Primary human osteoblasts in response to 25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D3and 24R,25-dihydroxyvitamin D3

The most biologically active metabolite 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) has well known direct effects on osteoblast growth and differentiation in vitro. The precursor 25-hydroxyvitamin D3 (25(OH)D3) can affect osteoblast function via conversion to 1,25(OH)2D3, however, it is largely unknown whether 25(OH)D3 can affect primary osteoblast function on its own. Furthermore, 25(OH)D3 is not only converted to 1,25(OH)2D3, but also to 24R,25-dihydroxyvitamin D3 (24R,25(OH)2D3) which may have bioactivity as well. Therefore we used a primary human osteoblast model to examine whether 25(OH)D3 itself can affect osteoblast function using CYP27B1 silencing and to investigate whether 24R,25(OH)2D3 can affect osteoblast function. We showed that primary human osteoblasts responded to both 25(OH)D3 and 1,25(OH)2D3 by reducing their proliferation and enhancing their differentiation by the increase of alkaline phosphatase, osteocalcin and osteopontin expression. Osteoblasts expressed CYP27B1 and CYP24 and synthesized 1,25(OH)2D3 and 24R,25(OH)2D3 dose-dependently. Silencing of CYP27B1 resulted in a decline of 1,25(OH)2D3 synthesis, but we observed no significant differences in mRNA levels of differentiation markers in CYP27B1-silenced cells compared to control cells after treatment with 25(OH)D3. We demonstrated that 24R,25(OH)2D3 increased mRNA levels of alkaline phosphatase, osteocalcin and osteopontin. In addition, 24R,25(OH)2D3 strongly increased CYP24 mRNA. In conclusion, the vitamin D metabolites 25(OH)D3, 1,25(OH)2D3 and 24R,25(OH)2D3 can affect osteoblast differentiation directly or indirectly. We showed that primary human osteoblasts not only respond to 1,25(OH)2D3, but also to 24R,25(OH)2D3 by enhancing osteoblast differentiation. This suggests that 25(OH)D3 can affect osteoblast differentiation via conversion to the active metabolite 1,25(OH)2D3, but also via conversion to 24R,25(OH)2D3. Whether 25(OH)D3 has direct actions on osteoblast function needs further investigation

The effects of binaural and monoaural beat stimulation on cognitive functioning in subjects with different levels of emotionality

Today, binaural and monaural beats are offered over the Internet or by mental health institutes to improve wellbeing or cognitive functioning. This improvement is explained by the assumption that the brain adapts its brainwave frequency to the frequency of the auditory beat. The present study examined the effects of binaural and monaural beat stimulation on attention and working memory in high and low emotional participants. A group of 24 participants (16 females, 8 males) between 19 and 31 years old (M = 22.33, SD = 3.42) performed a Flanker task to measure attention and a Klingberg task to measure working memory while listening to white noise (WN), 40 Hz gamma binaural beat (BB) and 40 Hz gamma monaural beat (MB). Speed of performance on all three levels of difficulty of the Flanker attention task was faster under the BB and MB condition than under WN. No differences were found between BB and MB conditions. With respect to the quality of performance on the Flanker attention task and the Klingberg working memory task no significant differences under the WN, MB, and BB condition were found. Finally, as participants with low or high emotionality did not respond differently to BB and MB under any of the conditions, effects of BB and MB seem similar in high and low emotional participants. The present study supports the notion that faster attention processing may equally be attributed to the influence of BB and MB. Further research is recommended to gain more insight in the role of factors such as duration of stimulation of BB and MB, frequency range, most appropriate carrier tones, and the role of personality traits

Primary Human Osteoblasts in Response to 25-Hydroxyvitamin D-3, 1,25-Dihydroxyvitamin D-3 and 24R, 25-Dihydroxyvitamin D-3

The most biologically active metabolite 1,25-dihydroxyvitamin D-3 (1,25(OH)(2)D-3) has well known direct effects on osteoblast growth and differentiation in vitro. The precursor 25-hydroxyvitamin D-3 (25(OH) D-3) can affect osteoblast function via conversion to 1,25(OH)(2)D-3, however, it is largely unknown whether 25(OH) D-3 can affect primary osteoblast function on its own. Furthermore, 25(OH) D-3 is not only converted to 1,25(OH)(2)D-3, but also to 24R, 25-dihydroxyvitamin D-3 (24R, 25(OH)(2)D-3) which may have bioactivity as well. Therefore we used a primary human osteoblast model to examine whether 25(OH) D-3 itself can affect osteoblast function using CYP27B1 silencing and to investigate whether 24R, 25(OH)(2)D-3 can affect osteoblast function. We showed that primary human osteoblasts responded to both 25(OH) D-3 and 1,25(OH)(2)D-3 by reducing their proliferation and enhancing their differentiation by the increase of alkaline phosphatase, osteocalcin and osteopontin expression. Osteoblasts expressed CYP27B1 and CYP24 and synthesized 1,25(OH)(2)D-3 and 24R, 25(OH)(2)D-3 dose-dependently. Silencing of CYP27B1 resulted in a decline of 1,25(OH)(2)D-3 synthesis, but we observed no significant differences in mRNA levels of differentiation markers in CYP27B1-silenced cells compared to control cells after treatment with 25(OH) D-3. We demonstrated that 24R, 25(OH)(2)D-3 increased mRNA levels of alkaline phosphatase, osteocalcin and osteopontin. In addition, 24R, 25(OH)(2)D-3 strongly increased CYP24 mRNA. In conclusion, the vitamin D metabolites 25(OH) D-3, 1,25(OH)(2)D-3 and 24R, 25(OH)(2)D-3 can affect osteoblast differentiation directly or indirectly. We showed that primary human osteoblasts not only respond to 1,25(OH)(2)D-3, but also to 24R, 25(OH)(2)D-3 by enhancing osteoblast differentiation. This suggests that 25(OH) D-3 can affect osteoblast differentiation via conversion to the active metabolite 1,25(OH)(2)D-3, but also via conversion to 24R, 25(OH)(2)D-3. Whether 25(OH) D-3 has direct actions on osteoblast function needs further investigation

Effects of 1,25(OH)₂D₃ and 25(OH)D₃ on VDR, CYP27B1 and CYP24 mRNA levels in primary human osteoblast.

<p>Osteoblasts were cultured in the presence of 0, 1, 10 and 100 nM 1,25(OH)₂D₃ or 0, 100, 200 and 400 nM 25(OH)D₃ for 24 hours and mRNA levels of VDR (<b>A</b>), CYP27B1 (<b>B</b>) and CYP24 (<b>C</b>) were determined. Results (mean ± SEM) are expressed as treatment versus control ratios (control was set at 1.0; dashed line) using cells from 5 or 6 different donors. Results were analysed using Friedman test followed by Dunn’s post hoc test (*p<0.05, **p<0.01, ***p<0.001).</p

Effects of 24R,25(OH)₂D₃ on VDR, CYP27B1 and CYP24 mRNA levels in primary human osteoblasts.

<p>Osteoblasts were cultured in the presence of 0, 100, 200 or 400 nM 24R,25(OH)₂D₃ and mRNA levels of VDR (<b>A</b>), CYP27B1 (<b>B</b>) and CYP24 (<b>C</b>) were determined after 72 hours. Results (mean ± SEM) are expressed as treatment versus control ratios (control was set at 1.0; dashed line) using cells from 4 different donors. Results were analysed using Friedman test followed by Dunn’s post hoc test (*p<0.05, **p<0.01, ***p<0.001).</p

Effects of 1,25(OH)₂D₃ and 25(OH)D₃ on primary human osteoblast proliferation.

<p>Osteoblasts were cultured in the presence of 0, 1, 10 or 100 nM 1,25(OH)₂D₃ (<b>A</b>) and 0, 100, 200 or 400 nM 25(OH)D₃ (<b>B</b>) and the proliferation was quantified at day 3 and 6. Results (mean ± SEM) are expressed as treatment versus control ratios (time-point 0 was set at 1.0) using cells from 4 (A) or 7 (B) different donors. Results were analysed using Friedman test followed by Dunn’s post hoc test for each timepoint (*p<0.05, **p<0.01, ***p<0.001).</p

Synthesis of 1,25(OH)₂D₃ and 24R,25(OH)₂D₃ by primary human osteoblasts.

<p>Osteoblasts were cultured in the presence of 0, 100, 200, 400 and 1.000 nM 25(OH)D₃ for 24 hours and 25(OH)D₃ (<b>A</b>) 1,25(OH)₂D₃ (<b>B</b>) and 24R,25(OH)₂D₃ (<b>C</b>) levels were measured in non-conditioned and conditioned culture medium. Results are expressed as mean ± SEM using cells from 3 different donors.</p

Effects of 1,25(OH)₂D₃ and 25(OH)D₃ on ALP activity, P1NP and osteocalcin secretion by primary human osteoblast.

<p>Osteoblasts were cultured in the presence of 0 or 100 nM 1,25(OH)₂D₃ and 0 or 400 nM 25(OH)D₃ and ALP activity (<b>A</b> and <b>B</b> respectively), P1NP (<b>C</b> and <b>D</b> respectively) and osteocalcin secretion (<b>E</b> and <b>F</b> respectively) were measured at day 3, 7, 10 and 14 of the differentiation. Results are expressed as mean ± SEM using cells from 5 different donors. Results were analyzed using Wilcoxon signed rank test for each timepoint (*p<0.05, **p<0.01, ***p<0.001).</p

Primer sequence.

<p>Primer sequence.</p

Effects of 24R,25(OH)₂D₃ on mRNA levels of genes involved in osteoblast differentiation.

<p>Osteoblasts were cultured in the presence of 0, 100, 200 or 400 nM 24R,25(OH)₂D₃ and mRNA levels of COL1α1 (<b>A</b>), ALP (<b>B</b>), osteocalcin (<b>C</b>) and osteopontin (<b>D</b>) were determined after 72 hours. Results (mean ± SEM) are expressed as treatment versus control ratios (control was set at 1.0; dashed line) using cells from 4 different donors. Results were analysed using Friedman test followed by Dunn’s post hoc test (*p<0.05, **p<0.01, ***p<0.001).</p