Proliferation and differentiation of osteoblast-like MC3T3-E1 cells on biomimetically and electrolytically deposited calcium phosphate coatings

Biomimetic and electrolytic deposition are versatile methods to prepare calcium phosphate coatings. In this article, we compared the effects of biomimetically deposited octacalcium phosphate and carbonate apatite coatings as well as electrolytically deposited carbonate apatite coating on the proliferation and differentiation of mouse osteoblast-like MC3T3-E1 cells. It was found that MC3T3-E1 cells cultured on the biomimetically deposited carbonate apatite coating demonstrated the greatest proliferation rate and the highest differentiation potential. Cells on the biomimetically deposited octacalcium phosphate coating had lower proliferation rate before day 7, but higher after that, than those on the electrolytically deposited carbonate apatite coating. There was no difference on the expression of early differentiation markers, that is, alkaline phosphatase activity and collagen content, between biomimetically deposited octacalcium phosphate and electrolytically deposited carbonate apatite coatings. However, higher expression of late differentiation markers, that is, osteocalcin and bone sialoprotein mRNA, was found on the biomimetically deposited octacalcium phosphate coating on day 14. These results suggest that the difference in in vitro osteoblast cell performance of calcium phosphate coatings might relate to their physicochemical properties. Biomimetic carbonate apatite coating is the most favorable surface for the proliferation and differentiation of MC3T3-E1 cells.     

Influence of multilayer rhBMP-2 DNA coating on the proliferation and differentiation of MC3T3-E1 cells seeded on roughed titanium surface

For bone morphogenetic protein (BMP) gene therapy to be a viable approach for enhancing implant osseointegration clinically, requires the development of efficient nonviral delivery vectors that can coat the implant. This study evaluated a multilayer cationic liposome-DNA complex (LDc) coating as a delivery vehicle for recombinant human BMP-2 (rhBMP-2). Multilayered coatings, comprising hyaluronic acid (HA) and LDc, were fabricated onto titanium using a layer-by-layer (LBL) assembly technique. Preosteoblastic MC3T3-E1 cells were cultured on the roughened titanium surfaces coated with multilayers of HA/LDc, or on uncoated or HA/liposome only surfaces as controls. The amount of rhBMP-2 secreted by the MC3T3-E1 cells and the effect of the various surfaces on cell viability, proliferation, alkaline phosphatase (ALP) activity, osteocalcin (OC) secretion, and calcium deposition were evaluated. Messenger RNA levels of OC, ALP, Runx2, and Osx were also investigated. The results demonstrated that rhBMP-2 protein secreted into culture medium at 3 days was significantly higher than control groups. MC3T3-E1 cells cultured on the HA/LDc coating displayed significantly higher ALP activity and OC secretion at 7 days and 14 days culture, respectively. MC3T3-E1 cells cultured on HA/LDc upregulated expression of the osteoblast differentiation markers, especially on days 12 for OC and on days 6 and 12 for ALP and Osx. In conclusion, MC3T3-E1 cell cultured on the multilayer HA/LDc coating surface can secret rhBMP-2 protein and the protein levels were effective in inducing early osteogenic differentiation. ? 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A 100A: 2766-2774, 2012.     

Poly(beta-hydroxybutyrate-co-beta-hydroxyvalerate) supports in vitro osteogenesis

Studies have demonstrated that polymeric biomaterials have the potential to support osteoblast growth and development for bone tissue repair. Poly(beta-hydroxybutyrate-co-beta-hydroxyvalerate) (PHBV), a bioabsorbable, biocompatible polyhydroxy acid polymer, is an excellent candidate that, as yet, has not been extensively investigated for this purpose. As such, we examined the attachment characteristics, self-renewal capacity, and osteogenic potential of osteoblast-like cells (MC3T3-E1 S14) when cultured on PHBV films compared with tissue culture polystyrene (TCP). Cells were assayed over 2 weeks and examined for changes in morphology, attachment, number and proliferation status, alkaline phosphatase (ALP) activity, calcium accumulation, nodule formation, and the expression of osteogenic genes. We found that these spindle-shaped MC3T3-E1 S14 cells made cell-cell and cell-substrate contact. Time-dependent cell attachment was shown to be accelerated on PHBV compared with collagen and laminin, but delayed compared with TCP and fibronectin. Cell number and the expression of ALP, osteopontin, and pro-collagen alpha1(I) mRNA were comparable for cells grown on PHBV and TCP, with all these markers increasing over time. This demonstrates the ability of PHBV to support osteoblast cell function. However, a lag was observed for cells on PHBV in comparison with those on TCP for proliferation, ALP activity, and cbfa-1 mRNA expression. In addition, we observed a reduction in total calcium accumulation, nodule formation, and osteocalcin mRNA expression. It is possible that this cellular response is a consequence of the contrasting surface properties of PHBV and TCP. The PHBV substrate used was rougher and more hydrophobic than TCP. Although further substrate analysis is required, we conclude that this polymer is a suitable candidate for the continued development as a biomaterial for bone tissue engineering.     

Enhancing effect of poly(L-lactide) on the differentiation of mouse osteoblast-like MC3T3-E1 cells

Poly(L-lactide) (PLLA) has bioabsorbability and biocompatibility, and it is used as biodegradable screws, pins and plates for internal bone fixation. The purpose of this study was to clarify the effects of low molecular weight (Mw) PLLA on the proliferation and differentiation of mouse osteoblast-like MC3T3-E1 cells. MC3T3-E1 cells were cultured with the concentration of 5-50 microg/ml of PLLA with weight average Mw of 5000 (PLLA-5k) and 10,000 (PLLA-10k) for 2 weeks using the micromass culture. Both PLLAs did not affect the proliferation of MC3T3-E1 cells. However, the calcifications of MC3T3-E1 cells were stimulated with increasing the concentration of the PLLAs. Then PLLA-5k increased the calcification of MC3T3-E1 cells more than PLLA-10k. Additionally, both PLLAs increased the alkaline phosphatase (ALP) activity and calcium content of MC3T3-E1 cells up to the similar level to the calcification. These results indicated that low Mw PLLA enhanced the differentiation of MC3T3-E1 cells with no effect on the proliferation. Moreover, it was suggested that the increase of the ALP activity was a key step to stimulate the calcification of MC3T3-E1 cells. The osteoconductivity of implanted PLLA would be based on the enhancing effect of low Mw PLLA on the differentiation of the osteoblasts.     

Abnormal Differentiation in MC3T3-E1 Preosteoblasts Expressing a Dominant-Negative Type I Collagen Mutation

To examine the effects that an organizing extracellular matrix might have on osteoblast precursors, we created MC3T3-E1 cell lines that stably incorporated a plasmid that expressed proαl(I) collagen chains having a truncated triple helical domain. Cells that had incorporated the proαl(I) expression plasmid (pMG155) efficiently secreted molecules with shortened prood(I) chains into culture media. Electron micrographs indicated that expression of the minigene dramatically interferes with normal type I collagen fibril architecture. The turnover of newly deposited collagenous matrix as measured by ³[H]-hydroxyproline release was 29% after a 14 day chase in cells expressing the mini-gene compared to essentially no turnover in control cultures. MC3T3-E1 cells in culture normally demonstrate a time dependent reduction of cell division followed by an increase in osteoblast characteristics. Cell number was consistently 20–25% higher than control in MC3T3-E1 cultures expressing the truncated prooαl(I) gene but ALP activity was only 45% of control. Secretion and steady state mRNA levels for osteocalcin were over fivefold higher than control cultures but expression of other extracellular matrix components was not changed. These findings demonstrate that osteoblasts require a normally structured collagenous matrix for inhibition of cellular proliferation and subsequent upregulation of ALP. However, in the presence of rapid turnover of osteoblast matrix, the gene for osteocalcin may be upregulated in response to local signals.     

Responses of MC3T3-E1 cells to three dental resin-based restorative materials

This study aimed to examine the influences of three dental resin-based restorative materials on cells associated with hard tissue regeneration using osteoblastic MC3T3-E1 cells. A Bis-GMA-based resin composite [Clearfil AP-X (APX)], an MMA-based resin cement [Superbond C&B (SB)], and a resin-modified glass-ionomer [Fuji Ionomer Type II LC (LC)] were tested. A zinc oxide eugenol cement [Super EBA (EBA)] was included in the study for comparison. MC3T3-E1 cells were cultured on set materials for 3, 7, 14, or 21 days. Cell attachment and proliferation were observed by scanning electron microscopy, and mitochondrial dehydrogenase and alkaline phosphatase (ALP) activities of the cells were evaluated. Cell cultures on polystyrene tissue culture dishes served as controls. On APX and SB, cells demonstrated attachment, spreading, and proliferation similar to the controls. In contract, cells adhered and proliferated poorly on LC and EBA. The mitochondrial function and ALP activity of the cells were significantly suppressed (p < 0.05, Scheffe's F test) throughout the experimental period when cultured on LC or EBA, although APX and SB exhibited less inhibition. The results indicate that APX and SB are less toxic to proliferation and differentiation of MC3T3-E1, suggesting that a smaller influence on cementogenesis on these materials can be expected.     

Asxl1在炎性微环境中对成骨细胞增殖分化的作用

背景:研究表明Asxl1的缺失可导致骨质发育不全、骨质缺损类疾病的发生,但目前在根尖周炎环境下该因子与骨破坏之间的关系暂无相关报道。目的:探讨炎性微环境下Asxl1对成骨细胞增殖分化的影响。方法:实验选用脂多糖刺激MC3T3-E1细胞建立体外炎性微环境,通过CCK-8实验筛取脂多糖最佳质量浓度和最佳作用时间,然后用20 mg/L脂多糖刺激MC3T3-E1细胞24 h,免疫荧光检测Asxl1的蛋白表达水平,Real Time-PCR检测Asxl1 mRNA的表达水平。为进一步验证Asxl1基因在炎性微环境中影响成骨细胞的增殖与分化,脂多糖刺激形成炎性微环境后转染Asxl1-SiRNA 24 h,采用CCK-8检测细胞增殖活性,RealTime-PCR检测Asxl1及成骨相关基因ALP和RUNX2 mRNA的表达水平。结果与结论:①脂多糖刺激MC3T3-E1细胞后,Asxl1蛋白和mRNA表达水平呈降低趋势;②脂多糖刺激MC3T3-E1细胞后,转染Asxl1-SiRNA 24 h,细胞增殖活性下降趋势明显,Asxl1基因及成骨相关基因ALP和RUNX2 mRNA的表达水平明显降低;③结果提示,Asxl1可能通过参与炎性反应过程,影响成骨细胞的增殖与分化,进而参与骨破坏进程。     

In vitro response of osteoblast-like and odontoblast-like cells to unsubstituted and substituted apatites

Different types of calcium phosphate compounds [calcium-deficient apatite (CDA); beta-tricalcium phosphate (beta-TCP); biphasic calcium phosphate (BCP)] are commercially available for medical and dental applications as bone substitute materials. Most of the reported in vitro studies on cell-material interactions have used osteoblast-like cells. The purpose of this study was to investigate the in vitro response of osteoblast-like (MC3T3-E1) and odontoblast-like (MDPC23) cells on unsubstituted (HA) and substituted (F-substituted) apatites. MC3T3-E1 and MDPC23 were cultured in alpha-modified medium containing 10% fetal bovine serum, ascorbic acid (50 microg/mL) and beta-glycerophosphate (2 mM). The cells were seeded on pellets made from HA, and FAp (with low, medium, and high F concentrations). Cell morphology was observed after 7 and 14 days using scanning electron microscopy (SEM). Cell attachment and differentiation were determined from the DNA content, alkaline phosphatase (ALP) activity, and total collagen content. Pellet surface composition was characterized by using Fourier Transform infrared spectroscopy. MC3T3-E1 and MDPC23 cells on HA were normal in shape and in fusion but not on FAp. Results of this study showed that the pattern of cell proliferation of osteoblast-like cells was different from that of the odontoblast-like cells. This study suggests that cell morphology, fusion, and proliferation on biomaterial surfaces depend on cell type (osteoblast-like vs odontoblast-like cell) and biomaterial composition (unsubstituted vs substituted F-apatites).     

The effect of calcium phosphate microstructure on bone-related cells in vitro

Microstructure is essential for inductive bone formation in calcium phosphate materials after soft tissue implantation. We hereby evaluated activities (cell attachment, proliferation, ALP/DNA and protein/DNA) of three types of cells cultured on three kinds of calcium phosphate ceramic discs to study how microstructure takes its role in inductive bone formation. Three kinds of biphasic calcium phosphate (BCP) ceramic discs with the same chemistry and the same dimension of 10.0 x 1.0 mm3 (BCP1150-P, BCP1150-D and BCP1300), either having similar micropore sizes and surface roughness but different surface area (BCP1150-P vs BCP1150-D) or having similar surface area but different micropore sizes and different roughness (BCP1150-D vs BCP1300), were prepared. Conventionally Culturing C2C12, human bone marrow stromal cells (HBMSC) and MC3T3-E1 cells on BCP discs showed that, surface roughness did not affect cell attachment, cell proliferation and ALP expression of all cell types evaluated, while surface area did affect cell functions. ALP/DNA of C2C12 on BCP1150-P, having larger surface area, was significantly higher than on BCP1300 and BCP1150-D. Furthermore, all cells cultured on all of the three kinds of BCPs pre-soaked in culture medium having additional rhBMP-2 had a higher ALP expression than the conventional cell culture. Comparing with on BCP1300 and BCP1150-D, ALP/DNA of all cells tested increased more on BCP1150-P after the discs were pre-soaked in culture medium with rhBMP-2. The results indicated that increasing surface areas, microstructured calcium phosphate materials might concentrate more proteins (including bone-inducing proteins) that differentiate inducible cells to osteogenic cells that form inductive bone.     

Icariin stimulates MC3T3-E1 cell proliferation and differentiation through up-regulation of bone morphogenetic protein-2

Previous studies suggest that icariin has anabolic effects on bone, but the mechanisms are unknown. We aimed to investigate the osteogenic effects of icariin in an undifferentiated osteoblast cell line by detecting cell morphology, viability, cell cycling and bone morphogenetic protein-2 (BMP-2) expression. We treated pre-osteoblastic MC3T3-E1 cells with different concentrations of icariin [0 (as a control), 10, 20 and 40 ng/ml] for 48, 72 and 96 h. Cell morphology, viability and the cell cycle were examined and measured using microscopy, the MTT assay or flow cytometry, respectively. BMP-2-positive cells and BMP-2 protein expression levels in icariin-treated MC3T3-E1 cells were examined using immunohistochemistry staining with fluorescence optical density analysis and Western blotting. MC3T3-E1 cells showed typical characteristics of osteoblasts in response to treatment with icariin. Cells treated with all concentrations of icariin had increased percentages of S-phase cells and decreased percentages of G1-phase cells, especially in the 10 and 20 ng/ml icariin groups. The number of BMP-2-positive cells and BMP-2 protein expression levels in the 10 and 20 ng/ml icariin treatment groups were greater compared to the 0 and 40 ng/ml groups. Treatment of icariin promotes osteoblast MC3T3-E1 proliferation and differentiation in vitro, potentially owing to its role in increasing BMP-2 protein expression. Icariin potentially can be used as a drug in clinical settings to treat osteoporosis.     

Effects of alpha-TCP and TetCP on MC3T3-E1 proliferation,differentiation and mineralization

Calcium phosphates with high solubility in water such as alpha-tricalcium phosphate (alpha-TCP) and tetracalcium phosphate (TetCP) have received considerable attention as components of bone-substitution materials. However, the osteoblast response to these materials has not yet been clearly understood. This study examined the effects of alpha-TCP and TetCP on osteoblast proliferation, differentiation and mineralization in the culture system of MC3T3-E1 cells. Cells were cultured in a differentiation medium with or without alpha-TCP or TetCP at 1 or 10 microM, and the number of cells attached to the culture plates was determined. To examine osteoblast differentiation, the alkaline phosphatase (ALP) activity was measured and the expression of osteoblastic markers analyzed by RT-PCR. In addition, mineralization was evaluated by staining the calcium deposit with Alizalin red. Culture in the presence of alpha-TCP or TetCP showed no significant influence on cell proliferation. ALP activities of the cells were enhanced by both calcium phosphates for 3d and the expression of type I collagen was promoted at 12h and 1d after incubation. Enhancement of bone-like tissue formation by the addition of alpha-TCP or TetCP at 10 microM was observed after 7d incubation and thereafter. The results of the present study indicate that alpha-TCP and TetCP promote osteogenesis by increasing collagen synthesis and calcification of the extra-cellular matrix.     

IL-37通过调控M-CSF和抑制IL-6-JAK2/STAT3信号通路抑制骨质疏松的机制研究

目的:探讨IL-37 在抑制骨质疏松过程中的作用机制。方法:选取本院2013 年1 月至2015 年12 月收治的97例骨质疏松患者及在本院行骨折手术的81 例无骨质疏松患者(对照组)为研究对象,检测两组血清中IL-37 及IL-6 的水平。构建IL-37 转基因小鼠,将C57BL/6J 小鼠、IL-37 转基因小鼠分别设置假手术(Sham)组,手术组(卵巢切除术,OVX 组)。8 周后,取小鼠血清,检测血清中雌激素水平、碱性磷酸酶水平(ALP)、血钙和血磷水平;同时取小鼠的双侧股骨、脊柱,病理切片分析股骨组织形态结构,骨密度仪检测脊柱骨密度变化。分离培养各组小鼠骨髓基质细胞(Bone marrow stromal cells,BMSCs), 检测BMSCs 的体外增殖能力,M-CSF 及IL-6 的表达及STAT3 的激活。IL-37 转染小鼠成骨细胞MC3T3-E1,转染后72 h,ELISA 检测上清中M-CSF 及IL-6,流式细胞术检测MC3T3-E1 细胞的凋亡,Western blot 检测STAT3 的激活。结果:骨质疏松患者血清中IL-37 水平显著低于对照组(P<0.05),而IL-6 则显著高于对照组;C57BL/6J 小鼠、IL-37 转基因小鼠OVX 组血清中雌激素、血钙和血磷显著低于假手术组,而ALP 水平显著高于假手术组(P<0.05),但IL-37 转基因小鼠OVX 组血钙和血磷则显著高于C57BL/6J 小鼠OVX 组(P<0.05)。股骨病理切片及脊柱骨密度结果显示,C57BL/6J 小鼠、IL-37 转基因小鼠OVX组均出现组织形态结构的破坏和骨密度下降,但IL-37 转基因小鼠明显优于C57BL/6J 小鼠(P<0.05)。IL-37 转基因小鼠OVX 组BMSCs 增殖能力显著高于C57BL/6J 小鼠OVX 组,而STAT3 的激活和M-CSF 的表达则显著低于C57BL/6J 小鼠OVX组(P<0.05)。MC3T3-E1 细胞转染IL-37 后能明显抑制M-CSF 及IL-6 的表达,而STAT3 的激活也明显被抑制,流式细胞检测显示转染IL-37 后能显著抑制MC3T3-E1 细胞的凋亡。结论:骨质疏松患者血清IL-37 水平显著降低,IL-37 可能是通过调控M-CSF 及IL-6-JAK2/ STAT3 信号通路促进BMSCs 增殖和抑制成骨细胞的凋亡从而抑制骨质疏松的进展。     

The effect of a bioactive collagen membrane releasing PDGF or GDF-5 on bone regeneration

Regenerative procedures using barrier membrane technology are presently well established in periodontal/endodontic surgery. The objective of this study was to compare the subsequent effects of the released platelet-derived growth factor (PDGF) and growth/differentiation factor 5 (GDF-5) from collagen membranes (CMs) on bone regeneration in vitro and in vivo. In vitro studies were conducted using MC3T3-E1 mouse preosteoblasts cultured with or without factors. Cell viability, cell proliferation, alkaline phosphatase (ALP) activity and bone marker gene expression were then measured. In vivo studies were conducted by placing CMs with low or high dose PDGF or GDF-5 in rat mandibular defects. At 4 weeks after surgery new bone formation was measured using μCT and histological analysis. The results of in vitro studies showed that CM/GDF-5 significantly increased ALP and cell proliferation activities without cytotoxicity in MC3T3-E1 cells when compared to CM/PDGF or CM alone. Gene expression analysis revealed that Runx2 and Osteocalcin were significantly increased in CM/GDF-5 compared to CM/PDGF or control. Quantitative and qualitative μCT and histological analysis for new bone formation revealed that although CM/PDGF significantly enhanced bone regeneration compared to CM alone or control, CM/GDF-5 significantly accelerated bone regeneration to an even greater extent than CM/PDGF. The results also showed that GDF-5 induced new bone formation in a dose-dependent manner. These results suggest that this strategy, using a CM carrying GDF-5, might lead to an improvement in the current clinical treatment of bone defects for periodontal and implant therapy.     

小分子肽对成骨前体细胞MC3T3-E1骨保护素和核转录因子κB受体活化因子配体表达的影响

背景：体内实验显示,小分子肽能明显增加去卵巢大鼠的骨钙含量,使其骨密度增加,能很好地预防骨质疏松。同时体外实验显示,小分子肽能促进小鼠成骨细胞和成骨前体细胞MC3T3-E1增殖、分化、矿化,并且可能是通过抑制核转录因子 p50和p65的表达来起作用。而小分子肽对骨保护素/核转录因子κB受体活化因子配体的影响尚不明确。 目的：观察小分子肽对MC3T3-E1在增殖、分化、矿化过程中骨保护素和RANKL表达的影响。 方法：以体积分数10%胎牛血清的DMEM培养液为空白对照组,50,100 mg/L质量浓度小分子肽作用小鼠成骨前体细胞MC3T3-E1,分别于作用3,6,12,18,24,30 d后,收集细胞提取蛋白,Western Blot检测骨保护素和核转录因子κB受体活化因子配体蛋白的表达。 结果与结论：50,100 mg/L小分子肽作用MC3T3-E1后能明显促进作用骨保护素的表达(P < 0.01),而对核转录因子κB受体活化因子配体无明显影响。小分子肽作用后MC3T3-E1中骨保护素/核转录因子κB受体活化因子配体的比值要明显高于空白对照组(P < 0.01)。因此,认为小分子肽可以通过增加骨保护素的表达来影响骨保护素/核转录因子κB受体活化因子配体系统,间接地抑制破骨细胞的数量和功能。     

Effect of gamma-ray irradiated poly(L-lactide) on the differentiation of mouse osteoblast-like MC3T3-E1 cells

The purpose of this study was to clarify the effects of gamma-ray irradiated poly(L-lactide) (PLLA) on the proliferation and differentiation of mouse osteoblast-like MC3T3-E1 cells. The PLLA was y-irradiated at the dose of 10, 25 or 50 kGy, and the molecular weight of irradiated PLLA decreased with increasing irradiation dose. The proliferation and differentiation of MC3T3-E1 cells cultured on irradiated PLLA for 2 weeks were evaluated using micromass culture. The y-irradiation of PLLA did not affect the proliferation, but stimulated the differentiation of MC3T3-E1 cells cultured on irradiated PLLA. These results suggested that lower change in the molecular weight of PLLA was responsible for stimulation of the differentiation of MC3T3-E1 cells cultured on irradiated PLLA. Furthermore, the proliferation and calcification of MC3T3-E1 cells cultured in the medium containing low molecular weight PLLA for 2 weeks were evaluated. The low molecular weight PLLA also stimulated the calcification of MC3T3-E1 cells with no effect on the proliferation. The y-irradiation was suitable for PLLA on the differentiation of mouse osteoblasts.     

Txndc5介导雌激素诱导的小鼠前成骨细胞MC3T3-E1增殖加快

 目的 研究Txndc5基因在小鼠前成骨细胞增殖中的作用。 方法 用雌激素诱导小鼠前成骨细胞系MC3T3-E1,或在MC3T3-E1细胞中分别用质粒载体过表达Txndc5和用siRNA抑制Txndc5的表达后,Western blot检测Txndc5和细胞周期蛋白水平,荧光实时定量PCR检测Cyclin A的mRNA水平,MTS法和细胞计数法检测细胞增殖速度,流式细胞术检测细胞周期。 结果 雌激素诱导MC3T3-E1增殖加快时,Txndc5的蛋白水平亦上升。抑制Txndc5的表达阻止雌激素的促细胞增殖作用。过表达Txndc5使MC3T3-E1细胞增殖速度加快,S期细胞比例增加,同步化细胞进入细胞周期18h时,过表达Txndc5组Cyclin A 的表达升高,且S期细胞比例为18.69%±4.08%,而对照组仅为8.15%±3.68%。抑制Txndc5的表达则使MC3T3-E1细胞增殖速度减慢,S期细胞比例减少,Cyclin A 的表达下降。抑制Cyclin A的表达减弱Txndc5的促细胞增殖作用。结论 Txndc5通过上调Cyclin A 的表达介导雌激素的促前成骨细胞增殖作用。     

人牙周膜干细胞来源外泌体干预成骨细胞MC3T3-E1的增殖和分化

背景:人牙周膜干细胞具有较强的成骨分化能力,人牙周膜干细胞来源外泌体作为牙周膜干细胞分泌的主要成分,对成骨细胞MC3T3-E1增殖和成骨分化的影响尚不明确。目的:探讨人牙周膜干细胞来源外泌体对MC3T3-E1细胞增殖和分化的影响。方法:采用酶消化法分离及培养人牙周膜干细胞,超速离心法提取人牙周膜干细胞来源外泌体,通过透射电镜、粒径分析及Western blot方法对人牙周膜干细胞来源外泌体进行鉴定;CCK8法检测不同质量浓度人牙周膜干细胞来源外泌体对MC3T3-E1细胞增殖的影响,茜素红染色观察100 mg/L人牙周膜干细胞来源外泌体对MC3T3-E1细胞成骨矿化的影响,Western blot检测100 mg/L人牙周膜干细胞来源外泌体干预前后MC3T3-E1细胞内MEK和ERK的磷酸化水平。结果与结论:①透射电镜观察可见外泌体为脂质双分子层形成的囊泡结构,粒径检测显示外泌体直径分布在50-120 nm,集中在79.86 nm,Western blot检测结果显示提取的外泌体中含有CD81,CD63,TSG101的表达;②与对照组相比,人牙周膜干细胞来源外泌体对MC3T3-E1细胞的增殖具有促进作用,且作用呈剂量依赖性;③与对照组相比,人牙周膜干细胞来源外泌体组MC3T3-E1细胞能够形成更多的钙结节;与对照组相比,人牙周膜干细胞来源外泌体组MC3T3-E1细胞内p-MEK及p-ERK蛋白表达量升高;④结果表明,人牙周膜干细胞来源外泌体可以显著促进MC3T3-E1增殖和成骨分化,推测可能与其激活MEK/ERK信号通路有关。     

Lipopolysaccharide (LPS) Induces the Apoptosis and Inhibits Osteoblast Differentiation Through JNK Pathway in MC3T3-E1 Cells

Bone degradation is a serious complication of chronic inflammatory diseases such as septic arthritis, osteomyelitis, and infected orthopedic implant failure. Up to date, effective therapeutic treatments for bacteria-caused bone destruction are limited. In our previous study, we found that LPS promoted osteoclast differentiation and activity through activation of mitogen-activated protein kinases (MAPKs) pathway such as c-Jun N-terminal kinases (JNK) and extracellular signal regulated kinase (ERK1/2). The current study was to evaluate the mechanism of LPS on the apoptosis and osteoblast differentiation in MC3T3-E1 cells. MC3T3-E1 osteoblasts were non-treated, treated with LPS. After treatment, the cell viability, the activity of alkaline phosphatase (ALP) and caspase-3 were measured. The expressions of osteoblast-specific genes and Bax, Bcl-2, and caspase-3 were determined by real-time quantitative polymerase chain reaction (qPCR). Protein levels of Bax, Bcl-2, caspase-3, and phosphorylation of MAPKs were measured using Western blotting assays. The MAPK signaling pathway was blocked by pretreatment with JNK inhibitor SP600125. LPS treatment induced a significant decrease in cell metabolism, viability, and ALP activity in MC3T3-E1 cells. LPS also significantly decreased mRNA expressions of osteoblast-related genes in MC3T3-E1 cells. On the other hand, LPS significantly upregulated mRNA expressions and protein levels of Bax and caspase-3 as well as activation of caspase-3, whereas decreased Bcl-2 expression in MC3T3-E1 cells. Furthermore, LPS significantly promoted MAPK pathway including the phosphorylation of JNK and the phosphorylation of ERK1/2; moreover, pretreatment with JNK inhibitor not only attenuated both of phosphorylation-JNK and ERK1/2 enhanced by LPS in MC3T3-E1 cells, but also reversed the downregulated expressions of osteoblast-specific genes including ALP and BSP induced by LPS. In conclusion, LPS could induce osteoblast apoptosis and inhibit osteoblast differentiation via activation of JNK pathway.     

盐酸小檗碱通过PI3K/Akt信号通路促进小鼠前成骨细胞MC3T3-E1的分化

目的:研究盐酸小檗碱对小鼠前成骨细胞系MC3T3-E1分化与矿化的调控作用及其机制。方法:MC3T3-E1细胞给予不同浓度(0、1、5、10和20 mg/L)的盐酸小檗碱刺激3 d,CCK-8法检测细胞活性。不同浓度的盐酸小檗碱分别干预3 d和7 d,检测细胞碱性磷酸酶(ALP)活性。进一步将实验随机分为4组:对照组、盐酸小檗碱组、盐酸小檗碱+LY249002(PI3K/Akt通路抑制剂)组及LY249002组。干预2 d后,采用real-time PCR检测成骨细胞分化相关因子ALP、骨钙素(OCN)、骨桥蛋白(OPN)及Runt相关转录因子2(Runx2)的mRNA表达情况,采用Western blot检测PI3K/Akt信号通路相关蛋白p-Akt的表达水平。将MC3TC-E1细胞用矿化培养基诱导21 d,茜素红染色检测其矿化情况。结果:与对照组相比,不同浓度的盐酸小檗碱对细胞活性的影响没有明显差异;不同浓度的盐酸小檗碱处理MC3T3-E1细胞后ALP活性有不同程度升高。Real-time PCR结果表明,盐酸小檗碱(5 mg/L)促进ALP、OCN、OPN及Runx2的mRNA表达(P 0. 01),而LY294002能抑制这些分化相关因子的表达。Western blot检测结果表明,盐酸小檗碱(5 mg/L)促进p-Akt蛋白的表达(P 0. 01),其作用被LY249002抑制。茜素红染色发现盐酸小檗碱组矿化明显,但LY294002能抑制盐酸小檗碱的促进作用。结论:盐酸小檗碱可以促进小鼠前成骨细胞的分化与矿化,其机制可能与其激活PI3K/Akt信号通路有关。