Comparison between static and semi-dynamic models for microcosm biofilm formation on dentin

Abstract Microcosm biofilm has been applied to induce carious lesions in dentin. However, no study has been done to compare the impact of the type of model for providing nutrients to microcosm biofilm formation on dentin. Objective This study compared the performance of two kinds of models (static and semi-dynamic) on the biofilm formation and the development of dentin carious lesions. Material and Methods In both models, biofilm was produced using inoculum from pooled human saliva mixed with McBain saliva for the first 8 h (5% CO2 and 37°C). Afterwards, for the static model, the samples were placed in 24-wells microplate containing McBain saliva with 0.2% sucrose, which was replaced at 24 h. In the semi-dynamic model, the samples were submitted to artificial mouth system with continuous flow of McBain saliva with 0.2% sucrose (0.15 ml/min, 37°C) for 10 h a day (for the other 14 h, no flow was applied, similarly to the static model). After 5 days, biofilm viability was measured by fluorescence and dentin demineralization by transverse microradiography. Results Biofilm viability was significantly lower for the static compared with semi-dynamic model, while dentin demineralization was significantly higher for the first one (p<0.05). The static model was able to produce a higher number of typical subsurface lesions compared with the semi-dynamic model (p<0.05). Conclusions The type of model (static and semi-dynamic) applied in the microcosm biofilm may have influence on it's viability and the severity/profile of dentin carious lesions.


Introduction
Dental caries is a disease that affects millions of people around the world, 1 generated by the instability created between the host and the microorganisms from dental biofilm due to the high and frequent consume of sugar, especially sucrose. 2 Other factors may interfere on biofilm development and increase the risk of root carious lesions, especially for adults and elderly individuals, who present low salivary flow and root exposure due to chronic periodontitis. It has been already established that the prevalence of carious lesions, involving dentin, increases with age. 3 To better understand the dynamic of biofilm on dentin and to test the protective effect of antimicrobial agents, in vitro models of dental caries formation have been applied. 4 The microcosm biofilm has been considered the biofilm model closest to the in vivo reality, making possible to more accurately simulate the complexity of a real dental biofilm in vitro. 5,6 Biofilm models can be further classified according to the availability of nutrients, as: 1) static model, which consists of limited supply of nutrients over time (e.g.: agar plates or multiple well plates); and 2) dynamic model that allows a continuous nutrients supply over time (e.g.: constant depth biofilm fermenter or artificial mouth). [4][5][6][7][8] However, there is no study comparing the impact of the type of model for providing nutrients (static and semi-dynamic) to microcosm biofilm on the development of carious lesion in dentin.
Therefore, this study aimed to compare two models (static and semi-dynamic) regarding the viability of a microcosm biofilm and its capacity of producing carious lesion in dentin. The null hypothesis is that the models do not differ in biofilm viability and capacity of inducing dentin demineralization.

Saliva collection
This study was firstly approved by the local Ethical Committee (CAAE: 58330616.7.0000.5417). Saliva was collected from 2 healthy donors only (the amount of saliva was enough for the experiment), who have followed the inclusion criteria: 1) normal salivary flow (stimulated saliva flow >1 ml/min and non-stimulated saliva flow >0.3 ml/min), 2) with previous history of caries, but not active caries (no active white spot and/ or cavitated lesions), 3) without gingivitis/periodontitis (gum bleeding or tooth mobility) and 4) who did not ingest antibiotics 3 months before the experiment. The donors were not allowed to brush their teeth in the last 24 h before saliva collection and to ingest food or drinks in the last 2 h before this procedure. 9,10 Saliva was collected under stimulation by chewing a gum for 10 min during the morning. The human saliva pool (70%) was mixed with glycerol (30%) and frozen at -80°C. 9,10 Tooth sample preparation Thirty-six dentin samples were prepared from eighteen bovine roots (4 mm x 4 mm, buccal and lingual surfaces) by using a semi-precision cutting machine (Buehler; Lake Bluff, Illinois, USA) and The biofilm cultivation was repeated threeindependent times (n=6 independent samples for each type of model per replicate). Figure 1 shows the experimental design.

Bacterial viability analysis
Samples from static and semi-dynamic models (36 in total) were transferred to new 24-and 6-well microplates and exposed to 1 and 9 ml of MTT dye (0.

Results
The biofilm viability was significantly lower for the static model compared with the semi-dynamic model ( Figure 2). On the other hand, the static model produced dentin lesions with higher values of the integrated mineral loss and lesion depth compared with the semi-dynamic model ( Table 1).
The semi-intact surface layer was often seen in samples from the static model (83%, n=15/18) compared with those from the semi-dynamic model (45%, n=8/18), which means that the static model was able to produce a significant higher number of Considering the limitations of the design and the interpretations of the results, the null hypothesis can be rejected. Both models are able to produce viable cariogenic biofilm and dentin carious lesions; however, semi-dynamic model tends to produce more lesions with loss of surface integrity than the static one, which can be a consequence of the availability of nutrients in each system. The response of both models to antimicrobial agents shall be analyzed in the future, especially concerning the type of microorganisms prevalent in both biofilms and their impact on carious lesions formation in dentin. Conclusion The type of model applied to supply nutrients may have influence on the microcosm biofilm viability and the production of carious lesions in dentin.