Biorepair Toothpaste Research Paper

Abstract

Objective

Dentine hypersensitivity is an increasing problem in dentistry. Several products are available that claim to occlude open dentine tubules and to reduce dentine hypersensitivity. The aim of this study was to investigate the effectiveness of several different products on dentine tubule occlusion using qualitative and quantitative methods.

Materials and methods

Dentine discs were prepared from extracted human premolars and molars. The dentine discs were brushed with 6 different experimental toothpastes, 1 positive control toothpaste and 1 negative control without toothpaste; the brushing simulated a total brushing time of 1 year. Half of the discs were etched with lemon juice after toothpaste application. Standardized scanning electron microphotographs were taken and converted into binary black and white images. The black pixels, which represented the open dentine tubules, were counted and statistically evaluated. Then, half of the dentine discs were broken, and the occlusion of the dentine tubules was investigated using energy dispersive X-ray spectroscopy (EDS).

Results

The number of open dentine tubules decreased significantly after brushing with 5 of the 6 tested toothpastes. A significant effect was observed after acid erosion for 3 of the 6 tested toothpastes. EDS revealed partly closed dentine tubules after brushing with 3 toothpastes; however, no partly closed dentine tubules were observed after acid erosion.

Conclusions

Some toothpastes are capable of partial dentine tubule occlusion. This occlusion is unstable and can be removed with acid erosion.

Clinical significance

Desensitizing toothpastes are the most common products that are used against dentine hypersensitivity, and these toothpastes affect dentine tubule occlusion.

Introduction

Increased life expectancy and growing consumption of acidic food, beverages and drugs increase the need for effective preventive measures to mitigate the effects of risky dietary habits on irreversible loss of hard tooth tissues due to their dissolution by acids without the involvement of bacteria, defined as tooth erosion. While educational efforts have been put forth to inform the public about tooth erosion, toothpastes and mouth rinses with various active ingredients and claimed anti-erosive properties have been recently developed. They contain fluorides in the traditional form of sodium fluoride (NaF) and amine fluoride (AmF) but also tin fluoride (SnF2) or chloride (SnCl2) (Young et al., 2006; Schlueter et al., 2009; Yu et al., 2010), as well as their combinations with biopolymers (Ganss et al., 2012; Carvalho and Lussi, 2014). Products with particles of crystalline (Roveri et al., 2009) or amorphous (Reynolds, 1997) nanoparticles of calcium phosphates (CaPs) represent different way to protect enamel against erosion. Because of a larger surface area they have increased affinity to the enamel surface (Hannig and Hannig, 2010) and provide higher concentrations of bioavailable calcium and phosphate ions for CaPs precipitation on enamel surface (Reynolds, 2008). Another group of products is based on formation of reactive amorphous CaPs after their mixing with saliva in the oral cavity. There are two commercially available technologies with different sources of calcium and phosphate ions: toothpastes with bioactive glass particles and toothpastes with a mixture of calcium and phosphate salts (Reynolds, 2008; Cochrane et al., 2010).

The anti-erosive properties of toothpastes are most often tested under in vitro conditions by immersion of specimens into the product slurry and evaluation of dental tissue loss profilometrically, by nano- or microhardness measurement, by autoradiographical methods, by micro-computed tomography and light, electron and AFM microscopy (Field et al., 2010; Hamba et al., 2011; Schlueter et al., 2011). The anti-erosive effect of toothpastes cannot be derived from their protective potential as assessed by immersion tests but rather from their ability to preserve protective properties upon toothbrush application when the treated enamel or dentin are exposed to abrasive components of the toothpaste under mechanical forces. As shown in previous studies (Hara et al., 2009; Ganss et al., 2011, 2013; Wiegand et al., 2014), this effect may act against anti-erosive properties of active ingredients of toothpastes. Due to differences in the testing methods and protocols, the results of published studies even on the same products are usually controversial.

When microscopic techniques are used for investigation of anti-erosive properties of oral hygiene products, it is suitable to characterise the same surface area before and after treatment. To identify this area, a small window is usually prepared on the tooth surface. Another possibility is to mark the area of interest with artificial defects such as well-defined indents. It is proposed that filling up the indents with precipitates and monitoring changes in their shape after toothpaste applications would make it possible to characterise the formation of protective deposits in greater detail. The purpose of this in vitro study is to investigate the ability of different toothpastes to form deposits on enamel and to test their resistance to acid challenge using light and scanning electron microscopy.

Materials and Methods

Toothpastes

The toothpastes with claimed anti-erosive properties were used in the study, Table I. The Sensodyne Pronamel toothpaste contains NaF, while a more complex combination of AmF and NaF with SnCl2 was represented by the Elmex Erosion Protection toothpaste. The group of CaP toothpastes included fluoride-free toothpastes BioRepair Plus Sensitivity Control with hydroxyapatite nanoparticles and SensiShield with calcium-sodium phosphosilicate particles. The Enamel Care toothpaste contained a mixture of calcium and phosphate salts in combination with fluoride. Two control groups were used: the negative group was exposed to artificial saliva while the positive control group received Elmex Erosion Protection mouth rinse with reported anti-erosive properties (Ganss et al., 2011).

Sensodyne pronamelAqua, sorbitol, hydrated silica, glycerin, 5 % KNO3, PEG-6, cocamidopropyl betaine, NaF, titanium dioxideGlaxoSmithKline, Brentford, UK20348KWC
 1450 ppm F  
Elmex erosion protectionAqua, glycerin, sorbitol, hydrated silica, cocamidopropyl betaine, titanium dioxide, Olaflur, sodium gluconate, SnCl2, alumina, chitosan, NaFGaba, Lörrach, Germany30451B
 1400 ppm F  
BioRepair plus sensitivity controlAqua, zinc hydroxyapatite, glycerin, sorbitol, PEG-32, silica, sodium myristoyl sarcosinate, sodium methyl cocoyl taurateCoswell Farma, Bologna, Italy109151002
 0 ppm F  
SensiShieldGlycerin, silica, PEG-400, Ca/Na phosphosilicate, sodium lauryl sulphatePeriproducts Ltd, Ruislip, UK08163
 0 ppm F  
Enamel care for sensitive teethSodium bicarbonate, PEG/PPG-38/8 copolymer, calcium sulphate, PEG/PPG-116/66 copolymer, silica, sodium lauryl sulphate, dipotassium phosphate, NaFChurch-Dwight Ltd, Folkestone, UKFE1109
 1100 ppm F  
Control groups
Artificial saliva13.7 mmol/L NaCl,Hospital laboratory
 16.1 mmol/L KCl, 0.68 mmol/L CaCl2.2H2O, 1.32 mmol/L K2HPO4.3H2O, 0.49 mmol/L MgCl2.6H2O, with pH= 7.0  
Elmex erosion mouth rinseAqua, glycerin, sodium gluconate, PEG-40, Olaflur (125 ppm F), SnCl2 (800 ppm), NaF (375 ppm F), cocamidopropyl betaineGaba, Lörrach, Germany0228CHG11A
 total concentration 500 ppm F  

Specimen Preparation and Experimental Procedure

The research has been conducted in full accordance with the ethical principles, including the World Medical Association Declaration of Helsinki and independently reviewed and approved by General Faculty Hospital IRB. Patients were informed about the use of their teeth for in vitro tests and written consent was obtained. Thirty five intact human third molars (five per group) extracted less than 6 months prior to the experiment were randomly selected from a pool of non-identifiable extracted teeth stored in a 0.5% chloramine T solution for one week and then in tap water at 5–8°C. The tooth roots were cut off using an Isomet low-speed saw (Buehler, Lake Bluff, USA), and the coronal parts were fixed with self-polymerising acrylic resin (Spofacryl, SpofaDental, Jicin, Czech Republic) in stainless steel rings with a diameter of 20 mm and 12 mm in height. Approximately 200–300 μm of the buccal or lingual sides was ground off with P1200 and P2500 SiC papers (Buehler, Lake Bluff, USA) under water cooling to prepare approximately 10 mm2 of flat enamel. The enamel surface was polished using a 6 μm diamond paste and 1 μm alumina suspension on Nylon and Micro Cloth II polishing cloth (Buehler, Lake Bluff, USA) with the aid of a MetaServ 250 grinder-polisher (Buehler, Lake Bluff, USA). The polished samples were rinsed with non-fluoridated tap water and cleaned in distilled water in a laboratory ultrasound bath (Whaledent BioSonic Jr., Mahwah, USA) 3 times for 2 min. A series of five rhomboid-shaped enamel indents of approximately 15–100 μm in length were made using a Knoop microhardness tester (IndentaMet 1600–1405D Buehler, Lake Bluff, USA) at 10–25–50–100–200 grams acting for 5 s on the indenter. The indents were documented using a Micrometrics 318CU camera (Micrometrics, Beijing, China) at 400-times magnification. Samples with a microhardness exceeding 300 KHN units were accepted for testing. A pea-size amount of toothpaste was applied on the polished enamel 2 min daily for a period of 30 days using a soft generously wetted SPOKAR Junior soft 3,431 toothbrush (Spokar, Pelhrimov, Czech Republic) by three different subjects rotating regularly after each product application. The load exerted on the samples during tooth brushing by each subject (PB 60–90, VF 30–60 and RV 50–80 grams) was measured using HF-200 g compact scales (A&D Instruments Ltd., Tokyo, Japan). Each tooth in the Elmex Erosion Protection mouth rinse control group was immersed in 10 mL of non-diluted rinse for 30 s as recommended by the manufacturer. After application, the teeth were rinsed with non-fluoridated tap water and placed into a plastic vessel with approximately 20 mL of artificial saliva with 0.5 mL of 0.5% chloramin T solution to prevent microbiological growth and were stored in a thermostat at 37°C until the next application. The saliva was replaced after each cycle. In the artificial saliva control group, the saliva was replaced daily without brushing. During applications the surfaces were checked regularly using light microscopy. After 15 applications a slice of approximately 2.0–3.0 mm2 was cut off from the treated area of each tooth using an Isomet low-speed saw equipped with a thin diamond blade of 0.2 mm in thickness under water cooling and the rest of the sample continued in the treatment. Each slice was cleaned twice for 1 min in distilled water in an ultrasound bath, rinsed with distilled water, air-dried and analysed using scanning electron microscopy (SEM). After 30 cycles, two more slices were prepared from each sample. One slice was analyzed using SEM, the other was demineralized in 5 mL of 0.2% citric acid solution prepared from citric acid monohydrate (Penta, Chrudim, Czech Republic) for 6 min under slow stirring at the ambient temperature and then investigated by SEM. The citric acid solution pH was adjusted to 3.30 with 1.0 N NaOH using an inoLab pH 730 pH-meter and combined glass electrode SenTix 21 (WTW, Weilheim, Germany) calibrated with WTW buffers at pH values of 4.0 and 7.0. These experimental conditions were suitable for protective effect differentiation as found in preliminary experiments. The scanning electron microscopy samples (JSM5500-LV, Jeol, Tokyo, Japan) were air-dried for 1 week, followed by 14 days at 37°C and then sputter-coated with gold (JFC-1200 Fine coater, Jeol, Japan) before analysis.

Results

Control Groups

Figure 1A shows an enamel surface with a series of indents and typical unidirectional scratches caused by abrasive particles of SiC papers during sample preparation. After exposition to artificial saliva, the indents remained sharp, Figure 1B, which indicates that neither erosion nor phosphates precipitation on the enamel surface had occurred in the artificial saliva used as a storage medium. Irregular deposits, shown in, Figure 1B, were most likely impurities or remnants of the bacteria settled on the surface during the tests. Exposure to citric acid resulted in a pronounced enamel demineralization as indicated by a distinct prismatic structure of the treated enamel, its porosity and disappearance of indents, Figure 1C. A completely different surface morphology was found when enamel was treated with the Elmex Erosion Protection mouth rinse, Figure 2. With this treatment, a deposit layer formation was observed on the enamel surface after 15 applications, Figure 2B. Unidirectional grinding scratches visible below the deposits suggested that only a thin layer of precipitated products covered the enamel surface. This layer, which was clearly visible after 30 applications on the periphery of a new indent, Figure 2C, seemed to protect enamel against demineralization environment used, Figure 2D.

Fluoride-Based Toothpaste

After 30 applications of Sensodyne Pronamel, the enamel surface was smooth, Figure 3B, with rounded indent edges, Figure 3C, suggesting low abrasivity of this toothpaste. Exposition to an erosive solution resulted in deep enamel demineralization indicated by its clearly visible prismatic structure, Figure 3D.

Tin-Containing Toothpaste

Numerous randomly oriented scratches and fractured indent edges indicated abrasive properties of the Elmex Erosion Protection toothpaste, Figure 4B–C. Although this toothpaste contained a rather high concentration of Sn and fluoride, the treated enamel was liable to demineralization, Figure 4D.

CaP Toothpastes

With the BioRepair Plus Sensitivity Control toothpaste, light microscopy did not detect any signs of deposits on the enamel surface even after 30 applications. SEM analysis at higher magnification, however, revealed deposition of irregular particle clusters on the treated enamel, Figure 5B. In an erosive solution, considerable enamel demineralization was observed, Figure 5C, where the distinct prismatic structure and porosity was reminiscent of the low acid resistance of the control group stored in artificial saliva. With the SensiShield toothpaste, irregular randomly oriented scratches on the enamel surface, fractured indent edges and an almost complete disappearance of the smallest indent even after 15 applications, as observed in Figure 6B, indicated its increased abrasivity. Distinct prismatic structure after demineralization, Figure 6C did not suggest protective effect of this toothpaste. On the other hand, with the Enamel Care, deposition of precipitates that completely filled up the indents and formed a compact layer on the enamel surface was clearly detected after a few applications. A control indent into this layer resulted in a “halo” formation around the indent visible in the light microscope, Figure 7B. SEM images showed a peeling off the layer from the enamel surface around an indent and layer fractures due to vacuum drying for SEM analysis, Figure 7C–D. The layer thickness reached micrometre dimensions, Figure 7D. After exposition to an erosive solution, neither a prismatic structure nor scratches after enamel grinding were found, Figure 7E.

Discussion

While numerous studies confirm a significant decline in the incidence and progression rate of tooth decay attributed to fluoride programmes and improved oral hygiene, a clear-cut prevention and treatment strategy for erosive damage of hard dental tissue is still lacking. Besides education, it is necessary to develop effective anti-erosive oral hygiene products. Currently, there are several technologies on the market with claimed anti-erosive properties and various erosion-inhibiting mechanisms. Conventional products with fluoride ions, which form fluoridated apatites in the enamel structure and accelerate precipitation of CaPs and CaF2-like material from supersaturated saliva (ten Cate and Featherstone, 1991; ten Cate, 1999), were represented in our study by Sensodyne Pronamel. The Elmex Erosion Protection toothpaste with SnCl2 and fluoride may create acid-resistant Sn-containing layers on the enamel surface (Babcock et al., 1978; Barbakow et al., 1985; Ellingsen, 1986) or Sn ions may be incorporated into hard dental tissues (Ganss et al., 2010). The CaP technology was used in BioRepair Plus Sensitivity Control with nanoparticles of Zn-carbonate hydroxyapatite (Roveri et al., 2009) and in SensiShield and Enamel Care toothpastes. The SensiShield toothpaste contains bioactive calcium-sodium phosphosilicate glass particles in which calcium and phosphate are enclosed in a silicate matrix of glass particles. The Enamel Care toothpaste contains a mixture of calcium sulphate and dipotassium phosphate. These active ingredients are dispersed in glycerol or polyglycols to maintain the stability of these toothpastes during storage. When mixed with saliva, the hydrolytically unstable glass disintegrates and releases calcium and phosphate ions (Hench, 1977). A simultaneous pH increase improves the conditions for precipitation of amorphous CaPs. Similarly, mixing Enamel Care with saliva results in dissolution of these salts and reaction between calcium and phosphate ions (Charig et al., 2009). The amorphous CaPs transform into crystalline, thermodynamically more stable crystalline CaPs on the enamel surface (Tung and Eichmiller, 2004; Burwell et al., 2009).

The choice of a testing protocol is a relevant issue in studying anti-erosion properties of oral hygiene products (Wiegand et al., 2011). Because of variability of the demineralization environment, exposition time, number of applications and demineralization cycles, application with or without a toothbrush, evaluation methods and other factors, contradictory results are frequently found within the literature. For that reason, simple experimental protocol was used in this study. It consisted of a 2 min toothbrush application of the product on polished enamel, without usual demineralization between the applications. It could be supposed that under these conditions, formation of protective deposits would not be disturbed by subsequent dissolution in the acidic environment and would thus provide clear evidence of their formation. Demineralization at the end of the tests would characterise resistance of the deposits to acid attacks. To eliminate a premature formation of CaPs during SensiShield and Enamel Care slurry preparation, all the toothpastes were applied in a non-diluted form with a generously wetted toothbrush.

The results showed pronounced differences in the ability of the test products to create protective deposits on enamel and, thus, to increase its resistance to acids. At the same time, they confirmed that abrasivity played an essential role in the anti-erosive properties of the toothpastes (Hara et al., 2009; Ganss et al., 2011, 2013; Hove et al., 2014). It was particularly observed with the Elmex Erosion Protection toothpaste when compared with Elmex Erosion Protection mouth rinse of similar active ingredients. Randomly oriented scratches and worn, fractured and broken-off edges of indents and clear demineralization of the treated enamel suggested that its abrasivity prevailed over ability of active ingredients to form deposit layers on the enamel surface. Due to the mechanical load induced by abrasive particles during tooth brushing, formation of acid-resistant deposits on the enamel surface, such as found with the mouth rinse, might be strongly inhibited. Abrasivity of the Elmex Erosion Protection toothpaste was surprising as studies investigating experimental toothpastes with similar active ingredients using profilometry and microhardness measurement demonstrated a significant tissue loss reduction in erosion/abrasion cycling models (Ganss et al., 2012; Carvalho and Lussi, 2014). In addition to other testing variables and differences in the composition of experimental and commercial products it might result from a longer brushing time (2 min) in our study, which enhances the toothpaste's abrasive effect, compared with a shorter brushing time (15 and 20 s) used in those studies. Another fluoride-based toothpaste, Sensodyne Pronamel, also failed in our experimental setup, but not because of its abrasivity. With this toothpaste, the enamel remained smooth and without any deposits. The enamel surface free of randomly oriented scratches after 30 applications and slightly rounded indent edges indicated its low abrasivity. Although SEM is a qualitative rather than a quantitative enamel demineralization assessment method, the typical prismatic structure of the treated enamel after citric acid challenge did not suggest pronounced anti-erosive properties of the toothpaste. As specified by the manufacturer, Sensodyne Pronamel contains substances quite commonly used in toothpaste formulations plus 5% KNO3 for tooth hypersensitivity reduction and provides lower abrasivity and higher concentrations of bioavailable fluoride ions, which should guarantee its higher anti-erosive potential. An independent study (Ganss et al., 2011), however, confirmed neither an increased level of free fluoride ions in the toothpaste slurry, nor enhanced anti-erosive properties compared to conventional toothpastes. It is not surprising as fluoride ions should protect the enamel surface at pH above 4.5 because of solubility of fluoridated apatites at lower pH. CaPs-based toothpastes represent an interesting group with favorable biological properties. The preceding generations of these products contained particles of amorphous or crystalline CaPs such as beta-tricalcium phosphates, sodium pyrophosphate, octacalcium phosphate or hydroxyapatites and others. Generally, they should form solutions supersaturated with calcium and phosphate ions to allow for their precipitation and formation of CaPs deposits on the enamel surface. Model calculations (Larsen and Pearce, 2003), however, show that at neutral pH, saliva is supersaturated with calcium and phosphate ions with respect to typical crystalline CaPs, which suggests that their ability to precipitate on enamel is limited. As a result, the protective effect of these products is questionable. On the other hand, technologies based on hydroxyapatite nanoparticles were reported to remineralize enamel by deposition of a new apatite mineral (Roveri et al., 2009). With BioRepair Plus Sensitivity Control, however, only isolated particle clusters most likely consisting of carbonated hydroxyapatite nanoparticles were found on the enamel surface. Due to their small size, absence of fluoride and presence of carbonates in their structure, these particles dissolved in the acidic environment, leaving the underlying enamel without effective protection against demineralization, agreeing with the findings of Ganss (Ganss et al., 2011). Abrasive properties were also important within this group of toothpastes. This was obvious with the SensiShield toothpaste containing calcium-sodium phosphosilicate particles. The products containing these bioactive glass particles are known for their ability to precipitate on dentin and close dentinal tubules and are recommended for tooth hypersensivity treatment (Gillam et al., 2002; Burwell et al., 2010). However, clear enamel abrasion features suggested that, as with Elmex Erosion Protection toothpaste, abrasion prevailed over its ability to form deposits with protective properties on the enamel surface. On the other hand, a very unique ability to form compact protective layers was observed with the Enamel Care toothpaste. This toothpaste is a one-component version of the Enamelon dual dispensing system, which was tested both in the laboratory and under in situ conditions, with varying results for caries inhibition and enamel remineralization summarized in another study (Reynolds, 2008). In our study, creation of a rather thick layer completely filling up the indents after a few applications indicated its high anti-erosive potential and, thus, ability to prevent erosive loss. However, as this layer consists of acid soluble CaPs it can provide temporary rather than long-term enamel protection.

Conclusion

Within the limitation of this in vitro study there are pronounced variations in the ability of the toothpastes to form anti-erosive deposits on the enamel surface. Besides low efficacy of active ingredients, these differences may result from too high abrasivity of some toothpaste components, which disturb settlement of deposits on the treated enamel surface.

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