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Bone morphogenetic protein-2 (BMP-2) is widely used in orthopedic surgery and bone tissue engineering because of its strong osteogenic activity. However, BMP-2 treatments have several drawbacks and many groups are actively exploring alternatives. Since BMP-6 has been demonstrated to be more osteoinductive, its use, either alone or together with other growth factors, might be an interesting option. In this work, we have compared the effect of BMP-2, BMP-6, or insulin-like growth factor-1 (IGF-1), either alone or in combination. Murine preosteoblasts were treated with 15 nM IGF-1 and/or 6 nM BMP-2 or -6 and the expression of osteogenic marker genes, proliferation, and alkaline phosphatase (ALP) activity in vitro were analyzed. The results showed that IGF-1 greatly enhanced the BMP-induced osteogenic differentiation of these cells in general and that the ALP activity in the cultures was higher when the combination was made with BMP-6 than with BMP-2. Furthermore, we tested the osteogenic potential of these treatments in vivo by loading 25 pmoles of IGF-1 and/or 10 pmoles of BMP-2 or -6 onto absorbable collagen sponges and implanting them into an ectopic bone formation model in rats. This study revealed that only BMP-6 was able to induce bone formation at the used dose and that the addition of IGF-1 contributed to an increase of the mineralization in the implants. Hence, the combination of BMP-6 with IGF-1 might be a better alternative than BMP-2 for orthopedic surgery or bone tissue engineering approaches. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1867-1875, 2017., (© 2017 Wiley Periodicals, Inc.)

Design of new osteoinductive biomaterials to reproduce an optimized physiological environment capable of recruiting stem cells and instructing their fate towards the osteoblastic lineage has become a priority in orthopaedic surgery. This work aims at evaluating the bioactivity of BMP combined with human plasma fibronectin (FN/BMP) delivered in solution or coated onto titanium-hydroxyapatite (TiHA) surfaces. Herein, we focus on the comparison of in vitro osteogenic efficacy in mouse C2C12 pre-osteoblasts of three BMP members, namely: BMP-2, BMP-6 and BMP-7. In parallel, we evaluated the molecular binding strength between each BMP with FN using the Surface Plasmon Resonance (SPR) technology. The affinity of BMPs for FN was found totally different and dependent on BMP type. Indeed, the combination of FN with BMP-2 on TiHA surfaces potentiates the burst of gene-mediated osteogenic induction, while it prolongs the osteogenic activity of BMP-6 and surprisingly annihilates the BMP-7 one. These results correlate with FN/BMP affinity for TiHA, since BMP-6>BMP-2>BMP-7. In addition, by analyzing the osteogenic activity in the peri-implant environment, we showed that osteoinductive paracrine effects were significantly decreased upon (FN/BMP-6), as opposed to (FN/BMP-2) coatings. Altogether, our results support the use of FN/BMP-6 to develop a biomimetic microenvironment capable to induce osteogenic activity under physiological conditions, with minimum paracrine signalization., Statement of Significance: The originality of our paper relies on the first direct comparison of the in vitro osteogenic potential of three osteogenic BMPs (BMP-2, -6 and -7) combined with native human plasma fibronectin delivered in solution or coated by laser transfer onto titanium hydroxyapatite surfaces. We confirm that BMP association with fibronectin enhances the osteogenic activity of BMP-2, -6 and -7, but with essential discrepancies, depending on the BMP member, and in agreement with the affinity of BMPs for fibronectin. Moreover, we bring elements to explain the origin of the BMP-2 medical life-threatening side-effects by analyzing in vitro paracrine effects. Finally, this work supports the alternative use of FN/BMP-6 to induce osteogenic activity under physiological conditions, with minimum side effects., (Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Recent studies indicated a lower osteogenic differentiation potential of adipose tissue-derived stromal cells (ASCs) compared to bone marrow derived mesenchymal stromal cells. The aim of this study was to evaluate the effects of potent combinations of highly osteogenic bone morphogenetic proteins (BMPs) in order to enhance the osteogenic differentiation potential of ASCs. Human ASCs were cultured for 10 days in the presence of osteogenic medium consisting of dexamethasone, ß-glycerophosphate and ascorbat-2-phosphate (OM) supplemented with BMP-2, BMP-6, BMP-9+IGF-2 and BMP-2,-6,-9 (day 1+2: 50 ng/ml, days 3-6: 100 ng/ml, days 7-10: 200 ng/ml). The formation of the osteoblast phenotype was evaluated by quantification of osteoblast-related marker genes using real-time polymerase chain reaction (RT-PCR). Matrix mineralization was assessed by Alizarin Red S staining. Statistical analysis was carried out using the one-way analysis of variance (ANOVA) followed by the Scheffe's post hoc procedure. Osteogenic medium (OM) significantly increased the expression of alkaline phosphatase (ALP) and osteocalcin (p < 0.05) and led to a stable matrix mineralization. Under the influence of BMP-9+IGF-2 and BMP-2,-6,-9 the ALP expression further increased compared to ASCs cultured with OM only (p < 0.01). However, multiple osteogenic markers showed no change or decreased under the influence of OM and BMP combinations (p < 0.05). The current results indicate a restricted osteogenic differentiation potential of ASCs and suggest careful reconsideration of their use in bone tissue engineering applications., (Copyright © 2016 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.)

While only two members of the bone morphogenetic protein subfamily (BMP-2 and -7) are approved to be used in combination with collagen type I in orthopaedic surgery, other BMPs are known to also be highly osteoinductive. Although all the osteogenic BMPs signal through Smad-1/-5/-8 phosphorylation, they show different preferences for the available BMP receptors. In this work we studied the effect of combining two osteogenic BMPs (-2 and -6), which belong to different groups within the subfamily and have different affinities to the existing BMP receptors. Both the growth and in vitro differentiation of MC3T3-E1 mouse preosteoblasts and rat bone marrow-derived mesenchymal stem cells (MSCs) were studied, as well as in vivo ectopic bone formation when the BMPs were intramuscularly implanted in rats with collagen type I sponges as carriers. The results show that these two growth factors have additive effects on the osteoblastic differentiation of cells in vitro and that their combination might be helpful to accelerate in vivo osteogenesis while reducing the amount of each individual BMP used., (© 2015 Wiley Periodicals, Inc.)

A variety of different growth factors, most notably bone morphogenetic proteins (BMPs), have been shown to stimulate the osteogenic differentiation of mesenchymal stromal cells (MSCs) in vitro. Yet, due to the lack of comparative studies it remains unclear which protocol is the most effective in the induction of osteogenesis in MSC cultures. The aim of this study was to compare the most potent growth factors in regard to their osteoinductive potential. Human MSCs were cultured for 10 days in the presence of BMP-2, BMP-6, BMP-9 + IGF-2 and BMP-2, -6, -9 (day 1 + 2: 50 ng/ml; days 3-6: 100 ng/ml; days 7-10: 200 ng/ml). The formation of the osteoblast phenotype was assessed by quantification of osteoblast-related marker genes using reverse transcription polymerase chain reaction (RT-PCR) and alkaline phosphatase (ALP) staining. Matrix mineralization was assessed by alizarin red S and von Kossa staining. Statistical analysis was carried out using the one-way analysis of variance (ANOVA) followed by Scheffe's post hoc procedure. Among the tested growth factors the combination of BMP-2 + BMP-6 + BMP-9 most effectively induced the upregulation of collagen type I, collagen type V, osteocalcin, alkaline phosphatase, RUNX2, BMP-2, osteonectin and DLX5 (p < 0.01) and resulted in a consistent matrix mineralization. The findings suggest the combined addition of BMP-2, BMP-6 and BMP-9 to the osteoinductive culture medium containing dexamethasone, β-glycerophosphate and ascorbate-2-phosphate produces more potent osteoblast differentiation of human MSCs in vitro., (Copyright © 2014 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.)

Aims: Microarray data were analyzed using bioinformatic tools to screen marker genes of human mesenchymal stem cells (hMSC) in response to bone morphogenetic protein 6 (BMP6)., Results: A total of 190 differentially expressed genes were identified. The interaction network was divided into three functional modules. These genes were connected with BMP signaling pathways and regulation of cell processes, while NOG and BMPR2 participated in the transforming growth factor-beta signal pathway. Besides, several related small molecules were acquired., Conclusion: Marker genes in osteogenic responses to BMP6 treatment for hMSC were screened with microarray data along with elaborate function analysis by bioinformatics. NOG and BMPR2 showed potential to become indicators to monitor the directed differentiation of hMSC into osteoblasts, which can be used for bone disease treatment. Moreover, small molecules such as W-13 were retrieved and provided directions for future drug design.

Bone morphogenetic proteins (BMPs) exert an important role in skeletal development, adult bone homeostasis, and fracture healing and have demonstrated clinical utility for bone regeneration. However, BMPs fall short as regenerative agents because high doses need to be used to obtain therapeutic effects. Determining the molecular mechanisms controlling BMP-induced bone formation may lead to the development of more effective BMP-based therapies. To identify kinases mediating BMP-induced osteoblast differentiation, we performed an siRNA screen to find kinases modulating BMP-6-induced alkaline phosphatase (ALP) activity. Surprisingly, although transforming growth factor β (TGF-β) generally is considered to antagonize BMP-induced osteoblast differentiation, C2C12  cells transfected with siRNAs targeting TGF-β receptors displayed reduced BMP-6-induced ALP activity. Furthermore, pharmacologic inhibitors blocking the TGF-β type I receptor impaired BMP-induced ALP activity in KS483 and C2C12 cells and mineralization of KS483 cells. Consistently, costimulation with BMPs and TGF-β further increased expression of osteoblast-specific genes, ALP activity, and mineralization of KS483 cells and primary mesenchymal stem cells compared with BMPs alone. The stimulatory and inhibitory effects of TGF-β were found to depend on timing and duration of the costimulation. TGF-β inhibited BMP-induced activation of a BMP-Smad-dependent luciferase reporter, suggesting that the stimulatory effect of TGF-β is not due to increased BMP-Smad activity. TGF-β also inhibited the BMP-induced expression of the BMP antagonist noggin and prolonged BMP activity. In conclusion, TGF-β, besides acting as an inhibitor, also can, by dampening the noggin-mediated negative-feedback loop, enhance BMP-induced osteoblast differentiation, which might be beneficial in fracture healing., (Copyright © 2011 American Society for Bone and Mineral Research.)

Bone regeneration is required for fracture healing. Various procedures have been used to promote osteogenesis with bone morphogenetic proteins (BMPs). We assessed the effects of BMP-2, BMP-5, and BMP-6 in isolated and combined use on the generation of osteoblasts and osteoclasts by comparing the osteoclastic potency of each on osteoclasts of primary murine bone marrow cells. Subsequently, cells were stained for tartrate-resistant acid phosphatase, and real time PCR analysis of receptor activator of NKκB ligand and osteoprotegerin was conducted. The same combination of BMPs was used to assess their potential to enhance osteoblasts, employing a mineralization assay and real-time PCR analysis of collagen type-1, runx2, and osterix. While BMP-2 alone and the combination of BMP-2 and BMP-5 significantly enhanced osteoclastogenesis, BMP-2, BMP-5, and BMP-6 in combination did not have additional effects. However, the combined use of BMP-2, BMP-5, and BMP-6 had an additive effect on matrix mineralization and osterix expression in osteoblasts. Our study shows that the combination of BMP-2, BMP-5, and BMP-6 stimulates osteoblasts but not osteoclastogenesis. Thus, the synergistic use of various BMPs might improve effective bone regeneration in the clinical setting., (© 2010 Orthopaedic Research Society.)

The anabolic effect of dynamic mechanical loading on skeletal architecture has been repeatedly demonstrated, but the cellular and molecular events occurring between load and ultimate bone formation remain obscure. The discovery of sclerostin, an antagonist of Wnt/Lrp5 signaling, and the sclerosing bone dysplasias that result from its mutation suggest its pivotal role in modulating bone formation. We examined expression of Sost mRNA across a variety of clonal cell lines spanning the osteogenic phenotype from immature osteoblast to mature osteocyte. No sclerostin expression was detected in immature MC3T3-E1 osteoblasts and, surprisingly, mature MLO-Y4 osteocytes, whereas immature MLO-A5 osteocytic cells expressed very low levels of Sost. Highest expression was observed in mature UMR 106.01 osteoblasts. We examined the influence of bone morphogenetic proteins on Sost expression. Treatment with BMP-2, -4 or -6 was without effect on Sost in mature MLO-Y4 osteocytes but elicited a robust increase in Sost expression in immature MLO-A5 osteocytes. Oscillatory fluid flow applied to mature UMR 106.01 osteoblasts transiently decreased expression of sclerostin at both the mRNA and protein level. Overall, our results indicate that BMP treatment and in vitro mechanical loading demonstrate opposite effects upon sclerostin expression.