Modal analysis of nanoindentation data, confirming that reduced bone turnover may cause increased tissue mineralization/elasticity

https://doi.org/10.1016/j.jmbbm.2018.05.014Get rights and content

Abstract

It is widely believed that the activities of bone cells at the tissue scale not only govern the size of the vascular pore spaces (and hence, the amount of bone tissue available for actually carrying the loads), but also the characteristics of the extracellular bone matrix itself. In this context, increased mechanical stimulation (in mediolateral regions of human femora, as compared to anteroposterior regions) may lead to increased bone turnover, lower bone matrix mineralization, and therefore lower tissue modulus. On the other hand, resorption-only processes (in endosteal versus periosteal regions) may have the opposite effect. A modal analysis of nanoindentation data obtained on femurs from the Melbourne Femur Research Collection (MFRC) indeed confirms that bone is stiffer in endosteal regions compared to periosteal regions (E̅endost = 29.34 ± 0.75 GPa > E̅periost = 24.67 ± 1.63 GPa), most likely due to the aging-related increase in resorption modeling on endosteal surfaces resulting in trabecularization of cortical bone. The results also show that bone is stiffer along the anteroposterior direction compared the mediolateral direction (E̅anteropost = 28.89 ± 1.08 GPa > E̅mediolat = 26.03 ± 2.31 GPa), the former being aligned with the neutral bending axis of the femur and, thus, undergoing more resorption modeling and consequently being more mineralized.

Introduction

As bones are the predominant load carriers in the vertebrate animal kingdom, their mechanical properties (in particular elasticity and strength) have always been of great interest for biomedicine and the scientific community at large. However, bone exhibits a hierarchical organization (Lakes, 1993, Katz et al., 1984, Weiner and Wagner, 1998), and mechanical properties involving quantities of the dimension "force per area" need to be assigned to a specific observation scale, on which these forces and areas are recorded. As regards elasticity and strength, two such observation scales are of particular interest for the biomedical field:

  • the macroscopic scale with a material volume measuring some hundreds of micrometers to milimeters cubed, where cortical and trabecular bone are distinguished. At this length scale bone exhibits pore spaces with characteristic sizes of tens to hundreds of micrometers, populated by biological cells (Buckwalter et al., 1995); and

  • the extracellular bone tissue, with material volumes at the tens of micrometers-scale, consisting of a nanocomposite made of hydroxyapatite, collagen, and water with non-collageneous organics (Lees, 1987).

It is widely believed that the activities of bone cells at the higher one of these two levels not only govern the size of these pore spaces (and hence, the amount of bone tissue available for actually carrying the loads), but also the characteristics of the extracellular bone matrix itself. In this context, several mechanisms have been proposed, and the present paper focusses on the following research question: Does excessive bone resorption cause extracellular tissue elasticity increase?

The relevance of this question is inferred from the following deliberations reported in literature:

  • Within a certain physiological load window (Frost, 1964, Frost, 1992, Rubin and Lanyon, 1985, Turner, 1998, Hsieh et al., 2001, Rubin et al., 2001), mechanical stimulation is known to increase the rate of bone remodeling or turnover, i.e. the cellular processes leading to resorption of old extracellular bone tissue and subsequent formation of new tissue. On the other hand, bone turnover is associated to the mineralization degree at the tissue scale (Boivin and Meunier, 2002, Meunier and Boivin, 1997, Boivin et al., 2000), with higher turnover resulting in lower mineralization. The "complete" (or secondary) mineralization of the tissue would last up to years (Marotti et al., 1972, Bala et al., 2010), and would be hindered simply by tissue resorption before its full "maturation". Conversely, the fingerprint of low turnover would be highly mineralized tissue.

  • Increased bone tissue mineralization causes an increase in the tissue elastic modulus, according to the micromechanical theory applied to, and experimentally validated for, extracellular bone tissue, as reported, among others, by Crolet et al., 1993, Hellmich et al., 2004, Fritsch and Hellmich, 2007, Grimal et al., 2011.

The present paper aims at giving an answer to the aforementioned research question, by presenting a correspondingly designed nanoindentation study and its evaluation. In this context, it is important that the tested samples all consist of very similar tissue, so that any differences between tissue properties from one tested sample to another result only from the resorption activities as described above, with any other causes remaining highly improbable. A short literature review, see e.g. the Appendix of Hellmich et al. (2008), evidences that bone tissue within adult bony organs is, when spatially averaged over long bone cross sections, constant over space and time. This was independently shown by both microscopic and radiographic analyses revealing mineral density distributions (Boivin and Meunier, 2002, Akkus et al., 2003, Roschger et al., 2003, Bossy et al., 2004), and by nanoindentation studies (Hoffler et al., 2000a, Rho et al., 2002, Feng and Jasiuk, 2011, Wolfram et al., 2010).

Accordingly, the current study was performed on mid-shafts from adult female human femurs provided by the Melbourne Femur Research Collection (MFRC), discriminating regions of potentially higher and lower mineralization, and hence, higher and lower tissue elastic modulus:

  • anteroposterior regions, being closely aligned with the neutral axis of the bending beam structure "human femur" (Feik et al., 2000, Thomas et al., 2005, Thomas et al., 2006), therefore undergoing lower mechanical stimulation, lower turnover, and exhibiting higher mineralization and tissue elasticity) versus mediolateral regions (with higher mechanical stimulation, higher turnover, lower mineralization and lower tissue elasticity); and

  • endosteal regions with significantly reduced formation activities indicated by trabecularization of cortical bone (Simmons et al., 1991, Cooper et al., 2007), and with expectedly higher mineralization and higher tissue modulus, versus periosteal regions (with expectedly lower mineralization and lower tissue modulus).

The correspondingly obtained nanoindentation data underwent a modal analysis as introduced by Furin et al., 2016, Kariem et al., 2015, in order to identify those indented half-spaces the stiffnesses of which were not affected by microcracks.

Section snippets

Selection and preparation of femoral mid shaft samples

Two femoral mid-shaft sections were obtained from bone samples collected in 1990–1993 and 1998 by the Victorian Institute of Forensic Medicine as part of the Melbourne Femur Research Collection (MFRC). Ethical approval was given by the Office for Research Ethics and Integrity as part of a larger study, conducted by the researchers at Melbourne Dental School, where the collection is housed. The femurs, coded 269 and 275, originated from female donors without diseases directly affecting bone, and

Results

We applied the deconvolution algorithm to all experimental data. The following results are expressed as the expected value and standard deviation of the right-most Gaussian distribution depicted in Fig. 2, Fig. 3.

The average modulus of the undamaged bone material on the endosteal surface of all four tested samples was E̅endost = 29.34 ± 0.74 GPa (Fig. 2A); while the intracortical modulus E̅intracort amounted to 26.23 ± 1.93 GPa (Fig. 2B); and the periosteal modulus E̅periost amounted to

Discussion

The present study confirmed that lower bone turnover leads to higher extracellular tissue elasticity, most likely through increased levels of bone mineralization. Thereby, the elasticity values were determined from a modal analysis of data obtained from a nanoindentation grid technique. This allowed for identification of the bone tissue elasticity at the observation scale of a few micrometers; as it is well known that the maximum indentation size (here 250 nm) needs to be less than one tenth of

Acknowledgements

The authors would like to acknowledge the kind, expert assistance of Dr. Tim Spelman in the statistical analysis of this project, as well as the assistance of David Thomas in the handling of the samples. We are grateful to the mortuary staff and the staff of the Donor Tissue Bank of the Victorian Institute of Forensic Medicine Australia for their assistance in the collection of the bone specimens used for this study, and we are particularly grateful to the next-of-kin of the donors for

Authors’ roles

Conceptual study design: all. Sample cutting and identification: RB. Nanoindentation sample preparation and tests: MIP. Data analysis: MIP. Manuscript drafting: MIP and CH. Revising manuscript content: all. Approving final version of manuscript: RB, JGC, PP, CH.

References (88)

  • S. Eppell et al.

    Shape and size of isolated bone mineralites measured using atomic force microscopy

    J. Orthop. Res.

    (2001)
  • Z. Fan et al.

    Anisotropic properties of human tibial cortical bone as measured by nanoindentation

    J. Orthop. Res.

    (2002)
  • L. Feng et al.

    Multi-scale characterization of swine femoral cortical bone

    J. Biomech.

    (2011)
  • A. Fritsch et al.

    Universal' microstructural patterns in cortical and trabecular, extracellular and extravascular bone materials: micromechanics-based prediction of anisotropic elasticity

    J. Theor. Biol.

    (2007)
  • A. Fritsch et al.

    Ductile sliding between mineral crystals followed by rupture of collagen crosslinks: Experimentally supported micromechanical explanation of bone strength

    Journal of Theoretical Biology

    (2009)
  • H.M. Frost

    Perspectives: bone's mechanical usage windows

    Bone Miner.

    (1992)
  • Q. Grimal et al.

    A two-parameter model of the effective elastic tensor for cortical bone

    J. Biomech.

    (2011)
  • T. Hassenkam et al.

    High-resolution AFM imaging of intact and fractured trabecular bone

    Bone

    (2004)
  • C. Hellmich et al.

    Mineral-collagen interactions in elasticity of bone ultrastructure – a continuum micromechanics approach

    Eur. J. Mech. A

    (2004)
  • S. Hengsberger et al.

    Nanoindentation discriminates the elastic properties of individual human bone lamellae under dry and physiological conditions

    Bone

    (2002)
  • C.E. Hoffler et al.

    Heterogeneity of bone lamellar-level elastic moduli

    Bone

    (2000)
  • S.S. Huja et al.

    Development of a fluorescent light technique for evaluating microdamage in bone subjected to fatigue loading

    J. Biomech.

    (1999)
  • H. Kariem et al.

    Micro-poro-elasticity of baghdadite-based bone tissue engineering scaffolds: a unifying approach based on ultrasonics, nanoindentation, and homogenization theory

    Mater. Sci. Eng. C

    (2015)
  • R. Khanna et al.

    Bone nodules on chitosan-polygalacturonic acid-hydroxyapatite nanocomposite films mimic hierarchy of natural bone

    Acta Biomater.

    (2011)
  • C. Kohlhauser et al.

    Ultrasonic contact pulse transmission for elastic wave velocity and stiffness determination: influence of specimen geometry and porosity

    Eng. Struct.

    (2013)
  • K. Luczynski et al.

    Extracellular bone matrix exhibits hardening elastoplasticity and more than double cortical strength: evidence from homogeneous compression of non-tapered single micron-sized pillars welded to a rigid substrate

    J. Mech. Behav. Biomed. Mater.

    (2015)
  • A. Malandrino et al.

    Anisotropic tissue elasticity in human lumbar vertebra, by means of a coupled ultrasound-micromechanics approach

    Mater. Lett.

    (2012)
  • P.J. Meunier et al.

    Bone mineral density reflects bone mass but also the degree of mineralization of bone: therapeutic implications

    Bone

    (1997)
  • M. Miller et al.

    Surface roughness criteria for cement paste nanoindentation

    Cem. Concr. Res.

    (2008)
  • C. Morin et al.

    Micromechanics of elastoplastic porous polycrystals: Theory, algorithm, and application to osteonal bone

    International Journal of Plasticity

    (2017)
  • F. O'Brien et al.

    The effect of bone microstructure on the initiation and growth of microcracks

    J. Orthop. Res.

    (2005)
  • A.G. Reisinger et al.

    Principal stiffness orientation and degree of anisotropy of human osteons based on nanoindentation in three distinct planes

    J. Mech. Behav. Biomed. Mater.

    (2011)
  • J.Y. Rho et al.

    Microstructural elasticity and regional heterogeneity in human femoral bone of various ages examined by nano-indentation

    J. Biomech.

    (2002)
  • J.-Y. Rho et al.

    Elastic properties of human cortical and trabecular lamellar bone measured by nanoindentation

    Biomaterials

    (1997)
  • P. Roschger et al.

    Constant mineralization density distribution in cancellous human bone

    Bone

    (2003)
  • M. Schaffler et al.

    Examination of compact bone microdamage using back-scattered electron microscopy

    Bone

    (1994)
  • M.B. Schaffler et al.

    Aging and matrix microdamage accumulation in human compact bone

    Bone

    (1995)
  • P. Tennis et al.

    A model for two types of calcium silicate hydrate in the microstructure of Portland cement pastes

    Cem. Concr. Res.

    (2000)
  • C.H. Turner

    Three rules for bone adaptation to mechanical stimuli

    Bone

    (1998)
  • B. van Rietbergen et al.

    A new method to determine trabecular bone elastic properties and loading using micromechanical finite-element models

    J. Biomech.

    (1995)
  • T. Wenzel et al.

    In vivo trabecular microcracks in human vertebral bone

    Bone

    (1996)
  • U. Wolfram et al.

    Rehydration of vertebral trabecular bone: influences on its anisotropy, its stiffness and the indentation work with a view to age, gender and vertebral level

    Bone

    (2010)
  • H.S. Yoon et al.

    Ultrasonic wave propagation in human cortical bone – II. Measurements of elastic properties and microhardness

    J. Biomech.

    (1976)
  • P.K. Zysset et al.

    Elastic modulus and hardness of cortical and trabecular bone lamellae measured by nanoindentation in the human femur

    J. Biomech.

    (1999)
  • Cited by (6)

    • Micromechanics of dental cement paste

      2021, Journal of the Mechanical Behavior of Biomedical Materials
    • Influence of surface roughness on concrete nanoindentation

      2022, European Journal of Environmental and Civil Engineering
    • AN ALTERNATIVE to PERIODIC HOMOGENIZATION for DENTIN ELASTIC STIFFNESS

      2020, Journal of Mechanics in Medicine and Biology
    View full text