Sulgin A A, Sidorova T N, Sidorov V Y
Siberian State Medical University, Moskovsky tract, Tomsk, 634050, Russia.
Division of Allergy, Pulmonary and Critical Care Medicine, Vanderbilt University Medical Center, 1211 Medical Center Dr, Nashville, 37232, TN, USA.
Biulleten Sib Meditsiny. 2020;19(2):85-95. doi: 10.20538/1682-0363-2020-2-85-95.
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.
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.
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.
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.
优化一种生物工程化的“I-Wire”平台，用于培养源自冠状动脉平滑肌细胞的组织工程构建体（TCs），并表征所培养TCs的机械弹性特性。
将基于纤维蛋白原的细胞混合物用移液管移至一个铸模中，该铸模有两根平行的钛锚定丝插入铸模相对两端的凹槽中以支撑TCs。铸模深度为3毫米，宽度为2毫米，长度为12毫米。为测量TCs的变形，使用了直径为365微米、长度为42毫米的柔性探针。利用倒置显微镜光学记录系统记录在对TCs施加不同拉力时探针尖端的偏转。基于为每个TC重建的拉伸应力图计算弹性模量。评估了对照TCs以及在异丙肾上腺素（Iso）、乙酰胆碱（ACh）、blebbistatin（Bb）和细胞松弛素D（Cyto-D）影响下的TCs的机械弹性特性。对平滑肌α-肌动蛋白、结蛋白和细胞核进行免疫组织化学染色，以对TCs进行结构表征。
在培养的第5 - 6天形成了TCs。在随后的7天内进行的后续测量未发现弹性有显著变化。在TCs形成后的培养第一天，弹性模量值为7.4±1.5千帕，第三天为7.9±1.4千帕，第七天为7.8±1.9千帕。在随后施加Bb和Cyto-D后，TCs机械弹性特性的变化呈两相模式，表明TC弹性的主动和被动成分可能存在分离。施加1微摩尔的Iso导致弹性模量值从7.9±1.5千帕增加到10.2±2.1千帕（p<0.05，n = 6）。ACh未引起弹性的显著变化。
该系统能够对TCs在药理学刺激下的机械弹性特性进行量化，可用于模拟血管平滑肌细胞的病理变化。
