1 and Smad5 in amino acid sequence. Smad2 and Smad3 act in the TGF-β/activin pathway.
Mechanism of BMPs
The binding of BMP to its cellular receptors follows a mechanism different from that established for TGF-βs or activins.36 The BMP receptors have a more flexible oligomerization pattern compared with TGF-β receptors, which are homodimeric. In live cells, a certain level of preexisting heteromeric and homodimeric receptors are present in the absence of the BMP-2 ligand.
BMP signaling is initiated by binding of ligand to type I and type II BMP receptors at the cell surface.53 Ligand-induced receptor activation initiates 2 types of signal transduction pathways: (1) the canonical Smad pathway in which receptor-specific Smad1, 5, and 8 are activated, form complexes with the common partner Smad 4, and translocate into the nucleus to regulate the transcription of target genes54 ; and (2) nonorthodox pathways that include mitogen-activated protein kinase (MAPK) pathways, such as the p38 via TAB1/TAK1 pathway, JNK pathway, Ras/MAPK/AP1 pathway, JAK–STAT pathway, Ca2+/calmodulin pathway, and extracellular signal-regulated kinase (ERK) pathways.53,55 The downstream targets of BMPs are runt-related transcription factor 2 (RUNX2) and other osteoblast-related transcription factors, such as DLX5 and osterix.56,57 RUNX2 (the osteoblast-specific product of the Cbfa1 gene) is a runt domain-containing transcription factor that is essential for osteoblast differentiation and bone formation. Cbfa1 is also the locus of cleidocranial dysplasia, an autosomal dominant disorder associated with defects in the cranial and appendicular skeleton.58,59 Overexpression of RUNX2 in mesenchymal cell lines and primary cultures of marrow stromal cells induces osteoblast-specific gene expression and mineralization.60 BMP-2 and dexamethasone regulate alkaline phosphate transcription by regulating osteogenic transcription factors, such as RUNX2, osterix, and the homeobox genes Dlx5 and Msx2, via Smad- or glucocorticoid receptor–dependent signaling pathways.54
Antagonists of BMPs
The presence of antagonists is necessary to control the level of BMPs. We know numerous antagonists for BMPs.61 Among them are noggin and chordin, which also play a crucial role in the dorsal ventral axis formation.62,63 Another one is follistatin, which is expressed during gastrulation.64 Cerberus and gremlin are other examples of BMP antagonists.65,66 All of these antagonists were shown to bind to BMP-2, BMP-4, and BMP-7.61 Dan, another member of the BMP antagonists, can bind BMP-2 in vitro, but experiments in Xenopus indicate that Dan does not affect the physiological function of BMP-2, BMP-4, and BMP-7.61,66 Another set of antagonists, sclerostatin and chordin-like, were found to bind to BMP-5 and BMP-6.41,61
Intracellular Signaling Pathways
Smad signaling pathway
R-Smads transiently associate with type-I receptors and undergo direct phosphorylation at their C-terminal SSXS motif.67,68 Activation of type-I receptor by type II receptor is required for direct interaction between type-I receptor and R-Smads.67 R-Smads are released rapidly from the type-I receptor after phosphorylation to interact with co-Smads. After interaction, the resulting hetero-oligomeric complexes translocate into the nucleus to regulate the transcription of various target genes. In the nucleus, Smad proteins exert transcriptional activity through direct binding to DNA and through association with other DNA-binding proteins.69,70 Ligand stimulation promotes R-Smads to form homo- or hetero-oligomers that are composed of R-Smads alone or together with co-Smads.41,67,71 The type-I receptors phosphorylate the MH2 domain (Smad homology domain) of Smads 1, 5, and possibly 8. Smad6 may block the phosphorylation cascade by binding the type-I receptor. After phosphorylation, Smads either bind to Smad4 and translocate to the nucleus or bind to Smad6, where the signal is stopped. The Smad complex may directly or indirectly initiate transcription of the osteoblast-specific factor-2 gene, which is shown in Figure 2.
Inhibition of Smad signaling
In the absence of ligand, I-Smads are located mainly in the nucleus. They are exported rapidly into the cytoplasm after ligand stimulation.72 I-Smads can antagonize the BMP signaling pathway by interacting with activated type-I receptors and thereby preventing access of R-Smads to the type-I receptor. Smad6 can inhibit signaling by competing with Smad4 for heteromeric complex formation with activated Smad1.73 A third mechanism has been described where Smad7 interacts with Smurf and Smurf2.74 This family of enzymes contain HECT catalytic domains characteristic of E3-ubiquitin ligases and modulate Smad proteins for proteosomal and lysosomal degradation.75 Expression of I-Smads is induced by various extracellular stimuli, including epidermal growth factor (EGF), TGF-β1, activin, and BMP-7 as well as mechanical stresses such as laminar shear stress in vascular endothelial cells.76 Recently, a new class of protein AMSH was discovered that inhibits the antagonistic effects of Smad6.41
Map kinase pathway activation via TAB1/TAK1 pathway
TAB1 (Tak binding protein) is able to bind to p38-α and is activated by autophosphorylation.77 TAK1 (transforming growth factor β–activated kinase 1) is a Map kinase kinase and is activated by BMP-4 and BMP-2 stimulation. Evidence suggests that the activation of the p38 pathway by BMP-2 or BMP-4 is due to the activation of TAK1/TAB1.78 BMP-2 and BMP-4 are also known to play a role in the apoptosis of cells.79,80 A summary of BMP receptor signaling pathways is shown in Figure 3.
Inhibition of TAK1/TAB1 pathway
Expression of Smad6 and Smad7 drastically inhibits BMP-2-induced neurite outgrowth and activation of p38 kinase. This demonstrates that Smad6 and Smad7 have an inhibitory effect on the TAK1-p38 kinase pathway.81 Smad6 physically interacts with TAK1. This was confirmed in a COS7 cell expression system and in MH60/Smad6 transfectant clones, where endogenous TAK1 was found to bind to Smad6.41
BMP-2 stimulation of osteoblasts leads to the activation of RAS and ERK. ERK is important for the upregulation of fibronectin and osteopontin. It has been suggested that the ERK activity can be modulated by Smads.82,83 Further investigation of the direct linkage of RAS to the BMP receptors is needed.
Smads and signal transducers and activators of transcription (STATs) are referred to as ‘‘fast-track'' signaling molecules because members of both families of proteins can directly transduce a signal from the plasma membrane to the gene. The leukemia inhibitory factor (LIF) acts through the gp130 receptor and STAT3. There is evidence that cooperative signaling between LIF and BMP-2 induces fetal neural progenitor cells to differentiate as astrocytes.84 It has been shown that interferon-γ, acting via Janus kinase (JAK) 1 and STAT1, induces the expression of Smad7.85
The interleukin-6 (IL-6) family of cytokines is also known to influence the differentiation of osteoblasts.86 The binding of IL-6 to its receptor activates JAKs.87 Activated JAKs phosphorylate STAT3.88 Activated STAT3 enters the nucleus and regulates the transcription of multiple genes that regulate cell proliferation and differentiation.
Calmodulin is involved in a wide range of diverse cellular processes such as cell cycle control, cell motility, smooth muscle contraction, and intercellular signaling. Calmodulin can bind Smads1–4 in a calcium-dependent manner; however, the effect on TGF-β signaling is uncertain.89
Co-activators/co-repressors of BMP signaling
Smad1, Smad5, and Smad8 can bind to DNA through their MH1 domain.90,91 Co-factors are needed for activation of the target genes. Co-factors are p300 or CBP, which can bind to the MH2 domain of Smad1, Smad4, or Smad5 and can activate transcription through their histone acetylase activity.92,93 Ski and Tob are co-repressors of transcription. Ski binds to the MH2 domain of Smad1, Smad4, and Smad5 and represses the transcription by recruiting histone acetylases.94 Tob is induced by BMP signaling and forms a negative feedback loop. Tob can bind to Smad1, Smad5, and Smad8 and can inhibit Smad-dependent transcription.95 SIP1 was isolated because of its binding to the MH2 domain of Smad1. SIP1 also contains several zinc finger clusters; its function is unknown, but it has been speculated that it might act in BMP signaling as a co-repressor.
Transcription factors involved in BMP signaling
Crosstalk between the BMP and the TGF-β-related signaling pathways
A splice variant of Smad8, Smad8B, lacks the C-terminal SSXS motif. Smad8B associates specifically with Smad8 and Smad4 in the cytoplasm; however, these complexes do not translocate to the nucleus, therefore, do not inhibit BMP signaling mediated by Smad8.98 The Erk–MAPK pathway is activated by peptide growth factors, including epidermal growth factor (EGF) and hepatocyte growth factor. Complexes formed by ERK phosphorylated R-Smads and Smad4 cannot translocate to the nucleus, which results in inhibition of signaling by BMPs.99 It has been shown that EGF can oppose the BMP-2 dependent induction of osteogenic differentiation and fibroblastic growth factor can oppose the ability of BMP-4 to induce interdigital apoptosis during digit formation.100,101
BMP-2 increases the level of β-catenin in the nucleus of preosteoblastic cells and induces the expression of Wnt15, 3a, 5b,102,103 which suggests the existence of an interconnection between Wnt and BMP-2 signaling. Wnt proteins constitute a family of secreted cysteine-rich glycosylated proteins. They are involved in a large variety of modeling and remodeling processes, including cell polarity, cell differentiation, and cell migration.104,105 Secreted Wnt's bind to and activate receptor complexes consisting of the frizzled family of G-protein coupled receptors and the low-density lipoprotein receptor-related protein 5 and 6.92 In the canonical Wnt/β-catenin pathway (Figure 4), activation of receptors results in the stabilization of β-catenin and its subsequent translocation into the nucleus where, in concert with such transcription factors as Tcf or Lef, it drives the transcription of target genes.106
Clinical Uses of BMPs
Successful bone regeneration mediated by biofactors will change the clinical management of musculoskeletal disorders and aid in fracture healing, spinal fusion, and dental bone grafting and implant placement.76 BMPs are multifunctional proteins; they stimulate proteoglycan synthesis in chondroblasts, alkaline phosphatase activity, the C-AMP response to parathyroid hormone, type I collagen synthesis in osteoblasts, chemotaxis of monocytes, and differentiation of neural cells. BMPs are also known to play critical roles in the morphogenesis during embryogenesis4 and are involved in recruiting the pluripotent mesenchymal stem cells and their development into osteoblasts, which later on makes the stable osteocytes. This process of osteocyte development is schematically presented in Figure 5.
BMPs and other growth differentiation factors are used in situations such as the healing of large bone defects (eg, after tumor excision) or osteolytic defects with polyethylene wear. Here an important concern has been expressed regarding the effect of BMPs on remaining tumor cells after resection.107 BMP-2 has been shown extensively to have the ability to improve or accelerate fracture healing.108–110 Recombinant human BMP-2 is highly osteoinductive. In vitro studies show that mesenchymal stem cells incubated with rhBMP-2 have increased alkaline phosphatase activity and undergo matrix mineralization.111 The bone formed has exactly the same composition as bone elsewhere in the body.12,112
The clinical uses of BMPs include spinal fusion, treatment of long bone defects and non-unions, dental and periodontal tissue engineering, craniofacial defects and diseases, fracture repair, improvement of osteointegration with metallic implants, musculoskeletal reconstructive surgery, and tendon and ligament reconstruction.113
Spinal fusions consist of nearly half of all grafting surgery. Furthermore, failure rates of up to 35% have been reported. Thus, there is interest in using rhBMPs to accelerate healing in patients with disk degenerative disease, removing the need for autograft harvesting and reducing morbidity. It may be more reliable and produce fusion of spinal vertebrae in less time. Additionally, it may reduce the need for the implantation of spinal rods and screws.
In periodontal and dental tissue engineering, rhBMPs are known to induce differentiation of pulp stem cells into odontoblasts and promote the regeneration of pulp tissue and teeth. Because the pulp is an organ known to have tremendous regenerative abilities, tissue engineering has been considered a promising approach for diverse clinical cases, such as caries, pulpitis, and apical periodontitis.114 In dentistry, BMPs have been tested in periodontal (regeneration of lost bone tissue due to periodontal disease), implant (increase in bone volume for placement of implants, maxillary sinus augmentation), and restorative endodontic (pulpotomies) procedures.115 The ultimate goal in dental tissue engineering using BMPs is achieving a complete restoration of the physiological, structural, and mechanical integrity of the native dentine–pulp complex, including nerve and vascular regeneration.113,114
BMP-2 and BMP-7 are superior to autologus bone grafting in patients with known risk factors.103 An autograft requires a second surgical site, which increases operative time, risk of morbidity, and patient pain and recovery time. The rate of infection and osteomyelitis is reduced in patients with use of BMPs compared with autologus bone grafting,116 and the donor-site morbidity associated with the harvesting of the bone graft is avoided.117
Another important issue to consider in the use of BMPs in humans is their side-effect profile.109 BMPs are reported to be safe if they are used appropriately and precisely. Reported side effects include local erythema and swelling, heterotopic ossification, and immune response.106,109 Antibodies to rhBMP-2 and the bovine collagen carrier have been reported to occur in approximately 6%9 and 5%–20%9 of patients with low titers and no consequences. Critical issues to consider include the potential risk that BMPs will induce heterotopic bone formation, especially when implanted adjacent to neural tissues. Cautions include use in children, pregnant women, and immunocompromised patients which represent a vast field to be explored.106
An unforeseen issue regarding BMPs that has come to light in recent years is their role in osteoclast activation and formation. When applied to an injury site, BMPs stimulate the osteoblast lineage and initiate the release of factors that promote the rapid production of osteoclasts. Osteoclasts are formed before osteoblasts. Therefore, large doses of BMP may lead to a wave of resorption that precedes the appearance and effect of the osteoblasts.110
Delivery Methods of BMPs
In the Western world, an estimated 5%–10% of all bone fractures show deficient healing, leading to delayed union or non-union and causing significant morbidity and psychological stress to the patients and elevated costs to society.107 The main role of a delivery system for BMPs is to retain the growth factors at the site of injury for a prolonged time.107 For this requirement to be satisfied an intense need for the ideal carrier is necessary.
An ideal carrier should possess the following characteristics: ability to induce optimal inflammatory responses, adequate porosity to allow the infiltration of cells, and ability to form blood vessels in the newly formed bone. It should also be biodegradable to allow the formation of an interface with the surrounding biological tissue or for complete invasion of healed tissues. The carrier should protect the BMPs from premature degradation and maintain its bioactivity while releasing the protein in a time- and space-controlled way to promote the formation of new bone at the treatment site. Finally, carriers should be conveniently sterilizable, easy to handle, stable over time, easy to store, and suitable for commercial production, thereby allowing for large-scale production and approval by regulatory authorities.113
Retention of BMP at the surgical site is affected by many parameters, such as the interaction between different biomaterials and influence of pH, temperature, porosity, and salt concentrations. Current applications include rhBMPs loaded in delivery systems made of synthetic or natural polymers and the differentiation of transplanted stem cells from the patient with rhBMPs for later body implantation.113
Extremes in release profiles, such as long low-amount release of BMPs or initial burst of BMPs are known to not be beneficial to bone formation or healing.113 Some researchers still believe that if BMPs can be generated naturally in any way inside the body at the site of injury or surgery, this would eliminate the need for a different carrier system. Anecdotal reports suggest that use of steroids, parathyroid hormones, and other substances can increase the production of BMPs in vivo. Such information provides the opportunity to explore the possible use of these substances in promoting BMP levels inside the body.
Future Challenges: A Global Perspective
Bone repair and regeneration with BMPs are ushering in a new era of orthopedics and dentistry. The past 10–15 years have seen practical demonstration of bone repair in a series of animal studies and subsequently in clinical trials. However, despite the significant evidence of potential for bone healing, future clinical investigations will be needed to redefine such variables as dose, scaffold, and route of administration. Sometimes impressive results of animal models are difficult to replicate in humans, perhaps because of the species-specific dose response and differences in biological behavior of the various species; thus, understanding the regulation between BMPs and BMP inhibitors may be a key issue. Moreover, different uses or treatment sites may require different dosages.9
Clearly, the use of BMPs in dentistry and orthopedics is still in its nascent stage, but the latest trials in humans suggest that an exciting and promising future will unfold in the development of novel tissue-engineering products for a wide range of clinical situations. It is possible that a cocktail of different BMPs with simultaneous or sequential release would be the most desirable approach to clinical uses, instead of a single stimulus or molecule.114 Raiche and Puleo115 have already explored the sequential release of rhBMP-2 in combination with insulin-like growth factor-1. Nevertheless, in the near future the emergent advances with recombinant production of BMPs will help researchers obtain larger amounts of bioactive rhBMPs, which could be used for tissue-engineering research and the development of novel products.
bone morphogenic protein
epidermal growth factor
extracellular signal-related kinase
c-jun-N terminal kinase
leukemia inhibitory factor
LDL receptor related protein
mitogen-activated protein kinase
runt-related transcription factor 2
signal transducer and activator of transcription
transforming growth factor beta