Development of bioprosthetic heart valve calcification in vitro and in animal models: morphology and composition

While calcification of bioprosthetic valves presents a clinical problem, th e mechanism of formation of calcific deposits in the leaflets remains uncle ar. A new method for in vitro calcification is employed, in parallel with a n in vivo (subcutaneous animal) model. The nature of crystal phases grown b y both methods on porcine bioprosthetic heart valves was investigated. Ligh t microscopy, scanning electron microscopy (SEM), X-ray energy dispersive s pectroscopy (SEM-EDS) and stoichiometric chemical analysis were used as too ls for the qualitative and quantitative assessment of the calcification pro cess. Photomicrographs from leaflets calcified early in vitro (24-48 h) and in vivo (5-10 days) confirmed that calcification initiated in the central region of the tissue, mainly in fibrosa, and extended with time (after 28 a nd 56 days) in vivo towards the outer surface of the tissue. SEM micrograph s from tissue sections of early in vivo and in vitro calcified leaflets sho wed coexistence of different sizes (1-30 mu m) of crystal phases in contact with tissue fibers. EDS analysis confirmed that the deposits were calcium phosphate salts. Chemical analysis of samples calcified in vivo for longer time periods, showed that the content of the salts was mainly calcium and p hosphate with a Ca/P molar ratio 1.78 and 1.94 after 28 and 56 days, respec tively. These results suggested that the calcification process is followed by a sequential hydrolytic transformation of CaHPO4. 2H(2)O (DCPD) to Ca4H( PO4)(3). 2.5H(2)O (OCP) and to Ca-5(PO4)(3)OH (HAP). Calcium phosphate crys tal phases grown in vitro under controlled conditions and at constant super -saturation provide a successful simulation of the calcification process in the animal model and may be applied in screening new biomaterials with res pect to their potential for calcification. (C) 1999 Elsevier Science B.V. A ll rights reserved.