A dual energy micro-CT methodology for visualization and quantification of biofilm formation and dentin demineralization

ObjectiveThe aim of this study was to induce artificial caries in human sound dentin by means of a microcosm model using human saliva as source of bacteria and to apply a novel dual-energy micro-CT technique to quantify biofilm formation and evaluate its demineralization potential.DesignEight sound third molars had the occlusal enamel removed by cutting with a diamond disk and five cylindrical cavities (±2 mm diameter; ±1.5 mm depth) were prepared over the dentin surface in each specimen (n = 40 cavities). After sterilization, each specimen received the bacterial salivary inoculum obtained from individuals without any systemic diseases presenting dentin caries lesions and were incubated in BHI added of with 5% sucrose for 96 h to allow biofilm formation. After that, two consecutive micro-CT scans were acquired from each specimen (40kv and 70kv). Reconstruction of the images was performed using standardized parameters. After alignment, registration, filtering and image calculations, a final stack of images containing the biofilm volume was obtained from each prepared cavity. Dentin demineralization degree was quantified by comparison with sound dentin areas. All data were analyzed using Shapiro-Wilk test and Spearman correlation using α=5%.ResultsDual-energy micro-CT technique disclosed biofilm formation in all cavities. Biofilm volume inside each cavity varied from 0.30 to 1.57 mm³. A positive correlation between cavity volume and volume of formed biofilm was obtained (0.77, p < 0.01). The mineral decrease obtained in dentin was high (± 90%) for all cavities and all demineralized areas showed mineral density values lower than a defined threshold for dentin caries (1.2 g/cm³).ConclusionDual-energy micro-CT technique was successful in the quantification of a microcosm human bacterial biofilm formation and to quantify its demineralization potential in vitro.