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J_ID: Z8H Customer A_ID: 07-0543.R1 Cadmus Art: JBMB 31059 Date: 12-FEBRUARY-08
Stage: I
Page: 1
Biomechanical and Immunohistochemical Properties of Meniscal
Cartilage After High Hydrostatic Pressure Treatment
AQ1
Florian D. Naal,1 Johannes Schauwecker,2 Erwin Steinhauser,2 Stefan Milz,3 Fabian von Knoch,1
Wolfram Mittelmeier,4 Peter Diehl4
1
Department of Orthopaedic Surgery, Schulthess Clinic, Lengghalde 2, 8008 Zurich, Switzerland
2
Department of Orthopaedic Surgery and Orthopaedic Sports Medicine, Technical University of Munich,
Ismaninger Street 22, 81675 Munich, Germany
3
AO Research Institute - Tissue Morphology, AO Foundation, Clavadeler Street 8, 7270 Davos, Switzerland
4
Department of Orthopaedic Surgery, University of Rostock, Doberaner Street 142, 18055 Rostock, Germany
Received 13 October 2007; revised 22 November 2007; accepted 10 December 2007
Published online 00 Month 2008 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.b.31059
Abstract: Meniscal allograft processing procedures, in particular gamma irradiation,
deteriorate the biomechanical and biological properties of the transplanted tissue. High
hydrostatic pressure (HHP) treatment, widely used in food technology to inactivate
microorganisms while preserving natural compounds, might serve as a gentle alternative to
gamma irradiation in the processing of meniscal allografts. We therefore investigated the
effects of HHP treatment on the biomechanical and immunohistochemical properties of
meniscal cartilage. Specimens of bovine menisci were treated with HHP for 10 min (208C) at
300 MPa and 600 MPa. Untreated control samples were left at room temperature and ambient
pressure. We performed repetitive cycling indentation-tests to assess the biomechanical
properties—in particular the viscoelastic behavior—of HHP treated and untreated meniscal
specimens. Immunohistochemical analysis for collagens type I, II, and III and for the
proteoglycans versican, aggrecan and for link-protein was performed by immunolabeling
cross-sections of untreated and at 600 MPa HHP treated specimens. Comparing untreated and
HHP treated meniscal specimens there were no significant differences for all tested
biomechanical parameters. All cross-sections of untreated and HHP treated specimens
stained positive for the collagens and proteoglycans. We demonstrated that meniscal cartilage
can be treated by HHP at levels as high as 600 MPa without affection of the biomechanical and
immunochistochemical properties. Therefore, HHP treatment might serve as a gentle
alternative to gamma irradiation in the processing of meniscal allografts. Further research
is necessary to verificate the present results in vivo. ' 2008 Wiley Periodicals, Inc. J Biomed Mater Res
Part B: Appl Biomater 00B: 000–000, 2008
Keywords: high hydrostatic pressure treatment; meniscal cartilage; allograft transplantation; tissue properties; processing technique
INTRODUCTION
The principal functions of the menisci are load transmission and shock absorption. Both functions account for the
protection of the tibiofemoral articular cartilage. Moreover
the menisci contribute to knee joint lubrication, proprioception, and stability.1 The meniscus transmits 50% of the
load with the knee in extension and up to 90% of the joint
load with the knee in flexion.2 The loss of meniscal carti-
Correspondence to: F. D. Naal, MD (e-mail: [email protected])
' 2008 Wiley Periodicals, Inc.
lage as a result of partial or complete meniscectomy leads
to articular cartilage damage and a higher incidence of
osteoarthritis due to increased pressures at certain regions
of the joint surface.3 Seedhom and Hargreaves demonstrated in an in vitro study that removal of 16–34% of
meniscal cartilage resulted in 350% increased joint contact
forces.4 Several studies have shown that transplantation of
meniscal allografts might provide at least partial protection
against articular cartilage damage and onset or progression
of knee osteoarthritis.5,6 Clinically, meniscal allograft transplantation has been reported to reduce pain and improve
knee joint function; most short- to long-term results were
encouraging.5,6
1
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The transplantation of allograft tissue may, nevertheless,
include the risk of disease transmission or microbial contamination.7 It has been shown that 27.7% of musculosceletal allografts were initially discarded by tissue banks
primarily due to bacterial contamination and that 5.8% of
the grafts still had positive cultures before processing.7 Despite the very low incidence of serious disease transmission
with allografts in general, there is considerable concern
regarding the risk of human immunodeficiency virus (HIV)
transmission.8 Therefore the processing of meniscal allografts often includes a sterilization procedure with gamma
irradiation being used in most cases.9 However, irradiated
tissues suffer from impaired biomechanical and biological
properties. The irradiation of bone-tendon-bone allografts
has been shown to produce severe biomechanical alterations of the graft tissue.10–12 Yahia et al. in 1993 and Yahia
and Zukor in 1994 found that gamma irradiation at dosages
commonly used for sterilization had significant adverse
effects on the biomechanical properties of meniscal grafts
in rabbits.13,14 Maintaining meniscal biomechanical and biological properties may be crucial to restore normal joint
function, to decrease articular cartilage contact stresses and
to improve the longevity of the transplant. Therefore alternative processing procedures should be developed leading
to an inactivation of microorganisms while leaving the
meniscal properties unchanged. High hydrostatic pressure
(HHP) treatment, widely used in food technology to inactivate microorganisms while preserving natural compounds
such as flavors, aromas or vitamins,15,16 might serve as
such an alternative. HHP has recently been investigated for
its potential use in orthopaedic tumor surgery.17,18 Several
studies demonstrated that HHP effectively inactivates different cell types, including malignant cells,17,19 while leaving the biomechanical and biological properties of bone,
tendons, and osteoarticular segments unchanged.20–22 The
aim of the present study was to investigate the effects of
HHP treatment on the biomechanical and immunohistochemical properties of meniscal cartilage to evaluate a
potential alternative to gamma irradiation in the processing
of meniscal allografts.
MATERIALS AND METHODS
Harvest and Sample Preparation
Meniscal cartilage samples were harvested from 15 fresh
bovine knee joints obtained from a local slaughterhouse.
We used bovine knee joints because cattle are young at the
time of slaughter and the joint offers relatively large
menisci. After preparation of the knee, the menisci and the
articular cartilage were studied macroscopically for degenerative and traumatic changes. Only specimens from joints
with intact cartilage and meniscal surfaces were investigated. Using a cylinder for ‘‘osteochondral autologous
transplantation’’ (OATS-cylinder) and a scalpel we harvested four samples from the medial and lateral menisci of
each joint, 10 mm in diameter and 4 mm in height. The
meniscal specimens were stored in arthroscopy-fluid (Purisole SM, Fresenius Kabi AG, Bad Homburg, Germany)
until treatment. Harvest of the specimens, high hydrostatic
pressure treatment and biomechanical tests were performed
on a single day to avoid storage effects on the samples.
High Hydrostatic Pressure Treatment
Samples were put in 15 mL tubes (Falcon Blue Max Tube
15 mL, Becton Dickinson Labware, Franklin Lakes). The
tubes were completely filled with arthroscopy-fluid,
plugged and the plugs additionally sealed with parafilm
(Parafilm M, American National CanTM, Greenwich). The
charged tubes then were placed into the high hydrostatic
pressure device (HDR 100-20, RECORD, Königsee, Germany) and treated for 10 min at 300 or 600 MPa (208C).
Control samples were placed into 15 mL tubes filled with
arthroscopy-fluid and left at ambient pressure and room
temperature. Twenty samples were treated at 300 MPa, 20
samples at 600, MPa, and 20 samples served as controls.
Biomechanical Testing
To assess the biomechanical properties of HHP-treated and
untreated meniscal samples we performed cyclic indentation tests as minimally constraint compression-relaxation
tests as previously described.23 We used a universal testing
machine (Zwicki1120, Zwick, Ulm, Germany) with a calibrated depth and load sensitive indenter (KAP-S, A.S.T.,
Dresden, Germany). The tip of the indenter consisted of a
steel ball with a diameter of 5 mm. By using ball geometry
for indentation, notch effects and stress concentrations
(shear stress) at the contact area of the indenter with the
specimens could be avoided. Such shear stresses would
have been provoked using a flat indenter in tensile reactions would have possibly influenced our results. The
meniscal specimens were placed horizontally under the
testing device onto a specially designed smooth and flat
metallic plate with a circular sink (diameter 10 mm, depth
0.2 mm) to achieve lateral stabilisation during axial loading
(minimally constraint to allow quasi-free evasion during
testing). The indenter position was calibrated prior to each
test; it was set to zero at the level of the base of the cavity.
A preload of 0.5 N was applied to the specimens. The testseries comprised five repetitive indentation test-cycles consisting of the following phases: (1) Specimen preloading
(0.5 N). (2) Dynamic compression of the sample with a
constant load velocity of 5 mm/min until a peak test load
of 7 N was achieved. (3) Static compression with the
indenter remaining in the achieved position for 60 s. (4)
Relaxation of the sample after 60 s with a constant velocity
of 1 mm/min until a load of 0.1 N. The indenter remained
in the achieved position for an interval of 60 s and
the described test-cycle was then repeated another four
times. Load, indenter position, and time were logged and
displayed online by the test software package TestXpert
Journal of Biomedical Materials Research Part B: Applied Biomaterials
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3
PROPERTIES OF MENISCAL CARTILAGE
F1
F2
(Version 8.1, Zwick). The stiffness (N/mm) of the meniscal
specimens was determined from the linear-elastic slope of
the loading curve between 2 N and 5 N. As parameters for
the viscous properties we assessed the indentation depth
(mm) and recorded the residual force (N) which was
defined as the measured load at the end of the static compression phase. The test-setup is schematically shown in
Figure 1. A typical loading curve obtained during testing is
presented in Figure 2.
AQ2
Immunohistochemistry
After HHP treatment at 600 MPa (10 min, 208C) the
meniscal cartilage samples were fixed in 100% methanol
for seven days at 48C. Afterwards the specimens were
rinsed with phosphate buffered saline (PBS), infiltrated
overnight with 5% sucrose in PBS, and cryosectioned into
12 lm slices on an HM 500 OMV Microm cryostat (Walldorf, Germany). Sections were immunolabelled with monoclonal antibodies against collagens (type I, II, and III) and
proteoglycans (versican, aggrecan, and the associated link
protein). Full details of the antibodies and pretreatment
procedures were previously published.24 The sections were
enzymatically treated for 30 min at 378C with hyaloronidase / chondroitinase ABC (1.5 units (U)/mL/0.25 U/mL;
Sigma) for collagens, with chondroitinase AC (0.25 U/mL;
Sigma) for versican and with chondroitinase ABC (0.25 U/
mL; Sigma) for aggrecan and link protein. Endogenous peroxidase activity was blocked by pretreatment with 0.3%
Figure 2. Load curve of an indentation test consisting of five repetitive cycles showing the graphical course of preload, dynamic and
static compression and relaxation. Note the linear-elastic slope during dynamic compression. [Color figure can be viewed in the online
issue, which is available at www.interscience.wiley.com.]
hydrogen peroxide in methanol for 30 min, any nonspecific
protein binding was reduced by incubating the sections
with an appropriate horse serum block for 60 min. We controlled for nonspecific binding of the secondary antibodies
by omitting the primary antibody. Specific antibody binding
was detected with an avidin-biotin-peroxidase-kit (Vectastain ABC-kit Elite, Vector Labs, Burlingame, CA). The
sections were finally counterstained with Meyer’s haematoxylin. One of the authors with broad expertise in immunohistochemistry (S. M.) compared the labeling pattern of
the collagens and proteoglycanes in HHP-treated and untreated sections using light microscopy.
Statistics
Statistical analysis was performed using the software package SPSS (Version 11.0, SPSS Inc., Chicago, Illinois).
After testing for normal distribution (Kolmolgorov-Smirnov
test) we used t-tests to compare the biomechanical parameters of HHP treated and untreated meniscal cartilage samples. Significance level was defined at p \ 0.05.
RESULTS
Biomechanics
Figure 1. Schematic diagram of the indentation test. The meniscal
specimens (diameter 10 mm, height 4 mm) were placed under the
testing machine. The tip of the indenter consisted of a steel ball
with a diameter of 5 mm. Five axial compression test-cycles were
performed with a maximum test load of 7 N.
All samples could be loaded to 7 N without signs of plastic
deformity. Slopes of the load curves were linear between 2
N and 5 N in all tests. The stiffness of all meniscal specimens increased significantly (p \ 0.05) throughout testing
(Figure 3). There were no significant differences between
control specimens and those treated at 300 or 600 MPa
(p [ 0.05). In control specimens, the stiffness increased
from 18.4 N/mm (61.3 SD) in the first test-cycle to 25.4
N/mm (62.1 SD) in the fifth. Accordingly, the stiffness for
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NAAL ET AL.
Figure 3. Stiffness (N/mm) of meniscal specimens throughout the
five indentation test-cycles. There were no significant (p [ 0.05) differences between untreated and at 300 MPa or 600 MPa HHP
treated samples.
Figure 5. Residual force (N) of meniscal specimens throughout the
five indentation test-cycles. There were no significant (p [ 0.05) differences between untreated and at 300 MPa or 600 MPa HHP
treated samples.
HHP treated specimens increased from 18.6 N/mm (61.9
SD) to 23.9 N/mm (62.7 SD) and from 18.7 N/mm (61.9
SD) to 24.8 N/mm (62.1 SD) after treatment at 300 and
600 MPa, respectively. The indentation depth of the specimens to achieve the peak test load of 7 N (at a velocity of
5 mm/min) decreased throughout testing (Figure 4). There
were no significant differences between control specimens
and those treated at 300 or 600 MPa (p [ 0.05). In control
specimens and in all HHP-treated specimens an indentation
depth of 0.5 mm was measured during the first test-cycle
that was decreased to 0.4 mm during the fifth test-cycle. In
all specimens the residual force significantly (p \ 0.05)
increased throughout testing (Figure 5). There were no differences between control specimens and those treated at
300 or 600 MPa (p [ 0.05). In control specimens, the residual force increased from 3.6 N (60.4 SD) in the first
test-cycle to 4.6 N (60.1 SD) in the fifth. Accordingly, the
load for HHP-treated specimens increased from 3.5 N
(60.6 SD) to 4.6 N (60.2 SD) and from 3.9 N (60.2 SD)
to 4.6 N (60.2 SD) after treatment at 300 and 600 MPa,
respectively.
tectable. This was also observed in all specimens of meniscal cartilage treated at 600 MPa. As determined by light
microscopy, there were no differences in the labelling pattern between untreated and HHP treated sections regarding
the labelling of collagen type I, II, and III (Figure 6) or of
versican, aggrecan and link-protein (Figure 7). Because of
the larger amount of collagen type I and II represented in
the meniscus,25 the staining was more intense than that
of collagen type III. No unspecific staining was observed
in control sections in which the primary antibody was
omitted.
Immunohistochemistry
All sections showed the typical meniscal histology with
fibers partially sectioned horizontally, transversely, or longitudinally. In all sections of the untreated specimens the
specifically stained collagens and proteoglycans were de-
Figure 4. Indentation depth (mm) to achieve the maximum test load
of 7 N in meniscal specimens throughout the five indentation testcycles. There were no significant (p [ 0.05) differences between
untreated and at 300 MPa or 600 MPa HHP treated samples.
DISCUSSION
To address the concerns regarding the risk of bacterial and
viral disease transmission by musculoskeletal allografts,
stringent donor selection and screening guidelines have
been recommended by the American Association of Tissue
Banks (AATB) and the Food and Drug Administration
(FDA) and many tissue banks perform an additional sterilization procedure, mostly in the form of gamma irradiation.9
In a survey of the AATBs in 1996, Vangsness et al.
reported that 18 of 36 tissue banks sterilized meniscal allografts by gamma irradiation following the initial preservation process.26 Gamma irradiation dosages ranged from 10
to 35 kGy (1.0 to 3.5 Mrad). However, the studies by
Fideler et al. and by Campbell and Li indicated that HIV is
not inactivated by gamma irradiation at dosages below 35
kGy (3.5 Mrad).27,28 Nevertheless gamma irradiation dosages exceeding 20 kGy (2.0 Mrad) caused significant deteriorations of the biomechanical properties of bone-tendonbone allografts10–12 and also of meniscal allografts. Two
studies by Yahia et al. investigated the effects of gamma
irradiation on the mechanical properties of meniscal allografts in a rabbit model.13,14 They found significantly
reduced biomechanical parameters of the meniscal grafts
after irradiation at 25 kGy (2.5 Mrad).13 Six months after
reimplantation of the irradiated menisci, two out of ten
grafts were almost totally removed by granulation tissue.
Gamma irradiation produced a significant reduction in the
compliance to long-term creep in the remaining grafts.14
The authors concluded that gamma irradiation is unsuitable
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Figure 6. Cross-section of untreated (upper row) and at 600 MPa HHP treated (lower row) meniscal cartilage samples, immunolabeled for collagen type I, II and III (from left to right). All samples
stained positive without differences in labeling pattern.
for clinical use, and that other techniques should be used
whenever possible.14
In contrast to these reports we demonstrated that HHP
treatment at 300 and 600 MPa had no adverse effects on
the biomechanical properties of bovine meniscal cartilage.
There were no differences between untreated and treated
specimens regarding the stiffness, indentation depth and residual force; all being important viscoelastic parameters.
The viscoelastic properties of meniscal cartilage were in
particular subject of the biomechanical investigation.
Figure 7. Cross-section of untreated (upper row) and at 600 MPa HHP treated (lower row) meniscal cartilage samples, immunolabeled for the proteoglycanes aggrecan, versican and for link-protein (from left to right). All samples stained positive without differences in labeling pattern.
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NAAL ET AL.
Therefore, we used a self-developed advanced experimental
setup—described previously by Maier at al.23—that allows
precise and reliable measurements of viscoelastic parameters (stiffness, compression and relaxation). Stiffness of the
meniscal specimens was calculated during the dynamic
compression phase which analyzes the elastic properties of
the menisci. As explained by Maier et al., high stiffness
characterizes high material elasticity and low stiffness low
material elasticity.23 The residual force characterizes the
viscoelastic properties of the specimens, high residual
forces indicate a more elastic than viscous behavior and
low residual forces a more viscous than elastic behaviour.23
The stiffening of the specimens, the decreased indentation
depth, and the increased recovery load observed throughout
testing can be explained by a compaction of the extracellular matrix.29 Nevertheless, there were no differences
between untreated and HHP-treated samples. Our results
are in line with those of Diehl et al. who reported that the
biomechanical behavior of articular cartilage remained
unchanged after HHP treatment of up to 600 MPa (10 min,
208C).20 Similar results were observed in HHP treated
Achilles tendons.21 Compared to untreated control tendons,
treatment at 600 MPa caused no significant differences in
Young’s modulus and tensile strength.21 Similar to these
results in collagenous tissues (meniscal and articular cartilage and tendons), there were no adverse effects of HHP
on bone biomechanics.22
The obvious differences between the effects of gamma
irradiation and HHP treatment on biomechanical tissue
properties may be explained by the different mode of
action of both methods. Irradiation acts by two mechanisms. The primary mechanism is the direct alteration of
nucleic acids in bacteria and viruses inducing genome dysfunction and destruction. The secondary mechanism is the
production of free radicals. Radicals however, interfere
with collagens and lead to a disruption of collagen fibers.30
Our results demonstrate, in contrast, that collagens and proteoglycans in the meniscus are not affected by HHP. This
finding is confirmed by the study of Diehl et al. who
showed the preservation of collagen type II, aggrecan and
link-protein in articular cartilage after HHP treatment at
600 MPa.20 In vitro adhesion tests demonstrated that HHP
treatment of up to 600 did not affect collagen type I.31 In
contrast to the mode of action of gamma irradiation, HHP
does not exert damaging effects on covalent molecular
bonds.15 This leads to a preservation of natural compounds
such as flavors, aromas, or vitamins15,16 and as shown in
the present study, to the preservation of the collagens type
I, II, and III and of the proteoglycans aggrecan, versican,
and link-protein. The preservation of these extracellular
matrix components therefore seems to be responsible for
the maintenance of the biomechanical properties of meniscal cartilage after HHP treatment.
HHP at levels of 600 MPa has been shown to kill several Gram-negative and Gram-positive microorgansims by
more than 6-log colony forming units.32 Many viruses
pathogenous to humans are inactivated at pressure levels of
400–600 MPa. This has been shown for, for example, hepatitis A and caliciviruses,33,34 herpes viruses such as herpes
simplex virus type 1 (HSV-1) and human cytomegalovirus
(CMV).35 Very important is the observed inactivation of
human immunodeficiency virus (HIV) by HHP treatment at
levels of 400 MPa.36 It should be emphasized that most of
these mentioned studies were performed under in vitro conditions or within food components. We are unaware of
studies that investigated the sterilization capability of HHP
in infected tissues utilized as allografts. Such investigations
still need to be performed. Furthermore our results were
obtained in bovine meniscal specimens and can therefore
not simply be transferred to the human meniscus.
In conclusion, the present study demonstrated for the
first time that the biomechanical properties of meniscal cartilage were not affected by HHP treatment at levels as high
as 600 MPa. Furthermore there were no differences in the
immunohistochemical labeling pattern of key extracellular
matrix proteins between untreated and HHP-treated specimens. Future research is necessary to verificate the present
results in vivo and to investigate the sterilization capacity
of HHP in allograft tissues. It would be recommendable to
design a direct comparison of HHP treatment, irradiation,
and autoclaving. In such an approach, tensile forces and
tear strengths should be assessed as well.
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