MB-102
Meniscus Structure in Human, Sheep, and Rabbit for Animal Models of
Meniscus Repair
Anik Chevrier,1 Monica Nelea,1 Mark B. Hurtig,2 Caroline D. Hoemann,1,3 Michael D. Buschmann1,3
1
Department of Chemical Engineering, Ecole Polytechnique, PO Box 6079, Station Centre-Ville, Montreal, Quebec, Canada, 2University of Guelph,
Guelph, Ontario, Canada, 3Institute of Biomedical Engineering, Ecole Polytechnique, PO Box 6079, Station Centre-Ville, Montreal, Quebec, Canada
Received 7 July 2008; accepted 26 January 2009
Published online 25 February 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jor.20869
ABSTRACT: Meniscus injury is a frequently encountered clinical orthopedic issue and is epidemiologically correlated to osteoarthritis. The
development of new treatments for meniscus injury is intimately related to the appropriateness of animal models for their investigation. The
purpose of this study was to structurally compare human menisci to sheep and rabbit menisci to generate pertinent animal models for
meniscus repair. Menisci were analyzed histologically, immunohistochemically, and by environmental scanning electron microscopy
(ESEM). In all species, collagen I appeared throughout most menisci, but was absent from the inner portion of the tip in some samples.
Collagen II was present throughout the inner main meniscal body, while collagen VI was found in pericellular and perivascular regions.
The glycosaminoglycan-rich inner portion of menisci was greater in area for rabbit and sheep compared to human. Cells were rounded in
central regions and more fusiform at the surface, with rabbit being more cellular than sheep and human. Vascular penetration in rabbit was
confined to the very outermost region (1% of meniscus length), while vessels penetrated deeper into sheep and human menisci (11–15%).
ESEM revealed a lamellar collagenous structure at the articulating surfaces of sheep and human menisci that was absent in rabbit. Taken
together, these data suggest that the main structural features that will influence meniscus repair—cellularity, vascularity, collagen
structure—are similar in sheep and human but significantly different in rabbit, motivating the development of ovine meniscus repair
models. ß 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:1197–1203, 2009
Keywords: meniscus; animal models; meniscus repair; histology; immunohistology; ESEM
Menisci are semilunar shaped fibrocartilagenous structures wedged between the femoral condyles and the
tibial plateau on the medial and lateral sides of
the knee.1,2 Menisci play central load-bearing roles in
the knee joint including joint stabilization, shock
absorption, and protection of articular cartilage from
excessive stress.3–5 Removing menisci leads to reduced
contact areas in the joint and increased peak stress on
articular cartilage load-bearing zones.6 Total or partial
meniscectomy therefore not surprisingly results in
supra-physiological stress on the articular cartilage,
which can lead to knee damage and osteoarthritis.7,8
Meniscal tissue is principally composed of water,
collagens, and proteoglycans in approximate bulk mass
proportions of 72%:22%:1%.9 Collagens predominate at
60–70% of the dry tissue weight where Type I collagen
has the highest concentration and Type II, III, V, and VI2
are also present. Scanning electron microscopy (SEM)
has previously revealed a complex arrangement of
human meniscal collagen in three distinct layers10:
(1) a fibril network covering the femoral and tibial
surfaces, (2) a lamellar layer oriented towards the
femoral and tibial surface, and (3) a central main portion
composed of circumferentially oriented fibers along with
occasional radial tie fibers.
A meniscus feature in adults that is crucial for repair
is that only its outer periphery is vascularized.11 The
degree of vascular penetration from the periphery was
previously determined to be 10–30% of the meniscus
length in human11 and 15–25% in dog.12 Because wound
healing in adult tissues is triggered by blood clotting,13
Correspondence to: Michael D. Buschmann (T: 514-340-4711 ext.
4931; F: 514- 340-2980; E-mail: [email protected])
ß 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.
the capacity for natural repair is optimal in the periphery
of the meniscus and diminished in the inner margins.
Surgical techniques have been developed to provide
access to vascularization for inner meniscal portions via
trephination, which induces bleeding and creates access
channels connecting lesions in the central avascular
portion to the vascularized periphery to aid repair.12
Tissue engineering techniques aiming at regenerating
menisci have also been developed.14,15
In addition to vascularity, overall cellularity and the
composition and structure of the extracellular matrix
(ECM) of the meniscus are expected to influence repair
processes. These and other features need to be assessed
in animal species prior to developing models for human
meniscus repair. Canine models16–18 have often been
used due to similarity to human in vascular supply,
anatomy, and biochemical composition. Other species
that can be less costly and more available than dogs
have also been used for meniscus repair, including
sheep16,19,20 and rabbit.16,19,21,22 Unfortunately, no
direct comparison of structural features of menisci for
these species versus the human counterpart exists,
which severely limits the ability of these models to
represent meniscus repair in humans.
The purpose of the current study was to structurally
characterize features of menisci that can influence
repair in human, sheep, and rabbit in order to assess
the appropriateness of these potential animal models for
meniscus repair. We hypothesized that these species
would differ in vascularization, cellularity, and in
the composition and ultrastructure of the ECM. We
therefore characterized menisci from skeletally mature
rabbit, sheep, and human using histological, immunohistochemical, and ultrastructural techniques. We found
that the adult sheep meniscus displays a high degree of
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CHEVRIER ET AL.
structural similarity to the human meniscus for features
that are important in repair processes, more so than the
rabbit, and should be pursued as an animal model that is
representative of human for meniscus repair.
MATERIALS AND METHODS
Lateral and medial menisci from skeletally mature New
Zealand White rabbits (n ¼ 26; average age 6.9 months),
Suffolk-Dorset sheep (n ¼ 18; average age 2.9 years), and
human (n ¼ 12 menisci from four human cadavers; average age
54.0 years) were processed for the study. The study protocol
was approved by the Ecole Polytechnique Ethics Committee.
Menisci were fixed in 10% neutral buffered formalin (NBF)
(245–684; Fisher Scientific, St-Laurent, Quebec, Canada) with
or without 2.5% (w/v) cetylpiridinium chloride (CPC) (C-0732;
Sigma, Oakville, Ontario, Canada) or in 4% (w/v) paraformaldehyde (P-6148; Sigma), 1% (v/v) glutaraldehyde (16520;
Cedarlane, Burlington, Ontario, Canada), 0.1 M sodium
cacodylate (BP 325-50; Fisher Scientific), pH 7.3. Transverse
2 mm thick blocks were trimmed from each of the cranial
(rabbit and sheep) or anterior (human), middle and caudal
(rabbit and sheep), or posterior (human) portions of each
meniscus for cryosectioning to obtain vertical sections (Fig. 1A).
For clarity in the remainder of the text, only the terms anterior,
middle, and posterior will be used. Samples were washed in
Figure 1. Schematic representation of the sectioning of the
menisci-producing vertical sections that span the radial width
of the meniscus in anterior (1), middle (2), and posterior (3) regions
(A) and producing horizontal sections in the anterior region (B).
PBS, infiltrated with a graded series of sucrose embedded in
OCT compound (Cedarlane), and frozen. Cryosections (6–8 mm
thickness) were collected on 1 adhesive-coated slides using
the CryoJane tape transfer system (475208 and 475205;
Instrumedics, St-Louis, MO) or on Superfrost plus slides
Figure 2. Safranin O staining
was detected in the inner body
of the menisci from rabbit (A),
sheep (E), and human (I). Panel
(I) has higher than average Safranin O staining, to illustrate localization of GAG when detected
in the human meniscus. Chondrocyte-like cells were observed in
the inner body of the menisci (white
arrowheads in B, F, J). Cells
were more fusiform near the
surface (black arrows in C, G, K).
Blood vessels were identified at
the outer periphery (black arrowheads in D, H, L) and further
quantified in Figure 3C. Rabbit
menisci appeared more cellular
(B, C) than sheep (F, G) and human
(J, K).
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MENISCUS FOR ANIMAL MODELS OF REPAIR
(12-550-15; Fisher Scientific) using a Microm Cryo-Star HM
560 M cryostat.
For Safranin O/Fast Green staining, sections were sequentially immersed in Weigert iron hematoxylin counterstain
(HT1079; Sigma), 0.04% (w/v) Fast Green (F-7252; Sigma),
and 0.2% (w/v) Safranin O (S-2255; Sigma) in water. Digital
images of stained sections were acquired with either a Zeiss
Axiolab microscope equipped with a digital Hitachi HV-F22F
camera or a Zeiss Stemi stereomicroscope equipped with a Sony
analogue camera.
For immunostaining, sections were subjected to antigen
retrieval by placing in a heated solution of 10 mM Tris (pH 10)
and allowing to cool to room temperature. Enzymatic treatment
was with 0.1% (w/v) pronase (EC 3.4.24.31) (P-8811; Sigma) in
PBS for 20 min at room temperature, followed by 2.5% (w/v)
hyaluronidase (EC 3.2.1.35) (H-3506; Sigma) in PBS for 30 min
at 378C. Sections were blocked with 20% (v/v) goat serum
(G-9023; Sigma) in PBS with 0.1% Triton X-100 for 60 min at
room temperature and incubated with the primary antibody of
interest diluted with 10% goat serum in PBS with 0.1% Triton
X-100 for 60 min at room temperature or overnight at 48C. The
primary antibodies were: (1) monoclonal anticollagen type I
IgG2a clone I-8H5 (631701; MP Biomedicals, Montreal,
Quebec, Canada) diluted to 20 mg/mL or monoclonal anticollagen type I IgG1 clone COL-1 diluted to 37 mg/mL (C-2456;
Sigma); (2) monoclonal anticollagen type II IgG1 diluted
1:10 (II-II6B3; DSHB, IA); and (3) monoclonal anticollagen
type VI IgG1 diluted 1:10 (5C6; DSHB). Sections were
incubated with biotinylated goat antimouse IgG (Fab specific)
(B-7151; Sigma) diluted to 22 mg/mL with 10% goat serum in PBS
with 0.1% Triton X-100 for 60 min at room temperature.
Histochemical detection was performed with the Vectastain
ABC-Alkaline Phosphatase (AP) system and AP Red Substrate
kit (AK-5000 and SK-5100; Vector Laboratories Inc., Burlington, Ontario, Canada). Some sections were counterstained with
Weigert Iron Hematoxylin (HT-1079; Sigma), dehydrated in
graded ethanol, and mounted in Permount mounting medium
(SP15-100; Fisher Scientific). Exclusion of primary antibodies
resulted in no staining.
To quantify the cross-sectional size of the menisci, the area
(mm2) of meniscal tissues excluding the adipose tissue at the
periphery of the meniscal body was measured with Northern
Eclipse software (Empix, Mississauga, Ontario, Canada).
To quantify the percentage of Safranin O-positive tissues in
the menisci, the area (mm2) of Safranin O-positive tissues was
measured with Northern Eclipse software and then normalized
to the total area (mm2) of meniscal tissues, excluding the
adipose tissue at the periphery of the meniscal body.
To quantify the extent of vascular penetration, the length
between the outer lateral edge of the menisci and the most
medial blood vessel found was measured with Northern Eclipse
software and then normalized to the total length of the menisci.
Presented results are the average from all anterior, middle,
and posterior sections. The Student’s t-test was performed
to compare groups, with p < 0.05 considered statistically
significant.
ECM ultrastructure was observed by environmental scanning electron microscopy (ESEM) of menisci that were fixed,
trimmed, and frozen as described above. Vertical cryosections
(30 mm thickness) were collected from the anterior portion of
menisci (Fig. 1A). Horizontal cryosections (30 mm thickness)
were also collected from the anterior portion of menisci
(Fig. 1B). Cryosections were then postfixed in 10% NBF and
washed in PBS prior to observation in the ESEM mode, where
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chamber pressure and temperature were 4.6 Torr and 08C
initially to create a relative humidity of 100%. After 2 min, the
pressure was reduced to 3.0 Torr, resulting in a relative
humidity of about 65%, to permit ultrastructural observations
without sample drying. Images were taken at 10 and 12.5 kV,
with a working distance of 6 mm.
RESULTS
All menisci appeared morphologically normal, smooth,
white, and glistening upon gross examination, except
for one left medial human meniscus that had a small
horizontal tear. When present, Safranin O staining
for glycosaminoglycans (GAG) was in the inner main
body of the menisci in all species (Fig. 2A,E,I). The
percentage of Safranin O-positive tissues varied substantially within all species but was significantly
greater for rabbit and sheep menisci than for human
(25.7 25.1% and 57.7 18.9% and 3.1 8.9%, respectively) (Fig. 3A). As expected, large round chondrocytelike cells were more prevalent in the inner main body of
Figure 3. The average areal percent of Safranin O-positive
tissue in rabbit and sheep menisci was significantly higher than
in human menisci (A). The average cross-sectional size of sheep and
human menisci were similar and both were significantly greater
than the average cross-sectional size of rabbit menisci (B). Vascular
penetration in the outer peripheral portion of menisci was
significantly higher for sheep and human menisci compared to
rabbit (C). *p < 0.05 using Student’s t-test. Data are expressed as
mean SD.
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Figure 4. Collagen I was found throughout the matrix of most menisci from rabbit (A), sheep (D), and human (G), but was absent from the
inner portion of the tip in some samples (black arrows in B, E, H). Discrete collagen I fibers were observed within the menisci matrix (black
arrowheads in C, F, I).
the menisci (Fig. 2B,F,J), while near the surface, cells
were more fusiform (Fig. 2C,G,K). The menisci from
rabbit appeared more cellular when compared to sheep
and human (Fig. 2B vs. 2F and 2J). As expected, cross-
sections of sheep and human menisci were similar
in size and significantly larger compared to rabbit
(25.6 7.4 mm2 and 34.7 9.6 mm2 and 3.1 1.0 mm2)
(Fig. 3B).
Figure 5. Collagen II was found in the inner body of rabbit (A), sheep (C), and human (E) menisci. Some collagen II fibers were arranged as
an intricate network (B, D, F).
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MENISCUS FOR ANIMAL MODELS OF REPAIR
Blood vessels were only found in the outer portion of
the meniscal body in all three species (Fig. 2D,H,L),
however blood vessels penetrated significantly deeper
into sheep and human menisci compared to rabbit
(vascular penetration was 11.2 5.6% and 14.4 5.8%
vs. 1.1 2.0%, respectively) (Fig. 3C). For sheep and
human menisci, vascular penetration did not differ
significantly between the anterior, middle, and posterior
portions. Vascular penetration in the posterior portion of
the rabbit menisci was slightly higher than in the middle
portion (1.7 2.7% vs. 0.4 1.0%), but still much lower
than that seen in human and sheep.
Collagen I was present throughout the matrix of most
rabbit, sheep, and human menisci (Fig. 4A,D,G). In 4 out
of 12 human samples, 7 out of 26 rabbit samples, and 10
out of 18 sheep samples, the inner portion of the tip of the
menisci was devoid of collagen I (Fig. 4B,E,H). Some
large immunostained collagen I fibers were visible
within the matrix (Fig. 4C,F,I).
Collagen II was detected in the inner main body of
rabbit, sheep, and human menisci, terminating abruptly
at the outer border (Fig. 5A,C,E). Some collagen II fibers
were arranged as an intricate network (Fig. 5B,D,F) that
was distinct from the collagen I pattern. Collagen II
immunostaining did not always overlap with Safranin O
staining and was often present in larger areas than
Safranin O was, particularly in human.
Collagen VI was detected throughout rabbit and
human menisci as well as in the adipose-rich tissue
peripheral to the menisci (Fig. 6A,D). Cellular and
pericellular staining was seen in and around the fusiform
cells, the chondrocyte-like cells (Fig. 6B,E), and some
cells in the adipose tissue adjacent to the menisci. The
vascular smooth muscle of large arteries was also
positive for collagen VI (Fig. 6C,F). Collagen VI immunostaining was not successful in sheep tissues with this
particular antibody.
In ESEM in horizontal sections (Fig. 1B), fibers were
parallel and oriented in a circumferential fashion
(Fig. 7A,D,G). In vertical transverse sections (Fig. 1A),
cross-sections of large fiber bundles were observed
(Fig. 7B,E,H), with some radially oriented collagen fibers
perpendicular to other meniscus fibers (Fig. 7B, white
arrow). Collagen fiber diameter ranged from 28 to
220 nm in horizontal sections. A distinct lamellar layer
of collagen with a thickness of 40 to 65 mm was identified
at the surface of menisci from sheep and human
(Fig. 7F,I) but not in rabbit. Chondrocyte lacunae
(Fig. 7B, empty white arrow) and chondrocytes
(Fig. 7A,C, white arrowheads) were more often observed
in rabbit menisci.
DISCUSSION
The purpose of this study was to structurally compare
human menisci to sheep and rabbit menisci in order to
assess the appropriateness of these animal species as
models for meniscus repair. Our results revealed
a global similarity in cell and tissue properties and
matrix composition for these three species. However,
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Figure 6. Collagen VI was found throughout rabbit (A) and
human (D) menisci as well as in the adipose-rich tissue adjacent to
the menisci. Staining was cellular and pericellular around
chondrocyte-like cells (black arrowheads in B, E). Collagen Type
VI was also immunodetected around some large blood vessels (black
arrows in C, F).
certain structural features of the human meniscus that
are critical for meniscus repair, such as vascularization
pattern, cellularity, and ECM collagen ultrastructure,
were similar in human and sheep but not in rabbit,
suggesting sheep to be advantageous as an animal
model for meniscus repair.
In the current study, Safranin O staining for the
presence of GAGs was found in the inner main body of the
menisci in all species, as reported previously.23,24
However, the average percentage of Safranin O-positive
tissue was lower in human menisci than both rabbit and
sheep, suggesting either an interspecies variation in
meniscus GAG content or an effectively older human
(average 54 years) menisci versus sheep (average 3 years)
and rabbit (average 7 months). There also may have been
pre-existing meniscal degeneration in the human samples used for this study, or postmortem tissue alterations. We found rabbit menisci to be more cellular,
which may promote an intrinsic repair process more
than in sheep and human. Although, in terms of cell
morphology, we found two cell types in all three species, a
fusiform morphology at the surface and a rounder
morphology in central regions, similar to previous
investigations.10,24,25,26
The patterns of vascularization we observed in human
agreed with previous observations11 and were similar to
sheep, while vascularization in the rabbit menisci was
distinctly lower and limited to the extreme periphery of
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CHEVRIER ET AL.
Figure 7. ESEM images of menisci from rabbit (A–C), sheep (D–F), and human (G–I) sectioned horizontally (A, C, D, G) and vertically (B,
E, F, H, I). In horizontal sections, fibers were mainly parallel and oriented in a circumferential fashion (A, D, G). In vertical sections, crosssections of large fiber bundles were observed (B, E, H) and some radial collagen fibers were oriented perpendicular to most other meniscus
fibers (white arrow in B). Chondrocyte lacunae (empty white arrow in B) and chondrocytes (white arrowheads in A, C) were more often
observed in rabbit menisci. A lamellar layer of collagen fibers (white double arrows) was identified at the surface of menisci from sheep (F) and
human (I) but could not be detected in rabbit.
the meniscal body. This finding is consistent with a
previous study, which stated parenthetically that few
vessels were seen penetrating the rabbit menisci postnatally.27 This finding suggests a fundamental difference in vascularization pattern in the smaller menisci,
which could significantly alter meniscus repair processes.
In the current study, global patterns of collagen
typing were similar for all species and consistent with
previously published data.23,27 Collagen I appeared
throughout most of the menisci while the inner portion
of the tip of some samples was devoid of collagen I.
Collagen II was detected throughout the inner region of
menisci. It has previously been reported in the rabbit
that collagen I and II overlap in younger animals while
discrete areas of collagen I or II appear with age.27
Interestingly, some collagen II fibers were organized into
a network similar to that described in the dog meniscus28
and quite distinct from that observed in articular
cartilage. The ability of fibrochondrocytes to form
JOURNAL OF ORTHOPAEDIC RESEARCH SEPTEMBER 2009
fibrillar networks of collagen II and collagen I appears
to be specific to the meniscus. Our ESEM observations
were in agreement with the above histological patterns
as well as with previous SEM work.10,25,26 ESEM
ultrastructure also allowed the observation of a lamellar
layer of collagen near the surface of the human and sheep
menisci as has been observed with SEM,10 but that was
not evident in the rabbit menisci of our study. This layer
could form a functional surface similar to that seen in the
articular cartilage in contact with the meniscal surface.
The pericellular immunolocalization of human and
rabbit collagen VI in our study is also consistent with
that observed previously in articular cartilage and
meniscus in rabbit,29 but in contrast with a previous
study of the canine meniscus,2 where collagen VI was
interterritorial, possibly due to a species dependence or
technical difference related to the staining procedure.
Interestingly we also found collagen VI in perivascular
regions. This location around the blood vessels in menisci
is compatible with a proposed role of collagen VI in
MENISCUS FOR ANIMAL MODELS OF REPAIR
binding von Willebrand factor in the vascular subendothelium.30
In summary, the discovery and development of
methods and products to repair the human meniscus
requires testing in animal models that allow translation
to human. The use of sheep models appears advantageous because they are highly representative of human
in terms of structural properties that are expected to
influence meniscus repair. More specifically, sheep
menisci bore greater similarity to human than rabbit in
terms of vascularization patterns, cell density, the
presence of an articulating lamellar collagen structure,
and in terms of cross-sectional size, which can be of
practical importance in surgical models. With reference
to the latter, it is important to note that the radial length
of the rabbit meniscus is 3 mm versus 9 and 10 mm for
sheep and human. In related work we have found that
the accurate radial placement of defects or tears in the
rabbit meniscus is very challenging due to its small
dimensions, and this aspect can again play a great role in
determining repair outcome. Taken together, our data
suggest that the main structural features that will
influence meniscus repair—cellularity, vascularity, collagen structure—are similar in sheep and human,
motivating the development of ovine meniscus repair
models.
ACKNOWLEDGMENTS
Financial support was provided by the Canadian Institutes of
Health Research (CIHR), the Canada Research Chairs
Program (CRC), the Fonds de la Recherche en Santé Québec
(FRSQ), and the Canadian Foundation for Innovation (CFI).
We thank Dr. Patrick Lavigne for providing human menisci
and Dr. Jun Sun and Geneviéve Picard for technical
assistance.
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