Ex vivo Detection of Amyloid-β in Naturally Formed Oral Biofilm

Abstract

Background: Oral infection has been implicated in the possible etiology of Alzheimer’s disease. Objective: To detect amyloid-β (Aβ) within microbial biofilms. Methods: Freshly extracted teeth (N = 87) with periodontal disease were separated into Group A (N = 11), with primary root canal infection and Group B (N = 21) with failed endodontic treatment identified by the presence of, gutta percha root filling. Biofilm characteristics were observed by scanning electron microscopy (SEM). Demineralized paraffin wax embedded tooth sections and mineralized calculus biofilm were immunostained with the anti-Aβ antibody. The gutta perchas were processed either for on-section acrylic resin tissue immunocolloidal gold silver staining (IGSS) using the anti-Aβ antibody or in Araldite resin for ultrastructure. Results: SEM demonstrated calculus and gutta percha in situ harboring a polymicrobial biofilm featuring extracellular polymeric substance (EPS) and water channels. Immunohistochemistry on rehydrated paraffin wax tooth sections from Group A, demonstrated Aβ staining on external (calculus and plaque) and all intracanal infected regions. In Group B, the gutta percha biofilm IGSS gave an inconclusive result for Aβ. Transmission electron microscopy of selected teeth with infected intra-canals (Group A) and 20% of gutta percha biofilm (Group B) EPS contained electron dense fibrils of variable sizes, some of which were typical of human Aβ fibrils. Conclusion: This study detected both soluble and insoluble Aβ fibrils within the EPS of periodontal and endodontic natural biofilm, strongly suggesting its role as an antimicrobial peptide in combatting local infection, with potential risk for cross-seeding into the brain for AD development.