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。     

周期性张应力促进兔髁状突软骨细胞的增殖

背景：髁状突是下颌最重要的生长区之一,终生具有生长改建能力。在体内条件下,细胞力学的功能研究因其所处生理环境的复杂性、刺激因素传导的不定向性、实验条件的不易控制性而很难得到满意结果,应力刺激对髁状突软骨细胞的直接影响需要进一步行体外研究。 目的：观察周期性张应力对体外培养兔髁状突软骨细胞生长增殖的影响。 方法：体外分离培养及鉴定兔髁状突软骨细胞,在细胞培养至第3代时使用细胞加力装置对细胞施加强度为10%,频率为6循环/min的周期性张应力,作用时间分别为1,6,12和24 h,并设置未加力组作为对照。应用流式细胞仪检测细胞生长周期,应用MTT法分析细胞的增殖活性。 结果与结论：在周期性张应力下,髁状突软骨细胞流式细胞仪检测结果显示在加力6 h和12 h,加力组细胞生长周期开始有显著性变化,在24 h达到实验最大值,差异有显著性意义(P < 0.01)。MTT检测结果示细胞生长活跃,在6,12 h与对照组有明显变化,在24 h达到实验最大值,差异有显著性意义(P < 0.01)。提示周期性张应力可明显促进髁状突细胞增殖,在24 h内具有持续促进作用。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

周期性应力刺激对成牙骨质样细胞增殖的影响

背景：成牙骨质细胞在牙根形成、牙周病牙骨质的再生以及正畸牙根吸收的修复中起着重要的作用,其在体外培养状态下对力学信号反应特性的变化,在国内外少见报道。 目的：分析正畸牙移动张力侧及压力侧两种不同受力形式与牙骨质改建的关系。 方法：采用四点弯曲细胞力学加载仪,对体外培养的成牙骨质样细胞OCCM30分别施加2 000 με张、压应力,应用流式细胞术检测应力刺激3,6,12,24 h后OCCM30细胞周期及增殖指数的变化。 结果与结论：2 000 με张、压应力加载3,6 h后,成牙骨质样细胞OCCM30在S期的细胞百分率明显减小,细胞增殖活性降低;随着加力时间的延长,S期细胞百分率回升,加载24 h后S期细胞百分率与对照组无明显差异,细胞增殖活性恢复。张、压应力对增殖活性的影响趋势一致,相互之间差别无显著性意义。     

周期性张应力下成肌细胞中半胱天冬氨酸蛋白酶9参与力学信号传导

背景:功能矫形使面颌部肌肉发生适应性改建,以纠正错颌畸形。成肌细胞是适应性改建的主要体现者,周期性牵张应力导致成肌细胞凋亡,而半胱天冬氨酸蛋白酶9是线粒体凋亡通路中的重要因子。目的:明确半胱天冬氨酸蛋白酶9在不同时间周期性张应力作用下的表达变化。方法:以大鼠L6成肌细胞为实验对象,在成功构建成肌细胞体外培养-力学刺激模型的基础上,通过多通道细胞牵张应力加载系统,分别对待测细胞持续施加1,6,12,24 h的周期性张应力,不加力组为对照组。倒置相差显微镜下观察成肌细胞的形态学改变和生长状况;运用RT-PCR和Western Blot技术检测周期性张应力作用下半胱天冬氨酸蛋白酶9的mRNA和蛋白含量变化。结果与结论:倒置相差显微镜观察结果显示成肌细胞施加周期性张应力后贴壁生长情况良好,细胞无变性并且脱落率极低,说明成功构建了成肌细胞体外培养-力学刺激模型。RT-PCR和Western Blot检测结果显示,随着加力时间的延长,半胱天冬氨酸蛋白酶9 mRNA含量和活化型半胱天冬氨酸蛋白酶9的蛋白含量均显著增加,而半胱天冬氨酸蛋白酶9前体蛋白含量随着加力时间的延长均显著降低。可见半胱天冬氨酸蛋白酶9参与了周期性张应力作用下的力学信号传导。     

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     

流动切应力对培养的血管内皮细胞形态的影响

为探索培养的血客内皮细胞承受流体冲击的能力，以便在人工心脏瓣膜表面形成一个抵抗血流切应力作用强的血管内皮细胞层，在我们研制的内皮细胞切应力反应测试装置上，观察了培养在盖玻片表现的牛主动脉内皮细胞形成单层细胞后在切应力分别在１５ｄｆｙｍｅｓ／ｃｍ＾２２４小时和１１５ｄｙｍｓ／ｃｍ＾２８小时作用下细胞的形态张脱落情况。     

流体切应力梯度对血管内皮细胞排列和形状的影响

目的研究不同梯度切应力作用下,血管内皮细胞(endothelial cells,ECs)排列和形状变化,旨在了解流体切应力梯度对ECs形态的影响,为进一步探讨其功能变化提供实验基础。方法建立可对体外培养ECs施加梯度切应力的流动腔装置,并应用该装置对人脐静脉ECs加载了大小在15dyn/cm2～6.6dyn/cm2(1dyn=10-5N)范围、梯度分别为1.5dyn/cm2和3dyn/cm2的切应力,加载时间均为6h。比较这两种不同切应力梯度对ECs的细胞方向角、细胞宽长比和细胞形态指数的影响。结果在不同切应力梯度作用下,ECs的细胞方向角分布散乱,细胞无排列规律。与3dyn/cm2相比,1.5dyn/cm2切应力梯度下ECs的宽长比和细胞形态指数明显减少,趋向于拉伸状态。结论在不同切应力梯度作用下,ECs均排列紊乱,无规律可循。然而,在相对较小的切应力梯度作用下,细胞容易被拉伸,细胞形状趋向于伸长,而较大切应力梯度作用下,细胞形状则趋向于圆形。     

张应力刺激大鼠髁突软骨细胞Ⅱ型胶原和聚集蛋白聚糖的表达*

背景：研究表明软骨细胞外基质合成的变化反映了外力对于颞下颌关节的影响和机体对于外力的适应性。 目的：观察周期性张应力对髁突软骨细胞主要细胞外基质合成的影响。 方法：采用FX-5000T应力加载系统对第3代大鼠髁突软骨细胞分别施加0,1,6,12和24 h的周期性张应力,应力刺激强度为10% 1 Hz。加力完成后收集加力细胞,提取总RNA反转录成cDNA,应用RT-PCR技术检测软骨细胞主要细胞外基质Ⅱ型胶原和聚集蛋白聚糖的表达变化情况。 结果与结论：与对照组(0 h组)相比,加力   6 h时Ⅱ型胶原和聚集蛋白聚糖表达均显著增加(P < 0.05);加力12 h时Ⅱ型胶原和聚集蛋白聚糖表达均开始下降;当加力至24 h时二者表达量均显著降低(P < 0.05)。结果表明：周期性张应力可以影响髁突软骨细胞主要细胞外基质的合成,随加力时间的延长基质合成逐渐增强;进一步延长加力时间,基质的合成受到明显抑制。      

细胞应变装置中硅橡胶膜的应力分析

目的对自行开发的动态机械应变细胞加载仪中的弹性底培养皿的硅橡胶膜的应力分布情况进行分析,从而明确膜上各部位细胞所处的应力环境,为进一步探讨应力对细胞生长的影响提供理论依据。方法应用三维有限元软件ANSYS8.0对硅橡胶膜加载后的应力和位移情况进行分析,根据有限元的轴对称原则进行建模,并进行边缘约束和位移加载,从而模拟硅橡胶膜接受球冠顶板的拉伸后所产生的应力变化。结果通过有限元分析得出了硅橡胶膜在牵张应变时的von Mises应力云图、位移云图及应力数值。结论1%、5%和10%应变组中,在膜的边缘区和中央区之间的环形区的应力值要低于前两个区,并且其应力值颜色无突变性。在20%和25%应变组中,中央区和环形区的应力值比较接近,但是从应力值看中央区仍稍高于环形区。     

间歇张应力对骨质疏松大鼠骨髓基质干细胞骨向分化中细胞骨架的影响

背景:细胞骨架在力学信号的传递中发挥着重要作用,间歇张应力可促进骨髓间充质干细胞的骨向分化,但关于骨质疏松大鼠骨髓间充质干细胞的细胞骨架在间歇张应力作用下骨向分化中的变化尚无相关报道。目的:探讨间歇性张应力在骨质疏松大鼠骨髓间充质干细胞骨向分化过程中对细胞骨架的影响。方法:建立大鼠骨质疏松模型,提取骨髓间充质干细胞进行体外培养,用FX-4000T Flexcell对细胞施加不同强度的间歇性张应力(5%,10%,15%),对照组不予施力,碱性磷酸酶染色明确骨向分化情况,激光共聚焦显微镜观察并应用Image ProPlus6.0软件进行分析,测定细胞面积、长宽比及骨架蛋白F-actin的积分荧光强度。结果与结论:力学作用下,骨质疏松大鼠骨髓间充质干细胞的排列基本垂直于加力方向,所选幅度皆可促进细胞骨向分化,以10%应变组碱性磷酸酶染色最深,15%张应力组细胞排列出现连续性中断。加力后,细胞骨架发生适应性改建,F-actin纤维束平行排列似栅栏状。图像分析显示:间歇性张应力刺激下,骨质疏松大鼠骨髓间充质干细胞面积减小(10%,15%应变组)、长宽比增加(10%,15%应变组)、F-actin表达量增加(5%,10%,15%应变组),与对照组比较差异有显著性意义(P0.05)。由此可得,力学刺激下,在骨质疏松大鼠骨髓间充质干细胞骨向分化的过程中,细胞骨架结构发生了相应改建。     

Cardiac looping in experimental conditions: effects of extraembryonic forces.

The chick embryo is a popular experimental model used to study the mechanisms of cardiac looping. To facilitate oxygen transport, researchers typically culture the embryo on the surface of the medium. Such preparations, however, expose the embryo and the heart to surface tension that is not present in ovo. This study investigates the influence that surface and extraembryonic membrane tensions have on looping morphology. To eliminate surface tension, we developed a technique in which the embryo is cultured under a thin layer of fluid. To eliminate membrane tension, the membrane was removed. Our results show that both tensions can affect looping, with surface tension potentially having a much greater effect. Moreover, we show that surface tension can alter results in one classic looping experiment.     

Evaluation of Lateral Mechanical Tension in Thin-Film Tissue Constructs

Fibroblast-populated collagen matrices provide a simplified tissue model for wound healing and development processes. A technology (CELLDRUM Technology) evaluating lateral mechanical tension in fibroblast-populated collagen matrices (tissue constructs) with a thickness of 1 mm was introduced. Defined mechanical boundary conditions together with the known number and orientation of the cells revealed precise data on the average tension exerted by a single cell. Circular cell-populated collagen gels were manufactured inside the CELLDRUM on top of a flexible membrane. The collagen matrix was then excited by a sound pulse. The resulting resonance oscillation was monitored by a laser-based deflection sensor and frequency and damping were analyzed giving information on mechanical properties of the tissue construct. Several evaluation experiments were performed. Calf serum enhanced contractile forces of fibroblasts dose dependently. After the gels were treated with cytochalasin D for 24 h, the cell forces were reduced by 42% of control. The remaining tension was attributed to the extracellular matrix remodeling occurring during cell growth and to other cytoskeletal structures like microtubules and intermediate filaments. We also found that only after a few hours of culture fibroblast-seeded collagen gels began developing significant mechanical tension. A mechanical tension profile of proliferating fibroblasts in collagen gels over culture time was obtained.     

Noradrenaline-induced depolarization is smaller in isobaric compared to isometric preparations of rat mesenteric small arteries

The hypothesis was tested that wall tension can influence the membrane potential response to noradrenaline (NA) using isometric and isobaric vessel preparations of rat mesenteric small arteries. The resting membrane potential was significantly less negative in the isobaric (–49.7±0.5 mV, S.E.M., n=12 vessels) compared to the isometric preparation (–56.1±0.7 mV, n=10), although there was no difference in wall tension. The depolarization induced by 10^–5 M NA was 2.6-fold smaller in the isobaric preparation, where wall tension decreased, compared to the isometric preparation, where wall tension increased. Since wall tension decreases under isobaric conditions, but increases under isometric conditions, the latter finding can be explained by assuming that part of the NA-induced membrane potential change is wall tension dependent.     

Studies of the contractility of mammalian myocardium at low rates of stimulation.

1. Measurements have been made of tension development in papillary muscles isolated from the right ventricles of young cats. In some cases membrane potentials have also been recorded, using micro electrodes. 2. Regular contractions at a stimulation rate of 20 min(-1) (the 'standard' rate used in this study) had the following characteristics (30 degrees C): peak tension developed, about 43mN mm(-2); time to peak tension and time to 80% repolarization of the cell membrane, about 400 ms. 3. The corresponding figures for the first contraction after a rest of several minutes (rested state contraction) were: tension developed, about 4mN mm(-2); time to peak tension and time to 80% repolarization of the cell membrane, about 560 ms. Sometimes there was also an early peak in the mechanical response, about 250 ms after stimulation. 4. The time course with which tension development declined when the muscle was allowed to rest was examined under various conditions. It was found to decline more slowly when the muscle was potentiated by raising the bathing Ca2+ concentration and by stimulation at rates above 20 min(-1). 5. Tension development in rested state contractions was found to depend on the Ca2+ and Na+ concentrations in the bathing solution. The full effect of a change in either could be produced by exposing the resting muscle to the altered ionic conditions. 6. These experimental findings have been interpreted in terms of a simple model of the calcium movements involved in excitation-contraction coupling in the myocardial cell.     

高压氧与张力下坐骨神经缝合后的神经再生

本文用QTM520全自动图像分析仪和人工计数方法,观察了30只SD雄性大鼠坐骨神经左侧横断无张力外膜缝合和右侧切除3mm有张力缝合后经高压氧治疗有髓纤维再生情况。发现高压氧不但能促进神经再生,而且可以克服一定程度张力对神经再生的阻碍作用。实验为临床周围神经损伤的手术和治疗提供了新的可能,有深入研究的价值。     

Mechanoregulation of valvular interstitial cell phenotype in the third dimension

A quantitative understanding of the complex interactions between cells, soluble factors, and the biological and mechanical properties of biomaterials is required to guide cell remodeling toward regeneration of healthy tissue rather than fibrocontractive tissue. In the present study, we characterized the combined effects of boundary stiffness and transforming growth factor-β1 (TGF-β1) on cell-generated forces and collagen accumulation. We first generated a quantitative map of cell-generated tension in response to these factors by culturing valvular interstitial cells (VICs) within micro-scale fibrin gels between compliant posts (0.15–1.05 nN/nm) in chemically-defined media with TGF-β1 (0–5 ng/mL). The VICs generated 100–3000 nN/cell after one week of culture, and multiple regression modeling demonstrated, for the first time, quantitative interaction (synergy) between these factors in a three-dimensional culture system. We then isolated passive and active components of tension within the micro-tissues and found that cells cultured with high levels of stiffness and TGF-β1 expressed myofibroblast markers and generated substantial residual tension in the matrix yet, surprisingly, were not able to generate additional tension in response to membrane depolarization signifying a state of continual maximal contraction. In contrast, negligible residual tension was stored in the low stiffness and TGF-β1 groups indicating a lower potential for shrinkage upon release. We then studied if ECM could be generated under the low tension environment and found that TGF-β1, but not EGF, increased de novo collagen accumulation in both low and high tension environments roughly equally. Combined, these findings suggest that isometric cell force, passive retraction, and collagen production can be tuned by independently altering boundary stiffness and TGF-β1 concentration. The ability to stimulate matrix production without inducing high active tension will aid in the development of robust tissue engineered heart valves and other connective tissue replacements where minimizing tissue shrinkage upon implantation is critical.     

Effect of low temperature on the membrane currents and tension components of bullfrog atrial muscle.

In order to clarify the nature of inotropic action of low temperature, the effects of cooling on the membrane currents and tension components were studied on the bullfrog atrial muscle under voltage clamped and unclamped conditions with double gap method. Cooling (in between 35 degrees-7 degrees C) produced an increase of overshoot and a prolongation of the action potential accompanied by a slight depolarization of the membrane, a decrease of basal tension and an increase of twitch contraction. Under voltage clamp, a marked augmentation of contraction also occurred despite a decrease of basal tension, suggesting that the inotropic effect of cooling is not merely dependent on the prolongation of action potential. When the components of membrane current and tension were isolated in modified Ringer solutions, it became clear that ICa and ICa -dependent tension markedly increased at low temperature, while all other currents (INaf, INas, IK1 Ix) and ICa-independent tension decreased. Leaky membrane current (I1) for hyperpolarizing pulses also diminished. Temperature coefficient (Q10) of the ICa-independent tension component was 1.2-1.5 between 7 degrees C and 17 degrees C, while that of ICa-dependent tension varied depending on depolarization voltages. These data were discussed in relation to possible alteration of Ca concentration at outer and inner layers of the membrane which may depend on temperature.     

Excitation-contraction coupling in frog ventricle

In frog ventricular muscle generation of tension was found to be under direct and continuous control of membrane potential. No phasic component of tension was found at any membrane potential. Developed tension depended only on the duration and amplitude of depolarization and was independent of previous contractile history. Developed tension, in part, depended on Ca²⁺ influx through a slowly inactivating component of I_si. Using long voltage clamp steps to achieve steady-state tension, no decline or reversal of developed tension was found atE _Ca. Increasing the [Ca]_o shifted the tension-voltage relation to more negative potentials and increased the net outward current at potentials positive to ?10 mV. The increase in tension seemed to be related to the increase in outward membrane current and K⁺ efflux, as estimated from post clamp K⁺ accumulations. Increasing [K]_o, either by clamp-induced K⁺-accumulation or by increasing the [K] of the bathing solution, decreased the developed tension. These results suggest that in frog ventricular muscle Ca²⁺ for activation of tension is transported primarily from the extracellular space. There was no trigger-release of internal stores or recirculation of sequestered Ca²⁺. Activator Ca²⁺ was transported in part by a slowly inactivating I_si channel and a coupled transport mechanism. The exact mechanism by which Ca²⁺ transport and K⁺ efflux were related could not be identified.     

Low Oxygen Tension is Critical for the Culture of Human Mesenchymal Stem Cells with Strong Osteogenic Potential from Haemarthrosis Fluid

Satisfactory osseous tissue integration of the soft tissue graft with bone is the mainstay of healing following surgical reconstruction of the anterior cruciate ligament (ACL). However, tissue remodelling is slow and significantly impacts on quality of life by delaying return to work and sport and accelerating the onset of degenerative diseases such as osteoarthritis. Delivery of multipotent human mesenchymal stem cells (hMSCs) at surgery could enhance osseous tissue integration. We aim to use hMSCs derived from haemarthrosis fluid (HF) (the intra-articular bleed accrued post-trauma) which is aspirated and discarded as clinical waste. With the aim of improving our bioprocessing methodologies for clinical translation we have investigated the effect of low oxygen tension on the derivation and osteogenic potential of this novel HF-hMSC population. Mononuclear cells were isolated from HF aspirated samples and divided for derivation and culture under normal or low oxygen tension. HF-hMSCs were derived from 100 % of cultures under low oxygen tension compared to 71 % for normal oxygen tension; this was coupled with increased CFU-Fs. We investigated the osteogenic potential and cellular health of HF-hMSC populations following ex vivo expansion. HF-hMSC populations showed enhanced matrix mineralisation and cellular health when differentiated under low oxygen tension. This positive effect of low oxygen on osteogenesis and cellular health was reduced with prolonged culture. These data demonstrate that derivation and culture of HF-hMSC populations under low oxygen tension will enable the translation of a cellular therapy for the treatment of broad patient numbers with optimal osteogenic potency and cellular vitality.