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Property variations in the prism and the organic sheath within enamel by nanoindentation
ARTICLE IN PRESS
Biomaterials 26 (2005) 3333–3339 www.elsevier.com/locate/biomaterials
Property variations in the prism and the organic sheath within enamel by nanoindentation J. Gea, F.Z. Cuia,, X.M. Wanga, H.L. Fengb a
Biomaterials Laboratory, Department of Materials Science and Engineering, Tsinghua University, Beijing 100084, China b School of Stomatology, Peking University, Beijing 100081, China Received 7 April 2004; accepted 30 July 2004 Available online 15 September 2004
Abstract Atomic force microscopy (AFM) combined with nanoindentation technique was used to definitely, site-specifically, test the nanomechanical properties, including nanohardness and elastic modulus, of the isolated domains within single enamel, the prisms and the surrounding sheaths, of mature human maxillary third molars. In this way, it is for the first time that evident differences of nanomechanical properties were revealed between these domains. The nanohardness and elastic modulus of the sheaths were about 73.6% and 52.7% lower than those of the prisms, respectively. Measuring the residual impressions with AFM supported the similar conclusion. The variations of mechanical properties in these domains are considered to be mainly relative to their different component and fibrils arrangement. r 2004 Elsevier Ltd. All rights reserved. Keywords: Nanohardness; Elastic modulus; Nanoindentation; Enamel prisms
1. Introduction Dental enamel is a particularly hard connective tissue of human body. It contains special structure of aligned inorganic prisms, which run approximately perpendicular to the tooth surface [1–3]. Each prism consists primarily of fibril-like hexagonal hydroxyapatite crystals [4,5]. Further investigation to the prism structure indicated that the crystals tend to align lengthways and lie parallel to the prism axis along the center [6], even though there is incoherence of combining crystals of different orientations at the prism boundary [7]. The interfacial area between prisms, termed sheaths, has a high protein contents. The variations of the microstructure and composition of enamel lead to alterations of nanomechanical properties of the isolated domains of enamel. Knowledge on the details should be of interest Corresponding author. Tel.: +86-10-62771556; fax: +86-1062772850. E-mail address: [email protected] (F.Z. Cui).
0142-9612/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2004.07.059
to many issues, including well understanding the unique mechanical characteristics of enamel, setting up mechanics model, developing novel restorative materials and dental clinic treatments [8,9]. Recently, nanoindentation has become an excellent tool to study these nanomechanical alterations, as the indent size is less than the size of measured area. This depth-sensing technique allows performing nanomechanical test, including nanohardness and elastic modulus of the enamel, in very small features and in different orientations [9–11]. A few works have been performed in investigating the variations of the nanomechanical properties in the prisms and the organic sheaths within enamel. Recently, the decline of the nanohardness and elastic modulus of the organic sheaths was noticed [9]. Habelitz et al. also found that the values of nanohardness and elastic modulus were not significantly different because the indents were placed along lines, which may result in that the indentations did not hit exclusively the small organic soft sheaths.
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In order to control the nanoindent exactly on the desired spot of prism, AFM observation to un-etched enamel surface before each nanoindentation was used in the present study. By this way, nanoindentation can hit exactly regions of enamel. On the other hand, suitable maximum load was selected so that the nanoindent size on the organic sheaths would not extend drastically outside the domain. Such improvements on the control of the site-specific nanoindention made possible to find an evident alteration of nanomechanical properties among these domains.
2. Materials and methods 2.1. Samples preparation Mature human maxillary third molars were extracted from individuals as part of their dental treatment. Protocols were provided by Dental Clinic, School of Stomatology of Peking University. Before the following processes, the molars were disinfected by immersing in X11% (w/v) NaClO solution for several days, washed in distilled water, and air-dried. The enamel specimens for nanoindentation tests were prepared by hard tissue microtome. The cut directions were selected so that the enamel prism direction was nearly vertical to the cut surface. The enamel specimens were embedded in polymethylmethacrylate at room temperature. The embedded samples were further polished through progressive grades of waterproof silicon carbide abrasive papers under de-ionized water and polished on microcloths with successively finer grades of alumina power by Phoenix 4000 Sample Preparation System. The finest was 0.05 mm [12]. Finally, the specimen was ultrasonically cleaned for 10 s to remove surface debris. Long-time ultrasonic cleaning was avoided, as it may destroy the microstructure of the samples. As well, no acid etching was used for the nanoindentation test samples because it could cause mechanical decline of the enamel [9]. To investigate the detailed structure within prisms, some enamel samples were etched in citric acid buffer (pH=3.8) for 5 min and scanned by AFM in contact mode.
[13,14]. The experimentally measured quantities, used to determine the mechanical properties, include the peak load (Pmax), the peak displacement (hmax), the contact depth (hc), the contact area (A), and the contact stiffness (S). The equations used to calculate the nanohardness (H) and the effective indentation elastic modulus (Er) are: Pmax ; A rffiffiffiffi p S Er ¼ ; A 2b
H¼
(1)
(2)
where b is an empirical factor to distinguish different indenter shapes. For triangular pyramid indenter, b=1.034 and 1 n2b 1 n2i 1 ¼ þ ; Er Es Ei
(3)
where n is Poisson’s ratio, which varies between 0 and 0.5 for most materials. The subscript s corresponds to the indented materials, and i refers to the indenter materials. The elastic properties of the diamond indenter, ni and E i ; are 0.07 and 1140 GPa. Eq. (2) is derived under the assumption that the material is elastically homogeneous and isotropic. This assumption does not limit nanoindentation as a technique for measurement of elastic modulus in anisotropic materials [15]. In this study, Triboscope (Hysitron Inc., Minnesota, USA) was used to assess the nanomechanical properties of unetched enamel samples at room temperature (23 1C) under the control of AFM. The time-dependent load curve is shown in Fig. 1. Ten load–unload circles at the same load speed and increasing maximum loads
2.2. Nanoindentation The nanoindentation attached with AFM has become an available technique, which is used to measure the mechanical properties including nanohardness and elastic modulus. During indentation, the applied indenter force load and the depth of penetration into the sample are continuously monitored, resulting in force–displacement curves to measure the nanohardness and elastic modulus with the Oliver–Pharr method
Fig. 1. Time-dependent load curves of nanoindentation. Ten load–unload circles were performed during once nanoindentation to acquire the nanomechanical properties at several depths from one nanoindent. The real line indicates the load applied in the prism with the maximum load 1000 mN and the dot line indicates the load applied in the sheath. The final maximum load is 300 mN.
ARTICLE IN PRESS J. Ge et al. / Biomaterials 26 (2005) 3333–3339
were performed during once nanoindentation to acquire the nanohardness and elastic modulus at several depths from one nanoindent. The final maximum load to indent in the prisms was 1000 mN. Since high load tends to cause large crack in the organic sheaths, which may extend drastically outside the area, the maximum load applied to indent here was only 300 mN. The load speeds were 200 and 60 mN/s, respectively. In total, 50% area of the unloading curve was chosen to determine the elastic modulus. Five enamel specimens were studied, and 30 nanoindentations were made in each specimen. The data were analyzed using standard routines to provide nanohardness and elastic modulus for each nanoindentation [13]. The attached AFM was used to scan on the specimen’s surface to control the nanoindenter on the desired spots of enamel. 2.3. AFM observations In this experiment, PicoScan AFM (MI Corp., AZ, USA) was used to explore the unetched enamel specimens before nanoindentation, the impression of nanoindents after nanomechanical measurements and acid-etched specimens at room temperature. The AFM was operated in contact mode in air using Si tips. The typical force constant was 0.6 N/m.
3. Results The AFM image of the polished and unetched enamel specimens is shown in Fig. 2a. The inorganic and organic domains of enamel show different resistance to the fracture during polishing; hence, different domains could be distinguished by the AFM exploration without acid etching. Characteristic keyhole-like prisms, evidently showing that the prisms run vertically to the sample surface, were arranged compactly and separated with each other by the organic sheaths. The typical diameter of the prism is 7 mm. From the profile line in Fig. 2a, the sheath was measured to be 10 nm lower than the prisms in z-direction, which was cause by polish. Fig. 2b show the AFM image of the specimen surface after it was etched by immersing in citric acid buffer. After the inorganic prisms were etched away, the surrounding organic sheaths were revealed to be 800–1000 nm in width. Fig. 3 presents typical sets of load–depth curve obtained in this experiment by indenting on polished enamel section surface. The load–depth curve 1 was measured when the indenter was located on the enamel prism under the control of AFM exploration, while the curve 2 was measured when it was placed on the sheath, respectively.
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Each nanoindentation can give the information on the alterations of nanohardness and elastic modulus at different depths of one nanoindent. All the data of nanohardness and elastic modulus were used for preliminary statistical analysis, respectively. The means and standard deviations of nanohardness and elastic modulus of the prisms and the sheaths were calculated. Fig. 4a showed the average nanohardness and elastic modulus of the prisms (H ¼ 4:3 0:8 GPa; E r ¼ 83:4 7:1 GPa) and organic sheaths (H ¼ 1:1 0:3 GPa; E r ¼ 39:5 4:1 GPa), which meant the arithmetic average with different depth, selected over the range of 30–80 nm displacements to avoid the inaccurate influence caused by the surface roughness. And the values of nanohardness and elastic modulus of the prisms (H ¼ 3:9 0:5 GPa; E r ¼ 77:1 5:7 GPa) and sheaths (H ¼ 1:4 0:4 GPa; E r ¼ 41:2 8:5 GPa) were depicted in Fig. 4b referred to the values acquired from unload. AFM was used to illuminate the variations of the nanoindent impressions impressed in the sample. Fig. 5a and b displays the typical AFM topographies of the impressions in the prism and the sheath, respectively.
4. Discussion Nanohardness and elastic modulus are two important nanomechanical properties of materials, referring to the resistance of the materials to plastic and elastic deformation, respectively. In this study, nanomechanical properties of the isolated domains within enamel, including nanohardness and elastic modulus were tested by nanoindentation attached with AFM. Similar approaches have been taken on enamel previously, but have so far not yet been quantified. The main difference in this approach is that both the loads and nanoindentation position were carefully controlled to measure nanomechancial properties. As a result, a significant decrease in nanohardness and elastic modulus of the sheaths versus prisms was observed. This is an expected finding, since the interred enamel contains a higher amount of protein. Statistically significant differences of the nanomechanical properties between the prisms and organic sheaths of enamel may be noted by inspecting the nanoindentation results from Fig. 4. Because the properties were compared between the prisms and organic sheaths, an unpaired t-test was performed to assess the differences. In general, the average nanohardness and average elastic modulus of the sheaths was about 73.6% (po0.002, unpaired t-test) and 52.7% (p o0.002, unpaired t-test) lower than that of the prisms, respectively. The AFM topographies of nanindents in the prism and sheath in Fig. 5 suggest evident differences. Here, two points can be noted. First, the impression in the prism is a rigid-edged equilateral triangle, a typical
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Fig. 2. (a) Shows the AFM investigation to the polished enamel surface without acid etching. Left side: the typical keyhole-like structure of enamel prisms, which is pointed by the white arrow, can be evidently noted. Since the enamel surface was not etched by acid, the sheath area, indicated by the black arrow, is lower than the prism in high due to the different resistibility to polish. The schematic positions of nanoindentation in the prism and sheath are pointed out with the triangles in white and black, respectively. Right side: profile on the AFM image shows the height distribution on the AFM image, indicating that the sheath is about 10 nm lower than the prisms in height. (b) Shows the AFM image of the acid-etched enamel surface. After the inorganic prisms were removed by etching, the organic sheaths with 800–1000 nm in width were revealed.
nanoindent impression in rigid homogeneous materials. While the impression in the sheath has isosceles triangle shape compared with the impression in the prism, implying different crack expanding velocity at different angles of the triangular impression in the sheath. The second notable variation of the topographies is the distribution and accumulating direction of the pile-up found around the impressions. The pile-up and its
accumulating directions are indicated by the white arrows in Fig. 5a and b. To the nanoindent in the prism, the pile-up accumulates at the three sides of the triangular impression, and the contact points, referring to the maximum points of the pile-up, are figured out by the schematic dash lines, which may be used to modify the measurement of nanomechanical properties [16]. While to the nanoindent in the sheath, pile-up concen-
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Fig. 3. Load–depth curves obtained by nanoindentation on polished enamel. Curve 1 is the load–depth curve applied in the prism. The maximum loads are 1000 mN. And Curve 2 is the load–depth curve applied in the sheath with 300 mN as the maximum load.
trates at a couple of the angles, where the crack expends slower as mentioned previously. Moreover, the accumulating directions of pile-up in Fig. 5b run along the sheath. Considering the topographic differences of the impressions can determine the conclusion consistent with the nanoindentation test. The nanomechanical properties of organic sheaths evidently decrease, compared with those of the prisms; hence, the impression in the sheath remains anisotropic including its shape and pile-up accumulating. In Fig. 2a, the topography difference between the AFM images of the sheaths and the prisms within unetched enamel can supply the potential nanomechanical variations between them. It can be noted that the sheaths is darker than that of the prisms, meaning lower in high. This can be attributed to the outstanding ability of AFM to reveal the height distribution in z-direction. More important, the topography difference implies their different resistibility to polish: the prisms have the better resistibility; therefore, it is difficult to be fractured by polishing. Such better resistibility corresponds to the higher nanohardness of the prisms. In the previous structural studies to enamel, it is reported that there is a much higher content of organic component, mainly deposited by protein fragments, and less inorganic crystal in the organic sheaths than that in the prisms, which can be considered the primary reason to the dramatic declines of nanohardness and elastic modulus of the organic sheaths [3]. Furthermore, it should be hereby stated that the nanomechanical properties determined from the sheaths are properties obtained from a composite of minerals and organic components; however, the mineralized fibrils array loosely and orientate confusing in the
Fig. 4. Variations of nanohardness and elastic modulus between the prism and the sheath: (a) Shows the arithmetic average of nanohardness and elastic modulus over the range of 30–80 nm hc. The means and standard deviations of nanohardness and elastic modulus acquired from unload were illustrated in (b). Error bars indicate standard deviations.
boundary. These unique structure and outstanding mechanical properties of enamel are putatively due to the extraordinary principle of enamel biomineralization [7,17]. Fincham and Simmer has advanced such a formation and calcification model of enamel [18]. The process can be split into several phases and is mediated by enamel matrix proteins (mainly amelogenins and enamelins). What is special for the formation of enamel is that once the cells stop secreting proteins, the apatite crystals fuse to generate the mature enamel, and the proteins ultimately degrade to small fragments and other unidentified products, leaving apatite crystals stacked in prisms with organic-rich sheaths between the prisms and thus constructing the unique structure: the prisms are separated by organic sheaths.
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evident differences of nanomechanical properties were for the first time revealed between the prisms and the surrounding sheaths within enamel by nanoindentation when the load was limited and the nanoindentation positions were controlled. The evident variation of nanomechanical properties among these isolated domains of enamel thus might be of importance for understanding mechanism, therapeutic strategies of enamel and novel biomimetic materials designs. The biomechanical functions of teeth generally result in stress, which is performed first to the outer hard enamel layer. The enamel is adapted to absorb essential mechanical and abrasive stress due to its great stiffness. This property is related to its microstructure. Understanding of the details of the characterization of inorganic and organic regions properties by nanoindentation methods, and their relation to the structure of the enamel, can help in developing a more accurate mechanical mode, which would provide deep insight into the manner in which teeth function. Further material-level studies would also help to develop this understanding and clarify the relationships between the various structures. Such investigations would provide possibly new ideas for novel materials design and synthesis.
Acknowledgements This work was in part supported by the National Nature Science Foundation of China (Project No. 20031010) and the analysis foundation of Tsinghua University.
References
Fig. 5. Comparison of AFM images of the nanoindent impressions in the prism and the sheath: (a) and (b) show the impression in the prism and in the sheath, respectively. The white arrows indicate pile-up and the accumulating directions in different impressions. In (b), the black arrow indicates the direction in which there is a higher cracking velocity and less pile-up accumulation.
5. Conclusion As expected in the dental research field and corresponding to the microstructural statements to enamel,
[1] Ten Cate AR. Oral histology: development, structure and function. 4th ed. Mosby, 1994. [2] Meckel AH, Griebstein WJ, Neal RJ. Structure of mature human dental enamel as observed by electron microscopy. Arch Oral Biol 1965;10:775–83. [3] Warshawsky H. Organization of crystals in enamel. Anat Rec 1989;224:242–62. [4] Lowenstam HA, Weiner S. On biomineralization. London: Oxford University Press; 1989. [5] Mann S. The biomimetics of enamel: Paradigm for organized biomaterials synthesis. In: Dental enamel, Proceedings of Ciba Foundation Symposium, vol. 205. Chichester: Wiley; 1997. p. 261–74. [6] Boyde A. Microstructure of enamel. In: Dental enamel, Proceedings of Ciba Foundation Symposium, vol. 205. New York: Wiley; 1997. p. 18–31. [7] Jones FH. Teeth and bones: applications of surface science to dental materials and related biomaterials. Surf Sci Rep 2001; 42:75–205. [8] Spears IR. A three-dimensional finite element model of prismatic enamel: a re-appraisal of the data on the Young’s modulus of enamel. J Dent Res 1997;76:1690–7.
ARTICLE IN PRESS J. Ge et al. / Biomaterials 26 (2005) 3333–3339 [9] Habelitz S, Marshall SJ, Marshall Jr GW, Balooch M. Mechanical properties of human dental enamel on the nanometer scale. Arch Oral Biol 2001;46:173–83. [10] Cuy JL, Mann AB, Livi KJ, Teaford MF, Weihs TP. Nanoindentation mapping of the mechanical properties of human molar tooth enamel. Arch Oral Biol 2002;47:281–91. [11] Finke M, Hughes JA, Parker DM, Jandt KD. Mechanical properties of in situ demineralised human enamel measured by AFM nanoindentation. Surf Sci 2001;491:456–67. [12] Wang XM, Cui FZ, Ge J, Zhang Y, Ma C. Variation of nanomechanical properties of bone by gene mutation in the zebrafish. Biomaterials 2002;23:4557–63. [13] Oliver WC, Pharr GM. An improved technique for determining hardness and elastic modulus using load and displacement sensing indentation experiments. J Mater Res 1992;7:1564–83.
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[14] Doerner MF, Nix WD. A method for interpreting the data from depth-sensing indentation instruments. J Mater Res 1986; 1:601–9. [15] Turner CH, Rho J, Takano Y, Tsui TY, Pharr GM. The elastic properties of trabecular and cortical bone tissue are similar: results from two microscopic measurement techniques. J Biomech 1999;32:437–41. [16] Beegan D, Schowdhury S, Laugier MT. A nanoindentation study of copper films on oxidised silicon substrates. Surf Coatings Technol 2003;176:124–30. [17] Fincham AG, Moradian-Oldak J, Simmer JP. The structural biology of the developing dental enamel matrix. J Struct Biol 1999;126:270–99. [18] Fincham AG, Simmer JP. Amelogenin proteins of developing dental enamel. Ciba Found Symp 1997;205:118–30.
Biomaterials 26 (2005) 3333–3339 www.elsevier.com/locate/biomaterials
Property variations in the prism and the organic sheath within enamel by nanoindentation J. Gea, F.Z. Cuia,, X.M. Wanga, H.L. Fengb a
Biomaterials Laboratory, Department of Materials Science and Engineering, Tsinghua University, Beijing 100084, China b School of Stomatology, Peking University, Beijing 100081, China Received 7 April 2004; accepted 30 July 2004 Available online 15 September 2004
Abstract Atomic force microscopy (AFM) combined with nanoindentation technique was used to definitely, site-specifically, test the nanomechanical properties, including nanohardness and elastic modulus, of the isolated domains within single enamel, the prisms and the surrounding sheaths, of mature human maxillary third molars. In this way, it is for the first time that evident differences of nanomechanical properties were revealed between these domains. The nanohardness and elastic modulus of the sheaths were about 73.6% and 52.7% lower than those of the prisms, respectively. Measuring the residual impressions with AFM supported the similar conclusion. The variations of mechanical properties in these domains are considered to be mainly relative to their different component and fibrils arrangement. r 2004 Elsevier Ltd. All rights reserved. Keywords: Nanohardness; Elastic modulus; Nanoindentation; Enamel prisms
1. Introduction Dental enamel is a particularly hard connective tissue of human body. It contains special structure of aligned inorganic prisms, which run approximately perpendicular to the tooth surface [1–3]. Each prism consists primarily of fibril-like hexagonal hydroxyapatite crystals [4,5]. Further investigation to the prism structure indicated that the crystals tend to align lengthways and lie parallel to the prism axis along the center [6], even though there is incoherence of combining crystals of different orientations at the prism boundary [7]. The interfacial area between prisms, termed sheaths, has a high protein contents. The variations of the microstructure and composition of enamel lead to alterations of nanomechanical properties of the isolated domains of enamel. Knowledge on the details should be of interest Corresponding author. Tel.: +86-10-62771556; fax: +86-1062772850. E-mail address: [email protected] (F.Z. Cui).
0142-9612/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2004.07.059
to many issues, including well understanding the unique mechanical characteristics of enamel, setting up mechanics model, developing novel restorative materials and dental clinic treatments [8,9]. Recently, nanoindentation has become an excellent tool to study these nanomechanical alterations, as the indent size is less than the size of measured area. This depth-sensing technique allows performing nanomechanical test, including nanohardness and elastic modulus of the enamel, in very small features and in different orientations [9–11]. A few works have been performed in investigating the variations of the nanomechanical properties in the prisms and the organic sheaths within enamel. Recently, the decline of the nanohardness and elastic modulus of the organic sheaths was noticed [9]. Habelitz et al. also found that the values of nanohardness and elastic modulus were not significantly different because the indents were placed along lines, which may result in that the indentations did not hit exclusively the small organic soft sheaths.
ARTICLE IN PRESS 3334
J. Ge et al. / Biomaterials 26 (2005) 3333–3339
In order to control the nanoindent exactly on the desired spot of prism, AFM observation to un-etched enamel surface before each nanoindentation was used in the present study. By this way, nanoindentation can hit exactly regions of enamel. On the other hand, suitable maximum load was selected so that the nanoindent size on the organic sheaths would not extend drastically outside the domain. Such improvements on the control of the site-specific nanoindention made possible to find an evident alteration of nanomechanical properties among these domains.
2. Materials and methods 2.1. Samples preparation Mature human maxillary third molars were extracted from individuals as part of their dental treatment. Protocols were provided by Dental Clinic, School of Stomatology of Peking University. Before the following processes, the molars were disinfected by immersing in X11% (w/v) NaClO solution for several days, washed in distilled water, and air-dried. The enamel specimens for nanoindentation tests were prepared by hard tissue microtome. The cut directions were selected so that the enamel prism direction was nearly vertical to the cut surface. The enamel specimens were embedded in polymethylmethacrylate at room temperature. The embedded samples were further polished through progressive grades of waterproof silicon carbide abrasive papers under de-ionized water and polished on microcloths with successively finer grades of alumina power by Phoenix 4000 Sample Preparation System. The finest was 0.05 mm [12]. Finally, the specimen was ultrasonically cleaned for 10 s to remove surface debris. Long-time ultrasonic cleaning was avoided, as it may destroy the microstructure of the samples. As well, no acid etching was used for the nanoindentation test samples because it could cause mechanical decline of the enamel [9]. To investigate the detailed structure within prisms, some enamel samples were etched in citric acid buffer (pH=3.8) for 5 min and scanned by AFM in contact mode.
[13,14]. The experimentally measured quantities, used to determine the mechanical properties, include the peak load (Pmax), the peak displacement (hmax), the contact depth (hc), the contact area (A), and the contact stiffness (S). The equations used to calculate the nanohardness (H) and the effective indentation elastic modulus (Er) are: Pmax ; A rffiffiffiffi p S Er ¼ ; A 2b
H¼
(1)
(2)
where b is an empirical factor to distinguish different indenter shapes. For triangular pyramid indenter, b=1.034 and 1 n2b 1 n2i 1 ¼ þ ; Er Es Ei
(3)
where n is Poisson’s ratio, which varies between 0 and 0.5 for most materials. The subscript s corresponds to the indented materials, and i refers to the indenter materials. The elastic properties of the diamond indenter, ni and E i ; are 0.07 and 1140 GPa. Eq. (2) is derived under the assumption that the material is elastically homogeneous and isotropic. This assumption does not limit nanoindentation as a technique for measurement of elastic modulus in anisotropic materials [15]. In this study, Triboscope (Hysitron Inc., Minnesota, USA) was used to assess the nanomechanical properties of unetched enamel samples at room temperature (23 1C) under the control of AFM. The time-dependent load curve is shown in Fig. 1. Ten load–unload circles at the same load speed and increasing maximum loads
2.2. Nanoindentation The nanoindentation attached with AFM has become an available technique, which is used to measure the mechanical properties including nanohardness and elastic modulus. During indentation, the applied indenter force load and the depth of penetration into the sample are continuously monitored, resulting in force–displacement curves to measure the nanohardness and elastic modulus with the Oliver–Pharr method
Fig. 1. Time-dependent load curves of nanoindentation. Ten load–unload circles were performed during once nanoindentation to acquire the nanomechanical properties at several depths from one nanoindent. The real line indicates the load applied in the prism with the maximum load 1000 mN and the dot line indicates the load applied in the sheath. The final maximum load is 300 mN.
ARTICLE IN PRESS J. Ge et al. / Biomaterials 26 (2005) 3333–3339
were performed during once nanoindentation to acquire the nanohardness and elastic modulus at several depths from one nanoindent. The final maximum load to indent in the prisms was 1000 mN. Since high load tends to cause large crack in the organic sheaths, which may extend drastically outside the area, the maximum load applied to indent here was only 300 mN. The load speeds were 200 and 60 mN/s, respectively. In total, 50% area of the unloading curve was chosen to determine the elastic modulus. Five enamel specimens were studied, and 30 nanoindentations were made in each specimen. The data were analyzed using standard routines to provide nanohardness and elastic modulus for each nanoindentation [13]. The attached AFM was used to scan on the specimen’s surface to control the nanoindenter on the desired spots of enamel. 2.3. AFM observations In this experiment, PicoScan AFM (MI Corp., AZ, USA) was used to explore the unetched enamel specimens before nanoindentation, the impression of nanoindents after nanomechanical measurements and acid-etched specimens at room temperature. The AFM was operated in contact mode in air using Si tips. The typical force constant was 0.6 N/m.
3. Results The AFM image of the polished and unetched enamel specimens is shown in Fig. 2a. The inorganic and organic domains of enamel show different resistance to the fracture during polishing; hence, different domains could be distinguished by the AFM exploration without acid etching. Characteristic keyhole-like prisms, evidently showing that the prisms run vertically to the sample surface, were arranged compactly and separated with each other by the organic sheaths. The typical diameter of the prism is 7 mm. From the profile line in Fig. 2a, the sheath was measured to be 10 nm lower than the prisms in z-direction, which was cause by polish. Fig. 2b show the AFM image of the specimen surface after it was etched by immersing in citric acid buffer. After the inorganic prisms were etched away, the surrounding organic sheaths were revealed to be 800–1000 nm in width. Fig. 3 presents typical sets of load–depth curve obtained in this experiment by indenting on polished enamel section surface. The load–depth curve 1 was measured when the indenter was located on the enamel prism under the control of AFM exploration, while the curve 2 was measured when it was placed on the sheath, respectively.
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Each nanoindentation can give the information on the alterations of nanohardness and elastic modulus at different depths of one nanoindent. All the data of nanohardness and elastic modulus were used for preliminary statistical analysis, respectively. The means and standard deviations of nanohardness and elastic modulus of the prisms and the sheaths were calculated. Fig. 4a showed the average nanohardness and elastic modulus of the prisms (H ¼ 4:3 0:8 GPa; E r ¼ 83:4 7:1 GPa) and organic sheaths (H ¼ 1:1 0:3 GPa; E r ¼ 39:5 4:1 GPa), which meant the arithmetic average with different depth, selected over the range of 30–80 nm displacements to avoid the inaccurate influence caused by the surface roughness. And the values of nanohardness and elastic modulus of the prisms (H ¼ 3:9 0:5 GPa; E r ¼ 77:1 5:7 GPa) and sheaths (H ¼ 1:4 0:4 GPa; E r ¼ 41:2 8:5 GPa) were depicted in Fig. 4b referred to the values acquired from unload. AFM was used to illuminate the variations of the nanoindent impressions impressed in the sample. Fig. 5a and b displays the typical AFM topographies of the impressions in the prism and the sheath, respectively.
4. Discussion Nanohardness and elastic modulus are two important nanomechanical properties of materials, referring to the resistance of the materials to plastic and elastic deformation, respectively. In this study, nanomechanical properties of the isolated domains within enamel, including nanohardness and elastic modulus were tested by nanoindentation attached with AFM. Similar approaches have been taken on enamel previously, but have so far not yet been quantified. The main difference in this approach is that both the loads and nanoindentation position were carefully controlled to measure nanomechancial properties. As a result, a significant decrease in nanohardness and elastic modulus of the sheaths versus prisms was observed. This is an expected finding, since the interred enamel contains a higher amount of protein. Statistically significant differences of the nanomechanical properties between the prisms and organic sheaths of enamel may be noted by inspecting the nanoindentation results from Fig. 4. Because the properties were compared between the prisms and organic sheaths, an unpaired t-test was performed to assess the differences. In general, the average nanohardness and average elastic modulus of the sheaths was about 73.6% (po0.002, unpaired t-test) and 52.7% (p o0.002, unpaired t-test) lower than that of the prisms, respectively. The AFM topographies of nanindents in the prism and sheath in Fig. 5 suggest evident differences. Here, two points can be noted. First, the impression in the prism is a rigid-edged equilateral triangle, a typical
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Fig. 2. (a) Shows the AFM investigation to the polished enamel surface without acid etching. Left side: the typical keyhole-like structure of enamel prisms, which is pointed by the white arrow, can be evidently noted. Since the enamel surface was not etched by acid, the sheath area, indicated by the black arrow, is lower than the prism in high due to the different resistibility to polish. The schematic positions of nanoindentation in the prism and sheath are pointed out with the triangles in white and black, respectively. Right side: profile on the AFM image shows the height distribution on the AFM image, indicating that the sheath is about 10 nm lower than the prisms in height. (b) Shows the AFM image of the acid-etched enamel surface. After the inorganic prisms were removed by etching, the organic sheaths with 800–1000 nm in width were revealed.
nanoindent impression in rigid homogeneous materials. While the impression in the sheath has isosceles triangle shape compared with the impression in the prism, implying different crack expanding velocity at different angles of the triangular impression in the sheath. The second notable variation of the topographies is the distribution and accumulating direction of the pile-up found around the impressions. The pile-up and its
accumulating directions are indicated by the white arrows in Fig. 5a and b. To the nanoindent in the prism, the pile-up accumulates at the three sides of the triangular impression, and the contact points, referring to the maximum points of the pile-up, are figured out by the schematic dash lines, which may be used to modify the measurement of nanomechanical properties [16]. While to the nanoindent in the sheath, pile-up concen-
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Fig. 3. Load–depth curves obtained by nanoindentation on polished enamel. Curve 1 is the load–depth curve applied in the prism. The maximum loads are 1000 mN. And Curve 2 is the load–depth curve applied in the sheath with 300 mN as the maximum load.
trates at a couple of the angles, where the crack expends slower as mentioned previously. Moreover, the accumulating directions of pile-up in Fig. 5b run along the sheath. Considering the topographic differences of the impressions can determine the conclusion consistent with the nanoindentation test. The nanomechanical properties of organic sheaths evidently decrease, compared with those of the prisms; hence, the impression in the sheath remains anisotropic including its shape and pile-up accumulating. In Fig. 2a, the topography difference between the AFM images of the sheaths and the prisms within unetched enamel can supply the potential nanomechanical variations between them. It can be noted that the sheaths is darker than that of the prisms, meaning lower in high. This can be attributed to the outstanding ability of AFM to reveal the height distribution in z-direction. More important, the topography difference implies their different resistibility to polish: the prisms have the better resistibility; therefore, it is difficult to be fractured by polishing. Such better resistibility corresponds to the higher nanohardness of the prisms. In the previous structural studies to enamel, it is reported that there is a much higher content of organic component, mainly deposited by protein fragments, and less inorganic crystal in the organic sheaths than that in the prisms, which can be considered the primary reason to the dramatic declines of nanohardness and elastic modulus of the organic sheaths [3]. Furthermore, it should be hereby stated that the nanomechanical properties determined from the sheaths are properties obtained from a composite of minerals and organic components; however, the mineralized fibrils array loosely and orientate confusing in the
Fig. 4. Variations of nanohardness and elastic modulus between the prism and the sheath: (a) Shows the arithmetic average of nanohardness and elastic modulus over the range of 30–80 nm hc. The means and standard deviations of nanohardness and elastic modulus acquired from unload were illustrated in (b). Error bars indicate standard deviations.
boundary. These unique structure and outstanding mechanical properties of enamel are putatively due to the extraordinary principle of enamel biomineralization [7,17]. Fincham and Simmer has advanced such a formation and calcification model of enamel [18]. The process can be split into several phases and is mediated by enamel matrix proteins (mainly amelogenins and enamelins). What is special for the formation of enamel is that once the cells stop secreting proteins, the apatite crystals fuse to generate the mature enamel, and the proteins ultimately degrade to small fragments and other unidentified products, leaving apatite crystals stacked in prisms with organic-rich sheaths between the prisms and thus constructing the unique structure: the prisms are separated by organic sheaths.
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evident differences of nanomechanical properties were for the first time revealed between the prisms and the surrounding sheaths within enamel by nanoindentation when the load was limited and the nanoindentation positions were controlled. The evident variation of nanomechanical properties among these isolated domains of enamel thus might be of importance for understanding mechanism, therapeutic strategies of enamel and novel biomimetic materials designs. The biomechanical functions of teeth generally result in stress, which is performed first to the outer hard enamel layer. The enamel is adapted to absorb essential mechanical and abrasive stress due to its great stiffness. This property is related to its microstructure. Understanding of the details of the characterization of inorganic and organic regions properties by nanoindentation methods, and their relation to the structure of the enamel, can help in developing a more accurate mechanical mode, which would provide deep insight into the manner in which teeth function. Further material-level studies would also help to develop this understanding and clarify the relationships between the various structures. Such investigations would provide possibly new ideas for novel materials design and synthesis.
Acknowledgements This work was in part supported by the National Nature Science Foundation of China (Project No. 20031010) and the analysis foundation of Tsinghua University.
References
Fig. 5. Comparison of AFM images of the nanoindent impressions in the prism and the sheath: (a) and (b) show the impression in the prism and in the sheath, respectively. The white arrows indicate pile-up and the accumulating directions in different impressions. In (b), the black arrow indicates the direction in which there is a higher cracking velocity and less pile-up accumulation.
5. Conclusion As expected in the dental research field and corresponding to the microstructural statements to enamel,
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