Cells, Materials, and Fabrication Processes for Cardiac Tissue Engineering

Cardiovascular disease is the number one killer worldwide, with myocardial infarction (MI) responsible for approximately 1 in 6 deaths. The lack of endogenous regenerative capacity, added to the deleterious remodelling programme set into motion by myocardial necrosis, turns MI into a progressively debilitating disease, which current pharmacological therapy cannot halt. The advent of Regenerative Therapies over 2 decades ago kick-started a whole new scientific field whose aim was to prevent or even reverse the pathological processes of MI. As a highly dynamic organ, the heart displays a tight association between 3D structure and function, with the non-cellular components, mainly the cardiac extracellular matrix (ECM), playing both fundamental active and passive roles. Tissue engineering aims to reproduce this tissue architecture and function in order to fabricate replicas able to mimic or even substitute damaged organs. Recent advances in cell reprogramming and refinement of methods for additive manufacturing have played a critical role in the development of clinically relevant engineered cardiovascular tissues. This review focuses on the generation of human cardiac tissues for therapy, paying special attention to human pluripotent stem cells and their derivatives. We provide a perspective on progress in regenerative medicine from the early stages of cell therapy to the present day, as well as an overview of cellular processes, materials and fabrication strategies currently under investigation. Finally, we summarise current clinical applications and reflect on the most urgent needs and gaps to be filled for efficient translation to the clinical arena.This work was supported by funds from the ISCIII Red TERCEL RETIC RD16/0011/0005, PI 19/01350, ERANET II (Nanoreheart) and Gobierno de Navarra Departamento de Salud GNa8/2019, co-funded by FEDER funds, MINECO (Program RETOS Cardiomesh RTC-2016-4911-1), Gobierno de Navarra 0011-1383-2019-000006 and 0011-1383-2018-000011, and European Union's H2020 Program under grant agreement No. 874827 (BRAV(sic))

Three-dimensional bioprinting in cardiovascular disease: current status and future directions

Three-dimensional (3D) printing plays an important role in cardiovascular disease through the use of personalised models that replicate the normal anatomy and its pathology with high accuracy and reliability. While 3D printed heart and vascular models have been shown to improve medical education, preoperative planning and simulation of cardiac procedures, as well as to enhance communication with patients, 3D bioprinting represents a potential advancement of 3D printing technology by allowing the printing of cellular or biological components, functional tissues and organs that can be used in a variety of applications in cardiovascular disease. Recent advances in bioprinting technology have shown the ability to support vascularisation of large-scale constructs with enhanced biocompatibility and structural stability, thus creating opportunities to replace damaged tissues or organs. In this review, we provide an overview of the use of 3D bioprinting in cardiovascular disease with a focus on technologies and applications in cardiac tissues, vascular constructs and grafts, heart valves and myocardium. Limitations and future research directions are highlighted

Generation of an induced pluripotent stem cell line (ESi107-A) from a transthyretin amyloid cardiomyopathy (ATTR-CM) patient carrying a p.Ser43Asn mutation in the TTR gene

Transthyretin (TTR) amyloid cardiomyopathy (ATTR-CM) is a life-threatening disease caused by the abnormal production of misfolded TTR protein by liver cells, which is then released systemically. Its amyloid deposition in the heart is linked to cardiac toxicity and progression toward heart failure. A human induced pluripotent stem cell (iPSC) line was generated from peripheral blood mononuclear cells (PBMCs) from a patient suffering familial transthyretin amyloid cardiomyopathy carrying a c.128G>A (p.Ser43Asn) mutation in the TTR gene. This iPSC line offers a useful resource to study the disease pathophysiology and a cell-based model for therapeutic discovery

Fabrication of human myocardium using multidimensional modelling of engineered tissues

Biofabrication of human tissues has seen a meteoric growth triggered by recent technical advancements such as human induced pluripotent stem cells (hiPSCs) and additive manufacturing. However, generation of cardiac tissue is still hampered by lack of adequate mechanical properties and crucially by the often unpredictable post-fabrication evolution of biological components. In this study we employ melt electrowriting (MEW) and hiPSC-derived cardiac cells to generate fibre-reinforced human cardiac minitissues. These are thoroughly characterized in order to build computational models and simulations able to predict their post-fabrication evolution. Our results show that MEW-based human minitissues display advanced maturation 28 post-generation, with a significant increase in the expression of cardiac genes such as MYL2, GJA5, SCN5A and the MYH7/MYH6 and MYL2/MYL7 ratios. Human iPSC-cardiomyocytes are significantly more aligned within the MEW-based 3D tissues, as compared to conventional 2D controls, and also display greater expression of C ×43. These are also correlated with a more mature functionality in the form of faster conduction velocity. We used these data to develop simulations capable of accurately reproducing the experimental performance. In-depth gauging of the structural disposition (cellular alignment) and intercellular connectivity (C ×43) allowed us to develop an improved computational model able to predict the relationship between cardiac cell alignment and functional performance. This study lays down the path for advancing in the development of in silico tools to predict cardiac biofabricated tissue evolution after generation, and maps the route towards more accurate and biomimetic tissue manufacture

Modelling, design, fabrication and characterization of engineered human myocardium made with melt electrowriting and cardiac cells derived from hiPSCs

The adult human heart has evolved to become a highly specialized organ, whose continuous pumping of blood is critical for survival. However, its ability to regenerate or self-repair following injury is very limited, so consequently any event or disease resulting in damage to the heart poses a serious threat to the patient. Moreover, cardiovascular diseases represent one of the most pressing healthcare concerns nowadays, as they are the leading cause of death worldwide, and the number of cases is only expected to increase in the following years. Despite great progress made over the years to treat cardiovascular diseases, to date there is no therapy able to fully cure a heart that has been damaged. In consequence, there is a dire need to generate new strategies to repair the heart damage and restore the lost cardiac function, as well as to develop accurate modelling platforms to advance in the understanding of disease progression and assess the effectiveness of new drugs. Since its advent, cardiac tissue engineering and regenerative medicine has been regarded as a promising candidate to realise this enormous challenge. Given its interdisciplinary nature, scientific breakthroughs in different areas such as cellular reprogramming, polymer chemistry, and additive manufacturing technologies have resulted in the advancement of cardiac tissue engineering and regenerative medicine over the years. One of such cornerstone discoveries was the generation of induced pluripotent stem cells and subsequent differentiation to different cardiac phenotypes, and the present Thesis revolves around their application to generate patient-specific cardiac disease models and humanised engineered functional cardiac minitissues. Firstly, we reprogrammed peripheral blood mononuclear cells from a transthyretin amyloid cardiomyopathy patient, resulting in the generation of a new cell line carrying a c.128G>A (p.Ser43Asn) mutation in the transthyretin gene. Experiments demonstrated the efficacy and safety of the approach, confirming the pluripotency of the cells, the presence of the disease-causing mutation, and the removal of reprogramming vectors. This cell line, which is now available in a repository, can be used to investigate disease biology, molecular mechanisms and progression; as well as an advanced cellular model to test novel therapeutic strategies. Secondly, we aimed to generate functional human minitissues by combining human cardiomyocytes derived from induced pluripotent stem cells and tridimensional fibrillar scaffolds generated with the technology of melt electrowriting. Compared to conventional two-dimensional cell culture, the cardiac minitissues demonstrated enhanced maturation, with a significant increase in conduction velocity, presence of connexin 43 and expression of cardiac-associated genes such as MYL2, GJA5 and SCN5A, and isoform ratios MYH7/MYH6 and MYL2/MYL7 after 28 days in culture. When investigating the effect of the scaffold fibres on the cells, we found that cardiomyocytes placed close to the fibre were arranged parallel to it, but that alignment was progressively lost towards the centre of the scaffold pore. We then used these data to develop simulations capable of accurately reproducing the experimental performance. In-depth gauging of the structural disposition and intercellular connectivity allowed us to develop an improved computational model able to predict the relationship between cardiac cell alignment and functional performance. This study lays down the path for advancing in the development of in silico tools to predict cardiac biofabricated tissue evolution after generation, and maps the route towards more accurate and biomimetic tissue manufacture. We next aimed at increasing the biological representativity of the cardiac minitissues, by implementing a few changes in cellular (addition of induced pluripotent stem cell-derived cardiac fibroblasts) and hydrogel (substitution of Matrigel for fibrin) composition. We also sought to control cardiomyocyte behaviour based on melt electrowritten scaffold geometry. For this, we hypothesized that diamond-based scaffolds would induce cardiomyocyte contraction in the direction of least mechanical resistance, i.e., the small diagonal of the diamonds. The characterization of the new cardiac minitissues demonstrated functional maturation consistent with the previous work in terms of gene expression and conduction velocity, although the observed low initial cell retention within the scaffold highlighted the need of new strategies to improve cell seeding efficiency. When comparing contractile dynamics between melt electrowritten scaffolds made with square, rectangular, and diamond-shaped pores, we found that the latter resulted in significantly faster, stronger and aligned contraction in the direction that we had anticipated. The potential use of the cardiac minitissues as therapy agents was tested by implanting the constructs in a murine model of chronic myocardial infarction. Compared to controls, implanted animals showed significant improvement, including higher left ventricular ejection fraction and greater wall thickness. Finally, in another attempt to enhance the biological representativity of the constructs, a proof of concept was made to generate melt electrowritten ellipsoidal scaffolds with controlled pore architecture. In summary, the present Thesis revolves around human induced pluripotent stem cells and melt electrowriting as cornerstone tools for cardiac tissue engineering and regenerative medicine efforts. By combining both and iteratively optimising the design and experimental conditions, we were able to generate human functional cardiac minitissues of increased biological relevance

Melt Electrospinning Writing of Poly-Hydroxymethylglycolide-co-ε-Caprolactone-Based Scaffolds for Cardiac Tissue Engineering

Current limitations in cardiac tissue engineering revolve around the inability to fully recapitulate the structural organization and mechanical environment of native cardiac tissue. This study aims at developing organized ultrafine fiber scaffolds with improved biocompatibility and architecture in comparison to the traditional fiber scaffolds obtained by solution electrospinning. This is achieved by combining the additive manufacturing of a hydroxyl-functionalized polyester, (poly(hydroxymethylglycolide-co-ε-caprolactone) (pHMGCL), with melt electrospinning writing (MEW). The use of pHMGCL with MEW vastly improves the cellular response to the mechanical anisotropy. Cardiac progenitor cells (CPCs) are able to align more efficiently along the preferential direction of the melt electrospun pHMGCL fiber scaffolds in comparison to electrospun poly(ε-caprolactone)-based scaffolds. Overall, this study describes for the first time that highly ordered microfiber (4.0-7.0 μm) scaffolds based on pHMGCL can be reproducibly generated with MEW and that these scaffolds can support and guide the growth of CPCs and thereby potentially enhance their therapeutic potential

Melt electrospinning writing of poly-Hydroxymethylglycolide-co-ε-Caprolactone-based scaffolds for cardiac tissue engineering

\u3cp\u3eCurrent limitations in cardiac tissue engineering revolve around the inability to fully recapitulate the structural organization and mechanical environment of native cardiac tissue. This study aims at developing organized ultrafine fiber scaffolds with improved biocompatibility and architecture in comparison to the traditional fiber scaffolds obtained by solution electrospinning. This is achieved by combining the additive manufacturing of a hydroxyl-functionalized polyester, (poly(hydroxymethylglycolide-co-ε-caprolactone) (pHMGCL), with melt electrospinning writing (MEW). The use of pHMGCL with MEW vastly improves the cellular response to the mechanical anisotropy. Cardiac progenitor cells (CPCs) are able to align more efficiently along the preferential direction of the melt electrospun pHMGCL fiber scaffolds in comparison to electrospun poly(ε-caprolactone)-based scaffolds. Overall, this study describes for the first time that highly ordered microfiber (4.0–7.0 µm) scaffolds based on pHMGCL can be reproducibly generated with MEW and that these scaffolds can support and guide the growth of CPCs and thereby potentially enhance their therapeutic potential.\u3c/p\u3

Melt Electrospinning Writing of Poly-Hydroxymethylglycolide-co-ε-Caprolactone-Based Scaffolds for Cardiac Tissue Engineering

Current limitations in cardiac tissue engineering revolve around the inability to fully recapitulate the structural organization and mechanical environment of native cardiac tissue. This study aims at developing organized ultrafine fiber scaffolds with improved biocompatibility and architecture in comparison to the traditional fiber scaffolds obtained by solution electrospinning. This is achieved by combining the additive manufacturing of a hydroxyl-functionalized polyester, (poly(hydroxymethylglycolide-co-ε-caprolactone) (pHMGCL), with melt electrospinning writing (MEW). The use of pHMGCL with MEW vastly improves the cellular response to the mechanical anisotropy. Cardiac progenitor cells (CPCs) are able to align more efficiently along the preferential direction of the melt electrospun pHMGCL fiber scaffolds in comparison to electrospun poly(ε-caprolactone)-based scaffolds. Overall, this study describes for the first time that highly ordered microfiber (4.0-7.0 μm) scaffolds based on pHMGCL can be reproducibly generated with MEW and that these scaffolds can support and guide the growth of CPCs and thereby potentially enhance their therapeutic potential

Melt Electrospinning Writing of Poly-Hydroxymethylglycolide-co-ε-Caprolactone-Based Scaffolds for Cardiac Tissue Engineering

Current limitations in cardiac tissue engineering revolve around the inability to fully recapitulate the structural organization and mechanical environment of native cardiac tissue. This study aims at developing organized ultrafine fiber scaffolds with improved biocompatibility and architecture in comparison to the traditional fiber scaffolds obtained by solution electrospinning. This is achieved by combining the additive manufacturing of a hydroxyl-functionalized polyester, (poly(hydroxymethylglycolide-co-ε-caprolactone) (pHMGCL), with melt electrospinning writing (MEW). The use of pHMGCL with MEW vastly improves the cellular response to the mechanical anisotropy. Cardiac progenitor cells (CPCs) are able to align more efficiently along the preferential direction of the melt electrospun pHMGCL fiber scaffolds in comparison to electrospun poly(ε-caprolactone)-based scaffolds. Overall, this study describes for the first time that highly ordered microfiber (4.0–7.0 µm) scaffolds based on pHMGCL can be reproducibly generated with MEW and that these scaffolds can support and guide the growth of CPCs and thereby potentially enhance their therapeutic potential

Three-dimensional bioprinting in cardiovascular disease: current status and future directions

Three-dimensional (3D) printing plays an important role in cardiovascular disease through the use of personalised models that replicate the normal anatomy and its pathology with high accuracy and reliability. While 3D printed heart and vascular models have been shown to improve medical education, preoperative planning and simulation of cardiac procedures, as well as to enhance communication with patients, 3D bioprinting represents a potential advancement of 3D printing technology by allowing the printing of cellular or biological components, functional tissues and organs that can be used in a variety of applications in cardiovascular disease. Recent advances in bioprinting technology have shown the ability to support vascularisation of large-scale constructs with enhanced biocompatibility and structural stability, thus creating opportunities to replace damaged tissues or organs. In this review, we provide an overview of the use of 3D bioprinting in cardiovascular disease with a focus on technologies and applications in cardiac tissues, vascular constructs and grafts, heart valves and myocardium. Limitations and future research directions are highlighted