Tenocytes express primary cilia, which elongate when tendon is maintained in the absence of biomechanical load. Previous work indicates differences in the morphology and metabolism of the tenocytes in the tendon fascicular matrix (FM) and the inter‐fascicular matrix (IFM). This study tests the hypothesis that primary cilia in these two regions respond differently to stress deprivation and that this is associated with differences in the biomechanical degradation of the extracellular matrix. Rat tail tendon fascicles were examined over a 7‐day period of either stress deprivation or static load. Seven days of stress deprivation induced cilia elongation in both regions. However, elongation was greater in the IFM compared to the FM. Stress deprivation also induced a loss of biomechanical integrity, primarily in the IFM. Static loading reduced both the biomechanical degradation and cilia elongation. The different responses to stress deprivation in the two tendon regions are likely to be important for the aetiology of tendinopathy. Furthermore, these data suggest that primary cilia elongate in response to biomechanical degradation rather than simply the removal of load. This response to degradation is likely to have important consequences for cilia signalling in tendon and as well as in other connective tissues. © 2016 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 34:2146–2153, 2016.

This study used isolated human tenocytes to test the hypothesis that cyclic mechanical strain directly stimulates primary cilia disassembly, and to elucidate the mechanisms involved. Cells were seeded onto flexible membranes and strained at 0–3%; 1 Hz, for up to 24 hours. Cilia length and prevalence progressively reduced with increasing strain duration but showed full recovery within 2 hours of strain removal. The response to loading was not influenced by actin organisation as seen in other cell types. However, the loading response could be recreated by treatment with TGFβ. Furthermore, treatment with the HDAC6 inhibitor Tubacin, or a TGFβ receptor inhibitor both prevented strain induced cilia disassembly. These data are the first to describe primary cilia expression in isolated tenocytes, showing that mechanical strain regulates cilia expression independent of changes in tendon extracellular matrix. Furthermore, we show that cilia disassembly is mediated by the activation of TGFβ receptors leading to activation of HDAC6. Previous studies have shown that cilia are required for TGFβ signalling and that tendon mechanosignalling is mediated by TGFβ. The present study therefore suggests a novel feedback mechanism whereby cilia disassembly inhibits prolonged TGFβ activation in response to continuous cyclic loading.

Introduction Primary cilia are eukaryotic organelles found singularly on almost every cell in the body. Primary cilia localise a number of developmental signalling pathways, and for some of these pathways, the rate of signalling is dependent on cilia length (Thompson et al. 2014). Tenocytes have previously been shown to have primary cilia, which increase in length when tendon is stress deprived (Gardner et al. 2011). The cilia within fascicles have also been shown to orient in the direction of applied load (Donnelly et al. 2010). Recent work from our group suggests two distinct populations of cells within tendon; those in the inter-fascicular matrix (IFM) or epitendon (IFM cells), and those in the fascicular matrix (FM cells). Preliminary data suggests IFM cells experience different strain fields to FM cells, and may also be more metabolically active. We therefore hypothesise that there will be a difference in the length of the primary cilia in IFM and FM cells under physiological strain conditions, and cilia orientation will also differ between cell types in relation to their different strain conditions. Further, we hypothesise that stress deprivation will result in a greater increase in primary cilia length in the more metabolically active IFM cells. Materials and methods Tails were collected from two 200 g female Wistar rats killed for unrelated reasons. From each tail, 4–6 fascicles were dissected and immediately fixed, whilst an additional 4–6 fascicles were dissected and incubated in media for 7 days in a stress deprived condition, prior to fixing. The fixed fascicles were fluorescently labelled using the antibody arl13b, a cilia specific membrane protein. A series of images 0.25μm apart in the z-plane were taken, either at a depth 20–40μm into the FM or through the entire thickness of the IFM. Images were taken using a Leica scanning confocal microscope, using a x63 lens and reconstructed into a 3D z-stack using imageJ. Cilia length and orientation was measured in 3D, using image J (n = 5–30/fascicle). Results Considering the fresh fixed samples, the primary cilia of IFM cells were significantly longer than those of FM cells in one rat tail (IFM = 1.8 ± 0.52 μm; FM = 1.4 ± 0.46 μm; p < 0.001), whilst the second tail showed no significant differences. After stress deprivation, the primary cilia of both cell populations were significantly longer than seen in the fresh tendon. However, the increase in length was significantly larger in the IFM cells, shown in Figure 1. In both rats, FM cell cilia were consistently aligned parallel to the tendon loading axis, as seen previously (Donnelly et al. 2010). However, the cilia of IFM cells were predominantly located in alignment with the boundary between the IFM and FM, but in multiple directions relative to the tendon loading axis. Abstract 89 Figure 1 Mean length of primary cilia in FM and IFM cells from two rat tail tendons. Data compares cilia length in freshly dissected fascicles, and fascicles subjected to 7 days of stress relaxation. Error bars represent...