Electrical and mechanical stimulation have both been used extensively to improve the function of cardiac engineered tissue as each of these stimuli is present in the physical environment during normal development in vivo. However, to date, there has been no direct comparison between electrical and mechanical stimulation and current published data are difficult to compare due to the different systems used to create the engineered cardiac tissue and the different measures of functionality studied as outcomes. The goals of this study were twofold. First, we sought to directly compare the effects of mechanical and electrical stimulation on engineered cardiac tissue. Second, we aimed to determine the importance of the timing of the two stimuli in relation to each other in combined electromechanical stimulation. We hypothesized that delaying electrical stimulation after the beginning of mechanical stimulation to mimic the biophysical environment present during isovolumic contraction would improve construct function by improving proteins responsible for cell-cell communication and contractility. To test this hypothesis, we created a bioreactor system that would allow us to electromechanically stimulate engineered tissue created from neonatal rat cardiac cells entrapped in fibrin gel during 2 weeks in culture. Contraction force was higher for all stimulation groups as compared with the static controls, with the delayed combined stimulation constructs having the highest forces. Mechanical stimulation alone displayed increased final cell numbers but there were no other differences between electrical and mechanical stimulation alone. Delayed combined stimulation resulted in an increase in SERCA2a and troponin T expression levels, which did not happen with synchronous combined stimulation, indicating that the timing of combined stimulation is important to maximize the beneficial effect. Increases in Akt protein expression levels suggest that the improvements are at least in part induced by hypertrophic growth. In summary, combined electromechanical stimulation can create engineered cardiac tissue with improved functional properties over electrical or mechanical stimulation alone, and the timing of the combined stimulation greatly influences its effects on engineered cardiac tissue.

Multi-material polymer scaffolds with multiscale pore architectures were characterized and tested with vascular and heart cells as part of a platform for replacing damaged heart muscle. Vascular and muscle scaffolds were constructed from a new material, poly(limonene thioether) (PLT32i), which met the design criteria of slow biodegradability, elastomeric mechanical properties, and facile processing. The vascular-parenchymal interface was a poly(glycerol sebacate) (PGS) porous membrane that met different criteria of rapid biodegradability, high oxygen permeance, and high porosity. A hierarchical architecture of primary (macroscale) and secondary (microscale) pores was created by casting the PLT32i prepolymer onto sintered spheres of poly(methyl methacrylate) (PMMA) within precisely patterned molds followed by photocuring, de-molding, and leaching out the PMMA. Pre-fabricated polymer templates were cellularized, assembled, and perfused in order to engineer spatially organized, contractile heart tissue. Structural and functional analyses showed that the primary pores guided heart cell alignment and enabled robust perfusion while the secondary pores increased heart cell retention and reduced polymer volume fraction.

A photocurable thiol-ene network polymer, poly(limonene thioether) (PLT32o), was synthesized, characterized, fabricated into tissue engineering scaffolds, and demonstrated in vitro and in vivo. Micromolded PLT32o grids exhibited compliant, elastomeric mechanical behavior similar to grids made of poly(glycerol sebacate) (PGS), an established biomaterial. Multilayered PL32o scaffolds with regular, geometrically defined pore architectures supported heart cell seeding and culture in a manner similar to multilayered PGS scaffolds. Subcutaneous implantation of multilayered PLT32o scaffolds with cultured heart cells provided long term 3D structural support and retained the exogenous cells, whereas PGS scaffolds lost both their structural integrity and the exogenous cells over 31 days in vivo. PLT32o membrane implants retained their dry mass, whereas PGS implants lost 70 percent of their dry mass by day 31. Macrophages were initially recruited to PLT32o and PGS membrane implants but were no longer present by day 31. Facile synthesis and processing in combination with the capability to support heart cells in vitro and in vivo suggest that PLT32o can offer advantages for tissue engineering applications where prolonged in vivo maintenance of 3D structural integrity and elastomeric mechanical behavior are required.

Mechanical stimulation has been used extensively to improve the function of cardiac engineered tissue, as it mimics the physical environment in which the tissue is situated during normal development. However, previous mechanical stimulation has been carried out under a constant frequency that more closely resembles a diseased heart. The goal of this study was to create a bioreactor system that would allow us to control the mechanical stimulation of engineered cardiac tissue on a cycle-by-cycle basis. This unique system allows us to determine the effects on cardiac construct function of introducing variability to the mechanical stretch. To test our bioreactor system, constructs created from neonatal rat cardiomyocytes entrapped in fibrin hydrogels were stimulated under various regimes for 2 weeks and then assessed for functional outcomes. No differences were observed in the final cell number in each condition, indicating that variability in frequency did not have a negative effect on viability. The forces were higher for all mechanical stimulation groups compared to static controls, although no differences were observed between the mechanically stimulated conditions, indicating that variable frequency on a cycle-by-cycle basis has limited effects on the resulting force. Although differences in the observed twitch force were not observed, differences in the protein expression indicate that variable-frequency mechanical stimulation had an effect on cell-cell coupling and growth pathway activation in the constructs. Thus, this bioreactor system provides a valuable tool for further development and optimization of engineered myocardial tissue as a repair or replacement strategy for patients undergoing heart failure. Copyright © 2014 John Wiley & Sons, Ltd.

This commentary discusses the rationale behind our recently reported work entitled "Mimicking isovolumic contraction with combined electromechanical stimulation improves the development of engineered cardiac constructs," introduces new data supporting our hypothesis, and discusses future applications of our bioreactor system. The ability to stimulate engineered cardiac tissue in a bioreactor system that combines both electrical and mechanical stimulation offers a unique opportunity to simulate the appropriate dynamics between stretch and contraction and model isovolumic contraction in vitro. Our previous study demonstrated that combined electromechanical stimulation that simulated the timing of isovolumic contraction in healthy tissue improved force generation via increased contractile and calcium handling protein expression and improved hypertrophic pathway activation. In new data presented here, we further demonstrate that modification of the timing between electrical and mechanical stimulation to mimic a non-physiological process negatively impacts the functionality of the engineered constructs. We close by exploring the various disease states that have altered timing between the electrical and mechanical stimulation signals as potential future directions for the use of this system.

This chapter details the creation of three-dimensional fibrin hydrogels as an engineered myocardial tissue and introduces a mechanical stretch bioreactor system that allows for the cycle-to-cycle variable amplitude mechanical stretch of the constructs as a method of conditioning the constructs to be more similar to native tissue. Though mechanical stimulation has been established as a standard method of improving construct development, most studies have been performed under constant frequency and constant amplitude, even though variability is a critical aspect of healthy cardiac physiology. The introduction of variability in other organ systems has demonstrated beneficial effects to cell function in vitro. We hypothesize that the introduction of variability in engineered cardiac tissue could have a similar effect.

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