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Additional Supporting Information may be found in the online version of this article.

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jbmr2222-sm-0001-SuppFig-S1.tif2702KSupplementary Figure 1: Loading increased the number of osteoblastic cells in the periosteum of young but not old mice. (A) Representative H&E sections from the posterior-lateral (high strain) region of the tibia of male mice 24 hours after artificial loading. Cells were counted in the periosteum which is indicated by the open brackets. (B) Periostin staining was used to confirm that the cells counted were osteoblastic as illustrated in a representative high power image. Scale bar = 25m.
jbmr2222-sm-0002-SuppFig-S2.tif421KSupplementary Figure 2: Cells cultured from aged mice are osteoblastic in nature as shown by their expression of alkaline phosphatase and their ability to form mineralized nodules. (A) ALP activity was measured between 7 and 21 days in osteoblastic cells cultured from the long bones of aged male and female mice. Results are corrected for protein content and expressed as a percentage of ALP activity at day 7. (B) After 21 days in culture in complete medium containing 50µM ascorbic acid and 10mM β-glycerol phosphate, cells from young and aged mice formed mineralized nodules as shown by Alizarin Red staining. Scale bar = 15mm.
jbmr2222-sm-0003-SuppFig-S3.tif365KSupplementary Figure 3: The effect of loading on trabecular bone in young and aged male and female mice. Percentage change [(right - left) / left] * 100 in trabecular number (A) and separation (B) were compared in young and aged mice. Data represents mean ± SEM, n=6 for each strain magnitude. Where it was possible to fit a two-stage linear regression line, this line is shown on the relevant graph. ***p<0.001: the gradient of the load-response regression line being different from zero. p<0.05: the overall difference between young and aged lines determined by regression analysis.
jbmr2222-sm-0004-SuppFig-S4.tif544KSupplementary Figure 4: The effect of loading on cortical bone in young and aged male and female mice. Percentage change [(right - left) / left] * 100 in medullary area (A), bone area fraction (B) and cortical thickness (C) were compared in young and aged mice. Data represents mean ± SEM, n=6 for each strain magnitude. Where it was possible to fit a two-stage linear regression line, this line is shown on the relevant graph. For medullary area in males, it was only possible to fit linear regression lines. *p<0.05, **p<0.01, ***p<0.001: the gradient of the load-response regression line being different from zero.
jbmr2222-sm-0005-SuppFig-S5.tif279KSupplementary Figure 5: The effect of loading and ageing on the proportion of cells in G1/S or S phase of the cell cycle. Proportion of Ki67 positive cells in G1/S- (A, B) and S-phase (C, D) of the cell cycle were calculated by analysis of Ki67 nuclear patterning using immunofluorescence. n=4 per group with 2 repeats in males and 3 repeats in females. Bars represent mean and SEM. **p<0.01 compared to aged static controls (by paired t-test).
jbmr2222-sm-0006-SuppTable-S1.doc55KSupplementary Table 1: Loading-engendered strains measured at the 37% site of the proximal tibia in representative mice were used to determine the magnitude of load required to engender strain magnitudes of 500, 1000, 1500, 1750, 2000, 2250 and 2500 on the medial surface of the tibia at the 37% site measured from the proximal end in young and aged, male and female mice. The load rate to apply an average strain rate of 30,000s-1 during loading and unloading was also calculated. Strain magnitudes are greater in the posterior-lateral region of the bone cortex where the magnitude is approximately 2.5 times higher[9].

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