Shear piezoelectricity in bone at the nanoscale

APPLIED PHYSICS LETTERS 97, 153127 共2010兲

Shear piezoelectricity in bone at the nanoscale Majid Minary-Jolandana兲 and Min-Feng Yu Department of Mechanical Science and Engineering, University of Illinois at Urbana-Champaign, 1206 West Green Street, Urbana, Illinois 61801, USA

共Received 7 September 2010; accepted 27 September 2010; published online 15 October 2010兲 Recent demonstration of shear piezoelectricity in an isolated collagen fibril, which is the origin of piezoelectricity in bone, necessitates investigation of shear piezoelectric behavior in bone at the nanoscale. Using high resolution lateral piezoresponse force microcopy 共PFM兲, shear piezoelectricity in a cortical bone sample was studied at the nanoscale. Subfibrillar structure of individual collagen fibrils with a periodicity of 60–70 nm were revealed in PFM map, indicating the direct contribution of collagen fibrils to the shear piezoelectricity of bone. © 2010 American Institute of Physics. 关doi:10.1063/1.3503965兴 Piezoelectricity, the linear electromechanical coupling, is considered as one of the fundamental properties of bone, and other biological materials such as tendon and dentin. The electric charges generated in these materials due to the piezoelectric effect under deformation has been proposed as the source of mechanoelectric stimulation for bone growth and remodeling.1,2 Collagen fibrils, as small as 50 nm in diameter and as the main protein constituent of bone, were proposed as the origin of piezoelectricity in bone.3 Due to the hierarchical structure of bone,4 the unequivocal investigation of piezoelectricity necessitates probing this property at the level of individual building blocks, i.e., collagen fibrils in bone, in their relevant functional mode. Recently, it was demonstrated that isolated collagen fibrils behave predominantly as shear piezoelectric materials and have a uniaxial polarization in their axial direction.5,6 With progress in nanoscale characterization techniques, piezoelectricity in biomaterials has been probed at the nanoscale with ever increasing details.7–9 For bone, however, only normal 共vertical兲 piezoelectric response has been investigated.7 In bone, collagen fibrils are randomly orientated on lateral planes in radial layers of osteons.10 As such and considering that collagen fibrils are shear piezoelectric materials polarized in their axial direction, it is expected that the shear piezoelectricity might be the dominant form of the linear electromechanical coupling in bone. Therefore, it is imperative to study the shear piezoelectric response in bone at the nanoscale to assess the direct correlation between the piezoelectric responses of collagen and bone. Bone at the nanoscale is a composite of mineral nanocrystals 共hydroxyapatite兲 and organic type I collagen fibrils.10 The mineral nanocrystals, due to their centrosymmetric crystal structure, are excluded to show piezoelectric effect. Therefore, collagen fibrils having hexagonal 共C6兲 crystal structure are responsible for piezoelectricity in bone.2 Collagen fibrils are distinguished by their characteristic periodic banding pattern of 60–70 nm 关as shown in the high resolution atomic force microscope 共AFM兲 tapping-mode image in Fig. 1共b兲兴. In this letter, applying lateral piezoresponse force microscopy 共PFM兲, we have probed shear pia兲

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ezoelectricity in bone with high resolution down to subfibrillar structure of individual collagen fibrils. The PFM is a demonstrated method to study piezoelectricity at the nanoscale, due to its high resolution capability and insensitivity to the topographical features.8,9 PFM is a technique based on the AFM that characterizes the converse piezoelectric effect with high spatial resolution.11–13 In PFM, a conductive AFM cantilever is brought into contact onto the

FIG. 1. 共a兲 Schematic depicting the lateral-PFM operation based on measuring the torsional twist of the cantilever. 共b兲 High resolution AFM tappingmode amplitude image of a cortical bone surface, revealing individual collagen fibrils randomly oriented on the bone surface.

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© 2010 American Institute of Physics

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substrate surface under a constant contact force. An AC voltage 关V0 sin共␻t兲兴 is applied between the cantilever and the grounded substrate. Deformation in the sample due to the converse piezoelectric effect, often in picometer range, is detected by the AFM cantilever through the sensitive photodetector. The induced deformation signal 关d0 sin共␻t + ␾兲兴 is deconvoluted in a lock-in amplifier to amplitude 共d0兲 and phase shift 共␾兲 in respect to the input ac voltage. The amplitude signal is indicative of the strength of the piezoelectric response and the phase shift is indicative of the polarization direction under the probe tip. PFM could be operated in vertical and lateral modes 关Fig. 1共a兲兴, where the vertical mode measures the out-of-plane piezoelectric response by detecting the bending of the AFM cantilever, and the lateral mode measures the in-plane shear piezoelectricity by detecting the torsional twist of the cantilever.9,14 A small piece 共⬃10 mm⫻ 10 mm兲 of animal cortical bone sample was prepared for the PFM study. The bone sample was first polished by a fine sand paper to obtain macroscopically flat surface; then it was partially demineralized by dipping in a diluted phosphoric acid to remove the surface mineral nanocrystals, and expose the surface collagen fibrils. The sample was then washed several times with distilled water and dried at room temperature. For PFM study, a Si chip was coated with a thin layer 共⬃20 nm兲 of Au/Pd to serve as the bottom electrode. The bone sample was placed on the coated substrate and the bottom and the corners of the bone sample were glued to the conductive substrate with a conductive silver paste. A Dimension 3100 AFM 共Veeco, Inc.兲 with a Nanoscope IV controller was used for the study. A custom designed PFM setup equipped with external electronics including a function generator 共Stanford Research System DS 340兲, and a lock-in amplifier 共Signal Recovery Model 5210兲, along with a LABVIEW program were used. Pt-coated Si AFM probes with a flexural stiffness of kb ⬃ 0.15 N / m, and torsional stiffness of kt ⬃ 40 N / m were used for the experiment. The radius of curvature of the tip of the probe was estimated to be ⬃15 nm from deconvoluting AFM images acquired from a tip-calibration sample. Relative humidity was kept below 12% by enclosing the setup in a humidity chamber regulated with a flow of dry nitrogen. The lateral sensitivity SL of the AFM cantilever was calibrated based on the measured vertical sensitivity SV and the geometry of the probe, based on R = SL / SV = 2L / 3h.5,6 For the cantilever used in this study, L = 445 ␮m, h = 25 ␮m, the measured vertical sensitivity was SV = 4.6 mV/ nm, and R ⬃ 12, which yields a lateral sensitivity of SL = 55 mV/ nm. For PFM experiment, the bone surface was first imaged with the tapping mode AFM to locate an area with collagen fibrils on the surface. Then the AFM was switched to contact mode for piezoelectric characterization. Point measurement of shear piezoresponse was conducted by sweeping the input ac voltage from zero up to 2.5 V with a step of 0.2 V at a fixed point on the bone surface, and acquiring the corresponding lateral PFM responses. For mapping of piezoelectricity on a bone surface, an ac voltage of 4 V pp with a frequency of 60 kHz was applied between the AFM tip and the sample, while the tip simultaneously scanned at 0.1 Hz over the surface. The particular ac frequency was indentified to be away from tip-sample contact resonance in order to eliminate interference from sample elasticity and topography on PFM imaging. The induced piezoelectric signal from tor-

Appl. Phys. Lett. 97, 153127 共2010兲

FIG. 2. High-resolution shear piezoresponse in cortical bone. 共a兲 AFM deflection image, and 共b兲 the simultaneously acquired lateral PFM response image showing the shear-piezoelectric response in bone with a spatial resolution down to the level of subfibrillar structures in individual collagen fibrils. Inset in 共a兲 is a line profile obtained from the line marked in 共b兲 showing the periodic modulation in shear piezoresponse with a periodicity of 60–70 nm.

sional twist of the probe was deconvoluted in the lock-in amplifier and acquired simultaneously with the contact mode image. Figure 1共b兲 shows a 5 ␮m2 high resolution AFM image of the cortical bone sample. Collagen fibrils, randomly oriented with their axis on the surface of the bone sample, were clearly resolved in the image. The collagen fibrils were ⬃50– 200 nm in diameters and were often several microns long. The fibrils were mostly individually isolated from each other, with a few of them crossing other collagen fibrils. The characteristic banding pattern 共60–70 nm兲 of individual collagen fibrils were apparent in the image. Figure 2共a兲 is the contact mode AFM image, simultaneously acquired with the PFM image on an area of the bone surface. Figure 2共b兲 shows a high resolution shear PFM image acquired from the same area as Fig. 2共a兲. Individual collagen fibrils were ostensibly resolved in the PFM image, as a direct evidence of being the source of shear piezoelectricity in bone. The inset in Fig. 2共a兲 is a line scan from Fig. 2共b兲 showing a modulation with a periodicity of 60–70 nm in the shear PFM response, correlating with the characteristic band-


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FIG. 3. Representative shear piezoresponse vs input amplitude curve acquired at a sample location on the bone surface showing the linear piezoelectric coupling. The slope provides a measure of the effective shear piezoelectric coefficient d ⬃ 0.3 pm/ V.

ing pattern of collagen fibrils. This electromechanical heterogeneity in bone was consistent with the reported piezoelectric heterogeneity in individual collagen fibrils.5 The resolution of the obtained PFM map was better than 10 nm. We chose an area where collagen fibrils were randomly orientated on the bone surface, with their axis parallel to the surface. For a collagen fibril oriented with an angle ␪ respect to the AFM probe, the transferred shear response is proportional to sin共␪兲. The maximum shear transfer from the surface to the AFM probe occurs when the probe is perpendicular to a collagen fibril. Figure 3 depicts the point measurements of shear piezoelectricity versus the input amplitude. The response is perfectly linear, as expected for a piezoelectric behavior. Furthermore, this linear behavior indicates that the electrostatic and electrostrictive interferences are minimal, and the obtained signal is a true piezoelectric response. The magnitude of the higher order signals due to electrostatic and electrostrictive effects were measured to be negligible with respect to the piezoelectric signal. Furthermore, lateral piezoresponse imaging is known to be less prone to electrostatic interference due to the symmetrical cancellation of the lateral components of electrostatic forces over the AFM cantilever. The slope of this curve is the shear piezoelectric constant of the bone sample at each point. Point measurements of piezoresponses collected from ten randomly selected points on the bone surface showed a variation in piezoelectric constant between 0.1–0.3 pm/V. The variation could be attributed to the random orientation of collagen fibrils on bone as well as the concentration of the fibrils on different locations on the surface. This coefficient is one order of magnitude smaller than the shear piezoelectricity of an isolated collagen fibril, pre-

sumably due to the confinement effect of collagen fibrils in bone. To our knowledge this is the highest resolution piezoelectricity map obtained on cortical bone samples. This could be due to the direct probing of the dominant piezoelectric mode in underlying collagen fibrils, i.e., the shear mode, in our study. Piezoelectricity is ubiquitous in biological materials. This study could be extended to other types of biological materials that show piezoelectric behavior due to presence of collagen in their structure such as tendon and dentin.9,14 The direct appearance of individual collagen fibrils in lateral PFM map not only confirms their role as the origin of piezoelectricity in bone, but also supports their shear piezoelectric structure, as revealed recently.6 Another implication of our finding is the relationship between structure and function in bone. It is known that the main deformation mechanism in bone involves shearing of collagen proteins by mineral nanocrystals.15,16 The obtained map of shear piezoelectricity in bone exhibits heterogeneous structure. This heterogeneity in electromechanical behavior might be relevant to modulation of the ionic environment of bone and may have an effect on bone mineralization process. In summary, employing lateral PFM, we have presented high resolution map of shear piezoelectricity on a cortical bone sample, revealing subfibrillar microstructure of individual collagen fibrils, consistent with the recent discovery of shear piezoelectricity in isolated collagen fibril. The work is supported by NSF Grant Nos. CMMI 0600583 and CBET 0731096. C. A. L. Bassett and R. O. Becker, Science 137, 1063 共1962兲. E. Fukada and I. Yasuda, J. Phys. Soc. Jpn. 12, 1158 共1957兲. 3 A. A. Marino, R. O. Becker, and S. C. Soderholm, Calcif. Tissue Res. 8, 177 共1971兲. 4 S. Weiner and H. D. Wagner, Annu. Rev. Mater. Sci. 28, 271 共1998兲. 5 M. Minary-Jolandan and M.-F. Yu, ACS Nano 3, 1859 共2009兲. 6 M. Minary-Jolandan and M.-F. Yu, Nanotechnology 20, 085706 共2009兲. 7 C. Halperin, S. Mutchnik, A. Agronin, M. Molotskii, P. Urenski, M. Salai, and G. Rosenman, Nano Lett. 4, 1253 共2004兲. 8 A. Gruverman, D. Wu, B. J. Rodriguez, S. V. Kalinin, and S. Habelitz, Biochem. Biophys. Res. Commun. 352, 142 共2007兲. 9 S. V. Kalinin, B. J. Rodriguez, S. Jesse, T. Thundat, and A. Gruverman, Appl. Phys. Lett. 87, 053901 共2005兲. 10 S. C. Cowin, Bone Mechanics Handbook, 2nd ed. 共CRC, New York, 2001兲. 11 O. Kolosov, A. Gruverman, J. Hatano, K. Takahashi, and H. Tokumoto, Phys. Rev. Lett. 74, 4309 共1995兲. 12 P. Güthner and K. Dransfeld, Appl. Phys. Lett. 61, 1137 共1992兲. 13 A. Gruverman, O. Auciello, and H. Tokumoto, Annu. Rev. Mater. Sci. 28, 101 共1998兲. 14 S. V. Kalinin, B. J. Rodriguez, J. Shin, S. Jesse, V. Grichko, T. Thundat, A. P. Baddorf, and A. Gruverman, Ultramicroscopy 106, 334 共2006兲. 15 H. S. Gupta, W. Wagermaier, G. A. Zickler, D. R.-B. Aroush, S. S. Funari, P. Roschger, H. D. Wagner, and P. Fratzl, Nano Lett. 5, 2108 共2005兲. 16 D. K. Dubey and V. Tomar, Ann. Biomed. Eng. 38, 2040 共2010兲. 1 2
