以甲殼素為基底之溫敏性水膠作為釋放人類脂肪幹細胞載體之研究

Abstract

在再生醫學研究中，運用溫敏性水膠將幹細胞進行包埋以增加細胞存活率是極有潛力的治療方式。然而，此法卻亦可能限制了細胞彼此交互作用的能力。因此本研究為了探討幹細胞之釋放是否能有助於傷口修復，使用在幾丁聚醣溫敏性水膠中加入明膠及凱索尼酸等親水性材料進行改質。期望藉由混摻親水性物質於材料中形成孔洞，進而探討幹細胞釋放應用於傷口修復和血管新生能力，是此研究最主要的目標。 在保有幾丁聚醣水膠之溫敏感性條件下，實驗中選擇混摻明膠4%以及凱索尼酸1%作為幾丁聚醣水膠之改質，並由掃描式電子顯微鏡（SEM）下觀察出混摻兩種親水性材料將使幾丁聚醣水膠形成較多孔洞。其中，明膠的混摻濃度又與孔洞的形成呈正相關。在計數每日細胞釋放量後，混摻明膠除了形成孔洞有助於脂肪幹細胞之釋放趨勢外，在提高細胞存活率及增生方面皆有較高之表現。 至於此細胞釋放機制是否能用於傷口修復，研究中以纖維母細胞（Hs68）以及包覆有脂肪幹細胞之幾丁聚醣-明膠材料進行共培養，可得脂肪幹細胞之釋放確實能使傷口較快修復。在血管新生方面，實驗中以體外共培養SVEC4-10觀察結果顯示，當脂肪幹細胞包覆於幾丁聚醣水膠中確實能使SVEC4-10形成網狀管柱結構，代表其確實具血管新生之潛力。在體內實驗部分，本實驗以雞胚絨毛尿囊膜（CAM）進行血管新生探討，在定量新生微血管後得出脂肪幹細胞的釋放確實能增加血管新生方面能力。因此，本研究推測幹細胞脂釋放將有助於應用在未來生醫材料發展。

In the research of regenerative medicine, it is promising treatment to apply thermosensitive hydrogel encapsulated with stem cells to improve the cell viability. However, the application might limit the effect of cell-cell interaction. The present study focuses on whether the adipose stem cells (ASC) release would benefit wound healing. Therefore, the thermosensitive chitosan hydrogel was blended with hydrophilic material such as gelatin and chitosonic acid (CA) in order to form porous structure. The research aims to construct the stable ASC release systems and to discuss the effect of wound healing and angiogenesis ability. To maintain thermosensitive character of chitosan hydrogel, the study blended chitosan with 4% gelatin and 1% chitosonic acid, and the hydrophilic materials resulted in porous structure under the scanning electron microscope (SEM) image. We found that the blending concentration of gelatin was proportional to the pores of chitosan hydrogel. According to the number of released cells from the hydrogel, the gelatin blending with porous structure was not only beneficial to the effect of ASC release profile, but also enhanced viability and proliferation of ASC. As for the function of wound healing to ASC release systems, the present study demonstrated that the release of ASC could facilitate wound closure by co-culturing fibroblast (Hs68) with ASC encapsulated in chitosan-gelatin hydrogel. In terms of angiogenesis, the in vitro study of co-culture endothelial cell (SVEC4-10) with ASC release systems observed SVEC4-10 differentiated into network tube formation, indicating it had potential in angiogenesis. For in vivo study, the angiogenesis ability was confirmed with chick embryo chorioallantoic membrane (CAM) assay. The quantification of the average capillary density showed that ASC released from chitosan hydrogel promoted angiogenesis and wound healing in tissue engineering.