Doron Steinberg

Mechanism of adsorption of human albumin to titanium in vitro

Our previous studies have shown that human albumin is one of the main salivary proteins that adsorb to titanium (Ti). The goal of the present study was to investigate the role of electrostatic interactions in the adsorption of human albumin to Ti-oxide (TiO2) in vitro. The binding profile of human albumin to Ti was analyzed according to an adsorption isotherm. Purified human serum albumin (HSA) was suspended with native, calcium-, magnesium-, or potassium-treated commercially pure Ti powders, at pH 3.0 and 7.0. The amount of unadsorbed protein in the supernatant fluid was measured. The maximum amount of adsorbed albumin was 0.13 mg/1.0 g Ti. The albumin-Ti association constant was 2.77 mL/mg. Pretreatment of Ti with calcium, or magnesium alone, or combined with increasing pH values (3.0-7.0) resulted in augmented adsorption of HSA to Ti. No increase in adsorption was observed following pretreatment of Ti with potassium. These results point to the involvement of electrostatic interactions in the adsorption of HSA to TiO2.

A high molecular mass cranberry constituent reduces mutans streptococci level in saliva and inhibits in vitro adhesion to hydroxyapatite

Previous investigations showed that a high molecular mass, non-dialyzable material (NDM) from cranberries inhibits the adhesion of a number of bacterial species and prevents the co-aggregation of many oral bacterial pairs. In the present study we determined the effect of mouthwash supplemented with NDM on oral hygiene. Following 6 weeks of daily usage of cranberry-containing mouthwash by an experimental group (n = 29), we found that salivary mutans streptococci count as well as the total bacterial count were reduced significantly (ANOVA, P < 0.01) compared with those of the control (n = 30) using placebo mouthwash. No change in the plaque and gingival indices was observed. In vitro, the cranberry constituent inhibited the adhesion of Streptococcus sobrinus to saliva-coated hydroxyapatite. The data suggest that the ability to reduce mutans streptococci counts in vivo is due to the anti-adhesion activity of the cranberry constituent.

Bacteriophage isolation from human saliva.

Aims: To detect bacteriophages for Gram‐positive oral pathogens in human saliva.

The Anticariogenic Effect of Amine Fluorides on Streptococcus sobrinus and Glucosyltransferase in Biofilms

Dental caries is a chronic infectious disease caused by the accumulation of bacterial plaque (biofilm) on tooth surfaces. Antibacterial agents, in addition to other preventive measures, can control dental plaque accumulation. Amine fluorides (AmF) are known anticaries agents for over 30 years. The purpose of our study was to assess the adsorption and desorption of AmF to experimental dental biofilm and to evaluate the effect of AmF on Streptococcus sobrinus 6715 and glucosyltransferase (GTF) activity in experimental dental biofilms. The experimental plaque model used in this study consists of hydroxyapatite beads coated with human saliva (sHA), followed by adsorption of S. sobrinus and synthesis of in situ polysaccharides. Our results show that the viability of S. sobrinus in biofilm decreased as the concentration of AmF and chlorhexidine (CHX) increased. The concentration of AmF and CHX required to kill S. sobrinus adherent to sHA is about 100 times greater than the concentration required to kill the same amount of planktonic bacteria. Adsorption of AmF to surfaces was more than 90% and the desorption of AmF from our experimental model was limited. Pre–adsorption of AmF on the surface increased adhesion of S. sobrinus but also resulted in surface killing of the adsorbed bacteria. At low concentrations AmF increased GTF activity in solution by about 10%, but at concentrations above 0.1 mM it inhibited GTF activity. Inhibition of GTF on the surface required about 100 times more AmF than in solution. Our results show that AmF retains its anticariogenic effects in solution and in biofilm systems.

Adsorption of human salivary proteins to titanium powder. I. Adsorption of human salivary albumin

Titanium (Ti) is among the most widely used implant materials in dentistry today. The success of Ti implants is associated with their interactions with the surrounding tissues and biological fluids. In the present study, the adsorption of salivary proteins to Ti and the effect of calcium (Ca) on this process were investigated. Untreated and Ca-treated Ti powders were suspended in human clarified whole saliva. After incubation, the supernatant fluid was collected for protein analysis. The powders were then washed and resuspended in EDTA to desorb proteins from Ti surfaces. Sodium dodecylsulphate polyacrylamide gel electrophoresis and Bradford protein assay were conducted to determine the concentration and type of proteins that adsorbed onto Ti surfaces. The presence of Ca ions enhanced the adsorption of salivary proteins to Ti. A 66 kDa protein, identified by immunoblotting as albumin, was found as the main adsorbed salivary protein. Adsorption of albumin to Ti pretreated with Ca was significantly greater than to native Ti. The Ca-dependent adsorption process was reversed by EDTA. The data suggest that salivary albumin is one of the main constituents of a salivary biofilm formed on Ti dental implants and its adsorption to Ti surfaces is Ca-dependent. The presence of albumin on Ti dental implants may affect plaque accumulation on the implants and the biocompatibility of Ti implants.

Bactericidal and Cytotoxic Effects of Sodium Hypochlorite and Sodium Dichloroisocyanurate Solutions In Vitro

The antimicrobial and cytotoxic effects of sodium hypochlorite (NaOCl) and sodium dichloroisocyanurate (NaDCC) were evaluated and compared in vitro. The minimal inhibitory concentration and minimal bactericidal concentration of NaOCl and NaDCC were tested for Streptococcus sobrinus, Streptococcus salivarius, Enterococcus faecalis, and Streptococcus mutans. The cytotoxic effect was assessed by using human fibroblast tissue culture. Survival rate was assessed by a protein determination method. Results showed that the minimal inhibitory concentration and minimal bactericidal concentration values of NaOCl and NaDCC for the tested bacteria were in a similar range. NaDCC in concentrations higher than 0.02%, and NaOCl in concentrations higher than 0.01% were lethal to fibroblasts. In conclusion it seems that both agents were very effective in killing bacteria, and their cytotoxicity to fibroblasts in tissue culture was similar.

Early formation of Streptococcus sobrinus biofilm on various dental restorative materials

OBJECTIVES To examine the formation of dental biofilm by Streptococcus sobrinus on different types of restorative materials, using a model consisting of host and bacterial constituents. METHODS The adsorption pattern of saliva to the restorative material was determined by means of gel electrophoresis coupled with computerized densitometry techniques. The amount of salivary proteins adsorbed onto the surfaces was measured using the Bradford method. Sucrose-dependent bacterial adhesion to the saliva-coated restorative material was tested by radioactive-labelled Streptococcus sobrinus, and viable counts of these bacteria in the biofilm was determined using bacterial culture techniques. RESULTS Different adsorption patterns by salivary proteins to restorative materials were recorded. Durafil and acrylic dental materials demonstrated the most affinity to salivary proteins. A surface dependent adhesion profile was recorded, showing a high affinity of albumin and amylase to Acrylic and Durafil materials. Bacterial accumulation was the highest with Fuji LC and Fuji GC, which also demonstrated the highest bacterial viability. CONCLUSIONS Our study demonstrates the specificity of biofilm formation on different brands of dental restorative materials. Formation of a variety of dental biofilms has a significant impact on the progression of dental diseases in the oral cavity.

The adhesion of oral bacteria to modified titanium surfaces: role of plasma proteins and electrostatic forces

OBJECTIVES Modifications of titanium (Ti) implant surfaces have a significant effect on early biofilm formation and the outcome of implant procedures. The aim of this study was to examine the role of plasma proteins and electrostatic forces in the adhesion mechanism of oral bacteria to modified Ti surfaces. MATERIALS AND METHODS Ti discs with three different types of surface modifications, machined, acid-etched, and acid-etched and blasted, were examined for adhesion of oral bacteria: Streptococcus mutans, Porphyromonas gingivalis, and Fusobacterium nucleatum. Following pretreatment of the Ti with ion rich solutions or coating by human serum albumin or fibronectin, bacterial adhesion was examined by scanning electron microscopy and assessed quantitatively by DNA analysis. Ti coating by proteins as well as bacterial adhesion and their interrelationships were further investigated through confocal scanning laser microscopy. RESULTS Acid-etched and blasted Ti surfaces exhibited significantly higher amounts of bacteria adhesion than the other two surfaces. Calcium was found to serve as a bridging agent in the adhesion process of S. mutans and F. nucleatum to Ti surfaces. Although albumin coating of the Ti reduced the adhesion of S. mutans to all surfaces, it had no influence on the adhesion of P. gingivalis or F. nucleatum. Coating the Ti with fibronectin enhanced P. gingivalis and F. nucleatum adhesion. CONCLUSIONS Bacterial adhesion to Ti surfaces is roughness-dependent, and the adhesion mechanism is influenced by ions and proteins of the initial coating derived from the blood.

Genetic adaptation of Streptococcus mutans during biofilm formation on different types of surfaces

BackgroundAdhesion and successful colonization of bacteria onto solid surfaces play a key role in biofilm formation. The initial adhesion and the colonization of bacteria may differ between the various types of surfaces found in oral cavity. Therefore, it is conceivable that diverse biofilms are developed on those various surfaces. The aim of the study was to investigate the molecular modifications occurring during in vitro biofilm development of Streptococcus mutans UA159 on several different dental surfaces.ResultsGrowth analysis of the immobilized bacterial populations generated on the different surfaces shows that the bacteria constructed a more confluent and thick biofilms on a hydroxyapatite surface compared to the other tested surfaces. Using DNA-microarray technology we identified the differentially expressed genes of S. mutans, reflecting the physiological state of biofilms formed on the different biomaterials tested. Eight selected genes were further analyzed by real time RT-PCR. To further determine the impact of the tested material surfaces on the physiology of the bacteria, we tested the secretion of AI-2 signal by S. mutans embedded on those biofilms. Comparative transcriptome analyses indicated on changes in the S. mutans genome in biofilms formed onto different types of surfaces and enabled us to identify genes most differentially expressed on those surfaces. In addition, the levels of autoinducer-2 in biofilms from the various tested surfaces were different.ConclusionsOur results demonstrate that gene expression of S. mutans differs in biofilms formed on tested surfaces, which manifest the physiological state of bacteria influenced by the type of surface material they accumulate onto. Moreover, the stressful circumstances of adjustment to the surface may persist in the bacteria enhancing intercellular signaling and surface dependent biofilm formation.

Genetic and Physiological Effects of Noncoherent Visible Light Combined with Hydrogen Peroxide on Streptococcus mutans in Biofilm

ABSTRACT Oral biofilms are associated with the most common infections of the oral cavity. Bacteria embedded in the biofilms are less sensitive to antibacterial agents than planktonic bacteria are. Recently, an antibacterial synergic effect of noncoherent blue light and hydrogen peroxide (H2O2) on planktonic Streptococcus mutans was demonstrated. In this study, we tested the effect of a combination of light and H2O2 on the vitality and gene expression of S. mutans embedded in biofilm. Biofilms of S. mutans were exposed to visible light (wavelengths, 400 to 500 nm) for 30 or 60 s (equivalent to 34 or 68 J/cm2) in the presence of 3 to 300 mM H2O2. The antibacterial effect was assessed by microbial counts of each treated sample compared with that of the control. The effect of light combined with H2O2 on the different layers of the biofilm was evaluated by confocal laser scanning microscopy. Gene expression was determined by real-time reverse transcription-PCR. Our results show that noncoherent light, in combination with H2O2, has a synergistic antibacterial effect through all of the layers of the biofilm. Furthermore, this treatment was more effective against bacteria in biofilm than against planktonic bacteria. The combined light and H2O2 treatment up-regulated the expression of several genes such as gtfB, brp, smu630, and comDE but did not affect relA and ftf. The ability of noncoherent visible light in combination with H2O2 to affect bacteria in deep layers of the biofilm suggests that this treatment may be applied in biofilm-related diseases as a minimally invasive antibacterial procedure.