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Ann Thorac Surg 1998;66:209-213
© 1998 The Society of Thoracic Surgeons

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Original articles: general thoracic

Tracheal replacement with cryopreserved tracheal allograft: experiment in dogs

^a Department of Surgery III, Nara Medical University, Nara, Japan

Accepted for publication January 27, 1998.

Address reprint requests to Dr Tojo, Department of Surgery III, Nara Medical University, 840, Shijo-cho, Kashihara, Nara, Japan 634

	Abstract

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Background. It has been difficult to perform tracheal allotransplantation without immunosuppression. To determine whether cryopreserved trachea can be used in tracheal replacement, we evaluated the viability of cryopreserved tracheal allografts in a canine model of immunosuppressant-free transplantation.

Methods. Cryopreserved tracheal allografts, which had been frozen to -80°C in a programmed freezer and then stored in liquid nitrogen (-196°C) (group 1, n = 6), fresh tracheal autografts (group 2, n = 5), and fresh tracheal allografts (group 3, n = 4) were transplanted into the thoracic segment of the trachea using an omental flap without immunosuppressive agents.

Results. All dogs in groups 1 and 2 survived, but in group 3, all 4 died of airway obstruction between 1 month and 2 months after operation. Histologically, the cryopreserved allografts displayed normal epithelium and cartilage, but the fresh allografts showed chronic inflammatory changes, no epithelium, and no cartilage.

Conclusions. Cryopreserved tracheal allografts maintain their structural integrity after transplantation. The cryopreservation process seems to reduce the allogenic response of the trachea in canine models. Therefore, we believe the cryopreserved tracheal allograft is an excellent choice for tracheal replacement.

	Introduction

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Tracheal reconstruction can be safely carried out by direct anastomosis after resection of up to half of the trachea in adults and a third of the trachea in infants. More extensive lesions are not amenable to conventional surgical intervention because there is no definitive strategy for reconstructing such large tracheal defects. To treat such lesions, tracheal interposition has been investigated [1], but the results have usually been unfavorable because of ischemia and rejection of the transplanted tracheal grafts.

As the technique of cryopreservation has developed, it has been applied to various tissues. Excellent clinical results with cryopreserved homograft valves [2] prompted us to evaluate cryopreserved trachea. Cryopreserved trachea has been demonstrated to maintain smooth muscle, cartilage, and epithelium [3]. If cryopreserved trachea can be used to replace diseased trachea, the operative indications will expand. To determine whether cryopreserved trachea can be a realistic substitute in replacement procedures, we evaluated the viability of and the histologic changes in cryopreserved tracheal allografts in a canine model of transplantation without immunosuppression.

	Material and methods

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Sixteen dogs weighing 13.9 ± 2.1 kg (range, 10 to 17 kg) were used. Anesthesia was induced with intravenous administration of sodium pentobarbital (10 mg/kg) and pancuronium bromide (0.1 mg/kg) after premedication with an intramuscular injection of ketamine hydrochloride (20 mg/kg). The dogs were orally intubated and ventilated with a mixture of oxygen and nitrous oxide. Using sterile technique, the recipient thoracic tracheas were exposed through a right thoracotomy, and tracheal transplantations were performed.

Tracheal allografts for cryopreservation were taken from healthy dogs used in other experiments. The freezing medium was TC-199 solution with a final concentration of 5% HEPES (hydroxy-ethyl-piperazine-ethane-sulfonic acid) buffer and 10% dimethyl sulfoxide. A sterile plastic bag containing the specimen was filled with the freezing medium, sealed, and frozen at a rate of -1°C per minute to -80°C in a programmed freezer. The bag was stored in liquid nitrogen (-196°C) for about 1 month. At implantation, the specimen was thawed by placing the bag in a 40°C water bath, and the freezing medium was then rinsed out.

In group 1 (n = 6), a five-ring thoracic tracheal segment was resected from each recipient, and airway continuity was reestablished with the interposition of a cryopreserved tracheal allograft. Anastomoses were performed with interrupted 4-0 polyglyconate sutures, and anastomotic sites were covered with an omental flap. During the proximal anastomosis, ventilation was maintained by inserting a separate endotracheal tube into the distal tracheal segment, and during performance of the distal anastomosis, the lungs were ventilated by the oral endotracheal tube passed through the graft.

In group 2 (n = 5), a five-ring thoracic tracheal segment was excised, reimplanted immediately as an autograft, and covered with an omental flap as in group 1. In group 3 (n = 4), the procedure was the same except that the tracheal segment was replaced with a fresh tracheal allograft.

Extubation was performed immediately after operation, and the chest tube was removed on the first postoperative day. No immunosuppressive agents or steroids were given. Examination with a flexible bronchoscope was performed on the seventh and 30th postoperative days and every 6 months thereafter, and each tracheal graft was examined grossly and microscopically when the animal died or was sacrificed.

Throughout the experiment, all animals received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research, the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health (NIH publication 86-23, revised 1985), and the "Guideline for Animal Experimentation" published by the Japanese Association for Laboratory Animal Science (Exp. Anim. 36(3), 285–288, 1987).

	Results

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All of the dogs survived the operation, and all of their wounds healed well. Tracheal stents were not necessary in any dog. In groups 1 and 2, the dogs were alive for the duration of the experiment and were subsequently killed for the histologic examinations. In group 3, however, all 4 dogs died of airway obstruction on the 32nd, 44th, 68th, and 78th postoperative days (Table 1).

View larger version (109K): [in this window] [in a new window]   Fig 1. Bronchoscopic and macroscopic findings in transplanted grafts. Airways were open and no stenoses were seen in (A, B) the cryopreserved group (535 days after operation) and (C, D) the autograft group (532 days). (E, F) In the allograft group, airways were almost completely obstructed, and the grafts showed extensive necrosis (44 days after operation).

View larger version (69K): [in this window] [in a new window]   Fig 2. Histologic findings in cross sections of transplanted tracheal grafts. Both (A) cryopreserved allografts (535 days) and (B) fresh autografts (532 days) displayed normal architecture. However, (C) fresh allografts (44 days) had chronic inflammatory and necrotic changes with massive infiltration of lymphocytes, apparently demonstrating allograft rejection. (All, x40 before 32% reduction.)

	Comment

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The use of cryopreserved trachea for tracheal replacement appears a reasonable alternative to the use of autologous tissues. Previous experiments have demonstrated that after transplantation of cryopreserved trachea, the functional and histologic characteristics of the epithelium are well preserved [3], but the cartilage undergoes some ischemic changes [3, 12, 13]. We think these ischemic changes are due to insufficient blood supply, and the ability of the omentum to revascularize free tracheal grafts rapidly may solve this problem [14]. In fact, in our experiment, the cryopreserved tracheal cartilage was well preserved and ischemic changes were minimized by the use of omental flaps.

The trachea is generally believed to induce only a weak graft rejection compared with other organs [15], but a tracheal allograft will develop rejection without immunosuppression [4, 5, 16]. Studies [17, 18] of antigenicity have shown that the human tracheal epithelium develops HLA-DR antigen, which activates T lymphocytes and may play an important role in graft rejection. Use of immunosuppressants to control immunologic reactions will certainly attenuate graft rejection but will also increase airway infection after tracheal replacement. Therefore, reducing the antigenicity of the allograft itself is an excellent method of control. For this purpose, cryopreservation [13], radiation therapy [19], and photodynamic therapy [20] have been reported. Cryopreservation techniques are favored for maintaining viability and structural integrity and reducing the immune response of several tissues [21–23]. It has been postulated that cryopreservation results in the loss of class II antigen expression during freezing and thawing, but it is still unclear why this phenomenon takes place.

In our experiment, after implantation of cryopreserved tracheal allografts, the epithelium and the cartilage maintained their structure. There was no lymphocytic infiltration of or under the epithelial layer after 6 months, and this condition has lasted as long as 2 years. On bronchoscopic examination, the tracheal lumen was open and no malacic changes were observed in the transplanted segments. Our results suggest that the immune response of tracheal allografts early and late after transplantation is greatly reduced by the cryopreservation process.

A few clinical tracheal allotransplantations have been reported. In one instance, a two-stage tracheal allotransplantation without immunosuppression was performed successfully [15]. In another with immunosuppression and revascularization by the omentum, graft stenosis developed, and a silicone stent was used for prophylaxis [24]. In one study [25], replacement of a long segment of trachea using a human tracheal homograft and a Dumon stent led to good results. Our experimental findings indicate that the cryopreserved tracheal allograft has excellent long-term tissue viability and reduced antigenicity of the graft. It is an excellent choice for tracheal replacement.

In conclusion, the epithelium, the smooth muscle, and the cartilage were preserved after the implantation of cryopreserved tracheal allografts revascularized by omentum and without immunosuppression. We think the cryopreservation process reduces the allogenic response to the trachea in the canine model.

	References

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