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.

Regional differences in cell shape and gap junction expression in rat Achilles tendon: relation to fibrocartilage differentiation.

Tendon cells have complex shapes, with many cell processes and an intimate association with collagen fibre bundles in their extracellular matrix. Where cells and their processes contact one another, they form gap junctions. In the present study, we have examined the distribution of gap junction components in phenotypically different regions of rat Achilles tendon. This tendon contains a prominent enthesial fibrocartilage at its calcaneal attachment and a sesamoid fibrocartilage where it is pressed against the calcaneus just proximal to the attachment. Studies using DiI staining demonstrated typical stellate cell shape in transverse sections of pure tendon, with cells withdrawing their cell processes and rounding up in the fibrocartilaginous zones. Coincident with change in shape, cells stopped expressing the gap junction proteins connexins 32 and 43, with connexin 43 disappearing earlier in the transition than connexin 32. Thus, there are major differences in the ability of cells to communicate with one another in the phenotypically distinct regions of tendon. Individual fibrocartilage cells must sense alterations in the extracellular matrix by cell/matrix interactions, but can only coordinate their behaviour via indirect cytokine and growth factor signalling. The tendon cells have additional possibilities--in addition to the above, they have the potential to communicate direct cytoplasmic signals via gap junctions. The formation of fibrocartilage in tendons occurs because of the presence of compressive as well as tensile forces. It may be that different systems are used to sense and respond to such forces in fibrous and cartilaginous tissues.

Characterization of collagens and proteoglycans at the insertion of the human Achilles tendon.

This study provides a unique correlation between a molecular biological and biochemical analysis of the extracellular matrix (ECM) macromolecules in one half of 28 human Achilles tendons with an immunohistochemical study of the other. Both the insertion site and the mid-tendon were studied. The insertion (enthesis) is characterized by three distinctive fibrocartilages, two in the tendon (enthesial and sesamoid) and one on the heel bone (periosteal). Thus, its structure contrasts markedly with the fibrous character of the mid-tendon. RT-PCR analyses were performed on RNA extracted from mid-tendon and from the tendon fibrocartilages to investigate transcription of collagens and proteoglycans. Western blotting was also used to identify and characterize these macromolecules, and immunohistochemistry to localize their distribution. The results demonstrate striking differences in the ECM between the mid-tendon and its insertion. Types I, III, V and VI collagens, decorin, biglycan, fibromodulin and lumican were found in both the mid-tendon and the fibrocartilages, although their precise distribution often differed with site. mRNA for type II collagen was constantly present in the fibrocartilages, but it was only found in the mid-tendon of one specimen. The patterns of distribution for versican and aggrecan mRNA were complimentary - versican mRNA was present in the mid-tendon and absent from the fibrocartilages, while aggrecan mRNA was present in the fibrocartilages and absent from the mid-tendon. The range and distribution of ECM molecules detected in the Achilles tendon reflect the differing forces acting on it - the mid-tendon largely transmits tension and is characterized by molecules typical of fibrous tissues, but the fibrocartilages must also resist compression and thus contain, in addition, molecules typical of cartilage.

Microfibrillar elements in the synovial joint: presence of type VI collagen and fibrillin-containing microfibrils.

OBJECTIVES: The aims were to isolate and positively identify the microfibrillar elements which have been observed in the synovial lining. In addition, synovial fluid was examined for these elements to improve the understanding of the role of these structures in health and disease. METHODS: Bacterial collagenase digestion of bovine synovial linings and human and bovine synovial fluids was used to release intact, non-denatured microfibrillar elements. The microfibrils were isolated by Sepharose CL-2B chromatography and viewed by rotary shadowing. They were characterised by immunogold labelling with specific antibodies. RESULTS: Intact type VI collagen microfibrils and fibrillin-containing microfibrils were isolated and positively identified in the synovial lining from bovine ankle joints by immunogold labelling. Type VI collagen microfibrils were also present in the synovial fluid. CONCLUSIONS: The role of the microfibrillar elements in vivo is not fully understood, but their distribution in the synovial lining suggests they have an important role in the mechanical and physical properties of this tissue. The presence of type VI collagen microfibrils in synovial fluid poses the intriguing possibility that it may represent a product of microfibril turnover and a potential early marker for rheumatoid arthritis. Alternatively, type VI collagen may be specifically secreted into the synovial fluid to interact with hyaluronan and form part of the structure of synovial fluid.