3D-bioprinting of patient-derived cardiac tissue models for studying congenital heart disease.

INTRODUCTION: Congenital heart disease is the leading cause of death related to birth defects and affects 1 out of every 100 live births. Induced pluripotent stem cell technology has allowed for patient-derived cardiomyocytes to be studied in vitro. An approach to bioengineer these cells into a physiologically accurate cardiac tissue model is needed in order to study the disease and evaluate potential treatment strategies. METHODS: To accomplish this, we have developed a protocol to 3D-bioprint cardiac tissue constructs comprised of patient-derived cardiomyocytes within a hydrogel bioink based on laminin-521. RESULTS: Cardiomyocytes remained viable and demonstrated appropriate phenotype and function including spontaneous contraction. Contraction remained consistent during 30 days of culture based on displacement measurements. Furthermore, tissue constructs demonstrated progressive maturation based on sarcomere structure and gene expression analysis. Gene expression analysis also revealed enhanced maturation in 3D constructs compared to 2D cell culture. DISCUSSION: This combination of patient-derived cardiomyocytes and 3D-bioprinting represents a promising platform for studying congenital heart disease and evaluating individualized treatment strategies

Spatiotemporal Characterizations of Beating Cardiomyocytes on Mechanical Loading Platform and Biomaterial Substrates

Cardiovascular diseases are a major cause of mortality resulting in serious health concerns and deteriorating quality of life. The minimal regeneration capacity of the myocardium revamps the need for an alternative therapeutic strategy such as myocardial tissue engineering. Human heart generates in a mechanically active environment beginning from organogenesis and persistent throughout adulthood. Hence, mechanical loading may have great utility in the control of cardiomyocyte (CM) development for cardiac tissue engineering and in the study of molecular mechanisms of CM function and pathology. This dissertation is aimed at utilizing mechanical stretch loading to physiologically mimic these CM developmental conditions and guide murine P19 pluripotent embryonic stem cell differentiation to CM lineage. The beating dynamics and genomics development of contracting CMs were improved or at comparable levels for stretch loading alone case compared with those from known soluble factor induction with 5-Azacytidine, suggesting that stretch loading may serve as a potent trigger to induce functional CM development. In addition, potential regulatory role of molecular mechanosensors such as focal adhesion kinase and Rho-associated protein kinase were tested as mechanotransduction pathways. The next part of the dissertation was focused on evaluating the spontaneous contraction of CMs, as a measure of their functional health. A non-invasive, robust, and unbiased method is presented for novel spatial-temporal characterization of CM contraction. The method developed enables to adaptively select reference frame when applying digital image correlation (DIC) to observe CM contraction, overcoming the lack of static reference frame and resultant noise buildup in conventional DIC methods. Based on the physiological criteria of individual sarcomere displacement length of 0.14 µm as a contraction threshold, novel spatiotemporal parameters were defined to account for the homogeneity, propagation, and synchronicity of CM contraction. To reiterate the elasticity of mechanically active native myocardium for the myocardial regenerative medicine, the final part of the dissertation attempted to develop cardiac tissue patches on electrospun polyurethane nanofiber scaffolds. Relative to aligned nanofibers or flat control, more number of contractile CM tissues were obtained on randomly oriented polyurethane nanofiber substrates, which may thus be better suited for the myocardial tissue engineering application

Spatiotemporal Characterizations of Beating Cardiomyocytes on Mechanical Loading Platform and Biomaterial Substrates

Cardiovascular diseases are a major cause of mortality resulting in serious health concerns and deteriorating quality of life. The minimal regeneration capacity of the myocardium revamps the need for an alternative therapeutic strategy such as myocardial tissue engineering. Human heart generates in a mechanically active environment beginning from organogenesis and persistent throughout adulthood. Hence, mechanical loading may have great utility in the control of cardiomyocyte (CM) development for cardiac tissue engineering and in the study of molecular mechanisms of CM function and pathology. This dissertation is aimed at utilizing mechanical stretch loading to physiologically mimic these CM developmental conditions and guide murine P19 pluripotent embryonic stem cell differentiation to CM lineage. The beating dynamics and genomics development of contracting CMs were improved or at comparable levels for stretch loading alone case compared with those from known soluble factor induction with 5-Azacytidine, suggesting that stretch loading may serve as a potent trigger to induce functional CM development. In addition, potential regulatory role of molecular mechanosensors such as focal adhesion kinase and Rho-associated protein kinase were tested as mechanotransduction pathways. The next part of the dissertation was focused on evaluating the spontaneous contraction of CMs, as a measure of their functional health. A non-invasive, robust, and unbiased method is presented for novel spatial-temporal characterization of CM contraction. The method developed enables to adaptively select reference frame when applying digital image correlation (DIC) to observe CM contraction, overcoming the lack of static reference frame and resultant noise buildup in conventional DIC methods. Based on the physiological criteria of individual sarcomere displacement length of 0.14 µm as a contraction threshold, novel spatiotemporal parameters were defined to account for the homogeneity, propagation, and synchronicity of CM contraction. To reiterate the elasticity of mechanically active native myocardium for the myocardial regenerative medicine, the final part of the dissertation attempted to develop cardiac tissue patches on electrospun polyurethane nanofiber scaffolds. Relative to aligned nanofibers or flat control, more number of contractile CM tissues were obtained on randomly oriented polyurethane nanofiber substrates, which may thus be better suited for the myocardial tissue engineering application

Spatiotemporal Characterizations of Spontaneously Beating Cardiomyocytes with Adaptive Reference Digital Image Correlation

We developed an Adaptive Reference-Digital Image Correlation (AR-DIC) method that enables unbiased and accurate mechanics measurements of moving biological tissue samples. We applied the AR-DIC analysis to a spontaneously beating cardiomyocyte (CM) tissue, and could provide correct quantifications of tissue displacement and strain for the beating CMs utilizing physiologically-relevant, sarcomere displacement length-based contraction criteria. The data were further synthesized into novel spatiotemporal parameters of CM contraction to account for the CM beating homogeneity, synchronicity, and propagation as holistic measures of functional myocardial tissue development. Our AR-DIC analyses may thus provide advanced non-invasive characterization tools for assessing the development of spontaneously contracting CMs, suggesting an applicability in myocardial regenerative medicine