Polarization of Macrophages, Cellular Adhesion, and Spreading on Bacterially Contaminated Gold Nanoparticle-Coatings in Vitro.

Biomaterial-associated infections often arise from contaminating bacteria adhering to an implant surface that are introduced during surgical implantation and not effectively eradicated by antibiotic treatment. Whether or not infection develops from contaminating bacteria depends on an interplay between bacteria contaminating the biomaterial surface and tissue cells trying to integrate the surface with the aid of immune cells. The biomaterial surface plays a crucial role in defining the outcome of this race for the surface. Tissue integration is considered the best protection of a biomaterial implant against infectious bacteria. This paper aims to determine whether and how macrophages aid osteoblasts and human mesenchymal stem cells to adhere and spread over gold nanoparticle (GNP)-coatings with different hydrophilicity and roughness in the absence or presence of contaminating, adhering bacteria. All GNP-coatings had identical chemical surface composition, and water contact angles decreased with increasing roughness. Upon increasing the roughness of the GNP-coatings, the presence of contaminating Staphylococcus epidermidis in biculture with cells gradually decreased surface coverage by adhering and spreading cells, as in the absence of staphylococci. More virulent Staphylococcus aureus fully impeded cellular adhesion and spreading on smooth gold- or GNP-coatings, while Escherichia coli allowed minor cellular interaction. Murine macrophages in monoculture tended toward their pro-inflammatory "fighting" M1-phenotype on all coatings to combat the biomaterial, but in bicultures with contaminating, adhering bacteria, macrophages demonstrated Ym1 expression, indicative of polarization toward their anti-inflammatory "fix-and-repair" M2-phenotype. Damage repair of cells by macrophages improved cellular interactions on intermediately hydrophilic/rough (water contact angle 30 deg/surface roughness 118 nm) GNP-coatings in the presence of contaminating, adhering Gram-positive staphylococci but provided little aid in the presence of Gram-negative E. coli. Thus, the merits on GNP-coatings to influence the race for the surface and prevent biomaterial-associated infection critically depend on their hydrophilicity/roughness and the bacterial strain involved in contaminating the biomaterial surface.

Bacterial Density and Biofilm Structure Determined by Optical Coherence Tomography.

Optical-coherence-tomography (OCT) is a non-destructive tool for biofilm imaging, not requiring staining, and used to measure biofilm thickness and putative comparison of biofilm structure based on signal intensity distributions in OCT-images. Quantitative comparison of biofilm signal intensities in OCT-images, is difficult due to the auto-scaling applied in OCT-instruments to ensure optimal quality of individual images. Here, we developed a method to eliminate the influence of auto-scaling in order to allow quantitative comparison of biofilm densities in different images. Auto- and re-scaled signal intensities could be qualitatively interpreted in line with biofilm characteristics for single and multi-species biofilms of different strains and species (cocci and rod-shaped organisms), demonstrating qualitative validity of auto- and re-scaling analyses. However, specific features of pseudomonas and oral multi-species biofilms were more prominently expressed after re-scaling. Quantitative validation was obtained by relating average auto- and re-scaled signal intensities across biofilm images with volumetric-bacterial-densities in biofilms, independently obtained using enumeration of bacterial numbers per unit biofilm volume. The signal intensities in auto-scaled biofilm images did not significantly relate with volumetric-bacterial-densities, whereas re-scaled intensities in images of biofilms of widely different strains and species increased linearly with independently determined volumetric-bacterial-densities in the biofilms. Herewith, the proposed re-scaling of signal intensity distributions in OCT-images significantly enhances the possibilities of biofilm imaging using OCT.

Biofilm composition and composite degradation during intra-oral wear.

OBJECTIVES: The oral environment limits the longevity of composite-restorations due to degradation caused by chewing, salivary and biofilm-produced enzymes and acids. This study investigates degradation of two resin-composites in relation with biofilm composition in vitro and in vivo. METHODS: Surface-chemical-composition of two Bis-GMA/TEGDMA composites was compared using X-ray-Photoelectron-Spectroscopy from which the number ester-linkages was derived. Composite-degradation was assessed through water contact angles, yielding surface-exposure of filler-particles. Degradation in vitro was achieved by composite immersion in a lipase solution. In order to evaluate in vivo degradation, composite samples were worn in palatal devices by 15 volunteers for 30-days periods in absence and presence of manually-brushing with water. PCR-DGGE analysis was applied to determine biofilm composition on the samples, while in addition to water contact angles, degradation of worn composites was assessed through surface-roughness and micro-hardness measurements. RESULTS: In vitro degradation by lipase exposure was highest for the high ester-linkage composite and virtually absent for the low ester-linkage composite. Filler-particle surface-exposure, surface-roughness and micro-hardness of both resin-composites increased during intra-oral wear, but filler-particle surface-exposure was affected most. However, based on increased filler-particle surface-exposure, the high ester-linkage composite degraded most in volunteers harvesting composite biofilms comprising Streptococcus mutans, a known esterase and lactic acid producer. This occurred especially in absence of brushing. SIGNIFICANCE: Degradation during intra-oral wear of a low ester-linkage composite was smaller than of a high ester-linkage composite, amongst possible other differences between both composites. S. mutans herewith is not only a cariogenic, but also a composite-degradative member of the oral microbiome.

Synergy of brushing mode and antibacterial use on in vivo biofilm formation.

OBJECTIVES: Orthodontic, multi-strand retention-wires are used as a generalized model for oral retention sites to investigate whether biofilm left-behind after powered toothbrushing in-vivo enabled better penetration of antibacterials as compared with manual brushing. METHODS: 2-cm multi-strand, stainless-steel retention-wires were placed in brackets bonded bilaterally in the upper arches of 10-volunteers. Volunteers used NaF-sodium-lauryl-sulphate-containing toothpaste and antibacterial, triclosan-containing toothpaste supplemented or not with an essential-oils containing mouthrinse. Opposite sides of the dentition including the retention-wires, were brushed manually or with a powered toothbrush. Health-care-regimens were maintained for 1-week, after which wires were removed and oral biofilm was collected. RESULTS: When powered toothbrushing was applied, slightly less bacteria were collected than after manual brushing, regardless whether an antibacterial-regimen was used or not. Powered-toothbrushing combined with antibacterial-regimens yielded lower biofilm viability than manual brushing, indicating better antibacterial penetration into biofilm left-behind after powered brushing. Major shifts in biofilm composition, with a decrease in prevalence of both cariogenic species and periodontopathogens, were induced after powered brushing using an antibacterial-regimen. CONCLUSION: Oral biofilm left-behind after powered brushing in-vivo enabled better penetration of antibacterials than after manual brushing. CLINICAL SIGNIFICANCE: Mechanical removal of oral biofilm is important for prevention of dental pathologies, but biofilm is always left-behind, such as in fissures, buccal pits, interproximal areas and gingival margins and around orthodontic appliances. Use of antibacterial toothpastes or mouthrinses can contribute to removal or killing of biofilm bacteria, but biofilm structure hampers antibacterial penetration. A synergy between brushing mode and antibacterial-regimen applied exists with clinically demonstrable effects.

In vivo biofilm formation on stainless steel bonded retainers during different oral health-care regimens.

Retention wires permanently bonded to the anterior teeth are used after orthodontic treatment to prevent the teeth from relapsing to pre-treatment positions. A disadvantage of bonded retainers is biofilm accumulation on the wires, which produces a higher incidence of gingival recession, increased pocket depth and bleeding on probing. This study compares in vivo biofilm formation on single-strand and multi-strand retention wires with different oral health-care regimens. Two-centimetre wires were placed in brackets that were bonded to the buccal side of the first molars and second premolars in the upper arches of 22 volunteers. Volunteers used a selected toothpaste with or without the additional use of a mouthrinse containing essential oils. Brushing was performed manually. Regimens were maintained for 1 week, after which the wires were removed and the oral biofilm was collected to quantify the number of organisms and their viability, determine the microbial composition and visualize the bacteria by electron microscopy. A 6-week washout period was employed between regimens. Biofilm formation was reduced on single-strand wires compared with multi-strand wires; bacteria were observed to adhere between the strands. The use of antibacterial toothpastes marginally reduced the amount of biofilm on both wire types, but significantly reduced the viability of the biofilm organisms. Additional use of the mouthrinse did not result in significant changes in biofilm amount or viability. However, major shifts in biofilm composition were induced by combining a stannous fluoride- or triclosan-containing toothpaste with the mouthrinse. These shifts can be tentatively attributed to small changes in bacterial cell surface hydrophobicity after the adsorption of the toothpaste components, which stimulate bacterial adhesion to the hydrophobic oil, as illustrated for a Streptococcus mutans strain.

Voice prosthetic biofilm formation and Candida morphogenic conversions in absence and presence of different bacterial strains and species on silicone-rubber.

Morphogenic conversion of Candida from a yeast to hyphal morphology plays a pivotal role in the pathogenicity of Candida species. Both Candida albicans and Candida tropicalis, in combination with a variety of different bacterial strains and species, appear in biofilms on silicone-rubber voice prostheses used in laryngectomized patients. Here we study biofilm formation on silicone-rubber by C. albicans or C. tropicalis in combination with different commensal bacterial strains and lactobacillus strains. In addition, hyphal formation in C. albicans and C. tropicalis, as stimulated by Rothia dentocariosa and lactobacilli was evaluated, as clinical studies outlined that these bacterial strains have opposite results on the clinical life-time of silicone-rubber voice prostheses. Biofilms were grown during eight days in a silicone-rubber tube, while passing the biofilms through episodes of nutritional feast and famine. Biofilms consisting of combinations of C. albicans and a bacterial strain comprised significantly less viable organisms than combinations comprising C. tropicalis. High percentages of Candida were found in biofilms grown in combination with lactobacilli. Interestingly, L. casei, with demonstrated favorable effects on the clinical life-time of voice prostheses, reduced the percentage hyphal formation in Candida biofilms as compared with Candida biofilms grown in absence of bacteria or grown in combination with R. dentocariosa, a bacterial strain whose presence is associated with short clinical life-times of voice prostheses.

Biofilm formation on stainless steel and gold wires for bonded retainers in vitro and in vivo and their susceptibility to oral antimicrobials.

OBJECTIVE: Bonded retainers are used in orthodontics to maintain treatment result. Retention wires are prone to biofilm formation and cause gingival recession, bleeding on probing and increased pocket depths near bonded retainers. In this study, we compare in vitro and in vivo biofilm formation on different wires used for bonded retainers and the susceptibility of in vitro biofilms to oral antimicrobials. MATERIALS AND METHODS: Orthodontic wires were exposed to saliva, and in vitro biofilm formation was evaluated using plate counting and live/dead staining, together with effects of exposure to toothpaste slurry alone or followed by antimicrobial mouthrinse application. Wires were also placed intra-orally for 72 h in human volunteers and undisturbed biofilm formation was compared by plate counting and live/dead staining, as well as by denaturing gradient gel electrophoresis for compositional differences in biofilms. RESULTS: Single-strand wires attracted only slightly less biofilm in vitro than multi-strand wires. Biofilms on stainless steel single-strand wires however, were much more susceptible to antimicrobials from toothpaste slurries and mouthrinses than on single-strand gold wires and biofilms on multi-strand wires. Also, in vivo significantly less biofilm was found on single-strand than on multi-strand wires. Microbial composition of biofilms was more dependent on the volunteer involved than on wire type. CONCLUSIONS: Biofilms on single-strand stainless steel wires attract less biofilm in vitro and are more susceptible to antimicrobials than on multi-strand wires. Also in vivo, single-strand wires attract less biofilm than multi-strand ones. CLINICAL SIGNIFICANCE: Use of single-strand wires is preferred over multi-strand wires, not because they attract less biofilm, but because biofilms on single-strand wires are not protected against antimicrobials as in crevices and niches as on multi-strand wires.

Composition and architecture of biofilms on used voice prostheses.

BACKGROUND: Biofilms on medical devices are a frequent reason for failure of the device. Voice prostheses in laryngectomized patients deteriorate within 3 to 4 months due to adhering biofilms, impeding proper functioning. Recently, we showed that these biofilms are dominated by Candida and lactobacilli. However, the early report of this finding lacked an in depth analysis of the species diversity and community structure. METHODS: Denaturing gradient gel electrophoresis (DGGE) and sequence analysis of amplified rRNA genes was used to identify microorganisms. Fluorescence in situ hybridization (FISH) was used to analyze biofilm architecture. RESULTS: Candida are the predominant fungi in the biofilms, while lactobacilli are the predominant bacteria in all investigated biofilms, followed by streptococci. FISH shows that lactobacilli and streptococci seem to have an important interaction with fungi. CONCLUSION: The results give a better understanding of biofilm formation and can be used in further development to prevent biofilm formation on voice prostheses.

Surface thermodynamic homeostasis of salivary conditioning films through polar-apolar layering.

Salivary conditioning films (SCFs) form on all surfaces exposed to the oral cavity and control diverse oral surface phenomena. Oral chemotherapeutics and dietary components present perturbations to SCFs. Here we determine the surface energetics of SCFs through contact angle measurements with various liquids on SCFs following perturbations with a variety of chemotherapeutics as well as after renewed SCF formation. Sixteen-hour SCFs on polished enamel surfaces were treated with a variety of chemotherapeutics, including toothpastes and mouthrinses. After treatment with chemotherapeutics, a SCF was applied again for 3 h. Contact angles with four different liquids on untreated and treated SCF-coated enamel surfaces were measured and surface free energies were calculated. Perturbations either caused the SCF to become more polar or more apolar, but in all cases, renewed SCF formation compensated these changes. Thus, a polar SCF attracts different salivary proteins or adsorbs proteins in a different conformation to create a more apolar SCF surface after renewed SCF formation and vice versa for apolar SCFs. This polar-apolar layering in SCF formation presents a powerful mechanism in the oral cavity to maintain surface thermodynamic homeostasis--defining oral surface properties within a narrow, biological range and influencing chemotherapeutic strategies. Surface chemical changes brought about by dietary or chemotherapeutic perturbations to SCFs make it more polar or apolar, but new SCFs are rapidly formed compensating for changes in surface energetics.

Surfactive and antibacterial activity of cetylpyridinium chloride formulations in vitro and in vivo.

AIM: To compare effects of three cetylpyridinium chloride (CPC) formulations with and without alcohol and Tween80 on physico-chemical properties of salivary pellicles, bacterial detachment in vitro and bacterial killing in vivo. MATERIAL AND METHODS: Adsorption of CPC to salivary pellicles in vitro was studied using X-ray photoelectron spectroscopy and water contact angle measurements. Adhesion and detachment of a co-adhering bacterial pair was determined in vitro using a flow chamber. Killing was evaluated after live/dead staining after acute single use in vivo on 24- and 72-h-old plaques after 2-week continuous use. RESULTS: The most pronounced effects on pellicle surface chemistry and hydrophobicity were observed after treatment with the alcohol-free formulation, while the pellicle thickness was not affected by any of the formulations. All CPC formulations detached up to 33% of the co-adhering pair from pellicle surfaces. Bacterial aggregate sizes during de novo deposition were enhanced after treatment with the alcohol-free formulation. Immediate and sustained killing in 24 and 72 h plaques after in vivo, acute single use as well as after 2-week continuous use were highest for the alcohol-free formulation. CONCLUSIONS: CPC bioavailability in a formulation without alcohol and Tween80 could be demonstrated through measures of pellicle surface properties and bacterial interactions in vitro as well as bacteriocidal actions on oral biofilms in vivo.

Influence of adhesion to activated carbon particles on the viability of waterborne pathogenic bacteria under flow.

In rural areas around the world, people often rely on water filtration plants using activated carbon particles for safe water supply. Depending on the carbon surface, adhering microorganisms die or grow to form a biofilm. Assays to assess the efficacy of activated carbons in bacterial removal do not allow direct observation of bacterial adhesion and the determination of viability. Here we propose to use a parallel plate flow chamber with carbon particles attached to the bottom plate to study bacterial adhesion to individual carbon particles and determine the viability of adhering bacteria. Observation and enumeration is done after live/dead staining in a confocal laser scanning microscope. Escherichiae coli adhered in higher numbers than Raoultella terrigena, except to a coconut-based carbon, which showed low bacterial adhesion compared to other wood-based carbon types. After adhesion, 83-96% of the bacteria adhering to an acidic carbon were dead, while on a basic carbon 54-56% were dead. A positively charged, basic carbon yielded 76-78% bacteria dead, while on a negatively charged coconut-based carbon only 32-37% were killed upon adhesion. The possibility to determine both adhesion as well as the viability of adhering bacteria upon adhesion to carbon particles is most relevant, because if bacteria adhere but remain viable, this still puts the water treatment system at risk, as live bacteria can grow and form a biofilm that can then be shedded to cause contamination.

Efficacy and mechanisms of non-antibacterial, chemical plaque control by dentifrices--an in vitro study.

OBJECTIVES: The provision of antiplaque benefits to dentifrices assists patients in improving hygiene and reducing susceptibility to gingivitis and caries. Chemical plaque control involves different mechanisms and is mostly associated with antibacterial effects, but also includes effects on pellicle surface chemistry to improve cleansing or discourage renewed plaque formation. It is the aim of this paper to analyze in vitro detachment of co-aggregating oral actinomyces and streptococci from pellicle surfaces by dentifrice supernates and to study subsequent de novo streptococcal deposition. METHODS: Detachment by dentifrices of a co-adhering bacterial pair was studied in the parallel plate flow chamber on a 16 h pellicle coated surface. After detachment by perfusing the chamber with a dentifrice, re-deposition was initiated by flowing with a fresh streptococcal suspension. The dentifrices included both a regular, SLS-fluoride based formulation as well a pyrophosphate, anticalculus and antimicrobial formulations. RESULTS: All dentifrice supernates containing SLS were effective in detaching co-adhering bacteria from pellicles surfaces, except in combination with SnF(2). When hexametaphosphate was added immediate detachment was relatively low, but continued even during re-deposition. The re-deposition of streptococci after detachment by other, NaF containing dentifrices involved relatively few large aggregates, presumably because fluoride was able to block bi-dentate calcium binding sites on the bacterial cell surfaces, mediating co-adhesion. When pyrophosphate was present in addition to NaF, re-deposition involved significantly more large aggregates, likely because pyrophosphate served as a bi-dentate bridge between calcium bound on the bacterial cell surfaces. CONCLUSION: Commercially available dentifrice formulations differ in their ability to stimulate bacterial detachment from pellicles and dependent on their composition yield the formation of large co-adhering aggregates of actinomyces and streptococci in de novo deposition.

Atomic force microscopy study on specificity and non-specificity of interaction forces between Enterococcus faecalis cells with and without aggregation substance.

Enterococcus faecalis is one of the leading causes of hospital-acquired infections, and indwelling medical devices are especially prone to infection. E. faecalis expressing aggregation substance (Agg) adheres to biomaterial surfaces by means of positive cooperativity, i.e. the ability of one adhering organism to stimulate adhesion of other organisms in its immediate vicinity. In this study, atomic force microscopy (AFM) was used to measure the specificity and non-specificity of interaction forces between E. faecalis cells with and without Agg. Bacteria were attached to a substratum surface and a tip-less cantilever. Two E. faecalis strains expressing different forms of Agg showed nearly twofold higher interaction forces between bacterial cells than a strain lacking Agg [adhesive force (F(adh)), -1.3 nN]. The strong interaction forces between the strains with Agg were reduced after adsorption of antibodies against Agg from -2.6 and -2.3 nN to -1.2 and -1.3 nN, respectively. This suggests that the non-specific interaction force between the enterococci amounts to approximately 1.2 nN, while the specific force component is only twofold stronger. Comparison of the results of the AFM interaction forces with the positive cooperativity after adhesion to a biomaterial in a parallel-plate flow chamber showed that in the absence of strong interaction forces between the cells, positive cooperativity was also absent. In conclusion, this is believed to be the first time that the influence of specific antibodies on interaction forces between E. faecalis cells has been demonstrated by AFM, thereby experimentally distinguishing between specific and non-specific force components.