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J Thorac Cardiovasc Surg 1994;108:985-987
© 1994 Mosby, Inc.

LETTERS TO THE EDITOR

Cryopreserved heart valve allografts can have a normal endothelium

Ravinder Mohan, MDa, Xiao J. Feng, MDa, Paul Walter, MD, PhD, FACSa, Arnold Herman, MD, PhDb


Department of Cardiac Surgerya

To the Editor:

The research of Lupinetti and associates (J THORAC CARDIOVASC SURG 1993;106:912-7) would suggest that after cryopreservation very few endothelial cells remain on heart valve allografts. This is not the case.

We have earlier shown that the fate of endothelium depends on the quality of cryopreservation. Grafts that are better cryopreserved have a functionally viable endothelium that produces prostacyclin similar to fresh valves. Under stimulation this production increases, further evidence that cellular processes are not only intact but actively responsive. This evidence is important inasmuch as prostacyclin production is reduced even by sublethal endothelial injury.Go 1

The low endothelial counts of valves cryopreserved by Cryolife Inc. (Marietta, Ga.) and investigated by Lupinetti and colleagues are particularly surprising because endothelial immunohistochemical staining (by either Ulex europeausor factor VIII–related antigen) is possible in aldehyde-fixed paraffin-embedded tissue, which means that all cells, dead or living, would be stained.Go 2 Unless the pre-fixation cryopreservation has been harsh enough to scrape away the endothelium altogether, a combination of stained dead and living cells must result in high cell counts. Our "poorer" cryopreservation technique also could maintain a confluent layer of abundant endothelial cells (Fig. 1), although they were probably dead because their prostacyclin production was negligible. However, their structural appearance and histometric counts were similar to those of both fresh valves (Fig. 2) and "better" cryopreserved valves (Fig. 3), which produced highly significantly more prostacyclin.



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  		Fig. 1. Scanning electron microscopic appearance of a porcine valve after undergoing a "poorer" cryopreservation technique. PMN, Polymorphonuclear leukocytes. (Original magnificationx500.)

 


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  		Fig. 2. Scanning electron microscopic appearance of endothelial cells of a fresh porcine aortic valve. (Original magnificationx500.)

 


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  		Fig. 3. Scanning electron microscopic appearance of a porcine valve after undergoing a "better" cryopreservation technique. BL, Basal lamina. (Original magnification x500).

 
In contrast to what Lupinetti and coworkers write, Yankah and associatesGo 3 were not the first to report viabilityof endothelial cells on cryopreserved valve allografts because they too could not differentiate between dead or living endothelium by staining factor VIII–related antigen. Van der Kamp and colleaguesGo 4 were the first to study endothelial viability, but after "optimal" cryopreservation of "fresh" valves they found 88% fibroblast viability and 0% endothelial viability. However, their technique of radiolabeled proline incorporation was suited more to functions of collagen laying fibroblasts.Go 4 Our research was the first to demonstrate that after better cryporeservation endothelial function is almost similar to that of normal endothelium.

Cryopreservation must aim to preserve a viable endothelial lining, inasmuch as its loss could be the main reason behind the accelerated thrombosis, calcification, and infection of commercial bioprosthetic valves.Go 5 WeGo 6 have recently shown that endothelial structure and function remains unaffected after exposure to an antibiotic combination with low-dose amphotericin B, and others showed earlier that with a 24-hour antibiotic exposure immunogenicity is also markedly reduced.Go 7

In any case, those who find a lack of viable endothelium on their cryopreserved grafts must review their technique of cryopreservation, because the quality of preservation of the remaining valve may also then be suspect.

References

  1. Feng XJ, van Hove CE, Mohan R, et al. Improved endothelial viability of heart valves cryopreserved by a new technique. Eur J Cardiothorac Surg 1992;6:251-5.[Abstract]
  2. Holden CA, Spaull J, Williams R, et al. The detection of endothelial cell antigens in cutaneous tissue using methacarn and periodate lysine paraformaldehyde fixation. J Immunol Methods 1986;91:45-52.[Medline]
  3. Yankah AC, Wottge HU, Muller-Hermelink HK, et al. Transplantation of aortic and pulmonary allografts, enhanced viability of endothelial cells by cryopreservation, importance of histocompatibility. J Cardiac Surg 1987;1(suppl):209-20.
  4. van der Kamp AWM, Visser WJ, van Dongen JM, Nauta J, Galjaard H. Preservation of aortic heart valves with maintenance of cell viability. J Surg Res 1981;30:47-56.[Medline]
  5. Ferrans VJ, Spray TL, Billingham ME, et al. Structural changes in glutaraldehyde treated porcine heterografts used as substitute cardiac valves. Am J Cardiol 1978;41:1159-84.[Medline]
  6. Feng XJ, van Hove CE, Mohan R, et al. Effects of different antibiotics on the endothelium of the porcine aortic valve. J Heart Valve Dis 1993;2:694-704.[Medline]
  7. Yacoub MH. Applications and limitations of histocompatibility in clinical cardiac valve allograft surgery. In: Yankah AC, Hetzer R, Miller DC, et al. eds. Cardiac valve allografts 1962-1987. New York, Springer, 1988:95-102.



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