Bone Morphogenetic Protein-2 Treatment Research Articles

Bone morphogenetic protein 2 alleviated intervertebral disc degeneration through mediating the degradation of ECM and apoptosis of nucleus pulposus cells via the PI3K/Akt pathway.

The present study aimed to explore the underlying mechanisms of bone morphogenetic protein 2 (BMP2) in alleviating intervertebral disc degeneration (IDD). A rat puncture IDD model was constructed, and the rats were randomly divided into six groups: Control; IDD (model); IDD+PBS [containing 1010 adeno‑associated virus serotype 2 (AAV)]; and IDD + AAV2‑BMP2 (106, 108 and 1010). IL‑1β was used to treat primary nucleus pulposus (NP) cells to mimic IDD in vitro. The effects of BMP2 in IDD were determined by magnetic resonance imaging (MRI), hematoxylin and eosin staining and Alcian Blue staining in vivo. The levels of collagen II, aggrecan, transcription factor SOX9 (SOX9) and matrix metalloproteinase 13 (MMP‑13) were examined using western blot analysis and reverse transcription quantitative polymerase chain reaction (RT‑qPCR) in NP tissues and cells. The expression of C‑telopeptide of type II collagen (CTX‑II) in the sera or cell supernatants was determined by ELISA. In addition, the levels of phosphorylation of phosphoinositide 3‑kinase (PI3K) and protein kinase B (Akt), and the levels of apoptosis‑associated proteins and apoptosis ratio of NP cells were also determined by western blot analysis and flow cytometry, respectively. LY29400, an inhibitor of PI3K, was used to additionally confirm the signal pathway mechanism of BMP2 treatment in IDD. BMP2 significantly extended the interval between discs and alleviated the fibrous ring rupture and the decrease in the levels of glycoproteins in IDD rats, as determined by MRI and histological staining. Additionally, BMP2 treatment significantly upregulated the levels of collagen II, aggrecan and SOX9, but downregulated the levels of MMP‑13 and CTX‑II in IDD rats and NP cells in a dose‑dependent manner. Concurrently, recombinant human (rh)BMP2 pretreatment also significantly decreased the apoptosis ratio of interleukin (IL)‑1β‑treated NP cells via downregulating the level of cleaved caspase‑3 and upregulating the level of uncleaved poly (adenosine 5'‑diphosphate‑ribose) polymerase. It was demonstrated that rhBMP2 also significantly decreased the inflammatory response in NP tissues and cells, based on levels of IL‑6, TNF‑α and IL‑10. In addition, rhBMP2 inhibited cell apoptosis via upregulating the phosphorylation levels of the PI3K/Akt signaling pathway, and LY29400 pretreatment inhibited the effects of BMP2 in IL‑1β treated NP cells. BMP2 alleviated IDD via the PI3K/Akt signaling pathway by inhibiting NP cell apoptosis and decreasing the levels of matrix proteins.

BMP-2 restoration aids in recovery from liver fibrosis by attenuating TGF-β1 signaling

Transforming growth factor-β (TGF-β) plays a central role in hepatic fibrogenesis. This study investigated the function and mechanism of bone morphogenetic protein-2 (BMP-2) in regulation of hepatic fibrogenesis. BMP-2 expression in fibrotic liver was measured in human tissue microarray and mouse models of liver fibrosis induced by bile duct ligation surgery or carbon tetrachloride administration. Adenovirus-mediated BMP-2 gene delivery was used to test the prophylactic effect on liver fibrosis. Primary hepatic stellate cells (HSC), HSC-T6 and clone-9 cell lines were used to study the interplay between BMP-2 and TGF-β1. Hepatic BMP-2 was localized in parenchymal hepatocytes and activated HSCs and significantly decreased in human and mouse fibrotic livers, showing an opposite pattern of hepatic TGF-β1 contents. BMP-2 gene delivery alleviated the elevations of serum hepatic enzymes, cholangiocyte marker CK19, HSC activation markers, and liver fibrosis in both models. Mechanistically, exogenous TGF-β1 dose dependently reduced BMP-2 expression, whereas BMP-2 significantly suppressed expression of TGF-β and its cognate type I and II receptor peptides, as well as the induced Smad3 phosphorylation levels in primary mouse HSCs. Aside from its suppressive effects on cell proliferation and migration, BMP-2 treatment prominently attenuated the TGF-β1-stimulated α-SMA and fibronectin expression, and reversed the TGF-β1-modulated epithelial-to-mesenchymal transition marker expression in mouse HSCs. The mutual regulation between BMP-2 and TGF-β1 signaling axes may constitute the anti-fibrogenic mechanism of BMP-2 in the pathogenesis of liver fibrosis. BMP-2 may potentially serve as a novel therapeutic target for treatment of liver fibrosis.The authors identified BMP-2 downregulation in human and experimental fibrotic livers, and found that adenovirus-mediated BMP-2 gene delivery ameliorates liver fibrosis and biliary injury in mice. Mechanistically, BMP-2 suppresses proliferation and migration of hepatic stellate cells by antagonizing TGF-β signaling and epithelial-to-mesenchymal transition. BMP-2 may therefore constitute a therapeutic target for liver fibrosis treatment.

Macrophage depletion by clodronate attenuates bone morphogenetic protein-7 induced M2 macrophage differentiation and improved systolic blood velocity in atherosclerosis

Bone morphogenetic protein-7 (BMP-7) affects the presence of macrophage subtypes in vitro and in vivo at an early stage of atherosclerosis (ATH); however, it remains unknown whether BMP-7 treatment affects the development and progression of ATH at a mid-stage of the disease. We therefore performed a Day 28 (D28) study to examine BMP-7's potential to affect monocyte differentiation. Atherosclerosis was developed in ApoE KO mice, and these animals were treated with intravenous injections of BMP-7 and/or liposomal clodronate (LC). BMP-7 significantly (P < 0.05) lowers plaque formation following induction of atherosclerosis. However, upon macrophage depletion, BMP-7 fails to significantly alter plaque progression suggesting a direct role of BMP-7 on macrophages. Immunohistochemical staining of carotid arteries was performed to determine BMP-7's effect on pro-inflammatory M1 inducible nitric oxide synthase and anti-inflammatory M2 (cluster of differentiation [CD]206, Arginase-1) macrophages, and monocytes ( CD14). BMP-7 significantly reduced pro-inflammatory M1 macrophages and increased anti-inflammatory M2 macrophages at D28, while BMP-7 showed no effect on M2 macrophage differentiation in animals treated with LC. Enzyme-linked immunosorbent assay data showed significant reduction in proinflammatory cytokines (Interleukin-6 [IL-6]), monocyte chemoattractant protein-1, and tumor necrosis factor-α) and a significant increase in anti-inflammatory cytokine (IL-10) in BMP-7 treated mice (P < 0.05).Western blot analysis of arterial tissue confirms a significant increase in pro-survival kinases extracellular-signal regulated kinase and SMAD and a reduction in pro-inflammatory kinases p38 and c-Jun N-terminal kinase in BMP-7 treated mice (P < 0.05). Overall, this study suggests that clodronate treatment inhibits BMP-7 induced differentiation of monocytes into M2 macrophages and improved systolic blood velocity.

The Effects of Age and Dose on Gene Expression and Segmental Bone Defect Repair After BMP-2 Delivery.

ABSTRACTAge is a well‐known influential factor in bone healing, with younger patients generally healing bone fractures more rapidly and suffering fewer complications compared with older patients. Yet the impact age has on the response to current bone healing treatments, such as delivery of bone morphogenetic protein 2 (BMP‐2), remains poorly characterized. It remains unclear how or if therapeutic dosing of BMP‐2 should be modified to account for age‐related differences in order to minimize potential adverse effects and consequently improve patient bone‐healing outcomes. For this study, we sought to address this issue by using a preclinical critically sized segmental bone defect model in rats to investigate age‐related differences in bone repair after delivery of BMP‐2 in a collagen sponge, the current clinical standard. Femoral defects were created in young (7‐week‐old) and adult (8‐month‐old) rats, and healing was assessed using gene expression analyses, longitudinal radiography, ex vivo micro‐computed tomography (µCT), as well as torsional testing. We found that young rats demonstrated elevated expression of genes related to osteogenesis, chondrogenesis, and matrix remodeling at the early 1‐week time point compared with adult rats. These early gene expression differences may have impacted long‐term healing as the regenerated bones of young rats exhibited higher bone mineral densities compared with those of adult rats after 12 weeks. Furthermore, the young rats demonstrated significantly more bone formation and increased mechanical strength when BMP‐2 dose was increased from 1 µg to 10 µg, a finding not observed in adult rats. Overall, these results indicate there are age‐related differences in BMP‐2‐mediated bone regeneration, including relative dose sensitivity, suggesting that age is an important consideration when implementing a BMP‐2 treatment strategy. © 2018 The Authors JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Drilling Combined with Adipose-derived Stem Cells and Bone Morphogenetic Protein-2 to Treat Femoral Head Epiphyseal Necrosis in Juvenile Rabbits.

This study was designed to evaluate the effects of drilling through the growth plate and using adipose-derived stem cells (ADSCs) and bone morphogenetic protein-2 (BMP-2) to treat femoral head epiphyseal ischemic necrosis, which can be done in juvenile rabbits. Passagefour bromodeoxyuridine (BrdU)-labeled ADSCs were cultured, assayed with MTT to determine their viability and stained with alizarin red dye to determine their osteogenic ability. Two-month-old, healthy male rabbits (1.2 to 1.4 kg, n=45) underwent ischemic induction and were randomly divided into five groups (group A: animal model control; group B: drilling; group C: drilling & ADSCs; group D: drilling & BMP-2; and group E: drilling & ADSCs & BMP-2). Magnetic resonance imaging (MRI), X-ray imaging, hematoxylin and eosin staining and BrdU immunofluorescence detection were applied 4, 6 and 10 weeks after treatment. Approximately 90% of the ADSCs were labeled with BrdU and showed good viability and osteogenic ability. Similar results were observed in the rabbits in groups C and E at weeks 6 and 10. The animals of groups C and E demonstrated normal hip structure and improved femoral epiphyseal quotients and trabecular areas compared with those of the groups A and B (P<0.01). Group D demonstrated improved femoral epiphyseal quotients and trabecular areas compared with those of groups A and B (P<0.05). In summary, drilling through the growth plate combined with ADSC and BMP-2 treatments induced new bone formation and protected the femoral head epiphysis from collapsing in a juvenile rabbit model of femoral head epiphyseal ischemic necrosis.

In silico clinical trials for pediatric orphan diseases

To date poor treatment options are available for patients with congenital pseudarthrosis of the tibia (CPT), a pediatric orphan disease. In this study we have performed an in silico clinical trial on 200 virtual subjects, generated from a previously established model of murine bone regeneration, to tackle the challenges associated with the small, pediatric patient population. Each virtual subject was simulated to receive no treatment and bone morphogenetic protein (BMP) treatment. We have shown that the degree of severity of CPT is significantly reduced with BMP treatment, although the effect is highly subject-specific. Using machine learning techniques we were also able to stratify the virtual subject population in adverse responders, non-responders, responders and asymptomatic. In summary, this study shows the potential of in silico medicine technologies as well as their implications for other orphan diseases.

Exogenous BMP7 in aortae of rats with chronic uremia ameliorates expression of profibrotic genes, but does not reverse established vascular calcification.

Hyperphosphatemia and vascular calcification are frequent complications of chronic renal failure and bone morphogenetic protein 7 (BMP7) has been shown to protect against development of vascular calcification in uremia. The present investigation examined the potential reversibility of established uremic vascular calcification by treatment of uremic rats with BMP7. A control model of isogenic transplantation of a calcified aorta from uremic rats into healthy littermates examined whether normalization of the uremic environment reversed vascular calcification. Uremia and vascular calcification were induced in rats by 5/6 nephrectomy, high phosphate diet and alfacalcidol treatment. After 14 weeks severe vascular calcification was present and rats were allocated to BMP7, vehicle or aorta transplantation. BMP7 treatment caused a significant decrease of plasma phosphate to 1.56 ± 0.17 mmol/L vs 2.06 ± 0.34 mmol/L in the vehicle group even in the setting of uremia and high phosphate diet. Uremia and alfacalcidol resulted in an increase in aortic expression of genes related to fibrosis, osteogenic transformation and extracellular matrix calcification, and the BMP7 treatment resulted in a decrease in the expression of profibrotic genes. The total Ca-content of the aorta was however unchanged both in the abdominal aorta: 1.9 ± 0.6 μg/mg tissue in the vehicle group vs 2.2 ± 0.6 μg/mg tissue in the BMP7 group and in the thoracic aorta: 71 ± 27 μg/mg tissue in the vehicle group vs 54 ± 18 μg/mg tissue in the BMP7 group. Likewise, normalization of the uremic environment by aorta transplantation had no effect on the Ca-content of the calcified aorta: 16.3 ± 0.6 μg/mg tissue pre-transplantation vs 15.9 ± 2.3 μg/mg tissue post-transplantation. Aortic expression of genes directly linked to extracellular matrix calcification was not affected by BMP7 treatment, which hypothetically might explain persistent high Ca-content in established vascular calcification. The present results highlight the importance of preventing the development of vascular calcification in chronic kidney disease. Once established, vascular calcification persists even in the setting when hyperphosphatemia or the uremic milieu is abolished.

Exogenous BMP-7 Facilitates the Recovery of Cardiac Function after Acute Myocardial Infarction through Counteracting TGF-β1 Signaling Pathway.

Myocardial fibrosis after acute myocardial infarction (AMI) is one of the main causes of myocardial remodeling and heart function abnormalities. Bone morphogenetic protein-7 (BMP-7) has been reported to play essential roles in anti-fibrosis. In this study, we demonstrated the role of exogenous BMP-7 on myocardial fibrosis and heart function recovery after AMI. A rat model of AMI was established via ligation of the left anterior descending coronary artery (LAD). Twenty rats were grouped into sham group which underwent chest open operation, but did not receive LAD ligation. Another 40 rats underwent LAD ligation were randomly grouped into saline-treated group (n = 20) and BMP-7-treated group (n = 20) which received saline treatment or exogenous BMP-7 treatment for 14 days, respectively. Two weeks after LAD ligation, the survival rate of BMP-7-treated AMI group was significantly improved compared to the saline group. Moreover, the cardiac function was preserved as shown by echocardiography examination, and the infarcted size was limited upon BMP-7 treatment. In addition, we investigated the role of TGF-β1 signaling pathway in BMP-7-mediated cardioprotective effects by analyzing the expression levels of TGF-β1, Smad 2 and Smad 3 in the infarct zone, border zone, and non-infarct zone. Western blot and quantitative PCR results suggested that BMP-7 attenuated myocardial fibrosis through counteracting TGF-β1 signaling pathway, thereby exerting cardioprotective effects. In conclusion, our data provide a potential therapeutic direction for preserving cardiac function and improving prognosis of AMI patients.

The anti-myeloma activity of bone morphogenetic protein 2 predominantly relies on the induction of growth arrest and is apoptosis-independent

Multiple myeloma (MM), a malignancy of the bone marrow, is characterized by a pathological increase in antibody-producing plasma cells and an increase in immunoglobulins (plasmacytosis). In recent years, bone morphogenetic proteins (BMPs) have been reported to be activators of apoptotic cell death in neoplastic B cells in MM. Here, we use bone morphogenetic protein 2 (BMP2) to show that the "apoptotic" effect of BMPs on human neoplastic B cells is dominated by anti-proliferative activities and cell cycle arrest and is apoptosis-independent. The anti-proliferative effect of BMP2 was analysed in the human cell lines KMS12-BM and L363 using WST-1 and a Coulter counter and was confirmed using CytoTox assays with established inhibitors of programmed cell death (zVAD-fmk and necrostatin-1). Furthermore, apoptotic activity was compared in both cell lines employing western blot analysis for caspase 3 and 8 in cells treated with BMP2 and FasL. Additionally, expression profiles of marker genes of different cell death pathways were analysed in both cell lines after stimulation with BMP2 for 48h using an RT-PCR-based array. In our experiments we observed that there was rather no reduction in absolute cell number, but cells stopped proliferating following treatment with BMP2 instead. The time frame (48–72 h) after BMP2 treatment at which a reduction in cell number is detectable is too long to indicate a directly BMP2-triggered apoptosis. Moreover, in comparison to robust apoptosis induced by the approved apoptotic factor FasL, BMP2 only marginally induced cell death. Consistently, neither the known inhibitor of apoptotic cell death zVAD-fmk nor the necroptosis inhibitor necrostatin-1 was able to rescue myeloma cell growth in the presence of BMP2.

Suppression of BMP-7 by histone deacetylase 2 promoted apoptosis of renal tubular epithelial cells in acute kidney injury

Cisplatin, a highly effective and widely used chemotherapeutic agent, has a major limitation for its nephrotoxicity. Currently, there are no therapies available to treat or prevent cisplatin nephrotoxicity. We recently identified a novel strategy for attenuating its nephrotoxicity in chemotherapy by histone deacetylase (HDAC) inhibitors via epigenetic modification to enhance bone morphogenetic protein 7 (BMP-7) expression. Cisplatin upregulated the activity of HDAC2 in the kidney. Inhibition of HDAC with clinically used trichostatin A (TSA) or valproic acid (VPA) suppressed cisplatin-induced kidney injury and epithelial cell apoptosis. Overexpression of HDAC2 promotes CP-treated tubular epithelium cells apoptosis. Chromatin immunoprecipitation assay clearly detected HDAC2 assosiation with BMP-7 promoter. Western blot and immunofluorescence results demonstrated that the expression of BMP-7 was clearly induced by TSA or VPA in vivo and in vitro. Interestingly, administration of recombinant BMP-7 (rhBMP-7) reduced cisplatin-induced kidney dysfunction. Moreover, BMP-7 treatment suppressed epithelial cell apoptosis and small interfering RNA-based knockdown of BMP-7 expression abolished HDAC inhibitors suppression of epithelial cell apoptosis in vitro. Results of current study indicated that TSA or VPA inhibited apoptosis of renal tubular epithelial cells via promoting the level of BMP-7 epigenetically through targeting HDAC2. Hence, HDAC inhibitors could be useful therapeutic agents for the prevention of cisplatin nephrotoxicity.

A small population of liver endothelial cells undergoes endothelial-to-mesenchymal transition in response to chronic liver injury.

Rising evidence points to endothelial-to-mesenchymal transition (EndMT) as a significant source of the mesenchymal cell population in fibrotic diseases. In this context, we hypothesized that liver endothelial cells undergo EndMT during fibrosis progression. Cirrhosis in mice was induced by CCl4 A transgenic mouse expressing a red fluorescent protein reporter under the control of Tie2 promoter (Tie2-tdTomato) was used to trace the acquisition of EndMT. Sinusoidal vascular connectivity was evaluated by intravital microscopy and high-resolution three-dimensional confocal microscopy. A modest but significant fraction of liver endothelial cells from both cirrhotic patients and CCl4-treated Tie2-tdTomato mice acquired an EndMT phenotype characterized by the coexpression of CD31 and α-smooth muscle actin, compared with noncirrhotic livers. Bone morphogenetic protein-7 (BMP-7) inhibited the acquisition of EndMT induced by transforming growth factor-β1 (TGF-β1) treatment in cultured primary mouse liver endothelial cells from control mice. EndMT was also reduced significantly in vivo in cirrhotic Tie2-tdTomato mice treated intraperitoneally with BMP-7 compared with untreated mice (1.9 ± 0.2 vs. 3.8 ± 0.3%, respectively; P < 0.05). The decrease of EndMT in cirrhotic livers correlated with a significant decrease in liver fibrosis (P < 0.05) and an improvement in the vascular disorganization rate (P < 0.05). We demonstrated the acquisition of the EndMT phenotype by a subpopulation of endothelial cells from cirrhotic livers in both animal models and patients. BMP-7 treatment decreases the occurrence of the EndMT phenotype and has a positive impact on the severity of disease by reducing fibrosis and sinusoidal vascular disorganization.NEW & NOTEWORTHY A subpopulation of liver endothelial cells from cirrhotic patients and mice with liver fibrosis undergoes endothelial-to-mesenchymal transition. Liver endothelial cells from healthy mice could transition into a mesenchymal phenotype in culture in response to TGF-β1 treatment. Fibrotic livers treated chronically with BMP-7 showed lower EndMT acquisition, reduced fibrosis, and improved vascular organization.

The periosteal requirement and temporal dynamics of BMP2‐induced middle phalanx regeneration in the adult mouse

Regeneration of mammalian limbs is restricted to amputation of the distal digit tip, the terminal phalanx (P3). The adjacent skeletal element, the middle phalanx (P2), has emerged as a model system to investigate regenerative failure and as a site to test approaches aimed at enhancing regeneration. We report that exogenous application of bone morphogenetic protein 2 (BMP2) stimulates the formation of a transient cartilaginous callus distal to the amputation plane that mediates the regeneration of the amputated P2 bone. BMP2 initiates a significant regeneration response during the periosteal‐derived cartilaginous healing phase of P2 bone repair, yet fails to induce regeneration in the absence of periosteal tissue, or after boney callus formation. We provide evidence that a temporal component exists in the induced regeneration of P2 that we define as the “regeneration window.” In this window, cells are transiently responsive to BMP2 after the amputation injury. Simple re‐injury of the healed P2 stump acts to reinitiate endogenous bone repair, complete with periosteal chondrogenesis, thus reopening the “regeneration window” and thereby recreating a regeneration‐permissive environment that is responsive to exogenous BMP2 treatment.

Insulin-like growth factor-1 (IGF-1) enhances the osteogenic activity of bone morphogenetic protein-6 (BMP-6) in vitro and in vivo, and together have a stronger osteogenic effect than when IGF-1 is combined with BMP-2.

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.

Bone morphogenetic protein-2 enhances the osteogenic differentiation capacity of mesenchymal stromal cells derived from human bone marrow and umbilical cord.

Mesenchymal stromal cells (MSCs) are multipotent cells that can give rise to different cell types of the mesodermal lineages. They are powerful sources for cell therapy in regenerative medicine as they can be isolated from various tissues, and can be expanded and induced to differentiate into multiple lineages. Recently, the umbilical cord has been suggested as an alternative source of MSCs. Although MSCs derived from the umbilical cord can be induced to differentiate into osteoblasts with a phenotypic similarity to that of bone marrow-derived MSCs, the differentiation ability is not consistent. In addition, MSCs from the umbilical cord require a longer period of time to differentiate into osteoblasts. Previous studies have demonstrated the benefits of bone morphogenetic protein-2 (BMP-2) in bone tissue regeneration. In addition, several studies have supported the use of BMP-2 in periodontal regeneration, sinus lift bone-grafting and non-unions in oral surgery. Although the use of BMP-2 for bone tissue regeneration has been extensively investigated, the BMP-2-induced osteogenic differentiation of MSCs derived from the umbilical cord has not yet been fully examined. Therefore, in this study, we aimed to examine the effects of BMP-2 on the osteogenic differentiation of MSCs derived from umbilical cord compared to that of MSCs derived from bone marrow. The degree of osteogenic differentiation following BMP-2 treatment was determined by assessing alkaline phosphatase (ALP) activity, and the expression profiles of osteogenic differentiation marker genes, osterix (Osx), Runt-related transcription factor 2 (Runx2) and osteocalcin (Ocn). The results revealed that BMP-2 enhanced the osteogenic differentiation capacity of MSCs derived from both bone marrow and umbilical cord as demonstrated by increased ALP activity and the upregulation of osteogenic differentiation marker genes. The enhancement of the osteogenic differentiation capacity of MSCs by BMP-2 suggests that these MSCs may be used as alternative sources for bone engineering or cell therapy in regenerative medicine.

Direct Gingival Fibroblast/Osteoblast Transdifferentiation via Epigenetics

Alveolar bone resorption caused by trauma or periodontal diseases has represented a challenge for both dental clinicians and researchers. In this study, we evaluate the osteogenic potential of human gingival fibroblasts (HGFs) through a direct transdifferentiation from HGFs to functional osteoblasts via epigenetic modification and osteogenic signaling with bone morphogenetic protein 2 (BMP2) in vitro and in vivo. HGF treatment with 5-aza-2′-deoxycytidine (5-aza-dC) induced demethylation in the hypermethylated CpG islands of the osteogenic lineage marker genes RUNX2 and ALP, and subsequent BMP2 treatment successfully drove the fibroblasts to the osteoblasts’ lineage. Cell morphological changes viewed under microscopy and alkaline phosphatase (ALP) and alizarin red S (ARS) staining confirmed the osteoblastic change mediated by epigenetic modification as did real-time polymerase chain reaction (PCR), methylation-specific PCR (MSP), and chromatin immunoprecipitation (ChIP) assay, which demonstrated the altered methylation patterns in the RUNX2 and ALP promoter regions and their effect on gene expression. Furthermore, micro–computed tomography (CT) analysis of in vivo mouse cell transplantation experiments showed high-density signal in the epigenetically modified HGF group; in addition, a significant amount of bone formation was observed in the transplanted material using hematoxylin and eosin (H&E) staining as well. Collectively, our results indicate that epigenetic modification permits the direct programming of HGFs into functional osteoblasts, suggesting that this approach might open a novel therapeutic avenue in alveolar bone regeneration.

Bone morphogenetic protein-2-mediated pain and inflammation in a rat model of posterolateral arthrodesis.

BackgroundBone morphogenetic protein-2 (BMP-2) is a pleiotropic, secreted molecule with diverse effects. The potent ability of BMP-2 to stimulate bone growth prompted its widespread clinical use for arthrodesis (spine fusion). However, elevated post-operative pain in patients treated with BMP-2 has been increasingly reported. Determining whether BMP-2 induces pain directly or whether it induces neuroinflammation, which could lower the threshold for pain, is important for developing therapeutic interventions. We therefore modeled the clinical use of BMP-2 for posterior lumbar fusion by implanting absorbable collagen sponges soaked with either recombinant human BMP-2 (rhBMP-2) or vehicle above the L4–L5 transverse processes of rat spine.ResultsUsing microarray analysis we found that implantation of rhBMP-2-soaked absorbable collagen sponges resulted in altered expression of numerous pro-inflammatory genes in the adjacent dorsal root ganglia (DRG) showing that implantation of rhBMP-2/absorbable collagen sponges triggers potent neuroinflammatory responses in the DRG-2. Interestingly, direct BMP-2 treatment of DRG explants resulted in changes in gene expression that were not specifically pro-inflammatory. Rats implanted with rhBMP-2 in absorbable collagen sponges also exhibited a transient change in thermal and mechanical sensitivity indicating that rhBMP-2 applied to the lumbar spine could increase pain sensitivity. Immunohistochemical analysis indicated macrophage infiltration in the DRG and spinal nerve in rats implanted with rhBMP-2/absorbable collagen sponges or absorbable collagen sponges alone, but not in rats that underwent surgery without implantation of the absorbable collagen sponges suggesting that the sponges contributed to the biological response. Indeed, analysis of DRGs taken from rats implanted with absorbable collagen sponges without rhBMP-2 showed a significant change in gene expression distinct from DRGs from rats undergoing surgery only.ConclusionsOur data indicate that implantation of rhBMP-2/absorbable collagen sponges on the lumbar spine triggers potent neuroinflammatory responses in the DRG. Importantly, however, these BMP-2 effects may be partially mediated through a response to the absorbable collagen sponges.

Do osteoporotic fractures constitute a greater recalcitrant challenge for skeletal regeneration? Investigating the efficacy of BMP-7 and zoledronate treatment of diaphyseal fractures in an open fracture osteoporotic rat model

SummaryOsteoporotic fractures may pose a challenge for skeletal regeneration. This study investigates if pharmaceutical interventions such as bone morphogenetic protein 7 (BMP-7) alone or in combination with Zoledronate have equivalent efficacy in osteoporotic bone? Our findings suggest they do and that an osteoporotic bone environment may increase sensitivity to BMP-7.IntroductionOsteoporosis is thought to contribute to delayed or impaired bone healing. Bone morphogenetic protein 7 (BMP-7) alone or synergistically combined with zoledronate (ZA) has proven effective in augmenting the regenerative response in healthy young male rats. Yet their comparative efficacy in an osteoporotic bone environment is unknown. Our study aimed to answer the following questions using the ovariectomized (OVX) rat model of osteoporosis: Do osteoporotic fractures pose a greater challenge for skeletal regeneration? Are interventions with BMP-7-alone or combined with ZA of equivalent efficacy in osteoporotic bone?MethodsSham operations (n = 33) or ovariectomies (n = 34) were performed in 12-week-old female Sprague-Dawley rats. Mid-diaphyseal open femoral osteotomies were created at 24 weeks of age and the rats allocated to either (i) untreated, (ii) BMP-7-only or (iii) BMP-7 + ZA treatment groups. At 6 weeks post-osteotomy, fracture healing was evaluated by radiography, μCT and 3-point bending mechanical tests.ResultsCumulatively, radiological, micro-structural and mechanical measures were equivalent in both healthy and osteoporotic environments. A reduced response to BMP-7-alone was observed in healthy rats that may be age/gender- or protocol/fracture-model dependent. Conversely, the BMP-7-only treated OVX group attained 100 % union in addition to significantly increased measures of mineralized bone volume, total callus volume, peak force and absorbed energy relative to untreated OVX fractures.ConclusionsOur findings refute the hypothesis that osteoporotic fractures constitute a greater recalcitrant challenge for skeletal regeneration. Furthermore, our results suggest that an oestrogen-deficient environment may in fact cause an increased sensitivity to BMP-7.

Enhanced Mandibular Bone Repair by Combined Treatment of Bone Morphogenetic Protein 2 and Small-Molecule Phenamil.

Growth factor-based therapeutics using bone morphogenetic protein 2 (BMP-2) presents a promising strategy to reconstruct craniofacial bone defects such as mandible. However, clinical applications require supraphysiological BMP doses that often increase inappropriate adipogenesis, resulting in well-documented, cyst-like bone formation. Here we reported a novel complementary strategy to enhance osteogenesis and mandibular bone repair by using small-molecule phenamil that has been shown to be a strong activator of BMP signaling. Phenamil synergistically induced osteogenic differentiation of human bone marrow mesenchymal stem cells with BMP-2 while suppressing their adipogenic differentiation induced by BMP-2 in vitro. The observed pro-osteogenic and antiadipogenic activity of phenamil was mediated by expression of tribbles homolog 3 (Trb3) that enhanced BMP-smad signaling and inhibited expression of peroxisome proliferator-activated receptor gamma (PPARγ), a master regulator of adipogenesis. The synergistic effect of BMP-2+phenamil on bone regeneration was further confirmed in a critical-sized rat mandibular bone defect by implanting polymer scaffolds designed to slowly release the therapeutic molecules. These findings indicate a new complementary osteoinductive strategy to improve clinical efficacy and safety of current BMP-based therapeutics.

BMP7 retards peripheral myelination by activating p38 MAPK in Schwann cells.

Schwann cell (SC) myelination is pivotal for the proper physiological functioning of the nervous system, but the underlying molecular mechanism remains less well understood. Here, we showed that the expression of bone morphogenetic protein 7 (BMP7) inversely correlates with myelin gene expression during peripheral myelination, which suggests that BMP7 is likely a negative regulator for myelin gene expression. Our experiments further showed that the application of BMP7 attenuates the cAMP induced myelin gene expression in SCs. Downstream pathway analysis suggested that both p38 MAPK and SMAD are activated by exogenous BMP7 in SCs. The pharmacological intervention and gene silence studies revealed that p38 MAPK, not SMAD, is responsible for BMP7-mediated suppression of myelin gene expression. In addition, c-Jun, a potential negative regulator for peripheral myelination, was up-regulated by BMP7. In vivo experiments showed that BMP7 treatment greatly impaired peripheral myelination in newborn rats. Together, our results established that BMP7 is a negative regulator for peripheral myelin gene expression and that p38 MAPK/c-Jun axis might be the main downstream target of BMP7 in this process.

An investigation of BMP-7 mediated alterations to BMP signalling components in human tenocyte-like cells

The incidence of tendon re-tears post-surgery is an ever present complication. It is suggested that the application of biological factors, such as bone morphogenetic protein 7 (BMP-7), can reduce complication rates by promoting tenogenic characteristics in in vitro studies. However, there remains a dearth of information in regards to the mechanisms of BMP-7 signalling in tenocytes. Using primary human tenocyte-like cells (hTLCs) from the supraspinatus tendon the BMP-7 signalling pathway was investigated: induction of the BMP associated Smad pathway and non-Smad pathways (AKT, p38, ERK1/2 and JNK); alterations in gene expression of BMP-7 associated receptors, Smad pathway components, Smad target gene (ID1) and tenogenic marker scleraxis. BMP-7 increases the expression of specific BMP associated receptors, BMPR-Ib and BMPR-II, and Smad8. Additionally, BMP-7 activates significantly Smad1/5/8 and slightly p38 pathways as indicated by an increase in phosphorylation and proven by inhibition experiments, where p-ERK1/2 and p-JNK pathways remain mainly unresponsive. Furthermore, BMP-7 increases the expression of the Smad target gene ID1, and the tendon specific transcription factor scleraxis. The study shows that tenocyte-like cells undergo primarily Smad8 and p38 signalling after BMP-7 stimulation. The up-regulation of tendon related marker genes and matrix proteins such as Smad8/9, scleraxis and collagen I might lead to positive effects of BMP-7 treatment for rotator cuff repair, without significant induction of osteogenic and chondrogenic markers.