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

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.

Figure 1

Scanning electron micrographs of subgingival dental plaque demonstrating a dense web of extracellular matrix material (solid arrow). Bead structures can be seen on the strands of extracellular material (broken arrow). Rod-shaped cells (r) were present with extracellular strands originating from the cell surface. In addition, polymeric ‘sweaters’ (s) can be seen encasing cells. ×35,000 magnification.

Figure 2

Scanning electron micrographs of subgingival dental plaque demonstrating a range of microorganisms but with the predominate species having a swollen rod morphology that could potentially be members of TM7 phylum (t). A Spirochaetal microorganism, probably Treponema sp., can also be seen (s). Bacterial septa can be clearly seen in the suspected TM7 microorganisms. Extensive membrane-associated vesicles can be seen associated with some septa (solid arrow), whereas other septa are totally devoid of vesicles (broken arrow). (a) ×35,000 magnification; (b) ×80,000 magnification.

Figure 3

Scanning electron micrographs of subgingival dental plaque. Large coccoid cells, straight rods and spirochaetal microorganisms can be seen. Chains of vesicles (solid arrows) can be seen in several places. Fine strands of extracellular material can also be seen (broken arrow). (a) ×20,000 magnification; (b) ×100,000 magnification.

Figure 4

Scanning electron micrographs of subgingival dental plaque. (a) Numerous ‘open tubule’ structures (solid arrow). An extensive web of extracellular strands can be seen (broken arrow) encompassing the tubules and also several chains of intact short rod-shaped microorganisms. Membrane-associated vesicles can be seen (wide ‘v’ arrow). (b) A single ‘open tubule’ (solid arrow) surrounded by intact cells (c). (a) ×20,000 magnification; (b) ×50,000 magnification.

Figure 5

Scanning electron micrographs demonstrating intact biofilm 100 microns from a sectioned sample edge. (a) The cut edge of the tooth root with the gouges from the diamond bur clearly visible. The dashed box indicates where higher-resolution images were obtained. (b) ×25,000 magnification demonstrating intact rod-like microorganisms in a biofilm. (c) ×80,000 magnification showing extensive extracellular matrix as fine strands between the microorganisms.

Figure 6

Secondary electron micrographs of subgingival dental plaque labelled with immune-gold antibodies (a) and control (no primary antibody) (b). (a) The complex nature of the subgingival biofilm with a range of rod- and spirochaetal-like microorganisms. A microorganism with flagella emerging from its underside can be seen (a, solid arrow). (b) Several bunches of cocci along with rod-shaped microorganisms. (a) ×20,000 magnification; (b) ×25,000 magnification.

Figure 7

Backscatter electron micrographs of subgingival dental plaque labelled with immune-gold antibodies (a) and control (no primary antibody) (b). The labelled sample (a) has more contrast within the image compared with the control (b) but it was not possible to discern individual immune-gold particles. (a) ×25,000 magnification; (b) ×20,000 magnification.