Safranin o and fast green

Human cartilage tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and made in sections. Mouse knee joints were fixed in 4% paraformaldehyde, decalcified in 0.5 µM EDTA, and embedded in paraffin. For Safranin-O and fast green staining, all sections were deparaffinized in xylene, hydrated with graded ethanol, and stained with Safranin-O and fast green (Sigma). Cartilage destruction, synovitis, and osteophyte formation were scored by three observe under blinded conditions using the OARSI (grade 0–6), synovitis (grade 0–3), and osteophyte (grade 0–3) scoring system. The OARSI, synovitis, and osteophyte scores are presented as the mean of the maximum score in each mouse, and each representative Safranin-O-stained image was selected from the most advanced lesion among serial sections. For immunohistochemistry, the slides were treated with EDTA repair solution in a boiling water bath for 30 min, and blocked in 5% BSA (MCE) for 1 h. Primary antibodies against MMP3 (1:100; Abcam, ab52915), MMP13 (1:100; Proteintech, 18165-1-AP), NFKB1 (Proteintech, 14220-1-AP), and p-p65 (Cell Signaling, 3033) were added into the slides at 4 °C overnight. The next day, the slides were washed with PBS thrice and incubated with secondary fluorescence antibodies for 1 h at room temperature. DAPI was used to stain the nuclei. Nikon Ti2-E was used to acquire the fluorescence images.

Osteochondral plugs (n = 5 native, n = 5 decellularised using process 1; n = 3 decellularised using processes 2–5) were fixed for 48 h in 10 % (v/v) neutral buffered formalin (NBF; Bios Europe Ltd), before being decalcified in 12.5 % (w/v) EDTA (pH 7: Fisher Scientific) for 4 weeks or until soft enough to be cut with a scalpel. Plugs were bisected before being dehydrated and paraffin wax and embedded using an automated process (Lecia TP 1020, Lecia Microsystems). Sections of 6 μm thickness were cut through the cartilage surface into the bone, encompassing the different cartilage zones and subchondral bone. Haematoxylin and eosin (H&E; Bios Europe Ltd) staining was used to assess tissue histoarchitecture. DAPI (Sigma) staining was used to visualise cell nuclei. Safranin O and Fast Green (Sigma) staining was used to visualise glycosaminoglycan (GAG) distribution.

One leg was randomly selected from each mouse and dissected for histology. The knee joints were collected and fixed in 4% paraformaldehyde solution. Fixed joints were decalcified in Decalcifying Solution Lite (Sigma‐Aldrich, St. Louis, MO, USA) for 6 h and embedded in paraffin. Joint tissues were sectioned by a microtome, and sections (5 μm) had been hydrated in 100%, 90%, 70%, and 50% ethanol in distilled water (DW) for 5 min each. Hydrated samples were stained with haematoxylin and eosin (H&E) (Sigma‐Aldrich, St. Louis, MO, USA) for morphological analysis and measurement of the infiltration of immune cells, or safranin O and fast green (Sigma‐Aldrich, St. Louis, MO, USA) for determining cartilage damage. Stained samples were dehydrated in 50%, 70%, 90% and 100% ethanol and xylene and mounted by using balsam. They were observed by using DM750 microscope (Leica, Wetzlar, Germany).