Morphological analysis of subgingival biofilm formation on synthetic carbonate apatite inserted into human periodontal pockets

Background: Details of the development of human subgingival biofilm are unknown due to the difficulties in conducting experiments and especially in obtaining undisturbed materials. Methods: This study was performed using deposits on carbonate apatite that had been inserted into human periodontal pockets for up to three weeks. Scanning electron microscopy using the vertically sectioned method and transmission electron microscopy using the freeze-substitution method were adopted. Results: The development of subgingival biofilm occurred in five sequential phases: pellicle formation, microbial adherence, initial colonization, microbial organization, and establishment. Certain species in each of the initial, secondary and tertiary colonizers were considered to have a predilection for biofilm formation. Gram-positive, bacillary initial colonizers and Gram-negative, filamentous secondary colonizers organized one stable structure that served as the framework for biofilm formation, and Gram-negative, rod-shaped tertiary colonizers with cell-surface vesicles showed multigeneric coaggregation. The microbiota in the tertiary colonizers underwent repeated microflora alteration. Conclusions: Subgingival biofilm is constituted by initial, secondary and tertiary colonizers. Microflora alteration which is suggested to be related to periodontal disease, frequently occurred in the tertiary colonizers.