BMP9-induced Ectopic Bone Formation Research Articles

Sonic Hedgehog potentiates BMP9-induced osteogenic differentiation of mesenchymal stem cells

Bone morphogenetic protein 9 (BMP9) has remarkable potential to induce the differentiation of mesenchymal stem cells (MSCs) towards the osteoblastic lineage. Additionally, research suggests that certain growth factors have the ability to potentiate BMP9-induced osteogenic differentiation of MSCs. Sonic Hedgehog (Shh) plays an indispensable role in the regulation of skeletal development. The objective of this research was to assess the potential influence of Shh on BMP9-induced osteogenic differentiation of MSCs. Our findings indicated that Shh effectively enhanced BMP9-induced early and late osteogenic differentiation of MSCs, and increased BMP9-induced expression/transcriptional activity of osteogenesis-related transcription factors. Besides, it was observed that Shh promoted BMP9-induced ectopic bone formation of MSCs in vivo. Moreover, BMP9 was able to facilitate the repair of bone defects in rats, while Shh further accelerated this reparative process. Mechanistically, Shh enhanced the activation of the Smad1/5/8 signaling pathway which was induced by BMP9. Furthermore, GANT-61, an inhibitor of Gli1 and Gli2, attenuated the enhancing effect of Shh on BMP9-induced osteogenic differentiation of MSCs. Collectively, the co-administration of BMP9 and Shh may present a promising therapeutic approach for the treatment of fracture nonunion, delayed fracture healing, and bone defects.

TAZ promotes osteogenic differentiation of mesenchymal stem cells line C3H10T1/2, murine multi-lineage cells lines C2C12, and MEFs induced by BMP9

Bone morphogenetic protein 9 (BMP9), also named as growth differentiation factor 2 (GDF-2), is the strongest cytokine that promotes osteogenic differentiation in the BMP family, and has broad clinical application value. Nevertheless, the mechanism of BMP9 promotes osteogenic differentiation remain unclear. TAZ, a transcriptional co-activator, has great effects on cell proliferation, differentiation, and stem cell self-renewal. In this research, we investigated the effects of TAZ in BMP9-induced osteogenic differentiation of mesenchymal stem cell line C3H10T1/2 (MSCs) and murine multi-lineage cell lines C2C12 and MEFs (MMCs) and explored its possible mechanisms. This study has found that BMP9 induces the expression of TAZ and promotes its nuclear translocation. Meanwhile, our study found that Ad-TAZ and TM-25659, a TAZ agonist, can enhance the osteogenic differentiation of MSCs and MMCs induced by BMP9. Conversely, Ad-si-TAZ and verteporfin, an inhibitor of TAZ, have the contradictory effect. Likewise, the promotion of TAZ to the BMP9-induced ectopic bone formation in vivo was confirmed by the subcutaneous transplantation of MSCs in nude mice. Furthermore, we have detected that TAZ might increase the levels of the phosphorylation of Smad1/5/8, p38, ERK1/2, and JNK induced by BMP9. Additionally, we also found that TAZ increased the total protein level of β-catenin induced by BMP9. In summary, our results strongly indicated that TAZ will promote the osteogenic differentiation in MSCs and MMCs induced by BMP9 through multiple signal pathways.

Selective Retinoic Acid Receptor γ Antagonist 7C is a Potent Enhancer of BMP-Induced Ectopic Endochondral Bone Formation.

Bone morphogenetic proteins (BMPs) have been clinically applied for induction of bone formation in musculoskeletal disorders such as critical-sized bone defects, nonunions, and spinal fusion surgeries. However, the use of supraphysiological doses of BMP caused adverse events, which were sometimes life-threatening. Therefore, safer treatment strategies for bone regeneration have been sought for decades. Systemic administration of a potent selective antagonist of retinoic acid nuclear receptor gamma (RARγ) (7C) stimulated BMP-induced ectopic bone formation. In this study, we developed 7C-loaded poly lactic nanoparticles (7C-NPs) and examined whether local application of 7C enhances BMP-induced bone regeneration. The collagen sponge discs that absorbed recombinant human (rh) BMP-2 were implanted into the dorsal fascia of young adult mice to induce ectopic bone. The combination of rhBMP-2 and 7C-NP markedly increased the total bone volume and thickness of the bone shell of the ectopic bone in a dose-dependent manner compared to those with rhBMP-2 only. 7C stimulated sulfated proteoglycan production, expression of chondrogenic marker genes, and Sox9 reporter activity in both chondrogenic cells and MSCs. The findings suggest that selective RARγ antagonist 7C or the related compounds potentiate the bone inductive ability of rhBMP-2, as well as support any future research to improve the BMP-2 based bone regeneration procedures in a safe and efficient manner.

Long non-coding RNA (LncRNA) HOTAIR regulates BMP9-induced osteogenic differentiation by targeting the proliferation of mesenchymal stem cells (MSCs).

Long non-coding RNAs are important regulators of biological processes, but their roles in the osteogenic differentiation of mesenchymal stem cells (MSCs) remain unclear. Here we investigated the role of murine HOX transcript antisense RNA (mHotair) in BMP9-induced osteogenic differentiation of MSCs using immortalized mouse adipose-derived cells (iMADs). Touchdown quantitative polymerase chain reaction analysis found increased mHotair expression in bones in comparison with most other tissues. Moreover, the level of mHotair in femurs peaked at the age of week-4, a period of fast skeleton development. BMP9 could induce earlier peak expression of mHotair during in vitro iMAD osteogenesis. Silencing mHotair diminished BMP9-induced ALP activity, matrix mineralization, and expression of osteogenic, chondrogenic and adipogenic markers. Cell implantation experiments further confirmed that knockdown of mHotair attenuated BMP9-induced ectopic bone formation and mineralization of iMADs, leading to more undifferentiated cells. Crystal violet staining and cell cycle analysis revealed that silencing of mHotair promoted the proliferation of iMAD cells regardless of BMP9 induction. Moreover, ectopic bone masses developed from mHotair-knockdown iMAD cells exhibited higher expression of PCNA than the control group. Taken together, our results demonstrated that murine mHotair is an important regulator of BMP9-induced MSC osteogenesis by targeting cell cycle and proliferation.

Contrary Roles of Wnt/β-Catenin Signaling in BMP9-Induced Osteogenic and Adipogenic Differentiation of 3T3-L1 Preadipocytes.

Our previous study revealed that 3T3-L1 preadipocytes can differentiate to either osteoblasts or adipocytes in response to bone morphogenic protein 9 (BMP9). In the present study, we try to further investigate whether the Wnt/β-catenin signaling plays a crucial role in this process. It was found that BMP9 effectively activated the Wnt/β-catenin signaling, and induced the expression levels of certain canonical Wnt ligands and their receptors in preadipocytes. Exogenous expression of β-catenin, Wnt1, Wnt3a, and Wnt10b potentiated BMP9-induced alkaline phosphatase (ALP) activity, while β-catenin knockdown or Dickkopf 1 (Dkk1) diminished BMP9-induced ALP activity. Moreover, it was demonstrated that β-catenin overexpression promoted BMP9-induced mineralization, and increased the expression levels of late osteogenic markers osteopontin and osteocalcin. Furthermore, β-catenin inhibited BMP9-induced lipid accumulation and the adipogenic marker adipocyte fatty acid binding protein (aP2). The cell-implantation assay results identified that β-catenin not only augmented BMP9-induced ectopic bone formation, but also blocked adipogenesis in vivo. Mechanistically, it was found that β-catenin and BMP9 synergistically stimulated the osteogenic transcription factors runt-related transcription factor 2 (Runx2) and Osterix (OSX). However, BMP9-induced adipogenic transcription factors, peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT enhancer-binding protein α (C/EBPα), were inhibited by β-catenin. Therefore, these findings suggested that the Wnt/β-catenin signaling, potentially via the modulation of osteogenic and adipogenic transcriptional factors, exerts an opposite effect on BMP9-induced osteogenic and adipogenic differentiation in preadipocytes.

LncRNA Rmst acts as an important mediator of BMP9-induced osteogenic differentiation of mesenchymal stem cells (MSCs) by antagonizing Notch-targeting microRNAs.

Understanding the bone and musculoskeletal system is essential to maintain the health and quality of life of our aging society. Mesenchymal stem cells (MSCs) can undergo self-renewal and differentiate into multiple tissue types including bone. We demonstrated that BMP9 is the most potent osteogenic factors although molecular mechanism underlying BMP9 action is not fully understood. Long noncoding RNAs (lncRNAs) play important regulatory roles in many physiological and/or pathologic processes. Here, we investigated the role of lncRNA Rmst in BMP9-induced osteogenic differentiation of MSCs. We found that Rmst was induced by BMP9 through Smad signaling in MSCs. Rmst knockdown diminished BMP9-induced osteogenic, chondrogenic and adipogenic differentiation in vitro, and attenuated BMP9-induced ectopic bone formation. Silencing Rmst decreased the expression of Notch receptors and ligands. Bioinformatic analysis predicted Rmst could directly bind to eight Notch-targeting miRNAs, six of which were downregulated by BMP9. Silencing Rmst restored the expression of four microRNAs (miRNAs). Furthermore, an activating Notch mutant NICD1 effectively rescued the decreased ALP activity caused by Rmst silencing. Collectively, our results strongly suggest that the Rmst-miRNA-Notch regulatory axis may play an important role in mediating BMP9-induced osteogenic differentiation of MSCs.

Wnt11 promotes BMP9-induced osteogenic differentiation through BMPs/Smads and p38 MAPK in mesenchymal stem cells.

Bone morphogenetic protein 9 (BMP9), as one of the most potent osteogenic factors, is a promising cytokine for bone tissue engineering. Wnt11 can regulate the development of the skeletal system and is related to high bone mass syndrome. However, the effect of Wnt11 on BMP9-induced osteogenic differentiation remains unknown. In this study, we investigated the relationship between Wnt11- and BMP9-induced osteogenic differentiation in mesenchymal stem cells (MSCs). We recapitulated the osteogenic potential of BMP9 in C3H10T1/2 cells. The messenger RNA expression of Wnt11 is detectable in the available progenitor cells, and BMP9 can obviously increase the protein level of Wnt11 in these cells. Exogenous Wnt11 potentiates the effect of BMP9 on increasing alkaline phosphatase (ALP) activities, the expression of osteopontin (OPN), and Runt-related transcription factor 2 (Runx2), so does matrix mineralization in C3H10T1/2 cells. Although Wnt11 cannot increase the BMP9-induced ectopic bone formation, it can increase the bone density induced by BMP9 apparently. Wnt11 increases the level of p-Smad1/5/8, as well as p-p38. Meanwhile, Wnt11 promotes the effect of BMP9 on increasing the levels of p-Smad1/5/8 and p-p38. Inhibition of p38 decreases the BMP9-induced ALP activities, the expression of OPN, and the mineralization in C3H10T1/2 cells. However, all of these effects of the p38 inhibitor on BMP9-induced osteogenic markers can be almost reversed by the overexpression of Wnt11. Our findings suggested that Wnt11 can enhance the osteogenic potential of BMP9 in MSCs, and this effect may be partly mediated through enhancing BMPs/Smads and the p38 MAPK signal, which was induced by BMP9.

Hmox1 promotes osteogenic differentiation at the expense of reduced adipogenic differentiation induced by BMP9 in C3H10T1/2 cells.

Mesenchymal stem cells (MSCs) are multipotent progenitors that can differentiate into a variety of cell types under proper stimuli. Bone morphogenetic protein 9 (BMP9) is able to simultaneously induce both adipogenic and osteogenic differentiation of MSCs although the regulatory molecules involved remain to be fully identified and characterized. Heme oxygenase 1 (Hmox1) plays an essential role not only in fat metabolism, but also in bone development. In the present study, we investigated the functional role of Hmox1 in BMP9-induced osteogenic/adipogenic differentiation in MSCs line C3H10T1/2 and probed the possible mechanism involved. We found that BMP9 promoted the endogenous expression of Hmox1 in C3H10T1/2 cells. Overexpression of Hmox1 or cobalt protoporphyrin (CoPP), an inducer of Hmox1, increased BMP9-induced osteogenic differentiation in vitro. Subcutaneous stem cell implantation in nude mice further confirmed that Hmox1 potentiated BMP9-induced ectopic bone formation in vivo. In contrast, Hmox1 reduced BMP9-induced adipogenic differentiation in C3H10T1/2 cells. Although had no obvious effect on BMP9-induced Smad1/5/8 phosphorylation, Hmox1 enhanced phosphorylation of p38, and AKT, while decreased phosphorylation of ERK1/2. Furthermore, Hmox1 increased total β-catenin protein level, and promoted the nuclear translocation of β-catenin in C3H10T1/2 cells. Taken together, our study strongly suggests that Hmox1 is likely to potentiate osteogenic differentiation and yet decrease adipogenic differentiation induced by BMP9 possibly through regulation of multiple signaling pathways.

Gelatin-Derived Graphene-Silicate Hybrid Materials Are Biocompatible and Synergistically Promote BMP9-Induced Osteogenic Differentiation of Mesenchymal Stem Cells.

Graphene-based materials are used in many fields but have found only limited applications in biomedicine, including bone tissue engineering. Here, we demonstrate that novel hybrid materials consisting of gelatin-derived graphene and silicate nanosheets of Laponite (GL) are biocompatible and promote osteogenic differentiation of mesenchymal stem cells (MSCs). Homogeneous cell attachment, long-term proliferation, and osteogenic differentiation of MSCs on a GL-scaffold were confirmed using optical microscopy and scanning electron microscopy. GL-powders made by pulverizing the GL-scaffold were shown to promote bone morphogenetic protein (BMP9)-induced osteogenic differentiation. GL-powders increased the alkaline phosphatase (ALP) activity in immortalized mouse embryonic fibroblasts but decreased the ALP activity in more-differentiated immortalized mouse adipose-derived cells. Note, however, that GL-powders promoted BMP9-induced calcium mineral deposits in both MSC lines, as assessed using qualitative and quantitative alizarin red assays. Furthermore, the expression of chondro-osteogenic regulator markers such as Runx2, Sox9, osteopontin, and osteocalcin was upregulated by the GL-powder, independent of BMP9 stimulation; although the powder synergistically upregulated the BMP9-induced Osterix expression, the adipogenic marker PPARγ was unaffected. Furthermore, in vivo stem cell implantation experiments demonstrated that GL-powder could significantly enhance the BMP9-induced ectopic bone formation from MSCs. Collectively, our results strongly suggest that the GL hybrid materials promote BMP9-induced osteogenic differentiation of MSCs and hold promise for the development of bone tissue engineering platforms.

Vibration acceleration promotes bone formation in rodent models.

All living tissues and cells on Earth are subject to gravitational acceleration, but no reports have verified whether acceleration mode influences bone formation and healing. Therefore, this study was to compare the effects of two acceleration modes, vibration and constant (centrifugal) accelerations, on bone formation and healing in the trunk using BMP 2-induced ectopic bone formation (EBF) mouse model and a rib fracture healing (RFH) rat model. Additionally, we tried to verify the difference in mechanism of effect on bone formation by accelerations between these two models. Three groups (low- and high-magnitude vibration and control-VA groups) were evaluated in the vibration acceleration study, and two groups (centrifuge acceleration and control-CA groups) were used in the constant acceleration study. In each model, the intervention was applied for ten minutes per day from three days after surgery for eleven days (EBF model) or nine days (RFH model). All animals were sacrificed the day after the intervention ended. In the EBF model, ectopic bone was evaluated by macroscopic and histological observations, wet weight, radiography and microfocus computed tomography (micro-CT). In the RFH model, whole fracture-repaired ribs were excised with removal of soft tissue, and evaluated radiologically and histologically. Ectopic bones in the low-magnitude group (EBF model) had significantly greater wet weight and were significantly larger (macroscopically and radiographically) than those in the other two groups, whereas the size and wet weight of ectopic bones in the centrifuge acceleration group showed no significant difference compared those in control-CA group. All ectopic bones showed calcified trabeculae and maturated bone marrow. Micro-CT showed that bone volume (BV) in the low-magnitude group of EBF model was significantly higher than those in the other two groups (3.1±1.2mm3 v.s. 1.8±1.2mm3 in high-magnitude group and 1.3±0.9mm3 in control-VA group), but BV in the centrifuge acceleration group had no significant difference compared those in control-CA group. Union rate and BV in the low-magnitude group of RFH model were also significantly higher than those in the other groups (Union rate: 60% v.s. 0% in the high-magnitude group and 10% in the control-VA group, BV: 0.69±0.30mm3 v.s. 0.15±0.09mm3 in high-magnitude group and 0.22±0.17mm3 in control-VA group). BV/TV in the low-magnitude group of RFH model was significantly higher than that in control-VA group (59.4±14.9% v.s. 35.8±13.5%). On the other hand, radiographic union rate (10% in centrifuge acceleration group v.s. 20% in control-CA group) and micro-CT parameters in RFH model were not significantly different between two groups in the constant acceleration studies. Radiographic images of non-union rib fractures showed cartilage at the fracture site and poor new bone formation, whereas union samples showed only new bone. In conclusion, low-magnitude vibration acceleration promoted bone formation at the trunk in both BMP-induced ectopic bone formation and rib fracture healing models. However, the micro-CT parameters were not similar between two models, which suggested that there might be difference in the mechanism of effect by vibration between two models.

Pro-Inflammatory Cytokine TNF-α Attenuates BMP9-Induced Osteo/ Odontoblastic Differentiation of the Stem Cells of Dental Apical Papilla (SCAPs)

Background/Aims: Periapical periodontitis is a common oral disease caused by bacterial invasion of the tooth pulp, which usually leads to local release of pro-inflammatory cytokines and osteolytic lesion. This study is intended to examine the effect of TNF-α on BMP9-induced osteogenic differentiation of the stem cells of dental apical papilla (SCAPs). Methods: Rat model of periapical periodontitis was established. TNF-α expression was assessed. Osteogenic markers and ectopic bone formation in iSCAPs were analyzed upon BMP9 and TNF-α treatment. Results: Periapical periodontitis was successfully established in rat immature permanent teeth with periapical lesions, in which TNF-α was shown to release during the inflammatory phase. BMP9-induced alkaline phosphatase activity, the expression of osteocalcin and osteopontin, and matrix mineralization in iSCAPs were inhibited by TNF-α in a dose-dependent fashion, although increased AdBMP9 partially overcame TNF-α inhibition. Furthermore, high concentration of TNF-α effectively inhibited BMP9-induced ectopic bone formation in vivo. Conclusion: TNF-α plays an important role in periapical bone defect during the inflammatory phase and inhibits BMP9-induced osteoblastic differentiation of iSCAPs, which can be partially reversed by high levels of BMP9. Therefore, BMP9 may be further explored as a potent osteogenic factor to improve osteo/odontogenic differentiation in tooth regeneration in chronic inflammation conditions.

Adenovirus-mediated expression of vascular endothelial growth factor-a potentiates bone morphogenetic protein9-induced osteogenic differentiation and bone formation.

Mesenchymal stem cells (MSCs) are suitable seed cells for bone tissue engineering because they can self-renew and undergo differentiation into osteogenic, adipogenic, chondrogenic, or myogenic lineages. Vascular endothelial growth factor-a (VEGF-a), an angiogenic factor, is also involved in osteogenesis and bone repair. However, the effects of VEGF-a on osteogenic MSCs differentiation remain unknown. It was previously reported that bone morphogenetic protein9 (BMP9) is one of the most important osteogenic BMPs. Here, we investigated the effects of VEGF-a on BMP9-induced osteogenesis with mouse embryo fibroblasts (MEFs). We found that endogenous VEGF-a expression was undetectable in MSCs. Adenovirus-mediated expression of VEGF-a in MEFs potentiated BMP9-induced early and late osteogenic markers, including alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN). In stem cell implantation assays, VEGF-a augmented BMP9-induced ectopic bone formation. VEGF-a in combination with BMP9 effectively increased the bone volume and osteogenic activity. However, the synergistic effect was efficiently abolished by the phosphoinositide 3-kinase (PI3K)/AKT inhibitor LY294002. These results demonstrated that BMP9 may crosstalk with VEGF-a through the PI3K/AKT signaling pathway to induce osteogenic differentiation in MEFs. Thus, our findings demonstrate the effects of VEGF-a on BMP9-induced bone formation and provide a new potential strategy for treating nonunion fractures, large segmental bony defects, and/or osteoporotic fractures.

BMP9-induced osteogenic differentiation is partially inhibited by miR-30a in the mesenchymal stem cell line C3H10T1/2.

In the bone morphogenetic protein (BMP) family, BMP9 is the strongest inducer of osteogenic differentiation in mesenchymal stem cells. Recent studies have suggested that the miR-30 family regulates cell proliferation and osteoblastic differentiation. In the present study, we found that expression of only one miR-30 family member, miR-30a, first decreased and then increased during BMP9-induced osteogenic differentiation. Cell proliferation assays revealed that miR-30a had no effect on the proliferation of C3H10T1/2 cells. However, over-expression of miR-30a led to expression of an early osteogenic marker and a reduction in Runx2 expression. In addition, we observed decreases in the expression of late osteogenic markers and osteopontin, as well as calcium deposition. Dual-luciferase reporter assays indicated that this process might be mediated by suppressing Runx2 protein expression. In vivo stem cell implantation revealed inhibition of BMP9-induced ectopic bone formation and matrix mineralization by miR-30a. This study provides a better understanding of the molecular mechanisms through which miR-30a negatively regulates BMP9-induced osteogenic differentiation.

Endoplasmic Reticulum (ER) Stress Inducible Factor Cysteine-Rich with EGF-Like Domains 2 (Creld2) Is an Important Mediator of BMP9-Regulated Osteogenic Differentiation of Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are multipotent progenitors that can undergo osteogenic differentiation under proper stimuli. We demonstrated that BMP9 is one of the most osteogenic BMPs. However, the molecular mechanism underlying BMP9-initiated osteogenic signaling in MSCs remains unclear. Through gene expression profiling analysis we identified several candidate mediators of BMP9 osteogenic signaling. Here, we focus on one such signaling mediator and investigate the functional role of cysteine-rich with EGF-like domains 2 (Creld2) in BMP9-initiated osteogenic signaling. Creld2 was originally identified as an ER stress-inducible factor localized in the ER-Golgi apparatus. Our genomewide expression profiling analysis indicates that Creld2 is among the top up-regulated genes in BMP9-stimulated MSCs. We confirm that Creld2 is up-regulated by BMP9 in MSCs. ChIP analysis indicates that Smad1/5/8 directly binds to the Creld2 promoter in a BMP9-dependent fashion. Exogenous expression of Creld2 in MSCs potentiates BMP9-induced early and late osteogenic markers, and matrix mineralization. Conversely, silencing Creld2 expression inhibits BMP9-induced osteogenic differentiation. In vivo stem cell implantation assay reveals that exogenous Creld2 promotes BMP9-induced ectopic bone formation and matrix mineralization, whereas silencing Creld2 expression diminishes BMP9-induced bone formation and matrix mineralization. We further show that Creld2 is localized in ER and the ER stress inducers potentiate BMP9-induced osteogenic differentiation. Our results strongly suggest that Creld2 may be directly regulated by BMP9 and ER stress response may play an important role in regulating osteogenic differentiation.

BMP9 and COX-2 form an important regulatory loop in BMP9-induced osteogenic differentiation of mesenchymal stem cells

Mesenchymal stem cells (MSCs) can self-renew and differentiate into osteogenic, chondrogenic, adipogenic and myogenic lineages. It's reported that bone morphogenetic protein 9 (BMP9) is one of the most potent osteogenic BMPs to initiate the commitment of MSCs to osteoblast lineage. Cyclooxygenase-2 (COX-2) is critical for bone fracture healing and osteogenic differentiation in MSCs. However, the relationship between COX-2 and BMP9 in osteogenesis remains unknown. Herein, we investigate the role of COX-2 in BMP9-induced osteogenesis in MSCs. We demonstrate that COX-2 is up-regulated as a target of BMP9 in MSCs. Both COX-2 inhibitor (NS-398) and COX-2 knockdown siRNAs can effectively decrease alkaline phosphatase (ALP) activities induced by BMP9 in MSCs. NS-398 also down-regulates BMP9-induced expression of osteopontin and osteocalcin, so does the matrix mineralization. The in vivo studies indicate that knockdown of COX-2 attenuates BMP9-induced ectopic bone formation. In perinatal limb culture assay, NS-398 is shown to reduce the hypertropic chondrocyte zone and ossification induced by BMP9. Mechanistically, knockdown of COX-2 significantly inhibits the BMP9 up-regulated expression of Runx2 and Dlx-5 in MSCs, which can be rescued by exogenous expression of COX-2. Furthermore, knockdown of COX-2 apparently reduces BMP9 induced BMPR-Smad reporter activity, the phosphorylation of Smad1/5/8, and the expression of Smad6 and Smad7 in MSCs. NS-398 blocks the expression of BMP9 mediated by BMP9 recombinant adenovirus. Taken together, our findings suggest that COX-2 plays an important role in BMP9 induced osteogenic differentiation in MSCs; BMP9 and COX-2 may form an important regulatory loop to orchestrate the osteogenic differentiation in MSCs.

Noggin resistance contributes to the potent osteogenic capability of BMP9 in mesenchymal stem cells

Mesenchymal stem cells (MSCs) are multipotent progenitors and can differentiate into osteogenic, chondrogenic, and adipogenic lineages. Bone morphogenetic proteins (BMPs) play important roles in stem cell proliferation and differentiation. We recently demonstrated that BMP9 is a potent but less understood osteogenic factor. We previously found that BMP9-induced ectopic bone formation is not inhibited by BMP3. Here, we investigate the effect of BMP antagonist noggin on BMP9-induced osteogenic differentiation. BMP antagonists noggin, chording, gremlin, follistatin, and BMP3 are highly expressed in MSCs, while noggin and follistatin are lowly expressed in more differentiated pre-osteoblast C2C12 cells. BMP9-induced osteogenic markers and matrix mineralization are not inhibited by noggin, while noggin blunts BMP2, BMP4, BMP6, and BMP7-induced osteogenic markers and mineralization. Likewise, ectopic bone formation by MSCs transduced with BMP9, but not the other four BMPs, is resistant to noggin inhibition. BMP9-induced nuclear translocation of Smad1/5/8 is not affected by noggin, while noggin blocks BMP2-induced activation of Smad1/5/8 in MSCs. Noggin fails to inhibit BMP9-induced expression of downstream targets in MSCs. Thus, our results strongly suggest that BMP9 may effectively overcome noggin inhibition, which should at least in part contribute to BMP9's potent osteogenic capability in MSCs.

Cross‐talk between EGF and BMP9 signalling pathways regulates the osteogenic differentiation of mesenchymal stem cells

Mesenchymal stem cells (MSCs) are multipotent progenitors, which give rise to several lineages, including bone, cartilage and fat. Epidermal growth factor (EGF) stimulates cell growth, proliferation and differentiation. EGF acts by binding with high affinity to epidermal growth factor receptor (EGFR) on the cell surface and stimulating the intrinsic protein tyrosine kinase activity of its receptor, which initiates a signal transduction cascade causing a variety of biochemical changes within the cell and regulating cell proliferation and differentiation. We have identified BMP9 as one of the most osteogenic BMPs in MSCs. In this study, we investigate if EGF signalling cross-talks with BMP9 and regulates BMP9-induced osteogenic differentiation. We find that EGF potentiates BMP9-induced early and late osteogenic markers of MSCs in vitro, which can be effectively blunted by EGFR inhibitors Gefitinib and Erlotinib or receptor tyrosine kinase inhibitors AG-1478 and AG-494 in a dose- and time-dependent manner. Furthermore, EGF significantly augments BMP9-induced bone formation in the cultured mouse foetal limb explants. In vivo stem cell implantation experiment reveals that exogenous expression of EGF in MSCs can effectively potentiate BMP9-induced ectopic bone formation, yielding larger and more mature bone masses. Interestingly, we find that, while EGF can induce BMP9 expression in MSCs, EGFR expression is directly up-regulated by BMP9 through Smad1/5/8 signalling pathway. Thus, the cross-talk between EGF and BMP9 signalling pathways in MSCs may underline their important roles in regulating osteogenic differentiation. Harnessing the synergy between BMP9 and EGF should be beneficial for enhancing osteogenesis in regenerative medicine.

The local administration of TNF-α and RANKL antagonist peptide promotes BMP-2-induced bone formation

Abstract Objectives Because high doses of bone morphogenetic proteins (BMPs) are required to achieve a certain level of bone regeneration in primates, thereby causing side effects such as inflammation, combination therapies using BMPs along with agents that can reduce the required amount of BMPs have been developed. Recently, we found that subcutaneous injections of W9 peptide (W9), which has been established as a tumor necrosis factor (TNF)-α and receptor activator of nuclear factor-κB ligand (RANKL) antagonist, promoted BMP-induced ectopic bone formation. Since TNF-α is known to reduce bone formation, we investigated the stimulatory mechanism of W9 on bone formation by using TNF-α-deficient, TNF type 1 receptor (TNFR1)-deficient, and wild-type (WT) mice. Methods Collagen discs containing either BMP-2 (1μg) alone or BMP-2 (1μg) with W9 (0.56mg) (BMP-2+W9) were implanted into the back muscles of 5-week-old male mice. The animals were sacrificed on day 12 after implantation. Radiographic and histomorphometric analyses were performed on the dissected ectopic bones. Results A significant increase in ectopic bone was observed in the BMP-2+W9 group compared to the BMP-2 group in WT mice. Bone histomorphometric technique revealed a significant increase of osteoblast surface and mineralized bone indices in the BMP-2+W9 group compared to the BMP-2 group in WT mice. Interestingly, W9 also increased the bone mineral content of ectopic bone induced by BMP-2 in both TNF-α deficient and TNFR1-deficient mice, to the same extent as in WT mice. Conclusion Our data suggest that W9 promotes bone formation by a mechanism other than antagonism of TNF-α action.

Osteoclastic bone resorption directly activates osteoblast function

Osteoclasts, the multinucleated cells that resorb bone, originate from cell cycle-arrested quiescent osteoclast precursors [1]. Mesenchymal osteoblastic cells are involved in osteoclast differentiation. Osteoclast precursors express RANK (a receptor of RANKL), recognize RANKL expressed by osteoblasts through cell-cell interaction and differentiate into osteoclasts in the presence of M-CSF. OPG, produced mainly by osteoblasts, is a soluble decoy receptor for RANKL. Deficiency of OPG in mice induces osteoporosis caused enhanced bone resorption. Elevated osteoblastic activity was suppressed by bisphosphonate administration in OPG-deficient mice. These results suggest that bone formation is accurately coupled with bone resorption. Collagen sponge disks containing BMP-2 were implanted into the dorsal muscle pouches in OPG-deficient mice. TRAP-positive osteoclasts and ALP-positive osteoblasts were observed in BMP-2-disks preceding the onset of calcification for one week. OPG and soluble RANK inhibited BMP-2-induced osteoclast formation but not the appearance of ALP-positive cells in OPG-deficient mice. We then examined how osteoblasts are involved in osteoclastogenesis other than RANKL expression, using RANKL-deficient mice. RANKL-deficient mice showed severe osteopetrosis due to loss of osteoclasts. Injection of RANKL into RANKL-deficient mice induced many osteoclasts in bone but not soft tissues [2]. These results suggest that osteoblasts determine the place of osteoclastogenesis from haemopoietic stem cells in bone. We next explored roles of osteoclasts in ectopic bone formation induced by BMP using op/op and c-fos-deficient osteopetrotic mice. The ectopic bones formed in op/op mice showed extremely rough surfaces, whereas those in wild-type mice showed smooth ones. Bone mineral density of BMP-induced ectopic bone in op/op mice was about 2-times higher than that in wild-type mice. TRAP-positive osteoclasts exhibit in outer of the ectopic bone in the wild-type mice. In op/op mice, although osteoclasts strongly exhibit in inside of the BMP-induced ectopic bone, TRAP-positive osteoclasts did not exhibit in outer of the BMP-induced ectopic bone. Furthermore, the accentuation of the BMP-induced ectopic bone formation did not exist in osteopetrotic c-Fos-deficient mice. In c-Fos-deficient mice, which are completely osteoclasts deficiency, the accentuation of the BMP-induced ectopic bone formation did not exist. Furthermore, there is no RANK-positive osteoclast progenitors in bone derived from c-Fos-deficient mice. These results suggest that RANK-positive osteoclast progenitors are positively regulate the signal of bone formation. In summary, osteoclastic bone resorption directly activates osteoblast function and osteoclasts are involved in normal bone morphogenesis.

The role of MyoD+ muscle progenitor cells in bone formation and repair

Bone morphogenetic proteins (BMPs) are able to induce osteogenic differentiation in many cells, including muscle cells. However, the actual contribution of muscle cells to bone formation and repair is unclear. Our objective was to examine the capacity of myogenic cells to contribute to BMP-induced ectopic bone formation and fracture repair. Osteogenic gene expression was measured by quantitative PCR in osteoprogenitors, myoblasts, and fibroblasts following BMP-2 treatment. The MyoD-Cre x ROSA26R and MyoD-Cre x Z/AP mouse strains were used to track the fate of MyoD+ cells in vivo. In these double-transgenic mice, MyoD+ progenitors undergo a permanent recombination event to induce reporter gene expression. Ectopic bone was produced by the intramuscular implantation of BMP-7. Closed tibial fractures and open tibial fractures with periosteal stripping were also performed. Cellular contribution was tracked at one, two and three week time points by histological staining. Osteoprogenitors and myoblasts exhibited comparable expression of early and late bone markers; in contrast bone marker expression was considerably less in fibroblasts. The sensitivity of cells to BMP-2 correlated with the expression of BMP receptor-1a (Bmpr1a). Pilot experiments using the MyoD-Cre x Rosa26R mice identified a contribution by MyoD expressing cells in BMP-induced ectopic bone formation. However, false positive LacZ staining in osteoclasts led us to seek alternative systems such as the MyoD-cre x Z/AP mice that have negligible background staining. Initially, a minor contribution from MyoD expressing cells was noted in the ectopic bones in the MyoD-cre x Z/AP mice, but without false positive osteoclast staining. Soft tissue trauma usually precedes the formation of ectopic bone. Hence, to mimic the clinical condition more precisely, physical injury to the muscle was performed. Traumatising the muscle two days prior to BMP-7 implantation: (1) induced MyoD expression in quiescent satellite cells; (2) increased ectopic bone formation; and (3) greatly enhanced the number of MyoD positive cells in the ectopic bone. In open tibial fractures the majority of the initial callus was MyoD+ indicating a significant contribution by myogenic cells. In contrast, closed fractures with the periosteum intact had a negligible myogenic contribution. Myoblasts but not fibroblasts were highly responsive to BMP stimulation and this was associated with BMP receptor expression. Our transgenic mouse models demonstrate for the first time that muscle progenitors can significantly contribute to ectopic bone formation and fracture repair. This may have translational applications for clinical orthopaedic therapies.