Representative KEGG Pathways with z-score >2.0.

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

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

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

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

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

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

Image_1_In vitro assessment of Neuronal PAS domain 2 mitigating compounds for scarless wound healing.JPEG

BackgroundThe core circadian gene Neuronal PAS domain 2 (NPAS2) is expressed in dermal fibroblasts and has been shown to play a critical role in regulating collagen synthesis during wound healing. We have performed high throughput drug screening to identify genes responsible for downregulation of Npas2 while maintaining cell viability. From this, five FDA-approved hit compounds were shown to suppress Npas2 expression in fibroblasts. In this study, we hypothesize that the therapeutic suppression of Npas2 by hit compounds will have two effects: (1) attenuated excessive collagen deposition and (2) accelerated dermal wound healing without hypertrophic scarring.Materials and methodsTo test the effects of each hit compound (named Dwn1, 2, 3, 4, and 5), primary adult human dermal fibroblasts (HDFa) were treated with either 0, 0.1, 1, or 10 μM of a single hit compound. HDFa behaviors were assessed by picrosirius red staining and quantitative RT-PCR for in vitro collagen synthesis, cell viability assay, in vitro fibroblast-to-myofibroblast differentiation test, and cell migration assays.ResultsDwn1 and Dwn2 were found to significantly affect collagen synthesis and cell migration without any cytotoxicity. Dwn3, Dwn4, and Dwn5 did not affect collagen synthesis and were thereby eliminated from further consideration for their role in mitigation of gene expression or myofibroblast differentiation. Dwn1 also attenuated myofibroblast differentiation on HDFa.ConclusionDwn1 and Dwn2 may serve as possible therapeutic agents for future studies related to skin wound healing.</p

Titanium biomaterials with complex surfaces induced aberrant peripheral circadian rhythms in bone marrow mesenchymal stromal cells

<div><p>Circadian rhythms maintain a high level of homeostasis through internal feed-forward and -backward regulation by core molecules. In this study, we report the highly unusual peripheral circadian rhythm of bone marrow mesenchymal stromal cells (BMSCs) induced by titanium-based biomaterials with complex surface modifications (Ti biomaterial) commonly used for dental and orthopedic implants. When cultured on Ti biomaterials, human BMSCs suppressed circadian <i>PER1</i> expression patterns, while <i>NPAS2</i> was uniquely upregulated. The Ti biomaterials, which reduced <i>Per1</i> expression and upregulated <i>Npas2</i>, were further examined with BMSCs harvested from <i>Per1</i>::<i>luc</i> transgenic rats. Next, we addressed the regulatory relationship between <i>Per1</i> and <i>Npas2</i> using BMSCs from <i>Npas2</i> knockout mice. The <i>Npas2</i> knockout mutation did not rescue the Ti biomaterial-induced <i>Per1</i> suppression and did not affect <i>Per2</i>, <i>Per3</i>, <i>Bmal1</i> and <i>Clock</i> expression, suggesting that the Ti biomaterial-induced <i>Npas2</i> overexpression was likely an independent phenomenon. Previously, vitamin D deficiency was reported to interfere with Ti biomaterial osseointegration. The present study demonstrated that vitamin D supplementation significantly increased <i>Per1</i>::<i>luc</i> expression in BMSCs, though the presence of Ti biomaterials only moderately affected the suppressed <i>Per1</i>::<i>luc</i> expression. Available <i>in vivo</i> microarray data from femurs exposed to Ti biomaterials in vitamin D-deficient rats were evaluated by weighted gene co-expression network analysis. A large co-expression network containing <i>Npas2</i>, <i>Bmal1</i>, and <i>Vdr</i> was observed to form with the Ti biomaterials, which was disintegrated by vitamin D deficiency. Thus, the aberrant BMSC peripheral circadian rhythm may be essential for the integration of Ti biomaterials into bone.</p></div

The role of <i>Npas2</i> in BMSC circadian rhythm.

<p><b>A</b>. A snapshot of circadian rhythm gene expression from rat BMSCs shows the transcriptional downregulation of <i>Per1</i>, <i>Per2</i>, <i>Bmal1</i> and <i>Id2</i> when the BMSCs were cultured on B-DAE-DCD Ti discs. A similar upregulation in <i>Npas2</i> was observed in human BMSCs. RT-PCR was performed in triplicates. *: p<0.05 by Student’s t-test. <b>B</b>. The role of the transcription factor <i>Npas2</i> on the steady state mRNA levels of circadian rhythm-related genes was examined by siRNA. <i>Npas2</i> knock down appeared to have a greater impact on circadian rhythm-related gene expression in BMSCs cultured on the polypropylene dishes. *: p<0.05 by Student’s t-test. <b>C</b>. To further clarify the role of <i>Npas2</i>, BMSCs were harvested from mice carrying the <i>Npas2</i> allele lacking Exon 3, which was replaced by the <i>LacZ</i> reporter gene cassette, resulting in a <i>Npas2</i> functional knockout mutation due to the lack of the basic helical-loop-helical (bHLH) domain. <b>D</b>. BMSCs were harvested from the femurs of wild-type, <i>Npas2</i>+/- and <i>Npas2</i>-/- male mice (n = 5 each). BMSCs (passage 4) were cultured and synchronized on polypropylene dishes, machined Ti discs or B-DAE-DCD discs (n = 2 in each group). Thirty-two hours after synchronization, <i>LacZ</i> reporter gene expression was increased in the D-DAE-DCD disc group. <b>E</b>. The expression of circadian rhythm genes were observed to increase in the <i>Npas2</i>+/- and <i>Npas2</i>-/- BMSCs maintained on polypropylene culture dishes for 32 hours after synchronization. <b>F</b>. <i>Npas2</i>+/- and <i>Npas2</i>-/- BMSC maintained on B-DAE-DCD discs for the same length of time demonstrated similar circadian rhythm gene expression patterns as wild-type mouse BMSCs.</p