Cryopreservation of the tracheal grafts

Abstract

Transplantation of the trachea may become the preferred method for the reconstruction of extensive tracheal defects, however, several unresolved problems must be addressed, such as immunosuppression, preservation, and donor shortage. In this manuscript, the cryopreservation of tracheal grafts is reviewed, which potentially is associated with a lessened immunological response. Cryopreservation may be used clinically for long-term preservation and may solve the donor shortage. It is very important to confirm the immunomodulatory effect of cryopreservation on tracheal allografts in order to expand the potential clinical application of tracheal transplantation in the future. The cartilage as well as the epithelium and lamina propria serve as targets for rejection. However, the effect of cryopreservation on chondrocytes could be associated with reduced allogenicity of the trachea. The long-term cryopreservation of cartilage must be investigated in basic research models of chondrocyte viability. Growth of cryopreserved tracheal allografts is less well understood. Further studies are needed to elucidate the mechanism of synergistic effects of both cryopreservation and adequate immunosuppression for tracheal xenografts.

Introduction

An extensive tracheal resection is often required in patients with malignancies involving the trachea,1,2 or with benign stenosis of the trachea secondary to accidental trauma, or to congenital,3 inflammatory4,5 or iatrogenic causes.6 When a primary anastomosis is not feasible following an extensive resection of the trachea, a terminal tracheal fistula is created. However, in certain cases it may be preferable to interpose a substitute trachea. While prosthetic and tissue grafts are used in repairing circumferential tracheal defects their use has achieved only limited success. Several experiments on the prosthetic replacement of large tracheal defects have been performed with the prospect of clinical application.7–9 However, most of these prosthetic tracheal grafts failed to reepithelialize.9–11 The repair of tracheal defects with autogenous tissue, such as free periosteal,12 jejunal,13 muscular,14 esophageal,15 bronchial16 and aortic17 grafts also has met with limited success because of difficulties in maintaining a patent airway. In contrast, a tracheal allograft contains native epithelium and has cartilaginous rings to maintain a patent airway. Therefore, a tracheal allograft is an ideal substitute for the defect. However, before the routinge clinical application of the tracheal allograft procedure can become a reality, several questions must be addressed, regarding revascularization, immunosuppression, preservation and donor shortage.

To clarify revascularization for tracheal grafts, the author has already reported several studies18–23 and a review of the literature.24 In this manuscript, cryopreservation of tracheal grafts is reviewed, which may potentially be used for long-term preservation and may be associated with a low immunological response.

Rejection Reaction and Immunosuppression

Tracheal transplants, in contrast to other organ transplants, induce only a weak graft-rejection reaction even in the presence of major histoincompatibility. Rose and co-workers first demonstrated that a tracheal allotransplant functioned without any evidence of rejection in man.25 However, several investigators have reported that the cartilage as well as the epithelium and lamina propria serve as targets for rejection.26,27 Beigel and colleagues demonstrated the antigenicity of rat tracheal transplants and found that the donor epithelium is rejected and that new epithelium migrated from recipient to the transplant.28–30 Thirteen years later, this was definitively shown to be correct using techniques of molecular biology.31 Human studies have demonstrated Human Leukocyte Antigen (HLA) class II subregion gene to be the antigenic profile of the trachea.32–34

To better characterize the immunobiology of the trachea, Genden and co-workers successfully established an orthotopic tracheal transplant model in the mouse.35 In their experiments using nonimmunosuppressed mice, the allogeneic recipients developed a persistent audible stridor two days after tracheal transplantation and allogeneic grafts demonstrated a loss of normal ciliated respiratory epithelium and a CD8/CD4-positive lymphocytic infiltrate. In nonimmunosuppressed dogs, the author demonstrated that heterotopic tracheal allotransplants display an early rejection reaction similar to that of Genden's murine model and developed a set of criteria for diagnosing rejection. The target for rejection was shown to be epithelium first, followed by lamina propria and cartilage.36

The chronic histopathologic features of tracheal allotransplant rejection remain to be fully elucidated. The author developed a reproducible model of a refractory tracheal allotransplant rejection using Tacrolimus in rats.37 However, it is difficult to develop a reliable chronic rejection model of the tracheal allotransplant because the chronic rejection represents neither just a refractory immunologic reaction nor a repeat of acute rejection.

Many researchers have presented immunosuppressive therapies for acute rejection of tracheal allografts. Delaere and colleagues demonstrated that tracheal allografts undergoing continuous immunosuppression with 10 mg/kg per day of Cyclosporin-A showed no rejection and remained functional with preservation of mucociliary activity.38,39 The author advocated that an appropriate dose of 15 mg/kg/day of Cyclosporin-A may be used to maintain rat tracheal allograft viability.40 Davreux and co-workers demonstrated that 2 mg/kg/day of methylprednisolone in addition to 15 mg/kg/day of Cyclosporin-A may improve epithelial viability in rat tracheal allografts.41 With regard to other agents, Moriyama and co-workers reported successful tracheal allotransplantation using 0.1 mg/kg/day of Tacrolimus in dogs.42

The author and colleagues made an attempt to minimize the dose of immunosuppressive agent in order to avoid side effects such as organ toxicity, carcinogenesis and infectious complications. A short-course of immunosuppression, such as 1.5 mg/kg/day of Tacrolimus administered for only 3 consecutive days after surgery in rats,43 5 mg/kg/day of Mizoribine for 21 days in dogs,44 and 150 to 225 mg/kg/day of Multiglycosidorum tripterygii for 3 days in rats, maintained the morphology of tracheal allografts.37,45 A short-course and a large dose of immunosuppressive agent seem to induce immunologic unresponsiveness.46 Acceptable efficacy of those agents was shown in animals although such methods cannot be applied directly to humans.

Other immunosuppression methods include denervation of the spleen,47 high dose radiation therapy,48 photodynamic therapy,49 medication (Cialit and Merthiolate,50 and t-octylphenoxy-polyethoxyethanol51) used in the treatment of reduction of graft antigenicity, or interleukin-10 gene transfer therapy to restrain an immune response of T cells.52 It is necessary to wait for future reports on the long-term results of those methods. However, Yokomise and co-workers have reported that tracheal allografts do not survive long term following high dose irradiation therapy.48,53 Recently, several researchers have reported that cryopreservation reduces antigenicity of the tracheal allografts and controls a rejection reaction in a large animal model. These will be reviewed below.

Long-Term Preservation

As transplantation of the trachea becomes a more preferred method for the reconstruction of extensive tracheal defects in the future, the shortage of donor trachea will remain a major obstacle. A long-term period of preservation is one of the methods to help solve this problem. In regard to cold ischemia time of the trachea, Macchiarini and associates demonstrated that the trachea may be safely preserved for as long as 15 hours of static storage in Euro-Collins solution at 4 degrees Celsius, as assessed by histology and mucous-secreting functions.54 However, the period of the preservation in this study is too short to solve the donor shortage. A longer-term preservation method is required.

The first experimental study on cryopreservation of the trachea was reported in 1951.55,56 Experimental work intensified after Deschamps and co-workers reported the functional and hisotological changes of the cryopreserved canine trachea.55 Yokomise and colleagues showed that structural reliability of a cryopreserved trachea was maintained for one month with a new solution including trehalose in addition to dimethyl sulfoxide.57 The author and associates assessed the maximal period of cryopreservation using an easy and cheap slow-freezing instrument for viable tracheal isograft in rats. As a result, the effect of cryopreservation on the trachea has been recognized as the degeneration of both the cartilage and the epithelium. The cartilage showed irreversible damage while the epithelium partially remained after cryopreservation for 6 months. The permissible period of cryopreservation to maintain the isograft viability was 3 months.58 This period of cryopreservation could help clinicians to greatly relieve the shortage of donors.

If the trachea can be retrieved for transplantation late after circulatory arrest, the shortage of donors may be further alleviated. The combined use of non beating-heart cadaveric donors and a long period of cryopreservation for tracheal grafting offers great hope for their eventual clinical use in humans. The author and associates therefore tried to elucidate the permissible period of warm ischemia before cryopreservation in rat tracheal isografts. The maximum permissible period of warm ischemia before a 3-month cryopreservation which maintained isograft viability was 18 hours.59 Concerning the method of cryopreservation, several researchers have reported the usefulness of lyophilization for tracheal allografts.56,60 Such a technique could expand the human tracheal donor pool. When a longer period of cryopreservation is required, investigators and clinicians may face decreased viability, but this may be balanced by the decreased antigenicity that results from cryopreservation.

Effect of Cryopreservation on Immunologic Reaction

The effect of cryopreservation on tissue allografts has been studied in basic research models for many years. However, its effect on immune responses remains controversial. Several investigators have reported that cryopreservation decreases tracheal allogenicity and prolongs survival of allografts in canine experimental models.61–63 Similar experiments using rats have suggested that denuded epithelium caused by cryopreservation is closely related to decreased antigenicity.64,65 However, the author and associates found that the epithelium does not always disappear after thawing of cryopreserved tracheal grafts. In each cryopreserved graft that we observed, approximately 10 to 40% of the epithelium remained immediately after thawing, although longer periods of cryopreservation resulted in more denuded epithelium. The remaining epithelium underwent degeneration, consisting of a single layer of non-ciliated epithelium (Fig. 1).58,66 After transplantation of the cryopreserved tracheal grafts, the remaining epithelium was gradually regenerated in the isografts. In the allografts, however, the remaining epithelium was rejected whereas other components of the trachea were maintained.66 These facts refute the speculation that the denuded epithelium caused by cryopreservation is related to decreased antigenicity, and suggest that the remaining epithelium after cryopreservation may possess antigenicity.

If the denudation of the mucosa including the epithelium and the mixed glandular tissue holds the key of treatment for immunologic rejection in the tracheal allotransplantation, allografts could maintain their structure without immunologic rejection in artificial mucosa denudation models. Liu and associates showed that the antigenicity of canine tracheal allografts could be reduced by removing the epithelium and mixed glands from the graft by detergent treatment.51 However, the detergent treatment using t-octylphenoxypolyethoxyethanol may reduce the antigenicity of the cartilage adventitia as well as the mucosa. The author found that fresh tracheal allografts undergoing an artificial removal of the epithelium could not maintain their patent structure in rats because of immunologic rejection (unpublished data) (Fig. 2). Therefore, loss of the epithelium only does not necessarily hold the key for treatment of immunologic rejection.

It is very important to confirm the immunomodulatory effect of cryopreservation on tracheal allografts in order to expand the potential clinical applications of tracheal transplantation in the future. The author and colleagues assessed second-set allograft rejection to demonstrate the credibility of the immunomodulatory effect of cryopreservation. In this study, second-set rejection occurred only in rats undergoing fresh primary allografting, not in rats undergoing cryopreserved primary allografting (Fig. 3).67 Thus, cryopreservation prevents enhanced allograft rejection and potentially has an immunomodulatory effect on the allograft. The mechanism for this immunomodulatory effect potentially includes delayed antigen sensitization and reduced antigenicity of the trachea. The antigen sensitization could be weakly induced by cryopreservation until the blood supply is restored to the transplanted graft. However, our experimental results showed that the cryopreserved allografts survived for a long period after transplantation and did not show delayed rejection.67 Therefore, cryopreservation does not seem to delay the antigen sensitization of tracheal allografts but probably decreases the antigenicity itself.

Similar effects of cryopreservation have been reported in other tissues. In skin flaps transplanted via microvascular anastomosis, Hirase and co-workers demonstrated a decrease in the antigenicity of dermis in cryopreserved allografts without immunosuppression.68 The history of cryopreserved cardiac valve allotransplantation has suggested that the denuded endothelium after cryopreservation may be associated with decreased antigenicity although the immunologic rejection is inevitable.69,70 The immunomodulatory effect of cryopreservation on tissue allografts is considered to be credible.

Other factors as well as reduced epithelium may be associated with decreased antigenicity in the cryopreserved tracheal allografts. Although the mucosa including the epithelium and the mixed glandular tissue is reported to express HLA class II subregion gene products,32 the chondrocytes are also thought to have a tissue-specific surface antigen.27 The author and associates demonstrated that a longer period of cryopreservation helps to maintain a better patency of tracheal allografts despite the decreased viability of chondrocytes.66 Messineo and co-workers also showed that cryopreserved tracheal allografts without immunosuppression have no viable chondrocytes, but the patency of the lumen can be adequately preserved.71 The effect of cryopreservation on chondrocytes may be associated with reduced allogenicity of the trachea. On the other hand, Ohlendorf and colleagues showed that the beneficial effect of cryoprotectant is confined to the chondrocytes in the superficial layer of the cartilage.72 The degeneration of the chondrocytes occurring in a major part of the cartilage in cryopreserved allografts may lead to decreased antigenicity because the cryoprotectant does not penetrate deeply into the cartilage. The chondrocytes could determine the fate of the cryopreserved tracheal allografts. The long-term cryopreservation of cartilage must therefore be investigated in basic research models of chondrocyte viability.

Although Murakawa and colleagues showed in vitro that cryopreservation does not downregulate the allogenicity of human fibroblasts,73 they did however demonstrate the successful allotransplantation of cryopreserved tracheal grafts in nonhuman primates.74 They reported that cryopreservation potentially reduces antigenicity by the downregulation of HLA class II antigen presentation of airway epithelial cells and also infiltration of various kinds of antigen presenting cells.74,75 The effect of cryopreservation on antigen presenting cells remains controversial, and further investigation is required to clarify the mechanism.

The growth potential of tracheal transplants is another important, unresolved issue. In an experimental study using autografts, Kubota and co-workers demonstrated the growth potential of tracheal autografts in a growing puppy model.76 On the other hand, Hisamatsu and colleagues studied growth of cryopreserved tracheal allotransplant using rabbits for the treatment of congenital tracheal stenosis, and demonstrated that patency of the cryopreserved tracheal allografts was favorable, but no growth occurred even with the use of an immunosuppressant.77 This issue will also be the subject of future investigated.

Xenotransplantation

As a possible solution to the donor shortage, xenotransplation of the trachea has been examined as an option to tracheal transplantation. Humoral immunity is always seen early after concordant xenogeneic transplantation, followed by T-cell mediated cellular immunity.78 In discordant xenogeneic transplantation, the hyperacute rejection reaction is caused by activation of antibodies and complement. Even if this reaction is averted, the delayed xenogeneic rejection occurs within five days after transplantation, followed by cellular rejection by T cell.79,80 Therefore, it is very difficult to control the course of xenogeneic rejection. However, several investigators have shown that immunosuppressive agents can maintain the morphology of xenogeneic transplants. Valdivia and associates reported the effectiveness of tacrolimus in suppressing antibody production in hamster-to-rat liver xenotransplantation.81 Reichenspurner and co-workers presented Leflunomide to potentially maintain the epithelial viability in hamster-to-rat heterotopic tracheal xenotransplantation.82 An appropriate immunosuppressive regimen could prolong xenograft survival.

The author and colleagues combined the effects of reduced immunogenicity by cryopreservation and inhibited T cell-derived rejection by immunosuppressive agents, and studied the feasibility of cryopreserved xenografts with the use of short-course immunosuppression. An observed synergistic effect of cryopreservation and adequate dose of tacrolimus therapy enabled tracheal xenografts to remain viable over a long period (Fig. 4).83 In tissue xenotransplantation, hyperacute rejection between discordant species may not occur because of low natural antibody titers.84 A primitive immunoreaction may be associated with tracheal xenotransplantation, as evidenced by the effect of immunosuppression.83 Further studies are needed to elucidate the mechanism of tracheal xenograft rejection.

Clinical Application

Rose and co-workers reported the first successful human tracheal allotransplantation in 1979.25 In 1993, one-staged allotransplantation of the trachea was performed in a patient with marked tracheal stenosis caused by idiopathic fibrosing mediastinitis, and successful results were achieved with adequate immunosuppression and omental revascularization.85 Thereafter, several investigators working with Herberhold reported successful tracheal transplantation using chemically treated allografts for pediatric patients with congenital tracheal stenosis.86–88 Such a treatment is now an established option for congenital tracheal reconstruction.

The study of tracheal transplantation using non-chemically treating allografts is proceding, as described above.89 Tracheal transplantation using cryopreserved and irradiated homograft for human laryngotracheal reconstruction has been reported to be a viable alternative.90 As the tracheal transplantation was used in the treatment of glottic cancer,91 an immunosuppressant-free allotransplantation was required. Although the clinical applications for cryopreserved allografts are limited at present, they will almost certainly expand with further advances in immunosuppression and preservation techniques.

Figures and Tables

Figure 1 Histologic findings of cryopreserved rat tracheal allograft immediately after thawing. The epithelium shows degeneration but not denuded. (Hematoxylin and eosin; original magnification, ×200).

Figure 2 Histologic findings of fresh rat tracheal allograft 3 months after transplantation, which was denuded of the epithelium. The graft shows narrowed patency because of thickened mucosa with mononuclear cell infiltration. (Hematoxylin and eosin; original magnification, ×25).

Figure 3 Histologic findings of rat tracheal allografts 28 days after transplantation. (A) a second allograft after transplantation of fresh allograft. The allograft is severely rejected. (B) a second allograft after transplantation of cryopreserved allograft. The patency of the allograft is narrow but the structure is maintained. (Hematoxylin and eosin; original magnification, ×25).

Figure 4 Histologic findings of cryopreserved tracheal xenograft 28 days after transplantation, which received short-course immunosuppression with 3.5 mg/kg/day of Tacrolimus. The epithelium shows degeneration but the cartilage is well maintained. (Hematoxylin and eosin; original magnification, ×200).