Fig. 4: CCN3 enhances osteogenesis, bone formation and fracture repair.
a–c, Osteogenic differentiation assays (14 days). a, Differentiation of mouse ocSSCs (from 2-week-old male and female mice) treated with mCCN3, met-ENK and Bam22P. Inset, cells stained with Alizarin Red (Al Red) (n = 3). Veh, vehicle. b,c, Human ocSSCs treated with human CCN3 (hCCN3) during in vitro osteogenesis. b, Human ocSSCs from a 14-year-old male treated with hCCN3, met-ENK, Bam22P, gastric-related peptide (GRP) and follistatin (FST). c, Additional ocSCCs from 15-year-old (left), 72-year-old (middle) and 61-year-old (right) patients treated with hCCN3 (red bars) (n = 3), with representative images of wells stained with Alizarin Red to the right of the graph. F, female; M, male. d–f, Whole femur bone cultures treated with plasma or mCCN3 daily for 5 days. d, Left, %BV/TV for control Esr1fl/fl female (red) and male (blue) 6–8-week-old femurs treated with plasma from Esr1Nkx2.1-cre females; contralateral femurs treated with plasma from Esr1fl/fl mice (N = 19, 10). Right, per cent change in %BV/TV of contralateral female (red bar) or male (blue bar) femurs. e, Representative µCT images from treated femurs. f, Left, %BV/TV control female (N = 11) and male (N = 8) 10–11-week-old femurs treated daily for 5 days with mCCN3 (3 nM) compared with untreated baseline contralateral femur control. Right, per cent change in female (red bar) or male (blue bar) %BV/TV treated with CCN3 or saline normalized to baseline (N = 5–11). g, Per cent change in %BV/TV in Esr1fl/fl mice following daily CCN3 injections (i.p. 7.5 µg kg–1) or saline for 21 days, normalized to mean %BV/TV of saline controls (N = 6, 8 females and 7, 6 males). h, Representative µCT images from treated femurs. i, %BV/TV (left) and mechanical strength of callus (right) 21 days after fracture in aged male mice after slow-release mCCN3 (1 or 2 µg) treatment (phosphate-buffered saline (PBS) N = 4; 1 µg N = 5, 6; and 2 µg N = 4). j, Representative µCT images and cross-sections of callus from 24-month-old C57BL/6 male femurs. One-way ANOVA in a–c and i (Dunnett’s (a, b, i) and Tukey’s (c) multiple-comparisons test). Paired Student’s t-test, one-tailed for left panels in d, f, and unpaired Student’s t-test, two-tailed for right panels in d, f and g. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Error bars ± s.e.m.
