幹細胞於功能性胜肽材料之軟骨分化培養

Abstract

近年來，再生醫學及組織修復的觀念逐漸興起，許多研究皆致力於幹細胞及奈米材料上的結合及應用，從高分子支架甚至是小分子水凝膠的研究都逐漸熱門起來。於本研究中，欲探討幹細胞於小分子自組裝之功能性胜肽序列是否具有促進軟硬骨分化之成效，我們進行一系列的共培養及分析來驗證。第一部分研究探討幹細胞培養於五種由N-caherin上延伸出來之功能性胜肽序列時的軟骨分化情況，首先探討細胞於二維胜肽混合溶液的分化情形，實驗過程中分別培養7、14、21天來觀察分化的情況表現，我們透過Alcian Blue的染色及qPCR的定量分析發現於二維胜肽混合溶液條件下，以HAVD及HAVDI這兩組序列能夠表現出較多的軟骨特異性基因，如Collagen type II及Collagen type X；然而，若當細胞培養於三維胜肽水凝膠體中時，HAVD及HAVDI之軟骨特異性基因表現量相對於HAVS組的表現量低。進一步我們藉由調控基材的堅韌度，發現基材堅韌度固定後，各組的軟骨特異性基因表現均上升，且以HAVS表現最為突出。從此結果我們推斷當細胞培養於三維環境時，HAVS胜肽序列相較於其他序列具有較強的驅動力，促使細胞趨向軟骨分化的潛力 。

Regenerative medicine is becoming more popular in tissue repair. Several researches have been focusing on combining stem cell and biomaterials for tissue regenerations. Materials ranges from polymer scaffold to even small molecular weight hydrogel all have been to have some tissue engineering applications. In this study, we focused on the ability of self-assembling peptides, and hypothesized that they can promote cell chondrogenesis and osteogenesis. We implemented a series of experiments and analysis to confirm our hypothesis. Firstly, we designed five different functional sequences derived from N-cadherin, and investigated their capability in inducing chondrogenesis after exposing them to a mesenchymal stem cell (MSC) line The levels of chondrogensis were determined by Alcian blue staining and qPCR analysis after culturing the cells with the peptides at a 2D environment for 7、14、21 days. The data showed that either HAVD or HAVDI treated cells expressed more chondrocyte specific genes such as Collagen type II and Collagen type X compared to negative control. However, when the cells were exposed to either HAVD or HAVDI in 3D hydrogel, lower expressions of chondrocyte specific genes were detected compared to HAVS treated cells. Furthermore, the expression of chondrocyte specific gene could increase by modulating the peptide-based material stiffness , in which HAVS containing hydrogel showed the best capability in inducing chondrogenesis after stiffness modulation.