Runx2

We harvested the left hind knees of mice after euthanasia at 2 or 4 weeks after ACLT and fixed them with 4% formaldehyde for 48 hours. Then, the knees were immersed in 1.5 M EDTA for 1-1.5 weeks for decalcification. Following dehydration in a gradient of alcohol solutions, the knees were embedded in OCT compound for sectioning (6 μm). For safranin-O & fast green staining, sections were dewaxed in water and immersed in fast green staining solution for 6 minutes. After washing with distilled water for 1 minute, the sections were immersed in safranin O solution for 4 minutes, rinsed with distilled water for 1 minute and differentiated in glacial acetic acid for 1 minute. The sections were sealed with neutral resin after dehydration. For immunohistochemical staining, sections were dewaxed in water and repaired in boiled antigen retrieval solution for 8 minutes. After blocking with a 3% BSA/PBS solution for 1 hour at room temperature, sections were incubated with anti-endomucin (Emcn; Santa Cruz, Dallas, Texas, USA), anti-matrix metalloproteinase 13 (MMP13; Abcam, Cambridge, UK) and anti- Runt-related transcription factor 2 (RUNX2; Servicebio, Wuhan, China) antibodies, followed by fluorescent dye-conjugated secondary antibodies (Abcam, Cambridge, UK) for fluorescence imaging and HRP-conjugated secondary antibodies (Servicebio, Wuhan, China) for DAB staining.

Femurs were harvested, fixed with 4% PFA and decalcificated with 10% EDTA for about 1 month. After dehydrated, specimens were embedded in paraffin. 3‐μm sections were prepared for haematoxylin and eosin (HE) staining and Immunohistochemistry (IHC) staining. All staining procedures were performed according to the standard procedures. IHC was performed using primary antibodies against Ocn (Servicebio Technology), Bmp2 (Servicebio Technology) and Runx2 (Servicebio Technology) at 1:200 dilution, followed by HRP‐conjugated secondary antibody (Invitrogen). Then, signal was detected by 3, 3′‐diaminobenzidine tetrahydrochloride (DAB) kit (Invitrogen).

Tibia, liver, spleen and heart of the male mice were immediately separated and pulverized in a cooled mortar with liquid nitrogen. The bone marrow in tibia was flushed out before pulverization. Primary osteoblasts were washed with ice‐cold PBS before protein extraction. Whole‐cell lysates for Western blot were extracted using RIPA Lysis buffer (Beyotime) with protease and phosphatase inhibitor cocktails (Calbiochem). The concentration of protein was analysed by BCA protein assay. Protein lysates were separated using 10% SDS‐PAGE at 120V for 1 hour and then electro‐transferred to nitrocellulose membranes at 100V for 2 hours. The membranes were blocked with 5% non‐fat milk in TBST and then incubated with antibodies against Macf1 (1:500, Abcam), Alp (1:1000, Santa Cruz Biotech), Col1 (1:1000, Cusabio Technology), Bmp2 (1:1000, Servicebio Technology), p‐Smad1/5/9 (1:1000, Cell Signalling Technology), Smad1 (1:1000, Cusabio Technology), Runx2 (1:500, Servicebio Technology) and Gapdh (1:2000, Servicebio Technology) overnight at 4°C, followed by incubation with corresponding secondary antibodies for 2 hours at room temperature. Protein bands were facilitated by Western Bright ECL Spray (Advansta San Jose) and visualized by a T5200 Multi chemiluminescence detection system (Tanon). Densitometric quantification of the bands was performed with Image J analysis software (Image J).