The Influence of PAA Substratum in Different with Stiffness with Identical Pore Size on the Behaviors of Adipose-Derived Stem Cells

• Main Authors: Po-HanChu, 朱柏翰
• Other Authors: Ming-Long Yeh
• Format: Others
• Language: zh-TW
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/f34e85

碩士 === 國立成功大學 === 生物醫學工程學系 === 105 === 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.