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

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 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

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

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