Crosstalk between Wnt and bone morphogenetic protein signaling during osteogenic differentiati

Mesenchymal stem cells(MSCs)originate from many sources,including the bone marrow and adipose tissue,and differentiate into various cell types,such as osteoblasts and adipocytes.Recent studies on MSCs have revealed that many transcription factors and signaling pathways control osteogenic development.Osteogenesis is the process by which new bones are formed;it also aids in bone remodeling.Wnt/β-catenin and bone morphogenetic protein(BMP)signaling pathways are involved in many cellular processes and considered to be essential for life.Wnt/β-catenin and BMPs are important for bone formation in mammalian development and various regulatory activities in the body.Recent studies have indicated that these two signaling pathways contribute to osteogenic differen-tiation.Active Wnt signaling pathway promotes osteogenesis by activating the downstream targets of the BMP signaling pathway.Here,we briefly review the molecular processes underlying the crosstalk between these two pathways and explain their participation in osteogenic differentiation,emphasizing the canonical pathways.This review also discusses the crosstalk mechanisms of Wnt/BMP signaling with Notch-and extracellular-regulated kinases in osteogenic differentiation and bone development.

AAV-mediated expression of p65shRNA and bone morphogenetic protein 4 synergistically enhances chondrocyte regeneration

BACKGROUND:Adeno-associated virus(AAV)gene therapy has been proven to be reliable and safe for the treatment of osteoarthritis in recent years.However,given the complexity of osteoarthritis pathogenesis,single gene manipulation for the treatment of osteoarthritis may not produce satisfactory results.Previous studies have shown that nuclear factorκB could promote the inflammatory pathway in osteoarthritic chondrocytes,and bone morphogenetic protein 4(BMP4)could promote cartilage regeneration.OBJECTIVE:To test whether combined application of AAV-p65shRNA and AAV-BMP4 will yield the synergistic effect on chondrocytes regeneration and osteoarthritis treatment.METHODS:Viral particles containing AAV-p65-shRNA and AAV-BMP4 were prepared.Their efficacy in inhibiting inflammation in chondrocytes and promoting chondrogenesis was assessed in vitro and in vivo by transfecting AAV-p65-shRNA or AAV-BMP4 into cells.The experiments were divided into five groups:PBS group;osteoarthritis group;AAV-BMP4 group;AAV-p65shRNA group;and BMP4-p65shRNA 1:1 group.Samples were collected at 4,12,and 24 weeks postoperatively.Tissue staining,including safranin O and Alcian blue,was applied after collecting articular tissue.Then,the optimal ratio between the two types of transfected viral particles was further investigated to improve the chondrogenic potential of mixed cells in vivo.RESULTS AND CONCLUSION:The combined application of AAV-p65shRNA and AAV-BMP4 together showed a synergistic effect on cartilage regeneration and osteoarthritis treatment.Mixed cells transfected with AAV-p65shRNA and AAV-BMP4 at a 1:1 ratio produced the most extracellular matrix synthesis(P<0.05).In vivo results also revealed that the combination of the two viruses had the highest regenerative potential for osteoarthritic cartilage(P<0.05).In the present study,we also discovered that the combined therapy had the maximum effect when the two viruses were administered in equal proportions.Decreasing either p65shRNA or BMP4 transfected cells resulted in less collagen II synthesis.This implies that inhibiting inflammation by p65shRNA and promoting regeneration by BMP4 are equally important for osteoarthritis treatment.These findings provide a new strategy for the treatment of early osteoarthritis by simultaneously inhibiting cartilage inflammation and promoting cartilage repair.

Cloning of Rabbit Bone Morphogenetic Protein 15 and Its Expression During in vitro Maturation of Rabbit Oocytes

Partial cDNA sequence of rabbit BMP15 was cloned by RT-PCR from rabbit ovaries, showing a similarity of 83%-90% with the BMP15 nucleotide sequences in humans, mice, ovine, sheep, cows and pigs. The expression of BMP15 in rabbit cumulus-oocyte complexs during oocytes in vitro maturation （IVM） was measured by fluorescent quantitative RT-PCR method. BMP 15 was expressed at low levels in immature oocytes and increased to the highest level at 16h of IVM, which coincides with the time of cumulus cell expansion, then declined slowly under IVM cultivation. The expression pattern of BMP 15 suggested that it might be important in cumulus expansion in rabbits.

Influence of bone morphogenetic protein type IA receptor conditional knockout in lens on expression of bone morphogenetic protein 4 in lens

AIM: To investigate the influence of bone morphogenetic protein type IA receptor [BMPR-IA(ALK3)] conditional knockout in lens on expression of bone morphogenetic protein 4(BMP4) in lens during the development of the vertebrate eye.METHODS: Cre-positive mice were mated with Crenegative mice to generate 50% Cre-positive(conditional knockout, CKO) 4 embryos, 8 eyes and 50% Cre-negative offspring(wild type, WT) 4 embryos, 8 eyes. The embryos were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned to a thickness of 4 μm.Removal of paraffin wax and dehydrating for sections,and then the procedure of in situ hybridization was processed, BMP4 MK1784-m(BOSTER) was used, and observed the expression of BMP4 in the lens in experimental group and control group. We selected SPSS11.0 software for statistical analysis, P【0.05 showed that the difference was statistically significant.· RESULTS: Four embryos of each genotype were examined, totally we had 8 embryos, 16 eyes. We got the uniform outcomes in all the embryos. We found ALK3 was required during lens growing, but was not essential for the formation of lens. We observed that the expression of BMP4 in the lens was significantly reduced in all 8 ALK3 CKO lens, BMP4 expression was normal in all the 8 WT lens, P 【0.01. This phenomenon became increasingly visible in accordance with embryo development. The most apparent alteration was present at stage E15.5.CONCLUSION: ALK3 is essential for lens growth. The influence of ALK3 on the expression of BMP4 is present during the development of mice lens.

Extraction, Purification and Identification of Bone Morphogenetic Protein in Conditioned Medium of Osteosarcoma Cell (MG-63)

Objective: To find out a method of extraction and purification of bone morphogenetic protein (BMP) from osteosarcoma cell conditioned medium, and evaluate the biological activity of BMP.Methods: Conditioned medium of osteosarcoma cell lines (MG-63) was collected, concentrated and dialyzed. The concentrated protein was purified through gel chromatography on Sephcryl-S-100. The purified protein was tested by BMP monoclonal antibody (McAb), its molecular weight (MW) was determined by SDS-PAGE and its biological activity was demonstrated by heterotopic ossification.Results: The purified protein was proved to be BMP by BMP McAb, had a satisfactory heterotopic ossification, and its MW was about 21 kD.Conclusion: BMP existed in the conditioned medium of osteosarcoma cell and had a satisfactory biological activity after purification. Because osteosarcoma cell can be cultured and grew for a long timein vitro, this method will be helpful to a vast extraction of BMP and clinical application. Key words osteosarcoma cell - conditioned medium - bone morphogenetic protein - protein purification This project was a key scientific and technological program of Hubei Provicial Scientific and Technological Committee (No. 002p1503).

Effect of Danshao Huaxian capsule on Gremlin and bone morphogenetic protein-7 expression in hepatic fibrosis in rats

AIM: To observe the effect of Danshao Huaxian capsule (DHC) on the expression of Gremlin and bone morphogenetic protein-7 (BMP-7) in the liver of hepatic fibrosis rats.

Expression of Vascular Endothelial Growth Factor in Bone Morphogenetic Protein-2 Induced Osteogenesis

An experimental model of femoral muscular pouch in 20 mice was adopted. The expression of VEGF was examined by in situ hybridization method and immunohistochemical method in bone morphogenetic protein- 2 induced osteogenesis . The experimental results demonstrated that the expression signals of VEGF mRNA and VEGF appeared in cytoplasm during condensation of mesenehymal cell. As the mesenchymal cells differentiated into precartilage, the expression signals decreased in mesenehymal cells, but increased in chondrocytes and kept getting denser in the process of cartilage maturity. The peak expression of VEGF mRNA and VEGF in the experimental group appeared on the 14 th day, accompanied by numerons hypertrophic chondrocytes. When mature cartilage calcified and neu, bone trabecula formed, the expression of VEGF mRNA and VEGF decreased in chondrocytes, but still expressed moderately in the osteoblasts and osteocytes. Signals of VEGF mRNA and VEGF can not be detected in the control groups.

The biological function of type I receptors of bone morphogenetic protein in bone

Bone morphogenetic proteins （BMPs） have multiple roles in skeletal development, homeostasis and regeneration. BMPs signal via type I and type II serine/threonine kinase receptors （BMPRI and BMPRII）. In recent decades, genetic studies in humans and mice have demonstrated that perturbations in BMP signaling via BMPRI resulted in various diseases in bone, cartilage, and muscles. In this review, we focus on all three types of BMPRI, which consist of activin-like kinase 2 （ALK2, also called type IA activin receptor）, activin- llke kinase 3 （ALK3, also called BMPRIA）, and activin-like kinase 6 （ALK6, also called BMPRIB）. The research areas covered include the current progress regarding the roles of these receptors during myogenesis, chondrogenesis, and osteogenesis. Understanding the physiological and pathological functions of these receptors at the cellular and molecular levels will advance drug development and tissue regeneration for treating musculoskeletal diseases and bone defects in the future.

Effect of transforming growth factor beta and bone morphogenetic proteins on rat hepatic stellate cell proliferation and transdifferentiation

AIM: To explore different roles of TGF-β (transforming growth factor beta) and bone morphogenetic proteins (BMPs)in hepatic stellate cell proliferation and trans-differentiation.METHODS: Hepatic stellate cells were isolated from male Sprague-Dawley rats. Sub-cultured hepatic stellate cells were employed for cell proliferation assay with WST-1 reagent and Western blot analysis with antibody against smooth muscle alpha actin (SMA).RESULTS: The results indicated that TGF-β1 significantly inhibited cell proliferation at concentration as low as 0.1 ng/ml, but both BMP-2 and BMP-4 did not affect cell proliferation at concentration as high as 10 ng/ml. The effect on hepatic stellate cell trans-differentiation was similar between TGFβ1 and BMPs. However, BMPs was more potent at transdifferentiation of hepatic stellate cells than TGF-β1. In addition, we observed that TGF-β1 transient reduced the abundance of SMA in hepatic stellate cells.CONCLUSION: TGF-β may be more important in regulation of hepatic stellate cell proliferation while BMPs may be the major cytokines regulating hepatic stellate cell transdifferentiation.

RETINOIC ACID DOWN-REGULATES BONE MORPHOGENETIC PROTEIN 7 EXPRESSION IN RAT WITH CLEFT PALATE

Objective To evaluate the effects of retinoic acid （RA） on expression of bone morphogenetic protein 7 （ BMP-7 ） in rat fetus with cleft palate, and the effects of RA on proliferation and apoptosis of osteoblasts. Methods All-trans RA （ATRA） was used to induce congenital cleft palate in Wistar rat. BMP-7 mRNA expression in maxillary bone tissue of fetal rats was measured by Northern blotting analysis. Flow cytometry and MTF assay were used to measure the apoptosis and proliferation of ATRA-treated MC-3T3-E1 cells. BMP-7 mRNA and protein expressions in ATRA-treated MC-3T3-E1 cells were detected by RT-PCR and Western blotting analysis. Remilts ATRA could induce cleft palate of rat fetus. The incidence rate of cleft palate induced by 100 mg/kg AT-RA （45.5%） was significantly higher than 50 mg/kg ATRA （ 12.5%, P 〈 0. 05 ）. BMP-7 mRNA expression decreased in maxillary bone tissue of rat fetus with cleft palate. MC-3T3-E1 cells proliferation treated with 1 × 10^-6 mol/L ATRA decreased by 60%, the cell apoptosis increased by 2 times. BMP-7 mRNA and protein levels in MC-3T3-E1 cells treated with 1 × 10^-6 mol/L ATRA decreased by 60% and 80%, respectively, compared with ATRA-untreated cells （ P 〈 0.05 ）. Conclusions BMP-7 may play an important role in embryonic palate development. RA may possess the ability to down-regulate cell proliferation through regulation of BMP-7 gene expression.

Bone morphogenetic proteins:Relationship between molecular structure and their osteogenic activity

Bone morphogenetic proteins(BMPs)are a family of potent,multifunctional growth factors belonging to transforming growth factor-(TGF-).They are highly conservative in structures.Over 20 members of BMPs with varying functions such as embryogenesis,skeletal formation,hematopoiesis and neurogenesis have been identified in human body.BMPs are unique growth factors that can induce the formation of bone tissue individually.BMPs can induce the differentiation of bone marrow mesenchymal stem cells into osteoblastic lineage and promote the proliferation of osteoblasts and chondrocytes.BMPs stimulate the target cells by specific membrane-bound receptors and signal transduced through mothers against decapentaplegic(Smads)and mitogen activated protein kinase(MAPK)pathways.It has been demonstrated that BMP-2,BMP-4,BMP-6,BMP-7,and BMP-9 play an important role in bone formation.This article focuses on the molecular characterization of BMPs family members,mechanism of osteogenesis promotion,related signal pathways of osteogenic function,relationships between structure and osteogenetic activity,and the interactions among family members at bone formation.

Bone morphogenetic protein 7 mediates stem cells migration and angiogenesis:therapeutic potential for endogenous pulp regeneration

Pulp loss is accompanied by the functional impairment of defense,sensory,and nutrition supply.The approach based on endogenous stem cells is a potential strategy for pulp regeneration.However,endogenous stem cell sources,exogenous regenerative signals,and neovascularization are major difficulties for pulp regeneration based on endogenous stem cells.Therefore,the purpose of our research is to seek an effective cytokines delivery strategy and bioactive materials to reestablish an ideal regenerative microenvironment for pulp regeneration.In in vitro study,we investigated the effects of Wnt3a,transforming growth factor-beta 1,and bone morphogenetic protein 7(BMP7)on human dental pulp stem cells(h-DPSCs)and human umbilical vein endothelial cells.2D and 3D culture systems based on collagen gel,matrigel,and gelatin methacryloyl were fabricated to evaluate the morphology and viability of h-DPSCs.In in vivo study,an ectopic nude mouse model and an in situ beagle dog model were established to investigate the possibility of pulp regeneration by implanting collagen gel loading BMP7.We concluded that BMP7promoted the migration and odontogenic differentiation of h-DPSCs and vessel formation.Collagen gel maintained the cell adhesion,cell spreading,and cell viability of h-DPSCs in 2D or 3D culture.The transplantation of collagen gel loading BMP7 induced vascularized pulp-like tissue regeneration in vivo.The injectable approach based on collagen gel loading BMP7 might exert promising therapeutic application in endogenous pulp regeneration.

Prolonged propagation of rat neural stem cells relies on inhibiting autocrine/paracrine bone morphogenetic protein and platelet derived growth factor signals

Continuous expansion of rat neural stem cell lines has not been achieved due to proliferation arrest and spontaneous differentiation in vitro. In the current study, neural precursor cells derived from the subventricular zone of adult rats spontaneously underwent astroglial and oligodendroglial differentiation after limited propagation. This differentiation was largely induced by autocrine or paracrine bone morphogenetic protein and platelet derived growth factor signals. The results showed that, by inhibiting bone morphogenetic protein and platelet derived growth factor signals, adult rat neural precursor cells could be extensively cultured in vitro as tripotent stem cell lines. In addition to adult rat neural stem cells, we found that bone morphogenetic protein antagonists can promote the proliferation of human neural stem cells. Therefore, the present findings illustrated the role of autocrine or paracrine bone morphogenetic protein and platelet derived growth factor signaling in determining neural stem cell self-renewal and differentiation. By antagonizing both signals, the long-term propagation of rat neural stem cell lines can be achieved.

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. Twomonth-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.

Construction of Adeno-associated Virus System for Human Bone Morphogenetic Protein 7 Gene

To construct the recombinant adeno-associated virus （rAAV） vector with human bone morphogenetic protein 7 （BMP7） and observe the BMP7 mRNA expression in vitro, BMP7 CDS sequence was cloned into expression plasmid pAAV-MCS of AAV Helper Free System. The recombinant plasmid was identified with enzyme digestion and sequencing. The recombinant plasmid, pAAV-RC, pHelper were co-transfected into AAV-293 cells according to the calcium phosphate-based protocol. The viral stock was collected by 4 rounds of freeze/thaw. After purified and concentrated, the recombinant virus titer was determined by dot-blot assay. HEK293 cells were transfected with the recombinant virus at different MOI, and the expression of BMP7 mRNA was detected by RT-PCR. The results showed rAAV-BMP7 was constructed and packaged successfully. The physical particle titer was 2.5×10^11 vector genomes/mL. There was different expression level of BMP7 mRNA after transfecton. These data suggested that recombinant AAV mediated a stable expression of hBMP7 mRNA in 293 cells. The AAV production method may pave the way of an effective strategy for the jaw bone defection around dental implants.

Regulation of scleral fibroblast differentiation by bone morphogenetic protein-2

Bone morphogenesis proteins(BMPs) are multi-functional growth factors. They are expressed in retina,retinal pigment epithelium(RPE) and sclera and serve as a regulator in the growth and development of the eye. This article reviewed the chondrogenic potency of the sclera,biochemical and pathological changes of myopic scleral tissue and the differentiation of chondrogenesis by BMP-2. We proposed the hypothesis that BMP-2 can regulate differentiate of scleral fibroblasts and affect the development of myopia.

Chondrogenesis of periodontal ligament stem cells by transforming growth factor-β3 and bone morphogenetic protein-6 in a normal healthy impacted third molar

The periodontal ligament-derived mesenchymal stem cell is regarded as a source of adult stem cells due to its multipotency.However, the proof of chondrogenic potential of the cells is scarce.Therefore,we investigated the chondrogenic differentiation capacity of periodontal ligament derived mesenchymal stem cells induced by transforming growth factor(TGF)-p3 and bone morphogenetic protein(BMP)-6.After isolation of periodontal ligament stem cells(PDLSCs) from human periodontal ligament,the cells were cultured in Dulbecco’s modified Eagle’s medium(DMEM) with 20%fetal bovine serum(FBS).A mechanical force initiated chondrogenic differentiation of the cells.For chondrogenic differentiation,10μg·LTGF-β3 or 100μg·LBMP-6 and the combination treating group for synergistic effect of the growth factors.We analyzed the PDLSCs by fluorescence-activated cell sorting and chondrogenesis were evaluated by glycosaminoglycans assay,histology,immunohistochemistry and genetic analysis.PDLSCs showed mesenchymal stem cell properties proved by FACS analysis.Glycosaminoglycans contents were increased 217%by TGF-β3 and 220%by BMP-6. The synergetic effect of TGF-β3 and BMP-6 were shown up to 281%compared to control.The combination treatment increased Sox9, aggrecan and collagen II expression compared with not only controls,but also TGF-P3 or BMP-6 single treatment dramatically.The histological analysis also indicated the chondrogenic differentiation of PDLSCs in our conditions.The results of the present study demonstrate the potential of the dental stem cell as a valuable cell source for chondrogenesis,which may be applicable for regeneration of cartilage and bone fracture in the field of cell therapy.

The Effect of Simvastatin on mRNA Expression of Transforming Growth Factor-β1,Bone Morphogenetic Protein-2 and Vascular Endothelial Growth Factor in Tooth Extraction Socket

Aim To determine the effect of local simvastatin application on the mRNA expression level of transforming growth factor-β1 （TGF-β1）, bone morphogenetic protein-2 （BMP-2） and vascular endothelial growth factor （VEGF） in the tooth sockets of rat. Methodology Forty-eight male Wistar rats were randomly divided into experimental and control groups （n=24）. Polylactic acid/polyglycolic acid copolymer carriers, with or without simvastatin, were implanted into extraction sockets of right mandibular incisors. The expression of TGF-β1, BMP-2 and VEGF mRNA was determined by in situ hybridization in the tooth extraction socket at five days, one week, two weeks and four weeks after implantation. Results The fusiform stroma cells in the tooth extraction socket began to express TGF-β1, BMP-2 and VEGF mRNA in both experimental and control groups from one week after tooth extraction until the end of experiment. The expression of TGF-131 and BMP-2 mRNA in the experimental group was significantly up-regulated after one, two and four weeks, and expression of VEGF mRNA was significantly increased after one and two weeks compared with that in the control group. Conclusion The findings indicate that local administration of simvastatin can influence alveolar bone remodeling by regulating the expression of a school of growth factors which are crucial to osteogenesis in the tooth extraction socket.

Recombinant human bone morphogenetic protein-7 expressed from CHO cells possessing the activity of bone-induced in vitro

Objective To express the recombinant human bone morphogenetic protein-7 (rhBMP-7) in Chinese hamster ovary (CHO) cells and to establish the in vitro biological activity assay of rhBMP-7. Methods Human BMP-7 cDNA was subcloned into pcDNA3.1 mammalian expression vector and transfected to CHO cells by using the lipofectin transfection method. BMP-7 expression cell culture supernatants were harvested and purified for target protein. To analyze the bioactivity of the secreted rhBMP-7, a novel in vitro assay was established by measuring its alkaline phosphatase (ALP) stimulating of osteoblast cell line, W-20-17. Results BMP-7 stably expressing cell clone was selected, which secreted mature disulfide-linked homodimer form of hBMP-7 and had an apparent molecular weight of 36kDa. rhBMP-7 with >95% purity was obtained using 3 step chromatography method. Bioactivity assay showed that the purified protein specifically stimulated W-20-17 cell producing ALP, with a 4-fold increase of ALP activity at 100ng/ml or more, and the EC50 of 15.6ng/ml. Conclusion Purified rhBMP-7 from this CHO expression system has significant biological activity in induction of osteoblast phenotype, which demonstrates potential bone regeneration activity.

Bone morphogenetic protein-7 induced bone marrow stromal cells differentiate into neuron-like cells

Bone morphogenetic protein-7 is widely accepted as an inducer for bone marrow stem cells differentiating into osteoblasts and chondrocytes. Whether bone marrow stromal cells differentiate into neuron-like cells remains unclear. The current study examined the presence of positive cells for intermediate filament protein and microtubule associated protein-2 in the cytoplasm of bone marrow stromal cells induced by bone morphogenetic protein-7 under an inverted microscope, while no expression of glial fibrillary acidic protein was found. Reverse transcription PCR electrophoresis also revealed a positive target band for intermediate filament protein and microtubule-associated protein 2 mRNA. These results confirmed that bone morphogenetic protein-7 induces rat bone marrow stromal cells differentiating into neuron-like cells.