Microindentation for in vivo measurement of bone tissue mechanical properties in humans.

Microindentation for In Vivo Measurement of Bone
Tissue Mechanical Properties in Humans
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Bone tissue mechanical properties are deemed a key component of bone strength, but their assessment requires invasive procedures.
Here we validate a new instrument, a reference point indentation (RPI) instrument, for measuring these tissue properties in vivo. The RPI
ORIGINAL ARTICLE JBMRAddress correspondence to: Paul K Hansma, PhD, Department of Physics, Un
E-mail: prasant@physics.ucsb.eduinstrument performs bone microindentation testing (BMT) by inserting a probe assembly through the skin covering the tibia and, after
displacing periosteum, applying 20 indentation cycles at 2 Hz each with a maximum force of 11N. We assessed 27 women with
osteoporosis-related fractures and 8 controls of comparable ages. Measured total indentation distance (46.0� 14 versus 31.7� 3.3mm,
p¼ .008) and indentation distance increase (18.1� 5.6 versus 12.3� 2.9mm, p¼ .008) were significantly greater in fracture patients than
in controls. Areas under the receiver operating characteristic (ROC) curve for the twomeasurements were 93.1% (95% confidence interval
[CI] 83.1–100) and 90.3% (95% CI 73.2–100), respectively. Interobserver coefficient of variation ranged from 8.7% to 15.5%, and the
procedure was well tolerated. In a separate study of cadaveric human bone samples (n¼ 5), crack growth toughness and indentation
distance increase correlated (r¼ –0.9036, p¼ .018), and scanning electron microscope images of cracks induced by indentation and by
experimental fractures were similar. We conclude that BMT, by inducing microscopic fractures, directly measures bone mechanical
properties at the tissue level. The technique is feasible for use in clinics with good reproducibility. It discriminates precisely between
patients with and without fragility fracture and may provide clinicians and researchers with a direct in vivo measurement of bone tissue
resistance to fracture. � 2010 American Society for Bone and Mineral Research.
KEY WORDS: BONE; FRACTURE; BONE QUALITY; INSTRUMENT; CLINICAL TRIALS
Introduction
As people age, their bone strength deteriorates, and theirbone becomes more susceptible to fracture.(1) The clinical
consequence of this, the fracture, contributes to the morbidity
and mortality of osteoporosis. Bone strength has been defined as
the integration of bone mass and bone quality.(2) Available
techniques for clinical estimation of bone strength or suscept-
ibility to fracture are based mainly on bone mineral density
(BMD) assessment(3) that can be reliably measured by
densitometry techniques, but its sensitivity and specificity are
modest.(3,4) Furthermore, its ability to predict the response to a
treatment is limited, and only a small proportion of fracture risk
reduction is explained by bone density increases.(5) Advanced
bone imaging and analysis technologies promise better
assessment of bone strength(6) but rely on potentially inaccurate
assumptions about the tissue-level mechanical properties. The
addition of other surrogates, such as biochemical markers,
results in very limited improvement on these strength predic-
tions.(7)
There is clinical and laboratory evidence that in addition to
BMD, the mechanical properties of bone tissue may play a critical
role in bone strength.(8-10) These mechanical properties would be
expected to play a significant role in bone fracture risk, even
Received in original form December 17, 2009; revised form January 21, 2010; accepted February 17, 2010. Published online February 23, 2010.
iversity of California, Santa Barbara, Santa Barbara, CA 93106-9530, USA.Adolfo Diez-Perez ,1,6 Roberto Gu¨erri ,1 Xavier No
Leonardo Mellibovsky,1,6 Connor Randall ,2 Danie
Davis Brimer ,4 Kurt J Koester ,5 Robert O Ritchie
1Hospital del Mar-IMIM-Universitat Auto´noma, Barcelona, Spain
2Department of Physics, University of California, Santa Barbara,
3Coastal Marine Biolabs, Ventura, CA, USA
4Active Life Scientific, Inc., Santa Barbara, CA, USA
5Department of Materials Science and Engineering, University o
6RETICEF, Instituto Carlos III, Madrid, Spain
ABSTRACTJournal of Bone and Mineral Research, Vol. 25, No. 8, August 2010, pp 1877–1885
DOI: 10.1002/jbmr.73
� 2010 American Society for Bone and Mineral Researchs ,1,6 Enric Ca´ceres ,1,6 Maria Jesus Pen˜a ,1
idges,2 James C Weaver,2,3 Alexander Proctor,4
nd Paul K Hansma2,4
SA
ifornia, Berkeley, CA, USA1877

though it has not been clear what mechanical properties are
propagation, the anatomic basis of fracture, in a series of women
head unit of the RPI instrument.(17) (2) Apply alcohol and localwith osteoporosis-related fractures and controls. Moreover, we
have performed exploratory studies on the anatomic substrate of
the technique.
Materials and Methods
Subjects
This study involves 27 women with osteoporotic fractures (25 hip
fractures and 2 multiple vertebral fractures) measured during the
hospitalization following the event in the acute-care orthopedics
ward and 8 controls of comparable age with no fractures from
the Hospital del Mar, Barcelona, Spain. Fracture patients were
excluded if there was some previous treatment with drugs
for osteoporosis, corticosteroids use, a previous diagnosis of
advanced renal or liver disease, neoplasia, malabsorption,
thyroid or parathyroid disorder, immobilization, or inability to
provide consent. Exclusion criteria for controls were identical, but
in addition, control individuals were required to have no
prevalent fracture. Thoracic and lumbar lateral radiographs
validated the absence of subclinical vertebral fractures.
Bone microindentation testing (BMT)
The reference point indentation (RPI) instrument (which was
called the tissue diagnostic instrument(17) and the bone diagnostic
instrument(18–20) in previous publications) can measure bone
mechanical properties, in particular, the resistance to fracture, at
the tissue level (Fig. 1A). The complete BMT protocol involves 10
steps: (1) Attach a presterilized, disposable probe assembly to themost important.(11–14) However, currently available methods for
direct estimates of these properties require invasive bone
sampling,(15) making routine use in clinics unfeasible.
Assessment of the intrinsic mechanical properties of bone
tissue, as a key component of the widely used concept of bone
quality, is limited. Besides the practical inconvenience of their
routine measurement, the term bone quality is poorly defined
and encompasses a series of geometric, microarchitectural, and
tissue-composition elements.(15) As a consequence, the poten-
tially relevant contribution of bone tissue strength to fracture risk
in clinical practice cannot be evaluated, even though it is known
that it deteriorates in osteoporosis and contributes to fracture
propensity.(16)
Therefore, there is a critical need to better quantify bone
mechanical properties at the tissue level, in particular, the ability
of bone to resist the growth of cracks that result in bone fracture.
This quantification is not only desirable for more complete
clinical assessment of fracture risk but eventually also for
treatment monitoring. Moreover, this development could help to
better assess the effect of drugs on bone strength without the
need for large and expensive prospective fracture trials.
Here we report the validation results of a novel microindenta-
tion technique capable of directly testing the mechanical
endurance of bone tissue and suitable for a repeated
measurement in patients. By measuring indentation distances,
we assess the ability of bone to resist crack generation and1878 Journal of Bone and Mineral Researchanesthesia to the testing site (midshaft of anterior tibia). (3) Use
the guidance arm with the vertical slider to position the head
unit over the midshaft anterior tibia. The head unit must be
perpendicular to bone’s surface within about 15 degrees.
Since the head unit is held vertical by the guidance arm with
the vertical slider, this is achieved by holding the patient’s foot
and leg such that the midshaft of the anterior tibia is level to
within an estimated 15 degrees or less. (4) Holding the sterile
probe assembly with a sterile glove, lower head unit vertically
along slider to insert the probe assembly through the skin to rest
on the bone surface. (5) Displace the periosteum from the
measurement area by moving the reference probe by
hand laterally along the surface of the bone a distance of
approximately 5mm for a series of five times, and then place it in
the center of this approximately 5-mm region for measurement.
(6) Release the probe assembly so that it rests with the full weight
of the head unit on the bone. (7) Actuate the measurement cycle,
which first removes an initial 2.5-N force on the test probe
(used to keep the test probe from sliding back into the reference
probe during insertion) and then begins a series of precycles at
4 Hz that incrementally increase up to a threshold force of order
2.5 N and then runs the 20 indentation cycles at 2 Hz each with a
maximum force of 11N. (8) Repeat steps 3 through 7 to obtain
measurements at five or more locations. Each measurement
location should be separated by at least 2mm from other
measurement locations. (9) After the final measurement, raise
the head unit away from tibia, and detach and discard the
disposable probe assembly. (10) Wipe the measurement site with
alcohol, and apply a bandage. Local edema or advanced skin
disorder and infection in the measurement area would have
precluded use of this technique. Warfarin treatment or severe
coagulation defects have to be considered for careful local
hemostasis.
The indentations are small, on the order of 375mm across
(Fig. 1B), so they are not harmful to the patient. They are large
enough, however, that the bone is fractured (Fig. 1C) as the test
probe indents the bone. The more easily the bone is fractured,
the farther the test probe will indent the bone. Thus we quantify
the bone fracture resistance by measuring the indentation
distances achieved in a measurement. The indentation has to be
performed by the test probe perpendicular to the bone surface,
with a tolerance of �15 degrees to obtain reliable results.
The control system for the reference point indentation
instrument supplies a modified triangular wave to its internal
force generator for the 20 indentation cycles used in measure-
ments. The modified triangular waveform consists of one-third of
a cycle of linear increase, followed by one-third of a cycle hold at
maximum force (for measuring creep), and then one-third of a
cycle of linear decrease. The total cycle time is 500ms. The
purpose of the hold at maximum force is to monitor creep effects
and to minimize the effect of the remaining creep during the
linear decrease. After the cycles are complete, a computer displays
the first and last (twentieth) force-versus-distance curves
(Fig. 2A). Three indentation parameters are defined in the figure.
Total time for the test is 10minutes. The patient experiences
minimal discomfort (only during the local anesthesia injection),
and no complications have been observed whatsoever.DIEZ-PEREZ ET AL.

DXA measurement of BMD
BMD with dual-energy X-ray absorptiometry (DXA) using a
Hologic QDR 4500 SR Bone Densitometer (Hologic, Inc., Waltham,
MA, USA) was measured at the nonfractured hip within 4 weeks
of admission in a subset of 14 individuals randomly chosen
(nine fracture cases and five controls) from our clinical cohort.
Statistical analysis
Normality of continuous variables was assessed by Q-Q plots.
Analysis of covariance was used to obtain and compare age-
adjusted means. Pearson correlation index was computed to
assess the relationship between continuous variables. The ability
of the indentation distance parameters to discriminate between
those who have a fracture and those who do not was assessed by
calculating the area under the receiver operating characteristic
(ROC) curve.
Preclinical experiments on cadaveric bone
To connect indentation distance increase (IDI), as determined by
the reference point indentation instrument, to a conventional
Fig. 1. Indentation procedure for measuring material properties of bone in vivo
the method for obtaining indentation measurements, including insertion of the
first-cycle indentation, and last-cycle indentation, which determines the IDI wit
dashed line) being compared to a dime (the smallest U.S. coin). (C) This magnif
repetitive loading cycles at a constant force.
IN VIVO MEASUREMENT OF BONE MECHANICAL PROPERTIESmeasure of fracture resistance on machined samples, we
measured both IDI and crack growth toughness on cadaveric
bone samples from a group of five donors (aged 17 to 74 years).
This is a totally different group from the clinical group discussed
earlier. There were eight samples, three for the 74-year-old male,
two for the 23-year-old male, and one for each of the other
three subjects that gave crack growth data. In the case of the
multiple measurements on one donor, the multiple measure-
ments were averaged together to give one data point for the
correlation calculation. For IDI data, there were 15 samples, 3
for each donor and 10 tests on each sample for a total of
150 measurements. Again, all measurements on one donor were
averaged together to give one data point for the correlation
calculation. We were able to do more measurements for the IDI
because we could do multiple measurements on each sample,
and no special machining was required. The samples were cut
from the tibia with dimensions of the order of 2 cm in length
and width and the full thickness of the cortical bone. The bone
samples were stored in a �808C freezer. Prior to testing, the
samples were brought to room temperature, gently stripped of
soft tissue, and placed in Hank’s balanced saline physiologic
buffer solution(21) to ensure hydration. The surface of the bone
and SEM imaging of an indent on a human bone sample. (A) Illustration of
test probe assembly, displacing the periosteum with the reference probe,
h respect to the first cycle. (B) SEM image of an indentation (encircled by
ied SEM image of the indentation shows microcracks created during the
Journal of Bone and Mineral Research 1879

was not polished. Figure 1C shows the microcracks opened by
the indentations. Microcracks are opened during RPI testing
just as cracks are opened on machined samples during R-
curve testing. Thus it is reasonable to compare the results of
RPI testing with the crack growth toughness from R-curve
testing.
Indentation testing was conducted by the RPI instrument.
The bone samples were held in a vice submerged in
physiologic buffer and tested under the buffer. The indenta-
tions were normal to the outside surface of the cortical shell.
Each sample had a minimum of 10 tests conducted in varying
locations. Three samples were tested from each donor. Each
individual test was analyzed by software that was written to
compute a variety of mechanical parameters such as IDI. The
second method used crack resistance curves (R curves) to
determine the crack growth toughness. Compact tension
samples were sectioned and notched transverse to the bones’
long axis. The notch orientation was such that the nominal
crack growth direction was transverse to the long axis of the
tibia. We used nonlinear elastic fracture mechanics testing of
Fig. 2. Parameters are calculated from force-versus-distance data obtained by t
(IDI), total indentation distance (total ID), and creep indentation distance (creep
indentation distance in the last cycle relative to the indentation distance in th
distance while the force is held constant at the maximum value for a duration of
distance the test probe is inserted into the bone from touchdown to the end of t
fracture (n¼ 27) and control (n¼ 8) patients. Note that fracture patients usually
that the parameters were measured with the Hospital del Mar protocol. Thi
measurement protocol.
1880 Journal of Bone and Mineral Researchthe bone samples under hydrated conditions in situ in an
environmental scanning electron microscope (ESEM) to permit
resistance curve measurements for growing short cracks in the
transverse orientation less than 1000mm in size. Additional
details on the testing method and procedure used in this
preclinical experiment are discussed by Koester and collea-
gues.(22) The stress intensity K and crack extension data were
linearly extrapolated to determine the growth toughness
DK/Da (MPaHm/mm), which is obtained from the slope of
the R curve.(22-24) Higher growth toughness signifies a bone
that is less prone to continued crack propagation.
Results
BMT clinical experiment
Two of the three measured indentation parameters are
significantly greater for patients with fractures than for control
patients (Figs. 2 and 3). Note also that there is no apparent
correlation between age and indentation values, at least in the
he RPI instrument. The parameters include indentation distance increase
ID) measured in the first cycle. (A) The IDI is defined as the increase in the
e first cycle (see Fig. 1A). The creep ID is determined by the increase in
one-third of the first indentation cycle. The total ID is defined as the total
he twentieth cycle. (B–D) Results from clinical trials of each parameter with
had higher indentation distances. The subscript H on the graphs indicates
s is important because the values of these parameters depend on the
DIEZ-PEREZ ET AL.

Table 1. Indentation Distance Increase and Crack Growthsmall population of elderly women investigated in this study
(Fig. 2). The ROC curve shows that the total indentation distance
(total ID) is a good discriminator between patients with and
without fractures.(25) The area under the ROC curve (AUC)
value(26) in this study for total ID was of 0.931 [95% confidence
interval (CI) 83.1–100], 90.3% for IDI (95% CI 73.2–100), and 73.6%
for creep ID (95% CI 56.4–90.9).
Interobserver variability was assessed by separated measure-
ments performed by two observers in 14 individuals. The
coefficient of variation ranged from 8.7% (for IDI) to 15.5%
(for total ID).
Differences between cases and controls are shown in Fig. 3A.
As expected, BMD differences were observed. However, the
correlation between total-hip BMD and IDI (r2¼�0.127, p¼ .211)
and total ID (r2¼�0.264, p¼ .06) was low, indicating, as might
be expected, that measurements of bone loss (DXA) alone
cannot predict bone tissue mechanical properties as measured
by the RPI instrument.(25)
Preclinical experiments on cadaveric bone
The results for the comparison between IDI and crack growth
toughness are shown in Table 1. The IDI is much greater for the
Fig. 3. Data results including statistics and a receiver operating char-
acteristic (ROC) curve. (A) Age-adjusted statistical results for IDI (mm),
creep ID (mm), total ID (mm), femoral neck bone mineral density (FN BMD,
g/cm2), and total-hip bone mineral density (TH BMD, g/cm2). (B) The ROC
curve displays the clinical results from Hospital Del Mar, Barcelona. The
area under the curve (AUC) is a scalar quantity to gauge the performance
of the curve. An AUC of 100% would represent a perfect model; however,
an area going along the line of discrimination (dashed diagonal) would be
a completely random model.
IN VIVO MEASUREMENT OF BONE MECHANICAL PROPERTIES74-year-old male subject with an IDI of 20.49� 6.88mm, whereas
it is very low for younger subjects. We measured the IDI of
cadaveric bone from additional older subjects but were unable
to generate an R curve for each of the subjects because of the
geometry of the bones and the requirements of our testing
method.(19) For example, with most of the older individuals who
had osteoporosis, there was very little cortical shell to work with
on the limited number of samples we had available. Since we had
only one older subject from whom we got multiple tests, our
results can only be regarded as preliminary. Future testing to
compare IDI and crack growth toughness on a wider range of
individuals would be valuable. This may require novel methods
for determining crack growth toughness.
Figure 4A–C shows scanning electron microscope (SEM)
images of human bone samples that were fractured and exhibit
crack bridging, which resists crack extension. The crack growth
toughness of the samples then was compared to the IDI. In
samples fractured in fluid,(27) microcracks were observed by SEM,
and their appearance was similar to microcracks created by the
RPI instrument during repetitive indentations. Comparisons
between IDI and the crack growth toughness(22) (slope of the R
curve) for samples from five donors showed that high IDI and low
crack growth toughness are associated with bones that are
prone to fracture. The graph shows this trend by relating high IDI
to low crack growth toughness and vice versa. Pearson’s
correlation coefficient between the IDI and crack growth
toughness is �0.9036, with p¼ .018 (Fig. 4D). The coefficient
is negative owing to the inverse relationship between IDI and
Toughness for Each Donor Sample Tested for Correlations
Age/sex IDI� SD (mm) (N) DK/Da (MPaHm/mm) (N)
74/M 20.49� 6.88 (3) 0.0365 (3)
23/M 14.75� 3.12 (3) 0.0428 (2)
17/F 13.97� 2.76 (3) 0.0405 (1)
44/F 12.89� 3.70 (3) 0.0426 (1)
22/F 12.43� 2.49 (3) 0.0455 (1)
Note: The number of samples tested from each donor n for each test is
shown next to the test result in parentheses. Note the inverse relationship
between IDI and DK/Da because high IDI and low DK/Da correspond to a
high fracture risk.crack growth toughness.
Discussion
Here we describe the validation study of a novel device that
performs bone microindentation testing (BMT) of bone in vivo
in a series of patients with and without osteoporotic fractures.
BMT discriminates between cases and controls and measures
parameters different from BMD. Preclinical studies in human
cadavers suggest that BMT induces separation of mineralized
collagen fibrils and initiation of cracks, very likely the basic
mechanism of fracture, thus directly measuring the mechanical
competence of bone tissue to resist fracture.
Journal of Bone and Mineral Research 1881