Graphene-based dental adhesive with anti-biofilm activity

Abstract

Background: Secondary caries are considered the main cause of dental restoration failure. In this context, anti-biofilm and bactericidal properties are desired in dental materials against pathogens such as Streptococcus mutans. To this purpose, graphene based materials can be used as fillers of polymer dental adhesives. In this work, we investigated the possibility to use as filler of dental adhesives, graphene nanoplatelets (GNP), a non toxic hydrophobic nanomaterial with antimicrobial and anti-biofilm properties.

Results: Graphene nanoplatelets have been produced starting from graphite intercalated compounds through a process consisting of thermal expansion and liquid exfoliation. Then, a dental adhesive filled with GNPs at different volume fractions has been produced through a solvent evaporation method. The rheological properties of the new experimental adhesives have been assessed experimentally. The adhesive properties have been tested using microtensile bond strength measurements (µ-TBS). Biocidal activity has been studied using the colony forming units count (CFU) method. The anti-biofilm properties have been demonstrated through FE-SEM imaging of the biofilm development after 3 and 24 h of growth.

Conclusions: A significantly lower vitality of S. mutans cells has been demonstrated when in contact with the GNP filled dental adhesives. Biofilm growth on adhesive-covered dentine tissues demonstrated anti-adhesion properties of the produced materials. µ-TBS results demonstrated no significant difference in µ-TBS between the experimental and the control adhesive. The rheology tests highlighted the necessity to avoid low shear rate regimes during adhesive processing and application in clinical protocol, and confirmed that the adhesive containing the 0.2%wt of GNPs possess mechanical properties comparable with the ones of the control adhesive.

Fig. 1

Schematics of the experimental dental adhesive production process. The wormlike expanded graphite (WEG) undergoes tip sonication to obtain GNP. Then, the obtained suspension is mixed with the standard dental adhesive. Finally, the obtained material consists in a good dispersion of the filler nanoparticles among the polymer chains

Fig. 2

FE-SEM top-view micrographs showing the detail of GNP interaction with the polymer matrix: a low magnification and b higher magnification of the polymer-nanostructures interface. GNPs (pointed out by white arrows) are well integrated into the adhesive, thus demonstrating the uniform dispersion achieved

Fig. 3

FE-SEM top-view micrographs showing different magnifications of the tested sample sets on different substrates. The micrographs illustrate the differences in nanofiller distribution and exposition over the adhesive surface (GNPs are pointed out by the white arrows and dotted frames): a, e, i represent the control adhesive on different substrates ( p₀, p₂₀ and p₁₅₀). GNPs are embedded in correspondence of the p₀ substrate (b–d); the nanofillers are well dispersed and exposed over the surface of adhesives filled with at 0.1 and 0.2%wt, applied on the substrates p₂₀ and p₁₅₀ (f, g, l, m); GNPs are exposed over the surface of the adhesive filled with at 0.5%wt, more densely when applied over the substrate with larger porosity p₁₅₀ (n) than over the substrated with smaller porosity p₂₀ (h). Scale bar 1 µm

Fig. 4

Shear stress (a) and viscosity (b) of the produced samples measured at room temperature (23 °C), as function of the shear rate

Fig. 5

CFU results of S. mutans in contact with the produced sample sets. Error bar indicates standard deviation

Fig. 6

a, b Two different magnitudes of the 3 h-growth of S. mutans biofilm on the teeth coated with the control adhesive A. c, d Represent two different magnitudes of the 3 h-growth of S. mutans biofilm on teeth-coated by experimental adhesive A02

Fig. 7

a, b Two different magnitudes of the 3 h-growth of S. mutans biofilm on the teeth coated with the control adhesive A. c, d Represent two different magnitudes of the 3 h-growth of S. mutans biofilm on teeth-coated by experimental adhesive A02

Fig. 8

a Photographs of control adhesive- and A02-covered teeth stained with CV. b Biofilm biomass analysis on CV stained teeth. Histograms are the mean of three independent experiments. Error bars indicate SD and Student’s t test was used to assess statistical significance (*p < 0.05 with respect to control adhesive)

Fig. 9

Cytosolic ROS quantification by measuring the dichlorofuorescein diacetate (H₂DCFDA) probe activation through ROS generation in S. mutans biofilm grown on adhesives containing or not GNPs. Data are expressed as ROS accumulation relative to commercial adhesive sample. As a positive control is shown ROS amount of hydrogen peroxide-treated biofilm. Statistical analysis was performed by one-way ANOVA method coupled with the Bonferroni post-test (ns not significant; *p < 0.05 with respect to control adhesive)

Fig. 10

Antimicrobial and antibiofilm action mechanism of the experimental adhesive developed in this work. Graphene is known to be an anti-adhesion material. GO are well-known antimicrobial materials thanks to the shape and to the presence of oxygen containing functional groups that increase its hydrophilicity and allow ROS production. GNPs possess biocidal properties typical of 2D-shaped graphene based materials and anti-adhesion properties typical of graphene thanks to the absence of basal plane functional groups. Moreover, GNPs do not produce ROS due to the absence of oxygen reacting species, and consequently are characterized by a much lower potential of cytotoxicity when compared with GOs