Abstract 2666: Insulin-like Growth Factor-I And Perfused Culture Independently Enhance Formation Of Tissue Engineered Cardiac Grafts
Tissue engineered cardiac grafts (TECG) have been proposed as a platform for systematic in vitro studies of complex stimuli that occur in vivo, and as a means to repair damaged or congenitally defective myocardium. However, clinical application of TECG is currently limited by suboptimal cardiomyocyte (CM) survival, differentiation, and contractility. We tested the hypothesis that insulin-like growth factor-I (IGF) and perfused culture can enhance formation of TECG.
Method: Three-dimensional porous scaffolds were placed individually in either Petri dishes or the chamber of a novel modular, perfused bioreactor, seeded with primary neonatal rat heart cells, and cultured in 10 mL of media supplemented or not with 100 ng/mL of IGF for 8 days. Two-factor ANOVA was used to assess significant effects of IGF and perfusion acting individually and in concert.
Results: IGF significantly enhanced contractile properties of TECG compared to unsupplemented controls, as assessed by contractile amplitude (p<0.05) and excitation threshold (p<0.01); IGF also significantly increased the amount of cardiac troponin-I (Tn-I) in TECG, as assessed by Western blot (p<0.01). Perfusion significantly enhanced CM survival and metabolic activity in TECG compared to non-perfused controls, as assessed by TUNEL (p<0.01) and MTT (p<0.01) assays. CM elongation and myofibrillogenesis were readily observed in perfused TECG but were rare without perfusion, as assessed by immunostaining for cardiac Tn-I. Spontaneous contractility was present only in perfused TECG, although contractions could be induced by electrical field stimulation of TECG cultured both with and without perfusion. In concert, perfused culture in IGF-supplemented media significantly increased contractile amplitude by 450% and decreased excitation threshold by 50%, as compared to static culture in unsupplemented media. Effects of perfused culture and IGF on TECG were significant (p<0.0001 for amplitude; p<0.005 for threshold), independent, and additive, suggesting at least partially different cell activation mechanisms.
Conclusion: Insulin-like growth factor-I and perfusion culture can improve the viability, differentiation, and contractility of cardiomyocytes in tissue engineered cardiac grafts.