Micro-CT analysis of mouse femurs

Following the removal of surrounding soft tissue, mouse femurs were fixed in 4% formaldehyde in phosphate buffered saline (PBS, pH 7.4) for 24 h at 4°C. Bones were then extensively washed in running tap water and stored in 70% ethanol until analysed. Comparisons of bone structure in intact femurs were performed between 16-week-old WT (n = 8), Htra1-heterozygous (Htra1-HET) (n = 8), and Htra1-KO (n = 7) mice; 52-week-old WT (n = 8) and Htra1-KO (n = 6) mice. Femurs were scanned on a microCT40 (Scanco Medical AG, Brüttisellen Switzerland) operated at 55 kVp and 145 μA with 200 ms integration time and 2-fold frame averaging. Images were reconstructed from 1000 projections at a nominal isotropic resolution of 10 μm. After application of a Gaussian Filter (sigma 0.8, support 1), image thresholds were set, and automated masks of full bone, cortex and metaphyseal trabecular bone created [41, 42].

Evaluation of bone volume in osteotomy sites stabilized with a flexible MouseFix plate was performed in WT and Htra1-KO mice at 21 days (WT, n = 11; Htra1-KO, n = 10) and 35 days (WT, n = 10; Htra1-KO, n = 13) post-surgery. Analysis of osteotomy sites stabilized with a rigid MouseFix plate was performed in WT and Htra1-KO mice at 21 days (WT, n = 9; Htra1-KO, n = 9) post-surgery. Following removal of the MouseFix plate, micro-CT measurements were performed using the same settings as the intact femurs described above. After image processing, a threshold of 25% of maximum grey value was applied, and a volume of interest (500 x 500 x 280 voxels) manually selected to accommodate the full callus volume between the inner screws in which the volume of mineralized tissue was calculated (S1 Fig). We chose not to distinguish between original and newly formed bone as no reliable thresholds could be determined. Analysis of osteotomy repair was not performed in cases where the MouseFix plates failed to attach correctly during surgery (WT, n = 6; Htra1-KO, n = 7), or showed signs of loosening or dislocation at the time of harvesting (WT, n = 4; Htra1-KO, n = 4).