(The FASEB Journal. 2003;17:823-828.)
© 2003 FASEB
A composite tissue-engineered trachea using sheep nasal chondrocyte and epithelial cells
KOJI KOJIMA,
LAWRENCE J. BONASSAR,
AMIT K. ROY,
HIROKAZU MIZUNO,
JOAQUIN CORTIELLA and
CHARLES A. VACANTI1
Center for Tissue Engineering, University of Massachusetts Medical School, Worcester, Massachusetts, USA
1Correspondence: Charles A. Vacanti, Laboratory for Tissue Engineering, Department of Anesthesiology, Brigham & Women’s Hospital, Harvard Medical School, 75 Frances St., Boston, MA 02115, USA. E-mail: cvacanti{at}partners.org
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ABSTRACT
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This study evaluates the feasibility of producing a composite engineered tracheal equivalent composed of cylindrical cartilaginous structures with lumens lined with nasal epithelial cells. Chondrocytes and epithelial cells isolated from sheep nasal septum were cultured in Ham’s F12 media. After 2 wk, chondrocyte suspensions were seeded onto a matrix of polyglycolic acid. Cell-polymer constructs were wrapped around silicon tubes and cultured in vitro for 1 wk, followed by implanting into subcutaneous pockets on the backs of nude mice. After 6 wk, epithelial cells were suspended in a hydrogel and injected into the embedded cartilaginous cylinders following removal of the silicon tube. Implants were harvested 4 wk later and analyzed. The morphology of implants resembles that of native sheep trachea. H&E staining shows the presence of mature cartilage and formation of a pseudo-stratified columnar epithelium, with a distinct interface between tissue-engineered cartilage and epithelium. Safranin-O staining shows that tissue-engineered cartilage is organized into lobules with round, angular lacunae, each containing a single chondrocyte. Proteoglycan and hydroxyproline contents are similar to native cartilage. This study demonstrates the feasibility of recreating the cartilage and epithelial portion of the trachea using tissue harvested in a single procedure. This has the potential to facilitate an autologous repair of segmental tracheal defects.—Kojima, K., Bonassar, L. J., Roy, A. K., Mizuno, H., Cortiella, J., Vacanti, C. A. A composite tissue-engineered trachea using sheep nasal chondrocyte and epithelial cells.
Key Words: chondrocytes • polyglycolic acid (PGA) • hydrogel • tracheal replacement
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INTRODUCTION
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THE SURGICAL MANAGEMENT of tracheal pathology poses one of the most challenging clinical problems facing thoracic surgeons. Extensive tracheal resection is often required for patients with malignant and benign diseases. This often includes patients with congenital or benign stenosis of the trachea resulting from trauma, inflammation, or iatrogenic cases. When reconstructing the trachea it is desirable to use autogenous tissue in order achieve better reliability, durability, and biocompatibility. Several approaches for tracheal replacement have been described, including the use of autogenous tissue (1)
, allografts (2)
, prosthetic materials (3
, 4)
, or a combination of approaches (5
, 6)
, but a completely satisfactory approach has not been achieved. None of the current approaches has the potential to grow with the patient; therefore, durability is limited in growing children.
Tissue engineering endeavors to combine concepts from biology, fundamental engineering, and polymer chemistry to produce new tissue replacements.
Reconstruction of other cartilaginous structures such as ear and nose has been accomplished using tissue engineering techniques (7
, 8)
. However, few studies have focused on tracheal cartilage reconstruction. Previously, we demonstrated the replacement of a resected rat trachea using cartilage tissue engineering principals (9)
.
Except for tracheal allografts, there has been no prosthetic tracheal replacement using composites of cartilage and epithelium. In this study, we demonstrate the ability to seed different material constructs with more than one cell type to create a composite tissue. Our efforts have generated engineered tracheal equivalents composed of cartilaginous tubes lined with tracheal epithelium. This study also demonstrates the feasibility of recreating the cartilage and epithelial portions of the trachea using tissue harvested from a single procedure.
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MATERIALS AND METHODS
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Cell isolation and culture
Samples (5x5 mm) of sheep nasal septum cartilage were obtained from 2-month-old sheep (N=6) (Fig. 1
). Nasal epithelial tissue was separated from the underlying nasal septum cartilage. Chondrocytes were isolated from the cartilage by digestion with 0.3% collagenase type II (Worthington Biochemical Corp., Freehold, NJ, USA) at 37°C for 5–8 h while shaking. The resulting cell suspension was passed through a 100 µm cell strainer (Becton Dickinson and Co.,Franklin Lakes, NJ, USA). Chondrocytes cells were cultured in Ham’s F-12 media (Gibco, Grand Island, NY, USA) including 10% fetal calf serum (FCS) (Gibco) with 292 µg/mL L-glutamine, 10,000 U/mL penicillin G, 10,000 U/mL streptomycin sulfate, 25 µg/mL amphotericin B, and 50 µg/mL ascorbic acid for 2 wk. Culture media was changed every 3 days (Fig. 2
a). After 2 wk, cells became confluent and chondrocytes were harvested via digestion with 0.05% trypsin-EDTA (Gibco). The isolated cells were counted and resuspended to a final concentration of 50 x 106/mL. Epithelial cells were also obtained from mucosal lining of the nasal septum tissue by culturing 2 mm x 2 mm explants in Ham’s F-12 media (Gibco), including 10% FCS (Gibco) with L-glutamine and antibiotic-antimycotic solution.
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Figure 1. Schematic diagram of methods for isolation, culture, and implantation of composite trachea constructs.
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Figure 2. Phase-contrast photomicrograph (200x) of sheep nasal chondrocytes in monolayer culture (a). Nonwoven PGA mesh used for the scaffold material (b). Chondrocytes seeded onto PGA on day 0 (c) and growth of cells and matrix on day 7 (d).
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Human apo-transferrin (5 µg/mL; Sigma, St. Louis, MO, USA), 5 µg/mL human recombinant insulin (Sigma), and 10 ng/mL epithelial growth factor (PeproTech, Rocky Hill, NJ, USA). Culture media was changed every 2 days and explant was removed at 2 wk The resulting cells were trypsinized, counted, and replated at a density of 2500 cell/cm2 in T175 flasks. Cells were subsequently passaged upon confluence for up to 4–6 wk. This technique has been shown to yield cells that retain epithelial morphology and cytokeratin expression throughout culture (10)
.
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Cell seeding and implantations
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Chondrocyte suspensions were seeded onto 30 mm x 40 mm x 2 mm nonwoven mesh of polyglycolic acid (PGA) fibers-14 µm in diameter (Davis and Geck, Danbury, CT, USA) (Fig. 2b, c
). Cell-polymer constructs were incubated in vitro for 1 wk (Fig. 2d
), wrapped around a 7 mm diameter x 30 mm length silicone tube (N=6) (Fig. 3
). These composite trachea were implanted subcutaneously in athymic mice and harvested after 6 wk (Fig. 4
a).
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Figure 3. Tissue-engineered trachea construct immediately after assembly. Seeded PGA mesh was wrapped around a cylindrical mandrel.
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Figure 4. Appearance of tissue-engineered trachea before (a) and during (b) 10 wk harvest. Both the radial (c) and axial (d) views reveal similarities to native trachea, with a distinct structure containing cartilage and epithelial lining (arrow).
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Pluronic F-127 NF (BASF, Mount Olive, NJ, USA) was dissolved at 4°C in culture media to a final concentration of 23% (w/w) and filtered through a cold 0.22 µm filter. Nasal epithelial cells at a concentration of 50 x 106 cells/mL were suspended in 23% Pluronic F-127 and injected into the embedded cylindrical tubes of cartilage generated in the nude mouse after the silicone tube had been removed. The implants were harvested at 4 wk after epithelial cell injection (Fig. 4b
). All animals received humane care in compliance with the "Principles of Laboratory Animals Care" formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Science and published by the National Institutes of Health (NIH publication 85-23, revised 1985). All animal procedures complied with the guidelines provided by the Institutional Animal Care and Use Committee of the University of Massachusetts Medical School.
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Histological examinations
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Samples were fixed in 10% phosphate-buffered formalin, embedded in paraffin, and sectioned. Sections were stained with either hematoxylin and eosin or safranin-O.
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Biochemical analysis
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Biochemical analysis was performed on harvested tissue to quantify the level of cartilage-specific ECM components. Samples were digested by the addition of 1.0 mL of 100 mM sodium phosphate (Na2HPO4), 10 mM sodium EDTA (Na2EDTA), 10 mM cysteine hydrochloride (Sigma), 5 mM EDTA, and 125 µg/mL papain (Sigma). The samples were incubated at 60°C for 24 h and stored at -20°C. The sulfated glycosaminoglycan (GAG) content of digests was quantified by described methods (11). Briefly, 10 µL of papain digest was added to 200 µL of 1,9-dimethylmethylene blue dye at pH 3.0. Absorbance at 525 nm was measured with by spectrophotometer immediately after addition of the dye. GAG content of the samples was calculated using a chondroitin-6-sulfate from shark cartilage (Sigma) as a standard. The hydroxyproline content of the samples were determined by the procedure of Stegemann and Stadler (12)
. The papain digests were hydrolyzed with equal volumes of 6N HCL at 115°C for 16–24 h. Chloramine T and p-dimethylamino-benzaldehyde were added to hydrolyzed samples and absorbances at 560 nm were measured with a spectrophotometer immediately after addition of the dye. DNA content of samples was determined by quantitating fluorescence (358/458 nm) of aliquots immediately after mixing with bisbenzimidazole dye (Hoechst 33258) using a fluorometer (13)
.
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RESULTS
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Gross morphology
Cell-polymer constructs formed de novo cartilage in the shape of cylinders lined with a pseudo-stratified columnar epithelium after 10 wk. The gross appearance of the tissue-engineered trachea looked very similar to native tracheal cartilage (Fig. 4c
). The lumen of inside were lined with epithelial structure (Fig. 4d
). Average epithelial coverage of the lumen was 
60%.
Histology
Histological examination of engineered trachea using hematoxylin and eosin (H&E) stains showed the presence of mature cartilage and formation of epithelial layer, with a distinct interface between them. Safranin-O staining showed that tissue engineering cartilage was organized into lobules with round, angular lacunae, each containing single chondrocytes (Fig. 5
).
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Figure 5. Hematoxylin and eosin staining of tissue-engineered trachea (a) and native trachea (b). Safranin-O staining of tissue-engineered trachea (c) and native trachea (d).
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Biochemical assays
Proteoglycan content of the tissue-engineered trachea was similar to that of native trachea (Fig. 6
). The total hydroxyproline content of tissue-engineered trachea was 1.4±0.3 µg/mg, similar to that of native trachea (Fig. 7
). Total tissue DNA content was measured to quantify cell density. The cell density tissue-engineered trachea was higher than native trachea (Fig. 8
); there is no difference between tissue-engineered trachea and native.
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DISCUSSION
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Tissue-engineered trachea has the potential of correcting many structural and functional defects resulting from disease or injury, especially congenital absence of the trachea (14)
. Several approaches for tracheal replacement have been described. However, these efforts have met with limited success due to stenosis, immunological complications, bacterial infection, and material failure. Experimental tracheal reconstruction has been attempted with autotransplants and xenografts. The problems associated with both of these approaches are the necessity for lifelong immunosuppression and a lack of sufficient donor tissue. Reconstruction of other cartilaginous structures such as the ear and nose have been accomplished using tissue engineering techniques (7
, 15)
, but few studies have focused on tracheal reconstruction from isolated chondrocytes. Except for tracheal allografts, there have been no prosthetic tracheal replacements that use composites of cartilage and epithelium. It has been reported in cases of tracheal allografts that epithelial denudation of the tracheal grafts will likely occur within 20 days after transplantation (16)
.
Tissue-engineered tracheal epithelium is also an attractive therapy for epithelial disease, such as smoke inhalation injury and Kartagener’s syndrome or immobile cilia syndrome. The goal of this study was to create an autologous organoid structure with a cartilaginous framework lined with stable epithelial mucosa to provide many potential advantages in tracheal surgery. This study demonstrates the feasibility of using tissue-engineered cartilage with an epithelial lining to repair potentially fatal tracheal defects created by long circumferential resections in a single procedure.
Gross inspection of our tissue-engineered trachea demonstrates our tissue was similar to the native tracheal cartilage and had excellent patency, rigidity, and cylindrical shape. When cut into cross section, the cartilage cylinder appeared similar to the native sheep tracheal ring (Fig. 4)
. Cartilage formation was uniform throughout the sample, with no fibrous formation, as evident from gross morphology (Fig. 4)
; proteoglycan content was similar to native cartilage (Fig. 6)
. From our results, there is good apposition of cartilage and epithelial layers in these implanted tissue-engineered trachea. Safranin-O and H&E stains demonstrated progressive maturing hyaline cartilage with no evidence of PGA. In terms of degradation, the PGA polymers can be engineered to degrade from as short a period as 4 wk to as long as 2 years (9)
.
Proteoglycans are a major structural component of the cartilage ECM and are responsible the tissue stiffness. GAG and hydroxyproline content are reflective of proteoglycan and collagen levels in tissue-engineered cartilage. From our results, GAG and hydroxyproline content of the tissue-engineered cartilage were similar to that of native trachea (Figs. 6
, 7)
. The proteoglycan and collagen content of native tracheal cartilage is less than articular cartilage (17)
, but similar to nasal cartilage (18)
. Since mechanical properties of cartilage are correlated with proteoglycan and collagen content, it is likely that samples would exhibit sufficient mechanical integrity.
Our initial experiments have examined which areas of the body provide the easiest, safest, and least invasive sources of cartilage for tissue engineering. The main sources of cartilage use for tissue engineering are tracheal, nasal septum, knee, and ear cartilage. Ear cartilage is the easiest, safest, and least invasive to obtain for tissue engineering purposes. However, ear cartilage is elastic and may not provide ideal mechanical characteristics for making tracheal tissue. We chose nasal cartilage as a source of cells for tracheal replacement because both are hyaline cartilage. The feasibility of generating tissue-engineered cartilage from human nasal chondrocytes was recently documented (19)
. This engineered cartilage had excellent mechanical properties, and this approach was shown to be feasible for wide range of patient ages. In addition, nasal septum provides a simultaneous source of epithelial cells. We have shown that with a single nasal septum biopsy, it is possible to obtain 100 x 106 chondrocytes and 10–20 x 106 epithelial cells within 3 wk. These results have demonstrated that nasal chondrocytes can be used to create a tissue-engineered trachea by growing cartilage in the form of a rigid tube with an epithelial lining attached to the lumen.
Future studies will incorporate the use of autologous cartilage for the creation of a functional trachea.
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ACKNOWLEDGMENTS
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This study was funded by the University of Massachusetts Medical School and Worcester Foundation for Biomedical Research.
Received for publication May 16, 2002.
Accepted for publication October 4, 2002.
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ABSTRACT
INTRODUCTION
