ORIGINAL PAPER

Radioprotection provides functional mechanics but delayshealing of irradiated tendon allografts after ACLreconstruction in sheep

Aaron U. Seto • Brian M. Culp •

Charles J. Gatt Jr. • Michael Dunn

Received: 19 February 2013 / Accepted: 22 June 2013

� Springer Science+Business Media Dordrecht 2013

Abstract Successful protection of tissue properties

against ionizing radiation effects could allow its use

for terminal sterilization of musculoskeletal allografts.

In this study we functionally evaluate Achilles tendon

allografts processed with a previously developed

radioprotective treatment based on (1-ethyl-3-(3-

dimethylaminopropyl)carbodiimide) crosslinking

and free radical scavenging using ascorbate and

riboflavin, for ovine anterior cruciate ligament recon-

struction. Arthroscopic anterior cruciate ligament

(ACL) reconstruction was performed using double

looped allografts, while comparing radioprotected

irradiated and fresh frozen allografts after 12 and

24 weeks post-implantation, and to control irradiated

grafts after 12 weeks. Radioprotection was successful

at preserving early subfailure mechanical properties

comparable to fresh frozen allografts. Twelve week

graft stiffness and anterior-tibial (A-T) translation for

radioprotected and fresh frozen allografts were com-

parable at 30 % of native stiffness, and 4.6 and 5 times

native A-T translation, respectively. Fresh frozen

allograft possessed the greatest 24 week peak load at

840 N and stiffness at 177 N/mm. Histological evi-

dence suggested a delay in tendon to bone healing for

radioprotected allografts, which was reflected in

mechanical properties. There was no evidence that

radioprotective treatment inhibited intra-articular graft

healing. This specific radioprotective method cannot

be recommended for ACL reconstruction allografts,

and data suggest that future efforts to improve allograft

sterilization procedures should focus on modifying or

eliminating the pre-crosslinking procedure.

Keywords Allograft � Sterilization �Ionizing radiation � ACL reconstruction � In vivo �Radioprotection � Crosslinking �Free radical scavenging

Introduction

Anterior cruciate ligament (ACL) injuries represent

the most common musculoskeletal injuries resulting in

complete tissue failure (Muneta et al. 1999), with an

occurrence estimated at 1 in 3,000 in the United States

(Fu et al. 1999; Ireland 2002; Shelbourne and Patel

1995; Soderman et al. 2002). Surgical reconstruction

has become the standard treatment due to insufficient

ability for the ACL to self repair. Use of graft material

for reconstructions has been primarily dominated by

autogenous tissue (Bartlett et al. 2001; Peterson et al.

2001; Harner et al. 1996; Stringham et al. 1996),

although greater consideration and use of allogenic

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

Department of Orthopaedic Surgery, Robert Wood

Johnson Medical School - Rutgers University,

51 French St MEB Rm 424, P.O. Box 19,

New Brunswick, NJ 08901, USA

e-mail: dunnmg@rwjms.rutgers.edu

123

Cell Tissue Bank

DOI 10.1007/s10561-013-9385-x

tissue as an alternative has developed in the past

decade. A major obstacle preventing reliance on

allografts has been the potential for disease transmis-

sion from donor tissues (Buck et al. 1989; Barber

2003). Ionizing radiation has been an established

sterilization method but high dose radiation is not

regularly accepted by tissue banks for use on allografts

(Prokopis and Schepsis 1999; Vaishnav et al. 2009).

Those that perform radiation, limit to lower doses

from 10 to 18 kGy (McAllister et al. 2007; Vangsness

2006). Others have sought alternative sterilization

methods (Schimizzi et al. 2007; Jones et al. 2007) or

relied solely on current screening protocols (McAll-

ister et al. 2007). Radiation at higher doses sufficient to

achieve sterilization over a range of bioburden have

been associated with detrimental effects on mechan-

ical properties (Salehpour et al. 1995; Gibbons et al.

1991; Seto et al. 2008) as well as graft healing (Seto

et al. 2008; Nguyen et al. 2007). These effects are

caused by radiation-generated free radical modifica-

tions to collagen microstructure (Halliwell and Gut-

teridge 1989).

We have previously shown that initial mechanical

properties could be fully maintained for tendon

allografts treated with a radioprotective process after

exposure to 50 kGy of ionizing radiation (Seto et al.

2008, 2009). This radioprotective treatment was

developed to counteract free radical effects by com-

bining a pre-irradiation crosslinking procedure and

free radical scavenging during irradiation. Radiopro-

tection also stabilized irradiated allografts in a colla-

genase challenge under mechanical stimulus,

simulating an in vivo environment (Seto et al. 2012).

Radioprotection also provided greater stability of

sterilized implants against host degradation compared

to untreated tendon after implantation in a rabbit knee

model (Seto et al. 2012).

Our previous data motivates a functional evalua-

tion of radioprotected allografts for ACL reconstruc-

tion in an ovine in vivo model. Studies investigating

in vivo performance of processed allografts are

limited, especially for large animal ACL reconstruc-

tions. In the current study, we hypothesized that our

treatment would provide initial radioprotective

effects for irradiated allografts, and knees recon-

structed with radioprotected allografts would possess

functional mechanical stability comparable to fresh

frozen allografts. Secondly, we hypothesized that

radioprotected allografts would display normal bone

tunnel healing and ligamentization. Demonstration of

competent knee function and absence of any irregu-

larities in graft healing would further validate the

potential for radioprotective methods to be used in

conjunction with ionizing radiation to improve allo-

graft safety.

Methods

We performed ACL reconstructions using Achilles

tendon allografts on left hindquarter sheep knees

comparing the following groups: control-irradiated

(CO), radioprotected-irradiated (RP), and fresh frozen

(FF). Upon sacrifice, sheep knee joints were assessed

mechanically or histologically in order to evaluate our

hypotheses. ACLs of right knees served as native

contralateral controls.

Achilles tendons were harvested from frozen hind-

limbs of Colombia Rambouillet sheep (Colorado

State, School of Veterinary Medicine, CO). Hindlimbs

were thawed at room temperature and careful dissec-

tion was done to expose the Achilles tendon from the

ankle to hoof. Sharp excision was done to remove

15 cm of tendon tissue proximal to the ankle inter-

section, which was then stored at -20 �C in saline

soaked gauze until processing.

Control irradiated tendons were soaked in saline

(pH 7.4) 1 tendon per 100 ml on an oscillating shaker

for 48 h at room temperature. They were then

individually placed in polystyrene sample tubes with

100 ml saline. All samples designated for radiation

were shipped to Sterigenics Inc. (Rockaway, NJ, USA)

for gamma radiation at 50 kGy at room temperature.

Upon return, sample tube contents were removed in a

sterile field and grafts were stored frozen at -20 �C.

For the crosslinking procedure, treated tendons

were soaked in 10 mM/5 mM EDC/NHS [(1-ethyl-3-

(3-dimethylaminopropyl)carbodiimide) and (N-hydro-

xyl succinimide)] solution (both Sigma-Aldrich, St.

Louis, MO, USA), 1 tendon per 100 ml on an

oscillating shaker for 6 h. The tendons were then

washed thoroughly 3 times with deionized water. They

were then soaked in 100 mM sodium phosphate

monohydrate (Sigma-Aldrich, St. Louis, MO, USA)

solution in deionized water for 2 h on the shaker. This

was followed by soaking in saline for 16 h to conclude

the crosslinking process. Finally they were transferred

to a free radical scavenger cocktail solution composed

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of 50 mM sodium ascorbate and 12.5 mM riboflavin-

5-phoshate (both Sigma-Aldrich, St Louis, MO, USA)

in deionized water for 24 h. After processing they were

stored in polystyrene sample tubes filled with 100 ml

ascorbate/riboflavin-5-phosphate solution and shipped

with control tendons for radiation. After return, the

tube solution was removed and allografts were stored

at -20 �C.

Fresh frozen tendons were aseptically harvested,

both instrument and field disinfection was done using

70 % ethanol. After harvest, tendons were placed in

90 % ethanol for 1 h with agitation, then in sterile

saline for 1 h. Finally in a sterile field, the tendons

were removed and rinsed once more in 90 % ethanol

for 1 min, then sterile saline for 5 min. Tendons were

packaged in sterile gauze and saline, and stored in a

-80 �C freezer until implantation. This procedure for

aseptic processing and storage is consistent with

current tissue banking procedures (Beebe et al. 2009;

Shelton et al. 1998).

There were 24 total sheep used for this study, with

n = 3 for CO (12 weeks), n = 10 for FF (5 each for

12 and 24 weeks), n = 10 for RP (5 each for 12 and

24 weeks). One sheep was lost prematurely due to

illness, but was replaced. Surgeries began with an

initial 12 week evaluation of CO allografts. After

analyzing this data, it was decided to reallocate the

samples to increase the size of the other groups (n = 4

to n = 5).

The ACL reconstruction was performed according

to an approved IACUC protocol. After the surgical leg

was prepped and draped, anterolateral and anterome-

dial arthroscopy portals were established. The arthro-

scope was introduced through the lateral portal and a

diagnostic examination of the knee joint was per-

formed. A partial resection of the fat pad was

performed to improve visualization. Next the native

ACL was resected. A notchplasty was then performed

with a motorized shaver to allow improved visualiza-

tion in the intercondylar notch and aid in the anatomic

placement of the implant. Using and Acufex tibial

ACL guide (Smith and Nephew, London, UK), a guide

wire was drilled through the anteromedial cortex into

the knee joint through the tibial insertion site of the

native ACL. This was then over reamed with a

cannulated reamer to create the tibial tunnel. A guide

wire was then inserted into the knee joint through the

medial portal, positioned on the femoral insertion site

of the native ACL, and drilled through the lateral

femoral condyle of the femur and out of the antero-

lateral thigh. A small incision was made where the

guide wire penetrated the skin and an 8 mm femoral

tunnel was created using a cannulated reamer. The

Achilles tendon allograft was looped over a #5

Ethibond suture (Ethicon, Somerville, NJ, USA).

Using arthroscopic assistance, the suture graft com-

plex was pulled into the tibial tunnel and pulled

through the knee joint into the femoral tunnel. The

implant was secured at the femoral end using an extra-

cortical polypropylene button. The graft was tensioned

and secured to the tibia with a 7 mm 9 25 mm RCI

interference screw (Smith and Nephew, London, UK).

Excess allograft length was cut to be flush with the

tibial surface. All incisions were then closed with 2-0

Vicryl suture (Ethicon, Somerville, NJ, USA).

After surgery, animals were returned to cages and

administered antibiotics and analgesics, while under

close observation. Gait, ambulation, and signs of pain

were monitored until sufficient recovery allowed

transportation to a local farm for uninhibited rehabil-

itation, while gait and weight bearing were monitored

in a daily basis. After 12 or 24 weeks post-op, animals

were returned and sacrificed. The surgical knee joint

including the femoral and tibial heads with the graft

and surrounding tissues were harvested. One animal

per group was used for histological analysis, and the

remaining four for mechanical testing. Contra-lateral

right knees were also removed to obtain native ACL

data.

Non-implanted Achilles tendon allografts were

tested in tension to determine initial mechanical

properties. Tendons were gripped in cryogenic clamps

with a 7 cm gauge length, allowing the remaining

4 cm on each side to pass fully through each grip with

1 cm protruding outside the grip for maximum contact

area. During this testing, tendons were in a single

strand orientation, but were looped over when

implanted. Preconditioning was performed for 10

cycles to 50 N at 60 mm/min. Tendons were then

pulled at 60 mm/min.

Knee joints were mechanically tested as a femoral-

ACL graft-tibial complex. The joint complex was

mounted on custom jigs allowing proper angling of the

femoral and tibial knee sections to align the ACL graft

parallel to the load axis. Femoral and tibial sections of

the knee were cleaned, while leaving the tissue

surrounding the joint capsule intact. These sections

of bone were trimmed to fit into the grip wells, and

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were then potted with methyl methacrylate into the

wells. Once dry, the wells were mounted with the grips

onto the mechanical tester (Instron, Canton, MA,

USA) at 60� flexion (Fig. 1). Careful dissection of the

tissues around the knee was done to leave only the

ACL or graft intact. Testing was finally performed first

through a non-destructive anterior-tibial (A-T) trans-

lation test, and then tension to failure.

While at 60� flexion, the graft was subjected to

50 N for 10 cycles at a speed of 60 mm/min. Anterior-

tibial translation was measured as the deformation

experienced by the graft during the last cycle. After

translational measurement, the grafts were pulled in

tension to failure at a speed of 60 mm/min. Failure

modality was characterized and inspection of graft

ends was done to assess the possibility of slippage. If

slippage occurred, the graft was re-gripped using cyro-

clamps (Bose Electroforce, Eden Prairie, MN, USA)

and retested. The following structural properties were

recorded: breaking load, deformation, stiffness, and

energy.

To prepare for histological analysis all tissues

surrounding the ACL graft were removed by dissec-

tion. Similarly sections of femur and tibia were cut

away using a slow cutting rotating saw (Buehler, Lake

Bluff, IL, USA) leaving only the bone tunnels. The

remaining bone and graft was fixed in 10 % buffered

formalin. The graft and bone tissue were then charac-

terized into 3 zones: the femoral tunnel with graft

tissue, the intra-articular zone with only graft tissue,

and the tibial tunnel with graft tissue. One represen-

tative section of each of the three zones, for each

sample designated for histology was sent for process-

ing and staining at AML Laboratories Inc. (Baltimore,

MD, USA). In addition, one sample from time zero

(unimplanted) tendon allografts were also included.

Longitudinal slices cut at 5 micron thickness were

taken from each section and were stained with

hematoxylin and eosin. Bone tunnel and intra-articular

zone images were assessed for key characteristics

pertinent to graft healing (Table 1) adapted from a

grading scheme developed by Lui et al. (2010). For

representative bone tunnel slides, we assessed pro-

gression of graft degradation, fibro-vascular tissue

presence, cellularity, and sharpey-like fiber presence.

In the intra-articular zone we similarly focused on graft

degradation and cellularity relative to native ACL.

Results

Radiation effects were evident in initial properties for

control untreated allografts, while radioprotected

allografts were comparable to fresh frozen. Control

irradiated allografts had the lowest stiffness (203.0 N/

mm), followed by fresh frozen (249.5 N/mm), and

treated irradiated (264.7 N/mm) allografts, although

differences were not significant (Fig. 2a). Failure load

of time zero tendon allografts could not be determined

accurately due to cyrogenic grip limitations. Achilles

tendons for each condition exhibited slippage at loads

beyond the load capacity of the grips (2,250 N, Bose

Electroforce).

Radioprotected allografts possessed similar early

subfailure properties to fresh frozen allografts, but this

trend deviated over time. Prior to sacrifice, all animals

returned to normal ambulation and activity during

rehabilitation, and there were no premature allograft

failures. After 12 weeks, subfailure properties such as

anterior-tibial (A-T) translation and stiffness were

Fig. 1 Custom built grips were used to mount reconstructed

knees to perform non-destructive anterior-tibial translation

testing at 60� flexion, followed by tension to failure to determine

structural properties

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similar for RP allografts compared to FF allografts

(Fig. 2c, d). Structural properties directly influenced

by point of failure, such as break load and energy

absorbed were greater for CO and FF allografts

compared to RP allografts after 12 weeks (Fig. 2b).

During testing, two of the RP grafts pulled through the

femoral tunnel at an average of 106 N. Of the two

grafts that pulled out, the femoral ends were gripped in

a cryo-clamp and retested, resulting in pullout from

the tibial tunnel. Finally, one strand of a looped graft

remained testable and was re-gripped using cryogenic

grips at both ends. Retesting of the remaining graft

material resulted in a break load of 320 N. Theoret-

ically, an intact double looped graft would possess a

break load of at least 640 N (Fig. 2b). At 24 weeks, FF

grafts had a break load of 840 N, which was signif-

icantly higher compared to 12 weeks at 289 N. FF

mechanical properties were superior to RP grafts,

which broke at 186 N at 24 weeks, which was similar

to CO and FF grafts at 12 weeks (Fig. 2b). After

24 weeks, tensile testing of RP grafts resulted in intra-

articular failure and no longer slippage through bone

tunnels. Sheep native ACL had a break load of 1952 N

and stiffness of 256 N/mm, which was determined

from contra-lateral limbs. After 24 weeks, break load

and stiffness of FF grafts were 43 and 69 % of native

ACL values, and for RP grafts 9 and 26 % of native

values. Trends observed for load and stiffness were

similar for energy absorbed.

Additionally, trends observed after histological

analyses were supportive of results obtained from

mechanical testing. At 12 weeks, FF graft material

within the bone tunnel showed a moderate level of

cellular infiltration through the tendon graft. A blended

zone of fibro-vascular tissue was evident between the

graft and bone interface (Fig. 3). Within this zone of

fibro-vascular tissue, fibers resembling Sharpey’s fibers

were present along the bone tissue interface. Control

irradiated grafts at 12 weeks showed a high cellular

presence throughout the graft. There was also a discrete

zone present between the graft and bone, more prom-

inent further from the articular end of the tunnel. Graft

degradation was most concentrated in this area, though

the presence of the original graft was substantial

sharpey-like fibers were also present but not uniformly

distributed. Cellular activity was much higher compar-

atively for RP grafts within the bone tunnels at

12 weeks. There was a buildup of inflammatory cells

Table 1 Grading schemes tendon to bone and intra-articular

healing

Histologic features-bone tunnels

Graft degeneration

None (100 % original graft) 0

Slight ([75 % original graft) 1

Moderate (\50 % original graft) 2

Major (\25 % original graft) 3

Graft remodeling

Major ([75 %) 0

Moderate (\50 %) 1

Slight (\25 %) 2

None (0 %) 3

Percentage of fibrous tissue

None (0 %) 0

Slight (\25 %) 1

Moderate (\50 %) 2

Major (100 %)

Sharpey-like fiber presence

None (0 %) 0

Slight (sporadic) 1

Moderate (abundant, non-continuous) 2

Major (abundant, continuous) 3

Cellularity

High (ovoid, mainly inflammatory) 0

Moderate (inflammatory/fibroblast presence) 1

Slight (localized pockets) 2

Normal (spindle shaped, mainly fibroblast) 3

Histologic features-intra-articular zone

Crimp presence

None 0

Slight (sporadic, depressed) 1

Moderate (abundant, unorganized) 2

Major (abundant, organized, uniform direction) 3

Graft remodeling

None (0 %) 0

Slight (\25 %) 1

Moderate (\50 %) 2

Major (100 %) 3

Cellularity

High (ovoid, mainly inflammatory) 0

Moderate (inflammatory/fibroblast presence) 1

Slight (localized pockets) 2

Similar to native ACL 3

This table describe the grading scheme used to judge tendon to

bone healing in the bone tunnels and ligamentization in the

intra-articular zones adapted from Lui et al. (2010)

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along the perimeter of the graft and bone interface. A

clearly defined fibrous zone was established between

the bone and graft, with distinct borders transitioning

between bone, fibrous zone, and original graft (Fig. 3).

The tendon graft was largely uninterrupted with areas

still possessing original crimp pattern.

After 24 weeks, for the FF grafts, vascular forma-

tion was increased while cellularity was greatly

decreased compared to 12 weeks. Additionally there

was a continuous and uniform presence of sharpey-

like fibers along the bone interface (Fig. 3). These

trends from 12 to 24 weeks were greatly lagged for RP

grafts. At 24 weeks there was a small decrease in

cellularity, which was distributed more uniformly

through the graft. In central regions of the graft

original tendon material was still evident, and the

emergence of sharpey-like fibers.

In the intra-articular zone of FF allografts at

12 weeks there was a moderate inflammatory pres-

ence. Cellular activity was high and concentrated

around areas of original graft material. Control

allografts displayed lower cellularity than other groups

with localized areas of high activity. At 24 weeks in FF

allografts, cellularity was decreased and approaching

native ACL. Tissue was mainly populated with spindle

shaped fibroblasts. There was moderate cellularity that

was distributed throughout the graft with pockets of

inflammatory cells. Compared to the response in the

bone tunnel, there were fewer intra-articular differ-

ences between treatment groups (Fig. 4). Overall graft

healing for RP allografts at 24 weeks was similar to a

level observed for FF and CO grafts at 12 weeks.

Discussion

Allograft safety and tissue banking procedures could

be greatly improved if terminal sterilization of allo-

grafts can be achieved. Radioprotection and successful

maintenance of mechanical and biological graft prop-

erties may be a potential solution to regular terminal

sterilization using ionizing radiation. This study

Fig. 2 a–d Bars are reported as percent post-operative

structural properties relative to native ACL at 12, 24 weeks.

Bar labels represent actual values for break load (newtons),

stiffness (newtons/millimeters), and anterior-tibial (A-T) trans-

lation (millimeters). mp \ .001 (compared to TR 24 weeks).

Both radiation and radioprotective effects were evident for

initial stiffness of sheep Achilles allografts (a). After 12 weeks,

failure properties of fresh frozen and control allografts were

superior than radioprotected, but subfailure mechanics were

comparable (c, d). After 24 weeks, the increase in break load in

radioprotected allografts compared to fresh frozen allografts

was significantly lower, indicating a delayed transition to

remodeling (b)

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evaluates radioprotected tendons as functional allo-

grafts for ACL reconstruction in sheep. We hypoth-

esized that radioprotection would stabilize initial graft

mechanical properties after radiation, and furthermore

radioprotected allografts would possess functional

post-implantation mechanical properties comparable

to fresh frozen allografts (representing the current gold

standard for allograft use). Finally, we hypothesized

that radioprotected allografts would follow regular

graft healing. Based on our results, our hypothesis was

not supported with regard to post-implantation

mechanics and graft healing.

There are several limitations to this study that must

be considered. Determination of initial failure

Fig. 3 Longitudinal bone tunnel images for control (CO),

radioprotected (RP), and fresh frozen (FF) grafts and fresh

frozen sharpey-like fibers, after 12 and 24 weeks post-implan-

tation. (magnification = 940, scale = 50 microns, magnifica-

tion = 9100, scale = 100 microns). Delayed tendon to bone

healing and ligamentization was observed for radioprotected

allografts after histological analysis, supporting mechanical

results. For radioprotected allografts at 12 weeks, there was

clear distinction between bone (B), fibro-vascular tissue (F), and

original tendon (T). The uniform presence of sharpey-like fibers

(SF) was most established in fresh frozen allografts after

24 weeks. Although graft healing was delayed, there was no

evidence that the process was inhibited

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properties was not possible due to load capacity

limitations of cryogenic clamps. The large thickness

and overall size of sheep Achilles tendons also

contributed to gripping difficulties. There is currently

no method to test large animal soft tissues with

regularity. The use of methyl methacrylate fixation is

not possible, unlike for bone-tendon-bone allografts.

Additionally, the 24 week control group was discon-

tinued. After observing a significant decline in mechan-

ical properties for control sterilized allografts after

12 weeks, the 24 week group was redistributed to avoid

the potential for premature failure while increasing

sample sizes of fresh frozen and radioprotected groups.

Radiation and radioprotective effects were evident

for initial graft mechanical properties. Radioprotection

provided successful stabilization of allograft stiffness

after exposure to radiation comparable to non-irradi-

ated allografts. Control sterilized allografts displayed

the lowest stiffness which confirmed known radiation

effects on tendon allografts. An 18 % reduction in

stiffness was observed for tendons irradiated at

50 kGy. Similar reductions in stiffness were observed

for goat bone-tendon-bone allografts, 17 % at 30 kGy

(Gibbons et al. 1991) and 18 % at 40 kGy (Salehpour

et al. 1995). Initial in vivo stiffness was expected to be

greater after implantation in the looped orientation. As

mentioned, failure properties could not be determined

due to grip load limitations, tendons were observed to

slip above 2,250 N. Weiler et al. observed an average

break load of Achilles tendons from merino sheep of

1,120 N (Weiler et al. 2002). The allografts used for

this study were obtained from larger sheep compared

to the study by Weiler et al. [average 82 kg compared

to 51 kg (Weiler et al. 2002)]. Although initial break

loads of irradiated allografts were greater than

observed by Weiler et al. and may seem acceptable,

radiation effects may be amplified by in vivo condi-

tions in the knee. In previous studies we have observed

greater sensitization to radiation effects with exposure

to protease components of synovial fluid and mechan-

ical stimulation (Seto et al. 2012). As a result, initial

mechanical properties cannot be an absolute predictor

of in vivo performance.

Despite irradiation effects, we observed compara-

ble mechanical properties for control allografts rela-

tive to fresh frozen allografts after 12 weeks. Based on

the comparison of 12 week mechanical data, we would

predict that a control 24 week group would also

observe a similar increase as fresh frozen. It is critical

for allografts to maintain sufficient mechanical stabil-

ity over a functional load range and over sufficient

time to allow neotissue formation and organization to

restore graft strength. It is possible that although initial

mechanical properties were weakened, control grafts

experienced loads below the threshold to cause failure

during early healing. As remodeling begins, mechan-

ical properties will rebound independent of initial

mechanical properties and radiation effects. Similarly,

Schwartz et al. showed that material properties of

BPTB allografts irradiated at 40 kGy were comparable

to unirradiated allografts 6 months after ACL recon-

struction in goats (Schwartz et al. 2006). Additionally,

the success of ACL reconstruction grafts in this study

also benefited from doubled over orientation. Greater

graft properties have been observed by inserting more

graft material into the ACL space (Grood et al. 1992;

Clancy et al. 1981; Noyes et al. 1983). Poor clinical

results have been observed with irradiated ACL

reconstruction tissues frequently involving premature

rupture (Sun et al. 2009; Rappe et al. 2007). As a result,

sterilization of allografts using ionizing radiation has

not been accepted among clinicians. Detrimental dose

dependent radiation effects on allograft properties

have been well established (Seto et al. 2008; Salehpour

et al. 1995). Radiation effects may show further

influence as mechanically compromised tissues are

further weakened under dynamic loading in an in vivo

environment.

Fresh frozen allografts displayed superior mechan-

ical properties compared to radioprotected. After

12 weeks, both control and fresh frozen allografts

0

2

4

6

8

10

12

14

16

18

CO 12 wk RP 12 wk RP 24 wk FF 12 wk FF 24 wk

His

tolo

gy

Sco

re

IA BT

Fig. 4 Total histology scores show the delayed healing

observed for radioprotected (RP) allografts compared to fresh

frozen (FF) and control (CO) allografts in the bone tunnels (BT)

and intra-articular zone (IA)

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possessed greater ultimate structural properties over

this time compared to radioprotected allografts. In

contrast, subfailure properties, specifically stiffness

and anterior-tibial translation, were comparable. Dur-

ing post-operative recovery, sheep were allowed

uninhibited motion of reconstructed knees and dis-

played active behavior without rupture of the grafts.

This suggests that subfailure mechanics of the radio-

protected allografts were sufficient to maintain knee

stability and functionality over a load range represen-

tative of normal activity. A slight rebound in mechan-

ical properties was observed for radioprotected

allografts from 12 to 24 weeks but was greater for

fresh frozen allografts. These findings suggest that

graft healing is slower for radioprotected allografts

compared to fresh frozen. Ultimate structural proper-

ties of ACL grafts indicate the weakest point where

failure occurs. Lower break loads were observed for

radioprotected allografts at early time points due to

immature tendon to bone anchoring for radioprotected

allografts, and was also indicated by slippage of the

grafts from the bone tunnel during mechanical testing.

Although delayed biological fixation was observed,

radioprotected allografts still possessed substantial

integrity as observed after retesting of slipped grafts.

This suggested radioprotection successfully counter-

acted radiation effects on initial graft properties,

however influence on tendon to bone healing was a

drawback.

Given a longer healing period, we would expect

improved biological fixation and subsequent mechan-

ical properties. Indeed, a slight recovery of mechanical

properties from 12 to 24 weeks for radioprotected

allografts suggests a transition to matrix synthesis and

remodeling. Full recovery of ACL reconstruction and

maturation of the ACL graft continues beyond

24 weeks in humans and similarly in this large animal

model. In this study we have focused on the initial

6 months during where any potential contributors of

catastrophic graft failure would be evident. Mechan-

ical properties of the mature graft were not determined

from this study.

Histological observations support delayed tendon to

bone fixation observed in mechanical data. Within the

bone tunnels breakdown of radioprotected allografts

was slower compared to fresh frozen. Graft pullout

during tensile testing was reflected by slower emer-

gence of sharpey-like fibers for radioprotected allo-

grafts. At later sacrifices, these fibers were more

established and slippage was no longer experienced

during testing. In fresh frozen allografts over

12–24 weeks, the establishment of denser tissue within

the bone tunnels and reduction of cellularity closer to

native density was indicative of active remodeling.

Scheffler and Dustmann et al. observed a similar

progression of graft healing over 12–52 weeks for

other ACL reconstructions in sheep using allogenic

tendon tissue (Scheffler et al. 2008; Dustmann et al.

2008). There was no evidence suggesting radioprotec-

tive processing would prevent graft healing, and there

were no indications of chronic inflammation or cytox-

icity. Prominent inflammatory presence for radiopro-

tected allografts at 24 weeks is characteristic of

cellular proliferation preceding remodeling. Similar

indications in the intra-articular zone were also

observed for graft degradation and cellularity.

Impeded inflammatory infiltration and initial graft

breakdown, ultimately influencing biological fixation

for radioprotected allografts was likely the result of

EDC surface crosslinking.

Potential improvements to tendon to bone healing

of radioprotected allografts can begin with directly

addressing the crosslinking procedure. EDC cross-

linking is an important radioprotective component

acting to counter radiation damage to collagen micro-

structure. The current data suggest there needs to be a

balance achieved between enhancing mechanical

properties and mediating cellular attachment and

infiltration. Ultimately a lower concentration of

crosslinker may be used to facilitate infiltration, while

still affording mechanical integrity. Alternatively, to

potentially reduce surface crosslinking while ensuring

bulk crosslinking, a crosslinker with slower reaction

kinetics or better penetration could be considered.

Additionally, several growth factors have been shown

to enhance integration, many of which have been

noted to be released by activated platelets in PRP

constructs (Taylor et al. 2011), which could be

included as a supplement to tendon allografts.

Potential mitigation of radiation-induced free radical

effects has also been investigated using a method

known as fractionation, more commonly used for

cancer treatment. Fractionation is most applicable for

ebeam radiation, which is more adept to applying

multiple doses quickly. Rather than applying a single

34 kGy dose, a succession of 3.4 kGy doses are

applied 10 times. This is thought to result in smaller

energies produced and therefore limit the amount of

Cell Tissue Bank

123

free radicals generated. Hoburg et al. has reported that

this method of radiation shows less deleterious effects

on mechanical properties of human BPTB allografts

(Hoburg et al. 2011). Further studies are required to

confirm equivalent sterilization efficiency.

Radioprotection of the Achilles tendon allografts

was successful in maintaining initial mechanical

properties. We observed normal but delayed graft

healing in radioprotected allografts, which also con-

tributed to delayed mechanical recovery compared to

fresh frozen allografts. This may have been the result

of EDC component of the treatment and over-cross-

linking of the graft surface may have caused impeded

cellular infiltration. There was no evidence against

achieving normal biological fixation over time.

Despite slower bone tunnel healing for knees recon-

structed with radioprotected allografts and resulting

structural properties, knee stability was still main-

tained over functional loads as grafts remained intact

during unrestricted rehabilitation. Based on the find-

ings from this study, the radioprotective treatment in

its current iteration, cannot be recommended for

allografts used for ACL reconstruction. Re-evaluation

of the radioprotective treatment for future studies will

require a balance between achieving protection of

mechanical properties and facilitating graft healing,

specifically cellular infiltration. The results of this

study do not deter the potential benefit of radiopro-

tective methods. With further modification, successful

radioprotection may allow terminal sterilization of

allografts using ionizing radiation.

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