Chapter 35

Mechanical Properties of Bone Ex Vivo

Simon R. Goodyear and Richard M. Aspden

Abstract
The primary functions of bone are to do with support and protection – mechanical functions. The aim of
this chapter is to set out some of the methods that can be used to measure these properties in cortical and
cancelleous bone from large (e.g. human or bovine) and small (e.g. mouse) animals. The difference
between properties of the sample (intrinsic properties) and properties of the material (extrinsic properties)
is introduced and techniques for measuring them suggested. The addition of other tests to give a complete
characterisation of a bone sample is presented.

Key words: Mechanical testing, Bone, Material properties, Mechanical properties

1. Introduction

The primary function of bone is to form the skeleton, which

provides support for the body and protection for vital organs.
These are primarily mechanical functions. To fulfil these, the bone
matrix has to have the right combination of stiffness and strength
to enable it to withstand the forces imposed upon it. These forces
may be repetitive and moderate, such as those generated during
walking, or high and transient, such as inflicted by a blow on the
head. The structure and composition of bone can adapt over time
to try to match the mechanical properties of the bone to the pre-
vailing demands being placed on it. How to measure some of these
mechanical properties is the aim of this chapter.
There are two types of bone which are considered in this
chapter: cortical or dense bone and cancellous or trabecular bone.
Cortical bone is a solid, compact material which forms the diaphy-
sis of long bones, and a shell around the metaphysis and the verte-
brae. Cancellous bone has an open, porous structure comprising
rods or plates. Hence, it is less dense than cortical bone and also
less stiff and strong. It makes up the centre of the vertebrae and the

Miep H. Helfrich and Stuart H. Ralston (eds.), Bone Research Protocols, Methods in Molecular Biology, vol. 816,
DOI 10.1007/978-1-61779-415-5_35, © Springer Science+Business Media, LLC 2012

555
556 S.R. Goodyear and R.M. Aspden

metaphysis. However, the two materials in combination form

structures which are strong and light and their properties in situ
may differ considerably from those measured from tests on isolated
samples (1, 2). In cancellous bone, the distinction between material
and structure is not always easy to define and treating it as a cellular
material has met with a considerable degree of success (3, 4).
Mechanical testing is one test in an armamentarium of meth-
ods that can be used to characterise bone, as illustrated in Fig. 1,
which shows a scheme we have developed for testing mouse bones.
Together, the methods measure the material, mechanical, and
geometrical properties to describe the factors that determine a

Fig. 1. A suggested flowchart for characterising mouse bones. The order of tests is deter-
mined by whether preparation irreversibly changes the bone and also the size of sample
required for the test. For example, mCT is positioned first as it is non-destructive but requires
an intact piece of bone, whereas although Raman is also non-destructive it can be used on
small samples, and so is at the bottom of the chart. The decision point half way through
assesses whether there is a phenotype by testing both geometrical and mechanical proper-
ties. If neither shows an effect of treatment, it is likely the factor does not affect bone.
 35 Mechanical Properties of Bone Ex Vivo 557

bone’s competence in fulfilling its primary function, outlined

above. In this chapter, methods to measure the intrinsic and
extrinsic mechanical properties of bone (the shaded methods in
Fig. 1) are described.
Although conceptually relatively simple, the mechanical prop-
erties of these tissues are not easy to measure. If only a comparison
is required between equivalent sites in different patient groups,
then careful use of the same technique on all samples will provide
consistent relative values. If, however, absolute values are required,
then great care must be taken as bone is not isotropic or homoge-
neous, water content is important, and its properties depend on
the rate at which it is deformed. In addition, sample preparation
can be difficult. Unlike engineering materials, which can be cut
and machined to predetermined sizes for testing and for which
there are officially recognised standards, bone samples are gener-
ally limited in size and shape by the site from which they are taken.
The size of the sample being tested may affect the measurements
being made.
This chapter describes methods of preparation and testing that
can be applied to cortical and cancellous bone from human, bovine,
or equine sources and goes on to present a method for testing
whole bones in small animals, such as that required to measure
bone properties in genetically modified mice. These are so small
that preparing and testing isolated samples of cortical or cancellous
bone becomes very difficult and tests are generally performed on
intact bones. Differences in approach that are required because of
the different natures of the materials are noted. The descriptions
are restricted to methods that can reasonably easily be applied by
anyone having access to a limited range of laboratory equipment,
such as a materials testing machine, a balance of suitable precision,
an ultrasound generator, an oven, and furnace. More sophisticated
techniques for mechanical and material characterisation include
micro- and nano-indentation, infrared spectroscopy [Fourier trans-
form infrared (FTIR) or Raman], backscattered electron micros-
copy, and atomic force microscopy. Researchers requiring these
techniques or more details of variations are referred to the book by
Cowin (5) and to the original papers.

2. Materials

1. Phosphate-buffered saline (PBS): Used to keep specimens

moist during preparation and testing (see Note 1).
2. Washing up liquid: For reducing surface tension.
3. Tweezers, scalpel, and scissors: For removing soft tissue and
manipulating samples.
558 S.R. Goodyear and R.M. Aspden

4. Hacksaw: For cutting large bone samples to manageable-sized

pieces prior to precision cutting.
5. Mineralogical saw [Accutom 2 (Struers Ltd., Rotherham) or
Isomet (Buehler, Lake Bluff, IL, USA)] fitted with either a
diamond or an aluminium oxide cut-off wheel: For precision
cutting of bone samples (see Note 2).
6. Lathe or milling machine, as might be found in any engineer-
ing workshop: To machine bone samples to desired shape.
7. Coring bits, 5- and 9-mm internal diameter and break-off
tools: Custom made by Bolton Surgical Ltd. (Sheffield, UK).
8. Materials testing machine (e.g. Instron, High Wycombe, Bucks
or Electroforce test Instruments, Bose, Minnesota): For
mechanical testing of samples.
9. Balance (Sartorius Gmbh, Göttingen, Germany) with specific
gravity determination kit Sartorius 6080/60801 Specific
Gravity Determination Kit: For density measurement by
Archimedes’ principle.
10. Ultrasound generator (Panametrics Pulser–Receiver model
5052PR (Panametrics Inc, Waltham, MA, USA) with a
V211BA transducer) and oscilloscope (Hitachi V-665A,
Tokyo, Japan): For measuring the speed of sound.
11. Oven (Hybaid, Teddington) and furnace (Carbolite, Sheffield):
For determining the relative composition.
12. Software for calculation of biomechanical variables: Origin
(OriginLab Corp., Northampton, MA, USA), Matlab (the
MathWorks Inc., Natick, MA, USA), or MathCad (Mathsoft
Engineering and Education Inc., Cambridge, MA, USA).

3. Methods

3.1. General All human tissues must be handled with due regard to health and
Considerations safety because of the risk of infection. In addition to gloves and
for the Preparation laboratory coat, a mask and eye shield are required since there is a
of Bone Samples risk of aerosols being generated during cutting, drilling, or machin-
ing. Samples must be kept moist at all times during the preparation
procedure. Bones must be dissected free of soft tissues, taking care
not to create notches in the bone that is going to be tested, since
this weakens it. General methods for the preparation of samples for
testing are described in the following sections.

3.2. Preparing Prisms 1. Cut the bone into manageable-sized pieces using a hacksaw or
of Cortical Bone a junior hacksaw.
2. Precision cut the bone samples to the desired size using a min-
eralogical saw rotating at 600–800 rpm. For compression and
 35 Mechanical Properties of Bone Ex Vivo 559

Fig. 2. Tensile test specimens are traditionally produced with a waist to ensure that failure
consistently occurs in the centre and not near the grips.

bending tests, the samples should be cut to give rectangular

parallelepiped-shaped specimens. For tensile testing, the speci-
mens should be milled to give a waisted configuration (Fig. 2).
3. If the samples are large enough, the samples should have cylin-
drical symmetry, but for thin specimens the waisting can be
done in two dimensions. This is to ensure that fracture occurs
consistently in the central part of the specimen away from the
gripped ends. Water should be run over the sample at all times
during sawing and machining.

3.3. Preparing 1. Obtain the bone core by drilling through the bone sample
Cancellous Bone with a coring bit (Fig. 3). To remove the core, it either has to
Cores be drilled right through a piece of bone or broken off the
underlying bone. Providing the core is not too deep; this can
be done using the break-off tool shown in Fig. 3. This is made
to the same specification as the coring bit, except it does not
require teeth and half of its circumference has been removed.
This is inserted in place of the coring bit and, by levering on
the end of it, the base of the core may be broken from the
underlying tissue (see Note 3).
2. Trim the ends of the cores plane and parallel using a mineral-
ogical saw. If the cores have been obtained from the articular
surface of joints, they will have a cartilage and subchondral
bone at one end which needs to be removed using the saw.

3.4. Preparation 1. Remove the hind limb from the animal by cutting through the
of Mouse Bones pelvis with scissors. Take care not to damage the head and neck
of the femur. Strip the fur from the leg. If not immediately
560 S.R. Goodyear and R.M. Aspden

Fig. 3. Coring bits and matching “break-off” devices. Unless cores can be drilled right
through a piece of bone, they need to be removed from the underlying bone to which they
are attached. Inserting the break-off device after drilling the core and levering it sideways
generally successfully snaps the core at its base after which it may be removed using
forceps.

required for testing, samples should now be immersed in PBS

and stored below −20°C.
2. Before testing, samples are defrosted and the soft tissue
removed. First, the foot is removed by cutting though the
ankle joint and then the soft tissue stripped from the bone
using a scalpel. The femur is then separated from the tibia and
pelvis, where present, and each bone placed in a separate vial of
fresh PBS. The fibula is also removed from the tibia at this
stage. If testing is to proceed in the next 24 h, bones are stored
in the fridge or returned to the freezer if a longer interval is
expected.
3. In mice, density and speed of sound measurements are made
in cortical bone and so the metaphysis are removed to sepa-
rate cortical from predominantly trabecular bone. The broken
end of each bone section from the bending test (see below) is
wrapped in PBS-soaked tissue paper and lightly clamped in a
mineralogical saw and the metaphysis cut-off. After removing
the distal metaphysis from the femur, a ~1.5-mm section is
cut off for ultrasonic testing and the remaining cortical bone
used for density determination. In the tibia, the proximal
metaphysis is removed first, with the ultrasound sample being
taken from the adjacent diaphysis. During cutting, the sample
is irrigated with distilled water to remove cutting debris and
keep it cool.
 35 Mechanical Properties of Bone Ex Vivo 561

3.5. General Before testing, it is important to decide what parameters are going
Considerations to be measured. Most testing machines apply a deformation to a
for Mechanical Testing sample, which normally increases linearly with time. This deforma-
tion is commonly referred to as displacement. The operator can
choose the rate at which the displacement occurs and the proper-
ties measured depend on this rate as bone is viscoelastic. Choosing
when to stop the test is also important depending on what is to be
done next with the specimen. In tension or bending, the machine
can be set to stop automatically on fracture, as the load drops
rapidly at this point. In compression, failure is not so easy to
determine and various methods can be used. The applied load
and displacement are recorded throughout the test. The load–
displacement curve represents the extrinsic properties of the
specimen under test and is, therefore, used to measure the prop-
erties of whole bones (Fig. 4a). These properties are important
for understanding why bones fracture. For prepared samples of

Fig. 4. A load–displacement curve (a) measures the extrinsic properties of a specimen,

e.g. an individual bone. The main parameters are stiffness, work to failure (shaded ), and
ultimate load and displacement. The stress–strain curve (b) is similar but, because it is
normalised for the sample dimensions, measures the intrinsic properties of the material
being tested. These are the elastic or Young’s modulus (E ), the yield stress and strain, the
ultimate stress and strain, and the energies to yield and failure.
562 S.R. Goodyear and R.M. Aspden

known dimensions, what are commonly required are the intrinsic

properties of the bone material itself. To obtain these, the load is
divided by the cross-sectional area of the specimen to produce the
stress, and the displacement is divided by the original length to
give the strain. The stress–strain curve is then very similar to the
load–displacement curve, but refers now to the material not the
structure (Fig. 4b).

3.6. Repeating It has been found that if a test is repeated several times on the
Tests on the Same same specimen within a short period then the resulting load–
Specimens displacement plots do not immediately coincide but appear to
converge towards a stable curve. Because of this, many investiga-
tors apply up to 5 or 6 loading cycles to a specimen, often at a
reduced load, before performing the actual test: a process called
preconditioning. This is a point of some contention because it is
not understood what happens during this process. It can be argued
that what is finally being measured is not the natural property of
the specimen but one that has somehow been modified by a series
of cyclically applied loads. Does repeatability necessarily imply
accuracy? We always apply a single test to a specimen.

3.7. Types There are four main types of test: tension, compression, bending,
of Mechanical Testing and torsion.
1. Tension and compression testing. These are most commonly
done to measure the intrinsic properties of bone matrix. These
tests use machined samples of known dimensions. Tension is
generally used for cortical bone, and compression is more com-
mon for cancellous bone because of the difficulty obtaining and
mounting suitably sized specimens for tensile testing. Bending
and torsion can be applied in this way, but the interpretation of
the results is more difficult. They are probably more commonly
applied to whole bones. In this section, descriptions are given
of tension and compression tests for machined specimens.
2. Bending. This is most commonly applied to whole long bones
from rodents because of the difficulties of machining tensile or
compressive specimens from such small bones. It is best for
measuring extrinsic properties of the whole bone and, there-
fore, is very useful for studies comparing the effects of different
drug therapies or genetic modifications. It is not easy to esti-
mate intrinsic properties from this test for a variety of reasons,
mainly related to the size and shape of the bone. More details
are given by Turner and Burr (6). There are two bending con-
figurations, called three-point and four-point loading (Fig. 5).
In three-point loading, there is a significant shear stress gener-
ated at the mid-point of the beam. For this reason, four-point
loading is sometimes preferred because in a uniform specimen
shear is minimised and a pure bending moment is applied.
 35 Mechanical Properties of Bone Ex Vivo 563

Fig. 5. Whole bones may be tested in either (a) three-point or (b) four-point loading con-
figuration. Force is applied through the upper plate and the resulting displacement
recorded. The span is the distance between the lower supports.

However, in whole bones, the non-uniform cross section

means that these assumptions do not apply. In addition, it is
difficult to load all four points identically and test results are
subject to large errors. Three-point and four-point loading jigs
can be bought or made to fit most materials testing machines.
It is important to be able to adjust the span, the distance
between the lower supports. For testing whole bones, this
should be maximised to reduce shear stresses but be constant
for all specimens to be compared. For machined specimens,
testing at different spans enables extrapolation to be made for
an infinite span to give a shear stress-free modulus (7).

1. Prepare the specimen as described in Subheading 3.2 or 3.3

3.8. Tension Testing
ensuring that it is at least 15–25-mm long and 5 mm across
(see Note 4).
2. Keep the sample moist by lightly wrapping in cling film or
moist tissue or gauze.
3. Fix the specimen in the testing machine: Most testing machines
have grips for holding specimens in tension. These can be
tightened on to the broader ends of the specimen and are
564 S.R. Goodyear and R.M. Aspden

adequate for testing cortical bone (see Note 5). If you are
testing cancellous bone cores, set the ends into cups using
dental or bone cement and the cups are then held in the testing
machine grips.
4. Set up the machine to apply the desired strain rate, and pro-
gram in any predetermined limits on load, displacement or
fracture detection, and the rate of data recording. It is advis-
able to set the machine to stop as soon as fracture occurs
because at this point the load falls to zero.
5. Start the testing machine.
6. After the test, transfer data from the instrument to a spread-
sheet for further analysis if the instrument does not already
have appropriate software.

3.9. Analysing Data 1. If intrinsic properties are required and not already calculated,
from Tension Testing convert load values to stress by dividing by the original cross-
sectional area. (No account is taken of changes in cross-sectional
area which occur during the test.)
2. Convert displacement values to strains by dividing by the
unstretched distance between the points of attachment or ref-
erence marks of the displacement transducer (see Note 5).
3. Plot stress as a function of strain: The elastic or Young’s, mod-
ulus, yield strength and strain, ultimate strength and strain,
and energies to yield and failure may then be calculated.
4. Calculate the elastic modulus by estimating the gradient of the
linear part of the curve. For linear relationships, this is often
calculated by the instrument software. If not, this can be found
crudely by hand calculation, better by fitting a straight line to
the data or for non-linear relationships by differentiating the
curve using software, such as Origin, Matlab, or MathCad.
5. Calculate the yield point by estimating when the elastic modu-
lus starts to reduce. This is not always easy to define, but one
way is to construct a line parallel with the linear part of the
curve but offset along the strain axis by a small amount. The
point at which this intersects the curve is defined as the offset
yield point and the stress and strain at this point are the yield
stress and strain.
6. Calculate the failure stress and strain from the point at which
fracture occurs.
7. Integrate the curve to either the yield point or failure point to
find the area beneath to estimate the energy needed to pro-
duce yield or failure, respectively, per unit volume of material.
The energy to failure is often referred to as the modulus of
toughness.
 35 Mechanical Properties of Bone Ex Vivo 565

3.10. Compression 1. Prepare a cylinder or rectangular parallelepiped of bone as

Testing described above. Try to ensure that the length of the specimen
is about twice the diameter (see Note 6).
2. Place the sample on the lower anvil of the testing machine. As
in the case of tensile testing, a universal joint must be used in
the loading path (see Note 4). Alternatively, a steel plate with
small indentation containing a ball bearing between the speci-
men and the upper loading anvil can be used; this allows for
any slight non-parallelism between the two faces.
3. Accurate measurements of strain require the use of a displace-
ment transducer, as for tension, but for most comparative
studies the displacement of the cross head is probably adequate
(see Note 5).
4. Lower the cross head of the machine until it is just in contact
with the specimen.
5. Set the strain rate and any predetermined load or displacement
limits as before.
6. Start the test, and stop the test when the chosen criterion is
met (see Note 7).
7. Calculate stress and strain and plot stress as a function of strain
as described above. Where testing is done between two anvils,
there is often a region near the origin, where the slope of the
curve starts small and increases rapidly before becoming
approximately linear, resulting in a J-shaped curve. This toe
region is an artefact of the method and is commonly found in
this sort of testing both of natural and synthetic polymeric
materials. Because it can be difficult to determine a straight
portion of the curve or to fit a straight line to it, it is best to
differentiate the curve to find the modulus as a function of
strain. The elastic modulus can then be taken to be the peak
value of this curve.
8. The yield stress may be defined as the stress at which the mod-
ulus starts to decrease. We used a reduction of 3% from its peak
value to provide a defined reference point (8).
9. Energy to yield is found by integration of the area under the
stress–strain curve up to the yield stress. Strains are ill-defined
in this testing protocol because of the toe region. Embedding
the ends of longer specimens and gripping as for a tensile test
can eliminate this problem. Failure cannot usually be defined
in the same way as for tensile testing because progressive crush-
ing of the material often occurs rather than a fracture.

3.11. Bending The method described here applies to testing whole bones, from a
mouse for instance, but with small alterations could be used for
regular sections cut from larger bones.
566 S.R. Goodyear and R.M. Aspden

1. Set the bending jig in the materials testing machine.

2. Adjust the spacing of the lower supports to be as close to the
ends of the diaphysis as possible and centred on the moving
anvil.
3. Place the bone in a stable position on the supports. For femur,
the anterior side is usually upwards and for tibia the fibula
insertion point is normally at the top.
4. Lower the cross head of the instrument so that the upper load-
ing anvil is just in contact with the bone surface.
5. Set the displacement rate, any load or displacement limits, and
the data collection rate.
6. Start the test.
7. The machine can be set to stop as soon as fracture occurs
because at this point the load falls very rapidly, though not
always to zero.
8. Load is plotted as a function of displacement and the stiffness,
yield load and failure load, and energy to fracture may be found
using the same criteria as used for stress–strain curves in the
tensile and compression tests (see Note 8).

3.12. The Elastic The elastic or Young’s modulus of cortical bone can be calculated
Modulus of Cortical from density and speed of sound measurements described below
Bone using equation (9):
E = rc 2 .

This provides a direct measure of the intrinsic or material stiff-

ness of the bone and can be applied to small samples, such as mouse
bone and suitably machined specimen from larger mammals.

3.13. Density of Bone This technique can be applied to both cortical and cancellous bones
by Archimedes’ and is particularly applicable to hollow, porous, or irregularly
Principle shaped specimen. With mouse bone, it is difficult to obtain repeat-
able results because the bone’s weight is small and comparable
with surface tension, evaporation, and excess water effects, which
must all be controlled.
1. The density of the bone is measured by comparing the weight
in air with the weight when suspended in a fluid, typically dis-
tilled water. We use a five-figure balance (Sartorius Gmbh,
Göttingen, Germany) for all measurements.
2. The true weight of each bone is measured first by weighing
wet in sealed containers to eliminate any loss of weight due to
evaporation. Prior to weighing, excess water is removed from
the bone surface by wiping on damp tissue and the marrow
canal emptied of fluid by passing air through it using a syringe
 35 Mechanical Properties of Bone Ex Vivo 567

and needle. Bones are then placed in a pre-weighed micro-

centrifuge tube containing PBS and weighed.
3. Each bone is then weighed suspended in distilled water using a
Sartorius 6080/60801 Specific Gravity Determination Kit.
The bone is suspended by a fine wire that passes through the
medullary canal. Larger specimens can be suspended in a bas-
ket. Buoyancy effects due to trapped air are eliminated by fill-
ing the medullary canal with fluid using a syringe and needle.
A droplet of detergent (washing-up detergent is adequate!) is
added to the fluid to reduce surface tension effects.
4. The fluid density is calculated by measuring the upthrust
exerted on a glass bob provided as part of the specific gravity
kit when suspended in the fluid and using the equation:
U = V rf ,
where U is the upthrust (weight in air – weight in fluid), V the
volume of the body immersed, and rf the fluid density.
5. Bone density is calculated from the formula:
rf m
r= ,
m − ms
where r f is the fluid density, m the true weight or weight in air,
and ms the weight suspended in fluid.
All weights are taken in triplicate and averaged. Where one
of the measurements is clearly different to the other two, it is
discarded and a repeat measurement made.

3.14. Speed of Sound Thin sections of cortical bone were prepared for ultrasound testing
Measured Using as described in Subheading 3.4, step 3.
Ultrasound
1. Measure the length of the bone section accurately using a digi-
tal micrometer, for example.
2. Measure the time taken for ultrasound pulses to traverse the
sample. We use pulse-echo mode, so the same transducer is
used to generate and receive ultrasound pulses and a second,
identical but unconnected transducer used as a reflecting sur-
face at the other bone surface, although any glass or metal
block would serve just as well as long as the impedance is dif-
ferent to that of the bone. The bone surface must be coupled
to the transducers ultrasonically using a fluid, such as water or
ultrasound gel. In our arrangement, pulses of ultrasound, of
10 MHz frequency, are generated using a Panametrics Pulser–
Receiver model 5052PR (Panametrics Inc, Waltham, MA,
USA) with a V211BA transducer in pulse-echo mode.
Ultrasound echoes are displayed on an oscilloscope (Hitachi
V-665A, Tokyo, Japan) and the time between echoes measured
using the oscilloscope’s internal callipers.
568 S.R. Goodyear and R.M. Aspden

3. The speed of sound is then calculated using the formula:

2d
c= ,
t
where d is the section thickness and t the time measured
between echoes (9).
4. The bone sections are subsequently rinsed in PBS to remove
any adhering ultrasound gel.

After density measurements have been made, the bone can be

3.15. Ashing
dehydrated and ashed to determine the gross composition in terms
of water, organic, and mineral content.
1. Crucibles are prepared by washing and drying in a furnace
(Carbolite, Sheffield) at 600°C for 24 h and allowed to cool in
a desiccator and then weighed. This ensures that they are free
from contamination and moisture.
2. Remove excess water from the bone surface and medullary
canal, as for density measurement, and add to the pre-weighed
crucibles to measure wet weight.
3. Heat at 100°C for 24 h in an oven (Hybaid, Teddington),
allow to cool (in a desiccator), and reweigh to determine the
dry weight.
4. Then, heat at 600°C for a further 24 h in the furnace and, after
cooling, weigh again to determine the ash weight. The bones
are cooled in a desiccator to prevent any rehydration.
5. Water content is found from wet weight – dry weight, the
organic content from dry weight – ash weight and mineral
content as the ash weight (10). Measurements are then
expressed as a percentage of the wet (or sometimes dry) weight
to allow comparison between bones of different sizes.

4. Notes

1. Storage of samples: Samples should be kept moist with PBS

prior to mechanical testing; formalin and other fixatives must
never be used as this affects the mechanical properties. If the
samples are not being tested immediately (within 2–4 h), the
specimens can be stored frozen at −20°C. They must be
wrapped in tissue or gauze dampened with PBS and vacuum
sealed into plastic freezer bags. A vacuum bag sealer can be
obtained for this purpose. Freezing is reported to have no
effect on the mechanical properties of bone (11, 12).
2. Instruments for precision cutting: We use an Accutom 2 min-
eralogical saw (Struers Ltd., Rotherham) and a 125-mm
 35 Mechanical Properties of Bone Ex Vivo 569

aluminium oxide wheel, but the most common saw cited in the
bone literature is the Isomet (Buehler, Lake Bluff, IL, USA).
Whatever saw is used, faster wheel speeds and slower specimen
feed result in a better surface finish. Where samples of a prede-
termined size are required, it is important to know how much
of the sample is lost during the cutting procedure. Any saw
removes an amount of material corresponding to the width of
the blade, termed the kerf. For an aluminium oxide wheel, the
kerf is about 0.5 mm compared with about 0.3 mm for a dia-
mond blade. When the kerf is known and the machine is fitted
with a micrometer or other precision positioning device, it is
possible to obtain samples of the desired size by adjusting the
cutting position appropriately. It is possible to cut specimens
100–150-mm thick using an aluminium oxide wheel. Care
must be taken to keep specimen feed speeds, i.e. the rate at
which the specimen is moved past the cutting wheel, fairly low,
as aluminium oxide wheels are brittle and shatter easily and
diamond wheels are thin and are easily bent. However, alu-
minium oxide wheels cost about £10, in contrast to about
£200–300 for a diamond cutter, depending on size.
3. Obtaining bone cores: When coring right through a block of
tissue, the sample is commonly left in the coring bit.
Occasionally, this can happen, too, if a core snaps off while
being drilled. In order to be able to remove this core, we have
had the bits made with a hole from end to end so that a close
fitting plunger may be inserted to push out the core. However,
this can damage the top of the core if it is tightly wedged into
the drill bit. For this reason, we prefer not to drill right through
and to use the break-off tool to snap the core from its base
after the coring bit has been removed.
4. Tension testing: The specimen must be large enough to get
consistent results; Keaveny’s methods start with a specimen
40-mm long, which highlights the difficulties that may some-
times be encountered obtaining suitable specimens (13). If the
grips are not exactly in line or the specimen is not precisely
machined, then applying a tensile load will also result in a
degree of bending. This adds uncertainty to the test data which
is different for each specimen and makes accurate comparison
between specimens impossible. A universal joint must be placed
in the path of the load to ensure that only tensile forces are
transmitted to the specimen. These are available from the
instrument manufacturers.
5. Measuring strain during tension tests: An estimate of the strain
can be calculated from the displacement of the cross head
recorded by the machine. However, for accurate studies, a
strain gauge or displacement transducer must be applied to the
central, often waisted, part of the specimen. This can be done
using either a clip-on device or video recording methods.
570 S.R. Goodyear and R.M. Aspden

6. Shape of samples for compression testing: It is desirable for the

length of the specimen to be approximately twice the diameter.
It is possible to conduct tests on samples, where these dimen-
sions are roughly equal without too much of a problem and in
many cases this may be unavoidable due to anatomical consid-
erations. Boundary conditions, e.g. friction between the sam-
ple and the loading platens, can become a problem if the sample
is not longer than it its width.
7. Setting limits for compression testing: Care needs to be taken
when setting applied loads and limits into certain testing
machines. Conventionally, engineers use positive numbers to
denote tensile loads and negative numbers for compressive
loads. The most commonly used test is tension and here there
is generally no problem as everything is positive. However, set-
ting a limit for a compressive load often needs to include a
negative sign. It sounds trivial but is easily overlooked, and
many students have stood watching the machine crush their
specimen wondering why the machine has not stopped! Only
after hitting the emergency stop button have they realised that
they omitted the negative sign in front of the load limit they
had set. There is often no clear end point for failure in com-
pression, unlike tension, and determining when to stop the test
has to be decided beforehand. In our studies, we have decided
that any reduction in stiffness, that is a reduction in the slope
of the load–displacement curve, is a sign of the beginning of
failure. As we usually wish to do further analysis on the speci-
mens and minimise the damage caused by the test, we watch
the load–displacement curve (which is plotted on the com-
puter monitor during the test) carefully during the test, and
stop the test as soon as it becomes obvious that the slope of the
curve is decreasing.
8. Calculating intrinsic properties of bone from bending tests:
Equations have been derived to calculate intrinsic properties
from these data but are fraught with difficulties when testing
whole bones because of the asymmetric cross section of the
bone. It is not recommended to use these without expert
advice.

References
1. Aspden, R. M. (1990) Constraining the lateral
dimensions of uniaxially loaded materials increases
the calculated strength and stiffness: application
to muscle and bone. Journal of Materials Science:
Materials in Medicine 1, 100–104.
2. Bryce, R., Aspden, R. M., Wytch, R., and
Langrana, N. A. (1995) Stiffening effects of
cortical bone on vertebral cancellous bone in
situ. Spine 20, 999–1003.
3. Gibson, L. J. (1985) The mechanical behaviour
of cancellous bone. Journal of Biomechanics 18,
317–328.
4. Gibson, L. J., and Ashby, M. F. (1988) Cellular
solids, Pergamon Press, Oxford.
 35
5. Cowin, S.C. (2001) Bone mechanics handbook,
CRC Press, Boca Raton.
6. Turner, C. H., and Burr, D. B. (2001)
Experimental techniques for bone mechanics,
in Bone mechanics handbook (Cowin S.C. ed.)
CRC Press, Boca Raton, pp. 7-1-7-35.
7. Spatz, H. -Ch., O’Leary, E. J., and Vincent, J.
F. V. (1996) Young’s moduli and shear moduli
in cortical bone. Proceedings of the Royal Society
B: Biological Sciences 263, 287–294.
8. Li, B., and Aspden, R. M. (1997) Composition
and mechanical properties of cancellous bone
from the femoral head of patients with osteo-
porosis or osteoarthritis. Journal of Bone and
Mineral Research 12, 641–651.
9. Lees, S., Heeley, J. D., and Cleary, P. F. (1979)
A study of some properties of a sample of
bovine cortical bone using ultrasound. Calcified
Tissue International 29, 107–117.
10. Mkukuma, L. D., Imrie, C. T., Skakle, J. M.
S., Hukins, D. W. L., and Aspden, R. M.
Mechanical Properties of Bone Ex Vivo 571
(2005) Thermal stability and structure of
cancellous bone mineral from the femoral
head of patients with osteoarthritis or osteo-
porosis. Annals of the Rheumatic Diseases 64,
222–225.
11. Nazarian, A., Hermannsson, B. J., Muller, J.,
Zurakowski, D., and Snyder, B. D. (2009)
Effects of tissue preservation on murine bone
mechanical properties. Journal of Biomechanics
42, 82–86.
12. van Haaren, E. H., van der Zwaard, B. C., van
der Veen, A. J., Heyligers, I. C., Wuisman, P. I.,
and Smit, T. H. (2008) Effect of long-term
preservation on the mechanical properties of
cortical bone in goats. Acta Orthopaedica 79,
708–716.
13. Keaveny, T. M., Guo, E., Wachtel, E. F.,
McMahon, T. A., and Hayes, W. C. (1994)
Trabecular bone exhibits fully linear elastic
behavior and yields at low strains. Journal of
Biomechanics 27, 1127–1136.