A Method to Trace Porphyromonas gingivalis Within Periodontal Ligament in 3D

Abstract

Background and Objective: Periodontitis is an inflammatory disease caused by bacterial dysbiosis that leads to tooth support loss and eventually, loss of tooth. There have been studies using micro-CT, histology, and electron microscopy to elucidate the pathological effect of the pathogens in periodontal disease; however, the exact trace of these bacteria in inflamed periodontal tissue remains unclear. The purpose of this study was to develop a 3D imaging method that elucidates the distribution of P. gingivalis within periodontal ligament (PDL) upon infection, combining an optical clearing method, fluorescent-labeling via click chemistry, and multi-photon microscopy. Materials and methods: Periodontal disease was induced by using 5-0 silk ligature in C57BL/6 mice. P.g was labeled with fluorophore via click chemistry, then introduced to the sulcus by tying 7-0 silk ligature. Optical clearing of dissected hemi-mandibles was achieved by ECi-based clearing method. Multi-photon microscopy was utilized to obtain 3D images of 2-photon signals from the fluorophore and SHG signals from type I collagen in PDL. Fluorescent bacteria in 2-photon images were detected by trained Zen Intellesis module. Results: ECi-based clearing of hemi-mandible resulted in optimal transparency for microscope imaging, and preserved fluorophore signals labeled with click chemistry method. Multi-photon microscopy imaging allowed 3D visualization of fluorescent-labeled P.g and collagen fibers from PDL. Significantly more bacteria were detected by Zen Intellesis module in the infection group compared to the non-infection group. Conclusion: This study presents a 3D imaging method that allows tracing of bacteria in murine model without sectioning. This method has the potential to be effectively utilized in different research fields of dentistry, such as in endodontics, oral pathology, or implantology.