ORIGINAL PAPER

Sterilization of tendon allografts: a method to improvestrength and stability after exposure to 50 kGy gammaradiation

Aaron U. Seto • Charles J. Gatt Jr •

Michael G. Dunn

Received: 12 June 2012 / Accepted: 30 July 2012 / Published online: 24 August 2012

� Springer Science+Business Media B.V. 2012

Abstract Terminal sterilization of tendon allografts

with high dose gamma irradiation has deleterious

effects on tendon mechanical properties and stability

after implantation. Our goal is to minimize these

effects with radio protective methods. We previously

showed that radio protection via combined crosslink-

ing and free radical scavenging maintained initial

mechanical properties of tendon allografts after irra-

diation at 50 kGy. This study further evaluates the

tissue response and simulated mechanical degradation

of tendons processed with radio protective treatment,

which involves crosslinking in 1-ethyl-3-(3-dimethyl-

aminopropyl) carbodiimide followed by soaking in an

ascorbate/riboflavin-5-phosphate solution. Control

untreated and treated tendons were irradiated at

50 kGy and implanted in New Zealand White rabbit

knees within the joint capsule for four and 8 weeks.

Tendons were also exposed to cyclic loading to 20 N

at one cycle per 12 s in a collagenase solution for 150

cycles, followed by tension to failure. Control irradi-

ated tendons displayed increased degradation in vivo,

and failed prematurely during cyclic processing at an

average of 25 cycles. In contrast, radio protected

irradiated tendons displayed greater stability follow-

ing implantation over 8 weeks, and possessed strength

at 59 % of native tendons and modulus equivalent to

that of native tendons after cyclic loading in collage-

nase. These results suggest that radio protective

treatment improves the strength and the stability of

tendon allografts.

Keywords Gamma radiation � Radio protection �Tendon allografts � Implantation � Cyclic loading �Collagenase

Introduction

Allograft safety, including development of terminal

sterilization methods, remains a major concern among

the tissue bank community (McAllister et al. 2007;

Suarez and Richmond 2007). This concern is justified

by the large demand for allograft tissues in the United

States and the potential for transmission of existing or

emerging diseases. At sufficiently high doses, ionizing

radiation can neutralize both bacterial and viral agents

through direct interaction with high energy particles

and chemical modifications driven by the formation of

free radicals (Halliwell and Gutteridge 1989; Block

1991). Unfortunately, it has been well established that

at high doses, radiation also negatively alters mechan-

ical (Block 1991; Salehpour et al. 1995; Gibbons et al.

1991) and biological properties of allografts (Nguyen

et al. 2007). Successful protection of musculoskeletal

allografts against damage from high dose ionizing

A. U. Seto � C. J. Gatt Jr � M. G. Dunn (&)

University of Medicine and Dentistry of

New Jersey–Robert Wood Johnson Medical School,

New Brunswick, NJ 08901, USA

e-mail: dunnmg@umdnj.edu

123

Cell Tissue Bank (2013) 14:349–357

DOI 10.1007/s10561-012-9336-y

radiation would permit its use as a standard method of

sterilization.

In previous studies, we demonstrated that a combined

crosslinking and free radical scavenging cocktail treat-

ment was capable of maintaining initial mechanical

integrity of tendon tissues in the presence of up to

50 kGy of ionizing radiation (Seto et al. 2009). This

combined treatment adds crosslinks and minimizes the

effects of oxygen free radicals that are generated during

the tendon iradiation process. The crosslinking compo-

nent utilizes 1-ethyl-3-(3-dimethylaminopropyl) carbo-

diimide (EDC), which has been shown to crosslink

collagen biomaterials (Caruso and Dunn 2005). The

scavenging component includes ascorbate, a potent

antioxidant (Halliwell and Gutteridge 1989; Zbikowska

et al. 2006), and riboflavin which has shown both

scavenging (Fuga et al. 2004) and crosslinking abilities

(Spoerl et al. 2004; Wollensak et al. 2003).

Tendon allografts must have sufficiently high initial

strength and strength retention after implantation, both

of which may be compromised by ionizing radiation.

After implantation, the graft experiences degradation

both biologically as it is broken down by infiltrating host

cells and enzymes, as well as mechanically due to cyclic

loading. Following ACL injury, MMP expression has

been shown to be elevated in the surrounding environ-

ment (Tang et al. 2009). Additionally, synovial cell

MMP activity has been associated with ACL loading

(Raif el 2008). In the current study we assess the ability

of radio protective treatment to stabilize irradiated

tendon allografts that are subjected to a simulated knee

environment challenged by both mechanical and bio-

logical degradation, and an in vivo knee environment.

We hypothesized that irradiated allografts receiving the

radio protective treatment would (1) maintain mechan-

ical properties better than control irradiated allografts in

response to the simulated knee environment (cyclic

loading in collagenase in vitro), and (2) maintain better

structural stability with comparable cellular compati-

bility following implantation in the in vivo environment.

If this hypothesis is correct, radio protected allografts

will subsequently be evaluated in a functional ACL

reconstruction model in large animals.

Methods

To estimate post-implantation stability of radio pro-

tected allografts after radiation exposure in response to

mechanical and biological degradation, we conducted

the following two studies. First we evaluated failure

properties of control untreated and radio protected

tendons after cyclic loading in a protease solution.

Secondly, we evaluated the host tissue response to

tendon allografts after implantation in an unloaded

rabbit knee model.

Cyclic loading in collagenase

Soleus tendons used for cyclic testing in the enzyme

solution were harvested from New Zealand White

rabbit hind limbs (Bioreclamation, Liverpool, NY).

A total of 50 tendons were harvested and randomized

into 5 groups of 10 tendons with different treatments

and exposure to different conditions:

1) Native tendon—no cycling,

2) Native tendon—cycled in saline,

3) Native tendon—cycled in collagenase,

4) Irradiated control tendon—cycled in collagenase,

and

5) Irradiated tendon treated with radio protective

protocol—cycled in collagenase

By this nomenclature, ‘native’ tendons remained

both untreated and unsterilized. Prior to sterilization,

control tendons were soaked in saline. Radio protected

tendons were first soaked in a 5/10 mM EDC/NHS (N-

hydroxyl succinimide) crosslinking solution (Sigma-

Aldrich) made with saline at pH 7.4 for 6 h. This was

followed by washing in saline for 2 h. These tendons

were then soaked in a 50/12.5 mM ascorbate/ribo-

flavin-5-phosphate (both Sigma-Aldrich) solution

made in deionized water for 48 h. Control and treated

tendons were sterilized with gamma radiation at a dose

of 50 kGy (Sterigenics, Rockaway, NJ) at room

temperature. The control tendons were irradiated in

saline; the treated tendons were irradiated in the radio

protective solution.

A custom test chamber was constructed to perform

cyclic tests in either a saline or enzyme solution. For

enzyme testing, the chamber was filled with collagenase

solution with an activity of 20 units/ml derived from

clostridiopeptidase A (Sigma-Aldrich, St. Louis, MO).

Compared to mammalian collagenase, bacterial colla-

genase digests a wider range of amino acid sequences.

The selection of bacterial collagenase, and its activity,

was based on pilot studies to ensure there was a

detectable effect on tendon mechanical properties. The

350 Cell Tissue Bank (2013) 14:349–357

123

solution was pumped at a rate of 300 cc/min in an

isolated loop through a heated water bath (Julabo 5,

Seelbach, Germany) to maintain a chamber temperature

of 37 �C. Tendons were clamped in custom built vise

grips with a gauge length of 3 cm. The bottom grip was

fixed to the base of the Instron while the top clamp was

attached to the Instron crosshead (Fig. 1c). The chamber

was mounted on the testing machine (Instron 5569,

Canton, MA), which performed a cyclic tension profile

at a rate of 1 cycle per 12 s up to a maximum load of

20 N, for a total of 150 cycles.

After cyclic loading, the tendons were removed

from the chamber and rinsed and soaked in saline for

4 h. Three measurements were taken using a laser

micrometer (Z-mike model 1202B, Dayton, OH) for

width and thickness of each tendon to determine cross-

sectional area using rectangular estimation. Tendons

were then tested in tension to failure at a rate of

60 mm/min on the Instron using cryogenic clamps

(Bose, Eden Prarie, MN). The gauge length for cyclic

tests and the failure tests was 3 cm. Material properties

(ultimate tensile stress (MPa), elastic modulus (MPa),

and toughness (MPa)) were determined from the

cross-sectional areas and load-deformation curves

obtained from Instron software (Canton, MA). Statis-

tical analysis was performed using a one-way

ANOVA post hoc Tukey test using SigmaStat soft-

ware to ascertain if there were statistically significant

Fig. 1 a Failure strength, b elastic modulus of tendons after

cyclic loading in collagenase, and c Biochamber used for cyclic

loading in solution. Rabbit soleus tendons were mounted on

custom acrylic vise grips and cyclically loaded while submerged

in circulating collagenase solution maintained at 37 �C. Native

tendons cycled in collagenase retained 41 % of their initial

strength. Radio protected irradiated tendons retained 24 % of

the initial strength of a native tendon, and 59 % of the value of

the native tendon cycled in collagenase. In contrast, control

irradiated tendons were untestable due to failure during cyclic

loading in collagenase. The difference between native and

treated tendons was significant for strength, but not for modulus.

(filled triangle p \ 0.001, filled diamond p \ 0.040 compared to

native)

Cell Tissue Bank (2013) 14:349–357 351

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differences between the properties of the various

groups.

Non-functional implantation study

Tendon implants for the non-functional rabbit surgery

were prepared from allograft tendons harvested from

New Zealand White rabbit hind limbs (Bioreclaima-

tion, Livingston, NY). Soleus tendons were harvested

and a 1 cm section was cut from the bone insertion end

at the intersection of the ankle joint. Typical implant

dimensions were 1 cm in length, 2 mm in width, and

1.5 mm thickness. Twenty tendon sections were

dissected and randomized into two groups: irradiated

(50 kGy) control, and irradiated treated with radio

protective protocol. Additionally two unimplanted

samples were reserved for histology.

Before surgery, the control and radio protected

tendon implants were rinsed in sterile saline for

30 min. The surgical procedure was performed fol-

lowing an IACUC approved protocol. One treated and

one control implant were randomly implanted bilater-

ally into in a surgically formed pocket within the joint

capsule in New Zealand White rabbit knees. A small

incision was made on the medial aspect of the knee

adjacent to the tibial condyle. The knee capsule was

located and opened. Presence of synovial fluid was

confirmed and a small pocket was opened alongside

the condyle for placement of the specimen. The

implant was sutured to the inside of the knee capsule.

The specimen was not introduced between the

condyles to avoid interfering with knee motion.

The structure of the knee was left undisturbed and

uninjured, and motion was not compromised. Animals

resumed normal ambulation and activity one day post

surgery and recovered normally to the sacrifice date. A

total of ten rabbits were used, and implants remained

in the knee for for 4 or 8 weeks. Upon harvest,

implants were placed in 10 % buffered formalin and

shipped for histological processing (AML Laborato-

ries, Bethesda, MD). Samples were paraffin embedded

and 5 micron axial slices were taken and stained with

hematoxylin and eosin. These slides were evaluated by

a trained pathologist to identify presence of various

inflammatory cells, multinucleated giants cells, extent

of cellular infiltration, and tendon ‘crimp’. Under

polarized light, collagen fascicles display a sinusoidal

pattern, where wave frequency is defined as crimp.

Two non-implanted irradiated samples from the

control and treated groups, as well as native non-

sterilized implants, were included for comparison.

Results

Cyclic loading in collagenase

Compared to control tendons, treated tendons retained

superior mechanical properties after cyclic loading in

collagenase solution. Overall there were significant

changes observed for radiation conditions and for

tendons cyclically loaded in collagenase. Native

tendons cycled in collagenase retained 41 % of their

initial strength (Fig. 1a). Strength of radio protected

irradiated tendons was 24 % of a native tendon, and

59 % of native tendon after cyclic loading in collage-

nase. In sharp contrast, control irradiated tendons were

untestable due to failure during the cyclic loading.

Control tendons never withstood more than 48 cycles

and failed at an average of 25 ± 13 cycles, whereas

treated tendons remained intact throughout the cyclic

loading in collagenase. Tendon toughness showed

similar trends as strength values (not shown). Inter-

estingly, the elastic moduli (Fig. 1b) of native and

radio protected irradiated tendons were similar after

cycling in collagenase, retaining approximately 50 %

of the value for a native tendon.

Non-function implantation study

Radiation affected the structure of tendons that were

examined prior to implantation. Compared to native

tendon, irradiation caused crimp loss for both control

and treated implants, and separation of collagen

bundles was greatest for control implants (Fig. 2a–

c). Histological evaluation of explanted irradiated

tendons showed greater degradation in control com-

pared to treated tendons from 4 to 8 weeks. The

borders of treated implants more closely resembled

the original implant shape, whereas control implants

became more frayed, indicative of degradation

(Fig. 3a, b). The type of tissue response was similar

for control and radio protected implants, although

there were differences in the rate of cellular infiltration

and degradation of the tendons. Inflammatory cells

were more prevalent within control implants at both 4

and 8 weeks, whereas among treated implants, their

presence was only moderate and increased slightly

352 Cell Tissue Bank (2013) 14:349–357

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from 4 to 8 weeks. The depth of cellular infiltration

was greater for control implants, as cells were more

concentrated on the periphery of treated implants,

especially at 4 weeks (Fig. 3c, d). At 8 weeks, there

was new collagen deposition for several of the control

implants. The characteristic crimp pattern was com-

pletely lost for nearly all implanted samples. There

were few multinucleated giant cells associated with

the 4 weeks control implants, but not for the other

groups.

Discussion

With improvements in radio protective methods, the

use of high dose ionizing radiation for terminal

sterilization could ensure distribution of musculoskel-

etal allografts without risk of bacterial or viral

contamination. Recently, several investigations have

shown radio protection of the initial mechanical

properties of musculoskeletal allografts (Akkus et al.

2005; Grieb et al. 2006). Previously, our lab demon-

strated excellent maintenance of the initial mechanical

properties of tendon radio protected allografts exposed

to high doses of ionizing radiation (Seto et al. 2009). In

the current study we sought to assess allograft stability

with regard to biological and mechanical degradation.

We hypothesized that the radio protective treatment

would improve the stability of irradiated tendons after

cyclic loading in a proteolytic challenge and in vivo

after non-functional implantation. Mechanical prop-

erties of radio protected tendons after cyclic loading,

as well as histologic data of the implantation study are

supportive of improved stability in treated tendons

compared to control irradiated tendons.

It is necessary for allografts to support functional

loads soon after implantation in order to sooner initiate

physical rehabilitation. Repetitive subfailure loading is

frequently used to model graft behavior under these

conditions. The response of allografts to cyclic loading

has been analyzed under a variety of conditions: dry, in

saline solution, or in tissue culture media, and at various

loads, speeds, and repetitions (Honl et al. 2002; Asundi

and Rempel 2008; Mihalko et al. 2010). To our

knowledge, this is the first study to perform cyclic

testing in collagenase solution, which roughly simulates

the loading and intra-articular environment of the joint

space following ACL injury and reconstruction. Colla-

genase I expression is elevated in patellar tendon ACL

Fig. 2 Polarized images of non-implanted tendons: a native

tendon; b control irradiated tendon; c treated irradiated tendon

(H&E stained, original magnification 920; bars = 500 lm).

Radiation effects were evident when comparing crimp and

separation between collagen bundles. For native tendon not

exposed to radiation, collagen bundles were tight and the crimp

pattern was uniform and continuous. Some crimp loss was

observed for both control and treated irradiated tendons, and

gaps between collagen bundles were most prominent for control

irradiated tendons

Cell Tissue Bank (2013) 14:349–357 353

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reconstructions in human patients, confirmed by real

time polymerase chain reaction (PCR) (Roseti et al.

2008). Compared to mammalian collagenase, the use of

bacterial collagenase provides an accelerated degrada-

tion environment. Radio protective treatment afforded

irradiated tendons with greater stability in the simulated

knee environment compared to control tendons which

were untestable. Although strength was diminished, the

modulus of radio protected tendons was not significantly

different from that of native tendon after cycling in

collagenase. This suggests that a sufficient amount of

crosslinks were present to resist deformation, and at

non-failure loads, the tissue was still functioning

comparably to native tendon. The significant difference

in strength between irradiated and non-irradiated ten-

dons suggests that mechanical degradation was accen-

tuated by cyclic loading in collagenase for tendons

exposed to radiation. The damage caused to the collagen

microstructure by free radical effects during irradiation

could result in greater access to dissociation sites

leading to accelerated protease degradation. In con-

junction, fissures and defects created by radiation

damage may be further propagated by mechanical

action. Consequently, radio protection and crosslinking

may be critical to prevent premature degradation, which

ultimately must be determined with long-term func-

tional implantation studies.

In previous studies investigating the initial mechan-

ical properties of irradiated tendons, we observed that

radio protected tendons maintained mechanical prop-

erties comparable to native tendons (Seto et al. 2009).

In contrast, after cyclic loading in collagenase, treated

tendons had significantly lower strength compared to

native tendons. Cyclic processing in an enzymatic

Fig. 3 Control irradiated tendon at a 4 weeks and b 8 weeks

post-implantation. Treated irradiated tendon at c 4 weeks and

d 8 weeks. (H&E stained, original magnification 920;

bars = 500 lm). After implantation, control tendons were more

rapidly infiltrated with inflammatory cells, and tendon degra-

dation was evident. Treated tendons remained largely intact, and

cell infiltration was delayed. Crimp was no longer present for

both groups at 8 weeks

354 Cell Tissue Bank (2013) 14:349–357

123

challenge as performed in this study might provide a

better estimate of biomechanical performance in vivo

compared to conducting routine tensile testing to

failure. This may have further implications for allo-

grafts that are regularly sterilized at lower doses

(10–35 kGy (Vangsness et al. 2003; Vangsness et al.

1996) which are believed to have no negative effects

on mechanical properties (Balsly et al. 2008).

In vivo studies are the only way to determine the

tissue response to allografts that have been processed

chemically or physically. Control untreated implants

displayed an enhanced inflammatory response com-

pared to treated implants. Greater inflammation,

cellular infiltration, and deformation of implant

borders was noted for control implants. Prior to

new matrix deposition, the original tissue is broken

down by protease producing inflammatory cells. This

process of tissue breakdown is likely accelerated by

the damage caused by radiation. In a study of

radiation effects on remodeling rat patellar tendon

allografts, Torisuka et al. determined that gamma

radiation accelerated the removal of radiolabeled

donor collagen along with synthesis of new collagen

(Toritsuka et al. 1997). Lomas et al. also showed that

peracetic acid disinfection resulted in the disruption

of collagen fibrils, which was reflected in increased

digestion by collagenase. These studies demonstrate

that irradiation or chemical treatment can cause

accelerated breakdown of collagen, consistent with

our results.

In addition to determining in vivo graft stability, the

implantation study also serves to identify the nature of

the tissue response to treated tendons with regard to

safety. There were no signs of an aggressive or unusual

inflammatory response to the control or treated

allografts. Treated implants demonstrated a normal

inflammatory response through 8 weeks, which was

consistent with several ACL reconstruction studies in

rabbits showing inflammation from 2 to 6 weeks (Li

et al. 2007; Xu and Ao 2009; Soon et al. 2007). In

contrast, tissue processing involving glutaraldehyde or

dehydrothermal crosslinking of collagen biomaterials

has shown to improve mechanical properties, although

each resulted in failure as a result of cytotoxicity

(Chvapil et al. 1983; Gibeault et al. 1989) or premature

degradation (Weadock et al. 1996), respectively.

Gibeault et al. attributed long-term inflammation,

distention of collagen bundles, and disintegration of

glutaraldehyde processed pericardium intervertebral

disc replacements to hydrolytic release of cytotoxic

residues after 3 months post-implantation in rabbits

(Gibeault et al. 1989). Conversely, EDC forms zero-

length crosslinks with no intermediates to be released

during an inflammatory response. Collagen based

materials crosslinked with EDC have been shown to

accommodate cultured fibroblasts over 8 weeks (Car-

uso and Dunn 2005). EDC crosslinked dermal substi-

tutes have also successfully managed wound closure in

athymic mice (Powell and Boyce 2007). These studies

have reported no negative effects attributable to

antioxidant treatment. Thiourea has been shown to

successfully serve as a free radical scavenger for

irradiated bone grafts with low toxicity, but may be

considered carcinogenic (Purves and Griesbach 1947;

Deichmann et al. 1967). Commercially developed

sterilization techniques including Clearant� and Bio-

cleanse� have also been investigated in vivo to

determine treatment performance and safety. Clea-

rant� sterilized DBM implants were shown to have no

adverse effects on in a rat spinal fusion model (Alanay

et al. 2008). The Clearant Process� includes a radio

protectant component and performs ionizing radiation

under strict conditions (Grieb et al. 2006). Addition-

ally the decellularization capability of Biocleanse�

has been investigated for xenogenic cortical and

cancellous bone grafts. It was reported that antigenic-

ity of bovine bone allografts was reduced for bio-

cleanse treated allografts after implantation in non-

immune compromised rats (Supronowicz et al. 2008).

The data strongly suggest that radiation destabilizes

the integrity of tendon allografts, causing higher

susceptibility to biological and mechanical degrada-

tion. Control irradiated tendons exhibited greater

degradation under mechanical load, which is consis-

tent with accelerated implant breakdown by inflam-

matory cells, both due to radiation-induced matrix

damage. Radio protection shows the ability to atten-

uate premature degradation and maintain load bearing.

Although radio protection was successful at prevent-

ing premature failure in these models, it is unknown

whether the radio protective allografts will be effica-

cious for ACL reconstruction. Although it is desirable

to prevent graft failure via premature degradation,

prolonged stability may have disadvantages as well,

perhaps inhibiting new tissue ingrowth, or delaying

graft remodeling. ACL reconstruction studies are

required to determine the efficacy of radio protected

allografts.

Cell Tissue Bank (2013) 14:349–357 355

123

The long-term goal of this work is to develop a

protective method that would allow use of ionizing

radiation as a terminal sterilization method for mus-

culoskeletal allografts without the associated negative

effects on graft properties. Results of this study

suggest that radiation damage results in an accelerated

inflammatory response that could be associated with

implant degradation. Additionally, radiation effects

are more pronounced both in the in vivo environment,

and the simulated in vivo environment during loading

in collagenase, beyond that observed in routine tensile

testing. Most importantly, the radio protective treat-

ment provides maintenance of strength and stability

during a critical time of healing when grafts are prone

to failure. These encouraging results motivate a larger

animal study to evaluate the efficacy of radio protected

tendon allografts in an ACL reconstruction model.

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