Ann Thorac Surg 2000;70:1876-1879
© 2000 The Society of Thoracic Surgeons
Original article: general thoracic
Effects of warm ischemia and cryopreservation on cartilage viability of tracheal allografts
Keiji Kushibe, MDa,
Takashi Tojo, MDa,
Hidehito Sakaguchi, MDa,
Makoto Takahama, MDa,
Kazuhiko Nishizaki, MDa,
Kunimoto Nezu, MDa,
Shigeki Taniguchi, MDa
a Department of Surgery III, Nara Medical University, Nara, Japan
Accepted for publication May 5, 2000.
Address reprint requests to Dr Kushibe, Department of Surgery III, Nara Medical University, 840 Shijo-cho, Kashihara, Nara, Japan, 634-8522
e-mail: kushikuk{at}nmu-gw.naramed-u.ac.jp
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Abstract
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Background. For clinical use of a cryopreserved tracheal allograft, it is important to evaluate cartilage viability. We assessed cell viability of the cartilage in a cryopreserved tracheal allograft by measurement of Na235SO4 incorporation. We also investigated the effects of warm ischemic time on tracheal cartilage viability.
Methods. The tracheas from Lewis rats were harvested and preserved at different warm ischemic times from cardiac death to preservation (0, 1, 2, 4, 6, 9, and 12 hours, each group n = 8). The cartilage was labeled with 4 µCi/mL of Na235SO4. The specimen was hydrolyzed in 0.5 mol/L NaOH, and a solution of the extracts was then counted by liquid scintillation counter. Tracheas were transplanted into Brown Norway rats.
Results. 35Sulfur incorporation in the cartilage decreased as warm ischemic time increased. In addition, 35Sulfur incorporation decreased from 76% to 67% after cryopreservation. Histologic examinations of the normal tracheal cartilage before preservation and after thawing were done in all the groups. After transplantation, the cartilage had severe fibrous changes, and its layer was almost nonobservable in the 9- and 12-hour groups.
Conclusions. The viability of the tracheal cartilage decreased with warm ischemic time and from 76% to 67% after cryopreservation. In the rat tracheal transplantation model, a cryopreserved tracheal allotransplant could be done safely with a graft that was cryopreserved within 6 hours of warm ischemic time.
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Introduction
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Many experimental studies [1–4] of cryopreserved tracheal allotransplantations have been reported with satisfactory results. Some investigators [5, 6] have evaluated the immunoreactive resopnse to tracheal epithelium in a tracheal allotransplantation with a cryopreserved graft. It appears that cartilage viability of a cryopreserved tracheal allograft affects the function and durability of a graft. For the clinical use of a tracheal allograft, it is important to evaluate cartilage viability. Some studies regarding the viability of the articular [7–10] or nasoseptal [11] cartilage have been reported. However, no study of tracheal cartilage viability has been reported. Therefore, we assessed cell viability of the cartilage in cryopreserved tracheal allografts by measurement of Na235SO4.
The warm ischemic time (WIT) from cardiac death to preservation is recognized as an important determinant of cell survival in a cryopreserved heart valve [12]. We hypothesized that WIT would be an important determinant of cartilage survival for a cryopreserved trachea and examined the relationship between WIT and tracheal cartilage viability by measurement of Na235SO4 incorporation. We also examined histologic changes in the cartilage before preservation, after thawing, and after tracheal transplantation [5].
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Material and methods
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Harvest and preservation of grafts
Fifty-six inbred male Lewis rats (200 to 250 g) were used for graft harvesting. Donor animals were sacrificed with intraperitoneal injection of 1 mL pentobarbital. The tracheas were harvested at different warm ischemic times (at 0, 1, 2, 4, 6, 9, and 12 hours, each group n = 8). The freezing medium was Tissue Culture Medium-199 solution with a final concentration of 5% hydroxy-ethyl-piperazone-ethane-sulfonic acid (HEPES) buffer and 10% dimethylsulfoxide. A sterile plastic bag containing a specimen was filled with the freezing medium, sealed, and frozen to –80°C at rate of –1°C per minute in a programmable freezer. The bag was stored in liquid nitrogen (-196°C) for approximately 2 months. The specimen was thawed by placing the bag in a 40°C water bath before transplantation or viability assessment. The freezing medium was then rinsed.
Assessment of cartilage viability by measurement of Na235SO4 incorporation
The cartilage viability was evaluated by proteoglycan synthesis. Three tracheal cartilage rings were resected from a graft by trimming. The cartilage was labeled with 4 µCi/mL of Na235SO4 in 1 mL of modified Eagle medium at 37°C for 4 hours. Next, each specimen was rinsed with 1 mL of phosphate-buffered saline three times, then incubated in 1 mL of modified Eagle medium containing 10% fetal bovine serum at 37°C for 24 hours. Then the cartilage was homogenized on ice with 1 mL of 10% trichloroacetic acid and centrifuged at 10,000 rpm for 10 minutes at 4°C. The sediment was hydrolyzed by shaking in 1 mL of 0.5 mol/L NaOH at room temperature for 1 week. Then the solution was centrifuged again at 10,000 rpm for 10 minutes at 4°C. Next, the supernatant was neutralized with 0.5 mol/L HCl and counted by a liquid scintillation counter. The protein’s weight of the final supernatant was measured with a BCA Protein Assay kit (Pierce, Rockford, IL). 35Sulfur incorporation was expressed as disintergration per minute per milligram of tissue protein (DPM/mg). 35Sulfur incorporations in the cartilage before and after cryopreservation were examined in each group.
Histologic assessment of the cartilage
Histologic changes in the cartilage before cryopreservation and after thawing were examined in each group. For tracheal transplantations, Lewis rats were the donors and brown Norway rats the recipients. After resection of a five-ring segment of a brown Norway rat’s cervical trachea, the trachea was reconstructed using the cryopreserved tracheal segment of a Lewis rat in each group [5]. Three months after tranplantation, histologic changes in the cartilage were examined.
Animal care
All animals received humane care in compliance with the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health (NIH publication no. 85-23, revised 1985).
Statistical analysis
All values are presented as mean and standard error of the mean. The significance of differences was determined by one-way analysis of variance. Probability values less than 0.05 were judged to be significant.
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Results
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35Sulfur incorporation in the cartilage at different warm ischemic times before cryopreservation is shown in Figure 1. The viability of tracheal cartilage decreased as warm ischemic time increased, and the relationships were statistically significant. The ratios of cartilage viability after cryopreservation to that before cryopreservation are shown in Table 1. The viability before cryopreservation was significantly higher (p < 0.05) than that after cryopreservation in each group.
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Fig 1. 35Sulfur incorporation decreased as warm ischemia time increased. Relationships were statistically significant. (DPM/mg = disintegration per minute per milligram of tissue protein.)
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Macroscopic findings of the tracheas before cryopreservation and after thawing showed that their structure was intact in all the groups. All recipient rats survived for 3 months, but tracheal stenosis was observed in 4 of 8 rats in the group that had had a tracheal graft harvested with 12 hours of WIT. In histologic examinations, the tracheal cartilage was intact before preservation and after thawing in all groups (Fig 2). After transplantation, histologic examinations showed a decrease in the nucleated cell number of the cartilage and its layer in the 0-, 1-, 2-, 3-, and 4-hour groups (Fig 3A and B). In the 6-hour group, the cartilage had fibrous changes (Fig 3C). In addition to a decrease in nucleated cell number of the cartilage and its layer in the 9- and 12-hour groups, the cartilage had severe fibrous change, its layer was almost nonobservable, (Fig 3D) and the remaining cartilage structure was compromised.
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Fig 2. Histologic findings of tracheal grafts before preservation and after thawing. The trachea had intact cartilage before preservation (A) and after thawing (B) even in the group with 12 hours of warm ischemic time. (Hematoxylin and eosin; original magnification x200.)
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Fig 3. Histologic findings of transplanted tracheal grafts. (A) In the 0-hour group, the nucleated cell number of the cartilage decreased. (B) In the 4-hour group, the nucleated cell number of the cartilage and its layer decreased. (C) In the 6-hour group, the cartilage had fibrous change. (D) In the 9-hour group, the cartilage had severe fibrous change and its layer was almost nonobservable. (Hematoxylin and eosin; original magnification x200.)
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Comment
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Cartilage viability of a cryopreserved tracheal allograft affects the function and durability of the graft. Although some studies of the viability of cryopreserved articular [7–10] or nasoseptal [11] cartilage have been reported, no study of tracheal cartilage viability has been reported. The most widely used tests in assessing cell viability are trypan blue dye-exclusion [11, 13] or fluorescein diacetate-propidium iodine staining [14–16] on isolated cells. For assessment of cell viability of the articular cartilage, measurement of Na235SO4 incorporation was often done [8–10, 17, 18]. We therefore measured Na235SO4 incorporation to assess cell viability of the cartilage in a cryopreserved tracheal allograft.
The WIT between cardiac death and preservation is recognized as an important determinant of cell survival in cryopreserved heart valves [12]. Niwaya and colleagues [16] found a significant negative correlation between fibroblast viability of the allograft valve and WIT by flow cytometry. We hypothesized that WIT would be an important determinant of cartilage survival for a cryopreserved trachea. Macchiarini and coworkers [19] demonstrated the maximal preservation time of a tracheal allograft in an immunosuppressed piglet model. Kawabe and Yoshinao [8] showed that the viability of an articular cartilage slice immersed at 4°C or 37°C in cryopreservative decreased steadily with time, when the viability was assessed by measurement of Na235SO4 incorporation. We found that the viability of the tracheal cartilage decreased as WIT increased.
Determination of viability after thawing is one of the major problems associated with cryopreservation experiments. Mankin and colleagues [7] showed a 20% viability rate of chondrocytes in intact articular cartilage frozen with glycerol and dimethylsulfoxide. Kawabe and Yoshinao [8] reported that the survival rate for cryopreserved articular cartilage in 10% dimethylsulfoxide was, on average, 19% for intact slices and 34% when holes were made in the slices. Schachar and associates [9] reported that the intact articular cartilage slow-frozen at approximately 0.4 to 0.5°C/minute yielded 30% to 40% survival. Similarly, Kuranobu [10] reported that slow-frozen discs yielded about 65% survival. In our experiments, the cryopreserved tracheal cartilage yielded 67% to 76% survival. The wide variation in viability of cryopreserved cartilage among the various reports might be related to differences in types of the cartilage used, freezing methods, cryoprotectives, and methods used to test cell function.
Although histologic examinations showed intact tracheal cartilage before preservation and after thawing in all the groups in our study, assessment of cartilage viability by measurement of Na235SO4 incorporation revealed that viability before cryopreservation was significantly higher than after cryopreservation in all groups. Tavakol and coworkers [20] studied the ultrastructural changes in the condrocytes of fresh versus frozen-thawed articular cartilage using transmission electron microscopy. Similarly, Bujia and associates [11] reported that transmission electron studies of postfrozen cartilage showed varying degrees of cellular damage in all cells. Histologic examinations showed that the cartilage of a graft had fibrous change after transplantation, and its layer decreased as WIT increased. We found that the tracheal grafts that had been harvested with 6 hours of WIT had less fibrous change than those with 9 or 12 hours of WIT. In the rat tracheal transplantation model, we demonstrated that cryopreserved tracheal allotransplantation could be done safely with a graft that had 6 hours of WIT or less. We studied the effect of WIT and cryopreservation on cartilage viability in a rat model, which complements the studies by Nakanishi and associates [21] and Aoki and coworkers [22] demonstrating the maximal period of cryopreservation in a rat model. Our experimental study helps to resolve the problem about the effect of warm ischemia and cryopreservation on cartilage viability of tracheal allografts in humans.
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Tracheal allotransplantation maintaining cartilage viability with long-term cryopreserved allografts
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May 1, 2001;
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Abstract
Introduction
