Assaying endogenous matrix metalloproteinases (MMPs) in acid-etched dentinal cavity walls.

Endogenous dentinal matrix metalloproteinases (MMPs) have been implicated in the auto-degradation of collagen fibrils within resin infiltrated layers of dentinal attachment. In order to target these proteinases, we must know which MMPs are produced and activated at the resin/dentin interface. In this study, we have optimized an extraction procedure and quantitated levels of endogenous MMPs in samples of dentin removed from the cavity walls of a single, extracted tooth. In our tooth-cavity model, an occlusal cavity (2×4×2 mm) was prepared and removed from the tooth crown, leaving surrounding dentinal walls of 1-mm-thick. The samples were pulverized with an analytic mill. Using enzyme-linked immunosorbent assay (ELISA), an average of 34.7 picograms of MMP-9 was detected in less than 300 mg of dentinal powder. This is the first study of its kind to quantitate endogenous levels of MMP in dentinal protein isolated from the cavity walls of a single, extracted tooth.

Evolution of resistance to cationic biocides in Streptococcus mutans and Enterococcus faecalis.

OBJECTIVES: The aim of this study was to investigate whether Streptococcus mutans and Enterococcus faecalis develop resistance to the cationic biocides chlorhexidine (CHX), cetylpyridinium chloride (CPC), and 12-methacryloyloxydodecylpyridinium bromide (MDPB). METHODS: The minimum inhibitory concentrations (MICs) of CHX, CPC, and MDPB were assessed after repeated exposure of S. mutans and E. faecalis to these biocides. Cell-surface hydrophobicity and protein expression profiles of bacterial cells were examined to elucidate possible resistance mechanisms. RESULTS: The MIC of CHX against E. faecalis showed constant increases up to 10 passages. No changes in the MICs of CPC and MDPB against E. faecalis were observed. The MICs of CHX, CPC, and MDPB against S. mutans did not increase. The surface hydrophobicity of E. faecalis significantly increased with increasing exposure to CHX and CPC. However, changes in protein expression profiles were only found in CHX-adapted E. faecalis, as evidenced by the emergence of a novel, approximately 19-kDa band following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. CONCLUSIONS: While E. faecalis and S. mutans did not exhibit increased resistance to CPC or MDPB, repeated exposure of E. faecalis to CHX led to resistance. It is likely that the acquisition of resistance is related to an altered protein composition. CLINICAL SIGNIFICANCE: Alkyl pyridinium compounds, such as CPC and MDPB, could have a lower risk to cause adaptation of E. faecalis, which is advantageous compared with CHX.

Development of sustained antimicrobial-release systems using poly(2-hydroxyethyl methacrylate)/trimethylolpropane trimethacrylate hydrogels.

Reconstructive materials with sustained antimicrobial effects could be useful for preventing infectious diseases in an environment containing indigenous bacteria or fungi such as the oral cavity. With the objective of applying a non-biodegradable hydrogel to resin-based materials as a reservoir for water-soluble antimicrobials, novel hydrogels consisting of 2-hydroxyethyl methacrylate (HEMA) and trimethylolpropane trimethacrylate (TMPT) were fabricated. Cetylpyridinium chloride (CPC) was loaded into five hydrogels comprising different ratios of HEMA/TMPT, and their ability to release as well as to be recharged with CPC was examined in vitro. A polyHEMA/TMPT hydrogel comprising 50% HEMA/50% TMPT could be effectively loaded and recharged with CPC by immersion into a CPC solution, demonstrating the longest release of CPC, above the concentration required to inhibit bacteria and fungi. The binding of CPC to the hydrogels was mainly through hydrophobic interaction. Loading of CPC into a hydrogel by mixing CPC powder with the HEMA/TMPT monomer before polymerization resulted in marked extension of the initial CPC-release period. The CPC-pre-mixed hydrogel was confirmed to exhibit antibacterial activity by agar diffusion tests. It is possible to achieve a sustained release system for antimicrobials by pre-mix loading and recharging CPC into a 50% HEMA/50% TMPT hydrogel.

Mechanism of detoxification of the cationic antibacterial monomer 12-methacryloyloxydodecylpyridiniumbromide (MDPB) by N-acetyl cysteine.

OBJECTIVES: The protective effects of N-acetyl cysteine (NAC) against cytotoxicity induced by conventional dental resin monomers have been widely documented. However, its effectiveness to detoxify cationic antibacterial monomers has not yet been elucidated. The aim of the present study was to investigate the possible protective effects of NAC against the cytotoxicity of 12-methacryloyloxydodecylpyridiniumbromide (MDPB) and explore the role of adduct formation in NAC-directed detoxification. METHODS: The influences of NAC on the cytotoxicity of MDPB were studied in mouse osteoblast-like MC3T3-E1 cells using the MTT assay. Ultra-performance liquid chromatography (UPLC) and liquid chromatography-mass spectrometry (LC-MS) analysis were performed to investigate the possible chemical reaction between NAC and MDPB. RESULTS: While only slight reduction in the cytotoxicity of MDPB by NAC was observed immediately after mixing with MDPB, remarkable protection against MDPB-induced cell death was detected when the mixture was tested after 24h of pre-incubation. UPLC and LC-MS analysis revealed that chemical binding of MDPB and NAC occurred under neutral conditions after 24h of pre-incubation. SIGNIFICANCE: Our findings suggest that NAC reduces the toxicity of the cationic antibacterial monomer MDPB, and adduct formation is partially responsible for the detoxification ability of NAC against MDPB-induced cell damage.

In vitro precision of fit of computer-aided design and computer-aided manufacturing titanium and zirconium dioxide bars.

OBJECTIVES: Optical scanners combined with computer-aided design and computer-aided manufacturing (CAD/CAM) technology provide high accuracy in the fabrication of titanium (TIT) and zirconium dioxide (ZrO) bars. The aim of this study was to compare the precision of fit of CAD/CAM TIT bars produced with a photogrammetric and a laser scanner. METHODS: Twenty rigid CAD/CAM bars were fabricated on one single edentulous master cast with 6 implants in the positions of the second premolars, canines and central incisors. A photogrammetric scanner (P) provided digitized data for TIT-P (n=5) while a laser scanner (L) was used for TIT-L (n=5). The control groups consisted of soldered gold bars (gold, n=5) and ZrO-P with similar bar design. Median vertical distance between implant and bar platforms from non-tightened implants (one-screw test) was calculated from mesial, buccal and distal scanning electron microscope measurements. RESULTS: Vertical microgaps were not significantly different between TIT-P (median 16µm; 95% CI 10-27µm) and TIT-L (25µm; 13-32µm). Gold (49µm; 12-69µm) had higher values than TIT-P (p=0.001) and TIT-L (p=0.008), while ZrO-P (35µm; 17-55µm) exhibited higher values than TIT-P (p=0.023). Misfit values increased in all groups from implant position 23 (3 units) to 15 (10 units), while in gold and TIT-P values decreased from implant 11 toward the most distal implant 15. SIGNIFICANCE: CAD/CAM titanium bars showed high precision of fit using photogrammetric and laser scanners. In comparison, the misfit of ZrO bars (CAM/CAM, photogrammetric scanner) and soldered gold bars was statistically higher but values were clinically acceptable.

Assessment of bactericidal effects of quaternary ammonium-based antibacterial monomers in combination with colloidal platinum nanoparticles.

Pretreatment of dentin using colloidal platinum nanoparticles (CPtN) can enhance the bond strength of dentin adhesives. However, the combination of CPtN, which is negatively charged, with cationic monomer-containing adhesive may reduce the antibacterial activity of the original material. Thus, the purpose of this study was to assess the effect of CPtN on the bactericidal activity of two cationic antibacterial monomers, 12-methacryloyloxydodecylpyridinium bromide (MDPB) and methacryloxylethyl cetyl dimethyl ammonium chloride (DMAE-CB). The rapid killing effects of the two monomers against planktonic or attached Streptococcus mutans in the presence or absence of CPtN were examined by viable cell counts. The measurement of minimum inhibitory and bactericidal concentrations demonstrated that CPtN up to 2.5 mM has no antibacterial activity. In the absence of CPtN, rapid killing of both planktonic and attached Streptococcus mutans were achieved by the two cationic monomers. Combination with 0.1 mM CPtN did not reduce the bactericidal effects of the two monomers, indicating that CPtN may be used as a pretreatment with antibacterial adhesives.

Evaluation of cytotoxic effects of six self-etching adhesives with direct and indirect contact tests.

This study evaluated the cytotoxicity of self-etching primers/adhesives by direct contact and dentin barrier tests. The three two-step self-etching systems Clearfil SE Bond (CSE), Clearfil Protect Bond (CPB), Prime&Bond NT/NRC (PB) and one-step self-etching systems Reactmer Bond (RB), Clearfil Tri-S Bond (CTS), and Adper Prompt L-Pop (AP) were examined. In direct contact tests, L929 cells were cultured in the presence of diluted solutions (50, 20, 10, and 1%) of primer/conditioner of adhesive systems. For dentin barrier tests, each system was applied onto 0.5 or 1.5 mm thick human dentin assembled in a simple pulp chamber device and incubated for 24 h at 37°C to make the diffusive components contact the L929 cells placed at the bottom of the chamber. The cytotoxic effects were assessed by MTT assay. Cell culture without application of any primers/adhesives served as the control for both tests. One-way ANOVA and Tukey HSD tests were used for statistical analyses. The direct contact tests demonstrated that CSE and CPB were less toxic than the other materials at all dilutions. In the dentin barrier tests, toxic effects of materials were reduced with an increase in thickness of intervening dentin. CSE and CPB showed less cytotoxicity than the other adhesives (p<0.05) when applied to 0.5 mm-thick dentin, and CSE was the least toxic in the 1.5 mm-dentin group (p<0.05). Dentin thickness positively affected biocompatibility of the tested bonding systems. Two-step self-etching systems with HEMA-based primers were more biocompatible than other self-etching adhesives.

Effects of the antibacterial monomer 12-methacryloyloxydodecylpyridinium bromide (MDPB) on bacterial viability and metabolism.

The antibacterial monomer 12-methacryloyloxydodecylpyridinium bromide (MDPB) is a strong bactericide when unpolymerized and has the potential to be utilized in various resinous biomaterials. To analyze the antibacterial characteristics of this monomer in detail, the ability of high concentrations of unpolymerized MDPB to kill Streptococcus mutans in planktonic or biofilm forms within a short time-period of contact, and the inhibitory effects of low concentrations of MDPB on the metabolic function of S. mutans, were examined. High concentrations of MDPB showed effective killing of planktonic and biofilm S. mutans cells within 60 s, and complete killing was obtained by contact with 1,000 µg ml(-1) of MDPB for 60 s. At a concentration of 4-8 µg ml(-1) , MDPB demonstrated growth inhibition, inducing elongation of the lag phase and of the doubling time, when the bacterial number was low. Inhibition of the production of acid from S. mutans by 8 µg ml(-1) of MDPB may have been caused by the inhibition of lactate dehydrogenase activity. At high concentrations, MDPB is lethal to both planktonic and biofilm forms of S. mutans in a short time-period, and at low concentrations, MDPB inhibits metabolic enzymatic activity.

Proliferation and differentiation potential of pluripotent mesenchymal precursor C2C12 cells on resin-based restorative materials.

This study investigated the proliferation and differentiation potential of pluripotent mesenchymal cells on three resin-based restoratives using a typical pluripotent mesenchymal precursor cell line, C2C12. C2C12 cells were cultured for 3-21 days on cured specimens of a Bis-GMA/TEGDMA-based composite resin (APX; Clearfil AP-X), a 4-META/MMA-based resin cement (SB; Superbond C&B) or a HEMA-containing resin modified glass-ionomer (LC; Fuji Ionomer Type II LC). To examine the influences on differentiation potential, alkaline phosphatase (ALP) activity of the cells cultured on each material was determined. On APX and SB, cells adhered and proliferated well, and no significant influences on ALP activity were observed. In contrast, poor cell proliferation and significant suppression of ALP activity were observed for cells cultured on LC, similar to those cultured on a zinc oxide EBA cement used as a control material. Bis-GMA/TEGDMA-based composite resin and 4-META/MMA-based resin exhibited better biocompatibility for C2C12 cells than HEMA-containing resin modified glass-ionomer, suggesting a potential advantage of the former two resins to show smaller influences on regeneration of periapical or periodontal tissue.

Association of salivary Streptococcus mutans levels determined by rapid detection system using monoclonal antibodies with prevalence of root surface caries.

PURPOSE: To examine the hypothesis that salivary Streptococcus mutans levels determined by a rapid detection system using monoclonal antibodies are associated with prevalence of root surface caries in a selected population of older adults. METHODS: Oral examinations were performed in 241 elderly people aged over 60 years with at least 10 teeth, and root surface caries were recorded. Populations of S. mutans in saliva were classified into three groups (Low: < 1 x 10(5) CFU/mL; Moderate: < or = 1 x 10(5) CFU/mL, < 1 x 10(6) CFU/mL; High: < or = 1 x 10(6) CFU/mL) using the analyzing kit with species-specific monoclonal antibodies. Stimulated whole saliva was collected; the flow rate and pH value were determined. RESULTS: About 38% of subjects had at least one decayed lesion (inactive, active, or secondary lesions). Subjects were grouped according to levels of S. mutans into Low (51.5%), Moderate (39.4%), and High (9.1%). Significant associations were observed between inactive, secondary, or total decayed lesions and salivary S. mutans levels by Kruskal-Wallis test (P < 0.05). Multiple ordinary regression analyses demonstrated that numbers of inactive, secondary, and total decayed lesions were significantly associated with S. mutans levels (P < 0.05) independent of age, gender, frequency of brushing, salivary flow rate, or pH value of saliva.