In vivo tissue engineering for airway replacement
Ingénierie tissulaire in vivo pour remplacement des voies respiratoires
D. Radu*, E. Martinod*
* Service de chirurgie thoracique et vasculaire, hôpital Avicenne, Bobigny, université Paris XIII ; laboratoire de recherches biochirurgicales, fondation Alain-Carpentier, université Paris-Descartes, Paris.
Background
I
t has been more than 70 years since the first attempts at airway replacement. The issue stirred enthusiasm in a great number of teams worldwide, and research work has been intense, but deceiving in most of the cases. The reason is that it is very difficult to find an implant that would reproduce the characteristics of trachea and bronchi and that would keep these characteristics in the long run.Presently, progress has been achieved and this was possible because the concept of airway replacement has changed.
Previously, researchers were looking for an “already made” structure that would replace the trachea.
Synthetic material, bioprosthesis, autologous tissues and airway transplantation, they mostly failed. The new concept is to find the ideal matrix that, whether pre-seeded with stem cells before implantation or not, would, within the receiver’s organism, transform and become a new airway. It is the tissue-engineered
tracheal and bronchi replacements that would use, at some point, the body as a bioreactor.
The last 15 years have been marked by a lot of human cases of airway replacements performed by several teams that used the concept of tissue engineering.
Most of these interventions were done for lesions that did not have any alternative valid solution. Tracheal substitutes obtained by different processes were used, all with longstanding research background.
Certain teams worked on in-vitro prepared implants with either synthetic or decellularized biological scaf- folds, others used mainly in-vivo tissue engineering methods. As the success of these procedures was variable, The Pulmonary Comity of the International Society for Cellular Therapy (1) suggested that all those teams should join their efforts in the search for the ideal tissue-engineered airway substitute and create an international registry for these patients.
In this article, we would like to present the state of the art in tissue-engineered airway replacements, particularly for those already tried on humans.
Summary Résumé
Tracheal and bronchial replacements with tissue- engineered airways are complex procedures limited to benign or malignant lesions with no valid treatment option. Several matrices have been tried: tracheal decellularized allografts or synthetic scaffolds seeded with cells in vitro, tracheal allografts and cryopreserved aortic allografts submitted exclusively to in vivo tissue engineering. In this article, we present the state of the art of human airway replacements with advantages and pitfalls of each technique.
Keywords: Tissue engineering – Trachea – Tissue scaffolds – Transplantation.
Les remplacements de trachée et de bronches par de nouvelles voies respiratoires créées par des techniques d’ingénierie tissulaire sont des procédures complexes, limitées à des lésions bénignes ou malignes sans possibilité de traitements classiques. Plusieurs matrices ont été évaluées : allogreffes trachéales décellularisées ou échafaudages synthétiques ensemencés avec des cellules in vitro, allogreffes trachéales et allogreffes aortiques cryoconservées soumises exclusivement à l’ingénierie tissulaire in vivo. Dans cet article, nous présentons l’état de l’art des remplacements des voies respiratoires humaines, avec les avantages et les inconvénients de chaque technique.
Mots-clés : Ingénierie tissulaire – Trachée – Matrices tissulaires – Transplantation.
long congenital tracheal defects are still in their expe- rimental phases. At Boston Children’s Hospital and Harvard Medical School, D.O. Fauza et al. have been working for a very long time on prenatal tracheal replacements in an ovine model. They first tried a syn- thetic biodegradable matrix (poliglycolic acid polymer mesh recovered by poly-L-lactic acid) [2] seeded with mesenchymal amniocytes labeled with green fluores- cent protein (GFP) and maintained in a bioreactor in chondrogenic medium for chondrogenic differentiation.
In-vitro-tissue-engineered tracheal substitutes were then implanted in vivo. Fetal tracheal replacements of either the anterior tracheal wall or of a circumferential segment were done at about 30 to 40 days prior to gestation term. After birth, the lambs breathed well, but, several days later, they developed respiratory difficulties. Although stenosis developed in all grafts, microscopically, tracheal conduits were completely epithelialized. Neo-chrondrogenesis from mesenchymal amniocytes was maintained within the grafts after the in-vivo implantation. In 2012, D.O. Fauza et al. published their work on tracheal circumferential replacement with an in-vitro-engineered trachea composed of a decellularized rabbit tracheal segment seeded with GFP-labeled amniotic mesenchymal stem cells (MSCs) exposed to chondrogenic medium (3). In this study, a second group of animals was implanted with a non- seeded decellularized rabbit trachea. Even though most of the animals breathed well at birth, stenosis invariably developed in both groups. Full epithelialization was present in all in-vitro-tissue-engineered grafts, while the acellular implants were only partially epithelia- lized. Neo-epithelialization originated from the native trachea ; interestingly, no goblet cell was identified.
As to the cartilage, cells were absent in some of the lacunae, however with more cells in the engineered grafts than in the acellular grafts. GFP expression was present, suggesting survival of cells originating from seeded amniotic MSCs.
In children, the first case of a tissue-engineered tracheal replacement was performed in London, in 2010, by M.J. Elliott et al. (4). The child was born with a long tracheal stenosis and had already needed tracheal reconstructions and stenting. Tracheal allografting (replacement with a tracheal homograft) was needed when the child was 3 years old, as an aortotracheal fistula developed. At the age of 10, a new intermittently active aortotracheal fistula appeared. Replacement was then performed with a tis-
Factor beta (TGF-β) to induce chondrogenesis. A bio- resorbable stent was also inserted. Malacia developed in the graft, and restenting was necessary several times.
Nevertheless, the child was well, growing and returned to school 6 months after surgery. Bronchoscopic examina- tion showed normal mucosa and complete epithelializa- tion with cytology of tracheal brushing showing normal ciliated epithelium, 15 months after the procedure. The stented graft remained stable up to about 3 years after the operation (5), when an infection with in-stent restenosis of the grafted trachea and of the native left main bronchus developed. Airway dilatation was again necessary with a good recovery. Imagistic studies showed that while the ends of the graft grew with the child, the middle portion did not. The reason would be repeated restenosis and stenting with finally stents ending up embedded in the tracheal mucosa. Late tracheal biopsies (at 42 months) showed a complete epithelium with areas of squamous and respiratory epithelium. There had never been any sign of rejection (no anti-donor HLA antibodies, no lymphocyte-associated epithelial damage and normal submucosal T-cell density).
The same type of matrix had already been used in 2008 for the replacement of a long post-tuberculous stenosis of the left main bronchus in a 30-year old woman (6) by P. Macchiarini et al. The decellularized human tracheal segment was seeded with recipient’s bronchial epithelial cells and autologous chondrocytes that were developed in vitro from bone marrow MSCs. The authors published the 5-year follow-up of this patient (7): the left lung was well expanded. However, the bronchial graft partially restenosed and regular endobrochial dilation and stenting were necessary.
Encouraged by these 2 cases, M.J. Elliott et al. (8) used again a similar tissue-engineered graft to reconstruct the trachea of a 15-year old girl. She was born with a single, left lung and a long tracheal stenosis. Several interven- tions were necessary to keep the tracheal patent over the years, the child ending up at 15 with a tracheostomy and ventilation-dependent at night. After a successfully resuscitated respiratory arrest, she was considered for compassionate tracheal replacement. The girl recovered very well after the complex reconstructive operation. The in-vitro-tissue-engineered graft was prepared according to strict standards of good manufacturing practice, com- pared to previous grafts, which did not fulfill these criteria.
Furthermore, to reduce the risk of in-stent graft stenosis, the team decided not to place a temporary stent within
the graft this time, only the tracheostomy tube. Repeated assessments showed only mild malacia within the graft.
Nevertheless, at postoperative day 15, the child deve- loped ventilator dysfunction with prolonged respiratory arrest, resulting in hypoxic brain damage, and died. For the authors, one the very probable reasons for this brutal event would be the loss of rigidity of the tissue-engineered graft during the early phase of remodeling and revasculari- zation after implantation, with consequent acute malacia.
In total, about 8 patients were implanted with in-vitro-re- seeded decellularized tracheal segments, but scientific communications were scarce on the subject (9, 10). It was probably the imperfect results of those airway reconstruc- tions that made P. Macchiarini et al., at Karolinska Institute in Stockholm, Sweden, to turn to synthetic in-vitro-tis- sue-engineered scaffolds. Indeed, it was K. Omori et al.
who published the first human case of tracheal replace- ment with a synthetic scaffold, in 2005 (11). The operation was performed for a thyroid papillary adenocarcinoma involving the cartilaginous tracheal wall. The replacement was small: only the right half of 3 cartilaginous rings. The airway substitute consisted of a Marlex® mesh covered in collagen and soaked in autologous blood at the time of implantation. At 2-year follow-up, there was no compli- cation and the epithelialization was complete. They also used the same type of prosthesis soaked in blood and fibroblast growth factor to repair the cartilaginous part in 3 patients with post-intubation tracheal stenosis (12).
Reported follow-up at 6 months was successful.
So, in 2011, P. Macchiarini et al. replaced the trachea of a 36-year-old man with a recurrence of a mucoepi- mermoid carcinoma of the trachea and main bronchi, already treated with debulking and radiotherapy (13).
The in-vitro-engineered implant was made of a nanocom- posite polymer (polyhedral oligomeric silsesquioxane covalently bonded to poly-carbonate-urea urethane [POSS-PCU]) seeded with recipients’ bone marrow MSCs.
Complications were due to insufficient vascularization and incomplete epithelialization of the graft that generated bacterial and fungi infections and stenotic granulomas at the anastomotic sites (1). Stenting was inevitable. The team tried to improve their synthetic scaffold implants and at least another 9 patients were operated on, 2 of them at Karolinska Institute (14) and others in Russia (1), as P. Macchiarini received a grant to do a clinical trial in this country (9). Several patients have died, others were still hospitalized in 2014 (1). Development of malacia seems to be an important cause of complications (8, 14). Up to 2013, P. Macchiarini’s fame rapidly increased (15), but the medical community started to question his work as scientific reports on the outcome of these patients were lacking. Polemic arose with other scientific teams arguing
the manner of his work, as scientific foundation of his work applied to human subjects was not considered solid. Thus, he is contested from a legal point of view because ethical review revealed a lack of authorization for these operations (as it should have been considered human research) [14], but also because of his manner of concealing certain fai- lures. The technique used is also contested (16), as it is considered that a synthetic matrix in a contaminated environment would only work temporarily and that the exuberant growth of granulomas is unavoidable with a permanent need for stenting.
During the same time, 2 other teams were also perfor- ming human airway replacements with other types of implants. These teams favored biological matrices and in-vivo tissue-engineering methods.
In 2010, P. Delaere et al. published their first human case of tracheal allograft revascularized and epithelialized in vivo (17). The patient had a long tracheal post-intubation stenosis refractory to endotracheal dilatation. The graft needed a long in-vivo preparation, for about 9 months.
First, the tracheal allograft was implanted in the patient’s forearm, completely wrapped in subcutaneous and fascial tissue. During this time, the patient was under immuno- suppressive therapy in order to allow the graft to be vas- cularized by recipient’s neo-vessels. The graft was checked every 2 weeks, and when the membranous part suffered avascular necrosis, this part was removed and patient’s own buccal mucosa was implanted. Thus, the luminal part of the graft contained a double epithelium: the donor’s respiratory epithelium and the recipient’s buccal epithe- lium. Immunosuppressive therapy was stopped when vascularization was optimal, and so the recipient’s buccal mucosa took over and completely covered the luminal side of the tracheal allograft. Then, the in-vivo-prepared graft was used to replace the tracheal stenotic segment.
Follow-up was uneventful, with a graft that remained rigid with no sign of rejection.
P. Delaere et al. tried to reduce the time of prepara- tion of the graft and the time of immunosuppression.
Four other patients were also operated on using the same technique, but with a reduced immunosuppres- sion time (18). Complications developed in 3 of these patients: 1 of the grafts necrosed after withdrawal of immunosuppression and so it was never implanted;
for 2 other patients, revascularization of the graft was not sufficient enough to sustain the growth of the reci- pient’s epithelium and so, stenosis of the graft appeared in the middle part. Therefore, it was concluded that in order to have a successful graft, it is necessary to give time to recipients’ vessels to develop into the graft.
A good submucosal vascularization is vital to sustain the growth of recipient’s epithelium during the rejection
gous epithelialization is optimal, the tracheal graft remains stable as the donor’s cartilage stays viable and keeps its functions (away from any possible rejection, being comple- tely embedded in recipient’s mucosa), as was the case for 1 of those 4 additional patients (19). Scientific reports show that a total of 6 patients have been treated by P. Delaere’s method (20). As one can easily realize, this is an effective technique of tracheal replacement, which can be used in case of benign tracheal stenosis or for patients with low-malignancy tumors (18). The most important limitation of the technique is that it cannot be applied to patients with malignant tumors, as immunosuppressive therapy is absolutely necessary for the preparation of the graft.
Airway-tissue engineering in France
In 2011, our team published the first case of human transplantation of a biological airway to replace the bronchus in order to preserve the right lower lobe, in a case of bronchopulmonary cancer (21). This was the result of a long experimental work in an animal model that showed that an aortic allograft supported by a stent in an airway position transformed itself and rege- nerated the airway, with newly formed epithelium but also cartilage (22-25). No immunosuppressive therapy was needed. Moreover, our team has shown that this regeneration was obtained with autologous aortic grafts, fresh or cryopreserved aortic allografts, but not with glutaraldehyde or decellularized aortic grafts (24). As cryopreserved aortic allografts are easily available in tissue banks, we favored their use.
In France, these experimental findings triggered com- passionate clinical applications in 6 patients with long tracheal or carinal malignant lesions, with promising results (26, 27). The tracheal substitute consisted of a stent-supported aortic allograft freshly harvested in 2 patients and cryopreserved in the last 4 patients. Large resections of the trachea were performed; carina was also resected in 4 patients. No immunosuppressive therapy was given. There was no postoperative mortality, but early postoperative course was difficult for 5 patients (27):
anastomotic dehiscence in 1 case, sternal dehiscence in 2 patients, severe pneumonia in 1 patient, 1 case of fungal infection of the aortic allograft necessitating allograft replacement, anterior spinal cord ischemia in 1 patient and development of a fistula between the carina and the esophagus in 1 patient. All patients sur-
metastatic disease, but no sign of local recurrence; the other died of hemoptysis at 26 months, with no sign of disease relapse (28). Stent withdrawal was not possible either because a tracheoesophageal fistula developed (in 3 patients), or because the new airway was not rigid enough (mature cartilage was not seen within the graft on imagistic examinations, only sparse calcifications) [27].
Our group’s first human bronchial replacement with a biological airway, consisting of a cryopreserved aortic allograft supported by a bronchial stent (21), was per- formed in 2009. After collegial discussion, this first case was approved by the French National Bioethics Advisory Commission (Agence française de sécurité sanitaire des produits de santé [Afssaps]). The patient was a 78-year-old man who had had neo-adjuvant che- motherapy for a large squamous-cell carcinoma and needed a right pneumonectomy for curative intent.
The patient refused the option of a pneumonectomy, as he was aware of the high risks of this operation in his case, but he agreed to a sleeve upper-bilobectomy with a bronchial airway reconstruction with an aortic allograft, in order to preserve his right lower lobe.
In the postoperative course, the patient developed supraventricular tachyarrhythmia with mild pulmonary edema, lower right lobe pneumonia and several recur- rent episodes of urinary retention, due to his prostatic adenoma. Treatment was successful, and the patient was discharged on postoperative day 16 with a normally functional right lower lobe. Pathological examination of the surgical specimen showed a pT4N0 cancer with negative resection margins. There was no pulmonary complication at 1-year follow-up, and the patient had a good quality of life, as confirmed by a 12% score at Saint George’s Respiratory Questionnaire. The graft was well integrated in the surrounding tissues, but no mature cartilage could be seen on CT-scan examina- tion. An intriguing question concerned cartilage forma- tion, as no new cartilage could be observed in human cases (21, 27), whereas in the animal model it already started to develop after a few months of follow-up (24, 25). Several hypotheses emerged: the young age of the animals in the research studies, which were therefore more prone to regeneration; cartilage regeneration in humans may simply be a delayed process; patients’
malignant disease with adjuvant treatments, postopera- tive complications or comorbidities may have impacted the potential for tissue regeneration. Nevertheless, our team recently made an amazing finding: it seems
that the aortic scaffold plays a central role for cartilage regeneration in humans (29). Our group continued to do human airway replacements in a phase I trial (TRACHEOBRONCHART study, NCT01331863) intended for patients with long malignant or benign lesions of the trachea or bronchi, not accessible to conven- tional sleeve resections. In this trial, we have used only − 80 °C cryopreserved human aortic allografts for airway replacement, as it has been demonstrated that − 80 °C cryopreserved arterial grafts have better mechanical characteristics than − 150 °C cryopreserved grafts (30). In the previous cases, − 150 °C cryopreserved aortic allografts were used. Thus, we analyzed the trans- formation of the airway graft in 2 patients from the TRACHEOBRONCHART study, who had long complex
post-intubation tracheal stenosis, refractory to other treatments (29). They were both implanted with − 80 °C cryopreserved aortic allografts supported by a tracheal stent. Indeed, in these patients cartilage regeneration was observed and tracheal stents were completely removed at 15 and 39 months, respectively (figure).
Neo-cartilage formation was detected on superficial biopsy samples: clusters of immature cartilage were seen, with positive immunodetection of type II collagen and Sox-9 specific markers. In biopsy samples taken from one of these patients (a woman who received a male aortic allograft), it was also possible to perform XX/ XY chimerism studies, which showed that neo- cartilage developed from recipient cells.
The study of the biological characteristics of those human − 80 °C cryopreserved aortic allografts showed that these matrices preserved aortic cells viability with active cytokine and growth-factor release (29).
This aortic scaffold made possible the right cell inter- actions in the human body, to be repopulated with the recipient’s appropriate stem cells and so to obtain an in-vivo-tissue-engineered airway, entirely made of recipient’s elements.
In light of these findings, we consider the aortic − 80 °C cryopreserved allograft to be the best scaffold to be used in airway replacements. It is a biological tissue, readily available in tissue banks, whose implantation does not require immunosuppression. Moreover, the viable cells of the aortic matrix release cytokines and growth factors (29) that promote cell interactions of major importance in sustaining the complex process of regeneration of a new airway.
Despite all the difficulties, these very encouraging recent results bring us closer than ever to the long- awaited tracheal replacement. However, numerous efforts are still required for achieving a successful in-vivo-tissue-engineered airway. ■
Les auteurs déclarent ne pas avoir de liens d’intérêts.
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Figure. Virtual bronchoscopy in a patient at long-term follow-up after tracheal replacement using a stented cryo- preserved aortic allograft. Cartilage regeneration (star) allowed stent removal at 39 months.
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