This study was conducted in accordance with the Declaration of Helsinki and was approved by the Institutional Review Committee of the National Hospital Organization Sagamihara Hospital. Prior to the acquisition of cartilage samples, informed consent was obtained in writing from each patient or family of the donor. OA cartilage was harvested from a total of 45 end-stage OA knee joints of 45 patients (8 males and 37 females; mean age 74.1 years old; range 63-89 years old) who underwent prosthetic surgery within 4 h after the operation. All knees had medial disease involvement, and the diagnosis of OA was based on the established criteria for knee OA .
Control cartilage was obtained from 9 non-arthritic knee joints from 9 donors (6 males and 3 females; mean age 81.1 years old; range 69-88 years old) within 24 h after death. The donors had no known history of joint disease, and the normality of the joint was confirmed macroscopically at the time of harvest.
LCM and quantitative gene expression analyses
In the analysis, human cartilage tissues were precisely divided into cartilage zones using an LCM device (PixCell IIe; Arcturus, Mountain View, CA, USA), and the gene expression was determined in respective zones. Cartilage obtained from 16 OA knees and 9 control knees was used for this analysis. In OA knees, cartilage samples were harvested from both macroscopically intact areas (preserved areas) and areas showing various degrees of cartilage degeneration (degenerated areas). In control knees, cartilage samples were obtained at 1-3 sites in each knee from the femoral condyles, confirming that the areas showed little sign of cartilage degeneration. At each site, approximately 20 mm × 5 mm of cartilage was obtained in full thickness above the tide mark. The tissue was immediately embedded in OCT compound (Sakura Finetek Japan, Tokyo, Japan), snap-frozen in liquid nitrogen, and stored at -80°C.
For analyses, cryosections were prepared from the OCT-embedded cartilage tissues, and cartilage zones were separated by LCM as described previously [3, 20]. In detail, the cryosections were cut into 20- to 40-mm-thick slivers, and first treated with 0.5M EDTA (pH 8.0) for 3 minutes, before being dehydrated with graded concentrations of ethanol, and clarified with xylene. All reagents were RNase-free, and the entire process was completed within 30 minutes to minimize RNA degradation. The sections were then placed on glass slides, set on an LCM device, and divided into cartilage zones by LCM based on their histological features  (Fig. 1). Immediately after LCM, RNA was extracted from the respective cartilage zones using an RNeasy Micro kit (Qiagen GmbH, Hiden, Germany) with the routine use of DNase I (Qiagen). cDNA was synthesized using Sensiscript reverse transcriptase (Qiagen). The gene expression was evaluated quantitatively by qPCR on a LightCycler (Roche Diagnostics, Basel, Switzerland), using gene-specific primers and probes. SYBR® Premix Ex Taq® Perfect Real Time (Takara Bio, Shiga, Japan) or Premix Ex Taq® Perfect Real Time (Takara Bio) was used for PCR. The cDNA levels were normalized by the expression of GAPDH.
Cartilage protein analyses
Cartilage tissues from 20 end-stage OA knees were used for this analysis. In each knee, cartilage tissues were obtained in pairs from preserved areas and degenerated areas on the tibial plateau. At each area, 400-700 mg wet-weight of tissue was harvested above the tide mark with a scalpel, which was rinsed thoroughly in ice-cold phosphate-buffered saline (PBS). Protein extraction was performed following a previously described method with some modifications [18, 23]. In brief, after being blotted dry and weighed, each cartilage tissue was finely diced and subjected to extraction in 10 ml urea buffer which consisted of 50 mM Tris-HCl buffer, pH 6.0, containing 8M urea, 0.3M NaCl, 0.05% Triton-X100, and proteinase inhibitors (1 mM AEBSF, 0.8 mM aprotinin, 40 mM bestatin, 14 mM E-64, 20 mM leupeptin, 15 mM pepstatin A). Extraction was carried out at 4°C for 48 h on an orbital shaker at 100 rpm. The urea buffer was then recovered and clarified by centrifugation, and the supernatant was dialyzed against PBS, pH 7.4, containing the proteinase inhibitors at 4°C for 72 h with daily change of the buffer. After dialysis, the supernatant was clarified by centrifugation and filtered through a 0.22-mm-pore polyethersulfone filter (Millipore Express; Millipore, Burlington, MA, USA). The filtrate was aliquoted and stored at -80°C until the analysis.
Protein concentrations in the supernatant were determined by a BioPlex 200 system (BioRad, Hercules, CA, USA) using commercially available kits (Milliplex MAP Total Akt/mTOR Magnetic Bead Kit, and Human IGF Binding protein Magnetic Bead Panel, Millipore). For the measurement, 25 ml of the dialyzed extracts was incubated with a suspension of capture antibody-conjugated magnetic microspheres following the manufacturer’s protocols. The microspheres were then incubated with biotinylated detection antibody and R-phycoerythrin (PE)-conjugated streptavidin, and the fluorescence intensity was measured. Protein concentrations were compared after normalization by the total protein concentration of the extract, which was determined by a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) using bovine serum albumin as a standard.
Cartilage explant culture
For explant culture, cartilage tissues were obtained from 5 OA knees in pairs from preserved areas and degenerated areas on the tibial plateaus, as described for the cartilage protein analysis. After being rinsed in sterilized PBS, each piece of cartilage tissue was diced into 30-50 cubes of 2-3 mm. The diced cartilage, or explants, were equally divided into two, which were placed onto respective wells of a 12-well plastic plate. These explants were cultured in Dulbecco's modified Eagle's medium (DMEM)/F‐12 supplemented with ITS Liquid Media Supplement (Sigma Aldrich, St. Louis, MO, USA) and 25 μg/ml ascorbic acid overnight, and then the media was replaced with those containing Akt inhibitor IV (Sigma Aldrich; 5 mM in dimethyl sulfoxide [DMSO]) or DMSO alone (vehicle control). After 48 h of culture, the explants were recovered, embedded in OCT compound, snap frozen in liquid nitrogen and stored at -80°C until analysis. Extraction of RNA from the explants was performed following a previously described method . In brief, 20 mm-thick cryosections were prepared from the explants embedded in OCT compound, which were immediately immersed in TRIzol (Thermo Fisher Scientific). RNA was first recovered from the TRIzol reagent in the aqueous phase, and then purified using the RNeasy Micro kit (Qiagen).
Primary culture human articular chondrocytes
To obtain primary culture human chondrocytes, cartilage tissue was obtained from macroscopically intact areas of four OA knee joints, which were subjected to serial enzymatic digestion using Pronase (Sigma Aldrich) and Collagenase P (Sigma Aldrich) [24, 25]. Following digestion, chondrocytes were plated onto a 12-well plastic culture plate at a density of 2 × 105/cm2, and maintained in the culture media described above for the explant culture. Chondrocytes were used for experiments starting two days after plating.
Generation of recombinant adenoviruses
Recombinant adenoviruses were constructed using a ViraPower Adenoviral Expression System (Thermo Fisher Scientific). For this, human SP1 complementary DNA (cDNA) were cloned into the adenoviral‐generating constructs . The construct was then transfected into 293A cells (Thermo Fisher Scientific) using FuGENE 6 (Roche Diagnostics), and the cells were subcultured to generate recombinant adenoviruses carrying these genes under the control of the human cytomegalovirus immediate‐early enhancer/promoter. The viruses were titrated by limiting dilution plaque titration on 293A cells, and used at 50–100 plaque‐forming units/cell. Our preliminary experiments showed that the efficiency of transduction by this method is almost 100%.
All small interfering RNAs (siRNAs) were purchased from Qiagen. The siRNAs were introduced into primary cultured chondrocytes by electroporation using a Nucleofector (Lonza, Basel, Switzerland), according to the manufacturer’s protocols with some modifications . In brief, immediately after enzymatic digestion of cartilage tissues, 1 × 106 chondrocytes were suspended in 100 μl of electroporation buffer containing 20 pmoles of siRNA, and the siRNAs were introduced into the cells by electroporation. Chondrocytes were then plated and cultured in DMEM/F‐12 containing 20% fetal bovine serum (FBS), which was replaced with DMEM/F‐12 containing 10% FBS the next day. Based on the result of our preliminary experiments, the effect of RNAi was evaluated three days after electroporation. In this study, 2 siRNAs with respective sequences were used to suppress the Sp1 expression, which were confirmed to reduce the expression by 43% and 56%, respectively, as evaluated by qPCR.
Data were compared by paired or unpaired t‐tests. For multiple comparisons, data were compared using a one‐way factorial analysis of variance (ANOVA), and when necessary, Fisher's protected least significant difference was used as a post hoc test. The correlation of the expression was evaluated by a linear regression analysis. P values less than 0.05 were considered significant.