GROWTH AND CHARACTERIZATION OF A TISSUE-ENGINEERED CONSTRUCT FROM HUMAN CORONARY ARTERY SMOOTH MUSCLE CELLS.
GROWTH AND CHARACTERIZATION OF A TISSUE-ENGINEERED CONSTRUCT FROM HUMAN CORONARY ARTERY SMOOTH MUSCLE CELLS.
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DOI：
10.20538/1682-0363-2020-2-85-95
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发表时间：
2020-07
期刊：
影响因子：
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通讯作者：
A. A. Sulgin-A.;T. Sidorova;V. Sidorov
中科院分区：
文献类型：
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作者：
A. A. Sulgin-A.;T. Sidorova;V. Sidorov
目的优化生物工程“I-Wire”平台，用于冠状动脉平滑肌细胞来源的组织工程构建物（tissue-engineered constructs，TC）的生长，并表征其力学弹性特性。材料和方法将基于纤维蛋白原的细胞混合物移液到铸模中，所述铸模具有两个平行的钛锚定丝，所述钛锚定丝插入到所述铸模的相对端上的凹槽中以支撑TC。铸模的深度为3 mm，宽度为2 mm，长度为12 mm。为了测量TC变形，使用直径为365 mm、长度为42 mm的柔性探针。使用倒置显微镜光学记录系统记录在施加到TC的各种张力下探针尖端的偏转。根据每个TC重建的拉伸-应力图计算弹性模量。评价对照TC和在异丙肾上腺素（Iso）、乙酰胆碱（ACh）、blebbistatin（Bb）和细胞松弛素D（Cyto-D）影响下的TC的机械弹性性质。平滑肌α-肌动蛋白、结蛋白和细胞核的免疫组织化学染色用于TC的结构表征。结果培养第5-6天形成TC。在随后的7天内进行的后续测量未显示弹性的显著变化。培养第1天、第3天和第7天的弹性模量分别为7.4 ± 1.5kPa、7.9 ± 1.4kPa和7.8 ± 1.9kPa。在随后的应用Bb和Cyto-D的TC的机械弹性性能的变化有两个阶段的模式，表明可能的分离的TC弹性的主动和被动元素。应用1 μM Iso导致弹性模量值从7.9 ± 1.5 kPa增加至10.2 ± 2.1 kPa（p<0.05，n = 6）。乙酰胆碱没有引起弹性的显着变化。结论该系统可以定量分析药物刺激下TC的力学弹性特性，可用于血管平滑肌细胞病理变化的模拟。
Objective To optimize a bioengineered «I-Wire» platform to grow tissue-engineered constructs (TCs) derived from coronary artery smooth muscle cells and characterize the mechano-elastic properties of the grown TCs. Materials and Methods A fibrinogen-based cell mixture was pipetted in a casting mold having two parallel titanium anchoring wires inserted in the grooves on opposite ends of the mold to support the TC. The casting mold was 3 mm in depth, 2 mm in width and 12 mm in length. To measure TC deformation, a flexible probe with a diameter of 365 mk and a length of 42 mm was utilized. The deflection of the probe tip at various tensile forces applied to the TC was recorded using an inverted microscope optical recording system. The elasticity modulus was calculated based on a stretch-stress diagram reconstructed for each TC. The mechano-elastic properties of control TCs and TCs under the influence of isoproterenol (Iso), acetylcholine (ACh), blebbistatin (Bb) and cytochalasin D (Cyto-D) were evaluated. Immunohistochemical staining of smooth muscle α-actin, desmin and the cell nucleus was implemented for the structural characterization of the TCs. Results The TCs formed on day 5-6 of incubation. Subsequent measurements during the following 7 days did not reveal significant changes in elasticity. Values of the elastic modulus were 7.4 ± 1.5 kPa at the first day, 7.9 ± 1.4 kPa on the third day, and 7.8 ± 1.9 kPa on the seventh day of culturing after TC formation. Changes in the mechano-elastic properties of the TCs in response to the subsequent application of Bb and Cyto-D had a two-phase pattern, indicating a possible separation of active and passive elements of the TC elasticity. The application of 1 μM of Iso led to an increase in the value of the elastic modulus from 7.9 ± 1.5 kPa to 10.2 ± 2.1 kPa (p<0.05, n = 6). ACh did not cause a significant change in elasticity. Conclusion The system allows quantification of the mechano-elastic properties of TCs in response to pharmacological stimuli and can be useful to model pathological changes in vascular smooth muscle cells.