Bone morphogenetic protein (BMP) homodimers are of significant osteoinductivity. However, their clinical application is limited because of high effective dosage. Recently, BMP heterodimers are reported to address the issue. This is a review of the researches on BMP heterodimers, including existent evidences, types and synthetic methods, biological activities in comparison to BMP homodimers and possible mechanisms, further research direction and future expectations.
Animals ; Biopolymers ; Bone Morphogenetic Protein 2 ; Bone Morphogenetic Protein 7 ; Bone Morphogenetic Proteins ; genetics ; pharmacology ; Humans ; Protein Multimerization

BACKGROUNDThe palate is differently regulated and developed along the anterior-posterior axis. The Bmp signal pathway plays a crucial role in palatogenesis. Conditioned-inactivation of Bmp type I receptor Alk2 or Alk3 in the neural crest or craniofacial region leads to palatal cleft in mice. However, how different Bmp members are involved in palatogenesis remains to be elucidated. In the present study, mRNA expression patterns of Bmp2, Bmp3 and Bmp4 in the developing anterior and posterior palates were examined and compared, focusing on the fusion stage.

METHODSTo detect the expression of Bmp mRNA, antisense riboprobes were synthesized by in vitro transcription. Radioactive in situ hybridization was performed on sagital and coronal sections of mice head from E13 to E18.

RESULTSThe expression of these Bmps were developmentally regulated in the anterior and posterior palates prior to, during and after palatal fusion. During palatal fusion, Bmp4 expression shifted from the anterior to the posterior palate, Bmp2 was highly expressed in both the anterior and posterior palates in this process, whereas Bmp3 was only localized in the posterior palate. They showed generally non-overlapping pattern in their expression domains. Thereafter, their expression was detected in both the anterior and posterior palates regulating osteogenesis and myogenesis respectively.

CONCLUSIONSBmp signalling is involved in palatogenesis in multiple stages and has multiple roles in regulating anterior and posterior palatal development. Disturbances of Bmp signalling during palatogenesis might be a possible mechanism of cleft palate.

Animals ; Bone Morphogenetic Protein 2 ; Bone Morphogenetic Protein 3 ; Bone Morphogenetic Protein 4 ; Bone Morphogenetic Proteins ; genetics ; Female ; Gene Expression Regulation, Developmental ; Mice ; Palate ; embryology ; metabolism ; RNA, Messenger ; analysis ; Signal Transduction ; Transforming Growth Factor beta ; genetics

OBJECTIVE: To screen for BMP2 mutation with functional impact in patients with congenital tooth agenesis and to make oral and skeletal phenotype record and functional analysis with in vitro experiments. METHODS: We enrolled eighteen patients with congenital tooth agenesis. The medical and dental history was collected,and clinical and dental examinations including the X-ray examination of oral-facial and skeletal bone were performed for the phenotypic analysis. Blood samples were collected to extract DNA and whole exome sequencing was conducted. The genes involved in oral-facial development and congenital skeletal diseases were investigated for mutation screening. The mutations with functional impact were then investigated. In one patient, the BMP2 mutation with putative functional impact was selected for functional analysis. Wild type and mutant BMP2 plasmids with green fluorescent protein (GFP) tag were constructed and transfected into HEK293T cells. Subcellular protein distribution was observed under laser scanning confocal microscope. The activation of downstream SMAD1/5/9 phosphorylation by BMP2 was detected by Western blotting to investigate the functional impact and genetic pathogenicity. RESULTS: BMP2 mutation NM_001200.3:c.393A>T (p.Arg131Ser), rs140417301 was detected in one patient with congenital tooth agenesis, while for other genes involved in oral-facial development and congenital skeletal diseases, no functionally significant mutation was found. The proband's parents didn't carry this mutation. The father had normal dentition, while the mother lacked one premolar, and both the parents showed normal palate and maxilla. The patient also had maxillary hypoplasia in both sagittal and coronal planes, palatal dysmorphology, and malocclusion, and was diagonsed with osteopenia after the X-ray examnination of his skeletal bone. Functional analysis showed this mutation had normal subcelluar localization but reduced phosphorylation of SMAD1/5/9 (reduction by 32%, 22%, and 27% in three independent replicates). Taken together with family co-segregation, this mutaion was considered as "likely pathogenic". CONCLUSION BMP2 mutation c.393A>T (p. Arg131Ser) affects bone morphogenetic protein signaling activity, and may affect the number of teeth, growth of maxilla and palate, and bone mineral density.
Bone Morphogenetic Protein 2/genetics* ; HEK293 Cells ; Humans ; Mutation ; Phenotype ; Plasmids ; Tooth

OBJECTIVETo characterize the role of Smads proteins in alpha 2 (I) collagen (COL1A2) gene expression induced by bone morphogenetic protein-2 (BMP-2) in odontoblast cell line MDPC-23.

METHODSEndogenous Smad protein expression was determined by immunocytochemistry. Smads function and their role in COL1A2 gene expression were investigated in cotransfection experiments using promoter-luciferase reporter gene construct.

RESULTSMDPC-23 cells expressed Smad1, Smad5 and Smad6. BMP-2 promoted the activation of COL1A2 promoter reporter construct. Transient overexpression of Smad1 or Smad5 was enhanced, while overexpression of Smad6 inhibited BMP-2-induced COL1A2 promoter activity. BMP-2 inducibility could be blocked by overexpression of Smad1 or Smad5 dominant negative mutant.

CONCLUSIONSSmad signaling is functioning and appears to be involved in BMP-2-induced COL1A2 collagen transcription in MDPC-23. Smad signaling may play an important role in odontoblast differentiation and dentin extracellular matrix formation mediated by BMP-2.

Animals ; Bone Morphogenetic Protein 2 ; Bone Morphogenetic Proteins ; genetics ; Cell Line ; Collagen ; genetics ; Collagen Type I ; Mice ; Odontoblasts ; cytology ; metabolism ; Smad Proteins ; physiology ; Transforming Growth Factor beta ; genetics

Optimization of cultivation condition of recombinant E. coli DH5 alpha/pDH-B2m and the condition suitable for expression of recombinant mature peptide of human bone morphogenetic protein-2 was carried out in 500 mL shaking flasks and then transferred to NBS Bioflo IV, a 20 L DO feed-back fed-batch culture system, to obtain rhBMP-2. The results indicate that keeping dissolved oxygen at 40% and controlling nutrient feeding rate with DO feed back strategy can obtain theoretically 3.59 g recombinant mature peptide of hBMP-2 per liter of broth, the final cell density OD600 reaches 57(22.8 g dry cell weight/L), and the expression of rhBMP-2 amounts to 30% of the total protein in E. coli.
Bone Morphogenetic Protein 2 ; Bone Morphogenetic Proteins ; biosynthesis ; genetics ; Culture Media ; Escherichia coli ; genetics ; Fermentation ; Glycerol ; pharmacology ; Recombinant Proteins ; biosynthesis ; Time Factors ; Transforming Growth Factor beta

BACKGROUNDNanobone putty is an injectable and bioresorbable bone substitute. The neutral-pH putty resembles hard bone tissue, does not contain polymers or plasticizers, and is self-setting and nearly isothermic, properties which are helpful for the adhesion, proliferation, and function of bone cells. The aim of this study was to investigate the osteogenic potential of human bone morphogenetic protein 2 (hBMP2) gene activated nanobone putty in inducing ectopic bone formation, and the effects of the hBMP2 gene activated nanobone putty on repairing bone defects.

METHODSTwenty four Kunming mice were randomly divided into two groups. The nanobone putty + hBMP2 plasmid was injected into the right thigh muscle pouches of the mice (experiment side). The nanobone putty + blank plasmid or nanobone putty was injected into the left thigh muscle pouches of the group 1 (control side 1) or group 2 (control side 2), respectively. The effects of ectopic bone formation were evaluated by radiography, histology, and molecular biology analysis at 2 and 4 weeks after operation. Bilateral 15 mm radial defects were made in forty-eight rabbits. These rabbits were randomly divided into three groups: Group A, nanobone putty + hBMP2 plasmid; Group B, putty + blank plasmid; Group C, nanobone putty only. Six rabbits with left radial defects served as blank controls. The effect of bone repairing was evaluated by radiography, histology, molecular biology, and biomechanical analysis at 4, 8, and 12 weeks after operation.

RESULTSThe tissue from the experimental side of the mice expressed hBMP2. Obvious cartilage and island-distributed immature bone formation in implants of the experiment side were observed at 2 weeks after operation, and massive mature bone observed at 4 weeks. No bone formation was observed in the control side of the mice. The ALP activity in the experiment side of the mice was higher than that in the control side. The tissue of Group A rabbits expressed hBMP2 protein and higher ALP level. The new bone formation rate and antibending strength of group A was significantly higher than those of group B and C. The defects in blank control were not healed.

CONCLUSIONSThe hBMP2 gene activated nanobone putty exhibited osteoinductive ability, and had a better bone defect repair capability than that of nanobone putty only.

Absorbable Implants ; Animals ; Bone Morphogenetic Protein 2 ; Bone Morphogenetic Proteins ; analysis ; genetics ; Female ; Male ; Mice ; Osteogenesis ; physiology ; Rabbits ; Transforming Growth Factor beta ; analysis ; genetics

OBJECTIVE: To explore the difference of bone formation potential between hypertrophic nonunion tissue and atrophic nonunion tissue, which may be beneficial to nonunion therapy. METHODS: From October 2010 to March 2014, 40 nonunion tissue samples were collected in Department of Orthopedics, Second Xiangya Hospital. The samples were divided into a hypertrophic nonunion group (n=20) and an atrophic nonunion group (n=20) according to nonunion character; or a 20 to 35 years old group (n=18), a 36 to 50 years old group (n=18), a more than 50 years old group (n=4) according to different ages; or a 9-12 months group (n=21), 13-24 months group (n=14) and a more than 24 months group (n=5) according to different nonunion time. Semi-quantification was performed by SP immunohistochemical method and IPP6.0 was used to analyze the expression of bone morphogenetic protein-2 (BMP-2) through measuring the mean optical density. RESULTS: The mean optical density of BMP-2 was 0.1540±0.0408 in hypertrophic nonunion tissue, 0.1372±0.0372 in atrophic nonunion tissue, there was no significant difference between the 2 groups (P>0.05). The mean optical density of BMP-2 was 0.1477±0.0379 in the 20 to 35 years old group, 0.1419±0.0399 in the 35 to 50 years old group, 0.1456±0.0595 in the more than 50 years old group, there was no significant difference among the three groups (P>0.05). The mean optical density of BMP-2 was 0.1449±0.0366 in the 9-12 months group, 0.1472±0.0400 in the 13-24 months group, 0.1445±0.0541 in the more than 24 months group, there was no significant difference among the 3 groups (P>0.05). CONCLUSION The present results suggest that the hypertrophic nonunion tissue share similar osteogenic potential with the atrophic nonunion tissue.
Adult ; Bone Morphogenetic Protein 2 ; genetics ; metabolism ; Fracture Healing ; Fractures, Ununited ; genetics ; metabolism ; Humans ; Middle Aged ; Osteogenesis ; Young Adult

OBJECTIVETo study the feasibility of osteogenic phenotype expression by human skin fibroblasts induced in polyglycolic acid (PGA) foams and the effect of tumor necrosis factor-alpha (TNF-alpha) on the expression of bone morphogenetic protein (BMP) receptors.

METHODSThe fibroblasts were isolated, purified from human skin. (1) Fibroblasts were seeded onto PGA foams. The cell-PGA complexes were cultured in RCCS for 6 weeks, in the media of TNF-alpha (50 U/ml) and BMP-2 (0.1 microg/ml). 1 d, 3 and 6 weeks later, cells and extracellular matrix were investigated by electron microscopic and histochemistry observation respectively. Secretion of osteogenic markers were analyzed by biochemical methods. (2) Fibroblasts were seeded on the glass fragments or culture flasks and treated with TNF-alpha (50 U/ml) in different usage (one-time, all-time). The RT-PCR method and the immunohistochemistry fluorescence staining were used to examine the influence of TNF-alpha on the mRNA expression and the protein expression of the type I BMP receptors at 2, 4, 6, 8 d after treatment.

RESULTSFibroblasts seeded on the PGA foams formed 3-dimensional matrix 3 weeks after seeding, which was demonstrated as osteo-tissue by tetracycline labeling and ARS staining. Cells secreted much more bone-specific alkaline phosphatase (B-AKP) and osteocalcin (OCN) into supernatant than the cells that were cultured in the media without TNF-a and BMP2. Eight days after all-time usage, the TNF-alpha (50 U/ml) increased the expression of the mRNA and protein of the type IB BMP receptor.

CONCLUSIONSFibroblasts on 3-D cell-foam structures can express osteoblastic phenotype under certain inducing conditions. The numerous fibroblasts in body would be a promising resource for cell seeds candidate of tissue- engineered bone. TNF-alpha provides the essential condition for BMP2's target effect on fibroblasts, and combined use of TNF-alpha and BMP2 is one of the regulating factors.

Bone Morphogenetic Protein 2 ; Bone Morphogenetic Protein Receptors, Type I ; biosynthesis ; genetics ; Bone Morphogenetic Protein Receptors, Type II ; biosynthesis ; genetics ; Bone Morphogenetic Proteins ; pharmacology ; Cell Culture Techniques ; methods ; Cell Differentiation ; drug effects ; Cells, Cultured ; Drug Synergism ; Fibroblasts ; cytology ; drug effects ; metabolism ; Humans ; Phenotype ; Polyglycolic Acid ; Transforming Growth Factor beta ; pharmacology ; Tumor Necrosis Factor-alpha ; pharmacology

OBJECTIVE: To investigate the effect of human bone morphogenetic protein (hBMPs) 2/3/6 and 12 on osteosarcoma cell UMR106. METHODS: Adenovirus-BMP2/3/6 and 12 (AdBMP2/3/6 and12) were used to treat the cell line. Their proliferation, apoptosis, and transmigration were detected by Trypan blue exclusion test, TdT-mediated biotinylated-dUTP nick end labeling (TUNEL), acridine orange-ethidium bromide (AO/EB) double fluorescent dye staining, and transwell-room test, respectively. The alkaline phosphatase (ALP) activity was detected to reflect the differentiation of tumors. RESULTS: Compared with the control groups, the cell survival rate of the experimental groups treated with AdBMP2/3/6 and 12 showed a significant time-dependent decrease (P<0.01). The apoptosis indexes were increased significantly (P<0.01) and the results from TUNEL and AO/EB method were consistent. The cell numbers of transmembrane significantly decreased at 24,48, and 72 h (P<0.01). AdBMP2/3/6 and 12 treatment enhanced the activity of ALP activity from day 3 and this effect might still be observed up to day 9 of the treatment (P<0.01). CONCLUSION hBMPs2/3/6 and 12 can inhibit the proliferation and transmigration, and induce their apoptosis and differentiation in osteosarcoma cell line UMR106.
Adenoviridae ; genetics ; metabolism ; Apoptosis ; drug effects ; Bone Morphogenetic Protein 2 ; pharmacology ; Bone Morphogenetic Protein 3 ; pharmacology ; Bone Morphogenetic Protein 6 ; pharmacology ; Bone Morphogenetic Proteins ; pharmacology ; Bone Neoplasms ; pathology ; Cell Line, Tumor ; Cell Transformation, Neoplastic ; drug effects ; Growth Differentiation Factors ; pharmacology ; Humans ; Osteosarcoma ; pathology ; Recombinant Proteins ; pharmacology

OBJECTIVE: To construct the recombined retroviral expression vector of BMP2/pLEGFP and investigate the bio-activity of the expressed chimeric protein. METHODS: The recombinant vector constructed by gene recombinant technology was analyzed by restriction enzyme digestion and PCR. BMP2/pLEGFP was transfected into COS-7 cells with liposome transfection reagents for transient expression. The expression of chimeric protein BMP2/EGFP was identified by fluorescent microscope and Western blotting. The bio-activity was examined by the cellular activity and animal heterotopic osteogenesis experiment. RESULTS: The recombinant plasmid proved successful by restriction enzyme digestion and PCR. The expression of the chimeric protein was shown by fluorescent microscope and Western blotting. The chimeric protein had the double bio-activities of BMP2 and EGFP identified by the cellular activity and animal heterotopic osteogenesis tests. CONCLUSION The recombinant vector of BMP2/pLEGFP is successfully constructed by the gene recombinant technology and its chimeric protein has double bioactivities of BMP2 and EGFP.
Animals ; Bone Morphogenetic Protein 2 ; Bone Morphogenetic Proteins ; biosynthesis ; genetics ; COS Cells ; Chlorocebus aethiops ; Eukaryotic Cells ; metabolism ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Transfection ; Transforming Growth Factor beta ; biosynthesis ; genetics