Microcarriers facilitate mineralization in MC3T3-E1 cells

Summary MC3T3-E1 cells showed mineral deposits after about 1 week of culture when incubated in the presence of microcarrier beads. These deposits appeared as white spots on the dish surface, and under light microscopy the cells showed multiple cell layers and mineralization around the microcarriers. The deposits stained positive with calcium-specific Von Kossa's method. Using conventional assay, alkaline phosphatase activity (ALP) and parathyroid hormone-stimulated intracellular cAMP production were lower in the microcarrier cultures than in the control, but using cytochemical methods, high alkaline phosphatase activity was found around the microcarriers. These results indicate that microcarriers facilitated the formation of multiple cell layers and provided a culture environment for mineralization.     

Matrix mineralization in MC3T3-E1 cell cultures initiated by β-glycerophosphate pulse

MC3T3-E1 cells, grown in the presence of serum and ascorbate, express alkaline phosphatase and produce an extensive collagenous extracellular matrix that can be mineralized by the addition of β-glycerophosphate (β-GP). In the present work, we study the influence of concentration and duration of β-GP treatment on the mineralization pattern in 4-week-old cell cultures. Amount and structure of mineral deposition were monitored by von Kossa staining, light, and electron microscopy, as well as small-angle X-ray scattering (SAXS) of unstained specimens. SAXS measures the total surface of the mineral phase and is therefore preferentially sensitive to very small crystals (typically <50 nm). It was used to determine the ratio (M) of small crystals to collagen matrix. A variety of mineralization patterns was observed to occur simultaneously, some associated with collagen within nodules or in deeper layers of the cultures and some independent of it. At a β-GP concentration of 10 mmol, mineralization was initiated after about 24 h and continued to increase, irrespective of whether the high level of β-GP was maintained or reduced to 2 mmol. With shorter pulses (<24 h), no significant mineralization was observed in the week following β-GP pulse. With continuous treatment at 5 mmol β-GP, the first signs of mineralization were detected 14 days after the beginning of treatment in the 4-week-old cultures, but no mineralization at all occurred at lower β-GP concentrations. When cells were grown without ascorbic acid for 4 weeks, only two cell layers without collagen matrix were found. In these cultures, no mineralization detectable by SAXS could be induced with β-GP. These data indicate that, in viable cells, high doses of β-GP are essential for the nucleation of mineral crystals, but not for the progression of mineralization once crystals had been nucleated. In contrast, when 4-week-old cell cultures were devitalized, M was found to increase immediately, even at 2 mmol β-GP. These results suggest that, in MC3T3-E1 cell cultures, cell viability is essential for prevention of spontaneous mineralization of the extracellular matrix.     

Stimulation by bone sialoprotein of calcification in osteoblast-like MC3T3-E1 cells

Bone sialoprotein (BSP) containing an Arg-Gly-Asp cell-binding sequence was purified from bovine bone 4 M guanidine-HCl extract after HCl demineralization by a series of chromatographic procedures. When this protein was coated on culture dishes in the presence of type I collagen, it increased both DNA content and alkaline phosphatase (ALP) activity in osteoblast-like MC3T3-E1 cells, and stimulated calcification in the cells, whereas fibronectin, another cell-binding protein, showed a marked increase in the DNA content but had little effect on the ALP activity. These findings suggest that BSP is mitogenic for preosteoblasts and differentiating the cells into osteoblasts, thereby stimulating bone calcification     

Effect of microfibrillar collagen on growth and differentiation of osteoblast-like MC3T3-E1 cells

Effect of microfibrillar collagen on the proliferation and differentiation of osteoblastic cells was studied using MC3T3-E1 (E1) cells. In order to achieve direct contact of microfibrillar collagen with E1 cells, they were embedded in denatured collagen gel, and DNA content, [³H] thymidine incorporation, alkaline phosphatase activity, and⁴⁵Ca accumulation were examined after long-term culture. Microfibrillar collagen embedded with E1 cells increased DNA content and stimulated DNA synthesis of E1 cells in a dose-dependent manner. In vitro mineralization induced by E1 cells was also stimulated by microfibrillar collagen in a dose-dependent manner: 1mg/ml of microfibrillar collagen stimulated⁴⁵Ca accumulation by about 3 fold, and 10 mg/ml by 5 fold. Alkaline phosphatase activity was not affected by the presence of microfibrillar collagen. Because the interaction of specific RGD tripeptide recognition site on collagen fiber with cell surface adhesion receptors is proposed to affect the proliferation and differentiation of various cells, it is suggested that the interaction of osteoblastic cells with collagen fibers plays an important role in the regulation of proliferation and the expression of osteoblastic phenotype in these cells.     

健骨胶囊对成骨样细胞MC3T3-E1增殖及分化作用机制

目的 探讨国医大师刘柏龄“健骨胶囊”对MC3T3-E1成骨细胞分化及增殖的影响。方法 制备健骨胶囊水提物,采用CCK-8法和细胞迁移实验检测健骨胶囊提取物对MC3T3-E1细胞增殖和细胞迁移的影响;茜素红染色检测MC3T3-E1细胞的矿化能力;实时荧光定量PCR检测成骨分化基因Runx2、OCN、OPN、Col1a1、ALP、Bcl2、RASSF1A等mRNA表达水平;蛋白质印迹法Western blot检测Col1a1、Bcl2的蛋白表达水平。结果 通过实验结果比对得出,健骨胶囊提取物能促进MC3T3-E1细胞增殖、使细胞迁移率提高;同时健骨胶囊提取物组能明显提高MC3T3-E1细胞钙化能力(P＜0.01),促进Runx2、OCN、OPN、Col1a1、ALP、Bcl2的mRNA表达(P＜0.05),上调Col1a1、Bcl2蛋白量的表达。结论 健骨胶囊能促进成骨细胞MC3T3-E1的增殖及细胞迁移能力,并通过上调成骨基因的表达水平如Runx2、OCN、OPN、Col1a1、ALP、Bcl2等,提高MC3T3-E1细胞的成骨能力。     

二苯乙烯苷对H2O2诱导的MC3T3-E1凋亡的保护作用及机制研究

目的研究二苯乙烯苷(tetrahydroxystilbene glucoside,TSG)对氧化应激导致的成骨前体细胞MC3T3-E1凋亡的保护作用,并初步探讨相关机制。方法不同浓度TSG预处理MC3T3-E1 24 h后,300μmol/L H_2O_2作用24 h。实验分组:①空白对照组;②单纯H_2O_2处理组;③H_2O_2+TSG 0.1μmol/L处理组;④H_2O_2+TSG 1μmol/L处理组;⑤H_2O_2+TSG 10μmol/L处理组;⑥阳性对照抗氧化剂NAC 1 mmol/L处理组,通过MTT法、Hoechst33258染色来评价细胞凋亡情况及TSG对氧化损伤的保护作用;利用荧光酶标仪和MDA检测试剂盒来检测细胞中ROS及MDA的水平来评价细胞的氧化应激状态;Western-blot和RT-PCR分别从蛋白水平和基因水平评价Bcl-2和Bax的表达水平。结果单纯H_2O_2处理可以导致细胞死亡,显微镜下可见许多细胞死亡,脱壁;Hoechst33258染色可见有较多的细胞出现核固缩、核浓集现象;不同浓度的TSG预处理后,细胞凋亡现象得到改善。单独H_2O_2作用可以导致成骨前体细胞出现较大程度的凋亡,不同浓度TSG预处理后,细胞凋亡率有所降低;单纯H_2O_2作用可以导致细胞水平的ROS和脂质氧化产物MDA产生增加,TSG(1～10μmol/L)可以显著降低细胞水平ROS和MDA水平(P0.05),改善细胞的氧化应激状态;Western-blotting和实时定量RT-PCR结果 TSG预处理可以减少H_2O_2处理后,促凋亡蛋白Bax的表达,增加抗凋亡蛋白Bcl-2的表达。结论氧化应激可以导致成骨前体细胞MC3T3-E1凋亡增加,TSG可以通过降低氧化应激水平。其机理可能是TSG通过促凋亡蛋白Bax的表达及增加抗凋亡蛋白Bcl-2的表达起到保护作用。     

Effects of triiodothyronine on morphology, growth behavior, and the actin cytoskeleton in mouse osteoblastic cells (MC3T3-E1)

We investigated the effects of thyroid hormone treatment on morphology, growth behavior, and cytoskeletal structures of long-term cultured MC3T3-El cells. Morphological investigations were carried out on native cells by phase contrast microscopy and on epon-embedded semithin sections. The area covered by the cell and matrix layers (tissue-like area), percent extracellular matrix, average height of tissue-like area, and length and height of single cells were measured histomorphometrically on the cross sections. F-actin was analyzed histochemically and quantitated after fluorochrome-labeled phalloidin staining using confocal microscopy and fluorometry. Significant differences between control and T₃-treated cells were found after confluency, but not in subconfluent cultures. Control cells continued to proliferate forming multilayers, and produced increasing amounts of extracellular matrix. In contrast, T₃-treated cells stopped to proliferate forming two cell layers at the maximum. These cells were flattened, distinctly enlarged, and polygonal in shape. Histochemical staining for F-actin revealed three different staining patterns, depending on the position of the cell within the multilayer of control cultures. Basal cells contained a large number of thick stress fibers in parallel arrangement. Intermediate cells exhibited only a few thick actin filament bundles located at the outermost periphery. The superficial cells were characterized by a large number of thin, parallel-oriented microfllament bundles extending across the entire cytoplasm. The actin pattern of T₃-treated cells resembled that of the basal cell layer of the control cells. The amount of F-actin increased with the prolonged T₃ treatment. We conclude from these data that the known specific cellular responses to T₃ treatment are accompanied by significant morphological alterations indicating pivotal effects of thyroid hormones on osteoblastic differentiation.     

牵拉应力下成骨细胞(MC3T3-E1)的增殖与matrilin-2 mRNA的表达

目的观察三维培养的克隆鼠颅骨成骨细胞(MC3T3-E1)在特定的周期性牵拉应力刺激下,其增殖能力、碱性磷酸酶(ALP)活性及matrilin-2mRNA表达的变化。方法以明胶海绵(2cm×2cm×0.25cm)作为MC3T3-E1的三维培养支架,每块明胶海绵种植细胞悬液100μl,细胞数目为1.25×105个。明胶海绵拉伸度为5%、频率60r/min、15min/h,牵拉后2、4、6、8、10d分别从牵拉组和对照组中各取3个样本,行细胞计数、细胞及培养液的ALP活性测定、细胞的matrilin-2mRNA测定。结果第2天牵拉组的细胞数目即多于对照组(P<0.05),细胞倍增时间从71h提前至55h。除第2天外,牵拉组细胞的ALP活性低于对照组(P<0.05),两组培养液中ALP活性均处于低水平,无显著差别。第4天牵拉组matrilin-2mRNA的表达水平高于对照组(P<0.01),之后保持高水平表达。结论周期性牵拉应力促进三维培养的MC3T3-E1的增殖,抑制细胞的ALP活性,促进matrilin-2mRNA的表达,细胞的分化能力增强。     

MC3T3-E1细胞在不同机械应力刺激下成骨表达机制的研究进展

机械应力是刺激骨组织生长的有效条件,它的加载和卸载通过影响成骨细胞的成骨进程和功能,以维持骨重建与动态平衡,从而对骨骼微环境和骨代谢产生重要影响。成骨样细胞系MC3T3-E1细胞作为力学刺激敏感细胞,常被用作应力环境下研究成骨机制的经典细胞。本文主要综述了5种体外不同机械应力刺激下的MC3T3-E1细胞成骨表达机制最新进展,以期阐明成骨细胞接收和传导力学刺激的转导网络,并对研究不同机械应力刺激下保持骨骼稳定和损伤重建的机制有所指导。     

铁调素对MC3T3-E1小鼠成骨细胞骨保护素和骨钙素基因表达的影响

目的研究铁调素对小鼠成骨细胞MC3T3-E1细胞骨保护素(OPG)和骨钙素(BGP)基因表达的影响。方法小鼠MC3T3-E1细胞体外培养后,以不同浓度(100 nmol/ml,200 nmol/ml,300 nmol/ml)的铁调素作用72 h,用RT-PCR方法检测OPG、BGP mRNA的表达水平。结果RT-PCR检测显示在不同浓度铁调素干预后,各组均有OPG mRNA和BGP mRNA表达;不同浓度组的OPG mRNA和BGP mRNA表达光密度比值不同,组间密度比值比较存在显著性差异(P0.05)。结论铁调素可上调MC3T3-E1细胞OPG及BGP mRNA表达,铁调素浓度增加转录水平逐渐增加,结果显示有浓度依赖性。     

二甲基乙二酰基甘氨酸对小鼠胚胎成骨细胞前体细胞增殖和分化的影响

目的 :探讨低氧模拟剂二甲基乙二酰基甘氨酸(dimethyloxalylglycine,DMOG)对小鼠胚胎成骨细胞前体细胞(MC3T3-E1)的增殖、成骨分化及血管内皮生长因子(vascular endothelial growth factor,VEGF)表达的影响。方法:将MC3T3-E1细胞接种到培养板24h后,实验组培养基中分别加入50μM(50μM组)和200μM(200μM组)的DMOG,对照组加入完全培养液。分别于培养1、3、5d时采用MTT法检测MC3T3-E1细胞增殖情况,5、10d行碱性磷酸酶(alkaline phosphatase,ALP)染色及ALP活性检测成骨细胞分化,21d用茜素红染色检测MC3T3-E1细胞钙结节的形成并进行定量分析,采用ELISA法检测MC3T3-E1培养3d情况时上清液中的VEGF蛋白含量,并用实时荧光定量PCR法检测MC3T3-E1细胞VEGF mRNA的相对表达量。结果:培养1d时对照组、50μM组和200μM组的MC3T3-E1的光密度(optical density,OD)值分别为0.041±0.009、0.074±0.019、0.086±0.044,3d时分别为0.123±0.027、0.148±0.020、0.224±0.061,5d时分别为0.297±0.044、0.325±0.084、0.354±0.038,1d、3d时50μM组和200μM组与同时间点对照组比较均有显著性差异(P0.05),3d时50μM组与200μM组有显著性差异(P0.05)。培养5d和10d时对照组ALP染色颜色较深,50μM组颜色中等,200μM组颜色较浅;5d时对照组ALP活性为1.943±0.072,50μM组为1.632±0.051,200μM组为1.319±0.065;10d时对照组ALP活性为3.734±0.067,50μM组为3.381±0.070,200μM组为2.831±0.086。三组间同时间点比较均有统计学差异(P0.05),同组10d时与5d时比较均有统计学差异(P0.05)。茜素红染色200μM组可见少量红色结节,50μM组可见中等量红色结节,对照组可见大量红色结节;对照组茜素红含量(μg/ml)为56.178±7.940,50μM组为41.922±2.438,200μM组为31.929±1.922,三组间比较均有统计学差异(P0.05)。培养3d时对照组细胞培养上清液中VEGF蛋白含量(ng/孔)为9.063±0.603,50μM组为12.123±0.870,200μM组为15.540±0.581,三组间比较均有统计学差异(P0.05);50μM组VEGF mRNA表达量为对照组的1.792±0.067,200μM组为对照组的3.963±0.092,三组间比较均有统计学差异(P0.05)。结论 :低氧模拟剂DMOG可促进MC3T3-E1细胞增殖和VEGF表达,抑制其成骨分化。     

MicroRNA-196a靶向调节HDAC9对MC3T3-E1细胞成骨分化的影响

目的 探究微小RNA（miR）-196a靶向调节组蛋白去乙酰化酶9（HDAC9）对MC3T3-E1细胞成骨分化的影响。方法 将MC3T3-E1细胞分为对照组（Cont）组、诱导组、miR-196a-mimics-NC组、miR-196a-mimics组、miR-196a-inhibitor-NC组、miR-196a-inhibitor组、miR-196a-mimics+pCMV-HDAC9-NC组、miR-196a-mimics+pCMV-HDAC9组,根据分组转染后进行成骨诱导。定量荧光PCR检测MC3T3-E1细胞中miR-196a、HDAC9表达量;试剂盒检测碱性磷酸酶（ALP）活性;茜素红染色观察矿化程度;Western blot检测HDAC9、ALP、Runt相关转录因子2（Runx2）、胶原蛋白I（COL1）、骨桥蛋白（OPN）、Histone H3、Histone H3（acetyl K9、K14和K23）表达量。结果 与Cont组相比,诱导组MC3T3-E1细胞中miR-196a表达、ALP、Runx2、COL1、OPN蛋白表达、ALP活性、矿化程度及Histone H3 K9、K14、K23位点乙酰化水平增高（P＜0.05）,HDAC9 mRNA和蛋白表达降低（P＜0.05）。转染miR-196a-mimics可明显增加miR-196a表达,降低HDAC9表达,并增加ALP、Runx2、COL1、OPN蛋白表达、ALP活性、矿化程度及Histone H3乙酰化,转染miR-196a-inhibitor则作用相反。miR-196a可靶向下调HDAC9表达,过表达HDAC9可部分逆转miR-196a mimics对MC3T3-E1细胞成骨分化的促进效应。结论 miR-196a可靶向下调HDAC9表达,增加组蛋白乙酰化水平,促进MC3T3-E1细胞成骨分化。     

Actin and microtubule cytoskeletons of the processes of 3D-cultured MC3T3-E1 cells and osteocytes

Cell shape is the most critical determinant of cell function and is potentially influenced by the organization of a cell's cytoskeletal components. It has been reported that three-dimensionally cultured osteoblasts have a morphology that closely resembles that of osteocytes, most notably including formation of processes. We have previously shown the critical differences between cytoskeletal components in osteoblasts and osteocytes in two-dimensional culture. We have now extended that investigation to the cytoskeletal components of 3D-cultured osteoblasts and osteocytes using 3D cultures of the osteoblast cell line, MC3T3-E1, and primary osteocytes grown in collagen gel. Three-dimensional fluorescent image reconstructions for actin, fimbrin, alpha-actinin, myosin, tropomyosin, and microtubules were made using IMARIS software. Actin, fimbrin, alpha-actinin, myosin, and tropomyosin all appeared in the processes of both cell types, but fimbrin and myosin showed differences in their distribution patterns between cell types. Microtubules were limited in distribution to the proximal region of osteocyte processes but extended the entire length of MC3T3-E1 cell processes. Microtubules were essential for the integrity and formation of MC3T3-E1 cell processes, but osteocyte processes were dependent on actin. These results showed that there are significant differences between the actin and microtubule cytoskeletons in the processes of 3D-cultured MC3T3-E1 cells and in the processes of 3D-cultured primary osteocytes. These differences in the cytoskeleton of the processes of 3D-cultured osteoblasts and of osteocyte dendrites suggest that osteoblast processes may have a different functional role than the osteocyte dendritic network. An erratum to this article is available at .     

不同浓度rhBMP-2对MC3T3-E1细胞增殖、ALP活性及成骨分化的影响

目的研究不同浓度rh BMP-2对MC3T3-E1细胞增殖、ALP活性及成骨分化的影响规律。方法 MC3T3-E1细胞接种到培养板24 h后,试验组分别加入1.25、2.5、5、10μg/ml的rh BMP-2进行药物干预,对照组加入全培。试验组应用CCK-8法检测细胞增殖活性,PNPP法检测细胞ALP活性,RT-PCR法检测成骨标志蛋白m RNA的表达情况。结果rh BMP-2浓度为2.5μg/ml时开始促进细胞的增殖(P0.05),rh BMP-2浓度为5μg/ml时促进作用达到峰值(P0.05)。rh BMP-2浓度为2.5μg/ml时ALP活性显著提高(P0.05);rh BMP-2浓度为5μg/ml和10μg/ml时,ALP活性较浓度为2.5μg/ml时继续升高,但差异无统计学意义(P0.05)。ALP、COL1-α以及转录因子RUNX-2的m RNA含量均在加入浓度5μg/ml rh BMP-2后明显升高(P0.05),继续加大rh BMP-2浓度,m RNA含量没有明显增加。结论在研究范围内,rh BMP-2对MC3T3-E1的影响呈浓度依赖性。     

Bone Morphogenetic Protein-6-loaded Chitosan Scaffolds Enhance the Osteoblastic Characteristics of MC3T3-E1 Cells

The purpose of this study is to investigate the convenience of bone morphogenetic protein-6 (BMP-6)-loaded chitosan scaffolds with preosteoblastic cells for bone tissue engineering. MC3T3-E1 cells were seeded into three different groups: chitosan scaffolds, BMP-6-loaded chitosan scaffolds, and chitosan scaffolds with free BMP-6 in culture medium. Tissue-engineered constructs were characterized by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide assay, scanning electron microscopy (SEM), mineralization assay (von Kossa), alkaline phosphatase (ALP) activity, and osteocalcin (OCN) assays. BMP-6-loaded chitosan scaffolds supported proliferation of the MC3T3-E1 mouse osteogenic cells in a similar pattern as the unloaded chitosan scaffolds group and as the chitosan scaffolds with free BMP-6 group. SEM images of the cell-seeded scaffolds revealed significant acceleration of extracellular matrix synthesis in BMP-6-loaded chitosan scaffolds. Both levels of ALP and OCN were higher in BMP-6-loaded chitosan scaffold group compared with the other two groups. In addition, BMP-6-loaded scaffolds showed strong staining in mineralization assays. These findings suggest that BMP-6-loaded chitosan scaffold supports cellular functions of the osteoblastic cells; therefore, this scaffold is considered as a new promising vehicle for bone tissue engineering applications.     

利用Label-free技术筛选MC3T3-E1细胞中miR-381-3p介导的靶蛋白和差异蛋白生物学功能分析

目的 比较小鼠前成骨细胞(MC3T3-E1)实验组(MC3-E1-in)与对照组(MC3-E1-NC-in)的蛋白质组学差异水平,以初步探究miR-381-3p所介导的靶蛋白和相关信号通路以及差异蛋白生物学功能分析.方法 通过细胞慢病毒转染构建实验模型,分为实验组与对照组;实时荧光定量PCR验证miR-381-3p在细...     

miR-381-3p通过靶向ERK1/2/ETS1信号通路影响MC3T3-E1细胞成骨分化

目的 探讨miR-381-3p对MC3T3-E1细胞成骨分化的影响和作用机制。方法 慢病毒转染细胞后实验分为NC mimic miR-381-3p组、mimic miR-381-3p组、NC inhibitor miR-381-3p组和inhibitor miR-381-3p组;运用实时荧光定量PCR(qR T-PCR)法检测不同组miR-381-3p的表达水平以验证转染效率;诱导MC3T3-E1细胞成骨分化后,检测各组细胞中碱性磷酸酶(ALP)活性水平;茜素红染色法检测各组细胞的成骨矿化能力; qR T-PCR测定各组细胞中ALP、Runx2、PPARγ和ETS1 mR NA表达水平;蛋白免疫印迹法测定各组细胞中collagenⅠ、ALP、Runx2、PPARγ、ETS1、P-ERK1/2水平;并用MAPK/ERK通路抑制剂(PD98059)干预各组细胞后进行目的蛋白检测。结果 与NC mimic miR-381-3p组、mimic miR-381-3p组及NC inhibitor miR-381-3p组比较,inhibitor miR-381-3p组中细胞中ALP、Runx2、ET...     

Restoration of Mineral Depositions by Dexamethasone in the Matrix of Nonmineralizing Osteoblastic Cells Subcloned from MC3T3-E1 Cells

The original osteoblastic cell line, MC3T3-E1, was derived from normal mouse bone tissue and mineralized without any specific factors in vitro. This cell line may be slightly unstable because of high differentiation, and some of these cells sometimes lost the ability for mineral deposition. In this study, a new cell line was cloned which lost the ability for mineral deposition from MC3T3-E1 cells. This cell line, termed MC3T3-NM4, was not observed to undergo mineral deposition for up to at least 36 days even in media containing beta-glycerophosphate. The alkaline phosphatase (ALP) activity was also not increased. The lack of calcifying ability was found to be restored by the addition of dexamethasone in the media. This restoration was accompanied by an increase in ALP activity and osteocalcin level. It was suggested that this restoration was not due to artificial mineralization resulting from cell death. Received: 3 March 1999 / Accepted: 25 May 2000 / Online publication: 22 September 2000