Adhesion of human gingival fibroblasts/<Emphasis Type="Italic">Streptococcus mitis</Emphasis> co-culture on the nanocomposite system Chitlac-nAg

Composite materials are increasingly used as dental restoration. In the field of biomaterials, infections remain the main reason of dental devices failure. Silver, in the form of nanoparticles (AgNPs), ions and salt, well known for its antimicrobial properties, is used in several medical applications in order to avoid bacterial infection. To reduce both bacterial adhesion to dental devices and cytotoxicity against eukaryotic cells, we coated BisGMA/TEGDMA methacrylic thermosets with a new material, Chitlac-nAg, formed by stabilized AgNPs with a polyelectrolyte solution containing Chitlac. Here we analyzed the proliferative and adhesive ability of human gingival fibroblasts (HGFs) on BisGMA/TEGDMA thermosets uncoated and coated with AgNPs in a coculture model system with Streptococcus mitis. After 48 h, HGFs well adhered onto both surfaces, while S. mitis cytotoxic response was higher in the presence of AgNPs coated thermosets. After 24 h thermosets coated with Chitlac as well as those coated with Chitlac-nAg exerted a minimal cytotoxic effect on HGFs, while after 48 h LDH release raised up to 20 %. Moreover the presence of S. mitis reduced this release mainly when HGFs adhered to Chitlac-nAg coated thermosets. The reduced secretion of collagen type I was significant in the presence of both surfaces with the co-culture system even more when saliva is added. Integrin β1 localized closely to cell membranes onto Chitlac-nAg thermosets and PKCα translocated into nuclei. These data confirm that Chitlac-nAg have a promising utilization in the field of restorative dentistry exerting their antimicrobial activity due to AgNPs without cytotoxicity for eukaryotic cells.     

Molecular mechanisms driving <Emphasis Type="Italic">Streptococcus mitis</Emphasis> entry into human gingival fibroblasts in presence of chitlac-nAg and saliva

The molecular mechanisms leading to Streptococcus mitis capability of entering oral cells were investigated in a co-culture of S. mitis and Human Gingival Fibroblasts (HGFs) in the presence of saliva. An innovative colloidal solution based on silver nanoparticles (Chitlac-nAg), a promising device for daily oral care, was added to the experimental system in order to study the effects of silver on the bacterial overgrowth and ability to enter non-phagocytic eukaryotic cells. The entry of bacteria into the eukaryotic cells is mediated by a signalling pathway involving FAK, integrin β1, and the two cytoskeleton proteins vinculin and F-actin, and down-regulated by the presence of saliva both at 3 and 48?h of culture, whereas Chitlac-n Ag exposure seems to influence, by incrementing it, the number of bacteria entering the fibroblasts only at 48?h. The formation of fibrillary extrusion from HGFs and the co-localization of bacteria and silver nanoparticles within the fibroblast vacuoles were also recorded. After longer experimental times (72 and 96?h), the number of S. mitis chains inside gingival cells is reduced, mainly in presence of saliva. The results suggest an escape of bacteria from fibroblasts to restore the microbial balance of the oral cavity.

Open image in new window

     

Cell-protection mechanism through autophagy in HGFs/<Emphasis Type="Italic">S. mitis</Emphasis> co-culture treated with Chitlac-nAg

Silver-based products have been proven to be effective in retarding and preventing bacterial growth since ancient times. In the field of restorative dentistry, the use of silver ions/nanoparticles has been explored to counteract bacterial infections, as silver can destroy bacterial cell walls by reacting with membrane proteins. However, it is also cytotoxic towards eukaryotic cells, which are capable of internalizing nanoparticles. In this work, we investigated the biological effects of Chitlac-nAg, a colloidal system based on a modified chitosan (Chitlac), administered for 24–48?h to a co-culture of primary human gingival fibroblasts and Streptococcus mitis in the presence of saliva, developed to mimic the microenvironment of the oral cavity. We sought to determine its efficiency to combat oral hygiene-related diseases without affecting eukaryotic cells. Cytotoxicity, reactive oxygen species production, apoptosis induction, nanoparticles uptake, and lysosome and autophagosome metabolism were evaluated. In vitro results show that Chitlac-nAg does not exert cytotoxic effects on human gingival fibroblasts, which seem to survive through a homoeostasis mechanism involving autophagy. That suggests that the novel biomaterial Chitlac-nAg could be a promising tool in the field of dentistry.     

S. sanguinis adhesion on rough titanium surfaces: Effect of culture media

Bacterial colonization plays a key role in dental implant failure, because they attach directly on implant surface upon implantation. Between different types of bacteria associated with the oral environment, Streptococcus sanguinis is essential in this process since it is an early colonizer. In this work the relationship between titanium surfaces modified by shot blasting treatment and S. sanguinis adhesion; have been studied in approached human mouth environment. Bacteria pre-inoculated with routinary solution were put in contact with titanium samples, shot-blasted with alumina and silicon carbide, and adhesion results were compared with those obtained when bacteria were pre-inoculated with modified artificial saliva medium and on saliva pre-coated titanium samples. Our results showed that bacterial adhesion on titanium samples was influenced by culture conditions. When S. sanguinis was inoculated in routinary culture media, colonies forming unities per square millimeter presented an increment correlated with roughness and surface energy, but separated by the type of particle used during shot-blasting treatment; whereas in modified artificial saliva only a relationship between bacteria adhered and the increment in both roughness and surface energy were observed, regardless of the particle type. Finally, on human saliva pre-coated samples no significant differences were observed among roughness, surface energy or particle.     

Translationally controlled tumor protein against apoptosis from 2-hydroxy-ethyl methacrylate in human dental pulp cells

2-Hydroxy-ethyl methacrylate (HEMA) is a major monomer released from resin-base dental restorative materials. HEMA is cytotoxic to pulp cells and leads to apoptosis. This study examined the effect of Translationally Controlled Tumor Protein (TCTP) against apoptosis from HEMA. TCTP from banana prawn (Penaeus merguiensis) was cloned and the protein was purified. It significantly increased the number of viable of HEMA-treated cells compared to HEMA-treated cells alone. Flow cytometry indicated the addition of TCTP at 10 μg/ml to 8 and 10 mM HEMA decreased the apoptotic cells from 20 to 10%. The proliferative property and anti-apoptotic activity against HEMA was concentration dependent. It was interesting that the added TCTP was not detected inside the cells and the native human TCTP was decreased after treated with HEMA and TCTP (20 μg/ml) + HEMA(10 mM) for 24 h. These results provided preliminary information, which may contribute to the development of less toxic dental materials.     

In vitro antimicrobial properties of silver–polysaccharide coatings on porous fiber-reinforced composites for bone implants

Biostable fiber-reinforced composite (FRC) implants prepared from bisphenol-A-dimethacrylate and triethyleneglycoldimethacrylate resin reinforced with E-glass fibers have been successfully used in cranial reconstructions in 15 patients. Recently, porous FRC structures were suggested as potential implant materials. Compared with smooth surface, porous surface allows implant incorporation via bone ingrowth, but is also a subject to bacterial attachment. Non-cytotoxic silver–polysaccharide nanocomposite coatings may provide a way to decrease the risk of bacterial contamination of porous FRC structures. This study is focused on the in vitro characterization of the effect porosity on the antimicrobial efficiency of the coatings against Staphylococcus aureus and Pseudomonas aeruginosa by a series of microbiological tests (initial adhesion, antimicrobial efficacy, and biofilm formation). Characterization included confocal laser scanning microscopy and scanning electron microscopy. The effect of porosity on the initial attachment of S. aureus was pronounced, but in the case of P. aeruginosa the effect was negligible. There were no significant effects of the coatings on the initial bacterial attachment. In the antimicrobial efficacy test, the coatings were potent against both strains regardless of the sample morphology. In the biofilm tests, there were no clear effects either of morphology or of the coating. Further coating development is foreseen to achieve a longer-term antimicrobial effect to inhibiting bacterial implant colonization.     

Adhesion of streptococci to saliva-coated and uncoated composite-based resins

This study was designed to evaluate the bacterial adhesion to five types of experimental composite-based resins and a commercial composite resin used as a control. Physicochemical surface characteristics of composite resins with and without an artificial saliva coating were measured. The relationship between the numbers of adhering cells (Streptococcus sanguis, S. mutans and S. sobrinus) and surface characteristics was analysed. The values of contact angles and the number of adhering cells were small with saliva coating. S. sanguis ATCC 10557 showed a positive correlation (r=0.835, p<0.05) with the contact angles of uncoated resins, whereas no relationship was observed for saliva-coated resins. With S. mutans Ingbritt the cell numbers adhering to resins correlated strongly (p<0.01) with the values of zeta potential of resins for either saliva coated or uncoated. Electrical repulsion forces had a strong contribution to adherence of cells such as S. mutans and S. sobrinus which show a high absolute zeta potential.     

Cellular uptake and cell-to-cell transfer of polyelectrolyte microcapsules within a triple co-culture system representing parts of the respiratory tract

Polyelectrolyte multilayer microcapsules around 3.4 micrometers in diameter were added to epithelial cells, monocyte-derived macrophages, and dendritic cells in vitro and their uptake kinetics were quantified. All three cell types were combined in a triple co-culture model, mimicking the human epithelial alveolar barrier. Hereby, macrophages were separated in a three-dimensional model from dendritic cells by a monolayer of epithelial cells. While passing of small nanoparticles has been demonstrated from macrophages to dendritic cells across the epithelial barrier in previous studies, for the micrometer-sized capsules, this process could not be observed in a significant amount. Thus, this barrier is a limiting factor for cell-to-cell transfer of micrometer-sized particles.     

Adhesion-dependent rupturing of Saccharomyces cerevisiae on biological antimicrobial nanostructured surfaces

Recent studies have shown that some nanostructured surfaces (NSS), many of which are derived from surfaces found on insect cuticles, rupture and kill adhered prokaryotic microbes. Most important, the nanoscale topography is directly responsible for this effect. Although parameters such as cell adhesion and cell wall rigidity have been suggested to play significant roles in this process, there is little experimental evidence regarding the underlying mechanisms involving NSS-induced microbial rupture. In this work, we report the NSS-induced rupturing of a eukaryotic microorganism, Saccharomyces cerevisiae. We show that the amount of NSS-induced rupture of S. cerevisiae is dependent on both the adhesive qualities of the yeast cell and the nanostructure geometry of the NSS. Thus, we are providing the first empirical evidence that these parameters play a direct role in the rupturing of microbes on NSS. Our observations of this phenomenon with S. cerevisiae, particularly the morphological changes, are strikingly similar to that reported for bacteria despite the differences in the yeast cell wall structure. Consequently, NSS provide a novel approach for the control of microbial growth and development of broad-spectrum microbicidal surfaces.     

The adherence of <Emphasis Type="Italic">Candida albicans</Emphasis> to acrylic resin reinforced with different fibers

One important aethiological factor in the pathogenesis of chronic atrophic candidosis is the presence of Candida albicans on the fitting surface of the dentures. Fibers may come into contact with oral mucosa during the finishing procedures of acrylic resins. The exposed fibers may provide mechanical retention for yeast cells at the interface of the components. The effect of two different glass fibers and two different environments were evaluated in respect of Candida albicans adhesion to the acrylic surface. Half of the acrylic samples reinforced with two different fibers (Sticknet and Eversticknet) were pretreated with phosphate-buffered saline (PBS) and the rest with unstimulated saliva. The test specimens were placed in yeast suspension. The adhered cells were examined with a scanning electron microscope. The amount of adhered cells in PBS was lower for Eversticknet but the difference was not significant (p > 0.05). The number of yeast cells decreased in saliva for both groups and the difference was statistically significant for the samples reinforced with Eversticknet (p < 0.01). The use of Sticknet or Eversticknet as reinforcing material for poly(methylmethacrylate) had no effect on surface topography due to the same adhesion state of Candida albicans. The presence of a salivary pellicle derived from unstimulated saliva reduced adhesion of Candida albicans.     

Development of compartmentalized antibacterial systems based on encapsulated alliinase

Allicin, an organic compound produced via the transformation of biologically inactive alliin by alliinase, an enzyme found in garlic, combines a strong antibacterial effect with suppressed development of bacterial resistance. However, because of the high reactivity and volatility, controlled in-situ production of allicin that mimics the natural synthesis is essential to achieve desired therapeutic effects. In this work, the spray-drying technique was employed for encapsulation of alliinase into polymer micro-carriers with an emphasis on the effect of process parameters, i.e., drying temperature, nozzle type, choice of the carrier materials, on the activity of encapsulated alliinase in soluble maltodextrin and swellable chitosan microparticles. The results show that maltodextrin is a suitable carrier for the effective protection of alliinase against thermal stress. On the other hand, we can control allicin production and release from chitosan carriers with immobilized alliinase by variation of the cross-linker amount. Antibacterial activity of in-situ formed allicin vapors was confirmed against Escherichia coli bacterial strain using customized sample holders preventing physical contact of powder sample with the inoculated agar plate.     

Effect of novel chitosan-fluoroaluminosilicate resin modified glass ionomer cement supplemented with translationally controlled tumor protein on pulp cells

Dental materials that can promote cell proliferation and function is required for regenerative pulp therapy. Resin modified glass ionomer cement (RMGIC), a broadly used liner or restorative material, can cause apoptosis to pulp cells mainly due to HEMA (2-hydroxyethyl methacrylate), the released residual monomer. Recent studies found that chitosan and albumin could promote release of protein in GIC while translationally controlled tumor protein (TCTP) has an anti-apoptotic activity against HEMA. The aim of this study was to examine the effect of chitosan and albumin modified RMGIC (Exp-RMGIC) supplemented with TCTP on pulp cell viability and mineralization. Exp-RMGIC+TCTP was composed of RMGIC powder incorporated with 15 % of chitosan, 5 % albumin and supplemented with TCTP mixed with the same liquid components of RMGIC. The effect of each specimen on pulp cells was examined using the Transwell plate. From the MTT assay, Exp-RMGIC+TCTP had the highest percentages of viable cells (P < 0.05) at both 24 and 74 h. Flow cytometry revealed that, after 24 h, Exp-RMGIC+TCTP gave the lowest percentages of apoptotic cells compared to other groups. There was no difference in alkaline phosphatase (ALP) activity among different formula of the specimens, while cells cultured in media with TCTP had higher ALP activity. Von Kossa staining revealed that RMGIC+TCTP, and Exp-RMGIC+TCTP had higher percentages of calcium deposit area compared to those without TCTP. It was concluded that Exp-RMGIC supplemented with TCTP had less cytotoxicity than RMGIC and can protect cells from apoptosis better than RMGIC supplemented with TCTP.     

Enhancing the grafting of poly(2-hydroxyethyl methacrylate) on silica nanoparticles (SiO2-g-PHEMA) by the sequential UV-induced graft polymerization with a multiple-UV irradiation

Grafting of poly(2-hydroxyethyl methacrylate) on silica nanoparticles was accomplished via the sequential UV-induced graft polymerization. Under UV-irradiation, the silica was functionalized with the surface initiator, benzophenone (BP) and subsequently graft-polymerized with 2-hydroxyethyl methacrylate (HEMA). The grafting on the silica particles was confirmed by DSC analysis which revealed a shift of the glass transition temperature (T_g) of grafted PHEMA to higher temperature than T_g of ungrafted PHEMA. A significant improvement in the grafting efficiency and the grafting percentage was achieved when a sequential grafting approach was taken, employing multiple UV exposures. Using this approach, the efficient chain extension from the grafted-PHEMA was possible without producing significant amounts of ungrafted PHEMA when low HEMA concentrations were used during each UV-exposure.     

Orthogonal Cell-Based Biosensing: Fluorescent, Electrochemical, and Colorimetric Detection with Silica-Immobilized Cellular Communities Integrated with an ITO-Glass/Plastic Laminate Cartridge

This is the first report of a living cell-based environmental sensing device capable of generating orthogonal fluorescent, electrochemical, and colorimetric signals in response to a single target analyte in complex media. Orthogonality is enabled by use of cellular communities that are engineered to provide distinct signals in response to the model analyte. Coupling these three signal transduction methods provides additional and/or complementary data regarding the sample which may reduce the impact of interferants and increase confidence in the sensor's output. Long-term stability of the cells was addressed via 3D entrapment within a nanostructured matrix derived from glycerated silicate, which allows the device to be sealed and stored under dry, ambient conditions for months with significant retention in cellular activity and viability (40% viability after 60 days). Furthermore, the first co-entrapment of eukaryotic and bacterial cells in a silica matrix is reported, demonstrating multianalyte biodetection by mixing disparate cell lines at intimate proximities which remain viable and responsive. These advances in cell-based biosensing open intriguing opportunities for integrating living cells with nanomaterials and macroscale systems.     

Influence of 2-hydroxyethyl methacrylate content on the optical properties of experimental 2-hydroxyethyl methacrylate-added dental glass ionomer

The influence of 2-hydroxyethyl methacrylate (HEMA) on the properties of HEMA-added dental glass ionomer (HAGI) should be determined systematically to develop a smart restorative material. The purposes of this study were to determine the influence of incrementally added HEMA in experimental HAGIs on the color, translucency, opalescence, fluorescence and compressive strength, and to compare with those of commercial resin-modified glass ionomer (RMGI) materials. A varied amount of HEMA (10–50 wt.%) was added into commercial glass ionomer liquid (Fuji II), which was then mixed with three shades of the same glass ionomer powder (shade nos. 21, 22 and 23). RMGIs from the same manufacturer were also investigated. Five specimens, 10 mm in diameter and 2 mm in thickness, were fabricated for each condition. Color of the HAGIs and RMGIs was measured with a reflectance spectrophotometer in the reflectance and the transmittance modes. Translucency, opalescence and fluorescence parameters were calculated. Compressive strength was determined. As the HEMA content increased, CIE L^* value (lightness) decreased while the chroma increased. CIE a^* value showed small and CIE b^* value showed high increase as the HEMA content increased. Increase in translucency was generally dependent on HEMA content except for 30% HEMA condition of shade no. 21. Opalescence decreased as the HEMA content increased while the trend of fluorescence change was shade-dependent. Compressive strength significantly increased after HEMA addition except 50% HEMA condition. The influence of HAMA on the optical properties of HAGIs varied by the shade of powder and the amount of HEMA. For the most relevant simulation of the optical properties of teeth, addition of 30–40% HEMA is recommended when formulating HEMA-added glass ionomers.     

Size-dependent antibacterial activities of silver nanoparticles against oral anaerobic pathogenic bacteria

Dental caries and periodontal disease are widespread diseases for which microorganism infections have been identified as the main etiology. Silver nanoparticles (Ag Nps) were considered as potential control oral bacteria infection agent due to its excellent antimicrobial activity and non acute toxic effects on human cells. In this work, stable Ag Nps with different sizes (~5, 15 and 55 nm mean values) were synthesized by using a simple reduction method or hydrothermal method. The Nps were characterized by powder X-ray diffraction, transmission electron microscopy and UV–vis absorption spectroscopy. The antibacterial activities were evaluated by colony counting assay and growth inhibition curve method, and corresponding minimum inhibitory concentration (MIC) against five anaerobic oral pathogenic bacteria and aerobic bacteria E. coli were determined. The results showed that Ag Nps had apparent antibacterial effects against the anaerobic oral pathogenic bacteria and aerobic bacteria. The MIC values of 5-nm Ag against anaerobic oral pathogenic bacteria A. actinomycetemcomitans, F. nuceatum, S. mitis, S. mutans and S. sanguis were 25, 25, 25, 50 and 50 μg/mL, respectively. The aerobic bacteria were more susceptible to Ag NPs than the anaerobic oral pathogenic bacteria. In the mean time, Ag NPs displayed an obvious size-dependent antibacterial activity against the anaerobic bacteria. The 5-nm Ag presents the highest antibacterial activity. The results of this work indicated a potential application of Ag Nps in the inhibition of oral microorganism infections.     

Poly(hydroxyethyl methacrylate-co-methacryloylamidotryptophane) nanospheres and their utilization as affinity adsorbents for porcine pancreas lipase adsorption

Novel nanospheres with an average size of 350 nm utilizing N-methacryloyl-(l)-tryptophane methyl ester (MATrp) as a hydrophobic monomer were prepared by surfactant free emulsion polymerization of 2-hydroxyethyl methacrylate (HEMA), (MATrp) conducted in an aqueous dispersion medium. MATrp was synthesized using methacryloyl chloride and (l)-tryptophane methyl ester. Specific surface area of the non-porous nanospheres was found to be 1902.3 m²/g. poly(HEMA–MATrp) nanospheres were characterized by Fourier Transform Infrared Spectroscopy (FTIR) and scanning electron microscopy (SEM). Average particle size and size distribution measurements were also performed. Elemental analysis of MATrp for nitrogen was estimated at 1.36 mmol/g nanospheres. Then, poly(HEMA–MATrp) nanospheres were used in the adsorption of porcine pancreas lipase in a batch system. Using an optimized adsorption protocol, a very high loading of 558 mg enzyme/g nanospheres was obtained. The adsorption phenomena appeared to follow a typical Langmuir isotherm. The K_m value for immobilized lipase (16.26 mM) was higher than that of free enzyme (10.34 mM). It was observed that enzyme could be repeatedly adsorbed and desorbed without significant loss in adsorption amount or enzyme activity. These findings show considerable promise for this material as an adsorption matrix in industrial processes.     

Antibacterial activity of a triclosan-containing resin composite matrix against three common oral bacteria

This study investigated the antibacterial effect of a resin composite matrix with or without incorporated triclosan (0.3 wt%) on Streptococcus mutans, Actinomyces viscosus and Lactobacillus casei. In the quantitative assay, bacterial suspensions were filled into 20-μl cavities within temporary restorative resins. After 0, 4, 8, 12, 24 and 48 h of incubation, the suspensions were removed from the restoratives and the numbers of viable bacteria were determined. Bacterial suspensions incubated without restoratives served as the controls. Ten replicates were carried out for each experiment. The resin composite containing triclosan demonstrated variable degrees of antibacterial activity against the microorganisms, revealing a significant inhibitory effect on S. mutans within 12 h compared to the control. The viable counts of A. viscosus significantly decreased after 24 h. A significant reduction of L. casei was observed after 48 h. The unloaded resin composite did not reveal a marked antibacterial effect. The resin composite loaded with triclosan might be beneficial in preventing cavity contamination and minimizing the risk of pulpal irritation in the short-term.     

Aggregation by depletion attraction in cultures of bacteria producing exopolysaccharide

In bacteria, the production of exopolysaccharides—polysaccharides secreted by the cells into their growth medium—is integral to the formation of aggregates and biofilms. These exopolysaccharides often form part of a matrix that holds the cells together. Investigating the bacterium Sinorhizobium meliloti, we found that a mutant that overproduces the exopolysaccharide succinoglycan showed enhanced aggregation, resulting in phase separation of the cultures. However, the aggregates did not appear to be covered in polysaccharides. Succinoglycan purified from cultures was applied to different concentrations of cells, and observation of the phase behaviour showed that the limiting polymer concentration for aggregation and phase separation to occur decreased with increasing cell concentration, suggesting a ‘crowding mechanism’ was occurring. We suggest that, as found in colloidal dispersions, the presence of a non-adsorbing polymer in the form of the exopolysaccharide succinoglycan drives aggregation of S. meliloti by depletion attraction. This force leads to self-organization of the bacteria into small clusters of laterally aligned cells, and, furthermore, leads to aggregates clustering into biofilm-like structures on a surface.     

Cellular uptake and cell-to-cell transfer of polyelectrolyte microcapsules within a triple co-culture system representing parts of the respiratory tract

Abstract

Polyelectrolyte multilayer microcapsules around 3.4 micrometers in diameter were added to epithelial cells, monocyte-derived macrophages, and dendritic cells in vitro and their uptake kinetics were quantified. All three cell types were combined in a triple co-culture model, mimicking the human epithelial alveolar barrier. Hereby, macrophages were separated in a three-dimensional model from dendritic cells by a monolayer of epithelial cells. While passing of small nanoparticles has been demonstrated from macrophages to dendritic cells across the epithelial barrier in previous studies, for the micrometer-sized capsules, this process could not be observed in a significant amount. Thus, this barrier is a limiting factor for cell-to-cell transfer of micrometer-sized particles.