*4.2. Histological Assessment of Disease Severity*

Injured and uninjured (contralateral) joints were collected at six-week post-injury and processed as previously described for histology [10]. Briefly, dissected joints were fixed in 4% paraformaldehyde, decalcified using 0.5 M EDTA, processed, and embedded intact into paraffin. The joints were sectioned in the sagittal plane and serial medial sections that included the femoral condyles, menisci, and tibial plateaus were cut at 4 μm; stained on glass slides using 0.1% Safranin-O (0.1%, Sigma, St. Louis, MO, USA; S8884) and 0.05% Fast Green (0.05%, Sigma, St. Louis, MO, USA; F7252) using standard procedures (IHC World, Woodstock, MD, USA); and imaged using a Leica DM5000 microscope. Blinded slides were evaluated by seven scientists (six with and one without expertise in OA) utilizing modified (sagittal) Osteoarthritis Research Society International (OARSI) scoring parameters because of the severity of the phenotype six weeks following TC injury. Modified scoring scores (0) for intact cartilage staining with strong red staining on the femoral condyle and tibia, (1) for minor fibrillation without cartilage loss, (2) for clefts below the superficial zone, (3) for cartilage thinning on the femoral condyle and tibia, (4) for lack of staining on the femoral condyle and tibia, (5) for staining present on 90% of the entire femoral condyle with tibial resorption, (6) for staining present on over 80% of the femoral condyle with tibial resorption, (7) for staining present on 75% of the femoral condyle with tibial resorption, (8) for staining present on over 50% of the femora condyle with tibial resorption, (9), for staining present in 25% of the femoral condyle with tibial resorption, and (10) for staining present in less than 10% of the femoral condyle with tibial resorption.
