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Studies on a novel combination polymer system: in vitro erosion prevention and promotion of fluoride uptake in human enamel
journal of dentistry 38, S3 (2010) S4–S11
available at www.sciencedirect.com
journal homepage: www.intl.elsevierhealth.com/journals/jden
Studies on a novel combination polymer system: in vitro erosion prevention and promotion of fluoride uptake in human enamel Louise H. Graciaa , Alan Brownb , Gareth D. Reesc , Christabel E. Fowlera, * a
GlaxoSmithKline Consumer Healthcare, Weybridge, Surrey, UK CERAM Surface and Materials Analysis, Penkhull, Stoke-on-Trent, UK c Genesis Oral Bioscience, Regents Mews, Horley, Surrey, UK b
article info
abstract
Keywords: Human enamel Erosion Mouthrinse Fluoride Profilometry Dynamic secondary ion mass spectrometry DSIMS
Objectives: Firstly, determine the effect of pre-treating sound human enamel with a hydrosoluble combination polymer system (TriHydra™) comprising 0.20% carboxymethylcellulose, 0.010% xanthan gum and 0.75% copovidone, alone or in combination with fluoride, on in vitro erosion by citric acid. Secondly, investigate the effect of the polymers on fluoride uptake by incipient erosive lesions. Methods: Study 1: Sound enamel specimens were treated (60 s, 20ºC, 150 rpm) with either (i) deionised water, (ii) polymers in deionised water, (iii) 300 mg/L fluoride or (iv) polymers in 300 mg/L fluoride. Specimen groups (n = 5) were then immersed in 1.0% citric acid (pH 3.8, 300 s, 20ºC, 50 rpm) and non-contact profilometry was used to determine surface roughness (Sa) and bulk tissue loss. Study 2: Incipient erosive lesions were similarly treated with (i)-(iv). Dynamic Secondary Ion Mass Spectrometry (DSIMS) was then used to determine the fluoride depth-distribution. Results: Study 1: Mean±SD Sa and erosion depths for treatment groups (i)−(iv) were a 657±243, b 358±50, c 206±72, d 79±16 nm and a 19.73±8.70, b 2.52±1.34, b 0.49±0.34 and b 0.31±0.21 mm respectively (matching superscripts denote statistically equivalent groups). Study 2: Lesions treated with (iii) and (iv) exhibited similar fluoride penetration depths (~60 mm). Mean fluoride intensity ratios based on F/(F+P) at 1 mm for treatment groups (i)−(iv) were a 0.010±0.004, a 0.011±0.004, b 0.803±0.148 and c 0.994±0.004 respectively. Conclusions: The combination polymer system exhibited anti-erosion efficacy in its own right. The polymer/fluoride admixture statistically significantly reduced Sa, however suppression of bulk tissue loss was not statistically significantly different versus either treatment alone. The presence of polymer appears to promote fluoride uptake by erosive lesions most noticeably in the first 6 mm. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction Erosive wear is a multifactorial process that, in vivo, involves erosion in combination with either abrasion or * Corresponding author. Dr Christabel Fowler, GlaxoSmithKline Consumer Healthcare, Weybridge, Surrey KT13 0DE, UK. Tel.: +44 1932 822000; fax: +44 1932 822100. E-mail address: [email protected] (C.E. Fowler).
attrition, or a combination of both. 1−4 These processes are of chemical and physical, but not microbiological, origin and do not involve dental plaque. 3,4 This is one of a number of key features distinguishing erosion and erosive wear from dental caries. Erosion is primarily a surface chemical dissolution phenomenon characterised by top-down demineralisation, whilst the latter involves sub-surface demineralisation. 4 The two types of incipi-
0300-5712/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
Journal of Dentistry 38, S3 (2010) S4–S11
ent lesion can be differentiated in vitro using a technique such as transverse microradiography (TMR) which will show a mineralised front in the case of the caries lesion. 5 The prophylaxis and treatment of dental caries has rightly been a major and important focus of dental research for over fifty years. The introduction and now almost ubiquitous availability of fluoride being the principal driver for the observed decline in caries incidence. 6 However there is growing evidence that the last 10−15 years have seen an increase in the prevalence of erosion and erosive wear. 7 With improved self-awareness and general improvements in healthcare, individuals are keeping their dentition for longer. Studies reporting erosion and erosive wear in adolescents are thus concerning, 8−10 albeit there is significant variability in the reported incidence, 6,11 and few longitudinal epidemiological studies. 12−14 Agreement within the research community regarding choice of clinical indices and study design would further understanding of the contributing aetiologies and true extent and severity of the problem. 4,6,15 To date, excluding intrinsic sources of acid associated with voluntary or involuntary regurgitation, the primary aetiological factors correlated with erosion and erosive wear are gender, socioeconomic status and dietary acid intake. 8−14 Chemical and biological factors that are known to be important include pH, titratable acidity and substantivity of the erosive challenge, as well as saliva flow rate, buffering capacity and the presence of enamel-binding pellicle proteins. 16,17 In vivo, the salivary pellicle provides a physical barrier that confers a degree of protection against an erosive challenge, however the pellicle is susceptible to desorption under acid conditions, and newly formed pellicle is believed to be less effective at mitigating the damaging effects of an erosive challenge on the underlying hard tissue. 18 This may in part explain why subjects with higher frequency and total intake of dietary acid exhibit a higher incidence of dental erosion. As with caries, fluoride has been shown to be effective in vitro and in situ at inhibiting dietary acidmediated erosion of dental hard tissues as well as promoting repair of demineralised incipient erosive lesions. 19−30 Fluoride has also been shown to reduce erosive wear both in enamel and dentine. 20,21 The mechanism of action of fluoride is similar in both cases, that is to suppress acid demineralisation and promote remineralisation through formation of less acidsoluble fluoridated mineral phases. However, a typical erosive challenge is at least an order of magnitude more severe than that posed by plaque acid, and consequently higher fluoride levels are recommended for the management of dental erosion. 31 As fluoride levels in conventional delivery formats available to the public, such as dentifrices and rinses, are controlled through the relevant monograph, there is considerable interest
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in potentiating fluoride efficacy, adopting regimes and identifying anti-erosion technologies that may act in concert with existing fluoride technologies. A variety of analytical techniques have been used to study hard tissue erosion in vitro that include nanoindentation, microindentation, microradiography and 3D non-contact profilometry. 32 All the aforementioned techniques have advantages as well as disadvantages as some are destructive whilst others are not, and many provide only an indirect measure of the extent of substrate mineralisation. The major drawback, however, is that none of the techniques are element specific. Consequently, and despite fluoride being the most widely studied active in these systems, the measurement endpoints tell us little concerning the fate of fluoride itself. Fluoride incorporation onto and into enamel surfaces has been studied using X-ray photoelectron spectroscopy (XPS), 33,34 however the detection sensitivity is poor and the depth profiling rate slow. Secondary ion mass spectrometry (SIMS) is an alternative elementspecific technique whose superior sensitivity to XPS has been exploited in the determination of elemental distributions in biominerals. 35,36 The technique provides detailed compositional analysis of materials on a depth scale ranging from nanometers to several hundred microns in depth profiling mode with sensitivity in the ppm–ppb region for all elements. Additionally, in imaging mode, SIMS provides elemental maps with lateral resolution on the sub-micron scale. The primary aim of the present study was to evaluate a trademarked hydrosoluble combination triple polymer system (Tri-Hydra™) comprising 0.20% carboxymethylcellulose (CMC), 0.010% xanthan gum and 0.75% copovidone as a fluoride-compatible anti-erosion agent. The combination polymer system was originally developed for its utility as a film-forming oral lubricant in mouthrinses designed to prevent and/or relieve symptoms associated with xerostomia. 37 The combination polymer system has been evaluated alone and as an admixture with sodium fluoride in a single-treatment enamel erosion demineralisation model. A secondary aim of the study is to employ SIMS to investigate the effect of the combination polymer system on fluoride uptake by incipient erosive lesions prepared from polished human enamel.
2. Materials and methods 2.1. Materials Citric acid monohydrate, sodium fluoride, sodium hydroxide and thymol were obtained from Sigma-Aldrich (Poole, Dorset, UK). Carboxymethylcellulose (Blanose® 7H3SXF) was supplied by Hercules (Wilmington, DE, USA), xanthan gum (Keltrol-F) was supplied by Nutrasweet Kelco Co. (Chicago, IL, USA) and copovidone (Plasdone® S-630) obtained from ISP (Tadworth, Surrey, UK). Stycast
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Journal of Dentistry 38, S3 (2010) S4–S11
acrylic resin was obtained from Hitek Electronic Materials (Scunthorpe, Lincolnshire, UK). 2.2. Preparation of enamel specimens Extracted human molars and premolars were stored in an aqueous solution of saturated thymol for up to 2 weeks after removal of the roots and pulp. Prior to use, teeth were thoroughly rinsed and visually examined for evidence of damage, decay and white spot lesions. Sound enamel sections were cut from the buccal and lingual sides of the teeth using a Microslice 2 diamond wire saw supplied by Metals Research (Cambridge, Cambridgeshire, UK) fitted with an annular saw, grit size 280, supplied by Ultratec (Santa Ana, CA, USA). The sections were mounted in acrylic resin and cured overnight, then polished flat using 1200 and 2400 grit silicon carbide paper. Where required, artificial incipient erosive lesions were created by immersing the sound enamel specimens into 1.0% citric acid solution pH 3.8 (300 s, 20ºC, 50 rpm). Enamel specimens were stored enamel side up in a humidified sealed container at 4ºC. 2.3. Surface roughness and bulk tissue loss An ADE PhaseShift MicroXAM white light interferometer (ADE Phase Shift Inc., Tucson, AZ, USA) was used to interrogate surface topography. This non-contact technique allows surface roughness parameters to be determined on the nanometer scale, and allows the calculation of bulk tissue loss following an erosive challenge. 38 Multiple scans (n = 3) were acquired for each specimen within the erosion window created using an acid-resistant tape. Two scan areas were employed (200 mm × 160 mm and 100 mm × 80 mm) in order to ensure calculated values of the mean surface roughness (Sa) were representative. Bulk tissue loss was calculated from the step height difference between the exposed and unexposed enamel surface following removal of the tape. 2.4. Dynamic secondary ion mass spectrometry (DSIMS) analysis and fluoride quantification Fluoride uptake by enamel erosive lesions was determined by DSIMS depth profiling and cross-sectional imaging using a Cameca ims 4f instrument (Cameca, Paris, France). Specimens were sputter-coated with gold to prevent excessive charging, and depth profile analysis performed using a 12.5 keV O− primary ion beam with beam current of ~100 nA into an area of 150 mm × 150 mm, detecting negatively charged secondary ions with an extraction field of −2.25 keV. The sputtered crater depths were measured using white light interferometry, facilitating calibration of the depth profile x-axis (time) to depth (in microns). Following depth profile analysis,
specimens were cross-sectioned to expose the central plane (surface to bulk) of the enamel. After polishing the cut surface to remove the smear layer, sections were gold-coated prior to SIMS imaging analysis using a 15 keV O+2 primary ion beam (~1 nA). Images were acquired from areas of typically 200 mm × 200 mm and 70 mm × 70 mm respectively. Negative secondary ion detection was used with an extraction field of −4.5 keV and a normalincidence electron gun for charge compensation. The intensity of the ionised phosphorus signal was used to normalise the data, thereby allowing comparison of the relative fluoride concentration at a given depth via the mean fluoride intensity ratio (F/F+P) for each treatment group. 2.5. Effect of a combination polymer system on enamel erosion The anti-erosion efficacy of a hydrosoluble combination polymer system comprising 0.20% CMC, 0.010% xanthan gum and 0.75% w/w copovidone was investigated in vitro in the presence and absence of fluoride. Twenty sound polished enamel specimens were randomly assigned to one of four treatment groups. Specifically, (i) deionised (DI) water, (ii) combination polymer system in DI water, (iii) 300 mg/L fluoride and (iv) a 300 mg/L fluoride solution containing the combination polymer system. Following treatment (60 s, 20ºC, 150 rpm), specimens were rinsed with DI water, then immersed in 1.0% citric acid (pH 3.8, 20ºC, 50 rpm), rinsed again and analysed by non-contact profilometry to determine Sa and bulk tissue loss. The pH of all treatment solutions fell in the range 6.5−7.0. 2.6. The effect of the combination polymer system on fluoride uptake by incipient enamel erosive lesions Twenty enamel specimens, each containing an incipient erosive lesion, were randomly assigned to one of the four treatment groups (n = 5) detailed in section 2.5. Following treatment (60 s, 20ºC, 150 rpm), specimens were rinsed with DI water, then analysed by DSIMS. Depth profiling mode was used initially to determine the relative fluoride concentration in the first few microns of the surface. Specimens were subsequently vertically sectioned through the lesion and fluoride distribution mapped to a depth of 170 mm. 2.7. Statistical analyses Means and standard deviation (SD) were calculated for Sa and erosion depth for each treatment group. Analysis of variance (ANOVA) and post hoc Student–Newman–Keuls were used to compare between-treatment statistical differences for each endpoint. Significance levels (p) were set at 0.05.
Journal of Dentistry 38, S3 (2010) S4–S11
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Table 1 – Mean±SD surface roughness (Sa) and erosion depth of enamel specimen groups following a 60 s treatment with either (i) DI water, (ii) 0.20% CMC, 0.010% xanthan and 0.75% copovidone (Polymers), (iii) 300 mg/L fluoride or (iv) 300 mg/L fluoride in the presence of the polymer system, prior to demineralisation with 1.0% citric acid pH 3.8 for 300 s Treatment
Mean±SD Sa (nm)
Mean±SD erosion depth (mm)
DI water
657±243 a
19.73±8.70 a
Polymers
358±49 b
2.52±1.34 b
300 mg/L fluoride
206±72 c
0.49±0.34 b
Polymers + fluoride
79±16 d
0.31±0.21 b
a−d Within
columns, different superscript letters indicate statistically significant differences between treatment groups (p < 0.05).
3. Results 3.1. Effect of a combination polymer system on enamel erosion The effects of the different pre-treatments on mean Sa and erosion depth following a subsequent citric acid challenge are summarised in Table 1. An increase in surface roughness was observed for all treatment groups. Mean Sa was statistically significantly lower for treatment groups (ii)−(iv) versus the DI-water control (p < 0.05). The 300 mg/L fluoride solution and the admix-
ture treatment groups conferred statistically significantly greater suppression of surface roughening than the combination polymer system alone (p < 0.05). The mean surface roughness of specimens treated with the admixture (79±16 nm) was statistically significantly lower than that associated with either the polymer only or fluoride only treatment groups (358±50 and 206±72 nm respectively). The surface damage caused by the erosive challenge is clearly apparent in Figures 1a−d which show representative topographic images for enamel specimens taken from the four treatment groups.
(a)
(b)
(c)
(d)
Fig. 1 – Representative 3D topographic images taken from enamel specimens pre-treated for 60 s with (a) DI water, (b) CMC/xanthan/copovidone polymer system, (c) 300 mg/L fluoride and (d) CMC/xanthan/copovidone polymer system plus fluoride, and subsequently demineralised with 1.0% citric acid pH 3.8.
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Journal of Dentistry 38, S3 (2010) S4–S11
(a)
(b)
Fig. 2 – Representative 2D line profiles for enamel specimens pre-treated for 60 s with either (a) DI water or (b) the CMC/ xanthan/copovidone polymer system plus fluoride, and subsequently demineralised with 1.0% citric acid pH 3.8.
Representative 2D line profiles for specimens selected from the DI water and polymer/fluoride admixture treatment groups are shown in Figures 2a and 2b respectively. Suppression of bulk tissue loss in treatment groups (ii)−(iv) was highly statistically significant versus the DI-water negative control (p < 0.001). Pre-treatment of specimens with the combination polymer system resulted in an eight-fold reduction in bulk tissue loss (2.52±1.34) compared to that observed for the DI-water treatment group (19.73±8.70). The mean bulk tissue loss of specimens pre-treated with fluoride or the polymer/ fluoride admixture were reduced to sub-micron levels (0.49±0.34 and 0.31±0.21 mm respectively), however these differences were not statistically significant (p > 0.05).
shown in Figures 3a and 3b respectively. Inspection of these images shows that lesions treated with the admixture of fluoride and polymer contained substantially more fluoride in the first 6 mm of the specimen than those treated with fluoride alone. Representative DSIMS elemental line scans for specimens from treatment groups (iii) and (iv) are shown in Figures 4a and 4b respectively. Mean±SD relative fluoride levels can be calculated as a function of depth based on the DSIMS intensity ratio F/(F+P). At 1 mm, the mean intensity ratios for treatment groups (i) to (iv) were a 0.010±0.004, a 0.011±0.004, b 0.803±0.148 and c 0.994±0.004 respectively [values with different superscript letters are statistically significantly different (p < 0.05)].
3.2. The effect of the Tri-Hydra polymers on fluoride uptake by incipient enamel erosive lesions
4. Discussion
DSIMS cross-sectional images of lesions from treatment groups (iii) and (iv) exhibited a fluoride band extending from the specimen surface to a depth of ~60 mm that was absent in specimens from treatment groups (i) and (ii). Representative cross-sectional images of the former are
(a)
(b)
Fig. 3 – DSIMS cross-sectional images of enamel specimens containing incipient erosive lesions following treatment for 60 s with a solution containing (a) 300 mg/L fluoride or (b) 300 mg/L fluoride containing the triple polymer (CMC/xanthan/copovidone) system. High fluoride intensity shows up as white in this greyscale image.
Formulation optimisation is an essential element in the development of effective drug delivery systems and affects retention, dissolution and bioavailability of actives. In particular, formats designed for the local delivery of actives to the oral cavity must overcome the challenges associated with clearance by salivary washout. The combination polymer system evaluated in the present study was originally developed for its utility as a film-forming oral lubricant in mouthrinses designed to prevent and/or relieve the symptoms associated with xerostomia. 36 The present study sought to investigate whether its film-forming properties might also confer an anti-erosion benefit in a single-treatment in vitro model using polished sound enamel where the effects of a subsequent erosive insult could be determined using 3D non-contact profilometry. The combination polymer system proved to be more effective than originally anticipated, as evidenced by suppression of bulk tissue loss that was reduced by almost an order of magnitude compared to its DI-water placebo control. Its anti-erosion efficacy based on suppression of surface roughening and bulk tissue loss was substantial, albeit the mean Sa for this treatment group was significantly higher than that
Journal of Dentistry 38, S3 (2010) S4–S11
Oxygen
(c/s)
Carbon Fluoride Phosphorus
(a)
Distance (μm)
(c/s) Oxygen
Carbon
Phosphorus
Fluoride
(b)
Distance (μm)
Fig. 4 – DSIMS elemental line scans for fluoride, phosphorus, oxygen and carbon as a function of depth in incipient erosive lesions treated for 60 s with (a) 300 mg/L fluoride and (b) 300 mg/L fluoride containing the triple polymer (CMC/xanthan/copovidone) system.
observed in the group pre-treated with 300 mg/L fluoride. The combination polymer system employed as an admixture with fluoride conferred significantly greater suppression of enamel surface etching, as reflected by Sa, in comparison to either component alone. Using erosion depth as the measurement endpoint, bulk tissue loss was almost completely suppressed by pre-treatment of enamel with either fluoride or the polymer/fluoride admixture. It is possible the two treatment groups
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would be statistically differentiable using a 5-day in vitro enamel erosion cycling model where levels of bulk tissue loss are greater. 39 The results obtained in the current study are consistent with a previously published study that showed that a range of polymers, including CMC and xanthan, were effective inhibitors of HA crystal dissolution in 50 mM (0.30% w/w) acetic acid pH 5.0. 40 The authors proposed formation of a barrier film as the most likely mode of action, however it should be noted that the aforementioned study was simulating a plaque acid challenge that is relatively benign in comparison to the 1.0% citric acid pH 3.8 challenge employed in the present study. The apparent improvement in anti-erosion efficacy obtained by combining the triple polymer system with 300 mg/L fluoride may be attributable solely to the combined effects of the barrier function of an adsorbed polymeric film and the known anti-erosion effect of the fluoride. The CMC and xanthan gum are anionic polysaccharides, and the copovidone is a non-ionic copolymer so it is unlikely that there is a specific interaction between the fluoride ion and the polymers that serves to transport fluoride to the surface of the enamel specimens. Nonetheless, the DSIMS analysis of erosive lesions treated with fluoride with and without the combination polymer system suggests that fluoride delivery is enhanced in the presence of the polymers, at least in regard to the upper ~6 mm of the specimen. Fluoride uptake by the lesions is clearly apparent in both treatment groups to a depth of ~60 mm. All specimens were rinsed with DI water following treatment. For specimens treated with the admixture of polymers and fluoride, the substantivity and microviscosity of the adsorbed polymer film may effectively “trap” fluoride close to the surface of the specimen. Further investigations are required to investigate this hypothesis and assess its potential in situ/in vivo relevance.
5. Conclusion A combination polymer system comprising CMC, xanthan and copovidone delivered as an aqueous rinse exhibits anti-erosion efficacy in a single-treatment in vitro demineralisation model. Furthermore, the antierosion efficacy of a fluoride rinse is augmented by the addition of the polymers. DSIMS analysis of erosive lesions treated with fluoride with/without polymer indicates enhanced delivery of fluoride to the surface of the lesion in the presence of the combination polymer system.
6. Acknowledgements This study was supported by GlaxoSmithKline Consumer Healthcare, Weybridge, Surrey, UK.
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Journal of Dentistry 38, S3 (2010) S4–S11
7. Conflict of interest statement LHG and CEF are employees of GlaxoSmithKline Consumer Healthcare; GDR was an employee of GSK at the time this research was conducted. Subsequent to this study, GDR received compensation from GSK for consulting and scientific writing services in his new role as Director of Genesis Oral Bioscience. AB is an employee of CERAM Surface and Materials Analysis and was engaged by GSK to undertake profilometric and DSIMS analysis of pre-coded specimens supplied by the study sponsor.
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available at www.sciencedirect.com
journal homepage: www.intl.elsevierhealth.com/journals/jden
Studies on a novel combination polymer system: in vitro erosion prevention and promotion of fluoride uptake in human enamel Louise H. Graciaa , Alan Brownb , Gareth D. Reesc , Christabel E. Fowlera, * a
GlaxoSmithKline Consumer Healthcare, Weybridge, Surrey, UK CERAM Surface and Materials Analysis, Penkhull, Stoke-on-Trent, UK c Genesis Oral Bioscience, Regents Mews, Horley, Surrey, UK b
article info
abstract
Keywords: Human enamel Erosion Mouthrinse Fluoride Profilometry Dynamic secondary ion mass spectrometry DSIMS
Objectives: Firstly, determine the effect of pre-treating sound human enamel with a hydrosoluble combination polymer system (TriHydra™) comprising 0.20% carboxymethylcellulose, 0.010% xanthan gum and 0.75% copovidone, alone or in combination with fluoride, on in vitro erosion by citric acid. Secondly, investigate the effect of the polymers on fluoride uptake by incipient erosive lesions. Methods: Study 1: Sound enamel specimens were treated (60 s, 20ºC, 150 rpm) with either (i) deionised water, (ii) polymers in deionised water, (iii) 300 mg/L fluoride or (iv) polymers in 300 mg/L fluoride. Specimen groups (n = 5) were then immersed in 1.0% citric acid (pH 3.8, 300 s, 20ºC, 50 rpm) and non-contact profilometry was used to determine surface roughness (Sa) and bulk tissue loss. Study 2: Incipient erosive lesions were similarly treated with (i)-(iv). Dynamic Secondary Ion Mass Spectrometry (DSIMS) was then used to determine the fluoride depth-distribution. Results: Study 1: Mean±SD Sa and erosion depths for treatment groups (i)−(iv) were a 657±243, b 358±50, c 206±72, d 79±16 nm and a 19.73±8.70, b 2.52±1.34, b 0.49±0.34 and b 0.31±0.21 mm respectively (matching superscripts denote statistically equivalent groups). Study 2: Lesions treated with (iii) and (iv) exhibited similar fluoride penetration depths (~60 mm). Mean fluoride intensity ratios based on F/(F+P) at 1 mm for treatment groups (i)−(iv) were a 0.010±0.004, a 0.011±0.004, b 0.803±0.148 and c 0.994±0.004 respectively. Conclusions: The combination polymer system exhibited anti-erosion efficacy in its own right. The polymer/fluoride admixture statistically significantly reduced Sa, however suppression of bulk tissue loss was not statistically significantly different versus either treatment alone. The presence of polymer appears to promote fluoride uptake by erosive lesions most noticeably in the first 6 mm. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction Erosive wear is a multifactorial process that, in vivo, involves erosion in combination with either abrasion or * Corresponding author. Dr Christabel Fowler, GlaxoSmithKline Consumer Healthcare, Weybridge, Surrey KT13 0DE, UK. Tel.: +44 1932 822000; fax: +44 1932 822100. E-mail address: [email protected] (C.E. Fowler).
attrition, or a combination of both. 1−4 These processes are of chemical and physical, but not microbiological, origin and do not involve dental plaque. 3,4 This is one of a number of key features distinguishing erosion and erosive wear from dental caries. Erosion is primarily a surface chemical dissolution phenomenon characterised by top-down demineralisation, whilst the latter involves sub-surface demineralisation. 4 The two types of incipi-
0300-5712/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
Journal of Dentistry 38, S3 (2010) S4–S11
ent lesion can be differentiated in vitro using a technique such as transverse microradiography (TMR) which will show a mineralised front in the case of the caries lesion. 5 The prophylaxis and treatment of dental caries has rightly been a major and important focus of dental research for over fifty years. The introduction and now almost ubiquitous availability of fluoride being the principal driver for the observed decline in caries incidence. 6 However there is growing evidence that the last 10−15 years have seen an increase in the prevalence of erosion and erosive wear. 7 With improved self-awareness and general improvements in healthcare, individuals are keeping their dentition for longer. Studies reporting erosion and erosive wear in adolescents are thus concerning, 8−10 albeit there is significant variability in the reported incidence, 6,11 and few longitudinal epidemiological studies. 12−14 Agreement within the research community regarding choice of clinical indices and study design would further understanding of the contributing aetiologies and true extent and severity of the problem. 4,6,15 To date, excluding intrinsic sources of acid associated with voluntary or involuntary regurgitation, the primary aetiological factors correlated with erosion and erosive wear are gender, socioeconomic status and dietary acid intake. 8−14 Chemical and biological factors that are known to be important include pH, titratable acidity and substantivity of the erosive challenge, as well as saliva flow rate, buffering capacity and the presence of enamel-binding pellicle proteins. 16,17 In vivo, the salivary pellicle provides a physical barrier that confers a degree of protection against an erosive challenge, however the pellicle is susceptible to desorption under acid conditions, and newly formed pellicle is believed to be less effective at mitigating the damaging effects of an erosive challenge on the underlying hard tissue. 18 This may in part explain why subjects with higher frequency and total intake of dietary acid exhibit a higher incidence of dental erosion. As with caries, fluoride has been shown to be effective in vitro and in situ at inhibiting dietary acidmediated erosion of dental hard tissues as well as promoting repair of demineralised incipient erosive lesions. 19−30 Fluoride has also been shown to reduce erosive wear both in enamel and dentine. 20,21 The mechanism of action of fluoride is similar in both cases, that is to suppress acid demineralisation and promote remineralisation through formation of less acidsoluble fluoridated mineral phases. However, a typical erosive challenge is at least an order of magnitude more severe than that posed by plaque acid, and consequently higher fluoride levels are recommended for the management of dental erosion. 31 As fluoride levels in conventional delivery formats available to the public, such as dentifrices and rinses, are controlled through the relevant monograph, there is considerable interest
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in potentiating fluoride efficacy, adopting regimes and identifying anti-erosion technologies that may act in concert with existing fluoride technologies. A variety of analytical techniques have been used to study hard tissue erosion in vitro that include nanoindentation, microindentation, microradiography and 3D non-contact profilometry. 32 All the aforementioned techniques have advantages as well as disadvantages as some are destructive whilst others are not, and many provide only an indirect measure of the extent of substrate mineralisation. The major drawback, however, is that none of the techniques are element specific. Consequently, and despite fluoride being the most widely studied active in these systems, the measurement endpoints tell us little concerning the fate of fluoride itself. Fluoride incorporation onto and into enamel surfaces has been studied using X-ray photoelectron spectroscopy (XPS), 33,34 however the detection sensitivity is poor and the depth profiling rate slow. Secondary ion mass spectrometry (SIMS) is an alternative elementspecific technique whose superior sensitivity to XPS has been exploited in the determination of elemental distributions in biominerals. 35,36 The technique provides detailed compositional analysis of materials on a depth scale ranging from nanometers to several hundred microns in depth profiling mode with sensitivity in the ppm–ppb region for all elements. Additionally, in imaging mode, SIMS provides elemental maps with lateral resolution on the sub-micron scale. The primary aim of the present study was to evaluate a trademarked hydrosoluble combination triple polymer system (Tri-Hydra™) comprising 0.20% carboxymethylcellulose (CMC), 0.010% xanthan gum and 0.75% copovidone as a fluoride-compatible anti-erosion agent. The combination polymer system was originally developed for its utility as a film-forming oral lubricant in mouthrinses designed to prevent and/or relieve symptoms associated with xerostomia. 37 The combination polymer system has been evaluated alone and as an admixture with sodium fluoride in a single-treatment enamel erosion demineralisation model. A secondary aim of the study is to employ SIMS to investigate the effect of the combination polymer system on fluoride uptake by incipient erosive lesions prepared from polished human enamel.
2. Materials and methods 2.1. Materials Citric acid monohydrate, sodium fluoride, sodium hydroxide and thymol were obtained from Sigma-Aldrich (Poole, Dorset, UK). Carboxymethylcellulose (Blanose® 7H3SXF) was supplied by Hercules (Wilmington, DE, USA), xanthan gum (Keltrol-F) was supplied by Nutrasweet Kelco Co. (Chicago, IL, USA) and copovidone (Plasdone® S-630) obtained from ISP (Tadworth, Surrey, UK). Stycast
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acrylic resin was obtained from Hitek Electronic Materials (Scunthorpe, Lincolnshire, UK). 2.2. Preparation of enamel specimens Extracted human molars and premolars were stored in an aqueous solution of saturated thymol for up to 2 weeks after removal of the roots and pulp. Prior to use, teeth were thoroughly rinsed and visually examined for evidence of damage, decay and white spot lesions. Sound enamel sections were cut from the buccal and lingual sides of the teeth using a Microslice 2 diamond wire saw supplied by Metals Research (Cambridge, Cambridgeshire, UK) fitted with an annular saw, grit size 280, supplied by Ultratec (Santa Ana, CA, USA). The sections were mounted in acrylic resin and cured overnight, then polished flat using 1200 and 2400 grit silicon carbide paper. Where required, artificial incipient erosive lesions were created by immersing the sound enamel specimens into 1.0% citric acid solution pH 3.8 (300 s, 20ºC, 50 rpm). Enamel specimens were stored enamel side up in a humidified sealed container at 4ºC. 2.3. Surface roughness and bulk tissue loss An ADE PhaseShift MicroXAM white light interferometer (ADE Phase Shift Inc., Tucson, AZ, USA) was used to interrogate surface topography. This non-contact technique allows surface roughness parameters to be determined on the nanometer scale, and allows the calculation of bulk tissue loss following an erosive challenge. 38 Multiple scans (n = 3) were acquired for each specimen within the erosion window created using an acid-resistant tape. Two scan areas were employed (200 mm × 160 mm and 100 mm × 80 mm) in order to ensure calculated values of the mean surface roughness (Sa) were representative. Bulk tissue loss was calculated from the step height difference between the exposed and unexposed enamel surface following removal of the tape. 2.4. Dynamic secondary ion mass spectrometry (DSIMS) analysis and fluoride quantification Fluoride uptake by enamel erosive lesions was determined by DSIMS depth profiling and cross-sectional imaging using a Cameca ims 4f instrument (Cameca, Paris, France). Specimens were sputter-coated with gold to prevent excessive charging, and depth profile analysis performed using a 12.5 keV O− primary ion beam with beam current of ~100 nA into an area of 150 mm × 150 mm, detecting negatively charged secondary ions with an extraction field of −2.25 keV. The sputtered crater depths were measured using white light interferometry, facilitating calibration of the depth profile x-axis (time) to depth (in microns). Following depth profile analysis,
specimens were cross-sectioned to expose the central plane (surface to bulk) of the enamel. After polishing the cut surface to remove the smear layer, sections were gold-coated prior to SIMS imaging analysis using a 15 keV O+2 primary ion beam (~1 nA). Images were acquired from areas of typically 200 mm × 200 mm and 70 mm × 70 mm respectively. Negative secondary ion detection was used with an extraction field of −4.5 keV and a normalincidence electron gun for charge compensation. The intensity of the ionised phosphorus signal was used to normalise the data, thereby allowing comparison of the relative fluoride concentration at a given depth via the mean fluoride intensity ratio (F/F+P) for each treatment group. 2.5. Effect of a combination polymer system on enamel erosion The anti-erosion efficacy of a hydrosoluble combination polymer system comprising 0.20% CMC, 0.010% xanthan gum and 0.75% w/w copovidone was investigated in vitro in the presence and absence of fluoride. Twenty sound polished enamel specimens were randomly assigned to one of four treatment groups. Specifically, (i) deionised (DI) water, (ii) combination polymer system in DI water, (iii) 300 mg/L fluoride and (iv) a 300 mg/L fluoride solution containing the combination polymer system. Following treatment (60 s, 20ºC, 150 rpm), specimens were rinsed with DI water, then immersed in 1.0% citric acid (pH 3.8, 20ºC, 50 rpm), rinsed again and analysed by non-contact profilometry to determine Sa and bulk tissue loss. The pH of all treatment solutions fell in the range 6.5−7.0. 2.6. The effect of the combination polymer system on fluoride uptake by incipient enamel erosive lesions Twenty enamel specimens, each containing an incipient erosive lesion, were randomly assigned to one of the four treatment groups (n = 5) detailed in section 2.5. Following treatment (60 s, 20ºC, 150 rpm), specimens were rinsed with DI water, then analysed by DSIMS. Depth profiling mode was used initially to determine the relative fluoride concentration in the first few microns of the surface. Specimens were subsequently vertically sectioned through the lesion and fluoride distribution mapped to a depth of 170 mm. 2.7. Statistical analyses Means and standard deviation (SD) were calculated for Sa and erosion depth for each treatment group. Analysis of variance (ANOVA) and post hoc Student–Newman–Keuls were used to compare between-treatment statistical differences for each endpoint. Significance levels (p) were set at 0.05.
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Table 1 – Mean±SD surface roughness (Sa) and erosion depth of enamel specimen groups following a 60 s treatment with either (i) DI water, (ii) 0.20% CMC, 0.010% xanthan and 0.75% copovidone (Polymers), (iii) 300 mg/L fluoride or (iv) 300 mg/L fluoride in the presence of the polymer system, prior to demineralisation with 1.0% citric acid pH 3.8 for 300 s Treatment
Mean±SD Sa (nm)
Mean±SD erosion depth (mm)
DI water
657±243 a
19.73±8.70 a
Polymers
358±49 b
2.52±1.34 b
300 mg/L fluoride
206±72 c
0.49±0.34 b
Polymers + fluoride
79±16 d
0.31±0.21 b
a−d Within
columns, different superscript letters indicate statistically significant differences between treatment groups (p < 0.05).
3. Results 3.1. Effect of a combination polymer system on enamel erosion The effects of the different pre-treatments on mean Sa and erosion depth following a subsequent citric acid challenge are summarised in Table 1. An increase in surface roughness was observed for all treatment groups. Mean Sa was statistically significantly lower for treatment groups (ii)−(iv) versus the DI-water control (p < 0.05). The 300 mg/L fluoride solution and the admix-
ture treatment groups conferred statistically significantly greater suppression of surface roughening than the combination polymer system alone (p < 0.05). The mean surface roughness of specimens treated with the admixture (79±16 nm) was statistically significantly lower than that associated with either the polymer only or fluoride only treatment groups (358±50 and 206±72 nm respectively). The surface damage caused by the erosive challenge is clearly apparent in Figures 1a−d which show representative topographic images for enamel specimens taken from the four treatment groups.
(a)
(b)
(c)
(d)
Fig. 1 – Representative 3D topographic images taken from enamel specimens pre-treated for 60 s with (a) DI water, (b) CMC/xanthan/copovidone polymer system, (c) 300 mg/L fluoride and (d) CMC/xanthan/copovidone polymer system plus fluoride, and subsequently demineralised with 1.0% citric acid pH 3.8.
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(a)
(b)
Fig. 2 – Representative 2D line profiles for enamel specimens pre-treated for 60 s with either (a) DI water or (b) the CMC/ xanthan/copovidone polymer system plus fluoride, and subsequently demineralised with 1.0% citric acid pH 3.8.
Representative 2D line profiles for specimens selected from the DI water and polymer/fluoride admixture treatment groups are shown in Figures 2a and 2b respectively. Suppression of bulk tissue loss in treatment groups (ii)−(iv) was highly statistically significant versus the DI-water negative control (p < 0.001). Pre-treatment of specimens with the combination polymer system resulted in an eight-fold reduction in bulk tissue loss (2.52±1.34) compared to that observed for the DI-water treatment group (19.73±8.70). The mean bulk tissue loss of specimens pre-treated with fluoride or the polymer/ fluoride admixture were reduced to sub-micron levels (0.49±0.34 and 0.31±0.21 mm respectively), however these differences were not statistically significant (p > 0.05).
shown in Figures 3a and 3b respectively. Inspection of these images shows that lesions treated with the admixture of fluoride and polymer contained substantially more fluoride in the first 6 mm of the specimen than those treated with fluoride alone. Representative DSIMS elemental line scans for specimens from treatment groups (iii) and (iv) are shown in Figures 4a and 4b respectively. Mean±SD relative fluoride levels can be calculated as a function of depth based on the DSIMS intensity ratio F/(F+P). At 1 mm, the mean intensity ratios for treatment groups (i) to (iv) were a 0.010±0.004, a 0.011±0.004, b 0.803±0.148 and c 0.994±0.004 respectively [values with different superscript letters are statistically significantly different (p < 0.05)].
3.2. The effect of the Tri-Hydra polymers on fluoride uptake by incipient enamel erosive lesions
4. Discussion
DSIMS cross-sectional images of lesions from treatment groups (iii) and (iv) exhibited a fluoride band extending from the specimen surface to a depth of ~60 mm that was absent in specimens from treatment groups (i) and (ii). Representative cross-sectional images of the former are
(a)
(b)
Fig. 3 – DSIMS cross-sectional images of enamel specimens containing incipient erosive lesions following treatment for 60 s with a solution containing (a) 300 mg/L fluoride or (b) 300 mg/L fluoride containing the triple polymer (CMC/xanthan/copovidone) system. High fluoride intensity shows up as white in this greyscale image.
Formulation optimisation is an essential element in the development of effective drug delivery systems and affects retention, dissolution and bioavailability of actives. In particular, formats designed for the local delivery of actives to the oral cavity must overcome the challenges associated with clearance by salivary washout. The combination polymer system evaluated in the present study was originally developed for its utility as a film-forming oral lubricant in mouthrinses designed to prevent and/or relieve the symptoms associated with xerostomia. 36 The present study sought to investigate whether its film-forming properties might also confer an anti-erosion benefit in a single-treatment in vitro model using polished sound enamel where the effects of a subsequent erosive insult could be determined using 3D non-contact profilometry. The combination polymer system proved to be more effective than originally anticipated, as evidenced by suppression of bulk tissue loss that was reduced by almost an order of magnitude compared to its DI-water placebo control. Its anti-erosion efficacy based on suppression of surface roughening and bulk tissue loss was substantial, albeit the mean Sa for this treatment group was significantly higher than that
Journal of Dentistry 38, S3 (2010) S4–S11
Oxygen
(c/s)
Carbon Fluoride Phosphorus
(a)
Distance (μm)
(c/s) Oxygen
Carbon
Phosphorus
Fluoride
(b)
Distance (μm)
Fig. 4 – DSIMS elemental line scans for fluoride, phosphorus, oxygen and carbon as a function of depth in incipient erosive lesions treated for 60 s with (a) 300 mg/L fluoride and (b) 300 mg/L fluoride containing the triple polymer (CMC/xanthan/copovidone) system.
observed in the group pre-treated with 300 mg/L fluoride. The combination polymer system employed as an admixture with fluoride conferred significantly greater suppression of enamel surface etching, as reflected by Sa, in comparison to either component alone. Using erosion depth as the measurement endpoint, bulk tissue loss was almost completely suppressed by pre-treatment of enamel with either fluoride or the polymer/fluoride admixture. It is possible the two treatment groups
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would be statistically differentiable using a 5-day in vitro enamel erosion cycling model where levels of bulk tissue loss are greater. 39 The results obtained in the current study are consistent with a previously published study that showed that a range of polymers, including CMC and xanthan, were effective inhibitors of HA crystal dissolution in 50 mM (0.30% w/w) acetic acid pH 5.0. 40 The authors proposed formation of a barrier film as the most likely mode of action, however it should be noted that the aforementioned study was simulating a plaque acid challenge that is relatively benign in comparison to the 1.0% citric acid pH 3.8 challenge employed in the present study. The apparent improvement in anti-erosion efficacy obtained by combining the triple polymer system with 300 mg/L fluoride may be attributable solely to the combined effects of the barrier function of an adsorbed polymeric film and the known anti-erosion effect of the fluoride. The CMC and xanthan gum are anionic polysaccharides, and the copovidone is a non-ionic copolymer so it is unlikely that there is a specific interaction between the fluoride ion and the polymers that serves to transport fluoride to the surface of the enamel specimens. Nonetheless, the DSIMS analysis of erosive lesions treated with fluoride with and without the combination polymer system suggests that fluoride delivery is enhanced in the presence of the polymers, at least in regard to the upper ~6 mm of the specimen. Fluoride uptake by the lesions is clearly apparent in both treatment groups to a depth of ~60 mm. All specimens were rinsed with DI water following treatment. For specimens treated with the admixture of polymers and fluoride, the substantivity and microviscosity of the adsorbed polymer film may effectively “trap” fluoride close to the surface of the specimen. Further investigations are required to investigate this hypothesis and assess its potential in situ/in vivo relevance.
5. Conclusion A combination polymer system comprising CMC, xanthan and copovidone delivered as an aqueous rinse exhibits anti-erosion efficacy in a single-treatment in vitro demineralisation model. Furthermore, the antierosion efficacy of a fluoride rinse is augmented by the addition of the polymers. DSIMS analysis of erosive lesions treated with fluoride with/without polymer indicates enhanced delivery of fluoride to the surface of the lesion in the presence of the combination polymer system.
6. Acknowledgements This study was supported by GlaxoSmithKline Consumer Healthcare, Weybridge, Surrey, UK.
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7. Conflict of interest statement LHG and CEF are employees of GlaxoSmithKline Consumer Healthcare; GDR was an employee of GSK at the time this research was conducted. Subsequent to this study, GDR received compensation from GSK for consulting and scientific writing services in his new role as Director of Genesis Oral Bioscience. AB is an employee of CERAM Surface and Materials Analysis and was engaged by GSK to undertake profilometric and DSIMS analysis of pre-coded specimens supplied by the study sponsor.
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