Influence of salivary conditioning and sucrose concentration on biofilm-mediated enamel demineralization

Abstract The acquired pellicle formation is the first step in dental biofilm formation. It distinguishes dental biofilms from other biofilm types. Objective To explore the influence of salivary pellicle formation before biofilm formation on enamel demineralization. Methodology Saliva collection was approved by Indiana University IRB. Three donors provided wax–stimulated saliva as the microcosm bacterial inoculum source. Acquired pellicle was formed on bovine enamel samples. Two groups (0.5% and 1% sucrose–supplemented growth media) with three subgroups (surface conditioning using filtered/pasteurized saliva; filtered saliva; and deionized water (DIW)) were included (n=9/subgroup). Biofilm was then allowed to grow for 48 h using Brain Heart Infusion media supplemented with 5 g/l yeast extract, 1 mM CaCl2.2H2O, 5% vitamin K and hemin (v/v), and sucrose. Enamel samples were analyzed for Vickers surface microhardness change (VHNchange), and transverse microradiography measuring lesion depth (L) and mineral loss (∆Z). Data were analyzed using two-way ANOVA. Results The two-way interaction of sucrose concentration × surface conditioning was not significant for VHNchange (p=0.872), ∆Z (p=0.662) or L (p=0.436). Surface conditioning affected VHNchange (p=0.0079), while sucrose concentration impacted ∆Z (p<0.0001) and L (p<0.0001). Surface conditioning with filtered/pasteurized saliva resulted in the lowest VHNchange values for both sucrose concentrations. The differences between filtered/pasteurized subgroups and the two other surface conditionings were significant (filtered saliva p=0.006; DIW p=0.0075). Growing the biofilm in 1% sucrose resulted in lesions with higher ∆Z and L values when compared with 0.5% sucrose. The differences in ∆Z and L between sucrose concentration subgroups was significant, regardless of surface conditioning (both p<0.0001). Conclusion Within the study limitations, surface conditioning using human saliva does not influence biofilm–mediated enamel caries lesion formation as measured by transverse microradiography, while differences were observed using surface microhardness, indicating a complex interaction between pellicle proteins and biofilm–mediated demineralization of the enamel surface.


Introduction
Dental caries is a multifactorial disease, in which acid-producing bacteria, dietary carbohydrates, time, and a susceptible host contribute to the disease initiation and progression. 1 The process starts when oral bacteria, present in an equilibrium state, ferment carbohydrates; this equilibrium shifts to increased populations of acidogenic (acid-producing) and aciduric (acid-tolerant) bacteria. 1 The consistent presence of acid in the environment disrupts the mineral equilibrium of the exposed dental structures (i.e. enamel and/or dentin), and, therefore, leads to carious lesions. 1,2 Dental biofilm has been defined as "matrixenclosed microbial communities in which cells adhere to each other and/or to surfaces or interfaces." 3 Over 700 bacterial species are present in the oral cavity. 4 They are in all oral hard and soft tissue structures.
These bacterial aggregations usually produce and become enclosed in extracellular polymeric substance (EPS). The formation of dental biofilm (or dental plaque) consists of several steps, which start with the formation of the acquired pellicle, followed by the initial adhesion of planktonic bacteria to the pellicle layer by binding sites, subsequent maturation of the bacterial biofilm, and, finally, the dispersion of biofilm with detachment of cells/clusters of cells. 5 The formation of the acquired pellicle is the first step in dental biofilm formation, and it is a unique step distinguishing it from other biofilm types. 5 It consists of several interactions between various salivary glycoproteins, and their interaction with the tooth surface. These biochemical interactions are based on Gibbs law of free enthalpy 5,6 ; they lead to the attachment of salivary glycoproteins to a surface (i.e. the enamel). The resulting formed layer is a protein-rich layer with binding sites; these sites are ready for early colonizers to attach. 6 Based on this unique process, some studies suggested a new intervention to prevent biofilm formation: this intervention is in the form of preventing pellicle formation. 7 Many microbial studies have explored and studied dental biofilm from many aspects using different cariogenic models. [8][9][10][11] However, they omitted the step of surface conditioning by the formation of acquired pellicle. This leads to less clinical relevance, especially for this area of study (the significance of including the pellicle) has not been researched previously.
Acquired enamel pellicle (AEP) has been explored previously for its composition and function. 12-16 Studies have explored pellicles and found differences between AEP formed in vitro, in vivo, and in situ. These studies have reported ultrastructural variations, intrinsic and extrinsic maturation variations, as well as variation in the AEP morphology. Studies have found that in vitro AEP were superior to in vivo, which contain higher amounts of proteins. They are also superior in the overall amounts produced (due to the difficulty in collecting in vivo AEP). [12][13][14][15][16] In in vitro studies, the salivary pellicle can typically form before exposure to bacteria-containing media, resulting in biofilm formation. Several methods have been used to form a salivary pellicle. [17][18][19] In general, the dental surface is exposed to saliva (sterilized, free from bacteria) for a specific amount of time (ranges from minutes to several hours) before being exposed to oral bacteria for biofilm formation. [17][18][19] The significance of surface conditioning before biofilm growth (to allow the formation of acquired enamel pellicle) in studying biofilm models was not evaluated previously and, therefore, needs to be explored. Hence, this study aims to explore the influence of salivary conditioning before biofilm formation on enamel demineralization.
The hypothesis was: 1) a significant difference between filtered/pasteurized saliva, filtered saliva, and deionized water (DIW; negative control) as conditioning agents on biofilm-mediated enamel demineralization; and 2) a significant difference between 0.5% and 1% sucrose-supplemented growth media on enamel demineralization.

Methodology Specimen preparation
Extracted bovine incisors were sectioned to obtain 5×5 mm enamel specimens using a Buehler Isomet TM low-speed saw (Buehler, Ltd., Lake Bluff, IL, USA).
Approximately 54 teeth were used to obtain 54 specimens. During preparation, the teeth were stored in deionized water with thymol. Using a Struers Rotopol 31/RotoForce 4 polishing unit (Struers Inc., Cleveland, PA, USA), all specimens were ground and polished to ensure flat parallel dentin/enamel surfaces. For the finishing process, the dentin side was ground using 500-grit silicon carbide grinding paper. Then, the Influence of salivary conditioning and sucrose concentration on biofilm-mediated enamel demineralization J Appl Oral Sci. 2020;28:e20190501 3/8 enamel side was serially ground using 1,200,2,400 and 4,000 grit papers. After that, specimens were polished using a 1-µm diamond polishing suspension on a polishing cloth to obtain a 5×5 mm polished enamel surface. All specimens were examined for cracks, white spots, or any other flaws that could exclude the specimen from the study, using Nikon SMZ 1500 stereomicroscope at ×20 magnification.
Baseline measurement and experimental groups

Biofilm model
After completing specimen preparation, specimens were mounted on the inside of a lid of a 6-well plate (FisherBrand, Fisher Scientific), with three specimens per well, using acrylic cubes to create an active attachment model and following a previously described protocol. 1,20 The model was disinfected using 70% ethanol prior to bacterial and/or pellicle inoculation. 21

Saliva collection
Ethical approval was obtained from the Indiana

Saliva pasteurization
The collected, pooled saliva was diluted in sterile saline at 1:10 dilution. The diluted solution was filtered using Whatman filter paper to remove large debris. This filtered saliva was used to create the salivary pellicle in subgroups exposed to filtered saliva.
For pasteurization, an additional sterilization step, pasteurization, was performed with the remaining filtered saliva, using a previously published protocol. 22 Briefly, after the diluted solution first filtration, it was centrifuged to remove mucin and bacteria (10 minutes, 4ºC, 27,000× g). The supernatant was retained and pasteurized at 60ºC for 30 minutes, then recentrifuged for 10 minutes. The prepared saliva was stored in aliquots of 50 mL and frozen at −80°C for further use.

Surface conditioning
All specimens were immersed in their corresponding solutions: filtered/pasteurized saliva, filtered saliva, or DIW as negative control. Specimens were incubated in their respective solution at 5% CO 2 and 37ºC for 5 minutes to allow surface conditioning.

Biofilm growth
Immediately after surface conditioning, specimens were transferred to a new, sterile 6-well plate containing growth culture media that was inoculated with the overnight bacterial culture (without washing the samples between the two steps). Microcosm biofilm was grown under anaerobic conditions at 37°C for 48 h. The growth media used to grow the biofilm was Brain Heart Infusion (BHI) broth, supplemented with 5 g/L yeast extract, 5% vitamin K and hemin (v/v) and supplemented with either 0.5% sucrose or 1% sucrose. After 48 h, the biofilm was collected by placing each specimen in an Eppendorf tube (containing 1 mL sterile saline), sonicated at 30 W for 10 seconds, and vortexed immediately for 10 seconds to completely Sound enamel was assumed to be 87% v/v mineral.
The data obtained from this analysis were integrated mineral loss (∆Z; %vol.μm) and lesion depth (L; μm).

Statistical analysis
All three variables (VHN change , ∆Z, L) were analyzed using two-way ANOVA, with factors for sucrose concentration and surface conditioning as well as the interaction between them. All pair-wise comparisons from ANOVA analysis were made using Fisher's Protected Least Significant Differences to control the overall significance level at 5%. Statistical analysis was performed using SAS version 9.4 (SAS Institute, Inc., Cary, NC).
Surface conditioning affected VHN change significantly (p=0.0079); however, it did not affect ∆Z (p=0.7383) or L (p=0.7323). Sucrose concentration impacted ∆Z (p<0.0001) and L (p<0.0001); however, it did not affect VHN change (p=0.2877). Table 1  Upper case letters indicate statistically significant differences between surface conditioning methods within sucrose concentrations Lower case letters indicate statistically significant differences between sucrose concentrations within surface conditioning methods

Discussion
This study aimed to evaluate the influence of surface conditioning using human saliva before biofilm formation in vitro on enamel demineralization. The statistical analysis results showed the hardness data were only affected by pellicle type, whereas the TMR data were only affected by sucrose concentration. To fully understand this contradiction, one should consider the differences between the variables studied.
Surface microhardness is a measurement of how a material responds to deformation. It is mainly influenced by surface integrity and rather than by structural characteristics or mineral content of the bulk substrate. One of the pellicle functions in the oral cavity is its masking effect: it coats dental surfaces and other structures, which may lead to different patterns of bacterial biofilm formation according to the presence/absence or the quality of the pellicle. [23][24][25] The presence or absence of a pellicle layer, therefore, will affect surface characteristics, and this may explain the significant differences between pellicle subgroups in our study. On the other hand, TMR measures are based on mineral content rather than structure. Therefore, the expectation is to observe differences only when carious lesions with different mineral contents and/or distributions form during demineralization. 26 Surface microhardness testing is straightforward and nondestructive. In some studies, it is coupled with transverse microradiography based on their objective.
The minerals loss within the outer enamel was found to be proportional with the degree of the indenter penetration. However, deeper lesions cannot be quantitatively measured using surface microhardness. 27 Moreover, surface microhardness is most effective in analyzing homogenous materials and shallow lesions only (e.g. enamel outer surface). 28 White 28 (1987) reported, in a study in which they evaluated the differences between surface microhardness and microradiography, that surface microhardness could detect remineralization in early lesions (or at least hardening of the surface without remineralization). 28 Evaluating mineral content within the outermost layers of the enamel using microradiography is difficult. Therefore, the two analyses are usually considered complementary to each other in demineralization/ remineralization studies. 28 In this study, an active attachment model adopted from a previously published model was used. 1,20 Despite still lacking more complex features that lead to more clinical relevance (e.g. pulsation of nutrients into the environment), 29 an active attachment has the advantage of ensuring that the bacterial layers Although including salivary pellicle in the model seems to be more clinically relevant, this step requires an  39 Consequently, the biofilm cariogenicity may also be affected. As mentioned before, acquired pellicle formation is an integral step that precedes bacterial attachment to dental and oral surfaces. The formation of acquired pellicle generally consists of two stages. 40 The first stage is very rapid and includes adsorption of salivary glycoproteins to the substrate.
However, the second stage occurs immediately after the first stage in vivo. 40 It is characterized by more adsorption of biomolecules, being the oral fluids the source of these biomolecules. 40 Therefore, two different conditions of sources for the salivary pellicle (filtered/ pasteurized and filtered saliva) were included in this study to represent these two stages and explore their influence in the pattern of demineralization. Although salivary pellicle formed from filtered/pasteurized saliva (which becomes free of viable bacteria) makes the in vitro study more controllable and the model more applicable if used in studies involving single/multiple species biofilm, using filtered saliva ensures more clinical relevance as the only eliminated element is food debris.
This study focused mainly on pellicle involvement in in vitro microbial studies, and on the influence of this factor on the hard tissue substrate characteristics. It did not test the influence of the presence of acquired pellicle on the cariogenicity of a microcosm biofilm.
This can be tested in a similar study by collecting 48-hour biofilm and analyzing cariogenicity (e.g., lactic acid production). The bacterial source (i.e., saliva vs. plaque samples) may also be tested, since it was already reported that biofilms formed from saliva vs.
plaque have different characteristics. 21 Furthermore, different incubation times may affect pellicle formation and maturation. Lastly, pellicle formation can also be achieved by exposing specimens to the oral cavity for different periods of time, which provides material for future research. Lastly, all the variables tested may be evaluated in a prolonged study (more than 48 h) to observe the lesion characteristics, especially TMR data.

Conclusion
Considering the limitations of this study, the presence or absence of an artificially induced acquired pellicle layer does not influence biofilm-mediated enamel caries lesion formation as measured by TMR. Some differences were observed using surface microhardness, indicating a complex interaction between pellicle proteins and biofilm-mediated demineralization of the enamel surface.