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

This study was aimed to compare the expression of BMP4 mRNA in the in-vivo tissue engineering bone constructed with Ca/P ceramics against the expression of BMP4 mRNA in the naturally healing bone. 20 porous Ca/P ceramics cylinders with Phi 5 mm x 8 mm were made and implanted into the dorsal muscles of 5 dogs. As control, one molar tooth was pulled out from each dog to create bone defect for the naturally healing bone at the same time. The specimens and the naturally healing bone were harvested at 1, 2, 4, 12 and 24 weeks post-implantation. After RNA extraction and reverse transcription, bone morphogenetic protein 4 (BMP4) and GAPDH mRNA were detected by real-time quantitative polymerase chain reaction (PCR) method. The results showed that the expression level of BMP4 mRNA of the in-vivo tissue engineering bone constructed with Ca/P ceramics was higher than that of the naturally healing bone in the period of experiment. However, the in-vivo tissue engineering bone had the same chronological order of BMP mRNA expression that the naturally healing bone did. As a bone substitute analogous to autologous bone, the in-vivo tissue engineering bone constructed with Ca/P ceramics has the potential for clinical application.
Animals ; Bone Morphogenetic Protein 4 ; genetics ; metabolism ; Bone Substitutes ; chemistry ; Calcium Phosphates ; chemistry ; Ceramics ; chemistry ; Dogs ; Humans ; Implants, Experimental ; RNA, Messenger ; genetics ; metabolism ; Tissue Engineering

OBJECTIVEBone marrow stromal cells (MSCs) were transfected with human bone morphogenetic protein-4 (hBMP-4) gene in vitro to provide BMP gene modified cells for tissue-engineered bone.

METHODSMSCs were cultured and transfected with pEGFP-hBMP4, pEGFP plasmids respectively or left uninfected as control. Transcription of BMP-4 gene as well as gene transfection efficiency was tested. Morphological and growth feature of the transfected cells were valued. Alkaline phosphatase (ALP), von Kossa, and Osteocalcin (OC) were tested to determine the phenotypes of osteoblast.

RESULTSThe gene transfection efficiency was 20%-30%, based on GFP expression. RT-PCR showed that MSCs had low transcription of BMP-4 that was enhanced by the gene transfer. Morphological feature of MSCs transfected with pEGFP-hBMP-4 changed but growth curves did not show much difference among the groups. In pEGFP-hBMP-4 group, ALP positive stain area and the number of calcium nodules were increased, as well as the expression of OC.

CONCLUSIONSA high transfer efficiency of MSCs was achieved under optimized conditions. The gene transfer technique strengthened the transcription of BMP-4 and promoted differentiation from MSCs to osteoblasts. hBMP-4 transferred MSCs may serve as an ideal cell source for tissue-engineered bone.

Animals ; Bone Marrow Cells ; cytology ; Bone Morphogenetic Protein 4 ; Bone Morphogenetic Proteins ; biosynthesis ; genetics ; Cell Differentiation ; Cells, Cultured ; Gene Transfer Techniques ; Osteoblasts ; cytology ; metabolism ; Osteocalcin ; biosynthesis ; genetics ; Osteogenesis ; Rabbits ; Stromal Cells ; cytology ; Tissue Engineering ; Transfection

BACKGROUNDTissue-engineering techniques combined with gene therapy have been recently reported to improve osteogenesis. In this study, tissue-engineered bone constructed by human Bone Morphogenetic Protein 4 (hBMP-4) gene-modified bone marrow stromal cells (bMSCs) was explored in an ectopic bone formation model in rabbits.

METHODSA pEGFP-hBMP-4 mammalian plasmid (EGFP: Enhanced Green Fluorescent Protein) was constructed by subcloning techniques. bMSCs obtained from rabbits were cultured and transfected with either pEGFP-hBMP-4, pEGFP or left uninfected in vitro. Transfer efficiency was detected through the expression of EGFP. Transcription of the target gene was detected by RT-PCR. Alkaline phosphatase (ALP) and Von Kossa tests were also conducted to explore the phenotypes of osteoblasts. The autologous bMSCs of the 3 groups were then combined with Natural Non-organic Bone (NNB), a porous hydroxyapatite implant with a dimension of 6 mm x 6 mm x 3 mm, at a concentration of 5 x 10(7) cells/ml. They were subsequently implanted into 6 rabbits subcutaneously using NNB alone as a blank control (6 implants per group). Four weeks after surgery, the implants were evaluated with histological staining and computerized analysis of new bone formation.

RESULTSpEGFP-hBMP-4 expression plasmid was constructed. Under optimal conditions, gene transfer efficiency reached more than 30%. Target gene transfer could strengthen the transcription of BMP-4, and increase the expression of ALP as well as the number of calcium nodules. In the ectopic animal model, NNB alone could not induce new bone formation. The new bone area formed in the bMSCs group was (17.2 +/- 7.1)%, and pEGFP group was (14.7 +/- 6.1)%, while pEGFP-hBMP-4 group was (29.5 +/- 8.2)%, which was the highest among the groups (F = 7.295, P < 0.01).

CONCLUSIONSThe mammalian hBMP-4 expression plasmid was successfully constructed and a comparatively high transfer efficiency was achieved. The gene transfer technique enhanced the expression of BMP-4 and promoted differentiation from bMSCs to osteoblasts. These in vivo results suggested that transfection of bMSCs with hBMP-4 might be a suitable method to enhance their inherent osteogenic capacity for bone tissue engineering applications.

Animals ; Bone Marrow Cells ; cytology ; metabolism ; Bone Morphogenetic Protein 4 ; Bone Morphogenetic Proteins ; genetics ; Cell Differentiation ; Gene Transfer Techniques ; Genetic Therapy ; Humans ; Osteogenesis ; Plasmids ; Rabbits ; Stromal Cells ; cytology ; metabolism ; Tissue Engineering

Distraction osteogenesis is currently a standard method of bone lengthening. It is a viable method for the treatment of short extremities as well as extensive bone defects, because large amounts of bone can be regenerated in the distraction gap. echanical stimulation by distraction induces biological responses of skeletal regeneration that is accomplished by a cascade of biologic processes that may include differentiation of pluripotential tissue, angiogenesis, mineralization, and remodeling. There are complex interactions between bone-forming osteoblasts and other cells present within the bone microenvironment, particularly vascular endothelial cells that may be pivotal members of a complex interactive communication network in bone. Regenerate bone forms by three modes of ossification, which include intramembranous, enchondral, and transchondroid ossifications, although intramembraneous bone formation is the predominant mechanism of ossification. In this review we discussed the coupling between angiogenesis and mineralization, the biological and mechanical factors affecting them, the cellular and molecular events occurring during distraction osteogenesis, and the emerging modalities to accelerate regenerate bone healing and remodeling.
Animals ; Biological Markers ; Bone Morphogenetic Protein 2 ; Bone Morphogenetic Protein 4 ; Bone Morphogenetic Proteins/genetics/metabolism ; Bone and Bones/radiography/ultrastructure ; Calcification, Physiologic/*physiology ; Collagen/metabolism ; Cytokines/metabolism ; Growth Substances/metabolism ; Humans ; Neovascularization, Physiologic/*physiology ; Osteoblasts/physiology ; *Osteogenesis, Distraction ; *Transforming Growth Factor beta

OBJECTIVETo investigate the role of bone morphogenetic protein 4 (BMP4) on the proliferation and apoptosis in glioma stem cells.

METHODSStem cells were isolated from a human glioma cell line U87 by using vincristine and characterized by immunofluorescence assay. Proliferation and apoptosis were determined by soft agar colony assay and flow cytometry; Cyclin D1, Bcl-2 and Bax were detected by Western blot analysis.

RESULTSBMP4 inhibited cell proliferation and promoted apoptosis in U87 glioma stem cells. Moreover, Bcl-2 and Cyclin D1 expression were decreased by BMP4, while Bax level was elevated.

CONCLUSIONBMP4 can inhibit U87 glioma stem cells proliferation through downregulating Cyclin D1 level, and promote apoptosis through induction of Bax expression and inhibition of Bcl-2 level. It suggests that BMP4 plays an important role in human glioma stem cell biology.

Apoptosis ; Bone Morphogenetic Protein 4 ; genetics ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Cyclin D1 ; genetics ; metabolism ; Gene Expression Regulation, Neoplastic ; Glioma ; genetics ; metabolism ; physiopathology ; Humans ; Neoplastic Stem Cells ; cytology ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; metabolism

OBJECTIVETo study the role of antenatal glucocorticoid (dexamethasone and betamethasone) on bone morphogenetic protein (BMP) signal transduction of the rat fetal lungs.

METHODSFifteen pregnant rats were randomly divided into five groups: the rats treated with dexamethasone for 1 day (1D-DEX) or 3 days (3D-DEX), with betamethasone for 1 day (1D-BEX) or 3 days (3D-BEX) or with normal saline (control group), followed cesarean section on the 19th day of gestation. The mRNA levels of BMP4, BMPR-II, Smad1 and ATF-2 of fetal rat lungs were ascertained by reverse transcriptase polymerase chain reaction (RT-PCR). The expression of BMP4, BMPR-II, Smad1 and ATF-2 antigen expression in fetal lungs was assessed by immune histochemical staining. The expression of BMP4 and BMPR-II was determined by Western blot.

RESULTSThe levels of BMP4, BMPR-II and Smad1 mRNA expression were up-regulated in the 1D-BEX, 3D-BEX and 3D-DEX groups compared with those in the control group (P<0.05). The immune histochemiscal analysis showed that the expression of BMP4, BMPR-II, Phospho-Smad1 (pSmad1) and ATF-2 in the 1D-BEX, 3D-BEX and 3D-DEX groups was significantly higher than that in the control group (P<0.01). The results of Western blot demonstrated that the expression of BMP4 and BMPR-II protein increased significantly in the 1D-BEX, 3D-BEX and 3D-DEX groups when compared with the control group (P<0.01).

CONCLUSIONSBetamethasone and dexamethasone may play important roles in the regulation of BMP signal transduction in the rat fetal lungs. Up-regulation of BMP4, BMPR-II and Smad1 might be one of crucial factors for the glucocorticoid-induced maturity of fetal lungs.

Activating Transcription Factor 2 ; analysis ; genetics ; Animals ; Betamethasone ; pharmacology ; Bone Morphogenetic Protein 4 ; analysis ; genetics ; physiology ; Bone Morphogenetic Protein Receptors, Type II ; analysis ; genetics ; Dexamethasone ; pharmacology ; Female ; Fetus ; drug effects ; metabolism ; Lung ; drug effects ; metabolism ; Pregnancy ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; drug effects ; Smad1 Protein ; analysis ; genetics

OBJECTIVETo investigate the effect of basic fibroblast growth factor(bFGF) on expression of protein and mRNA of bone morphogenetic protein 4 in hypoxic-ischemic brain damage (HIBD) in newborn rats.

METHODOne hundred and twenty 7 days old neonatal rats were randomly divided into control group, hypoxic-ischemic brain damage and interventional group of bFGF, each having forty neonatal rats. After HIBD model was established, bFGF was given to interventional group by peritoneal injection for 5 continuous days. Every group was randomly divided into 7 days, 14 days, 21 days and 28 days group, according to the time of sacrifice. BMP4 protein in hippocampus was determined with immunohistochemical method. Messenger RNA of BMP4 were determined with in situ hybridization. Apoptosis of nerve cell was determined with TUNEL. Intergroup or intragroup comparisons were performed with analysis of variance.

RESULTOn the days 7 and 14, expression of BMP4 protein in hippocampus was higher in interventional group of bFGF than in HIBD while expression of BMP4 protein in interventional group of bFGF and HIBD was lower on day 7 than on day 14. Expression of BMP4 protein on the days 21 and 28 had no significant difference among three groups. mRNA expression of BMP4 in interventional group of bFGF and HIBD was significantly higher in hippocampus than in control group. On the day 14, BMP4 mRNA in hippocampus widely expressed in HIBD while BMP4 mRNA only expressed in CA1 in interventional group of bFGF. Expression of BMP4 mRNA in hippocampus on the affected side decreased from the time of killing on 28th day while there was no significant change in interventional group of bFGF. Apoptosis of neural cells at the time of sacrifice on day 7 was lower in interventional group of bFGF than that in HIBD group (F=9.010, P<0.01). Apoptotic neural cells was higher in bFGF and HIBD groups at the time of killing on days 14, 21 and 28 than that on day 7 but that the bFGF group had less apoptotic neural cells than HIBD group (F=9.202, 7.932, 14.985, P<0.01).

CONCLUSIONSbFGF has a neurorestoration effect, which promotes expression of BMP4 protein and BMP4 mRNA in hippocampus of HIBD and inhibit apoptosis of neural cells.

Animals ; Animals, Newborn ; Apoptosis ; drug effects ; Bone Morphogenetic Protein 4 ; metabolism ; Fibroblast Growth Factor 2 ; pharmacology ; Hippocampus ; drug effects ; metabolism ; Hypoxia-Ischemia, Brain ; metabolism ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley

Embryonic stem cells (ESCs) can be propagated in vitro on feeder layers of mouse STO fibroblast cells. The STO cells secrete several cytokines that are essential for ESCs to maintain their undifferentiated state. In this study, we found significant growth inhibition of mouse ESCs (mESCs) cultured on STO cells infected with adenovirus containing a dominant-negative mutant form of IkappaB (rAd-dnIkappaB). This blockage of the NF-kappaB signal pathway in STO cells led to a significant decrease in [3H]thymidine incorporation and colony formation of mESCs. Expression profile of cytokines secreted from the STO cells revealed an increase in the bone morphogenetic protein4 (BMP4) transcript level in the STO cells infected with adenoviral vector encoding dominant negative IkappaB (rAd-dnIkappaB). These results suggested that the NF-kappaB signaling pathway represses expression of BMP4 in STO feeder cells. Conditioned medium from the rAd-dnIkappaB-infected STO cells also significantly reduced the colony size of mESCs. Addition of BMP4 prevented colony formation of mESCs cultured in the conditioned medium. Our finding suggested that an excess of BMP4 in the conditioned medium also inhibits proliferation of mESCs.
Animals ; *Bone Morphogenetic Protein 4/genetics/metabolism ; Cell Differentiation/genetics ; Cell Proliferation ; Culture Media, Conditioned ; *Embryonic Stem Cells/cytology/metabolism ; *Feeder Cells/cytology/metabolism ; *Fibroblasts/cytology/metabolism ; Gene Expression Regulation/genetics ; *I-kappa B Proteins/genetics/metabolism ; Mice ; Mutation ; NF-kappa B/genetics/metabolism ; Signal Transduction

Bone morphogenic protein 4 (BMP4), a member of the TGF-beta superfamily, induced neural differentiation of neural stem cells (NSCs) grown in a medium containing basic fibroblast growth factor (bFGF). The Ras protein level and the activities of the downstream ERKs were increased by transfection of BMP4 or treatment with recombinant BMP4. The effects of BMP4, including activation of the Ras-ERK pathway and induction of the neuron marker beta-tubulin type III (Tuj1), were blocked by co-treatment of the BMP4 antagonist, noggin. The roles of the Ras-ERK pathway in neuronal differentiation by BMP4 were revealed by measuring the effect of the ERK pathway inhibition by dominant negative Ras or PD98059, the MEK specific inhibitor. BMP4 is a transcriptional target of Wnt/beta-catenin signaling, and both the mRNA and protein levels of BMP4 were increased by treatment of valproic acid (VPA), a chemical inhibitor of glycogen synthase kinase 3beta (GSK3beta) activating the Wnt/beta-catenin pathway. The BMP4- mimicking effects of VPA, activation of the Ras-ERK pathway and induction of Tuj1, also were blocked by noggin. These results indicate the potential therapeutic usage of VPA as a replacement for BMP4.
Animals ; Bone Morphogenetic Protein 4/genetics/*metabolism ; Cell Differentiation/drug effects ; Cells, Cultured ; Cerebral Cortex/cytology/embryology ; Extracellular Signal-Regulated MAP Kinases/*metabolism ; Neurons/*cytology ; Rats ; Rats, Sprague-Dawley ; Stem Cells/*cytology ; Up-Regulation/drug effects ; Valproic Acid/pharmacology ; beta Catenin/metabolism ; ras Proteins/genetics/metabolism