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