Bioartificial heart: a human-sized porcine model - the way ahead

BACKGROUND: A bioartificial heart is a theoretical alternative to transplantation or mechanical left ventricular support. Native hearts decellularized with preserved architecture and vasculature may provide an acellular tissue platform for organ regeneration. We sought to develop a tissue-engineered whole-heart neoscaffold in human-sized porcine hearts. METHODS: We decellularized porcine hearts (n = 10) by coronary perfusion with ionic detergents in a modified Langendorff circuit. We confirmed decellularization by histology, transmission electron microscopy and fluorescence microscopy, quantified residual DNA by spectrophotometry, and evaluated biomechanical stability with ex-vivo left-ventricular pressure/volume studies, all compared to controls. We then mounted the decellularized porcine hearts in a bioreactor and reseeded them with murine neonatal cardiac cells and human umbilical cord derived endothelial cells (HUVEC) under simulated physiological conditions. RESULTS: Decellularized hearts lacked intracellular components but retained specific collagen fibers, proteoglycan, elastin and mechanical integrity; quantitative DNA analysis demonstrated a significant reduction of DNA compared to controls (82.6+/-3.2 ng DNA/mg tissue vs. 473.2+/-13.4 ng DNA/mg tissue, p<0.05). Recellularized porcine whole-heart neoscaffolds demonstrated re-endothelialization of coronary vasculature and measurable intrinsic myocardial electrical activity at 10 days, with perfused organ culture maintained for up to 3 weeks. CONCLUSIONS: Human-sized decellularized porcine hearts provide a promising tissue-engineering platform that may lead to future clinical strategies in the treatment of heart failure

Reendothelialization of Human Heart Valve Neoscaffolds Using Umbilical Cord-Derived Endothelial Cells.

Background: Heart valve tissue engineering represents a concept for improving the current methods of valvular heart disease therapy. The aim of this study was to develop tissue engineered heart valves combining human umbilical vein endothelial cells (HUVECs) and decellularized human heart valve matrices. Methods and Results: Pulmonary (n=9) and aortic (n=6) human allografts were harvested from explanted hearts from heart transplant recipients and were decellularized using a detergent-based cell extraction method. Analysis of decellularization success was performed with light microscopy, transmission electron microscopy and quantitative analysis of collagen and elastin content. The decellularization method resulted in full removal of native cells while the mechanical stability and the quantitative composition of the neoscaffolds was maintained. The luminal surface of the human matrix could be successfully recellularized with in vitro expanded HUVECs under dynamic flow conditions. The surface appeared as a confluent cell monolayer of positively labeled cells for von Willebrand factor and CD 31, indicating their endothelial nature. Conclusions: Human heart valves can be decellularized by the described method. Recellularization of the human matrix resulted in the formation of a confluent HUVEC monolayer. The in vitro construction of tissue-engineered heart valves based on decellularized human matrices followed by endothelialization using HUVECs is a feasible and safe method, leading to the development of future clinical strategies in the treatment of heart valve disease

Images of native (A, C, E, G) and decellularized ventricular tissue (B, D, F, H) stained with DAPI, Masson's Trichrome Stain (Masson), Herovici's Stain (Herovici) and Movat's Pentachrome Stain (Movat) revealed absence of nuclear staining after decellularization (B) and stable preservation of extracellular matrix collagen, elastic fibers including large coronary vessels (D, H, red arrow) after the decellularization procedure.

<p>Scale bars, 200 µm.</p

Demonstration of multi-electrode array electric voltage undulations of up to 200 mV in a time scale from ca. 500–1000 ms (red arrows) as a measure of myocardial electrical activity.

<p>Demonstration of multi-electrode array electric voltage undulations of up to 200 mV in a time scale from ca. 500–1000 ms (red arrows) as a measure of myocardial electrical activity.</p

Results of biomechanical measurements.

<p>Left ventricular peak pressure vs. volume. Decellularized hearts showed similar mechanical stability as native hearts with no significant differences in biomechanical behavior. All values are expressed as mean ± SEM.</p

Photomicrographs of unstained tissue samples demonstrating intact coronary vasculature (A, B) with intact third- and fourth-level vessels (A, red arrows).

<p>The extracellular matrix composition of the aortic wall (<b>C</b>) and aortic valve leaflet (<b>D</b>) was preserved after decellularization and showed no remnant nuclear material as demonstrated by hematoxylin and eosin (HE) staining (C, D) and TEM analysis (<b>box in D</b>). Also the aortic valve remained competent after decellularization (<b>E, F</b>).</p

Representative images of a porcine heart before (A) and after (B) decellularization with sodium dodecyl sulfate (SDS).

<p>All structures including the coronary vasculature (B, red arrow) are preserved. Hematoxylin and eosin (HE) staining of ventricular tissue before (C) and after perfusion decellularization (D) showing no remnant nuclear structures after treatment with SDS, with maintained extracellular matrix and coronary vessels (D, black arrow). Scale bars, 200 µm.</p

Results of Extracellular Matrix Analysis.

<p>All values are expressed as mean ± SEM and adjusted to 1 mg lyophilized tissue sample.</p><p>Results of Extracellular Matrix Analysis.</p

Whole-heart bioreactor: BIOSTAT B-DCU II and BioPAT DCU control tower (Sartorius Stedim Biotech GmbH, Germany).

<p>Whole-heart bioreactor: BIOSTAT B-DCU II and BioPAT DCU control tower (Sartorius Stedim Biotech GmbH, Germany).</p