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Original Research: NEOPLASTIC DISEASE |

Tracheal Replacement With Cryopreserved Allogenic Aorta FREE TO VIEW

Demosthènes Makris, MD, PhD; Muriel Holder-Espinasse, MD, PhD; Alain Wurtz, MD; Agathe Seguin, MD; Thomas Hubert, DVM, PhD; Sophie Jaillard, MD, PhD; Marie Christine Copin, MD, PhD; Ramadan Jashari, MD; Martine Duterque-Coquillaud, PhD; Emmanuel Martinod, MD, PhD; Charles-Hugo Marquette, MD, PhD
Author and Funding Information

From the Critical Care Department (Dr Makris), University Hospital Larisa, University of Thessaly, Larisa, Greece; JE2490 Institut de Médecine Prédictive et de Recherche Thérapeutique (Drs Hubert and Marquette), IFR 114 Université de Lille II, Lille, France; UMR8161 CNRS (Drs Holder-Espinasse and Duterque-Coquillaud), Institut de Biologie de Lille, Université Lille Nord de France, Lille, France; Service de Chirurgie Thoracique (Dr Wurtz) and Pôle de Pathologie (Dr Copin), CHRU de Lille, Lille, France; Laboratoire d’Etude des Greffes et Prothèses Cardiaques (Drs Seguin and Martinod), Hôpital Broussais, Université Paris 6, Paris, France; Département de Chirurgie (Dr Jaillard), Polyclinique du Bois, Lille, France; European Homograft Bank (Dr Jashari), Brussels, Belgium; Service de Chirurgie Thoracique et Vasculaire (Dr Martinod), Hôpital Avicenne, Bobigny, France; and INSERM ER1-21 (Dr Marquette), University of Nice Sophia Antipolis, Nice, France.

Correspondence to: Charles-Hugo Marquette, Service de Pneumologie, Hôpital Pasteur, Centre Hospitalier Universitaire de Nice, 30 avenue de la voie Romaine, BP 1069, 06002, NICE, cedex 1 France; e-mail: marquette.ch@chu-nice.fr


Funding/Support: This work was supported by the Agence de la Biomédecine (Saint Denis la Plaine, France) and by the RESPIR Foundation (Lille, France).

Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (www.chestjournal.org/site/misc/reprints.xhtml).


© 2010 American College of Chest Physicians


Chest. 2010;137(1):60-67. doi:10.1378/chest.09-1275
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Background:  Radical resection of primary tracheal tumors may be challenging when more than one-half of the tracheal length is concerned. The present study evaluated the use of cryopreserved aortic allografts (CAAs) to replace long tracheal segments.

Methods:  Sixteen adult minipigs underwent tracheal replacement with a CAA. A silicone stent was used to splint the CAA for the first 12 months. Animals were followed-up using bronchoscopic evaluation and killed at predetermined times, for a period up to 18 months long.

Results:  Intense inflammation and progressive disappearance of typical histologic structures of the aorta were seen within the first 3 months. All animals studied for more than 3 months showed progressive transformation of the graft into a chimerical conduit sharing aortic and tracheal histologic patterns (eg, islands of disorganized elastic fibers/mature respiratory ciliated epithelium, respiratory glands, islets of cartilage). Stent removal was attempted after 12 months in 10 animals, and critical tracheal stenosis was found in six animals and moderate asymptomatic stenosis in four. Clinical course in these latter animals was uneventful until they were killed at 15 to 18 months. In situ hybridization showed that collagen2a1 mRNA was expressed in the cartilage islets at 1 year. Polymerase chain reaction analysis of the SRY gene demonstrated that the newly formed cartilage cells derived from the host.

Conclusions:  CAA may be considered as a valuable tracheal substitute for patients with extensive tracheal tumors. Prolonged stenting will be probably mandatory for the clinical application of the procedure in humans.

Figures in this Article

Radical resection of primary tracheal tumors is recommended but may be challenging when more than one-half of the tracheal length is concerned.1,2 Although selected reports suggest that some primary tracheal tumors may respond to chemoradiation,3 only palliative treatments are currently available for long-segment tracheal tumors.4-6

Experimental studies have recently shown that the replacement of both extrathoracic and intrathoracic trachea using fresh allogenic aortic allograft is feasible and may overcome the shortcomings of previous methods applied in central airway replacement.7,8 Fresh allogenic aortas can be placed as long-segment grafts in the trachea, providing a scaffold to support cellular ingrowth of recipient progenitor cells participating in guided tissue regeneration. The technique was initially demonstrated in sheep,7 and results were secondarily confirmed in another mammal (minipig).8 These consistent results led to the performance of tracheal replacement using fresh aortic allograft for salivary gland-type tumors in humans.9 However, questions remain concerning the exact mechanisms of airway regeneration and the outcome of this technique in the long-term.10 Furthermore, it is important to verify whether tracheal regeneration could be reproduced within readily available cryopreserved aortic allografts (CAAs).

In the present investigation, we therefore evaluated the replacement of trachea with CAA in a mammal. We examined morphohistologic changes in the graft over a period of 18 months and assessed cellular ingrowths using in situ hybridization. Prior to considering tracheal replacement in other patients, it was essential to verify whether tracheal regeneration could be reproduced within readily available CAA, and the present experimentation was conducted to address this question.

Experimental Design

Large thoracic (n = 13) or cervical (n = 3) tracheal replacements with CAA were performed in 16 male nonsyngeneic adult minipigs (Pannier SA; Wylder, France) weighing 25 to 30 kg. Tracheal size in adult minipigs is equivalent to that of a human adult, permitting bronchoscopy using the same instruments as used in humans.11,12 Animals received care in accordance to French regulations and institutional ethical committee guidelines for animal research, and were housed in our institution at the University Hospital Department of Experimental Research.

Harvest and Cryopreservation of the CAA

A 10-cm-long segment of the descending thoracic aorta was harvested under general anesthesia through a left thoracotomy in 25 female, large, white-landrace piglets weighing 35 to 45 kg. Preparation, packaging, labeling, processing, preservation, and storage in vapor of liquid nitrogen ( − 140°C/ − 170°C) of CAA were performed according to the European Homograft Bank (Brussels, Belgium) manual of procedures for allografts processing.13 Bacteriologic and mycologic controls were performed in all steps of the preparation, and six out of 25 CAAs were discarded after final positive controls.

Anesthesia, Surgical Procedure, and Postoperative Management

Anesthesia, surgical technique, and postoperative management of thoracic and cervical tracheal replacement with fresh aortic allograft in the pig have been previously described.8 Replacements with CAA were performed in a similar way. Thirteen pigs underwent a large thoracic tracheal resection (9-10 rings) and reconstruction, up to the thoracic inlet and down to the main carina, through a right thoracotomy, with minimal dissection of surrounding tissues whenever possible, to facilitate the revascularization process into the graft. Three pigs underwent a large cervical tracheal replacement in a similar way, through cervicotomy combined with partial sternotomy. To prevent airway collapse, the CAA was splinted by means of a 10-cm-long/13-mm-diameter Y silicone stent (Endoxane; Novatech; Aubagne, France) inserted into the lumen of the CAA, under direct bronchoscopic guidance in 14 animals. In order to check whether the stent could be placed without the help of a bronchoscope, a subtotal longitudinal incision of the graft was performed in the latter two animals undergoing a thoracic replacement. Cross-field stent insertion was carried out, followed by an approximation of the incision with an absorbable monofilament running suture. To avoid postoperative displacement, the stent was fixed proximally and distally to the native trachea with nonabsorbable sutures (Dafilon; Aesculap; Tuttlingen, Germany). All animals were extubated in the recovery room. No immunosuppressive therapy was given at any time.

Follow-up and Bronchoscopy Evaluation

Monitoring was performed daily until the 10th operative day and then monthly. Data on overall status, weight, and respiratory status were collected. Follow-up bronchoscopies were performed as previously described,11,12 to assess graft viability and anastomosis healing at 1, 6, and 12 months or at any other time if the animals showed signs of respiratory distress.

Animal killing, performed under general anesthesia using embutramide, mebezonium, and tetracaine (T61; Intervet; Beaucouzé, France), was scheduled at regular intervals up to 18 months after tracheal replacement for microscopic evaluation and genetic study. Animals showing signs of respiratory distress before the predetermined killing time underwent systematic bronchoscopy and, if abnormalities were present, were killed and underwent postmortem examination.

Histologic Examination

The trachea and main bronchi were carefully excised. Transverse and longitudinal sections were cut at the level of the graft following 2 days of formalin fixation. Additional sections sampled proximally and distally to the grafted aorta served as controls. Specimens were embedded in paraffin, cut into 3-μm sections for slides, and stained with hematoxylin-eosin-saffron (HES), eosin/Alcian blue (for acidic proteoglycans) and elastic fiber stain (Orcein) for microscopic examination.

In Situ Hybridization and Polymerase Chain Reaction Assay

In situ hybridization was performed at 12 to 15 months, as described previously.8 Transplantation of an aorta from a female pig into a male recipient enabled us to analyze the newly formed tissues, especially cartilage, using the polymerase chain reaction (PCR) technique for detection of the SRY gene (attesting to the presence of the Y chromosome), as previously described.8

Clinical Evaluation

Although the pigs did not tolerate apnea for > 1 min, despite preoxygenation, we did not observe peroperative hypoxemic death, and all animals survived the surgical procedure. We did not observe anastomosis dehiscence, fistulation, rejection or necrosis of a graft, or stent migration in this series of animals. Unexpected deaths occurred in three animals at 3, 4, and 7 weeks after the procedure, respectively (Table 1). In pig 1, postmortem examination showed a postobstructive pneumonia resulting from granulation tissue obstructing the right limb of the stent. Pig 2 died in a similar way as the result of obstructive granulation tissue at the edge of the stent. These two cases lead us to systematically use stents with chamfered edges, in order to avoid granulation tissue due to microtrauma coming from sharp stent edges. Pig 3, undergoing a cervical replacement, died of airway collapse, likely due to a too short stent with a lack of proximal anastomosis overlapping. This last case led us to use a Y stent with a vertical limb overlapping the proximal suture by at least 15 mm in the rest of the animals undergoing a cervical tracheal replacement.

Table Graphic Jump Location
Table 1 —Clinical and Pathologic Findings of Animals That Underwent Tracheal Replacement with CAAs

NA = not assessed.

a 

Not assessed at these early times.

b 

Extrathoracic tracheal replacement.

c 

Reduction of the airway lumen by more than 50% or presence of dyspnea.

d 

Reduction of the airway lumen by less than 30% and clinically asymptomatic.

At 3 months, both animals (pigs 4 and 5) whose grafts were longitudinally incised underwent stent removal as scheduled and were killed for further microscopic examination of the CAA. At this time, frank collapse of the graft was still obvious, as expected. In contrast, the longitudinal suture of the graft was not visible (Table 1).

At 6 months, one animal (pig 6) underwent stent removal as scheduled. At that time, there was still collapse of the CAA, and the animal was killed.

The remaining 10 animals underwent stent removal at 12±1 months. Critical stenosis of the airway that reduced the lumen by more than 50% was observed in six animals (pigs 7, 8, 9, 10, 14, and 15). Four of them (pigs 7, 8, 9, and 10) were killed as scheduled, whereas two of them (pigs 14 and 15) received a new Y stent for 3 additional months and were then killed at 15 months. The remaining four animals (pigs 11, 12, 13, and 16) showed moderate strictures that were clinically asymptomatic. The clinical course was uneventful until they were killed at 15 to 18 months (see Table 1)

Macroscopic Evaluation and Pathologic Findings

Macroscopic examination at 2 months showed a typical whitish graft. Grafts consistently showed a thickened, pink, and flaccid aspect resembling an esophagus up to 6 months. At that time, a cleavage plane could be found between the graft and the surrounding mediastinal tissues. Examination of the grafts, which had been incised longitudinally 3 months before, did not find any sequel of the incision.

After 11 to 13 months, animals showed a neo-trachea, which could be dissected from surrounding mediastinal structures with major difficulties. In most cases, there was a longitudinal contraction of the graft. Typical folds of a longitudinally oriented “posterior membrane” as well as cartilage-like fragments, most of which transversally oriented, could be seen. Cartilage located at the caudal part of the graft was not as regular as normal rings (Fig 1).

Figure Jump LinkFigure 1. Macroscopic view of a trachea 15 months after replacement with a CAA. Left side of the main bronchus (A), right side of the main bronchus (B). Arrows delineate the regenerated trachea. Right arrow also shows the level of the stricture, just above the main carina. CAA = cryopreserved aortic allograft.Grahic Jump Location

At that time, there was evidence for newly formed cartilage. Whereas the trachea in adult minipigs bears a total of 29 to 30 rings and nine to 10 tracheal rings were removed from each animal (Table 1), a median number of 25 (range, 24-29) cartilage rings could be seen. In animals undergoing thoracic tracheal replacement, the proximal anastomosis was difficult to distinguish, whereas the caudal anastomosis showed strictures of varying degrees of severity and stiffness. Animals that underwent cervical tracheal replacement and lived for more than 12 months showed hourglass stenosis; one of them was clinically asymptomatic.

Microscopic examination showed that the typical histologic structure of the aorta progressively disappeared within the first 3 months after transplantation. The aorta was replaced by an extensive inflammatory tissue containing islands of disorganized elastic fibers and covered by scattered spots of metaplastic squamous epithelium on the luminal face of the graft (Fig 2). Later on, at 3 to 6 months postoperatively, the amount of collagen tissue increased, with a persistence of inflammatory cells and islands of elastic fibers. The internal face of the graft was lined with metaplastic surface epithelium. For all pigs that passed 12 months of follow-up, numerous islets of cartilage were evident (Fig 3), while remaining islets of elastic fibers could be identified at the same location. Islets of cartilage were frequently found close to large and thickened vessels in the external part of the graft. Few of the cartilaginous formations showed also osteoblastic activity and osseous metaplasia.

Figure Jump LinkFigure 2. Microscopic view of a trachea 2 months after replacement with a CAA (HES, original magnification ×25). Note islands of disorganized elastic fibers (long arrows) within the fibrosis, lymphoid follicles (°), and vessels (short arrow); lumen (*). HES = hematoxylin-eosin-saffron. See Figure 1 legend for expansion of other abbreviations.Grahic Jump Location
Figure Jump LinkFigure 3. Microscopic view of a neo-trachea 14 months after tracheal replacement (HES, original magnification ×25). The trachea was covered with mature respiratory epithelium (short arrows). Lumen (*) and mature cartilage (long arrow) can be seen as well as residual islands (double arrows) of elastic fibers, which are only observed in the aortic graft. See Figure 2 legend for expansion of the abbreviation.Grahic Jump Location

Surface epithelialization of the graft with mature respiratory ciliated epithelial cells was present in all cases at 12 months, interrupted occasionally by squamous metaplastic epithelium. Notably, formation of respiratory glands was observed in three cases (at 13, 15, and 17 months, respectively) (Fig 4). The lower part of the graft, close to the graft-trachea anastomosis, was characterized by the coexistence of respiratory epithelium and inflammatory and fibrotic elements. Clinical and pathologic findings are summarized in Table 1.

Figure Jump LinkFigure 4. Microscopic view of a neo-trachea 14 months after tracheal replacement (same slide as Fig 3). Lumen (*), mature respiratory ciliated epithelium at the surface (short arrow), cartilage in the deep portion (long arrow), and formation of respiratory glands (°) (HES, original magnification ×25) (A). Respiratory glands (°), lumen (*), and residual elastic fibers (double arrows) (HES, original magnification ×100) (B). See Figure 2 legend for expansion of the abbreviation.Grahic Jump Location
In Situ Hybridization and PCR for the SRY Gene on Trachea and Graft

Because collagen2a1 gene is expressed very early during the cartilage formation, collagen2a1 mRNA in situ hybridization was performed on several aortic graft slides, revealing expression in the cartilage islets at 12 and 15 months after grafting (Fig 5). Gender prediction on SRY gene assay revealed positive results for samples from a male control minipig and negative results for a female control minipig. IGF1 amplification, used as a positive control of the PCR assay, was positive in both controls. SRY gene amplification was positive in aortas from female pigs grafted into male minipigs studied at 7 weeks and 3, 12, and 15 months (Fig 6).

Figure Jump LinkFigure 5. Collagen2a1 mRNA in situ using digoxigenin-labeled antisense riboprobe hybridization revealed expression in the cartilage islets (short arrows) 15 months after grafting. Note coexistence of aortic elastic fibers (long arrows) and cartilage (original magnification ×4 [A]; original magnification ×20 [B]). Both images are from the same slide. See Figure 2 legend for expansion of the abbreviation.Grahic Jump Location
Figure Jump LinkFigure 6. SRY and IGF1 gene amplification on a 2% agarose gel. PHA = phytohemagglutinin.Grahic Jump Location

The present investigation shows that extensive replacement of pig trachea with CAAs has the potential to retain viability and continue functioning for a period of up to 18 months. Epithelial ingrowths and cartilage formation were documented. Furthermore, in the present investigation, CAAs showed no evidence of ischemia or acute rejection, despite the absence of blood or tissue compatibility and without the use of any immunosuppressive agent. These findings suggest that the biologic process observed previously in mammals when the trachea was replaced with fresh aortic allografts7,8 can be reproduced when the trachea is substituted by CAAs.

Previous investigations have shown that replacing the trachea with various viable or nonviable substitutes is challenging, but results have been disappointing.14-17 Despite progress in surgical techniques, tissue typing, and immunogenetic and immunosuppressive therapy, replacement of the trachea by fresh tracheal autografts or allografts has been unsuccessful.18-20 Regeneration of the local blood supply is too slow to nourish the complex tissues of the trachea, and rejection responses are frequent.21-24 Other methods involving tracheal replacement by autogenous tissues, nonviable tissues, or synthetic prostheses have been complicated by necrosis, infection, granulation tissue, or scar formation, and have not led to any safe and practicable solution.25-27 Heterotopic tracheal transplantation with fresh aortic homograft or allografts7,8,28 can overcome the shortcomings of previous techniques. Fresh aortic allografts produce a respiratory conduit that shares fundamental elements of the trachea.7,8

In the present investigation, we used CAAs as a tracheal substitute. Compared with fresh allografts, cryopreserved tissues are readily available in tissue banks and may offer the advantage over fresh allografts of reducing the probabilities for graft rejection.29,30 Despite the potential advantages of cryopreserved allografts, CAAs have been rarely tested as tracheal substitutes. Previous experimental studies conducted in rabbits tested CAAs wrapped by means of the epiploon as a tracheal substitute.31 Neoangiogenesis was observed as soon as day 7, but no relevant conclusions could be drawn because of the very short follow-up (<21 days). In a second study with the CAAs placed within an expanded polytetrafluoroethylene vascular prosthesis, the lack of a revascularization process arising from the surrounding tissues led to graft necrosis; thus, definitive conclusions for the utility of CAAs in central airway replacement could not be drawn in this model.32 In an attempt to determine the best way to preserve the aortic allograft, we recently showed in the sheep model that CAAs transformed into a neo-trachea, whereas decellularized and glutaraldehyde-treated aortic allografts did not.33 Another substantial advantage of the CAA is its bacteriologic safety. Indeed, bacterial or fungal quality controls are hardly compatible with immediate processing and transplantation of fresh grafts, whereas cryopreservation and storage allow such investigations.

The capacity of transformation of a CAA into tracheal tissue in the present study was impressive. Major inflammatory reactions in the graft were observed within the first 3 months. Typical structural aspects of an aorta, including the lamellar structure, progressively vanished with time, while morphologic structures of a neotrachea progressively appeared. At 12 months, the CAA had transformed in a chimerical cylindrical organ with a mature respiratory epithelium, a typical longitudinal posterior membrane, fragments or cartilage, or sometimes well-formed cartilaginous arches, but contained residual islets of disorganized elastic aortic fibers. In a previous investigation in the pig,8 graft epithelial cell ingrowths and cartilage formation was incomplete. This might have raised the question whether epithelial reconstruction was not similar in all mammals. However, the follow-up period was short in that investigation.8 In addition, the use of airway stenting might have delayed the development of mature epithelium. In the present investigation, we confirmed the formation of mature mucociliary epithelium and glands about 12 months after tracheal replacement in the minipig. These findings are in agreement with previous findings where fresh aortic allografts were used in the sheep7 and suggest that these results could be reproduced by using CAAs in other mammals.

The origin of the cells promoting the epithelial and cartilage reconstruction remains unclear.34,35 Bone-marrow-derived mesenchymal stem cells are able to differentiate into fat, bone, cartilage, and other mesenchymal tissues. Recent reports suggest that these cells may be recruited to the lung and participate in the tissue repair processes.36 Moreover, it has recently been shown in a human model that mesenchymal stem-cell-derived chondrocytes can colonize a decellularized donor tracheal scaffold.37

In the present investigation, using the PCR technique for chromosome Y identification in male mammals who received a female aorta, it was demonstrated that the cells present in the CAA were derived from the recipient. At early stages, these cells could be the recipient’s infiltrating mononuclear cells. Later on, most of these cells were probably mesenchymal stem cells, triggered by local signals of differentiation. Indeed, as we showed in a previous report,28 similar results were obtained when using the PCR technique for chromosome Y identification in the cartilage (free of mononuclear cells) and not on the whole neo-trachea. Interestingly, although we documented the formation of complete, mature cartilaginous rings at 12 months after replacement, this phenomenon was almost always present in the central and upper part of the graft. At the lower part of the graft, more irregular cartilaginous islands were present close to large vessels. Thus, airway regeneration is not homogenous along the grafted tissue, nor it is inhibited in the caudal part, possibly as the result of initial ischemia or factors that were not assessed in the present study, leading to moderate or critical, short, hour-glass-shaped airway stenoses very similar to the stenoses seen either after prolonged intubations or after sleeve resection. Based on this analogy, one may hypothesize that the underlying mechanism for the stenoses is ischemia. Such transversal contraction, together with longitudinal contraction, has been described in previous experiments and was considered part of a healing process.38

Our findings also allow us to consider that it is unlikely that neocartilage seen in the neotracheal conduit arises from both edges of the native trachea pulled into the graft by scar contraction, as has been speculated in the past. We speculate that cartilage regeneration follows a pattern related to angiogenesis, which is also present during this process. Further investigations are required to explore the mechanisms that guide and the factors that regulate the development of epithelium and cartilage formation in the walls of the aortic graft. Nevertheless, despite unanswered questions regarding the exact mechanism of the process, regeneration of the epithelium has been well documented in this study, which confirms previous investigations reporting epithelial regeneration after elective destruction of the tracheal epithelium or replacement using homografts or autografts.29,39 In the present study, airway stenting was combined with the technique of tracheal replacement with aortic allograft. This is mandatory to prevent airway collapse of the initially compliant graft.40 In addition, we observed that removal of the stent in this setting may result in an adverse outcome. In this study, the stents were removed approximately 12 months after tracheal replacement, which proved to be too early in the majority of animals. Thus, prolonged stenting will be probably mandatory for the clinical application of the procedure in humans. Furthermore, the occurrence of lower graft stenosis observed in animals is another concern, and in this respect, the technique in humans will require close follow-up.

In conclusion, the present study showed that in pigs, a CAA progressively transforms into a structure resembling tracheal tissue without evidence of acute or late rejection. Tracheal replacement with CAAs may be considered as a potential therapy for extensive tracheal tumors in humans.

Author contributions:Dr Makris: contributed to the performance of the bronchoscopies and drafting the article.

Dr Holder-Espinasse: contributed to performing genetic analysis and reviewing data.

Dr Wurtz: contributed to surgical procedures and reviewing data.

Dr Seguin: contributed to reviewing data.

Dr Hubert: contributed to animal care.

Dr Jaillard: contributed to surgical procedures.

Dr Copin: contributed to pathologic analysis and reviewing data.

Dr Jashari: contributed by performing the whole cryopreserved graft processing.

Dr Duterque-Coquillaud: contributed to genetic analysis and reviewing data.

Dr Martinod: contributed to reviewing data.

Dr Marquette: contributed to bronchoscopies, reviewing data, and drafting the article.

Financial/nonfinancial disclosures: The authors have reported to CHEST that no potential conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article.

Other contributions: The “minipig custom-made” bifurcated silicone stents were kindly provided by the Novatech Laboratory (Aubagne, France). We acknowledge Dr Yves Goffin from the European Homograft Bank (Brussels, Belgium) for his help in checking the histologic condition of the donor aortas.

CAA

cryopreserved aortic allografts

HES

hematoxylin-eosin-saffron

PCR

polymerase chain reaction

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Martinod E, Seguin A, Pfeuty K, et al. Long-term evaluation of the replacement of the trachea with an autologous aortic graft. Ann Thorac Surg. 2003;755:1572-1578. [CrossRef] [PubMed]
 
Murakawa T, Nakajima J, Motomura N, Murakami A, Takamoto S. Successful allotransplantation of cryopreserved tracheal grafts with preservation of the pars membranacea in nonhuman primates. J Thorac Cardiovasc Surg. 2002;1231:153-160. [CrossRef] [PubMed]
 
Mukaida T, Shimizu N, Aoe M, Andou A, Date H, Moriyama S. Origin of regenerated epithelium in cryopreserved tracheal allotransplantation. Ann Thorac Surg. 1998;661:205-208. [CrossRef] [PubMed]
 
Carbognani P, Spaggiari L, Solli P, et al. Experimental tracheal transplantation using a cryopreserved aortic allograft. Eur Surg Res. 1999;312:210-215. [CrossRef] [PubMed]
 
Feito BA, Rath AM, Kambouchner M, et al. Replacement of a tracheal segment by a mixed graft (aorta and prosthesis): an experimental study in rabbits. Eur J Surg. 1999;16512:1175-1181. [CrossRef] [PubMed]
 
Seguin A, Radu D, Holder-Espinasse M, et al. Tracheal replacement with cryopreserved, decellularized, or glutaraldehyde-treated aortic allografts. Ann Thorac Surg. 2009;873:861-867. [CrossRef] [PubMed]
 
Abedin M, Tintut Y, Demer LL. Mesenchymal stem cells and the artery wall. Circ Res. 2004;957:671-676. [CrossRef] [PubMed]
 
Etheridge SL, Spencer GJ, Heath DJ, Genever PG. Expression profiling and functional analysis of wnt signaling mechanisms in mesenchymal stem cells. Stem Cells. 2004;225:849-860. [CrossRef] [PubMed]
 
Loebinger MR, Janes SM. Stem cells for lung disease. Chest. 2007;1321:279-285. [CrossRef] [PubMed]
 
Macchiarini P, Jungebluth P, Go T, et al. Clinical transplantation of a tissue-engineered airway. Lancet. 2008;3729655:2023-2030. [CrossRef] [PubMed]
 
Pressman JJ, Simon MB. Observations upon the experimental repair of the trachea using autogenous aorta and polyethylene tubes. Surg Gynecol Obstet. 1958;1061:56-62. [PubMed]
 
Letang E, Sánchez-Lloret J, Gimferrer JM, Ramírez J, Vicens A. Experimental reconstruction of the canine trachea with a free revascularized small bowel graft. Ann Thorac Surg. 1990;496:955-958. [CrossRef] [PubMed]
 
Martinod E, Zakine G, Fornes P, et al. Metaplasia of aortic tissue into tracheal tissue. Surgical perspectives [in French]. C R Acad Sci III. 2000;3235:455-460. [CrossRef] [PubMed]
 

Figures

Figure Jump LinkFigure 1. Macroscopic view of a trachea 15 months after replacement with a CAA. Left side of the main bronchus (A), right side of the main bronchus (B). Arrows delineate the regenerated trachea. Right arrow also shows the level of the stricture, just above the main carina. CAA = cryopreserved aortic allograft.Grahic Jump Location
Figure Jump LinkFigure 2. Microscopic view of a trachea 2 months after replacement with a CAA (HES, original magnification ×25). Note islands of disorganized elastic fibers (long arrows) within the fibrosis, lymphoid follicles (°), and vessels (short arrow); lumen (*). HES = hematoxylin-eosin-saffron. See Figure 1 legend for expansion of other abbreviations.Grahic Jump Location
Figure Jump LinkFigure 3. Microscopic view of a neo-trachea 14 months after tracheal replacement (HES, original magnification ×25). The trachea was covered with mature respiratory epithelium (short arrows). Lumen (*) and mature cartilage (long arrow) can be seen as well as residual islands (double arrows) of elastic fibers, which are only observed in the aortic graft. See Figure 2 legend for expansion of the abbreviation.Grahic Jump Location
Figure Jump LinkFigure 4. Microscopic view of a neo-trachea 14 months after tracheal replacement (same slide as Fig 3). Lumen (*), mature respiratory ciliated epithelium at the surface (short arrow), cartilage in the deep portion (long arrow), and formation of respiratory glands (°) (HES, original magnification ×25) (A). Respiratory glands (°), lumen (*), and residual elastic fibers (double arrows) (HES, original magnification ×100) (B). See Figure 2 legend for expansion of the abbreviation.Grahic Jump Location
Figure Jump LinkFigure 5. Collagen2a1 mRNA in situ using digoxigenin-labeled antisense riboprobe hybridization revealed expression in the cartilage islets (short arrows) 15 months after grafting. Note coexistence of aortic elastic fibers (long arrows) and cartilage (original magnification ×4 [A]; original magnification ×20 [B]). Both images are from the same slide. See Figure 2 legend for expansion of the abbreviation.Grahic Jump Location
Figure Jump LinkFigure 6. SRY and IGF1 gene amplification on a 2% agarose gel. PHA = phytohemagglutinin.Grahic Jump Location

Tables

Table Graphic Jump Location
Table 1 —Clinical and Pathologic Findings of Animals That Underwent Tracheal Replacement with CAAs

NA = not assessed.

a 

Not assessed at these early times.

b 

Extrathoracic tracheal replacement.

c 

Reduction of the airway lumen by more than 50% or presence of dyspnea.

d 

Reduction of the airway lumen by less than 30% and clinically asymptomatic.

References

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Martinod E, Seguin A, Pfeuty K, et al. Long-term evaluation of the replacement of the trachea with an autologous aortic graft. Ann Thorac Surg. 2003;755:1572-1578. [CrossRef] [PubMed]
 
Murakawa T, Nakajima J, Motomura N, Murakami A, Takamoto S. Successful allotransplantation of cryopreserved tracheal grafts with preservation of the pars membranacea in nonhuman primates. J Thorac Cardiovasc Surg. 2002;1231:153-160. [CrossRef] [PubMed]
 
Mukaida T, Shimizu N, Aoe M, Andou A, Date H, Moriyama S. Origin of regenerated epithelium in cryopreserved tracheal allotransplantation. Ann Thorac Surg. 1998;661:205-208. [CrossRef] [PubMed]
 
Carbognani P, Spaggiari L, Solli P, et al. Experimental tracheal transplantation using a cryopreserved aortic allograft. Eur Surg Res. 1999;312:210-215. [CrossRef] [PubMed]
 
Feito BA, Rath AM, Kambouchner M, et al. Replacement of a tracheal segment by a mixed graft (aorta and prosthesis): an experimental study in rabbits. Eur J Surg. 1999;16512:1175-1181. [CrossRef] [PubMed]
 
Seguin A, Radu D, Holder-Espinasse M, et al. Tracheal replacement with cryopreserved, decellularized, or glutaraldehyde-treated aortic allografts. Ann Thorac Surg. 2009;873:861-867. [CrossRef] [PubMed]
 
Abedin M, Tintut Y, Demer LL. Mesenchymal stem cells and the artery wall. Circ Res. 2004;957:671-676. [CrossRef] [PubMed]
 
Etheridge SL, Spencer GJ, Heath DJ, Genever PG. Expression profiling and functional analysis of wnt signaling mechanisms in mesenchymal stem cells. Stem Cells. 2004;225:849-860. [CrossRef] [PubMed]
 
Loebinger MR, Janes SM. Stem cells for lung disease. Chest. 2007;1321:279-285. [CrossRef] [PubMed]
 
Macchiarini P, Jungebluth P, Go T, et al. Clinical transplantation of a tissue-engineered airway. Lancet. 2008;3729655:2023-2030. [CrossRef] [PubMed]
 
Pressman JJ, Simon MB. Observations upon the experimental repair of the trachea using autogenous aorta and polyethylene tubes. Surg Gynecol Obstet. 1958;1061:56-62. [PubMed]
 
Letang E, Sánchez-Lloret J, Gimferrer JM, Ramírez J, Vicens A. Experimental reconstruction of the canine trachea with a free revascularized small bowel graft. Ann Thorac Surg. 1990;496:955-958. [CrossRef] [PubMed]
 
Martinod E, Zakine G, Fornes P, et al. Metaplasia of aortic tissue into tracheal tissue. Surgical perspectives [in French]. C R Acad Sci III. 2000;3235:455-460. [CrossRef] [PubMed]
 
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