Andrea Völpel

Full-Mouth Antimicrobial Photodynamic Therapy in Fusobacterium nucleatum― Infected Periodontitis Patients

BACKGROUND The purpose of this study is to clinically and microbiologically evaluate the effect of photodynamic therapy (PDT) as a full-mouth procedure in Fusobacterium nucleatum-infected patients with periodontitis. METHODS In the present study, PDT is administered adjuvantly after scaling and root planing (SRP) to patients with localized chronic periodontitis (LCP). Twenty-four patients, in whom only F. nucleatum was detected by baseline polymerase chain reaction (PCR) after SRP, were randomly assigned to PDT and control groups. PDT was carried out once as a full-mouth disinfection in the test group. The control group was treated with the photosensitizer solution, but without laser irradiation. In all subjects, the clinical parameters plaque index, reddening, bleeding on probing (BOP), probing depth (PD), gingival recession, and clinical attachment level (CAL) were determined at the baseline examination and at 1, 4, and 12 weeks after PDT. Quantitative analysis of the F. nucleatum DNA concentration was performed by competitive PCR. All clinical indices were calculated for each test and control subject as were the median and interquartile range of each group. RESULTS In patients with LCP who received PDT treatment, significant reductions in reddening, BOP, and mean PD and CAL were observed during the observation period and with respect to controls. Four and 12 weeks after PDT, the mean PD and CAL showed significant differences from baseline values and from those of the control group. In the PDT group, 12 weeks after treatment, the F. nucleatum DNA concentration was found to be significantly reduced compared to the baseline level. CONCLUSION The results of this study show that the adjuvant application of the described PDT method is appropriate to reduce periodontal inflammatory symptoms and to successfully treat infection with F. nucleatum.

Release of metronidazole from electrospun poly(l-lactide-co-d/l-lactide) fibers for local periodontitis treatment

OBJECTIVES We aimed to achieve detailed biomaterials characterization of a drug delivery system for local periodontitis treatment based on electrospun metronidazole-loaded resorbable polylactide (PLA) fibers. METHODS PLA fibers loaded with 0.1-40% (w/w) MNA were electrospun and were characterized by SEM and DSC. HPLC techniques were used to analyze the release profiles of metronidazole (MNA) from these fibers. The antibacterial efficacy was determined by measuring inhibition zones of drug-containing aliquots from the same electrospun fiber mats in an agar diffusion test. Three pathogenic periodontal bacterial strains: Fusobacterium nucleatum, Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis were studied. Cytotoxicity testing was performed with human gingival fibroblasts by: (i) counting viable cells via live/dead staining methods and (ii) by exposing cells directly onto the surface of MNA-loaded fibers. RESULTS MNA concentration influenced fiber diameters and thus w/w surface areas: diameter being minimal and area maximal at 20% MNA. HPLC showed that these 20% MNA fibers had the fastest initial MNA release. From the third day, MNA release was slower and nearly linear with time. All fiber mats released 32-48% of their total drug content within the first 7 days. Aliquots of media taken from the fiber mats inhibited the growth of all three bacterial strains. MNA released up to the 28th day from fiber mats containing 40% MNA significantly decreased the viability of F. nucleatum and P. gingivalis and up to the 2nd day also for the resistant A. actinomycetemcomitans. All of the investigated fibers and aliquots showed excellent cytocompatibility. SIGNIFICANCE This study shows that MNA-loaded electrospun fiber mats represent an interesting class of resorbable drug delivery systems. Sustained drug release properties and cytocompatibility suggest their potential clinical applicability for the treatment of periodontal diseases.

The influence of various light curing units on the cytotoxicity of dental adhesives

OBJECTIVES The objective of this study was to test the hypothesis that various light curing units (LCUs) have an influence on the cytotoxic action of adhesive systems. METHODS Samples of the dental adhesives (Syntac), iBondTM, Clearfil Protect Bond, Prime & Bond NT, Adper Prompt L-Pop) were prepared in microwell plates, making use of the LCUs Voco Polofil Lux (VPL), EMS Swiss Master Light (SML) and the LED prototype developed by the IMT of Jena University. To obtain extracts, the samples were topped with cell culture medium, which was changed daily on the 1st to 7th days and then on the 14th, 21st and 28th day, and stored for further use at -20 degrees C. Human gingival fibroblasts (HGFs) were cultivated in the extract-containing medium for 48 h. The viability of the HGFs was determined by the neutral red (NR) uptake test. The statistical test was performed by one-way ANOVA according to Bonferroni. RESULTS During the first few days, reduction of the viability rates of the HGFs by 85-90% were observed in all adhesives. A rise up to a plateau phase was observed at different times depending on the materials. The influence of the LCUs on the cytotoxic action of the dental adhesives was clearly evident for the adhesives Syntac and Clearfil Protect Bond. In case of the Syntac extracts, cytotoxicity after polymerization with the VPL was statistically significant reduced compared to the other LCUs used (p<0.001). A comparison between all the adhesives used proved that Adper Prompt L-Pop and Prime & Bond NT had the lowest overall cytotoxicities. SIGNIFICANCE In practice, one should use combinations of dental adhesive and LCU in which the material has the least toxic influences.

Resin-composite cytotoxicity varies with shade and irradiance

OBJECTIVES The study was aimed at investigating the cytotoxicity of different composites as a function of composite shade and the light curing unit (LCU) employed. METHODS Non-polymerized and polymerized samples of the composites Grandio(®) (VOCO, Cuxhaven), Solitaire(®) (Heraeus Kulzer, Hanau) and Filtek Z 250(®) (3M/Espe, Seefeld) in two markedly differing shades (A2, C2) were prepared. Polymerization was performed with two LCUs: Heliolux II (Ivoclar/Vivadent, Ellwangen) and Swiss Master Light (EMS, Nyon, Switzerland). To obtain composite extracts, the samples were immersed in cell culture medium (DMEM--Dulbeccos Modified Eagle Medium), which was replaced daily up to the 7th day of the experiment, and then on the 14th, 21st and 28th day. After incubation of human gingival fibroblasts (HGF) with the extracts obtained, cytotoxicity was determined using the MTT test. RESULTS With the non-polymerized samples, essentially no influence of the composite shades investigated on HGF viability was detected, with the exception of the Solitaire material, where a higher cytotoxicity of the shade C2 in the non-polymerized state was found at the end of the observation period. After polymerization of the different composites, the cytotoxic reaction observed for the extracts of shade C2 was stronger than that observed for A2. After polymerization with the Heliolux II (HLX) LCU, the extracts of composites Grandio and Solitaire C2 were significantly more toxic than those of the A2 shade (p<0.01). Polymerization with the Swiss Master Light (SML) reduces this cytotoxic effect. The extracts of the Grandio composite showed the least cytotoxic effect throughout the observation period, irrespective of the LCU used. For the extracts of the Z250 specimens, the cytotoxicity observed was generally higher. SIGNIFICANCE The results show that the shade of the composite has an influence on its cytotoxicity and that this cytotoxicity is also influenced by the light curing unit used. It was observed that composites of the darker shade (C2) had a higher cytotoxicity, which varied with the LCU employed.

Photodynamic suppression of Enterococcus faecalis using the photosensitizer mTHPC

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.

Antibacterial effect of different root canal sealers on three bacterial species

OBJECTIVE Bacteria persisting in the root canal system may thwart endodontic therapy. It is therefore interesting to know whether clinically available root canal sealers have an antimicrobial effect. The objective of the present in vitro study was to investigate the antibacterial effect of various sealers on the endodontologically detectable species Enterococcus faecalis (E. faecalis), Fusobacterium nucleatum (F. nucleatum) and Porphyromonas gingivalis (P. gingivalis). METHODS The antibacterial effectiveness of the sealers was tested by means of the agar diffusion test (ADT) and the direct contact test (DCT). Eight different sealers (AH Plus(®), Hermetic(®), RoekoSeal(®), Sealapex(®), Apexit Plus(®), 2Seal(®), EndoREZ(®) and ProRoot MTA(®)) and two temporary sealers (Calxyl(®) and Gangraena Merz(®)) were tested. At first, 100μl of bacterial suspension (BS) of each individual micro-organism (optical density (OD) 0.5) was applied separately to Schaedler agar plates for the ADT. Subsequently, freshly mixed and set sealer was applied. After 48h of incubation, the inhibition zones were measured. Further, 18mg of sealer were put into each well of 48-well cell culture plates and overlaid with 400μl of Schaedler liquid medium and 100μl of BS (OD 0.5) in monoculture. Bacterial growth was determined by the DCT from the optical density of the liquid by photospectrometry after 2, 4, 6, 8, 10, 12 and 24h. RESULTS For the application, the sealer Hermetic(®), a significant suppression of the species E. faecalis, F. nucleatum and P. gingivalis was detected in both the ADT and the DCT. AH Plus(®) showed a suppressive effect on E. faecalis and F. nucleatum in the DCT. With all other sealers tested, E. faecalis was not suppressible. RoekoSeal(®), Calxyl(®) and Gangraena-Merz(®) showed no antibacterial effect on the tested species whatsoever. SIGNIFICANCE We have shown in both ADT and DCT that some root canal sealers suppress the growth of E. faecalis in vitro.

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.

Colonization of Enterococcus faecalis in a new SiO/SiO2-microtube in vitro model system as a function of tubule diameter

OBJECTIVES Endodontic pathogens can penetrate deep into dentinal tubules and therefore survive the chemo-mechanical disinfection procedures. Bacterial penetration has been mainly studies using sliced infected human teeth which, besides creating artifacts, can hinder the observation of the inner tubules due to the dense and opaque dentin structure. The aim of the present study was to develop a standardized dentin model by using artificial SiO/SiO2-microtubes of different diameters and lengths to test the penetration ability of Enterococcus faecalis. METHODS E. faecalis was grown in Schaedler fluid media for 24h and thereafter cell density was settled to 10(3)cells/ml by addition of fresh media. The bacterial solution was then incubated for 2, 3, 5 and 10 days with the SiO/SiO2-microtubes of different diameters (2-5.5μm) and lengths (100-500μm). The colonization of the tubes was evaluated by phase-contrast microscopy and the amount of colonization was determined by using a colonization index (CI; 0-none, 1-mild, 2-moderate, 3-heavy). RESULTS The diameter of the tubes strongly influences the microbial colonization. After 2 days of cultivation the 5.5μm tubes showed a moderate to heavy colonization (CI 2-3). In comparison, the 2 and 3μm tubes were clearly less colonized at the same point in time. In detail: at day 3, only mild to moderate bacteria colonization (CI 1-2) were found in the 3μm tubes and at day 10 penetration of the 2μm tubes just started. The colonization of the 5.5μm tubes was also influenced by their length. In case of the longer microtubes, though, a smaller share of heavily colonized tubes was observed. SIGNIFICANCE Our results show that E. faecalis was able to penetrate and reproduce within the standardized SiO/SiO2-microtubes in a short time. To examine the mechanisms of bacterial adhesion and invasion into tubular structures the 2μm tubes could serve as a model system because the diameters are similar to those of dentinal tubules.

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.

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

BackgroundIt was the aim of this study to evaluate the antibacterial impact of two silver(I) carbohydrate complexes with tripodal thioglycosides, namely tris[2-(β-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).MethodsMinimum 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.ResultsMIC 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.ConclusionsThe 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.