利用明膠泡泡支架探討孔徑大小對於人類脂肪幹細胞軟骨分化的影響

Abstract

人體中的軟骨組織在遭遇到創傷導致受損時並不具有自瘉的能力，所以有許多關於自體軟骨細胞移植的研究產生。但是，這樣的移植需要相當高的細胞數量，而軟骨細胞在一般細胞培養過程中容易喪失其分化的表現型，因此，以幹細胞進行分化並生成大量軟骨細胞及組織便成為一個可能的解答。在本實驗中，我們選用人類脂肪幹細胞(hASC)做為細胞模型。 在組織工程中，3D多孔性材料是一個常用的模版，有許多研究在談討細胞最佳的分化條件。在這些議題中，孔徑大小時常被提出來討論，因為在傳統研究中生醫材料的製備多採用凍乾或者是顆粒浸出的方式，因此製備出的多孔性生醫材料在孔徑大小並不是那麼的精確。在這份研究中，我們利用微流體技術製作出四種不同孔洞大小的均一圓球狀之三維明膠泡泡支架以模仿細胞在生物體內的生長環境，並比較在不同孔洞大小對於細胞在生長及分化上的影響。 在過去的研究中，曾經有探討過此材料對於hASC硬骨及脂肪分化的影響，因此這份研究專注在孔徑大小對於軟骨分化表現的影響，首先我們利用Live/Dead染色來判斷這些孔徑大小皆能讓細胞有良好的存活率，接著觀察細胞的型態、生長速率並比較孔徑大小對於細胞生長的影響，再對細胞進行分化並以螢光免疫染色和基因表現的定量來比較及觀察孔徑大小對於細胞分化的影響。結果顯示，在最大孔徑，也就是在孔洞直徑為200μm下有著最好的分化效果，並由分化的細胞型態來分析，可能是由於hASC在軟骨分化的型態為聚集成球狀，而大孔徑給予細胞足夠的空隙來聚集進而分化成軟骨細胞，未來將對於這樣的推測進行驗證。

In human bodies, cartilage tissue lacks in the ability to heal when encounters trauma or lesion. This inability of self-repair motivates all sorts of study among autologous chondrocyte transplantation. However, the difficulty of high chondrocytes concentration is hard to be overcome due to the loss of differentiated phenotype of chondrocytes during cell culture. The use of stem cells to differentiate into chondrocytes has been a possible solution to provides large number of differentiated chondrocytes. In this study, human adipose-derived stem cells (hASC) is chosen as the model to further differentiate into chondrocytes. In tissue engineering, 3D porous scaffolds are frequently used as the substrate due to its similarity with microenvironments in organisms. Studies about the influence of porous biomaterials toward cell behavior were made to discuss the best condition for stem cell differentiation. Among these topics, pore size is a factor which is commonly discussed. In traditional studies, the preparation of porous biomaterials was either by freeze-drying or particle leaching, which leads to non-uniformity in pore size. In our study, we fabricate four gelatin microbubble scaffolds with different pore size but is uniform and spherical in pores by microfluidic techniques. Then, we compare the influence of pore size toward cell growth and differentiation. In a previous study, adipogenesis and osteogenesis of hASC in this scaffold had been studied. Therefore, we focus on the influence of pore size toward chondrogenesis in this study. Live/Dead was used to confirm the high cell viability of hASC in our scaffolds. Then, cell morphology and proliferation was observed. Finally, chondrogenesis was induced to hASC. Immunofluorescence staining and qPCR was performed to investigate the influence of pore size toward chondrogenic differentiation. According to the experimental results, the largest pore size, which is 200 μm in diameter shows the best chondrogenesis result. This result is possibly due to the aggregation during chondrogenesis. The vacancy in large pore size provides sufficient space for hASC to aggregate. Our future work will focus on the confirmation of this speculation and myogenesis in the scaffolds.