Luminescent and paramagnetic properties of nanoparticles shed light on their interactions with proteins.

Nanoparticles have been recognized as promising tools for targeted drug-delivery and protein therapeutics. However, the mechanisms of protein-nanoparticle interaction and the dynamics underlying the binding process are poorly understood. Here, we present a general methodology for the characterization of protein-nanoparticle interaction on a molecular level. To this end we combined biophysical techniques including nuclear magnetic resonance (NMR), circular dichroism (CD), resonance energy transfer (RET) and surface plasmon resonance (SPR). Particularly, we analyzed molecular mechanisms and dynamics of the interaction of CaF2 nanoparticles with the prototypical calcium sensor calmodulin (CaM). We observed the transient formation of an intermediate encounter complex involving the structural region linking the two domains. Specific interaction of CaM with CaF2 NPs is driven by the N-terminal EF-hands, which seem to recognize Ca2+ on the surface of the nanoparticle. We conclude that CaF2 NP-CaM interaction is fully compatible with potential applications in nanomedicine. Overall, the methods presented in this work can be extended to other systems and may be useful to quantitatively characterize structural and dynamic features of protein-NP interactions with important implications for nanomedicine and nano-biotechnology.

CO2 laser irradiation enhances CaF2 formation and inhibits lesion progression on demineralized dental enamel-in vitro study.

This study evaluated if Carbon dioxide (CO2) (λ 10.6 µm) laser irradiation combined with acidulated phosphate fluoride gel application (APF gel) enhances "CaF2" uptake by demineralized enamel specimens (DES) and inhibits enamel lesion progression. Thus, two studies were conducted and DES were subjected to APF gel combined or not with CO2 laser irradiation (11.3 or 20.0 J/cm(2), 0.4 or 0.7 W) performed before, during, or after APF gel application. In study 1, 165 DES were allocated to 11 groups. Fluoride as "CaF2 like material" formed on enamel was determined in 100 DES (n = 10/group), and the surface morphologies of 50 specimens were evaluated by scanning electron microscopy (SEM) before and after "CaF2" extraction. In study 2, 165 DES (11 groups, n = 15), subjected to the same treatments as in study 1, were further subjected to a pH-cycling model to simulate a high cariogenic challenge. The progression of demineralization in DES was evaluated by cross-sectional microhardness and polarized light microscopy analyses. Laser at 11.3 J/cm(2) applied during APF gel application increased "CaF2" uptake on enamel surface. Laser irradiation and APF gel alone arrested the lesion progression compared with the control (p < 0.05). Areas of melting, fusion, and cracks were observed. CO2 laser irradiation, combined with a single APF application enhanced "CaF2" uptake on enamel surface and a synergistic effect was found. However, regarding the inhibition of caries lesion progression, no synergistic effect could be demonstrated. In conclusion, the results have shown that irradiation with specific laser parameters significantly enhanced CaF2 uptake by demineralized enamel and inhibited lesion progression.

Calcium fluoride nanoparticles induced suppression of Streptococcus mutans biofilm: an in vitro and in vivo approach.

Biofilm formation on the tooth surface is the root cause of dental caries and periodontal diseases. Streptococcus mutans is known to produce biofilm which is one of the primary causes of dental caries. Acid production and acid tolerance along with exopolysaccharide (EPS) formation are major virulence factors of S. mutans biofilm. In the current study, calcium fluoride nanoparticles (CaF2-NPs) were evaluated for their effect on the biofilm forming ability of S. mutans in vivo and in vitro. The in vitro studies revealed 89 % and 90 % reduction in biofilm formation and EPS production, respectively. Moreover, acid production and acid tolerance abilities of S. mutans were also reduced considerably in the presence of CaF2-NPs. Confocal laser scanning microscopy and transmission electron microscopy images were in accordance with the other results indicating inhibition of biofilm without affecting bacterial viability. The qRT-PCR gene expression analysis showed significant downregulation of various virulence genes (vicR, gtfC, ftf, spaP, comDE) associated with biofilm formation. Furthermore, CaF2-NPs were found to substantially decrease the caries in treated rat groups as compared to the untreated groups in in vivo studies. Scanning electron micrographs of rat's teeth further validated our results. These findings suggest that the CaF2-NPs may be used as a potential antibiofilm applicant against S. mutans and may be applied as a topical agent to reduce dental caries.

Cold shock induces apoptosis of dorsal root ganglion neurons plated on infrared windows.

The chemical status of live sensory neurons is accessible with infrared microspectroscopy of appropriately prepared cells. In this paper, individual dorsal root ganglion (DRG) neurons have been prepared with two different protocols, and plated on glass cover slips, BaF2 and CaF2 substrates. The first protocol exposes the intact DRGs to 4 °C for between 20-30 minutes before dissociating individual neurons and plating 2 hours later. The second protocol maintains the neurons at 23 °C for the entire duration of the sample preparation. The visual appearance of the neurons is similar. The viability was assessed by means of trypan blue exclusion method to determine the viability of the neurons. The neurons prepared under the first protocol (cold exposure) and plated on BaF2 reveal a distinct chemical signature and chemical distribution that is different from the other sample preparations described in the paper. Importantly, results for other sample preparation methods, using various substrates and temperature protocols, when compared across the overlapping spectral bandwidth, present normal chemical distribution within the neurons. The unusual chemically specific spatial variation is dominated by a lack of protein and carbohydrates in the center of the neurons and signatures of unraveling DNA are detected. We suggest that cold shock leads to apoptosis of DRGs, followed by osmotic stress originating from ion gradients across the cell membrane leading to cell lysis.

A calcium prerinse required to form calcium fluoride in plaque from a sodium fluoride rinse.

The purpose of this study is to determine whether a calcium (Ca) prerinse used before a 228 µg/g (ppm) fluoride (F) rinse would induce the formation of 'calcium fluoride-like' (CaF2-like) deposits in human dental plaque. Sixty minutes after the use of the Ca prerinse/F rinse, plaque samples were collected from 10 volunteers, homogenized, and split into 2 aliquots. The plaque mass from one aliquot was then extracted with a 'plaque-like' solution that extracted all the CaF2-like deposits. The total F in both aliquots was then determined and compared. The results demonstrated that, as in previous studies, the Ca prerinse induced large increases in plaque fluid and total plaque F. However, unlike previous results without the Ca prerinse, 30% of the plaque F deposits were CaF2 or CaF2-like. Given that maintaining an elevated F concentration in the vicinity of a developing lesion may play an important role in the cariostatic effect of this ion, and the potential advantages of CaF2-like deposits as an F source, these results suggest that a Ca prerinse may increase the cariostatic effect of topical agents.

Influence of Er,Cr:YSGG laser on CaF2 -like products formation because of professional acidulated fluoride or to domestic dentifrice application.

This study evaluated the synergy of professional acidulated fluoride gel (APF) or fluoridated dentifrice application combined with Er,Cr:YSGG laser irradiation on the formation of CaF2 -like products (CaF2 ), in vitro. Thus, 272 bovine enamel slabs were randomly distributed among eight groups: G1: untreated enamel; G2: treated with fluoridated dentifrice (NaF, 1,100 µgF/g); G3: treated with acidulated phosphate fluoride gel (APF, 1.23% F(-) ); G4: irradiated with Er,Cr:YSGG laser at 8.5 J/cm(2) ; G5 and G6: combination of pre-irradiation with Er,Cr:YSGG followed by dentifrice or APF application, respectively; G7: combination of dentifrice application followed by Er,Cr:YSGG irradiation; G8: combination of APF application followed by Er,Cr:YSGG irradiation. After treatments, samples were evaluated by scanning electron microscopy, and the content of CaF2 was determined by an ion specific electrode. Both APF and dentifrice application promoted the formation of CaF2 on enamel, whereas Er,Cr:YSGG irradiation promoted an increase of roughness of the enamel, increasing the surface area. Laser irradiation before fluoridated products increased the content of CaF2 formed when compared to groups that APF or dentifrice were applied isolated. However, the content of CaF2 formed when irradiation was performed after APF or dentifrice was not statically significant when compared to the control groups. In conclusion, Er,Cr:YSGG laser increases the formation of CaF2 on enamel when the irradiation is performed before the application of APF or dentifrice. The association of laser with APF is most promissory for caries prevention because of the higher concentration of CaF2 formation and also the chemical changes promoted by laser irradiation demonstrated in literature.

Fluorides - mode of action and recommendations for use.

Various authors have shown that the caries decline in the industrialized countries during recent decades is based on the use of fluorides, of which local fluoride application in the form of fluoridated toothpastes is of primary importance. The caries-protective potential of fluorapatite is quite low; in contrast, dissolved fluorides in the vicinity of enamel are effective both in promoting remineralization and inhibiting demineralization. Considering the fact that the caries decline occurred at the same time that local fluoridation measures became widely used, the conclusion seems justified that regular application of F⁻ can inhibit caries.

Oral fluoride reservoirs and the prevention of dental caries.

Current models for increasing the anti-caries effects of fluoride (F) agents emphasize the importance of maintaining a cariostatic concentration of F in oral fluids. The concentration of F in oral fluids is maintained by the release of this ion from bioavailable reservoirs on the teeth, oral mucosa and - most importantly, because of its association with the caries process - dental plaque. Oral F reservoirs appear to be of two types: (1) mineral reservoirs, in particular calcium fluoride or phosphate-contaminated 'calcium-fluoride-like' deposits; (2) biological reservoirs, in particular (with regard to dental plaque) F held to bacteria or bacterial fragments via calcium-fluoride bonds. The fact that all these reservoirs are mediated by calcium implies that their formation is limited by the low concentration of calcium in oral fluids. By using novel procedures which overcome this limitation, the formation of these F reservoirs after topical F application can be greatly increased. Although these increases are associated with substantive increases in salivary and plaque fluid F, and hence a potential increase in cariostatic effect, it is unclear if such changes are related to the increases in the amount of these reservoirs, or changes in the types of F deposits formed. New techniques have been developed for identifying and quantifying these deposits which should prove useful in developing agents that enhance formation of oral F reservoirs with optimum F release characteristics. Such research offers the prospect of decreasing the F content of topical agents while simultaneously increasing their cariostatic effect.

Fluoride in dental erosion.

Dental erosion develops through chronic exposure to extrinsic/intrinsic acids with a low pH. Enamel erosion is characterized by a centripetal dissolution leaving a small demineralized zone behind. In contrast, erosive demineralization in dentin is more complex as the acid-induced mineral dissolution leads to the exposure of collagenous organic matrix, which hampers ion diffusion and, thus, reduces further progression of the lesion. Topical fluoridation inducing the formation of a protective layer on dental hard tissue, which is composed of CaF(2) (in case of conventional fluorides like amine fluoride or sodium fluoride) or of metal-rich surface precipitates (in case of titanium tetrafluoride or tin-containing fluoride products), appears to be most effective on enamel. In dentin, the preventive effect of fluorides is highly dependent on the presence of the organic matrix. In situ studies have shown a higher protective potential of fluoride in enamel compared to dentin, probably as the organic matrix is affected by enzymatical and chemical degradation as well as by abrasive influences in the clinical situation. There is convincing evidence that fluoride, in general, can strengthen teeth against erosive acid damage, and high-concentration fluoride agents and/or frequent applications are considered potentially effective approaches in preventing dental erosion. The use of tin-containing fluoride products might provide the best approach for effective prevention of dental erosion. Further properly designed in situ or clinical studies are recommended in order to better understand the relative differences in performance of the various fluoride agents and formulations.

New insights into structural alteration of enamel apatite induced by citric acid and sodium fluoride solutions.

Attenuated total reflectance infrared spectroscopy and complementary scanning electron microscopy were applied to analyze the surface structure of enamel apatite exposed to citric acid and to investigate the protective potential of fluorine-containing reagents against citric acid-induced erosion. Enamel and, for comparison, geological hydroxylapatite samples were treated with aqueous solutions of citric acid and sodium fluoride of different concentrations, ranging from 0.01 to 0.5 mol/L for citric acid solutions and from 0.5 to 2.0% for fluoride solutions. The two solutions were applied either simultaneously or consecutively. The citric acid-induced structural modification of apatite increases with the increase in the citric acid concentration and the number of treatments. The application of sodium fluoride alone does not suppress the atomic level changes in apatite exposed to acidic agents. The addition of sodium fluoride to citric acid solutions leads to formation of surface CaF2 and considerably reduces the changes in the apatite P-O-Ca framework. However, the CaF2 globules deposited on the enamel surface seem to be insufficient to prevent the alteration of the apatite structure upon further exposure to acidic agents. No evidence for fluorine-induced recovery of the apatite structure was found.

Fluorosis y deporte. A propósito de un caso / Fluorosis and sport. By the way of a case

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Calcium fluoride uptake by human enamel after use of fluoridated mouthrinses / Incremento de CaF2 sobre o esmalte dental humano após utilização de soluções fluoretadas

O presente estudo teve como objetivo verificar, através da microscopia eletrônica de varredura (MEV), a formação e retenção de fluoreto de cálcio (CaF2) sobre a superfície do esmalte dental humano in situ após tratamento tópico com diferentes soluções fluoretadas disponíveis para bochecho no mercado, durante 5 e 10 minutos. Foram selecionados três produtos: solução fluoretada em pH neutro (Fluordent), solução fluoretada acidulada (Fluorgard) e uma solução fluoretada associada à clorexidina (Duplak). Cinqüenta e seis secções, obtidas de 14 terceiros molares hígidos, foram divididas em dois grupos: grupo A: 5 minutos de tratamento e grupo B: 10 minutos de tratamento. Através da análise dos resultados foi possível observar que as amostras tratadas por 5 min com solução associada à clorexidina demonstraram maior formação do produto semelhante ao CaF2. No grupo tratado por 10 min, as amostras em pH neutro demonstraram maior incremento de CaF2. De qualquer forma pôde-se perceber que o produto reacional foi formado e retido em quantidades significantes em todas as situações. Estes resultados demonstraram que os produtos utilizados neste experimento foram capazes de formar e depositar CaF2 sobre as superfícies do esmalte dental, podendo ser recomendados como coadjuvantes na prevenção e no controle da cárie dentária

Calcium fluoride uptake by human enamel after use of fluoridated mouthrinses.

The aim of this study was to evaluate, using scanning electron microscopy (SEM), the calcium fluoride uptake by human enamel in situ after topical application using three fluoridated mouthrinses: a neutral pH solution (Fluordent), an acidulated solution (Fluorgard) and a solution associated with chlorhexidine (Duplak). Twenty-four samples from 6 third sound human molars were divided into two groups: 5-min treatment and 10-min treatment. In both the 5-min and 10-min treatment samples, those treated with the chlorhexidine-associated solution showed higher formation of CaF2-like material. In the 10-min treatment group, the samples treated with the neutral solution showed higher formation of CaF2. These results indicated that the products used deposited CaF2 on the enamel surface and may be recommended to aid the prevention and control of dental caries.

The cariostatic mechanism of fluoride.

The benefits of using fluoride to prevent caries have been known for many years, but a complete understanding of this mechanism is still being researched. The fluoride concentration in the apatitic structure of enamel does not have as significant an effect on reducing caries as a continuous presence of fluoride in the plaque liquid. Concentrated, topical fluoride agents (such as in toothpaste, fluoride mouth rinses, gels, or varnishes) have a different mechanism of fluoride protection than low-concentration applications (such as fluoridated water). In initial caries lesions and plaque, concentrated agents form globules of a calcium fluoride-like material on the enamel surface. This material is fairly insoluble, possibly because it is coated with phosphates or proteins. This mechanism explains how the topical application of a fluoride varnish, two or three times a year, can result in caries reduction.

Fluoride uptake in situ after the use of dental floss with fluoride.

The possibility of having a commercial product such as waxed floss with an additional agent for controlling and preventing caries is promising. The aim of this research was to determine the uptake of fluoride on tooth enamel in situ after the utilization of a dental floss with fluoride incorporated into the wax. One hundred blocks of bovine enamel were artificially demineralized and randomly separated into a Control Group (C) and a Test Group (T). The dental blocks in group T were mounted two-by-two simulating proximal contacts and were fixed into intra-oral lower arch devices. Eight volunteers with a similar salivary flow and buffer capacity wore devices with the enamel blocks for eight days. During this period of time the subjects applied a 25 cm long portion of a mint waxed floss with fluoride (0.15 mgF/m, Oral-B) between the blocks, 3 times a day for 2 minutes after each meal. At the same time, brushing was carried out with a fluoride-free toothpaste. The alkali-soluble fluoride (CaF2) formed on the enamel was extracted using the Caslavska et al. method and measured with the Orion 96-09 electrode-specific and the EA 720 ion analyzer. The results showed that the group that had the most CaF2 on the enamel (median, minimum and maximum in microF/cm2) was Group T (3.00, 2.11 and 4.00), which differed significantly (p < 0.01) from group C (0.26, 0.10 and 0.69). It was concluded that fluoride uptake on enamel was 11.54 times higher after use of dental floss with fluoride in this study.

Determination of fluoride from fluoride-releasing elastomeric ligature ties.

Unaesthetic white spot lesions or larger unsightly areas of decalcification around orthodontic brackets remain a significant problem during fixed appliance treatment. This study determined the in vitro fluoride release from 200 fluoride-containing elastomeric ligature ties. With the potentiometric analytic method, the fluoride release was determined in distilled water, for 10 groups of 20 elastomerics, representing the clinical usage in a patient. Readings were taken every 24 hours for 5 days and then every second week for 6 months. The data were analyzed with the Wilcoxon matched pairs signed ranks test. Fluor-I-Ties (Ortho Arch Company Inc., Hoffman Estates, III.) released significant amounts of fluoride compared with the control readings. The fluoride release was characterized by an initial burst of fluoride during the first day and second day, followed by a logarithmic decrease. By the end of the second week 88% of the total fluoride had been leached from the elastomerics, but adequate magnitudes of fluoride were released over the remainder of the test period to aid theoretically in the prevention of demineralization and enhance remineralization of enamel through calcium fluoride and fluorapatite formation. For optimum clinical benefit, Fluor-I-Ties should be replaced monthly. Future prospective longitudinal clinical studies are indicated.

Formation of phosphate-containing calcium fluoride at the expense of enamel, hydroxyapatite and fluorapatite.

During the caries process complex reactions involving calcium, phosphate, hydrogen and fluoride ions as main species take place. In this study the precipitation and dissolution reactions occurring in suspensions of enamel, hydroxyapatite (HAP) and fluorapatite (FAP) on addition of fluoride were investigated under well-defined conditions. pH and pF were monitored; calcium and phosphate concentrations were measured at selected times; the solid phases were examined by infra-red, X-ray diffraction and transmission electron microscopy. Precipitation of phosphate-containing calcium fluoride crystals, CaF2(P), can cause severe reduction in the calcium ion concentration and release of hydrogen ions from the precipitated phosphate. These reactions result in considerable dissolution of enamel, HAP and even of FAP. More of the added mineral dissolves with 50 mmol/l fluoride than with 10 mmol/l fluoride, mainly due to the greater reduction in calcium ion concentration. This work shows that phosphate-containing calcium fluoride is most likely an important compound to be considered in the caries process.

Professional topical fluoride applications--clinical efficacy and mechanism of action.

All currently used topical fluoride agents deposit soluble fluoride as calcium fluoride on enamel or in lesions. Calcium fluoride serves as a source of fluoride for the formation of fluorapatite. The latter phase is formed when pH drops in plaque, not during topical application. The potential for calcium fluoride formation should probably be increased in topical fluoride agents. In countries with low caries prevalence, the clinical recommendations for topical fluoride need to be reconsidered. Toothpaste is the basic fluoride regimen recommended for everybody. The need for additional fluoride supplementation depends on caries activity. There is no distinct difference in the caries-preventive effects of concentrated fluoride solutions, gels, or varnishes. Thus, the choice of method depends on costs, convenience, patient acceptance, and safety. The use of fluoride varnishes has proven to be a feasible and safe method of fluoride application. With fluoride varnishes, the amounts of fluoride exposure can be better controlled, and less chair-time is required compared with conventional solutions and gels. No dose-response effect to concentrated fluoride agents is apparent, and the benefit of frequent application is not clearly established. In individuals with the most severe cariogenic challenge, combinations of fluoride and antimicrobials may give better clinical effects than fluoride alone.

Experiments on the initiation of calcium fluoride formation with reference to the solubility of dental enamel and brushite.

As calcium fluoride formation following topical application of fluoride may be responsible for at least some of the caries-reducing effect of fluoride, the concentration of fluoride necessary to induce its formation were examined. The aim was to determine the degree of supersaturation with respect to calcium fluoride necessary for inducing its spontaneous precipitation, with close reference to its possible formation and retention on dental hard tissue when topical fluoride solutions are used clinically. Powdered enamel or brushite were suspended for 4 h in aqueous solutions buffered at pH 7.2 and 5.0. After the equilibration, ion concentrations were determined and degrees of saturation with respect to apatite and brushite were calculated. In aqueous solutions at pH 7.2 and 5.0 with similar concentrations as those found in the equilibrated suspensions the fluoride concentration was adjusted to from 5 to 500 parts/10(6). After 2 h of gentle agitation the supernatant was analysed, the precipitate (if any) isolated and examined by X-ray diffraction. Initiation of spontaneous calcium fluoride formation required a calcium fluoride ion-activity product of 10(-7.6) or more, 300 parts/10(6) fluoride were necessary to initiate calcium fluoride formation in neutral solution saturated with enamel, and the increased solubility of enamel apatite at low pH allowed calcium fluoride formation from solutions with as low as 100 parts/10(6) fluoride. When phosphate was present in the solution a competing apatite formation could mask the calcium fluoride formation. In neutral solutions saturated with respect to brushite, spontaneous fluorapatite formation was initiated by 100 pats/10(6) fluoride. With 200 parts/10(6) fluoride a further competing formation of calcium fluoride occurred.

Calcium fluoride formation in enamel from semi- or low-concentrated F agents in vitro.

The formation of CaF2 was measured on sound enamel and in artificial, standardized (acidified gelatin, pH 4.5) caries-like enamel lesions after exposure to: (a) dentifrice/saliva slurries adjusted to relevant F-concentrations of approximately 100 or 8 ppm by appropriate addition of 1,000 ppm F NaF or Na monofluorophosphate (MFP) dentifrice, respectively, or (b) a mixture of saliva with a 0.2% NaF solution obtained by a usual 1-min rinse procedure or an aqueous solution of 0.2% NaF. CaF2 was determined after extraction with KOH and fluoride analysis by gas chromatography. Only negligible amounts of CaF2 were produced on sound enamel ranging from (mean +/- SEM) 0.76 +/- 0.14 micrograms F/cm2 with the 0.2% NaF solution to as little as 0.04 +/- 0.06 with the MFP dentifrice slurry. In caries-like enamel lesions, the CaF2 production with the 0.2% NaF solution/saliva mixture corresponded to 3.7 +/- 0.4 micrograms F/cm2 and corresponding amounts obtained with the dentifrice/saliva slurries were 1.5 +/- 0.19 micrograms F/cm2 with NaF, but only 0.19 +/- 0.04 with MFP. It was suggested that the deposition of CaF2 in the micropores of early lesions can be expected to be an important mechanism with F rinses, probably to some extent with NaF dentifrices, but barely with MFP dentifrices. The formation of CaF2 on sound enamel is unlikely to play a significant role in the caries-reducing effect of F rinses and F dentifrices.