藉由調控3D基材的軟硬度來誘導間葉幹細胞朝向神經細胞系之分化

Control of Mesenchymal Stem Cell Fate toward Neural Lineages with Tunable 3D Substrate Modulus

吳繼禾(GOH JIH HER)

指導教授 ： 王子威

國立清華大學/工學院/材料科學工程學系/碩士(2012年)

https://doi.org/10.6843/NTHU.2012.00006

摘要

幹細胞的無限再生與其強大的分化能力使它在組織工程與再生醫學領域上的應用備受矚目。幹細胞可分化成各種不同的組織細胞，誘導幹細胞分化的因素有許多，其中包括以組織替代物為最終目標之仿生材料其本身的基本特性, 如物理性質、化學特性、生物因子等等。我們希望藉由了解幹細胞與仿生材料之間的交互作用並利用以上特性，期望得以控制幹細胞分化的方向，進而替換在生物體成長過程中不會再生或損傷的細胞，最終達到治療損傷組織的效果。在本研究當中，我們利用天然材料－透明質酸作為主要的材料並與第一型膠原蛋白結合，透過真空冷凍乾燥技術製造成一個三維多孔性結構的細胞支架，藉由控制交聯劑濃度改變基材軟硬度，使培養在此細胞支架內的人體間葉幹細胞可朝向神經細胞系的方向生長與分化。
本實驗分為二大部分，第一部分為材料製作與各特性分析，第二部份為細胞培養並利用此細胞支架特性來誘導間葉幹細胞朝向神經細胞系方向分化，探討不同軟硬程度的細胞支架對於間葉幹細胞朝向神經細胞系方向分化的影響。不同軟硬程度細胞支架的調控，我們利用不同濃度的EDC碳二亞胺交聯劑來調控材料的彈性係數(E)，使此細胞支架的彈性係數落在1kPa以及10 kPa的範圍，以定義細胞支架軟材質和硬材質兩大範圍。在細胞支架的製作過程，透過材料的物化性分析，如微拉力試驗機測試不同交聯濃度的細胞支架之機械性質、掃描式電子顯微鏡以觀察各細胞支架的微結構、TNBS測試以取得各EDC交聯濃度的細胞支架之交聯程度、Carbazole測試來測量細胞支架內透明質酸的釋放量等等。與此同時，我們將人體間葉幹細胞培養在細胞支架上，以檢測細胞的型態、生長、增生與分化的情形。
本實驗主要的目的是利用經由各濃度的EDC交聯劑所形成的不同軟硬程度之透明質酸－第一型膠原蛋白細胞支架來誘導與探討人體間葉幹細胞分化成神經細胞的可行性。藉由此種可調控軟硬程度的仿生材料，我們得以探討細胞與基材的交互作用並控制幹細胞分化的結果，如朝向神經細胞系方向分化，對於目前幹細胞治療的研究以及對損傷的神經組織再生修復，非常具有臨床應用價值。

關鍵字

間葉幹細胞 ； 透明質酸 ； 細胞支架 ； 軟硬度 ； 神經細胞

並列摘要

Unlimited self-renewal of stem cells and their multipotency ability lead a great potential in the application of tissue engineering and regenerative medicine. The induction of stem cells can be controlled by multiple factors including physical, chemical and biological cues. By knowing the interaction between stem cells and their vicinity biomimetic microenvironment, we may manipulate stem cell’s fate. In this study, three dimensional porous scaffolds were synthesized by type I collagen (Col) and hyaluronic acid (HA). The elastic modulus (E) of the 3D substrates was modified by adjustable concentrations of 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) crosslinking agent. The purpose of this study is to investigate the matrix stiffness on the influence of neurogenic differentiation of human mesenchymal stem cells (hMSCs). The mechanical property of Col-HA scaffolds was evaluated and the induction and characterization of hMSCs differentiation toward neural lineages by different substrate stiffness were studied. With different EDC crosslinking concentration, the stiffness of the matrices can be tunable in the ranges of 1 kPa to 10 kPa for soft and stiff substrates. The results found that MSCs tend to differentiate into neuronal lineage in substrate at 1 kPa, while they transform into glial cells in matrix with 10 kPa. The morphology and proliferation behavior of hMSCs were corresponded to substrate stiffness. By using this modifiable matrix, we can investigate the relationship between stem cell behavior and substrate mechanical properties in ECM-based biomimetic 3D scaffolds. A tunable substrate stiffness that would induce hMSCs specifically toward neuronal differentiation may also be very useful as tissue-engineered construct or substitute for delivering hMSCs in brain and spinal cord regeneration.

並列關鍵字

mesenchymal stem cell ； mechanical property ； 3D scaffold ； matrix stiffness ； neuronal differentiation

參考文獻

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