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Preservation of aortic heart valves with maintenance of cell viability
JOURNAL
OF SURGICAL
Preservation
30, 47-56 (1981)
RESEARCH
of Aortic
Heart Valves with Maintenance
of Cell Viability
ARTHUR W. M. VAN DER KAMP, M. D.,*,’ WILLEM J. VISSER,* JOHANNES M. VAN DONGEN, PH. D.,* JAN NAUTA, M. D.,t AND HANS GALJAARD, M. D.* *Department
of Cell Biology and Genetics and tDepartment of Thoracic and Cardiovascular Erasmus University, Rotterdam, The Netherlands
Surgery,
Submitted for publication January 15, 1980 The goal of the present study was to develop a procedure for sterilization and infinite preservation of aortic heart valves with maintenance of cell viability. For this purpose the influence of a number of methods of controlled freezing on the viability of the fibroblasts of the aortic valve was tested. Cell viability was assessed quantitatively by the incorporation of [3H]proline by the valve fibroblasts. Controlled freezing at a rate of l”C/min under protection of 10% dimethylsulfoxide yielded the highest number of viable fibroblasts (88%). This preservation method was also tested for its influence on the structural and functional integrity of the valve matrix, which was found to be preserved throughout sterilization and storage. This study is the first to present quantitative data on cell survival, stress-strain characteristics, and the electronmicroscopic structure of collagenic fibrils after sterilization and controlled freezing of aortic valves.
ervation of various cell types and tissues [14, 15, 28, 441, has been used to preserve Transplantation of allovital aortic valves aortic heart valves, but cell survival was offers the possibility for maintenance of the only assessed by qualitative techniques, valve matrix by the fibroblasts [24, 321. i.e., cell culture [34, 431. Several series of such allovital transplantaWe have compared the influence of tions have been reported [3-5,8,10,38,45]. several controlled freezing methods on the Although these transplantations were carmaintenance of viability of aortic valve ried out with fresh valves no qualitative fibroblasts, endothelial cells, and cells of the or quantitative assessment of their viability aortic wall. The capacity for protein synthewas performed. The preservation procedure sis, monitored by the incorporation of [3H]reported by Al-Yanabi et al. can only be proline, was used as a parameter for cell used for short-term storage [ 1, 21, while the viability after preservation. Effects of validity of the assay by which cell survival preservation on the structural and funcwas tested can be criticized [20, 321. tional characteristics were investigated by Long-term preservation will be needed to light microscopy, electron microscopy of assure a proper match between donor and isolated collagenic fib&s, and stress-strain acceptor orifice diameter. Controlled freezmeasurements. Throughout the experiing with cryoprotective agents to low temments sterilization by antibiotics as deperatures has been used successfully as a scribed by Lackey et al. [27] was used, and long-term preservation method maintaining its validity with respect to sterilizing cell viability. The method introduced by capacity and its effect on cell survival were Polge et al. [41], and applied for the prestested. INTRODUCTION
1 To whom requests for reprints should be Sent at: Department of Cell Biology and Genetics, Erasmus University Rotterdam, P.O. Box 1738,3OOO DR Rotterdam, The Netherlands.
MATERIALS
AND METHODS
Both canine and human aortic heart valves were used. The aortic valves were 47
0022-48O4/81/OlOO47-10$01.00/O Copyright All rights
0 1981 by Academic Press. Inc. of reproduction in any form reserved.
48
JOURNAL
OF SURGICAL
RESEARCH:
divided into six equal parts one of which was taken as an internal control. Test for cell viability. Aortic valve tissue attached to a piece of aortic wall was incubated at 37°C under air containing 5% CO, during 2, 4, 6, or 8 hr in Ham’s FlO nutrient medium. The medium was supplemented with fetal calf serum (15%) and 15 PCi [3H]proline per milliliter (sp act 10 mCi . mmole-’ , Radiochemical Centre, Amersham). After incubation the tissue was washed three times for I5 min with cold proline to remove all nonincorporated [3H]proline. After fixation (10% formalin) paraffin sections (5 pm) were processed for autoradiography (Ilford K-2 emulsion). After staining (hematoxylineosin) the microscopic localization and percentage of labeled fibroblasts were determined in five random fields of view (microscopic magnification 400x) of three widely separated sections of each valve piece. The ratio of the percentages for preserved and control valves rendered the percentage of cell survival. Survival of endothelial cells and cells in the aortic wall was determined in material with optimal survival of the valve fibroblasts. To determine whether the presence of [3H]proline in the valves was dependent on cell viability or was related to inadequate removal of nonincorporated proline, incorporation studies were performed with valve pieces which were rapidly frozen and thawed to destroy the cells. To determine whether protein synthesis was essential for the incorporation of r3H]proline such protein synthesis was blocked by cycloheximide (20 pg.cm3-l, Serva). Aortic valve sterilization. Human and canine aortic valves were obtained from autopsy performed without any attempt at sterility within 24 hr after death. The dissected valve together with six pieces of aortic wall was kept in a solution of antibiotics as described by Lackey et al. [271, during 24 hr, at 4°C. Subsequently the pieces of aortic wall and 5 cm3 of nutrient
VOL. 30, NO. 1, JANUARY
1981
medium were tested for growth of aerobic and anaerobic bacteria as well as fungi and yeasts at 4, 20, and 37°C for 3 weeks. Sterility tests were performed both before and after removal of the antibiotics. Sterility was also tested after controlled freezing. The effect of sterilization on cell survival was tested as described above. Preservation procedures. The aortic valve pieces were immersed in Ham’s FlO nutrient medium supplemented with 15% fetal calf serum. After addition of the cryoprotective agent; dimethylsulfoxide, glycerol, or ethylene glycol (Merck) at different concentrations (5, 10, 15, and 20%), the valves were incubated during various time intervals (1, 15, and 30 min) and at different temperatures (4,20 and 37°C). The valves were frozen at a rate of l”C/min, controlled by a Cryoson biological freezer, Type BV-4, connected with a Cryoson pressure tank, Type 200 VLR. At -90°C the valves were rapidly cooled to - 196°C(liquid nitrogen). After storage at - 196°C for a period of 1 week to 9 months the valves were thawed rapidly followed by stepwise dilution of the cryoprotective agent three times for 5 or 10 min in 50, 25 and 0% of the cryoprotective agent or dropwise dilution during 60 min. Subsequently the valves were tested for cell viability. Effects of preservation on the valve matrix. For light microscopy, valves were fixed in 10% formalin and paraffin sections (5 pm) were stained with hematoxylinazophloxine-saffran. Isolated collagenic fibrils were prepared and stained according to the method of Olsen [40]. These fibrils were examined with a Zeiss 9,s. electron microscope. The magnification of the electron microscope was calibrated with acrossgrating replica (2 160lines/mm). The banding pattern of the microfibrils was studied by measurement of at least 1000 periods on electron micrographs of only straight parts of the fibrils. Six valve pieces were investigated for every condition tested. Stress-strain measurements. Such measurements were performed on human thoracic
VAN DER KAMP ET AL.: PRESERVATION
49
OF AORTIC HEART VALVES
FIG. 1. Autoradiograph of a canine aortic valve after incubation with [3H]proline for 6 hr. Note the large number of labeled fibroblasts with thin cytoplasmic extensions. obj. x40.
aortas excised within 24 hr after death [ 171. The tissue was preserved in saline solution before testing. With a blade, model strips of 3.5cm length, l.O-cm width at the ends and 0.6-cm width in the middle part were cut in the radial and circumferential direction. Control strips and strips to be preserved were taken from adjacent locations. The strips were placed on a rifling bench with a cross-head speed of 1.Omm per minute, connected, via a transducer-strain indicator, to a writer with a chart speed of 1.0 cm per minute, yielding a magnification of 10x . The initial length was adjusted to 2.4 cm without buckling or crimping of the tissue. A total of 20 strips, taken from five different aortas, were tested.
Sterility of Aortic Heart Valves Fourteen human aortic valves were sterilized according to the method of Lackey et al. [27]. All valves except one were found to be sterile. Five sterilized valves were tested for contamination after controlled freezing and storage in liquid nitrogen; all were found to be sterile. The effect of sterilization on INCUBATION %Labclled 100 1
TIME WITH 3H- PROLINE cells
RESULTS
Determination of Cell Viability The percentage fibroblasts of fresh canine valves labeled with [3H]proline as shown by autoradiography (Fig. 1) was determined after incubation periods of 2,4,6, and 8 hr. Figure 2 shows that the percentage of labeled fibroblasts was maximal after incubation for 6 hr. Incorporation of 13H]proline was prevented by repeated freeze-thawing of aortic valves as well as by treatment with cycloheximide.
50
0
2
4
6
0
hours
FIG. 2. Percentage labeled cells in canine aortic valves after incubation with [3H]proline for different periods. Six valve pieces were used for every period tested. Standard error of the mean is indicated.
50
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OF SURGICAL
RESEARCH:
VOL. 30, NO. 1, JANUARY
1981
b
5
to
15
20
%
15
30
60 ml”
FIG. 3. Effect of controlled freezing with ethylene glycol on cell survival. (a) Effect of incubation temperature. (b) Effect of incubation time. (c) Effect of ethylene glycol concentration. (d) Effect of duration of the removal period. Mean values of five canine valve pieces from different valve systems for every condition tested are represented with standard error of the mean.
cell viability was tested on 10 canine valves. Each valve was dissected in two pieces, both of which were incubated with [3H]pro1ine either before or after sterilization. The percentage of labeled fibroblasts in the nonsterilized valve pieces was 87 k 7%, while in the sterilized valve pieces it was 74 k 7%. The results show that an average loss of 15% viable fibroblasts is due to the sterilization procedure. Injuence of Different Cryoprotective Agents on Cell Survival after Controlled Freezing (I”Clmin) of Canine Aortic Valves Ethylene glycol. The effect on cell survival in valves incubated for 30 min at 4,20, or 37°C with 10% v/v ethylene glycol followed by controlled freezing was estimated.
The valves were thawed rapidly with subsequent removal by dilution of the cryoprotective agent during a 60-min period. Figure 3a illustrates that the highest percentage of cell survival (70%) was scored when incubation with ethylene glycol was performed at 20°C. The influence of incubation time with 10% v/v ethylene glycol on cell survival was tested for the periods of 1, 15, and 30 min at 20°C. Controlled freezing, thawing, and removal of ethylene glycol were carried out as described. Figure 3b indicates that optima1 results are achieved (79%) with a 15-min incubation period. The influence of ethylene glycol concentration on cell survival was estimated for the concentrations of 5, 10, 1.5, and 20% v/v at 20°C for 15 min. Figure 3c illustrates
VAN DER KAMP ET AL.: PRESERVATION
OF AORTIC HEART VALVES
51
0
0 5
IO
15
20 7.
15
30
60
mln
FIG. 4. Effect of controlled freezing with glycerol on cell survival. (a) Effect of incubation temperature. (b) Effect of incubation time. (c) Effect of glycerol concentration. (d) Effect of duration of the removal period. Mean values of five canine valve pieces from different valve systems for every condition tested are represented with standard error of the mean.
that a concentration of 10% v/v yields the best results in terms of cell survival (79%). Finally, the influence on cell survival of the time of removal by dilution of the cryoprotective agent after thawing of the valve was determined. The valves were incubated with 10% v/v ethylene glycol for 15 min at 20°C; the cryoprotective agent was removed by dilution at 15,30, and 60 min. The results show (Fig. 3d) that a dilution period of 60 min is required to obtain optimal survival (79%). From these experiments with ethylene glyco1 as cryoprotective agent it can be concluded that cell survival is optimal (79%) if ethylene glycol is used in a 10% v/v concentration, incubated for 15 min at 2O”C, and removed after freezing by dilution for 60 min. Glycerol. Experiments similar to those described for ethylene glycol were carried
out using glycerol as cryoprotective agent. The conditions were varied as described for ethylene glycol. The data illustrated in Figs. 4a-d indicate that optimal cell survival (78%) using glycerol as cryoprotective agent could be realized with incubation at 37°C for 30 min, a glycerol concentration of 10% v/v, and removal by dilution for 60 min. Dimethylsulfoxide (DMSO). Experiments identical to those described for ethylene glyco1and glycerol were carried out with DMSO as cryoprotective agent. The data represented in Figs. 5a-d show that optimal cell survival (81%) could be achieved with incubation at 4°C for 1 min with a concentration of 10% v/v DMSO, while removal of the DMSO had to be carried out by dilution for 15 min. This method allowed an average survival of 50% of the endothelial cells.
52
JOURNAL OF SURGICAL RESEARCH: VOL. 30, NO. 1, JANUARY
1981
7. Crll-s”r”,“cll
d
100 i
FIG. 5. Effect of controlled freezing with DMSO on cell survival. (a) Effect of incubation temperature. (b) Effect of incubation time. (c) Effect of DMSO concentration. (d) Effect of duration of the removal period. Mean values of five canine valve pieces from different valve systems for every condition tested are presented with standard error of the mean.
Throughout these experiments the total number of tritium-labeled and nonlabeled fibroblasts in frozen valves was comparable to that in control valves. The optimal conditions of controlled freezing with ethylene glycol, glycerol, and DMSO found for canine valves were tested for cell survival in human material. The valves were placed in a glass vial, as illustrated in Fig. 6, constructed to withstand both high and low temperatures necessary for sterilization of the vial and preservation. Because limitation of the dilution time is mandatory during actual valve transplantation, to avoid a nondesirable prolongation of surgery, the dilution period was restricted to 15 min in spite of the fact that this short period was found to be far from optimal for valves frozen with ethylene glycol and glyc-
erol. The results represented in Fig. 7 show that the percentages of cell survival are 16, 23, and 88% for ethylene glycol, glycerol, and DMSO, respectively. For controlled freezing with DMSO, cell survival in the aortic wall was also measured and found to be around 40%, while most of the endothelial cells were lost. Preservation Controlled
of the Valve Matrix Freezing
after
Human aortic valves were control-frozen with DMSO under the optimal conditions described. Light microscopic studies revealed no changes in the distribution or structure of the collagenic and elastic fibers. However, even with careful handling most of the endothelial cells were disrupted from the basal membrane. Measurement of the
VAN DER KAMP ET AL.: PRESERVATION
53
OF AORTIC HEART VALVES
FIG. 6. Glass vial for controlled freezing of aortic valves, constructed of Pyrex glass, a screw cap of Bakelite with a silicone center part to form a pressure valve, necessary whenever liquid nitrogen has entered the vial. A Teflon ring is used to assure good closure.
periodicity length of collagenic fibrils prepared for electron microscopy (see Fig. 8) revealed no significant changes between fresh aortic valves (641 _t 8 A) and controlled frozen specimens (639 2 11 A). The stress- strain characteristics of strips of aortic wall of fresh human thoracic aortas and strips sterilized in antibiotics and preserved by controlled freezing under optimal conditions with DMSO were determined. Data of initial expansion at low stress, stress and elongation at transition, and rupture are plotted in Fig. 9. From these data it can be concluded that this method of sterilization and preservation only slightly alters human aortic wall material in its initial expansion and posttransitional stress-strain characteristics.
to use preservation methods which have been successfully applied for the long-term storage of various human cell types, i.e., controlled freezing. Several authors used controlled freezing for heart valve preservation [23, 34, 431. Controlled freezing offers the advantage of infinite storage at - 196°Cwhich enables the collection of suitable autopsy material with respect to the valve diameter and matching for HLA-defined antigens. Sterilization of the autopsy material, accord% Cell-survival 100
m
ethylene
@g
glycerol
glycol
DISCUSSION
Transplantation of viable allograft valves necessitates long-term storage of the valves with maintenance of cell viability and matrix integrity. A number of investigators have observed the presence of viable cells in aortic valves after short-term preservation in Hanks’ solution. Long-term storage in nutrient medium [ 1, 61 does not seem to result in the maintenance of cell viability [32]. To maintain cell viabilitv it seems more aooronriate I
I
-r------
FIG. 7. Effect of controlled freezing with ethylene glycol, glycerol, and DMSO on cell survival in human aortic valves. Mean values of five human valve pieces from different valve systems for every condition tested are represented with standard error of the mean.
54
JOURNAL OF SURGICAL RESEARCH: VOL. 30, NO. 1, JANUARY
1981
FIG. 8. Electron micrograph of an isolated collagenic microfibril. ~39,000.
ing to Lackey et al. [27], was found to be effective and allowed an 86% cell survival. In spite of what is known about the action of cryoprotective agents the optimal conditions for controlled freezing have to be determined for each tissue separately [26, 28, 3 1, 351. Methods developed for cryopreservation of whole skin [41], cornea [ 161, developing teeth [ 111,and bone marrow stem cells [44] give guidelines for the type of cryoprotective agent, the rate of freezing and thawing, and the stepwise removal of the cryoprotective agent after thawing. Intracellular acting cryoprotective agents were found to be effective in the maintenance of cell viability in heart valves provided optimal conditions were applied. The differences in cell survival between ethylene glycol, glycerol, and dimethylsulfoxide are partly based on differences in penetration rate and cytotoxicity [30, 351. Similar observations have been made for cultured hamster cells [26], bone marrow stem cells [39], and whole skin [7, 251. In our experiments we observed that cryopreservation of human aortic heart valves under optimal conditions for leaflet fibroblasts (88%) resulted in 44% cell survival of aortic wall cells and no survival of endothelial cells. This differential susceptibility is a well-known phenomenon which is likely to be due to tissue and cell penetration of the cryoprotective agent, cell volume, and cell membrane permeability [3 1, 421. In this study cell viability was assessed by [3H]proline incorporation followed by autoradiography. This enables one to deter-
mine the exact number of surviving cells and their intravalvular localization and at the same time to test whether the fibroblasts are still capable of synthesizing protein (collagen). The viability of aortic valves at the time of procurement is of primary importance. The results show that human aortic valves obtained from patients who, within 2 to 10 hr after death, were placed in a cold room and sectioned within 24 hr after death, were viable at the time of procurement. The time period between death and cold-room storage seemsto be essential. This is in agreement with the data reported by McGregor et al. [33], showing a lack of cell outgrowth in tissue culture in those valves that were subjected to autolysis for periods of about 12 hr or longer. By the method of [3H] proline incorporation it was shown that controlled freezing
FIG. 9. Plot of average values of stress and strain for fresh and controlled frozen human aortic wall strips. Twenty strips taken from five different aortas were tested for each condition.
VAN DER KAMP ET AL.: PRESERVATION
OF AORTIC HEART VALVES
55
of human heart valves permits infinite stor- action which might be prevented by tissue typing for HLA-defined tissue antigens. To age with survival of 88% of the fibroblasts, the majority of which were actively engaged collect the larger number of valves necesin protein synthesis after thawing, a pre- sary for the selection of the proper match requisite for optimal maintenance of the in terms of orifice diameter and the HLAstructural and functional integrity of the valve defined antigens the long-term preservation matrix after transplantation [24]. The proce- procedure presented in this paper seems dure developed had no damaging effect on suitable. the microscopic structure of the valve and ACKNOWLEDGMENTS the ultrastructural pattern of the collagenic The authors gratefully acknowledge Dr. M. F. fibrils. Finally, the stress-strain characMichel for performing the sterility tests, Mr. T. M. teristics and hence the aortic wall elasticity van OS for preparation of the illustrations, and Mrs. remained unaffected by the cryopreservation R. J. Boucke for typing the manuscript. procedure. The loss of the endothelial lining of human aortic valves as observed after REFERENCES controlled freezing is not caused primarily 1. Al-Yanabi, N., Gonzalez-Lavin, Neirotti, R., and by the preservation procedure itself but more Ross, D. N. Viability of fresh aortic valve homolikely is due to autolytic processes during grafts: A quantitative assessment. Thorax 27: 83, 1972. the period between death of the individual and freezing of the valve. This can be con- 2. Al-Yanabi, N., and Ross, D. N. Enhancedviability of fresh aortic homografts stored in nutrient mecluded from the fact that fresh canine valves dium. Cardiovasc. Res. 7: 817, 1973. are lined by a substantial number of viable 3. Angell, W. W., Wuerllein, R. D., and Shumway, endothelial cells after preservation (data N. E. Mitral valve replacement with the fresh aortic valve homograft: Experimental results and not shown). clinical application. Surgery 62: 807, 1967. With the use of viable allografts, graft reAngel], W. W., Stinson, E. B., Iben, A. B., and jection may obviously be a problem. The long- 4. Shumway, N. E. Multiple valve replacement with term results obtained with allovital valve the fresh aortic homograft. .I. Thorac. Cardiovasc. transplants suggest that such heart valves Surg. 56: 232, 1968. do not incite a severe rejection process 5. Angell, W. W., Shumway, N. E., and Kosek, J. C. A five year study of viable aortic valve homo[5, 93. In experiments with allovital canine J. Thorac. Cardiovasc. Surg. 64: 329, 1972. valves, however, observations suggestive of 6. grafts. Angel], W. W., Wuerllein, R. D., Chum, C. W., a mild graft rejection process were made and Shumway, N. E. Antibiotic sterilization of [3, 13, 19,291. This rejection process seems aortic homografts. N. Z. Med. J. 77: 1973. 7. Athreya, B. H., Grimes, E. L., Lehr, H. B., to be dependent on contact of the valve with Greene, A. E., and Coriell, L. L. Differential susthe host circulation [ 181.Additional expericeptibility of epithelial cells and fibroblasts of humental evidence for valve antigenicity has man skin to freeze injury. Cryobiology 5: 262, been reported by several investigators who 1969. transplanted allovital aortic valves into sub- 8. Barratt-Boyes, B. G. Homograft aortic valve replacement in aortic incompetence and stenosis. cutaneous or intramuscular positions [ 12, Thorax 19: 131, 1964. 22, 361. The absence of endothelial lining 9. Barratt-Boyes, B. G., Roche, A. H. G., Brandt, may result in the formation of microthrombi P. W. T., Smith, J. C., and Lowe, J. B. Aortic and in insufficient nutrition of the valve fibrohomograft valve replacement. Circulation 40: 763, blasts. It seems therefore essential that af1969. ter transplantation the endothelial lining of 10. Barratt-Boyes, B. G., Roche, A. H. G., and Whitlock, R. M. L. Six year review of the results of the valve will be restored. In principle this freehand aortic valve replacement using an antican be accomplished by overgrowth of endobiotic sterilized homograft valve. Circulation 55: thelium from the graft-host junction [5, 19, 353, 1977. 21, 371. The overgrowth of endothelium 11. Bartlett, P. F., and Reade, P. C. Cryopreservation of developing teeth. Cryobiology 9: 20.5, 1972. could be hampered by an immunological re-
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12. Baue, A. E., Donawick, W. J., and Blakemore, W. S. The immunological response to heterotopic allovital aortic valve transplants in presensitized and non-sensitized recipients. J. Thorac. Cardiovase. Surg. 56: 775, 1968. 13. Beall, A. C., Morris, G. C., Cooley, D. A., and de Bakey, N. E. Homotransplantation of the aortic valve. J. Thorac. Cardiovasc. Surg. 42: 497, 1961. 14. Billingham, R. E., and Medawar, P. B. The freezing, drying and storage of mammalian skin. J. Exp. Biol. 30: 454, 1952.
15. Bunge, R. G., and Sherman, J. K. Fertilizing capacity of frozen human spermatozoa. Nature (Land.) 172: 767, 1953. 16. Capella, J. A., Kaufman, H. E., and Robbins, J. E. Preservation of viable cornea. Arch. Ophthal. 74: 669, 1966. 17. Clark, R. E. Stress-strain characteristics of fresh and frozen human aortic and mitral leaflets and chordea tentinea. J. Thorac. Cardiovasc. Surg. 66: 202, 1973. 18. Donawick, W. J., and Baue, A. E. The immunological response to intravascular skin allografts. Surgery 67: 667, 1970.
19. Duran, C. M. G., and Whitehead, R. Experimental homologous aortic valve transplantation. Brir. J. Surg. 53: 1049, 1966. 20. Gavin, J. B., Monro, J. L., Wall, F. M., and Chalcroft, S. C. W. Fine structural changes in the fibroblasts of canine heart valves prepared for grafting. Thorax 28: 748, 1973. 21. Gavin, J. B., Barratt-Boyes, B. G., Hitchcock, G. C., and Herdson, P. B. Histopathology of “fresh” human aortic valve allografts. Thorax 28: 482, 1973. 22. Heslop, B. F., Wilson, S. E., and Hardey, B. E. Antigenicity of aortic valve allografts. Ann. Surg. Res. 177: 301, 1973. 23. van der Kamp, A. W. M., Nauta, J., and Galjaard, H. Effect of preservation and sterilization methods on structure and function of aortic-valves. Eur. J. Surg. Res. 4: 361, 1972. 24. van der Kamp, A. W. M., and Nauta, J. Fibroblast function and the maintenance of the aorticvalve matrix. Cardiovasc. Res. 13: 167, 1979. 25. Lehr, H. B., Berggren, R. B., Lotke, P. A., and Coniell, L. L. Permanent survival of preserved skin autografts. Surgery 56: 742, 1964. 26. Leibo, S. P., and Mazur, P. The role of cooling rates in low-temperature preservation. Ctyobiology 8: 447, 1971. 27. Lackey, E., Al-Yanabi, N., Gonzalez-Lavin, L., and Ross, D. N. A method of sterilizing and preserving fresh allograft heart valves. Thorax 27: 398, 1972. 28. Lovelock, Y. E. The mechanism of the protective action of glycerol against haemolysis by freezing and thawing. Biochim. Biophys. Acta 2: 28, 1953. 29. Lower, R. R., Pillsbury, R. C., Angell, W. W.,
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and Kosek, J. C. Homograft heartvalves. Circufation 33: 135, 1966.
30. Malinin, G. L. Cytotoxic effect of dimethylsulfoxide on the ultrastructure of cultured rhesus kidney cells. Cryobiology 10: 22, 1973. 3 1. Mazur, P. Cryobiology: The freezing of biological systems. Science 168: 939, 1970. 32. McGregor, C. G. A., Bradley, J. F., McGee, J. O’D., and Wheatley, D. J. Tissue culture, protein and collagen synthesis in antibiotic sterilized canine heart valves. Cardiovasc. Res. 10: 389, 1976. 33. McGregor, C. G. A., Bradley, J. F., McGee, J.O’D. and Wheatley, D. J. Viability in human heart valves prepared for grafting. Cardiovasc. Res. 10: 394, 1976. 34. Mermet, B., Angell, W. W., and Dor, V. Viabilite des homogreffes fraiches sterilistes par les antibiotiques et conservees au grand froid (-196°C). Ann. Chir. Thorac. Cardiovasc. 10: 463, 1971. 35. Meryman, H. T. Cryoprotective agents. Cryobiology 8: 173, 1971. 36. Mohri, H., Reichenbach, D. D., Barnes, R. W., and Merendino, A. K. A biological study of the homologous aortic valve in dogs. J. Thorac. Cardiovasc. Surg. 54: 622, 1967. 37. Mohri, H., Reichenbach, D. D., Barnes, R. W., and Merendino, A. K. Homologous aortic valve transplantation. J. Thoruc. Cardiovasc. Surg. 56: 767, 1968. 38. Murray, G. D. Q. Homologous aortic-valve-segment transplants as surgical treatment for aortic and mitral insufficiency. Angiology 7: 466, 1956. 39. O’Grady, L. F., and Lewis, J. P. The long-term preservation of bone marrow. Trnnsfusion 12: 312, 1972. 40. Olsen, B. R. Electron microscope studies on collagen. Z. Zellforsch. Mikrosk. Anat. 59: 184, 1963. 41. Polge, C., Smith, A. U., and Parkes, A. S. Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature (Lond.) 164: 666, 1949. 42. Pribor, D. B. PVP contrasted with dextran and a multifactor theory of cryoprotection. Cryobiology 2: 60, 1974. 43. Rajotte, R. V., Shnitka, T. K., Liburd, E. M., Dossetor, J. B., and Voss, W. A. G. Histological studies on cultured canine heart valves recovered from - 196°C. Cryobiology 14: 15, 1977. 44. Schaeffer, U. W., Dicke, K. A., and van Bekkum, D. W. Recovery of haemopoiesis in lethally irradiated monkeys by frozen allogeneic bone marrow grafts. Eur. J. C/in. Biol. Res. 17: 483, 1972. 45. Thompson, R., Knight, E., Ahmed, M., Somerville, W., Towers, M., and Yacoub, M. The use of “fresh” unstented homograft valves for replacement of the aortic valve. Circulation 56: 837, 1977.
OF SURGICAL
Preservation
30, 47-56 (1981)
RESEARCH
of Aortic
Heart Valves with Maintenance
of Cell Viability
ARTHUR W. M. VAN DER KAMP, M. D.,*,’ WILLEM J. VISSER,* JOHANNES M. VAN DONGEN, PH. D.,* JAN NAUTA, M. D.,t AND HANS GALJAARD, M. D.* *Department
of Cell Biology and Genetics and tDepartment of Thoracic and Cardiovascular Erasmus University, Rotterdam, The Netherlands
Surgery,
Submitted for publication January 15, 1980 The goal of the present study was to develop a procedure for sterilization and infinite preservation of aortic heart valves with maintenance of cell viability. For this purpose the influence of a number of methods of controlled freezing on the viability of the fibroblasts of the aortic valve was tested. Cell viability was assessed quantitatively by the incorporation of [3H]proline by the valve fibroblasts. Controlled freezing at a rate of l”C/min under protection of 10% dimethylsulfoxide yielded the highest number of viable fibroblasts (88%). This preservation method was also tested for its influence on the structural and functional integrity of the valve matrix, which was found to be preserved throughout sterilization and storage. This study is the first to present quantitative data on cell survival, stress-strain characteristics, and the electronmicroscopic structure of collagenic fibrils after sterilization and controlled freezing of aortic valves.
ervation of various cell types and tissues [14, 15, 28, 441, has been used to preserve Transplantation of allovital aortic valves aortic heart valves, but cell survival was offers the possibility for maintenance of the only assessed by qualitative techniques, valve matrix by the fibroblasts [24, 321. i.e., cell culture [34, 431. Several series of such allovital transplantaWe have compared the influence of tions have been reported [3-5,8,10,38,45]. several controlled freezing methods on the Although these transplantations were carmaintenance of viability of aortic valve ried out with fresh valves no qualitative fibroblasts, endothelial cells, and cells of the or quantitative assessment of their viability aortic wall. The capacity for protein synthewas performed. The preservation procedure sis, monitored by the incorporation of [3H]reported by Al-Yanabi et al. can only be proline, was used as a parameter for cell used for short-term storage [ 1, 21, while the viability after preservation. Effects of validity of the assay by which cell survival preservation on the structural and funcwas tested can be criticized [20, 321. tional characteristics were investigated by Long-term preservation will be needed to light microscopy, electron microscopy of assure a proper match between donor and isolated collagenic fib&s, and stress-strain acceptor orifice diameter. Controlled freezmeasurements. Throughout the experiing with cryoprotective agents to low temments sterilization by antibiotics as deperatures has been used successfully as a scribed by Lackey et al. [27] was used, and long-term preservation method maintaining its validity with respect to sterilizing cell viability. The method introduced by capacity and its effect on cell survival were Polge et al. [41], and applied for the prestested. INTRODUCTION
1 To whom requests for reprints should be Sent at: Department of Cell Biology and Genetics, Erasmus University Rotterdam, P.O. Box 1738,3OOO DR Rotterdam, The Netherlands.
MATERIALS
AND METHODS
Both canine and human aortic heart valves were used. The aortic valves were 47
0022-48O4/81/OlOO47-10$01.00/O Copyright All rights
0 1981 by Academic Press. Inc. of reproduction in any form reserved.
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divided into six equal parts one of which was taken as an internal control. Test for cell viability. Aortic valve tissue attached to a piece of aortic wall was incubated at 37°C under air containing 5% CO, during 2, 4, 6, or 8 hr in Ham’s FlO nutrient medium. The medium was supplemented with fetal calf serum (15%) and 15 PCi [3H]proline per milliliter (sp act 10 mCi . mmole-’ , Radiochemical Centre, Amersham). After incubation the tissue was washed three times for I5 min with cold proline to remove all nonincorporated [3H]proline. After fixation (10% formalin) paraffin sections (5 pm) were processed for autoradiography (Ilford K-2 emulsion). After staining (hematoxylineosin) the microscopic localization and percentage of labeled fibroblasts were determined in five random fields of view (microscopic magnification 400x) of three widely separated sections of each valve piece. The ratio of the percentages for preserved and control valves rendered the percentage of cell survival. Survival of endothelial cells and cells in the aortic wall was determined in material with optimal survival of the valve fibroblasts. To determine whether the presence of [3H]proline in the valves was dependent on cell viability or was related to inadequate removal of nonincorporated proline, incorporation studies were performed with valve pieces which were rapidly frozen and thawed to destroy the cells. To determine whether protein synthesis was essential for the incorporation of r3H]proline such protein synthesis was blocked by cycloheximide (20 pg.cm3-l, Serva). Aortic valve sterilization. Human and canine aortic valves were obtained from autopsy performed without any attempt at sterility within 24 hr after death. The dissected valve together with six pieces of aortic wall was kept in a solution of antibiotics as described by Lackey et al. [271, during 24 hr, at 4°C. Subsequently the pieces of aortic wall and 5 cm3 of nutrient
VOL. 30, NO. 1, JANUARY
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medium were tested for growth of aerobic and anaerobic bacteria as well as fungi and yeasts at 4, 20, and 37°C for 3 weeks. Sterility tests were performed both before and after removal of the antibiotics. Sterility was also tested after controlled freezing. The effect of sterilization on cell survival was tested as described above. Preservation procedures. The aortic valve pieces were immersed in Ham’s FlO nutrient medium supplemented with 15% fetal calf serum. After addition of the cryoprotective agent; dimethylsulfoxide, glycerol, or ethylene glycol (Merck) at different concentrations (5, 10, 15, and 20%), the valves were incubated during various time intervals (1, 15, and 30 min) and at different temperatures (4,20 and 37°C). The valves were frozen at a rate of l”C/min, controlled by a Cryoson biological freezer, Type BV-4, connected with a Cryoson pressure tank, Type 200 VLR. At -90°C the valves were rapidly cooled to - 196°C(liquid nitrogen). After storage at - 196°C for a period of 1 week to 9 months the valves were thawed rapidly followed by stepwise dilution of the cryoprotective agent three times for 5 or 10 min in 50, 25 and 0% of the cryoprotective agent or dropwise dilution during 60 min. Subsequently the valves were tested for cell viability. Effects of preservation on the valve matrix. For light microscopy, valves were fixed in 10% formalin and paraffin sections (5 pm) were stained with hematoxylinazophloxine-saffran. Isolated collagenic fibrils were prepared and stained according to the method of Olsen [40]. These fibrils were examined with a Zeiss 9,s. electron microscope. The magnification of the electron microscope was calibrated with acrossgrating replica (2 160lines/mm). The banding pattern of the microfibrils was studied by measurement of at least 1000 periods on electron micrographs of only straight parts of the fibrils. Six valve pieces were investigated for every condition tested. Stress-strain measurements. Such measurements were performed on human thoracic
VAN DER KAMP ET AL.: PRESERVATION
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OF AORTIC HEART VALVES
FIG. 1. Autoradiograph of a canine aortic valve after incubation with [3H]proline for 6 hr. Note the large number of labeled fibroblasts with thin cytoplasmic extensions. obj. x40.
aortas excised within 24 hr after death [ 171. The tissue was preserved in saline solution before testing. With a blade, model strips of 3.5cm length, l.O-cm width at the ends and 0.6-cm width in the middle part were cut in the radial and circumferential direction. Control strips and strips to be preserved were taken from adjacent locations. The strips were placed on a rifling bench with a cross-head speed of 1.Omm per minute, connected, via a transducer-strain indicator, to a writer with a chart speed of 1.0 cm per minute, yielding a magnification of 10x . The initial length was adjusted to 2.4 cm without buckling or crimping of the tissue. A total of 20 strips, taken from five different aortas, were tested.
Sterility of Aortic Heart Valves Fourteen human aortic valves were sterilized according to the method of Lackey et al. [27]. All valves except one were found to be sterile. Five sterilized valves were tested for contamination after controlled freezing and storage in liquid nitrogen; all were found to be sterile. The effect of sterilization on INCUBATION %Labclled 100 1
TIME WITH 3H- PROLINE cells
RESULTS
Determination of Cell Viability The percentage fibroblasts of fresh canine valves labeled with [3H]proline as shown by autoradiography (Fig. 1) was determined after incubation periods of 2,4,6, and 8 hr. Figure 2 shows that the percentage of labeled fibroblasts was maximal after incubation for 6 hr. Incorporation of 13H]proline was prevented by repeated freeze-thawing of aortic valves as well as by treatment with cycloheximide.
50
0
2
4
6
0
hours
FIG. 2. Percentage labeled cells in canine aortic valves after incubation with [3H]proline for different periods. Six valve pieces were used for every period tested. Standard error of the mean is indicated.
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b
5
to
15
20
%
15
30
60 ml”
FIG. 3. Effect of controlled freezing with ethylene glycol on cell survival. (a) Effect of incubation temperature. (b) Effect of incubation time. (c) Effect of ethylene glycol concentration. (d) Effect of duration of the removal period. Mean values of five canine valve pieces from different valve systems for every condition tested are represented with standard error of the mean.
cell viability was tested on 10 canine valves. Each valve was dissected in two pieces, both of which were incubated with [3H]pro1ine either before or after sterilization. The percentage of labeled fibroblasts in the nonsterilized valve pieces was 87 k 7%, while in the sterilized valve pieces it was 74 k 7%. The results show that an average loss of 15% viable fibroblasts is due to the sterilization procedure. Injuence of Different Cryoprotective Agents on Cell Survival after Controlled Freezing (I”Clmin) of Canine Aortic Valves Ethylene glycol. The effect on cell survival in valves incubated for 30 min at 4,20, or 37°C with 10% v/v ethylene glycol followed by controlled freezing was estimated.
The valves were thawed rapidly with subsequent removal by dilution of the cryoprotective agent during a 60-min period. Figure 3a illustrates that the highest percentage of cell survival (70%) was scored when incubation with ethylene glycol was performed at 20°C. The influence of incubation time with 10% v/v ethylene glycol on cell survival was tested for the periods of 1, 15, and 30 min at 20°C. Controlled freezing, thawing, and removal of ethylene glycol were carried out as described. Figure 3b indicates that optima1 results are achieved (79%) with a 15-min incubation period. The influence of ethylene glycol concentration on cell survival was estimated for the concentrations of 5, 10, 1.5, and 20% v/v at 20°C for 15 min. Figure 3c illustrates
VAN DER KAMP ET AL.: PRESERVATION
OF AORTIC HEART VALVES
51
0
0 5
IO
15
20 7.
15
30
60
mln
FIG. 4. Effect of controlled freezing with glycerol on cell survival. (a) Effect of incubation temperature. (b) Effect of incubation time. (c) Effect of glycerol concentration. (d) Effect of duration of the removal period. Mean values of five canine valve pieces from different valve systems for every condition tested are represented with standard error of the mean.
that a concentration of 10% v/v yields the best results in terms of cell survival (79%). Finally, the influence on cell survival of the time of removal by dilution of the cryoprotective agent after thawing of the valve was determined. The valves were incubated with 10% v/v ethylene glycol for 15 min at 20°C; the cryoprotective agent was removed by dilution at 15,30, and 60 min. The results show (Fig. 3d) that a dilution period of 60 min is required to obtain optimal survival (79%). From these experiments with ethylene glyco1 as cryoprotective agent it can be concluded that cell survival is optimal (79%) if ethylene glycol is used in a 10% v/v concentration, incubated for 15 min at 2O”C, and removed after freezing by dilution for 60 min. Glycerol. Experiments similar to those described for ethylene glycol were carried
out using glycerol as cryoprotective agent. The conditions were varied as described for ethylene glycol. The data illustrated in Figs. 4a-d indicate that optimal cell survival (78%) using glycerol as cryoprotective agent could be realized with incubation at 37°C for 30 min, a glycerol concentration of 10% v/v, and removal by dilution for 60 min. Dimethylsulfoxide (DMSO). Experiments identical to those described for ethylene glyco1and glycerol were carried out with DMSO as cryoprotective agent. The data represented in Figs. 5a-d show that optimal cell survival (81%) could be achieved with incubation at 4°C for 1 min with a concentration of 10% v/v DMSO, while removal of the DMSO had to be carried out by dilution for 15 min. This method allowed an average survival of 50% of the endothelial cells.
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7. Crll-s”r”,“cll
d
100 i
FIG. 5. Effect of controlled freezing with DMSO on cell survival. (a) Effect of incubation temperature. (b) Effect of incubation time. (c) Effect of DMSO concentration. (d) Effect of duration of the removal period. Mean values of five canine valve pieces from different valve systems for every condition tested are presented with standard error of the mean.
Throughout these experiments the total number of tritium-labeled and nonlabeled fibroblasts in frozen valves was comparable to that in control valves. The optimal conditions of controlled freezing with ethylene glycol, glycerol, and DMSO found for canine valves were tested for cell survival in human material. The valves were placed in a glass vial, as illustrated in Fig. 6, constructed to withstand both high and low temperatures necessary for sterilization of the vial and preservation. Because limitation of the dilution time is mandatory during actual valve transplantation, to avoid a nondesirable prolongation of surgery, the dilution period was restricted to 15 min in spite of the fact that this short period was found to be far from optimal for valves frozen with ethylene glycol and glyc-
erol. The results represented in Fig. 7 show that the percentages of cell survival are 16, 23, and 88% for ethylene glycol, glycerol, and DMSO, respectively. For controlled freezing with DMSO, cell survival in the aortic wall was also measured and found to be around 40%, while most of the endothelial cells were lost. Preservation Controlled
of the Valve Matrix Freezing
after
Human aortic valves were control-frozen with DMSO under the optimal conditions described. Light microscopic studies revealed no changes in the distribution or structure of the collagenic and elastic fibers. However, even with careful handling most of the endothelial cells were disrupted from the basal membrane. Measurement of the
VAN DER KAMP ET AL.: PRESERVATION
53
OF AORTIC HEART VALVES
FIG. 6. Glass vial for controlled freezing of aortic valves, constructed of Pyrex glass, a screw cap of Bakelite with a silicone center part to form a pressure valve, necessary whenever liquid nitrogen has entered the vial. A Teflon ring is used to assure good closure.
periodicity length of collagenic fibrils prepared for electron microscopy (see Fig. 8) revealed no significant changes between fresh aortic valves (641 _t 8 A) and controlled frozen specimens (639 2 11 A). The stress- strain characteristics of strips of aortic wall of fresh human thoracic aortas and strips sterilized in antibiotics and preserved by controlled freezing under optimal conditions with DMSO were determined. Data of initial expansion at low stress, stress and elongation at transition, and rupture are plotted in Fig. 9. From these data it can be concluded that this method of sterilization and preservation only slightly alters human aortic wall material in its initial expansion and posttransitional stress-strain characteristics.
to use preservation methods which have been successfully applied for the long-term storage of various human cell types, i.e., controlled freezing. Several authors used controlled freezing for heart valve preservation [23, 34, 431. Controlled freezing offers the advantage of infinite storage at - 196°Cwhich enables the collection of suitable autopsy material with respect to the valve diameter and matching for HLA-defined antigens. Sterilization of the autopsy material, accord% Cell-survival 100
m
ethylene
@g
glycerol
glycol
DISCUSSION
Transplantation of viable allograft valves necessitates long-term storage of the valves with maintenance of cell viability and matrix integrity. A number of investigators have observed the presence of viable cells in aortic valves after short-term preservation in Hanks’ solution. Long-term storage in nutrient medium [ 1, 61 does not seem to result in the maintenance of cell viability [32]. To maintain cell viabilitv it seems more aooronriate I
I
-r------
FIG. 7. Effect of controlled freezing with ethylene glycol, glycerol, and DMSO on cell survival in human aortic valves. Mean values of five human valve pieces from different valve systems for every condition tested are represented with standard error of the mean.
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1981
FIG. 8. Electron micrograph of an isolated collagenic microfibril. ~39,000.
ing to Lackey et al. [27], was found to be effective and allowed an 86% cell survival. In spite of what is known about the action of cryoprotective agents the optimal conditions for controlled freezing have to be determined for each tissue separately [26, 28, 3 1, 351. Methods developed for cryopreservation of whole skin [41], cornea [ 161, developing teeth [ 111,and bone marrow stem cells [44] give guidelines for the type of cryoprotective agent, the rate of freezing and thawing, and the stepwise removal of the cryoprotective agent after thawing. Intracellular acting cryoprotective agents were found to be effective in the maintenance of cell viability in heart valves provided optimal conditions were applied. The differences in cell survival between ethylene glycol, glycerol, and dimethylsulfoxide are partly based on differences in penetration rate and cytotoxicity [30, 351. Similar observations have been made for cultured hamster cells [26], bone marrow stem cells [39], and whole skin [7, 251. In our experiments we observed that cryopreservation of human aortic heart valves under optimal conditions for leaflet fibroblasts (88%) resulted in 44% cell survival of aortic wall cells and no survival of endothelial cells. This differential susceptibility is a well-known phenomenon which is likely to be due to tissue and cell penetration of the cryoprotective agent, cell volume, and cell membrane permeability [3 1, 421. In this study cell viability was assessed by [3H]proline incorporation followed by autoradiography. This enables one to deter-
mine the exact number of surviving cells and their intravalvular localization and at the same time to test whether the fibroblasts are still capable of synthesizing protein (collagen). The viability of aortic valves at the time of procurement is of primary importance. The results show that human aortic valves obtained from patients who, within 2 to 10 hr after death, were placed in a cold room and sectioned within 24 hr after death, were viable at the time of procurement. The time period between death and cold-room storage seemsto be essential. This is in agreement with the data reported by McGregor et al. [33], showing a lack of cell outgrowth in tissue culture in those valves that were subjected to autolysis for periods of about 12 hr or longer. By the method of [3H] proline incorporation it was shown that controlled freezing
FIG. 9. Plot of average values of stress and strain for fresh and controlled frozen human aortic wall strips. Twenty strips taken from five different aortas were tested for each condition.
VAN DER KAMP ET AL.: PRESERVATION
OF AORTIC HEART VALVES
55
of human heart valves permits infinite stor- action which might be prevented by tissue typing for HLA-defined tissue antigens. To age with survival of 88% of the fibroblasts, the majority of which were actively engaged collect the larger number of valves necesin protein synthesis after thawing, a pre- sary for the selection of the proper match requisite for optimal maintenance of the in terms of orifice diameter and the HLAstructural and functional integrity of the valve defined antigens the long-term preservation matrix after transplantation [24]. The proce- procedure presented in this paper seems dure developed had no damaging effect on suitable. the microscopic structure of the valve and ACKNOWLEDGMENTS the ultrastructural pattern of the collagenic The authors gratefully acknowledge Dr. M. F. fibrils. Finally, the stress-strain characMichel for performing the sterility tests, Mr. T. M. teristics and hence the aortic wall elasticity van OS for preparation of the illustrations, and Mrs. remained unaffected by the cryopreservation R. J. Boucke for typing the manuscript. procedure. The loss of the endothelial lining of human aortic valves as observed after REFERENCES controlled freezing is not caused primarily 1. Al-Yanabi, N., Gonzalez-Lavin, Neirotti, R., and by the preservation procedure itself but more Ross, D. N. Viability of fresh aortic valve homolikely is due to autolytic processes during grafts: A quantitative assessment. Thorax 27: 83, 1972. the period between death of the individual and freezing of the valve. This can be con- 2. Al-Yanabi, N., and Ross, D. N. Enhancedviability of fresh aortic homografts stored in nutrient mecluded from the fact that fresh canine valves dium. Cardiovasc. Res. 7: 817, 1973. are lined by a substantial number of viable 3. Angell, W. W., Wuerllein, R. D., and Shumway, endothelial cells after preservation (data N. E. Mitral valve replacement with the fresh aortic valve homograft: Experimental results and not shown). clinical application. Surgery 62: 807, 1967. With the use of viable allografts, graft reAngel], W. W., Stinson, E. B., Iben, A. B., and jection may obviously be a problem. The long- 4. Shumway, N. E. Multiple valve replacement with term results obtained with allovital valve the fresh aortic homograft. .I. Thorac. Cardiovasc. transplants suggest that such heart valves Surg. 56: 232, 1968. do not incite a severe rejection process 5. Angell, W. W., Shumway, N. E., and Kosek, J. C. A five year study of viable aortic valve homo[5, 93. In experiments with allovital canine J. Thorac. Cardiovasc. Surg. 64: 329, 1972. valves, however, observations suggestive of 6. grafts. Angel], W. W., Wuerllein, R. D., Chum, C. W., a mild graft rejection process were made and Shumway, N. E. Antibiotic sterilization of [3, 13, 19,291. This rejection process seems aortic homografts. N. Z. Med. J. 77: 1973. 7. Athreya, B. H., Grimes, E. L., Lehr, H. B., to be dependent on contact of the valve with Greene, A. E., and Coriell, L. L. Differential susthe host circulation [ 181.Additional expericeptibility of epithelial cells and fibroblasts of humental evidence for valve antigenicity has man skin to freeze injury. Cryobiology 5: 262, been reported by several investigators who 1969. transplanted allovital aortic valves into sub- 8. Barratt-Boyes, B. G. Homograft aortic valve replacement in aortic incompetence and stenosis. cutaneous or intramuscular positions [ 12, Thorax 19: 131, 1964. 22, 361. The absence of endothelial lining 9. Barratt-Boyes, B. G., Roche, A. H. G., Brandt, may result in the formation of microthrombi P. W. T., Smith, J. C., and Lowe, J. B. Aortic and in insufficient nutrition of the valve fibrohomograft valve replacement. Circulation 40: 763, blasts. It seems therefore essential that af1969. ter transplantation the endothelial lining of 10. Barratt-Boyes, B. G., Roche, A. H. G., and Whitlock, R. M. L. Six year review of the results of the valve will be restored. In principle this freehand aortic valve replacement using an antican be accomplished by overgrowth of endobiotic sterilized homograft valve. Circulation 55: thelium from the graft-host junction [5, 19, 353, 1977. 21, 371. The overgrowth of endothelium 11. Bartlett, P. F., and Reade, P. C. Cryopreservation of developing teeth. Cryobiology 9: 20.5, 1972. could be hampered by an immunological re-
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15. Bunge, R. G., and Sherman, J. K. Fertilizing capacity of frozen human spermatozoa. Nature (Land.) 172: 767, 1953. 16. Capella, J. A., Kaufman, H. E., and Robbins, J. E. Preservation of viable cornea. Arch. Ophthal. 74: 669, 1966. 17. Clark, R. E. Stress-strain characteristics of fresh and frozen human aortic and mitral leaflets and chordea tentinea. J. Thorac. Cardiovasc. Surg. 66: 202, 1973. 18. Donawick, W. J., and Baue, A. E. The immunological response to intravascular skin allografts. Surgery 67: 667, 1970.
19. Duran, C. M. G., and Whitehead, R. Experimental homologous aortic valve transplantation. Brir. J. Surg. 53: 1049, 1966. 20. Gavin, J. B., Monro, J. L., Wall, F. M., and Chalcroft, S. C. W. Fine structural changes in the fibroblasts of canine heart valves prepared for grafting. Thorax 28: 748, 1973. 21. Gavin, J. B., Barratt-Boyes, B. G., Hitchcock, G. C., and Herdson, P. B. Histopathology of “fresh” human aortic valve allografts. Thorax 28: 482, 1973. 22. Heslop, B. F., Wilson, S. E., and Hardey, B. E. Antigenicity of aortic valve allografts. Ann. Surg. Res. 177: 301, 1973. 23. van der Kamp, A. W. M., Nauta, J., and Galjaard, H. Effect of preservation and sterilization methods on structure and function of aortic-valves. Eur. J. Surg. Res. 4: 361, 1972. 24. van der Kamp, A. W. M., and Nauta, J. Fibroblast function and the maintenance of the aorticvalve matrix. Cardiovasc. Res. 13: 167, 1979. 25. Lehr, H. B., Berggren, R. B., Lotke, P. A., and Coniell, L. L. Permanent survival of preserved skin autografts. Surgery 56: 742, 1964. 26. Leibo, S. P., and Mazur, P. The role of cooling rates in low-temperature preservation. Ctyobiology 8: 447, 1971. 27. Lackey, E., Al-Yanabi, N., Gonzalez-Lavin, L., and Ross, D. N. A method of sterilizing and preserving fresh allograft heart valves. Thorax 27: 398, 1972. 28. Lovelock, Y. E. The mechanism of the protective action of glycerol against haemolysis by freezing and thawing. Biochim. Biophys. Acta 2: 28, 1953. 29. Lower, R. R., Pillsbury, R. C., Angell, W. W.,
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30. Malinin, G. L. Cytotoxic effect of dimethylsulfoxide on the ultrastructure of cultured rhesus kidney cells. Cryobiology 10: 22, 1973. 3 1. Mazur, P. Cryobiology: The freezing of biological systems. Science 168: 939, 1970. 32. McGregor, C. G. A., Bradley, J. F., McGee, J. O’D., and Wheatley, D. J. Tissue culture, protein and collagen synthesis in antibiotic sterilized canine heart valves. Cardiovasc. Res. 10: 389, 1976. 33. McGregor, C. G. A., Bradley, J. F., McGee, J.O’D. and Wheatley, D. J. Viability in human heart valves prepared for grafting. Cardiovasc. Res. 10: 394, 1976. 34. Mermet, B., Angell, W. W., and Dor, V. Viabilite des homogreffes fraiches sterilistes par les antibiotiques et conservees au grand froid (-196°C). Ann. Chir. Thorac. Cardiovasc. 10: 463, 1971. 35. Meryman, H. T. Cryoprotective agents. Cryobiology 8: 173, 1971. 36. Mohri, H., Reichenbach, D. D., Barnes, R. W., and Merendino, A. K. A biological study of the homologous aortic valve in dogs. J. Thorac. Cardiovasc. Surg. 54: 622, 1967. 37. Mohri, H., Reichenbach, D. D., Barnes, R. W., and Merendino, A. K. Homologous aortic valve transplantation. J. Thoruc. Cardiovasc. Surg. 56: 767, 1968. 38. Murray, G. D. Q. Homologous aortic-valve-segment transplants as surgical treatment for aortic and mitral insufficiency. Angiology 7: 466, 1956. 39. O’Grady, L. F., and Lewis, J. P. The long-term preservation of bone marrow. Trnnsfusion 12: 312, 1972. 40. Olsen, B. R. Electron microscope studies on collagen. Z. Zellforsch. Mikrosk. Anat. 59: 184, 1963. 41. Polge, C., Smith, A. U., and Parkes, A. S. Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature (Lond.) 164: 666, 1949. 42. Pribor, D. B. PVP contrasted with dextran and a multifactor theory of cryoprotection. Cryobiology 2: 60, 1974. 43. Rajotte, R. V., Shnitka, T. K., Liburd, E. M., Dossetor, J. B., and Voss, W. A. G. Histological studies on cultured canine heart valves recovered from - 196°C. Cryobiology 14: 15, 1977. 44. Schaeffer, U. W., Dicke, K. A., and van Bekkum, D. W. Recovery of haemopoiesis in lethally irradiated monkeys by frozen allogeneic bone marrow grafts. Eur. J. C/in. Biol. Res. 17: 483, 1972. 45. Thompson, R., Knight, E., Ahmed, M., Somerville, W., Towers, M., and Yacoub, M. The use of “fresh” unstented homograft valves for replacement of the aortic valve. Circulation 56: 837, 1977.