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

Titanium (Ti)-based biomaterials significantly modulated the expression patterns of circadian rhythm genes from human bone marrow stromal cells (BMSCs).

<p><b>A</b>. Scanning electron microscopy was used to characterize the surface of the Ti discs used in this study. The machined-polished Ti discs showed a smooth surface, whereas the Ti disc treated by sand-blasting followed by double acid etching and discrete calcium phosphate nanoparticle deposition (B-DAE-DCD) showed a complex surface topography at the micrometer and nanometer range. <b>B</b>. The surface topography was quantitatively evaluated by optical photometry (n = 3 in each group). The B-DAE-DCD surface was approximately 10x rougher than the machined-polished surface. <b>C</b>. Human BMSCs cultured on conventional polypropylene culture dishes (n = 4 per time point) or B-DAE-DCD Ti discs (n = 4 per time point) were synchronized by forskolin and exposed to 1 nM 1,25(OH)2D3 vitamin D-supplemented culture medium. The number of BMSCs was determined using a live/dead assay at 24 hours and 72 hours of culture. <b>D</b>. Calcein-stained live BMSCs cultured on polypropylene dishes maintained a fibroblastic morphology after 72 hours of culture. By contrast, the live BMSCs on the B-DAE-DCD discs were widely spread and made contacts with adjacent cells, resulting in the establishment of confluency. <b>E</b>. The steady state mRNA levels of circadian rhythm-related genes were determined by PCR every 4 hours starting from 24 hours to 72 hours after synchronization (n = 4 per time point in each group). <i>PER1</i>, <i>PER2</i> and <i>PER3</i> from human BMSCs cultured on polypropylene culture dishes and machined-polished discs exhibited a circadian expression pattern. When cultured on the B-DAE-DCD discs, the circadian expression pattern was diminished, while <i>NPAS2</i> was upregulated compared to the polypropylene control.</p

The weighted gene co-expression network analysis (WGCNA).

<p>The microarray data were obtained previously from rat femur bone marrow tissue after osteotomy surgery with or without DAE-DCD Ti implants with sufficient or deficient serum vitamin D levels (ITV+, ITV-, OSV+ and OSV- groups, respectively) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183359#pone.0183359.ref020" target="_blank">20</a>]. <b>A</b>. WGCNA identified 47 modules of co-expressed genes. The Turquoise and Blue modules contained a disproportionately large number of genes. <b>B</b>. A dendrogram of the WGCNA analysis suggested an unusually large co-expressed gene network of hierarchical clustering among Blue and Turquoise modules, which contained 9,202 and 11,511 genes, respectively. <b>C</b>. Association of the module eigengenes with experimental bone marrow tissue traits responding to osteotomy surgery with or without Ti implant placement (IT and OS, respectively) in vitamin D-sufficient and -deficient rats (V+ and V-, respectively). Out of the 47 modules identified, the Blue and Turquoise modules (arrows) showed the highest eigengene correlation in OS/IT. Within each cell, upper values indicate correlation coefficients between the module eigengene and the traits, while the lower values indicate the corresponding p-value. <b>D</b>. Scatter plots of module membership (eigengene-based connectivity) and gene significance for the Blue module for each of the therapeutic traits (ITV+, ITV-, OSV+ and OSV-). The highest correlation between module membership and gene significance was in ITV+; this was noticeably decreased in ITV-.</p

Gene networks formed around the biomaterial implant.

<p><b>A</b>. The protein-protein interaction network of a total of 36 unique hub genes from the Blue module suggested interacting core networks between circadian rhythm/E-box binding proteins and nuclear steroid hormone receptors, including the vitamin D receptor (<i>Vdr</i>). <b>B</b>. The interaction between circadian rhythm-related genes and connective tissue extracellular matrix genes in the comparison between OSV+ and ITV+ indicated that the trait-significant Blue module contained <i>Npas2</i>, <i>Bmal1</i> (<i>Arntl</i>) and <i>Vdr</i>, whereas the Turquoise module contained <i>Per1</i>, <i>Per3</i> and <i>Per3</i>. <b>C</b>. The comparison between OSV+ and OSV- without implanted biomaterials did not detect <i>Vdr</i> and <i>Npas2</i> was found to be significantly downregulated. <b>D</b>. The comparison between ITV+ and ITV- revealed that the Blue module lost its trait-significance and Vdr. <i>Per1</i>, <i>Per2</i> and <i>Per3</i> were also moved from the Turquoise module and to the Purple module.</p

Circadian expression of the <i>Per1</i>::<i>luc</i> reporter gene in BMSCs.

<p>Femur-derived BMSCs were harvested from 3 male <i>Per1</i>::<i>luc</i> rats (5 months old). BMSCs from passages 3 to 5 were used for the experiments. <b>A</b>. Time-lapse microscopy depicted the periodic activation of <i>Per1</i>::<i>luc</i> reporter gene expression in the BMSCs. <b>B</b>. <i>Per1</i>::<i>luc</i> reporter gene expression was measured by luminometry. The experiment was performed with three dishes in each group. The baseline-subtracted luminometry data demonstrated the circadian activation of <i>Per1</i>::<i>luc</i> expression when BMSCs were cultured on polypropylene dishes with 1 nM vitamin D supplementation; this was reduced on the Ti discs. Strikingly, the circadian rhythm of <i>Per1</i>::<i>luc</i> expression was nearly completely lost on the B-DAE-DCD discs. <b>C</b>. The loss of <i>Per1</i>::<i>luc</i> circadian expression on the B-DAE-DCD discs was depicted by the decreased peak-to-trough amplitude and peak-to-peak period. **: p<0.01 by Wilks Lambda for the amplitude and period. <b>D</b>. The raw amplitude measurements depicted the stable expression of the <i>Per1</i>::<i>luc</i> reporter gene after the initial forskolin shock when the BMSCs were cultured on the polypropylene dishes or machined-polished Ti discs. In contrast, <i>Per1</i>::<i>luc</i> reporter gene expression remained low when the BMSCs were cultured on the B-DAE-DCD discs. <b>E</b>. The average raw amplitude of the <i>Per1</i>::<i>luc</i> reporter gene was significantly lower in the B-DAE-DCD group than the polypropylene and machine-polished groups; however, it was above the background level of the no cell negative control. **: p<0.01 by ANOVA. <b>F</b>. When BMSCs were cultured on B-DAE-DCD Ti discs without vitamin D supplementation, the lost <i>Per1</i>::<i>luc</i> circadian expression was partially recovered. <b>G</b>. The raw amplitudes from the luminometry data indicated that vitamin D supplementation did not affect Per1 expression levels in BMSCs cultured on the machined-polished Ti discs or B-DAE-DCD discs.</p