Nrecon version 1

Left hindlimbs of 14-week-old mice were harvested. X-rays of knee joints were taken with a Faxitron X-ray machine (Edimex) to evaluate the integrity and calcification of the knee surface. Besides, the knee joints were also scanned with 2 K resolution, 10-μm voxel size, 0.5 Al filter at 60 kV, and 167 μA. Images were reconstructed using NRecon version 1.1.11 (Bruker micro-CT) and analyzed using CTAn, v1.15 (SkyScan1176 in vivo micro-CT; Bruker).

Left hindlimbs of 12-week-old mice were harvested and soaked in neutral formalin. Bone morphology and microarchitecture were assessed at the distal femoral metaphysis for trabecular parameters. Femurs were scanned with 2 K resolution, 10-μm voxel size, 0.5 Al filter at 60 kV, and 167 μA. Trabecular bone in the 1000 μm above the growth plate and extending proximally for 1000 μm was measured for analysis. Images were reconstructed using NRecon version 1.1.11 (Bruker micro-CT) and analyzed using CTAn, v1.15 (SkyScan1176 in vivo micro-CT; Bruker).

Rat knee joints were scanned using a SkyScan 1276 Micro-CT (Bruker, Kontich, Belgium) and NRecon version 1.6 software (Bruker). Data were then analyzed using the CTAn version 1.9 software (Bruker), and a three-dimensional model was generated in CTVol version 2.0 (Bruker). Quantitative morphometry indexes based on three-dimensional morphometry were determined using microtomographic data. Bone volume (BV), BV/total tissue volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp) were determined for the region of interest between the proximal tibia growth plate and tibial plateau.

Knee joint images were captured by X-ray (MX-20, Faxitron X-ray, Corp., Lincolnshire, IL, United States) and SkyScan 1276 Micro-CT (Bruker, Kontich, Belgium) and NRecon version 1.6 software (Bruker) and Ingenia3.0 T MRI system (Philips, United States). The rats were anesthetized by intraperitoneal injection of pentobarbital sodium (40 mg/kg) and fixed in supine position. The bilateral ankles were fixed on the tray with adhesive tape. The lens was focused at an appropriate focal length on the knee joint of the rat and the exposure time was set to apropriate minutes to ensure a clear image. The extent of osteoarthritis was assessed by imaging findings, including joint space narrowing and articular surface calcification, as well as articular cartilage damage, according to the imaging scoring system used in previous literature with macroscopic score which was based on surface roughness and erosin. Using imaging techniques, we quantified the tibial plateau or femoral condyle surface by calculating the ratio of the lesion area to the total surface area. Both the tibial and femoral joints were evaluated based on a maximum score of 10 (Gerwin et al., 2010 (link); Kohn et al., 2016 (link); Lin et al., 2021 (link)). The scoring was performed by two experienced observers who were blinded to the study groups.