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J Thorac Cardiovasc Surg 1995;110:248-257
© 1995 Mosby, Inc.
SURGERY FOR ACQUIRED HEART DISEASE |
Leiden, The Netherlands, and Brussels, Belgium
Financial support for this study was given by Bio Implant Services/Eurotransplant Foundation, Leiden, The Netherlands, and by grant NHS 93.130 of the Dutch Heart Foundation.
Received for publication April 27, 1994. Accepted for publication Oct. 31, 1994. Address for reprints: M. G. Hazekamp, MD, Afdeling Thoraxchirurgie, Academisch Ziekenhuis Leiden, Postbus 9600, 2300 RC Leiden, The Netherlands.
Abstract
Tissue degeneration reduces the durability of cryopreserved homografts. Earlier studies indicated that the presence of fibroblasts in homograft leaflets may contribute to increased valve longevity. These fibroblasts may be of recipient origin or represent surviving donor cells. We developed a method, based on in situ hybridization, to determine the origin of fibroblasts in homograft explants. In young pigs we performed aortic valve replacement with a cryopreserved porcine aortic homograft. A male homograft was implanted in a female pig, whereas two male recipients received a female homograft. After 3 to 4 months the homografts were explanted. Frozen sections were made and alternately examined with hematoxylin-eosin staining and in situ hybridization. With a biotinylated porcine Y chromosome-specific deoxyribonucleic acid probe, male fibroblasts could be clearly distinguished from female fibroblasts. In all leaflets we observed both donor and recipient fibroblasts. The distribution of these populations was marked in schematic drawings. Recipient fibroblasts mostly spread onto the leaflet surface but also penetrated the leaflet tissue. Remaining donor fibroblasts did not show morphologic signs of decreased viability on hematoxylin-eosin staining. In situ hybridization may become a useful technique in homograft research. In this porcine model, the fibroblasts in the aortic homograft explants were of both donor and recipient origin. (J THORACCARDIOVASCSURG1995;110:248-57)
The use of valvular homografts in heart surgery is increasing, but as experience grows the main disadvantage of homografts, which is their limited durability, becomes more and more clear. Further research is therefore necessary to overcome the problem of structural degeneration of the leaflet tissue. The exact nature of homograft failure is not known, but it is multifactorial. Analyzing explanted homografts helps in understanding the different factors that eventually lead to graft dysfunction. The fate of the cellular elements in the transplanted valves has been the object of much discussion and speculation. The presence of living, remaining donor cells in explanted cryopreserved grafts has been reported.
1-3 Other studies underline the importance of host cellingrowth into the graft tissue. 4-6 In this study, in situ hybridization (ISH) is presented as a new technique that may have a role in the analysis of explanted homograft valves. The technique was used to distinguish between recipient and donor fibroblasts in explanted cryopreserved porcine aortic homografts. The valve grafts were implanted in young pigs.
METHODS
One female and two male domestic pigs received a cryopreserved aortic homograft from a donor of the opposite sex. The weight at implantation of the recipient animals averaged 69 kg (range 64.7 to 71.5 kg). Donor weight averaged 64 kg (range 58.6 to 73.7 kg). All animals 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 Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH Publication No. 86-23, revised 1985). Donor procedures were performed after premedication (azaperone 2 mg/kg intramuscularly, atropine 0.5 mg/10 kg intramuscularly, and metomidate 20 mg/kg intraperitonally) and with the pig under general anesthesia (endotracheal intubation, mechanical ventilation with isoflurane 0.4% to 1.0% in a mixture of oxygen and nitrous oxide, 33%:66%). After sterile blood donation, cardiectomy was performed under sterile conditions. The aortic grafts were dissected and made suitable for subcoronary implantation. Both coronary sinuses were scalloped whereas the noncoronary sinus was preserved. The valves were placed in a mixture of 98 ml medium TC 199 (Imperial Laboratories, Endover, United Kingdom) and 2 ml antibiotic solution (5-flucytosine, final concentration 30 µg/ml; vancomycin, 12 µg/ml; amikacin, 12 µg/ml; ciprofloxacin, 3 µg/ml; and metronidazole, 12 µg/ml). In this solution the valves were stored at 4º C for 24 hours, after which they were cryopreserved in a 10% dimethylsulfoxide solution according to the protocol of the Dutch Heart Valve Bank (Rotterdam, The Netherlands). The grafts were then stored in vapor-phase liquid nitrogen (-180º C) until implantation. One day before implantation the homografts were transported on frozen carbon dioxide pellets. They were thawed immediately before insertion in accordance with the protocol for human homograft valves. After overnight fasting the recipient animals received premedication (azaperone 2 mg/kg intramuscularly, atropine 0.5 mg/10 kg intramuscularly, and ketamine 10% 30 mg/kg intraperitoneally). Anesthesia was induced with halothane and oxygen, administered by a face mask. After endotracheal intubation, anesthesia was maintained by mechanical ventilation with a mixture of isoflurane in oxygen and nitrous oxide. Pancuronium was used as a muscle relaxant. Antibiotic prophylaxis consisted of intravenous gentamicin and intramuscular benzylpenicillin. The right jugular vein and carotid artery were cannulated. Cannulas were left in situ for the first postoperative week.
The heart was approached through a median sternotomy and cardiopulmonary bypass was started after heparinization and cannulation of the ascending aorta and the right auricle. Left heart decompression was through the left ventricular apex. The body temperature was lowered to 29º C and St. Thomas' Hospital cardioplegic solution was given after aortic crossclamping. The ascending aorta was transected approximately 5 mm above top-commissural level. The native aortic valve was removed and a homograft was implanted in the subcoronary mode with preservation of the noncoronary sinus. The valve was not rotated. The lower and upper suture lines were performed with continuous running 5-0 Prolene sutures (Ethicon, Inc., Somerville, N.J.). Size mismatches were not encountered. After the aortotomy was closed, the clamp was removed and the heart was de-aired. At a normal body temperature, cardiopulmonary bypass was discontinued and protamine was given. The wound was closed with two suction drains in situ. The animals were extubated and returned to the recovery stable. Phenylbutazone/isopropylaminophenazone (1 ml/10 kg subcutaneously) was used for postoperative analgesia. During the first postoperative week, intravenous gentamicin and intramuscular procaine benzylpenicillin were given daily; after removal of the intravascular cannulas, prophylaxis was continued with oral co-trimoxazol for 5 more days. Two weeks after the operation the animals were returned to the farm. Two animals were electively put to death 125 and 132 days after homograft implantation and the third one died suddenly after 95 days.
The homografts were explanted together with the aortic root of the recipient animal. The roots were opened longitudinally between the right and the left coronary cusps, with care taken to avoid damage to the valve cusps. Macroscopic photographs were made and abnormalities were noted when present. The pulmonary valves were removed and were used as a control for the ISH procedure. From each aortic leaflet two 5 mm wide tissue blocks were cut for microscopic examination. Each block contained a radial section of leaflet together with donor and recipient aortic wall tissue. The pulmonary valve was treated in the same way with the difference that only one cusp was used. The tissue blocks were packed in small plastic bags that contained an embedding fluid for frozen tissue specimens (Tissue Tek OCT Compound; Miles Scientific, Elkhart, Ind.). After proper orientation of the tissue block, the bags were heat-sealed and transferred to a tube containing cold isopentane, which was then immersed in liquid nitrogen for 30 seconds. The specimens were stored at -70º C until they were cut.
From each block several radial 6 to 8 µm sections were made with the use of a Reichert-Jung 2800 Frigocut cryostat (Leica Instruments GmbH, Nussloch, Germany), starting at the level of the leaflet belly. Sections for routine stainings were applied to regular glass slides whereas the sections for ISH were put on Starfrost precoated slides (Knittel Glaser, Braunschweig, Germany) to reduce the risk of tissue detachment during the ISH procedure. The slides were stored at -20º C until further use. Tissue was fixed in a 1% solution of formaldehyde in phosphate-buffered saline. Sections were stained with hematoxylin and eosin (HE). Other sections were used for the ISH procedure. The same valve areas were both treated by routine staining and by ISH.
The technique of ISH enables the detection of specific nucleic acid sequences ("target" sequences) in histologic sections. 7 For this purpose, a chromosome-specific labeled "probe" (a small strand of nucleic acids complementary to the target sequence) is applied to the tissue section and will bind to the target deoxyribonucleic acid (DNA) under the right circumstances. After the ISH procedure has been carried out, the presence of the probe in a cell indicates the presence of the target sequence (and the chromosome on which it resides) in that specific cell. In this study, a porcine Y chromosome-specific library probe was used to distinguish male from female porcine cells. The probe was kindly provided by Drs. D. Milan and M. Yerle, Laboratoire de Genetique Cellulaire, INRA, Castanet-Tolosan, France. 8
The ISH procedure involves several phases. The probe should first be labeled with a hapten, which at the end of the procedure is detected by immunocytochemical methods. In this study, biotin is used for this purpose by incorporating biotin-11-dUTP (Sigma Chemical Co., St. Louis, Mo.) into the probe. The use of a so-called library probe means that the probe contains many nonspecific repetitive nucleic acid sequences that occur throughout the genome. These sequences have to be masked by adding an excess amount of competitor DNA (which we extracted from male porcine spleen tissue), because the probe would otherwise bind aspecifically to any other chromosome. The next phase involves denaturation of probe DNA and the DNA in the tissue sections, after which the probe DNA can be applied to the tissue slides. The slides are then incubated at 37º C to allow hybridization of target and probe DNA. After hybridization, washings of various stringencies are performed to remove the probe molecules that did not take part in hybridization. The remaining probe molecules can then be stained, for which we used FITC-conjugated avidin (Vector Laboratories, Inc., Burlingame, Calif.). Avidin binds to the biotin molecules incorporated in the probe; FITC (fluorescein isothiocyanate) is a green fluorochrome that can be detected through a fluorescence microscope. To facilitate microscopic evaluation, we counterstained the nuclei of the cells with propidium iodide (Sigma), a red dye.
To test the specificity of the DNA probe and to establish an appropriate ISH protocol before starting the animal implant study, we applied the probe to smear preparations of male porcine lymphocytes and to frozen tissue sections made from several control tissues, which included male porcine spleen tissue, native male and female porcine aortic valves, and an unimplanted cryopreserved male porcine homograft. For evaluation of the ISH material, a Leitz Diaplan fluorescence microscope was used with an HBO 100 W vapor mercury lamp and Leitz filters A and K3 (Ernst Leitz Wetzlar GmbH, Wetzlar, Germany). Cells containing one clear bright green fluorescent spot were regarded as being of male origin. Nuclei without a spot or with a small vague signal were interpreted as of female origin. The distribution pattern of acellular zones, areas containing the green signal, and zones without the signal was marked in schematic drawings. Photomicrographs of relevant material were taken with a Leitz Vario Orthomat 2 camera, with Scotch 3M 640-T color slide film (3M, St. Paul, Minn.). Inasmuch as ISH sections were always paired to histologic sections treated by HE staining, cell structure could be related to cell origin.
RESULTS
One female and two male pigs received an aortic homograft from a donor of the opposite sex. One male animal died before it was scheduled to be put to death (B272, 95 days after implantation). Endocarditis was the cause of death. The two other animals were electively put to death after 125 and 132 days. Weight gain was approximately 100% in this relatively short period. This weight gain is normal in young growing pigs. A summary of relevant data concerning the animals that were used in this study is given in Table I.
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Microscopy of the HE-stained sections
Evaluation of the HE-stained sections did not reveal any unknown findings. In all cases the typical three-layered appearance as encountered in the native aortic valve leaflet was no longer present. No endothelium was found on the leaflet surfaces. The macroscopically observed thickening at the hinge area was due to the presence of young fibroblasts. In this region some neovascularization was present. In most cases a pannuslike intimal fibrous sheathing was present on the leaflets. The sheaths were more prominent on the ventricular surfaces and ran along the leaflets for various distances. At some places, cells originating from these sheaths appeared to infiltrate deeply into the graft tissue. At the donor-recipient interface some mild chronic inflammatory reaction was frequently observed. The leaflets of the infected valve showed large acellular areas; one leaflet (the right coronary cusp) was almost completely acellular. Focal accumulations of bacteria were found on almost all leaflets of this valve. The noncoronary cusp was damaged by the endocarditis, but the left coronary cusp was relatively intact.
Microscopy of the ISH sections
To test the specificity and sensitivity of ISH, we applied the probe for the porcine Y chromosome to several control tissues. In smear preparations of male porcine blood lymphocytes, all nuclei carried a bright green signal; in metaphase chromosomal spreads there was always one chromosome that was stained with FITC. Nearly all the cells in the cryopreserved unimplanted male porcine aortic valves were found to be Y positive, and similar observations were made in ISH-treated sections from the pulmonary valves that were explanted from the male recipient animals that were put to death. None of the cells in a native female porcine aortic valve carried a bright green spot after ISH.
Pig B268—female recipient/male homograft (Fig. 1)
Noncoronary cusp.
A large acellular area occupied the central region of the leaflet. In the homograft aortic wall, most cells were of host origin and had replaced the male graft fibroblasts. These host fibroblasts (the nature of the cells was derived from their appearance at light microscopic evaluation in the paired section) extended to the base of the leaflet. Remaining Y-positive (i.e., donor) fibroblasts were found in the distal part of the leaflet, where they formed the majority of the cells. These donor cells showed no morphologic signs of decreased viability in the paired HE-stained sections. Cellular or nuclear lysis was not observed. On the ventricular side of the leaflet a layer of host fibroblasts was present, extending almost to the free edge and at some places infiltrating deeply into the graft tissue. Comparison with the paired HE section revealed that these recipient fibroblasts had actually grown into the graft tissue.
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Left coronary cusp.
Cellularity of the left coronary cusp was good throughout the whole leaflet. The homograft aortic wall contained predominantly host cells. On the ventricular side an intimal fibrous sheath containing host fibroblasts extended along the leaflet for one third of its length. Distally, a thin layer of mononuclear host cells was found both on the aortic and on the ventricular edges of the cusp. The remainder of this leaflet was occupied by donor fibroblasts, which again on HE-stained sections did not show signs of cell death.
Pig B270—male recipient/female homograft (Fig. 2)
Noncoronary cusp.
This map may not represent a good radial section of the leaflet and the drawing therefore does not reflect the actual localization of the different cells in this leaflet. Nevertheless, the conclusion can be made that both donor cells and recipient cells have remained in significant amounts in this leaflet. Examination with a light microscope showed that the majority of the cells had the structure of fibroblasts without signs of decreased viability.
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Left coronary cusp.
Nearly all the fibroblasts in the graft aortic wall contained the Y chromosome (Fig. 7). Host cell infiltration extended into the proximal part of the cusp and was bounded by a large acellular area. The majority of the cells in the distal two thirds of the leaflet were donor fibroblasts with a viable structure. Some mononuclear host cells were present on the valve surface but did not penetrate any deeper.
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The clinical results of heart valve replacement by aortic or pulmonary valve homografts are good. 2, 9-13 Homografts have a low incidence of thromboembolic events. They are less susceptible to endocarditis than other heart valve prostheses. 14 Their function is good with essentially low transvalvular gradients. It is generally accepted that the durability of homografts is better than that of porcine aortic or bovine pericardial bioprostheses. However, homograft valves still have a limited life span with an unavoidable need for reoperation. Throughout the years, many efforts have been made to improve the durability of the homograft by altering methods of preimplantation processing. After disappointing early experiences with freeze drying, irradiation, and different chemical methods for sterilization and conservation, two methods have remained. The first is antibiotic sterilization and storage in tissue culture medium at 4º C. The major drawback of this method lies in the fact that storage is limited to a few weeks because longer conservation will lead to decreased durability after implantation. 12 The second method is antibiotic sterilization followed by cryopreservation and storage in the vapor phase of liquid nitrogen. So far as we know, conservation is not limited, which makes cryopreservation the method of choice for most homograft valve banks. In this study wet storage will no longer be considered.
Improving the durability of homograft valves is desirable. Long periods of warm ischemia are detrimental to cellularity, fiber structure, and ultimately longevity of the homograft valve prosthesis. 15 Ideally, the valve should be harvested within 6 to 12 hours after death. Some antibiotics are cytotoxic and should be avoided. Research in this field has led to recommendations on the composition of antibiotic solutions and on the maximal time during which homografts should be exposed to this antibiotic solution. 6, 16
The so-called viability of homograft valves has drawn much attention. Viability in this respect means that the homograft contains living cells after processing. Inasmuch as endothelial cells disappear almost completely in the sequence of dissection, sterilization, and wet storage or cryopreservation, 15, 17, 18 viability always means fibroblast viability. Although modern homograft banks can deliver homograft valves containing living fibroblasts, the importance of this viability remains unclear. Two contrasting opinions concerning the role of the fibroblast in determining homograft durability can be roughly distilled from the literature: first, that the presence of viable donor fibroblasts is essential to homograft survival; 1, 10, 19 second, that the ingrowth of recipient fibroblasts into the homograft tissue plays an important role in maintaining good homograft valve function. 4-6, 12 Some authors state that viable homografts give rise to host-versus-graft reactions that decrease the durability of the implanted homograft valve. 20-22 No conclusive evidence is present to support this statement, just as no convincing evidence has been presented to prove that retained viability after cryopreservation will lead to enhanced graft survival. The presence of viable donor fibroblasts after cryopreservation, but before implantation, cannot predict that the donor cells will remain viable after years of implantation. Several authors state that the importance of living fibroblasts in valvular homografts at the moment of transplantation is no more than speculation. 23, 24 Before extensive changes in valve processing—such as adenosine catabolism inhibition, which decreases the metabolic injury at various steps in the sequence of graft processing—are recommended, 25 further studies should prove that the durability of transplanted grafts benefits from retained leaflet interstitial cell viability.
To test the hypothesis that viability improves homograft durability, one must study the fate of the fibroblasts in explanted homograft valves. Many observations have been made that great differences exist with respect to cellularity and condition of the fiber structures in the explanted homograft leaflets. 1-6, 17, 26 Despite this variability, the general tendency appears to be that of a decreasing cellularity and a loss of normal fiber structures.
The use of valid viability assays is necessary to bring more light to this matter. Fibroblast viability can be assessed in different ways, using cell morphology criteria, 15 tissue culture techniques(which are not quantitative), 1 autoradiographic studies (showing the uptake of tritiated amino acids into collagen), 27 or determination of glucose use and pH changes as indications for metabolic activity. 28, 29
At this moment, the origin of the fibroblasts can be exactly defined only if donor and recipient are of the opposite sex. Cytogenetic analysis of cultured fibroblasts by means of chromosome banding is one technique 1 and demonstrating Barr bodies is the other method that is available at present. Chromosome banding is a reliable method to determine the chromosomal content of a cell and allows a distinction to be made between male and female cells. However, this technique cannot say anything about the distribution and the total number of examined cells. The same reasoning is valid for the method of Barr body determination because, to our knowledge, sex chromatin analysis has not been applied to tissue sections made from explanted valves. In addition to that, determination of sex chromatin is known to have a low sensitivity and specificity, varying from tissue to tissue. 30 Gavin and associates 4 in 1973 stated that for this reason the results of sex chromatin analysis should be interpreted with care. They pointed out the need for a more specific and sensitive method to clarify the role of donor and host fibroblasts in the heart valve homograft.
In this study we present ISH as a new approach to an old problem in heart valve research. The high sensitivity, specificity, and resolution of ISH, together with the fact that the technique can be directly applied to tissue sections, offer clear advantages over the aforementioned techniques. In all cases in which donor and recipient are of the opposite sex, ISH done with a Y chromosome-specific probe can reliably determine the origin of cells in situ, that is, in their actual intravalvular location, which has not been possible thus far.
ISH is not a new technique, but so far as we know it has never been used in homograft heart valve research. The technique is commonly used in oncology and in other fields of pathology. ISH detects specific nucleic acid sequences in histologic sections. The technique can be applied both to metaphase and to interphase cells, which allows a good preservation of valve structure. Before using the technique in explanted human homograft valves, we have gathered experience in a growing pig model. ISH is a time-consuming method, and for each new application a protocol has to be established by a trial and error method. Furthermore, from previous studies that included porcine aortic homografts, we knew that large fibroblast populations are present in porcine cryopreserved aortic homografts explanted after 5 to 6 months of implantation in growing pigs. 31
Although this first animal experience is small, the technique was well reproducible and the specificity and sensitivity of the ISH method were excellent. Pairing the ISH-treated sections to HE-stained sections allows for determination of cell structure and origin. In this study no attempts were made to ensure that the fibroblasts were viable, but cell structure did not show signs of death or cell damage. Recipient fibroblast ingrowth is much more pronounced in the rapidly growing pig model than in the human situation. The graft aortic wall had invariably been occupied by host fibroblasts, whereas the donor fibroblasts at that site had disappeared almost completely. This may be a consequence of the fact that cellular viability in homograft walls is less well maintained by cryopreservation than it is in homograft leaflets. 32, 33 Recipient fibroblast ingrowth always extended into the proximal half of the leaflet and could be distinguished from the pannus that covered the surfaces of the leaflets. Many times recipient fibroblasts originating from the surface pannus were seen to penetrate deep into the leaflet tissue. Another form of host cellular ingrowth was sometimes observed on the distal parts of the cusp where small islands of recipient cells had formed. Inasmuch as these islets did not communicate with the fibrous sheath or with other fields of recipient cells, they must have been created by blood-borne cells. Apart from small foci in the basal cusp regions, the remaining donor fibroblasts resided in the distal parts of the leaflet where they mostly had remained in abundance.
Thus in this study both recipient and donor fibroblasts were present in the explanted homografts. No knowledge exists on the fate of the donor cells in this model. At present we are repeating this experiment with an extended follow-up. No conclusions can be made with respect to what this observation means to the durability or functioning of the graft. In our opinion it is premature to state that the presence of either donor or recipient fibroblasts leads to an enhanced graft longevity. The main outcome of this study is that ISH may become an important tool in future observations on explanted homograft valves in clinical medicine. Species differences between pigs and human beings are no problem: the same technique can be used for the human Y chromosome. We recently started studying explanted human homografts.
Acknowledgments
We gratefully acknowledge the contributions made to this project by members of the Laboratory for Experimental Surgery, University Hospital, Leiden, The Netherlands, and members of the Dutch Heart Valve Bank, Dijkzigt University Hospital, Rotterdam, The Netherlands. We thank Drs. Denis Milan and Martine Yerle, Laboratoire de Génétique Cellulaire, Institut National de la Recherche Agronomique, Castanet-Tolosan, France, for kindly providing the DNA probe.
Footnotes
From the Departments of Cardiothoracic Surgerya and Pathology,b University Hospital, Leiden, The Netherlands, and the European Homograft Bank,c Brussels, Belgium. 
References
This article has been cited by other articles:
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  E. Hickey, S. M. Langley, O. Allemby-Smith, S. A. Livesey, and J. L. Monro Subcoronary Allograft Aortic Valve Replacement: Parametric Risk-Hazard Outcome Analysis to a Minimum of 20 Years Ann. Thorac. Surg., November 1, 2007; 84(5): 1564 - 1570. [Abstract] [Full Text] [PDF]
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 D. R. Koolbergen, M. G. Hazekamp, E. de Heer, E. F. Bruggemans, H. A. Huysmans, R. A. E. Dion, and J. A. Bruijn The pathology of fresh and cryopreserved homograft heart valves: An analysis of forty explanted homograft valves J. Thorac. Cardiovasc. Surg., October 1, 2002; 124(4): 689 - 697. [Abstract] [Full Text] [PDF]
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