Hierarchical cluster analysis.

<p><b>A</b>. Two-way ANOVA resulted in a total of 103 genes, which were further grouped into 5 clusters (Cluster 1 to 5). <b>B</b>. Cluster 2 contained NPAS2 (arrow), one of the most significantly affected genes, as well as cartilage ECM genes (arrowheads). <b>C</b>. Directional hierarchical clustering revealed that the NPAS2 and Per2 clusters behaved in a complementary fashion.</p

Unadjusted t-test of significantly modulated genes between ITV+ (n = 4) and ITV- (n = 4).

<p>*Average gene expression ratio from microarray data analyzed by GeneSifter. D =  down-regulated in ITV-; U =  up-regulated in ITV-.</p><p>**n.d.  =  not detected within the threshold of 1.5-fold change.</p

Immunological identification of type X collagen associated with implant osseointegration.

<p><b>A</b>. Mouse bone marrow stromal cells (D1 cells) were cultured on implant disk (IT) or glass dish with or without 1,25D supplementation (V). Confocal laser scanning micrographs depicted cell nuclei (DAPI: blue), cytoskeleton (β-actin: red) and type X collagen (green). <b>B</b>. The size of D1 cells was determined by the β-actin positive area per nucleus. The average cell size under different culture conditions was compared. *: p<0.05. <b>C</b>. T-shape implants were harvested 2 weeks after the placement in femur of V+ rats and bone tissues were carefully removed from the external implant surface. In the highly cellular region depicted by a cluster of nuclei (arrows; DAPI), type X collagen was not observed (upper left panel). In the transition region, type X collagen (green) showed a similar appearance as in the <i>in vitro</i> culture (upper right panel). In the areas of exposed bone-implant interface, where no cells remained (lower panels), type X collagen appeared to be involved in extracellular matrix with defined hexagonal structures (arrowhead).</p

Circadian parameters of activity in LD and DD in WT, Q175 Het and Hom mice from 3 months to 12 months of age.

<p>Parameters were analyzed using one-way ANOVA, and the <i>F</i> and <i>P</i> statistics are reported for genotype comparisons within each age group. <i>H</i> values are reported from ANOVA on ranks comparisons in the event of failed normality or equal variance tests. * indicates that <i>post hoc</i> Bonferroni’s <i>t</i>-tests detected significant differences compared to WT, and ^∧ indicates significant differences were detected between Het and Hom groups.</p

Representative KEGG Pathways with z-score >2.0.

<p>Representative KEGG Pathways with z-score >2.0.</p

Age-related decline in circadian rhythms of locomotor activity in Q175 mutants under DD conditions.

<p><b>A.</b> Free-running period (tau) is no different between the three genotypes. <b>B.</b> Power, as measured by the X² periodogram, declines dramatically in Q175 Hom mutants at 9 months. C. The cycle-to-cycle activity onset is less precise in Q175 Hom mutants from 9 months. D. The amount of activity (wheel revolutions per hour, rev/hr) plummets in Q175 Hom mutants at 9 months. * indicates significance to <i>P</i><0.05 in <i>post hoc</i> pairwise comparisons with WT within each age group after the two way ANOVA revealed an effect of genotype. Effect of age within each genotype is indicated with #. <i>F</i> and <i>P</i> statistics are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069993#pone-0069993-t002" target="_blank"><b>Table 2</b></a>.</p

The effect of implant disk and 1,25D supplementation on mouse bone marrow stromal cells (D1 cells) <i>in vitro</i>.

<p><b>A</b>. D1 cells cultured on polystyrene dish or implant disk with or without 1,25D supplementation (V+ and V−, respectively) were subjected to RT-PCR evaluation of bone-and cartilage-related genes. <b>B</b>. The steady state mRNA levels of NPAS2 were assessed by RT-PCR under the same conditions. <b>C</b>. The commercially available siRNA effectively knocked down the targeted NPAS2 in D1 cells cultured on polystyrene dish or implant disk with 1,25D supplementation. The RT-PCR data were evaluated against the average control value in each group. *: p<0.05. <b>D</b>. The effect of NPAS2 knockdown on the expression of type II and type X collagens. The RT-PCR data were evaluated using the average control value of D1 cells cultured on plastic dish as the reference. *: p<0.05, against the control in each group.</p

Neuron number and PER2 expression are unaffected in the SCN of Q175 mutants.

<p><b>A.</b> Representative images of Nissl stains at 10X magnification of coronal sections of the SCN from WT (left), Q175 Het (middle) and Hom (right) mice. 3V: 3^rd ventricle. oc: optic chiasm. <b>B.</b> Quantification of Nissl+cells in the SCN from each genotype was determined by two independent observers blind to the experimental conditions. <b>C.</b> SCN expression of the circadian protein, PER2, is unaltered in Q175 mutants. Mice at 12m of age were sacrificed at ZT 2 and ZT 14, the respective trough and peak of SCN PER2 expression. Representative images of ZT 14 SCN, 40X magnification, are shown from each genotype. <b>D.</b> The day/night difference in PER2 expression is unaltered in Q175 mutants. Quantification of PER2+cells in the SCN was determined by 2 independent observers blind to the experimental conditions.</p

Significantly modulated genes identified by Two-way ANOVA for the vitamin D deficiency effect.

<p>Significantly modulated genes identified by Two-way ANOVA for the vitamin D deficiency effect.</p

T-shaped implant placed in vitamin D sufficient (V+) and deficient (V-) rats.

<p><b>A</b>. T-shaped experimental implant with a hollow inner chamber was fabricated with Ti6Al4V and the implant surface was treated with dual acid-etching and discrete deposition of HA nanoparticles. T-shaped implant was placed in the osteotomy site of the distal end of rat femur. <b>B</b>. Serum chemistry evaluations of 25-hydroxy vitamin D3 (25D), parathyroid hormone (PTH), calcium (Ca), and phosphorous (P). *: p<0.05. <b>C</b>. Non-decalcified histology of bone tissues grew in the inner chamber of T-shaped implant. The bone tissue (arrows; green in Goldner's Masson Trichrome staining) was closely adhered to the implant surface in V+ rats. In V- rats, the intimate bone-implant was not observed; and instead, implant surface was associated with fibrous connective tissue (arrowheads; red staining). <b>D</b>. Histomorphometric characterization of bone volume and bone-implant contact within the inner chamber. *p<0.05.</p