Transmural myocardial repair with engineered heart muscle in a rat model of heterotopic heart transplantation - A proof-of-concept study.

Engineered heart muscle (EHM) can be implanted epicardially to remuscularize the failing heart. In case of a severely scarred ventricle, excision of scar followed by transmural heart wall replacement may be a more desirable application. Accordingly, we tested the hypothesis that allograft (rat) and xenograft (human) EHM can also be administered as transmural heart wall replacement in a heterotopic, volume-loaded heart transplantation model. We first established a novel rat model model to test surgical transmural left heart wall repair. Subsequently and in continuation of our previous allograft studies, we tested outcome after implantation of contractile engineered heart muscle (EHM) and non-contractile engineered connective tissue (ECT) as well as engineered mesenchymal tissue (EMT) allografts as transmural heart wall replacement. Finally, proof-of-concept for the application of human EHM was obtained in an athymic nude rat model. Only in case of EHM implantation, remuscularization of the surgically created transmural defect was observed with palpable graft vascularization. Taken together, feasibility of transmural heart repair using bioengineered myocardial grafts could be demonstrated in a novel rat model of heterotopic heart transplantation.

Highly conductive stretchable and biocompatible electrode-hydrogel hybrids for advanced tissue engineering.

Hydrogel-based, molecular permeable electronic devices are considered to be promising for electrical stimulation and recording of living tissues, either in vivo or in vitro. This study reports the fabrication of the first hydrogel-based devices that remain highly electrically conductive under substantial stretch and bending. Using a simple technique involving a combination of chemical polymerization and electropolymerization of poly (3,4-ethylenedioxythiophene) (PEDOT), a tight bonding of a conductive composite of PEDOT and polyurethane (PU) to an elastic double-network hydrogel is achieved to make fully organic PEDOT/PU-hydrogel hybrids. Their response to repeated bending, mechanical stretching, hydration-dessication cycles, storage in aqueous condition for up to 6 months, and autoclaving is assessed, demonstrating excellent stability, without any mechanical or electrical damage. The hybrids exhibit a high electrical conductivity of up to 120 S cm(-1) at 100% elongation. The adhesion, proliferation, and differentiation of neural and muscle cells cultured on these hybrids are demonstrated, as well as the fabrication of 3D hybrids, advancing the field of tissue engineering with integrated electronics.

Myocardial tissue engineering and heart muscle repair.

Cardiovascular diseases, including myocardial infarction and heart failure, are the main causes of death worldwide. Classical pharmacological treatment may halt, but cannot reverse the underlying disease process. Cellular cardiomyoplasty has the potential to reconstruct myocardium in situ; yet, it is hampered by poor cell survival, engraftment, and differentiation. Tissue engineering has emerged as an alternative cell-based approach, aiming at partial or full replacement of damaged organs with in vitro generated tissue equivalents. However, limited availability of therapeutic cardiomyocytes poses a major challenge on cell-based and in particular tissue engineering-based therapies. Rapidly evolving stem cell technologies, enabling mass cultures may overcome this limitation. Translating available experimental concepts into clinical reality will be the ultimate challenge. This review discusses potentially therapeutic cells for cardiac repair, current stem cell-based myocardial tissue engineering strategies, and the requirements for a translation of myocardial tissue engineering into clinical practice.

Terminal differentiation, advanced organotypic maturation, and modeling of hypertrophic growth in engineered heart tissue.

RATIONALE: Cardiac tissue engineering should provide "realistic" in vitro heart muscle models and surrogate tissue for myocardial repair. For either application, engineered myocardium should display features of native myocardium, including terminal differentiation, organotypic maturation, and hypertrophic growth. OBJECTIVE: To test the hypothesis that 3D-engineered heart tissue (EHT) culture supports (1) terminal differentiation as well as (2) organotypic assembly and maturation of immature cardiomyocytes, and (3) constitutes a methodological platform to investigate mechanisms underlying hypertrophic growth. METHODS AND RESULTS: We generated EHTs from neonatal rat cardiomyocytes and compared morphological and molecular properties of EHT and native myocardium from fetal, neonatal, and adult rats. We made the following key observations: cardiomyocytes in EHT (1) gained a high level of binucleation in the absence of notable cytokinesis, (2) regained a rod-shape and anisotropic sarcomere organization, (3) demonstrated a fetal-to-adult gene expression pattern, and (4) responded to distinct hypertrophic stimuli with concentric or eccentric hypertrophy and reexpression of fetal genes. The process of terminal differentiation and maturation (culture days 7-12) was preceded by a tissue consolidation phase (culture days 0-7) with substantial cardiomyocyte apoptosis and dynamic extracellular matrix restructuring. CONCLUSIONS: This study documents the propensity of immature cardiomyocytes to terminally differentiate and mature in EHT in a remarkably organotypic manner. It moreover provides the rationale for the utility of the EHT technology as a methodological bridge between 2D cell culture and animal models.

PDGF-BB protects cardiomyocytes from apoptosis and improves contractile function of engineered heart tissue.

Platelet-derived-growth-factor-BB (PDGF-BB) can protect various cell types from apoptotic cell death, and induce hypertrophic growth and proliferation, but little is known about its direct or indirect effects on cardiomyocytes. Cardiac muscle engineering is compromised by a particularly high rate of cardiomyocyte death. Here we hypothesized that PDGF-BB stimulation can (1) protect cardiomyocytes from apoptosis, (2) enhance myocyte content in and (3) consequently optimize contractile performance of engineered heart tissue (EHT). We investigated the effects of PDGF-receptor activation in neonatal rat heart monolayer- and EHT-cultures by isometric contraction experiments, cytomorphometry, (3)H-thymidine and (3)H-phenylalanine incorporation assays, quantitative PCR (calsequestrin 2, alpha-cardiac and skeletal actin, atrial natriuretic factor, alpha- and beta-myosin heavy chain), immunoblotting (activated caspase 3, Akt-phosphorylation), and ELISA (cell death detection). PDGF-BB did not induce hypertrophy or proliferation in cardiomyocytes, but enhanced contractile performance of EHT. This effect was concentration-dependent (E(max) 10 ng/ml) and maximal only after transient PDGF-BB stimulation (culture days 0-7; total culture duration: 12 days). Improvement of contractile function was associated with higher cardiomyocyte content, as a consequence of PDGF-BB mediated protection from apoptosis (lower caspase-3 activity particularly in cardiomyocytes in PDGF-BB treated vs. untreated EHTs). We confirmed the anti-apoptotic effect of PDGF-BB in monolayer cultures and observed that PI3-kinase inhibition with LY294002 attenuated PDGF-BB-mediated cardiomyocyte protection. We conclude that PDGF-BB does not induce hypertrophy or proliferation, but confers an anti-apoptotic effect on cardiomyocytes. Our findings suggest a further exploitation of PDGF-BB in cardiomyocyte protection in vivo and in vitro.

Development of a biological ventricular assist device: preliminary data from a small animal model.

BACKGROUND: Engineered heart tissue (EHT) can be generated from cardiomyocytes and extracellular matrix proteins and used to repair local heart muscle defects in vivo. Here, we hypothesized that pouch-like heart muscle constructs can be generated by using a novel EHT-casting technology and applied as heart-embracing cardiac grafts in vivo. METHODS AND RESULTS: Pouch-like EHTs (inner/outer diameter: 10/12 mm) can be generated mainly from neonatal rat heart cells, collagen type I, and serum containing culture medium. They contain a dense network of connexin 43 interconnected cardiomyocytes and an endo-/epicardial surface lining composed of prolylhydroxylase positive cells. Pouch-like EHTs beat spontaneously and show contractile properties of native heart muscle including positive inotropic responses to calcium and isoprenaline. First implantation studies indicate that pouch-like EHTs can be slipped over uninjured adult rat hearts to completely cover the left and right ventricles. Fourteen days after implantation, EHT-grafts stably covered the epicardial surface of the respective hearts. Engrafted EHTs were composed of matrix and differentiated cardiac muscle as well as newly formed vessels which were partly donor-derived. CONCLUSIONS: Pouch-like EHTs can be generated with structural and functional properties of native myocardium. Implantation studies demonstrated their applicability as cardiac muscle grafts, setting the stage for an evaluation of EHT-pouches as biological ventricular assist devices in vivo.