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Ann Thorac Surg 1998;66:205-208
© 1998 The Society of Thoracic Surgeons

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Original articles: general thoracic

Origin of regenerated epithelium in cryopreserved tracheal allotransplantation

^a Department of Surgery II, Okayama University School of Medicine, Okayama, Japan

Accepted for publication December 1, 1997.

Address reprint requests to Dr Mukaida, Department of Surgery II, Okayama University School of Medicine, 2-5-1 Shikata-cho, Okayama, 700, Japan

	Abstract

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Background. Our previous study showed that a cryopreserved tracheal allograft could be transplanted using omentopexy without immunosuppression. The present study investigated, by the polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) method, whether the regenerated epithelia were of recipient origin or donor origin in a cryopreserved tracheal allotransplantation model.

Methods. Twenty-nine mongrel dogs were classified by preoperative peripheral blood PCR-RFLP analysis. The cryopreserved tracheal allografts were implanted into recipient animals that showed a different phenotype from donor animals. A small specimen of epithelia excised from the allograft of animals postmortem was analyzed with the modified PCR-RFLP method.

Results. The animals were separated into 16 phenotypes by preoperative PCR-RFLP results, and cryopreserved tracheal allografts transplanted into 8 animals. PCR-RFLP analysis of graft epithelia at 10 days after transplantation showed the donor blood phenotype and analysis of graft epithelia taken from the animals that survived more than 20 days after operation showed the recipient blood and epithelial phenotype.

Conclusions. The donor epithelia in the grafts were no longer present within about 20 days after transplantation. The recipient epithelia migrated gradually from the anastomotic site, and the regenerated epithelia that are of recipient origin covered the allograft within about 50 days after transplantation.

	Introduction

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Tracheal reconstruction is necessary in patients with extensive tracheal stenosis caused by neoplasm, trauma, and congenital disease. Previous experiments demonstrated that cryopreserved tracheal allotransplantation could achieve satisfactory results with omentopexy and without immunosuppression. One question that must be answered is whether the graft epithelia remain allogenic forever or whether the growth of recipient epithelia from the tracheal anastomotic sites eventually replace donor epithelia. We studied the regenerated epithelial phenotype by using the polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) technique.

	Material and methods

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Preparation
Preoperatively, genomic DNA was extracted from the peripheral blood of 29 adult mongrel dogs, weighing 8 to 16 kg. PCR amplification was performed by using two sets of primers (DRBAMP-A and DRBAMP-B), and the modified PCR-RFLP procedure was as follows. A final 33-µL reaction mixture that contained extracted DNA, reaction buffer (10 mmol/L Tris-HCl pH 8.3, 50 mmol/L KCl, 2.0 mmol/L MgCl₂, and 0.1% Triton X-100), 200 µmol/L of dNTP, 0.15 µmol/L of each primers, and 1.5 U of Taq DNA polymerase was subjected to a total of 30 cycles of amplification with 96°C denaturation for 1 minute, 53°C annealing for 1 minute, 72°C extension for 2 minutes.

Aliquots of each primer product were digested at 37°C for 1.5 hours with 5 U each of restriction enzymes as follows: HinfI (779 652, Boehringer Mannheim Biochemica, Mannheim, Germany), HphI (158L, BioLabs, Beverly, MA), and PstI (621 625, Boehringer Mannheim). The amplified DNA fragments were electrophoresed in 10% polyacrylamide gel, and the bands were visualized after staining with SyBR Green I (S-7567, Molecular Probes, Inc, Eugene, OR).

Cryopreserved tracheal allotransplantation method
The selected animals (n = 8) were anesthetized with intramuscular ketamine, intravenous sodium pentobarbital (30 mg/kg), and pancronium bromide (2 mg). The animals were orally intubated and connected to a volume-limited respirator. The animals were placed in the lateral decubitus position and the chest was opened through a right fourth intercostal thoracotomy. The seven thoracic tracheal rings were excised in continuity, and immersed in the preservative solution at 4°C for 4 hours. The preservation solution consisted of Dulbecco’s modified Eagle medium (Gibco, Grand Island Biological Co, New York, NY), 20% fetal calf serum, 10% dimethyl sulfoxide, and 0.1 mol/L sucrose. A Cell Freeze bag (7039-2, Chartermed Inc, Lakewood, NJ) containing the trachea was filled with the preservative solution and sealed. The bag was surrounded with cotton, placed in a plastic case, and frozen at -80°C for 24 hours. The bag was then stored in the gaseous liquid nitrogen (-196°C) for 14 more days. The cryopreserved trachea was thawed rapidly in a water bath maintained at 37°C. An allograft of five tracheal rings was excised from the thawed trachea and determined by PCR-RFLP analysis to be of a different phenotype from the recipient animals. Recipient animals (n = 8) were anesthetized and intubated in the same manner as the donors. The chest was also opened in the same manner and the recipient seven tracheal rings were removed circumferentially. The recipient trachea was repaired by insertion of the five rings tracheal graft with an over-and-over continuous suture of 4-0 Prolene (Ethicon Inc, Somerville, NJ). After the anastomoses, the omental pedicle was brought into the right hemithorax through the incision at the right diaphragm and wrapped around the grafts including both anastomotic sites.

Postoperatively, animals received intramuscular antibiotics for the first 3 days. No immunosuppressive agents or steroids were given. Follow-up bronchofiberoscopy was performed weekly for 1 month to examine graft viability. When animals died or were sacrificed, all grafts were excised and examined grossly and microscopically.

All animals received humane care in compliance with the "Principles of Laboratory Animal Care" of 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 85-23, revised 1985).

PCR-RFLP analysis of regenerated epithelia
A small specimen (5 by 5 mm) of regenerated epithelia was excised from the allograft of postmortem animals. Genomic DNA was extracted from the specimen and washed well with saline solution. The specimen was analyzed with the modified PCR-RFLP method.

	Results

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
The animals were separated into 16 phenotypes (A to P type) by preoperative PCR-RFLP results from peripheral blood, and cryopreserved tracheal allografts transplanted into 8 animals. All animals except dog 8 survived until sacrificed. Dog 8 died of prostration due to continuous diarrhea. The posttransplantation PCR-RFLP results from the regenerated allograft epithelia are shown in Table 1. Dog 1 was sacrificed at day 10 and the allograft appeared almost normal. Histologic examination of the epithelia demonstrated pseudostratified columnar features without cilia, and PCR-RFLP analysis of the graft epithelia showed the same phenotype as the donor peripheral blood samples. Dog 2 was sacrificed at day 20 and the graft showed no epithelia grossly or histologically, which was the reason that PCR-RFLP analysis was not performed. Dog 3 was also sacrificed at day 20 and the remaining or regenerated graft epithelia were observed near the anastomotic sites. PCR-RFLP analysis of these epithelia showed the same phenotype as the recipient blood and epithelia. Dog 4 was sacrificed at day 40 and the partially defective graft epithelia were seen (Fig 1A). The pseudostratified columnar ciliary epithelia were observed near the anastomotic site (Fig 1B), but one epithelial layer without cilia existed next to the epithelial defect (Fig 1C). PCR-RFLP analysis of both epithelial layers showed the same phenotype as the recipient blood and epithelia (Fig 1D). The grafts taken from the animals that survived more than 70 days after operation showed normal tracheal architecture (Fig 2A). Histologic examination revealed that these mucosa were composed of pseudostratified columnar ciliary epithelia (Fig 2B). PCR-RFLP analysis of epithelia in the middle of the graft showed the recipient blood and epithelial phenotype (Fig 2C).

View larger version (108K): [in this window] [in a new window]   Fig 1. (A) Gross examination of the graft of dog 4. No stenosis and no sign of necrosis is seen in the graft. Partial defect of epithelia is observed. (B) Histologic findings near the anastomotic site. Pseudostratified columnar ciliary epithelia are seen. (C) Histologic findings next to the epithelial defect. One epithelial layer without cilia is seen. (D) The result of PCR-RFLP. The graft epithelium phenotype is the same as the recipient blood and epithelium. (B and C, hematoxylin and eosin stain; x100 before 53% reduction.)

View larger version (49K): [in this window] [in a new window]   Fig 2. (A) Gross examination of dog 8 shows normal tracheal architecture. No stenosis of the graft is seen. (B) Histologic examination shows that the mucosa is composed of pseudostratified columnar ciliary epithelium and the tracheal secretory glands are regenerated (hematoxylin and eosin stain; x100 before 53% reduction). (C) The result of PCR-RFLP. The graft epithelium phenotype is the same as the recipient blood and epithelium.

	Comment

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

Twenty-nine animals were classified preoperatively into 16 phenotypes by PCR-RFLP analysis of peripheral blood. These preoperative peripheral blood results agreed with the posttransplantation results of PCR-RFLP analysis of recipient tracheal epithelium in five animals. This method has already been used for HLA-DRB1 genotyping in human tissue [9] and should also be useful in mongrel dogs to classify each genotype.

All cryopreserved tracheal allografts survived without evidence of stenosis, and maintained sufficient rigidity to keep the lumen completely open. The epithelia of allografts that remained allogenic were no longer present 20 days after transplantation. The recipient epithelia gradually migrated from the anastomosis site to the transplanted graft. The allograft was covered with regenerated epithelia of the recipient phenotype within about 50 to 60 days after operation. These results show that the origin of regenerated epithelia in cryopreserved allograft was recipient tracheal epithelia.

These facts suggest that the most important point in tracheal reconstruction is not that the grafts had epithelia but that grafts had the structure-keeping lumen and the graft materials caused no rejection response. Although many experimental studies on prosthetic tracheal replacement have been reported [10–13], to date no satisfactory prosthesis has been established. From the standpoints of physiology and histocompatibility, we believe that no material can be superior to the trachea itself as a graft. The cryopreserved tracheal allograft is presently the material that best meets all clinical criteria.

One more problem remains to be solved, the antigenicity of cryopreserved tracheal allograft. Yokomise and colleagues [14] reported that allotransplantation of the trachea was possible without immunosuppressive agents if the graft was exposed to preoperative irradiation. Our previous study and the present study, however, have showed the possibility of cryopreserved tracheal allotransplantation without irradiation and immunosuppressive agents. Presently, we are conducting experiments on the appearance of major histocompatibility complex class II antigen in the cryopreserved tracheal allograft as part of the next investigative step. If these antigenic problems are solved, cryopreserved tracheal allotransplantation can be applied to the clinical situation.

	Acknowledgments

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
We thank Dr Yuji Yamamoto and Dr Hideo Ishizu at the Department of Legal Medicine, Okayama University Medical School, for PCR-RFLP analysis, and Tesuo Kawakami for the expert technical assistance.

	References

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Nobuyoshi Shimizu Shigeharu Moriyama Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Mukaida, T. Articles by Moriyama, S. Search for Related Content PubMed PubMed Citation Articles by Mukaida, T. Articles by Moriyama, S.