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Type VI collagen in mouse masseter tendon from osseous attachment to myotendinous junction.

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THE ANATOMICAL RECORD 243:294-302 (1995)
Type VI Collagen in Mouse Masseter Tendon, From Osseous
Attachment to Myotendinous Junction
Departments of Oral Surgery (K.S., H.H., K.Y., H.M., M.U.) and Anatomy 11 (M.K., T.H.),
Nagoya University School of Medicine, Nagoya 466, Japan
Background and Methods: The association of masseter tendon type VI collagen with other extracellular matrix (ECM) components
was examined from osseous attachment to myotendinous junction by immunohistochemistry and transmission electron microscopy with ATP
treatment and enzyme digestion.
Results: In the tendon proper, fibrocytes extended their processes among
bundles of striated collagen fibrils and associated with adjacent cells
through amorphous materials, thus forming a three-dimensional network.
The amorphous or filamentous material was observed around the fibrocyte
cell body and along the cell processes, where the localization of type VI
collagen was confirmed by immunohistochemistry using anti-type VI collagen antibody. After treatment with 20 mM adenosine 5’4riphosphate
(ATP), 100 nm periodic fibrils, an aggregated form of type VI collagen, were
formed in the place where amorphous or filamentous material was present
before the treatment. In myotendinous junction, the ATP-aggregated periodic fibrils were observed to associate with the external lamina of the muscle cells as well as among junctional tendon collagen fibrils. In the tendonbone boundary, ATP-aggregated periodic fibrils were observed around
fibrocartilage-like cells in the uncalcifying area but not in the calcification
front. Prolonged ATP treatment or hyaluronidase predigestion caused the
formation of type VI collagen periodic fibrils in the area near the calcified
Conclusions: The distribution of type VI collagen in mouse masseter tendon is different in different anatomical position. This may reflect the different functional demand for this collagen. o 1995 Wiley-Liss, Inc.
Key words: Type VI collagen, Mouse, Masseter tendon, Electron microscopy, ATP treatment
Tendons are dense connective tissue structures hav- junction is a highly specializedregion where the plasma
ing great tensile strength and a degree of pliability or membrane of the muscle cell is folded and invaginates
elasticity. They are composed of striated thick collagen to form finger-like structures; thus terminal cell profibrils which are regularly arranged in bundles and cesses interdigitate with tendon collagen fibers (Trotter
extend from myotendinous junction to tendon-bone et al., 1981; Tidball et al., 1986; Law, 1993).There were
joint. Structural and mechanical properties of the ten- some reports on macromolecular composition at this
don have been studied in relation to its function (Evans junction (Trotter et al., 1983; Chiquet and Fambrough,
and Barbenel, 1975).
1984; Tidball et al., 1986; Tidball, 1992). It was sugA tendon fascicle consists of a group of collagen fiber gested that a high concentration of macromolecular
bundles. Within each fascicle, rows of elongated and components at this junction might increase the adheflattened fibrocytes are scattered among thick collagen sive property and be important in improving the elastic
fibrils. Using scanning and transmission electron buffer capacity against loading (Jarvien et al., 1991).
microscopes, the ultrastructure was characterized: The tendon-bonejoint has been classified into four zones
crimped architecture of tendon fibers (Rowe, 1985b),the
chain-like arrangement of many fibrils and bridges between adjacent fibrils in human tendon (Dyer and
Received May 11, 1995; accepted July 11, 1995.
Enna, 1976), three-dimensional organization of fibroAddress reprint requests to Dr. Katsuhiro Senga, Department of
blasts (Squier and Bausch, 1984), and a network made Oral Surgery, Nagoya University School of Medicine, 65 Tsurumaby collagen fibers (Jozsa et al., 1991).The myotendinous cho, Showa-ku, Nagoya 466, Japan.
(Schneider, 1956; Benjamin et al., 1986): tendon, fibrocartilage, calcified fibrocartilage, and bone. The fibrocartilage ensures that tendon fibers do not bend, splay
out, or become compressed a t the hard tissue interface,
and thereby offers some protection from wear and tear.
Type VI collagen is a macromolecular component of
extracellular matrices and is widely distributed in connective tissues (Timpl and Chu, 1994). Electron microscopic examinations and analysis of the amino acid sequences imply that type VI collagen may work as an
anchor in cell-matrix or matrix-matrix interaction
(Doane et al., 1992; Keene et al., 1988), though the
precise role of this collagen has not been fully understood. Type VI collagen, which consists of a relatively
short triple helix and large N- and C-terminal globular
domains, exists in the form of thin, beaded filaments.
When the tissues are treated with acidic ATP, type VI
collagen aggregates to form 100 nm periodic fibrils
(Bruns et al., 1986; Hirano et al., 1989; Yasue et al.,
1994), which can be observed by electron microscopy.
ATP treatment is useful for the detection of type VI
collagen in the tissue. Type VI collagen is widely distributed in the tendon (Swasdison and Mayne, 1989;
Fleischmajer et al., 1991; Neurath, 1993). However, its
entire distribution from osseous attachment to myotendinous junction has not been fully investigated.
In this study, we examined localization and ultrastructure of type VI collagen in mouse masseter muscle
tendon by immunohistochemistry and electron microsCOPY.
Male and female mice of Balb/C strain were used at
8 weeks after birth. Care of the animals in this investigation conformed to the Guide for Animal Research,
Nagoya University School of Medicine. After ether anaesthesia, tendons of masseter rostral superficialis
were removed and processed for light and electron microscopy as follows.
Light and Electron Microscopy
Untreated control just after removal and ATPtreated specimens were placed immediately in Karnovsky’s fixative and fixed for 24 hours a t 4°C. They
were rinsed in 0.1 M phosphate buffered saline (PBS),
pH 7.4, and decalcified in 10% ethylene diamine tetraacetic acid (EDTA) . 4Na-glycerol solution, pH 7.4 at
4°C for 15 days. After washing in PBS, they were postfixed in 1%osmium tetroxide buffered with PBS at
room temperature for 90 minutes. After washing in
PBS, they were dehydrated in a graded series of ethanol and embedded in Quetol 812 (Nissin EM, Tokyo).
Semi-thin sections 2 to 2.75 pm thick were stained
with 1%toluidine blue in 0.1M sodium borate for light
microscopy. Then adjacent ultrathin sections were cut
on an ultramicrotome (Porter-Blum MT-1) and doublestained with uranyl acetate and lead citrate and observed by a transmission electron microscope (lOOCX,
JEOL, Tokyo).
washed in 0.1 M PBS, and then decalcified, dehydrated,
and embedded in Spurr’s resin. Sections 1to 2 pm thick
were mounted on poly-L-lysine coated glass slides, and
resin was removed by an ethanolic solution of sodium
ethoxide (Mizoguchi et al., 1990). They were incubated
with a 1:200 dilution of anti-type VI collagen polyclonal
antibody (Heyl, Inc., Germany) at room temperature for
6 hours in moisture chamber. The sections were rinsed
with PBS and were subsequently reacted with a 1:lOO
dilution of biotinylated goat anti-rabbit IgG (E.Y. Laboratories, Inc.,) a t room temperature for 60 minutes,
incubated with Vectastain ABC Reagent (Vector Laboratories, Inc., Burlingame, CA) at room temperature
for 60 minutes, and washed in cool PBS. Then they were
reacted with DAB solution and observed with a light
microscope. Sections incubated with non-immune sera
or PBS instead of anti-type VI collagen polyclonal antibody served as controls.
ATP Treatment
Specimens were incubated in PBS containing 20 mM
ATP . 2 Na (Sigma, A-6144, Sigma Chemical, Inc., St.
Louis, MO), pH 4.1, at 37°C for 3 or 24 hours before
fixation with Karnovsky’s fixative (Hirano et al.,
1989). Then they were decalcified, postfixed, dehydrated, embedded, and observed by light and electron
Hyaluronidase Digestion
Specimens were digested with 25 mg/ml of testicular
hyaluronidase (H3631 Sigma Chemical Co., Inc. St.
Louis, MO) a t 37°C for 3 hours before ATP treatment.
Testicular hyaluronidase solution contained 0.005 M
sodium acetate, 0.5 M sodium chloride, 0.5 mg/ml bovine serum albumin at pH 6.5 with 0.8 mM benzamidine, 1.6 mM EDTA, and 1.6 mg/ml of soybean trypsin
inhibitor. Then they were treated with ATP, fixed, and
processed for electron microscopy.
Anatomy of Mouse Masseter Tendon
The origin and insertion of mouse rostral superficialis masseter is shown in Figure l . The tendon was thick
at the origin and became thinner toward the insertion
covering the muscle fibers. By light microscopy, thick
collagen was regularly arranged in bundles from tendon-bone joint to myotendinous junction. In longitudinal sectional profile, rows of elongated and flattened
fibrocytes were among collagen fibrils (not shown).
Interaction of Type VI Collagen, Fibrocytes, and Thick
Collagen Fibrils in Tendon Proper
Among the thick collagen fibrils, fibrocytes in the
tendon proper extended their processes radially and
associated with adjacent cells (Figs. 2A,C, 3A). The
processes were among the interspace of striated collagen fibrils tapering off to the tip. Cell bodies and cell
processes were surrounded by amorphous or filamenlrnrnunohistochernistry
tous material (Fig. 3A). Island-like sectional profiles of
Specimens were fixed immediately after removal cell processes were seen among the collagen fibrils
with periodate-lysine-paraformaldehydefixative (0.01 (Fig. 2C). The processes were linked to neighboring cell
M NaI0,-0.075 M lysine-0.0375M phosphate buffer-2% processes directly or by amorphous material (Figs. 2C,
paraformaldehyde) at 4°C for 6 hours. They were 3A). Immunoreaction for type VI collagen was strongly
Fig, 1, Skeleton of a male Balbic mouse of 8 weeks after birth. Rostra1 superficialis masseter origins
from constricted area in maxilla (arrow) and inserts into caudal lower margin of mandible (white arrowheads). Thick tendon has a characteristic shiny white appearance in the origin and it becomes
thinner in the insertion covering muscle fibers (asterisk).
positive around the cell body and along the cell processes (Fig. 2B).
After ATP treatment for 3 hours, periodic fibrils of
100 nm interval were observed (Fig. 3B,D,E); they are
known as the aggregated form of type VI collagens.
Since there were no periodic structures in control specimens or those treated with PBS only, ATP treatment
could detect type VI collagen in the tendon proper. The
aggregated form of type VI collagen was observed
around the cell bodies and cell processes where amorphous or filamentous material was present before ATP
treatment (Fig. 3B). At the region where fibrocytes
connected with each other, the periodic structures were
formed along the cell membrane (Fig. 3D,E). In longitudinal section, type VI collagen periodic fibrils were
observed between cells and striated collagen fibrils, but
the periodic structure was less frequently observed
among the thick collagen fibrils in the bundle (not
along the collagen fibrils more removed from the myotendinous junction.
Type VI Collagen on the Border of Tendon-BoneJoint
The calcification front was clearly observed when decalcification stopped a t 15 days (Fig. 5A). Striated
thick collagen fibrils inserted into the bone cortex as
Sharpey’s fibers. Cells with osteoblastic, fibrocartilagelike, or fibroblastic appearances were observed in this
area (not shown). On the border of the tendon-bone
joint, thin collagen fibrils in the matrix of fibrocartilage-like cell were randomly arranged (Fig. 5B), where
the immunoreaction for type VI collagen was positive
(not shown). After ATP treatment, 100 nm periodic
fibrils were formed in the matrix around the fibrocartilage-like cell at this junction, especially in the uncalcified area (Fig. 5C). In the calcified bone matrix or
among the tendon collagen fibrils, no periodic structures were observed by 3 hour ATP treatment (not
shown). If the tissue was treated with ATP for 24
Type VI Collagen in Myotendinous Junction
hours, or was digested with testicular hyaluronidase
In the myotendinous junction, tendon collagen fibers before ATP treatment, numerous periodic structures
inserted into muscle cells and linked to endomysium were observed in the area near the calcified matrix
(Fig. 4A). Immunoreaction for type VI collagen was (Fig. 6A,B). No periodic structures were observed
strongly positive at this junction (Fig. 4B). The muscle among thick collagen fibrils of Sharpey’s fibers (not
cells of this junction had anastomotic processes like shown).
fingers, and the tendon collagen fibrils inserted into
the interspace of the finger-like processes and were
In the tendon proper, fibrocytes formed a three-dilined with external lamina of the muscle cell processes
(Fig. 4C). Around the anastomotic processes and along mensional network; thus tendon collagen fibrils were
the basal lamina, electron dense amorphous material grouped into fibers as previously reported (Rowe,
was seen. Type VI collagen periodic structures were 1985a). We observed amorphous or filamentous mateabundantly formed close to the external lamina of mus- rial around these fibrocytes. The material was identicle cells as well as among the collagen fibrils inserting cal to the thin electron-dense seams mentioned by Bray
into the finger-like muscle cell processes after ATP et al. (1990, 1993). By ATP treatment, we have contreatment for 3 hours (Fig. 4D,E). Hardly any of them firmed that type VI collagen abundantly exists in the
were observed in the interspace of muscle fibers or pericellular amorphous or filamentous material. The
Fig. 2. A Light microscopy of masseter tendon. Two micrometer
thick cross section of tendon proper was stained with toluidine blue.
Fibrocytes associate with each other. B: Immunohistochemistry for
type VI collagen in a cross section of mouse masseter tendon proper
shows the strongly positive reaction around the fibrocytes and along
the cell processes. C: Low magnification transmission electron micro-
graph of a cross section of the tendon proper. Fibrocytes (F) extend
their processes radially and are associated with adjacent cells. The
processes taper off to a point among the interspace of collagen fibrils
(arrowheads). Some detached processes like islands are seen among
the collagen fibrils (arrows). Tc, tendon collagen fibrils. Bars indicate
10 p,m (A, B) and 1 p,m (C).
Fig. 3.A Higher magnification of cell processes of tendon fibrocyte
from Figure 2C. Amorphous materials associate with the cell bodies
and processes (arrows). B: Periodic type VI collagen fibrils are formed
by ATP treatment in tendon proper (arrowheads). Fibrocytes were
destroyed by the strong acidity of the 20 mM ATP solution (open
arrows). C: Longitudinal section shows amorphous materials between
the cells (arrows). D Periodic type VI collagen (curved arrows) formed
by ATP treatment are between the cells destroyed by strong acidity of
ATP. E: Higher magnification of D. F, fibrocyte; Tc, tendon collagen
fibrils. Bars indicate 1 Fm (A, B, C, D) and 100 nm (El.
three-dimensional network was consequently composed of fibrocytes and the type VI collagen around
them. This indicates, in addition to a n adhesive role in
cell-cell interaction, type VI collagen may have a buffer
action to reduce mechanical stress to fibrocytes in the
tendon collagens.
At myotendinous junction, type VI collagen was associated with the external lamina of the finger-like
processes of muscle cells as well as with the inserting
tendon collagen fibrils. Rittig et al. (1990) have reported that immunostaining for anti-type VI collagen
was positive adjacent to the basal lamina of the human
Fig. 4. A A longitudinal section profile of myotendinous junction
stained with toluidine blue. Tendon collagen fibers (T) inserted into
muscle cells (m) and linked to endomysium. B: Immunohistochemistry for type VI collagen at the myotendinous junction. The reaction is
strongly positive a t this junction. C . Electron micrograph of myotendinous junction of mouse masseter muscle. The muscle cells have
anastomotic processes like fingers and terminal cell processes interdigitate with tendon collagen fibrils. The amorphous materials are
seen along the external lamina of the anastomotic processes (arrows).
D Formation of periodic type VI collagen fibrils by ATP treatment
(curved arrows) among the collagen fibrils in the tendon. The periodic
structures were at about 100 nm intervals. The fibrils were also associated with external lamina (small arrows). E: Higher magnification of 100 nm periodic fibrils in D. F, fibrocyte; Tc, tendon collagen
fibrils; mF, myofibril. Bars indicate 100 pm (A, B), 1 p m (C, D),and
100 nm (E).
Fig, 5. A. A longitudinal section profile of tendon (T) -bone (B)joint
stained with toluidine blue. B: Transmission electron micrograph of
the matrix of fibrocartilage-like cell. Collagen fibrils of the matrix are
thinner than tendon collagen fibrils and run at random. C: Formation
of periodic type VI collagen fibrils by 3 hour ATP treatment (curved
arrow). Higher magnification of the periodic fibrils (inset; arrowheads). Calcified matrix is seen (open arrow). F, fibrocartilage-like
cell; Tc, tendon collagen fibrils. Bars indicate 100 pm (A), 1 pm (B, C),
and 100 nm (C inset).
mouse masseter tendon could be common from osseous
attachment to the myotendinous junction, withstanding the pressure, tension, and twisting as well as sharing the stress.
The participation of PGs/GAGs is important to study
the interaction of type VI collagen with striated collagen fibrils or cell-matrix interactions. Takahashi and
Tohyama (1991) showed that amorphous material,
which resembled the “amorphous material” in the
present study, was located among the collagen fibrils of
sclera. They also showed the material in the pericellular regions of keratocytes in bovine cornea included
PGs. Nakamura et al. (1994) have demonstrated the
possibility that GAGs or PGs mediated the type VI
collagen fibril-striated collagen fibril interaction in
mouse cornea. It is also supposed that PGs/GAGs are
closely related in cell-type VI collagen and fibril-type
VI collagen interactions. The interaction of PGs/GAGs
with type VI collagen in mouse masseter tendon, from
osseous attachment to myotendinous junction, is presently under investigation in our laboratory.
Fig. 6. A Formation of periodic type VI collagen fibrils by enzyme
predigestion and ATP treatment. Numerous periodic fibrils are seen
(curved arrow). B: Higher magnification of (A). Tc, tendon collagen
fibrils; F, fibrocartilage-like cell. Bars indicate 1 pm.
ciliary muscle cells. Iida et al. (1994) also reported that
type VI collagen was localized close to the trophoblastic
and endothelial basal lamina in the human term placenta by light and electron microscopy. Type VI collagen in this area may have a role in cell-matrix interaction, a n arrangement of ECM environments for the
exchange of the metabolites and protection of inserting
thick collagen from mechanical stresses.
In the tendon-bone joint, the localization of type VI
collagen was confirmed in this study. Immunohistochemistry for type VI collagen has revealed a positive
reaction in the matrices of fibrocartilage-like cells near
the calcification front of osseous attachment, in which
type VI collagen may protect against mechanical
stresses to the cell. Since prolonged ATP treatment or
predigestion with testicular hyaluronidase successfully
formed a larger amount of type VI collagen periodic
fibrils, type VI collagen in this area could be more
closely associated with proteoglycans (PGs) or glycosaminoglycans (GAGs) than at other positions. We
have recently reported that testicular hyaluronidase
predigestion facilitated the formation of periodic fibrils
in fibrous zone of mouse mandibular condyle (Yasue et
al., 1994). Thus, the function of type VI collagen in
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junction, mouse, attachment, tendon, masseter, typed, collagen, osseous, myotendinous
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