每一年約有2.8% 周邊神經損傷的病患，部分的病患於神經受損的位置會失去運動及感覺功能，如果不接受進一步的治療可能會導致神經病變或神經瘤的形成。自體神經移植是目前常用於周邊神經修復的方法，但自體神經移植還是有許多缺點，如:捐贈神經位置是有限的、捐贈的神經尺寸不一定能與接受的神經尺寸相符以及需要進行二次手術。由於組織工程的進步，目前有許多的文獻致力於開發人工神經導管，但能夠有效修復神經並且恢復神經功能的研究少之又少。這些研究無法有效修復神經的原因，來自於神經導管中缺乏物理性之順向性引導及空間分佈結構、化學性的生物活性因子誘導刺激及生物性之細胞支持作用。 因此，本研究擬開發新穎性之人工神經導管，使其具多孔道結構，合併順向性纖維及神經滋養因子，期望能更有效的修復周邊神經的損傷。製備的基材選用天然明膠作為神經導管的主要材料，具有生物相容性及降解性，並且結合四種策略(1) 順向性靜電紡絲纖維作為物理性的引導，引導神經細胞軸突生長之方向；(2)神經滋養因子梯度作為生物化學性引導，增加神經軸突生長速率、保護受損軸突及提供合適的修復環境；(3)在基材中直接包覆NGF及奈米顆粒上搭載BDNF，兩種不同包覆方式，使其能夠長時間緩慢並且階段性地釋放，提供神經階段性修復所需因子；(4) 多孔道結構，提供適當的生長空間並模擬神經結構。 研究結果顯示，我們成功採用靜電紡絲技術收集具有順向性的纖維，並由掃描式電子顯微鏡觀察其順向性；以梯度製作器製作神經滋養因子之濃度梯度，搭配分光光度計鑑定其濃度梯度的分佈;在體外材料降解實驗中，觀察到神經導管藉由mTG酵素交聯後能達到長時間之降解，與神經修復週期能相互匹配。在體外細胞實驗中，結果顯示已分化的神經幹細胞培養在順向性纖維上，可觀察到其細胞沿著纖維方向生長；而當細胞培養於具生長因子濃度梯度的環境下，則會影響細胞密度及軸突生長速度。將分化的神經幹細胞及許旺氏細胞共培養在NGF/GN-BDNF濃度梯度支架的環境下，可觀察到BDNF能夠有效的使許旺氏細胞分泌更多髓鞘鹼性蛋白(MBP)，進而促進許旺氏細胞髓鞘化；由實驗中也可以發現NGF及BDNF具有協同效應能刺激許旺氏細胞產生更多的MBP。在體內動物實驗中，使用紐西蘭白兔作為實驗對象，將白兔坐骨神經截斷15毫米，而後將神經導管縫合於神經斷端。在植入後8及24週，觀察到結合神經滋養因子梯度與奈米形貌之多孔道明膠支架(MC/AN/NG scaffold)能夠幫助神經軸突再生，藉由電生理數據觀察到神經再生後能傳遞電訊號，神經導管具有幫助神經功能性回復的功效。同時，神經導管植入後，由於神經對肌肉的再支配，所以能夠有效減少腓腸肌之萎縮。在肌肉組織切片上，觀察到肌肉纖維直徑也有顯著提升。

Peripheral nerve injuries affect a great amount of trauma patients annually. Development of nerve conduits will likely allow scientific and medical communities to improve functional recovery after nerve injuries. However, the efficacy of nerve conduits is often compromised by the lack of cells within the conduit, molecular factors enriched microenvironment and the extracellular matrix (ECM) mimetic spatial arrangement for nerve regeneration. In this study, a multi-channeled scaffold combined with aligned nanofibers and neurotrophic gradient (MC/AN/NG) was developed to attract axon outgrowth and mimic the fascicular architecture of ECM. In mechanical test, the result confirmed that a multi-channeled (MC) scaffold crosslinked with microbial transglutaminase (mTG) was stronger as demonstrated by the higher ultimate tensile strength and Young's modulus compared to untreated one. Nerve growth factor (NGF) release profile exhibited a discontinuous concentration gradient from 6.6 ng/mL to 107.2 ng/mL. In in vitro study, differentiated neural stem cells (dNSCs) could extend their neurites along the aligned nanofibrous structure. The cell density increased in higher NGF concentration region of gradient membrane. BDNF promoted myelination more significantly than the non-treated and NGF-treated groups, evidenced by the immunostaining. In in vivo study, the MC/AN/NG scaffold was used for bridging a 15 mm gap in a rabbit sciatic nerve transection model. The MC/AN/NG scaffold achieved functional recovery comparable to autograft as evidenced by significantly improved nerve function and fascicular morphology. From the above result findings, we suggests that the MC/AN/NG scaffold could be a promising nerve guidance conduit for peripheral nerve regeneration.

摘要 I
Abstract III
Table of content I
List of figures V
List of tables VII
Chapter 1 Introduction 1
1.1 Peripheral nerve injury (PNI) 1
1.2 Treatment of peripheral nerve injuries 3
1.3 Materials for nerve guidance conduit 5
1.4 Gelatin 7
1.5 Growth factor 8
1.5.1 Nerve growth factor (NGF) 9
1.5.2 Brain derived neurotrophic factor (BDNF) 10
1.6 Motivation and purpose 11
Chapter 2 Literature review 13
2.1 Commercialized nerve conduits 13
2.2 Development of nerve guidance conduit 14
2.3 Experimental researches for nerve conduit 15
2.3.1 Guided nerve regeneration: the use of structural guidance cues 15
2.3.2 Guided nerve regeneration: the use of electrospun fibers as topographical cue 17
2.3.3 Guided nerve regeneration: the use of biomolecules gradient environment 19
2.3.4 Multiple growths factor delivery for axonal regeneration 20
Chapter 3 Theoretical basis 22
3.1 Electrospinning 22
3.1.1 The electrospinning setup 23
3.1.2 Production of aligned electrospun fibers 24
3.1.3 The electrospinning process 26
3.1.4 Electrospinning parameters 27
3.2 Crosslinking of gelatin 30
3.3 Chemotaxis and haptotaxis in directing neurite outgrowth 32
3.4 Incorporation of neurotrophic factors into nerve guidance conduit 33
3.4.1 Dual Growth factor delivery designs 34
Chapter 4 Materials and methods 37
4.1 Experimental design 39
4.2 Methods 40
4.2.1 Electrospinning of aligned nanofibrous membrane 40
4.2.2 Fabrication of enzymatic crosslinked multi-channeled scaffold 41
4.2.3 Fabrication of rhodamine B (Rhod) gradient scaffold 42
4.2.4 Synthesis of gelatin nanoparticles (GNs) 43
4.2.5 Fabrication of neurotrophic gradient scaffold 43
4.2.6 Fabrication of multi-channeled scaffold combined with aligned nanofibers and neurotrophic gradient (MC/AN/NG scaffold) 44
4.3 Characterization of various scaffold 45
4.3.1 Scanning electron microscopy (SEM) on the morphology of electrospun nanofibers and observation under dissecting microscope for the structure of multi-channeled gelatin scaffolds 45
4.3.2 Determination of crosslinking degree (the number of non-crosslinked ε-amino groups) in gelatin by trinitrobenzene sulfonic acid (TNBS) assay 45
4.3.3 Mechanical property of gelatin scaffolds 46
4.3.4 Degradation rate of gelatin scaffolds 47
4.3.5 Quantification of neurotrophic gradient in gelatin scaffolds 47
4.3.6 Dynamic light scattering for nanoparticle size analysis 48
4.3.7 Release profile of neurotrophic gradient in gelatin scaffolds 48
4.4 In vitro study 48
4.4.1 Scaffold preparation 48
4.4.2 Cell culture 49
4.4.3 Cell morphology of differentiated neural stem cells (dNSCs) on aligned nanofibers by ICC 49
4.4.4 Differentiated neural stem cells (dNSCs) cultured on neurotrophic gradient scaffold 50
4.4.5 Effect of the neurotrophic gradient on SCs myelination 50
4.5 In vivo study of composite scaffolds 51
4.5.1 Animals and experimental groups 51
4.5.2 Surgical procedure 51
4.5.3 Histological analyses on regenerated sciatic nerve 51
4.5.4 Electrophysiological recovery of the regenerated sciatic nerve 52
4.5.5 Histological analyses and relative gastrocnemius muscle weight 52
Chapter 5 Results 54
5.1 Characterization of aligned nanofibrous membrane 54
5.2 Characterization of multi-channeled scaffold with aligned nanofibers (MC/AN scaffold) 55
5.3 Crosslinking degree of gelatin scaffolds 56
5.4 Mechanical property of gelatin scaffolds 57
5.5 Degradation of gelatin scaffolds 58
5.6 Characterization of concentration gradient on scaffold 59
5.7 Characterization of gelatin nanoparticles 61
5.8 Release profile of neurotrophic gradient 61
5.9 Neurite outgrowth on aligned nanofibers 63
5.10 dNSCs response to neurotrophic gradient 64
5.11 Effect of the neurotrophic gradient on SCs myelination 65
5.12 In vivo rabbit animal study group and successful rate of nerve regeneration 67
5.13 Histological analysis for regenerated sciatic nerve 68
5.14 Electrophysiological recovery of the regenerated sciatic nerve 73
5.15 Histological analyses and muscle weight for gastrocnemius muscle 75
Chapter 6 Discussion 80
6.1 The effect of rotational speed on fiber orientation 80
6.2 Characterization of gelatin scaffolds 80
6.3 Cumulative release of neurotrophic factors 82
6.4 dNSCs induction and growth on the orientated fiber matrice 83
6.5 dNSCs behaviors in response to neurotrophic gradient 83
6.6 Effect of the neurotrophic gradient on SC myelination 84
6.7 Histological and electrophysiological recovery of the regenerated sciatic nerve 84
6.8 Prevention of muscle atrophy and maintenance of muscle fibers 85
Chapter 7 Conclusion 86
Reference 87

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