本研究目的是要製備含有親疏水表面的奈米纖維支架，並探討其物理特性和生物相容性。利用電紡絲和製膜技術，製備親水性的聚乙烯醇(polyvinyl alcohol, PVA)及疏水性的聚苯乙烯(polystyrene, PS)奈米纖維膜和薄膜，並結合兩者形成雙層支架(bilayer scaffold)。希望可以藉由調控PVA和PS的支架結構與比例來改變支架表面親疏水性(hydrophilic/hydrophobic)、形態(topography)和粗糙度(roughness)以探討其對細胞貼附和生長的影響。
我們使用掃描式電子顯微鏡(SEM)觀察纖維支架的形態及在水中的穩定性；利用黏度計及動態機械分析儀(DMA)之量測來觀察戊二醛與PVA進行交聯反應，其溶液黏度、纖維形態及纖維支架的機械強度的影響；利用原子力顯微鏡(AFM)量測支架表面的粗糙度，再藉由量測支架表面的接觸角(contact angle)和吸濕性(water uptake)，來觀察表面上PVA和PS的分布與形態對其粗糙度的變化與表面親疏水性之關聯性。
在生物相容性的探討方面，我們使用老鼠纖維母細胞(Murine NIH-3T3 fibroblasts)在支架上進行體外的(in vitro)細胞培養，以MTT assay分析細胞於支架上的活性及增生，並利用SEM觀察細胞於支架表面上培養一到七天後的貼附和生長形態。由MTT assay和SEM結果顯示，當表面的形態改變時會影響細胞的生長行為。由本實驗發現支架表面的親水性和粗糙度的提升，對細胞的貼附和生長情形有所改善，以下層為PS纖維結合上層為PVA纖維之雙層支架的效果為最佳。

The aim of this study is to prepare several scaffolds containing hydrophilic/hydrophobic bilayer surface and investigate the physical properties and biocompatibility of the scaffold as prepared. By using electrospinning and film formation technique, the nanofibers and thin films made by hydrophilic polyvinyl alcohol (PVA) and hydrophobic polystyrene (PS) were prepared. A variety of bilayer scaffolds was then fabricated by combining these hydrophilic/hydrophobic nanofiber and thin film structure. By changing the morphology of these hydrophilic and hydrophobic combinations, the surface characteristics as well as cell attachment and growth behavior on these bilayer scaffolds were analyzed.
Scanning electron microscopy (SEM) was utilized to examine the surface morphology of bilayer scaffold in ambient environment as well as being immersed in water. From the results of viscometer and dynamic mechanical analyzer (DMA), the crosslinking reaction of PVA by glutaraldehyde (GA) would affect the viscosity of solution, fibrous morphology and mechanical strength of scaffolds. The correlations between the preparation scheme and roughness and wettability of bilayer scaffold as resulted was determined by atomic force microscopy (AFM), water contact angle and water uptake.
For biocompatibility evaluation, in vitro cell culture using murine NIH-3T3 fibroblasts was utilized. MTT assay was used to assess the cell viability and proliferation. Cell morphology was determined by SEM for those attached on the bilayer scaffold for 1 and 7 days. It was clearly noted that the cell morphology and cell proliferation were affected by the surface characteristics of the bilayer scaffold. By increasing the surface roughness and hydrophilicity, the cell attachment and proliferation on the bilayer scaffold is also increased. It was concluded the bilayer scaffold formed by the PS nanofibers on the bottom and PVA nanofibers on the top was the most biocompatible one in this investigation.

[1] Molly M. Stevens JHG. Exploring and Engineering the Cell Surface Interface. Science 2005;310:1135-8
[2] Ji W, Sun Y, Yang F, van den Beucken JJ, Fan M, Chen Z, et al. Bioactive electrospun scaffolds delivering growth factors and genes for tissue engineering applications. Pharmaceutical research 2011;28:1259-72.
[3] Rahaman MN, Day DE, Bal BS, Fu Q, Jung SB, Bonewald LF, et al. Bioactive glass in tissue engineering. Acta biomaterialia 2011;7:2355-73.
[4] You-Xiong Wang JLR, William B. Spillman J, and Richard O. Claus. Effects of the Chemical Structure and the surface properties of polymeric biomaterials on their biocompatibility. Pharmaceutical research 2004;21.
[5] Borges AMG, Benetoli LO, Licínio MA, Zoldan VC, Santos-Silva MC, Assreuy J, et al. Polymer films with surfaces unmodified and modified by non-thermal plasma as new substrates for cell adhesion. Materials Science and Engineering: C 2013;33:1315-24.
[6] Kasemo B. Biological surface science. Surface Science 2002;500:656-77.
[7] CAMERON J. WILSON BE, RICHARD E. CLEGG, , DAVID I. LEAVESLEY aMJP. Mediation of Biomaterial–Cell Interactions by Adsorbed. TISSUE ENGINEERING 2005;11.
[8] Cheng Z, Teoh S-H. Surface modification of ultra thin poly (ε-caprolactone) films using acrylic acid and collagen. Biomaterials 2004;25:1991-2001.
[9] Oh SH, Lee JH. Hydrophilization of synthetic biodegradable polymer scaffolds for improved cell/tissue compatibility. Biomedical Materials 2013;8:014101.
[10] Luo C, Li L, Li J, Yang G, Ding S, Zhi W, et al. Modulating cellular behaviors through surface nanoroughness. Journal of Materials Chemistry 2012;22:15654.
[11] Milleret V, Hefti T, Hall H, Vogel V, Eberli D. Influence of the fiber diameter and surface roughness of electrospun vascular grafts on blood activation. Acta biomaterialia 2012;8:4349-56.
[12] J. I. Sheppard WGM, and I. A. Feuerstein. adherent platelet morphology on adsorbed fibrinogen - effects of protein incubation-time and albumin addition. Journal of Biomedical Materials Research 1994;28.
[13] Gentile F, Tirinato L, Battista E, Causa F, Liberale C, di Fabrizio EM, et al. Cells preferentially grow on rough substrates. Biomaterials 2010;31:7205-12.
[14] Colette S. Ranucci PVM. Substrate microtopography can enhance cell adhesive and migratory responsiveness to matrix ligand density. Journal of Biomedical Materials Research 2001;54: 149-61.
[15] Goh Y-F, Shakir I, Hussain R. Electrospun fibers for tissue engineering, drug delivery, and wound dressing. Journal of Materials Science 2013;48:3027-54.
[16] Lee HJ, Kim HS, Kim HO, Koh WG. Micropatterns of double-layered nanofiber scaffolds with dual functions of cell patterning and metabolite detection. Lab on a chip 2011;11:2849-57.
[17] Bhardwaj N, Kundu SC. Electrospinning: a fascinating fiber fabrication technique. Biotechnology advances 2010;28:325-47.
[18] Baumgart.Pk. Electrostatic Spinning of Acrylic Microfibers. J Colloid Interf Sci 1971;36:71-&.
[19] Gupta P, Elkins C, Long TE, Wilkes GL. Electrospinning of linear homopolymers of poly(methyl methacrylate): exploring relationships between fiber formation, viscosity, molecular weight and concentration in a good solvent. Polymer 2005;46:4799-810.
[20] H. Fong IC, D.H. Reneker. Beaded nanofibers formed during electrospinning. polymer 1999;40:4585.
[21] Du J, Zhang X. Role of polymer–salt–solvent interactions in the electrospinning of polyacrylonitrile/iron acetylacetonate. Journal of Applied Polymer Science 2008;109:2935-41.
[22] Haghi AK, Akbari M. Trends in electrospinning of natural nanofibers. physica status solidi (a) 2007;204:1830-4.
[23] Sill TJ, von Recum HA. Electrospinning: applications in drug delivery and tissue engineering. Biomaterials 2008;29:1989-2006.
[24] Vaquette C, Cooper-White JJ. Increasing electrospun scaffold pore size with tailored collectors for improved cell penetration. Acta biomaterialia 2011;7:2544-57.
[25] Van der Schueren L, De Schoenmaker B, Kalaoglu ÖI, De Clerck K. An alternative solvent system for the steady state electrospinning of polycaprolactone. European Polymer Journal 2011;47:1256-63.
[26] Pakravan M, Heuzey M-C, Ajji A. A fundamental study of chitosan/PEO electrospinning. Polymer 2011;52:4813-24.
[27] Stankus JJ, Guan J, Fujimoto K, Wagner WR. Microintegrating smooth muscle cells into a biodegradable, elastomeric fiber matrix. Biomaterials 2006;27:735-44.
[28] Yang W, Yang F, Wang Y, Both SK, Jansen JA. In vivo bone generation via the endochondral pathway on three-dimensional electrospun fibers. Acta biomaterialia 2013;9:4505-12.
[29] Ortega I, Ryan AJ, Deshpande P, MacNeil S, Claeyssens F. Combined microfabrication and electrospinning to produce 3-D architectures for corneal repair. Acta biomaterialia 2013;9:5511-20.
[30] Franco RA, Nguyen TH, Lee BT. Preparation and characterization of electrospun PCL/PLGA membranes and chitosan/gelatin hydrogels for skin bioengineering applications. Journal of materials science Materials in medicine 2011;22:2207-18.
[31] Kai D, Prabhakaran MP, Jin G, Ramakrishna S. Guided orientation of cardiomyocytes on electrospun aligned nanofibers for cardiac tissue engineering. Journal of biomedical materials research Part B, Applied biomaterials 2011;98:379-86.
[32] Fathi-Azarbayjani A, Chan SY. Single and Multi-Layered Nanofibers for Rapid and Controlled Drug Delivery. Chemical & Pharmaceutical Bulletin 2010;58:143-6.
[33] Zhou Y, Yang D, Chen X, Xu Q, Lu F, Nie J. Electrospun water-soluble carboxyethyl chitosan/poly(vinyl alcohol) nanofibrous membrane as potential wound dressing for skin regeneration. Biomacromolecules 2008;9:349-54.
[34] Jannesari M, Varshosaz J, Morshed M, Zamani M. Composite poly(vinyl alcohol)/poly(vinyl acetate) electrospun nanofibrous mats as a novel wound dressing matrix for controlled release of drugs. International journal of nanomedicine 2011;6:993-1003.
[35] Gao C, Gao Q, Li Y, Rahaman MN, Teramoto A, Abe K. Preparation and in vitro characterization of electrospun PVA scaffolds coated with bioactive glass for bone regeneration. Journal of biomedical materials research Part A 2012;100:1324-34.
[36] Vrana NE, Liu Y, McGuinness GB, Cahill PA. Characterization of Poly(vinyl alcohol)/Chitosan Hydrogels as Vascular Tissue Engineering Scaffolds. Macromolecular Symposia 2008;269:106-10.
[37] Mansur HS, Sadahira CM, Souza AN, Mansur AAP. FTIR spectroscopy characterization of poly (vinyl alcohol) hydrogel with different hydrolysis degree and chemically crosslinked with glutaraldehyde. Materials Science and Engineering: C 2008;28:539-48.
[38] Tang C, Saquing CD, Harding JR, Khan SA. In SituCross-Linking of Electrospun Poly(vinyl alcohol) Nanofibers. Macromolecules 2010;43:630-7.
[39] Lee SY, Jang DH, Kang YO, Kim OB, Jeong L, Kang HK, et al. Cellular response to poly(vinyl alcohol) nanofibers coated with biocompatible proteins and polysaccharides. Applied Surface Science 2012;258:6914-22.
[40] Yang JH, Yoon NS, Park JH, Kim IK, Cheong IW, Deng Y, et al. Electrospinning fabrication and characterization of poly(vinyl alcohol)/waterborne polyurethane nanofiber membranes in aqueous solution. Journal of Applied Polymer Science 2011;120:2337-45.
[41] Curtis AS, Forrester JV, McInnes C, Lawrie F. Adhesion of cells to polystyrene surfaces. The Journal of cell biology 1983;97:1500-6.
[42] Baker SC, Atkin N, Gunning PA, Granville N, Wilson K, Wilson D, et al. Characterisation of electrospun polystyrene scaffolds for three-dimensional in vitro biological studies. Biomaterials 2006;27:3136-46.
[43] Kidoaki S, Kwon IK, Matsuda T. Mesoscopic spatial designs of nano- and microfiber meshes for tissue-engineering matrix and scaffold based on newly devised multilayering and mixing electrospinning techniques. Biomaterials 2005;26:37-46.
[44] Gupta S, Webster TJ, Sinha A. Evolution of PVA gels prepared without crosslinking agents as a cell adhesive surface. Journal of materials science Materials in medicine 2011;22:1763-72.
[45] Kim CH, Khil MS, Kim HY, Lee HU, Jahng KY. An improved hydrophilicity via electrospinning for enhanced cell attachment and proliferation. Journal of biomedical materials research Part B, Applied biomaterials 2006;78:283-90.