From March 2017 to June 2018, articular cartilage specimens were collected from 8 clinically confirmed DNOAP patients, and the articular cartilage of matched joints from 8 amputation patients without underlying disease were as control group.
Inclusion criteria for DNOAP patients are as follows: (1) DNOAP was definitely diagnosed according to the diabetes mellitus history, clinical and imaging manifestations, (2) Type I- IIIA according to Brodsky classification, (3) Phase III judged by Eichenholtz classification system, (4) patients underwent middle or posterior foot fusion surgery. Exclusion criteria: (1) Patients with peripheral arterial disease, (2) active infection and ulcer, (3) unclear diagnosis of DNOAP. The 8 patients with DNOAP were 3 males and 5 females, aged 20~66 (55.7 ± 3.8) years, diabetes mellitus duration of 6~14 (11.7 ± 3.1) years, DNOAP duration of 5~13 (7.3 ± 4.3) months. Two cases were Brodsky type I, 4 cases were type II, and 2 cases of Type IIIA.
Enrolled controls were patients without diabetes and peripheral neuropathy who underwent amputation due to traffic accident or serious trauma. Exclusion criteria: (1) osteoarthritis, Rheumatoid arthritis, and other degenerative joint disease, (2) open injury or contamination of affected joints. Eight cases of traffic accident or severe injury amputation were recruited as the controls, including 4 males and 4 females, aged 19-65 (57.6 ± 3.7) years old.
The articular cartilages of tibiotalar joint, subtalar joint, and talonavicular joint were taken from two groups. The use of human samples was approved by the Ethical Committee of Honghui Hospital of Xi’an Jiaotong University (approval No.201702003), and informed consent was obtained from each participant.
The articular cartilage biopsies from donors of DNOAP group and controls were fixed in 10% formaldehyde for 24 hours, decalcified by 15% neutral EDTA-2Na for 15 days, then dehydrated by alcohol gradient of different concentrations and embedded with paraffin. Cut the paraffin-embedded specimens into 5μm consecutively. Then, the tissue sections were stained with Masson's Trichrome Stain Kit and Modified Safranine O-Fast Green FCF Cartilage Stain Kit (Beijing Solarbio Science & Technology Co., Ltd), according to the manufacturer’s instructions. Finally, the pathological changes of cartilage, calcified layer and subchondral bone were observed directly under the optical microscope.
Transmission Electron Microscopy
The fresh cartilage specimens of DNOAP group and controls were fixed with 2.5% glutaraldehyde at 4 ℃ in a volume of 1 mm3 overnight. Next day, the cartilage samples were rinsed with ddH2O, fixed with 1% osmium acid for 1h, stained with 2% uranium acetate for 30min, dehydrated gradiently with 50%, 70%, 90%, 100% ethanol and 100% acetone. After infiltration, embedding and polymerization, the sections were sliced to a thickness of 70nm by ultramicrotome, and then stained with uranium acetate lead citrate. The ultrastructure of organelles in chondrocytes were observed under HITACHIH-7650 transmission electron microscope (Hitachi, Tokyo, Japan).
The cartilage samples were segmented into 3~5mm3 pieces, and washed into the phosphate buffer solution (PBS) containing penicillin (100U/L) and streptomycin (100mg/L). Then the small pieces were digested with 0.25% trypsin at room temperature for 15-20min, and centrifugated at 1000×g for 5min. After removing the supernatant, the deposit was washed with PBS for three times. Next, the deposit was digested at 37 ℃ for 8~10h with 0.2% Collagenase Type II (C2-BIOC, Millipore Sigma, USA) in a constant temperature shaker. After filtration by aseptic cell sieves, the cells were washed and collected through centrifugation (1000×g, 10 min). The chondrocytes were cultured with DMEM medium (Invitrogen, USA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, USA) at 37°C in a humidified atmosphere containing 5% CO2.
Detection of reactive oxygen species (ROS) in chondrocytes
Reactive oxygen species (ROS) production was detected with a Reactive Oxygen Species Assay Kit (Shanghai Beyotime Biotechnology Co., Ltd). Inoculate the chondrocytes into 6-well plate (1×106 cells per well) and 3 auxiliary holes were set for each group. When the cell fusion rate reached 70% ~ 80%, the culture medium was removed, and DCFH-DA with the concentration of 10μmol/L was added into each well, and then the plate was placed in dark for 20min at 37℃. Wash the cells with serum-free DMEM for three times. ROS was measured by fluorescence microscope at an excitation wavelength of 485nm and an emission of 525nm. The intensity of each group was analyzed by Image Pro Plus image analysis software.
Protein concentration was detected using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, USA) in accordance with the manufacturer’s instructions. Total proteins were electrophoretically separated on sodium dodecyl sulfate polyacrylamide gels (8-15%) according to the molecular size of the target protein, and were subsequently transferred onto polyvinylidene difluoride membranes. After being blocked with 5% skim milk, the membranes were incubated at 4℃ overnight with the following primary antibodies: anti-RANKL, anti-OPG, anti-IL-1β, anti-IL-6, anti-TNF-α and anti-β-actin (Proteintech, USA).
Then, the membranes were washed thoroughly and incubated with secondary antibodies (1:2000 anti-mouse/ rabbit, Santa Cruz Biotechnology, USA) at room temperature for 2 hours. The signals were visualized using the enhanced chemiluminescence method (Immobilon Western Chemiluminescent HRP Substrate, Millipore). The samples were analyzed in duplicate, and the experiment was performed three times.
Detection of apoptosis by flow cytometry
The percentage of apoptotic cells was ascertained through Annexin V/FITC-PI apoptosis detection kit (Beijing Sizhengbai Biotechnology Co., Ltd).
Cells were prepared with the concentration of 1×106/ml in 10% bovine serum albumin, according to the manufacturer’s instructions. After cultured in the incubator with 5% CO2 at 37℃ for 24h, the cells were collected in a 10 ml centrifuge tube, centrifuged at 1000×g for 5 min, and washed with precooled PBS twice. Added Annexin V-FITC/ PI and PI (100ug/mL) working solution into cells, and stained for 15min at room temperature in dark. Flow cytometry was used to detect the apoptosis rate of cells, and Cell Quest software was used to obtain and analyze parameters. The experiment was repeated three times.
High glucose induced expression of RANKL and OPG in chondrocytes
Hyperglycemia is a common feature of DNAOP patients, thus, we simulated anomalous level of blood sugar in vitro by treating normal chondrocytes with a wide range of glucose concentrations (5mM, 10mM, 15mM, 20mM, 25 mM, 33mM) for 24 hours. Western blot analyses were used to detect the expression of RANKL and OPG in chondrocytes.
Statistical analyses were performed with SPSS 19.0 (SPSS Inc., Chicago, IL, USA). Data from cell experiments were analyzed using unpaired Student’s t-tests, and images based on the statistical analyses were made in GraphPad Prism 5.0 (GraphPad Software Inc, La Jolla, CA, USA). All hypothetical tests were two-sided, and P-values less than 0.05 were considered statistically significant in all tests.