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Figure 1.

Sculptured tissue-engineered cartilage. The ruler is in centimeters.

Sculptured tissue-engineered cartilage. The ruler is in centimeters.

Figure 2.

The tissue-engineered cartilage graft being sutured between the distracted cricoid plates.

The tissue-engineered cartilage graft being sutured between the distracted cricoid plates.

Figure 3.

The final position of the tissue-engineered cartilage as an anterior cricoid graft.

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).

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).

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).

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).

1.
de Jong  ALPark  AHRaveh  ESchwartz  MRForte  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.
PubMed
2.
McGuirt Jr  WFLittle  JPHealy  GB Anterior cricoid split: use of hyoid as autologous grafting material. Arch Otolaryngol Head Neck Surg.1997;123:1277-1280.
PubMed
3.
Cotton  RTEvans  JN Laryngotracheal reconstruction in children: five-year follow-up. Ann Otol Rhinol Laryngol.1981;90:516-520.
PubMed
4.
Cotton  RT Pediatric laryngotracheal stenosis. J Pediatr Surg.1984;19:699-704.
PubMed
5.
Gustafson  LMHartley  BELiu  JH  et al Single-stage laryngotracheal reconstruction in children: a review of 200 cases. Otolaryngol Head Neck Surg.2000;123:430-434.
PubMed
6.
Jacobs  BRSalman  BACotton  RTLyons  KBrilli  RJ Postoperative management of children after single-stage laryngotracheal reconstruction. Crit Care Med.2001;29:164-168.
PubMed
7.
Ludemann  JPHughes  CANoah  ZHolinger  LD Complications of pediatric laryngotracheal reconstruction: prevention strategies. Ann Otol Rhinol Laryngol.1999;108:1019-1026.
PubMed
8.
Hubbell  RNZalzal  GCotton  RTMcAdams  AJ Irradiated costal cartilage graft in experimental laryngotracheal reconstruction. Int J Pediatr Otorhinolaryngol.1988;15:67-72.
PubMed
9.
Albert  DMCotton  RTConn  P The use of alcohol-stored cartilage in experimental laryngotracheal reconstruction. Int J Pediatr Otorhinolaryngol.1989;18:147-155.
PubMed
10.
Atala  ACima  LGKim  W  et al Injectable alginate seeded with chondrocytes as a potential treatment for vesicoureteral reflux. J Urol.1993;150:745-747.
PubMed
11.
Kamil  SHKojima  KVacanti  MPBonassar  LJVacanti  CAEavey  RD In vitro tissue engineering to generate a human-sized auricle and nasal tip. Laryngoscope.2003;113:90-94.
PubMed
12.
Saim  ABCao  YWeng  Y  et al Engineering autogenous cartilage in the shape of a helix using an injectable hydrogel scaffold. Laryngoscope.2000;110:1694-1697.
PubMed
13.
Arevalo-Silva  CAEavey  RDCao  YVacanti  MWeng  YVacanti  CA Internal support of tissue-engineered cartilage. Arch Otolaryngol Head Neck Surg.2000;126:1448-1452.
PubMed
14.
Kamil  SHAminuddin  BSBonassar  LJ  et al Tissue-engineered human auricular cartilage demonstrates euploidy by flow cytometry. Tissue Eng.2002;8:85-92.
PubMed
15.
Dunham  BPKoch  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.
PubMed
16.
Arevalo-Silva  CACao  YWeng  Y  et al The effect of fibroblast growth factor and transforming growth factor-β on porcine chondrocytes and tissue-engineered autologous elastic cartilage. Tissue Eng.2001;7:81-88.
PubMed
17.
Gooch  KJBlunk  TCourter  DL  et al IGF-I and mechanical environment interact to modulate engineered cartilage development. Biochem Biophys Res Commun.2001;286:909-915.
PubMed
18.
Arevalo-Silva  CACao  YVacanti  MWeng  YVacanti  CAEavey  RD Influence of growth factors on tissue-engineered pediatric elastic cartilage. Arch Otolaryngol Head Neck Surg.2000;126:1234-1238.
PubMed
19.
Sims  CDButler  PECasanova  R  et al Injectable cartilage using polyethylene oxide polymer substrates. Plast Reconstr Surg.1996;98:843-850.
PubMed
Original Article
September 2004

Tissue-Engineered Cartilage as a Graft Source for Laryngotracheal ReconstructionA Pig Model

Author Affiliations

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.

Arch Otolaryngol Head Neck Surg. 2004;130(9):1048-1051. doi:10.1001/archotol.130.9.1048
Abstract

Objective  To evaluate the feasibility of using tissue-engineered cartilage for laryngotracheal reconstruction in the pig model.

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.

Setting  An animal research facility.

Subjects  Three young Yorkshire swine.

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.

Conclusion  Tissue-engineered auricular cartilage served as a viable graft in the pig model and might be an alternative cartilage source for laryngotracheal reconstruction.

The 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.35

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 alcohol-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.1012 In this study, cartilage was generated as a firm mass in the subcutaneous tissue by injection of a biodegradable 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

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 146-426; 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 L-glutamine, 50-mg/L L-ascorbic acid, 100-U/L penicillin, 100-mg/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.

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

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 presence 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 cartilage 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 tissue-engineered 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

The use of growth factors, multiples passages, and recycling of the culture media has generated the critical number of chondrocytes needed to grow tissue-engineered cartilage.1518 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 donor-site 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.

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Article Information

Correspondence: Roland D. Eavey, MD, Department of Otolaryngology, Massachusetts Eye and Ear Infirmary, 243 Charles St, Boston, MA 02114 (roland_eavey@meei.harvard.edu).

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.

References
1.
de Jong  ALPark  AHRaveh  ESchwartz  MRForte  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.
PubMed
2.
McGuirt Jr  WFLittle  JPHealy  GB Anterior cricoid split: use of hyoid as autologous grafting material. Arch Otolaryngol Head Neck Surg.1997;123:1277-1280.
PubMed
3.
Cotton  RTEvans  JN Laryngotracheal reconstruction in children: five-year follow-up. Ann Otol Rhinol Laryngol.1981;90:516-520.
PubMed
4.
Cotton  RT Pediatric laryngotracheal stenosis. J Pediatr Surg.1984;19:699-704.
PubMed
5.
Gustafson  LMHartley  BELiu  JH  et al Single-stage laryngotracheal reconstruction in children: a review of 200 cases. Otolaryngol Head Neck Surg.2000;123:430-434.
PubMed
6.
Jacobs  BRSalman  BACotton  RTLyons  KBrilli  RJ Postoperative management of children after single-stage laryngotracheal reconstruction. Crit Care Med.2001;29:164-168.
PubMed
7.
Ludemann  JPHughes  CANoah  ZHolinger  LD Complications of pediatric laryngotracheal reconstruction: prevention strategies. Ann Otol Rhinol Laryngol.1999;108:1019-1026.
PubMed
8.
Hubbell  RNZalzal  GCotton  RTMcAdams  AJ Irradiated costal cartilage graft in experimental laryngotracheal reconstruction. Int J Pediatr Otorhinolaryngol.1988;15:67-72.
PubMed
9.
Albert  DMCotton  RTConn  P The use of alcohol-stored cartilage in experimental laryngotracheal reconstruction. Int J Pediatr Otorhinolaryngol.1989;18:147-155.
PubMed
10.
Atala  ACima  LGKim  W  et al Injectable alginate seeded with chondrocytes as a potential treatment for vesicoureteral reflux. J Urol.1993;150:745-747.
PubMed
11.
Kamil  SHKojima  KVacanti  MPBonassar  LJVacanti  CAEavey  RD In vitro tissue engineering to generate a human-sized auricle and nasal tip. Laryngoscope.2003;113:90-94.
PubMed
12.
Saim  ABCao  YWeng  Y  et al Engineering autogenous cartilage in the shape of a helix using an injectable hydrogel scaffold. Laryngoscope.2000;110:1694-1697.
PubMed
13.
Arevalo-Silva  CAEavey  RDCao  YVacanti  MWeng  YVacanti  CA Internal support of tissue-engineered cartilage. Arch Otolaryngol Head Neck Surg.2000;126:1448-1452.
PubMed
14.
Kamil  SHAminuddin  BSBonassar  LJ  et al Tissue-engineered human auricular cartilage demonstrates euploidy by flow cytometry. Tissue Eng.2002;8:85-92.
PubMed
15.
Dunham  BPKoch  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.
PubMed
16.
Arevalo-Silva  CACao  YWeng  Y  et al The effect of fibroblast growth factor and transforming growth factor-β on porcine chondrocytes and tissue-engineered autologous elastic cartilage. Tissue Eng.2001;7:81-88.
PubMed
17.
Gooch  KJBlunk  TCourter  DL  et al IGF-I and mechanical environment interact to modulate engineered cartilage development. Biochem Biophys Res Commun.2001;286:909-915.
PubMed
18.
Arevalo-Silva  CACao  YVacanti  MWeng  YVacanti  CAEavey  RD Influence of growth factors on tissue-engineered pediatric elastic cartilage. Arch Otolaryngol Head Neck Surg.2000;126:1234-1238.
PubMed
19.
Sims  CDButler  PECasanova  R  et al Injectable cartilage using polyethylene oxide polymer substrates. Plast Reconstr Surg.1996;98:843-850.
PubMed
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