一种新型机械应变细胞加载装置的研制

目的研究开发一套具有自主知识产权的数控机械应变细胞加载装置,为细胞力学研究提供必要的研究手段。方法加载装置基于圆形基底形变技术,采用数字式测控系统和基于PC机平台的专用软件,实现对体外培养细胞加载牵张应变。采用MTT比色实验检测人牙周膜细胞在弹性硅橡胶细胞培养膜上的附着生长能力。采用该装置对体外培养人牙周膜细胞加载1%、10%、20%拉伸应变0.5、1和24 h,倒置相差显微镜观察细胞形态、排列的变化。结果数控机械应变细胞加载装置可对体外培养细胞加载不同强度、频率和时间的拉伸应变,具有输出应变范围大、精度高、操作方便、显示直观等优点。硅橡胶膜和对照细胞培养板接种细胞1、2、4、7、8 d后,MTT比色实验光吸收值之间无统计学差异(P>0.05),显示弹性硅橡胶膜具有良好的细胞附着生长能力。人牙周膜细胞加载10%、20%拉伸应变24 h后,细胞形态、排列发生改变,胞体呈长梭形,并成栅栏状平行排列,细胞长轴垂直于拉伸应变方向。结论数控机械应变细胞加载装置可有效地对体外培养细胞加载动态机械拉伸应变,为体外细胞力学研究提供了必要的研究手段。     

一种血管张应力体外加载装置的实验研究

目的研制一种血管张应力体外加载装置,研究弹性基底(硅胶片)上的张应力、张应变分布。方法基于基底形变加载技术,研制一种接近人体血液动力学环境的血管张应力体外加载装置。利用装置中的摄像机拍摄硅胶片拉伸前后硅胶片网格点的图像并转化为数字图像,使用Matlab软件对网格点的位置特征进行计算,从而得到硅胶片的应变分布。利用万能材料试验机对硅胶片进行实验和计算得到硅胶片的力学参数,根据力学参数建立有限元模型,并对硅胶片的张应力、张应变分布进行模拟计算。将实验结果和模拟结果进行比较。结果有限元结果和实验结果基本一致,张应力、张应变的最大值均出现在加载点处,中间区域应力、应变较为均匀。硅胶片中间60%面积区域可视为均匀应变场。结论研究结果可为后期血管壁内皮细胞的动态培养以及细胞力学研究提供实验技术。     

血管张应力体外加载装置的研制

背景：国内外已经研制出多种体外细胞张应力加载装置,主要拉伸方法有矩形基底拉伸法、圆形基底变形法和4点弯曲梁加载法3种,其中圆形基底变形法虽能够很好的反映体内如肺泡的扩张、血管的脉动等真实情况,但该种加载过程中膜的应变是辐射对称的;4点弯曲梁加载法能够提供的应变范围很小,加载时间有限,应变调节比较困难。 目的：采用矩形基底拉伸法研制血管张应力体外加载装置。 方法：采用机电一体化设计研制血管张应力体外加载装置,由电源模块、控制模块、传动模块和数据采集模块4个部分组成,以硅胶片为基底材料,通过对电机旋转角度和转动速度的高精度控制,实现对硅胶膜片上的拉伸控制。 结果与结论：通过测试和试验,该装置可以满足试验所需的参数范围,能够在体外模拟出人体张应力环境,初步认为该张应力加载装置的研制是成功的,实现了：①装置有两种工作模式：应力模式和应变模式,解决了基底加载装置的硅胶片材还没有实现标准化的问题。②能实现张应力在0-5×105 Pa范围内的调节。③能实现张应变在0-40%范围内的调节。④能实现0-80次/min的拉伸频率的变化,并能控制拉伸时间。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程     

基底应变对肺癌细胞形态调整的影响

目的从肺腺癌A549细胞对拉伸应力响应时释放出的细胞形变调整行为,解读肿瘤细胞骨架异常与其生物学行为的相关性。方法使用单向等轴应变加载装置对培养的肺腺癌A549细胞施以周期性基底拉伸加载,通过计算机图像处理并与对照组进行比较。结果(1)A549细胞的形态在加载中有明显的变化,铺展面积明显增大,细胞外形变得非常不规则,即,细胞不规则度增加;(2)与对照组比较,加载组细胞细胞核的铺展面积增大,拉伸引起细胞核形态的变化比较小,加载组与对照组细胞核的形态差别不大;(3)周期性拉伸引起了细胞取向的调整,细胞调整到与主应变近似垂直的方向,许多细胞拉长首尾连成一线,线的取向趋于与主应变方向垂直。结论A549细胞的细胞骨架各组份间对应力的响应协同反应下降;肺腺癌A549细胞对应力方向的呼应尚属正常。     

拉伸基底加载成骨类细胞内应变张量分析

根据黏附在基底材料膜上成骨类细胞的形态学特征，从细胞固体模型和细胞液滴模型讨论了细胞应变张量的均匀性，导出了反映单、双轴拉伸加载下细胞的应变特征张量。引入标量场参数λ描述细胞应变的非均匀性，给出了非均匀范围与细胞黏附形态几何参数的定量关系。结果有一定的理论和实验意义。     

一种用于骨组织工程研究的加载装置

力学环境对骨内细胞的生物学行为有重要影响。无论是体内还是体外培养,骨组织的生长、发育都依赖力学环境。根据骨组织生理调节中的力学感知、传导、传质机理,研制一种用于骨组织工程研究的加载装置。该装置可为体外骨组织培养提供生理范围内不同大小、频率的应变及不同的应变波形,特别是对有一定强度的硬支架(与松质骨、密质骨强度相当)也能满足生理应变的要求。采用弹性较优的合成塑料作为参考支架进行有效性检测,结果表明在支架材料上此装置产生正常的生理应变,且重复精度高。装置使用智能材料-压电陶瓷作为动力来源,通过计算机控制实现了骨支架材料骨生理水平应变的精确控制。此装置将为工程化骨组织构建中载荷影响的研究提供方便。     

硅橡胶膜细胞载体的应力分析及生物相容性

目的 报道一种自制的硅橡胶膜细胞载体,通过三维有限元方法计算其表面应变分布情况,并作细胞生物相容性分析,对材料进行全面评价,为细胞的应力刺激实验提供理论依据。方法 将硅橡胶材料制作成0.1 cm厚的透明薄膜,结合硅橡胶材料的泊松比以及弹性模量,应用三维有限元分析软件对数据进行处理,模拟硅橡胶受到牵张应变后产生的形变;通过MTT方法比较细胞在硅橡胶和标准培养板上的生长情况,并采取体外皮下包埋实验验证硅橡胶材料是否具有生物学毒性。结果 在对材料加载0.5%~20%的过程中,有效应变范围集中在硅橡胶膜的中心区域,约占总面积的90%;同时,硅橡胶材料虽然在生物相容性方面与标准培养板存在一定差异,但其本身无生物学毒性。结论 这种自制的硅橡胶细胞载体表面应力分布良好,生物相容性尚可满足细胞培养,但表面需进一步改善,可满足细胞的牵张应变实验。     

间歇性机械拉伸对体外培养兔关节软骨细胞微管蛋白-β取向的影响

目的探讨间歇性机械拉伸对兔关节软骨细胞生长和细胞形态及骨架的影响,为软骨细胞内力学信号传导研究的提供参考。方法采用FX-4000TM柔性基底拉伸系统对体外单层原代培养的兔软骨细胞实施正弦波、0.5Hz、05%间歇性周期应变加载,每日拉伸3h,共加载3天。对照组静态培养。3天后检测细胞活力、基质合成情况以及微管蛋白-β细胞免疫荧光染色,并对细胞周期进行检测。结果加载组软骨细胞生长良好,氨基多聚糖和蛋白多糖的合成与对照组无明显差异;但细胞增殖指数显著增高(P<0.05);微管蛋白-β沿细胞长轴发生重排,细胞形态和取向趋于一致。结论间歇性机械拉伸对细胞生长起到积极的作用,并且通过微管蛋白-β的重排影响细胞形态和取向。     

微振动在骨髓间充质干细胞成骨向分化中的作用机制研究进展

微振动是指系统相对平衡位置幅度很小的周期性偏离,而高频率、低振幅的微振动(low-magnitude high-frequency vibration, LMHFV)对骨骼系统细胞的作用力与肌肉运动时对骨骼产生的力学刺激相似。骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs)作为力学敏感细胞,存在于骨髓基质,具有多向分化潜能。在体外适当机械刺激下,BMSCs增殖、分化等生物学特性发生功能性变化,对力学刺激做出适应性应答。LMHFV可促进BMSCs向成骨细胞分化,探明其机制有助于将微振动应用于骨质疏松、骨折、成骨不全症、肥胖症等疾病的治疗以及正畸牙移动的加速等方面。综述微振动对BMSCs成骨向分化的影响以及可能的作用机制,为研究微振动刺激下BMSCs的力学生物学改变提供思路。     

用于骨力生物学研究的加载装置

背景：关节软骨组织细胞对力学刺激产生反馈来维持它的形态和结构,进而适应环境。加载装置能提供研究关节软骨力生物学的合适的力学环境。 目的：根据组织工程仿生的原理,建立一种用于软骨力学生物学研究的双频加载装置。 方法：该力学环境采用双频加载方式实现,通过控制可调凸轮和压电陶瓷振幅和频率来实现低频-高幅载荷耦合高频-低幅载荷,同时使用有限元法对受载体进行力学分析。 结果与结论：按照仿真的原理,研制出用于软骨力生物学研究的加载装置。在双频加载的条件下,软骨浅表层受到的力学载荷最大,其次是中间层,深层受到的力学应力最小;在高频10 Hz和20 Hz与低频1 Hz和2 Hz叠加时,软骨表现出不同的力学响应,但是二者引起的差异很小。该力学环境的构建可能有助于组织工程的发展和临床医学的应用,未来将采用生物学实验进行检验。     

多通道细胞牵张应变加载系统的建立及应用

本研究自行开发了单片机控制的多通道细胞牵张应力加载仪,控制与真空室相连的真空泵,使真空室内的负压产生周期性变化,使置于真空室上的弹性膜培养板底壁的弹性膜发生变形,对培养于弹性膜上的细胞施加牵张应变,实现对细胞施加周期性拉伸变形的作用。控制系统可以同时进行三通道不同形变率的对比实验,而且体积小,便于携带,形变率、频率可以精确调节,操作简单,可以提供1%～21%范围的形变率,应变频率可在0～0.5Hz范围内可调。系统运行稳定,达到了设计要求,提供了对贴壁生长细胞施加基底膜牵张应力的体外实验方法。     

Development of a device to stretch tissue-like materials and to measure their mechanical properties by scanning probe microscopy

We have developed a new stretch device to investigate the biomechanical responses to an external loading force on a tissue-like material consisting of cells and a collagen gel. Collagen gel, a typical matrix found abundantly in the connective tissue, was attached to an elastic chamber that was precoated with a thin layer of collagen. Madin-Darby canine kidney cells that were cultured on the collagen gel were stretched in a uniaxial direction via deformation of the elastic chamber. Changes in the morphology and stiffness of the tissue-like structure were measured before and after the stretch using wide-range scanning probe microscopy (WR-SPM). The change in cellular morphology was heterogeneous, and there was a twofold increase in the intercellular junction due to the stretch. In addition to the WR-SPM measurements, this device enables observation of the spatial distribution of cytoskeletal proteins such as vimentin and alpha-catenin using immunofluorescent microscopy. We concluded that the stretch device we have reported in this paper is useful for measuring the mechanical response of a tissue-like material over a range of cell sizes when exposed to an external loading force.     

体外细胞培养的加力装置及压力测量的研究

目的：在一定力学作用下,机体的器官、组织、细胞和生物大分子会发生相应的形态和功能改变,这对于维持正常生理功能具有重要作用。细胞力学是组织工程和细胞工程的基础之一,在离体培养过程中对细胞施加不同的机械力以研究应力对细胞的影响是细胞力学的一个重要研究领域,也是细胞力学的重要研究手段。本研究是为了模拟在体细胞的力学环境,实现在体外培养的条件下对细胞施加力的作用,设计了一种力加载装置和相应的压力检测模块。方法：力加载装置包括应力加载模块、细胞培养室、步进电机传动模块组成。计算机通过软件驱动步进电机控制活塞在培养室内直线往复运动,实现细胞培养室内压力大小、频率和持续时间的可控变化。应力检测模块可以实时监测培养室内压力大小的变化,并与预期参数对比后通过反馈系统调节各模块的运行,实现压力加载的精准控制。结果：系统压力加载的频率调控范围为0 Hz~1Hz,压力加载范围为-71 kPa~60 kPa。结论：该系统为研究压力对细胞的影响提供了一种简单、可行的方法,实验证明系统压力加载方式准确、可行,能对离体培养的细胞进行有效的压力加载。     

Anisotropic, Three-Dimensional Deformation of Single Attached Cells Under Compression

Quantifying three-dimensional deformation of cells under mechanical load is relevant when studying cell deformation in relation to cellular functioning. Because most cells are anchorage dependent for normal functioning, it is desired to study cells in their attached configuration. This study reports new three-dimensional morphometric measurements of cell deformation during stepwise compression experiments with a recently developed cell loading device. The device allows global, unconfined compression of individual, attached cells under optimal environmental conditions. Three-dimensional images of fluorescently stained myoblasts were recorded with confocal microscopy and analyzed with image restoration and three-dimensional image reconstruction software to quantify cell deformation. In response to compression, cell width, cross-sectional area, and surface area increased significantly with applied strain, whereas cell volume remained constant. Interestingly, the cell and the nucleus deformed perpendicular to the direction of actin filaments present along the long axis of the cell. This strongly suggests that this anisotropic deformation can be attributed to the preferred orientation of actin filaments. A shape factor was introduced to quantify the global shape of attached cells. The increase of this factor during compression reflected the anisotropic deformation of the cell.     

The effect of timing of mechanical stimulation on proliferation and differentiation of goat bone marrow stem cells cultured on braided PLGA scaffolds

Bone marrow stromal cells (BMSCs) have been shown to proliferate and produce matrix when seeded onto braided poly(L-lactide/glycolide) acid (PLGA) scaffolds. Mechanical stimulation may be applied to stimulate tissue formation during ligament tissue engineering. This study describes for the first time the effect of constant load on BMSCs seeded onto a braided PLGA scaffold. The seeded scaffolds were subjected to four different loading regimes: Scaffolds were unloaded, loaded during seeding, immediately after seeding, or 2 days after seeding. During the first 5 days, changing the mechanical environment seemed to inhibit proliferation, because cells on scaffolds loaded immediately after seeding or after a 2-day delay, contained fewer cells than on unloaded scaffolds or scaffolds loaded during seeding (p<0.01 for scaffolds loaded after 2 days). During this period, differentiation increased with the period of load applied. After day 5, differences in cell content and collagen production leveled off. After day 11, cell number decreased, whereas collagen production continued to increase. Cell number and differentiation at day 23 were independent of the timing of the mechanical stimulation applied. In conclusion, static load applied to BMSCs cultured on PLGA scaffolds allows for proliferation and differentiation, with loading during seeding yielding the most rapid response. Future research should be aimed at elucidating the biomechanical and biochemical characteristics of tissue formed by BMSCs on PLGA under mechanical stimulation.     

Effects of substrate characteristics on bone cell response to the mechanical environment

The effect of substrate characteristics on primary human bone cell response to mechanical loading was investigated in this study. The substrates comprised organic and inorganic materials with a range of hydrophilic and hydrophobic features. Substrate surface topography varied from smooth to particulate to porous. It was found that hydrophilic substrates such as borosilicate glass facilitated bone cell adhesion, in contrast to hydrophobic substrates such as poly(L-lactic acid), in which clumps of cells grew unevenly across the substrate surface. All primary bone cells cultured in the various collagen-coated substrates were responsive to mechanical stimulation. The study showed that, at a low strain level of 1000μstrain, mechanical stimulation enhanced bone cell differentiation rather than proliferation. Coating the substrates with collagen type l enhanced cell adhesion and promoted an elongated cell morphology, indicating that the presence of specific binding sites on a substrate may be more important than its hydrophilic properties, regardles of the substrate topography.     

Strain measurements in cultured vascular smooth muscle cells subjected to mechanical deformation

Early work in the field of biomechanics employed rigorous application of the principles of mechanics to the study of the macroscopic structural response of tissues to applied loads. Interest in the functional response of tissues to mechanical stimulation has lead researchers to study the biochemical responses of cells to mechanical loading. Characterization of the experimental system (i.e., specimen geometry and boundary conditions) is no less important on the microscopic scale of the cell than it is for macroscopic tissue testing. We outline a method for appropriate characterization of cell deformation in a cell culture model; describe a system for applying a uniform, isotropic strain field to cells in culture; and demonstrate a dependence of cell deformation on morphology and distribution of adhesion sites. Cultured vascular smooth-muscle cells were mechanically deformed by applying an isotropic strain to the compliant substrate to which they were adhered. The state of strain in the cells was determined by measurement of the displacements of fluorescent microspheres attached to the cell surface. The magnitude and orientation of principal strains were found to vary spatially and temporally and to depend on cell morphology. These results show that cell strain can be highly variable and emphasize the need to characterize both the loading conditions and the actual cellular deformation in this type of experimental model.     

Characterization of the Flexcell Uniflex cyclic strain culture system with U937 macrophage-like cells

Mechanical forces alter many cell functions in a variety of cell types. It has been recognized that stimulation of cells in culture may be more representative of some physiologic conditions. Although there are commercially available systems for the study of cells cultured in a mechanical environment, very little has been documented on the validation techniques for these devices. In this study, Flexcell's recently introduced Uniflex cyclic strain system was programmed to apply 10% longitudinal sinusoidal strain (0.25 Hz) for 48 h to U937 cells cultured on Uniflex plates. Image analysis was employed to characterize the actual strain field. For a chosen amplitude of 10% the applied strain was highly reproducible and relatively uniform (10.6+/-0.2%) in a central rectangular region of the membrane (dimensions of 9.2+/-2 x 13.6+/-0.8 mm2). The strain increased the release of IL-6, esterase and acid phosphatase activity (p<0.05) from adherent U937 cells. Cells also displayed altered morphology, aligning and lengthening with the direction of strain, whereas static cells maintained a round appearance showing no preferred orientation. These data indicate that cyclic mechanical strain applied by the Uniflex strain system modulates U937 cell function leading to selective increases in enzymatic activities as well as orientation in a favored direction.     

全骨髓法体外培养分离大鼠骨髓间充质干细胞及PKH26标记

背景：要获得动物实验需要的标记大鼠骨髓间充质干细胞,体外培养、扩增和示踪已成为实验的关键环节。 目的：采用全骨髓培养分离大鼠骨髓间充质干细胞,以及PKH26对其体外标记,建立一种方便、实用的分离培养并示踪骨髓间充质干细胞的方法。 方法：通过全骨髓培养分离法纯化大鼠骨髓间充质干细胞。经传代扩增,细胞进一步纯化。取第3代大鼠骨髓间充质干细胞按PKH26标记程序进行标记后培养,荧光显微镜下观察标记后细胞生长状态、萤光强度变化和传代培养效果。利用四唑盐比色法测定标记后骨髓间充质干细胞的生长曲线。 结果与结论：全骨髓培养分离法能成功获得纯度高的骨髓间充质干细胞,用PKH26标记后的骨髓间充质干细胞呈红色荧光,体外连续传代培养3代后,细胞荧光强度逐渐减弱。PKH26标记骨髓间充质干细胞的生长形态、生长活力不发生改变。结果证实全骨髓培养分离法简便易行,能获取较高纯度的生长增殖状态良好的骨髓间充质干细胞,PKH26荧光标记大鼠骨髓间充质干细胞是一种有效、实用的方法。     

持续张应力对骨髓基质干细胞增殖及骨向分化的影响

目的通过体外加力装置研究持续牵张应力对大鼠骨髓基质干细胞(bone marrow stromal cells,BMSCs)增殖及骨向分化能力的影响。方法选用3月龄健康SD雌性大鼠,采用全血贴壁培养法分离及培养BMSCs。取生长良好的第3～5代细胞接种于Flexercell应力加载系统(10%、1 Hz),根据应力作用时间不同分为1、6、12、24 h组和48 h组。观察并分析持续牵张力对于大鼠BMSCs形态、增殖活性以及成骨能力变化的影响。结果 (1)随着加力时间的延长,与对照组相比,实验组细胞形态呈现一定规律性,细胞长轴多垂直于受力径向。(2)10%持续张应力作用可抑制BMSCs增殖活性。(3)持续张应力可增高碱性磷酸酶(alkaline phosphatase,ALP)、Ⅰ型胶原(collagenI,COLⅠ)、核心结合因子Cbfa1(core binding factor a1,又名Runx2)mRNA的表达量,且呈现时间依赖性。其中实验组ALP表达量在24 h明显高于相应对照组,COLⅠ表达量在24 h及48 h均明显高于对照组,Runx2表达量在6 h与对照组相比显著增高(P<0.05)。骨钙素(osteocalcin,OC)含量在加力起始阶段显著高于对照组,随时间推移逐渐下降,48 h时明显低于对照组(P<0.05)。(4)持续张力可以促使Runx2蛋白水平增高,且在6 h实验组明显高于对照组(P<0.05)。之后缓慢下降,在24 h时显著低于对照组水平(P<0.05)。结论持续牵张力作用下BM-SCs细胞形态呈现一定规律性排列,其增殖活性受到抑制,但早期成骨向分化能力却显著提高。