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Improvement of tracheal autograft survival with transplantation into the greater omentum
Improvement of Tracheal Autograft Survival With Transplantation Into the Greater Omentum Jian Li, MD, Peizhang Xu, MD, Hongyi Chen, MD, Zequan Yang, MD, and Qilian Zhang, MD Departments of Cardiothoracic Surgery and Endoscopy, The First Hospital of Beijing Medical University, Beijing, China
Background. Reconstruction of tracheal defects with tracheal grafts is not practicable clinically because the problem of tracheal graft revascularization has not been solved successfully. We conducted experiments to investigate efficacy of implanting tracheal graft into the greater omentum for revascularization and possibility of adopting staged transplantation procedure for repair of tracheal defect. Methods. Twenty-four mongrel dogs were randomly and equally divided into groups I and II. Six-ring cervical tracheal segments were harvested as autografts. The grafts were wrapped with the omentum and placed into the peritoneal cavity in group I, and reimplanted with omentopexy in group II. Four grafts were examined
macroscopically, microscopically, and35S-autoradiographically on postoperative days 3, 7, and 14, respectively. Results. Epithelium loss was evident in the mucosas of the grafts except the 4 from group I. Percentages of viable chondrocytes assessed with 35S-autoradiography were significantly higher in tracheal grafts from group I than group II. All tracheal grafts with their own omental pedicles could be brought to any portions of the trachea. Conclusions. We conclude that prior implantation of tracheal graft in the omentum is beneficial for preservation of its structure, and reconstruction of a tracheal defect with a tracheal graft implanted first into the omentum is feasible.
he reconstruction of extensive circumferential tracheal defect with a graft has not been solved successfully [1]. From the viewpoints of physiology and histocompatibility, no other material w o u l d be superior to the trachea itself as a graft if the p r o b l e m s of revascularization of the tracheal graft and rejection were solved. The experimentation concerned with these p r o b l e m s has been conducted for m o r e than four decades. Up to now, only limited i m p r o v e m e n t has been achieved [2-7[. It has b e e n discovered that by m e a n s of one-stage tracheal transplantation p r o c e d u r e with omentopexy, the tracheal graft can be r e v a s c u l a r i z e d [3-7]. Past e x p e r i m e n t s s h o w e d that not all of the tested animals could survive long, and p o s t m o r t e m studies showed that the animals d i e d of ischemic necrosis of the tracheal grafts. It is evident that reliable a p p r o a c h e s to tracheal transplantation need to be found for tracheal transplantation to be a p p l i e d to h u m a n beings. Studies by Nakanishi a n d associates [7] s h o w e d that all of the tracheal autografts i m p l a n t e d into the greater o m e n t u m survived. We a s s u m e d that tracheal transplantation might be further i m p r o v e d if a revascularized and viable tracheal graft first i m p l a n t e d into the greater o m e n t u m for revascularization could be used to repair the tracheal defect. In the p r e s e n t study, we used the tracheal autotransplantation m o d e l s to avoid the immunorejection, and
used a u t o r a d i o g r a p h y with 3sS-Na2SO4, in addition to macroscopic and microscopic examinations, to d e t e r m i n e the viability of tracheal graft. W e evaluated w h e t h e r implantation of a tracheal graft into the greater o m e n t u m is better for tracheal graft survival than implantation of tracheal graft in situ using omentopexy. Moreover, we investigated the feasibility of using the tracheal graft i m p l a n t e d first into the o m e n t u m for reconstruction of tracheal defect.
T
Accepted for publication July 22, 1995. Address reprint requests to Dr Li, Department of Cardiothoracic SurgeD,, The First Hospital, Beijing Medical University, No. 8 Xishiku Str, Beijing 100034, China.
© 1995 by The Society of Thoracic Surgeons
(Ann Thorac Surg 1995;60:1592-6)
Material and M e t h o d s Twenty-four adult m o n g r e l dogs w e i g h i n g from 10.4 to 15.2 kg were r a n d o m l y and equally assigned into two groups. All dogs were anesthetized with intravenous injection of p e n t o b a r b i t a l s o d i u m (20 mg/kg) and placed in the supine position. Then oral intubation was performed, and the tube was connected to a pressurelimiting respirator. The following experiments were performed.
Operative Procedure GROUP I (N -- 12): TRACHEAL AUTOGRAFT IMPLANTED INTO
THE GREATER OMENTUM. After a midline cervical incision was made, a six-ring s e g m e n t of the trachea was excised and placed in physiologic saline solution containing penicillin G (200 unit p e r milliliter). Intubation was carried out across the operative field with sterile tube for ventilation. Tracheal reconstruction was p e r f o r m e d by e n d - t o - e n d anastomosis with i n t e r r u p t e d silk sutures. After a small m i d d l e laparotomy, the greater o m e n t u m was b r o u g h t out of the peritoneal cavity. The tracheal 0003-4975/95/$9.50 SSDI 0003-4975(95)00839-X
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animals received h u m a n e care in compliance with the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health (NIH publication 85-23, revised 1985). Examinations On postoperative days 3, 7, and 14, four dogs from each group were reanesthetized with injection of pentobarbital sodium intravenously (20 m g per kg) and placed in the supine position. Then intubations were performed and connected to a pressure-limiting respirator.
Fig, 1. Excision of a six-ring segment qfl the trachea and implantation of the graft into the left lower portion of the greater omentum.
autografts were wrapped with the lower portion of the greater o m e n t u m and placed into the peritoneal cavity (Fig 1). During the entire process, care was taken to avoid leaving talc and unnecessary foreign materials in the peritoneal cavity. All incisions were closed in layers. GROUP II (N -- 12): T R A C H E A L A U T O T R A N S P L A N T A T 1 O N W I T H
OMENTOPEXY.As in group I, a six-ring segment of cervical trachea was also excised. A sterile endotracheal tube was positioned into the lower trachea for ventilation. The excised segment was then reimplanted in its original position. The upper anastomosis was sutured first, then the m e m b r a n o u s portion of the lower anastomosis was sutured similarly. The lower anastomosis was completed after the oral endotracheal tube was inserted across both upper and lower anastomoses. Then, after the midline upper laparotomy, an omental pedicle, which was fed by the right gastroepiploic artery, was formed as Messineo and his associates reported [6]. The omental pedicle was brought to the cervical area through the right thoracic cavity by a diaphragmatic defect. This pedicle was used to wrap the autograft, including each anastomosis. Finally, both the cervical and the abdominal incisions were closed in the usual fashion. Postoperative Care Animals were given penicillin G, 1 million units intramuscularly per day for three postoperative days. All
GROUP I. The abdomens of the dogs were reopened through the upper midline incision. The grafts were found. The lumens of the grafts were opened and inspected macroscopically. A laser Doppler flowmeter (Periflux PF3, Stockholm, Sweden) was used to monitor mucosal blood flow of the tracheal grafts, and the greater omentums with the grafts in their tips were dissected from the spleen and the left edge of the stomach (Fig 2). Whether the tracheal graft with omental pedicle flap could be brought to the neck of dog was evaluated. The state of adherence between the graft and the greater o m e n t u m was investigated. The grafts and partial normal tracheas were excised and flushed with physiologic saline solution containing penicillin G (200 units per milliliter). From the cartilage ring of normal tracheas and midportion of the grafts, a small specimen of the full thickness of the tracheal wall (1 × 3 m m 2) was excised and placed in cold Hanks' solution for autoradiographic study. The remains of the grafts and normal trachea were fixed with 10% formalin solution for histologic study. GROUP II. The grafts and partial normal tracheas were removed for histologic and autoradiographic studies as in group I. Light Microscopic A u t o r a d i o g r a p h y Using 35S-Na2S04 Light microscopic autoradiography was used in both groups. The specimens were washed with Hanks" solu-
~.~-
-
J
Fig 2. Operative procedure for extension of the greater omentum with the tracheal graft at its top.
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tion, then placed in culture m e d i u m RPMI 1640 (Gibco, New York) containing 10% fetal calf serum, 200 u/mL of penicillin G, 50/~g/mL of gentamicin, and 20/zCi/mL of 35S-Na2SO4 (specific activity, 150 millicuries per millimole) (Institute of Atomic Energy of China, Beijing, China) and incubated at 37°C for 24 hours. Thereafter, the specimens were flushed with water, fixed in 10% formalin solution for 10 hours, and then washed with running water for 3 hours. Following the standard histologic procedures, 5-p,m histology sections were prepared and m o u n t e d on microscope slides. By the dipping technique, the slides were covered with liquid nuclear emulsion (N4) (Institute of Atomic Energy of China), laid on the exposure box, and exposed at 4°C in a refrigerator for 9 days. The autoradiographs were developed in D19 developer (Beijing Medical University, Beijing, China) for 3 minutes at 19°C and fixed in F5 solution (Beijing Medical University) for 8 minutes, and then stained with hematoxylin and eosin. From five unspecified sections of each specimen, three fields of full-thickness tracheal cartilage were examined microscopically for Ca) histologic appearance, (b) n u m b e r of chondrocytes labeled with 35S-Na2SO4, and (c) n u m b e r of unlabeled chondrocytes. The percentage of labeled chondrocytes was calculated according to the following formula: {Total no. of (b)/Total no. of [(b) + (c)]} × 100 = % labeled chondrocytes. Student's t test for unpaired data was used for statistical analysis.
Ann Thorac Surg 1995;60:1592-6
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Results Macroscopic Assessment GROUP I. The four tracheal grafts from the dogs examined on postoperative day 3 were intact and were w r a p p e d by the greater omentums. The adhesions between the tracheal grafts and the omentums were not sound. Brown mucous-like fluid was packed in the lumens of all the autografts, and there were red ecchymoses in the mucosas of the grafts. Except that there was more fluid in the lumens of the grafts and that the adhesions between the tracheal grafts and the omentums was tight, the findings on postoperative day 7 were similar to those on day 3. The four tracheal grafts examined on postoperative day 14 were intact and had normal appearance in the mucosas. There was more brown fluid in their lumens than in the grafts examined on postoperative day 7.
B Fig 3. Light microscopic sSS-autoradiographs of specimens from the dogs sacrificed on postoperative day 14 in group I (A) and group II (B). (A) The airway is lined by normal mucociliated epithelium and 78% chondrocytes labeled with 35S-Na2S04. (B) The airway is lined by single-layered epithelium and only 67% chondrocytes labeled with 35S-Na2S04. (Hematoxylin and eosin; ×100 before 10% reduction.)
lium was seen in all of the tracheal grafts on postoperative day 7, whereas normal mucociliated epithelium was seen on postoperative day 14 (Fig 3A).
GROUP lI. The ornental flaps were adherent to the membranous portions of the autograft on postoperative day 3 and were adherent circumferentially to the grafts on postoperative days 7 and 14. The changes in mucosal ecchymoses in all the grafts in the group were more severe than in group I.
GROUP II. Phenomena including epithelium loss and tracheal glands, low-grade inflammation, and hemorrhage in the submucosa and lamina propria were observed by light microscopy on postoperative days 3 and 7. Differing from group I, the four grafts examined on postoperative day 14 showed a confluent multilayered epithelium in the intercartilaginous area and a single-layered epithelium in the area underlying the cartilage rings (Fig 3B).
Light Microscopic Aspect GROUP I. Phenomena including epithelium loss and tracheal glands, low-grade inflammation, and hemorrhage in the submucosa and lamina propria were observed by light microscopy in tracheal grafts on postoperative days 3 and 7. A confluent, single-layered, nonciliated epithe-
Light Microscopic Autoradiographic Assessment In all the tracheal grafts of both groups, the chondrocytes in the area close to the o m e n t u m were labeled with 35S-Na2SO4 (see Fig 3), but the chondrocytes in the area far from the o m e n t u m were not labeled. Table I shows the percentage of labeled cells of the grafts in the two
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LI ET AL TRACHEAL AUTOGRAFT SURVIVAL IMPROVED
Table 1. Percentages of Living Chondrocytes in the Tracheal Autografts Assessed With "3SS-Autoradiography PLC of TAS (%) Postoperative Day 3
Group I
80
II
68 70 58 55
73
72
78
Postoperative Day 7 70
75
50 68
76
78
Postoperative Day 14 78
76
70 52 68 67
PLC - p e r c e n t a g e of l a b e l e d c h o n d r o c y t e s ; tografts.
TAS
80
73
70 58
tracheal au-
groups. The results in group I were significantly better than those of group II (p < 0.01). The percentage of labeled chondrocytes of the tested normal tracheas was 100%. This indicates that every normal tracheal chondrocyte has the ability to metabolize the sulfate (Fig 4).
State of the Peritoneal Cavity at Relaperotomy (Group I Only) In group I, no adhesions were found in the peritoneal cavities of any of the dogs. Without impairing the mucosal blood flow of the tracheal grafts, the greater omental pedicles with the tracheal grafts at their tips were formed successfully. All 12 tracheal grafts with their own omental pedicle could be brought to any portion of the trachea. Comment
Many attempts at tracheal replacement have been made experimentally and clinically. Investigative efforts have been made in two general categories: tissue grafts I2-8] and prosthetic grafts [9, 10]. To date, only limited success has been achieved for either [1]. Ideally, a tracheal
Fig 4. Light microscopic3SS-autoradiograph of normal trachea. (Hematoxylin and eosin; ×200 before 10% reduction.)
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allograft would be an excellent substitute for an abnormal trachea if the problems of revascularization of the tracheal graft and rejection were solved. The blood vessels of the trachea arising from the lateral tracheal vascular pedicles are numerous and very fine [11[. Once the trachea is separated from these pedicles, it is impossible to restore blood supply to the trachea immediately. Therefore, after transplantation, the tracheal graft would inevitably experience a period of ischemia. Neville and associates [12] reported that a tracheal autograft, even a segment including three cartilage rings, could not survive after orthotopic transplantation. Morgan and associates [13] reported in 1982 that application of the greater omental flap to an avascular bronchial autograft 2 cm in length resulted in early revascularization and prevented bronchial graft necrosis. Since that report, many experiments concerned with tracheal transplantation using omentopexy have been clone and showed omentopexy can promote revascularization of tracheal grafts. Whether omental wrapping can maintain the viability of tracheal graft is still controversial. Canine studies by Balderman and Weinblatt [3] showed negative data. Although Moriyama and associates [5] and Messineo and associates [6] obtained improved results, still some animals died of airway obstruction due to ischemic necrosis of cartilage rings of the tracheal grafts, as their report showed. Rose and associates [2] reported the only successful clinical tracheal allotransplantation. They placed a tenring allograft in the bed of the recipient's sternocleidomastoid muscle for 3 weeks. An eight-ring abnormal trachea of a 21-year-old male patient was replaced by the graft with the muscular pedicle. It is obvious that this muscular flap cannot be extended to the thoracic portion of trachea. The study by Nakanishi and associates [7] showed that all of the tracheal grafts e m b e d d e d into greater o m e n t u m survived. In the present studies, macroscopic, microscopic, and 3SS-autoradiographic studies were used to evaluate the viability of the tracheal graft. All findings indicate that tracheal grafts e m b e d d e d into the greater o m e n t u m can regain their blood supply earlier, compared with implantation in situ using omentopexy, and suggest that using a staged procedure for tracheal transplantation is reasonable. We assume that the possibility of the improved results in group I, compared with group II, can be attributed to the operative procedures in group I giving less trauma to tracheal gr?ft and leaving the greater o m e n t u m intact, and immobiliz~ ing the tracheal grafts at the early period after transplantation. As the studies revealed, there was no adhesion in t!e peritoneal cavities and the greater omentums with tracheal grafts could be extended to any portion of the trachea. These suggest that using a tracheal graft implanted first into the greater o m e n t u m to repair tracheal defect is feasible. From the findings in group I, ~e consider the optimal time for the second operation to be about 2 weeks after the first operation, because only after
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14 postoperative days did mucosal regenerate a n d the adhesion between the tracheal graft and the o m e n t u m become sound. As a next step, the tracheal autograft a n d allograft should be studied with the staged approach. Using a laser Doppler flowmeter to measure blood flow on various tissues is noninvasive and easy. In a clinical report, Jones a n d Mayou demonstrated the usefulness of the method for skin flap blood flow monitoring after plastic surgery where early diagnosis of vascular insufficiency is of p a r a m o u n t value [14]. In their study, the laser Doppler flowmeter was used to monitor the process of dissection and extension of the greater o m e n t u m to prevent damage to the mucosal blood flow of the tracheal graft. With the laser Doppler flowmeter monitoring, the staged tracheal transplantation procedure used in the studies appeared more reliable. In previous studies, the viability of tracheal graft was d e t e r m i n e d only with the light microscopy, electron microscopy, and long-term observation methods. By these methods, the early changes of the cartilage rings of tracheal graft could not be demonstrated. Autoradiography is an old and useful technique. It was applied to determine the viability of aortic graft [15] a n d osteochondral grafts [16]. We used autoradiography with radioactive sulfate to observe the early changes of the tracheal cartilage after transplantation, a n d found that the living status of the cartilage rings of the tracheal grafts can be determined quantitatively by using this 35S-autoradiographic and histologic method. In conclusion, compared with tracheal transplantation using omentopexy, implantation of tracheal graft into the greater o m e n t u m is an effective method for tracheal graft revascularization a n d survival. It is feasible by using a living tracheal graft after revascularization in the greater o m e n t u m to repair a tracheal defect. The optimal time for the second operation is about 2 weeks after implantation of the tracheal graft into the great o m e n t u m .
Ann Thorac Surg 1995;60:1592-6
References 1. Grillo HC. Tracheal replacement. Ann Thorac Surg 1990;49: 864-5. 2. Rose KG, Sesterhenn K, Wustrow F. Tracheal allotransplantation in man. Lancet 1979;1:433. 3. Balderman SC, Weinblatt G. Tracheal autograft revascularization. J Thorac Cardiovasc Surg 1987;94:434-41. 4. Nagasawa H. Experimental tracheal reconstruction with the use of homograft covered with omental flap. Nippon Kyobu Geka Gakkai Zasshi 1988;36:337-47. 5. Moriyama S, Shimizu N, Teramoto S. Experimental tracheal aUotransplantation using omentopexy. Transplant Proc 1989; 21:2596- 600. 6. Messineo A, Filler RM, Bahoric B, Smith C, Bahoric A. Successful tracheal autotransplantation with a vascularized omental flap. J Pediatr Surg 1991;26:1296-300. 7. Nakanishi R, Shirakusa T, Takachi T. Omentopexy for tracheal autografts. Ann Thorac Surg 1994;57:841-5. 8. Papp C, McCraw JB, Arnold PG. Experimental reconstruction of the trachea with autogenous materials. J Thorac Cardiovasc Surg 1985;90:13-20. 9. Neville WE, Bolanowski PJP, Soltanzadeh H. Prosthetic reconstruction of the trachea and carina. J Thorac Cardiovasc Surg 1976;72:525-38. 10. Mendak SH Jr, Jensik RJ, Haklin MF, Roseman DL. The evaluation of various bioabsorbable materials on the titanium fiber metal tracheal prosthesis. Ann Thorac Surg 1984;38:488-93. 11. Salassa JR, Pearson BW, Payne WS. Gross and microscopical blood supply of the trachea. Ann Thorac Surg 1977;24:100-7. 12. Neville WE, Bolanoski PJP, Scoltazadeh H. Homograft replacement of the trachea using immunosuppression. J Thorac Cardiovasc Surg 1976;72:596-601. 13. Morgan E, Lima O, Goldberg M, Ferdman A, Luk SK, Cooper JD. Successful revascularization of totally ischemic bronchial autografts with omental pedicle flaps in dogs. J Thorac Cardiovasc Surg 1982;84:204-10. 14. Jones BM, Mayou BJ. The laser Doppler flowmeter for microvascular monitoring: a preliminary report. Br J Plast Surg 1982;35:147-9. 15. A1-Janabi N, Gonzalez-Lavin L, Neirotti R, Rose DN. Viability of fresh aortic homografts: a quantitative assessment. Thorax 1972;27:83-6. 16. Depalma AF, Tsaltas TT, Mauler GG. Viability of osteochondral grafts as determined by uptake of 35S. J Bone Joint Surg 1963;45A:1565-78.
Background. Reconstruction of tracheal defects with tracheal grafts is not practicable clinically because the problem of tracheal graft revascularization has not been solved successfully. We conducted experiments to investigate efficacy of implanting tracheal graft into the greater omentum for revascularization and possibility of adopting staged transplantation procedure for repair of tracheal defect. Methods. Twenty-four mongrel dogs were randomly and equally divided into groups I and II. Six-ring cervical tracheal segments were harvested as autografts. The grafts were wrapped with the omentum and placed into the peritoneal cavity in group I, and reimplanted with omentopexy in group II. Four grafts were examined
macroscopically, microscopically, and35S-autoradiographically on postoperative days 3, 7, and 14, respectively. Results. Epithelium loss was evident in the mucosas of the grafts except the 4 from group I. Percentages of viable chondrocytes assessed with 35S-autoradiography were significantly higher in tracheal grafts from group I than group II. All tracheal grafts with their own omental pedicles could be brought to any portions of the trachea. Conclusions. We conclude that prior implantation of tracheal graft in the omentum is beneficial for preservation of its structure, and reconstruction of a tracheal defect with a tracheal graft implanted first into the omentum is feasible.
he reconstruction of extensive circumferential tracheal defect with a graft has not been solved successfully [1]. From the viewpoints of physiology and histocompatibility, no other material w o u l d be superior to the trachea itself as a graft if the p r o b l e m s of revascularization of the tracheal graft and rejection were solved. The experimentation concerned with these p r o b l e m s has been conducted for m o r e than four decades. Up to now, only limited i m p r o v e m e n t has been achieved [2-7[. It has b e e n discovered that by m e a n s of one-stage tracheal transplantation p r o c e d u r e with omentopexy, the tracheal graft can be r e v a s c u l a r i z e d [3-7]. Past e x p e r i m e n t s s h o w e d that not all of the tested animals could survive long, and p o s t m o r t e m studies showed that the animals d i e d of ischemic necrosis of the tracheal grafts. It is evident that reliable a p p r o a c h e s to tracheal transplantation need to be found for tracheal transplantation to be a p p l i e d to h u m a n beings. Studies by Nakanishi a n d associates [7] s h o w e d that all of the tracheal autografts i m p l a n t e d into the greater o m e n t u m survived. We a s s u m e d that tracheal transplantation might be further i m p r o v e d if a revascularized and viable tracheal graft first i m p l a n t e d into the greater o m e n t u m for revascularization could be used to repair the tracheal defect. In the p r e s e n t study, we used the tracheal autotransplantation m o d e l s to avoid the immunorejection, and
used a u t o r a d i o g r a p h y with 3sS-Na2SO4, in addition to macroscopic and microscopic examinations, to d e t e r m i n e the viability of tracheal graft. W e evaluated w h e t h e r implantation of a tracheal graft into the greater o m e n t u m is better for tracheal graft survival than implantation of tracheal graft in situ using omentopexy. Moreover, we investigated the feasibility of using the tracheal graft i m p l a n t e d first into the o m e n t u m for reconstruction of tracheal defect.
T
Accepted for publication July 22, 1995. Address reprint requests to Dr Li, Department of Cardiothoracic SurgeD,, The First Hospital, Beijing Medical University, No. 8 Xishiku Str, Beijing 100034, China.
© 1995 by The Society of Thoracic Surgeons
(Ann Thorac Surg 1995;60:1592-6)
Material and M e t h o d s Twenty-four adult m o n g r e l dogs w e i g h i n g from 10.4 to 15.2 kg were r a n d o m l y and equally assigned into two groups. All dogs were anesthetized with intravenous injection of p e n t o b a r b i t a l s o d i u m (20 mg/kg) and placed in the supine position. Then oral intubation was performed, and the tube was connected to a pressurelimiting respirator. The following experiments were performed.
Operative Procedure GROUP I (N -- 12): TRACHEAL AUTOGRAFT IMPLANTED INTO
THE GREATER OMENTUM. After a midline cervical incision was made, a six-ring s e g m e n t of the trachea was excised and placed in physiologic saline solution containing penicillin G (200 unit p e r milliliter). Intubation was carried out across the operative field with sterile tube for ventilation. Tracheal reconstruction was p e r f o r m e d by e n d - t o - e n d anastomosis with i n t e r r u p t e d silk sutures. After a small m i d d l e laparotomy, the greater o m e n t u m was b r o u g h t out of the peritoneal cavity. The tracheal 0003-4975/95/$9.50 SSDI 0003-4975(95)00839-X
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animals received h u m a n e care in compliance with the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health (NIH publication 85-23, revised 1985). Examinations On postoperative days 3, 7, and 14, four dogs from each group were reanesthetized with injection of pentobarbital sodium intravenously (20 m g per kg) and placed in the supine position. Then intubations were performed and connected to a pressure-limiting respirator.
Fig, 1. Excision of a six-ring segment qfl the trachea and implantation of the graft into the left lower portion of the greater omentum.
autografts were wrapped with the lower portion of the greater o m e n t u m and placed into the peritoneal cavity (Fig 1). During the entire process, care was taken to avoid leaving talc and unnecessary foreign materials in the peritoneal cavity. All incisions were closed in layers. GROUP II (N -- 12): T R A C H E A L A U T O T R A N S P L A N T A T 1 O N W I T H
OMENTOPEXY.As in group I, a six-ring segment of cervical trachea was also excised. A sterile endotracheal tube was positioned into the lower trachea for ventilation. The excised segment was then reimplanted in its original position. The upper anastomosis was sutured first, then the m e m b r a n o u s portion of the lower anastomosis was sutured similarly. The lower anastomosis was completed after the oral endotracheal tube was inserted across both upper and lower anastomoses. Then, after the midline upper laparotomy, an omental pedicle, which was fed by the right gastroepiploic artery, was formed as Messineo and his associates reported [6]. The omental pedicle was brought to the cervical area through the right thoracic cavity by a diaphragmatic defect. This pedicle was used to wrap the autograft, including each anastomosis. Finally, both the cervical and the abdominal incisions were closed in the usual fashion. Postoperative Care Animals were given penicillin G, 1 million units intramuscularly per day for three postoperative days. All
GROUP I. The abdomens of the dogs were reopened through the upper midline incision. The grafts were found. The lumens of the grafts were opened and inspected macroscopically. A laser Doppler flowmeter (Periflux PF3, Stockholm, Sweden) was used to monitor mucosal blood flow of the tracheal grafts, and the greater omentums with the grafts in their tips were dissected from the spleen and the left edge of the stomach (Fig 2). Whether the tracheal graft with omental pedicle flap could be brought to the neck of dog was evaluated. The state of adherence between the graft and the greater o m e n t u m was investigated. The grafts and partial normal tracheas were excised and flushed with physiologic saline solution containing penicillin G (200 units per milliliter). From the cartilage ring of normal tracheas and midportion of the grafts, a small specimen of the full thickness of the tracheal wall (1 × 3 m m 2) was excised and placed in cold Hanks' solution for autoradiographic study. The remains of the grafts and normal trachea were fixed with 10% formalin solution for histologic study. GROUP II. The grafts and partial normal tracheas were removed for histologic and autoradiographic studies as in group I. Light Microscopic A u t o r a d i o g r a p h y Using 35S-Na2S04 Light microscopic autoradiography was used in both groups. The specimens were washed with Hanks" solu-
~.~-
-
J
Fig 2. Operative procedure for extension of the greater omentum with the tracheal graft at its top.
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tion, then placed in culture m e d i u m RPMI 1640 (Gibco, New York) containing 10% fetal calf serum, 200 u/mL of penicillin G, 50/~g/mL of gentamicin, and 20/zCi/mL of 35S-Na2SO4 (specific activity, 150 millicuries per millimole) (Institute of Atomic Energy of China, Beijing, China) and incubated at 37°C for 24 hours. Thereafter, the specimens were flushed with water, fixed in 10% formalin solution for 10 hours, and then washed with running water for 3 hours. Following the standard histologic procedures, 5-p,m histology sections were prepared and m o u n t e d on microscope slides. By the dipping technique, the slides were covered with liquid nuclear emulsion (N4) (Institute of Atomic Energy of China), laid on the exposure box, and exposed at 4°C in a refrigerator for 9 days. The autoradiographs were developed in D19 developer (Beijing Medical University, Beijing, China) for 3 minutes at 19°C and fixed in F5 solution (Beijing Medical University) for 8 minutes, and then stained with hematoxylin and eosin. From five unspecified sections of each specimen, three fields of full-thickness tracheal cartilage were examined microscopically for Ca) histologic appearance, (b) n u m b e r of chondrocytes labeled with 35S-Na2SO4, and (c) n u m b e r of unlabeled chondrocytes. The percentage of labeled chondrocytes was calculated according to the following formula: {Total no. of (b)/Total no. of [(b) + (c)]} × 100 = % labeled chondrocytes. Student's t test for unpaired data was used for statistical analysis.
Ann Thorac Surg 1995;60:1592-6
A
Results Macroscopic Assessment GROUP I. The four tracheal grafts from the dogs examined on postoperative day 3 were intact and were w r a p p e d by the greater omentums. The adhesions between the tracheal grafts and the omentums were not sound. Brown mucous-like fluid was packed in the lumens of all the autografts, and there were red ecchymoses in the mucosas of the grafts. Except that there was more fluid in the lumens of the grafts and that the adhesions between the tracheal grafts and the omentums was tight, the findings on postoperative day 7 were similar to those on day 3. The four tracheal grafts examined on postoperative day 14 were intact and had normal appearance in the mucosas. There was more brown fluid in their lumens than in the grafts examined on postoperative day 7.
B Fig 3. Light microscopic sSS-autoradiographs of specimens from the dogs sacrificed on postoperative day 14 in group I (A) and group II (B). (A) The airway is lined by normal mucociliated epithelium and 78% chondrocytes labeled with 35S-Na2S04. (B) The airway is lined by single-layered epithelium and only 67% chondrocytes labeled with 35S-Na2S04. (Hematoxylin and eosin; ×100 before 10% reduction.)
lium was seen in all of the tracheal grafts on postoperative day 7, whereas normal mucociliated epithelium was seen on postoperative day 14 (Fig 3A).
GROUP lI. The ornental flaps were adherent to the membranous portions of the autograft on postoperative day 3 and were adherent circumferentially to the grafts on postoperative days 7 and 14. The changes in mucosal ecchymoses in all the grafts in the group were more severe than in group I.
GROUP II. Phenomena including epithelium loss and tracheal glands, low-grade inflammation, and hemorrhage in the submucosa and lamina propria were observed by light microscopy on postoperative days 3 and 7. Differing from group I, the four grafts examined on postoperative day 14 showed a confluent multilayered epithelium in the intercartilaginous area and a single-layered epithelium in the area underlying the cartilage rings (Fig 3B).
Light Microscopic Aspect GROUP I. Phenomena including epithelium loss and tracheal glands, low-grade inflammation, and hemorrhage in the submucosa and lamina propria were observed by light microscopy in tracheal grafts on postoperative days 3 and 7. A confluent, single-layered, nonciliated epithe-
Light Microscopic Autoradiographic Assessment In all the tracheal grafts of both groups, the chondrocytes in the area close to the o m e n t u m were labeled with 35S-Na2SO4 (see Fig 3), but the chondrocytes in the area far from the o m e n t u m were not labeled. Table I shows the percentage of labeled cells of the grafts in the two
A n n Thorac S u r g 1995;60:1592-6
LI ET AL TRACHEAL AUTOGRAFT SURVIVAL IMPROVED
Table 1. Percentages of Living Chondrocytes in the Tracheal Autografts Assessed With "3SS-Autoradiography PLC of TAS (%) Postoperative Day 3
Group I
80
II
68 70 58 55
73
72
78
Postoperative Day 7 70
75
50 68
76
78
Postoperative Day 14 78
76
70 52 68 67
PLC - p e r c e n t a g e of l a b e l e d c h o n d r o c y t e s ; tografts.
TAS
80
73
70 58
tracheal au-
groups. The results in group I were significantly better than those of group II (p < 0.01). The percentage of labeled chondrocytes of the tested normal tracheas was 100%. This indicates that every normal tracheal chondrocyte has the ability to metabolize the sulfate (Fig 4).
State of the Peritoneal Cavity at Relaperotomy (Group I Only) In group I, no adhesions were found in the peritoneal cavities of any of the dogs. Without impairing the mucosal blood flow of the tracheal grafts, the greater omental pedicles with the tracheal grafts at their tips were formed successfully. All 12 tracheal grafts with their own omental pedicle could be brought to any portion of the trachea. Comment
Many attempts at tracheal replacement have been made experimentally and clinically. Investigative efforts have been made in two general categories: tissue grafts I2-8] and prosthetic grafts [9, 10]. To date, only limited success has been achieved for either [1]. Ideally, a tracheal
Fig 4. Light microscopic3SS-autoradiograph of normal trachea. (Hematoxylin and eosin; ×200 before 10% reduction.)
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allograft would be an excellent substitute for an abnormal trachea if the problems of revascularization of the tracheal graft and rejection were solved. The blood vessels of the trachea arising from the lateral tracheal vascular pedicles are numerous and very fine [11[. Once the trachea is separated from these pedicles, it is impossible to restore blood supply to the trachea immediately. Therefore, after transplantation, the tracheal graft would inevitably experience a period of ischemia. Neville and associates [12] reported that a tracheal autograft, even a segment including three cartilage rings, could not survive after orthotopic transplantation. Morgan and associates [13] reported in 1982 that application of the greater omental flap to an avascular bronchial autograft 2 cm in length resulted in early revascularization and prevented bronchial graft necrosis. Since that report, many experiments concerned with tracheal transplantation using omentopexy have been clone and showed omentopexy can promote revascularization of tracheal grafts. Whether omental wrapping can maintain the viability of tracheal graft is still controversial. Canine studies by Balderman and Weinblatt [3] showed negative data. Although Moriyama and associates [5] and Messineo and associates [6] obtained improved results, still some animals died of airway obstruction due to ischemic necrosis of cartilage rings of the tracheal grafts, as their report showed. Rose and associates [2] reported the only successful clinical tracheal allotransplantation. They placed a tenring allograft in the bed of the recipient's sternocleidomastoid muscle for 3 weeks. An eight-ring abnormal trachea of a 21-year-old male patient was replaced by the graft with the muscular pedicle. It is obvious that this muscular flap cannot be extended to the thoracic portion of trachea. The study by Nakanishi and associates [7] showed that all of the tracheal grafts e m b e d d e d into greater o m e n t u m survived. In the present studies, macroscopic, microscopic, and 3SS-autoradiographic studies were used to evaluate the viability of the tracheal graft. All findings indicate that tracheal grafts e m b e d d e d into the greater o m e n t u m can regain their blood supply earlier, compared with implantation in situ using omentopexy, and suggest that using a staged procedure for tracheal transplantation is reasonable. We assume that the possibility of the improved results in group I, compared with group II, can be attributed to the operative procedures in group I giving less trauma to tracheal gr?ft and leaving the greater o m e n t u m intact, and immobiliz~ ing the tracheal grafts at the early period after transplantation. As the studies revealed, there was no adhesion in t!e peritoneal cavities and the greater omentums with tracheal grafts could be extended to any portion of the trachea. These suggest that using a tracheal graft implanted first into the greater o m e n t u m to repair tracheal defect is feasible. From the findings in group I, ~e consider the optimal time for the second operation to be about 2 weeks after the first operation, because only after
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LI ET AL TRACHEALAUTOGRAFTSURVIVALIMPROVED
14 postoperative days did mucosal regenerate a n d the adhesion between the tracheal graft and the o m e n t u m become sound. As a next step, the tracheal autograft a n d allograft should be studied with the staged approach. Using a laser Doppler flowmeter to measure blood flow on various tissues is noninvasive and easy. In a clinical report, Jones a n d Mayou demonstrated the usefulness of the method for skin flap blood flow monitoring after plastic surgery where early diagnosis of vascular insufficiency is of p a r a m o u n t value [14]. In their study, the laser Doppler flowmeter was used to monitor the process of dissection and extension of the greater o m e n t u m to prevent damage to the mucosal blood flow of the tracheal graft. With the laser Doppler flowmeter monitoring, the staged tracheal transplantation procedure used in the studies appeared more reliable. In previous studies, the viability of tracheal graft was d e t e r m i n e d only with the light microscopy, electron microscopy, and long-term observation methods. By these methods, the early changes of the cartilage rings of tracheal graft could not be demonstrated. Autoradiography is an old and useful technique. It was applied to determine the viability of aortic graft [15] a n d osteochondral grafts [16]. We used autoradiography with radioactive sulfate to observe the early changes of the tracheal cartilage after transplantation, a n d found that the living status of the cartilage rings of the tracheal grafts can be determined quantitatively by using this 35S-autoradiographic and histologic method. In conclusion, compared with tracheal transplantation using omentopexy, implantation of tracheal graft into the greater o m e n t u m is an effective method for tracheal graft revascularization a n d survival. It is feasible by using a living tracheal graft after revascularization in the greater o m e n t u m to repair a tracheal defect. The optimal time for the second operation is about 2 weeks after implantation of the tracheal graft into the great o m e n t u m .
Ann Thorac Surg 1995;60:1592-6
References 1. Grillo HC. Tracheal replacement. Ann Thorac Surg 1990;49: 864-5. 2. Rose KG, Sesterhenn K, Wustrow F. Tracheal allotransplantation in man. Lancet 1979;1:433. 3. Balderman SC, Weinblatt G. Tracheal autograft revascularization. J Thorac Cardiovasc Surg 1987;94:434-41. 4. Nagasawa H. Experimental tracheal reconstruction with the use of homograft covered with omental flap. Nippon Kyobu Geka Gakkai Zasshi 1988;36:337-47. 5. Moriyama S, Shimizu N, Teramoto S. Experimental tracheal aUotransplantation using omentopexy. Transplant Proc 1989; 21:2596- 600. 6. Messineo A, Filler RM, Bahoric B, Smith C, Bahoric A. Successful tracheal autotransplantation with a vascularized omental flap. J Pediatr Surg 1991;26:1296-300. 7. Nakanishi R, Shirakusa T, Takachi T. Omentopexy for tracheal autografts. Ann Thorac Surg 1994;57:841-5. 8. Papp C, McCraw JB, Arnold PG. Experimental reconstruction of the trachea with autogenous materials. J Thorac Cardiovasc Surg 1985;90:13-20. 9. Neville WE, Bolanowski PJP, Soltanzadeh H. Prosthetic reconstruction of the trachea and carina. J Thorac Cardiovasc Surg 1976;72:525-38. 10. Mendak SH Jr, Jensik RJ, Haklin MF, Roseman DL. The evaluation of various bioabsorbable materials on the titanium fiber metal tracheal prosthesis. Ann Thorac Surg 1984;38:488-93. 11. Salassa JR, Pearson BW, Payne WS. Gross and microscopical blood supply of the trachea. Ann Thorac Surg 1977;24:100-7. 12. Neville WE, Bolanoski PJP, Scoltazadeh H. Homograft replacement of the trachea using immunosuppression. J Thorac Cardiovasc Surg 1976;72:596-601. 13. Morgan E, Lima O, Goldberg M, Ferdman A, Luk SK, Cooper JD. Successful revascularization of totally ischemic bronchial autografts with omental pedicle flaps in dogs. J Thorac Cardiovasc Surg 1982;84:204-10. 14. Jones BM, Mayou BJ. The laser Doppler flowmeter for microvascular monitoring: a preliminary report. Br J Plast Surg 1982;35:147-9. 15. A1-Janabi N, Gonzalez-Lavin L, Neirotti R, Rose DN. Viability of fresh aortic homografts: a quantitative assessment. Thorax 1972;27:83-6. 16. Depalma AF, Tsaltas TT, Mauler GG. Viability of osteochondral grafts as determined by uptake of 35S. J Bone Joint Surg 1963;45A:1565-78.