Proteins and bioprosthetic calcification in the rat model.

Background and aim of the study: The mechanism of valvular bioprostheses calcification is still unknown, but early studies showed increased Gla-protein content in calcified valves, Using an experimental model, which reproduces the clinical process, we therefore analyzed the role of minerals and proteins in bioprosthetic valvular calcification. Methods: Glutaraldehyde pretreated porcine valves were studied before and after implantation in rats by X-ray, calcium (Ca) and phosphorus (P) measurement, Fourier Transform Infrared (FTIR) spectroscopy, SDS-PAGE and Ca-45 ligand blotting of the extracted proteins. Results: Before implantation there was no X-ray calcification with very little Ca and P content. After implantation, X-ray calcifications appeared on day seven with increased Ca and P up to day 35 (p<0.05, ANOVA). FTIR revealed structural proteins alone before implantation, plus minor proportions of lipids on day two, which always preceded Ca and P appearance. Ca and P increased up to day 35, first as amorphous and changed in carbapatite over time. SDS-PAGE before implantation revealed two proteins (66-kD and 54-kD) alone, which were sustained up to day 35. The 66-kD had Ca-45 affinity. On day two, many other proteins appeared on SDS-PAGE, four of which (52, 45, 14 and below 14-kD) with Ca-45 affinity. Protein pattern did not change from day two to 35. Conclusions: Valvular bioprosthesis calcification is associated with progressive increase in Ca and P content and at least five calcium-binding proteins: one intrinsic valvular protein, pre-existing to implantation, plus four other, extrinsic valvular proteins adsorbed within the tissue after implantation.