Sage Journals: Discover world-class research

Abstract

CD44 is a multifunctional adhesion molecule that binds to hyaluronan (HA), type I collagen, and fibronectin. We investigated localization of CD44 and HA in mandibular condylar cartilage compared with the growth plate and the articular cartilage, to clarify the characteristics of chondrocytes. We also performed Western blotting using a lysate of mandibular condyle. In mandibular condyle, CD44-positive cells were seen in the surface region of the fibrous cell layer and in the proliferative cell layer. Western blotting revealed that the molecular weight of CD44 in condyle was 78 to 86 kD. Intense reactivity for HA was detected on the surface of the condyle and the lacunae of the hypertrophic cell layer. Moderate labeling was seen in cartilage matrix of the proliferative and maturative layer. Weak labeling was also seen in the fibrous cell layer. In growth plate and articular cartilage, HA was detected in all cell layers. However, chondrocytes of these cartilages did not exhibit reactivity for CD44. These results suggest that chondrocytes in the mandibular condylar cartilage differ in expression of CD44 from those in tibial growth plate and articular cartilage. Cell-matrix interaction between CD44 and HA may play an important role in the proliferation of chondrocytes in the mandibular condyle.

Keywords

CD44 hyaluronan condylar cartilage growth plate immunohistochemistry

The temporomandibular joint is a highly specialized diarthrodial joint that is under multidirectional compressive and tensile forces during mandibular movement such as mastication. Mandibular condyle and joints of limbs are covered with hyaline cartilage to reduce friction. The growth plate cartilage and articular cartilage of long bones are classified as primary cartilage. In contrast, mandibular condylar cartilage, which is formed by aggregated mesenchymal cells after mandibular body development, is classified as secondary cartilage (Durkin 1972; Durkin et al. 1973; Hall 1984). Mandibular condylar cartilage differs from other articular cartilage in its cellular organization and population (Blackwood 1966; Luder et al. 1988; Shibata et al. 1993; Koike et al. 1995; Ohashi et al. 1997; Suda et al. 1999; Visnapuu et al. 2000). It contains a fibrous cell layer and a wide proliferative cell layer, neither of which has been observed in other articular cartilage or growth plate cartilage. Mandibular condyle does not contain a growth plate, the structure responsible for the longitudinal growth of long bones. Immunohistochemistry and in situ hybridization have revealed differences between the mandibular condylar cartilage and articular cartilage of long bones in localization of type I collagen (Mizoguchi et al. 1990,1996), tenascin (Mackie et al. 1987; Thesleff et al. 1988), connective tissue growth factor (Fukunaga et al. 2003), brain-derived growth factor and its receptor, TrkB (Yamashiro et al. 2001). However, the importance of these differences is unclear.

CD44 is an adhesion molecule distinct from cadherin and integrins (Underhill 1992; Goodison et al. 1999; Bajorath 2000; Turley et al. 2002). This protein functions as a hyaluronan (HA) receptor and exhibits homology in its extracellular domain with cartilage link and proteoglycan core proteins (Goldstein et al. 1989; Aruffo et al. 1990; Wolffe et al. 1990; Haynes et al. 1991). CD44 is involved in cell migration (Jacobson et al. 1984; Stamenkovic et al. 1989; Spessotto et al. 2002), metastatic spread (Günthert et al. 1991), and attachment of hematopoietic stem cells to stromal cells (Miyake et al. 1990). Biochemical and histochemical studies indicate that cartilage matrix contains HA, a ligand for CD44 (Asari et al. 1992,1994; Pakkinen et al. 1996; Melrose et al. 2002; Teramoto et al. 2003). Additionally, in vitro studies suggest that CD44 is expressed in chondrocytes (Ishida et al. 1997; Knudson 2003; Wong and Carter 2003). Ostergaard et al. (1997) reported that CD44 expressed in human osteoarthritic cartilage. However, in our previous studies, we did not observe CD44 immunoreactivity in chondrocytes of the growth plate cartilage or articular cartilage of tibia (Nakamura et al. 1995; Nakamura and Ozawa 1996).

In the present study we investigated distribution of CD44-positive chondrocytes and localization of HA in rat mandibular condyle to clarify their characteristics in the process of chondrocyte differentiation by comparison with tibial articular and growth plate cartilage.

Materials and Methods

All animal procedures were performed in accordance with The Guidelines for Animal Experiments, Okayama University Graduate School of Medicine and Dentistry.

Antibody against CD44

A cysteine-conjugated peptide corresponding to c-terminal of CD44 (DQFMTADETRNLQSVDMKIGV) was synthesized and coupled via a terminal cysteine residue to keyhole limpet hemocyanin according to Okamoto et al. (1999). This antigen was subcutaneously injected into rabbits, and antisera were collected. Specific antibody to CD44 was purified using Affi-Gel 10 (Bio-Rad; Hercules, CA) coupled with the peptide.

Protein Extraction, Sodium Dodecyl Sulfate (SDS)-Polyacrylamide Gel Electrophoresis and Western Blotting

Mandibular condyles of 8-week-old rats were resected and then cut into small pieces using razor blades. Extraction of organic material was performed over a 24 hr period at 4C using 1 ml of RIPA buffer (1% NP-40, 0.5% deoxycholic acid and 0.1% SDS in 50 mM Tris-HCl buffer, pH (8.2) containing 10 mg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, and 2 mM sodium orthovanadate. The supernatant was separated by centrifugation at 14,000 × g for 10 min and concentrated with acetone. Precipitates were dissolved in 100 μl of sample buffer containing 4% SDS, 20% glycerol and 12% mercaptoethanol in 100 mM Tris-HCl (pH 6.8), followed by heating at 100C. SDS-polyacrylamide gel electrophoresis was performed using 7.5% polyacrylamide gel. Samples were electrophoresed at 150 V for 90 min and then transferred to nitrocellulose membrane. The membrane was immersed in 10% skim milk in 10 mM phosphate-buffered saline (PBS) for 1 hr to block nonspecific binding and was then washed with PBS containing 0.05% Tween 20. The membrane was incubated with rabbit anti-CD44 antibody diluted 1:1000 for 12 hr at 4C, followed by incubation with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Sigma; St Louis, MO) for 1 hr at room temperature. Immunoreactivity was visualized using ECL Western blotting detection reagents (Amersham Pharmacia Biotech UK Ltd.; Buckinghamshire, England) according to the manufacturer's instructions.

Preparation of Tissue

Five 8-week-old Wistar rats were used for immunohistochemical and histochemical experiments. The rats were anesthetized with sodium pentobarbital and perfused through the left ventricle with 4% paraformaldehyde and 0.1% glutaraldehyde in 0.05 M phosphate buffer (pH 7.3). Mandibular condyles were resected, immersed in perfusion fixative for 12 hr at 4C, and then decalcified in 5% ethylenediamine-tetraacetic acid (pH 7.3) for 2 weeks at 4C.

Light Microscopic CD44 Immunohistochemistry and HA Histochemistry

Specimens were dehydrated in graded ethanol, embedded in paraffin, and cut into 7-μm-thick sections. For the detection of HA, dewaxed sections were treated with 0.1% trypsin (Sigma) in PBS for 15 min at room temperature or 2 units of chondroitinase ABC (Sigma) in 50 mM Tris-HCl buffer (pH 8.0) containing 60 mM sodium acetate for 1 hr at 37C. They were then immersed in PBS containing 10% bovine serum albumin (BSA) for 15 min and then incubated in mouse anti-rat CD44 monoclonal antibody (Pharmingen; San Diego, CA) diluted 1:500 and biotinylated hyaluronic-acid-binding protein (HABP) (Seikagaku; Tokyo, Japan) diluted to 2 μg/ ml for 12 hr at 4C. They were immersed in AlexaFluor-594-conjugated anti-mouse Ig G (Molecular Probes, Inc.; Eugene, OR) and AlexaFluor-488-conjugated streptavidin (Molecular Probes) diluted 1:200. Fluorescence of specimens was observed under an Axioplan 2 (Carl Zeiss; Oberkochen, Germany) with the appropriate filter combinations.

Electron Microscopic Immunohistochemistry

Approximately 50-μm-thick sections were cut using a Mi-croslicer (D.S.K.; Kyoto, Japan). These sections were incubated in anti-CD44 monoclonal antibody for 24 hr at 4C, followed by incubation with HRP-conjugated anti-mouse Ig G (Sigma) for 24 hr at 4C. Immunoreactivity was visualized by immersion in DAB-H₂O₂ solution. After postfixation with 1% O_sO₄ in 0.1 M phosphate buffer (pH 7.4) for 1 hr at 4C, the tissue slices were dehydrated in graded ethanol and embedded in Epon 812 (TAAB; Berkshire, England). Ul-trathin sections were cut using an Ultracut E ultramicrotome (Reichert-Jung; Vienna, Austria) and stained with 1% tannic acid and uranyl acetate. These sections were observed under a Hitachi H-800 transmission electron microscope (Hitachi; Tokyo, Japan) at an accelerating voltage of 100 kV.

Figure 1

Western blotting with anti-CD44 antibody under reduced condition. An intense active band at 78 to 86 kD is detected in the mandibular condyle extract. A weak band is also seen at 42 kD.

Controls

Control sections were incubated with normal mouse Ig G or without a primary antibody. Otherwise, immunohistochemi-cal procedures were performed in the same manner as for the non-control sections. Control sections for HA were digested with 25 mg/ml hyaluronidase (Sigma) in PBS at room temperature for 30 min prior to the incubation with HABP.

Results

Western Blotting

Anti-CD44 antibody reacted with a 78- to 86-kD band and a 42-kD band in the mandibular condyle extract. The reactivity of the 78- to 86-kD band was more intense than that of the 42-kD band (Figure 1).

Localization of CD44 and HA

The mandibular condylar cartilage is divided into four layers: (a) fibrous cell layer, (b) proliferative cell layer, (c) maturative cell layer, and (d) hypertrophic cell layer. We examined the anterior region of sagittal sections of the condyle because these layers were clearly distinguishable in that region.

CD44 immunoreactivity was most pronounced in the proliferative cell layer of mandibular condyle (Figure 2A). Reactivity was detected mainly along plasma membrane of chondrocytes in the proliferative cell layer. The superficial cells in the fibrous cell layer also exhibited moderate reactivity. Several cells in the fibrous cell layer and the maturative cell layer exhibited weak reactivity. Very little immunoreactivity was detected in cells of the hypertrophic cell layer. Strong HA staining was observed on the surface of the condyle and the lacunae of the hypertrophic cell layer. Moderate labeling was also detected in cartilage matrix of the proliferative and maturative layer. Weak labeling was seen in the fibrous cell layer (Figure 2B). Colocalization of CD44 and HA was observed in the surface region of condyle and the proliferative cell layer (Figure 2C).

Figure 2

Fluorescent micrographs indicating CD44 and HA in a mandibular condyle. (A) Fluorescent micrograph indicating the localization of CD44 in the mandibular condyle. Intense immunoreactivity is detected in cells under the surface of condyle (arrowheads) and in the proliferative cell layer. A chondroclast or an osteoclast (∗) also shows strong reactivity. Weak labeling is also seen in the fibrous cell layer. Bar = 20 μm. Inset: Immunoreactivity is mainly detected along plasma membrane of chondrocytes in the proliferative cell layer. Bar = 10 μm. (B) Fluorescent micrograph indicating the localization of HA in the mandibular condyle. Intense labeling is detected on the surface of the condyle and the lacunae of the hypertrophic cell layer. Moderate reactivity is also seen in cartilage matrix of the proliferative and the mat-urative cell layers. Bar = 20 μm. (C) Merged micrograph of (A) and (B). Colocalization of CD44 and HA is seen on the surface of condyle and in the proliferative cell layer. F: fibrous cell layer, P: proliferative cell layer, M: maturative cell layer, H: hypertrophic cell layer. Bar = 20 μm.

Figure 3

Fluorescent micrographs indicating CD44 and HA in an articular cartilage and a growth plate cartilage. (A) Fluorescent micrograph indicating the localization of CD44 in the articular cartilage. Immunoreactivity is detected in osteocytes (arrowheads) and in a chon-droclast or an osteoclast (∗) of the articular cartilage. (B) Fluorescent micrograph indicating the localization of HA in the articular cartilage. Labeling is detected on the surface of the articular cartilage and on the pericellular matrix around chondrocytes. The lacunae of the hypertrophic cell layer show intense reactivity. (C) Fluorescent micrograph indicating the localization of CD44 in the growth plate cartilage. No immunoreactivity is detected in chondrocytes of the growth plate. Osteoclasts (∗) in epiphysis show CD44-labeling. (D) Fluorescent micrograph indicating the localization of HA in the growth plate cartilage. Labeling is detected on the cartilage matrix. Intense reactivity is seen on the pericellular matrix around chondrocytes and the lacunae of the hypertrophic chondrocytes. Bars = 20 μm.

In articular cartilage, no reactivity for CD44 was detected in chondrocytes (Figure 3A). Unmasking procedure, such as trypsin, chondroitinase ABC, and hyaluronidase pretreatment, did not change the stainability. HA labeling was most pronounced on the surface of the articular cavity and the hypertrophic cell layer. Other layers showed moderate positive reactivity (Figure 3B). The reactivity was intense in the pericellular matrix around chondrocytes.

In growth plate, no positive CD44 labeling was detected in chondrocytes at any stage of differentiation (Figure 3C). The staining pattern was not changed after unmasking procedure. HA labeling was observed in cartilage matrix of proliferative, maturative, and hypertrophic cell layers (Figure 3D). Particularly, pericellular matrix around chondrocytes showed intense reactivity.

No specific immunoreactivity was detected in the control sections. Staining by HABP was diminished by pretreatment with hyaluronidase.

Immunoelectron Microscopy for CD44

CD44 labeling was detected on the plasma membrane of the superficial cells in the fibrous cell layer (Figure 4A). Immunoreactivity was also detected on the plasma membrane of cells in the proliferative cell layer (Figure 4B). Mitotic cells exhibited CD44 immunoreactivity (Figure 4C). Some maturative chondrocytes adjacent to the proliferative cell layer showed weak CD44 reactivity.

Figure 4

Electron micrographs indicating CD44 localization in a mandibular condyle. (A) Electron micrograph of the surface of the mandibular condyle. Immunoreactivity is seen on the plasma membrane of a cell (∗) under the surface region of the condyle. (B) Electron micrograph of the proliferative cell layer. The cells in the proliferative cell layer show positive reactivity on their plasma membrane. (C) Immunoreactivity is seen on the plasma membrane of a mitotic chondrocyte (MC). Bars = 1 μm.

Discussion

In the present study, the pattern of CD44 expression in mandibular condylar cartilage differed from that of tibial cartilage. In mandibular condylar cartilage, CD44-positive cells were seen in the proliferative cell layer below the fibrous cell layer. However, chondrocytes in the proliferative zone of the growth plate and the articular cartilage of tibia did not exhibit CD44 immunoreactivity. Cranial bone and cartilage, including mandibular condylar cartilage, originate in neural-crest-derived mesenchyme (Helms and Schneider 2003). In contrast, bones and cartilage in limbs are derived from lateral plate mesoderm. Thus, mandibular condylar cartilage has a different developmental origin from tibial cartilage. The different patterns of CD44 expression observed in these two types of cartilage may be due to their different origins. CD44 is used as a marker of neural-crest-derived cells and embryonic stem cells (Corbel et al. 2000). Although differentiated cells such as osteoclasts and osteocytes also express CD44, CD44-positive cells in the proliferative cell layer of mandibular condyle may be undifferentiated mesenchymal cells.

The present Western blotting finding demonstrates that chondrocytes in the condylar cartilage express CD44. The molecular weight of a core protein of CD44 is reportedly ~50 kD in tunicamycin-treated cells (Okamoto et al. 1999). In addition, CD44 could be cleaved by metalloproteinase (Okamoto et al. 1999; Kajita et al. 2001). Therefore, the present 42-kD band corresponds to the core protein or a fragment of CD44. We assume that the 78- to 86-kD band corresponds to CD44 containing glycosaminoglycans. Chondroitin sulfate and/or heparan sulfate chains may be present on CD44 (Brown et al. 1991; Faassen et al. 1992). The type of glycosaminoglycan present in chondrocytes of the proliferative layer has yet to be determined.

Trypsin and chondroitinase ABC pretreatments revealed that HA localized in cartilage matrix at all stages of chondrocytes. Without an unmasking procedure, reactivity for HA was restricted in the surface of condyle and articular cartilage and in the pericellular matrix of the hypertrophic chondrocytes (data not shown). The unmasking procedure is essential for the detection of HA in cartilage as reported by Pakkinen et al. (1996). In the case of CD44 detection, chondroitinase ABC pretreatment slightly increased reactivity in the proliferative layer. However, unmasking procedure did not change the staining pattern for CD44.

Colocalization of CD44 and HA was observed in the proliferative layer of the mandibular condyle, although HA staining was less pronounced in the proliferative cell layer than on the surface of the condyle or the hypertrophic cell layer. CD44 is thought to participate in the internalization and degradation of HA (Knudson et al. 2002). Moreover, the CD44/HA pathway of chondrocytes seems to be involved in cellular signaling that induces cell survival (Brun et al. 2003), cell aggregation (Toole et al. 1972; Knudson et al. 1996), proliferation and expression of c-myc mRNA (Ishida et al. 1997). Furthermore, CD44 is involved in a cytoskeletal organization via the ERM family (Tsukita et al. 1994). Thus, cell-matrix interaction via CD44 and HA in the proliferative cell layer of the condylar cartilage may play an important role in the proliferation of chondrocytes.

We also found colocalization of CD44 and HA in the superficial area of the mandibular condyle. HA in the articular cavity is thought to reduce friction and facilitate joint movement. Thus, HA in the superficial area may play an important role in smooth movement and protection of articular cartilage. HA bound to CD44 in the superficial cells of the fibrous cell layer may serve as a reservoir of HA. Another possibility is that these cells internalize and degrade HA via a CD44-mediated mechanism.

We observed that HA was prominently present in the pericellular matrix around chondrocytes, as reported by previous research (Asari et al. 1992,1994; Pakkinen et al 1996; Melrose et al. 2002). However, chondrocytes in the growth plate and the articular cartilage did not exhibit expression of CD44. These results indicate that CD44 and HA are not involved in cell-matrix interaction of chondrocytes in these cartilage. HA mediates hydrostatic pressure by adsorbing water (Gakunga et al. 2000), indicating that HA is associated with increase in lacunae size of chondrocytes. Calcified cartilage matrix is subsequently resorbed by chondroclasts and osteoclasts in the process of endochondral ossification. These cells express CD44 on their plasma membrane (Nakamura et al. 1995; Nakamura and Ozawa 1996; Spessotto et al. 2002). Hence, HA in the pericellular matrix around chondrocytes may be involved in recognition and adhesion by chondroclasts and osteoclasts during cartilage resorption.

The differences between mandibular condylar cartilage and the articular and growth plate cartilage of long bones observed in the present study are clearly relevant to basic and pathological research of the temporomandibular joint.

Footnotes

Acknowledgements

This study was supported in part by a grant (No. 15591934) for scientific research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

We would like to thank Dr Noriyuki Nagaoka and Tomoko Yamamoto for their technical support.

References

Aruffo

Stamenkovic

Melnick

Underhill

Seed

(1990) CD44 is the principal cell surface receptor for hyaluronate. Cell 61:1303–1313.

Asari

Miyauchi

Miyazaki

Hamai

Horie

Takahashi

Sekiguchi

. (1992) Intra-and extracellular localization of hyaluronic acid and proteoglycan constituents (chondroitin sulfate, keratan sulfate, and protein core) in articular cartilage of rabbit tibia. J Histochem Cytochem 40:1693–1704.

Asari

Miyauchi

Kuriyama

Machida

Kohno

Uchiyama

(1994) Localization of hyaluronic acid in human articular cartilage. J Histochem Cytochem 42:513–522.

Bajorath

(2000) Molecular organization, structural features, and ligand binding characteristics of CD44, a highly variable cell surface glycoprotein with multiple functions. Proteins 39:103–111.

Blackwood

(1966) Growth of the mandibular condyle of the rat studied with tritiated thymidine. Arch Oral Biol 11:493–500.

Brown

Bouchard

St John

Wayner

Carter

(1991) Human keratinocytes express a new CD44 core protein (CD44E) as a heparan-sulfate intrinsic membrane proteoglycan with additional exons. J Cell Biol 113:207–221.

Brun

Panfilo

Daga Gordini

Cortivo

Abatangelo

(2003) The effect of hyaluronan on CD44-mediated survival of normal and hydroxyl radical-damaged chondrocytes. Osteoarthritis Cartilage 11:208–216.

Corbel

Lehmann

Davison

(2000) Expression of CD44 during early development of the chick embryo. Mech Dev 96:111–114.

Durkin

(1972) Secondary cartilage: a misnomer? Am J Orthod 62:15–41.

10.

Durkin

Heeley

Irving

(1973) The cartilage of the mandib-ular condyle. Oral Sci Rev 2:29–99.

11.

Faassen

Schrager

Klein

Oegema

Couchman

McCarthy

(1992) A cell surface chondroitin sulfate proteogly-can, immunologically related to CD44, is involved in type I collagen-mediated melanoma cell motility and invasion. J Cell Biol 116:521–531.

12.

Fukunaga

Yamashiro

Oya

Takeshita

Takigawa

Ta-kano-Yamamoto

(2003) Connective tissue growth factor mRNA expression pattern in cartilages is associated with their type I collagen expression. Bone 33:911–918.

13.

Gakunga

Kuboki

Opperman

(2000) Hyaluronan is essential for the expansion of the cranial base growth plates. J Craniofac Genet Dev Biol 20:53–63.

14.

Goldstein

Zhou

Picker

Minty

Bargatze

Ding

Butcher

(1989) A human lymphocyte homing receptor, the hermes antigen, is related to cartilage proteoglycan core and link proteins. Cell 56:1063–1072.

15.

Goodison

Urquidi

Tarin

(1999) CD44 cell adhesion molecules. Mol Pathol 52:189–196.

16.

Günthert

Hofmann

Rudy

Reber

Zöller

Haussmann

Matzku

. (1991) A new variant of glycoprotein CD44 confers metastatic potential to rat carcinoma cells. Cell 65:13–24.

17.

Hall

(1984) Genetic and epigenetic control of connective tissues in the craniofacial structures. Birth Defects Orig Artic Ser 20:1–17.

18.

Haynes

Liao

Patton

(1991) The transmembrane hyal-uronate receptor (CD44): multiple functions, multiple forms. Cancer Cells 3:347–350.

19.

Helms

Schneider

(2003) Cranial skeletal biology. Nature 423:326–331.

20.

Ishida

Tanaka

Morimoto

Takigawa

Eto

(1997) Chon-drocytes are regulated by cellular adhesion through CD44 and hyaluronic acid pathway. J Bone Miner Res 12:1657–1663.

21.

Jacobson

O'Dell

Holifield

Murphy

August

(1984) Redistribution of a major cell surface glycoprotein during cell movement. J Cell Biol 99:1613–1623.

22.

Kajita

Itoh

Chiba

Mori

Okada

Kinoh

Seiki

(2001) Membrane-type 1 matrix metalloproteinase cleaves CD44 and promotes cell migration. J Cell Biol 153:893–904.

23.

Koike

Ejiri

Hanada

Ozawa

(1995) Age-related histological changes in rat mandibular condyle. J Bone Miner Metab 13:10–16.

24.

Knudson

(2003) Hyaluronan and CD44: strategic players for cell-matrix interactions during chondrogenesis and matrix assembly. Birth Defects Res Part C Embryo Today 69:174–196.

25.

Knudson

Chow

Knudson

(2002) CD44-mediated uptake and degradation of hyaluronan. Matrix Biol 21:15–23.

26.

Knudson

Aguiar

Hua

Knudson

(1996) CD44-anchored hyaluronanrich pericellular matrices: an ultrastructural and biochemical analysis. Exp Cell Res 228:216–228.

27.

Luder

Leblond

von der Mark

(1988) Cellular stages in cartilage formation as revealed by morphometry, radioautography and type II collagen immunostaining of the mandibular condyle from weanling rats. Am J Anat 182:197–214.

28.

Mackie

Thesleff

Chiquet-Ehrismann

(1987) Tenascin is associated with chondrogenic and osteogenic differentiation in vivo and promotes chondrogenesis in vitro. J Cell Biol 105:2569–2579.

29.

Melrose

Tammi

Smith

(2002) Visualisation of hyaluronan and hyaluronanbinding proteins within ovine vertebral cartilages using biotinylated aggrecan G1-link complex and biotinylated hyaluronan oligosaccharides. Histochem Cell Biol 117:327–333.

30.

Miyake

Underhill

Lesley

Kincade

(1990) Hyaluronate can function as a cell adhesion molecule and CD44 participates in hyaluronate recognition. J Exp Med 172:69–75.

31.

Mizoguchi

Nakamura

Takahashi

Kagayama

Mitani

(1990) An immunohistochemical study of localization of type I and type II collagens in mandibular condylar cartilage compared with tibial growth plate. Histochemistry 93:593–599.

32.

Mizoguchi

Takahashi

Nakamura

Sasano

Sato

Kagayama

Mitani

(1996) An immunohistochemical study of regional differences in the distribution of type I and type II collagens in rat mandibular condylar cartilage. Arch Oral Biol 41:863–869.

33.

Nakamura

Kenmotsu

Sakai

Ozawa

(1995) Localization of CD44, the hyaluronate receptor, on the plasma membrane of osteocytes and osteoclasts in rat tibiae. Cell Tissue Res 280:225–233.

34.

Nakamura

Ozawa

(1996) Immunolocalization of CD44 and the ERM family in bone cells of mouse tibiae. J Bone Miner Res 11:1715–1722.

35.

Ohashi

Ejiri

Hanada

Ozawa

(1997) Changes in type I, II, and X collagen immunoreactivity of the mandibular condylar cartilage in a naturally aging rat model. J Bone Miner Metab 15:77–83.

36.

Okamoto

Kawano

Tsuiki

Sasaki

Nakao

Matsumoto

Suga

. (1999) CD44 cleavage induced by a membrane-associated metalloprotease plays a critical role in tumor cell migration. Oncogene 18:1435–1446.

37.

Ostergaard

Salter

Andersen

Petersen

Bendtzen

(1997) CD44 expression is up-regulated in the deep zone of os-teoarthritic cartilage from human femoral heads. Histopathology 31:451–459.

38.

Pakkinen

Hakkinen

Savolainen

Wang

Tammi

Agren

Lammi

. (1996) Distribution of hyaluronan in articular cartilage as probed by a biotinylated binding region of aggrecan. Histochem Cell Biol 105:187–194.

39.

Shibata

Suzuki

Yamashita

Ichijo

(1993) A comparative ultrastructural study of the mitotic chondrogenic cells in the mandibular condyle and tibial growth plate of the rat. Arch Oral Biol 38:845–851.

40.

Spessotto

Rossi

Degan

Di Francia

Perris

Colombatti

Gattei

(2002) Hyaluronan-CD44 interaction hampers migration of osteoclast-like cells by down-regulating MMP-9. J Cell Biol 158:1133–1144.

41.

Stamenkovic

Amiot

Pesando

Seed

(1989) A lymphocyte molecule implicated in lymph node homing is a member of the cartilage link protein family. Cell 56:1057–1062.

42.

Suda

Shibata

Yamazaki

Kuroda

Senior

Beck

Hammond

(1999) Parathyroid hormone-related protein regulates proliferation of condylar hypertrophic chondrocytes. J Bone Miner Res 14:1838–1847.

43.

Teramoto

Kaneko

Shibata

Yanagishita

Soma

(2003) Effect of compressive forces on extracellular matrix in rat mandibular condylar cartilage. J Bone Miner Metab 21:276–286.

44.

Thesleff

Kantomaa

Mackie

Chiquet-Ehrismann

(1988) Immunohistochemical localization of the matrix glycoprotein tenascin in the skull of the growing rat. Arch Oral Biol 33:383–390.

45.

Toole

Jackson

Gross

(1972) Hyaluronate in morphogenesis: inhibition of chondrogenesis in vitro. Proc Natl Acad Sci USA 69:1384–1386.

46.

Tsukita

Oishi

Sato

Sagara

Kawai

Tsukita

(1994) ERM family members as molecular linkers between the cell surface glycoprotein CD44 and actin-based cytoskeletons. J Cell Biol 126:391–401.

47.

Turley

Noble

Bourguignon

(2002) Signaling properties of hyaluronan receptors. J Biol Chem 277:4589–4592.

48.

Underhill

(1992) CD44: the hyaluronan receptor. J Cell Sci 103:293–298.

49.

Visnapuu

Peltomaki

Isotupa

Kantomaa

Helenius

(2000) Distribution and characterization of proliferative cells in the rat mandibular condyle during growth. Eur J Orthod 22:631–638.

50.

Wolffe

Gause

Pelfrey

Holland

Steinberg

August

(1990) The cDNA sequence of mouse Pgp-1 and homol-ogy to human CD44 cell surface antigen and proteoglycan core/link proteins. J Biol Chem 265:341–347.

51.

Wong

Carter

(2003) Articular cartilage functional histomorphology and mechanobiology: a research perspective. Bone 33:1–13.

52.

Yamashiro

Fukunaga

Yamashita

Kobashi

Takano-Yamamoto

(2001) Gene and protein expression of brain-derived neurotrophic factor and TrkB in bone and cartilage. Bone 28:404–409.

Localization of CD44 (Hyaluronan Receptor) and Hyaluronan in Rat Mandibular Condyle

Abstract

Keywords

Materials and Methods

Antibody against CD44

Protein Extraction, Sodium Dodecyl Sulfate (SDS)-Polyacrylamide Gel Electrophoresis and Western Blotting

Preparation of Tissue

Light Microscopic CD44 Immunohistochemistry and HA Histochemistry

Electron Microscopic Immunohistochemistry

Controls

Results

Western Blotting

Localization of CD44 and HA

Immunoelectron Microscopy for CD44

Discussion

Footnotes

Acknowledgements

References