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. 2017 Aug 17;12(8):e0183359.
doi: 10.1371/journal.pone.0183359. eCollection 2017.

Titanium Biomaterials With Complex Surfaces Induced Aberrant Peripheral Circadian Rhythms in Bone Marrow Mesenchymal Stromal Cells

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Titanium Biomaterials With Complex Surfaces Induced Aberrant Peripheral Circadian Rhythms in Bone Marrow Mesenchymal Stromal Cells

Nathaniel Hassan et al. PLoS One. .
Free PMC article


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 PER1 expression patterns, while NPAS2 was uniquely upregulated. The Ti biomaterials, which reduced Per1 expression and upregulated Npas2, were further examined with BMSCs harvested from Per1::luc transgenic rats. Next, we addressed the regulatory relationship between Per1 and Npas2 using BMSCs from Npas2 knockout mice. The Npas2 knockout mutation did not rescue the Ti biomaterial-induced Per1 suppression and did not affect Per2, Per3, Bmal1 and Clock expression, suggesting that the Ti biomaterial-induced Npas2 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 Per1::luc expression in BMSCs, though the presence of Ti biomaterials only moderately affected the suppressed Per1::luc expression. Available in vivo 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 Npas2, Bmal1, and Vdr 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.

Conflict of interest statement

Competing Interests: IN received a research grant from Biomet3I and in-kind research materials from Biomet3I and Zimmer Biomet for this study. This does not alter our adherence to PLOS ONE policies on sharing data and materials. All other authors have declared that no competing interests exist.


Fig 1
Fig 1. Titanium (Ti)-based biomaterials significantly modulated the expression patterns of circadian rhythm genes from human bone marrow stromal cells (BMSCs).
A. 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. 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. C. 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. D. 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. E. 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). PER1, PER2 and PER3 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 NPAS2 was upregulated compared to the polypropylene control.
Fig 2
Fig 2. Circadian expression of the Per1::luc reporter gene in BMSCs.
Femur-derived BMSCs were harvested from 3 male Per1::luc rats (5 months old). BMSCs from passages 3 to 5 were used for the experiments. A. Time-lapse microscopy depicted the periodic activation of Per1::luc reporter gene expression in the BMSCs. B. Per1::luc 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 Per1::luc 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 Per1::luc expression was nearly completely lost on the B-DAE-DCD discs. C. The loss of Per1::luc 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. D. The raw amplitude measurements depicted the stable expression of the Per1::luc reporter gene after the initial forskolin shock when the BMSCs were cultured on the polypropylene dishes or machined-polished Ti discs. In contrast, Per1::luc reporter gene expression remained low when the BMSCs were cultured on the B-DAE-DCD discs. E. The average raw amplitude of the Per1::luc 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. F. When BMSCs were cultured on B-DAE-DCD Ti discs without vitamin D supplementation, the lost Per1::luc circadian expression was partially recovered. G. 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.
Fig 3
Fig 3. Characterization of the Per1::luc circadian rhythm in BMSCs.
All the luminometry experiments were performed in triplicate for each group. A. The baseline-subtracted luminometry data demonstrated the steady circadian expression of Per1::luc of BMSC, which progressively decreased over the culture period. B. The average peak-to-trough amplitude and peak-to-peak period by day showed the tightly regulated Per1::luc circadian expression, which was not affected by 1 nM vitamin D supplementation. C. The raw amplitude data suggested that there was the steady decline in BMSC Per1::luc expression over the culture period after the initial forskolin shock. D. BMSC vitamin D supplementation was found to increase Per1::luc expression. **: p<0.01 by ANOVA. E. When BMSCs were exposed to osteogenic differentiation medium, both the baseline-subtracted peak-to-trough amplitude and the peak-to-peak period were modulated. Vitamin D supplementation appeared to accelerate the effects of osteogenic medium. **: p<0.01 by Wilks Lambda ANOVA for the amplitude and period. F. Vitamin D supplementation with osteogenic medium sustained Per1::luc expression throughout the culture period. **: p<0.01 by ANOVA.
Fig 4
Fig 4. The role of Npas2 in BMSC circadian rhythm.
A. A snapshot of circadian rhythm gene expression from rat BMSCs shows the transcriptional downregulation of Per1, Per2, Bmal1 and Id2 when the BMSCs were cultured on B-DAE-DCD Ti discs. A similar upregulation in Npas2 was observed in human BMSCs. RT-PCR was performed in triplicates. *: p<0.05 by Student’s t-test. B. The role of the transcription factor Npas2 on the steady state mRNA levels of circadian rhythm-related genes was examined by siRNA. Npas2 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. C. To further clarify the role of Npas2, BMSCs were harvested from mice carrying the Npas2 allele lacking Exon 3, which was replaced by the LacZ reporter gene cassette, resulting in a Npas2 functional knockout mutation due to the lack of the basic helical-loop-helical (bHLH) domain. D. BMSCs were harvested from the femurs of wild-type, Npas2+/- and Npas2-/- 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, LacZ reporter gene expression was increased in the D-DAE-DCD disc group. E. The expression of circadian rhythm genes were observed to increase in the Npas2+/- and Npas2-/- BMSCs maintained on polypropylene culture dishes for 32 hours after synchronization. F. Npas2+/- and Npas2-/- 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.
Fig 5
Fig 5. The weighted gene co-expression network analysis (WGCNA).
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) [20]. A. WGCNA identified 47 modules of co-expressed genes. The Turquoise and Blue modules contained a disproportionately large number of genes. 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. C. 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. D. 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-.
Fig 6
Fig 6. Gene networks formed around the biomaterial implant.
A. 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 (Vdr). 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 Npas2, Bmal1 (Arntl) and Vdr, whereas the Turquoise module contained Per1, Per3 and Per3. C. The comparison between OSV+ and OSV- without implanted biomaterials did not detect Vdr and Npas2 was found to be significantly downregulated. D. The comparison between ITV+ and ITV- revealed that the Blue module lost its trait-significance and Vdr. Per1, Per2 and Per3 were also moved from the Turquoise module and to the Purple module.

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    1. McAfee PC, Farey ID, Sutterlin CE, Gurr KR, Warden KE, Cunningham BW. The effect of spinal implant rigidity on vertebral bone density. A canine model. Spine (Phila Pa 1976). 1991;16(6 Suppl):S190–7. . - PubMed
    1. Long M, Rack HJ. Titanium alloys in total joint replacement—a materials science perspective. Biomaterials. 1998;19(18):1621–39. . - PubMed
    1. Abdel-Hady Gepreel M, Niinomi M. Biocompatibility of Ti-alloys for long-term implantation. J Mech Behav Biomed Mater. 2013;20:407–15. doi: 10.1016/j.jmbbm.2012.11.014 . - DOI - PubMed
    1. Davies JE. Understanding peri-implant endosseous healing. J Dent Educ. 2003;67(8):932–49. . - PubMed
    1. Veronesi F, Giavaresi G, Fini M, Longo G, Ioannidu CA, Scotto d'Abusco A, et al. Osseointegration is improved by coating titanium implants with a nanostructured thin film with titanium carbide and titanium oxides clustered around graphitic carbon. Mater Sci Eng C Mater Biol Appl. 2017;70(Pt 1):264–71. doi: 10.1016/j.msec.2016.08.076 . - DOI - PubMed