Cardiovascular diseases remain the leading cause of death worldwide. Our research focuses in building cardiac microtissues that resemble the native heart as closely as possible, in terms of both structure and function. Across its thickness, native myocardium is built of several thin tissue sheaths, and the cardiomyocytes (CMs) in each of the layers are aligned, and thus contract, in a specific direction. Herein we aim at mimicking the laminar architecture of the myocardium, by producing tissue engineered constructs which show a preferential direction of contraction, and can be assembled to achieve relevant tissue thickness.
Fibrous scaffolds with a diamond pattern were manufactured by melt electrospinning writing (MEW) of medical grade polycaprolactone, mechanically characterized and subsequently seeded with a mixture of 90% CMs and 10% cardiac fibroblasts, both obtained from the differentiation of human induced pluripotent stem cells. Samples were kept in culture and fully characterized, and their beating compared to that of other pore geometries.
Myocardial tissue from porcine samples was histologically characterized in order to determine the variation of the fiber orientation from epicardium to endocardium, showing an angle variation of 6.39°/mm. A design with diamond-shaped pores was predicted to produce an in-plane contraction. MEW proved to be a reproducible and accurate method for printing these scaffolds, which showed adequate levels of compliance when mechanically tested. Engineered cardiac microtissues exhibited relevant cardiac-like features, including beating rate, sarcomere length and gene expression, as well as good viability and metabolic activity. When compared to other pore geometries, diamond-patterned scaffolds not only contracted along a preferential direction we had anticipated (that of lower mechanical resistance, i.e., the short axis of the diamonds), but also displayed greater magnitude and velocity of contraction than squared and rectangular scaffolds. Furthermore, optical mapping of the constructs showed better electrophysiological properties for the diamond-patterned samples, with values closer to native human cardiac tissue. Individual constructs could be stacked to a total thickness of ≈800 µm, and showed good cohesion of the distinct layers as well as more complex beating patterns.
In this work we developed a diamond patterned, melt electrospun scaffold, and show how this particular architecture favours the biomimetic contraction of seeded CMs along the short axis of the diamonds. By subsequently stacking several scaffolds with distinctly oriented diamonds, we obtained cardiac microtissues with increased biological representativity, in terms of their thickness, their multi-layered structure, and the varying principal orientation of each of the layers. Constructs demonstrated an adequate performance in vitro and were also tested in vivo, and show great potential for cardiac tissue engineering and regenerative medicine applications.