Figure 4.

Bone-targeted AAV gene silencers promote bone regeneration in mice

(A) rAAV9.DSS.egfp (5 × 10¹³ vg/kg) was injected i.v. into 2-month-old mice, and, 2 weeks later, a 3-mm length of cortical bone defect was generated on the lateral aspect of the left femur. GFP expression in the cryo-sectioned femurs was monitored by fluorescence microscopy 2 weeks post surgery (n = 3). Scale bar: 100 μm. (B–D) Two-month-old mice were i.v. injected with rAAV9.DSS (5 × 10¹³ vg/kg) carrying amiR-ctrl, amiR-shn3, amiR-sost, or amiR-sost/shn3, and, 2 weeks later, cortical bone defect surgery was performed on the lateral aspect of the left femur. Newly formed bones in the defect areas were assessed by microCT and histology 2 weeks after the surgery (B and C). Representative trichrome-stained longitudinal sections of femurs (B, top), 2D cross-sectional microCT images (B, bottom), and relative quantification of bone volume, region of interest (ROI, C), Ob.S/BS (and osteoclast numbers per bone surface (Oc.N/BS) (D) are displayed (n = 8). Scale bars: (B, top) 200 μm, (B, bottom) 1 mm. (E) Three-month-old mice were i.v. injected with rAAV9.DSS.egfp (5 × 10¹³ vg/kg), and, 2 weeks later, femoral osteotomy and intramedullary fixation were performed. GFP expression on the cryo-sectioned femurs was assessed by fluorescence microscopy 1, 2, and 4 weeks postoperatively. Scale bars: (B) 200 μm, (E) 25 μm. (F–K) Three-month-old mice were i.v. injected with rAAV9.DSS (5 × 10¹³ vg/kg) carrying amiR-ctrl, amiR-shn3, amiR-sost, or amiR-sost/shn3, and, 2 weeks later, femoral osteotomy and intramedullary fixation were performed. Six weeks after the surgery, mRNA levels of Shn3 and Sost (F) and Axin2 (G) in the tibia were assessed by RT-PCR and normalized to Gapdh (n = 8). Tb.BV/TV in the contralateral femurs was assessed by microCT (H, n = 8). Representative radiography and microCT images of the fractured femurs 2 and 6 weeks post surgery are displayed, respectively (I). Union rate at the fracture sites was quantitated by microCT (J). Representative H&E-stained longitudinal sections of femurs at the fracture sites 6 weeks post surgery are displayed (K). Asterisk (∗) indicates fibrous tissues at persistent non-union sites. Scale bars: (I) 1 mm, (K) 200 μm. Values represent mean ± SD by a one-way ANOVA test (C, D, F–H, J).