Original Full Length Article
Microcrack density and nanomechanical properties in the subchondral region of the
immature piglet femoral head following ischemic osteonecrosis
Olumide O. Aruwajoyea,b, Mihir K. Patelb, Matthew R. Allenc, David B. Burrc,d,
Pranesh B. Aswathb, Harry K.W. Kima,e,⁎
aCenter for Excellence in Hip Disorders, Texas Scottish Rite Hospital for Children, Dallas, TX, USA
bMaterials Science and Engineering Department, University of Texas at Arlington, 501 West First Street, ELB Rm 231, Arlington, TX 76019, USA
cDepartment of Anatomy and Cell Biology, Indiana University School of Medicine, 635 Barnhill Dr, MS 5035, Indianapolis, IN 46202, USA
dDepartment of Biomedical Engineering, Indiana University–Purdue University at Indianapolis, USA
eDepartment of Orthopaedic Surgery, University of Texas Southwestern Medical Center, Dallas, TX, USA
a b s t r a c ta r t i c l ei n f o
Received 11 June 2012
Revised 26 July 2012
Accepted 27 July 2012
Available online 3 August 2012
Edited by: Thomas Einhorn
Development of a subchondral fracture is one of the earliest signs of structural failure of the immature femoral
head following ischemic osteonecrosis, and this eventually leads to a flattening deformity of the femoral head.
The mechanical and mineralization changes in the femoral head preceding subchondral fracture have not been
elucidated. We hypothesized that ischemic osteonecrosis leads to early material and mechanical alterations in
the bone of the subchondral region. The purpose of this investigation was to assess the bone of the subchondral
region for changes in the histology of bone cells, microcrack density, mineral content, and nanoindentation
properties at an early stage of ischemic osteonecrosis in a piglet model. This large animal model has been
shown to develop a subchondral fracture and femoral head deformity resembling juvenile femoral head
osteonecrosis. The unoperated, left femoral head of each piglet (n=8) was used as a normal control, while the
right side had a surgical ischemia induced by disrupting the femoral neck vessels with a ligature. Hematoxylin
and eosin (H&E) staining and TUNEL assay were performed on femoral heads from 3 piglets. Quantitative back-
scattered electron imaging, nanoindentation, and microcrack assessments were performed on the subchondral
region of both control and ischemic femoral heads from 5 piglets. H&E staining and TUNEL assay showed
extensive cell death and an absence of osteoblasts in the ischemic side compared to the normal control.
Microcrack density in the ischemic side (3.2±0.79 cracks/mm2) was significantly higher compared to the
normal side (0.27±0.27 cracks/mm2) in the subchondral region (pb0.05). The weighted mean of the weight
percent distribution of calcium (CaMean) also was significantly higher in the ischemic subchondral region
(pb0.05). Furthermore, the nanoindentation modulus within localized areas of subchondral bone was signifi-
cantly increased in the ischemic side (16.8±2.7 GPa) compared to the normal control (13.3±3.2 GPa)
(pb0.05). Taken together, these results support the hypothesis that the nanoindentation modulus of the
subchondral trabecular bone is increased in the early stage of ischemic osteonecrosis of the immature femoral
head and makes it more susceptible to microcrack formation. We postulate that continued loading of the hip
joint when there is a lack of bone cells to repair the microcracks due to ischemic osteonecrosis leads to
microcrack accumulation and subsequent subchondral fracture.
© 2012 Published by Elsevier Inc.
Juvenilefemoralheadosteonecrosis resultsfroma loss of blood flow
to the femoral head and leads to a flattening deformity. This disruption
of bloodflowcauses extensivecell death.Alackof bonecellsleads to an
inability to remodel and repair the damaged bone . Consequently,
ischemic osteonecrosis of the immature femoral head can lead to a
subchondral fracture and subsequent femoral head collapse [2,3].
Some studies have examined the pathogenesis of subchondral fracture
and femoral head deformity following osteonecrosis, but the studies
are based on adult patients [4,5]. In a juvenile model of osteonecrosis,
one of the mechanisms involved with the pathogenesis of a femoral
head deformity is excessive bone resorption , which occurs during
the revascularization phase of the repair process. During this phase,
an increased osteoclastic activity with a decrease in trabecular
bone volume is observed. Surprisingly, a significant decrease in the
stiffness of the necrotic femoral head and its bony component has
Bone 52 (2013) 632–639
⁎ Corresponding author at: Center for Excellence in Hip Disorders, Texas Scottish Rite
Hospital for Children, Dallas, 2222 Welborn Street, Dallas, TX 75219, USA. Fax: +1 214
E-mail addresses: firstname.lastname@example.org (O.O. Aruwajoye),
email@example.com (M.K. Patel), firstname.lastname@example.org (M.R. Allen),
email@example.com (D.B. Burr), firstname.lastname@example.org (P.B. Aswath), email@example.com
8756-3282/$ – see front matter © 2012 Published by Elsevier Inc.
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been observed in experimental studies even before the revasculari-
zation phase when the bone loss occurs [6,7]. In these studies, the
whole femoral head was mounted and indented with a 4 mm spher-
ical indenter. For isolated bone studies, uniaxial compressive testing
was performed on 3.9 mm diameter×4.7 mm length bone cores,
which included the subchondral and central epiphyseal trabecular
bone. The decrease in the mechanical properties was unexpected at
an early stage (2 weeks post ischemia) and prompted an investigation
of the material and mechanical properties of necrotic bone before any
bone resorption occurs within the femoral head.
Subchondral fracture is one of the earliest radiographic signs seen
in patients with juvenile femoral head osteonecrosis . Since it rep-
resents the earliest sign of mechanical insufficiency of the infarcted
femoralhead,itisimportant to understandthemechanismsunderlying
the mechanical failure of the subchondral bone that leads to the
subchondral fracture. The early mechanical and mineralization changes
in the subchondral region have not been elucidated. Several studies
have highlighted the role of microdamage in bone homeostasis  in
the context of metabolic bone disorders, as well as its association with
various bone failures [9–11]. The role of microdamage in the develop-
ment of the femoral head deformity following ischemic osteonecrosis
has not been investigated. We hypothesized that ischemic osteonecrosis
leads to early mechanical and mineralization alterations in the sub-
chondral region, which predisposes it to fracture. The purpose of this in-
of bone cells, microcrack number, mineral content, and nanoindentation
properties in an early stage of ischemic osteonecrosis (two weeks post
ischemia induction) using a well-established large animal (piglet)
model of juvenile osteonecrosis.
Materials and methods
The local Institutional Animal Care and Use Committee (IACUC) ap-
proved the study. Eight 5–6-week old Yorkshire male piglets were
used. Ischemic osteonecrosis was induced surgically by transecting the
ligamentum teres and placing a ligature tightly around the femoral
neck that stops blood flow to the femoral head. The unoperated contra-
lateral side was used as a normal control. All animals were sacrificed at
two weeks post ischemia induction when the femoral heads are
avascular and no bone resorption is observed [2,6]. Following re-
trieval, all femoral heads were bisected, fixed in formalin, and stored
in 70% ethanol. The femoral heads from five animals were used for
microcrack quantification, quantitative backscattered imaging, and
nanoindentation testing. Two millimeter sections were obtained
from each femoral head of the five piglets. Each section was embedded
in methyl methacrylate using standard methods. For nanoindentation
and quantitative backscattered imaging, the sections were polished
using increasing grades of silicon carbide paper (500, 800, 1200, 2500,
and 4000 grit), and polished by 0.25 μm and 0.05 μm diamond suspen-
sion.Inbetween eachpolishingstep,the samples were rinsed for5 min
hematoxylin and eosin (H&E) staining and terminal deoxynucleoidyl
transferase-mediated dUTP nick end-labeling (TUNEL) assay to assess
Sample preparation for H&E and TUNEL assay has been previously
bedded in paraffin, sectioned 6 μm thick, and stained with H&E or used
for a TUNEL assay. Sections from femoral head pairs, non-operated (left)
mentation was detected by performing terminal deoxynucleotidyl
transferase-mediated digoxigenin-deoxy-UTP nick end-labeling using
the ApopTag Peroxidase In Situ Apoptosis Detection Kit (Intergen, USA).
TUNEL reaction was visualized using diaminobenzidine as peroxidase
substrate. Hematoxylinwas used as a counterstain. DNAsedigested carti-
lage sections were used as positive controls. Normal, unoperated femoral
heads from contralateral sides and the sections withomissionof terminal
section of the ischemic femoral head, the unaffected region of the
proximal femur, the metaphysis, served as negative internal controls.
Portions of the femoral head (1.5 cm×1.5 cm×2 mm thick) were
processed for microdamage assessment by bulk staining in calcein
. Specimens were transferred from 70% ethanol to 0.9% saline
for 5 h and then soaked in calcein (0.3 g/100 mL saline) for 18 h
under vacuum (20 in Hg). Following calcein staining, bones were
washed for 10 min with reverse osmosis water and then embedded in
methyl methacrylate using standard methods. Central (8 μm) sections
were cut using a Reichert-Jung 2050 microtome (Magee Scientific).
Microdamage quantification was performed using a semiautomatic
analysis system (Bioquant OSTEO, Bioquant Image Analysis Co.) at-
tached to a microscope equipped with a florescent light source (Nikon
Optiphot 2 microscope, Nikon). Measurements were carried out on
two sections per femoral head . A 5 mm2region of interest, located
thetwo sectionsresulting in a total tissue area of 10 mm2for each fem-
oral head. Microdamage was assessed using ultraviolet fluorescence.
Microcracks were identified by their typical linear shape, relative size
(greater than canaliculi, smaller than vascular channels), and positive
fluorescence (due to filling of the microcrack with stain), as previously
described [16,17]. Bone was visually inspected for microcracks at 100×
magnificationandwhenfound;eachcrack lengthwasmeasured at200×
number (Cr.N), with calculations of crack density (Cr.Dn, #/mm2; Cr.N/
bone area) and crack surface density (Cr.S.Dn, μm/mm2; Cr.N∗Cr.Le/
Quantitative backscattered electron imaging
A backscattered electron image of the subchondral and calcified
cartilage regions was taken at a magnification of 70× for all samples
during the same scanning electron microscopy session. An electron
beam energy of 25 kV and a beam current of 92 μA were maintained.
The working distance was 10 mm in all cases. The images were used
for quantifying distribution of mineralization in bone. The calcified
cartilage (0.5 mm2) and subchondral (1 mm2) regions were analyzed
separately. This type of quantification for mineralization in bone has
been previously described [18,19]. Briefly, carbon, aluminum, and
hydroxyapatite were used as standards to calibrate a bone mineral
density distribution (BMDD) from a backscattered electron signal. A
calibration line was drawn to correlate the respective gray level values
and atomic numbers for each standard. The gray levels were then
converted to weight percent of calcium based on the molecular formula
weight percent value with the most frequency (CaPeak) were obtained
from each BMDD.
A 2×2 mm superolateral area that included the calcified cartilage
andsubchondralbone regionswas consistentlyidentifiedforallsamples
for nanoindentation. The subchondral region of the femoral head bone
was identified through an optical microscope and an exposed area of
trabecular bone within the region was selected for nanoindentation. A
Hysitron Ubi-1 Nanoindenter (Hysitron, Minneapolis, MN) with a
Berkovich tip was used for nanoindentation. The location for the
nanoindentation area was verified by view through the optical camera
O.O. Aruwajoye et al. / Bone 52 (2013) 632–639
in the nanoindentation chamber that showed both the Berkovich tip
(r=150 nm) and the region of interest. A bony structure within the
subchondral region was identified. However, the exact location of the
indenter tip on the trabecular bone could not be confirmed in relation
to its porous structures such as canaliculi and lacunae. A scanning
probe microscopy image and respective nanoindentation values were
embedding material that surrounded the bone consistently had a re-
duced modulus less than 7 GPa. The term “reduced” is used to describe
the calculation as referenced to the Young's modulus of the diamond
nanoindentation tip. We, thus, analyzed indents above the threshold
of 7 GPa. Only nanoindentations with a reduced modulus above
7 GPa were used for statistical analysis as lower moduli were
assumed to be nanoindentations in the plastic embedding substrate.
One area (3600 μm2) was analyzed per femoral head. At least 21
nanoindentations were analyzed in the area. The following loading
function was performed for each indent; ramped to 250 μN at
50 μN/s, held for 5 s, and then unloaded at 50 μN/s. The unloading
point of the loading curve was used for reduced modulus (1) and
hardness (2) calculations based on the Oliver–Pharr method .
The reduced modulus was calculated from
Eris definedasthereduced modulus, Sastheslopeof themostlinear
part of the upper portion of the unloading curve, and Acas the projected
contact area of the Berkovich tip. The projected contact area of the
Berkovich tip was determined by calibrating the tip at various indenta-
tion depths with fused quartz. For hardness, the values are calculated
H is defined as the hardness, which is calculated by the maximum
load, Pm, divided by the projected contact area of the Berkovich tip.
The crack density (Cr.Dn, #/mm2) and crack surface density
(Cr.S.Dn, μm/mm2; Cr.N∗Cr.Le/bone area) were averaged for both
ischemic (n=5) and normal control (n=5) groups. The averaged
was used to obtain an average for all of the femoral heads in each re-
spective group of ischemic (n=5) and normal control (n=5) femoral
heads. More specifically, comparisons were made using averaged
nanoindentation values from each head. CaMean and CaPeak values
for each respective group were averaged similarly. Given the small
Fig. 1. A scanning probe microscopy image with a dashed line depicting the plastic-bone
interface. The plastic is on the left and the bony area is on the right. The numbers on the
image indicate the reduced modulus in GPa units obtained at each respective location.
Fig. 2. (A) The subchondral region of normal and ischemic bone stained with H&E. An arrow indicates osteoblast lining the trabecular bone of the normal subchondral region. The
ischemic side shows a lack of osteoblast lining the trabeculae. (B) Normal and ischemic subchondral regions assessed with terminal deoxynucleotidyl transferase mediated dUTP
biotin nick end labeling (TUNEL) assay. The normal subchondral region shows a negative TUNEL staining whereas the ischemic bone shows diffuse TUNEL staining of the cells in the
bone and the marrow space. The original magnification is 100× (scale bar=200 μm). The boxed regions are at 400× magnification (scale bar=50 μm).
O.O. Aruwajoye et al. / Bone 52 (2013) 632–639
sample size, Wilcoxon sign-rank test (non-parametric testing) was
used. A pb0.05 was defined as being statistically significant.
Histological comparison of control and ischemic sides (Figs. 2A
and B) showed extensive cell death in the ischemic side with an ab-
sence of osteoblasts lining the trabeculae. The finding of extensive
cell death in H&E staining was further supported by TUNEL assay
which showed diffuse positive staining of the cells in the marrow
space and the trabecular bone in the ischemic side compared to the
normal control side.
Microcracks were rarely present in the subchondral region of the
normal control sections whereas they were present in significant num-
bersintheischemic group (Figs.3Aand B).Thecalculatedcrackdensity
(Cr.Dn, #/mm2) and the crack surface density (Cr.S.Dn, μm/mm2;
Cr.N∗Cr.Le/bone area) in the subchondral region were significantly in-
creased in the ischemic side compared to the normal control side
(pb0.05) (Figs. 4A and B).
Quantitative backscattered imaging showed increased mineraliza-
tion in the ischemic bone samples. BMDD in each respective pair
shows a higher calcium weight percent in the ischemic side (Fig. 5)
(Table 1). The CaMean was significantly higher in the subchondral
(pb0.05) and calcified cartilage regions (pb0.05) in the ischemic
side. The CaPeak was also significantly higher in the calcified cartilage
region (pb0.05) and trends toward significance in the subchondral
On average, the penetration depth of the indenter was higher in the
normal control (191±11.1 nm) compared to the ischemic nano-
indentations (164±16.7 nm) (pb0.05). The load displacement curves
of the ischemic subchondral bone showed a shallow penetration depth
from nanoindentations compared to the normal control (Fig. 6A).
Tables 2 and 3 show averaged reduced modulus and hardness values
from each respective femoral head. In Table 2, four of five ischemic fem-
oral heads showed higher mean values of reduced modulus. In Table 3,
ness. Fig. 6B shows a typical scatter plot of the reduced moduli found in
the subchondral bone. The scatter plot showed a much larger spread in
the reduced moduli of the ischemic side (7.2–25.5 GPa) compared to
the normal control side (7.1–17.1 GPa). A significant increase in the re-
duced modulus was found in the subchondral region of the ischemic
femoral head compared to the normal femoral head (pb0.05) (Fig. 6C).
The mean hardness value was higher in the ischemic side compared to
tically significant (p=0.23).
To date, much of the research on the subchondral region of the
femoral head has focused on the specimens from adult patients due to
the lack of availability of surgical and pathological specimens from chil-
model of femoral head osteonecrosis provided an alternative approach
to investigate the early changes in the subchondral region following
ischemic osteonecrosis. This study sheds light on the complexity of
the mechanical compromise following ischemic osteonecrosis in
the immature femoral head which includes higher mineral content,
changes in the nanoindentation properties, cell death leading to an
inability to repair microdamage, microdamage accumulation, and lack
of cells to form new bone. The results of the study support the hypoth-
esis that ischemic osteonecrosis of the immature femoral head leads to
an early increase in nanoindentation modulus of the subchondral re-
gion and microdamage accumulation, resulting in compromise of the
mechanical properties of the subchondral region.
Fig. 3. (A) Fluorescent microscopy image of the ischemic femoral head stained with
calcein captured at 20× magnification. The dashed white line indicates the subchondral
dividual trabeculae captured at 200× magnification with a microcrack (indicated by an
Fig. 4. Bar graphs showing (A) crack density and (B) crack surface density. These parameters were significantly greater in the ischemic subchondral region compared to the normal
side. (*) pb0.05, by Wilcoxon signed-rank test, n=5 per group.
O.O. Aruwajoye et al. / Bone 52 (2013) 632–639
The purpose of this investigation was to assess the subchondral
region for changes in histologic appearance, microcrack number,
mineral content, and nanoindentation properties in an early stage of
ischemic osteonecrosis. Histological findings showed diffuse cell
death within the marrow space and bone in the subchondral region
with an absence of osteoblasts. This region also showed an increased
presence of microcracks and increased nanoindentation moduli in the
ischemic femoral heads. To our knowledge, this is the first study to
describe these findings in the subchondral region of the immature
femoral head in an early stage of osteonecrosis. These findings pro-
vide further insight into the decrease in the mechanical properties
of the femoral head prior to the occurrence of the revascularization
phase and bone resorption. They also provide a potential mechanism
for the development of a subchondral fracture following ischemic
osteonecrosis. Since microcracks develop with normal daily activities
[21,22], we believe that daily loading of the femoral head in absence
of bone cells to repair microcracks led to microcrack accumulation
and eventual mechanical compromise of the trabecular bone in the
infarcted femoral head.
Microdamage accumulation in canine femurs has been shown to be
chemic osteonecrosis, a decrease in the macroindentation stiffness of
the infarcted femoral head (indentation measurements using a spheri-
cal indenter on the articular surface of the whole femoral head) was
found at two weeks post ischemia induction using wet, non-fixed
of the bone cores from the infarcted femoral heads was also observed at
two weeks post ischemia induction . The reason for the decreases in
the mechanical properties, however, was unknown, which prompted
compromise. Indeed, the finding of increased presence of microcracks in
the subchondral region supports the findings of a decrease in bone stiff-
ness following ischemic osteonecrosis reported in the previous studies
using whole femoral heads and bone cores [6,7].
Fig. 5. (A) Representative normal and (B) ischemic backscattered scanning electron microscopy images. The dashed line in both images separates the calcified cartilage region (CC) and
the subchondral bone region (SB). The soft tissue and marrow space was removed by a common threshold and set to a gray level of zero (black). The brightness and contrast of both im-
both normal and ischemic bone.
The weighted mean of the bone mineral density (CaMean) and the weight percent value with the most frequency (CaPeak) from subchondral bone of each femoral head.
Calcified cartilage regionSubchondral region
NormalIschemicNormalIschemicNormal IschemicNormal Ischemic
Femoral head 1
Femoral head 2
Femoral head 3
Femoral head 4
Femoral head 5
O.O. Aruwajoye et al. / Bone 52 (2013) 632–639
Interestingly, the results of nanoindentation testing using a
Berkovich tip showed increased nanoindentation modulus of the ne-
crotic trabecular bone in the subchondral region, which seem contrary
tothedecreased bonestiffness reportedpreviouslyusingwholefemoral
heads and bone cores. The key difference is that the measurements in
thepreviousstudies defined the stiffness at a larger scale, testingthere-
gions of the femoral head that included a greater bone volume. The de-
crease in indentation stiffness in the previous studies was most likely
influenced by the presence of microcracks and porosity within the ne-
crotic bony epiphysis. More specifically, the spherical indenter used in
the previous study  had a much larger interaction volume with the
necrotic bony epiphysis due to the size indenter (4 mm) and depth of
indentation (0.5 mm). A study by Paietta et al. has shown that increas-
denter used was many magnitudes smaller and indented at depths
reaching ~200 nm in bone.
The increase in the nanoindentation modulus of the bone is consis-
tent with the finding of higher mineral content of the subchondral
bone reported in this study. Similarly, higher mineral content has
been reported previously at four and eight weeks post ischemia using
the piglet model . An increase in the mineral content of the bone
has been shown to be associated with increased stiffness in normal tra-
becular bone of mandibular condyles in newborn pigs , and normal
femoral cortical bonein anadulthuman . Due tothe higher mineral
content in the calcified cartilage and subchondral bone region of the is-
chemicbonecompared tothecontrol, higherstress concentrationsmay
be present in the transitional zone between the articular cartilage and
subchondral bone during loading of the joint.
The reason for higher mineral content following ischemic osteo-
necrosis can only be speculated.The process inwhichbonematrixmin-
eralization is altered following ischemia may be partly explained by
changes in the extracellular and intracellular mineralization associated
with apoptosis [26,27]. The normal subchondral region has higher cell
mia induction, the cells in the subchondral region become apoptotic as
our results indicate extensive cell death. Apoptotic cells are thought to
become more permeable to calcium and phosphate ions leading to in-
tracellular mineralization . In fact, there is a correlation between
Fig. 6. (A) A graph showing representative load displacement curves of ischemic (solid line) and the normal (dashed line) bone from the subchondral region. The load displacement
curve for normal nanoindentations typically show greater penetration depth compared to the ischemic nanoindentation. (B) A representative scatter plot of the reduced moduli at
different locations in both subchondral regions of normal (white diamond) and ischemic bone (black circle). A larger range of moduli is seen in the ischemic bone nanoindentations
compared to the normal bone nanoindentations. (C) A graph showing the mean reduced moduli±standard deviation of the normal and ischemic groups. The reduced modulus was
significantly increased in the ischemic subchondral region compared to the normal side. (*) pb0.05, by Wilcoxon signed-rank test, n=5 per group. (D) A graph showing the mean
hardness±standard deviation of the normal and ischemic groups. The mean hardness was higher in the ischemic side but the difference was not statistically significant.
The reduced modulus (Er) from subchondral bone of each femoral head.
Femoral head pair #12345
Normal IschemicNormal IschemicNormalIschemicNormalIschemicNormal Ischemic
Reduced modulus Mean±SD (GPa)
Number of indents>7 GPa
O.O. Aruwajoye et al. / Bone 52 (2013) 632–639
mineralization and apoptosis . Magne et al. suggested that matrix
vesicles and apoptotic bodies contribute to extracellular calcification
. Apoptotic bodies have been suggested to act as nucleating miner-
chemia induction contributes to higher mineral content seen in the
subchondralregioninthis study. Wepostulatethathighermineralcon-
tent of the necrotic bone makes it more prone to microdamage, which
may facilitate the process of microdamage accumulation because the
bone is less compliant. Positive associations between higher mineral
content and microdamage have been observed with both aging and
pharmacological treatment . One can further postulate that micro-
crack accumulation makes the bone more susceptible to fracture in
this region. In the piglet model of osteonecrosis, subchondral fractures
are observed around four weeks post ischemia induction in some of
the animals . Since this study observed the presence of increased
microcracks at two weeks post ischemia induction, it is reasonable to
speculate that the microcrackswill continue toaccumulate and eventu-
ally lead to a fracture.
This study does have some limitations. Local measurements of
nanoindentation properties and microcrack density were done on
dehydrated and embedded bone. While dehydration has been shown
drated sample of bone , this increase in the nanoindentation prop-
erties has been shown not to affect comparative trends between
samples that are prepared under similar conditions . The samples
in both normal and ischemic groups were dehydrated in ethanol and
embedded in PMMA under the exact same conditions. We assumed
that the tissue preparation procedures had the same effect on the
nanomechanical properties of bone tissue from ischemic and normal
femoral heads. While it has been discussed that microcracks develop
due to ethanol dehydration , a study by Burr and Stafford demon-
strated that the en bloc staining technique does not cause artifactual
cracking from dehydration . In our study, the en bloc staining tech-
nique was applied, as the samples were placed in calcein and PBS prior
to dehydration and embedding. For the purpose of comparisons, the
nanomechanical and microcrack density differences seen between the
groups are probable results of ischemic osteonecrosis. We also made
and controlled for nanoindentations in the embedding material.
In summary, this study provides new evidence of increased stiffness
of the trabecular bone at a nanoscale level using nanoindentation and
increased presence of microcracks in the subchondral region of the im-
mature femoral head in the early stage of ischemic osteonecrosis. The
results support the hypothesis that early alterations of the material
properties of the infarcted femoral head render the subchondral bone
more prone to microcrack development, with continued loading of the
femoral head leading to microcrack accumulation in the subchondral re-
gion and possible fracture.
The research work was funded by the Texas Scottish Rite Hospital
for Children. The authors thank Keith Condon for microcrack sample
preparation. The authors are grateful for the nanoindentation and
backscattered electron imaging made possible at the Characterization
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