Biofabrication of functional tissues using surfaces with complex stiffness gradients created through 3D printing

2022 Fiscal Year Annual Research Report

• Project/Area Number: 22F22373
• Allocation Type: Single-year Grants
• Research Institution: Osaka University
• Principal Investigator: 境 慎司 大阪大学, 大学院基礎工学研究科, 教授 (20359938)
• Co-Investigator(Kenkyū-buntansha): MUBAROK WILDAN 大阪大学, 基礎工学研究科, 外国人特別研究員
• Project Period (FY): 2022-11-16 – 2025-03-31
• Keywords: バイオプリンティング / 組織工学 / 生体材料 / 再生医療

Outline of Annual Research Achievements

本研究は、過酸化水素のインクジェットパターニングによって得られる不均一な剛性を持つハイドロゲルを作製することを目的とする。このシステムは、過酸化水素が西洋ワサビペルオキシダーゼ（HRP）を介した架橋において、架橋を誘導して剛性を高める一方で、酸化剤としてポリマーを分解し、ハイドロゲルの剛性を下げるという矛盾した役割を果たすことを利用したものである。
2.0w/v%フェノール化ゼラチンとヒアルロン酸を含む溶液に、過酸化水素を含む16ppmの空気を15分間暴露した後、得られたハイドロゲルに10mM過酸化水素をパターニングして非一様な硬さのヒドロゲルを作製した。その結果、ハイドロゲル上にパターン化された過酸化水素によりハイドロゲルを構成する高分子が部分的に分解され、パターン化されていない領域（1.5 kPa）と比較して低い剛性（0.5 kPa）を示し、ハイドロゲルに剛-柔-剛領域を効果的に形成することができた。この技術を用いて生成された不均一な硬さのハイドロゲルは、骨軟骨（軟骨から骨）組織、筋肉と腱、腱と骨の接合部、胚発生時の神経管に見られるような、一般的に使用される均一な硬さのバルクハイドロゲルでは得られない、本来の組織の状態を模倣することができた。
このシステムを、ヒト間葉系幹細胞（UE7T-13）、マウス線維芽細胞（10T1/2）、マウス骨格筋芽細胞（C2C12）を用いた細胞-基板相互作用の評価に適用したところ、細胞の生存に影響を与えず（生存率95％以上）、細胞への毒性を持つアクリルアミド系材料を用いて不均一な硬さのハイドロゲルを作製する既報の手法に代わるものであることを明らかにすることができた。

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 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.

Strategy for Future Research Activity

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.