Explant cultures were utilized to further investigate the role of both estradiol and A2m on condylar fibrocartilage degradation. Entire mandibles with condyle and condylar fibrocartilage intact were harvested from 13-week old WT female mice and cultured in a 24-well plate with 1.5 mL of FBS-free BGJb medium supplemented with 1% Penicillin-Streptomycin (ThermoFisher Scientific) overnight. Following overnight incubation, media was removed, samples were washed 2 × with sterile PBS, and fresh media with either 0 M, 10−8 M, or 10−6 M 17β-estradiol (17β-estradiol, Sigma E2758, for the estradiol study), 100 nM and 200 nM A2m (from human plasma, Sigma SRP6314), or 50 nM and 100 nM Pi15 (synthetic – based on human peptide sequence, abcam ab23016) was added and incubated for 48 hours. Estradiol concentrations were chosen based on similar studies21 (link) and further substantiated by evidence of circulating estrogen levels in humans59 (link). A2m concentrations were determined based on previous evidence in a knee osteoarthritis model49 (link). After 48 hours of treatment, supernatant was collected for zymogram assays and collagen type I and II cleavage ELISAs. Messenger RNA from both left and right samples was pooled together and utilized for RTPCR. For gelatin zymography and ELISA analysis, supernatant from only the left specimen was utilized.
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