In situ UV-crosslinked Electrospun Gelatin Fibers for Tissue Engineering

使用天然高分子材料製作電紡絲時，通常會具備易使細胞貼附、生物相容性高及具生物可分解性等優點，但較弱的機械性質則是其共有的缺點，而高度的水溶性也會限制天然高分子材料電紡絲在生醫材料方面的應用，現今的研究主要是利用各種物理或化學的交聯方法來改善這些缺點。但目前對於電紡絲的交聯方法研究仍存在著生物毒性不易去除或效率過低等問題。本研究使用聚丙烯酸接枝疊氮基(PAA-Az)與明膠(Gelatin) 混摻並進行電紡絲實驗，並在製程中以紫外光照射使其交聯，藉以建立一快速且低生物毒性之新型電紡絲交聯方法。並以纖維母細胞培養及混摻氫氧基磷灰石(HAp)後骨母細胞礦化結果來分析其細胞貼附和增生之表現及進一步修飾應用之可能性。最後於此明膠電紡絲中分別及共同混摻入氫氧基磷灰石、聚乙烯亞胺接枝RGD序列(PEI-RGD)及骨型態蛋白-2 (BMP-2)，並在其上培養間葉幹細胞，以製作具骨組織工程發展潛力之生物支架。 結果發現，以聚丙烯酸接枝疊氮基交聯之明膠電紡絲機械性質及玻璃轉換溫度均較未交聯者為高;不具明顯細胞毒性，且纖維母細胞貼附和增生之表現也較以戊二醛交聯者為佳;混摻氫氧基磷灰石後骨母細胞之礦化表現有所上升(第14天約132%)。間葉幹細胞在含有氫氧基磷灰石或聚乙烯亞胺接枝RGD序列的電紡絲上也有鹼性磷酸酶活性(第7天約44%)和礦化表現提升(第14天約83%)或細胞貼附和增生之表現提升的相應表現。實驗結果說明此交聯方法應用於天然及水溶性高分子電紡絲製備與組織工程支架製作上具有相當的潛力。Electrospun fibers fabricated by natural polymer usually consisted of high cell adhesion ability with many beneficial characteristics, including their biocompatibility and biodegradability. However, weak mechanical property and high dissolution property are some of the drawbacks for natural polymer in limiting its application in electrospun fiber for biomedical usage. In addition, the biological toxicity was noted during the crosslinkage process of electrospun fibers remained a crucial problem in current research with either precipitation of toxic substances after crosslinkage or inefficient methods for removing them. Besides, the commonly used two-step crosslinking method (crosslinked after fiber fabrication) may cause un-even crosslinking of interior and exterior of the electrospun scaffolds. In this research, UV crosslinker poly(acrylic acid)-g-azide (PAA-Az) combined with an in situ crosslinking method was used for crosslinkage of gelatin to provide a low biological toxic route in fabricating electrospun fibers with high fibroblast proliferation. Hydroxyapatite (HAp) was also mixed in the solution for fabrication of the crosslinked fibers, resulting in high mineralization of osteoblasts. Finally, HAp, PEI-RGD, and BMP-2 were mixed in the solution for fabrication crosslinked electrospun gelatin fibers, resulting in high cell proliferation and mineralization of mesenchymal stem cells. As result, gelatin electronspun fibers crosslinked by PAA-Az exhibited higher mechanical strength and glass transition temperature in compared to un-crosslinked gelatin. When comparing gelatin electrospun fibers crosslinked by glutaraldeyde, low cell toxicity was observed with enhancement in fibroblast adhesion for PAA-Az crosslinked gelatin fibers. In addition, when blending HAp in the solution, higher mineralization was also observed in osteoblasts on PAA-Az crosslinked gelatin fibers in compared to crosslinked gelatin fibers without HAp. Finally, PEI-RGD incorporation indeed enhance 3A6 cell attachment and proliferation. HAp incorporation increase the calcium deposited amount of 3A6 cell seeded on the electrospun gelatin fibers. The research presents potential of the in situ PAA-Az crosslinking method in the application of natural and high dissoluble polymeric electrospun fibers for tissue engineering