Cell responses to bone morphogenetic proteins (BMP) depend on the expression and surface localisation of transmembrane receptors BMPR-IA, -IB and -II. The present study shows that all three antigens are readily detected in human bone cells. However, only BMPR-II was found primarily at the plasma membrane, whereas BMPR-IA was expressed equally in the cytoplasm and at the cell surface. Notably, BMPR-IB was mainly intracellular, where it was associated with a number of cytoplasmic structures and possibly the nucleus. Treatment with transforming growth factor beta1 (TGF-beta1) caused rapid translocation of BMPR-IB to the cell surface, mediated via the p38 mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) pathways. The TGF-beta1-induced increase in surface BMPR-IB resulted in significantly elevated BMP-2 binding and Smad1/5/8 phosphorylation, although the receptor was subsequently internalised and the functional response to BMP-2 consequently down-regulated. The results show, for the first time, that BMPR-IB is localised primarily in intracellular compartments in bone cells and that TGF-beta1 induces rapid surface translocation from the cytoplasm to the cell surface, resulting in increased sensitivity of the cells to BMP-2.

Formation of highly organized dental hard tissues is a complex process involving sequential and ordered deposition of an extracellular scaffold, followed by its mineralization. Odontoblast and ameloblast differentiation involves reciprocal and sequential epithelial-mesenchymal interactions. Similar to early tooth development, various Bmps are expressed during this process, although their functions have not been explored in detail. Here, we investigated the role of odontoblast-derived Bmp2 for tooth mineralization using Bmp2 conditional knockout mice. In developing molars, Bmp2LacZ reporter mice revealed restricted expression of Bmp2 in early polarized and functional odontoblasts while it was not expressed in mature odontoblasts. Loss of Bmp2 in neural crest cells, which includes all dental mesenchyme, caused a delay in dentin and enamel deposition. Immunohistochemistry for nestin and dentin sialoprotein (Dsp) revealed polarization defects in odontoblasts, indicative of a role for Bmp2 in odontoblast organization. Surprisingly, pSmad1/5/8, an indicator of Bmp signaling, was predominantly reduced in ameloblasts, with reduced expression of amelogenin ( Amlx), ameloblastin ( Ambn), and matrix metalloproteinase ( Mmp20). Quantitative real-time polymerase chain reaction (RT-qPCR) analysis and immunohistochemistry showed that loss of Bmp2 resulted in increased expression of the Wnt antagonists dickkopf 1 ( Dkk1) in the epithelium and sclerostin ( Sost) in mesenchyme and epithelium. Odontoblasts showed reduced Wnt signaling, which is important for odontoblast differentiation, and a strong reduction in dentin sialophosphoprotein ( Dspp) but not collagen 1 a1 ( Col1a1) expression. Mature Bmp2-deficient teeth, which were obtained by transplanting tooth germs from Bmp2-deficient embryos under a kidney capsule, showed a dentinogenesis imperfecta type II-like appearance. Micro-computed tomography and scanning electron microscopy revealed reduced dentin and enamel thickness, indistinguishable primary and secondary dentin, and deposition of ectopic osteodentin. This establishes that Bmp2 provides an early temporal, nonredundant signal for directed and organized tooth mineralization.

Interleukin 17 (IL-17) stimulates the osteogenic differentiation of progenitor cells in vitro through a synergy with bone morphogenetic protein (BMP)-2. This study investigates whether the diverse responses mediated by IL-17 in vivo also lead to enhanced BMP-2-induced bone formation. Since IL-17 is known to induce osteoclastogenesis, we studied the interactions between IL-17 and BMP-2 in ceramic scaffolds either or not carrying a coating with the bisphosphonate zoledronic acid (ZOL). Histological evaluation revealed that IL-17 alone did not induce any osteoclasts at day 10. On the other hand, BMP-2 clearly stimulated early tissue ingrowth and osteoclastogenesis. Both of these processes were blocked in presence of ZOL. IL-17 signaling restored early vascularized connective tissue formation and osteoclastogenesis induced by BMP-2 in ZOL-coated scaffolds. After 12 weeks, the bone volume induced by co-delivery of BMP-2 and IL-17 was doubled as compared to that induced by BMP-2 alone. We conclude that IL-17 has osteo-stimulatory effects through a synergy with bone-inductive BMP-2. Although local and single application of IL-17 does not mediate osteoclast formation, it could promote other processes involved in bone formation such as connective tissue ingrowth. The use of IL-17 may contribute to the development of improved bone graft substitutes.

The aim of the study was to investigate the effect of a sustained release of bone morphogenetic protein2 (BMP-2) incorporated in a polymeric implant coating on bone healing. In vitro analysis revealed a sustained, but incomplete BMP-2 release until Day 42. For the in vivo study, the rat tibia osteotomy was stabilized either with control or BMP-2 coated wires, and the healing progress was followed by micro computed tomography (µCT), biomechanical testing and histology at Days 10, 28, 42 and 84. MicroCT showed an accelerated formation of mineralized callus, as well as remodeling and an increase of mineralized/total callus volume (p=0.021) at Day 42 in the BMP-2 group compared to the control. Histology revealed an increased callus mineralization at Days 42 and 84 (p=0.006) with reduced cartilage at Day 84 (p=0.004) in the BMP-2 group. Biomechanical stiffness was significantly higher in the BMP-2 group (p=0.045) at Day 42. In summary, bone healing was enhanced after sustained BMP-2 application compared to the control. Using the same drug delivery system, but a burst release of BMP-2, a previous published study showed a similar positive effect on bone healing. Distinct differences in the healing outcome might be explained due to the different BMP release kinetics and dosages. However, further studies are necessary to adapt the optimal release profiles to physiological mechanisms.

The use of the FDA-approved osteoinductive growth factor BMP2 is widespread for bone regeneration. However, its clinical application has been hindered by limitations in cell permeability and a short half-life in circulation. To address this issue, we have developed a modified version of BMP2, referred to as Cell Permeable (CP)-BMP2, which possesses improved cell permeability. CP-BMP2 incorporates an advanced macromolecular transduction domain (aMTD) to facilitate transfer across the plasma membrane, a solubilization domain, and recombinant human BMP2. Compared to traditional rhBMP2, CP-BMP2 exhibits enhanced cell permeability, solubility, and bioavailability, and activates Smad phosphorylation through binding to BMP receptor 2. The effectiveness of CP-BMP2 was evaluated in three animal studies focusing on bone regeneration. In the initial study, mice and rabbits with critical-size calvarial defects received subcutaneous (SC) injections of CP-BMP2 and rhBMP2 (7.5 mg/kg, 3 injections per week for 8 weeks).Following 8 weeks of administration, CP-BMP2 demonstrated a remarkable 65 % increase in bone formation in mice when compared to both the vehicle and rhBMP2. Moreover, rabbits exhibited faster bone formation, characterized by a filling pattern originating from the center. In a subsequent study involving injured horses, hind limb bones treated with CP-BMP2 exhibited an 85 % higher bone regeneration rate, as evidenced by Micro-CT results, in contrast to horses treated with the vehicle or rhBMP2 (administered at 150 μg/defect, subcutaneously, once a week for 8 weeks, without a scaffold). These results underscore the potential of CP-BMP2 to facilitate rapid and effective healing. No noticeable adverse effects, such as ectopic bone formation, were observed in any of the studies. Overall, our findings demonstrate that CP-BMP2 holds therapeutic potential as a novel and effective osteogenic agent.

The use of growth factors for the regeneration of soft and hard tissues has been utilized extensively in dental medicine over the past decade. Recently our group found that recombinant human bone morphogenetic protein 9 (rhBMP9) was more osteopromotive than recombinant human bone morphogenetic protein 2 (rhBMP2) when combined with a deprotenized bovine bone mineral bone grafting material. The aim of the present in vitro study was to evaluate the regenerative potential of an absorbable collagen sponge(ACS) specifically designed for extraction socket healing loaded with rhBMP9 when compared to rhBMP2. The adsorption and release kinetics of rhBMP2 and rhBMP9 were first investigated by enzyme-linked immunosorbent assay quantification. Then, the cellular effects of stromal cell line (ST2) preosteoblasts were investigated utilizing four groups including rhBMP2 and rhBMP9 at both low(10 ng/ml) and high(100 ng/ml) concentrations loaded onto ACS. Cellular attachment(8 hours) and proliferation(1, 3, and 5 days) as well as osteoblast differentiation were investigated by real-time polymerase chain reaction (PCR) at 3 and 14 days, alkaline phosphatase (ALP) activity at 7 days, and alizarin red staining at 14 days. ACS fully adsorbed both rhBMP2 and rhBMP9 that were slowly released up to 10 days. Although neither rhBMP2 nor rhBMP9 had any effects on cell attachment or proliferation, pronounced effects were observed on osteoblast differentiation. ALP activity was increased seven-fold with rhBMP2-high, whereas a marked 10-fold and 20-fold increase was observed with rhBMP9-low and high loaded to ACS, respectively. Furthermore, mRNA levels of collagen1, ALP, bone sialoprotein, and osteocalcin were all significantly higher for rhBMP9 when compared to control or rhBMP2 groups. Alizarin red staining further confirmed that rhBMP9-low and high demonstrated marked increases in mineralization potential when compared to rhBMP2-high. The results demonstrate the marked effect of rhBMP9 on osteoblast differentiation when combined with ACS in comparison to rhBMP2 at doses as much as 10 times lower. Further in vivo studies are necessary to investigate whether the regenerative potential is equally as potent.

The present study investigated the synergistic effect of co‑administering fibroblast growth factor‑2 (FGF‑2) and bone morphogenetic protein‑2 (BMP‑2) on osteoblastic differentiation in C2C12 cells and in rats. C2C12 murine myoblast cells represent a well‑accepted in vitro model system to study the ability of BMP‑2 to alter cell lineage from the myogenic to the osteogenic phenotype. The osteoblastic differentiation potency was determined by alkaline phosphatase (ALP) and Alizarin red S staining. ALP activity and calcium concentrations were colorimetrically measured. Simultaneous administration of 4 µg/ml recombinant human BMP‑2 with 2 ng/ml FGF‑2 markedly enhanced ALP activity (an early marker of osteogenesis) of C2C12 cells. This combination also increased extracellular signal‑regulated kinase1/2 mitogen activated protein kinase signaling that is involved in the promoting effect of FGF‑2 on BMP‑2‑induced osteoblastic differentiation in C2C12 cells. Calcium deposition (a late marker of osteogenesis) and the expression of CD34 (a marker of new vessels) were promoted optimally by simultaneous local sustained administration of FGF‑2 and BMP‑2 using collagen and chitosan‑coated antigen‑extracted porcine cancellous implants in a rat ectopic implantation model. The synergistic effects of a combination of BMP‑2 and FGF‑2 may have potential for bone regenerative therapeutics.

Bone Morphogenetic Protein-2 (BMP-2) may offer the potential to enhance allograft-host osseous union in limb-salvage surgery following osteosarcoma resection. However, there is concern regarding the effect of locally applied BMP-2 on tumor recurrence and metastasis. The purpose of this project was to evaluate the effect of exogenous BMP-2 on osteosarcoma migration and invasion across a panel of tumor cell lines in vitro and to characterize the effect of BMP-2 on pulmonary osteosarcoma metastasis within a xenograft model.

Lowering the efficacious dose of bone morphogenetic protein-2 (BMP-2) for the repair of critical-sized bone defects is highly desirable, as supra-physiological amounts of BMP-2 have an increased risk of side effects and a greater economic burden for the healthcare system. To address this need, we explored the use of heparan sulfate (HS), a structural analog of heparin, to enhance BMP-2 activity. We demonstrate that HS isolated from a bone marrow stromal cell line (HS-5) and heparin each enhances BMP-2-induced osteogenesis in C2C12 myoblasts through increased ALP activity and osteocalcin mRNA expression. Commercially available HS variants from porcine kidney and bovine lung do not generate effects as great as HS5. Heparin and HS5 influence BMP-2 activity by (i) prolonging BMP-2 half-life, (ii) reducing interactions between BMP-2 with its antagonist noggin, and (iii) modulating BMP2 distribution on the cell surface. Importantly, long-term supplementation of HS5 but not heparin greatly enhances BMP-2-induced bone formation in vitro and in vivo. These results show that bone marrow-derived HS effectively supports bone formation, and suggest its applicability in bone repair by selectively facilitating the delivery and bioavailability of BMP-2.

Primordial follicles (PF) are formed when somatic cells differentiate into flattened pregranulosa cells, invaginate into the oocyte nests and encircle individual oocytes. We hypothesize that BMP2 regulates PF formation by promoting the transition of germ cells into oocytes and somatic cells into pregranulosa cells. E15 hamster ovaries were cultured for 8 days corresponding to postnatal day 8 (P8) in vivo, with or without BMP2, and the formation of PF was examined. BMP2 was expressed in the oocytes as well as ovarian somatic cells during development. BMP2 exposure for the first two days or the last two days or the entire 8 days of culture led to increase in PF formation suggesting that BMP2 affected both germ cell transition and somatic cell differentiation. Whereas an ALK2/3 inhibitor completely blocked BMP2-induced PF formation, an ALK2-specific inhibitor was partially effective, suggesting that BMP2 affected PF formation via both ALK2 and ALK3. BMP2 also reduced apoptosis in vitro. Further, more meiotic oocytes were present in BMP2 exposed ovaries. In summary, the results provide the first evidence that BMP2 regulates primordial follicle formation by promoting germ cell to oocyte transition and somatic cell to pre-granulosa cells formation and it acts via both ALK2 and ALK3.

This Classic Article is a reprint of the original work by Marshall R. Urist and Basil S. Strates, Bone Morphogenetic Protein. An accompanying biographical sketch of Marshall R. Urist, MD is available at DOI 10.1007/s11999-009-1067-4; a second Classic Article is available at DOI 10.1007/s11999-009-1069-2; and a third Classic Article is available at DOI 10.1007/s11999-009-1070-9. The Classic Article is copyright 1971 by Sage Publications Inc. Journals and is reprinted with permission from Urist MR, Strates BS. Bone morphogenetic protein. J Dent Res. 1971;50:1392-1406.

The bone morphogenetic protein (BMP) signaling pathway comprises the largest subdivision of the transforming growth factor (TGFβ) superfamily. BMP signaling plays essential roles in both embryonic development and postnatal tissue homeostasis. Dysregulated BMP signaling underlies human pathologies ranging from pulmonary arterial hypertension to heterotopic ossification. Thus, understanding the basic mechanisms and regulation of BMP signaling may yield translational opportunities. Unfortunately, limited tools are available to evaluate this pathway, and genetic approaches are frequently confounded by developmental requirements or ability of pathway components to compensate for one another. Specific inhibitors for type 2 receptors are poorly represented. Thus, we sought to identify and validate an antibody that neutralizes the ligand-binding function of BMP receptor type 2 (BMPR2) extracellular domain (ECD).

In idiopathic portal hypertension (IPH) typical vascular lesions are present in the branches of the portal vein or in the perisinusoidal area of the liver. Similar histological alterations have been reported in the pulmonary vasculature of patients with idiopathic pulmonary artery hypertension (IPAH). As IPAH is associated with mutations of the bone morphogenetic protein receptor 2 (BMPR2) gene, the aim of this study was to investigate whether this association might also be found in patients with IPH. Twenty-three samples belonging to 21 unrelated caucasian patients with IPH followed in the hepatic haemodynamic laboratory of the Hospital Clinic in Barcelona were included in the study. All patients were studied for the entire open reading frame and splice site of the BMPR2 gene by direct sequencing and multiple ligation probe amplification (MLPA) in order to detect large deletions/duplications. None of the 23 patients had pulmonary artery hypertension. Four patients presented one single nucleotide polymorphism (SNP) in intron 5, four patients had a SNP in exon 12 and a SNP in exon 1 was found in two cases. Two patients had both intron 5 and exon 12 polymorphisms. All SNPs were previously described. Except for these three SNPs, neither mutations nor rearrangements have been identified in the BMPR2 gene in this population. We did not detect mutations or rearrangements in the coding region of the BMPR2 gene in our patients with IPH. These findings suggest that, in contrast to IPAH, mutations in BMPR2 are not involved in the pathogenesis of IPH.

Latexin is the only known carboxypeptidase A inhibitor in mammals. We previously demonstrated that BMP-2 significantly induced latexin expression in Runx2-deficient mesenchymal cells (RD-C6 cells), during chondrocyte and osteoblast differentiation. In this study, we investigated latexin expression in the skeleton and its role in chondrocyte differentiation. Immunohistochemical studies revealed that proliferating and prehypertrophic chondrocytes expressed latexin during skeletogenesis and bone fracture repair. In the early phase of bone fracture, latexin mRNA expression was dramatically upregulated. BMP-2 upregulated the expression of the mRNAs of latexin, Col2a1, and the gene encoding aggrecan (Agc1) in a micromass culture of C3H10T1/2 cells. Overexpression of latexin additively stimulated the BMP-2-induced expression of the mRNAs of Col2a, Agc1, and Col10a1. BMP-2 treatment upregulated Sox9 expression, and Sox9 stimulated the promoter activity of latexin. These results indicate that latexin is involved in BMP-2-induced chondrocyte differentiation and plays an important role in skeletogenesis and skeletal regeneration.

Bone Morphogenetic Protein-2 (BMP-2) is a cysteine rich growth factor expressed in homodimeric form and has a pivotal role in osteochondral development and fracture healing. Recent studies have benefited more from recombinant BMP-2 in osteochondral tissue engineering. Cost-effective and easy production at large scale makes Escherichia coli (E. coli) the first choice for recombinant protein expression programs. However, inclusion body aggregation and refolding process limits production and purification of recombinant BMP-2 in bacterial systems.

Supraphysiologic doses of bone morphogenetic protein-2 (BMP-2) are used clinically to promote bone formation in fracture nonunions, large bone defects, and spinal fusion. However, abnormal bone formation (i.e., heterotopic ossification) caused by rapid BMP-2 release from conventional collagen sponge scaffolds is a serious complication. We leveraged the strong affinity interactions between heparin microparticles (HMPs) and BMP-2 to improve protein delivery to bone defects. We first developed a computational model to investigate BMP-2-HMP interactions and demonstrated improved in vivo BMP-2 retention using HMPs. We then evaluated BMP-2-loaded HMPs as a treatment strategy for healing critically sized femoral defects in a rat model that displays heterotopic ossification with clinical BMP-2 doses (0.12 mg/kg body weight). HMPs increased BMP-2 retention in vivo, improving spatial localization of bone formation in large bone defects and reducing heterotopic ossification. Thus, HMPs provide a promising opportunity to improve the safety profile of scaffold-based BMP-2 delivery.

Recombinant human bone morphogenetic protein-2 (rhBMP-2) is one of the most commonly used osteogenic agents in the craniofacial skeleton. This study reviews the safety and efficacy of rhBMP-2 as applied to craniofacial reconstruction and assesses the level of scientific evidence currently available.

Bone morphogenetic proteins (BMPs) regulate adipogenesis but it is not clear whether they influence regional adipose tissue (AT) development in humans.

Bone morphogenetic proteins (BMPs) play a key role in embryonic differentiation for osteoblast and bone formation. Kielin/chordin-like protein (Kcp) is known to enhance the effects of BMP signaling. Here, we present ALP activity, gene expression, and calcification data demonstrating that Kcp affects the differentiation of C2C12 myoblasts into osteoblasts. We report that the presence of Kcp enhances the ability of BMP-2 to induce the differentiation of C2C12 myoblasts into osteoblasts. Additionally, BMP-2-mediated stimulation of phosphorylated Smad1/5 was apparently enhanced in the presence of Kcp. The present findings may contribute to progression toward the clinical use of BMPs for treatment of bone fracture, osteoarthritis, and other similar conditions.

Bone morphogenetic proteins (BMP) stimulate osteoblast differentiation by signal transduction via three BMP receptors (BMPR-IA, -IB and -II), whereas the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) has been shown to suppress osteoblast differentiation. Although the mechanisms which regulate the BMPR are not yet known, it is possible that they may be negatively controlled by TNF-alpha, thereby inhibiting BMP-induced osteoblast differentiation. To test this hypothesis, we have examined the effects of TNF-alpha on BMPR-IA, -IB and -II expression and the functional consequences of this cytokine on BMPR-mediated functions in human bone cells. The results showed that although TNF-alpha down-regulated BMPR-IA and -II transcripts, it increased the level of BMPR-IB mRNA via a MAPK-dependent pathway. In marked contrast, however, TNF-alpha nevertheless caused marked down-regulation of the expression of the BMPR-IB surface antigen specifically. Moreover, the cytokine-induced decrease in BMPR-IB expression was found to be associated with the concurrent presence of a 'soluble' form of this antigen in supernatants of TNF-alpha-treated cultures. Furthermore, the TNF-alpha-induced loss of BMPR-IB was found to ablate BMP-2-stimulated bone cell functions, including phosphorylation of Smad1/5/8, alkaline phosphatase activity and osteocalcin expression. In conclusion, our study has provided evidence, for the first time, that BMPR can be differentially modulated by TNF-alpha at both the post-transcriptional and post-translational levels, with the TNF-alpha-induced shedding of the BMPR-IB antigen associated with a significantly diminished response to BMP-2 in vitro.