幾丁聚醣/明膠/聚氧化乙烯電紡複合纖維膜之製備、滲透率及強度之改善、及其於細胞培養之應用

Abstract

本研究於幾丁聚醣(簡稱C)/明膠(簡稱G)複合溶液中添加少量的超高分子量聚氧化乙烯(PEO，簡稱P)，藉由靜電紡絲法將上述溶液製成以幾丁聚醣/明膠為主之電紡纖維。本研究利用無毒性及腐蝕性較低之醋酸水溶液作為幾丁聚醣之溶劑，取代常用於靜電紡絲的高毒性及高腐蝕性之溶劑，如三氟乙酸(TFA)或六氟異丙醇(HFIP)等。其次則利用兩種不同溶液同時進行電紡，形成粗、細兩種纖維，進而提高電紡纖維膜之滲透率，藉此有助於養分及代謝廢物進出纖維膜，以提供細胞較佳生長環境，並提升細胞增殖速率。在研究中探討最適之電紡溶液組成及電紡參數，並分析電紡纖維膜之顯微結構、機械性質、水相環境中穩定性及滲透性質等特性，最後以人類骨髓間葉幹細胞(KP-hMSCs)及人類纖維母細胞(WS1)進行纖維膜之細胞相容性測試及探討纖維膜滲透性質改變對細胞增殖速率之影響，據以評估此電紡纖維膜在組織工程相關應用之潛力。 首先利用不同成分比例及濃度之幾丁聚醣/明膠/聚氧化乙烯複合溶液進行靜電紡絲以得到奈米纖維，並觀察溶液組成及電紡參數對電紡纖維的影響。研究結果發現若在總濃度為3 wt%幾丁聚醣/明膠複合溶液中添加0.3 wt%之聚氧化乙烯即可在以5 wt%醋酸水溶液為溶劑之條件下以靜電紡絲法製備出電紡纖維，但此時所製備出之電紡絲纖維仍具有紡錘狀之結構。若將複合溶液總濃度提升至5 wt%及10.5 wt%，即可在以5 wt%及20 wt%醋酸水溶液作為溶劑之條件下得到良好且無顆粒及紡錘狀結構之電紡纖維。若針對不同組成所得到之電紡纖維進行分析，則可發現電紡纖維直徑及纖維膜之抗拉強度及極限應力大致隨著複合溶液濃度增加而上升。其中複合溶液總濃度10.5 wt%(幾丁聚醣：5 wt%、明膠：5 wt%、聚氧化乙烯：0.5 wt%)且以20 wt%醋酸水溶液為溶劑之組別(簡稱CGP)具有最大之纖維直徑及抗拉強度與極限應力。若利用Reynolds number (Re)、Capiilary number (Ca)及Weber number (We)三個無因次群對各種不同成分組成及濃度之複合溶液靜電紡絲結果進行分析，則可發現，可以經由靜電紡絲法得到較佳電紡纖維之複合溶液，在We-Re之關係圖上，呈現一斜直線之分布。 其次將CGP電紡纖維膜利用不同之物理及化學方式進行抗拉強度、極限應力、穩定性及滲透性之改善。經熱處理後，CGP纖維膜之抗拉強度、極限應力及伸長率分別由265 N/g、2.68 MPa及4.5%，上升至430 - 450 N/g、4.1 - 4.4 MPa及7.2% - 8.0%。若利用改變收集器轉速使電紡纖維具方向性排列，則在收集器轉速為1000 rpm的情形下，其平行纖維排列方向之抗拉強度及極限應力大幅上升至660 N/g及8.1 MPa，約為不具方向性排列纖維膜抗拉強度(265 N/g及2.68 MPa)的2.5- 3.0倍。除了以上兩種方法外，也利用戊二醛交聯CGP電紡纖維膜，同時提升纖維膜之抗拉強度及在水相環境中之穩定性。結果發現，在利用戊二醛蒸氣交聯2.5小時後，纖維膜之抗拉強度及極限應力可由未交聯之265 N/g及2.68 MPa提升至450 N/g及4.59 MPa，浸泡磷酸緩衝液(PBS)後之殘餘重量(代表纖維膜在水相環境下之穩定性)也由未交聯之19%上升至72%，顯示利用戊二醛交聯可有效同時提升纖維膜之抗拉強度及在水相環境中之穩定性。為改善CGP纖維膜之滲透性，本研究利用Eudragut®溶液(簡稱為E)與CGP複合溶液同時進行雙針電紡(dual-needle electrospinning)，而得到兩種粗細不同纖維混紡而成之CGP/E纖維膜，其中CGP纖維較細，而E纖維較粗。CGP/E纖維膜其孔隙度約為88.1%，大於CGP纖維膜之84.8%，顯示將E纖維混紡於CGP纖維中可以增加纖維膜中孔洞大小，提供更多空間。若利用95%乙醇溶解並去除纖維膜中之E纖維，則可以降低纖維膜中之纖維密度，形成更鬆散之CGP (E removed)纖維膜。根據滲透率測定結果顯示，牛血清白蛋白(bovine serum albumin，BSA)滲透通過CGP纖維膜之滲透率約為1.50  10-11 m2/s，若與E纖維進行混紡，形成之CGP/E纖維膜之滲透率數提升至6.19 - 10-11 m2/s。以95%乙醇去除E纖維後所形成之CGP (E removed)纖維膜之滲透率可進一步提升至1.53 - 10-10 m2/s，約為CGP纖維膜之滲透率之10倍。 最後是利用間葉幹細胞(KP-hMSCs)測試CGP纖維膜之細胞相容性，並利用將纖維膜覆蓋於KP-hMSCs及人類纖維母細胞(WS1)之細胞培養方式(模擬細胞在纖維膜內部之微環境)，測定細胞增殖情況，以觀察上述CGP纖維膜之滲透性質改善技術對細胞生長及增殖之影響。結果發現，KP-hMSCs培養於CGP纖維膜上呈現紡錘狀，顯示貼附及生長情況良好，表示CGP纖維膜對細胞沒有毒性，適宜作為細胞培養之基材。而透過將纖維膜覆蓋於細胞上方之細胞培養方式，可以發現覆蓋CGP (E removed)纖維膜組别由於該纖維膜滲透率較大，養分及代謝物較易通過，因此KP-hMSCs及WS1細胞皆有較大之增殖速率，和覆蓋CGP纖維膜組别相比，在覆蓋4層纖維膜(濕膜厚度約160 m)之情形下，覆蓋CGP (E removed)纖維膜組别之KP-hMSCs及WS1細胞之細胞數量分別約為覆蓋CGP纖維膜組别之1.6  1.7及1.9倍。 總而言之，本研究利用靜電紡絲法在以低濃度醋酸水溶液(20 wt%)為溶劑之條件下，成功製備幾丁聚醣/明膠/聚氧化乙烯電紡複合纖維，且聚氧化乙烯所佔比例極低，約為材料組成的5 wt%。該電紡纖維經戊二醛交聯後具有較佳之抗拉強度、水相環境中之穩定性及良好之細胞相容性，並可利用與Eudragit®纖維混紡甚至再移除E纖維等方式，大幅改善纖維膜之滲透性質，以提高細胞增殖速率，因此該電紡纖維具有在組織工程方面應用之良好潛力。

In this research, a small amount of ultra-high molecular weight polyethylene oxide (PEO) was added to chitosan (C)/gelatin (G) composite solution in order to obtain chitosan/gelatin based nanofibers by electrospinning. Besides, non-toxic and low-corrosive acetic acid was used as the solvent of chitosan to replace toxic and corrosive solvents commonly used in electrospinning, such as trifluoroacetic acid (TFA) or hexafluoroisopropanol (HFIP). Also, two different solutions were co-electrospun to form fibers of large and small diameters simutaneously, thereby increasing the permeability of the electrospun nanofiber membrane. The more permeable nanofiber membrane was beneficial to nutrients and metabolic wastes transport and thus provided a better cell growth environment. The optimal conditions for electrospinning were investigated, and the microstructure, mechanical properties, stability in aqueous environment and permeability of the nanofiber membrane were analyzed. Finally, the human mesenchymal stem cells (KP-hMSCs) and human fibroblasts (WS1) were cultured for cytocompatibility test of the membrane, and also for exploring the influence of the more permeable membrane on the cell proliferation rate, so as to evaluate the potential of this electrospun nanofiber membrane in tissue engineering applications. First, chitosan/gelatin/polyethylene oxide composite solution with different compositions and concentrations were used to find the optimal condition for preparing nanofibers by electrospinning. The diameter of the electrospun nanofibers, tensile strength and ultimate stress of the nanofiber membranes increased with the increase in the concentration of the composite solution. The use of the composite solution with total concentration of 10.5 wt% (chitosan: 5 wt%, gelatin: 5 wt%, polyethylene oxide: 0.5 wt%) and a 20 wt% acetic acid aqueous solution used as a solvent gave the largest average fiber diameter and the highest tensile strength and ultimate stress. Further, three dimensionless groups, Reynolds number (Re), Capillary number (Ca) and Weber number (We), were used to analyze the electrospinning results under various conditions. It was found that uniform electrospun nanofibers were obtained in a slash-like area on the diagrams of (We vs. Re). Second, different physical and chemical methods were used to enhance the tensile strength, ultimate stress, stability and permeability of electrospun chitosan/gelatin/polyethylene oxide (CGP) nanofiber membranes. After heat treatment, the tensile strength, ultimate stress and elongation of CGP nanofiber membranes increased from 265 N/g, 2.68 MPa and 4.5% to 430  450 N/g, 4.1  4.4 MPa and 7.2%  8.0%, respectively. By increasing the collector rotating speed to 1000 rpm to obtain aligned nanofiber membranes, the tensile strength and ultimate stress in the fiber direction was drastically increased to 660 N/g and 8.1 MPa, which is approximately 2.5  3.0 times the tensile strength and ultimate stress of random nanofiber membrane (265 N/g and 2.68 MPa). Moreover, glutaraldehyde was also used to crosslink electrospun CGP nanofiber membranes for enhancing their tensile strength and stability. The tensile strength and ultimate stress increased from 265 N/g and 2.68 MPa (uncrosslinked) to 450 N/g and 4.59 MPa, and the stability (remaining weight after immersion in phosphate buffered saline) rose from 19% (uncrosslinked) to 72% after crosslinking for 2.5 hours. In order to improve the permeability of CGP nanofiber membrane, Eudragut® (E) solution and CGP composite solution were co-electrospun by dual-needle electrospinning, and a CGP/E nanofiber membrane containing small-diameter CGP and large-diameter E fibers was obtained. The porosity of CGP/E nanofiber membrane was about 88.1%, greater than that of CGP nanofiber membrane (84.8%), indicating that blending the large-diameter E fibers with the CGP fibers provided more space in the nanofiber membrane. Further, after using 95% ethanol to dissolve and remove the E fibers from CGP/E membrane, the fiber density was reduced to form a looser CGP (E removed) nanofiber membrane. The permeability of bovine serum albumin (BSA) through CGP membrane was about 1.50  10-11 m2/s, which increased to 6.19  10-11 m2/s for CGP/E membrane, and increased further to 1.53  10-10 m2/s for CGP (E removed) membrane. Finally, KP-hMSCs cultured on CGP nanofiber membrane showed a regular spindle shape with normal cell adhesion and growth, indicating that CGP nanofiber membrane was non-toxic to cells. Besides, by culturing cells underneath the nanofiber membranes, it was found that the KP-hMSCs and WS1 underneath CGP (E removed) nanofiber membrane exhibited higher proliferation rate since it was easier for nutrients and metabolic wastes to transport through the more permeable CGP (E removed) nanofiber membrane. The cell number of KP-hMSCs and WS1 underneath CGP (E removed) nanofiber membrane were about 1.6  1.7 and 1.9 times that underneath CGP nanofiber membrane, respectively. In conclusion, electrospun CGP composite nanofibers were fabricated successfully using a low concentration aqueous acetic acid (20 wt%) as a solvent, and the amount of PEO added was very small (5 wt% of the nanofibers). After being crosslinked by glutaraldehyde, the electrospun nanofiber membrane showed enhanced tensile strength, and stability. Furthermore, the CGP nanofibers were co-electrospun with E nanofibers, followed by removing the E nanofibers to obtain more permeable CGP (E removed) nanofibers for improved cell proliferation. Therefore, the electrospun CGP (E removed) nanofiber membrane has great potential for tissue engineering applications.