The effects of vitamin D on bone health have been widely described, but the precise direct actions of vitamin D on bone forming osteoblasts have yet to be fully elucidated. Vitamin D can act to promote innate immune responses. Non-classical vitamin D functions are described in monocytes and macrophages. Stress and damage signals, as well as infection, may influence bone function and fracture healing. The current PhD project hypothesizes that innate immune response to vitamin D contributes to its effects on bone. Mouse osteoblasts obtained from primary calvaria and MC3T3-E1 cell lines were used and differentiated for 5 days, 14 days and 21 days to represent different stages of osteoblast differentiation. Cells were treated with vitamin D metabolites 25(OH)D3 and 1,25(OH)2D3 to observe vitamin D effects on differentiated osteoblasts. Vitamin D metabolites did not alter vitamin D metabolism genes Vdr and Cyp27b1 in differentiation osteoblasts. In addition, LPS as Toll-like receptor 4 agonist and Pam3CysK as Toll-like receptor 2 agonist were stimulated in differentiated osteoblasts to evaluate osteoblastic responses. Western blot and RT-PCR analyses confirmed expression of TLR2 and TLR4 and TLR4-specific signaling factors TRIF at all stages of differentiation. Mouse osteoblasts displayed divergent responses to LPS and Pam3CysK stimulations in osteoblast differentiation markers and osteoclastogenesis markers. Although both LPS and Pam3CysK promote inflammatory cytokines, only Pam3CysK stimulation demonstrated potent modulation to promote osteoclastogenesis through increased expression of Rankl and suppression of Ocn. TLR2 and TLR4 share an MyD88 signaling pathway to induce inflammation and osteoclastogeneis, but only TLR4 can signal via the intracellular TRIF/TRAM pathway. TrifsiRNA was introduced to 14 days differentiated MC3T3-E1 cells. RT-PCR analysis showed suppression of Vdr, Alp, Opg and Il-6 by TrifsiRNA. In addition, TLRs signaling activates MAPK phosphorylation, Pam3CysK induced higher phosphorylation of p38 MAPK and inhibition of p38 by TLCK (NF-κB inhibitor) neutralized suppression of osteoblast differentiation markers by Pam3CysK. In conclusion, TLR2 and TLR4 stimulation showed differential effects in osteoblasts possibly due to distinct MAPK phosphorylation pattern.

Objectives: In skin wounds, inflammation associated cytokines and growth factors induce fibroblasts to undergo transdifferentiation allowing them to contract the wound. Though wounds in the oral cavity heal with a lesser degree of inflammation, they contract at a faster pace and exhibit reduced scarring compared to the skin. FGFR1OP2/wit3.0 is a small cytoplasmic peptide highly expressed by oral wound fibroblasts, but not by skin wound fibroblasts. In this study we aim to determine the exogenous factors possibly inducing FGFR1OP2/wit3.0 expression in skin fibroblasts through the development of a high throughput screening assay.

Methods: This study examined the use of direct mRNA measurement assays for use in high throughput applications to find chemical agents that up or downregulate FGFR1OP2/wit3.0 expression. Using mouse clonal fibroblastic cell lines we characterized genetic expression, phenotype, and si-RNA knockdown of wit3.0. We then developed and optimized a protocol for high throughput screening on a bDNA platform. A pilot screening was performed at the UCLA MSSR.

Results: Confocal microscopy revealed that the cytoskeletal arrangement of actin and wit3.0 were phenotypically different in the heterozygous knockout stem cell derived fibroblasts. wit3.0 (+/-) cells exhibited disorganized actin fibers, while the wildtype showed a more organized archetype. We found that wit3.0 was expressed in both L-929 and C3H/10T1/2 and appears in immunostaining as a cytoplasmic, granular protein. We were able to successfully perform a high throughput screening of 320 chemicals as a pilot study, and identified 7 compounds as modulators of wit3.0.

Conclusion: This pilot study has identified biologically functional chemical compounds which could provide a novel clue in elucidating the mechanism of fibroblast-driven wound closure, and presents a new therapeutic potential in the field of wound healing.

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.

BACKGROUND: Previous studies suggested that vitamin D played a significant role in bone regeneration, facilitating the establishment of implant osseointegration. A whole genome microarray study further suggested that the vitamin D axis might involve circadian rhythm gene expression in the bone peripheral tissue.

OBJECTIVES: To identify key gene networks involved with vitamin D receptor in the bone regeneration process and to explore any correlation with circadian rhythm gene expression in bone marrow mesenchymal stromal/stem cells (BMSC).

METHODS: The available whole gene microarray data was analyzed using the weighted gene correlation network analysis (WGCNA) R package and Cytoscape software. Any gene expression correlation was examined for vitamin D receptor (VDR) as well as circadian rhythm genes. Separately, Per 1 luciferase transgenic Wistar rats were then applied for in vitro evaluation on BMSC circadian rhythm. Per1::luc BMSCs were seeded on 35mm dishes and forskolin-synchronized luminescence was recorded across different media conditions. The recording media included growth medium containing F12 (10% Fetal Bovine Serum, 1% Pen-Strep and antibiotic) supplemented with or without 1 nM 1,25D. Luminescence was also recorded in F12 growth medium containing bone differentiation factors (beta glycerophosphate, Ascorbic acid and Dexamethasone) supplemented with 0, 1 or 10 nM 1,25D.

RESULTS: A total of 47 gene network modules, based on the correlation between gene significance values and module membership, were generated. The "Blue" module contained VDR. With a threshold of 17 and median of 13, VDR exhibited 35 gene connections as well as correlation values of 0.31 and 0.55 for OSV+ (vitamin D sufficient condition) and OSV- (vitamin D insufficient condition) respectively. NPAS2, CLOCK, BMAL1 and casein kinase 1 were directly connected to VDR. Per1-3 and cry1-2 were each found in a different module with generally low correlation values. In vitro luminescence measures of Per1::luc rat BMSC showed a dose dependent sustained increase in Per 1 expression with the addition of 1,25D to the F12 growth medium as well as osteogenic differentiation medium. The baseline subtracted luminescence data indicated that the circadian pattern of BMSC was maintained with 1,25D supplementation.

CONCLUSION: The WGCNA data suggests that VDR may serve as the main coordinator, or hub gene, strongly correlated with the identified circadian rhythm genes. The forskolin-synchronized expression of Per1::luc exhibited circadian expression in BMSC in vitro. 1,25D supplementation significantly and dose dependently increased the baseline expression of Per1::luc, whereas the circadian rhythm was maintained. We postulate that the physiological regulation of vitamin D may mediate gene network organization via circadian rhythm gene hubs during the bone regeneration or remodeling process.

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