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 × 25 mm) and seeded onto the submucosal side with neonatal rat CMs at a density of 1.8 × 10(5) cells cm(-2). CM sheets were prepared using temperature-responsive dishes by seeding CMs at a density of 4.0 × 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 μm and 500 μm, 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.