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J Thorac Cardiovasc Surg 1997;113:1014-1021
© 1997 Mosby, Inc.
SURGERY FOR ACQUIRED HEART DISEASE |
Supported in part by Programa CIENCIA/JNICT, Fundação Calouste Gulbenkian, and the Portuguese Health Ministery.
Received for publication May 6, 1996 revisions requested July 9, 1996; revisions received Jan. 16, 1997 accepted for publication Jan. 22, 1997. Address for reprints: José Neves, MD, Instituto do Coração, 27, Av. Reinaldo dos Santos, 2795 Carnaxide, Portugal.
Abstract
Objective: To analyze the mechanism(s) underlying homograft degeneration, we designed an experimental model in which the behavior of cryopreserved autografts and homografts, as well as fresh autografts, implanted in the same animal was compared. Methods: A cryopreserved homograft was implanted in the aorta of 14 sheep. The excised aortic autologous segment was then subjected to cryopreservation, and 1 to 8 weeks later it was implanted 1 to 2 cm below the cryopreserved homograft. The intermediate segment of the native aorta, the fresh autograft, was dissected at this point. Animals were put to death at different times and the implanted segments were harvested together with a portion of native aorta. Histologic and immunohistochemical analyses, as well as cell viability assessments, were then performed on the explanted segments. Similar studies were also conducted on fragments of cryopreserved autografts and homografts before implantation. Results: With the exception of a partial loss of the endothelium, cryopreserved specimens retained cell viability and morphologic integrity before implantation. Explanted cryopreserved homografts showed profound changes affecting all strata, as well as a decline in cell viability. Lymphocyte infiltrates were found up to 12 months after implantation. Endothelium was always absent in cryopreserved homografts. However, a reendothelialization of the cryopreserved autografts was observed. After an initial period of neuronal degeneration, reenervation of the cryopreserved autograft segment occurred 6 to 12 months after the operation. Findings regarding the fresh autografts were similar to those of the cryopreserved autografts. Conclusion: Our results suggest that the immunologic reaction rather than the cryopreservation process is responsible for the degenerative process occurring in cryopreserved homografts.
Cryopreserved homograft heart valves and conduits are frequently used in the surgical correction of several diseases, in which they remain the substitutes of choice. However, the mechanisms underlying the degenerative process that ultimately leads to their failure remain unknown.
Recent studies comparing heart valve homografts and valves of transplanted hearts showed that whereas the latter contained fibroblasts of both donor and recipient origin, the former were acellular.
1-3 These differences could be due to either the occurrence of an immune response in homograft valve recipients, which was prevented or abrogated by the immunosuppressive therapy administered to heart transplant recipients but not to homograft valve recipients, or to the cryopreservation process to which the homograft valves were subjected. To distinguish between these two alternatives, we designed an experimental model in which the behavior of cryopreserved autografts and homografts, implanted in the same animal, was compared. Fresh autografts were used to analyze the role of denervation and devascularization. The biologic alterations induced by the different procedures were assessed by evaluation of cell viability and by immunohistochemical analysis of the endothelium, perivascular innervation, and medial smooth muscle cells. Genetic profiles of donor and recipient cells were used to determine the origin of the cells in explanted cryopreserved homografts.
Material and methods
Animals.
Fifteen Merino Branco sheep, eight ewes and seven rams, aged 2 to 18 months, were used.
Surgery.
Standard anesthesia technique with thiopental sodium (Pentothal) and halothane was used. Humane care was provided to all animals during the experiment, in compliance with the "Guide for the Care and Use of Laboratory Animals" (NIH publication No. 86-23, revised 1985). Cryopreserved homografts of the descending thoracic aorta were prepared as previously described. 1,2 In the first operation, a 1 to 2 cm segment of thoracic aorta was replaced by a cryopreserved homograft. The excised aortic segment was then subjected to the same cryopreservation process used for treatment of the homografts. One to 8 weeks later, the cryopreserved autograft was implanted 1 to 2 cm below the cryopreserved homograft. The intermediate segment of the native aorta was, at this point, completely dissected from the surrounding tissues and used as a fresh autograft. Animals were put to death at different times (2 weeks, 1, 3, 6, 12, and 24 months) after the second operation and the implanted segments were harvested together with a portion of native aorta to be used as control tissue. With regard to the latter segment, care was taken to analyze areas that were far from the surgical sites.
Immunohistochemistry.
Immediately after excision, the various segments were fixed by immersion according to the method of Steffanini, De Martino, and Zamboni 4 for 18 to 24 hours at 4° C. After being rinsed in several changes of phosphate-buffered saline solution (0.01 mol/L, pH 7.2) containing 15% sucrose, tissues were processed as cryostat sections for indirect immunofluorescence staining together with antisera raised against 
-smooth muscle actin and neuronal markers (protein gene product 9.5), endothelial markers (von Willebrand factor), and Schwann cell markers (S-100). Primary antisera were obtained from Sigma Chemical Company, St. Louis, Missouri, except for the antiserum to protein gene product 9.5, which was obtained from Ultraclone, Isle of White, United Kingdom.
Cell viability.
Initially, cell viability was evaluated by culturing tissue fragments in 10 ml flasks for 3 to 4 weeks in Dulbecco's modified medium at 37° C in an atmosphere containing 10% carbon dioxide and graded as 0 to 5, according to the rate of fibroblast growth. Explanted fragments were also analyzed by a new method, devised to ascertain a less subjective evaluation of cell viability. Thus 8 to 12 fragments of 1 mm diameter, excised from each portion of the explanted conduit, were cultured in RPMI medium (Roswell Park Memorial Institute, Buffalo, N.Y.) containing 10% fetal calf serum, in 96-well microtiter plates, at 37° C in an atmosphere containing 5% carbon dioxide. Fibroblast growth was evaluated microscopically on days 4, 8, and 15 after initiation of cultures. Wells with five or more fibroblasts were considered positive. The results were then expressed in percent of positive wells.
Analysis of DNA hypervariable regions.
Cell origin was determined as previously described 1,2 by analyzing the hypervariable (CA)n repeat regions from DNA of native aorta and cryopreserved homografts. In brief, DNA obtained from tissues by the standard proteinase K method, followed by phenol-chloroform extraction, was amplified by polymerase chain reaction with the use of oligonucleotide primers for TGLA53, TGLA126, TGLA263, MGTG7, and MGTG4B loci (kindly provided by Applied Biosystems Division of Perkin-Elmer, Foster City, Calif.). The polymerase chain reaction amplification was performed in a model PE 9600 thermocycler and the products were electrophoresed in an automated DNA sequencer (model 373A, ABI Analytical). The size of the (CA)n amplimers was determined with the use of the GENESCAN 672 software (ABI Analytical).
Results
With the exception of a partial loss of endothelial cells, cryopreserved specimens retained cell viability and morphologic integrity before implantation.
The results obtained with the various markers 3 months and 2 years after the last operation are shown in Figs. 1 and 2, respectively. These two time points illustrate the degenerative and regenerative processes that occurred during the course of the experiment (2 years). As can be seen, explanted cryopreserved homografts showed profound histologic changes that affected all strata, as well as decline in cell viability (Fig. 3). After an initial period of nonspecific inflammatory reaction, which in most cases subsided after 1 to 3 months, progressive neuronal and smooth muscle degeneration, as well as fragmentation of elastic fibers, was observed, leading in later stages (6 to 12 months) to the disappearance of the perivascular innervation, fibrosis hyalinization, and calcification. Most likely as a result of this process, one cryopreserved homograft ruptured after 17 months. Polymorphonuclear cells disappeared after 1 to 3 months; lymphocyte infiltrates, however, persisted up to 2 years after implantation (Fig. 4). Although endothelial cells were always absent in cryopreserved homografts, reendothelialization occurred in the cryopreserved autografts. After an initial inflammatory reaction, as in all other segments, nerve degeneration was observed in cryopreserved autografts. At this point (15 to 30 days), signs of smooth muscle alteration were also present, as judged by the weakness of the immunoreactive staining for actin. At 3 to 6 months and thereafter, smooth muscle cells and myofibrils had, however, recovered their normal structure, and progressive reenervation was observed, with reestablishment of the normal nervous tissue pattern being achieved 6 to 12 months after the operation. Histologically, a single alteration, consisting of an intimal thickening, was patent in these explants (see Fig. 2, D). Cell viability was similar to that of native aorta.
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Origin of the cells in the cryopreserved homografts was determined in fragments explanted 1 and 2 years after the operation. The results from these experiments showed that cells of both donor and recipient origin were present in the tissue in the first animal; and in the second only the latter were found (Table I and Fig. 5).
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Even when performed under optimal conditions, harvesting, handling, cryopreservation, and antibiotic treatment of homografts affects the degree of cell survival, the metabolic rates of the viable cells, and, consequently, matrix degradation and synthesis. 5-14 Therefore, and despite the abundant evidence of immunogenicity of the valve homograft, 1-3,15-27 the concept that the durability of a homograft is related to the degree of cell viability at the time of implantation prevailed. 28-30
By creating conditions in which autologous tissue was treated in the same way as homologous tissue, we could ascertain that the preparation procedures, including ischemic time, antibiotic treatment, and cryopreservation, were not responsible for the long-term deterioration found in homografts.
Of particular interest were the findings that loss of the endothelium and degeneration of the neuronal tissue smooth muscle and vasa vasorum occurred in the cryopreserved autografts as it did in the homografts. Thus the evidence that these processes were followed by regeneration in the former but not the latter further strengthens the concept that the mechanism underlying the failure of the homografts is most likely immunologic rather than physical. Whether the immune response induced by the allogeneic tissue led to the destruction of progenitor cells or prevented the release of angiogenic and neurogenic factors required for the regeneration process remains to be determined.
More information on the origin of the cells in the homograft is needed before conclusions can be drawn. However, it is possible that at 2 years the donor cells have been destroyed and replaced by recipient cells. These findings are not consistent with those obtained in human heart valves. 1-3 This discrepancy might be due to the structural differences between the two tissues, in particular the presence of muscular tissue in the aorta but not in the valve leaflets.
Some of the findings in the present study may be clinically relevant. Thus it became clear that denervation and devascularization did not affect the vitality of the tissue, inasmuch as regeneration of both systems could occur, provided factors such as the immune response did not hinder the process. Such is the case of pulmonary autografts in the aortic root position (Ross operation). Also worth notice was the single alteration found in the cryopreserved autologous grafts, the thickening of the intima. Similar findings have been reported for cryopreserved rat aortic isografts. 26 Because intimal thickening did not occur in the fresh autologous fragments, it is tempting to hypothesize that this is the sole transformation induced by cryopreservation.
Appendix: Discussion
Dr. Patrick G. Hogan (Brisbane, Australia).
I am asking my questions from the perspective of an immunologist. First, were you transplanting across a major histocompatibility complex barrier? What is known about the immunogenetics of the major histocompatibility complex in sheep?
Dr. Neves.
Yes. We were transplanting across the major histocompatibility complex.
Dr. Hogan.
The sheep are not likely to be inbred. They would be outbred. Might they be polymorphic for major histocompatibility complex loci?
Dr. Neves.
Yes, but the same condition occurs in human beings.
Dr. Hogan.
I am interested in the S-100 staining. A recent paper from St. Vincent's Hospital in Australia, discussing the use of S-100, purports to show that there are dendritic cells in the human aorta. If immunologic responses are important, do you think any of your staining could have been produced by dendritic cells? I do note that there was no staining in the cryopreserved tissue.
Dr. Neves.
I am a surgeon, not an immunologist, and the information that I have from my colleagues is that S-100 stains the Schwann cells. I cannot answer that.
Dr. Hogan.
In your abstract, lymphocytic infiltrates are documented as one point in favor of an immune response. Could you expand on that? Were the lymphocytic infiltrates different in intensity or nature in the homografts as opposed to the autografts?
Dr. Neves.
All of the segments in the first weeks have an inflammatory reaction. After 1 or 2 months, the homograft persists with lymphocytes. We tried to mark them for T cells, B cells, and activations, but we had problems with the specific markers for sheep. It was difficult to get conclusions. However, we have performed these studies in human beings and we know that there is an immunologic response with activated T cells.
Acknowledgments
We are grateful to M. Castelo, M. R. Alpiarça, R. Santos, P. Dias, G. Horak, and R. Cunha Ferreira for excellent technical assistance. We are also grateful to Dr. J. Ziegle from Perkin-Elmer Applied Biosystems Division for kindly providing the primers.
Footnotes
From the Instituto do Coração/Hospita de Santa Cruz, Carnaxide, Portugala; Departamento de Biologia Celular Instituto Gulbenkian de Ciência, Oeiras, Portugalb; Eatação Zootécnica Nacional, Santarem, Portugalc; and Departamento de Genética, Faculdade de Ciências Médicas UNL, Lisboa, Portugal.d 
Read at the Seventy-sixth Annual Meeting of The American Association for Thoracic Surgery, San Diego, Calif., April 28-May 1, 1996.
References
This article has been cited by other articles:
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  M. Peltz, B. A. Sandoval, and M. E. Jessen Traumatic Rupture of a Descending Thoracic Aortic Homograft Ann. Thorac. Surg., August 1, 2005; 80(2): 710 - 712. [Abstract] [Full Text] [PDF]
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 W. J. Wells, H. Arroyo Jr, R. M. Bremner, J. Wood, and V. A. Starnes Homograft conduit failure in infants is not due to somatic outgrowth J. Thorac. Cardiovasc. Surg., July 1, 2002; 124(1): 88 - 96. [Abstract] [Full Text] [PDF]
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J. A. Hawkins, J. P. Breinholt, L. M. Lambert, T. C. Fuller, T. Profaizer, E. C. McGough, and R. E. Shaddy CLASS I AND CLASS II ANTI-HLA ANTIBODIES AFTER IMPLANTATION OF CRYOPRESERVED ALLOGRAFT MATERIAL IN PEDIATRIC PATIENTS J. Thorac. Cardiovasc. Surg., February 1, 2000; 119(2): 324 - 330. [Abstract] [Full Text] [PDF]
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