Introduction: Tendons consist of longitudinally running parallel bundles of collagen with rows of tendon cells between them. The tendon cells are linked to one another via gap junctions1 and cytoskeletally associated adherens junctions. Recent in vitro studies indicate that the two types of gap junction present, made of connexin 32 and connexin 43, regulate tendon cell responses to mechanical load2. In view of the importance of cell-cell interactions in tendon cell behaviour, we describe the behaviour of tendon cells in a novel 3-dimensional culture system designed to allow cells to establish cell-cell contacts and deposit matrix in the absence of scaffolds, which would favour cell-substrate interactions, and without disturbance by medium changes.
Methods: Tendon fibroblasts were isolated from chicken tendons by protease and collagenase digestion3, grown to passage 3 in HEPES buffered DMEM/5% foetal calf serum/1% antibiotic/1% L-glutamine at 370C, in 5% CO2 in air. Cells were suspended at 3x107 cells/ ml and 1 ml placed in a Spectrapor DispoDialyzerTM tube (MW cutoff 300,000). This was then placed into a 60 ml centrifuge tube with 40 ml DMEM containing ascorbate (1mg/ml) on a roller. Medium in the large tube was changed every 2 days. At 24 hours, 7, 14 and 21 days the cell aggregates were fixed in 90% methanol (4°C), frozen on dry ice and cryosections cut at 10–15 μm. Sections were labelled by indirect immunofluorescence with monoclonal type I, II, and III collagen, actin, vimentin and decorin, connexins 32 and 43, vinculin, Pan cadherin and N-cadherin.
Results: Cells in suspension culture formed elongated aggregates up to 3cm long. Immunolabels showed that at 7 days type I and III collagens were present, predominantly in the periphery. At 14 days the collagens were uniformly distributed throughout the aggregates and showed parallel longitudinal organisation. It is also clear from propidium iodide label that the cell nuclei have distinct areas of alignment. The aggregates labelled positively for actin stress fibres, N-cadherin, decorin and connexin 32. Type II collagen and connexin 43 showed no conclusive label.
Discussion: The suspension cultures clearly show that tendon cells can form large scale, organised structures in this culture system, and are capable of assembling an organised extracellular matrix in the absence of a scaffold to support them. The structures formed were similar to tendons in their cell and matrix organization. The amount of collagen deposited by the cells increases over time. Therefore, tendon cells could be used in a tissue engineering context to form well organised tissue in the absence of scaffolds in suitable culture systems. The presence of connexin 32 gap junctions and absence of connexin 43 shows the cell aggregates favouring matrix synthesis pathways – in cell cultures connexin 32 junctions promote collagen sythesis whereas connexin 43 inhibits it2.
