Evaluation of three-dimensional biofilms on antibacterial bonding agents containing novel quaternary ammonium methacrylates

Antibacterial adhesives are promising to inhibit biofilms and secondary caries. The objectives of this study were to synthesize and incorporate quaternary ammonium methacrylates into adhesives, and investigate the alkyl chain length effects on three-dimensional biofilms adherent on adhesives for the first time. Six quaternary ammonium methacrylates with chain lengths of 3, 6, 9, 12, 16 and 18 were synthesized and incorporated into Scotchbond Multi-Purpose. Streptococcus mutans bacteria were cultured on resin to form biofilms. Confocal laser scanning microscopy was used to measure biofilm thickness, live/dead volumes and live-bacteria percentage vs. distance from resin surface. Biofilm thickness was the greatest for Scotchbond control; it decreased with increasing chain length, reaching a minimum at chain length 16. Live-biofilm volume had a similar trend. Dead-biofilm volume increased with increasing chain length. The adhesive with chain length 9 had 37% live bacteria near resin surface, but close to 100% live bacteria in the biofilm top section. For chain length 16, there were nearly 0% live bacteria throughout the three-dimensional biofilm. In conclusion, strong antibacterial activity was achieved by adding quaternary ammonium into adhesive, with biofilm thickness and live-biofilm volume decreasing as chain length was increased from 3 to 16. Antibacterial adhesives typically only inhibited bacteria close to its surface; however, adhesive with chain length 16 had mostly dead bacteria in the entire three-dimensional biofilm. Antibacterial adhesive with chain length 16 is promising to inhibit biofilms at the margins and combat secondary caries.     

One-year water-ageing of calcium phosphate composite containing nano-silver and quaternary ammonium to inhibit biofilms

Dental composites are commonly used restorative materials; however, secondary caries due to biofilm acids remains a major problem. The objectives of this study were (1) to develop a composite containing quaternary ammonium dimethacrylate (QADM), nanoparticles of silver (NAg), and nanoparticles of amorphous calcium phosphate (NACP), and (2) to conduct the first investigation of the mechanical properties, biofilm response and acid production vs water-ageing time from 1 day to 12 months. A 4 × 5 design was utilized, with four composites (NACP-QADM composite, NACP-NAg composite, NACP-QADM-NAg composite, and a commercial control composite), and five water-ageing time periods (1 day, and 3, 6, 9, and 12 months). After each water-ageing period, the mechanical properties of the resins were measured in a three-point flexure, and antibacterial properties were tested via a dental plaque biofilm model using human saliva as an inoculum. After 12 months of water-ageing, NACP-QADM-NAg had a flexural strength and elastic modulus matching those of the commercial control (P40.1). Incorporation of QADM or NAg into the NACP composite greatly reduced biofilm viability, metabolic activity and acid production. A composite containing both QADM and NAg possessed a stronger antibacterial capability than one with QADM or NAg alone (Po0.05). The anti-biofilm activity was maintained after 12 months of water-ageing and showed no significant decrease with increasing time (P40.1). In conclusion, the NACP-QADM-NAg composite decreased biofilm viability and lactic acid production, while matching the load-bearing capability of a commercial composite. There was no decrease in its antibacterial properties after 1 year of water-ageing. The durable antibacterial and mechanical properties indicate that NACP-QADM-NAg composites may be useful in dental restorations to combat caries.     

Antimicrobial activity of alexidine alone and associated with N-acetylcysteine against Enterococcus faecalis biofilm

The purpose of this study was to assess the efficacy of alexidine（ALX）,alone and combined with N-acetylcysteine（NAC）,in eradicating two Enterococcus faecalis strain biofilms.The biofilms of E.faecalis ATCC 29212 and the clinical isolate E.faecalis D1 were grown in the MBEC-high-throughput device for 24 h and were exposed to five twofold dilutions of ALX（2%–0.007 8%）alone and combined with100 mg？mL21NAC,for 1 and 5 min.Eradication was defined as 100%kill of biofilm bacteria.The Student’s t-test was used to compare the efficacy of the associations of the two irrigants.After 1-min contact time,ALX eradicated the biofilms at all concentrations except for 0.007 8%and 0.015 6%–0.007 8%with E.faecalis ATCC 29212 and E.faecalis D1,respectively.Similar results for eradication and concentration were obtained when it was combined with 100 mg？mL21NAC.After 5 min of contact time,ALX alone and combined with NAC eradicated all enterococci biofilms.ALX showed antimicrobial properties against the two E.faecalis strain biofilms tested at very low concentrations,and its combined use with NAC was not seen to enhance its activity.     

Incidence of C-shaped root canal systems in mandibular second molars in the native Chinese population by analysis of clinical methods

The aims of the study were to investigate the incidence of C-shaped root canal systems in mandibular second molars in a native Chinese population using radiography and clinical examination under microscope and to compare the relative efficacies of these methods.For the recognition of C-shaped root canal system,1 146 mandibular second molars were selected and examined.Teeth with C-shaped canal systems were categorized by using the radiographic classification criteria and the modified Melton’s method.C-shaped canals were identified in 397(34.64%) mandibular second molars by radiography(type I,31.23%;type II,38.29%;type III,30.48%).Clinical examination showed that 449(39.18%) cases exhibited C-shaped canal systems(C1,22.94%;C2,48.11%;C3a,15.59%;C3b,13.36%).As for the result of the radiographic and clinical combined examination,C-shaped root canals were found in 473(41.27%) mandibular second molars(C1,21.78%;C2,45.67%;C3a,16.70%;C3b,15.86%).The incidence of C-shaped root canal diagnosed by radiographic method was statistically different from that by clinical examination and the combined examination(P,0.05).The study indicated a high incidence of C-shaped canal system in a Chinese population.The combination of microscopic and radiographic examination is an effective method in identifying the C-shaped root canal system.     

In vitro analysis of riboflavin-modified,experimental, two-step etch-and-rinse dentin adhesive:Fourier transform infrared spectroscopy and micro-Raman studies

To modify two-step experimental etch-and-rinse dentin adhesive with different concentrations of riboflavin and to study its effect on the bond strength,degree of conversion,along with resin infiltration within the demineralized dentin substrate,an experimental adhesive-system was modified with different concentrations of riboflavin(mlm,0,1%,3%,5%and 10%).Dentin surfaces were etched with 37%phosphoric acid,bonded with respective adhesives,restored with restorative composite-resin,and sectioned into resin-dentin slabs and beams to be stored for 24 h or 9 months in artificial saliva.Micro-tensile bond testing was performed with scanning electron microscopy to analyse the failure of debonded beams.The degree of conversion was evaluated with Fourier transform infrared spectroscopy(FTIR) at different time points along with micro-Raman spectroscopy analysis.Data was analyzed with one-way and two-way analysis of variance followed by Tukey’s for pair-wise comparison.Modification with 1%and 3%riboflavin increased the micro-tensile bond strength compared to the control at 24 h and 9-month storage with no significant differences in degree of conversion(P<0.05).The most predominant failure mode was the mixed fracture among all specimens except 10%riboflavin-modified adhesive specimens where cohesive failure was predominant.Raman analysis revealed that 1%and 3%riboflavin adhesives specimens showed relatively higher resin infiltration.The incorporation of riboflavin in the experimental two-step etch-and-rinse adhesive at 3%(mlm) improved the immediate bond strengths and bond durability after 9-month storage in artificial saliva without adversely affecting the degree of conversion of the adhesive monomers and resin infiltration.     

Influence of acrylamide monomer addition to the acrylic denture-base resins on mechanical and physical properties

The aim of the study was to evaluate the effect of adding acrylamide monomer （AAm） on the characterization, flexural strength, flexural modulus and thermal degradation temperature of poly（methyl methacrylate） （PMMA） denture-base resins. Specimens （n= 10） were fabricated from a conventional heat-activated QC-20 （Qc-） and a microwave heat-activated Acron MC （Ac-） PMMA resins. Powder/ liquid ratio followed the manufacturer＇s instructions for the control groups （Qc-c and Ac-c） and for the copolymer groups, the resins were prepared with 5% （-5）, 10% （- 10）, 15% （- 15） and 20% （-20） acrylamide contents, according to the molecular weight ratio, respectively. The flexural strength and flexural modulus were measured by a three-point bending test. The data obtained were statistically analyzed by Kruskal-Wallis test （a=O.05） to determine significant differences between the groups, The chemical structures of the resins were characterized by the nuclear magnetic resonance spectroscopy. Thermal stabilities were determined by thermogravimetric analysis （TGA） with a heating rate of 10 ~C.min-1 from 35 ~C to 600 ~C. Control groups from both acrylic resins showed the lowest flexural strength values. Qc-15 showed significant increase in the flexural strength when compared to Qc-c （P〈O.01）. Ac-10 and Ac-15 showed significance when compared to Ac-c （P〈O.01）. Acrylamide incorporation increased the elastic modulus in Qc-10, Qc-15 and Qc-20 when compared to Qc-c （P〈0.01）. Also significant increase was observed in Ac-10, Ac-15 and Ac-20 copolymer groups when compared to Ac-c （P〈0.01）. According to the 1H-nuclear magnetic resonance （NMR） results, acrylamide copolymerization was confirmed in the experimental groups. TGA results showed that the thermal stability of PMMA is increased by the insertion of AAm.     

Isolation and characterisation of human gingival margin-derived STRO-1/MACS~+ and MACS~-cell populations

Recently,gingival margin-derived stem/progenitor cells isolated via STRO-1/magnetic activated cell sorting(MACS) showed remarkable periodontal regenerative potential in vivo.As a second-stage investigation,the present study’s aim was to perform in vitro characterisation and comparison of the stem/progenitor cell characteristics of sorted STRO-1-positive(MACS~+) and STRO-1-negative(MACS~-) cell populations from the human free gingival margin.Cells were isolated from the free gingiva using a minimally invasive technique and were magnetically sorted using anti-STRO-1 antibodies.Subsequently,the MACS~+ and MACS~- cell fractions were characterized by flow cytometry for expression of CD14,CD34,CD45,CD73,CD90,CD105,CD146/MUC18 and STRO-1.Colony-forming unit(CFU) and multilineage differentiation potential were assayed for both cell fractions.Mineralisation marker expression was examined using real-time polymerase chain reaction(PCR).MACS~+ and MACS- cell fractions showed plastic adherence.MACS~+ cells,in contrast to MACS- cells,showed all of the predefined mesenchymal stem/progenitor cell characteristics and a significantly higher number of CFUs(P<0.01).More than 95%of MACS~+ cells expressed CD105,CD90 and CD73;lacked the haematopoietic markers CD45,CD34 and CD14,and expressed STRO-1 and CD146/MUC18.MACS- cells showed a different surface marker expression profile,with almost no expression of CD14 or STRO-1,and more than 95%of these cells expressed CD73,CD90 and CD146/MUC18,as well as the haematopoietic markers CD34 and CD45 and CD105.MACS~+ cells could be differentiated along osteoblastic,adipocytic and chondroblastic lineages.In contrast,MACS- cells demonstrated slight osteogenic potential.Unstimulated MACS~+ cells showed significantly higher expression of collagen I(P<0.05) and collagen III(P<0.01),whereas MACS~- cells demonstrated higher expression of osteonectin(P<0.05;MannWhitney).The present study is the first to compare gingival MACS~+ and MACS- cell populations demonstrating that MACS~+ cells,in contrast to MACS- cells,harbour stem/progenitor cell characteristics.This study also validates the effectiveness of the STRO-l/MACS~+technique for the isolation of gingival stem/progenitor cells.Human free gingival margin-derived STRO-1/MACS~+ cells are a unique renewable source of multipotent stem/progenitor cells.     

Oral microbiota and host innate immune response in bisphosphonate-related osteonecrosis of the jaw

Bacterial biofilms have emerged as potential critical triggers in the pathogenesis of bisphosphonate（BP）-related osteonecrosis of the jaw（ONJ） or BRONJ. BRONJ lesions have shown to be heavily colonized by oral bacteria, most of these difficult to cultivate and presents many clinical challenges. The purpose of this study was to characterize the bacterial diversity in BRONJ lesions and to determine host immune response. We examined tissue specimens from three cohorts（n530）; patients with periodontal disease without a history of BP therapy（Control, n510）, patients with periodontal disease having history of BP therapy but without ONJ（BP, n55） and patients with BRONJ（BRONJ, n515）. Denaturing gradient gel electrophoresis of polymerase chain reaction（PCR）-amplified 16 S r RNA gene fragments revealed less bacterial diversity in BRONJ than BP and Control cohorts. Sequence analysis detected six phyla with predominant affiliation to Firmicutes in BRONJ（71.6%）, BP（70.3%） and Control（59.1%）. Significant differences（P,0.05） in genera were observed, between Control/BP, Control/BRONJ and BP/BRONJ cohorts. Enzyme-linked immunosorbent assay（ELISA）results indicated that the levels of myeloperoxidase were significantly lower, whereas interleukin-6 and tumor necrosis factor-alpha levels were moderately elevated in BRONJ patients as compared to Controls. PCR array showed significant changes in BRONJ patients with downregulation of host genes, such as nucleotide-binding oligomerization domain containing protein 2, and cathepsin G, the key modulators for antibacterial response and upregulation of secretory leukocyte protease inhibitor, proteinase 3 and conserved helix–loop–helix ubiquitous kinase. The results suggest that colonization of unique bacterial communities coupled with deficient innate immune response is likely to impact the pathogenesis of ONJ.     

Porous titanium granules in critical size defects of rabbit tibia with or without membranes

Recently, porous titanium granules （PTGs） have been indicated for the preservation of the dimensions of post-extraction sockets, as a filler in sinus lift procedures and for the treatment of peri-implant and periodontal defects, based on the osteoconductivity and dimensional stability of the titanium granules. However, there is a lack of information regarding the use of this material in larger defects and in conjunction with membranes. The objective of this study is to test the behavior of PTGs used to fill critical size defects in rabbit tibiae, with and without membranes. Critical defects were created in both tibiae of rabbits, divided randomly into three groups： Group A （defect filled with PTG）, Group B （defect filled with PTG＋collagen membrane） and a control group （empty defect）. After six weeks, histomorphometric analysis was performed. The results showed more defect closures at the cortical area （87.37%±2.2%） and more bone formation at the marrow area （57.6%± 1.3%） in Group B, in comparison with the other groups （P〈0.05）; the use of membranes improved the material stability expressed as more percentages of the original material when membranes were used （P〈0.05）. Finally, inflammatory reactions were observed when the granules were not protected by membranes. In spite of the limitations of this animal study, it may be concluded that PTG particles are osteoconductive and allow bone growth. The PTG particles must be covered by a membrane, especially when grafting larger defects, in order to control particle migration, promote clot stabilization and separate the PTG graft from undesired soft tissue cells.     

Role of sortase in Streptococcus mutans under the effect }f nicotine

Streptococcus mutans is a common Gram-positive bacterium and plays a significant role in dental caries. Tobacco and/or nicotine have documented effects on S. mutans growth and colonization. Sortase A is used by many Gram-positive bacteria, including S. mutans, to facilitate the insertion of certain cell surface proteins, containing an LPXTGX motif such as antigen 1/11. This study examined the effect of nicotine on the function of sortase A to control the physiology and growth of S. mutans using wild-type S. mutans NG8, and its isogenic sortase-defective and -complemented strains. Briefly, the strains were treated with increasing amounts of nicotine in planktonic growth, biofilm metabolism, and sucrose-induced and saliva-induced antigen I/ll-dependent biofilm formation assays. The strains exhibited no significant differences with different concentrations of nicotine in planktonic growth assays. However, they had significantly increased （P~〈 0.05） biofilm metabolic activity （2- to 3-fold increase） as the concentration of nicotine increased. Furthermore, the sortase-defective strain was more sensitive metabolically to nicotine than the wild-type or sortase-complemented strains. All strains had significantly increased sucrose-induced biofilm formation （2- to 3-fold increase） as a result of increasing concentrations of nicotine. However, the sortase-defective strain was not able to make as much sucrose- and saliva-induced biofilm as the wild-type NG8 did with increasing nicotine concentrations. These results indicated that nicotine increased metabolic activity and sucrose-induced biofilm formation. The saliva-induced biofilm formation assay and qPCR data suggested that antigen 1/11 was upregulated with nicotine but biofilm was not able to be formed as much as wild-type NG8 without functional sortase A.     

Successful Treatment of Postpeak Stage Patients with Class Ⅱ Division 1 Malocclusion Using Non-extraction and Multiloop Edgewise Archwire Therapy： A Report on 16 Cases

Aim To determine cephalometrically the mechanism of the treatment effects of non-extraction and multiloop edgewise archwire （MEAW） technique on postpeak Class Ⅱ Division 1 patients.
Methodology In this retrospective study, 16 postpeak Class Ⅱ Division 1 patients successfully corrected using a non-extraction and MEAW technique were cephalometrically evaluated and compared with 16 matched control subjects treated using an extraction technique. Using CorelDRAW software, standardized digital cephalograms preand post-active treatments were traced and a reference grid was set up. The superimpositions were based on the cranial base, the mandibular and the maxilla regions,and skeletal and dental changes were measured. Changes following treatment were evaluated using the paired-sample t-test. Student＇s t-test for unpaired samples was used to assess the differences in changes between the MEAW and the extraction control groups.
Results The correction of the molar relationships comprised 54% skeletal change （mainly the advancement of the mandible） and 46% dental change. Correction of the anterior teeth relationships comprised 30% skeletal change and 70% dental change.
Conclusion The MEAW technique can produce the desired vertical and sagittal movement of the tooth segment and then effectively stimulate mandibular advancement by utilizing the residual growth potential of the condyle.     

Influence of fiber posts on the fracture resistance of endodontically treated premolars with different dental defects

This study aimed to evaluate the influence of quartz fiber post placement on the fracture resistance of endodontically treated premolars with different dental defects under dynamic loading.Fifty extracted single-rooted mandibular premolars were randomized into five groups.Each group was prepared according to numbers of residual walls ranged from 0 to 4.Then each group was divided into two subgroups with one restored with quartz fiber posts and the other without posts.In no-post groups,gutta percha point 2 mm below cemento-enamel junction was removed.Composite resin was adapted to the well and used to shape the core directly.Each tooth was restored with a complete metal crown.Dynamic loading was carried out in a masticatory simulator with a nominal load of 50 N at 2 Hz for 300 000 loading cycles.Then a quasi-statically load was applied in a universal testing machine 306 to the long axis with a crosshead speed of 1 mm？min21until fracture.Data were analyzed with one-way analysis of variance and pairwise comparison（P,0.05）.No specimens failed during dynamic loading.The fracture resistance enhanced with the increase of numbers of coronal walls and the differences were significant（P,0.05）.Placement of fiber posts had a significant effect when fewer than two walls remained（P,0.05）,but it had no significant influence in groups with two,three or four walls（P.0.05）.Fiber post did not change failure mode,and the fracture pattern was mainly favorable.More dentin walls need to be retained in clinic.When no less than two walls remained,a fiber post is not always necessary.     

Accuracy evaluation of a new three-dimensional reproduction method of edentulous dental casts, and wax occlusion rims with jaw relation

The article introduces a new method for three-dimensional reproduction of edentulous dental casts,and wax occlusion rims with jaw relation by using a commercial high-speed line laser scanner and reverse engineering software and evaluates the method’s accuracy in vitro.The method comprises three main steps：（i）acquisition of the three-dimensional stereolithography data of maxillary and mandibular edentulous dental casts and wax occlusion rims;（ii）acquisition of the three-dimensional stereolithography data of jaw relations;and（iii）registration of these data with the reverse engineering software and completing reconstruction.To evaluate the accuracy of this method,dental casts and wax occlusion rims of 10 edentulous patients were used.The lengths of eight lines between common anatomic landmarks were measured directly on the casts and occlusion rims by using a vernier caliper and on the three-dimensional computerized images by using the software measurement tool.The direct data were considered as the true values.The paired-samples t-test was used for statistical analysis.The mean differences between the direct and the computerized measurements were mostly less than 0.04 mm and were not significant（P.0.05）.Statistical significance among 10 patients was assessed using one-way analysis of variance（P,0.05）.The result showed that the 10 patients were considered statistically no significant.Therefore,accurate three-dimensional reproduction of the edentulous dental casts,wax occlusion rims,and jaw relations was achieved.The proposed method enables the visualization of occlusion from different views and would help to meet the demand for the computer-aided design of removable complete dentures.     

Optimal continuous positive airway pressure level in korean patients with obstructive sleep apnea syndrome

Objectives

The aim of this study was to investigate optimal continuous positive airway pressure (CPAP) level, to examine the factors affecting optimal CPAP level, and to develop a predictive equation for optimal CPAP level in Korean patients with obstructive sleep apnea syndrome (OSAS).

Methods

A total of 202 patients with OSAS who underwent successful manual titration for CPAP treatment were included in this study. Correlations between the optimal CPAP level and baseline data including anthropometric and polysomnographic variables were analyzed. A predictive equation for optimal CPAP level was developed based on anthropometric and polysomonographic data.

Results

The mean optimal CPAP level in 202 patients with OSAS was 7.8±2.3 cm H₂O. The mean optimal CPAP level in the mild, moderate, and severe OSAS groups was 6.0±1.3, 7.4±1.9, and 9.1±2.1 cm H₂O, respectively. The apneahypopnea index (AHI) (r=0.595, P<0.001), arousal index (r=0.542, P<0.001), minimal SaO₂ (r=-0.502, P<0.001), body mass index (BMI) (r=0.494, P<0.001), neck circumference (r=0.265, P<0.001), and age (r=-0.164, P=0.019) were significantly correlated with optimal CPAP level. The best predictive equation according to stepwise multiple linear regression analysis was: Optimal CPAP level (cm H₂O)=0.681+(0.205×BMI)+(0.040×AHI). Forty-two percent of the variance in the optimal CPAP level was explained by this equation (R²=0.42, P<0.001).

Conclusion

A predictive equation for optimal CPAP level in Korean patients with OSAS was developed using AHI and BMI, which can be easily measured during the diagnostic process.     

Effects of quaternary ammonium chain length on the antibacterial and remineralizing effects of a calcium phosphate nanocomposite

Composites containing nanoparticles of amorphous calcium phosphate (NACP) remineralize tooth lesions and inhibit caries. A recent study synthesized quaternary ammonium methacrylates (QAMs) with chain lengths (CLs) of 3–18 and determined their effects on a bonding agent. This study aimed to incorporate these QAMs into NACP nanocomposites for the first time to simultaneously endow the material with antibacterial and remineralizing capabilities and to investigate the effects of the CL on the mechanical and biofilm properties. Five QAMs were synthesized: DMAPM (CL3), DMAHM (CL6), DMADDM (CL12), DMAHDM (CL16), and DMAODM (CL18). Each QAM was incorporated into a composite containing 20% NACP and 50% glass fillers. A dental plaque microcosm biofilm model was used to evaluate the antibacterial activity. The flexural strength and elastic modulus of nanocomposites with QAMs matched those of a commercial control composite (n 5 6; P . 0.1). Increasing the CL from 3 to 16 greatly enhanced the antibacterial activity of the NACP nanocomposite (P , 0.05); further increasing the CL to 18 decreased the antibacterial potency. The NACP nanocomposite with a CL of 16 exhibited biofilm metabolic activity and acid production that were 10-fold lesser than those of the control composite. The NACP nanocomposite with a CL of 16 produced 2-log decreases in the colony-forming units (CFU) of total microorganisms, total streptococci, and mutans streptococci. In conclusion, QAMs with CLs of 3–18 were synthesized and incorporated into an NACP nanocomposite for the first time to simultaneously endow the material with antibacterial and remineralization capabilities. Increasing the CL reduced the metabolic activity and acid production of biofilms and caused a 2-log decrease in CFU without compromising the mechanical properties. Nanocomposites exhibiting strong anti-biofilm activity, remineralization effects, and mechanical properties are promising materials for tooth restorations that inhibit caries.     

Laser disruption and killing of methicillin-resistant Staphylococcus aureus biofilms

Objective

The aim of the study was to study the efficacy of 2 different lasers in vitro, in disrupting biofilm and killing planktonic pathogenic bacteria.

Materials and methods

Biofilms of a stable bioluminescent of Staphylococcus aureus Xen 31 were grown in a 96-well microtiter plate for 3 days. The study included 7 arms: (a) control; (b) ciprofloxacin (3 mg/L, the established minimum inhibitory concentration [MIC]) alone; (c) shock wave (SW) laser alone; (d) near-infrared (NIR) laser alone; (e) SW laser and ciprofloxacin; (f) SW and NIR lasers; (g) SW, NIR lasers, and ciprofloxacin. The results were evaluated with an in vivo imaging system (IVIS) biophotonic system (for live bacteria) and optical density (OD) for total bacteria.

Results

Without antibiotics, there was a 43% reduction in OD (P < .05) caused by the combination of SW and NIR suggesting that biofilm had been disrupted. There was an 88% reduction (P < .05) in live biofilm. Ciprofloxacin alone resulted in a decrease of 28% of total live cells (biofilm remaining attached) and 58% of biofilm cells (both P > .05). Ciprofloxacin in combination with SW and SW + NIR lasers caused a decrease of more than 60% in total live biomass and more than 80% of biofilm cells, which was significantly greater than ciprofloxacin alone (P < .05).

Conclusions

We have demonstrated an effective nonpharmacologic treatment method for methicillin-resistant Staphylococcus aureus (MRSA) biofilm disruption and killing using 2 different lasers. The preferred treatment sequence is a SW laser disruption of biofilm followed by NIR laser illumination. Treatment optimization of biofilm is possible with the addition of ciprofloxacin in concentrations consistent with planktonic MIC.     

Role of saliva proteinase 3 in dental caries

Salivary analysis can be used to assess the severity of caries. Of the known salivary proteins, a paucity of information exists concerning the role of proteinase 3 (PR3), a serine protease of the chymotrypsin family, in dental caries. Whole, unstimulated saliva was collected from children with varying degrees of active caries and tested using a Human Protease Array Kit and an enzyme-linked immunosorbent assay. A significantly decreased concentration of salivary PR3 was noted with increasing severity of dental caries (P<0.01); a positive correlation (r=0.87; P<0.01; Pearson''s correlation analysis) was also observed between salivary pH and PR3 concentration. In an antibacterial test, a PR3 concentration of 250 ng·mL⁻¹ or higher significantly inhibited Streptococcus mutans UA159 growth after 12 h of incubation (P<0.05). These studies indicate that PR3 is a salivary factor associated with the severity of dental caries, as suggested by the negative relationship between salivary PR3 concentration and the severity of caries as well as the susceptibility of S. mutans to PR3.     

Effect of Galla chinensis on the remineralization of two bovine root lesions morphous in vitro

The present study aims to evaluate the effect of Galla chinensis compounds on the remineralization of two artificial root lesions morphous in vitro.Sixty bovine dentine blocks were divided into two groups and individually treated with two levels of demineralization solutions to form erosive and subsurface artificial carious lesions in vitro.Each group was then divided into three subgroups,each of which were treated with a remineralization solution(positive control),deionized water(negative control),or 4 000 mg?L 21 aqueous solutions of Galla chinensis extract.The dentine blocks were then subjected to a pH-cycling regime for 7 days.During the first 4 days,the daily cycle included 21-h deal and 3-h demineralization applications.The dentine blocks were dealt with the entire day during the remaining 3 days.Two specimens from each of the treatment groups were selected and observed under a polarized light microscope.Data collected using a laser scanning confocal microscope were computerized and analyzed.Galla chinensis extract clearly enhanced the remineralization of both erosive lesion and subsurface lesion patterns in the specimens(P,0.05).The level of remineralization of the erosive lesion by Galla chinensis extract was lower than that of the subsurface lesion(P,0.05).In addition,the remineralization of the subsurface lesion by Galla chinensis extract was higher than that of the remineralization solution(P,0.05).No significant difference between the remineralization of erosive lesions by Galla chinensis extract and the remineralization solution was observed(P.0.05).So Galla chinensis extract has the potential to improve the remineralization of artificial root lesions under dynamic pH-cyclic conditions,indicating its potential use as a natural remineralization medicine.     

In VitroStreptococcus pneumoniae Biofilm Formation and In Vivo Middle Ear Mucosal Biofilm in a Rat Model of Acute Otitis Induced by S. pneumoniae

Objectives

Streptococcus pneumoniae is one of the most common pathogens of otitis media (OM) that exists in biofilm, which enhances the resistance of bacteria against antibiotic killing and diagnosis, compared to the free-floating (planktonic) form. This study evaluated biofilm formation by S. pneumoniae on an abiotic surface and in the middle ear cavity in a rat model of OM.

Methods

In vitro biofilm formation was evaluated by inoculation of a 1:100 diluted S. pneumoniae cell suspension in a 96-well microplate. Adherent cells were quantified spectrophotometrically following staining with crystal violet by measurement of optical density at 570 nm. The ultrastructure of pneumococcal biofilm was assessed by scanning electron microscopy (SEM). For in vitro biofilm study, S. pneumoniae cell suspensions containing 1×10⁷ colony forming units were injected through transtympanic membrane into the middle ear cavity of Sprague Dawley rats. The ultrastructure of middle ear mucus was observed by SEM 1 and 2 weeks post-inoculation.

Results

The in vitro study revealed robust biofilm formation by S. pneumoniae after 12-18 hours of incubation in high glucose medium, independent of exogenously supplied competence stimulating peptide and medium replacement. Adherent cells formed three-dimensional structures approximately 20-30 µm thick. The in vivo study revealed that ciliated epithelium was relatively resistant to biofilm formation and that biofilm formation occurred mainly on non-ciliated epithelium of the middle ear cavity. One week after inoculation, biofilm formation was high in 50% of the treated rats and low in 25% of the rats. After 2 weeks, biofilm formation was high and low in 25% and 37.5% of rats, respectively.

Conclusion

The results imply that glucose level is important for the S. pneumoniae biofilm formation and S. pneumoniae biofilm formation may play important role in the pathophysiology of OM.     

A Genome-Wide Association Study of Chronic Otitis Media with Effusion and Recurrent Otitis Media Identifies a Novel Susceptibility Locus on Chromosome 2

Chronic otitis media with effusion (COME) and recurrent otitis media (ROM) have been shown to be heritable, but candidate gene and linkage studies to date have been equivocal. Our aim was to identify genetic susceptibility factors using a genome-wide association study (GWAS). We genotyped 602 subjects from 143 families with 373 COME/ROM subjects using the Illumina Human CNV370-Duo DNA Bead Chip (324,748 SNPs). We carried out the GWAS scan and imputed SNPs at the regions with the most significant associations. Replication genotyping in an independent family-based sample was conducted for 53 SNPs: the 41 most significant SNPs with P < 10⁻⁴ and 12 imputed SNPs with P < 10⁻⁴ on chromosome 15 (near the strongest signal). We replicated the association of rs10497394 (GWAS discovery P = 1.30 × 10⁻⁵) on chromosome 2 in the independent otitis media population (P = 4.7 × 10⁻⁵; meta-analysis P = 1.52 × 10⁻⁸). Three additional SNPs had replication P values < 0.10. Two were on chromosome 15q26.1 including rs1110060, the strongest association with COME/ROM in the primary GWAS (P = 3.4 ×10⁻⁷) in KIF7 intron 7 (P = 0.072), and rs10775247, a non-synonymous SNP in TICRR exon 2 (P = 0.075). The third SNP rs386057 was on chromosome 5 in TPPP intron 1 (P = 0.045). We have performed the first GWAS of COME/ROM and have identified a SNP rs10497394 on chromosome 2 is significantly associated with COME/ROM susceptibility. This SNP is within a 537 kb intergenic region, bordered by CDCA7 and SP3. The genomic and functional significance of this newly identified locus in COME/ROM pathogenesis requires additional investigation.