Bone Morphogenetic Protein-2 mRNA Expression Research Articles

Fabrication of a coral-like barium titanate nano-piezoelectric coating and its effect on promoting osteogenic differentiation of rat bone marrow mesenchymal stem cells in vitro

Objective: To investigate the biocompatibility of coral-like barium titanate nano-piezoelectric coatings and the influence of ultrasound-excited piezoelectric effect on the early osteogenic differentiation. Methods: The barium titanate nano-piezoelectric coating (the coating group) was prepared on the surface of titanium metal by anodic oxidation, hydrothermal reaction and high-temperature annealing, and polished titanium specimens were used as control group. The surface morphology, composition, and crystal phase and hydrophilicity of the two groups of titanium specimens were characterized using scanning electron microscopy, X-ray photoelectron spectroscopy, Raman spectroscopy and contact angle meter. The piezoelectric properties of the materials were characterized by piezoresponse force microscopy. Rat bone marrow mesenchymal stem cells (BMSC) were cultured and identified and seeded the surface of titanium specimens in two groups. The cells seeded on blank culture plates were used as blank group. After low intensity pulsed ultrasound intervention, cell proliferation and live/dead staining were detected to evaluate cytocompatibility of the coatings. Alkaline phosphatase (ALP) activity of each group was detected by ALP staining kit, and the expression of osteogenesis-related genes [integrin, bone morphogenetic protein 2 (BMP-2), Runt-related transcription factor 2 (RUNX2)] was detected by real-time fluorescent quantitative PCR (RT-qPCR) to evaluate the effect of the coating on promoting the early osteogenic differentiation of BMSC. Results: The surface of titanium specimens in the coating group showed a uniform coral-like morphology, and the diameter of the coral tentacles was 70-100 nm. The main component was tetragonal barium titanate. The surface hydrophilicity of the coating group (water contact angle 10.12°± 0.93°) was significantly better than that of the control group (water contact angle 78.32°±0.71°) (F= 10 165.91, P<0.001). The coating has a stable piezoelectric property with a piezoelectric constant of about 5 pC/N. Cell experiments showed that, with or without ultrasound, the cell proliferation activity of the coating group was significantly lower than that of the blank group and the control group on the third day (P<0.05). On the fifth day, with or without ultrasound, there was no significant difference in cell proliferation activity between the three groups (P>0.05). After 7 days of culture, the ALP activity of the coating group was significantly higher than that of the blank group and the control group (P<0.05). The results of RT-qPCR showed that the mRNA expression of integrin and BMP-2 in the coating group with ultrasound was significantly higher than that in the other groups with ultrasound, and was higher than that of the coating group without ultrasound (P<0.05). The expression of integrin mRNA in the control group with ultrasound was significantly higher than that in the control group without ultrasound (P<0.05). The expression of RUNX2 mRNA in the coating group with ultrasound was significantly higher than that in the coating group without ultrasound (P<0.05). Conclusions: The coral-like barium titanate nano-piezoelectric coating exhibits favorable biocompatibility and stable piezoelectric property, and facilitates the early osteogenic differentiation of BMSC under the excitation of low-intensity pulsed ultrasound.

Panax quinquefolius saponin inhibits vascular smooth muscle cell calcification via activation of nuclear factor-erythroid 2-related factor 2

BackgroundPanax quinquefolius saponin (PQS) is the main active component of Panax quinquefolius. Emerging evidence suggests that PQS exerts beneficial effects against cardiovascular diseases. However, the role and mechanism of PQS in vascular calcification are not unclear. The present study investigated the effects of PQS on the calcification of vascular smooth muscle cell (VSMCs).MethodsThe present study used calcification medium containing 3 mM inorganic phosphate (Pi) to induce rat VSMCs calcification. We investigated the effects of PQS on VSMCs calcification using alizarin red staining and alkaline phosphatase (ALP) activity assays. The intracellular reactive oxygen species (ROS) levels and the transcriptional activity of nuclear factor-erythroid 2-related factor 2 (Nrf2) were determined. The mRNA and protein expression levels of Nrf2, the antioxidant gene heme oxygenase-1 (HO-1), osteogenic markers, including runt-related transcription factor 2 (Runx2) and bone morphogenetic protein 2 (BMP2), and Kelch-like ECH-associated protein 1 (Keap1) were also measured.ResultsTreatment with Pi significantly increased intracellular calcium deposition and ALP activity, which were suppressed by PQS in a concentration-dependent manner. During VSMCs calcification, PQS inhibited the mRNA and protein expression of Runx2 and BMP2. PQS treatment reduced intracellular ROS production and significantly upregulated Nrf2 transcriptional activity and the expression of Nrf2 and its target antioxidant gene HO-1. PQS suppressed the Pi-induced protein expression of Keap1, which is an endogenous inhibitor of Nrf2. Keap1 siRNA treatment induced Nrf2 expression and downregulated Runx2 expression in the presence of Pi and PQS.ConclusionTaken together, these findings suggest that PQS could effectively inhibit VSMCs calcification by ameliorating oxidative stress and regulating osteogenic genes via the promotion of Nrf2 expression.

Possible roles of bone morphogenetic protein 4 in regulating endometrial function in cows.

This study investigated the expression dynamics of bone morphogenetic protein 4 (BMP4) and its receptors (BMPR1A, BMPR1B, and BMPR2) in bovine endometrium and examined the physiological function and regulatory mechanism of BMP4 expression. The messenger RNA (mRNA) expression of BMP4 and its receptors was detected in bovine endometrium of both ipsilateral (corpus luteum [CL]-side) and contralateral (non-CL-side) uterine horns during the estrous cycle and early pregnancy. BMP4 protein levels were higher in the endometrial tissues obtained from those cows in early pregnancy than in the estrous cycle. Immunohistochemical analysis showed that BMP4 and its receptors were localized in endometrial epithelial cells. The addition of BMP4 to cultured endometrial epithelial cells did not affect caspase-3/-8 mRNA expression, whereas it significantly inhibited cell proliferation. Both prostaglandin (PG) E2 and PGF2α concentrations in the culture supernatant were decreased when stimulated by BMP4. Furthermore, BMP4 mRNA expression was increased by stimulation with tumor necrosis factor-α (TNF) and interferon-γ (IFNG). In conclusion, BMP4 is produced in bovine endometrial epithelial cells and may contribute to the regulation of cell proliferation and suppression of PG secretion through autocrine or paracrine mechanisms. BMP4 expression in the bovine endometrium may be regulated by TNF and IFNG.

The regulatory role of BMP4 in testicular Sertoli cells of Tibetan sheep.

This study aimed to determine the regulatory mechanism of bone morphogenetic protein 4 (BMP4) gene in the testes of Tibetan sheep and its role in the blood-testis barrier (BTB). First, we cloned BMP4 gene for bioinformatics analysis, and detected the mRNA and protein expression levels of BMP4 in the testes of Tibetan sheep pre-puberty (3-mo-old), during sexual maturity (1-yr-old), and in adulthood (3-yr-old) by qRT-PCR and Western blot. In addition, the subcellular localization of BMP4 was analyzed by immunohistochemical staining. Next, BMP4 overexpression and silencing vectors were constructed and transfected into primary Sertoli cells (SCs) to promote and inhibit the proliferation of BMP4, respectively. Then, CCK-8 was used to detect the proliferation effect of SCs. The expression of BMP4 and downstream genes, pathway receptors, tight junction-related proteins, and cell proliferation and apoptosis-related genes in SCs were studied using qRT-PCR and Western blot. The results revealed that the relative expression of BMP4 mRNA and protein in testicular tissues of 1Y group and 3Y group was dramatically higher than that of 3M group (P < 0.01), and BMP4 protein is mainly located in SCs and Leydig cells at different development stages. The CDS region of the Tibetan sheep BMP4 gene was 1,229 bp. CCK-8 results demonstrated that the proliferation rate of BMP4 was significantly increased in the overexpression group (pc-DNA-3.1(+)-BMP4; P < 0.05). In addition, the mRNA and protein expressions of SMAD5, BMPR1A, and BMPR1B and tight junction-related proteins Claudin11, Occludin, and ZO1 were significantly increased (P < 0.05). The mRNA expression of cell proliferation-related gene Bcl2 was significantly enhanced (P < 0.05), and the expression of GDNF was enhanced (P > 0.05). The mRNA expression of apoptosis-related genes Caspase3 and Bax decreased significantly (P < 0.05), while the mRNA expression of cell cycle-related genes CyclinA2 and CDK2 increased significantly (P < 0.05). It is worth noting that the opposite results were observed after transfection with si-BMP4. In summary, what should be clear from the results reported here is that BMP4 affects testicular development by regulating the Sertoli cells and BTB, thereby modulating the spermatogenesis of Tibetan sheep.

The effect of simvastatin-loaded methoxy poly(ethylene glycol)-polylactic acid nanoparticles on osteoblasts

ObjectiveTo investigate the effect of simvastatin-loaded polymer nanoparticles on osteoblast proliferation and differentiation, and to seek a better pharmaceutical dosage form to improve the osteogenic effect of simvastatin and provide a more practical drug carrier for its clinical application. Methodspolymer nanoparticles were prepared via the modified spontaneous emulsifying solvent diffusion (SESD) method, and the effects of simvastatin-loaded methoxy poly(ethylene glycol)-polylactic acid nanoparticles on osteoblasts were investigated. A 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay was adopted to detect the proliferation abilities of the cells. An enzyme-linked immunosorbent assay was used to detect the amount of alkaline phosphatase (ALP) in the cells. A real-time polymerase chain reaction was used to detect the expression of osteocalcin (OC) and bone morphogenetic protein-2 (BMP-2) messenger ribonucleic acid (mRNA). ResultsThe study found that the proliferation of osteocytes was inhibited in the simvastatin-loaded nanoparticle (Sim-NP) group, and this inhibition was stronger in the Sim-NP than in the simvastatin group; the ALP activity of osteoblasts was increased in both groups, and this increase was stronger in the Sim-NP than in the simvastatin group. The expression of ALP, OC and BMP-2 mRNA was increased in both groups, and this increase was stronger in the Sim-NP than in the simvastatin group. ConclusionThe authors thus posited that the modified Sim-NPs SESD preparation method was simple and reproducible, and the prepared nanoparticles, with uniform distribution, a regular morphology and spherical shape could greatly improve the solubility of simvastatin. The polymer nanoparticle carrier system improved the effect of simvastatin on the proliferation and differentiation of osteoblasts so that the Sim-NPs were systematically applied to target bone tissues. These results provide a new direction for research on the drugs and treatment methods for osteoporosis.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Lysine (K)-specific demethylase 5C regulates the incidence of severe preeclampsia by adjusting the expression of bone morphogenetic protein-7

ABSTRACT This study aimed to investigate the roles of the lysine (K)-specific demethylase 5C (KDM5C)–bone morphogenetic protein-7 (BMP-7) signaling pathway in the pathogenesis of severe preeclampsia (sPE). A total of 180 pregnant patients were enrolled in the study and classified into three groups: an early-onset sPE group (EOsPE) (n = 60), a late-onset sPE group (LOsPE) (n = 60), and a control group (normal pregnancy; n = 60). The messenger RNA (mRNA) and protein expression levels of bone morphogenetic protein receptor II (BMPRII), BMP-7, and KDM5C were detected in placenta samples from the two sPE groups, and their sites were evaluated using immunohistochemistry (IHC). The sPE groups showed an increased KDM5C mRNA expression, and the EOsPE group showed a decreased BMP-7 and BMPRII mRNA expression compared with the LOsPE group. However, contradictory results were discovered in terms of protein expression. Immunostaining of KDM5C, BMP-7, and BMPRII was observed in villous trophoblast and extravillous trophoblast cells. Compared with the control group, the staining intensity of KDM5C in the placental tissue trophoblast cell nucleus and vascular endothelial cells of the sPE groups was weaker, while that of BMP-7 and BMPRII was stronger, and the staining intensity was more subjective in the LOsPE group. Consistent findings were obtained by IHC and Western blot analysis. KDM5C nuclear-cytoplasmic translocation may regulate sPE through BMP-7 and its receptors. The KDM5C–BMP-7 signaling pathway may also lead to less invasion and increased apoptosis of the trophoblast cells, which is involved in the pathogenesis of sPE.

High BMP4 expression in low/intermediate risk BCP-ALL identifies children with poor outcomes

AbstractPediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) outcome has improved in the last decades, but leukemic relapses are still one of the main problems of this disease. Bone morphogenetic protein 4 (BMP4) was investigated as a new candidate biomarker with potential prognostic relevance, and its pathogenic role was assessed in the development of disease. A retrospective study was performed with 115 pediatric patients with BCP-ALL, and BMP4 expression was analyzed by quantitative reverse transcription polymerase chain reaction in leukemic blasts at the time of diagnosis. BMP4 mRNA expression levels in the third (upper) quartile were associated with a higher cumulative incidence of relapse as well as a worse 5-year event-free survival and central nervous system (CNS) involvement. Importantly, this association was also evident among children classified as having a nonhigh risk of relapse. A validation cohort of 236 patients with BCP-ALL supported these data. Furthermore, high BMP4 expression promoted engraftment and rapid disease progression in an NSG mouse xenograft model with CNS involvement. Pharmacological blockade of the canonical BMP signaling pathway significantly decreased CNS infiltration and consistently resulted in amelioration of clinical parameters, including neurological score. Mechanistically, BMP4 favored chemoresistance, enhanced adhesion and migration through brain vascular endothelial cells, and promoted a proinflammatory microenvironment and CNS angiogenesis. These data provide evidence that BMP4 expression levels in leukemic cells could be a useful biomarker to identify children with poor outcomes in the low-/intermediate-risk groups of BCP-ALL and that BMP4 could be a new therapeutic target to blockade leukemic CNS disease.

Platelet-rich plasma combined with ozone prevents cartilage destruction and improves weight-bearing asymmetry in a surgery-induced osteoarthritis rabbit model.

Osteoarthritis (OA) is the most common degenerative disease in older adults and its treatment remains unsatisfactory. This study aimed to evaluate the effectiveness and explore the therapeutic mechanisms of the combination of platelet-rich plasma (PRP) and ozone (O3) for knee OA. Thirty male rabbits were randomly divided into five groups (Control group, OA group, PRP group, O3 group, and PRP + O3 group). Rabbit model of OA were induced by improved Hulth surgery. Gross articular observation, histopathological examination and cartilage scoring system were used to assess the articular cartilage destruction. The bone morphogenetic protein-2 (BMP-2) mRNA expression in joint fluid was determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The expression of type II collagen, matrix metalloproteinase-1 (MMP-1) of cartilage was detected via Immunohistochemistry. Pain behavior was observed by percent ipsilateral weight-bearing (PIW) asymmetry. The content of platelet in PRP was increased by 6.2-times that in whole blood. Among induced OA groups (the OA, PRP, O3 and PRP + O3 group), PRP + O3 significantly inhibited the surgically induced increase in gross articular alterations, histopathological damage of cartilage and Mankin score when compared to the OA, PRP and O3 groups (P<0.05). Observed pain behavior by weight-bearing asymmetry, in the PRP + O3 group was reversed at 3 and 6 weeks after the administration of PRP + O3 (PIW asymmetry: -10.66%±1.172%). In addition, surgery-induced BMP-2 mRNA expression was significantly downregulated after the treatment of PRP, O3 and PRP + O3 (P<0.01). PRP + O3 group significantly increased the expression of type II collagen but decreased MMP-1 of cartilage in comparison to OA, PRP and O3 groups (P<0.05) by immunohistochemical analysis. PRP combined with O3 may prevent cartilage destruction and improve weight-bearing asymmetry by restoring homeostasis between anabolism and catabolism of extracellular matrix in progressive OA. Furthermore, a combination of PRP and O3 might achieve even better results than the two agents alone.

Cytotoxicity and bioactive potential of new root repair materials for use with BMP-2 transfected human osteoblast cells.

Modified formulations of calcium silicate repair materials with additives have been developed to enhance handling, consistency, biocompatibility and bioactivity. Considering the relevance of osteoblastic cell response to mineralized tissue repair, human osteoblastic cells (Saos-2 cells overexpressing BMP-2) were exposed to mineral trioxide aggregate (MTA) (with calcium tungstate - CaWO4), MTA HP Repair, Bio-C Repair and Bio-C Pulpo. Cell viability was assessed by 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide (MTT) and neutral red (NR), and cell death, by flow cytometry. Gene expression of bone morphogenetic protein 2 (BMP-2), runt-related transcription factor 2 (RUNX-2), and alkaline phosphatase (ALP) osteogenic markers were evaluated by real-time polymerase chain reaction (RT-qPCR). ALP activity and alizarin red staining (ARS) were used to detect mineralization nodule deposition. Bioactive cements presented no cytotoxic effect, and did not induce apoptosis at the higher dilution (1:12). MTA, Bio-C Repair and Bio-C Pulpo exhibited higher ALP activity than the control group (P < 0.05) after 7 days. MTA, MTA HP and Bio-C Pulpo affected the formation of mineralized nodules (p < 0.05). Exposure to all cement extracts for 1 day increased BMP-2 gene expression. RUNX-2 mRNA was greater in MTA, MTA HP and Bio-C Repair. MTA, MTA HP and Bio-C Pulpo increased the ALP mRNA expression, compared with BMP-2 unexposed cells (P < 0.05). Calcium silicate cements showed osteogenic potential and biocompatibility in Saos-2 cells transfected BMP-2, and increased the mRNA expression of BMP-2, RUNX-2, and ALP osteogenic markers in the BMP-2 transfected system, thereby promoting a cellular response to undertake the mineralized tissue repair.

Silicate/zinc-substituted strontium apatite coating improves the osteoinductive properties of \u03b2-tricalcium phosphate bone graft substitute

Backgroundβ-Tricalcium phosphate (β-TCP) is a popular synthetic bone graft substitute with excellent osteoconductive properties and bioabsorbability. However, its osteoinductive properties are inferior to those of autologous or allogeneic bone. Trace elements such as strontium (Sr), silica (Si), and zinc (Zn) have been reported to promote osteogenesis in materials. In this study, we aimed to determine whether a Si/Zn-substituted Sr apatite coating of β-TCP could enhance osteoinductive properties.MethodsThe apatite-coated β-TCP disks were prepared using nanoparticle suspensions of silicate-substituted Sr apatite (SrSiP) or silicate- and Zn-co-substituted Sr apatite (SrZnSiP).Bone marrow mesenchymal cells (BMSCs) from rat femur were cultured and subsequently seeded at a density of 1.0 × 106/cm2 onto apatite-coated and non-coated β-TCP disks.In vitro, the β-TCP disks were then placed in osteogenic medium, and lactate dehydrogenase (LDH) activity was measured from supernatants after culture for 2 days. Additionally, after culture for 14 days, the mRNA expression of genes encoding osteocalcin (OC), alkaline phosphatase (ALP), bone morphogenetic protein-2 (BMP-2), and vascular endothelial growth factor (VEGF) was evaluated by qRT-PCR. In vivo, the β-TCP disks were transplanted subcutaneously into rats that were sacrificed after 4 weeks. Then, the harvested disks were evaluated biochemically (ALP activity, OC content, mRNA expression of OC, ALP, BMP-2, and VEGF measured by qRT-PCR), radiologically, and histologically.ResultsSignificantly higher mRNA expression of almost all evaluated osteogenic and angiogenic genes was observed in the SrZnSiP and SrSiP groups than in the non-coated group, with no significant cytotoxicity elicited by the apatite coating in vitro. Moreover, in vivo, the SrZnSiP and SrSiP groups showed significantly higher osteogenic and angiogenic gene expression and higher ALP activity and OC content than the non-coated group (P < 0.05). Radiological and histopathological findings revealed abundant bone formation in the apatite-coated group.ConclusionsOur findings indicate that apatite coating of β-TCP improves osteoinductive properties without inducing significant cytotoxicity.

Simvastatin Enhances the Chondrogenesis But Not the Osteogenesis of Adipose-Derived Stem Cells in a Hyaluronan Microenvironment.

Directing adipose-derived stem cells (ADSCs) toward chondrogenesis is critical for ADSC-based articular cartilage regeneration. Simvastatin (SIM) was reported to promote both chondrogenic and osteogenic differentiation of ADSCs by upregulating bone morphogenetic protein-2 (BMP-2). We previously found that ADSC chondrogenesis is initiated and promoted in a hyaluronan (HA) microenvironment (HAM). Here, we further hypothesized that SIM augments HAM-induced chondrogenesis but not osteogenesis of ADSCs. ADSCs were treated with SIM in a HAM (SIM plus HAM) by HA-coated wells or HA-enriched fibrin (HA/Fibrin) hydrogel, and chondrogenic differentiation of ADSCs was evaluated. SIM plus HAM increased chondrogenesis more than HAM or SIM alone, including cell aggregation, chondrogenic gene expression (collagen type II and aggrecan) and cartilaginous tissue formation (collagen type II and sulfated glycosaminoglycan). In contrast, SIM-induced osteogenesis in ADSCs was reduced in SIM plus HAM, including mRNA expression of osteogenic genes, osteocalcin and alkaline phosphatase (ALP), ALP activity and mineralization. SIM plus HAM also showed the most effective increases in the mRNA expression of BMP-2 and transcription factors of SOX-9 and RUNX-2 in ADSCs, while these effects were reversed by CD44 blockade. HAM suppressed the levels of JNK, p-JNK, P38 and p-P38 in ADSCs, and SIM plus HAM also decreased SIM-induced phosphorylated JNK and p38 levels. In addition, SIM enhanced articular cartilage regeneration, as demonstrated by implantation of an ADSCs/HA/Fibrin construct in an ex vivo porcine articular chondral defect model. The results from this study indicate that SIM may be an enhancer of HAM-initiated MSC-based chondrogenesis and avoid osteogenesis.

Evaluation of Three Growth Factors with the Most Controversial Expression Pattern in Osteoarthritic and Non-osteoarthritic Cartilage

There is no clear consensus regarding the expression pattern of many growth factors in healthy and osteoarthritic cartilage. In this study, the expression pattern of three controversial cartilage growth factors was compared between osteoarthritic (n=27) ‎and non-osteoarthritic cartilage samples (n=14). The candidate growth factor included Insulin-like ‎growth factor 1 (IGF-1), Fibroblast Growth Factor 2 (FGF-2), and Bone Morphogenetic Protein 2 (BMP-2). The candidate markers' gene and protein expression were analyzed using relative quantification relative to real-time PCR and ELISA, respectively. The mean IGF-I ‎mRNA and protein expression were 11.1, and 2 folds greater in osteoarthritic cartilage, respectively (p&lt;0.001 for both). The mean FGF-2 ‎mRNA and protein expression were 6.7 and 1.3 folds greater in osteoarthritic cartilage, respectively (p&lt;0.001 and p=0.003). The mean BMP-2 ‎mRNA and protein expression were 3.48 and 1.51 folds greater in osteoarthritic cartilage, respectively (p=0.001 and p&lt;0.001). A significant positive correlation was found between the mRNA and protein expression of all candidate growth factors. In conclusion, the gene and protein expression of IGF-I, FGF-2, and BMP-2 were significantly higher in the ‎osteoarthritic cartilages when compared with non-osteoarthritic, suggesting them as‎‏ a therapeutic ‏target in OA treatment.

A new glioma grading model based on histopathology and Bone Morphogenetic Protein 2 mRNA expression

Glioma, the most common form of primary malignant brain tumors, is graded based solely on histopathological appearance, which has led to prognostic discrepancies. This study aimed to establish a new glioma grading model by analyzing the expression of Bone Morphogenetic Protein 2 (BMP2) mRNA in patients with gliomas as well, named the Histopathological-BMP2 (HB) system. Clinical information was collected from 692 patients from the Chinese Glioma Genome Atlas database. According to pathological glioma subtypes and the expression of BMP2 mRNA in tumor tissues, the new subtypes HBs, HBh, HBm and HB1 were established, with BMP2 expression highest in HBs and lowest in HB1. Survival periods were analyzed. Based on this, the expression of three BMP2 receptors (BMPR1A, BMPR1B, and BMPR2) was also analyzed, which was related to the prognosis of patients. This new classification model was validated in further groups of patients from the CGGA database (n = 291) and the Cancer Genome Atlas (n = 625). A new glioma grade (HB grade) based on histopathology and BMP2 expression can predict the prognosis of glioma patients, with BMPR1B and BMPR2 expression indicating a different prognosis in different types of gliomas. The higher the concentration of BMP2, the better the prognosis of patients.

Daidzein induces bone morphogenetic protein-2 and runt-related transcription 2 on periodontal ligament cells after experimental tooth movement

ABSTRACT Purpose To analysis, daidzein increases the expression of bone morphogenetic protein-2 (BMP-2) and runt-related transcription factor 2 (Runx2) after experimental tooth movement (EXTM). Materials and Methods 36 Wistar rats were randomly divided into the EXTM group and EXTM with daidzein group (EXTM+DZ group). After EXTM, the rats in the EXTM+DZ group were treated with daidzein (10 mg/kg/day). Micro-computed tomography (CT) analysis revealed the bone mineral density (BMD), ratio of bone volume to tissue volume (BV/TV), and ratio of relapse. Histopathological changes were examined by immunohistochemical staining for BMP-2 and Runx2. Human PDLCs were subjected to tension force and treated with 10 μg/mL daidzein. The expression of BMP-2 and Runx2 by daidzein was confirmed by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Results The EXTM+DZ group showed a significantly decreased ratio of relapse and increased BMD and BV/TV as compared to the EXTM group. The ratio of BMP-2 – and Runx-2 –positive cells in the EXTM+DZ group was higher than that in the EXTM group. Moreover, daidzein upregulated the mRNA expression and protein levels of BMP-2 and Runx2 in a time-dependent manner. Conclusion These results suggested that daidzein decreased the ratio of relapse through BMP-2 and Runx2 expression.

The protective effects of renin-angiotensin system componts on vascular calcification.

Renin-angiotensin system (RAS) has important roles in cardiovascular disease. Angiotensin II (Ang II) and angiotensin-(1-7) (Ang-(1-7)) are major effector peptides of RAS. However, the roles of Ang II type 2 receptor (AT2R) need to be further explored and the roles of Ang-(1-7) are still not very clear on vascular calcification (VC). Therefore, we hypothesized they have effects on preventing VC in vivo and in vitro. VC model is established by inorganic phosphate (IP) cultured with vascular smooth muscle cells (VSMC) for in vitro study and by 5/6 nephrectomy in mice for in vivo study. Increased calcified nodules by Alizarin Red S staining and mRNA expressions of bone morphogenetic protein-2 (BMP-2) and osteocalcin (OCN) by reverse transcription polymerase chain reaction in calcified WT VSMC were significantly inhibited in calcified AT2R overexpression (SmAT2) VSMC or after Ang-(1-7) treatment. After 5/6 nephrectomy, the ratio of positive and total area by Alizarin Red S and von Kossa staining and mRNA expressions of BMP-2 and OCN were significantly increased in ApoE/AT2R knockout mice compared with apolipoprotein E knockout mice, and which were significantly inhibited with Ang-(1-7) administration. Both AT2R and Ang-(1-7) have the effects on preventing VC induced by IP, at least in part through inhibiting BMP-2, OCN expressions, and in which Ang-(1-7) had protective roles mainly through Mas receptor rather than AT2R.

Construction and characterization of an adenoviral vector encoding human bone morphogenetic protein-2.

ObjectivesConstruction of adenoviral vectors can be complicated and time-consuming. The aim of this study was to construct an adenoviral vector expressing human bone morphogenetic protein 2 (BMP-2).MethodsAn adenoviral vector expressing human BMP-2 was constructed using the Gateway™ technique based on site-specific recombination. Briefly, BMP-2 cDNA was obtained by polymerase chain reaction, inserted into the pMD19-T cloning vector, and subcloned into a shuttle vector. The DNA sequence encoding BMP-2 was then subcloned from pEC3.1-BMP2 into the pAd/BLOCK-iT™-DEST adenoviral vector genome, which was then linearized and used to transfect 293 cells for adenovirus packaging. Adenovirus particles were generated with a titer of 1 × 1010 infectious units/mL.ResultsPotent expression of BMP-2 mRNA and protein was detected in adenovirus-infected 293 cells, confirming that the adenoviral vector encoding BMP-2 was successfully constructed.ConclusionsThis recombinant adenoviral vector encoding BMP-2 can be applied in future studies to explore the roles of BMP-2 in various cell types and tissues.

Analysis of Wnt7B and BMP4 expression patterns in congenital pulmonary airway malformation.

Congenital pulmonary airway malformation (CPAM) is a rare disorder characterized by aberrant overgrowth of terminal bronchioles. The objective of this study was to describe wingless-type MMTV integration site family 7B (Wnt7B) and bone morphogenetic protein 4 (BMP4) expression patterns in human CPAM lesions and to explore the possible roles of Wnt7B and BMP4 in the pathogenesis of CPAM. Fifteen tissue samples from patients with CPAM were obtained from the Pathology Department of Shengjing Hospital of China Medical University. Samples representing CPAM lesions and adjacent normal lung tissues were collected and Wnt7B and BMP4 expression was detected through immunohistochemical (IHC) staining, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blotting. IHC revealed that Wnt7B immunopositive cells were only detected in epithelial cells, whereas BMP4 immunopositive cells were detected in epithelial and mesenchymal cells. Expression of Wnt7B and BMP4 immunopositive cells was higher in CPAM lesions than that in adjacent normal lung tissue. qRT-PCR and Western blotting showed that Wnt7B and BMP4 mRNA and protein expression were significantly higher in CPAM lesions than in adjacent normal lung tissue (P < .05). Overall, the level of BMP4 was higher than that of Wnt7B. Increased expression of Wnt7B and BMP4 appear to be related to the pathogenesis of CPAM and abnormal pulmonary development. Upregulation of Wnt7B and BMP4 could play an important role in the development of the bronchial-alveolar structures that characterize CPAM.

Bone morphogenetic protein 6 expression in cumulus cells is negatively associated with oocyte maturation

Bone morphogenetic protein 6 (BMP6) is a regulatory peptide secreted by oocytes and granulosa cells that locally regulates folliculogenesis and follicular development. To determine BMP6 location, we studied BMP6 expression in human follicles using immunohistochemistry, real-time polymerase chain reaction (RT-PCR) and western blot analysis. RT-PCR was performed on 354 individual cumulus cell (CC) masses from 48 women to investigate the relationship between BMP6 mRNA expression in CCs and oocyte developmental potential. Results showed that BMP6 protein was mainly located in oocytes from preantral follicles and in granulosa cells from antral follicles. BMP6 mRNA expression was much higher in oocytes than in CCs and mural granulosa cells (mGCs) from preovulatory follicles (p < 0.01), and BMP6 protein level was higher in CCs than in mGCs (p < 0.05). BMP6 mRNA expression was higher in CCs from immature oocytes than in those from mature oocytes (p < 0.05). However, BMP6 mRNA expression in CCs was not associated with oocyte fertilization, embryo morphological grading, or implantation. In conclusion, BMP6 was mainly expressed in oocytes at all human follicular developmental stages and BMP6 mRNA expression in CCs may be negatively correlated with oocyte maturation. BMP6 expression could therefore be used as a biomarker of oocyte maturation.

Angiogenesis and bone regeneration by mesenchymal stem cell transplantation with danshen in a rabbit model of avascular necrotic femoral head

The present study aimed to explore the potential of combined treatment with mesenchymal stem cells (MSCs) and danshen for angiogenesis and bone regeneration in a rabbit model of avascular necrosis of femoral head (ANFH). A rabbit model of ANFH was established using the Shwartzman reaction with methylprednisolone and Escherichia coli endotoxin injection. Magnetic resonance imaging (MRI) and histopathological examination were used to evaluate the rabbit model of ANFH. The rabbits were randomly divided into the danshen group, the MSCs group, the danshen combined with MSCs group and the model group (treated with physiological saline). The expression level of monocyte chemoattractant protein-1 (MCP-1) and stromal cell-derived factor-1 (SDF-1) were determined by reverse transcription polymerase chain reaction (RT-PCR). The expression level of bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) were detected by immunofluorescence and the mRNA expression of BMP-2 and VEGF were detected by RT-PCR. Typical osteonecrosis occurred in the rabbit model of ANFH, which indicated that the model was successfully established. MCP-1 and SDF-1 were significantly increased in the model group compared with the normal group (P<0.05). Following the administration of MSCs and Salvia miltiorrhiza (danshen), MSCs labeled with 5-bromo-2-deoxyuridine were observed to be gathered in the necrotic area. The increased migration of MSCs to the necrotic area may be due to the upregulated expression of the chemokines MCP-1 and SDF-1. ANFH treated with danshen combined with MSCs may promote revascularization by increasing the expression of VEGF and BMP-2 in the femoral head, promoting re-ossification and revascularization. Danshen combined with the transplantation of MSCs may be regarded as a novel therapy for the treatment of ANFH in a clinical setting.