Implantation protocol for nude mice was approved by the committee on the Ethics of Animal Experiments in Osaka University(Authorization number; 27–065-010).
Harvest and isolation of articular chondrocyte
Cell isolation protocol for mini pig articular chondrocytes was followed as described below. Total number of mini pigs used for the study was 6. Porcine cartilage was obtained aseptically from the knee joints of skeletally mature male mini pigs (24 months of age) within 24 h of death. Knee joint articular cartilage was minced into small pieces and pre-treated with 0.1% pronase (Roche Diagnostics GmbH, Germany) in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco BRL, Grand Island, NY), containing 10% fetal bovine serum (FBS) (Sigma-Aldrich, St Louis, MO, USA) for 20 min incubating at 37 °C. After centrifugation, participated tissue fragments were resuspended in DMEM (Gibco) containing 10% FBS (Sigma-Aldrich, St Louis, MO, USA) supplemented with 400 unit/mL collagenase type II (Worthington, Lakewood, NJ, USA) and incubated at 37 °C. After 12 h, dissociated cells were washed three times and cultured in tissue culture dish (Corning, Corning, NY, USA) with high glucose-DMEM containing 10% FBS and 1% antibiotic-antimitotic (Sigma-Aldrich) at 37 °C with humidified 5% CO2. Cells were passaged with 0.25% trypsin/EDTA at 80% confluency (about once per week) and replated at a density of 10,000 cells/cm2. Media changes were performed twice a week [13, 16]. Expanded articular chondrocytes were used for experiments at passage 3 (P3; at day 28). Articular chondrocytes at passage 0 (P0; at day 7) isolated from mini pig #4 were used for evaluation of gene expression level.
3-D pellet culture
3-D pellets were cultured with chondrogenic medium for two weeks in the presence of TD198946 1 nM to 100 nM for histological evaluation, and 1 nM to 1000 nM for qualification of glycosaminoglycan (GAG). To obtain cell aggregated pellets, 2 × 105 cells (P3) were centrifuged in 96 deep well polypropylene plate (Evergreen Scientific, Vernon, CA, USA) and cultured in chondrogenic medium: high glucose-DMEM, 1 mM pyruvate (Gibco), 1% ITS +Premix (Corning), 50 μg/mL ascorbic acid (Sigma-Aldrich), 40 μg/mL L-proline (Wako, Osaka, Japan), 100 nM dexamethasone (Sigma-Aldrich), 1% antibiotic-antimitotic (Sigma-Aldrich). Experimental groups were divided by supplement with vehicle (0.1 μL/mL DMSO, serving as vehicle control), various concentrations of TD198946 (1 to 1000 nM), and/or 50 ng/mL BMP-2 (Medtronic, Dublin, Ireland). The pellets were maintained with 0.5 mL medium at 37 °C with humidified 5% CO2. The medium was replaced twice per week. Pellet sizes were measured from microscopic images in the same manner we reported, using NIS Elements (Nikon Instech, Tokyo, Japan) and size were described as area (mm2) [1, 2] (n = 5). For evaluation of gene expression level, 3-D pellets were cultured with chondrogenic medium for 7 days with or without 10 nM TD198946.
3-D scaffold culture for engineered cartilage
In Supplemental Table 1, all different combination of medium of the present study is shown. 2.5 × 106 articular chondrocytes (P3) in 100 μL of culture medium (High glucose Dulbecco’s Modified Eagle Medium; DMEM, 10% fetal bovine serum and 1% antibiotic/antimycotic solution) were seeded in type I collagen scaffolds (15 mm in diameter and 3 mm in thickness, KURABO INDUSTRIES LTD., Japan) and cultured in chondrogenic medium containing with TD198946 (10 nM) and/or BMP-2 (50 or 100 ng/mL). Scaffolds were maintained with 2 mL medium at 37 °C with humidified 5% CO2 for two or four weeks. The medium was replaced three times per week.
Biochemical analysis - sulfated glycosaminoglycan (sGAG) measurement
To quantitatively measure sulfated glycosaminoglycan (sGAG), the pellets (n = 5) were initially digested with 0.05% papain (Sigma-Aldrich) for 3 h at 65 °C with shaking and the sGAG content was measured using dimethylmethylene blue dye binding assay (Blyscan™ Glycosaminoglycan Assay Kit, Biocolor, Westbury, NY, USA) with chondroitin sulfate as a standard (kit contained). The cellularity was measured based on the double strand DNA (dsDNA) content using Qubit® 3.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) and Qubit® dsDNA HS Assay kit (Thermo Fisher Scientific).
RNA and qRT-PCR
Samples (n = 5) were homogenized with zirconia beads and TissueLyser (QIAGEN, Hilden, Germany) in NucleoZol (MACHEREY-NAGEL, Düren, Germany), then total RNA was extracted according to their manufacture’s protocol. RNA clean & concentrator kit (Zymo Research, Irvine, CA, USA) was used for further purification of total RNA and removal of genomic DNA. Total RNA was reverse transcribed to complementally DNA using ReverTra Ace® (Toyobo, Osaka, Japan). Quantitative RT-PCR was performed using Applied Biosystems™ Taqman® Gene Expression Assays (Thermo Fisher Scientific), Taqman® Fast Advanced Master Mix (Thermo Fisher Scientific) and StepOnePlus™ (Thermo Fisher Scientific). Target transcriptional levels were normalized to the level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. The results were calculated using the relative quantitation standard curve method. The expression levels of each target genes were represented by the calculated value (2−ΔΔct). The information of primer is listed in Table S2.
For implantation, the engineered cartilage was developed in four different chondrogenic medium. Specifically, supplementing with or without 10 nM TD198946 and 100 ng/mL BMP-2 for two weeks. Twelve-week-old male athymic nude mice (BALB/cAJcl-nu/nu; CLEA japan) were anesthetized by intraperitoneal injection of a mixture of 0.3 mg/kg of medetomidine, 4.0 mg/kg of midazolam and 5.0 mg/kg of butorphanol. Total number of nude mice was 10 for the study. Bilateral subcutaneous pockets were made in the back and engineered cartilage were implanted. Origin of these cells were mini pig #2. We made four different groups (n = 5); vehicle alone, vehicle + BMP-2, TD198946 alone, TD198946 + BMP-2. Total number of implanted engineered cartilage was 20. At four weeks after surgery, the mice were sacrificed and the implanted engineered cartilage was used for histological analysis.
Histological evaluation and immunohistochemistry
The samples were fixed in 4% paraformaldehyde, followed by dehydrating with ethanol, clearing with xylene, embedding in paraffin wax and 5 μm sections were prepared. The sections were stained with safranin-O/fast green/iron hematoxylin staining for sulfated GAGs. Immunohistochemical (IHC) analyses for type I collagen, type II collagen, and type X collagen were performed on sections using primary antibodies (type I collagen; Abcam plc, UK, type II collagen; Kyowa Pharma Chemical, Japan, type X collagen; Abcam plc, UK) . The deparaffinized sections were incubated in 3% H2O2 for 10 min to block endogenous peroxidase activity, followed by incubation in proteinase K (DAKO, Glostrup, Denmark) for 5 min for antigen retrieval, and then in 10% goat serum for 30 min to avoid non-specific binding of primary antibodies. Primary antibody was applied to each section and incubated for 1 h. Detection was then performed using Histofine® Simple Stain™ MAX PO (MULTI; Nichirei Bioscience, Tokyo, Japan) and Simple Stain™ DAB Solution (Nichirei Bioscience). All procedure was done at room temperature, and during each step, the sections were washed three times with 0.1% Tween 20 in PBS for 5 min. Immunostaining for type I collagen, type II collagen, and type X collagen were assessed with Aperio CS2 (Leica Microsystems, Wetzlar, Germany) based on positive stained area.
Student T-test or two-way ANOVA test or Tukey-Kramer test were performed for comparison of 2 groups or multiple groups. All statistical analysis was performed using BellCurve for Excel® (Social Survey Research Information Co., Ltd., Tokyo, Japan). All data are presented as mean ± standard deviation. For analyses, p < 0.05 was used to indicate statistical significance.