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Ann Thorac Surg 2004;78:444-448
© 2004 The Society of Thoracic Surgeons
a Department of Thoracic and Vascular Surgery, Heidehaus Hospital, Hannover, Germany
b Tissue Engineering Network, Medical School Hannover, Hannover, Germany
Accepted for publication February 10, 2004.
* Address reprint requests to Dr Walles, Department of Thoracic and Vascular Surgery, Heidehaus Hospital, Am Leineufer 70, D-30149 Hannover, Germany
e-mail: twalles{at}yahoo.com
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Abstract |
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METHODS: We obtained tracheal, costal, and auricular cartilage from porcine donor animals (n = 10). The chondrocytes were cultured two-dimensionally in cell flasks or mixed with a liquid collagen solution forming a three-dimensional culture system. Labeling with carboxy fluorescein diacetate succinimidyl ester (CFDA SE) and biochemical reduction of formazan served to determine cell viability and proliferation. The extracellular matrix produced by the chondrocytes was characterized by Western blot.
RESULTS: The CFDA SE labeling proved viability and the MTT assays documented a proliferation of the chondrocytes over time in vitro. While the chondrocytes in the three-dimensional cell culture system produced hyaline cartilage composed of collagen II, the two-dimensional culture conditions resulted in nonspecific collagen synthesis.
CONCLUSIONS: Chondrocytes grown in a three-dimensional matrix can effectively proliferate and produce cartilage and are viable for more than 2 weeks. Costal chondrocytes are suitable for tracheal cartilage tissue engineering.
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Introduction |
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Tissue engineering may represent an alternative to the shortage of suitable grafts for reconstructive surgery [3]. Through tissue engineering, human-made functional biological organs or tissue replacements can be created from biodegradable carrier structures, so-called matrices, and autologous cells [4]. The properties of these matrices include biocompatibility, bioabsorbability, nonimmunogenity, support of cell attachment and growth, and an ability to induce angiogenesis. Compared with biodegradable synthetic scaffolds [5], natural decellularized xenogenic and allogenic scaffolds maintain the natural extracellular matrix (ECM) composition, do not produce toxic biodegradation products, do not induce inflammation, and have already been successfully used for tissue engineering of heart valves, blood vessels, and urinary bladder [6].
Tracheal reconstruction applying tissue-engineering techniques has recently been attempted by several groups. They reported the dedifferentiation of two-dimensional chondrocyte cultures in vitro and a lack of chondrocyte viability after implantation [7–10]. Knowledge of the specific in vitro characteristics of chondrocytes from different anatomical sites is fragmentary. In our study we compared the expansion potential of porcine chondrocytes of different explantation sites. The influence of two- and three-dimensional culture conditions on cellular growth and cartilage production was evaluated. We focused on cultural metabolic activity and in vitro cartilage production to quantify chondrocyte viability and function.
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Material and methods |
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Cell harvesting
German Landrace pigs (n = 10; body weight 18 to 25 kg) were used to explant costal, tracheal and auricular cartilage and a jejunal segment for the generation of the biological carrier matrix. Graft harvesting was performed under sterile conditions. All animals received human care in compliance with the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health (National Institutes of Health publication 85-23, revised 1996).
Cell isolation
Chondrocytes were harvested from porcine auricular (ACh), tracheal (TCh), and costal (CCh) cartilage under sterile conditions, minced into 1-mm3 pieces, and washed in phosphate-buffered saline (PBS) solution containing 100 mg/L penicillin and streptomycin. Cartilage specimens were enzymatically digested with 0.2% collagenase II at 37°C for 20 hours. The digested cartilage suspension was filtered using a sterile 250-nm nylon filter and centrifuged at 6,000 rpm. The cells were washed twice with BGJ medium containing 10% fetal calf serum (FCS) and centrifuged at 6,000 rpm for 10 minutes. Cell number and viability were determined by cell counts using a hemocytometer and Trypan blue vital dye. Resulting cell pellets with a viability more than 85% were used for cell culture.
Liquid matrix generation
In the donor animals, a median laparotomy was used to isolate a 15- to 20-cm long segment of the jejunum, including its own artery and vein. The superior mesenteric artery was cannulated with a 6F catheter and flushed with 1,000 mL 4°C cold NaCl 0.9%. The intestinal lumen was flushed with 500 mL 4°C cold NaCl 0.9% containing antibiotic solution. The graft was stored at 4°C. The graft was decellularized after mechanical removal of the small bowl mucosa using a modified method of Meezan and associates [11] and kept in cell-type-specific medium at 37°C. Decellularized porcine matrix segments were incubated with 0.2% collagenase II solution for 12 hours at 37°C. The reaction was stopped by adding FCS (10 µL/mL). After filtration and centrifugation with 1,500 rpm (10 minutes) the cell pellet was dissolved in 45 mL Dulbecco's modified essential medium (DMEM) solution containing FCS, penicillin, and streptomycin and stored at 4°C until further processing.
Cell cultures
Chondrocytes were cultured in two different culture conditions using DMEM as culture medium: The first consisted of a two-dimensional chondrocyte monolayer in a 25-cm2 cell flask seeded with 1 x 105 cells. In the second, 1 x 106 cells were mixed with 500 µL liquid matrix and distributed in a single well on a 24-well plate, thus forming a three-dimensional culture system.
Carboxy fluorescein diacetate succinimidyl ester labeling
Carboxy fluorescein diacetate succinimidyl ester (CFDA SE; Molecular Probes, Leiden, Netherlands) was used to label viable cells; 500 µg of CFDA SE was dissolved in 90 µL of DMSO obtaining a 10 mmol/L stock solution. For labeling a 10 µmol/L solution in a total volume of 10 mL PBS/5 mmol/L EDTA was used. Chondrocytes were trypsinized and centrifuged at 1,200 rpm for 10 minutes. The supernatant was removed and the pellet was resuspended in the prewarmed labeling solution and incubated with 5% CO2 at 37°C for 15 minutes. The reaction was stopped by FCS. After an additional washing step the cell pellet was resuspended in 5 mL PBS, 5 mmol/L EDTA, 0.1% bovine serum albumin. This procedure was repeated twice. Labeled cells were resuspended in culture medium and cultured in the liquid biological matrix.
MTT assay
After 3, 7, and 14 days in culture cells were tested for metabolic activity. The MTT measurements were performed by replacing the DMEM culture medium with a 20% MTT-DMEM solution and incubation for 90 minutes. Extinction was measured at 490 nm.
Western blotting
Proteins were isolated and separated after the NuPAGE Bis-Tris Gel instructions (Invitrogen Life Technologies, Madison, WI) on days 1, 3, 7, 10, and 14 of the culture period. Quantitative signal detection was performed according the instructions of Electrochemiluminescence. Western Blotting detection and analysis system of Amersham Bioscience (Freiburg, Germany). A prestained sodium dodecyl sulfate-polyacrylamide gel electrophoresis standard marker with a broad range from 6,000 to 196,000 kDa was used (Bio-Rad, Munich, Germany). Native porcine cartilage served as positive control, and porcine endothelial cells and liquid biological matrix served as negative controls.
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Results |
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Metabolic activity was identical for all three chondrocyte subtypes (Fig 2). The TCh and CCh demonstrated an increased cellular viability compared with ACh after 2 weeks in culture. This difference did not reach statistical significance (Fig 2).
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Cartilage production
The ECM that was synthesized by ACh, TCh, and CCh chondrocyte subtypes grown in the two-dimensional cell culture system consisted of collagen III and collagen X proteins, and no collagen II. In the three-dimensional biological liquid matrix, collagen II was synthesized by all chondrocyte subtypes beginning after 7 days and increasing over time. The three-dimensional biological liquid matrix had no evidence of collagen III and collagen X production (Fig 3).
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Comment |
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The first attempts at engineering tracheal bioprostheses have been made with chondrocyte cell cultures [7–10]. Very recently, Yang and coworkers [7] noticed a dedifferentiation of human TCh in a two-dimensional environment in vitro and their redifferentiation when seeded on a synthetic scaffold. Lee and coworkers [8] stimulated isolated costal rabbit chondrocyte cultures with ß-fibroblast growth factor and insulin-like growth factor I to amplify growth. They encountered difficulties to get phenotypically stable chondrocytes. After seeding a poly-lactic glycolic acid polymer scaffold they obtained no viable chondrocytes [8]. Kojima and associates [9] generated tracheal implants containing cartilage and connective tissue from ovine nasal chondrocytes seeded on polyglycolic acid mesh. They reported no dedifferentiation of chondrocytes in two-dimensional culture systems. Currently no consensus exists about the suitable cell type [14] and culture condition for tracheal cartilage generation [7, 15]. Therefore, we cultured in a porcine model three chondrocyte subtypes in a two- and three-dimensional cell culture system to investigate their ability to produce cartilage.
Our standardized cell isolation and cultivation procedures resulted in equal cellular viabilities for the three tested chondrocyte subtypes in the two- and three-dimensional culture conditions. Also, we were able to show equal metabolic activities for auricular, costal, and tracheal porcine chondrocytes in both cell culture systems. Interestingly, the metabolic activity of the chondrocyte subtypes seeded in two-dimensional conditions was fourfold increased compared with the three-dimensional cultures. Only the chondrocytes cultivated in three-dimensional conditions synthesized functional hyaline cartilage composed of collagen II.
We seeded a tenfold smaller cell number in the fast proliferating two-dimensional culture system to preclude a cellular overgrowth during the 2-week culture period. However, the three-dimensional cell culture system showed one-fourth of the metabolic activity, despite the tenfold amount of cells. The MTT assay resembles the metabolism of the tetrazolium salt MTS into formazan by viable cells. For the MTT assay the medium was removed after 90 minutes and analyzed for its formazan content. We hypothesize that in our three-dimensional culture system the formazan salt attached to the collagen fibers and was thereby removed from the culture medium. Subsequent quantification therefore showed decreased formazan values. The adsorbed formazan can be visualized macroscopically after multiple repetitive measurements. We hypothesized that the chondrocytes in two-dimensional cultures were exposed to nonphysiologic environmental stimuli and their cellular activity shifted toward surplus proliferation instead of forming a cell-specific ECM. It has been shown previously that chondrocytes undergoing serial passages in monolayer cultures exhibit cellular dedifferentiation [7, 15]. These findings are supported by our protein biochemical data in the Western blot analyses: Collagen II represented the major collagen fraction in hyaline cartilage, whereas collagen III and collagen X were generally not found. Therefore we used the detection of collagen III and collagen X as markers for chondrocyte dedifferentiation. While regular cartilage production was detectable in the three-dimensional cultures, all chondrocyte subtypes showed functional dedifferentiation in the two-dimensional culture system. Further studies are planned to quantify the ECM composition by specific immunohistochemical and protein biochemical techniques [16–18].
Kafienah and coworkers [14] demonstrated that chondrocytes of different explantation sites show unequal differentiation patterns. However, our findings did not confirm these data: ACh, CCh, and TCh showed equal metabolic activities and almost identical viability scores. In the clinical setting CCh can be obtained from the patient by a small, safe biopsy procedure. In contrast, the isolation of a sufficient number of TCh and ACh from a patient would demand a more invasive procedure. Based on our findings we therefore favor the use of CCh as a source for hyaline tracheal cartilage.
We herein have introduced a new method to seed isolated chondrocytes in vitro in a three-dimensional fashion. The generated three-dimensional cultures were viable and functional more than 2 weeks. Our presented approach might facilitate the generation of cartilaginous bioartificial implants such as tracheal autografts. However, our generated bioartificial cartilage tissue samples are currently too small for clinical applications. We intend to apply our described techniques to generate larger tissues suitable for biomechanical testing and animal experiments. Therefore, we are seeding CCh on acellular biological carrier matrices. Considering the influence of the ECM composition on cellular differentiation and tissue formation [19], the ideal matrix for tracheal tissue engineering would be decellularized tracheobronchial tissue. However, due to the different wall diameters of the muscular pars membranacea and the tracheal ring its cartilaginous portion could not reliably be decellularized and repopulated with chondrocytes in previous experiments (data not shown). Allogenic or xenogenic matrices would trigger graft rejection, whereas acellular grafts experience early degeneration [12]. Additionally, human and porcine acellular tracheobronchial matrices exceed the critical wall thickness of 0.8 mm, thus requiring vascularization for tissue nutrition and graft survival [20]. Recently, we generated a unique biological vascularized matrix (BioVaM; ARTISS, Hannover, Germany) that supplies a capillary bed for the nutrition of complex human-made biological implants (unpublished data). The matrix is generated from porcine tissue by removal of all cellular components. The preserved vascular structures are then restored in vitro by autologous endothelial cells. In ongoing studies, CCh are injected into this vascularized matrix to form cartilaginous structures. The ECM components generating the liquid biological matrix are also found in the vascularized BioVaM scaffold.
We conclude that costal chondrocytes are a promising cell type for tracheal cartilage tissue engineering. Our introduced liquid biological matrix facilitates their three-dimensional cultivation in vitro, resulting in the synthesis of differentiated hyaline cartilage. The generated three-dimensional culture systems can be transferred onto scaffold matrices to form, for example, tracheal brooches.
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This article has been cited by other articles:
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  T. Walles, B. Giere, M. Hofmann, J. Schanz, F. Hofmann, H. Mertsching, and P. Macchiarini Experimental generation of a tissue-engineered functional and vascularized trachea J. Thorac. Cardiovasc. Surg., December 1, 2004; 128(6): 900 - 906. [Abstract] [Full Text] [PDF]
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