臺灣博碩士論文加值系統

透過機械性或化學性之細胞外刺激訊號施加於幹細胞，所產生一連串由細胞外基質傳遞進入細胞核的反應可以控制幹細胞分化命運。常見的高分子基材通過調配基材硬度已經被驗證出具有決定人類間葉幹細胞分化命運的能力。然而，具有奈米表面形貌矽基基材對於人類間葉幹細胞的效果則仍屬未知。本研究將採用無電鍍金屬沉積法來製備不同尺度的矽奈米線基材，且矽奈米線基材所呈現不同的彈性係數將作為唯一刺激人類間葉幹細胞的生物物理性刺激來源。藉此，本研究將證實矽奈米線基材如何將彈性係數的刺激導入，並驅使人類間葉幹細胞走向不同的分化方向。
首先，使用六個不同反應時間（第一組為最短反應時間依序到最長反應時間之第六組）於無電鍍金屬沉積法製備出六種具有不同尺度、立體表面形貌、緻密、垂直排列且單晶的矽基奈米線基材。實驗結果指出無電鍍金屬沉積法在固定的硝酸銀與氫氟酸電解液配方下對矽基平板基材呈現約1.06 um/min的線性蝕刻率。特別的是，當這些單根矽基奈米線生成後會因為毛細黏滯現象（Capillary stiction）聚合成一叢叢的矽基奈米線束。因此，針對這六組矽基奈米線基材的單根矽基奈米線以及矽基奈米線束取樣，並將量測其長度及直徑數據並投入Beam theory進行理論彈性係數之計算（KTheo, SiNW 以及 KTheo, bundles）。我們更進一步使用即時影像觀測用之穿透式顯微鏡（In-situ transmission electron microscope，In-situ TEM）進行奈米壓痕試驗儀（Picoindentor）量測出實際矽基奈米線束的彈性係數（KReal, bundles）。第一組到第六組之KTheo, SiNW、KTheo, bundles以及KReal, bundles全都呈現一致的趨勢：彈性係數與矽奈米線長度呈現反比的關係。也就是說憑藉對於無電鍍金屬沉積法的精準控制下，我們有能力調配出特定尺度以及彈性係數的矽基奈米線基材。然而在表面自由能方面，所有六組矽基奈米線基材都表現超親水性之表面特性，並不因為矽基奈米線基材之尺度變化有所差異。這超親水性表面將有助於細胞貼附的能力。
第二，當人類間葉幹細胞培養於矽基奈米線基材上約三天時，我們發現於各組矽基奈米線基材上所表現之存活率皆大於90%。這也就代表了矽基奈米線基材對於人類間葉幹細胞是不具備有細胞毒性的，這樣的結果也跟矽基平板基材相似。我們首先探討人類間葉幹細胞在沒有任何誘導分化成分的維持培養基下，矽基奈米線基材對於誘導人類間葉幹細胞進行成骨分化的機制。人類間葉幹細胞培養於六組矽基奈米線基材上均呈現圓球狀並收斂的細胞生長形貌。相反的，人類間葉幹細胞培養於矽基平板基材則是呈現平坦且外擴的細胞生長形貌。此外，第一組矽基奈米線基材（最短奈米線長度）顯示對於人類間葉幹細胞成骨基因表現量COL1a1以及RUNX2顯著的高於其他五組矽基奈米線基材以及矽基平板基材。深入發現培養於第一組矽基奈米線基材的人類間葉幹細胞具有最多的F-肌動蛋白（F-actin）、磷酸化局部黏著酵素（phosphorylated focal adhesion kinase，pFAK）、黏著斑蛋白（vinculin）以及alpha 2整聯蛋白（alpha 2 integrin）。這些蛋白的表現也就代表著當人類間葉幹細胞生長於第一組矽基奈米線基材時，進行了相當高程度的細胞骨骼的重整。也就是說第一組矽基奈米線基材所提供的最高程度的彈性係數，由細胞外開始提供有效的刺激，並驅使了人類間葉幹細胞進行細胞骨骼的重構，最後透過alpha 2/beta 1 異二聚體整聯蛋白誘導人類間葉幹細胞成功進行了成骨分化。
知悉第一組矽基奈米線基材能夠誘導人類間葉幹細胞成功走向成骨分化，其他五組矽基奈米線基材是否存在著其他可能性。因此我們便挑出成骨基因表現因子（COL1a1以及RUNX2）以及脂肪基因表現因子（PPARr 以及fatty acid-binding protein 4，FABP4）來了解六組矽基奈米線基材是否也存在有其他可能的人類間葉幹細胞分化方向。值得一提的是，無論在一般的維持培養基下或是含有脂肪分化因子的培養基下，第一組矽基奈米線基材都依舊顯著的驅動人類間葉幹細胞走向成骨分化。這也就證實第一組矽基奈米線基材具備強烈且穩定的成骨分化能力。另一方面，在一般的維持培養基下，各組的脂肪分化能力表現不夠顯著。因此，當我們將一般培養基換成含有脂肪分化因子的培養基後，發現PPARr 以及FABP4在第四組矽基奈米線基材才具備有顯著性差異的提升。這項結果說明了在不同的彈性係數的刺激下，矽基奈米線基材能夠操縱人類間葉幹細胞分化的方向。
最後，我們運用了原子力顯微鏡術定量的量測出成骨分化與脂肪分化之人類間葉幹細胞的細胞硬度。當人類間葉幹細胞培養並長在不同彈性係數的矽基奈米線基材上，不同彈性係數的刺激會讓人類間葉幹細胞進行不同程度的重構、再組合細胞骨骼、細胞形貌的改變乃至細胞基因表現的變化。因此我們發現人類間葉幹細胞生長於最大彈性係數的第一組矽基奈米線基材所表現的細胞硬度為最大。相反的，人類間葉幹細胞生長於最小彈性係數的第六組矽基奈米線基材所表現的細胞硬度為最小。其中，人類間葉幹細胞生長於第四組矽基奈米線基材所表現的細胞硬度居中。

Extracellular stimuli through mechanical and/or chemical approaches, imposed on stem cells and initiated the transductive signalings from extracellular matrix (ECM) to cell nuclei, determine the stem cell fates. Polymeric matrices, one of the major biophysical stimulating sources, are known to significantly affect the fates of human mesenchymal stem cells (hMSCs) by controlling the matrix stiffness. However, the stimulatory effects of nanotopographical silicon-based matrices on hMSCs have not been thoroughly investigated. In this study, the the electroless metal deposition method (EMD) was utilized to design the silicon-nanowires (SiNWs) matrices with various SiNWs dimnesions and the spring constant (KX), served as the unique and the only biophysical source to the hMSCs. This study will investigate how these SiNWs impose the unique biophysical signals to hMSCs and ultimately determine the fates of hMSCs.
First of all, six SiNWs groups, prepared by EMD, were designed to present various dimensions under six reaction periods. The experimental results revealed that stereo-topographical, dense, vertically aligned, single-crystalline SiNWs matrices were prepared by EMD and the EMD etching rate on Si was approximately 1.06 um/min under the constant concerntration of AgNO3/HF electrolyte. These SiNWs due the capillary stiction is easily formed the bundles while hMSCs were cultured under liquid envrionment. Then, these six SiNWs groups were measured their length and diameters of single SiNW and SiNWs bundles and calculated their theoretical spring constants based on the Beam theory (KTheo, SiNW and KTheo, bundles). Not only the theoretically calculations, but also the real spring constant performed from these SiNWs groups were considered in this study. In-situ transmission electron microscope (TEM) picoindenter was applied to measure the spring constant from these real SiNWs bundles (KReal, bundles). The respective KTheo, SiNW, KTheo, bundles and KReal, bundles values reinforce the fact that there is an inverse correlation between the spring constant and SiNWs length. For the surface free energy, all these SiNWs groups are all presented as superhydrophilicity no matter their SiNWs length or KX. This superhydrophilic surface on SiNWs will benefit in the cell adhesion of hMSCs.
Secondly, the hMSCs viability (cytotoxicity) on SiNWs matrices was demonstrated and found that living hMSCs cultured on SiNWs matrice after 72 h in percentage are above 90% and this performance proved the SiNWs matrix is non-cytotoxici and is quite similar to that of cells grown on 2D flat Si. After confirmed SiNWs is non-cytotoxic, hMSCs was subsequently cultured on these SiNWs groups with maintenance medium and decode the possibility of osteogenicity and the pathway of KX stimulating signals of SiNWs to hMSCs. hMSCs cultured on stereo SiNWs of different lengths in the absence of biochemical osteogenic induction cues displayed a spherical and less-elongated morphology and displayed an approximately 10% loss of cell viability compared to those grown on two-dimensional (2-D) flat Si. Moreover, osteogenic gene expression of COL1a1 and RUNX2 in hMSCs cultured on the shortest SiNWs (Group I) was significantly higher than those grown on the longer SiNWs and 2-D flat Si. hMSCs grown on shorter SiNWs also demonstrated higher expression levels for F-actin, phosphorylated focal adhesion kinase (pFAK), vinculin and alpha 2 integrin. Stereo-topographical cues provided by SiNWs are able to regulate osteogenic differentiation of hMSCs via cytoskeleton remodeling and this is correlated with the differential expression of alpha 2/beta 1 integrin heterodimers and the focal adhesion molecules pFAK and vinculin.
Moreover, this study is looking for more possible differentiating fates of hMSCs induced by SiNWs groups. Thereafter, to decode the fate regulations of SiNWs groups, the osteogenic markers (COL1a1 and RUNX2) and adipogenic markers [PPARr and fatty acid-binding protein 4 (FABP4)] were selected to determine the fates of hMSCs after SiNW stimulation. For oeteogenicity of hMSCs, COL1a1 and RUNX2 significantly presented the highest gene expression level on Group I SiNWs as well, even under adipogenic medium. For adipogenicity of hMSCs, PPARr and FABP4 were significantly enhanced by Group IV SiNWs under adipogenic medium. The results indicate that the SiNWs with different spring constants selectively triggered osteogenicity and adipogenicity in hMSCs.
Finally, the quantitatively measured cell stiffness from those fate-regulated fixed and living hMSCs on SiNWs matrices were evaluated by the atomic-force microscopy (AFM). While hMSCs grown on SiNWs groups with different stiffness, hMSCs received stress and stimulations and subsequently adapted to remodel and re-assemble their cytoskeleton, cell morphology, and gene expression. After this adapting process, the stiffness of such differentiated cells is also affected by substrate stiffness. The above results indicate that SiNWs with controllable spring constants are capable of regulating the osteogenicity and adipogenicity of hMSCs in vitro.

Aknowledgement I
中文摘要 III
Abstract VI
Content X
List of Figures XIII
List of Tables XVIII
Symbols XIX
Chapter 1 Introduction 1
Chapter 2 Literature review 3
2.1 Stem cells 3
2.1.1 Structure and types of stem cells 4
2.1.2 Human mesenchymal stem cells and its functionalities 6
2.2 Extracellular stimulations to stem cells 9
2.2.1 Mechanical approach 9
2.2.2 Chemical approach 11
2.2.3 Electric field approach 14
2.3 Nanomaterials 16
2.3.1 0-D nanomaterials 17
2.3.2 1-D nanomaterials 20
2.3.3 2-D nanomaterials 23
2.3.4 3-D nanomaterials 24
2.4 Silicon-nanowires 26
2.4.1 Methods of silicon-nanowires fabrication 28
2.5 Motivation 36
Chapter 3 Experimental method 37
3.1 SiNWs fabrication and dimensional evaluations 37
3.1.1 Details of the SiNWs fabrication 37
3.1.2 Dimensional evaluations of six SiNWs groups 39
3.2 Characteristics of SiNWs 40
3.2.1 Wettability of flat Si and SiNWs 40
3.2.2 Sample preparation and spring constant of SiNWs measured by in-situ TEM picoindentor 41
3.2.3 Sample preparation and spring constant of SiNWs measured by in-situ SEM picoindentor 45
3.3 hMSCs culture process 47
3.3.1 hMSCs obtainment and filtration 47
3.3.2 Cell viability and adhesion ssay 48
3.3.3 Reverse transcription and quantitative real-time polymerase chain reaction (PCR) 49
3.3.4 Immunofluorescence staining 53
3.3.5 Quantification of immunofluorescence staining 54
3.3.6 Data analysis 54
3.3.7 hMSCs fixation process and cell morphology observations 54
3.4 Measurements of elasticity of hMSCs adhered on SiNWs 55
3.4.1 Immuno-staining living hMSCs on SiNWs groups 55
3.4.2 Measurements of elasticity of the fixed and living hMSCs on SiNWs groups 57
Chapter 4 Results and discussion 60
4.1 Morphological observations of SiNWs 60
4.1.1 SEM observations and dimensions of SiNWs 60
4.1.2 Wettability of flat Si and SiNWs 62
4.1.3 Theoretical calculations of spring constant among six SiNWs groups 63
4.1.4 Real measurements of spring constant among six SiNWs groups 65
4.2 hMSCs viability and adhesion on SiNWs 67
4.2.1 hMSCs viability on SiNWs 67
4.3 Osteogenic differentiation of hMSCs cultured on SiNWs group 68
4.3.1 Cellular morphology and spread area of the hMSCs growth on SiNWs stereotopography 68
4.3.2 Effect of SiNWs on the osteogenic differentiation of hMSCs 71
4.3.3 Cytoskeletal rearrangement of hMSCs grown on SiNWs groups 73
4.3.4 Integrin and focal adhesion kinase gene expression 75
4.3.5 Discussion–Osteogenic differentiated hMSCs on SiNWs groups 77
4.4 Fates of hMSCs stimulated by SiNWs groups 84
4.4.1 Osteogenicity and adipogenicity 84
4.5 Cell morphology and stiffness of hMSCs on SiNWs groups 87
4.6 Fate-regulation map of hMSCs fates triggered by SiNWs groups 90
Chapter 5 Conclusions 95
Reference 97
Achievements 115
• Education 115
• Work experience & expertise 115
• Research expertise 116
• Studying projects 116
• Lecturing Experiences 116
• Memberships/Honors 117
• Publication 118

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