TRACHEAL ALLOTRANSPLANTATION AND REGENERATION

INTRODUCTION

The trachea is one of the few organs that are exceptionally difficult to transplant because of the technical difficulty to restore the blood supply to the graft. The blood supply of the 12 cm-long trachea depends in its entirety on small blood vessels branching out into numerous even smaller vessels, each of them subsequently penetrating the trachea in between the cartilage rings to provide blood supply to segments of the mucosal lining. If a part of the trachea is removed from the airway, all blood supply is interrupted. The removed part of the trachea cannot survive, even if it was to be placed straightaway back into the airway. Our group has a 20-year long research record in the field of tracheal revascularization and holds a leading position in the development of tracheal transplantation by means of vascularized segmental units.

A tracheal transplant may be necessary to repair surgical defects of the laryngotracheal airway tract that are unsuitable for segmental resection and autologous tissue repair. With the exception of some anecdotal, poorly documented cases performed without blood supply restoration¹ or immunosuppressive medication², no clinical tracheal allotransplants have been transplanted orthotopically as an isolated composite tissue graft. In tracheal allotransplantation, it is important to deal with both immunosupression and indirect revascularization in a heterotopic position. The first documented preserved viability of a heterotopically revascularized allotransplant was published by Klepetko et al. in 2004³. The graft was revascularized in the omentum of a patient who underwent lung transplantation from the same donor. Ultimately, the trachea transplant was not used, but its viability was documented for at least 60 days. The first documented revascularized tracheal allotransplant to be reported was published in 2010⁴.

Our approach to tracheal heterotopic revascularization, orthotopic transplantation, and withdrawal of immunosuppressive medication is based on a series of six cases⁵. For tracheal allotransplantation, we consider a “good match” to mean that the donor is of the same blood group as the patient.

SURGICAL TECHNIQUE

Revascularization of the trachea is the first step towards successful tracheal transplantation. The typical arterial and venous blood supply, consisting of several small tracheo-esophageal branches, does not enable direct tracheal transplantation. Currently, the only reliable way to achieve tracheal revascularization is to wrap the isolated avascular trachea with a well-vascularized soft tissue flap perfused by a vascular pedicle, which then allows transfer of the revascularized trachea to an airway defect. The forearm fascia flap pedicled on the radial artery and vein has proven to be reliable for tracheal revascularization⁵. The forearm skin is incised and dissected away from the underlying fascia and subcutaneous tissue. After removal of the membranous part, the trachea is wrapped with the radial forearm fascia and the forearm skin flaps are sutured to the incised trachea. It is important to ensure complete immobility between the trachea and the surrounding recipient’s vascular bed to obtain a fast revascularization of the blood vessels of the tracheal adventitia (Fig. 1). It usually occurs within three – four days.

Revascularization has to be achieved by the outgrowth of capillary buds from the fascia flap (recipient blood vessels) connecting with those within the adventitia (donor blood vessels) of the tracheal segment (Fig. 2). Inosculation is the establishment of direct vascular anastomoses between the vascularized soft tissue flap and the adventitia of the trachea.

Compared to a free skin graft, there are two additional barriers to revascularization for a tracheal allograft. The cartilage rings and intercartilaginous ligaments may interfere with the revascularization of the mucosal lining of the cartilaginous trachea. Cartilaginous tissue does not allow for the ingrowth of blood vessels. Revascularization of the mucosal layer of an avascular tracheal segment occurs through the intercartilaginous ligaments (Fig. 3). Full revascularization and mucosal regeneration of the cartilaginous trachea can be achieved within 2–4 months of the trachea being implanted in the forearm. In our initial patient series of tracheal transplantations, it became clear that the intercartilaginous ligaments formed an obstruction for the ingrowth of native blood vessels. The placement of intercartilaginous incisions at the time of forearm implantation was an important adaptation. The incisions of the intercartilaginous ligaments facilitated revascularization, enabling the ingrowth of recipient vessels into the submucosal space of the transplant. When incisions through the intercartilaginous ligaments were made at regular intervals, full revascularization and mucosal regeneration of the cartilaginous allotransplant could be obtained in a shorter period of time. Incision of the intercartilaginous ligaments will accelerate the revascularization process by bringing the recipient blood vessels closer to the submucosal capillaries (Fig. 4).

The tracheal allotransplant is a composite tissue transplant that may be used to restore the airway, with the goal of improving quality of life. The benefits achieved by tracheal allotransplantation have to be balanced against the morbidity of long-term immunosuppression therapy. Immunosuppressive medication should be withdrawn before immunosuppressant-related complications occur. The cartilage tissue seems to escape immunologic rejection owing to the absence of blood vessels, and because the chondrocytes are protected within a matrix^4,6,7. Regularly spaced intercartilaginous incisions provide routes for angiogenic recipient vessels to penetrate the ligamentous barrier and thus grow into the submucosal space of the transplant tissue after withdrawal of immunosuppressants.

CLINICAL EXAMPLES AND RESULTS

Eight transplants were used in 6 patients. Two of the initial transplants were lost after withdrawal of immunosuppressive therapy. Of the 6 patients treated so far, 5 patients were treated for a long-segment stenosis and 1 patient was transplanted to treat a long-segment laryngotracheal involvement by a chondrosarcoma. Two stage surgery – heterothopic allotransplantation and consequently orthotopic transplantation of a tracheal transplant to treat a long-segment (6 cm) airway stenosis is described as an example of our technique.

An eight cm long tracheal allotransplant is implanted to the forearm and wrapped with well-vascularized antebrachial fascia and skin (Fig. 5). During the first weeks the luminal site of the transplant is protected by the application of fibrin glue. After revascularization, a buccal mucosa graft from the recipient can be applied to the midportion of the transplant for replacement of donor mucosa and this allows safe withdrawal of immunosuppressive drugs (Fig. 6). A mucosal defect is created in the central part of the transplant and the midportion is grafted with a full-thickness mucosal graft from the recipient’s buccal area. The surviving recipient mucosal graft also allows secondary healing of the areas of donor epithelial lining that underwent necrosis. The recipient’s long-segment tracheal stenosis is incised longitudinally. After full revascularization and mucosal regeneration have been achieved (Fig. 7), the tracheal allotransplant is transplanted from the forearm to the airway defect on a radial vascular pedicle (Fig. 8). The radial blood vessels are sutured to the neck vessels to facilitate revascularization. The cartilaginous trachea is sutured into the airway defect to restore the concavity of the airway lumen. Withdrawal of immunosuppressive therapy can start one year after orthotopic transplantation.

Tracheal allotransplantation was also used in the treatment of a patient with an extended laryngotracheal chondrosarcoma. The patient was a 63-year-old man. The tumor developed over a period of more than 10 years. His airway could be preserved by the placement of a silicone stent. Due to the stagnation of secretions, he required periodical bronchoscopic cleaning of the stent. Since the last time, he had developed several acute episodes of stent blockages, which made definitive treatment necessary. The potential for tumor progression while under immunosuppression for a low-grade malignancy was considered to be low and was confirmed by CT scan at the time of orthotopic transplantation, which demonstrated a nearly unchanged tumor mass. Three months after implantation of a suitable allograft in the left forearm, the tumor was resected through an anterior cervical incision with a sternotomy extension and the tracheal allotransplant was used to repair the laryngotracheal defect. The lengths of the tracheal resection were 9 cm (right) and 6 cm (left side). Immunosuppressive medication was gradually withdrawn between 15 and 18 months after orthotopic transplantation. The transplant’s morphology remained intact after withdrawal of immunosuppressive therapy. It seems that the mucosal repopulation of the transplant after cessation of immunosuppressants can occur with minimal loss of airway lumen.

DISCUSSION

The relative contribution of tissue regeneration versus scarring in the healing of the airway mucosal lining depends on the extent of injury inflicted. A superficial epithelial wound can heal by way of regeneration of the surface epithelium⁸. Indeed tissues with a high proliferative capacity, such as airway tract epithelia, renew themselves continuously and, after injury, can regenerate above the basal membrane as long as the stem cells in these tissues have not been destroyed.

If a tissue injury is severe and involves damage of both epithelial cells and the submucosal layer, healing cannot be accomplished by regeneration alone. Under these conditions, the main healing process is repair by deposition of collagen, causing the formation of a scar. Future therapies should aim to promote regeneration and reduce scar tissue formation when dealing with full-thickness mucosal tracheal defects. Research of the potential use of stem cells for true regenerative healing is ongoing. The present challenge for regenerative medicine is to overcome the barriers to regeneration of the mucosal and epithelial lining in full-thickness epithelial defects. However, regeneration of full-thickness mucosal defects is not yet possible.

Tracheal segments destruction shorter than 5 cm may be treated by segmental resection. Post-intubation airway stenosis and malignant tumors are the two most common indications to perform a surgical resection of a part of the trachea.

Definitive prosthetic replacement of the airway wall is not possible. The internal side of the airway tract belongs to the outside world and bacterial contamination of the prosthetic’s internal surface prevents its usage as a definite solution.

The tracheal replacement is however necessary for longer partial or circumferential defects. Tracheal allotransplantation can be used but it is impossible to perform it directly using a single vascular pedicle due to irregular, segmental blood supply of tracheal wall. The native blood vessels are too small for microvascular anastomosis and the blood supply comes from several small sources. Tracheoesophageal branches from the inferior thyroid artery supply the upper half of the trachea. The bronchial arteries provide consistent blood supply to the carina and lowermost trachea. The best option seems to be the heterothopic transplantation of avascularised trachea into a well-nourished tissue bed and secondary orthotopic transplantation to the tracheal defect on the neck. The protocol for circumferential allotransplantation may be based on a bilateral transplantation of the cartilaginous trachea. The full length of the trachea and main bronchi can be used for allotransplantation. Two cartilaginous tracheal segments may be implanted at two forearm sites. By suturing the two allotransplants together, a tube may be created for circumferential airway repair. The first transplant is used to restore the posterior and lateral walls of the airway. A part of the forearm skin can be included as a temporary reconstruction of the anterior wall. In a second operation, the second transplant can be used to replace the forerarm skin and to further augment the airway lumen.

Since 2008 the trachea has been called the first human organ that can be man-made using acellular natural or synthetic scaffold and stem cells.⁹ De-vascularized native trachea was taken from deceased donor. As a first step towards a presumed stem-cell engineered regenerated trachea, a detergent was used to destroy all viable cells, leaving a scaffold of connective tissue. It was hypothesized that stem cells penetrate the connective tissue and subsequently cartilage, blood vessels and respiratory mucosa. This presumed regenerated trachea was implanted without restoration of any blood supply. It was also hypothesized that stem cell-mediated re-cellularization of a synthetic scaffold may also lead to a fully regenerated trachea that can be transplanted inside the airway.

Meanwhile an engineered trachea has been implanted in several patients. This achievement has received a lot of attention in medical journals as well as in the press. Indeed, the engineered windpipe was seen to be the first step towards other forms of organ regeneration. Classic organ transplantations with their typical side effects due to anti-rejection medication could then be replaced by growing organs from the body’s own cells. However, the optimism surrounding organ regeneration has proved to be completely unfounded. In fact, the engineered trachea is an example of obvious scientific deception.

The engineered trachea was represented as a regenerated trachea after applying bone marrow cells to a de-cellularized¹⁰ or synthetic scaffold¹¹. There is no scientific foundation whatsoever to assume why stem cells would support airway tissue regeneration in this setting. In addition, even if a trachea-like organ would be generated, it would irrefutably fail after implantation if adequate blood supply had not been restored. As expected, the implantation of de-cellularized and synthetic scaffolds resulted in extremely high morbidity and mortality rates¹². At this point in time, this form of airway regeneration should be regarded as hypothetical and scientifically unfounded^13,14,15.

CONCLUSION

Partial or circumferential airway repair may be necessary after long intubation, neck injury or resection of malignant tumors. Tracheal allotransplantation seems to be an option but still there are a lot of questions that have to be resolved before this becomes a routine technique. Tracheal allotransplantation at the time of tumor resection will be possible only for low-grade malignancies and not for other malignant tumors, because of the risk of tumor progression in the 3-month period of pretransplant immunosuppression. A circumferential defect left by tumor resection can be reconstructed temporarily with a stent wrapped in vascularized tissue. This type of reconstruction must be considered temporary due to inevitable stent-related complications. Tracheal allotransplantation may be considered in those patients with a temporary repair who remain tumor-free.

As a conclusion, tracheal transplantation can be safely performed in selected cases after heterotopic revascularization. Important are the partial incisions of the intercartilaginous ligaments for advancing the revascularization process and for safe withdrawal of immunosuppressive therapy. Growth factors may eventually be used for speeding up the revascularization process.

Declaration of interest: Author has no financial or other interests related to the content of the article.

Corresponding author:

Martin Molitor, M.D., PhD.

Department of Plastic Surgery

Hospital na Bulovce and First Faculty of Medicine, Charles University

Budínova 2, Prague 180 81

Czech Republic

E-mail: martinmolitor1@gmail.com