| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
Currently, the Fellow has successfully fabricated a hydrogel substrate comprised of phenolated gelatin (Gel-Ph) and hyaluronic acid (HA-Ph) with gradient stiffness obtained by patterning hydrogen peroxide (H2O2). This study exploited the contradictory role of H2O2 in the horseradish peroxidase (HRP)-mediated crosslinking, in which it can induce the crosslinking, which increase the stiffness, while also degrading the polymer as an oxidant, which can reduce the stiffness of the hydrogels. The H2O2 role in degrading the hydrogels resulted in a lower stiffness in the area in which H2O2 was patterned, thus, creating the gradient stiffness.
The cell behaviour on the substrate with gradient stiffness was investigated using a human bone marrow mesenchymal stem cell line (UE7T-13) and a mouse fibroblast cell line (10T1/2). Viability analysis using Calcein-AM/PI staining showed that the cells had >90% viability on the substrate, demonstrating the cytocompatibility of the method. Both UE7T-13 and 10T1/2 cells showed a stiffness-dependent difference in cell adhesion. Cells in the region patterned with H2O2 showed a small and circular morphology, while cells in the non-patterned area exhibited a large and elongated shape. The cells showed a smaller and circular morphology on the area patterned with H2O2, which was possibly due to the lower stiffness of the hydrogel caused by the H2O2-mediated degradation of the Gelatin-Ph/HA-Ph polymer backbone.
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| 今後の研究の推進方策 |
In the future, for the inkjet printing study, the mechanical property of the hydrogels will be measured to prove that H2O2-mediated degradation can indeed reduce the stiffness, creating a stiffness gradient. Currently, a single-line H2O2 pattern has been achieved. In the future, to fabricate a complex gradient stiffness, H2O2 will be patterned in two or three lines, fabricating double or triple parabolical gradient stiffness. To investigate the role of hydrogel compositions, hydrogel substrate will be fabricated by varying the concentrations of Gelatin-Ph and HA-Ph, to better mimic the native tissue condition. Regarding the cell study, in addition to the current cell viability and adhesion analysis, cell migration and differentiation will be evaluated. To demonstrate the effect on various tissues, in addition to the bone marrow mesenchymal stem cells and fibroblast cells, a study using myoblasts (C2C12 cells) and cervical cancer cells (HeLa) will be conducted. In addition, the mechanotransduction of the cells in response to the stiffness gradient will be elucidated by F-actin and Yes-associated proteins (YAP) cytoplasm-nuclear localization analysis. Finally, the result of this study will be published as research articles and presented at international conferences.
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