Question

Assignment5 One page summary of cardiac tissue engineering

Answer 1

Cardiovascular diseases are the major cause of death worldwide. The heart has limited capacity of regeneration, therefore, transplantation is the only solution in some cases despite presenting many disadvantages. Tissue engineering has been considered the ideal strategy for regenerative medicine in cardiology. It is an interdisciplinary field combining many techniques that aim to maintain, regenerate or replace a tissue or organ. The main approach of cardiac tissue engineering is to create cardiac grafts, either whole heart substitutes or tissues that can be efficiently implanted in the organism, regenerating the tissue and giving rise to a fully functional heart, without causing side effects, such as immunogenicity. In this review, we systematically present and compare the techniques that have drawn the most attention in this field and that generally have focused on four important issues: the scaffold material selection, the scaffold material production, cellular selection and in vitro cell culture. Many studies used several techniques that are herein presented, including biopolymers, decellularization and bioreactors, and made significant advances, either seeking a graft or an entire bioartificial heart. However, much work remains to better understand and improve existing techniques, to develop robust, efficient and efficacious methods.

Tissue engineering is a set of biomedical, biotechnological and engineering techniques that aim to maintain, regenerate or replace tissues or organs. Advances in tissue engineering are evident, and the application of this technology to the regeneration of myocardium has been increasingly explored, and presented encouraging results. The main approach of this scientific field is the creation of scaffolds, which contain cells that can be applied as cardiac grafts in the body, to have the desired recovery.

CARDIAC GRAFTS

The techniques used to obtain cardiac grafts focus on four important issues : (1) scaffold material selection; (2) scaffold material production; (3) cell selection; and (4) in vitro cell culture.

Tissue engineering involves the development of functional replacements for damaged tissues or organs . A common approach to produce engineered tissues is to add cells to a natural or synthetic extracellular matrix, which provides mechanical support and biochemical cues. Scaffold-free tissues are prepared by growing cells on thermally responsive polymers to facilitate the cell monolayers that form and then adding layers together or rolling the sheets. By addition of small molecules that activate specific differentiation pathways, three-dimensional organoids can be derived from human pluripotent stem cells . The engineered tissue may be prepared wholly or partially before implantation to activate and localize the body’s regenerative capacity to populate the implanted scaffold. Tissue engineering is a subset of the broader field of regenerative medicine, which seeks to repair or replace damaged organs. This could occur by direct injection of cells or modifying cellular processes to initiate repair and regrowth. In spite of significant research advances and insightful application of developmental cell biology, cell mechanics, and biomaterials, few products have emerged from these efforts to date, pointing to the challenges to develop truly functional tissues.

Key design goals to produce functional tissues in vitro are to reproduce the tissue structure and cell densityin vivo, identify suitable sources of cells, promote growth and differentiation of cells, design a construct that reproduces the extracellular matrix with the appropriate molecular cues and suitable mechanical properties, and create a vasculature within the construct to enable oxygenation and integration with host vasculature after implantation.

Several strategies that have emerged to address these challenges include the use of induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs) that can differentiate into the cells of interest, the reprogramming of primary cells to the cell type of interest, various ways to engineer the structural support for the cells to mimic the extracellular matrix, and efforts to promote vascular network formation . Over the past few years, a new use of tissue engineering has emerged in which microscale human tissue-engineered systems or microphysiological systems are used to model normal and disease states in vitro and assess drug responses.

A scaffold-free cardiac patch consisting of three layers of rat cardiomyocytes was successfully engrafted onto heart tissue by overlaying the patch over a vascular supply, enhancing the ability of endothelial cells (ECs) in the patch to form a functional tubular vascular network connected to the host blood supply ⁹. Recently, the development of a porous patch with an electroactive polypyrrole incorporating electronics for sensing and stimulating electrophysiological activity and release of various biological molecules offers a new level of control of cardiac patches while permitting incorporation of cells attached to a bioactive scaffold ¹⁰.

While tissue-engineered blood vessels (TEBVs) constructed from natural matrix components such as collagen  and fibrin  have traditionally exhibited poor mechanical strength, plastic compression of collagen gels embedded with smooth muscle cells (SMCs) increases the collagen fiber density and yields rapidly producible tubular structures with high mechanical strength . By plastic compression of collagen, TEBVs with burst pressures exceeding 1600 mmHg can be prepared in a few hours . After one week of perfusion at physiological shear stresses, the medial cells exhibited differentiation and contracted in response to phenylephrine. While these TEBVs have not been studied in vivo, this rapid method of fabrication could significantly reduce the time to produce functional TEBVs.

Stem cells for tissue engineering

iPSCs offer the potential to develop engineered tissues of individual human cardiovascular disease states and avoid ethical issues associated with ESCs. iPSCs can be induced to differentiate into a large number of cell types including cardiomyocytes, SMCs , and ECs . The formation of teratomas  can be reduced using non-integrating methods  and immunogenicity is low ²⁸. An exciting new development has been the creation of mouse iPSCs using small molecules that activate specific transcription factors , although this approach has not yet been demonstrated with human iPSCs. A challenge with the use of iPSCs in tissue engineering is that differentiation is often limited and the resulting structures do not display a mature phenotype .

Vascular cells can be obtained from iPSCs or ESCs by first activating the Wnt signaling pathway. Early activation of Wnt and β-catenin by inhibition of glycogen synthase kinase 3 (GSK3) before differentiation on surfaces with serum produces cardiomyocytes . Following Wnt pathway activation with GSK3 inhibitors, ECs can be obtained by addition of vascular endothelial growth factor (VEGF) and forskolin, while SMCs can be obtained using platelet-derived growth factor-BB (PDGF-BB) and ActivinA . Interestingly, a recent report indicated that by culturing murine iPSCs on gelatin-coated polycaprolactone nanofibrous scaffolds, Wnt/β-catenin can be transiently activated to induce differentiation towards cardiomyocytes . Combining GSK3 inhibitors with specific modification of substrate properties may lead to more robust differentiation.

One week of electrical stimulation at 0.5, 1, or 2 Hz of human ESCs or iPSCs in three-dimensional engineered tissues facilitates differentiation to cardiomyocytes by producing hypertrophy, an increase in connexin-43 gap junctions, and increased expression of hERG, the potassium channel which regulates cardiomyocyte repolarization ³⁴. Some connexin-40 is expressed, indicating that rapidly conducting cells can be stimulated; however, it is not yet possible to regulate the relative expression of the various connexins though selection of a specific stimulation protocol. The stimulated cells responded to chronotropic drugs and the cells maintained synchrony to the rate of applied stimulation for two weeks after the stimulation ended.

Cardiomyocytes generated by selecting for Nkx2-5-positive cells among mouse iPSCs exhibit a number of markers found in mature cardiomyocytes, and the resting membrane potential approaches physiological levels . Three-dimensional engineered tissues produced aligned cardiomyocytes that exhibited adherens and gap junctions, although the electrophysiological responses were similar to those exhibited by fetal cardiomyocytes

Vascularization

The density and thickness of engineered tissues is limited by the transport of nutrients to the cells. Oxygen is often the limiting nutrient, since it is consumed at the highest rate and is critical for producing the energy needed for normal cell function. In vivo, capillary distances range from 15–50 µm depending on the cell density and the metabolic demands . In vitro, cell densities are lower, but uniform cell densities can be achieved only for thicknesses of about 100 µm owing to consumption of oxygen in the engineered tissue. Perfusion can lead to somewhat thicker tissues. However, without its own microvascular network that could integrate with the host network after implantation, only thin tissue-engineered constructs can remain viable after implantation.

While addition of VEGF can initiate the formation of new capillaries or branches from existing capillariesin vitro, the resulting structures are unstable and last at most a few days. New vessel formation involves several discrete stages. Initially, exogenous VEGF causes the release of matrix metalloproteinases, which degrade the extracellular matrix, enabling migration of the newly forming vessel buds. The newly forming vessel secretes growth factors to recruit mural cells, such as fibroblasts, pericytes, or SMCs, which interact with the newly formed microvessels, stabilizing them.

Although MSCs can stabilize EC networks in vitro and exhibit pericyte-like behavior , the heterogeneity of MSCs from various sources or by different isolation methods leads to variable responses . The cells must be characterized and tested for their ability to stabilize networks when developing a system to create microvascular networks. iPSCs could provide a ready source of pericytes , although SMCs or fibroblasts derived from iPSCs may be suitable.

A potentially useful model system to identify conditions that promote vascularization of tissue-engineered systems involves creating microvascular networks in a synthetic extracellular matrix hydrogel. The hydrogel contains matrix metalloproteinase degradation sites and peptide sequences of extracellular matrix proteins to elicit specific cell binding . Photopolymerization of polyethylene glycol gels enables straightforward incorporation of cells, and the networks are robust and sensitive to perfusion in the extracellular space . The direction of flow is very critical for improving mass transfer and enabling microvessels to stabilize . The extracellular matrix peptide sequences provided influence the extent of network formation, with addition of cell binding sequences from both fibronectin (RGD) and laminin (YIGSR) producing the most robust network formation in the hydrogel . Adding macrophages enhanced new vessel formation in synthetic hydrogels, consistent with their role in vivo . Other factors to enhance microvessel network formation in hydrogels involve regulating growth factor delivery  and a hypoxic environment .