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Changes in integrin expression during chondrogenesis in vitro: an immunomorphological study. M Shakibaei, B Zimmermann and H J Merker J Histochem Cytochem 1995 43: 1061 DOI: 10.1177/43.10.7560884 The online version of this article can be found at: http://jhc.sagepub.com/content/43/10/1061

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Original Article

I Changes in Integrin Expression During Chondrogenesis In Vitro: An Immunomorphological Study' MEHDI SHAKIBAEI, BERND ZIMMERMANN, and HANS-JOACHIM MERKER Institute of Anatomy, Free University of Berlin, Berlin, Germany.

Received for publication November 8, 1994 and in revised form March 13. 1995 and May 17, 1995; accepted May 31, 1995 (4A3529).

I Integrins are receptors composed of ligand-specifica-chains and cell type-specificp-chains which are involved in cell-cell and cell-matrix interactions. The distribution of al- and a3integrins as well as collagen Types I and 11, was investigated by immunofluorescence and immunoelectron microscopy during chondrogenesisin organ culture after various culture periods. Mesenchymal cells from limb buds of Day 12 mouse embryos were grown at high density. Within the first 2 days of the culture period, only al-integrin could be detected. Formation of cartilage-specificmatrix on Day 3 was accompanied by the Occurrence of a3-integrin. On Day 7, a 3 was present only in cartilage nodules, whereas a1 was strongly expressed in the perichondrium and was more or less homo-

Introduction Integrins are dimeric cell membrane receptors composed of one a- and one B-subunit. They bind components of the connective tissue matrix or ligands on the membrane of other cells. They are composed of three segments: an extracellular,a transmembranous. and a cytoplasmic domain that binds to the cytoskeleton. This construction enables them to connect the cell specifically with intercellular matrix components (cell-matrix interaction) and to inform the cell on matrix composition, thus regulating the behavior of the cells (Hynes, 1987,1992;McDonald and Mecham, 1991; Albelda and Buck, 1990; Hemler, 1990,1991;Akiyama et al., 1989; Ruoslahti and Pierschbacher, 1987). Integrins play an important role in cell migration, proliferation, differentiation, and cell adhesion (Hynes, 1992; Ruoslahti, 1991; Sommarin et al., 1989). Integrins of the fil-type (VLA, very late activation genes) are predominantly responsible for the binding to the matix. They have a common pl-subunit but varying a-subunits. Each integrin of this group preferentiallybinds certain matrix components: alp1 (VLA-1) and a2fil (VLA-2) to collagen and laminin (Albelda, 1993; Languino et al., 1989; Wayner and Carter, 1987), a3p1 (VLA-3) additionally to fibronectin (Wayner et al., 1988), a4pl (VLA-4) to

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Supported by grants from the Deutsche Forschungsgemeinschaft 174 and 465/1-1. * Correspondence to: Dr. Mehdi Shakibaei, Institut fiir Anatomie, Freie Universitat Berlin, Konigin-Luise-Str. 15, D-14195 Berlin, Germany.

geneously distributed in the surrounding mesenchyme. On Day 14, al-integrin was again detectable in cartilage. We suggest that the change in collagen formation from Type I to Tppe II during chondrogenesis is accompaniedby a change in integrin expression from a1 to a3. Conversely, d e r e n tiation of chondrocytes in aging cartilage is accompanied by the occurrence of collagen Type I and al-integrin. Therefore, a strict correlation between the collagen type synthesized by the cells and the appropriate receptor presented by the cells is suggested. (JHisrochemCytochem 43:106-1069, 1995) Integrins; Cartilage; Immunomorphology; Chondrogenesis; Organ culture. KEY WORDS:

fibronectin but also cellular ligands (Albelda, 1993), a5pl (VLA5) to fibronectin and other components with RGD segments (Albelda, 1993; Ruoslahti and Pierschbacher, 1987), and both a6pl (VLA-6) and a7pl (VLA-7) to laminin (Song et al., 1992; Sonnenberg et al., 1988). However, the ligands and their preferential affinity may vary in dependence on the cell type, the available cations (Mg2', Mn2', Ca2'), and the functional state of the cells (Hynes, 1992; Hemler, 1990,1991; Albelda and Buck, 1990). Several authors have demonstrated the occurrence of p l and 03integrins in cartilage tissue from different species in vivo and in vitro (Diirr et al., 1993; Enomoto et al., 1993; Shakibaei et al., 1993a; Ramachandrula et al., 1992; Salter et al., 1992; Woods et al., 1991). It has been suggested that these integrins are responsible for binding of chondrocytes to collagen Type 11, vitronectin, and fibronectin (Ramachandrula et al., 1992; Sommarin et al., 1989). However, it is not yet known which a-chain participates in this matrix-specific binding. The most promising candidate for binding to collagen Type I1 is the a3-chain, because it has been demonstrated to be present in cartilage by Salter et al. (1992), Enomoto et al. (1993), and Shakibaei (1995). On the other hand, it has been shown that alfi1-integrin is a receptor for collagen Type I (Enomoto et al., 1993; Syfrig et al., 1991; Ignatius et al., 1990; Kramer and Marks, 1989),which is present before chondrogenesis and after dedifferentiation. We therefore investigated the distribution of al- and a3-integrins and their co-localizationwith collagen Types I and I1 during chondrogenesis in cartilage organ cultures. 1061

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Materials and Methods Cartilage Organ Culture. Upper and lower limb buds of Day 12 mouse embryos were excised, rinsed with Hank’s solution, and incubated for 20 min in 0.1% dispase in Ca- and Mgfree solution (Zimmermann et al., 1990,1992a,b; Schroter-Kermani et al., 1991). After rinsing in growth medium, a single-cellsuspension was obtained by repeated pipetting and filtration through a nylon mesh of 20-pm pore width. Sedimentation of the cells was performed by centrifugation at 600 rpm. Ten p1of the cell sediment containing about 2 x lo6 cells was placed on a membrane filter that rested on a stainless steel grid at the medium-air interphase. Because of the high cell density, pronounced cell contacts developed in addition to sortingout processes leading to the formation of chondrogenic blastemal areas where chondrocyte differentiation occurred (Zimmermann et al., 1990,1992a,b).The medium consisted of DMEM supplemented with 15% fetal calf serum, antibiotics, and 50 kg ascorbic acidlml. It was changed every 3-4 days. The cells were grown at 37°C with 5 % CO2 in air. The cultures were investigated after 1, 2, 3, 4, 7, and 14 days by light and immunoelectron microscopy. Immunofluorescence. The cultureswere immersed in OCT embedding material (Bayer; Munich, Germany) and immediately frozen in liquid nitrogen. Eight-pn thick sections were cut perpendicularlyand processed as follows. The sections were placed on gelatin-covered slides and allowed to thaw at room temperature (RT) for 20 min, fixed in methanol for 10 min, and rinsed three times for 5 min in PBS. They were treated with collagenase (1 mg/ml) to unmask the integrin receptor as a prerequisite for immunolabeling of the integrins for 10 min at RT. After washing, the sections were incubated with the primary antibodies anti-integrin p1, al, a3, and aSp1 (diluted 140 in PBS) for 1 hr at RT. After rinsing three times for 5 min, the sections were incubated with the secondary antibody GAR-FITC (goat anti-rabbit, diluted 1:30)for 1hr at RT. To demonstratechondrogenic blastemata, sections were incubated with FIE-labeled PNA (peanut agglutinin, diluted 1:20 in PBS) for 1 hr at RT. Finally, the sections were rinsed and air-dried, coverslipped with glycerin, and examined and photographed with a light microscope (Axiophot 100; Zeiss, Oberkochen, Germany). Transmission Electron Microscopy (TEM). The cultures were fixed in 1% glutaraldehyde plus 1% tannic acid in 0.1 M phosphate buffer, pH 7.4,

post-fixed in 2% os04 in the same buffer, dehydrated in ethanol, and embedded in Epon. Ultra-thin sections were contrasted with 2% uranyl acetate and lead citrate and investigated under a transmission electron microscope (Zeiss EM 10). Immunoelectron Microscopy. After fixation for 1 hr with a mixture of 3% paraformaldehyde and 0.25% glutaraldehydein PBS, the cultures were rinsed overnight with PBS plus 1% BSA. This was followed by dehydration in ethanol and embedding in LR White (London Resin; Plano, Marburg, Germany). Sections were cut with an Ultracut E (Reichert) and placed on nickel grids. Immunolabeling was performed as follows. (a) Sections were treated with Clostridium histofytzcum collagenase 1 mglml (Sigma; Munich, Germany) for demonstration of integrins and with testicular hyaluronidase 1 mglml (Serva; Heidelberg, Germany) for immunolabeling of collagen Types I and I1 for 10 min at RT. (b) The sections were then washed and incubated with I% bovine serum albumin (BSA) in 0.01 M PBS, pH 7.0, at RT for 10 min and (c) incubated with primary antibodies (anti-integrin 01, al, a3, asp1 1:40, anti-collagen Types I and I1 130 in PBS-lOh BSA-0.5% Tween) overnight at 4°C.This was followed by (d) rinsing in PBS-BSA-Tween three times for 5 min at RT, (e) incubation with secondary antibody (goat anti-rabbit labeled with 10-nm gold particles 1:50 in PBS-BSA-Tween) for 60 min at RT, ( f ) rinsing in PBS-BSA-Tween three times for 5 min and in PBS three times for 5 min at RT, (g) fixation (1% glutaraldehyde) for 10 min at RT, and (h) rinsing in PBS twice for 5 min at RT. Contrasting was carried out with 1% 0 s 0 4 for 5 min. an aqueous saturated solution of 5 % uranyl acetate for 20 min. lead citrate for 10 min, and 1% tannic acid for 30 at RT. Examinaton was performed with a Zeiss EM 10 TEM.

SHAKIBAEI, ZIMMERMANN, MERKER

Antibodies and Lectins. Collagen Types I and I1 were isolated and purified according to Engvall and Ruoslahti (1977). Polyclonal antibodies against these collagenswere raised in rabbits. Specificitywas controlled by the ELISA technique; no crossreactivity between Type I and Type I1 collagen could be detected (Gosslau and Barrach, 1979). The polyclonal antibody CPHBOl against asp1 was purchased from Telios Pharmaceuticals(San Diego, CA). Argraves et al. (1987) have reported on the specificityof this antibody. The polyclonal antibody against a 3 was obtained from Chemicon (Temecula. CA). Ruoslahti and Pienchbacher (1987) and Plantefaber and Hynes (1989) have reported on the specificity of this antibody. The polyclonal antibodies against p1 and a1 were a kind gift of Prof. Reutter (Institute of Molecular Biology and Biochemistry, Free University of Berlin). The specificity of these antibodieswas tested with the ELISA technique (LLister et al., 1994). FIX-conjugated goat anti-rabbit immunoglobulin (GAR-FIX) was purchased from Dianova (Hamburg, Germany). Gold-conjugated goat antirabbit immunoglobulin with 10-nm gold particles (GAR-10 nm) was purchased from Amersham (Braunschweig,Germany). FIX-lectin from Armhis hypogaea (peanut, PNA) was purchased from Sigma.

Results In Vitro Chondrogenesis Cartilage formation started after 3 days of cultivation, which was recognizable by the appearance of round chondrocytes enclosed within the typical matrix. Many cartilage nodules had developed after 6 days. These nodules were surrounded by a perichondrium consisting of two to five layers of flat fibroblast-like cells. Between Days 9 and 14 in vitro, the cartilage nodules had become larger; many chondrocytes contained glycogen deposits and showed the typical features of hypertrophy. These characteristicshave already been demonstrated previously (Zimmermann et al., 1992a; Schroter-Kermani et al., 1991).

Immunofluorescence Microscopy Investigation of the distribution of al- and a3-integrins was performed by immunofluorescenceand immunoelectron microscopy. After 1 and 2 days in vitro, only al-integrin was detectable in the cultures;0.3-integrin could not be found. Distribution of al-integrin was more or less homogeneous; the reticular pattern of labeling indicated 1ocaliza.tionat the cell surface (Figures 1 and 2). After 3 days, formation of young cartilage with a specificmatrix occurred and revealed positive labeling with a3-integrin. al-Integrin was clearly present in the perichondrium and sparsely in cartilage (Figures 3a and 3b). To prove the presence of cartilage-specificmatrix, labeling with FIX-PNA was performed. PNA binds to galactose residues on glycoproteins and has been reported to specifically stain premature matrix in vitro and in vivo (Aulthouse and Solursh, 1987; Zimmermann and Thies, 1984). Lectin was localized in distinctly separated cell groups, indicating the formation of cartilage in blastemata (Figure 3c). Immunolabeling with integrins on Day 4 of the culture period showed that a1 was present in the perichondrium and the surrounding mesenchyme, whereas a3 was predominantly present in cartilage nodules (Figures4a and 4b). After 7 days in culture, cartilage nodules of various sizes were surrounded by a perichondrium of fibroblast-likecells. al-Integrin could be observed solely in the perichondrium and the space between cartilage nodules. a3-Integrin was present only in cartilage, but some punctate fluorescencewas also visible between the nod-

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i~ i Figures 1-6. Immunofluorescencemicroscopic demonstratlon of (11- and rr3-chains of integrins during chondrogenesis of limb bud mesenchymal cells from 12day-old mouse embryos grown in organ culture. Figure 1. One-day culture. lntegrin a1 is more or less uniformly distributed within the mesenchymal cell mass. Original magnification x 160. Bar = 20 vm. Figure 2. Two-day culture. lntegrin a1 is homogeneously distributed within the mesenchymal cell mass. The reticular pattern of labeling indicates localization at the cell surface. Original magnification x 160. Bar = 20 Wm. Figure 3. Three-day culture. (a) lntegrin a1 is mainly detectable in the perichondrium and mesenchyme (arrows). Stars, blastemata. (b) lntegrin a3 is found in cartilage blastemata (stars). Faint staining is Seen between nodules. (c) Labeling with FITC-labeled cartilage-specific lectin PNA revealed binding in small cell clusters, indicating the formation of cartilage-specific matrix (star). Original magnification x 160. Bars = 20 um.

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Figure 4 Four-day culture (a) lnlegrin (11 is found in the perichondrium(arrows) and (b) integrin (13predominanlly in the cartilage (star). Original magnification x 160. Bar = 20 pm. Figure 5. Seven-day culture. (a) lntegrin a1 is found in the perichondrium (arrows) and mesenchyme; no label is present in cartilage. (b) lntegrin a3 is found predominantly in cartilage nodules (star), but some punctate fluorescence, perhaps the result of nonspecific reactivity, is also visible between the nodules. No label is present in the perichondrium. Original magnification x 160. Bars = 20 pm. Figure6. Fourteen-day culture. (a) lntegrin a1 is present in the perichondrium(thick arrows), but it is also detectable around chondrocytes in cartilage (thin arrows). (b) lntegrin a3 is present predominantly in cartilage nodules (star). Original magnification x 160. Bars = 20 vm.

Figure 7. (a) Electron micrograph of the periphery of a cartilage nodule in an organ culture after 7 days. The flat fibroblast-like cells (F) of the perichondrium are embedded in dense bundles of thick collagen fibrils (star). Typical chondrocytes (C) are embedded in the cartilage matrix (M).A perichondral cell (P) appears to be a transitional stage between fibroblast-like cells and chondrocytes. (b) lmmunolabeling with collagen Type I.Gold particles are detectable only on collagenous fibrils of the perichondrium (arrowheads). Gold particles are not present in the cartilage matrix (M). (c) lmmunolabeling with collagen Type II is only demonstrable in cartilage matrix (arrowheads), not in the perichondrium (star). P, perichondral cell; F,fibroblast-like cell. Original magnifications: a x 6500; b,c x 67.000.Bars: a = 2 pm; b,c = 0.1 pm.

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SHAKIBAEI, ZIMMERMANN, MERKER

Figure 8. lmmunoelectron microscopic demonstratton of (a) al- and (b) a3integrins in cartilage organ culture after 7 days using immunogold labeling. (a) Positive labeling(IO-nmgold particles) against integrin a1 is present at the plasma membrane (arrowheads)of fibroblast-likecells (F) in the outer zone of the perichondrium, at the cell-fibril contacts, and at pericellular collagen fibrils; gold particles are absent at the plasma membrane of perichondralcells (P) in the inner zone. (Inset) A patch of gold particles on the cell membrane. (b) Positive labeling against a34ntegrin is present at the plasma membrane (arrowheads) of perichondral cells (P) in the transitional zone of a peripheral chondrocyte (C) and at cell-fibril contacts; gold particles are absent at the plasma membrane of fibroblast-like cell (F) in the outer zone of the perichondrium. (Inset) A patch of gold particles on the cell membrane. Original magnifications:8 x 36,000; Inset x 94,000; b x 35.000; inset x 75.000. Bars: a,b = 0.2 pm; Insets = 0.1 p n .

INTEGRIN CHANGES DURING CHONDROGENESIS

ules (Figures 5a and 5b). After 14 days in culture, the same distri-

bution of integrins occurred as described for the 7-day culture. In addition, al-integrin again appeared in cartilage (Figures 6a and 6b). pl- and aSPl-integrins were found in both the perichondrium and the cartilage at all stages of chondrogenesis in this culture system (not shown).

Electron Microscopy and Immunoelectron Microscopy Electron microscopy was performed only on Day 7 of the culture period. Typical cartilaginous tissue with chondrocytes and appropriate matrix had developed, which was surrounded by several layers of fibroblast-like cells in the perichondrium (Figure 7a). Labeling with anti-Type I collagen antibodies revealed the gold particles to be localized to collagen in the matrix of the perichondrium (Figure 7b). Collagen Type I1 was located solely in cartilaginousmatrix (Figure 7c). Gold label demonstrating al-integrin could be observed only on fibroblast-like cells and in the pericellular matrix of the outer zone of the perichondrium (Figure 8a), but did not occur at the perichondral cells in the inner zone of the perichondrium and on chondrocytes in cartilage nodules. a3-Integrin was demonstrated on the cell membrane of perichondral cells in the inner zone and of chondrocytes at the sites of contact with collagen fibrils (Figure 8b). It was hardly recognizable in the outer zone of the perichondrium. Both gold-labeled integrin antibodies were quite irregularly distributed on the plasma membranes of the different cell types. Gold particles were concentrated predominantly at collagen-cell contacts. In addition, they were observed at various distances from the cell surface and formed clusters at the pericellular collagen fibrils.

Discussion The occurrence and localization of different integrins in cartilage and perichondrium during chondrogenesis in organ cultures from mouse limb bud mesenchymal cells were demonstrated by immunofluorescence and immunoelectron microscopy. The following results were obtained. (a) al-integrin was present on mesenchymal cells during a culture period of 1-3 days. (b) Cartilage formation was accompanied by the occurrence of a3-integrin together with PNA binding in the matrix. al-integrin remained in the mesenchyme and became more concentrated in the perichondrium during chondrogenesis. (c) Mature cartilage (Day 7) showed the presence of a3-integrin at chondrocytes and at perichondral cells in the inner zone of the perichondrium. Fibroblast-like cells in the outer tone of the perichondrium exhibited al-integrin. (d) In older cartilage, al-integrin again appeared in cartilage nodules. ( e )Immunoelectron microscopy revealed integrins in patches on the cell membrane at cell-collagen contact sites and at pericellular collagen fibrils. (f) 81- and a5pl-integrins were homogeneously distributed in the perichondrium as well as in cartilage during chondrogenesis from Day 1 of cultivation onwards. Occurrence and distribution of al- and a3-integrins have been investigated by Salter et al. (1992),Diirr et al. (1993), and Enomoto

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et al. (1993) in human and chick cartilage by biochemical techniques and immunofluorescence. These investigators found that al-integrin was not present in cartilage but was present in fibroblasts. a3-integrin was revealed by Enomoto et al. and Salter et al. in cartilage but not by Durr et al. In addition, a3-integrin was shown by Enomoto et al. in fibroblasts. Our light and electron microscopic results confirm the presence of a3-integrin at chondrocytes and al-integrin at fibroblast-like cells in the perichondrium. The discrepanciesbetween these results and those of other authors may be due to species differences and possibly to different masking by proteoglycansof a3-chains as well as to methodological differences (e.g., pre-treatment with different enzymes: hyaluronidase by Diirr et al., trypsin by Enomoto et al., collagenase used in our laboratory) and different culture techniques. The cartilage organ culture system allows investigations during chondrogenesis starting from early blastema condensations until cartilage maturation. This chondrogenicdevelopment is accompanied by enlargement of cartilage nodules, which is not due to cell proliferation but rather is due to appositional growth, which includes the transition of perichondral cells into chondrocytes.These conclusions are based on the almost complete absence of mitotic figures in cartilage and perichondrium and on the continuous increase in nodule size during cultivation (Zimmermann et al., 1991a; Schroter-Kermani et al., 1991). It is also indicated by the present results demonstrating that al-integrin-positive cells in the outer part of the perichondrium differentiate into a3-bearing cells in the inner part of the perichondrium. In our material, differentiation of blastemal cells and perichondral cells into chondrocytes was accompanied by changes in collagen expression from Type I to Type I1 and simultaneously in integrin expression from al- to a3-chains. It has been shown that alal-integrin is a receptor for collagen Type I (Syfrig et al., 1991; Ignatius et al., 1990; Kramer and Marks, 1989),which is confirmed by our study. In aging cultures a change in integrin expression from a 3 to a1 again occurred. This change has already been demonstrated in monolayer cultures of epiphyseal chondrocytesfrom 17-day-old mouse embryos after dedifferentiation into fibroblast-likecells, accompanied by transition from Type I1 to Type I collagen synthesis (Shakibaei, 1995). This was paralleled by a switch of collagen synthesis from Type I1 to Type I (Shakibaei et al., 1993a,b). The co-localization of al-integrin and collagen Type I and of a3-integrin and collagen Type 11, and their changes during development and dedifferentiation, suggests a coupling in expression or activation of integrins with collagen synthesis. However, it can be suggested that changing the ligand (from collagen Type I to Type 11, and vice versa) induces the correspondingexpression or presentation of the receptor for the new ligand. It has been reported that the regulation of integrin receptor distribution on the surface of cells can be influenced by ligand occupancy (LaFlamme et al., 1992). This was indicated by a simultaneous decrease in ligand (fibronectin) and receptor (a5pl-integrin) expression in oncogenically transformed cells (Plantefaber and Hynes, 1989). Obviously, al- and a3-integrins are recognizable at collagen fibrils in the pericellular matrix. This integrin localization can be explained by shedding, which has also been shown to be a property of cell membrane-bound heparan sulfate proteoglycan (syn-

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decan) and receptors for cytokines and growth factors (Rose-John and Heinrich, 1994; Jalkanen et al., 1987; Rapraeger et al., 1986,1987;Hook et al., 1984; Rapraeger and Bernfield, 1982). Shedding includes the cleavage of the extracellularpart of the receptor and, in the case of al- and a3-integrin, tight binding to the collagen ligand. This may simply reflect a turnover process. A functional implication is not yet clear, but it could serve as an indicator of the collagen source. The observed correlation between the collagen type synthesized by the cells during differentiation and dedifferentiation and the expression of the appropriate receptor reveals a basic mechanism of regulation and control of the matrix by the cells. Integrins may be the key structure in transmitting information about matrix composition into the cells.

Acknowledgments We thank Prof Dr Reutter (Institute of Molecular Biology and Biochemistry, Free University ofBerlin) for his kindgift of the anti-integrin antibodies, Ms Barbara Steyn for he& in preparing the manuscript, Ms Ingrid Wolf for expertphotographic work, andMr Phil+pe De SouzaandMs Angelika Steuer for technical assistance.

SHAKIBAEI, ZIMMERMANN, MERKER

Reichardt L (1990): Molecular cloning of the rat integrin al-subunit: a receptor for laminin and collagen. J Cell Biol 111:709 Jalkanen M. Rapraeger A. Saunders S, Bernfield M (1987): Cell surface proteoglycan of mouse mammary epithelial cells is shed by cleavage of its matrixbinding ectodomain from its membrane-associated domain. J Cell Biol 105:3087 Kramer R, Marks N (1989): Identification of integrin collagen receptors on human melanoma cells. J Biol Chem 264:4684 LaFlamme SE, Akiyama SK, Yamada KM (1992): Regulation of fibronectin receptor distribution. J Cell Biol 117437 Languino LR, Gehlsen K, Wayner E, Carter W, Engvall E, Ruoslahti E (1989): Endothelial cells use a281 integrin as alaminin receptor.J Cell Biol 109:2455 Iijster K, Voigt S, Heidrich C, Hofmann W, Reutter W (1994): Cell-collagen adhesion is inhibited by monoclonal antibody 33.4 against the rat al-integrin subunit. Exp Cell Res 212:155 McDonald JA, Mecham RP, eds (1991): Receptors for extracellular matrix. San Diego, Academic Press Plantefaber LC, Hynes RO (1989): Changes in integrin receptors on oncogenically transformed cells. Cell 56:281 Ramachandmla A, Tiku K, Tiku ML (1992): Triopeptide RGD-dependent adhesion of articular chondrocytes to synovial fibroblasts. J Cell Sci lOl:859

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