Human cartilage tissue

Healthy human articular cartilage tissue was obtained from the victims of traffic accidents, with no history of arthritic diseases (n = 5, age 30.6 ± 9.22 years, four males and one females). OA cartilage tissue was obtained from OA patients (n = 5, age: 64 ± 5.75 years, two males, three females) undergoing total knee joint replacements. Other joint diseases were excluded from the study. All samples were from the Department of Orthopedics, the Third Affiliated Hospital of Southern Medical University. All recruited patients contributed their informed consent and were identified and approved by the Ethics Committee of the Third Affiliated Hospital of Southern Medical University.

Animals and the mouse model

All animal experiments were approved by the Animal Care and Use Committee of Southern Medical University. Six-week-old male C57BL/6 mice were purchased from the Experimental Animal Center of Southern Medical University, Guangzhou, China.

In the instability of medial meniscus OA model (DMM, Kamekura et al., 2005) [52], mice were anesthetized by intraperitoneal injection of 5% chloral hydrate, and the skin was cut along the medial collateral ligament. The joint capsule was opened to expose the femoral condyle. Then, the connection between the medial meniscus and the tibial plateau was cut open to release the medial meniscus. The joint capsule and skin were sutured after surgery.

Animal treatment and specimen preparation

After surgery, 9 × 108 TU/mL (total volume of approximately 5 µL) was injected into the joint once a week using OeFBP1 lentivirus. The right leg was then collected from 4 to 8 weeks after surgery (n = 5 in each group). The knee joints of mice in different experimental groups were fixed in 4% paraformaldehyde for 48 h and decalcified for 21 days. The specimens were then embedded in paraffin, and 4 μm serial sections were cut from the sagittal portion through the inner side of the knee.

Cell and cartilage explants

The prechondral cell line, ATDC5 (Tsukuba, Japan), was cultured in DMEM/F12 (Gibco, Grand Island, NY, USA), containing 10% fetal bovine serum (FBS; Gibco), 100 U/mL penicillin, and 100 mg/mL streptomycin sulfate (Gibco) at 37 °C, in an incubator containing 5% CO2. We dissected the rib cartilages of newborn mice (24 − 72 h of age) under a stereoscopic optical microscope to obtain primary chondrocytes. After trypsin digestion for 30 min, primary chondrocytes were isolated and purified, then digested at 37 °C for 4 − 6 h using 0.1% type II collagenase (Sigma-Aldrich, St. Louis, MO, USA) containing 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin sulfate. The primary chondrocytes were then resuspended and inoculated in 24-well plates and cultured in DMEM/F 12 containing 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin sulfate at 37 °C and 5% CO2. The femoral head was separated from 8-week-old mice. The explants cultured in DMEM/F12 were treated with 10 mM phosphate-buffered saline (PBS) for 1 h, followed by 50 ng/mL recombinant mouse or human interleukin (IL-1β) in PBS/0.1% bovine serum albumin (BSA) or PBS/0.1% BSA alone. The explants were then harvested and the culture medium was collected after 3 days of culture.

Real‐time polymerase chain reaction

Total RNA was isolated from and cartilage of the human tibial plateau, then ground using TRIzol reagent. For mRNA quantification, 1 mg of total RNA was purified with gDNA remover and transcribed using 5 × HiScript II qRT SuperMix II (Vazyme Biotech, Nanjing, China). Each PCR reaction consisted of 10 μL 2 × ChamQ SYBR qPCR main mixture, 10 μM forward and reverse primers, and 500 ng cDNA. For miRNA quantification, 1 mg of total RNA was purified using a gDNA wipe mixture, followed by a Hiscript II enzyme mixture of 10 × RT mixture and specific stem ring primers for reverse transcription. Template DNA was combined with 2 × Mix miRNA Universal SYBR qPCR Master Mix, specific primers, and mQ primer R. All reactions were in triplicate. Primer sequences were of mice and are listed below:

Fbp1.

forward 5′- TGCTGAAGTCGTCCTACGCTAC-3′,

reverse 5′- TTCCGATGGACACAAGGCAGTC-3′;

Sox9.

forward 5′- CACACGTCAAGCGACCCATGAA-3′,

reverse 5′- TCTTCTCGCTCTCGTTCAGCAG-3′;

Col2.

forward 5′- GCTGGTGAAGAAGGCAAACGAG-3′,

reverse 5′- CCATCTTGACCTGGGAATCCAC-3′;

Acan.

forward 5′- CAGGCTATGAGCAGTGTGATGC-3′,

reverse 5′- GCTGCTGTCTTTGTCACCCACA-3′;

Mmp13.

forward 5′- GATGACCTGTCTGAGGAAGACC-3′,

reverse 5′- GCATTTCTCGGAGCCTGTCAAC-3′;

ColX.

forward 5′- GTACCAAACGCCCACAGGCATA-3′,

reverse 5′- GGACCAGGAATGCCTTGTTCTC-3′;

Crik.

forward 5′- TGTCTGGCTGTCTGGAACTC-3′,

reverse 5′- GAAGGACAATGGGCATCATGG-3′;

Fgf2.

forward 5′- AGCGGCTCTACTGCAAGAAC-3′,

reverse 5′- GTTGGCACACACTCCCTTGA-3′;

Apon.

forward 5′- AGGCTGATGAGTAGCCCAGA-3′,

reverse 5′- CGTCTAGCTACACACCGTGG-3′;

Pcdha4.

forward 5′- CTGATTCAAGGGACAGAGAGGA-3′,

reverse 5′- CTGGACCAGCCCGTAGAATG-3′;

Pcdhga4.

forward 5′- CCAGCGCTTGCTTCTTTCTT-3′,

reverse 5′- GCCTCCTCAGGGATGGAGTA-3′;

Nalcn.

forward 5′- CAACAGCAAAAGGCAAGCGA-3′,

reverse 5′- ACCACAGTCTGTAACCGCAG-3′;

Serpina9.

forward 5′- TGAGGTGAGCACTCAGACAC-3′,

reverse 5′- TGTCCCTAACCCTGAACCGT-3′;

Atp8a2.

forward 5′- TTCTGCGGGCTACAAGAAGG-3′,

reverse 5′- TACTGATCCGGTTGTCGCAG-3′;

Crb3.

forward 5′- GGGTGACTAAACTTTCCGGGT-3′,

reverse 5′- GACTTCGCTCAGGTTCCCAA-3′;

Grin3a.

forward 5′- CCGCAACTCCCTCACCTATC-3′,

reverse 5′- GAATGGCTTGGAGTGTGGGA-3′;

Western blotting

Pyrolysis buffer was comprised of 10% glycerol, 2% sodium dodecyl sulfate (SDS), 10 mM dithiothreitol, 10 mM Tris–HCl (pH 6.8), 1 mM phenymethylsulfonlyfloride, and 10% ethanethiol. The proteins were incubated in pyrolysis buffer at 98 °C for 10 min. The sample was resolved using SDS-PAGE for 90 min. The sample was then transferred to a nitrocellulose membrane for 1 h and incubated with primary antibody (in 5% BSA, 0.2% NaN3) at 4 °C overnight. The primary antibodies are mouse anti-β-Actin (1:10,000), rabbit anti-SOX9 (1:2000), rabbit anti-MMP13 (1:2000), rabbit anti-COLX (1:2000), mouse anti-P21 (1:1000), mouse anti-P16 (1:1000), rabbit anti-FBP1 (1:200), and mouse anti-CRB3 (1:1000). Samples were then incubated with secondary antibodies (mouse or rabbit, 1:4000) at 37 °C for 1 h.

Histology and immunohistochemical/IF staining

Morphological analysis was performed on tissue sections using Safranine O-Fast Green staining. Immunohistochemical (IHC) and immunofluorescence (IF) staining were performed on 0.004-mm-thick tissue sections. The glass slide was dewaxed and rehydrated and washed three times in PBS for 5 min each time. The slide was then soaked in citric acid buffer and heated in a microwave for 2 min to recover the antigen. After washing three times in PBS, the slide was quenched at room temperature in 3% hydrogen peroxide for 10 min and washed three more times with PBS. Then, the slides were blocked with 10% BSA for 1 h at room temperature (IHC staining). Slides were then incubated with primary antibodies at 4 °C overnight. A secondary antibody for IHC or a fluorescent secondary antibody for IF was then used at room temperature for 1 h. Then, IHC slides were stained with diaminobenzidine and hematoxylin, dehydrated, and fixed. The IF slide was then treated with 4,6-diamino-2-phenylindole staining solution and fixed with a cover glass. Antibodies used for IHC/IF staining were as follows: rabbit anti-FBP1, rabbit anti-SOX9, rabbit anti-MMP13, rabbit anti- P21, rabbit anti-P16, rabbit anti-Aggrecan, rabbit anti-ColX, mouse anti-CRB3, species-matched horseradish peroxidase-conjugated secondary antibodies, and species-matched Alexa-488-or 594-labeled secondary antibody (rabbit 1:200).

Statistical analysis

Data are expressed as the mean ± standard deviation. Unpaired Student’s t test was used for experiments comparing two groups of data. One-way analysis of variance was performed for data involving multiple groups, followed by Tukey’s post hoc test. A value of p < 0.05 was considered statistically significant.