高重力下成骨细胞的力学生物学响应

骨适应性力学环境中,成骨细胞作为骨形成的主要功能细胞,也是感应力学载荷的主要细胞之一。目前随着科技的发展,越来越多的航天员、飞行员等暴露于高重力环境中。为了更好了解和认识高重力下成骨细胞的力学生物学响应,综述高重力下成骨细胞形态及增殖、基因表达、细胞因子分泌以及信号转导通道等力学生物学方面的研究进展,为高重力环境下骨组织的力学生物学研究提供思路和准备。     

大鼠成骨细胞对动态拉伸刺激的生理响应

应用体外周期性机械拉伸装置研究机械拉伸对大鼠成骨细胞增殖及分化的影响.测试了在应变15%,频率20次/min的拉伸刺激下不同加载时间对成骨细胞增殖及分化的影响.发现在受载12h时,细胞相对增殖指数最大,随受载时间的增加而逐渐趋向于1,受载36h后,碱性磷酸酶活性的相对指数最显著.说明成骨细胞对周期性机械拉伸刺激的响应与受载时间相关联,并且受载的成骨细胞经过自身调控后,逐渐适应于新的力学环境,保持一种新的平衡状态.     

茶多酚对脂多糖刺激下成骨细胞增殖分化的影响

背景：研究表明,茶多酚在牙周炎等疾病的研究中能发挥抗炎、抗氧化作用。目的：探讨茶多酚在脂多糖刺激下对小鼠成骨细胞增殖分化及氧化应激的影响。方法：体外培养小鼠成骨细胞MC3T3-E1,分4组处理：对照组常规培养;脂多糖组加入10μg/mL脂多糖,茶多酚组加入1μg/mL茶多酚,联合组加入10μg/mL脂多糖+1μg/mL茶多酚。采用MTT法检测细胞增殖情况,碱性磷酸酶试剂盒检测细胞碱性磷酸酶活性,偶氮偶联法检测细胞碱性磷酸酶染色程度,钙检测试剂盒检测细胞钙沉积量,超氧化物试剂盒检测细胞超氧化物含量,丙二醛试剂盒检测细胞丙二醛含量。结果与结论：(1)与对照组相比,脂多糖组成骨细胞增殖活性、碱性磷酸酶活性、钙沉积量降低(P <0.05),碱性磷酸酶染色程度降低(P <0.01),超氧化物和丙二醛含量增加(P <0.05);茶多酚组成骨细胞增殖活性、碱性磷酸酶活性、钙沉积量升高(P <0.05),碱性磷酸酶染色程度提高(P <0.01),超氧化物和丙二醛含量减少(P <0.05);(2)与脂多糖组相比,联合组成骨细胞增殖活性、钙沉积量升高(P <0.0...     

流体剪切力作用下无间隙连接成骨细胞阵列内的钙响应

目的 研究间隙连接和ATP在力致胞间钙传递过程中各自的作用。方法 首先应用微模式化方法建立无间隙连接的成骨细胞阵列,再利用流动腔对细胞施加流体剪切力,观察并分析细胞的钙响应特征参数。结果 细胞间无物理连接时,仍会出现多个钙响应峰,但第1峰的响应时间与有间隙连接时相比明显加长。除去胞外或胞内钙时,只有约40%的细胞发生钙响应,且单峰与多峰约各占一半。阻断胞外ATP通路后,只有约20%的细胞有钙响应,且大多为单峰。结论 细胞间的间隙连接不存在时,ATP是介导细胞间钙传递的主要途径,说明间隙连接不是细胞间力致钙响应的必需途径。     

低氧条件下成骨细胞的增殖与分化

背景：低氧可通过多种途径作用于成骨细胞影响骨代谢,对骨生成、骨愈合等产生负面影响。 目的：观察低氧对体外培养大鼠成骨细胞增殖、分化的影响,并探讨其分子机制。 方法：分离培养新生Wistar大鼠颅盖骨成骨细胞,取第2代细胞分别在常氧(体积分数20%O₂)与低氧(体积分数3%O₂)条件下培养。 结果与结论：低氧组成骨细胞增殖、碱性磷酸酶活性、骨钙素水平及茜素红结节形成数量均明显低于常氧组(P < 0.05或  P < 0.01),说明缺氧条件对成骨细胞的增殖、分化及功能有抑制作用;低氧组骨形成发生蛋白2及Runx2表达低于常氧组(P < 0.05或P < 0.01),说明低氧条件下大鼠成骨细胞Runx2、骨形成发生蛋白2的表达受抑制。结果表明低氧可通过抑制大鼠成骨细胞的Runx2、骨形成发生蛋白2的表达进一步抑制成骨细胞的增殖与分化。     

成骨细胞分化与信号转导研究进展

成骨细胞在骨的生长、发育及形成过程中起重要作用,其分化受严格调控,通过多种信号途径实现对成骨细胞的定向分化。综述了G蛋白信号调节因子、BMP/Smads、MAPK、PI3-k以及细胞因子等信号通路在成骨细胞定向分化方面的研究进展。     

骨的应力集中效应

骨的最主要的功能是给身体提供支撑框架并协助机体的运动。其重要特点是具有应力适应性 ,当其受到扭曲、拉伸、压缩等力作用时 ,它可改变自身的结构和质量以适应功能性应变。由于生命体时刻处于力学环境中 ,因而应力对骨的生长、重建和成形起着重要的调节作用。然而骨也有一个最适应的应力范围 ,应力过低或过高都会使骨逐渐萎缩 ,甚至破坏。骨处于应力区内会增加其密度和硬度 ,而处于没有力学刺激的区域则会弱化密度乃至失去钙化特征。在含有几何不连续区的弹性体中 ,应力集中点的应力状态是力学研究的一个课题。在应力集中点及其邻近区域上…     

空间失重环境力学生物学2023年度研究进展

航空航天活动过程中特殊力学环境导致的生理变化一直是力学生物学研究的重要组成部分。本文综述了2023年度空间失重环境力学生物学研究进展,主要聚焦失重的生物学效应,包括在细胞、模式动物和人体水平在空间真实环境和地面模拟失重条件下得到的研究结果,以期助力航空航天力学生物学的发展研究,并为航天员或地面相关人群的健康防护或对抗措施研究提供帮助。     

基于转录组测序分析微重力环境下MC3T3-E1成骨细胞miRNA/mRNA表达谱及功能变化

目的探讨微重力环境对MC3T3-E1成骨细胞分化的影响。方法采用转录组测序技术(RNA-seq)观察微重力培养前后MC3T3-E1成骨细胞miRNA/mRNA表达谱变化,测序结果采用q-PCR验证。采用生物信息学方法进一步研究差异性表达的miRNA/mRNA。结果与对照组(CON)相比,模拟微重力组(SMG)共有160条miRNA和1 912个mRNAs发生显著改变;根据生物信息学结果,筛选出10个关键性基因(3条miRNA、7个mRNA),其中miR-9_6666-5p为微重力敏感miRNA,可能在微重力环境下成骨细胞分化过程中起到重要的作用。结论在微重力环境下,成骨细胞分化受到抑制可能与miRNA/mRNA表达谱的改变有关。研究结果将有助于深入理解miRNA与mRNA在微重力环境下调控成骨分化与骨生成的分子机制。     

动态轴向压应变促进丝素蛋白支架内MC3T3-E1细胞成骨分化

目的 观察动态轴向压应变对三维丝素蛋白支架内成骨细胞成骨相关基因表达的影响。方法 应用动态力学加载仪对实验组小鼠胚胎成骨细胞MC3T3-E1加载动态轴向压应变（5%应变幅度,1 Hz,30 min/d,共21 d）,对照组细胞常规静置培养,不施加力学刺激。应用定量PCR检测细胞成骨基因碱性磷酸酶（ALP）、I型胶原（COLⅠ）、骨特异性转录因子（Runx2）、成骨相关转录因子（Osx）、骨钙蛋白（OCN） mRNA表达量。结果 成骨细胞在周期性轴向压应力刺激下,Runx2、Osx及COLⅠ表达分别增加280%、68.9%和79.6%,ALP及OCN表达也分别增加10.7%和26.9%。实验组成骨相关基因mRNA表达与对照组比较,差异具有统计学意义（P<0.05）。结论 成骨细胞复合丝素蛋白生物支架材料在周期性轴向压应力刺激下,成骨基因COLⅠ、Runx2、Osx及OCN表达明显上调,可能是生理状态下压应力刺激促进骨折愈合的重要机制之一。研究结果对于以力学信号为基础的细胞疗法修复骨缺损等疾病具有重要临床价值。     

A comparative analysis of the osteogenic capacity of osteoblasts from newborn and two-week-old rats

AimTo compare the proliferation and osteogenic differentiation of osteoblasts between newborn rats (1d group) and two-week-old rats (14d group) and to clarify the mechanism underlying these effects.MethodThe endogenous expression of osteogenic marker genes was detected by qPCR, including ALP, OCN, Col1a1, and Runx2. The osteoblasts proliferation was evaluated by EdU assay and Western Blotting [PCNA and Cyclin D1]. ALP activities in osteoblasts were detected using a PNPP kit, ALP staining and qPCR. Mineralized nodule formation and intracellular calcium levels were assessed by Alizarin Red staining and calcium colorimetric assay respectively while OCN, Col1a1 and Runx2 levels in osteoblasts were analyzed by immunostaining. Osteogenesis-associated pathways including Wnt/β-Catenin, Akt/PPAR and Smad were analyzed via Western Blotting.ResultEndogenous ALP, OCN, Col1a1, and Runx2 expression levels were significantly higher in osteoblasts from 14d group than those from 1d group. After treatment with osteogenic induction medium, osteoblast proliferation, ALP activity, mineralized nodule formation, and intracellular calcium levels were markedly increased in osteoblasts from 1d group, with similar results also being observed for the expression of OCN, Col1a1, and Runx2. Wnt3a, β-catenin, p-Akt, p-Smad1/5/8, and p-Smad5 protein levels were also higher in osteoblasts from 1d group relative to those from 14d group, while the expression of PPARγ was lower.ConclusionThe superior osteogenic differentiation capacity in osteoblasts was associated with the higher activation levels of Wnt/β-Catenin, Akt/PPAR and Smad signaling pathways, and the enhanced proliferative activity in osteoblasts from 1d group.     

The effect of substrate surface nanotopography on the behavior of multipotnent mesenchymal stromal cells and osteoblasts

Hexagonally arranged Gold nanoparticles with controllable diameters and inter-particle distances were deposited on thick SiO₂ layers on top of Si wafers and used as masks during subsequent reactive ion etching. In this way, arrays of nanopillars are obtained with well-defined diameters (10/30 nm), inter-pillar distances (50–120 nm) and heights (20–35 nm), all on the nanoscale. Such nanotopographies served as substrate for multipotent mesenchymal stromal cells (MSC) and human osteoblasts (OB) allowing to study cellular responses to purely topographically patterned interfaces. Focus was put on adhesion, proliferation and differentiation of the cells. It turned out experimentally that adhesion is comparable for both cell types practically independent of topographical details at the substrate surface. Topography induced proliferation enhancement, however, is again independent of geometrical details in case of MSC, but significantly sensitive to pillar height in case of OB with a clear preference towards short nanopillars (20 nm). A high sensitivity to topographic details is also observed for osteogenic differentiation of MSC, in that case with a preference towards higher nanopillars (50 nm). The present experimental data also allow the important conclusion that cell proliferation and differentiation can be optimized simultaneously by fine-tuning nanoscaled topographical parameters.     

钛颗粒负荷对成骨细胞释放细胞因子的影响

假体周围的溶骨性因子是造成无菌性松动的主要原因之一。前期的研究表明,磨损颗粒抑制成骨细胞分化和矿化能力,为探索不同直径的磨损颗粒对成骨细胞释放细胞因子的影响,我们采用双夹心抗体ELISA、RT-PCR法分析3种直径钛颗粒对兔成骨细胞表达IL-6I、L-10和破骨细胞分化因子(ODF)的影响。结果显示:0.9μm钛颗粒刺激成骨细胞产生大量促骨吸收因子(IL-6、ODF)的同时快速而短暂地释放抑骨吸收因子(IL-10);2.7μm和6.9μm钛颗粒,尤其是后者主要是刺激成骨细胞缓慢而强烈地产生促骨吸收因子。提示:磨屑颗粒作用成骨细胞的生物学反应是双相的,且反应程度因颗粒大小和作用时间的不同而又有所不同。可见如何抑制假体周围溶骨性因子的产生以及人工材料的优化选择是提高人工假体寿命的关键。     

低强度高频率振动对成骨细胞生物学特性的影响

目的　研究低强度高频率振动（low-magnitude high-frequency vibration, LMHFV）对成骨细胞生物学特性的影响。 方法　建立LMHFV加载MC3T3-E1细胞模型,观察不同频率LMHFV对MC3T3-E1细胞OPG/RANKL浓度比的影响,获得OPG/RANKL浓度比最高的频率（F）为后续研究频率;以0 Hz为对照,观察LMHFV对MC3T3-E1细胞碱性磷酸酶 (ALP)、骨钙素(OCN) mRNA和蛋白活性,及钙化结节形成的影响;LMHFV加载形成的条件培养液（CMF）孵育RAW264.7细胞,观察CMF对破骨细胞抗酒石酸酸性磷酸酶（TRAP）染色、多核破骨细胞形成、TRAP mRNA及蛋白活性的影响;观察LMHFV对MC3T3-E1细胞环氧化酶2(COX-2)蛋白水平的表达及COX-2抑制剂NS-398对LMHFV影响MC3T3-E1细胞分化的作用。 结果　30 Hz LMHFV获得OPG/RANKL浓度比最高,促进ALP、OCN mRNA及蛋白活性增加,增加钙化结节形成。30 Hz LMHFV形成的CM抑制RAW264.7细胞向多核破骨细胞分化,抑制TRAP mRNA及活性;LMHFV可诱导COX-2蛋白水平增加,NS-398能抑制LMHFV促进成骨细胞分化。 结论　30 Hz的LMHFV对MC3T3-E1细胞OPG/RANKL浓度比及成骨分化具有积极的影响,通过调控成骨细胞OPG/RANKL浓度比间接抑制骨吸收,COX-2通路参与了LMHFV对成骨细胞生物学特性的调节作用。     

Initial responses of human osteoblasts to sol-gel modified titanium with hydroxyapatite and titania composition

Sol-gel thin films of hydroxyapatite (HA) and titania (TiO(2)) have received a great deal of attention in the area of bioactive surface modification of titanium (Ti) implants. Sol-gel coatings were developed on Ti substrates of pure HA and TiO(2) and two composite forms, HA+10% TiO(2) and HA+20% TiO(2), and the biological properties of the coatings were evaluated. All the coating layers exhibited thin and homogeneous structures and phase-pure compositions (either HA or TiO(2)). Primary human osteoblast cells showed good attachment, spreading and proliferation on all the sol-gel coated surfaces, with enhanced cell numbers on all the coated surfaces relative to uncoated Ti control at day 1, as observed by MTT assay and scanning electron microscopy. Cell attachment rates were also enhanced on the pure HA coating relative to control Ti. The pure HA and HA+10% TiO(2) composite coating furthermore enhanced proliferation of osteoblasts at 4 days. Moreover, the gene expression level of several osteogenic markers including bone sialoprotein and osteopontin, as measured by RT-PCR at 24h, was shown to vary according to coating composition. These findings suggest that human primary bone cells show marked and rapid early functional changes in response to HA and TiO(2) sol-gel coatings on Ti.     

犬骨髓间充质干细胞的生长特点和在诱骨条件下的成骨特性

为了研究犬骨髓间充质干细胞（canine mesenchymal stem cells，cMSCs）的生长特点和在诱导条件下的成骨特性。使用密度梯度法分离成年犬骨髓间充质干细胞进行培养，保留贴壁细胞传代，观察，以地塞米松、β甘油磷酸钠、抗坏血酸为成骨诱导剂。利用倒置光学显微镜和透射电镜观察细胞形态特征，四甲基偶氮盐（MTT）比色测定增殖，用碱性磷酸酶（Alkaline phosphatase，ALP）活性及骨钙素（Osteocalcin，OCN）含量来研究细胞分化情况。形态学观察表明，cMSCs贴壁细胞呈集落生长，有成纤维细胞样外观，透射电镜可见成骨诱导后cMSCs具有分泌型细胞的特征；推测成骨诱导剂可促进骨髓间充质干细胞成骨，表现为ALP活性、OCN含量明显升高。本实验表明所培养的cMSCs保持了未分化状态，并在成骨诱导剂的作用下可向成骨细胞分化。     

Cognitive and emotional alterations in periadolescent mice exposed to 2 g hypergravity field

The development of the nervous system is a dynamic process where epigenetic factors play a fundamental role. Both ground-based and space research indicate that exposure to an altered gravitational environment affects rodent neurobehavioral profile and stage of development as well as duration of exposure appear to be critical for the observed effects. The behavioral profile of adolescent (28-day-old) male and female CD-1 mice upon acute 2 g exposure was characterized and emotional/anxiety responses (plus-maze), as well as spatial learning performance (Morris water-maze), were assessed respectively 24 and 48 h after hypergravity exposure. Behavioral observation indicated a transient mild sickness associated with hypergravity, with a decrease in spontaneous activity. Rotation per se induced an increase in emotional/anxious responses and a deterioration of spatial learning acquisition, while hypergravity specifically improved flexibility of spatial orientation.