膠原蛋白-透明質酸複合電紡奈米纖維包覆多種可階段性釋放之血管生長因子應用於慢性傷口修復

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研究生：	賴奐如
論文名稱：	膠原蛋白-透明質酸複合電紡奈米纖維包覆多種可階段性釋放之血管生長因子應用於慢性傷口修復 Electrospun Collagen-Hyaluronic Acid Composite Nanofibers with Programmable Release of Multiple Angiogenic Growth Factors for Chronic Wound Healing
指導教授：	王子威
口試委員:	洪士杰林頌然
學位類別：	碩士 Master
系所名稱：	工學院 - 材料科學工程學系 Materials Science and Engineering
論文出版年：	2013
畢業學年度：	101
語文別：	英文
論文頁數：	84
中文關鍵詞：	靜電紡織、天然高分子、奈米顆粒、血管生長因子、傷口修復
外文關鍵詞：	electrospinning, natural polymer, nanoparticle, multiple angiogenic growth factors, would healing
相關次數：	點閱：4 下載：0
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對慢性傷口(例: 糖尿病傷口、壓瘡、褥瘡等)而言，其中一個重要的課題便是血管再生。當血液無法運輸養份到末端的傷口，除了傷口復原可能延緩之外，還有可能使患部惡化，組織壞死導致截肢。由於血管新生過程需要多種的蛋白質、生長因子，以及酵素的配合，因此如何適時並持續補充生長因子是加速慢性傷口癒合的重要關鍵。截至目前為止，商業化販售的創傷敷料與人工皮膚產品，鮮少添加促進血管生長因子，只有一項由Johnson & Johnson開發的產品becaplermin (Regranex) 具有此功能，然而該產品只含一種因子。有鑑於此，本研究欲開發同時包覆多種血管生長因子的天然高分子奈米纖維人工皮膚敷料。所製備的基材具有透明質酸與膠原蛋白交錯之奈米纖維結構，構造上與細胞外間質有極高相似度，且奈米孔洞的結構能幫助傷口滲出液排除以及氣體運輸;除此之外，有利於細胞遷移到基材內部，讓血管新生以及組織的再生更加容易。再者，奈米纖維內所包覆的多種生長因子，藉由不同的載體包覆，能夠長時間緩慢並且階段性地釋放，幫助傷口修復。當透明質酸或膠原蛋白奈米纖維降解後，能幫助細胞增生及皮膚再生，所釋放出來多種血管生長因子，與細胞作用後可促使血管新生、穩定並成熟，對於表皮以及真皮也有加速修復的效果。
本研究包含四大部分:第一部分為利用靜電紡織技術製備出奈米複合纖維，找出電紡參數的最佳條件，並將多種血管新生因子分別包覆在奈米纖維與明膠奈米顆粒中，找出最佳載藥條件; 第二部份為支架物化性分析，利用場發射電子顯微鏡及共軛焦顯微鏡觀察奈米纖維之結構與所包覆奈米顆粒的大小，用微拉伸試驗機檢測基材的機械性質，以及測定生長因子的釋放曲線與活性；第三部分在體外建立細胞培養模型，檢驗基材的生物相容性、與生長因子在促進細胞生長速率與生成環狀血管構造的差異；第四部分利用糖尿病老鼠當作實驗對象，觀察所製備的添加多種血管新生因子天然奈米複合纖維人工皮膚是否能進一步促進慢性傷口修復及血管新生。

Table of contents:
摘要    I
Abstract    II
List of Figures    IV
List of Tables    VI
Chapter 1 Introduction
1    Wound healing stages     1
2    Clinical wounds and treatments    3
3    Chronic wounds    4
4    Vascularization    7
4.1    Angiogenesis    7
4.2    Angiogenic growth factors    10
4.2.1    Vascular endothelial cell growth factor (VEGF)     11
4.2.2    Fibroblast growth factor (FGF)     11
4.2.3    Platelet-derived growth factor (PDGF)     12
4.2.4    Other angiogenic growth factors    12
5    Motivation and objective of this study    13
Chapter 2 Literature reviews
1 State of art in skin substitutes and bioactive wound dressings    15
1.1 Commercialized skin substitutes    15
1.2 Commercial products for chronic wounds    17
2 Experimental researches for novel skin constructs    20
2.1 Natural materials based wound dressing    20
2.2 Three-dimensional scaffold for cellular infiltration    21
3 Strategies towards the vascularization inside 3D scaffolds    24
3.1 Angiogenic growth factors delivery    26
Chapter 3 Theoretical basis
1 Electrospinning    28
1.1 Setups    29
1.2 Process    29
1.3 Variables    30
1.3.1 Solution parameter    31
1.3.2 Process parameter    31
2 Drug delivery from electrospun scaffold    33
2.1 Electrospun scaffolds with biomolecule within fibers    33
2.2 Dual release designs based on electrospun fibrous structure    36
Chapter 4 Materials and methods
1 Materials (List)    38
2 Instruments (List)    39
3 Experimental design    39
4 Methods    40
4.1 Synthesis of gelatin nanoparticles    40
4.2 Dual source dual power electrospinning of Col-HA scaffold    40
4.3 Fabrication of electrospun Col-HA-GN scaffold with multiple growth factors    41
5 Characterization of nanofibrous membrane    42
5.1 Scanning electron microscopy (SEM) and confocal microscopy    42
5.2 Dynamic light scattering    43
5.3 Release profile from gelatin nanoparticles    43
5.4 Release profiles of multiple growth factors from composite Col-HA-GN scaffolds    44
5.5. Tensile test    44
6 In vitro studies of composite nanofibrous scaffolds    45
6.1 Scaffold preparing    45
6.2 Cell culture    45
6.3 Cell proliferation    45
6.4 Angiogenesis assay in vitro    46
7 In vivo studies of composite nanofibrous scaffolds    47
7.1 Wound healing on diabetic rats examination    47
7.2 Histological and immunohistochemical staining    48
7.3 Quantitative PCR    48
8 Statistical analysis    49
Chapter 5 Results-----------------------------------------------------------------------------------50
1 Characterization of gelatin nanoparticles    50
2 Characterization of nanofibrous membrane    50
3 Release pattern from gelatin nanoparticles    53
4 In vitro release profiles of multiple growth factors    55
5 Mechanical property of nanofibrous scaffolds    56
6 Cell growth behaviors on nanofibrous scaffolds    57
7 Angiogenesis assay in vitro    57
8 Gross observation of wounds    59
9 Histological staining    61
10 Neovascularization after implantation of nanofibrous membranes    64
11 Gene expression level analysis    67

Chapter 6 Discussions    69
Chapter 7 Conclusion    75
Reference    

                                

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