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Ivtigation of the Effects of Mechanical Strain in Human Tenocytes

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Ivtigation of the Effects of Mechanical Strain in Human Tenocytes Investigation of the effects of Mechanical Strain in Human Tenocytes Eleanor Rachel Jones In partial fulfilment of the requirements for the Degree of Doctor of Philosophy University of East Anglia, Biological Sciences September 2012 This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and that use of any information derived there from must be in accordance with current UK Copyright Law. In addition, any quotation or extract must be included full attribution. Abstract Tendinopathies are a range of diseases characterised by pain and insidious degeneration. Although poorly understood, onset is often associated with physical activity. Metalloproteinases are regulated differentially in tendinopathy causing disruptions in extracellular matrix (ECM) homeostasis. An increase in the anti-inflammatory cytokine TGFβ has also been documented. This project aims to investigate the effect of cyclic tensile strain loading and TGFβ stimulation on protease and ECM protein expression by human tenocytes and begin to characterise the pathway of mechanotransduction. Human tenocytes were seeded at 1.5x106 cells/ml into collagen gels (rat tail type I, 1mg/ml) and stretched using a sinusoidal waveform of 0-5% at 1Hz using the Flexcell FX4000T™ system. Cultures were treated with or without 1ng/ml TGFβ1 or inhibitors of TGFβRI, metalloproteinases, RGD, Mannose-6-phosphate, integrin β1 and a thrombospondin as appropriate. qRT-PCR and a cell based luciferase assay were used to assess RNA and TGFβ activity respectively. The prolonged application of 5% cyclic mechanical strain in a 3D culture system induced an anabolic response in protease and matrix genes. In most genes changes in gene expression with loading was mirrored with TGFβ stimulation. We also demonstrated that the inhibition of the TGFβRI abrogated the strain induced changes in mRNA. TGFβ activity was increased with 48 hours mechanical strain although there was no increase in mRNA or total TGFβ. This indicates that TGFβ activation plays an important role in the mechanoregulation of gene expression. Inhibition of potential mechanisms of TGFβ activation including; serine protease or metalloproteinase activity, integrin or thrombospondin interaction with latent TGFβ showed no effect upon TGFβ activation or gene regulation with strain. Therefore strain mediated TGFβ activation may occur via a novel mechanism. By understanding mechanotransduction, we may be able to determine whether dysregulation of this system is involved in the development of tendinopathy. Table of Contents Abstract .................................................................................................................................. 2 List of Tables.......................................................................................................................... 7 Table of Figures ..................................................................................................................... 9 Acknowledgements .............................................................................................................. 12 CHAPTER 1: Introduction ...................................................................................................... 13 1.1. Tendon Structure ....................................................................................................... 14 1.1.1. Collagens of tendon............................................................................................ 16 1.1.2. Proteoglycans of tendon ..................................................................................... 20 1.1.3. Glycoproteins of tendon ..................................................................................... 24 1.2. Tendon cell population .............................................................................................. 28 1.2.1. Tendon cell markers ........................................................................................... 28 1.3. Vascularisation .......................................................................................................... 30 1.4. Tendon Function ....................................................................................................... 31 1.5. Tendinopathy............................................................................................................. 34 1.5.1. Collagen Expression with Tendinopathy ........................................................... 35 1.5.2. GAG and Tendinopathy ..................................................................................... 35 1.5.3. Proteoglycan and Glycoprotein regulation with Tendinopathy ......................... 35 1.5.4. Inflammatory cytokine regulation with Tendinopathy ...................................... 36 1.5.5. Tendon ‘marker’ regulation with Tendinopathy ................................................ 37 1.5.6. Age and tendinopathy ........................................................................................ 37 1.6. Extracellular Matrix Degradation In Tendinopathy .................................................. 39 1.6.1. Matrix Metalloproteinases (MMP) .................................................................... 40 1.6.2. Tissue Inhibitors of Metalloproteinases ............................................................. 44 1.6.3. Disintegrin and Metalloproteinase domain (ADAM) ........................................ 45 1.6.4. A Disintegrin and Metalloproteinase domain with Thrombospondin motifs (ADAMTS) .................................................................................................................. 47 1.7. Mechanical Strain...................................................................................................... 50 1.7.1. MMP1mRNA regulation with mechanical load ................................................ 51 1.7.2. MMP2 mRNA regulation with mechanical load ............................................... 55 1.7.3MMP3 mRNA regulation with mechanical load ................................................. 57 1.7.4. MMP9 mRNA regulation with mechanical load ............................................... 59 1.7.5. MMP13 mRNA expression with mechanical load............................................. 61 Table 1.10. MMP13 Regulation with mechanical strain ............................................. 62 1.7.6. Rat Collagenase .................................................................................................. 63 1.7.7. Other MMPs regulated with mechanical load .................................................... 65 1.7.8. ADAMTS and TIMP regulation with mechanical load ..................................... 65 1.7.9. Matrix protein regulation with mechanical strain .............................................. 66 1.7.10. Cytokine regulation in response to Mechanical load ....................................... 66 1.7.11. Mechanical regulation of ‘markers’ of tendon ................................................. 67 1.7.12. Mechanical load and tendinopathy................................................................... 68 1.8. Mechanosensation and transduction ......................................................................... 70 1.8.1. Cytoskeletal detection of mechanical strain ....................................................... 72 1.8.2. Stress deprivation ............................................................................................... 75 1.8.3. Growth factors .................................................................................................... 76 1.8.4. Gap Junction communication ............................................................................. 77 1.8.5. Calcium signalling ............................................................................................. 78 1.9. Transforming Growth Factor beta ............................................................................. 83 1.9.1. TGFβ Synthesis and secretion ............................................................................ 84 1.9.2. Activation of latent TGFβ .................................................................................. 86 1.9.3. Disruption of TGFβ regulation .......................................................................... 97 1.9.4. TGFβ Signalling ................................................................................................. 98 1.9.5. TGFβ and mechanical load .............................................................................. 100 1.9.6. TGFβ and tendinopathy ................................................................................... 101 CHAPTER 2; Materials and Methods ................................................................................... 102 2.1. Materials .................................................................................................................. 102 2.2 Cell culture Methods ................................................................................................ 106 2.2.1 Cell culture ........................................................................................................ 106 2.2.2 Tissue Train cell culture protocol ..................................................................... 106 2.2.3 Cell viability testing – Calcein AM and Ethidium Homodimer staining .......... 109 2.2.4 Cell viability testing – tenocyte extraction by collagenase digestion ............... 109 2.2.5 siRNA knockdown ...........................................................................................
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