Bone matrix material properties on the micro- and nanoscale

Abstract

The evaluation of fracture risk in osteoporotic patients, which is still mostly based on bone mineral density (BMD) measurements, constitutes a major clinical challenge. Despite the fact that BMD is highly correlated with fracture risk in large populations, it unfortunately fails to be an accurate predictor for the individual. To increase the accuracy of fracture risk evaluation, a better understanding of all factors affecting bone fracture behaviour, including (but not limited to) BMD, is needed. This means a deeper understanding of bone’s material properties and structure-function relationship is required.

Mammalian bones are composed of mineralized type I collagen fibrils immersed in a matrix of non-collagenous proteins (NCPs). This fundamental unit assembles into progressively larger features in a hierarchical manner, supplying bone with various “lines of defence” against catastrophic failure. Optimal load transfer and energy dissipation mechanisms have, to some extent, been discovered within bone’s nanostructure on which NCPs have been proposed to play a crucial role. Yet, it is largely unknown if integration of such mechanisms occurs to any other hierarchical level. This thesis attempts to answer this question for the osteonal level of cortical bone. The feature dominating this level is a hollow tubular structure of a few hundred micrometres in diameter, the osteons, consisting of concentric aligned lamellae. Lamellae range from 3 -10 ?m in thickness and between them lie interlamellar areas (often referred to as thin lamellae).

This thesis is the outcome of three studies. The first focuses on the aforementioned osteonal features, namely lamellae and interlamellar areas, studying their structure, composition and their mechanical behaviour during loading. For this purpose a series of experimental techniques were used including ?-RAMAN microscopy, atomic force microscopy (AFM), AFM cantilever-based nanoindentation and in-situ AFM analysis during microtensile testing. It was shown that interlamellar areas differ from lamellae by (a) being collagen-deficient and NCP-rich, (b) having a different arrangement of collagen fibres, (c) being more compliant when no load is applied to the bone and (d) exhibiting higher strains under loading conditions. Finally, stable crack propagation was for the first time captured in a time-lapsed fashion within the interlamellar areas by means of AFM, further proving their significant contribution towards fracture toughness.

The second is a technical study for the development of a method capable for generating full fracture-resistance curves (R-curves) of small bone samples where crack propagation cannot easily be observed. The outcome was the development of a novel computer-aided videography method, “whitening-front tracking” method, which uses the whitening effect formed by the development of the damage in front of the crack-tip (frontal process zone) to indirectly track the crack propagation which is needed for the generation of the R-curve.

The new method was then applied in the third study to correlate the ultimate toughness of human cortical bone, i.e. “fracture toughness” and “crack growth resistance” at the tissue-level, with the elasticity inhomogeneity between lamella and interlamellar areas at the osteonal-level and the damage-formation resistance at the micro-level. In this study the mechanical properties of bone in tissue-, micro- and osteonal-level were measured by means of the “whitening front tracking”, reference point indentation (RPI) and AFM cantilever-based nanoindentation methods respectively. The results revealed a correlation of toughness and crack-growth resistance at the tissue-level with the elastic inhomogeneity between the sub-osteonal features. That is, the higher the difference between the moduli of lamellae and interlamellar areas the higher the toughness of the tissue. Furthermore, toughness and crack-growth resistance correlated with bone’s “resistance to damage” as it was characterised by RPI at the micro-level. Finally, these measured suggested age-related degradation of the mechanical properties at all three levels measured.

Overall, the results presented in this thesis propose that osteons are the principal component of a previously unknown proactive mechanism which transfers load and movement in a manner analogous to engineered “elastomeric bearing pads”. This ability originates from the elastic inhomogeneity between the lamellae and the interlamellar areas which allows osteons to adapt to high stresses without damage formation.

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Identifiers

ORCID for Orestis Katsamenis: orcid.org/0000-0003-4367-4147

ORCID for Philipp J. Thurner: orcid.org/0000-0001-7588-9041

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