Lubrication by salivary conditioning films Veeregowda, Deepak Halenahally
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Lubrication by Salivary Conditioning Films
Deepak Halenahally Veeregowda
Lubrication by Salivary Conditioning Films
University Medical Center Groningen, University of Groningen Groningen, The Netherlands
Printing of this thesis is sponsored by UMCG and Ducom Instruments (Europe) B.V.
Copyright © 2012 by Deepak Halenahally Veeregowda ISBN (printed version): 978‐90‐367‐5944‐1
ISBN (electronic version): 978‐90‐367‐5945‐8
Lubrication by Salivary Conditioning Films
ter verkrijging van het doctoraat in de Medische Wetenschappen aan de Rijksuniversiteit Groningen
op gezag van de
Rector Magnificus, dr. E. Sterken, in het openbaar te verdedigen op
maandag 7 januari 2013 om 16.15 uur
Deepak Halenahally Veeregowda geboren op 16 februari 1984
te Bangalore, India
Prof. dr. H.C. van der Mei Prof. dr. A. Vissink
Copromotor: Dr. P.K. Sharma
Beoordelingscommissie : Prof. dr. E. Veerman
Prof. dr. A. Herrmann
Prof. dr. G. Raghoebar
Paranimfen: Katya Ovchinnikova Shariyar Sharifi
For my mom and dad….
Chapter 1 General Introduction 1
Chapter 2 Influence of fluoride‐detergent combinations on the visco‐
elasticity of adsorbed salivary protein films (European Journal of Oral Science 2010; 119: 21‐26)
Chapter 3 Role of structure and glycosylation of adsorbed protein films in biolubrication
(PlOS One 2012; 7(8): e42600)
Chapter 4 Boundary lubrication by brushed salivary conditioning films and their degree of glycosylation
(Clinical Oral Investigations 2012; 16:1499‐1506)
Chapter 5 Recombinant cationic proteins to improve biolubrication
Chapter 6 Stannous fluoride increases the protection offered by salivary conditioning films against erosion
Chapter 7 General discussion
155 Acknowledgements 163
Articulating surfaces in the healthy human body like hip and knee joints, eye lids and the tongue are well lubricated and encourage social activities, whereas absence of this lubrication greatly deteriorates the quality of life, as observed in patients with Sjögrens syndrome [1‐3]. Lubrication at the articulating surfaces is attributed to interstitial fluids like synovial fluid secreted in the knee and hip joint spaces , tears secreted onto the cornea  and saliva secreted in the oral cavity . The mechanism of biolubrication between the articulating body surfaces is a function of the sliding velocity and contact pressures and depends on the friction coefficient, i.e. ratio of the friction to normal force. In Figure 1, three regimes of lubrication mechanisms are shown, i.e. boundary, elasto‐hydrodynamic and hydrodynamic lubrication that can all exist during the articulation of the various surfaces [5, 7, 8]. Boundary lubrication is expected in the oral cavity especially at molar surfaces  with a contact pressure of 87 MPa . Elasto‐hydrodynamic lubrication is suggested for oral soft tissues [8, 10, 11] and eye lids moving over the cornea  operating at contact pressures of 50 KPa  and 1 KPa , respectively. A combination of elasto‐hydrodynamic and hydrodynamic lubrication is observed for cartilage surfaces in knee and hip joints [5, 7] at contact pressure of 7.5 MPa . Boundary lubrication becomes the dominating lubrication mechanism after exhaustion of hydrodynamic and elasto‐
hydrodynamic lubrication [15‐18]. Therefore, boundary lubrication is considered as the final protective physiological lubrication mechanism. In boundary lubrication, the sliding surfaces are in contact (see Fig. 1) and the friction coefficient is lowered by an adsorbed thin film of boundary lubricants like a salivary conditioning film on oral surfaces , hyaluronic acid and proteoglycans from synovial fluid , and ocular mucins from tears . However, the
3 mechanism of boundary lubrication by these naturally occurring conditioning films is not well understood.
Figure 1 Stribeck curve indicating different lubrication regimes during articulation at varying sliding velocities and contact pressure.
Boundary lubrication: Sliding surfaces (A and B) in a fluid medium (blue) are in contact and shear the adsorbed thin protein film (orange). The friction coefficient is high and stable, as expected for between hard surfaces (teeth) in the oral cavity.
Elasto‐hydrodynamic lubrication: Sliding surfaces with adsorbed protein films are well separated in the presence of a fluid medium formed from elastic deformation of the surfaces. The friction coefficient is low and decreases with increasing ratio of (viscosity x velocity) over pressure, as for example in soft contacts between the cornea and the eye lids and between opposing joint surfaces.
Hydrodynamic lubrication: Sliding surfaces with adsorbed protein films are completely separated by a thick fluid medium and there is a low friction coefficient. This friction coefficient increases due to an increase in velocity, leading to dissipation of the fluid medium. For example, a high viscous synovial fluid separates cartilage surfaces at knee joints. Due to (patho)physiological
activities the viscosity of the synovial fluid can become lower resulting in an increase in friction at the knee joints.
Interestingly, boundary lubrication in the oral cavity has to persist under severe and ever changing conditions. It is only in the oral cavity that articulating surfaces like tooth enamel, tongue, palate, cheeks and floor of the mouth are exposed to either chemical or mechanical perturbations almost on an hourly basis, i.e. during eating, drinking, chewing, swallowing, speech, use of oral and pharmaceutical products and toothbrushing. Fascinatingly, in a healthy person, the adsorbed salivary proteins exert a lubricating action on all oral surfaces under these varying physiological conditions.
Because of the interesting daily perturbations of salivary conditioning films, the ease of accessibility of saliva and the oral cavity in general, we have chosen oral salivary conditioning films as a model for our biolubrication studies.
SALIVARY CONDITIONING FILMS AND ORAL HEALTH
A salivary conditioning film is a mixture of adsorbed salivary proteins like mucins, proline‐rich proteins, histatins and statherins [19, 21, 22] found on all surfaces in the oral cavity [23, 24]. These conditioning films have an enormous influence on oral health due to their role in protecting oral surfaces against erosion, caries, periodontitis [26‐27], mechanical abrasion [28, 29] and noxious influences from, e.g., oral flora, drinks and food. Erosion and caries protection is due to the buffering capacity of salivary conditioning films, including its selective ion permeable like network . The abrasion resistance created by the salivary conditioning films is through wear resistant lubrication between oral sliding surfaces [6, 8, 10, 28, 30‐33]. The lubricating effect by salivary conditioning films adsorbed on glass was demonstrated in vitro against a sliding silica ball and
5 showed a 10 times decrease in coefficient of friction at a contact pressure of 380 MPa when compared to coefficient of friction between a silica ball sliding on glass without an adsorbed salivary conditioning film . However, it is unknown which proteins in salivary conditioning films contribute most to boundary lubrication [11, 35‐38].
Patients with hyposalivation, where the salivary flow is hampered by disfunctioning salivary glands as e.g. after radiation therapy, may struggle with severe pain and oral dryness due to an in complete salivary conditioning film and therewith reduced biolubrication [39, 40]. Glycosylated mucins are known for immobilizing water molecules during articulation of opposing surfaces , especially in the oral cavity during speaking, mastication and swallowing, and assist in comfort during social activities. Whether there is a relationship between degree of glycosylation and lubrication is unknown, however.
EFFECT OF ORAL PRODUCTS ON SALIVARY CONDITIONING FILM AND SENSORY FEELING
Oral health care products like toothbrushes and toothpastes, are designed to remove biofilms in the oral cavity in order to improve dental hygiene and prevent disease, but they also affect the adsorbed salivary conditioning film covering all surfaces in the oral cavity. Toothpastes with detergents like sodium lauryl sulfate or sodium hexametaphosphate are increasing the wettability of salivary conditioning films [41, 42] and, simultaneously desorb adsorbed salivary proteins from the enamel surface . Not surprisingly, these changes in the wettability and adsorbed mass of the salivary conditioning films can be restored by saliva due to a naturally occurring mechanisms called “polar‐apolar layering” in salivary conditioning film formation . However, the secondary salivary conditioning
film can have different mechanical and physico‐chemical structures that may lead to changes in sensory perception, as often experienced after tooth brushing conditions.
Oral sensory perception is a collective response by various mechanoreceptors facing chemical and mechanical perturbations in the oral cavity . The mechano‐receptors on the tongue can be triggered by either gustatory perception and tactile perception or both. However, in the PhD research presented in this thesis we will concentrate on the tactile perception by the tongue. There are very few studies that identify physical parameters associated with the sensory feeling [45‐47]. One can speculate that the salivary conditioning film acts as an interface between tongue and tooth enamel and triggers the sensory feelings like smoothness, cleanliness and dryness. Interestingly, a sensory perception study in dry mouth patients evaluating various saliva substitutes, showed that a saliva substitute with glycosylated mucins components provided the best moist feeling and comfort for speech and mastication [48, 49]. Identifying the role of salivary conditioning film in lubrication and sensory perception of smoothness, cleanliness and dryness can provide clues for the development of improved therapeutics for dry mouth patients and, in designing the consumer preferred oral products.
PHYSICAL PARAMETERS THAT CAN INFLUENCE SALIVARY CONDITIONING FILM LUBRICATION
Friction forces between sliding surfaces can be controlled by tuning the physical parameters like adhesion force, roughness, toughness, contact area and thickness of the lubricating film separating the sliding surfaces. Nanotribology, the science of friction, wear and lubrication at nanoscale, sheds light on the relationships between the friction force and various physical parameters, as described below:
7 1) Relationship between friction force and adhesion force: Amonton’s law states that friction force at a sliding interface is directly proportional to the applied normal force at macroscale. At nanoscale friction experiments, the friction force is not only proportional to the applied normal force but also depends on the adhesion force, as described in equation 1 :
where, f is the friction force (nN), µ is the coefficient of friction (constant), Fn the applied normal force (nN) and Fadh is the surface adhesion force (nN). From equation 1 it follows that decreasing adhesion forces reduce friction forces.
2) Relationship between friction force and contact area: Friction force is directly proportional with the contact area, according to equation 2 :
f = τ*AC (2)
where, τ is the interfacial shear stress (Pa) and Ac the contact area (m2). At constant interfacial shear stress and decreasing contact area, the friction force will reduce. It has to be noted that the contact area depends on the surface roughness and elastic modulus of the conditioning film.
3) Relationship between friction force and separation between the contacting surfaces: In rheology the shear stress between sliding bodies in a Newtonian fluid is proportional with the shear rate at the interface, as described in equation 3:
f = μ *[F + F ]
. . V
τ = η*γ , and γ
where, η is the viscosity of the fluid (Pa s), γ. the shear rate (s‐1), V the sliding velocity (m s‐1), and D is the separation between the sliding bodies (m). By combining equations 2 and 3, we can deduce an inversely proportional relationship between the friction force and the separation distance between the sliding bodies, as described in equation 4:
This relationship has been demonstrated experimentally on biomimetic lubricants like polymer brushes [52, 53].
Examining the role of these relationships in naturally occurring lubricating films like salivary conditioning films is novel. Most importantly it will give us the intricate details for designing the biomimetic lubricants that may alleviate pain and discomfort in diseased lubrication conditions like arthritis and hyposalivation.
AIM OF THE THESIS
The aim of this thesis is to provide a comprehensive analysis of biolubrication in the oral cavity at a molecular level and to identify the role of salivary conditioning films in lubrication and oral tactile perception. Influences of chemical and mechanical perturbation of salivary conditioning films on biolubrication will be analyzed and effects of recombinant proteins adsorbed into adsorbed salivary protein films on biolubrication are determined, to provide a clue to improve current saliva substitutes.
f = D
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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 .
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.
Salivary film structure
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 . 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 . 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 . 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  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 , 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 . 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 . A higher characteristic film frequency implies a higher packing density in the adsorbed layer and an increased rigidity of the film.
Salivary film structure
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 . 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
Salivary film structure
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 .
Thicknesses and characteristic film frequencies obtained after treatment of a primary film and after secondary film formation were compared with those of
Salivary film structure
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.
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.
Salivary film structure
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
0 20 40 60 80
Primary film Paste treated film Secondary film Characteristic Frequency (MHz)
NaF-NaHMP SnF2-SLS SnF2-NaHMP
Au HAP 0
10 20 30 40 50 60
0 20 40 60 80
Primary film Paste treated film Secondary film Characteristic Frequency (MHz)
NaF-NaHMP SnF2-SLS SnF2-NaHMP
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.
Salivary film structure
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.
SPF on enamel
HAP SPF on
NaF‐SLS NaF‐NaHMP SnF2‐SLS SnF2‐NaHMP
56.6 66.9 66.1 62.3 52.0 60.4
53.1 59.9 59.3 44.5 60.2
26.7 22.3 23.9 25.4 28.7 23.7
28.2 25.1 27.0 34.2 27.2
0 0 0 0 0 0
0 0.7 0.6 0.4 0.4
0 0 0 0.6 0 2.2
0 0 0 2.0 0
Salivary film structure
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 . 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.  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
Salivary film structure
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  , 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 , which may reduce enamel demineralization and explain the role of SnF2 in erosion protection, as observed by Wiegand et al .
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.
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.