Design and fabrication of heart muscle using scaffold-based tissue engineering.

Cardiac tissue engineering strategies are based on the development of functional models of heart muscle in vitro. Our research is focused on evaluating the feasibility of different tissue engineering platforms to support the formation of heart muscle. Our previous work was focused on developing three-dimensional (3D) models of heart muscle using self-organization strategies and biodegradable hydrogels. To build on this work, our current study describes a third tissue engineering platform using polymer-based scaffolding technology to engineer functional heart muscle in vitro. Porous scaffolds were fabricated by solubilizing chitosan in dilute glacial acetic acid, transferring the solution to a mold, freezing the mold at -80 degrees C followed by overnight lyophilization. The scaffolds were rehydrated in sodium hydroxide to neutralize the pH, sterilized in 70% ethanol and cellularized using primary cardiac myocytes. Several variables were studied: effect of polymer concentration and chitosan solution volume (i.e., scaffold thickness) on scaffold fabrication, effect of cell number and time in culture on active force generated by cardiomyocyte-seeded scaffolds and the effect of lysozyme on scaffold degradation. Histology (hematoxylin and eosin) and contractility (active, baseline and specific force, electrical pacing) were evaluated for the cellularized constructs under different conditions. We found that a polymer concentration in the range 1.0-2.5% (w/v) was most suitable for scaffold fabrication while a scaffold thickness of 200 microm was optimal for cardiac cell functionality. Direct injection of the cells on the scaffold did not result in contractile constructs due to low cell retention. Fibrin gel was required to retain the cells within the constructs and resulted in the formation of contractile constructs. We found that lower cell seeding densities, in the range of 1-2 million cells, resulted in the formation of contractile heart muscle, termed smart material integrated heart muscle (SMIHMs). Chitosan concentration of 1-2% (w/v) did not have a significant effect on the active twitch force of SMIHMs. We found that scaffold thickness was an important variable and only the thinnest scaffolds evaluated (200 microm) generated any measurable active twitch force upon electrical stimulation. The maximum active force for SMIHMs was found to be 439.5 microN while the maximum baseline force was found to be 2850 microN, obtained after 11 days in culture. Histological evaluation showed a fairly uniform cell distribution throughout the thickness of the scaffold. We found that lysozyme concentration had a profound effect on scaffold degradation with complete scaffold degradation being achieved in 2 h using a lysozyme concentration of 1 mg/mL. Slower degradation times (in the order of weeks) were achieved by decreasing the lysozyme concentration to 0.01 mg/mL. In this study, we provide a detailed description for the formation of contractile 3D heart muscle utilizing scaffold-based methods. We demonstrate the effect of several variables on the formation and culture of SMIHMs.