Tracheal Allografts Research Articles

Assessment of the Therapeutic Potential of Enhancer of Zeste Homolog 2 Inhibition in a Murine Model of Bronchiolitis Obliterans Syndrome.

Bronchiolitis obliterans syndrome (BOS) is a chronic complication following lung transplantation that limits the long-term survival. Although the enhancer of zeste homolog 2 (EZH2) is involved in post-transplantation rejection, its involvement in BOS pathogenesis remains unclear. We aimed to investigate the therapeutic potential of EZH2 inhibition in BOS. 3-deazaneplanocin A (DZNep) was administered intraperitoneally to heterotopic tracheal transplant recipient model mice. Tracheal allografts were obtained on days 7, 14, 21, and 28 after transplantation. The obstruction ratios of the DZNep and control groups on days 7, 14, 21, and 28 were 15.1% ± 0.8% vs. 20.4% ± 3.6% (p = 0.996), 16.9% ± 2.1% vs. 67.7% ± 11.5% (p < 0.001), 47.8% ± 7.8% vs. 92.2% ± 5.4% (p < 0.001), and 60.0% ± 9.6% vs. 95.0% ± 2.3% (p < 0.001), respectively. The levels of interleukin (IL)-6 and interferon-γ on day 7 and those of IL-2, tumor necrosis factor, and IL-17A on days 14, 21, and 28 were significantly reduced following DZNep treatment. DZNep significantly decreased the number of infiltrating T-cells on day 14. In conclusion, DZNep-mediated EZH2 inhibition suppressed the inflammatory reactions driven by pro-inflammatory cytokines and T cell infiltration, thereby alleviating BOS symptoms.

Tracheal transplantation: lessons learned that may apply to lung transplantation.

The purpose of this review is to explore the lessons learned from experimental and human tracheal transplantation to determine if this information may be applied to lung transplantation. Experimental work in animal models and the recent human tracheal transplantation suggests that a robust tracheal vascular supply prevents anastomotic complications. Further, this work demonstrates that tracheal allografts undergo a progressive chimerism as recipient epithelium repopulates the allograft. In contrast to most vascularized composite allografts such as hand and face transplantation that experience high rates of rejection, the tracheal allograft did not demonstrate rejection. This may suggest that tissue chimerism plays a role in evading immune-mediated allograft rejection. While anastomotic complications and chronic allograft rejection are the most common complications related to lung transplantation, the findings associated with tracheal transplantation may have implications for both reducing complications associated with lung transplantation.

Tracheal Replacement: A Scoping Review

Abstract Objective To summarize patient characteristics and outcomes for the historical and current methods of long-segment tracheal replacement in humans. Materials and Methods A single reviewer screened the abstracts and full texts using Covidence for file management. Studies published in English that reported human subjects with circumferential or near-circumferential (&gt;270 degrees) cervical tracheal replacements were included. Articles with subjects treated with primary anastomosis alone, retracted articles, abstracts, expert opinion articles, and conference presentations were excluded. Results A total of 32 articles were included in the review reporting 156 cases of long-segment tracheal replacement including synthetic (alive at 1–8 years n = 6/64), regenerative medicine (dead at 15 days–55 months n = 4, not reported n = 6), cadaveric tracheal allograft (alive at 5 months–10 years n = 32/38), aortic allograft (alive at 6–85 months n = 12/16), free tissue transfer (alive at 6–108 months n = 13/21), allotransplantation (alive at 6–24 months n = 5/8), and vascular composite allograft (VCA) (alive at 20 months n = 1/1). Conclusion Silicone and Marlex prostheses have poor long-term outcomes. The cadaveric tracheal allograft can only replace near-circumferential tracheal defects and is therefore limited to benign tracheal pathology. Inadequate structural support plagues the aortic allograft and often requires numerous invasive procedures and maintenance of an intraluminal stent. A lack of mucociliary clearance exists in all methods of tracheal replacement except cadaveric tracheal allograft and VCA and can cause fatal mucous plugging and chronic pulmonary infections. VCA and allotransplantation require long-term immunomodulation therapy.

Case Report: Human Tracheal Transplantation Undergoes Progressive Reepithelialization Over Time.

Tracheal transplantation is an ideal option for the reconstruction of long-segment circumferential tracheal defects. Our group performed the first successful vascularized single-staged tracheal transplantation in January 2021. Although a rigid biocompatible structure is necessary for a functioning tracheal replacement, the importance of ciliated epithelium, which allows for critical mucociliary clearance, is now being appreciated. Here, we examined the histological changes of the first single-staged human tracheal transplant from serial endoscopic biopsies. Biopsies of the tracheal mucosa were serially obtained since the time of the tracheal transplantation. Samples were examined via hematoxylin and eosin, electron microscopy, and immunohistochemistry. One week after transplantation, there is loss of ciliated epithelium and seromucinous cells, with only a basal layer of epithelium remaining. By 2 weeks, however, the epithelium begins to recover, albeit differently depending on the location of the biopsy. Near the site of tracheal anastomosis, there is epithelial proliferation, with the appearance of early ciliated cells. However, in the midgraft, there appears to be evidence of squamous metaplasia. Over time, however, normal ciliated epithelium and mucous cells appear without signs of chronic inflammation. Critically, the tracheal allograft regained normal appearing respiratory epithelium after initial ischemic injury. The histologic differences at the midgraft versus anastomosis may suggest unique mechanisms of reepithelialization. At the recipient-donor interface, there may be a faster direct migration of recipient-derived epithelial cells, in line with preclinical studies. The midgraft, in contrast, responds with epithelial proliferation from the donor basal cells or dedifferentiated mucous cells. N/A Laryngoscope, 134:2664-2671, 2024.

Targeting PD-1/PD-L1 inhibits rejection in a heterotopic tracheal allograft model of lung transplantation.

Immune checkpoint molecules such as programmed death-1 (PD-1) and programmed death ligand-1 (PD-L1) have revolutionized the field of lung cancer treatment. As part of our study, we examined the role of these proteins in acute rejection in a mouse model of heterotopic tracheal transplantation. Recipient mice were untreated (Allo group) or treated with anti-PD-L1 (aPDL1 group) or PD-L1 Fc recombinant protein (PD-L1 Fc group). A further group of C57BL/6 mice received isografts (Iso group). The occlusion rate was significantly higher in the Allo group than in the Iso group (p = 0.0075), and also higher in the aPD-L1 group (p = 0.0066) and lower in the PD-L1 Fc group (p = 0.030) than in the Allo group. PD-L1 Fc recombinant protein treatment significantly decreased interleukin-6 and interferon-γ levels and reduced the CD4+/CD8+ T cell ratio, without increasing PD-1 and T-cell immunoglobulin mucin 3 expression in CD4+ T cells. These data suggest that PD-L1 Fc recombinant protein decreases the levels of inflammatory cytokines and the proportion of CD4+ T cells without exhaustion. The PD-L1-mediated immune checkpoint mechanism was associated with rejection in the murine tracheal transplant model, suggesting a potential novel target for immunotherapy in lung transplantation.

Pre-epithelialized cryopreserved tracheal allograft for neo-trachea flap engineering.

Background: Tracheal reconstruction presents a challenge because of the difficulty in maintaining the rigidity of the trachea to ensure an open lumen and in achieving an intact luminal lining that secretes mucus to protect against infection. Methods: On the basis of the finding that tracheal cartilage has immune privilege, researchers recently started subjecting tracheal allografts to "partial decellularization" (in which only the epithelium and its antigenicity are removed), rather than complete decellularization, to maintain the tracheal cartilage as an ideal scaffold for tracheal tissue engineering and reconstruction. In the present study, we combined a bioengineering approach and a cryopreservation technique to fabricate a neo-trachea using pre-epithelialized cryopreserved tracheal allograft (ReCTA). Results: Our findings in rat heterotopic and orthotopic implantation models confirmed that tracheal cartilage has sufficient mechanical properties to bear neck movement and compression; indicated that pre-epithelialization with respiratory epithelial cells can prevent fibrosis obliteration and maintain lumen/airway patency; and showed that a pedicled adipose tissue flap can be easily integrated with a tracheal construct to achieve neovascularization. Conclusion: ReCTA can be pre-epithelialized and pre-vascularized using a 2-stage bioengineering approach and thus provides a promising strategy for tracheal tissue engineering.

Partial decellularization eliminates immunogenicity in tracheal allografts.

There is currently no suitable autologous tissue to bridge large tracheal defects. As a result, no standard of care exists for long-segment tracheal reconstruction. Tissue engineering has the potential to create a scaffold from allografts or xenografts that can support neotissue regeneration identical to the native trachea. Recent advances in tissue engineering have led to the idea of partial decellularization that allows for the creation of tracheal scaffolds that supports tracheal epithelial formation while preserving mechanical properties. However, the ability of partial decellularization to eliminate graft immunogenicity remains unknown, and understanding the immunogenic properties of partially decellularized tracheal grafts (PDTG) is a critical step toward clinical translation. Here, we determined that tracheal allograft immunogenicity results in epithelial cell sloughing and replacement with dysplastic columnar epithelium and that partial decellularization creates grafts that are able to support an epithelium without histologic signs of rejection. Moreover, allograft implantation elicits CD8+ T-cell infiltration, a mediator of rejection, while PDTG did not. Hence, we establish that partial decellularization eliminates allograft immunogenicity while creating a scaffold for implantation that can support spatially appropriate airway regeneration.

De-Epithelialization Protocol with Tapered Sodium Dodecyl Sulfate Concentrations Enhances Short-Term Chondrocyte Survival in Porcine Chimeric Tracheal Allografts

Background: Tracheal transplantation is indicated in cases where injury exceeds 50% of the organ in adults and 30% in children. However, transplantation is not yet considered a viable treatment option partly due to high morbidity and mortality associated with graft rejection. Recently, decellularization (decell) has been explored as a technique for creating bioengineered tracheal grafts. However, risk of post-operative stenosis increases due to the death of chondrocytes, which are critical to maintain the biochemical and mechanical integrity of tracheal cartilage. In this project, we propose a new de-epithelialization protocol that adequately removes epithelial, mucosal, and submucosal cells while maintaining a greater proportion of viable chondrocytes. Methods: The trachea of adult male outbred Yorkshire pigs were extracted, decontaminated, and decellularized according to the original and new protocols before incubation at 37 °C in DMEM for 10 days. Chondrocyte viability was quantified immediately following post-decellularization and on days 1, 4, 7, and 10. Histology was performed pre-decellularization, post-decellularization, and post-incubation. Results: The new protocol showed a significant (p &lt; 0.05) increase in chondrocyte viability up to four days after de-ep when compared to the original protocol. We also found that the new protocol preserves ECM composition to a similar degree as the original protocol. When scaffolds created using the new protocol were re-epithelialized, cell growth curves were near identical to published data from the original protocol. Conclusion: While not without limitations, our new protocol may be used to engineer chimeric tracheal allografts without the need for cartilage regeneration.

Critical Care and Postoperative Management of the First Human Long Segment Orthotopic Tracheal Transplantation.

The recipient required reconstruction of a long segment tracheal defect from a previous prolonged intubation. A male donor was chosen for a female recipient to enable analysis of the reepithelialization kinetics using fluorescence in situ hybridization to analyze the source of the new ciliated epithelium. Transplant ICU at the Mount Sinai Hospital, New York, NY. The female recipient was previously intubated for an asthma exacerbation and subsequently developed long segment tracheal stenosis and failed conventional management including dilatation, stenting, and six major surgical procedures rendering her chronically tracheostomy-dependent. The male donor suffered a massive subarachnoid hemorrhage and was subsequently pronounced brain dead. Organ procurement occurred after obtaining appropriate consent from the patient's family. The patient received a deceased donor tracheal allograft that included the thyroid gland, parathyroid glands, and the muscularis of the cervical and thoracic esophagus. Triple therapy immunosuppression (tacrolimus, mycophenolate mofetil, and a corticosteroid taper) was maintained. The patient was initially managed postoperatively with deep sedation on ventilator via armored/reinforced endotracheal tube placed through a small tracheostomy located along the superior tracheal anastomosis. Serial bronchoscopies were performed for graft assessment, pulmonary toilet, and biopsies, which initially showed acute inflammatory changes but no features of acute allograft rejection. A euthyroid state was maintained but hypercalcemia developed. The ICU management of this first long segment orthotopic tracheal transplant required a multidisciplinary approach involving critical care, otolaryngology, transplant surgery, interventional pulmonary, endocrinology, 1:1 nursing throughout the recipient's transplant ICU stay, and respiratory therapy that resulted in the successful establishment of a viable tracheal airway and heralded the end of chronic tracheostomy dependence.

Whole-organ deceased donor tracheal allograft transplantation for the treatment of refractory tracheal stenosis: a case report

Tracheal stenosis involving &gt;5 cm of trachea is debilitating for patients, impedes their quality of life and may even become life-threatening. Large, open defects often result in these patients because of multiple failed tracheal resections and endoscopic dilatations. Tracheal transplantation has been proposed as a curative solution, but revascularization of the trachea as an allograft has generally been considered prohibitively challenging due to a tenuous blood supply. We report a multidisciplinary effort resulting in the first vascularized, single-stage, deceased donor tracheal allograft transplantation in a woman with long-segment tracheal stenosis.

Nintedanib reduces alloimmune-induced chronic airway changes in murine tracheal allografts

BackgroundThe major obstacle for long-term survival after successful lung transplantation is the development of bronchiolitis obliterans (BO) which is one phenotype of chronic lung allograft dysfunction (CLAD). Nintedanib has beneficial effects treating neoplastic diseases and idiopathic pulmonary fibrosis by blocking tyrosine kinase receptors. These receptors play an important role in alloimmune-mediated proliferative diseases. The aim of this study was to determine the effect of nintedanib on proliferative airway changes after orthotopic trachea transplantation in mice. MethodsC57BL/6 mice (H-2b) donor tracheas were orthotopically transplanted into CBA/J mice (H-2k). After transplantation, recipients were daily treated with nintedanib (60 mg/kg; p.o.). Histological and immunofluorescence analysis were performed after 30 days and intragraft gene expression measurements after 14 days of treatment, respectively. ResultsTracheal allografts from mice treated with nintedanib showed significantly less features of chronic rejection than untreated allografts reflected in a higher epithelium/lamina propria ratio (ELR) [ELR: 0.65 ± 0.13 nintedanib vs. 0.50 ± 0.07 untreated controls; p < 0.05] and a reduced submucosal smooth muscle actin (SMA) content [SMA: 1.26% ± 0.78% nintedanib vs. 2.18% ± 1.01% untreated controls; p < 0.01]. Furthermore, lower T cell, macrophage and dendritic cell infiltration was detected in the nintedanib treated grafts. The protein and intragraft mRNA expression of receptor subtypes was considerably decreased in grafts of nintedanib treated mice. The mRNA expression of relevant immune mediators was affected by nintedanib treatment. ConclusionReceptor blocking by nintedanib reduced alloimmune-induced inflammation and chronic airway changes in mouse trachea allografts and might be a promising approach to diminish the development of BO in lung transplants.

Bioinspired carbon monoxide delivery using artificial blood attenuates the progression of obliterative bronchiolitis via suppression of macrophage activation by IL-17A

Carbon monoxide (CO) is expected to attenuate the progression of obliterative bronchiolitis (OB), which is a serious complication after lung transplantation. However, issues in terms of feasible exogenous CO supply, such as continuousness and safety, remain unsolved. Here, we applied nano red blood cells, namely hemoglobin vesicles (Hb-V), as a CO cargo based on the biomimetic concept and investigated the therapeutic potential of CO-loaded Hb-V on OB in orthotopic tracheal transplant model mice. The CO-loaded Hb-V was comprised of negatively charged liposomes encapsulating carbonylhemoglobin with a size of ca. 220 nm. The results of histological evaluation showed that allograft luminal occlusion and fibrosis were significantly ameliorated by treatment with CO-loaded Hb-V compared to treatment with saline, cyclosporine, and Hb-V. The therapeutic effects of CO-loaded Hb-V on OB were due to the suppression of M1 macrophage activation in tracheal allografts, resulting from decreased IL-17A production. Furthermore, the expression of TNF-α and TGF-β in tracheal allografts was decreased by CO-loaded Hb-V treatment but not saline and Hb-V treatment, indicating that CO liberated from CO-loaded Hb-V inhibits epithelial-mesenchymal transition. These findings suggest that CO-loaded Hb-V exerts strong therapeutic efficacy against OB via the regulation of macrophage activation by IL-17A and TGF-β-driven epithelial-mesenchymal transition.

Inhibition of Vascular Endothelial Growth Factor Receptors 1 and 2 Attenuates Natural Killer Cell and Innate Immune Responses in an Experimental Model for Obliterative Bronchiolitis

Obliterative bronchiolitis (OB) after lung transplantation is a nonreversible, life-threatening complication. Herein, the role of vascular endothelial growth factor receptor (Vegfr)-1 and -2 was investigated in the development of obliterative airway disease (OAD), an experimental model for OB. The nonimmunosuppressed recipients underwent transplantation with fully major histocompatibility complex mismatched heterotopic tracheal allografts and received Vegfr1 and -2-specific monoclonal antibodies either alone or in combination, or rat IgG as a control. The treatment with Vegfr1- or -2-blocking antibody significantly decreased intragraft mRNA expression of natural killer cell activation markers early after transplantation. This was followed by reduced infiltration of Cd11b+ cells and Cd4+ T cells as well as down-regulated mRNA expression of proinflammatory chemokines and profibrotic growth factors. However, blocking of both Vegfr1 and -2 was necessary to reduce luminal occlusion. Furthermore, concomitant inhibition of the calcineurin activation pathway almost totally abolished the development of OAD. This study proposes that blocking of Vegf receptors blunted natural killer cell and innate immune responses early after transplantation and attenuated the development of OAD. The results of this study suggest that further studies on the role of Vegfr1 and -2 blocking in development of obliterative airway lesions might be rewarding.

Recipient bone marrow-derived IL-17 receptor A-positive cells drive allograft fibrosis in a mouse intrapulmonary tracheal transplantation model

IL-17A is implicated in the pathogenesis of chronic lung allograft dysfunction, which limits survival after lung transplantation. While many cells express the IL-17 receptor A (IL-17RA) which is the main receptor for IL-17A, the cellular targets of IL-17A in development of post-transplant fibrosis are unknown. The purpose of this study was to determine whether IL-17RA expression by donor or recipient structural or bone marrow (BM) cells is required for the development of allograft fibrosis in a mouse intrapulmonary tracheal transplantation (IPTT) model. BM chimeras were generated using C57BL/6 and IL-17RA-knockout mice. After engraftment, allogeneic IPTTs were performed using the chimeric and BALB/c mice as donors or recipients. This allowed us to assess the effect of IL-17RA deficiency in recipient BM, recipient structural, donor BM, or donor structural compartments separately. Tracheal grafts, the surrounding lung, and mediastinal lymph nodes were assessed 28 days after IPTT. Only recipient BM IL-17RA deficiency resulted in attenuation of tracheal graft obliteration. In the setting of recipient BM IL-17RA deficiency, T cells and neutrophils were decreased in mediastinal lymph nodes. Additionally, recipient BM IL-17RA deficiency was associated with increased B220+PNAd+ lymphoid aggregates, consistent with tertiary lymphoid organs, in proximity to the tracheal allograft. In this IPTT model, recipient BM-derived cells appear to be the primary targets of IL-17RA signaling during fibrotic obliteration of the tracheal allograft.

In vivo performance of decellularized tracheal grafts in the reconstruction of long length tracheal defects: Experimental study

The repair of long-segment tracheal lesions remains an important challenge. Nowdays no predictable and dependable substitute has been found. Decellularized tracheal scaffolds have shown to be a promising graft for tracheal transplantation, since it is non-immunogenic. Evaluate in vivo decellularized tracheal allografts performance to replace long tracheal segment. Forty-five swines underwent surgery as follows: Fifteen trachea donors and 30 receptors of decellularized trachea allografts. The receptors were randomly divided in five groups (n = 6). In groups I and II, donor trachea segment was decellularized by 15 cycles with sodium deoxycholate and deoxyribonuclease, in group II, the allograft was reinforced with external surgical steel wire. Groups, III, IV, and V decellularization was reduced to seven cycles, supplemented with cryopreservation in group IV and with glutaraldehyde in group V. A 10 rings segment was excised from the receptor swine and the decellularized trachea graft was implanted to re-establish trachea continuity. Both decellularization cycles caused decreased stiffness. All trachea receptors underwent euthanasia before the third post-implant week due to severe dyspnea and trachea graft stenosis, necrosis, edema, inflammation, hemorrhage, and granulation tissue formation in anastomotic sites. Histologically all showed total loss of epithelium, separation of collagen fibers, and alterations in staining. Both decellularization techniques severely damaged the structure of the trachea and the extracellular matrix of the cartilage, resulting in a no functional graft, in spite of the use of surgical wire, cryopreservation or glutaraldehyde treatment. An important drawback was the formation of fibrotic stenosis in both anastomosis.

The History of Engineered Tracheal Replacements: Interpreting the Past and Guiding the Future.

The development of a tracheal graft to replace long-segment defects has thwarted clinicians and engineers alike for over 100 years. To better understand the challenges facing this field today, we have consolidated all published reports of engineered tracheal grafts used to repair long-segment circumferential defects in humans, from the first in 1898 to the most recent in 2018, totaling 290 clinical cases. Distinct trends emerge in the types of grafts used over time, including repair using autologous fascia, rigid tubes of various inert materials, and pretreated cadaveric allografts. Our analysis of maximum clinical follow-up, as a proxy for graft performance, revealed that the Leuven protocol has a significantly longer clinical follow-up time than all other methods of airway reconstruction. This method involves transplanting a cadaveric tracheal allograft that is first prevascularized heterotopically in the recipient. We further quantified graft-related causes of mortality, revealing failure modes that have been resolved, and those that remain a hurdle, such as graft mechanics. Finally, we briefly summarize recent preclinical work in tracheal graft development. In conclusion, we synthesized top clinical care priorities and design criteria to inform and inspire collaboration between engineers and clinicians toward the development of a functional tracheal replacement graft.Impact statementThe field of tracheal engineering has floundered in recent years due to multiple article retractions. However, with recent advances in biofabrication and tissue analysis techniques, the field remains ripe for advancement through collaboration between engineers and clinicians. With a long history of clinical application of tracheal replacements, engineered tracheas are arguably the regenerative technology with the greatest potential for translation. This work describes the many phases of engineered tracheal replacements that have been applied in human patients over the past 100 years with the goal of carrying forward critical lessons into development of the next generation of engineered tracheal graft.

Overexpression of the MSK1 Kinase in Patients With Chronic Lung Allograft Dysfunction and Its Confirmed Role in a Murine Model.

Chronic lung allograft dysfunction (CLAD) and its obstructive form, the obliterative bronchiolitis (OB), are the main long-term complications related to high mortality rate postlung transplantation. CLAD treatment lacks a significant success in survival. Here, we investigated a new strategy through inhibition of the proinflammatory mitogen- and stress-activated kinase 1 (MSK1) kinase. MSK1 expression was assessed in a mouse OB model after heterotopic tracheal allotransplantation. Pharmacological inhibition of MSK1 (H89, fasudil, PHA767491) was evaluated in the murine model and in a translational model using human lung primary fibroblasts in proinflammatory conditions. MSK1 expression was graded over time in biopsies from a cohort of CLAD patients. MSK1 mRNA progressively increased during OB (6.4-fold at D21 posttransplantation). Inhibition of MSK1 allowed to counteract the damage to the epithelium (56% restoration for H89), and abolished the recruitment of MHCII+ (94%) and T cells (100%) at the early inflammatory phase of OB. In addition, it markedly decreased the late fibroproliferative obstruction in allografts (48%). MSK1 inhibitors decreased production of IL-6 (whose transcription is under the control of MSK1) released from human lung fibroblasts (96%). Finally, we confirmed occurrence of a 2.9-fold increased MSK1 mRNA expression in lung biopsies in patients at 6 months before CLAD diagnosis as compared to recipients with stable lung function. These findings suggest the overall interest of the MSK1 kinase either as a marker or as a potential therapeutic target in lung dysfunction posttransplantation.

Microvasculature in murine tracheal allografts after combined therapy with clopidogrel and everolimus.

Survival after lung transplantation is mainly limited by the development of chronic lung allograft dysfunction. Previous studies have suggested T-cell mediated proliferation and microvascular changes in experimental small airways models as potential therapeutic targets. The aim of this study was to assess microvascular changes in murine orthotopic tracheal allografts after treatment with everolimus alone or in combination with clopidogrel. C57Bl/6 (H-2b) donor tracheas were orthotopically transplanted into CBA (H-2k) recipients. Mice received daily injections of everolimus (0.05 mg/kg) alone or combined with clopidogrel (1 mg/kg). Twenty-eight days after transplantation, ratio of the thickness of tracheal epithelium and lamina propria was measured as an indicator for chronic rejection. Additionally, graft oxygenation and graft perfusion were detected on postoperative days 4, 10 and 28. Quantitative reverse transcription polymerase chain reaction analysis was used for gene expression analysis. While syngeneic grafts showed a stable tissue pO2 and undisturbed microvascular perfusion, rejecting allografts had a drastic decline in both parameters as well as a flattened epithelium and an increased thickness of the lamina propria. Treatment with everolimus reduced allogeneic fibroproliferation, but had no protective effects on the microvasculature; polymerase chain reaction analysis indicated hypoxic stress and inflammation. Combining everolimus with clopidogrel improved microvascular integrity in the tracheal grafts, but had no synergistic effect in preventing obliterative bronchiolitis development. These data demonstrate that the ability of everolimus to reduce the development of post-transplant obliterative bronchiolitis is not caused by microvascular protection and has no synergistic effects with clopidogrel in acute airway rejection.

Mechanical properties of de-epithelialized tracheal allografts.

BackgroundMany studies have shown that mucosa-treated tracheas can be used for allotransplantation without the use of immunosuppressants. However, no literature has reported whether the transplanted trachea has normal biomechanics. In this study, fresh dog trachea was used as a control to determine the mechanical properties of allograft trachea after mucous membrane removal.MethodsCervical tracheas (~10 cm in length, consisting of the cartilage rings down to the tracheal carina) and the surrounding connective tissues were removed from 20 mixed-breed dogs and then divided into 4 groups with 5 samples in each group. The groups were defined as follows: group A, fresh canine tracheas without any treatment; group B, canine tracheas after sodium dodecyl sulphate (SDS) de-epithelialization; group C, canine tracheas implanted into the dorsal fascia of beagle dogs for 1 month after SDS de-epithelialization; group D, canine tracheas implanted into the dorsal fascia of beagle dogs for 6 months after SDS de-epithelialization. All heterotopically implanted beagle dogs were free of any immunosuppressive treatment. Uniaxial tensile (n=20) and compressive (n=20) experiments were performed on the 4 groups of trachea using a universal testing machine; the tensile load was recorded at 150% of the original tracheal length (50% displacement), and the compressive load was recorded at 50% of the initial anteroposterior diameter (50% displacement). The average difference in tensile and compressive loads between groups was compared by analysis of variance.ResultsNo significant difference was found in the mean compressive strength among groups (group A =2.38±0.87 N, group B =3.33±1.03 N, group C =5.39±2.44 N, group D =3.04±2.12 N; P=0.07 for all groups). The mean tensile strength of group C was 9.68±0.86 N, resulting in a significant difference in tensile strength compared to group A (22.51±3.91 N) and B (24.24±7.46 N) (P<0.001). No significant difference in tensile strength was observed among groups A, B, and D.ConclusionsThe de-epithelialized trachea showed no significant change in mechanical properties after 6 months of implantation.

Evaluation of Antigenicity of Components of Tracheal Allotransplant and Effect of Immunosuppressant Regime in a Rodent Model.

Background Tracheal transplantation seems to be the logical step in the process of reconstruction of the trachea following a long-segment resection, which is usually done to treat malignant disease or benign stenosis of the airway caused by a traumatic, congenital, inflammatory, or iatrogenic lesion. Immunosuppression following transplant is essential but not ideal after oncoresection. Methods The tracheal allografts, harvested from Sprague Dawley rats, were implanted in the Wistar strain rat. The harvested tracheal grafts were divided into groups and subgroups, based on the layers of trachea, method of decellularization, and immunosuppression. The antigenicity of different layers of trachea and the effect of various decellularization methods were studied within three time frames, that is, day 3, 9, and 15. Result On structural analysis, the day 3 and day 15 samples showed no meaningful comparison could be made, due to extensive neutrophil infiltration in all three layers. The day 9 tracheal grafts showed loss of epithelium, with no signs of regeneration in most of the allografts. The subepithelial lymphoid infiltration was found to be severe in nonimmunosuppressed allografts. The group in which both inner and outer layers were removed showed moderate-to-severe infiltrate of lymphoid cells in all the allografts, but there was no cartilage loss, irrespective of the method of decellularization. The irradiated specimens retained the cartilage but showed extensive ischemic damage. Conclusion Rat trachea is a good model for tracheal transplant research but not adequately sturdy to sustain mechanical debridement. Irradiation and chemical decellularization eliminates the immune response but causes intense ischemic damage. Out of the three time frames, day 9 seemed to be the best to study the immune response. To substantiate the results obtained in this study, the immunohistochemical study of the allografts is needed to be performed among a larger group of animals.