Critical parameters in cultivation of experimental biofilms using the example of Pseudomonas fluorescens.

Formation and treatment of biofilms present a great challenge for health care and industry. About 80% of human infections are associated with biofilms including biomaterial centered infections, like infections of prosthetic heart valves, central venous catheters, or urinary catheters. Additionally, biofilms can cause food and drinking water contamination. Biofilm research focusses on application of experimental biofilm models to study initial adherence processes, to optimize physico-chemical properties of medical materials for reducing interactions between materials and bacteria, and to investigate biofilm treatment under controlled conditions. Exploring new antimicrobial strategies plays a key role in a variety of scientific disciplines, like medical material research, anti-infectious research, plant engineering, or wastewater treatment. Although a variety of biofilm models exist, there is a lack of standardization for experimental protocols, and designing experimental setups remains a challenge. In this study, a number of experimental parameters critical for material research have been tested that influence formation and stability of an experimental biofilm using the non-pathogenic model strain of Pseudomonas fluorescens. These parameters include experimental time frame, nutrient supply, inoculum concentration, static and dynamic cultivation conditions, material properties, and sample treatment during staining for visualization of the biofilm. It was shown, that all tested parameters critically influence the experimental biofilm formation process. The results obtained in this study shall support material researchers in designing experimental biofilm setups.

Antibacterial effect of silver (I) carbohydrate complexes on oral pathogenic key species in vitro.

BACKGROUND: It was the aim of this study to evaluate the antibacterial impact of two silver(I) carbohydrate complexes with tripodal thioglycosides, namely tris[2-(ß-D-thio-glucopyranosyl)ethyl]-amine-silver(I)-nitrate (3) and tris[2-(α-D-thio-manno-pyranosyl)ethyl]-amine-silver(I)-nitrate (4), on five oral pathogenic bacterial strains. Furthermore, cytocompatibility was tested using human gingival fibroblasts (HGF). METHODS: Minimum inhibitory concentrations (MIC) were determined on five oral pathogenic bacterial strains by using the broth microdilution method: Fusobacterium nucleatum (ATCC 10953), Aggregatibacter actinomycetemcomitans (ATCC 33384), Porphyromonas gingivalis (ATCC 33277), Streptococcus mutans (ATCC 25175) and Enterococcus faecalis (DSMZ 20376). Furthermore, antimicrobial efficiency was tested using agar diffusion assays. To evaluate cytocompatibility, human gingival fibroblasts (HGFs) were exposed to AgNO3 and complex 3 followed by a live/dead staining. RESULTS: MIC of the silver(I) complexes ranged between 0.625 and 5.0 mmol/L. The silver complexes 3 and 4 showed higher antibacterial efficiency against all tested species than AgNO3. Antibacterial efficiency of complexes 3 and 4 on F. nucleatum (≥18 mm) and A. actinomycetemcomitans (≥23 mm) was more pronounced than against P. gingivalis (≥15 mm). Complex 3 (20 mM) induced the largest inhibition zones (30 to 31 mm) on Gram-negative strains. For Gram-positive strains, the largest inhibition zones were achieved by complex 3 (20 mM/S. mutans: 28 mm, E. faecalis: 18 mm). Complex 3 had a lower cytotoxic impact on HGFs compared to AgNO3 by the power of ten. CONCLUSIONS: The findings suggest that silver(I) carbohydrate complexes 3 and 4 might function as novel antimicrobial agents for the treatment of periodontal, carious or endodontic diseases.

Antibacterial photodynamic treatment of periodontopathogenic bacteria with indocyanine green and near-infrared laser light enhanced by Trolox(TM).

BACKGROUND AND OBJECTIVES: It has been shown that certain vitamins can significantly enhance the effect of photodynamic anti-tumor therapy. Unfortunately, there is no sufficient information available about the impact of those antioxidants on antimicrobial Photodynamic Therapy (aPDT). The present study is aimed at investigating the antimicrobial effect of the dye indocyanine green (ICG) in the presence of Trolox(TM) , a vitamin E analogue, upon irradiation with near-infrared (NIR) laser light (808 nm) on the gramnegative periodontopathogenic bacteria Aggregatibacter actinomycetemcomitans (A.a.), Porphyromonas gingivalis (P.g.) and Fusobacterium nucleatum (F.n.). METHODS: Bacteria solved in PBS were incubated with ICG (50-500 µg/ml) in the presence and absence of Trolox(TM) (2 mM). Irradiation was performed after 10 minutes of dark-incubation with NIR-laser-light (25-100 J/cm(2) , 810 nm). During treatment, temperature was also recorded inside the bacterial solutions. The treated suspensions were serial diluted and plated onto blood agar plates. After anaerobe cultivation for 5 days the colony-forming units (CFU/ml) were determined. RESULTS: The antibacterial effect was ICG-concentration and exposure dependent. It was found that high ICG-concentrations and light fluence rates caused bacterial reduction due to hyperthermia. Where low ICG-concentrations (<250 µg/ml) and fluence rates only induced minor regression, additional Trolox(TM) -administration significantly enhanced the photodynamic effect. While treatment of A.a. (250 µg/ml ICG, 100 J/cm(2) ) without Trolox(TM) caused no bacterial reduction, additional administration led to total eradication. In the presence of Trolox(TM) reduction to one-fifth of the original ICG-concentration (50 µg/ml) still induced total suppression of P.g. and F.n. at identical fluence (100 J/cm(2) ). Treatment with ICG, NIR-light or Trolox(TM) alone showed no remarkable bactericidal effect. Application of high ICG-concentrations (500 µg/ml) and exposure values (100 J/cm(2) ) caused peak temperatures of 64.53°C. CONCLUSIONS: The results clearly show that Trolox(TM) significantly enhanced the antibacterial effect of ICG upon irradiation with NIR-laser-light. Additional administration of Trolox(TM) may also increase the efficiency of other aPDT systems.

Photodynamic killing of Enterococcus faecalis in dentinal tubules using mTHPC incorporated in liposomes and invasomes.

OBJECTIVES: The present in vitro study investigates the antimicrobial photodynamic efficiency of the photosensitizer 5,10,15,20-tetra(m-hydroxyphenyl)chlorin (mTHPC) incorporated in liposomes (LIP) and highly flexible invasomes (INV) on the endodontopathogenic species Enterococcus faecalis in infected dental root canals. MATERIALS AND METHODS: A total of 48 root canals were prepared mechanically to file size ISO 50 and inoculated with E. faecalis for 48 h. In the test groups, the infected root canals were subjected to aPDT with either mTHPC linked to LIP or INV. The controls were either incubated with 1 % chlorohexidine gel (CHX, positive control) or root canals were irrigated with normal saline (NaCl, negative control). After treatment all canals were mechanically enlarged (ISO 50-110), and the debris of each filing process was subjected to bacterial culture analysis. RESULTS: Both mTHPC formulations showed a significant antimicrobial effect. A bacterial reduction by up to 3.6 log-steps was ascertained for INV directly at the root canal wall. aPDT using INV (ISO 60) was more effective than CHX, which caused a decrease in only 1.2 log-steps. It was found that both liposomal mTHPC formulations were capable to suppress E. faecalis inside the dentinal tubules up to 300 µm. CONCLUSIONS: The results show that mTHPC linked to LIP and INV is capable of efficiently reducing E. faecalis in dental root canals. CLINICAL RELEVANCE: As evidenced, E. faecalis is resistant to several conventional antibacterial treatment measures. In this context, photodynamic treatment with mTHPC delivered by INV is superior to temporary dressing with 1 % CHX gel applied for 24 h.

Photodynamic antimicrobial effect of safranine O on an ex vivo periodontal biofilm.

BACKGROUND AND OBJECTIVE: The increasing resistance of oral pathogens against antibiotic measures urgently requires new therapeutic strategies. In this context, antimicrobial photodynamic therapy (aPDT) may play a crucial part in the future. The aim of the present study was to compare the antibacterial efficiency of aPDT using the photosensitizer safranine O with that of chlorhexidine (0.2% CHX) on an ex vivo biofilm. METHODS: First the antibacterial activity of both measures against planktonic cultures of Streptococcus gordonii ATCC 33399, Streptococcus mutans ATCC 25175, Fusobacterium nucleatum ATCC 10953, Aggregatibacter actinomycetemcomitans ATCC 33384 and Porphyromonas gingivalis ATCC 33277 was observed. Then a patient specific ex vivo biofilm was established from plaque and saliva samples of patients (n = 19) with chronic periodontitis. The antibacterial effects of aPDT and of 0.2% CHX were determined on the ex vivo biofilms cultivated for 24 and 72 hours. After cultivation of the treated samples on blood agar (2 days) the results were quantified by counting the colony forming units (cfu/ml). RESULTS: Photodynamic treatment with safranine O showed a distinct antibacterial effect on F. nucleatum and P. gingivalis. Whereas S. gordonii was suppressed completely by aPDT, treatment with 0.2% CHX caused only a partial reduction. In the ex vivo biofilm model (24-hour biofilm), aPDT caused a significantly higher bacterial killing than treatment with 0.2% CHX. Compared to the untreated control, there was no significant difference on the 72-hour biofilm for both methods. CONCLUSIONS: The results show that oral-pathogenic species in planktonic solution can be suppressed significantly by aPDT with safranine O. Especially for bacteria in a 24-hour ex vivo biofilm, this method is more effective than treatment with 0.2% CHX. Both antibacterial treatments did not show any significant effect on the biofilm cultivated for 72 hours.

A 4-week clinical comparison of an oscillating-rotating power brush versus a marketed sonic brush in reducing dental plaque.

PURPOSE: To assess the plaque removal efficacy of an oscillating-rotating power brush relative to a newly-introduced sonic power brush. METHODS: This study used a randomized, examiner-blind, single-center, two-treatment, parallel group 4-week design. Subjects with pre-existing plaque scores of at least 1.75 on the Turesky Modification of the Quigley-Hein Plaque Index (TMQHPI) were evaluated for baseline whole mouth and approximal plaque scores. They received either the oscillating-rotating brush (Oral-B Professional Care 1000, sold as Oral-B Professional Care 600 in some regions, with the Oral-B Precision Clean brush head, D16u/EB20) or the sonic brush (Colgate ProClinical C200 with Colgate Triple Clean brush head) and brushed twice-daily with the assigned brush and a standard fluoride dentifrice for 4 weeks before returning for plaque measurements. Prior to baseline and the Week 4 measurements, participants abstained from oral hygiene for 12 hours and from eating, chewing gum and drinking for 4 hours. RESULTS: A total of 131 subjects were enrolled in the study at baseline, with all completing the study: 65 in the oscillating-rotating group, and 66 in the sonic group. Both brushes significantly reduced plaque over the 4-week study period. The oscillating-rotating brush was statistically significantly more effective in reducing plaque (P < 0.001) than the sonic brush. Compared to the sonic power brush, the adjusted mean plaque reduction scores for the oscillating-rotating power brush were more than five times greater for whole mouth and approximal areas.

Influence of Layer Thickness and Shade on the Transmission of Light through Contemporary Resin Composites.

BACKGROUND: Material-dependent parameters have an important impact on the efficiency of light polymerization. The present in vitro study aimed to investigate the influence of the increment thickness and shade of nano- and nanohybrid resin composites on the transmission of curing light. METHODS: Three contemporary resin composites were evaluated: Tetric EvoCeram® (TEC); Venus Diamond® (VD); and Filtek Supreme XTE® (FS XTE). Light transmission (LT) was recorded in accordance with the sample thickness (0.5 to 2.7 mm) and the shade. Polymerized samples were irradiated for 10 s each using the high-power LED curing light Celalux 2 (1900 mW/cm2). LT was simultaneously recorded using the MARC Patient Simulator (MARC-PS). RESULTS: LT was strongly influenced by the composite layer thickness. For 0.5 mm-thick samples, a mean power density of 735 mW/cm2 was recorded at the bottom side. For the 2.7 mm samples, a mean power density of 107 mW/cm2 was measured. Only LT was markedly reduced in the case of darker shades. From A1 to A4, LT decreased by 39.3% for FS XTE and 50.8% for TEC. Dentin shades of FS XTE and TEC (A2, A4) showed the lowest LT. CONCLUSIONS: The thickness and shade of resin composite increments strongly influences the transmission of curing light. More precise information about these parameters should be included in the manufacture manual.

Coaxial electrospun nanofibers as drug delivery system for local treatment of periodontitis.

OBJECTIVES: The aim of the present study was to prepare resorbable polylactide fibers for periodontitis treatment using coaxial electrospinning to optimize the release of metronidazole (MNA) by reducing the initial burst effect. METHODS: Poly(L-lactide-co-D,L-lactide) (PLA) fibers mats with different distributions of metronidazole (MNA) were manufactured by coaxial electrospinning (COAX). By COAX spinning the central core of the fiber was enriched with 40% MNA (m/m), while the sheath of the fiber consisted of PLA only (test group). In contrast, fibers of the control group were prepared by conventional electrospinning with the same amount of MNA but with a homogenous drug distribution (HDD - homogenously distributed drug). The release of MNA was determined by analyzing aliquots from the fiber mats using UV-VIS spectroscopy. Agar diffusion tests were carried out to determine the antibacterial effect on periodontopathogenic bacteria. Biocompatibility was tested in direct contact to human gingival fibroblasts (HGF) for two days. RESULTS: The COAX mats showed a retarded drug release compared to the conventional HDD fibers. After 24 h, 64% of total MNA was released cumulatively from the COAX fibers while 90% of the MNA was released from the HDD fibers (controls). The antibacterial effect of COAX fibers was significantly higher after 24 h compared to the HDD fibers. Cell cultivation revealed significant higher numbers of vital cells among the COAX mats. SIGNIFICANCE: COAX fibers showed improved sustained MNA release compared to conventional fibers and can be seen as potential drug delivery systems in local periodontitis treatment.

Photodynamic suppression of Enterococcus faecalis using the photosensitizer mTHPC.

BACKGROUND AND OBJECTIVES: Enterococcus faecalis is frequently found in persistent endodontic infections. In this context, the antimicrobial photodynamic therapy (aPDT) could become a modern alternative to existing antibacterial treatment approaches. The aim of this study was to investigate the effect of aPDT on E. faecalis using the photosensitizer (PS) 5,10,15,20-tetra(m-hydroxyphenyl)chlorin (mTHPC) enriched in liposomes. MATERIALS AND METHODS: Enterococcus faecalis was cultivated in Schaedler submerged culture for 24 hours, then isolated and adjusted in PBS to 10(8) cells/ml. The bacterial suspension was pipetted into a black microtitration plate and incubated for 15 minutes in the dark with mTHPC in various concentrations (10, 30, and 50 µM). The photosensitized suspensions were subjected to laser light (652 nm) at a light fluence of 100 J cm(-2) (test group A). In addition, the suspension sensitized with 50 µM mTHPC was irradiated with 25, 50, and 75 J cm(-2) (test group B). The following controls were used: non-irradiated bacterial suspension in the absence of mTHPC (C); irradiated bacterial suspension in the absence of mTHPC (D); non-irradiated bacterial suspension incubated with mTHPC (E). Dilution series (10(0)-10(-6)) were made of all groups and applied on Schaedler agar. After anerobic cultivation (4 days), the colony-forming units (CFU/ml) were determined. RESULTS: Enterococcus faecalis was suppressed completely after incubation with 50 µM mTHPC and illumination with 100 J cm(-2). Photodynamic treatment with 10 and 30 µM mTHPC caused reduction in CFU by 5.8 and 6.7 log-units. The application of an energy fluence <100 J cm(-2) resulted in a decline of antibacterial efficiency. Irradiation of the non-photosensitized solution showed no suppressing impact. Incubation of the PS without additional irradiation caused a maximal reduction in CFU by 1.5 log-units. CONCLUSION: The results show that aPDT using the PS mTHPC incorporated in liposomes could be a new approach to adjuvant treatment of endodontic infections with E. faecalis.

Effectiveness of Casein Phosphopeptide-Amorphous Calcium Phosphate (CPP-ACP) Compared to Fluoride Products in an In-Vitro Demineralization Model.

The goal of this study was to evaluate the effectiveness of the toothpaste Tooth Mousse compared to conventional fluoride-based versions in the prevention of enamel and dentin demineralization. Human enamel and dentin samples (n = 120 each) were exposed to artificial demineralization at pH 4.92. During the demineralization process, the samples in the test groups were periodically treated with Tooth Mousse (TM) containing casein-phosphopeptide -amorphous-calcium-phosphate (CPP-ACP) and Tooth Mousse Plus (TMP) containing amorphous-calcium-fluoride-phosphate (CPP-ACPF) to evaluate their protective properties. Fluoride toothpastes containing 1400 ppm amine fluoride (AmF) and 1450 ppm sodium fluoride (NaF) were applied in the positive control groups. Treatment with distilled water (group C-W) or demineralization without treatment (group C-D) served as negative controls. After the demineralization and treatment process, all samples were cut longitudinally and lesion depths were determined at six locations using polarized light microscopy. In TM/TMP groups (enamel: 80/86 µm, dentin: 153/156 µm) lesion depths were significantly smaller compared to the negative control groups C-W/C-D (enamel: 99/111 µm, dentin: 163/166 µm). However, TM and TMP compared to the positive controls AmF/NaF (enamel: 58/63 µm, dentin: 87/109 µm) showed higher lesion depths. The application of TM/TMP (89%/78%) during demineralization led to a reduced number of severe lesions compared to the negative controls C-W/C-D (100%/95%). In this study we demonstrate that Tooth Mousse is less effective regarding prevention of enamel and dentin demineralization compared to fluoride containing toothpastes.

Effect of biomimetic mineralization on enamel and dentin: A Raman and EDX analysis.

OBJECTIVE: To investigate the effect of an experimental biomimetic mineralization kit (BIMIN) on the chemical composition and crystallinity of caries-free enamel and dentin samples in vitro. METHODS: Enamel and dentin samples from 20 human teeth (10 for enamel; 10 for dentin) were divided into a control group without treatment and test samples with BIMIN treatment. Quantitative analysis of tissue penetration of fluoride, phosphate, and calcium was performed using energy-dispersive X-ray spectroscopy (EDX). Mineralization depth was measured by Raman spectroscopy probing the symmetric valence vibration near 960cm-1 as a marker for crystallinity. EDX data was statistically analyzed using a paired t-test and Raman data was analyzed using the Student's t-test. RESULTS: EDX analysis demonstrated a penetration depth of fluoride of 4.10±3.32µm in enamel and 4.31±2.67µm in dentin. Calcium infiltrated into enamel 2.65±0.64µm and into dentin 5.58±1.63µm, while the penetration depths for phosphate were 4.83±2.81µm for enamel and 6.75±3.25µm for dentin. Further, up to 25µm of a newly mineralized enamel-like layer was observed on the surface of the samples. Raman concentration curves demonstrated an increased degree of mineralization up to 5-10µm into the dentin and enamel samples. SIGNIFICANCE: Biomimetic mineralization of enamel and dentin samples resulted in an increase of mineralization and a penetration of fluoride into enamel and dentin.

Lipid peroxidation and antioxidant activity in saliva of periodontitis patients: effect of smoking and periodontal treatment.

The aim of this study was to measure lipid peroxidation (as an end product of oxidative stress) and corresponding antioxidant activity in patients with periodontitis and assess the influence of smoking and periodontal treatment on these parameters. Thirty healthy subjects (including 15 smokers) were compared to periodontitis patients (n = 30, including 15 smokers). Malondialdehyde (MDA), glutathione peroxidase (GSHPx) and the total antioxidant capacity (TAOC) were recorded in saliva. The lowest level of lipid peroxidation (MDA) was measured in saliva in the non-smoking periodontally healthy subjects (0.065 +/- 0.05 micromol/l). MDA levels were significantly higher in periodontitis patients who smoked (0.123 +/- 0.08 micromol/l) compared to non-smoking controls (0.065 +/- 0.05 micromol/l; p < 0.05). The periodontally healthy subjects demonstrated significantly lower levels of GSHPx (antioxidative parameter) than the periodontitis group (p < 0.05). The TAOC flow rate (delivered antioxidant components within saliva) was significantly lower in patients with periodontitis (0.34 +/- 0.26 micromol/ml) in comparison to the controls (0.62 +/- 0.24 micromol/ml; p < 0.05). Patients with periodontitis demonstrate more lipid peroxidation than healthy subjects, and this effect is enhanced by smoking. Imbalance between oxidative stress and antioxidant capacity may play a role in the pathogenesis of periodontal disease. Non-surgical periodontal treatment leads to a reduction of MDA and GSHPx to levels comparable to healthy controls.

Critical physiological factors influencing the outcome of antimicrobial testing according to ISO 22196 / JIS Z 2801.

Bactericidal materials gained interest in the health care sector as they are capable of preventing material surfaces from microbial colonization and subsequent spread of infections. However, commercialization of antimicrobial materials requires proof of their efficacy, which is usually done using in vitro methods. The ISO 22196 standard (Japanese test method JIS Z 2801) is a method for measuring the antibacterial activity of daily goods. As it was found reliable for testing the biocidal activity of antimicrobially active materials and surface coatings most of the laboratories participating in this study used this protocol. Therefore, a round robin test for evaluating antimicrobially active biomaterials had to be established. To our knowledge, this is the first report on inaugurating a round robin test for the ISO 22196 / JIS Z 2801. The first round of testing showed that analyses in the different laboratories yielded different results, especially for materials with intermediate antibacterial effects distinctly different efficacies were noted. Scrutinizing the protocols used by the different participants and identifying the factors influencing the test outcomes the approach was unified. Four critical factors influencing the outcome of antibacterial testing were identified in a series of experiments: (1) incubation time, (2) bacteria starting concentration, (3) physiological state of bacteria (stationary or exponential phase of growth), and (4) nutrient concentration. To our knowledge, this is the first time these parameters have been analyzed for their effect on the outcome of testing according to ISO 22196 / JIS Z 2801. In conclusion, to enable assessment of the results obtained it is necessary to evaluate these single parameters in the test protocol carefully. Furthermore, uniform and robust definitions of the terms antibacterial efficacy / activity, bacteriostatic effects, and bactericidal action need to be agreed upon to simplify communication of results and also regulate expectations regarding antimicrobial tests, outcomes, and materials.

Influence of different light curing units on the cytotoxicity of various dental composites.

OBJECTIVES: To evaluate the dependence of the toxicity of various dental composites on the use of high- and low-power light curing units (LCUs). METHODS: The composites Filtek Z 250, Durafill VS, Solitaire 2 and Grandio were polymerized using different light densities from three LCUs, namely Heliolux II, Swiss Master Light (SML) and a prototype LED. The toxicity of polymerized samples was tested by exposing them to the cell culture medium up to 28 days. The extracts of the composites were collected daily and used for incubation in human gingival fibroblasts cultures. RESULTS: Slow, low-intensity curing using the LED or the Heliolux II showed similar characteristics for all four composites, regarding the cell viability rate of human gingival fibroblasts. After 1 day of storage suboptimal results could be observed for the SML/Durafill and optimal results for SML/Grandio combination (approximately 100% cell viability). In addition, the composite Solitaire the SML yielded significantly better results than the other LCUs (cell viability, p < or = 0.001: SML 60.5%, Heliolux 44.5%, LED 44.2%). Furthermore, the combination of the SML with Z 250 composite showed, after the first day and up to day 28, statistically significantly higher cell viability rates than the combination with the LED or Heliolux II. SIGNIFICANCE: This study shows that the combination of a high power LCU with some composites positively influences the HGF cell viability effected by the investigated composite extracts. Moreover, there is further indication that a reduction of composite toxicity is possible if the curing mode is adapted to the used composite.

Influence of heat from light curing units and dental composite polymerization on cells in vitro.

OBJECTIVES: The aim of the present study was to test the hypothesis that the use of a light curing unit (LCU) with high light power would result in a higher temperature and therefore a statistically significant lower number of living human gingiva fibroblasts within a pulp chamber model than the use of a light emitting diode (LED) LCU. MATERIALS AND METHODS: The composites Admira, Grandio, Filtek Supreme and Filtek Z250 were polymerized with the LCUs Swiss Master Light, Optilux 501 and an LED LCU prototype in a mould on top of a pulp chamber model. The temperature was recorded within the pulp chamber with a thermocouple. The cytotoxicity of the polymerized samples was tested by using the MTT test. RESULTS: In general there was no considerable difference in the temperature increase within the pulp chamber model for the different LCUs and composites. There was no statistically significant difference in the cell number (p=0.3767) when the different LCUs were used. CONCLUSIONS: Using a high power halogen LCU for a short time or a standard halogen or LED LCU for a longer time did not result in a considerable difference in the temperature increase or the number of living cells within a pulp chamber model. This study indicates not only that the temperature may have an effect on the living cells, but also that cells may be negatively affected by the unpolymerized composite or light of the LCUs.

Development of novel electrospun dual-drug fiber mats loaded with a combination of ampicillin and metronidazole.

OBJECTIVE: Our study was performed with the aim of preparing electrospun polylactide fibers with a combination of ampicillin (AMP) and metronidazole (MNZ) and investigating their drug release behavior and the antibacterial effect on Aggregatibacter actinomycetemcomitans and other oral pathogens. METHODS: AMP and MNZ were integrated as a combination in two separate fibers (dual fiber mats - DFW mix) of electrospun PLA fiber mats by means of multijet electrospinning and in a single fiber (single fiber mats - SFW mix). HPLC (high-performance liquid chromatography) was used to measure the released drug quantities. Agar diffusion tests were used to determine the antibacterial effect of the eluates on A. actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis and Enterococcus faecalis. The neutral red test was made to examine the cytocompatibility of the eluates with human gingival fibroblasts (hGFs). RESULTS: The release of the active agents varied with the antibiotic concentrations initially used in the fiber mats, but also with the distribution of the active agents in one or two fibers. Of the total quantity of MNZ (AMP), the SFW mix fiber mats released >60% (>70%) within a span of 5min, and 76% (71%) after 96h. With these drug concentrations released by the fiber mats (≥5m%), an antibacterial effect was achieved on A. actinomycetemcomitans and on all other species tested. Fiber mats and their eluates have no cytotoxic influence on human gingival fibroblasts (hGFs). SIGNIFICANCE: Electrospun AMP/MNZ-loaded polymer fibers are a potential drug delivery system for use in periodontal and endodontic infections.

Study of energy transfer by different light curing units into a class III restoration as a function of tilt angle and distance, using a MARC Patient Simulator (PS).

OBJECTIVE: The MARC Patient Simulator (MARC PS) enables researchers to observe the influence of handling errors on the radiant exposure that is delivered by light curing units (LCUs). Changes in the tilt angle and distance of the light guide exit face in relation to the surface of the composite increment have a distinct effect on the total amount of light delivered during polymerization and may cause insufficient conversion of the material. Therefore, the aim of the present study was to determine the influence of the tilt angle and distance of irradiance on the efficiency of light application by recording the total amount of energy using the anterior tooth sensor of the MARC PS. METHODS: The influence of the tilt angle and distance of the light guide to the sensor surface on the delivered radiant exposure was examined for three different LCUs (Celalux 2 [C2], Bluephase [BP] and Translux Powerblue [TPB]). The measurements were performed for 20 s each with five different tilt angles (α=0°, 5°, 10°, 15°, 20°) and nine different distances (L=1, 2, 3, 4, 5, 6, 7, 8, 9 mm). RESULTS: For all LCUs, a distinct influence of the tilt angle on the delivered amount of fluence was found. At 0° tilt the C2 delivered a total light energy of 38.55 J/cm(2). By increasing the tilt of the light guide the amount of energy applied significantly decreased. At 20° tilt a reduction by 31.2% of the original light fluence was recorded. However, the C2 was the most powerful LCU measured. Even under optimum measurement conditions, the BP delivered a fluence of only 14.8 J/cm(2). At a tilt angle of 20°, though, the light sensor still registered 92.7% of the original output power. Under the same conditions, the TPB delivered 81.4%. With increasing distance of the light guide exit face to the surface of the sensor all LCUs showed a significant loss in delivered light energy. At a distance of 2mm the C2 showed a reduction by 46.7%, whereas total fluence of BP and TPB were reduced by 3.8% and 4.8%, respectively. SIGNIFICANCE: The choice of LCU and the application of an appropriate curing time are important for successful polymerization. Nevertheless, a perpendicular positioning of the light guide as close as possible to the surface of the composite increment is of essential importance in order to ensure sufficient delivery of light.

Template assisted surface microstructuring of flowable dental composites and its effect on microbial adhesion properties.

OBJECTIVES: Despite their various advantages, such as good esthetic properties, absence of mercury and adhesive bonding to teeth, modern dental composites still have some drawbacks, e.g., a relatively high rate of secondary caries on teeth filled with composite materials. Recent research suggests that microstructured biomaterials surfaces may reduce microbial adhesion to materials due to unfavorable physical material-microbe interactions. The objectives of this study were, therefore, to test the hypotheses that (i) different surface microstructures can be created on composites by a novel straightforward approach potentially suitable for clinical application and (ii) that these surface structures have a statistically significant effect on microbial adhesion properties. METHODS: Six different dental composites were initially tested for their suitability for microstructuring by polydimethylsiloxane (PDMS) templates. Each composite was light-cured between a glass slide and a microstructured PDMS template. The nano-hybrid composite Grandio Flow was the only tested composite with satisfying structurability, and was therefore used for the bacterial adhesion tests. Composites samples were structured with four different microstructures (flat, cubes, linear trapezoid structures, flat pyramids) and incubated for 4h in centrifuged saliva. The bacterial adherence was then characterized by colony forming units (CFUs) and scanning electron microscopy (SEM). RESULTS: All four microstructures were successfully transferred from the PDMS templates to the composite Grandio Flow. The CFU-test as well as the quantitative analysis of the SEM images showed the lowest bacterial adhesion on the flat composite samples. The highest bacterial adhesion was observed on the composite samples with linear trapezoid structures, followed by flat pyramids and cubes. The microstructure of dental composite surfaces statistically significantly influenced the adhesion of oral bacteria. SIGNIFICANCE: Modifying the composite surface structure may be a clinically suitable approach to control the microbial adhesion and thus, to reduce the risk of secondary caries at dental composite restorations. Smaller composite surface structures may be useful for accomplishing this.

Efficacy of photodynamic therapy on inflammatory signs and two selected periodontopathogenic species in a beagle dog model.

BACKGROUND: Current research aims to find alternatives to conventional methods for suppressing periodontopathogenic bacteria. Photodynamic therapy (PDT) could be a suitable treatment procedure of periodontal infections. METHODS: In the present study, the PDT method was tested with two photosensitizers, chlorine e6 and BLC1010, in an experiment on beagle dogs. The animals were infected with Porphyromonas gingivalis (Pg) and Fusobacterium nucleatum (Fn) in all subgingival areas. After infection, we observed clinical signs of gingival inflammation, including an increase of redness and bleeding on probing. Microbiological monitoring before and after treatment was performed using polymerase chain reaction (PCR). PDT was conducted with a diode laser with a wavelength of 662 nm using a power of 0.5 W and the photosensitizers. RESULTS: The PDT procedure carried out with either of the photosensitizers caused a significant reduction in the clinical inflammation signs of redness and BOP, compared to the controls (laser only and no treatment). Furthermore, PDT with chlorine e6 caused a significant reduction in P. gingivalis-infected sites, whereas there was a lack in suppression after PDT with BLC1010. F. nucleatum could hardly be reduced with chlorine e6, and only to a certain extent with BLC 1010 and laser only. In the control groups, the Pg-infected test sites did not change. CONCLUSIONS: This study demonstrated that the photodynamic therapy using photosensitizer and a 662 nm laser light source is distinctly advantageous in reducing the periodontal signs of redness and bleeding on probing. The procedure also appears to significantly suppress P. gingivalis.

Enhanced root planing and systemic metronidazole administration improve clinical and microbiological outcomes in a two-step treatment procedure.

BACKGROUND: Recently we described a non-surgical two-step treatment concept, in which we distinguished between a first scaling and root planing step (SRP) and an additional second enhanced root planing step (ERP). Until now it is difficult to determine how often a root surface should be instrumented during ERP. METHODS: The aim of the present study was to investigate the outcomes after different root planing intensities during ERP in 37 patients with aggressive periodontitis after SRP. During ERP a full-mouth root planing was performed. The patients were randomly assigned to one of two root planing regimens (group 1, N = 12 and group 2, N = 11), based on number of curet strokes per root surface (instrumentation frequency, IF) and probing depth. Group 1: PD 1 to 3.5 mm, 4 IF (for group 2, 8 IF); PD 4 to 6 mm, 8 IF (group 2, 14 IF); PD 6.5 to 9 mm, 12 IF (group 2, 20 IF); and PD >9 mm, 16 IF (group 2, 24 IF). Group 3 patients (controls; N = 14) received only the initial SRP. All three groups received the same adjunctive systemic antibiotic treatment. RESULTS: In all groups, the results showed statistically significant differences in PD and clinical attachment level (CAL) after 6 and 24 months compared to baseline data. Compared with the controls, a significant reduction in PD was observed in groups 1 and 2. The reduction in mean PD was distinctly greater in group 2 (higher IF). Furthermore, Porphyromonas gingivalis (Pg) and Actinobacillus actinomycetemcomitans (Aa) were completely suppressed in group 2 after 24 months. CONCLUSIONS: The present results show that the extent of root planing has a distinct influence on treatment outcomes. Patients treated with the highest instrumentation frequency showed the best long-term results.