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

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 Liguori et al. Tissue-Specific Hydrogels Drive Cell Plasticity 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.

Engineering New Microvascular Networks On‐Chip: Ingredients, Assembly, and Best Practices

“…Further, dECM has been reported as promoting angiogenesis within constructs in vitro or post-implantation. [128][129][130][131] Interestingly, one report proved that the mechanical stiffness and viscosity of a dECM hydrogel can be adjusted by addition of vitamin B2. [132] Other reports detailed that dECM modification changed the resulting microvascular network phenotype, highlighting both successful use of dECM for vascularization and methods of tuning the material for a desired microvascular network quality.…”

Cell-Based Therapies for Myocardial Regeneration in Heart Failure: 20 Years of Debate

“…Lastly, hydrogels-based approaches for tissue engineering are also a promising alternative for cardiac regeneration in models of HF. The hydrogels can be obtained from multiple sources such as collagen, proteoglycans, synthetic polymers, and from specific cardiac compartments [ 11 ] . The application form is mainly related to direct delivery to the myocardium, stimulating angiogenesis, and providing cell growth factors.…”

Architecture and Composition Dictate Viscoelastic Properties of Organ-Derived Extracellular Matrix Hydrogels

“…Discrepancies exist with our colleagues, where a fourth Maxwell element in LV-ECM hydrogels is reported [26]. Compared to the protocol from our colleagues, we employed two hours more for pepsin digestion [26]. The pepsin digestion time influences organ-derived ECM hydrogel mechanics, which could explain the differences observed [29].…”

“…Overall, these findings demonstrate that organ-specific ECM composition idiosyncrasies remain present after decellularization. Discrepancies exist with our colleagues, where a fourth Maxwell element in LV-ECM hydrogels is reported [26]. Compared to the protocol from our colleagues, we employed two hours more for pepsin digestion [26].…”