Radioprotection provides functional mechanics but delays healing of irradiated tendon allografts...
-
Upload
michael-dunn -
Category
Documents
-
view
216 -
download
1
Transcript of Radioprotection provides functional mechanics but delays healing of irradiated tendon allografts...
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: [email protected]
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
Cell Tissue Bank
123
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
Cell Tissue Bank
123
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
Cell Tissue Bank
123
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)
Cell Tissue Bank
123
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)
Cell Tissue Bank
123
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
Cell Tissue Bank
123
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)
Cell Tissue Bank
123
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.
References
Barber FA (2003) Should allografts be used for routine anterior
cruciate ligament reconstructions? Arthroscopy 19(4):421.
doi:10.1053/jars.2003.50130
Bartlett RJ, Clatworthy MG, Nguyen TN (2001) Graft selection
in reconstruction of the anterior cruciate ligament. J Bone
Joint Surg Br 83(5):625–634
Beebe KS, Benevenia J, Tuy BE, DePaula CA, Harten RD,
Enneking WF (2009) Effects of a new allograft processing
procedure on graft healing in a canine model: a preliminary
study. Clin Orthop Relat Res 467(1):273–280. doi:
10.1007/s11999-008-0444-8
Buck BE, Malinin TI, Brown MD (1989) Bone transplantation
and human immunodeficiency virus. An estimate of risk of
acquired immunodeficiency syndrome (AIDS). Clin Ort-
hop Relat Res 240:129–136
Clancy WG Jr, Narechania RG, Rosenberg TD, Gmeiner JG,
Wisnefske DD, Lange TA (1981) Anterior and posterior
cruciate ligament reconstruction in rhesus monkeys. J Bone
Joint Surg Am 63(8):1270–1284
Dustmann M, Schmidt T, Gangey I, Unterhauser FN, Weiler A,
Scheffler SU (2008) The extracellular remodeling of free-
soft-tissue autografts and allografts for reconstruction of
the anterior cruciate ligament: a comparison study in a
sheep model. Knee Surg Sports Traumatol Arthrosc
16(4):360–369. doi:10.1007/s00167-007-0471-0
Fu FH, Bennett CH, Lattermann C, Ma CB (1999) Current
trends in anterior cruciate ligament reconstruction. Part 1:
biology and biomechanics of reconstruction. Am J Sports
Med 27(6):821–830
Gibbons MJ, Butler DL, Grood ES, Bylski-Austrow DI, Levy
MS, Noyes FR (1991) Effects of gamma irradiation on the
initial mechanical and material properties of goat bone-
patellar tendon-bone allografts. J Orthop Res 9(2):209–218
Grood ES, Walz-Hasselfeld KA, Holden JP, Noyes FR, Levy
MS, Butler DL, Jackson DW, Drez DJ (1992) The corre-
lation between anterior–posterior translation and cross-
sectional area of anterior cruciate ligament reconstructions.
J Orthop Res 10(6):878–885. doi:10.1002/jor.1100100617
Halliwell B, Gutteridge J (1989) Free radicals in biology and
medicine. Clarendon Press, Oxford
Harner CD, Olson E, Irrgang JJ, Silverstein S, Fu FH, Silbey M
(1996) Allograft versus autograft anterior cruciate liga-
ment reconstruction: 3- to 5-year outcome. Clin Orthop
Relat Res 324:134–144
Hoburg A, Keshlaf S, Schmidt T, Smith M, Gohs U, Perka C,
Pruss A, Scheffler S (2011) Fractionation of high-dose
electron beam irradiation of BPTB grafts provides signifi-
cantly improved viscoelastic and structural properties
compared to standard gamma irradiation. Knee Surg Sports
Traumatol Arthrosc 19(11):1955–1961. doi:10.1007/
s00167-011-1518-9
Ireland ML (2002) The female ACL: why is it more prone to
injury? Orthop Clin North Am 33(4):637–651
Jones DB, Huddleston PM, Zobitz ME, Stuart MJ (2007)
Mechanical properties of patellar tendon allografts sub-
jected to chemical sterilization. Arthroscopy 23(4):400–
404. doi:10.1016/j.arthro.2006.11.031
Lui P, Zhang P, Chan K, Qin L (2010) Biology and augmenta-
tion of tendon-bone insertion repair. J Orthop Surg Res
5:59. doi:10.1186/1749-799X-5-59
McAllister DR, Joyce MJ, Mann BJ, Vangsness CT Jr (2007)
Allograft update: the current status of tissue regulation,
procurement, processing, and sterilization. Am J Sports
Med 35(12):2148–2158. doi:10.1177/0363546507308936
Muneta T, Sekiya I, Yagishita K, Ogiuchi T, Yamamoto H,
Shinomiya K (1999) Two-bundle reconstruction of the
anterior cruciate ligament using semitendinosus tendon
with endobuttons: operative technique and preliminary
results. Arthroscopy 15(6):618–624. doi:10.1053/ar.1999.
v15.0150611
Nguyen H, Morgan DA, Forwood MR (2007) Sterilization of
allograft bone: effects of gamma irradiation on allograft
biology and biomechanics. Cell Tissue Bank 8(2):93–105.
doi:10.1007/s10561-006-9020-1
Noyes FR, Butler DL, Paulos LE, Grood ES (1983) Intra-
articular cruciate reconstruction. I: perspectives on graft
strength, vascularization, and immediate motion after
replacement. Clin Orthop Relat Res 172:71–77
Cell Tissue Bank
123
Peterson RK, Shelton WR, Bomboy AL (2001) Allograft versus
autograft patellar tendon anterior cruciate ligament recon-
struction: a 5-year follow-up. Arthroscopy 17(1):9–13
Prokopis PM, Schepsis AA (1999) Allograft use in ACL
reconstruction. Knee 6:75–85
Rappe M, Horodyski M, Meister K, Indelicato PA (2007)
Nonirradiated versus irradiated Achilles allograft: in vivo
failure comparison. Am J Sports Med 35(10):1653–1658.
doi:10.1177/0363546507302926
Salehpour A, Butler DL, Proch FS, Schwartz HE, Feder SM,
Doxey CM, Ratcliffe A (1995) Dose-dependent response
of gamma irradiation on mechanical properties and related
biochemical composition of goat bone-patellar tendon-
bone allografts. J Orthop Res 13(6):898–906
Scheffler SU, Schmidt T, Gangey I, Dustmann M, Unterhauser
F, Weiler A (2008) Fresh-frozen free-tendon allografts
versus autografts in anterior cruciate ligament reconstruc-
tion: delayed remodeling and inferior mechanical function
during long-term healing in sheep. Arthroscopy 24(4):
448–458. doi:10.1016/j.arthro.2007.10.011
Schimizzi A, Wedemeyer M, Odell T, Thomas W, Mahar AT,
Pedowitz R (2007) Effects of a novel sterilization process
on soft tissue mechanical properties for anterior cruciate
ligament allografts. Am J Sports Med 35(4):612–616
Schwartz HE, Matava MJ, Proch FS, Butler CA, Ratcliffe A,
Levy M, Butler DL (2006) The effect of gamma irradiation
on anterior cruciate ligament allograft biomechanical and
biochemical properties in the caprine model at time zero
and at 6 months after surgery. Am J Sports Med 34(11):
1747–1755
Seto A, Gatt CJ Jr, Dunn MG (2008) Radioprotection of tendon
tissue via crosslinking and free radical scavenging. Clin
Orthop Relat Res 466(8):1788–1795
Seto A, Gatt CJ Jr, Dunn MG (2009) Improved tendon radio-
protection by combined cross-linking and free radical
scavenging. Clin Orthop Relat Res 467(11):2994–3001.
doi:10.1007/s11999-009-0934-3
Seto AU, Gatt CJ Jr, Dunn MG (2012) Sterilization of tendon
allografts: a method to improve strength and stability after
exposure to 50 kGy gamma radiation. Cell Tissue Bank.
doi:10.1007/s10561-012-9336-y
Shelbourne KD, Patel DV (1995) Timing of surgery in anterior
cruciate ligament-injured knees. Knee Surg Sports Trau-
matol Arthrosc 3(3):148–156
Shelton WR, Treacy SH, Dukes AD, Bomboy AL (1998) Use of
allografts in knee reconstruction: I. Basic science aspects
and current status. J Am Acad Orthop Surg 6(3):165–168
Soderman K, Pietila T, Alfredson H, Werner S (2002) Anterior
cruciate ligament injuries in young females playing soccer
at senior levels. Scand J Med Sci Sports 12(2):65–68
Stringham DR, Pelmas CJ, Burks RT, Newman AP, Marcus RL
(1996) Comparison of anterior cruciate ligament recon-
structions using patellar tendon autograft or allograft.
Arthroscopy 12(4):414–421
Sun K, Tian SQ, Zhang JH, Xia CS, Zhang CL, Yu TB (2009)
ACL reconstruction with BPTB autograft and irradiated
fresh frozen allograft. J Zhejiang Univ Sci B 10(4):
306–316. doi:10.1631/jzus.B0820335
Taylor DW, Petrera M, Hendry M, Theodoropoulos JS (2011) A
systematic review of the use of platelet-rich plasma in
sports medicine as a new treatment for tendon and ligament
injuries. Clin J Sport Med 21(4):344–352. doi:10.1097/
JSM.0b013e31821d0f65
Vaishnav S, Thomas Vangsness C Jr, Dellamaggiora R (2009)
New techniques in allograft tissue processing. Clin Sports
Med 28(1):127–141. doi:10.1016/j.csm.2008.08.002
Vangsness CT Jr (2006) Soft-tissue allograft processing con-
troversies. J Knee Surg 19(3):215–219
Weiler A, Peine R, Pashmineh-Azar A, Abel C, Sudkamp NP,
Hoffmann RF (2002) Tendon healing in a bone tunnel. Part
I: biomechanical results after biodegradable interference fit
fixation in a model of anterior cruciate ligament recon-
struction in sheep. Arthroscopy 18(2):113–123
Cell Tissue Bank
123