A Novel in vitro Microbial-Based Model for Studying Caries Formation – Development and Initial Testing

Abstract

We report on the development and the initial testing of a new microbial-based caries model. Specimens were fixed on a rotating mount within a reaction chamber hermetically surrounded by a sterilised glove box. A cariogenic environment was obtained by inoculation with Streptococcus mutans (ATCC 25175) combined with a continuously repeating supply of sucrose solution, trypticase soy broth and artificial saliva applied by dripping. Twenty-five caries-free upper premolars were used. The mesial parts of the occlusal fissures had been sealed with a resin-based fissure sealant (test group 1). To produce marginal gaps, the distal parts had been moistened with saliva before resin application (test group 2). Five teeth served as control and were exposed to all fluids under sterile conditions before being removed from the system after 7 days. Test specimens were infected with S. mutans and were incubated for another 14 days. No unintentional contamination occurred during the 3-week period of operation. Demineralizations were evaluated by using confocal laser scanning microscopy. Only the test specimens showed clearly visible signs of biofilm formation and caries-like lesions. The mean primary lesion depth did not differ significantly between test groups. Wall lesion depths and surface areas of demineralizations underneath the fissure sealants were significantly higher in test group 2. Thus, our model allows the simultaneous production of primary and secondary caries-like enamel lesions in a considerable number of specimens and facilitates the possibility to manipulate and transfer them without necessarily terminating the experiment, opening new possibilities for in vitro caries research.

Isolated development of inner (wall) caries like lesions in a bacterial-based in vitro model

“…The correct gap width was checked with a calibrated light microscope (Laborlux 12, Ernst Zeitz). Prior to entry in the microbial-based in vitro caries model, the specimens were sterilised with ethylene dioxide as described elsewhere [17].…”

“…A detailed description of the microbial-based in vitro caries model has recently been published [17,18]. Briefly, the initially sterilised specimens were inoculated with Streptococcus mutans (ATCC 25175) for biofilm development and incubated in this caries model for 21 days.…”

“…The lateral surfaces (perpendicular to the gap) of the specimens were polished with silicon carbide paper (4000 grit) and were examined with confocal laser scanning microscopy ("Zeiss LSM 510", Carl Zeiss; Fig. 1) [17]. An argon laser (wavelength, 488 nm; intensity, 46%), a 2.5× Fluar objective with a numerical aperture of 0.075 and the following beam path configuration was used: channel 1, long pass (LP650), 386 μm pinhole; channel 2, band pass (BP 505-530), 386-μm pinhole and channel 3, band pass (BP 560-615), 374-μm pinhole.…”

“…HFT 488 was taken as "main dichroic beam splitter" and NFT 545 as "secondary dichroic beam splitter". There was no need to stain the specimens [17]. The area of the inner lesion (in μm 2 ) was determined with the help of a special software (Zeiss LSM 510 Image Browser rel.…”

Sealing of Minimally Invasive Class II Fillings (slot) Using an Adhesive Patch: Sealant Margin Extension for Prevention

“…A biofilm formed from Streptococcus mutans medium ensured the development of caries-like enamel lesions (for details see Seemann & others). 15 The specimens were separated from the SEM mounts, cut in half bucco-orally and mounted on the specimen turntable of the device to expose the approximal surface. A stainless steel pin was fixed in the pulp chamber with resin composite (Tetric, Ivoclar Vivadent).…”