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ORIGINAL PAPER
The effect of sterilization on the mechanical propertiesof intact rabbit humeri in three-point bending, four-pointbending and torsion
Nicholas A. Russell • Alain Rives •
Matthew H. Pelletier • Warwick J. Bruce •
William R. Walsh
Received: 13 March 2012 / Accepted: 11 May 2012
� Springer Science+Business Media B.V. 2012
Abstract Load bearing bone allografts are used to
replace the mechanical function of bone that has been
removed or to augment bone that has been damaged in
trauma. In order to minimize the risk of infection and
immune response, the bone is delipidated and termi-
nally sterilized prior to implantation. The optimal
method for bone graft sterilization has been the topic
of considerable research. Recently, supercritical car-
bon dioxide (SCCO2) treatments have been shown to
terminally sterilize bone against a range of bacteria
and viruses. This study aimed to evaluate the effect of
SCCO2 treatment compared with two doses of gamma
irradiation, on the mechanical properties of whole
bone. Paired rabbit humeri were dissected and ran-
domly assigned into either SCCO2 control, SCCO2
additive or gamma irradiation at 10 or 25 kGy
treatment groups. The bones were mechanically tested
in three-point and four-point bending and torsion, with
the lefts acting as controls for the treated rights.
Maximum load, energy to failure and stiffness were
evaluated. This study found that SCCO2 treatment
with or without additive did not alter maximum load,
energy to failure or stiffness significantly under any
loading modality. Gamma irradiation had a deleterious
dose dependant effect, with statistically significant
decreases in all mechanical tests at 25 kGy; while at
10 kGy there were reductions in all loading profiles,
though only reaching statistical significance in torsion.
This study highlights the expediency of SCCO2
treatment for bone allograft processing as terminal
sterilization can be achieved while maintaining the
intrinsic mechanical properties of the graft.
Keywords Allograft � Sterilization � Supercritical
fluid � Gamma irradiation � Mechanical
Introduction
The use of bone graft is widespread in orthopaedic
surgeries, with more than 2.2 million bone graft
transplants performed every year (Lewandrowski et al.
2000). They are used in a wide variety of applications;
from the reconstruction of skeletal defects caused by
trauma, tumour, non-union and failed joint arthro-
plasty, to spinal surgery for segmental fusion or
deformity. Ideally the graft should exhibit mechanical
properties comparable to the host bone, acting as a
load bearing scaffold by mimicking the mechanical
N. A. Russell � A. Rives � M. H. Pelletier �W. R. Walsh (&)
Surgical and Orthopaedic Research Laboratories,
Prince of Wales Clinical School, Prince of Wales
Hospital, University of New South Wales, Level 1
Clinical Sciences Building, Avoca St Randwick,
Sydney, NSW 2031, Australia
e-mail: [email protected]
W. J. Bruce
Concord Repatriation General Hospital, Sydney, Australia
123
Cell Tissue Bank
DOI 10.1007/s10561-012-9318-0
function of the bone that is replaced, while also
actively participating in the healing process.
Autograft bone is often considered the ‘gold
standard’ as it naturally provides all the necessary
factors to promote bone repair, without the risk of
disease transmission or immunogenicity. However,
the limited availability of autograft bone and associ-
ated donor site morbidity has resulted in the need for
an alternative. Allograft bone is the logical option due
to its’ architectural similarities, the allowance for
anatomical matching and availability in numerous
preparations. However, rigorous processing and ter-
minal sterilization are required prior to use to mini-
mize the possibility of an immune response or disease
transmission. While effective at removing these
infectious agents, these treatments have been shown
to have a deleterious effect on the biological and
mechanical properties of the graft (Akkus and
Belaney 2005; Akkus and Rimnac 2001; Anderson
et al. 1992; Cornu et al. 2000; Currey et al. 1997;
DePaula et al. 2005; Thoren and Aspenberg 1995).
A processing and sterilization method that preserves
the mechanical and biological performance of allo-
graft bone is necessary.
Gamma irradiation is the most prevalent method of
terminally sterilizing allograft bone used by bone
banks due to its efficacy against bacteria and viruses
(Campbell et al. 1994; Nguyen et al. 2007a, b). A dose
of 25 kGy is generally accepted as the minimum
required dosage to achieve the quality assurance level
of 106 bacterial log reductions required by the
American Association of Tissue Banks (AATB) and
Food and Drug Administration (FDA). At this level,
gamma irradiation significantly diminishes the
mechanical (Akkus and Belaney 2005; Balsly et al.
2008; Mitchell et al. 2004; Nguyen et al. 2007b) and
biological (Dziedzic-Goclawska et al. 2005; Dziedzic-
Goclawska et al. 1991; Ijiri et al. 1994; Voggenreiter
et al. 1996) properties of the graft, with these effects
amplified as dose is increased (Godette et al. 1996).
During gamma irradiation, gamma rays split the
collagen backbone of the bone matrix (Dziedzic-
Goclawska et al. 2005), while radiolysis of water
causes free radicals to induce cross-links in the bone
matrix collagens (Akkus et al. 2005). These changes
affect the osteogenic ability of the graft, where the
activation of growth factors such as bone morphogenic
proteins (BMP) and Transforming Growth Factor-b(TGF-b) are essential for effective osteoinduction.
These growth factors require a carrier to provide an
osteoconductive scaffold for the recruitment of osteo-
clasts to begin the resorption process. Collagen is the
carrier of BMP in the bone matrix (Ijiri et al. 1994);
consequently changes in the fibrillar network caused
during gamma sterilization disrupt the process of bone
remodeling and graft-host healing (Dziedzic-Go-
clawska et al. 2005; Ijiri et al. 1994). Mechanically
these changes have their most considerable effect on
the post-yield properties of bone, where collagen
fibers provide a bridging and reinforcement function
to crack propagation (Akkus et al. 2005; Anderson
et al. 1992; Hamer et al. 1996, 1999; Triantafyllou
et al. 1975). This translates to a significant reduction in
the post-yield (plastic) properties of cortical bone
while the pre-yield (elastic) behaviour is unaffected
(Akkus and Rimnac 2001; Anderson et al. 1992;
Currey et al. 1997; Hamer et al. 1996). This behaviour
can be explained by the dependency of pre-yield
properties of cortical bone on the mineral phase, and
the post-yield properties on collagen (Burstein et al.
1975). It also manifests itself clinically with age and
disease related alterations in collagen biochemistry
causing bone brittleness (Vashishth et al. 2001;
Zioupos et al. 1999). This is a major clinical consid-
eration for bone allograft used in load bearing
applications (Davy 1999).
Recently, supercritical fluid (SCF) technology has
been investigated as a potential alternative to gamma
irradiation. Supercritical Fluids are substances that
exist as both liquid and gas above their critical
temperature and pressure; resulting in unique proper-
ties different to both liquid and gas under standard
conditions (Gerd 2005). These properties allow them
to penetrate substances easily; dissolve materials into
their component parts; and to function as an organic
solvent. Supercritical carbon dioxide (SCCO2) is
particularly expedient due to its low critical temper-
ature and pressure (Tc = 31.1 �C, Pc = 73.4 bar),
which allows it to be used to treat biological tissues
without deleterious effects on proteins and enzymes
(White et al. 2006). Alloimmunogenicity is strongly
correlated with the presence of human leukocyte
antigen (HLA) and major histocompatibility com-
plexes (MHC) which are found in bone components
such as collagen, lipids and matrix proteins. SCCO2
has been shown to effectively delipidate bone (Fages
et al. 1994), thus removing some of these immuno-
logical concerns. Furthermore, a number of studies
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have also reported the inactivation of bacteria, spores
and enzymes using high pressure supercritical carbon
dioxide at a range of experimental pressures and time
(Bertoloni et al. 2006; Dillow et al. 1999; Spilimbergo
and Bertucco 2003; Spilimbergo et al. 2003; Watanabe
et al. 2003). Sporicidal and bactericidal additives such
as hydrogen peroxide and ethanol has also been
incorporated to improve the efficacy of such treat-
ments (Hemmer 2007; Qiu et al. 2009; Shieh et al.
2009; Zhang et al. 2006).
The effects of SCF sterilization on the mechanical
properties of bones are not well documented. In the
most significant study to date, Nichols et al. (2009)
found that SCCO2 treatment with the addition of
active sterilant did not significantly affect human
cortical bone when tested in three point bending.
Moreover, they found that under these same testing
conditions terminal sterilization (SAL6) was achieved
for B. atrophaeus. In view of these findings, this study
aimed to evaluate the effect of SCCO2 treatment
compared with a low and moderate dose gamma
irradiation on the mechanical properties of paired
whole bones under a range of loading situations; and in
doing so investigate the expediency of these treat-
ments for processing of load bearing allograft bone.
Materials and methods
One hundred and twenty paired (left and right) humeri
were dissected fresh from the carcasses of 120
9 month old New Zealand white rabbits used in other
studies conducted by this laboratory. The rabbits used
were from ethically approved studies that did not
involve the hind or forelimbs, and where movement
was not impaired. Following dissection, the bones
were cleaned of residual soft tissue, wrapped in
phosphate buffered saline (PBS) soaked gauze and
stored at -20 �C until treatment.
Treatment
From the 120 pairs, groups of thirty pairs (n = 30
pairs per treatment group) were randomly assigned to
gamma irradiation at 10 or 25 kGy, or supercritical
fluid treatment with or without additive. The left
humeri in each pair acted as a control, while the rights
were treated.
Gamma irradiation
The specimens were placed on dry ice and sealed in a
Styrofoam box to maintain temperatures during treat-
ment between -20 and -50 �C. Irradiation at low
temperatures has been shown to minimize collagen
damage (Hamer et al. 1999) and reduces the genera-
tion and diffusion of free radicals (Grieb et al. 2005).
The bones were irradiated at doses of 10 and 25 kGy
using a cobalt60 irradiation source under well defined
operating procedures (Steritech, Wetherill Park, Aus-
tralia). After treatment the specimens were thawed out
at room temperature ready for mechanical testing.
Supercritical fluid
SCCO2 experiments were performed with an in-house
supercritical fluid rig (Fig. 1). For both SCCO2
experiments, the humeri were thawed out at room
temperature prior to testing. They were then loaded
into the pressure vessel with sterilant (SCF Additive)
or without (SCF Control) and thermally equilibrated at
an operating temperature of 37 �C. In the case of the
SCF Additive group, 1.04 mL of sterilant containing
active bactericidal/sporicidal products peracetic acid
(14.1 %) and hydrogen peroxide (4.9 %) was pipetted
onto a cellulose pad that was loaded into the bottom of
the pressure vessel. The carbon dioxide gas was then
pressurized incrementally past its critical point
(Pc = 73.8 bar) and into the supercritical phase region
using a high pressure pump (TharSFC, MA, USA),
where it was statically isolated at 100 bar for an hour.
The system was depressurized (100 bar-0) linearly
over 45 min by releasing a valve. The bones were then
extracted and stored in PBS before mechanical testing.
These experimental parameters were adapted from a
sterilization technique used by NovaSterilis (NovaS-
terilis, Lansing, NY, USA), which employed the
overkill methodology to ensure SAL6 compliance.
The treatments employed in this study were purely a
means to evaluate the mechanical effect of SCCO2
when utilized as a terminal sterilization methodology,
and treated bone samples were not completely
defatted.
Mechanical testing
From the thirty pairs of humeri in each treatment
group, 10 were randomly selected and placed into
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3-point bending, 4-point bending and torsion testing
groups (n = 10 pairs per group). All mechanical tests
were performed using an MTS 858 Bionix servo
hydraulic testing machine (MTS, Eden Prairie, MN,
USA). The parameters measured in this study
included:
Maximum load: The maximal force (N)
at which catastrophic failure of the specimen
occurred.
Energy to failure: The total amount of energy
absorbed (J) by the specimen to catastrophic failure,
calculated as the area under the load–displacement
curve to maximum load.
Stiffness: The specimen’s resistance to deformation
(N.mm) under the applied loading conditions,
calculated as the gradient of the load–displacement
curve in the linear elastic region.
Three-point bending
Specimens were placed on loading platens with a
2 mm radius of curvature and a 60 mm gauge span.
All humeri (left and right) were tested in unconfined
bending, with the anatomical orientation kept consis-
tent with the deltoid tuberosity facing downwards
(Fig. 2). Bones were failed at 5 mm/min in displace-
ment control. From the load–displacement output,
maximum load, energy to failure and stiffness were
calculated for each test using a custom-made Matlab
script (Matlab 7.11.0, The Mathworks, Inc.).
Fig. 1 Schematic showing
the supercritical fluid rig set
up used for treatment in this
study
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Four-point bending
A 60 mm gauge span was used for four-point bending,
with 20 mm spacing between each loading platen. The
humeri were positioned with the deltoid tuberosity
facing downwards and were unconstrained. This
orientation provided a flat surface where all points
were loaded simultaneously, and thus no shear stress
was produced in the central third (Fig. 3). Samples
were tested at 5 mm/min in displacement control, with
maximum load, energy to failure and stiffness calcu-
lated from the output using a custom-made Matlab
script (Matlab 7.11.0, The Mathworks, Inc.).
Torsion
The specimens were mounted in pots using a custom
jig that ensured vertical alignment and a consistent
gauge length of 50 mm. Low melting point alloy was
used to fix the specimens in place to avoid slippage.
Testing was performed in load and displacement
control to ensure zero axial loading during rotation.
The bone first underwent 20 preconditioning cycle
(±2 �) at 0.35 Hz, before being internally rotated at
1.5 deg/s to failure. A custom Matlab script (Matlab
7.11.0, The Mathworks, Inc.) was again used to
calculate maximum torque, energy to failure and
stiffness from the angle-torque output.
Statistical analysis
Statistical differences within the treatment groups
were determined using PASW Statistics (18.0.3, SPSS
Inc) for windows. The Shapiro–Wilk test was used to
confirm normality of the results, then paired two-tailed
t tests were carried out to determine the differences
and levels of significance for each of the measured
parameters (maximum load, stiffness, and energy to
failure) between the anatomically paired humeri. A
one-way ANOVA followed by a Games Howell Post
Hoc Test was used to assess differences between
groups for each testing modality.
Results
Tables 1, 2, 3 contain the results for maximum load,
energy to failure and stiffness for all loading
(A)
(B)
Fig. 2 Schematic showing the three-point bending test set up;
and representative shear force (a) and bending moment
diagrams (b)
(A)
(B)
Fig. 3 Schematic showing the four-point bending test set up;
and representative shear force (a) and bending moment
diagrams (b)
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modalities. Figures 4, 5, 6 graphical display the results
with the treated samples expressed as a percentage of
the untreated controls.
Three point bending
There was a dose dependant decrease in both maxi-
mum load and energy to failure observed in the
Gamma irradiated treatment groups (Fig. 4). At a dose
of 10 kGy, there was a 12 and 26 % decrease in
maximum load and energy to failure respectively,
though these were not statistically significant. At the
higher dose of 25 kGy, this effect was more pro-
nounced with statistically significant reductions of
18 % (P = 0.02) and 44 % (P = 0.005) in maximum
load and energy to failure respectively. Interestingly,
stiffness increased by 10 % (P = 0.04) in the 25 kGy
treatment group (Table 3).
Supercritical fluid treatment with or without addi-
tive had no significant effect on any of the measure
mechanical parameters in 3-point bending.
The one-way ANOVA for stiffness showed there
was no significant difference in any treatment group
compared with the control. However, post hoc analysis
revealed a statistically significant decrease in stiffness
in the 10 kGy gamma irradiation group compared with
a 25 kGy dose (P = 0.004) and the SCF Additive
treatment (P = 0.023). SCF treatment alone did not
significantly alter stiffness compared to any of the
other treatment groups.
Four point bending
Consistent with the 3-point bending results, there were
dose dependant reductions in both maximum load and
energy to failure in 4-point bending (Fig. 5). Maxi-
mum load decreased by 7 % (P = 0.06) and 31 %
(P = 0.000); and energy to failure by 39 % (P = 0.06)
and 57 % (P = 0.000) at treatment doses of 10 and
25 kGy respectively. There was no considerable
change in stiffness at either treatment dose (Table 3).
There was no significant effect seen in either
supercritical fluid treatment group for on any of the
measure mechanical parameters in 4-point bending.
ANOVA results for stiffness found no significant
effect of treatment compared to the control samples.
Furthermore, both gamma irradiation doses and SCF
treatments were statistically similar.
Torsion
Gamma irradiation had the most deleterious effect on
the mechanical properties of the bone in torsion
(Fig. 6). At the low dose of 10 kGy, there was 30 %
(P = 0.047), 38 and 26 % (P = 0.007) reductions in
maximum torque, energy to failure and stiffness
respectively. The higher 25 kGy dose exacerbated
these results with decreases of 64 % (P = 0.02), 75 %
(P = 0.02) and 45 % (P = 0.06) in the corresponding
parameters.
The supercritical fluid treatment alone (SCF Con-
trol) had no significant effect on any of the measured
torsional mechanical properties. The addition of
sterilant (SCF Additive) resulted in a small 8 %
increase in maximum torque, though this was not
statistically significant (P = 0.08). Energy to failure
and stiffness were preserved compared to the controls.
Consistent with 4-point bending, stiffness was not
significantly altered in torsion compared to control
sample or between either SCF or gamma treatments.
Discussion
The development of optimal processing and steriliza-
tion techniques for bone allograft has been the focus of
Table 1 Maximum load and torque results for the four treatment groups
Group Three-point bending Four-point bending Torsion
Control Treated Control Treated Control Treated
SCF Control 413.7 (51.7) 395.8 (68.8) 349.5 (52.3) 352.1 (57.1) 2,152.8 (485.9) 1,874.5 (732.7)
SCF Additive 418.1 (38.8) 405.4 (48.0) 422.5 (59.0) 401.2 (74.0) 2,087.7 (267.9) 2,257.9 (310.2)
Gamma 15 kGy 283.2 (67.3) 248.9 (42.5) 387.6 (75.1) 361.8 (75.5) 1,805.0 (900.4) 1,263.9 (730.9)*
Gamma 25 kGy 391.8 (81.6) 322.3 (23.8)* 419.9 (50.5) 292.0 (35.5)* 2,101.6 (248.0) 757.5 (563.1)*
* Denotes significance (P \ 0.05) compared with the control, values are mean (standard deviation)
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Table 2 Energy to failure results for the four treatment groups
Energy to failure
Group Three-point bending (J) Four-point bending (J) Torsion (J)
Control Treated Control Treated Control Treated
SCF Control 340.43 (39.4) 315.4 (54.4) 304.2 (97.6) 309.0 (112.8) 13,314.8 (4,839.1) 11,058.4 (5,226.9)
SCF Additive 276.5 (59.5) 273.1 (65.1) 384.7 (93.0) 410.1 (145.6) 14,504.1 (3,060.5) 14,934.7 (4,076.8)
Gamma 10 kGy 191.0 (87.5) 140.8 (44.7) 441.7 (272.4) 271.0 (80.1) 10,802.9 (8,126.7) 6,637.2 (5,666.6)
Gamma 25 kGy 270.4 (75.1) 150.4 (19.7)* 388.8 (64.2) 166.5 (42.5)* 13,772.8 (1,444.4) 3,370.5 (3,214.0)*
* Denotes significance (P \ 0.05) compared with the control, values are mean (standard deviation)
Table 3 Stiffness results for the four treatment groups
Stiffness
Group Three-point bending (N.mm) Four-point bending (N.mm) Torsion (N.mm/deg)
Control Treated Control Treated Control Treated
SCF control 430.3 (60.4) 452.7 (79.1) 334.5 (47.0) 347.3 (43.5) 205.2 (38.4) 173.9 (43.7)
SCF additive 473.8 (67.4) 476.3 (69.9) 354.5 (52.9) 339.3 (53.6) 178.7 (31.7) 172.3 (34.1)
Gamma 10 kGy 391.4 (65.8) 379.2 (56.4) 307.4 (68.1) 326.0 (87.7) 178.2 (48.8) 130.48 (47.6)*
Gamma 25 kGy 454.2 (72.7) 502.1 (54.4)* 345.8 (78.2) 330.4 (53.1) 172.0 (26.5) 94.7 (42.7)*
* Denotes significance (P \ 0.05) compared with the control, values are mean (standard deviation)
Fig. 4 Three-point bending results for maximum load and
energy to failure for each treatment group. Treated samples
(black) shown as a percentage of the untreated controls (white).
* Denotes significance (P \ 0.05) compared to controls
Fig. 5 Four-point bending results for maximum load and
energy to failure for each treatment group. Treated samples
(black) shown as a percentage of the untreated controls (white).
* Denotes statistical significance (P \ 0.05) compared to
controls; ^ denotes an almost statistical significance (P = 0.06)
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extensive investigation due to the limitations posed by
autograft and the inability of synthetic bone graft
materials to replicate the complex architecture and
hierarchical arrangement of natural bone. Supercriti-
cal carbon dioxide treatment has significant expedi-
ency for bone allograft processing and sterilization
due to its unique extraction properties and efficacy in
killing bacteria and viruses (Fages et al. 1994, 1998;
Qiu et al. 2009). However, little research has been
conducted examining the effect of these treatments on
the mechanical properties of bone allograft. Further-
more, current accepted practice utilises gamma irra-
diation at a range of doses that have been shown to be
detrimental to the mechanical integrity of the graft,
raising concerns over there in vivo performance. This
study aimed to evaluate the effect of SCCO2 treatment
compared with two doses of dose gamma irradiation
on the mechanical properties of paired whole bones
under different loading situations; and in doing so
investigate the expediency of these sterilization
methods for the processing of load bearing allograft
bone.
The loading modalities utilized in this study were
chosen to represent physiological loading conditions,
and as a means to isolate the effect of treatment on
different bone constituents. Three-point bending
compromises both shear and bending; while four-
point bending is pure bending with no shear, and
finally torsion measures only shear properties. In
addition, this study tested between anatomically
paired bones, eliminating variation between samples
and increasing the validity of the findings.
The results of this study showed that SCCO2
treatment alone had no significant effect on any of the
measured parameters, demonstrating the resistance of
bone to SCF conditions. It has previously been shown
that the rapid expansion of the CO2 during depressur-
ization can induce cracking and nucleation in a range
of polymers (Barry et al. 2006; Davies et al. 2008), and
this effect is accentuated when depressurization
occurs from high pressures. It may be that the forces
induced by the depressurization are in the elastic
region and are unable to overcome the mineral-organic
bonding in the bone; thus when the treatment is
completed there is no permanent damage. This would
also explain the retention of the bones post-yield
properties as seen by minimal change in energy to
failure after treatment.
The results of the SCF Additive group are consis-
tent with those of Nichols et al. (2009) under similar
treatment conditions, with the three-point bending
properties of the bone preserved. Additionally, this
study found that the four-point bending properties of
the bone were also maintained. Interestingly, the
maximum torque and energy to failure in torsion
increased by 8 % (P = 0.07) and 3 % respectively
compared with controls. This would suggest the
addition of the hydrogen peroxide solution may have
had a toughening or drying effect, as SCF treatment
alone decreased maximum torque and energy to
failure by 13 and 17 %. Studies have shown that
aqueous hydrogen peroxide does not affect collagen
structure or content in bone (Freeman and Silva 2002).
Therefore given the increases seen, this would suggest
hydrogen peroxide treatment may influence the bond-
ing between the mineral and organic phases. This
interfacial bonding is responsible for the bridging and
reinforcement function during loading in the plastic
region, increasing the resistance to crack propagation
(Akkus and Belaney 2005). However, further inves-
tigation is required before such a conclusion can be
made accurately.
The findings of this study are in agreement with
those reported in previous literature regarding the dose
dependant decrease in mechanical properties of bone
Fig. 6 Torsion results for maximum torque and energy to
failure for each treatment group. Treated samples (black) shown
as a percentage of the untreated controls (white). * Denotes
significance (P \ 0.05) compared to controls
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following gamma irradiation (Anderson et al. 1992;
Currey et al. 1997; Fideler et al. 1995; Gibbons et al.
1991; Hamer et al. 1999; Salehpour et al. 1995). There
was a significant deterioration in both maximum load
and energy to failure from a dose of 10–25 kGy in all
loading modalities.
At a moderate dose of 25 kGy, the most significant
reductions were seen in torsion where there was a
64 % decrease in maximum torque. This decrease was
consistent with recent results published by Zhou et al.
(2011). At the same dose they reported a statistically
significant 55 % decrease in torsional shear stress in
cortical struts. Such drastic reduction should be
expected in torsion as the preferential alignment of
bone mineral is perpendicular to the applied torque.
Consequently, it is mineral-collagen bonding and the
collagen matrix itself, both which are damaged during
gamma irradiation, that principally resists the applied
forces. Ionizing radiation damages collagen directly
through the splitting of polypeptide bonds by chain
scission (Dziedzic-Goclawska et al. 2005). Indirectly,
it causes radiolysis of water that leads to the release of
collagen-targeting free radicals that induce intermo-
lecular cross-linking and consequent changes in the
structural properties of the collagen (Akkus et al.
2005). Furthermore, de-carboxylation of collagen side
chains has been observed in irradiated bone, resulting
in a reduction of mineral-collagen bonding (Hubner
et al. 2005). These structural and biochemical changes
to bone collagen impair its bridging and reinforcement
function during loading, decreasing resistance to crack
propagation, and thus maximum load (Akkus and
Belaney 2005).
In three-point bending, this study reported statisti-
cally significant reductions of 18 and 44 % in max-
imum load and energy to failure respectively at a
treatment dose of 25 kGy. Compared with similar
studies these values are somewhat less substantial
(Currey et al. 1997; Hamer et al. 1996; Zhou et al.
2011). At a dose of 29.5 kGy Currey et al. (1997)
reported a 21.5 and 69 % fall in maximum load and
energy to failure. Correspondingly, Hamer et al.
(1996) recorded 36 and 66 % reductions respectively.
However, these variations could be explained by a
smaller sample variation due to the use of anatomi-
cally paired bones in this study; or the synergistic
effect of processing with ethanol and hydrogen
peroxide. Additionally, the gamma irradiation in these
studies was performed at room temperature, compared
with -50 �C in the present study. It has been shown
that irradiation at lower temperatures provides partial
protection against embrittlement by inhibiting the
movement of water and therefore the formation of free
radicals that can destroy collagen alpha chains (Cornu
et al. 2000, 2011; Hamer et al. 1999).
The effects of gamma irradiation on the mechanical
properties of bone in four-point bending have not been
extensively studied. In symmetric four-point bending,
loading results in uniform reaction forces at each
loading platen. This produces an area of zero shear
force in the central region (Fig. 3), resulting in failure
by pure bending when tested. In the present study, the
results for four-point bending were consistent with
those seen in three-point bending with statistically
significant reductions in both maximum load and
energy to failure under standard dose conditions.
In an effort to preserve the intrinsic mechanical and
biological properties of allograft bone studies are
investigating the use of a lower standard dose of
gamma irradiation (Balsly et al. 2008; Campbell et al.
1994; Currey et al. 1997; Jinno et al. 2000). There are
however, concerns over the effectiveness of low dose
gamma irradiation at reducing the bioburden and
inactivating viruses to the level required of SAL6.
This has prompted the use of other processing and
cleansing steps prior to final gamma treatment.
Nevertheless, the issue still remains whether a lower
dose can maintain the allograft biological and
mechanical capabilities. In one study, Jinno et al.
(2000) found a dose of 15 kGy did not significantly
affect the revascularization or graft incorporation of
gamma treated allograft in a rat segmental femoral
defect model in rats. Mechanically, Hamer et al.
(1996) and Simonian et al. (1994) showed that does of
9.5 and 15.7 kGy respectively, did not significantly
alter bone compared with untreated controls. These
results are difficult to compare due to the different
testing methodologies and treatment doses. The use of
10 kGy in the present study preserved the mechanical
properties of the bone in both three and four-point
bending. However, in torsion there was a statistically
significant 30 % decrease in maximum toque, and a
decline in energy to failure of 38 % that approached
significance. This would suggest that even with a
lower final treatment dose, gamma irradiation com-
promises the mechanical integrity of the bone.
Whether this translates to poorer clinical outcomes is
a topic for further research.
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There are several limitations that need to be
considered when interpreting these results. First of
all, quasi static mechanical testing is not truly
representative of physiological loading. The loads
typically experienced by bone are cyclic in nature, and
therefore fatigue loading may have more accurately
represented the physiological loading experienced by
load bearing allograft. These modes of testing are
however, pertinent at quantifying the bulk properties
of the bone that may have been altered during
treatment. Moreover, testing on whole bones provides
a representative model of clinical scenarios. For
example, torsional testing of whole humeri is analo-
gous to spiral fractures experienced in vivo, where
strut allografts are often used for repair and support.
Secondly, as bone is a viscoelastic material the
elastic properties are rate dependant. At lower strain
rates the bone accumulates more damage as stress
increases, resulting in a lower recorded yield stress.
The use of a range of strain rates may more completely
answer questions over the efficacy of the tested
treatments for load bearing allograft. However, the
paired nature of the samples, and consistency of
treatment and testing mean conclusions drawn are still
valid for these conditions.
Finally, the present study only addressed the effects
of SCF and gamma irradiation treatments alone, and
not in conjunction with common allograft processing
procedures. A number of studies have tested SCF and
gamma irradiation coupled with novel and standard
processing procedures (Balsly et al. 2008; Currey et al.
1997; Fages et al. 1998; Jinno et al. 2000; Mikhael
et al. 2008; Mitton et al. 2005; Schwiedrzik et al. 2011;
Vastel et al. 2004), with varying results. However, it is
difficult to draw conclusions and make comparisons
between such studies due the variation in treatment
methodology and inadequately reported conditions.
Fages et al. (1994) and Mitton et al. (2005) showed
that SCCO2 treatment followed by hydrogen peroxide
soaking preserved the compressive mechanical prop-
erties of cancellous bone. Though the pressure and
temperature used in the SCF treatment in each study
was different. Similarly, Vastel et al. (2004) and
Balsly et al. (2008) found that there was also no
significant effect on the compressive properties of
cancellous bone following defatting and gamma
irradiation at doses ranging between 20 and 30 kGy.
Conversely, Schwiedrzik et al. (2011) found that SCF
defatting followed by sterilization with gamma
irradiation at 31 kGy significantly reduced the com-
pressive strength properties and ability to absorb
energy. However, they found these reductions strongly
correlated to a reduced BV/TV which is major
determinant of the mechanical properties of cancel-
lous bone.
Comparisons with these studies are difficult as
they did not test cortical bone as in the present study.
In the most relevant study, Mikhael et al. (2008)
examined the effects of gamma irradiation in
conjunction with a novel chemical sterilization
technique on the mechanical properties of cadaveric
cortical allograft under a range of loading profiles.
Their study reported statistically significant reduc-
tions in ultimate shear stress, which is agreement
with the present study. Conversely, they found no
significant decrease in bending stress. The likely
source of this discrepancy is the chemical steriliza-
tion step prior to irradiation, or the slightly lower
dose used. We reported a non significant 12 %
decrease in maximum bending at 10 kGy, and
significant 18 % decrease at 25 kGy. Mikhael et al.
(2008) reported a 13.5 % decrease in bending stress
at a dose of 20–23 kGy, suggesting that under similar
conditions the results would be consistent.
The results of this study suggest that SCCO2
treatment has considerable expediency for processing
of load bearing allograft bone. Given the previously
reported extraction capabilities of SCCO2 treatment
(Fages et al. 1994, 1998) and ability to terminally
sterilize a range of bacteria and viruses (Hemmer
2007; Qiu et al. 2009; Shieh et al. 2009; Zhang et al.
2006), coupled with the preservation of mechanical
properties seen in this study, further in vivo investi-
gation should be pursued. This study also reaffirmed
the deleterious effect of gamma irradiation at a dose of
25 kGy on the mechanical properties of bone, with
significant reductions in all measured parameters.
Furthermore, whilst a dose of 10 kGy preserved the
bending properties of the bone, there were significant
reductions in torsion strength. This raises questions
over the utility of even low dose gamma irradiation for
allograft processing from a mechanical perspective.
However, whether in vitro mechanical results translate
negatively in a clinical setting is a topic for further
investigation.
Conflict of interest The authors declare that there is no
conflict of interest in the preparation of this manuscript.
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123
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