Engineering a novel three-dimensional contractile myocardial patch with cell sheets and decellularised matrix

Objectives: A persistent problem in generating a functional myocardial patch is maintaining contractions in a thicker construct. Thus far, we have successfully created contracting constructs with a defined directionality by seeding neonatal rat cardiomyocytes (CMs) on decellularised porcine small-intestinal submucosa (SIS). Here, we report our efforts in generating a thicker contracting construct by combining CM cell sheets with CM-seeded SIS. Methods: Porcine SIS was decellularised, opened along the longitudinal axis, fixed in a metal frame (45 mm x 25 mm) and seeded onto the submucosal side with neonatal rat CMs at a density of 1.8 x 10(5) cells cm(-2). CM sheets were prepared using temperature-responsive dishes by seeding CMs at a density of 4.0 x 10(5) cells cm-2. Three days after CM seeding, one- or three-layered CMs sheet(s) were stacked onto seeded SIS. Construct contraction was observed for an additional 10 days followed by histological analysis. Results: Stacked CM sheets contracted spontaneously and synchronously with seeded SIS after adherence. A large portion of analysed constructs showed a defined contraction direction, parallel to the longitudinal axis (seeded SIS: 83%, seeded SIS + 1 sheet: 70%, seeded SIS + 3 layered sheets: 71%). This finding was in agreement to the histological finding of aligned CMs parallel to the longitudinal axis. The thickness of seeded SIS with and without three-layered sheets was approximately 800 mu m and 500 mu m, respectively. Conclusions: By combining layered CM sheets with CM-seeded SIS, a three-dimensional myocardial patch with contraction in a defined direction was successfully generated. This may represent an intermediate step to a multiple layered, vascularised contractile myocardial graft. (C) 2010 European Association for Cardio-Thoracic Surgery. Published by Elsevier By. All rights reserved.