Aim: The availability of human teeth for in vitro testing of antimicrobial properties of substances for endodontic use is limited. The aim of the present study was to develop an in vitro method, based on bovine teeth, which allows for the testing of antimicrobials in artificially infected root canals and to evaluate its suitability by testing known antibacterial substances.
Materials and Methods: Roots from bovine incisors were cleaned, cut horizontally into three parts and autoclaved (121°C, 25min) in Tryptic Soy Broth(TBS). Apical parts served as sterility controls. Cervical and median parts were fixed (RelyX Unicem, 3MEspe) in a Petri dish, canals were filled with Enterococcus faecalis(ATCC 29212) suspension, covered with glass plates and incubated (7days, 37°C) on a shaker. Median parts were filled with test substances (NaOCl(0.5%,1.0%,3.0%), CHX(0.2%,1.0%); immersion periods 30s, 10min), cervical parts served as maximum bacterial growth controls. Specimens were cut along the long axis into halves and kept in aqua dest. After cleaning (microbrush, 2ml saline) biopsies of 0.5mm,1.0mm, and 1.5mm depth were taken from the root canal walls perpendicular to their surfaces (sterile round bur; 0.8mm). Resulting dentin chips and the bur were vortexed in 2ml medium for 10s, plated on TSA, incubated (20h,37°C), and colony-forming units(CFU) were counted. Mann-Whitney-Test and Error-Rates-Method were applied for statistical analysis of five samples per group.
Results: Sterility control showed zero CFU indicating an aseptic procedure. Growth controls showed CFU from 2x104 to 106, increasing with increasing depth, indicating an effective artificial infection. Antimicrobials killed bacteria significantly to less than 0.5% CFU related to matching untreated controls(100%). Reductions were significantly influenced by test substances (0.5mm depth).
Conclusions: The results are in accordance with clinical data. Thus, the new method using artificially infected root canals can be considered suitable for in vitro testing of antibacterial agents for endodontic use.