Biulleten' Sibirskoi meditsiny最新文献

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GROWTH AND CHARACTERIZATION OF A TISSUE-ENGINEERED CONSTRUCT FROM HUMAN CORONARY ARTERY SMOOTH MUSCLE CELLS. 人冠状动脉平滑肌细胞组织工程构建物的生长和表征。

Biulleten' Sibirskoi meditsiny

Pub Date : 2020-01-01 DOI: 10.20538/1682-0363-2020-2-85-95

A A Sulgin, T N Sidorova, V Y Sidorov

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.

目的:优化生物工程“I-Wire”平台，培养冠状动脉平滑肌细胞衍生的组织工程构建体(TCs)，并表征培养的TCs的力学弹性特性。材料和方法:将纤维蛋白原为基础的细胞混合物移液到铸造模具中，在模具两端的凹槽中插入两根平行的钛锚丝来支撑TC。铸型深3毫米，宽2毫米，长12毫米。为了测量TC变形，使用了一个直径为365 mk，长度为42 mm的柔性探头。利用倒置显微镜光学记录系统记录了在不同拉力作用下探针尖端的挠度。根据重建的拉伸-应力图计算弹性模量。在异丙肾上腺素(Iso)、乙酰胆碱(ACh)、blebbistatin (Bb)和细胞松弛素D (Cyto-D)的影响下，评价了对照TCs和TCs的力学弹性性能。采用平滑肌α-肌动蛋白、desmin和细胞核免疫组化染色对TCs进行结构表征。结果:TCs在孵育5 ~ 6 d形成。随后7天的测量没有发现弹性的显著变化。弹性模量第1天为7.4±1.5 kPa，第3天为7.9±1.4 kPa，第7天为7.8±1.9 kPa。随后应用Bb和Cyto-D后，TC的力学弹性性能发生了两相变化，表明TC弹性的主动和被动元素可能分离。应用1 μM的Iso可使TCs的弹性模量从7.9±1.5 kPa增加到10.2±2.1 kPa(结论:该系统可量化TCs对药物刺激的力学弹性特性，可用于血管平滑肌细胞病理变化的建模。

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