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The wear behaviour of remineralised human dental enamel: An in vitro study
Journal Pre-proof The wear behaviour of remineralised human dental enamel: An in vitro study Joseph A. Arsecularatne, Mark J. Hoffman PII:
S0043-1648(19)31372-9
DOI:
https://doi.org/10.1016/j.wear.2019.203165
Reference:
WEA 203165
To appear in:
Wear
Received Date: 14 September 2019 Revised Date:
18 November 2019
Accepted Date: 18 December 2019
Please cite this article as: J.A. Arsecularatne, M.J. Hoffman, The wear behaviour of remineralised human dental enamel: An in vitro study, Wear (2020), doi: https://doi.org/10.1016/j.wear.2019.203165. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier B.V.
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THE WEAR BEHAVIOUR OF REMINERALISED HUMAN DENTAL ENAMEL: AN IN VITRO STUDY
Joseph A. Arsecularatne1 and Mark J. Hoffman School of Materials Science and Engineering, UNSW Sydney, Sydney 2052, Australia [email protected]; [email protected]
ABSTRACT This paper investigates the wear behaviour of remineralised human dental enamel. Polished enamel flat surface samples were first demineralised in an acid solution and then remineralised in whole human saliva. They were opposed by enamel cusps in wear tests. Wear depth was measured by a profilometer and the wear surface and subsurface were examined by electron microscopy. The results show that the pores in the top surface layer collapse during wear and form a particle layer which is subsequently removed by ploughing/delamination. For enamel underneath, the dominating wear mechanism is delamination. Remineralised enamel is extremely vulnerable since the measured wear is approximately four times that for sound enamel. KEY WORDS: human dental enamel; remineralisation; demineralisation; wear mechanism
1. INTRODUCTION Tooth enamel is the hardest and most mineralised tissue of the human body. It is comprised of ~97 wt% calcium deficient hydroxyapatite crystals and the remainder ~2 wt% water and ~1 wt% protein and lipid. An acidic environment will dissolve enamel’s inorganic hydroxyapatite crystals and/or organic protein and lipid, causing enamel corrosion/softening (Barbour and Rees, 2006; Zheng et al., 2009). In the oral environment, enamel softening occurs due to extrinsic acids from acidic food/drinks and intrinsic acid from regurgitation processes. Current soft drinks contain food 1
Corresponding author. School of Materials Science and Engineering, UNSW Sydney, Sydney 2052, Australia. Tel.: +61 401 192 789.
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acids such as phosphoric, citric, lactic and malic having a range of pH 1 - 6 (West et al., 2001; Lewis and Dwyer-Joyce, 2005). In situ and in vitro studies have shown that acidic (low pH) beverages cause enamel softening (Hughes et al., 2000) which is becoming an significant issue with the increase in soft drinks consumption (Ashcroft and Joiner, 2010; Wu et al., 2017) and the prevalence of corrosive wear of teeth (West et al., 2011). In vitro studies also show an increase in enamel dissolution in a solution with a higher acid concentration (Hughes et al., 2000; West et al., 2000). Enamel dissolution increased further with increase in exposure time and temperature. Atomic force microscopy (AFM) and scanning electron microscopy (SEM) investigations of enamel following exposure to citric acid revealed different dissolution rates for enamel prism and inter-prism regions (Cheng et al., 2009; Zheng et al., 2009). The calcium and phosphate ion contents of the enamel surface (from energy dispersive X-ray (EDX) measurements) also decreased with an increase in dissolution time. Dissolution is known to adversely affect enamel’s mechanical properties with decreases in nanoindentation elastic modulus and hardness with increase in enamel dissolution (Barbour et al., 2006). The softened enamel layer formed is approximately 1 – 5 µm thick (Lippert et al. 2004; Barbour and Rees, 2006). Focused ion beam (FIB), SEM and transmission electron microscopy (TEM) subsurface analyses have shown numerous pores and corrosion cracks in the softened enamel layer which degrade its nanoindentation mechanical properties (Arsecularatne and Hoffman, 2014). Enamel dissolution by an acid causes softening of the surface layer, loss of its structure and demineralisation. The damage to this layer can be repaired by remineralisation. Salivary calcium and phosphate in the oral environment lead to this remineralisation (Hemingway et al., 2006). In vitro/situ studies have revealed the deposition of minerals from artificial or natural (e.g., saliva) remineralising solutions on the enamel surface and subsurface pores/cracks formed during dissolution (Tohda et al., 1990; Collys et al., 1993; Lippert et al., 2004; Zheng et al., 2011) and improvements in nanoindentation hardness and/or elastic modulus following remineralisation (Zheng et al., 2011; Arsecularatne and Hoffman, 2014).
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One major limitation of most of the previous studies is that the remineralisation intervals used were too long, above 6 hours (Lippert et al., 2004). This is because continuous remineralisation in vivo occurs between dietary ingestion and it is generally less than 4 hours during daytime. In addition, most of the previous studies used artificial remineralising solutions that could not simulate the vital role played by the proteins in saliva during remineralisation. These salivary proteins are particularly important for the control of the mineral deposition by the salivary pellicle formation (Zahradnik, 1979). Thus, a comparison of the previous in vitro findings to the in vivo remineralisation is problematic. In a recent in vitro study on the interrelationship between mechanical properties, microstructure and composition of human dental enamel remineralised for 0.5 – 2 hours with whole
human
saliva,
the
authors
used
electron
microscopy
techniques
such
as
SEM/FIB/TEM/EDX and nanoindentation (Arsecularatne and Hoffman, 2014). Their results revealed incomplete remineralisation of the softened surface enamel layer with some unrepaired pores. Consequently,
while the nanoindentation elastic modulus improved due to
remineralisation, hardness did not. Additionally, their nanoindentation elastic modulus and hardness results, and FIB/SEM/TEM/EDX analyses did not reveal any significant difference between enamel samples remineralised for 0.5 hours and those remineralised for 2 hours. It was reported that a complete remineralisation of the demineralised enamel is rarely detected as the remineralised top enamel surface hampers repair of pores/cracks underneath and that from a clinical viewpoint, complete restoration of the softened enamel layer is not essential (Larsen and Fejerskov, 1989). Since no reported in vitro/vivo investigations involving the wear of remineralised enamel are available in the literature, it was postulated that existence of pores in the surface zone (because of partial remineralisation) can adversely affect the wear behaviour of enamel (Arsecularatne and Hoffman, 2014). Recent in vitro wear studies with sound enamel revealed surface/subsurface crack formation (Arsecularatne and Hoffman 2010, 2012) as observed with common ceramics (Kato and Adachi, 2002). Moreover, pores in a ceramic may cause crack initiation and/or crack propagation (Lawn 1993; Arsecularatne et al., 2011). Additionally, scratch testing (with a diamond tip) revealed greater material loss in remineralised enamel samples compared to sound enamel (Zheng et al., 2011).
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Advanced microscopy methods such as SEM, TEM, EDX and FIB are increasingly used for subsurface, microstructure and/or chemical analyses. These techniques were also used recently in enamel and/or dental materials related studies, such as investigation of the wear scar subsurface of sound enamel and dental ceramic/composite following in vitro testing (Arsecularatne and Hoffman, 2012; Arsecularatne
et al., 2015) and the property-structure
relationships and nanoindentation deformation of remineralised, hypomineralised and sound enamel (Xie et al., 2007 and 2008; Arsecularatne and Hoffman, 2014). In particular, FIB/SEM can be used for subsurface imaging and to prepare foils for TEM analysis which would reveal the subsurface microstructure in great detail. In addition, FIB milling permits preparation of TEM foils with uniform thickness and from a particular region of interest. The present study investigates the wear behaviour (and the involved wear mechanisms) of remineralised human dental enamel in vitro. Bidirectional wear tests under lubricated conditions with an enamel cusp sample sliding on a flat surface enamel sample were carried out. FIB milling and, SEM, TEM and EDX analyses were the used to reveal the wear related processes that would occur below the surface. It is anticipated that valuable information related to the wear mechanisms/processes can be uncovered by this method since the enamel wear surfaces are normally concealed by accumulated wear particles (Eisenburger and Addy, 2002; Zheng and Zhou, 2007). Additionally, the above electron microscopy and related techniques are also utilised to establish the variations in enamel composition/structure following remineralisation and their influence on wear.
2. MATERIALS & METHODS 2.1 Enamel specimen preparation Ethics approval was obtained from the Human Research Ethics Committee of UNSW for collecting and testing of extracted human teeth for the present study (HC-11403). Details of the procedures for sterilization and storage of teeth, preparation of enamel samples for testing, equipment used, etc were given in previous work (Arsecularatne and Hoffman, 2010 and 2014).
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Accordingly, they are discussed briefly but the changes made to the above procedures elaborated. The collected teeth for this study were first sterilized in 10% formalin solution for 7 days (Arsecularatne and Hoffman, 2014). They were then washed with tap water and stored in a fridge (at 4ºC) in Hanks’ balanced salt solution (Sigma Aldrich Co, Sydney, Australia) to reduce possible demineralisation. A few thymol crystals were also added to the solution to prevent any bacterial growth. Only the teeth with minimum/no noticeable wear or damage during extraction were chosen for the tests which was accomplished within 4 months. For the present experiments, six molar teeth from three male/female donors were selected. The age range of the donors was 20 – 25 years. This relatively small number of teeth selected for the tests minimized possible interpersonal and/or intertooth variability and enabled an in-depth analysis of the tested teeth. For the preparation of required enamel samples for wear tests, larger enamel pieces (for making flat surface specimens) and selected cusps were cut from teeth using a diamond slicing wheel mounted on a dental drill. The enamel surface opposite to the occlusal surface of a sample was then ground flat and used to glue it vertical onto a Perspex holder. The occlusal surface of each larger enamel sample was then ground and polished to obtain a flat occlusal surface ~ 3 mm in length (Fig.1a). With the enamel cusp specimens, they were first ground to a conical shape. The contacting tips were then carefully ground/polished to obtain a circular contact area of approximately 0.5 mm diameter (Fig. 1b). The grinding/polishing processes were accomplished under hydrated conditions. The flat occlusal surface specimen and cusp specimen polished surfaces were considered to be unaffected by the initial sterilization of teeth in 10% formalin solution due to negligible formalin penetration (Sorenson and Gatewood, 1966). In vivo observations reveal that, depending on their location in the mouth, some teeth are more affected by acid attack than others (Mehta et al., 2012). This indicates that, due to consumption of acidic foods/drinks, teeth can be demineralised to varying degrees. Thus while some teeth are highly demineralised, others can remain nearly sound. This can subsequently lead to sliding contact where either the two opposing surfaces are remineralised or one surface is remineralised and the other remains nearly sound. In order to represent these situations in the
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current experiments, both remineralised and sound cusps were included. However, all the flat surface specimens for testing were remineralised since it is the focus of the present investigation. In order to obtain the necessary remineralised enamel cusp and flat surfaces for the wear tests, the original specimens were first demineralised in acetic acid (5 ml, 0.5% v/v concentration, pH 3) for 10 minutes followed by remineralisation using 5 ml of unstimulated fresh human saliva for 30 minutes. Since the composition of human saliva can vary depending on the time of day, etc. (Arends and Ten Cate, 1981), the required fresh saliva samples were obtained from a 52-year-old healthy male individual with normal salivation approximately same time of day and used immediately after its collection. Both demineralisation and remineralisation procedures were conducted at a temperature of 37 °C (approximate mean human body temperature). Further details of these procedures are provided in authors’ previous work (Arsecularatne and Hoffman, 2014). The surface roughness of remineralised and sound enamel were measured using a profilometer (Mitutoyo Surftest SV-600, Mitutoyo Corporation, Tokyo, Japan) with a diamond tip (radius 5 µm) traversing at a velocity 0.1 mm/s.
2.2 Wear testing A tribometer attached with a reciprocating module (CSEM, Peseux, Switzerland) was selected for the tests (Fig. 1c). For the test setup, a Perspex holder with a sound/remineralised enamel cusp sample was mounted on the stationary tribometer arm. Another Perspex holder with remineralised enamel flat-surface sample was held on the reciprocating module. A mounted pair of enamel cusp and flat surface specimens are shown in Fig.1c inset. Wear tests where an enamel cusp was sliding on an enamel flat surface were carried out for 44, 200 or 500 cycles (approximately 0.7, 3 or 7 mins) under a vertical load of 2 or 5 N. For these loads and cusp contact area with mean diameter of 0.5 mm, the corresponding initial contact stress values are in the range 10 – 25 MPa which are comparable to those for the natural dentition during mastication (Waters, 1980). During testing the sliding speed sinusoidally varied within the stroke (2 mm) at speed 66 cycles per minute which was also the reciprocating frequency. As the lubricant, distilled water at 37 ℃ was used. It was injected to the sliding
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surfaces by a hypodermic needle attached to an infusion pump at flow rate 90 ml/h and the lubricant was not recycled. Wear testing was carried out under five different conditions and the corresponding contact load, number of cycles and cusp type used under each condition are given in Table 1. Two tests were carried out under each condition. In addition, two preliminary tests were carried out to determine the suitable ranges of the above parameters for obtaining the wear measurements for the remineralised layer. Thus 12 wear tests were carried out in total. A laser confocal scanning microscope (VK-X200, Keyence corporation, Osaka, Japan) was used to obtain 3D images of a wear scar. An optical microscope (Nikon Eclipse ME600L, Japan) was used for imaging the contact area of a cusp before and after each wear test. These images were then used to measure the corresponding area of contact. The substance loss of the remineralised enamel flat-surface samples was quantified using the specific wear volume (in mm3/Nm). Required wear volume was determined using average values of the wear scar depth, width and length. The wear scar depth/width were obtained using depth profiles (Fig. 2a) measured perpendicular to the sliding direction (Fig. 2b) by the above profilometer. To calculate the average depth and width of a wear scar, five profiles obtained at different locations of a wear scar were used. When calculating the wear volume, a wear test sliding distance of 2 mm was taken as the average length of a wear scar. The number of samples selected for testing was based on the experimental conditions given in Table 1: two contact loads (2 and 5 N); three cyclic durations (44, 200, 500 cycles) and two cusp types (sound and remineralised). It was not possible to test all the resulting combinations since an in-depth investigation of the microscopic wear processes (wear mechanisms) of remineralised enamel using electron microscopy analyses was the focus of the present study. Thus, five combinations of the above parameters as given in Table 1 were selected and two tests per each combination were carried out. This resulted in a total of 10 wear tests which required 10 flat surface remineralised enamel specimens and 10 sound/remineralised enamel cusps. Two preliminary tests (with 2 flat surface remineralised enamel specimens and 2 sound enamel cusps) were also carried out as noted above. Thus, a total of 24 flat surface and cusp enamel specimens were prepared using six molar teeth.
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In order to carry out the electron microscopy (e.g., SEM, FIB/SEM, etc) analyses, enamel specimens were dried under ambient conditions for 12-15 hours. 2.3 SEM, FIB, TEM, EDX The wear scars on all enamel flat surface and cusp samples were analysed using electron microscopy procedures (SEM, FIB/SEM and TEM). EDX analyses were also carried out on selected TEM foils. To start with a gold coating of approximately 50 nm thickness was deposited on the enamel flat surface samples or on the contact surface of cusp samples ~ 15 minutes before the electron microscopy analyses for protecting the surfaces from ion beam damage and/or for minimising charge build-up.
2.3.1 SEM and FIB/SEM Imaging of an enamel surface and sectioning of the subsurface were carried out using an FIB/electron dual-beam equipment (Nova Nanolab 200, FEI Company, USA). When imaging with SEM, cross sections were made perpendicular to the direction of sliding and the wear surface which was also parallel to the direction of enamel prisms. For comparison, sections were also prepared radially on the remineralised surface outside of the wear scar. Prior to FIB milling of an enamel surface, a platinum layer of approximately1 µm thickness was also applied on the area of interest for greater protection of the surface from damage during FIB milling. Further details of the procedures used for subsurface sectioning and/or imaging of these enamel surfaces are given in (Arsecularatne and Hoffman, 2012; Arsecularatne and Hoffman, 2014).
2.3.2 TEM Preparation of the required TEM foils was carried out on the aforementioned electron/FIB dualbeam system. TEM foils were made radially on the wear scar of all flat surface enamel specimens and selected enamel cusp specimens perpendicular to the direction of sliding.
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Moreover, with flat surface specimens, foils were also made radially on the remineralised surface (outside of a wear surface) for comparison. Further details of the procedures used for the preparation of these TEM foils and to transport them to a carbon coated copper grid for TEM imaging can be found in (Arsecularatne and Hoffman, 2014; Arsecularatne et al., 2015). The TEM analyses were carried out on a field emission transmission electron microscope (FEI Philips CM200) with EDX spectroscopy. The latter allowed the chemical analysis of an area of interest of a TEM specimen.
3. RESULTS In this section, quantitative wear results obtained for the remineralised enamel specimens from the wear tests discussed above are first considered. Representative images of the wear surfaces and subsurfaces obtained from SEM and FIB/SEM analyses are then given. Finally, representative TEM images obtained for these wear scar subsurface sections are discussed.
3.1 Wear loss Some of the measured wear depth profiles for remineralised enamel revealed a large variation in wear depth across the profile (e.g., Fig. 2a) with left hand side of the profile showing higher wear than the right-hand side. The mean specific wear volume for remineralised enamel measured under different test conditions in the present study are shown in Fig.3. The experimental specific wear volume for sound enamel (under 5 N load for 2250 cycles) taken from Wu et al. (2015), is also included for comparison. Wu et al. (2015) carried out their wear tests and measured the wear volume using the same experimental set-up and equipment as used in the present study. As noted earlier the mean specific wear volume values shown in Fig. 3 for remineralised enamel were obtained using ten wear depth measurements (five measurements per wear test and two wear tests for each test condition in Table 1). The mean specific wear volume for sound
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enamel is the average value of 25 measurements (5 wear tests and five measurements per test). The error bars indicate the standard deviation. The specific wear rate parameter used in Fig. 3 accounts for the different loads and number of cycles used in the tests (Table 1). Fig. 3 shows that, at 44 and 200 cycles (under 5 N load), specific wear rate values are higher than those measured at 500 cycles. This high wear rate up to 200 cycles is due to the rapid wear of porous remineralised layer at the top enamel surface. During this phase, the contact load also profoundly influences the wear rate as much higher wear was observed under 5 N load compared to 2 N load. Once this layer is worn the wear rate reduces because of sound enamel underneath. With continued contact sliding the wear rate seems to approach that for sound enamel.
3.2 Wear scar surface A typical SEM image of a remineralised flat surface enamel specimen with both wear scar and unworn surfaces is depicted in Fig. 4a. It also shows an FIB section prepared on unworn surface (right inset) with a TEM specimen made on wear surface (left inset) for subsurface observations. The direction of enamel rods in these TEM and FIB sections is anticipated to be nearly perpendicular to the remineralised surface. The roughness (the maximum peak-to-valley height, Rt) values of the unworn remineralised surfaces measured using the profilometer were in the range 150 – 320 nm. The corresponding value for the original polished enamel surface (before demineralisation and subsequent remineralisation) was in the range 20 – 40 nm. Figure 4b shows a region of the remineralised enamel wear surface with agglomerated wear particles and surface cracks. Such a wear surface is an indication of possible delamination wear with agglomerated wear particles. This type of surface topography is observed in regions of the wear scar that showed relatively less wear, e.g., the right-hand side of wear scar profile in Fig.3a. Figure 4c shows a region of the remineralised enamel wear surface with few agglomerated wear particles. However, different stages in the formation and removal of a flakeshaped wear particle can be clearly observed: early stage of propagation of a crack (A); a well
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formed flake shaped wear particle nearing detachment (B); a location where such a wear particle was recently removed (C); a location where such a wear particle was removed but the surface has become smooth due to repeated sliding of the cusp (D). This type of wear surface topography was observed in regions of the wear scar that showed a relatively high wear rate, e.g., left-hand side of Fig. 3a.
3.3 FIB/SEM Figures 5a - 5d illustrate SEM images of subsurface sections for remineralised enamel outside the wear scar (Fig. 5a) and on the wear scar (Figs. 5b – 5d). The remineralised layer (Fig. 5a) shows only partial healing following immersion of demineralised enamel in human saliva for 30 minutes. The remineralised zone thickness determined from these FIB/SEM images was 1.4 ± 0.2 µm. A corrosion crack (arrowed), which was formed because of the preferential dissolution of enamel’s weak inter-rod areas by acid is clearly seen beyond the remineralised layer extended into sound enamel underneath. Figure 5b shows the subsurface of a wear scar region where no accumulated wear particles were observed. The remineralised enamel layer seems to be completely worn out because of the sliding of the cusp repeatedly under applied load. Figure 5c shows the subsurface of a flake shaped wear particle that is being formed (B in Fig. 5c). In this image, propagation of subsurface crack (arrowed) can be seen. Crack deflection and crack bridging can also be observed. These phenomena can be attributed to protein binding phase and enamel’s hierarchical structure which give enamel a higher fracture toughness, e.g., compared to geological HAP, enamel’s fracture toughness is three times higher. Figure 5d shows the subsurface of an area of the wear scar with agglomerated wear particles on the surface (Fig. 4b). A large lateral crack appears to have propagated (arrowed) beneath the remineralised layer which can result in the removal of the complete remineralised enamel layer. In the left-hand side of the subsurface (arrow heads), no remineralised layer is seen, possibly due to its removal during earlier sliding passes of the cusp.
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3.4 TEM Figure 6a is a bright field TEM image of a region of a foil obtained from a remineralised sample surface from a location away from the wear scar. The specimen surface is rough and pores can be seen in the remineralised layer. A corrosion crack (arrow heads) can be seen extended below the remineralised layer. The measured calcium and phosphorous (two main elements contained in HAP crystallites in enamel) contents using EDX (line scans) were negligible at these pores and corrosion cracks. In Fig. 6a, Pt indicates the platinum deposited on the specimen surface prior to FIB milling. In this image, individual hydroxyapatite crystals are not clearly observed since the specimen was obtained from mature sound enamel. Figure 6b is a TEM image of a remineralised enamel wear scar from a test under 2 N load and 200 cycles. Accumulated wear particles can be seen on the wear surface (arrowed), which are due to collapse of pores in the remineralised layer (Fig. 6a). Some intact pores are observed in the layer but well below the sliding surface (arrow heads) since the load and/or number of reciprocating cycles are too low to affect them. Figure 6c shows a TEM image of a remineralised enamel wear scar from a test under 5 N load and 500 cycles (with sound enamel cusp). A relatively thick layer of accumulated wear particles can be seen on the wear surface (arrowed) which was observed in the regions of the wear scar that exhibited lower wear, e.g., right-hand side of wear scar depth profile in Fig.3a. Evidence of the removal of these enamel particles through ploughing was also observed in some of the TEM images. Figure 6d shows a TEM image from a foil of a wear scar for test conditions identical to those for Fig.6c but with a remineralised cusp. In this case, a crack propagating through the layer of particles (arrowed) can be seen. This indicates that the particles accumulated on a wearing enamel surface can be strongly attached to each other and the intact surface underneath. Figure 6e shows a TEM image of a remineralised enamel wear scar from a test under 5 N load and 500 cycles with sound enamel cusp (test 4 in Table 1). No accumulated wear particles can be seen on the enamel surface. A subsurface crack (arrowed) can be seen to propagate
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perpendicular to the orientation of HAP crystals (white dashed line). Since this crack and the particle layer (in Fig. 6c) were formed well below the remineralised enamel layer (the wear scar depth in this region was in the range 3 - 4 µm, whereas the remineralised layer thickness measured using the TEM images was 1.4 ± 0.5 µm), the particles are likely to be formed by crushing of enamel above the horizontal crack (arrowed in Fig. 6e). The wear surface in Fig. 6e is rough and uneven due to delamination and the associated high wear rate. This type of wear surface and subsurface were observed in the regions of the wear scar where a relatively higher wear rate was observed e.g., left hand side of wear scar depth profile in Fig. 3a. Figure 6f shows a TEM image from a remineralised enamel cusp wear surface which opposed a remineralised flat surface specimen in a test under 5 N load and 500 cycles (test 5 in Table 1). It reveals subsurface cracks (arrowed) in the cusp specimen which are less severe compared to cracking seen in the flat surface specimens e.g., in Fig. 6e. The less severe cracking seen in the cusp specimen can be attributed to the fact that the cusp was in continuous contact with the flat surface specimen under compressive stress whereas the flat surface was under cyclic contact with sliding cusp.
4 DISCUSSION The present study investigated the dominant wear mechanism of remineralised enamel in vitro. The enamel flat surface specimens and some of the cusps used in the wear tests were demineralised in an acetic acid solution (at pH 3) for 10 minutes followed by remineralisation in whole human saliva for 30 minutes. Whole human saliva was used for remineralising so that the remineralisation of demineralised enamel would closely simulate the process that occurs in vivo. Human saliva containing ions (such as calcium, phosphate, etc) and multiple proteins (such as cystatins, histatins, satherins, proline-rich proteins (PRPs), lactoferrin, lysozyme, salivary peroxidase, sIgA) is a complex fluid (Kaplan and Baum, 1993; Lamkin and Oppenheim, 1993; Lenander-Lumikari and Loimaranta, 2000; Castagnola et al., 2011). The major salivary constituent proteins (cystatins, histatins, satherins and PRPs) are considered to regulate the remineralisation of demineralised human teeth (Lamkin and Oppenheim, 1993).
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When an enamel sample is immersed in saliva, e.g., through in vitro remineralisation during present experiments, a layer of pellicle is formed (Zahradnik, 1979; Kaplan and Baum, 1993; Lamkin and Oppenheim, 1993; Lendenmann et al., 2000; Lenander-Lumikari and Loimaranta, 2000; Castagnola et al., 2011). The pellicle behaves as a perm-selective film on the sample surface and influences ion transport. Moreover, the presence of pellicle decreases the remineralisation rate and impedes precipitation on the enamel surface which would enable mineral deposition over a greater depth of the demineralised zone (Zahradnik, 1979). Such mineral deposition including deeper demineralised region was observed in FIB/SEM analyses carried out on demineralised enamel following exposure to human saliva for 30 minutes and reported in the authors’ previous study (Arsecularatne and Hoffman, 2014). Moreover, their nanoindentation results revealed a statistically significant enhancement in the elastic modulus for remineralised enamel which was ascribed to the partial repair of the demineralised layer. Their results also indicated that the areas of enamel with partially dissolved hydroxyapatite crystals and smaller pores were repaired before those areas with heavily dissolved crystals and larger pores. Since the remineralised enamel specimens used in the current experiments were also prepared in an identical manner to those in Arsecularatne and Hoffman (2014), similar mineral deposition can be expected. Present FIB/SEM analysis of remineralised enamel (e.g., Fig. 5(a)) revealed only partial healing of the pores following immersion of demineralised enamel in human saliva for 30 minutes. This is in agreement with the results reported in the authors’ previous study (Arsecularatne and Hoffman, 2014). Present TEM analysis also revealed porous regions present in the enamel subsurface following remineralisation (e.g., Fig. 6a), particularly in the upper part of the remineralised zone. The authors’ previous TEM analysis of remineralised enamel (Arsecularatne and Hoffman 2014) also revealed similar subsurface microstructure with relatively large pores and was attributed to the FIB technique used for preparing the TEM specimens which involved removal of the material from front as well as back sides of an enamel section. Thus, all or a part of the lightly bound and recently deposited material in relatively larger pores was likely to be removed from the foil. During the preparation of FIB/SEM sections, however, material was sputtered only from the front of a section. Accordingly, their FIB/SEM analysis seemed to reveal greater subsurface repair during remineralisation compared to their
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TEM analysis. It was also argued that such newly deposited material was most likely to exist in the upper part of the demineralised region with relatively larger pores and severely dissolved crystals due to attack by the acid during demineralisation (Arsecularatne and Hoffman 2014). Conversely, the lower part of the demineralised zone was likely to suffer only mild acid attack (since its exposure to acid was shorter compared to the upper part) and hence contained smaller pores with partially dissolved crystals which enabled crystal growth and hence repair of pores during remineralisation. The present study also revealed that the presence of pores and deep corrosion cracks in remineralised enamel adversely affects its wear behaviour. Collapse of these pores during sliding contact under load caused the accumulation of enamel particles on the sliding surface. These particles seem to protect the surface temporarily, possibly acting as a solid lubricant (Kaidonis et al. 1998), since the measured wear scar profiles revealed lower wear rates in these regions. Removal of this layer of particles occurred through ploughing and/or delamination. Once this particle layer was removed, the dominant wear mechanism was delamination2. The measured specific wear rate for remineralised enamel (Fig. 3) was approximately 4 - 15 times higher than that measured for sound enamel with delamination was again identified as the dominant wear mechanism for sound enamel (Wu et al., 2015). A similar high wear rate for remineralised enamel compared to sound enamel was also reported by Zheng et al., (2011) following their nano-scratch tests (with diamond indenter) on enamel remineralised using artificial saliva. These very high wear rates measured show that the remineralised enamel is vulnerable. While Fig. 3 allowed a comparison of the wear rates for remineralised and sound enamel, a higher enamel sample number is likely to reduce the standard deviations provided that the teeth were obtained from individuals within a narrow age range. This is because the mechanical and tribological properties of human teeth are influenced by the age of an individual (Kruzic et al., 2018). The present and authors’ previous TEM analyses (Arsecularatne and Hoffman, 2014) revealed the presence of deep corrosion cracks in remineralised enamel which extend below the porous top surface region. These deep corrosion cracks appear to assist delamination thus 2
The wear surface/subsurface processes revealed by SEM, FIB/SEM and TEM analyses (e.g., Figs. 4b, 4c, 5c and 6e) of the present study for delamination wear of remineralised enamel (e.g., removal of flake shape wear particles, subsurface crack formation, etc) are similar to those reported in previous studies for sound enamel (Arsecularatne and Hoffman, 2012; Wu et al., 2015; Wu et al., 2018).
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increasing the wear rate of enamel. Once this corrosion affected region is worn out, the wear rate reduced and approach that of sound enamel. However, those individuals who consume acidic food/beverages on a regular basis can experience rapid enamel wear since a softened or partially remineralised enamel layer is frequently present on their teeth. This is in line with the in vivo results of Jarvinen et al., (1991), who reported a very high (37 times) risk of tooth wear3 in people because of the consumption of two or more portions of citrus fruits in a day compared to individuals who consumed less of these fruits. Moreover, the individuals who consumed four or more acidic beverages per week displayed 4 times higher risk of tooth wear compared those who did not. The results of the authors’ previous in vitro study (Arsecularatne and Hoffman, 2014) also revealed that the nanoindentation mechanical properties and microstructure/composition of enamel remineralised in whole human saliva for 30 minutes and 120 minutes were similar. Thus, the wear mechanisms identified in the present study for enamel remineralised for 30 minutes should be equally valid for enamel remineralised up to 120 minutes in vitro. The present study investigated the tribological behaviour of remineralised human dental enamel in vitro and used electron microscopy and quantitative wear results. It was possible to identify the dominant wear mechanism(s) involved and the influence of the microstructure of remineralised enamel, especially the top surface layer with pores. The methodology used allowed reducing the individual and between teeth differences (because of the use of six molar teeth from three donors) and to carry out an in-depth study for revealing the wear behaviour (and the associated wear mechanisms) of remineralised human enamel. Moreover, the use of whole human saliva and clinically relevant remineralisation intervals render the present results pertinent to in vivo remineralisation. However, there are some constraints associated with the present study: the polished specimens used in testing is subsurface enamel which is less mineralized and prismatic compared to surface enamel which is aprismatic; the tests used an in vitro environment which is far less
3
In this investigation, identification of tooth wear due to corrosion was based on: (i) absence of enamel developmental ridges which would result in a smooth surface; (ii) concave regions on the buccal enamel cervical surfaces with higher width than depth; (iii) edges of a tooth restoration are above the level of the surface of an adjacent tooth; (iv) appearance of depressions (cupping) on posterior teeth; (v) exposure of dentin (when corrosive wear was severe).
17
complex than the general oral environment with cyclic loads, thermal conditions and fluids with constantly changing composition and pH; the relatively small number of saliva samples and teeth used in the tests reduced the ability to consider the interpersonal enamel and/or saliva property variations of a wider population. Moreover, considering the scatter often associated with experimental wear measurements (Ramalho, 2010) and the relatively small number of tests carried out per test condition (with three independent variables within each test condition), the wear data obtained in the present study were considered insufficient for a statistical analysis. Hence, further research aimed at obtaining a comprehensive set of remineralised enamel wear data and a statistical analysis will be required to confirm whether the observed differences are statistically significant.
5 CONCLUSIONS The results presented in this paper identified the dominating mechanisms during the wear of remineralised human dental enamel. Based on the in vitro study carried out in the present work and discussed above, following conclusions can be drawn. 1. When remineralised enamel was in sliding contact under load, the pores in the remineralised enamel layer collapsed and formed a layer of particles on the sliding surface. 2. The layer of particles temporarily protected the enamel surface as a lower wear scar depth was measured in these regions. This accumulated layer of particles was removed by ploughing and/or delamination. 3. Once the layer of particles was removed, the dominating wear mechanism for enamel underneath was delamination. 4. The measured specific wear rate for remineralised enamel which was 4 - 15 times higher than that measured for sound enamel reveals that remineralised enamel is vulnerable.
ACKNOWLEDGEMENTS The authors wish to thank Mrs. D Rose and Mrs. F Westlake (Randwick Day Surgery) for providing freshly extracted teeth and the anonymous patients for their consent for using extracted teeth in the experiments, Dr G Yang and Mr W Joe for their assistance with the experimental
18
work and Mr J Platt for taking images of the experimental set-up. They also acknowledge the use of equipment at the UNSW node of the Australian Microscopy & Microanalysis Research Facility (AMMRF).
REFERENCES Arends J, Ten Cate JM, 1981. Tooth enamel remineralisation. Journal of crystal growth 53, 135 – 147. Arsecularatne JA, Hoffman M, 2010. On the Wear Mechanism of Human Dental Enamel, Journal of the Mechanical Behaviour of Biomedical Materials 3, 347-356. Arsecularatne JA, Hoffman M, O’Kelly K, Payraudeau N, 2011. FIB tomographic analysis of subsurface indentation crack interactions with pores in alumina. J. Am. Ceram. Soc. 94 40174024. Arsecularatne JA, Hoffman M, 2012. Ceramic-like Wear Behaviour of Human Dental Enamel, Journal of the Mechanical Behaviour of Biomedical Materials, 8 47-57. Arsecularatne JA and Hoffman M, 2014. An in vitro study of the microstructure, composition and nanoindentation mechanical properties of remineralised human dental enamel. J. Phys. D: Appl. Phys. 47 315403 Arsecularatne JA, Dingeldein JP, Hoffman M, 2015. An in vitro study of the wear mechanism of a leucite glass dental ceramic. Biosurface and Biotribology 1 50 – 61. Ashcroft AT, Joiner A (2010). Tooth cleaning and tooth wear: a review. Proc. IMechE Part J: J. Engineering Tribology 224: 539-548. Barbour ME, Finke M, Parker DM, Hughes JA, Allen GC, Addy M, 2006. The relationship between enamel softening and erosion caused by soft drinks at a range of temperatures. Journal of Dentistry 34 207 – 213. Barbour ME, Rees GD, 2006. The role of erosion, abrasion and attrition in tooth wear. Journal of Clinical Dentistry, 17, 88-93. Boyde A, 1997. Microstructure of enamel. In: Chadwick DJ and Cardew G, editors; Dental enamel: Ciba Foundation Symposium 205, New York, John Wiley & Sons, 18-31. Castagnola M, Picciotti PM, Messana I, et al., 2011. Potential applications of human saliva as diagnostic tool. Acta Otorhinolaryngol Ital 31 347-357. Cheng ZJ, Wang XM, Cui FZ, Ge J and Yan JX (2009). The enamel softening and loss during early erosion studied by AFM, SEM and nanoindentation. Biomed Mater 4: 1-7. Collys K, Cleymaet R, Coomans D, Michotte Y, Slop D, 1993. Rehardening of surface softened and surface etched enamel in vitro and by intraoral exposure. Caries Res 27 15 – 20. Eisenburger M and Addy M, 2002. Erosion and attrition of human enamel in vitro Part I: interaction effects. Journal of Dentistry 30, 341-347. Hamilton GM (1983), Explicit equations for the stresses beneath a sliding spherical contact, Proc. Inst. Mech. Engrs. Part C 197: 53-58. Hemingway CA, Parker DM, Addy M, Barbour ME, 2006. Erosion of enamel by non-carbonated soft drinks with and without toothbrushing abrasion. British Dental Journal, 201, 447-450.
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Hughes JA, West NX, Parker DM, van Den Braak MH, Addy M, 2000. Effects of pH and concentration of citric, malic and lactic acids on enamel, in vitro. J Dent 28, 147-152. Jarvinen VK, Rytomaa II, Heinonen OP, 1991. Risk factors in dental erosion. J Dent Res 70(6): 942-947. Kaidonis JA, Richards LC, Townsend GC and Tansley GD, 1998. Wear of human enamel: a quantitative in vitro assessment. J Dent Res 77, 1983-1990. Kaplan MD, Baum BJ, 1993. The functions of saliva. Dysphagia 8 225-229. Kato K and Adachi K, 2002. Wear of advanced ceramics. Wear 253, 1097-1104. Kruzic JJ, Arsecularatne JA, Tanaka CB, Hoffman MJ, Cesar PF, 2018. Recent advances in understanding the fatigue and wear behaviour of dental composites and ceramics. Journal of the mechanical behaviour of biomedical materials, 88, 504-533. Lamkin MS, Oppenheim FG, 1993. Structural features of salivary function. Critical Reviews in Oral Biology and Medicine 4, 251-259. Larsen MJ, Fejerskov O, 1989. Chemical and structural challenges in remineralisation of dental enamel lesions. Scand J Dent Res 97 285 – 296. Lawn B, 1993. Fracture of Brittle Solids (2nd Edition), Cambridge University Press, UK. Lenander-Lumikari M, Loimaranta V, 2000. Saliva and dental caries. Adv Dent Res 14 40-47. Lendenmann U, Grogan J, Oppenheim FG, 2000. Saliva and dental pellicle – A review. Adv Dent Res 14 22-28. Lewis R and Dwyer-Joyce RS, 2005. Wear of human teeth: a tribological perspective. Proceedings of the Institution of Mechanical Engineers Part J: J Engineering Tribology 219, 118. Lippert F, Parker DM, Jandt KD, 2004. In vitro demineralisation and remineralisation cycles at human tooth enamel surfaces investigated by AFM and nanoindentation. J Colloid Interface Sci 280 442 – 448. Mehta SB, Banerji S, Millar BJ, Suarez-Feito JM, 2012. Current concepts on the management of tooth wear: part 1. Assessment, treatment planning and strategies for the prevention and the passive management of tooth wear. British Dental Journal 212 17-27. Ramalho A. A reliability model for friction and wear experimental data 2010. Wear 269 213– 223. Sorenson FM and Gatewood DC, 1966. Formalin penetration of extracted teeth. J Dent Res 45 1413-1418. Tohda H, Yanagisawa T, Tanaka N, Takuma S, 1990. Growth and fusion of apatite crystals in the remineralised enamel. J. Electron Microsc 39 238 – 244. Waters NE, 1980. Some mechanical and physical properties of teeth. In Vincent J, Curry J (Eds). The mechanical properties of biological materials, Cambridge: Cambridge University Press, 99134. West NX, Hughes JA, Addy M, 2000. Erosion of dentine and enamel in vitro by dietary acids: The effect of temperature, acid character, concentration and exposure time. Journal of Oral Rehabilitation 27 875-880.
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West NX, Hughes JA, Addy M, 2001. The effect of pH on the erosion of dentine and enamel by dietary acids in vitro. Journal of Oral Rehabilitation 28 860-864. West NX, Davies M and Amaechi BT (2011). In vitro and in situ erosion models for evaluating tooth substance loss. Caries Res 45 (Suppl 1) 43-52. Wu YQ, Arsecularatne JA, Hoffman M, 2015. Effect of acidity upon attrition-corrosion of human dental enamel, Journal of the Mechanical Behaviour of Biomedical Materials 44 23-34. Wu YQ, Arsecularatne JA, Hoffman M, 2017. Attrition-corrosion of human dental enamel: a review, Biosurface and Biotribology 3 196-210. Wu YQ, Arsecularatne JA, Warton DI, Hoffman M, 2018. Effect of contact load upon attritioncorrosion of human dental enamel, Wear 414-415 101-108. Zahradnik RT, 1979. Modification by salivary pellicles of in vitro enamel remineralisation. J Dent Res 58 2066-2073. Zheng J and Zhou, ZR 2007. Friction and wear behaviour of human teeth under various wear conditions. Tribology International 40 278-284. Zheng J, Xiao F, Qian LM and Zhou ZR (2009). Erosion behaviour of human tooth enamel in citric acid solution. Tribology International 42 1558-1564. Zheng L, Zheng J, Weng LQ, Qian LM and Zhou ZR (2011). Effect of remineralisation on the nanomechanical properties and microtribological behaviour of acid-eroded human tooth enamel. Wear 271 2297 – 2304.
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FIGURE CAPTIONS Fig.1. Tribometer and reciprocating module set-up for wear testing. Double ended arrow indicates the sliding directions. Also shown (inset) are cusp (top) and flat surface (bottom) enamel specimens. Fig.2. Wear analysis: (a) measured wear scar profile at the location of dashed line in Fig.2b; (b) a 3D image of the middle part of the wear scar from a wear test (test condition 5, Table 1) with double sided arrow indicating reciprocating directions. Fig.3. Measured specific wear rate versus test conditions for remineralised enamel and comparison with that for sound enamel. Fig.4. SEM analysis: (a) A remineralised enamel surface and wear scar with TEM foil location (left inset) and FIB/SEM location (right inset) with double sided arrow illustrating the reciprocating directions (test condition 4, Table 1); (b) A region of wear surface with agglomerated particles (test condition 5, Table 1); (c) a region of wear surface with different stages in the formation and removal of a flake shape wear particle (test condition 5, Table 1). Fig.5. Remineralised enamel subsurfaces: (a) Outside the wear scar; (b) On the wear scar at a location without agglomerated wear particles; (c) under a partially formed flake shaped wear particle; (d) under agglomerated wear particles. Fig.6. Bright field TEM images of various surface and subsurface locations of remineralised enamel: (a) before wear; (b) surface accumulated wear particles and subsurface pores during wear; (c) a region of wear surface with a thick particle layer; (d) a crack propagating through the particle layer; (e) subsurface crack propagation in sound enamel during wear; (f) wearing cusp surface and subsurface cracking.
Table 1. Different load/wear-cycles/cusp combinations used under each test condition Test condition
Load (N)
Number of cycles
Cusp specimen
Flat surface specimen
1
2
200
Sound
R-30
2
5
44
Sound
R-30
3
5
200
Sound
R-30
4
5
500
Sound
R-30
5
5
500
R-30
R-30
HIGHLIGHTS •
Presents an in vitro investigation of the wear behaviour of remineralised human dental enamel and no such study is found in the literature.
•
The use of human saliva and clinically relevant remineralisation durations render the results of the present study relevant to in vivo remineralisation.
•
SEM and TEM analyses of enamel wear surfaces/subsurfaces reveal the dominant wear mechanisms
I declare that the work described has not been published previously, that it is not under consideration for publication elsewhere, that its publication is approved by all authors and tacitly or explicitly by the responsible authorities where the work was carried out, and that, if accepted, it will not be published elsewhere in the same form, in English or in any other language, including electronically without the written consent of the copyright-holder.
S0043-1648(19)31372-9
DOI:
https://doi.org/10.1016/j.wear.2019.203165
Reference:
WEA 203165
To appear in:
Wear
Received Date: 14 September 2019 Revised Date:
18 November 2019
Accepted Date: 18 December 2019
Please cite this article as: J.A. Arsecularatne, M.J. Hoffman, The wear behaviour of remineralised human dental enamel: An in vitro study, Wear (2020), doi: https://doi.org/10.1016/j.wear.2019.203165. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier B.V.
1
THE WEAR BEHAVIOUR OF REMINERALISED HUMAN DENTAL ENAMEL: AN IN VITRO STUDY
Joseph A. Arsecularatne1 and Mark J. Hoffman School of Materials Science and Engineering, UNSW Sydney, Sydney 2052, Australia [email protected]; [email protected]
ABSTRACT This paper investigates the wear behaviour of remineralised human dental enamel. Polished enamel flat surface samples were first demineralised in an acid solution and then remineralised in whole human saliva. They were opposed by enamel cusps in wear tests. Wear depth was measured by a profilometer and the wear surface and subsurface were examined by electron microscopy. The results show that the pores in the top surface layer collapse during wear and form a particle layer which is subsequently removed by ploughing/delamination. For enamel underneath, the dominating wear mechanism is delamination. Remineralised enamel is extremely vulnerable since the measured wear is approximately four times that for sound enamel. KEY WORDS: human dental enamel; remineralisation; demineralisation; wear mechanism
1. INTRODUCTION Tooth enamel is the hardest and most mineralised tissue of the human body. It is comprised of ~97 wt% calcium deficient hydroxyapatite crystals and the remainder ~2 wt% water and ~1 wt% protein and lipid. An acidic environment will dissolve enamel’s inorganic hydroxyapatite crystals and/or organic protein and lipid, causing enamel corrosion/softening (Barbour and Rees, 2006; Zheng et al., 2009). In the oral environment, enamel softening occurs due to extrinsic acids from acidic food/drinks and intrinsic acid from regurgitation processes. Current soft drinks contain food 1
Corresponding author. School of Materials Science and Engineering, UNSW Sydney, Sydney 2052, Australia. Tel.: +61 401 192 789.
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acids such as phosphoric, citric, lactic and malic having a range of pH 1 - 6 (West et al., 2001; Lewis and Dwyer-Joyce, 2005). In situ and in vitro studies have shown that acidic (low pH) beverages cause enamel softening (Hughes et al., 2000) which is becoming an significant issue with the increase in soft drinks consumption (Ashcroft and Joiner, 2010; Wu et al., 2017) and the prevalence of corrosive wear of teeth (West et al., 2011). In vitro studies also show an increase in enamel dissolution in a solution with a higher acid concentration (Hughes et al., 2000; West et al., 2000). Enamel dissolution increased further with increase in exposure time and temperature. Atomic force microscopy (AFM) and scanning electron microscopy (SEM) investigations of enamel following exposure to citric acid revealed different dissolution rates for enamel prism and inter-prism regions (Cheng et al., 2009; Zheng et al., 2009). The calcium and phosphate ion contents of the enamel surface (from energy dispersive X-ray (EDX) measurements) also decreased with an increase in dissolution time. Dissolution is known to adversely affect enamel’s mechanical properties with decreases in nanoindentation elastic modulus and hardness with increase in enamel dissolution (Barbour et al., 2006). The softened enamel layer formed is approximately 1 – 5 µm thick (Lippert et al. 2004; Barbour and Rees, 2006). Focused ion beam (FIB), SEM and transmission electron microscopy (TEM) subsurface analyses have shown numerous pores and corrosion cracks in the softened enamel layer which degrade its nanoindentation mechanical properties (Arsecularatne and Hoffman, 2014). Enamel dissolution by an acid causes softening of the surface layer, loss of its structure and demineralisation. The damage to this layer can be repaired by remineralisation. Salivary calcium and phosphate in the oral environment lead to this remineralisation (Hemingway et al., 2006). In vitro/situ studies have revealed the deposition of minerals from artificial or natural (e.g., saliva) remineralising solutions on the enamel surface and subsurface pores/cracks formed during dissolution (Tohda et al., 1990; Collys et al., 1993; Lippert et al., 2004; Zheng et al., 2011) and improvements in nanoindentation hardness and/or elastic modulus following remineralisation (Zheng et al., 2011; Arsecularatne and Hoffman, 2014).
3
One major limitation of most of the previous studies is that the remineralisation intervals used were too long, above 6 hours (Lippert et al., 2004). This is because continuous remineralisation in vivo occurs between dietary ingestion and it is generally less than 4 hours during daytime. In addition, most of the previous studies used artificial remineralising solutions that could not simulate the vital role played by the proteins in saliva during remineralisation. These salivary proteins are particularly important for the control of the mineral deposition by the salivary pellicle formation (Zahradnik, 1979). Thus, a comparison of the previous in vitro findings to the in vivo remineralisation is problematic. In a recent in vitro study on the interrelationship between mechanical properties, microstructure and composition of human dental enamel remineralised for 0.5 – 2 hours with whole
human
saliva,
the
authors
used
electron
microscopy
techniques
such
as
SEM/FIB/TEM/EDX and nanoindentation (Arsecularatne and Hoffman, 2014). Their results revealed incomplete remineralisation of the softened surface enamel layer with some unrepaired pores. Consequently,
while the nanoindentation elastic modulus improved due to
remineralisation, hardness did not. Additionally, their nanoindentation elastic modulus and hardness results, and FIB/SEM/TEM/EDX analyses did not reveal any significant difference between enamel samples remineralised for 0.5 hours and those remineralised for 2 hours. It was reported that a complete remineralisation of the demineralised enamel is rarely detected as the remineralised top enamel surface hampers repair of pores/cracks underneath and that from a clinical viewpoint, complete restoration of the softened enamel layer is not essential (Larsen and Fejerskov, 1989). Since no reported in vitro/vivo investigations involving the wear of remineralised enamel are available in the literature, it was postulated that existence of pores in the surface zone (because of partial remineralisation) can adversely affect the wear behaviour of enamel (Arsecularatne and Hoffman, 2014). Recent in vitro wear studies with sound enamel revealed surface/subsurface crack formation (Arsecularatne and Hoffman 2010, 2012) as observed with common ceramics (Kato and Adachi, 2002). Moreover, pores in a ceramic may cause crack initiation and/or crack propagation (Lawn 1993; Arsecularatne et al., 2011). Additionally, scratch testing (with a diamond tip) revealed greater material loss in remineralised enamel samples compared to sound enamel (Zheng et al., 2011).
4
Advanced microscopy methods such as SEM, TEM, EDX and FIB are increasingly used for subsurface, microstructure and/or chemical analyses. These techniques were also used recently in enamel and/or dental materials related studies, such as investigation of the wear scar subsurface of sound enamel and dental ceramic/composite following in vitro testing (Arsecularatne and Hoffman, 2012; Arsecularatne
et al., 2015) and the property-structure
relationships and nanoindentation deformation of remineralised, hypomineralised and sound enamel (Xie et al., 2007 and 2008; Arsecularatne and Hoffman, 2014). In particular, FIB/SEM can be used for subsurface imaging and to prepare foils for TEM analysis which would reveal the subsurface microstructure in great detail. In addition, FIB milling permits preparation of TEM foils with uniform thickness and from a particular region of interest. The present study investigates the wear behaviour (and the involved wear mechanisms) of remineralised human dental enamel in vitro. Bidirectional wear tests under lubricated conditions with an enamel cusp sample sliding on a flat surface enamel sample were carried out. FIB milling and, SEM, TEM and EDX analyses were the used to reveal the wear related processes that would occur below the surface. It is anticipated that valuable information related to the wear mechanisms/processes can be uncovered by this method since the enamel wear surfaces are normally concealed by accumulated wear particles (Eisenburger and Addy, 2002; Zheng and Zhou, 2007). Additionally, the above electron microscopy and related techniques are also utilised to establish the variations in enamel composition/structure following remineralisation and their influence on wear.
2. MATERIALS & METHODS 2.1 Enamel specimen preparation Ethics approval was obtained from the Human Research Ethics Committee of UNSW for collecting and testing of extracted human teeth for the present study (HC-11403). Details of the procedures for sterilization and storage of teeth, preparation of enamel samples for testing, equipment used, etc were given in previous work (Arsecularatne and Hoffman, 2010 and 2014).
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Accordingly, they are discussed briefly but the changes made to the above procedures elaborated. The collected teeth for this study were first sterilized in 10% formalin solution for 7 days (Arsecularatne and Hoffman, 2014). They were then washed with tap water and stored in a fridge (at 4ºC) in Hanks’ balanced salt solution (Sigma Aldrich Co, Sydney, Australia) to reduce possible demineralisation. A few thymol crystals were also added to the solution to prevent any bacterial growth. Only the teeth with minimum/no noticeable wear or damage during extraction were chosen for the tests which was accomplished within 4 months. For the present experiments, six molar teeth from three male/female donors were selected. The age range of the donors was 20 – 25 years. This relatively small number of teeth selected for the tests minimized possible interpersonal and/or intertooth variability and enabled an in-depth analysis of the tested teeth. For the preparation of required enamel samples for wear tests, larger enamel pieces (for making flat surface specimens) and selected cusps were cut from teeth using a diamond slicing wheel mounted on a dental drill. The enamel surface opposite to the occlusal surface of a sample was then ground flat and used to glue it vertical onto a Perspex holder. The occlusal surface of each larger enamel sample was then ground and polished to obtain a flat occlusal surface ~ 3 mm in length (Fig.1a). With the enamel cusp specimens, they were first ground to a conical shape. The contacting tips were then carefully ground/polished to obtain a circular contact area of approximately 0.5 mm diameter (Fig. 1b). The grinding/polishing processes were accomplished under hydrated conditions. The flat occlusal surface specimen and cusp specimen polished surfaces were considered to be unaffected by the initial sterilization of teeth in 10% formalin solution due to negligible formalin penetration (Sorenson and Gatewood, 1966). In vivo observations reveal that, depending on their location in the mouth, some teeth are more affected by acid attack than others (Mehta et al., 2012). This indicates that, due to consumption of acidic foods/drinks, teeth can be demineralised to varying degrees. Thus while some teeth are highly demineralised, others can remain nearly sound. This can subsequently lead to sliding contact where either the two opposing surfaces are remineralised or one surface is remineralised and the other remains nearly sound. In order to represent these situations in the
6
current experiments, both remineralised and sound cusps were included. However, all the flat surface specimens for testing were remineralised since it is the focus of the present investigation. In order to obtain the necessary remineralised enamel cusp and flat surfaces for the wear tests, the original specimens were first demineralised in acetic acid (5 ml, 0.5% v/v concentration, pH 3) for 10 minutes followed by remineralisation using 5 ml of unstimulated fresh human saliva for 30 minutes. Since the composition of human saliva can vary depending on the time of day, etc. (Arends and Ten Cate, 1981), the required fresh saliva samples were obtained from a 52-year-old healthy male individual with normal salivation approximately same time of day and used immediately after its collection. Both demineralisation and remineralisation procedures were conducted at a temperature of 37 °C (approximate mean human body temperature). Further details of these procedures are provided in authors’ previous work (Arsecularatne and Hoffman, 2014). The surface roughness of remineralised and sound enamel were measured using a profilometer (Mitutoyo Surftest SV-600, Mitutoyo Corporation, Tokyo, Japan) with a diamond tip (radius 5 µm) traversing at a velocity 0.1 mm/s.
2.2 Wear testing A tribometer attached with a reciprocating module (CSEM, Peseux, Switzerland) was selected for the tests (Fig. 1c). For the test setup, a Perspex holder with a sound/remineralised enamel cusp sample was mounted on the stationary tribometer arm. Another Perspex holder with remineralised enamel flat-surface sample was held on the reciprocating module. A mounted pair of enamel cusp and flat surface specimens are shown in Fig.1c inset. Wear tests where an enamel cusp was sliding on an enamel flat surface were carried out for 44, 200 or 500 cycles (approximately 0.7, 3 or 7 mins) under a vertical load of 2 or 5 N. For these loads and cusp contact area with mean diameter of 0.5 mm, the corresponding initial contact stress values are in the range 10 – 25 MPa which are comparable to those for the natural dentition during mastication (Waters, 1980). During testing the sliding speed sinusoidally varied within the stroke (2 mm) at speed 66 cycles per minute which was also the reciprocating frequency. As the lubricant, distilled water at 37 ℃ was used. It was injected to the sliding
7
surfaces by a hypodermic needle attached to an infusion pump at flow rate 90 ml/h and the lubricant was not recycled. Wear testing was carried out under five different conditions and the corresponding contact load, number of cycles and cusp type used under each condition are given in Table 1. Two tests were carried out under each condition. In addition, two preliminary tests were carried out to determine the suitable ranges of the above parameters for obtaining the wear measurements for the remineralised layer. Thus 12 wear tests were carried out in total. A laser confocal scanning microscope (VK-X200, Keyence corporation, Osaka, Japan) was used to obtain 3D images of a wear scar. An optical microscope (Nikon Eclipse ME600L, Japan) was used for imaging the contact area of a cusp before and after each wear test. These images were then used to measure the corresponding area of contact. The substance loss of the remineralised enamel flat-surface samples was quantified using the specific wear volume (in mm3/Nm). Required wear volume was determined using average values of the wear scar depth, width and length. The wear scar depth/width were obtained using depth profiles (Fig. 2a) measured perpendicular to the sliding direction (Fig. 2b) by the above profilometer. To calculate the average depth and width of a wear scar, five profiles obtained at different locations of a wear scar were used. When calculating the wear volume, a wear test sliding distance of 2 mm was taken as the average length of a wear scar. The number of samples selected for testing was based on the experimental conditions given in Table 1: two contact loads (2 and 5 N); three cyclic durations (44, 200, 500 cycles) and two cusp types (sound and remineralised). It was not possible to test all the resulting combinations since an in-depth investigation of the microscopic wear processes (wear mechanisms) of remineralised enamel using electron microscopy analyses was the focus of the present study. Thus, five combinations of the above parameters as given in Table 1 were selected and two tests per each combination were carried out. This resulted in a total of 10 wear tests which required 10 flat surface remineralised enamel specimens and 10 sound/remineralised enamel cusps. Two preliminary tests (with 2 flat surface remineralised enamel specimens and 2 sound enamel cusps) were also carried out as noted above. Thus, a total of 24 flat surface and cusp enamel specimens were prepared using six molar teeth.
8
In order to carry out the electron microscopy (e.g., SEM, FIB/SEM, etc) analyses, enamel specimens were dried under ambient conditions for 12-15 hours. 2.3 SEM, FIB, TEM, EDX The wear scars on all enamel flat surface and cusp samples were analysed using electron microscopy procedures (SEM, FIB/SEM and TEM). EDX analyses were also carried out on selected TEM foils. To start with a gold coating of approximately 50 nm thickness was deposited on the enamel flat surface samples or on the contact surface of cusp samples ~ 15 minutes before the electron microscopy analyses for protecting the surfaces from ion beam damage and/or for minimising charge build-up.
2.3.1 SEM and FIB/SEM Imaging of an enamel surface and sectioning of the subsurface were carried out using an FIB/electron dual-beam equipment (Nova Nanolab 200, FEI Company, USA). When imaging with SEM, cross sections were made perpendicular to the direction of sliding and the wear surface which was also parallel to the direction of enamel prisms. For comparison, sections were also prepared radially on the remineralised surface outside of the wear scar. Prior to FIB milling of an enamel surface, a platinum layer of approximately1 µm thickness was also applied on the area of interest for greater protection of the surface from damage during FIB milling. Further details of the procedures used for subsurface sectioning and/or imaging of these enamel surfaces are given in (Arsecularatne and Hoffman, 2012; Arsecularatne and Hoffman, 2014).
2.3.2 TEM Preparation of the required TEM foils was carried out on the aforementioned electron/FIB dualbeam system. TEM foils were made radially on the wear scar of all flat surface enamel specimens and selected enamel cusp specimens perpendicular to the direction of sliding.
9
Moreover, with flat surface specimens, foils were also made radially on the remineralised surface (outside of a wear surface) for comparison. Further details of the procedures used for the preparation of these TEM foils and to transport them to a carbon coated copper grid for TEM imaging can be found in (Arsecularatne and Hoffman, 2014; Arsecularatne et al., 2015). The TEM analyses were carried out on a field emission transmission electron microscope (FEI Philips CM200) with EDX spectroscopy. The latter allowed the chemical analysis of an area of interest of a TEM specimen.
3. RESULTS In this section, quantitative wear results obtained for the remineralised enamel specimens from the wear tests discussed above are first considered. Representative images of the wear surfaces and subsurfaces obtained from SEM and FIB/SEM analyses are then given. Finally, representative TEM images obtained for these wear scar subsurface sections are discussed.
3.1 Wear loss Some of the measured wear depth profiles for remineralised enamel revealed a large variation in wear depth across the profile (e.g., Fig. 2a) with left hand side of the profile showing higher wear than the right-hand side. The mean specific wear volume for remineralised enamel measured under different test conditions in the present study are shown in Fig.3. The experimental specific wear volume for sound enamel (under 5 N load for 2250 cycles) taken from Wu et al. (2015), is also included for comparison. Wu et al. (2015) carried out their wear tests and measured the wear volume using the same experimental set-up and equipment as used in the present study. As noted earlier the mean specific wear volume values shown in Fig. 3 for remineralised enamel were obtained using ten wear depth measurements (five measurements per wear test and two wear tests for each test condition in Table 1). The mean specific wear volume for sound
10
enamel is the average value of 25 measurements (5 wear tests and five measurements per test). The error bars indicate the standard deviation. The specific wear rate parameter used in Fig. 3 accounts for the different loads and number of cycles used in the tests (Table 1). Fig. 3 shows that, at 44 and 200 cycles (under 5 N load), specific wear rate values are higher than those measured at 500 cycles. This high wear rate up to 200 cycles is due to the rapid wear of porous remineralised layer at the top enamel surface. During this phase, the contact load also profoundly influences the wear rate as much higher wear was observed under 5 N load compared to 2 N load. Once this layer is worn the wear rate reduces because of sound enamel underneath. With continued contact sliding the wear rate seems to approach that for sound enamel.
3.2 Wear scar surface A typical SEM image of a remineralised flat surface enamel specimen with both wear scar and unworn surfaces is depicted in Fig. 4a. It also shows an FIB section prepared on unworn surface (right inset) with a TEM specimen made on wear surface (left inset) for subsurface observations. The direction of enamel rods in these TEM and FIB sections is anticipated to be nearly perpendicular to the remineralised surface. The roughness (the maximum peak-to-valley height, Rt) values of the unworn remineralised surfaces measured using the profilometer were in the range 150 – 320 nm. The corresponding value for the original polished enamel surface (before demineralisation and subsequent remineralisation) was in the range 20 – 40 nm. Figure 4b shows a region of the remineralised enamel wear surface with agglomerated wear particles and surface cracks. Such a wear surface is an indication of possible delamination wear with agglomerated wear particles. This type of surface topography is observed in regions of the wear scar that showed relatively less wear, e.g., the right-hand side of wear scar profile in Fig.3a. Figure 4c shows a region of the remineralised enamel wear surface with few agglomerated wear particles. However, different stages in the formation and removal of a flakeshaped wear particle can be clearly observed: early stage of propagation of a crack (A); a well
11
formed flake shaped wear particle nearing detachment (B); a location where such a wear particle was recently removed (C); a location where such a wear particle was removed but the surface has become smooth due to repeated sliding of the cusp (D). This type of wear surface topography was observed in regions of the wear scar that showed a relatively high wear rate, e.g., left-hand side of Fig. 3a.
3.3 FIB/SEM Figures 5a - 5d illustrate SEM images of subsurface sections for remineralised enamel outside the wear scar (Fig. 5a) and on the wear scar (Figs. 5b – 5d). The remineralised layer (Fig. 5a) shows only partial healing following immersion of demineralised enamel in human saliva for 30 minutes. The remineralised zone thickness determined from these FIB/SEM images was 1.4 ± 0.2 µm. A corrosion crack (arrowed), which was formed because of the preferential dissolution of enamel’s weak inter-rod areas by acid is clearly seen beyond the remineralised layer extended into sound enamel underneath. Figure 5b shows the subsurface of a wear scar region where no accumulated wear particles were observed. The remineralised enamel layer seems to be completely worn out because of the sliding of the cusp repeatedly under applied load. Figure 5c shows the subsurface of a flake shaped wear particle that is being formed (B in Fig. 5c). In this image, propagation of subsurface crack (arrowed) can be seen. Crack deflection and crack bridging can also be observed. These phenomena can be attributed to protein binding phase and enamel’s hierarchical structure which give enamel a higher fracture toughness, e.g., compared to geological HAP, enamel’s fracture toughness is three times higher. Figure 5d shows the subsurface of an area of the wear scar with agglomerated wear particles on the surface (Fig. 4b). A large lateral crack appears to have propagated (arrowed) beneath the remineralised layer which can result in the removal of the complete remineralised enamel layer. In the left-hand side of the subsurface (arrow heads), no remineralised layer is seen, possibly due to its removal during earlier sliding passes of the cusp.
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3.4 TEM Figure 6a is a bright field TEM image of a region of a foil obtained from a remineralised sample surface from a location away from the wear scar. The specimen surface is rough and pores can be seen in the remineralised layer. A corrosion crack (arrow heads) can be seen extended below the remineralised layer. The measured calcium and phosphorous (two main elements contained in HAP crystallites in enamel) contents using EDX (line scans) were negligible at these pores and corrosion cracks. In Fig. 6a, Pt indicates the platinum deposited on the specimen surface prior to FIB milling. In this image, individual hydroxyapatite crystals are not clearly observed since the specimen was obtained from mature sound enamel. Figure 6b is a TEM image of a remineralised enamel wear scar from a test under 2 N load and 200 cycles. Accumulated wear particles can be seen on the wear surface (arrowed), which are due to collapse of pores in the remineralised layer (Fig. 6a). Some intact pores are observed in the layer but well below the sliding surface (arrow heads) since the load and/or number of reciprocating cycles are too low to affect them. Figure 6c shows a TEM image of a remineralised enamel wear scar from a test under 5 N load and 500 cycles (with sound enamel cusp). A relatively thick layer of accumulated wear particles can be seen on the wear surface (arrowed) which was observed in the regions of the wear scar that exhibited lower wear, e.g., right-hand side of wear scar depth profile in Fig.3a. Evidence of the removal of these enamel particles through ploughing was also observed in some of the TEM images. Figure 6d shows a TEM image from a foil of a wear scar for test conditions identical to those for Fig.6c but with a remineralised cusp. In this case, a crack propagating through the layer of particles (arrowed) can be seen. This indicates that the particles accumulated on a wearing enamel surface can be strongly attached to each other and the intact surface underneath. Figure 6e shows a TEM image of a remineralised enamel wear scar from a test under 5 N load and 500 cycles with sound enamel cusp (test 4 in Table 1). No accumulated wear particles can be seen on the enamel surface. A subsurface crack (arrowed) can be seen to propagate
13
perpendicular to the orientation of HAP crystals (white dashed line). Since this crack and the particle layer (in Fig. 6c) were formed well below the remineralised enamel layer (the wear scar depth in this region was in the range 3 - 4 µm, whereas the remineralised layer thickness measured using the TEM images was 1.4 ± 0.5 µm), the particles are likely to be formed by crushing of enamel above the horizontal crack (arrowed in Fig. 6e). The wear surface in Fig. 6e is rough and uneven due to delamination and the associated high wear rate. This type of wear surface and subsurface were observed in the regions of the wear scar where a relatively higher wear rate was observed e.g., left hand side of wear scar depth profile in Fig. 3a. Figure 6f shows a TEM image from a remineralised enamel cusp wear surface which opposed a remineralised flat surface specimen in a test under 5 N load and 500 cycles (test 5 in Table 1). It reveals subsurface cracks (arrowed) in the cusp specimen which are less severe compared to cracking seen in the flat surface specimens e.g., in Fig. 6e. The less severe cracking seen in the cusp specimen can be attributed to the fact that the cusp was in continuous contact with the flat surface specimen under compressive stress whereas the flat surface was under cyclic contact with sliding cusp.
4 DISCUSSION The present study investigated the dominant wear mechanism of remineralised enamel in vitro. The enamel flat surface specimens and some of the cusps used in the wear tests were demineralised in an acetic acid solution (at pH 3) for 10 minutes followed by remineralisation in whole human saliva for 30 minutes. Whole human saliva was used for remineralising so that the remineralisation of demineralised enamel would closely simulate the process that occurs in vivo. Human saliva containing ions (such as calcium, phosphate, etc) and multiple proteins (such as cystatins, histatins, satherins, proline-rich proteins (PRPs), lactoferrin, lysozyme, salivary peroxidase, sIgA) is a complex fluid (Kaplan and Baum, 1993; Lamkin and Oppenheim, 1993; Lenander-Lumikari and Loimaranta, 2000; Castagnola et al., 2011). The major salivary constituent proteins (cystatins, histatins, satherins and PRPs) are considered to regulate the remineralisation of demineralised human teeth (Lamkin and Oppenheim, 1993).
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When an enamel sample is immersed in saliva, e.g., through in vitro remineralisation during present experiments, a layer of pellicle is formed (Zahradnik, 1979; Kaplan and Baum, 1993; Lamkin and Oppenheim, 1993; Lendenmann et al., 2000; Lenander-Lumikari and Loimaranta, 2000; Castagnola et al., 2011). The pellicle behaves as a perm-selective film on the sample surface and influences ion transport. Moreover, the presence of pellicle decreases the remineralisation rate and impedes precipitation on the enamel surface which would enable mineral deposition over a greater depth of the demineralised zone (Zahradnik, 1979). Such mineral deposition including deeper demineralised region was observed in FIB/SEM analyses carried out on demineralised enamel following exposure to human saliva for 30 minutes and reported in the authors’ previous study (Arsecularatne and Hoffman, 2014). Moreover, their nanoindentation results revealed a statistically significant enhancement in the elastic modulus for remineralised enamel which was ascribed to the partial repair of the demineralised layer. Their results also indicated that the areas of enamel with partially dissolved hydroxyapatite crystals and smaller pores were repaired before those areas with heavily dissolved crystals and larger pores. Since the remineralised enamel specimens used in the current experiments were also prepared in an identical manner to those in Arsecularatne and Hoffman (2014), similar mineral deposition can be expected. Present FIB/SEM analysis of remineralised enamel (e.g., Fig. 5(a)) revealed only partial healing of the pores following immersion of demineralised enamel in human saliva for 30 minutes. This is in agreement with the results reported in the authors’ previous study (Arsecularatne and Hoffman, 2014). Present TEM analysis also revealed porous regions present in the enamel subsurface following remineralisation (e.g., Fig. 6a), particularly in the upper part of the remineralised zone. The authors’ previous TEM analysis of remineralised enamel (Arsecularatne and Hoffman 2014) also revealed similar subsurface microstructure with relatively large pores and was attributed to the FIB technique used for preparing the TEM specimens which involved removal of the material from front as well as back sides of an enamel section. Thus, all or a part of the lightly bound and recently deposited material in relatively larger pores was likely to be removed from the foil. During the preparation of FIB/SEM sections, however, material was sputtered only from the front of a section. Accordingly, their FIB/SEM analysis seemed to reveal greater subsurface repair during remineralisation compared to their
15
TEM analysis. It was also argued that such newly deposited material was most likely to exist in the upper part of the demineralised region with relatively larger pores and severely dissolved crystals due to attack by the acid during demineralisation (Arsecularatne and Hoffman 2014). Conversely, the lower part of the demineralised zone was likely to suffer only mild acid attack (since its exposure to acid was shorter compared to the upper part) and hence contained smaller pores with partially dissolved crystals which enabled crystal growth and hence repair of pores during remineralisation. The present study also revealed that the presence of pores and deep corrosion cracks in remineralised enamel adversely affects its wear behaviour. Collapse of these pores during sliding contact under load caused the accumulation of enamel particles on the sliding surface. These particles seem to protect the surface temporarily, possibly acting as a solid lubricant (Kaidonis et al. 1998), since the measured wear scar profiles revealed lower wear rates in these regions. Removal of this layer of particles occurred through ploughing and/or delamination. Once this particle layer was removed, the dominant wear mechanism was delamination2. The measured specific wear rate for remineralised enamel (Fig. 3) was approximately 4 - 15 times higher than that measured for sound enamel with delamination was again identified as the dominant wear mechanism for sound enamel (Wu et al., 2015). A similar high wear rate for remineralised enamel compared to sound enamel was also reported by Zheng et al., (2011) following their nano-scratch tests (with diamond indenter) on enamel remineralised using artificial saliva. These very high wear rates measured show that the remineralised enamel is vulnerable. While Fig. 3 allowed a comparison of the wear rates for remineralised and sound enamel, a higher enamel sample number is likely to reduce the standard deviations provided that the teeth were obtained from individuals within a narrow age range. This is because the mechanical and tribological properties of human teeth are influenced by the age of an individual (Kruzic et al., 2018). The present and authors’ previous TEM analyses (Arsecularatne and Hoffman, 2014) revealed the presence of deep corrosion cracks in remineralised enamel which extend below the porous top surface region. These deep corrosion cracks appear to assist delamination thus 2
The wear surface/subsurface processes revealed by SEM, FIB/SEM and TEM analyses (e.g., Figs. 4b, 4c, 5c and 6e) of the present study for delamination wear of remineralised enamel (e.g., removal of flake shape wear particles, subsurface crack formation, etc) are similar to those reported in previous studies for sound enamel (Arsecularatne and Hoffman, 2012; Wu et al., 2015; Wu et al., 2018).
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increasing the wear rate of enamel. Once this corrosion affected region is worn out, the wear rate reduced and approach that of sound enamel. However, those individuals who consume acidic food/beverages on a regular basis can experience rapid enamel wear since a softened or partially remineralised enamel layer is frequently present on their teeth. This is in line with the in vivo results of Jarvinen et al., (1991), who reported a very high (37 times) risk of tooth wear3 in people because of the consumption of two or more portions of citrus fruits in a day compared to individuals who consumed less of these fruits. Moreover, the individuals who consumed four or more acidic beverages per week displayed 4 times higher risk of tooth wear compared those who did not. The results of the authors’ previous in vitro study (Arsecularatne and Hoffman, 2014) also revealed that the nanoindentation mechanical properties and microstructure/composition of enamel remineralised in whole human saliva for 30 minutes and 120 minutes were similar. Thus, the wear mechanisms identified in the present study for enamel remineralised for 30 minutes should be equally valid for enamel remineralised up to 120 minutes in vitro. The present study investigated the tribological behaviour of remineralised human dental enamel in vitro and used electron microscopy and quantitative wear results. It was possible to identify the dominant wear mechanism(s) involved and the influence of the microstructure of remineralised enamel, especially the top surface layer with pores. The methodology used allowed reducing the individual and between teeth differences (because of the use of six molar teeth from three donors) and to carry out an in-depth study for revealing the wear behaviour (and the associated wear mechanisms) of remineralised human enamel. Moreover, the use of whole human saliva and clinically relevant remineralisation intervals render the present results pertinent to in vivo remineralisation. However, there are some constraints associated with the present study: the polished specimens used in testing is subsurface enamel which is less mineralized and prismatic compared to surface enamel which is aprismatic; the tests used an in vitro environment which is far less
3
In this investigation, identification of tooth wear due to corrosion was based on: (i) absence of enamel developmental ridges which would result in a smooth surface; (ii) concave regions on the buccal enamel cervical surfaces with higher width than depth; (iii) edges of a tooth restoration are above the level of the surface of an adjacent tooth; (iv) appearance of depressions (cupping) on posterior teeth; (v) exposure of dentin (when corrosive wear was severe).
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complex than the general oral environment with cyclic loads, thermal conditions and fluids with constantly changing composition and pH; the relatively small number of saliva samples and teeth used in the tests reduced the ability to consider the interpersonal enamel and/or saliva property variations of a wider population. Moreover, considering the scatter often associated with experimental wear measurements (Ramalho, 2010) and the relatively small number of tests carried out per test condition (with three independent variables within each test condition), the wear data obtained in the present study were considered insufficient for a statistical analysis. Hence, further research aimed at obtaining a comprehensive set of remineralised enamel wear data and a statistical analysis will be required to confirm whether the observed differences are statistically significant.
5 CONCLUSIONS The results presented in this paper identified the dominating mechanisms during the wear of remineralised human dental enamel. Based on the in vitro study carried out in the present work and discussed above, following conclusions can be drawn. 1. When remineralised enamel was in sliding contact under load, the pores in the remineralised enamel layer collapsed and formed a layer of particles on the sliding surface. 2. The layer of particles temporarily protected the enamel surface as a lower wear scar depth was measured in these regions. This accumulated layer of particles was removed by ploughing and/or delamination. 3. Once the layer of particles was removed, the dominating wear mechanism for enamel underneath was delamination. 4. The measured specific wear rate for remineralised enamel which was 4 - 15 times higher than that measured for sound enamel reveals that remineralised enamel is vulnerable.
ACKNOWLEDGEMENTS The authors wish to thank Mrs. D Rose and Mrs. F Westlake (Randwick Day Surgery) for providing freshly extracted teeth and the anonymous patients for their consent for using extracted teeth in the experiments, Dr G Yang and Mr W Joe for their assistance with the experimental
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work and Mr J Platt for taking images of the experimental set-up. They also acknowledge the use of equipment at the UNSW node of the Australian Microscopy & Microanalysis Research Facility (AMMRF).
REFERENCES Arends J, Ten Cate JM, 1981. Tooth enamel remineralisation. Journal of crystal growth 53, 135 – 147. Arsecularatne JA, Hoffman M, 2010. On the Wear Mechanism of Human Dental Enamel, Journal of the Mechanical Behaviour of Biomedical Materials 3, 347-356. Arsecularatne JA, Hoffman M, O’Kelly K, Payraudeau N, 2011. FIB tomographic analysis of subsurface indentation crack interactions with pores in alumina. J. Am. Ceram. Soc. 94 40174024. Arsecularatne JA, Hoffman M, 2012. Ceramic-like Wear Behaviour of Human Dental Enamel, Journal of the Mechanical Behaviour of Biomedical Materials, 8 47-57. Arsecularatne JA and Hoffman M, 2014. An in vitro study of the microstructure, composition and nanoindentation mechanical properties of remineralised human dental enamel. J. Phys. D: Appl. Phys. 47 315403 Arsecularatne JA, Dingeldein JP, Hoffman M, 2015. An in vitro study of the wear mechanism of a leucite glass dental ceramic. Biosurface and Biotribology 1 50 – 61. Ashcroft AT, Joiner A (2010). Tooth cleaning and tooth wear: a review. Proc. IMechE Part J: J. Engineering Tribology 224: 539-548. Barbour ME, Finke M, Parker DM, Hughes JA, Allen GC, Addy M, 2006. The relationship between enamel softening and erosion caused by soft drinks at a range of temperatures. Journal of Dentistry 34 207 – 213. Barbour ME, Rees GD, 2006. The role of erosion, abrasion and attrition in tooth wear. Journal of Clinical Dentistry, 17, 88-93. Boyde A, 1997. Microstructure of enamel. In: Chadwick DJ and Cardew G, editors; Dental enamel: Ciba Foundation Symposium 205, New York, John Wiley & Sons, 18-31. Castagnola M, Picciotti PM, Messana I, et al., 2011. Potential applications of human saliva as diagnostic tool. Acta Otorhinolaryngol Ital 31 347-357. Cheng ZJ, Wang XM, Cui FZ, Ge J and Yan JX (2009). The enamel softening and loss during early erosion studied by AFM, SEM and nanoindentation. Biomed Mater 4: 1-7. Collys K, Cleymaet R, Coomans D, Michotte Y, Slop D, 1993. Rehardening of surface softened and surface etched enamel in vitro and by intraoral exposure. Caries Res 27 15 – 20. Eisenburger M and Addy M, 2002. Erosion and attrition of human enamel in vitro Part I: interaction effects. Journal of Dentistry 30, 341-347. Hamilton GM (1983), Explicit equations for the stresses beneath a sliding spherical contact, Proc. Inst. Mech. Engrs. Part C 197: 53-58. Hemingway CA, Parker DM, Addy M, Barbour ME, 2006. Erosion of enamel by non-carbonated soft drinks with and without toothbrushing abrasion. British Dental Journal, 201, 447-450.
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Hughes JA, West NX, Parker DM, van Den Braak MH, Addy M, 2000. Effects of pH and concentration of citric, malic and lactic acids on enamel, in vitro. J Dent 28, 147-152. Jarvinen VK, Rytomaa II, Heinonen OP, 1991. Risk factors in dental erosion. J Dent Res 70(6): 942-947. Kaidonis JA, Richards LC, Townsend GC and Tansley GD, 1998. Wear of human enamel: a quantitative in vitro assessment. J Dent Res 77, 1983-1990. Kaplan MD, Baum BJ, 1993. The functions of saliva. Dysphagia 8 225-229. Kato K and Adachi K, 2002. Wear of advanced ceramics. Wear 253, 1097-1104. Kruzic JJ, Arsecularatne JA, Tanaka CB, Hoffman MJ, Cesar PF, 2018. Recent advances in understanding the fatigue and wear behaviour of dental composites and ceramics. Journal of the mechanical behaviour of biomedical materials, 88, 504-533. Lamkin MS, Oppenheim FG, 1993. Structural features of salivary function. Critical Reviews in Oral Biology and Medicine 4, 251-259. Larsen MJ, Fejerskov O, 1989. Chemical and structural challenges in remineralisation of dental enamel lesions. Scand J Dent Res 97 285 – 296. Lawn B, 1993. Fracture of Brittle Solids (2nd Edition), Cambridge University Press, UK. Lenander-Lumikari M, Loimaranta V, 2000. Saliva and dental caries. Adv Dent Res 14 40-47. Lendenmann U, Grogan J, Oppenheim FG, 2000. Saliva and dental pellicle – A review. Adv Dent Res 14 22-28. Lewis R and Dwyer-Joyce RS, 2005. Wear of human teeth: a tribological perspective. Proceedings of the Institution of Mechanical Engineers Part J: J Engineering Tribology 219, 118. Lippert F, Parker DM, Jandt KD, 2004. In vitro demineralisation and remineralisation cycles at human tooth enamel surfaces investigated by AFM and nanoindentation. J Colloid Interface Sci 280 442 – 448. Mehta SB, Banerji S, Millar BJ, Suarez-Feito JM, 2012. Current concepts on the management of tooth wear: part 1. Assessment, treatment planning and strategies for the prevention and the passive management of tooth wear. British Dental Journal 212 17-27. Ramalho A. A reliability model for friction and wear experimental data 2010. Wear 269 213– 223. Sorenson FM and Gatewood DC, 1966. Formalin penetration of extracted teeth. J Dent Res 45 1413-1418. Tohda H, Yanagisawa T, Tanaka N, Takuma S, 1990. Growth and fusion of apatite crystals in the remineralised enamel. J. Electron Microsc 39 238 – 244. Waters NE, 1980. Some mechanical and physical properties of teeth. In Vincent J, Curry J (Eds). The mechanical properties of biological materials, Cambridge: Cambridge University Press, 99134. West NX, Hughes JA, Addy M, 2000. Erosion of dentine and enamel in vitro by dietary acids: The effect of temperature, acid character, concentration and exposure time. Journal of Oral Rehabilitation 27 875-880.
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West NX, Hughes JA, Addy M, 2001. The effect of pH on the erosion of dentine and enamel by dietary acids in vitro. Journal of Oral Rehabilitation 28 860-864. West NX, Davies M and Amaechi BT (2011). In vitro and in situ erosion models for evaluating tooth substance loss. Caries Res 45 (Suppl 1) 43-52. Wu YQ, Arsecularatne JA, Hoffman M, 2015. Effect of acidity upon attrition-corrosion of human dental enamel, Journal of the Mechanical Behaviour of Biomedical Materials 44 23-34. Wu YQ, Arsecularatne JA, Hoffman M, 2017. Attrition-corrosion of human dental enamel: a review, Biosurface and Biotribology 3 196-210. Wu YQ, Arsecularatne JA, Warton DI, Hoffman M, 2018. Effect of contact load upon attritioncorrosion of human dental enamel, Wear 414-415 101-108. Zahradnik RT, 1979. Modification by salivary pellicles of in vitro enamel remineralisation. J Dent Res 58 2066-2073. Zheng J and Zhou, ZR 2007. Friction and wear behaviour of human teeth under various wear conditions. Tribology International 40 278-284. Zheng J, Xiao F, Qian LM and Zhou ZR (2009). Erosion behaviour of human tooth enamel in citric acid solution. Tribology International 42 1558-1564. Zheng L, Zheng J, Weng LQ, Qian LM and Zhou ZR (2011). Effect of remineralisation on the nanomechanical properties and microtribological behaviour of acid-eroded human tooth enamel. Wear 271 2297 – 2304.
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FIGURE CAPTIONS Fig.1. Tribometer and reciprocating module set-up for wear testing. Double ended arrow indicates the sliding directions. Also shown (inset) are cusp (top) and flat surface (bottom) enamel specimens. Fig.2. Wear analysis: (a) measured wear scar profile at the location of dashed line in Fig.2b; (b) a 3D image of the middle part of the wear scar from a wear test (test condition 5, Table 1) with double sided arrow indicating reciprocating directions. Fig.3. Measured specific wear rate versus test conditions for remineralised enamel and comparison with that for sound enamel. Fig.4. SEM analysis: (a) A remineralised enamel surface and wear scar with TEM foil location (left inset) and FIB/SEM location (right inset) with double sided arrow illustrating the reciprocating directions (test condition 4, Table 1); (b) A region of wear surface with agglomerated particles (test condition 5, Table 1); (c) a region of wear surface with different stages in the formation and removal of a flake shape wear particle (test condition 5, Table 1). Fig.5. Remineralised enamel subsurfaces: (a) Outside the wear scar; (b) On the wear scar at a location without agglomerated wear particles; (c) under a partially formed flake shaped wear particle; (d) under agglomerated wear particles. Fig.6. Bright field TEM images of various surface and subsurface locations of remineralised enamel: (a) before wear; (b) surface accumulated wear particles and subsurface pores during wear; (c) a region of wear surface with a thick particle layer; (d) a crack propagating through the particle layer; (e) subsurface crack propagation in sound enamel during wear; (f) wearing cusp surface and subsurface cracking.
Table 1. Different load/wear-cycles/cusp combinations used under each test condition Test condition
Load (N)
Number of cycles
Cusp specimen
Flat surface specimen
1
2
200
Sound
R-30
2
5
44
Sound
R-30
3
5
200
Sound
R-30
4
5
500
Sound
R-30
5
5
500
R-30
R-30
HIGHLIGHTS •
Presents an in vitro investigation of the wear behaviour of remineralised human dental enamel and no such study is found in the literature.
•
The use of human saliva and clinically relevant remineralisation durations render the results of the present study relevant to in vivo remineralisation.
•
SEM and TEM analyses of enamel wear surfaces/subsurfaces reveal the dominant wear mechanisms
I declare that the work described has not been published previously, that it is not under consideration for publication elsewhere, that its publication is approved by all authors and tacitly or explicitly by the responsible authorities where the work was carried out, and that, if accepted, it will not be published elsewhere in the same form, in English or in any other language, including electronically without the written consent of the copyright-holder.