2023 Fiscal Year Research-status Report
Functional Tissue Fabrication on Surfaces with Complex Stiffness Gradient developed by 3D Printing
| Project/Area Number |
22KF0247
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| Allocation Type | Multi-year Fund |
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| Research Institution | Osaka University |
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Principal Investigator |
境 慎司 大阪大学, 大学院基礎工学研究科, 教授 (20359938)
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| Co-Investigator(Kenkyū-buntansha) |
MUBAROK WILDAN 大阪大学, 大学院基礎工学研究科, 外国人特別研究員
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| Project Period (FY) |
2023-03-08 – 2025-03-31
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| Keywords | 3Dプリント / 組織工学 / 再生医療 |
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| Outline of Annual Research Achievements |
The objective of this study is to create a hydrogel with anisotropic stiffness obtained by inkjet patterning of catalase. In horseradish peroxidase (HRP)-mediated cross-linking, H2O2 is used to induce cross-linking between the phenol groups in the polymer. Since catalase can degrade the H2O2, the area patterned with catalase should have lower H2O2 presence to induce the HRP-catalyzed crosslinking, resulting in a softer hydrogel, compared to the non-patterned area. Herein, the anisotropic hydrogel was fabricated by patterning catalase (8 kU/mL) to hydrogels containing phenolated hyaluronic acid (HA-Ph; 2.0 w/v%) and gelatin (Gel-Ph; 0.1 w/v%) followed by H2O2 exposure for 45 min. The catalase-patterned area had lower stiffness (1.0 kPa) compared to the non-patterned area (4.8 kPa), effectively forming anisotropic stiffness in the hydrogel. The non-uniform stiffness hydrogels generated using this technique were able to mimic the native tissue conditions that are not available with the commonly used uniform stiffness bulk hydrogels. This system can be applied to evaluate the network formation of human vascular endothelial cells (HUEhT-1), in which the cells in the lower stiffness region showed a network formation. Taken together, the inkjet patterning of catalase can be a promising technique for fabricating hydrogels with anisotropic stiffness. In the future, this system will be applied to study the differentiation of mesenchymal stem cells into tissue mimicking the native conditions.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
Currently, the Fellow has successfully fabricated hydrogel comprised of HA-Ph and gelatin Gel-Ph with anisotropic stiffness obtained by patterning catalase. This technique is an alternative to the previously reported method in which H2O2 is patterned on the hydrogels to induce polymer degradation. H2O2 possesses a cytotoxic risk and a high concentration of H2O2 can damage the equipment. Catalase patterning offers a more cell-friendly reaction. In principle, catalase activity to quench H2O2 can reduce the H2O2 presence that is needed to induce the HRP-catalyzed crosslinking. Thus, the area patterned with catalase had lower crosslinking compared to the non-patterned area. Herein, the hydrogel sheet was fabricated by exposing a solution containing 2.0 w/v% HA-Ph, 0.1 w/v% Gel-Ph, and 50 U/mL HRP for 15 min. Catalase (8 kU/mL) was then patterned using an inkjet printing system on the hydrogels. Following catalase patterning, air containing H2O2 was exposed for an additional 45 min, to further induce the crosslinking. Using this technique, hydrogels with anisotropic stiffness could be fabricated, in which Young’s modulus value in the patterned area was 6 times lower than those in the non-patterned area. Study on the behavior of the human vascular endothelial cells (HUEhT-1) showed a stiffness-dependent network formation of the cells on the anisotropic hydrogel. The stiffness of the catalase-patterned area (1.0 kPa) is within the optimum range to induce network formation, while the non-patterned area is too stiff (4.8 kPa) which resulted in no network formation.
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| Strategy for Future Research Activity |
In the future, the influence of catalase concentrations on the hydrogel stiffness and crosslinking density will be evaluated. The effect of dots and line patterning of the catalase as well as the gaps between the pattern will be investigated. Based on the current Young’s modulus value, the current hydrogels can be applied to differentiate the MSCs into neurons (in the softer patterned area) and muscle (in the stiffer non-patterned area) forming a neuromuscular junction. The method could be by culturing the cells in a mixture of neurogenic and myogenic medium. In addition, the durotaxis or stiffness gradient-based migration tendency of the MSCs to the stiffer region will be evaluated.
The Fellow will also fabricate hydrogels by using extrusion-based printing. Two different inks, one which contained only Gel-Ph, and one with the addition of HA-Ph, were alternately extruded in a specific pattern. The addition of HA-Ph can induce not only anisotropic stiffness, since HA-Ph can increase the stiffness but also anisotropic properties, considering that cells can interact with HA-Ph via its receptor CD44 and RHAMM. This anisotropic property will be applied to control the differentiation of stem cells into osteochondral tissue, in which the stiffer part of the hydrogels may induce bone differentiation, while the softer part may induce chondrogenic differentiation. Finally, the results of these studies will be published as research articles and presented at international conferences.
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