調控支架孔洞性於血管增生應用暨肝纖維化修復治療之研究

Effects of scaffold porosity on angiogenesis and its potential application on hepatic fibrosis therapy

吳宜真(Yi-Jhen Wu)

指導教授 ： 郭士民

共同指導教授 ： 莊景文(Chin Wen Chuang)

義守大學/電機資訊學院/電機工程學系/博士(2018年)

摘要

一般而言，具孔洞的3D支架可提供較多的表面積與其通透性的優點，目前也常被用來培養細胞，以發展成類再生組織。此一3D支架除了材料本身的性質外，孔洞的大小、分佈與形式均會影響細胞貼附生長與分化。目前，大孔洞（＞300 µm）及擁有良好連通性的3D支架，製備上有其挑戰及研究價值，尤其是天然高分子膠原蛋白材料。論文實驗中研製不同孔洞尺寸及孔洞分佈的膠原蛋白支架，並進行細胞培養、血管新生於肝臟組織修復之研究。研究結果顯示，以一般冷凍乾燥方式可製備出最大約80 μm孔洞尺寸之具連通性膠原蛋白支架。為增加膠原蛋白支架的孔洞尺寸，論文中利用電場系統製備褐藻酸微粒作為致孔劑，以褐藻酸微粒作為致孔劑所製備的膠原蛋白支架其最大孔洞尺寸可達約500 μm。論文中所製備的膠原蛋白支架連通性佳、含水量可達99%，改變褐藻酸微粒致孔劑的尺寸可製備出孔隙率70%-90%之膠原蛋白支架。細胞培養之SEM觀察結果顯示單純人類脂肪幹細胞在大孔洞膠原蛋白支架上生長狀況較，超音波刺激後其細胞數目無明顯增加。但超音波刺激在人類脂肪幹細胞/肝細胞/血管內皮細胞共培養中卻可促進細胞之吸附及生長，混合孔洞膠原蛋白支架與小孔洞膠原蛋白支架較利於細胞吸附生長。綜合以上結果，幹細胞生長與其分化所需的孔洞大小不盡相同，或可能是用於共培養的特性造成。此外，超音波刺激會表現出較明顯的細胞生長現象，但膠原蛋白支架經超音波刺激後會快速破碎分解。雷射掃描共軛焦顯微鏡觀察人類脂肪幹細胞在培養28天後仍出現其未分化特異性蛋白CD49d表現。在血管新生的動物實驗中，受到超音波刺激的支架在植入SD大鼠體內4週後有明顯的血管增生現象。另外，在肝臟修復的動物實驗中，小孔洞之膠原蛋白支架上有新生肝細胞及血管新生的現象。

關鍵字

孔洞性支架 ； 膠原蛋白 ； 血管新生 ； 肝修復 ； 超音波刺激

並列摘要

In general, Porous 3D scaffold has merits of providing larger surface and interconnection for cell growth and thereafter to generate tissues. The properties, pore size, pore geometry and the interconnection of pores all affect the cellular growth and differentiation of culture cells. Till today, It is very difficulty to prepare big pore size （＞300 µm）and good interconnection 3D porous scaffolds, especially for collagen biomaterials. In this present study, we prepared collagen scaffolds with various pore sizes and geometries and to evaluate the effects on cultured cells, angiogenesis and liver fibrosis repair. The obtained results indicated that we could produce average 80 µm pore size and good interconnection 3D collagen scaffold. Furthermore, we could produce more large pore size, about 500 µm, collagen scaffold by using alginate microspheres as porogen materials. The prepared collagen scaffolds all with good interconnection and with 99% water content. The porosity could be varied by using different alginate microspheres porogen and with a range of 70% -90%. The cell culture results showed that ADSCs grew well on large pore size collagen scaffolds. The ADSCs number did not change significantly after ultrasound stimulus. From SEM observation, the cell number increased obviously on ADSCs/Liver cell/HUVECs co-culture groups after ultrasound stimulus. The co-cultured cells grew better on the small pore size/mixed pore size collagen scaffolds; besides, ultrasound stimulus would influence the growth of cells. From confocal microscopy observation, the ADSCs expressed un-differentiated CD49d protein expression after 28-d culturing. The SD rats had obvious angiogenesis on the collagen scaffolds that implanted in the dorsal site after 4-w ultrasound stimulus. The liver repair and angiogenesis also appealed on rats that fibrotic liver implanted with small pore size collagen scaffolds.

並列關鍵字

Porous scaffold ； collagen ； angiogenesis ； liver repair ； ultrasound stimulus

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