Cell Tissue Bank (2009) 10:333–340
DOI 10.1007/s10561-009-9138-z
Storage conditions do not have detrimental effect
on allograft collagen or scaffold performance
E. L. Abreu Æ M. P. Palmer Æ M. M. Murray
Received: 10 April 2009 / Accepted: 22 May 2009 / Published online: 9 June 2009
Ó Springer Science+Business Media B.V. 2009
Abstract Musculoskeletal allografts are a valuable
alternative to autograft tissue in orthopaedic surger-
ies. However, the effects of the allografts’ storage
history on the collagen and subsequent allograft
scaffold properties are unknown. In this study, we
hypothesized that freezing and refrigeration of allo-
grafts for 1 week would alter the biologic perfor-
mance and mechanical properties of the allograft
collagen. Allograft collagen was characterized by
SDS–PAGE migration pattern, amino acid profile and
measured denaturation. Scaffolds made from allo-
graft collagen were evaluated for fibroblast prolifer-
ation, platelet activation and scaffold retraction.
Collagen gelation kinetics (elastic and inelastic
moduli and the viscous-elastic transition point) were
also evaluated. Fibroblast proliferation, platelet acti-
vation and scaffold retraction results showed only
minor, though statistically significant, differences
between the storage groups. In addition, there were
no significant differences in rheological properties or
collagen biochemistry. In conclusion, this study
suggests that freezing or refrigeration for 1 week
does not appear to have any detrimental effect on the
mechanical properties and biologic performance of
the collagen within allografts.
E. L. Abreu (&)
M. P. Palmer
M. M. Murray
Department of Orthopaedic Surgery, Children’s Hospital
of Boston, 300 Longwood Ave, Enders 1022, Boston, MA
02115, USA
e-mail: Eduardo.Abreu@childrens.harvard.edu
Keywords Allograft
Storage
Collagen

Scaffold
Introduction
Musculoskeletal allografts may represent an advan-
tageous graft choice for orthopaedic reconstructive
surgeries. Some of the benefits of using allografts are
shorter operative times, decreased postoperative pain,
lack of donor site morbidity and earlier rehabilitation
(Caldwell and Shelton 2005; Cohen and Sekiya
2007). However, the storage of tissue, often required
between procurement and implantation (Williams
et al. 2005), is thought to have the potential to
adversely affect the allograft .
Previous studies have shown variable results
regarding the effect of storage on graft quality
(Viidik and Lewin 1966; Brighton 1979; Woo et al.
1986; Schachar et al. 1994; Oates et al. 1995; Smith
et al. 1996; Sammarco et al. 1997; Williams et al.
2003; Rohde et al. 2004; Williams et al. 2005;
Malinin et al. 2006; Pennock et al. 2006; Lightfoot
et al. 2007; Williams et al. 2007; Giannini et al.
2008). Optimum refrigeration times for osteochondral
allografts have been reported in the literature to be
less than 5–7 days (Schachar et al. 1994; Rohde et al.
2004) or as long as 42 days (Williams et al. 2007).
Also, while some investigations have shown that
freezing has a deleterious effect on the mechanical
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334
properties of musculoskeletal allografts (Viidik and
Lewin 1966; Smith et al. 1996; Giannini et al. 2008),
others did not find statistically significant differences
between the mechanical properties of fresh and
frozen ligaments (Woo et al. 1986).
In this study, we hypothesized that storage under
refrigeration or freezing conditions would have
significant effects on the collagen within the graft—
specifically, that the soluble collagen fraction known
to be efficacious in stimulating cell proliferation and
collagen production in tissue engineering applications
would be altered with one or both of these storage
conditions.
To test this hypothesis, tendons were procured and
the acid-soluble collagen fraction was obtained either
immediately or after 1 week of storage, frozen or
under refrigeration. The acid soluble collagen frac-
tion was analyzed for biochemical collagen content
and rheologic behavior. In addition, the biologic
functionality of the solubilized collagen fraction was
evaluated by creating 3-D collagen-platelet compos-
ite (CPC) gels, seeding these with anterior cruciate
ligament (ACL) cells and measuring the cellular
proliferation rates and gel retraction, as well as the
platelet activation which is known to be stimulated by
the collagen fraction.
Materials and methods
Storage conditions
Rat tail tendon collagen was used for all experimental
groups. Tails were obtained from breeder rats used in
other Institutional Animal Care and Use Committee
approved studies and that had been euthanized for not
more than 2 h prior to tail retrieval. The tails were
divided into three groups according to storage
method: fresh (FRESH), frozen (FROZEN) and
refrigerated (REFRIG). For the FRESH group, ten-
dons were promptly dissected out under sterile
conditions and processed for collagen acid-solubili-
zation, as described in the next section. For the
FROZEN and REFRIG groups, the tails were stored
for 1 week at -20 and 4°C, respectively, before
tendon harvesting and processing. Tails of the FRO-
ZEN group were wrapped in gauze moisten in PBS
prior to freezing and were allowed to thaw at room
temperature for 20 min before tendon harvesting.
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Cell Tissue Bank (2009) 10:333–340
Manufacturing of the collagen solutions
After sterile harvesting, the rat tail tendons for each
group were independently solubilized in 0.01 N
hydrochloric acid (HCl) and digested in pepsin to
obtain atelocollagen solutions, which were analyzed
for potential effects of storage on the allograft
collagen. The collagen content of each group was
indirectly quantified by measuring hydroxyproline
(Hyp).
Collagen characterization: SDS–PAGE
The collagen solutions of the groups FRESH, FRO-
ZEN and REFRIG were analyzed using sodium
dodecylsulfate (SDS) 4–12% polyacrylamide gel
(PAGE) electrophoresis. Three parts of the diluted
collagen solutions were mixed with one part of
NuPAGEÒ LDS Sample Buffer (Invitrogen, Carls-
bad, CA) and each gel lane was loaded with 20 lg of
the respective mixtures. After electrophoresis, the gel
was stained with SimplyBlue SafeStain (Invitrogen,
Carlsbad, CA) overnight and destained in water.
Collagen characterization: denatured collagen
(Hyp vs. SIRCOL methods)
The SIRCOL Collagen Assay (Biocolor, Carrickfer-
gus, UK) quantifies acid-soluble collagen and is
based on the specific affinity of the dye picrosirius
red to the intact collagen molecule. Since this affinity
is affected by the loss of the triple helix structure
resulting from the exposure to collagenases, this
method can be used to estimate collagen denaturation
when its collagen values are compared to the collagen
values obtained through an amino acid quantification
method (i.e., hydroxyproline) (Li et al. 2000).
Collagen characterization: amino acid profile
Samples were hydrolyzed using 6 M HCl at 110°C
for 24 h. HCl was vaporized, the residues were
resuspended in dilution buffer and applied to a
Beckman Model 7300 Amino Acid Analyzer. The
amino acids were separated by ion-exchange chro-
matography, followed by post-column derivitization
using ninhydrin for detection. The amino acid
composition of collagen is highly characteristic. Four
amino acids, glycine (Gly), alanine (Ala), proline
Cell Tissue Bank (2009) 10:333–340
(Pro) and hydroxyproline (Hyp), account for 2/3 of
all residues of the collagen molecule (Eastoe 1967).
The hydroxylation of proline (Pro), practically seen
only in collagen, is essential to the folding and to the
stabilization of the collagen triple-helix (Vitagliano
et al. 2001; Kotch et al. 2008). The hydroxylation of
lysine (Lys) is basic for the formation of intermolec-
ular crosslinks and is closely related to the glycosyl-
ation level of the collagen molecule (Eastoe 1967;
Batge et al. 1997). Therefore, we chose the amounts
of glycine (Gly), alanine (Ala), hydroxyproline
(Hyp), hydroxylysine (Hyl) (number of residues/
1,000 residues) and the ratios Hyp/Pro and Hyl/Lys as
representative of the amino acid profiles of the
FRESH, FROZEN and REFRIG collagen solutions.
These amino acid profiles were compared among
themselves and also to the known profile of purified
collagen type I from rat tails (Eastoe 1967).
Provisional scaffolds: collagen solution
neutralization
To make the scaffolds, all collagen solutions were
adjusted to 10 mg/ml of collagen concentration by
adding different amounts of 0.01 N HCl. Then each
collagen solution was mixed with sterile HEPES and
Dulbecco’s Phosphate Buffered Saline (DPBS) (both
from Cellgro, Mediatech Inc., Herndon, VA) buffer.
The solution was neutralized to a pH of 7.4 using
7.5% sodium bicarbonate (Cambrex BioScience
Walkersville, Inc., Walkersville, MD) and kept on
ice until adding the platelet-fibroblast suspension.
Provisional scaffolds: platelet solution preparation
A platelet solution was produced using the Harvest
Smart PreP2 System (Harvest Technologies, Plym-
outh, MA). Briefly, 54 ml of human whole blood was
drawn into a 60 ml syringe containing 6 ml acid-
citrate dextrose (ACD) (Harvest Technologies, Plym-
outh, MA). The blood was centrifuged to allow for the
decantation of plasma, platelets and white blood cells
from the red blood cells. A second centrifugation step
was used to form a pellet of platelet concentrate at the
bottom of the decantation chamber. Excess plasma
was removed and the platelet pellet resuspended in the
remaining plasma to obtain a platelet concentrate
(PC). This method resulted in a 49 increase in platelet
335
concentration, from 2 9 108 platelets/ml in the whole
blood to 8.1 9 108 platelets/ml in the PC.
Provisional scaffolds: cell solution preparation
Human ACL fibroblasts were obtained by culturing
ACL tissue explants, retrieved at the time of ACL
reconstruction, in Dulbecco’s Minimum Essential
Medium (DMEM) (Cellgro, Mediatech Inc., Hern-
don, VA) supplemented with 10% defined fetal
bovine serum (FBS) (Hyclone, Logan, UT) and 1%
antibiotic-antimycotic solution (Cellgro, Mediatech
Inc., Herndon, VA). Media was changed twice a
week and the cells were passaged twice before being
used in this experiment. The fibroblasts were sus-
pended in the platelet solution at 1.5 9 106 cells/ml
just prior to incorporation into the scaffolds.
Provisional scaffolds: manufacturing
of the scaffolds and culture
Scaffolds were prepared by mixing the collagen
solutions of each group and the platelet-fibroblast sus-
pension, with final densities of 5 9 105 fibroblasts/ml
and 3.1 9 108 platelets/ml, and collagen concentration
of 3.8 mg/ml. Control scaffolds (without fibroblasts)
were identically prepared for each group. Using a repeat
pipettor, gel aliquots of 0.5 ml were transferred onto
3 cm long silicone semitubular molds with anchors of
polyester mesh at each end, placed on the wells of a
6-well plate, and cultured for up to 12 days (n = 7 and
3 for the fibroblast-seeded and unseeded constructs,
respectively). The plates were placed in the incubator
for 1 h at 37°C before adding enough complete medium
(DMEM ? 10% FBS ? 1% antibiotic/antimycotic
solution) to cover the constructs. Cells were allowed
to equilibrate in the scaffold overnight before the
baseline cell number was determined. On days 5 and 12,
cultures were interrupted and the scaffolds used for the
cell proliferation assay. Media was collected at 12 and
24 h and on days 3, 6 and 12 for the platelet activation
assay. On days 1, 7 and 12, the cultures were photo-
graphed for the gel retraction analysis.
Fibroblast proliferation: MTT assay
Fibroblast proliferation was evaluated using the
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide) assay. The MTT assay quantifies the
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conversion of yellow, soluble MTT salt into purple
formazan salt by mitochondrial dehydrogenase
enzymes. Under similar culture conditions, the
increase in total mitochondrial activity could be
correlated to an increase in cell number, indirectly
measuring cell proliferation. The MTT solution was
prepared fresh at a concentration of 1 mg/ml in
serum-free DMEM from a sterile 5 mg/ml MTT
(Sigma–Aldrich Inc., St. Louis, MO) stock solution in
sterile 1X DPBS. The scaffolds were transferred to
12-well plates. MTT solution (1 ml) was added to
each well and the plates incubated for 3 h (at 37°C,
5% CO2). Subsequently, the excess MTT solution
was removed and 1 ml of sterile 1X DPBS added to
each well. The plates were placed on a horizontal
shaker (Fisher Scientific Clinical Rotator, 100 rpm)
at room temperature for 30 min. Rinses were
repeated until the absorbances of the washes were
less than 0.100. Then the scaffold were transferred
into 3 ml centrifuge tubes with 1 ml of a detergent
reagent obtained by mixing (1:1 volume ratio) 20%
Sodium Dodecyl Sulfate (SDS) (Teknova, Hollister,
CA) and Formamide (VWR, West Chester, PA), and
incubated overnight at 37°C. Finally, 200 ll of the
supernatant from each tube were transferred onto a
sterile 96-well plate. Absorbance was measured at
562 nm. Fibroblast proliferation was calculated based
on the increase in absorbance between days 1, 5 and
12 (n = 7 for each time point).
Platelet activation: PDGF-AB
Platelet activation was determined by measuring the
levels of the heterodimer PDGF-AB, which com-
prises 70% of all PDGF isoforms purified from
human platelets (Vassbotn et al. 1994), in the
conditioned medium at 12, 24 h, 3, 6 and 12 days
(time between previous medium change and medium
collection were 12, 12 h, 3, 3 and 6 days respec-
tively). Concentrations of human PDGF-AB were
determined using the commercially available Quan-
tikine colorimetric sandwich ELISA kit (R&D Sys-
tems, Minneapolis, MN). Assays were performed in
duplicate on media samples (n = 6 at 12, 24 h and
3 days; n = 3 at days 6 and 12) according to the
instructions of the manufacturer. A standard curve
was produced by a twofold serial dilution of a known
concentration of PDGF-AB provided in the kit to
make final concentrations of 0, 31.2, 62.5, 125, 250,
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Cell Tissue Bank (2009) 10:333–340
500, 1,000 and 2,000 pg/ml. The color changes of the
final reactions were measured at 450 nm and the
standard curve was used to calculate the PDGF-AB
concentrations in the conditioned media.
Provisional scaffold retraction
Digital pictures of the cultures (n = 7) were taken on
days 1, 7 and 12, and scaffold dimensions measured
using a public domain image processing software
(ImageJ 1.37V, NIH). Retraction, for each construct,
was evaluated as the percent decrease of a particular
construct’s area at days 7 and 12 with respect to that
construct’s area at day 1.
Rheological properties
For rheological properties, 1 ml of the collagen-
platelet solution (n = 5) was placed onto the center
of the plate of a AR 1000 Rheometer (TA Instru-
ments, New Castle, DE) at 25°C, fitted with a 60 mm,
1° angle acrylic cone. The collagen-platelet hydrogels
were cyclically loaded to 1% strain with angular
frequency equal to 6.3 rad/sec. The shear viscoelastic
parameters elastic and inelastic moduli (G0 , G00 ,
respectively) were recorded as a function of time. The
elastic modulus represents the elastic component of
the provisional scaffold, while the inelastic modulus
represents its viscous component. A phase angle d,
obtained by the relation tan d = G00 /G0 , equal to 45°
(i.e., when G0 (t) and G00 (t) intersect) defined the
viscous-elastic transition during the gelation process.
Statistical analysis
The statistical analysis was performed using SPSS
version 15.0 (SPSS Inc., Chicago, IL). The rheolog-
ical properties of the provisional scaffolds were
compared using one-factor ANOVA model. The
MTT absorbancies were compared using two-factor
ANOVA mixed model. PDGF-AB levels in condi-
tioned media were compared using a repeated
measures two-factor ANOVA mixed model. Scaffold
areas were compared using a repeated measures
three-factor ANOVA mixed model. Post-hoc multiple
comparisons, when necessary, were adjusted using
the Bonferroni correction. All values of P \ 0.05
were considered statistically significant.
Cell Tissue Bank (2009) 10:333–340
Results
Collagen characterization: SDS–PAGE
SDS–PAGE showed similar migration patterns for all
groups, with bands characteristics of collagen type I
(i.e., a1 and a2 for monomeric chains, b11 and b12
for dimeric chains and c for trimeric chains bands;
Fig. 1). No bands suggesting collagen degradation
were detected in any of the groups.
Collagen characterization: denatured collagen
(Hyp vs. SIRCOL methods)
Also, for all groups the paired collagen concentra-
tions calculated using the Hyp and the SIRCOL
methods were not significantly different (t-test,
P = 0.09 for FROZEN and P C 0.17 for FRESH
and REFRIG; Table 1).
Collagen characterization: amino acid profile
There was no statistically significant difference
between the amino acid profiles of the FRESH,
FROZEN and REFRIG groups (P [ 0.5; Table 2).
Fibroblast proliferation: MTT assay
The ability of scaffolds made with the allograft
collagen to support fibroblast proliferation was
Fig. 1 The collagen solutions of the FRESH, FROZEN and
REFRIG group were qualitatively analyzed by SDS–PAGE,
confirming that the solutions consist mostly of collagen type I.
Also, no bands suggesting collagen degradation were detected
in any of the groups
337
Table 1 There was no difference between the paired collagen
concentrations measured by Hyp and SIRCOL, P = 0.09
(FROZEN) and P C 0.17 for other groups
Storage condition Hyp (mg/ml) SIRCOL (mg/ml)
FRESH 13.1 ± 1.8 13.9 ± 0.3
FROZEN 12.2 ± 0.8 11.5 ± 0.4
REFRIG 12.3 ± 0.6 12.7 ± 0.6
Values expressed as mean ± SD (n = 6 or 7)
significantly affected by storage condition
(P = 0.02), with 12.1 and 19.8% greater cell number
increase in the REFRIG group after 12 days com-
pared to the FRESH and FROZEN groups respec-
tively (Fig. 2). Mean absorbance values at day 1
represent initial cell seeding of approximately
5 9 105 cells/ml and were similar for all groups
(P = 0.65).
Platelet activation: PDGF-AB
The ability of the allograft collagen to stimulate
PDGF-AB release was significantly affected by
storage condition, with less PDGF-AB released by
the FRESH group at 12 h (15.9% less than the
REFRIG group, P = 0.04) and at 12 days (23.4 and
18.8% less than the FROZEN and REFRIG groups
respectively, P \ 0.001; Fig. 3). PDGF-AB eluted
into the conditioned medium was also time dependent
(P \ 0.001), with higher PDGF-AB levels at 12 h
after platelet activation by collagen (519 ± 88,
580 ± 34 and 617 ± 89 pg/ml, mean ± SD, for the
FRESH, FROZEN and REFRIG groups, respec-
tively). At 24 h and at 3 days, the PDGF-AB levels
for all groups had already decreased 69.6 ± 2.1 and
86.9 ± 1.0%, respectively, in relation to the 12 h
concentrations. At days 6 and 12, PDGF-AB mea-
surements of all groups were only 5–7% of the 12 h
concentrations.
Provisional scaffold retraction
Scaffold retraction, measured as the decrease in
allograft collagen scaffold area was significantly
affected by storage condition and time. At day 12,
scaffolds in the REFRIG group retracted 44.5 and
38.9% less than the FRESH and FROZEN groups
respectively (P \ 0.01). Percent decreases in area per
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Table 2 The amino acid profile of the collagen solutions was not affected by storage, P [ 0.5
Storage condition Gly (res/1,000) Ala (res/1,000) Hyp (res/1,000) Hylys (res/1,000) Hyp/Pro ratio Hylys/Lys ratio

FRESH 331.8 ± 11.0 105.9 ± 2.7 83.4 ± 5.2 6.6 ± 0.5 0.32 ± 0.01 0.73 ± 0.01
FROZEN 330.5 ± 9.6 105.2 ± 2.6 85.4 ± 4.1 6.6 ± 0.4 0.32 ± 0.01 0.75 ± 0.04
REFRIG 329.3 ± 10.5 104.8 ± 2.4 84.5 ± 3.5 6.5 ± 0.3 0.32 ± 0.01 0.75 ± 0.04
Values expressed as mean ± SD, n = 6 (FRESH) or 7 per group

1.2 group with respect to their initial areas are shown in

Fresh Frozen Refrigerated
Fig. 4.
*

Rheological properties
Absorbance

0.8

The storage conditions did not significantly affect the

viscous-elastic transition time (P = 0.053) and final
0.4
strength (P = 0.56 for elastic modulus and P = 0.15
for inelastic modulus) of the provisional scaffolds
(Table 3).
0.0
Day 1 Day 5 Day 12
Time
Discussion
Fig. 2 Proliferation of fibroblasts seeded into collagen-platelet
scaffolds, n = 7 per group and per data point (* denotes Allografts may be an advantageous option in recon-
significantly higher REFRIG absorbance reading at day 12
when compared to FRESH, P = 0.049, or FROZEN, structive orthopedics, for example, in ACL recon-
P = 0.001). Day 1 absorbance values reflect initial cell struction, but current methods of storage have been
seeding of 5 9 105 cells/ml reported to affect the material and functional prop-
erties of the allograft (Schachar et al. 1994; Rohde
et al. 2004; Giannini et al. 2008). The main objectives
800 of this work were to evaluate how the current
*

Fresh
600 Frozen
Day 7 Day 12
PDGF-AB (pg/ml)

Refrigerated
Scaffold Area (% of Day 1 Area)

*
100

400
75

200
50

**
0 25
12 hours 24 hours 3 days 6 days 12 days

Fig. 3 PDGF-AB eluted into surrounding medium from 0

collagen-platelet scaffolds at 12, 24 h and 3 days, n = 6 per
group, and at 6 and 12 days (n = 3 per group). Post-hoc
comparisons showed that the FRESH group released signifi-
cantly less PDGF-AB in the medium when compared to the
REFRIG group at 12 h (* P = 0.038) and when compared to
both other groups at 12 days (** P \ 0.001). No other
comparison was statistically significant (P [ 0.3). Error bars
represent ±SD
Fresh Frozen Refrigerated
Fig. 4 Retraction of cultured collagen-platelet scaffolds,
n = 7 per group, shown as percent decrease with respect to
initial area. Initial areas of all groups were similar (P = 0.31)
and interrupted line represents 100% at day 1 for all groups.
* denotes statistically significant difference with respect to
scaffolds of the FRESH and FROZEN groups (P \ 0.01)

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Table 3 The rheological parameters were not affected by
storage (P = 0.053 for the time to viscous-elastic transition
and P C 0.15 for the other parameters)
Storage Time to viscous- G0 max (Pa) G00 max (Pa)
condition elastic transition
(min)
FRESH 3.3 ± 0.1 230.1 ± 39.8 71.3 ± 14.6
FROZEN 3.1 ± 0.3 207.5 ± 51.3 58.0 ± 12.6
REFRIG 3.2 ± 0.2 205.3 ± 21.3 57.8 ± 4.9
Values expressed as mean ± SD, n = 5 per group
methods of storage (i.e., refrigeration and freezing)
affect the biologic and rheologic properties of the
allograft collagen.
Collagen was solubilized for the assessment of the
storage effect and to make the scaffolds. Collagen is
normally insoluble, but rat tail tendons are a good
source for collagen solubilization because of the
predominance of intermolecular aldimine cross-links
that are readily opened at acid pH (Chandrakasan
et al. 1976). In addition, digestion of the telopeptide
portions of the collagen molecules further increases
the collagen solubility (Traub and Piez 1971; Chand-
rakasan et al. 1976), while maintaining the triple-
helices conformation (Djabourov et al. 1993).
In this study we found minor, but statistically
significant, functional differences between the scaf-
folds of the FRESH, FROZEN and REFRIG groups.
At 12 days, the REFRIG group had higher cell
proliferation (7.5-fold increase with respect to mean
initial cell seeding vs. 6.7- and 6.3-fold of the FRESH
and FROZEN groups, respectively) and less scaffold
retraction (12.9% of retraction with respect to the
initial area vs. 29.0 and 33.2% of the FRESH and
FROZEN groups, respectively), while the FRESH
group had less PDGF-AB released at 12 h (10.5 and
15.9% less than the FROZEN and REFRIG groups,
respectively). No difference was noted in the rheo-
logical properties. Higher cell proliferation and
platelet activation could mean that a scaffold in vivo
would not only be more rapidly populated by cells,
but also that those cells would be more stimulated to
promote proper matrix remodeling.
Storage does not seem to have affected the
allografts’ collagen. SDS–PAGE gels have been used
to determine collagen composition (Bashey et al.
1978; Samuel et al. 1998) and identify ’-length or
other minor fragments that result from collagen
339
digestion (Kafienah et al. 1998; Christiansen et al.
2007). In this study, the SDS–PAGE gels of all
groups showed the bands characteristic of collagen
type I, but no signs of collagen degradation. Further-
more, the amino acid profiles of all groups are nearly
identical and match other amino acid profiles found
in the literature for collagen type I (Eastoe 1967;
Bashey et al. 1978). In addition, collagen concentra-
tions estimated by Hyp were similar to those found
using the SIRCOL assay. These results suggest that
the functional differences listed above were not
caused by changes in collagen.
Notwithstanding the variable results in the litera-
ture regarding the effect of the storage conditions on
diverse aspects of graft performance, it is unlikely
that the slight differences seen between the groups in
this work would hinder the in vivo use of allografts
stored frozen or under refrigeration for up to 1 week.
Similarly, scaffolds made from allograft collagen
should also be relatively unaffected by this length
of storage, with the exception of lower retraction
for scaffolds made with collagen extracted from
allografts stored under refrigeration conditions for
1 week.
Some limitations of this study are: (1) the lack of
an in vivo evaluation of the scaffolds; (2) the use of
rodent, and not human, tissue allograft; and (3) the
lack of evaluation of changes at the level of
the collagen morphological organization in the allo-
graft, for example by using transmission electronic
microscopy.
In terms of future research, animal studies can
show if the slight differences seen in this work (e.g.,
the higher cell proliferation and lower retraction seen
in the REFRIG group) will affect the in vivo
outcomes. The availability of human tissue for
collagen acid solubilization should also be reevalu-
ated. The length of storage time is a critical parameter
for refrigeration, since freezing temperatures are
more effective in delaying degradation by proteases.
Hence, it would be important to determine for how
long a tissue can be stored at 4°C and the acid
solubilized collagen still be used for scaffold
production.
Acknowledgments This work was supported by a grant from
the Musculoskeletal Transplant Foundation. The authors thank
Marie Torres (amino acid analysis), Yin Yin Lin and Zachary
Waldon (SDS–PAGE), and the help of Dr. David Zurakowski
with the statistical analyzes.
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