Cryopreservation of Rat Aortic Valves Results in Increased Structural Failure

Abstract

Background-The cause of valve allograft failure is most likely multifactorial and may include mechanical, immunological, and other factors. Cryopreservation of these valves is often used to extend storage times. However, there has been considerable confusion as to the effects of cryopreservation on valve durability. Our objective was to determine the effects of cryopreservation on histopathological changes in rat aortic valve grafts. Methods and Results-Syngeneic rat aortic valve grafts (Lewis to Lewis; nϭ24) and allogeneic rat aortic valve grafts (Brown Norway to Lewis; nϭ24) were implanted infrarenally, either fresh or after cryopreservation. At 7, 14, and 28 days, the valves were explanted, and histological and immunohistochemical examinations were performed in a blinded fashion. Fresh syngeneic graft leaflets retained their normal structure for the 28-day period of observation. Cryopreserved syngeneic grafts showed retrovalvar thrombus formation, with leaflet destruction at 7, 14, and 28 days. Fresh allogeneic graft leaflets showed significant leaflet thickening and progressive destruction at 14 and 28 days. Cryopreserved allogeneic grafts had evidence of retrovalvar thrombus formation with leaflet destruction at 7, 14, and 28 days. Cryopreserved syngeneic grafts resulted in significant infiltration of mononuclear (ED1 ϩ ) cells not seen with fresh syngeneic grafts but similar to fresh allogeneic grafts. All allogeneic grafts resulted in significant infiltration of T-lymphocytes (CD3 ϩ , CD8 ϩ , CD43 ϩ ). Conclusions-Cryopreservation appears to predispose syngeneic and allogeneic rat aortic valve leaflets to accelerated injury and destruction.

Fresh Homografts Obtained through a National Organ-Sharing Programme for Repair of Congenital Heart Disease

“…13 Experiments in animals have demonstrated that viable homografts induce immunological activation, and cryopreservation might result in increased structural failure. 14,15 Studies in humans have so far failed to show a relationship between HLA antibodies and homograft survival, but decellularized homografts with diminished antigenicity are currently being studied. 16,17 It is possible that non-viable antibiotic-preserved homografts might also have reduced antigenicity and antibody reaction.…”

HMG-COA Reductase Inhibitors Do not Attenuate the Inflammatory Response Associated With Glutaraldehyde-Fixed Bioprosthetic Heart Valve Conduits

“…Based on previous studies, a 28-day study period was sufficient to demonstrate negative infiltrative changes within the implanted valve. 40,[68][69][70] During the first post-operative week, the recipient animals underwent a baseline rodent ultrasonographic analysis. During the last week of the study period, the recipient once again underwent a follow-up ultrasonographic analysis.…”

“…The degree of cellularity/inflammation of the valve, media, and adventitia were scored on a four-point scale, similar to what has been previously reported ( Figure 6). 40,[68][69][70] The scoring scheme was as follows: 0, no inflammation/infiltration (<5%); +, mild inflammation/infiltration (5-24%); ++, moderate inflammation/infiltration (25-49%); +++, severe inflammation/infiltration (50-74%); and media and commissural areas of the explanted valve conduit were also commented on.…”

“…Only a handful of studies have been performed using small animal in vivo methodology to address questions relating to prosthesis durability for VHD. 40,[68][69][70]118 To address clinical applicability (Figures 4 & 5) and physiologic variability with in vivo work (Figures 6 & 7), the following techniques were employed. …”

“…129 Furthermore, studies that have employed similar in vivo rodent models have varied in their approach to quantifying their histological findings and their specific protocols remain unclear. 40,[68][69][70] To ensure integrity, our protocol followed a very conservative approach. Quantification of immunohistochemistry remains even more challenging, particularly if differences are subtle.…”