| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
a Laboratoire de Recherches Biochirurgicales, Fondation Alain Carpentier, Université Paris V, Paris, France
b Assistance Publique-Hôpitaux de Paris, Hôpital Avicenne, Département de Chirurgie Thoracique et Vasculaire, Université Paris XIII, Faculté de Médecine SMH, Bobigny, France
c Institut de Biologie, Lille, France
d Assistance Publique-Hôpitaux de Paris, Hôpital Européen George Pompidou, Département d'Anatomopathologie, Université Paris V, Faculté de Médecine, Paris, France
e Assistance Publique-Hôpitaux de Paris, Hôpital Henri Mondor, EFS Ile de France, Banque de Tissus, Créteil, France
Accepted for publication November 17, 2008.
* Address correspondence to Dr Seguin, Service de Chirurgie Thoracique et Vasculaire, Hôpital Avicenne, 125 route de Stalingrad, Bobigny Cedex, 93009, France (Email: agathe.seguin{at}avc.aphp.fr).
 |
Abstract |
|---|
 Top Abstract IntroductionMaterial and MethodsResultsCommentFootnotesAcknowledgmentsReferences |
|---|
Methods: Twenty-two sheep underwent tracheal replacement using cryopreserved (n = 10), decellularized (n = 7) or glutaraldehyde-treated (n = 5) allografts, supported by a temporary stent to prevent airway collapse. Aortic segments were retrieved at regular intervals up to 12 months after implantation to analyze the regenerative process.
Results: All animals survived the operation. Major complications such as infection, stent migration, or obstruction were predominantly encountered in the decellularized group. The lack of major inflammatory response within the aortic graft observed in the glutaraldehyde group was associated with the absence of tracheal regeneration. Histologic examinations showed a progressive transformation of the aorta into a tracheal tissue comprising respiratory epithelium and cartilage only in the cryopreserved group.
Conclusions: This study demonstrated that regeneration of a functional tissue could be obtained after tracheal replacement with a cryopreserved aortic allograft. The regenerative process followed the same pattern as previously described for fresh allografts. Cryopreserved aortic allografts present major advantages: availability in tissue banks, permanent storage, and no need for immunosuppression. This offers a new field of perspectives for clinical application in patients with extensive tracheal cancer.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterial and MethodsResultsCommentFootnotesAcknowledgmentsReferences |
|---|
In previous experiments, we demonstrated that replacement of the trachea with an aortic autograft or allograft led to the regeneration of a functional neotrachea [2–7]. This induced biologic repair could offer a new therapeutic option for patients with extensive tracheal tumors. To facilitate clinical application, it would be more convenient to use allografts from a tissue bank instead of fresh aortic allografts, as proposed in our previous studies [2–7]. However, confirmation is needed whether aortic transformation into a newly formed trachea would still occur with preserved allografts. Thus, we conducted this study to assess the use of cryopreserved, decellularized, or glutaraldehyde-treated aortic allografts as tracheal substitutes.
The aim of cryopreservation is to maintain cell viability even if some immune responses may occur. Decellularization treatment with sodium dodecylsulfate (SDS) leads to a complete loss of cellular structures from the three layers of the arterial wall, with conservation of the main component of the extracellular matrix [8]. Therefore, the aortic decellularized allograft acts as a biologic scaffold. Glutaraldehyde treatment protects the arterial wall from cell penetration and limits the inflammatory reaction by the cross-linking of collagen. Moreover, the use of these three methods of preservation, modulating inflammatory response, and aortic cells presence constitutes a good model to study the regenerative process involved.
|
Material and Methods |
|---|
|
TopAbstractIntroductionMaterial and MethodsResultsCommentFootnotesAcknowledgmentsReferences |
|---|
Cryopreservation protocol: Aortic segments to be cryopreserved (n = 10) were harvested and transported at 4°C in a Euro-Collins solution containing antimicrobial agents (vancomycin, clindamycin, and gentamicin). The same day, under sterile conditions, a 4% dimethylsulfoxide and albumin solution was added, and the cryopreservation process was conducted up to –80°C (n = 5) or –150°C (n = 5).
Decellularization protocol: Aortic segments to be decellularized (n = 7) were incubated for 15 hours at 37° in distilled water with 0.1% SDS, with gentle stirring. The SDS-treated grafts were rinsed five times in 0.9% saline solution with gentle stirring and then frozen at –20°C. Cell removal was verified by examination of fixed sections by light and electron microscopy. The detailed protocol is presented in Table 1.
|
Experimental Design
The study used 22 sheep that weighed 25 to 30 kg and were a mean age of 8 months. All animals received care in compliance with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Research Councils; National Academy Press, revised 1996). Animals were randomly assigned to one of the three types of tracheal replacement: group 1, cryopreserved aortic allograft (n = 10); group 2, decellularized aortic allograft (n = 7); and group 3, glutaraldehyde-treated aortic allograft (n = 5).
Anesthesia
Anesthesia was induced with intravenous propofol (1%, 8 mg/kg). After endotracheal intubation, ventilation was performed with a Siemens 900C ventilator (Siemens Medical Systems Inc, Iselin, NJ), with tidal volume of 10 mL/kg and 24 breaths/min. Anesthesia was maintained with inhaled 60% oxygen and 1% to 2% isoflurane. All animals were perfused with a crystalloid solution (10 mg/kg/h).
Surgical Procedure
The cervical trachea was exposed through a median cervical incision. A 7-cm circular segment of the cervical trachea was resected. After insertion of a cross-field endotracheal tube into the distal tracheal segment to maintain ventilation, the aortic allograft was interposed. Distal intubation was used. The proximal end-to-end anastomosis and the posterior wall of the distal end-to-end anastomosis were made with running 4-0 polydioxanone suture (PDS; Ethicon Inc, Sommerville, NJ).
In all animals, a silicone Tracheobronxane Dumon (Novatech, La Ciotat, France) stent (length, 10 cm; diameter, 15 mm) was placed in the lumen of the aortic graft under direct vision to prevent collapse of the new airway. After removal of the cross-field ventilation tube, the endotracheal tube was guided through the stented aortic allograft and inserted into the distal trachea. The distal anastomosis was then completed. A myoplasty was done to protect the graft and guide the vascularization process. The cervical incision was closed without drainage.
Clinical Evaluation
Once awake, the animals were immediately extubated. They received a daily intramuscular injection of 1 g of cefazolin for the first 2 postoperative days. No immunosuppressive therapy was given. Clinical examination was performed daily until operative day 10, then monthly. Data on overall status, weight, and respiratory status were collected. Postoperative evaluation included fiberscopic examination (band pass filter 40, 10 mm, Olympus, Rungis, France) under heavy sedation using propofol at 1, 3, 6, and 12 months to assess the patency of the graft.
Histologic Evaluation
Animal euthanasia was scheduled at 1, 3, 6, and 12 months, with an intravenous injection of potassium chloride and thiopental sodium, to explant the grafts. En bloc resection of the trachea with surrounding tissues was performed, and specimens underwent macroscopic and microscopic examination. After removal of the stent and macroscopic evaluation of the graft, the specimens were immediately placed in a 10% formaldehyde solution. Specimens were embedded in paraffin and cut into 4-µm slides, from the proximal native trachea to the distal native trachea (longitudinal slides). Additional transversal slides were made at each anastomosis and in the middle of the graft. The slides were stained with hematoxylin-eosin and saffron (HES) and elastic fiber stain (orcein) for microscopic examination. Special attention was given to microscopic evidence of inflammation and to morphologic transformation of the graft.
|
Results |
|---|
|
TopAbstractIntroductionMaterial and MethodsResultsCommentFootnotesAcknowledgmentsReferences |
|---|
|
Group 1 (cryopreservation group, n = 10): Two animals experienced complications during the follow-up period. The first died from stent migration and airway collapse at 2.5 months. The second animal was euthanized at 2 months for thymic abscess. No anastomotic leakage was found within the graft.
Group 2 (decellularization group, n = 7): Three animals died of local infection with graft leakage and cervical abscess at 2 months. One died at 4 months without explanation on necropsy examination. One animal died of distal stent obstruction by a granuloma at 5 months.
Group 3 (glutaraldehyde treatment group, n = 5): Two animals died of stent migration and airway collapse at 2.5 months.
For the remaining animals in all groups, long-term follow-up showed no complications: animals were in good general status and all gained weight. Tracheal specimens removed at different intervals after the operation (euthanasia or death of the animal) allowed us to monitor the histologic transformation of the grafted aortic tissue. No stenosis was observed among all animals with a silicone stent still present inside the graft (Fig 1).
|
Group 1: At 1 month, an inflammatory reaction was seen within the graft, consisting of a combination of cells (neutrophils and macrophages). At 3 months, the inflammatory reaction was less important, and a nonkeratinizing metaplastic squamous epithelium was observed. At 6 months, the reparation process was completed with the formation of a continuous mucociliary epithelium and immature cartilage (Fig 2). At 12 months, the reparation process was almost achieved. Disorganized elastic fibers were observed within the aortic graft at all stages (Fig 3). A significant contraction, representing about 30% of the total graft length, was observed in all specimens. No difference was noted between the subgroups with cryopreservation at –80°C and –150°C.
|
|
Group 3: No elastic fibers were observed within the graft at all stages. Inflammation was moderate. The aortic graft was replaced by a fibrous vascularized tissue with a squamous interrupted epithelium (Fig 4). A possible island of immature cartilage was observed in only 1 animal at 3 months.
|
|
Comment |
|---|
|
TopAbstractIntroductionMaterial and MethodsResultsCommentFootnotesAcknowledgmentsReferences |
|---|
More recently, efforts have been made to induce the formation of cartilaginous tubes covered with epithelial cells, but this type of tissue engineering has not provided reliable results to date. Finally, tracheal allotransplantation has also been disappointing due to major complications such as necrosis or stenosis of the graft [10, 11]. In addition, immunosuppressive therapy, which is mandatory because of tracheal immunogenicity, does not permit a clinical application in the treatment of cancer.
Tissue-engineered airway and guided tissue regeneration are virtually the only techniques that seem to offer any real promise. A bioartificial trachea with smooth muscle cells, ciliated respiratory epithelium, and extracellular cartilaginous matrix can be engineered on a vascularized scaffold, but still lacks the three-dimensional aspect and the biomechanical profile of a functioning trachea [12, 13].
In previous experiments, we reported that an aortic segment could be a valuable tracheal substitute for periods up to 3 years [2, 3]. We first investigated living autologous aortic conduit to avoid immunologic reactions. Instead of the expected successful grafting of the aorta, we actually found an inflammatory reaction leading to the destruction of the aortic tissue and its progressive replacement by a tracheal structure with mucociliary epithelium and newly formed cartilage. This technique, however, had a limited value in clinical practice because it required that an aortic segment be removed from the patient.
As a consequence, we investigated fresh aortic allograft implanted in the tracheal environment and found that the allograft was replaced after a few months by a newly formed trachea comprising mucociliary epithelium, cartilage rings, and a posterior membrane. No acute or chronic graft rejection was observed. This is consistent with the experience of orthotopic aortic transplantation in which only discrete immunologic reactions were noted despite the absence of immunosuppressive treatment [2–7]. More recently, these results were confirmed in pig model [14].
To explain tracheal regeneration, we hypothesized the participation of stem cells. The traditional view of adult stem cell differentiation believed that stem cell progeny progressed in a linear, irreversible manner that eliminated stem cell propensity and restricted its fate to within a germ line. A new evolving theory of differentiation proposed that stem cell progeny differentiates in a more graded manner, giving rise to more progressively restricted daughter cells that possess transgerm potential. There is precedence for this belief: Clonal strains of marrow adipocytes can be directed to form bone [15], and chondrocytes can dedifferentiate toward the osteogenic lineage [16].
Recent studies confirming the neurogenic potential of mesenchymal stem cells, the induction of hematopoietic stem cells into hepatocytes [17], and the conversion of neurogenic precursors into muscle and blood [18, 19] have contributed to this theory and may be a beginning of a paradigm shift. These observations strongly imply a critical influence of microenvironmental signals on cell fate. Evidence suggests that the pluripotent stem cell, once thought to be restricted to the fates of a lineage hierarchy, is capable of transdifferentiation [20].
Equally exciting is the emerging concept that stem cells may be found in multiple organs, including muscle, heart, liver [21, 22], and lung [23], as well as in tissues, including skin [24], placenta, and fat [25]. Moreover, mesenchymal stem cells reside in virtually all postnatal organs and tissues, as has been shown in a recent research article [26]. These cells act as a reservoir of undifferentiated cells to supply cellular demands of the tissue they belong to, acquiring local phenotypic characteristics. When necessary and after signs from the microenvironment, they give rise to committed progenitors that gradually integrate into the tissue. The model does not exclude the possible existence of other tissue specific stem cells or participation of circulating stem cells. To explain tracheal regeneration, some reports suggest presence of stem cell niches capable of giving rise to a complete mucociliary epithelium in tracheal injury [27]. But there is no report of specific stem cell isolated in the trachea and leading to cartilage regeneration.
In our model, we postulated that the environment has a major effect in inducing tissue transformation, leading to a structure as complex as a trachea with cartilage and epithelium. Using polymerase chain reaction technique and chromosome Y identification in female sheep having received a male aorta, we previously demonstrated that the cartilage cells derived from the recipient [2]. We thus hypothesized that recipient multipotent mesenchymal stem cells triggered by environmental signals colonized the graft and differentiated into chondrocytes.
Even if not entirely explained, this induced tissue transformation may open the way to important clinical applications. In humans, the use of a preserved aortic allograft from a tissue bank instead of a fresh aorta would facilitate the procedure. Therefore, the transformation process of the preserved aortic graft into a newly formed trachea after implantation needed to be confirmed. Experimental evidence indicates that a decellularized matrix becomes populated with functional recipient cells [28, 29]. A tissue-engineered vascular scaffold that recellularizes appropriately has numerous theoretic advantages over nonviable materials, including but not limited to the ability to grow and to repair itself. Unfortunately in our study, we did not observe these promising results. All animals but 2 died of local infection or stent-related complications. As evidenced by the pathologic examination, the aortic graft did not transform into a new trachea. Therefore, in our opinion, decellularized aortic grafts should not be used for clinical application. Decellularization resulted in preservation of the medial elastin network but also suppressed allograft adventitial inflammatory infiltration. We thus hypothesize that cell invasion mediated by a modulated inflammatory reaction is a critical step for the metaplastic process to take place.
Glutaraldehyde treatment also suppresses antigenicity of the grafts. It has been used to prepare valvular heterografts in humans for the past 30 years [30, 31]. Long-term follow-up studies have shown that certain histologic changes in glutaraldehyde-treated grafts impair their function. Nevertheless, because considerable clinical experience has been gained with valvular xenografts, glutaraldehyde remains the reference tanning reagent. In our experiments, a modulated inflammatory response was observed within the graft. No aortic elastic fibers were encountered, and the graft was replaced by a scar tissue.
These results are different from those of valvular experiments where the bioprosthesis maintains its structural appearance for the first months. Here, the aortic graft, heterotopically placed in a tracheal environment, was progressively destroyed and colonized by fibroblasts. Possible islands of cartilage were seen in only 1 animal at 3 months, but not confirmed at 6 months of follow-up. As a consequence, we conclude that the use of glutaraldehyde-treated grafts did not give appropriate results enough for clinical application.
Finally, cryopreservation is the only method preserving the graft like a fresh one, giving us the ability to constitute a tissue bank. Cryopreservation is now widely used as a long-term preservation technique of biologic tissues and seems to be the best method to maintain graft viability [32]. Cryopreservation has also been reported to reduce the immunogenicity of allografts, probably by diminishing viability of endothelial cells. Early results in our experiments showed no evidence of ischemia or acute rejection despite the absence of blood or tissue compatibility, without any immunosuppressive treatment.
As observed in our previous experiments [2–7], the use of cryopreserved aortic grafts allowed continuous epithelialization and cartilage development. Although only few animals could be studied long enough to show cartilage formation, our results are consistent with these experiments. Even if a degree of scar contraction appeared, the 7-cm length of resection reduced the possibility that the neocartilage seen in the graft would arise from the edges of native trachea and be pulled into the graft. We hypothesize that the cryopreserved graft was colonized by recipient multipotent mesenchymal stem cells that were triggered by environmental signals and differentiated into chondrocytes.
Experiments are currently underway to establish the origin of these cells. These experiments have been made in rabbits in which their mesenchymal stem cells have been labelled by a GFP lentivirus to track these specifics cells in vivo. On the basis of experimental studies on sheep and pig models [2, 3, 14], tracheal replacement using aortic allograft was applied in 4 selected patients with extensive tracheal cancer for whom only a palliative treatment was proposed [33]. With a maximal follow-up of 3 years, 3 are still alive, at home, with normal breathing.
As a consequence, a national multicenter study on tracheal replacement using cryopreserved aortic allograft has just started in France (le Projet Hospitalier de Recherche Clinique RTA No 1929) after approval of the ethic committee. Even if we need to remain extremely careful, this represents a real hope for patients with extensive tracheal tumors.
These experiments confirm our first results on tracheal replacement using aortic allograft, which can be sure and valuable. For clinical application, however, it would be interesting to know how to preserve and store the aortic allograft to simplify the protocol. Decellularized allografts and glutaraldehyde-treated grafts seem to give disappointing results and, in our opinion, are not recommended for clinical use. Cryopreserved aortic allografts represent the most promising solution, even if only 1 sheep had 12 months of follow-up.
On the other hand, our experiments permit a better understanding of the phenomena responsible for tracheal regeneration within the aortic allograft. Because the decellularized aortic allograft did not allow regeneration of a tracheal conduit, we believe that the initial presence of donor aortic cells is indispensable for the regeneration to be achieved, possibly by delivering signals when dying, thus leading stem cells to home inside the graft. Alternatively, as previously shown in the sex-mismatch experiments, recipient cells have an essential contribution to this process [2].
Some authors underlined the metaplastic potential of aortic muscle cells. It has been reported that transplanted fresh allogenic rat abdominal aortic grafts developed a cartilaginous metaplasia, whereas transplanted homografts did not [34]. In this view, we believe that the inflammatory response is crucial for the tracheal regeneration to take place. A recent report showed the modulating effect of inflammation on tissue remodeling and regeneration [35]. This is supported by the fact that the use of glutaraldehyde-treated aortic grafts in which no inflammation was noticed led to disappointing results. Other experiments are under investigation to explain the mechanisms of tracheal regeneration. Clinical perspectives are serious and will give hope to patients so far untreated for extensive tracheal lesions. Nevertheless, for the time being, clinical application should be conducted only in the setting of controlled trials.
|
Acknowledgments |
|---|
|
TopAbstractIntroductionMaterial and MethodsResultsCommentFootnotesAcknowledgmentsReferences |
|---|
|
Footnotes |
|---|
|
TopAbstractIntroductionMaterial and MethodsResultsCommentFootnotesAcknowledgmentsReferences |
|---|
|
References |
|---|
|
TopAbstractIntroductionMaterial and MethodsResultsCommentFootnotesAcknowledgmentsReferences |
|---|
Related Article
This article has been cited by other articles:
|
|
 |
|
  M. B. Shadmehr, R. Farzanegan, P. Graili, M. Javaherzadeh, M. Arab, S. Pejhan, M. B. Karam, and A. Abbasidezfouli Primary major airway tumors; management and results Eur J Cardiothorac Surg, May 1, 2011; 39(5): 749 - 754. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Martinod Editorial comment: Primary major airway tumors; management and results Eur J Cardiothorac Surg, May 1, 2011; 39(5): 754 - 755. [Full Text] [PDF]
|
|
|
|
 |
|
 E. Martinod, D. M. Radu, K. Chouahnia, A. Seguin, A. Fialaire-Legendre, P.-Y. Brillet, M.-D. Destable, G. Sebbane, S. Beloucif, D. Valeyre, et al. Human Transplantation of a Biologic Airway Substitute in Conservative Lung Cancer Surgery Ann. Thorac. Surg., March 1, 2011; 91(3): 837 - 842. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Tsukada, S. Gangadharan, R. Garland, F. Herth, M. DeCamp, and A. Ernst Tracheal Replacement With a Bioabsorbable Scaffold in Sheep Ann. Thorac. Surg., December 1, 2010; 90(6): 1793 - 1797. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Martinod Invited Commentary Ann. Thorac. Surg., December 1, 2010; 90(6): 1797 - 1798. [Full Text] [PDF]
|
|
|
|
|
|
C.-H. Marquette and A. Wurtz Tracheal Replacement With Aortic Allografts in the Pig Ann. Thorac. Surg., December 1, 2010; 90(6): 2091 - 2091. [Full Text] [PDF]
|
|
|
|
 |
|
 E. A. Agathos, P. Tomos, E. Lachanas, H. Gakiopoulou, A. Pantopoulou, and D. Perrea Experimental Replacement of Pig Trachea with Novel Bioprosthesis from Harp Seal Asian Cardiovasc Thorac Ann, December 1, 2010; 18(6): 557 - 562. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Wurtz, H. Porte, M. Conti, C. Dusson, J. Desbordes, M.-C. Copin, and C.-H. Marquette Surgical technique and results of tracheal and carinal replacement with aortic allografts for salivary gland-type carcinoma J. Thorac. Cardiovasc. Surg., August 1, 2010; 140(2): 387 - 393. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. M. Radu, A. Seguin, P. Bruneval, A. Fialaire Legendre, A. Carpentier, and E. Martinod Bronchial Replacement With Arterial Allografts Ann. Thorac. Surg., July 1, 2010; 90(1): 252 - 258. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Wurtz Tracheal Replacement With Banked Cryopreserved Aortic Allograft Ann. Thorac. Surg., June 1, 2010; 89(6): 2072 - 2072. [Full Text] [PDF]
|
|
|
|
|
|
T. F. Molnar and J. E. Pongracz Tissue engineering and biotechnology in general thoracic surgery Eur J Cardiothorac Surg, June 1, 2010; 37(6): 1402 - 1410. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Makris, M. Holder-Espinasse, A. Wurtz, A. Seguin, T. Hubert, S. Jaillard, M. C. Copin, R. Jashari, M. Duterque-Coquillaud, E. Martinod, et al. Tracheal Replacement With Cryopreserved Allogenic Aorta Chest, January 1, 2010; 137(1): 60 - 67. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. F. Badylak and T. Gilbert Invited Commentary Ann. Thorac. Surg., March 1, 2009; 87(3): 868 - 868. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ANN THORAC SURG | ASIAN CARDIOVASC THORAC ANN | EUR J CARDIOTHORAC SURG |
| J THORAC CARDIOVASC SURG | ICVTS | ALL CTSNet JOURNALS |
