The influence of oral cavity physiological parameters: temperature, pH, and swelling on the performance of denture adhesives - in vitro study.

BACKGROUND: The various physical and chemical conditions within the oral cavity are hypothesized to have a significant influence on the behavior of denture adhesives and therefore the overall comfort of denture wearers. As such, this study aims to understand the influence of oral cavity physiological parameters such as temperature (17 to 52 °C), pH (2, 7, 10), and denture adhesive swelling due to saliva (20-120%) on the behavior of denture adhesives. This study further aims to emphasize the need for a collective approach to modelling the in-situ behavior of denture adhesives. METHODS: Rheological measurements were carried out using the Super Polygrip Ultra fresh brand denture adhesive cream to evaluate its storage modulus (G´) and loss modulus (G´´) values at a range of physiologically relevant temperatures, pH values, and degrees of swelling, to represent and characterize the wide variety of conditions that occur within the oral cavity. RESULTS: Rheological data was recorded with respect to variation of temperature, pH, and swelling. Overall, it can be seen that the physiological conditions of the oral cavity have an influence on the rheological properties of the denture adhesive cream. Specifically, our data indicates that the adhesive's mechanical properties are weakly influenced by pH, but do change with respect to the temperature in the oral cavity and the swelling rate of the adhesive. CONCLUSIONS: Our results suggest that the collective inter-play of the parameters pH, temperature and swelling ratio have an influence on the behavior of the denture adhesive. The results clearly highlight the need for developing a multi-parameter viscoelastic material model to understand the collective influence of physiological parameters on the performance of denture adhesives. Multi-parameter models can also potentially be utilized in numerically simulating denture adhesives using finite element simulations.

High-Resolution Characterization of Enamel Remineralization using Time-of-Flight Secondary Ion Mass Spectrometry and Electron Microscopy.

The aim of this in vitro study was to assess the suitability of high-resolution time-of-flight secondary-ion mass spectrometry (ToF-SIMS) for visualizing cross-sectional changes in human enamel microstructure and chemical composition during treatment and remineralization cycling of artificially generated caries lesions underneath an artificial plaque. Treatments consisted of exposure to twice daily toothpaste/water slurries prepared from 0, 1100, and 5000 µg/g fluoride (F) NaF/Silica toothpastes. In addition, treatments with slurries prepared from 1100 µg/g F SnF2/Silica toothpastes were done using 44Ca in the remineralization solution to allow for differentiation of newly formed mineral and exploration of incorporated metal dopants using ToF-SIMS. Complementary microhardness, scanning electron microscopy, and high-resolution transmission electron microscopy (HR-TEM) investigations were performed on enamel cross-sections. HR-TEM was used for the first time to determine the change in crystallinity during remineralization revealing distinct microstructural zones within one lesion. Chemical mapping using ToF-SIMS demonstrated that the distribution of F, while observed primarily in the new mineral phase, was widespread throughout the lesion with 44Ca substantially limited to the remineralizing mineral. Both penetrated the inter-rod spaces of the sound enamel illustrating how acid damage propagates into the native mineral as the caries lesion deepens. HR-TEM examination revealed different regions within the lesion characterized by distinct micro- and ultra-structures. Importantly, HR-TEM revealed a return of crystallinity following remineralization. Fluoride dose response observations verified the ability of these high-resolution techniques to differentiate remineralization efficacy. The collective results provided new insights such as the visualization of fluoride or calcium penetration pathways, as well as new tools to study the caries process.

In Vitro Model to Evaluate the Development of Discolorations on Human Enamel Caused by Treatment with Mouth Rinses and Black Tea Considering Brushing.

OBJECTIVES: The study aimed to develop and test an in vitro model to investigate the staining potential of mouth rinses on human enamel, considering alternating intake of black tea and tooth brushing, thus mimicking the situation in the oral cavity more realistically. MATERIALS AND METHODS: Eight mouth rinses with six different active ingredients (benzydamine hydrochloride [BNZ], polyhexamethylene biguanide hydrochloride [PHMB], chlorhexidine digluconate [CHX], hexetidine gluconate [HEX], octenidine dihydrochloride [OCT] and octenidine dihydrochloride + 2-phenoxyethanol [OCTP]) and concentrations were tested. Sets of six halved human molar crowns were initially pretreated by soaking in artificial saliva (30 min). Afterward, the cyclic treatment was started by soaking in artificial saliva (2 min), staining with black tea (1 min), brushing with toothpaste (5 s), and soaking in the mouth rinse (30 s). Samples were rinsed with distilled water after each treatment step. The cyclic treatment was repeated 30 times, mimicking the consumer behavior after 15 days. Photographic images were taken after 0, 10, 20, and 30 cycles. Color measurements were conducted after each staining and brushing step using a VITA-Easyshade spectrophotometer to determine the difference in lightness ∆L and the total color difference ∆E. STATISTICAL ANALYSIS: Analysis of variance and post-hoc Tukey test (α = 0.05) were applied. RESULTS: The new testing model with included brushing sequences allowed to assess the staining behavior on human teeth and provided a clear differentiation between the different investigated products. In detail, up to cycle 10, ΔE values increased for all mouth rinses with each additional cycle number. However, while ΔE values continued to increase for 0.15% BNZ, 0.1% PHMB, and 0.2% CHX between treatment cycle 10 and 30, ΔE values only slightly increased after treatment with 0.08% OCTP, 0.1% OCTP, 0.1% OCT, and 0.1% HEX. After 20 and 30 cycles, significantly less staining was found for 0.08% OCTP, 0.1% OCT, 0.1% HEX as compared to 0.2% CHX, 0.15% BNZ, and 0.1% PHMB (p < 0.05). ΔE-values were significantly lower after treatment with 0.1% OCTP as compared to 0.2% CHX1 and 0.2% CHX2 (p < 0.05). CONCLUSION: The proposed new methodology was found to be appropriate for assessing the staining progression of mouth rinses over a simulated application period of 15 days. The model allows differentiation of products with different active ingredients and concentrations.

Mapping the role of oral cavity physiological factors into the viscoelastic model of denture adhesives for numerical implementation.

Physiological parameters of the oral cavity have a profound impact on any restorative solutions designed for edentulous patients including denture adhesives. This study aims to mathematically quantify the influence of three such variables, namely: the temperature, pH, and the swelling of such adhesives under the influence of saliva on its mechanical behavior. The mathematical quantification is further aimed to implement a material model for such adhesives which considers the impact of such physiological factors. The denture adhesive is experimentally investigated by means of rheological steady state frequency sweep tests to obtain the relaxation spectrum of the material. The relaxation behavior is measured for a wide range of oral cavity temperatures and pH. Also, the adhesive is hydrated and upon swelling to different levels again tested to understand the impact of swelling on the mechanical behavior. The experimentally measured continuous relaxation spectrum is modeled as a viscoelastic material using a discrete set of points based on the Prony series discretization technique. The relaxation spectrums for various temperatures are compared and the possibility of a time-temperature superposition is explored for the model. Similarly, the measured values of Storage and loss modulus are investigated to understand the role of pH and swelling. The results in this study clearly indicated a horizontal shift in the relaxation behavior with increase in temperature. And hence, the time-temperature shift factor was calculated for the adhesive. The relaxation spectrum also showed a strong correlation with swelling of the adhesive and the pH. The influence of these two parameters were captured into the model based on the relaxation time parameter in the Prony series approach. Based on this study the impact of these parameters could be appreciated on the performance and mechanical behavior of denture adhesives and implemented into a Prony series based viscoelastic material model which can be used with numerical simulations.

Nano-CT characterization of dentinal tubule occlusion in SDF-treated dentin.

Dentin hypersensitivity is an oral health concern affecting a large percentage of the world's adult population. Occlusion of the exposed dentinal tubules is among the treatment options available, and silver diammine fluoride (SDF) is an occluding agent used for interrupting or dampening the stimulus of the dental pulp nerves that produce pain. In addition to dentin permeability testing, the evaluation of desensitizing agents occluding dentinal tubules strongly relies on microscopic techniques, such as scanning electron microscopy (SEM). Limitations of SEM are that it provides only surface images that lack detailed information on the depth of penetration and amount of material present within the treated specimen, and it is prone to sample preparation artifacts. Here, we present high-resolution X-ray computed tomography (nano-CT) as a potential method for investigating dentin specimens with occluded tubules. We studied human dentin treated with SDF as an exemplary dentinal occlusion treatment option. We evaluated the silver deposits formed on the dentin surface region near the dentinal tubules and in the tubular regions using cross-section SEM, Energy Dispersive X-ray (EDX) analysis, and nano-CT. The resulting images obtained by SEM and nano-CT had comparable resolutions, and both techniques produced images of the tubules' occlusion. Nano-CT provided three-dimensional images adequate to quantitate tubule size and orientation in space. Moreover, it enabled clear visualization of dentinal tubules in any virtual plane and estimation of the amount and depth of occluding material. Thus, nano-CT has the potential to be a valuable technique for evaluating the occluding effects of virtually any material applied to dentinal tubules, supporting deciding between the best occluding treatment options.

Enamel Fluoride Uptake Determined Using the Microbiopsy Technique and Time-of-Flight Secondary Ion Mass Spectrometry: A Pilot Study.

This study evaluated the suitability of time-of-flight secondary ion mass spectrometry (ToF-SIMS) to assess enamel fluoride uptake (EFU) in comparison with the microbiopsy technique. Enamel specimens were exposed to equimolar solutions of fluoride prepared from sodium fluoride (NaF), stannous fluoride (SnF2), or amine fluoride (AmF). EFU was quantified by both techniques on the same specimens. EFU was found to be highest for samples treated with AmF, followed by SnF2 and NaF. Both methods yielded clearly interpretable, highly correlating (r = 0.95) data. ToF-SIMS can be considered a promising alternative to the microbiopsy technique for near-surface EFU assessment.

Influence of the Amount of Toothpaste on Cleaning Efficacy: An In Vitro Study.

OBJECTIVES: The aim of this in vitro study was to test the influence of the amount of toothpaste on enamel cleaning efficacy. MATERIALS AND METHODS: The hydrated silica-based test toothpaste (radioactive dentin abrasion: 60.19 ± 1.35) contained all ingredients of a regular fluoride toothpaste. The cleaning efficacy of four different toothpaste amounts (1.00 g, 0.50 g [both "full length of brush"], 0.25 g ["pea-size"], and 0.125 g ["grain of rice-size"]) diluted in 1.00 mL water were each tested for different brushing times (10, 30, 60, 120, 180, and 300 seconds) using a standardized staining model on human molars with a brushing machine. Photographic documentation and colorimetric measurements were conducted, respectively, initially, after staining and after each brushing step. Colorimetric measurements were used to calculate the stain removal (in %). STATISTICAL ANALYSIS: Results were analyzed by one-way analysis of variance with post hoc Tukey test and Levene's test for analysis of homogeneity of variance. The level of significance α was set at ≤ 0.05. RESULTS: The cleaning efficacy decreased significantly when using smaller toothpaste amounts. Stain removal after 120 seconds brushing time was: 77.4 ± 5.0% (1.00 g toothpaste), 75.7 ± 3.4% (0.50 g toothpaste), 54.1 ± 6.7% (0.25 g toothpaste), and 48.2 ± 7.1% (0.125 g toothpaste), respectively. CONCLUSION: In this in vitro study the cleaning efficacy of a medium-abrasive, hydrated silica-based toothpaste was analyzed. Note that 1.00 g toothpaste showed for all brushing times a significantly higher cleaning efficacy than 0.25 g toothpaste and 0.125 g toothpaste.

Robot and mechanical testing of a specialist manual toothbrush for cleaning efficacy and improved force control.

BACKGROUND: Toothbrushes require flexibility to access all dental surfaces and remove plaque effectively, but they should also aim to prevent or limit overbrushing and consequent damage to teeth and gums. In two studies, the physical properties and cleaning performance of specialist test toothbrushes with flexible necks were compared to a reference rigid-necked toothbrush. METHODS: In Study 1, a universal testing machine (Instron E 10,000) with a specially designed setup was used to test the deflection behaviour of toothbrush head and neck. Untufted toothbrushes were fixed in a custom holder and force was applied to the head while the deflection was measured. In Study 2, one control and five test toothbrushes were assessed using a robot system to simulate the cleaning of artificial plaque from defined surfaces of artificial replicated human teeth in a model oral cavity (typodonts). RESULTS: Study 1 showed that the flexible-neck toothbrush deflected 2 to 2.5 times more than the rigid-neck reference toothbrush when same force was applied to the toothbrush head. Study 2 revealed that all five test toothbrushes showed statistically superior simulated plaque removal to the reference toothbrush. This superiority was observed for all test toothbrushes employing horizontal and rotating brushing action (all p = 0.001) but only three of the five toothbrushes when vertical brushing was employed (all p = 0.001). Cleaning efficacy of the test toothbrushes was demonstrated both interdentally and at the gumline locations. The Complete Protection toothbrush showed the most effective cleaning performance followed by the Repair and Protect and Rapid Relief toothbrushes. CONCLUSION: The addition of a flexible-neck component to the toothbrush designs helped to reduce stiffness and may allow more effective cleaning compared to rigid designs with controlled force distribution on the teeth and gums. This may help to provide plaque control at all potential risk areas in an in vitro robot model and could support good oral hygiene in-use.

Numerical study of the stress state on the oral mucosa and abutment tooth upon insertion of partial dentures in the mandible.

The introduction of a removable partial denture onto the dental arch significantly influences the mechanical stress characteristics of both the jawbone and oral mucosa. The aim of this study was to analyze the stress state caused by biting forces upon insertion of partial dentures into the assembly, and to understand the influence of the resulting contact pressure on its retention behavior. For this purpose, a numerical model of a removable partial denture is proposed based on 3D models developed using computer tomography data of the jawbone and the removable partial denture. The denture system rests on the oral mucosa surface and three abutment teeth. The application of bite forces on the denture generated a stick condition on the loaded regions of the denture-oral mucosa interface, which indicates positive retention of the denture onto the oral mucosa surface. Slip and negative retention were observed in the regions of the contact space that were not directly loaded. The contact pressures observed in the regions of the oral mucosa in contact with the denture were below the clinical pressure pain threshold value for soft tissue, which potentially lowers the risk of pain being experienced by denture users. Further, the variation of the retention behavior and contact pressures across different regions of the denture assembly was observed. Thus, there is a need for adhesives or restraining mechanisms for the denture system in order to avoid bending and deformation of sections of the denture as a consequence of the applied bite force.

The Impact on Dental Staining Caused by Beverages in Combination with Chlorhexidine Digluconate.

OBJECTIVES: There are several hypotheses regarding how chlorhexidine (CHX) digluconate causes staining with the role of beverages, specifically the precipitation of anionic dietary chromogens onto adsorbed cations, the most probable cause. The aim of this study was to investigate and compare the staining potential of common beverages using an in vitro staining and brushing model to better understand the interactions between chromogens from different beverage categories and the teeth. MATERIALS AND METHODS: Human enamel samples were exposed to a cyclic treatment of artificial saliva and 0.2% CHX mouthwash combined with a range of beverages, with and without brushing, simulating a period equivalent to 2 weeks. Eleven beverages were tested: diet coke, diet lemonade, white wine, red wine, lager beer, black tea, coffee, black tea with milk, coffee with milk, ginger and lemon infusion, and water. Toothbrushing was performed in a brushing simulator with toothpaste and also with water. Colorimetric differences were determined by ΔE using a VITA Easyshade dental spectrophotometer. Statistical analyses were performed by one-way analysis of variance with post hoc Tukey's honestly significant difference test and Levene's test. RESULTS: Black tea and red wine produced highest staining, which agrees with the literature. Significant staining was also observed for a ginger and lemon infusion, coffee, coffee with milk, tea with milk, and lager beer compared with water (p < 0.05). The staining potential of diet coke in combination with brushing appeared to be connected to its low pH. Both white wine and diet lemonade produced stain comparable to the water control. After treatment with high staining beverages, scanning electron microscope evaluation confirmed the formation of a surface layer. The mechanical resistance of the stain differed depending on the beverage, black tea stain was the most resistant. The addition of milk to tea and coffee considerably modified the stain layer and the adhesion to the tooth surface. CONCLUSION: The data may help demonstrate that appropriate user guidance can avoid stain and in turn help improve user compliance during short-term use of this gold standard antimicrobial treatment.

Dentin tubule occlusion by a 38% silver diamine fluoride gel: an in vitro investigation.

OBJECTIVE: Silver diamine fluoride (SDF) is effective in treatment of dentin hypersensitivity and caries lesions. However, the non-viscous solution does not easily allow clinicians to control the application area. A 38% SDF experiment gel was compared in vitro to commercial SDF for its ability to penetrate and occlude dentinal tubules. MATERIALS AND METHODS: Human root surface dentin specimens were treated with gelled or standard 38% SDF or negative control. Penetration behavior was established by Drop Shape Analysis. Precipitates at the surface and within tubules were analyzed by SEM and EDX after treatment; Results: penetration depths up to 500 µm were observed for both SDF formulations. Both formulations occluded dentinal tubules similarly. Precipitates on the dentin surface and within dentinal tubules were found for both SDF formulations, with a slight tendency for the experimental gel SDF product to be more abundant than the commercially available one. DISCUSSION: behavior of the experimental 38% SDF gel formulation appeared indistinguishable from the commercial 38% SDF product with respect to dentinal tubule penetration and occlusion. CONCLUSIONS: The experimental 38% SDF gel may be a suitable intervention for the prevention of dentin hypersensitivity.

Finite Element Evaluation of the Effect of Adhesive Creams on the Stress State of Dentures and Oral Mucosa.

The base fit between a removable partial denture (RPD) and the underlying soft tissue plays a significant role in its performance. The application of a denture adhesive is hypothesized to result in better retention of RPDs and, as a result, contribute to lower stress on the oral mucosa. The objectives of this study were to observe and compare the distribution of simulated bite forces applied to the RPD through the abutments and soft tissue for models with and without the use of a denture adhesive. Furthermore, we evaluated the possible benefit of using a denture adhesive in lowering stresses on the oral mucosa. The RPD, mandible, oral mucosa, abutment teeth supporting the RPD, and the corresponding abutment periodontal ligaments (PDLs) were modelled as 3D volumes based on computer tomography (CT) datasets. A viscoelastic adhesive layer between the RPD and oral mucosa was incorporated into this base model using Prony series approximation. The layer was developed as a volume extract using the denture surface. Finite element (FE) simulations were performed for the bite force on one of the RPD segments, with the resulting force and moments experienced by the dental structures and oral mucosa compared between the model with the adhesive layer and the base model without. As a result, the contact pressure on the oral mucosa for the model with the denture adhesive decreased to 0.15 MPa as compared to 0.25 MPa for the model without the adhesive. The potential role of denture adhesives in leading to a better fit between the RPD and oral mucosa as well as lowering contact pressures could be used to improve comfort in patients wearing RPDs.

In Vitro Surface Investigation of Calcium Fluoride-like Precipitation on Human Enamel after Topical Treatment with the Organic Fluoride Nicomethanol Hydrofluoride.

PURPOSE: The topical fluoride treatment of teeth can lead to a formation of CaF2-like material, which is considered to play a significant role in caries prevention. Different types of fluoride sources are applied. The aim of this study was to analyse the in vitro fluoridation effect of the lesser known organic fluoride compound nicomethanol hydrofluoride (NH) regarding fluoride accumulation and morphological changes on dental enamel surfaces. Materials and Methods: The fluoridation effect was investigated by scanning electron microscopy (SEM) and energy dispersive x-ray analysis (EDX) after treatment with fluoride solutions at a concentration of 1350 ppm F - and a pH value of 5.5. NH was tested against inorganic sodium fluoride (NaF) as reference. Fluoridation was done on pellicle-free and pellicle-covered enamel. Results: Formation of globular CaF2-like material was observed for both fluoride types. However, NH led to considerably higher calcium fluoride accumulation on the enamel surface as shown by both EDX and SEM. The globule diameters varied between 0.2 and 0.8 µm. Cross-sectional analysis revealed that the globular precipitates lay directly on the enamel surface; only the very surface-near volume was affected. No statistically significant difference of the fluoridation effect was measured with vs without saliva pre-treatment. Conclusion: The experiments showed a 6 times greater F - surface uptake on dental enamel with NH compared to sodium fluoride, thus suggesting an important role of NH during remineralization phases, fostering equilibrium between de- and remineralization.

In Vitro Whitening Effect of a Hydroxyapatite-Based Oral Care Gel.

OBJECTIVE: Oral care formulations aim to prevent oral diseases such as dental caries and gingivitis. Additionally, desire for white teeth still exists across all age groups. It is known that most whitening toothpastes are highly abrasive and can be harmful to teeth and gingiva. Therefore, a gel formulation with biomimetic hydroxyapatite (HAP; Ca5[PO4]3[OH]) as active ingredient was developed. This formulation was tested with respect to its tooth whitening properties in an in vitro study. MATERIALS AND METHODS: Enamel samples were allocated to either group (a) HAP gel, (b) whitening mouth rinse with phosphates, or (c) negative control (distilled water). Test products were applied by finger (a) or were rinsed (b, c) for 1, 3, and 9 (b and c only) cycles, respectively. RESULTS: Color changes (ΔE) were measured spectrophotometrically. Group (a) showed a significant increase in color changes with respect to whitening after one cycle (mean ΔE = 5.4 [±2.66], p ≤ 0.006) and three cycles (mean ΔE = 11.2 [±3.11], p < 0.0001) compared to groups (b) and (c). For group (b), a significant increase in color change was measured after three (mean ΔE = 2.77 [±1.01], p = 0.02) and nine cycles (mean ΔE = 3.27 [±1.61], p = 0.006) compared to (c). Group (c) showed only minor and statistically insignificant color changes. CONCLUSION: This in vitro study demonstrated a significantly higher ad hoc whitening effect of the HAP gel compared to the mouth rinse and water after short-time application.

Retentive Force of Glass-Ceramic Soldered Customized Zirconia Abutment Copings with Prefabricated Titanium Bases.

Two-piece abutments consisting of customized zirconia abutment copings and prefabricated titanium bases are popular due to their biological and esthetic advantages. Glass-ceramic solder (GS) is an alternative biocompatible connective agent. This in vitro study evaluated the retentive force of GS in comparison to classical resin composite cements (RC) after artificial aging and autoclaving. Ninety specimens consisting of prefabricated titanium bases and zirconia abutment copings were fabricated. The two parts of each specimen were fixed either by RC (n = 30) or GS with a luting space of either 30 µm (n = 30) or 100 µm (n = 30). Ten specimens of each group underwent autoclaving before artificial aging (water storage, thermocycling). Twenty specimens (including the 10 autoclaved specimens) of each group were exposed to a mechanical load. The retentive force between the zirconia and titanium in all specimens was determined. A fractographic analysis was performed to analyze the fracture surfaces of the GS specimens. The RC- and GS-connected two-piece abutments showed no relevant differences, independent of the luting space. RC appears to be more vulnerable to the thermal and mechanical loads than GS. Thus, GS may be an appropriate alternative to RC for two-piece abutments, especially for patients with enhanced biocompatibility requirements.

Controlled release minocycline-lipid-complex extrudates for the therapy of periodontitis with enhanced flexibility.

We describe the development of flexible minocycline-lipid-complex extrudates with optimized mechanical and drug release properties. These extrudates contain a minocycline - magnesium stearate chelate complex with a higher stability in aqueous media, which has now been incorporated in a PEG-PLGA (polyethylene glycol - poly(lactic-co-glycolic acid)) matrix. PEG 1500 has been utilized in different concentrations to serve as plasticizer. The novel formulations have been characterized by texture analysis, X-Ray powder diffraction (XRPD) and differential scanning calorimetry (DSC). Extrudates with a reduced diameter of 300 µm (previously 600 µm) were introduced, and a more sensitive quantification method with a tandem-mass spectrometry detector was developed. From all tested formulations, the extrudates consisting of Expansorb DLG 50 - 6P (PEG-PLGA, molar weight 30-60 kDa) paired with 10% PEG 1500 emerged as best formulation. These extrudates feature a drug content of 11.5% and a controlled release over at least 42 days. The release profile is without a lag time and shows initially a slightly higher release rate, which is desired. Compared to previous developments, the extrudates now offer a high flexibility combined with a large mechanical resilience, which will ease the handling and administration.

In Vitro Evaluation of Antimicrobial Activity of Minocycline Formulations for Topical Application in Periodontal Therapy.

Periodontal therapy using antimicrobials that are topically applied requires slow or controlled release devices. The in vitro antimicrobial activity of biodegradable polymer formulations that contain a new minocycline lipid complex (P-MLC) was evaluated. The new P-MLC formulations that contained 11.5% minocycline were compared with pure minocycline or an existing commercial formulation, which included determination of minimal inhibitory concentration (MIC) values against two oral bacteria and activity on six-species periodontal biofilm. Moreover, the flow of gingival crevicular fluid (GCF) was modeled up to 42 d and the obtained eluates were tested both for MIC values and inhibiting biofilm formation. In general, MICs of the P-MLC formulations were slightly increased as compared with pure minocycline. Biofilm formation was clearly inhibited by all tested formulations containing minocycline with no clear difference between them. In 3.5 d old biofilms, all formulations with 250 µg/mL minocycline decreased bacterial counts by 3 log10 and metabolic activity with no difference to pure antimicrobials. Eluates of experimental formulations showed superiority in antimicrobial activity. Eluates of one experimental formulation (P503-MLC) still inhibited biofilm formation at 28 d, with a reduction by 1.87 log10 colony forming units (CFU) vs. the untreated control. The new experimental formulations can easily be instilled in periodontal pockets and represent alternatives in local antimicrobials, and thus warrant further testing.

Extrudates of lipophilic tetracycline complexes: A new option for periodontitis therapy.

The objective of this study was to develop an improved drug delivery system for the local antimicrobial treatment of periodontitis, that offers enhanced drug stability, easy application and controlled release over several weeks. Chelate complexes consisting of a tetracycline antibiotic and a fatty acid salt were developed. Minocycline and doxycycline were paired with magnesium- and calcium stearate in different molar ratios. These chelate complexes stabilize the active pharmaceutical ingredient and enable the incorporation into a PLGA (poly(lactic-co-glycolic acid)) polymer matrix via hot melt extrusion. The chelate complexes were characterized via UV/Vis- and IR-spectroscopy. A high antibiotic activity of the complex was observed in a disc diffusion test. The drug complex was mixed with different PLGA-polymers and cryomilled in advance of the extrusion. The hot melt extrusion yielded homogeneous extrudates with a diameter from 600 to 900 µm. They contain 11.5% of minocycline, are adjustable in length and are easy to handle. In vitro release studies revealed a controlled release of the drug over 42 days. In conclusion, the developed extrudates are promising systems to improve the treatment of periodontitis.

Two genes in a pathogenicity gene cluster encoding secreted proteins are required for appressorial penetration and infection of the maize anthracnose fungus Colletotrichum graminicola.

To avoid pathogen-associated molecular pattern recognition, the hemibiotrophic maize pathogen Colletotrichum graminicola secretes proteins mediating the establishment of biotrophy. Targeted deletion of 26 individual candidate genes and seven gene clusters comprising 32 genes of C. graminicola identified a pathogenicity cluster (CLU5) of five co-linear genes, all of which, with the exception of CLU5b, encode secreted proteins. Targeted deletion of all genes of CLU5 revealed that CLU5a and CLU5d are required for full appressorial penetration competence, with virulence deficiencies independent of the host genotype and organ inoculated. Cytorrhysis experiments and microscopy showed that Δclu5a mutants form pressurized appressoria, but they are hampered in forming penetration pores and fail to differentiate a penetration peg. Whereas Δclu5d mutants elicited WT-like papillae, albeit at increased frequencies, papillae induced by Δclu5a mutants were much smaller than those elicited by the WT. Synteny of CLU5 is not only conserved in Colletotrichum spp. but also in additional species of Sordariomycetes including insect pathogens and saprophytes suggesting importance of CLU5 for fungal biology. Since CLU5a and CLU5d also occur in non-pathogenic fungi and since they are expressed prior to plant invasion and even in vegetative hyphae, the encoded proteins probably do not act primarily as effectors.

Morphological Characterization of the Self-Assembly of Virus Movement Proteins into Nanotubes in the Absence of Virus Particles.

One infection mechanism of plant viruses is the generation of nanotubes by viral movement proteins, allowing cell-to-cell virus particle transport. Previously, it was assumed that viral nanotubes extend directly from the host-cell plasma membrane. In virus-infected plants, these nanotubes reach an extraordinary diameter:length ratio (≈100 nm:µm or mm range). Here, viral nanotubes are produced in a transient protoplast system; the coding sequence for alfalfa mosaic virus movement protein is translationally fused to green fluorescent protein. The maximum extension of viral nanotubes into the culture medium is achieved 24-48 h posttransfection, with lengths in the micro- and millimeter ranges. Scanning electron microscopy and transmission electron microscopy show that strong inhomogeneous viral nanotubes are formed compared to particle-filled systems. The nanotubes have similar length, but fluctuating wall thickness and diameter and are susceptible to entanglement and recombination. Indirect methods demonstrate that movement proteins assemble independently at the top of the nanotube. These viral nanotubes grow distinctly from previously known natural particle-filled systems and are a unique biological tubular nanomaterial that has the potential for micro- or nanoapplications as a mechanically stable structural component.