Figure 6: RhNELL-1 intravenous injection increases bone formation in osteoporotic mice.

(a) Induction of osteoporosis was performed by OVX and ensuing bone loss over a 5-week period, confirmed by DXA analysis of the lumbar vertebrae (N=13 and 15 mice, respectively). Confirmation of OVX was also performed post mortem by demonstration of uterine atrophy (Supplementary Fig. 7b,c). (b,c) RhNELL-1 was next administered by tail vein injection (1.25 mg kg−1 q48 hr) over a period of 4 weeks, and compared with PBS control treatment (N=6, 7, 6 and 9 mice, respectively). DXA analysis of the lumbar vertebrae was performed weekly. (d) Representative images after 4 weeks of rhNELL-1 treatment, including live CT, 18F-PET and microCT. MicroCT indices have been compared with the published norms to ensure accuracy of analysis and reporting59,60,61. (e–i) MicroCT quantification after 4-week treatment, including (e) BMD, (f) BV, (g) BV/TV, (h) Tb.Th, (i) Tb.N and (j) Tb.Sp (N=6, 7, 6 and 9 mice, respectively). (k) Osteocalcin (OCN) immunohistochemical staining, and quantification of OCN+ bone-lining cells per B.Pm (N=18, 23, 25 and 30 images, respectively). (l) TRAP staining and quantification of TRAP+, multinucleated, bone-lining cells per B.Pm (N=28, 34, 28 and 40 images, respectively). (m) Calcein/Alizarin red complex on bone labelling and quantification of mineral apposition rate (MAR) and BFR. Red and green arrows highlight the space between fluorochrome labels (N=3 mice and 6 measurement fields per treatment group). (n) FEA and quantification of von Mises stress. Red colour indicates areas of high stress (N=6 per treatment group). Black scale bars, 25 μm; red scale bar, 10 mm; yellow scale bar, 100 μm. Data points indicate the means, while error bars represent one s.e.m. In vivo experiments were performed without replicate, unless otherwise described. Parametric data were analysed using an appropriate Student’s t-test or a one-way ANOVA, followed by a post hoc Tukey’s test. Nonparametric data were analysed with a Mann–Whitney U-test or a Kruskal–Wallis one-way analysis. *P<0.05, **P<0.01 in comparison with PBS control, unless otherwise indicated.