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J Thorac Cardiovasc Surg 2001;121:500-509
© 2001 The American Association for Thoracic Surgery

Surgery for Acquired Cardiovascular Disease

Dynamic in vitro calcification of bioprosthetic porcine valves: Evidence of apatite crystallization

Elena Pettenazzo, PhDa, Michael Deiwick, MDb, Gaetano Thiene, MDa, Gianmario Molin, BScc, Birgit Glasmacher, PhDd, Fedora Martignago, BScc, Tomaso Bottio, MDa, Helmut Reul, PhDd, Marialuisa Valente, MDa

From the Departments of Pathologya and Mineralogy & Petrology,c University of Padua Medical School, Padua, Italy; the Department of Cardiothoracic Surgery,b Münster, Germany; and the Helmholtz-Institute for Biomedical Engineering,d Aachen, Germany.

This study was supported by the Istituto Superiore di Sanità, Progetto "Protesi Endovascolari," by MURST, and by the Veneto Regione, Rome and Venice, Italy.

Received for publication April 26, 2000. Revisions requested July 24, 2000, revisions received Oct 11, 2000. Accepted for publication Oct 19, 2000. Address for reprints: Marialuisa Valente, MD, Department of Pathology, University of Padua Medical School, Via A. Gabelli, 61, 35121 Padova, Italy (E-mail: mvalente{at}ux1.unipd.it).

Objective: Calcification is the most important cause of structural deterioration of glutaraldehyde-fixed bioprosthetic valves. Devitalization of tissue favors calcium deposits in the shape of apatite crystals. Host factors influence the extent and progression of calcification, but the phenomenon can also occur in vitro in the absence of a viable milieu. Whether calcific deposits obtained in vitro are similar to those found in vivo is unknown.
Methods: Four porcine frame-mounted bioprostheses (St Jude Medical Bioimplant; St Jude Medical, Inc, St Paul, Minn) were tested in vitro by using a pulsatile accelerated calcification testing device at a frequency of 300 cycles per minute at 37°C for 19 x 106 cycles with a rapid synthetic calcification solution (final product [calcium x phosphate], 130 mg/dL2). Three of the same type of xenografts explanted from human subjects because of calcific failure (time in place, 108 ± 25.63 mo) served as control grafts. Each sample underwent gross and x-ray examination, histology, transmission and scanning electron microscopy, atomic absorption spectroscopy, electron microprobe analysis, and x-ray powder diffraction methods.
Results: All in vitro bioprostheses were heavily calcific, with intrinsic Von Kossa stain–positive deposits and a mean calcium content of 205.285 ± 64.87 mg/g dry weight. At transmission electron microscopy, nuclei of calcification involved mostly collagen fibers and interfibrillar spaces and, more rarely, cell debris and nuclei. Electron microprobe analysis showed a Ca/P atoms ratio of 4.5:3, a value intermediate between hydroxyapatite and its precursor, octacalciumphosphate. X-ray powder diffraction showed a well-separated and sharp peak, which is typical of hydroxyapatite. Aggregates of plate-like crystals up to 8 µm in size were observed at scanning electron microscopy, with a typical tabular hexagonal shape consistent with apatite. The morphologic and chemical findings in human explants were similar.
Conclusions: Intrinsic calcification of glutaraldehyde-fixed porcine valves was induced in vitro. Electron microprobe analysis and x-ray powder diffraction findings were in keeping with apatite crystallization, such as that occurring in valve xenografts implanted in vivo. The model may be of value to accelerate the screening of anticalcific agents and may reduce the need for animal experiments.


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J. Thorac. Cardiovasc. Surg. 2001 121: 428-430. [Extract] [Full Text] [PDF]



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