Biofilm three-dimensional architecture influences in situ pH distribution pattern on the human enamel surface

Abstract

To investigate how the biofilm three-dimensional (3D) architecture influences in situ pH distribution patterns on the enamel surface. Biofilms were formed on human tooth enamel in the presence of 1% sucrose or 0.5% glucose plus 0.5% fructose. At specific time points, biofilms were exposed to a neutral pH buffer to mimic the buffering of saliva and subsequently pulsed with 1% glucose to induce re-acidification. Simultaneous 3D pH mapping and architecture of intact biofilms was performed using two-photon confocal microscopy. The enamel surface and mineral content characteristics were examined successively via optical profilometry and microradiography analyses. Sucrose-mediated biofilm formation created spatial heterogeneities manifested by complex networks of bacterial clusters (microcolonies). Acidic regions (pH<5.5) were found only in the interior of microcolonies, which impedes rapid neutralization (taking more than 120 min for neutralization). Glucose exposure rapidly re-created the acidic niches, indicating formation of diffusion barriers associated with microcolonies structure. Enamel demineralization (white spots), rougher surface, deeper lesion and more mineral loss appeared to be associated with the localization of these bacterial clusters at the biofilm-enamel interface. Similar 3D architecture was observed in plaque-biofilms formed in vivo in the presence of sucrose. The formation of complex 3D architectures creates spatially heterogeneous acidic microenvironments in close proximity of enamel surface, which might correlate with the localized pattern of the onset of carious lesions (white spot like) on teeth.

Figure 1

Biofilm structure and Enamel hard surface characteristics. Light microscopic images (a-1-a-4), enamel roughness (b-1-b-4), lesion depth of the enamel (c-1-c-4) and 3D reconstructed confocal microscopic images (d-1-d-4) were shown. A selected sectional image of the biofilm formed in 1% sucrose (d-1) was shown in (e) and (f-1-f-3). The white arrow labeled in images (f-1-f-3) indicated the microcolony structure in the biofilms. 3D, three-dimensional; EPS, exopolysaccharides.

Figure 2

Intact mixed-species biofilms formed on teeth in vivo using the intra-oral (in situ) model. Representative 3D rendering images of plaque-biofilms formed in the presence of glucose+fructose (a) or sucrose (b). (c) Projection images of structured plaque-biofilms. All bacterial cells labeled with EUB338-Cy3 are depicted in green, while Streptococcus labeled with STR405-Cy5 is in red. Microcolonies (see yellow arrows) were observed in sucrose grown biofilms formed in vivo using the intra-oral human plaque model. (c) demonstrated the typical microlonies structure that were enlarged from (b).

Figure 3

Biofilm structure and real-time pH profile. (a) Real-time pH distribution within biofilms; (b) pH value at selected zones within microcolony; (c) pH at the Interface between biofilm and enamel surface vs microcolony diameter; (d) pH at the Interface between biofilm and enamel surface vs height. EPS, exopolysaccharides.