JJOD-2220; No. of Pages 9 journal of dentistry xxx (2014) xxx–xxx
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Remineralizing efficacy of different calcium-phosphate and fluoride based delivery vehicles on artificial caries like enamel lesions Dina Elkassas a, Abla Arafa b,* a
Department of Operative Dentistry, Faculty of Oral and Dental Medicine, Misr International University, Egypt Department of Pediatric Dentistry and Dental Public Health, Faculty of Oral and Dental Medicine, Misr International University, Egypt b
article info
abstract
Article history:
Objectives: To assess enamel remineralization of different calcium-phosphate and fluoride
Received 9 October 2013
delivery systems.
Received in revised form
Methods: Artificial caries lesions were created on 115 extracted human molars. Specimens
30 December 2013
were assigned according to remineralizing agent into five groups: G1: Control (artificial
Accepted 31 December 2013
saliva), G2: ClinproTM white varnish, G3: Relief, G4: Tooth Mousse Plus, G5: VanishTMXT.
Available online xxx
Surface micro-hardness (SMH), surface roughness (Ra) and surface topography by scanning electron microscope (SEM) were evaluated at baseline, after demineralization, after 2 and 4
Keywords:
weeks remineralization and after acid challenge.
Acid challenge
Results: Demineralized enamel showed the lowest SMH. By 2 weeks remineralization, SMH
Calcium-phosphate-based systems
were ranked as follows: G2 (282.14 6.82) > G3 (269.37 7.25) > G5 (263.00 6.49) = G4
Microhardness
(251.83 8.26) > G1 (226.5 9.34). However, 4 weeks remineralization showed the following:
Remineralization
G2 (304.09 6.65) > G3 (293.1 5.24) = G4 (285 7.29) > G5 (272.43 4.89) > G1 (233.33
Scanning electron microscopy
9.12). By exposure to acid challenge, groups presented order of: G2 (279.71 5.99) = G3
Surface roughness
(275.51 5.59) > G4 (262.29 6.65) > G5 (245.43 6.43) > G1 (190.27 8). Surface roughness showed the following mattress after 2 weeks remineralization: G1 (0.2488 0.0016) = G2 (0.2487 0.0007) = G3 (0.2476 0.0006) > G4 (0.2442 0.0004) > G5 (0.2396 0.0009). After 4 weeks remineralization: G1 (0.2469 0.0017) > G4 (0.244 0.0004) > G5 (0.2413 0.0008) = G3 (0.2405 0.0007) = G2 (0.2399 0.0006). After acid challenge; G1 (0.2582 0.0027) > G5 (0.2556 0.0007) > G4 (0.2484 0.0009) > G3 (0.2463 0.0007) > G2 (0.2443 0.0004). SEM revealed mineralized coating on the surfaces which resists dissolution by acid challenge at variable degrees according to remineralization regimen applied. Conclusions: Remineralizing agents containing different calcium-phosphate formulas and fluoride have increased remineralization potential compared to artificial saliva. ClinproTM varnish presented the highest remineralization tendency with greatest resistance for acid challenge. Clinical significance: This in vitro study imitated the application of different calcium phosphorous and fluoride based delivery vehicles to enamel tooth surfaces in the mouth. The new therapeutic techniques based on different calcium phosphate formulas containing fluoride provide a new avenue for remineralization of non-cavitated and early carious lesions. # 2014 Elsevier Ltd. All rights reserved.
* Corresponding author at: Department of Pediatric Dentistry, Faculty of Oral and Dental Medicine, Misr International University, 28 CairoEl Ismailia Road, Cairo, Egypt. Tel.: +20 2 22594539; fax: +20 2 23635413; mobile: +20 1100670307. E-mail address: [email protected] (A. Arafa). 0300-5712/$ – see front matter # 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jdent.2013.12.017 Please cite this article in press as: Elkassas D, Arafa A, Remineralizing efficacy of different calcium-phosphate and fluoride based delivery vehicles on artificial caries like enamel lesions. Journal of Dentistry (2014), http://dx.doi.org/10.1016/j.jdent.2013.12.017
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1.
Introduction
Dental caries is still one of the most prevalent diseases affecting humans. A goal of modern dentistry is to manage non-cavitated carious lesions non-invasively in an attempt to prevent further disease progression and preserve integrity of healthy tooth substrate. Non-invasive management of noncavitated carious lesions comprises several strategies; among these is healing the demineralized lesions using remineralizing agent or sealing them using resinous based materials.1,2 Remineralization of hard dental tissues is defined as the process whereby calcium and phosphate ions are supplied from a source external to the tooth to promote ion deposition into crystal voids in demineralized enamel to produce net mineral gain. Fluoride is considered the cornerstone in the remineralization process, yet its ability to promote net remineralization is limited by the availability of calcium and phosphate ions.3–5 Recently, a range of novel calcium phosphate based delivery systems were introduced into the dental market. The manufacturers claim that these products provide new avenues for the remineralization of noncavitated caries lesions.6 They can be categorized into one of the three types-crystalline, unstabilized amorphous, or stabilized amorphous formulations. Crystalline systems as tricalcium phosphate (TCP) showed potential to deliver calcium and phosphorous to enamel lesions.6 In those systems, tricalcium phosphate is functionalized ( fTCP) by being ball milled with sodium lauryl sulphate. Unstabilized amorphous calcium phosphate (ACP) remineralizing system provides the benefit of having both calcium and phosphate ions close to each other in an amorphous phase. The ACP technology requires a two-phase delivery system to keep the calcium and phosphorus components from reacting with each other before use. Some studies reported that this system can enhance remineralization, decrease demineralization or provide a combination of both during an acid challenge to the tooth surface.7,8 In stabilized amorphous formulations, casein phosphopeptide (CPP), have a remarkable ability to stabilize calcium phosphate (ACP) in metastable stage. Through the multiple phosphoseryl residues, the CPP binds to form nanoclusters of ACP, preventing their growth to the critical size required for nucleation and phase transformation.6,9,10 The proposed mechanism for the CPP-ACP is to localize ACP in dental plaque, which buffers the free calcium and phosphate ion activities, thereby helping to maintain a stage of supersaturation with respect to tooth enamel depressing demineralization and enhancing remineralization.8 Several authors reported the remineralizing potential of stabilized casein phosphopeptide amorphous calcium phosphate (CPPACP).11,12 Owing to the overwhelming role of fluoride in the remineralization process, it was added to most of calciumphosphate delivery products, in different forms, and concentrations. The remineralization efficacy of fluoride containing materials is related to fluoride content, fluoride matrices, setting mechanisms and other materials components.13 For the sealing strategy, the resin modified glass ionomer varnish is launched into the market. It is claimed by the
manufacturer that this material provides unique ability of healing and sealing incipient demineralized lesions. The sealing ability arises from the penetrative capability of the resinous component incorporated in the material and chemical bonding activity of the glass ionomer component, while its healing ability emerges from long term release of calcium, phosphorous and fluoride ions.14,15 Hitherto, no study compared the impact of strategies of delivery of calciumphosphate and fluoride ions on the remineralizing potential of incipient carious lesions. Furthermore, the stability of the built up mineral product following acid challenge were scantly studied in the literature. Thus, the aim of this study is to assess the effect of different calcium-phosphorous and fluoride delivery systems on enamel remineralization using surface micro-hardness, scanning electron microscopy and surface roughness methods. The null hypotheses tested; first: Functionalized Tricalcium phosphate (ClinproTM white varnish, fTCP), Amorphous calcium phosphate (Relief, ACP), Casein phosphopeptide amorphous calcium phosphate (Tooth Mousse Plus, CPP-ACPF), and Resin modified glass ionomer (VanishTMXT, RMGI), based remineralizing agents, would have no additional benefits for enhancement of enamel remineralization compared to artificial saliva. Second, there is no difference in the remineralizing efficacy of the aforementioned compounds.
2.
Materials and methods
2.1.
Materials
Four remineralizing agents were evaluated in this study: (a) ClinproTM white varnish (3 M ESPE, USA) as functionalized tricalcium phosphate containing 22,600 ppm fluoride ( fTCP), (b) Relief (Discus Dental, USA) as amorphous calcium phosphate containing 1100 ppm fluoride (ACPF), (c) Tooth Mousse Plus (GC Dental, Japan) as casein phosphopeptide amorphous calcium phosphate containing 900 ppm fluoride (CPP-ACPF) and (d) VanishTMXT (3M ESPE, USA) as extended contact varnish based on resin modified glass ionomer technology (RMGI).
2.2.
Specimens preparation
Total of 115 extracted human mandibular molars free from clinically visible abnormality were stored in an aqueous solution of saturated thymol for 2 weeks after removal of the roots. The teeth were thoroughly rinsed and examined under the stereomicroscope (Nikon, Japan), any tooth with defects, erosions, or micro-cracks on their enamel surfaces or visible stains was excluded. The coronal part of each tooth was longitudinally sectioned in mesio-distal direction using diamond disk (Komet, Rock Hill, USA) in low speed under water cooling. Custom-made plastic cylindrical molds were prepared and self-cured acrylic resin (Acrostone Dental Factor, England) was poured on them. Each buccal section was mounted horizontally in acrylic resin and cured overnight, and then polished flat using 800, 1200 and 2400 grit silicon carbide paper. An acid resistant nail varnish was applied around the exposed enamel surface, leaving four equal overtures of approximately (2 mm 2 mm) each.
Please cite this article in press as: Elkassas D, Arafa A, Remineralizing efficacy of different calcium-phosphate and fluoride based delivery vehicles on artificial caries like enamel lesions. Journal of Dentistry (2014), http://dx.doi.org/10.1016/j.jdent.2013.12.017
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2.3.
Demineralization
Following the proposal suggested by Pulido et al.16 artificial caries like lesions were created by individually immersing acrylic-mounted enamel specimens in continuously stirred, daily renewed demineralization solution. This solution contained 2.2 mM CaCl22H2O, 2.2 mM KH2PO4, 0.05 M acetic acid and pH adjusted to 4.4 with 10 M KOH. The molars were kept in this solution for 5 days until a uniform white spot lesion was created on the surface of the window. Enamel specimens were washed carefully and then the first window was coated with acid resistant nail varnish to act as control for demineralization.
2.4.
Remineralization
Artificial saliva was prepared according to the formulation of Torres et al.17 and consisted of 22.1 mM hydrogen carbonate, 16.1 mM potassium, 14.5 mM sodium, 2.6 mM hydrogen phosphate, 0.8 mM boric acid, 0.7 mM calcium, 0.2 mM thiocyanate, and 0.2 mM magnesium. The pH was between 7.4 and 7.8. Specimens were randomly divided into five groups (n = 23/ gp), according to the treatment employed:
acid resistance of the treated surfaces. The nail varnish was peeled off from all of the specimens carefully. The 23 specimens of each group were divided into three groups and tested by the following methods: assessment of the surface micro-hardness (n = 10), scanning electron microscopy examination (n = 3) and assessment of the surface roughness (n = 10).
2.5.
For each specimen, a control window was painted with nail varnish after demineralization. The second window was covered with nail varnish after 2 weeks remineralization, while the third window was covered following 4-week remineralization. Prior to covering the second and third windows, the remineralizing agents were removed. The specimens of G3 and G4, that received Relief and Tooth Mousse Plus agents, the surfaces were cleaned using cotton tip immersed in deionized water.18 In G2 and G5, that received Clinpro white varnish and Vanish extended contact varnish, the varnishes were carefully removed using surgical blade followed by cleaning off the surface with cotton tip immersed in deionized water.19 No chemical substance was used to clean the surfaces in order not to alter the enamel surface.18 After these periods, the samples were immersed again in the previously described demineralizing solution to evaluate the
Assessment of Surface Micro-hardness (SMH)
Enamel surface micro-hardness was measured at baseline of sound untreated enamel, after surface softening, after 2 weeks remineralization, after 4 weeks remineralization and after being subjected to acid challenge. The surface micro-hardness was measured using micro-hardness tester with Vickers diamond indenter in different regions of the specimens (Vickers diamond, 100 g, 5 s, HMV 2; Shimadzu Corporation, Tokyo, Japan). All readings were performed by the same examiner using the same calibrated machine. In each reading, four indentations were made and their average was taken to represent the specimen’s hardness value.
2.6. G1 (Control group): No treatment was given to the enamel surface and specimens were kept in artificial saliva which renewed every day. G2 ( fTCP group): Single application of ClinproTM white varnish was performed, specimens rinsed and dried for 10 s, and then material coating was applied to cover specimen surface and left for 20 s, then stored in artificial saliva. G3 (ACPF group): Relief coating was applied twice/day, specimens were rinsed and dried for 10 s, then thin layer of remineralizing agent was applied to cover specimen surface and left for 3 min, then wiped gently with gauze, washed and stored in artificial saliva. G4 (CPP-ACPF group): Tooth Mousse Plus was applied twice/ day as G3. G5 (RMGI group): Single application of VanishTMXT was performed, specimens rinsed and dried for 10 s, then material coating was applied to cover specimen surface and light cured for 20 s (EliparTM S10 LED Curing Light, 1200 mW/cm2, 3 M ESPE), then stored in artificial saliva.
3
Scanning electron microscope examination (SEM)
For the SEM examination, three specimens in each group were treated and then air dried. Each specimen was mounted on SEM stud and then sputter coated under vacuum with gold by the Ladd sputter coater (BAL-TEC, SCD 005, Germany). They were examined under Scanning Electron Microscope (Philips, Holland). SEM photomicrographs were captured at 1000 magnification.
2.7.
Assessment of Surface Roughness (Ra)
The optical methods tend to fulfil the need for quantitative characterization of surface topography without contact. Specimens were photographed using USB Digital microscope with a built-in camera (Scope Capture Digital Microscope, Guangdong, China) connected with an IBM compatible computer using fixed magnification of 50. The images were recorded with a resolution of 1280 1024 pixels per image and then cropped to 350 400 pixels using Microsoft office picture manager to specify and standardize area of roughness measurement. Cropped images were analyzed using WSxM software (Version 5 develop 4.1, Nanotec, Electronica, SL). Within the WSxM software, all limits, sizes, frames and measured parameters are expressed in pixels. Therefore, system calibration was done to convert the pixels into absolute real world units. Calibration was made by comparing an object of known size (a ruler in this study) with a scale generated by the software. Subsequently, a 3D image of the surface profile of the specimens was created. Three 3D images were collected per each window for each specimen of an area 10 mm 10 mm. WSxM software was used to calculate average surface roughness (Ra) expressed in mm.20
2.8.
Statistical analyses
The mean (SD) SMH values and the mean (SD) Ra values were statistically analyzed. Repeated measure ANOVA was
Please cite this article in press as: Elkassas D, Arafa A, Remineralizing efficacy of different calcium-phosphate and fluoride based delivery vehicles on artificial caries like enamel lesions. Journal of Dentistry (2014), http://dx.doi.org/10.1016/j.jdent.2013.12.017
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performed to compare remineralizing potential of each product at different time interval, 2 and 4 weeks remineralization as well as exposure to acid challenge, followed by multiple comparisons Tukey’s post hoc test. Comparison between different products at different time interval to monitor the remineralizing efficacy was carried out using one-way ANOVA followed by multiple comparisons Tukey’s post hoc test. The significance level was set at 0.05. All data were processed by the SPSS (version 16.0) software package (SPSS Inc., Chicago, IL, USA).
3.
Results
3.1.
Enamel surface micro-hardness
hidden by mineral deposition. ACPF specimens showed evidence of thickening of the inter-rod substance early by 2week remineralization; however, extended remineralization period enhanced the growth of enamel crystals to cover over fish scale appearance of demineralized surface. In fTCP group, after 4 weeks remineralization, some of enamel crystals were even fused together and arranged homogenously with no obvious inter-crystalline spaces on the surface. After exposure to acid challenge, the experimental groups showed more resistance to dissolution as compared to the control. The treated surfaces retained calcified deposits but with the presence of some potholes on the surface.
3.3.
The mean (SD) SMH values of surfaces at different enamel treatment points are shown in Table 1. The groups presented significantly the highest SMH at the baseline ( p < 0.05). The demineralized enamel showed significant reduction in SMH values ( p < 0.05). The SMH values of all treated groups demonstrated significant increase after 2 and 4 weeks of application of remineralization regimen ( p < 0.05). All of the groups showed significant reduction in SMH values indicating mineral loss as exposed to acid challenge ( p < 0.05). SMH values in the fTCP group were significantly the highest, early after 2 weeks remineralization and even after 4 weeks of remineralization ( p < 0.05). In addition, after 4 weeks remineralization, there were no significant differences between the ACPF and CPP-ACPF groups ( p > 0.05). After exposure to acid challenge, the fTCP and ACPF groups showed statistically significant higher SMH mean values compared to CPP-ACPF and RMGI groups ( p < 0.05). However, all groups presented statistically significant higher resistance to softening than the control ( p < 0.05).
3.2.
SEM morphological characters
Sound enamel has homogeneous smooth appearance. However, demineralized enamel showed increased porosity with fish-scale pattern as depicted in Fig. 1. The test groups showed increased density of crystals after 2 weeks remineralization. While after 4 weeks remineralization, enamel surfaces revealed new coating progressively filled the pits and scratches where the prismatic enamel structures became
Enamel surface roughness
The mean (SD) Ra values of surfaces at different enamel treatment points are presented in Table 2. At the baseline measure, the mean Ra value of specimens was within the range of 0.2361–0.2389 mm. After demineralization, significant increase in Ra values was evident in all tested groups ( p < 0.05). After 2 weeks remineralization, all experimental groups showed significant reduction in Ra values ( p < 0.05). After 4 weeks remineralization, further significant reduction in the Ra values was evident in the fTCP and ACPF groups; however, no significant difference in Ra values was evident in control, CPPACPF and RMGI groups. Interestingly, RMGI group showed increase in Ra values by 4 weeks remineralization ( p > 0.05). Following the exposure to acid challenge, all tested groups showed significant increase in Ra values when compared to 4 weeks remineralization ( p < 0.05). Comparing between the remineralizing agents examined at different intervals, it was evident that RMGI group showed significantly lowest Ra values after 2 weeks ( p < 0.05). Meanwhile, the fTCP group showed significantly the lowest Ra values after 4 weeks and after exposure to acid challenge ( p < 0.05). The differences in surface roughening mediated of fTCP group among different time points are apparent when inspecting the 3D topographical images as illustrated in Fig. 2.
4.
Discussion
The present study was carried out to compare the remineralizing efficacy of five regimens on artificial caries like enamel lesions, in terms of change in surface micro-hardness,
Table 1 – Analysis of surface microhardness (SMH-VHN) at baseline (B), after demineralization (D), after 2 weeks and 4 weeks remineralization (WR) and after acid challenge (AC) of the groups (n = 10/gp). Group
G1 G2 G3 G4 G5
(control) ( fTCP) (ACPF) (CPP-ACPF) (RMGI)
Enamel treatment SMH-B
SMH-D
SMH-2WR
SMH-4WR
SMH-AC
304.17 10.89a,A 316.43 10.94a,A 317.31 6.22a,A 314.57 9.86a,A 310.29 8.59a,A
216.00 9.29c,A 225.17 10.24c,A 225.20 7.11d,A 212.46 6.82e,A 219.36 11.27d,A
226.50 9.34b,c,D 282.14 6.82b,A 269.37 7.25c,B 251.83 8.26d,C 263.00 6.49b,B,C
233.33 9.12b,D 304.09 6.65a,A 293.10 5.24b,B 285.00 7.29b,B 272.43 4.89b,C
190.27 8.00d,D 279.71 5.99b,A 275.51 5.59c,A 262.29 6.65c,B 245.43 6.43c,C
Different upper- and lower-case superscript letters indicate significant difference between tested groups at p < 0.05. Lower case superscript letters are used for comparison within the same row and upper case letters are used for comparison within each column.
Please cite this article in press as: Elkassas D, Arafa A, Remineralizing efficacy of different calcium-phosphate and fluoride based delivery vehicles on artificial caries like enamel lesions. Journal of Dentistry (2014), http://dx.doi.org/10.1016/j.jdent.2013.12.017
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Fig. 1 – SEM images of (B) sound enamel surface at baseline, (D) demineralized enamel surface, and the Control, fTCP, ACPF, CPP-ACPF and RMGI groups at (2 W) two weeks remineralization, (4 W) 4 weeks remineralization, and (AC) after acid challenge (1000T).
ultrastructure and surface roughness. The specimens were polished for two motifs: first: to remove the prismless enamel layer that arises at the end of amelogenesis. Although this layer is not frequently found on the surface of permanent teeth compared to their predecessors, it is known that the prism-free enamel is gradually worn off during mastication but it is retained in protected areas. Flat and polished specimens were used in the present study in an attempt to standardize specimens and remove natural variations on surface enamel between teeth and between different tooth
sites and types, which may result in different responses to acid dissolution.21 Second: Micro-hardness assessment needs flat polished surface to enable accurate measurements.22 Thus, the area subjected for demineralization did not represent the ideal enamel surface, as removal of the outer surface layer made the enamel more susceptible for demineralization.18,23 Micro-hardness testing is considered to be a relatively simple and reasonably reliable method for the provision of indirect information about the mineral content changes of hard dental tissues. Vickers surface micro-hardness
Please cite this article in press as: Elkassas D, Arafa A, Remineralizing efficacy of different calcium-phosphate and fluoride based delivery vehicles on artificial caries like enamel lesions. Journal of Dentistry (2014), http://dx.doi.org/10.1016/j.jdent.2013.12.017
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Table 2 – Analysis of surface roughness (Ra, mm) at baseline (B), after demineralization (D), after 2 weeks and 4 weeks remineralization (WR) and acid challenge (AC) of the groups (n = 10/gp). Group
G1 G2 G3 G4 G5
(control) ( fTCP) (ACPF) (CPP-ACPF) (RMGI)
Enamel treatment Ra – B
Ra – D
Ra – 2WR
Ra – 4WR
Ra – AC
0.2387 0.0029a,A 0.2389 0.0043a,A 0.2361 0.0023a,A 0.2386 0.0024a,A 0.2381 0.0024a,A
0.2602 0.0043c,A 0.2610 0.0011d,A 0.2602 0.0003d,A 0.2604 0.0007d,A 0.2577 0.0033c,A
0.2488 0.0016b,D 0.2487 0.0007c,C,D 0.2476 0.0006c,C 0.2442 0.0004b,B 0.2396 0.0009a,b,A
0.2469 0.0017b,D 0.2399 0.0006a,A 0.2405 0.0007b,A,B 0.2440 0.0004b,C 0.2413 0.0008b,B
0.2582 0.0027c,E 0.2443 0.0004b,A 0.2463 0.0007c,B 0.2484 0.0009c,C 0.2556 0.0007c,D
Different upper- and lower-case superscript letters indicate significant difference between tested groups at p < 0.05. Lower case superscript letters are used for comparison within the same row and upper case letters are used for comparison within each column.
technique has been utilized as an indirect mineral content assessment method in several laboratory models simulating the effect of application of various commercial products on in vitro.24–26 Surface roughness assessment is an important aspect as it may not only affect aesthetic properties but also reflects the bacterial adhesion and plaque formation potentials in the oral environment. 3D Non-Contact Optical Profile techniques tend to fulfil the need for quantitative characterization of surface topography without contact.27 SEM is a complementary tool, which helps to elucidate the surface ultra-morphological changes induced by different remineralizing agents. Constant remineralization model was used to screen the remineralizing potential of tested calcium phosphate compounds. While pH-cycling experiments (including demineralizing periods) might mimic the clinical dynamics more adequately, remineralization-only models offer the opportunity to effectively monitor caries-preventive products on dental hard tissues on a short-time basis, thus simulating a best-case scenario, even if the breadth of relevant biological aspects was limited.28 The initial mean VHN values for the five groups ranged from 304.17 to 317.9 for human teeth enamel. After demineralization, the VHN values apparently decreased, surface
roughness significantly increased, and enamel surface micro-porosities increased as depicted in Fig. 1. The net remineralization produced by saliva is small and is a slow process, with a tendency for the mineral gain to be in the surface layer of the lesion due to the low ion concentration gradient from saliva into the lesion.29 Thus, the control group showed the least values of remineralization based on the limited availability of calcium and phosphorous ions compared to the remineralizing effect of other regimens applied in accordance to Cochran et al.6 In addition, the formula of the artificial saliva used in the present study did not contain fluoride component which could explain the limited ability for remineralization by the control group. Despite of different forms of calcium and phosphorous compounds incorporated in these products, all remineralizing agents examined showed higher remineralizing potential compared to their control (demineralized) in terms of increase in VHN and reduction in Ra values with reduction in the surface micro-porosities as presented in SEM photos. In fTCP group, the fTCP technology has been customized to deliver targeted and sustained fluoride, calcium and phosphate. Where tricalcium phosphate particles have been ball milled with sodium lauryl sulphate and included in a tooth varnish with 5% sodium fluoride. During the manufacturing
Fig. 2 – Representative surface roughness image for enamel specimen of fTCP group (B) at baseline, (D) demineralized, (2 W) 2 weeks remineralization, (4 W) 4 weeks remineralization and (AC) acid challenge. Please cite this article in press as: Elkassas D, Arafa A, Remineralizing efficacy of different calcium-phosphate and fluoride based delivery vehicles on artificial caries like enamel lesions. Journal of Dentistry (2014), http://dx.doi.org/10.1016/j.jdent.2013.12.017
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process, a protective barrier is created around the calcium allowing it to coexist with the fluoride. After application, it comes in contact with saliva, releasing calcium and fluoride. CPP-ACPF and ACPF technologies deliver ACP and fluoride compounds to the tooth structure that readily solubilize to calcium, phosphate and fluoride ions when comes in contact with saliva, creating a supersaturated state of calcium, phosphate and fluoride around the tooth.16 In CPP-ACPF technology, ACP is stabilized by CPP casein-derived peptides. CPP contains the amino acid cluster sequence –Ser(P)-Ser(P)Ser(P)-Glu-Glu– and have been reported to bind amorphous calcium phosphate, forming small clusters of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP). This helps to prevent these clusters from reaching the critical size for precipitation, thereby, stabilizing calcium phosphates in solution in close proximity of the tooth making it available when needed. These nanocomplexes act as calcium and phosphate reservoirs when incorporated into the dental plaque and on the tooth surface.3,5,11 In ACPF technology two phase delivery system is used to keep the calcium and phosphorus components from reacting with each other before use. The source of calcium and phosphates are two salts, calcium sulfate and dipotassium phosphate. When the salts are mixed, they rapidly form ACPF that precipitate on tooth surface. The precipitated ACPF readily dissolves into saliva to be available for tooth remineralization. RMGI group is a resin modified glass ionomer vanish, extended contact varnish, based on the patented methacrylate modified polyalkenoic acid. The liquid component consists primarily of polyalkenoic acid, HEMA (2-hydroxethylmethacrylate), water and initiators (including camphorquinone) plus calcium glycerophosphate. The paste is a combination of HEMA, BIS-GMA, water, initiators and fluoroaluminosilicate glass (FAS glass). VanishTMXT varnish is claimed by the manufacturer to have dual benefits of both sealing and healing remineralizing agent. Its sealing ability arises from the adhesive potential of glass ionomer component and its resin content that infiltrates into the demineralized lesion and prevents further demineralization. Its remineralizing potential arises from sustained fluoride, calcium and phosphate release. The fluoride resides in fluoroaluminosilicate glass particles: reaction at the surface provides the immediate release, while the bulk provides a reservoir of fluoride for sustained release. The calcium glycerophosphate in VanishTMXT provides continual release of calcium and phosphate over the life of the coating. During enamel remineralization process different sequential crystalline phases can be produced: brushite, octacalcium phosphate, hydroxyapaptite and fluoroapatite. The solubility parameter of the formed phases is ordered phosphafluoroapatite > hydroxyapaptite > octacalcium te > brushite. During enamel remineralization and crystal voids repair, the degree of remineralization and the repaired crystalline phase formed will depend on the concentration of bioavailable fluoride, calcium and phosphate ions. However several studies reported that the bioavailability of active fluorine ions is the critical factor promoting remineralization and inhibition of demineralization in calcium- and phosphaterich circumstance.13,18,25,30 Fluoride catalysis the diffusion of
7
calcium and phosphate into the tooth surface, which in turn remineralize the crystalline structures in dental cavities leading to fluorapatite crystals, which represent the most acid resistant phase. Taking all the previous elements in account might help to interpret the obtained results. It was found that fTCP group showed the highest remineralizing potential and the greatest inhibition to softening by exposure to acid challenge compared to all other groups, in terms of highest VHN, lowest Ra values, and reduction in the surface micro-porosities as depicted in SEM Fig. 1. This could be attributed to high fluoride content of this product. As it contains 22,600 ppm of fluoride in comparison to CCP-ACPF and ACPF products which contain 900 and 1100 ppm, respectively. The dose-dependent effect of active fluoride ion in the remineralization efficacy in calcium, phosphate rich media was addressed by several authors.13,18,25,31 ten Cate et al.31 attributed this to elevated external fluoride-level. Higher F-gradient promotes driving fluoride deeper into the lesion. In addition, advanced lesions have large crystalline surface area for fluoride adsorption. In fTCP products, functionalizing the b-TCP is prepared with silica, which may provide linking opportunities with hard-tissue defects under acidic condition. It can permeate throughout enamel without attacking the inter-prismatic organic material, which may encourage greater calcium, phosphate, and fluoride uptake in demineralized lesions.32 The results are in contrary to Shen et al.1 who found CCPACPF product showed higher remineralization potential compared to fTCP product. This contradiction could be attributed to difference in form and composition of the used products. Tooth cre`me containing fTCP and 950 ppm fluoride was used in the former study while in the present study; the used varnish form was containing 22,600 ppm fluoride. In addition, the results of the present study could be attributed to the regimen of application of the test products. In the present study CPP-ACPF and ACPF products were applied twice daily according to the manufacturer, while the fTCP varnish is recommended twice a year. Considering that CPPACPF and ACPF products contains low level of fluoride and is recommended twice a day, 4 weeks might be relatively short for CPP-ACPF and ACPF to act sufficiently. So, supposing that this study goes for long period, the result of CPP-ACPF and ACPF groups may be better than that of this study, so the effect of different application methods may be considerable.18 RMGI group product showed similar remineralizing potential to CPP-ACPF group after 2 weeks, and lowest remineralizing potential compared to fTCP, ACPF and CPP-ACPF groups after 4 weeks and further after exposure to acidic challenge. Two mechanisms have been proposed by which fluoride may be released from glass ionomer into an aqueous environment. One mechanism is a short-term reaction, which involves rapid dissolution from outer surface into the solution (wash out), whereas the second is more gradual and resulted in the sustained long-term diffusion of ions through its bulk.33 In this product the initial burst effect of fluoride release might be responsible for producing remineralizing potential comparable to CPP-ACPF when tested after 2 weeks. However the short-term gradual fluoride release from the bulk of the cement did not reach the remineralizing effect produced by the other tested compounds. It was found that this product
Please cite this article in press as: Elkassas D, Arafa A, Remineralizing efficacy of different calcium-phosphate and fluoride based delivery vehicles on artificial caries like enamel lesions. Journal of Dentistry (2014), http://dx.doi.org/10.1016/j.jdent.2013.12.017
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produced a sealing capacity as depicted in SEM figure of 2 weeks remineralization. However, the degradation of this sealed layer after 4 weeks owing to its acid–base reaction through continuous dissolution of the siliceous hydrogel layer or the polysalt matrix,34 lead to surface voids and cracks as depicted in SEM of 4 weeks with increase in surface roughness.35,36 Furthermore, following the exposure to acidic challenge, partial dissolution of coating layer was evident (SEM-AC) with the existence of demineralized surface. This might denote that this material provided neither optimal remineralization nor efficient infiltration to complete depth of demineralized lesion leading to the lowest VHN compared to other tested products after 4 weeks remineralization or exposure to acidic challenge. The results of the present study are in contrary to Zhou et al., 2013 study,37 who found no significant difference between fTCP product and RMGI sealant. This contradiction could be attributed to the difference in the formulas used to induce enamel demineralization, as in Zhou et al. study the formula was used to induce enamel erosion resulting in different lesion depths. In addition to the difference in nature of examined substrates, as in the former study, bovine specimens were evaluated, while human molars were used in the present study.
5.
Conclusion
Under the limitations of the present study, it can be concluded that: 1-Calcium phosphate based remineralizing agents provide superior remineralization effects and greater resistance to acid softening as compared to artificial saliva. 2-Extended period of time had helped to attain more benefits of remineralization regimens application. 3-ClinproTM remineralizing system confers the highest remineralization tendency with the highest potential for inhibition of surface softening by acid challenge.
Conflict of interest The authors received no financial support and declare no potential conflicts of interest with respect to authorship and/ or publication of this article.
references
1. Shen P, Manton DJ, Cochrane NJ, Walker GD, Yuan Y, Reynolds C, et al. Effect of added calcium phosphate on enamel remineralization by fluoride in a randomized controlled in situ trial. Journal of Dentistry 2011;39:518–25. 2. Paris S, Meyer-Lueckel H. Inhibition of caries progression by resin infiltration in situ. Caries Research 2010;44:47–54. 3. Reynolds C, Cai F, Cochrane J, Shen P, Walker G, Morgan M. Fluoride and casein phosphopeptide-amorphous calcium phosphate. Journal of Dental Research 2008;87:344–8. 4. ten Cate JM. New agents for caries prevention: introduction to ICNARA 2. Advances in Dental Research 2012;24:27.
5. Vogel GL, Schumacher GE, Chow LC, Takagi S, Carey CM. Ca pre-rinse greatly increases plaque and plaque fluid F. Journal of Dental Research 2008;87:466–9. 6. Cochrane N, Cai F, Hug N, Burrow M, Reynolds E. New approaches to enhanced remineralization of tooth enamel. Journal of Dental Research 2010;89:1187–97. 7. Shen P, Cai F, Nowicki A, Vincent J, Reynolds E. Remineralization of enamel subsurface lesions by sugar free chewing gum containing casein phosphopeptideamorphous calcium phosphate. Journal of Dental Research 2001;80:2066–70. 8. Reynolds C, Cai F, Shen P, Walker D. Retention in plaque and remineralization of enamel lesions by various forms of calcium in a mouth rinse or sugar-free chewing gum. Journal of Dental Research 2003;82:206–11. 9. Bowen W, Pearson S. Effect of milk on cariogenesis. Caries Research 1993;27:461–6. 10. Jensen M, Wefel J. Effects of processed cheese on human plaque pH and demineralization and remineralization. American Journal of Dentistry 1999;3:217–23. 11. Cross KJ, Huq NL, O’Brien-Simpson NM, Perich JW, Attard TJ, Reynolds EC. The role of multiphosphorylated peptides in mineralized tissue regeneration. International Journal of Peptide Research and Therapeutics 2007;13:479–95. 12. Lata S, Varghese N, Varghese J. Remineralization potential of fluoride and amorphous calcium phosphate-casein phosphor peptide on enamel lesions: an in vitro comparative evaluation. Journal of Conservative Dentistry 2010;13:42–6. 13. Yamazaki H, Litman A, Margolis HC. Effect of fluoride on artificial caries lesion progression and repair in human enamel: regulation of mineral deposition and dissolution under in vivo-like conditions. Archives of Oral Biology 2007;52:263–6. 14. Nicholson JW, Czarnecka B. The biocompatibility of resin modified glass-ionomer cements for dentistry. Dental Materials 2008;24:1702–8. 15. Rafeek RN. The effects of heat treatment on selected properties of a conventional and a resin-modified glass ionomer cement. Journal of Materials Science Materials in Medicine 2008;19:1913–20. 16. Pulido M, Wefel J, Hernandez M, Denehy G, GuzmanArmstrong G, Chalmers J, et al. The inhibitory effect of MI paste, fluoride and a combination of both on the progression of artificial caries-like lesions in enamel. Operative Dentistry 2008;33:550–5. 17. Torres CR, Rosa PC, Ferreira NS, Borges AB. Effect of caries infiltration technique and fluoride therapy on microhardness of enamel carious lesions. Operative Dentistry 2012;37:363–9. 18. Kim MJ, Lee SH, Lee NY, Lee IH. Evaluation of the effect of PVA tape supplemented with 2.26% fluoride on enamel demineralization using micro-hardness assessment and scanning electron microscopy: in vitro study. Archives of Oral Biology 2012;58:160–6. 19. Lee YE, Baek HJ, Choi YH, Jeong SH, Park YD, Song KB. Comparison of remineralization effect of three topical fluoride regimens on enamel initial carious lesions. Journal of Dentistry 2010;38:166–71. 20. Kakaboura A, Fragouli M, Rahiotis C, Silikas N. Evaluation of surface characteristics of dental composites using profilometry, scanning electron, atomic force microscopy and gloss-meter. Journal of Materials Science Materials in Medicine 2007;18:155–63. 21. Poggio C, Lombardini M, Dagna A, Chiesa M, Bianchi S. Protective effect on enamel demineralization of a CPP-ACP paste: an AFM in vitro study. Journal of Dentistry 2009;37: 949–54.
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22. Srinivasan N, Kavitha M, Loganathan SC. Comparison of the remineralization potential of CPP-ACP and CPP-ACP with 900 ppm fluoride on eroded human enamel: an in situ study. Archives of Oral Biology 2010;55:541–4. 23. Ranjitkar S, Rodriguez JM, Kaidonis JA, Richards LC, Townsend GC, Bartlett DW. The effect of casein phosphopeptide-amorphous calcium phosphate on erosive enamel and dentin wear by toothbrush abrasion. Journal of Dentistry 2009;37:250–4. 24. Featherstone JD. Modeling the caries-inhibitory effects of dental materials. Dental Materials 1996;12:194–7. 25. Diamanti I, Koletsi-Kounari H, Mamai-Homata E, Vougiouklakis G. Effect of fluoride and of calcium sodium phosphosilicate toothpastes on pre-softened dentin demineralization and remineralization in vitro. Journal of Dentistry 2010;38:671–7. 26. Diamanti I, Koletsi-Kounari H, Mamai-Homata E, Vougiouklakis G. In vitro evaluation of fluoride and calcium sodium phosphosilicate toothpastes, on root dentine caries lesions. Journal of Dentistry 2011;39:619–28. 27. Bui S, Vorburger T. Surface metrology algorithm testing system. Precision Engineering 2007;31:218–25. 28. Tschoppe P, Zandim D, Martus P, Kielbassa A. Enamel and dentine remineralization by nano-hydroxyapatite toothpastes. Journal of Dentistry 2011;39:430–7. 29. Zhang Q, Zou J, Yang R, Zhou X. Remineralization effects of casein phosphopeptide-amorphous calcium phosphate cre`me on artificial early enamel lesions of primary teeth. International Journal of Pediatric Dentistry 2011;21:374–81.
30. Hamba H, Nikaido T, Inoue G, Sadr A, Tagami J. Effects of CPP-ACP with sodium fluoride on inhibition of bovine enamel demineralization: a quantitative assessment using micro-computed tomography. Journal of Dentistry 2011;39:405–13. 31. ten Cate JM, Buijs MJ, Miller CC, Exterkate RA. Elevated fluoride products enhance remineralization of advanced enamel lesions. Journal of Dental Research 2008;87:943–7. 32. Karlinsey RL, Pfarrer AM. Fluoride plus functionalized bTCP: a promising combination for robust remineralization. Advances in Dental Research 2012;24:48–52. 33. Dionysopoulos D, Koliniotou-Koumpia E, HelvatzoglouAntoniades M, Kotsanos N. Fluoride release and recharge abilities of contemporary fluoride-containing restorative materials and dental adhesives. Dental Materials 2013;32:296–304. 34. Yu F, Kubo S, Yakushiji M. Effect of three fluoride agents on remineralization and fluoride uptake on enamel lesion. Pediatric Dental Journal 2005;15:165–70. 35. Roulet JF, Walti C. Influence of oral fluid on composite resin and glass-ionomer cement. Journal of Prosthetic Dentistry 1984;52:182–9. 36. Bala O, Arisu HD, Yikilgan I, Arslan S, Gullu A. Evaluation of surface roughness and hardness of different glass ionomer cements. European Journal of Dentistry 2012;6:79–86. 37. Zhou S, Zhou J, Watanabe S, Watanabe K, Wena L, Xua K. In vitro study of the effects of fluoride-releasing dental materials on remineralization in an enamel erosion model. Journal of Dentistry 2012;40:255–63.
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Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.intl.elsevierhealth.com/journals/jden
Remineralizing efficacy of different calcium-phosphate and fluoride based delivery vehicles on artificial caries like enamel lesions Dina Elkassas a, Abla Arafa b,* a
Department of Operative Dentistry, Faculty of Oral and Dental Medicine, Misr International University, Egypt Department of Pediatric Dentistry and Dental Public Health, Faculty of Oral and Dental Medicine, Misr International University, Egypt b
article info
abstract
Article history:
Objectives: To assess enamel remineralization of different calcium-phosphate and fluoride
Received 9 October 2013
delivery systems.
Received in revised form
Methods: Artificial caries lesions were created on 115 extracted human molars. Specimens
30 December 2013
were assigned according to remineralizing agent into five groups: G1: Control (artificial
Accepted 31 December 2013
saliva), G2: ClinproTM white varnish, G3: Relief, G4: Tooth Mousse Plus, G5: VanishTMXT.
Available online xxx
Surface micro-hardness (SMH), surface roughness (Ra) and surface topography by scanning electron microscope (SEM) were evaluated at baseline, after demineralization, after 2 and 4
Keywords:
weeks remineralization and after acid challenge.
Acid challenge
Results: Demineralized enamel showed the lowest SMH. By 2 weeks remineralization, SMH
Calcium-phosphate-based systems
were ranked as follows: G2 (282.14 6.82) > G3 (269.37 7.25) > G5 (263.00 6.49) = G4
Microhardness
(251.83 8.26) > G1 (226.5 9.34). However, 4 weeks remineralization showed the following:
Remineralization
G2 (304.09 6.65) > G3 (293.1 5.24) = G4 (285 7.29) > G5 (272.43 4.89) > G1 (233.33
Scanning electron microscopy
9.12). By exposure to acid challenge, groups presented order of: G2 (279.71 5.99) = G3
Surface roughness
(275.51 5.59) > G4 (262.29 6.65) > G5 (245.43 6.43) > G1 (190.27 8). Surface roughness showed the following mattress after 2 weeks remineralization: G1 (0.2488 0.0016) = G2 (0.2487 0.0007) = G3 (0.2476 0.0006) > G4 (0.2442 0.0004) > G5 (0.2396 0.0009). After 4 weeks remineralization: G1 (0.2469 0.0017) > G4 (0.244 0.0004) > G5 (0.2413 0.0008) = G3 (0.2405 0.0007) = G2 (0.2399 0.0006). After acid challenge; G1 (0.2582 0.0027) > G5 (0.2556 0.0007) > G4 (0.2484 0.0009) > G3 (0.2463 0.0007) > G2 (0.2443 0.0004). SEM revealed mineralized coating on the surfaces which resists dissolution by acid challenge at variable degrees according to remineralization regimen applied. Conclusions: Remineralizing agents containing different calcium-phosphate formulas and fluoride have increased remineralization potential compared to artificial saliva. ClinproTM varnish presented the highest remineralization tendency with greatest resistance for acid challenge. Clinical significance: This in vitro study imitated the application of different calcium phosphorous and fluoride based delivery vehicles to enamel tooth surfaces in the mouth. The new therapeutic techniques based on different calcium phosphate formulas containing fluoride provide a new avenue for remineralization of non-cavitated and early carious lesions. # 2014 Elsevier Ltd. All rights reserved.
* Corresponding author at: Department of Pediatric Dentistry, Faculty of Oral and Dental Medicine, Misr International University, 28 CairoEl Ismailia Road, Cairo, Egypt. Tel.: +20 2 22594539; fax: +20 2 23635413; mobile: +20 1100670307. E-mail address: [email protected] (A. Arafa). 0300-5712/$ – see front matter # 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jdent.2013.12.017 Please cite this article in press as: Elkassas D, Arafa A, Remineralizing efficacy of different calcium-phosphate and fluoride based delivery vehicles on artificial caries like enamel lesions. Journal of Dentistry (2014), http://dx.doi.org/10.1016/j.jdent.2013.12.017
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1.
Introduction
Dental caries is still one of the most prevalent diseases affecting humans. A goal of modern dentistry is to manage non-cavitated carious lesions non-invasively in an attempt to prevent further disease progression and preserve integrity of healthy tooth substrate. Non-invasive management of noncavitated carious lesions comprises several strategies; among these is healing the demineralized lesions using remineralizing agent or sealing them using resinous based materials.1,2 Remineralization of hard dental tissues is defined as the process whereby calcium and phosphate ions are supplied from a source external to the tooth to promote ion deposition into crystal voids in demineralized enamel to produce net mineral gain. Fluoride is considered the cornerstone in the remineralization process, yet its ability to promote net remineralization is limited by the availability of calcium and phosphate ions.3–5 Recently, a range of novel calcium phosphate based delivery systems were introduced into the dental market. The manufacturers claim that these products provide new avenues for the remineralization of noncavitated caries lesions.6 They can be categorized into one of the three types-crystalline, unstabilized amorphous, or stabilized amorphous formulations. Crystalline systems as tricalcium phosphate (TCP) showed potential to deliver calcium and phosphorous to enamel lesions.6 In those systems, tricalcium phosphate is functionalized ( fTCP) by being ball milled with sodium lauryl sulphate. Unstabilized amorphous calcium phosphate (ACP) remineralizing system provides the benefit of having both calcium and phosphate ions close to each other in an amorphous phase. The ACP technology requires a two-phase delivery system to keep the calcium and phosphorus components from reacting with each other before use. Some studies reported that this system can enhance remineralization, decrease demineralization or provide a combination of both during an acid challenge to the tooth surface.7,8 In stabilized amorphous formulations, casein phosphopeptide (CPP), have a remarkable ability to stabilize calcium phosphate (ACP) in metastable stage. Through the multiple phosphoseryl residues, the CPP binds to form nanoclusters of ACP, preventing their growth to the critical size required for nucleation and phase transformation.6,9,10 The proposed mechanism for the CPP-ACP is to localize ACP in dental plaque, which buffers the free calcium and phosphate ion activities, thereby helping to maintain a stage of supersaturation with respect to tooth enamel depressing demineralization and enhancing remineralization.8 Several authors reported the remineralizing potential of stabilized casein phosphopeptide amorphous calcium phosphate (CPPACP).11,12 Owing to the overwhelming role of fluoride in the remineralization process, it was added to most of calciumphosphate delivery products, in different forms, and concentrations. The remineralization efficacy of fluoride containing materials is related to fluoride content, fluoride matrices, setting mechanisms and other materials components.13 For the sealing strategy, the resin modified glass ionomer varnish is launched into the market. It is claimed by the
manufacturer that this material provides unique ability of healing and sealing incipient demineralized lesions. The sealing ability arises from the penetrative capability of the resinous component incorporated in the material and chemical bonding activity of the glass ionomer component, while its healing ability emerges from long term release of calcium, phosphorous and fluoride ions.14,15 Hitherto, no study compared the impact of strategies of delivery of calciumphosphate and fluoride ions on the remineralizing potential of incipient carious lesions. Furthermore, the stability of the built up mineral product following acid challenge were scantly studied in the literature. Thus, the aim of this study is to assess the effect of different calcium-phosphorous and fluoride delivery systems on enamel remineralization using surface micro-hardness, scanning electron microscopy and surface roughness methods. The null hypotheses tested; first: Functionalized Tricalcium phosphate (ClinproTM white varnish, fTCP), Amorphous calcium phosphate (Relief, ACP), Casein phosphopeptide amorphous calcium phosphate (Tooth Mousse Plus, CPP-ACPF), and Resin modified glass ionomer (VanishTMXT, RMGI), based remineralizing agents, would have no additional benefits for enhancement of enamel remineralization compared to artificial saliva. Second, there is no difference in the remineralizing efficacy of the aforementioned compounds.
2.
Materials and methods
2.1.
Materials
Four remineralizing agents were evaluated in this study: (a) ClinproTM white varnish (3 M ESPE, USA) as functionalized tricalcium phosphate containing 22,600 ppm fluoride ( fTCP), (b) Relief (Discus Dental, USA) as amorphous calcium phosphate containing 1100 ppm fluoride (ACPF), (c) Tooth Mousse Plus (GC Dental, Japan) as casein phosphopeptide amorphous calcium phosphate containing 900 ppm fluoride (CPP-ACPF) and (d) VanishTMXT (3M ESPE, USA) as extended contact varnish based on resin modified glass ionomer technology (RMGI).
2.2.
Specimens preparation
Total of 115 extracted human mandibular molars free from clinically visible abnormality were stored in an aqueous solution of saturated thymol for 2 weeks after removal of the roots. The teeth were thoroughly rinsed and examined under the stereomicroscope (Nikon, Japan), any tooth with defects, erosions, or micro-cracks on their enamel surfaces or visible stains was excluded. The coronal part of each tooth was longitudinally sectioned in mesio-distal direction using diamond disk (Komet, Rock Hill, USA) in low speed under water cooling. Custom-made plastic cylindrical molds were prepared and self-cured acrylic resin (Acrostone Dental Factor, England) was poured on them. Each buccal section was mounted horizontally in acrylic resin and cured overnight, and then polished flat using 800, 1200 and 2400 grit silicon carbide paper. An acid resistant nail varnish was applied around the exposed enamel surface, leaving four equal overtures of approximately (2 mm 2 mm) each.
Please cite this article in press as: Elkassas D, Arafa A, Remineralizing efficacy of different calcium-phosphate and fluoride based delivery vehicles on artificial caries like enamel lesions. Journal of Dentistry (2014), http://dx.doi.org/10.1016/j.jdent.2013.12.017
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2.3.
Demineralization
Following the proposal suggested by Pulido et al.16 artificial caries like lesions were created by individually immersing acrylic-mounted enamel specimens in continuously stirred, daily renewed demineralization solution. This solution contained 2.2 mM CaCl22H2O, 2.2 mM KH2PO4, 0.05 M acetic acid and pH adjusted to 4.4 with 10 M KOH. The molars were kept in this solution for 5 days until a uniform white spot lesion was created on the surface of the window. Enamel specimens were washed carefully and then the first window was coated with acid resistant nail varnish to act as control for demineralization.
2.4.
Remineralization
Artificial saliva was prepared according to the formulation of Torres et al.17 and consisted of 22.1 mM hydrogen carbonate, 16.1 mM potassium, 14.5 mM sodium, 2.6 mM hydrogen phosphate, 0.8 mM boric acid, 0.7 mM calcium, 0.2 mM thiocyanate, and 0.2 mM magnesium. The pH was between 7.4 and 7.8. Specimens were randomly divided into five groups (n = 23/ gp), according to the treatment employed:
acid resistance of the treated surfaces. The nail varnish was peeled off from all of the specimens carefully. The 23 specimens of each group were divided into three groups and tested by the following methods: assessment of the surface micro-hardness (n = 10), scanning electron microscopy examination (n = 3) and assessment of the surface roughness (n = 10).
2.5.
For each specimen, a control window was painted with nail varnish after demineralization. The second window was covered with nail varnish after 2 weeks remineralization, while the third window was covered following 4-week remineralization. Prior to covering the second and third windows, the remineralizing agents were removed. The specimens of G3 and G4, that received Relief and Tooth Mousse Plus agents, the surfaces were cleaned using cotton tip immersed in deionized water.18 In G2 and G5, that received Clinpro white varnish and Vanish extended contact varnish, the varnishes were carefully removed using surgical blade followed by cleaning off the surface with cotton tip immersed in deionized water.19 No chemical substance was used to clean the surfaces in order not to alter the enamel surface.18 After these periods, the samples were immersed again in the previously described demineralizing solution to evaluate the
Assessment of Surface Micro-hardness (SMH)
Enamel surface micro-hardness was measured at baseline of sound untreated enamel, after surface softening, after 2 weeks remineralization, after 4 weeks remineralization and after being subjected to acid challenge. The surface micro-hardness was measured using micro-hardness tester with Vickers diamond indenter in different regions of the specimens (Vickers diamond, 100 g, 5 s, HMV 2; Shimadzu Corporation, Tokyo, Japan). All readings were performed by the same examiner using the same calibrated machine. In each reading, four indentations were made and their average was taken to represent the specimen’s hardness value.
2.6. G1 (Control group): No treatment was given to the enamel surface and specimens were kept in artificial saliva which renewed every day. G2 ( fTCP group): Single application of ClinproTM white varnish was performed, specimens rinsed and dried for 10 s, and then material coating was applied to cover specimen surface and left for 20 s, then stored in artificial saliva. G3 (ACPF group): Relief coating was applied twice/day, specimens were rinsed and dried for 10 s, then thin layer of remineralizing agent was applied to cover specimen surface and left for 3 min, then wiped gently with gauze, washed and stored in artificial saliva. G4 (CPP-ACPF group): Tooth Mousse Plus was applied twice/ day as G3. G5 (RMGI group): Single application of VanishTMXT was performed, specimens rinsed and dried for 10 s, then material coating was applied to cover specimen surface and light cured for 20 s (EliparTM S10 LED Curing Light, 1200 mW/cm2, 3 M ESPE), then stored in artificial saliva.
3
Scanning electron microscope examination (SEM)
For the SEM examination, three specimens in each group were treated and then air dried. Each specimen was mounted on SEM stud and then sputter coated under vacuum with gold by the Ladd sputter coater (BAL-TEC, SCD 005, Germany). They were examined under Scanning Electron Microscope (Philips, Holland). SEM photomicrographs were captured at 1000 magnification.
2.7.
Assessment of Surface Roughness (Ra)
The optical methods tend to fulfil the need for quantitative characterization of surface topography without contact. Specimens were photographed using USB Digital microscope with a built-in camera (Scope Capture Digital Microscope, Guangdong, China) connected with an IBM compatible computer using fixed magnification of 50. The images were recorded with a resolution of 1280 1024 pixels per image and then cropped to 350 400 pixels using Microsoft office picture manager to specify and standardize area of roughness measurement. Cropped images were analyzed using WSxM software (Version 5 develop 4.1, Nanotec, Electronica, SL). Within the WSxM software, all limits, sizes, frames and measured parameters are expressed in pixels. Therefore, system calibration was done to convert the pixels into absolute real world units. Calibration was made by comparing an object of known size (a ruler in this study) with a scale generated by the software. Subsequently, a 3D image of the surface profile of the specimens was created. Three 3D images were collected per each window for each specimen of an area 10 mm 10 mm. WSxM software was used to calculate average surface roughness (Ra) expressed in mm.20
2.8.
Statistical analyses
The mean (SD) SMH values and the mean (SD) Ra values were statistically analyzed. Repeated measure ANOVA was
Please cite this article in press as: Elkassas D, Arafa A, Remineralizing efficacy of different calcium-phosphate and fluoride based delivery vehicles on artificial caries like enamel lesions. Journal of Dentistry (2014), http://dx.doi.org/10.1016/j.jdent.2013.12.017
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performed to compare remineralizing potential of each product at different time interval, 2 and 4 weeks remineralization as well as exposure to acid challenge, followed by multiple comparisons Tukey’s post hoc test. Comparison between different products at different time interval to monitor the remineralizing efficacy was carried out using one-way ANOVA followed by multiple comparisons Tukey’s post hoc test. The significance level was set at 0.05. All data were processed by the SPSS (version 16.0) software package (SPSS Inc., Chicago, IL, USA).
3.
Results
3.1.
Enamel surface micro-hardness
hidden by mineral deposition. ACPF specimens showed evidence of thickening of the inter-rod substance early by 2week remineralization; however, extended remineralization period enhanced the growth of enamel crystals to cover over fish scale appearance of demineralized surface. In fTCP group, after 4 weeks remineralization, some of enamel crystals were even fused together and arranged homogenously with no obvious inter-crystalline spaces on the surface. After exposure to acid challenge, the experimental groups showed more resistance to dissolution as compared to the control. The treated surfaces retained calcified deposits but with the presence of some potholes on the surface.
3.3.
The mean (SD) SMH values of surfaces at different enamel treatment points are shown in Table 1. The groups presented significantly the highest SMH at the baseline ( p < 0.05). The demineralized enamel showed significant reduction in SMH values ( p < 0.05). The SMH values of all treated groups demonstrated significant increase after 2 and 4 weeks of application of remineralization regimen ( p < 0.05). All of the groups showed significant reduction in SMH values indicating mineral loss as exposed to acid challenge ( p < 0.05). SMH values in the fTCP group were significantly the highest, early after 2 weeks remineralization and even after 4 weeks of remineralization ( p < 0.05). In addition, after 4 weeks remineralization, there were no significant differences between the ACPF and CPP-ACPF groups ( p > 0.05). After exposure to acid challenge, the fTCP and ACPF groups showed statistically significant higher SMH mean values compared to CPP-ACPF and RMGI groups ( p < 0.05). However, all groups presented statistically significant higher resistance to softening than the control ( p < 0.05).
3.2.
SEM morphological characters
Sound enamel has homogeneous smooth appearance. However, demineralized enamel showed increased porosity with fish-scale pattern as depicted in Fig. 1. The test groups showed increased density of crystals after 2 weeks remineralization. While after 4 weeks remineralization, enamel surfaces revealed new coating progressively filled the pits and scratches where the prismatic enamel structures became
Enamel surface roughness
The mean (SD) Ra values of surfaces at different enamel treatment points are presented in Table 2. At the baseline measure, the mean Ra value of specimens was within the range of 0.2361–0.2389 mm. After demineralization, significant increase in Ra values was evident in all tested groups ( p < 0.05). After 2 weeks remineralization, all experimental groups showed significant reduction in Ra values ( p < 0.05). After 4 weeks remineralization, further significant reduction in the Ra values was evident in the fTCP and ACPF groups; however, no significant difference in Ra values was evident in control, CPPACPF and RMGI groups. Interestingly, RMGI group showed increase in Ra values by 4 weeks remineralization ( p > 0.05). Following the exposure to acid challenge, all tested groups showed significant increase in Ra values when compared to 4 weeks remineralization ( p < 0.05). Comparing between the remineralizing agents examined at different intervals, it was evident that RMGI group showed significantly lowest Ra values after 2 weeks ( p < 0.05). Meanwhile, the fTCP group showed significantly the lowest Ra values after 4 weeks and after exposure to acid challenge ( p < 0.05). The differences in surface roughening mediated of fTCP group among different time points are apparent when inspecting the 3D topographical images as illustrated in Fig. 2.
4.
Discussion
The present study was carried out to compare the remineralizing efficacy of five regimens on artificial caries like enamel lesions, in terms of change in surface micro-hardness,
Table 1 – Analysis of surface microhardness (SMH-VHN) at baseline (B), after demineralization (D), after 2 weeks and 4 weeks remineralization (WR) and after acid challenge (AC) of the groups (n = 10/gp). Group
G1 G2 G3 G4 G5
(control) ( fTCP) (ACPF) (CPP-ACPF) (RMGI)
Enamel treatment SMH-B
SMH-D
SMH-2WR
SMH-4WR
SMH-AC
304.17 10.89a,A 316.43 10.94a,A 317.31 6.22a,A 314.57 9.86a,A 310.29 8.59a,A
216.00 9.29c,A 225.17 10.24c,A 225.20 7.11d,A 212.46 6.82e,A 219.36 11.27d,A
226.50 9.34b,c,D 282.14 6.82b,A 269.37 7.25c,B 251.83 8.26d,C 263.00 6.49b,B,C
233.33 9.12b,D 304.09 6.65a,A 293.10 5.24b,B 285.00 7.29b,B 272.43 4.89b,C
190.27 8.00d,D 279.71 5.99b,A 275.51 5.59c,A 262.29 6.65c,B 245.43 6.43c,C
Different upper- and lower-case superscript letters indicate significant difference between tested groups at p < 0.05. Lower case superscript letters are used for comparison within the same row and upper case letters are used for comparison within each column.
Please cite this article in press as: Elkassas D, Arafa A, Remineralizing efficacy of different calcium-phosphate and fluoride based delivery vehicles on artificial caries like enamel lesions. Journal of Dentistry (2014), http://dx.doi.org/10.1016/j.jdent.2013.12.017
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Fig. 1 – SEM images of (B) sound enamel surface at baseline, (D) demineralized enamel surface, and the Control, fTCP, ACPF, CPP-ACPF and RMGI groups at (2 W) two weeks remineralization, (4 W) 4 weeks remineralization, and (AC) after acid challenge (1000T).
ultrastructure and surface roughness. The specimens were polished for two motifs: first: to remove the prismless enamel layer that arises at the end of amelogenesis. Although this layer is not frequently found on the surface of permanent teeth compared to their predecessors, it is known that the prism-free enamel is gradually worn off during mastication but it is retained in protected areas. Flat and polished specimens were used in the present study in an attempt to standardize specimens and remove natural variations on surface enamel between teeth and between different tooth
sites and types, which may result in different responses to acid dissolution.21 Second: Micro-hardness assessment needs flat polished surface to enable accurate measurements.22 Thus, the area subjected for demineralization did not represent the ideal enamel surface, as removal of the outer surface layer made the enamel more susceptible for demineralization.18,23 Micro-hardness testing is considered to be a relatively simple and reasonably reliable method for the provision of indirect information about the mineral content changes of hard dental tissues. Vickers surface micro-hardness
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Table 2 – Analysis of surface roughness (Ra, mm) at baseline (B), after demineralization (D), after 2 weeks and 4 weeks remineralization (WR) and acid challenge (AC) of the groups (n = 10/gp). Group
G1 G2 G3 G4 G5
(control) ( fTCP) (ACPF) (CPP-ACPF) (RMGI)
Enamel treatment Ra – B
Ra – D
Ra – 2WR
Ra – 4WR
Ra – AC
0.2387 0.0029a,A 0.2389 0.0043a,A 0.2361 0.0023a,A 0.2386 0.0024a,A 0.2381 0.0024a,A
0.2602 0.0043c,A 0.2610 0.0011d,A 0.2602 0.0003d,A 0.2604 0.0007d,A 0.2577 0.0033c,A
0.2488 0.0016b,D 0.2487 0.0007c,C,D 0.2476 0.0006c,C 0.2442 0.0004b,B 0.2396 0.0009a,b,A
0.2469 0.0017b,D 0.2399 0.0006a,A 0.2405 0.0007b,A,B 0.2440 0.0004b,C 0.2413 0.0008b,B
0.2582 0.0027c,E 0.2443 0.0004b,A 0.2463 0.0007c,B 0.2484 0.0009c,C 0.2556 0.0007c,D
Different upper- and lower-case superscript letters indicate significant difference between tested groups at p < 0.05. Lower case superscript letters are used for comparison within the same row and upper case letters are used for comparison within each column.
technique has been utilized as an indirect mineral content assessment method in several laboratory models simulating the effect of application of various commercial products on in vitro.24–26 Surface roughness assessment is an important aspect as it may not only affect aesthetic properties but also reflects the bacterial adhesion and plaque formation potentials in the oral environment. 3D Non-Contact Optical Profile techniques tend to fulfil the need for quantitative characterization of surface topography without contact.27 SEM is a complementary tool, which helps to elucidate the surface ultra-morphological changes induced by different remineralizing agents. Constant remineralization model was used to screen the remineralizing potential of tested calcium phosphate compounds. While pH-cycling experiments (including demineralizing periods) might mimic the clinical dynamics more adequately, remineralization-only models offer the opportunity to effectively monitor caries-preventive products on dental hard tissues on a short-time basis, thus simulating a best-case scenario, even if the breadth of relevant biological aspects was limited.28 The initial mean VHN values for the five groups ranged from 304.17 to 317.9 for human teeth enamel. After demineralization, the VHN values apparently decreased, surface
roughness significantly increased, and enamel surface micro-porosities increased as depicted in Fig. 1. The net remineralization produced by saliva is small and is a slow process, with a tendency for the mineral gain to be in the surface layer of the lesion due to the low ion concentration gradient from saliva into the lesion.29 Thus, the control group showed the least values of remineralization based on the limited availability of calcium and phosphorous ions compared to the remineralizing effect of other regimens applied in accordance to Cochran et al.6 In addition, the formula of the artificial saliva used in the present study did not contain fluoride component which could explain the limited ability for remineralization by the control group. Despite of different forms of calcium and phosphorous compounds incorporated in these products, all remineralizing agents examined showed higher remineralizing potential compared to their control (demineralized) in terms of increase in VHN and reduction in Ra values with reduction in the surface micro-porosities as presented in SEM photos. In fTCP group, the fTCP technology has been customized to deliver targeted and sustained fluoride, calcium and phosphate. Where tricalcium phosphate particles have been ball milled with sodium lauryl sulphate and included in a tooth varnish with 5% sodium fluoride. During the manufacturing
Fig. 2 – Representative surface roughness image for enamel specimen of fTCP group (B) at baseline, (D) demineralized, (2 W) 2 weeks remineralization, (4 W) 4 weeks remineralization and (AC) acid challenge. Please cite this article in press as: Elkassas D, Arafa A, Remineralizing efficacy of different calcium-phosphate and fluoride based delivery vehicles on artificial caries like enamel lesions. Journal of Dentistry (2014), http://dx.doi.org/10.1016/j.jdent.2013.12.017
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process, a protective barrier is created around the calcium allowing it to coexist with the fluoride. After application, it comes in contact with saliva, releasing calcium and fluoride. CPP-ACPF and ACPF technologies deliver ACP and fluoride compounds to the tooth structure that readily solubilize to calcium, phosphate and fluoride ions when comes in contact with saliva, creating a supersaturated state of calcium, phosphate and fluoride around the tooth.16 In CPP-ACPF technology, ACP is stabilized by CPP casein-derived peptides. CPP contains the amino acid cluster sequence –Ser(P)-Ser(P)Ser(P)-Glu-Glu– and have been reported to bind amorphous calcium phosphate, forming small clusters of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP). This helps to prevent these clusters from reaching the critical size for precipitation, thereby, stabilizing calcium phosphates in solution in close proximity of the tooth making it available when needed. These nanocomplexes act as calcium and phosphate reservoirs when incorporated into the dental plaque and on the tooth surface.3,5,11 In ACPF technology two phase delivery system is used to keep the calcium and phosphorus components from reacting with each other before use. The source of calcium and phosphates are two salts, calcium sulfate and dipotassium phosphate. When the salts are mixed, they rapidly form ACPF that precipitate on tooth surface. The precipitated ACPF readily dissolves into saliva to be available for tooth remineralization. RMGI group is a resin modified glass ionomer vanish, extended contact varnish, based on the patented methacrylate modified polyalkenoic acid. The liquid component consists primarily of polyalkenoic acid, HEMA (2-hydroxethylmethacrylate), water and initiators (including camphorquinone) plus calcium glycerophosphate. The paste is a combination of HEMA, BIS-GMA, water, initiators and fluoroaluminosilicate glass (FAS glass). VanishTMXT varnish is claimed by the manufacturer to have dual benefits of both sealing and healing remineralizing agent. Its sealing ability arises from the adhesive potential of glass ionomer component and its resin content that infiltrates into the demineralized lesion and prevents further demineralization. Its remineralizing potential arises from sustained fluoride, calcium and phosphate release. The fluoride resides in fluoroaluminosilicate glass particles: reaction at the surface provides the immediate release, while the bulk provides a reservoir of fluoride for sustained release. The calcium glycerophosphate in VanishTMXT provides continual release of calcium and phosphate over the life of the coating. During enamel remineralization process different sequential crystalline phases can be produced: brushite, octacalcium phosphate, hydroxyapaptite and fluoroapatite. The solubility parameter of the formed phases is ordered phosphafluoroapatite > hydroxyapaptite > octacalcium te > brushite. During enamel remineralization and crystal voids repair, the degree of remineralization and the repaired crystalline phase formed will depend on the concentration of bioavailable fluoride, calcium and phosphate ions. However several studies reported that the bioavailability of active fluorine ions is the critical factor promoting remineralization and inhibition of demineralization in calcium- and phosphaterich circumstance.13,18,25,30 Fluoride catalysis the diffusion of
7
calcium and phosphate into the tooth surface, which in turn remineralize the crystalline structures in dental cavities leading to fluorapatite crystals, which represent the most acid resistant phase. Taking all the previous elements in account might help to interpret the obtained results. It was found that fTCP group showed the highest remineralizing potential and the greatest inhibition to softening by exposure to acid challenge compared to all other groups, in terms of highest VHN, lowest Ra values, and reduction in the surface micro-porosities as depicted in SEM Fig. 1. This could be attributed to high fluoride content of this product. As it contains 22,600 ppm of fluoride in comparison to CCP-ACPF and ACPF products which contain 900 and 1100 ppm, respectively. The dose-dependent effect of active fluoride ion in the remineralization efficacy in calcium, phosphate rich media was addressed by several authors.13,18,25,31 ten Cate et al.31 attributed this to elevated external fluoride-level. Higher F-gradient promotes driving fluoride deeper into the lesion. In addition, advanced lesions have large crystalline surface area for fluoride adsorption. In fTCP products, functionalizing the b-TCP is prepared with silica, which may provide linking opportunities with hard-tissue defects under acidic condition. It can permeate throughout enamel without attacking the inter-prismatic organic material, which may encourage greater calcium, phosphate, and fluoride uptake in demineralized lesions.32 The results are in contrary to Shen et al.1 who found CCPACPF product showed higher remineralization potential compared to fTCP product. This contradiction could be attributed to difference in form and composition of the used products. Tooth cre`me containing fTCP and 950 ppm fluoride was used in the former study while in the present study; the used varnish form was containing 22,600 ppm fluoride. In addition, the results of the present study could be attributed to the regimen of application of the test products. In the present study CPP-ACPF and ACPF products were applied twice daily according to the manufacturer, while the fTCP varnish is recommended twice a year. Considering that CPPACPF and ACPF products contains low level of fluoride and is recommended twice a day, 4 weeks might be relatively short for CPP-ACPF and ACPF to act sufficiently. So, supposing that this study goes for long period, the result of CPP-ACPF and ACPF groups may be better than that of this study, so the effect of different application methods may be considerable.18 RMGI group product showed similar remineralizing potential to CPP-ACPF group after 2 weeks, and lowest remineralizing potential compared to fTCP, ACPF and CPP-ACPF groups after 4 weeks and further after exposure to acidic challenge. Two mechanisms have been proposed by which fluoride may be released from glass ionomer into an aqueous environment. One mechanism is a short-term reaction, which involves rapid dissolution from outer surface into the solution (wash out), whereas the second is more gradual and resulted in the sustained long-term diffusion of ions through its bulk.33 In this product the initial burst effect of fluoride release might be responsible for producing remineralizing potential comparable to CPP-ACPF when tested after 2 weeks. However the short-term gradual fluoride release from the bulk of the cement did not reach the remineralizing effect produced by the other tested compounds. It was found that this product
Please cite this article in press as: Elkassas D, Arafa A, Remineralizing efficacy of different calcium-phosphate and fluoride based delivery vehicles on artificial caries like enamel lesions. Journal of Dentistry (2014), http://dx.doi.org/10.1016/j.jdent.2013.12.017
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produced a sealing capacity as depicted in SEM figure of 2 weeks remineralization. However, the degradation of this sealed layer after 4 weeks owing to its acid–base reaction through continuous dissolution of the siliceous hydrogel layer or the polysalt matrix,34 lead to surface voids and cracks as depicted in SEM of 4 weeks with increase in surface roughness.35,36 Furthermore, following the exposure to acidic challenge, partial dissolution of coating layer was evident (SEM-AC) with the existence of demineralized surface. This might denote that this material provided neither optimal remineralization nor efficient infiltration to complete depth of demineralized lesion leading to the lowest VHN compared to other tested products after 4 weeks remineralization or exposure to acidic challenge. The results of the present study are in contrary to Zhou et al., 2013 study,37 who found no significant difference between fTCP product and RMGI sealant. This contradiction could be attributed to the difference in the formulas used to induce enamel demineralization, as in Zhou et al. study the formula was used to induce enamel erosion resulting in different lesion depths. In addition to the difference in nature of examined substrates, as in the former study, bovine specimens were evaluated, while human molars were used in the present study.
5.
Conclusion
Under the limitations of the present study, it can be concluded that: 1-Calcium phosphate based remineralizing agents provide superior remineralization effects and greater resistance to acid softening as compared to artificial saliva. 2-Extended period of time had helped to attain more benefits of remineralization regimens application. 3-ClinproTM remineralizing system confers the highest remineralization tendency with the highest potential for inhibition of surface softening by acid challenge.
Conflict of interest The authors received no financial support and declare no potential conflicts of interest with respect to authorship and/ or publication of this article.
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