Tracheal Replacement With a Silicone-Stented, Fresh Aortic Allograft in Sheep

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1 Tracheal Replacement With a Silicone-Stented, Fresh Aortic Allograft in Sheep Hisashi Tsukada, MD, PhD, Armin Ernst, MD, Sidhu Gangadharan, MD, Simon Ashiku, MD, Robert Garland, RTT, Diana Litmanovich, MD, and Malcolm DeCamp, MD Chest Disease Center and Department of Radiology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts Background. Tracheal tissue regeneration after allogeneic aortic transplants in sheep has been reported. We sought to confirm these findings and elucidate the mechanism of this transformation. Methods. Ten male sheep underwent cervical tracheal replacement with fresh, descending thoracic aortic allografts, 8 cm long, from female sheep, without postoperative immunosuppressive therapy. A 10-cm silicone stent was placed to prevent airway collapse. Graft evaluations with flexible bronchoscopy and computed tomography were conducted between 2 weeks and 1 year after surgery. Results. There were no procedural deaths, but 6 animals died or required euthanasia between 12 days and 3 months postoperatively owing to severe tracheitis, cervical lymphadenitis, pneumonia, graft necrosis, stent migration, or airway obstruction after stent removal. The 4 remaining sheep were euthanized as planned at 6 to 12 months after surgery. Harvested tracheas revealed no evidence of graft incorporation into the surrounding tissue, and there was no histologic evidence of any neocartilage within or around the graft at any point. Bronchoscopy revealed marked graft necrosis in the 4 animals surviving to planned euthanasia. In all sheep, computed tomography imaging revealed that the graft was replaced by connective tissue without any signs of cartilage regeneration. Image analysis also indicated profound shortening of the grafted area up to 87.5% at 1 year after implantation, secondary to axial shift of the native trachea. Conclusions. Fresh aortic allografts appear to be unsuitable for primary tracheal replacement. However, the observed graft shortening may allow for two-staged, end-to-end reconstruction of large tracheal defects with temporary grafting techniques. (Ann Thorac Surg 2010;89:253 8) 2010 by The Society of Thoracic Surgeons The management of long-segment tracheal disease remains challenging. Currently, pathologic processes affecting more than half of the length of the trachea, such as adenoid cystic carcinoma, are managed with palliative radiation or stenting as a result of limitations on the maximal length of tracheal resection or a suitable replacement construct [1]. Attempts to create long grafts have resulted in necrosis or extensive granulation tissue obstructing airflow and secretion clearance. An ideal graft construct must be biocompatible, nontoxic, nonimmunogenic, and noncarcinogenic [1]. Numerous tracheal substitutes have been reported with limited success [2], but an optimal long tracheal bioprosthesis has yet to be identified. The current trend in tracheal replacement research focuses on regenerative medicine using tissue-engineering techniques [3]. However, Martinod and colleagues [4, 5] reported tracheal regeneration in sheep after replacing the trachea with fresh, autologous, and allogeneic aortic grafts. This group also reported successfully replacing the carina Accepted for publication Sept 1, Address correspondence to Dr DeCamp, Chest Disease Center, Beth Israel Deaconess Medical Center, Harvard Medical School, 185 Pilgrim Rd, Deaconess Suite 201, Boston, MA 02215; mdecamp@bidmc. harvard.edu. using a similar method [6]. Jaillard and associates [7] confirmed these results in a pig model. Wurtz and others [8] have also reported successful total tracheal replacements with aortic allografts harvested from brain-dead donors in a patient with mucoepidermoid carcinoma and in another with adenoid cystic carcinoma. The mechanism of tracheal regeneration in these cases is unknown. We sought to confirm the above results in sheep and to document the endoscopic, histologic, and radiologic steps of tracheal regeneration in sheep using freshly procured aortic allografts. Material and Methods The study was approved by the Beth Israel Deaconess Medical Center s Institutional Animal Care and Use Committee. All animals were cared for by a veterinarian in accordance with US Department of Agriculture regulations and the Guidelines for the Care and Use Committee of Laboratory Animals of Beth Israel Deaconess Medical Center and the National Research Council s Guide for the Care and Use of Laboratory Animals, prepared by the Institute of Laboratory Animals and published by the National Institutes of Health (Publication No , 1996) by The Society of Thoracic Surgeons /10/$36.00 Published by Elsevier Inc doi: /j.athoracsur

2 254 TSUKADA ET AL Ann Thorac Surg TRACHEAL REPLACEMENT WITH FRESH AORTA 2010;89:253 8 Table 1. Result of Tracheal Replacement With a Fresh Aortic Allograft Sheep No. Complication Follow-up Grafted Area Contraction (%) Epithelialization Outcome Other 1 Tracheitis 12 days 0... Euthanized mo 87.5 Mucociliary Planned sacrificed Stent replacement at 6mo 3 Stent migration 3 mo Euthanized Stent removal at 3 mo 4 Stent migration 3 mo Dead... 5 Allograft infection 1 mo Euthanized... 6 Aspiration 1 mo Dead mo 82 Squamous Planned sacrificed mo 75 Mixed Planned sacrificed... 9 Allograft infection 14 days 0... Euthanized mo 87.5 Mucociliary Planned sacrificed Stent replacement at 6mo Allograft Preparation and Experimental Design The descending aorta was harvested from 10 female sheep (range, 70 to 90 kg) through a left lateral thoracotomy immediately after the animal was euthanized. The harvested aortic allograft was simply washed in 1 L of normal (0.9%) saline and preserved in saline solution at room temperature. After less than 1 hour, the allografts were transplanted into 10 male sheep (range, 30 to 40 kg), where they replaced sections of the cervical trachea without postoperative immunosuppressive therapy. Anesthesia The recipient sheep received 40 mg ketamine (Fort Dodge Animal Health, Fort Dodge, IA) intramuscularly and 25 mg/kg thiopental (HOSPIRA, Inc, Lake Forest, IL) intravenously and were then intubated. Ventilation was maintained (model 2000 ventilator; Hallowell EMC, Pittsfield, MA) at a tidal volume of 10 ml/kg and 10 to 15 breaths/min. General anesthesia was maintained with inhaled 60% oxygen and 1% to 3% isoflurane (Webster Veterinary, Sterling, MA). The animals also received a crystalloid solution (10 ml kg 1 h 1 ) during the surgery. Oxygen concentration (pulse oximetry), heart rate, and body temperature were monitored throughout surgery. Enrofloxacin (Baytril; Bayer Corp, Leverkusen, Germany) was administered intramuscularly at 5 mg/kg at anesthesia induction and at 2.5 mg/kg intramuscularly per day until postoperative day 4. Postoperative analgesia was provided by a fentanyl transdermal patch (ALZA Corp, Mountain View, CA) (1 g/kg). Surgical Procedure With the animal supine, the cervical trachea was exposed through a median cervical incision, and a tracheotomy tube (6 mm outer diameter) was placed three rings distal to the proposed tracheal resection site through a small tracheotomy. Anesthesia was maintained through the tracheotomy during tracheal resection and implantation of the aortic allograft. A 6-cm length (10 rings) of circumferential cervical trachea was excised. An 8-cm length of the aortic allograft was then implanted by performing end-to-end anastomoses with 4-0 nonabsorbable monofilament (Prolene; Ethicon, Somerville, NJ) running sutures. A silicone stent, 10 cm long and 14 mm in diameter (Tracheobronxane Dumon; Novatech, La Ciotat, France), was inserted before completing the distal anastomosis. Ventilation was resumed through the oral endotracheal tube, the tracheotomy tube was removed, and the tracheotomy was closed with interrupted sutures. Internal coverage of the entire aortic graft by the silicone stent was confirmed by flexible bronchoscopy. To secure the stent and prevent migration, two sutures were placed at both ends of the stent under direct bronchoscopic visualization. Metallic markers were placed on the outside of the trachea to indicate the proximal and distal ends of the performed tracheal resection. The cervical incision was closed, and the animal was immediately extubated after emerging from anesthesia. Follow-Up and Evaluation All animals were examined daily after surgery. Bronchoscopy (model BF 200; Olympus America, Inc, Center Fig 1. Sheep #3 at 3 months. (A) Computed tomography revealed distal airway migration of a silicone stent. (B) The migrated stent with graft was removed by rigid bronchoscope.

3 Ann Thorac Surg TSUKADA ET AL 2010;89:253 8 TRACHEAL REPLACEMENT WITH FRESH AORTA 255 Fig 2. Parallel bronchoscopic (left) and computed tomography (right) images in sheep 10. (A) Yellowish-white aortic graft was seen through the stent and computed tomography revealed thick reactive tissue surrounding the stent at 2 weeks after surgery. (B) Partial aortic graft defect was seen and hypervascular reaction tissue was observed at 2 months. (C) Aortic graft invisible and grafted area was replaced with connective tissue at 1 year. Valley, PA) and computed tomography (CT; Aquilion 64 TSX-101A, Toshiba America Medical Systems, Inc, Tustin, CA) were performed at 2 weeks and at 1, 2, 3, 6, and 12 months to evaluate the graft and stent. For these examinations, animals were intubated and received general anesthesia. If the animal showed any respiratory symptoms, bronchoscopy was performed. Animals that did not respond to treatment or were determined to be suffering based on veterinary examination were euthanized. Animals were planned to be humanely killed at 3, 6, 9, and 12 months after the surgery for macroscopic and microscopic examination. Histologic Examination Tissue from excised tracheal allografts were fixed in 10% phosphate-buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin and safranin O to evaluate the degree of epithelialization and neocartilage formation. Results Morbidity and Mortality A summary of the results is given in Table 1. All sheep tolerated the surgical procedure well and there was no perioperative mortality. However, 6 of 10 sheep (60%) had complications. Sheep 1 had severe tracheitis, which did not respond to medication. As a result, sheep 1 was euthanized on postoperative day 12. In sheep 3, the CT scan performed at 3 months revealed that the silicone stent had migrated to the proximal trachea (Fig 1A). The migrated stent was removed with a rigid bronchoscope, and the aortic graft was unexpectedly extracted with the stent (Fig 1B). Sheep 3 exhibited pneumonia 1 week after stent removal owing to airway stenosis at the grafted area and was therefore sacrificed. Sheep 4 had an unremarkable 3-month postoperative follow-up examination, but died of airway obstruction as a result of a migrated stent immediately after returning to the farm. Sheep 5 was euthanized at 1 month after surgery owing to cervical lymphadenitis and graft aortitis refractory to intravenous antibiotics. Sheep 6 expired after severe aspiration after premedication before the intubation during the 1-month follow-up examination. Sheep 9 was euthanized at 2 weeks after implantation because of pneumonia secondary to the graft necrosis. The remaining 4 sheep (sheep 2, sheep 7, sheep 8, and sheep 10) were euthanized according to the protocol at 12, 6, 9, and 12 months, respectively. To prevent stent migration, sheep 2 and 10 were given a Fig 3. Bronchoscopic findings of sheep 8 at 1 month after surgery. (A) Yellowish-white specimen was observed at the end of the silicone stent. (B) Same specimen confirmed as necrotic aorta with calcification (hematoxylin and eosin; original magnification, 100).

4 256 TSUKADA ET AL Ann Thorac Surg TRACHEAL REPLACEMENT WITH FRESH AORTA 2010;89:253 8 using reconstructed CT images. There was no difference in the number of cartilage rings at each of the follow-up examinations after surgery. Pathologic Allograft Evaluation The gross morphology of the graft harvested from sheep 1 at postoperative day 12 and the removed stent with graft from sheep 3 (Fig 1) at 3 months showed that neither graft had been incorporated into the surrounding tissue. The length of the grafted area was visibly shortened secondary to axial contraction of the native trachea (Fig 5). Contraction rate (%) was calculated by the following formula: original aortic graft length (8 cm) subtracted by the measured longitudinal length of grafted area between proximal and distal end of the native trachea, Fig 4. Computed tomography traced metal marker in sheep 10 at 2 weeks (A) and 1 year (B). Distal native trachea shifted up toward the proximal; no neocartilage ring was detected at grafted area. replacement silicone stent (18 mm diameter by 4 cm long) at 6 months under general anesthesia with rigid bronchoscopy. Endoscopic Allograft Evaluation Bronchoscopic images of sheep 10, which survived for 12 months, showed that the yellowish-white color of the aortic allograft was initially visible through the clear silicone stent 2 weeks after surgery (Fig 2A). A partial graft defect was observed at 2 months, and the graft itself could no longer be seen at 6 or 12 months (Figs 2B and 2C). A floating yellowish-white specimen, taken by bronchoscopic forceps from the distal side of the silicone stent, was histologically confirmed to be necrotic aortic tissue (Fig 3). Radiographic Evaluation Atypical bilateral lower lobe pneumonias were observed for the first 3 months after surgery in surviving animals. Additionally in sheep 10, CT images compared with the bronchoscopic images revealed that the grafted area had been replaced by reactive connective tissue without any signs of regenerative cartilage (Fig 2). The thickness of the connective tissue noticeably decreased with time in parallel and in response to inflammatory changes. The location of the metal markers along the sheep tracheas indicated a profound shortening of the grafted area (Fig 4). The number of cartilage rings between the first tracheal ring and the distal end of the native trachea, and between the proximal end of the native trachea and the first branch of the right tracheal bronchus, were counted Fig 5. Gross morphology of a harvested specimen in sheep 7 at 6 months. (A) Suture materials were confirmed at the both ends of native trachea. (B) Grafted area length was 1.5 cm in diameter. (C) No evidence of neocartilage formation between proximal and distal native tracheal cartilage ring.

5 Ann Thorac Surg TSUKADA ET AL 2010;89:253 8 TRACHEAL REPLACEMENT WITH FRESH AORTA 257 Fig 6. Necrotic aortic graft without any incorporation into surrounding tissue at 12 days after tracheal replacement (hematoxylin and eosin; original magnification, 50; from sheep 1). Fig 8. Mucociliary epithelium was confirmed on the fibrous connective tissue at grafted area on 1 year after the surgery (hematoxylin and eosin; original magnification, 630; from sheep 2). divided by the original aortic graft length (8 cm). The maximum grafted area of contraction was 87.5% in both sheep 2 and 10 at 1 year after the implantation. The histologic findings for the grafted area harvested from sheep 1 revealed grafted aorta necrosis with calcification with no signs of angiogenesis into the graft (Fig 6). Evidence of grafted area epithelialization was confirmed in all sheep that survived beyond 6 months, suggesting that the transformation from squamous metaplasia to mucociliary epithelium is time dependent. In addition, no confirmation of cartilage regeneration at the grafted area was visible by safranin O staining (Fig 7). The grafted area for both sheep 2 and 10 was completely replaced by fibrous tissue with mucociliary epithelium at 1 year (Fig 8). Comment Fig 7. Graft harvested from sheep 10 at 1 year after tracheal replacement (original magnification, 6). (A) Mucociliary epithelialization was observed at the grafted area without signs of neocartilage formation (hematoxylin and eosin staining). (B) Safranin-O stains were strongly positive only at the native tracheal cartilage. Several approaches to tracheal reconstruction using a graft interposition have been reported with limited success [2]. Tracheal regeneration after fresh aortic allograft without immunosuppressive therapy has been reported by two groups [4, 7]. These reports seemed to be breakthroughs in this field. We therefore sought to replicate the results and to begin to understand the sequence of events resulting in tracheal regeneration from the aortic construct. If successful, we had subsequent plans to test the utility of cryopreserved aortic allografts for logistical convenience and to reduce graft immunogenicity [9] and bacterial resistance [10]. Our experimental design was based on the published prior studies. The harvested grafts were expected to serve as a scaffold for tracheal regeneration and were therefore simply preserved in saline solution according to the protocol by Jaillard and colleagues [7]. Minor procedural details were intentionally changed, such as using nonabsorbable sutures to confirm the anastomosis sites at necropsy and adding postoperative CT examinations to hopefully detect stepwise tracheal regeneration. Also, we replaced a 6-cm length of trachea defect with an 8-cm aortic graft. We used a longer length of aortic graft as compared with the length of tracheal defect to minimize graft tension caused by the distal trachea pulling the graft down into the thoracic inlet. The disposition of replaced aortic tissue is unknown. Martinod and colleagues [4] mentioned that the replaced autologous aorta had almost completely disappeared and had been replaced by extensive inflammatory tissue 1 month after implantation. Jaillard and associates [7] reported that the implanted allogeneic aorta progressively vanished. These descriptions seem to contradict their conclusions of tracheal regeneration from the aortic graft and transforms into a conduit containing the major tracheal components. In the harvested grafts from sheep 1 and 5 and stent with aortic graft from sheep 3, all aortic allografts were visibly not connected into the host tissue. Suspicious sections of the aortic graft found loose in the distal airway were histologically confirmed to be

6 258 TSUKADA ET AL Ann Thorac Surg TRACHEAL REPLACEMENT WITH FRESH AORTA 2010;89:253 8 necrotic aortic tissue. These observations may represent graft rejection or simply ischemic necrosis as there was no evidence for graft incorporation. The unusual bilateral, lower-lobe pneumonia detected by CT scans performed up to 3 months after surgery may have been caused by aspiration of the necrotic aortic graft debris. In one sheep, the CT scan detected a dehisced, rolled-up aortic graft at the proximal end of the stent. Our observations support the hypothesis that the grafts may become necrotic and undergo autolysis with internal drainage or external expectoration and are entirely consistent with the vanished graft observations in the prior studies. Axial shift of the native trachea after circumferential tracheal grafting has been reported [11, 12]. The native rings may have been displaced toward the reconstructed trachea by shifting as healing progressed. Tsukada and Osada [13] reported tracheal shift caused by accordionlike prosthetic shrinkage owing to the spiral configuration of the stent in a canine mediastinal tracheal reconstruction model. Unfortunately, radiologic follow-up was not used in their study to confirm native tracheal axial contraction. In our study, CT images traced the movement of metallic markers in 2 animals, revealing axial shortening of the grafted area secondary to the axial shifting of the native trachea. Graft anastomoses sites were clearly visible at the time of tissue excision by the presence of nonabsorbable anastomotic sutures and the metallic markers. These data suggest that tracheal axial shift is a more likely phenomenon than tracheal regeneration. In normal sheep, the width of a cartilage ring is 2 to 3 mm at 30 kg and 4 to 6 mm at 70 kg. The mechanism of native tracheal axial shift is at least partially explained by normal tracheal growth accompanied by increased width of the cartilage rings. Further investigation may be needed to confirm tracheal shifting process using a mature animal model. We observed axial shift of the trachea most likely as a result of natural healing of tracheal defects (with and without growth of the animal). Complete, spontaneous reconnection of the native trachea was not observed in sheep that reached 1 year of follow-up. However, endto-end anastomosis could be safely performed at the 1-year follow-up time given the close proximity of the native tracheal ends. A two-stage reconstruction of the trachea with temporary grafting could be effective for repairing large tracheal defects. An appropriate temporary tracheal graft or scaffold should be determined. On the basis of these results, we conclude that fresh aortic allografts are not feasible tracheal replacement conduits. We found no evidence of tracheal regeneration after aortic allograft interposition. Our data suggest that aortic allografts induce intense paratracheal inflammation yet eventually become necrotic, slough, and are expectorated, aspirated, or swallowed. The native trachea undergoes maturation of the paratracheal inflammatory process with subsequent axial shortening. This phenomenon may allow for safe, two-stage end-to-end reconstruction of larger tracheal defects using a first-stage temporary grafting technique. References 1. Grillo HC. Tracheal replacement: a critical review. Ann Thorac Surg 2002;73: Osada H. Artificial trachea. J Bronchol 2006;13: Kojima K, Bonassar LJ, Roy AK, Vacanti CA, Cortiella J. Autologous tissue-engineered trachea with sheep nasal chondrocytes. J Thorac Cardiovasc Surg 2002;123: Martinod E, Seguin A, Pfeuty K, et al. Long-term evaluation of the replacement of the trachea with an autologous aortic graft. Ann Thorac Surg 2003;75: Martinod E, Seguin A, Holder-Espinasse M, et al. Tracheal regeneration following tracheal replacement with an allogenic aorta. Ann Thorac Surg 2005;79: Seguin A, Martinod E, Kambouchner M, et al. Carinal replacement with an aortic allograft. Ann Thorac Surg 2006; 81: Jaillard S, Holder-Espinasse M, Hubert T, et al. Tracheal replacement by allogenic aorta in the pig. Chest 2006;130: Wurtz A, Porte H, Conti M, et al. Tracheal replacement with aortic allografts. N Engl J Med 2006;355: Motomura N, Imakita M, Yutani C, et al. Histologic modification by cryopreservation in rat aortic allografts. Ann Thorac Surg 1995;60(Suppl):S Lizler P-Y, Thomas P, Danielou E, et al. Bacterial resistance of refrigerated and cryopreserved aortic allografts in an experimental virulent infection model. J Vasc Surg 1999;29: Pressman JJ, Simon MB. Tracheal stretching and metaplasia of the tracheal rings from cartilage to bone following the use of aortic homografts. Am Surg 1959;25: Bryant LR. Replacement of tracheobronchial defects with autogenous pericardium. J Thorac Cardiovasc Surg 1964;48: Tsukada H, Osada H. Experimental study of a new tracheal prosthesis: pored Dacron tube. J Thorac Cardiovasc Surg 2004;127: