Polymicrobial biofilms related to dental implant diseases: unravelling the critical role of extracellular biofilm matrix.

Biofilms are complex tri-dimensional structures that encase microbial cells in an extracellular matrix comprising self-produced polymeric substances. The matrix rich in extracellular polymeric substance (EPS) contributes to the unique features of biofilm lifestyle and structure, enhancing microbial accretion, biofilm virulence, and antimicrobial resistance. The role of the EPS matrix of biofilms growing on biotic surfaces, especially dental surfaces, is largely unravelled. To date, there is a lack of a broad overview of existing literature concerning the relationship between the EPS matrix and the dental implant environment and its role in implant-related infections. Here, we discuss recent advances in the critical role of the EPS matrix on biofilm growth and virulence on the dental implant surface and its effect on the etiopathogenesis and progression of implant-related infections. Similar to other biofilms associated with human diseases/conditions, EPS-enriched biofilms on implant surfaces promote microbial accumulation, microbiological shift, cross-kingdom interaction, antimicrobial resistance, biofilm virulence, and, consequently, peri-implant tissue damage. But intriguingly, the protagonism of EPS role on implant-related infections and the development of matrix-target therapeutic strategies has been neglected. Finally, we highlight the need for more in-depth analyses of polymicrobial interactions within EPS matrix and EPS-targeting technologies' rationale for disrupting the complex biofilm microenvironment with more outstanding translation to implant applications in the near future.

Bacterial-derived extracellular polysaccharides reduce antimicrobial susceptibility on biotic and abiotic surfaces.

OBJECTIVE: Extracellular biofilm matrix plays a role in reducing bacterial susceptibility against antimicrobials. Since the surface where biofilm is growing modulates microbial accumulation and bacterial-derived exopolysaccharides (EPS) synthesis, this study compared the role of EPS to reduce antimicrobial susceptibility on biotic (dental surface) and abiotic (titanium (Ti) material) surfaces and the effect of remaining matrix-enriched biofilms to promote bacterial recolonization. DESIGN: 48 h Streptococcus mutans UA159 strain biofilms were grown on enamel and Ti surfaces. The medium was supplemented with 1% sucrose, substrate for EPS synthesis, or with 0.5% glucose + 0.5% fructose as control. Chlorhexidine (CHX) 0.2% was used for antimicrobial treatment. Biofilms were collected and the following analyses were considered: viable bacterial counts, biofilm pH, EPS content, and biofilm structure by scanning electron microscopy and confocal laser scanning microscopy (CLSM). Substrate surfaces were analyzed by 3D laser scanning confocal microscope. RESULTS: Enamel surface showed a higher amount of EPS content (p < 0.05), which may be explained by the higher bacterial biomass compared to Ti material. EPS content reduced bacterial susceptibility against antimicrobial treatments for both substrates, compared to EPS control (p < 0.05). However, sucrose-treated cells presented the same magnitude of reduction for Ti or enamel. Interestingly, matrix-enriched biofilms favored bacterial recolonization for both substrates. CONCLUSION: The surface where the biofilm is growing modulates the amount of EPS synthesized and matrix content plays a key role in reducing antimicrobial susceptibility and promoting bacterial recolonization.

Fitting pieces into the puzzle: The impact of titanium-based dental implant surface modifications on bacterial accumulation and polymicrobial infections.

Polymicrobial infection is the main cause of dental implant failure. Although numerous studies have reported the ability of titanium (Ti) surface modifications to inhibit microbial adhesion and biofilm accumulation, the majority of solutions for the utilization of Ti antibacterial surfaces have been testedin in vitro and animal models, with only a few developed surfaces progressing into clinical research. Motivated by this huge gap, we critically reviewed the scientific literature on the existing antibacterial Ti surfaces to help understand these surfaces' impact on the "puzzle" of undesirable dental implant-related infections. This manuscript comprises three main sections: (i) a narrative review on topics related to oral biofilm formation, bacterial-implant surface interactions, and on how implant-surface modifications can influence microbial accumulation; (ii) a critical evidence-based review to summarize pre-clinical and clinical studies in an attempt to "fit pieces into the puzzle" to unveil the best way to reduce microbial loads and control polymicrobial infection around dental implants showed by the current in vivo evidence; and (iii) discussion and recommendations for future research testing emerging antibacterial implant surfaces, connecting basic science and the requirements for future clinical translation. The findings of the present review suggest no consensus regarding the best available Ti surface to reduce bacterial colonization on dental implants. Smart release or on-demand activation surface coatings are a "new piece of the puzzle", which may be the most effective alternative for reducing microbial colonization on Ti surfaces, and future studies should focus on these technologies.

Cariogenic Potential of Human and Bovine Milk on Enamel Demineralization.

The higher cariogenicity of human milk when compared with bovine milk is still a debatable subject. Therefore, we evaluated the effect of human or bovine milk exposure on biofilm composition and enamel demineralization using a validated cariogenic biofilm model. Streptococcus mutans UA159 biofilms (n = 8) were grown on human saliva-coated bovine enamel slabs of known surface hardness. The biofilms were exposed 8×/day to 0.9% NaCl (negative control), human milk, bovine milk, 7.0% lactose (active human milk control), 4.5% lactose (active bovine milk control), or 10% sucrose (positive control). The culture medium was changed twice daily, and the pH was analyzed as an indicator of biofilm acidogenicity. After 120 h of growth, biofilms were harvested to evaluate viable cells, and soluble and insoluble extracellular polysaccharides (EPS). Enamel demineralization was assessed by the percentage of surface hardness loss (%SHL). Data were analyzed by one-way ANOVA/Tukey's test (α = 5%). In terms of %SHL, negative control (7.7 ± 3.1), human milk control (13.3 ± 7.5), bovine milk control (15.3 ± 8.2), human milk (7.5 ± 5.0), and bovine milk (8.7 ± 6.3) did not differ among them (p > 0.05) but differed (p < 0.05) from sucrose (55.1 ± 5.4). The findings of enamel demineralization (%SHL) were statistically supported by the data of biofilm acidogenicity, bacterial counts and EPS biofilm composition. This experimental study suggests that human and bovine milk have low cariogenic potential to provoke caries lesions in enamel.

Titanium particles and ions favor dysbiosis in oral biofilms.

OBJECTIVE: To evaluate the effect of titanium (Ti) particles and ions on oral biofilm growth and composition. BACKGROUND: Particles and ions of Ti released from dental implants can trigger unfavorable biological responses in human cells. However, their effect on oral biofilms composition has not been tested. METHODS: In this blind in situ study, volunteers wore a palatal appliance containing Ti disks for 7 days to allow biofilm formation. Disks were then collected and biofilms were treated, in vitro, with Ti particles (0.75% and 1%), ions (10 and 20 ppm), or a combination of both (1% particles + 20 ppm ions). Biofilms exposed only to medium was used as control group. After 24 hours, biofilms were collected and analyzed by checkerboard DNA-DNA hybridization. Direct effects of Ti particles and ions on biofilm/cellular morphology were evaluated by transmission electron microscopy (TEM). RESULTS: Ti particles affected biofilm composition, increasing population of four bacterial species (P < .05), while Ti ions showed higher levels of putative pathogens from the orange complex with reduction in species from the yellow complex (P < .05), compared with control. The combination of particles + ions increased green complex and reduced yellow complex proportions (P < .05). TEM showed clusters of particles agglomerated in extracellular environment, while Ti ions were precipitated in both extracellular and intracellular sites. CONCLUSIONS: Ti products, especially Ti ions, have the potential to change the microbiological composition of biofilms formed on Ti surfaces. Therefore, the presence of Ti products around dental implants may contribute to microbial dysbiosis and peri-implantitis.

Contextual and Individual Determinants of Root Caries in Older People.

The aim of this study was to identify the association of the presence of root caries in older people with contextual and individual determinants using a multilevel model. Data from the National Survey of Oral Health collected in Brazil were used. A sample of older Brazilians (aged 65-74 years) was included and selected through multistage probability cluster sampling, using probability proportional to size. Contextual variables of municipalities and individual variables of older people were included. Descriptive, bivariate, and multilevel analyses were conducted. Of the 3,926 older people included in the study, 934 (21.8%) had at least 1 tooth with root caries. There seemed to be no pattern of involvement between the anterior and posterior teeth in the dental arches. Multilevel analysis showed a higher presence of root caries among older people resident in municipalities that were noncapital cities (OR = 1.50), who were over 70 years of age (odds ratio, OR = 1.22), had nonwhite skin color (OR 1.35), had coronal caries (OR = 5.58), were dissatisfied with their teeth and mouth (OR = 1.47), and had self-perceived dental treatment needs (OR = 1.33). Contextual and individual determinants were associated with the occurrence of root caries in older people. Lesion presence demonstrated a profile of social inequality.

Three-species biofilm model onto plasma-treated titanium implant surface.

In this study, titanium (Ti) was modified with biofunctional and novel surface by micro-arc oxidation (MAO) and glow discharge plasma (GDP) and we tested the development of a three-species periodontopatogenic biofilm onto the treated commercially-pure titanium (cpTi) surfaces. Machined and sandblasted surfaces were used as control group. Several techniques for surface characterizations and monoculture on bone tissue cells were performed. A multispecies biofilm composed of Streptococcus sanguinis, Actinomyces naeslundii and Fusobacterium nucleatum was developed onto cpTi discs for 16.5h (early biofilm) and 64.5h (mature biofilm). The number of viable microorganisms and the composition of the extracellular matrix (proteins and carbohydrates) were determined. The biofilm organization was analyzed by scanning electron microscopy (SEM) and Confocal laser scanning microscopy (CLSM). In addition, MC3T3-E1 cells were cultured on the Ti surfaces and cell proliferation (MTT) and morphology (SEM) were assessed. MAO treatment produced oxide films rich in calcium and phosphorus with a volcano appearance while GDP treatment produced silicon-based smooth thin-film. Plasma treatments were able to increase the wettability of cpTi (p<0.05). An increase of surface roughness (p<0.05) and formation of anatase and rutile structures was noted after MAO treatment. GDP had the greatest surface free energy (p<0.05) while maintaining the surface roughness compared to the machined control (p>0.05). Plasma treatment did not affect the viable microorganisms counts, but the counts of F. nucleatum was lower for MAO treatment at early biofilm phase. Biofilm extracellular matrix was similar among the groups, excepted for GDP that presented the lowest protein content. Moreover, cell proliferation was not significantly affected by the experimental, except for MAO at 6days that resulted in an increased cell proliferative. Together, these findings indicate that plasma treatments are a viable and promising technology to treat bone-integrated dental implants as the new surfaces displayed improved mechanical and biological properties with no increase in biofilm proliferation.