In vitro calcification of bioprosthetic heart valves: test method validation on prosthetic heart valves

A major reason for the failure of bioprosthetic heart valves is calcification. Various pretreatment methods are developed to reduce the calcification behavior. The effectiveness of these methods has so far been investigated in expensive and time-consuming large animal studies. In order to provide a cost-, animal- and possibly also time-saving method, we developed an accelerated dynamic in vitro calcification test method. In the present study, we validated this method using a comparative test of two differently pretreated groups of porcine heart valve bioprostheses (T6 sodium dodecyl sulfate surfactant versus No-T6 treatment). Each group, whose calcification behavior was known from an animal study, contained N = 4 identical surgically implantable aortic bioprostheses. Calcification onsets, progression and extent were detected by high-speed video (HSV) documentation and microscopy. Structural identification of the deposits was carried out by X-ray powder diffraction (XRD). Semidestructive quantification of the calicifications was done by µ-CT as well as destructive chemical quantification of calcium masses per g tissue dry weight via colorimetry and compelxometry. The histomorphologic localization of the calcifications was examined by von Kossa staining. Structural analysis of the investigated deposits indicated “biological apatite” for both test groups. Histological examination revealed sandwich-like localization of the calcifications in the spongiosa zone of the cusps. Quantification of the calcifications showed a distinctly stronger calcification tendency of the No-T6 compared to the anti-calcifying pretreated T6 bioprostheses. We developed and validated a novel and unique test method for in vitro calcification assessment. The quantitative calcification tendencies of the two test groups are comparable with the results of an in vivo study in sheep of these two test groups, after a total of 20 weeks of testing. The structural findings are in line with published in vivo observations and the histomorphological localization appears as known for porcine prostheses.