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Asian Cardiovasc Thorac Ann 2003;11:237-244
© 2003 Asia Publishing EXchange Ltd

ORIGINAL CONTRIBUTION

Rejection and Indirect Revascularization of Glycerin-Preserved Tracheal Implant

Maurício G Saueressig, MSc, Alexandre H Moreschi, MSc, Gilberto V Barbosa, MD, Maria IA Edelweiss, PhD, Felipe H de Souza, JD, Fabrício L Savegnago, JD, Amarílio V de Macedo Neto, PhD

Department of Thoracic Surgery and Lung Transplantation, Hospital de Clínicas de Porto Alegre, Federal University of Rio Grande do Sul, Porto Alegre - Brazil

For reprint information contact: Maurício G Saueressig, MSc Tel: 55-51-3321-2101 Fax: 55-51-3316-8684 email: mguidi{at}zaz.com.br 2171, Ramiro Barcelos St., Ap.21, 90035-007, Porto Alegre - Brazil.


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The objective of the following study was to evaluate antigenicity, malacia and revascularization in glycerin-preserved canine tracheal allografts. Trachea with six cartilage rings (2.4 to 3.1 cm) were distributed in three study groups: autograft (21), allograft (18) and glycerin-preserved (22). We implanted two segments from different groups in the greater omentum of dogs. After 28 days, latex was injected in the canine aorta before the segments were harvested. We evaluated number of sectors with functional vessels, number of vessels dyed in the submucosa, acute arteritis score, incidence of acute rejection, cartilage lesion score, and malacia. The autograft group had a larger number of dyed vessels than the glycerin-preserved group. The autograft group also had a higher average number of quadrants with functional vessels than the allograft group and the glycerin-preserved group. The allograft group had a higher mean score for acute arteritis than the autograft group and more acute rejection than the glycerin-preserved group. The cartilage lesion score did not show any significant difference between groups. Malacia was not observed in any tracheal segment. Overall, the glycerin-preserved tracheal implant had low antigenicity and good rigidity, but showed incomplete revascularization.


   INTRODUCTION
TOP
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tracheoplasty is the most common surgical treatment of benign and malignant tracheal diseases. However, there is still no definitive solution for the problem of reconstruction of defects larger than 60% of the trachea.1 The best anatomical and physiological replacement of the trachea is its own allograft2 since the use of prostheses and autografts to replace the trachea has shown contradictory results that are difficult to reproduce.1

The routine use of tracheal transplants depends on the solution of three problems: revascularization, rejection and preservation of grafts.2 Tracheal graft ischemia is the main cause of transplant failure. Therefore, the graft has to be revascularized with flaps of the greater omentum, lateral thoracic fascia, muscle, or direct vascular anastomosis with en bloc cervicothoracic exenteration.1–3 Arteritis characterizes acute vascular rejection of the trachea and results in thrombosis of microcirculation in the submucosa and, consequently, in ischemic necrosis of the respiratory epithelium, of the interstitium and, finally, of the cartilage.4

The denudation of respiratory epithelium, where Major Histocompatibility Complex (MHC) class II is found, as well as the degeneration of chondrocytes in the tracheal allograft, are effects of the method of preservation, which reduces tracheal antigenicity.11 Therefore, besides reducing the allogenic stimulus, another important property of these methods is the maintenance of the tissue structure and the formation of a trachea bank.5 Cryopreservation of tracheas is the most frequently studied preservation method.5 However, disagreements about its effect on chronic rejection, about the variability of the length of time of cryopreservation, which ranges from three to 24 months, and the viability of chondrocytes raise questions about the long-term use of this preservation method.

For these reasons, another method of preservation that may be easily applicable and offer unlimited preservation time for the tracheal segment should be researched. This motivated us to research the use of glycerin, an easily obtained and manipulated trialcohol with antiseptic and preservative properties.6 We previously tested glycerin’s preservation capacity in different tissues, especially in dura mater6 and the trachea.7 The studies demonstrated the qualities of glycerin in reducing the antigenic response and maintaining the structure of tissues. These results and concepts formed the basis for the formulation of the following hypothesis: the tracheal segment preserved in 99% glycerin and implanted in the omentum of dogs would present vascularization similar to an autograft, preserving tracheal lumen rigidity, and would present immunological tolerance better than an allograft.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
This controlled blind randomized trial was conducted at Veterinary Clinic Hospital, Federal University of Rio Grande do Sul, Brazil. The sample was composed of 61 canine tracheal segments of six cartilage rings each. They were divided into the following groups:

  1. autografts (n = 21);
  2. allografts (n = 18);
  3. glycerin-preserved grafts (n = 22).

These tracheal segments were harvested from 51 adult mongrel dogs of both sexes kept under adequate sanitary control and provided by the kennel at Centro de Zoonoses, Secretaria Municipal de Saúde, Porto Alegre, Brazil. The dogs were randomly assigned to one of the three destinations presented in Figure 1Go. The animals weighed 9.497 ± 1.975 kg, and there was no statistically significant difference between destinations (p = 0.915). We housed and handled the dogs in accordance with Normative Resolution 196/96 issued by the Conselho Nacional de Saúde8 (Brazilian Health Council). This trial was approved by the Research and Ethics in Health Committee at Clinical Hospital of Porto Alegre under number 99411.



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  		Figure 1. Origin of the sample and of the research groups (autograft, allograft and glycerin-preserved) out of the group of 51 dogs. #One dog with an autograft and a glycerin-preserved graft died. These two results were not included in results analysis. *Tracheal segments composed of 6 cartilage rings.

 
The animals were premedicated with subcutaneous atropine sulphate (0.044 mg•kg-1) and intramuscular acepromazine (0.2 mg•kg-1), and anesthetized with intravenous thiopental sodium (15 mg•kg-1). After orotracheal intubation with a 7 mm tube (Rüschelit®, Rüsch, Montevideo, Uruguay), anesthesia was maintained with inhaled halothane (0.5% to 1%) with the dogs spontaneously breathing a mixture of room air and oxygen (3 L•min-1). All dogs except those in group B received intravenous ampicillin (20 mg•kg-1) during anesthesia induction. No immunosuppressive drug was used.

The 20 dogs donating allografts (group B) were killed with an overdose of intravenous thiopental sodium. After that, a cervical incision in the midline was made. We used an aseptic technique, and excised the cervical trachea. Each trachea provided two six-ring segments 2.4 to 3.1 cm long each. After resection of the adventitia, we placed 18 of these tracheal segments in 0.9% saline solution until their implantation into the greater omentum (allograft group). A further 22 segments were placed in glass jars containing 99% bidistilled glycerin (glycerin group), which were hermetically closed. We stored the glycerin group tracheal segments at room temperature for two to six months.7 At the time of implantation, the glycerin-preserved grafts were rehydrated with 0.9% saline solution for 30 minutes before they were implanted into the greater omentum.

We repeated the same cervical approach used for the previous group in the 21 dogs donating autografts and receiving allografts or glycerin-preserved grafts. The immediate reconstruction of airways in these dogs followed steps similar to those followed for tracheoplasty in clinical practice. During the same surgery, these animals received implantation of an autograft (autograft group) and an allograft, or a glycerin-preserved graft into the greater omentum.

Implantation into the greater omentum was performed in 31 dogs, 21 from group A and 10 from group C. Following a supra-umbilical median laparotomy of about 10 cm, we brought the greater omentum out of the peritoneal cavity. Each of the two segments of the trachea was wrapped with the greater omentum and fixed with a few stitches of 3-0 polyglactin suture 2 cm from the great gastric curvature and at least 10 cm from the other graft. The random implantation of the 61 tracheal segments in these 31 dogs formed three combinations of research groups (Table 1Go).


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  		Table 1. Total number of dogs with one of the three combinations of two different tracheal segments from the different research groups (autograft, allograft and glycerin-preserved)
 
The other 34 tracheal segments (17 animals) were perfused with latex on the 28th day of implantation. We used 100 ml latex9 (Labsynth, Diadema, SP, Brazil) for each 50 ml green dye (Suvinil®, São Bernardo do Campo, SP, Brazil) diluted at 50% in 0.9% saline solution. The latex injection technique had the following stages:

  1. anticoagulation with intravenous sodium heparin (300 U•kg-1), dissection and clamping of thoracic and infrarenal aorta;
  2. 2)puncture of aorta above the celiac trunk with an intravenous catheter (Jelco®, Johnson & Johnson, São José dos Campos, SP, Brazil) with an external diameter of 2.1 mm;
  3. injection of 250 ml saline solution at 37°C followed by infusion of 45 ml latex under 150 mm Hg pressure.

Twenty-eight days after operation we anesthetized the dogs from groups A and C again. The laparotomy was reopened and the implanted tracheal segments were excised. Tests of resistance to deformation and macroscopic examination were carried out, and the tracheal segments were then fixed in 10% formalin solution.

We modified the technique described by Vacanti et al.10 to test the resistance to collapse of the last seven tracheal segments (one autograft, three allografts and three glycerin-preserved grafts). One segment was cross-sectioned from each tracheal segment and placed in a condom (Jontex®, Johnson & Johnson, São José dos Campos, SP, Brazil). After that, we ran a multiperforated no. 16 tracheal aspiration catheter (Markmed, São Paulo, SP, Brazil) through the condom and the tracheal segment lumen. With a 60 ml syringe (Becton, Dickinson e Co, Franklin Lakes, NJ, USA) connected to the catheter, we applied a pressure of -200 mmHg. We considered that malacia only existed when the tracheal wall collapsed during negative pressure.

The tracheal specimens were cross-sectioned in their middle portion, specimens included in paraffin, and 5 µm thick cross-sections prepared and stained with hematoxylin-eosin for histological analysis. We counted the number of latex stained vessels in the tracheal submucosa. The circumferential revascularization of the submucosa was also quantified by dividing the area of the transversal section in four equal sectors, and counting only the functional vessels (independent of latex filling), that is, the endothelium with nucleated cells and absence of thrombus. Therefore, the values of this parameter ranged from zero to four sectors with functional vessels. We excluded from indirect revascularization analysis the tracheal segments that, at histological examination of sections, showed loss of structure of the mucosa, submucosa, cartilage and adventitia, since loss of structure would hinder the correct identification of vessels.

The pattern of vascular rejection of the trachea is similar to that of other solid organs, such as the kidney and the heart.4,11 Therefore, we decided to adopt the Banff 9712 criteria for assessment of renal rejection to define the presence and quantify the intensity of acute and chronic arteritis in the implanted tracheas. Acute vascular rejection was defined by the presence of acute arteritis in at least one vessel of the tracheal segment under examination, because such identification reveals immunological reaction against the graft. Acute arteritis was quantified according to the highest intensity vascular lesion present in at least one artery in the histological section under examination. The values of the intensity of arterial lesions were distributed according to the score of acute arteritis:

(v0)no arteritis;

(v1)light-to-moderate intimal arteritis defined by permeation of the intima by lymphocytes;

(v2) severe intimal arteritis, confirmed by the occlusion of at least 25% of the artery lumen by mononuclear cells;

(v3) transmural arteritis, shown by the loss of vascular architecture and by necrosis of the endothelium and the middle layer of muscle, associated with infiltration of the vascular wall by mononuclear, polymorphonuclear cells, with fibrinoid changes.

We also assessed the relation between indirect revascularization (number of quadrants with functional vessels and number of stained vessels in the submucosa) and score of acute arteritis.

The presence of chronic arteritis (cv) was also assessed. The criteria to define chronic vascular lesion were: fibrous thickening of the arterial intima with delamination or discontinuity of the internal elastic membrane; and / or the presence of foamy and / or mononuclear cells in the fibrosed intima, observed in at least one vessel in the histological section under examination.

We assessed the signs of ischemia or degeneration of the tracheal cartilage according to the modified Kumon et al.13 score: grade 0 – absence of ischemic lesion; grade 1 – ischemic lesion in up to 50% of the cartilage ring; grade 2 – ischemic lesion in 50% or more of the cartilage ring; and grade 3 – ischemic lesion in all of the cartilage ring. Tracheal cartilage was classified as ischemic when at least one of the following changes was observed: hyalinization, metachromasia, fibrosis or reabsorption of the cartilage matrix, or karyolysis in chondrocytes.

To investigate the possibility of infection in the tracheal segments, we assessed the presence of leukocyte infiltration, which was defined as the presence of polymorphonuclear cells in over 25% of the area of the interstitium under examination.4,12

We used the Fisher exact test and the chi-square test for the comparison of proportions. The Kruskal-Wallis test was used for the comparison of means. The Spearman rank correlation test was used to study the correlation between revascularization and acute arteritis. The significance level was defined at 5% ({alpha} <= 0.05) and all statistical calculations were performed with the Pepi 3.0.


   RESULTS
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We evaluated the results of 20 autografts, 18 allografts and 21 glycerin-preserved grafts, since one dog (autograft and glycerin group) did not survive anesthesia. Another dog received the implant of only the tracheal segment from the glycerin group, as the tracheal segment from the allograft group was missing at the time of the study. The main results are presented in Table 2Go.


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  		Table 2. Histological examination and presence of malacia in the tracheal segments 28 days after implantation into the greater omentum
 
Macroscopic examination showed that 88.13% of the tracheal segments had some material in the lumen, similar to a clot (glycerin) or mucopurulent liquid (allograft and autograft). The greater omentum blocked the lumen but did not invade the interior of the tracheal segments. Two allografts presented stenosis with 50% of the lumen obstructed by histologically confirmed fibrosis. No tracheal segment had a soft tissue consistency. None of the seven tracheal segments examined (one autograft, three allografts and three glycerin-preserved grafts) presented malacia when tested for resistance to collapse under negative pressure (-200 mm Hg).

Of the 34 tracheal segments perfused with latex, we excluded two allografts and one autograft from final analysis. Therefore, we studied the number of latex-stained vessels in ten autografts, nine allografts and 12 glycerin-preserved grafts. The autograft group had a higher mean number of stained vessels than the glycerin group (p = 0.029) (Figure 2Go), but that number was not statistically higher than the mean for the allograft group. We analyzed the number of quadrants with submucosal functional vessels in 53 tracheal segments (one autograft and five allografts were excluded). The autograft group had a higher mean number of quadrants with functional vessels than the allograft (p = 0.013) and the glycerin (p = 0.005) groups.



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  		Figure 2. Photomicrographs of glycerin-preserved trachea 28 days after implantation into the greater omentum. Submucosa (s), cartilage (C), adventitia (a), respiratory epithelium (re), submucosal glands (g), vessel filled with latex (*). Note epithelium denudation (ed) in photo 4 (hematoxylin-eosin; original magnification: photos 1 and 3 = 40 X; photo 2 = 100 X; photo 4 = 400 X).

 
The incidence of acute rejection was higher in the allograft (p = 0.001) and glycerin (p = 0.01) groups when compared with the absence of rejection in the autograft group. Glycerin-preserved grafts showed a frequency rate of rejection that was half the rate for the allografts (p = 0.039). The mean acute arteritis score in the allograft group was higher than in the autograft group (p = 0.001), but not statistically higher than the mean for the glycerin group (Figure 3Go). In the glycerin group, 28.57% of the implants had mild-to-moderate intimal arteritis (v1), and none had severe (v2) or transmural (v3) arteritis. In the allograft group, 50% of the tracheal segments had mild-to-moderate intimal arteritis (v1), and 16.7% had severe intimal arteritis (v2) (Figure 4Go).



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  		Figure 3. Mean scores of acute arteritis in the autograft, allograft and glycerin-preserved groups. The small vertical bars represent the standard deviation in these groups. The symbol (*) indicates the means in the autograft and allograft groups that were statistically different (p = 0.001).

 


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  		Figure 4. Photomicrographs of mild-to-moderate intimal arteritis (v1) in photos 1 and 2, severe intimal arteritis (v2) in photo 3, and chronic arteritis (cv) in photo 4, in tracheal allografts implanted into the greater omentum for 28 days. In photo 2, the presence of latex in the arteriolar lumen does not hinder the identification of arteritis. Arrows indicate the vessel with arteritis (hematoxylin-eosin; original amplification: 400 X).

 
The analysis of the relation between the number of quadrants with functional vessels and the intensity of acute arteritis revealed a negative correlation of 0.308, which was statistically significant (p = 0.025). However, there was no significant correlation (p = 0.259) between the number of stained vessels and the intensity of the acute vascular lesion. Chronic arteritis was identified in only two glycerin-preserved tracheas and in one allograft (Figure 4Go). All vasculitis observed was located only in the tracheal adventitia.

We did not find any significant differences between the mean cartilage lesion scores for the three groups (p = 0.646). The means were low for the three groups, and most lesions affected less than 50% of the cartilage ring. The most frequent ischemic and focal degenerative lesion was the hyalinization of the cartilage matrix. Leukocyte infiltration was more frequently found in the tracheal segments in the autograft and allograft groups (30% in autografts and 33.3% in allografts) than in the glycerin-preserved grafts (0%, p = 0.0063).

The glycerin-preserved tracheas presented form and consistency similar to fresh tracheas after hydration and before implantation. Histological examination showed that all the implants preserved the tracheal architecture, the gland structure in the submucosa, and the nuclei of the chondrocytes (Figure 2Go). The adventitia vessels in the glycerin-preserved tracheas were filled with latex.


   DISCUSSION
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
As the antigenic reaction was controlled with the inclusion of an autograft group, we may suggest that acute arteritis is responsible for the lack of revascularization. Therefore, the obviously better revascularization in the autograft group in both parameters measured may be justified. However, there was a significant difference in the comparison with the glycerin group when we examined the number of stained vessels separately.

We selected allografts with a better immunological tolerance, and a consequently better preserved vascularization, for the analysis of the number of stained vessels in the submucosa. This resulted in an artificially higher mean for this parameter and may therefore explain the lack of statistically significant differences from the autografts, and suggest a better revascularization than in the glycerin group. However, this apparently superior vascularization in the allografts in comparison with the glycerin-preserved tracheas was corrected when we included a higher number of tracheal segments in the quantification of the number of quadrants with functional vessels. These two groups had functional vessels in only half of the quadrants in a cross section, which was statistically lower than the almost complete circumferential revascularization of the autografts.

Nickeleit et al.14 showed that the severity of acute vasculitis was the main prognostic factor for the functionality of the renal graft. In the allograft and glycerin groups in our study, the lower revascularization of the submucosa is justified by the statistically significant correlation between a lower number of functional vessels and the higher intensity of acute arteritis. However, acute vasculitis showed low intensity in the glycerin group, which does not fully explain the lack of revascularization. In the glycerin-preserved tracheas, we observed the maintenance of the structure associated with the absence of cell viability. Therefore, only the granulation tissue would respond for the new vessels observed, since there is no recanalization of thrombosed vessels in the absence of an endothelium with cell viability, as was demonstrated by Moriyama et al.15 in cryopreserved tracheas. We studied indirect revascularization for 28 days, which may have been too short a period of time for a better penetration of vessels in the glycerin-preserved structures. Tracheas preserved in 2% glutaraldehyde showed the first functional vessels in the submucosa only after 45 days,16 which reinforces the premise in favor of slow revascularization in devitalized tissues.

There was acute rejection in 66.7% of the allografts, which is more than twice the incidence rate observed in the glycerin-preserved grafts and very similar to the 50% vasculitis observed in transplants of other organs.17 Nakanishi et al.4 described perivasculitis in the adventitia 28 days after implantation. However, there are no other studies that describe in detail the intensity and the type of vasculitis in tracheal rejection. In our study, allografts had a higher incidence of mild-to-moderate intimal (v1) and severe (v2) arteritis. The relevance of this finding indicates more severe rejection and leads to failure of graft by stenosis,4–5,12,18 as was observed in the obstruction of the lumen by fibrosis in two of our allografts. The short period of implantation, when compared to the 78 days in the study with 100 % obstruction of the grafts,19 explains the reduced number of graft stenosis in our study.

Chronic arteritis is an obstructive and immunologically indolent lesion that compromises the function of the transplanted organ.19 Moriyama et al.15 described signs of this chronic rejection (vascular thrombosis) in cryopreserved tracheas three months after transplantation. Transplantation of other organs revealed 30% chronic arteritis in periods as long as five years.19 Therefore, the incidence of chronic vascular lesion in our study (three tracheal segments) may be underestimated.

The denudation of most of the respiratory epithelium,7 – where allopeptides from MHC class II were identified – the absence of immediate recanalization of antigenic endothelium with cell viability, and the non-existence of allogenic hematolymphoid cells19 – which hinder the direct recognition of antigens – contributed to a lower antigenic stimulation. This was demonstrated in the glycerin-preserved tracheas of our study by the reduction in the intensity of acute arteritis, the low incidence of rejection, and the non-existence of the mononuclear infiltrate characteristic of antigenic reactions.

However, acute and chronic arteritis, as seen in some glycerin-preserved grafts, suggest that the persistence of the antigenic stimulus originated in the preserved structures, such as the chondrocyte nuclei, the submucosal glands or even the small areas of no denudation of respiratory epithelium. Chondrocyte nutrition is based on the diffusion of solutes by the cartilage matrix, which makes it more resistant to ischemia.10,11 Therefore, its degeneration is slow and takes two to three months to develop histological changes typical of necrosis, such as karyolysis, metabolic dysfunction, destruction of the perichondrium and replacement by fibrosis, resulting in malacia.5,9 Our histological findings confirm the higher resistance of cartilage to ischemia. In this study, the hyalinization of the cartilage matrix was the predominant change. In most of the cartilages examined we observed only restricted signs of ischemic lesion (less than 50% of the tracheal ring), in agreement with findings by Nakanishi et al.,4 who reported a limited atrophy of chondrocytes and destruction of the perichondrium in allografts. The mechanical evaluation by the test of resistance to deformation10 also confirms our histological findings, since we demonstrated the absence of structural deformation of the airways (malacia) under a pressure of -200 mm Hg. In our study, most chondrocyte nuclei were preserved no matter how long the trachea was preserved in glycerin, and there were no diffuse signs of degeneration, unlike in tracheas cryopreserved for prolonged periods of time (two to six months), when karyolysis (degeneration) occurs in 30–90% of the chondrocytes. We may thus suggest that a bank of glycerin-preserved tracheas may be more advantageous because of the better preservation of the cartilage structure.

The importance of preserving the viability of the cartilage during the period of preservation of a tracheal segment13,15,19 is challenged by the good results obtained with devitalized tracheal segments preserved in acetone20 or in 2% glutaraldehyde.15 This is further supported by the reduction of antigenicity associated with the degeneration of chondrocytes with concurrent malacia in allografts cryopreserved for longer than six months.5 On the other hand, retention of viability of tracheas cryopreserved for 60 days contributes to the preservation of the antigenic stimulus from the endothelium and of viable chondrocytes, which results in loss of structure and fibrosis of these allografts probably due to chronic rejection (three months after transplantation).15

In our study, the 28 days of implantation and the obstruction of the extremities of the tracheal segments by the greater omentum may have contributed to the accumulation of secretion in the lumen and to infection (infiltration of polymorphonuclear cells). The antiseptic characteristic of glycerin6 explains the absence of leukocyte infiltration in the glycerin-preserved implants in our study. This advantage also allows the glycerin-preserved trachea to remain in the greater omentum for over one month until full revascularization is achieved, with a reduced risk of infection.

From this study, a number of conclusions can be drawn. Firstly, autografts had the most complete revascularization. In comparison, the new vascularization of the glycerin-preserved graft was more difficult, slower and incomplete. Secondly, the lower intensity of acute arteritis suggests that antigenicity is reduced in the glycerin-preserved trachea, although more time is needed for definitive conclusion. Thirdly, the glycerin-preserved trachea had a lower rate of infection while implanted into the greater omentum. Lastly, the non-existence of malacia confirmed the possibility of preserving lumen rigidity of the glycerin-preserved trachea even with the devitalization of its cartilage.


   Acknowledgments
 
This study was supported by grants from FIPE (Fundo de Incentivo à Pesquisa e Eventos do Hospital de Clínicas de Porto Alegre), FAPERGS (Fundação de Amparo à Pesquisa do Rio Grande do Sul) and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico). We are grateful for the editorial support provided by the Post-Graduate Research Group (GPPG) at Hospital de Clínicas de Porto Alegre.


   REFERENCES
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Grillo HC. Tracheal replacement. Ann Thorac Surg 1990;49:864–5.[Medline]

  2. Li J, Xu PZ, Chen HY, Yang ZQ, Zhang QL. Improvement of tracheal autograft survival with transplantation into the greater omentum. Ann Thorac Surg 1995;60:1592–6.[Abstract/Free Full Text]

  3. Macchiarini P, Lenot B, de Montpreville V, Dulmet E, Mazmanian GM, Fattal M, et al. Heterotopic pig model for direct revascularization and venous drainage of tracheal allografts. J Thorac Cardiovasc Surg 1994;108:1066–75.[Abstract/Free Full Text]

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  7. Lenot B. De Ia greffe de trachee conservee a Ia transplantation tracheale: etude d’un modele experimental [Diplome D’Etudes Approndies]. Paris (IF): Universite de Franche Comte de Besancon. Universite de Bourgogne; 1992.

  8. Resolução Normativa número 196/96 do Conselho Nacional de Saúde. 1996. www.saude.gov.br (10 Março 2002).

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  15. Moriyama H, Sasajima T, Hirata S, Yamazaki K, Yatsuyanagi E, Kubo Y. Revascularization of canine cryopreserved tracheal allografts. Ann Thorac Surg 2000;69:1701–6.[Abstract/Free Full Text]

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  20. Jacobs JP, Quintessenza JA, Andrews T, Burke RP, Spektor Z, Delius RE, et al. Tracheal allograft reconstruction: the total North American and worldwide pediatric experiences. Ann Thorac Surg 1999;68:1043–51.[Abstract/Free Full Text]





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ANN THORAC SURG ASIAN CARDIOVASC THORAC ANN EUR J CARDIOTHORAC SURG
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