新型体外细胞加压培养装置的构建及应用

目的:设计一种新型简易细胞加压培养装置模拟在体外培养条件下对细胞施加流体静压,为进一步研究恒定流体静压条件下体外细胞变化打下基础。方法:装置包括压力培养罐、微压传感器、可编程序控制器以及微型气泵。通过在可编程序控制器上编写控制程序来控制微型气泵的运行,进而调节培养罐内的气体压力,从而控制细胞受到的流体静压。结果:装置的压力加载范围为0～120 mmHg,波动幅度为±2 mmHg。可将常规细胞培养板及培养瓶平稳放置在压力培养罐中并施加预设的流体静压。结论:该装置具有良好的准确性与可靠性,可用于在体外研究恒定流体静压下细胞变化。     

离体培养细胞的力学实验方法

细胞力学是组织工程和细胞工程的基础之一，在细胞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。本文综述了离体培养细胞的力学实验方法，根据不同的载荷施加方式可分为：微吸管吸吮技术、压力载荷法、基底拉伸法和流体剪切法等，并指出了各种方法存在的利与弊。     

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。     

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。     

一种用于体外培养细胞的静磁场加载装置

磁性附着体均存在漏磁,应用于患者口腔中时将对邻近组织施加一定强度的静磁场。磁性附着体磁场对机体组织的生物学效应尚鲜见研究报道。本研究研制了一套细胞静磁场加载装置,并模拟磁性附着体静磁场对体外培养人牙周膜细胞加载。该装置能产生恒定的静磁场且磁场强度可调,体积小巧,可对培养皿、培养孔板等多种规格细胞培养器皿中的细胞加载静磁场,便于对细胞的多项生物学指标进行研究。对人牙周膜细胞加载静磁场的结果显示该装置可以有效地对体外培养细胞进行不同强度、不同时间的静磁场加载。该装置为研究静磁场细胞生物学效应提供了必要的手段。     

可调压式细胞培育箱的研制

为研究离体培养细胞在静压力作用时的生长行为,研制了可调压式细胞培育箱。本装置包括持续性加压系统、间歇性加压系统、静压力控制测量系统、变形测量显示系统等。该系统不仅能模拟出细胞在密封培养箱内所承受的静压力效果,也可模拟出牵张力的力学作用效果,对研究细胞的应力生长关系十分有用。     

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     

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。     

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。     

张应力作用下体外细胞培养膜上应力分布的三维有限元分析

在自行研制细胞张应力施加装置的基础上，建立细胞张应力施加装置中生物膜三维有限元模型，模拟施加不同大小的静水压力，采用有限元应力分析的方法计算生物膜（硅胶膜）上的张应力及位移分布及其规律，为相关的细胞力学实验提供理论依据。     

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.     

张应力对成骨分化骨髓间充质干细胞ODF mRNA表达的影响

目的观察大鼠骨髓间充质干细胞骨向诱导分化来源的成骨细胞在张应力刺激下破骨细胞分化因子(osteo-clast differentiation factor,ODF)基因表达的变化,探讨正畸牙移动骨改建过程中成骨细胞调控破骨细胞分化成熟的机制。方法分离培养大鼠股骨骨髓间充质干细胞来源的成骨细胞,采用膜式动态张应力加载体系按张应力大小及作用方式不同分为静态1、3、5 kPa组,动态3、5 kPa组(频率0.017 Hz)及对照不加力组6个实验组和按不同作用时间分为0、3、6、9、12、24和48 h 7个时间段进行体外细胞加力,RT-PCR技术检测不同强度、不同性质、不同作用时间张应力对成骨细胞ODF mRNA表达的影响。结果张应力刺激抑制成骨细胞ODF的表达,静态张应力的抑制效应弱;抑制作用与刺激强度无明显关系;ODF mRNA在动态张应力作用6 h后表达逐渐下降,9 h后显著下降,之后维持在一较低水平,48 h最低,具有时效性。结论机械张应力刺激通过抑制成骨细胞ODF mRNA的表达限制破骨细胞的分化成熟。     

The impact of substrate stiffness and mechanical loading on fibroblast-induced scaffold remodeling

Fibroblasts as many other cells are known to form, contract, and remodel the extracellular matrix (ECM). The presented study aims to gain an insight into how mechanical boundary conditions affect the production of ECM components, their remodeling, and the feedback of the altered mechanical cell environment on these processes. The influence of cyclic mechanical loading (f=1?Hz, 10% axial compression) and scaffold stiffness (E=1.2 and 8.5?kPa) on the mechanical properties of fibroblast-seeded scaffold constructs were investigated in an in vitro approach over 14 days of culture. To do so, a newly developed bioreactor system was employed. While mechanical loading resulted in a clear upregulation of procollagen-I and fibronectin production, scaffold stiffness showed to primarily influence matrix metalloproteinase-1 (MMP-1) secretion and cell-induced scaffold contraction. Higher stiffness of the collagen scaffolds resulted in an up to twofold higher production of collagen-degrading MMP-1. The changes of mechanical parameters like Young's modulus, maximum compression force, and elastic portion of compression force over time suggest that from initially distinct mechanical starting conditions (scaffold stiffness), the construct's mechanical properties converge over time. As a consequence of mechanical loading a shift toward higher construct stiffness was observed. The results suggest that scaffold stiffness has only a temporary effect on cell behavior, while the impact of mechanical loading is preserved over time. Thus, it is concluded that the mechanical environment of the cell after remodeling is depending on mechanical loading rather than on initial scaffold stiffness.     

静压力对脂肪干细胞与静电纺丝纳米纤维细胞相容性的影响

BACKGROUND: Electrospun polylactic acid/polycaprolactone nanofibers (ENF) are a kind of self-synthesized biodegradable material. Our preliminary studies have indicated that the biomaterial exhibits excellent biocompatibility; however, the research about its mechanics is still little. OBJECTIVE: To explore the effects of static pressure on the cytocompatibility of adipose-derived stem cells on the ENF scaffold. METHODS: Adipose-derived stem cells were seeded onto the ENF scaffold, and then cultured in the low-glucose DMEM supplemented with 10% fetal bovine serum. The mixed constructs were submitted to the static pressure at 0, 15, 30, and 45 kPa for 4 hours using a static pressure device, respectively. Subsequently, the proliferation, adhesion and viability of adipose-derived stem cells on the ENF scaffold were detected using MTT assay and living/dead staining to evaluate the cytocompatibility. RESULTS AND CONCLUSION: MTT assay showed that there were significant differences in absorbance values among groups by one-way analysis of variance after 4 hours of loading with different static pressures in vitro. Under 0-30 kPa static pressure, the absorbance values increased with static pressure, but the absorbance values declined until the pressure reached 45 kPa, and multiple comparisons between groups showed significant difference. The significant differences in the cell attachment percentage by MTT assay could be found among groups. The living/dead staining results supported the above findings. Furthermore, the significant differences in percentage of living cells among groups were shown using either one-way analysis of variance or paired t test. In conclusion, the appropriate static pressure can promote the cytocompatibility, proliferation, adhesion and viability of adipose-derived stem cells on the ENF scaffold. But the excessive pressure is likely to inhibit the cellular biological behaviors, thus affecting cytocompatibility of adipose-derived stem cells with the ENF scaffold.     

压应力对体外培养的表皮干细胞增殖分化的影响

目的： 初步观察间歇压应力对表皮干细胞增殖分化的影响并探讨其可能的作用机制。 方法： Ⅳ型胶原分离培养表皮干细胞，并用免疫组织化学PowervisionTM二步法和细胞周期分析进行鉴定；采用不同压应力（4 kPa、6 kPa、8 kPa、10 kPa、12 kPa）细胞培养体系装置培养表皮干细胞和进行细胞加压，利用表皮干细胞特异表达角蛋白19及终末分化细胞表达角蛋白10的特点和免疫组织化学PowervisionTM二步法检测加压前后表皮干细胞和终末分化细胞的变化。 结果： 分离、培养的表皮干细胞K19免疫组化染色阳性，细胞周期分析有84.80%的细胞处于G1期；8 kPa以上的间歇压应力作用粘附于硅胶膜上的表皮干细胞1周后，其数量明显增多，免疫组化染色发现其中有角蛋白10阳性细胞。 结论： 8 kPa以上的间歇压应力能诱导表皮干细胞增殖分化，表皮干细胞对机械应力的响应机制可能是其主要的生物学基础。     

成骨细胞的体外剪切应力加载实验的变形分析及信号转导区域探讨

目的 根据已有体外培养鼠成骨细胞的参数实验数据,估算剪切应力加载实验中细胞整体剪切形变,借以研究细胞的主要转导区域.方法 计算过程采用黏弹性力学理论,对细胞运用了标准黏弹性模型,并简化其膜所受剪切力为均匀.结果 细胞剪切力产生的细胞变形大约是引起成骨细胞相同生物学响应的拉伸加载变形的十分之一.结论 从细胞总的力学刺激生物学响应来看,剪切应力加载实验中细胞的整体变形所产生的力学转导是可以忽略的,主要转导区域在承受剪切应力的细胞膜.     

The stiffness of bone marrow cell-knit composites is increased during mechanical load

A novel device for mechanical stimulation of primary adult rat bone marrow cells cultured on three-dimensional knitted textiles has been prototyped. A method has been developed ensuring a well-defined, high-density, and reproducible cell seeding on the knitted fabric. After culturing for 18-52 days the cell-knit composites were subjected to uniaxial 2% stretching and relaxation. The frequency was altered between 0.1 Hz (196 min, loading phase) and 0.01 Hz (360 min, resting phase). Identically treated knits without cells exhibited a slight stiffness reduction, whereas the stiffness of knits with cells increased from cycle to cycle. The stiffness increase was found to depend on the duration of the culture period before mechanical loading. Our data suggest that the extracellular matrix deposited by the cells on the knit and intact microtubuli of living cells cause the observed stiffness increase. In comparison to the unstrained static cell-knit composites cell proliferation and bone cell differentiation were reduced by the mechanical load.