Cellular Responses of Corneal Fibroblasts to Cyclic Stretching Loads

機械應力是維持結締組織的重要因子之一。在許多組織器官中，機械應力對於維持組織正常生理作用抑或是病理的產生都有重要的影響。眼角膜基質組織是一具有特化排列膠原蛋白結構的透明組織，其正常的結構代謝循環是個相當緩慢的過程。任何病變導致此膠原蛋白結構的平衡破壞，將會導致角膜組織失去其透明度，而導致病患的視力受損。在眼部，眼壓被認為對於正常眼球的生長是有決定的影響性。而在眼角膜組織中，眼角膜纖維細胞所產生的應力可決定周邊組織的排列。而臨床中，揉眼的機械外力是被認為導致圓錐角膜疾病惡化的重要因素之一。所以在本項研究中，我們試圖探討機械應力對於眼角膜的影響。 在這系列的研究中，我們運用非侵入性多光子影像系統來分析圓錐角膜及運用交聯處理後的眼角膜組織的結構變化，尤其是基質的膠原蛋白排列的變化。從此項研究，我們不只證實了多光子影像系統在探討眼角膜結構變化的可行性，我們發現到在圓錐角膜中，膠原蛋白結構排列的變化。膠原蛋白纖維排列變成向心的排列方向。這結果提供了一個重要訊息讓我們了解到機械應力對角膜組織是可能產生影響的。所以我們進一步開始研究機械應力對角膜組織的影響。我們從探討週期性應力對於角膜纖維細胞的影響開始。我們發現雖然眼角膜纖維細胞對於週期性應力後並無顯著的形變及排列上的改變， MMP-2及微量的MMP-9確實有增加表現。MMP-2的基因表現，蛋白質分泌量，及酵素活性都有增加，而其訊號路徑是MAPK路徑調控的。這結果證實了角膜纖維細胞受到週期性機械應力後會對周邊基質組織進行重建作用。這結果也同時提供一個可能的佐證來連結揉眼外力與圓錐角膜惡化的關聯。而在中，我們證實了多光子系統可用於分析運用在組織工程中的高分子材料結構。這項結果是對於在包括眼角膜的組織工程研究中，探討細胞與高分子架構，及周邊基質組織的交互作用將可提供重要的訊息。未來我們會延續探討機械應力對角膜組織的影響，將運用立體細胞培養系統，以模仿角膜組織的反應。而同時也將延續運用多光子影像系統進行分析。結果預計將可提供資訊來了解機械應力於圓錐角膜致病機轉，也將可提供角膜組織工程相關研究之必要資訊。Mechanical signaling is one of the essential factors contributing to the homeostasis of connective tissues. It is important both in the maintenance of normal physiology as well as the development of pathology within many tissues. The normal turnover of mesenchymal extracellular matrix (ECM) in cornea is at a very slow balanced way in life and the integrity of highly organized collagen network that keeps cornea transparent is maintained in life. Any pathology disturbing the balanced homeostasis of the collagen network can lead to the structural perturbation and loss of corneal transparency, and therefore compromises the vision. In eyes, intraocular pressure has been proven to be essential for the normal development of eyeballs. In cornea, physical forces induced by corneal fibroblasts organize the production of unique extracellular matrices (ECM). In addition, eye rubbing, one of the commonly encountered extrinsic mechanical stimuli in daily scenario, have been shown to be strongly correlated to the development and progression of keratoconus clinically. Therefore in this work, we attempt to investigate the potential role of mechanical cues in cornea. In this serial works, we firstly demonstrated the morphological changes of keratoconus, as well as the alterations in corneal collagenous stroma following ribroflavin/UVA crosslinking treatment, using a noninvasive MPM imaging system. We not only demonstrated the feasibility of MPM imaging system for disclosing cellular and structural information within cornea noninvasively, we also found that the architecture of stroma was altered in keratoconus, with collagen fibers directed toward the apex of cone. The result provided a clue for the role of mechanical cues in cornea. We then investigated the cellular responses of corneal fibroblasts to cyclic stretching, as the entry for deciphering the effect of mechanical cues in cornea. We found that although the morphology and alignment of corneal fibroblasts were not affected, MMP-2 and weakly MMP-9 expression were enhanced in corneal fibroblasts. The levels of MMP-2 in gene expression, protein secretion, and enzyme activity were all enhanced, and regulated through MAPK pathway. The results implicated a process of ECM modulation by corneal fibroblasts in response to cyclic stretch developed, which may therefore provide a mechanism for the correlation between eye rubbing and keratoconus. And in the supplementary works, we demonstrated the capability of noninvasive MPM system for disclosing structural information of polymeric scaffolds applied in tissue engineering, which may potentially be of valuable in studying cell-matrix interaction in the field of tissue engineering, including cornea. Further works will be done to investigate thorough ECM modulation processes within 3D cell-seeded corneal constructs, in responses to mechanical cues, using MPM system as an effective noninvasive monitoring system. And the results may not only provide information about the role of mechanical cues in the pathogenesis of keratoconus, but also valuable information in the field of corneal tissue engineering.口試委員會審定書 I 誌謝 II 中文摘要 III-IV 英文摘要 V-VII Chapter 1 Morphological Changes in Keratoconus 1-35 1.1 Introduction of Keratoconus 1-3 1.1.1 Etiology of keratoconus 1 1.1.2 Treatment of keratoconus 2-3 1.2 Non-invasive multiphoton microscopic (MPM) imaging of Keratoconus 3-8 1.2.1 The application of MPM in the field of biomedicine 3-4 1.2.2 The application of MPM in cornea 4-5 1.2.3 The application of MPM in keratoconus 6-8 1.3 MPM imaging of UVA/riboflavin crosslinked cornea 9-35 1.3.1 Introduction 9-12 1.3.1.1 UVA/Riboflavin crosslinking treatment of cornea 9 1.3.1.2 Clinical and laboratory methods for evaluating the efficacy of crosslinking treatment 10-12 1.3.1.3 Previous work of the application of MPM imaging in evaluating the UVA/riboflavin crosslinking effect in corneas 12 1.3.2 Aim of this work 13 1.3.3 Materials and Methods 13 1.3.3.1 Collagen crosslinking treatments of porcine cornea 13-14 1.3.3.2 SHG microscopic imaging and histological comparison 14-15 1.3.3.3 2D-Fast Fourier Transform (2D- FFT) Analysis for SHG Imaging 15-20 1.3.4 Results 20-30 1.3.4.1 Qualitative SHG analysis of ribroflavin/UVA crosslinked cornea 20-24 1.3.4.2 Quantitative analysis of ribroflavin/UVA crosslinked cornea using FT-SHG imaging technique 24-30 1.3.5 Discussion 30-32 1.3.6 Limitations of this work 34-35 1.3.7 Conclusion 35 Chapter 2 The Cellular Responses of Corneal fibroblasts to Cyclic Stretching Loads 36-58 2.1 Introduction 36-39 2.1.1 The importance of homeostasis of corneal stroma 35 2.1.2 The importance of mechanical signaling in connective tissue 36-37 2.1.3 The effects of cyclic stretching in cell behaviors 37-38 2.1.4 The effect of mechanical forces in scleral fibroblasts 38 2.1.5 The potential effect of mechanical loads in cornea 39 2.2 Aim of this work 39 2.3 Materials and Methods 39-46 2.3.1 Cell culture 39-41 2.3.2 Application of cyclic mechanical stretch 41 2.3.3 Treatment of mitogen-activated protein kinases (MAPKs) inhibitors 41-42 2.3.4 Immunofluorescence assay 42 2.3.5 Metabolic assay 42-43 2.3.6 Cell cycle assay 43 2.3.7 MMP activities determined by zymography 43-44 2.3.8 Western blot analysis 44-45 2.3.9 Gene Expression 45 2.3.10 Data analysis 46 2.4 Results 46-53 2.4.1 Effects of cyclic stretching loads on cell morphology of corneal fibroblasts 46-48 2.4.2 Effects of cyclic stretching loads on the metabolic and proliferative activities in corneal fibroblasts 48-49 2.4.3 Effects of cyclic stretching loads on protein expression of corneal fibroblasts 49-51 2.4.4 The role of MAPK pathway in mechanotransduction of corneal fibroblasts 52 2.4.5 The role of MAPK pathway in regulating cyclic stretching-induced MMP-2 expression in corneal fibroblasts 52 2.5 Discussion 54-57 2.5.1 Eye rubbing and keratoconus 54 2.5.2 The effect of cyclic stretching in eyes 54-55 2.5.3 The signaling pathway of mechanotransduction in corneal fibroblasts 56-57 2.6 Conclusion 58 Chapter 3 Summary and Future Works 59-60 Supplementary Works: Multiphoton Microscopy (MPM) For Imaging Tissue Engineered Scaffolds…………………….………………………………………61-94 S-1 Introduction 62 S1-1 The application of MPM in the field of tissue engineering 62 S1-2 The application of polymeric blends in the field of tissue engineering 62-63 S1-3 Other imaging technique for investigating tissue engineered scaffolds 63-64 S-2 Aim of this work 64-65 S-3 Methodology 65-68 S3-1 Part I experiment – imaging commercial available polymeric scaffolds 65-66 S3-2 Part II for imaging phase-separated polymeric blended scaffolds 66-68 S3-3 Part III experiment – imaging miscible polymeric blends 68 S-4 Materials 68-70 S4-1 Part I – commercial available scaffolds 68-69 S4-2 Part II – preparing phase-separated nylon/chitosan blended membrane 69-70 S4-3 Part III – preparing miscible collagen/chitosan blended sponges 70 S-5 Results 71-88 S5-1 Part I – MPM imaging of commercially available scaffolds 71-72 S5-2 Part II – MPM imaging of phase-separated nylon/chitosan blended membranes 73-82 S5-2-1 SEM photography 73-75 S5-2-2 MPM and reflected confocal photography 75-82 S5-2-2-1 pure Nylon and Pure Chitosan Membrane 75-77 S5-2-2-2 Nylon/Chitosan Blended Membrane 78-82 S5-3 Part III – MPM imaging of miscible collagen/chitosan blended sponges 83-88 S-6 Discussion 88-94 S-7 Conclusion 94 References 95-10