Diffusion of sugars and carboxylic acids through human dental plaque in vitro

Diffusion through plaque was measured with an apparatus consisting of 3 identical diffusion cells contained in an aluminium alloy block thermostatically controlled at 35 °C. In two of the cells, measurements were made of diffusion through a composite membrane consisting of 18 h dental plaque contained between bacteriological grade filters and support screens. In the third cell, diffusion occurred through a reference membrane from which the bulk of the plaque was absent. Subtraction of the diffusion resistance of this reference membrane allowed calculation of apparent tracer diffusion coefficients in the bulk of the plaque alone. Diffusion of ¹⁴C-labelled sugars and carboxylic acids was compared with that of tritiated water using dual-channel liquid scintillation counting. The diffusion coefficient of tritiated water in the plaque samples varied from 0.81 to 1.02 × 10^?9 m²s^?1 depending on the wet wt used, with coefficients of variation of ~ 15 per cent. This is between $\text{1}\text{4}$ and $\text{1}\text{3}$ of the value in aqueous solution. Diffusion coefficients relative to that of tritiated water in plaque ranged from 0.154 ± 0.014 for sucrose to 0.363 ± 0.029 for acetate. Permselectivity was low, and diffusion coefficients of I he test molecules were all between $\text{1}\text{4}$ and $\text{1}\text{5}$ of their values in free aqueous solution.     

Influence of gender on the metabolism of alcohols in human saliva in vitro

Objective

The aim of this study was to investigate possible gender differences in salivary metabolism of two alcohols, ethanol and 2-propanol. Ethanol and its metabolite acetaldehyde may play important roles in tumour development, especially in the upper digestive tract. 2-Propanol is tested to elucidate our previous findings, where gender-specific differences in salivary acetone levels were seen after exposure to this alcohol.

Design

Saliva was collected from 25 females and 22 males for in vitro exposure to 2-propanol. In the experiments with ethanol, saliva samples were collected from 17 females and 18 males. The saliva was exposed in vitro to 2-propanol or ethanol. The metabolites acetone, derived from 2-propanol, and acetaldehyde, derived from ethanol, together with the maternal substance were analysed by headspace gas chromatography.

Results

No differences related to gender, age, medication or tobacco intake in the acetone concentration in the saliva samples were found. Gender, age or tobacco intake did not result in difference in the production of acetaldehyde in saliva. However, in the pre-exposure samples the men had a significantly higher concentration of acetaldehyde compared to the women (p = 0.04). Also, there was a tendency (p = 0.05) to higher concentrations of acetaldehyde in the samples (at 1 mM ethanol exposure) from subjects who take medications.

Conclusion

No gender difference in the metabolism of 2-propanol and ethanol in human saliva in vitro were found.     

A photochemical method for in vitro evaluation of fluid flow in human dentine

Objective

To evaluate the flow dynamics of dentine fluid using a chemiluminescence method in vitro.

Materials and methods

Horizontally sliced coronal dentine specimens with thicknesses of 1.4, 1.6, 1.8, and 2.0 mm (n = 10 each) were prepared from extracted human third molars. After cleaning with EDTA, a mounted specimen was clamped between 2 acrylic chambers attached to both the occlusal and pulpal sides. The occlusal chamber, which was closed with a glass coverslip, was filled with a chemiluminescent solution (0.02% luminol and 1% sodium hydroxide in water). A trigger solution of 1% hydrogen peroxide and 1% potassium ferricyanide was injected into the pulpal chamber at a constant pressure of 2.5 kPa, and allowed to immediately flow into the patent dentinal tubules. Four consecutive measurements (T1–T4) were performed on each sample by recording the emission of chemiluminescence with a photodetector. The relationship between the crossing time of the liquid through the slice and dentine thickness was examined.

Results

An apparent time delay was detected between the starting points of the trigger solution run and photochemical emission at T1. Dt (Dt, s) values of each thickness group were 13.6 ± 4.25 for 1.4 mm, 18.1 ± 2.38 for 1.6 mm, 28.0 ± 2.46 for 1.8 mm, and 39.2 ± 8.61 for 2.0 mm, respectively. Dt significantly decreased as dentine became thinner towards the pulp chamber (P < 0.001).

Conclusions

The velocity of fluid flow increased both with increasing dentine depth or reduction of remaining dentine thickness.     

The effect of sucrose on plaque and saliva urease levels in vivo

Objective

Dietary sugar exposures induce an immediate drop of the plaque pH. Based on in vitro observations, it was hypothesized that oral bacteria may rapidly respond to this environmental change by increasing the activity or expression of alkali-generating pathways, such as the urease pathway. The objective of this exploratory in vivo study was to determine the short-term effect of a brief sucrose exposure on plaque and saliva urease activity and expression, and to relate this effect to caries experience.

Methods

Urease activity levels were measured in plaque and saliva samples collected from 20 children during fasting conditions and 30 min after rinsing with a sucrose solution. Streptococcus salivarius ureC-specific mRNA in saliva was quantified using real-time RT-PCR. The impact of host-related factors, such as age, gender, sugar consumption, salivary mutans streptococci levels and caries status on urease activity was evaluated.

Results

Plaque urease activity under fasting conditions was higher in subjects with low caries and mutans streptococci levels. This difference was not observed after the sucrose exposure. The response of urease to sucrose in vivo did not depend on caries experience or salivary mutans levels. Significant increase in urease activity of plaque and saliva after exposure to sucrose was observed only in the subjects who had low urease levels at baseline.

Conclusions

The findings of this exploratory study suggest that plaque urease activity may have an important long-term influence in caries development but not during a cariogenic challenge.     

Repairability of dental siloranes in vitro

Objectives

Aim of the study was the investigation of the repairability of a silorane (Filtek Silorane, 3M Espe, Seefeld, Germany) after different surface pretreatments in vitro.

Methods

54 silorane specimens (5 mm × 5 mm × 5 mm) were fabricated and stored in saline solution (24 h/37 °C). Their surface was polished with abrasive paper (600 grit), etched with phosphoric acid (10 s) and rinsed with water (30 s). Repair was performed with a silorane based on one of the 9 treatment protocols (each n = 6): no additional treatment (NAT), silorane primer (P) and silorane bond (B), B only, sandblasting (SB), SB plus P/B, SB plus B, CoJet and silane (CJ), CJ plus P/B, CJ plus B. Whole silorane specimens (5 mm × 5 mm × 10 mm) with no repair served as control. Specimens were sectioned and microtensile bond strength (μTBS) was measured (30 beams per group, surface area approx. 1.2 mm², crosshead-speed 1 mm/min) statistical analysis (ANOVA, Tukey HSD, p < 0.05) was performed.

Results

μTBS of the specimens was significantly influenced by the surface pretreatment (p < 0.001). The highest μTBS was determined for CJ/B and SB/B, which were not significantly different from the control. NAT, SB and CJ benefited from an additional treatment with B (p < 0.01). The additional use of P did not improve μTBS, but was detrimental for the SB and CJ groups (p < 0.05).

Significance

Siloranes can be repaired with either SB or CJ in combination with a silorane bond, the additional use of silorane primer is disadvantageous.     

Validation of enamel erosion in vitro

Objectives

Many tools are available to quantify dental erosion, but each technique has its own inherent disadvantages. This study aims to validate the use of quantitative light-induced fluorescence (QLF) and non-contacting surface profilometry compared to the gold standard transverse microradiography (TMR) for the quantification of enamel erosion in vitro.

Methods

This was an in vitro laboratory based study. 60 bovine incisors were divided into 6 groups of 10. Each tooth's labial surface was completely varnished except for a window of enamel approximately 3 mm × 5 mm. Each was baseline imaged with QLF and non-contacting surface profilometry before being subjected to an erosive solution (pH 3.4) for up to 36 h. The lesions were imaged using non-contacting surface profilometry and QLF, sectioned and analysed with TMR. Correlation coefficients were calculated to assess the validity of the methods of measurement as compared to TMR.

Results

A range of lesion severities resulted. Mineral loss measured as ΔQ (QLF) and step height (profilometry), was recorded and confirmed by TMR. A correlation was found between ΔZ (TMR) and profilometry lesion depth of r = 0.648 (p < 0.001). A poorer correlation was found between ΔZ and ΔQ: r = 0.217 (p = 0.096).

Conclusions

Profilometry lesion depth and ΔZ correlated significantly. Both methods allow for quantification of erosive crater depth. QLF correlated poorly with ΔZ, but is useful for measuring subsurface loss of mineralisation. TMR is valuable but is destructive and can only be used in vitro. Currently only QLF can be used in vivo. Advances in these technologies may allow the development of non-destructive in vivo measurements of mineral loss, combining the positive features of each measurement method.     

Metabolism of testosterone by human healthy and inflamed gingiva in vitro

Testosterone was incubated with homogenate and mitochondrial, microsomal and soluble-fraction preparations of healthy and inflamed gingiva in human subjects of both sexes. In the inflamed preparations, the metabolic activity was higher than in the samples from healthy gingiva. The gingiva contained the following enzymic activities: Δ⁴-5α-steroid hydrogenase, Δ⁴-5β-steroid hydrogenase, and 3α-, 3β- and 17β-hydroxysteroid dehydrogenase, principally in the soluble fraction. Mitochondrial and microsomal preparations were less active. Both the healthy and especially the inflamed samples were able to convert testosterone to 17/gb-hydroxy -5α-androstan-3-one, suggesting that gingiva may be a target tissue for androgens.     

Effects of fluoride on locomotion of human blood leucocytes in vitro

The effect of NaF on the locomotion and chemotaxis of human blood neutrophils and monocytes was studied using two assays: the micropore filter assay and a time-lapse cinematographic assay in which the chemotaxis of cells in response to spores of Candida albicans was filmed. At high concentrations (> 10^?4 M), NaF inhibited locomotion of both cell types, but no inhibition of locomotion of either cell-type was seen in either assay using NaF at ? 10^?4 M, whether or not the cells were responding to a chemotactic source. This was so, even for monocytes incubated for 48 h in the presence of NaF. It is therefore improbable that fluoride, at levels added to drinking water or found in the body fluids of persons drinking fluoridated water, has any deleterious effect on the locomotor capacity of phagocytic cells or on their capacity to detect and home on to chemotactic sources.     

The effect of epigallocatechin-3-gallate (EGCG) on human alveolar bone cells both in vitro and in vivo

Objective

The effects of epigallocatechin-3-gallate (EGCG), a major catechin in green tea, on human and mouse osteoblasts remain controversial. This study investigated the direct effects of EGCG on human alveolar bone-derived cells (hABCs) both in vitro and in vivo.

Design

hABCs which were collected from eight children (aged 7–9 years, seven males and one female) were treated with EGCG at various concentrations (1, 5, 10, 25, and 50 μM), and a proliferation assay, flow cytometric analysis for apoptosis evaluation, migration assay, and in vitro osteogenic differentiation were performed. hABCs that were pretreated with 10 μM EGCG and mixed with calcium phosphate carrier combined with EGCG (0.1, 0.5, or 1.5 mg) in vivo were transplanted into immunodeficient mouse. Histological staining, quantitative gene expressions, and alkaline phosphatase activity were evaluated in the retrieved transplants.

Results

The proliferation and migration were decreased when EGCG was present at over 25 μM. The osteogenic differentiation increased slightly when EGCG was present at up to 10 μM, and clearly decreased for higher concentrations of EGCG. In vivo, the potential for hard-tissue formation was slightly higher for the group with 0.1 mg of EGCG than for the control group, and decreased sharply for higher concentrations of EGCG.

Conclusion

The present observations suggest that EGCG at a low concentration can slightly enhance the osteogenic effect in vivo, whereas at a higher concentration it can prevent the osteogenic differentiation of hABCs both in vitro and in vivo.     

The structure of experimental in vitro lesions around silicate fillings in human teeth

“Secondary caries” produced in 33 extracted human teeth by exposure to acidified gelatin were studied on 60–120 μm thick longitudinal ground sections by polarized light microscopy and microradiography. The silicate material caused a characteristic effect on the cavity walls. The “secondary caries” pattern consisted of an outer lesion and a cavity wall lesion as found previously in corresponding experiments with amalgam fillings. However, with silicate cement the lesions were less demineralized, and outer lesions were less frequent (44.3%), due, it is suggested, to the action of fluoride released from the silicate.     

Contamination of human gingival crevicular fluid by plaque and saliva.

Dental plaque and saliva are both possible contaminants of gingival crevicular fluid (GCF). Plaque samples from 12 sites in four subjects who had allowed plaque to accumulate for 24-48 h were quantified using the Plaque Index and then transferred to filter paper strips for fluid volume determination using the Periotron 6000. The mean volume of fluid for plaque scores of 0 was 0.02 (+/- 0.01) microliter, whereas for plaque scores of 3 the mean volume was 0.15 (+/- 0.07) microliters. In a clinical study, GCF samples, from 19/184 subjects (10.4%) were assessed as contaminated or suspected to be contaminated with saliva, but only 28/1740 strips (1.6%) were placed in these categories of contamination. An assay to confirm salivary contamination was established, using an immunochemical, double-antibody method with sheep anti-human salivary alpha-amylase followed by peroxidase-conjugated rabbit anti-sheep immunoglobulin. The detection threshold of the assay was 7.5 ng of alpha-amylase, which represents approx. 15-25 nl of saliva. The assay was evaluated on 90 GCF samples categorized as 'known' (n = 16), 'suspected' (n = 16), or 'not' contaminated (n = 58) with saliva; 81.25, 50-61.5 and 5.2-8.6%, respectively, were positive for salivary alpha-amylase. In some GCF samples salivary contamination was in excess of 50%. It was concluded that GCF samples seen to be contaminated with saliva should be discarded, whereas samples considered free from contamination may be used with confidence. Samples suspected of contamination may require an alpha-amylase assay before further analysis.     

The adhesion of the yeast Candida albicans to epithelial cells of human origin in vitro

Monolayers of HeLa cells were incubated with Candida suspensions and the number of adherent yeasts per unit area of epithelial cells counted microscopically. Adhesion was proportional to the incubation time and the yeast concentration in the suspension. Preincubation of Candida with sucrose facilitated their adhesion, there being a linear enhancement with increasing sucrose concentrations. The sucrose-facilitated adhesion disappeared when the yeasts were killed prior to incubation in a sucrose-containing medium, suggesting that an extracellular metabolic product could be responsible. On comparison of the adhesion of yeasts to viable and non-viable HeLa cells, greater adhesion to non-viable cells was observed. This experimental system can be used to study parameters influencing candidal adhesion to epithelial surfaces.     

Original and repair bond strength of fiber-reinforced composites in vitro

Objective

A great benefit of FRC technology is that, in case of minor failure events, restorations can be repaired or reinserted. However, various FRC materials are available, that differ in matrix composition and fiber pre-treatment. The aim of this investigation was, therefore, to evaluate original and repair bond strength of FRC materials.

Methods

Five fully pre-impregnated, unidirectional FRCs were selected, one semi-interpenetrating polymer network FRC and four cross-linked-polymer FRCs. The primary endpoint was the evaluation of shear bond strength (SBS) between FRC and composite resin, which was performed by a universal testing machine. For each FRC specimens were divided into control (original SBS, resin to fresh FRC with oxygen inhibition layer (OIL), n = 30) and test groups (repair SBS, resin to FRC after removal of OIL and adhesive infiltration, n = 30).

Results

The cross-linked-polymer FRC GrandTec^® (12.4 ± 5.4 MPa) yielded the highest control SBS, followed by the semi-interpenetrating polymer network FRC (everStick^®, 9.2 ± 3.5 MPa). With everStick^®, repair led to a significant increase in the test SBS (14.6 ± 5.8 MPa, p = 0.01).

Significance

Control SBS was best with GrandTec^® indicating that the material is superior in direct clinical application. Test SBS was significantly increased with everStick^® which points at potential reparability and advantages in semi-direct or indirect fabrication of fiber-reinforced fixed partial dentures.     

Three-dimensional fit of lithium disilicate partial crowns in vitro

Objectives

A novel three-dimensional scanning technique was used to investigate the effects a one-step and a two-step impression methods can have on the three-dimensional fit of ceramic partial crowns.

Methods

An acrylic model of a mandibular first molar was prepared to receive a partial coverage all-ceramic crown (mesio-occlusal-distal inlay preparation with reduction of all cusps and rounded shoulder finish line of buccal wall). Type IV plaster replicates were cast based on one-step single viscosity (OS/SV), one-step dual viscosity (OS/DV), and two-step dual viscosity (TS/DV) impressions. Five partial crowns were fabricated per impression method using hot-pressed lithium disilicate ceramics. Then, preparation and restorations were digitized using a non-contact, white-light scanner featuring self-calibrating optics (overall measurement uncertainty of <5 μm). Data were entered into quality inspection software which superimposed the records (best-fit-algorithm), calculated fit-discrepancies for every pixel, and colour-coded the results to aid visualization. Furthermore, mean quadratic deviations (RMS) were computed and analyzed statistically with a 1-way ANOVA. Scheffé’s procedure was applied for multiple comparisons (α = 0.05).

Results

Mean RMS-values for marginal (internal) surfaces were: OS/SV 70 (20) μm, OS/DV 78 (34) μm, and TS/DV 107 (52) μm. Differences among impression techniques were statistically significant at p = 0.006 (0.001). Qualitatively, occlusal ridges and preparation finish lines were over contoured, whereas inner occlusal boxes and the proximal–occlusal isthmus were under contoured.

Conclusions

OS/SV and OS/DV impressions resulted in statistically significantly smaller marginal and internal discrepancies than the two-step technique.

Clinical significance

Marginal and internal fit of hot-pressed lithium disilicate partial crowns depended on the employed impression technique. One-step impressions are preferred over two-step techniques in many day-to-day clinical situations, especially for the fabrication of partial coverage crown restorations.     

The effects of endotoxin on cell detachment in vitro

The effects of endotoxin from E. coli and F. polymorphum on cell detachment was studied in vitro. L929 mouse fibroblasts or Ehrlich ascites carcinoma cells, cultured adherent to glass surfaces, were incubated with doses of endotoxin ranging from 5 to 200 μg/ml in serum-free media for 30 min. Cell detachment was assessed following exposure of cells to a known shearing force. Statistically significant facilitation of cell detachment was detected with doses of endotoxin of 10 μg/ml and higher. This detachment occurred in absence of demonstrable complement.     

Measurement of pH in human dental plaque in vivo with an ion-sensitive transistor electrode

A transistor pH electrode mounted on a removable partial denture was used to measure the pH of 2- and 4-day-old approximal and occlusal plaques in 2 adults. Direct application or a mouth-rinse of sucrose, glucose or maltose to the plaque covering the electrode caused the pH to fall within 2–3 min to approx. 4.4–5.2 in both subjects. Glucosylsucrose and lactose also lowered the pH, but not below 5.5. The transistor pH electrode was found suitable to record the pH of dental plaque in vivo, and to evaluate the fermentability of sugar substitutes.     

Marginal integrity and secondary caries of selectively excavated teeth in vitro

Objectives

Selective caries removal involves sealing of carious dentine beneath restorations, which might decrease their marginal integrity and increase the susceptibility for secondary caries and microleakage. The present study compared these marginal characteristics of restorations in selectively and completely excavated teeth.

Methods

In 32 premolars, shallow and deep artificial lesions were created on pulpo-axial walls of mesial-distal-occlusal cavities, with mesial and distal margins located in enamel and dentine, respectively. Demineralised dentine was either removed or left before adhesively restoring the teeth (n = 8), which were then submitted to thermo-mechanical cycling. The integrity of gingivo-cervical margins was assessed using scanning electron microscopy. In half of each margin, caries was induced adjacent to restorations using a continuous-culture biofilm model, and resulting lesions were evaluated using transversal microradiography. The other half of each margin was used to assess microleakage.

Results

Integrity or microleakage of margins located in enamel did not differ significantly between groups, and bacterial biofilms did not induce distinct caries lesions in enamel. Dentinal margins in teeth with deep compared with shallow lesions showed a significantly higher proportion of marginal imperfections, gaps and microleakage (p ≤ 0.05, Mann–Whitney/χ²-test). In contrast, neither marginal integrity nor microleakage differed significantly between completely and selectively excavated teeth (p > 0.05). Dentinal mineral loss adjacent to restorations did not differ significantly between groups (p > 0.80).

Conclusions

The marginal characteristics of restorations were affected by the depth of sealed or excavated lesions, but not by the performed caries excavation. This study did not find selective excavation detrimental for restoration integrity in vitro.

Clinical significance

Selective excavation of deep lesions was shown to reduce pulpal risks, whilst leaving caries beneath restorations is feared to compromise the marginal characteristics of the subsequently placed restoration. Based on the present in vitro study, such assumptions cannot be supported.     

Fluoride uptake by powdered human enamel treated with prolonged acting fluoride pellets in vitro

A preparation for the prolonged release of fluoride for topical use was developed. The delayed release of the fluoride was achieved by dispersing NaF or CaF₂ in ethyl cellulose with or without a stearic acid matrix. Orthodontic plates were used as delivery systems. The kinetics of fluoride release was controlled by diffusion mechanisms. The major compound resulting from enamel treatment with prolonged acting fluoride is fluoridated apatite.     

Bromodeoxyuridine-DNA interactions associated with arrest of rat odontogenesis in vitro

To better characterize the molecular mechanism responsible for the bromodeoxyuridine (BrdU)-mediated arrest of mammalian odontogenesis in vitro, the nature of nuclear DNA-analogue interactions was determined. Bioactive doses of the radiolabelled analogue were added to the tissue culture medium of 16-day old embryonic rat incisor primordia. Control rudiments were similarly exposed to equimolar, radiolabelled thymidine. After 16–18 h, DNA was isolated and purified from the labelled organ cultures. Following sedimentation to equilibrium through neutral CsCl density gradients, [³H]-BrdU-labelled DNA revealed a buoyant density indicative of a 12–15 per cent level of substitution in place of thymidine. Furthermore, similar centrifugation of DNA through alkaline density gradients suggested that the substitution was localized predominantly within a single-strand. DNA-DNA reassociation kinetics subsequently revealed that disproportionately more radiolabelled BrdU was concentrated within repetitive DNA nucleotide sequences in contrast to the more random distribution of [³H]-thymidine moieties. Thus it is likely that BrdU exerts its inhibitory effects on odontogenic differentiation through a relatively small proportion of rat embryo nuclear DNA.     

Physical chemical effects of zinc on in vitro enamel demineralization

Objectives

Zinc salts are formulated into oral health products as antibacterial agents, yet their interaction with enamel is not clearly understood. The aim was to investigate the effect of zinc concentration [Zn²⁺] on the in vitro demineralization of enamel during exposure to caries-simulating conditions. Furthermore, the possible mechanism of zinc's action for reducing demineralization was determined.

Methods

Enamel blocks and synthetic hydroxyapatite (HAp) were demineralized in a range of zinc-containing acidic solutions (0–3565 ppm [Zn²⁺]) at pH 4.0 and 37 °C. Inductively coupled-plasma optical emission spectroscopy (ICP-OES) was used to measure ion release into solution. Enamel blocks were analysed by Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy (ATR-FTIR), and HAp by X-ray diffraction (XRD) and neutron diffraction (ND).

Results

ICP-OES analysis of the acidic solutions showed a decrease in [Ca²⁺] and [PO₄³⁻] release with increasing [Zn²⁺]. FTIR revealed a α-hopeite (α-Zn₃(PO₄)₂.4H₂O)-like phase on the enamel surfaces at >107 ppm [Zn²⁺]. XRD and ND analysis confirmed a zinc-phosphate phase present alongside the HAp.

Conclusions

This study confirms that zinc reduces enamel demineralization. Under the conditions studied, zinc acts predominantly on enamel surfaces at PO₄³⁻ sites in the HAp lattice to possibly form an α-hopeite-like phase.

Clinical significance

These results have a significant implication on the understanding of the fundamental chemistry of zinc in toothpastes and demonstrate its therapeutic potential in preventing tooth mineral loss.