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1.  Development of a Multispecies Oral Bacterial Community in a Saliva-Conditioned Flow Cell 
Microbial communities within the human oral cavity are dynamic associations of more than 500 bacterial species that form biofilms on the soft and hard tissues of the mouth. Understanding the development and spatial organization of oral biofilms has been facilitated by the use of in vitro models. We used a saliva-conditioned flow cell, with saliva as the sole nutritional source, as a model to examine the development of multispecies biofilm communities from an inoculum containing the coaggregation partners Streptococcus gordonii, Actinomyces naeslundii, Veillonella atypica, and Fusobacterium nucleatum. Biofilms inoculated with individual species in a sequential order were compared with biofilms inoculated with coaggregates of the four species. Our results indicated that flow cells inoculated sequentially produced biofilms with larger biovolumes compared to those biofilms inoculated with coaggregates. Individual-species biovolumes within the four-species communities also differed between the two modes of inoculation. Fluorescence in situ hybridization with genus- and species-specific probes revealed that the majority of cells in both sequentially and coaggregate-inoculated biofilms were S. gordonii, regardless of the inoculation order. However, the representation of A. naeslundii and V. atypica was significantly higher in biofilms inoculated with coaggregates compared to sequentially inoculated biofilms. Thus, these results indicate that the development of multispecies biofilm communities is influenced by coaggregations preformed in planktonic phase. Coaggregating bacteria such as certain streptococci are especially adapted to primary colonization of saliva-conditioned surfaces independent of the mode of inoculation and order of addition in the multispecies inoculum. Preformed coaggregations favor other bacterial strains and may facilitate symbiotic relationships.
PMCID: PMC444820  PMID: 15240317
2.  Osteopontin Reduces Biofilm Formation in a Multi-Species Model of Dental Biofilm 
PLoS ONE  2012;7(8):e41534.
Combating dental biofilm formation is the most effective means for the prevention of caries, one of the most widespread human diseases. Among the chemical supplements to mechanical tooth cleaning procedures, non-bactericidal adjuncts that target the mechanisms of bacterial biofilm formation have gained increasing interest in recent years. Milk proteins, such as lactoferrin, have been shown to interfere with bacterial colonization of saliva-coated surfaces. We here study the effect of bovine milk osteopontin (OPN), a highly phosphorylated whey glycoprotein, on a multispecies in vitro model of dental biofilm. While considerable research effort focuses on the interaction of OPN with mammalian cells, there are no data investigating the influence of OPN on bacterial biofilms.
Methodology/Principal Findings
Biofilms consisting of Streptococcus oralis, Actinomyces naeslundii, Streptococcus mitis, Streptococcus downei and Streptococcus sanguinis were grown in a flow cell system that permitted in situ microscopic analysis. Crystal violet staining showed significantly less biofilm formation in the presence of OPN, as compared to biofilms grown without OPN or biofilms grown in the presence of caseinoglycomacropeptide, another phosphorylated milk protein. Confocal microscopy revealed that OPN bound to the surface of bacterial cells and reduced mechanical stability of the biofilms without affecting cell viability. The bacterial composition of the biofilms, determined by fluorescence in situ hybridization, changed considerably in the presence of OPN. In particular, colonization of S. mitis, the best biofilm former in the model, was reduced dramatically.
OPN strongly reduces the amount of biofilm formed in a well-defined laboratory model of acidogenic dental biofilm. If a similar effect can be observed in vivo, OPN might serve as a valuable adjunct to mechanical tooth cleaning procedures.
PMCID: PMC3413689  PMID: 22879891
3.  Salivary pellicles on titanium and their effect on metabolic activity in Streptococcus oralis 
BMC Oral Health  2013;13:32.
Titanium implants in the oral cavity are covered with a saliva-derived pellicle to which early colonizing microorganisms such as Streptococcus oralis can bind. The protein profiles of salivary pellicles on titanium have not been well characterized and the proteins of importance for binding are thus unknown. Biofilm bacteria exhibit different phenotypes from their planktonic counterparts and contact with salivary proteins may be one factor contributing to the induction of changes in physiology. We have characterized salivary pellicles from titanium surfaces and investigated how contact with uncoated and saliva-coated titanium surfaces affects metabolic activity in adherent cells of S. oralis.
Salivary pellicles on smooth titanium surfaces were desorbed and these, as well as purified human saliva, were subjected to two-dimensional gel electrophoresis and mass spectroscopy. A parallel plate flow-cell model was used to study binding of a fresh isolate of S. oralis to uncoated and saliva-coated titanium surfaces. Metabolic activity was assessed using the BacLight CTC Vitality Kit and confocal scanning laser microscopy. Experiments were carried out in triplicate and the results analyzed using Student’s t-test or ANOVA.
Secretory IgA, α-amylase and cystatins were identified as dominant proteins in the salivary pellicles. Selective adsorption of proteins was demonstrated by the enrichment of prolactin-inducible protein and absence of zinc-α2-glycoprotein relative to saliva. Adherence of S. oralis to titanium led to an up-regulation of metabolic activity in the population after 2 hours. In the presence of a salivary pellicle, this effect was enhanced and sustained over the following 22 hour period.
We have shown that adherence to smooth titanium surfaces under flow causes an up-regulation of metabolic activity in the early oral colonizer S. oralis, most likely as part of an adaptation to the biofilm mode of life. The effect was enhanced by a salivary pellicle containing sIgA, α-amylase, cystatins and prolactin-inducible protein which was, for the first time, identified as an abundant component of salivary pellicles on titanium. Further studies are needed to clarify the mechanisms underlying the effect of surface contact on metabolic activity as well as to identify the salivary proteins responsible for enhancing the effect.
PMCID: PMC3726426  PMID: 23866104
Bacteria; Microbial biofilm; Dental implant; Streptococci
4.  Oral Streptococci Biofilm Formation on Different Implant Surface Topographies 
BioMed Research International  2015;2015:159625.
The establishment of the subgingival microbiota is dependent on successive colonization of the implant surface by bacterial species. Different implant surface topographies could influence the bacterial adsorption and therefore jeopardize the implant survival. This study evaluated the biofilm formation capacity of five oral streptococci species on two titanium surface topographies. In vitro biofilm formation was induced on 30 titanium discs divided in two groups: sandblasted acid-etched (SAE- n = 15) and as-machined (M- n = 15) surface. The specimens were immersed in sterilized whole human unstimulated saliva and then in fresh bacterial culture with five oral streptococci species: Streptococcus sanguinis, Streptococcus salivarius, Streptococcus mutans, Streptococcus sobrinus, and Streptococcus cricetus. The specimens were fixed and stained and the adsorbed dye was measured. Surface characterization was performed by atomic force and scanning electron microscopy. Surface and microbiologic data were analyzed by Student's t-test and two-way ANOVA, respectively (P < 0.05). S. cricetus, S. mutans, and S. sobrinus exhibited higher biofilm formation and no differences were observed between surfaces analyzed within each species (P > 0.05). S. sanguinis exhibited similar behavior to form biofilm on both implant surface topographies, while S. salivarius showed the lowest ability to form biofilm. It was concluded that biofilm formation on titanium surfaces depends on surface topography and species involved.
PMCID: PMC4529887  PMID: 26273590
5.  The Exopolysaccharide Matrix Modulates the Interaction between 3D Architecture and Virulence of a Mixed-Species Oral Biofilm 
PLoS Pathogens  2012;8(4):e1002623.
Virulent biofilms are responsible for a range of infections, including oral diseases. All biofilms harbor a microbial-derived extracellular-matrix. The exopolysaccharides (EPS) formed on tooth-pellicle and bacterial surfaces provide binding sites for microorganisms; eventually the accumulated EPS enmeshes microbial cells. The metabolic activity of the bacteria within this matrix leads to acidification of the milieu. We explored the mechanisms through which the Streptococcus mutans-produced EPS-matrix modulates the three-dimensional (3D) architecture and the population shifts during morphogenesis of biofilms on a saliva-coated-apatitic surface using a mixed-bacterial species system. Concomitantly, we examined whether the matrix influences the development of pH-microenvironments within intact-biofilms using a novel 3D in situ pH-mapping technique. Data reveal that the production of the EPS-matrix helps to create spatial heterogeneities by forming an intricate network of exopolysaccharide-enmeshed bacterial-islets (microcolonies) through localized cell-to-matrix interactions. This complex 3D architecture creates compartmentalized acidic and EPS-rich microenvironments throughout the biofilm, which triggers the dominance of pathogenic S. mutans within a mixed-species system. The establishment of a 3D-matrix and EPS-enmeshed microcolonies were largely mediated by the S. mutans gtfB/gtfC genes, expression of which was enhanced in the presence of Actinomyces naeslundii and Streptococcus oralis. Acidic pockets were found only in the interiors of bacterial-islets that are protected by EPS, which impedes rapid neutralization by buffer (pH 7.0). As a result, regions of low pH (<5.5) were detected at specific locations along the surface of attachment. Resistance to chlorhexidine was enhanced in cells within EPS-microcolony complexes compared to those outside such structures within the biofilm. Our results illustrate the critical interaction between matrix architecture and pH heterogeneity in the 3D environment. The formation of structured acidic-microenvironments in close proximity to the apatite-surface is an essential factor associated with virulence in cariogenic-biofilms. These observations may have relevance beyond the mouth, as matrix is inherent to all biofilms.
Author Summary
Virulent biofilms formed on surfaces are associated with many human infections. The disease dental caries, expressed as cavities, is a prime example of the consequences arising from interactions between bacteria and sugars on tooth-surfaces. When Streptococcus mutans metabolize sugars, they produce a glue-like polymer termed glucan, helping them to adhere firmly to teeth. Glucan is also formed on bacterial surfaces in the mouth, and will accumulate and enmesh additional microorganisms creating the gelatinous formation known as dental plaque-biofilm. We found unique islets of bacteria within these biofilms, particularly close to the tooth-surface, providing safe havens in which bacteria thrive and produce acids that erode teeth. One intriguing mystery is why acids accumulate on the tooth-surface when there is an abundance of neutral-pH saliva surrounding the teeth. We found that bacterial-islets are particularly protected by glucan, which retards neutralization. We noticed that, within biofilms, the interiors of these islets are acidic, where only acid-tolerant bacteria can prosper, ensuring continued localized acid production. Our study demonstrates that construction of biofilms mediated by glucans forms complex 3D architectures, creating a variety of acidic-microenvironments that are essential for virulence expression. These results may aid in the development of enhanced methods to modulate biofilm formation.
PMCID: PMC3320608  PMID: 22496649
6.  Influence of topography and hydrophilicity on initial oral biofilm formation on microstructured titanium surfaces in vitro 
Clinical oral implants research  2011;23(3):301-307.
The aim of this study was to analyse the influence of the microtopography and hydrophilicity of titanium (Ti) substrates on initial oral biofilm formation.
Materials and methods
Nine bacterial species belonging to the normal oral microbiota, including: Aggregatibacter actinomycetemcomitans, Actinomyces israelii, Campylobacter rectus, Eikenella corrodens, Fusobacterium nucleatum, Parvimonas micra, Porphyromonas gingivalis, Prevotella intermedia, and Streptococcus sanguinis were tested on Ti surfaces: pretreatment (PT [Ra<0.2 μm]), acid-etched (A [Ra<0.8 μm]), A modified to be hydrophilic (modA), sand-blasted/acid-etched (SLA [Ra = 4 μm]), and hydrophilic SLA (modSLA). Disks were incubated for 24 h in anaerobic conditions using a normal culture medium (CM) or human saliva (HS). The total counts of bacteria and the proportion of each bacterial species were analysed by checkerboard DNA–DNA hybridization. Results: Higher counts of bacteria were observed on all surfaces incubated with CM compared with the samples incubated with HS. PT, SLA, and modSLA exhibited higher numbers of attached bacteria in CM, whereas SLA and modSLA had a significant increase in bacterial adhesion in HS. The proportion of the species in the initial biofilms was also influenced by the surface properties and the media used: SLA and modSLA increased the proportion of species like A. actinomycetemcomitans and S. sanguinis in both media, while the adhesion of A. israelii and P. gingivalis on the same surfaces was affected in the presence of saliva.
The initial biofilm formation and composition were affected by the microtopography and hydrophilicity of the surface and by the media used.
PMCID: PMC4287405  PMID: 21492236
biofilm; hydrophilicity; microstructure; titanium
7.  Introducing a Semi-Coated Model to Investigate Antibacterial Effects of Biocompatible Polymers on Titanium Surfaces 
Peri-implant infections from bacterial biofilms on artificial surfaces are a common threat to all medical implants. They are a handicap for the patient and can lead to implant failure or even life-threatening complications. New implant surfaces have to be developed to reduce biofilm formation and to improve the long-term prognosis of medical implants. The aim of this study was (1) to develop a new method to test the antibacterial efficacy of implant surfaces by direct surface contact and (2) to elucidate whether an innovative antimicrobial copolymer coating of 4-vinyl-N-hexylpyridinium bromide and dimethyl(2-methacryloyloxyethyl) phosphonate (VP:DMMEP 30:70) on titanium is able to reduce the attachment of bacteria prevalent in peri-implant infections. With a new in vitro model with semi-coated titanium discs, we were able to show a dramatic reduction in the adhesion of various pathogenic bacteria (Streptococcus sanguinis, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis), completely independently of effects caused by soluble materials. In contrast, soft tissue cells (human gingival or dermis fibroblasts) were less affected by the same coating, despite a moderate reduction in initial adhesion of gingival fibroblasts. These data confirm the hypothesis that VP:DMMEP 30:70 is a promising antibacterial copolymer that may be of use in several clinical applications.
PMCID: PMC4346959  PMID: 25690041
antimicrobial surface; polymer coating; bacteria; biofilm; implants; experiments in vitro
8.  The evaluation of the impact of titania nanotube covers morphology and crystal phase on their biological properties 
The highly ordered titanium dioxide nanotube coatings were produced under various electrochemical conditions on the surface of titanium foil. The anodization voltage changes proved to be a main factor which directly affects the nanotube morphology, structure, and wettability. Moreover we have noticed a significant dependence between the size and crystallinity of TiO2 layers and the adhesion/proliferation of fibroblasts and antimicrobial properties. Cellular functionality were investigated for up to 3 days in culture using a cell viability assay and scanning electron microscopy. In general, results of our studies revealed that fibroblasts adhesion, proliferation, and differentiation on the titania nanotube coatings is clearly higher than on the surface of the pure titanium foil. The formation of crystallic islands in the nanotubes structure induced a significant acceleration in the growth rate of fibroblasts cells by as much as ~200 %. Additionally, some types of TiO2 layers revealed the ability to the reduce of the staphylococcal aggregates/biofilm formation. The nanotube coatings formed during the anodization process using the voltage 4 V proved to be the stronger S. aureus aggregates/biofilm inhibitor in comparison to the uncovered titanium substrate. That accelerated eukaryotic cell growth and anti-biofilm activity is believed to be advantageous for faster cure of dental and orthopaedic patients, and also for a variety of biomedical diagnostic and therapeutic applications.
Graphical Abstract
The highly ordered titanium dioxide nanotube coatings were produced under various electrochemical conditions on the surface of titanium foil. The anodization voltage changes proved to be a main factor which directly affects the nanotube morphology, structure, and wettability. Moreover we have noticed a significant dependence between the size and crystallinity of TiO2 layers and the adhesion/proliferation of fibroblasts and antimicrobial properties.
PMCID: PMC4366560  PMID: 25791457
9.  Nanometer-thin TiO2 enhances skeletal muscle cell phenotype and behavior 
The independent role of the surface chemistry of titanium in determining its biological properties is yet to be determined. Although titanium implants are often in contact with muscle tissue, the interaction of muscle cells with titanium is largely unknown. This study tested the hypotheses that the surface chemistry of clinically established microroughened titanium surfaces could be controllably varied by coating with a minimally thin layer of TiO2 (ideally pico-to-nanometer in thickness) without altering the existing topographical and roughness features, and that the change in superficial chemistry of titanium is effective in improving the biological properties of titanium.
Methods and results
Acid-etched microroughened titanium surfaces were coated with TiO2 using slow-rate sputter deposition of molten TiO2 nanoparticles. A TiO2 coating of 300 pm to 6.3 nm increased the surface oxygen on the titanium substrates in a controllable manner, but did not alter the existing microscale architecture and roughness of the substrates. Cells derived from rat skeletal muscles showed increased attachment, spread, adhesion strength, proliferation, gene expression, and collagen production at the initial and early stage of culture on 6.3 nm thick TiO2-coated microroughened titanium surfaces compared with uncoated titanium surfaces.
Using an exemplary slow-rate sputter deposition technique of molten TiO2 nanoparticles, this study demonstrated that titanium substrates, even with microscale roughness, can be sufficiently chemically modified to enhance their biological properties without altering the existing microscale morphology. The controllable and exclusive chemical modification technique presented in this study may open a new avenue for surface modifications of titanium-based biomaterials for better cell and tissue affinity and reaction.
PMCID: PMC3215160  PMID: 22114483
nanotechnology; orthopedic implants; molten TiO2 nanoparticles; surface chemistry
10.  Streptococcus mutans Protein Synthesis during Mixed-Species Biofilm Development by High-Throughput Quantitative Proteomics 
PLoS ONE  2012;7(9):e45795.
Biofilms formed on tooth surfaces are comprised of mixed microbiota enmeshed in an extracellular matrix. Oral biofilms are constantly exposed to environmental changes, which influence the microbial composition, matrix formation and expression of virulence. Streptococcus mutans and sucrose are key modulators associated with the evolution of virulent-cariogenic biofilms. In this study, we used a high-throughput quantitative proteomics approach to examine how S. mutans produces relevant proteins that facilitate its establishment and optimal survival during mixed-species biofilms development induced by sucrose. Biofilms of S. mutans, alone or mixed with Actinomyces naeslundii and Streptococcus oralis, were initially formed onto saliva-coated hydroxyapatite surface under carbohydrate-limiting condition. Sucrose (1%, w/v) was then introduced to cause environmental changes, and to induce biofilm accumulation. Multidimensional protein identification technology (MudPIT) approach detected up to 60% of proteins encoded by S. mutans within biofilms. Specific proteins associated with exopolysaccharide matrix assembly, metabolic and stress adaptation processes were highly abundant as the biofilm transit from earlier to later developmental stages following sucrose introduction. Our results indicate that S. mutans within a mixed-species biofilm community increases the expression of specific genes associated with glucan synthesis and remodeling (gtfBC, dexA) and glucan-binding (gbpB) during this transition (P<0.05). Furthermore, S. mutans up-regulates specific adaptation mechanisms to cope with acidic environments (F1F0-ATPase system, fatty acid biosynthesis, branched chain amino acids metabolism), and molecular chaperones (GroEL). Interestingly, the protein levels and gene expression are in general augmented when S. mutans form mixed-species biofilms (vs. single-species biofilms) demonstrating fundamental differences in the matrix assembly, survival and biofilm maintenance in the presence of other organisms. Our data provide insights about how S. mutans optimizes its metabolism and adapts/survives within the mixed-species community in response to a dynamically changing environment. This reflects the intricate physiological processes linked to expression of virulence by this bacterium within complex biofilms.
PMCID: PMC3458072  PMID: 23049864
11.  Adherence of human oral keratinocytes and gingival fibroblasts to nano-structured titanium surfaces 
BMC Oral Health  2014;14:75.
A key element for long-term success of dental implants is integration of the implant surface with the surrounding host tissues. Modification of titanium implant surfaces can enhance osteoblast activity but their effects on soft-tissue cells are unclear. Adherence of human keratinocytes and gingival fibroblasts to control commercially pure titanium (CpTi) and two surfaces prepared by anodic oxidation was therefore investigated. Since implant abutments are exposed to a bacteria-rich environment in vivo, the effect of oral bacteria on keratinocyte adhesion was also evaluated.
The surfaces were characterized using scanning electron microscopy (SEM). The number of adhered cells and binding strength, as well as vitality of fibroblasts and keratinocytes were evaluated using confocal scanning laser microscopy after staining with Live/Dead Baclight. To evaluate the effect of bacteria on adherence and vitality, keratinocytes were co-cultured with a four-species streptococcal consortium.
SEM analysis showed the two anodically oxidized surfaces to be nano-structured with differing degrees of pore-density. Over 24 hours, both fibroblasts and keratinocytes adhered well to the nano-structured surfaces, although to a somewhat lesser degree than to CpTi (range 42-89% of the levels on CpTi). The strength of keratinocyte adhesion was greater than that of the fibroblasts but no differences in adhesion strength could be observed between the two nano-structured surfaces and the CpTi. The consortium of commensal streptococci markedly reduced keratinocyte adherence on all the surfaces as well as compromising membrane integrity of the adhered cells.
Both the vitality and level of adherence of soft-tissue cells to the nano-structured surfaces was similar to that on CpTi. Co-culture with streptococci reduced the number of keratinocytes on all the surfaces to approximately the same level and caused cell damage, suggesting that commensal bacteria could affect adherence of soft-tissue cells to abutment surfaces in vivo.
PMCID: PMC4083866  PMID: 24952379
Oral keratinocytes; Gingival fibroblasts; Cell attachment; Dental implant; Surface modification; Oral bacteria
12.  Interactions between Lactobacillus rhamnosus GG and oral micro-organisms in an in vitro biofilm model 
BMC Microbiology  2016;16:149.
Probiotics have shown favourable properties in maintaining oral health. By interacting with oral microbial communities, these species could contribute to healthier microbial equilibrium. This study aimed to investigate in vitro the ability of probiotic Lactobacillus rhamnosus GG (L.GG) to integrate in oral biofilm and affect its species composition. Five oral strains, Streptococcus mutans, Streptococcus sanguinis, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum and Candida albicans were involved. The group setup included 6 mono-species groups, 3 dual-species groups (L.GG + S. mutans/S. sanguinis/C. albicans), and 4 multi-species groups (4/5 species and 4/5 species + L.GG, 4 species were all the tested strains except S. mutans). Cell suspensions of six strains were pooled according to the group setup. Biofilms were grown on saliva-coated hydroxyapatite (HA) discs at 37 °C in anaerobic conditions for 64.5 h. Biofilm medium was added and refreshed at 0, 16.5, and 40.5 h. The pH of spent media was measured. Viable cells of the 16.5 h and 64.5 h biofilms were counted. 64.5 h biofilms were stained and scanned with confocal laser scanning microscopy.
Our results showed that L.GG and S. mutans demonstrated stronger adhesion ability than the other strains to saliva-coated HA discs. L.GG, C. albicans, S. mutans and F. nucleatum, with poor ability to grow in mono-species biofilms demonstrated better abilities of adhesion and reproduction in dual- and/or multi-species biofilms. L.GG slightly suppressed the growth of C. albicans in all groups, markedly weakened the growth of S. sanguinis and F. nucleatum in 4sp + L.GG group, and slightly reduced the adhesion of S. mutans in L.GG+ S. mutans group.
To conclude, in this in vitro model L.GG successfully integrated in all oral biofilms, and reduced the counts of S. sanguinis and C. albicans and lowered the biofilm-forming ability of F. nucleatum, but only slightly reduced the adhesion of S. mutans. C. albicans significantly promoted the growth of L.GG.
PMCID: PMC4942979  PMID: 27405227
Probiotics; Biofilm; Oral pathogen; Oral health
13.  Mutualism versus Independence: Strategies of Mixed-Species Oral Biofilms In Vitro Using Saliva as the Sole Nutrient Source 
Infection and Immunity  2001;69(9):5794-5804.
During initial dental plaque formation, the ability of a species to grow when others cannot would be advantageous, and enhanced growth through interspecies and intergeneric cooperation could be critical. These characteristics were investigated in three coaggregating early colonizers of the tooth surface (Streptococcus gordonii DL1, Streptococcus oralis 34, and Actinomyces naeslundii T14V). Area coverage and cell cluster size measurements showed that attachment of A. naeslundii and of S. gordonii to glass flowcells was enhanced by a salivary conditioning film, whereas attachment of S. oralis was hindered. Growth experiments using saliva as the sole carbon and nitrogen source showed that A. naeslundii was unable to grow either in planktonic culture or as a biofilm, whereas S. gordonii grew under both conditions. S. oralis grew planktonically, but to a much lower maximum cell density than did S. gordonii; S. oralis did not grow reproducibly as a biofilm. Thus, only S. gordonii possessed all traits advantageous for growth as a solitary and independent resident of the tooth. Two-species biofilm experiments analyzed by laser confocal microscopy showed that neither S. oralis nor A. naeslundii grew when coaggregated pairwise with S. gordonii. However, both S. oralis and A. naeslundii showed luxuriant, interdigitated growth when paired together in coaggregated microcolonies. Thus, the S. oralis-A. naeslundii pair formed a mutualistic relationship, potentially contact dependent, that allows each to grow where neither could survive alone. S. gordonii, in contrast, neither was hindered by nor benefited from the presence of either of the other strains. The formation of mutually beneficial interactions within the developing biofilm may be essential for certain initial colonizers to be retained during early plaque development, whereas other initial colonizers may be unaffected by neighboring cells on the substratum.
PMCID: PMC98697  PMID: 11500457
14.  Adhesion Forces and Composition of Planktonic and Adhering Oral Microbiomes 
Journal of Dental Research  2014;93(1):84-88.
The oral microbiome consists of a planktonic microbiome residing in saliva and an adhering microbiome (the biofilm adhering to oral hard and soft tissues). Here we hypothesized that possible differences in microbial composition of the planktonic and adhering oral microbiome on teeth can be related to the forces by which different bacterial species are attracted to the tooth surface. The relative presence of 7 oral bacterial species in saliva and biofilm collected from 10 healthy human volunteers was determined twice in each volunteer by denaturing-gradient-gel electrophoresis. Analysis of both microbiomes showed complete separation of the planktonic from the adhering oral microbiome. Next, adhesion forces of corresponding bacterial strains with saliva-coated enamel surfaces were measured by atomic force microscopy. Species that were found predominantly in the adhering microbiome had significantly higher adhesion forces to saliva-coated enamel (-0.60 to -1.05 nN) than did species mostly present in the planktonic microbiome (-0.40 to -0.55 nN). It is concluded that differences in composition of the planktonic and the adhering oral microbiome are due to small differences in the forces by which strains adhere to saliva-coated enamel, providing an important step in understanding site- and material-specific differences in the composition of biofilms in the oral cavity.
PMCID: PMC3872853  PMID: 24186560
atomic force microscopy; denaturing gradient gel electrophoresis; biofilm; dental plaque; saliva; bacterial adhesion
15.  Effects of the surface characteristics of nanoporous titanium oxide films on Ti-24Nb-4Zr-8Sn alloy on the initial adhesion of osteoblast-like MG-63 cells 
The aim of the present study was to investigate the effects of the surface characteristics of nanoporous titanium oxide films, formed by anodization on Ti-24Nb-4Zr-8Sn (Ti2448) alloy, on the early adhesion of osteoblast-like MG-63 cells. Nanoporous titanium oxide films with two different pore sizes (30 and 90 nm) were formed by anodization in NH4F solution on Ti2448 alloy. The surface roughness of the nanoporous titanium oxide films was determined using a Surftest Formtracer and field emission scanning electron microscopy (FESEM). Cell viability was evaluated at different time points using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. To investigate the regulatory mechanisms involved in the focal adhesion of osteoblasts to Ti2448 alloy, we quantified the expression levels of integrin β1 and paxillin mRNAs on the nanoporous titanium oxide films during early osteoblast adhesion using real-time RT-PCR. Samples with a 30-nm nanoporous film exhibited a greater number of overlapping microporous structures with microprojections compared with the 90-nm nanoporous film samples. The MTT assay indicated that cell viability on the 30-nm nanoporous surface following 24 and 48 h of cell culture was higher than those observed on the unanodized control and 90-nm nanoporous surfaces. Integrin β1 mRNA expression levels on the 30-nm nanoporous surface following cell culture for 48 h were also significantly higher compared with those on the unanodized control and 90-nm nanoporous surfaces. The results demonstrated that a 30-nm nanoporous titanium oxide film on Ti2448 alloy may provide the optimum bioactive implant surface for the initial adhesion of osteoblasts.
PMCID: PMC3735869  PMID: 23935754
titanium alloy; nanoporous surface; osteoblasts; early adhesion
16.  Effects of titania nanotubes with or without bovine serum albumin loaded on human gingival fibroblasts 
Modifying the surface of the transmucosal area is a key research area because this process positively affects the three functions of implants: attachment to soft tissue, inhibiting bacterial biofilm adhesion, and the preservation of the crestal bone. To exploit the potential of titania nanotube arrays (TNTs) with or without using bovine serum albumin (BSA) to modify the surface of a dental implant in contact with the transmucosal area, BSA was loaded into TNTs that were fabricated by anodizing Ti sheets; the physical characteristics of these arrays, including their morphology, chemical composition, surface roughness, contact angle, and surface free energy (SFE), were assessed. The effect of Ti surfaces with TNTs or TNTs-BSA on human gingival fibroblasts (HGFs) was determined by analyzing cell morphology, early adhesion, proliferation, type I collagen (COL-1) gene expression, and the extracellular secretion of COL-1. The results indicate that early HGF adhesion and spreading behavior is positively correlated with surface characteristics, including hydrophilicity, SFE, and surface roughness. Additionally, TNT surfaces not only promoted early HGF adhesion, but also promoted COL-1 secretion. BSA-loaded TNT surfaces promoted early HGF adhesion, while suppressing late proliferation and COL-1 secretion. Therefore, TNT-modified smooth surfaces are expected to be applicable for uses involving the transmucosal area. Further study is required to determine whether BSA-loaded TNT surfaces actually affect closed loop formation of connective tissue because BSA coating actions in vivo are very rapid.
PMCID: PMC3949701  PMID: 24623977
titania nanotubes; bovine serum albumin; modified surface; transmucosal area; human gingival fibroblast
17.  Reduced adhesion of macrophages on anodized titanium with select nanotube surface features 
One of the important prerequisites for a successful orthopedic implant apart from being osteoconductive is the elicitation of a favorable immune response that does not lead to the rejection of the implant by the host tissue. Anodization is one of the simplest surface modification processes used to create nanotextured and nanotubular features on metal oxides which has been shown to improve bone formation. Anodization of titanium (Ti) leads to the formation of TiO2 nanotubes on the surface, and the presence of these nanotubes mimics the natural nanoscale features of bone, which in turn contributes to improved bone cell attachment, migration, and proliferation. However, inflammatory cell responses on anodized Ti remains to be tested. It is hypothesized that surface roughness and surface feature size on anodized Ti can be carefully manipulated to control immune cell (specifically, macrophages) responses. Here, when Ti samples were anodized at 10 V in the presence of 1% hydrofluoric acid (HF) for 1 minute, nanotextured (nonnanotube) surfaces were created. When anodization of Ti samples was carried out with 1% HF for 10 minutes at 15 V, nanotubes with 40–50 nm diameters were formed, whereas at 20 V with 1% HF for 10 minutes, nanotubes with 60–70 nm diameters were formed. In this study, a reduced density of macrophages was observed after 24 hours of culture on nanotextured and nanotubular Ti samples which were anodized at 10, 15, and 20 V, compared with conventional unmodified Ti samples. This in vitro study thus demonstrated a reduced density of macrophages on anodized Ti, thereby providing further evidence of the greater efficacy of anodized Ti for orthopedic applications.
PMCID: PMC3184936  PMID: 21980239
anodization; titanium implants; TiO2 nanotube
18.  Improving cytocompatibility of Co28Cr6Mo by TiO2 coating: gene expression study in human endothelial cells 
Cobalt-based materials are widely used for coronary stents, as well as bone and joint implants. However, their use is associated with high corrosion incidence. Titanium alloys, by contrast, are more biocompatible owing to the formation of a relatively inactive titanium oxide (TiO2) layer on their surface. This study was aimed at improving Co28Cr6Mo alloy cytocompatibility via sol–gel TiO2 coating to reduce metal corrosion and metal ion release. Owing to their role in inflammation and tissue remodelling around an implant, endothelial cells present a suitable in vitro model for testing the biological response to metallic materials. Primary human endothelial cells seeded on Co28Cr6Mo showed a stress phenotype with numerous F-actin fibres absent on TiO2-coated material. To investigate this effect at the gene expression level, cDNA microarray analysis of in total 1301 genes was performed. Compared with control cells, 247 genes were expressed differentially in the cells grown on Co28Cr6Mo, among them genes involved in proliferation, oxidative stress response and inflammation. TiO2 coating reduced the effects of Co28Cr6Mo on gene expression in endothelial cells, with only 34 genes being differentially expressed. Quantitative real-time polymerase chain reaction and protein analysis confirmed microarray data for selected genes. The effect of TiO2 coating can be, in part, attributed to the reduced release of Co2+, because addition of CoCl2 resulted in similar cellular responses. TiO2 coating of cobalt-based materials, therefore, could be used in the production of cobalt-based devices for cardiovascular and skeletal applications to reduce the adverse effects of metal corrosion products and to improve the response of endothelial and other cell types.
PMCID: PMC3730696  PMID: 23825117
cobalt alloy; titanium oxide; endothelial cell; biocompatibility; gene expression
19.  Surface-associated MUC5B mucins promote protease activity in Lactobacillus fermentum biofilms 
BMC Oral Health  2013;13:43.
Mucosal surfaces are coated with layers of mucus gel that protect the underlying tissues and promote colonization by members of the commensal microflora. Lactobacillus fermentum is a common inhabitant of the oral cavity, gastrointestinal and reproductive tracts and is one of the most important lactic acid bacteria contributing to the formation of a healthy intestinal microflora. We have investigated the proteolytic activity in L. fermentum in response to interactions with the MUC5B mucin, which is a major component of mucus gels at sites colonized by this micro-organism.
Biofilms of Lactobacillus fermentum were established in mini-flow cells in the presence or absence of human salivary MUC5B. The proteolytic activity of biofilm cells was examined in a confocal scanning laser microscope with a fluorescent protease substrate. Degradation of MUC5B by L. fermentum was analysed using SDS-PAGE followed by Western blotting with antisera raised against the MUC5B peptide. Cell surface proteins differentialy expressed in a MUC5B-rich environment were identified with the aid of comparative two-dimensional electrophoresis followed by LC-MS/MS.
Lactobacillus fermentum adhered well to surfaces coated with MUC5B mucin and in biofilms of L. fermentum formed in a MUC5B environment, the proportion of proteolytically-active cells (47 ± 0.6% of the population), as shown by cleavage of a fluorescent casein substrate, was significantly greater (p < 0.01) than that in biofilms formed in nutrient broth (0.4 ± 0.04% of the population). Thus, the presence of MUC5B mucins enhanced bacterial protease activity. This effect was mainly attributable to contact with surface-associated mucins rather than those present in the fluid phase. Biofilms of L. fermentum were capable of degrading MUC5B mucins suggesting that this complex glycoprotein can be exploited as a nutrient source by the bacteria.
Comparison of the surface proteomes of biofilm cells of L. fermentum in a MUC5B environment with those in nutrient broth using two-dimensional electrophoresis and mass spectroscopy, showed that the enhanced proteolytic activity was associated with increased expression of a glycoprotease; O-sialoglycoprotein endopeptidase, as well as chaperone proteins such as DnaK and trigger factor.
Adhesion to mucin-coated surfaces leads to a shift towards a more protease-active phenotype within L. fermentum biofilms and proteases produced within the biofilms can degrade MUC5B mucins. The enhanced proteolytic activity was associated with an increase in O-sialoglycoprotein endopeptidase on the cell surface. We propose that the upregulation of chaperone proteins in the mucin environment may contribute to the protease-active phenotype through activation of the glycopeptidase. This would represent one way for commensal lactobacilli e.g. L. fermentum to exploit complex substrates in their local environment in order to survive on mucosal surfaces.
PMCID: PMC3847627  PMID: 24010726
Lactobacilli; Proteolytic activity; Proteolysis; Mucus glycoprotein
20.  Inhibition of Staphylococcus epidermidis Biofilm by Trimethylsilane Plasma Coating 
Antimicrobial Agents and Chemotherapy  2012;56(11):5923-5937.
Biofilm formation on implantable medical devices is a major impediment to the treatment of nosocomial infections and promotes local progressive tissue destruction. Staphylococcus epidermidis infections are the leading cause of biofilm formation on indwelling devices. Bacteria in biofilms are highly resistant to antibiotic treatment, which in combination with the increasing prevalence of antibiotic resistance among human pathogens further complicates treatment of biofilm-related device infections. We have developed a novel plasma coating technology. Trimethylsilane (TMS) was used as a monomer to coat the surfaces of 316L stainless steel and grade 5 titanium alloy, which are widely used in implantable medical devices. The results of biofilm assays demonstrated that this TMS coating markedly decreased S. epidermidis biofilm formation by inhibiting the attachment of bacterial cells to the TMS-coated surfaces during the early phase of biofilm development. We also discovered that bacterial cells on the TMS-coated surfaces were more susceptible to antibiotic treatment than their counterparts in biofilms on uncoated surfaces. These findings suggested that TMS coating could result in a surface that is resistant to biofilm development and also in a bacterial community that is more sensitive to antibiotic therapy than typical biofilms.
PMCID: PMC3486604  PMID: 22964248
21.  Sol-Gel-Derived Hydroxyapatite-Carbon Nanotube/Titania Coatings on Titanium Substrates 
In this paper, hydroxyapatite-carbon nanotube/titania (HA-CNT/TiO2) double layer coatings were successfully developed on titanium (Ti) substrates intended for biomedical applications. A TiO2 coating was firstly developed by anodization to improve bonding between HA and Ti, and then the layer of HA and CNTs was coated on the surface by the sol-gel process to improve the biocompatibility and mechanical properties of Ti. The surfaces of double layer coatings were uniform and crack-free with a thickness of about 7 μm. The bonding strength of the HA-CNT/TiO2 coating was higher than that of the pure HA and HA-CNT coatings. Additionally, in vitro cell experiments showed that CNTs promoted the adhesion of preosteoblasts on the HA-CNT/TiO2 double layer coatings. These unique surfaces combined with the osteoconductive properties of HA exhibited the excellent mechanical properties of CNTs. Therefore, the developed HA-CNT/TiO2 coatings on Ti substrates might be a promising material for bone replacement.
PMCID: PMC3344277  PMID: 22606041
hydroxyapatite; carbon nanotubes; titania; anodization; sol-gel process
22.  Effects of nanoporous anodic titanium oxide on human adipose derived stem cells 
The aim of current bone biomaterials research is to design implants that induce controlled, guided, successful, and rapid healing. Titanium implants are widely used in dental, orthopedic, and reconstructive surgery. A series of studies has indicated that cells can respond not only to the chemical properties of the biomaterial, but also, in particular, to the changes in surface topography. Nanoporous materials remain in focus of scientific queries due to their exclusive properties and broad applications. One such material is nanostructured titanium oxide with highly ordered, mutually perpendicular nanopores. Nanoporous anodic titanium dioxide (TiO2) films were fabricated by a three-step anodization process in propan-1,2,3-triol-based electrolyte containing fluoride ions. Adipose-derived stem cells offer many interesting opportunities for regenerative medicine. The important goal of tissue engineering is to direct stem cell differentiation into a desired cell lineage. The influence of nanoporous TiO2 with pore diameters of 80 and 108 nm on cell response, growth, viability, and ability to differentiate into osteoblastic lineage of human adipose-derived progenitors was explored. Cells were harvested from the subcutaneous abdominal fat tissue by a simple, minimally invasive, and inexpensive method. Our results indicate that anodic nanostructured TiO2 is a safe and nontoxic biomaterial. In vitro studies demonstrated that the nanotopography induced and enhanced osteodifferentiation of human adipose-derived stem cells from the abdominal subcutaneous fat tissue.
PMCID: PMC5072627  PMID: 27789947
adipose-derived stem cells; anodic titanium oxide; nanotopography; osteogenic differentiation; biomaterials
23.  Exopolysaccharides Produced by Streptococcus mutans Glucosyltransferases Modulate the Establishment of Microcolonies within Multispecies Biofilms▿  
Journal of Bacteriology  2010;192(12):3024-3032.
Streptococcus mutans is a key contributor to the formation of the extracellular polysaccharide (EPS) matrix in dental biofilms. The exopolysaccharides, which are mostly glucans synthesized by streptococcal glucosyltransferases (Gtfs), provide binding sites that promote accumulation of microorganisms on the tooth surface and further establishment of pathogenic biofilms. This study explored (i) the role of S. mutans Gtfs in the development of the EPS matrix and microcolonies in biofilms, (ii) the influence of exopolysaccharides on formation of microcolonies, and (iii) establishment of S. mutans in a multispecies biofilm in vitro using a novel fluorescence labeling technique. Our data show that the ability of S. mutans strains defective in the gtfB gene or the gtfB and gtfC genes to form microcolonies on saliva-coated hydroxyapatite surfaces was markedly disrupted. However, deletion of both gtfB (associated with insoluble glucan synthesis) and gtfC (associated with insoluble and soluble glucan synthesis) is required for the maximum reduction in EPS matrix and biofilm formation. S. mutans grown with sucrose in the presence of Streptococcus oralis and Actinomyces naeslundii steadily formed exopolysaccharides, which allowed the initial clustering of bacterial cells and further development into highly structured microcolonies. Concomitantly, S. mutans became the major species in the mature biofilm. Neither the EPS matrix nor microcolonies were formed in the presence of glucose in the multispecies biofilm. Our data show that GtfB and GtfC are essential for establishment of the EPS matrix, but GtfB appears to be responsible for formation of microcolonies by S. mutans; these Gtf-mediated processes may enhance the competitiveness of S. mutans in the multispecies environment in biofilms on tooth surfaces.
PMCID: PMC2901689  PMID: 20233920
24.  Modulation of human dermal microvascular endothelial cell and human gingival fibroblast behavior by micropatterned silica coating surfaces for zirconia dental implant applications 
Dental ceramic implants have shown superior esthetic behavior and the absence of induced allergic disorders when compared to titanium implants. Zirconia may become a potential candidate to be used as an alternative to titanium dental implants if surface modifications are introduced. In this work, bioactive micropatterned silica coatings were produced on zirconia substrates, using a combined methodology of sol–gel processing and soft lithography. The aim of the work was to compare the in vitro behavior of human gingival fibroblasts (HGFs) and human dermal microvascular endothelial cells (HDMECs) on three types of silica-coated zirconia surfaces: flat and micropatterned (with pillars and with parallel grooves). Our results showed that cells had a higher metabolic activity (HGF, HDMEC) and increased gene expression levels of fibroblast-specific protein-1 (FSP-1) and collagen type I (COL I) on surfaces with pillars. Nevertheless, parallel grooved surfaces were able to guide cell growth. Even capillary tube-like networks of HDMEC were oriented according to the surface geometry. Zirconia and silica with different topographies have shown to be blood compatible and silica coating reduced bacteria adhesion. All together, the results indicated that microstructured bioactive coating seems to be an efficient strategy to improve soft tissue integration on zirconia implants, protecting implants from peri-implant inflammation and improving long-term implant stabilization. This new approach of micropatterned silica coating on zirconia substrates can generate promising novel dental implants, with surfaces that provide physical cues to guide cells and enhance their behavior.
PMCID: PMC5090413  PMID: 27877662
Biomaterials; Micropatterning; Soft lithography; Human dermal microvascular endothelial cells (HDMECs); Human gingival fibroblasts (HGFs); Hemolysis; Bacteria adhesion
25.  Synthesis, characterization, and performance evaluation of multilayered photoanodes by introducing mesoporous carbon and TiO2 for humic acid adsorption 
Nanostructured photoanodes were prepared via a novel combination of titanium dioxide (TiO2) nanoparticles and mesoporous carbon (C). Four different photoanodes were synthesized by sol–gel spin coating onto a glassy substrate of fluorine-doped tin oxide. The photocatalytic activities of TiO2, TiO2/C/TiO2, TiO2/C/C/TiO2, and TiO2/C/TiO2/C/TiO2 photoanodes were evaluated by exposing the synthesized photoanodes to UV–visible light. The photocurrent density observed in these photoanodes confirmed that an additional layer of mesoporous carbon could successfully increase the photocurrent density. The highest photocurrent density of ~1.022 mA cm−2 at 1 V/saturated calomel electrode was achieved with TiO2/C/C/TiO2 under an illumination intensity of 100 mW cm−2 from a solar simulator. The highest value of surface roughness was measured for a TiO2/C/C/TiO2 combination owing to the presence of two continuous layers of mesoporous carbon. The resulting films had a thickness ranging from 1.605 µm to 5.165 µm after the calcination process. The presence of double-layer mesoporous carbon resulted in a 20% increase in the photocurrent density compared with the TiO2/C/TiO2 combination when only a single mesoporous carbon layer was employed. The improved performance of these photoanodes can be attributed to the enhanced porosity and increased void space due to the presence of mesoporous carbon. For the first time, it has been demonstrated here that the photoelectrochemical performance of TiO2 can be improved by integrating several layers of mesoporous carbon. Comparison of the rate of removal of humic acid by the prepared photoanodes showed that the highest performance from TiO2/C/C/TiO2 was due to the highest photocurrent density generated. Therefore, this study showed that optimizing the sequence of mesoporous carbon layers can be a viable and inexpensive method for enhanced humic acid removal.
PMCID: PMC4993268  PMID: 27574426
renewable energy; photocatalysis; mesoporous carbon; TiO2 nanoparticle; multilayer photoelectrode; humic acid

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