以甲殼素生物水膠為基底之微組織培養技術之開發

Abstract

利用生物3D列印技術在體外建立具有功能性的3D仿生組織作為藥物學與生物研究的模型是組織工程領域中的一大目標。其中，如何建立出仿生組織具有與體內相似的微血管網絡，並具有提供組織氧氣、營養與代謝的功能，至今依然是一大挑戰。在組織工程中，具有適當的基材及三維細胞支架結構的生物水膠，在體外可培養出具有功能性的血管網絡成為類組織尤為重要。目前建構血管網絡的方法是將血管內皮細胞及基質細胞加入具有細胞外基質的水膠，於體外培養出血管組織，例如: fibrin gel、collagen gel、matrigel等等，前述的水膠機械特性皆不適合用做3D列印水膠。然而目前適合使用在3D列印技術的生物水膠配方，鮮少具有誘導血管新生的潛力。 本研究主要探討可3D列印之甲殼素水膠作為基底與細胞外基質fibrin gel 、collagen gel與aprotinin混合之微組織培養性質，並發展出具有良好機械性質、及具有誘導血管新生潛能的生物水膠。另外此研究亦探討本水膠的各成份摻混順序影響微血管組織生長型態。此外，本研究開發了一組織培養系統。以具有生物相容性的聚二甲基矽氧烷(PDMS)作為水膠的注膠模具，並設計壓克力材質的底座與培養液槽，最後使用不鏽鋼螺絲將之固定。實驗結果顯示第五天時，對組織進行螢光染色可觀察到血管組織。本研究的新型生物水膠未來將結合組織晶片與微流道的概念發展出可作為藥物篩檢之微系統裝置、及作為生物3D列印技術之材料。

The aim of the 3-D bio-printing is to build functional 3-D biomimetic tissues in vitro. However, the current challenge is o build biomimetic tissues that can provide functional vasculature to supply oxygen, nutrition and metabolism similar to microvascular network in vivo. In tissue engineering, it is important that 3-D gel contains appropriate matrices, and 3-D cell scaffold has functional vessel network in vitro. Currently, the method of building microvascular network is to use hydrogel composed with extracellular matrix (ECM) embedded with endothelial cells and stromal cells. However, common ECM-based hydrogel like fibrin gel, collagen gel, and matrigel are not suitable for 3-D printing process. On the other hand, not all of 3-D printable gels have the potential for induction of angiogenic and vasculogenic process. In this thesis, the 3-D printable chitosan gel was used as the base of the 3-D printable hydrogel, and different extracellular matrix is added to study their contribution to the development of vasculature. The extracellular matrix been studied includes Fibrinogen and collagen-I. It is found that vasculogenic process is hard to be induced in a mixture of chitosan and fibrin gel. But, the level of vessel formation is increased with mixtures composed of both fibrinogen and collagen-I fibers. Using a bioreactor developed in this study to create a hypoxic environment for stimulating vasculogenesis, it is found that the addition sequence of chitosan, fibrinogen, and collagen-I can influence the development of vasculogenic process. After comparing the developed vessel structures on Day-5, it is found that a better vessel formation can be created by adding fibrinogen between chitosan and collagen-I. Discussion on the experimental findings and statistical analysis on the developed vasculature are detailed in this thesis.