*2.2. Injury-Induced Knee Joint Degeneration was Accelerated in Old Mice*

To understand how aging impacts PTOA development after injury, we investigated structural changes in the knee joints of 62-week-old and 10-week-old mice six weeks after an ACL injury. Ten-week-old mice represent young adult humans whose cartilage is normally healthy, whereas 62-week-old mice represent a ~50–60 year-old human, an age group in which OA is prevalent. By six-week post-injury, both 10-week-old and 62-week-old mice exhibited severe cartilage degradation in the injured joints (Figure 2A,B). OA lesions were more severe in 62-week-old mice than in 10-week-old mice, where the majority of the femoral head was lacking the articular cartilage layer in the injured 62-week old mice (Figure 2A). Osteophyte formation was observed in both age groups by six-week post-injury, and 62-week-old mice had significantly more osteophytes than 10-week-old mice (Figure 2C). ACL injury also resulted in a significant reduction in subchondral bone volume in the femoral epiphysis in both age groups (Figure 2D). Older, 62-week-old mice displayed a significantly lower subchondral bone volume than 10-week-old mice before injury, and they also lost more subchondral bone (25% loss) than younger mice (18% loss) by six weeks after injury (Figure 2D).

**Figure 2.** Characterization of post-traumatic osteoarthritis (PTOA)-associated structural changes in 10-week-old and 62-week-old mice. (**A**) Histological evaluation of uninjured contralateral joints and injured joints at six-week post-injury using Safranin-O and Fast Green staining which stains cartilage in red and surrounding tissue in green (5× magnification). Scale bars: 200 μm. (**B**) Osteoarthritis Research Society International (OARSI) scoring of histological sections of injured and uninjured contralateral joints at six-week post-injury. (**C**) Osteophyte volume at six-week post-injury. (**D**) Epiphyseal trabecular bone volume fraction (BV/TV) of the distal femur was quantified using μCT and analyzed between injured and uninjured contralateral joints at six-week post-injury. 10W: 10-week-old; 62W: 62-week-old. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001.
