Successful decellularization of thick-walled tissue: Highlighting pitfalls and the need for a multifactorial approach

Abstract

Introduction: Decellularization of thick tissue is challenging and varying. Therefore, we tried to establish a multifactorial approach for reliable aortic wall decellularization. Methods: Porcine aortic walls were decellularized according to different procedures. Decellularization was performed for 24 (G1), 48 (G2), and 72 h (G3) with a solution of 0.5% desoxycholate and 0.5% dodecyl sulfate. The procedure was characterized using intermittent washing steps, the inclusion of sonication as well as DNase and α-galactosidase treatment. The decellularization efficiency was measured by the evaluation of 4′,6-diamidino-2-phenylindole and hematoxylin and eosin staining and quantitative DNA assays. Pentachrome and picrosirius red staining, scanning electron microscopy as well as glycosaminoglycan assays were performed to evaluate the effect of the procedure on the extracellular matrix. Results: 4′,6-Diamidino-2-phenylindole and hematoxylin and eosin staining revealed a large amount of remaining nuclei in all groups. However, consecutive DNase treatment had a significant effect. While the remaining DNA was detected in some samples of G1 and G2, samples of G3 were fully decellularized. Glycosaminoglycan content was significantly reduced to 50% after 24 h (G1) but remained constant for G2 and G3. Picrosirius red staining revealed an intact and stable collagen network without any visible defects. Pentachrome staining substantiated these results. Nonetheless, the fiber network remains intact, which could be confirmed by reflection electron microscopy analysis. Conclusion: In this study, we developed a procedure that grants successful decellularization of porcine aortic wall while maintaining the fibrous microstructure. We highlighted the significant effect of DNase and α-galactosidase treatment. In addition, we could show the need for a multifactorial treatment and comprehensive evaluation protocols for thick tissue decellularization.

Re‐endothelialization of non‐detergent decellularized porcine vessels

“…Of note, H&E staining was less sensitive than DAPI staining when analyzing cellular remnants in decellularized tissues (Figure 1). The insensitivity of H&E staining compared to DAPI staining was reported by others as well 30‐32 and is most likely caused by the different mechanisms of action. Hematoxylin stains the acidic part of the cell, that is, the nucleic acids, by a complex, incompletely understood reaction, whereas DAPI specifically intercalates in the AT regions of dsDNA and experiences an enhancement of fluorescence upon intercalation with DNA.…”

Recent Trends in Decellularized Extracellular Matrix Bioinks for 3D Printing: An Updated Review

“…Small amounts of DNA such as 50 ng DNA are usually considered as decellularized properly [52,97,179,180,181,182]. The amount of collagen and other ECM components can also be determined through assays such as the hydroxyproline assay [179,180,181,182]. The bioprinting process can adversely affect cells and therefore cell viability assays must be performed.…”

Angiogenesis and Re-endothelialization in decellularized scaffolds: Recent advances and current challenges in tissue engineering

“…After perfusing endothelial cells within the acellular scaffold, the results represented a significant advancement in endothelialization and whole-organ bioengineering ( Baptista et al, 2011 ). Detergents with charged hydrophilic headgroups (Ionic agents) are remarkably effective during decellularization and DNA/cytoplasmic membrane solubilization ( Weymann et al, 2013 ; Peloso et al, 2015 ; Hussein et al, 2016 ; Mao, 2017 ; Schmitt et al, 2017 ; Koenig et al, 2019 ). Compared to Triton X-100, SDS, an ionic detergent, is more capable of removing the pathogenic and antigenic epitopes, leading to more complete decellularization ( Hussein et al, 2016 ).…”