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Development and Characterization of a Decellularised Xenogeneic Mitral Valve Scaffold
Marisa Granados, Lucrezia Morticelli, Pavel Yablonski, Andres Hilfiker, Igor Tudorache, Sergei Cebotari, Axel Haverich, Sotirios Korossis.
Department of Cardiothoracic, Transplantation and Vascular Surgery Hannover Medical School, Hannover, Germany.

OBJECTIVE: The aim of this work was to develop and characterize a decellularised mitral valve scaffold for mitral valve replacement.
METHODS: Mitral valves from 6 month old pigs were disinfected, placed in hypotonic buffer and treated with SDS and sodium deoxycholate for 36 hours, followed by extensive washing cycles and nucleic acid digestion. Radial sections comprising annulus, leaflets, chordae tendinae, and papillary muscle were analyzed histologically by H&E and DAPI staining, immunohistochemically by collagen IV, and by alpha-gal fluorescence staining. DNA was extracted from the annulus, anterior leaflet, chordae and papillary muscle, and quantified using a NanoDrop spectrophotometer. Sections of the treated leaflets were analyzed under transmission electron microscopy (TEM), whereas fresh and treated leaflet strips were subjected to uniaxial tensile loading to failure.
RESULTS: Following decellularisation, no cell nuclei were observed under H&E or DAPI staining. There was also no change in the presence of collagen IV. The treatment resulted in a significant decrease of alpha-gal, as observed under fluorescence staining (Fig. 1). DNA content was significantly reduced compared to the native tissue (99%). TEM showed a cell-free decellularised tissue, with a conserved histoarchitecture. The decellularised tissue also demonstrated a grossly-maintained mechanical integrity.

Figure 1. H&E, collagen IV and alpha-gal staining of fresh (A,B,C) and decellularised leaflet (D,E,F).
CONCLUSIONS: A protocol that effectively removed cells and DNA, whilst maintaining the native valve histoarchitecture and mechanical integrity was developed. Although some alpha-gal was still detectable after decellularisation, the significant reduction observed was encouraging. The presence of alpha-gal could potentially be overcome in the clinical setting by the use of alpha-gal knockout porcine tissue. However, analyzing the effect of decellularisation on alpha-gal in wild-type porcine tissue could provide an insight on whether other sugars, also potentially immunogenic, are removed. Future work will focus on optimizing the protocol in order to further decrease the alpha-gal content.

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