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

The first branchial arch (BA1), which is derived from cranial neural crest (CNC) cells, gives rise to various orofacial tissues. Cre mice are widely used for the determination of CNC and exploration of gene functions in orofacial development. However, there is a lack of Cre mice specifically marked BA1's cells. Pax2-Cre allele was previously generated and has been widely used in the field of inner ear development. Here, by compounding Pax2-Cre and R26R-mTmG mice, we found a specific expression pattern of Pax2
Animals ; Bone Morphogenetic Protein 4 ; Branchial Region ; Mesenchymal Stem Cells ; Mice ; Skull

Animals ; Bone Morphogenetic Protein 2 ; metabolism ; Bone Morphogenetic Protein 4 ; metabolism ; Carrier Proteins ; pharmacology ; Liver ; metabolism ; Liver Regeneration ; Male ; Rats ; Rats, Wistar

OBJECTIVETo determine the expression of bone morphogenetic protein 2 and 4 (BMP-2 and BMP-4) in prostatic carcinoma (PCa) and investigate their relationship with clinical stage and Gleason score of tumor.

METHODSForty-eight PCa cases and 5 normal prostatic tissue were analysed for the expressions of BMP-2 and BMP-4 by Western bolt assay.

RESULTSThe optical densities of BMP-2 expressions in the tumor with Gleason score < or =5, 6-8, and > or = 9 were 7547.1 +/- 1964.12, 9657.4 +/- 2010.54, 12467.7 +/- 2496.75 and of BMP-4 expressions were 5174.4 +/- 1400.54, 5940.3 +/- 1587.42, 6332.1 +/- 1647.83, respectively. The optical densities of BMP-2 expressions in the tumor in T1 - T2 and T3 - T4 stages were 8003.37 +/- 1889.23, 12385.55 +/- 2506.72 and of BMP4 expressions were 5267.41 +/- 1 464.19, 6543.75 +/- 1668.46, respectively. There were significant differences between tissues with Gleason score < or =5 and > or =9 (P <0.01), and tissues in T1 - T2 and T3 - T4 stages, in expressions of BMP-2 protein. The expression of BMP-2 protein was significantly high in the PCa with bone metastasis compared with that without bone metastasis.

CONCLUSIONThe expressions of BMP-2 and BMP-4 increase with the progression of clinical stage and Gleason score compared with normal prostatic tissue. The expression of BMP-2 protein is significantly upregulated in bone metastasis of PCa, which indicates a poor prognosis.

Aged ; Aged, 80 and over ; Blotting, Western ; Bone Morphogenetic Protein 2 ; Bone Morphogenetic Protein 4 ; Bone Morphogenetic Proteins ; biosynthesis ; Humans ; Male ; Middle Aged ; Neoplasm Metastasis ; Neoplasm Staging ; Prostate ; metabolism ; Prostatic Neoplasms ; metabolism ; pathology ; Transforming Growth Factor beta ; biosynthesis

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

BACKGROUND: The scales of bony fish represent a significant reservoir of calcium and calcification of the elasmoid scale is known to be associated with deposition of mineral crystals from the epidermis to dermis. However, little is known about the exact mechanisms of calcium deposition, mobilization and regeneration occurring in the zebrafish skin. OBJECTIVE: The purpose of this study was to investigate the expression of calcification-related molecular mediators in both the epidermis and dermis of the zebrafish (Danio rerio), using immunohistochemical study. METHODS: We examined the skin of zebrafish in four populations of different ages (i.e. 20 days post-fertilization (dpf), 35 dpf, 50 dpf, and the adult zebrafish), using several immunohistochemical markers, including bone morphogenetic protein 4 (BMP-4), beta-catenin, osteocalcin, osteopontin and osteonectin. RESULTS: BMP-4, osteopontin and osteonectin were moderately expressed in the epidermis of zebrafish after 35 dpf. Also, some of the cells in the upper dermis showed strong positivity for BMP-4, osteocalcin, osteopontin and osteonetin. CONCLUSION: Our results suggest that BMP-4, osteocalcin, osteopontin and osteonectin may play a role in the process of calcification of the elasmoid scale.
Adult ; beta Catenin ; Bone Morphogenetic Protein 4 ; Calcium ; Dermis ; Epidermis ; Humans ; Osteocalcin ; Osteonectin ; Osteopontin ; Regeneration ; Skin ; Weights and Measures ; Zebrafish

OBJECTIVE: To explore the effect of bone morphogenetic protein 4(BMP4) on the cell cycle and apoptosis of hemaropoictic stem and progenitor cells (HSPC) in conditions of 5-fluorouracil (5-FU)-inducing bone marrow suppression and stress hemogenesis, and its possible mechanism. METHODS: The C57BL transgenic mice with BMP4 overexpression were established and were enrolled in transgenic group (BMP4 group), at the same time the wild type mice matching in age, sex and body weight were selected and were enrolled in control group (WT group). The bone marrow suppression was induced by injection with 5-FU in dose of 150 mg/kg, then the nucleated cells were isolated from bone marrow. After the HSPCs were markered with C-kit/sca-1 fluorescent antibodies, the changes of cell cycle and apoptosis of HSPC were detected by Aunexin V/PI and Ki67/DAPI double staining; the cell cycle-essociated hemotopoietic regulatory factors were detected by RT-qPCR. RESULTS: Under physiologic status, there were no significant differences in cell cycle and apoptotic rate of HSPC between WT group and BMP-4 group. After the bone marrow was suppressed, the ratio of HSPC at G0 phase in BMP4 group significantly decreased(P＜0.05); the apoptosis rate of HSPC significantly increased(P＜0.05); the mRNA expression levels of hypoxia-inducing factor Hif-1α and chemotactic factor CXCL12 in stroma of BMP4 group were down-regulated significanfly(P＜0.05). CONCLUSION Under non-physiologic conditions such as stress hemogenesis or bone marrow suppression, the up-regulation of BMP4 can promote HSPC into cell cycle and apoptosis of HSPC, moreover, the BMP4 may play a regulatory role for cell cycle of HSPC through direct or indirect down-regulation of Hif-1α and CXCL-12 expressions.
Animals ; Antineoplastic Agents ; Apoptosis ; Bone Morphogenetic Protein 4 ; Cell Cycle ; Hematopoietic Stem Cells ; Mice ; Mice, Inbred C57BL

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

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