BIOMECHANICS OF ENGINEERED SMOOTH MUSCLE MICROTISSUES

Mechanical forces play a large role in biological processes. While individual cells are the basic unit of force generation in organisms, it is only at the tissue level of organization where the interplay between groups of cells and the surrounding extracellular matrix (ECM) can be studied. To investigate how mechanical forces affect tissues, a microfabricated substrate consisting of two flexible cantilevers was used in order to simultaneously apply forces to and measure responses from engineered microtissues. These constructs consisted of a few hundred bovine pulmonary smooth muscle cells contained in a collagen/fibrin matrix which spanned the gap between the two cantilevers. A nickel microsphere bonded to the top of one of the cantilevers enabled the application of force to the system through the transduction of a magnetic field generated by a magnetic tweezer apparatus. To investigate the mechanical properties of these microtissues, a quasistatic stretching protocol was applied and the tissues’ stress-strain response was measured. The ECM component of this response was ascertained by lysing the cells in select microtissues before applying the stretching protocol. A mathematical model was developed to help quantify these microtissue mechanical properties, and it was found that the cells contribute the bulk of the viscoelastic response while the ECM contributes most of the viscoplastic response. Notably, the presence of the cells seems to shield the microtissue from yielding under tension. The effect of periodic forces was also investigated. Successive quasistatic actuations as well as continuous cyclic strains were applied to the microtissues. The results from the repeated quasistatic stretches showed an active reinforcement of tissue stiffness following the first stretch, revealed a time scale of approximately 10 minutes during which the microtissues reverted to their pre-stretch states, and reinforced the dichotomy between the cell and ECM contributions to microtissue mechanical properties. Continuous cyclic stimulation led to two distinct initial responses from the microtissues: a relatively linear viscoelastic response as well as a strain-stiffening response. The strain-stiffening of the tissue diminished over the course of the continuous stimulation and microtissue behavior converged, indicating an active response. In order to apply magnetic forces to multiple microtissues simultaneously, a microfabricated device was designed and validated, and it was shown to reproduce the amount of force generated by the magnetic tweezer. Lastly, human embryonic stem cell-derived cardiomyocytes were used to construct microtissues, and they were able to mimic the pacing and Frank-Starling characteristics of native cardiac tissue. By elucidating the interplay between cell and extracellular matrix biomechanics, the experimental work reported with these microtissues provides new understanding of fundamental biophysical properties of cells and tissues and can potentially provide new input for applications such as tissue engineering and drug discovery and testing. Advisor: Daniel H. Reic