Lubrication by salivary conditioning films Veeregowda, Deepak Halenahally
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Influence of fluoride‐detergent combinations on the visco‐elasticity of adsorbed salivary protein films
Veeregowda DH, Van der Mei HC, Busscher HJ, Sharma PK (2011) Eur J Oral Sci 119:21‐26 .
ABSTRACT
Visco‐elasticity of salivary‐protein‐films relates with mouthfeel, lubrication, biofilm formation and protection against erosion and is influenced by adsorption of toothpaste components. Hydrated film thickness and visco‐elasticity, determined by a Quartz Crystal Microbalance, of 2 h old in vitro adsorbed salivary‐
protein‐films were 43.5 nm and 9.4 MHz, whereas the dehydrated thickness, measured using X‐ray photoelectron spectroscopy, was 2.4 nm. Treatment with toothpaste slurries decreased the film thickness depending on fluoride‐detergent combination involved. Secondary exposure to saliva increased the film thickness to much of its original thickness, although no relation existed between hydrated and de‐hydrated film thicknesses indicating differences in film structure.
Treatment with stannous fluoride – sodium lauryl sulphate (SnF2‐SLS) containing toothpaste slurries yielded a strong, immediate two‐fold increase in characteristic film frequency with respect to untreated films, indicating cross‐linking in adsorbed salivary‐protein‐films by Sn2+ that was absent when SLS was replaced by sodium hexametaphosphate (NaHMP). Secondary exposure to saliva of films treated with SnF2 caused a strong six‐fold increase in characteristic frequency compared with primary salivary‐protein‐films, regardless whether SLS or NaHMP was the detergent. This suggests that ionized stannous, is not directly available for cross‐
linking in combination with highly negatively charged NaHMP, but becomes slowly available after initial treatment to cause cross‐linking during secondary exposure to saliva.
INTRODUCTION
Adsorbed salivary protein films form on all surfaces exposed to the oral cavity and consist of a wide variety of different proteins and glycoproteins [1‐3]. Important functions of adsorbed salivary protein films are lubrication [4‐6], as well as protection against erosion [7, 8] and abrasion [9]. Moreover, adhesion of oral pathogens nearly always takes place to an adsorbed protein film and not to nascent enamel surfaces. Oral hygiene products, including toothpastes, are mainly designed for biofilm control, but their detergents and other active ingredients also affect the properties of adsorbed salivary protein films [10, 11]. Additionally, the kinetics of action of these ingredients may be influenced by the formulation chemistry of the toothpaste, i.e. either being aqueous or non‐aqueous in nature.
A non‐aqueous formulation is sometimes applied to ensure stability of components that would interact in aqueous systems, like for instance the combination of SnF2 with NaHMP as a detergent.
X‐ray photoelectron spectroscopy (XPS) and contact angle studies on salivary protein films exposed to different oral health care products have indicated that components of these products may be adsorbed to the adsorbed protein film to affect their hydrophobicity [12]. Also in vivo, water contact angles on the front incisors of human volunteers decreased from 64 to 47 degrees after use of a NaF‐
SLS containing toothpaste, while inclusion of NaHMP as a detergent caused an even further reduction to 43 degrees. Alternatively, products containing SnF2‐SLS caused a reduction in water contact angles to only 54 degrees [10]. During the day, water contact angles recovered to their initial values. In line, in vitro studies have demonstrated that secondary exposure of treated adsorbed films to saliva tends to drive its surface free energy [13, 14] and ellipsometric thickness [14] back to their original values. However, whether this implies that the adsorbed salivary
protein film has recovered also with respect to its visco‐elastic properties is not known. In vitro, SnF2 containing toothpastes protect enamel more extensively against erosion than NaF containing ones [15], but the use of SnF2 in combination with SLS as a detergent has been associated with discoloration. Discoloration while using SnF2 containing toothpaste is avoided by combination with NaHMP as a detergent [16]. However, because stannous ions can interact with the highly negative phosphate groups in NaHMP, this requires a non‐aqueous toothpaste formulation.
Protection against erosion as well as discoloration can be envisaged as phenomena relating with the hydrated thickness of the adsorbed protein‐film and cross‐linking of proteins within the film. A high degree of cross‐linking will not only impede acids to reach the enamel surface, but at the same time will prevent chromophores from leaching out of the adsorbed salivary protein film. Visco‐
elastic properties of adsorbed salivary protein films, and their hydrated thickness have been measured using a quartz crystal microbalance with dissipation (QCM‐D) [17‐19], and can be related with the degree of cross‐linking in the protein films using the measured shear modulus (G) and viscosity () [20‐22] according to
fc = G/ (1)
in which (fc) is the characteristic film frequency. The characteristic film frequency relates to the visco‐elastic deformability of the layer and its relaxation under stress, as determined amongst others by the packing density of the adsorbed molecules, the hydration state of the film and cross‐linking within the film [23]. A higher characteristic film frequency implies a higher packing density in the adsorbed layer and an increased rigidity of the film.
The aim of this study was to determine the hydrated and dehydrated thickness, characteristic frequency and chemical composition of adsorbed salivary protein films prior to and after exposure to SnF2 and NaF containing toothpaste slurries, based on either SLS or NaHMP as a detergent, and constituted into aqueous or non‐aqueous formulations.
MATERIALS AND METHODS
Preparation of saliva and toothpaste slurry
Human whole saliva from 20 volunteers of both genders was collected into ice‐
cooled beakers after stimulation by chewing Parafilm® and then pooled, centrifuged, dialyzed, and lyophilized for storage. Prior to lyophilization, phenylmethylsulfonylfluoride was added to a final concentration of 1 mM as a protease inhibitor in order to reduce protein breakdown and preserve high‐
molecular weight mucins. Note that recently it has been shown that freeze‐
thawing does not alter a saliva which has been stored at ‐80ºC for a period of 6 months [24]. For experiments, lyophilized saliva was reconstituted at 1.5 mg ml‐1 in buffer (50 mM potassium chloride, 2 mM potassium phosphate, 1 mM calcium chloride, pH 6.8). Thus reconstituted saliva will be referred to as “saliva”.
Volunteers gave their consent to saliva donation, in agreement with the Ethics Committee at UMCG (approval no. M09.069162).
Toothpastes used were selected on the basis of the absence or presence of SnF2 and NaF, in combination with either SLS or NaHMP as a detergent and included Crest regular® (NaF‐SLS), Crest vivid white night® (NaF‐NaHMP), Crest gum care® (SnF2‐SLS) and Crest pro health® (SnF2‐NaHMP). Note that all pastes are aqueous formulations with the exception of Crest pro health® which is a non‐aqueous formulation. All toothpastes were manufactured by Proctor and Gamble
(Cincinnati‐USA) and obtained commercially. Throughout this study, 25% w/v toothpaste slurries in buffer were used, prepared after removal of abrasive particles by centrifugation at 5000g, 10oC.
Formation of adsorbed salivary protein films and QCM‐D
The kinetics of salivary protein adsorption was studied using a QCM‐D device, model Q‐sense E4 (Q‐sense, Gothenburg, Sweden). Gold (Au) plated AT‐cut quartz crystals and hydroxyapatite (HAP) coated crystals, with a sensitivity constant of 17.7 ng cm‐2 for a 5 MHz sensor crystal, were used as substratum. Before each experiment, the Au crystals were cleaned by 10 min UV/ozone treatment, followed by immersion into a 3:1:1 mixture of ultrapure water, NH3 and H2O2 at 70°C for 10 min, drying with N2 and another UV/ozone treatment. HAP coated crystals were rinsed in ethanol (100%) for 15 min, followed by drying with N2 and an UV/ozone treatment. The QCM‐D flow chamber is disc shaped with a volume of approximately 40 l and a diameter of 11.1 mm with the inlet and outlet facing the crystal surface. At the start of each experiment, the crystal sensor was incubated in adhesion buffer under flow. When stable base lines for both oscillating frequency, f and dissipation, D were achieved, saliva was introduced in the system by perfusion from the inlet to the outlet reservoir by a peristaltic pump (Ismatec SA, Glattbrugg, Switzerland), without recirculation. Experiments were conducted at 20°C for 2 h at a flow rate of 50 l min‐1, corresponding with a shear rate of approximately 3 s‐1. After primary film formation, a toothpaste slurry or buffer control was passed through the chamber for 2 min to form a paste‐
treated or untreated film, followed by another 2 h of salivary flow to form a secondary adsorbed film. After each step, the chamber was treated with buffer for 15 min to remove un‐adsorbed salivary proteins or toothpaste components.
The four chambers available in the E4 unit were simultaneously used in parallel
for primary salivary protein film formation and subsequently, salivary protein films on the different Au crystals were treated with different toothpaste slurries.
HAP coated crystals were included in some experiments to investigate a possible influence of the substrate material on the primary salivary protein film structure.
Data were acquired using QSoft 401 2.0.1.
Voigt model fitting
A Voigt model containing a spring and dash‐pot was fitted to the frequency (∆f) and dissipation (∆D) shifts using Q‐Tools 3.0.6, in order to calculate the adsorbed salivary protein film thickness, its shear modulus (G) and viscosity (η). The Voigt model parameters were derived from the response of the adsorbed film, in terms of frequency (∆f) and dissipation (∆D) shifts due to the deformation induced by the oscillatory shear motion of the sensor surface, in a similar fashion as the modulus of elasticity is derived by dividing the stress response to an induced strain. The shear modulus (G) and viscosity (η) were then converted into a characteristic film frequency using equation 1. Note that, the frequency shift is the change in frequency of the crystal due to adsorbed salivary protein film, whereas the characteristic film frequency (fc = G/) purely quantifies the visco‐
elasticity of the adsorbed salivary protein film. Fluid viscosities used in the modeling were 1.15 mPa s for saliva, 2 mPa s for NaF‐SLS, SnF2‐SLS and SnF2‐ NaHMP and 3mPa s for NaF‐NaHMP containing toothpaste slurries, as measured using a viscometer (Brookfield DV‐II+Pro‐USA), at 50 rpm and 20°C. Saliva and toothpaste slurry densities were always assumed to be 1000 kg m‐3. During buffer flow, the viscosity and density of water were employed, i.e. 1 mPa s and 1000 kg m‐3, respectively. All overtones (3, 5, 7, 9, 11 and 13) were used for fitting and determining the hydrated thickness and characteristic frequency of the adsorbed protein films.
X‐ray photoelectron spectroscopy
Chemical compositions and dehydrated layer thicknesses (nm) of the adsorbed salivary protein films on bovine enamel were evaluated using XPS (S‐Probe spectrometer; Surface Science Instruments, Mountain View, CA, USA), while Au and HAP coated crystals were included in some experiments to investigate a possible influence of the substrate material on the primary salivary protein film composition and dehydrated thickness. Bovine enamel samples were prepared by grinding the surface with abrasive paper (800 and 1200 grit size), and polishing with Al2O3 powder (particle diameter of 0.05 µm) in demineralized water. Crystals with primary adsorbed salivary protein films were removed from the QCM chamber, and immediately subjected to XPS measurements. Adsorbed salivary protein films were formed on enamel surfaces and treated as described above for QCM‐D.
XPS was conducted at a photoelectron collection angle of 55 degrees with the sample and an electron flood gun setting of 10 eV. Elemental surface concentrations were calculated from overall scans in the binding energy range of 1‐1100 eV with a 1000 x 250 µm spot and pass energy of 150 eV accounting for instrumental sensitivity factors. The dehydrated layer thickness was calculated from an overlayer model, based on attenuation of the Ca2p electron count arising from the enamel surface with respect to N1s electron count from the overlaying adsorbed salivary protein film [25].
Statistical analysis
Thicknesses and characteristic film frequencies obtained after treatment of a primary film and after secondary film formation were compared with those of
primary films using a two tailed Student t‐test. p < 0.05 was taken as statistically significant. All the experiments described were performed in triplicate.
RESULTS
The QCM‐D output ratio ΔD/Δf was greater than 10‐7 in all experiments indicating that the films studied were highly dissipative and supporting the application of the Voigt model. Frequency shift and dissipation changes after 2 h primary salivary protein adsorption on Au coated crystals yielded a hydrated film thickness and characteristic film frequency of 43.5 ± 0.1 nm and 9.4 ± 0.1 MHz with no significant effects of treatment with a buffer (i.e. untreated films) or secondary film formation, as shown in Figure 1 (left panel).
Note that the use of an HAP coated crystal yielded a similar hydrated thickness (38.0 ± 5.0 nm) and characteristic frequency (7.0 ± 2.0 MHz) for the primary adsorbed protein film as did the use of a Au coated crystal (see Fig. 2). Treatment with a NaF‐NaHMP toothpaste slurry caused a decrease in film thickness to 20.0 nm with respect to untreated films (see for an example Fig. 1, right panel), with a minor effect on characteristic film frequency (7.5 MHz after treatment).
Secondary adsorption of salivary proteins restored the hydrated film thickness to 41.0 nm and increased its characteristic frequency to 11.0 MHz.
Figure 1 Examples of the adsorption kinetics of salivary proteins over a period of 340 min, including primary film formation, untreated (left panel) or treated with toothpaste slurry containing NaF‐NaHMP (right panel) and secondary film formation.
A and B: Frequencies and dissipation changes, C and D: Changes in hydrated thickness, E and F: Changes in characteristic frequency.
Figure 2 Thickness of hydrated, adsorbed salivary protein films on a gold crystal (Au) and a hydroxyapatite coated crystal (HAP) and on gold crystals with a adsorbed salivary protein film after treatment with a toothpaste slurry, as well as after secondary exposure to saliva (top), and accompanying changes in characteristic frequencies (bottom). The error bars indicate the standard deviations over triplicate measurements. Statistically significant differences (p<0.05) of treated and secondary films with respect to the properties of primary films are indicated by (*), while significant differences of secondary films with respect to treated films are indicated by (#).
Treatment of primary adsorbed films with toothpaste slurries on Au coated crystals decreased the hydrated film thickness and this decrease was largest after
0 10 20 30 40 50 60
Thickness (nm)
0 20 40 60 80
Primary film Paste treated film Secondary film Characteristic Frequency (MHz)
*
*
*
*
* *
*
*
*#
#
#
*#
*#
*#
# NaF-SLS
NaF-NaHMP SnF2-SLS SnF2-NaHMP
Au HAP 0
10 20 30 40 50 60
Thickness (nm)
0 20 40 60 80
Primary film Paste treated film Secondary film Characteristic Frequency (MHz)
*
*
*
*
* *
*
*
*#
#
#
*#
*#
*#
# NaF-SLS
NaF-NaHMP SnF2-SLS SnF2-NaHMP
Au HAP
NaF‐NaHMP and SnF2‐SLS containing slurries (thicknesses equal to 16.0 ± 3.8 and 21.2 ± 1.8 nm, respectively; Fig. 2). Secondary adsorption restored these hydrated film thicknesses only partially, whereas after secondary film formation following treatment with SnF2‐NaHMP, a high secondary film thickness (48.1 ± 1.9 nm; Fig.
2) exceeding the one of the primary film, was obtained. Treatment with a SnF2‐SLS containing toothpaste slurry immediately increased the characteristic film frequency to 25.4 ± 7.8 MHz, but none of the other toothpaste slurries significantly altered the characteristic film frequency. Secondary salivary protein adsorption on primary films treated with SnF2‐SLS or SnF2–NaHMP containing slurries yielded a significant increase in the characteristic frequency to 61.6 ± 1.1 and 67.1 ± 0.7 MHz, respectively (Fig. 2).
Carbon was the main element detected by XPS regardless of substrate material or treatment. Calcium was detected as the major component of the enamel and HAP surfaces, while nitrogen was indicative of adsorbed protein films (Table 1). XPS analyses clearly indicated incorporation of stannous in treated primary and secondary salivary protein films, while sodium was only retained immediately after treatment, but never found in secondary films. Variations in the observed %Ca and %N, yielded the dehydrated film thickness which amounted 2.4 nm for untreated primary adsorbed salivary protein films on enamel surfaces and decreased strongest after exposure to a NaF‐NaHMP containing formulation (0.9 nm). Note, that the dehydrated thicknesses of adsorbed primary salivary protein films on Au and HAP coated crystals, calculated on the basis of the attenuation of the Au4f and Ca2p electron counts with respect to N1s electron counts (2.1 and 2.7 nm, respectively), were comparable to the dehydrated film thickness on enamel.
In general, secondary film formation restored the dehydrated thickness of the adsorbed salivary protein film to much of its original values.
Figure 3 Relation between hydrated (derived from QCM‐D on gold crystals) and dehydrated (derived from XPS on enamel) thickness of salivary protein films, immediately after treatment with different toothpaste slurries (closed symbols) and after secondary film formation (open symbols). Hydrated and dehydrated thicknesses of the adsorbed primary salivary protein films on gold (Au) and hydroxyapatite (HAP) coated crystals are included for comparison.
Table 1 Chemical composition and dehydrated thicknesses of untreated, adsorbed salivary protein films (SPF) on bovine enamel, hydroxyapatite (HAP) and gold (Au) substrates and on bovine enamel after exposure to toothpaste slurries and secondary salivary protein film formation.
Composition
SPF on enamel
SPF on
HAP SPF on
Au
NaF‐SLS NaF‐NaHMP SnF2‐SLS SnF2‐NaHMP
Paste Treated
Film
Secondary Film
Paste Treated
Film
Secondary
Film
Paste Treated
Film
Secondary Film
Paste Treated
Film
Secondary Film
%C
56.6 66.9 66.1 62.3 52.0 60.4
53.1 59.9 59.3 44.5 60.2
%O
26.7 22.3 23.9 25.4 28.7 23.7
28.2 25.1 27.0 34.2 27.2
%N
5.9
9.1
6.2
3.3
6.8
3.2
2.5
3.3
8.2
1.9
8.5
%Ca
7.5
1.6
0
3.5
7.3
8.9
2.8
0.9
2.8
2.3
1.4
%P
3.3
0
0
1.8
4.3
3.6
4.2
1.2
2.3
4.7
1.6
%S
0
0
0
2.6
0
0.4
2.6
3.5
0
3.1
0
%Sn
0 0 0 0 0 0
0 0.7 0.6 0.4 0.4
%Na
0 0 0 0.6 0 2.2
0 0 0 2.0 0
%Au
‐
‐
3.8
‐
‐
‐
‐
‐
‐
‐
‐
Film thickness
(nm)
2.4
2.7
2.1
2.9
2.1
0.9
2.4
2.2
3.2
1.8
2.8
DISCUSSION
QCM‐D and XPS have been used to study influences of different fluoride‐
detergent combinations on the thickness (hydrated and dehydrated) and visco‐
elastic properties of adsorbed salivary protein films. Both techniques show that the underlying substrate material, i.e. enamel, HAP or Au, has little influence on the thickness and visco‐elastic properties of primary adsorbed salivary protein films, which is supported by a comparative QCM‐D study using HAP and silica substrates [26]. Hence, in the remainder of this discussion, we will assume that the substrate material does not affect the conclusions regarding thickness and visco‐elastic properties of the adsorbed salivary protein films. Hydrated film thickness decreased depending on the fluoride‐detergent combination applied, but secondary film formation on treated films restored the film thickness to much of its original thickness. This restoration in film thickness was also seen in dehydrated conditions, as obtained by XPS although there was no direct correspondence between hydrated and dehydrated film thicknesses, as can be seen in Figure 3. Characteristic frequencies of the films increased immediately after treatment with a SnF2‐SLS containing slurry from an aqueous formulation due to an increase in the shear modulus, indicating cross‐linking in the film. When SnF2 was combined with NaHMP as a detergent in a non‐aqueous formulation, no immediate increase in characteristic frequency was observed. However, secondary film formation on primary salivary protein films treated with SnF2 had consistently higher characteristic frequencies, regardless of whether applied in combination with SLS or NaHMP.
Dehydrated film thicknesses decreased upon treatment of films, depending on the toothpaste slurry applied and were much smaller than measured in a hydrated state as shown in Figure 3. Difference between hydrated and dehydrated
thicknesses were also observed by Macakova et al. [17] using QCM and surface plasmon resonance, although their thicknesses were much smaller than measured in the current study, likely because no protease inhibitor was reportedly added to their saliva, which generally yields breakdown of high molecular weight proteins at room temperature within 20 min. However, Figure 3 does indicate a pattern of increasing thickness in hydrated and dehydrated state of secondary salivary protein film. The lack of a unique relationship between hydrated and dehydrated thickness suggests structural differences between adsorbed salivary protein films after exposure to different chemical formulations as reflected by the observed differences in characteristic frequencies as well. HMP from an aqueous formulation (NaF‐NaHMP combination) yielded the largest reduction in film thickness (both hydrated and dehydrated ones). In a non‐aqueous formulation (SnF2‐NaHMP combination) however, HMP causes a smaller reduction in film thickness probably because its availability in ionized form is not immediate, as it is in an aqueous formulation.
The immediate stiffening of the paste treated film after application of SnF2‐SLS must be attributed to cross‐linking of adsorbed proteins by divalent Sn2+‐cations.
Monovalent Na+‐cations are not able to cross‐link, explaining the absence of stiffening after NaF‐detergent combinations. The cross‐linking by Sn 2+‐cations is confirmed by the stiffening of secondary films formed after treatment with both SnF2‐SLS and SnF2‐NaHMP combinations, since intermediate buffer rinsing will further increase ionization of stannous after treatment with the non‐aqueous SnF2‐NaHMP combination. Thus Sn2+‐cations becoming slowly available may diffuse from the treated primary film and cross‐link the adsorbed proteins in the secondary film to increase its characteristic frequency. XPS indeed not only indicated stannous in treated primary films but also in secondary films, as shown
in Table 1. Also detachment of adhering oral bacteria from salivary protein films induced by exposure to a non‐aqueous SnF2‐NaHMP toothpaste slurry continued for much longer time intervals than the actual slurry exposure time indicating a slower release [27] , whereas detachment stimulated by SLS containing slurries was confined to the duration of exposure. This attests to the fact that both SnF2 and NaHMP can be absorbed in salivary protein films, and slowly ionize to exert their cross‐linking and detergent actions. Cross‐linking of the salivary protein films will decrease their porosity and therewith their ion‐permeability [7], which may reduce enamel demineralization and explain the role of SnF2 in erosion protection, as observed by Wiegand et al [15].
In summary, the influence of fluoride‐detergent combination on the thickness and characteristic frequency of salivary protein films is dependent on the aqueous or non‐aqueous paste formulation. SnF2 in a non‐aqueous formulation has a prolonged cross‐linking action compared with the immediate cross‐linking action by SnF2 in an aqueous formulation. Strong detergent effects of NaHMP could only be observed in the aqueous formulation.
ACKNOWLEDGEMENTS
This study was funded by the UMCG, Groningen, The Netherlands. Purchase of QCM‐D apparatus was made possible by grant no. 91107008 from ZonMW, The Netherlands. The authors thank Brandon Peterson for helpful discussions.
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