Cell-Induced Alignment Augments Twitch Force in Fibrin Gel–Based Engineered Myocardium via Gap Junction Modification

Abstract

A high-potential therapy for repairing the heart post-myocardial infarction is the implantation of tissueengineered myocardium. While several groups have developed constructs that mimic the aligned structure of the native myocardium, to date no one has investigated the particular functional benefits conferred by alignment. In this study we created myocardial constructs in both aligned and isotropic configurations by entrapping neonatal rat cardiac cells in fibrin gel. Constructs were cultured statically for 2 weeks, and then characterized. Histological staining showed spread cells that express typical cardiac cell markers in both configurations. Isotropic constructs had higher final cell and collagen densities, but lower passive mechanical properties than aligned constructs. Twitch force associated with electrical pacing, however, was 181% higher in aligned constructs, and this improvement was greater than what would be expected from merely aligning the cells in the isotropic constructs in the force measurement direction. Our hypothesis was that this was due to improved gap junction formation=function facilitated by cell alignment, and further analyses of the twitch force data, as well as Western blot results of connexin 43 expression and phosphorylation state, support this hypothesis. Regardless of the specific mechanism, the results presented in this study underscore the importance of recapitulating the anisotropy of the native tissue in engineered myocardium.

Tissue Contraction Force Microscopy for Optimization of Engineered Cardiac Tissue

“…Finally, constrained gel compaction is the primary effector of cell and fiber alignment in threedimensional engineered tissues, and comparisons between aligned and isotropic ECTs show that alignment leads to increased twitch force. 16 All these factors associated with differential gel thickness can and will play into the contractile force measured for gel-based ECTs by any method [16][17][18][19][20][21][22] and need to be considered when interpreting the data.…”

“…This is possible because dishes are coated with the same mass of PDMS-microsphere mixture and experiments are cast using dishes from the same batch of PDMS. To verify that the relative comparisons of beat strength are valid, the twitch force produced by our hemispherical constructs was measured on a modified, custom-built force testing system 16 (Fig. 1B).…”

“…Tissue-level assays have also been developed and include free-floating tissue samples, as well as anchored tissues of both macro-and microscales that have proven useful for investigating the role of alignment, 16 matrix stiffness, 17 and matrix composition. 18 However, free-floating assays do not measure cell forces directly, instead rely on theory to relate the tissue compaction to cell forces.…”

“…18 However, free-floating assays do not measure cell forces directly, instead rely on theory to relate the tissue compaction to cell forces. 19,20 In addition, while macrosized tissues 16 are easy to prepare and conducive to force measurement without a highly specialized equipment, they are low throughput, and screening many factors quickly becomes cumbersome and taxing on resources. Conversely, microsized tissues enable high-throughput screening but generally require microfabrication 17,21 or the use of specialized equipment.…”

“…Since fibrin gel is a common choice for ECTs, 16,23,24 to demonstrate the utility of this approach, we compared microsphere displacements generated by ECTs fabricated from hiPSC-derived cardiomyocytes (hiPSC-CMs) in fibrin gels at two different fibrin concentrations as well as two different cell concentrations and then directly measured the forces in the same samples as a validation of the assay. These ECTs were also characterized histologically to provide insight as to how gel compaction might affect the measured bead displacements.…”