The transmembrane heparan sulphate proteoglycan syndecan-4 is involved in establishment of the lamellar structure of the annulus fibrosus of the intervertebral disc.

The annulus fibrosus of the intervertebral disc unites adjacent vertebral bodies along the length of the spine and provides tensile resistance towards compressive, twisting and bending movements arising through gait. It consists of a nested series of oriented collagenous lamellae, arranged in cross-ply circumferentially around the nucleus pulposus. The organisation of oriented collagen in the annulus is established during foetal development by an identical arrangement of oriented fibroblasts that are precisely organised into cell sheets, or laminae. These provide a template for ordered deposition of extracellular matrix material on cell surfaces, by means of a poorly understood mechanism involving the actin cytoskeleton. In this study, we investigate the role of two cell surface heparan sulphate proteoglycans (HSPGs), glypican-6 and syndecan-4, in the matrix assembly process in the developmental rat intervertebral disc. We compare their expression patterns with those of heparan sulphate and the interactive, cell-surface adhesive glycoprotein, fibronectin, and relate these to the stage-specific collagenous architectures present within the annulus at both light and electron microscopic levels. We show that both proteoglycans are strongly associated with the development, growth and aging of the intervertebral disc. Furthermore, the immunohistochemical labelling patterns suggest that syndecan-4, in particular, plays a potentially-significant role in annulus formation. We propose that this HSPG mediates interaction between the actin cytoskeleton and nascent extracellular matrix in the lamellar organisation of annulus tissue. These data add considerably towards an understanding of how cells organise and maintain complex, oriented extracellular matrices and has particular clinical relevance to the fields of tissue engineering and repair.

Enhancing inflammatory and chemotactic signals to regulate bone regeneration.

Stem cells have become the fundamental element in regenerative medicine due to their inherent potential to differentiate into various cell types, and the ability to produce various bioactive molecules, including growth factors, cytokines and extracellular matrix molecules. In vivo, the secretion of tropic factors is modulated by chemotactic and inflammatory factors. In this study, we analysed the influence of a 2 h stimulation of mesenchymal stem cells (MSCs) with interleukin-1ß (IL1ß), granulocyte-colony stimulating factor (GCSF), stromal cell-derived factor 1 (SDF1) and stem cell factor (SCF). Our results demonstrated that this short stimulation exerts pronounced effects on the expression of multiple cytokine genes and proteins in MSC cells 48 and 72 h later. IL1ß strongly promoted the secretion of a wide range of proteins with chemotactic, proinflammatory and angiogenic properties, whereas SCF regulated the expression of proteins involved in proliferation, chondrogenesis and ECM regulation. This demonstrates that the changes in secretome can be directed towards a desired final functional outcome by selection of the most appropriate cytokine. Moreover, the expression pattern of Wnt signalling pathway components suggested the differential regulation of this pathway by IL1ß and SCF. Altogether, the robust paracrine action of MSCs can be achieved within a just 2 h treatment, which would be feasible within the operating theatre during a single surgical procedure. These results suggest that integrating inflammatory modulation in bone tissue engineering, by modifying the MSC secretome by way of a short stimulus, would provide a more targeted approach than administering unmodified MSCs alone.

A phenotypic comparison of osteoblast cell lines versus human primary osteoblasts for biomaterials testing.

Immortalized cell lines are used more frequently in basic and applied biology research than primary bone-derived cells because of their ease of access and repeatability of results in experiments. It is clear that these cell models do not fully resemble the behavior of primary osteoblast cells. Although the differences will affect the results of biomaterials testing, they are not clearly defined. Here, we focused on comparing proliferation and maturation potential of three osteoblast cell lines, SaOs2, MG-63, and MC3T3-E1 with primary human osteoblast (HOb) cells to assess their suitability as in vitro models for biomaterials testing. We report similarities in cell proliferation and mineralization between primary cells and MC3T3-E1. Both, SaOs2 and MG-63 cells demonstrated a higher proliferation rate than HOb cells. In addition, SaOs2, but not MG-63, cells demonstrated similar ALP activity, mineralization potential and gene regulation to HOb's. Our results demonstrate that despite SaOs-2, MG63, and MC3T3 cells being popular choices for emulating osteoblast behavior, none can be considered appropriate replacements for HOb's. Nevertheless, these cell lines all demonstrated some distinct similarities with HOb's, thus when applied in the correct context are a valuable in vitro pilot model of osteoblast functionality, but should not be used to replace primary cell studies.

Collagen fibrillogenesis in the development of the annulus fibrosus of the intervertebral disc.

The annulus fibrosus of the intervertebral disc is a complex, radial-ply connective tissue consisting of concentric lamellae of oriented collagen. Whilst much is known of the structure of the mature annulus, less is known of how its complex collagenous architecture becomes established; an understanding of which could inform future repair/regenerative strategies. Here, using a rat disc developmental series, we describe events in the establishment of the collagenous framework of the annulus at light and electron microscopic levels and examine the involvement of class I and II small leucine rich proteoglycans (SLRPs) in the matrix assembly process. We show that a period of sustained, ordered matrix deposition follows the initial cellular differentiation/orientation phase within the foetal disc. Fibrillar matrix is deposited from recesses within the plasma membrane into compartments of interstitial space within the outer annulus - the orientation of the secreted collagen reflecting the initial cellular orientation of the laminae. Medially, we demonstrate the development of a reinforcing 'cage' of collagen fibre bundles around the foetal nucleus pulpous. This derives from the fusion of collagen bundles between presumptive end-plate and inner annulus. By birth, the distinct collagenous architectures are established and the disc undergoes considerable enlargement to maturity. We show that fibromodulin plays a prominent role in foetal development of the annulus and its attachment to vertebral bodies. With the exception of keratocan, the other SLRPs appear associated more with cartilage development within the vertebral column, but all become more prominent within the disc during its growth and differentiation.

Chondroitin sulphate sulphation motif expression in the ontogeny of the intervertebral disc.

Chondroitin sulphate chains on cell and extracellular matrix proteoglycans play important regulatory roles in developing systems. Specific, developmentally regulated, sulphation motifs within the chondroitin glycosaminoglycan structure may help bind, sequester or present bioactive signalling molecules to cells thus modulating their behaviour. Using monoclonal antibodies 3B3(-), 4C3, 6C3 and 7D4, we have mapped the distribution of different chondroitin sulphation epitopes in a rat intervertebral disc developmental series. The sulphation epitopes had complex, dynamic and specific distributions in the disc and vertebral tissues during their differentiation, growth and ageing. At embryonic day [E]15, prior to disc differentiation, 4C3 and 7D4 occurred within the cellular disc condensations whilst 6C3 was present in the notochordal sheath. At E17, post disc differentiation, 4C3 and 7D4 occurred within the nucleus pulposus, inner annulus and vertebral bodies; 3B3(-) in the nucleus, inner annulus, annulus/vertebral body interface and perichondrium; and 6C3, ventrally, within the perichondrium. At E19, 3B3(-), 4C3 and 7D4 became further restricted to the nucleus, inner annulus, annulus/vertebral body interface and perichondrium. Prior to birth, all four epitopes occurred within the inner annulus and nucleus, with 6C3 and 7D4 also occurring within the future end-plate. Postnatal expression of the sulphation epitopes was more widespread in the disc and also within the growth plate. At 4 months, the epitopes were associated with chondrocyte clusters within the nucleus; and at 24 months, with annular lesions. Overall, our data suggests that differential sulphation of chondroitin correlates with significant events in development, growth and aging of the rat intervertebral disc.

Characterization of Candida albicans infection of an in vitro oral epithelial model using confocal laser scanning microscopy.

INTRODUCTION: Oral candidosis presents as several distinct forms and one of these, chronic hyperplastic candidosis, is distinguished by penetration of the epithelium by Candida. The aim of this study was to use confocal laser scanning microscopy to examine invasion of the oral epithelium by Candida albicans from different oral conditions and to determine whether inherent strain differences exist that could relate to infection type. Reverse transcription-polymerase chain reaction was also used to detect products from virulence gene families. METHODS: C. albicans (n = 19) was used to infect reconstituted human oral epithelium, which was incubated for 12 h. One half of the reconstituted human oral epithelium was then fixed and stained with concanavalin A-Alexa 594, pan-cytokeratin antibody-Alexa 488 and Hoechst nucleic acid dye. RNA was extracted from the remaining tissue for reverse transcription-polymerase chain reaction targeting secreted aspartyl proteinase, phospholipase and agglutinin-like sequence genes of C. albicans. RESULTS: Confocal laser scanning microscopy revealed strain-dependent tissue invasion, with differences evident in surface colonization, C. albicans morphology and the extent and pattern of tissue penetration. Hyphae were seen to directly penetrate epithelial cells and migrate between keratinocytes with yeast budding also evident in the reconstituted human oral epithelium. A relationship between 'high tissue invasion' and expression of secreted aspartyl proteinase genes 4-6 was noted. Interestingly, four of the five 'high invaders' originated from chronic hyperplastic candidosis. CONCLUSIONS: Confocal laser scanning microscopy permitted high resolution analysis of reconstituted human oral epithelium invasion by C. albicans and identified strain differences in the invasion process. Association between extensive hyphal morphology, direct epithelial penetration and high surface colonization were made with the 'highly invasive' strains.

Where tendons and ligaments meet bone: attachment sites ('entheses') in relation to exercise and/or mechanical load.

Entheses (insertion sites, osteotendinous junctions, osteoligamentous junctions) are sites of stress concentration at the region where tendons and ligaments attach to bone. Consequently, they are commonly subject to overuse injuries (enthesopathies) that are well documented in a number of sports. In this review, we focus on the structure-function correlations of entheses on both the hard and the soft tissue sides of the junction. Particular attention is paid to mechanical factors that influence form and function and thus to exploring the relationship between entheses and exercise. The molecular parameters indicative of adaptation to mechanical stress are evaluated, and the basis on which entheses are classified is explained. The application of the 'enthesis organ' concept (a collection of tissues adjacent to the enthesis itself, which jointly serve the common function of stress dissipation) to understanding enthesopathies is considered and novel roles of adipose tissue at entheses are reviewed. A distinction is made between different locations of fat at entheses, and possible functions include space-filling and proprioception. The basic anchorage role of entheses is considered in detail and comparisons are explored between entheses and other biological 'anchorage' sites. The ability of entheses for self-repair is emphasized and a range of enthesopathies common in sport are reviewed (e.g. tennis elbow, golfer's elbow, jumper's knee, plantar fasciitis and Achilles insertional tendinopathies). Attention is drawn to the degenerative, rather than inflammatory, nature of most enthesopathies in sport. The biomechanical factors contributing to the development of enthesopathies are reviewed and the importance of considering the muscle-tendon-bone unit as a whole is recognized. Bony spur formation is assessed in relation to other changes at entheses which parallel those in osteoarthritic synovial joints.

Biology of fibrocartilage cells.

Fibrocartilage is an avascular tissue that is best documented in menisci, intervertebral discs, tendons, ligaments, and the temporomandibular joint. Several of these sites are of particular interest to those in the emerging field of tissue engineering. Fibrocartilage cells frequently resemble chondrocytes in having prominent rough endoplasmic reticulum, many glycogen granules, and lipid droplets, and intermediate filaments together with and actin stress fibers that help to determine cell organization in the intervertebral disc. Fibrocartilage cells can synthesize a variety of matrix molecules including collagens, proteoglycans, and noncollagenous proteins. All the fibrillar collagens (types I, II, III, V, and XI) have been reported, together with FACIT (types IX and XII) and network-forming collagens (types VI and X). The proteoglycans include large, aggregating types (aggrecan and versican) and small, leucine-rich types (decorin, biglycan, lumican, and fibromodulin). Less attention has been paid to noncollagenous proteins, although tenascin-C expression may be modulated by mechanical strain. As in hyaline cartilage, matrix metalloproteinases are important in matrix turnover and fibrocartilage cells are capable of apoptosis.

The skeletal attachment of tendons--tendon "entheses".

Tendon entheses can be classed as fibrous or fibrocartilaginous according to the tissue present at the skeletal attachment site. The former can be "bony" or "periosteal", depending on whether the tendon is directly attached to bone or indirectly to it via the periosteum. At fibrocartilaginous entheses, the uncalcified fibrocartilage dissipates collagen fibre bending and tendon narrowing away from the tidemark; calcified fibrocartilage anchors the tendon to the bone and creates a diffusion barrier between the two. Where there are additional fibrocartilaginous specialisations in the tendon and/or bone next to the enthesis, an "enthesis organ" is created that reduces wear and tear. Little attention has been paid to bone at entheses, despite the obvious bearing this has on the mechanical properties of the interface and the clinical importance of avulsion fractures. Disorders at entheses (enthesopathies) are common and occur in conditions such as diffuse idiopathic skeletal hyperostosis and the seronegative spondyloarthropathies. They are also commonly seen as sporting injuries such as tennis elbow and jumper's knee.

Three-dimensional reconstructions of the Achilles tendon insertion in man.

The distribution of type II collagen in sagittal sections of the Achilles tendon has been used to reconstruct the three-dimensional (3D) shape and position of three fibrocartilages (sesamoid, periosteal and enthesis) associated with its insertion. The results showed that there is a close correspondence between the shape and position of the sesamoid and periosteal fibrocartilages--probably because of their functional interdependence. The former protects the tendon from compression during dorsiflexion of the foot, and the latter protects the superior tuberosity of the calcaneus. When the zone of calcified enthesis fibrocartilage and the subchondral bone are mapped in 3D, the reconstructions show that there is a complex pattern of interlocking between pieces of calcified fibrocartilage and bone at the insertion site. We suggest that this is of fundamental importance in anchoring the tendon to the bone, because the manner in which a tendon insertion develops makes it unlikely that many collagen fibres pass across the tissue boundary from tendon to bone. When force is transmitted to the bone from a loaded tendon, it is directed towards the plantar fascia by a series of highly orientated trabeculae that are clearly visible in 3D in thick resin sections.

Actin stress fibres and cell-cell adhesion molecules in tendons: organisation in vivo and response to mechanical loading of tendon cells in vitro.

Tendons consist of parallel longitudinal rows of cells separated by collagen fibres. The cells are in intimate contact longitudinally within rows, and laterally via sheet-like lateral cell processes between rows. At points of contact, they are linked by gap junctions. Since tendons stretch under load, such cell contacts require protection. Here we describe the organisation of the actin cytoskeleton and actin-based cell-cell interactions in vivo and examine the effect of cyclic tensile loading on tendon cells in vitro. Cells within longitudinal rows contained short longitudinally running actin stress fibres. Each fibre was aligned with similar fibres in the cells longitudinally on either side, and fibres appeared to be linked via adherens junctions. Overall, these formed long oriented rows of stress fibres running along the rows of tendon cells. In culture, junctional components n-cadherin and vinculin and the stress fibre component tropomyosin increased in strained cultures, whereas actin levels remained constant. These results suggest that: (1) cells are linked via actin-associated adherens junctions along the line of principal strain; and (2) under load, cells appear to attach themselves more strongly together, and assemble more of their cytoplasmic actin into stress fibres with tropomyosin. Taken together, this suggests that cell-cell contacts are protected during stretch, and also that the stress fibres, which are contractile, may provide an active mechanism for recovery from stretch. In addition, stress fibres are ideally oriented to monitor tensile load and thus may be important in mechanotransduction and the generation of signals passed via the gap junction network.

Entheses--the bony attachments of tendons and ligaments.

Most tendons and ligaments have fibrocartilaginous entheses where there are 4 zones of tissue at their bony attachments--dense fibrous connective tissue, uncalcified fibrocartilage, calcified fibrocartilage and bone. Such entheses leave smooth, circumscribed markings on dried bones. The uncalcified fibrocartilage dissipates the bending of collagen fibres away from the bone, ensures that a stretched tendon or ligament does not narrow too close to the bone and acts as a mini growth plate. The zone is thickest at entheses where a great deal of bending of the tendon/ligament accompanies joint movement. The calcified fibrocartilage anchors the tendon/ligament to the bone and enables it to withstand shear. Enthesis fibrocartilage may be accompanied by sesamoid and periosteal fibrocartilages that similarly protect the enthesis from wear and tear and dissipate stress. Nevertheless, each fibrocartilage can show distinctive pathological changes. A wide variety of ECM molecules has been reported in enthesis fibrocartilage, but it is best characterised by its content of type II collagen and aggrecan which account for its compression-tolerance properties.

Fibrocartilage in the transverse ligament of the human atlas.

STUDY DESIGN: Immunohistochemical investigation. OBJECTIVE: To determine whether molecules typical of articular cartilage are present in the transverse ligament and whether the ligament may be a target for an autoimmune response in rheumatoid arthritis. SUMMARY OF BACKGROUND DATA: In chronic rheumatoid arthritis there is often a marked instability of the atlantoaxial complex, and the transverse ligament can show degenerative changes that compromise its mechanical function. In some rheumatoid patients there can be an autoimmune response to cartilage link protein, aggrecan, and Type II collagen. METHODS: Transverse ligaments were removed from 13 cadavers and fixed in 90% methanol. Cryosections were immunolabeled with antibodies against proteoglycans (aggrecan, link protein, and versican), glycosaminoglycans (chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, and keratan sulfate), and collagens (Types I, II, III, and VI). RESULTS: Labeling for aggrecan and link protein was characteristic of the fibrocartilages, but versican was only detected in the fibrous regions. Equally, Types I, III, and VI collagens and keratan, dermatan, and chondroitin-4-sulfates were found throughout the ligament, but labeling for Type II collagen and chondroitin-6-sulfate was restricted to the fibrocartilages. CONCLUSION: The presence of molecules typical of articular cartilage (aggrecan, link protein, and Type II collagen) in the transverse ligament explains why it can be a target for destruction in rheumatoid arthritis and also suggests that it is subject to constant compression against the dens rather than only at the extremes of movement.

Extracellular matrix in development of the intervertebral disc.

Intervertebral discs allow bending and twisting of the spine whilst resisting compression from gravity and muscle action, and are composite structures of the peripheral annulus fibrosus enclosing the nucleus pulposus. Their development is complex, involving several different connective tissue types, yet little is known of the developing extracellular matrix (ECM). We report the ECM composition of foetal rat discs from their first appearance to birth. The earliest collagen detected was type III, which was subsequently replaced by type II in the cartilaginous inner annulus and joined by type I in the fibrous outer annulus. Type IV collagen appeared in outer annulus, associated with myofibroblast-like cells of the orienting collagenous lamellae. Laminin and fibronectin co-distributed here in later stages, although overall they had a wider distribution. Aggrecan occurred in early nucleus pulposus and then appeared in the inner annulus, in association with cartilage differentiation. Versican appeared later in the inner annulus, and also in the dorsal region of the outer annulus. Comparisons of glycosaminoglycan and proteoglycan label allowed extrapolations to be made as to likely glycosaminoglycan components of the large proteoglycans, and of other proteoglycans that may be present - thus differential distribution of aggrecan and keratan sulfate label suggested the presence of fibromodulin and/or lumican. Functionally aggrecan would confer compression resistance to cartilaginous structures. Versican may also contribute, but along with the small proteoglycans is likely to be associated with various stages of control of cell differentiation, tissue morphogenesis and collagen fibre formation in the assembly of the annulus fibrosus.

Fibrocartilage in the transverse ligament of the human acetabulum.

Biomechanical experiments on isolated hip joints have suggested that the transverse ligament acts as a bridle for the lunate articular surface of the acetabulum during load bearing, but there are inherent limitations in such studies because the specimens are fixed artificially to testing devices and there are no modifying influences of muscle pull. Further evidence is thus needed to substantiate the theory. Here we argue that if the horns of the lunate surface are forced apart under load, the ligament would straighten and become compressed against the femoral head. It would thus be expected to share some of the features of tendons and ligaments that wrap around bony pulleys and yet previous work has suggested that the transverse ligament is purely fibrous. Transverse ligaments were removed from 8 cadavers (aged 17-39 y) and fixed in 90% methanol. Cryosections were immunolabelled with antibodies against collagens (types I, II, III, VI), glycosaminoglycans (chondroitins 4 and 6 sulphate, dermatan sulphate, keratan sulphate) and proteoglycans (aggrecan, link protein, versican, tenascin). A small sesamoid fibrocartilage was consistently present in the centre of each transverse ligament, near its inner surface at the site where it faced the femoral head. Additionally, a more prominent enthesis fibrocartilage was found at both bony attachments. All fibrocartilage regions, in at least some specimens, labelled for type II collagen, chondroitin 6 sulphate, aggrecan and link protein, molecules more typically associated with articular cartilage. The results suggest that the ligament should be classed as containing a 'moderately cartilaginous' sesamoid fibrocartilage, adapted to withstanding compression. This supports the inferences that can be drawn from previous biomechanical studies. We cannot give any quantitative estimate of the levels of compression experienced. All that can be said is that the ligament occupies an intermediate position in the spectrum of fibrocartilaginous tissues. It is more cartilaginous than some wrap-around tendons at the wrist, but less cartilaginous than certain other wrap-around ligaments, e.g. the transverse ligament of the atlas.

The mechanism of formation of bony spurs (enthesophytes) in the achilles tendon.

OBJECTIVE: To investigate the early stages in the formation of bony spurs in relation to normal enthesis development. METHODS: Histologic sections of rat Achilles tendons, stained with toluidine blue or Masson's trichrome, were examined in animals ranging from 2 weeks to 1 year of age. Further material prepared for immunohistochemistry was labeled with monoclonal antibodies for laminin and type IV collagen to highlight the presence of small blood vessels at the enthesis. Sections of small spurs from the Achilles tendons of elderly humans were also examined for comparison. RESULTS: As a part of normal development, bone grows into the Achilles tendon as the calcaneus enlarges. Ossification is preceded by vascular invasion, which occurs along rows of enthesis fibrocartilage cells. Small bony spurs develop when ossification at one point on the enthesis outstrips that on either side. CONCLUSION: Bony spurs can develop in the Achilles tendon without the need for preceding microtears or any inflammatory reaction, and they form by endochondral ossification of enthesis fibrocartilage. The increased surface area created at the tendon-bone junction may be an adaptive mechanism to ensure the integrity of the interface in response to increased mechanical loads.

The cell and developmental biology of tendons and ligaments.

We have sought to create, for the first time in a single comprehensive review, a modern synthesis of opinion on the cell, developmental, and molecular biology of tendons, ligaments, and their associated structures (tendon sheaths, vinculi, and retinacula). Particular attention has been paid to highlighting new data on the early development of tendons, the signaling molecules involved in their patterning, and the diversity of specialized regions (entheses, wrap-around regions, and myotendinous junctions) that characterize fully formed tendons and ligaments. We have emphasized the complexities of adult tendon and ligament cell shape and related these to their early development. The importance of gap junctions in allowing cell communication throughout an extensive extracellular matrix (ECM) has also been highlighted, particularly in relation to understanding how tendon and ligament cells respond to changes in mechanical load. Finally, we have considered the influence of growth factors and related molecules on cell proliferation and ECM synthesis.

Fibrocartilage in the extensor tendons of the human metacarpophalangeal joints.

The extensor tendons of the fingers cross both the metacarpophalangeal (MCP) and interphalangeal joints. Previous studies have shown that where the extensor tendons replace the capsule of the proximal interphalangeal (PIP) joint, they contain a sesamoid fibrocartilage that articulates with the proximal phalanx during flexion. The fibrocartilage labels immunohistochemically for a variety of glycosaminoglycans and collagens. In the current study, we investigate the molecular composition of the extensor tendons at the level of the MCP joints. This is of particular interest because the tendon has a greater moment arm at this location (and might thus be subject to greater compression), but is separated from the joint cavity by the capsule and peritendinous tissue. Six hands were removed from elderly cadavers (39-85 years of age) and the MCP joints were fixed in 90% methanol. The extensor tendons were dissected from all fingers, cryosectioned, and immunolabelled with a panel of monoclonal and polyclonal antibodies for types I, II, III, and VI collagens, chondroitin 4 and 6 sulphates, dermatan, and keratan sulphate and aggrecan. Antibody binding was detected with the Vectastain ABC 'Elite' avidin/biotin/peroxidase kit. The extensor tendons in all the fingers had a metachromatic sesamoid fibrocartilage on their deep surface which immunolabelled for types I, III, and VI collagens, and for all glycosaminoglycans and aggrecan. Labelling for type II collagen was also seen in some fibrocartilages and was a constant feature of all index fingers. This probably relates to the greater use of that digit and the higher loads to which its tendons are subject. Chondroitin 6 sulphate and type II collagen are the most consistent markers of the fibrocartilage phenotype and most of the chondroitin 6 sulphate is probably associated with aggrecan. It is concluded that the labelling profile of the tendon fibrocartilage in the different fingers at the MCP joints is broadly similar to that at the PIP joints. Thus, the potentially greater level of compression on the extensor tendons may be counterbalanced by the lack of fusion of the tendon with the joint capsule. It is suggested that the maintenance of a similar level of fibrocartilage differentiation at two different points along the length of the extensor tendon ensures that the tensile strength is the same in the two regions and that no weak link is present.

Role of actin stress fibres in the development of the intervertebral disc: cytoskeletal control of extracellular matrix assembly.

Orientation of collagen fibrils is a key event in the development of many tissues. In the intervertebral disc, the outer annulus fibrosus comprises lamellae of parallel collagen fibres, the direction of orientation of the long axis of which alternates in angle between lamellae. In development, this organisation is preceded by the formation of sheets of oriented fibroblasts, which then deposit the oriented lamellae. Here, using fluorescent labelling, confocal and electron microscopic techniques on developmental series, we show that the orientation of cells in lamellae is associated with the formation of adherens junctions intercellularly, involving cadherins and vinculin, and longitudinal stress fibres (label for filamentous actin and tropomyosin) intracellularly. The stress fibres direct the initial elongation of cells and control the deposition of oriented extracellular matrix via junctional complexes with the matrix involving vinculin and alpha 5 beta 1 integrins, which in turn promote the formation of oriented fibronectin at the cell surface; oriented collagen is deposited between cells at the same stages. Shortly after birth, the stress fibres disappear, probably because cells now gain orientational cues from the matrix, and are undergoing differentiation-related changes to form fibrocartilage cells. Dev Dyn 1999;215:179-189.

Organisation of the chondrocyte cytoskeleton and its response to changing mechanical conditions in organ culture.

Articular cartilage undergoes cycles of compressive loading during joint movement, leading to its cyclical deformation and recovery. This loading is essential for chondrocytes to perform their normal function of maintenance of the extracellular matrix. Various lines of evidence suggest the involvement of the cytoskeleton in load sensing and response. The purpose of the present study is to describe the 3-dimensional (3D) architecture of the cytoskeleton of chondrocytes within their extracellular matrix, and to examine cytoskeletal responses to experimentally varied mechanical conditions. Uniformly sized explants of articular cartilage were dissected from adult rat femoral heads. Some were immediately frozen, cryosectioned and labelled for filamentous actin using phalloidin, and for the focal contact component vinculin or for vimentin by indirect immunofluorescence. Sections were examined by confocal microscopy and 3D modelling. Actin occurred in all chondrocytes, appearing as bright foci at the cell surface linked to an irregular network beneath the surface. Cell surface foci colocalised with vinculin, suggesting the presence of focal contacts between the chondrocyte and its pericellular matrix. Vimentin label occurred mainly in cells of the deep zone. It had a complex intracellular distribution, with linked networks of fibres surrounding the nucleus and beneath the plasma membrane. When cartilage explants were placed into organ culture, where in the absence of further treatments cartilage imbibes fluid from the culture medium and swells, cytoskeletal changes were observed. After 1 h in culture the vimentin cytoskeleton was disassembled, leading to diffuse labelling of cells. After a further hour in culture filamentous vimentin label reappeared in deep zone chondrocytes, and then over the next 48 h became more widespread in cells of the explants. Actin distribution was unaffected by culture. Further experiments were performed to test the effects of load on the cytoskeleton. Explants were placed in culture and immediately subjected to static uniaxial radially unconfined compressive loads of 0.5, 1, 2 or 4 MPa for 1 h using a pneumatic loading device. Loads greater than 0.5 MPa maintained the vimentin organisation over the culture period. At 0.5 MPa, the chondrocytes within the explant behaved as in free-swelling culture. The rapid change in vimentin organisation probably relates to rapid swelling of the explants--under free-swelling conditions, these reached their maximum swollen size in just 15 min of culture. The chondrocytes' response to change in tissue dimensions, and thus to their relationship to their immediate environment, was to disassemble their vimentin networks. Loading probably counteracts the swelling pressure of the tissue. Overall, this work suggests that chondrocytes maintain their actin cytoskeleton and modify their vimentin cytoskeleton in response to changing mechanical conditions.