A reproducible oral microcosm biofilm model for testing dental materials

Abstract

Aims: Most studies of biofilm effects on dental materials use single-species biofilms, or consortia. Microcosm biofilms grown directly from saliva or plaque are much more diverse, but difficult to characterize. We used the Human Oral Microbial Identification Microarray (HOMIM) to validate a reproducible oral microcosm model.

Methods and results: Saliva and dental plaque were collected from adults and children. Hydroxyapatite and dental composite discs were inoculated with either saliva or plaque, and microcosm biofilms were grown in a CDC biofilm reactor. In later experiments, the reactor was pulsed with sucrose. DNA from inoculums and microcosms was analysed by HOMIM for 272 species. Microcosms included about 60% of species from the original inoculum. Biofilms grown on hydroxyapatite and composites were extremely similar. Sucrose pulsing decreased diversity and pH, but increased the abundance of Streptococcus and Veillonella. Biofilms from the same donor, grown at different times, clustered together.

Conclusions: This model produced reproducible microcosm biofilms that were representative of the oral microbiota. Sucrose induced changes associated with dental caries.

Significance and impact of the study: This is the first use of HOMIM to validate an oral microcosm model that can be used to study the effects of complex biofilms on dental materials.

Figure 1

(A) CDC biofilm reactor sampling rods with disks of Z100 composite resin, LS composite resin, and hydroxyapatite (HA). (B) Experimental set-up for the CDC biofilm reactor.

Figure 2

(A) Representative scanning electron micrograph of a biofilm grown on LS composite after 72 h in the CDC biofilm reactor. Dense multi-layered biofilm structures were separated by deep channels. (B) The same biofilm at higher magnification. A variety of bacterial morphotypes were visible, and linked by direct coaggregation, as well as a web-like polysaccharide matrix revealed by staining with Alcian Blue.

Figure 3

Dendrogram showing cluster analysis results for HOMIM data from the four adult subjects. Sample labels indicate first the source of the inoculum (saliva or plaque, or a stock prepared from a previous plaque microcosm), then whether or not sucrose pulsing was used, then the nature of the substrate the biofilm was grown on (HA, or LS; if no substrate is indicated, then DNA was extracted directly from the inoculum), then the subject identifier (Sub1, Sub2, Sub3, or Sub4), and finally the repeat number for microcosms grown from the same subject on different weeks (rep1, rep2, or rep3). Sample labels for major clusters are enclosed in boxes labeled with vertical text indicating the contents of the cluster (the abbreviations used are PMS1 for Plaque Microcosms Subject 1, SMS1 For Saliva Microcosms Subject 1, and S for Saliva. Inner boxes within each major cluster indicate samples from the same subject that were grouped together. Saliva_Sub1_rep1 and Saliva_Sub4_rep 3 are not in the dendrogram because they failed to meet HOMIM quality control criteria when they were run at the Forsyth Dental Center, and hence were excluded from all statistical analyses. Saliva_Sub2_rep4 denotes an additional sample from a preliminary experiment that was available only for Subject 2. It was included in the analysis to determine whether it would cluster with repeats 1 – 3 from Subject 2.

Figure 4

Dendrogram showing cluster analysis results for HOMIM data from 10 pediatric subjects. Only plaque was used as an inoculum for those experiments, and all microcosms were sucrose pulsed. Sample labels indicate the subject identifier Pxxx, followed by plaque for DNA extracted directly from inoculums, or HA, LS, or Z100 to indicate the substrate on which microcosm samples were grown. Sample labels for major clusters are enclosed in boxes labeled with vertical text indicating the contents of the cluster (the abbreviations used are P for Plaque, and SPM for Sucrose Pulsed Microcosm). Inner boxes within each major cluster indicate samples from the same subject that were grouped together.

Figure 5

Representative real-time pH measurements obtained for experiments with frozen stocks made from microcosms obtained from pediatric subjects. Results are shown for the frozen stock from subject 782. The control (blue line) denotes sterile media that was incubated in the reactor under the same protocol used for biofilm growth, and pulsed 5 times with sterile sucrose on each of the flow days. The red line indicates the pH curve for the 782 frozen stock grown with the same sucrose pulsing schedule, and the green line indicates the pH curve when the same stock was grown without sucrose pulsing. The red arrows on the X-axis indicate the times when sucrose pulses were administered.