paper

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

References

Akkus O, Belaney RM (2005) Sterilization by gamma radiation

impairs the tensile fatigue life of cortical bone by two

orders of magnitude. J Orthop Res 23(5):1054–1058. doi:

10.1016/j.orthres.2005.03.003

Akkus O, Rimnac CM (2001) Fracture resistance of gamma

radiation sterilized cortical bone allografts. J Orthop Res

19(5):927–934. doi:10.1016/S0736-0266(01)00004-3

Akkus O, Belaney RM, Das P (2005) Free radical scavenging

alleviates the biomechanical impairment of gamma radia-

tion sterilized bone tissue. J Orthop Res 23(4):838–845.

doi:10.1016/j.orthres.2005.01.007

Anderson MJ, Keyak JH, Skinner HB (1992) Compressive

mechanical properties of human cancellous bone after

gamma irradiation. J Bone Joint Surg Am 74(5):747–752

Balsly C, Cotter A, Williams L, Gaskins B, Moore M, Wolfin-

barger L (2008) Effect of low dose and moderate dose

gamma irradiation on the mechanical properties of bone

and soft tissue allografts. Cell Tissue Bank 9(4):289–298

Barry JJ, Silva MM, Popov VK, Shakesheff KM, Howdle SM

(2006) Supercritical carbon dioxide: putting the fizz into

biomaterials. Philos Transact A Math Phys Eng Sci

364(1838):249–261. doi:10.1098/rsta.2005.1687

Bertoloni G, Bertucco A, De Cian V, Parton T (2006) A study on

the inactivation of micro-organisms and enzymes by high

pressure CO2. Biotechnol Bioeng 95(1):155–160. doi:

10.1002/bit.21006

Burstein AH, Zika JM, Heiple KG, Klein L (1975) Contribution

of collagen and mineral to the elastic-plastic properties of

bone. J Bone Joint Surg Am 57(7):956–961

Campbell DG, Li P, Stephenson AJ, Oakeshott RD (1994)

Sterilization of HIV by gamma irradiation. A bone allo-

graft model. Int Orthop 18(3):172–176

Cornu O, Banse X, Docquier PL, Luyckx S, Delloye C (2000)

Effect of freeze-drying and gamma irradiation on the

mechanical properties of human cancellous bone. J Ortho-

paed Res 18(3):426–431. doi:10.1002/jor.1100180314

Cornu O, Boquet J, Nonclercq O, Docquier PL, Van Tomme J,

Delloye C, Banse X (2011) Synergetic effect of freeze-

drying and gamma irradiation on the mechanical properties

of human cancellous bone. Cell Tissue Bank 12(4):

281–288. doi:10.1007/s10561-010-9209-1

Currey JD, Foreman J, Laketic I, Mitchell J, Pegg DE, Reilly GC

(1997) Effects of ionizing radiation on the mechanical

properties of human bone. J Orthopaed Res 15(1):111–117.

doi:10.1002/jor.1100150116

Davies OR, Lewis AL, Whitaker MJ, Tai H, Shakesheff KM,

Howdle SM (2008) Applications of supercritical CO2 in

the fabrication of polymer systems for drug delivery and

tissue engineering. Adv Drug Deliv Rev 60(3):373–387.

doi:10.1016/j.addr.2006.12.001

Davy DT (1999) Biomechanical issues in bone transplantation.

Orthop Clin North Am 30(4):553–563

DePaula CA, Truncale KG, Gertzman AA, Sunwoo MH, Dunn

MG (2005) Effects of hydrogen peroxide cleaning proce-

dures on bone graft osteoinductivity and mechanical

properties. Cell Tissue Bank 6(4):287–298. doi:10.1007/

s10561-005-3148-2

Dillow AK, Dehghani F, Hrkach JS, Foster NR, Langer R (1999)

Bacterial inactivation by using near- and supercritical

carbon dioxide. Proc Natl Acad Sci USA 96(18):10344–

10348

Dziedzic-Goclawska A, Ostrowski K, Stachowicz W, Michalik

J, Grzesik W (1991) Effect of radiation sterilization on the

osteoinductive properties and the rate of remodeling of

bone implants preserved by lyophilization and deep-

freezing. Clin Orthop Relat Res 272:30–37

Dziedzic-Goclawska A, Kaminski A, Uhrynowska-Tyszkiewicz

I, Stachowicz W (2005) Irradiation as a safety procedure in

tissue banking. Cell Tissue Bank 6(3):201–219. doi:

10.1007/s10561-005-0338-x

Fages J, Marty A, Delga C, Condoret JS, Combes D, Frayssinet

P (1994) Use of supercritical CO2 for bone delipidation.

Biomaterials 15(9):650–656

Fages J, Poirier B, Barbier Y, Frayssinet P, Joffret ML, Ma-

jewski W, Bonel G, Larzul D (1998) Viral inactivation of

human bone tissue using supercritical fluid extraction.

ASAIO J 44(4):289–293

Fideler BM, Vangsness CT Jr, Lu B, Orlando C, Moore T (1995)

Gamma irradiation: effects on biomechanical properties of

human bone-patellar tendon-bone allografts. Am J Sports

Med 23(5):643–646

Freeman JJ, Silva MJ (2002) Separation of the Raman spectral

signatures of bioapatite and collagen in compact mouse

bone bleached with hydrogen peroxide. Appl Spectrosc

56(6):770–775

Gerd B (2005) Supercritical fluids: technology and application

to food processing. J Food Eng 67(1–2):21–33. doi:

10.1016/j.jfoodeng.2004.05.060

Gibbons MJ, Butler DL, Grood ES, Bylski-Austrow DI, Levy

MS, Noyes FR (1991) Effects of gamma irradiation on the

initial mechanical and material properties of goat bone-

patellar tendon-bone allografts. J Orthop Res 9(2):209–

218. doi:10.1002/jor.1100090209

Godette GA, Kopta JA, Egle DM (1996) Biomechanical effects

of gamma irradiation on fresh frozen allografts in vivo.

Orthopedics 19(8):649–653

Grieb TA, Forng R-Y, Stafford RE, Lin J, Almeida J, Bogdan-

sky S, Ronholdt C, Drohan WN, Burgess WH (2005)

Effective use of optimized, high-dose (50 kGy) gamma

irradiation for pathogen inactivation of human bone allo-

grafts. Biomaterials 26(14):2033–2042

Hamer AJ, Strachan JR, Black MM, Ibbotson CJ, Stockley I,

Elson RA (1996) Biochemical properties of cortical allo-

graft bone using a new method of bone strength measure-

ment. A comparison of fresh, fresh-frozen and irradiated

bone. J Bone Joint Surg Am 78(3):363–368

Hamer AJ, Stockley I, Elson RA (1999) Changes in allograft

bone irradiated at different temperatures. J Bone Joint Surg

Am 81(2):342–344

Hemmer J (2007) Sterilization of bacterial spores by using

supercritical carbon dioxide and hydrogen peroxide.

J Biomed Mater Res A 80B:511–518

Hubner W, Blume A, Pushnjakova R, Dekhtyar Y, Hein HJ

(2005) The influence of X-ray radiation on the mineral/

organic matrix interaction of bone tissue: an FT-IR

microscopic investigation. Int J Artif Organs 28(1):66–73

Cell Tissue Bank

123

Ijiri S, Yamamuro T, Nakamura T, Kotani S, Notoya K (1994)

Effect of sterilization on bone morphogenetic protein.

J Orthop Res 12(5):628–636. doi:10.1002/jor.1100120505

Jinno T, Miric A, Feighan J, Kirk SK, Davy DT, Stevenson S

(2000) The effects of processing and low dose irradiation

on cortical bone grafts. Clin Orthop Relat Res 375:275–285

Lewandrowski KU, Gresser JD, Bondre S, Silva AE, Wise DL,

Trantolo DJ (2000) Developing porosity of poly(propylene

glycol-co-fumaric acid) bone graft substitutes and the

effect on osteointegration: a preliminary histology study in

rats. J Biomater Sci Polym Ed 11(8):879–889

Mikhael MM, Huddleston PM, Zobitz ME, Chen Q, Zhao KD,

An K-N (2008) Mechanical strength of bone allografts

subjected to chemical sterilization and other terminal

processing methods. J Biomech 41(13):2816–2820. doi:

10.1016/j.jbiomech.2008.07.012

Mitchell EJ, Stawarz AM, Kayacan R, Rimnac CM (2004) The

effect of gamma radiation sterilization on the fatigue crack

propagation resistance of human cortical bone. J Bone Joint

Surg Am 86-A(12):2648–2657

Mitton D, Rappeneau J, Bardonnet R (2005) Effect of a super-

critical CO2 based treatment on mechanical properties of

human cancellous bone. Eur J Orthop Surg Traumatol

15(4):264–269. doi:10.1007/s00590-005-0250-x

Nguyen H, Morgan DA, Forwood MR (2007a) Sterilization of

allograft bone: effects of gamma irradiation on allograft

biology and biomechanics. Cell Tissue Bank 8(2):93–105.

doi:10.1007/s10561-006-9020-1

Nguyen H, Morgan DA, Forwood MR (2007b) Sterilization of

allograft bone: is 25 kGy the gold standard for gamma

irradiation? Cell Tissue Bank 8(2):81–91. doi:10.1007/

s10561-006-9019-7

Nichols A, Burns D, Christopher R (2009) The sterilization of

human bone and tendon musculoskeletal allograft tissue

using supercritical carbon dioxide. J Orthopaed 6(2):9–17

Qiu Q-Q, Leamy P, Brittingham J, Pomerleau J, Kabaria N,

Connor J (2009) Inactivation of bacterial spores and viru-

ses in biological material using supercritical carbon diox-

ide with sterilant. J Biomed Mater Res B 91B(2):572–578.

doi:10.1002/jbm.b.31431

Salehpour A, Butler DL, Proch FS, Schwartz HE, Feder SM,

Doxey CM, Ratcliffe A (1995) Dose-dependent response

of gamma irradiation on mechanical properties and related

biochemical composition of goat bone-patellar tendon-

bone allografts. J Orthop Res 13(6):898–906. doi:

10.1002/jor.1100130614

Schwiedrzik JJ, Kaudela KH, Burner U, Zysset PK (2011)

Fabric-mechanical property relationships of trabecular

bone allografts are altered by supercritical CO2 treatment

and gamma sterilization. Bone 48(6):1370–1377. doi:

10.1016/j.bone.2011.03.768

Shieh E, Paszczynski A, Wai CM, Lang Q, Crawford RL (2009)

Sterilization of Bacillus pumilus spores using supercritical

fluid carbon dioxide containing various modifier solutions.

J Microbiol Meth 76(3):247–252

Simonian PT, Conrad EU, Chapman JR, Harrington RM,

Chansky HA (1994) Effect of sterilization and storage

treatments on screw pullout strength in human allograft

bone. Clin Orthop Relat Res 302:290–296

Spilimbergo S, Bertucco A (2003) Non-thermal bacterial inac-

tivation with dense CO(2). Biotechnol Bioeng 84(6):627–

638. doi:10.1002/bit.10783

Spilimbergo S, Dehghani F, Bertucco A, Foster NR (2003)

Inactivation of bacteria and spores by pulse electric field

and high pressure CO2 at low temperature. Biotechnol

Bioeng 82(1):118–125. doi:10.1002/bit.10554

Thoren K, Aspenberg P (1995) Ethylene oxide sterilization

impairs allograft incorporation in a conduction chamber.

Clin Orthop Relat Res 318:259–264

Triantafyllou N, Sotiropoulos E, Triantafyllou JN (1975) The

mechanical properties of the lyophylized and irradiated

bone grafts. Acta Orthop Belg 41(suppl 1):35–44

Vashishth D, Gibson GJ, Khoury JI, Schaffler MB, Kimura J,

Fyhrie DP (2001) Influence of nonenzymatic glycation on

biomechanical properties of cortical bone. Bone 28(2):

195–201. doi:10.1016/S8756-3282(00)00434-8

Vastel L, Meunier A, Siney H, Sedel L, Courpied JP (2004)

Effect of different sterilization processing methods on the

mechanical properties of human cancellous bone allo-

grafts. Biomaterials 25(11):2105–2110. doi:10.1016/j.bio

materials.2003.08.067

Voggenreiter G, Ascherl R, Blumel G, Schmit-Neuerburg KP

(1996) Extracorporeal irradiation and incorporation of

bone grafts. Autogeneic cortical grafts studied in rats. Acta

Orthop Scand 67(6):583–588

Watanabe T, Furukawa S, Hirata J, Koyama T, Ogihara H,

Yamasaki M (2003) Inactivation of Geobacillus stearo-

thermophilus spores by high-pressure carbon dioxide

treatment. Appl Environ Microbiol 69(12):7124–7129

White A, Burns D, Christensen TW (2006) Effective terminal

sterilization using supercritical carbon dioxide. J Biotech-

nol 123(4):504–515

Zhang J, Burrows S, Gleason C, Matthews MA, Drews MJ, La-

berge M, An YH (2006) Sterilizing Bacillus pumilus spores

using supercritical carbon dioxide. J Microbiol Methods

66(3):479–485. doi:10.1016/j.mimet.2006.01.012

Zhou Z, Qin T, Yang J, Shen B, Kang P, Peil F (2011)

Mechanical strength of cortical allografts with gamma

radiation versus ethylene oxide sterilization. Acta Orthop

Belg 77(5):670–675

Zioupos P, Currey JD, Hamer AJ (1999) The role of collagen in

the declining mechanical properties of aging human cor-

tical bone. J Biomed Mater Res 45(2):108–116. doi:

10.1002/(SICI)1097-4636(199905)45:2\108:AID-JBM5[3.0.CO;2-A

Cell Tissue Bank

123