創傷性腦損傷(Traumatic brain injury, TBI)主要指外力撞擊導致的腦部損傷，嚴重者可能會永久性殘疾或具死亡的風險，損傷患處會累積大量活化的神經膠細胞(Microglia)與星形膠質細胞(Astrocyte)，兩者的活化使活性氧物質(Reactive oxygen species, ROS)過量累積以至於傷口持續發炎與造成神經細胞凋亡，而星狀膠質細胞的活化也會促使疤痕產生阻礙組織修復；故本實驗利用微流道設計出雙層微球(簡稱為ExoABPDA)，外層修飾上聚多巴胺(Polydopamine)透過氫鍵包埋硼烷氨化合物(Ammonia borane)，硼烷氨化合物能快速釋放氫氣捕獲ROS，與聚多巴胺一起達到雙重抗發炎作用，協同緩釋內層脂肪幹細胞(Adipose-derived mesenchymal stem cells, adMSC)分泌之外泌體促進神經細胞再生治療TBI。
聚多巴胺是多巴胺(Dopamine)的聚合物，除了製備方法簡單外，聚多巴胺具有高度的生物相容性常被應用於材料表面改質，又因為結構上具有許多特殊官能基像是鄰苯二酚(Catechol)、醌類(Quinone)，這些結構具有抗氧化的特性使聚多巴胺成為一個良好的去除活性氧物質材料；此外，氫氣能快速地捕抓氫氧自由基(Hydroxyl radical)並轉換成水，氫氧自由基是活性氧物質種類中最具細胞毒性的，故氫氣在創傷性腦損傷的應用是十分有潛力的，但除了機制探討的研究外，少數研究將氫氣與生醫材料結合用於治療TBI。
外泌體(Exosome)則是細胞分泌出的奈米級微小囊泡，囊泡雙層的磷脂結構內部攜帶核酸、蛋白質等多種訊號因子，作為細胞間溝通傳遞與調控生理機制，相較於傳統細胞治療，外泌體能免於植入手術風險也不易產生免疫排斥，惟外泌體治療多透過局部注射或靜脈注射，無法有效的將其滯留於患處治療，為解決此問題，許多研究將外泌體包埋於材料中使其緩釋治療；此外，脂肪幹細胞分泌之外秘體已知具有抑制發炎、促進血管新生、刺激細胞增生與調節細胞基質等功能，近期研究更顯示其內部含有神經生長因子能促進神經再生及功能恢復。
於本實驗結果中發現ExoABPDA除了具有良好的水凝膠可注射性質外，也具備良好的生物相容性，作為細胞支架充填於TBI患處，於最後的動物實驗中，ExoABPDA治療組別相較於其他組別，擁有顯著的抑制發炎與神經修復能力，有望開啟新的TBI治療策略。

Traumatic brain injury (TBI) is a major cause of disability and dead annually and so far, the effective treatment has not developed yet. Excessive reactive oxygen species (ROS) accumulation in the lesion site can cause secondary brain injury and further result in neurodegeneration, hindering the recovery. In this study, aiming to treat TBI, adMSC-derived exosomes incorporated into hydrogen-releasing double-layer microspheres (ExoABPDA) were developed. The surface of microspheres modified with ammonia borane (AB) linked to polydopamine (PDA), synergistically scavenged intracellular ROS. Further, adMSC-derived exosomes sustained released from the inner core were capable of improving neuroregeneration.
As an effective ROS scavenger, polydopamine (PDA) has been widely applied to reduce inflammation. Ammonia borane as a hydrogen storage material can rapidly release hydrogen to capture hydroxyl radical, most toxic oxidant among ROS. Adipose-derived mesenchymal stem cells (adMSC)-derived exosomes are growing attention and have been proved effective for nerve injury repair through IV or in situ injection. However, the difficulty of retaining exosomes at the targeted site remains obstacle to treat nerve injury. Through in situ injection of ExoABPDA sustained releasing adMSC-derived exosomes hydrogen not only showed hydrogel-like injectable property but also acted as scaffold allowing cell adhesion. Together, ExoABPDA significantly reduced inflammation, promoted neural regeneration and consequently, accelerated recovery from TBI.

中文摘要 II
Abstract III
致謝 IV
Table of contents V
List of Figures VII
Chapter 1 Introduction 1
Chapter 2 Literature review and theory 4
2.1 Traumatic brain injury (TBI) 4
2.1.1 Pathological changes in TBI and treatment 4
2.1.2 Microglial activation and inflammation 6
2.1.3 Astrocyte activation and glial scar formation 7
2.1.4 Oxidative stress and ROS scavenging strategy 8
2.2 Hydrogel-based study in TBI 11
2.2.1 Injectable hydrogels and microspheres 12
2.2.2 Anti-inflammatory hydrogel and polydopamine 13
2.2.3 Neuroregeneration-promoting hydrogel 15
2.3 Exosome therapy 16
2.3.1 MSC-derived exosome and nerve regeneration 18
2.4 Gas therapy 20
2.4.1 Hydrogen (H2) therapy 20
2.4.2 Ammonia borane (AB) 21
Chapter 3 Experimental Section 23
3.1 Materials 23
3.2 Method 23
3.2.1 Fabrication of microfluidic system 23
3.2.2 Synthesis of double-layer microspheres (PEGMP, ExoPEG) 24
3.2.3 Surface modification of PEGMP with PDA and AB (PDAMP, ExoPDA, ABPDA, ExoABPDA) 25
3.2.4 TMB and ABTS assay for evaluation of ROS 26
3.2.5 Gas chromatography for hydrogen detection 26
3.2.6 Exosome-release profile 27
3.2.7 Rheological test 27
3.2.8 Cytotoxicity analyzed with PrestoBlue assay and Live&Dead kit 27
3.2.9 Cell adhesion study 28
3.2.10 Exosome uptake 29
3.2.11 In vitro ROS determination 29
3.2.12 In vitro mitochondrial hydroxyl radical determination 30
3.2.13 Neural stem cell harvest 30
3.2.14 Neural differentiation assessment 31
3.2.15 TBI model and brain collection 31
3.2.16 In vivo analysis with immunofluorescence staining 32
3.2.17 Animal behavioral study 32
Chapter 4 Results and Discussion 34
4.1 Synthesis and morphology of double-layer microsphere 34
4.2 Synthesis and characterization of PDA and AB coated microsphere 35
4.3 ROS scavenging capacity 38
4.4 Self-healing property 39
4.5 Exosome sustained releasing profile 39
4.6 In vitro cytotoxicity of microspheres 40
4.7 In vitro ROS scavenging microspheres 41
4.8 Exosome uptake and improve neural differentiation 43
4.9 In vivo microglia and astrocyte activation assessment 46
4.10 In vivo neuroregeneration and angiogenesis 49
4.11 Animal behavioral evaluation 52
4.12 In vivo degradation and toxicity of microspheres 52
Chapter 5 Conclusion & Future perspective 55
Chapter 6 Reference 57

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