Figure 2. Cellular senescence is induced during femoral fracture healing.

(A) Femoral fractures (Fx) were induced in male C57BL/6 mice with contralateral (Contra), intact control femora. Callus and controls were analyzed 4, 8, 14, and 28 days after fracture (n = 4 per time point). (B) Cdkn1a^Cip1 and Cdkn2a^Ink4a mRNA expression levels during fracture healing were determined at each time point using rt-qPCR. Cdkn1a^Cip1 was strongly induced 8–14 days and Cdkn2a^Ink4a 14 days post fracture. (C) Senescence-associated distension of satellites (SADS) were detected using immune-fluorescent in situ hybridization (FISH) Top: representative image of the contralateral side. Below: fractured side with four SADS. (D) The total number of SADS as well as percentage of SADS-positive cells ( ≥ 4 SADS/cell) at all time points was increased in the callus compared to the contralateral control femur. (E) Telomere-associated foci (TAF) were determined using FISH to detect telomeres (red) and immunofluorescent staining for γH2A.X (green). TAF (yellow, see arrowheads) were defined as sites of γH2A.X-associated DNA damage co-localized with telomeres (n = 70 cells were analyzed per bone). (F) The highest percentage of TAF were detected on day 14 of fracture healing. The number of TAF-positive senescent cells ( ≥ 3 TAF/cell) was increased in fractured bones throughout the entire healing period. B: n = 24 (n = 24 in Contra, n = 24 in Fx, n = 6 per timepoint), all male. C-D: n = 16 (n = 16 in Contra, n = 16 in Fx), all male. E-F: n = 20 (n = 4 per time point and n = 4 in contra on day 14), all male. Mean ± SEM. One-way ANOVA or Student’s t-test for pairwise comparisons; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.