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

OBJECTIVEBone marrow stromal cells (bMSCs) of rabbits transferred with mammalian hBMP-4 expression plasmid were used to construct tissue-engineered bone. Gene therapy combined with tissue-engineering technique was explored to further improve osteogenesis.

METHODSpEGFP-hBMP-4 plasmid was constructed by subcloning technique. bMSCs were then transferred with either pEGFP-hBMP-4, pEGFP plasmid by lipofectamine or left uninfected in vitro. The cells from the 3 groups were combined with natural non-organic bone (NNB) to construct tissue-engineered bones, which were subcutaneously implanted into nude mice (6 implants per group) for 4 weeks. Specimens were evaluated through histological and computerized new bone formation analysis.

RESULTSpEGFP-hBMP-4 plasmid was successfully constructed. bMSCs could attach and proliferate on the surface on NNB. In vivo experiment showed that new bone formation in pEGFP-hBMP-4 group was higher than those of the control groups.

CONCLUSIONSTissue-engineered bone using hBMP-4 gene modified bMSCs might be an ideal alternative for the repair of bone.

Animals ; Bone Morphogenetic Protein 4 ; Bone Morphogenetic Proteins ; genetics ; Genetic Therapy ; Humans ; Mice ; Mice, Nude ; Osteogenesis ; Rabbits ; Tissue Engineering

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

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

OBJECTIVETo determine whether an adenoviral construct containing bone morphogenetic protein-4 (BMP-4) gene can be used for lumbar spinal fusion.

METHODSTwelve New Zealand white rabbits were randomly divided into two groups, 8 in the experimental group and 4 in the control group. Recombinant, replication-defective type 5 adenovirus with the cytomegalovirus (CMV) promoter and BMP-4 gene (Ad-BMP-4) was used. Another adenovirus constructed with the CMV promoter and beta-galactosidase gene (Ad-beta-gal) was used as control. Using collagen sponge as a carrier, Ad-BMP-4 (2.9 multiply 10(8) pfu/ml ) was directly implanted on the surface of L(5)-L(6) lamina in the experimental group, while Ad-beta-gal was implanted simultaneously in the control group. X-ray was obtained at 3, 6, and 12 weeks postoperatively to observe new bone formation. When new bone formation was identified, CT scans and three-dimensional reconstruction were obtained. After that, the animals were killed and underwent histological inspection.

RESULTSIn 12 weeks after operation, new bone formation and fusion were observed on CT scans in the experimental group, without the evidence of ectopic calcification in the canal. Negative results were found in the control group. Histological analysis demonstrated endochondral bone formation at the operative site and fusion at early stage was testified.

CONCLUSIONSIn vivo gene therapy using Ad-BMP-4 for lumbar posterolateral spinal fusion is practicable and effective.

Adenoviridae ; genetics ; Animals ; Bone Morphogenetic Protein 4 ; Bone Morphogenetic Proteins ; therapeutic use ; Genetic Therapy ; methods ; Humans ; Lumbar Vertebrae ; surgery ; Rabbits ; Spinal Fusion

OBJECTIVESTo construct an eucaryotic expression plasmid carrying the BMP7 gene and express in MSCs.

METHODSThe BMP7 gene was cloned into the eucaryotic expression vector pcDNA3.1. At the same time, mesenchymal stem cells (MSCs) were isolated and cultured in vitro. The plasmid carrying the BMP7 gene was transfected into MSCs.

RESULTSPCR and digesting demonstrated that the eucaryotic expression plasmid -pcDNA-BMP7 was obtained. RT-PCR and immunohistochemical methods showed that the BMP7 gene was expressed in MSCs.

CONCLUSIONConstruction of an eucaryotic expression plasmid carrying BMP7 gene and expression in MSCs provide a sound basis for gene therapy using the BMP7 gene and the ideal seeds for tissue engineering.

Bone Morphogenetic Protein 7 ; Bone Morphogenetic Proteins ; genetics ; Genetic Therapy ; Humans ; Mesenchymal Stromal Cells ; metabolism ; Plasmids ; Polymerase Chain Reaction ; Tissue Engineering ; Transforming Growth Factor beta

Animals ; Bone Morphogenetic Protein Receptors ; Bone Morphogenetic Proteins ; genetics ; physiology ; Cell Differentiation ; Gene Expression Regulation ; Hair Follicle ; cytology ; embryology ; physiology ; Humans ; Receptors, Growth Factor ; analysis ; Stem Cells ; cytology

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