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Endothelial integrity after venous cryopreservation
JOURNAL
OF SURGICAL
RESEARCH
Endothelial STEPHEN
32, 218-227 ( 1982)
Integrity
M. SACHS,
M.D.,’ AND
Deparrmenrs
after Venous
JOHN J. RICOTTA, JAMES A. DEWEESE,
Cryopreservation M.D., M.D.
of Surgery and Anaromy, University of Rochesrer Medical Rochester, New York, 14642
D. E. SCOTT,
PH.D.,
Center, 601 Elmwood
Avenue,
Presented at the Annual Meeting of the Association for Academic Surgery, Chicago, Illinois, November 8-l 1, 1981 Veins treated with dimethyl sulfoxide (DMSO) prior to freezing were compared with fresh veins with respect to fibrinolytic activity, morphologic changes, and endothelial viability. After storage for 3 to 8 weeks frozen veins retained their basic morphologic structure and fibrinolytic activity, but cell viability was lost. When such veins were placed in the canine femoral venous circulation, a high rate of thrombosis was noted. This could be prevented by treatment of the graft recipient with aspirin and dipyridamole. Frozen veins removed 5 days after grafting showed loss of endothelium and librinolytic activity. Implications of these findings with reference to “vein banking” are discussed.
Although autogenous saphenous vein remains the graft material of choice for peripheral vascular reconstruction, alternatives continue to be sought. Chronic venous insufficiency, previous removal for grafting or vein stripping, inadequate vein length or diameter, massive trauma, and desire to preserve usable vein for future small arterial grafting are all important limitations on the use of autogenous vein. The possibility of using a frozen venous allograft as an arterial substitute remains intriguing. In 1966 Barner et al. [2] reported 73% patency for frozen homologous vein followed to 30 weeks in the canine femoral artery. Similarly Weber et al. [ 171 have reported >60% 1 year patency of dimethyl sulfoxide (DMSO)-preserved vein grafts in the canine carotid artery. However, Calhoun et al. [ 31 found that cryopreservation resulted in marked endothelial damage with a high rate of thrombosis in frozen vein grafts (only 11% patent at 30 days). Malone et al. [8] have reported preservation of fibrinolytic activity in veins stored up to 3 months with only minimal endothelial changes in cryopreserved veins. Questions remain as to the changes in endothelial viability, ultrastructure, fibrinolytic activity of cryopreserved veins after implantation, and how these changes will effect
patency in a relatively thrombogenic system. To evaluate this we studied the ultrastructure, fibrinolytic activity, and patency of frozen autografts before and 5 days after implantation in the canine femoral venous circulation. MATERIALS
Preparation of frozen veins. Under general anesthesia using sterile technique, a loto 1S-cm segment of canine jugular vein was harvested, rinsed with normal saline, incubated at room temperature for 1 hr in 15% dimethyl sulfoxide (DMSO), and then placed in a -70°C freezer. Determination of$brinolytic activity. The fibrin plate method of Todd [ 161 was used to quantitate the fibrinolytic activity of venous endothelium. A fibrin monolayer was formed using thrombin 0.1 ml (0.2 pg/ml), bovine fibrinogen 1.5 ml (7.5 mg/ml), and plasminogen, 0.1 ml (1.6 pg/ml) buffered with Tris buffer to pH of 8. A 6-mm punch biopsy of vein was obtained, washed with normal saline, and placed endothelial side down onto the fibrin surface. The fibrin plates were incubated at room temperature for 18 hr and a zone of lysis was measured in square millimeters by planimetry. Determination of endothelial viability of cell culture. Canine endothelial cells were
’ To whom requests for reprints should be addressed. 0022-4804/82/030218-10.%01.00/O Copyright 0 1982 by Academic Press. Inc. All rights of reproduction in any form reserved.
AND METHODS
218
SACHS
ET AL.: VENOUS
harvested and grown using techniques previously described by Graham et al. [6] and Herring [7]. Ten centimeters of canine jugular vein was everted over a glass rod and washed in Hanks’ balanced salt solution (HBSS) free of Ca*+/Mg*+. The vein was incubated in 0.2% collagenase for 20 min at 37°C and jet-rinsed with HBSS. The cell suspension was centrifuged at 400g for 5 min. The cell button was washed once and suspended in culture medium (McCoy’s 5A) with 10% fetal bovine serum, penicillin/ streptomycin ( 1O4 pg/liter), and fungizone (0.25 mg/lOO ml). Cells were incubated at 37°C with 5% CO*. Each flask was examined daily for growth using a Zeiss tissue microscope and photomicrographs taken with a Wild inverted phase contrast microscope. Light
and scanning electron microscopy.
Sections of fresh and frozen veins were fixed in 10% formalin, sectioned to 4-pm thickness, and stained with hematoxylin and eosin. Sections for scanning electron microscopy (SEM) were fixed in Karnovsky’s fixative and scanned on an American Optical field and scanning electron microscope. Experiment 1. Firbinolytic activity and ultrastructural changes were compared in 20 frozen and 20 fresh canine jugular veins. Endothelial viability, i.e., ability to grow endothelial cells in tissue culture, was compared in a series of 10 fresh and 10 frozen veins. Experiment 2. One fresh and one frozen autograft were placed end to end in the femoral vein of 14 mongrel dogs using standard vascular techniques. This method has been used by one of us in the past to evaluate vascular grafts [ 11, 121. Anastomoses were performed with 6-O Prolene without magnification. Immediately prior to implantation segments of vein were taken for measurement of fibrinolytic activity. Seven dogs served as control while 7 received aspirin ( 1300 mg/day) and dipyridamole ( 100 mg/ day) beginning on the day prior to surgery. Grafts were removed 5 days after implan-
219
CRYOPRESERVATION
tation and studied for fibrinolytic activity, ultrastructural changes, and patency. Statistical methods. The two-sided exact two-sample test of difference of proportions was used to analyze the results of endothelial viability in 10 fresh versus 10 frozen veins and also in the patency data. All other data were analyzed using Student’s t test. RESULTS Experiment
1
Fibrinolytic activity. Both fresh and frozen veins reproducibly exhibited fibrinolytic activity. There was no statistically significant difference between the fibrinolytic activity of the 20 fresh veins (124 mm + 14) and the frozen veins ( 124 mm + 19). Light and scanning electron microscopy.
Fresh and frozen veins appeared virtually identical under light microscopy (Figs. 1a and b). Endothelial cells in fresh veins were normal under SEM whereas frozen veins revealed small focal disruption of the endothelial surface (Fig. 2). Endothelial viability. Endothelial cells were identified as polygonal cells growing in a confluent monolayer with evidence of intercellular bridge formation (Fig. 3). Endothelial viability, defined as the ability to harvest and grow cells in tissue culture, was markedly different when fresh and frozen veins were compared. We were able to grow viable cells in 8 of 10 fresh veins (Fig. 3). However, none of 10 frozen grafts yielded any cells which would grow in tissue culture (P < 0.001). Experiment
2
Fresh versus frozen autografts in the femoral venous system. Fibrinolytic activity in
fresh and frozen vein grafts 5 days after implantation is shown in Table 1. Treatment of the animals with aspirin and dipyridamole did not affect fibrinolytic activity, therefore, treated and control animals are considered together. There was a significant reduction in fibrinolytic activity in both fresh and fro-
220
JOURNAL OF SURGICAL
RESEARCH: VOL. 32, NO. 3, MARCH
1982
SACHS
ET AL.: VENOUS
221
CRYOPRESERVATION
FIG. 2. Scanning electron micrograph (I 100X) of a DMSO cryopreserved canine jugular vein. Note that most of the endothelial surface is intact. However, in the lower aspect note the focal endothelial cell disruption.
zen grafts 5 days after implantation. Fresh veins showed a 50% reduction in fibrinolytic activity. However, in frozen veins fibrinolytic activity was virtually absent. This difference was statistically significant (P < 0.001). Patency results are shown in Table 2. All fresh autografts remained patent in both control and treated animals. Frozen autografts in control animals all thrombosed by 5 days. In animals treated with aspirin and
dipyridamole, frozen autografts all remained patent. Untreated frozen autografts fared significantly less well (P < 0.001) than the other three groups. At 5 days fresh vein grafts were normal under light and scanning electron microscopy (Figs. 4a and 5a). However, frozen grafts showed marked endothelial loss with exposure of the subendothelial collagen layer, macrophage infiltration, and fibrin deposition (Figs. 4b and 5b).
FIG. la. Fresh canine jugular vein (H&E, 84X). Note intact endothelium thickening or cellular infiltrate. FIG. 1b. Cryopreserved canine jugular vein. Notice that there is minimal endothelial surface and that of a fresh vein (H&E, 84X).
and absence of medial difference
between this
222
JOURNAL OF SURGICAL
RESEARCH: VOL. 32, NO. 3, MARCH
1982
FIG. 3. Phase contrast micrograph (76X) of endothelial cells in tissue culture. Note the confluent monolayer of polygonal cells. Intercellular bridges can be seen in some areas. These characteristics identify the cells as endothebum. Cultures such as this were obtained from 8 of 10 fresh jugular veins.
DISCUSSION
The autogeneous vein is the only graft material with consistent long-term patency in small vessel arterial reconstruction. HowTABLE FIBRINOLYTIC
ever, in many patients the saphenous vein may not be usable because of prior venous disease, inadequate size, surgical stripping, or previous arterial graft procedures. Recently, suggestions have been made to spare autogenous saphenous veins in above-
1
ACTIVITY
TABLE
(mm’)
2
PATENCY 5 DAYS POSTIMPLANTATION Preimplant Fresh veins
122 + 4
Frozen veins
11.5 f 5
Postimplant Control group
Aspirin and dipyridamole group
Fresh
71’
717
Frozen
o/7*
717
60 f 4*
4 r 3**
* Significantly reduced (P < 0.001) when compared with same vein preimplantation. ** Significantly reduced (P < 0.001) when compared to fresh veins postimplantation.
* P < 0.001 when compared to fresh veins or frozen veins treated with ASA/Persantine.
SACHS
ET AL.: VENOUS
CRYOPRESERVATION
223
FIG. 4b. Photomicrograph (H&E, 55~) of a frozen vein graft removed 5 days after implantation. In contrast to the fresh graft, note the marked subintimal infiltration of inflammatory cells with sloughing of the endothelial cell layer.
SACHS ET AL.: VENOUS CRYOPRESERVATION
225
FIG. 5a. SEM (71X) of a fresh vein graft 5 days after implantation. Note the intact endothelial surface.
the knee-arterial reconstruction, saving it for tibia1 or coronary grafting. Synthetic prostheses have had limited successin small vessel reconstruction. A practical biologic “shelf graft” would be a great advance in coronary and peripheral arterial reconstruction. Previous work by one of us has shown promising results using venous allografts for venous reconstruction in the dog model [ 121. Current studies are aimed at evaluating the possible role of cryopreserved veins with the aim of developing a “vein bank.” The idea of using cryopreserved venous allografts is not new. Pretreatment with DMSO followed by cryopreservation has been successfully reported using parathyroid tissue, with good functional results [4, 191. Barner et al. studied cryopreserved auto-
grafts in 1966 with promising results in the canine femoral artery [2]. He was able to grow fibroblasts in tissue culture, suggesting that cell viability was preserved by incubation with DMSO. Weber et al. showed acceptable patency (60%) and normal intimal morphology in frozen autografts studied 1 year after implantation [ 171. This contrasts with results by Calhoun et al. who showed marked endothelial damage and graft thrombosis at 30 days [3] and Giordano et al. [5] who found a high thrombosis rate in the canine venous system. Tice et al., reporting their clinical experience with cryopreserved venous allografts in peripheral and coronary arterial reconstruction, noted patency in 33/46 cryopreserved vein grafts followed up to 3 years [ 151. There has been
226
JOURNAL
OF SURGICAL
RESEARCH:
VOL. 32, NO. 3, MARCH
FIG. 5b. SEM (924X) of a frozen vein graft 5 days postimplantation. gone with collagen exposed and macrophages infiltrating the wall.
considerable experience with arterial homografts which indicated a significant incidence of late aneurysm formation, but to date this has not been reported using venous homografts. It has been suggested that aneurysmal dilatation would be less likely in the venous homograft since it does not depend on the vasa vasorum for nutrient supply as does its arterial counterpart. However, no long-term studies exist which address this question. If cryopreserved allografts are to be of potential use in vascular surgery, more detailed study of morphologic changes which occur early after, implantation is needed. We decided to investigate three variables: fibrinolytic activity, endothelial viability, and morphologic changes shortly after implantation of allografts. We also investigated the
1982
Note that the endothelium
is
effect of these variables on graft patency in a stringent experimental model, the canine femoral venous system. To avoid immunologic considerations we employed venous autografts. Our results in Experiment 1 indicated that freezing after incubation with DMSO conserves vein architecture and fibrinolytic activity. These results agree with those previously published by Malone and Moore et al. [ 81. Interestingly, we were unable to culture endothelial cells from frozen veins, although we were successful in 8/ 10 fresh veins using a standard collagenase digestion technique. This indicated to us that endothelial cells were more fastidious than fibroblast or parathyroid tissue and did not survive freezing, even when pretreated with DMSO. We postulated that the minor intimal changes seen
SACHS
ET AL.: VENOUS
under SEM would progress and result in massive endothelial loss in the early postimplant period with significant graft thrombosis. This hypothesis was borne out in Experiment 2, where we observed marked loss of endothelium and fibrinolytic activity within 5 days of graft implantation. In untreated animals this resulted in graft thrombosis. Treatment of the recipient with a regimen designed to inhibit platelet aggregation maintained graft patency. This result supports our earlier observations [ 121 and those of other authors [lo]. Work using fresh venous allografts showed that marked endothelial loss, presumably immunologically mediated, occurred within 3 weeks of grafting and resulted in a high rate of thrombosis when allografts were placed in the canine venous circulation [ 11, 121. We have shown that patency rates can be improved in this situation by treating the recipient animal with aspirin and dipyridamole, graft repopulation occurs with host cells, and long-term patency is feasible [ 121. It is conceivable that similar endothelial destruction which accompanies cryopreservation could be compensated for by antiplatelet therapy. If so, intimal repopulation could occur as in the case of fresh allografts. Should these postulates prove correct, frozen venous allografts might provide an acceptable alternative to saphenous vein.
CRYOPRESERVATION
total parathyroidectomy and parathyroid autograft. Ann. Surg. 193: 777, 1981. 5. Giordano, J. M., Lamoy, R. E., Wright, C. B., and Hobson, R. W. A comparison of autografts and frozen irradiated homografts in canine femoral venous reconstruction. Surgery 81: 100, 1977. 6. Graham, L. M., Burkel, W. E., Ford, J. W., Vinter, D. W., Kahn, R. H., and Stanley, J. C. Immediate seeding of enzymatically derived endothelium in dacron vascular grafts. Arch. Surg. 114: 1289, 1980. 7. Herring, 8. Malone,
9.
10.
Il.
12.
13.
14.
15.
16.
REFERENCES 1. Axthelm, S. C., Porter, J. M., Strickland, S., and Baur, G. M. Antigenicity of venous allografts. Ann. Surg. 189: 290, 1979. 2. Barner, H. B., DeWeese, J. A., and Schenk, E. A. Fresh and frozen homologous venous grafts for arterial repair. Angiology 17: 389, 1966. 3. Calhoun, A. D., Baur, G. M., Porter, J. M., Houghton, D. H., and Templeton, J. W. Fresh and cryopreserved venous allografts in genetically characterized dogs. J. Surg, Res. 22: 687, 1977. 4. Diethelm, A. G., Adam, P. L., Murad, T. M., Daniel, W. W., Whelchel, J. D., Rutsky, E. A., and Rostand, S. G. Treatment of secondary hyperparathroidism in patients with chronic renal failure by
227
17.
M. Personal communication. J. M., Moore, W. S., Kischer, C. W., Keawn, K., and Conine, R. Venous cryopreservation: endothelial fibrinolytic activity and histology. J. Surg. Res. 29: 209, 1980. Oblath, R. W., Buckley, F. O., Green, R. M., Schwartz, S. I., and DeWeese, J. A. Prevention of platelet aggregation and adherence to prosthetic vascular grafts by aspirin and dipyridamole. Surgery 84: 37, 1978. PerloB, L. J., Anderson, R. T., and Barker, C. F. Endothelial repopulation in venous allografts. J. Surg. Res. 18: 131, 1975. Ricotta, J. J., Collins, G. J., Rich, N. M., and Reynolds, D. G. Failure of immunosuppression to prolong venous allograft survival. Arch. Surg. 115: 99, 1980. Ricotta, J. J., Schaff, H. V., and Gadacz, T. R. The effect of aspirin and dipyridamole on the patency of allograft veins. J. Surg. Rex 26: 262, 1979. Schwartz, S. I., Kutner, F. R., Neistadt, A., Barner, H., Resnicoff, S., and Vaughn, J. Antigenicity of homografted veins. Surgery 61: 47 1, 1967. Tice, D. A., and Zerbino, V. Clinical experience with preserved human allografts for vascular reconstruction. Surgery 72: 260, 1972. Tice, D. A., Zerbino, V., Isam, 0. W., Cunningham, J. N., and Engelman, R. M. Coronary artery bypass with freeze-preserved saphenous vein allografts. J. Thorac. Cardiovasc. Surg. 71: 378, 1976. Todd, A. S. Localization of fibrinolytic activity in tissues. Brit. Med. Bull. 20: 210, 1964. Weber, T. R., Dent, T. L., Lindenaur, S. M., Allen, E., Weatherbee, L., Spencer, H. H., and Gleick, P. Viable vein graft preservation. J. Surg. Res. 18:
247, 1975. 18. Weber, T. R., Lindenauer,
S. M., Dent, T. L., Allen, E., Salles, C. A., and Weatherbee, L. Long-term patency of vein grafts preserved in liquid nitrogen in dimethyl sulfoxide. Ann. Surg. 184: 709, 1976. 19. Wells, S. A., Gunnells, J. C., Shelburne, J. D., et nl. Transplantation of parathyroid glands in man: Clinical indications and results. Surgery 78: 34, 1975. 20. Williams,
G. M., TerHaar, A., Krajewski, C., Parks, L. C., and Roth, J. Rejection and repair of endothelium in major vessel transplants. Surgery 78: 694,
1975.
OF SURGICAL
RESEARCH
Endothelial STEPHEN
32, 218-227 ( 1982)
Integrity
M. SACHS,
M.D.,’ AND
Deparrmenrs
after Venous
JOHN J. RICOTTA, JAMES A. DEWEESE,
Cryopreservation M.D., M.D.
of Surgery and Anaromy, University of Rochesrer Medical Rochester, New York, 14642
D. E. SCOTT,
PH.D.,
Center, 601 Elmwood
Avenue,
Presented at the Annual Meeting of the Association for Academic Surgery, Chicago, Illinois, November 8-l 1, 1981 Veins treated with dimethyl sulfoxide (DMSO) prior to freezing were compared with fresh veins with respect to fibrinolytic activity, morphologic changes, and endothelial viability. After storage for 3 to 8 weeks frozen veins retained their basic morphologic structure and fibrinolytic activity, but cell viability was lost. When such veins were placed in the canine femoral venous circulation, a high rate of thrombosis was noted. This could be prevented by treatment of the graft recipient with aspirin and dipyridamole. Frozen veins removed 5 days after grafting showed loss of endothelium and librinolytic activity. Implications of these findings with reference to “vein banking” are discussed.
Although autogenous saphenous vein remains the graft material of choice for peripheral vascular reconstruction, alternatives continue to be sought. Chronic venous insufficiency, previous removal for grafting or vein stripping, inadequate vein length or diameter, massive trauma, and desire to preserve usable vein for future small arterial grafting are all important limitations on the use of autogenous vein. The possibility of using a frozen venous allograft as an arterial substitute remains intriguing. In 1966 Barner et al. [2] reported 73% patency for frozen homologous vein followed to 30 weeks in the canine femoral artery. Similarly Weber et al. [ 171 have reported >60% 1 year patency of dimethyl sulfoxide (DMSO)-preserved vein grafts in the canine carotid artery. However, Calhoun et al. [ 31 found that cryopreservation resulted in marked endothelial damage with a high rate of thrombosis in frozen vein grafts (only 11% patent at 30 days). Malone et al. [8] have reported preservation of fibrinolytic activity in veins stored up to 3 months with only minimal endothelial changes in cryopreserved veins. Questions remain as to the changes in endothelial viability, ultrastructure, fibrinolytic activity of cryopreserved veins after implantation, and how these changes will effect
patency in a relatively thrombogenic system. To evaluate this we studied the ultrastructure, fibrinolytic activity, and patency of frozen autografts before and 5 days after implantation in the canine femoral venous circulation. MATERIALS
Preparation of frozen veins. Under general anesthesia using sterile technique, a loto 1S-cm segment of canine jugular vein was harvested, rinsed with normal saline, incubated at room temperature for 1 hr in 15% dimethyl sulfoxide (DMSO), and then placed in a -70°C freezer. Determination of$brinolytic activity. The fibrin plate method of Todd [ 161 was used to quantitate the fibrinolytic activity of venous endothelium. A fibrin monolayer was formed using thrombin 0.1 ml (0.2 pg/ml), bovine fibrinogen 1.5 ml (7.5 mg/ml), and plasminogen, 0.1 ml (1.6 pg/ml) buffered with Tris buffer to pH of 8. A 6-mm punch biopsy of vein was obtained, washed with normal saline, and placed endothelial side down onto the fibrin surface. The fibrin plates were incubated at room temperature for 18 hr and a zone of lysis was measured in square millimeters by planimetry. Determination of endothelial viability of cell culture. Canine endothelial cells were
’ To whom requests for reprints should be addressed. 0022-4804/82/030218-10.%01.00/O Copyright 0 1982 by Academic Press. Inc. All rights of reproduction in any form reserved.
AND METHODS
218
SACHS
ET AL.: VENOUS
harvested and grown using techniques previously described by Graham et al. [6] and Herring [7]. Ten centimeters of canine jugular vein was everted over a glass rod and washed in Hanks’ balanced salt solution (HBSS) free of Ca*+/Mg*+. The vein was incubated in 0.2% collagenase for 20 min at 37°C and jet-rinsed with HBSS. The cell suspension was centrifuged at 400g for 5 min. The cell button was washed once and suspended in culture medium (McCoy’s 5A) with 10% fetal bovine serum, penicillin/ streptomycin ( 1O4 pg/liter), and fungizone (0.25 mg/lOO ml). Cells were incubated at 37°C with 5% CO*. Each flask was examined daily for growth using a Zeiss tissue microscope and photomicrographs taken with a Wild inverted phase contrast microscope. Light
and scanning electron microscopy.
Sections of fresh and frozen veins were fixed in 10% formalin, sectioned to 4-pm thickness, and stained with hematoxylin and eosin. Sections for scanning electron microscopy (SEM) were fixed in Karnovsky’s fixative and scanned on an American Optical field and scanning electron microscope. Experiment 1. Firbinolytic activity and ultrastructural changes were compared in 20 frozen and 20 fresh canine jugular veins. Endothelial viability, i.e., ability to grow endothelial cells in tissue culture, was compared in a series of 10 fresh and 10 frozen veins. Experiment 2. One fresh and one frozen autograft were placed end to end in the femoral vein of 14 mongrel dogs using standard vascular techniques. This method has been used by one of us in the past to evaluate vascular grafts [ 11, 121. Anastomoses were performed with 6-O Prolene without magnification. Immediately prior to implantation segments of vein were taken for measurement of fibrinolytic activity. Seven dogs served as control while 7 received aspirin ( 1300 mg/day) and dipyridamole ( 100 mg/ day) beginning on the day prior to surgery. Grafts were removed 5 days after implan-
219
CRYOPRESERVATION
tation and studied for fibrinolytic activity, ultrastructural changes, and patency. Statistical methods. The two-sided exact two-sample test of difference of proportions was used to analyze the results of endothelial viability in 10 fresh versus 10 frozen veins and also in the patency data. All other data were analyzed using Student’s t test. RESULTS Experiment
1
Fibrinolytic activity. Both fresh and frozen veins reproducibly exhibited fibrinolytic activity. There was no statistically significant difference between the fibrinolytic activity of the 20 fresh veins (124 mm + 14) and the frozen veins ( 124 mm + 19). Light and scanning electron microscopy.
Fresh and frozen veins appeared virtually identical under light microscopy (Figs. 1a and b). Endothelial cells in fresh veins were normal under SEM whereas frozen veins revealed small focal disruption of the endothelial surface (Fig. 2). Endothelial viability. Endothelial cells were identified as polygonal cells growing in a confluent monolayer with evidence of intercellular bridge formation (Fig. 3). Endothelial viability, defined as the ability to harvest and grow cells in tissue culture, was markedly different when fresh and frozen veins were compared. We were able to grow viable cells in 8 of 10 fresh veins (Fig. 3). However, none of 10 frozen grafts yielded any cells which would grow in tissue culture (P < 0.001). Experiment
2
Fresh versus frozen autografts in the femoral venous system. Fibrinolytic activity in
fresh and frozen vein grafts 5 days after implantation is shown in Table 1. Treatment of the animals with aspirin and dipyridamole did not affect fibrinolytic activity, therefore, treated and control animals are considered together. There was a significant reduction in fibrinolytic activity in both fresh and fro-
220
JOURNAL OF SURGICAL
RESEARCH: VOL. 32, NO. 3, MARCH
1982
SACHS
ET AL.: VENOUS
221
CRYOPRESERVATION
FIG. 2. Scanning electron micrograph (I 100X) of a DMSO cryopreserved canine jugular vein. Note that most of the endothelial surface is intact. However, in the lower aspect note the focal endothelial cell disruption.
zen grafts 5 days after implantation. Fresh veins showed a 50% reduction in fibrinolytic activity. However, in frozen veins fibrinolytic activity was virtually absent. This difference was statistically significant (P < 0.001). Patency results are shown in Table 2. All fresh autografts remained patent in both control and treated animals. Frozen autografts in control animals all thrombosed by 5 days. In animals treated with aspirin and
dipyridamole, frozen autografts all remained patent. Untreated frozen autografts fared significantly less well (P < 0.001) than the other three groups. At 5 days fresh vein grafts were normal under light and scanning electron microscopy (Figs. 4a and 5a). However, frozen grafts showed marked endothelial loss with exposure of the subendothelial collagen layer, macrophage infiltration, and fibrin deposition (Figs. 4b and 5b).
FIG. la. Fresh canine jugular vein (H&E, 84X). Note intact endothelium thickening or cellular infiltrate. FIG. 1b. Cryopreserved canine jugular vein. Notice that there is minimal endothelial surface and that of a fresh vein (H&E, 84X).
and absence of medial difference
between this
222
JOURNAL OF SURGICAL
RESEARCH: VOL. 32, NO. 3, MARCH
1982
FIG. 3. Phase contrast micrograph (76X) of endothelial cells in tissue culture. Note the confluent monolayer of polygonal cells. Intercellular bridges can be seen in some areas. These characteristics identify the cells as endothebum. Cultures such as this were obtained from 8 of 10 fresh jugular veins.
DISCUSSION
The autogeneous vein is the only graft material with consistent long-term patency in small vessel arterial reconstruction. HowTABLE FIBRINOLYTIC
ever, in many patients the saphenous vein may not be usable because of prior venous disease, inadequate size, surgical stripping, or previous arterial graft procedures. Recently, suggestions have been made to spare autogenous saphenous veins in above-
1
ACTIVITY
TABLE
(mm’)
2
PATENCY 5 DAYS POSTIMPLANTATION Preimplant Fresh veins
122 + 4
Frozen veins
11.5 f 5
Postimplant Control group
Aspirin and dipyridamole group
Fresh
71’
717
Frozen
o/7*
717
60 f 4*
4 r 3**
* Significantly reduced (P < 0.001) when compared with same vein preimplantation. ** Significantly reduced (P < 0.001) when compared to fresh veins postimplantation.
* P < 0.001 when compared to fresh veins or frozen veins treated with ASA/Persantine.
SACHS
ET AL.: VENOUS
CRYOPRESERVATION
223
FIG. 4b. Photomicrograph (H&E, 55~) of a frozen vein graft removed 5 days after implantation. In contrast to the fresh graft, note the marked subintimal infiltration of inflammatory cells with sloughing of the endothelial cell layer.
SACHS ET AL.: VENOUS CRYOPRESERVATION
225
FIG. 5a. SEM (71X) of a fresh vein graft 5 days after implantation. Note the intact endothelial surface.
the knee-arterial reconstruction, saving it for tibia1 or coronary grafting. Synthetic prostheses have had limited successin small vessel reconstruction. A practical biologic “shelf graft” would be a great advance in coronary and peripheral arterial reconstruction. Previous work by one of us has shown promising results using venous allografts for venous reconstruction in the dog model [ 121. Current studies are aimed at evaluating the possible role of cryopreserved veins with the aim of developing a “vein bank.” The idea of using cryopreserved venous allografts is not new. Pretreatment with DMSO followed by cryopreservation has been successfully reported using parathyroid tissue, with good functional results [4, 191. Barner et al. studied cryopreserved auto-
grafts in 1966 with promising results in the canine femoral artery [2]. He was able to grow fibroblasts in tissue culture, suggesting that cell viability was preserved by incubation with DMSO. Weber et al. showed acceptable patency (60%) and normal intimal morphology in frozen autografts studied 1 year after implantation [ 171. This contrasts with results by Calhoun et al. who showed marked endothelial damage and graft thrombosis at 30 days [3] and Giordano et al. [5] who found a high thrombosis rate in the canine venous system. Tice et al., reporting their clinical experience with cryopreserved venous allografts in peripheral and coronary arterial reconstruction, noted patency in 33/46 cryopreserved vein grafts followed up to 3 years [ 151. There has been
226
JOURNAL
OF SURGICAL
RESEARCH:
VOL. 32, NO. 3, MARCH
FIG. 5b. SEM (924X) of a frozen vein graft 5 days postimplantation. gone with collagen exposed and macrophages infiltrating the wall.
considerable experience with arterial homografts which indicated a significant incidence of late aneurysm formation, but to date this has not been reported using venous homografts. It has been suggested that aneurysmal dilatation would be less likely in the venous homograft since it does not depend on the vasa vasorum for nutrient supply as does its arterial counterpart. However, no long-term studies exist which address this question. If cryopreserved allografts are to be of potential use in vascular surgery, more detailed study of morphologic changes which occur early after, implantation is needed. We decided to investigate three variables: fibrinolytic activity, endothelial viability, and morphologic changes shortly after implantation of allografts. We also investigated the
1982
Note that the endothelium
is
effect of these variables on graft patency in a stringent experimental model, the canine femoral venous system. To avoid immunologic considerations we employed venous autografts. Our results in Experiment 1 indicated that freezing after incubation with DMSO conserves vein architecture and fibrinolytic activity. These results agree with those previously published by Malone and Moore et al. [ 81. Interestingly, we were unable to culture endothelial cells from frozen veins, although we were successful in 8/ 10 fresh veins using a standard collagenase digestion technique. This indicated to us that endothelial cells were more fastidious than fibroblast or parathyroid tissue and did not survive freezing, even when pretreated with DMSO. We postulated that the minor intimal changes seen
SACHS
ET AL.: VENOUS
under SEM would progress and result in massive endothelial loss in the early postimplant period with significant graft thrombosis. This hypothesis was borne out in Experiment 2, where we observed marked loss of endothelium and fibrinolytic activity within 5 days of graft implantation. In untreated animals this resulted in graft thrombosis. Treatment of the recipient with a regimen designed to inhibit platelet aggregation maintained graft patency. This result supports our earlier observations [ 121 and those of other authors [lo]. Work using fresh venous allografts showed that marked endothelial loss, presumably immunologically mediated, occurred within 3 weeks of grafting and resulted in a high rate of thrombosis when allografts were placed in the canine venous circulation [ 11, 121. We have shown that patency rates can be improved in this situation by treating the recipient animal with aspirin and dipyridamole, graft repopulation occurs with host cells, and long-term patency is feasible [ 121. It is conceivable that similar endothelial destruction which accompanies cryopreservation could be compensated for by antiplatelet therapy. If so, intimal repopulation could occur as in the case of fresh allografts. Should these postulates prove correct, frozen venous allografts might provide an acceptable alternative to saphenous vein.
CRYOPRESERVATION
total parathyroidectomy and parathyroid autograft. Ann. Surg. 193: 777, 1981. 5. Giordano, J. M., Lamoy, R. E., Wright, C. B., and Hobson, R. W. A comparison of autografts and frozen irradiated homografts in canine femoral venous reconstruction. Surgery 81: 100, 1977. 6. Graham, L. M., Burkel, W. E., Ford, J. W., Vinter, D. W., Kahn, R. H., and Stanley, J. C. Immediate seeding of enzymatically derived endothelium in dacron vascular grafts. Arch. Surg. 114: 1289, 1980. 7. Herring, 8. Malone,
9.
10.
Il.
12.
13.
14.
15.
16.
REFERENCES 1. Axthelm, S. C., Porter, J. M., Strickland, S., and Baur, G. M. Antigenicity of venous allografts. Ann. Surg. 189: 290, 1979. 2. Barner, H. B., DeWeese, J. A., and Schenk, E. A. Fresh and frozen homologous venous grafts for arterial repair. Angiology 17: 389, 1966. 3. Calhoun, A. D., Baur, G. M., Porter, J. M., Houghton, D. H., and Templeton, J. W. Fresh and cryopreserved venous allografts in genetically characterized dogs. J. Surg, Res. 22: 687, 1977. 4. Diethelm, A. G., Adam, P. L., Murad, T. M., Daniel, W. W., Whelchel, J. D., Rutsky, E. A., and Rostand, S. G. Treatment of secondary hyperparathroidism in patients with chronic renal failure by
227
17.
M. Personal communication. J. M., Moore, W. S., Kischer, C. W., Keawn, K., and Conine, R. Venous cryopreservation: endothelial fibrinolytic activity and histology. J. Surg. Res. 29: 209, 1980. Oblath, R. W., Buckley, F. O., Green, R. M., Schwartz, S. I., and DeWeese, J. A. Prevention of platelet aggregation and adherence to prosthetic vascular grafts by aspirin and dipyridamole. Surgery 84: 37, 1978. PerloB, L. J., Anderson, R. T., and Barker, C. F. Endothelial repopulation in venous allografts. J. Surg. Res. 18: 131, 1975. Ricotta, J. J., Collins, G. J., Rich, N. M., and Reynolds, D. G. Failure of immunosuppression to prolong venous allograft survival. Arch. Surg. 115: 99, 1980. Ricotta, J. J., Schaff, H. V., and Gadacz, T. R. The effect of aspirin and dipyridamole on the patency of allograft veins. J. Surg. Rex 26: 262, 1979. Schwartz, S. I., Kutner, F. R., Neistadt, A., Barner, H., Resnicoff, S., and Vaughn, J. Antigenicity of homografted veins. Surgery 61: 47 1, 1967. Tice, D. A., and Zerbino, V. Clinical experience with preserved human allografts for vascular reconstruction. Surgery 72: 260, 1972. Tice, D. A., Zerbino, V., Isam, 0. W., Cunningham, J. N., and Engelman, R. M. Coronary artery bypass with freeze-preserved saphenous vein allografts. J. Thorac. Cardiovasc. Surg. 71: 378, 1976. Todd, A. S. Localization of fibrinolytic activity in tissues. Brit. Med. Bull. 20: 210, 1964. Weber, T. R., Dent, T. L., Lindenaur, S. M., Allen, E., Weatherbee, L., Spencer, H. H., and Gleick, P. Viable vein graft preservation. J. Surg. Res. 18:
247, 1975. 18. Weber, T. R., Lindenauer,
S. M., Dent, T. L., Allen, E., Salles, C. A., and Weatherbee, L. Long-term patency of vein grafts preserved in liquid nitrogen in dimethyl sulfoxide. Ann. Surg. 184: 709, 1976. 19. Wells, S. A., Gunnells, J. C., Shelburne, J. D., et nl. Transplantation of parathyroid glands in man: Clinical indications and results. Surgery 78: 34, 1975. 20. Williams,
G. M., TerHaar, A., Krajewski, C., Parks, L. C., and Roth, J. Rejection and repair of endothelium in major vessel transplants. Surgery 78: 694,
1975.