以電紡奈米纖維紡絲技術製作組織工程細胞支架; Fabricate a nanofibrous scaffold for tissue engineering using electrospinning

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Title:	以電紡奈米纖維紡絲技術製作組織工程細胞支架;Fabricate a nanofibrous scaffold for tissue engineering using electrospinning
Authors:	梁仁溢;Jen-I Liang
Contributors:	中國醫藥大學：臨床醫學研究所
Keywords:	電紡;奈米纖維;細胞支架;聚乙烯醇;幾丁聚醣;纖維母細胞;electrospinning;nanofiber;scaffold;PVA;Chitosan;fibroblast
Date:	2008-07-05
Issue Date:	2009-08-12 14:12:06 (UTC+8)
Abstract:	研究背景與目的：電紡為一種紡織技術，可用來製造出直徑為數十到數百奈米大小的纖維。許多具有生物可降解性之高分子聚合物，包括天然聚合物及人工合成聚合物，皆可使用電紡之方式製作出奈米纖維細胞支架。本研究使用兩種聚合物做為材料，聚乙烯醇為一水溶性、無毒、且具生物相容性之高分子聚合物；幾丁聚醣為一天然高分子聚合物，具有生物可降解性、生物相容性及抗菌性，此兩種材料在過去皆廣泛應用於細胞支架之研究。然而，大部分的研究都只採用單一高分子聚合物來製作奈米纖維細胞支架。因此，本研究之目的為以電紡技術製作出適合組織工程應用之聚乙烯醇及聚乙烯醇/幾丁聚醣奈米纖維細胞支架並觀察基材對細胞之相容性。材料與方法：以電紡技術製作出不織布形態之聚乙烯醇及聚乙烯醇/幾丁聚醣奈米纖維細胞支架。細胞支架以第一型膠原蛋白進行表面改質處理，將3T3纖維母細胞培養於細胞支架中，以光學顯微鏡及電子顯微鏡觀察細胞之形態，使用H&E染色技術來計算出細胞貼附與攤平之比例，利用MTS測試量測出細胞生長於細胞支架時之細胞活性，細胞之基因表現以即時聚合酶鏈鎖反應測試。結果與討論：以電紡製作出直徑大小為316±63.4奈米之聚乙烯醇奈米纖維及直徑大小為109.4±16.5奈米之聚乙烯醇/幾丁聚醣奈米纖維，生長於聚乙烯醇奈米纖維之細胞形態主要為圓形，生長於聚乙烯醇/幾丁聚醣奈米纖維之細胞形態主要為紡錘形。在細胞對於基材之貼附性、攤平性、細胞活性、胞外基質分泌的表現，聚乙烯醇/幾丁聚醣奈米纖維都比聚乙烯醇奈米纖維來得好。而第一型膠原蛋白表面改質處理，對於細胞貼附率有明顯之提升，但對於長期之細胞活性變化並無影響。結論：本研究認為聚乙烯醇/幾丁聚醣奈米電紡絲本身對於纖維母細胞擁有非常好之相容性。在未來研究中，將針對建立系統性參數、收集器之改良、加強基材強度、研發生物反應器及動物試驗做更進一步之研究。 Background and purpose: Electrospinning is one of the process techniques to produce fiber in nanoscale diameter. Various biodegradable polymers were used to develop nanoscaffolds including natural and synthetic type. Poly(vinyl alcohol) (PVA) is a non-toxic, hydrophilic, and biocompatible material. Chitosan is a natural polymer, which is biodegradable, biocompatible, and antibacterial. In the previous studies, PVA and chitosan have been used for many tissue engineering applications. However, most of the studies use only single polymer to fabricate nanofibers. Therefore, the purpose of this study is to fabricate PVA and PVA/Chitosan nanofiber scaffold for further tissue engineering applications. Materials and methods: PVA and PVA/Chitosan were used as the material to fabricate scaffold. The 3D nanofiber scaffold was produced via electrospinning. Surface of nanofiber scaffolds were modified using Type I collagen coating. NIH 3T3 fibroblast cell line was used as the cell source in this study. Fibroblast cells were seeded onto 3D scaffolds for different time points. Cellular morphology characterization was observed using inverted light microscopy and scanning electron microscopy. The cell adhesion and spreading rate were determined by H&E Stain. The cell viability was determined by MTS assay. RT-PCR was used to examine the cellular gene expression after culturing for 5 days. Results and discussion: PVA nanofiber of 316±63.4 nm in diameter and PVA/Chitosan nanofiber of 109.4±16.5 nm in diameter were obtained. The fibroblast cell was attached with round shape on the PVA nanofiber scaffold and with spindle shape on the PVA/Chitosan nanofiber scaffold. The result shows that the fibroblast exhibit excellent cell adhesion rate, spreading rate, cell viability and gene expression on the PVA/Chitosan nanofiber scaffold. The cell adhesion rate was increased when matrices coated with Type I collagen. Conclusion: In present study, PVA/Chitosan nanofiber scaffold shows better biocompatibility than PVA nanofiber scaffold. Further coating collagen on nanofiber scaffold does not display further improvement in cell compatibility except cell adhesion rate. Future study will focus on systematic parameter setting, platform modification, increase strength of scaffold, bioreactor, and in-vivo experiment.
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