以聚異丙基丙烯醯胺為基材之溫感性水膠材料於三維細胞培養之應用

本研究主要是以三類溫感型水膠材料做為體外三維細胞培養之材料評估，除了進行物化性質探討之外，也結合了老鼠骨髓幹細胞株D1來進行細胞活性與分化能力之比較。此三類溫感型水膠是以異丙基丙烯醯胺(N-isopropylacrylamide, NIPPAM)為主要材料，第一類是以甲基丙烯酸(Methylacrylic acid, MAA)將p(NIPPAM)修飾上Carboxyl group形成具溫感特性的線性高分子p(NIPPAM-co-MAA)。第二類則利用第一類p(NIPPAM-co-MAA)與多胜肽聚離胺酸(polylysine, PL)交聯形成網狀的複合高分子p(NIPPAM-co-MAA)-PL。最後第三類是以第二類p(NIPPAM-co-MAA)-PL再結合透明質酸高分子 (Hyaluronic acid, HA) 共價形成p(NIPPAM-co-MAA)-PL-HA複合型水膠。其中第二、三類溫感型水膠是結合生物性多胜肽及透明質酸來增加材料的生物相容性與細胞分化活性。水膠合成之化學結構是以核磁共振(NMR)、紅外線光譜儀(FTIR)及分子量以液相層析(LC-MS)來分析。在水膠物理性質方面是利用UV/VIS及黏度儀探討了水膠的相轉變溫度及其過程的黏度變化，並利用了電子顯微鏡(SEM) 觀察水膠內部細微結構，同時也探討了三種不同水膠的含水率。三維水膠對於體外細胞培養之細胞毒性、細胞骨分化與軟骨分化評估是以老鼠骨髓幹細胞株D1細胞株進行，在不需使用任何酵素時，使細胞脫附於水膠表面將細胞回收評估。本研究初步結果顯示此三類水膠材料之相轉變溫度約為攝氏32℃~33℃，且10%以上的水膠濃度具有較穩定的凝膠性。SEM觀察凝膠後的三種溫感水膠內部均為一高孔洞隙之材料，且第三類溫感水膠p(NIPPAM-co-MAA)-PL-HA，其10%濃度時含水率可高達90%以上。細胞活性評估顯示此三類溫感水膠均無細胞毒性，但以結合HA溫感水膠p(NIPPAM-co-MAA)-PL-HA之細胞活性最高。初步幹細胞分化能力實驗顯示D1細胞在PNM-PL水膠環境中的骨化能力較好，但軟骨化能力則在三類水膠中則都可明顯觀察出，其中含HA溫感水膠p(NIPPAM-co-MAA)-PL-HA之軟骨化似乎更好。

關鍵字

溫感型水膠；三維細胞培養；聚異丙基丙烯醯胺；聚離胺酸；透明質酸；硬骨化；軟骨化

並列摘要

In this research, I have synthesized thermo-responsive hydrogel for three-dimensional cell culture. I have characterized the properties of hydrogel, and the proliferation of mouse bone marrow stem cell in the hydrogels was compared. Poly(N-isopropyacrylamide)(pNIPPAM), a thermo-responsive hydrogel, was modified with carboxyl group by methylacrylic acid(MAA) to give p(NIPPAM-co-MAA) linear polymer. In addition, the productwas grafted with polylysine (PL) to network copolymer was p(NIPPAM-co-MAA)-PL, finally, it combined with hyaluronic acid(HA) to p(NIPPAM-co-MAA)-PL-HA for biodegradable. The chemical structure analysized by NMR and FTIR, the molecular weight determined by LC-MS. For physical property, we used UV/VIS spectrometry and viscometer to LCST and its vary of viscosity. The hydrogel microsturcture inside observed by scanning electron microscope(SEM), and the water content of hydrogel. Moreover, we used mouse bone marrow stem cell, D1 cell line to biological in three-dimensional cell culture and performed MTS assay to cototoxicity, and cell differentiation. These result shown that LCST of hydrogel was about 32℃, and the gelationwas stable. the hydrogel was high porous structure. The p(NIPPAM-co-MAA)-PL-HA hydrogel was higher water content to 90% and highest growth activity for D1 cell line. The PNM-PL hydrogel has best capacity of osteogenesis, and chondrogenesis in all hydrogel, and combine with HA seems better.

並列關鍵字

thermo-responsive hydrogel ； three-dimensional cell culture ； Poly(N-isopropyacrylamide) ； polylysine ； hyaluronic acid ； osteogenesis ； chondrogenesis

參考文獻

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以聚異丙基丙烯醯胺為基材之溫感性水膠材料於三維細胞培養之應用

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