In this study, the mutual fusion of chondrocyte pellets was promoted in order to produce large-sized tissue-engineered cartilage with a three-dimensional (3D) shape. Five pellets of human auricular chondrocytes were first prepared, which were then incubated in an agarose mold. After 3 weeks of culture in matrix production-promoting medium under 5.78 g/cm 2 compression, the tissue-engineered cartilage showed a sufficient mechanical strength. To confirm the usefulness of these methods, a transplantation experiment was performed using beagles. Tissue-engineered cartilage prepared with 50 pellets of beagle chondrocytes was transplanted subcutaneously into the cell-donor dog for 2 months. The tissue-engineered cartilage of the beagles maintained a rod-like shape, even after harvest. Histology showed fair cartilage regeneration. Furthermore, 20 pellets were made and placed on a beta-tricalcium phosphate prism, and this was then incubated within the agarose mold for 3 weeks. The construct was transplanted into a bone/cartilage defect in the cell-donor beagle. After 2 months, bone and cartilage regeneration was identified on micro-computed tomography and magnetic resonance imaging. This approach involving the fusion of small pellets into a large structure enabled the production of 3D tissue-engineered cartilage that was close to physiological cartilage tissue in property, without conventional polyper scaffolds.
Cartilage is present not only in the limbs and vertebrae, but also in the face. It is present in the nose, ears, eyelids, and mandibular condyles, and maintains the facial morphology and movement. Facial cartilage may be lost due to congenital malformations such as cleft lip nasal deformities, or through trauma or extensive resection of cancer. This may result in functional losses in the face, markedly impairing quality of life. Furthermore, arthritis of the mandibular cartilage also leads to a deterioration of activities of daily life due to severe pain and trismus. Therefore, the treatment of impaired cartilage in the face is an important issue in the field of oral and maxillofacial surgery. Although autologous cartilage transplantation has been performed for these diseases involving the facial cartilage, there are related problems; for example, a sufficient volume of tissue cannot always be obtained and the procedure is highly invasive at the donor site. Therefore, cartilage tissue engineering is being investigated.
As regenerative medicine for facial cartilage, chondrocytes collected from auricular cartilage and cultured have been injected to fill spaces generated after the removal of silicon used for cosmetic rhinoplasty. Since auricular chondrocytes show a high rate of division and can be cultured relatively easily, they represent a favorable cell source for cartilage regenerative medicine. However, significant hypoplasia or severe deformation in congenital malformations and arthritis cannot be treated by this method. Thus, the present investigators developed a method to produce regenerative cartilage of a specific shape and hardness by incorporating porous poly- l -lactic acid (PLLA). This has been applied to cleft lip nasal deformity patients.
Since PLLA is a biodegradable polymer, the transplant using porous PLLA is eventually organized into the host tissue. However, the absorption process may be similar to that of foreign body reactions. Moreover, the volume reduced by absorption may lead to a risk of graft deformation in the future. Thus, it is necessary to develop a method that creates mechanical strength and a three-dimensional (3D) morphology without using a scaffold of biodegradable polymers, such as porous PLLA. This may be achieved by producing a cartilage matrix of cultured auricular chondrocytes in vitro and subsequently reconstructing the cartilage tissue. However, chondrocytes cultured in a monolayer condition lose their original cartilage matrix-producing ability and dedifferentiate. In order to re-start cartilage matrix production, cultured chondrocytes should form small aggregates (pellets), because the mesenchymal stem cells (MSC) – the progenitors of chondrocytes – undergo a mesenchymal condensation before differentiating into cartilage. However, cultured chondrocytes alone may not be able to form pellets if their cell-to-cell adhesion force is too weak and insufficient.
Thus, an initial attempt was made by the present investigators to apply atelocollagen, a type of medical hydrogel. It was found that cartilage matrix accumulation was promoted by incubation with a matrix production-promoting medium. However, it was known empirically that only pellets of approximately 1 mm in size could be prepared using this method, due to the limitations of substance exchange. To overcome this, it was next attempted to mass-produce 1-mm pellets. An agarose mold was developed in which many pellets could be cultured simultaneously while maintaining a sufficient substance exchange ( Fig. 1 ). Compressive force was applied to these pellets ( Fig. 1 ) for the purpose of bringing them into close contact, thereby inducing interactions among the matrices produced and adhesion between the matrices and cells, fusing many pellets.
The aim of the present study was to establish a method to produce large-sized tissue-engineered cartilage with a 3D shape, without using any polymer scaffold, by promoting the mutual fusion of chondrocyte pellets in the agarose mold. The conditions for preparing and fusing the chondrocyte pellets were investigated. In addition, subcutaneous or articular transplantation of the tissue-engineered cartilage was performed in beagles to verify its utility.
Materials and methods
Human auricular chondrocytes were isolated from microtia patients undergoing surgery to the ear. After obtaining informed consent, the perichondrium was detached from about 2–3 g of excised residual auricular cartilage, and the cartilage was cut into 1-mm 3 pieces using a surgical knife. These pieces were incubated in 0.15% collagenase solution (Worthington Biochemical Corporation, Lakewood, NJ, USA) at 37 °C for 24 h in a thermostat with shaking. The lysate was filtered through a cell strainer with a pore size of 100 μm. After removing the residue, the filtrate was centrifuged at 500 × g for 5 min, and the human auricular chondrocytes were isolated. The isolated chondrocytes were seeded in a collagen-coated plastic tissue culture dish (Iwaki, Scitech Division, Asahi Techno Glass Co. Ltd, Chiba, Japan) at a density of 2500 cells/cm 2 and were cultured in chondrocyte growth medium (Dulbecco’s modified Eagle medium nutrient mixture F-12 (DMEM/F12) containing 5% human serum, 100 ng/ml fibroblast growth factor 2 (FGF-2), and 5 μg/ml insulin) in an incubator at 37 °C/5% CO 2 . The medium was changed twice a week. Before reaching confluence, the cells were treated with trypsin–ethylenediaminetetraacetic acid (EDTA) solution and passaged. These chondrocytes were used in the experiments after two passages.
Canine auricular and articular chondrocytes were isolated from beagle dogs (8 months old, body weight about 10 kg; Tokyo Laboratory Animals Science Co., Ltd, Tokyo, Japan). Anesthesia was induced intravenously with 1 mg/kg propofol and maintained with 2% halothane in a non-breathing circuit. The unilateral auricular cartilage (approximately 2–3 g) and articular cartilage from the radius head (approximately 0.3 g) of the beagle was harvested by aseptic surgical technique. The samples were treated in the same manner as described above. The isolated chondrocytes (2.0 × 10 5 ) were seeded in a collagen-coated dish and cultured in DMEM/F12 containing 5% autologous serum, 100 ng/ml FGF-2, and 5 μg/ml insulin in an incubator at 37 °C/5% CO 2 . Autologous blood was collected from the cervical vein and allowed to clot over 5 h at room temperature. The serum was separated by centrifugation of the clotted blood at 1719 × g for 30 min. The medium was changed two times per week. Passages were performed by treatment with a trypsin–EDTA solution when the cells were approaching confluence. The cells were used in the experiments after two passages.
In order to prepare chondrocyte pellets and to enhance the matrix production of the chondrocytes, these cells were suspended in 0.8% atelocollagen solution (Koken Co. Ltd, Tokyo, Japan) at a density of 10 7 cells/ml. In the atelocollagen, 20 μl of the cell/material suspension (total 2 × 10 5 cells) was placed in the bottom of a 15-ml conical tube to form a gel on incubation for 1 h at 37 °C. For comparison, cultured chondrocytes alone (2 × 10 5 cells), without any scaffolds, were also placed in a conical tube. A matrix production-promoting medium containing 5 μg/ml insulin, 200 ng/ml bone morphogenetic protein 2 (BMP-2), and 100 nM 3,3′,5-triiodothyronine (T3), or basic medium without any growth factors, was used at a volume of 2 ml for each gel and cultured in an incubator at 37 °C/5% CO 2 . Throughout the experiment, the medium was changed twice per week.
The agarose mold was prepared using agarose (Takara Bio Inc., Otsu, Japan) ( Fig. 1 ). The agarose was mixed with minimum essential medium (MEM) at 2% and dissolved at 121 °C. In order to determine the optimal compression strength for pellet fusion, the 2% agarose solution was sterilized using an autoclave. Five milliliters of the agarose solution was added to the well of a six-well plate, and a 5-mm deep cylindrical concavity of 5 mm in diameter was made. Five pellets of the human auricular chondrocytes were placed in the concavity. Agarose of a 5-mm deep cylindrical shape and 5 mm in diameter was prepared, placed on the pellets, and compressed using sterilized glass to make the fusion-type tissue-engineered cartilage. Ten milliliters of the matrix production-promoting medium or the basic medium was used.
For the subcutaneous transplantation into beagles, a 50 × 5 × 5 mm concavity was made in a tall glass dish with agarose. Fifty pellets of the beagle chondrocytes were placed in the concavity. Agarose with a shape fitted to the concavity was prepared, placed on the pellets, compressed by sterilized glass, and cultured with 80 ml of the matrix production-promoting medium.
For articular transplantation into beagles, 10 ml of the 2% agarose solution was placed into the well of a six-well plate, and a 6-mm deep concavity with a regular hexagonal prism shape (side of the base 2 mm) was made. Porous beta-tricalcium phosphate (β-TCP) was chosen as an artificial scaffold, as it shows high bone conductivity. It is effectively absorbed in vivo and is rapidly replaced by bone. A hexagonal prism made of β-TCP (sides 2 mm and height of the base 3 mm; porosity 75%) was inserted into the concavity in the agarose, and 20 pellets were placed on the β-TCP prism. Another hexagonal prism with sides of 2 mm and a base of 3 mm in height was prepared with agarose and placed on the pellets of beagle articular chondrocytes and compressed with sterilized glass to form a tissue-engineered cartilage for the joint. Ten milliliters of medium was used.
Subcutaneous and articular transplantation in beagles
Fifty pellets of the beagle chondrocytes were cultured in the agarose mold for 3 weeks (total incubation period 6 weeks, including the pellet preparation period). The sample was removed from the concavity of the agarose mold and transplanted under the dorsal skin of the cell-donor beagle under anesthesia in the same manner as described above.
For the articular transplantation, the tissue-engineered cartilage with β-TCP prism was removed from the concavity of the agarose mold and transplanted into a joint cartilage defect model prepared in the cell-donor beagle. Anesthesia was induced in a similar manner as described above. The joint cartilage defect model was prepared as follows: A longitudinal skin incision was made at a site slightly medial to the median of the knee joint. The patella was dislocated laterally. The patella groove for the femur was exposed, and a hole of about 5 mm in depth was made in the groove using a metal drill with a diameter of 5 mm. The tissue-engineered cartilage was placed in the joint defects. As control, only a β-TCP scaffold was transplanted.
Two months after transplantation, the animals were anesthetized and euthanized with intravenous KCl administration. The transplants were harvested by operation. Each experiment was performed twice, using two beagles.
Biomechanical and histological evaluations
For the biomechanical evaluation, measurements were made using a Venustron tactile sensor (Axiom, Inc., Fukushima, Japan). During measurement using the Venustron tactile sensor, a motor-driven sensor unit automatically presses the surface of the material under computer control, and the compression strength and reduction in resonance frequency (tactile value) are measured. The resonance frequency of the sensor was set at 50 Hz, and the maximum press-in depth was 1 mm. Young’s modulus was calculated from the compression strength and the tactile value based on the principle reported by Aoyagi and Yoshida. Software, Venus 42, provided by the manufacturer was used for the calculation of Young’s modulus. Each sample was measured five times.
For the histological evaluation, the samples were fixed in 4% paraformaldehyde solution/0.1 M phosphate buffer (pH 7.4) for 2 h, then immersed in 10% sucrose/phosphate buffered saline (PBS), 20% sucrose/PBS, and 1:2 mixtures of optimal cutting temperature (OCT) compound (Sakura Finetek Japan, Tokyo, Japan) and 20% sucrose/PBS at 4 °C overnight. The samples were then rapidly frozen in liquid nitrogen. Frozen sections (10 μm in thickness) were prepared using a microtome (CM1850 Cryostat; Leica, Solms, Germany), stained with toluidine blue-O and hematoxylin–eosin, and observed under a light microscope (Olympus DP70; Olympus, Tokyo, Japan).
Computed tomography (CT) using a high-resolution 3D micro X-ray CT device was built at the National Institute of Advanced Industrial Science and Technology specifically for the inspection of bones and cartilage and consists of a 7 μm focus X-ray tube (Kevex PXS5-925EA; Thermo Fisher Scientific K.K., Yokohama, Japan) and a 100 μm pixel flat panel detector (C7942CA-02; Hamamatsu Photonics K.K., Hamamatsu, Japan).
The measurement position included the whole joint in the detection area. The sample was wrapped in Parafilm (Pechiney Plastic Packaging Company, Chicago, IL, USA) to avoid drying during the approximate 30 min of imaging and was fixed to the sample table. Parafilm is transparent to X-rays; this did not influence the imaging. Eliminating artifacts, such as beam hardening, 1000 × 600 pixel image data were collected at 1.86 times magnification. Referring to the histogram of the image, the tube voltage and current were set to maximize the contrast (56 kV and 60 μA, respectively). Three hundred and sixty images were acquired and subjected to 3D image reconstruction, and a 3D image comprising 501 slices (900 × 900 pixels per slice) was prepared. The spatial resolution was 52.9 μm in the xyz direction.
Magnetic resonance imaging
Quantitative magnetic resonance imaging (MRI) evaluation of the tissue-engineered cartilage on scaffolds and the control scaffolds embedded in the beagle femur joint was performed using a 2.0-T Biospec 20/30 System with a B-GA20 Gradient System (Bruker, Karlsruhe, Germany), with a maximum gradient strength of 100 mT/m. A 72-mm inside diameter birdcage coil tuned to 85 MHz for proton resonance was used for all measurements. The MRI data acquisition and reconstruction were performed using ParaVision software (Bruker). The parameters measured included the apparent diffusion coefficient (ADC). An ADC map was calculated from the images obtained using diffusion-weighted imaging (DWI) with conventional spin-echo method (SE-DWI). The imaging parameters for the ADC map were a repeat time (TR) of 7500 ms, an echo time (TE) of 39 ms, diffusion gradient strengths of 0, 23, 45, 68, 90 mT/m, diffusion gradient interval ( Δ ) of 19 ms, diffusion gradient duration ( δ ) of 11 ms, and b -values of 0, 92, 321, 688, 1194 s/mm 2 . All sequences were performed with a field of view (FOV) of 50 mm × 50 mm, matrix size of 128 × 128, and slice thickness of 3 mm. The ADC values were calculated as the average of the specimen from the ADC maps obtained. All MRI measurements were carried out with no contrast agent at room temperature.
Conditions for the fusion of chondrocyte pellets
In order to produce tissue-engineered cartilage, the conditions for fusion of the chondrocyte pellets were investigated. The five pellets of human auricular chondrocytes were placed in the agarose mold. A compression of 0.71, 2.00 or 5.78 g/cm 2 was applied for 3 weeks (total incubation period 6 weeks, including the pellet preparation period). Fusion was most favorable at 5.78 g/cm 2 compression in the culture with the matrix production-promoting medium ( Fig. 2 ). On mechanical evaluation, the condition of applying 5.78 g/cm 2 compression led to the highest compression strength and Young’s modulus, while the tactile value under this condition was low ( Fig. 3 ).