Objective: To preliminarily explore the mechanism of tensile stress regulating endochondral osteogenesis of condyle by analyzing the expression profiles of significantly different microRNAs (miRNAs) in exosomes of rat mandibular condylar chondrocytes (MCC) under quiescent and cyclic tensile strain (CTS) conditions. Methods: Rat condylar chondrocytes were cultured under static and CTS conditions respectively (10 SD rats, male, 2 weeks old), and exosomes were extracted. The two groups of exosomes were named as control group and CTS group respectively. The differential expression miRNAs were screened by high-throughput sequencing. Bioinformatics analysis and prediction of target genes related to osteogenesis were performed by TargetScan and miRanda website. Results: The exosomes of rat condylar chondrocytes cultured under tensile stress showed a "double concave disc" monolayer membrane structure, the expression of CD9 and CD81 were positive, and the particle size distribution accorded with the characteristics of exosomes, which was consistent with that of static cultured rat condylar chondrocytes. A total of 85 miRNAs with significantly different expression were detected by high-throughput sequencing (P<0.05). The main biological processes and molecular functions of differential miRNAs were biological processes and protein binding, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) database pathway enrichment analysis showed that there was significant enrichment in mammalian target of rapamycin (mTOR) signal pathway. The candidate target genes of miR-199a-5p include bone morphogenetic protein 3 (BMP3), endothelin converting enzyme 1, and miR-186-5p may target Smad8 and BMP3 to exert osteogenesis-related functions. Conclusions: Compared with static state, tensile stress stimulation can change the expression of miRNAs such as miR-199a-5p, miR-186-5p in the exocrine body of rat condylar chondrocytes, which can be considered as a mean to regulate the application potential of the exosomes.
Animals ; Male ; Rats ; Bone Morphogenetic Protein 3 ; Chondrocytes/metabolism* ; Mandibular Condyle ; MicroRNAs/metabolism* ; Rats, Sprague-Dawley ; Signal Transduction ; Stress, Mechanical

OBJECTIVE: This study aims to investigate the effect of neurotrophin 3 (NT-3) on the osteogenic differentiation of human dental follicle cells (hDFCs). METHODS: hDFCs were isolated and cultured in vitro. Immunocytochemical staining was used to identify the origin of hDFCs. The effects of different NT-3 concentrations on hDFCs proliferation were detected by using CCK-8 assay. The alkaline phosphatase (ALP) activities and mRNA expression levels of bone morphogenetic protein-2 (BMP-2) and osteocalcin (OCN) were determined to investigate the effects of NT-3 on hDFCs osteogenesis. The difference in the number of mineralized nodules was detected using alizarin red staining. RESULTS: Vimentin and cytokeratin staining results showed that hDFCs originated from the mesenchymal cells. NT-3 exerted no evident effect on hDFCs proliferation. The ALP activity and the BMP-2 and OCN mRNA expression levels of hDFCs were significantly improved under treatment with different NT-3 concentrations (25, 50, and 100 ng·mL ⁻¹) compared with those in the control group. BMP-2 and OCN mRNA relative expression levels of hDFCs reached the highest when the NT-3 concentration was 100 ng·mL ⁻¹. The number of mineralized nodules reached the maximum when the hDFCs were treated with 50 and 100 ng·mL ⁻¹ NT-3. CONCLUSIONS Appropriate mass concentration of NT-3 can promote the osteogenic differentiation of hDFCs.
Alkaline Phosphatase ; Bone Morphogenetic Protein 2 ; metabolism ; Cell Differentiation ; Cells, Cultured ; Dental Sac ; Humans ; Mesenchymal Stem Cells ; Neurotrophin 3 ; pharmacology ; Osteocalcin ; metabolism ; Osteogenesis

Aortic valve calcification is a common disease in the elderly, but its cellular and molecular mechanisms are not clear. In order to verify the hypothesis that Wnt/β-catenin signaling pathway is involved in the process of calcification of aortic valve, porcine aortic valve interstitial cells (VICs) were isolated, cultured and stimulated with oxidized low density lipoprotein (ox-LDL) for 48 h to induce the differentiation of VICs into osteoblast-like cells. The key proteins and genes of Wnt/β-catenin signaling pathway, such as glycogen synthase kinase 3β (GSK-3β) and β-catenin, were detected by using Western blotting and real-time polymerase chain reaction (PCR). The results showed that the VICs managed to differentiate into osteoblast-like cells after the stimulation with ox-LDL and the levels of proteins and genes of GSK-3β and β-catenin were increased significantly in VICs after stimulation for 48 h (P<0.05). It is suggested that Wnt/β-catenin signaling pathway may play a key role in the differentiation of VICs into osteoblast-like cells and make great contribution to aortic valve calcification.
Alkaline Phosphatase ; genetics ; metabolism ; Animals ; Aortic Valve ; metabolism ; pathology ; Aortic Valve Stenosis ; Blotting, Western ; Bone Morphogenetic Protein 2 ; genetics ; metabolism ; Calcinosis ; Cell Differentiation ; drug effects ; genetics ; Cells, Cultured ; Gene Expression ; drug effects ; Glycogen Synthase Kinase 3 ; genetics ; metabolism ; Lipoproteins, LDL ; pharmacology ; Osteoblasts ; drug effects ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Swine ; Wnt Signaling Pathway ; genetics ; physiology ; beta Catenin ; genetics ; metabolism

OBJECTIVETo compare the differentiation ability difference of hematopoietic, mesenchymal and endothelial potential between CD41⁺ cells derived from the mouse aorta-gonadmesonephros (AGM) region, yolk sac (YS) and embryonic circulating blood (CB).

METHODSCD41⁺ cells were sorted from AGM, YS and CB. The CD45 and c-kit expression were studied in CD41⁺ cells by flow cytometry. IL-3 and bone morphogenetic protein 4 (BMP-4) treatment together with semi solid culture were used to assess hematopoietic potential difference of CD41⁺ cells. Immunofluorescence staining of α-SMA was used to assess mesenchymal potential difference. The endothelial cell induction system was used to assess endothelial potential difference.

RESULTSThe proportions of CD45+ cells in CD41⁺ population were 51.9% (AGM), 45.8% (YS) and 22.2% (CB), respectively, while those of c-kit⁺ cells were 40.0% (AGM), 39.6% (YS) and 36.2% (CB), respectively. After stimulated by IL-3 factor, the number of total colonies increased in all three groups-derived CD41⁺ cells compared to that of unstimulated group[(14.1±1.9) vs (1.2±0.2), (32.4±1.1) vs (18.4±2.2) and (41.8±0.9) vs (10.4±1.8)], (P<0.01). After stimulated by BMP-4 factor, compared to unstimulated group, CFU-Mix colony number in CD41⁺ cells from AGM region and YS were significantly decreased[(0.5±0.6) vs (3.2±0.8), (1.3±0.7) vs (7.4±1.7)](P<0.01), but there was no difference in CB group[(2.5±0.5) vs (3.9±1.5)](P>0.01). The mesenchymal marker α-SMA was highly expressed in CD41⁺ cells from AGM region and YS, but lowly expressed in CD41⁺ cells from CB.

CONCLUSIONThere are some differences between CD41⁺ cells in AGM region, YS and CB on hematopoietic cell surface marker expression, hematopoietic colony formation with IL-3 and BMP-4 stimulation.

Animals ; Aorta ; cytology ; Bone Morphogenetic Protein 4 ; pharmacology ; Cell Differentiation ; Gonads ; cytology ; Interleukin-3 ; pharmacology ; Mesonephros ; cytology ; Mice ; Platelet Membrane Glycoprotein IIb ; metabolism ; Proto-Oncogene Proteins c-kit ; metabolism ; Yolk Sac ; cytology

In mammals, ovarian follicle is made of an oocyte with its surrounding granulosa cells and theca cells. Follicular growth and development is a highly coordinated programmable process, which guarantees the normal oocyte maturation and makes it having the fertilizing capacity. The paracrine and autocrine between oocytes and granulosa cells are essential for the follicular development to provide a suitable microenvironment. Phosphatidylinositol-3 kinase /protein kinase B is one of these important regulatory signaling pathways during this developmental process, and bone morphogenetic protein-15 an oocyte-specific secreted signal molecule, which regulates the follicular development by paracrine in the mammalian ovary. The present article overviewed the role of phosphatidylinositol-3 kinase / protein kinase B signaling during the follicular development based on our previous investigation about protein kinase B /forkhead transcription factor forkhead family of transcription factors -3a, and then focused on the regulatory effects of bone morphogenetic protein-15, as a downstream signal molecule of phosphatidylinositol-3 kinase / forkhead family of transcription factors -3a pathway, on ovarian follicular development, which helped to further understand the molecular mechanism regulating the follicular development and to treat ovarian diseases like infertility.
Animals ; Bone Morphogenetic Protein 15 ; physiology ; Female ; Granulosa Cells ; physiology ; Humans ; Mammals ; Ovarian Follicle ; growth & development ; Ovary ; growth & development ; Phosphatidylinositol 3-Kinase ; physiology ; Proto-Oncogene Proteins c-akt ; physiology ; Signal Transduction

OBJECTIVETo investigate the effect of recombinant adenovirus-mediated bone morphogenetic protein (BMP)-2 and -3 over expressions on chondrogenesis and osteogenesis of prenatal mouse intervertebral disc cells and provide experimental evidences for the application of BMPs in the therapy of disc diseases.

METHODSThe prenatal mouse intervertebral disc cells were infected with a recombinant adenovirus expressing BMP-2 and BMP-3 for 5-7 days, and the expressions of collagen type I (Col I), collagen type II (Col II), aggrecan, osteocalcin, osteoprotegerin and osteopontin mRNAs were detected with RT-PCR. The expression of cartilage matrix was evaluated with toluidine blue staining, and alkaline phosphatase (ALP) activity was detected with ALP reading and ALP staining.

RESULTSBMP-2 and -3 overexpression did not enhance chondrogenesis and osteogenesis of annulus fibrosus (AF) cells or cause significant increases in the expressions of Col I, Col II or aggrecan mRNA in nucleus pulposus (NP) cells. Adenovirus-mediated overexpression of BMP-2 and BMP-3, however, promoted osteogenesis of NP cells and significantly increased the expression of osteocalcin mRNA; the overexpression of BMP-2, but not BMP-3, enhanced the mRNA expressions of osteoprotegerin and osteopontin. Toluidine blue staining demonstrated that BMP-2 and BMP-3 overexpression did not obviously affect the secretion of cartilage matrix. In NP cells, BMP-2 and -3 overexpression significantly enhanced ALP activity, which was not observed in AF cells.

CONCLUSIONAdenovirus-mediated BMP-2 and -3 overexpression can promote the osteogenic differentiation of NP cells but can not affect osteogenesis of AF cells or chondrogenesis of NP cells.

Animals ; Bone Morphogenetic Protein 2 ; pharmacology ; Bone Morphogenetic Protein 3 ; pharmacology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Chondrogenesis ; Intervertebral Disc ; cytology ; drug effects ; Mice ; Osteogenesis

OBJECTIVE: Supplementation with vitamin E is able to protect bone against free radical-induced elevation of bone-resorbing cytokines. We examined gene expression by microarray analysis during the differentiation of human mesenchymal stem cells treated with vitamin E into osteoblasts in vitro. METHODS: Human bone marrow stem cells were cultured in osteogenic differentiation medium and vitamin E was added. A colorimetric immunoassay for the quantification of cell proliferation was used to measure osteoblast differentiation. Gene expression was analyzed using a microarray technique. We also used a real time reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: It was found that vitamin E enhanced cell proliferation when compared to cells cultured in media without vitamin E. We focused on 68 genes which are related to osteogenesis and osteoclastogenesis. Alkaline phosphatase, transforming growth factor-beta 1, fibroblast growth factor receptor 1, matrix metalloproteinase 2, muscle segment homeobox 2, bone morphogenetic protein 1, biglycan, vascular endothelial growth factor B, dentin sialophosphoprotein, cartilage oligomeric matrix protein, runt-related transcription factor 2, fibroblast growth factor receptor 3, and SMAD2 were upregulated > 2-fold compared to the control. Conversely, osteopetrosis-associated transmembrane protein 1, microphthalmia-associated transcription factor, and epidermal growth factor receptor were downregulated > 2-fold compared to the control. Vitamin E produced a 1.5-fold increase in the expression of runt-related transcription factor 2 and transforming growth factor-beta 1 as determined by real time RT-PCR. CONCLUSION: Vitamin E had a positive effect on the gene expressions regarding osteogenic differentiation of mesenchymal stem cells.
Alkaline Phosphatase ; Biglycan ; Bone Marrow ; Bone Morphogenetic Protein 1 ; Cartilage ; Cell Proliferation ; Cytokines ; Dentin ; Durapatite ; Extracellular Matrix Proteins ; Gene Expression ; Genes, Homeobox ; Glycoproteins ; Humans ; Immunoassay ; Matrix Metalloproteinase 2 ; Mesenchymal Stromal Cells ; Microarray Analysis ; Microphthalmia-Associated Transcription Factor ; Muscles ; Osteoblasts ; Osteogenesis ; Phosphoproteins ; Receptor, Epidermal Growth Factor ; Receptor, Fibroblast Growth Factor, Type 1 ; Receptor, Fibroblast Growth Factor, Type 3 ; Sialoglycoproteins ; Stem Cells ; Transcription Factors ; Vascular Endothelial Growth Factor B ; Vitamin E ; Vitamins

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

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

To investigate the function of ALK3 gene, the gene regulation and the signaling pathway related to ventricular septum defect during heart development. The model mice with ALK3 gene knock-out via alpha-MHC-Cre/lox P system were bred. The mRNA expression level of control group was compared with that of experiment group and ALK3 downstream genes were screened using PCR-select cDNA subtraction microarray. The mRNA of control group was extracted from E11.5 normal mouse hearts, and that of experiment group, from E11.5 hearts of mice with alpha-MHC Cre(+/-) ALK3(F/+) genotype. It was found that the mice with ALK3 gene knock-out produced heart defects involving the interventricular septum. The platelet-activating factors acetylhydrolase and the transcription factor Pax-8 and so on, were down-regulated. However, the Protein Tyrosine Kinase (PTK) of Focal Adhesion Kinase (FAK) subfamily and beta subtype protein 14-3-3 were up-regulated in the alpha-MHC Cre(+/-) ALK3(F/-) mice. These data provide support that ALK3 gene played an important role during heart development. The platelet-activating factors acetylhydrolase and Pax-8 genes could be important ALK3 downstream genes in the BMP signaling pathway during interventricular septum development. PTK and beta subtype protein 14-3-3 might be regulatory factors in this pathway.
1-Alkyl-2-acetylglycerophosphocholine Esterase ; genetics ; metabolism ; 14-3-3 Proteins ; genetics ; metabolism ; Animals ; Bone Morphogenetic Protein Receptors, Type I ; genetics ; metabolism ; Genotype ; Heart Septal Defects, Ventricular ; genetics ; Mice ; Mice, Knockout ; Oligonucleotide Array Sequence Analysis ; PAX8 Transcription Factor ; Paired Box Transcription Factors ; genetics ; metabolism ; Protein-Tyrosine Kinases ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; genetics ; physiology