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1.  Enamel Matrix Derivative Promote Primary Human Pulp Cell Differentiation and Mineralization 
Enamel matrix derivative (EMD) has been found to induce reactive dentin formation; however the molecular mechanisms involved are unclear. The effect of EMD (5–50 μg/mL) on primary human pulp cells were compared to untreated cells and cells incubated with 10−8 M dexamethasone (DEX) for 1, 2, 3, 7, and 14 days in culture. Expression analysis using Affymetrix microchips demonstrated that 10 μg/mL EMD regulated several hundred genes and stimulated the gene expression of proteins involved in mesenchymal proliferation and differentiation. Both EMD and DEX enhanced the expression of amelogenin (amel), and the dentinogenic markers dentin sialophosphoprotein (DSSP) and dentin matrix acidic phosphoprotein 1 (DMP1), as well as the osteogenic markers osteocalcin (OC, BGLAP) and collagen type 1 (COL1A1). Whereas, only EMD had effect on alkaline phosphatase (ALP) mRNA expression, the stimulatory effect were verified by enhanced secretion of OC and COL1A from EMD treated cells, and increased ALP activity in cell culture medium after EMD treatment. Increased levels of interleukin-6 (IL-6), interleukin-8 (IL-8), and monocyte chemoattractant proteins (MCP-1) in the cell culture medium were also found. Consequently, the suggested effect of EMD is to promote differentiation of pulp cells and increases the potential for pulpal mineralization to favor reactive dentine formation.
doi:10.3390/ijms15057731
PMCID: PMC4057702  PMID: 24857913
pulp cells; enamel matrix derivative (EMD); bone; dentin; epigenetic factors
2.  Enhanced in vitro osteoblast differentiation on TiO2 scaffold coated with alginate hydrogel containing simvastatin 
Journal of Tissue Engineering  2013;4:2041731413515670.
The aim of this study was to develop a three-dimensional porous bone graft material as vehicle for simvastatin delivery and to investigate its effect on primary human osteoblasts from three donors. Highly porous titanium dioxide (TiO2) scaffolds were submerged into simvastatin containing alginate solution. Microstructure of scaffolds, visualized by scanning electron microscopy and micro-computed tomography, revealed an evenly distributed alginate layer covering the surface of TiO2 scaffold struts. Progressive and sustained simvastatin release was observed for up to 19 days. No cytotoxic effects on osteoblasts were observed by scaffolds with simvastatin when compared to scaffolds without simvastatin. Expression of osteoblast markers (collagen type I alpha 1, alkaline phosphatase, bone morphogenetic protein 2, osteoprotegerin, vascular endothelial growth factor A and osteocalcin) was quantified using real-time reverse transcriptase–polymerase chain reaction. Secretion of osteoprotegerin, vascular endothelial growth factor A and osteocalcin was analysed by multiplex immunoassay (Luminex). The relative expression and secretion of osteocalcin was significantly increased by cells cultured on scaffolds with 10 µM simvastatin when compared to scaffolds without simvastatin after 21 days. In addition, secretion of vascular endothelial growth factor A was significantly enhanced from cells cultured on scaffolds with both 10 nM and 10 µM simvastatin when compared to scaffolds without simvastatin at day 21. In conclusion, the results indicate that simvastatin-coated TiO2 scaffolds can support a sustained release of simvastatin and induce osteoblast differentiation. The combination of the physical properties of TiO2 scaffolds with the osteogenic effect of simvastatin may represent a new strategy for bone regeneration in defects where immediate load is wanted or unavailable.
doi:10.1177/2041731413515670
PMCID: PMC3927861  PMID: 24555011
Simvastatin; TiO2 scaffold; alginate hydrogel; osteocalcin; bone morphogenetic protein 2; vascular endothelial growth factor A
3.  Ameloblastin upstream region contains structural elements regulating transcriptional activity in a stromal cell line derived from bone marrow 
European Journal of Oral Sciences  2011;119(Suppl 1):286-292.
Ameloblastin (AMBN) was originally described as a tooth-specific extracellular matrix protein, but current data have shown that AMBN is present in many different tissues of mesenchymal origin. The identification of regulatory elements in the promoter region of the Ambn gene would assist in identifying potential mesenchymal-specific transcriptional factors. In this study we subcloned a 3,788-bp region upstream (and a 54-bp region downstream) of the mouse Ambn transcriptional start site into a LacZ reporter construct and called this construct 3788-Ambn-lacZ. In silico analysis of the 3,788-bp Ambn promoter region identified 50 potential cis-regulatory elements, 29 of which are known to be functional in cell populations of mesenchymal origin. The reporter construct was activated in transfected bone marrow cells, and the promoter activity was induced in cell cultures following addition of recombinant AMBN, interferon-γ, serotonin, or dexamethasone. We discuss the relative significance of the potential cis-acting gene-regulatory elements of Ambn in relation to bone morphogenesis. Knowledge of Ambn gene-regulatory elements will be of importance when developing strategies for bone repair and replacement in a clinical surgical setting.
doi:10.1111/j.1600-0722.2011.00910.x
PMCID: PMC3370392  PMID: 22243258
ameloblastin; bone; mesenchymal cells; promoter elements
4.  Gene Expression Profiling during Murine Tooth Development 
Frontiers in Genetics  2012;3:139.
The aim of this study was to describe the expression of genes, including ameloblastin (Ambn), amelogenin X chromosome (Amelx), and enamelin (Enam) during early (pre-secretory) tooth development. The expression of these genes has predominantly been studied at post-secretory stages. Deoxyoligonucleotide microarrays were used to study gene expression during development of the murine first molar tooth germ at 24 h intervals, starting at the 11th embryonic day (E11.5), and up to the 7th day after birth (P7). The profile search function of Spotfire software was used to select genes with similar expression profile as the enamel genes (Ambn, Amelx, and Enam). Microarray results where validated using real-time reverse transcription-polymerase chain reaction (real-time RT-PCR), and translated proteins identified by Western-blotting. In situ localization of the Ambn, Amelx, and Enam mRNAs were monitored from E12.5 to E17.5 using deoxyoligonucleotide probes. Bioinformatics analysis was used to associate biological functions with differentially expressed (DE; p ≤ 0.05) genes. Microarray results showed a total of 4362 genes including Ambn, Amelx, and Enam to be significant DE throughout the time-course. The expression of the three enamel genes was low at pre-natal stages (E11.5–P0) increasing after birth (P1–P7). Profile search lead to isolation of 87 genes with significantly similar expression to the three enamel proteins. These mRNAs were expressed in dental epithelium and epithelium derived cells. Although expression of Ambn, Amelx, and Enam were lower during early tooth development compared to secretory stages enamel proteins were detectable by Western-blotting. Bioinformatic analysis associated the 87 genes with multiple biological functions. Around 35 genes were associated with 15 transcription factors.
doi:10.3389/fgene.2012.00139
PMCID: PMC3408794  PMID: 22866057
tooth development; ameloblastin; amelogenin; enamelin
5.  The peroxisome proliferator-activated receptor (PPAR) alpha agonist fenofibrate maintains bone mass, while the PPAR gamma agonist pioglitazone exaggerates bone loss, in ovariectomized rats 
Background
Activation of peroxisome proliferator-activated receptor (PPAR)gamma is associated with bone loss and increased fracture risk, while PPARalpha activation seems to have positive skeletal effects. To further explore these effects we have examined the effect of the PPARalpha agonists fenofibrate and Wyeth 14643, and the PPARgamma agonist pioglitazone, on bone mineral density (BMD), bone architecture and biomechanical strength in ovariectomized rats.
Methods
Fifty-five female Sprague-Dawley rats were assigned to five groups. One group was sham-operated and given vehicle (methylcellulose), the other groups were ovariectomized and given vehicle, fenofibrate, Wyeth 14643 and pioglitazone, respectively, daily for four months. Whole body and femoral BMD were measured by dual X-ray absorptiometry (DXA), and biomechanical testing of femurs, and micro-computed tomography (microCT) of the femoral shaft and head, were performed.
Results
Whole body and femoral BMD were significantly higher in sham controls and ovariectomized animals given fenofibrate, compared to ovariectomized controls. Ovariectomized rats given Wyeth 14643, maintained whole body BMD at sham levels, while rats on pioglitazone had lower whole body and femoral BMD, impaired bone quality and less mechanical strength compared to sham and ovariectomized controls. In contrast, cortical volume, trabecular bone volume and thickness, and endocortical volume were maintained at sham levels in rats given fenofibrate.
Conclusions
The PPARalpha agonist fenofibrate, and to a lesser extent the PPARaplha agonist Wyeth 14643, maintained BMD and bone architecture at sham levels, while the PPARgamma agonist pioglitazone exaggerated bone loss and negatively affected bone architecture, in ovariectomized rats.
doi:10.1186/1472-6823-11-11
PMCID: PMC3127763  PMID: 21615901
6.  Different skeletal effects of the peroxisome proliferator activated receptor (PPAR)α agonist fenofibrate and the PPARγ agonist pioglitazone 
Background
All the peroxisome proliferator activated receptors (PPARs) are found to be expressed in bone cells. The PPARγ agonist rosiglitazone has been shown to decrease bone mass in mice and thiazolidinediones (TZDs) have recently been found to increase bone loss and fracture risk in humans treated for type 2 diabetes mellitus. The aim of the study was to examine the effect of the PPARα agonist fenofibrate (FENO) and the PPARγ agonist pioglitazone (PIO) on bone in intact female rats.
Methods
Rats were given methylcellulose (vehicle), fenofibrate or pioglitazone (35 mg/kg body weight/day) by gavage for 4 months. BMC, BMD, and body composition were measured by DXA. Histomorphometry and biomechanical testing of excised femurs were performed. Effects of the compounds on bone cells were studied.
Results
The FENO group had higher femoral BMD and smaller medullary area at the distal femur; while trabecular bone volume was similar to controls. Whole body BMD, BMC, and trabecular bone volume were lower, while medullary area was increased in PIO rats compared to controls. Ultimate bending moment and energy absorption of the femoral shafts were reduced in the PIO group, while similar to controls in the FENO group. Plasma osteocalcin was higher in the FENO group than in the other groups. FENO stimulated proliferation and differentiation of, and OPG release from, the preosteoblast cell line MC3T3-E1.
Conclusion
We show opposite skeletal effects of PPARα and γ agonists in intact female rats. FENO resulted in significantly higher femoral BMD and lower medullary area, while PIO induced bone loss and impairment of the mechanical strength. This represents a novel effect of PPARα activation.
doi:10.1186/1472-6823-9-10
PMCID: PMC2678137  PMID: 19331671
7.  Plasma levels of leptin and mammographic density among postmenopausal women: a cross-sectional study 
Breast Cancer Research  2006;8(5):R55.
Introduction
Obesity has been linked to increased risk of breast cancer in postmenopausal women. Increased peripheral production of estrogens has been regarded as the main cause for this association, but other features of increased body fat mass may also play a part. Leptin is a protein produced mainly by adipose tissue and may represent a growth factor in cancer. We examined the association between leptin plasma levels and mammographic density, a biomarker for breast cancer risk.
Methods
We included data from postmenopausal women aged 55 and older, who participated in a cross-sectional mammography study in Tromsø, Norway. Mammograms, plasma leptin measurements as well as information on anthropometric and hormonal/reproductive factors were available from 967 women. We assessed mammographic density using a previously validated computer-assisted method. Multiple linear regression analysis was applied to investigate the association between mammographic density and quartiles of plasma leptin concentration. Because we hypothesized that the effect of leptin on mammographic density could vary depending on the amount of nondense or fat tissue in the breast, we also performed analyses on plasma leptin levels and mammographic density within tertiles of mammographic nondense area.
Results
After adjusting for age, postmenopausal hormone use, number of full-term pregnancies and age of first birth, there was an inverse association between leptin and absolute mammographic density (Ptrend = 0.001). When we additionally adjusted for body mass index and mammographic nondense area, no statistically significant association between plasma leptin and mammographic density was found (Ptrend = 0.16). Stratified analyses suggested that the association between plasma leptin and mammographic density could differ with the amount of nondense area of the mammogram, with the strongest association between leptin and mammographic absolute density in the stratum with the medium breast fat content (Ptrend = 0.003, P for interaction = 0.05).
Conclusion
We found no overall consistent association between the plasma concentration of leptin and absolute mammographic density. Although weak, there was some suggestion that the association between leptin and mammographic density could differ with the amount of fat tissue in the breast.
doi:10.1186/bcr1603
PMCID: PMC1779493  PMID: 17010200

Results 1-7 (7)