*4.2. Micro-Computed Tomography (*μ*CT)*

Injured joints, contralateral joints from injured mice, and bilateral joints from uninjured mice were collected six weeks post injury for all groups. Samples were dissected and fixed for 72 h at 4 ◦C using 10% neutral buffer formalin; samples were stored in 70% ethanol at 4 ◦C until scanned. Whole knees were scanned using a SCANO μCT 35 (Bassersdorf, Switzerland) according to the rodent bone structure analysis guidelines (X-ray tube potential = 55 kVp, intensity = 114 mA, 10 μm isotropic nominal voxel size, integration time = 900 ms) [34]. Trabecular bone in the distal femoral epiphysis was analyzed by manually drawing contours on 2D transverse slides to designate the region of trabecular bone enclosed by the growth plate and subchondral cortical bone plate. We quantified trabecular bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp) [38]. Mineralized osteophyte volume in injured and contralateral joints was also quantified by drawing contours around all heterotopic mineralized tissue attached to the distal femur and proximal tibia as well as the whole fabellae, menisci, and patella. Total mineralized osteophyte volume was then determined as the volumetric difference in mineralized tissue between injured and uninjured joints. Statistical analysis was performed using two-way ANOVA and a Student's *t*-test with a two-tailed distribution, with two-sample equal variance (homoscedastic test). For all tests, *p* < 0.05 was considered statistically significant.

#### *4.3. Histological Assessment of Articular Cartilage and Joint Degeneration*

VEH-, AB-, and LPS-treated injured, contralateral, and uninjured joints were dissected six weeks post injury, then fixed, dehydrated, paraffin-embedded, and sectioned as previously described [74]. The cartilage was visualized in sagittal 6-μm paraffin serial using Safranin-O (0.1%, Sigma; S8884; St. Louis, MO, USA) and Fast Green (0.05%, Sigma; F7252; St. Louis, MO, USA) as previously described [35]. OA severity was evaluated using a modified Osteoarthritis Research Society International (OARSI) scoring scale as previously described [85]. Cartilage scoring began ~0.4 mm out from the start of synovium to the articular cartilage. Blinded slides were evaluated by seven scientists (six with and one without expertise in OA) utilizing modified (sagittal) OARSI scoring parameters due to the severe phenotype caused by TC loading-destabilization that promotes mechanical-induced tibial degeneration on injured joints [74,85]. Modified scores: (0) for intact cartilage staining with strong red staining on the femoral condyle and tibia; (1) minor fibrillation without cartilage loss; (2) clefts below the superficial zone; (3) cartilage thinning on the femoral condyle and tibia; (4) lack of staining on the femoral condyle and tibia; (5) staining present on 90% of the entire femoral condyle with tibial degeneration; (6) staining present on over 80% of the femoral condyle with tibial degeneration; (7) staining present on 75% of the femoral condyle with tibial degeneration; (8) staining present on over 50% of the femora condyle with tibial degeneration; (9) staining present in 25% of the femoral condyle with tibial degeneration; (10) staining present in less than 10% of the femoral condyle with tibial degeneration.

#### *4.4. Immunofluorescent Staining*

Six-micrometer sections from injured samples from both treatment groups of *C57Bl*/*6J* were used. Unitrieve was used as an antigen retrieval method for 30 min at 65 ◦C [86]. Primary antibodies: Anti-F4/80 (Abcam, ab16911(1:50)), Anti-CD206 (Abcam, ab64693(1:500)), and Anti-iNOS (Abcam, ab15323(1:100)) were used and incubated overnight at room temperature in a dark humid chamber. Negative control slides were incubated with secondary antibody only. Stained slides were mounted with Prolong Gold with DAPI (Molecular Probes, Eugene, OR, USA). Slides were imaged using a Leica DM5000 microscope. ImagePro Plus V7.0 Software and a QIClick CCD camera (QImaging, Surrey, BC, Canada) were used for imaging and photo editing.
