Abstract Gamma irradiation from Cobalt 60

sources has been used to terminally sterilize bone

allografts for many years. Gamma radiation

adversely affects the mechanical and biological

properties of bone allografts by degrading the

collagen in bone matrix. Specifically, gamma rays

split polypeptide chains. In wet specimens irra-

diation causes release of free radicals via radiol-

ysis of water molecules that induces cross-linking

reactions in collagen molecules. These effects are

dose dependent and give rise to a dose-dependent

decrease in mechanical properties of allograft

bone when gamma dose is increased above

25 kGy for cortical bone or 60 kGy for cancellous

bone. But at doses between 0 and 25 kGy (stan-

dard dose), a clear relationship between gamma

dose and mechanical properties has yet to be

established. In addition, the effects of gamma

radiation on graft remodelling have not been

intensively investigated. There is evidence that

the activity of osteoclasts is reduced when they

are cultured onto irradiated bone slices, that

peroxidation of marrow fat increases apoptosis of

osteoblasts; and that bacterial products remain

after irradiation and induce inflammatory bone

resorption following macrophage activation.

These effects need considerably more investiga-

tion to establish their relevance to clinical out-

comes. International consensus on an optimum

dose of radiation has not been achieved due to a

wide range of confounding variables and indi-

vidual decisions by tissue banks. This has resulted

in the application of doses ranging from 15 to

35 kGy. Here, we provide a critical review on the

effects of gamma irradiation on the mechanical

and biological properties of allograft bone.

Keywords Allograft bone Æ Bone allograft

healing/remodelling Æ Allograft osteoinduction ÆDose-dependent effect Æ Gamma radiation ÆMechanical properties Æ Terminal sterilization

Introduction

Bone banks supply a wide range of tissue,

including massive bone allografts cortical bone

allografts, and milled bone. These products play

an important role in filling bone defects during

surgery aimed at improving the mobility of pa-

H. Nguyen Æ M. R. Forwood (&)Department of Anatomy and DevelopmentalBiology, The University of Queensland, School ofBiomedical Sciences, Brisbane, Qld 4072, Australiae-mail: m.forwood@uq.edu.au

D. A. F. MorganThe University of Queensland, Brisbane PrivateHospital, Brisbane, Australia 4000

H. Nguyen Æ D. A. F. MorganQueensland Bone Bank, Brisbane, Australia

Cell Tissue Banking (2007) 8:93–105

DOI 10.1007/s10561-006-9020-1

123

REVIEW PAPER

Sterilization of allograft bone: effects of gamma irradiationon allograft biology and biomechanics

Huynh Nguyen Æ David A. F. Morgan ÆMark R. Forwood

Received: 18 December 2005 / Accepted: 10 May 2006 / Published online: 25 October 2006� Springer Science+Business Media B.V. 2006

tients, and reducing the disability associated with

bone and joint diseases. However, the use of bone

allografts carries a risk of transferring bacteria,

viruses or prions from donor to recipient. To

eliminate potential infection, donor screening is

important along with aseptic surgical technique

during tissue retrieval, processing and storage

(AATB 2002; Andre and Liz 2000; Angermann

and Jepsen 1991; Boyce et al. 1999; Eastlund and

Strong 2003; IAEA 2002). Following tissue pro-

cessing, many banks consider it essential for

allografts to be terminally sterilized using gamma

irradiation from Cobalt 60 sources (Kennedy

et al. 2005). This issue has become controversial

in the bone bank community as the industry

debates whether it is necessary to use radiation to

sterilize bone. If so, the question is whether

25 kGy (standard dose) is justified as a gold

standard, or whether a lower dose for sterilization

can still minimize the risk of infection but reduce

the adverse effects of radiation? The aim of this

paper is to critically analyse the effects of gamma

radiation on mechanical and biological properties

of banked bones.

Effects of gamma radiation on mechanical

properties of bone allografts

When massive bone allografts are implanted, they

replace the functions of the removed bone. They

are often used to provide a scaffold to support

weak parts of the skeleton, to act as a bridge to

cover bone defects, and to provide a frame for

modeling new bone (Triantafyllou et al. 1975). It

usually takes several months, or even years, for

grafts to incorporate and be remodelled into new

bone. Therefore, mechanical properties of the

bone allografts must be as close as possible to the

original bone to reproduce its mechanical support

and structural integrity.

Multiple factors contribute to the biomechan-

ical performance of bone allografts (Davy 1999).

These may be the properties of the graft itself, the

interfaces between grafts and the host bone, and

the nature of the applied loads to the graft-host

bone complex. Unfortunately, in the process of

sterilization, the mechanical properties of bone

allografts can be impaired by gamma radiation

(Anderson et al. 1992; Cornu et al. 2000; Currey

et al. 1997; Gibbons et al. 1991; Hamer et al.

1996; Rasmussen et al. 1994; Vastel et al. 2004);

and these effects are dose-dependent (Fideler

et al. 1995; Hamer et al. 1996; Salehpour et al.

1995). The mechanism of mechanical impairment

has been explained, but the degree of change

varies among reports.

Mechanism of tissue damage by gamma

radiation

The tissue damage caused by gamma radiation

occurs through two mechanisms (Akkus et al.

2005; Colwell et al. 1996; Dziedzic-Goclawska

et al. 2005; Hamer et al. 1999). In the dry state,

splitting of polypeptide chains occurs under the

direct influence of gamma rays (Dziedzic-Go-

clawska et al. 2005). Indirectly, gamma rays cause

radiolysis of water molecules. These release free

radicals, which target the collagen rather than

minerals in the bone matrix (Akkus et al. 2005).

Consequently, a cross-linking reaction of bone

matrix collagens appears, and the biochemical

and structural properties of collagen fibres are

altered (Fig. 1).

The collagens of the bone matrix have their

most significant effect on the post-yield properties

of bone tissue (Akkus et al. 2005). Structural

changes to collagen in bone allografts under

gamma irradiation, therefore, result in significant

reductions in plastic properties such as ultimate

strength and toughness, rather than the elastic

properties (such as stiffness and yield strength)

(Akkus et al. 2005; Anderson et al. 1992; Currey

et al. 1997; Hamer et al. 1999; Hamer et al. 1996;

Triantafyllou et al. 1975).

During loading into the plastic region, intact

collagen fibres provide a bridging and reinforce-

ment function to the bone matrix, increasing the

resistance to crack propagation. This is similar to

many non-biological fibre composites, like fibre

glass. Using scanning electron microscopy, Akkus

et al. (2005) found that fracture surfaces of control

specimens exhibited tortuous fracture surfaces

with lamellar extrusions indicative of this func-

tion. Conversely, flat and regular fracture surfaces

of irradiated bone showed that collagen fibres

failed to provide bridging at the ultrastructural

94 Cell Tissue Banking (2007) 8:93–105

123

level (Fig. 2). These data indicated that when the

integrity of the collagen matrix is damaged by

irradiation, individual molecules collapse under

loading instead of transferring the load to the

whole lamella (Akkus et al. 2005).

Experiments using SDS–PAGE gel electro-

phoresis of collagen a-chains have provided fur-

ther evidence for the mechanical failure of

irradiated collagen (Akkus et al. 2005), illustrat-

ing that collagen fibres were degraded into mol-

ecules with different molecular weights (Fig 2).

Chemical protection of the bone microstructure

from these damaging effects can lead to signifi-

cant maintenance of mechanical properties

Fig. 1 Simplified schemeillustrating the direct andindirect effects of ionizingradiation on bonecollagen molecules.Adapted from Diedzic-Goclawsca et al. (2005)

Fig. 2 (A). SEM images of failed osteons in control bones(top) show irregular and tortuous surfaces associated withmechanical resistance from collagen; and smooth surfacesin irradiated bones (bottom) where collagen fibres fail toresist loading. (B). SDS-PAGE gel electrophoresis showsthe difference in contribution of collagen molecules

between control and irradiated samples. In control speci-mens, type I collagen molecules show strong demarcationinto 2 bands, adjacent to the molecular weight marker of97 kDa. In contrast, irradiated specimens provide veryweak signal, and the lanes showed a high degree of proteindegradation. (Figure adapted from Akkus et al. 2005)

Cell Tissue Banking (2007) 8:93–105 95

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(Grieb et al. 2005), but the safety of such radio-

protectants for use in humans needs greater

scrutiny in comparison to simply reducing the

radiation dose.

Mechanical changes in irradiated allograft

bone—a controversial issue

The effects of gamma radiation on biomechanical

properties of bone allografts are well documented,

and irradiation is suspected as the most damaging

factor for mechanical properties of bone allografts

during processing and sterilization (Triantafyllou

et al., 1975). For example, frozen bovine cortical

bone specimens (sized 8 · 0.5 · 0.5 cm), irradi-

ated at a dose of 30 ± 3 kGy, display reductions in

strength of 25–50% compared to control bone;

and similar reductions in maximum torque and

stress of rabbit tibiae of 23 and 25%, respectively,

have been observed (Godette et al. 1996),

Currey et al. (1997) reported that the standard

dose of 25 kGy significantly reduces bone

strength. They observed minimal differences in

the elastic Young’s modulus, but progressive

decreases in the plastic behaviour between bones

irradiated at 29.5 kGy and control specimens.

The bending strength of bone irradiated at the

standard dose, for example, was around 20–30%

lower than control groups. More importantly,

irradiation severely diminished work-to-fracture

by more than 70%. The results support the fact

that irradiation has little influence on the stiffness

of bone, but that brittleness is significantly

increased (Currey et al. 1997). This pattern of

failure is consistently observed (Akkus et al.

2005; Cornu et al. 2003; Hamer et al. 1999). For

example, Hamer et al. (1996) reported decreases

in the energy absorption capacity and bone

strength at doses between 28–30 kGy, while the

elastic properties were only mildly affected. The

maximum load declined by 36% and the work to

fracture by 68%, while the yield strength was only

reduced by 17%. Similarly, post-yield properties

such as work to fracture and post-yield energy of

the grafts were reduced by 70 and 87%, respec-

tively, following irradiation at 36.4 kGy, while

elastic energy was decreased by only 26% com-

pared to control (Akkus et al. 2005). Irradiation

also affects the fatigue behaviour of allografts

with evidence that it causes dramatic reductions

of 87% in fatigue life (cycles/1,000) at 36.4 kGy

(Akkus and Belaney 2005), and reduces the

resistance of bone to crack propagation (Mitchell

et al. 2004).

In clinical practice, of course, what is important

is the performance of the allograft during its

working life in patients. In a 3 year follow-up of

127 massive allografts sterilized by gamma irra-

diation (25 kGy), the rates of mechanical com-

plications such as fracture and non-union were

only 6 and 5.5%, respectively (Hernigou et al.

1993). These did not differ from other reports

using non-irradiated grafts. They concluded,

therefore, that irradiating allograft bone 25 kGy

was the most convenient and acceptable method

for sterilization. In contrast, Lietman et al. (2000)

found that the fracture rate in a patient group

implanted with irradiated grafts was more than

double that of a control group (38% vs. 18%).

Although they may have some observational rel-

evance, such case control studies are less rigorous

than clinically controlled trials, and more difficult

to interpret. This is because they are confounded

by factors such as recipient age, types of graft,

stage of original diseases, adjuvant therapy, fixa-

tion techniques, co-morbidity and weight bearing,

rather than gamma radiation alone (Hornicek

et al. 2001).

The use of massive osteochondral allografts

such as the proximal femur, distal femur and

proximal tibia is increasing in clinical practice.

These allografts contain both cortical and can-

cellous bone and are indicated for reconstruction

of large joints such as the hip and knee. In these

cases, cancellous bone is predominately loaded in

compression (Anderson et al. 1992). Therefore,

the effects of irradiation on mechanical properties

of cancellous bone also need to be considered.

Cancellous bone is more resistant to gamma

irradiation than cortical bone. Using cancellous

bone cubes from the tibial plateau, Anderson

et al. (1992) found no significant differences

between a control group and groups irradiated at

10, 31 and 51 kGy in failure stress. The failure

stress of these groups remained 78.9, 88.5 and 102

% of control value, respectively. However, it was

significantly reduced when radiation dose

increased to 60 kGy, only 23.9%. Similarly, iliac

96 Cell Tissue Banking (2007) 8:93–105

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crest wedges irradiated up to 20–25 kGy showed

minimal change in elastic modulus, compressive

strength and strain to failure, compared to non-

irradiated bone (Zhang et al. 1994).

With lower doses, such as 15–20 kGy, there is

very little affect of radiation on the properties of

bone allografts (Jinno et al. 2000; Pelker et al.

1989). When irradiated at 15 kGy, bone allografts

implanted into rat femora showed no deleterious

change in the bone strength after 4 months post-

transplantation (Jinno et al. 2000). Furthermore,

the strength of processed allogeneic grafts was

significantly higher than fresh allogeneic grafts.

Similarly, the force for screw pullout between

control and irradiated grafts (15.7–18.7 kGy) is

not significantly different (Simonian et al. 1994).

Unfortunately, the range of studies using doses

less than 25 kGy, is relatively small and further

work is required to verify the effects of lower

doses, while still achieving adequate sterilization.

The dose-response range, therefore, requires

further scrutiny.

Dose-dependent effect of irradiation

There is a general consensus that the strength of

allograft bone decreases in a dose-dependent

manner (Anderson et al. 1992; Currey et al. 1997;

Fideler et al. 1995; Gibbons et al. 1991; Hamer

et al. 1996; Salehpour et al. 1995) (Fig. 3).

For example, the bending strength of bone

allografts decreased by 10%, 20% and more than

50% as the gamma dose increased from 17 kGy

(low dose), to 29.5 kGy (standard) and 94.7 kGy

(high), respectively (Currey et al. 1997). More-

over, work-to-fracture decreased by 30–40%,

50–60%, and 90%. With a lower dose series, 0,

6.8, 9.5, 16, 23 and 60 kGy, a small dose-response

reduction was observed in work done to ultimate

stress up to 23 kGy, but a bigger decrease was

observed at 60 kGy (Fig. 4). (Hamer et al., 1996).

Similar trends have been observed from bone-

tendon-bone allografts in which the maximum

stress decreased by 37% for those irradiated at

40 kGy, 68% at 60 kGy and 76% at 80 kGy

(Salehpour et al. 1995); whereas lower doses of 20

and 30 kGy reduced the ultimate strength by

11–27% compared with controls (Gibbons et al.

1991; Hernigou et al. 2000). Increasing the

gamma dose with numerous small increments

shows that this relationship is non-linear with a

dose of 27 kGy causing a reduction in strength of

20%, but a dose of 37 kGy causing a reduction of

65% (Loty et al. 1990).

This dose-response reduction in allograft

strength is not only observed ex vivo, but affects

the success of the implanted bone. After being

implanted in rabbits for 6 weeks, the non-union

rate of irradiated graft-host bone was positively

related to the irradiation dose of the allografts

with rates increasing from 0 to 12.5 and 33% as

the gamma dose increased from 0 to 25 and

75 kGy (Godette et al. 1996).

Confounding variables for interpretation of

biomechanical studies

The evidence is consistent that gamma irradiation

causes a dose-response reduction in the mechanical

properties of allograft bone. Nonetheless, while

most authors agree that high doses of gamma

radiation (e.g. 40–60 kGy or more) cause deleteri-

ous effects, the influence of lower doses remains

debatable. Furthermore, interpretation of data is

confounded by the range of gamma doses applied,

and by other factors such as the age and sex of

donors, anatomical position of specimens, size of

specimens and especially direction of specimen and

type of mechanical tests (Zioupos et al. 2000).

Interpretation of different studies is very

difficult when the range of gamma doses, and the

definition of ‘‘standard dose’’, is very wide. The use

of ‘‘standard dose’’ has varied from 25–35 kGy,

with some authors adopting 25–30 kGy (Cornu

et al. 2000; Currey et al. 1997; Hamer et al. 1996;

Smith et al. 1996), but others reporting even

higher ‘‘standard’’ doses (Akkus and Rimnac 2001;

Dziedzic-Goclawska et al. 1991). The higher doses

adopted are those expected to eliminate viral

infection, with doses ranging from 40 to 50, 60 and

even 94 kGy (Campbell and Li 1999; Currey et al.

1997; Hernigou et al. 2000). This is because the

D10 value of HIV virus was very different among

studies. It may range from 4 to 8.3 kGy depending

on the irradiation environment (Dziedzic-

Goclawska et al. 1991). Moreover, there are

limited studies in which a small, but continuous,

Cell Tissue Banking (2007) 8:93–105 97

123

increase in dose across a large range has been

investigated (Hamer et al. 1996). Hence, a clear

and unequivocal relationship between gamma

dose and the mechanical properties of allograft

bone has yet to be established.

The age of donors from which samples are

collected is also a confounding variable that is

generally overlooked (Zioupos et al. 2000). The

age of donors may range from 51 to 67 in one

study (Anderson et al. 1992), while extending

from 19 to 60 in another (Vastel et al. 2004).

Bone mineral content of the skeleton increases

during growth to a peak at about 18 years in girls

and 20 years of age in boys (Bailey et al. 2000), at

which time the strength of bone becomes opti-

mum (Forwood et al. 2004). In contrast, during

aging, the changes in bone properties undergo a

progressive decrease (Mosekilde 1986). As a

result, samples taken from young donors would

have different mechanical properties from those

taken from aged individuals, even though the test

conditions may be the same. These changes may

not simply be exemplified in maximum strength,

but in the mode of failure and post-yield behav-

iour of bone (Diab et al. 2005); factors that are

also influenced by gamma irradiation.

Because bone is an adaptable tissue, the ana-

tomical position from which samples are retrieved

can also provide another source of variability

among studies (An 2000). That is, there are site-

specific variations in the strength and mechanical

behaviour of bone specimens. Samples taken

from the iliac region, for example (Zubillaga

et al. 2003) may not be as strong as cancellous

bone retrieved from the proximal tibia (Anderson

et al. 1992). Similarly, the strength of femoral

heads and necks is not the same as in the femoral

shaft (Currey et al. 1997; Vastel et al. 2004).

Even at the same anatomic position, different

orientation of the specimens can significantly

affect the results of the mechanical tests (Hamer

et al. 1996). For cortical bone, specimens with a

long axis aligned parallel to the long axis of the

bone are frequently used for bending tests

(Akkus and Rimnac 2001; Currey et al. 1997).

But the use of specimens machined with their

long axis perpendicular to the long axis of the

long bone would certainly confound simple com-

parisons and reduce the external validity of these

data. For instance, with the same dose (27.5 kGy)

Fig. 3 Values from theliterature show a generaltrend for dose-dependentdegradation ofmechanical properties ofallograft bone aftergamma irradiation. Forcomparison, degradationof mechanical propertiesare shown as percentagereduction compared tocontrol specimens, andthe mechanical variablemeasured is indicated inparentheses

Fig. 4 Dose dependent decrease in biomechanical prop-erties of cortical bone following gamma irradiation is non-linear. (Adapted from Hamer et al. 1996)

98 Cell Tissue Banking (2007) 8:93–105

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and from the same donors, the work-to-fracture

of transverse specimens was as low as 38% com-

pared to controls, but was up to 51% in specimens

with the axis aligned longitudinally with the bone

axis (Akkus and Rimnac 2001). Therefore, the

results from Currey’s study (Currey et al. 1997),

using longitudinal beams is more difficult to

compare with Hamer et al. (1996), who used

transverse sections.

In conclusion, gamma irradiation causes a

dose-dependent decrease in mechanical proper-

ties of allograft bone when the doses are signifi-

cantly and highly increased. However, at doses

between 0 and 25 kGy (our standard dose), a

clear relationship between gamma dose and

mechanical properties has not been established,

partly due to a range of confounding variables.

Therefore, there is still considerable justification

for further studies on the influence of gamma

irradiation on the mechanical properties of allo-

graft bone in which variability due to age, sex,

anatomical position, and range of gamma doses

has been controlled.

The correlation between bone allograft biology

and gamma radiation sterilization

Mechanism of bone allograft healing/

remodelling

To replace the functions of removed bone, the

mineralized allograft must facilitate biological

activity at the graft site. This activity includes the

provision of an osteoconductive surface for

remodelling, incorporation with the host skeleton,

facilitation of bone resorption and formation

phases of remodelling and biological acceptability

with the host. The biology of bone graft healing

has been well investigated (Aspenberg et al. 1996;

Enneking and Campanacci 2001; Enneking and

Mindell 1991; Heiple et al. 1987; Heyligers and

Klein-Nulend 2005; Kalfas 2001; Khan et al. 2005;

Kingsmill et al. 1999; Lee et al. 2000), but the role

of gamma irradiation on these biological functions

has not been clearly established in the literature.

Two processes occur during repair of massive

bone allografts: surface repair and internal repair.

Both of them start with bone resorption, in which

the activation of osteoclasts plays a central role.

During this process, un-resorbed surfaces of the

allograft serve as an anchor for the attachment of

the host soft tissues. Within the first 6 months, a thin

seam of appositional lamellar bone forms from the

host, and continues to cover about half of the outer

cortical surface by the end of the first year (Enne-

king and Campanacci 2001; Enneking and Mindell

1991). The other half of the surface is resorbed and

covered by new lamellar bone by remodelling dur-

ing the second year. Overall, resorption only re-

moves several millimetres of superficial cortical

bone. Therefore, the graft is not completely re-

placed by the new bone, and its remaining tissue is

surrounded by new lamellae (Enneking and Cam-

panacci 2001; Enneking and Mindell 1991). At that

time, the walls of Haversian canals start to be

remodeled and then filled with new bone formation.

This pattern replaces the allograft bone by viable

host bone (Schachar et al. 2002).

The repair of bone allografts described above

involves both osteoinduction and osteoconduction

(Kalfas 2001; Khan et al. 2005). Osteoinduction

refers to the adhesion of host mesenchymal stem

cells onto graft surfaces to differentiate and start

the remodeling process. Osteoconductivity is the

ability of the graft to provide a scaffold for bone

healing by recruitment of the mesenchymal stem

cells of the host, to stabilize on their bone surfaces

and stimulate cell differentiation and subsequent

remodelling (Kalfas 2001; Khan et al. 2005). Mej-

dahl et al. (1998) hypothesized that signals from

the allograft bone matrix are mediated by the

interaction of adhesion molecules with osteoblast-

like cells forming contacts with the graft surface.

These cells release factors that signal the osteo-

clastic cell-line and initiate bone resorption. When

bone mineral is resorbed, matrix-derived growth

factors from the graft, such as bone morphogenetic

protein –2, –4 and –7, are released to activate and

amplify the osteoinduction process (Kalfas 2001;

Khan et al. 2005).

Obviously, allografts in optimum condition

contribute to the success of transplantation sur-

geries. Although a great focus is placed on new

bone formation, the ability of the graft to be re-

sorbed by osteoclasts plays a crucial role in the

incorporation of bone allografts, because this is the

primary step in remodeling (Kluger et al. 2003).

Cell Tissue Banking (2007) 8:93–105 99

123

The influences of gamma radiation in bone

allograft remodelling

The activity of growth factors such as bone mor-

phogenetic proteins (BMP) and transforming

growth factor-b (TGF-b) is essential for effective

osteoinduction, but may be degraded during

irradiation (Ijiri et al. 1994; Puolakkainen et al.

1993). It is clear that BMP play an important role

in bone remodelling, and to express their potency,

they need a carrier. Collagen is the best carrier

for BMP (Dziedzic-Goclawska et al. 2005; Ijiri

et al. 1994). However, sterilization by gamma

irradiation degrades collagen and destroys the

fibrillar network of the bone matrix. When this

carrier is destroyed by gamma rays, the process of

bone remodelling, and the graft-recipient healing

process is affected (Dziedzic-Goclawska et al.

2005; Ijiri et al. 1994). Ijiri et al. (1994) investi-

gated the effects of sterilization methods on os-

teoinductivity, by implanting pellets of BMP and

collagen, or pellets of collagen only, into animals.

No bone formation was observed on pellets im-

planted with collagen only. Moreover, when the

combined BMP and collagen pellets were irradi-

ated at 25 kGy, much less bone formation was

observed compared to non-irradiated pellets.

More conclusively, when non-irradiated BMP was

combined with irradiated collagen pellets, the

bone formation was almost zero. In contrast,

compared to control groups, the bone formation

was not significantly different when pellets were

implanted with irradiated BMP combined with

non-irradiated collagen. The calcium yield and

alkaline phosphatase activity measured from re-

trieved pellets followed the same trend as bone

formation. While the calcium yield from pellets

with irradiated BMP/non-irradiated collagen was

reduced to 76% that of control, it was almost zero

from those with non-irradiated BMP/irradiated

collagen. These data supported the argument that

it was the collagen carrier that was more labile to

gamma irradiation than the BMP protein itself

(Dziedzic-Goclawska et al. 2005; Ijiri et al. 1994),

which is also consistent with maintenance of

TGF-b activity at doses of radiation lower than

25 kGy (Puolakkainen et al. 1993).

Studies have focused on the impairment of

osteoinduction caused by gamma irradiation. By

using radiographic, histologic and histochemical

methods, Buring and Urist (1967) investigated the

effect of radiation on bone induction induced by

bone matrix implanted into the muscle of rats and

rabbits. Bone induction was eliminated when the

allografts were irradiated at the doses from 20 to

40 kGy. Specifically, the implants were almost

resorbed and new bone formation was absent for

every one of 300 implants irradiated with doses

from 20 to 37 kGy. Furthermore, irradiated col-

lagen was much more soluble and absorbable.

The authors concluded that allografts only serve

as an allo-structural function when osteoinduction

is inhibited by gamma doses higher than 20 kGy.

These conclusions are supported by work showing

reduced calcium content of irradiated implants

after 2 months (Munting et al. 1988), and signifi-

cant delay in incorporation of bone grafts irradi-

ated at 25 kGy (Voggenreiter et al. 1996).

In contrast, Wientroub and Reddi (1988) con-

cluded that a standard dose of gamma irradiation

did not alter the osteoinductivity of demineral-

ized bone matrix implanted into rat muscle. In

fact, higher doses (30–50 kGy) even increased

these properties. Likewise, Glowacki (2005)

reported that demineralized bone irradiated at

doses of 20–40 kGy retained 80% of its activity,

and Hallfeldt et al. (1995) showed that bridging of

defects in drill holes was only 94% of controls

when demineralized bone matrix was irradiated

at 25 kGy. The question is how to reconcile these

observations with those above. First, the time

scale varied in these latter studies. In the former

ones (Buring and Urist 1967; Munting et al. 1988;

Voggenreiter et al. 1996), time for retrieval

implants ranged from 1 to 8 weeks; whereas in

the latter experiments (Hallfeldt et al., 1995;

Wientroub and Reddi, 1988), it only ranged

12–18 days. Second, it is clear that the tempera-

ture during irradiation could affect the quality of

bone allografts. Buring and Urist (1967) and

Munting et al. (1988) irradiated their specimens

at room temperature, while Wientroub and Reddi

(1988) and Hallfeldt et al. (1995) packed their

bone specimens in dried ice. Therefore, compar-

ison of studies under standard conditions of con-

temporary bone banks is required to reach

unequivocal conclusions on the biological conse-

quences of secondary sterilization.

100 Cell Tissue Banking (2007) 8:93–105

123

Dziedzic-Goclawska et al. (1991) compared the

effect of irradiation on osteoinduction of bone

allografts preserved by lyophilization or deep-

freezing. Samples were irradiated at 35 or 50 kGy

at room temperature for lyophilization, and on dry

ice for frozen groups and then implanted into

parietal bones of adult rabbits. After 12 weeks,

deep-frozen bone was much more resorbed, and

then replaced, by new bone than lyophilized bone.

Fifty percent of frozen implants were resorbed

and replaced with new bone, compared to 20%

resorption and 10% new bone formation in

lyophilized implants. After 26 weeks, 70% of

frozen allografts were remodeled and compared

to less than 40% for the lyophilized bone

(Dziedzic-Goclawska et al. 1991).

In frozen massive bone allografts, the lipid

content of the marrow cavity is not removed. This

may change their properties after exposure to

gamma irradiation, causing them to become toxic

to the osteoblasts (Moreau et al. 2000). When

osteoblast-like cells are cultured with irradiated

bone slices, considerable cell death is induced

around the bone slices, when compared to non-

irradiated bone (Moreau et al. 2000). Biochemi-

cal analysis revealed that the peroxidized lipids:

total lipids ratio was 2–3 times higher in the

gamma irradiation group. This analysis provided

evidence that bone marrow lipids were peroxi-

dized by gamma radiation, a process that releases

free radicals that can induce necrosis and osteo-

blast death. Peroxidation, therefore, may be

another mechanism by which remodelling of

allograft bone is impaired by gamma irradiation.

Moreover, peroxidation of lipids may also induce

giant cell reactions to release cytokines and

prostaglandins, leading to an inflammatory med-

iated bone resorption (Moreau et al. 2000).

Although experimental studies provide evidence

for a degradation of biological properties of allo-

grafts, this does not appear to be translated into

worse clinical outcomes. Clinically, there is evi-

dence that rates of non-union or fracture in bone

grafted with irradiated bone (25 kGy) do not differ

from those reported for non-irradiated grafts

(Hernigou et al. 1993; Lietman et al. 2000). The

apparent maintenance of osteoinductive properties

at 25 kGy is observed in mineralized bone allo-

grafts (Hernigou et al. 1993), as well as deminer-

alized bone matrix (Urist and Hernandez 1974;

Wientroub and Reddi 1988). Although these data

suggest that the osteoinduction and osteoconduc-

tion of bone allografts are not altered by irradia-

tion, it is difficult to compare with experimental

studies without strong evidence from randomized

controlled trials. Until these are performed, or

irradiated allografts are compared in well-designed

studies in large animals, the clinical performance of

irradiated allografts remains equivocal.

Where allograft bone has been grafted into ani-

mal models, a dose-dependent effect is generally

observed (Godette et al. 1996; Jinno et al. 2000;

Urist and Hernandez 1974; Voggenreiter et al.

1996). For example, allograft bone sterilized at

15 kGy did not compromise allograft incorporation

at 4 or 6 months following surgery (Jinno et al.

2000); yet, bone grafts irradiated at 25 kGy and

implanted into rabbit femora or rat tibiae showed

dramatically reduced rates of incorporation when

compared to fresh frozen specimens (Godette et al.

1996; Voggenreiter et al. 1996). These data are

more highly controlled than human clinical obser-

vations and provide evidence for dose-related

impairment of allograft performance, but would be

strengthened by experiments in a large animal

model, and randomised controlled trials in humans.

Changes in osteoconduction of irradiated

allografts

Resorption is the first stage of bone remodeling

during the incorporation of bone allografts.

Osteoclasts, therefore, play an initial and essen-

tial role (Kluger et al. 2003). Moreover, the cells

coordinate with osteoblasts to stimulate bridging

and facilitate union (Akkus and Rimnac 2001;

Chapman and Villar 1992; Enneking and Cam-

panacci 2001). Therefore, the effectiveness of

graft-host incorporation depends on effective

bone resorption by osteoclasts recruited from the

host. The better the quality of bone allografts, the

more host cells will be attracted and remodelling

facilitated (Kluger et al. 2003).

However, studies utilizing osteoclasts to

determine the quality of irradiated bone are few.

Using the resorption assay on bone slices Kluger

et al. (2003) investigated the effects of irradiation

on osteoclast activity. Transverse femoral cortical

Cell Tissue Banking (2007) 8:93–105 101

123

bone slices, 8–10 mm, were irradiated at 25 kGy,

and then incubated with osteoclasts isolated from

rabbit bone for 50 h. Full processing of bone

allografts, including de-fatting, freeze-drying and

gamma irradiation reduced osteoclast activity by

57% compared with fresh frozen bone. They also

recognized that processing damaged bone matrix

proteins such as integrins, which lead to the

impairment of osteoclast attachment and resorp-

tion (Kluger et al. 2003).

In short, sterilization by gamma irradiation af-

fects bone allograft remodelling by impairing the

organic components of allografts such as proteins

and lipids. However, the levels of damage differ

among the studies. These differences are caused by

variations in studies such as temperature conditions,

allograft materials and gamma dose. In addition,

aspects of bone formation have been more inten-

sively studied than those of bone resorption, al-

though bone resorption and osteoclast activation

play a fundamental role in bone remodelling.

The role of macrophages in bone allograft

implantation

When implanted into the new host, allograft tissue

stimulates cellular immune reactions (Muldashev

et al. 2005). These reactions appear as important

factors initiating osteoclast activation and trans-

plant resorption. During allograft remodelling, re-

lease of TNFa and activation of macrophages play a

key role in the resorption and formation process of

biomaterial implantation (Muldashev et al. 2005).

In infectious bone disease, products released

from bacteria may cause chronic inflammation

and subsequence bone loss (Jiang et al. 2002).

There is sound evidence for the hypothesis that

this bone resorption is bacteria-mediated (Hong

et al. 2004; Jiang et al. 2002; Meghji et al. 1998;

Moreau et al. 2000; Nair et al. 1995; Nair et al.

1996; Schuster et al. 2000). Nair and colleagues

have listed three mechanisms of bacterially

induced bone destruction (Nair et al. 1995, 1996):

1. The bone matrix is directly resorbed by

release of acids and proteases from bacteria.

2. The osteoclastic cell line is stimulated by

bacterial products; and

3. Bacterial products inhibit bone formation.

Of these, the two latter ones play a more

important role in bone loss, given that bacteria

can release many capsule factors during lysis.

Lipopolysaccharide (LPS) secreted from gram-

negative bacteria can promote bone resorption by

triggering macrophages to release osteolytic

mediators (Hong et al. 2004; Jiang et al. 2002).

LPS also directly stimulates the formation of

osteoclast-like cells without the involvement of

osteoblasts, and these cells form resorption pits in

vitro on bone slices (Jiang et al. 2002). Moreover,

this inflammatory bone resorption is bacterial

dose-dependent.

Surface-associated proteins (SAPs) from some

gram-positive bacteria such as Staphylococcus

epidermidis and aureus can also promote bone

resorption by directly activating osteoclasts

(Arora et al. 1998; Meghji et al. 1998; Meghji

et al. 1997a, 1997b; Nair et al. 1995). These bac-

terial infections, in orthopaedic clinics, are asso-

ciated with rapid bone loss. These gram-positive

anaerobic cocci rarely cause infectious diseases in

healthy humans, but it has emerged as an

important pathogen in foreign body implantation

(James and Gower 2002; Liu et al. 2002; Som-

merville et al. 2000).

Several experiments have used bone resorption

assays and calcium release to investigate the

osteoclast activation potential of SAPs extracted

from S. epidermidis and S. areaus (Arora et al.

1998; Meghji et al. 1998; Meghji et al. 1997a).

These bacterial products caused a dose-depen-

dent stimulation of calcium release and pit

resorption. Furthermore, there is evidence that

LPS and SAPs prevent osteoblasts from synthe-

sizing bone matrix (Schuster et al. 2000). The

mechanism of this inhibition is still not clear, but

proteins from certain bacteria can inhibit cell-

cycle progression by blocking cells in the G2

phase of the cell cycle. These experiments also

demonstrated that the bacterial products were

easily released by elution in saline solutions, and

were also heat and trypsin sensitive (Meghji et al.

1998; Meghji et al. 1997a).

Therefore, when irradiation is used for sterili-

zation of bone allografts, the destruction of bac-

teria may also release these products. Given that

the gamma dose is high enough to kill all bacteria

present in the bone allograft, their products may

102 Cell Tissue Banking (2007) 8:93–105

123

still remain. If the grafts are heavily contami-

nated, it could contain a large amount of bacterial

particles following irradiation. Consequently, we

hypothesize that the particles could activate a

cellular immune reaction and cause excess bone

resorption. To date there are no published studies

testing this hypothesis.

In conclusion, bone allografts provide replace-

ment and reinforcement for defective skeletal

structures caused by bone disease, and act as a

stimulus for new bone remodelling. For optimal

activation of these processes, biological and bio-

mechanical properties of bone allografts must be

preserved. However, the risk of disease transmis-

sion leads to the use of terminal/secondary sterili-

zation using gamma irradiation. This sterilization

method negatively alters the properties of bone al-

lografts. While the biomechanical properties of

bone allografts irradiated by gamma rays have been

widely studied, the biological properties such as the

ability for bone resorption and formation have not

been equally considered. Due to developments in

the bone bank industry, manufacturing procedures

have markedly improved bone quality. Therefore,

the trend towards application of lower gamma do-

ses for terminal sterilization is becoming a greater

concern in tissue banking. However, to substantiate

the lower dose, there is a need for global investi-

gation of bone allograft qualities such as sterility,

mechanical properties and biological functions

after gamma irradiation. Such studies should be

undertaken using a wide range of doses and under

tightly controlled conditions of bone banking in

which non-irradiation factors can be minimized.

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