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Repair of long tracheal defects with cryopreserved cartilaginous allografts
Repair of Long Tracheal Defects With Cryopreserved Cartilaginous Allografts By Antonio Messineo, Robert M. Filler, Andrej Bahoric, and Charles R. Smith Toronto, Ontario l Tracheoplasties osteum,
with various autografts
pericardium)
(cartilage,
peri-
have been used in the treatment
of
long-segment
tracheal stenosis. Previous studies have shown
that cartilage
allografts
survive transplantation
on a long-
term basis in various sites of the body. In this study we set out to determine
if cryopreserved
cartilage
and cryopre-
served tracheal allografts would survive when used to cover tracheal
defects in animals. A rectangular
cm long and incorporating
defect (2.8 2 0.3
50% of tracheal
circumference)
was created in the thoracic trachea of 18 piglets. The defect was covered with the excised tracheal segment in 8 (group A, control group), with a cryopreserved
tracheal allograft
in 8
(group B), and with a cryopreserved cartilage allograft harvested from the scapula in 8 (group C). The allografts were cryopreserved, by a standard slow-freezing technique, at -80°C for more than 21 days. All animals survived the grafting procedure and were killed after 2 months. None had signs of airway
obstruction.
defect as the standard,
Using the trachea
the mean sagittal
above the
narrowing
of the
airway in the repaired trachea was 0.4 mm in group A, 0.7 mm in group B, and 0.8 mm in group C; the coronal diameter normal
and grafted
trachea
was similar.
in
The lumen of all
grafts was lined by regenerating respiratory epithilium, and cilia were seen in many. Some cartilage was reabsorbed in group A and B but cartilage islands were present in all. In group A, reabsorption of cartilage was minimal. These findings suggest that segments of trachea or cartilage allografts can be cryopresewed, stored, and, subsequently, used when necessary for tracheoplasty. Copyright o 1992 by W.B. Saunders Company INDEX WORDS:
Cryopreservation;
tracheoplasty.
I
N THE TREATMENT of children with congenital or acquired tracheal narrowing, resection of the affected segment and primary anastomosis is possible only when the stenosis involves a short segment.l In long defects, various tracheoplasties with cartilage, pericardium, and periosteum have been used to increase the diameter of tracheal lumen.z-4 Although successes have been reported, none of the materials completely reproduces the characteristics of the native trachea.5 Thirty years ago, Gibson et al pointed out that transplanted cartilage homografts behave like autografts. In our previous pig experiments implanting cryopreserved tubular tracheal allografts in the airways, we noted that most chondrocytes did not survive and the cryopreserced implanted trachea was transformed into a fibrous, semirigid structure.7 In contrast, Kawabe and Yoshinao* showed that slices of cartilage partially survive freezing and thawing, and Journalofk’ediatric
Surgery, Vol27, No 8 (August), 1992: pp 1131-l 135
the chondrocyte-poor matrix has the physical characteristics of normal cartilage. The purpose of the current experiment was to determine if a cryopreserved patch of tracheal allograft or of cartilage allograft could survive and provide a suitable wall to cover long defects created by tracheoplasty. If successful, a bank of cryopreserved cartilage or tracheas could supply the needed tissue at the time of tracheal reconstruction. MATERIALS AND METHODS Piglets between 4 and 5 weeks of age (weighing 9.2 ? 2.1 kg) were chosen because their rapid growth (from about 10 to 35 kg in 2 months) allows the development of the patched airways to be tested. A tracheal defect was created in all animals, simulating the first stage of tracheoplasty for tracheal stenosis. In each of three groups of six animals, the defect was covered by cryopreserved cartilage, cryopreserved trachea, or the segment of trachea excised. The protocol for this study was approved by the Animal Care Committee of The Hospital for Sick Children. General anesthesia was used in ah animals. Atropine sulfate (0.03 mg/kg) was given as premeditation. The piglets were orally intubated with a polyvinyl chloride tube no. 6.0 (outside diameter, 8.1 mm), and a mixture of nitrous oxide, halothane, and oxygen was administered for anesthesia. Cefazolin (50 mgikg) was given 30 minutes before surgery through an ear vein, and 5% dextrose with 0.2% normal saline (10 ml/kg/h) was infused during the operation through the same route. Through a right posterior thoracotomy in the fourth intercostal space, the thoracic trachea was exposed and a rectangular portion of tracheal wall excised. An anterolateral defect incorporating 50% of tracheal circumference was created. The inferior margin of this defect was about 1 cm above the right upper lobe bronchus. The actual size of the defect in these animals was 2.8 2 0.3 cm. A tubular, silastic stent was inserted into the tracheal lumen and hxed with two 5/O Prolene sutures. Experimental Design The defect was covered with a graft as noted below. In group A (6 animals), the excised segment of trachea was resutured into place (control group). In group B (6 animals), the tracheal defect was covered with cryopreserved tracheal allograft (2.6 ? 0.2 cm
From the Departments of General Surgery and Pathology, The
Hospitalfor Sick Children. Toronto, Ontario. Presented at the 43rd Annual Meeting of the Surgical Section of the American Academy of Pediattics, New Orleans, Louisiana, October 26-27, 1991. Address reprint requests to Robert M. Filler, MD, Suite 1526, The Hospital for Sick Children, 555 UniversityAve, Toronto, Ontario M5G IX& Canada. Copyright 6 I992 by W.B. Saunders Company 0022-3468/92/2708-0039$03.OOlO 1131
MESSINEO ET AL
1132
long x0.7 f 0.2 cm wide). And in group C (6 animals), the tracheal defect was covered with a cryopreserved cartilage allograft harvested from the scapula of a donor animal (2.7 2 0.2 cm long x 0.7 f 0.2 cm wide). The grafts were fixed with interrupted sutures of 5/O Vicryl and the thoracotomy was closed. In the presence of an air leak, a chest tube was inserted through a separate stab incision and connected to a Heimlich valve (Bard-Parker, Lincoln Park, NJ) for 24 hours.
Cryopreservation
Table 2. Graft Defect as a Percentage of Tracheal Circumference Group No.
At Implant
At Autopsy
A (6)
50%
27%
S (6)
50%
18%
C (6)
50%
19%
None of the differences in diameter between the graft site and above it were significant. The graft defect as a percentage of the tracheal circumference at autopsy is shown in Table 2. Detailed findings by group are presented below.
The technique of cryopreserving cartilage has been described in a previous paper.8 The freezing solution contained 162 mL of balanced buffered salt solution with L-glutamine (RPM1 1640 medium) (JRM Biosciences, Lenexa, KS) with 18 mL of fetal calf serum. Twenty milliliters of dimethyl sulfoxide (DMSO) (Tara Pharmaceuticals Inc, Buena Park, CA) was added as a cryoprotectant, and the solution was sealed in a double plastic bag. The graft was maintained in this solution for 20 minutes at 4°C to allow penetration of the cryoprotectant into the tissue, and then the temperature was slowly decreased to -80°C over 200 minutes. Grafts were stored at - 196°C in the gas phase of liquid nitrogen for more than 21 days. The cartilage from the scapula and the tracheal segments were obtained from cadaveric pigs donated by different breeders, The scapular cartilage was shaved to 2 mm in thickness and shaped to fit the tracheal defect.
All grafts appeared grossly normal. The mean coronal narrowing of the airways was 0.4 mm; the sagittal diameter was not modified (Table 1). The graft circumference was reduced to 27% of the total circumference (Table 2). Histologically (Fig l), a normal ciliated columnar epithelium lined the lumen. The hyaline cartilage showed only minimal reabsorption; its shape was maintained and vital chondrocytes were present in normal numbers.
Postoperative Care
Group B
All piglets received two dose8 of buprenorphine (0.03 mg/kg) every 4 hours as an analgesic. Within 4 hours of the operation, the animals were ambulatory and eating. A combination of procainepenicillin G (10,000 U/kg), dihydro-streptomycin (15 mg/kg), and gentamycin (2.5 mgikg) was administered intramuscularly daily for a week. Animals were killed after 2 months with ketamine (0.33 mg/kg) given intramuscularly and pentobarbital (30 mglkg) given intravenously. The tracheas were retrieved and examinated grossly and histologically. The coronal and sagittal tracheal diameters were measured at the level of the graft and in the trachea above the graft. Values in each group were compared between the grafted area and above it, using one-way analysis of variance. Pvalues < .05 were accepted as statistically significant. Histologically, the proportion of the tracheal circumference composed of normal and grafted area was measured.
All specimens showed a slightly reduced coronal lumen (mean narrowing, 0.7 mm), while the sagittal diameter was the same in the normal and the grafted areas. The tracheal allografts maintained sufficient rigidity to keep the lumen completely open. The circumference of the grafted trachea was reduced to 18% (Table 2). Microscopically, no tissue rejection processes were observed. All airways were lined with regenerating epithelium, with some cases showing mature, ciliated, respiratory epithelium. In all cases, islands of cartilage were seen within the largely fibrous graft. Within the matrix there were no viable chondrocytes, only empty lacunae (Fig 2).
Group A (Control Group)
RESULTS
No animals suffered from respiratory distress and all survived. Stems were still inside the lumen in 12 cases. The others had been coughed out. Results of the comparison of tracheal diameters after reconstructive efforts are presented in Table 1. Table 1. Comparison of Tracheal Diameters After Reconstruction Sagittal(mm) Group (No.)
Above Graft Site
At Tracheoplasty
Coronal(mm) Above Graft Sits
At Tracheoplasty
A (6)
13.2 k 0.6
12.8 2 0.6
12.7 2 0.8
12.5 r 0.8
S (6)
12.9 2 0.5
12.2 * 0.7
11.9 2 1.4
11.8 _t ‘I.3
C (6)
13.1 2 0.5
12.5 r 0.5
12.5 + 0.7
12.4 k 0.7
NOTE.
Data
given as mean r SD. None of the differences were
statistically significant.
Fig 1. Group A (control group): a normal epithelium lines the lumen. The hyaline cartilage shows only minimal reabsorption. The shape of cartilage is maintained. Vital condrocytes are present.
CRYOPRESERVED CARTILAGE FORTRACHEALDEFECTS
1133
I
Fig 2. Group B: the tracheal lumen is preserved. The airway is lined with regenerating epithelium. Islands of cartilage can be obsewed within a fibrous graft.
Group
C
problem when rib cartilage is used.ll Rib cartilage has warped when used for nasal bridge reconstruction.12 In experiments with human volunteers, Gibson et al showed that cartilage allografts act as autografts, and emphasized the possible extensive applications of homologous cartilage in reconstructive surgety.6 Today, the use of cartilage allograft in joint reconstruction is well documented in the orthopedic literature.13 The rationale lies in the supposed immunologic privilege enjoyed by cartilage allograft.14 Studies reported by Gertzbein et all5 suggest that chondrocytes have tissue-specific antigen on their surface. However, these cells are surrounded by a weakly antigenic matrix that acts as a biological barrier to protect the chondrocytes.16 Previous experiments in this laboratory by Dykes et ali7 showed that when an elliptical defect was created in the cervical trachea and repaired with the excised tracheal patch, the graft necrosed in few days. In contrast, in the present study the graft in group A animals survived and appeared normal. This difference in outcome may be explained by the differences between these two experiments, ie, site of surgery (thoracic v cervical), surgical technique (closure with interrupted v continous sutures), type of defect (rectangular v elliptical), defect length (2.8 cm v 3.6 cm), and stent used (silastic tubular v silastic coated metal coil). Many attempts to preserve cartilage have been
These tracheas appeared similar to those in group B. The wall was rigid and the lumen open. The coronal size was slightly reduced (mean narrowing, 0.6 mm). The sagittal lumen was identical to that of the remaining trachea. The tracheal circumference at the level of the graft was reduced to 19% (Table 2). Histologically, cartilage had partially disappeared and was replaced by fibrous tissue; scattered islands of cartilage were observed. Regenerating epithelium and, in many cases, mature ciliated epithelium lined the tracheal lumina (Fig 3). DISCUSSION
Tracheoplasties using autologous cartilage, pericardium, and periosteum have been used in the treatment of long, congenital tracheal stenosis (CTS) with varying degrees of success.2-4 The choice of autograft has depended on the length of graft available, the need for airway stability, and surgeon’s preference.s Costa1 cartilage has been the autograft most extensively used.9 However, failures such as formation of granulation tissue, necrosis of the graft, and restenosis at the suture line have been reported with this technique.lO Furthermore, the creation of a shape suitable to the tracheal wall sometimes presents a
Fig 3. Group C: regenerating epithelium lines the tracheal lumen, which has preserved its caliber. The cartilage has partially disappeared and is replaced by fibrous tissue.
MESSINEO ET AL
1134
reported, Kawabe and Yoshinao,8 investigating the viability and function of cells in cryopreserved cartilage slices, showed a difference in the survival of chondrocytes by changing the concentration and time of exposure to DMSO. They concluded that the best technique used 10% DMSO and an exposure at 4°C for 4 hours. In the current experiment, the thin cartilages were exposed to 10% DMSO for 20 minutes at 4°C. In the three groups, the coronal tracheal diameter was only slightly smaller than in the same trachea above the defect, Although no statistically significant differences in caliber were demonstrated among these groups, the percent of the circumference of the tracheal graft was always less than the original 50% at placement, indicating that normal tracheal growth is much greater than that of the graft. These data
confirm BurringtorN findings, which indicated that each tracheal ring has a growth constant that is uninterrupted when a vertical defect is created. In groups B and C, the graft cartilage was partially reabsorbed and substituted with fibrous tissue. A similar observation was made by Tsugawa et all9 at autopsy in a child with CTS repaired with autologous rib cartilage. The framework of the trachea was maintained, although the major part of the cartilage was reabsorbed. However, it is possible that such a fibrous tube could be a satisfactory, stable conduit for ventilation. Although the cryopreservation process could be improved to allow more living chondrocytes to be transplanted, this experiment suggests that segments of cryopreserved tracheal and cartilage allograft could be used in the repair of tracheal defects.
REFERENCES 1. Kimura K, Mukohara N, Tsugawa C, et al: Tracheoplasty for congenital stenosis of the entire trachea. J Pediatr Surg 17869871, 1982 2. Nakayama DK, Harrison MR, de Lorimier AA, et al: Reconstructive surgery for obstructing lesions of the intrathoracic trachea in infants and small children. J Pediatr Surg 17:854-868,1982 3. Cosentino CM, Backer CL, Idriss FS, et al: Pericardial patch tracheoplasty for severe trachea1 stenosis in children: Intermediate results. J Pediatr Surg 26:879-885,199l 4. Cohen RC, Filler RM, Konuma K, et al: A new model of tracheal stenosis and its repair with free periosteal grafts. J Thorac Cardiovasc Surg 92:296-304,1986 5. Loeff DS, Filler RM, Vinograd I, et al: Congenital tracheal stenosis: A review of 22 patients from 1965 to 1987. J Pediatr Surg 23~744-7481988 6. Gibson T, Davis WB, Curran RC: The long-term survival of cartilage homografts in man. Br J Plast Surg 11:177-187,1959 7. Messineo A, Filler RM, Bahoric A, et al: Cryopreservation of pig trachea. J Pediatr Surg (in press) 8. Kawabe N, Yoshinao M: Cryopreservation of cartilage. Int Orthop 14:231-235,199O 9. Zalzal GH, Cotton RT, McAdams AJ: The survival of costal cartilage graft in laryngotracheal reconstruction. Otolaryngol Head Neck Surg 94:204-211,1986 10. Tsugawa C, Kimura K, Muraji T, et al: Congenital stenosis
involving a long segment of trachea: Further experience in reconstructive surgery. J Pediatr Surg 23:471-475,1988 11. Zalzal GH, Barber CS, Chandra R: Tracheal reconstruction using irradiated homologous grafts in rabbits. Otolaryngol Head Neck Surg 100:119-125,1989 12. Gibson T, Davis WB: The distortion of autogenous cartilage grafts: Its cause and prevention. Br J Plast Surg 10:257-274,1958 13. Hartog JM, Slavin AB, Kline SN: Reconstruction of the temporomandibular joint with cryopreserved cartilage and freezedried dura: A preliminary report. J Oral Maxillofac Surg 48:919925,199O 14. Langer F, Gross AE: Immunogenicity of allograft articular cartilage. J Bone Joint Surg [Am] 56297-304, 1974 15. Gertzbein SD, Tait JH, Devlin SR, et al: The antigenicity of chondrocytes. Immunology 33:141-145,1977 16. Gibson T: The transplantation of cartilage. J Clin Pathol 20:513-517,1967 17. Dykes EH, Bahoric A, Smith C, et al: Reduced trachea1 growth after reconstruction with pericardium. J Pediatr Surg 25:25-29,199O 18. Burrington JD: Tracheal growth and healing. J Thorac Cardiovasc Surg 76:453-458,1978 19. Tsugawa C, Nishijima E, Muraji T, et al: The use of omentai pedicle flap for tracheobronchial reconstruction in infants and children. J Pediatr Surg 26:762-765, 1991
Discussion C.D. Smith (Charleston, SC): I am actually bringing greetings and prepared remarks from Dr Biemann Othersen in Charleston. In this study, Drs Messineo, Filler, and their associates have continued the long-standing tradition of excellent investigations from the Hospital for Sick Children regarding tracheobronchial reconstruction. It has been shown in their previous studies and in our
clinical experience that pericardium is an excellent material to cover a tracheal defect, but eventually it contracts and causes recurrent stenosis. The same process occurs in the esophagus. When costal cartilage is utilized, the cartilage itself maintains structural integrity, but an epithelial lining often doesn’t occur and granulation tissue forms on the bare cartilage. For that reason, a composite graft of
CRYOPRESERVED
CARTILAGE
FOR TRACHEAL
DEFECTS
cartilage with adherent perichondrium has been utilized. Autologous material works best with the least contracture, but often gradual narrowing and granulation occurs. Therefore, the search for an ideal material continues. Cryopreserved cartilage is useful for maintaining immediate structural integrity of the trachea, but it would appear that a lining material, such as perichondrium or pericardium would still be beneficial. We know from previous clinical studies by investigators here that autografts and allografts of cartilage can be stored subcutaneously in patients until needed for reconstruction. The allografts and cryopreserved cartilages maintain some integrity and shape but gradually contract. Dr Othersen congratulates the Toronto group on their continuing studies and asks: (1) whether they have attempted to cryopreserve cartilage with attached pericardium, or (2) whether any of their studies have included the use of a lining material such
1135
as pericardium for the tracheal lumen, utilizing the preserved cartilage only to maintain immediate structural integrity and prevent collapse. Ken Kimura (Iowa City, 01): Exactly 10 years ago, we introduced a surgical technique to reconstruct the trachea using a costal cartilage graft. The l-year-old infant with congenital stenosis involving the entire tracheal length is now in the fifth grade and free of disease. At annual endoscopies, the site of the cartilage graft does not look rigid; rather it looks membranous. The implanted cartilage graft seems to have been absorbed and replaced by fibrous tissue. As a graft, the costal cartilage has a great advantage because of its consistency, elasticity and durability. A. Messineo (response): In another experiment I conducted in Toronto, we used the pericardium, and this is the subject of a further communication, which will illustrate its successful use.
with various autografts
pericardium)
(cartilage,
peri-
have been used in the treatment
of
long-segment
tracheal stenosis. Previous studies have shown
that cartilage
allografts
survive transplantation
on a long-
term basis in various sites of the body. In this study we set out to determine
if cryopreserved
cartilage
and cryopre-
served tracheal allografts would survive when used to cover tracheal
defects in animals. A rectangular
cm long and incorporating
defect (2.8 2 0.3
50% of tracheal
circumference)
was created in the thoracic trachea of 18 piglets. The defect was covered with the excised tracheal segment in 8 (group A, control group), with a cryopreserved
tracheal allograft
in 8
(group B), and with a cryopreserved cartilage allograft harvested from the scapula in 8 (group C). The allografts were cryopreserved, by a standard slow-freezing technique, at -80°C for more than 21 days. All animals survived the grafting procedure and were killed after 2 months. None had signs of airway
obstruction.
defect as the standard,
Using the trachea
the mean sagittal
above the
narrowing
of the
airway in the repaired trachea was 0.4 mm in group A, 0.7 mm in group B, and 0.8 mm in group C; the coronal diameter normal
and grafted
trachea
was similar.
in
The lumen of all
grafts was lined by regenerating respiratory epithilium, and cilia were seen in many. Some cartilage was reabsorbed in group A and B but cartilage islands were present in all. In group A, reabsorption of cartilage was minimal. These findings suggest that segments of trachea or cartilage allografts can be cryopresewed, stored, and, subsequently, used when necessary for tracheoplasty. Copyright o 1992 by W.B. Saunders Company INDEX WORDS:
Cryopreservation;
tracheoplasty.
I
N THE TREATMENT of children with congenital or acquired tracheal narrowing, resection of the affected segment and primary anastomosis is possible only when the stenosis involves a short segment.l In long defects, various tracheoplasties with cartilage, pericardium, and periosteum have been used to increase the diameter of tracheal lumen.z-4 Although successes have been reported, none of the materials completely reproduces the characteristics of the native trachea.5 Thirty years ago, Gibson et al pointed out that transplanted cartilage homografts behave like autografts. In our previous pig experiments implanting cryopreserved tubular tracheal allografts in the airways, we noted that most chondrocytes did not survive and the cryopreserced implanted trachea was transformed into a fibrous, semirigid structure.7 In contrast, Kawabe and Yoshinao* showed that slices of cartilage partially survive freezing and thawing, and Journalofk’ediatric
Surgery, Vol27, No 8 (August), 1992: pp 1131-l 135
the chondrocyte-poor matrix has the physical characteristics of normal cartilage. The purpose of the current experiment was to determine if a cryopreserved patch of tracheal allograft or of cartilage allograft could survive and provide a suitable wall to cover long defects created by tracheoplasty. If successful, a bank of cryopreserved cartilage or tracheas could supply the needed tissue at the time of tracheal reconstruction. MATERIALS AND METHODS Piglets between 4 and 5 weeks of age (weighing 9.2 ? 2.1 kg) were chosen because their rapid growth (from about 10 to 35 kg in 2 months) allows the development of the patched airways to be tested. A tracheal defect was created in all animals, simulating the first stage of tracheoplasty for tracheal stenosis. In each of three groups of six animals, the defect was covered by cryopreserved cartilage, cryopreserved trachea, or the segment of trachea excised. The protocol for this study was approved by the Animal Care Committee of The Hospital for Sick Children. General anesthesia was used in ah animals. Atropine sulfate (0.03 mg/kg) was given as premeditation. The piglets were orally intubated with a polyvinyl chloride tube no. 6.0 (outside diameter, 8.1 mm), and a mixture of nitrous oxide, halothane, and oxygen was administered for anesthesia. Cefazolin (50 mgikg) was given 30 minutes before surgery through an ear vein, and 5% dextrose with 0.2% normal saline (10 ml/kg/h) was infused during the operation through the same route. Through a right posterior thoracotomy in the fourth intercostal space, the thoracic trachea was exposed and a rectangular portion of tracheal wall excised. An anterolateral defect incorporating 50% of tracheal circumference was created. The inferior margin of this defect was about 1 cm above the right upper lobe bronchus. The actual size of the defect in these animals was 2.8 2 0.3 cm. A tubular, silastic stent was inserted into the tracheal lumen and hxed with two 5/O Prolene sutures. Experimental Design The defect was covered with a graft as noted below. In group A (6 animals), the excised segment of trachea was resutured into place (control group). In group B (6 animals), the tracheal defect was covered with cryopreserved tracheal allograft (2.6 ? 0.2 cm
From the Departments of General Surgery and Pathology, The
Hospitalfor Sick Children. Toronto, Ontario. Presented at the 43rd Annual Meeting of the Surgical Section of the American Academy of Pediattics, New Orleans, Louisiana, October 26-27, 1991. Address reprint requests to Robert M. Filler, MD, Suite 1526, The Hospital for Sick Children, 555 UniversityAve, Toronto, Ontario M5G IX& Canada. Copyright 6 I992 by W.B. Saunders Company 0022-3468/92/2708-0039$03.OOlO 1131
MESSINEO ET AL
1132
long x0.7 f 0.2 cm wide). And in group C (6 animals), the tracheal defect was covered with a cryopreserved cartilage allograft harvested from the scapula of a donor animal (2.7 2 0.2 cm long x 0.7 f 0.2 cm wide). The grafts were fixed with interrupted sutures of 5/O Vicryl and the thoracotomy was closed. In the presence of an air leak, a chest tube was inserted through a separate stab incision and connected to a Heimlich valve (Bard-Parker, Lincoln Park, NJ) for 24 hours.
Cryopreservation
Table 2. Graft Defect as a Percentage of Tracheal Circumference Group No.
At Implant
At Autopsy
A (6)
50%
27%
S (6)
50%
18%
C (6)
50%
19%
None of the differences in diameter between the graft site and above it were significant. The graft defect as a percentage of the tracheal circumference at autopsy is shown in Table 2. Detailed findings by group are presented below.
The technique of cryopreserving cartilage has been described in a previous paper.8 The freezing solution contained 162 mL of balanced buffered salt solution with L-glutamine (RPM1 1640 medium) (JRM Biosciences, Lenexa, KS) with 18 mL of fetal calf serum. Twenty milliliters of dimethyl sulfoxide (DMSO) (Tara Pharmaceuticals Inc, Buena Park, CA) was added as a cryoprotectant, and the solution was sealed in a double plastic bag. The graft was maintained in this solution for 20 minutes at 4°C to allow penetration of the cryoprotectant into the tissue, and then the temperature was slowly decreased to -80°C over 200 minutes. Grafts were stored at - 196°C in the gas phase of liquid nitrogen for more than 21 days. The cartilage from the scapula and the tracheal segments were obtained from cadaveric pigs donated by different breeders, The scapular cartilage was shaved to 2 mm in thickness and shaped to fit the tracheal defect.
All grafts appeared grossly normal. The mean coronal narrowing of the airways was 0.4 mm; the sagittal diameter was not modified (Table 1). The graft circumference was reduced to 27% of the total circumference (Table 2). Histologically (Fig l), a normal ciliated columnar epithelium lined the lumen. The hyaline cartilage showed only minimal reabsorption; its shape was maintained and vital chondrocytes were present in normal numbers.
Postoperative Care
Group B
All piglets received two dose8 of buprenorphine (0.03 mg/kg) every 4 hours as an analgesic. Within 4 hours of the operation, the animals were ambulatory and eating. A combination of procainepenicillin G (10,000 U/kg), dihydro-streptomycin (15 mg/kg), and gentamycin (2.5 mgikg) was administered intramuscularly daily for a week. Animals were killed after 2 months with ketamine (0.33 mg/kg) given intramuscularly and pentobarbital (30 mglkg) given intravenously. The tracheas were retrieved and examinated grossly and histologically. The coronal and sagittal tracheal diameters were measured at the level of the graft and in the trachea above the graft. Values in each group were compared between the grafted area and above it, using one-way analysis of variance. Pvalues < .05 were accepted as statistically significant. Histologically, the proportion of the tracheal circumference composed of normal and grafted area was measured.
All specimens showed a slightly reduced coronal lumen (mean narrowing, 0.7 mm), while the sagittal diameter was the same in the normal and the grafted areas. The tracheal allografts maintained sufficient rigidity to keep the lumen completely open. The circumference of the grafted trachea was reduced to 18% (Table 2). Microscopically, no tissue rejection processes were observed. All airways were lined with regenerating epithelium, with some cases showing mature, ciliated, respiratory epithelium. In all cases, islands of cartilage were seen within the largely fibrous graft. Within the matrix there were no viable chondrocytes, only empty lacunae (Fig 2).
Group A (Control Group)
RESULTS
No animals suffered from respiratory distress and all survived. Stems were still inside the lumen in 12 cases. The others had been coughed out. Results of the comparison of tracheal diameters after reconstructive efforts are presented in Table 1. Table 1. Comparison of Tracheal Diameters After Reconstruction Sagittal(mm) Group (No.)
Above Graft Site
At Tracheoplasty
Coronal(mm) Above Graft Sits
At Tracheoplasty
A (6)
13.2 k 0.6
12.8 2 0.6
12.7 2 0.8
12.5 r 0.8
S (6)
12.9 2 0.5
12.2 * 0.7
11.9 2 1.4
11.8 _t ‘I.3
C (6)
13.1 2 0.5
12.5 r 0.5
12.5 + 0.7
12.4 k 0.7
NOTE.
Data
given as mean r SD. None of the differences were
statistically significant.
Fig 1. Group A (control group): a normal epithelium lines the lumen. The hyaline cartilage shows only minimal reabsorption. The shape of cartilage is maintained. Vital condrocytes are present.
CRYOPRESERVED CARTILAGE FORTRACHEALDEFECTS
1133
I
Fig 2. Group B: the tracheal lumen is preserved. The airway is lined with regenerating epithelium. Islands of cartilage can be obsewed within a fibrous graft.
Group
C
problem when rib cartilage is used.ll Rib cartilage has warped when used for nasal bridge reconstruction.12 In experiments with human volunteers, Gibson et al showed that cartilage allografts act as autografts, and emphasized the possible extensive applications of homologous cartilage in reconstructive surgety.6 Today, the use of cartilage allograft in joint reconstruction is well documented in the orthopedic literature.13 The rationale lies in the supposed immunologic privilege enjoyed by cartilage allograft.14 Studies reported by Gertzbein et all5 suggest that chondrocytes have tissue-specific antigen on their surface. However, these cells are surrounded by a weakly antigenic matrix that acts as a biological barrier to protect the chondrocytes.16 Previous experiments in this laboratory by Dykes et ali7 showed that when an elliptical defect was created in the cervical trachea and repaired with the excised tracheal patch, the graft necrosed in few days. In contrast, in the present study the graft in group A animals survived and appeared normal. This difference in outcome may be explained by the differences between these two experiments, ie, site of surgery (thoracic v cervical), surgical technique (closure with interrupted v continous sutures), type of defect (rectangular v elliptical), defect length (2.8 cm v 3.6 cm), and stent used (silastic tubular v silastic coated metal coil). Many attempts to preserve cartilage have been
These tracheas appeared similar to those in group B. The wall was rigid and the lumen open. The coronal size was slightly reduced (mean narrowing, 0.6 mm). The sagittal lumen was identical to that of the remaining trachea. The tracheal circumference at the level of the graft was reduced to 19% (Table 2). Histologically, cartilage had partially disappeared and was replaced by fibrous tissue; scattered islands of cartilage were observed. Regenerating epithelium and, in many cases, mature ciliated epithelium lined the tracheal lumina (Fig 3). DISCUSSION
Tracheoplasties using autologous cartilage, pericardium, and periosteum have been used in the treatment of long, congenital tracheal stenosis (CTS) with varying degrees of success.2-4 The choice of autograft has depended on the length of graft available, the need for airway stability, and surgeon’s preference.s Costa1 cartilage has been the autograft most extensively used.9 However, failures such as formation of granulation tissue, necrosis of the graft, and restenosis at the suture line have been reported with this technique.lO Furthermore, the creation of a shape suitable to the tracheal wall sometimes presents a
Fig 3. Group C: regenerating epithelium lines the tracheal lumen, which has preserved its caliber. The cartilage has partially disappeared and is replaced by fibrous tissue.
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reported, Kawabe and Yoshinao,8 investigating the viability and function of cells in cryopreserved cartilage slices, showed a difference in the survival of chondrocytes by changing the concentration and time of exposure to DMSO. They concluded that the best technique used 10% DMSO and an exposure at 4°C for 4 hours. In the current experiment, the thin cartilages were exposed to 10% DMSO for 20 minutes at 4°C. In the three groups, the coronal tracheal diameter was only slightly smaller than in the same trachea above the defect, Although no statistically significant differences in caliber were demonstrated among these groups, the percent of the circumference of the tracheal graft was always less than the original 50% at placement, indicating that normal tracheal growth is much greater than that of the graft. These data
confirm BurringtorN findings, which indicated that each tracheal ring has a growth constant that is uninterrupted when a vertical defect is created. In groups B and C, the graft cartilage was partially reabsorbed and substituted with fibrous tissue. A similar observation was made by Tsugawa et all9 at autopsy in a child with CTS repaired with autologous rib cartilage. The framework of the trachea was maintained, although the major part of the cartilage was reabsorbed. However, it is possible that such a fibrous tube could be a satisfactory, stable conduit for ventilation. Although the cryopreservation process could be improved to allow more living chondrocytes to be transplanted, this experiment suggests that segments of cryopreserved tracheal and cartilage allograft could be used in the repair of tracheal defects.
REFERENCES 1. Kimura K, Mukohara N, Tsugawa C, et al: Tracheoplasty for congenital stenosis of the entire trachea. J Pediatr Surg 17869871, 1982 2. Nakayama DK, Harrison MR, de Lorimier AA, et al: Reconstructive surgery for obstructing lesions of the intrathoracic trachea in infants and small children. J Pediatr Surg 17:854-868,1982 3. Cosentino CM, Backer CL, Idriss FS, et al: Pericardial patch tracheoplasty for severe trachea1 stenosis in children: Intermediate results. J Pediatr Surg 26:879-885,199l 4. Cohen RC, Filler RM, Konuma K, et al: A new model of tracheal stenosis and its repair with free periosteal grafts. J Thorac Cardiovasc Surg 92:296-304,1986 5. Loeff DS, Filler RM, Vinograd I, et al: Congenital tracheal stenosis: A review of 22 patients from 1965 to 1987. J Pediatr Surg 23~744-7481988 6. Gibson T, Davis WB, Curran RC: The long-term survival of cartilage homografts in man. Br J Plast Surg 11:177-187,1959 7. Messineo A, Filler RM, Bahoric A, et al: Cryopreservation of pig trachea. J Pediatr Surg (in press) 8. Kawabe N, Yoshinao M: Cryopreservation of cartilage. Int Orthop 14:231-235,199O 9. Zalzal GH, Cotton RT, McAdams AJ: The survival of costal cartilage graft in laryngotracheal reconstruction. Otolaryngol Head Neck Surg 94:204-211,1986 10. Tsugawa C, Kimura K, Muraji T, et al: Congenital stenosis
involving a long segment of trachea: Further experience in reconstructive surgery. J Pediatr Surg 23:471-475,1988 11. Zalzal GH, Barber CS, Chandra R: Tracheal reconstruction using irradiated homologous grafts in rabbits. Otolaryngol Head Neck Surg 100:119-125,1989 12. Gibson T, Davis WB: The distortion of autogenous cartilage grafts: Its cause and prevention. Br J Plast Surg 10:257-274,1958 13. Hartog JM, Slavin AB, Kline SN: Reconstruction of the temporomandibular joint with cryopreserved cartilage and freezedried dura: A preliminary report. J Oral Maxillofac Surg 48:919925,199O 14. Langer F, Gross AE: Immunogenicity of allograft articular cartilage. J Bone Joint Surg [Am] 56297-304, 1974 15. Gertzbein SD, Tait JH, Devlin SR, et al: The antigenicity of chondrocytes. Immunology 33:141-145,1977 16. Gibson T: The transplantation of cartilage. J Clin Pathol 20:513-517,1967 17. Dykes EH, Bahoric A, Smith C, et al: Reduced trachea1 growth after reconstruction with pericardium. J Pediatr Surg 25:25-29,199O 18. Burrington JD: Tracheal growth and healing. J Thorac Cardiovasc Surg 76:453-458,1978 19. Tsugawa C, Nishijima E, Muraji T, et al: The use of omentai pedicle flap for tracheobronchial reconstruction in infants and children. J Pediatr Surg 26:762-765, 1991
Discussion C.D. Smith (Charleston, SC): I am actually bringing greetings and prepared remarks from Dr Biemann Othersen in Charleston. In this study, Drs Messineo, Filler, and their associates have continued the long-standing tradition of excellent investigations from the Hospital for Sick Children regarding tracheobronchial reconstruction. It has been shown in their previous studies and in our
clinical experience that pericardium is an excellent material to cover a tracheal defect, but eventually it contracts and causes recurrent stenosis. The same process occurs in the esophagus. When costal cartilage is utilized, the cartilage itself maintains structural integrity, but an epithelial lining often doesn’t occur and granulation tissue forms on the bare cartilage. For that reason, a composite graft of
CRYOPRESERVED
CARTILAGE
FOR TRACHEAL
DEFECTS
cartilage with adherent perichondrium has been utilized. Autologous material works best with the least contracture, but often gradual narrowing and granulation occurs. Therefore, the search for an ideal material continues. Cryopreserved cartilage is useful for maintaining immediate structural integrity of the trachea, but it would appear that a lining material, such as perichondrium or pericardium would still be beneficial. We know from previous clinical studies by investigators here that autografts and allografts of cartilage can be stored subcutaneously in patients until needed for reconstruction. The allografts and cryopreserved cartilages maintain some integrity and shape but gradually contract. Dr Othersen congratulates the Toronto group on their continuing studies and asks: (1) whether they have attempted to cryopreserve cartilage with attached pericardium, or (2) whether any of their studies have included the use of a lining material such
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as pericardium for the tracheal lumen, utilizing the preserved cartilage only to maintain immediate structural integrity and prevent collapse. Ken Kimura (Iowa City, 01): Exactly 10 years ago, we introduced a surgical technique to reconstruct the trachea using a costal cartilage graft. The l-year-old infant with congenital stenosis involving the entire tracheal length is now in the fifth grade and free of disease. At annual endoscopies, the site of the cartilage graft does not look rigid; rather it looks membranous. The implanted cartilage graft seems to have been absorbed and replaced by fibrous tissue. As a graft, the costal cartilage has a great advantage because of its consistency, elasticity and durability. A. Messineo (response): In another experiment I conducted in Toronto, we used the pericardium, and this is the subject of a further communication, which will illustrate its successful use.