Influence of biofilm formation on the mechanical properties of enamel after treatment with CPP-ACP crème

The study aimed to investigate the effects of bacterial biofilms on changes in the surface microhardness of enamel treated with casein phosphopeptide–amorphous calcium phosphate (CPP-ACP) with and without fluoride. Human enamel blocks with incipient caries-like lesions were divided into four groups of 13: G1: Saliva (Control); G2: fluoride dentifrice (CrestTM, 1100 ppm as NaF); G3: CPP-ACP (MI Paste; RecaldentTM); and G4: CPP-ACPF (MI Paste Plus; RecaldentTM 900 ppm as NaF). The specimens were soaked in demineralizing solution for 6 h and remineralized in artificial saliva for 18 h alternately for 10 days. The dentifrice was prepared with deionized water in a 1 : 3 ratio (w/w) or applied undiluted in the case of the CPP-ACP group. The surface microhardness (SMH) was evaluated at baseline, after artificial caries, after pH cycling and treatment with dentifrices, and after incubation in media with Streptococcus mutans for biofilm formation. The biofilms were exposed once a day to 2% sucrose and the biofilm viability was measured by MTT reduction. The percentage of change in surface microhardness (%SMHC) was calculated for each block. The data were analyzed by nonparametric test comparisons (α = 0.05). The %SMHC values observed in G2 were different from those of G1, G3, and G4 (p < 0.05). After biofilm formation, %SMHC was positive in G2 and G4 when compared to G1 and G3, but resistance to demineralization after biofilm formation was similar in all groups. In conclusion, the presence of biofilms did not influence the treatment outcomes of anticaries products.


Introduction
Dental caries is initiated by acid-producing bacteria on dental biofilms, which cause carious lesions in the presence of fermentable carbohydrates.Dental biofilms play a crucial role in cariogenesis.They do not only function as acid producers during caries formation, but they may also serve as reservoirs and diffusion barriers for caries-preventive components. 1 Several novel remineralization agents, including casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) nanocomplexes, have shown great anticaries potential. 2,3,4,5CPP-calcium phosphate complexes are anticariogenic and capable of remineralizing the early stages of enamel lesions in in situ and in vivo studies. 3,4,6The beneficial effect obtained with CPP-ACP is associated Declaration of Interests: The authors certify that they have no commercial or associative interest that represents a conflict of interest in connection with the manuscript.
with the ability to localize calcium and phosphate on dental biofilm close to the tooth, thus making it available when needed.In the presence of an acidic environment, such as after eating, when the pH of the mouth decreases, casein phosphopeptide proteins release amorphous calcium and phosphate, creating a supersaturated state of calcium and phosphate around the tooth.During a cariogenic challenge, fluoride, calcium, and phosphate present on the biofilm may diffuse into the enamel, promoting remineralization. 6In addition, significantly high levels of calcium and phosphate have been found in both biofilm and subsurface incipient carious lesions and in lower level demineralization of enamel or dentin surfaces previously treated with CPP-ACP-based compounds. 7In terms of its mechanism of action, it has been suggested that the presence of the CPP-ACP agent delays biofilm formation and favors nucleation and crystallization of calcium phosphates, possibly in the form of apatite, in mature biofilms. 8emineralized enamel treated with fluoride and CPP-ACP can be more resistant to a new cariogenic attack.Thus, Ekassas and Arafa 9 and Piagnatelli et al. 10 determined the stability of remineralized surfaces treated with CPP-ACP against acid attack and, therefore, a second sequence of demineralization was used to assess the stability of the newly formed precipitate.Furthermore, following the exposure to acid challenge, partial dissolution of the coating layer was evident with the existence of a demineralized surface, but CPP-ACPF, fTCP, and ACPF presented significantly higher resistance to softening than did the control. 9In another study, Piagnatelli et al. 10 showed that fluoride dentifrice offers fast remineralization when compared to CPP-ACP, but the precipitates formed following its application dissolved 2.7 times faster than those formed by CPP-ACP.
pH-cycling models are chemical models that are not able to estimate the antimicrobial effect of fluoride or other substances on caries. 1,11,12Thus, biofilm models have been used to evaluate the in vitro effect of toothpastes containing antimicrobial compounds such as fluoride on Streptococcus mutans biofilm formation. 12herefore, the aim of this in vitro study was to evaluate the effect of biofilm formation on surfaces treated with CPP-ACP and CPP-ACPF on enamel demineralization when compared to controls (fluoride dentifrice and no treatment).The null hypothesis was that the treatment of enamel surface with CPP-ACP and CPP-ACPF topical crème would not significantly reduce enamel surface microhardness following biofilm formation when compared to controls.

Sample preparation
In this study, 35 human third molar teeth, which had been extracted for surgical reasons, were used.This study was approved by the Medical Sciences Research Ethics Committee, Fluminense Federal University (process no.580.849).The samples presented caries-free, fluorotic, or hypomineralized lesions and any other visible defects.The teeth were stored in 0.1% thymol during sample preparation.Four enamel blocks were obtained from each tooth.After embedding the blocks in acrylic resin, the buccal and lingual surfaces of the enamel specimens (2 mm × 2 mm × 2 mm) were ground with SiC paper (400, 600 and 1,200 grit) (Struers S/A, Ballerup, Denmark) in order to obtain flat surfaces.The specimens were then polished using a 1-μm diamond polishing suspension with a polishing cloth (Arotec Ind & Com, Cotia, SP, Brazil).The surface hardness was measured using a 2001 MicroMet microhardness tester (Buehler, Lake Bluff, USA) with a Knoop indenter, and with a static load of 25 g for 15 s.The Knoop hardness number (KHN) was calculated from the length of the indentation and the applied load.An increase in length in µm indicates softening of the enamel due to demineralization.Five indentations separated by a 100-μm distance were made in the central region of each block (SMH baseline ).The average of the five indentations made on each specimen was used as the SMH baseline value (SMH baseline ).After SMH baseline measurements, 52 enamel blocks were selected, with a KHN ranging from 289.7 to 399.7. 13he KHN was used to monitor enamel surfaces in three phases: Phase 1: artificial caries; Phase 2: pH cycling and treatment with dentifrices; and Phase 3: demineralization with S. mutans biofilm formation on the newly treated surface (Figure 1).

Phase 1 -artificial caries
Each enamel block was immersed in 10 mL of a demineralizing solution for 72 h (2 mM Ca(Ca(NO 3 )) 2 , 2 mM PO 4 (KH 2 PO 4 ), and 75 mM of acetate at 4.3 pH) 13 and washed at the end in distilled water for 1 min.This demineralizing solution was used for both the pH-cycling model (demineralization) and caries-like lesion formation on enamel blocks. 13The exposed area was 2 mm long (y axis) and 2 mm wide (x axis).After induction of artificial caries, five other indentations were made at 100 μm from the SMH baseline .As described previously, the averages obtained from each sample were used as the enamel treatment value, SMH phase1 .

Phase 2 -pH cycling and treatment with dentifrices
The enamel blocks were distributed into four groups of 13 blocks each: Group 1: Control; Saliva; Group 2: FD -Crest ™ Cavity Protection (1,100 ppm F as NaF, Procter & Gamble), used as a positive control; Group 3: CPP-ACP (MI Paste; Recaldent ™ GC Corporation Tokyo, Japan); and Group 4: CPP-ACPF (MI Paste Plus; Recaldent ™ 900 ppm as NaF, GC Corporation Tokyo, Japan).The formulations of each product are described in Table 1.The enamel blocks were subjected to four pH cycles/day for 10 days, at 37°C (Figure 1).After sonication and rinsing with distilled water, the specimens were immersed separately in 10 mL of demineralizing solution (8-10 h, 12-14 h, 16-18 h), and in the remaining hours (18 h/day) they were transferred to an artificial saliva solution (10 mL).Standard pH-cycling conditions were used in a daily schedule of three cycles (2 h of demineralization and 2 h of remineralization each, followed by a period in artificial saliva (6 h in demineralizing solution and 18 h in saliva).The artificial saliva was replaced daily and consisted of 0.67 g/L NaCl; 0.1168 g/L CaCl 2 ; 8 g/L CMC; 0.0408 g/L MgCl 2 ; 0.96 g/L KCl; 1 g/L C 8 H 8 O 3 ; 24 g/L C 6 H 14 O 6 ; 964.938 mL/L H 2 O and 0.274 g/L KH 2 PO 4 .
In each transfer between the different solutions, the enamel specimens were rinsed in distilled water for 1 min at 37 °C.The fluoride dentifrice (G2) was applied in a slurry at a dentifrice to deionized water ratio of 1:3 for 60 s.A standardized volume (0.15 mL) was applied to each sample using a syringe.The treatment was carried out before the first, second, and third demineralization cycles.Specimens in the CPP-ACP and CPP-CPF (G3 and G4) groups received a topical application (0.03g) on their surface for the same amount of time. 13The negative control (Group 1) was only subjected to the cariogenic challenge.After the last demineralization challenge, the enamel specimens were rinsed in distilled water for 1 min and then immersed in 10 mL of artificial saliva.The solutions were changed every day.As described previously, five indentations were made and the averages  obtained from each sample were used as the enamel treatment value, SMH phase2 .The indentations were made at a distance of 100 μm from the SMH phase1 .

Phase 3 -Streptococcus mutans biofilm formation
In this phase, the samples subjected to Phases 1 and 2 were placed in 24-well plates (TPP, 24 Zellkultur Testplatte F) and sterilized by ethylene oxide.The mature biofilm was then formed by S. mutans, which is a cariogenic bacterium and the primary causative agent of caries.
Briefly, S. mutans ATCC 25175 (American Type Culture Collection, Fiocruz, Rio de Janeiro, RJ, Brazil) was cultured overnight in brain heart infusion (BHI, Difco, Sparks, MD, USA) broth at 37°C under anaerobic conditions.The bacterial inoculum was adjusted to an optical density (OD) of 0.5 at 550 nm in accordance with the McFarland standard.The bacterial suspension was diluted 1 : 100 and then 10 µL was inoculated into each well with BHI supplemented with 2% sucrose. 14he 24-well plates were then incubated at 37°C under anaerobic conditions for 48 h.During the 2 days of biofilm formation, the growth medium was changed every 24 h.The biofilm formed on the specimens was used in the subsequent experiments.

Metabolic activity of Streptococcus mutans biofilms
At the end of biofilm formation, the metabolic activity of S. mutans biofilms formed on the enamel specimens was analyzed by thiazolyl blue tetrazolium bromide (MTT, Sigma Aldrich, St. Louis, MO, USA) reduction assay. 14,15MTT is a colorimetric assay that measures the enzymatic reduction of MTT, a yellow tetrazole, to purple formazan.The specimens were transferred to microtubes with 1 mL of sterile saline solution (NaCl 0.85%) and the biofilms were removed by vortex mixing for three 1-minute periods.After that, the enamel specimens were removed for future analysis and the S. mutans suspensions were centrifuged for cell separation.Then, 100 µL of sterile MTT (1 mg/mL in PBS) was added to each microtube with biofilm cells and incubated at 37°C under anaerobic conditions for 1 h.Then, 100 µL of dimethyl sulfoxide (DMSO) was added to each microtube, which were incubated for 20 min at room temperature in the dark and with gentle agitation.The DMSO solutions were placed in a microplate reader at 540 nm.A higher absorbance is related to a higher formazan concentration, which indicates higher metabolic activity of the biofilm. 14The results were calculated as OD units.
After that, five indentations were made and the averages obtained from each sample were used as the enamel treatment value, SMH phase3 .The indentations were made at a distance of 100 μm from the SMH phase2 .

Determination of changes in surface microhardness (%SMHC)
Enamel SMH was measured at baseline in sound untreated enamel after artificial caries induction (Phase 1), after pH cycling and remineralization (Phase 2), and after biofilm formation (Phase 3).The percentage change of SMH (%SMHC) was calculated by

Statistical analysis
The data were analyzed using the SPSS statistical software package for Windows, version 20.0 (IBM Corporation, New York, NY, USA).Initially, all data (SMH baseline , SMH phase1 , SMH phase1 , SMH phase3 , and %SMHC) were checked by the Shapiro-Wilk test and Levene's test.Based on these preliminary analyses, the SMH phase1 , SMH phase2 and SMH phase3 data were subjected to one-way analysis of variance and Tukey's HSD post-hoc test and the %SMHC data were analyzed by the Kruskal Wallis and Mann-Whitney tests.%SMHC before and after treatment in the same group was analyzed using Student's t-test.All analyses were performed at a significance level of α = 0.05.

Results
The mean and standard deviation for the SMH (KHN) for the study groups and phases are presented in Table 2.The pH-cycling model utilized in this study demonstrated the ability to demineralize the tooth surface.The specimens demonstrated a significant decrease in microhardness after treatment (Student's t-test p < 0.05).One-way ANOVA showed a statistically significant difference in the mean enamel SMH between the groups in phases 2 and 3 (p < 0.05), with a higher mean SMH value in the group that was treated with 1100 ppm F dentifrice (p < 0.05).

Discussion
The purpose of this study was to evaluate the remineralization efficacy of CPP-ACP with and without fluoride in artificial caries-like enamel lesions.
To determine the stability of the treated surface against acid attack, a second demineralization sequence was performed to assess the stability of the newly formed precipitate with S. mutans biofilm formation.
This study was carried out in three phases.In the second phase, the demineralized enamel blocks were underwent pH cycling, and in the third phase, S. mutans biofilm formation was used to evaluate the metabolic activity of the biofilm formed on enamel treated with fluoride dentifrice, CPP-ACP, and CPP-ACPF crème during pH cycling.Changes in the enamel were evaluated by assessing SMH. 9,11,13,16,17Microhardness indentation measurements can provide indirect evidence of mineral loss or gain.The drawback with the technique (SMH) used in the study is that it cannot quantify the amount of mineral loss or gain; however, it is very sensitive to changes in mineral density. 18The standardization of enamel KHN in artificial caries allowed establishing the %SMHC among the groups after treatment (Souza et al., 17 Oliveira et al., 13 Fernández et al. 11 ).As an indicator of enamel mineral loss or gain, surface hardness has been widely employed for shallow lesions. 9,11,13,16,17,18,19he %SMHC (phase 2) values obtained after using FD (G2) differed from those observed in the saliva (G1), CPP-ACP (G3), and CPP-ACPF (G4) groups (p < 0.05).The results of our study are consistent with prior studies conducted by Pulido et al., 20 Kumar et al., 21 and Oliveira et al. 13 where CPP-ACP induced significantly less remineralization compared to FD. Pulido et al. 20 suggested that a longer CPP-ACP application time may be necessary to find remineralization when CPP-ACP is used.In fact, Souza et al. 17 observed that increasing the frequency of CPP-ACP application inhibits more demineralization at cyclic pH.In their study, CPP-ACP crème was applied five times a day in the form of a slurry made from 1 g of CPP-ACP in 3 mL of distilled deionized water.The results found may be due to methodological differences.However, regarding CPP-ACPF, previous studies showed that CPP-ACPF has better remineralization effects than saliva 9,13 or distilled water. 22MH and %SMHC did not differ between CPP-ACP and CPP-ACPF, being in agreement with the results of Oliveira et al. 13 and Hamba et al. 22 These results may be due to the presence of the fluoride ion in CPP-ACPF, which could interact with the ACP component of the casein complex, rendering both inorganic components ineffective, or due to saturation of the medium.17,19 In addition, the efficacy of CPP-ACP may be influenced by saturation of saliva, 13,23 time of application, 17,22,24 or by biofilm formation.3 However, in the presence of CPP, which prevents rapid transformation of the calcium phosphate phases, the ions would be stabilized and maintained such that molecules would spontaneously move from a region of higher concentration to one of lower concentration in the subsurface lesion.4 In fact, microradiography of the remineralized lesions demonstrated that the fluoride dentifrice (1100 ppm F) remineralized predominantly in the surface layer, whereas the 2% CPP-ACP dentifrice produced more homogenous remineralization throughout the body of the lesion.4 Few data are available in the literature on the resistance of surfaces treated with CPP-ACP with and without fluoride.4,9,10,22 The resistance of the precipitate formed after the application of remineralizing agents has been obtained in in vitro studies with demineralizing solutions.4,9,10,22 In this study, the S. mutans biofilm was used with this purpose.The model has been validated recently, enabling the assessment of anticaries effects of fluoride on demineralization of dental substrates and also analyzing the effect of treatments on the biofilm. 11S. mutans biofilm formation after 48 h of incubation in the presence of sucrose was assessed using the MTT assay, which is an indirect biochemical assay Table 3. Metabolic activity of biofilm formation on enamel surfaces after 48 h of incubation (phase 3) measured by MTT reduction assay.G1 (Saliva); G2 (FD); G3 (CPP-ACP), and G4 (CPP-ACPF). Meian values and confidence interval (CI); Kruskal-Wallis; p > 0.05.
Biofilm metabolic activity (A 540 ) 0.16 a 0.17 a 0.18 a 0.18 a 95%CI 0.12-0.20 0.15-0.190.17-0.20 0.17-0.19 Values with same superscript lowercase letter are statistically similar (p > 0.05).and a good evaluation of viable bacteria. 14,15So, MTT results demonstrate the metabolic activity of the viable cells present in the biofilm.Additionally, in the presence of sucrose, it is possible to assume that the more viable bacteria in the biofilm, the greater the acid production.
During pH cycling and remineralization treatment, loss of hardness was significantly lower in the FD group (phase 2), whereas no difference was found between phases 2 and 3 (Table 2) after biofilm formation.Thus, G1, G3, and G4 showed significant demineralization (phase 2), but the deposited material provided similar protection after biofilm formation.In addition, following the exposure to S. mutans biofilm, %SMHC (phase 3) was positive only in G2 and G4, but did not differ in comparison with the other groups (p < 0.05).The %SMHC results were consistent with the concentration of fluoride in the product (G2 and G4), which is an indicator of the effect of fluoride in demineralization and remineralization.In our model, the biofilm was treated once a day with 2% sucrose; therefore, the protocol may have benefited remineralization and this partially justifies the observed results.In these favorable conditions for remineralization (phase 3), no difference was found between the groups, regardless of the difference in hardness (phase 2).With this biofilm model, we were not able to show that the presence of a biofilm greatly affected the resistance of the precipitate formed after treatment with remineralizing agents.
The role of fluoride in the remineralization process was found to be rather complex.Fluoride acts by inhibiting mineral loss on the crystal surface and by enhancing the reconstruction or calcium and phosphate remineralization in a form that is more resistant to subsequent acid attack. 18,26The increased mineral levels observed from %SMHC in G2 and G4, especially those exposed to 1,100 ppm F, were supported by the highest fluoride incorporation into enamel found in these groups in the second phase.In addition, enamel treated with fluoride has been associated with higher saturation than of fluorapatite and more mineral forms precipitated on the enamel surface. 25Subsequent acid challenges must be very strong and long enough to dissolve the remineralized enamel. 26,27In addition, although none of the groups differed in terms of microhardness, it seems that the enamel treated with CPP-ACP and FD produced a reservoir of fluoride ions available for inhibiting future demineralization processes.Under such conditions, the enamel could have been enriched with fluoride (G2 and G4) due to precipitation of fluoride apatite in enamel during de-remineralization and dentifrice treatment (phase 2). 25,26,27he effect of CPP-ACPF products on the enamel surface had previously been shown to be superior after demineralization when compared to saliva. 9he greater protection against demineralization by CPP-ACPF when compared to CPP-ACP has been noted. 22In the current study, despite the presence of fluoride in the CPP-ACPF product, both remineralizing agents examined showed similar %SMHC after phases 2 and 3.In CPP-ACP technology, ACP is stabilized by CPP casein-derived peptides.CPP contains the amino acid cluster sequence -Ser(P)-Ser(P)-Ser(P)-Glu-Glu -and this has been reported to bind amorphous calcium phosphate, forming small clusters of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP). 4Thus, the surface formed after the use of CPP-ACP is different from that formed only in the presence of fluoride dentifrice. 4,10,13,17According to Zhang et al., 28 numerous particles and amorphous crystals were arranged on the surface in the NaF group, but in the CPP-ACP group, those crystals seemed to be more homogeneous than those in the NaF group, and there was no obvious intercrystalline space.So, dissolution of the new precipitate formed during remineralization by the CPP-ACP product is far more resistant to acid attack. 9,10,22 he precipitates formed by the CCP-ACP product were found to demineralize 2.7 times slower than those produced by the fluoride dentifrice (1.1% NaF). 10 In addition, the residual remineralization after acid challenge was significantly greater for the dentifrice containing CPP-ACP plus 1100 ppm fluoride when compared with NaF alone. 4D and CPP-ACPF treatments did lead to observable remineralization of the demineralized enamel, but the metabolic activity of the biofilm was the same in all groups (p > 0.05).This result was quite different from that of a previous report, which revealed fluoride treatment could significantly reduce the metabolic activity of biofilms compared with the control group. 12owever, our model differs from previous biofilm models, where biofilm formation was assayed after a 2-minute application of dentifrice to rigid surface enamel without pH cycling 12 and formation of biofilm before pH cycling. 1 So, precipitation on enamel after application of remineralizing agents can be different and influence metabolic activity.
The SMH result after FD treatment was superior to that of the other groups in both phases (2 and 3), but the metabolic activity of the S. mutans biofilm on the enamel specimens did not differ between groups.Nevertheless, in contrast to the observations of Brambilla et al., 12 our results demonstrated that enamel specimen treatment with fluoride cannot inhibit the metabolic activity of S. mutans biofilm, but it can change the diffusion of ions. 1 Additionally, although the amount of acid produced by this biofilm was not measured, all enamel specimens underwent the same cariogenic challenge generated by the S. mutans biofilm in vitro (phase 3).So, pH assessment could help clarify the results.However, in a study, the pH value of the culture medium, used as an indicator of biofilm acidogenicity, decreased after daily sucrose exposure, but no differences were observed among treatments. 11urther studies are needed to evaluate our hypothesis of the metabolic activity of S. mutans biofilm and its interference with resistance to demineralization in enamel treated with remineralizing agents.To achieve that, an important step will be the quantification of the acids produced by this biofilm induced in vitro.In addition, according to Zhang et al., 1 biofilms might act as diffusion barriers to slow the influx of acid and outflux of calcium and phosphate released from the enamel surfaces, resulting in protection of the underlying demineralized enamel. 1 So, analysis of calcium and fluoride concentrations could also be useful. 11nother limitation of the present study could be the use of monospecific biofilm; however, experimental studies have already demonstrated that the amount of lactic acid produced in vitro by polymicrobial biofilms is very similar to that produced by S. mutans biofilms alone. 29

Conclusion
We conclude that the CPP-ACP and CPP-ACPF tested in this in vitro study do not prevent demineralization in human enamel.Furthermore, there were no significant differences in microhardness in enamel treated with FD (1100), CPP-ACP, and CPP-ACPF after biofilm formation.Therefore, the presence of biofilms clearly does not influence the treatment outcomes of CPP-ACP products.Thus, the null hypothesis was accepted.

Figure 1 .
Figure 1.Schematic illustration of the procedure used in pH cycling and remineralization treatment.

Table 1 .
Tested products and their respective composition.

Table 2 .
Mean±SD for sound enamel (SMH baseline ), artificial caries (SMH phase1 ), post-treatment (SMH phase2 ), and post-biofilm (SMH phase3 ) surfaces in various experimental phases.n = 13 bCDifferent uppercase and superscript lowercase letters indicate significant difference between tested groups at p < 0.05 (ANOVA and Tukey's HSD post-hoc test).Superscript lowercase letters are used for comparison within the same column and uppercase letters are used for comparison within each row.Comparison before and after treatment in the same group was analyzed by Student's t-test.