Effects of two weeks of dietary calcium depletion followed by one week of dietary calcium repletion on dynamic bone formation histomorphometric parameters of at the endosteal surface of the secondary spongiosia of the distal femur.

<p>E = endosteal; L.Pm = tetracycline labeling surface (dL.Pm + ½ sL.Pm); B.Pm = bone surface; MAR = mineral apposition rate; BFR = bone formation rate.</p><p>P = N.S. (not significant), when P>0.05.</p><p>* P<0.05; ** P<0.01; *** P<0.001, when compared to respective control mice on the control diet.</p><p>Effects of two weeks of dietary calcium depletion followed by one week of dietary calcium repletion on dynamic bone formation histomorphometric parameters of at the endosteal surface of the secondary spongiosia of the distal femur.</p

Sequence of PCR primer sets for the test mouse genes.

<p>Sequence of PCR primer sets for the test mouse genes.</p

Comparison of cortical cross-sectional bone parameters at the mid-shaft of femurs between WT and <i>Igf1</i> cKO mice after two-week dietary calcium depletion and/or after one-week dietary calcium repletion.

<p>Results are shown as mean ± SEM.</p><p>^# The control group are mice receiving normal calcium-containing diet throughout.</p><p>^a P<0.001; ^b P<0.01; ^c P<0.05; when compared to corresponding WT littermates.</p><p>P = N.S., not significant, when P>0.05.</p><p>Comparison of cortical cross-sectional bone parameters at the mid-shaft of femurs between WT and <i>Igf1</i> cKO mice after two-week dietary calcium depletion and/or after one-week dietary calcium repletion.</p

Effects of dietary calcium depletion and subsequent repletion on dynamic histomorphometric parameters of bone formation in femurs of osteocyte <i>Igf1</i> cKO mutants and corresponding WT mice.

<p>Left panel: the bone mineralizing surface, shown as relative percentage of TLS of corresponding controls (mice of corresponding mouse strain fed the calcium-containing diet throughout); middle panel: the bone mineralization apposition rate; and right panel: the calculated bone formation rate per bone surface. Results are shown as mean ± SEM. The number of mice per group is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115897#pone.0115897.t004" target="_blank">Table 4</a>. *P<0.05.</p

Proposed cellular mechanisms contribute to bone repletion-induced bone formation (A) or to loading-induced bone formation (B). Please refer to text for details.

<p>Proposed cellular mechanisms contribute to bone repletion-induced bone formation (A) or to loading-induced bone formation (B). Please refer to text for details.</p

Schematic representation of the experimental design of the dietary calcium depletion and repletion experiment.

<p>The indicated numbers of 4-week-old female <i>Igf1</i> osteocytic cKO mutants and age- and gender-matched WT littermates were each divided into four groups: 1) depletion control groups, which were fed the calcium sufficient diet (with 1.2% calcium) for 2 weeks; 2) depletion experimental groups, which were fed the calcium deficient diet (with <0.01% calcium) for the same 2 weeks; 3) repletion control groups, which were fed the calcium sufficient diet for 3 weeks; and 4) repletion experimental groups, which were fed the calcium deficient diet for two weeks followed by the calcium sufficient diet for one week. At the end of each regimen, all mice were sacrificed and blood drawn for measurements of plasma parameters. Femurs were isolated, cleaned, fixed with formalin and subjected pQCT and histomorphometry measurements. Tibias were isolated, and total RNA was isolated for real-time RT-PCR measurements.</p

Comparison of effects of the two-week dietary calcium depletion on plasma levels of Ca, P, PTH, and FGF-23 level in 4 weeks-old female osteocyte <i>Igf1</i> conditional knockout (cKO) mice with those in age- and sex-matched WT control mice.

<p>^a P<0.05, when compared to WT control mice on basal calcium-containing diet.</p><p>* P<0.05, ** P<0.01; and ***P<0.001, compared to corresponding controls on calcium-containing diet of each respective mouse strain.</p><p>P = N.S. (not significant), when P>0.05.</p><p>Comparison of effects of the two-week dietary calcium depletion on plasma levels of Ca, P, PTH, and FGF-23 level in 4 weeks-old female osteocyte <i>Igf1</i> conditional knockout (cKO) mice with those in age- and sex-matched WT control mice.</p

Effects of calcium depletion and repletion on BMD parameters in femurs of osteocyte <i>Igf1</i> cKO mutants and corresponding WT mice.

<p>BMD parameters were determined by pQCT. Total BMD was performed on the entire bone; cortical BMD was measured at the mid-diaphysis; and Trabecular BMD was determined at the site corresponding to secondary spongiosa. Results are shown as relative percentage of corresponding control mice (mean ± S.E.M.). The number of mice per group is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115897#pone.0115897.t006" target="_blank">Table 6</a>. *P<0.05; and ***P<0.001.</p

Effects of one-week of dietary calcium repletion on plasma P1NP level in osteocyte <i>Igf1</i> cKO mutants and in corresponding WT mice.

<p>Four-week-old osteocyte <i>Igf1</i> cKO mice (n = 8) and age- and gender-matched WT control mice (n = 10) were fed either the calcium-deficient or the calcium-containing diet for two weeks followed by 1 week calcium-containing diet. Plasma samples were obtained at the beginning of the experiment (basal), and after three days and 7 days of calcium repletion. Results are shown as mean ± SEM. A shows the actual basal level of P1NP; and B shows the effect of the dietary calcium repletion in the two strains, which is reported as relative percentage changes of the basal level of WT control mice fed the calcium-containing diet for east indicated test time period. *P<0.05.</p

Effects of two-week dietary calcium restriction on plasma calcium (A), PTH (B), phosphorus (C), and FGF23 (D) of osteocyte <i>Igf1</i> cKO mice and those of WT mice.

<p>Four-week-old female osteocyte <i>Igf1</i> cKO mice and age- and gender-matched WT control mice were fed a mouse diet containing either <0.01% calcium (calcium-deficient diet) or 1.2% calcium (control diet) for two weeks. At the end of the calcium depletion, blood samples were collected from each mouse for measurements of calcium (Ca), PTH, phosphorus, and FGF23. Results are shown in relative percentage of the respective level of each metabolite in corresponding control mice that were fed the control calcium-containing diet (mean ± S.E.M). The number of animals in each test group is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115897#pone.0115897.t002" target="_blank">Table 2</a>. *P<0.05; **P<0.01; and ***P<0.001. P = N.S. (not significant), where P>0.05. </p