Fig. 6: Effects of rhPRG4 on chondrogenesis in vitro and in vivo.

Sca1⁺CD140a⁺ cells were isolated from the ear and bone marrow, exposed to increasing concentrations of rhPRG4 and assayed by qPCR a and alican blue staining b–g. Ear derived progenitors demonstrated a maximal beneficial chondrogenic effect at 50 µg/ml rhPRG4 in both marker expression a and pellet size b–h, while marrow derived progenitors demonstrated a maximal beneficial chondrogenic effect at 75 µg/ml rhPRG4 a. Hic1⁺ progenitors were permanently labeled in vivo with TdTomato by Tamoxifen induction pre-injury and their localization was examined 4 weeks post-injury h in presence/absence of rhPRG4 i–o (n = 6 mice per group). With rhPRG4 treatment, Hic1⁺ progenitors had given rise to auricular cartilage (overlap of TdTomato (red) with Collagen II (blue)) i–l, while in DMSO treated injuries, on minimal TdTomato⁺ signal was observed co-localized with Collagen II⁺ signal and this staining was not morphological consistent with auricular cartilage m–o. In uninjured ear tissue, few TdTomato⁺ cells were observed dispersed throughout the dermis and adjacent to the auricular cartilage. Few to no TdTomato⁺ Collagen II⁺ cells were observed p–r. In vitro experiments were undertaken on 3 biological and 3 technical replicates, in vivo experiments were undertaken on n = 4 animals per group. Bars represent mean with SD. n.s. = not significant. Error bars equal mean ± SD (a, h – 1 way ANNOVA).