Cyclic cryopreservation affects the nanoscale material properties of trabecular bone

Abstract

Tissues such as bone are often stored via freezing, or cryopreservation. During an experimental protocol, bone may be frozen and thawed a number of times. For whole bone, the mechanical properties (strength and modulus) do not significantly change throughout five freeze-thaw cycles. Material properties at the trabecular and lamellar scales are distinct from whole bone properties, thus the impact of freeze-thaw cycling at this scale is unknown. To address this, the effect of repeated freezing on viscoelastic material properties of trabecular bone was quantified via dynamic nanoindentation. Vertebrae from five cervine spines (1.5-year-old, male) were semi-randomly assigned, three-to-a-cycle, to 0–10 freeze-thaw cycles. After freeze-thaw cycling, the vertebrae were dissected, prepared and tested. ANOVA (factors cycle, frequency, and donor) on storage modulus, loss modulus, and loss tangent, were conducted. Results revealed significant changes between cycles for all material properties for most cycles, no significant difference across most of the dynamic range, and significant differences between some donors. Regression analysis showed a moderate positive correlation between cycles and material property for loss modulus and loss tangent, and weak negative correlation for storage modulus, all correlations were significant. These results indicate that not only is elasticity unpredictably altered, but also that damping and viscoelasticity tend to increase with additional freeze-thaw cycling.

Introduction

Bone tissue is frequently used in research studies. Many laboratories store bone specimens via freezing, often at the −18 °C to −20 °C range typical of industrial freezers (Langton and Njeh, 2004, Repositories, 2005). These specimens frequently undergo more than one freeze. The effect of freezing on bone material properties has been previously investigated (Borchers et al., 1995, Jung et al., 2011, Linde and Sørensen, 1993, Moreno and Forriol, 2002, Reikerås et al., 2010, Shaw et al., 2012) and has shown that changes in properties after a single freeze are not statistically significant in whole bone or cancellous bone cores. Whether additional freezing and thawing alters the tissue is unclear: a significant decrease in material properties subsequent to freeze-thaw cycling has been reported (Boutros et al., 2000, McElderry et al., 2011), while others have indicated insignificant changes (Borchers et al., 1995, Jung et al., 2011, Linde and Sørensen, 1993). Previous studies on freeze-thawing bone have been quasi-static and at the macro-scale; thus, significant changes in small-scale properties may adversely affect results of small-scale tests (e.g. Pathak et al., 2011, Polly et al., 2012).

Nanoindentation has been used to evaluate, material-scale characteristics of bone such as inter-trabecular variation (Giambini et al., 2012, Oyen, 2010), differences between cortical and cancellous structures (Bayraktar et al., 2004), and osteonal and lamellar differences (Faingold et al., 2012, Rho et al., 1997, Zysset et al., 1999). The micro-scale viscoelastic characteristics of bone have been measured via dynamic nanoindentation (Polly et al., 2012, Rodriguez-Florez et al., 2013, Shepherd et al., 2011), in which a contact stiffness measurement method (Asif et al., 1999) is employed to calculate the material elasticity, damping, and viscoelasticity.

Given the dependence on intrinsic variables of bone material properties (Lewis and Nyman, 2008), the fundamental differences between structural and material properties of cancellous bone (Rho et al., 1998), and the impact of freeze-thawing on soft tissue, the associated damage mechanics may change at the material scale. Thus, the purpose of this work is to quantify and compare the material-scale viscoelastic properties of cancellous bone following 0 (fresh bone) to 10 freeze-thaw cycles, under the hypothesis that no significant change will be discernible between any two cycles.