GROWTH AND CHARACTERIZATION OF A TISSUE-ENGINEERED CONSTRUCT FROM HUMAN CORONARY ARTERY SMOOTH MUSCLE CELLS.

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.