Type VI collagen in mouse masseter tendon from osseous attachment to myotendinous junction.код для вставкиСкачать
THE ANATOMICAL RECORD 243:294-302 (1995) Type VI Collagen in Mouse Masseter Tendon, From Osseous Attachment to Myotendinous Junction KATSUHIRO SENGA, MIYA KOBAYASHI, HISASHI HATTORI, KAZUKI YASUE, HIDEKI MIZUTANI, MINORU UEDA, AND TAKESHI HOSHINO 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 ABSTRACT 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 matrix. 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. 0 1995 WILEY-LISS. INC TYPE VI COLLAGEN IN MOUSE MASSETER TENDON (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. MATERIALS AND METHODS 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). 295 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 microscopy. 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. RESULTS 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 296 K. SENGA ET AL. 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 shown). 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 DISCUSSION 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 TYPE VI COLLAGEN IN MOUSE MASSETER TENDON 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- 297 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). 298 K. SENGA ET AL. 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 TYPE VI COLLAGEN IN MOUSE MASSETER TENDON 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 299 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). 300 K. SENGA ET AL. 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). TYPE VI COLLAGEN IN MOUSE MASSETER TENDON 301 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. LITERATURE CITED 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 Benjamin, M., E.J. Evans, and L. Copp 1986 The histology of tendon attachments to bone in man. J . Anat., 149:89-100. Bruns, R.R., W. Press, E. Engvall, R. Timpl, and J . Gross 1986 Type VI collagen in extracellular, 100-nm periodic filaments and fibrils: Identification by immunoelectron microscopy. J . Cell Biol., 103:393-404. Bray, D.F., C.B. Frank, and R.C. Bray 1990 Cytochemical evidence for the proteoglycan-associated filamentous network in ligament extracellular matrix. J . Orthopaedic Res., 8:l-12. Bray, D.F., R.C. Bray, and C.B. Frank 1993 Ultrastructural immunolocalization of type VI collagen and chondroitin sulphate in ligament. J . Orthopaedic Res., lIt677-685. Chiquet, M., and D.M. Fambrough 1984 Chick myotendinous antigen. 11. A novel extracellular glycoprotein complex consisting of large disulfide-linked subunits. J. Cell Biol., 98t1937-1946. Doane, K.J., G. Yang, and D.E. Birk 1992 Corneal cell-matrix interactions: Type VI collagen promotes adhesion and spreading of corneal fibroblasts. Exp. Cell Res., 200:490-499. Dyer, R.F. and C.D. Enna 1976 Ultrastructural features of adult human tendon. Cell Tiss. Res., 168:247-259. Evans, J.H. and J.C. Barbenel 1975 Structural and mechanical properties of tendon related to function. Equine Vet. J . , 7:l-8. Fleischmajer, R., J.S. Perlish, and T. Faraggiana 1991 Rotary shadowing of collagen monomers, oligomers, and fibrils during tendon fibrillogenesis. J . Histochem. Cytochem., 39:51-58. Hirano, K., M. Kobayashi, K., Kobayashi, T. Hoshino, and S. Awaya 1989 Experimental formation of 100 nm periodic fibrils in the mouse corneal stroma and trabecular meshwork. Invest. Ophthalmol. Vis. Sci., 30:869-874. Iida, K., M. Kobayashi, K. Kobayashi, S. Saga, T. Hoshino, and M. Matsuyama 1994 Experimental in vivo and in vitro formation of Type VI collagen periodic fibrils in chorionic villi of human placenta. J . Electron Microsc., 43:367-372. Jarvien, M., P. Kannus, M. Kvist, J . Isola, M. Lehto, and L. Jozsa 1991 Macromolecular composition of the myotendinous junction. Exp. Mol. Pathol., 55230-237. Jozsa, L., P. Kannus, J.B. Balint, and A. Reffy 1991 Three-dimensional ultrastructure of human tendons. Acta Anat., 142:306312. Keene, D.R., E. Engvall, and R.W. Glanville 1988 Ultrastructure of type VI collagen in human skin and cartilage suggests a n anchoring function for this filamentous network. J . Cell Biol., 107: 1995-2006. Law, D.J. 1993 Ultrastructural comparision of slack and stretched myotendinous junctions, based on a three-dimensional model of the connecting domain. J . Muscle Res. Cell Motil., 14t401-411. Mizoguchi, I., Nakamura, M., Takahashi, I., Kagayama, M., and Mitani, H 1990 An immunohistochemical study of localization of type 1and type I1 collagen in mandibular condylar cartilage compared with tibia1 growth plate. Histochemistry, 93t593-599. Nakamura, M., M. Kobayashi, K. Hirano, K. Kobayashi, T. Hoshino, and S. Awaya 1994 Glycosaminoglycan and collagen fibrillar in- 302 K. SENGA ET AL. teractions in the mouse corneal stroma. Matrix Biol., 14r283286. Neurath, M.F. 1993 Detection of Luse bodies, spiralled collagen, dysplastic collagen, and intracellular collagen in rheumatoid connective tissues: An electron microscopic study. Ann. Rheumatic Diseases., 52t278-284. Rittig, M., E. Lutjen-Drecoll, J . Rauterberg, R. Jander, and J. Mollenhauer 1990 Type VI collagen in the human iris and ciliary body. Cell Tissue Res., 259:305-312. Rowe, R.W.D. 1985a The structure of rat tail tendon. Connective Tissue Res., 14:9-20. Rowe, R.W.D. 198513 The structure of rat tail tendon fascicles. Connective Tissue Res., 1 4 2 - 3 0 . Schneider, H. 1956 Zur Struktur der Sehnenansatzzonen. Zeitschrift fur Anatomie und Entwicklungsgeschichte, 119t431-456. Squier, C.A. and W.H. Bausch 1984 Three-dimensional organization of fibroblasts and collagen fibrils in rat tail tendon. Cell Tiss. Res., 238:319-327. Swasdison, S. and R. Mayne 1989 Location of the integrin complex and extracellular matrix molecules in the chicken myotendinous junction. Cell Tissue Res., 257537-543. Takahashi, T. and K. Tohyama 1991 Electron microscopic study of distribution of proteoglycan in bovine cornea and sclera. Jpn. J . Ophthalmol., 35:211-225. Tidball, J.G., T. OHalloran, and K. Burridge 1986 Talin a t myotendinous junctions. J . Cell Biol., 103:1465-1472. Tidball, J.G. 1992 Desmin at myotendinous junctions. Exp. Cell Res., 199:206-212. Timpl, R., and M.-L Chu 1994 Microfibrillar collagen type VI. In: Extracellular Matrix Assembly and Structure. Academic Press, New York, pp. 207-242. Trotter, J.A., K. Corbett, and B.P. Avner 1981 Structure and function of the murine muscle-tendon junction. Anat. Rec., 201t293-302. Trotter, J.A., S. Eberhard, and A. Samora 1983 Structural domains of the muscle-tendon junction. 1. The internal lamina and the connecting domain. Anat. Rec., 207.573-591. Yasue, K., M. Kobayashi, H. Hattori, T. Teramoto, K. Senga, H. Mizutani, M. Ueda, T. Kaneda, and T. Hoshino 1994 An ultrastructural study of extracellular fibrillar components of developing mouse mandibular condyle with special reference to type VI collagen. Arch. Oral Biol., 39:689-694.