本論文成功藉由層疊法(layer by layer)製備奈米木質纖維複合薄膜，並在薄膜中添加二氧化鈣(CaO2, CPO)與過氧化氫酶(catalase)使所製備的纖維薄膜能夠在水相環境中連續釋放過氧化氫與氧氣。在材料分析方面，利用掃描式電子顯微鏡(SEM)觀察薄膜的表面形態，發現所製備的薄膜具有多孔性的表面結構，能夠促進物質與氣體的傳遞與交換，電子能譜化學分析儀(ESCA)結果顯示並未有鈣的訊號被偵測到，表示二氧化鈣與過氧化氫酶成功的被包含於纖維薄膜中，此外，過氧化氫的釋放曲線及動力學也被探討。為了探討材料的生物反應與過氧化氫及氧氣釋放對細胞毒性的影響，將L-929老鼠纖維母細胞直接培養於薄膜表面上，細胞存活率結果顯示過氧化氫的釋放降低了細胞於薄膜上的生長，而氧氣的釋放能夠提高細胞的存活率，其結果甚至高於市售的組織培養孔盤，因此本薄膜具有極大潛力應用於組織工程薄膜應用之上。
本論文並利用所製備的纖維薄膜塗佈於PDMS材質上來測試動態釋放過氧化氫與氧氣的可能性，並將L-929細胞培養於其中，細胞存活率結果與直接培養於薄膜上的結果相似，表示此方法可應用於生物微流道晶片上，以達到連續釋放之效果。
金屬奈米粒子因為具有特殊的物理化學特性，因此常被應用於光學、感測器、奈米科學與催化反應上，然而目前結合金屬粒子與基材的方式，如表面自組裝單層膜(SAM)或化學接枝法(chemical grafting)，都需複雜程序與多步驟才能完成，且大多會使用到有機溶劑，對環境生態影響甚大。本論文利用薄膜釋放的過氧化氫，成功的將三種貴重金屬(金、銀、銅)還原並固定在纖維素表面之上，此種方法具有簡單、低成本，及對環境友善製程之優點，可廣為應用於製備新型金屬/高分子複合材料、奈米科學、感測器與催化反應。

The utilizations and applications of natural materials have attracted tremendous attention in recent years due to the serious shortage of the fossil energy and therefore the highly demands on the sustainable and renewable materials. Microfibrillated cellulose (MFC), extracted mainly from wood pulp, was applied to various fields in paper industry, food engineering and biomedical applications due to its nanostructure morphology, great biocompatibility, and excellent mechanical property. This study reported a cost-effective and green approach for the preparation of MFC nanocomposites by layer by layer (LbL) deposition method and the additions of calcium peroxide (CPO) and catalase which modulated the sustained releases of hydrogen peroxide and oxygen, respectively. The highly porous structure of the nanocomposites provides a three-dimensional microenvironment which facilitates the mass diffusion and offers great niches for cell growth. The release of H2O2 is controlled by the amount of added CPO while the catalase further converts the generated H2O2 into O2. L-929 fibroblasts responses were modulated by the amount of embedded CPO and catalase. The cell viability results demonstrated that the proliferation of L-929 cells was delayed due the release of H2O2. On the other hand, both cell growth and viability were effectively promoted by the generation of oxygen. To the best of our knowledge, this study is among the first work to report the modulation for the release of H2O2 and O2 by designing the scaffolds using microfibrillated cellulose based membranes. The proposed microfibrillated composites show great potential for the applications in tissue engineering and biomedical device.
MFC/CPO and [MFC+catalase]/CPO composites were coated on PDMS to investigate possibility of develop dynamic release system of H2O2. Cell responses showed similar results with that directly cultivated on composites. L-929 cell survival was promoted effectively on [MFC+catalase]/CPO-15 coated PDMS both in serum-containing and serum-free condition, showing potential applications in bio-micro chip fields.
In the second part, released H2O2 was employed as a reducing agent for reducing metal ions meanwhile immobilizing metal particles on cellulose surface. Three metal (Ag, Au and Cu) were successfully reduced on composites and named for Ag-MFC/CPO, Au-MFC/CPO and Cu-MFC/CPO, respectively. The prepared metal-MFC/CPO was characterized by scanning electron microscope (SEM), electron spectroscope for chemical analysis (ESCA) and inductively coupled plasma (ICP) This method provide a novel way for preparing metal/polymer composites and showed great potential in optical, sensing devices and catalysis applications.

[1] Azizi Samir MAS, Alloin F, Dufresne A. Review of Recent Research into Cellulosic Whiskers, Their Properties and Their Application in Nanocomposite Field. Biomacromolecules. 2005;6:612-26.
[2] Simon J, Muller HP, Koch R, Muller V. Thermoplastic and biodegradable polymers of cellulose. Polymer Degradation and Stability. 1998;59:107-15.
[3] Payen A. Memoir on the composition of the tissue of plants and of woody [material]. Comptes Rendus. 1838:1052-6.
[4] Siqueira G, Bras J, Dufresne A. Cellulosic Bionanocomposites: A Review of Preparation, Properties and Applications. Polymers. 2010;2:728-65.
[5] Alila S, Besbes I, Vilar MR, Mutje P, Boufi S. Non-woody plants as raw materials for production of microfibrillated cellulose (MFC): A comparative study. Industrial Crops and Products. 2013;41:250-9.
[6] Herrick FW, Casebier RL, Hamilton JK, Sandberg KR. Microfibrillated cellulose: morphology and accessibility1983.
[7] Turbak AF, Snyder FW, Sandberg KR. Microfibrillated cellulose, a new cellulose product: properties, uses, and commercial potential1983.
[8] Stenstad P, Andresen M, Tanem B, Stenius P. Chemical surface modifications of microfibrillated cellulose. Cellulose. 2008;15:35-45.
[9] Lavoine N, Desloges I, Dufresne A, Bras J. Microfibrillated cellulose – Its barrier properties and applications in cellulosic materials: A review. Carbohydrate Polymers. 2012;90:735-64.
[10] Grande CJ, Torres FG, Gomez CM, Carmen Bano M. Nanocomposites of bacterial cellulose/hydroxyapatite for biomedical applications. Acta Biomaterialia. 2009;5:1605-15.
[11] Klemm D, Schumann D, Udhardt U, Marsch S. Bacterial synthesized cellulose — artificial blood vessels for microsurgery. Progress in Polymer Science. 2001;26:1561-603.
[12] Svensson A, Nicklasson E, Harrah T, Panilaitis B, Kaplan DL, Brittberg M. Bacterial cellulose as a potential scaffold for tissue engineering of cartilage. Biomaterials. 2005;26:419-31.
[13] Czaja W, Krystynowicz A, Bielecki S, Brown Jr RM. Microbial cellulose—the natural power to heal wounds. Biomaterials. 2006;27:145-51.
[14] Chiappone A, Nair JR, Gerbaldi C, Jabbour L, Bongiovanni R, Zeno E. Microfibrillated cellulose as reinforcement for Li-ion battery polymer electrolytes with excellent mechanical stability. Journal of Power Sources. 2011;196:10280-8.
[15] Qiu K, Netravali AN. Fabrication and characterization of biodegradable composites based on microfibrillated cellulose and polyvinyl alcohol. Composites Science and Technology. 2012;72:1588-94.
[16] Lu J, Askeland P, Drzal LT. Surface modification of microfibrillated cellulose for epoxy composite applications. Polymer. 2008;49:1285-96.
[17] Walboomers XF, Jansen JA. Cell and tissue behavior on micro-grooved surfaces. Odontology. 2001;89:0002-11.
[18] Karageorgiou V, Kaplan D. Porosity of 3D biomaterial scaffolds and osteogenesis. Biomaterials. 2005;26:5474-91.
[19] Dolatshahi-Pirouz A, Jensen T, Kraft DC, Foss M, Kingshott P, Hansen JL. Fibronectin Adsorption, Cell Adhesion, and Proliferation on Nanostructured Tantalum Surfaces. ACS Nano. 2010;4:2874-82.
[20] Santiago LY, Nowak RW, Peter Rubin J, Marra KG. Peptide-surface modification of poly(caprolactone) with laminin-derived sequences for adipose-derived stem cell applications. Biomaterials. 2006;27:2962-9.
[21] Tsai W-B, Chen Y-R, Li W-T, Lai J-Y, Liu H-L. RGD-conjugated UV-crosslinked chitosan scaffolds inoculated with mesenchymal stem cells for bone tissue engineering. Carbohydrate Polymers. 2012;89:379-87.
[22] Chien H-W, Xu X, Ella-Menye J-R, Tsai W-B, Jiang S. High Viability of Cells Encapsulated in Degradable Poly(carboxybetaine) Hydrogels. Langmuir. 2012;28:17778-84.
[23] Hauser J, Koeller M, Bensch S, Halfmann H, Awakowicz P, Steinau H-U. Plasma mediated collagen-I-coating of metal implant materials to improve biocompatibility. Journal of Biomedical Materials Research Part A. 2010;94A:19-26.
[24] Liu X, Smith LA, Hu J, Ma PX. Biomimetic nanofibrous gelatin/apatite composite scaffolds for bone tissue engineering. Biomaterials. 2009;30:2252-8.
[25] Chen W, Villa-Diaz LG, Sun Y, Weng S, Kim JK, Lam RHW. Nanotopography Influences Adhesion, Spreading, and Self-Renewal of Human Embryonic Stem Cells. ACS Nano. 2012;6:4094-103.
[26] Ho Q-P, Wang S-L, Wang M-J. Creation of Biofunctionalized Micropatterns on Poly(methyl methacrylate) by Single-Step Phase Separation Method. ACS Applied Materials & Interfaces. 2011;3:4496-503.
[27] Zhang X, Reagan MR, Kaplan DL. Electrospun silk biomaterial scaffolds for regenerative medicine. Advanced Drug Delivery Reviews. 2009;61:988-1006.
[28] Stevens MM, George JH. Exploring and Engineering the Cell Surface Interface. science. 2005;310:1135-8.
[29] Dash M, Chiellini F, Ottenbrite RM, Chiellini E. Chitosan—A versatile semi-synthetic polymer in biomedical applications. Progress in Polymer Science. 2011;36:981-1014.
[30] Miyata T, Taira T, Noishiki Y. Collagen engineering for biomaterial use. Clinical Materials. 1992;9:139-48.
[31] Bodin A, Backdahl H, Petersen N, Gatenholm P. Bacterial Cellulose as Biomaterial. In: Editor-in-Chief: Paul D, editor. Comprehensive Biomaterials. Oxford: Elsevier; 2011. p. 405-10.
[32] Yamaguchi I, Itoh S, Suzuki M, Sakane M, Osaka A, Tanaka J. The chitosan prepared from crab tendon I: the characterization and the mechanical properties. Biomaterials. 2003;24:2031-6.
[33] Chen ZG, Wang PW, Wei B, Mo XM, Cui FZ. Electrospun collagen–chitosan nanofiber: A biomimetic extracellular matrix for endothelial cell and smooth muscle cell. Acta Biomaterialia. 2010;6:372-82.
[34] Ku SH, Park CB. Human endothelial cell growth on mussel-inspired nanofiber scaffold for vascular tissue engineering. Biomaterials. 2010;31:9431-7.
[35] Bhattacharya M, Malinen MM, Lauren P, Lou Y-R, Kuisma SW, Kanninen L. Nanofibrillar cellulose hydrogel promotes three-dimensional liver cell culture. Journal of Controlled Release. 2012;164:291-8.
[36] Haroun A, Gamal-Eldeen A, Harding DK. Preparation, characterization and in vitro biological study of biomimetic three-dimensional gelatin–montmorillonite/cellulose scaffold for tissue engineering. J Mater Sci: Mater Med. 2009;20:2527-40.
[37] Muller FA, Muller L, Hofmann I, Greil P, Wenzel MM, Staudenmaier R. Cellulose-based scaffold materials for cartilage tissue engineering. Biomaterials. 2006;27:3955-63.
[38] Jia W, Guo M, Zheng Z, Yu T, Rodriguez EG, Wang Y. Electrocatalytic oxidation and reduction of H2O2 on vertically aligned Co3O4 nanowalls electrode: Toward H2O2 detection. Journal of Electroanalytical Chemistry. 2009;625:27-32.
[39] Moreno T, Garcia-Serna J, Plucinski P, Sanchez-Montero MJ, Cocero MJ. Direct synthesis of H2O2 in methanol at low pressures over Pd/C catalyst: Semi-continuous process. Applied Catalysis A: General. 2010;386:28-33.
[40] Duarte TL, Almeida GM, Jones GDD. Investigation of the role of extracellular H2O2 and transition metal ions in the genotoxic action of ascorbic acid in cell culture models. Toxicology Letters. 2007;170:57-65.
[41] Bladier C, Wolvetang EJ, Hutchinson P, Haan JBd, Kola I. Response of a primary human fibroblast cell line to H2O2: senescence-like growth arrest or apoptosis? Cell Growth & Differentiation. 1997;8:589-98.
[42] Goldstein LE, Leopold MC, Huang X, Atwood CS, Saunders AJ, Hartshorn M. 3-Hydroxykynurenine and 3-Hydroxyanthranilic Acid Generate Hydrogen Peroxide and Promote α-Crystallin Cross-Linking by Metal Ion Reduction†. Biochemistry. 2000;39:7266-75.
[43] Huang X, Atwood CS, Hartshorn MA, Multhaup G, Goldstein LE, Scarpa RC. The Aβ Peptide of Alzheimer's Disease Directly Produces Hydrogen Peroxide through Metal Ion Reduction†. Biochemistry. 1999;38:7609-16.
[44] Campos-Martin JM, Blanco-Brieva G, Fierro JLG. Hydrogen Peroxide Synthesis: An Outlook beyond the Anthraquinone Process. Angewandte Chemie International Edition. 2006;45:6962-84.
[45] AH A. Heart Disease and Stroke Statistics. 2004 Update Dallas. 2004.
[46] Oh SH, Ward CL, Atala A, Yoo JJ, Harrison BS. Oxygen generating scaffolds for enhancing engineered tissue survival. Biomaterials. 2009;30:757-62.
[47] Harrison BS, Eberli D, Lee SJ, Atala A, Yoo JJ. Oxygen producing biomaterials for tissue regeneration. Biomaterials. 2007;28:4628-34.
[48] Basolo F, Hoffman BM, Ibers JA. Synthetic oxygen carriers of biological interest. Accounts of Chemical Research. 1975;8:384-92.
[49] Nicholls P. Classical catalase: Ancient and modern. Archives of Biochemistry and Biophysics. 2012;525:95-101.
[50] Raducan A, Cantemir A, Puiu M, Oancea D. Kinetics of hydrogen peroxide decomposition by catalase: hydroxylic solvent effects. Bioprocess Biosyst Eng. 2012;35:1523-30.
[51] OGURA Y, YAMAZAKI I. Steady-State Kinetics of the Catalase Reaction in the Presence of Cyanide. Journal of Biochemistry. 1983;94:403-8.
[52] Holmes RS, Masters CJ. Species specific features of the distribution and multiplicity of mammalian liver catalase. Archives of Biochemistry and Biophysics. 1972;148:217-23.
[53] Vazquez-Fernandez MA, Bermejo MR, Fernandez-Garcia MI, Gonzalez-Riopedre G, Rodriguez-Douton MJ, Maneiro M. Influence of the geometry around the manganese ion on the peroxidase and catalase activities of Mn(III)–Schiff base complexes. Journal of Inorganic Biochemistry. 2011;105:1538-47.
[54] Wang J, Zhu Y, Bawa HK, Ng G, Wu Y, Libera M. Oxygen-Generating Nanofiber Cell Scaffolds with Antimicrobial Properties. ACS Applied Materials & Interfaces. 2010;3:67-73.
[55] Masaro L, Zhu XX. Physical models of diffusion for polymer solutions, gels and solids. Progress in Polymer Science. 1999;24:731-75.
[56] Lian W, Liu S, Yu J, Xing X, Li J, Cui M, et al. Electrochemical sensor based on gold nanoparticles fabricated molecularly imprinted polymer film at chitosan–platinum nanoparticles/graphene–gold nanoparticles double nanocomposites modified electrode for detection of erythromycin. Biosensors and Bioelectronics. 2012;38:163-9.
[57] Scire S, Crisafulli C, Giuffrida S, Mazza C, Riccobene PM, Pistone A. Supported silver catalysts prepared by deposition in aqueous solution of Ag nanoparticles obtained through a photochemical approach. Applied Catalysis A: General. 2009;367:138-45.
[58] Yallapu MM, Othman SF, Curtis ET, Gupta BK, Jaggi M, Chauhan SC. Multi-functional magnetic nanoparticles for magnetic resonance imaging and cancer therapy. Biomaterials. 2011;32:1890-905.
[59] Gupta RK, Srinivasan MP, Dharmarajan R. Synthesis of 16-Mercaptohexadecanoic acid capped gold nanoparticles and their immobilization on a substrate. Materials Letters. 2012;67:315-9.
[60] Tankhiwale R, Bajpai SK. Graft copolymerization onto cellulose-based filter paper and its further development as silver nanoparticles loaded antibacterial food-packaging material. Colloids and Surfaces B: Biointerfaces. 2009;69:164-8.
[61] Li S-M, Jia N, Zhu J-F, Ma M-G, Xu F, Wang B, et al. Rapid microwave-assisted preparation and characterization of cellulose–silver nanocomposites. Carbohydrate Polymers. 2011;83:422-9.
[62] Boufi S, Ferraria AM, do Rego AMB, Battaglini N, Herbst F, Vilar MR. Surface functionalisation of cellulose with noble metals nanoparticles through a selective nucleation. Carbohydrate Polymers. 2011;86:1586-94.
[63] Chien H-W, Kuo W-H, Wang M-J, Tsai S-W, Tsai W-B. Tunable Micropatterned Substrates Based on Poly(dopamine) Deposition via Microcontact Printing. Langmuir. 2012;28:5775-82.
[64] Sureshkumar M, Lee P-N, Lee C-K. Stepwise assembly of multimetallic nanoparticles via self-polymerized polydopamine. Journal of Materials Chemistry. 2011;21:12316-20.
[65] Han X, Gelein R, Corson N, Wade-Mercer P, Jiang J, Biswas P. Validation of an LDH assay for assessing nanoparticle toxicity. Toxicology. 2011;287:99-104.
[66] Peng X, Zhang L, Kennedy JF. Release behavior of microspheres from cross-linked N-methylated chitosan encapsulated ofloxacin. Carbohydrate Polymers. 2006;65:288-95.
[67] Wu Y, Qiu L-G, Wang W, Li Z-Q, Xu T, Wu Z-Y. Kinetics of oxidation of hydroquinone to p-benzoquinone catalyzed by microporous metal-organic frameworks M3(BTC)2 [M = copper(II), cobalt(II), or nickel(II); BTC = benzene-1,3,5-tricarboxylate] using molecular oxygen. Transition Met Chem. 2009;34:263-8.