Combined Bone Morphogenetic Protein-2 Research Articles

Is NO the Answer? The Nitric Oxide Pathway Can Support Bone Morphogenetic Protein 2 Mediated Signaling.

The growth factor bone morphogenetic protein 2 (BMP2) plays an important role in bone development and repair. Despite the positive effects of BMP2 in fracture healing, its use is associated with negative side effects and poor cost effectiveness, partly due to the large amounts of BMP2 applied. Therefore, reduction of BMP2 amounts while maintaining efficacy is of clinical importance. As nitric oxide (NO) signaling plays a role in bone fracture healing and an association with the BMP2 pathway has been indicated, this study aimed to investigate the relationship of BMP2 and NO pathways and whether NO can enhance BMP2-induced signaling and osteogenic abilities in vitro. To achieve this, the stable BMP reporter cell line C2C12BRELuc was used to quantify BMP signaling, and alkaline phosphatase (ALP) activity and gene expression were used to quantify osteogenic potency. C2C12BRELuc cells were treated with recombinant BMP2 in combination with NO donors and substrate (Deta NONOate, SNAP & L-Arginine), NOS inhibitor (LNAME), soluble guanylyl cyclase (sGC) inhibitor (LY83583) and activator (YC-1), BMP type-I receptor inhibitor (LDN-193189), or protein kinase A (PKA) inhibitor (H89). It was found that the NOS enzyme, direct NO application, and sGC enhanced BMP2 signaling and improved BMP2 induced osteogenic activity. The application of a PKA inhibitor demonstrated that BMP2 signaling is enhanced by the NO pathway via PKA, underlining the capability of BMP2 in activating the NO pathway. Collectively, this study proves the ability of the NO pathway to enhance BMP2 signaling.

Peroxisome Proliferator-Activated Receptor-γ Knockdown Impairs Bone Morphogenetic Protein-2–Induced Critical-Size Bone Defect Repair

The Food and Drug Administration-approved clinical dose (1.5 mg/mL) of bone morphogenetic protein-2 (BMP2) has been reported to induce significant adverse effects, including cyst-like adipose-infiltrated abnormal bone formation. These undesirable complications occur because of increased adipogenesis, at the expense of osteogenesis, through BMP2-mediated increases in the master regulatory gene for adipogenesis, peroxisome proliferator-activated receptor-γ (PPARγ). Inhibiting PPARγ during osteogenesis has been suggested to drive the differentiation of bone marrow stromal/stem cells toward an osteogenic, rather than an adipogenic, lineage. We demonstrate that knocking down PPARγ while concurrently administering BMP2 can reduce adipogenesis, but we found that it also impairs BMP2-induced osteogenesis and leads to bone nonunion in a mouse femoral segmental defect model. In addition, invitro studies using the mouse bone marrow stromal cell line M2-10B4 and mouse primary bone marrow stromal cells confirmed that PPARγ knockdown inhibits BMP2-induced adipogenesis; attenuates BMP2-induced cell proliferation, migration, invasion, and osteogenesis; and escalates BMP2-induced cell apoptosis. More important, BMP receptor 2 and 1B expression was also significantly inhibited by the combined BMP2 and PPARγ knockdown treatment. These findings indicate that PPARγ is critical for BMP2-mediated osteogenesis during bone repair. Thus, uncoupling BMP2-mediated osteogenesis and adipogenesis using PPARγ inhibition to combat BMP2's adverse effects may not be feasible.

Controlled release of FGF-2 and BMP-2 in tissue engineered periosteum promotes bone repair in rats

The aim of this study was to prepare chitosan–collagen (CS/COL) scaffolds that could release fibroblast growth factor-2 (FGF-2) and bone morphogenetic protein 2 (BMP-2), and to study the effect of this scaffold on bone repair. By improving the double emulsion/solvent evaporation technique, BMP-2 was encapsulated in poly(lactic acid)–poly(ethylene glycol)–poly(lactic acid) (PELA) microcapsules, to the surface of which FGF-2 was attached. The CS/COL scaffold carrying the microcapsules was prepared by freeze-drying. Periosteum derived cells (PDCs) were extracted and cultured on the scaffolds to study their proliferation and differentiation on the scaffolds. In addition, the effects of the scaffolds were investigated on rats with skull defects by micro-computed tomography and histology. We successfully prepared PELA microcapsules with external adherence to FGF-2 and encapsulated with BMP-2. The CS/COL scaffolds were porous and PDCs adhered, proliferated and underwent osteogenic differentiation on the scaffolds. The sequential release of FGF-2/BMP-2 had better osteogenic efficacy than other groups. Our results suggest that CS/COL scaffolds that bind FGF-2 and BMP-2 in combination with PDCs could be a promising new strategy for tissue engineering periosteum.

BMP-2, PDGF-BB, and bone marrow mesenchymal cells in a macroporous β-TCP scaffold for critical-size bone defect repair in rats

The aim of this work was to study the bone repair induced by bone morphogenetic protein-2 (BMP-2), rat mesenchymal stem cells (rMSCs), and platelet-derived growth factor (PDGF-BB) incorporated in a macroporous beta-tricalcium phosphate (β-TCP) system fabricated by robocasting, and to identify the most beneficial combination in a critical rat calvaria defect. BMP-2 was formulated in microspheres to provide a prolonged, local concentration, whereas PDGF-BB, which acts during the initial stage of defect repair, was incorporated in a thin layer of crosslinked alginate. Approximately 80% of PDGF-BB and 90% of BMP-2 were released into the defect during the first 2 d and 3 weeks, respectively. Histological analyses indicated a minor synergistic effect in the BMP-2-MSC groups. In contrast, significant antagonism was found with combined BMP-2 and PDGF-BB defect treatment. The high-grade repair induced by BMP-2 rules out any advantage from combining BMP-2 with PDGF-BB or MSCs, at least with this scaffold and defect model.

Enhanced osteoinductivity of recombinant human bone morphogenetic protein-2 in combination with epidermal growth factor in a rabbit tibial defect model

This study aims to explore the effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) on bone formation when treated with epidermal growth factor (EGF) using human mesenchymal stem cells (hMSCs) and a rabbit tibial defect model. The rhBMP-2 (250 ng/ml)+EGF (10 ng/ml) group showed higher alkaline phosphatase (ALP) activity, ALP expression, increased calcium amount than rhBMP-2 group. In micro-CT and histology results of animal experiments, the rhBMP-2+EGF group showed more amount of bone bridging compared to the rhBMP-2 group. Among the 8-week groups, the rhBMP-2+EGF group showed significantly higher percent bone volume and trabecular number compared to the rhBMP-2 group. The combined treatment with EGF and rhBMP-2 induced significantly higher bone formation compared to that of rhBMP-2 only in both hMSCs and a rabbit tibial defect model. Therefore, EGF is expected to facilitate bone formation effect of rhBMP-2 when both factors are treated in combination.

Smurf1 Knocked-Down, Mesenchymal Stem Cells and BMP-2 in an Electrospun System for Bone Regeneration

A sandwich-like system, fabricated with electrospun, poly(lactic-co-glycolic-acid) (PLGA) membranes incorporating either human recombinant bone morphogenetic protein 2 (BMP-2) enriched microspheres, rat bone marrow mesenchymal stem cells (rMSC), or rMSC with their Smurf1 (SMAD ubiquitin regulatory factor-1) expression knocked down by means of siRNA (rMSC573) at varying densities was evaluated in a rat calvarial, critical-size defect. The behavior of four membrane varieties, fabricated with different PLGA copolymers, was initially studied in rMSC cultures to decide on optimal membrane degradation and cell proliferation and differentiation characteristics. PLGA75:25 provided the most stable structure, and favored the cell environment. Radiological and histological analyses indicated bone repair in animals treated with the PLGA75:25 bioactivated systems. We found no synergist interaction between BMP-2 and rMSC 8 to 12 weeks postimplantation. By contrast, synergistic defect repair of around 85% was detected after 8 weeks of combined BMP-2 and rMSC573 treatment.

Effects of BMP2 and VEGF165 on the osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells

Bone marrow-derived mesenchymal stem cells (MSCs) are dominant seed cell sources for bone regeneration. Bone morphogenetic proteins (BMPs) initiate cartilage and bone formation in a sequential cascade. Vascular endothelial growth factor (VEGF) is an essential coordinator of extracellular matrix remodeling, angiogenesis and bone formation. In the present study, the effects of the vascular endothelial growth factor 165 (VEGF165) and bone morphogenetic protein 2 (BMP2) genes on bone regeneration were investigated by the lentivirus-mediated cotransfection of the two genes into rat bone marrow-derived MSCs. The successful co-expression of the two genes in the MSCs was confirmed using quantitative polymerase chain reaction (qPCR) and western blot analysis. The results of alizarin red and alkaline phosphatase (ALP) staining at 14 days subsequent to transfection showed that the area of staining in cells transfected with BMP2 alone was higher than that in cells transfected with BMP2 and VEGF165 or untransfected control cells, while the BMP2 + VEGF165 group showed significantly more staining than the untransfected control. This indicated that BMP2 alone exhibited a stronger effect in bone regeneration than BMP2 in combination with VEGF165. Similarly, in inducing culture medium, the ALP activity of the BMP2 + VEGF165 group was notably suppressed compared with that of the BMP2 group. The overexpression of VEGF165 inhibited BMP2-induced MSC differentiation and osteogenesis in vitro. Whether or not local VEGF gene therapy is likely to affect bone regeneration in vivo requires further investigation.

Guiding the osteogenic fate of mouse and human mesenchymal stem cells through feedback system control

Stem cell-based disease modeling presents unique opportunities for mechanistic elucidation and therapeutic targeting. The stable induction of fate-specific differentiation is an essential prerequisite for stem cell-based strategy. Bone morphogenetic protein 2 (BMP-2) initiates receptor-regulated Smad phosphorylation, leading to the osteogenic differentiation of mesenchymal stromal/stem cells (MSC) in vitro; however, it requires supra-physiological concentrations, presenting a bottleneck problem for large-scale drug screening. Here, we report the use of a double-objective feedback system control (FSC) with a differential evolution (DE) algorithm to identify osteogenic cocktails of extrinsic factors. Cocktails containing significantly reduced doses of BMP-2 in combination with physiologically relevant doses of dexamethasone, ascorbic acid, beta-glycerophosphate, heparin, retinoic acid and vitamin D achieved accelerated in vitro mineralization of mouse and human MSC. These results provide insight into constructive approaches of FSC to determine the applicable functional and physiological environment for MSC in disease modeling, drug screening and tissue engineering.

Synergistic effect of Indian hedgehog and bone morphogenetic protein-2 gene transfer to increase the osteogenic potential of human mesenchymal stem cells

IntroductionTo stimulate healing of large bone defects research has concentrated on the application of mesenchymal stem cells (MSCs).MethodsIn the present study, we induced the overexpression of the growth factors bone morphogenetic protein 2 (BMP-2) and/or Indian hedgehog (IHH) in human MSCs by adenoviral transduction to increase their osteogenic potential. GFP and nontransduced MSCs served as controls. The influence of the respective genetic modification on cell metabolic activity, proliferation, alkaline phosphatase (ALP) activity, mineralization in cell culture, and osteogenic marker gene expression was investigated.ResultsTransduction had no negative influence on cell metabolic activity or proliferation. ALP activity showed a typical rise-and-fall pattern with a maximal activity at day 14 and 21 after osteogenic induction. Enzyme activity was significantly higher in groups cultured with osteogenic media. The overexpression of BMP-2 and especially IHH + BMP-2 resulted in a significantly higher mineralization after 28 days. This was in line with obtained quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analyses, which showed a significant increase in osteopontin and osteocalcin expression for osteogenically induced BMP-2 and IHH + BMP-2 transduced cells when compared with the other groups. Moreover, an increase in runx2 expression was observed in all osteogenic groups toward day 21. It was again more pronounced for BMP-2 and IHH + BMP-2 transduced cells cultured in osteogenic media.ConclusionsIn summary, viral transduction did not negatively influence cell metabolic activity and proliferation. The overexpression of BMP-2 in combination with or without IHH resulted in an increased deposition of mineralized extracellular matrix, and expression of osteogenic marker genes. Viral transduction therefore represents a promising means to increase the osteogenic potential of MSCs and the combination of different transgenes may result in synergistic effects.

Bone marrow stromal cells with a combined expression of BMP-2 and VEGF-165 enhanced bone regeneration**All animals used in this study were treated in accordance with the ‘Guiding Principles for Research Involving Animals and Human Beings’ as adopted by The American Physiological Society, and were approved by the Ethics Committee of Shanghai Ninth People's Hospital affiliated to Shanghai JiaoTong University, School

Bone graft substitutes with osteogenic factors alone often exhibit poor bone regeneration due to inadequate vascularization. Combined delivery of osteogenic and angiogenic factors from biodegradable scaffolds may enhance bone regeneration. We evaluated the effects of bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor (VEGF), combined with natural coral scaffolds, on the repair of critical-sized bone defects in rabbit orbits. In vitro expanded rabbit bone marrow stromal cells (BMSCs) were transfected with human BMP2 and VEGF165 genes. Target protein expression and osteogenic differentiation were confirmed after gene transduction. Rabbit orbital defects were treated with a coral scaffold loaded with BMP2-transduced and VEGF-transduced BMSCs, BMP2-expressing BMSCs, VEGF-expressing BMSCs, or BMSCs without gene transduction. Volume and density of regenerated bone were determined by micro-computed tomography at 4, 8, and 16 weeks after implantation. Neovascularity, new bone deposition rate, and new bone formation were measured by immunostaining, tetracycline and calcein labelling, and histomorphometric analysis at different time points. The results showed that VEGF increased blood vessel formation relative to groups without VEGF. Combined delivery of BMP2 and VEGF increased new bone deposition and formation, compared with any single factor. These findings indicate that mimicking the natural bone development process by combined BMP2 and VEGF delivery improves healing of critical-sized orbital defects in rabbits.

Differentiation, engraftment and functional effects of pre-treated mesenchymal stem cells in a rat myocardial infarct model

Background — Mesenchymal stem cells (MSCs) offer a novel therapeutic option in the treatment of acute myocardial infarction. MSCs are able to differentiate into myogenic cells after 5-azacytitdine treatment. However, 5-azacytidine might have genotoxic effects. Recently, it was reported that combined treatment with bone morphogenetic protein-2(BMP-2) and fibroblast growth factor-4(FGF-4) caused cardiac differentiation in non-precardiac mesoderm explants. Therefore, we investigated whether MSCs treated with combined BMP-2 and FGF-4 showed evidence of myogenic differentiation in vitro, and whether these cells resulted in sustained engraftment, myogenic differentiation, and improved cardiac function after implantation in infarcted myocardium.Methods and results — In vitro study: MSCs were treated with BMP-2 + FGF-4 (GF-MSCs) and myogenic phenotype was evaluated immunohistochemically. Cell growth curve was used to compare MSC proliferative capacity between the growth factors and 5-azacytidine treatments. In vivo study: two weeks after coronary artery occlusion, GF-MSCs (n = 15), MSCs (n = 5) labelled with PKH26 were injected into infarcted myocardium. Control animals (n = 5) received a culture medium into the infarcted myocardium.Two weeks after implantation, some engrafted GF-MSCs or MSCs expressed sarcomeric-a-actinin and cardiac myosin heavy chain, as was observed in culture. Echocardiography showed that the GF-MSC group had a better (p < 0.05) left ventricular performance than the other groups.Conclusion — GF-MSCs induced myogenic differentiation in vitro. Moreover, GF-MSCs engrafted into the infarcted myocardium increased myogenic differentiation, prevented dilation of the infarcted region, and eventually improved heart function.

Expression of bone morphogenetic protein-2 messenger ribonucleic acid in cholesteatoma fibroblasts.

The aim of the study was to evaluate the role of bone morphogenetic protein-2 (BMP-2) in the pathology of middle ear cholesteatoma. Middle ear cholesteatoma is a chronic inflammatory disease associated with destruction of the temporal bone and marked by increased expression levels of diverse cytokines. Bone remodeling associated with this disease is mainly caused by the action of osteoclasts. It has been shown that BMP-2 expression is inducible by interleukin 1 in synovial fibroblasts and that BMP-2 in combination with interleukin 1alpha is able to stimulate the formation of osteoclast-like multinucleated cells in co-cultures of osteoblast-like cells and hematopoietic cells. By using Northern hybridizations, we examined the messenger ribonucleic acid expression of BMP-2 in keratinocytes and fibroblasts derived from normal external ear canal skin (EACS) and from cholesteatoma, respectively. We show that normal EACS fibroblasts do not express BMP-2, whereas keratinocytes of both EACS and cholesteatoma origin are positive for the BMP-2 transcript. In contrast to EACS fibroblasts, BMP-2 is clearly expressed in cholesteatoma perimatrix fibroblasts. Incubation of normal fibroblasts with cholesteatoma extracts caused the transcription of BMP-2. Interleukin 1alpha, bacterial endotoxin, or bovine keratin, however, were not able to initiate BMP-2 expression in normal fibroblasts. In view of the above data, it is tempting to speculate that BMP-2 expression might play a role in cholesteatoma pathology.

Combined BMP-2 and FGF-4, but Neither Factor Alone, Induces Cardiogenesis in Non-Precardiac Embryonic Mesoderm

Previous work in this laboratory has shown that endoderm cells in the heart forming region (HFR endoderm) of the chicken embryo induce terminal cardiac differentiation in explanted precardiac mesoderm cells. Immunostaining patterns indicating that HFR endoderm cells expressDrosophiladecapentaplegic (dpp)-like antigens prompted a degenerate polymerase chain reaction (PCR) screen to identify cDNAs in the dpp subgroup of the transforming growth factor-β (TGF-β) family. Among 50 clones of PCR products that have been sequenced, over half have identity with bone morphogenetic protein-2 (BMP-2). No other TGF-β cDNAs have been detected, suggesting that BMP-2 is the major dpp subgroup protein synthesized by HFR endoderm cells. However, BMP-2 protein did not promote survival of either precardiac or non-precardiac mesoderm cells in culture. Whereas FGF-4 supports cardiogenesis in precardiac mesoderm, it did not induce cardiogenesis in non-precardiac mesoderm, although explant viability was maintained. In contrast to the isolated effects of these growth factors, treatment of non-precardiac mesoderm with combined BMP-2 and FGF-4 induced cardiogenesis in the majority of explants, as revealed by the formation of a rhythmically contractile multicellular vesicle that expresses sarcomeric α-actin. These findings suggest that BMP-2 and FGF-4 possess respective differentiative and proliferative activities, the combination of which specifies cells to the cardiac lineage.