Remineralization effects of enamel binding peptide, WGNYAYK, on enamel subsurface demineralization in vitro. Enamel binding peptide, WGNYAYK effect remineralization of enamel.

Objectives: We investigated remineralization effects of enamel binding peptide (EBP), WGNYAYK, on enamel subsurface demineralization in vitro.Methods: Bovine lower incisor crowns were used as subsurface enamel demineralization samples, and changes of EBP binding, remineraliztion rate, hardness and microstructure were investigated. Binding of EBP, remineralization rate, hardness and structural changes were investigated. Fluorescein isothiocyatate (FITC)-labeled EBPs (0.4 mM, 4.0 mM, and 7.0 mM) were applied to the samples for 30 min at 37 °C, with sample surfaces and cross-sections observed by confocal laser scanning microscope (CLSM). Mineralization analysis samples were divided into 4 experimental groups; distilled water (DW), EBP 0.4 mM, EBP 4.0 mM, and EBP 7.0 mM. Mineral density changes were measured by micro-CT with hardness measured by nano-indentation. Samples were also observed by scanning electron microscope (SEM) for surface and longitudinal microstructure. Results: CLSM images indicated that increased fluorescence was observed in the surface layer and up to about 20 µm below the surface layer. The remineralization rate was significantly higher for EBP 7.0 mM compared to DW (p = 0.008). Enamel surface hardness was significantly higher in all EBP groups compared to DW (p < 0.05) and was highest in the 7.0 mM group. SEM images showed obscuring of the superficial columnar structure in the 7.0 mM EBP group, indicating subsurface crystalline structure recovery. Conclusion: The results of this study suggest that EBP binds to demineralized enamel and promotes remineralization.

Effects of bovine milk osteopontin on in vitro enamel remineralization as a topical application prior to immersion in remineralizing solutions with/without fluoride.

The aim of the present study was to investigate the effects of bovine milk osteopontin (OPN) on enamel remineralization as a topical application prior to immersion in remineralizing solutions with/without fluoride. Bovine enamel blocks were demineralized then were divided into the following 3 groups: OPN (2.7 and 5.4 µM) solutions and deionized water (control). Each group was divided into 2 groups (remineralizing solution with or without 1 ppm of fluoride (F)). The specimens were analyzed by micro-CT and scanning electron microscope (SEM). The percentage of remineralization was higher in remineralization solution with than without F (p<0.05). The present results suggest that bovine milk OPN inhibits remineralization in solution without F, but 5.4 µM bovine milk OPN does not inhibit remineralization of the demineralized body using solution containing F by interrupting mineral deposition on the enamel surface.

In vitro remineralization of enamel with a solution containing casein and fluoride.

The aim of the present study was to investigate the effects of casein in a remineralization solution on enamel remineralization. Bovine blocks were demineralized for 21 days, then, allocated into four groups. The specimens were remineralized for 21 days in the following artificial saliva solutions: 1) 0 µg/mL casein, 0 ppm fluoride (F) (C0-F0); 2) 0 µg/mL casein, 1 ppm F (C0-F1); 3) 10 µg/mL casein, 0 ppm F (C10-F0); and 4) 10 µg/mL casein, 1 ppm F (C10-F1). Micro-CT analyses were performed once a week. Specimens were characterized by scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). The present results suggest that casein by itself inhibits remineralization, whereas the coexistence of casein and F promotes the remineralization of caries bodies by interrupting mineral deposition on the enamel surface.

Role of ATF7-TAF12 interactions in the vitamin D response hypersensitivity of osteoclast precursors in Paget's disease.

Osteoclast (OCL) precursors from many Paget's disease (PD) patients express measles virus nucleocapsid protein (MVNP) and are hypersensitive to 1,25-dihydroxyvitamin D2 (1,25-(OH)2D3; also know as calcitriol). The increased 1,25-(OH)2D3 sensitivity is mediated by transcription initiation factor TFIID subunit 12 (TAF12), a coactivator of the vitamin D receptor (VDR), which is present at much higher levels in MVNP-expressing OCL precursors than normals. These results suggest that TAF12 plays an important role in the abnormal OCL activity in PD. However, the molecular mechanisms underlying both 1,25-(OH)2D3's effects on OCL formation and the contribution of TAF12 to these effects in both normals and PD patients are unclear. Inhibition of TAF12 with a specific TAF12 antisense construct decreased OCL formation and OCL precursors' sensitivity to 1,25-(OH)2D3 in PD patient bone marrow samples. Further, OCL precursors from transgenic mice in which TAF12 expression was targeted to the OCL lineage (tartrate-resistant acid phosphatase [TRAP]-TAF12 mice), formed OCLs at very low levels of 1,25-(OH)2D3, although the OCLs failed to exhibit other hallmarks of PD OCLs, including receptor activator of NF-κB ligand (RANKL) hypersensitivity and hypermultinucleation. Chromatin immunoprecipitation (ChIP) analysis of OCL precursors using an anti-TAF12 antibody demonstrated that TAF12 binds the 24-hydroxylase (CYP24A1) promoter, which contains two functional vitamin D response elements (VDREs), in the presence of 1,25-(OH)2D3. Because TAF12 directly interacts with the cyclic adenosine monophosphate-dependent activating transcription factor 7 (ATF7) and potentiates ATF7-induced transcriptional activation of ATF7-driven genes in other cell types, we determined whether TAF12 is a functional partner of ATF7 in OCL precursors. Immunoprecipitation of lysates from either wild-type (WT) or MVNP-expressing OCL with an anti-TAF12 antibody, followed by blotting with an anti-ATF7 antibody, or vice versa, showed that TAF12 and ATF7 physically interact in OCLs. Knockdown of ATF7 in MVNP-expressing cells decreased cytochrome P450, family 24, subfamily A, polypeptide 1 (CYP24A1) induction by1,25-(OH)2D3, as well as TAF12 binding to the CYP24A1 promoter. These results show that ATF7 interacts with TAF12 and contributes to the hypersensitivity of OCL precursors to 1,25-(OH)2D3 in PD.

ADAM8 enhances osteoclast precursor fusion and osteoclast formation in vitro and in vivo.

ADAM8 expression is increased in the interface tissue around a loosened hip prosthesis and in the pannus and synovium of patients with rheumatoid arthritis, but its potential role in these processes is unclear. ADAM8 stimulates osteoclast (OCL) formation, but the effects of overexpression or loss of expression of ADAM8 in vivo and the mechanisms responsible for the effects of ADAM8 on osteoclastogenesis are unknown. Therefore, to determine the effects of modulating ADAM expression, we generated tartrate-resistant acid phosphatase (TRAP)-ADAM8 transgenic mice that overexpress ADAM8 in the OCL lineage and ADAM8 knockout (ADAM8 KO) mice. TRAP-ADAM8 mice developed osteopenia and had increased numbers of OCL precursors that formed hypermultinucleated OCLs with an increased bone-resorbing capacity per OCL. They also had an enhanced differentiation capacity, increased TRAF6 expression, and increased NF-κB, Erk, and Akt signaling compared with wild-type (WT) littermates. This increased bone-resorbing capacity per OCL was associated with increased levels of p-Pyk2 and p-Src activation. In contrast, ADAM8 KO mice did not display a bone phenotype in vivo, but unlike WT littermates, they did not increase RANKL production, OCL formation, or calvarial fibrosis in response to tumor necrosis factor α (TNF-α) in vivo. Since loss of ADAM8 does not inhibit basal bone remodeling but only blocks the enhanced OCL formation in response to TNF-α, these results suggest that ADAM8 may be an attractive therapeutic target for preventing bone destruction associated with inflammatory disease.