Interstitial cellular and matrix restoration of cardiac valves after cryopreservation

Objectives: We previously characterized the porcine aortic leaflet intersti tial cell phenotype as having both synthetic and contractile characteristic s; that is, it is a myofibroblast, In this study we hypothesized (1) that t he cryopreservation of aortic valves causes a significant reduction in cell density, (2) that it simultaneously causes alterations in representative c omponents of extracellular matrix, and (3) that both of these processes are reversible. Methods: Seventy-two leaflets from 24 porcine aortic valves we re studied. Whole valves were subjected to variable lengths of preharvest i schemia (group 1), ischemia followed by processing analogous to clinical me thods (group 2), and ischemia followed processing with an organ culture typ e of resuscitation (group 3), Vital dye exclusion by cells enzymatically di spersed from leaflets was used to quantify viability. Electron and light mi croscopy, immunohistochemical assay, and a silicone rubber substratum contr actility assay were used both in dispersed cell preparations and in leaflet cross sections to examine structural, ultrastructural, and functional chan ges across the 3 groups through a range of preharvest ischemic times. Resul ts: Results indicated that harvest ischemic periods between 2 and 24 hours after donor death were not responsible for cell number reductions. During t his interval overt dissolution of chondroitin sulfate simultaneous with a r elative sparing of fibronectin was evidenced by immunohistochemical stainin g. Although not reduced in number, ischemic interstitial cells did show sig nificant ultrastructural evidence of injury and suppressed monoclonal bindi ng to vimentin and cl-smooth muscle actin, After cryopreservation, viable c ell numbers were always markedly reduced at all ischemic intervals and dama ge to both soluble extracellular matrix components and cell ultrastructure was increased. At all time and processing points, however, some retention o f matrix secretory and cellular contractile capabilities was observed among the surviving cells. After the extended periods of preharvest ischemia (2- 24 hours) followed by processing, a restitution of functioning cells was ac complished by means of whole-leaflet incubation in 15% fetal bovine serum, Conclusions: After application of the described methods, new cells within r estored intact leaflets as well as in single-cell preparations demonstrated normal ultrastructure and contractile and synthetic functions (normal phen otypic expression), If functioning leaflet interstitial cells can contribut e to homograft durability, bioengineering methods for pretransplantation ce ll repopulation could be refined with these techniques and applied to clini cal valve transplantation.