TY - JOUR
AU - Watari, Fumio
AB - Abstract Etching is one of the most fundamental steps in the restoration of teeth by adhesion of composite resin in dental clinics. Atomic force microscope (AFM) was used for the in situ observation of the etching process of human enamel, dentin and synthetic hydroxyapatite in the three different acid agents, 2% phosphoric acid, 10% citric acid and 10% polyacrylic acid. To measure the absolute depth from the initial level before etching and to correlate the surface height between the changing AFM images obtained, the depth profiles were recorded with etching time by carrying out the line scan consecutively at the representative place of the observed area. These chronological series of depth profiles enabled us to perform quantitative analysis of etched amount in addition to the surface roughness obtained from relative depth profile within one image. The course of etching process from the dissolution of smear layer, appearance of enamel prizms or dentinal tubules to progress of demineralization could clearly be observed. The depth profile, surface roughness, etching amount, etching rate and smear layer thickness could then be evaluated. The different etching characteristics of three acid agents and the effect of surface roughness produced by different mechanical prepolish were compared and discussed. atomic force microscope, in situ observation, tooth, etching, surface roughness, smear layer Introduction Although the observation in air [1–3] and in vacuum is most frequently done, the most unique feature of atomic force microscope (AFM) would be the observation in liquid [4] when compared with the conventional microscopes, such as scanning electron microscope (SEM) [5–11], transmission electron microscope (TEM) [12–14] and electron probe microanalyzer [15–17], and the other new comer microscopes, such as confocal laser scanning microscope [18] and X-ray scanning analytical microscope [19–23]. AFM can also provide us the quantitative data regarding surface morphology. Composite resin with fillers of ceramic powders in polymer matrix has aestheticity close to natural teeth color and is widely used for restoration of teeth after treatments of caries [24]. The adhesion step of resin includes etching, priming and bonding followed by filling of the composite resin. The binding force between composite resin and teeth is mainly supported by physical–mechanical interlocking effect and additionally by chemical bonding such as chelate bonding, hydrogen bonding and Van der Waals force. The commercial adhesion system has recently been adopting the fewer step system such as self-etching primer system to perform etching and priming in one agent and further, the single step system or all-in-one system to perform etching, priming and bonding in one agent. Despite of that the pretreatment to make etching of teeth using acid agents is still one of the most fundamental step in the resin restoration process to obtain good adhesivity. Therefore the surface morphology resulting from etching treatment is important for evaluation and further improvement of the adhesion system. To observe the morphology change with etching time in acid agent by electron microscopy, it is necessary to discontinue etching at a certain time and make a series of specimens to see the whole chronological etching process [25]. If AFM is used for in situ observation in acid agent, etching process can be observed continuously without changing the specimen. This contributes to eliminate the ambiguity due to the individual difference, which is an essential feature in biological specimens. One can make quantitative analysis of surface roughness induced by etching in each image. As etching proceeds, the specimen height as well as surface roughening is changing from the initial level at the start of etching. However, in the current AFM system there is no reference point to correlate the absolute depth from one image to another. To analyze the etched amount the information about the absolute depth is necessary. In this study AFM was applied for the in situ observation of the etching process for human enamel, dentin and synthetic HAP, and quantitative analysis to correlate the changing surface height of the consecutive plural AFM images was done. The results of etching amount, etching rate and smear layer thickness were compared for the three different acid agents and discussed. Experimental procedure Specimen Enamel in the labial surface of upper and lower central incisors was cut using a diamond disc from permanent teeth conserved in water after extraction. The surface was mechanically polished up to #2000 roughness using a water-proof abrasive paper of silicon carbide. The average size of abrasive particles on the #2000 paper is 7.9 μm. Some specimens were further polished using a 0.05 μm alumina emulsion. Each tooth was cut into four pieces of the size 3 × 3 mm2 and they were used for comparison of different etching agents and surface roughness. Observation Observation was carried out in a constant force mode by AFM (Topometrix TMX-2000 Explorer). The scanner coated with teflon on its surface for wide area observation in liquid was used with a standard tip of silicon nitride in a pyramidal shape. The scanner can cover an area of 150 × 150 μm2 at the maximum and has the dynamic range of 12 μm in the vertical direction. The specimen was fixed with an epoxy adhesive to the specimen holder. After an AFM image was taken from an area of 20 × 20 μm2 with a scanning speed of 80 μm s−1 in air, distilled water was supplied through a needle pipe using a syringe into the bottom of the cylindrical tub where the specimen was located. Observation in water was done in the same scanning conditions as in air. Then without interrupting the operation of the scanner water was drained out through another pipe and immediately acid agent was introduced through the supplier pipe. By using this method the scanning can start ∼10 s after the agent was put in and the in situ observation in acid agent was carried out up to 10 min immersion. For recording of the changing morphology with immersion time a relatively small numbers of pixels, 200 × 200, were chosen to obtain one AFM image in a shorter time. In this condition it took ∼1.5 min to acquire one image. As acid agents the most fundamental etchants were adopted: 2% phosphoric acid, 10% citric acid and 10% polyacrylic acid. To correlate the surface height or the absolute depth from the initial height for the changing plural AFM images obtained during etching process in acid agent, depth profile was recorded by carrying out the one-dimensional (1-D) line scan at the representative place of the observed area consecutively with etching time. After the absolute depth profile was measured for certain etching time the AFM image at the final state was observed. This method was also applied for the whole process, including the exchange from in-water (before etching), through the inlet of acid agent and during etching for certain time. Then, the dissolution of smear layer could be observed and its thickness was estimated. Results Figure 1 shows the AFM images of in situ etching process of human enamel in 10% citric acid. The observed area is 20 × 20 μm2, which was pre-polished with alumina emulsion of 0.05 μm. Figure 1a, b, c and d represent the images in water before etching, and etched for 1, 3 and 5 min in 10% citric acid, respectively. Fig. 1 View largeDownload slide In situ observation of etching process of human enamel in a 20 × 20 µm2 area pre-polished with alumina emulsion of 0.05 µm in water before etching (a), and 1 min (b), 3 min (c) and 5 min (d) after immersion in 10% citric acid. Fig. 1 View largeDownload slide In situ observation of etching process of human enamel in a 20 × 20 µm2 area pre-polished with alumina emulsion of 0.05 µm in water before etching (a), and 1 min (b), 3 min (c) and 5 min (d) after immersion in 10% citric acid. In each image it is possible to perform quantitative analysis of surface roughness formed by etching process. However, there is no reference point to correlate the height or depth from one image to another, or the absolute depth from the initiation level of etching. To analyze the etched amount the information about this absolute depth is necessary. Figure 2 shows the AFM images in a top view mode before and after etching in the left and the corresponding relation of these relative depths (surface roughness) within one AFM image and the changing absolute depth between the consecutive AFM images, schematically. Fig. 2 View largeDownload slide Relation of the relative depth (surface roughness) within one AFM image and the changing absolute depth between in situ consecutive AFM images. Fig. 2 View largeDownload slide Relation of the relative depth (surface roughness) within one AFM image and the changing absolute depth between in situ consecutive AFM images. To pursue the change of this absolute depth, we measured the 1-D line scan at the representative place of the observed area consecutively with etching time. Then we could follow the change of absolute depth from the starting height. Figure 3 gives the schematic figure of this method. This will then lead us to the 3-D expression of surface height or the relation of absolute depth (z) with position (x) and etching time (y). Figure 4 shows the AFM image of enamel etched with 2% phosphoric acid, before etching (A) and after etching for 3 min in 2% phosphoric acid (C). The specimen was pre-polished with alumina emulsion of 0.05 μm. Figure 4B shows 3-D expression of time dependence of depth profile. The course of deepening from initial height to the final state can be recognized continuously. Fig. 3 View largeDownload slide Schematic representation of depth profiles obtained from each AFM image and their mutual relation changing with etching time. Fig. 3 View largeDownload slide Schematic representation of depth profiles obtained from each AFM image and their mutual relation changing with etching time. Fig. 4 View largeDownload slide AFM image of enamel pre-polished with alumina emulsion of 0.05 µm, before etching (A), after etching for 3 min in 2% phosphoric acid (C) and time dependent depth profile (B), measured consecutively with etching time. Fig. 4 View largeDownload slide AFM image of enamel pre-polished with alumina emulsion of 0.05 µm, before etching (A), after etching for 3 min in 2% phosphoric acid (C) and time dependent depth profile (B), measured consecutively with etching time. Figure 5 shows the absolute depths extracted using data from Fig. 4B at a certain etching time, where the results with three different etchants are shown comparatively from the top: 10% citric acid, 2% phosphoric acid and 10% polyacrylic acid, for etching time 0 min (in water before etching), 1.5 and 3 min. Both the change of surface roughness and absolute depth were quantitatively drawn here. Fig. 5 View largeDownload slide Change of etched depth profile with etching time, 0 min (in water before etching), 1.5 and 3 min in enamel pre-polished with alumina emulsion of 0.05 µm by three different acid agents. (a) 10% citric acid, (b) 2% phosphoric acid, (c) 10% polyacrylic acid. Fig. 5 View largeDownload slide Change of etched depth profile with etching time, 0 min (in water before etching), 1.5 and 3 min in enamel pre-polished with alumina emulsion of 0.05 µm by three different acid agents. (a) 10% citric acid, (b) 2% phosphoric acid, (c) 10% polyacrylic acid. Figure 6 shows the comparison of the 3-D expression of depth profile in enamel, Fig. 7 shows the mean depth changing with etching time, and Fig. 8 shows the AFM images at the final state, after etching for 3.5 min, for the following three different acid agents: a, 10% citric acid; b, 2% phosphoric acid and c, 10% polyacrylic acid. In Fig. 7 shows the linear increase of etching depth in proportion to etching time, and etching speed is the largest for 2% phosphoric acid and the mildest for 10% polyacrylic acid. In Fig. 8 enamel prisms of the size ∼5 μm are recognized. The morphology is different, depending on used acid agents. Figs 5, 6 and 8 reveal clearly the different characteristics of etched morphology for three different acid agents. Fig. 6 View largeDownload slide 3-D expression of depth profile changing with etching time, in enamel by three different acid agents: (a) 10% citric acid, (b) 2% phosphoric acid and (c) 10% polyacrylic acid, which are corresponding to Fig. 5, respectively. Fig. 6 View largeDownload slide 3-D expression of depth profile changing with etching time, in enamel by three different acid agents: (a) 10% citric acid, (b) 2% phosphoric acid and (c) 10% polyacrylic acid, which are corresponding to Fig. 5, respectively. Fig. 7 View largeDownload slide Mean depth with etching time, in enamel pre-polished with alumina emulsion of 0.05 µm by three different acid agents: (a) 10% citric acid, (b) 2% phosphoric acid and (c) 10% polyacrylic acid. Fig. 7 View largeDownload slide Mean depth with etching time, in enamel pre-polished with alumina emulsion of 0.05 µm by three different acid agents: (a) 10% citric acid, (b) 2% phosphoric acid and (c) 10% polyacrylic acid. Fig. 8 View largeDownload slide AFM images of enamel after etching for 3.5 min by three different acid agents: (a) 10% citric acid, (b) 2% phosphoric acid and (c) 10% polyacrylic acid, corresponding to the state at 3.5 min shown in Fig. 5. Fig. 8 View largeDownload slide AFM images of enamel after etching for 3.5 min by three different acid agents: (a) 10% citric acid, (b) 2% phosphoric acid and (c) 10% polyacrylic acid, corresponding to the state at 3.5 min shown in Fig. 5. Figure 9 shows the similar sets of AFM images for dentin pre-polished with alumina emulsion of 0.05 μm and etched with 10% citric acid. The broadening of dentinal tubules is observed. Fig. 9 View largeDownload slide AFM image of dentin pre-polished with alumina emulsion of 0.05 µm, before etching (A), after etching for 3 min in 10% citric acid (C) and time dependent depth profile (B). Fig. 9 View largeDownload slide AFM image of dentin pre-polished with alumina emulsion of 0.05 µm, before etching (A), after etching for 3 min in 10% citric acid (C) and time dependent depth profile (B). Figure 10 shows the linear increase of mean depth with etching time for 10% citric acid for the different materials, enamel (d), HAP (e) and dentin (f). Figure 11 shows the comparison of morphology corresponding to Fig. 10 after etching for 3.5 min. In synthetic HAP [26–28] the secondary structure, such as enamel prism and dentinal tubule, does not exist. Only the morphology originating from sintered powders was recognized. Fig. 10 View largeDownload slide Mean depth with etching time for enamel (d), HAP (e) and dentin (f) after etching by 10% citric acid. Fig. 10 View largeDownload slide Mean depth with etching time for enamel (d), HAP (e) and dentin (f) after etching by 10% citric acid. Fig. 11 View largeDownload slide Comparison of morphology for enamel (d), HAP (e), dentin (f) after etching for 3.5 min by 10% citric acid corresponding to the final state shown in Fig. 10. Fig. 11 View largeDownload slide Comparison of morphology for enamel (d), HAP (e), dentin (f) after etching for 3.5 min by 10% citric acid corresponding to the final state shown in Fig. 10. Up to here, the first image in the in situ consecutive observation was recorded as quickly as possible after the acid agent was injected. However, it took at least about 10 s for starting the scanning of cantilever. The next problem is to record the very beginning of etching. This is important because the estimation of smear layer and the observation of its dissolution, which would occur within a few tens of seconds, is one of the most concerned matters for the adhesion of resin to teeth. Figure 12 shows the 3-D expression of change of depth profile in enamel, measured with etching time consecutively for the exchange from in-water to 10% citric acid, and up to 2 min immersion. In water, surface height is flat for all the places and unchanged with time. When 10% citric acid was introduced, the height was deepened instantly with nearly equal thickness for all the places and then etching proceeded, preferably or non-preferably, depending on the places. The instant drop of surface height could be attributed to the dissolution of smear layer, which is the accumulated layer of mechanically polished debris. The amount of instant height drop was estimated as 55 nm. Fig. 12 View largeDownload slide 3-D expression of change of depth profile in enamel measured with etching time consecutively from in-water to 10% citric acid and immersion up to 2 min. Fig. 12 View largeDownload slide 3-D expression of change of depth profile in enamel measured with etching time consecutively from in-water to 10% citric acid and immersion up to 2 min. In Figs 13 and 14, AFM images of enamel, before etching (A), after etching for 2 min in 10% citric acid (C) and time dependence depth profile (B) are shown for the prepolish with alumina emulsion of 0.05 μm and with the water-proof abrasive paper of #2000, respectively. Change of depth profile was measured consecutively with etching time from in-water to 10% citric acid, and in its immersion up to 2 min (B). The difference of surface roughness dependent on mechanical prepolish before etching and its influence on morphology after etching could be recognized. In Fig. 14 the instant drop at the acid inlet was estimated as ∼330 nm. The change of surface roughness (Ra) values from before etching to after etching was 20–176 nm, and 216–322 nm for the 0.05 μm and #2000 prepolish, respectively. Fig. 13 View largeDownload slide AFM image of enamel pre-polished with alumina emulsion of 0.05 µm, before etching (A), after etching for 2 min in 10% citric acid (C) and time dependent depth profile (B), measured consecutively with etching time from in-water to 10% citric acid and immersion up to 2 min. Fig. 13 View largeDownload slide AFM image of enamel pre-polished with alumina emulsion of 0.05 µm, before etching (A), after etching for 2 min in 10% citric acid (C) and time dependent depth profile (B), measured consecutively with etching time from in-water to 10% citric acid and immersion up to 2 min. Fig. 14 View largeDownload slide AFM image of enamel pre-polished with the water-proof abrasive paper of #2000, before etching (A), after etching for 2 min in 10% citric acid (C) and time dependent depth profile (B), measured consecutively with etching time from in-water to 10% citric acid and immersion up to 2 min. Fig. 14 View largeDownload slide AFM image of enamel pre-polished with the water-proof abrasive paper of #2000, before etching (A), after etching for 2 min in 10% citric acid (C) and time dependent depth profile (B), measured consecutively with etching time from in-water to 10% citric acid and immersion up to 2 min. Figure 15 shows the mean depth with etching time corresponding to Fig. 13 (alumina emulsion of 0.05 μm) and Fig. 14 (the water-proof abrasive paper of #2000). We may regard that the extrapolation to 0 min corresponds to the thickness of smear layer. The estimated smear layer thickness was 50 and 270 nm for the prepolish with alumina emulsion of 0.05 μm and the water-proof abrasive paper of #2000, respectively. We also notice that the etching speed, which is expressed by the tangent of the curve, is nearly the same for the surfaces with different roughness. Fig. 15 View largeDownload slide Mean depth with etching time by 10% citric acid for enamel pre-polished with alumina emulsion of 0.05 µm and the water-proof abrasive paper of #2000. Extrapolation to 0 min corresponds to the thickness of smear layer. Fig. 15 View largeDownload slide Mean depth with etching time by 10% citric acid for enamel pre-polished with alumina emulsion of 0.05 µm and the water-proof abrasive paper of #2000. Extrapolation to 0 min corresponds to the thickness of smear layer. Discussion Measurement of absolute depth The pursuit of dynamical change during etching process in acid agent by in situ consecutive observation is the unique feature of AFM, which could not be performed by conventional microscopes. This enables us to observe the same spot in the same specimen consecutively, which eliminates the ambiguity that originates from individual difference among specimens. In the SEM and TEM, each specimen must be prepared for each etching time to pursue the chronological change. This inevitably leads to ambiguity due to individual difference, which is usual in biological specimens, and it is necessary to make statistical analysis to obtain the reliable conclusions. The quantitative analysis of surface roughness obtainable within one image is sufficient for most analyses. However, in the case of in situ observation, where the surface height and morphology are changing chronologically, it is necessary to know the height relation between images. This is usually not considered for most of AFM instrument systems. Here we achieved this by adopting the chronologically consecutive depth profile, where the 1-D line scan at the representative place of the observed area was measured consecutively with etching time. This could lead to the quantitative evaluation of etched amount (Figs 7, 10 and 15), then etching rate and smear layer thickness. The absolute etched depth profile shows the difference of character of each etchant (Fig. 5). Dependence of etched tooth morphology on acid agent For phosphoric acid as the etching agent, 37% phosphoric acid has been used in clinics. We tried the observation in 37% phosphoric acid. However, the gas bubbles produced by reaction of enamel with 37% phosphoric acid were attached to the cantilever and the scanning of the cantilever became impossible due to the floating power. After trials with diluted agents the observation was possible with the milder reaction in 2% phosphoric acid solution. In spite of using diluted solutions 2% phosphoric acid showed the highest etching rate in demineralization than both 10% citric acid and 10% polyacrylic acid (Fig. 7). Enamel prisms, revealed after the removal of smear layer, were mostly in similar size, ∼5 μm. The etching pattern of enamel prism shown was the so-called type I defined by Silverstone et al. [29], where the periphery remained high and the inside of enamel prism was preferably etched deeper. The hollowed prisms with intact enamel peripheries make approximately a honeycomb close packed pattern (Fig. 8). The difference of morphology by different acid agents is shown in Fig. 8. The difference of etching rate is shown in Fig. 7. Etched morphology is recognized from depth profile given in Fig. 5. Etching rate, depth profile and AFM topographic image (Figs 5–8) are all in agreement with the order of decalcification power of three different acid agents, as 2% phosphoric acid > 10% citric acid > 10% polyacrylic acid. The forms of etching mode were also different for citric acid and phosphoric acid. The morphology evaluated from depth profile given in Fig. 5 shows that in citric acid the inside of enamel prism was etched preferentially and the peripheral parts remained relatively near the original height, while in phosphoric acid the peripheral parts were also etched with similar etching rate although the etching of the inside of enamel prism proceeded in advance. In 10% polyacrylic acid, demineralization power was weak to reveal slightly the enamel prism after dissolution of smear layer. Estimation of smear layer thickness The smear layer is the accumulated layer of mechanically polished debris. One of the main purposes of making etching as a pretreatment before adhesion of composite resin is the removal of smear layer, which effects on exposure of fresh tooth surface and contributes to the direct contact of adhesive and also to hydrogen bonding in adhesion. Another effect is to enhance mechanical bonding by interlocking effect in the roughened zigzag interface between resin and tooth. Thus the evaluation of smear layer is one of the most concerned matters in the process of adhesion. Smear layer was dissolved already to some extent before scanning started in the usual observation mode after specimen was immersed in acid agent. It took ∼10 s to start scanning of cantilever for obtaining the first image. For the beginning part of the first image, which takes ∼1.5 min to complete, one could sometimes observe the quickly changing depth surface inside one figure, probably reflecting the dissolution of smear layer. The time loss is already significant for recording the change by the dissolution of smear layer. In this study the smear layer could be estimated by two methods. One was obtained from the chronologically consecutive depth profile, where the instant drop was observed at the inlet of acid agent exchanged from water. The amount of drop is ∼55 nm for the enamel pre-polished with alumina emulsion of 0.05 μm (Figs 12 and 13) and 330 nm for the water-proof abrasive paper of #2000 (Fig. 14). The other was obtained from the extrapolation of the graph to 0 min in the etched depth–etching time curve (Fig. 15). The estimated smear layer thickness was 50 and 270 nm for the prepolish with alumina emulsion of 0.05 μm and the water-proof abrasive paper of #2000, respectively. These values estimated by both methods were in the same order and close to each other. The values obtained here would be reasonable when the roughness of mechanical polish is taken into account, where alumina emulsion of 50 nm gave rise to the surface roughness Ra ∼20 nm and #2000 paper 216 nm. The estimated values were found approximately in the same order as the Ra values before etching formed by mechanical polishing. From 1-D to 2-D analysis In the present study the absolute depth was obtained from the chronologically consecutive depth profile, which is the 1-D line scan in the representative place. To make the analysis more complete it is desirable to perform the chronologically consecutive 2-D area scan. To achieve this it is necessary to measure the height at least at one fixed point continuously. This is the target to be attained in the future. Conclusions AFM was applied for the in situ observation of the etching process of human enamel, dentin and synthetic HAP in the three different acid agents. Quantitative analysis to correlate the changing absolute surface height (depth) of the consecutive plural AFM images could be performed, as well as the relative surface roughness could be obtained within one image by using the chronologically consecutive line scan method, and the following were also deduced: The etched depth increased linearly with etching time up to 3 min. The surface roughness produced by mechanical polish affects the smear layer thickness, but has very little effect on etching rate. The dissolution of smear layer could be clearly recognized in the chronologically consecutive depth profile and its thickness was estimated. Acid agents showed different demineralization power, etching amount, etching rate and also different etched morphology. Demineralization is the largest in 2% phosphoric acid followed by 10% citric acid and the smallest in 10% polyacrylic acid. The study was performed under the support of Health and Labour Sciences Research Grants in Research on Advanced Medical Technology from the Ministry of Health, Labour and Welfare, Japan (H14-nano-021). References 1 Kobayashi Y, Ohshima Y, Ikeda T, Komatsu H, Watari F, and Shimokobe H. ( 1995) AFM and SEM observation of morphological changes of human dentin surface by treatment of acidic agents. Dent. Jpn (Tokyo)  32: 46–51. 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TI - In situ quantitative analysis of etching process of human teeth by atomic force microscopy
JF - Journal of Electron Microscopy
DO - 10.1093/jmicro/dfi056
DA - 2005-09-07
UR - https://www.deepdyve.com/lp/oxford-university-press/in-situ-quantitative-analysis-of-etching-process-of-human-teeth-by-W4Gb9f32Zy
SP - 299
EP - 308
VL - 54
IS - 3
DP - DeepDyve
ER -