Development of nanofiber sheets composed of liver-derived extracellular matrix for liver tissue engineering applications

九州大学学術情報リポジトリ

Kyushu University Institutional Repository

Development of nanofiber sheets composed of liver-derived extracellular matrix for liver tissue engineering applications

ブアル, ロナルド, ペローチョ

https://doi.org/10.15017/2534413

出版情報：九州大学, 2019, 博士（工学）, 課程博士 バージョン：

権利関係：

（様式２）

氏 名 ：ブアル ロナルド ペローチョ

論 文 名 ：

Development of nanofiber sheets composed of liver-derived extracellular matrix for liver tissue engineering applications

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区 分 ：甲

論 文 内 容 の 要 旨

At present, liver transplantation is the only radical treatment shown to improve survival in patients suffering from serious liver failure. However, this approach is limited by the availability of liver donor. Tissue engineering approaches, consisting of a scaffold, growth factors and cells, are under investigation as an alternative treatment or as a bridge for patients who are awaiting a donor liver. Choosing the suitable scaffold material that better mimic the normal in vivo condition will have a major impact on the realization of the tissue engineering strategy. The scaffold should provide support for the growing tissue, channels for cell migration and mass transport, and surface signaling receptors for cell interaction and attachment.

Recently, the use of organ-derived ECM such as liver-derived extracellular matrix (L-ECM) were shown to support liver-specific cell populations, maintain site-appropriate phenotypes, and improve liver-specific functions in in vitro culture systems. The main problem, however, is the poor mechanical characteristics.

In this study, a scaffold composed of L-ECM, gelatin, and polycaprolactone was developed using electrospinning technique. The resulting scaffold is expected to provide superior characteristics in terms of biocompatibility, bioactivity, and mechanical stability. The effectiveness of the L-ECM nanofiber scaffold was investigated by in vitro culture and in vivo implantation to CCl4-induced fibrosis rat liver.

As a summary, the thesis is composed of six chapters, wherein, the main studies and findings are found in Chapter 3, Chapter 4, and Chapter 5.

In Chapter 1, the current problems and solutions about liver tissue engineering were discussed and how the study was conceptualized. In addition, research objectives and main hypothesis were itemized in order to establish a clear target and well-defined concept.

In Chapter 2, related studies were presented to provide in-depth understanding of the study. Furthermore, previous reports and recent advances in the use of liver-specific extracellular matrix (L-ECM) were shown to emphasize the novelty and the much-needed technology for liver tissue engineering.

In Chapter 3, the protocol for producing solubilized L-ECM was modified to create an L-ECM with intact ECM components. The resulting L-ECM scaffold had higher collagen content, denser fiber network, and higher compression strength. The efficiency of this L-ECM was investigated in three-dimensional (3D) and two-dimensional (2D) culture in vitro using primary rat hepatocytes. Results showed the formation of a wider hepatocyte spreading in 2D culture and enhanced liver-specific functions in 3D culture.

In Chapter 4, the solubilized L-ECM, gelatin, polycaprolactone were used to fabricate non-woven fabrics using conventional electrospinning. We termed the scaffold as “L-ECM nanofiber” because L-ECM was incorporated to enhance the bio-functionality as substrate for in vitro culture. Primary rat hepatocytes were seeded to L-ECM nanofibers to determine how L-ECM influence cell interactions in vitro. For in vivo implantation, the 100 µm thick L-ECM nanofiber sheet was made into thicker scaffold by incorporating L-ECM hydrogel using layer-by-layer method. Three layers of L-ECM nanofibers sheet were assembled to create a 1 mm thick scaffold, herein, termed as “L-ECM gel sheet”. The L-ECM gel sheet was implanted to partial hepatectomy treated rat and CCl4-induced fibrosis rat model to determine the effectiveness.

In Chapter 5, coaxial electrospinning using the solubilized L-ECM, gelatin, polycaprolactone was used to fabricate a L-ECM core shell nanofiber sheet. The shell part is composed of L-ECM and gelatin while the core part is composed of polycaprolactone. The shell component contains important molecules that will be directly in contact with the cells and/or tissues and the core component will act as a mechanical support. Mesenchymal stem cells were seeded to the scaffold to determine the efficacy in in vitro culture.

In Chapter 6, conclusions were drawn to summarize the important results of the study.

Also, future plan was discussed and how future experiments will be conducted.

In Chapter 7, relevant references were listed.