Biohybrid thin films for measuring contractility in engineered cardiovascular muscle

Abstract

In vitro cardiovascular disease models need to recapitulate tissue-scale function in order to provide in vivo relevance. We have developed a new method for measuring the contractility of engineered cardiovascular smooth and striated muscle in vitro during electrical and pharmacological stimulation. We present a growth theory-based finite elasticity analysis for calculating the contractile stresses of a 2D anisotropic muscle tissue cultured on a flexible synthetic polymer thin film. Cardiac muscle engineered with neonatal rat ventricular myocytes and paced at 0.5 Hz generated stresses of 9.2 ± 3.5 kPa at peak systole, similar to measurements of the contractility of papillary muscle from adult rats. Vascular tissue engineered with human umbilical arterial smooth muscle cells maintained a basal contractile tone of 13.1 ± 2.1 kPa and generated another 5.1 ± 0.8 kPa when stimulated with endothelin-1. These data suggest that this method may be useful in assessing the efficacy and safety of pharmacological agents on cardiovascular tissue.

Introduction

Muscle in the cardiovascular system pumps blood and regulates vascular resistance, qualifying the contractile function of these tissues as critically important in development and disease. Currently however, there is no simple and repeatable method for measuring functional mechanical output of cardiovascular (CV) muscle cells in vitro that can be compared with traditional biochemical assays of protein and gene expression. Tissue engineered CV muscle provides a high-fidelity means of studying their diseases and potential therapeutic interventions. Recent reports demonstrate the ability to organize muscle cells into CV tissues that recapitulate a broad range of in vivo functions. Kleber and colleagues [1], [2] first developed patterned cell cultures of ventricular myocytes to study the physics of excitation wave front propagation through custom designed microenvironments. These studies were the first attempts to recapitulate the ventricular microarchitecture in vitro. Later, the application of soft lithography techniques to building 2D anisotropic monolayers of ventricular myocytes by Tung and colleagues [3], [4] allowed for higher fidelity studies of action potential propagation. Comparable approaches, such as growing ventricular myocytes on grooved substrata, have allowed for studies on calcium metabolism in anisotropic cultures [5]. Similarly, vascular smooth muscle cells have been patterned into fibrils and aligned monolayers using grooved substrates [6], [7] to control muscular alignment and differentiation. However, efforts to adapt the same culture methods to measure contractility have yet to emerge due to isometric constraint to a rigid or semi-rigid substrate. Three-dimensional cardiac [8], [9] and vascular [10], [11], [12] tissue engineering techniques enable stress measurement, but require physical attachment to force transducers and cannot direct microscale aligned tissue structure. Thus, we sought to combine the repeatable structures of previous 2-D techniques with the stress measuring capabilities of recent 3-D systems.

Previously, we reported on a muscular thin film (MTF) technique that enables cardiac muscle monolayers, engineered on a thin, flexible 2-D substrate, to undergo 3-D deformation [13]. The MTF is a bilaminate of polydimethylsiloxane (PDMS) and a cell monolayer, whose stress state is indicated by its radius of curvature. To determine the stress in the cell layer, we have developed a new elasticity based analysis that is more accurate and robust than previous analyses [13], [14], [15], enabling concurrent measurement of the orthotropic contraction stress in the muscle and the stress-free shortening that the muscle would undergo if unconstrained. Additionally, we build on the MTF technique by expanding to vascular smooth muscle to create microstructured CV tissues in vitro and directly measure stress generation due to electrical and pharmacological stimulation.