藉由調控3D基材的軟硬度來誘導間葉幹細胞朝向神經細胞系之分化

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研究生：	吳繼禾 GOH JIH HER
論文名稱：	藉由調控3D基材的軟硬度來誘導間葉幹細胞朝向神經細胞系之分化 Control of Mesenchymal Stem Cell Fate toward Neural Lineages with Tunable 3D Substrate Modulus
指導教授：	王子威 WANG, TZU-WEI
口試委員:	邱信程 Chiu, Hsin-Cheng 姚俊旭 Yao, Chun-Hsu
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：	2012
畢業學年度：	100
語文別：	英文
論文頁數：	68
中文關鍵詞：	間葉幹細胞、透明質酸、細胞支架、軟硬度、神經細胞
外文關鍵詞：	mesenchymal stem cell, mechanical property, 3D scaffold, matrix stiffness, neuronal differentiation
相關次數：	點閱：2 下載：0
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幹細胞的無限再生與其強大的分化能力使它在組織工程與再生醫學領域上的應用備受矚目。幹細胞可分化成各種不同的組織細胞，誘導幹細胞分化的因素有許多，其中包括以組織替代物為最終目標之仿生材料其本身的基本特性, 如物理性質、化學特性、生物因子等等。我們希望藉由了解幹細胞與仿生材料之間的交互作用並利用以上特性，期望得以控制幹細胞分化的方向，進而替換在生物體成長過程中不會再生或損傷的細胞，最終達到治療損傷組織的效果。在本研究當中，我們利用天然材料－透明質酸作為主要的材料並與第一型膠原蛋白結合，透過真空冷凍乾燥技術製造成一個三維多孔性結構的細胞支架，藉由控制交聯劑濃度改變基材軟硬度，使培養在此細胞支架內的人體間葉幹細胞可朝向神經細胞系的方向生長與分化。
本實驗分為二大部分，第一部分為材料製作與各特性分析，第二部份為細胞培養並利用此細胞支架特性來誘導間葉幹細胞朝向神經細胞系方向分化，探討不同軟硬程度的細胞支架對於間葉幹細胞朝向神經細胞系方向分化的影響。不同軟硬程度細胞支架的調控，我們利用不同濃度的EDC碳二亞胺交聯劑來調控材料的彈性係數(E)，使此細胞支架的彈性係數落在1kPa以及10 kPa的範圍，以定義細胞支架軟材質和硬材質兩大範圍。在細胞支架的製作過程，透過材料的物化性分析，如微拉力試驗機測試不同交聯濃度的細胞支架之機械性質、掃描式電子顯微鏡以觀察各細胞支架的微結構、TNBS測試以取得各EDC交聯濃度的細胞支架之交聯程度、Carbazole測試來測量細胞支架內透明質酸的釋放量等等。與此同時，我們將人體間葉幹細胞培養在細胞支架上，以檢測細胞的型態、生長、增生與分化的情形。
本實驗主要的目的是利用經由各濃度的EDC交聯劑所形成的不同軟硬程度之透明質酸－第一型膠原蛋白細胞支架來誘導與探討人體間葉幹細胞分化成神經細胞的可行性。藉由此種可調控軟硬程度的仿生材料，我們得以探討細胞與基材的交互作用並控制幹細胞分化的結果，如朝向神經細胞系方向分化，對於目前幹細胞治療的研究以及對損傷的神經組織再生修復，非常具有臨床應用價值。

Unlimited self-renewal of stem cells and their multipotency ability lead a great potential in the application of tissue engineering and regenerative medicine. The induction of stem cells can be controlled by multiple factors including physical, chemical and biological cues. By knowing the interaction between stem cells and their vicinity biomimetic microenvironment, we may manipulate stem cell’s fate. In this study, three dimensional porous scaffolds were synthesized by type I collagen (Col) and hyaluronic acid (HA). The elastic modulus (E) of the 3D substrates was modified by adjustable concentrations of 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) crosslinking agent. The purpose of this study is to investigate the matrix stiffness on the influence of neurogenic differentiation of human mesenchymal stem cells (hMSCs). The mechanical property of Col-HA scaffolds was evaluated and the induction and characterization of hMSCs differentiation toward neural lineages by different substrate stiffness were studied. With different EDC crosslinking concentration, the stiffness of the matrices can be tunable in the ranges of 1 kPa to 10 kPa for soft and stiff substrates. The results found that MSCs tend to differentiate into neuronal lineage in substrate at 1 kPa, while they transform into glial cells in matrix with 10 kPa. The morphology and proliferation behavior of hMSCs were corresponded to substrate stiffness. By using this modifiable matrix, we can investigate the relationship between stem cell behavior and substrate mechanical properties in ECM-based biomimetic 3D scaffolds. A tunable substrate stiffness that would induce hMSCs specifically toward neuronal differentiation may also be very useful as tissue-engineered construct or substitute for delivering hMSCs in brain and spinal cord regeneration.

Contents
摘   要    I
Abstract    II
Contents    III
Figure index    VI
Table Index    VIII
Abbrevations    IX
Chapter 1    – Introduction    1
1.1    Background Information    1
1.1.1    Mesenchymal Stem Cell    1
1.1.2    Hyaluronic Acid    2
1.1.3    Collagen    3
1.1.4    Matrix Compliance Affects Cell Behavior.    4
1.2    Statements of Problem    5
1.3    The Purpose of This Study    6
Chapter 2    - Literature Review    7
2.1    The Origin of Tissue Engineering    7
2.2    Optimal Scaffold Properties    7
2.3    Introduction of Stem Cell    8
2.4    Factors that Influence Stem Cells Fate    11
2.5    Future Perspective of Stem Cells in Neuroregeneration    16
Chapter 3    – Theoretical Basis    19
3.1    3D Hyaluronic acid－Collagen Porous Scaffold Preparation by Freeze-drying Technique    19
3.2    EDC Crosslinking Reaction    20
3.3    Mechanoregulation Towards Cell Functions    22
Chapter 4    – Materials and Methods    25
4.1    Experimental Design    25
4.2    Preparation of Collagen Type I Scaffold    26
4.3    Preparation of Hyaluronic Acid Scaffold    26
4.4    Fabrication of Collagen－Hyaluronic Acid Scaffold (1:1)    27
4.5    Crosslinking Reaction for Hyaluronic Acid－Collagen with EDC Carbodiimide    27
4.6    Scaffold Morphology Examination by Scanning Electron Microscopy    28
4.7    Pore Size & Porosity Characterization of Scaffolds    28
4.8    Scaffold’s Water Absorption Test    29
4.9    Crosslinking Degree Determination (The Number of Non-Crosslinked Amino Groups) of Scaffolds by Trinitrobenzene Sulfonic Acid (TNBS) Assay    30
4.10    Mechanical Property Characterization    31
4.11    Determination of Hyaluronic Acid Release by Carbazole Reaction Assay    32
4.12    Cell Culture of Human Mesenchymal Stem Cells    33
4.13    hMSCs Culture with Different Neural Induction Medium in 2D Condition    33
4.14    3D Culture of Human Mesenchymal Stem Cells in Col-HA Scaffolds    34
4.15    WST-1 Assay for Cell Proliferation Rate of hMSCs Culture in 3D Col-HA Scaffolds.    34
4.16    Examination of hMSCs in 3D Scaffolds by Scanning Electron Microscopy    35
4.17    Hematoxylin & Eosin Staining for Cell-seeded Scaffolds    35
4.18    Gene Expression of hMSCs in 3D Col-HA Scaffolds    36
4.18.1    RNA Extraction    36
4.18.2    Reverse Transcription Reaction of Total RNA to cDNA    37
4.18.3     mRNA Quantification of hMSCs with qPCR    37
4.19    Immunohistochemistry(IHC) for Characterization of Neural Lineage Cells    38
4.20    Statistical Analysis    39

Chapter 5    – Results    40
5.1    3D Scaffold Morphology Characterization : Pore Size and Porosity Analysis    40
5.2    Water Absorption Test of Scaffolds    41
5.3    TNBS Assay for Determination of Crosslinking Degree and Free Amino Groups in   Scaffolds    41
5.4    Scaffold’s Mechanical Property Characterization    42
5.5    Carbazole Assay for Determination of Hyaluronic Acid Released From Scaffolds    44
5.6    Summary for Physical and Chemical Characterization of 3D Scaffolds    45
5.7    hMSCs Culture with Different Neural Induction Medium in 2D Condition    46
5.8    Cell Proliferation Rate of hMSCs Culture in 3D Col-HA Scaffolds    49
5.9    hMSCs Distribution and Morphology in 3D Col-HA Scaffolds    50
5.10    mRNA Quantification for The Determination of hMSCs Neural Differentiation in 3D  Culture Conditions    52
5.11    Immunostaining for hMSCs Neural Differentiation in 3D Culture Conditions    54
Chapter 6    – Discussions    56
6.1    The Assessment of 3D Col-HA Scaffolds for Neural Tissue Replacement    56
6.2    hMSCs Induction Toward Neural Differentiation by Different Culture Medium    56
6.3    Effect of Substrate Stiffness Against hMSC Cell Behavior    57
6.4    The Phenomenon of hMSCs Differentiation Towards Neural Lineage in Different    Stiffness of 3D Col-HA Scaffolds    58
Chapter 7    – Conclusions    61
References        62


Figure index
Figure 1 1.  Mesenchymal stem cells (MSCs) have the potential to differentiate through a number of different pathways to form mesenchymal tissues.    1
Figure 1 2.  Molecular structure of hyaluronic acid.    2
Figure 1 3.  Structure of collagen].    4
Figure 1 4.  Effects of substrate stiffness on cell morphology].    5
Figure 1 5.  The concept of this study.    6
Figure 2 1.  The concept of tissue engineering.    7
Figure 2 2.  Matrix stiffness affects MSC lineage.    12
Figure 2 3.  vmIPN substrate with different stiffness direct neural stem cell’s neural fate.    13
Figure 3 1.  The mechanism of freeze-drying process.    19
Figure 3 2.  The mechanism of (a) EDC carbodiimide crosslinking reaction (b) blocking method of EDC crosslinking reaction.    21
Figure 3 3.  The hypothesis of mechanosensing within stem cell.    23
Figure 3 4.  Mechanosensitive ion channel within cell membrane.    23
Figure 3 5.  Cells experienced physical signaling from microenvironment.    24
Figure 4 1.  Test sample for porosity test.    29
Figure 4 2.  Mechanism of TNBS assay.    30
Figure 4 3.  Standard test sample for tensile test characterization.    31
Figure 4 4.  Mechanism of WST-1 assay.    35
Figure 5 1.  SEM characterization for the microstructure of (a) pure HA, (b) pure Col, and (c) Col-HA(1:1) mixture scaffolds. The higher magnification of each scaffold was shown in (d), (e), (f), respectively.    40
Figure 5 2.  (a) Crosslinking degree and (b) free amino groups of different groups of 3D scaffolds. (**p<0.05)    42
Figure 5 3.  Results of mechanical compression test. (a) Compressive profiles of 70% strain and  (b) Elastic modulus at 10% strain for all hydrated scaffolds.    43
Figure 5 4.  Results of mechanical tension test. (a) Tensile profiles and (b) Elastic modulus at 10% strain for all hydrated scaffolds.    44
Figure 5 5.  The quantification of hyaluronic acid released in 28 days.    45
Figure 5 6.  OM images of different induction mediums to hMSCs on tissue culture plate for day 1. (a) NIM_1 (b) NIM_2 (c) NIM_3 (d) NIM_4 (e) NIM_5 (f) NIM_6 (g) NIM_7 (h) NIM_8. Scale bars = 200μm.    47
Figure 5 7.  OM images of different induction mediums to hMSCs on tissue culture plate for day 14. (a) NIM_1 (b) NIM_2 (c) NIM_3 (d) NIM_4 (e) NIM_5 (f) NIM_6 (g) NIM_7 (h) NIM_8. Scale bars = 200μm.    48
Figure 5 8.  OM images of neural-like cells from differentiated hMSCs in NBM for day 14. Scale bars = 50μm.    49
Figure 5 9.  Cell proliferation rate of hMSCs in 3D scaffolds with (a) growth medium (GM) and (b) serum-free neurobasal medium (NBM). Data was normalized with the cell numbers at day 1. (n=3)    50
Figure 5 10. Morphology of hMSCs in 3D porous Col-HA scaffolds. (a),(b) H&E staining for day 1 and day 7. (c),(d) SEM at day 1, and (e),(f) SEM at day 3 of respective soft and stiff substrates. Yellow arrow represented neural-like morphology.    51
Figure 5 11. Characterization of neural lineage for hMSCs in 3D scaffolds. Quantitative real-time polymerase chain reaction results for representative (a) MSC stem cell genes and (b) neural lineage specific genes.    53
Figure 5 12. Immunohistochemistry of neural lineage protein markers for hMSCs in Col-HA_EDC 0.1% and Col-HA_EDC 2.0% scaffolds. DAPI in blue was stained for nuclei.    55
 
Table Index
Table 2 1.  Adults stem cells and primary differentiation lineage[34].    10
Table 2 2.  Promises & limitations of stem cell population[35].    10
Table 2 3.  Adult stem cell therapies for neurodegenerative diseases[35].    16
Table 2 4.  Examples of clinical studies with biomaterials for treating CNS diseases[47].    18
Table 3 1.  Mechanical properties of tissue-specific ECM[19].    22
Table 4 1.  Lyophilization condition of fabricating 3D porous scaffold.    26
Table 4 2.  Neural induction medium for hMSCs neural differentiation.    33
Table 4 3.  Primer sequences of hMSCs target gene for qPCR.    38
Table 4 4.  Antibodies for immunohistochemistry.    39
Table 5 1.  Summary for physical and chemical characterization of 3D scaffolds.    45

                                

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