可注射型/連續孔洞之異電微凝膠球支架系統於周邊神經損傷與腦創傷之應用

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研究生：	許如秀 Hsu, Ru-Siou
論文名稱：	可注射型/連續孔洞之異電微凝膠球支架系統於周邊神經損傷與腦創傷之應用 Injectable Isoelectric MicroBead-Gels with Swiftly Controllable Continuous Pores for Peripheral Nerve Injury and Trauma Brain Injury
指導教授：	胡尚秀 Hu, Shang- Hsiu
口試委員:	陳三元 Chen, San-yuan 葉秩光 Yeh, Chih-Kuang 張建文 Chang, Chien-Wen 李奕棋 ‪Lee, I Chi 陳冠宇 Chen, Guan-Yu
學位類別：	博士 Doctor
系所名稱：	原子科學院 - 生醫工程與環境科學系 Department of Biomedical Engineering and Environmental Sciences
論文出版年：	2021
畢業學年度：	109
語文別：	英文
論文頁數：	128
中文關鍵詞：	微凝膠、可注射性、可適應性、多孔性水凝膠、周邊神經損傷、腦創傷
外文關鍵詞：	microgel, injectable, adaptable, microporous hydrogel, peripheral nerve injury, trauma brain injury
相關次數：	點閱：2 下載：0
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再生醫學中的可注射水凝膠可以潛在地模仿分層的自然生物組織，並通過微創植入程序來填補形狀複雜的缺陷，在組織工程更提供了良好應用的優勢，其可同時封裝細胞和生長因子，並將細胞與成長因子與特定部位，進行組織再生。然而，許多臨床實驗發現常發現，傳統水凝膠之微孔結構不足及不可控的藥物釋放過程。導致細胞穿透率低與氧氣和營養交換供應不足，損害了再生細胞的存活率。然而，利用傳統發泡劑概念於聚合物本體產生孔隙率的具有一定可行性，但往往無法兼顧細胞封裝相容性，但大多數這些孔洞型支架不具有注射性，且表現出較差的相連孔。我們最近開發的一種可流動的細胞支架，即一種可適應的微孔微凝膠，通過基於液滴的微流體技術以進行自下而上的製造，不僅克服傳統水膠之無孔洞性或無法控制孔洞結構組成之缺點，同時為可商品化之新型態之可注射式微孔水凝膠。本論文研究依照生物醫學應用區分為二大部份: (一) 周邊神經修復 (二) 腦瘤術後與腦創傷之修復。
在第一部分中，我們提供一種未來治療方式，解決周圍神經損傷之關鍵問題，該損傷具有一定的軸突受損和較大的間隙缺損，導致缺損神經的再生速度緩慢，並延遲了功能恢復。在這項研究中，我們提出了一種適應性微孔水凝膠（Adaptable Microporous Hydrogel, AMH），基於獨特類型的微型構建基塊來加速和引導周圍神經，該微型構建基塊自發形成相互連接的毛孔，組裝神經生長因子之濃度梯度，調整剛度並控制孔隙率。通過微流體製造，通過自下而上的合成分別使用光可交聯的甲基丙烯酸明膠（GelMA）和殼聚醣低聚物-甲基丙烯酸酯（ChitoMA）作為帶負電荷和帶正電荷的結構單元來構建結構單元。 GelMA是由改質膠原蛋白成分組成的光交聯水凝膠。得益於變性膠原蛋白，GelMA保留了天然的細胞結合基序，例如細胞粘附肽（精氨酰-糖基葡萄糖酸，RGD）以及基質金屬蛋白酶肽（MMP）序列，可實現細胞控制的材料降解和隨後的吸收。 AMH透過剪切稀疏力和強大的電荷吸引特性進行再組裝，從而有助於形成穩定的3D多孔支架。具有合適的微孔的互連的可注射多孔支架可用於迅速的細胞遷移，提供機械支持並運輸生物分子線索來控制細胞的粘附和生長。同時構建細胞可穿透孔徑的適應性微孔支架，與神經管中NGF的傳播梯度相結合，可在4天內引導神經軸突向外生長4.7 mm，並在30天內使軸突排列良好並恢復功能後手術。快速物理性交聯鍵結，精確微孔控制和可調的分子濃度梯度，所形成之可注射AMH具此協同效應，並有效地為組織再生應用創造了新的視野。
在第二部分中，我們開發了一種基於電磁波的金奈米紗球（Gold Yarn Balls, GYBs）的適應性導電介孔水凝膠（Conductive Microporous Hydrogel, CMH），有獨特的微結構，可以自發形成100％互連孔隙並應用於腦部外傷。外傷性腦損傷（Trauma Brain Injury, TBI）在造成發展中國家的長期殘疾方面超過了許多疾病，因此在全世界構成了沉重的負擔。 TBI後，迅速的炎症和神經膠質疤痕的形成會阻礙腦組織的修復，導致自發加重組織損傷，而造成血管生成和腦功能恢復困難。透過微創植入，CMH能夠填補複雜形狀的缺損，調節TBI環境並增強細胞滲透以減輕炎症。在高頻磁場（High Frequency Magnetic Field, HFMF）下，GYBs上產生的渦電流，促進神經幹細胞（Neural Stem Cells, NSCs）的細胞分化和神經突向外生長，從而導致神經元細胞從神經球遷移出來的數量增加了2.5倍。在動物實驗中，利用可注射型CMH填充在梗死周圍的TBI區，經磁電刺激後2週後，顯著增強了成熟的腦源性神經營養因子分泌增加（mature Brain-Derived Neurotrophic Factor, mBDNF）180％，創傷區血管生成增加250％；這些作用使神經元存活和運動功能恢復相較於控制組則提高了50％。並藉由血液氧合水平依賴性（Blood Oxygenation Level-Dependent, BOLD）功能性神經影像學檢查顯示皮層中腦功能成功恢復。這使該研究引起了材料界的極大興趣。

Despite the significant advances in designing injectable bulk hydrogels, the inability to control the pore interconnectivity and material degradation with tissue regrowth has tremendously limited the applicability of stiff, flowable hydrogels for 3D cellular engineering. To overcome this persistent challenge. Our recent development of a flowable cell-scaffold, that is, an adaptable microporous microgels, provides a new approach to make the balance more feasible. The microgel building blocks are designed to enable LEGO-type bottom-up assembly of complex structures. The topic of this research is divided into two parts according to the application of biomedicine. (1) Peripheral nerve repair (2) Postoperative applications related to brain trauma injury.
In the first part, we addressed critical issue and future treatments of peripheral nerve injury with a certain axonal loss and sizable gap defect exhibits a slowly regenerative rate of the defected nerve and delays functional recovery. In this study, we proposed an adaptable microporous hydrogel (AMH) to accelerate and direct peripheral nerves based on a unique type of microsized building block that spontaneously forms interconnected pores, propagates the gradients of neuron growth factors, tailors the stiffness, and controls the pore sizes in nerve conduits. Through microfluidic fabrication, building blocks are constructed by a bottom-up synthesis employing photocrosslinkable gelatin methacrylate (GelMA) and chitosan oligmer-methacrylate (ChitoMA) as negatively and positively charged building blocks, respectively. GelMA is a photo-crosslinking hydrogel composed of modified collagen components. With the benefit of denatured collagen, GelMA retains natural cellbinding motifs, such as cell adhesive peptide (arginyl-glycylaspartic acid, RGD) as well as matrix metalloprotease peptide (MMP) sequences that allowed cell controlled material degradation and subsequent resorption. This AMH is reshapable and reassembles through shear-thinning force and strong cohesive properties, facilitating the formation of a stable 3D porous scaffold. Such an interconnected injectable porous scaffold with suitable micropores for prompt cell migration as well as offer mechanical support and transports biomolecular cues to manage cell adhesion and growth. The adaptable microporous scaffold constructing cell-penetrable pore sizes in real time, integrated with a propagating gradient of a NGF in a nerve tube, directs axon outgrowth of up to 4.7 mm in 4 days in vivo and well aligned axons with functional recovery within 30 days postsurgery. Such synergistic effects of injectable AMHs of rapid bonding, precise pore control, and tunable molecular cue gradient formation effectively create a new horizon for applications in tissue regeneration.
In the second part, an adaptable conductive mesoporous hydrogel (CMH) based on electromagnetized gold nanoyarn balls (GYBs) coated with a unique microsized building block that can spontaneously form 100% interconnected pores in traumatic brain lesions was developed. Traumatic brain injury (TBI) surpasses many diseases in causing long-term disability in developing countries and thus constitutes a substantial burden worldwide. Following TBI, prompt inflammation and glial scar formation hinder the repair of brain tissue, and the frequent difficulty of stimulating angiogenesis and recovery of brain function usually causes the spontaneous exacerbation of tissue damage. By minimally invasive implantation, the CMH was able to fill defects with complex shapes, regulate the TBI environment and enhance cell penetration to reduce inflammation. Under a high-frequency magnetic field (HFMF), the eddy currents generated on the GYBs promote cell differentiation and neurite outgrowth of neural stem cells (NSCs) ex vivo, resulting in a 2.5-fold increase in neuronal cells migrating away from neurospheres. In animals, placement of this injectable CMH at peri-infarct TBI regions significantly enhanced mature brain-derived neurotrophic factor (mBDNF) by 180% and improved angiogenesis by 250% in vivo within 2 weeks after magneto-electrical stimulation; these effects facilitated neuron survival and motor function recovery by 50%. Blood oxygenation level-dependent (BOLD) functional neuroimaging also revealed the successful restoration of brain functional connectivity in the corticostriatal and corticolimbic circuits. More broadly, this aspect of functional adaptable conductive mesoporous hydrogel also represents a major paradigm advance in the design of tissue engineering, making this manuscript of great interest to the materials community.

Table of content
摘要    i
Abstract    iii
Table of Content    v
致謝    vi
Figure Captions    x
Table Captions    xiii
Chapter 1 Introduction    1
Chapter 2 Literature Review and Theory    2
1 Introduction of peripheral nerve Injury    2
1.1 Intrinsic axonal factors - Retrograde injury signalling and Distal axon degeneration    4
1.2 The role of Schwann cells - Injury-associated phenotypic changes    4
1.3 Nerve Guide Conduit (NGC)    6
1.4 Neurotrophic factors for peripheral nerve repair: Toward targeted therapeutics.    8
1.5 Gradient structure design and their fabrication    9
2 Introduction of traumatic brain injury    10
2.1 The kinetics of the immune response to brain injury    12
2.2 Astrocytes in traumatic brain injury    13
2.3 Microglia in traumatic brain injury    14
2.4 Interleukin-6    15
2.5 Electro-magnetized gold nanoparticles    15
2.6 Electrical Stimulation (ES) and Neuron modulation    17
3 Introduction of injectable materials - Hydrogel    17
3.1 Synthesis, properties, and biomedical applications of gelatin methacryloyl (GelMA) hydrogels    20
3.2 The challenge of hydrogel and Microgels (MPs) for biomedical applications    21
3.3 Fabrication of Microgels    22
3.4 Designing adaptable scaffolds from microgels    24
3.5 Droplet Based microfluidics system    26
3.6 Recent advances in microgel scaffold    28
Chapter 3 Experimental Section    32
1 Microfluidic Chip Fabrication    32
2 Synthesis of GelMA    33
3 Synthesis of ChitoMA    33
4 Zeta Potential Measurement.    34
5 Fabrication of Building Blocks    34
6 Measurement of the Porosity of AMH    35
7 Measurement of the Median Void Volume Size in AMH    36
8 Elastic Modulus Measurement    36
9 Degradation of The GelMa Building Blocks    36
10 In Vitro NGF Release Study    37
11 Rheology Technique for The Gel Measurement    37
12 Cell Lines and Animals    38
13 Cell Culture on Building Blocks    38
14 Cell Proliferation Analysis    39
15 Preparation of GelMA Nerve-Guiding Conduit    39
16 Animal and Surgical Procedure    39
17 Functional Assessment of Walking Track Analysis    41
18 Electrophysiological Analysis    41
19 Morphology of Regenerated Distal Nerves    41
20 Transmission Electron Microscopy of the Regenerated Nerves    42
21 Relative Gastrocnemius Muscle Weight (RGMW)    42
22 Tissue Section Immunofluorescence at 4 And 7 Days Postinjection    42
23 Synthesis and Modification of Gold Nanoyarn-Ball (GYBs)    43
24 Characterizations of GYBs    44
25 Preparation of Conductive Mesoporous Microgels    44
26 Electrical Conductivity and LED Emitting Test    44
27 In Vitro Cell culture    45
28 In Vitro Experiments    45
29 Cell Viability Assay    46
30 Immunocytochemistry of Differcientation    47
31 Analysis of Differentiation Percentage of Neural Cells    47
32 Quantification of Sprouts in the NSC spheroid    48
33 In Vivo Experiment    48
34 Brain Collection and Immunofluorescence Staining    48
35 BOLD-fMRI Acquisition and Data Analysis    49
36 Western Blotting    51
37 Animal Behavior Test    52
38 In Vivo Magnetic Resonance Imaging (MRI) Protocol    52
39 Statistical Analysis    53
Chapter 4    54
Adaptable Microporous Hydrogels of Propagating NGF-Gradient by Injectable Building Blocks for Accelerated Axonal Outgrowth    54
1 Abstract    54
2 Introduction    55
3. Results and Discussions    59
3.1. Synthesis and Physicochemical Characterization of AMH    59
3.4. In Vivo Study of Regenerated Nerve    72
3.5. Functional Recovery of a Regenerated Sciatic Nerve In Vivo    80
3.6. Regenerated Myelinated Nerve Fibers    83
4 Conclusions    85
Chapter 5    86
Electromagnetized-Field-Mediated Adaptable Conductive Microporous Hydrogels for Directing Nerve Repair and Brain Function Recovery    86
1 Abstract    86
2 Introduction    87
3 Result and discussion    90
3.1 Synthesis and Characterization of CMH    90
3.2 Rheological and Self‐Healing Properties of CMH    95
3.3 In Vitro Electromagnetized GYBs Stimulate Neuron Differentiation.    96
3.4 In Vivo Study of Regenerated Nerve in TBI    101
3.5 In Vivo Cell Infiltration and Vascular Formation    104
3.6 In Vivo BOLD fMRI Evaluation    107
3.7 In Vivo Motor and Somatosensory in Brain Functions    110
4 Conclusion    114
References:    116
Curriculum Vitae    124


                                

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