Periodic Reporting for period 1 - G-CYBERHEART (Computationally and experimentallY BioEngineeRing the next generation of Growing HEARTs)
Reporting period: 2022-09-01 to 2025-02-28
Congenital Heart Disease (CHD) results when the heart abnormally develops before birth. With diminished cardiac function, the heart fails to supply the required oxygenated blood to the body, causing a cascade of failures at different scales that prevent the newborn from growing normally. Even with improved surgical and medical treatments, CHD remains a lifelong risk factor for many diseases.
An emerging application in cardiac tissue engineering is the 3D bioprinting of human hearts, or their parts, for clinical transplantation, with CHD representing a potential therapeutic target. Advances in tissue engineering and 3D-printing techniques allow the precise deposition of biomaterials, cells, and biochemicals to fabricate tissue-like structures to recapitulate the biological function and mechanical properties of tissues and organs. Whereas the fabrication of bioartificial hearts is currently feasible, significant scientific and technological challenges remain to be overcome. The development of novel experimental approaches is fundamental. At the same time, there is a pressing need for complementary computational methods to efficiently assist in the design of biophysically feasible and printable hearts, which must grow with the CHD patient's body while adapting to lifelong changes in hemodynamic conditions.
The EU-funded G-CYBERHEART project focuses on the fabrication of bioartificial cardiac ventricles based on novel and interrelated experimental and computational approaches for reproducing and predicting the evolution of the bioengineered ventricles. The experimental design aims to enhance cardiac muscle maturation and the biomechanical function of bioprinted ventricles. The coupled multi-physics computational platform being developed aims to describe these complex processes under multiple stimulated conditions to predict the critical adaptation and evolution of bioengineered ventricles potentially implanted in CHD patients. By closely interrelating these approaches, G-CYBERHEART will drive advances in regenerative medicine and tissue engineering that will help accelerate the development of the bioengineered heart of the future.
An emerging application in cardiac tissue engineering is the 3D bioprinting of human hearts, or their parts, for clinical transplantation, with CHD representing a potential therapeutic target. Advances in tissue engineering and 3D-printing techniques allow the precise deposition of biomaterials, cells, and biochemicals to fabricate tissue-like structures to recapitulate the biological function and mechanical properties of tissues and organs. Whereas the fabrication of bioartificial hearts is currently feasible, significant scientific and technological challenges remain to be overcome. The development of novel experimental approaches is fundamental. At the same time, there is a pressing need for complementary computational methods to efficiently assist in the design of biophysically feasible and printable hearts, which must grow with the CHD patient's body while adapting to lifelong changes in hemodynamic conditions.
The EU-funded G-CYBERHEART project focuses on the fabrication of bioartificial cardiac ventricles based on novel and interrelated experimental and computational approaches for reproducing and predicting the evolution of the bioengineered ventricles. The experimental design aims to enhance cardiac muscle maturation and the biomechanical function of bioprinted ventricles. The coupled multi-physics computational platform being developed aims to describe these complex processes under multiple stimulated conditions to predict the critical adaptation and evolution of bioengineered ventricles potentially implanted in CHD patients. By closely interrelating these approaches, G-CYBERHEART will drive advances in regenerative medicine and tissue engineering that will help accelerate the development of the bioengineered heart of the future.
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.
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.
In collaboration with other groups, we have started to tackle the development and integration of valves on the bioprinted ventricles to obtain closed-chamber pressure-generator TECVs, representing one of the potential breakthroughs of the project. Still, after successfully designing and building the first prototype, the silicone used for the valves seems toxic to the cardiomyocytes, so improvements are forthcoming.
The first step in establishing growth and remodeling (G&R) computations of a TECV is to numerically determine its initial homeostatic stress state. After facing some issues in establishing this state, we are currently developing a more robust and general prestressing algorithm suitable for G&R using constrained mixture models and, particularly, to help the computational initialization of the TECV. So far, the numerical results align with analytical solutions in both two-dimensional (slab) and three-dimensional (cylindrical) test cases. Once fully validated, we anticipate this approach will provide a robust and generalizable method for initializing G&R simulations, not only for TECVs but also for native cardiac tissue.
The first step in establishing growth and remodeling (G&R) computations of a TECV is to numerically determine its initial homeostatic stress state. After facing some issues in establishing this state, we are currently developing a more robust and general prestressing algorithm suitable for G&R using constrained mixture models and, particularly, to help the computational initialization of the TECV. So far, the numerical results align with analytical solutions in both two-dimensional (slab) and three-dimensional (cylindrical) test cases. Once fully validated, we anticipate this approach will provide a robust and generalizable method for initializing G&R simulations, not only for TECVs but also for native cardiac tissue.