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. 2011 Jun;5(6):e115-25.
doi: 10.1002/term.377. Epub 2011 Jan 10.

Optimization of electrical stimulation parameters for cardiac tissue engineering

Nina Tandon  1 , Anna Marsano, Robert Maidhof, Leo Wan, Hyoungshin Park, Gordana Vunjak-Novakovic
Affiliations

Optimization of electrical stimulation parameters for cardiac tissue engineering

Nina Tandon et al. J Tissue Eng Regen Med. 2011 Jun.

Abstract

In vitro application of pulsatile electrical stimulation to neonatal rat cardiomyocytes cultured on polymer scaffolds has been shown to improve the functional assembly of cells into contractile engineered cardiac tissues. However, to date, the conditions of electrical stimulation have not been optimized. We have systematically varied the electrode material, amplitude and frequency of stimulation to determine the conditions that are optimal for cardiac tissue engineering. Carbon electrodes, exhibiting the highest charge-injection capacity and producing cardiac tissues with the best structural and contractile properties, were thus used in tissue engineering studies. Engineered cardiac tissues stimulated at 3 V/cm amplitude and 3 Hz frequency had the highest tissue density, the highest concentrations of cardiac troponin-I and connexin-43 and the best-developed contractile behaviour. These findings contribute to defining bioreactor design specifications and electrical stimulation regime for cardiac tissue engineering.

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Figures

Figure 1
Figure 1. Experimental setup and modeling for electrical stimulation bioreactor
Experimental setup: (A) An electrical stimulator generates the pulses which are transmitted to bioreactors located inside an incubator maintained at 37° C. Bioreactors are in form of modified 60 mm diameter Petri dishes outfitted with electrodes 4 cm in length placed 1 cm apart. 3-D collagen scaffolds are placed in between the electrodes. Modeling of electrical fields in bioreactor at the initiation of a 5V pulse: (B) Top view. (C) Cross-sectional view with traces of pathways between electrodes, where Trace A corresponds to the longest and Trace B corresponds to the shortest distance between the two electrodes. (D) Plot of voltage versus distance from the center of the Petri dish for Trace A (blue) and Trace B (pink).
Figure 2
Figure 2. Electrode Material Optimization
(A) Sample bioreactor current-versus-time trace, illustrating the concepts of injected charge (the total amount of charge transduced into the bioreactor during a stimulus pulse) and recovered charge (the amount of charge that was injected into the system via reversible processes). (B) Current traces for a 5 V/cm, 2 ms monophasic square wave pulse delivered to bioreactors with electrodes 4 cm in length placed 1 cm apart but differing electrode materials (carbon, stainless steel, titanium-nitride coated titanium, and titanium). (C, D) Contractile activity of tissue-engineered cardiac constructs cultured either under conditions of no stimulation (control) or with pulsatile electric-field stimulation (square-wave monophasic pulses, amplitude 5V/cm, duration 2 ms) for 5 days with electrodes of varying materials (C) Excitation threshold (electrical field voltage gradient that needs to be applied to induce synchronous contractions of cultured tissue constructs) (D) maximum capture rate (the maximum frequency at which tissue constructs can be induced to beat). * significantly different with one-way ANOVA test (p<0.05) n=3 stimulated, from one experiment; n=9 control, from two experiments.
Figure 3
Figure 3. Optimization of stimulation amplitude
Contractile activity of tissue-engineered cardiac constructs cultured either without stimulation (control) or with pulsatile stimulation (square-wave monophasic pulses, duration 2 ms) for 5 days with increasing amplitudes of stimulation. Pink shading indicates the range of applied electrical stimulation regimes identified as having produced enhanced performance with respect to the particular metric (i.e. excitation threshold, maximum capture rate, amplitude of contraction: see discussion for details) (A) Excitation threshold (electrical field that needs to be applied to induce synchronous contractions of cultured tissue constructs) (B) maximum capture rate (the maximum frequency at which tissue constructs can be induced to beat). (C) Amplitude of contraction. * significantly different from control group with one-way ANOVA test (p<0.05) n=5–10 each group from 2 separate experiments.
Figure 4
Figure 4. Optimization of stimulation frequency
Contractile activity of tissue-engineered cardiac constructs cultured either under conditions of no stimulation (control) or with pulsatile electric-field stimulation (square-wave monophasic pulses, duration 2 ms) for 5 days with increasing frequencies of stimulation (A) Excitation threshold (electrical field that needs to be applied to induce synchronous contractions of cultured tissue constructs) (B) maximum capture rate (the maximum frequency at which tissue constructs can be induced to beat). (C) Amplitude of contraction ¥ significantly different from 5 V/cm group, * significantly different from control group, § significantly different from all other groups with one-way ANOVA test (p<0.05) n=5–10 from 3 separate experiments. (D) Strength-Duration Curve for control (blue) and stimulated (pink) constructs (n=3 each group).
Figure 5
Figure 5. Strain Analysis
Data are shown for a typical stimulated and control cardiac tissue constructs during relaxed and contracted states in a sample contraction. Dark blue corresponds to 0 % contraction, and red to 4% contraction. Scale bar corresponds to 3 mm.
Figure 6
Figure 6. Tissue structure
(A, B) Hematoxylin and Eosin staining of engineered tissue consttructs that were (A) unstimulated, or (B) stimulated with monophasic square wave pulses of 3 V amplitude, 3 Hz frequency and 2 ms duration. (Scale bar indicates 1 mm). (C, D) Tramsmission electron microscopy images of engineered tissue either (C) unstimulated during culture, or (D) stimulated with monophasic square wave pulses of 3 V amplitude, 3 Hz frequency and 2 ms duration, with insets of sarcomeres (scale bar indicates 2 um in main image, 500 nm in inset). (E) Length of intercalated disc. (F) Distance between desmosomes per each unit of intercalated disc (E, F) * p < 0.06 denotes statistical difference via Wilcoxon-Mann-Whitney U-test (n=4 each group) (G, H) Protein analysis of stimulated and control tissue for selected cardiac proteins. (G) Western blot for Connexin-43 (Cx-43), cardiac troponin-I, beta myosin heavy chain (β-MHC), alpha myosin heavy chain (α -MHC), muscle-type creatine kinase (CK-MM), and β-actin. (H) Band intensity as quantified by image analysis. * p<0.06 denotes statistical difference via Wilcoxon-Mann-Whitney U-test (n=2 control, n=3 stimulated).
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