Effect of the Fluoride Gels and Varnishes Comparing to CPP-ACP Complex on Human Enamel Demineralization/Remineralization

Vesna Ambarkova (1), Kristina Gorseta (2), Mira Jankulovska (1),Domagoj Glavina (2), Ilija Skrinjaric (2)

Affiliation:

1 - Faculty of Dentistry, University Sv.Cyril & Methodius, FYR Macedonia
2 - Department of Pedodontics, School of Dental Medicine University of Zagreb, Croatia

Address for correspondence:

Professor Domagoj Glavina
University of Zagreb
School of Dental Medicine
Department of pedodontics
Gunduliceva 5, 10 000 Zagreb, Croatia
Tel: + 384 1 4802 111
glavina@sfzg.hr

Received: January 3, 2013

Accepted: May 2, 2013

Available online: June 15, 2013

Acta stomatol Croat. 2013;47(2):99-110.

Original scientific article

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Abstract

Aim: This in vitro study was conducted to investigate the effect of fluoride gels and varnishes comparing to CPP-ACP complex on the inhibition of enamel demineralization. Material and Methods: Enamel blocks were ground flat, allocated into eight groups and subjected to a daily cycling regime. Three groups were treated within the period of 10 minutes with fluoride gels: Fluorogal, Fluor Protector Gel and Cervitec Gel, one was treated only with GC Tooth Mousse and one was treated with GC Tooth Mousse (Recaldent CPP-ACP 10.0%) The remaining three groups were treated with fluoride varnishes: Fluoridin Gel N5, Bifluorid 12 and Fluor Protector. They were coated once a week before the demineralization period. All specimens were stored in artificial saliva between and after cycles. The surface microhardness (SMH) of the specimens was determined at baseline and after 12 days using HMV-2000 (Shimadzu, Japan).The percentage of SMH change (% SMC) was calculated before and after cycling regime. Data were analyzed by t-test for individual comparisons (p<0.05). Results: Statistical analysis by t-test showed significant difference between SMH before and after fluoride treatment in all groups. All the groups treated with fluoride gels, varnishes and GC Tooth Mousse showed increase in SMH. There was no significant statistical difference between the percentages of SMH of the enamel between groups. There was no statistically significant difference between the fluoride gels, varnishes and GC Tooth Mousse. Conclusion: The results obtained in the present study showed that high fluoride varnishes, gels and Tooth Mousse effectively inhibit demineralization under experimental conditions.

Key Words:

Fluorides, Topical; Dental Enamel; Hardness; Tooth Demineralization

 

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Introduction

Fluoride therapy has been the centerpiece of caries-preventive strategies since the introduction of water fluoridation schemes over five decades ago (1). Protective factors are present in the oral environment, an extrinsic supply of these factors such as fluoride and bioavailable calcium and phosphate will favor an equilibrium shift towards enamel remineralization (2, 3).
Topically applied fluoride products are used extensively as an operator-applied caries-preventive intervention. The most important anti-caries effect of fluoride is considered to result from its action on the tooth/plaque interface, by promotion of remineralization of early caries lesions and reducing tooth enamel solubility (4,5).
 Varnishes were originally developed to prolong the contact time between fluoride and dental enamel, as they adhere to the tooth surface for longer periods (12 hours or more) in a thin layer, and prevent the immediate loss of fluoride after application. After application the varnish acts as a slow-releasing reservoir of fluoride (6-9). Fluoride ion can be incorporated into the hydroxylapatite structure of tooth enamel by the replacement of hydroxyl groups or by redeposition of dissolved hydroxyl-apatite as less soluble fluoridated forms, such as fluorapatite or fluorhydroxyl-apatite. Topical fluoride agents have been shown to decrease enamel demineralization in vitro and in clinical studies (10).
Calcium and phosphate technologies currently incorporated into dental products include amorphous calcium phosphate (ACP), casein phosphopeptide-amorphous calcium phosphate (CPP-ACP), calcium sodium phosphosilicate (CSPS) and tricalcium phosphate (TCP). The overall intent of these technologies is to increase the amount of available calcium and phosphate, typically together with fluoride. In the case of professional products, ACP, CSPS and TCP technologies have variously been incorporated and are currently available in restorative materials, sealants, orthodontic cement, prophylaxis pastes, in-office fluoride varnishes and in-office fluoride gels (11, 12).
Apart from the varnishes, the CPP-ACP complex in the form of paste has a strong anticariogenic effect which was attributed to the phosphoprotein casein and calcium phosphate (13). Casein phosphopeptide (CPP) containing the sequence Ser(P)-Ser(P)-Ser(P)-Glu-Glu has the ability to stabilize calcium phosphate in solution by binding amorphous calcium phosphate (ACP) with their multiple phosphoserine residues (14). The anticariogenicity of the CPP-ACP nanocomplexes has been demonstrated in animal and in vitro caries models (15-17). Further studies using human in situ caries models have shown that the CPP-ACP could prevent enamel demineralization and promote remineralization (14, 18, 19, 20).
The use of CPP-ACP with fluoride and its synergistic effect on enamel remineralization have been attributed to the formation of CPP-stabilized amorphous calcium fluoride phosphate (21), resulting in the increased incorporation of fluoride ions into plaque, together with increased concentrations of bioavailable calcium and phosphate ions. This synergistic effect of CPP-ACP and fluoride in reducing caries was investigated in animals, in in-vitro and in situ studies (16, 21, 22).
The aim of this study was to determine the effect of the fluoride gels and varnishes comparing to CPP-ACP complex on surface microhardness of early enamel lesions in pH-cycling model enamel (demineralization/remineralization).

 

 

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Materials and methods

Study design
Extracted sound human teeth were stored in 2% formaldehyde solution (pH 7.0) at room temperature. After removal of the roots and pulp, sound enamel sections were cut from the buccal and lingual sides of the teeth using a diamond saw. The sections were mounted in acrylic resin (Acryl Fix Kit- Struers) and cured overnight. Enamel surface of the blocks is ground flat with water-cooled carborundum discs (320, 600 and 1200 grit of Al2O3 papers; Buehler, Lake Bluff, IL, USA). These procedures are conducted to form parallel planar surfaces for the Vickers microhardness tests. Specimens were re-hydrated in deionised water for at least 60 minutes prior to use. For standardization of the blocks, a previous selection of specimens for the initial microhardness was made (five indentations in different regions of the blocks, 50 g (490.3 mN or 0.05 Hv), 10 s, HMV-2000; Shimadzu Corporation, Tokyo, Japan). Enamel blocks with a mean surface microhardness between 142 and 462 VHN were randomly divided into eight experimental groups.
A total of 34 sound enamel slabs were divided into experimental and control groups. Within eight experimental groups, six contained four slabs each, and two of them contained three slabs. The control group contained four slabs and did not receive any treatment during the experiment. The following fluoride gels and varnishes  were applied: Fluoridin Gel N5 (Fluoride gel; Voco-GmbH, Cuxhaven, Germany), Fluorogal forte (NaF 8 400 ppm, FH2 2600 ppm, Galenika, Belgrade, Serbia), Fluor Protector Gel (1450 ppmF, Ivoclar Vivadent), Bifluorid 12 (NaF 27 100 ppm, CaF 29 200) (Fluoride gel;Voco-GmbH, Cuxhaven, Germany), Fluor Protector varnish (fluorsilane, NaF 22.600 ppm, Ivoclar Vivadent AG FL-9494 Schaan, Cervitec Gel (900 ppm F, Ivoclar Vivadent) and Tooth Mousse (GC Corporation, Itabashi-Ku, Tokyo, Japan) , Table 1. Three groups were treated with fluoride varnishes (Fluoridin Gel N5, Bifluorid 12 and Fluor Protector).They were coated once a week before the demineralization period. Agents were left undisturbed for five minutes on the tooth surfaces. After 12 days, pH cycling regime the fluoride film was carefully removed with a scalpel. Also, the teeth were cleaned of any remaining resinous sediments with distilled water.
From the remaining five experimental groups, three of them were treated with fluoride gels (Fluorogal, Fluor Protector Gel and Cervitec Gel), one was treated only with GC Tooth Mousse and the last group was treated with GC Tooth Mousse and with Cervitec Gel. All the five experimental groups were treated within the period of 10 minutes, with the exception of the group which was treated with GC Tooth Mousse and Cervitec Gel. This treatment lasted longer, which means 10 minutes of treatment with GC Tooth Mousse and 2 minutes with Cervitec Gel. To study the cycles of demineralization and remineralization of enamel that occur under dental plaque in the mouth, a laboratory pH cycling model was previously developed. This model tries to mimic the process of acid attack (demineralization) and remineralization by saliva in the mouth (23). Human enamel specimens were subjected to a daily cycling regime comprising: two ten minutes treatments with fluoride gels, one before and one after the demineralization period of 6 hours using demineralization solution and 18 hours remineralization in artificial saliva. Daily pH cycling regime was repeated during 12 days. The test scheme was designed to model a daily challenge of a 6-hour demineralization and an 18-hour remineralisation repair by artificial saliva. Three experimental groups received one fluoride varnish treatment per week. Following the pH-cycling regime, the fluoride film was carefully removed with a scalpel.  All slabs were rinsed with distilled water for 15s before and after any DM/RM solution change or treatment period and were wiped dry with soft tissue paper. All samples were then immersed in remineralizing solution at pH 8.0 for 18 hours at 37°C (Cultura Vivacare Diagnostic Line-Vivadent).
SMN was determined by measuring the lengths of the indentations (µm) using the following image analysis system. Samples were examined with reflected light using an Olympus BH-2 microscope. The image was transmitted via a video camera to a monitor connected to an IBM-XT computer (Olympus CUE-2 software). The SMH of each enamel block was determined at baseline and again after the 12 days pH cycling regime.
The final SMH minus the baseline SMH gives the change in hardness (∆ SMH) as change in indentation length (µm).The indentation length was used as a direct measure of change rather than the VHN which is derived from, and inversely proportional to, the square of the indentation length.  

De/remineralising solutions
The composition of demineralizing solution was: sodium acetate (0.1 mM CH3COONa), potassium chloride (150 mM KCl2), calcium chloride (1.5 mM CaCl2) and potassium dihydrogen phosphate (0.9 mM KH2PO4). The pH was adjusted to 4.5 using hydrochloric acid (0.1 mol/l). Slight elevations were corrected with hydrochloric acid 0.1 mol/l to maintain a constant pH value between 4.35 and 4.65 during the demineralization period. The artificial saliva (BSI-24) contained: sodium chloride (0.50 gr/l), sodium bicarbonate (4.2 g/l), sodium nitrate (0.03 g/l) and potassium chloride (0.20 g/l), shown in Table 2. The pH of artificial saliva was 8.0.The pH values of demineralization and remineralization solutions were measured every day using pH meter (HI 8014, HANNA instruments, Bioblock Scientific, Illkirch, France)(24).

 

Testing procedure
The Vickers hardness number (VHN) was determined from the mean values obtained from six indentations on the surface of each specimen. Microhardness of enamel surface was measured before and after pH-cycling regime in each tested group. The obtained data on SMH change before/after treatment in each group were analyzed using Student t-test for dependent samples at 95% level of confidence. Differences between the different groups of materials were tested with one-way ANOVA and Tukey HSD test. Commercially available software (Sigma Stats, SPSS) was used for the analysis.

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Table 1 Formulation details for fluoride gels, varnishes and GC Tooth Mousse tested in microhardness study

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Table 2 Composition of artificial saliva

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Results

The means and standard deviations for the SMH are presented in Table 3. After 12 days cycling, specimens treated with fluoride gels, dental crème Tooth Mousse and combination of Tooth Mousse and Cervitec Gel exhibited statistically higher SMH than the control group (p<0.05).SMH analysis showed that enamel microhardness after treatment with combination of Tooth Mousse and Cervitec Gel in comparison with the surface microhardness of the enamel blocks treated only with Tooth Mousse was higher but it was not statistically significant (p>0.05). ANOVA did not show statistically significant differences in the effect on remineralization between fluoride containing gels and varnishes and CPP-ACP complex (p>0.05).
The highest values of percentage SMHR were observed for the Fluoridin N5 and the lowest with Fluor Protector Gel, Table 4.

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Table 3 Statistical analysis of rates of enamel surface microhardness change during 12 days pH cycling (p-value, Student t-test)

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Table 4 The percentage of microhardness changes as a difference between SMH measurements before and after 12 days pH cycling regime

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Discussion

Minerals, primarily calcium and phosphate, leak out from the hydroxyapatite crystals during demineralization and in situations where demineralization outpaces remineralization, this leads to the development of subsurface lesions. These initially involve only the enamel and often result in the appearance of white spots where sufficient subsurface mineral content has been lost to alter the optical properties of the dental hard tissues.
Saliva is supersaturated with calcium and phosphate, which helps to prevent demineralization until the critical pH is reached during an acid attack.   
For more than a decade, individual investigators and expert panels have recommended that professional topical fluoride use be limited to those individuals with moderate-to-high caries risk (8, 11, 25).
The role of topical fluoride in caries prevention and enhancing tooth mineral resistance to a cariogenic challenge is well known. The mechanism for reducing demineralization and facilitating remineralization involves modification of the mineral structure of enamel with creation of fluoridated calcium and phosphate mineral phases, and increasing the surface enamel fluoride content. The presence of low-level fluoride influences the transformation of less stable, more soluble mineral phases (dicalcium phosphate dehydrate [DCPD], octacalcium phosphate [OCP], tricalcium phosphate [TCP] to more stable, less soluble mineral phases hydroxyapatite [HAP], fluorhydroxyapatite [FHAP], fluorapatite [FAP].
The exposure to a relatively high fluoride content most likely led to calcium fluoride formation initially on the enamel surface. With the subsequent treatment of the enamel slabs with fluoride protective gel, fluoride, calcium and phosphate ions became available, which may have allowed for transformation of the surface calcium fluoride to FHAP.
Sustained-release vehicles such as varnishes may exert a long-term prophylactic effect. The agent’s efficacy depends on its degree and rate of release from the carrying material. Fluoride varnishes have been found to be effective (8, 21).  Sustained-release systems, including varnishes, generally show an initial burst, with rapid release of the active agent, followed by a slower phase osf release (26).  
One in vitro study of two 5% sodium fluoride varnishes conducted in 2001 found that varnish coated on enamel slabs was then immersed in buffered calcium phosphate solution (to mimic the intraoral environment) and continued to release fluoride for five to six months (27).
In this study, chlorhexidine gel was used (Cervitec gel) alone or in combination with dental crème Tooth Mousse.
In the study of Erdem AP, the effect of two fluoride varnishes and one fluoride/chlorhexidine varnish on Streptococcus mutans and Streptococcus sobrinus on biofilm formation, was evaluated and compared in vitro (28). In that study, bifluoride 12 (6% naF and 6% Ca F2) had the lowest inhibitory effect during the experimental period, although it had the highest fluoride concentration compared to Fluor Protector varnish (1% difluorsilan).   These differences between the fluoride varnishes may be explained by the characteristics of the different varnishes and mechanisms of action. Bifluoride 12 has a higher viscosity than the other tested varnishes, which may have resulted in a thicker layer on the enamel slabs. Fluor Protector contains the polyurethane-based compound difluorosilane, has a low pH and forms a thin transparent film on the enamel surface. Fluor Protector contained a lower fluoride concentration than Bifluoride 12, its remineralization effect was better, and this may be explained by the silane content.
Cervitec gel used in our study contains chlorhexidine, which is chemically bis-biguanide with antibacterial, anticariogenic and remineralizing actions and few toxic effects. The addition of Cervitec gel after treatment of enamel slabs with dental crème Tooth Mousse increased the remineralisation effect of Tooth Mousse against demineralization. Also, pH values of the test varnishes and gels are parameters that should be considered.  
The inhibition of demineralization and the promotion of remineralization both require the presence of sufficient quantities of calcium, phosphate and fluoride. If a higher level of these minerals can be maintained at the tooth surface prior to and during an acid attack, their increased concentration helps prevent migration of calcium and phosphate from the tooth. It is known that supersaturation with calcium and phosphate ions intraorally results in increased resistance to demineralization and that saliva is supersaturated with these ions. During remineralization, fluoride is present on the surface of the demineralized enamel on the surface of the demineralized enamel crystals and attracts calcium and phosphate ions, thereby aiding remineralization of the crystals.
Following the application of topical fluorides, calcium fluoride-like globules are formed on the tooth surface. In addition, the surface coatings of phosphates on these calcium fluoride-like deposits have been found to reduce their solubility in saliva. The calcium fluoride-like globular deposit is believed to create a fluoride reservoir, with subsequent release of calcium, phosphate and fluoride.
A higher concentration of topical fluoride and a more prolonged application increase the amount of fluoride released as well as the deposition and availability of these globules (29, 30).
The amount of calcium fluoride-like deposit has also been found to be related to the availability of calcium and fluoride ions on the tooth surface.
Loosely bound fluoride is also known as KOH-soluble or alkali-soluble fluoride, and inhibits demineralization of the enamel crystals. The calcium fluoride-like globules and ionic fluoride available intraorally are loosely bound fluoride. The other category is firmly bound fluoride, which is also known as alkali-insoluble fluoride, KOH insoluble fluoride or apatitically bound fluoride, which is the fluoride, incorporated into the apatite crystals. Cruz et al. found this to be minimal in sound enamel during in vitro testing following brief exposures with topical fluorides (31).
Attin et al. found that demineralized samples treated with 5% sodium fluoride varnish in one study acquired both KOH-soluble and KOH-insoluble fluoride subsequent to fluoride application, at the fluoridated sites (32). Firmly bound fluoride also requires time for its acquisition, which first involves diffusion of available fluoride into the enamel.
In this study, it was necessary to polish the enamel blocks flat for reliable SMH testing. The SMH test does not appear to be overly influenced by the inherent porosity of the enamel blocks and is not as sensitive to operator technique. The SMH test has sufficient sensitivity to detect the early stages of enamel demineralization. Overall, the SMH test appears to be less susceptible to operator-related error, and is not overly influenced by the baseline porosity of the enamel blocks, thus eliminating the necessity for screening large numbers of enamel blocks to obtain a few acceptable blocks (33).
A 50-gram load was chosen because indentations made with this load have previously been shown to be a sensitive measure of mineral change in cross-sectional microhardness testing (34). Previous studies using the SMH test have generally used higher loads, such as 500 g, applied to the Vickers diamond to evaluate changes in mineral content. Since the focus of our study was on the incipient lesion, we chose a 50-gram load to evaluate SMH based on experience with 50-gram load in our previous study (35). The 50-gram load proved to be an acceptable means of following mineral loss. We consider the use of a higher load, such as the 500-gram load used by Koulourides et al. to be too high to accurately differentiate changes in hardness occurring during 12 pH cycling regime with daily 6-hour acid challenge in  our experiment (36).
The SMH test cannot be used to give accurate details of the hardness changes occurring below the enamel surface (34). SMH test gives a measure of the change in hardness occurring at multiple minisites at different locations on the enamel surface. The effectiveness of the topical applications of concentrated fluorides is dependent on the diffusion process and how much of the agent is loaded into the enamel during a given amount of time.
The fluoridated products assessed in the present study are the following, fluoride gels: Fluorogal, Fluor Protector Gel and Cervitec Gel and fluoride varnishes: Fluoridin Gel N5, Bifluorid 12 and Fluor Protector which are commercially available and commonly used in many countries. Topical application of the fluoride varnishes was done once a week to simulate professional application by a dentist. The increase of microhardness observed in the groups who received fluoride gels or fluoride varnishes indicates that both fluoride gels and varnishes were able to significantly inhibit the demineralization of enamel in vitro. These results are in accordance with those of previous studies (37, 38, 39) that investigated the effect of fluoride gels and fluoride varnishes on enamel demineralization.
Because fluoride varnishes had a greater concentration of fluoride and was left in contact with the teeth for a longer period of time, they had a better protective effect against demineralization, which is not statistically significant.
Fluoridin N5 was the most efficient recovering 40.49% of the enamel hardness (% SMH).The enamel pretreated with Bifluorid 12 every week during the pH cycling had a lower percentage SMH than the other groups.
The Tooth Mousse has been investigated in many studies. Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) involves the incorporation of the nano-complexes into dental plaque and onto the tooth surface, thereby acting as a calcium and phosphate reservoir. (37). Studies have shown that CPP-ACP incorporated into dental plaque can significantly increase the levels of plaque calcium and phosphate ions (40, 41). The therapy with dental crème Tooth Mousse directed at correcting demineralization-remineralization imbalances is based on the promise that calcium phosphate concentrations in plaque, plaque fluid and saliva play an important role in caries prevention. The lost mineral ions due to demineralization must be replaced with ions of the same shape, size and electrical charge. Since CPP-CP have been found to increase the levels of calcium and phosphate in plaque up to fivefold in human in situ caries models and short-term mouthwash studies (17, 41), the proposed mechanism of their anticariogenicity is that they act as a calcium-phosphate reservoir, buffering the activities of free calcium and phosphate ions in the plaque fluid helping to maintain a state of supersaturation with respect to enamel minerals, thereby depressing enamel demineralization and enhancing remineralisation (17).
Many authors have found that CPP-ACP can reduce the size of demineralized areas and promote the remineralization of enamel, but a combined application with fluoride strengthens the effect.  
In the study of Uysal et al., (37) mineral loss was assessed in vitro by cross-sectional microhardness, a recognized analytical method. In his in vivo and in vitro study the subjects were randomly divided into two equal groups and each subject received only one tested material (Aegis Ortho ®ACP-containing orthodontic composite or Concise ® resin-based composite) because the baseline clinical, radiological, salivary and laser fluorescence examinations showed that the patients were equal in regards to caries risk or demineralization activity. In this study, microhardness results show that teeth bonded with ACP-containing orthodontic composite have significantly less enamel mineral loss when compared with teeth bonded with conventional composite resin in a group of orthodontic adolescent patients.
The results of this study show that dental crème Tooth Mousse (10% CPP-ACP) was able to increase the surface hardness of enamel and remineralize subsurface lesions in human enamel in vitro. The use of Cervitec gel combined with Tooth Mousse in our study demonstrated 26.88% increase of SMH compared with the used of Tooth Mousse alone demonstrating 21.7% increase of SMH. Our findings are in agreement with the findings of Sudjalim et al. and Kargul et al. (42, 43).
According to Sudjalim et al., four-hourly application of topical medicaments corresponds to approximately twice-weekly applications. Enamel slabs from the control group in our study were immersed in demineralization/remineralization solution without exposure to any treatments with Tooth Mousse or fluoride products (42).
 It is likely that both fluoride gels and fluoride varnishes used within the current in vitro study could provide a substantial and prolonged increase in fluoride levels within saliva and dental plaque in the oral environment. With the fluoride varnishes used in this study, only periodic professional application at the dental office would be possible.
There are currently three calcium phosphate-based remineralization technologies that claim to promote remineralization: CPP-ACP; amorphous calcium phosphate (ACP); and synthetic minerals composed of calcium, sodium, phosphorus and silica that release a crystalline hydroxyl-carbonate apatite (HCA) layer that is chemically and structurally the same as tooth mineral.

 

 

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Conclusions

The results of the present study obtained under experimental conditions show that high fluoride gels, high fluoride varnishes, and Tooth Mousse alone and in combination with Cervitec gel effectively inhibit enamel demineralization. Clinical studies are required to check whether similar results can be obtained in the more complex oral environment. Further investigation should be carried out to develop strategies for using such products to prevent dental caries.

 

 

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Acknowledgement

This research was supported by Erasmus Mundus External Cooperation Window Project Basileus – Balkans Academic Scheme for the Internalization of Learning in cooperation with EU universities.

 

 

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