Precision of nanoindentation protocols for measurement of viscoelasticity in cortical and trabecular bone Hanna Isaksson a,n , Shijo Nagao b , Marta Ma"kiewicz a,b , Petro Julkunen c , Roman Nowak b , Jukka S. Jurvelin a a Department of Physics and Mathematics, University of Eastern Finland, PO Box 1627, Kuopio 70211, Finland b The Nordic Hysitron Laboratory, Faculty of Chemistry and Materials Science, Aalto University, FI-00076 Aalto, Finland c Department of Clinical Neurophysiology, Kuopio University Hospital, Kuopio, Finland article info Article history: Accepted 16 April 2010 Keywords: Nanoindentation Viscoelastic Creep Strain-hardening Dissipated energy Semi-dynamic Bovine abstract Nanoindentation has recently gained attention as a characterization technique for mechanical properties of biological tissues, such as bone, on the sub-micron level. However, optimal methods to characterize viscoelastic properties of bones are yet to be established. This study aimed to compare the time-dependent viscoelastic properties of bone tissue obtained with different nanoindentation methods. Bovine cortical and trabecular bone samples (n ¼8) from the distal femur and proximal tibia were dehydrated, embedded and polished. The material properties determined using nanoinden- tation were hardness and reduced modulus, as well as time-dependent parameters based on creep, loading-rate, dissipated energy and semi-dynamic testing under load control. Each loading protocol was repeated 160 times and the reproducibility was assessed based on the coefficient of variation (CV). Additionally, three well-characterized polymers were tested and CV values were calculated for reference. The employed methods were able to characterize time-dependent viscoelastic properties of bone. However, their reproducibility varied highly (CV 9–40%). The creep constant increased with increasing dwell time. The reproducibility was best with a 30 s creep period (CV 18%). The dissipated energy was stable after three repeated load cycles, and the reproducibility improved with each cycle (CV 23%). The viscoelastic properties determined with semi-dynamic test increased with increase in frequency. These measurements were most reproducible at high frequencies (CV 9–10%). Our results indicate that several methods are feasible for the determination of viscoelastic properties of bone material. The high frequency semi-dynamic test showed the highest precision within the tested nanoindentation protocols. & 2010 Elsevier Ltd. All rights reserved. 1. Introduction The mineral phase in bone controls its stiffness, whereas the collagen contributes to the ultimate strength and toughness of the bone (Boivin et al., 2008; Wang et al., 2001). Bone becomes more brittle with age (Burr, 2002). Most likely, this is due to decreased ductility, which may be related to viscoelasticity of the collagen fibers (Les et al., 2004, 2005). Hence, the changes in the collagen phase can contribute significantly to increased fragility of bone (Banse et al., 2002; Knott and Bailey, 1998; Wang et al., 2002). Optimal methods should be determined to sensitively evaluate the time-dependent properties of bone, and to assess effectively the effects of experimental bone interventions. Although nanoindentation is widely used in material science, it is a relatively new tool for testing biological tissues (Lewis and Nyman, 2008; Oyen and Cook, 2009). The obtained pertinent parameters, which have previously been quantified in bone (Hengsberger et al., 2002; Oyen et al., 2008; Rho et al., 1997; Roy et al., 1999; Zysset et al., 1999), include hardness and reduced elastic modulus. In contrast, determination of the viscoelastic properties of bone or mineralized tissues by nanoindentation is less covered. The creep behavior of human enamel (He and Swain, 2009) and bone (Bembey et al., 2006b) and the determination of dissipated energy in cortical bone using repeated loading (Fan and Rho, 2003), are the only subjects investigated so far. Also, the viscoelastic properties of a collagen scaffold were quantified with quasi-static indentation (Chaudhry et al., 2009). The aforemen- tioned studies used different indentation protocols to characterize different physical parameters on various types of tissues. A systematic extensive characterization of bone viscoelastic proper- ties and the reliability and precision of the different methods using nanoindentation have not yet been presented. Bone is an inhomogeneous, anisotropic and viscoelastic material. Hence, measurements on the nanoscale add new aspects Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/jbiomech www.JBiomech.com Journal of Biomechanics 0021-9290/$ - see front matter & 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.jbiomech.2010.04.017 n Corresponding author. Tel.: + 358 40 355 2079; fax: + 358 1716 3032. E-mail address: hanna.isaksson@uef.fi (H. Isaksson). Journal of Biomechanics 43 (2010) 2410–2417