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HISTOLOGICAL AND BIOCOMPABILITY EVALUATION OF THE BOVINE PERICARDIUM AFTER SOFT DESCELLULARIZARION PROCESS FOR THE USE IN VALVE BIOPROTHESIS
Bioprosthetic heart valves are extensively applied in patients with valvulopathies requiring valve replacement. However, conventional bioprosthesis present limitations related to its rupture and calcification, mainly by the use of chemical crosslinkers.
Objetivos - Metodologia - Resultados - Discussão dos Resultados/Objectives - Methodology - Results - Discussion of Results/Objetivos - Metodología - Resultados - Discusión de los resultados
In this context, aiming the development of a valvular bioprosthesis that overcome these limitations, the purpose of this study is to evaluate the structural integrity and biocompatibility of the bovine pericardium (BP) after a soft decellularization process with a 0.1% sodium dodecyl sulfate (SDS) solution. The efficacy of BP decellularization was accessed by extracting genomic DNA followed by agarose gel electrophoresis, Hematoxylin/Eosin staining (H&E) and nuclear labeling with 4',6-diamino-2-phenylindole (DAPI). Immunofluorescence was performed in order to visualize the proteins of the extracellular matrix (ECM) and the immunogenic xenoantigen galactose-alpha-1,3-galactose (alpha-gal), found on the membrane of mammalian cells except humans and higher primates. The biocompatibility of the tissue was evaluated by its culture with mesenchymal stem cells derived from human adipose tissue (hASCs). Thus, decellularization process promoted a mean reduction of 77% of the amount of DNA in the samples. Cell nuclei were not visible in H&E and DAPI staining, despite the presence of DNA bands larger than 200 bp in the agarose gel. The content and arrangement of type I, III, IV collagens, decorin and elastin were not altered with the decellularization. However, laminin and lumicam exhibited reduced tissue labeling, as well as the cytoskeleton protein vimentin. Scanning electron microscopy showed a loosening of the fibers on the fibrous face of the decellularized tissue compared to the fresh one. In addition, the decellularization protocol has proven to be efficient in removing the xenoantigen alpha-gal. The decellularized BP was non-cytotoxic in vitro and allowed the adhesion of hASCs after 24 h in culture. Finally, after 7 days in culture the tissue scaffold became repopulated by hASCs and after 30 days the ECM protein pro-collagen I was seen in the repopulated scaffold. Taken together, these results strengthen the idea that a balance between elimination of imunnogenic components, resorting to soft chemical treatment, and preservation of the tissue structure is desirable to ultimately achieve an active pericardium prone to recellularization.
Considerações Finais/Final considerations/Consideraciones finales
These characteristics indicate that soft BP decellularization with 0,1% SDS solution allows the acquirement of a suitable scaffold for cell repopulation and for the development of valvular bioprostheses.
Palavras-chave/Key words/Palabras clave
Valvular bioprothesis, bovine pericardium, extracellular matrix, tissue decellularization
MARCO AUGUSTO STIMAMIGLIO, Marina Augusto Heuschkel, João Gabriel Roderjan, Paula Hansen Suss, César Augusto Oleinik Luzia, Francisco Diniz Affonso da Costa, Alejandro Correa