Development of a new decellularization protocol for the whole porcine heart

Abstract

Background: Cardiovascular diseases are the leading cause of death in many countries. Advances in technology have been promoted in this regard, especially in tissue engineering, to meet the need for tissue or organ grafts. In this way, the porcine model has been used due to its morphophysiological similarity between the human species, mainly regarding the cardiovascular system. Tissue engineering is employed using biological scaffolds that are currently derived from porcine. These scaffolds are produced by decellularization, a process to remove cells aiming to maintain only its three-dimensional structure, formed by extracellular matrix (ECM). Its main objective is to produce organs through recellularized scaffolds that could eventually substitute the ones with impaired functions.

Aim: In this way, the present study aimed to establish a new protocol for porcine heart decellularization with potential application on tissue engineering.

Methods: A porcine heart aorta was cannulated with a silicon tube, and the organ was washed in 0.1% phosphate-buffered saline through a peristaltic pump (Harvard Peristaltic Pump - Harvard Apparatus). After that, deionized water was introduced in the same system. The decellularization procedure was carried out using ionic and non-ionic detergents, namely 4% sodium dodecyl sulfate (SDS) and 1% Triton X-100, respectively. SDS was perfused through myocardial circulation at 400 mL/min for 24 h for 6 days. Subsequently, the heart was infused with Triton X-100 and washed by PBS and water for 24 h. The heart volume was measured before and after the recellularization. After macroscopic evaluation, the heart samples were processed and stained by Hematoxylin and Eosin, Masson's Trichrome, Weigert-Van Gieson, Alcian Blue, and Pricrosirius Red techniques for microscopic analysis. To observe the cell adhesion, the recellularization was provided in this scaffold, which was analyzed under immunofluorescence and scanning electronic microscopy.

Results: The protocol provided cells remotion, with adequate concentration of remaining DNA. ECM components as collagen type I, elastin, and glycosaminoglycans were successfully maintained. The scaffold showed a high cells adherence and proliferation in the recellularization process.

Conclusion: According to results, the protocol described in this work preserved the ECM components and the organ architecture, minimizing ECM loss and being possible to state that it is a promising approach to tissue bioengineering.

Relevance for patients: This study provides a protocol for whole porcine heart decellularization, which will ultimately contribute to heart bioengineering and may support further studies on biocompatibility relationship of new cells with recellularized scaffolds.

Keywords: heart; porcine; protocol; scaffold; tissue engineering.

Figure 1. Macroscopic images. (A) Native porcine heart. (B) Decellularized porcine heart, showed that the heart was decellularized, with a greater volume left ventricular retained.

Figure 2. DNA measurement in native and decellularized heart samples. T tests analysis, with significant differences for **p≤0.05. Values expressed as mean and standard deviation.

Figure 3. Microscopic analysis of native and decellularized porcine hearts. (A) Native heart – Cardiomyocyte nucleus shown in an arrow, stained by hematoxylin and eosin; (B) Decellularized heart – collagen shown in an arrow, stained by hematoxylin and eosin; (C) Native heart – staining of collagen fibers (dark red) and cardiomyocytes (reddish-pink), stained by Masson’s trichrome; (D) Decellularized heart – collagen fibers highlighted in blue stained by Masson’s trichrome; (E) Native heart – discrete tissue staining, arrow showing discrete area of GAGs, stained by Alcian Blue; (F) Decellularized heart – areas with evidenced GAGs highlighted in greenish-blue, stained by Alcian Blue; (G) Native heart – arrow pointing to a discrete area highlighted in dark red, corresponding to the collagen fiber, interspersed with cardiomyocytes, stained by Weigert-Van Gieson; (H) Decellularized heart – collagen fibers evidenced in dark red, stained by Weigert-Van Gieson; (I) Native heart – collagen fiber highlighted in dark red indicated by the arrow, peripheral to cardiomyocytes, stained with Picrosirius red; (J) Decellularized heart – collagen fibers intensely stained in dark red, stained by Picrosirius red; (K) Native heart – polarized light microscopy highlighting collagen fibers in bright red, stained by Picrosirius red; (L) Decellularized heart – polarized light microscopy with intense highlighting of collagen fibers in pale red, stained by Picrosirius red.

Figure 4. Immunofluorescence images from porcine sample hearts. (A) Native heart – positive immunofluorescence for DAPI; (B) Native heart – positive immunofluorescence for collagen type I; (C) Native heart – positive immunofluorescence for elastin; (D) Decellularized heart – negative immunofluorescence for DAPI; (E) Decellularized heart – positive immunofluorescence for collagen type I; (F) Decellularized heart – positive immunofluorescence for elastin.

Figure 5. Immunofluorescence of recellularized scaffold. (A) Positive immunofluorescence for DAPI; (B) Positive immunofluorescence for vimentin; (C) Merge for DAPI+Vimentin immunostaining; (D) Positive immunofluorescence for DAPI; (E) Positive immunofluorescence for laminin; (F) Merge for DAPI+Laminin immunostaining.

Figure 6. Scanning electron microscopy images. (A) Native heart showing the presence of cells and tissue integrity (10 μm); (B) Decellularized scaffold showing absence of cells and presence of extracellular matrix (5 μm); (C) SEM image from decellularized scaffold by equine muscle fibroblasts 24 h after incubation (10 μm); (D) SEM image from decellularized scaffold by equine muscle fibroblasts 48 h after incubation (10 μm); (E) SEM image from decellularized scaffold by equine muscle fibroblasts 72 h after incubation, with cells well adhered at its surface (10 μm).

Supplementary Figure 1. Experimental design for organ compliance analysis

Supplementary Figure 2. Complacency. Relation of the volume injected into the organ and the pressure exerted on it, both in the heart before and after being decellularized (scatter plot). Statistical analysis t-test did not show significant differences between them.

Supplementary Figure 3. Total collagen. Density in percentage volume of total collagen in native heart and decellularized heart

Supplementary Figure 4. Total GAGs density in percentage volume of GAGs in native heart and decellularized heart