Decellularization of allogeneic and xenogeneic tissues for cardiovascular reconstruction; assessment of a NaOH based process and perspectives

Abstract

Current biological cardiovascular substitutes are hindered by inability to grow and fast degeneration of these tissues occurs in the adult and more precociously in child. This tissue deterioration is probably related to host immune reaction and glutaraldehyde cytotoxicity. Tissue decellularization/desantigenization is an interesting way to reduce immune reaction and to avoid the need of glutaraldehyde to mask antigenicity. Clinical results with decellularized patches, vessels or valves are promising for some studies while poor results may be associated with inadequate decellularization. No Gold standard decellularization process exists. Most of used processes are detergent-based. Our primary experimental work aims to evaluate a sodium hydroxide-based process as a decellularization agent. This process possess an additional propriety which is to inactivate conventional and non-conventional pathogens (such as prions) that other processes do not include. We assessed the efficacy of this processing in xenogeneic and allogeneic patches (patch study), porcine vessels (conduit study) and porcine valves (valve study). In each study, the tissues were firstly investigated in vitro for decellularization (cellularity was assessed by Hematoxylin and eosin-DAPI-DNA; antigenicity by IHC for alpha-Gal/MHC-I) and for mechanical resistance (by elongation stress test or compression test). Secondly, tissues were assessed in in vivo models (subcutaneous and intravascular) for inflammation (IHC for CD3/CD68), calcifications (von Kossa/Ca2+ tissue content) and vascular remodeling (IHF asma and CD31). In the patch study, adequate decellularization of porcine, bovine pericardia, peritoneum and human pericardium was obtained. In vitro, our Decellularized bovine pericardium (DBP) showed better decellularization in comparison to otherpericardia (two commercially available pericardia: Synovis pericardium (decellularized with another NaOH based-process and fixed in glutaraldehyde) and the Edwards pericardium (not decellularized et glutaraldehyde-fixed). After 4 months, in a vascular rodent model (n=15), the DBP used as a patch on the abdominal aorta, showed lower postoperative complications, lower calcifications, lower inflammation and better vascular remodeling than Synovis pericardium. In the conduit study, the entire porcine carotid artery was well decellularized. After 1 month, in a rodent vascular model (n=5), its showed low inflammation, partial recellularization and vascular remodeling. In the porcine carotid interposition model (n=2), the decellularized carotid was partially recellularized and showed less calcification, less inflammation, less intimal hyperplasia and better remodeling than PTFE/ synthetic prosthesis (control). Eventually, in our valve study, the porcine pulmonary valve was better decellularized with the NaOH-based process in comparison to a detergent process. In vivo, in a growing orthotopic implantation porcine model (n=3), after 1 month (n=1) and 3 months (n=2), the processed valves showed lower inflammation, partial recellularization and absence of major structural deterioration. In conclusion, we demonstrated the sodium hydroxide-based process can be proposed in order to obtain a decellularization of a wide range of cardiovascular substitutes. This tissue processing must therefore, be considered as an effective tool in the field of vascular tissue reconstruction.</p