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Partial replacement of mitral valve by homograft
Partial replacement of mitral valve by homograft An experimental study Satisfactory long-term clinical results with heart valves have renewed the interest in the use of mitral homografts, despite the technical difficulties with their surgical implantation. This report describes the behavior and viability of the partial mitral homograft in the ortotopic position in a chronic sheep model (n = 25). The 20 surviving animals were studied hemodynamically and were anesthetized and electively put to death 3, 6, 9, and 12 months after the operation. All specimens had a normal mitral valve without signs of infection or thrombosis. Light, scanning, and transmission electron microscopy showed the presence of viable endothelial cells from the recipient covering the graft, signs of reendothelialization, and organized dense collagen tissue. The structural integrity was more evident in the fresh mitral homografts. This method may provide consider improvement in the viability of the mitral homograft, and it could be a valid alternative for repair of mitral valve localized pathology. (J THORAC CARDIDVASC SURG 1992;104:1274-9)
Jose M. Revuelta, MD, Juan C. Cagigas, MD, Jose M. Bernal, MD, Fernando Val, MD, Jose M. Rabasa, MD, and Maria A. Lequerica, MD, Santander, Spain
Initial experiments in partial transplantation of the cardiac valves were carried out in the canine model by Robicsek and colleagues. I Those early experiments proved that homograft implantation was technically possible, and the results demonstrated remarkable performance and durability.o ' The search for an ideal mitral homograft heart valve substitute has been the objective of a considerable investigative effort." However, methods of sterilization and preservation of homografts and the surgical technique of replacement of the mitral valve by mitral homograft have not been satisfactory. The aim of this study was to evaluate, in a chronic animal model, the viability and behavior of the mitral homograft by replacing a segment of the mitral valve with an original technique. Material and methods Thirty-five mitral homografts were harvested immediately after death of donor young sheep with a mean weight of From the Department of Cardiovascular Surgery, HospitalUniversitario Valdecilla, Universidad de Cantabria, Santander, Spain. Received for publication Sept. 5, 1991. Acceptedfor publication April 9, 1992. Addressfor reprints: JoseM. Revuelta,MD,PhD, Professor ofSurgery, Chairman of Cardiovascular Surgery, Hospital Universitario "Marques de Valdecilla," 39008Santander, Spain.
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24.7 ± 2.3 kg (range 21 to 32 kg.). Ten homografts were washed in distilled water and then in a modified Hanks solution before being immersed for 24 hours at 4° C in an antibiotic solution' composed of cefoxitin (240 mg/rnl), lincomycin (120 mg/rnl), polymyxin B (100 rng/rnl), vancomycin (50 mg/rnl), and amphotericin B (25 mg/rnl), in modified Hanks solution (I L), with a pH of 6.8 to 7.0. The fresh mitral homografts were then moved to a TC 199 culture medium for another 24 hours (group A). After immersion for 24 hours at 4°C in the same antibiotic solution, 15 mitral homografts were cryopreserved in a controlled-rate liquid nitrogen freezer at -I ° C per minute up to -40° C, and later to -196° C, with 10% dimethyl sulfoxide as cryoprotectant (group B). Five of them were refused because of contamination, and the remaining five mitral homografts are still cryopreserved ready to be used in another experiment. Aortic wall fragments and samples of the used solutions were introduced into a thioglycolate and Sabouraud's dextrose medium solution for cultures. Twenty-five young sheep, with a mean weight of 23.4 ± 2 kg (range 17 to 30 kg) and an average age of 12 months, were used for this study. All animals in our experiment received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH Publication No. 85-23, revised 1985). The sheep were sheared the day before the operation and fasted for 24 hours. Anesthesia was induced with thiopental sodium (2 rug/kg) and halothane; a mechanical ventilator was used for respiratory support. The heart was exposed via a left thoracotomy through the fourth intercostal space. Cardiopulmonary bypass was established by cannulation of the right atri-
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Partial replacement of mitral valve by homograft
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B Fig. 1. Partial mitral homograft implanted in the orthotopic position. A, A segment of the anterior leaflet and subvalvular apparatus of the mitral valve has been resected and substituted by a partial mitral homograft (H). B, Implanted mitral homograft (H).
qA H-F-P-(l.-9 Fig. 2. Specimen of a sheep killed 9 months after cryopreserved mitral homograft implantation in the anterior leaflet. The graft appears almost like the native mitral valve. urn and the femoral artery. An apical left ventricular vent was insertedand connected to the arterial return line and used to test mitral valve competence. During cardiopulmonary bypass the heart was maintained beating under normothermic conditions. Thus the aorta was not crossclamped and cardioplegic solution was not used. A wide left atriotomy was performed from the atrial appendage to the left lower pulmonary vein. Partial resection of the anterior (n = 20) or posterior (n = 5) leaflet and their corre-
sponding subvalvular apparatus was carried out. Partial mitral homografts were inserted into the recipient sheep of similar weight and age as the donor sheep. The amount of the anterior leaflet of homograft to be replaced should be at least 4 to 6 mm wider than the resected leaflet to have enough tissue to allow an adequate suture line overlapping to native leaflet. A doublearmed 2-0 pledget-supported Ti-Cron suture (Davis & Geck, Danbury, Conn.) was passed through the tip of the homograft papillary muscle and then through the recipient anterior papil-
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Revuelta et al.
Fig. 3. Histologic section ofthe implanted mitral homograft. A, Fresh mitral homograft (H) totally integrated into the valve (V), explanted 6 months after transplantation. (Masson's stain; original magnification XSO.) B, Endothelial surface around the chordae tendineae in the fresh mitral homograft, explanted 6 months after implantation. (Hematoxylin and eosin stain; original magnification X32.)
lary muscle (Fig. 1). The implantation of the donor papillary muscle to the recipient papillary muscle is done in the dome in a way that allows a normal coaptation of the leaflets and does not interfere with the native subvalvular apparatus. In our experience a homograft 2 to 4 mm larger in the subvalvular apparatus avoids an eventual shrinkage of the graft. The mitral homograft leaflet fragment, including two marginal chordae on average, was then sutured to the recipient valve defect with a running 5-0 polypropylene suture, from the free edge of the leaflet toward the mitral anulus. Blood under pressure was allowed to enter the left ventricular cavity through the vent connected to the arterial line to test valve competence. The suture holding the homograft subvalvular apparatus to the papillary muscle must be tied when the adequacy of the valve is checked. One gram of cefamandole was administered subcutaneously before and after the operation and was maintained for 3 days postoperatively. The sheep were closely observed at the Surgical Research Unit for 7 days after operation and then were transferred to the farm, where they lived an active normal life. The animals were checked for evidence of new murmurs by cardiac auscultation and were observed for signs of heart failure. A hemodynamic study was performed in all survivingsheep before they were electivelyput to death 3, 6, 9, and 12 months postoperatively.
Results Five sheep died within the first 3 months after operation because of residual severe mitral insufficiency (n = 3), anesthesia (n = 1), and accident at the farm (n = 1). The residual valve incompetence was due to malposition of the subvalvular apparatus of the mitral homograft at a lower level than its normal location. Twenty-four animals survived the operation. Nine animals from group A (n = 10) and II animals from group B (n = 15) survived and were electively killed. Mean follOW-Up period was 8 ± 2.5 months (range 3 to 12 months). The animals were killed between 3 and 6 months (n = 5), 6 and 9 months (n = 10), and 9 and 12 months (n = 5). The hemodynamic study performed immediately before each animal was killed demonstrated a mean left atrial pressure of9.5 ± 5 mm Hg, and mean peak systolic left ventricular pressure was 113 ± 3 mm Hg. Residual mitral incompetence was detected during the postoperative hemodynamic study in three patients. Mean heart rate was 97 ± 10 beats/min (range 80 to 160).
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Fig. 4. Transmission electron microscopicviewshowsthe dense organized collagen fibers with a uniform distribution in a fresh preserved mitral homograft explanted 6 months after operation.
Fig. 5. Scanning electron microscopyshowsa cryopreservedmitral homograft (H) denuded of endothelial cellswith an ingrowth tissue of the viable endothelial cells from the recipient (V), 9 months after implantation. (Original magnification 160 X 1.1.) All mitral homografts had cultures positive for infection at dissection from the donor heart. Contaminating organisms included Candida albicans, Pseudomonas
aeruginosa, Pseudomonas maltophilia, Acinetobacter, Escherichia coli, Moraxella, genus and non fermenting
species. After 24 hours of antibiotic immersion, 30 of the previously contaminated grafts had cultures negative for infection, while 5 homograftscontinued to grow P. aeruginosa, E. coli, M oraxella genus, and non fermenting species. No new types of organisms were detected after
The Journal of Thoracic and Cardiovascular Surgery
I 2 7 8 Revuelta et al.
Fig. 6. Scanning electron microscopic view of a mitral fresh homograft 9 monthsafter operation, with chordal (C) integration clearlyidentified. (Originalmagnification 20 X 1.) homograft manipulation. Twenty-five homograft valves were implanted. All cultures obtained at the time of implantation were negative for infection. There have been no instances of late infection in this series. Macroscopic examination of the heart showed no intracardiac thrombi and an apparently normal functioning repaired mitral valve without thrombosis, infective endocarditis, or degenerative failure signs (perforations, ruptures, or calcifications). The segments of the mitral homografts implanted were totally incorporated into the recipient valves and subvalvular apparatus, retaining their original flexibility and length. It was difficult to identify the replaced homografts because they appeared almost like the recipient valve tissue (Fig. 2). A technical error was the cause of valve insufficiency in the three animals studied hemodynamically late after operation. The error consisted in an inappropriate location of the subvalvular apparatus of the partial homograft in the recipient
papillary muscle. No rupture of the chordae tendineae was found in these specimens. Microscopic evaluation, 3 to 12 months after implantation, revealed the presence of viable endothelial cells from the recipient valves invading the mitral homografts and connective tissue fibroblasts in fresh and cryopreserved homografts with signs of reendothelialization (Fig. 3, A). A sheath of connective tissue around the chordae tendineae in the fresh homografts was observed (Fig. 3, B). Organized dense collagen tissue was detected in all implanted homografts without signs of degeneration. A total structural integrity of the homograft with cellular viability was microscopically demonstrated in all specimens from groups A and B. Mitral homograft was completely acellular within ground substance (Fig. 3), and no signs of infiltrations of mononuclear cells neither areas of fibroblast necrosis in the subendothelial tissues. Fibrous sheathing was minimal when it was present. These microscopic findings were similar in both groups of mitral homografts. Valve morphology detected by transmission electron microscopy showed dense organized collagen fibers with a uniform distribution with cellular rests of fibroblasts (Fig. 4). Scanning electron microscopy showed the grafts denuded of endothelial cells, with an ingrowth tissue of viable recipient endothelial cells trying to cover the homograft surface and a normal appearance of the remaining denuded graft (Fig. 5). Organized collagen fibers without cellular infiltration and chordae tendineae reendothelialization were also observed with scanning electron microscopy in fresh and cryopreserved homografts implanted at least 6 months in the sheep (Fig. 6). Discussion Although the use of mitral homografts to substitute mitral valves was introduced by Rastelli, Berguis, and Swan' experimental results were not satisfactory. Trying to solve the technical difficulties of mitral homograft implantation, various surgical procedures have been described with unpredictable results." 7 Hubka and colleagues? used the mitral homograft in the tricuspid position in the dog, finding that the homograft, being avascular, did not induce any immunologic reaction. Robicsek and coworkers' demonstrated that the partial transplantation of the atrioventricular valves in the tricuspid position was technically possible, and the homografts remained viable and properly functioning, as the subvalvular apparatus, for a period up to 3.5 years. Our technique for partial mitral homograft implantation in the mitral position, although it requires a learning period, could avoid the complex measurements and sur-
Volume 104 Number 5 November 1992
gical maneuvers. In this experimental study we have tested the behavior and viability of the mitral homograft. The need for an adequate sterilization during preparation and storage of homografts has been widely recognized,8,9 particularly in the animal, because it was not possible to collect mitral valves under sterile conditions. In our experience all postmortem cultures were positive for infection but were negative at the time of implantation. No late infection was detected in this series. Although fresh and cryopreserved homografts have been used for more than two decades 10, 11, with use of a variety of acceptable protocols, the studies to evaluate their behavior and viability are scarce. 12 Because methods of cryopreservation are still poorly understood, we have divided this experimental study into two different groups of homograft preservation (fresh and cryopreserved grafts), trying to analyze the viability. As Yankah and Hetzer!' described, we have obtained the mitral homograft within 24 hours after animal death and have maintained it at 4 0 C, since warm ischemia represents the main cause of ongoing viability loss during preservation. Scanning, transmission, and light microscopy showed, 3 to 12 months after implantation, viable endothelial cells from the recipient valves invading the mitral homografts and connective tissue fibroblasts with signs of reendothelialization in fresh and cryopreserved homografts. Explanted fresh homograft sections, 9 months after operation, demonstrated still the presence of their own endothelial cells, a finding never seen in cryopreserved grafts. Fresh and cryopreserved mitral homografts explanted early after implantation were almost completely acellular. Dense organized collagen fibers with a uniform distribution were found in group A, and disorganized fibers were found in group B. Hematoxylin and eosin-stained sections of these cryopreserved homografts showed acid mucopolysaccharide, which appeared to be the precursor of microcalcification, as Gonzalez-Lavin and colleagues 14 described. This finding was not seen in the fresh homografts. A complete structural integrity of the homograft with cellular viability was demonstrated in all specimens from groups A and B. A denuded surface of endothelial cellswith and without ingrowth tissue of viable recipient cellswas observed in the histologic preparations studied, with this denuded surface being more irregular in the cryopreserved homografts. No cellular component of inflammatory infiltration demonstrating evidence of a cellular immunologic homograft rejection was found. In conclusion, this experimental study demonstrates that this partial replacement of the mitral valve with a mitral homograft represents a stable technique that could be a valid alternative for repair of local pathology of this valve. Mitral homografts showed structural integrity in
Partial replacement of mitral valve by homograft
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both groups, but the cellular viability was more evident in the fresh grafts. We are indebted to Professor Jose L. Ojeda for giving valuable information and figures to analyze the mitral homografts. REFERENCES 1. Robicsek F, Sanger PW, Taylor, et al. Transplantability of heart valves. Arch Surg 1962;84: 150. 2. Rastelli GC, BerguisJ, Swan HJC. Evaluation of the function of the mitral valve after homotransplantation in the dog. J THORAC CARDIOVASC SURG 1965;49:459. 3. Hubka M, Siska K, Brozman M, Holec V. Replacement of mitral and tricuspid valvesby mitral homograft. J THORAC CARDIOVASC SURG 1966;51:195. 4. Ross DN. The versatile homograft and autograft valve. Ann Thorac Surg 1989;48:569-70. 5. KirklinJW, Barratt-Boyes BG. Method of homograft valve preparation. In: Kirklin JW, Barratt-Boyes BG, eds. Cardiac surgery, 1st ed. New York: John Wiley, 1986:421-9. 6. Shievers HH, Lange PE, Wessel A, Bernhard A. Allogenous transplantation of the mitral valve.An open question. Thorac Cardiovasc Surg 1985;33:227-9. 7. Clarke CP, Barratt-Boyes BG, Sims FH. The fate of preserved homograft pericardium and autogenous pericardium within the heart. Thorax 1988;23:111-20. 8. Barratt-Boyes BG, Roche AHG, Whitlock RML. Six year review of the results of freehand aortic valve replacement using an antibiotic sterilized homograft valve. Circulation 1977;55:353-61. 9. Gonzalez-LavinL, McGrath L, Alvarez M, GrafD. Antibiotic sterilization in the preparation of homovital homograft valves: is it necessary? In: Yankah AC, Hetzer R, Miller DC, et al., eds. Cardiac valve allografts 1962-1987. New York: Springer, 1987:17-23. 10. Strickett MG, Barratt-Boyes BG, Macculloch D. Disinfection of human valve allografts with antibiotics in low concentration. Pathology 1983;15:457. 11. Hopkins RA. Rationale for use of cryopreservedallograft tissue for cardiac reconstructions. In: Hopkins RA. Cardiac reconstructionswith allograft valves.New York: Springer, 1989:15-21. 12. Yankah AC, Wottge HU, Miiller-Ruchholtz W. Antigenity and fate of cellular componentsof heart valveallografts. In: Yankah AC, Hetzer R, Miller DC, Ross DN, Somerville J, Yacoub MH, eds. Cardiac valve allografts 19621987. New York: Springer, 1987:77-89. 13. Yankah AC, Hetzer R. Procurement and viability of cardiac valveallografts. In: Yankah AC, Hetzer R, Miller DC, Ross DN, Somerville J, Yacoub MH, eds. Cardiac valve allografts 1962-1987.New York: Springer, 1987:23-7. 14. Gonzalez-LavinL, BianchiJ, Graf D, Amini S, Gordon CI. Homograft valvecalcification: evidencefor an immunological influence. In: Yankah AC, Hetzer R, Miller DC, Ross DN, Somerville J, Yacoub MH, eds. Cardiac valve allografts 1962-1987.New York: Springer, 1987:69-75.
Jose M. Revuelta, MD, Juan C. Cagigas, MD, Jose M. Bernal, MD, Fernando Val, MD, Jose M. Rabasa, MD, and Maria A. Lequerica, MD, Santander, Spain
Initial experiments in partial transplantation of the cardiac valves were carried out in the canine model by Robicsek and colleagues. I Those early experiments proved that homograft implantation was technically possible, and the results demonstrated remarkable performance and durability.o ' The search for an ideal mitral homograft heart valve substitute has been the objective of a considerable investigative effort." However, methods of sterilization and preservation of homografts and the surgical technique of replacement of the mitral valve by mitral homograft have not been satisfactory. The aim of this study was to evaluate, in a chronic animal model, the viability and behavior of the mitral homograft by replacing a segment of the mitral valve with an original technique. Material and methods Thirty-five mitral homografts were harvested immediately after death of donor young sheep with a mean weight of From the Department of Cardiovascular Surgery, HospitalUniversitario Valdecilla, Universidad de Cantabria, Santander, Spain. Received for publication Sept. 5, 1991. Acceptedfor publication April 9, 1992. Addressfor reprints: JoseM. Revuelta,MD,PhD, Professor ofSurgery, Chairman of Cardiovascular Surgery, Hospital Universitario "Marques de Valdecilla," 39008Santander, Spain.
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24.7 ± 2.3 kg (range 21 to 32 kg.). Ten homografts were washed in distilled water and then in a modified Hanks solution before being immersed for 24 hours at 4° C in an antibiotic solution' composed of cefoxitin (240 mg/rnl), lincomycin (120 mg/rnl), polymyxin B (100 rng/rnl), vancomycin (50 mg/rnl), and amphotericin B (25 mg/rnl), in modified Hanks solution (I L), with a pH of 6.8 to 7.0. The fresh mitral homografts were then moved to a TC 199 culture medium for another 24 hours (group A). After immersion for 24 hours at 4°C in the same antibiotic solution, 15 mitral homografts were cryopreserved in a controlled-rate liquid nitrogen freezer at -I ° C per minute up to -40° C, and later to -196° C, with 10% dimethyl sulfoxide as cryoprotectant (group B). Five of them were refused because of contamination, and the remaining five mitral homografts are still cryopreserved ready to be used in another experiment. Aortic wall fragments and samples of the used solutions were introduced into a thioglycolate and Sabouraud's dextrose medium solution for cultures. Twenty-five young sheep, with a mean weight of 23.4 ± 2 kg (range 17 to 30 kg) and an average age of 12 months, were used for this study. All animals in our experiment received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH Publication No. 85-23, revised 1985). The sheep were sheared the day before the operation and fasted for 24 hours. Anesthesia was induced with thiopental sodium (2 rug/kg) and halothane; a mechanical ventilator was used for respiratory support. The heart was exposed via a left thoracotomy through the fourth intercostal space. Cardiopulmonary bypass was established by cannulation of the right atri-
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Partial replacement of mitral valve by homograft
I 275
B Fig. 1. Partial mitral homograft implanted in the orthotopic position. A, A segment of the anterior leaflet and subvalvular apparatus of the mitral valve has been resected and substituted by a partial mitral homograft (H). B, Implanted mitral homograft (H).
qA H-F-P-(l.-9 Fig. 2. Specimen of a sheep killed 9 months after cryopreserved mitral homograft implantation in the anterior leaflet. The graft appears almost like the native mitral valve. urn and the femoral artery. An apical left ventricular vent was insertedand connected to the arterial return line and used to test mitral valve competence. During cardiopulmonary bypass the heart was maintained beating under normothermic conditions. Thus the aorta was not crossclamped and cardioplegic solution was not used. A wide left atriotomy was performed from the atrial appendage to the left lower pulmonary vein. Partial resection of the anterior (n = 20) or posterior (n = 5) leaflet and their corre-
sponding subvalvular apparatus was carried out. Partial mitral homografts were inserted into the recipient sheep of similar weight and age as the donor sheep. The amount of the anterior leaflet of homograft to be replaced should be at least 4 to 6 mm wider than the resected leaflet to have enough tissue to allow an adequate suture line overlapping to native leaflet. A doublearmed 2-0 pledget-supported Ti-Cron suture (Davis & Geck, Danbury, Conn.) was passed through the tip of the homograft papillary muscle and then through the recipient anterior papil-
1276
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Revuelta et al.
Fig. 3. Histologic section ofthe implanted mitral homograft. A, Fresh mitral homograft (H) totally integrated into the valve (V), explanted 6 months after transplantation. (Masson's stain; original magnification XSO.) B, Endothelial surface around the chordae tendineae in the fresh mitral homograft, explanted 6 months after implantation. (Hematoxylin and eosin stain; original magnification X32.)
lary muscle (Fig. 1). The implantation of the donor papillary muscle to the recipient papillary muscle is done in the dome in a way that allows a normal coaptation of the leaflets and does not interfere with the native subvalvular apparatus. In our experience a homograft 2 to 4 mm larger in the subvalvular apparatus avoids an eventual shrinkage of the graft. The mitral homograft leaflet fragment, including two marginal chordae on average, was then sutured to the recipient valve defect with a running 5-0 polypropylene suture, from the free edge of the leaflet toward the mitral anulus. Blood under pressure was allowed to enter the left ventricular cavity through the vent connected to the arterial line to test valve competence. The suture holding the homograft subvalvular apparatus to the papillary muscle must be tied when the adequacy of the valve is checked. One gram of cefamandole was administered subcutaneously before and after the operation and was maintained for 3 days postoperatively. The sheep were closely observed at the Surgical Research Unit for 7 days after operation and then were transferred to the farm, where they lived an active normal life. The animals were checked for evidence of new murmurs by cardiac auscultation and were observed for signs of heart failure. A hemodynamic study was performed in all survivingsheep before they were electivelyput to death 3, 6, 9, and 12 months postoperatively.
Results Five sheep died within the first 3 months after operation because of residual severe mitral insufficiency (n = 3), anesthesia (n = 1), and accident at the farm (n = 1). The residual valve incompetence was due to malposition of the subvalvular apparatus of the mitral homograft at a lower level than its normal location. Twenty-four animals survived the operation. Nine animals from group A (n = 10) and II animals from group B (n = 15) survived and were electively killed. Mean follOW-Up period was 8 ± 2.5 months (range 3 to 12 months). The animals were killed between 3 and 6 months (n = 5), 6 and 9 months (n = 10), and 9 and 12 months (n = 5). The hemodynamic study performed immediately before each animal was killed demonstrated a mean left atrial pressure of9.5 ± 5 mm Hg, and mean peak systolic left ventricular pressure was 113 ± 3 mm Hg. Residual mitral incompetence was detected during the postoperative hemodynamic study in three patients. Mean heart rate was 97 ± 10 beats/min (range 80 to 160).
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Fig. 4. Transmission electron microscopicviewshowsthe dense organized collagen fibers with a uniform distribution in a fresh preserved mitral homograft explanted 6 months after operation.
Fig. 5. Scanning electron microscopyshowsa cryopreservedmitral homograft (H) denuded of endothelial cellswith an ingrowth tissue of the viable endothelial cells from the recipient (V), 9 months after implantation. (Original magnification 160 X 1.1.) All mitral homografts had cultures positive for infection at dissection from the donor heart. Contaminating organisms included Candida albicans, Pseudomonas
aeruginosa, Pseudomonas maltophilia, Acinetobacter, Escherichia coli, Moraxella, genus and non fermenting
species. After 24 hours of antibiotic immersion, 30 of the previously contaminated grafts had cultures negative for infection, while 5 homograftscontinued to grow P. aeruginosa, E. coli, M oraxella genus, and non fermenting species. No new types of organisms were detected after
The Journal of Thoracic and Cardiovascular Surgery
I 2 7 8 Revuelta et al.
Fig. 6. Scanning electron microscopic view of a mitral fresh homograft 9 monthsafter operation, with chordal (C) integration clearlyidentified. (Originalmagnification 20 X 1.) homograft manipulation. Twenty-five homograft valves were implanted. All cultures obtained at the time of implantation were negative for infection. There have been no instances of late infection in this series. Macroscopic examination of the heart showed no intracardiac thrombi and an apparently normal functioning repaired mitral valve without thrombosis, infective endocarditis, or degenerative failure signs (perforations, ruptures, or calcifications). The segments of the mitral homografts implanted were totally incorporated into the recipient valves and subvalvular apparatus, retaining their original flexibility and length. It was difficult to identify the replaced homografts because they appeared almost like the recipient valve tissue (Fig. 2). A technical error was the cause of valve insufficiency in the three animals studied hemodynamically late after operation. The error consisted in an inappropriate location of the subvalvular apparatus of the partial homograft in the recipient
papillary muscle. No rupture of the chordae tendineae was found in these specimens. Microscopic evaluation, 3 to 12 months after implantation, revealed the presence of viable endothelial cells from the recipient valves invading the mitral homografts and connective tissue fibroblasts in fresh and cryopreserved homografts with signs of reendothelialization (Fig. 3, A). A sheath of connective tissue around the chordae tendineae in the fresh homografts was observed (Fig. 3, B). Organized dense collagen tissue was detected in all implanted homografts without signs of degeneration. A total structural integrity of the homograft with cellular viability was microscopically demonstrated in all specimens from groups A and B. Mitral homograft was completely acellular within ground substance (Fig. 3), and no signs of infiltrations of mononuclear cells neither areas of fibroblast necrosis in the subendothelial tissues. Fibrous sheathing was minimal when it was present. These microscopic findings were similar in both groups of mitral homografts. Valve morphology detected by transmission electron microscopy showed dense organized collagen fibers with a uniform distribution with cellular rests of fibroblasts (Fig. 4). Scanning electron microscopy showed the grafts denuded of endothelial cells, with an ingrowth tissue of viable recipient endothelial cells trying to cover the homograft surface and a normal appearance of the remaining denuded graft (Fig. 5). Organized collagen fibers without cellular infiltration and chordae tendineae reendothelialization were also observed with scanning electron microscopy in fresh and cryopreserved homografts implanted at least 6 months in the sheep (Fig. 6). Discussion Although the use of mitral homografts to substitute mitral valves was introduced by Rastelli, Berguis, and Swan' experimental results were not satisfactory. Trying to solve the technical difficulties of mitral homograft implantation, various surgical procedures have been described with unpredictable results." 7 Hubka and colleagues? used the mitral homograft in the tricuspid position in the dog, finding that the homograft, being avascular, did not induce any immunologic reaction. Robicsek and coworkers' demonstrated that the partial transplantation of the atrioventricular valves in the tricuspid position was technically possible, and the homografts remained viable and properly functioning, as the subvalvular apparatus, for a period up to 3.5 years. Our technique for partial mitral homograft implantation in the mitral position, although it requires a learning period, could avoid the complex measurements and sur-
Volume 104 Number 5 November 1992
gical maneuvers. In this experimental study we have tested the behavior and viability of the mitral homograft. The need for an adequate sterilization during preparation and storage of homografts has been widely recognized,8,9 particularly in the animal, because it was not possible to collect mitral valves under sterile conditions. In our experience all postmortem cultures were positive for infection but were negative at the time of implantation. No late infection was detected in this series. Although fresh and cryopreserved homografts have been used for more than two decades 10, 11, with use of a variety of acceptable protocols, the studies to evaluate their behavior and viability are scarce. 12 Because methods of cryopreservation are still poorly understood, we have divided this experimental study into two different groups of homograft preservation (fresh and cryopreserved grafts), trying to analyze the viability. As Yankah and Hetzer!' described, we have obtained the mitral homograft within 24 hours after animal death and have maintained it at 4 0 C, since warm ischemia represents the main cause of ongoing viability loss during preservation. Scanning, transmission, and light microscopy showed, 3 to 12 months after implantation, viable endothelial cells from the recipient valves invading the mitral homografts and connective tissue fibroblasts with signs of reendothelialization in fresh and cryopreserved homografts. Explanted fresh homograft sections, 9 months after operation, demonstrated still the presence of their own endothelial cells, a finding never seen in cryopreserved grafts. Fresh and cryopreserved mitral homografts explanted early after implantation were almost completely acellular. Dense organized collagen fibers with a uniform distribution were found in group A, and disorganized fibers were found in group B. Hematoxylin and eosin-stained sections of these cryopreserved homografts showed acid mucopolysaccharide, which appeared to be the precursor of microcalcification, as Gonzalez-Lavin and colleagues 14 described. This finding was not seen in the fresh homografts. A complete structural integrity of the homograft with cellular viability was demonstrated in all specimens from groups A and B. A denuded surface of endothelial cellswith and without ingrowth tissue of viable recipient cellswas observed in the histologic preparations studied, with this denuded surface being more irregular in the cryopreserved homografts. No cellular component of inflammatory infiltration demonstrating evidence of a cellular immunologic homograft rejection was found. In conclusion, this experimental study demonstrates that this partial replacement of the mitral valve with a mitral homograft represents a stable technique that could be a valid alternative for repair of local pathology of this valve. Mitral homografts showed structural integrity in
Partial replacement of mitral valve by homograft
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both groups, but the cellular viability was more evident in the fresh grafts. We are indebted to Professor Jose L. Ojeda for giving valuable information and figures to analyze the mitral homografts. REFERENCES 1. Robicsek F, Sanger PW, Taylor, et al. Transplantability of heart valves. Arch Surg 1962;84: 150. 2. Rastelli GC, BerguisJ, Swan HJC. Evaluation of the function of the mitral valve after homotransplantation in the dog. J THORAC CARDIOVASC SURG 1965;49:459. 3. Hubka M, Siska K, Brozman M, Holec V. Replacement of mitral and tricuspid valvesby mitral homograft. J THORAC CARDIOVASC SURG 1966;51:195. 4. Ross DN. The versatile homograft and autograft valve. Ann Thorac Surg 1989;48:569-70. 5. KirklinJW, Barratt-Boyes BG. Method of homograft valve preparation. In: Kirklin JW, Barratt-Boyes BG, eds. Cardiac surgery, 1st ed. New York: John Wiley, 1986:421-9. 6. Shievers HH, Lange PE, Wessel A, Bernhard A. Allogenous transplantation of the mitral valve.An open question. Thorac Cardiovasc Surg 1985;33:227-9. 7. Clarke CP, Barratt-Boyes BG, Sims FH. The fate of preserved homograft pericardium and autogenous pericardium within the heart. Thorax 1988;23:111-20. 8. Barratt-Boyes BG, Roche AHG, Whitlock RML. Six year review of the results of freehand aortic valve replacement using an antibiotic sterilized homograft valve. Circulation 1977;55:353-61. 9. Gonzalez-LavinL, McGrath L, Alvarez M, GrafD. Antibiotic sterilization in the preparation of homovital homograft valves: is it necessary? In: Yankah AC, Hetzer R, Miller DC, et al., eds. Cardiac valve allografts 1962-1987. New York: Springer, 1987:17-23. 10. Strickett MG, Barratt-Boyes BG, Macculloch D. Disinfection of human valve allografts with antibiotics in low concentration. Pathology 1983;15:457. 11. Hopkins RA. Rationale for use of cryopreservedallograft tissue for cardiac reconstructions. In: Hopkins RA. Cardiac reconstructionswith allograft valves.New York: Springer, 1989:15-21. 12. Yankah AC, Wottge HU, Miiller-Ruchholtz W. Antigenity and fate of cellular componentsof heart valveallografts. In: Yankah AC, Hetzer R, Miller DC, Ross DN, Somerville J, Yacoub MH, eds. Cardiac valve allografts 19621987. New York: Springer, 1987:77-89. 13. Yankah AC, Hetzer R. Procurement and viability of cardiac valveallografts. In: Yankah AC, Hetzer R, Miller DC, Ross DN, Somerville J, Yacoub MH, eds. Cardiac valve allografts 1962-1987.New York: Springer, 1987:23-7. 14. Gonzalez-LavinL, BianchiJ, Graf D, Amini S, Gordon CI. Homograft valvecalcification: evidencefor an immunological influence. In: Yankah AC, Hetzer R, Miller DC, Ross DN, Somerville J, Yacoub MH, eds. Cardiac valve allografts 1962-1987.New York: Springer, 1987:69-75.