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Title: An investigation of tendon fibroblast responses to tensile loading in vitro
Author: Zaman, Tunku Kamarul
ISNI:       0000 0004 2736 9617
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2011
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Tears in the rotator cuff tendon are a common disabling and painful condition affecting the elderly population. Despite being well recognized, the aetiology that underpins this condition has not been explained adequately. Tendons focus muscle power into bone movement. In simple tendons, both the collagen and tendon fibroblasts (tenocytes) are aligned in the direction of force. The rotator cuff is a multilayered, multidirectional conjoined tendon, which support the wide range of movement at the shoulder. Aberrant mechanical loading, which may be the result of inappropriate rate or direction of loading, has been suggested to play a role in the pathogenesis of rotator cuff tears. The translation of mechanical load into biochemical signal at the cellular level is known as mechanotransduction. In this thesis, the mechanobiology of tenocytes was investigated through experiments which examined the responses of cultured human primary tenocytes subjected to either no loading (static) or different rates of tensile loading for different durations. In many experiments the cells were seeded with no constraint on their alignment to the direction of loading, or their ability to change alignment. In others, alignment was determined by micro grooves. The data has improved understanding of the relationship between loading and tenocyte numbers and alignment, especially the proliferative and constructive outcome of loading in the direction of cell alignment in contrast to when cells are aligned perpendicular to the direction of loading. Methods: This study is composed of 4 inter-related series of experiments. Study 1: Primary human tenocytes cultured on collagen coated silicone culture flasks were subjected to OHz (static), 0.5Hz, 1Hz, and 2Hz at 8% strain loading regimes. Changes in cell orientation and numbers were measured using image analysis and haemocytometer counts respectively at 0, 6, 24 and 48 hours of strain. Study2: Cell culture medium collected from a subset of the experiments in study 1 were tested for matrix metalloproteinase 1 (MMP-1) and fibronectin levels at each time points. Study 3: The role of calcium supply modification in these cultures was examined by introducing BAPTA, EGTA or Thapsigargin into cell culture medium at the start of the loading period. These cultures were subjected to 1Hz cyclic loading at 8% tensile strain. Cell orientation and numbers were measured using image analysis at 0, 6, 24 and 48 hours. Study 4: Before loading, cells were seeded on microgroove-patterned silicone flask surfaces aligned in a direction parallel or perpendicular to the direction of tensile loading. The loading regimen employed was the same as that used in study 3. Changes in cell numbers and morphology were determined by image analysis. Results: Proliferation: Cyclic tensile loading at 8% strain and 1Hz produced the highest proliferation rate (128% increase). Loss of cell numbers were observed at 6 and 48 hours for cultures subjected to 0.5Hz (52% decrease) and 2Hz (98% decrease) loading respectively. At the end of 48 hours, cell cultures subjected to 1 Hz experienced significantly higher proliferation than static (77% increase), 0.5Hz (61 % increase) and 2Hz (21 % increase) cultures. Orientation: Significant change in cell re-orientation towards perpendicular direction to the direction of loading was only observed in cells subjected to 1Hz cyclic loading with a mean increase of 4.16°. Cells in static, 0.5 Hz and 2 Hz cultures experienced an increase in mean perpendicular arrangement of 0.23°, 1.52° and 1.42° respectively. Matrix components: The increase in total fibronectin and MMP-1 was highest at this same 1Hz loading regimen (432% and 234% respectively). In comparison, the fibronectin increase in static, 0.5Hz and 2Hz cultures were 202%, 207% and 189% whilst for the MMP-1 increase in static, 0.5Hz and 2Hz cultures were 115%, 112% and 178% respectively. At 48 hours, the MMP-1 to fibronectin ratio was approximately 0.6, indicating a net increase in fibronectin, which is a matrix synthesis protein as opposed to MMP-1, which is a degradation enzyme. In all other cultures, this ratio was only observed to be between 0.9 to 1. Calcium channels manipulation: In static cultures depletion of calcium supplies extracellularly (EGT A), intracellularly (BAP TA) or through the calcium stores (Thapsigargin), results in loss of cell proliferation. Tensile loading at 1Hz protected cells from the inhibitory effect of EGTA and Thapsigargin as by 300% and 50% higher concentrations respectively were needed to halt both cell proliferation and reorientation. This did not apply to BAPTA treated cultures, in which the concentration effective in static cultures was also effective in 1Hz loaded cultures. This suggests that loading may increase calcium influx into the cytoplasm from extracellular sources and/or intracellular stores. Intracellular chelation abolished the consequences of this loading related calcium signal. Microgrooves: The highest proliferation rate was observed in cells trapped in a parallel alignment to the direction loading (82% increase). Cells that were trapped in a perpendicular alignment demonstrated an even lower proliferation rate than unconstrained static cells (22% versus 42% increase). Conclusion: These studies demonstrate that tenocyte proliferation, reorientation and matrix remodelling proteins production are modulated both by the rate of cyclic tensile loading; with the most significant increases observed in cultures subjected to 1Hz loading, and the direction of the loading relative to the orientation of the cells. Depleting calcium supplies results in reduced proliferative and reorientation responses. This data indicates that the alignment of tenocytes in the direction of loading within tendons serves to optimise the constructive outcome of tendon function. The data from this thesis suggests that in a complex tendon such as the rotator cuff, where the directions of loading and of collagen fibres are multiple, multiple directional usage will be necessary to keep the tendon in good condition.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available