488 Annals of the Rheumatic Diseases 1994; 53: 488-496

REVEW Ann Rheum Dis: first published as 10.1136/ard.53.8.488 on 1 August 1994. Downloaded from

Cartilage collagens: strategies for the study of their organisation and expression in the extracellular matrix

J Terrig Thomas, Shirley Ayad, Michael E Grant

The collagens constitute a family of proteins mediate the growth and development of the that are assembled into a variety of supra- skeleton. The critical role of collagen II in molecular structures in extracellular matrices. maintaining cartilage integrity has been Research conducted over the last 20 years has demonstrated by studies on certain heritable led to the identification of at least 16 chondrodysplasias, whereby aberrant collagen genetically distinct collagen types encoded by II synthesis gives rise to a spectrum of clinical over 30 different genes. The structure, symptoms ranging from mild dwarfism, function and distribution of these collagens are myopia and secondary osteoarthritis (OA) in still the subject of intense investigation, but on some forms of spondyloepiphyseal dysplasia the basis of their known gene structure and (SED) to several lethal forms of achondro- amino acid sequences it is possible to classify plasia.2 It is clear that the cartilage collagens are them into different groups. The most studied crucial determinants of the mechanical group is that comprising the collagens capable properties of all articulating joints and any of forming classical fibrous structures (types I, disturbance of the complex collagenous II, III, V and XI), whose main function is to resist tensile stresses exerted on tissues. Another class of collagenous molecules Type Molecular Chain includes types IX, XII and XIV which are form composition believed to be associated with the fibrous collagens and have been assigned the name of http://ard.bmj.com/ Fibril Associated Collagens with Interrupted 11 e O [al (1)13 Triple-helices (FACIT). Collagen types VIII and X appear to form another class of matrix proteins for they exhibit remarkable homology at the levels of gene organisation and amino acid sequence and have the ability to form xi * al (XI) a2(XI) a3(XI) regular hexagonal lattice structures. A detailed on September 24, 2021 by guest. Protected copyright. review of all collagen types is published elsewhere. 1 IX Individual collagen types are rarely found in isolation in extracellular matrices and generally al(VI) a2(VI) a3(VI) occur in conjunction with several others. For example, collagen types I, III, V and VI are widely distributed in connective tissues such as skin, tendon, aorta, gut etc. In contrast, collagen IV is localised exclusively to basement al(IX) a2(IX) a3(IX) membranes and collagen VII has only been described in the anchoring fibrils underlying the lamina densa of many epithelia. During School ofBiological evolution a separate group of collagens has Sciences, Stopford Building, arisen to provide cartilaginous tissues with x .-. [la (X)]3 University of their own distinct macromolecular organis- Manchester, ation. Here we find the cartilage-specific Manchester, X with United Kingdom collagens (types II, IX, and XI) together J T Thomas the more ubiquitous collagen type VI, and S Ayad possibly types XII and XIV (fig 1). There is still Cl IN M E Grant much to be discovered about the role of these 0)A Correspondence to: in and about their 3 3 3 Professor M E Grant, collagens cartilage especially 3 School of Biological interactions with the highly hydrated proteo- Figure 1 The molecularform and chain composition of the Sciences, of the matrix. The The molecular are Stopford Building, glycans cartilage collagens cartilage collagens. forms represented by University of Manchester, must function not only to resist tensile forces schematic scale diagrams with the triple helical domains Oxford Road, and to distribute load on bones in drawn as thick black lines and the non-triple helical Manchester Ml 3 9PT, articulating domains (NC) illustrated byfilled or shaded circles. The United Kingdom. cartilaginous and synovial joints but also to molecules are orientated with their NH2-termini to the right. Cartilage collagens: strategies for the study of their organisation and expression in the extracellular matrix 489

architecture is likely to lead to dysfunction and the remaining X and Y residues of the

arthritic disease. repeating sequence [Gly-X-Y]n are proline and Ann Rheum Dis: first published as 10.1136/ard.53.8.488 on 1 August 1994. Downloaded from In this review we briefly discuss current hydroxyproline respectively, hydroxyproline knowledge of cartilage collagens and highlight being essential for the formation of hydrogen how modem technologies are providing greater bonds that stabilise the helix. insight into cartilage collagen structure and The fibril-forming collagens are synthesised function in health and disease. as large precursor (procollagen) forms with non-collagenous propeptides at the N- and C-termini of an uninterrupted 300 nm triple- 1) Collagen: a definition helix. The propeptides are removed completely Over the past decade it has become in the case of types I, II and III collagens by increasingly difficult to define what is a specific N- and C-proteinases before/during collagen and what is not. Many ofthe collagens self-assembly and crosslinking of the triple- that do not constitute the major fibres of the helical molecules into stable fibrils. Such fibrils extracellular matrix are hybrid molecules, exhibit the classical x ray diffraction patterns of where the collagenous domain may only be collagen.3 Many of the newly discovered small in comparison to the non-collagenous collagens do not fit entirely with the above part of the molecule. Others, for example type criteria. For example, some collagens contain IX collagen, can be considered as protein cores interrupted triple-helices and proteolysis leads of proteoglycans. However, the triple-helix, in to their fragmentation into shorter helical which three left-handed helices (a chains) twist domains (types IV, VII, IX, XII and XIV). around each other to form a right-handed Others do not undergo the procollagen to superhelix, is common to all collagen types. collagen processing but retain their terminal Each at chain must contain glycine (Gly) at non-collagenous domains which influence the every third residue as this is the only amino supramolecular structures that they assume in acid small enough to fit into the centre of the the extracellular matrix (types IV, VI, VII, triple-helix. Approximately 10-12% of each of VIII, IX, X, XII and XIV).

Collagen Supramolecular A) THE FIBROUS COLLAGENS: TYPES II AND XI type structure Collagen type II is the major fibrous collagen found in cartilage as well as in other related tissues such as the vitreous humour of the eye 11 and the intervertebral disc; and normally represents approximately 80-90% of the collagen content of the cartilage matrix. Type II collagen is closely associated with type XI http://ard.bmj.com/ which accounts for less than 5% ofthe cartilage Xl + 11 collagen. As stated above type II and XI collagens are initially synthesised as soluble procollagen precursor molecules with non- collagenous amino- and carboxy-termini. In the case of type II collagen these peptides are VI removed by N- and C-proteinase, respectively, on September 24, 2021 by guest. Protected copyright. before fibril formation,4 whereas, in type XI collagen all the C-terminal non-collagenous domain, but only a proportion of the c N N-terminal non-collagenous domain, is N - _ _:_ ~ < removed (fig 2). IX + 11 Type II collagen is a homotrimer [t I(II)]A , -Q-0 -"",I whereas type XI collagen is a heterotrimer [aol(XI)ao2(XI)ao3(XI)].5 The a3 chain of type XI collagen is probably the product ofthe same gene (COL2A1) as that of aol (II), although it is more glycosylated. Comparisons of the x cDNA and gene sequences of the al (XI)6 and a2(XI)7 chains with those of type V collagen8 9 have demonstrated striking similarities. Both type XI and type V collagen have been shown to form heterotypic fibrils with type II and type Figure 2 Diagramatic representation ofthe supramolecular assemblies adopted by the I collagen respectively">'2 and their retention cartilage collagens. Unless otherwise indicated the molecules are orientated with their of the NH2-termini to the left. Type II collagen is organised intofibrils iUustrated as an array N-terminal non-collagenous domains is ofindividual triple helices with a quarter-stagger of 67 nm. Type XI collagen forms thought to restrict the overall diameter of these heterotypicfibrils with type II collagen (see text), but is distinguishedfrom type II collagen fibrils. by retaining the N-terminal NC domains as illustrated. Type VI collagen forms beaded The structural microfibrils by antiparallel association of monomers into dimers, lateral association ofdimers similarities observed for the into tetramers and end-on association of tetramers. Type IX collagen does notform fibril-forming collagens are reflected at the multimeric aggregates on its own, but associates, via cross-links, with the surface of type II gene level. They contain many discrete-sized collagen fibrils in an antiparallelfashion (see text). The globular NC4 domain and the exons of 54 triple helical COL3 domain project outfrom thefibril surface. Type X collagen molecules bp or repeats thereof, with each 54 are thought to associate laterally and via their C-terminal NC domains toform a regular bp encoding 6 Gly-X-Y repeats. Consequently, hexagonal lattice. it has been suggested that the fibrillar collagen 490 Thomas, Ayad, Grant

genes evolved from a common 54 bp ancestral and IX collagens has been shown to be coding unit.'3 14 stabilised via cross-links2' and recent evidence Ann Rheum Dis: first published as 10.1136/ard.53.8.488 on 1 August 1994. Downloaded from Early studies on type II collagen demon- suggests that the type IX and type II collagen strated it had a higher hydroxylysine:lysine molecules are arranged in an anti-parallel ratio and increased levels of glycosylated fashion22 (fig 2). It has become apparent that hydroxylysine than collagen types I and 111.15 the NC domains are of important structural The function of the carbohydrate moieties significance. The large NC4 domain on the remains unclear but there appears to be an (x 1 (IX) chain is very basic, offering the inverse relationship between glycosylation and possibility of interaction with acidic proteo- fibril diameter.'6 Fibril diameter is clearly glycan molecules. The o2(IX) chain contains under careful control and glycosylation may a NC3 domain which is 5 amino acids longer explain, in part, why type II collagen forms than the other two chains providing a flexible much finer fibrils than type I collagen. hinge region to the molecule and an However, in mammalian articular cartilage, attachment site for a glycosaminoglycan fine fibrils (10-25 nm) are only found in the (GAG) side chain consisting of either pericellular matrix immediately surrounding chondroitin sulphate or dermatan sulphate. the chondrocytes, with much thicker fibrils The GAG chain length is highly variable, is not occurring in the territorial and interterritorial present on all type IX collagen molecules in matrices. 17 cartilage,23 and its role is presently unknown. It has recently been discovered that there are Types XII and XIV collagen are partially two alternatively spliced forms of type II homologous to type IX collagen and their collagen. A 207 bp 5'terminal exon (exon2) proposed function is to associate with type I coding for a 69 amino acid sequence can either collagen-containing fibrils in an analogous be spliced in (collagen type IIA) or out manner to the association between type IX and (collagen type IIB) of the procollagen mRNA type II collagen. However, the precise nature in a tissue-specific manner. 8 The domain of the interaction remains to be established. encoded by the exon is conserved between the Types XII and XIV collagen are homotrimers fibrillar collagens and may have a functional containing two triple-helical domains (COLl role in fibrillogenesis. In situ hybridisation and COL2) and three non-collagenous studies have revealed that the exon is absent domains (NC1 to NC3).24 25 The COLl from the proot 1 (II) mRNA species in fully domain and part of the NC3 domain are differentiated cartilage and collagen IIA homologous to the COLl and NC4 domains appears to be preferentially expressed by the of type IX collagen respectively. However, the cells of the perichondrium and in non- NC3 domain of each a chain is very large, chondrogenic embryonic tissues such as being 190 K in non-cartilaginous tissues, nucleus pulposus of the developing inter- giving the molecules their characteristic vertebral disc.'8 Studies in the chick system cross-shape when viewed under the electron http://ard.bmj.com/ have demonstrated collagen type IIA microscope by rotary shadowing. In cartilage, expression in pre-cartilage limb mesenchyme the NC3 domain of type XII collagen has and also in non-cartilage tissues including recently been shown to be at least 200 K embryonic calvaria, skin, heart, skeletal muscle greater.2' There is also evidence indicating the and brain.'9 No studies have yet been carried presence of GAG in the NC3 domain of type out to determine whether collagen type IIA is XII collagen and in one of the other NC expressed in OA tissue. domains of type XIV collagen.26 Type XII on September 24, 2021 by guest. Protected copyright. collagen has been localised to the peri- chondrium at the articular surface and around B) THE FACIT COLLAGENS: TYPES IX, XII AND cartilage canals whereas type XIV collagen is XIV found throughout the matrix.26 Much is yet to The major role of the collagen fibres of be learned about the functional roles of this cartilage is to form a stable meshwork to unusual family of collagenous extracellular counteract the swelling pressure generated by matrix proteins. the hydrated proteoglycan aggregates. The achievement of stability requires the inter- action of the collagen fibres with other matrix C) TYPE X COLLAGEN components and here the first FACIT collagen Type X collagen is a developmentally regulated to be described (type IX collagen) may play a short-chain collagen which is transiently significant role. expressed by hypertrophic chondrocytes at Type IX collagen is a heterotrimer composed sites of endochondral bone formation.27 28 of three different ot chains [ot1(IX)a2(IX) Such tissue-specific expression makes type X oL3(IX)] and is found associated with type II collagen a useful marker for both normal and collagen. It contains three triple-helical aberrant bone formation. For example, in collagenous domains (COL 1, 2 and 3) and addition to its synthesis by developing long four non-collagenous domains (NC 1, to 4). bones, type X collagen has also been localised Electron microscopical studies have in fracture repair callus29 and osteoarthritic demonstrated the localisation of type IX joints. 30 31 Type X collagen is unusual in that collagen along the surface of type II collagen it is encoded by a condensed gene with fibrils with the COL3 domain and the large approximately 95% of the coding sequence (243 amino acids) NC4 domain projecting out being contained in a large single exon.32 33 from the fibril surface into the perifibrillar Type X collagen is a homotrimer with ot chains matrix.'0 20 The interaction between types II of Mr 59 000 and a helical domain of Mr Cartilage collagens: strategies for the study of their organisation and expression in the extracellular matrix 491

45 000, approximately half the size e Ao1(II) beaded microfibrils which occur in cartilage

chains. Studies in vitro carried out mlainly on and in a number of other tissues including Ann Rheum Dis: first published as 10.1136/ard.53.8.488 on 1 August 1994. Downloaded from chick type X collagen have demonstr,ated that aorta, placenta, uterus, skin, tendon and it has a triple-helical domain of appro)ximately cornea. It has a relatively short triple-helical 130 nm34 and is capable offorming a hexagonal domain (105 nm) flanked by exceptionally lattice-like structure35 similar to that (observed large N- and C-terminal globular domains that for the genetically-related type VIII collagen in account for more than two thirds of the mass Descemet's membrane.36 Unlike the fibrillar of the molecule (fig 1). Three distinct at chains collagens, type X collagen retains its non- have been attributed to type VI collagen collagenous C- and N-terminal domains and although there is evidence that all three are not the hexagonal lattice-like structure can be coordinately expressed and there may be a visualised as regularly spaced nocJules of family of collagen VI isoforms.37 The most aggregated C-termini interconnectced with common form is the heterotrimer [x 1 (VI) filamentous structures formed via intceractions a2(VI)c3(VI)], where the al(VI) and ot2(VI) between adjacent triple-helices (fig 2!).3" The chains have M, 140 K and share some potential involvement of such structur-es in the sequence homology, and the unrelated a3(VI) mineralisation of hypertrophic cartil[age and chain has Mr 240-280 K. The larger size of the the endochondral ossification process remains a3(VI) chain is accounted for by a much larger to be defined. N-terminal non-collagenous domain which is subject to variation as a result of alternative splicing of the a3(VI) mRNA.38 39 D) TYPE VI COLLAGEN The structure adopted by type VI collagen Type VI collagen forms a distinct ssub-class is very different from other collagen types. within the collagen family and assemlbles into Following its synthesis, monomers assemble intracellularly into antiparallel staggered Non-collagenous proteins isolatedfrom cartilage dimers, two of which in turn align in register to form a tetramer. Following secretion, end to Protein Localisation Function Reference end polymerisation of the tetrameric units Anchorin CII Chick chondrocyte cell Binds type II collagen 43, 44 results in the formation of typical microfibrillar (Mr 34 000) surface networks (fig 2). Cartilage matrix protein Bovine tracheal and Interacts with CMP epiphyseal cartilage, proteoglycans and binds 45,46 The function of type. VI collagen is ill- (M, 148 000 homotimer nasal septum, not type II collagen defined, but it possesses a number of subunit M, 54 000) articular cartilage interactive Cartilage matrix protein Articular cartilage Not known 45 properties which suggest it may (M, 36 000 monomeric) have a bridging role between cells and their Chondrocalcin (C- Hypertrophic cartilage Binds hydroxyappatite. 47 extracellular matrix. The triple-helical domain propeptide of type II Possible role in cartilage of collagen) mineralisation type VI collagen contains a number of (M, 105 000 homo- sequences capable of supporting cell binding http://ard.bmj.com/ trimer, subunits M, 35 000) (Arg-Gly-Asp or RGD) but although it has Collagen-binding Swarm chondrosarcoma Binds type II collagen 48 been shown to bind to cells, at present it is not protein CBP Cartilage and interacts with known which of the RGD sequences are (Mr 54 000) proteoglycans. Possible role in involved. The type VI collagen microfibrils regulating collagen fibril have been shown to be stabilised by specific diameter non-covalent interactions with hyaluronan40 Cartilage matrix Articular cartilage Not known 49 on September 24, 2021 by guest. Protected copyright. glycoprotein (CMGP) Fibrocartilage and type VI collagen has also been shown to (PA 550 000 tetramer, Vitreous interact with collagen types I and II, and the subunits M, 1 16 000- 130 000) small proteoglycan decorin.4' The globular Cartilage oligomeric Swarm chondrosarcoma Not known 50 domains of type VI collagen contain repetitive matrix protein (COMP) Cartilage 200 amino acid motifs [three in a1(VI) and Similar/identical to CMGP ot2(VI) and 11 in a3(VI)] which share a (PA 524 000 pentamer, high degree of homology to the fibrillar subunits M, 100 000) collagen-binding A domains in von Willebrand Ch21 protein Chondrocytes and Not known (M,21 000) granulocytes undergoing (binding and transport factor.42 Identical to p20K terminal differentiation of small hydrophobic protein molecules?) Chondronectin Cartilage and serum Mediates attachment of 52 E) NON-COLLAGENOUS PROTEINS (PA 180 000, subunits chondrocytes to type II 56 000-77 000) collagen Collagens, virtually by definition, are assigned Fibromodulin Cartilage and other Collagen binding 53, 54 a structural (PA 59 000) connective tissues regulation of role in cartilaginous tissues but in Structurally related to fibrillogenesis addition to the proteoglycans there also exists decorin and PG-S 1 a number of other proteins which may play key Fibronectin Most tissues Binds to chondrocytes 55,56 structural roles. Some of the proteins have (PA 440 000) and type II collagen PA 58 000 Bovine articular cartilage Not known 53 defined functions but, for many, their function Other tissues? remains unknown. It is beyond the scope ofthe M, 55 000 (monomer) Adult human articular Not known 57 present review to give a detailed account of all Similar to M, 58 000 cartilage (Immunoglobulin binding?) the proteins isolated to date but some brief PARP (Proline/Arginine Cartilage Not known 58 information is included in the table. The Rich Protein) defined ultrastructure of the cartilage matrix PA 24 000 monomer 49% identical to therefore is undoubtedly complex and there is N-term. end of a 1 (XI) still much work to be done to determine the Tenascin Binds to aggrecan (PA > 106 59 specific mechanisms by which matrix assembly daltons) takes place. 492 Thomas, Ayad, Grant

2) Current strategies in the study of the enzymic system coupled to a develop-

coliagens in normal and diseased ment of a coloured end product which is Ann Rheum Dis: first published as 10.1136/ard.53.8.488 on 1 August 1994. Downloaded from cartilage recognised by light microscopy. To achieve To enhance our understanding of cartilage greater definition, antigenic epitopes can be structure and the complex interplay between recognised by electron microscopy by using cells and matrix that occur in this avascular a second antibody coupled to colloidal tissue, a variety of techniques have been gold. developed. A brief synopsis of the basic Immunolocalisation can provide valuable strategies currently in use in the study of information on the temporal and spatial cartilage collagens is outlined below. deposition of various matrix proteins. The extraction and subsequent purification of particular collagen types has permitted A) BIOCHEMICAL EXTRACTION collagen type-specific antibody production for Although the existence of 'collagen' in use in studies such as immunolocalisation. connective tissues has been known since the Although antibodies to type I and II collagens nineteenth century it was not until the 1950s are now commercially available, the pro- that the structure of type I collagen was duction of antibodies which specifically defined,60 followed by type II collagen in the recognise the other minor cartilage collagens late 1960s.6' The early studies on chick sternal has proved to be a more difficult task. The cartilage demonstrated that type II collagen, helical domains of collagen molecules usually which is not readily solubilised in neutral salt give only a weak immunological response when solutions, can be extracted following limited injected into rabbits or other species. However, pepsin digestion.62 Such a relatively crude the non-collagenous extension peptides have procedure relies on the fact that collagens proved to be much more immunogenic. comprise three polypeptides in a triple-helical Polyclonal antibodies can be readily raised to conformation which is resistant to proteolytic purified collagens but the structural similarity cleavage. The other cartilage collagens have between the triple helical domains requires that since been successfully extracted using similar, they be cross-absorbed against other known but more elaborate adaptations. Extraction of collagen types. This approach is becoming collagens from mature mammalian cartilages increasingly difficult as more and more requires preliminary extraction of the proteo- collagens are being discovered. Monoclonal glycans with dissociative agents such as 4 M antibodies, demonstrating high specificity guanidinium hydrochloride to ensure a more and affinity for the initial immunogen, efficient solubilisation of the collagens by represent an alternative strategy in anti- pepsinisation. Differential salt fractionation of body production technology. Unfortunately, the pepsin digests at acid pH can readily most monoclonal antibodies raised to the separate out the different collagen types. A various collagen types also show a high http://ard.bmj.com/ detailed breakdown of the fractionation of degree of species specificity so that anti- pepsin-solubilised collagens from cartilage has bodies raised against a collagen type from one been given elsewhere.63 Analyses of the major species may not cross-react/recognise its fibrillar cartilage collagen types are particularly human counterpart. Caution must always amenable to such extraction procedures. be exercised when using monoclonal anti- However, the helical domain of collagen VI bodies in immunolocalisation studies as it is represents less than 30% ofthe native molecule possible that the single epitope recognised may on September 24, 2021 by guest. Protected copyright. and fragmentation of collagen IX arises due to be masked for example, by interactions proteolytic cleavage at the non-collagenous between other matrix macromolecules or by (NC) domains separating the helical segments. mineral. It is often a prerequisite to de-calcify Consequently, alternative methods for the tissue sections before subsequent immuno- extraction of the intact forms of these cartilage localisation. Due to epitopes being con- collagens have had to be developed.64 65 formationally determinant, it is essential that Biochemical extraction has allowed much the antibody is known to recognise the native information to been gained into the macro- protein. This will depend largely on the molecular composition and gross quantities of biochemical extraction procedure adopted particular collagen types in normal and during the initial purification of the diseased cartilage, but the approach does have immunogen. its limitations. The procedures are lengthy and Biochemical and immunolocalisation require relatively large amounts of tissue which analyses of surgically removed joints from restricts the application of studies on small patients with later stages of OA give some animal model systems of cartilage disease insight into the resultant effect of cumulative where early phases of damage and repair can changes within the joint over a number of be observed. In contrast, human material is years. Although this information is useful, normally available only from severely affected in order to identify the root cause of the it tissue at a joints and therefore it is only the resultant end- disease is necessary to obtain can be stage of the disease that is being studied. much earlier stage. Animal models used, but it is very often difficult to extrapolate the results back to humans. The removal B) IMMUNOLOCALISATION of small cartilage samples by arthroscopy Immunocytochemical studies commonly from patients with early OA should allow use indirect immunofluorescence in which subsequent analyses by in situ hybridisation the antigenic epitopes are visualised by an and immunolocalisation. Cartilage collagens: strategies for the study of their organisation and expression in the extracellular matrix 493

C) MEASUREMENT OF COLLAGEN BREAKDOWN shown to exhibit a rounded cell morphology,

There are comprehensive data documenting elaborate an extracellular matrix containing Ann Rheum Dis: first published as 10.1136/ard.53.8.488 on 1 August 1994. Downloaded from proteoglycan degradation in OA but there is cartilage collagens and maintain their little understanding of what happens to the phenotype for longer periods than those collagens. The distinctive features of cultured on plastic. Manipulation of the collagenous proteins such as their relatively surrounding matrix by incorporation of other high content of hydroxyproline, hydroxylysine components into the collagen gel system such and pyridinium cross-links have provided as collagen II, proteoglycan aggregates and useful urinary and/or serum markers for calcium salts have been shown to have detecting abnormal cartilage/bone degra- profound effects on embryonic chondrocyte dation. Hydroxyproline, the most common collagen gene expression72-74 It remains to be marker studied,66 is extremely non-specific due established whether cells from mature cartilage to the possibility of it arising in body fluids can respond to external factors in a similar from several sources including the C 1 q manner. However, studies on OA cartilage complement component and from the high have demonstrated the presence of at least proportions of newly synthesised collagen three different chondrocyte phenotypes which are degraded intracellularly.67 Urinary expressing different patterns of collagenous levels of pyridinium cross-links, which are polypeptides.32 There is undoubtedly a need to present in mature collagen fibrils, have been develop in vitro systems whereby the effects of shown to correlate with severity of OA68 and factors, particularly those known to be elevated may provide a means of monitoring disease in OA tissue, can be examined on immortalised progression and therapy. Analysis of synovial chondrocytes exhibiting a stable phenotype. fluid for such markers can indicate collagen The availability of stable chondrocyte cell lines breakdown in specific joints since the would also benefit molecular biological fragments formed following degradation are approaches such as the study of DNA-protein not interacting with other matrix macro- interactions controlling and modulating molecules and are free to diffuse out into the collagen gene expression. The isolation of synovial fluid. Assays have not yet been sufficient nuclear proteins for performing developed to detect degradation products of DNA footprinting and gel retardation assays collagen types VI, IX, X, XI, XII or XIV which requires large numbers of cells with a defined may appear in synovial fluid or serum following phenotype. Cell lines have been produced by the breakdown of the fibrillar collagen infection of fetal rat costal chondrocytes with meshwork. viruses carrying the myc and rafoncogenes75 or A major limitation in detecting and studying by transfection of rabbit articular chondrocytes collagen degradation in normal and OA joints with a plasmid containing the SV40 early has been the lack of a technique for identifying function segment.76 However, the immortal- collagen breakdown in situ. Recently, ised cells produced only maintain some of the http://ard.bmj.com/ antibodies have been raised which specifically properties of differentiated chondrocytes. The recognise only fragmented and denatured type main problem with current methods of cell II collagen ox chains.69 Immunolocalisation immortalisation is that they rely on the random studies have demonstrated that staining for integration of the responsible gene into the denatured type II collagen is much greater in host genome leading to the possibility of it arthritic cartilage and has a different having adverse effects on the expression of distribution pattern to that in normal tissue. It certain host cell genes. Studies on the on September 24, 2021 by guest. Protected copyright. is conceivable that the levels of denatured molecular mechanisms involved in cellular fragments of type II collagen in the synovial immortalisation will hopefully provide a means fluid of affected joints may correlate well with of producing more reliable cell lines in the the extent of disease. If so, then the ability to future. Alternatively, cells from chondro- detect them specifically would provide a sarcomas may be of value to in vitro analyses sensitive marker system. provided they are able to respond to external stimuli in a manner similar to normal chondrocytes. Karyotyping of such cells is D) IN VITRO SYSTEMS essential due to a number of chondrosarcomas In vitro culture experiments on chondrocytes having been shown to contain chromosomal or cartilage explants accompanied by SDS- translocations.77 PAGE analysis of [3H]proline-labelled bio- The elucidation of the mechanisms regu- synthetic products have been extremely lating the complex interactions between valuable in providing an insight into the nature different collagen types and between collagens of collagen precursors, their processing and and other extracellular matrix components in deposition in the extracellular matrix.63 The vivo is still in its infancy. For example, effects of various growth factors and cytokines although the longitudinal packing of fibrillar on collagen gene expression are most readily collagen molecules into a quarter-stagger investigated using in vitro systems but are array has been known for a long time, much subject to the criticism that chondrocytes in less is known about the lateral packing of vivo do not exist in monolayers on plastic collagen molecules to form circular cross- bathed in serum. Consequently attempts have sectioned fibrils ofuniform diameter. However, been made to develop culture systems having advances have been made with the somewhat more biologically-relevant environ- development of an in vitro system for ments. Chondrocytes cultured within 3-D generating collagen fibrils by enzymic cleavage collagen gels70 or over agarose7' have been of type I procollagen with purified procollagen 494 Thomas, Ayad, Grant

C-terminal proteinase.78 79 The technique has sections. Since the functional half-life of many

provided an insight into fundamental aspects mRNAs is relatively short (minutes or hours) Ann Rheum Dis: first published as 10.1136/ard.53.8.488 on 1 August 1994. Downloaded from of collagen fibril formation and has been their detection in situ can allow for an accurate successfully applied to the study of abnormal assessment of the temporal and spatial type I collagen fibrils produced by patients with expression of a given gene. It should be osteogenesis imperfecta.80 It is conceivable that emphasised, however, that the transcription of similar studies could be carried out on type II a gene and its subsequent translation are two collagen from patients with genetically independently controlled events and inherited OA showing linkage to COL2A1. consequently any data obtained by in situ Whether such systems can be exploited to gain hybridisation can only be substantiated by a better understanding of overall matrix concomitant immunolocalisation of the gene assembly involving multiple macromolecular product. This should also be taken into components remains to be established. account with results from quantitative PCR and Northern and slot blot analyses. To ascertain whether the level of gene E) MOLECULAR BIOLOGICAL APPROACHES transcription is equivalent to its translation in Reports of the first cloned collagen DNA situ hybridisation and immunolocations should sequences appeared over a decade ago8' and be performed on serial sections31 32 and ideally, since then rapid progress has been made in the the techniques should be combined to allow for isolation, characterisation and chromosomal simultaneous localisation of the mRNAs and localisation of all the genetically distinct their respective gene products. It is not yet collagens discovered to date.82 Complementary possible to carry out such a study on DNA (cDNA) probes have proved to be an mineralised cartilage tissue, although a method extremely useful and sensitive tool for has been described for a cell culture system.85 determining the levels of collagen gene The application of recombinant DNA expression (as measured by the level of specific technology to the study of cartilage collagen mRNA) and the sites of collagen synthesis (by gene expression has so far led to only a in situ hydridisation). relatively basic understanding of the Steady-state mRNA levels for a given gene mechanisms involved in collagen gene can be estimated by transferring known regulation and many areas remain to be amounts of either total RNA or polyA RNA investigated. For example, little is known of extracted from a tissue sample onto nylon on how multiple DNA sequences and nitrocellulose membranes (Northern or slot transcription factors interact to modulate the blot) followed by hydridisation to a labelled level of gene transcription. An understanding probe. The technique is limited by its of such regulatory mechanisms is essential for requirement of microgram quantities of RNA both elucidating normal patterns of collagen which may not be readily available from a gene expression in endochondral bone http://ard.bmj.com/ clinically removed sample. However, the formation and aberrant gene expression in OA. development of the polymerase chain reaction Ultimately, it may be possible to alter the (PCR) has provided a method for the detection controlling mechanisms of and stimulate the of mRNA species from small amounts of formation/regeneration of cartilage in diseased tissue.83 Essentially, RNA extracted from the tissue. tissue is reverse transcribed into cDNA and amplified by consecutive rounds of annealing on September 24, 2021 by guest. Protected copyright. of specific oligonucleotide primers, elongation F) TRANSGENIC ANIMALS of the target sequence using Taq DNA The latest and perhaps most far reaching polymerase and denaturation of the double- technology which will benefit the study of OA stranded product. Due to the primer extension is that of transgenics. Any gene which has been products from one cycle acting as templates in cloned and sequenced can potentially be the next, the copy number approximately manipulated in the germ line, usually of mice, doubles at every cycle so that 20 cycles of PCR enabling more precise information to be gained yields about a million-fold amplification. about its function. Homologous recombi- Unfortunately, the sensitivity and simplicity of nation of the gene of interest into embryonic the technique is not matched by quantitative stem cells is currently the most effective accuracy. Anything capable of interfering with method for ensuring that the gene is expressed the exponential amplification effectively at the correct genomic locus.86 The mice which nullifies any quantitative assessment of the end subsequently develop from this procedure are products. However, PCR can be a quantitative chimeric heterozygotes; mating of siblings procedure if appropriate internal controls are allows the creation of a homozygous founder used and precautions are taken to minimise the line carrying the mutant gene. amount ofinterference with the doubling ofthe The mechanical properties of bone and target sequence.84 Quantitative PCR therefore, cartilage are functions of the molecular could potentially be of great use in measuring organisation oftheir extracellular matrices, and the gene expression of a variety of collagenous OA involves a malfunction of that structure. and non-collagenous proteins from limited Consequently, the creation of transgenic mice amounts of OA tissue. harbouring defects in specific structural In situ hybridisation is also a highly sensitive components may predispose them to early technique, allowing the detection of gene onset of OA. However, the use of transgenics expression by individual cells by hybridisation in the study of cartilage matrix function is still of cDNA probes to suitably prepared tissue in its infancy. To date transgenic mice have Cartilage collagens: strategies for the study of their organisation and expression in the extracellular matrix 495

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