Col 1

Protein expression was assessed by western blot analysis as we described previously (Xiao et al., 2015b (link)). In brief, protein (50 μg) samples were resolved by 10% SDS–PAGE and transferred to PVDF membranes, then probed with various primary antibodies against E-cadherin (1:500), α-SMA (1:500), COL-I (1:500) (Santa Cruz Biotechnology) and β-catenin (1:500) (Sigma-Aldrich, St Louis, MO, USA). After incubating with an appropriate secondary antibody, the protein expression was detected by western blot analysis, as described previously (He et al., 2009 (link)). β-actin (Santa Cruz Biotechnology, 1:1,000) was used as an internal control.

For western blot analysis, PDLSC and JBMSC sheets were lysed in lysis buffer (Sigma-Aldrich) supplemented with 1 mM of PMSF (Roche, Basel, Switzerland). The total protein concentrations were then measured using a bicinchoninic acid protein assay kit (Beyotime). Next, 40 μg of cell lysates were added to each lane of 10% SDS-PAGE gels, separated based on molecular weight, and electrotransferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). After blocking with 5% non-fat milk for 2 h, the membranes were incubated in specific primary antibodies against COL I, COL III, OPN, OCN, CEMP1 and CAP (all from Santa Cruz Biotechnologies) overnight at 4 °C. The membranes were then incubated in a horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibody (CoWin Biotech Co., Ltd., Beijing, China) for 1 h at room temperature. The membranes were developed using a Western Light Chemiluminescent Detection System (Peiqing, Shanghai, China). All assays were repeated in triplicate.

The knee joints were harvested and fixed in 4% paraformaldehyde overnight. Decalcification was conducted in 4% ethylene diamine tetraacetic acid (EDTA) for one month, with the decalcifying solution changed every 3 days. Decalcified joints were embedded in paraffin and used to cut 4 μm sections of the medial femoral condyle. The sections were stained using hematoxylin and eosin (HE), safranin O, and toluidine blue.
The severity of cartilage degeneration was assessed in histological sections using the modified Mankin score [35 (link)], which was scored based on: (1) surface integrity (score 0–10), (2) cellularity (score 0–4), (3) cell clones (score 0–4), and (4) Safranin O staining (score 0–5). A higher score indicated a greater level of cartilage degeneration.
Immunohistochemical staining was conducted using the histological sections. Briefly, after deparaffinization, sections were incubated with 0.3% hydrogen peroxide for 30 min and treated with hyaluronidase for 60 min. The sections were then incubated with COL-I or COL-II monoclonal antibodies (Santa Cruz Biotechnology, Dallas, TX, USA). All antibody dilutions were made in PBS. After an overnight reaction with the primary antibody at 4 °C, sections were incubated with labelled polymer-HRP anti-mouse IgG at room temperature for 30 min.