The Effect of Biomimetic Remineralization of Calcium Phosphate Ion Clusters-Treated Enamel Surfaces on Bracket Shear Bond Strength.

Purpose: To evaluate the remineralization effect of calcium phosphate ion clusters (CPICs) on demineralized enamel surfaces and their effects on bracket shear bond strength. Patients and Methods: Extracted premolars were prepared in resin blocks. The samples in the form of resin blocks were divided into five experimental groups: control group, demineralized group, and groups of CPIC solution treatment for 30, 60, and 90s. The specimens were examined using scanning electron microscopy (SEM), energy-dispersive spectroscopy (EDX), microhardness testing, micro-computed tomography (micro-CT) assessment, shear bond strength (SBS) test, and adhesive remnant index (ARI) score. Results: The SEM images revealed epitaxial growth of enamel and a decrease in the thickness of the demineralized enamel layer when treated with CPIC solution. The EDX analysis revealed an increase in the Ca/P ratio in the CPIC-treated groups. The microhardness value significantly increased when treated with CPICs; however, it showed a lower value than that of the sound enamel groups. As a result of the micro-CT test, radiolucency decreased gradually as the CPIC treatment time increased. The SBS test and ARI score results showed an improvement in bonding stability after treatment with CPICs. Conclusion: We demonstrated an enamel biomodification approach using CPIC solution treatment, which is a promising strategy for enamel remineralization. Specifically, remineralization of demineralized enamel improves the orthodontic bracket SBS.

The effect of kaempferol on the dentin bonding stability through matrix metalloproteinases inhibition and collagen crosslink in dentin biomodification.

Background/purpose: Naturally derived collagen crosslinkers with matrix metalloproteinases (MMPs) inhibitory activity for dentin bonding have been previously studied. One of these crosslinkers is flavonoids. The purpose of this study was to investigate whether dentin pretreatment with kaempferol (KEM), one of the flavonoids, enhances dentin bond stability and nanoleakage at the dentin-resin interface through MMPs inhibition and collagen crosslinking. Materials and methods: The experimental KEM-containing solution was used to pretreat demineralized dentin prior to the application of a universal adhesive. KEM is a natural flavonoid and those which did not take the experimental solution served as the control group (CON). Microtensile bond strength (µTBS) and nanoleakage tests were conducted before and after the thermocycling to evaluate the influence of KEM on dentin bond strength. The MMPs inhibition activity of KEM was analyzed via MMPs zymography using a confocal microscopy. Fourier-transform infrared (FTIR) spectroscopy was used to demonstrate that KEM inhibits MMPs and enhances collagen crosslinking. Results: The µTBS values of KEM group exhibited a higher bond strength after thermocycling. At the resin-dentin interface, the KEM group did not exhibit any signs of nanoleakage after thermocycling. Furthermore, MMPs zymography confirmed that there was a relatively low activity of MMPs in the presence of KEM. In FTIR analysis, the PO4 peak representing the cross-link between dentin and collagen was significantly higher in the KEM group. Conclusion: Our findings suggest that pretreatment with KEM enhances the dentin bonding stability at the resin-dentin interface by acting as a collagen crosslinker and MMPs inhibitor.

A remineralizing orthodontic etchant that utilizes calcium phosphate ion clusters.

This study aimed to investigate whether a phosphoric acid (H3PO4) solution containing calcium phosphate ion clusters (CPICs) could minimize enamel damage during long-term bracket bonding by dissolving the enamel surface and promoting enamel remineralization. The experimental design is as follows: first, three experimental etchants (H3PO4, CPICs-incorporated H3PO4 solution-I, and CPICs-incorporated H3PO4 solution-II) and two bonding resins (conventional orthodontic resin and self-adhesive orthodontic resin) were used in combination to create six groups, respectively. Each of these six groups was then divided into two sub-groups based on the presence or absence of thermocycling (TC). Twenty samples were assigned to each of the 12 groups (independent variables), and thus a total of 240 metal bracket-attached human premolars were used in this experiment. Bracket debonding was performed on each of 20 premolars in 12 groups, and shear bond strength (SBS) and adhesive remnant index (ARI) values were measured as dependent variables. Next, the three experimental etchants were applied (independent variables) to each of the three enamel samples, and the remineralization of the enamel surface was investigated as a dependent variable. The enamel surface was observed using electron scanning and atomic force microscopy. Furthermore, X-ray diffraction, energy dispersive spectroscopy (EDX) spectrum X-ray spectroscopy, and elemental mapping were performed, and the Knoop microhardness scale was measured. Therefore, the experiment was performed in two steps: SBS and ARI measurements for 12 groups, followed by observation of the enamel surface and microhardness measurements, according to the three types of etchants. As a result of the experiment, first, when the bracket was debonded, SBS did not decrease, and residual adhesive was hardly observed in the C2A group (before TC), C2A, and C1C groups (after TC) (p < 0.001). Second, the experimental etchant containing CPICs achieved remineralization while demineralizing the enamel. This was verified through SEM/EDX, element mapping, XRD, and AFM. Also, the roughness and microhardness of the enamel surface were better in the remineralized surface by the experimental etchant containing CPICs (p < 0.017). The CPICs-incorporated H3PO4 solution reduced ARI while maintaining SBS during bracket debonding, regardless of whether TC was performed or the type of resin. The etchant containing CPICs was also shown to remineralize the enamel and increase its microhardness.

Fibronectin regulates anoikis resistance via cell aggregate formation.

The loss of cell-matrix interactions induces apoptosis, known as anoikis. For successful distant metastasis, circulating tumor cells (CTCs) that have lost matrix attachment need to acquire anoikis resistance in order to survive. Cell aggregate formation confers anoikis resistance, and CTC clusters are more highly metastatic compared to single cells; however, the molecular mechanisms underlying this aggregation are not well understood. In this study, we demonstrated that cell detachment increased cell aggregation and upregulated fibronectin (FN) levels in lung and breast cancer cells, but not in their normal counterparts. FN knockdown decreased cell aggregation and increased anoikis. In addition, cell detachment induced cell-cell adhesion proteins, including E-cadherin, desmoglein-2, desmocollin-2/3, and plakoglobin. Interestingly, FN knockdown decreased the levels of desmoglein-2, desmocollin-2/3, and plakoglobin, but not E-cadherin, suggesting the involvement of desmosomal junction in cell aggregation. Accordingly, knockdown of desmoglein-2, desmocollin-2, or plakoglobin reduced cell aggregation and increased cell sensitivity to anoikis. Previously, we reported that NADPH oxidase 4 (Nox4) upregulation is important for anoikis resistance. Nox4 inhibition by siRNA or apocynin decreased cell aggregation and increased anoikis with the downregulation of FN, and, consequently, decreased desmoglein-2, desmocollin-2/3, or plakoglobin. The coexpression of Nox4 and FN was found to be significant in lung and breast cancer patients, based on cBioPortal data. In vivo mouse lung metastasis model showed that FN knockdown suppressed lung metastasis and thus enhanced survival. FN staining of micro tissue array revealed that FN expression was positive for human lung cancer (61%) and breast cancer (58%) patients. Furthermore, the expression levels of FN, desmoglein-2, desmocollin-2, and plakoglobin were significantly correlated with the poor survival of lung and breast cancer patients, as per the Kaplan-Meier plotter analysis. Altogether, our data suggest that FN upregulation and enhanced desmosomal interactions are critical for cell aggregation and anoikis resistance upon cell detachment.

Effect of Remineralized Collagen on Dentin Bond Strength through Calcium Phosphate Ion Clusters or Metastable Calcium Phosphate Solution.

This study aimed to investigate whether dentin remineralization and micro-tensile bond strength increase when using calcium phosphate ion clusters (CPICs) or metastable Ca-P. After being etched, each dentin specimen was designated into four groups and treated with the appropriate solution for 1 min: 100% ethanol, 2 and 1 mg/mL of CPICs, and metastable Ca-P. The specimens were then prepared for scanning electron microscopy (SEM), transmission electron microscropy (TEM) imaging, a matrix metalloproteinases inhibition assay, and the micro-tensile bond strength test. To compare among the groups, one-way analysis of variance was performed. In the SEM imaging, with a rising concentration of CPICs, the degree of remineralization of dentin increased significantly. The metastable Ca-P treated specimens showed a similar level of remineralization as the 1 mg/mL CPICs treated specimens. The TEM imaging also revealed that dentin remineralization occurs in a CPICs concentration-dependent manner between the demineralized dentin and the resin layer. Furthermore, the results of micro-tensile bond strength showed the same trend as the results confirmed by SEM and TEM. We demonstrated that a 1 min pretreatment of CPICs or metastable Ca-P in etched dentin collagen fibril can achieve biomimetic remineralization and increase micro-tensile bond strength.

Regulation of anoikis resistance by NADPH oxidase 4 and epidermal growth factor receptor.

BACKGROUND: Normal cells are sensitive to anoikis, which is a cell detachment-induced apoptosis. However, cancer cells acquire anoikis resistance that is essential for successful metastasis. This study aimed to demonstrate the function and potential mechanism of NADPH oxidase 4 (NOX4) and EGFR activation in regulating anoikis resistance in lung cancer. METHODS: Cells were cultured either in the attached or suspended condition. Cell viability was measured by cell counting and live and dead cell staining. Expression levels of NOX4 and EGFR were measured by PCR and immunoblotting. Reactive oxygen species (ROS) levels were measured by flow cytometry. Effects of NOX4 overexpression or NOX4 knockdown by si-NOX4 on anoikis sensitivity were explored. Levels of NOX4 and EGFR in lung cancer tissues were evaluated by IHC staining. RESULTS: NOX4 was upregulated but EGFR decreased in suspended cells compared with attached cells. Accordingly, ROS levels were increased in suspended cells, resulting in the activation of Src and EGFR. NOX4 knockdown decreased activation of Src and EGFR, and thus sensitised cells to anoikis. NOX4 overexpression increased EGFR levels and attenuated anoikis. NOX4 expression is upregulated and is positively correlated with EGFR levels in the lung cancer patient tissues. CONCLUSIONS: NOX4 upregulation confers anoikis resistance by ROS-mediated activation of EGFR and Src, and by maintaining EGFR levels, which is critical for cell survival.

Anoikis resistance: an essential prerequisite for tumor metastasis.

Metastasis is a multistep process including dissociation of cancer cells from primary sites, survival in the vascular system, and proliferation in distant target organs. As a barrier to metastasis, cells normally undergo an apoptotic process known as "anoikis," a form of cell death due to loss of contact with the extracellular matrix or neighboring cells. Cancer cells acquire anoikis resistance to survive after detachment from the primary sites and travel through the circulatory and lymphatic systems to disseminate throughout the body. Because recent technological advances enable us to detect rare circulating tumor cells, which are anoikis resistant, currently, anoikis resistance becomes a hot topic in cancer research. Detailed molecular and functional analyses of anoikis resistant cells may provide insight into the biology of cancer metastasis and identify novel therapeutic targets for prevention of cancer dissemination. This paper comprehensively describes recent investigations of the molecular and cellular mechanisms underlying anoikis and anoikis resistance in relation to intrinsic and extrinsic death signaling, epithelial-mesenchymal transition, growth factor receptors, energy metabolism, reactive oxygen species, membrane microdomains, and lipid rafts.

Intracellular expression of cytokines and granzyme B in auricular lymph nodes draining skin exposed to irritants and sensitizers.

The murine local lymph node assay (LLNA) has been extensively utilized to evaluate sensitizing chemicals. However, there have been some concerns that its use to discriminate between classes of chemicals is minimal. It is thus desirable to identify better or alternative immune endpoints with in LLNA itself. Here, we evaluated the protein and/or mRNA levels of cytokines and granzyme B (GzmB), a cytotoxic lymphocyte product, to discriminate between sensitizers and irritants and to characterize the chemical sensitizers when used as supplemental indicators in LLNA endpoints. For this, CBA/N mice were topically treated daily with a well-known chemical sensitizer such as a strong contact sensitizer 1-chloro-2,4-dinitrobenzene (DNCB), a skin contact sensitizer 2-phenyl-4-ethoxymethylene-5-oxazolone (OXA), and a skin or respiratory sensitizer toluene 2,4-diisocyanate (TDI), and the non-sensitizing irritants, croton oil (CRO) and nonanoic acid (NA), for 3 consecutive days. The protein and/or mRNA levels in auricular lymph nodes draining the ear skin were then analyzed by real-time RT-PCR and immunoassay. The sensitizers, but not the irritants, evoked pronounced interleukin (IL)-2, IL-3 and IL-4 or interferon (IFN)-gamma. Significantly, different sensitizers evoked different cytokine patterns of IL-4 and IFN-gamma, as DNCB strongly up-regulated both IFN-gamma and IL-4, OXA up-regulated IFN-gamma strongly but IL-4 weakly, and TDI up-regulated IL-4 strongly but IFN-gamma weakly. The sensitizers also strongly up-regulated GzmB mRNA, while the irritants had a much weaker effect. Thus, these cytokines and GzmB mRNA may be useful as additional endpoints for discriminating between irritants and sensitizers or contact and respiratory sensitizers in the LLNA.