Effect of anaerobic or/and microaerophilic atmosphere on microcosm biofilm formation and tooth demineralization

Abstract Microcosm biofilms can reproduce the complexity of a dental biofilm. However, different forms of cultivation have been used. The impact of the culture atmosphere on the development of microcosm biofilms and their potential to cause tooth demineralization has not yet been deeply studied. Objective This study analyzes the effects of three experimental cultivation models (microaerophile vs. anaerobiosis vs. experimental mixed) on the colony-forming units (CFU) of the cariogenic microorganisms and tooth demineralization. Methodology 90 bovine enamel and 90 dentin specimens were distributed into different atmospheres: 1) microaerophilia (5 days, 5% CO2); 2) anaerobiosis (5 days, jar); 3) mixed (2 days microaerophilia and 3 days anaerobiosis), which were treated with 0.12% chlorhexidine (positive control – CHX) or Phosphate-Buffered Saline (negative control – PBS) (n=15). Human saliva and McBain’s saliva containing 0.2% sucrose were used for microcosm biofilm formation, for 5 days. From the second day to the end of the experiment, the specimens were treated with CHX or PBS (1x1 min/day). Colony-forming units (CFU) were counted, and tooth demineralization was analyzed using transverse microradiography (TMR). Data were subjected to two-way ANOVA and Tukey’s or Sidak’s test (p<0.05). Results CHX was able to reduce total microorganism’s CFU compared to PBS (differences of 0.3–1.48 log10 CFU/mL), except for anaerobiosis and microaerophilia in enamel and dentin biofilm, respectively. In the case of dentin, no effect of CHX on Lactobacillus spp. was observed. CHX significantly reduced enamel demineralization compared to PBS (78% and 22% reductions for enamel and dentin, respectively). Enamel mineral loss did not differ when compared with the other atmospheres; however, the enamel lesion depth was greater under anaerobiosis. Dentin mineral loss was lower under anaerobiosis when compared with the other atmospheres. Conclusion The type of atmosphere has, in general, little influence on the cariogenic ability of the microcosm biofilm.


Introduction
Dental caries is a multifactorial disease of great clinical relevance, which occurs due to the presence of a biofilm in dysbiosis, induced by the frequent ingestion of sugar, which is rich in acidogenic, aciduric, and extracellular polysaccharide-producing microorganisms. These microorganisms can metabolize different types of sugars from the diet, but mainly sucrose, producing acids that change the biofilm pH and cause tooth demineralization. [1][2][3] The most common microorganisms involved in the development of dental caries are Streptococcus mutans and lactobacilli. 4,5 Due to the high degree of controlled conditions and reproducibility, in vitro models have been well accepted to mimic cariogenic biofilms. 6 In vitro models can be produced from monospecies (namely, S. mutans) or multispecies (namely, S. mutans + Lactobacillus casei) biofilms by using microbial strains or from microorganisms derived from human saliva or dental biofilms, called microcosm biofilm. 7,8 Microcosm biofilms are able to reproduce the complexity of a dental biofilm. [9][10][11] However, different forms of cultivation have been used comprising 24-well microtiter plates, where specimens can be attached to the bottom of the wells; 12,13 the application of meshes for the retention of microorganisms; 14 or the suspension of specimens to prevent the formation of biofilm only by microorganism precipitation. 15 Some microcosm biofilm models use artificial mouths for programmed or continuous nutrient flow. 6,16 Studies also differ with respect to the sucrose concentrations and form of exposure, which can be either continuous or intermittent. 17,18 Cultivation time can vary from 2-14 days depending on the selected response variables. 13,19 Incubation atmosphere is another important factor, 13 which can vary between microaerophilia (5%-10% CO 2 ); 12,14,16,19 and anaerobiosis with the use of jars, candles, or anaerobic cabins (O 2 <0.1% and 5%-10% CO 2 ; 10% CO 2 , 10% H 2 , and 80% N 2 ). 17,20 Oral dental biofilms are often subjected to fluxes of environmental conditions, such as fast pH challenges, nutrient abundance or scarcity, different carbohydrates exposures, and variations in redox potentials due to atmospheric conditions. These environmental parameters can define the biofilms microbiome 21,22 and, consequently, the cariogenic potential of biofilm. 10,23 Despite this wide methodological variation between studies, the impact of the culture atmosphere on the development of microcosm biofilms and their potential to cause tooth demineralization has not yet been deeply studied. Therefore, this study aims to compare the three experimental cultivation models two-thirds of the surface was covered with red nail polish (Estreia-Colorama, Rio de Janeiro, RJ, Brazil) to create two sound areas. All lateral areas were also covered with red nail polish, which is needed for the transverse microradiography (TMR) analysis.
Subsequently, the specimens were sterilized by exposure to ethylene oxide and randomly distributed into three groups and two subgroups (n=15) according to their mean Ra values (Enamel Ra: 0.140 ± 0.029 µm and dentin Ra: 0.288 ± 0.056 µm). Specimens with Ra values below 0.100 µm or above 0.300 µm for enamel and below 0.200 µm and above 0.400 µm for dentin were excluded from the study. This was done to provide similar mean baseline Ra values among the groups.

Study conditions and sample size calculation
The experimental models differed regarding the atmosphere, namely, microaerophilia (greenhouse with 5% Carbonic Dioxide -CO 2 ), anaerobiosis

Preparation of artificial saliva and microcosm biofilm formation
McBain artificial saliva was prepared according to study by Braga et al. 11 (2021). In a 24-wells microtiter plate, each enamel or dentin specimen was exposed to 1.5 mL of inoculum (human saliva-glycerol + McBain saliva, 1:50) for 8 h. After the first 8 h, the inoculum was removed, the specimens washed with PBS (5 s) and then exposed to 1. The plates were then incubated under the same conditions as the microcosm biofilm. The plates from mixed atmosphere group were incubated separately using two atmospheres, half under anaerobiosis conditions and the other half of plates

PBS (negative control) --
The groups were divided in subgroups (n = 15). under microaerophilic conditions. After 48 h, CFU was computed and transformed to log 10 CFU/mL.
Ethylene glycol (Sigma-Aldrich, Steinheim, Germany) was applied on the dentin specimens for 24 h to avoid shrinkage. 29 A microradiograph was obtained using an X-ray generator (Softex, Tokyo, Japan) on a glass plate at 20 kV and 20 mA (at a distance of 42 cm) for Distribution and homogeneity were tested using the Kolmogorov-Smirnov and Bartlett's tests, respectively. A two-way ANOVA (factors: atmospheres and treatments), was applied, followed by Tukey's or Sidak's test. The level of significance was set at 5%.

CFU counting
Enamel  CFU), which did not differ from the anaerobic and mixed-anaerobic CFU.
Regarding mutans streptococci, lower microbial growth was observed in the microaerophilic atmosphere (p=0.0041), which was similar to the mixed atmosphere (microaerophilic CFU). However, the microaerophilic atmosphere significantly differed from the anaerobic atmosphere when considering PBS and from the mixed atmosphere (anaerobic CFU) when considering CHX. Capital letters show significant difference between treatments, for each type of atmosphere (Example: comparation between rows 3 and 4). Lowercase letters show significant difference between atmospheres, for each type of treatment (Example: comparation of between CHX rows 2, 4, 6 and 8). For total microorganisms, two-way ANOVA was applied, followed by Tukey's test (treatment p<0.0001, atmosphere p<0.1222 and interaction p=0.0099). For Lactobacillus spp., two-way ANOVA was applied, followed by the Tukey's test (treatment p=0.0602, atmosphere p=0.0002, no interaction, p=0.5955). For mutans streptococci, two-way ANOVA was applied, followed by the Tukey's test (treatment p<0.0001, atmosphere p=0.4706 and interaction p=0.4637).   Table 5-Mean ± SD of the integrated mineral loss (ΔZ, vol%. μm), lesion depth (LD, μm) and average mineral loss (R, vol%) of the dentin specimens all the tested atmospheres (p<0.0001). Similar carious lesions were produced in enamel in all atmospheres (p=0.7627), except for microaerophilia, which induced a shallow lesion compared to the anaerobic atmosphere, both similar to the mixed atmosphere (Table 4 and Figure 1).

Dentin
Chlorhexidine was also able to reduce dentin demineralization in all tested atmospheres (p < 0.0001), except the average mineral loss in the case of microaerophilia and anaerobiosis.
Microaerophilic and mixed atmospheres produced similar carious lesions in dentin with greater mineral loss (integrated and mean) than those produced under anaerobiosis (lesion depth p = 0.0809; average mineral loss p = 0.0093) ( Table 5 and Figure 2).

Discussion
Although the microcosm biofilm model is a wellestablished practice, there are several differences between the protocols, as for example the growth atmosphere. Accordingly, the atmosphere that better represents oral conditions needs to be investigated.
This study did not aim to validate a type of atmosphere based on the oral environment since no comparison was made between the tested protocol and in vivo conditions. This study compared three experimental cultivation models (microaerophile vs. anaerobiosis vs. experimental mixed) on the colony-forming units (CFU) of the cariogenic microorganisms and tooth demineralization. Some minor differences were observed among the atmospheric conditions, but they were able to produce very similar carious lesions in both enamel and dentin. Furthermore, most atmospheres were able to differentiate CHX vs. PBS, which is an essential outcome since the antimicrobial effect of CHX has been well established. 30 Based on the findings of the CFU counting, a lower number of Lactobacillus spp. and mutans streptococci was observed under the microaerophilic than in the anaerobic environment, especially for enamel. The differences of 0.2 log 10 CFU/mL might be clinically irrelevant; in fact, they were not significant when TMR data were taken into account.
Both bacteria were chosen because they are highly established cariogenic species. 33 A limitation of the study is that other species, which might be present in the microcosm biofilm, were not examined. Therefore, future studies using "omics" are desirable, especially to understand the reason that CHX did not reduce Lactobacillus spp. in microcosm biofilms produced on dentin. Interestingly, the number of total microorganisms was reduced under anaerobic conditions for CHX. This led to speculations regarding the contribution of other species. Herein, it is important to consider that the term total microorganism does not include species that require supplementation for their growth on BHI agar.
The association between lactobacilli and dental caries dates back to one century ago. 34 In another study, 0.2% Chlorhexidine, used in an in situ model, was not able to reduce the CFU counting for lactobacilli on dentin specimens when compared with the control. 35 The author suggested that dentin can act as a "shelter" for this bacterium against the action of CHX. 35 The outside atmosphere had little impact on the microbiological analysis of the microcosm biofilms because the biofilm itself can create its own atmosphere, with the deeper layers rich in strictly anaerobic microorganisms and the superficial layers rich in facultative microorganisms. 36 Although dental biofilms are composed primarily of obligate anaerobe species with preferential growth in the presence of Carbon Dioxide (CO 2 ), these microorganisms may be protected from the toxic effects of oxygen, enabling their growth under microaerophilic conditions. 10 Notably, the findings of this study cannot be extrapolated to monospecies or other multispecies models. Regarding enamel demineralization, the mineral loss (integrated and mean) was similar among different atmospheres; however, the anaerobic condition induced a deeper lesion, showing that, in this case, the acids could have penetrated deeper into the environment, which may be related to the metabolic profile of the biofilm. While dentin lesion depth was similar among the atmospheres, dentin mineral loss (integrated and mean) was lower for anaerobic than for microaerophilic and mixed atmospheres, which behaved similarly. Therefore, as previously discussed, the metabolic products (primarily acids) induced by the anaerobic biofilm may have been neutralized by the organic content of the dentin. 3 The mixed biofilm model was first performed by Braga, et al. 11 (2021) but in a different sequence: the first three days in an anaerobic and the last two days in a microaerophilic atmosphere. In their study, the biofilm model was able to differentiate CHX from PBS.
The results of this study warrant further metabolome analysis to better explain the slight differences found in CFU counting and TMR analysis and to signalize that the mixed model can be applied in the future.
For both tissues, especially the enamel, lesions induced in the absence of treatment (PBS group) were highly demineralized, and most of them were cavitated (about 85-100% for enamel and 31-55% for dentin), with no differences among the atmospheres.
It is suggested that the higher cavitation of enamel is due to its lower organic content compared to dentin, which plays an important role in the modulation of de-remineralization progression. Also, the percentage of prevention fraction of CHX was much higher for enamel (~78%) than for dentin (~22%), which may be related to the high degree of de-remineralization of the enamel and the type of interaction of CHX with the biofilm, in agreement with previous reports. 16,18 Our results provide new information about the effect of the atmosphere on microcosm biofilm growth and its potential to induce tooth demineralization.
These different protocols should be analyzed in the future to identify their impact on the results and interpretation of metabolome. Moreover, the biofilm should be analyzed by laser scanning microscopy or scanning electron microscopy to map the 3D architecture of the biofilm and thus identify if the specific organization of microbial communities could be created by different atmospheric conditions.

Conclusion
In conclusion, this study identified some minor differences among the atmospheric conditions; however, in general, microcosm biofilms produced under microaerophilia, anaerobiosis, or mixed models produced very similar artificial carious lesions in both enamel and dentin. Considering a general overview, mixed model may be interesting since it does not differ from the classical ones; however, researchers must be prepared to combine both methods in the Laboratory.
Nevertheless, any of them could be applied since all were able to produce dental caries and to differentiate CHX and PBS.

Conflicts of interest
There are no conflicts of interest.

Data availability statement
All data generated or analyzed during this study are included in this article. Further enquiries can be directed to the corresponding author.