Post on 01-May-2023
Royal College of Surgeons in Irelande-publications@RCSI
Anatomy Articles Department of Anatomy
2009
Biomechanical properties across trabeculae fromthe proximal femur of normal and ovariectomisedsheep.Orlaith BrennanRoyal College of Surgeons in Ireland
Oran D KennedyRoyal College of Surgeons in Ireland
T Clive LeeRoyal College of Surgeons in Ireland
Susan M RackardUniversity College Dublin
Fergal J O'BrienRoyal College of Surgeons in Ireland
This Article is brought to you for free and open access by the Departmentof Anatomy at e-publications@RCSI. It has been accepted for inclusion inAnatomy Articles by an authorized administrator of e-publications@RCSI.For more information, please contact epubs@rcsi.ie.
CitationBrennan O, Kennedy OD, Lee TC, Rackard SM, O'Brien FJ. Biomechanical properties across trabeculae from the proximal femur ofnormal and ovariectomised sheep. Journal of Biomechanics. 2009;42(4):498-503.
— Use Licence —
Attribution-Non-Commercial-ShareAlike 1.0
You are free:• to copy, distribute, display, and perform the work.• to make derivative works.
Under the following conditions:• Attribution — You must give the original author credit.• Non-Commercial — You may not use this work for commercial purposes.• Share Alike — If you alter, transform, or build upon this work, you may distribute the resulting work onlyunder a licence identical to this one.
For any reuse or distribution, you must make clear to others the licence terms of this work. Any of theseconditions can be waived if you get permission from the author.
Your fair use and other rights are in no way affected by the above.
This work is licenced under the Creative Commons Attribution-Non-Commercial-ShareAlike License. Toview a copy of this licence, visit:
URL (human-readable summary):• http://creativecommons.org/licenses/by-nc-sa/1.0/
URL (legal code):• http://creativecommons.org/worldwide/uk/translated-license
This article is available at e-publications@RCSI: http://epubs.rcsi.ie/anatart/13
Elsevier Editorial System(tm) for Journal of Biomechanics
Manuscript Draft
Manuscript Number:
Title: Biomechanical Properties across Trabeculae from the Proximal Femur of Normal and Ovariectomised
Sheep
Article Type: Full Length Article (max 3000 words)
Section/Category:
Keywords: Nanoindentation; Ovariectomy; Biomechanical Properties; Animal Model; Trabecular Bone
Corresponding Author: Dr Orlaith Brennan, Ph.D.
Corresponding Author's Institution: Royal College of Surgeons in Ireland
First Author: Orlaith Brennan, Ph.D.
Order of Authors: Orlaith Brennan, Ph.D.; Oran D Kennedy, Ph.D.; T C Lee, Ph.D.; Susan M Rackard, Ph.D.;
Fergal J O'Brien, Ph.D.
Manuscript Region of Origin:
Conflict of Interest Statement
None of the authors has any conflict of interest to report.
Conflict of Interest Statement
RCSIRoyal College of Surgeons in IrelandColáist e Ríoga na Máinlia in Éir inn RCSIRCSIRoyal College of Surgeons in IrelandColáist e Ríoga na Máinlia in Éir inn
RE: Biomechanical Properties across Trabeculae from the Proximal Femur of Normal and Ovariectomised SheepOrlaith Brennan, Oran D Kennedy, T Clive Lee, Susan M Rackard and Fergal J O’Brien
31st July 2008
Dear Sir/Madam,
On behalf of my co-authors, I am submitting the enclosed original article for consideration for publication in the Journal of Biomechanics. It has not been submitted for publication elsewhere nor has it been published in whole or in part elsewhere. I attest to the fact that all authors listed on the title page were fully involved in the study and preparation of the manuscript and agree on its submission to the Journal of Biomechanics. No written assistance was obtained in the preparation of this manuscript.
Regards,
Orlaith Brennan
Orlaith BrennanDepartment of Anatomy123 St. Stephen’s Green, Dublin 2, IrelandTel: +353 1 402 2147 Fax: +353 1 402 2355Email: obrennan1@rcsi.ie
* Cover Letter
1
Biomechanical Properties across Trabeculae from the Proximal Femur of Normal and
Ovariectomised Sheep
Brennan Oa,b*, Kennedy ODa,b, Lee TCa,b, Rackard SMc, O’Brien FJa,b
a Department of Anatomy, Royal College of Surgeons in Ireland, Dublin 2,
Ireland
b Trinity Centre for Bioengineering, Trinity College Dublin, Dublin 2, Ireland
c School of Agriculture, Food Science and Veterinary Medicine, University
College Dublin, Dublin 4, Ireland
* Corresponding Author
Orlaith Brennan
Department of Anatomy,
Royal College of Surgeons in Ireland,
123, St Stephen’s Green,
Dublin 2,
Ireland
Phone: +353 1 4022147
Fax: +353 1 4022355
E-mail: obrennan1@rcsi.ie
Word Count: 2,837
Submission: Original Article
* Manuscript
2
Introduction
Older bone is continually removed and replaced by new bone through the remodelling
process, also known as bone turnover. At any one time in normal bone, about 20% of
the trabecular surfaces are undergoing remodelling (Eriksen et al., 1994; Ott, 1996).
In general, estrogens inhibit bone resorption by decreasing both osteoclast numbers
and activity (Krassas and Papadopoulou, 2001). High-turnover, or type I, osteoporosis
occurs in women after the age of 50 due to the sudden postmenopausal decrease in
estrogen levels. The ovariectomy procedure is a recognised model for inducing
postmenopausal osteoporosis by reducing the level of circulating hormones and
increasing bone turnover. Following the formation of osteoid by osteoblasts,
mineralisation occurs in two phases. Primary mineralisation occurs when the bone
mineral is deposited during the bone remodelling cycle. Secondary mineralisation
describes the process of further mineralisation after the remodelling cycle has been
completed and occurs from the periphery of the bone (Compston, 2006). During
primary mineralisation, about half of the bone mineral accumulates within a few days.
Secondary mineralisation proceeds more slowly during 6 months or more and further
increases the density (Lips, 2001). The degree of secondary mineralisation is critically
dependent on the rate of remodelling or bone turnover. When this is low, there is
more time for mineralisation to proceed whereas in high turnover states, recently
formed bone is removed before prolonged secondary mineralisation can occur
(Meunier and Boivin, 1997). As mineralisation is a determinant of Young’s modulus
and hardness, bone turnover is an influential factor in bone biomechanical properties.
Traditionally, when the term ‘mechanical properties’ is used in relation to bone, it is
taken to mean purely structural properties (An and Draughn, 2000). However, the
mechanical strength of any material is dependent not only on structural properties but
3
also on the properties of its constituent material. Trabecular bone is a hierarchical
material (Paris et al., 2000), the structure of which guarantees that the mechanical
properties will also vary at different levels. In order to understand why it fails, it is
necessary to examine its mechanical behaviour at a number of different levels. At the
lowest level of hierarchy, the bone matrix consists of mineralized collagen fibrils
aligned primarily in a longitudinal direction, with some variation within lamellar
interface regions (Weiner et al., 1997). These fibrils form lamellae a few microns
thick and finally these lamellae are arranged into trabeculae (Rokitaa et al., 2005).
In an effort to address the issue of scaling, mechanical tests of trabeculae have been
carried out to determine the modulus of individual trabeculae (Runkle and Pugh,
1975; Townsend et al., 1975). Since then, numerous measurements of the elastic
modulus of individual trabeculae have been reported (Kuhn et al., 1989; Mente and
Lewis, 1989; Ryan and Williams, 1989; Choi et al., 1990; Rho et al., 1993; Bini et al.,
2002; McNamara et al., 2006). These studies have involved a variety of micro-
mechanical tests, including tensile, three-point bending, four-point bending and
buckling. Reported values for elastic modulus of single human trabeculae from these
studies have ranged from 1.89 to 14 GPa. However, to fully understand the origins of
the strength of a material, investigation at a finer scale is required.
Recently the technique of nanoindentation has been employed to determine the
mechanical properties of bone tissue. Unlike previous work which determined the
elastic modulus of bone from a structural test, nanoindentation examines bone at the
material level and can have a resolution of 1µm (Turner et al., 1999). The elastic
behaviour of trabecular bone is a function of not only quantity and architecture, but
also of tissue material properties (Bourne and van der Meulen, 2004). It has also been
suggested that tissue modulus variation may have a substantial effect on the
4
biomechanical properties of trabecular bone (Jaasma et al., 2002). Nanoindentation
provides the ideal means by which to examine these changes. Due to the high
resolution, it is possible to not only obtain a modulus for trabecular bone tissue, but
also to profile the modulus across the width of a trabecula. In bone, the modulus has
been shown to correlate positively with calcium content (Currey, 1988) and
subsequently with the degree of mineralisation (Choi et al., 1990; Silva et al., 2004;
Mulder et al., 2007). In iliac crest biopsies, bone was found to become more
mature/crystalline moving from the periphery towards the centre of trabeculae
(Paschalis et al., 1997). In human mandibular trabecular bone, the degree of
mineralisation has also been shown to increase with distance from the surface of the
trabeculae to their cores (Renders et al., 2006). This is consistent with the process of
bone formation on the surface of trabeculae after which it becomes more mineralised
and rigid with age (Ziv et al., 1996).
Indentation tests are performed by driving a diamond indenter into the specimen
surface and dynamically collecting the applied force and displacement data. Material
properties are then derived from these data. As the indenter is driven into the material,
both elastic and plastic deformations cause the material to form a hardness impression
matching the shape of the indenter tip. As the indenter tip is removed, only the elastic
portion of the displacement is recovered and it is this recovery which allows the
elastic properties be determined.
Objectives
The aim of this study was to determine how modulus and hardness vary across the
width of trabeculae extracted from the ovine proximal femur. Furthermore the effect
of ovariectomy on these biomechanical properties was also examined.
5
Materials and Methods
Thirty-eight skeletally mature aged female sheep were randomly assigned into one of
two groups, ovariectomy (OVX, n=19) or control (n=19) on which no operative
procedure was carried out. Under an animal licence granted by the Irish Department
of Health (B100/2443), ovariectomy was performed through a ventral midline
laparotomy under general anaesthesia with sodium thiopentone (Pentanol, Abbot Ltd,
Sligo, Ireland) and maintained on 3-4% halothane (Merial Animal Health, Harlow,
UK) in oxygen. Animals were maintained at pasture for 12 months and feeding and
activity levels were the same for both groups. Following euthanasia, all bones were
harvested and frozen at -20oC.
Individual trabeculae were then excised from the left proximal femur. The femur was
cut 20cm proximal to the mid-point of the shaft. A second cut was made directly
below the head of the femur and the section of bone between these two cuts was
bisected along the coronal plane. The section was then cleaned of bone marrow using
a water jet. A dissecting microscope was used to locate trabeculae on the surface of
the anteromedial region of the medullary cavity. The trabeculae were randomly
selected and excised using a scalpel blade and forceps under 30X magnification. The
individual trabeculae were embedded in non-infiltrating dental stone (Suprastone,
Kerr UK Ltd, England). Samples were mounted upright in the stone and were cut
using a diamond saw to create a cross section of the trabeculum. The surface was then
polished with a series of graded polishing cloths until finally a 0.25µm diamond
suspension was used. In Figure 1(a) a representative cross section is visualised using
scanning electron microscopy.
All testing was carried out in a temperature controlled room at 20°C. A Nano Indenter
XP (MTS Systems, Oakridge, TN) was used to assess the Young’s modulus (E) and
6
contact hardness (Hc). The Nano Indenter XP has load and displacement resolutions
of 0.05µN and 0.01nm respectively. The indenter tip chosen was an AccuTip™
Berkovich diamond indenter tip, with defined elastic modulus of 1141GPa, a
Poisson’s ratio equal to 0.07 and a radius of <50nm. The Berkovich pyramid
geometry is preferred for measurements of elastic modulus and hardness (Hay and
Pharr, 2000). To examine the cross section of the trabeculae, 10 indents were made
around the edge of the trabeculae (Superficial). An inner circle of 6 indents was made
half way along the radius (Intermediate) and finally 4 indents were made in the centre
(Core) of the trabeculae (Figure 1(b)). A permanent hardness impression was made by
driving the indenter into the sample for 90 seconds to a maximum load of 20mN,
holding for 120 seconds and then unloading. This cycle was repeated 3 times at each
location and the modulus and hardness were determined from the unloading segment
of the final indent. The effects of time-dependent plasticity are diminished by multiple
loading-unloading cycles and a long holding period at maximal load (Fan and Rho,
2003).
From the data collected, a load-displacement curve was obtained (Figure 2). The slope
of the initial unloading curve determines the elastic contact stiffness, S. The load (P)
can be expressed according to a power law relationship which is valid for the upper
portion of the unloading curve. This relation is of the form:
Equation 1
( )mfhhP −= α
where α and m are power law fitting constants. When this equation is differentiated at
the maximum contact depth hmax, the contact stiffness, S is given by:
7
Equation 2
dh
dPS =
To determine the modulus and hardness it is necessary to know the contact area, A. In
traditional microhardness tests, this is determined by examining the indent optically.
In nanoindentation, knowing the type of indenter tip that is used allows the area be
calculated. Figure 3 is a schematic of the contact geometry and shows how the various
parameters are related.
The contact stiffness is used to calculate the reduced modulus of elasticity Er using
(Oliver and Pharr, 1992):
Equation 3
A
SEr 2
1 πβ
=
where β is a constant dependent on the geometry of the indenter tip, which for a
Berkovich tip is 1.034.
The reduced modulus of elasticity takes into consideration not only the fact that the
indenter is causing a deformation in the material being tested, but also that the test
material is causing a deformation of the indenter tip. The reduced modulus is
expressed by:
Equation 4
( ) ( )EEE i
i
r
22 111 νν −+−
=
8
where Ei and νi are the modulus and Poisson’s ratio respectively of the indenter
material. The Poisson’s ratio for bone, ν is taken as 0.3 (Rho et al., 1997; Turner et
al., 1999; Zysset et al., 1999; Rho et al., 2002).
This equation can be solved for E to give:
Equation 5
( )( )
−−
−=
i
i
r EE
E2
2
11
1
νν
The contact hardness (Hc) is defined as the hardness at peak load, Pmax (Oliver and
Pharr, 1992):
Equation 6
A
PH c
max=
Statistical Analysis
Results are presented as the mean ± standard deviation. For statistical analyses, results
were tested for normality using the SPSS software package (SPSS Inc, Chicago, IL).
One-way ANOVA and the Mann-Whitney rank sum tests were used to determine
statistical significance.
Results
This study demonstrates that both modulus and hardness vary across the width of
trabeculae. In the control group, as the distance from the surface increases towards the
9
core of the trabecula so too does the modulus. The module in the core (23.4±2.8GPa)
and intermediate locations (21.7±3.1GPa), were significantly greater (p<0.005) than
in the superficial region (17.2±1.3GPa) (Figure 4). Hardness followed a similar trend,
increasing towards the centre of the trabecula. In the control group (Figure 5) the
hardness was greater (p<0.005) in the core (0.47±0.11GPa) and intermediate
(0.41±0.14GPa) locations than in the superficial location (0.29±0.05GPa).
Similar trends were observed in the OVX group with both modulus (Figure 6) and
hardness (Figure 7) increasing towards the centre of the trabeculae. The modulus was
less (p<0.005) in the superficial location (14.8±1.6GPa) than in the intermediate
region (17.8±1.3GPa). The modulus in the core (19.4±1GPa) was greater (p<0.05)
than in the intermediate location in this group. A similar trend was seen in hardness.
In the superficial region (0.31±0.03GPa) the hardness was less (p<0.005) than in the
intermediate (0.40±0.04GPa) or core locations (0.46±0.04GPa).
Comparison between groups found that the modulus in the OVX group at the core,
intermediate and superficial locations was less (p<0.01) than in the control group at
each of those locations (Figure 8). Comparison between the groups found no
significant difference in hardness (Figure 9) between the control and OVX groups.
Discussion
The nature of tissue property differences within a single trabeculum is poorly
understood (Bourne and van der Meulen, 2004). The main focus of this study was to
examine the variation in modulus across the width of trabeculae. In the control group,
the modulus increased towards the centre of the trabeculae and a similar pattern was
seen in the ovariectomised group. Similarly the hardness increased with distance from
10
the edge of the trabeculae in both groups. The second objective of this work was to
examine the difference between healthy and diseased tissue. Comparison between the
two groups found that the modulus was less in the OVX group than in the control
group at all three locations. This is consistent with previous work on these animals
where we showed using a series of fluorochrome dyes, that following ovariectomy
there was a significant increase in trabecular bone turnover (Kennedy et al., 2008).
This result also suggests that diseased bone tissue has diminished biomechanical
properties.
While no direct measurement of mineralisation was made in this study, a positive
correlation between mineralisation and modulus has been shown in the past (Choi et
al., 1990; Follet et al., 2004; Silva et al., 2004; Mulder et al., 2007). Recently in
human mandibular trabecular bone, the degree of mineralisation has been shown to
increase with the distance from the surface of the trabeculae to their cores (Renders et
al., 2006) and a similar trend has been observed in porcine trabecular bone (Mulder et
al., 2007). Variation in Young’s modulus of bone tissue is mainly due to changes in
mineralisation. As calcium content and hardness are strongly related, it has been
proposed that a relationship also exists between hardness and Young's modulus
(Hodgskinson et al., 1989). Subsequent to this it has been observed in human
trabecular bone that hardness follows a similar distribution to elastic modulus, but
with lower statistical contrast (Zysset et al., 1999).
Increasingly sophisticated imaging techniques are providing more accurate inputs on
trabecular architecture; however the material properties of any structure are also
important in determining strength. In whole vertebrae, it has been shown that
11
variations in modulus of trabecular bone play a significant role in determining whole
vertebral strength (Kim et al., 2007). Changes in strength cannot be solely attributed
to the overall material properties, as small scale variations within a sample are also
crucial. Using computational models, it has been shown that if trabecular tissue
inhomogeneities are accounted for, a lower apparent modulus is predicted than if
homogeneity is assumed (van der Linden et al., 2001; Jaasma et al., 2002). The
majority of finite element (FE) models assume a homogenous modulus. The findings
of the current study disagree with this, as the modulus of elasticity increases
significantly towards the centre of the trabecula. Therefore the assumption that
trabecular bone has a homogeneous modulus is incorrect and previous work has
concluded that neglect of the inhomogeneity of the mineral distribution results in a
large underestimation of the stresses and strains in trabecular bone of more than 50%
(van Ruijven et al., 2007). Along with providing evidence of the variation in modulus
in both healthy and ovariectomised bone, this work has also provided values which
may be used in future FE models of trabecular bone.
In the current study, the average modulus across the width of control trabeculae was
20.78±2.4GPa. This result is consistent with a recent study which found that samples
of human trabecular bone from the proximal femur had a modulus which ranged from
19.6 to 22.3 GPa (Chevalier et al., 2007). In human trabecular bone from the distal
femoral condyles the modulus was 18.14±1.7GPa (Turner et al., 1999). The average
modulus across trabeculae measured in this study was also consistent with previous
work from the human femoral neck where the modulus was 21-25 GPa (Hengsberger
et al., 2002). These results indicate that, at a material level the mechanical properties
12
of ovine femoral trabecular bone are very similar to those of human femoral
trabecular bone.
Few studies have examined the material properties of osteoporotic bone using
nanoindentation, and those that have, have used rats (Guo and Goldstein, 2000; Lane
et al., 2003; Hengsberger et al., 2005; Sheng et al., 2006). In the current study, the
ovariectomy procedure significantly reduced the average modulus of trabecular bone
tissue after 12 months. As new bone is less mineralised than older bone; it is not
surprising that the modulus was significantly less in the 12 month OVX group
compared with the controls. A reduction in the ratio of mineral to matrix in the centre
of bone trabeculae has also previously been observed in OVX cynomolgus monkey
(Gadeleta et al., 2000).
In summary, this study provides valuable information on the variation of mechanical
properties in healthy trabecular bone tissue. It has previously been demonstrated that
if trabecular tissue inhomogeneities are accounted for, a lower apparent modulus is
predicted than if a homogeneous assumption is made (van der Linden et al., 2001;
Jaasma et al., 2002). The results of the current study will therefore be useful in finite
element modelling where more accurate values of trabecular bone modulus will be
able to better predict the macroscale behaviour of trabecular bone. The reduction of
modulus in trabecular bone tissue due to ovariectomy has also been quantified.
13
Acknowledgements
This project was funded by the Health Research Board of Ireland under Grant number
RP/2004/229 and the Higher Education Authority in Ireland under the PRTLI Cycle
III.
References
An, Y.H. and R.A. Draughn, 2000. Mechanical testing of bone and the bone-implant interface. Boca Raton, CRC Press.
Bini, F., A. Marinozzi, F. Marinozzi and F. Patane, 2002. Microtensile measurements of single trabeculae stiffness in human femur. Journal of Biomechanics, 35(11): 1515-9.
Bourne, B.C. and M.C. van der Meulen, 2004. Finite element models predict cancellous apparent modulus when tissue modulus is scaled from specimen CT-attenuation. Journal of Biomechanics, 37(5): 613-21.
Chevalier, Y., D. Pahr, H. Allmer, M. Charlebois and P. Zysset, 2007. Validation of a voxel-based FE method for prediction of the uniaxial apparent modulus of human trabecular bone using macroscopic mechanical tests and nanoindentation. Journal of Biomechanics, 40(15): 3333-40.
Choi, K., J.L. Kuhn, M.J. Ciarelli and S.A. Goldstein, 1990. The elastic moduli of human subchondral, trabecular, and cortical bone tissue and the size-dependency of cortical bone modulus. Journal of Biomechanics, 23(11): 1103-13.
Compston, J., 2006. Bone quality: what is it and how is it measured? Andrology of the Brazilian Society of Endocrinology and Metabolism, 50(4): 579-85.
Currey, J.D., 1988. The effect of porosity and mineral content on the Young's modulus of elasticity of compact bone. Journal of Biomechanics, 21(2): 131-9.
Eriksen, E.F., D.W. Axelrod and F. Melsen, 1994. Bone Histomorphometry. New York, Raven Press.
Fan, Z. and J.Y. Rho, 2003. Effects of viscoelasticity and time-dependent plasticity on nanoindentation measurements of human cortical bone. Journal of Biomedical Materials Research Part A, 67(1): 208-14.
Follet, H., G. Boivin, C. Rumelhart and P.J. Meunier, 2004. The degree of mineralization is a determinant of bone strength: a study on human calcanei. Bone, 34(5): 783-9.
Gadeleta, S.J., A.L. Boskey, E. Paschalis, C. Carlson, F. Menschik, T. Baldini, M. Peterson and C.M. Rimnac, 2000. A physical, chemical, and mechanical study of lumbar vertebrae from normal, ovariectomized, and nandrolone decanoate-treated cynomolgus monkeys (Macaca fascicularis). Bone, 27(4): 541-50.
Guo, X.E. and S.A. Goldstein, 2000. Vertebral trabecular bone microscopic tissue elastic modulus and hardness do not change in ovariectomized rats. Journal of Orthopaedic Research, 18(2): 333-6.
Hay, J.L. and G.M. Pharr, 2000. ASM Handbook: Mechanical Testing and Evaluation, ASM International.
14
Hengsberger, S., P. Ammann, B. Legros, R. Rizzoli and P. Zysset, 2005. Intrinsic bone tissue properties in adult rat vertebrae: modulation by dietary protein. Bone, 36(1): 134-41.
Hengsberger, S., A. Kulik and P. Zysset, 2002. Nanoindentation discriminates the elastic properties of individual human bone lamellae under dry and physiological conditions. Bone, 30(1): 178-84.
Hodgskinson, R., J.D. Currey and G.P. Evans, 1989. Hardness, an indicator of the mechanical competence of cancellous bone. Journal of Orthopaedic Research, 7(5): 754-8.
Jaasma, M.J., H.H. Bayraktar, G.L. Niebur and T.M. Keaveny, 2002. Biomechanical effects of intraspecimen variations in tissue modulus for trabecular bone. Journal of Biomechanics, 35(2): 237-46.
Kennedy, O.D., O. Brennan, N.J. Mahony, S.M. Rackard, F.J. O'Brien, D. Taylor and T.C. Lee, 2008. The Effects of High Bone Turnover on the Biomechanical Properties of the L3 Vertebra in a Model of Early Stage Osteoporosis. Spine (In Press).
Kim, D.G., C.A. Hunt, R. Zauel, D.P. Fyhrie and Y.N. Yeni, 2007. The Effect of Regional Variations of the Trabecular Bone Properties on the Compressive Strength of Human Vertebral Bodies. Annals of Biomedical Engineering.
Krassas, G.E. and P. Papadopoulou, 2001. Oestrogen action on bone cells. Journal of Musculoskeletal and Neuronal Interactions, 2(2): 143-51.
Kuhn, J.L., S.A. Goldstein, K. Choi, M. London, L.A. Feldkamp and L.S. Matthews, 1989. Comparison of the trabecular and cortical tissue moduli from human iliac crests. Journal of Orthopaedic Research, 7(6): 876-84.
Lane, N.E., W. Yao, J.H. Kinney, G. Modin, M. Balooch and T.J. Wronski, 2003. Both hPTH(1-34) and bFGF increase trabecular bone mass in osteopenic rats but they have different effects on trabecular bone architecture. Journal of Bone and Mineral Research, 18(12): 2105-15.
Lips, P., 2001. Vitamin D deficiency and secondary hyperparathyroidism in the elderly: Consequences for bone loss and fractures and therapeutic implications. Endocrine Reviews, 22: 477-501.
McNamara, L.M., A.G. Ederveen, C.G. Lyons, C. Price, M.B. Schaffler, H. Weinans and P.J. Prendergast, 2006. Strength of cancellous bone trabecular tissue from normal, ovariectomized and drug-treated rats over the course of ageing. Bone, 39(2): 392-400.
Mente, P.L. and J.L. Lewis, 1989. Experimental method for the measurement of the elastic modulus of trabecular bone tissue. Journal of Orthopaedic Research, 7(3): 456-61.
Meunier, P.J. and G. Boivin, 1997. Bone mineral density reflects bone mass but also the degree of mineralization of bone: therapeutic implications. Bone, 21(5): 373-7.
Mulder, L., J.H. Koolstra, J.M. den Toonder and T.M. van Eijden, 2007. Intratrabecular distribution of tissue stiffness and mineralization in developing trabecular bone. Bone, 41(2): 256-65.
Mulder, L., J.H. Koolstra, J.M. den Toonder and T.M. van Eijden, 2007. Relationship between tissue stiffness and degree of mineralization of developing trabecular bone. Journal of Biomedical Materials Research Part A, 84(2): 508-15.
Oliver, W.C. and G.M. Pharr, 1992. An improved technique for determining hardness and elastic modulus using load and displacement sensing indentation experiments. Journal of Materials Research, 7(6): 1564-1583.
15
Ott, S.M. (1996). Theoretical and methodological approach. Principles of Bone Biology. J. P. Bilezikian, L. G. Raisz and G. R. Rodan. San Diego, CA, Academic Press: 231-241.
Paris, O., I. Zizak, H. Lichtenegger, P. Roschger, K. Klaushofer and P. Fratzl, 2000. Analysis of the hierarchical structure of biological tissues by scanning X-ray scattering using a micro-beam. Cellular and Molecular Biology (Noisy-le-grand), 46(5): 993-1004.
Paschalis, E.P., F. Betts, E. DiCarlo, R. Mendelsohn and A.L. Boskey, 1997. FTIR microspectroscopic analysis of normal human cortical and trabecular bone. Calcified Tissue International, 61(6): 480-6.
Renders, G.A., L. Mulder, L.J. van Ruijven and T.M. van Eijden, 2006. Degree and distribution of mineralization in the human mandibular condyle. Calcified Tissue International, 79(3): 190-6.
Rho, J.Y., R.B. Ashman and C.H. Turner, 1993. Young's modulus of trabecular and cortical bone material: ultrasonic and microtensile measurements. Journal of Biomechanics, 26: 111-119.
Rho, J.Y., T.Y. Tsui and G.M. Pharr, 1997. Elastic properties of human cortical and trabecular lamellar bone measured by nanoindentation. Biomaterials, 18(20): 1325-30.
Rho, J.Y., P. Zioupos, J.D. Currey and G.M. Pharr, 2002. Microstructural elasticity and regional heterogeneity in human femoral bone of various ages examined by nano-indentation. Journal of Biomechanics, 35(2): 189-98.
Rokitaa, E., P. Chevallierb, P.H.A. Mutsaersc, Z. Tabora and A. Wróbeld, 2005. Studies of crystal orientation and calcium distribution in trabecular bone Nuclear Instruments and Methods in Physics Research Section B: Beam Interactions with Materials and Atoms 240(1-2): 69-74
Runkle, J.C. and J. Pugh, 1975. The micro-mechanics of cancellous bone. II. Determination of the elastic modulus of individual trabeculae by a buckling analysis. Bulletin of the Hospital for Joint Diseases, 36(1): 2-10.
Ryan, S.D. and J.L. Williams, 1989. Tensile testing of rodlike trabeculae excised from bovine femoral bone. Journal of Biomechanics, 22(4): 351-5.
Sheng, Z.F., R.C. Dai, P. Wang, X.F. Yao, X.Q. Feng, L.N. Fang, H.J. Fan, X.P. Wu and E.Y. Liao, 2006. Nanomechanical properties of vertebral trabeculae in ovariectomized rats. Zhonghua Yi Xue Za Zhi, 86(8): 515-9.
Silva, M.J., M.D. Brodt, Z. Fan and J.Y. Rho, 2004. Nanoindentation and whole-bone bending estimates of material properties in bones from the senescence accelerated mouse SAMP6. Journal of Biomechanics, 37(11): 1639-46.
Townsend, P.R., R.M. Rose and E.L. Radin, 1975. Buckling studies of single human trabeculae. Journal of Biomechanics, 8(3-4): 199-201.
Turner, C.H., J. Rho, Y. Takano, T.Y. Tsui and G.M. Pharr, 1999. The elastic properties of trabecular and cortical bone tissues are similar: results from two microscopic measurement techniques. Journal of Biomechanics, 32(4): 437-441.
van der Linden, J.C., D.H. Birkenhager-Frenkel, J.A. Verhaar and H. Weinans, 2001. Trabecular bone's mechanical properties are affected by its non-uniform mineral distribution. Journal of Biomechanics, 34(12): 1573-80.
van Ruijven, L.J., L. Mulder and T.M. van Eijden, 2007. Variations in mineralization affect the stress and strain distributions in cortical and trabecular bone. J Biomech, 40(6): 1211-8.
16
Weiner, S., T. Arad, I. Sabanay and W. Traub, 1997. Rotated plywood structure of primary lamellar bone in the rat: orientations of the collagen fibril arrays. Bone, 20(6): 509-14.
Ziv, V., H.D. Wagner and S. Weiner, 1996. Microstructure-microhardness relations in parallel-fibered and lamellar bone. Bone, 18(5): 417-28.
Zysset, P.K., X.E. Guo, C.E. Hoffler, K.E. Moore and S.A. Goldstein, 1999. Elastic modulus and hardness of cortical and trabecular bone lamellae measured by nanoindentation in the human femur. Journal of Biomechanics, 32(10): 1005-12.
Conflict of Interest Statement
None of the authors have any conflict of interests to report.
Suggested Referees
Dr Gwendolen Reilly
Lecturer in Tissue Engineering
Department of Engineering Materials
Kroto Research Institute
Broad Lane
University of Sheffield
Sheffield, S3 7HQ
Tel: +44 114 222 5986, Fax: +44 114 222 5945
e-mail: g.reilly@sheffield.ac.uk
Dr Glenn Dickson
Senior Lecturer
School of Medicine, Dentistry and Biomedical Sciences
Queens University
73 University Road
Belfast
BT7 1NN
Tel: +44 28 9097 2253, Fax: +44 28 9097 1445
Email: g.dickson@qub.ac.uk
* Referee Suggestions
Abstract
The elastic behaviour of trabecular bone is a function not only of bone volume and
architecture, but also of tissue material properties. Variation in tissue modulus can
have a substantial effect on the biomechanical properties of trabecular bone.
However, the nature of tissue property variation within a single trabecula is poorly
understood. This study uses nanoindentation to determine the mechanical properties
of bone tissue in individual trabeculae. Using an ovariectomised ovine model, the
modulus and hardness distribution across trabeculae were measured. In both normal
and ovariectomised bone, the modulus and hardness were found to increase towards
the centre of the trabeculae. Across the width of the trabeculae, the modulus was
significantly less in the ovariectomised bone than in the control bone. However in
contrast to this hardness was found not to differ significantly between the two groups.
This study provides valuable information on the variation of mechanical material
properties in healthy and diseased trabecular bone tissue. The results of the current
study will be useful in finite element modelling where more accurate values of
trabecular bone modulus will enable the prediction of the macroscale behaviour of
trabecular bone.
Abstract
Figure 1: (a) Scanning electron microscopy image of a trabecula. (b) Schematic of indent locations
across the width of a trabecula. ▲indicates indent location.
Figure 2: Typical load-displacement curve for an instrumented nanoindentation test.
Figure 3: Schematic of the contact geometry during loading and unloading [49].
Figure 4: Modulus distribution across trabeculae from controls.
* Significantly greater than Superficial region (p<0.005)
Figure 5: Hardness variation across the width of trabeculae from the control group.
* Significantly greater than Superficial region, p<0.005
Figure 6: Modulus distribution across trabeculae in the ovariectomised group.
* Greater than Superficial (p<0.005), ** Greater than Intermediate (p<0.05)
Figure 7: Hardness variation across the width of trabeculae from the OVX group.
* Significantly greater than Superficial region, p<0.005
Figure 8: Modulus distribution across trabeculae in both the control and OVX groups.
* Significantly less than control (p<0.01)
Figure 9: Hardness variation across the width of trabeculae in the control and OVX groups at 12
months.
Figure Legend(s)