It is important to evaluate the inhibitory effect on lesion progression of CPP-ACP when delivered in a mousse vehicle, commercially known as MI Paste, and compare it to actual remineralization products already established.
This in-vitro study evaluated the inhibition of demineralization in enamel sections produced by MI paste, fluoride and a combination of both, compared to artificial saliva and NaF 5000 ppm in a caries progression pH-cycling model. Twenty-one teeth were demineralized to create subsurface enamel lesions (approximately 200 microns in depth). The teeth were sectioned and characterized using polarized-light-microscopy (PLM). A single section from each lesion was assigned to a treatment group: Artificial saliva, NaF 5000 ppm (Prevident, Colgate), MI paste (Recaldent, GC America Inc), NaF 1100 ppm (Crest, Procter & Gamble) and NaF 1100 ppm plus MI paste. The sections were covered with varnish except for an exposed window on the external surface of the lesion and placed in a six-day pH-cycling model with two daily treatment applications of two minutes each. The sections were characterized by PLM, and the lesion areas were measured using a digital image analysis system. Based on a paired-sample t-test, significant differences (p<.05) in percentage of change in lesion size were found between the high fluoride group and all the other groups. No significant difference was found between the artificial saliva and MI paste group, neither was there any significant difference between the NaF 1100 ppm, the combined application group or the MI paste group alone. In conclusion, the higher concentration of NaF (5000 ppm) reduced lesion progression to the greatest extent. The MI paste group did not show any effect on the inhibition of lesion progression. Further studies on the preventive effect and longer treatment applications are recommended.
New methods for early caries diagnosis have been developed to detect lesions in the early stages before cavitation is produced and a restoration is needed. Different preventive therapies have been studied to enhance remineralization, decrease demineralization and, therefore, arrest the active carious lesions. Recently, studies have focused on the concentration of calcium and phosphate present in the tooth. Since both ions are major components of the tooth and are ultimately related to demineralization, most of the efforts have been directed toward their deposition or enhancement in the dental structure.
Amorphous calcium phosphate (ACP) provides the benefit of having both calcium and phosphate ions close to each other in an amorphous phase. They can enhance remineralization, decrease demineralization or provide a combination of both during an acid challenge to the tooth surface. ACP has been used for several years; but it has been challenging to avoid the formation of clusters, the precipitation of these ions in crystals and the formation of calculus and its inability to bind to the tooth surface.1–2
On the other hand, milk and milk-derived products have been shown to produce a beneficial effect in preventing and inhibiting the initiation of dental caries.3–4 Casein phosphopeptide (CPP), a milk protein, binds to calcium and phosphate, creating complexes of both ions as amorphous calcium phosphate. This helps to solve the major problem of ACP applications, since CPP does not allow the combination of calcium and phosphate into crystals and prevents the growth of these clusters from reaching the critical size for precipitation. CPP stabilizes calcium and phosphate solutions by forming CPP-ACP under alkaline and neutral conditions, creating a potentially supersaturated state compared with the basic calcium and phosphate phases. The beneficial effect obtained with CPP-ACP is associated with the ability to localize calcium and phosphate in dental plaque in the proximity of the tooth, thus making it available when needed. In the presence of an acid environment, such as after eating, when the pH of the mouth decreases, the casein phosphopeptide proteins release amorphous calcium and phosphate, creating a supersaturated state of calcium and phosphate around the tooth. The CPP-calcium phosphate complexes are anticariogenic and capable of remineralizing the early stages of enamel lesions.5
One possible application of CPP-ACP is as a home-care product, similar to the use of fluoride toothpaste. This method is commercially available as MI Paste (GC America Inc). There is not sufficient scientific evidence related to the remineralization effectiveness of this method of application. In contrast, over the years, high fluoride-containing products have demonstrated producing enhanced remineralization by forming fluorapatite crystals in the tooth.6–10 The main effect of fluoridated toothpaste is produced by elevating the levels of fluoride in the oral environment.6 Page found that five minutes of exposure to 100 and 1000 ppm fluoride delivery via a dentifrice significantly inhibited the demineralization of enamel.10 Higher concentrations of fluoride, such as 5000 ppm, have been shown to significantly reduce the risk and incidence of caries among high-risk populations.11 There is also a lack of data showing the effect of CPP-ACP when applied with fluoridated toothpaste. This is mostly due to the recent development of the CPP-ACP combination.
Therefore, it is necessary to evaluate this product's effect on the inhibition of lesion progression compared with other existing treatments before recommending its use to patients. This in vitro study evaluated the inhibitory effect produced by MI paste, fluoride and the combination of both compared to artificial saliva when used on artificially created enamel caries-like lesions.
The null hypothesis to be tested is that, in a pH cycling model system, remineralization treatments, such as fluoride, MI paste or a combination of both, do not result in a greater reduction in lesion progression compared to a placebo (artificial saliva) when applied to artificial subsurface enamel lesions.
METHODS AND MATERIALS
Twenty-one extracted human molars free of caries and enamel defects from The University of Iowa College of Dentistry were used in this in-vitro study. The teeth were sterilized by being immersed in Streck Tissue Fixative for two weeks and rinsed with copious amounts of water. After sterilization, the teeth were kept in 0.2% thymol solution prior to the study. The teeth had all surfaces covered with an acid resistant varnish (Revlon 730 Valentine, Revlon Consumer Products Corporation, New York, NY, USA) except for a facial window approximately 1 mm wide and 5 mm in length above the CEJ. The teeth were then suspended in a continuously stirred demineralization solution.12–13 This solution contained 2.2 mM CaCl2·2H2O, 2.2 mM KH2PO4, 0.05 M acetic acid and a pH adjusted to 4.4 with 10 M KOH. The molars were kept in this solution for five days until a uniform white spot lesion was created on the surface of the window.
The molars were longitudinally sectioned using a Silverstone/Taylor microtome (Sci Fab, Littleton, CO, USA) across the prepared window. The sections were approximately 140 to 160 microns thick. Five sections from each tooth were selected for the study. The criteria of acceptance were a lesion about 200 microns deep, no loss of surface and good integrity of the adjacent structure around the lesion.
The selected sections were wetted and photographed using a polarized light microscope to record initial lesion size. One section of each tooth was randomly assigned to each of the following groups (n=21) depending on the type of treatment: fluoride 5000 ppm paste (Prevident, Colgate-Palmolive Co, New York, NY, USA), fluoride 1100 ppm paste (Crest Cavity Protection, Procter & Gamble, Cincinnati, OH, USA), Recaldent MI Paste (GC America Inc, Alsip, IL, USA), fluoride 1100 ppm paste and MI paste, and artificial saliva. The treatment was applied in a slurry formed by one part treatment product and three parts deionized water.
The sections were covered with an acid-resistant varnish (Revlon 730 Valentine) using a brush under the microscope; all surfaces were covered except for a window the width of the artificial lesion on the natural outer surface of the section. The sections were then subjected to six days of pH-cycling at 20°C. The pH-cycling consisted of a two-minute treatment application, followed by a demineralizing solution for three hours, artificial saliva for two hours, demineralizing solution for three hours and a second treatment application for two minutes. The sections were stored overnight in artificial saliva. In-between all the steps of pH-cycling, each group was rinsed using distilled water.
The artificial saliva prepared for this study consisted of 20 mM NaHCO3, 3 mM NaH2PO4 and 1mM CaCl2. The demineralization and remineralization solutions were changed on the fourth day of pH-cycling, so that the concentration of calcium and phosphate ions in the solutions would not affect the results. The duration and design of the pH-cycling was calculated to increase the lesion in the group without treatment and double its initial size. The effect of the treatment groups was measured as an inhibition of demineralization. Two extra sections were included in the artificial saliva group to check on the progression of the lesion during the pH-cycling study. Once the pH-cycling was finished, the acid-resistant varnish was removed using acetone (99.6%) and a brush. The sections were kept in plastic containers with deionized water for less than 24 hours until they were photographed using polarized light microscopy.
The lesions were quantitatively measured using the images generated from the polarized light microscope (Olympus BX50, Olympus America, Center Valley, PA, USA). The images were captured using a 4× objective lens and a 10× eyepiece for magnification. The area in microns (μm2) of the initial and final size of each lesion was carefully measured and recorded with an image analysis system (Image-Pro Plus). The area measurements were conducted by drawing a line following the external contour of the lesion. Two perpendicular lines to this line were drawn on the margins of the lesion. A final line was drawn following the margin of the body of the lesion.
The difference in area between the initial and final lesion was calculated using the percentage of change in the lesion area. This was calculated by subtracting the initial area from the final area and dividing the result by the initial area of the lesion to obtain the percent of change. One sample of each tooth was placed in each of the treatment groups, then compared as paired measurements. A paired-samples t-test was conducted to determine differences among the five treatment groups. SAS for Windows (v9.1, SAS Institute Inc, Cary, NC, USA) was used for the data analysis, and the level of significance was set at α=0.05.
From the initial 105 sections used in this study, four were rejected after pH-cycling, due to extensive loss of the external surface of the lesion and it being too difficult to measure.
Of the remaining sections (Table 1), the group treated with Fluoride 5000 ppm (Figure 1) showed the least percentage change in lesion size (26.8 ± 12.6), being statistically significantly different (p<0.05) from all the other treatment groups. A definitive reduction in progression of lesion size was observed in relation to the fluoride dose, as the Fluoride 1100 ppm group had a change in lesion size of 37.1 ± 7.8% and the control group without any fluoride treatment had a change in lesion size of 51.2 ± 24.4%.
When comparing the experimental groups of Fluoride 1100 ppm, the combined MI paste and Fluoride treatment group and the MI paste group, no statistically significant difference was found among them (p≤0.14). On the other hand, the combined treatment group and the Fluoride 1100 ppm group, both containing fluoride, were statistically different from the artificial saliva group (p≤0.013). Meanwhile, the percentage change of lesion size of the MI paste group was not statistically different from the artificial saliva group (p=0.62).
The greatest increase in lesion size after pH-cycling was obtained by the control group or artificial saliva (51.2 ± 24.4) followed by the MI paste treatment group (49.1 ± 24.4). Although not statistically significantly different, the combined treatment group (35.6 ± 14.9) had a minor increase in lesion size when compared to the Fluoride 1100 ppm alone (37.1 ± 17.8).
Currently, fluoride is the most effective preventive treatment to remineralize early stages of carious lesions. Products containing low fluoride concentrations, such as fluoride toothpastes and mouth rinses, are recommended to patients for home care use. MI Paste is recommended by its manufacturer as a home-care product that claims to remineralize early enamel demineralization in a non-cavitated situation. Therefore, it was interesting to compare the effect of MI Paste against fluoride in a fluoride dose-related model system. One of the in-vitro models used to study the effects of fluoride in reducing caries progression has been a pH-cycling model, simulating the conditions in the oral cavity. Sections were used to reduce the variability between teeth and their effect on the mean percentage of change of the groups, although the variability between areas of the same tooth is still present. Also, the use of sections as samples in this study provided an accurate measurement of pre-/post-lesion size. The two fluoride groups tested and the artificial saliva (non-containing fluoride) group had the expected dose-related protective effect against caries progression.8,10,14 This difference and the dose-related response observed from the results confirm the accuracy of the use of this in-vitro model. On the other hand, the lesion-progression model used in this study may have been too fast. This is shown in the high percentage of sections that had some kind of erosion present on the external surface of the lesion at the end of treatment. This erosion could have hidden a mild effect on the inhibition of the demineralization achieved with any of the test products.
One possible reason for the results obtained with MI Paste may be due to the short treatment application time. All the treatment regimens were applied for two minutes. Also, the surface of the sections was rinsed, and all remnants of the products were removed immediately after the treatment application. It might be necessary to have a longer application time to be able to detect some deposition of calcium and phosphate in a remineralized lesion.
Another aspect to evaluate when considering the duration of treatment application is the clearance time from the oral cavity. Duckworth and Morgan15 showed that fluoride treatments had a rapid clearance from saliva; they state that, after 30 to 60 seconds, the major fraction of fluoride content is lost from the mouth when excess saliva is spit out of the mouth. This procedure is also recommended by the manufacturers of MI Paste, so that it can be assumed that a similar reduction in CPP-ACP content in saliva could be expected, therefore, not being available to interact with the bacteria and tooth surface. When comparing this rapid clearance with remineralizing CPP-ACP studies, the delivery vehicle employed was either lozenges or gum, thus it was not necessary to spit out of the excess, and longer times of oral exposure and possibly retention might have occurred. In addition, it is important that these delivery vehicles also increased salivary flow, and its remineralizing effect was not differentiated from the one produced by the CPP-ACP active ingredient.1–2,16
Several studies have shown remineralization of initial enamel lesions by using CPP-ACP in lozenges, gums and mouth rinses.1–2,16–17 These studies used CPP-ACP treatment applications at different concentrations and different frequencies. The results obtained in the current study might be different, because the treatment product was MI Paste with an already diluted CPP-ACP concentration at 10% and numerous other ingredients.
Reynolds and Walsh18 suggested that CPP-ACP molecules need an acid challenge to be activated and it should separate ACP from the casein. In the current study, the pH-cycling design was performed to simulate the frequency of tooth brushing and acid challenges. Therefore, immediately after the first treatment application, the sections were immersed in a demineralization solution following the suggestions by Reynolds and Walsh.18 On the other hand, Shen and others1 designed their study to remove the appliances from the mouth after treatment application using a sugar-free gum, not allowing time for an acid challenge. These authors were able to show some remineralizing effect of the CPP-ACP product on enamel subsurface lesions as measured by microradiography.
The results of the current study did not show any effect of MI Paste on reducing progression of the lesion. These results might be different due to shorter treatment applications and immediate acid challenge. Therefore, CPP-ACP may have been incorporated into the lesion but not activated when it was necessary or even washed away in the demineralizing solution. This may be due to a different time between the release of ACP from CPP during the acid challenge and the timing of a gradient necessary to deposit calcium and phosphate into the lesion during remineralization.
It is also interesting to consider that one of the beneficial effects of fluoride during an acid challenge is to be able to dissolve from the tooth surface into the solution and slow the demineralization process.8 An increased buffer capacity is also claimed by using CPP-ACP products18 but it has not been shown. This buffer effect of CPP-ACP may be related to an inhibition of demineralization, having more effect as a preventive treatment against caries lesions than remineralizing already formed subsurface enamel lesions. Hicks and Flaitz19 demonstated a protective effect against demineralization when MI paste was applied before an acid challenge. However, the application of MI paste on the sections was performed for 14 days without any acid challenge during that time. Yamaguchi and others20 found a protective effect against demineralization after an application of 10-minute treatments with CPP-ACP. Haderlie and others21 also found a protective effect against secondary caries along the enamel margins in amalgam and composite restorations when MI paste was applied in conjunction with fluoride, although a high fluoride concentration provided the highest protection, as occurred in the current study.
One of the reservoirs of CPP-ACP in the mouth may be plaque and bacteria. Reynolds and others2 used a mouth rinse as a delivery vehicle to analyze the retention of CPP-ACP in plaque. These authors were able to detect CPP levels using an anti-casein antibody to locate CPP on the bacteria surface and in the intercellular matrix. The current in-vitro study might not have shown the same remineralization effect as previous studies, because there was no plaque involved. The design of this study measured the demineralization inhibition effect of CPP-ACP that binds to the tooth surface. It can be suggested that the major part of CPP-ACP binds to bacteria instead of the tooth surface, but, in that case, the application time should be prior to tooth brushing. Therefore, it is necessary to evaluate how long before brushing and how the acid produced by bacteria metabolism might affect tissue demineralization and the remineralization produced by CPP-ACP.
The results obtained from this study showed no remineralizing effect of MI paste on early lesions in enamel, although slightly less demineralization was observed on MI paste when compared with artificial saliva. On the other hand, high fluoride concentration demonstrated having the most protective effect against demineralization; therefore, it should be still considered the most important method against caries progression. More research using MI paste with different approaches as preventive treatment, such as longer treatment time or using a model simulating normal oral conditions with natural saliva and plaque as an in situ model or with a clinical study, need to be performed to provide a scientifically supported clinical recommendation for its use.
The authors greatly appreciate the technical help provided by J Harless and M Hogan.
None of the authors had any conflict of interest in the current study. No funding was provided by any manufacturer. The material employed in this study was donated by the company.
Maria Teresa Pulido, DDS, MS, assistant professor, Department of Restorative and Prosthetic Dentistry, College of Dentistry, The Ohio State University, Columbus, OH, USA
James S Wefel, PhD, professor, Department of Pediatric Dentistry, College of Dentistry, The University of Iowa, Iowa City, IA, USA
Maria Marcela Hernandez, DDS, MS, assistant professor, Department of Operative Dentistry, College of Dentistry, The University of Iowa, Iowa City, IA, USA
Gerald E Denehy, DDS, MS, professor, Department of Operative Dentistry, College of Dentistry, The University of Iowa, Iowa City, IA, USA
Sandra Guzman-Armstrong, DDS, MS, assistant professor, Department of Operative Dentistry, College of Dentistry, The University of Iowa, Iowa City, IA, USA
Jane M Chalmers, BDSc, MS, PhD, associate professor, Department of Preventive and Community Dentistry, College of Dentistry, The University of Iowa, Iowa City, IA, USA
Fang Qian, MA, PhD, associate research scientist, Department of Preventive and Community Dentistry, College of Dentistry, The University of Iowa, Iowa City, IA, USA