Impact of cryopreservation on extracellular matrix structures of heart valve leaflets

Background. Transplantation of cryopreserved allografts represents a well-established valve replacement option. Despite their clinical use for more than 40 years, the integrity of the extracellular matrix (ECM) of these valves after thawing has not been determined. The purpose of this study was to investigate and compare ECM structures of fresh and cryopreserved porcine heart valve leaflets with special emphasis on the condition of collagenous and elastic fibers. Methods. Pulmonary valves were excised from unprocessed porcine hearts under sterile conditions. After treatment with antibiotics, the valves were incubated in a cryoprotective solution, cryopreserved stepwise, and stored at -196 degrees C for 1 week. Two groups of heart valves (fresh untreated and thawed cryopreserved [each, n = 8]) were analyzed using biochemical (collagen, elastin, desmosine), histologic (hematoxylin-eosin, Movat-pentachrome, resorcin-fuchsin), and immunohistochemical (antibodies against collagen I, III, IV, and elastin) methods. Near-infrared femtosecond multiphoton laser scanning microscopy and second harmonic generation were used for high-resolution three-dimensional imaging of ECM structures. Results. Biochemical testing demonstrated similar amounts of collagen and desmosine, but a minor loss of elastin in the cryopreserved specimens. Conventional histology revealed almost comparable cell and ECM formations in fresh and cryopreserved valve leaflets. In contrast, laser-induced autofluorescence imaging showed substantial ultrastructural deterioration and disintegration of most collagenous structures. Second harmonic generation was not inducible. Conclusions. Conventional cryopreservation of heart valves is accompanied by serious alterations and destruction of leaflet ECM structures, specifically demonstrated by multiphoton imaging. Further in-depth studies to clarify the impact of alternative cryopreservation techniques proposed for clinical use, such as vitrification, are crucial.