Cardiac tissue engineering could be valuable for the reconstruction of malformed hearts in children, and the regeneration of failing hearts in adults. In addition, cardiac tissue engineering can allow the testing of new drugs, and the study of general cardiac tissue development and function. A biomimetic approach to cardiac tissue engineering attempts to recapitulate any number of aspects of the in vivo environment, including 3-dimensional structure and/or biochemical cues, and physical forces (electrical and/or mechanical). Recent studies investigating the possible role of biomimetic electrical stimulation in cardiac tissue assembly led us to hypothesize that cells exhibit differential responses when subjected to electrical stimulation and this response is not only dependent on signal parameters such as amplitude and frequency, but also on certain bioreactor characteristics such as electrode material and geometry.
In this thesis work, studies were performed at both relevant stages of maturity (development and adult) as well as with both pulsatile and direct-current biomimetic electrical stimulation. Rational experimental design principles were first developed for the electrodes and electrical stimulation regime for cardiac tissue engineering. Electrode geometries and protocols were optimized and a safety range of 8 V/cm was identified. To optimize electrical stimulation conditions for 3-dimensional culture of neonatal rat cardiomyocytes, electrode material, amplitude and frequency of stimulation were varied. Conditions of electrical stimulation of amplitude 3 V/cm, frequency 3 Hz and stimulated with carbon graphite electrodes produced cardiac tissues with the best functional properties, and produced denser tissue, higher concentrations of cardiac troponin-I and connexin-43, and more uniform contractions of higher amplitude compared to control.
To investigate the effects of pulsatile stimulation on human adipose-derived stem cells (hASCs), a microscale culture system incorporating an interdigitated microarray of electrodes composed of excimer-laser-ablated indium tin oxide was developed, validated with neonatal rat cardiomyocytes, and used to stimulate cultures of hASCs. hASCs grown in this system and exposed to electrical stimulation exhibited higher proliferation, higher amounts of Connexin-43, and elongated in a direction perpendicular to the applied electric field.
To investigate the use of single pulses of electrical stimulation to stimulate cardiogenesis in embryoid bodies (EBs) derived from human embryonic stem cells via stimulation of intracellular reactive oxygen species, electrode material, length of stimulus, and age of EBs at the onset of electrical stimulation were varied. The highest rate of ROS generation was observed for stainless steel electrodes, signal duration of 90 s and 4-day old EBs, and cardiac differentiation was substantially enhanced by electrical stimulation, as evidenced by levels of spontaneous contractions, expression of Troponin T, and sarcomeric organization.
Finally the use of direct current electric fields (such as those present during embryonic development and wound healing) for human adipose tissue-derived stem cells derived from lipoaspirate (hASCs) or human epicardial fat (heASCs) was investigated. Upon stimulation, stem cells derived both from liposuction aspirates and from cardiac fat were observed to demonstrate morphological and phenotypic changes including alignment, elongation, and gene expression.