UCLA

BACKGROUND: Endosseous implant osseointegration has been primarily studied from a wound healing-model. The molecular pathway of implant osseointegration is not known. Previous animal studies have shown vitamin D insufficiency impairs the establishment of implant osseointegration. Recent microarray data found an interesting connection between vitamin D receptor and circadian rhythm genes using a vitamin D insufficient animal model (implant failure model). Per1 is one of eight circadian genes that has widely been used as a molecular marker of circadian rhythm. Previous experiments with forskolin-synchronized expression of Per1 in bone marrow mesenchymal stromal cells (BMSCs) in vitro showed vitamin D supplementation significantly and dose dependently increased the baseline expression of Per1::luc, but preserved the circadian rhythm. We have thus postulated that the circadian rhythm of BMSCs may be influenced by titanium dental implants.

OBJECTIVES: To determine the effect of titanium implant substrates with different surface characteristics on BMSCs peripheral circadian rhythm.

METHODS: In this study, a series of in vitro studies were designed to characterize the peripheral circadian rhythm of BMSCs harvested from Per1::luc transgenic Wistar rats. Per1 transgenic rats genomic locus of Per1 allele were modified by inserting a beetle luciferase reporter gene (Per1::luc). The peripheral circadian rhythms over a 24-hour period were monitored by the real time activation of luciferase-mediated bioluminescence. Lumicycle was the instrument used to record the luciferase activity (Per1 expression) every ten minutes over five days. Data was evaluated both in the raw form (photons/second) Per1 gene expression and baseline subtracted data. The baseline subtracted data provides the peaks, troughs and periods of the BMSCs peripheral circadian rhythm over 5 days. The first experiments were completed on plastic cell culture dishes with and without vitamin D supplementation.

In the second project, two titanium substrates with polished (smooth) surface or complex (rough) surface with blasted, acid etched and discrete HA crystalline deposition were characterized. Surface topography: Sa (average surface roughness) and Sdr (hybrid parameter that presents information about the number and height of peaks of a given surface) were determined by a 3D surface profilometry. To ensure BMSC survival and proper attachment a WST-1 assay was completed. To determine the level of osteoblast differentiation capabilities of BMSCs on all three surfaces (plastic, smooth and rough) a calcium mineralization assay was preformed.

In the third project, BMSC peripheral circadian rhythm studies were then executed on smooth and rough titanium surfaces with and without vitamin D supplementation. This study was accomplished using Per1:;luc BMSCs and Lumicyle.

In the forth project, Taqman- based quantitative reverse transcription polymerase chain reaction (RT-PCR) was used to validate gene expression of 6 circadian rhythm-related molecules (Per1, Per2, Id2, Bmal1, Clock, and NPas2).

RESULTS: Bioluminescent raw measurement of Per1 expression on plastic with and without vitamin D showed that vitamin D increased Per1 expression over days 2, 3 and 4. The baseline subtracted data suggested that vitamin D suppressed the amplitude (peak to trough) but maintained the period.

The WST-1 assay revealed that over 4 days the three different surfaces had a comparable number of viable cells. The calcium assay also suggested that the different substrate surfaces did not have large influence on osteogenic differentiation of BMSCs.

The raw measurement of Per1::luc on titanium implant substrates indicated that the rough titanium surface had a significant reduction in Per1 expression (more than 50%) of photons per second in comparison to the smooth surface. Furthermore, rough titanium with vitamin D had a lower Per1 expression when compared to the rough titanium with no vitamin D. The smooth titanium baseline subtracted data showed a consistent rhythm having developed peaks and troughs. The most dramatic difference was seen in the rough surface baseline subtracted data. The rough surface almost completely eliminated the amplitude and period of the peripheral circadian rhythm.

RT-PCR validated that five of the six circadian rhythm genes were downregulated by the rough titanium surface. The only gene upregulated on rough titanium was NPAS2.

CONCLUSION: Titanium material had a negative effect on Per1 expression and amplitude of BMSCs peripheral circadian rhythm. Osseointegration pathway is unknown, however, the peripheral circadian rhythm of BMSCs may play a role in the establishment of implant-bone integration. NPAS2 was found to be highly expressed with the rough titanium substrate. Although speculative, the modulation of peripheral circadian rhythm may lead to successful osseointegration.