Differentially expressed genes and signalling pathways are involved in mouse osteoblast-like MC3T3-E1 cells exposed to 17-β estradiol

Oestrogen is essential for maintaining bone mass, and it has been demonstrated to induce osteoblast proliferation and bone formation.In this study, complementary DNA（cDNA） microarrays were used to identify and study the expression of novel genes that may be involved in MC3T3-E1 cells’ response to 17-b estradiol. MC3T3-E1 cells were inoculated in minimum essential media alpha（a-MEM）cell culture supplemented with 17-b estradiol at different concentrations and for different time periods. MC3T3-E1 cells treated with1028mol？L2117-b estradiol for 5 days exhibited the highest proliferation and alkaline phosphatase（ALP） activity; thus, this group was chosen for microarray analysis. The harvested RNA was used for microarray hybridisation and subsequent real-time reverse transcription polymerase chain reaction（RT-PCR） to validate the expression levels for selected genes. The microarray results were analysed using both functional and pathway analysis. In this study, microarray analysis detected 5 403 differentially expressed genes,of which 1 996 genes were upregulated and 3 407 genes were downregulated, 1 553 different functional classifications were identified by gene ontology（GO） analysis and 53 different pathways were involved based on pathway analysis. Among the differentially expressed genes, a portion not previously reported to be associated with the osteoblast response to oestrogen was identified. These findings clearly demonstrate that the expression of genes related to osteoblast proliferation, cell differentiation, collagens and transforming growth factor beta（TGF-b）-related cytokines increases, while the expression of genes related to apoptosis and osteoclast differentiation decreases, following the exposure of MC3T3-E1 cells to a-MEM supplemented with 17-b estradiol. Microarray analysis with functional gene classification is critical for a complete understanding of complementary intracellular processes. This microarray analysis provides large-scale gene expression data that require further confirmatory studies.

雌二醇通过雌激素受体α/糖原合酶激酶-3β/β联蛋白信号通路调节成骨细胞增殖

目的研究观察雌二醇对小鼠成骨细胞前体细胞增殖影响并研究经典wnt通路在其增殖过程中的作用。方法本研究利用小鼠胚胎成骨细胞前体细胞Mc3T3-E1为细胞模型,不同浓度雌二醇处理Mc3T3-E1细胞,四甲基偶氮唑蓝(MTT)检测细胞增殖率,蛋白印迹法和免疫荧光技术检测经典Wnt信号通路信号分子表达。结果研究表明不同浓度雌二醇处理Mc3T3-E1细胞,MTT显示随着雌二醇浓度增加,Mc3T3-E1细胞增殖率增加,其中以10-7 mol/L浓度处理后细胞获得最佳增殖率。蛋白印迹法结果显示随着雌激素浓度增加,小鼠Mc3T3-E1细胞中p-GSK-3β,β联蛋白(catenin),细胞周期蛋白(Cyclin)D1的表达增加。经雌激素受体(ER)α沉默后,相对于空白对照组小鼠Mc3T3-E1细胞p-GSK-3β,β-catenin及Cyclin D1表达下降,而ERβ沉默后,同对照组相比成骨细胞中各分子表达则无明显变化。结论雌二醇可通过ERα/GSK-3β/β-catenin信号通路调节小鼠成骨细胞增殖。

Sustained release of Semaphorin 3A from a-tricalcium phosphate based cement composite contributes to osteoblastic differentiation of MC3T3-E1 cells

The reinforcement of calcium phosphate materials with silk fibroin （SF） has been one of the strategies to overcome the brittleness. However, the lack of osteoinductivity may still restrict their further use. This study aimed to investigate the biocompatibility and osteogenesis capacity of a novel Semaphorin 3A-loaded chitosan microspheres/SF/a-tricalcium phosphate composite （Sema3A CMs/SF/a-TCP） in vitro. Sema3A was first incorporated into CMs, and the Sema3A CMs/SF/a-TCP composite was then prepared. The morphology of the CMs was observed using SEM. The in vitro release kinetics, cytotoxicity, and cell compatibility were evaluated, and the real-time quantitative polymerase chain reaction （RT-qPCR） and activity of alkaline phosphatase （ALP） were used to evaluate the osteogenesis capacity of the composite. The in vitro release of Sema3A from the Sema3A CMs/SF/a-TCP composite showed a temporally controlled manner. The extract of the Sema3A CMs/SF/a-TCP composite presented no obvious side effect on the MC3T3-E1 cell proliferation, nor promote cell proliferation. The MC3T3-E1 cells were well-spread and presented an elongated shape on the Sema3A CMs/SF/a-TCP composite surface; the ALP activity and the osteogenic-related gene expression were higher than those seeded on the surface of the CMs/SF/a-TCP and SF/a-TCP composites. In conclusion, Sema3A CMslSF/a-TCP has excellent biocompatibility and contributes to the osteoblastic differentiation of MC3T3-E1 cells.

In Vitro Biocompatibility of MC3T3-E1 Osteoblast-like Cells on Arg-Gly-Asp Acid Peptides Immobilized Graphite-like Carbon Coating on Carbon/Carbon Composites

Carbon/carbon （C/C） composites were deposited with graphite-like carbon （GLC） coating, and then, Arg-Gly- Asp acid （RGD） peptides were successfully immobilized onto the functionalized GLC coating. GLC coating was utilized to prevent carbon particles releasing and create a uniform surface condition for C/C composites. RGD peptides were utilized to improve biocompatibility of GLC coating. Surface chemical characterizations of functionalized GLC coating were detected by contact angle measurement, X-ray photoelectron spectroscopy and Raman spectra. Optical morphology of GLC coatings was observed by confocal laser scanning microscopy. In vitro biological performance was determined using samples seeded with MC3T3-E1 osteoblast-like cells and cultured for 1 week. Surface characterizations and morphological analysis indicated that C/C composites were covered by a dense and uniform GLC coating. Contact angle of GLC coating was reduced to 27.2° when it was functionalized by H202 oxidation at 40 ℃ for 1 h. In vitro cytological test showed that the RGD peptides immobilized GLC coating had a significant improvement in biocompatibility. It was suggested that RGD peptides provided GLC coating with a bioactive surface to improve cell adhesion and proliferation on C/C composites.

In vitro Biological Effects of Ti2448 Alloy Modified by Micro-arc Oxidation and Alkali Heatment

The purpose of this study was to test the hypothesis that the combination of micro-arc oxidation and alkali heatment （MAH） would improve the cytocompatibility of a newly designed Ti-24Nb-4Zr-8Sn alloy. In this study, commercially pure titanium （cp Ti） and Ti-24Nb-4Zr-8Sn were used. Surface modification of Ti-24Nb- 4Zr-8Sn by a two-step treatment of micro-arc oxidation （MAO） and alkali heatment was reported. Surface characterizations were performed by scanning electron microscopy （SEM）, thin film X-ray diffraction （TF-XRD） and X-ray photoelectron spectroscopy （XPS）. The MAH layer consisted of finer crystals and possessed a higher degree of crystallity and stability than the MAO layer. A biocompatibility study on treated and untreated Ti- 24Nb-4Zr-8Sn in comparison with cp Ti was carried out to investigate the effect of the different surfaces on the bone integration property in vitro. The cellular assays revealed that the MAO and MAH layer favored the initial adhesion of MC3T3-E1 cells and that the growth rate of MC3T3-E1 cells on MAH layer was significantly higher than that on the conventional MAO-treated layer after 3-day and 5-day incubation, demonstrating the greater potential of the hybrid treatment of micro-arc oxidation followed with alkali heatment as a novel surface modification method for implanting materials.