Elsevier

Journal of Biomechanics

Volume 46, Issue 4, 22 February 2013, Pages 696-701
Journal of Biomechanics

Changes in the viscoelastic properties of cortical bone by selective degradation of matrix protein

https://doi.org/10.1016/j.jbiomech.2012.11.038Get rights and content

Abstract

We have studied stress relaxation of bovine femoral cortical bone specimens treated with KOH aqueous solution which had been known to degrade selectively protein molecules in bone. With the KOH treatment, we found an increase in specimens' volume. This increase was regarded as swelling of the bone specimen, presumably due to matrix protein network degradation including that of collagen. In an analogy of bone to gel structure, an increasing ratio of specimen volume was used as an indicating parameter for the matrix protein network degradation by the treatment. Although an empirical equation with a linearly combined form of two Kohlrausch–Williams–Watts (KWW) functions has been shown to describe the stress relaxation of bone specimens, a single KWW function was suitable for the bone specimens treated with KOH solution for as little as 3 h. In KOH treated specimens, both the initial modulus and the relaxation time decreased with the volume-increasing ratio, while the relaxation time distribution did not change. A chemo-rheological consideration attributed the reduction of modulus values to the network degradation in the organic matrix phase. The relaxation time of KOH treated specimens was thought to be related to the longer relaxation time of untreated bones, although there was a discontinuity between the extrapolated relaxation time values for KOH treated specimens and untreated specimens. This discontinuity may have originated from the release of residual stress existing in the bone by the matrix protein degradation. The results of the present study suggest that the state of matrix protein is crucial for integrating the mechanical properties of bone.

Introduction

Bone has been often regarded mechanically as a composite material of hydrated organic matrix mainly composed of collagen and hydroxyapatite (HAp)-like mineral phase. It is thought that the pliant collagen is reinforced by stiff mineral particles, and, as a composite, the brittleness of the mineral is compensated for by the viscoelasticity of the collagen. Recently, the existence of non-collagenous glue proteins that connect mineralized collagen fibers has been revealed (Recker, 1992, Braidotti et al., 1997, Fanter et al., 2005). Because of the viscoelasticity of collagen fibers and non-fibrous proteins in the bone matrix, bone itself has noticeable viscoelasticity (Currey, 1965, Sasaki, 2000). As for the mechanical role of organic phase in bone, Ji and Gao (2004) predicted on the basis of the tension–shear chain model (Jäger and Fratzl, 2000) that the organic phase endows bones with important characteristics such as crack shielding, and energy dissipation, that is, the strength and the toughness. From the result, it can be easily expected that a change in organic phase would affect the mechanical properties of bone, in particular its viscoelasticity. However, there have been not so many studies that treat detailed or thorough experimental results on the relationship between matrix protein degradation and the mechanical properties of bone. Wynnyckyj et al. (2009) studied the change in toughness of emu femoral bone after the selective degradation of bone collagen by KOH treatment. Following the treatment their stress–strain curves indicated an increase in the elastic energy needed for bone destruction. As the increment was contributed mainly by the stress–strain curve after partial fracture, the toughness increase is considered as a mechanism caused by something like a sacrificial structure. In order to relate such a structural feature to the toughness of bone, direct measurements of viscoelasticity would provide useful information about the relationship. The aim of this study was to obtain the relationship between the state of matrix protein and the viscoelastic properties of bone. For this, we prepared bone specimens with variously degraded matrix proteins. As KOH is known to affect protein molecules without changing the mineral phase in bone (Abe et al., 1992, Wynnyckyj et al., 2009), we used KOH solution for the selective degradation of matrix protein in bone. We monitored the degree of degradation of collagen in bone by hydroxyproline assays (Reddy and Enwemeka, 1996) for a measure of matrix protein degradation.

In our previous papers, as a new empirical equation for the description of stress relaxation of cortical bone, we proposed that stress relaxation of cortical bone could generally be described by a linear combination of two Kohlraush–Williams–Watts (KWW) functions (Iyo et al., 2004, Iyo et al., 2006),E(t)=E0{Aexp[(t/τ1)β]+(1A)exp[(t/τ2)γ]},[0<A,β,γ<1],where E0 is the initial modulus value, E(0).τ1 and τ2τ1) are characteristic times of the relaxation processes, A is the fractional contribution of the fast relaxation to the whole relaxation process, and β and γ are parameters describing the shape of the relaxation modulus. It has been revealed that the first term represents the relaxation in the collagen matrix in bone and the second term is related to the change in a higher-order structure of bone that is responsible for the anisotropic mechanical properties (Iyo et al., 2004). It seems to be possible to relate the viscoelastic properties and the hierarchical structure of bone by investigating these mechanical parameters. The expected change in mechanical properties of bone due to the degradation of matrix proteins would be quantified by the parameters in Eq. (1).

Section snippets

Materials

The bone samples used in this study were obtained from the anterior area of the mid-diaphysis of 18-month-old bovine femoral cortical bone. Optical microscopic examination showed that all of the samples were generally plexiform but partly transformed into Haversian bone. The samples were cut using a diamond saw. The cut sections were shaped by emery paper under tap water into rectangular plates approximately 0.5 cm wide, 5.0 cm long and 0.1 cm thick. The longer edge of the specimen plate was

Hydroxyproline assay

Fig. 1 shows the weight ratio of collagen dissolved in the reactor solution against collagen in bone, [Dcol], as a function of reaction time. [Dcol] was estimated from the hydroxylproline concentration, [Hyp], in the solution (Wynnyckyj et al., 2009, Reddy and Enwemeka, 1996). [Hyp] was determined by the hydroxylproline assay indicated above. The [Dcol] values for the initial two points, the reactions for 3 and 6 hours, were not different from those of controls and seemed to be below the

Matrix protein degradation

As shown in Fig. 1, the deviation of [Dcol] values of specimens treated for 3 and 6 h from that of untreated specimens was too small to be detected by the hydroxyproline assay. However, even though the increment in [Dcol] value is negligibly small, the mechanical properties of bone specimens after only 3 h of treatment with KOH drastically changed. This fact indicates that for specimens treated for 3 h and 6 h changes that cannot be reflected on [Dcol] would be resulted. There are at least two

Conflict of interest statement

The authors do not have any conflict of interest about the material within the manuscript submitted.

Acknowledgments

A part of this work was supported by the Grant-in-Aid for Scientific Research (A) (No. 19200035) and (C) (No. 21500401) both from JSPS.

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