Aggrecan

Decalcified bone sections were deparaffinized with xylene, and endogenous peroxidase activity was quenched by treatment with 3% H₂O₂ for 15 min, followed by antigen retrieval by trypsinization for 10 min. Sections were then blocked with normal goat serum for 30 min and incubated at 4 °C overnight with primary antibody followed by the appropriate biotinylated secondary antibody and horseradish peroxidase-conjugated streptavidin-biotin staining. Immunoreactivity was visualized with a 3,3′-diaminobenzidine tetrahydrochloride kit (ZSGB-BIO, Beijing, China) followed by counterstaining with Methyl green. Primary antibodies against the following proteins were used: collagen II (1:400; Chondrex, Redmond, WA, USA), FGFR3 (1:200; Santa Cruz Biotechnology, Dallas, Texas, USA), Osteocalcin (1:200; Santa Cruz Biotechnology), PCNA (1:200; Epitomics, Burlingame, CA, USA), Collagen X (1:200; Abcam, Cambridge, MA, USA), Aggrecan (1:200; Millipore, Billerica, MA, USA), MMP13 (1:200; Proteintech, Chicago, IL, USA), ADANTS5(1:200; Abcam), lubricin (1:600; Abcam), IHH (1:100; Abcam) and RUNX2 (1:200; Santa Cruz Biotechnology). The number of immunoreactive cells in three central regions of condylar cartilage section was counted by using Image-Pro Plus 5.1.

Western blot analysis was carried out as described previously.17 Briefly, total proteins were isolated from normal or OA chondrocytes by RIPA buffer (Beyotime Biotechnology, Beijing, China) containing protease inhibitors (Abcam, Cambridge, UK) to obtain whole cell extracts. Membranes were incubated with primary antibodies against RUNX2 (1:1000 dilution; Cell Signalling Technology, Boston, USA); β‐actin (1:3000; Cell Signalling Technology); HDAC2, HDAC8, COL2A1 and MMP13 (1:1000; Abcam); aggrecan and SOX9 (1:2000; Millipore, Bedford, USA). β‐actin was used as an internal control. Immunohistochemical analysis was performed as described previously.17 Cartilage tissue sections were blocked in PBS plus 0.025% Tween 20 with 10% foetal bovine serum, followed by incubation with rabbit anti‐human HDAC2/8 antibody (1:200; Abcam).

C57BL/6 female mice at 5 days (d), 10 weeks (w), and 24 w of age were obtained from Institute of Laboratory Animal Sciences (CAMS&PUMC, Beijing, China). All experimental protocols were approved by the Institute of Materia Medica Animal Authorities (No. 00003787, 27 May 2019).
Antibodies to the following antigens were used: aggrecan (Millipore, AB1031, Burlington, MA, USA), CD44 (BioLegend, 103028, San Diego, CA, USA), Chondroitin Sulfate (Abcam, ab11570, Cambridge, UK), collagen I (Abcam, ab34710, Cambridge, UK), collagen II (Santa Cruz, sc-52658, Dallas, TX, USA), collagen IX (Santa Cruz, sc-376969, Dallas, TX, USA), collagen XI (Lifespan, LS-C-352032, Seattle, WA, USA), hyaluronan binding protein (Millipore, 385911, Burlington, MA, USA), osteocalcin (Santa Cruz, sc-376835, Dallas, TX, USA), syndecan-1 (BioLegend, 142511, San Diego, CA, USA), and donkey anti-rabbit (Jackson, 34213ES60, West Grove, PA, USA) and goat anti-mouse IgG2b (Thermo, A-21144, Waltham, MA, USA).

Cartilage extracts (n ≥ 3 biological replicates per genotype) were incubated in SDS sample buffer (62.5 mM Tris buffer, pH 6.8, 2% SDS, 10% glycerol, 0.04% bromophenol blue, 0.5% β-mercaptoethanol, 50 mM DTT) for 15 min at 65 °C, resolved on 8% SDS polyacrylamide gels and transferred onto nitrocellulose (Whatman, Sigma-Aldrich, St. Louis, MI, USA). Primary antibodies against PRG4 (1/150, Invitrogen, Carlsbad, CA, USA), IBSP (1/500, Immundiagnostik, Bensheim, Germany), SOX6 (A4, 1/1000, Santa Cruz Biotechnology, Dallas, TX, USA), SOX9 (1/1000, Millipore, Burlington, MA, USA), aggrecan (1/1000, Millipore, Burlington, Massachusetts, USA), collagen IX (1/2000, [32 (link)]), collagen II (1/100, Abcam, Cambridge, UK), collagen X (X53, 1/500, [33 (link)]) and GAPDH (14C10, 1/1000, Cell Signaling Technology, Danvers, MA, USA) were added overnight at 4 °C. Primary antibodies were detected by corresponding secondary antibodies labeled with horseradish peroxidase (DAKO, Jena, Germany) and visualized by enhanced chemoluminescence using 10 mM Tris, pH 8.8, 12.5 μM luminol, 2.3 μM coumarin acid, 5.3 μM hydrogen peroxide solution. Band intensities were quantified by ImageJ software [34 (link)]. A density profile line graph was obtained and the area under each peak was measured as the number of pixels, normalized to GAPDH and calibrated by fixed point of control [35 (link)].

Immunoistochemistry was used for the semiquantitative assessment of collagen I and III, aggrecan and versican, for evaluation of angiogenesis at the site of injury and for the detection of the presence of hMSCs in the tendon tissue. Detection of collagen II was used to confirm cartilage formation at the site of injury. Staining was performed against collagen I (1:500, ABcam), II (1:200, Sigma), III (1:800, ABcam), versican (1:500, Millipore) and aggrecan (1:1000, Millipore). Neovascularisation was assessed by staining using RECA-1 (ABcam, 1:50). Immunofluorescent staining for human mitochondria (anti-Cytochrome c oxidase subunit II antibody, MTCO2, ABcam) was used to identify possible surviving transplanted cells. Antigen-antibody complexes were visualized using secondary antibodies conjugated with Alexa-Fluor 488 or 594 (Molecular Probes).

Embryos of ddY mice (Japan SLC, Japan) were obtained at the 14th day of gestation. The dura was removed, and cerebral cortices were isolated, minced, and dispersed. Cells were cultured on 8-well chamber slides (Corning, NY), coated with poly-D-Lysine (PDL; 5 μg/mL; Sigma-Aldrich, St. Louis, MO), at a density of 1.5 × 10^{4 (link)} cells/well, in Neurobasal medium (Thermo Fisher Scientific, Waltham, MA) containing 12% horse serum, L-glutamine, and 0.6% D-glucose at 37°C in a humidified incubator with 10% CO₂. Five hours after seeding, the medium was replaced with fresh Neurobasal medium containing 2% B-27 supplement (Thermo Fisher Scientific), 0.6% D-glucose, and 2 mmol/L L-glutamine. For CSPG coating, culture dishes were coated with 5 μg/mL PDL (Sigma-Aldrich) and 2 μg/mL aggrecan (Sigma-Aldrich) in Hank's buffered salt solution (HBSS; Thermo Fisher Scientific) overnight at 37°C.