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J Thorac Cardiovasc Surg 1995;110:282-284
© 1995 Mosby, Inc.

LETTERS TO THE EDITOR

Invited letter concerning: Biochemical and cellular characterization of cardiac valve tissue after cryopreservation or antibiotic preservation (J THORAC CARDIOVASC SURG 1994;108:63-7)

Auckland, New Zealand

To the Editor:

The vexing question of whether aortic valve allografts survive longer when their donor cell population is viable at implantation has been debated now for more than 20 years. Truly conclusive evidence in favor of maintaining cell viability during the preparation and storage of grafts is still lacking. Most follow-up studies of so-called "viable" valve grafts have not attempted to establish the persistence of donor cells in the explanted valves or to define the histologic status of the grafts. A notable exception is the paper of O'Brien and colleagues, ¹ who demonstrated persistent donor cells in one single case of the series they studied. Despite good cellularity, however, this graft did not exhibit normal leaflet architecture. Without adequate cytologic and histologic information on the "viable" graft at the end of its useful life, the possibility remains ² that the presence of live cells at implantation simply indicates a better overall condition of the connective tissue, especially the collagen.

Very recently, we have shown that significant changes in the radial, elastic stretch properties of aortic valve leaflets occur both during life in the normal human population ³ and in aorticallografts after implantation. ⁴ These biomechanical changes are of such large magnitude that they must be an important cause of central incompetence and, ultimately, of allograft failure. We found that the mode of preparation of the allograft and its degree of cellularity at explantation had little bearing on the loss of leaflet stretch that occurred during implantation. However, the elastic stretch for aortic valve leaflets was strongly dependent on the donor age, which suggests that other properties of the leaflet tissue may also undergo significant change with age.

Whatever inherent advantages "viable" valve grafts may possess in respect of their clinical durability, it is advisable to remember that the doctrine of "immunologic privilege," on which the viability theory was founded, has now been seriously challenged, and it can no longer be assumed that donor cells alive at implantation will necessarily survive and function indefinitely. Experimental studies have clearly shown that aortic valves are indeed antigenic and that the degree of antigenicity is related to the degree of histocompatibility between donor and recipient. ^5,6 Because it is not practicable to tissue-match valve grafts to their recipients, it is likely that donor cells persist after implantation only if there has been a fortuitously good tissue match. Noteworthy in this regard is the frequently overlooked investigation of Wheatley and McGregor. ⁷ Using a dog model, these investigators showed that the cell population of known viable allograft valves explanted after 1, 4, or 8 weeks could be of either donor or recipient origin. It is also significant that recipient cells have been detected in a clinical graft explanted at 10 months for prolapse of a leaflet, ² although the viability of this valve at implantation is not certain. Thus, when donor cells are detected in clinical explants, the degree of donor-recipient tissue match should also be determined.

The long controversy regarding valve graft viability has been due in large part to the fact that preparation procedures vary from institution to institution, so that accurate comparisons are difficult or impossible to make. Confusion could be minimized if each group determined the preimplantation viability status of its own grafts as routinely prepared. The recent paper by Lang and associates ⁸ in this Journal therefore represents a welcome move in this direction. Two specific aspects of their study raise particularly interesting considerations.

First, the finding that 12 to 24 hours of treatment with an antibiotic solution containing an exceptionally high concentration of amphotericin B, together with an aminoglycoside (gentamicin), does not alter viability, proliferation, or metabolic status of the valves. Both the aminoglycosides and amphotericin are known to be markedly toxic to valve leaflet cells. ⁹ We ¹⁰ have found that cell viability declines markedly or is completely lost after 24 hours of exposure to an antibiotic solution without any aminoglycoside and with only one-twentieth the concentration of amphotericin used by Lang's group. ¹¹ Therefore, factors other than the composition of the sterilizing solution and the duration of exposure to it must be involved in determining the effects on cell viability. The interval between donor death and procurement of the valve (i.e., the period of autolysis) is a probable major influence, as hypoxically injured cells can be expected to be more susceptible to noxious substances than normal ones. The valves used by Lang and associates were all collected within 9 hours of donor death, whereas ours were collected for up to 30 hours after death.

The second point of interest is the fact that all cells cultured were identified as endothelial cells, even in the control tissue samples. Inasmuch as the latter had only 9 hours or less of postmortem autolysis, not only the dominant intracuspal cells of the valve leaflet (fibroblasts) but also the smooth muscle cells of the aortic wall could have been expected to remain viable in sufficient numbers to produce cultures. ^10,12 It is difficult to speculate on the possible reasons for the failure of these cell types to grow when details of the culturing technique are not provided in the paper. It is perhaps significant that the range of immunocytochemical probes used for the cell-type recognition did not include the commercially available antibody specific for cultured fibroblasts.

The paper of Lang and associates ⁸ focuses our attention on the precise significance of viability relative to the function of the aortic allograft. Key issues that still require clarification are as follows:

1. How should "viability" be defined and assessed? Are persistent live cells that survive preparation and storage procedures and are capable of giving rise to cultures still necessarily fully functional?
   
2. What is the actual value of "viable" cells in the preimplantation and postimplantation contexts? Do these cells serve simply to indicate the overall quality of valve leaflet connective tissue before implantation? If they do survive implantation in the long term, what interrelationships exist between cell function and the physical and mechanical conditions to which valve grafts are subjected?
   
3. What is the likely relevance of these considerations to the overall biomechanical function of the allografts?
   

References

1. O'Brien MF, Stafford EG, Gardner MAH, et al. Comparison of aortic valve replacement with viable cryopreserved and fresh allograft valves, with a note on chromosomal studies. J THORAC CARDIOVASC SURG 1987;94:812-23.
   
2. Gonzalez-Lavin L, Spotnitz AJ, Mackenzie JW, et al. Homograft valve durability: Host or donor influence? Heart Vessels 1990;5:102-6.
   
3. Christie GW, Barratt-Boyes BG. Age-dependent changes in the radial stretch of human aortic valve leaflets determined by biaxial testing techniques. Ann Thorac Surg [In press].
   
4. Christie GW, Barratt-Boyes BG. The biaxial mechanical properties of explanted aortic allograft leaflets. Ann Thorac Surg [In press].
   
5. Thiede A, Timm C, Bernhard A, Muller-Ruchholtz W. Studies on the antigenicity of vital allogeneic valve leaflet transplants in immunogenetically controlled strain combinations. Transplantation 1978;26:391-5.
   
6. El Khatib H, Lupinetti FM. Antigenicity of fresh and cryopreserved rat valve allografts. Transplantation 1990;49:765-7.
   
7. Wheatley DJ, McGregor CGA. Post implantation viability in canine allograft heart valves. Cardiovasc Res 1977;11:78-85.
   
8. Lang SJ, Giordano MS, Cardon-Cardo C, Summers BD, Saiano-Coico L, Hajjar DP. Biochemical and cellular characterization of cardiac valve tissue after cryopreservation or antibiotic preservation. J THORAC CARDIOVASC SURG 1994;108:63-7.
   
9. Hu J-F, Gilmer L, Hopkins R, Wolfinbarger L. Effects of antibiotics on cellular viability in porcine heart valve tissue. Cardiovasc Res 1989;23:960-4.
   
10. Armiger LC. "Viability" studies of human valves prepared for use as allografts. Ann Thorac Surg [In press].
    
11. Strickett MG, Barratt-Boyes BG, MacCulloch D. Disinfection of human heart valve allografts with antibiotics in low concentration. Pathology 1983;15:457-62.
    
12. Poole CA, Brookes NH, Gilbert RT, et al. Detection of viable and non-viable cells in connective tissue explants using the fixable fluoroprobes 5-chloromethylfluorescein diacetate and ethidium homodimer-1. Connect Tissue Res [In press].
    

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