去細胞豬膀胱基質之生醫應用發展｜國立臺灣科技大學博碩士論文系統

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研究生：	阮黃光耀 NGUYEN HUYNH QUANG DIEU
論文名稱：	去細胞豬膀胱基質之生醫應用發展 Decellularization of Porcine Bladder Matrix and The Development of Its Biomedical Applications
指導教授：	高震宇 Chen-Yu Kao
口試委員:	楊銘乾 Ming-Chien Yang 蔡協致 Hsieh-Chih Tsai 羅俊民 Chun-Min Lo 徐善慧 Shan-Hui Hsu 高震宇 Chen-Yu Kao
學位類別：	博士 Doctor
系所名稱：	應用科技學院 - 應用科技研究所 Graduate Institute of Applied Science and Technology
論文出版年：	2022
畢業學年度：	110
語文別：	英文
論文頁數：	135
中文關鍵詞：	膀胱、細胞外基質、水凝膠、牙髓幹細胞、免疫調節
外文關鍵詞：	bladder, extracellular matrix, hydrogel, dental pulp stem cell, immunomodulation
相關次數：	點閱：437 下載：2
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上一筆

細胞外基質（Extracellular matrix, ECM）是所有組織與器官中無細胞的三維結構，其成分主要由細胞分泌與維持。此結構為高度動態的微環境，調節細胞增生，附著，移動，極性，分化等重要行為特徵。在組織工程中，膀胱基質（Urinary bladder matrix, UBM）為一種被廣泛研究的ECM。儘管大部分UBM水膠均是由過氧乙酸（Peracetic acid, PAA）去細胞製成，然而近期的研究卻顯示，以PAA去細胞並不是一個可靠的方法。若以更強的洗滌劑，例如十二烷基硫酸鈉（Sodium dodecyl sulfate, SDS）去細胞，也許是更加可行的辦法，但其對水膠特性的影響尚未明朗。因此第一篇研究的目標為以SDS為基礎，制定一個更可靠的去細胞水膠製程，並與廣泛使用的PAA法進行水膠性質的比較。研究結果顯示，SDS比PAA有更加的去細胞效力；然而除成膠動力學外，兩種製程的水膠在生化組成，表面型態，流變學等性質上並無顯著差異。由SDS去細胞製成的水膠，在體外試驗中展現了良好的細胞相容性，此結果表明，SDS為取得去細胞UBM更好的洗滌劑，因為它有較低的免疫誘導性。此研究中以SDS為基礎建立的水膠製程，所得水膠在各項性質與廣泛使用的PAA製程相似，(且有更佳的細胞相容性)，是組織重建中相當有潛力的材料。
儘管可將膀胱分成UBM和其他基質（Subtype ECM, sECM），大部分研究僅使用UBM製備水膠而忽略了sECM的潛力。另外，將膀胱分離為UBM和sECM是一件相當耗時的流程，因此直接運用全膀胱（Whole bladder, WB）可能可以提高產率。基於以上觀察，第二部分的研究目標在比較以UBM，sECM和WB所得之水膠。結果顯示三種不同分層製成的水膠，在生化組成上沒有顯著差異；然而在成膠動力學，流變學，型態學方面，UBM和sECM有部分差異，UBM和WB水膠之間卻意外的相似。體外細胞實驗中，進行L929細胞活性測試與C2C12分化測試，三種水膠呈現相似的結果。此結果可初步推論sECM不應該被忽視，WB的運用可替代僅使用UBM的流程，簡化分層的麻煩，達到工業化大量製造的可能。
近年全球遭遇COVID-19大流行，此病毒攻擊肺部與呼吸道使免疫系統失調，造成細胞激素釋放症候群（Cytokine release syndrome, CRS）。間質幹細胞(Mesenchymal Stem Cells , MSCs) 具有免疫調節功能，能促進內生性的再生作用，因此幹細胞療法被視為有潛力治療COVID-19的方法。然而，幹細胞無法停留在肺部中夠長時間以達到其治療效果是一大問題。豬膀胱來源的細胞外基質（Extracellular matrix from porcine bladders, B-ECM）已被發現能調節細胞活動，並具有免疫調節特性，因此可以推測B-ECM水膠也許是讓MSCs生長的良好介質，並可以使MSCs停留在肺部夠久以達治療效果。第三篇研究中，建立了ECM分解物和MSCs與巨噬細胞的共培養系統，用以研究感染情況下ECM和MSCs的免疫調節特性。結果顯示B-ECM分解物可減低巨噬細胞誘發促發炎反應，並增加抗發炎激素的釋放。在仿體內實驗的共培養系統中，培養在B-ECM上的MSCs在基因和蛋白質層面都顯示出免疫調節的特性。B-ECM分解物和MSCs條件培養基均促使肺泡上皮的癒合。此研究結果提供ECM和MSCs免疫調節特性研究的初步結果，可做為未來體內研究參考，進而應用於再生醫學。

Extracellular matrix (ECM) is a three-dimensional lattice structure secreted and maintained by the cells, also known as the non-cellular component of all tissues and organs. It is a highly dynamic micro-environment which determines and modulates the most essential behaviors and characteristics of cells such as proliferation, adhesion, migration, polarity, differentiation, and apoptosis. Among different types of ECM, urinary bladder matrix (UBM) is one of the most studied ECM in the tissue engineering field. Although almost all of the UBM hydrogels were prepared by using peracetic acid (PAA), recent studies indicated that PAA was not a trustworthy way to decellularize UBM. A stronger detergent, such as sodium dodecyl sulfate (SDS), may help tackle this issue; however, its effects on the hydrogels’ characteristics remain unknown. Therefore, the objective of the first study of this thesis was to develop a more reliable protocol to decellularize UBM, using SDS, and to compare the characteristics of hydrogels obtained from this method to the widely employed technique, using PAA. The results indicated that SDS was superior to PAA in decellularization efficacy. Different decellularization methods led to dissimilar gelation kinetics; however, the methods did not affect other hydrogel characteristics in terms of biochemical composition, surface morphology and rheological properties. The SDS-treated hydrogels possessed excellent cytocompatibility in vitro. These results showed that the SDS decellularization method could offer a more stable and safer way to obtain acellular UBM, due to reducing immunogenicity. The hydrogels prepared from this technique had comparable characteristics as those from PAA and could be a potential candidate as a scaffold for tissue remodeling.
Although there are two different ECM types inside a bladder, i.e. urinary bladder matrix (UBM) and a subtype ECM (sECM), most of the studies only employed UBM for hydrogel fabrication, and overlooked the potential use of sECM. In another aspect, the delamination of UBM from bladders is a time-consuming process; consequently, utilization of the whole bladder (WB) will likely increase production yield. Therefore, the objective of the second study of this dissertation was to fabricate hydrogels from sECM and WB and compared them to UBM. The results indicated that different layers of the bladder shared almost the same biochemical composition. In term of gelation kinetics, rheology and morphology, although hydrogels from UBM and sECM exhibited some discrepancies, those from UBM and WB interestingly possessed almost the same characteristics. In in vitro studies, all the hydrogels possessed nearly the same biochemical effects towards L929 viability and C2C12 differentiation. These results could preliminarily indicate that the use of sECM should no longer be ignored, and WB could be a promising substitution for UBM hydrogels, eliminating the need for time-costing delamination process, as well as increasing the possibility for mass production.
Currently, almost every country in the world has ever suffered from COVID-19 pandemic. COVID-19 primarily attacks the lungs and respiratory tract through dysregulate the immune system which causes a cytokine release syndrome (CRS). Mesenchymal stem cells (MSCs) possess immunomodulatory properties and capacity for endogenous regeneration. Therefore, MSC therapy is a promising treatment strategy for COVID-19. However, the cells cannot stay in the lung long enough to exert their function. The extracellular matrix from porcine bladders (B-ECM) has been shown not only to regulate cellular activities but also to possess immunoregulatory characteristics. Therefore, it can be hypothesized that B-ECM hydrogel could be an excellent scaffold for MSCs to grow and could anchor MSCs long enough in the lung so that they can exhibit their immunomodulatory functions. In this study, ECM degradation products and a co-culture system of MSCs and macrophages were developed to study the immunomodulatory properties of ECM and MSCs under septic conditions. The results showed that B-ECM degradation products could decrease pro-inflammatory and increase anti-inflammatory cytokines from macrophages. In an in vivo mimicking co-culture system, MSCs cultured on B-ECM hydrogel exhibited immunomodulatory properties at both gene and protein levels. Both B-ECM degradation products and MSC conditioned medium supported the wound healing of alveolar epithelial cells. The results from the study could offer a basis for investigation of immunomodulation by ECM and MSCs before conducting in vivo experiments, which could later be applied in regenerative medicine.

CHAPTER 1: INTRODUCTION    1
1 Background    1
2 Objectives    3
3 Materials, Reagents and Instruments    4
3.1 Materials and Reagents    4
3.2 Instruments    5
3.3 Statistical analysis    5
CHAPTER 2: LITERATURE REVIEW    7
1 Extracellular matrix    7
1.1 Major ECM components    7
1.2 Structure the ECM    8
1.3 Functions of the ECM    10
2 Decellularization    10
2.1 Rationale for decellularization of ECM  [5]    10
2.2 Decellularization strategies    11
2.3 Assessment of Decellularization    15
3 ECM hydrogel    16
3.1 ECM hydrogel formation    18
3.2 ECM hydrogel characterization    20
4 Porcine bladder    22
4.1 The bladder ECM architecture    22
4.2 Urinary bladder matrix    23
5 Immunomodulation property of ECM materials    24
6 Immunomodulation property of dental pulp stem cells    25
CHAPTER 3: EXPERIMENTAL METHODS    27
1 Bladder decellularization    27
1.1 Bladder delamination and pre-processing    27
1.2 Decellularization of UBM using PAA    27
1.3 Decellularization of bladder ECM using SDS    27
2 Decellularization Efficiency    27
2.1 dsDNA Quantification    27
2.2 DAPI Staining    28
2.3 Histological Staining    28
2.4 DNA Electrophoresis    28
2.5 Residual SDS Detection    28
3 Biochemical Characterization    29
3.1 Collagen and sGAG    29
3.2 Relative quantification of protein profile by liquid chromatography mass spectrometry (LC-MS/MS)    29
4 ECM Hydrogel Preparation    30
5 Turbidimetric Gelation Kinetics    30
6 Rheologic Testing    30
7 Hydrogel Surface Morphology    31
8 Cytocompatibility    31
8.1 In Vitro Cytotoxicity    31
8.2 2D Surface Culture    31
8.3 3D Cell Encapsulation    32
9 Effect on C2C12 Myogenic Differentiation    32
9.1 C2C12 cell culture    32
9.2 Immunocytochemistry    33
9.3 Western blot    33
10 Effect on C2C12 Osteoblast Differentiation    34
10.1 Osteoblast differentiation    34
10.2 Alizarin red staining (ARS)    34
10.3 Alkaline phosphatase (ALP) activity    34
11 ECM Hydrogels In Vitro Degradation    35
12 Preparation of ECM degradation products    35
13 Mesenchymal Stem Cell Verification    35
14 DPSC conditioned-medium collection    36
15 Culture of RAW 264.7 with digested B-ECM supplement    36
16 Culture of RAW 264.7 with conditioned-medium supplement    36
17 DPSC Growth rate on hydrogel surface    36
18 “In vitro mimicking” co-culture of RAW 264.7 mouse macrophages with human DPSC    37
19 Macrophage Response Assays    37
20 Immunomodulatory Assay    38
21 Real-Time Quantitative PCR (qPCR)    38
22 Enzyme-Linked Immunosorbent Assays (ELISA)    38
23 Wound healing assay    39
CHAPTER 4: CHARACTERIZATION OF PORCINE URINARY BLADDER MATRIX HYDROGELS FROM SODIUM DODECYL SULFATE DECELLULARIZATION METHOD    40
1 Introduction    40
2 Results    42
2.1 Decellularization Efficiency    42
2.2 Residual SDS Detection    43
2.3 Biochemical Characterization    44
2.4 Turbidimetric Gelation Kinetics    44
2.5 Rheology Study    46
2.6 Hydrogel Surface Morphology    47
2.7 Cytocompatibility    49
3 Discussion    50
4 Conclusions    55
CHAPTER 5: EVALUATING THE EFFECT OF TISSUE SELECTION ON THE CHARACTERISTICS OF EXTRACELLULAR MATRIX HYDROGELS FROM DECELLULARIZED PORCINE BLADDERS    57
1 Introduction    57
2 Results    59
2.1 Decellularization confirmation    59
2.2 Biochemical Composition    60
2.3 Turbidity Measurement    62
2.4 Rheological Measurements    64
2.5 Scanning Electron Microscopy    66
2.6 Effect on L292 Fibroblast in vitro Cell Viability    67
2.7 Coating confirmation    68
2.8 Effect On C2C12 Myogenic Differentiation    69
2.9 Effect on C2C12 Osteoblast Differentiation    70
3 Discussion    71
4 Conclusions    77
CHAPTER 6: INVESTIGATING THE IMMUNOMODULATORY POTENTIAL OF DENTAL PULP STEM CELLS CULTURED ON DECELLULARIZED PORCINE BLADDER HYDROGEL TOWARDS MACROPHAGE RESPONSE IN VITRO    78
1 Introduction    78
2 Results    81
2.1 Decellularization Confirmation    81
2.2 Mesenchymal Stem Cell Verification    81
2.3 B-ECM Hydrogels In Vitro Degradation    82
2.4 DPSC Proliferation on Hydrogel Surface    83
2.5 Macrophage Response Assays    83
2.6 Immunomodulatory Effect of Digested B-ECM towards RAW Cells    84
2.7 Immunomodulatory Effect of DPSC on B-ECM Hydrogel towards RAW Cell under “In Vivo Mimicking” Co-Culture System    85
2.8 Wound Healing Assay    86
3 Discussion    87
4 Conclusion    93
CHAPTER 7: SUMMARY AND OUTLOOK    94
1 Summary    94
2 Outlook    94
REFERENCES    95
APPENDIX    113


                                

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