Polarization-Sensitive Optical Coherence Tomography for Monitoring De- and Remineralization of Bovine Enamel In Vitro.

Early caries diagnosis still challenges dentistry. Polarization-sensitive optical coherence tomography (PS-OCT) is promising to detect initial lesions non-invasively in depth-resolved cross-sectional visualization. PS-OCT with determined degree of polarization (DOP) imaging provides an intuitive demineralization contrast. The aim of this study is to evaluate the suitability of DOP-based PS-OCT imaging to monitor controlled de- and remineralization progression for the first time and to introduce it as a valid, non-destructive in vitro detection method. Twelve standardized bovine enamel specimens were divided in different groups and demineralized with hydrochloric acid (HCl) as well as partly remineralized with fluoride over a 14-day pH-cycling experiment. The specimens were stored in artificial saliva and sodium chloride (NaCl), respectively. Progress measurements with PS-OCT were made with polarization-sensitive en faceand B-scan mode for qualitative evaluation. The specimens demineralized in HCl showed the most pronounced surface change (lowest DOP) and the most significant increase in depolarization. Additional fluoride treatment and the storage in artificial saliva resulted in the opposite (highest DOP). Therefore, DOP-based PS-OCT imaging appears to be a valuable technique for visualization and monitoring of enamel demineralization and remineralization processes in vitro. However, these findings need to be confirmed in human teeth ex vivo or in situ.

Olive Oil as a Transport Medium for Bioactive Molecules of Plants?-An In Situ Study.

(1) Caries and erosions still remain a challenge for preventive dentistry. Certain plant extracts have shown beneficial effects in preventive dentistry. The aim of this study was to evaluate the antibacterial, anti-adherent and erosion-protective properties of ellagic acid (EA) as a polyphenolic agent. The combination with olive oil was investigated additionally to verify a possible improved bioactive effect of EA. (2) An in situ study was carried out with six subjects. Individual splints were prepared with bovine enamel specimens. The splints were worn for 1 min (pellicle formation time). Thereafter, 10 min rinses were performed with EA in water/in oil. Bacterial adherence was evaluated by fluorescence microscopy (DAPI, ConA, BacLight) after an 8 h oral exposition time. Additionally, the splints were worn for 30 min to quantify demineralization processes. The ultrastructure of the pellicle was investigated after an oral exposure time of 2 h under a transmission electron microscope. Statistical analysis was performed by Kruskal-Wallis tests, Mann-Whitney U tests and Bonferroni-Holm correction. (3) Rinsing with EA led to a significant reduction of adherent vital and dead bacteria. The combination with olive oil did not improve these outcomes. The assessment of glucan structures after rinsing with EA in water showed significant effects. Significant differences were observed for both rinses in calcium release at pH 3.0. After rinsing with EA in oil, significantly less calcium was released compared to rinsing with EA in water (pH = 3.0). (4) Olive oil is not suitable as a transport medium for lipophilic polyphenols. EA has anti-adherent and antibacterial properties in situ. EA also shows erosion-protective effects, which can be enhanced in combination with olive oil depending on the pH value. Ellagic acid has a neutral pH and could be an opportunity in the treatment of specific patient groups (xerostomia or mucositis).

Efficacy of Endodontic Disinfection Protocols in an E. faecalis Biofilm Model-Using DAPI Staining and SEM.

The aim of this study was to investigate the antimicrobial efficacy of different disinfection protocols in a novel Enterococcus faecalis biofilm model based on a visualization method and to evaluate the potential alteration of dentinal surface. A total of 120 extracted human premolars were allocated to 6 groups with different irrigation protocols. The assessment of the effectiveness of each protocol and the alteration of dentinal surface were visualized by using SEM and fluorescence microscopy (DAPI). A dense E. faecalis biofilm with a penetration depth of 289 µm (medial part of the root canal) and 93 µm (apical part) validated that the biofilm model had been successfully implemented. A significant difference between the 3% NaOCl groups and all the other groups in both observed parts of the root canal (p < 0.05) was detected. However, the SEM analysis revealed that the dentinal surface in the 3% NaOCl groups was severely altered. The established biofilm model and the visualization method based on DAPI are appropriate for bacterial quantification and evaluation of the depth effect of different disinfection protocols in the root canal system. The combination of 3% NaOCl with 20% EDTA or MTAD with PUI allows the decontamination of deeper dentine zones within the root canal but simultaneously alters the dentinal surface.

Direct and indirect effects of different dentifrices on the initial bacterial colonization of enamel in situ overnight.

OBJECTIVE: The aim of this study was to investigate the direct and indirect influence of fluoridated toothpastes and fluoride-free toothpaste with hydroxyapatite (HAP) as active ingredient on initial bacterial colonization on enamel in situ. METHODS: For this clinical-experimental pilot study, eight subjects were instructed to brush their teeth with three different toothpastes (Elmex® : 1400 ppm AmF, Meridol® : 1400 ppm AmF +SnF2, Karex® : HAP), using each for two consecutive days. As a control, brushing without toothpaste was performed. To evaluate bacterial colonization, subject wore splints with buccally placed bovine enamel platelets overnight. Two modes were tested. In a first pass (regimen A), the splints were inserted after toothbrushing to examine the indirect effects of the dentifrices. In order to investigate the direct effects, the specimens were brushed in situ in a second pass (regimen B). Biofilm formation was visualized and quantified using fluorescence microscopy (DAPI and BacLight) and transmission electron microscopy (TEM). RESULTS: For brushing regimen A (indirect effect of dentifrices), no statistical differences were detected between any of the tested dentifrices or the control. Likewise, no statistically significant differences were recorded for brushing regimen B (direct effect of dentifrices). Furthermore, no differences between the different brushing techniques were determined with regard to the ultrastructure of the overnight biofilm. CONCLUSION: Within the limitations of the present pilot study, it can be concluded that in patients with good oral hygiene, dentifrices and their chemical composition have no statistically significant effect on the initial bacterial colonization of enamel platelets in situ, irrespectively of the mode of application.

Quantification of Bacterial DNA from Infected Human Root Canals Using qPCR and DAPI after Disinfection with Established and Novel Irrigation Protocols.

The removal of bacterial infections within the root canal system is still a challenge. Therefore, the cleansing effect of established and new irrigation-protocols (IP) containing silver diamine fluoride (SDF) 3.8% on the whole root canal system was analyzed using quantitative PCR (qPCR) and 4',6-diamidino-phenylindole-(DAPI)-staining. Extracted human premolars were instrumented up to F2 (ProTaper Gold) under NaCl 0.9% irrigation and incubated with Enterococcus faecalis for 42 days. Subsequently, different ultrasonically agitated IP were applied to the roots: control (no irrigation), 1. NaOCl 3%, EDTA 20%, CHX 2%, 2. NaOCl 3%, EDTA 20%, 3. NaOCl 3%, EDTA 20%, SDF 3.8%, 4. SDF 3.8%, and 5. NaCl 0.9%. One half of the root was investigated fluorescent-microscopically with DAPI. The other half was grinded in a cryogenic mill and the bacterial DNA was quantified with qPCR. The qPCR results showed a statistically significant reduction of bacteria after the application of IP 1, 2, and 3 compared to the control group. While IP 4 lead to a bacterial reduction which was not significant, IP 5 showed no reduction. These data corresponded with DAPI staining. With qPCR a new molecular-biological method for the investigation of the complete root canal system was implemented. The novel IP 3 had an equally good cleansing effect as the already established IP.

Bioadhesion on Textured Interfaces in the Human Oral Cavity-An In Situ Study.

Extensive biofilm formation on materials used in restorative dentistry is a common reason for their failure and the development of oral diseases like peri-implantitis or secondary caries. Therefore, novel materials and strategies that result in reduced biofouling capacities are urgently sought. Previous research suggests that surface structures in the range of bacterial cell sizes seem to be a promising approach to modulate bacterial adhesion and biofilm formation. Here we investigated bioadhesion within the oral cavity on a low surface energy material (perfluorpolyether) with different texture types (line-, hole-, pillar-like), feature sizes in a range from 0.7-4.5 µm and graded distances (0.7-130.5 µm). As a model system, the materials were fixed on splints and exposed to the oral cavity. We analyzed the enzymatic activity of amylase and lysozyme, pellicle formation, and bacterial colonization after 8 h intraoral exposure. In opposite to in vitro experiments, these in situ experiments revealed no clear signs of altered bacterial surface colonization regarding structure dimensions and texture types compared to unstructured substrates or natural enamel. In part, there seemed to be a decreasing trend of adherent cells with increasing periodicities and structure sizes, but this pattern was weak and irregular. Pellicle formation took place on all substrates in an unaltered manner. However, pellicle formation was most pronounced within recessed areas thereby partially masking the three-dimensional character of the surfaces. As the natural pellicle layer is obviously the most dominant prerequisite for bacterial adhesion, colonization in the oral environment cannot be easily controlled by structural means.

Antifungal and Surface Properties of Chitosan-Salts Modified PMMA Denture Base Material.

Chitosan (CS) and its derivatives show antimicrobial properties. This is of interest in preventing and treating denture stomatitis, which can be caused by fungi. Therefore, the aim of this study was the development of a novel antifungal denture base material by modifying polymethyl methacrylate (PMMA) with CS-salt and characterizing its antifungal and surface properties in vitro. For this purpose, the antifungal effect of chitosan-hydrochloride (CS-HCl) or chitosan-glutamate (CS-G) as solutions in different concentrations was determined. To obtain modified PMMA resin specimens, the CS-salts were added to the PMMA before polymerization. The roughness of these specimens was measured by contact profilometry. For the evaluation of the antifungal properties of the CS-salt modified resins, a C. albicans biofilm assay on the specimens was performed. As solutions, both the CS-G and CS-HCl-salt had an antifungal effect and inhibited C. albicans growth in a dose-dependent manner. In contrast, CS-salt modified PMMA resins showed no significant reduced C. albicans biofilm formation. Furthermore, the addition of CS-salts to PMMA significantly increased the surface roughness of the specimens. This study shows that despite the antifungal effect of CS-salts in solution, a modification of PMMA resin with these CS-salts does not improve the antifungal properties of PMMA denture base material.

Author Correction: Influence of pure fluorides and stannous ions on the initial bacterial colonization in situ.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Influence of pure fluorides and stannous ions on the initial bacterial colonization in situ.

The present clinical-experimental study aims to examine the effect of pure experimental fluoride solutions and stannous chloride on the initial oral bioadhesion under in situ conditions. After 1 min of pellicle formation on bovine enamel slabs, 12 subjects rinsed with 8 ml of the fluoride test solutions (NaF, Na2PO3F, AmF, SnF2,) with 500 ppm fluoride concentration each for 1 min. Additionally, rinsing without a solution (control) and rinsing with 1563 ppm SnCl2 solution took place for 1 min. Afterwards, fluorescence microscopy took place to visualize bacterial adhesion and glucan formation (8 h oral exposition) with DAPI and ConA and the BacLight method. TEM was performed to visualize the pellicle ultrastructure together with EDX to detect stannous ions. The rinsing solutions with pure SnF2 and SnCl2 reduced significantly the initial bacterial colonization (DAPI). While, NaF and Na2PO3F showed no significant effect compared to the control. There was no significant difference between AmF, SnF2 and SnCl2. All tested experimental solutions showed no reducing effect on the glucan formation. Considerable alterations of the pellicle ultrastructure resulted from rinsing with the Sn-containing solutions. SnF2 appears to be the most effective type of fluoride to reduce initial bacterial colonization in situ. The observed effects primarily have to be attributed to the stannous ions' content.

Is it really penetration? Part 2. Locomotion of Enterococcus faecalis cells within dentinal tubules of bovine teeth.

OBJECTIVE: The aim of the present vitro study was to examine the question whether devitalized Enterococcus faecalis (E. faecalis) cells can migrate into dentinal tubules and if that process takes place in a time-dependent manner. DESIGN: Sixty bovine root canals were incubated with devitalized and vital streptomycin-resistant E. faecalis strains after root canal enlargement (size 80, taper .02) with 3% NaOCl solution. Incubation times 7, 14, 21, 28, 35, and 42 days. Samples were processed for analysis by scanning electron microscopy (SEM) and DAPI (4',6-diamidino-2-phenylindole) staining. The penetration depth was calculated with the measurement tool of the Axio Vision program (Zeiss, Jena, Germany). Statistical analysis was performed by Kruskal-Wallis (α = 0.05) and Mann-Whitney U test (p < 0.05). RESULTS: Devitalized E. faecalis strains were able to migrate into dentinal tubules. The total number and penetration depth of devitalized E. faecalis cells was lower compared to the vital suspension of E. faecalis. It was noted, that bacterial penetration was not common to all of the dentinal tubules in the vital E. faecalis control and especially in the devitalized control. The migration took place in a time-dependent migration characteristic. CONCLUSIONS: Devitalized E. faecalis cells are still able to migrate into the dentinal tubules due to possible electrokinetic and osmotic processes. Thereby, increased exposure times lead to a time-dependent penetration characteristic. CLINICAL RELEVANCE: Since devitalized bacteria can migrate as well into dentinal tubules, the presence of bacteria within dentinal tubules cannot be interpreted as a failure of tested preparation regimens.

Initial microbial colonization of enamel in children with different levels of caries activity: An in situ study.

PURPOSE: To investigate patterns of overnight in situ microbial colonization of enamel in children. METHODS: Overall, 29 children (aged 5-9 years) participated in the study. Nine were caries-free with no decayed, missing, or filled teeth (DMFT), 11 were caries-rehabilitated (DMFT ≥ 2, no active carious lesions), and nine were caries-active (DMFT ≥ 2, at least two carious lesions). Bovine enamel samples were fixed on individual upper jaw splints stored overnight in situ. 4',6-diamidino-2-phenylindole (DAPI) combined with Concanavalin A staining was applied for fluorescence microscopic visualization of total adherent bacteria and glucans. Fluorescence in situ hybridization (FISH) was used for distinction of eubacteria, streptococci, and Candida albicans. Salivary samples were investigated for Streptococcus mutans (S. mutans) by using CRT bacteria test and yeasts with Calcofluor white (CFW) staining. RESULTS: With all fluorescence methods, bacteria but not Candida albicans were detected on enamel samples. No statistically significant differences were observed in distribution patterns of the adherent bacteria between the groups. CFW staining indicated fungal structures in saliva samples of all participants. Based on CRT test results, the lowest amount of S. mutans were observed in caries-free children. Thus, initial microbial colonization patterns of enamel in children are not influenced by caries activity. CLINICAL SIGNIFICANCE: Caries activity in children may influence the process of initial bioadhesion and thus distribution patterns of bacterial attachment to the enamel surface. Investigation of in situ biofilm formation might provide valuable insights regarding the varying caries susceptibility in children.

Is it really penetration? Locomotion of devitalized Enterococcus faecalis cells within dentinal tubules of bovine teeth.

OBJECTIVE: The aim of the present study was to evaluate the penetration characteristics of devitalized and vital E. faecalis cells into root dentinal tubules. DESIGN: Thirteen root canals were incubated with devitalized (4days, 7days, 14days, 28days) and vital (28days) E. faecalis strains (streptomycin-resistant strains) after root canal enlargement (size 80, taper 0.02) with 3 % NaOCl solution. The smear layer was intentionally removed with 20 % EDTA before inoculation. Samples were processed for analysis by scanning electron microscopy (SEM) and DAPI (4',6-diamidino-2-phenylindole) staining. DAPI was conducted for fluorescence microscopic visualization of the bacterial penetration into dentinal tubules. The penetration depth was calculated with the measurement tool of the Axio Vision program (Zeiss, Jena, Germany). RESULTS: Devitalized E. faecalis strains were able to penetrate into dentinal tubules of the root canal. Apikal penetration depths of the devitalized cells were 100.67µm±26.54µm after 7days, 230.67µm±111.5µm after 14days and 266.5µm±92.63µm after 28days of incubation. The total number and penetration depth of E. faecalis cells was lower compared to a vital suspension of E. faecalis (1002.45µm) after 28days. It was noted that bacterial penetration was not common to all of the dentinal tubules in the vital E. faecalis control and especially in the devitalized control. CONCLUSIONS: Increased exposure times of devitalized bacteria into root canals lead to an increased number of penetrated dentinal tubules as well as to a deeper penetration.

The chemical composition of the pharmacologically active Thymus species, its antibacterial activity against Streptococcus mutans and the antiadherent effects of T. vulgaris on the bacterial colonization of the in situ pellicle.

The pharmacological active genus Thymus L. comprises over 200 species. Besides its traditional pharmacological use, thyme may reduce the risk of caries disease, however, there is very little respective literature. The pharmacological effects can be attributed to the secondary plant metabolites. The composition of the essential oil and the polyphenols is important for the evaluation of the pharmacological activity. Nevertheless, there are no studies regarding a comparative analysis of the different pharmacological thyme species. In the present study, four different pharmacology Thymus species were cultivated under comparable conditions, and the volatile compounds as well as the polyphenols were characterized. In addition, the in vitro antibacterial activity against S. mutans, one of the primary cariogenic bacterial species, as well as of the essential oil and of the polyphenols were investigated. Furthermore, the bacterial viability and its effect on the initial bacterial adhesion under oral conditions were evaluated in situ for the essential oil and the polyphenols. By GC-MS, 69 volatile compounds, and by LC-DAD-MS/MS, 46 polyphenols could be identified. The comprehensive examination of the essential oils and the polyphenols revealed that the main compounds were equal. However, the yield of the essential oil and the polyphenol content differed clearly. The essential oils of the four investigated Thymus species exhibited an antibacterial activity against S. mutans in vitro, in contrast to the polyphenols of T. vulgaris. Rinsing with polyphenol-rich infusions reduced the initial bacterial colonization while the essential oil inhibited the bacterial growth on dental enamel in situ.

Enzymology and Ultrastructure of the in situ Pellicle in Caries-Active and Caries-Inactive Patients.

AIM: The present study aimed to evaluate the impact of caries activity on the key enzymes and the ultrastructure of the in situ pellicle. METHODS: Pellicle formation was performed on bovine enamel slabs. Intraoral exposure (3, 30, and 120 min) was accomplished by 14 caries-active (DMFS: 22.7 ± 12.1) and 13 caries-inactive (DMFS: 1.5 ± 1.8) individuals. The enzyme activities (lysozyme, peroxidase, α-amylase, glycosyltransferase [GTF]) in the in situ pellicle and resting saliva of all participants were analyzed directly after oral exposure. In addition, a simultaneous visualization of these enzymes, extracellular glucans, and adherent bacteria was carried out. Fluorescent patterns were analyzed with fluorescence labeling and 4',6-diamidino-2-phenylindole/concanavalin A staining. In addition, the distribution of GTF B, C, and D and the ultrastructure of the pellicle were examined by gold immunolabeling and transmission electron microscopy with selected samples. RESULTS: Enzyme activities of amylase, peroxidase, lysozyme, and GTF were detected on all enamel slabs in an active conformation. Neither exposure time nor caries activity had an impact on the enzyme activities. Gold immunolabeling indicated that the pellicle of caries-active subjects tends to more GTF D molecules. The pellicles of caries-inactive and -active individuals revealed a similar ultrastructural pattern. CONCLUSION: The enzyme activities as well as the pellicle's ultrastructure are of high similarity in caries-active and -inactive subjects. Thereby, oral exposure time has no significant influence. This reflects a high uniformity during the initial phase of bioadhesion (3-120 min) concerning enzymatic functions. However, there is a tendency towards more GTF D in caries-active individuals.

Effect of Tannic Acid on the Protective Properties of the in situ Formed Pellicle.

OBJECTIVES: In the present in situ/ex vivo study the impact of tannic acid on the erosion-protective properties of the enamel pellicle was tested. Additionally, the antiadherent and antibacterial effects of tannic acid were evaluated. METHODS: The pellicle was formed in situ on bovine enamel samples fixed on individual splints worn by 6 subjects. Following 1 min of pellicle formation the volunteers rinsed for 10 min with tannic acid. After further oral exposure for 19 min, 109 min, and 8 h overnight, respectively, slabs were incubated in HCl ex vivo (pH 2.0, 2.3, 3.0) over 120 s. Subsequently, kinetics of calcium and phosphate release were measured photometrically. Samples after a 1-min fluoride mouth rinse as well as enamel samples with and without a 30-min in situ pellicle served as controls. Antiadherent effects were evaluated after a 1-min rinse with tannic acid and oral exposure of the slabs overnight. DAPI (4',6-diamidino-2-phenylindole) combined with concanavalin A staining and live/dead staining was used for fluorescence microscopic visualization and quantification of adherent bacteria and glucans. Modification of the pellicle's ultrastructure by tannic acid was evaluated by transmission electron microscopy (TEM). RESULTS: Tannic acid significantly improved the erosion-protective properties of the pellicle in a pH-dependent manner. Bacterial adherence and glucan formation on enamel were significantly reduced after rinses with tannic acid as investigated by fluorescence microscopy. TEM imaging indicated that rinsing with tannic acid yielded a sustainable modification of the pellicle; it was distinctly more electron dense. CONCLUSION: Tannic acid offers an effective and sustainable approach for the prevention of caries and erosion.

Effect of Inula viscosa on the pellicle's protective properties and initial bioadhesion in-situ.

OBJECTIVES: The present in situ study investigated the effect of Inula viscosa tea on the pellicle's acid protective properties and on initial oral biofilm formation. DESIGN: Biofilm formation was performed on bovine enamel slabs on individual maxillary splints. Following 1min of pellicle formation, eight subjects rinsed for 10min with Inula viscosa tea and the splints remained for 8h intraorally. Samples carried after 1-min rinsing with CHX (0.2%) or without rinse served as controls. BacLight™ staining, 4',6-diamidino-2-phenylindole (DAPI)-staining and fluorescence in situ hybridization (FISH) were used for fluorescence microscopic detection of adherent bacteria. For investigation of acid protective properties, three subjects rinsed for 10min with Inula viscosa tea after 1min pellicle formation and kept the splints intraorally for further 19min. Physiological 30-min pellicles and native enamel samples served as controls. After HCl incubation of the samples ex-vivo over 120s (pH 2.0, 2.3, 3.0) calcium- and phosphate release were quantified photometrically. Potential influences on the pellicle's ultrastructure by Inula viscosa tea were evaluated by transmission electron microscopy (TEM). RESULTS: Application of Inula viscosa tea yielded a significant reduction of adherent bacteria on all enamel samples as detected by fluorescence microscopy. For calcium- and phosphate release no significant effect was recorded. TEM investigation indicated a modification of the pellicle's ultrastructure, but no enhanced protection against erosive noxae. CONCLUSION: Rinsing with Inula viscosa tea influences the bacterial colonization on enamel in situ over 8h but has no impact on acid protective properties of the pellicle.

The Polyphenolic Composition of Cistus incanus Herbal Tea and Its Antibacterial and Anti-adherent Activity against Streptococcus mutans.

The Mediterranean plant Cistus incanus is rich in polyphenols and has shown several pharmacological activities, mainly antibacterial effects. Furthermore, in situ studies revealed that a C. incanus infusion reduces the initial bacterial adhesion in the oral cavity due to the polyphenols, an indication that C. incanus might reduce the risk of caries disease. In the present study, the polyphenols from four different commercial C. incanus herbal teas were extracted by standardized accelerated solvent extraction for in vitro tests and by an infusion for in situ tests. Both extracts were characterized qualitatively and quantitatively by high-performance liquid chromatography and only the polyphenol content differed slightly. By means of diode array detection and mass spectrometry, 29 polyphenols, including ellagitannins, flavanols, and glycosylated flavonols, were identified. Thereby, only quantitative but no qualitative differences between the four samples were detected. Furthermore, the in vitro antibacterial activity of the C. incanus accelerated solvent extracts against Streptococcus mutans, one of the primary cariogenic bacterial species, was examined using a live/dead assay (BacLight®). With this approach, C. incanus yielded antibacterial properties. Additional in situ experiments indicated that rinses with a C. incanus infusion reduced the initial bacterial colonization of enamel samples exposed to oral fluids for over eight hours. Furthermore, it was shown by transmission electron microscopy that the application of a C. incanus infusion modifies the ultrastructure of the acquired enamel pellicle, yielding a more electron-dense morphology. It can be assumed that the polyphenols are responsible for the observed effects.

Effect of CPP/ACP on initial bioadhesion to enamel and dentin in situ.

The present in situ study investigated the influence of a preparation containing CPP/ACP (caseinphosphopeptide-amorphous calcium phosphate) (GC Tooth mousse) on initial bacterial colonization of enamel and dentin. Therefore, pellicle formation was performed in situ on bovine enamel and dentin specimens fixed to individual upper jaw splints worn by 8 subjects. After 1 min of pellicle formation GC Tooth mousse was used according to manufacturer's recommendations. Rinses with chlorhexidine served as positive controls. Specimens carried without any rinse served as negative controls. After 8 h overnight exposure of the splints, bacterial colonization was quantified by fluorescence microscopy (DAPI and BacLight live/dead staining). Additionally, the colony forming units (CFU) were determined after desorption. Furthermore, the effects on Streptococcus mutans bacteria were tested in vitro (BacLight). There was no significant impact of CPP/ACP on initial bacterial colonization proved with DAPI and BacLight. Determination of CFU showed statistical significance for CPP/ACP to reduce bacterial adherence on enamel. The in vitro investigation indicated no antimicrobial effects for CPP/ACP on Streptococcus mutans suspension. Under the chosen conditions, CPP/ACP (GC Tooth mousse) had no significant impact on initial biofilm formation on dental hard tissues. The tested preparation cannot be recommended for biofilm management.