Eur J Cardiothorac Surg 2003;23:15-20
© 2003 Elsevier Science NL
Heterotopic transplantation of cryopreserved tracheae in a rat model
Erich Stoelbena*,
Holger Harperinga,
Joerg Haberstrohb,
Antonio di Filippoa,
Eckhard Wellensc
a Department of Thoracic Surgery, University Hospital Freiburg, Hugstetter Strasse 55, 79106 Freiburg, Germany
b Department of Experimental Surgery, University Hospital Freiburg, Hugstetter Strasse 55, 79106 Freiburg, Germany
c Institute of Pathology, University Hospital Freiburg, Hugstetter Strasse 55, 79106 Freiburg, Germany
Received 17 May 2002;
received in revised form 14 September 2002;
accepted 7 October 2002.
* Corresponding author. Tel.: +49-761-2702-457; fax: +49-761-2702-499
e-mail: stoelben{at}ch11.ukl.uni-freiburg.de
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Abstract
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Introduction: The successful use of cryopreserved tracheal allografts in canine models suggests their use in humans. The grade of genetic difference, the mechanism of revascularisation and the method of cryopreservation are not clearly defined. The purpose of our study was to investigate the rejection of tracheal transplants in a standardised heterotopic rat model using different forms of cryopreservation. Methods: Tracheae from Brown Norway rats were implanted into the omentum from Brown Norway rats or Lewis rats. We transplanted fresh isografts or allografts and pretreated isografts or allografts. Cryopreservation was performed in a medium containing 10% dimethyl sulphoxide at -80°C for 28 days (I) or -196°C for 84 days (II) or without medium at -80°C for 28 days (III). The transplants were excised after 7 and 21 days, respectively. Results: Histological examinations revealed normal structure and function of isografts after 21 days. In the cryopreserved isograft, the epithelium had disappeared and the tracheal lumen was partially obstructed by a non-compact fibrous tissue. In the fresh allografts, the epithelium was replaced by aggressive fibrous tissue, infiltrating the membranous part of the trachea and occluding the tracheal lumen. The cartilage was vital without any sign of rejection. In the cryopreserved allografts, the tracheal lumen was obstructed by dense fibrous tissue with an inflammatory reaction. The cartilage of cryopreserved allografts (II) and (III) had lost the nuclei corresponding to non-vital tissue. Only in the cryopreserved allografts (I) did we find nodular regeneration at the edges of the cartilaginous bow. Conclusions: The heterotopic transplantation model allows the study of the mechanisms leading to tracheal obstruction. Cryopreservation was found to have no clear advantage in reducing transplant immunogenicity. Cryopreservation leads to significant damage to the cartilage, the intensity of which is dependent on the mode of cryopreservation.
Key Words: Trachea • Transplantation • Cryopreservation • Rat
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1. Introduction
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Successful tracheal transplantation in man by means of a vital allograft was reported only once in 1979 [1]. In the last few years, an increasing number of reports about the use of cryopreserved allografts in orthotopic settings in animal models have been published [2–9]. The conditions of cryopreservation varied markedly between the studies. The authors used different cryoprophylactic solutions or no medium at all to store the transplants. Temperatures of -80 to -196°C were applied for 1 week to 3 months. The studies were performed in dogs, piglets and rats. Only in one study [8] was the genetic relationship between the donors and recipients (two inbred rat strains) well defined. The results of the studies varied between success through cryopreservation, moderate results for fresh and cryopreserved allografts or failure of both transplants. Although in clinical use long segment tracheal transplantation would be necessary, the authors used five to ten rings of the trachea for orthotopic transplantation. Five rings represent about 2 cm of the canine trachea [3]. Tracheal autografts longer than 4 cm in a canine model with orthotopic transplantation with omental wrapping resulted in transplant necrosis [10]. Revascularisation can be achieved by heterotopic transplantation into the omentum or muscle. The second-step orthotopic transplantation leads to successful healing of the transplant [11,12]. The tracheal mucosa disappeared after cryopreservation and allotransplantation and regenerated from the anastomotic borders [13,14]. Until 3–6 weeks, when the transplant is lined with epithelium from the recipient, the allograft is exposed to the danger of infection. In conclusion, the use of cryopreserved allografts for clinical settings is not yet possible. Questions of immunological rejection, revascularisation and epithelisation still need to be answered. The purpose of our study was to investigate the rejection of cryopreserved tracheal allografts in a standardised heterotopic rat model using different forms of cryopreservation. This model [15,16] was introduced to study lung transplant rejection and the effects of immunosuppressants. Tracheal allograft transplantation into the omentum leads to the proliferation of fibrotic tissue occluding the tracheal lumen mimicking obliterative bronchiolitis. This form of rejection can be avoided using immunosuppressive drugs. Since extended tracheal resection would be necessary in children and patients with malignomas we endeavour after the possibility of tracheal transplantation without systemic immunosuppression. The intensity and form of rejection may be depending on the location of implantation, e.g. heterotopic or orthotopic transplantation. We used heterotopic transplantation because it allows revascularisation of the transplant for a second-step orthotopic transplantation. At the same time, re-epithelialisation may be performed with autologous cells. Using this heterotopic model, death of experimental animals by asphyxia can be avoided.
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2. Materials and methods
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2.1. Experimental design
Experiments were performed using male (175–200 g) Brown-Norway and Lewis rats (Charles River, Sulzfeld, Germany). The inbred rat strains possess different RT1 genes (Brown-Norway: RT1n, Lewis: RT1l), representing MHC mismatches in humans. Tracheae from Brown Norway rats were implanted into the omentum from Brown Norway rats (isografts, fresh, n=8) or Lewis rats (allograft, fresh, n=8) without pretreatment. After cryopreservation (I) (n=16) equal numbers of tracheae from Brown Norway rats were transplanted into Brown Norway rats (isograft, cryo, n=8) or Lewis rats (allograft, cryo, n=8). The transplants were examined after 7 and 21 days respectively. After cryopreservation (II) (n=8) and (III) (n=8) only allograft transplantations were performed and examinations of the transplants were carried out after 21 days.
The experimental protocol was approved by the animal care department of the local national authorities. All animals received human care in compliance with the German Law of Animal Protection (BGBL 1998, Part 1, Nr. 30, p. 1105 ff.) and the European Convention on Animal Care.
2.2. Donor procedure
Rats were anaesthetised in a narcotic chamber with isoflurane in oxygen and then killed with carbon dioxide. Using a sterile technique, the trachea was transected distally to the larynx and proximally to the carina after midline incision and blunt dissection. Adherent tissue was carefully removed. The trachea was wrapped in a piece of normal saline-soaked gauze for immediate transplantation or stored in a cryopreservation medium.
2.3. Recipient procedure
Rats were anaesthetised in a narcotic chamber with isoflurane in oxygen. For the surgical procedure anaesthesia was maintained by inhalation of isoflurane in oxygen through a breathing mask. The animals received novaminsulfon subcutaneously (10 mg) for postoperative pain relief. Using a sterile technique, the trachea was implanted into the greater omentum after an upper abdomen midline incision. The trachea was fixed to the omentum by two single knots. The abdominal wall and the skin were closed by continuous sutures. Polypropylene (5-0) was used for all sutures. After recovery from the surgical procedure the animals received standard rat food and water ad libitum. No immunosuppressants were given. For histological investigations, the animals were killed (see above) and the trachea was excised with the surrounding omentum.
2.4. Cryopreservation (I)
We followed the procedure for the cryopreservation of aortic homografts [17]. The tracheae were immersed in Dulbecco's modified Eagle medium (Sigma) containing antibiotics (Cefoxitin 140 µg/l, Lincomycin 120 µg/l, Vancomycin 50 µg/l and Amphotericin B 25 µg/l) for 24 h at 4°C. Subsequently, the transplants were passed into a medium containing 10% dimethyl sulphoxide, frozen at -20°C for 24 h and stored at -80°C for 28 days. Before transplantation the tracheae were thawed in a water bath (30°C) and rinsed with a large amount of medium.
2.5. Cryopreservation (II)
The tracheae were immersed in Dulbecco's modified Eagle medium containing 10% dimethyl sulphoxide, frozen at -20°C for 24 h and stored at -196°C for 84 days. Before transplantation the tracheae were thawed in a water bath (30°C) and rinsed with a large amount of medium.
2.6. Cryopreservation (III)
The tracheae were placed in freezing bags without any medium at -20°C for 24 h and stored at -80°C for 28 days. Before transplantation the tracheae were thawed in a water bath (30°C).
2.7. Histological assessment
The tracheal specimens were fixed in buffered formalin (4%) and embedded in paraffin. Three cross sections were obtained from the middle part of each specimen and stained with haematoxylin/eosin. The following pathological changes were assessed using semiquantitive scales: The normal ciliated epithelium could be reduced to a single layer, metaplastic or disappeared (1=no epithelium, 2=single layer, 3=metaplastic squamous epithelium, 4=normal ciliated epithelium). The cartilage could be vital without signs of damage or showed areas of regeneration. In avital cartilage the cells have disappeared and in this cases the matrix could be calcified (1=calcified, 2=non-vital, 3=vital, partial vital, 4=vital, regeneration). The revascularisation was estimated by the number of capillaries in the submucosa which were filled by erythrocytes. To describe the degree of inflammation, the number of lymphocytes in the submucosa of the membranaceous part of the transplant was counted. In both cases the number per high-power field was assessed. The tracheal lumen was normal without infiltration or obstructed by various degree by granulation tissue. The degree of obstruction was estimated using five steps beginning with total obstruction (1=100%, 2=<75%, 3=<50%, 4=<25%, 5=0%).
2.8. Statistical analysis
Differences between isografts and allografts and between the different allograft preparations were evaluated with the Kruskal–Wallis test and the pairwise Wilcoxon test [18].
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3. Results
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3.1. Results after 7 days (Table 1)
At the time of reoperation, the transplant was well incorporated without significant adhesions between the greater omentum and the parietal peritoneum. Histological examinations revealed a reduction in the epithelium to a single layer for the fresh transplanted isografts. The tracheal lumen was free, the cartilage vital and we found an inflammatory response neither in the transplant nor in the surrounding tissue. The major difference between fresh isografts and fresh allografts consisted of a strong inflammatory response in the surrounding greater omentum of the allografts. The tracheal lumen was free, the epithelium reduced to a single layer and the cartilage vital.
Cryopreservation led to some changes in the graft before transplantation. The epithelium was reduced to a single layer and the nucleus gave a pyknotic impression. The structure of the cartilage was normal, whereas the nuclei of the chondrocytes were also pyknotic like the epithelium.
Seven days after cryopreservation and transplantation in iso- and allografts a very flat epithelium was recognizable in some areas of the inner tracheal surface. The tracheal lumen was partially obstructed by non-compact fibrous tissue. The lymphocyte infiltration of the membranous part of the transplant was marked in the allografts compared to the isografts. The cartilage was partially calcified and avital.
In contrast to the fresh grafts, the cryopreserved grafts showed significant damage to the cartilage and the epithelium with partial necrosis and replacement by granulation tissue. In all specimens, capillaries containing red blood cells could be detected in the membranaceous part of the trachea.
Statistically significant differences existed between fresh and cryopreserved grafts with respect to the epithelium, cartilage and luminal obstruction.
3.2. Results after 21 days (Table 2)
At the time of explantation the transplant was enveloped by the greater omentum without peritoneal adhesions or inflammatory reaction.
The fresh isografts were filled by eosinophilic secretions produced by a multilayer ciliated epithelium. The cartilage was vital without shrinkage of the fibrous tissue or any remarkable inflammatory response (Fig. 1)
. In the fresh allografts, the epithelium was replaced by fibrous tissue containing a high number of fibroblasts which infiltrates the membranaceous part of the trachea. The tracheal lumen was obstructed by about 75%. The cartilage was vital with some regenerating nodules. The marked inflammatory response in the greater omentum after 7 days was translocated to the connective tissue of the transplant and into the lumen of the trachea (Fig. 2)
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Fig. 1. Isograft 21 days after heterotopic transplantation with vital cartilage and epithelium with secretion into the tracheal lumen (HE, x100).
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Fig. 2. Allograft without pretreatment 21 days after heterotopic transplantation with vital cartilage. The tracheal lumen is obstructed by aggressive fibrous tissue which invades the trachea via the membranous part (HE, x10).
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In the cryopreserved isograft, the epithelium has disappeared and the tracheal lumen was partially obstructed by non-compact fibrous tissue. The cartilage showed calcified areas beneath nodules of regenerated tissue with a basophilic reaction. In contrast to the fresh allograft, we found no inflammatory reaction (Fig. 3)
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Fig. 3. Isograft after cryopreservation (I) 21 days after heterotopic transplantation with partially vital cartilage. The epithelium is replaced by non-compact fibrous tissue (HE, x25).
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In the cryopreserved allografts, the obstruction of the tracheal lumen was completed by dense fibrous tissue accompanied by an inflammatory reaction. The cartilages of cryopreserved allografts (II) and (III) lost their nuclei, corresponding to non-vital tissue (Fig. 4)
. Only in the cryopreserved allografts (I) we did find nodular regeneration at the edges of the cartilaginous bow. The cartilage after preservation (I) resembled that of the cryopreserved cartilage of the isografts.
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Fig. 4. Allograft after cryopreservation (I) 21 days after heterotopic transplantation with calcified and partially vital cartilage. The tracheal lumen is obstructed by aggressive fibrous tissue which invades the trachea via the membranous part (HE, x10).
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The difference between fresh isografts versus cryopreserved isografts and all allografts with respect to the epithelium is significant. The inflammatory response in the isografts is significantly lower than in all allografts.
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4. Discussion
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Our results of heterotopic transplantation of fresh isogenic trachea confirm the experience [11,15] that the transplants recover to full function within 21 days. The multilayer epithelium with a ciliated surface produced esosinophilic secretions into the tracheal lumen and the cartilage was vital. In contrast, the transplantation of fresh allogeneic tracheae led to an intense inflammatory reaction with destruction of the epithelium and luminal obstruction by fibrous tissue. The cartilage remained vital over a period of 21 days without any signs of rejection. In most publications cited above, only the changes concerning the epithelium and luminal obstruction were described, since these examinations deal with a bronchiolitis-obliterans model for lung transplantation. The fact that the epithelium may be the major antigenic structure was suggested by immunohistochemical investigations [19,20] in humans and rats. The antigenic role of the epithelium was confirmed in successful orthotopic allografting after removing the epithelium in dogs [21].
The cryopreservation of the isografts led to a reduction in the respiratory epithelium from ciliated multilayer to a monolayer. Furthermore, we found the nuclei pyknotic. After transplantation the epithelium disappeared and the tracheal lumen is occluded by non-compact fibrous tissue that is different from inflammatory aggressive granulation tissue in allografts. The disappearence of the epithelium is confirmed by other authors using cryopreservation [8,14]. Since the method of cryopreservation has been developed and is used for storing vital tissue, e.g. parathyroid tissue [22], we would expect vital epithelium and cartilage in the tracheal transplants. Probably the combined damage of cryopreservation and ischaemia caused cell death.
The cryopreservation of the allografts induced the same damage to the tissue as in the isografts. The higher number of lymphocytes in the specimen after cryopreservation might be the result of the tissue damage induced by the pretreatment and not only by the rejection of the transplant. After 7 days the tracheal lumen is partially filled by non-compact tissue. After 21 days the tracheal lumen was occluded by connective tissue rich in fibroblasts, which is not different from fresh allografts. This fibroproliferative tissue entered the tracheal lumen via the membranous part and resembles the description of bronchiolitis obliterans [15]. Cryopreservation is not able to prevent this reaction. In contrast to heterotopic transplantation, in orthotopic transplantation the inner surface of the trachea is remodelled by the recipient's epithelium. In an orthotopic rat model, the inflammatory response and luminal obstruction stopped when the tracheal surface was completely covered by host epithelium [23]. Nevertheless, in most publications involving fresh allografts without pretreatment, the animals (dogs) died of respiratory insufficiency due to luminal obstruction. We suppose that in the small trachea of the rat the epithelium can completely cover the inner surface of the trachea before the fibroproliferative reaction leads to occlusion of the tracheal lumen. The beneficial effect of the cryopreservation of the allografts in canine models may be based on the destruction of the donor epithelium, which reduces the antigenic inflammatory response. In the heterotopic environment, epithelial covering of the transplant by the recipient's epithelium is not possible. Subsequently, the inner surface of the transplant is filled up by connective tissue in isografts and allografts. Although the heterotopic transplantation is quite different from orthotopic transplantation concerning microenvironment and reepithelialisation, the two-step procedure using heterotopic implantation first is necessary for revascularisation. In the heterotopic setting, the epithelialisation of the transplant with the recipient's epithelium may be performed by a recently reported technique using cell culture of tracheal epithelial cells [24]. Thus the orthotopic transplantation could be imitated partially in the heterotopic setting.
In our experiments, over a period of 21 days we found no aggressive reactions against the cartilage in isografts and allografts. The intact cartilage induces a weak antigenic reaction, whereas isolated chondrocytes are immunogenic [25]. For tracheal transplantation the rejection of cartilage may not be the major problem. Vitality of the cartilage was completely preserved in the fresh transplants, was significantly reduced after cryopreservation (I) and destroyed after cryopreservation (II) and (III). In the publications involving canine tracheae, the cartilage is described as being vital or having a normal structure. In fact, the vitality of the cartilage is essential for long-term patency of the graft, since necrotic cartilage will be resorbed.
In conclusion, the heterotopic transplantation model allows the study of the mechanisms leading to tracheal obstruction. The technique has to be developed using recipient's epithelial cell transplantation into the transplant. Cryopreservation has no clear advantage in reducing transplant immunogenicity. Since cryopreservation of the tracheal transplant would facilitate transplant accessibility, systematic investigations on cryopreservation of the trachea and vitality of the cartilage would be necessary. The two-step procedure with re-epithelisation and revascularisation and secondary orthotopic transplantation may be feasible.
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Abstract
1. Introduction
