Author's Accepted Manuscript
Effect of adsorption time on the adhesion strength between salivary pellicle and Human tooth enamel Y.F. Zhang, J. Zheng, L. Zheng, Z.R. Zhou
www.elsevier.com/locate/jmbbm
PII: DOI: Reference:
S1751-6161(14)00376-2 http://dx.doi.org/10.1016/j.jmbbm.2014.11.024 JMBBM1329
To appear in: Journal of the Mechanical Behavior of Biomedical Materials
Received date:15 July 2014 Revised date: 17 November 2014 Accepted date: 22 November 2014 Cite this article as: Y.F. Zhang, J. Zheng, L. Zheng, Z.R. Zhou, Effect of adsorption time on the adhesion strength between salivary pellicle and Human tooth enamel, Journal of the Mechanical Behavior of Biomedical Materials, http://dx.doi.org/10.1016/j.jmbbm.2014.11.024 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Effect of adsorption time on the adhesion strength between salivary pellicle and human tooth enamel Y.F. Zhang, J. Zheng*, L. Zheng, Z.R. Zhou Tribology Research Institute, Key Laboratory of Advanced Technologies of Materials, Ministry of Education, Southwest Jiaotong University, Chengdu 610031, China Abstract Salivary pellicle is a biofilm that is formed by the selective adsorption of salivary proteins. Almost all the functions of the salivary pellicle (lubricating properties, anti-caries properties, etc) are closely associated with its adhesion strength to tooth surface. The objective of this study was to investigate the effect of adsorption time on the adhesion strength between salivary pellicle and human tooth enamel, aiming to understand what act as the determinant of the interfacial adhesion. In this study, human tooth enamel samples were immersed in human whole saliva in vitro to obtain a salivary pellicle on the surface of enamel. Immersion treatments lasting up to 1, 3, 10 and 60 min were conducted, respectively. Nano-scratch tests were conducted on the surface of enamel after different adsorption times. The wettability of enamel surface was measured through water contact angle. Results showed that the shear energy between salivary pellicle and enamel surface increased exponentially with the adsorption time. The adhesion force between salivary pellicle and bare enamel surface was more than twice that between salivary pellicle and salivary pellicle. It was found that both the wettability and zeta potential of enamel increased obviously after 1 min saliva-adsorption treatment, and then they almost kept stable as the adsorption time further increased. In summary, the adhesion strength between initial salivary pellicle and enamel surface was much higher than that between initial salivary pellicle and outer salivary pellicle. It seemed that electrostatic interaction contributed to the adhesion between the initial salivary pellicle and enamel surface, but not to the adhesion between the initial and outer salivary pellicle. The results would be helpful to extend the understanding of the adhesion mechanism of salivary pellicle and then to develop new artificial saliva and dental restorative materials. Keywords: Human tooth enamel, Salivary pellicle, Adhesion strength, Adsorption time *Corresponding author. Tel.: +86-28-87634037; fax: +86-28-87603142. E-mail address: [email protected] (J. Zheng)
1
1.
Introduction Human saliva is secreted from salivary glands. It consists of approximately 98% water and a
variety of electrolytes and proteins (De Almeida et al., 2008), such as secretory IgA, acidic proline-rich protein (PRP), cystatin SA-I high molecular weight mucin, lactoferrin, lysozyme, and amylase (Lendenmann et al., 2000). Those proteins can be selectively adsorbed onto all solid substrates exposed to oral environment. Thus, an extended and highly hydrated proteinaceous biofilm is formed, which is generally called acquired salivary pellicle (Zhang et al., 2013). The pellicle plays an important role in oral physiology. First of all, the pellicle serves as boundary lubricant between hard (enamel) and soft (mucosal) tissues to decrease tooth wear, reduce the friction of oral mucosa and tongue surfaces, and make swallowing easier. Mastication, deglutition and the faculty of speech depend strongly on the lubrication from salivary pellicle (De Almeida et al., 2008). Secondly, the role of salivary pellicle is also thought to protect the underlying tooth surface from acid attack because it is a buffer to acids introduced into the mouth (Hannig, 2002). The functions of saliva in the mouth depend strongly upon the adhesion strength of salivary pellicle on the substrate. It has been widely accepted that the adhesion strength between salivary pellicle and tooth surface is critical in determining whether the pellicle would be detached from the enamel surface under masticatory force or not. Once the pellicle is detached, the masticatory force is applied to the surfaces of the opposing teeth by tooth-tooth contact, which could result in excessive toothwear. Moreover, without the protection of salivary pellicle, the naked tooth surface is infinitely more vulnerable to acid-erosion. Previous studies suggested that generally the salivary pellicle could adhere tightly to tooth surface. As a result, the pellicle is really hard to be detached away and then can act as both an excellent lubricant and a protection against acid attack in oral environment (Berg et al., 2003; Zhang et al., 2013). Thus, it is very necessary to investigate the adhesion mechanism of pellicle on tooth surface, which could help develop new artificial saliva with similar adhesion/lubrication performance to natural human saliva, and then give valuable insights into the clinical treatments for tooth wear and dental erosion. Two parameters “pull-off adhesion strength” and “shear adhesion strength” have been used by various researchers, respectively, to evaluate the adhesion strength of biofilms (Schwender et al., 2005; Sotres et al., 2011). The pull-off adhesion strength, characterized with adhesion force (which is also called pull-off force), was reported to be the preferred parameter to assess the adhesion 2
strength of the biofilms which were mainly subjected to tension and compression stresses. For the biofilms subjected to shear stress, shear adhesion strength was considered a required parameter. Shear adhesion strength is generally characterized with critical shear force. Some works have been done to study the adhesion forces of salivary pellicle adsorbed onto substrates, such as human tooth enamel, silica, PMMA, etc (Bowen et al., 1998; Schwender et al., 2005). In these studies, salivary proteins were firstly adsorbed onto a tip, and then the coated tip was brought to close the surface of a substrate without any modifications along the vertical direction. After a defined interaction time, the tip was pulled away. During the whole process, the force-distance curve was automatically recorded using a scanning force microscopy or an atomic force microscope. Thus, the adhesion forces of salivary pellicle on substrates were measured (Bowen et al., 1998; Schwender et al., 2005). These studies mainly focused on the effect of pH-value or substrate properties on the pull-off adhesion strength. N. Schwender et al. used an atomic force microscopy to investigate adhesion force between salivary proteins and enamel surface at different pH-value (Schwender et al., 2005). It was found that adhesion forces showed an evident pH-value dependency. Xu et al. observed the adhesion force between salivary proteins and polyethylene surface with various levels of water wettability (Xu and Siedlecki, 2007). However, few efforts have been made to investigate the shear adhesion strength of salivary pellicle with human tooth enamel. Due to oral physiological functions such as mastication and swallow, the salivary pellicle adsorbed on tooth surface is usually subjected to the loading actions of normal and tangential forces in the mouth (Zhang et al., 2013). Especially the tangential force produced during mastication, often called shear force, was considered to be a main cause of the detachment of salivary pellicle. Therefore, studying the shear adhesion strength of salivary pellicle is really necessary to reveal the adhesion mechanism of pellicle on tooth surface. Additionally, both the thickness and structure of salivary pellicle were found to vary with the increase of adsorption time (Zhang et al., 2013). However, up to now, almost no research work has been done to study the variation of the pull-off/shear adhesion strength of salivary pellicle with adsorption time. In this paper, the shear and pull-off adhesion strength of the salivary pellicle formed on human tooth enamel surface after different adsorption times were investigated using a nano-scratch tester and an atomic force microscopy, respectively. In vitro adsorption was conducted by immersing the enamel samples in unstimulated human whole saliva for different times. The surface properties of 3
enamel samples were analyzed by means of contact angle and zeta potential measurements. Particular attention was paid to the effect of adsorption time on the adhesion strength of salivary pellicle to human tooth enamel.
2. Materials and methods
2.1. Sample preparation The chemical composition of human saliva is affected by stress or other stimuli (Krieger, 1975; Rohleder and Nater, 2009), and then saliva samples collected following proper collection procedures help obtain high quality data (Salimetrics and Europe, 2011). Therefore, human whole saliva (HWS) used in this study were collected from a single male donor in a convenient, non-invasion manner by passive drool into a small vial. The donor has no dental care and oral health problems. Moreover, in order to avoid the influence of contaminating substances introduced from foods or beverages to saliva, the samples were collected in the morning before breakfast. The donor was required to rinse mouth with water 10 minutes before saliva collection. In order to keep the natural performance of saliva, the collected saliva was used to do experiments immediately without any treatments (Zhang et al., 2013). Human tooth enamel samples were prepared from freshly extracted human mandibular second permanent molars (M2) without caries, which were from individuals aged between 20 and 22 (Zhang et al., 2013). Each tooth was cut into four parts, and each part was embedded with self-setting plastic to obtain an enamel sample with the exposed occlusal surface. The samples were ground and polished to obtain a flat testing surface (about 2×2 mm). The average roughness Ra of enamel samples was measured to be about 0.10 µm using a high resolution surface profilometer (TALYSURF6, England). The detailed preparation method of enamel samples referred to our previous study (Zhang et al., 2013).
2.2 Saliva-adsorption treatment
Adsorption treatments were conducted in vitro using human whole saliva. To investigate the effect of adsorption time on the shear adhesion strength of salivary pellicle to enamel, enamel 4
samples were divided into four groups to do adsorption treatments lasting up to 1, 3, 10 and 60 min, respectively. For each sample, a drop of previously collected HWS was dropped upon about half of its exposed enamel surface to obtain a salivary pellicle, as shown in Fig. 1. After a defined period of adsorption, the sample was carefully washed with deionised water to remove any residual saliva on its surface. Thus, two different areas appeared on the surface of each sample. One half was the bare surface without saliva-adsorption treatment, while the other half was the saliva-adsorbed surface. 2.3 Contact angle characterization The contact angles of both the bare and saliva-adsorbed enamel surfaces were measured using a Drop Analysis System (DSA100, Krüss Corp., Germany). Deionized water was used as a probe liquid. The results of our prepare experiments showed that an equilibrium contact angle was attained after 20 s and then changed little with time. Thus, the droplet which rested on the sample surface was photographed after 20 s. Given that free water may increase the wettability of a solid surface (Sipahi et al., 2001), the enamel samples were dried in ambient temperature before measurement. For each surface, the contact angle was determined through a minimum of eight independent measurements. 2.4 Zeta potential characterization For protein adsorption and the formations of pellicle and plaque, zeta potential is an important parameter used to describe surface interaction (Young et al., 1997). Electrophoresis technique is a traditional method used to measure zeta potentials of materials, and the measured samples need to be ground into particles before measurements. Recently, some experimental techniques have been developed to measure zeta potential on solid surfaces which would avoid grinding (Sze et al., 2003). It was found that for a solid surface, the zeta potential obtained by electrophoresis technique was similar to those obtained by other experimental techniques (Sze et al., 2003). In this study, the zeta potentials of both the bare enamel and the enamel subjected to saliva-adsorption treatment were measured by traditional electrophoresis technique at room temperature using Zetasizer (Nano-ZS90, Malvern, Britain). Before measurements, the enamel was ground into particles with a diamond saw under water-cooling condition. The particle-containing water was statically settled for 24 h, and 5
then the sedimentated enamel particles were collected. These particles were divided equally into five groups. One group was suspended in 6 mL 40 mmol/L sodium phosphate buffer (its pH value ranged from pH 7.7 to 7.9), which was referred to as “control”. The other four groups were incubated in 20 mL previously collected HWS for 1, 3, 10 and 60 min, respectively, at room temperature. These solutions were then centrifuged at 1100 g for 10 min. The obtained enamel particles were firstly washed with 1 mmol/L sodium phosphate buffer and then suspended in 6 ml 40 mmol/L sodium phosphate buffer. Subsequently, the suspensions were incubated with agitation for 1 h at room temperature. In order to remove large particles, the suspensions were statically settled for 45 min prior to zeta potential measurements. Only the enamel particles suspended in the upper half of the suspensions were used in the measurements. 2.5 Nano-scratch tests Nanoscratch technique has been widely used to study the shear adhesion strength of coating to substrate (Chalker et al., 1991; Lahiri et al., 2011; Sotres et al., 2011). Therefore, the shear adhesion strength between salivary pellicle and enamel was investigated using a nano-scratch tester (TI 900, Hysitron Corp., USA) in this study. The nano-scratch tester has a lateral resolution of 0.5 µN and a lateral displacement resolution of 3 nm. The thermal drift is maintained below 0.05 nm/s for all scratch tests. All the unidirectional scratch tests were conducted at a constant load mode using a standard conical tip with a radius of 1 µm. A normal load of 1.5 mN and a scratch length of 8 µm were used. The results of our prepare experiments showed that when the scratching was conducted under the normal load of 1.5 mN, the salivary pellicles on the saliva-adsorbed enamel surface were completely removed while only very slight plastic deformation occurred on the bare enamel surface. As mentioned above, there existed two different areas on the surface of each enamel sample. One half was the bare enamel surface, and another half was the saliva-adsorbed enamel surface. As shown in Fig.2.a, a scratch was started in the bare enamel surface, traversed through the salivary pellicle, and finally ended at the saliva-adsorbed surface. Thus, a scratch, about half in the bare enamel surface while another half in the adsorbed enamel surface, was obtained, which was referred to as “measured”. Subsequently, another scratch was done at the same location, which was referred to as “control”. The variations of shear force versus scratch displacement were recorded 6
automatically for each scratch, as shown in Fig.2.b. For the measured scratch, when the scratch tip moved through the pellicle, an extra shear force was applied to the tip, which could cause an increase of the shear force recorded automatically. As mentioned above, when the scratching was conducted under the applied normal load of 1.5 mN, the salivary pellicles on the saliva-adsorbed surface were completely removed while only very slight plastic deformation occurred on the bare enamel surface. So, for the control scratch, no obvious increase of shear force appeared in the shear force-displacement curve due to that the salivary pellicles on the saliva-adsorbed surface had been completely removed by the measured scratching. Thus, for each sample, the shear energy between salivary pellicle and enamel can be evaluated by calculating the area between the shear force-displacement curves of measured scratch and control scratch. The surface morphologies of scratches were examined by a Scanning Electronic Microscopy (SEM) (QUANTA200, FEI Corp., England). 2.6 Adhesion force measurements The adhesion forces of salivary pellicle on both the bare enamel surface and the enamel surface subjected to saliva-adsorption treatment were examined using an Atomic Force Microscopy (AFM) (SPI 3800N, Seiko, Japan). A silicon dioxide tip with a nominal radius of 2 µm was used. As shown in Fig. 3, for each sample, the adhesion force between bare tip and bare enamel surface was measured firstly, which was referred to as “control”. Subsequently, the tip was incubated in the previously collected HWS for 1 min to obtain a tip coated with salivary pellicle. Then the adhesion force of the coated tip on the bare enamel surface was measured, which represented the adhesion force of salivary pellicle on enamel surface. Afterwards, the enamel surface was subjected to different times of saliva-adsorption treatment. The adhesion force of the coated tip on the saliva-adsorbed enamel surface was measured, which represented the adhesion force of salivary pellicle on salivary pellicle. As a result, the adhesion forces of three different interfaces were measured in turn for each sample. For each interface, 8 measurements were made at different locations on a sample. It should be noted that either for the interface of coated tip and bare enamel surface or for the interface of coated tip and saliva-adsorbed enamel surface, no significant difference was observed among the 8 test results from a sample. This means that the saliva constitutes did not detach either from the tip or from the enamel surface within the test procedure. 7
3. Results
3.1. Surface morphology The surfaces of the enamel samples subjected to different times of saliva-adsorption treatment were firstly examined by SEM, and the typical micrographs are shown in Fig. 4. The enamel surface without any saliva-adsorption treatments was observed to be compact and smooth (Fig.4.a). After 1 min saliva-adsorption treatment, a dense salivary pellicle appeared on the surface of enamel (Fig.4.b). With the adsorption time further increasing, a few irregularly shaped agglomerates were formed on the dense salivary pellicle (Fig.4.c). The agglomerates increased with the adsorption time (Fig.4.d), and finally the initial salivary pellicle was completely covered by the agglomerates (Fig.4.e). 3.2 Shear adhesion strength As mentioned above, nano-scratch technique was used to examine the shear adhesion strength between salivary pellicle and enamel. Based on the two shear force-displacement curves of measured scratch and control scratch, the shear energy was calculated for each sample. For the enamel samples subjected to different times of saliva-adsorption treatment, the typical shear force-displacement curves are shown in Fig. 5. For a defined adsorption time, two curves obtained from an enamel sample were given. One curve was from the measured scratch which ran through the bare enamel surface and the adsorbed enamel surface, while another curve was from the control scratch. SEM images of the typical measured scratches are inserted on the top of the shear force-displacement curve figures. It was seen that for any one defined adsorption time, the salivary pellicle on enamel surface was completely scraped off under the applied normal load of 1.5 mN, while only very slight plastic deformation occurred on the bare enamel surface. As shown in Fig.5.a, the shear force of the enamel sample treated with 1 min saliva-adsorption increased from 200 up to 280 µN when the scratch tip moved from the juncture of the bare and saliva-adsorbed enamel surfaces to the end. And the value of shear force increased from 200 up to 300 µN after 3 min adsorption (Fig.5.b), from 200 up to 320 µN after 10 min adsorption (Fig.5.c), and from 200 up to 8
380 µN after 60 min adsorption, respectively (Fig.5.d). The corresponding shear energy between salivary pellicle and enamel surfaces subjected to different times of saliva-adsorption treatment and one-way analysis of variance (ANOVA) result are shown in Table 1. One-way ANOVA revealed a significant difference between the shear energy for enamel surfaces subjected to different times of saliva-adsorption treatment (P
Effect of adsorption time on the adhesion strength between salivary pellicle and Human tooth enamel Y.F. Zhang, J. Zheng, L. Zheng, Z.R. Zhou
www.elsevier.com/locate/jmbbm
PII: DOI: Reference:
S1751-6161(14)00376-2 http://dx.doi.org/10.1016/j.jmbbm.2014.11.024 JMBBM1329
To appear in: Journal of the Mechanical Behavior of Biomedical Materials
Received date:15 July 2014 Revised date: 17 November 2014 Accepted date: 22 November 2014 Cite this article as: Y.F. Zhang, J. Zheng, L. Zheng, Z.R. Zhou, Effect of adsorption time on the adhesion strength between salivary pellicle and Human tooth enamel, Journal of the Mechanical Behavior of Biomedical Materials, http://dx.doi.org/10.1016/j.jmbbm.2014.11.024 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Effect of adsorption time on the adhesion strength between salivary pellicle and human tooth enamel Y.F. Zhang, J. Zheng*, L. Zheng, Z.R. Zhou Tribology Research Institute, Key Laboratory of Advanced Technologies of Materials, Ministry of Education, Southwest Jiaotong University, Chengdu 610031, China Abstract Salivary pellicle is a biofilm that is formed by the selective adsorption of salivary proteins. Almost all the functions of the salivary pellicle (lubricating properties, anti-caries properties, etc) are closely associated with its adhesion strength to tooth surface. The objective of this study was to investigate the effect of adsorption time on the adhesion strength between salivary pellicle and human tooth enamel, aiming to understand what act as the determinant of the interfacial adhesion. In this study, human tooth enamel samples were immersed in human whole saliva in vitro to obtain a salivary pellicle on the surface of enamel. Immersion treatments lasting up to 1, 3, 10 and 60 min were conducted, respectively. Nano-scratch tests were conducted on the surface of enamel after different adsorption times. The wettability of enamel surface was measured through water contact angle. Results showed that the shear energy between salivary pellicle and enamel surface increased exponentially with the adsorption time. The adhesion force between salivary pellicle and bare enamel surface was more than twice that between salivary pellicle and salivary pellicle. It was found that both the wettability and zeta potential of enamel increased obviously after 1 min saliva-adsorption treatment, and then they almost kept stable as the adsorption time further increased. In summary, the adhesion strength between initial salivary pellicle and enamel surface was much higher than that between initial salivary pellicle and outer salivary pellicle. It seemed that electrostatic interaction contributed to the adhesion between the initial salivary pellicle and enamel surface, but not to the adhesion between the initial and outer salivary pellicle. The results would be helpful to extend the understanding of the adhesion mechanism of salivary pellicle and then to develop new artificial saliva and dental restorative materials. Keywords: Human tooth enamel, Salivary pellicle, Adhesion strength, Adsorption time *Corresponding author. Tel.: +86-28-87634037; fax: +86-28-87603142. E-mail address: [email protected] (J. Zheng)
1
1.
Introduction Human saliva is secreted from salivary glands. It consists of approximately 98% water and a
variety of electrolytes and proteins (De Almeida et al., 2008), such as secretory IgA, acidic proline-rich protein (PRP), cystatin SA-I high molecular weight mucin, lactoferrin, lysozyme, and amylase (Lendenmann et al., 2000). Those proteins can be selectively adsorbed onto all solid substrates exposed to oral environment. Thus, an extended and highly hydrated proteinaceous biofilm is formed, which is generally called acquired salivary pellicle (Zhang et al., 2013). The pellicle plays an important role in oral physiology. First of all, the pellicle serves as boundary lubricant between hard (enamel) and soft (mucosal) tissues to decrease tooth wear, reduce the friction of oral mucosa and tongue surfaces, and make swallowing easier. Mastication, deglutition and the faculty of speech depend strongly on the lubrication from salivary pellicle (De Almeida et al., 2008). Secondly, the role of salivary pellicle is also thought to protect the underlying tooth surface from acid attack because it is a buffer to acids introduced into the mouth (Hannig, 2002). The functions of saliva in the mouth depend strongly upon the adhesion strength of salivary pellicle on the substrate. It has been widely accepted that the adhesion strength between salivary pellicle and tooth surface is critical in determining whether the pellicle would be detached from the enamel surface under masticatory force or not. Once the pellicle is detached, the masticatory force is applied to the surfaces of the opposing teeth by tooth-tooth contact, which could result in excessive toothwear. Moreover, without the protection of salivary pellicle, the naked tooth surface is infinitely more vulnerable to acid-erosion. Previous studies suggested that generally the salivary pellicle could adhere tightly to tooth surface. As a result, the pellicle is really hard to be detached away and then can act as both an excellent lubricant and a protection against acid attack in oral environment (Berg et al., 2003; Zhang et al., 2013). Thus, it is very necessary to investigate the adhesion mechanism of pellicle on tooth surface, which could help develop new artificial saliva with similar adhesion/lubrication performance to natural human saliva, and then give valuable insights into the clinical treatments for tooth wear and dental erosion. Two parameters “pull-off adhesion strength” and “shear adhesion strength” have been used by various researchers, respectively, to evaluate the adhesion strength of biofilms (Schwender et al., 2005; Sotres et al., 2011). The pull-off adhesion strength, characterized with adhesion force (which is also called pull-off force), was reported to be the preferred parameter to assess the adhesion 2
strength of the biofilms which were mainly subjected to tension and compression stresses. For the biofilms subjected to shear stress, shear adhesion strength was considered a required parameter. Shear adhesion strength is generally characterized with critical shear force. Some works have been done to study the adhesion forces of salivary pellicle adsorbed onto substrates, such as human tooth enamel, silica, PMMA, etc (Bowen et al., 1998; Schwender et al., 2005). In these studies, salivary proteins were firstly adsorbed onto a tip, and then the coated tip was brought to close the surface of a substrate without any modifications along the vertical direction. After a defined interaction time, the tip was pulled away. During the whole process, the force-distance curve was automatically recorded using a scanning force microscopy or an atomic force microscope. Thus, the adhesion forces of salivary pellicle on substrates were measured (Bowen et al., 1998; Schwender et al., 2005). These studies mainly focused on the effect of pH-value or substrate properties on the pull-off adhesion strength. N. Schwender et al. used an atomic force microscopy to investigate adhesion force between salivary proteins and enamel surface at different pH-value (Schwender et al., 2005). It was found that adhesion forces showed an evident pH-value dependency. Xu et al. observed the adhesion force between salivary proteins and polyethylene surface with various levels of water wettability (Xu and Siedlecki, 2007). However, few efforts have been made to investigate the shear adhesion strength of salivary pellicle with human tooth enamel. Due to oral physiological functions such as mastication and swallow, the salivary pellicle adsorbed on tooth surface is usually subjected to the loading actions of normal and tangential forces in the mouth (Zhang et al., 2013). Especially the tangential force produced during mastication, often called shear force, was considered to be a main cause of the detachment of salivary pellicle. Therefore, studying the shear adhesion strength of salivary pellicle is really necessary to reveal the adhesion mechanism of pellicle on tooth surface. Additionally, both the thickness and structure of salivary pellicle were found to vary with the increase of adsorption time (Zhang et al., 2013). However, up to now, almost no research work has been done to study the variation of the pull-off/shear adhesion strength of salivary pellicle with adsorption time. In this paper, the shear and pull-off adhesion strength of the salivary pellicle formed on human tooth enamel surface after different adsorption times were investigated using a nano-scratch tester and an atomic force microscopy, respectively. In vitro adsorption was conducted by immersing the enamel samples in unstimulated human whole saliva for different times. The surface properties of 3
enamel samples were analyzed by means of contact angle and zeta potential measurements. Particular attention was paid to the effect of adsorption time on the adhesion strength of salivary pellicle to human tooth enamel.
2. Materials and methods
2.1. Sample preparation The chemical composition of human saliva is affected by stress or other stimuli (Krieger, 1975; Rohleder and Nater, 2009), and then saliva samples collected following proper collection procedures help obtain high quality data (Salimetrics and Europe, 2011). Therefore, human whole saliva (HWS) used in this study were collected from a single male donor in a convenient, non-invasion manner by passive drool into a small vial. The donor has no dental care and oral health problems. Moreover, in order to avoid the influence of contaminating substances introduced from foods or beverages to saliva, the samples were collected in the morning before breakfast. The donor was required to rinse mouth with water 10 minutes before saliva collection. In order to keep the natural performance of saliva, the collected saliva was used to do experiments immediately without any treatments (Zhang et al., 2013). Human tooth enamel samples were prepared from freshly extracted human mandibular second permanent molars (M2) without caries, which were from individuals aged between 20 and 22 (Zhang et al., 2013). Each tooth was cut into four parts, and each part was embedded with self-setting plastic to obtain an enamel sample with the exposed occlusal surface. The samples were ground and polished to obtain a flat testing surface (about 2×2 mm). The average roughness Ra of enamel samples was measured to be about 0.10 µm using a high resolution surface profilometer (TALYSURF6, England). The detailed preparation method of enamel samples referred to our previous study (Zhang et al., 2013).
2.2 Saliva-adsorption treatment
Adsorption treatments were conducted in vitro using human whole saliva. To investigate the effect of adsorption time on the shear adhesion strength of salivary pellicle to enamel, enamel 4
samples were divided into four groups to do adsorption treatments lasting up to 1, 3, 10 and 60 min, respectively. For each sample, a drop of previously collected HWS was dropped upon about half of its exposed enamel surface to obtain a salivary pellicle, as shown in Fig. 1. After a defined period of adsorption, the sample was carefully washed with deionised water to remove any residual saliva on its surface. Thus, two different areas appeared on the surface of each sample. One half was the bare surface without saliva-adsorption treatment, while the other half was the saliva-adsorbed surface. 2.3 Contact angle characterization The contact angles of both the bare and saliva-adsorbed enamel surfaces were measured using a Drop Analysis System (DSA100, Krüss Corp., Germany). Deionized water was used as a probe liquid. The results of our prepare experiments showed that an equilibrium contact angle was attained after 20 s and then changed little with time. Thus, the droplet which rested on the sample surface was photographed after 20 s. Given that free water may increase the wettability of a solid surface (Sipahi et al., 2001), the enamel samples were dried in ambient temperature before measurement. For each surface, the contact angle was determined through a minimum of eight independent measurements. 2.4 Zeta potential characterization For protein adsorption and the formations of pellicle and plaque, zeta potential is an important parameter used to describe surface interaction (Young et al., 1997). Electrophoresis technique is a traditional method used to measure zeta potentials of materials, and the measured samples need to be ground into particles before measurements. Recently, some experimental techniques have been developed to measure zeta potential on solid surfaces which would avoid grinding (Sze et al., 2003). It was found that for a solid surface, the zeta potential obtained by electrophoresis technique was similar to those obtained by other experimental techniques (Sze et al., 2003). In this study, the zeta potentials of both the bare enamel and the enamel subjected to saliva-adsorption treatment were measured by traditional electrophoresis technique at room temperature using Zetasizer (Nano-ZS90, Malvern, Britain). Before measurements, the enamel was ground into particles with a diamond saw under water-cooling condition. The particle-containing water was statically settled for 24 h, and 5
then the sedimentated enamel particles were collected. These particles were divided equally into five groups. One group was suspended in 6 mL 40 mmol/L sodium phosphate buffer (its pH value ranged from pH 7.7 to 7.9), which was referred to as “control”. The other four groups were incubated in 20 mL previously collected HWS for 1, 3, 10 and 60 min, respectively, at room temperature. These solutions were then centrifuged at 1100 g for 10 min. The obtained enamel particles were firstly washed with 1 mmol/L sodium phosphate buffer and then suspended in 6 ml 40 mmol/L sodium phosphate buffer. Subsequently, the suspensions were incubated with agitation for 1 h at room temperature. In order to remove large particles, the suspensions were statically settled for 45 min prior to zeta potential measurements. Only the enamel particles suspended in the upper half of the suspensions were used in the measurements. 2.5 Nano-scratch tests Nanoscratch technique has been widely used to study the shear adhesion strength of coating to substrate (Chalker et al., 1991; Lahiri et al., 2011; Sotres et al., 2011). Therefore, the shear adhesion strength between salivary pellicle and enamel was investigated using a nano-scratch tester (TI 900, Hysitron Corp., USA) in this study. The nano-scratch tester has a lateral resolution of 0.5 µN and a lateral displacement resolution of 3 nm. The thermal drift is maintained below 0.05 nm/s for all scratch tests. All the unidirectional scratch tests were conducted at a constant load mode using a standard conical tip with a radius of 1 µm. A normal load of 1.5 mN and a scratch length of 8 µm were used. The results of our prepare experiments showed that when the scratching was conducted under the normal load of 1.5 mN, the salivary pellicles on the saliva-adsorbed enamel surface were completely removed while only very slight plastic deformation occurred on the bare enamel surface. As mentioned above, there existed two different areas on the surface of each enamel sample. One half was the bare enamel surface, and another half was the saliva-adsorbed enamel surface. As shown in Fig.2.a, a scratch was started in the bare enamel surface, traversed through the salivary pellicle, and finally ended at the saliva-adsorbed surface. Thus, a scratch, about half in the bare enamel surface while another half in the adsorbed enamel surface, was obtained, which was referred to as “measured”. Subsequently, another scratch was done at the same location, which was referred to as “control”. The variations of shear force versus scratch displacement were recorded 6
automatically for each scratch, as shown in Fig.2.b. For the measured scratch, when the scratch tip moved through the pellicle, an extra shear force was applied to the tip, which could cause an increase of the shear force recorded automatically. As mentioned above, when the scratching was conducted under the applied normal load of 1.5 mN, the salivary pellicles on the saliva-adsorbed surface were completely removed while only very slight plastic deformation occurred on the bare enamel surface. So, for the control scratch, no obvious increase of shear force appeared in the shear force-displacement curve due to that the salivary pellicles on the saliva-adsorbed surface had been completely removed by the measured scratching. Thus, for each sample, the shear energy between salivary pellicle and enamel can be evaluated by calculating the area between the shear force-displacement curves of measured scratch and control scratch. The surface morphologies of scratches were examined by a Scanning Electronic Microscopy (SEM) (QUANTA200, FEI Corp., England). 2.6 Adhesion force measurements The adhesion forces of salivary pellicle on both the bare enamel surface and the enamel surface subjected to saliva-adsorption treatment were examined using an Atomic Force Microscopy (AFM) (SPI 3800N, Seiko, Japan). A silicon dioxide tip with a nominal radius of 2 µm was used. As shown in Fig. 3, for each sample, the adhesion force between bare tip and bare enamel surface was measured firstly, which was referred to as “control”. Subsequently, the tip was incubated in the previously collected HWS for 1 min to obtain a tip coated with salivary pellicle. Then the adhesion force of the coated tip on the bare enamel surface was measured, which represented the adhesion force of salivary pellicle on enamel surface. Afterwards, the enamel surface was subjected to different times of saliva-adsorption treatment. The adhesion force of the coated tip on the saliva-adsorbed enamel surface was measured, which represented the adhesion force of salivary pellicle on salivary pellicle. As a result, the adhesion forces of three different interfaces were measured in turn for each sample. For each interface, 8 measurements were made at different locations on a sample. It should be noted that either for the interface of coated tip and bare enamel surface or for the interface of coated tip and saliva-adsorbed enamel surface, no significant difference was observed among the 8 test results from a sample. This means that the saliva constitutes did not detach either from the tip or from the enamel surface within the test procedure. 7
3. Results
3.1. Surface morphology The surfaces of the enamel samples subjected to different times of saliva-adsorption treatment were firstly examined by SEM, and the typical micrographs are shown in Fig. 4. The enamel surface without any saliva-adsorption treatments was observed to be compact and smooth (Fig.4.a). After 1 min saliva-adsorption treatment, a dense salivary pellicle appeared on the surface of enamel (Fig.4.b). With the adsorption time further increasing, a few irregularly shaped agglomerates were formed on the dense salivary pellicle (Fig.4.c). The agglomerates increased with the adsorption time (Fig.4.d), and finally the initial salivary pellicle was completely covered by the agglomerates (Fig.4.e). 3.2 Shear adhesion strength As mentioned above, nano-scratch technique was used to examine the shear adhesion strength between salivary pellicle and enamel. Based on the two shear force-displacement curves of measured scratch and control scratch, the shear energy was calculated for each sample. For the enamel samples subjected to different times of saliva-adsorption treatment, the typical shear force-displacement curves are shown in Fig. 5. For a defined adsorption time, two curves obtained from an enamel sample were given. One curve was from the measured scratch which ran through the bare enamel surface and the adsorbed enamel surface, while another curve was from the control scratch. SEM images of the typical measured scratches are inserted on the top of the shear force-displacement curve figures. It was seen that for any one defined adsorption time, the salivary pellicle on enamel surface was completely scraped off under the applied normal load of 1.5 mN, while only very slight plastic deformation occurred on the bare enamel surface. As shown in Fig.5.a, the shear force of the enamel sample treated with 1 min saliva-adsorption increased from 200 up to 280 µN when the scratch tip moved from the juncture of the bare and saliva-adsorbed enamel surfaces to the end. And the value of shear force increased from 200 up to 300 µN after 3 min adsorption (Fig.5.b), from 200 up to 320 µN after 10 min adsorption (Fig.5.c), and from 200 up to 8
380 µN after 60 min adsorption, respectively (Fig.5.d). The corresponding shear energy between salivary pellicle and enamel surfaces subjected to different times of saliva-adsorption treatment and one-way analysis of variance (ANOVA) result are shown in Table 1. One-way ANOVA revealed a significant difference between the shear energy for enamel surfaces subjected to different times of saliva-adsorption treatment (P
