Dentin matrix components extracted with phosphoric acid enhance cell proliferation and mineralization.

OBJECTIVE: Acids, such as those used in adhesive dentistry, have been shown to solubilize bioactive molecules from dentin. These dentin matrix components (DMC) may promote cell proliferation and differentiation, and ultimately contribute to dentin regeneration. The objective of this study was to evaluate the potential for varying concentrations of DMC extracted from human dentin by phosphoric acid of a range of pHs to stimulate proliferation and mineralization of two different cultured pulp cell populations. METHODS: DMC were solubilized from powdered human dentin (7 days - 4°C) by phosphoric acid of pH 1, 3, and 5 and also, EDTA. Extracts were dialyzed for 7 days against distilled water and lyophilized. Undifferentiated mouse dental pulp cells (OD-21) and cells of the odontoblast-like cell line (MDPC-23) were seeded in six-well plates (1×10(5)) and cultured for 24h in DMEM (Dulbecco's modified Eagle's medium) containing 10% (v/v) FBS (fetal bovine serum). The cells were washed with serum-free medium and then treated with different concentrations of DMC (0.01, 0.1, 1.0 and 10.0µg/ml) daily in serum free medium for 7 days. After 3, 5 (MDPC-23 only), and 7 days of treatment, cell proliferation was measured using 10vol% Alamar blue solution, which was added to each well for 1h. Cell numbers were first measured by cell counting (Trypan blue; n=5) and Alamar blue fluorescence to validate the assay, which was then used for the subsequent assessments of proliferation. Mineralization was assessed by Alizarin Red S assay after 12 days exposure to DMC (n=5). Controls were media-only (DMEM) and dexamethasone (DEX; positive control). Results were analysed by ANOVA/Tukey's (p≤0.05). RESULTS: There was a linear correlation between cell counts and Alamar blue fluorescence (R(2)>0.96 for both cell types) , verifying the validity of the Alamar blue assay for these cell types. In general, there was a dose-dependent trend for enhanced cell proliferation with higher concentration of DMC for both cell lines, especially at 10.0µg/ml. DEX exposure resulted in significantly higher mineralization, but did not affect cell proliferation. DMC exposure demonstrated significantly greater mineralization than media-only control for 10µg/ml for all extracts, and at lower concentrations for EDTA and pH 5 extracts. SIGNIFICANCE: Human dentin matrix components solubilized by acids at pH levels found in commercial dentin adhesives enhanced cell proliferation and mineralization of mouse and rat undifferentiated dental pulp cells when presented in adequate concentration.

Biofilm formation affects surface properties of novel bioactive glass-containing composites.

OBJECTIVES: This study investigated the effects of bacterial biofilm on the surface properties of novel bioactive glass (BAG)-containing composites of different initial surface roughness. METHODS: BAG (65 mol% Si; 4% P; 31% Ca) and BAG-F (61% Si; 31% Ca; 4% P; 3% F; 1% B) were synthesized by the sol-gel method and micronized (size ∼0.1-10 µm). Composites with 72wt% total filler load were prepared by replacing 15% of the silanized Sr glass with BAG, BAG-F, or silanized silica. Specimens (n=10/group) were light-cured and divided into 4 subgroups of different surface roughness by wet polishing with 600 and then up to 1200, 2400, or 4000 grit SiC. Surface roughness (SR), gloss, and Knoop microhardness were measured before and after incubating in media with or without a Streptococcus mutans (UA 159) biofilm for 2 weeks. Results were analyzed with ANOVA/Tukey's test (α=0.05). RESULTS: The SR of the BAG-containing composites with the smoothest surfaces (2400/4000 grit) increased in media or bacteria; the SR of the roughest composites (600 grit) decreased. The gloss of the smoothest BAG-containing composites decreased in bacteria and media-only, but more in media-alone. The microhardness of all of the composites decreased with exposure to media or bacteria, with BAG-containing composites affected more than the control. SIGNIFICANCE: Exposure to bacterial biofilm and its media produced enhanced roughness and reduced gloss and surface microhardness of highly polished dental composites containing a bioactive glass additive, which could affect further biofilm formation, as well as the esthetics, of restorations made from such a material.

Sol-gel-derived bioactive glasses demonstrate antimicrobial effects on common oral bacteria.

PURPOSE: To determine the antibacterial effect of nano-structured, sol-gel processed bioactive glasses that may be used for implants, coatings, and as adjuncts to dental restorative materials. METHODS: Six bioactive glasses (BAG), three made with differing amounts of silica (65, 75 and 85 mole%), and three with different amounts of silica (61, 71, and 81 mole%) and 3 mole% fluoride were prepared by a sol-gel synthesis method and tested against clinically important bacteria species, Streptococcus sobrinus (ATCC33478), Streptococcus mutans (ATCC25175) and Enterococcus faecalis (ATCC19433). Bacterial suspensions were independently incubated with bioactive glass in particulate form (< 3 µm) for 4 and 24 hours. Viability was determined by colony-forming units. RESULTS: At 4 hours, all BAG produced an order of magnitude reduction in all three bacteria. After 24 hours, all BAG produced a significant reduction in S. sobrinus colonies, but no further reduction in S. mutans; all BAG, except BAG 61-F, significantly reduced E. faecalis compared to the control. At 4 hours, an increase in the pH of the BAG groups (to pH 9) could also have contributed to the bactericidal effect. In further experiments it was found that the viability of S. sobrinus was significantly reduced following exposure to an extract of BAG in media adjusted to a pH of 7.4. Additionally media with pH adjusted to 9 exerted a significant antibacterial effect against S. sobrinus after 4 hours. To determine the influence of the calcium ions released from the BAG in the absence of the pH effect, a typical dose response was demonstrated after 4 hours of exposure of S. sobrinus to media containing various levels of calcium. The results of this study clearly suggest that the effect of BAG extract on bacteria is not only related to a pH effect, but is also linked to an effect of liberated ions, such as calcium, extracted from the surface of the bioactive glasses.

Cytotoxicity of resin composites containing bioactive glass fillers.

OBJECTIVE: To determine the in vitro cytotoxicity of dental composites containing bioactive glass fillers. METHODS: Dental composites (50:50 Bis-GMA/TEGDMA resin: 72.5wt% filler, 67.5%Sr-glass and 5% OX50) containing different concentrations (0, 5, 10 and 15wt%) of two sol-gel bioactive glasses, BAG65 (65mole% SiO2, 31mole% CaO, 4mole% P2O5) and BAG61 (3mole% F added) were evaluated for cytotoxicity using Alamar Blue assay. First, composite extracts were obtained from 7 day incubations of composites in cell culture medium at 37°C. Undifferentiated pulp cells (OD-21) were exposed to dilutions of the original extracts for 3, 5, and 7 days. Then freshly cured composite disks were incubated with OD-21 cells (n=5) for 2 days. Subsequently, fresh composite disks were incubated in culture medium at 37°C for 7 days, and then the extracted disks were incubated with OD-21 cells for 2 days. Finally, fresh composites disks were light cured for 3, 5, and 20s and incubated with OD-21 cells (n=5) for 1, 3, 5, and 7 days. To verify that the three different curing modes produced different levels of degree of conversion (DC), the DC of each composite was determined by FTIR. Groups (n=5) were compared with ANOVA/Tukey's (α≤0.05). RESULTS: Extracts from all composites significantly reduced cell viability until a dilution of 1:8 or lower, where the extract became equal to the control. All freshly-cured composites showed significantly reduced cell viability at two days. However, no reduction in cell viability was observed for any composite that had been previously soaked in media before exposure to the cells. Composites with reduced DC (3s vs. 20s cure), as verified by FTIR, showed significantly reduced cell viability. SIGNIFICANCE: The results show that the composites, independent of composition, had equivalent potency in terms of reducing the viability of the cells in culture. Soaking the composites for 7 days before exposing them to the cells suggested that the "toxic" components had been extracted and the materials were no longer cytotoxic. The results demonstrate that the cytotoxicity of composites with and without BAG must predominantly be attributed to the release of residual monomers, and not to the presence of the BAG.

Berberine possesses muscarinic agonist-like properties in cultured rodent cardiomyocytes.

Berberine, a natural product alkaloid, has been shown to display a wide array of pharmacological effects. Generally, the mechanism of action of each of these effects has not been well described. The aim of the present study is to test the hypothesis that some of berberine's cardiovascular effects are mediated through activation of cardiac M2 muscarinic cholinergic receptors. In our studies, we tested the ability of berberine to alter the contraction rate of cultured neonatal rodent cardiomyocytes. In these spontaneously contracting primary cultured cells, berberine reduced the contraction rate in a manner independent of ß-adrenergic receptor blockade but sensitive to pertussis toxin, a Gi/o G protein inhibitor. Muscarinic antagonists completely blocked the effect of berberine on contraction rate of cardiomyocytes, whereas the effect of berberine was not opposed by antagonists to opioid, adenosine or α-adrenergic receptors. Further, berberine bound to muscarinic receptors of adult mouse heart membranes with relatively high affinity (K(i)=5.4×10(-6)M) comparable to that of the classic muscarinic agonist, carbachol, and to muscarinic M2 receptors exogenously expressed in HEK 293 cells (K(i)=4.9×10(-6)M). Therefore, the findings of the present study suggest that berberine is a muscarinic agonist at M2 receptors, potentially explaining some of its reported cardiovascular effects.

Hawthorn (Crataegus monogyna Jacq.) extract exhibits atropine-sensitive activity in a cultured cardiomyocyte assay.

Hawthorn (Crataegus spp.) plant extract is used as a herbal alternative medicine for the prevention and treatment of various cardiovascular diseases. Recently, it was shown that hawthorn extract preparations caused negative chronotropic effects in a cultured neonatal murine cardiomyocyte assay, independent of beta-adrenergic receptor blockade. The aim of this study was to further characterize the effect of hawthorn extract to decrease the contraction rate of cultured cardiomyocytes. To test the hypothesis that hawthorn is acting via muscarinic receptors, the effect of hawthorn extract on atrial versus ventricular cardiomyocytes in culture was evaluated. As would be expected for activation of muscarinic receptors, hawthorn extract had a greater effect in atrial cells. Atrial and/or ventricular cardiomyocytes were then treated with hawthorn extract in the presence of atropine or himbacine. Changes in the contraction rate of cultured cardiomyocytes revealed that both muscarinic antagonists significantly attenuated the negative chronotropic activity of hawthorn extract. Using quinuclidinyl benzilate, L-[benzylic-4,4'-(3)H] ([(3)H]-QNB) as a radioligand antagonist, the effect of a partially purified hawthorn extract fraction to inhibit muscarinic receptor binding was quantified. Hawthorn extract fraction 3 dose-dependently inhibited [(3)H]-QNB binding to mouse heart membranes. Taken together, these findings suggest that decreased contraction frequency by hawthorn extracts in neonatal murine cardiomyocytes may be mediated via muscarinic receptor activation.