Molecular and Biomechanical Clues From Cardiac Tissue Decellularized Extracellular Matrix Drive Stromal Cell Plasticity.

Gabriel Romero Liguori,Joris A Van Dongen,Luiz Felipe Pinho Moreira,Sérgio Rodrigues De Moraes,Vincenzo Terlizzi,Prashant K Sharma,Viktor Sinkunas,Martin Conrad Harmsen,Tácia Tavares Aquinas Liguori

doi:10.3389/fbioe.2020.00520

Abstract

Decellularized-organ-derived extracellular matrix (dECM) has been used for many years in tissue engineering and regenerative medicine. The manufacturing of hydrogels from dECM allows to make use of the pro-regenerative properties of the ECM and, simultaneously, to shape the material in any necessary way. The objective of the present project was to investigate differences between cardiovascular tissues (left ventricle, mitral valve, and aorta) with respect to generating dECM hydrogels and their interaction with cells in 2D and 3D. The left ventricle, mitral valve, and aorta of porcine hearts were decellularized using a series of detergent treatments (SDS, Triton-X 100 and deoxycholate). Mass spectrometry-based proteomics yielded the ECM proteins composition of the dECM. The dECM was digested with pepsin and resuspended in PBS (pH 7.4). Upon warming to 37°C, the suspension turns into a gel. Hydrogel stiffness was determined for samples with a dECM concentration of 20 mg/mL. Adipose tissue-derived stromal cells (ASC) and a combination of ASC with human pulmonary microvascular endothelial cells (HPMVEC) were cultured, respectively, on and in hydrogels to analyze cellular plasticity in 2D and vascular network formation in 3D. Differentiation of ASC was induced with 10 ng/mL of TGF-β1 and SM22α used as differentiation marker. 3D vascular network formation was evaluated with confocal microscopy after immunofluorescent staining of PECAM-1. In dECM, the most abundant protein was collagen VI for the left ventricle and mitral valve and elastin for the aorta. The stiffness of the hydrogel derived from the aorta (6,998 ± 895 Pa) was significantly higher than those derived from the left ventricle (3,384 ± 698 Pa) and the mitral valve (3,233 ± 323 Pa) (One-way ANOVA, p = 0.0008). Aorta-derived dECM hydrogel drove non-induced (without TGF-β1) differentiation, while hydrogels derived from the left ventricle and mitral valve inhibited TGF-β1-induced differentiation. All hydrogels supported vascular network formation within 7 days of culture, but ventricular dECM hydrogel demonstrated more robust vascular networks, with thicker and longer vascular structures. All the three main cardiovascular tissues, myocardium, valves, and large arteries, could be used to fabricate hydrogels from dECM, and these showed an origin-dependent influence on ASC differentiation and vascular network formation.

Highlights

Decellularized organ-derived extracellular matrix has been used for many years in tissue engineering and regenerative medicine (Conklin et al, 2002; Dahl et al, 2003; Schenke-Layland et al, 2003)
The tissues were washed at room temperature (RT) in phosphate buffer saline (PBS) and triturated in a commercial blender until pieces were smaller than 1 mm
Porcine cardiac components i.e., left ventricle, mitral valves, and aorta were subjected to a rigorous decellularization procedure to generate cell-free extracellular matrix

Summary

Introduction

Decellularized organ-derived extracellular matrix (dECM) has been used for many years in tissue engineering and regenerative medicine (Conklin et al, 2002; Dahl et al, 2003; Schenke-Layland et al, 2003). Decellularization of the extracellular matrix can be achieved through different physical, chemical or biological processes, including freeze/thaw cycles, use of organic detergents, and mild treatment with proteolytic enzymes (Crapo et al, 2011; Hrebikova et al, 2015; Keane et al, 2015; Leonel et al, 2017; Kawecki et al, 2018). Smaller ECM constituents such as growth factors, chemokines, and other small signaling molecules are largely washed out

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