一种体外贴壁细胞应变加载装置的研制

目的开发一套新型的应变加载装置,用于贴壁细胞力学生物学研究。方法该装置基于基底形变加载技术,采用可控制编程器驱动步进器,引起硅橡胶小室变形,实现多单元大应变的细胞加载;研制该装置,检测机械性能;建立硅橡胶小室的三维模型,利用有限元技术对硅橡胶小室进行仿真,分析该小室的应变场均匀性问题;采用该装置对骨髓间充质干细胞(bone marrow stromal cells,BMSCs)加载5%机械应变,频率0.5 Hz,2 h/d,持续5 d,并在倒置显微镜下观察细胞形态的变化。结果所研制的适用于体外细胞加载装置可对3组细胞加载基底实现最大至50%机械单向应变;在10%应变范围内,硅橡胶小室底部的均匀应变场面积占比保持在50%以上,保证了细胞受力均匀; BMSCs形态发生明显变化,排列方向趋于垂直主应变加载方向。结论该装置运行可靠,应变范围宽,频率可调,操作方便,可同时对多组细胞培养基底进行应变加载,为细胞力学生物学研究提供了便利条件。     

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。     

Human Mesenchymal Stem Cells Form Multicellular Structures in Response to Applied Cyclic Strain

Mesenchymal stem cells (MSCs) are a component of many cardiovascular cell-based regenerative medicine therapies. There is little understanding, however, of the response of MSCs to mechanical cues present in cardiovascular tissues. The objectives of these studies were to identify a model system to study the effect of well-defined applied cyclic strain on MSCs and to use this system to determine the effect of cyclic equibiaxial strain on the cellular and cytoskeletal organization of MSCs. When exposed to 10%, 1 Hz cyclic equibiaxial strain for 48 h, MSCs remained viable, retained characteristic gene and protein markers, and rearranged to form multicellular structures defined as clusters and knobs. This novel observation of cluster (overlapping cells surrounded by radial cellular projections) and knob (more dome-like structure containing significantly more cells than a cluster) formation did not involve changes in cytoskeletal proteins and resulted from cellular rearrangements initiated within 8 h of applied strain. Observed cellular responses were found to be dependent on substrate coating, but not on cell density for the 8-fold ranges tested. This system can thus be used to study the mechanoresponse over hours to days of MSCs exposed to applied cyclic strain in the context of cell–cell and cell–matrix interactions. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Roles of Hemodynamic Forces in Vascular Cell Differentiation

The pulsatile nature of blood flow is a key stimulus for the modulation of vascular cell differentiation. Within the vascular media, physiologic stress is manifested as cyclic strain, while in the lumen, cells are subjected to shear stress. These two respective biomechanical forces influence the phenotype and degree of differentiation or proliferation of smooth muscle cells and endothelial cells within the human vasculature. Elucidation of the effect of these mechanical forces on cellular differentiation has led to a surge of research into this area because of the implications for both the treatment of atherosclerotic disease and the future of vascular tissue engineering. The use of mechanical force to directly control vascular cell differentiation may be utilized as an invaluable engineering tool in the future. However, an understanding of the role of hemodynamics in vascular cell differentiation and proliferation is critical before application can be realized. Thus, this review will provide a current perspective on the latest research and controversy behind the role of hemodynamic forces for vascular cell differentiation and phenotype modulation. Furthermore, this review will illustrate the application of hemodynamic force for vascular tissue engineering and explicate future directions for research.     

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。     

Assessment of Strain Field in Endothelial Cells Subjected to Uniaxial Deformation of Their Substrate

A stretch chamber has been developed in order to visualize the deformation of cells subjected to controlled uniaxial stretch of their substrate. A rectangular, custom-made, transparent silicone channel is used as a deformable substrate. Bovine aortic endothelial cells are plated at the bottom of the channel whose lateral deformation is controlled by two piezoelectric translators. The system is mounted on the stage of a confocal microscope where three-dimensional (3D) images of the cells can be acquired simultaneously in the three RGB channels. The first channel provides images of 216 nm fluorescent beads embedded in the cytoskeleton (used as internal markers). The second is used to image the shape of the nucleus revealed by live cell nucleic acid staining. The third one provides a transmitted light image of the cell outline. 3D images of the cell are taken before deformation, after uniaxial deformation of the substrate (up to 25%) and after relaxation. Results indicate that: (a) the cell closely follows the deformation imposed by the substrate with no measurable residual strain after relaxation, and (b) there is a clear mechanical coupling between the extracellular matrix and the nucleus, which deforms significantly under the applied substrate stretch. Suggesting that the nucleus can directly sense the mechanical environment of the cell, the latter result has potentially important implications for signal transduction.     

A Device for Long Term, <Emphasis Type="Italic">In Vitro</Emphasis> Loading of Three-Dimensional Natural and Engineered Tissues

In vitro studies of mechanical loads applied to three-dimensional tissue constructs are important to the design and production of functional, engineered bone tissue. This study reports the development and characterization of a mechanical device capable of subjecting a three-dimensional section of natural or engineered tissue to precise, reproducible four-point bending deformations over a range of programmable magnitudes and frequencies. To test the biological and mechanical capabilities of the system, a low-cycle (360 cycles/day), medium-range strain (2500 microstrain), long-term (16 day) loading regime was applied to rat bone marrow stromal cells cultured in porous DL-polylactic acid scaffolds. Cells proliferated in culture throughout the experiment, and with time showed an increase in alkaline phosphatase expression per cell. Calcium and phosphorus mineral deposition by the unloaded group was significantly greater (p < 0.05) than that deposited by the loaded group. The molar ratio of calcium to phosphorus in the unloaded group (0.94:1) was significantly greater (p < 0.05) than that of the loaded group (0.41:1). The loading device presented here is a tool which can be used to help elucidate contributions of mechanical loading/fatigue on biodegradable materials, as well as study the effects of mechanical loading on natural or engineered tissues in vitro. © 2003 Biomedical Engineering Society. PAC2003: 8768+z, 8780Rb     

Role of Rac and Rho-GDI Alpha in the Frequency-dependent Expression of h1-calponin in Vascular Smooth Muscle Cells under Cyclic Mechanical Strain

Phenotype transformation of vascular smooth muscle cells (VSMCs) has been reported to be directly influenced by the frequency of mechanical strain. This study explored the effects of different frequencies of mechanical strain on expression of phenotype marker h1-calponin and the possible mechanism. VSMCs were subjected to cyclic strains of 10% elongation at 1 and 2 Hz for 24 h by using a Flexercell strain unit. The protein expression of h1-calponin was assessed by Western blotting and the possible protein kinases involved were evaluated by their specific inhibitor or targeted siRNA ‘knock-down.’ The results showed that cyclic strains modulated the expressions of h1-calponin, phospho-p38, Rac and Rho-guanine nucleotide dissociation inhibitor alpha (Rho-GDIα) in nonlinear frequency-dependent manners. This nonlinear frequency-dependent change of h1-calponin expression could be blocked by a specific p38 inhibitor, SB202190. The changed expression of phospho-p38 induced by the frequencies of cyclic strain was reversed by targeted siRNA ‘knock-down’ of Rac, while enhanced by targeted siRNA ‘knock-down’ of Rho-GDIα. These results suggest that the frequency-dependent expression of h1-calponin under cyclic strain is mediated at least partly by the regulation of Rac and Rho-GDIα expression on the activation of p38 pathway. Ming-Juan Qu and Bo Liu contributed equally to this work.     

Qualitative Electron Microscopic Analysis of Cultured Chick Embryonic Cardiac and Skeletal Muscle Cells: The Cellular Effect of Coenzyme Q10 After Exposure to Triton X-100

The numerous protective effects of coenzyme Q10 (CoQ10) evoked the question of whether it might be able to elicit protection to cell membranes after being challenged by the membrane disrupter Triton X-100. Cardiac and skeletal muscle tissue from chick embryos was cultured and exposed to increasing concentrations of CoQ10 and Triton X-100. Scanning electron microscopy was used to study cell morphology. Results suggested the ability of CoQ10 to offer protection to cells challenged by Triton X-100. The authors suggest that CoQ10 may offer protection to muscle cells, by enhancing membrane repair via patch formation by an unknown mechanism that possibly involves Ca²⁺-dependent ion channel activation.