Parathyroid hormone's enhancement of bones' osteogenic response to loading is affected by ageing in a dose- and time-dependent manner

Abstract

Decreased effectiveness of bones' adaptive response to mechanical loading contributes to age-related bone loss. In young mice, intermittent administration of parathyroid hormone (iPTH) at 20-80μg/kg/day interacts synergistically with artificially applied loading to increase bone mass. Here we report investigations on the effect of different doses and duration of iPTH treatment on mice whose osteogenic response to artificial loading is impaired by age. One group of aged, 19-month-old female C57BL/6 mice was given 0, 25, 50 or 100μg/kg/day iPTH for 4weeks. Histological and μCT analysis of their tibiae revealed potent iPTH dose-related increases in periosteally-enclosed area, cortical area and porosity with decreased cortical thickness. There was practically no effect on trabecular bone. Another group was given a submaximal dose of 50μg/kg/day iPTH or vehicle for 2 or 6weeks with loading of their right tibia three times per week for the final 2weeks. In the trabecular bone of these mice the loading-related increase in BV/TV was abrogated by iPTH primarily by reduction of the increase in trabecular number. In their cortical bone, iPTH treatment time-dependently increased cortical porosity. Loading partially reduced this effect. The osteogenic effects of iPTH and loading on periosteally-enclosed area and cortical area were additive but not synergistic. Thus in aged, unlike young mice, iPTH and loading appear to have separate effects. iPTH alone causes a marked increase in cortical porosity which loading reduces. Both iPTH and loading have positive effects on cortical periosteal bone formation but these are additive rather than synergistic.

Keywords: Ageing; Bone; Mechanical loading; Osteoporosis; Parathyroid hormone.

Supplementary Fig. 1

Mice were treated with vehicle or 50 μg/kg/day for 28 days. The fibula was sectioned and immunostained for sclerostin. Scale bars equals 100 μm.

Supplementary Fig. 2

Mice were treated with vehicle or 50 μg/kg/day for 14 days and the effect on bone architecture examined using μCT (a). Further sections of bone were stained with H&E (b) or immunostained for cathepsin K (c) or sclerostin (d). Large images are 10 times and insert images are 40 times magnification. Scale bars in large images equals 100 μm and in insert images 50 μm. Osteoclasts were defined as large, strongly cathepsin K stained cells on a bone surface and are indicated by arrows in figure c. Examples of sclerostin positive osteocytes are indicated by arrows in figure d.

Fig. 1

Timeline of experiments. a) In the dose response study mice were injected with 0, 25, 50 or 100 μg/kg/day by subcutaneous injection for 28 days and killed on day 29. For the loading studies, mice were injected with vehicle or 50 μg/kg/day for either b) 15 or c) 40 days with loading three times per week for the final two weeks.

Fig. 2

The effect of various doses of iPTH on trabecular and cortical bone mass and architecture after 28 days of treatment. Mice were administered 0, 25, 50 or 100 μg/kg/day and bone analysed by μCT. iPTH had no clear dose response effect on trabecular BV/TV (a), Tb.Th (b) or Tb.N (c) but did cause mild variation in Tb.Sp (d). Conversely there was a marked increase in Tt.Ar (e), Ct.Ar (f) and Ct.Po (h) with a corresponding decrease in Ct.Th (g) as shown in representative images (i). Analysis was by one-way ANOVA with p-values indicated on the graphs. Where significant, post-hoc Bonferroni was performed. Bars with the same letter above were not significantly different from each other.

Fig. 3

Mice were treated with vehicle or 50 μg/kg/day for 28 days and the effect on bone architecture examined using μCT (a) and fluorescent bone labels (b). Further sections of bone were stained with H&E (c) or immunostained for cathepsin K (d), sclerostin (e) or periostin (f). Large images are 10 times and insert images are 40 times magnification. Scale bars in large images equals 100 μm and in insert images 50 μm. Osteoclasts were defined as large, strongly cathepsin K stained cells on a bone surface and are indicated by arrows in figure d. Examples of sclerostin positive osteocytes are indicated by arrows in figure e.

Fig. 4

The effect of iPTH on the load strain relationship. Tibial stiffness was unaffected by treatment with iPTH.

Fig. 5

The effect of 2- and 6-weeks of treatment with vehicle or 50 μg/kg/day iPTH on the response to loading for trabecular BV/TV (a), Tb.Th (b), Tb.N (c) and Tb.Sp (d). Representative 3D reconstructions showing the effect of 6-weeks iPTH on the response to loading in the region of trabecular bone analysed by μCT (e-h). P-values represent results of the two-way repeated measures ANOVA for the 2- and 6-week loading experiments for the effect of PTH, loading and their interaction. Significant results are indicated in bold.

Fig. 6

The effect of 2- and 6-weeks of treatment with vehicle or 50 μg/kg/day iPTH on the response to loading for Tt.Ar (a), Ct.Ar (b), Ct.Th (c) and Ct.Po (d). Representative 3D reconstructions showing the effect of 6-weeks iPTH on the response to loading in the region of cortical bone analysed by μCT (e-h). P-values represent results of the two-way repeated measures ANOVA for the 2- and 6-week loading experiments for the effect of PTH, loading and their interaction. Significant results are indicated in bold.

Fig. 7

The combination of iPTH and loading additively increased Ct.Ar (a) and Tt.Ar (b) predominantly over the proximal half of the tibia. iPTH, but not loading, increased Ct.Ar (a) distal to the tibia/fibula junction (approximately 60% of the tibial length). iPTH decreased, whereas loading increased Ct.Th (c) in the proximal tibia.