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Ann Thorac Surg 1995;60:1592-1596
© 1995 The Society of Thoracic Surgeons

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Original Articles: General Thoracic

Improvement of Tracheal Autograft Survival With Transplantation Into the Greater Omentum

Departments of Cardiothoracic Surgery and Endoscopy, The First Hospital of Beijing Medical University, Beijing, China

Accepted for publication July 22, 1995.

	Abstract

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 
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, and ³⁵S-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.

	Introduction

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 
The 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 would be superior to the trachea itself as a graft if the problems of revascularization of the tracheal graft and rejection were solved. The experimentation concerned with these problems has been conducted for more than four decades. Up to now, only limited improvement has been achieved [2–7]. It has been discovered that by means of one-stage tracheal transplantation procedure with omentopexy, the tracheal graft can be revascularized [3–7]. Past experiments showed that not all of the tested animals could survive long, and postmortem studies showed that the animals died of ischemic necrosis of the tracheal grafts. It is evident that reliable approaches to tracheal transplantation need to be found for tracheal transplantation to be applied to human beings.

Studies by Nakanishi and associates [7] showed that all of the tracheal autografts implanted into the greater omentum survived. We assumed that tracheal transplantation might be further improved if a revascularized and viable tracheal graft first implanted into the greater omentum for revascularization could be used to repair the tracheal defect.

In the present study, we used the tracheal autotransplantation models to avoid the immunorejection, and used autoradiography with ³⁵S-Na₂SO₄, in addition to macroscopic and microscopic examinations, to determine the viability of tracheal graft. We evaluated whether implantation of a tracheal graft into the greater omentum 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 implanted first into the omentum for reconstruction of tracheal defect.

	Material and Methods

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 
Twenty-four adult mongrel dogs weighing from 10.4 to 15.2 kg were randomly and equally assigned into two groups. All dogs were anesthetized with intravenous injection of pentobarbital sodium (20 mg/kg) and placed in the supine position. Then oral intubation was performed, and the tube was connected to a pressure-limiting 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 segment of the trachea was excised and placed in physiologic saline solution containing penicillin G (200 unit per milliliter). Intubation was carried out across the operative field with sterile tube for ventilation. Tracheal reconstruction was performed by end-to-end anastomosis with interrupted silk sutures. After a small middle laparotomy, the greater omentum was brought out of the peritoneal cavity. The tracheal autografts were wrapped with the lower portion of the greater omentum 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.

View larger version (27K): [in this window] [in a new window]   Fig 1. . Excision of a six-ring segment of the trachea and implantation of the graft into the left lower portion of the greater omentum.

Postoperative Care
Animals were given penicillin G, 1 million units intramuscularly per day for three postoperative days. All animals received humane 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 mg per kg) and placed in the supine position. Then intubations were performed and connected to a pressure-limiting respirator.

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 omentum 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 x 3 mm²) 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.

View larger version (28K): [in this window] [in a new window]   Fig 2. . Operative procedure for extension of the greater omentum with the tracheal graft at its top.

Light Microscopic Autoradiography Using ³⁵S-Na₂SO₄
Light microscopic autoradiography was used in both groups. The specimens were washed with Hanks' solution, then placed in culture medium RPMI 1640 (Gibco, New York) containing 10% fetal calf serum, 200 u/mL of penicillin G, 50 µg/mL of gentamicin, and 20 µCi/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-µm histology sections were prepared and mounted 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 (a) histologic appearance, (b) number of chondrocytes labeled with ³⁵S-Na₂SO₄, and (c) number of unlabeled chondrocytes. The percentage of labeled chondrocytes was calculated according to the following formula: {Total no. of (b)/Total no. of [(b) + (c)]} x 100 = % labeled chondrocytes. Student's t test for unpaired data was used for statistical analysis.

	Results

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 
Macroscopic Assessment
GROUP I.
The four tracheal grafts from the dogs examined on postoperative day 3 were intact and were wrapped 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.

GROUP II.
The omental 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.

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 epithelium was seen in all of the tracheal grafts on postoperative day 7, whereas normal mucociliated epithelium was seen on postoperative day 14 (Fig 3A).

View larger version (109K): [in this window] [in a new window]   Fig 3. . Light microscopic 35S-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-Na2SO4. (B) The airway is lined by single-layered epithelium and only 67% chondrocytes labeled with 35S-Na2SO4. (Hematoxylin and eosin; x100 before 10% reduction.)

Light Microscopic Autoradiographic Assessment
In all the tracheal grafts of both groups, the chondrocytes in the area close to the omentum were labeled with ³⁵S-Na₂SO₄ (see Fig 3), but the chondrocytes in the area far from the omentum were not labeled. Table 1 shows the percentage of labeled cells of the grafts in the two 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).

View larger version (142K): [in this window] [in a new window]   Fig 4. . Light microscopic 35S-autoradiograph of normal trachea. (Hematoxylin and eosin; x200 before 10% reduction.)

	Comment

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 
Many attempts at tracheal replacement have been made experimentally and clinically. Investigative efforts have been made in two general categories: tissue grafts [2–8] and prosthetic grafts [9, 10]. To date, only limited success has been achieved for either [1]. Ideally, a tracheal 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 done 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 ten-ring 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 embedded into greater omentum survived. In the present studies, macroscopic, microscopic, and ³⁵S-autoradiographic studies were used to evaluate the viability of the tracheal graft. All findings indicate that tracheal grafts embedded into the greater omentum 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 graft and leaving the greater omentum intact, and immobilizing the tracheal grafts at the early period after transplantation.

As the studies revealed, there was no adhesion in the 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 omentum to repair tracheal defect is feasible. From the findings in group I, we consider the optimal time for the second operation to be about 2 weeks after the first operation, because only after 14 postoperative days did mucosal regenerate and the adhesion between the tracheal graft and the omentum become sound. As a next step, the tracheal autograft and 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 and Mayou demonstrated the usefulness of the method for skin flap blood flow monitoring after plastic surgery where early diagnosis of vascular insufficiency is of paramount value [14]. In their study, the laser Doppler flowmeter was used to monitor the process of dissection and extension of the greater omentum 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 determined 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] and osteochondral grafts [16]. We used autoradiography with radioactive sulfate to observe the early changes of the tracheal cartilage after transplantation, and 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 omentum is an effective method for tracheal graft revascularization and survival. It is feasible by using a living tracheal graft after revascularization in the greater omentum 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 omentum.

	Footnotes

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 
Address reprint requests to Dr Li, Department of Cardiothoracic Surgery, The First Hospital, Beijing Medical University, No. 8 Xishiku Str, Beijing 100034, China.

	References

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 

1. Grillo HC. Tracheal replacement. Ann Thorac Surg 1990;49:864–5.[Medline]
2. Rose KG, Sesterhenn K, Wustrow F. Tracheal allotransplantation in man. Lancet 1979;1:433.[Medline]
3. Balderman SC, Weinblatt G. Tracheal autograft revascularization. J Thorac Cardiovasc Surg 1987;94:434–41.[Abstract]
4. Nagasawa H. Experimental tracheal reconstruction with the use of homograft covered with omental flap. Nippon Kyobu Geka Gakkai Zasshi 1988;36:337–47.[Medline]
5. Moriyama S, Shimizu N, Teramoto S. Experimental tracheal allotransplantation using omentopexy. Transplant Proc 1989;21:2596–600.[Medline]
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.[Medline]
7. Nakanishi R, Shirakusa T, Takachi T. Omentopexy for tracheal autografts. Ann Thorac Surg 1994;57:841–5.[Abstract]
8. Papp C, McCraw JB, Arnold PG. Experimental reconstruction of the trachea with autogenous materials. J Thorac Cardiovasc Surg 1985;90:13–20.[Abstract]
9. Neville WE, Bolanowski PJP, Soltanzadeh H. Prosthetic reconstruction of the trachea and carina. J Thorac Cardiovasc Surg 1976;72:525–38.[Abstract]
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.[Abstract]
11. Salassa JR, Pearson BW, Payne WS. Gross and microscopical blood supply of the trachea. Ann Thorac Surg 1977;24:100–7.[Abstract]
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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.[Abstract]
14. Jones BM, Mayou BJ. The laser Doppler flowmeter for microvascular monitoring: a preliminary report. Br J Plast Surg 1982;35:147–9.[Medline]
15. Al-Janabi N, Gonzalez-Lavin L, Neirotti R, Rose DN. Viability of fresh aortic homografts: a quantitative assessment. Thorax 1972;27:83–6.[Abstract/Free Full Text]
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