Comparison of Viable and Betapropiolactone-Treated Orthotopic Homologous Aortic Valves in Dogs

Transcription

1 THE ANNALS OF THORACIC SURGERY Journal of The Society of Thoracic Surgeons and the Southern Thoracic Surgical Association VOLUME 13 NUMBER 3 * MARCH 1972 Comparison of Viable and Betapropiolactone-Treated Orthotopic Homologous Aortic Valves in Dogs A Four-Year Follow-up Hitoshi Mohri, M.D.,* Dev R. Manhas, M.D., Dennis D. Reichenbach, M.D., and K. Alvin Merendino, M.D. ABSTRACT Orthotopic homograft single-cusp replacement of the aortic valve was done in 40 dogs. The report is focused on alterations in 12 late specimens procured between 20 and 40 months after transplantation. Connective tissue coverings on leaflets became shorter and thinner in later specimens, and tissue appearance became more compact and dense. Despite initial excellent results and absence of destructive host connective tissue invasions in the leaflet even in late periods after transplantation, betapropiolactone-strilbed valves underwent severe degeneration of leaflet collagen and elastic fibers with marked round-cell infiltrations. Viable grafts demonstrated mild changes in collagen and elastic fibers and showed the presence of donor leaflet cells for 31 months. However, an increase in round cells in the leaflet and shifts of cartilaginous metaplastic areas toward the leaflet base from the host-graft junction created some concern. From the Department of Surgery and the First Surgical Service of the University Hospital, University of Washington School of Medicine, Seattle, Wash. Aided by U.S. Public Health Service Grants HE and GM We wish to acknowledge the valuable assistance of Mr. Murray P. Sands and Mr. Rix Kuester. 'Established investigator of the American Heart Association. Accepted for publication Aug. 8, Address reprint requests to Dr. Merendino, Department of Surgery, University of Washington School of Medicine, Seattle, Wash '99

2 MOHRI ET AL. A n increasing number of reports of late homologous aortic valve failure [2, 3, 61 have caused some disappointment regarding the use of chemically sterilized homografts despite their initial excellent performance. The fresh, viable or untreated preserved graft has been advocated as the valve of choice [l, 31. We have reported details of histological alterations in canine homograft aortic valves followed up to 23 months (fresh, viable) or 12.5 months (sterilized, nonviable) [9]. This series has now been followed for up to four years, and significant differences in histological alterations between viable and nonviable grafts have been discovered. The purpose of this paper is to depict mainly the detailed analysis of late alterations observed in 12 specimens procured between 20 and 40 months after transplantation, with special attention to graft failures and donor leaflet cell viability. Materials and Methods Single-cusp homograft replacement of the aortic valve was performed in 40 adult mongrel dogs utilizing a method described elsewhere [81. Males were used as recipients and females were donors. Twenty dogs received fresh, viable grafts procured under sterile conditions and transplanted within 90 minutes after harvest. A total of 20 dogs received betapropiolactone-sterilized (BPL) grafts, 10 of which had been stored at 4 C. in isotonic saline solution and 10 of which had been stored frozen at -8O"C., all for up to four weeks. None of the experimental animals received antithrombogenic or immunosuppressive drugs. The origin of leaflet cells was determined by tissue culture of the leaflet free edge and by subsequent sex-chromatin body analysis [51. Results There were no operative deaths, but a total of 11 dogs later died from thromboemboli or graft infection. Twenty-six dogs were sacrificed under sterile conditions at intervals between one week and 40 months postoperatively. One dog with a fresh, viable graft and 2 dogs with BPL-frozen grafts are still under observation in their fifth year following transplantation. COMPLICATIONS Thromboemboli. Nearly all specimens obtained within 2 to 3 months demonstrated thrombus or large fibrin deposits over the grafts as contrasted to specimens obtained thereafter. Thromboembolism to major organs was found in 13 of 29 dogs with noninfected grafts, 2 of whom died from this complication. The kidney was the most commonly affected organ, and the spleen was occasionally involved. 200 THE ANNALS OF THORACIC SURGERY

3 Viable and BPL-Treated Aortic Valves in Dogs In most animals sacrificed past 12 months after transplantation, the infarcted lesions were scarred, suggesting that emboli usually developed in the early postoperative period. The incidence of thromboembolism was 7 of 17 with viable grafts, 4 of 7 with BPL grafts, and 2 of 5 with BPL-frozen valves. Infection. Eight dogs developed graft infection. Five of them died within 13 days. In the remaining 3 dogs the infection apparently developed later, with death occurring respectively at 12 months (viable graft), 6 months (BPL graft), and 5 months (BPL-frozen graft). The incidence of proved graft infection was 2 of 20 dogs with fresh grafts, 3 of 10 dogs with BPL grafts, and 3 of 10 dogs with BPL-frozen grafts. Tear or Perforation of the Leaflet. Five dogs demonstrated leaflet tears or perforations in the absence of graft infection. A leaflet tear was found in 1 specimen obtained at 23 months (BPL graft) and another at 40 months (viable graft). Three leaflet perforations occurred in BPL-frozen grafts procured at 25, 26, and 37 months following transplantation. There were 12 specimens obtained between 20 and 40 months after transplantation-5 viable grafts, 3 BPL grafts, and 4 BPL-frozen grafts. Thus the incidence of leaflet tears or perforations in the later specimens was 1 of 5 in viable grafts, 1 of 3 in BPL grafts, and 3 of 4 in BPL-frozen grafts. Scarring of the Leaflet. In 1 specimen (viable graft) procured at 31 months after transplantation, the leaflet was scarred and adherent to the sinus wall of the aorta. A small leaflet tissue remnant remained beneath the coronary artery orifice. Leaflet Calcification. Leaflet calcification was found in only 1 specimen in the absence of graft infection. The specimen was a BPL-frozen graft obtained 37 months after transplantation from a dog that had developed severe pneumonia 12 months postoperatively. However, no evidence of graft infection was found at sacrifice. There was a perforation in the leaflet at its peripheral portion, and the area of calcification was located slightly distal to the perforation. DONOR CELL SURVIVAL Sex-chromatin body analysis of cultured leaflet tissue explants revealed the persistent survival of donor leaflet fibroblasts even at 31 months after transplantation. Populations of the cells positive for sex chromatin, however, decreased to 8% of total fibroblasts in cultured explants from the control value of 37%. STRUCTURAL ALTERATIONS OF VALVE GRAFTS Histological alterations in homograft valves are summarized in Figure 1. Alterations in grafts followed up to 23 months (viable grafts) and 12 months (nonviable grafts) have been reported elsewhere [9]. Briefly, homograft aortic valves revealed a lack of immunological cellular responses, but they gradually VOL. 13, NO. 3, MARCH, 1972 ZOI

4 ~~ MOHRI ET AL. Mo. I BPL Endothe lial covering Inflammatory cellular reaction at the host-graft junction Fresh - Edematous, (hick leaflet Increased ground substance Donor cell proliferation Seporation of elastica Connective tissue covers graft base Leaf let cel lul a ri t y normal Normol.thickness leaflet New elastic fibers in the sheath Leaflet cells from host - ~ Dersistent donor cells Segmentol ocellulority Loose structured sheoth covers part of leaflet Leaflet thin and slightly mobile Collagen less distinct Increased macrophage and plasma cells in the leaflet Loose structured )onor leaflet cells Thick sheath covers the most ventricular side of t h Ieoflet New elastic layer Leaflet immobile Collagen fair Thin compact sheoth recessed in length Severe macrophage infiltration Collagen homogenous Protein deposit Cartilage near leaflet Donor cells Thin, short, compact sheath Leaflet mobile Collogen-some change Slight increase in mocrodhage Cartiloge near leaflet FIG. 1. Schema of histological alterations in homograft aortic valves. (C = cartilage; H = host; P = protein deposit; S = host connective tissue covering (sheath); arrows = extent of host connective tissue infiltration with some vasculature; large dots and M = macrophage, plasma cell, and lymphocyte infiltration; small dots and B = leaflet fibroblastic cells.) gained steady host tissue supports and maintained their original leaflet supporting structure well. Lymphocytes and plasma cells rarely infiltrated the leaflets, and in viable grafts donor leaflet fibroblasts persisted in leaflets at least up to 12 months. Host connective tissues gradually extended and covered the ventricular surface nearly entirely by 12 months in nonviable grafts or by 23 months in viable grafts, but on the aortic surface of the leaflet host connective tissue covered only one-half of the leaflet. In these connective 202 THE ANNALS OF THORACIC SURGERY

5 Viable and BPL-Treated Aortic Valves in Dogs tissue coverings, new elastic fibers were formed along the ventricular surface of the leaflets and host fibroblastic cells infiltrated into the leaflets through the covering. Host substitutions, however, did not progress any further in later specimens. In nonviable homografts, severe destruction of the original leaflet supporting structures gradually developed. Graft Base. Connective tissues at the graft base appeared to be more mature in late specimens. Vascular channel formations became more intense and extended further into the graft base, but neither connective tissues nor vascular channels invaded the leaflet itself up to 40 months. Cartilage formations of the invaded connective tissues appeared to become more extensive as time passed up to around 12 months but increased little thereafter. However, in the late specimens, affected areas shifted toward the leaflet base from the host-graft junction. Plasma cells and lymphocytes in the host-graft junction gradually decreased as time passed and nearly disappeared in nonviable grafts procured later than 23 months. However, in 4 of 5 viable grafts procured at comparable times there was mild but positive infiltration of these cells along with foreign-body giant cells around the suture materials. Connective Tissue Covering. Remarkable alterations of the covering developed in late specimens. The connective tissue, which once nearly entirely covered the ventricular side of the leaflet at 12 months in nonviable grafts or 23 months in viable grafts, appeared to regress in later specimens. On the graft base the covering was still thick, and new elastic-layer formations progressed more. However, the connective tissue covered only onefourth to two-thirds of the ventricular side of the leaflet in both types of grafts in later specimens. The appearance of the covering was remarkably altered. In earlier specimens, the covering consisted of a loose connective-tissue network and was thick (Fig. 2A). In later specimens the connective tissue structure became compact, dense, and remarkably thin with relatively less cellularity in areas overlying the leaflet itself (Fig. 2B). Occasionally the distal end of the covering was represented only by a few layers of elastic fibers, making judgment of the actual limits of sheath extension difficult. This thinning of the fibrous sheath apparently made once immobile viable leaflets mobile, although the leaflets were still slightly thicker than normal and there was still some restriction in movement. These overlying connective tissues were usually covered by endothelial extensions. In viable grafts, endothelial linings occasionally extended further to the peripheral, uncovered portion of the leaflet, but in nonviable grafts there was no endothelial lining over the peripheral, acellular leaflet. Collagen and Elastic Fibers. Supporting structures such as collagen and elastic fibers altered as time passed. Viable grafts at 23 months and nonviable grafts at 12 months revealed fraying and interruption of the elastic lamella, but collagen bundles usually remained well preserved. VOL. 13, NO. 3, MARCH,

6 MOHRI ET AL. A FIG. 2. Histological alterations in host connective tissue covering. (A) The connective tissue sheath extending over this aortic valve homograft left in place for 12 months occupies the major portion of the figure. On the left is the elastic tissue (E) of the original leaflet. To the right of this is the loose connective tissue sheath which has extended up over the ventricular surface of the homograft from the host. Numerous cell nuclei are evident within the connective tissue sheath, which is lined by a flattened surface layer of endothelial cells. Several small elastic filaments are seen in the outer portions of the sheath (arrows). (Verhoeff-van Gieson stain; X 100 before 2S% reduction.) (B) The elastic tissue (E) of the homograft aortic valve is seen in the midportion of this fresh homograft aortic valve removed after 40 months. The connective tissue sheath extending over the ventricular surface of the leaflet seen to the right of the elastic tissue is composed of rather dense collagen in comparison with the sheath seen in (A). Connective tissue cell nuclei (arrows) are seen in the fibrous intimal sheath as well as in the homograft aortic valve. (Verhoeff-van Gieson stain; X 100 before 25% reduction.) B At around 25 to 30 months in nonviable grafts, fraying or interruption of the elastic lamella progressed. Deposits of eosinophilic substances, probably protein, increased in the distal portion of the leaflet. The outlines of each collagen fiber in its bundle became indistinct, but alterations were still mild at this stage. In viable grafts, changes in the elastic lamella were nearly identical to those in nonviable grafts, but the collagen bundles were usually better preserved. In specimens of both viable and nonviable grafts procured earlier, there were scattered infiltrates of macrophages and polymorphonuclear leukocytes in superficial portions of the leaflet and the covering sheath. Cellular infiltrations in nonviable grafts became prominent at around 25 to 30 months with plasma cells and lymphocytes superimposed. These cellular infiltrations were mild in viable grafts at this stage. 204 THE ANNALS OF THORACIC SURGERY

7 Viable and BPL-Treated Aortic Valves in Dogs Striking structural alterations were seen in nonviable grafts obtained at 37 months (Fig. 3A). The elastic layers not only were interrupted in many places but also focally disappeared from leaflets. Collagen bundles, especially in the peripheral portion of the leaflet, had a homogeneous appearance, and a vast part of the peripheral portion was occupied by protein deposits. Focal and diffuse infiltrations of pigment-laden macrophages, lymphocytes, and plasma cells increased and involved the proximal one-half of the leaflet. Cellular reactions appeared to be much more extensive in a BPL-frozen graft than in a BPL graft. On the other hand, in viable grafts recovered at 40 months the collagen bundles showed only some staining alterations, but the elastic tissue was frayed (Fig. 3B). Mild to moderate scattered foci of macrophages and round cells, far less in degree than in nonviable grafts, were seen in the connective tissue covering and leaflet. Protein deposits were also present between collagen bundles, but to a minor degree. Leaflet Cellularity. The leaflet cellularity of viable grafts usually decreased as time passed, although there were some areas with fair numbers of elongated cells or contrary focal acellular areas. In specimens obtained 40 months following transplantation there were oval and elongated cells throughout the leaflet stroma. Nonviable grafts that had scattered host cells of the fibroblastic type in A B FIG. 3. Histological alterations in the grafted leaflet. (A) This homograft aortic valve leaflet which prior to use had been preserved by freezing and which had been left in place for 37 months shows acellular donor leaflet connective tissue in the upper left and host inflammatory reaction on the right extending into the elastic tissue of the graft. The inflammatory infiltrate is composed of lymphocytes and plasma cells and is associated with fragmentation of the elastic tissue filaments. (Verhoefl-van Gieson stain; X258 before?o% reduction.) (B) This fresh homograft aortic valve left in place for 40 months shows the donor leaflet with its elastic tissue (El on the left and the fibrous intimal sheath on the right. Nuclei of connective tissue cells (arrows) are seen in the fibrous intimal sheath. (Verhoeffvan Gieson stain; X 250 before 30% reduction.) VOL. 13, NO. 3, MARCH,

8 MOHRI ET AL. the proximal portion of the leaflet at around 12 months still revealed the presence of these cells at 25 months. However, such fibroblastic host cells did not extend further to distal portions of the leaflet. In specimens obtained at 37 months, the presence of this fibroblastic-type cell was indistinguishable due to the aforementioned severe infiltrations of inflammatory cells. Comment The striking destruction of the leaflet supporting structures in late BPL-sterilized grafts is disappointing. Examination of earlier specimens led us to expect possible progressive repopulation with host cells of the fibroblastic type and extension of the host connective tissue sheath. Apparently neither of these seemingly desirable alterations progressed. Between two and four years after transplantation, changes somehow developed either in the recipient (immunological origin or foreign-body reaction) or in the transplants (fatigue of supporting structures). Later changes in thickness and appearance of the connective tissue covering, which made viable graft leaflets somewhat mobile, suggests possible reversibility of initial host connective tissue reactions. The mechanism of collagen-bundle destruction is not exactly known; however, continuous stress to the supporting structures of the original leaflets, especially if these structures are not replaced by the recipient, might be a contributing factor, combined with destruction possibly due to the phagocytic action of infiltrating cells. Late round-cell reactions in leaflets combined with tremendous macrophage infiltrations were difficult to interpret in the absence of roundcell reactions along the host-graft junction of nonviable grafts. These cellular infiltrations may be attributed to either immunological factors or foreign-body responses, but it is unknown whether they initiated leaflet structure destruction or appeared in areas where degeneration had already progressed. The persistent presence of round cells in the host-graft junction of the viable grafts even at 40 months suggests a higher immunological POtential of viable grafts over nonviable homografts. If the structural destruction is related to an immunological factor, viable grafts would have displayed greater destruction. This study has demonstrated that there is persistent function and minimum deterioration of the leaflet supporting structures of viable aortic valve homografts for at least 40 months. However, a gradual increase of macrophages and the appearance of some round cells in later specimens is worrisome. Whatever the cause of these cellular reactions, viable homograft aortic valve leaflets may, at some future time, undergo destruction similar to that seen in nonviable grafts. Gradual inward shifts of cartilaginous metaplastic areas toward the leaflet in both types of grafts with the passage of time is also of concern. Judging from this shift toward the leaflet, it is possible that host fibrous tissue may eventually infiltrate the leaflet and develop cartilag- 206 THE ANNALS OF THORACIC SURGERY

9 Viable and BPL-Treated Aortic Valves in Dogs inous metaplasia later in the transplantation period. Viable heterografts, which have stronger antigenicity, revealed connective tissue invasion and later severe cartilaginous metaplasia in the leaflet at 23 months after transplantation [lo]. The progressive decrease of positive sex chromatin cells in viable graft leaflets is attributed to recipient fibroblastic cell infiltrations in the leaflet. One viable leaflet from a donor whose sex was not known was transplanted into a female recipient and recovered at 24 months. This specimen demonstrated a nearly normal population of cells positive for sex chromatin. This fact definitely proved the presence of host-cell repopulations, since the transplanted leaflet should demonstrate no (male donor) or decreased (female donor) cell populations positive for sex chromatin at 24 months if no hostcell infiltration had occurred. It is uncertain which is the more important factor for long-term function of valve grafts, viable leaflet cells or unaltered leaflet structures without viable cells. If viable cells are important, the accelerated cellular proliferation seen in earlier specimens creates another concern. Diploid connective tissue cells are said to have genetically determined numbers of cell doublings [4, 71. In the human skin fibroblast it is about 50. The accelerated cell doublings in early viable homografts may conceivably shorten the life of donor leaflet fibroblasts. References 1. Angell, W. W. Long-Term Results-Mitral Homografts. Presented at the American College of Cardiology, Aspen, Colo., Jan , Barnes, R. W., Rittenhouse, E. A., Mohri, H., and Merendino, K. A. A clinical experience with the betapropiolactone-sterilized homologous aortic valve followed up to four years. J. Thorac. Cardiovasc. Surg. 59:785, Barratt-Boyes, B. G., and Roche, A. H. G. A review of aortic valve homografts over a six and one-half year period. Ann. Surg. 170:483, Hayflick, L. The limited in vitro life time of human diploid cell strain. Exp. Cell Res. 37:614, Loder, R. M., and Martin, G. M. Establishment of human skin fibroblast cultures with an automatic wedge biopsy instrument. J. Invest. Dermatol. 47: 603, Malm, J. R. Testing and Preservation of Valve Grafts. Presented at the American College of Cardiology, Aspen, Colo., Jan , Martin, G. M., Sprague, C. A., and Epstein, C. J. Replicative life span of cultivated human cells. Lab. Invest. 23:86, Mohri, H., Reichenbach, D. D., Barnes, R. W., and Merendino, K. A. A bioiogic study of the homologous aortic valve in dogs. J. Thorac. Cardiovasc. Surg. 54:622, Mohri, H., Reichenbach, D. D., Barnes, R. W., and Merendino, K. A. Homologous aortic valve transplantation: Alterations in viable and non-viable valves. J. Thornc. Cardiovasc. Surg. 56:767, Mohri, H., Reichenbach, D. D., and Merendino, K. A. Alterations of transplanted viable sheep aortic valves in dogs: Follow-up studies to 23 months. Surgery 59:762, VOL. 13, NO. 3, MARCH,