ORIGINAL ARTICLE
Tissue-Engineered Cartilage as a Graft Source
for Laryngotracheal Reconstruction
A Pig Model
Syed H. Kamil, MD; Roland D. Eavey, MD; Martin P. Vacanti, MD;
Charles A. Vacanti, MD; Christopher J. Hartnick, MD
Objective: To evaluate the feasibility of using tissueengineered cartilage for laryngotracheal reconstruction
in the pig model.
Setting: An animal research facility.
Design: Auricular cartilage was harvested from 3 young
swine. The cartilage was digested, processed, and suspended and a cell culture was obtained. The cells were then
suspended in 3 mL of a 30% solution of a biodegradable
polymer (Pluronic F-127) (polyethylene oxide/
polypropylene oxide copolymer) at a cellular concentration of 50⫻106 cells/mL. This suspension was then implanted subcutaneously into each pig’s dorsum. Eight weeks
after implantation, the cartilage was harvested with the surrounding perichondrial capsule. An anterior cartilage graft
laryngotracheal reconstruction was performed. Bronchoscopy was performed at 3 postoperative weeks to demonstrate airway patency. The animals were killed at 3 months,
and specimens were obtained for histological analysis.
Results: All 3 pigs survived to the 3-month postoperative interval with no evidence of stridor or airway distress. Interval bronchoscopy revealed a normal patent airway with a mucosalized graft. Histopathologic analysis
revealed incorporation of the tissue-engineered cartilage graft in the cricoid area, which correlated with results of bronchoscopic evaluation.
T
From the Department of
Otolaryngology, Pediatric
Otolaryngology Service,
Massachusetts Eye and Ear
Infirmary (Drs Kamil, Eavey,
and Hartnick), the Department
of Pathology, Massachusetts
General Hospital
(Dr M. Vacanti), the
Department of Anesthesiology,
Perioperative and Pain
Medicine, Brigham and
Women’s Hospital
(Dr C. Vacanti), and Harvard
Medical School, Boston. The
authors have no relevant
financial interest in this article.
Subjects: Three young Yorkshire swine.
Conclusion: Tissue-engineered auricular cartilage served
as a viable graft in the pig model and might be an alternative cartilage source for laryngotracheal reconstruction.
Arch Otolaryngol Head Neck Surg. 2004;130:1048-1051
HE SURGICAL TREATMENT OF
subglottic stenosis has
evolved during the past
century to encompass procedures designed to enlarge the airway diameter by (1) allowing
the stenotic segment to remain in situ
while augmenting the subglottic region or
(2) resecting the subglottic scar with anastomosis of the patent airway segments. The
most popular current approach for the laryngotracheal augmentation is the use of
autologous cartilage, although local tissue such as hyoid bone has been used successfully.1,2 Cartilage has been harvested
from the auricle, the thyroid ala, or the costal area.1 Rib cartilage grafting has become the standard procedure for pediatric laryngotracheal reconstruction.3-5
The disadvantages of the rib graft donor source include donor site pain and
scarring,6 limited tissue availability, and
the potential complication of pneumothorax during the harvest of rib cartilage.7 In
an effort to diminish these disadvantages, irradiated costal cartilage8 and al-
(REPRINTED) ARCH OTOLARYNGOL HEAD NECK SURG/ VOL 130, SEP 2004
1048
cohol-stored auricular cartilage9 have been
used in animal models. Both demonstrate significant resorption. Autologous
auricular and thyroid ala cartilage grafts
have been used with some success, but remain useful primarily for anterior grafting where wide cricoid distraction in not
needed. For more complex repairs, autologous rib cartilage remains the standard source of graft material for the cricoid implant. The sculpturing of a graft to
fit within the airway lumen and to afford
wide distraction requires a larger cartilage tissue block. The theoretical potential to generate a sizable block of rigid cartilage with perichondrium that can be
sculpted to provide distraction was the motivation for the present experiment.
Tissue engineering combines living
cells with biocompatible and biodegradable polymers to produce new tissue. Cartilage can be generated successfully in vitro
and in vivo by using animal and human
chondrocytes.10-12 In this study, cartilage
was generated as a firm mass in the subcutaneous tissue by injection of a biodeWWW.ARCHOTO.COM
Downloaded from www.archoto.com by ChristopherHartnick, on January 11, 2006
©2004 American Medical Association. All rights reserved.
Figure 1. Sculptured tissue-engineered cartilage. The ruler is in centimeters.
gradable polymer (Pluronic F-127; BASF, Mount Olive,
NJ) seeded with autologous auricular chondrocytes.
Pluronic F-127 is a thermosensitive polymer liquid at 4°C
that polymerizes to a thick gel at body temperature and
degrades as the cartilage is generated in the subcutaneous tissues.13,14
Figure 2. The tissue-engineered cartilage graft being sutured between the
distracted cricoid plates.
IN VIVO IMPLANTATIONS
Under general anesthesia, the dorsal surfaces of the pigs were
cleaned and draped. A mixture of chondrocytes and polymer
(Pluronic F-127) was injected into the dorsal subdermal space
by means of a 5-mL syringe.
HARVESTING OF CARTILAGE AND
CRICOID IMPLANTATION
METHODS
Autologous auricular cartilage was harvested from 3 pigs under general anesthesia. Perichondrium was removed under sterile conditions, and the cartilage was fragmented into small pieces;
washed in phosphate-buffered saline solution containing 100
U/L of penicillin, 100 mg/L of streptomycin, and 0.25 mg/L of
amphotericin B (GIBCO, Grand Island, NY); and digested with
0.3% collagenase II (Worthington Biochemical Corp, Lakewood, NJ) for 8 to 12 hours. The resulting cell suspension was
passed through a sterile 250-µm mesh filter (Spectra/Mesh 146426; Spectrum Medical Industries Inc, Rancho Dominguez,
Calif). The filtrate was centrifuged, and the resulting cell pellet was washed twice with copious amounts of Dulbecco phosphate-buffered saline solution. Cell number and viability were
determined by means of cell count using a hemocytometer and
trypan blue dye. These chondrocytes were suspended in Ham
F12 culture medium (Invitrogen, Carlsbad, Calif) with Lglutamine, 50-mg/L L-ascorbic acid, 100-U/L penicillin, 100mg/L streptomycin, and 0.25-mg/L amphotericin B and supplemented with 10% fetal bovine serum (Sigma-Aldrich Corp, St
Louis, Mo). The chondrocyte suspensions demonstrated cell
viability in excess of 85% and were suspended in 3 mL of polymer (Pluronic F-127) at a concentration of 50 ⫻ 106 cells/mL.
PLURONIC F-127 AND CHONDROCYTES
Pluronic F-127 consists by weight of approximately 70% ethylene oxide and 30% propylene oxide. This material is soluble
in water and becomes a hydrogel at room temperature. An aliquot of chondrocyte suspension was mixed at 4°C with a 30%
solution of Pluronic F-127 at a cellular concentration of 50⫻106
cells/mL. A total of 3 mL of the polymer was used. At room
temperature, this mixture of chondrocytes and Pluronic F-127
became gellike in consistency.
Implants were removed after 8 to 12 weeks, and the specimens harvested were examined for the formation of cartilage.
No signs of inflammatory reaction around the injected sites were
observed. The cartilage was harvested as a block with its surrounding perichondrial capsule and was sculpted as illustrated (Figure 1) before its implantation in the cricoid area.
Specimens were also sent for histological examination. An anterior cartilage-graft laryngotracheal reconstruction was performed. No prior effort had been made to create subglottic stenosis. A horizontal incision was made in the midline cervical
neck region, and the cervical musculature was lateralized to
expose the laryngotracheal skeleton. An incision was made in
the anterior cricoid plate and the cricoid was slit open in the
middle. The tissue-engineered cartilage graft was then sutured between the distracted cricoid plates (Figure 2 and
Figure 3). The incision was closed in layers. The animals were
extubated at the end of the procedure and were observed for
signs of respiratory distress. All the animals were monitored
closely for the first 12 hours.
BRONCHOSCOPY
Bronchoscopy was performed on each animal at the 3-week postoperative interval to evaluate the laryngeal mucosa after implantation of tissue-engineered cricoid and to demonstrate graft
viability or the presence of any adverse effects such as inflammatory reaction or granulation tissue formation.
SPECIMEN ANALYSIS
All animals were killed 8 to 12 weeks after implantation, and
each larynx was removed and analyzed grossly for the pres-
(REPRINTED) ARCH OTOLARYNGOL HEAD NECK SURG/ VOL 130, SEP 2004
1049
WWW.ARCHOTO.COM
Downloaded from www.archoto.com by ChristopherHartnick, on January 11, 2006
©2004 American Medical Association. All rights reserved.
Figure 3. The final position of the tissue-engineered cartilage as an anterior
cricoid graft.
Figure 4. Histological examination of the tissue-engineered cartilage grown
using a biodegradable polymer (Pluronic F-127; BASF, Mount Olive, NJ)
demonstrates lobular elastic cartilage (safranin O, original magnification ⫻100).
ence of tissue-engineered cartilage and its integration with the
normal cricoid cartilage. Samples were obtained for histological analysis and were fixed in 10% phosphate-buffered formalin (Fisher Chemicals, Fairlawn, NJ). Once fixed for at least 24
hours, specimens were embedded in paraffin and sectioned using standard histochemical techniques. Slide sections were
stained with hematoxylin-eosin, safranin O, Verhoeff, and trichrome.
RESULTS
All 3 pigs survived to the 3-month postoperative mark
with no evidence of stridor or airway distress. Interval
bronchoscopy revealed a normal patent airway with a mucosalized graft in all animals. There was no evidence of
any adverse effects such as inflammatory reaction or
granulation tissue formation.
Results of the histological examination (Figure 4)
of the tissue-engineered cartilage graft grown as part of
the graft in subcutaneous tissue demonstrated lobular cartilage. Hematoxylin-eosin staining demonstrated the car-
Figure 5. Histological findings of the junction of native hyaline cricoid
cartilage (left) with implanted tissue-engineered elastic cartilage
demonstrating elastin in the matrix as a dark brown stain (right) (Verhoeff,
original magnification ⫻100).
tilage to be highly cellular with round-to-oval lacunae
containing binucleate and single forms. The cytoplasm
was abundant and contained condensed linear eosinophlic fragments of materials suggestive of elastin. Areas
bordering the lobular cartilage demonstrated flattened collagenous tissue suggestive of perichondrium. Inflammation and foreign-body reaction were not noted. Safranin
O staining of the same specimen showed strong, even positivity throughout, suggesting proteoglycans production. The fibrous perichondrium tissue was highlighted
by the absence of safranin O staining.
Gross examination by means of palpation of the laryngeal cartilaginous skeleton revealed no defects in the
cricoid area, and the grafts appeared well incorporated
with the surrounding native cartilage.
Histological examination of the graft and the laryngeal complex demonstrated incorporation of tissueengineered cartilage with the native cartilage. There was
no evidence of inflammatory reaction or necrosis of the
graft. The blending of elastic tissue-engineered cartilage
with the native hyaline cartilage was evident by the histological findings. Safranin O staining of elastic cartilage demonstrated even positivity for proteoglycan presence in the extracelluar matrix. Binucleate forms of
chondrocytes were present. The native cartilage was less
cellular with single-lacuna nuclei. Proteoglycan content
appeared to be similar in both types of cartilage. These
differences between native (hyaline) and implanted (elastic) cartilage were noted after staining the specimens with
trichome and Verhoeff stains (Figure 5).
COMMENT
The purpose of this novel technique was to evaluate the
potential to generate tissue-engineered cartilage in sufficient thickness and size for use in reconstructive surgery. It is hoped that one day a small biopsy specimen of
auricular cartilage from a patient might be able to generate enough cartilage for implantation into the larynx
to enlarge the airway. Human cartilage has properties exploitable for tissue engineering.14
(REPRINTED) ARCH OTOLARYNGOL HEAD NECK SURG/ VOL 130, SEP 2004
1050
WWW.ARCHOTO.COM
Downloaded from www.archoto.com by ChristopherHartnick, on January 11, 2006
©2004 American Medical Association. All rights reserved.
The use of growth factors, multiples passages, and
recycling of the culture media has generated the critical
number of chondrocytes needed to grow tissueengineered cartilage.15-18 In our experience, Pluronic F-127
consistently has given the best histological quality of tissue-engineered cartilage.12,19 By using the mixture of
Pluronic F-127 and chondrocytes in this study, we were
able to generate cartilage in the required rigidity and volume. The tissue-engineered cartilage was easily sculptured for use in the anterior cartilage graft laryngotracheal reconstruction procedure. The cartilage generated
was viable and structurally stable.
The preliminary demonstration that the cartilage graft
that had been grown from elastic ear cartilage blended
seamlessly with the native hyaline cartilage was surprising and encouraging. No evidence existed of a cleft or a
boundary zone between the graft and the native tissue. The
junction of the 2 types of cartilage was demonstrated only
by the elastin stain, which highlighted the auricular donor source (Figure 5). The chief histological difference between the hyaline (cricoid) and ear (elastic) cartilages is
the presence or absence of the protein elastin. Verhoeff
elastic stain highlights auricular cartilage, the original
source of the tissue-engineered cartilage, with the brown
matrix stain. The abutting cartilage with pink matrix demonstrates the hyaline (cricoid) cartilage without any elastin stain.
The use of tissue-engineered cartilage as a substitute for costal cartilage would not be an initial graft choice
for every patient undergoing airway reconstruction. Tissue-engineered cartilage would seem to have an application when multiple grafts are needed or when previous costal grafts have been harvested. A major advantage
of tissue-engineered cartilage would be reduced donorsite morbidity. Disadvantages of tissue-engineered cartilage graft include cost and the time interval to allow for
the growth of an adequate piece of cartilage. An additional surgical procedure would be required. However,
the ability to generate the cartilage in a desired thickness and size can be regarded as an important alternative step on the road to the successful surgical treatment
of subglottic stenosis.
Submitted for publication August 25, 2003; final revision
received December 12, 2003; accepted March 2, 2004.
We thank Betty Treanor for her excellent word processing.
Correspondence: Roland D. Eavey, MD, Department
of Otolaryngology, Massachusetts Eye and Ear Infirmary,
243 Charles St, Boston, MA 02114 (roland_eavey@meei
.harvard.edu).
REFERENCES
1. de Jong AL, Park AH, Raveh E, Schwartz MR, Forte V. Comparison of thyroid,
auricular, and costal cartilage donor sites for laryngotracheal reconstruction in
an animal model. Arch Otolaryngol Head Neck Surg. 2000;126:49-53.
2. McGuirt WF Jr, Little JP, Healy GB. Anterior cricoid split: use of hyoid as autologous grafting material. Arch Otolaryngol Head Neck Surg. 1997;123:12771280.
3. Cotton RT, Evans JN. Laryngotracheal reconstruction in children: five-year followup. Ann Otol Rhinol Laryngol. 1981;90:516-520.
4. Cotton RT. Pediatric laryngotracheal stenosis. J Pediatr Surg. 1984;19:699704.
5. Gustafson LM, Hartley BE, Liu JH, et al. Single-stage laryngotracheal reconstruction in children: a review of 200 cases. Otolaryngol Head Neck Surg. 2000;123:
430-434.
6. Jacobs BR, Salman BA, Cotton RT, Lyons K, Brilli RJ. Postoperative management of children after single-stage laryngotracheal reconstruction. Crit Care Med.
2001;29:164-168.
7. Ludemann JP, Hughes CA, Noah Z, Holinger LD. Complications of pediatric laryngotracheal reconstruction: prevention strategies. Ann Otol Rhinol Laryngol.
1999;108:1019-1026.
8. Hubbell RN, Zalzal G, Cotton RT, McAdams AJ. Irradiated costal cartilage graft
in experimental laryngotracheal reconstruction. Int J Pediatr Otorhinolaryngol.
1988;15:67-72.
9. Albert DM, Cotton RT, Conn P. The use of alcohol-stored cartilage in experimental laryngotracheal reconstruction. Int J Pediatr Otorhinolaryngol. 1989;18:147155.
10. Atala A, Cima LG, Kim W, et al. Injectable alginate seeded with chondrocytes as
a potential treatment for vesicoureteral reflux. J Urol. 1993;150:745-747.
11. Kamil SH, Kojima K, Vacanti MP, Bonassar LJ, Vacanti CA, Eavey RD. In vitro
tissue engineering to generate a human-sized auricle and nasal tip. Laryngoscope. 2003;113:90-94.
12. Saim AB, Cao Y, Weng Y, et al. Engineering autogenous cartilage in the shape of
a helix using an injectable hydrogel scaffold. Laryngoscope. 2000;110:16941697.
13. Arevalo-Silva CA, Eavey RD, Cao Y, Vacanti M, Weng Y, Vacanti CA. Internal support of tissue-engineered cartilage. Arch Otolaryngol Head Neck Surg. 2000;126:
1448-1452.
14. Kamil SH, Aminuddin BS, Bonassar LJ, et al. Tissue-engineered human auricular cartilage demonstrates euploidy by flow cytometry. Tissue Eng. 2002;8:8592.
15. Dunham BP, Koch RJ. Basic fibroblast growth factor and insulinlike growth factor I support the growth of human septal chondrocytes in a serum-free environment. Arch Otolaryngol Head Neck Surg. 1998;124:1325-1330.
16. Arevalo-Silva CA, Cao Y, Weng Y, et al. The effect of fibroblast growth factor and
transforming growth factor-␤ on porcine chondrocytes and tissue-engineered
autologous elastic cartilage. Tissue Eng. 2001;7:81-88.
17. Gooch KJ, Blunk T, Courter DL, et al. IGF-I and mechanical environment interact
to modulate engineered cartilage development. Biochem Biophys Res Commun. 2001;286:909-915.
18. Arevalo-Silva CA, Cao Y, Vacanti M, Weng Y, Vacanti CA, Eavey RD. Influence of
growth factors on tissue-engineered pediatric elastic cartilage. Arch Otolaryngol Head Neck Surg. 2000;126:1234-1238.
19. Sims CD, Butler PE, Casanova R, et al. Injectable cartilage using polyethylene
oxide polymer substrates. Plast Reconstr Surg. 1996;98:843-850.
(REPRINTED) ARCH OTOLARYNGOL HEAD NECK SURG/ VOL 130, SEP 2004
1051
WWW.ARCHOTO.COM
Downloaded from www.archoto.com by ChristopherHartnick, on January 11, 2006
©2004 American Medical Association. All rights reserved.