- Email: [email protected]
Transplantation of the decellularized tracheal allograft in animal model (rabbit)
+
MODEL
Asian Journal of Surgery (2017) xx, 1e5
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.e-asianjournalsurgery.com
ORIGINAL ARTICLE
Transplantation of the decellularized tracheal allograft in animal model (rabbit) Reza Ershadi*, Mohamadbagher Rahim, Siavash Jahany, Siamak Rakei Department of Thoracic Surgery, Valiasr Hospital, Tehran University of Medical Science, Iran Received 3 January 2017; received in revised form 15 February 2017; accepted 20 February 2017
KEYWORDS decellularized allograft; rabbits; tracheal transplantation
Summary Background: It has been difficult to perform tracheal allotransplantation without immunosuppression. To determine whether decellularized trachea can be used in tracheal replacement, we evaluated the viability of decellularized tracheal allografts in a rabbit model of immunosuppressant-free transplantation. Method: Half allograft (Group 1, n Z 7) was harvested from adult New Zealand white rabbits, subjected to a detergenteenzymatic method (containing sodium deoxycholate/DNase lavations) of decellularization for as many cycles as needed, and the other half was stored in phosphate-buffered saline at 4 C as a control (Group 2, n Z 7). Bioengineered and control tracheas were then implanted in 14 age-matched rabbits. Results: In Group 1 (decellularized), all rabbits survived, whereas in Group 2(control), all rabbits died of airway obstruction between 20 days and 45 days after operation. Histologically, the decellularized allografts displayed complete regeneration of epithelium and cartilage, but the fresh allografts showed inflammatory changes, no epithelium, and no cartilage. Conclusions: Complete regeneration of epithelium and cartilage tracheal rings occurred after the implantation of decellularized tracheal allografts without immunosuppression. We demonstrate that the decellularized process reduces the allogeneic response to the trachea. Therefore, we believe that the decellularized tracheal allograft is an excellent choice for tracheal replacement. To our knowledge, this is the first study to observe the long-term (1 year) prognosis of this transplanted trachea. ª 2017 Asian Surgical Association and Taiwan Robotic Surgical Association. Publishing services by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).
* Corresponding author. Department of Thoracic Surgery, Valiasr Hospital, Bagherkhan Street, Tehran, Iran. E-mail address: [email protected] (R. Ershadi). http://dx.doi.org/10.1016/j.asjsur.2017.02.007 1015-9584/ª 2017 Asian Surgical Association and Taiwan Robotic Surgical Association. Publishing services by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article in press as: Ershadi R, et al., Transplantation of the decellularized tracheal allograft in animal model (rabbit), Asian Journal of Surgery (2017), http://dx.doi.org/10.1016/j.asjsur.2017.02.007
+
MODEL
2
R. Ershadi et al.
1. Introduction
2.1. Matrix bioengineering
Tracheal resection with primary reconstruction is the only curative treatment in patients with a variety of benign or malignant tracheal lesions.1 Unfortunately, the resectable length of the diseased trachea is usually restricted to approximately 30% of the total length in children and around 6 cm in adults, and any further increase in this resection rate depends on the development of a safe tracheal replacement.2 This is not yet clinically available because almost every attempt to provide an autologous or synthetic safe and reproducible tracheal graft has been disappointing thus far,1 casting doubt on the future of longsegment tracheal replacement.3 Tissue bioengineering has emerged as the most promising technique to create a nearnormal trachea,1,4 and encouraging early experimental results5 and clinical applications6 have been reported. To determine whether decellularized trachea can be a realistic substitute in replacement procedures, we evaluated the viability of and the histologic changes in decellularized tracheal allografts in rabbit model of transplantation without immunosuppression.
The overlying tissue of the harvested tracheas was stripped off, deprived of trachealis muscle, and rinsed four times (for 4 hours each) in PBS containing 1% antibiotic and antimycotic solution. Thereafter, the protocol continued as previously reported.7 Briefly, the tissue was processed with multiple treatment cycles, including the following steps: the tissue was stored in Aqua milliQ (Millipore) for 48 hours at 4 C and then incubated in 4% sodium deoxycholate for 4 hours and 2000 kU of DNase-I in 1 mol/L NaCl (Sigma Chemical Company) for 4 hours. The presence of cellular elements and major histocompatibility complex (MHC) cells were verified histologically after each cycle. The bioengineering process lasted for 35 days, corresponding to 17 detergenteenzymatic method (DEM) cycles, after which the bioengineered tracheal matrices showed few residual nuclei of chondrocytes but a complete removal of MHC class I and II antigens (Figure 1A).8 In contrast, MHC class I and II expression was ubiquitous in control tracheas recipient.8 The transplantation was performed with recipient rabbits of matched age. Under anesthesia, the recipient was placed in a supine position and a six-ring tracheal segment was resected through a midline cervical incision. Allograft transplantation (control, n Z 7; decellularize, n Z 7) was performed in an end-to-end continuous fashion using absorbable monofilament (5-0 PDS, Ethicon, USA) (Figure 1B), and the wound was closed in the usual manner. Postoperatively,all the 14 recepient rabbits were observed for 3 hours before being returned to their individual cages. They received standard feed and water. Analgesics (buprenorphine, 0.05 mg/kg) were administered twice a day on postoperative Day 1. Antibioprophylaxis (enrofloxacin, 10 mg/kg) was administered postoperatively over 5 days. No immunosuppressive agents or steroids were given. Examination with a flexible bronchoscope was performed on the 5th and 15th postoperative days and every 3 months thereafter, and each tracheal graft was examined grossly and microscopically (pathologic examination with hematoxylin and eosin staining)when the animal died or was sacrificed from Day 20 to Day 365.
2. Material and methods Special humane animal care measures were taken in compliance with the “Principles of laboratory animal care” formulated by the National Society for Medical Research and the “Guide for the care and use of laboratory animals” prepared by the Institute of Laboratory Animal Resources, National Research Council, and published by the National Academy Press, revised 1996. Each allograft was harvested from adult male New Zealand white rabbits weighing 3170e4575 g. The animals were anesthetized with intravenous administration of pentobarbital (30 mg/kg). They were placed in a supine position under spontaneous ventilation without an endotracheal tube, and either harvesting or transplantation was performed. The cervical trachea was exposed through a midline cervical incision and then harvested from the cricoid cartilage to the carina. To obtain maximum graft viability, grafts were harvested from beating heart donors without warm ischemia. The harvested segment was then divided into two groups. One half (control) was placed in a stock solution made of phosphate-buffered saline (PBS), containing 1% antibiotic and antimycotic solution. The other half was similarly stored for 24 hours but then submitted to the bioengineering protocol, as described below.
Figure 1
3. Results Table 1 summarizes the results for the 14 transplant recipients. In the decellularized group (Group 1), the rabbits were alive for the duration of the experiment and were subsequently killed for the histologic examinations. In control group (Group 2), however, all the seven rabbits died
(A) A decellularized trachea. (B) Transplantation of decellularized tracheal allograft.
Please cite this article in press as: Ershadi R, et al., Transplantation of the decellularized tracheal allograft in animal model (rabbit), Asian Journal of Surgery (2017), http://dx.doi.org/10.1016/j.asjsur.2017.02.007
+
MODEL
Decellularized tracheal allograft transplantation Table 1
3
Findings of 14 rabbits undergoing tracheal allotransplantation
Group
Rabbit n
Death or sacrifice day
Epithelium regeneration
Cartilage
Lymphocyte infiltration
Mononuclear cell infiltration
1-Decellularized allograft
1 2 3 4 5 6 7 1 2 3 4 5 6 7
200 Sacrificed 220 Sacrificed 280 Sacrificed 320 Sacrificed 340 Sacrificed 365 Sacrificed 365 Sacrificed 20 Died 25 Died 29 Died 34 Died 39 Died 41 Died 45 Died
þ þ þ þ þ þ þ
þ þ þ þ þ þ þ
þ þ þ þ þ þ þ
þ þ þ þ þ þ þ
2-Control allograft
of airway obstruction on the 20th, 25th, 29th, 34th, 39th, 41st, and 45th postoperative days. On bronchoscopy, signs of epithelial regeneration appeared early in the posterior wall of the grafts and then around the entire circumference, and airways were open and no stenosis or malacic changes were seen in Group 1 (Figure 2). However, in Group 2, airways were almost completely obstructed by fibrosis and granulation tissue. Histologically, remarkable lymphocytic and mononuclear cell infiltration, severe fibrosis, and destruction of the cartilaginous rings were observed in Group 2 (Figure 3A). In Group 1, complete epithelial regeneration (well-differentiated respiratory epithelium) and regeneration of cartilage tracheal rings were observed. Lymphocytic and mononuclear cell infiltration were not observed in Group 1 (Figure 3B).
4. Discussion In contrast with other successful organ replacements, which take place in sterile mesenchymal environments
(e.g., the liver, kidney, and heart), the airway represents an interface between the mammal and the external environment. Unsurprisingly, its mucosa holds immunologically active cells playing a key role in airway transplantation,9,10 and these contribute to acute allograft rejection, requiring high-dose immunosuppression.11,12 Replacement of the trachea has many unresolved surgical problems. Various efforts have been made to accomplish this using prosthetic materials, different autologous tissues, and homografts in both experimental and clinical settings.13,14 A variety of prosthetic materials have been tested, but clinically suitable prostheses have not yet been developed because of insufficient epithelialization, bacterial infection, obstruction caused by excessive granulation, and anastomotic leakage. Reconstruction with various autogenous tissues, such as pericardium,15 periosteum,16 esophagus,17 and small bowel,18 all reinforced with a stent, has been reported. However, their clinical application must be pursued with caution. Studies19,20 of antigenicity have shown that the human tracheal epithelium develops HLA-DR antigen, which activates T lymphocytes and may play an important
Figure 2 (A) Bronchoscopic examination of decellularized tracheal allograft groups showing a whitish aspect of the graft lumen on Day 5. (B) Epithelial regeneration appearing early on Day 15. (C) Final bronchoscopy on Day 365 with complete epithelial regeneration.
Please cite this article in press as: Ershadi R, et al., Transplantation of the decellularized tracheal allograft in animal model (rabbit), Asian Journal of Surgery (2017), http://dx.doi.org/10.1016/j.asjsur.2017.02.007
+
MODEL
4
R. Ershadi et al. segments. Our experimental findings indicate that the decellularized tracheal allograft has excellent long-term tissue viability and reduced antigenicity of the graft. It is an excellent choice for tracheal replacement. This study is the first to observe the long-term (1 year) prognosis of this transplanted trachea. In conclusion, complete regeneration of epithelium and cartilage tracheal rings occurred after the implantation of decellularized tracheal allografts without immunosuppression. Thus, the decellularized process reduces the allogeneic response to the trachea.
Conflicts of interest The authors have no conflicts of interest to declare.
References
Figure 3 Histologic findings of the control and decellularized tracheal allotransplantation. (A) Remarkable cellular infiltration (lymphocyte and mononuclear cell), fibrosis and destruction of cartilaginous rings in the transplanted graft of the control group. (B) Respiratory epithelial regeneration (block arrow and inset), cartilage ring, and graft revascularization (small arrow) in the decellularized tracheal allograft. Hematoxylineeosin staining 200. Inset 400.
role in graft rejection. The use of immunosuppressants to control immunologic reactions will certainly attenuate graft rejection but will also increase airway infection after tracheal replacement. Therefore, reducing the antigenicity of the allograft itself is an excellent method of control. For this purpose, cryopreservation,21 radiation therapy,22 and photodynamic therapy23 have been reported. The present results demonstrate that the DEM process is a simple and effective method to bioengineer tracheal matrices lacking any MHC antigens while maintaining a structural integrity similar to that of native tracheas and, most importantly, of sufficient length to have potential clinical application. In our experiment, after implantation of decellularized tracheal allografts, complete regeneration of epithelium and cartilage was observed. There was no lymphocytic infiltration of or under the epithelial layer. On bronchoscopic examination, the tracheal lumen was open and no malacic changes were observed in the transplanted
1. Grille HC. Tracheal replacement: a critical review. Ann Thorax Surg. 2002;73:1995e2004. 2. Gasser HA, Grille HC, Shadier MB, et al. Long-term survival after resection of primary adenoid cystic and squamous cell carcinoma of the trachea and carina. Ann Thorax Surg. 2004; 78:1889e1896. 3. Birchall M, Macchiarini P. Airway transplantation: a debate worth having? Transplantation. 2008;85:1075e1080. 4. Macchiarini P. Trachea-guided generation: de ´ja ` vu all over again? J Thorax Cardiovasc Surg. 2004;128:14e16. 5. Walles T, Giere B, Hofmann M, et al. Experimental generation of a tissue-engineered functional and vascularized trachea. J Thorax Cardiovasc Surg. 2004;128:900e906. 6. Macchiarini P, Walles T, Biancosino C, Mertsching H. First human transplantation of a bioengineered airway tissue. J Thorax Cardiovasc Surg. 2004;128:638e641. 7. Conconi MT, De Coppi P, Di Liddo R, et al. Tracheal matrices, obtained by a detergent-enzymatic method, support in vitro the adhesion of chondrocytes and tracheal epithelial cells. Transpl Int. 2005;18:727e734. 8. Jungebluth P, Asnaghi A, Bellini S, et al. Structural and morphologic evaluation of a novel detergent enzymatic tissueengineered tracheal tubular matrix. J Thorax Cardiovasc Surg. 2009;138:586e593. 9. Barker E, Murison P, Macchiarini P, et al. Early immunological changes associated with laryngeal transplantation in a major histocompatibility complex-matched pig model. Clin Exp Immunol. 2006;146:503e508. 10. Tojo T, Niwaya K, Sawabata N, et al. Tracheal replacement with cryopreserved tracheal allograft: experiment in dogs. Ann Thorax Surg. 1998;66:209e213. 11. Macchiarini P, Lenot B, de Montpre ´ville VT, et al. Heterotopic pig model for direct revascularization and venous drainage of tracheal allografts. Paris-Sud University Lung Transplantation Group. J Thorax Cardiovasc Surg. 1994;108:1066e1075. 12. Macchiarini P, Mazmanian GM, de Montpre ´ville VT, et al. Experimental tracheal and tracheoesophageal allotransplantation. Paris-Sud University Lung Transplantation Group. J Thorax Cardiovasc Surg. 1995;110:1037e1046. 13. Alonso WA, Bridger GP, Bordley JE. Tracheal transplantation in dogs. Laryngoscope. 1972;82:204e209. 14. Grille HC. Tracheal replacement [Editorial]. Ann Thorax Surg. 1990;49:864e865. 15. Idriss FS, DeLeon SY, Ilbawi MN, Gerson CR, Tucker GF, Holinger L, et al. Tracheoplasty with pericardial patch for extensive tracheal stenosis in infants and children. J Thorax Cardiovasc Surg. 1984;88:527e535.
Please cite this article in press as: Ershadi R, et al., Transplantation of the decellularized tracheal allograft in animal model (rabbit), Asian Journal of Surgery (2017), http://dx.doi.org/10.1016/j.asjsur.2017.02.007
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MODEL
Decellularized tracheal allograft transplantation 16. Kimura K, Mukohara N, Tsugawa C, et al. Tracheoplasty for congenital stenosis of the entire trachea. J Pediatr Surg. 1982; 17:869e871. 17. Kato R, Onuki AS, Watanabe M, et al. Tracheal reconstruction by esophageal interposition: an experimental study. Ann Thorax Surg. 1990;49:951e954. 18. Letang E, Sa ´nchez-Lloret J, Gimferrer JM, Ramı´rez J, Vicens A. Experimental reconstruction of the canine trachea with a free revascularized small bowel graft. Ann Thorax Surg. 1990;49: 955e958. 19. Bujia J, Wilmes E, Hammer C, Kastenbauer E. Tracheal transplantation: demonstration of HLA class II subregion gene products on human trachea. Acta Otolaryngol (Stockh). 1990; 110:149e154.
5 20. Kalb TH, Chuang MT, Marom Z, Mayer L. Evidence for accessory cell function by class II MHC antigen-expressing airway epithelial cells. Am J Respir Cell Mol Biol. 1991;4:320e329. 21. Messineo A, Filler RM, Bahoric A, Smith CR. Repair of tracheal defects with cryopreserved cartilaginous allografts. J Pediatr Surg. 1992;27:1131e1135. 22. Yokomise H, Inui K, Wada H, et al. High-dose irradiation prevents rejection of canine tracheal allografts. J Thorax Cardiovasc Surg. 1994;107:1391e1397. 23. LaMuraglia GM, Adili F, Schmitz-Rixen T, Michaud NA, Flotte TJ. Photodynamic therapy inhibits experimental allograft rejection. Circulation. 1995;92:1919e1926.
Please cite this article in press as: Ershadi R, et al., Transplantation of the decellularized tracheal allograft in animal model (rabbit), Asian Journal of Surgery (2017), http://dx.doi.org/10.1016/j.asjsur.2017.02.007
MODEL
Asian Journal of Surgery (2017) xx, 1e5
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.e-asianjournalsurgery.com
ORIGINAL ARTICLE
Transplantation of the decellularized tracheal allograft in animal model (rabbit) Reza Ershadi*, Mohamadbagher Rahim, Siavash Jahany, Siamak Rakei Department of Thoracic Surgery, Valiasr Hospital, Tehran University of Medical Science, Iran Received 3 January 2017; received in revised form 15 February 2017; accepted 20 February 2017
KEYWORDS decellularized allograft; rabbits; tracheal transplantation
Summary Background: It has been difficult to perform tracheal allotransplantation without immunosuppression. To determine whether decellularized trachea can be used in tracheal replacement, we evaluated the viability of decellularized tracheal allografts in a rabbit model of immunosuppressant-free transplantation. Method: Half allograft (Group 1, n Z 7) was harvested from adult New Zealand white rabbits, subjected to a detergenteenzymatic method (containing sodium deoxycholate/DNase lavations) of decellularization for as many cycles as needed, and the other half was stored in phosphate-buffered saline at 4 C as a control (Group 2, n Z 7). Bioengineered and control tracheas were then implanted in 14 age-matched rabbits. Results: In Group 1 (decellularized), all rabbits survived, whereas in Group 2(control), all rabbits died of airway obstruction between 20 days and 45 days after operation. Histologically, the decellularized allografts displayed complete regeneration of epithelium and cartilage, but the fresh allografts showed inflammatory changes, no epithelium, and no cartilage. Conclusions: Complete regeneration of epithelium and cartilage tracheal rings occurred after the implantation of decellularized tracheal allografts without immunosuppression. We demonstrate that the decellularized process reduces the allogeneic response to the trachea. Therefore, we believe that the decellularized tracheal allograft is an excellent choice for tracheal replacement. To our knowledge, this is the first study to observe the long-term (1 year) prognosis of this transplanted trachea. ª 2017 Asian Surgical Association and Taiwan Robotic Surgical Association. Publishing services by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).
* Corresponding author. Department of Thoracic Surgery, Valiasr Hospital, Bagherkhan Street, Tehran, Iran. E-mail address: [email protected] (R. Ershadi). http://dx.doi.org/10.1016/j.asjsur.2017.02.007 1015-9584/ª 2017 Asian Surgical Association and Taiwan Robotic Surgical Association. Publishing services by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article in press as: Ershadi R, et al., Transplantation of the decellularized tracheal allograft in animal model (rabbit), Asian Journal of Surgery (2017), http://dx.doi.org/10.1016/j.asjsur.2017.02.007
+
MODEL
2
R. Ershadi et al.
1. Introduction
2.1. Matrix bioengineering
Tracheal resection with primary reconstruction is the only curative treatment in patients with a variety of benign or malignant tracheal lesions.1 Unfortunately, the resectable length of the diseased trachea is usually restricted to approximately 30% of the total length in children and around 6 cm in adults, and any further increase in this resection rate depends on the development of a safe tracheal replacement.2 This is not yet clinically available because almost every attempt to provide an autologous or synthetic safe and reproducible tracheal graft has been disappointing thus far,1 casting doubt on the future of longsegment tracheal replacement.3 Tissue bioengineering has emerged as the most promising technique to create a nearnormal trachea,1,4 and encouraging early experimental results5 and clinical applications6 have been reported. To determine whether decellularized trachea can be a realistic substitute in replacement procedures, we evaluated the viability of and the histologic changes in decellularized tracheal allografts in rabbit model of transplantation without immunosuppression.
The overlying tissue of the harvested tracheas was stripped off, deprived of trachealis muscle, and rinsed four times (for 4 hours each) in PBS containing 1% antibiotic and antimycotic solution. Thereafter, the protocol continued as previously reported.7 Briefly, the tissue was processed with multiple treatment cycles, including the following steps: the tissue was stored in Aqua milliQ (Millipore) for 48 hours at 4 C and then incubated in 4% sodium deoxycholate for 4 hours and 2000 kU of DNase-I in 1 mol/L NaCl (Sigma Chemical Company) for 4 hours. The presence of cellular elements and major histocompatibility complex (MHC) cells were verified histologically after each cycle. The bioengineering process lasted for 35 days, corresponding to 17 detergenteenzymatic method (DEM) cycles, after which the bioengineered tracheal matrices showed few residual nuclei of chondrocytes but a complete removal of MHC class I and II antigens (Figure 1A).8 In contrast, MHC class I and II expression was ubiquitous in control tracheas recipient.8 The transplantation was performed with recipient rabbits of matched age. Under anesthesia, the recipient was placed in a supine position and a six-ring tracheal segment was resected through a midline cervical incision. Allograft transplantation (control, n Z 7; decellularize, n Z 7) was performed in an end-to-end continuous fashion using absorbable monofilament (5-0 PDS, Ethicon, USA) (Figure 1B), and the wound was closed in the usual manner. Postoperatively,all the 14 recepient rabbits were observed for 3 hours before being returned to their individual cages. They received standard feed and water. Analgesics (buprenorphine, 0.05 mg/kg) were administered twice a day on postoperative Day 1. Antibioprophylaxis (enrofloxacin, 10 mg/kg) was administered postoperatively over 5 days. No immunosuppressive agents or steroids were given. Examination with a flexible bronchoscope was performed on the 5th and 15th postoperative days and every 3 months thereafter, and each tracheal graft was examined grossly and microscopically (pathologic examination with hematoxylin and eosin staining)when the animal died or was sacrificed from Day 20 to Day 365.
2. Material and methods Special humane animal care measures were taken in compliance with the “Principles of laboratory animal care” formulated by the National Society for Medical Research and the “Guide for the care and use of laboratory animals” prepared by the Institute of Laboratory Animal Resources, National Research Council, and published by the National Academy Press, revised 1996. Each allograft was harvested from adult male New Zealand white rabbits weighing 3170e4575 g. The animals were anesthetized with intravenous administration of pentobarbital (30 mg/kg). They were placed in a supine position under spontaneous ventilation without an endotracheal tube, and either harvesting or transplantation was performed. The cervical trachea was exposed through a midline cervical incision and then harvested from the cricoid cartilage to the carina. To obtain maximum graft viability, grafts were harvested from beating heart donors without warm ischemia. The harvested segment was then divided into two groups. One half (control) was placed in a stock solution made of phosphate-buffered saline (PBS), containing 1% antibiotic and antimycotic solution. The other half was similarly stored for 24 hours but then submitted to the bioengineering protocol, as described below.
Figure 1
3. Results Table 1 summarizes the results for the 14 transplant recipients. In the decellularized group (Group 1), the rabbits were alive for the duration of the experiment and were subsequently killed for the histologic examinations. In control group (Group 2), however, all the seven rabbits died
(A) A decellularized trachea. (B) Transplantation of decellularized tracheal allograft.
Please cite this article in press as: Ershadi R, et al., Transplantation of the decellularized tracheal allograft in animal model (rabbit), Asian Journal of Surgery (2017), http://dx.doi.org/10.1016/j.asjsur.2017.02.007
+
MODEL
Decellularized tracheal allograft transplantation Table 1
3
Findings of 14 rabbits undergoing tracheal allotransplantation
Group
Rabbit n
Death or sacrifice day
Epithelium regeneration
Cartilage
Lymphocyte infiltration
Mononuclear cell infiltration
1-Decellularized allograft
1 2 3 4 5 6 7 1 2 3 4 5 6 7
200 Sacrificed 220 Sacrificed 280 Sacrificed 320 Sacrificed 340 Sacrificed 365 Sacrificed 365 Sacrificed 20 Died 25 Died 29 Died 34 Died 39 Died 41 Died 45 Died
þ þ þ þ þ þ þ
þ þ þ þ þ þ þ
þ þ þ þ þ þ þ
þ þ þ þ þ þ þ
2-Control allograft
of airway obstruction on the 20th, 25th, 29th, 34th, 39th, 41st, and 45th postoperative days. On bronchoscopy, signs of epithelial regeneration appeared early in the posterior wall of the grafts and then around the entire circumference, and airways were open and no stenosis or malacic changes were seen in Group 1 (Figure 2). However, in Group 2, airways were almost completely obstructed by fibrosis and granulation tissue. Histologically, remarkable lymphocytic and mononuclear cell infiltration, severe fibrosis, and destruction of the cartilaginous rings were observed in Group 2 (Figure 3A). In Group 1, complete epithelial regeneration (well-differentiated respiratory epithelium) and regeneration of cartilage tracheal rings were observed. Lymphocytic and mononuclear cell infiltration were not observed in Group 1 (Figure 3B).
4. Discussion In contrast with other successful organ replacements, which take place in sterile mesenchymal environments
(e.g., the liver, kidney, and heart), the airway represents an interface between the mammal and the external environment. Unsurprisingly, its mucosa holds immunologically active cells playing a key role in airway transplantation,9,10 and these contribute to acute allograft rejection, requiring high-dose immunosuppression.11,12 Replacement of the trachea has many unresolved surgical problems. Various efforts have been made to accomplish this using prosthetic materials, different autologous tissues, and homografts in both experimental and clinical settings.13,14 A variety of prosthetic materials have been tested, but clinically suitable prostheses have not yet been developed because of insufficient epithelialization, bacterial infection, obstruction caused by excessive granulation, and anastomotic leakage. Reconstruction with various autogenous tissues, such as pericardium,15 periosteum,16 esophagus,17 and small bowel,18 all reinforced with a stent, has been reported. However, their clinical application must be pursued with caution. Studies19,20 of antigenicity have shown that the human tracheal epithelium develops HLA-DR antigen, which activates T lymphocytes and may play an important
Figure 2 (A) Bronchoscopic examination of decellularized tracheal allograft groups showing a whitish aspect of the graft lumen on Day 5. (B) Epithelial regeneration appearing early on Day 15. (C) Final bronchoscopy on Day 365 with complete epithelial regeneration.
Please cite this article in press as: Ershadi R, et al., Transplantation of the decellularized tracheal allograft in animal model (rabbit), Asian Journal of Surgery (2017), http://dx.doi.org/10.1016/j.asjsur.2017.02.007
+
MODEL
4
R. Ershadi et al. segments. Our experimental findings indicate that the decellularized tracheal allograft has excellent long-term tissue viability and reduced antigenicity of the graft. It is an excellent choice for tracheal replacement. This study is the first to observe the long-term (1 year) prognosis of this transplanted trachea. In conclusion, complete regeneration of epithelium and cartilage tracheal rings occurred after the implantation of decellularized tracheal allografts without immunosuppression. Thus, the decellularized process reduces the allogeneic response to the trachea.
Conflicts of interest The authors have no conflicts of interest to declare.
References
Figure 3 Histologic findings of the control and decellularized tracheal allotransplantation. (A) Remarkable cellular infiltration (lymphocyte and mononuclear cell), fibrosis and destruction of cartilaginous rings in the transplanted graft of the control group. (B) Respiratory epithelial regeneration (block arrow and inset), cartilage ring, and graft revascularization (small arrow) in the decellularized tracheal allograft. Hematoxylineeosin staining 200. Inset 400.
role in graft rejection. The use of immunosuppressants to control immunologic reactions will certainly attenuate graft rejection but will also increase airway infection after tracheal replacement. Therefore, reducing the antigenicity of the allograft itself is an excellent method of control. For this purpose, cryopreservation,21 radiation therapy,22 and photodynamic therapy23 have been reported. The present results demonstrate that the DEM process is a simple and effective method to bioengineer tracheal matrices lacking any MHC antigens while maintaining a structural integrity similar to that of native tracheas and, most importantly, of sufficient length to have potential clinical application. In our experiment, after implantation of decellularized tracheal allografts, complete regeneration of epithelium and cartilage was observed. There was no lymphocytic infiltration of or under the epithelial layer. On bronchoscopic examination, the tracheal lumen was open and no malacic changes were observed in the transplanted
1. Grille HC. Tracheal replacement: a critical review. Ann Thorax Surg. 2002;73:1995e2004. 2. Gasser HA, Grille HC, Shadier MB, et al. Long-term survival after resection of primary adenoid cystic and squamous cell carcinoma of the trachea and carina. Ann Thorax Surg. 2004; 78:1889e1896. 3. Birchall M, Macchiarini P. Airway transplantation: a debate worth having? Transplantation. 2008;85:1075e1080. 4. Macchiarini P. Trachea-guided generation: de ´ja ` vu all over again? J Thorax Cardiovasc Surg. 2004;128:14e16. 5. Walles T, Giere B, Hofmann M, et al. Experimental generation of a tissue-engineered functional and vascularized trachea. J Thorax Cardiovasc Surg. 2004;128:900e906. 6. Macchiarini P, Walles T, Biancosino C, Mertsching H. First human transplantation of a bioengineered airway tissue. J Thorax Cardiovasc Surg. 2004;128:638e641. 7. Conconi MT, De Coppi P, Di Liddo R, et al. Tracheal matrices, obtained by a detergent-enzymatic method, support in vitro the adhesion of chondrocytes and tracheal epithelial cells. Transpl Int. 2005;18:727e734. 8. Jungebluth P, Asnaghi A, Bellini S, et al. Structural and morphologic evaluation of a novel detergent enzymatic tissueengineered tracheal tubular matrix. J Thorax Cardiovasc Surg. 2009;138:586e593. 9. Barker E, Murison P, Macchiarini P, et al. Early immunological changes associated with laryngeal transplantation in a major histocompatibility complex-matched pig model. Clin Exp Immunol. 2006;146:503e508. 10. Tojo T, Niwaya K, Sawabata N, et al. Tracheal replacement with cryopreserved tracheal allograft: experiment in dogs. Ann Thorax Surg. 1998;66:209e213. 11. Macchiarini P, Lenot B, de Montpre ´ville VT, et al. Heterotopic pig model for direct revascularization and venous drainage of tracheal allografts. Paris-Sud University Lung Transplantation Group. J Thorax Cardiovasc Surg. 1994;108:1066e1075. 12. Macchiarini P, Mazmanian GM, de Montpre ´ville VT, et al. Experimental tracheal and tracheoesophageal allotransplantation. Paris-Sud University Lung Transplantation Group. J Thorax Cardiovasc Surg. 1995;110:1037e1046. 13. Alonso WA, Bridger GP, Bordley JE. Tracheal transplantation in dogs. Laryngoscope. 1972;82:204e209. 14. Grille HC. Tracheal replacement [Editorial]. Ann Thorax Surg. 1990;49:864e865. 15. Idriss FS, DeLeon SY, Ilbawi MN, Gerson CR, Tucker GF, Holinger L, et al. Tracheoplasty with pericardial patch for extensive tracheal stenosis in infants and children. J Thorax Cardiovasc Surg. 1984;88:527e535.
Please cite this article in press as: Ershadi R, et al., Transplantation of the decellularized tracheal allograft in animal model (rabbit), Asian Journal of Surgery (2017), http://dx.doi.org/10.1016/j.asjsur.2017.02.007
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Please cite this article in press as: Ershadi R, et al., Transplantation of the decellularized tracheal allograft in animal model (rabbit), Asian Journal of Surgery (2017), http://dx.doi.org/10.1016/j.asjsur.2017.02.007