||The Influence of PAA Substratum in Different with Stiffness with Identical Pore Size on the Behaviors of Adipose-Derived Stem Cells
||Department of BioMedical Engineering
Adipose-derived stem cells
先前的研究有提到細胞外基質會藉由化學刺激及物理刺激的方式來調控細胞的行為，如細胞爬行、增生、分化、貼附等。其中化學刺激的方式主要是以可溶性因子以及細胞與基材接觸進而活化細胞表面受體的方式來調控細胞，而物理刺激則是以細胞外基質的硬度、形貌、形狀等來做為調控細胞行為的因子。在硬度的部分，有研究指出當幹細胞被培養於不同硬度的基材上時，幹細胞會相對應於基材硬度而分化成不同的細胞，例如當幹細胞被培養於0.1-1 kPa的基材上時會分化成神經細胞，在8-19 kPa的基材上則分化成肌肉細胞，在25-40 kPa則是分化成骨細胞。然而在近期的研究發現，在製備不同硬度的基材同時也會造成基材表面孔洞大小不一的情形，並有研究更進一步去驗證細胞的行為是由基材表面的孔洞大小去調控而非基材硬度。
本研究的主要目的是利用丙烯酰胺(40%)及雙丙烯酰胺(2%)以同一比例混合再藉由波長254 nm的紫外光照射不同的時間來製備出具有不同硬度而孔洞大小上沒有顯著差異的聚丙烯酰胺水膠(35秒: 0.5 kPa，8分鐘:20 kPa)，再將脂肪誘導幹細胞(ADSCs)培養於水膠及玻片上去更精確的探討基材硬度對於細胞行為的影響。
免疫螢光染色的結果發現，細胞培養於硬度較高的基材上時細胞粘著斑(focal adhesion)和纖維應力(stress fiber)的表現量都會比軟基材來的高，同時，細胞的貼附面積也比軟基材還要大。
Studies had showed that extracellular matrix (ECM) would regulate cells behaviors, like proliferation, differentiation, migration, and adhesion through the biochemical and biophysical cues. The biochemical induction was mainly based on the soluble factors and the activation of cell surface receptor by the cell-matrix interaction, however, the biophysical induction was by the stiffness, topography, and pattern to regulate cells behaviors. Researches had pointed out that stem cells cultured on the specific stiffness substrate would differentiate into the specific lineages cells, for example, human mesenchymal stem cells would differentiate into neurons, myocytes, and osteocytes when cultured on the stiffness 0.1-1 kPa, 8-17 kPa, and 25-40 kPa, respectively. However, the substrate with different stiffness usually had different pore size. This study intended to validate the cells behaviors was regulated by the substrate pore size instead of pure stiffness. The gel with different stiffness but had similar porosity was prepared to test the cell behavior.
This study used the same ratio of acrylamide (40%) and bis-acrylamide (2%) but with different ultraviolet light (254nm) irradiated time to fabricate the polyacrylamide (PAA) gel with different stiffness (35s: 0.5 kPa;8min: 20 kPa) and identical pore size. Then cultured the adipose-derived stem cells (ADSCs) on the PAA gel and glass coverslip to investigate how the substrate stiffness influence on the ADSCs behaviors.
The cell proliferation result from WST1-assay by culturing ADSCs on the different stiffness substrate showed no significant difference in proliferation rate between two substrate; however, the proliferation rate was slight higher in the stiff group. However, the wound healing assay showed the speed of cell migration on stiff substrate was faster than that in soft substrate.
Adipogenic differentiation by Oil Red O staining was positive in ADSCs cultured on the 0.5 kPa PAA gel with adipogenic induction medium; however, osteogenic differentiation by Alizarin Red staining were positive in ADSCs cultured on the both 20 kPa PAA gel and glass coverslip with osteogenic induction medium. Immunofluorescence images showed the formation of focal adhesion and stress fiber; and the spreading area of ADSCs were proportional to the substrate stiffness. It means ADSCs differentiation and adhesion would direct by the substrate stiffness.
In this study, PAA gel with different stiffness and identical pore size was fabricated and this structure could be used to differentiate the influence of stiffness on stem cells behaviors by either pure stiffness effect or porosity. The result of this study showed differentiation, proliferation, and spreading area of ADSCs were regulated by the substrate stiffness not pore size.
Table of Contents VI
List of Tables VII
List of Figures VIII
Chapter 1: Introduction 1
1.1 Stem cells classification and repair potential 1
1.2 Unique stem cell niche 2
1.3 Influence of stem cell niche on stem cell behaviors 3
1.3.1 Influence of biochemical cues on stem cells differentiation 3
1.3.2 Influence of biophysical and biochemical cues on stem cells differentiation 4
1.4 Study aims and purposes 12
Chapter 2: Materials and Methods 13
2.1 Experiment flow chart 13
2.2 Reagents and instruments 13
2.3 Polyacrylamide gel fabrication 15
2.3.1 Glass coverslip treatment 15
2.3.2 PAA gel fabrication 16
2.3.3 Coating ECM 16
2.4 PAA gel mechanical property characterize 18
2.5 PAA gel microstructure characterize 18
2.6 Adipogenic and osteogenic induction medium formulation 19
2.7 Adipose-derived stem cells isolation 19
2.8 Cells culture 20
2.9 Cells differentiation assay 20
2.10 Cells proliferation assay 21
2.11 Cells migration assay 22
2.12 Cells adhesion assay 23
Chapter 3: Results and Discussion 24
3.1 Mechanical properties of PAA gel 24
3.2 Effect of substrate stiffness on ADSCs proliferation 26
3.3 Effect of substrate stiffness on ADSCs differentiation 28
3.4 Effect of substrate stiffness on ADSCs adhesion 34
3.5 Effect of substrate stiffness on ADSCs migration 38
Chapter 4: Conclusions 41
Chapter 5: Limitations and Future works 42
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