Bacterial biofilms are related to various dental and periodontal infectious diseases, and the characterization of this biological structure with micro-computed tomography (micro-CT) may offer valuable information for clinical and research applications. In this study, we aimed to develop a model to visualize three-dimensionally the biofilm structure on dentin using micro-CT. Dentin blocks were prepared and incubated in tryptic soy broth with Enterococcus faecalis (ATCC 29212). The control group did not receive any staining procedure, while groups 1 and 2 were stained with 100% and 50% barium sulfate, respectively. Transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM) were used to detect biofilm formation, barium sulfate penetration, and microbial cell density in the biofilm. Micro-computed tomography (micro-CT) (SkyScan 1172, Bruker Co., Belgium) was used to visualize biofilm formation on the dentin blocks. Biofilm thicknesses were measured from 10 different locations on the specimen surfaces, using CTAn v.1.14.4 software. Obtained data were statistically analyzed using Kruskal–Wallis and Dunn’s tests. TEM photomicrographs showed that barium sulfate could penetrate the biofilm structure. CLSM analysis showed that viable and total cell densities were similar between the control and barium sulfate-treated groups ( P > 0.05), indicating barium sulfate had no significant influence on cell density. In barium sulfate-treated blocks, biofilm could be discriminated from the dentin, and its thickness could be measured with micro-CT. This study showed that bacterial biofilm on dentin could be characterized by micro-CT after barium sulfate staining without causing any significant side effect on viable and total cell densities.

中文翻译：

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牛牙本质中粪肠球菌生物膜结构的可视化和表征使用 2D 和 3D 显微技术

细菌生物膜与各种牙科和牙周传染病有关，用微计算机断层扫描 (micro-CT) 表征这种生物结构可能为临床和研究应用提供有价值的信息。在这项研究中，我们旨在开发一个模型，使用显微 CT 对牙本质上的生物膜结构进行三维可视化。制备牙本质块并在含有粪肠球菌 (ATCC 29212) 的胰蛋白酶大豆肉汤中培养。对照组不接受任何染色程序，而第 1 组和第 2 组分别用 100% 和 50% 硫酸钡染色。透射电子显微镜 (TEM) 和共聚焦激光扫描显微镜 (CLSM) 用于检测生物膜形成、硫酸钡渗透和生物膜中的微生物细胞密度。微计算机断层扫描（micro-CT）（SkyScan 1172，Bruker Co.，Belgium）用于观察牙本质块上生物膜的形成。使用 CTAn v.1.14.4 软件从样本表面的 10 个不同位置测量生物膜厚度。使用 Kruskal-Wallis 和 Dunn 检验对获得的数据进行统计分析。TEM 显微照片显示硫酸钡可以穿透生物膜结构。CLSM 分析显示，对照组和硫酸钡处理组之间的活细胞密度和总细胞密度相似（ P > 0.05），表明硫酸钡对细胞密度没有显着影响。在硫酸钡处理的块中，生物膜可以与牙本质区分开来，其厚度可以用微 CT 测量。