The ultrastructure of subgingival dental plaque, revealed by high-resolution field emission scanning electron microscopy

Richard Holliday,Leon Bowen,Nicholas S Jakubovics,Philip M Preshaw

doi:10.1038/bdjopen.2015.3

Abstract

Objectives/Aims:To explore the ultrastructure of subgingival dental plaque using high-resolution field emission scanning electron microscopy (FE-SEM) and to investigate whether extracellular DNA (eDNA) could be visualised in ex vivo samples.Materials and Methods:Ten patients were recruited who fulfilled the inclusion criteria (teeth requiring extraction with radiographic horizontal bone loss of over 50% and grade II/III mobility). In total, 12 teeth were extracted using a minimally traumatic technique. Roots were sectioned using a dental air turbine handpiece, under water cooling to produce 21 samples. Standard fixation and dehydration protocols were followed. For some samples, gold-labelled anti-DNA antibodies were applied before visualising biofilms by FE-SEM.Results:High-resolution FE-SEMs of subgingival biofilm were obtained in 90% of the samples. The sectioning technique left dental plaque biofilms undisturbed. Copious amounts of extracellular material were observed in the plaque, which may have been eDNA as they had a similar appearance to labelled eDNA from in vitro studies. There was also evidence of membrane vesicles and open-ended tubular structures. Efforts to label eDNA with immune-gold antibodies were unsuccessful and eDNA was not clearly labelled.Conclusions:High-resolution FE-SEM images were obtained of undisturbed subgingival ex vivo dental plaque biofilms. Important structural features were observed including extracellular polymeric material, vesicles and unusual open tubule structures that may be remnants of lysed cells. The application of an eDNA immune-gold-labelling technique, previously used successfully in in vitro samples, did not clearly identify eDNA in ex vivo samples. Further studies are needed to characterise the molecular composition of the observed extracellular matrix material.

Highlights

A biofilm is a collection of microbial cells that forms on a surface or interface, and is encased within an extracellular polymeric matrix
In vitro models vary from simple monospecies biofilms to complex high-throughput microfluidic systems
The samples are required to be dehydrated for field emission scanning electron microscopy (FE-scanning electron microscopy (SEM)) and the biofilm will be observed in a collapsed state

Summary

Introduction

A biofilm is a collection of microbial cells that forms on a surface or interface, and is encased within an extracellular polymeric matrix. Dental plaque is a complex biofilm, with around 700 ‘natural colonisers’.2. Understanding the composition and structure of dental plaque is key to developing new techniques for improving the treatment of biofilm-related pathologies in patients. Subgingival dental plaque, a major initiating aetiological factor in periodontal diseases, is much more challenging to study due to its protected location beneath the gumline. A range of in vitro and ex vivo techniques have been utilised in studies of subgingival dental plaque biofilms. In vitro models vary from simple monospecies biofilms to complex high-throughput microfluidic systems.. In vitro models vary from simple monospecies biofilms to complex high-throughput microfluidic systems.4 These are limited in their ability to replicate the physiological situation in the mouth as several species are unculturable, some progress is being made in this area In vitro models vary from simple monospecies biofilms to complex high-throughput microfluidic systems. These are limited in their ability to replicate the physiological situation in the mouth as several species are unculturable, some progress is being made in this area

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