Ultrasensitive Poly(boric acid) Hydrogel-Coated Quartz Crystal Microbalance Sensor by Using UV Pressing-Assisted Polymerization for Saliva Glucose Monitoring.

Quartz crystal microbalance (QCM) has attracted extensive attention in the field of biological analysis and detection because of its high sensitivity, fast response, real-time measurement, good operability, and low-cost production. However, to detect the trace amounts of small molecules, such as low-concentration saliva glucose under physiological conditions, is still a major challenge. Herein, the surface of a QCM chip was coated with a poly(boric acid)-based hydrogel using UV pressing-assisted polymerization to obtain a simple device for glucose detection. The designed QCM sensor shows a record-low detection limit of glucose (3 mg/L at pH 7.5), which is ∼30 times lower than that of sensors fabricated by conventional surface initiation-spin coating. The outperformance of the poly(boric acid) hydrogel-coated QCM sensor is probably due to the uniform and compact microstructure, as well as the presence of sufficient glucose-binding sites resulting from the hydrogel coating generated by UV pressing-assisted polymerization. This method provides an important solution to detect the trace amounts of small organic molecules or ions and has the potential to push forward the practical applications of QCM sensors.

Cellulose cone tip as a sorbent material for multiphase electrical field-assisted extraction of cocaine from saliva and determination by LC-MS/MS.

A porous and hydrophilic sorbent material was used in an extraction system, assisted by electric fields, for the extraction of cocaine in saliva and subsequent determination by ultra-high-performance liquid chromatography associated with sequential triple quadrupole mass spectrometry (UHPLC-MS/MS). The cellulose-based material was characterized by scanning electron microscopy, infrared spectroscopy, thermogravimetric analysis, and X-ray diffraction. The time and voltage variables applied in the extraction process were investigated through a Doehlert experimental design, and with the best conditions found (35min and 300 V) some validation parameters were evaluated. The established working range was 1-100 µg L-1 (R2 > 0.99), and the detection and quantification limits determined were 0.3 and 0.8 µg L-1, respectively. Recoveries from 80 to 115% and coefficient of variation ≤15 and 16% for intra-day and inter-day assays, respectively, were obtained for sample concentrations of LOQ, 5, 25, and 75 µg L-1, indicating satisfactory accuracy and precision for the proposed method. In addition, the method presented no matrix effect, and the extraction efficiency was between 56 and 70%. The results showed that the material used has adequate physicochemical characteristics and can be applied as a sorbent and electrolyte support in multiphase extractions using electric fields.

Commercial Porcine Gastric Mucin Preparations, also Used as Artificial Saliva, are a Rich Source for the Lectin TFF2: In Vitro Binding Studies.

Mucous gels (mucus) cover internal body surfaces. The secretory mucins MUC5AC and MUC6 and the protective peptide TFF2 are characteristic constituents of gastric mucus; TFF2 is co-secreted with MUC6. Herein, we investigated two commercial mucin preparations by FPLC and proteomics, because they are model systems for studying the rheology of gastric mucins. One preparation is also used as a saliva substitute, for example, after radiation therapy. We show that both preparations contain TFF2 (≈0.6 to 1.1 %, w/w). The majority of TFF2 is strongly bound noncovalently to mucin in a manner that is resistant to boiling in SDS. First overlay assays with 125 I-labeled porcine TFF2 revealed that mucin binding is modulated by Ca2+ and can be blocked by the lectin GSA-II and the antibody HIK1083, both recognizing the peripheral GlcNAcα1→4Galß1→R moiety of MUC6. TFF2 binding was also inhibited in the presence of Me-ß-Gal but less so by the α anomer. TFF2 may play a role in the oligomerization and secretion of MUC6, the rheology of gastric mucus, and the adherence of gastric microbiota. TFF2 in artificial saliva may be of benefit. TFF2 might also interact with the sugar moiety of various receptors.

Laboratory investigations into the potential anticaries efficacy of fluoride varnishes.

PURPOSE: The purpose of this study was to investigate the potential anticaries efficacy of fluoride varnishes (FVs) by studying their ability to reharden and deliver fluoride to carious lesions and to release fluoride into saliva. METHODS: Enamel carious lesions were created and allocated to 24 groups (11 FVs with two FV incubation times and two control groups) based on Knoop microhardness test values. FVs were applied to lesions, which were incubated in artificial saliva for two or six hours, with saliva being renewed hourly. FV was removed and lesions were remineralized in artificial saliva for 22 hours. Microhardness was measured and enamel fluoride uptake (EFU) was determined. Saliva samples (six-hour groups) were analyzed to determine fluoride release characteristics. Data were analyzed using analysis of variance. RESULTS: FVs differed considerably in their ability to reharden and deliver fluoride to carious lesions and in their fluoride release characteristics. Little consistency was found between investigated study variables for virtually all tested FVs. For example, a particular FV showed the highest EFU and fluoride release values but the lowest rehardening value. A longer FV contact time led to increased EFU for five of the 11 FVs. Some FVs delivered more fluoride to lesions in two hours than others did in six hours. CONCLUSION: Fluoride varnishes differ greatly in their in vitro anticaries efficacy.

Measurement of salivary cortisol by a chemiluminescent organic-based immunosensor.

A highly sensitive chemiluminescent immunoassay (CLIA) using a sensitive organic photodetector was developed to detect human cortisol, an important biomarker for stress-related diseases. The developed CLIA was performed onto gold-coated glass chips, on which anti-cortisol antibodies were immobilised and chemiluminescent horseradish peroxidase-luminol-peroxide reactions were generated. Using cortisol-spiked artificial saliva samples, the CLIA biosensor showed a linear range of detection between 0.1 ng/mL and 175 ng/mL and a detection limit of 80 pg/mL. The sensor response was highly specific to cortisol and did not vary significantly between assays. The results indicate the potential clinical application of the CLIA sensor. Furthermore, the simple layered structure of the organic photodetector may encourage the realisation of integrated optical biosensors for point-of-use measurement of salivary cortisol levels.

An in vitro analysis of disintegration times of different formulations of olanzapine orodispersible tablet: a preliminary report.

BACKGROUND: Orodispersible tablets (ODTs) are tablet or wafer forms of medication that disintegrate in the mouth, aided only by saliva. ODTs rely on different fast dissolve/disintegration manufacturing technologies. OBJECTIVES: Disintegration time differences for several olanzapine ODT forms were investigated. Risperdal M-Tab(®) was included as a non-olanzapine ODT comparator. RESEARCH DESIGN AND METHODS: Eleven olanzapine ODT examples and orodispersible risperidone strengths were evaluated in vitro for formulation composition, manufacturing method, disintegration and dissolution characteristics, and formulation differences in comparison with freeze dried Zydis(®) ODT. Automated dissolution test equipment captured ODT dissolution rates by measuring real-time release of active ingredient. A high-speed video camera was used to capture tablet disintegration times in warm simulated saliva. MAIN OUTCOME MEASURE: The main outcome measure was the disintegration and dissolution characteristics of the ODT formulations. RESULTS: The ODT manufacturing method was associated with time to disintegrate; the fastest were freeze dried tablets, followed by soft compressed tablets and then hard/dense tablets. Olanzapine Zydis(®) was the only ODT that completely disintegrated in less than 4 s for all strengths (5, 10, 15, and 20 mg), followed by 5-mg Prolanz FAST(®) (12 s) and then risperidone ODT 4 mg (40 s). Reasons for slow dissolution of the olanzapine generics may include low product potency, excipient binding, excipient solubility, active ingredient particle size and incomplete disintegration. CONCLUSIONS: Differences in the formulation and manufacturing process of olanzapine ODTs appear to have a strong influence on the disintegration time of the active compound; differences that may potentially impact their use in clinical practice.

Migration of fluoride ions from the permanent teeth into saliva in children with glass ionomer cement restorations: an in vitro study.

BACKGROUND/AIM: Glass ionomer cements (GIC) belong to the group of polycarboxyl cements, and one of the principal characteristics of these materials is their anticariogenic potential of fluorine release into saliva and enamel-dentin substance. The aim of this study was to examine the content of released fluorine from GIC restorations (Fuji IX, GC, Japan) of young permanent teeth in the medium of artificial saliva and similar releases in the same medium by the restorations of these teeth treated with a low concentration fluoride solution. METHODS: We examined 12 premolars extracted from orthodontic reasons. The GIC restored teeth were divided into the group treated daily with low concentration fluoride solution (334 ppm) and the control, not treated group. The samples of artificial saliva were analyzed for fluorine ion content using an ion selective electrode. RESULTS: Our comparative analysis of the mean values using the Student's t-test demonstrated a statistically significant difference in fluorine ion concentration in artificial saliva of fluoridated and non-fluoridated teeth with GIC fillings after 14 and 21 days (p < 0.05), while the difference detected after 7 days was with no statistical significance. CONCLUSION: The results of this in vitro study indicated that low-concentration fluoride solutions could serve to refluoridate GIC fillings and contribute to an increased fluorine content in saliva. The process of refluoridation of GIC fillings should be advised 2-3 weeks after the restoration, since the release of fluorine from GIC fillings diminishes in time.

Effect of sintering atmosphere on properties of porous stainless steel for biomedical applications.

This study discusses manufacturing of metallic biomaterials by means of powder metallurgy with consideration for their unquestionable advantages, i.e. opportunities of obtaining materials with controllable porosity. The paper focuses on properties of 316 L stainless steel obtained using the method of powder metallurgy with respect to compacting pressure and sintering atmosphere. All the specimens were compacted at 700, 400 and 225 MPa, and sintered at 1250 °C. In order to analyze the sintering atmosphere, three different media were used: dissociated ammonia, hydrogen and vacuum. The study covered sintering density, porosity, microstructure analysis and corrosion resistance. The proposed method of powder metallurgy allowed for obtaining materials with predictable size and distribution of pores, depending on the parameters of sinter preparation (compaction force, sinter atmosphere). High corrosion resistance of the materials (sintering in the atmosphere of hydrogen and in vacuum) and high porosity in the sinters studied offer opportunities for using them for medical purposes.

Release of metal ions from orthodontic appliances: an in vitro study.

In this paper, we report the results of an in vitro experiment on the release of metal ions from orthodontic appliances composed of alloys containing iron, chromium, nickel, silicon, and molybdenum into artificial saliva. The concentrations of magnesium, aluminum, silicon, phosphorus, sulfur, potassium, calcium, titanium, vanadium, manganese, iron, cobalt, copper, zinc, nickel, and chromium were significantly higher in artificial saliva in which metal brackets, bands, and wires used in orthodontics were incubated. In relation to the maximum acceptable concentrations of metal ions in drinking water and to recommended daily doses, two elements of concern were nickel (573 vs. 15 µg/l in the controls) and chromium (101 vs. 8 µg/l in the controls). Three ion release coefficients were defined: α, a dimensionless multiplication factor; ß, the difference in concentrations (in micrograms per liter); and γ, the ion release coefficient (in percent). The elevated levels of metals in saliva are thought to occur by corrosion of the chemical elements in the alloys or welding materials. The concentrations of some groups of dissolved elements appear to be interrelated.

Salivary enzymes and exhaled air affect Streptococcus salivarius growth and physiological state in complemented artificial saliva.

To better understand the phenomena governing the establishment of the oral bacterium Streptococcus salivarius in the mouth, the effect of some environmental factors has been studied in complemented artificial saliva, under oral pH and temperature conditions. Three salivary enzymes at physiological concentrations were tested: peroxidase, lysozyme and amylase, as well as injection of exhaled air. Injection of air containing 5% CO2 and 16% O2 induced a deleterious effect on S. salivarius K12, mainly by increasing redox potential. Addition of lysozyme slightly affected the physiological state of S. salivarius by altering membrane integrity. In contrast, peroxidase was not detrimental as it made it possible to decrease the redox potential. The addition of amylase reduced the specific growth rate of S. salivarius by formation of a complex with amylase and mucins, but led to high final biomass, as a result of enzymatic degradation of some nutrients. Finally, this work demonstrated that salivary enzymes had a slight impact on S. salivarius behaviour. It can thus be concluded that this bacterium was well adapted to in-mouth conditions, as it was able to resist certain salivary enzymes, even if tolerance to expired air was affected, as a result of an increased redox potential.

Improved artificial saliva for studying the cariogenic effect of carbohydrates.

Saliva is a complex fluid that possesses many important functions regarding oral health. Many in vitro studies require relatively large quantities of saliva. While natural saliva would be the material of choice, it is difficult to obtain in sufficient quantities and varies in composition. Substitutes mimicking the physicochemical properties of saliva have been developed, but these are not appropriate to study the growth of mutans streptococci. Brain Heart Infusion (BHI) has been commonly used for this, but this medium is richer in nutrients than saliva. We therefore developed artificial saliva (AS) with nutrient levels resembling those in natural saliva as a substitute for natural human saliva (HS) to study the influence of different carbon sources on mutans streptococci growth. Growth of a wild-type Streptococcus mutans strain and S. mutans ATCC 15175 in BHI, HS, and AS was monitored anaerobically. Growth of S. mutans in the modified AS was very similar to the growth in HS, both in the absence and presence of different carbon sources. We therefore conclude that the developed AS is suitable for in vitro tests on S. mutans growth.

Effect of nano-topographical features of Ti/TiO(2) electrode surface on cell response and electrochemical stability in artificial saliva.

The paper is a new approach which aims to evaluate the relation between surface aspects (wettability and roughness) of materials based on titanium with native passive TiO(2) as untreated samples and TiO(2) nanotubes as treated discs respectively, their electrochemical stability in artificial saliva, and fibroblast cell behavior. Ti/TiO(2) modified electrodes as nanotubes with 120 nm as diameter were obtained using an electrochemical method as anodizing and surface analysis as SEM, AFM and contact angle measurements were performed to obtain topographical features and wettability. The TiO(2) nanotube structured oxide films electrochemical growth increases the stability of titanium surfaces. The electrochemical behavior of the Ti/TiO(2) nanotube surface was evaluated by corrosion parameters obtained from Tafel plots and electrical parameters for proposed circuits from electrochemical impedance spectroscopy were analyzed. The cell results indicated a slight preference in terms of cell survival and adhesion for nanostructure TiO(2) with a more hydrophilic character and the electrochemical data revealed that such features are connected with better stability in artificial saliva. The roughness seems to be not conclusive for this case.

Aluminium and fluoride release into artificial saliva from dental restoratives placed in teeth.

This study examined the release of aluminium and fluoride from restorative materials placed in either deciduous or young permanent immature teeth stored in artificial saliva for 1 month. Cavities were prepared in extracted teeth, then filled with a fluoride releasing restorative (glass-ionomer, compomer or composite resin), with and without conditioning as appropriate. The teeth were then stored in artificial saliva for 1 month, after which the amount of aluminium and fluoride released was determined spectrophotometrically. With all materials tested, both aluminium and fluoride were released in all cases. Young immature teeth were associated with lower level of ion release which was attributed to the absorption of ions by the enamel. However, unconditioned samples were usually associated with similar ion release to conditioned ones, suggesting that the loss of mineral phase on conditioning has only a marginal effect on the capacity for ion uptake. The ratio of aluminium to fluoride released varied with the type of tooth, deciduous conditioned teeth generally absorbing proportionately less aluminium than young immature teeth. The overall conclusion is that interaction with ions released by restorative materials is influenced by type of tooth.

Long-term assessment of the implant titanium material--artificial saliva interface.

This paper studies the long-term (20,000 exposure hours) behavior of titanium and Ti-5Al-4V alloy-Carter-Brugirard saliva interface and the short-term (500 exposure hours) resistance of titanium and Ti-5Al-4V alloy-Tani&Zucchi saliva interface. Potentiodynamic polarization method was applied for the determination of the main electrochemical parameters. Linear polarization measurements for to obtain the corrosion rates were used. Monitoring of the open circuit potentials (E(oc)) for long-term have permitted to calculate the potential gradients due to the pH, DeltaE(oc)(pH) and to the saliva composition DeltaE(oc)(c) changes which can appear "in vivo" conditions and can generate local corrosion. Atomic force microscopy (AFM) has analyzed the surface roughness. Ion release was studied by atomic absorption spectroscopy (AAS). In Carter-Brugirard saliva both titanium and Ti-5Al-4V alloy present very stable passive films, long-term stability, "very good" resistance, low values of the open circuit potential gradients, which cannot generate local corrosion. In Tani&Zucchi artificial saliva, pitting corrosion and noble pitting protection potentials (which cannot be reached in oral cavity) were registered; titanium ion release is very low; surface roughness increase in time and in the presence of the fluoride ions, denoting some increase in the anodic activity.

Influence of 0.05% sodium fluoride solutions on microhardness of resin-modified glass ionomer cements.

This study aimed to evaluate the influence of fluoride-containing mouthrinse solutions (Fluorgard and Oral B) on the superficial microhardness of two resin-modified glass ionomer cements (Vitremer and Fuji II LC). Fifteen discs-shaped specimens of each glass ionomer cement (Ø10 mm; 2 mm thick) were prepared, thereby forming two groups. After 24-hour storage in artificial saliva, the microhardness was measure and the data were recorded. Next, each group was divided into three subgroups (n = 5), according to the solution to be immersed in. Control specimens were kept in artificial saliva along the whole experiment. The test specimens were kept in mouthrinse solution for 30 days. Vickers surface microhardness was analyzed at predetermined evaluation periods: 24 h, 48 h, 7, 14, 21 and 30 days after specimens' preparation. Data were subjected to three-way ANOVA and to Tukey test (p<0.05). A better behavior of Fuji II LC was observed and Fluorgard affected most the characteristics of the tested materials. It may be concluded that fluoride-containing solutions influenced the tested characteristics of materials, mainly of Vitremer.

Construction and evaluation of an inexpensive device that simulates oral clearance.

The clearance pattern of a specific substance is very important to estimate its oral availability. Devices or models that simulate clearance in the mouth are important to study the effects and retention time of foods and drugs. This report describes an efficient device which was assembled with low-cost materials in our laboratory and that can be used to study the clearance of cariogenic substrates, mouthwashes, programmed-release drugs as well as adsorption of drugs to enamel. The device can have up to three chambers with varying minimum and maximum volumes that can be eluted simultaneously at different flows. The simulated swallowed volumes are adjustable and the ratio between the maximum and minimum volumes can be programmed. We also present the results of an evaluation study using the device to determine the clearance of fluoride from a fluoride-containing mouthwash, the clearance of a 1% glucose solution and the programmed release of fluoride from a methacrylate bioadhesive using artificial saliva as eluent.

Oral bioavailability of lead and arsenic from a NIST standard reference soil material.

The oral bioavailability of soil contaminants is measured using in vitro or in vivo techniques. Current efforts in our laboratory are focused on the comparisons of in vitro methods for bioavailability estimation with the presently employed in vivo techniques, such as animal models. We present a comparison of two techniques for oral bioavailability estimation: in vitro dissolution and in vivo rat feeding using a standard reference soil. Lead (Pb) and arsenic (As) were chosen because of the range of concentration in this soil as well as the large historical database of bioavailability values for these metals. Metal solubility was measured using a sequential soil extraction in synthetic analogues of human saliva, gastric and intestinal fluids. The soluble metal was defined as the bioaccessible fraction. Oral bioavailability of Pb and As was measured in Sprague Dawley rats by determining metal levels in the major organs and urine, feces, and blood at 1-, 2-, and 3-day time points. Extractions to determine bioaccessibility yielded a gastric component of 76.1% and 69.4% for Pb and As, respectively, and intestinal components were 10.7% and 65.9%. The oral bioavailability of the standard reference soil was 0.7% and 37.8% for Pb and As, respectively. Bioaccessibility was greater than bioavailability for both metals in both gastrointestinal compartments. Although Pb had the highest soil concentration of the selected metals, it was the least bioavailable, while As was highly available in both the in vitro and in vivo method. These types of data allow for an in vitro-in vivo comparison of a soil whose metal concentrations have been certified and validated.

An in vitro system for the analysis of changes in depth distribution of diffusates in bacterial films.

No suitable technique exists which allows simultaneous analysis of time- and depth-dependent concentrations of all components of interest in single samples of intercellular fluid from dental plaque biofilms. We have developed an in vitro model which allows detailed study of these interactions by analysis of intercellular fluid and pH measurement at the film base. Compact, defined-depth films of Streptococcus mutans were formed in a micrometer-controlled, variable-depth well and bathed in synthetic saliva. Films exposed to synthetic saliva containing glucose for 2 min followed by clearance with glucose-free synthetic saliva gave typical 'Stephan-type' pH profiles. Intercellular fluid isolated from successive 200-microm-thick sections of 600-microm-deep films was analysed by ion chromatography. A concentration gradient of lactate, falling with depth, was measured. The experimental system described here, the first of its type to be described, can easily be used to analyse pH changes and the depth-dependent distribution of diffusates in a model bacterial film. Although this bacterial film is far removed from a natural biofilm, the apparatus has potential for the study of grown biofilms and is an important advance towards position-dependence analysis of diffusates in biofilms.

Metallic ion release in artificial saliva of titanium oral implants coupled with different metal superstructures.

In this work the metallic ion release in oral implants with superstructures of different metals and alloys used in clinical dentistry has been determined. This study has been realized in a saliva environment at 37 degrees C. The measurements of the ion release were carried out by means of the Inductively Coupled Plasma Mass Spectrometry technique. The titanium oral implant coupled with a chromium-nickel alloy releases a high quantity of ions and the implant coupled with the titanium superstructure presents a low value of ion release.

Effect of nutrients on defined bacterial plaques and Streptococcus mutans C67-1 implantation in a model mouth.

Actinomyces viscosus WVU 627, Streptococcus oralis LPA-1 and Veillonella dispar OMZ 193 were cocultured on teeth in a model mouth for 66 h. Synthetic saliva containing bovine salivary glycoprotein supported bacterial growth, although the delivery of an intermittent nutrient supplement, containing 1% (w/v) glucose or sucrose, gave greater bacterial cell and viable counts. When Streptococcus mutans C67-1 was super-inoculated onto 24-hour mixed plaques, it became established under all regimens, but there was pronounced colonization resistance. With saliva only, the proportion of S. mutans at 66 h was less than 0.5% of the total cultivable microflora. When a glucose supplement was delivered for 1 h every 6 h, S. mutans attained a final proportion of 2.4%. With sucrose, both S. mutans C67-1 and its non-cariogenic glucan-deficient mutant, C67-25, attained similar proportions of 15-20%. These experiments indicate how this model can be used to study the factors influencing colonizing ability and microbial interactions in biofilms under controlled conditions.