Chondrons from articular cartilage - CiteSeerX

Journal of Cell Science 103, 1101-1110 (1992) Printed in Great Britain © The Company of Biologists Limited 1992

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Chondrons from articular cartilage V.* Immunohistochemical evaluation of type VI collagen organisation in isolated chondrons by light, confocal and electron microscopy

C. ANTHONY POOLE1,†, SHIRLEY AYAD2 and RAYMOND T. GILBERT1 1Department 2Department

of Anatomy, University of Auckland, School of Medicine, Private Bag 92019, Auckland, New Zealand of Biochemistry and Molecular Biology, University of Manchester Medical School, Manchester, M13 NPT, England

*Paper IV in this series is Poole et al. (1991) †Author for correspondence

Summary The pericellular microenvironment around articular cartilage chondrocytes must play a key role in regulating the interaction between the cell and its extracellular matrix. The potential contribution of type VI collagen to this interaction was investigated in this study using isolated canine tibial chondrons embedded in agarose monolayers. The immunohistochemical distribution of an anti-type VI collagen antibody was assessed in these preparations using fluorescence, peroxidase and gold particle probes in combination with light, confocal and transmission electron microscopy. Light and confocal microscopy both showed type VI collagen concentrated in the pericellular capsule and matrix around the chondrocyte with reduced staining in the tail region and the interconnecting segments between adjacent chondrons. Minimal staining was recorded in the territorial and interterritorial matrices. At higher resolution, type VI collagen appeared both as microfibrils and as amorphous deposits that accumulated at the junction of intersecting capsular fibres and microfibrils. Electron microscopy also showed type VI collagen anchored to

the chondrocyte membrane at the articular pole of the pericellular capsule and tethered to the radial collagen network through the tail at the basal pole of the capsule. We suggest that type VI collagen plays a dual role in the maintenance of chondron integrity. First, it could bind to the radial collagen network and stabilise the collagens, proteoglycans and glycoproteins of the pericellular microenvironment. Secondly, specific cell surface receptors exist, which could mediate the interaction between the chondrocyte and type VI collagen, providing firm anchorage and signalling potentials between the pericellular matrix and the cell nucleus. In this way type VI collagen could provide a close functional interrelationship between the chondrocyte, its pericellular microenvironment and the load bearing extracellular matrix of adult articular cartilage.

Introduction

microenvironment are thought to represent the chondron, arguably the primary functional and metabolic unit of hyaline cartilages (for review see Poole, 1992). Slow-speed homogenisation techniques have now been developed to separate significant numbers of chondrons from the bulk extracellular matrix of a variety of normal mammalian articular cartilages (Poole et al. 1988a,b; Poole, 1992). With the identification of the chondron as a true microstructure of articular cartilage, current studies have focused on defining the composition and organisation of the chondron, and its role in chondrocyte - matrix interactions. Using a variety of immunohistochemical techniques, the chondron has been shown to contain collagen types II, VI and IX (Poole et al. 1988a,c), the aggrecan components chondroitin 4-sul-

The interaction between the chondrocyte and its extracellular matrix is of critical importance in regulating the development, maintenance and repair of articular cartilage. However, it is now generally accepted that the chondrocytes in adult articular cartilage are surrounded by a specialised microenvironment, which effectively insulates the chondrocyte and physically separates the cell from direct interaction with the bulk of the load-bearing matrix. The pericellular microenvironment must therefore play an important role in mediating the interaction between the chondrocyte and its extracellular matrix. Collectively, the chondrocyte and its pericellular

Key words: articular cartilage, isolated chondrons, type VI collagen, pericellular microenvironment, cell-matrix interactions, immunohistochemistry, confocal microscopy, ultrastructure.

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C. A. Poole and others

phate, chondroitin 6-sulphate, keratan sulphate, core protein and hyaluronan binding region (Poole et al. 1991), and the glycoprotein fibronectin (Poole, 1990). Type VI collagen was originally identified as a highly disulphide bonded aggregate in pepsin digests of aortic intima (Chung et al. 1976), but is now considered a component of most, if not all, connective tissues (for reviews see Engel et al. 1985; Rauterberg et al. 1986; Timpl and Engel, 1987). In bovine articular cartilage, type VI collagen accounts for only 1-2% of the total collagens present (Eyre et al. 1987), but its preferential localisation in the chondron (Poole et al. 1988a) suggests it represents a significant component of the pericellular microenvironment. The type VI collagen monomer consists of a short triplehelical domain (105 nm) at each end of which is a large globular domain (Engel et al. 1985: Rauterberg et al. 1986; Timpl and Engel, 1987). Biosynthetic studies have demonstrated that monomers assemble intracellularly into dimers and tetrameres, which are then secreted into the extracellular matrix and assemble into microfibrillar structures by end-to-end association (Engvall et al. 1986). Ultrastructurally, these microfibrils have a characteristic beaded appearance with a periodicity of approximately 100 nm, and have been observed in association with cell surfaces as well as the major collagen fibres of the extracellular matrix (Bruns et al. 1986). Lateral assembly of these microfibrillar structures is also believed to give rise to the “unusual banded (FLS-like) structures” previously reported in the nucleus pulposus (Cornah et al. 1970) and more recently in the pericellular capsule of human articular cartilage (Poole et al. 1987). Fluorescein, peroxidase and gold particle probes, in conjunction with light, confocal and transmission electron microscopy, were used in this study to detail the organisation and structure of type VI collagen in isolated chondrons embedded in agarose gel. The results show that type VI collagen interacts with both the chondrocyte plasma membrane and the fibrous components of the pericellular capsule to provide a cohesive interaction between the chondrocyte, its pericellular microenvironment and the extracellular matrix.

Materials and methods Sample collection Mongrel dogs (2-6 years, 18-30 kg), killed under veterinary supervision with lethal pentobarbital injection, were randomly selected from a local pound and the stifle joint examined macroscopically. Both the lateral and medial tibial surfaces were resected at the tidemark and stored in sterile phosphate-buffered saline (PBS: 0.14 M sodium chloride; 10 mM phosphate buffer; pH 7.4) at 4oC for 15-30 minutes. Pooled samples were diced into 1-2 mm3 chips and suspended in sterile PBS.

Isolation of chondrons The techniques used to liberate chondrons from articular cartilage have been widely published (Poole et al. 1988b; Poole, 1990, 1992). Briefly, 1 g samples of the diced cartilage were serially homogenized in 20 ml of PBS at relatively slow speeds (4000-

6000 r.p.m.), for short periods (1-10 min), and the flocculent supernatants collected. Pooled supernatants were filtered, collected by centrifugation and the final pellet resuspended in 10 ml of PBS at 4 oC.

Chondron-agarose gel preparation Methods to embed chondrons in agarose gels have recently been published (Poole et al. 1991). Briefly, a 1.5% solution of low melting point agarose gel (Sea Plaque, F.M.C. Bioproducts, USA) was thoroughly mixed with the filtered chondron homogenate to give a final preparation containing isolated chondrons, small cartilage chips and collagen debris in a 1% solution of agarose. This solution was cast directly into 90 mm culture dishes to create a chondron-agarose monolayer 0.5-0.8 mm thick, which had sufficient content, strength and optical clarity for subsequent experimentation. The majority of chondron-agarose gels were fixed for 30 minutes in either 1% glutaraldehyde/2% paraformaldehyde or acid ethanol, washed in PBS and held at 4oC as previously described (Poole et al. 1991). In some experiments, unfixed cultures were used to compare the native distribution of the epitope with that found in fixed preparations. A core punch was used to cut small plugs (6-10 mm diameter) from the centre of the culture dish where the gel was thinnest and most uniform. Large numbers of plugs were prepared in this manner, and could be used over several months if stored appropriately (Poole et al. 1991). For immunohistochemistry, stored plugs were transferred to a multiwell culture plate and washed extensively in Tris-saline (0.14 M sodium chloride; 10 mM Tris-HCl; pH 7.4).

Anti-type VI collagen antibody A rabbit polyclonal antibody was raised against a less extensively pepsinised form of bovine type VI collagen. The antibody reacts preferentially with epitopes in the globular domain of the type VI collagen as shown by immunoblotting (Ayad et al. 1989) and rotary shadowing (S. Ayad, unpublished observations). It is specific for type VI collagen up to dilutions of 1:50 000, and shows no cross-reactivity with collagen types I, II, III, IV, V, IX and XI, or fibronectin.

Immunohistochemical preparation of chondron-agarose gels The pre-embedding immunohistochemical techniques used in this study have also been described in detail (Poole et al. 1991). Prior to labelling, all plugs were digested with testicular hyaluronidase (Type 1, Sigma Chemicals, USA) to remove proteoglycans, which otherwise mask antigenic sites on the collagen molecule. Anti-type VI collagen antibody was prepared at dilutions of 1:100-1:1000 in Tris-saline containing BSA and the plugs labelled for extended periods (18-60 h) at 4oC with continuous agitation. Specific probes were selected according to the microscopic technique to be employed and included; (i) streptavidinfluorescein isothiocyanate (FITC) (Amersham, UK); (ii) streptavidin-biotinylated horseradish peroxidase (HRP) complex (Amersham, UK); (iii) Peroxidase-anti-peroxidase (PAP) complex (Dakopatts, Denmark); (iv) Auroprobe One-streptavidin (1 nm gold particles) or Auroprobe EM-streptavidin G10 (10 nm gold particles; Amersham, UK); (v) Protein A-gold G10 (10 nm gold particles; Amersham, UK). Plugs probed with FITC, HRP or PAP were prepared for light microscopy as previously described (Poole et al. 1991). In a new development, chondron-agarose plugs labelled with 1 nm gold particles were silver enhanced (IntenS EM, Amersham, UK), while those labelled with 10 nm gold particles were divided into two groups, one receiving silver intensification, the other receiving no further treatment. Silver enhancment is a time depen-

Type VI collagen in cartilage chondrons dent process and 6-10 minutes intensification of 1 nm gold particles was required to produce silver grains just visible at the light microscope level. Shorter times (4-6 minutes) were required for 10 nm gold particles, while longer intensification regimes (20-30 minutes) produced intensely black images that were routinely used for light microscopic confirmation of a gold particle reaction. Resin embedding techniques were also developed during this study to improve the quality of sections for light and electron microscopy. Both Procure 812 (Probing and Structure, Australia) and LR White (London Resin Company, Hampshire, England) were tested on chondron-agarose plugs labelled with HRP, PAP or gold particle probes. All plugs were postfixed with 1% OsO4 for 1-2 hours to improve structural preservation and intensify the HRP reaction product. For Procure 812, osmicated plugs were dehydrated through graded ethanols, infiltrated with increasing concentrations of resin over a 48-hour period, and embedded flat between a slide and a coverslip, previously coated with 1% (v/v) dimethylchlorosilane (Sigma Chemicals, USA) in benzene for 15 minutes and air dried. For LR White resin, plugs were dehydrated to 70% ethanol, slowly infiltrated with increasing concentrations of resin, embedded flat and polymerized at 60oC for 21 hours. Finally, the coverslip was carefully removed from the chondronagarose wafer, which remained mounted on the glass slide, for correlative light and electron microscopy. By this method large numbers of resin-embedded chondrons were examined by light microscopy and specific chondrons selected for ultrastructural evaluation.

Microscopy Light microscopy All light microscopy was performed on a Leitz Dialux 20 microscope equipped with phase contrast, differential interference contrast (DIC) and brightfield lenses ranging from 10× to 100×, a 50 W epi-illumination system fitted with 488 nm filter block, and an automated Wild camera system. For aqueous and permanent mounts, chondrons were routinely examined with a 100× oil immersion objective and photographed on T max 100 black and white film (Kodak, USA). For resin mounts, the chondron was photographed and its position inscribed with a diamond-tipped objective. Selected chondrons were cut from the wafer, mounted on Epon stubs, sectioned at 1-2 µm, and examined for the distribution of HRP reaction product.

Confocal microscopy A BioRad MRC 600 confocal microscope (Bio Rad, Sydney, Australia) has recently been included in our studies of chondronagarose plugs labelled with FITC or silver-enhanced 1 nm gold particles. Since only 25 isolated chondrons have been examined under fluorescence or reflectance modes to date, these results must be viewed as preliminary in nature, and will be the subject of further detailed investigations. Nevertheless, the optical sectioning capability and high resolution afforded by the confocal microscope combined to give a unique view of type VI collagen distribution in isolated canine chondrons, and further highlights the potential of this new imaging technology in the study of articular cartilage microstructure (Wotton et al. 1991).

Electron microscopy Chondrons suitable for transmission electron microscopy were serially sectioned at 100 nm, mounted on Formvar-coated grids, stained with 2% aqueous uranyl acetate at 60oC and examined on an Hitachi H7000 electron microscope at accelerating voltages of 100 to 125 KV. Some peroxidase-labelled preparations were not treated with uranyl acetate to contrast the distribution of label. Data in the form of photomicrographs was recorded on TechPan (Kodak, USA) black and white film.

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Results Light microscopy Fluorescein probes Chondron-agarose plugs labelled with FITC showed type VI collagen concentrated in the pericellular capsule and matrix around the chondrocyte, with a less intense, sometimes patchy distribution in the tail region (Fig. 1A). Background levels of FITC were generally low in the extracellular matrix and absent from the supporting agarose gel. Peroxidase probes HRP complex was used routinely in these studies and penetrated the agarose gels sufficiently to selectively stain the multiple chondrons columns aligned within large cartilage chips (Fig. 1B). Using the oil immersion objective, peroxidase staining of whole chondrons appeared similar to that seen with FITC, while differential interference contrast microscopy highlighted unstained collagen bundles woven into the capsule and tail (Fig. 1C). Identical results were found with PAP (results not shown). Resin sections examined by light microscopy showed improved detail and a distribution of HRP similar to that seen in whole-mount preparations. Dense granular deposits were concentrated around the articular pole and lateral margins of the pericellular capsule and merged to form a compact enclosure around the chondrocyte (Fig. 1D). A number of large deposits were also found at the distal end of the tail and combined with amorphously stained material to give the tail a patchy, granular appearance (Fig. 1D). The transition region between the pericellular capsule and the tail usually contained fewer granular deposits and significantly more amorphous material. Label distribution was clearly delineated at the outer margin of the capsule, with occasional punctate deposits randomly distributed amongst the radial collagen bundles associated with the chondron. Gold particle probes None of the probes used in this aspect of the study could be visualised at the light microscopic level without silver intensification. Differential interference contrast microscopy gave the best images and showed silverenhanced gold particles distributed throughout the pericellular microenvironment, with minimal background staining (Fig. 1E). Most particles were concentrated around the articular pole and lateral margins of the pericellular capsule with less intense staining adjacent to the chondrocyte and in the tail. A control sample from which the primary antibody was omitted is shown in Fig. 1F. Confocal microscopy Fluorescence mode The distribution of fluorescently labelled type VI collagen is shown in Fig. 2A,B, a stereo pair reconstructed from twenty optical sections 1 µm apart. The increased resolution afforded by confocal microscopy provides significantly more detail than conventional whole mounts (cf. Fig. 1A). In addition to the expected distribution of type VI collagen, close examination of individual optical sections revealed

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Fig. 1. Light microscopy. (A) In chondrons labelled with FITC, each negatively contrasted chondrocyte (c) was surrounded by an intense staining reaction with obvious divisions between adjacent chondrons (arrowheads). Bar, 10 µm. (B) In cartilage chips labelled with HRPDAB, intensely stained chondrons (arrowheads) were aligned in parallel with the radial collagen network. Bar, 100 µm. (C) Detail from B showing intense staining around the chondrocyte at the articular pole of the chondron (large arrowhead) and lighter staining of the tapered tail (t) at the basal pole. Bundles of type II collagen fibres show no obvious staining reaction (small arrowheads). Bar, 10 µm. (D) In thick resin sections, chondrons labelled with HRP-DAB-OsO4 showed granular deposits concentrated at the periphery of the pericellular capsule but continuous with the cell surface (small arrowheads). Amorphous staining characterised the proximal region of the tail, while its distal region showed an intense clumped reaction (large arrowhead). Bar, 10 µm. (E) Adjacent chondrons labelled with silver enhanced Protein A-10 nm gold showed strong labelling at the articular pole (large arrowhead), but diminished staining towards the cell surface. The tail was diffusely stained and terminated abruptly at the junction with the subjacent chondron (small arrowheads). Bar, 10 µm. (F) Control preparation in which the primary antibody was omitted. The intact chondrocyte (c), fine cell processes (small arrowhead), the pericellular capsule (large arrowhead) and the tail (t) were all identified by DIC in the absence of staining. Bar, 10 µm.

the inner margin of the pericellular capsule was generally less intensely stained than the outer margin of the capsule. The distal region of the tail showed a strong granular reaction similar to that seen in thick epon sections (cf. Fig. 1D), while serial reconstruction and stereo observations suggest the tail tapers like an inverted cone from the basal pole of the chondron (Fig. 2A,B). Although background fluorescence levels were low in these preparations, a punctate reaction persisted in the collagen bundles attached to isolated chondrons (Fig. 2A,B).

Reflectance mode When operated in this mode, the laser scans the subject with the detectors set to record light reflected from within the subject. Silver-enhanced gold particles proved to be particularly suited to this scanning mode, which showed significantly greater resolution than conventional optical sections (cf. Figs 2C and 1E). Fig. 2C shows a single optical section through a double and a single chondron labelled with silver-enhanced 1 nm gold particles. The stippled reaction product was concentrated in the pericellular matrix and

Type VI collagen in cartilage chondrons

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Fig. 2. Confocal microscopy. (A,B) Stereo reconstruction of 20 optical sections 1 µm apart through a single chondron labelled with FITC and imaged in fluorescence mode. Staining was most intense in the pericellular capsule and matrix around the chondrocyte with less intense staining in the ‘cone-shaped’ tail. A strong granular reaction was evident in the distal region of the tail. Bar, 10 µm. (C) A single optical section through two chondrons at the edge of a cartilage chip labelled with silver-intensified 1 nm gold particles and imaged in reflectance mode. Silver/gold particles were concentrated in the chondron, with minimal staining in the extracellular matrix (m) and no reaction in the supporting agarose (a). Bar, 10 µm.

capsule with less intense staining in the tail, and a clear boundary between the two chondrons. Higher background staining levels were evident in the extracellular matrix of the chip, but were absent from the supporting agarose gel (Fig. 2C). Transmission electron microscopy Peroxidase probes Ultrastructural examination of isolated chondrons showed the chondrocyte occupied most of the volume defined by the pericellular capsule (Fig. 3A). Characteristically, the outer margin of the pericellular capsule was densely stained, while the pericellular matrix adjacent to the chondrocyte was more diffusely stained. Frequently, chondrocytes formed intimate contacts with some regions of the capsule, while other areas of the cell seemed detached from the capsule but maintained contact through a series of extended microfibrils (Fig. 3A). A detail of the articular pole of a pericellular capsule is shown in Fig. 3B. The HRP reaction product consists of dense granules of variable size, concentrated and compacted at the outer margin of the capsule. Banded type II collagen fibres adjacent to the capsule showed no evidence of staining. Towards the inner margin of the capsule, HRP granules were generally smaller and more loosely arranged, forming an interwoven network of irregular microfibrils connecting small clumps of granular reaction product (Fig. 3B). The staining of these microfibrils and granular clumps is detailed in Fig. 3C. Microfibrils showed a variety of profiles, some displaying obvious curves and bends, others having a rather straight profile, which intersected several adjacent microfibrils. Some microfibrils showed small peroxidase deposits periodically distributed at approximately 100 nm intervals along the microfibril (Fig. 3C). In contrast, granular HRP deposits tended to accumulate at the

junctions of several microfibrils but obscured the detailed ultrastructure of this area. PAP produces a smaller reaction product than HRP and was introduced to improve visualisation of the fine pericellular capsule fibres. In a comparative example of PAP labelling (Fig. 3D), capsular fibres were coated with a fine label product to form an amorphous network, which accumulated at the junction of several fibres. The junction between the chondrocyte and the pericellular capsule is illustrated in Fig. 3E,F. In regions of intimate contact (Fig. 3E), the loosely woven staining pattern of the inner capsular margin formed a continuum with the chondrocyte membrane and cell processes. Frequently, the junction between type VI collagen and the plasma membrane was characterised by small dense particles and increased membrane density (Fig. 3E). In regions of less intimate contact (Fig. 3F), extended microfibrils labelled with HRP showed a distinct period of approximately 100 nm, and impinged directly onto the cell processes and the surface of the chondrocyte (Fig. 3F). In the distal region of the tail, numerous unlabelled fibres pervaded the tail but were totally obscured by the HRP reaction product (Fig. 3G). The margin of the tail was defined by several large aggregates, which extended around the typical type II collagen fibres of the territorial matrix. Gold particles Three gold particle probes were evaluated in this study to improve the ultrastructural resolution of labelled material. Whereas silver intensification of streptavidin-linked 1 nm or 10 nm gold particles proved suitable for confocal microscopy, ultrastructural examination of these preparations showed a lower than expected distribution of silverenhanced gold particles. Those silver/gold aggregates present varied significantly in size but were invariably asso-

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Fig. 3. Transmission electron microscopy: horseradish peroxidase probes. (A) Plan view of an intact chondron showing both intimate (large arrowhead) and attenuated (small arrowhead) contacts between the chondrocyte and the densely labelled pericellular capsule. Large and small circled areas are detailed in E and F respectively. Bar, 5 µm. (B) Detail showing the junction between the pericellular capsule and the territorial matrix collagen fibres. Compacted granular reaction product creates a distinct boundary with the unlabelled type II collagen fibres (large arrowheads), while closer to the cell the capsular weave loosens and consists of clumped reaction product connected by labelled microfibrils (small arrowheads). Bar, 0.5 µm. (C) Detail from a capsular ghost. Several periodically labelled microfibrils intersect (small arrowheads), and connect small clumps of HRP reaction product (large arrowheads). Bar, 0.2 µm. (D) Comparative detail of a pericellular capsule labelled with PAP-DAB-OsO4. Fine capsular fibres were coated with amorphous reaction product (small arrowheads) which was concentrated at the junction of several fibres (large arrowheads). Bar, 0.2 µm. (E) Detail from large circle in A showing the intimate contact between the chondrocyte and the pericellular capsule. Compact aggregates define the outer margin of the capsule (large arrowhead), and were continuous with fine granular deposits and microfibrils in contact with cell processes and the chondrocyte surface (small arrowheads). Note the increased membrane densities at the junction with type VI collagen microfibrils (small arrowheads). Bar, 0.5 µm. (F) Detail from the small circle in A showing labelled microfibrils intersecting with adjacent microfibrils (large arrowhead), cell processes (medium arrowheads), and the dense chondrocyte membrane (small arrowheads). Bar, 0.2 µm. (G) Detail from the distal region of a tail. Large dense clumps of reaction product dominate (large arrowhead) and obscure the fibrous substructure. Several unlabelled collagen fibres adjacent to the tail interact directly with the labelled deposits (small arrowheads). Bar, 0.5 µm.

ciated with amorphous material located at the junction of several capsular fibres (results not shown). Protein A-10 nm gold showed substantially improved levels of gold particle distribution. At low magnification (Fig. 4A), gold particles were distributed throughout the capsule, but were more concentrated at its outer margin. At

intermediate magnifications (Fig. 4B), gold particles were associated with a moderately dense amorphous material, which extended towards, and in places surrounded, unlabelled type II collagen fibres at the margin of the capsule. At high magnification (Fig. 4C), labelled microfibrils appeared to crosslink and brace the fine, unlabelled fibres

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Fig. 4. Transmission electron microscopy: protein A 10 nm gold particle probes. (A) Low-power view of gold particles distributed throughout the fine filamentous material of the pericellular capsule (arrowheads). Label density was highest at the outer margin of the capsule, but was absent from the supporting agarose gel (a). Bar, 0.5 µm. (B) Intermediate magnification showing gold particles located on fine microfibrils which adhere to type II collagen fibres at the outer margin of the capsule (large arrowheads). Labelled microfibrils were continuous with amorphously stained material displaying a microfibrillar substructure and a degree of periodic organisation (small arrowheads). Bar, 0.2 µm. (C) High magnification showing the interaction between labelled microfibrils and unlabelled fibres of the pericellular capsule. Gold particles were concentrated at the junction between several microfibrils and the unlabelled capsular fibres (large arrowheads), but also decorated individual microfibrils (small arrowheads) which connect and brace intersecting capsular fibres. Bar, 0.1 µm.

of the pericellular capsule, while amorphous deposits were frequently located at the junction between microfibrillar strands and the fine capsular fibres. Discussion This study shows that a range of microscopy techniques coupled with a variety of immunoprobes combine to give an accurate definition of type VI collagen distribution and organisation in the pericellular microenvironment of isolated chondrons embedded in agarose monolayers. The results suggest that type VI collagen acts as a reliable chondron marker, and are consistent with previous studies reporting a pericellular distribution of type VI collagen in articular cartilage (Ayad et al. 1984), in suspension labelled chondrons (Poole et al. 1988a), and in the microenvironment around chondrocytes and osteocytes of the intervertebral disc (Roberts et al. 1991). Conventional light microscopy showed a discrete and reproducible distribution of type VI collagen in the cellular microenvironment with all labels concentrated in the articular pole and lateral margins of the pericellular capsule. By comparison, the basal pole of the capsule was weakly stained and frequently tapered for several micrometres to form a distinct tail. Tails up to 100 µm have been identified (Poole et al. 1988b), but generally ranged from 10 to 40 µm in length. Frequently, the junction between the articular pole of one chondron and the tail of its subjacent neighbour was defined by a virtual absence of type VI collagen staining. Basal levels of type VI collagen were seen in the extracellular matrix of cartilage chips, where it presented as occasional punctate deposits randomly distributed

in the territorial and interterritorial matrices between adjacent chondrons. Confocal microscopy provided a much higher degree of resolution than conventional light microscopy, and in particular highlighted the ‘‘cone-like’’ morphology and granular staining pattern of the tail. Confocal microscopy has recently been used to show type IX collagen is specifically localised in the pericellular microenvironment around articular cartilage and rat swarm chondrosarcoma chondrocytes (Wotton et al. 1991). These observations confirmed earlier light microscopy studies showing type IX collagen preferentially localised in the pericellular capsule of isolated porcine and rat swarm chondrosarcoma chondrons (Poole et al. 1988c). Current concepts now suggest that types II, IX, and XI collagens together form fine heterotypic fibres comprising a central type XI collagen core surrounded by type II collagen and decorated on the surface by type IX collagen molecules (Vaughan et al. 1988; Mendler et al. 1989; Eikenberry et al. 1992; see Fig. 5). Although the molecular interactions between type VI collagen and these heterotypic fibres is still unclear, they are thought to have presented ultrastructurally in this study as the fine, unlabelled, occasionally banded fibres, typically associated with the pericellular capsule. In a recent hypothesis, Smith and Brandt (1992) have also suggested that these fibres may bind or “glue” adjacent fibres together via ionic interactions between the cationic NC4 domain of type IX collagen and the anionic matrix proteoglycans, also concentrated in the pericellular microenvironment (Poole et al. 1991). The results of transmission electron microscopy accurately reflected those reported by light and confocal microscopy. However, the increased resolution afforded by

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Fig. 5. Diagrammatic representation of the interaction between type VI collagen, pericellular capsule collagens and the chondrocyte membrane. Type VI collagen microfibrils and heterotypic fibres consisting of a type XI collagen core, periodically banded type II collagen and a surface coat of type IX collagen were drawn to scale in a linear dimension, but their width was exaggerated for clarity. A variety of type VI collagen configurations are shown based on the data presented. Chondrocyte receptors known to interact with type VI collagen are schematically represented and not drawn to scale.The hyaluronan receptor (CD-44) has two TYPE II COLLAGEN TYPE XI COLLAGEN heparan sulphate/chondroitin sulphate chains attached to the core protein and is TYPE VI COLLAGEN TYPE IX COLLAGEN 100 nm thought to mediate type VI collagen binding via hyaluronan. Integrins may bind directly to type VI collagen as illustrated or via intermediate matrix macromolecules, which are not shown for clarity. Anchorin CII is shown binding a heterotypic type II collagen fibre to which type VI collagen is anchored via its globular domain. The receptor NG-2 is expressed by immature and proliferating chondrocytes, contains several chondroitin sulphate chains on its protein core, and interacts directly with type VI collagen. HYALURONAN RECEPTOR

INTEGRIN RECEPTOR

ANCHORIN CII

electron microscopy showed a gradation in the distribution of immunolabel in the pericellular capsule. At its outer margin, type VI collagen was concentrated in dense aggregates that formed a distinct boundary with the radial type II collagen fibres of the territorial matrix. In contrast, the inner margin of the capsule became progressively less compacted and more fibrillar towards the chondrocyte before terminating at the plasma membrane. At the distal end of the tail, large dense aggregates of type VI collagen totally filled the spaces created by the weave of fine territorial matrix fibres which traverse the tail. These observation suggest that type VI collagen plays a role in binding the tail and pericellular capsule to the radial collagen network, ultimately anchored at the tidemark in the calcified cartilage matrix. In summary, the results indicate that type VI collagen is concentrated around and anchored to the chondrocyte at the articular pole of the pericellular capsule, and is tethered to the radial collagen network through the tail at the basal pole of the capsule. Functionally, this arrangement would not only ensure a close interrelationship between the chondrocyte and its pericellular microenvironment, but would also serve to maintain the position of the chondron complex during the deformation and displacement that occurs in loaded articular cartilage (Broom and Poole, 1982; Poole et al. 1984). These interpretations are consistent with previous studies which suggest that type VI collagen plays a key role in anchoring the cell and stabilising the extracellular matrix to which it adheres (Linsenmayer et al. 1986; Timpl and Engel, 1987; Keene et al. 1988; Bonaldo et al. 1990; Bidanset et al. 1992). Articular cartilage, however, has a paucity of cells and thus the interaction between the chondrocyte and its pericellular matrix must be particularly important in regulating chondrocyte metabolism. The high pericellular concentrations of type VI collagen reported here clearly suggest that this collagen plays an important role in regulating cell-matrix interactions in articular cartilage.

NG-2 RECEPTOR

Type VI collagen is a hybrid molecule composed of a short triple helix with large N- and C-terminal globular regions, which have a unique mosaic structure containing several domains that show a striking homology to those present in adhesive extracellular matrix proteins such as von Willebrand factor, cartilage matrix protein and fibronectin (Chu et al. 1990; Bonaldo and Colombatti, 1989; Bonaldo et al. 1990; Koller et al. 1989). These structural features, together with potential arginine-glycine-aspartic acid (RGD) cell binding sequences in the triple-helical domain (Aumailley et al. 1989), make type VI collagen ideally suited to a role in cell-matrix and matrix-matrix interactions. At an ultrastructural level, many type VI collagen microfibrils impinged directly onto the cell membrane, which showed increased membrane density and the presence of small transmembrane particles (see Fig. 3E,F). Both integrin receptors (Humphries, 1990; Salter et al. 1992) and non-integrin receptors such as anchorin CII (Mollenhauer et al. 1984; Pfäffle et al. 1990), the hyaluronan receptor CD-44 (Aruffo et al. 1990; Culty et al. 1990; Toole, 1991) and the developmental receptor NG2 (Stallcap et al. 1990; Nishiyama et al. 1991) have all been identified on the chondrocyte surface and could be involved in anchoring type VI collagen to the cell membrane (see Fig. 5). To date however, the cell binding motif in type VI collagen has not been identified unequivocally, but is thought to involve both the triple-helical and globular regions of the molecule, and to include both RGD-dependent and RGD-independent binding mechanisms (Aumailley et al. 1989; Aumailley et al. 1991). Cell surface receptors may therefore interact directly with type VI collagen microfibrils as suggested by our ultrastructural data, or indirectly via intermediate macromolecules such as the small proteoglycan decorin, recently colocalised in the chondron (Poole et al. 1993) and shown to bind both typeVI collagen and fibronectin (Schmidt et al.

Type VI collagen in cartilage chondrons 1991; Bidanset et al. 1992). Further, in vitro studies have shown that type II collagen can bind directly to type VI collagen, but this interaction was inhibited by decorin, which binds via its protein core, at or near the globular domain of type VI collagen molecule (Bidanset et al. 1992). It is therefore possible that decorin could mediate the interaction between type VI collagen, fibronectin and the dominant integrin receptor (α5β1) expressed by articular cartilage chondrocytes (Salter et al. 1992). Alternatively, type II collagen bound to the globular region of type VI collagen could secure attachment via anchorin CII (see Fig. 5), a chondrocyte specific member of the annexin V family of proteins known to mediate binding on type II collagen substrates (Mollenhauer et al. 1984; Pfäffle et al. 1990). Although type VI collagen distribution was the subject of this investigation, no unlabelled capsular fibres were observed anchoring the pericellular capsule to the chondrocyte membrane, and further studies will be required to confirm if such anchoring mechanisms exist in articular cartilage chondrons. The non-sulphated glycosaminoglycan hyaluronan also interacts with type VI collagen (Wu et al. 1987; McDevitt et al. 1991) and binds via the globular domains of the intact type VI collagen microfibril (Kielty et al. 1991, 1992). Since hyaluronan forms a pericellular coat around chondrocytes (Knudson and Knudson, 1991; Toole, 1991), and the cell surface receptor for hyaluronan, CD-44, is well established (Aruffo et al. 1990; Culty et al. 1990; Toole, 1991), it is probable that one of the main interactions between the chondrocyte and type VI collagen occurs via hyaluronan (see Fig. 5). In this study, however, isolated chondrons were first digested with testicular hyaluronidase to expose the collagen epitopes, and it is possible that partial disruption or extraction of the type VI collagen-hyaluronan complex could explain the consistently lower levels of type VI collagen observed adjacent to the chondrocyte. In conclusion, we suggest that type VI collagen plays a dual role in the maintenance of chondron integrity. First, it could bind to the radial collagen network and stabilise the collagens, proteoglycans and glycoproteins of the pericellular microenvironment, ensuring the structural and functional integrity of the chondron. Secondly, several cell surface receptors exist, which could mediate the interaction between the chondrocyte and type VI collagen, providing firm anchorage and signalling potentials between the pericellular matrix and the cell nucleus. Further studies are required to elucidate the complexity of mechanisms responsible for the attachment of the chondrocyte to its pericellular microenvironment, and their combined interaction with the load-bearing extracellular matrix of adult articular cartilage. Supported and funded by the Health Research Council of New Zealand (C.A.P.; R.T.G.) and the Arthritis and Rheumatism Council (United Kingdom; S.A.). We wish to thank Ms K. Higginson for technical assistance and Mr A. Ellis for the illustration.

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