Ann Thorac Surg 2003;76:1884-1888
© 2003 The Society of Thoracic Surgeons
Original article: general thoracic
Comparison of tracheal and nasal chondrocytes for tissue engineering of the trachea
Koji Kojima, MD, PhDa*,
Lawrence J. Bonassar, PhDb,
Ronald A. Ignotz, PhDb,
Kamil Syed, MDa,
Joaquin Cortiella, MDb,
Charles A. Vacanti, MDa
a Laboratory for Tissue Engineering and Regenerative Medicine, Brigham & Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA
b Center for Tissue Engineering, University of Massachusetts Medical School, Boston, Massachusetts, USA
Accepted for publication June 30, 2003.
* Address reprint requests to Dr Kojima, Laboratory for Tissue Engineering and Regenerative Medicine, Department of Anesthesiology, Brigham & Women's Hospital, 75 Francis St, Boston, MA 02115, USA
e-mail: kojima{at}zeus.bwh.harvard.edu
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Abstract
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BACKGROUND: This study was undertaken to evaluate the feasibility of creating engineered tracheal equivalents grown in the shape of cylindrical cartilaginous structures using sheep nasal cartilage–derived chondrocytes. We also tested sheep tracheal and nasal septum for cell yield and quality of the engineered cartilage each produced.
METHODS: Nasal septum and tracheal tissue were harvested from sheep. Chondrocytes from each were separately isolated from the tissues and suspended in culture media. Tracheal and nasal chondrocytes were seeded onto separate polyglycolic acid matrices. Cell-polymer constructs were cultured for 1 week and then wrapped around a 7-mm diameter x 30-mm length silicon tube and implanted subcutaneously on the back of nude mice for 8 weeks (each, n = 6). Both of the tissue-engineered tracheas (TET) were harvested and analyzed for histological, biochemical, and biomechanical properties. These values were compared with native sheep trachea.
RESULTS: The morphology and histology of both tracheal-chondrocyte TET and nasal-chondrocyte TET closely resembled that of native sheep trachea. Safranin-O staining showed that tissue-engineered cartilage was organized into lobules with round, angular lacunae, each containing a single chondrocyte. Chondrocytes from the trachea or nasal septum produced tissue with similar mechanical properties and had similar glycosaminoglycan and hydroxyproline content.
CONCLUSIONS: This study demonstrates that the property of TET using nasal chondrocytes is similar to that obtained using tracheal chondrocytes. This has the potential benefit of facilitating an autologous approach for repair of segmental tracheal defects using an easily obtained chondrocyte population.
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Introduction
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A variety of tracheal replacements have been demonstrated, such as allograft replacement, autologous tissue reconstruction, and prosthetic replacement, including a combination of the above approaches. To date, none of these approaches has attained a place in human use. Application of tissue engineering techniques is an ideal solution to overcome the shortage of reliable tissue for transplant by using a patient's own cells to generate new structural tissues. Tissue engineering endeavors to combine concepts from biology, fundamental engineering, and polymer chemistry to produce new tissue replacements [1]. Since the advent of tissue engineering technology, many researchers have attempted to engineer mammalian tissue using cells combined with various natural or biomaterials. We have already created other cartilaginous structures, such as ear [2] and nose [3], using tissue engineering techniques, but few studies have focused on reconstruction of cartilage in trachea. A main issue in cartilage tissue engineering is to determine which tissue harvesting technique is the easiest, safest, and minimally invasive. An optimal source of cartilage must also be determined. In this study, we tested both sheep tracheal and nasal septum chondrocytes for cell yield and quality of engineered cartilage produced. This study was also designed to evaluate the feasibility of making tissue-engineered cartilage shaped into cylinders to mimic a structural similar to native trachea.
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Material and methods
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Cell isolation and culture
Samples (5 x 5 mm) of sheep nasal septum cartilage (n = 6) and two to three tracheal rings (n = 6) were obtained from 2-month-old sheep. Chondrocytes were isolated from each cartilage sample by digestion in 0.3% collagenase type II (Worthington Biochemical Corp, Freehold, NJ) at 37°C in a shaker for 5 to 8 hours with gentle shaking. The resulting cell suspensions were passed through a 70-µm cell strainer (Becton Dickinson and Company, Franklin Lakes, NJ). Both chondrocyte preparations were cultured in Ham's F-12 media (Gibco, Grand Island, NJ) containing 10% fetal calf serum (Gibco) with 292 µg/mL L-glutamine, 10,000 U/mL penicillin G, 10,000 U/mL streptomysin sulfate, 25 µg/mL amphotericin B, and 50 µg/mL ascorbic acid for 2 weeks. Culture media was changed every 3 days (Fig 1a).
After 2 weeks, a confluent monolayer was formed. Chondrocytes were then harvested via digestion with 0.05% Trypsin-EDTA (Gibco). Each isolated cell preparation was counted using a hemocytometer and viability determined using the trypan-blue (Sigma-Aldrich, Irvine, CA) exclusion method.
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Fig 1. Phase contrast photomicrograph (x200) of sheep nasal chondrocytes in monolayer culture (a). Nonwoven polyglycolic acid (PGA) mesh used for the scaffold material (b). Chondrocytes seeded onto PGA on day 7 (c), and seeded PGA mesh wrapped around a cylindrical mandrel (d). An identical process was followed using tracheal-derived chondrocyte.
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Cell seeding and implantations
Both chondrocyte suspensions were concentrated at 50 x 106 cell/mL and seeded onto a 30 x 40-mm non-woven mesh of polyglycolic acid (PGA) fibers (Davis & Geck, Danbury, CT) (Fig 1b). Both cell-polymer constructs were incubated in vitro for 1 week (Fig 1c) and then wrapped around a 7-mm diameter x 30-mm length silicone tube (Fig 1d) and implanted into subcutaneous pockets on nude mice (Tracheal cartilage, n = 6; nasal septum cartilage, n = 6). The implants were harvested at 8 weeks, and analyzed by histology, biochemistry, and biomechanics. Tissue was assayed for cartilage-specific extracellular matrix components, including glycosaminoglycan (GAG) and hydroxyproline. 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.
Histological examinations
The specimens for histology were fixed in 10% phosphate-buffered formalin, embedded in paraffin, and sectioned. Sections were stained with hematoxylin and eosin or safranin-O.
Biochemical analysis
Biochemical analysis was performed on harvested tissue to quantify the level of cartilage-specific extracellular matrix 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 (BDH), and 125 µg/mL papain (Sigma). The samples were incubated at 60°C for 24 hours, then stored at -20°C. The sulfated GAG content of digests was quantified by previously described methods [4]. Briefly, 10 µL of the papain digest was added to 200 µL of 1,9-dimethylmethylene blue dye at pH 3.0 with absorbencies detected at 525 nm with a spectrophotometer immediately after the addition of the dye. Glycosaminoglycan content of the samples was determined using a C-6-S from shark cartilage (Sigma) as a standard. The hydroxyproline contents of digests were determined by the procedure of Stegemann and Stadler [5]. Briefly, the papain digests were hydrolyzed with equal volumes of 6N HCL in 60°C for 16 to 24 hours. Chloramine T and p-dimethylamino-benzaldehyde were added to hydrolyzed samples, and absorbances were detected at 560 nm with a spectrophotometer immediately after the addition of the dye.
Biomechanical testing
Previously frozen samples of native and both types of tissue-engineered trachea were thawed at room temperature while immersed in phosphate-buffered saline (Gibco) for approximately 30 minutes. Circumferential strips of tissue were cut from all samples with a razor blade with care to ensure that the specimens consisted primarily of cartilage. Rectangular strips were cut to approximately 20 x 5 mm, and the precise length, width, and thickness were determined to within 0.1 mm using calipers.
Samples were tested within 30 minutes of cutting, and all samples remained hydrated until the times of testing. Specimens were placed in stainless steel serrated tensile grips (Harvard Apparatus, Holliston, MA) mounted in a Dynastat mechanical spectrometer (IMASS, Hingham, MA). Specimens were mounted with an initial grip-to-grip distance of 10 mm and were subjected to ramp displacements at a rate of 0.020 mm/s for 150 seconds up to a maximum of 3 mm total displacement. Resultant loads were recorded to within 10 mN at a frequency of 2.5 Hz for the duration of testing.
Applied displacements were normalized to initial grip-to-grip distance to yield values for tissue strain. Sample width and thickness were used to calculate cross-sectional area, which was used to convert measured loads to stresses. The tensile modulus was determined from the stress-strain data by calculating the slope of the linear region of the curve (generally 15% to 25% strain). Moduli for native and both groups of tissue-engineered trachea compared by single-factor analysis of variance, using a t test with Bonferroni correction for pairwise comparison, with p = 0.05 as the minimum level of significance. The statistical power of these studies was 0.84, with a minimum detectable difference of 0.5 MPa
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Results
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Cell-polymer and gross morphology
Cell-polymer constructs formed cartilage de novo in the shape of cylinders after 8 weeks. The gross appearance of both the tracheal-chondrocyte–derived cartilage and nasal septum-chondrocyte–derived cartilage tissue-engineered trachea (tracheal TET, nasal TET) looked very similar to native tracheal cartilage (Fig 2).
Each exhibited a translucent white appearance reminiscent of hyaline cartilage of native trachea. The consistency and elasticity were also comparable.
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Fig 2. Appearance of tracheal tissue-engineered trachea (TET) (a) and cross-section (b) and nasal TET (c) and cross-section (d) at 8 weeks. The gross appearance was very similar to that of native trachea (not shown). Each of the TET specimens had similar appearances.
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Histology
Histological examination of both tracheal TET and nasal TET, using hematoxylin & eosin stains, showed the presence of mature cartilage. Safranin-O staining showed that for both tissue-engineered cartilage samples, cells were organized into lobules with large round, angular lacunae, each containing a single chondrocyte (Fig 3).
Each had cartilaginous histology similar to that of native tracheal cartilage.
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Fig 3. Safranin-O staining of native tracheal cartilage (a), tracheal tissue-engineered trachea (TET) (b), and nasal TET (c). Histologically, the tracheal TET and nasal TET were indistinguishable from native tracheal cartilage. Similar appearances were noted with hematoxylin & eosin staining (not shown).
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Biochemical analysis
Six samples of each engineered trachea and native trachea were analyzed for GAG and hydroxyproline content. Glycosaminoglycan content of the tracheal TET, nasal TET, and native tracheal cartilage were 84.3 ± 7.5, 97.1 ± 3.2, and 120.0 ± 9.2 µg/mg, respectively (Fig 4).
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Fig 4. Glycosaminoglycan (GAG) content of native tracheal cartilage, tracheal TET, and nasal TET (n = 6 ± SEM). Glycosaminoglycan content was assayed as described in Material and Methods on six samples of each tissue type. (TET = tissue-engineered trachea.)
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The hydroxyproline content of the tracheal TET, nasal TET, and native tracheal cartilage were 1.25 ± 0.21, 1.28 ± 0.20, and 1.36 ± 0.13 µg/mg, respectively (Fig 5).
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Fig 5. Hydroxyproline content of native tracheal cartilage, tracheal TET, and nasal TET (n = 6 ± SEM). Hydroxyproline content was determined as an indirect measurement of total collagen content. Six samples of each tissue were assayed as described in Material and Methods. No significant difference was noted between the three tissue types. (TET = tissue-engineered trachea.)
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Biomechanical analysis
The stress-strain response for both normal and tissue-engineered tracheas were nonlinear, with an initial soft "toe" region at low strains followed by a stiffer, more linear region at higher strains (Fig 6a).
The modulus of native trachea was 10.6 ± 1.8 MPa, significantly higher than that of tracheal tissue engineered from tracheal (1.4 ± 0.4 MPa) or nasal chondrocytes (1.4 ± 0.5 MPa). There was not a significant difference between the moduli of tracheal tissue engineered from tracheal or nasal chondrocytes (Fig 6b).
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Fig 6. (a) Representative stress-strain curves for native trachea and tracheal tissue engineered from nasal and trachea chondrocytes. (b) Tensile modulus of native trachea and tracheal tissue engineered from nasal and trachea chondrocytes calculated from the linear portions of stress-strain curves such as those represented in Figure a. Data are presented as mean ± standard deviation for all groups. (TET= tissue-engineered trachea.)
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Comment
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Tissue-engineered cartilage has the potential of correcting tracheal structural and functional defects resulting from benign or malignant diseases [6] or as the result of trauma. Multiple cell types (ie, mature chondrocytes, adult stem cells, embryonic stem cells, marrow stromal stem cells, etc [7–9]) may be useful in tissue engineering. However, it is important to have a readily available and easily obtained cell source. Thus it might be possible to use stromal stem cell–derived bone marrow. However, these cells are not available for making cartilaginous structure in humans. Currently, we still need to harvest native tissue from the patients. Here we evaluated two cartilage tissues for the ability of making good quality cartilage. Ear cartilage is the easiest to harvest for the purpose for tissue engineering, because it can be done safely and is minimally invasive. However, its use as a source of the cells for generation of hyaline cartilage found in the trachea is not optimal. Ear cartilage is elastic cartilage and thus is more flexible than the hyaline cartilage of the trachea. A tissue-engineered trachea made from elastic cartilage would lack the rigidity of hyaline cartilage and collapse easily. In contrast, nasal septum is very similar to tracheal cartilage. Furthermore, we will be able to harvest both epithelial cells and connective tissue from the same small nasal septum tissue. Thus, it will be possible to make a composite engineered tracheal equivalent composed of cylindrical structure with a lumen lined by nasal epithelial cells. It will be also possible to shape the TET into a helix to form the structural component of a functional trachea replacement.
Gross morphology showed that the tracheal TET and nasal TET were similar to the native tracheal cartilage and had excellent rigidity and patency. There was significant angiogenesis in the area surrounding the implant, providing the necessary vascular supply to support the new tissue. Histological evaluation reveals mature hyaline cartilage with evenly distributed lacunae containing single chondrocytes. Safranin-O staining was deeply positive, indicative of abundant proteoglycan production in the matrix. There was no evidence of polyglycolic acid (PGA) or of inflammatory reaction to the implants.
The major structural components of the trachea are proteoglycans and collagen. Glycosaminoglycan content of the tracheal TET was 84.3 ± 7.5 µg/mg, approximately 70% of that of native tracheal cartilage. Nasal TET contained 97.1 ± 3.2 µg/mg GAG, approximately 81% of that of native tracheal cartilage. Nasal TETs were much stiffer than the tracheal TET. Whereas both tracheal chondrocyte–derived TET and nasal chondrocyte–derived TET contained less GAG than native trachea, these samples were "grown" for only a total of 9 weeks, 1 week in culture and 8 weeks in vivo. Longer "growth" times may allow GAG content to more closely approximate native tracheal cartilage. The hydroxyproline content of the tracheal TET and nasal TET were 1.25 ± 0.21 and 1.28 ± 0.20 µg/mg, respectively, approximately 91% and 94% of that of native tracheal cartilage. Thus, the biochemical results demonstrate that the properties of tissue-engineered cartilage are similar, when generated from either tracheal or nasal chondrocytes.
The values for modulus of native sheep tracheal cartilage reported here (Fig 6b) are consistent with previous reports for tensile testing [10] and bending [11] of human tracheal cartilage. Prior studies indicated linearity in the stress-strain behavior of tracheal cartilage [10], in contrast to the present study, which demonstrates a distinct toe region at low strains, as noted in other types of cartilage [12]. The tensile modulus of tracheal tissue engineered from nasal and tracheal chondrocytes was only about 15% that of native trachea in these studies. Despite this difference, the TETs were stiff to the touch and were easily handled with no damage to the tissue. Furthermore, it should be noted that these values do fall in the range of the less stiff regions of native trachea examined in other studies [10], suggesting this technique may ultimately produce tissue capable of withstanding the normal mechanical environment of the trachea. In this model, chondrocytes from the trachea or nasal septum produced tissue with similar mechanical properties and had similar GAG and hydroxyproline content. The fractional amounts of GAG and hydroxyproline were relatively higher than the mechanical properties, compared with native tissue. This may suggest that other compositional features of the extracellular matrix, such as collagen crosslinking, may be important for proper mechanical function [13].
The single procedure of harvesting a 5-mm portion of nasal septum tissue produced enough cells to make a cartilaginous-shaped cylinder in 2 weeks. However, some patients such as burn patients or children would pose a difficulty in harvesting nasal septum. At present, we are working with stromal cells derived from bone marrow. It is our hope in the future that we will be able to use marrow stromal cells for the production of tissue-engineered trachea.
We conclude that this study clearly shows it is possible to make tissue-engineered trachea using nasal septum cartilage. Tissue-engineered trachea is an ideal method to overcome the shortage of available reconstructive trachea while using a patient's own cells to regenerate new structural tissue.
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Acknowledgments
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The authors thank Michelle Maynard for her expert technical assistance and preparation of materials for these experiments. This work was founded in the part by University of Massachusetts Medical School and Worcester Foundation For Biomedical Research.
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Abstract
Introduction
