Ann Thorac Surg 2001;71:1666-1669
© 2001 The Society of Thoracic Surgeons
Original article: general thoracic
Tracheal allotransplantation maintaining cartilage viability with long-term cryopreserved allografts
Keiji Kushibe, MDa,
Kunimoto Nezu, MDa,
Kazuhiko Nishizaki, MDa,
Makoto Takahama, MDa,
Shigeki Taniguchi, MDa
a Department of Surgery III, Nara Medical University, Nara, Japan
Accepted for publication February 4, 2001.
Address reprint requests to Dr Kushibe, Department of Surgery III, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8522, Japan
e-mail: Kushikuk{at}naramed-u.ac.jp
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Abstract
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Background. Cartilage viability of a cryopreserved tracheal allograft seems to affect graft function and durability. We previously reported the influence of warm ischemia and cryopreservation on cartilage viability of tracheal allografts. For the clinical application of tracheal allotransplantation, it is essential to preserve grafts for a long time. In this study, we assessed cartilage viability of tracheal allografts after long-term cryopreservation in transplantation models.
Methods. The tracheas were harvested from Lewis rats. The grafts were frozen to -80°C in a programmable freezer immediately after being harvested and were then stored in liquid nitrogen (-196°C) for different lengths of preservation (1, 2, 6, 9, 12, 18, and 24 months; n for each group = 8). Cartilage viability was evaluated by estimating proteoglycan synthesis. After harvest or thawing of the tracheas, the cartilage was labeled with 4 µCi/mL of Na235SO4. Specimens were then hydrolyzed in 0.5 mol/L NaOH, and a solution of the extracts was then counted by a liquid scintillation counter. 35Sulfur incorporation before and after cryopreservation was examined in each group. Tracheal allotransplantation was performed using Lewis rats as donors and Brown Norway rats as recipients.
Results. The average 35S incorporation in the cartilage before cryopreservation was 224 ± 17 disintegrations per minute per milligram of tissue protein. The average 35S incorporation in the cartilage after cryopreservation decreased to 67% to 76% compared with that before cryopreservation. There were no significant differences among the groups in 35S incorporations after cryopreservation. Histologic examination after transplantation revealed normal tracheal cartilage in all groups.
Conclusions. The viability of tracheal cartilage after cryopreservation decreased to 67% to 76%. There were no significant differences in viability of cartilage among the tracheas after different lengths of cryopreservation. Tracheal allotransplantation after long-term cryopreservation can be safely performed in the rat model.
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Introduction
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In tracheal allotransplantation with cryopreserved allografts, cartilage viability of a cryopreserved tracheal allograft seems to affect graft function and durability. In most experimental studies [1–5] of cryopreserved tracheal transplantations, the transplantation has been performed after about 1 month of cryopreservation. For the clinical application of tracheal allografts, grafts must be cryopreserved for a long time. We previously documented the influence of warm ischemia and cryopreservation on cartilage viability of tracheal allografts by measurement of Na235SO4 incorporation [6]. In the present study, we assessed cell viability of the cartilage of tracheal allografts after long-term cryopreservation by measurement of Na235SO4 incorporation.
We also evaluated the histologic changes in tracheal cartilage in a rat allotransplantation model and examined whether the tracheas after long-term cryopreservation could be used clinically.
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Material and methods
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Groups and preservation of grafts
Inbred male Lewis rats were premedicated with anesthetic ether. The donor rats were killed with an intraperitoneal injection of 1 mL of pentobarbital. The tracheas were removed and immersed immediately in the freezing medium of Tissue Culture Medium 199, a solution containing 5% hydroxy-ethyl-piperazone-ethane-sulfonic acid buffer and 10% dimethyl sulfoxide. A specimen was sealed in a sterile plastic bag containing the freezing medium and frozen to -80°C at a rate of -1°C per minute in a programmable freezer. The bag was stored in liquid nitrogen (-196°C) for different lengths of preservation (1, 2, 6, 9, 12, 18, and 24 months; n for each group = 8). For transplantation or viability assessment, the specimen was thawed by placing the bag in a 40°C water bath for 5 minutes. The freezing medium was then rinsed off.
Assessment of cartilage viability
After harvesting, three tracheal cartilage rings were resected from a graft by trimming. After harvest or thawing, the cartilage was labeled with 4 µCi/mL of Na235SO4 in 1 mL of modified Eagle’s medium. 35Sulfur incorporation in the cartilage before and after cryopreservation was examined by the method described previously [6]. 35Sulfur incorporation was expressed as disintegrations per minute per milligram of tissue protein (DPM/mg).
Allotransplantation of trachea
The cryopreserved Lewis rat tracheal segments were thawed and transplanted into Brown Norway rats by the technique described previously [7]. After resection of a five-ring segment of a Brown Norway rat’s cervical trachea, the trachea was reconstructed using the five-ring thawed tracheal segment of a Lewis rat in the 2-, 6-, 12-, 18-, and 24-month preservation groups. As controls, noncryopreserved autografts (n = 8) were transplanted into Lewis rats, and noncryopreserved allografts (n = 8) were transplanted into Brown Norway rats. The recipient rats were sacrificed 3 months after transplantation. The animals received anesthesia by ether and were killed with an intraperitoneal injection of 1 mL of pentobarbital. All animals received humane care in compliance with the "Guide for the Care and Use of Laboratory Animals" (NIH publication 85-23, revised 1985).
After the specimens were embedded in paraffin, 5-µm-thick sections were stained with hematoxylin and eosin. We assessed the epithelium and cartilage histologically. A normal epithelium structure is regarded as possessing ciliated, mucous, and basal cells. Also, a normal nucleated cell in the cartilage is regarded as richly stained and possessing a clear nuclear membrane.
Statistical methods
Continuous data were expressed as the mean ± standard deviation of the mean. Significance of differences was determined by one-way analysis of variance. p values less than 0.05 were considered significant.
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Results
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The average 35S incorporation in the cartilage before cryopreservation was 224 ± 17 DPM/mg. 35Sulfur incorporations after different lengths of cryopreservation are shown in Table 1. There were no significant differences in 35S incorporation among the groups. 35Sulfur incorporation decreased to 67% to 76% after cryopreservation.
Gross examination after thawing revealed an intact tracheal structure in all groups. All rats with noncryopreserved autografts and cryopreserved allografts survived for 3 months. No tracheal stenosis or tracheomalacia was observed in these rats. All rats with noncryopreserved allografts died before 50 days after transplantation because of tracheal stenosis. Histologic examination after thawing revealed depletion of the epithelium and normal cartilage in all groups (Fig 1A, 1B). Noncryopreserved autografts transplanted showed normal cartilage without decrease of the nucleated cell number (Fig 2). Noncryopreserved allografts transplanted showed defective epithelium and necrotic cartilage with fibrosis (Fig 3). In all groups of cryopreserved allografts, transplanted grafts exhibited normal epithelium and decrease in the only nucleated cell number in the cartilage (Fig 4A, 4B).
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Fig 1. Histologic findings for tracheal grafts after thawing. The trachea had normal cartilage in all groups after thawing. (A) Two-month group. (B) Twenty-four-month group. (Hematoxylin and eosin, x200.)
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Fig 2. Histologic findings for noncryopreserved autografts transplanted. The trachea showed normal cartilage without decrease of the nucleated cell number. (Hematoxylin and eosin, x200.)
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Fig 3. Histologic findings for noncryopreserved allografts transplanted. The trachea showed defective epithelium and acellular cartilage with fibrosis. (Hematoxylin and eosin, x200.)
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Fig 4. Histologic findings for cryopreserved allografts transplanted. In all groups, the tracheas exhibited decrease in the only nucleated cell number in the cartilage. (A) Two-month group. (B) Twenty-four-month group. (Hematoxylin and eosin, x200).
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Comment
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Many experimental studies [1–5, 7–11] of cryopreserved tracheal allotransplantations have been reported. Some problems regarding the clinical application have already been resolved: techniques of graft preservation [1, 5, 12], operative methods of transplantation [13, 14], immunosuppression regimen [1, 11], and extent of graft resection [15, 16]. In most studies [1–5], the tracheal transplantation was performed after about 1 month of cryopreservation. Clinically, long-term cryopreservation of tracheas would be essential. In this study, we examined whether tracheas could be used clinically after long-term cryopreservation. Yokomise and associates [11] showed that transplanted grafts survived more than 2 months with preservation of graft viability after 9 months of cryopreservation in a trehalose cryopreservation solution. In our experiments, tracheal allotransplantation with 24-month cryopreserved allografts could be safely performed. In contrast, Nakanishi and associates [10] reported that the permissible period of cryopreservation with the Bicell biofreezing device was 3 months. There were some differences in the freezing techniques, cryoprotectants, freezing machines, and storage systems used among these studies. With regard to the freezing technique and freezing machine, Lange and Hopkins [17] suggested that -1°C per minute would be the best freezing rate for cryopreservation using a programmable freezer. VanderKamp and colleagues [18] also theorized that -1°C per minute would maximize fibroblast viability in a heart valve allograft. Concerning storage temperature, Karow and Pegg [19] showed that the rate of intracellular ice recrystallization accelerated as the frozen tissue temperature rose above -130°C and that nearly all biological materials could be stored at liquid nitrogen temperatures (-196°C) for approximately 10 years. Yokomise and coworkers [11] reported that long-term cryopreservation (9 months) was possible with the combined use of dimethyl sulfoxide and trehalose as cryoprotective agents. Although we used only dimethyl sulfoxide as a cryoprotectant, we could successfully perform tracheal allotransplantation with long-term cryopreserved (24 months) allografts by freezing the grafts with a programmable freezer and storing them in liquid nitrogen (-196°C). Freezing injury is normally classified as being caused by either direct or indirect chilling injury [20]. Direct chilling injury depends on the rate of cooling, and indirect chilling injury occurs after a long period at the reduced temperature [20]. Also, the viability of the cryopreserved tracheal cartilage seems to vary based on the storage methods, freezing machine, and storage systems. We demonstrated that 35S incorporation in the cartilage after cryopreservation decreased to 67% to 76% with our method in this model.
Cryopreservation techniques are favored for maintaining viability and structured integrity and reducing the immunologic response of several tissues [12, 21, 22]. Bujita and associates [23] demonstrated human leukocyte antigen class II subregion gene products on human tracheal epithelium and mixed glandular tissue. Depletion of the tracheal epithelium seems to play an important role in the success of allotransplantation with cryopreserved tracheal allografts [3, 11]. We previously reported that the epithelium of the transplanted cryopreserved tracheal segment originated from the recipient epithelium, whereas the cartilage retained the structure of the donor trachea [7]. We think that there are some changes of antigenicity of cartilage after cryopreservation and that transplantation of a cryopreserved trachea leads to the growth of the recipient’s epithelium over the donor trachea, thereby reducing the antigenicity of the transplant.
Cartilage viability of a cryopreserved tracheal allograft seems to affect graft function and durability. We previously documented the influence of warm ischemia and cryopreservation on cartilage viability of tracheal allografts by measurement of Na235SO4 incorporation [6]. We reported in the past study that 35S incorporations in the cartilage decreased as warm ischemic time increased. We also found that 35S incorporation in the cartilage at 48 hours of warm ischemic time was 4 ± 1 DPM/mg (unpublished data). Therefore, we think that 35S uptake does not occur if the cells are dead and that measurement of Na2SO4 incorporation is a reliable method as a marker of cell viability in the cartilage. In the present study, we assessed cell viability of the cartilage of the tracheas after long-term cryopreservation by the same method. Cartilage viability of cryopreserved tracheal grafts was maintained during cryopreservation for at least 24 months. We also previously demonstrated that the cartilage of a cryopreserved allograft decreased in the nucleated cell number and had fibrous change after transplantation as warm ischemic time from cardiac death to preservation increased, and that tracheal stenosis was observed in a graft that had fibrous change [6]. All the grafts transplanted after different lengths of preservation exhibited decreases in the only nucleated number in the cartilage. Therefore, these grafts would maintain good function and durability. The heart valves after long-term cryopreservation can be used clinically with good results [22, 24]. Cryopreserved semen [25] and pancreatic islets [26] have also maintained good function. In this study, all rats with cryopreserved allografts survived for 3 months, and the transplanted grafts exhibited decrease in the only nucleated cell number in the cartilage. We think that cartilage viability will also be maintained in cryopreserved tracheas for more than 2 years and that tracheas after long-term cryopreservation can be used clinically.
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Abstract
Introduction
