Regarding experiments, we acquired a human-induced pluripotent stem cell (hiPSC) and successfully differentiated it into cardiomyocytes (hiPSC-CMs) to 3D bioprint different cardiac constructs. We have used a bio-ink made of gelatine and collagen methacrylate, with the hiPSC-CMs embedded in it, obtaining the highest resolution when printing with FRESH (Freeform Reversible Embedding of Suspended Hydrogels).
To study the growth and remodeling response in tissue-engineered cardiac ventricles (TECV), we have designed an idealized cardiac ventricle as a truncated ellipsoid with a high density of hiPSC-CMs. These cells, cultured for several weeks, grouped in clusters of beating cells that did not form a connected network, providing poor contractility of the TECV. Also, although hiPSC-CMs were positive for cardiac markers, they did not have well-developed sarcomeres. Finally, preliminary optical mapping studies did not show calcium waves due to poor cell connections.
Since these initial constructs had poor contractility and cardiomyocyte connections, we enhanced the cell composition by adding human cardiac fibroblasts (hCFs). Cardiac patches with and without hCFs were bioprinted for cardiac function assessment. We observed stronger cardiac contractions of the patches including hCFs via photographic and video registries at 2, 4, and 6 weeks after printing. Immunostaining analysis showed better-organized sarcomeres and an improved connection between cardiomyocytes of the patches with hCFs, confirming that cardiomyocytes remain isolated in beating clumps in patches with only hiPSC-CMs. Furthermore, the sarcomere length of hiPSC-CMs in the patches with hCFs was higher, indicative of an improved cardiac maturity.
In addition, by determining the elastic properties of patches obtained from TECVs via biaxial testing, we concluded that constructs with cells are stiffer than those without cells and that constructs with and without hCFs present similar stiffness.
The computational development of this project has progressed in parallel with experimental work, focusing primarily on finite element analysis of an idealized left ventricle (LV) with dimensions matching those of the bioprinted tissue-engineered cardiovascular tissues (TECVs). A key challenge has been establishing an initial homeostatic stress state, which is a critical requirement for growth and remodeling (G&R) simulations that remains an ongoing difficulty in the field. To address this, we developed a prestressing approach based on the virtual work balance principle, combined with an iterative algorithm to determine the unstressed configuration of elastin while prescribing pre-stretches for collagen and cardiomyocyte fibers (Figure 6). This method is currently being evaluated by comparing it with alternative algorithms and benchmarking it against analytical solutions.
In parallel, a mechanobiologically equilibrated growth and remodeling constitutive and computational model has been extended from the vascular to the cardiac settings and has been implemented in an open-source multiphysics software. Studies with this model are being performed to evaluate the growth and remodeling response of the idealized LV geometry in terms of parameters that can be tuned experimentally, going, therefore, in the right direction to interrelate both approaches, as planned.
