软刻蚀方法制备壳聚糖/牛血清白蛋白复合微图形及其相关性质研究
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摘要
制备具有微图形的材料基底来控制细胞的行为已经成为了生物医学工程领域的研究热点。多种细胞可以响应微米尺度,甚至于纳米尺度的材料表面拓扑结构,细胞的形态、取向、生长和分化亦受到表面结构的影响。复合微图形就能实现这样的目标,复合微图形是指将两种或多种类型的分子共同结合在同一块基底上,利用其不同的化学性质,产生不同的作用,达到利用同一块材料能产生不同功效的目的。
本研究采用软刻蚀技术在硅表面制备出规则的,有序的,两种成分复合的壳聚糖/牛血清白蛋白(CS/BSA)复合微图形。壳聚糖作为自然界中唯一的碱性多糖,具有良好的生物降解性、可再生性和抗菌性。牛血清白蛋白具有较好的生物相容性。在软刻蚀中,用聚二甲基硅氧烷(PDMS)复制具有微图形的硅模板,得到具有微沟槽和点状图案PDMS弹性印章。采用微转移模塑法和复制模塑法制备CS/BSA复合微图形、BSA沟槽、CS沟槽微图形及点状微图形。
利用抗菌实验来评价CS/BSA复合微图形的抗菌效果。结果表明,CS/BSA复合微图形对大肠杆菌和白色葡萄球菌都有着一定的抗菌能力,抗菌率在30%左右。体外成骨细胞培养实验表明CS/BSA复合微图形能在细胞生长初期约束细胞的生长方向,并且会先选择粘附在复合微图形中的CS区域,之后才逐渐铺展到BSA区域。BSA沟槽和CS沟槽对细胞生长方向有很强的诱导力。由于微图形凸起的影响,细胞会首先吸附在微图形脊上,并沿着微图形的方向生长,随后铺展到微图形的底部。小尺寸沟槽对细胞生长方向的诱导作用较强。沟槽微图形表面细胞的增殖和分化能力弱于平板样品表面,强于点状微图形表面；BSA比CS利于细胞增殖和分化。
Controlling cell orientation and morphology by micropatterned substrates is one of hot topic in biomedical engineering. Cells can respond to micrometer scale, and even nano-scale, surface topography. Cell shape, orientation, growth and differentiation are also affected by surface texture of materials. Composite micropatterns were multifunctional biomaterials that co-patterned two or more types of molecules on one substrate. Thus, it can employ the advantage of components.
In this study, we developed a new process that can prepare chitosan/bovine serum albumin (CS/BSA) composite micropatterns by soft-lithography on silicon substrates. CS is a basic polysaccharide in the nature that has good biodegradability, renewable ability and antibacterial ability. BSA is a serum protein that has numerous biochemical applications. Polydimethylsiloxane (PDMS) microstamps were used to transfer micropatterns from the micromachined silicon mold to the hydroxylated silicon wafers. CS/BSA composite micropatterns, BSA micropatterns and CS micropatterns were successfully fabricated on silicon surfaces by micro-transfer molding and replica molding technique.
The antibacterial test was used to evaluate the antibacterial effect of CS/BSA composite micropatterns. The results showed that the CS/BSA composite micropatterns were effectively against two types of bacteria, Escherichia coli and Staphylococcus albus. The osteoblast (Sprague-Dawley rat) culture indicated that CS/BSA composite micropatterns could strongly influence cell behavior. Cells selectively adhered in the CS region at intial culturing stage, and then they gradually spread to the BSA region. The microgrooved surfaces had strong contact guidance effect on cell orentation. Cells tended to be parallel to the microgrooves when they were cultured on BSA microgrooves and CS microgrooves surfaces, They began to attach on the ridges of microgrooves, grew along the direction of microgrooves, and then spread to the bottom of microgrooves. This tendency was stronger on the microgrooves with smaller width. The cells on plane samples had random growth directions. The proliferation and differential rate of cells on microgrooved surfaces is lower than that on plane surface and higher than that on microdot-patterned surfaces, which indicated that micropatterns had complicated effects on cell behaviors. This study provides valuable guidance for the design of the biomaterial surfaces.
本研究采用软刻蚀技术在硅表面制备出规则的,有序的,两种成分复合的壳聚糖/牛血清白蛋白(CS/BSA)复合微图形。壳聚糖作为自然界中唯一的碱性多糖,具有良好的生物降解性、可再生性和抗菌性。牛血清白蛋白具有较好的生物相容性。在软刻蚀中,用聚二甲基硅氧烷(PDMS)复制具有微图形的硅模板,得到具有微沟槽和点状图案PDMS弹性印章。采用微转移模塑法和复制模塑法制备CS/BSA复合微图形、BSA沟槽、CS沟槽微图形及点状微图形。
利用抗菌实验来评价CS/BSA复合微图形的抗菌效果。结果表明,CS/BSA复合微图形对大肠杆菌和白色葡萄球菌都有着一定的抗菌能力,抗菌率在30%左右。体外成骨细胞培养实验表明CS/BSA复合微图形能在细胞生长初期约束细胞的生长方向,并且会先选择粘附在复合微图形中的CS区域,之后才逐渐铺展到BSA区域。BSA沟槽和CS沟槽对细胞生长方向有很强的诱导力。由于微图形凸起的影响,细胞会首先吸附在微图形脊上,并沿着微图形的方向生长,随后铺展到微图形的底部。小尺寸沟槽对细胞生长方向的诱导作用较强。沟槽微图形表面细胞的增殖和分化能力弱于平板样品表面,强于点状微图形表面；BSA比CS利于细胞增殖和分化。
Controlling cell orientation and morphology by micropatterned substrates is one of hot topic in biomedical engineering. Cells can respond to micrometer scale, and even nano-scale, surface topography. Cell shape, orientation, growth and differentiation are also affected by surface texture of materials. Composite micropatterns were multifunctional biomaterials that co-patterned two or more types of molecules on one substrate. Thus, it can employ the advantage of components.
In this study, we developed a new process that can prepare chitosan/bovine serum albumin (CS/BSA) composite micropatterns by soft-lithography on silicon substrates. CS is a basic polysaccharide in the nature that has good biodegradability, renewable ability and antibacterial ability. BSA is a serum protein that has numerous biochemical applications. Polydimethylsiloxane (PDMS) microstamps were used to transfer micropatterns from the micromachined silicon mold to the hydroxylated silicon wafers. CS/BSA composite micropatterns, BSA micropatterns and CS micropatterns were successfully fabricated on silicon surfaces by micro-transfer molding and replica molding technique.
The antibacterial test was used to evaluate the antibacterial effect of CS/BSA composite micropatterns. The results showed that the CS/BSA composite micropatterns were effectively against two types of bacteria, Escherichia coli and Staphylococcus albus. The osteoblast (Sprague-Dawley rat) culture indicated that CS/BSA composite micropatterns could strongly influence cell behavior. Cells selectively adhered in the CS region at intial culturing stage, and then they gradually spread to the BSA region. The microgrooved surfaces had strong contact guidance effect on cell orentation. Cells tended to be parallel to the microgrooves when they were cultured on BSA microgrooves and CS microgrooves surfaces, They began to attach on the ridges of microgrooves, grew along the direction of microgrooves, and then spread to the bottom of microgrooves. This tendency was stronger on the microgrooves with smaller width. The cells on plane samples had random growth directions. The proliferation and differential rate of cells on microgrooved surfaces is lower than that on plane surface and higher than that on microdot-patterned surfaces, which indicated that micropatterns had complicated effects on cell behaviors. This study provides valuable guidance for the design of the biomaterial surfaces.
引文
[1]Hench LL. Biomaterials:a forecast for the future. Biomaterials.1998,19(16):1419-1423.
[2]俞耀庭,王连永,王深琪.生物医用材料发展状况与对策.北京：科学出版社,2003：13-19.
[3]袁佐平.生物医用材料的研究进展.新经济导刊.2003,(17)：56-57.
[4]刘敬肖,杨大智,王伟强,陈吉华,蔡英骥.表面改性在生物医用材料研究中的应用.材料研究学报.2000,14(3)：225.
[5]曾晟宇,赵乃勤,崔振铎,朱胜利,杨贤金.金属生物材料表面改性研究的进展.材料保护.2000,33(1)：5-8.
[6]张丽,成炜,李铁军.钛种植体表面改性常用方法及其研究进展.社区医学杂志.2010,8(6)：11-13.
[7]Toner M, Irimia D. Blood-on-a-chip. Annual Review of Biomedical Engineering. 2005,7:77-103.
[8]Bransky A, Korin N, Nemirovski Y, Dinnar U. Correlation between erythrocytes deformability and size:a study using a microchannel based cell analyzer. Microvascular Research.2007,73(1):7-13.
[9]姜海,郑小林,彭承琳,翁璇.基于MEMS技术的红细胞变形性测量系统的研究.中国医疗器械信息.2006,12(12)：28-38.
[10]王小章,王朝晖,蒋庄德,叶鹏.硅微通道内血细胞流动特性测试.西安交通大学学报.2009,43(5)：128-132.
[11]Nguyen NT, Wereley S. Fundamentals and applications of microfluidics. USA:Artech House Press,2003:27-45.
[12]Wang FG, Song SY, Zhang JY. Surface texturing of porous silicon with capillary stress and its superhydrophobicity. Chemical Communications.2009,(28):4239-4241.
[13]谈国强,博海洋,苗鸿雁,夏傲,贺中亮.硅基图案化Hf02表面微结构的制备和表征.功能材料.2009,40(10)：1660-1662.
[14]陈长琦,王艳,王旭迪,汪力.软刻蚀技术及其应用.真空.2003,(6)：11-14.
[15]Xia Y, Whitesides GM. Soft lithography. Annual Review of Materials Research. 1998,28:153-184.
[16]Xia Y, Whitesides GM. Soft lithography. Angewandte Chemie International Edition. 1998,37(5):550-575
[17]张冬梅.基于全息光刻及软刻蚀技术制备二维光子晶体模板.浙江大学硕士学位论文.2007：17-19.
[18]赵小力.基于PDMS的自组装及转移印刷装备微结构的研究.哈尔滨工业大学博士学位论文.2007：17-21.
[19]Sotomayor TCM. Alternative lithography. USA:Springer Press,2003:169-204.
[20]石锦霞.软刻蚀技术在高分子科学中的应用.中国科学技术大学博士学位论文.2008：3-5.
[21]罗运军,桂红军.有机硅树脂及其应用.北京：化学工业出版社,2001：6-17.
[22]Mark JE. Polymer data handbook. USA:Oxford University Press,1999:411-435.
[23]潘力佳,何平笙.纳米器件制备的新方法：微接触印刷术.化学通报.2000,(12)：12-17.
[24]Kumar A, Whitesides GM. Features of gold having micrometer to centimeter dimensions can be formed through a combination of stamping with an elastomeric stamp and an alkanethiol "ink" followed by chemical etching. Applied Physics Letters. 1993,63(14):2002-2004.
[25]崔婧怡,马莒生,王广龙.微接触印刷术：制备纳米器件的新技术.电子元件与材料.2005,24(8)：56-69.
[26]洪吉,刘伟庭,陈裕泉.软光刻技术.国外医学(生物医学工程分册).2001,34(3)：134-137.
[27]周明,郑傲然,杨加宏.复制模塑法制备超疏水表面及其应用.物理化学学报.2007,23(8)：1296-1300.
[28]王哲,邢汝博,韩艳春,李滨耀.聚合物三维微图案加工的转移微模塑新方法.高等学校化学学报.2003,24(5)：946-948.
[29]吴福贵,杨海洋,王政,何平笙.毛细微模塑和转移微模塑制备PVP/Ru(dpphen)3Cl2微图形.高分子材料科学与工程.2006,22(6)：188-196.
[30]陈默.微全分析系统中微传递成模软光刻技术研究.重庆大学硕士论文.2004：18.
[31]Rogers JA, Paul KE, Jackman RJ. Using an elastomeric phase mask for sub-100 nm photolithography in the optical near field. Applied Physics Letters. 1997,70(20):2658-2660.
[32]蒋稼欢.生物医学微系统技术及应用.北京：化学工业出版社,2006：80-81,375.
[33]Krsko P, McCann TE, Thach TT, Laabs TL, Geller HM, Libera MR. Length-scale mediated adhesion and directed growth of neural cells by surface-patterned poly(ethylene glycol) hydrogels. Biomaterials.2009,30(5):721-729.
[34]Charest JL, Garci'a AJ, King WP. Myoblast alignment and differentiation on cell culture substrates with microscale topography and model chemistries. Biomaterials. 2007,28(13):2202-2210.
[35]Recknora JB, Recknorb JC, Sakaguchi DS, Mallapragada SK. Oriented astroglial cell growth on micropatterned polystyrene substrates. Biomaterials.2004,25(14):2753-2767.
[36]巩沅鑫,赵先英.成骨细胞生物矿化过程中的调控机制.重庆医学.2009,38(5)：596-599.
[37]Chen W, Oh S, Ong AP, Oh N, Liu Y, Courtney HS, et al. Antibacterial and osteogenic properties of silver-containing hydroxyapatite coatings produced using a sol gel process. Journal of biomedical materials research. Part A.2007,82(4):899-906.
[38]Ravi Kumar MNV. A review of chitin and chitosan applications. Reactive and Functional Polymers.2000,46(1):1-27.
[39]Sorlier P, Denuziere A, Viton C, Domard A. Relation between the degree of acetylation and the electrostatic properties of chitin and chitosan. Biomacromolecules. 2001,2(3):765-772.
[40]Constantine CA, Mello SV, Dupont A, Cao XH, Santos D, Oliveira ON, et al. Layer-by-Layer self-assembled chitosan/poly(thiophene-3-acetic acid) and organophosphorus hydrolase multilayers. Journal of the American Chemical Society. 2003,125 (7):1805-1809.
[41]Camilo CS, Santos DS, Rodrigues JJ, Jr, Vega ML, Campana Filho SP, et al. Surface-relief gratings and photoinduced birefringence in layer-by-layer films of chitosan and an azopolymer. Biomacromolecules.2003,4(6):1583-1588.
[42]付寅,姚桂君,董宇平,曹宇静,段恩奎.重氮树脂与牛血清白蛋白共价结合自组装膜及其表面细胞培养.高等学校化学学报.2003,24(1)：181-183.
[43]Feng J, Gao CY, Wang B, Shen JC. Co-patterning chitosan and bovine serum albumin on an aldehyde-enriched glass substrate by microcontact printing. Thin Solid Films. 2004,460(1-2):286-290.
[44]Xia YN, Rogers JA, Paul KE, Whitesides G. Unconventional methods for fabricating and patterning nanostructures. Chemical Reviews.1999,99(7):1823-1848.
[45]Franssila S. Introduction to microfabrication. Chen D, Liu JQ, Zhu J. Beijing:Electronics Industry Press,2006:154-163
[46]Tavana H, Simon F, Grundke K, Kwok DY, Hair ML, Neumann AW. Interpretation of contact angle measurements on two different fluoropolymers for the determination of solid surface tension. Journal of Colloid and Interface Science.2005,291(2):497-506.
[47]丁晓峰,陈沛智,管蓉.接触角测量技术的应用.分析试验室.2008,27：72-75.
[48]Kim CJ, Muller RS, Pisano AP. Residual strain measurement of thin films using microfabricated vernier gauges. Ensors and Materials.1993,4(4):291-304.
[49]薛晓康.电化学表面增强红外光谱的应用与发展.复旦大学博士学位论文.2008：17-35.
[50]谢佳,鲁雄,张红平,周先礼,屈树新,冯波,等.硅表面CS/BSA复合微图形的制备及表征.高等学校化学学报.2009,30(10)：2319-2325.
[51]陈瑜,吴俊徐,郭平生,王连卫,Zwan M, Sarro PM.一种用于硅基MEMS加工的深刻蚀技术.微细加工技术.2005,4：37-41.
[52]张莉,卓馨,王红艳,王聪.层层静电自组装构筑壳聚糖/磷钨酸复合膜的研究.无机化学学报.2007,23(11)：1988-1993.
[53]Fukuzaki S, Urano H, Nagata KJ. Adsorption of bovine serum albumin onto metal oxide surfaces. Journal of Fermentation and Bioengineering.1996,81(2):163-167.
[54]Kopac T, Bozgeyik K, Yener J. Effect of pH and temperature on the adsorption of bovine serum albumin onto titanium dioxide. Colloids and Surfaces A. 2008,322(1-3):19-28.
[55]Kurrat R, Prenosil JE, Ramsden JJ. Kinetics of human and bovine serum albumin adsorption at silica-titania surfaces. Journal of Colloid and Interface Science. 1997,185(1):1-8.
[56]沈玉华,杨展澜,吴瑾光.BSA-羟基磷灰石可溶性复合物的FTIR光谱.北京大学学报自然科学版.1999,35(4)：431-436.
[57]包志明,潘继伦,吴晨,俞耀庭.半乳糖修饰大孔壳聚糖支架的制备及表征.功能材料.2004,35：2454-2456.
[58]季君晖,史维明.抗菌材料.北京：化学工业出版社,2003.
[59]徐孝华.微生物世界.北京：农业大学出版社,1989.
[60]余贺,龙振洲.医学微生物学.北京：人民卫生出版社,1986.
[61]Onishi H, Machida Y. Biodegradation and distribution of water-soluble chitosan in mice. Biomaterials.1999,20(2):175-182.
[62]Dong WR, Hong FY, Ma XJ, Wei W.The enzymatic degradation and swelling properties of chitosan matrices with different degree of N-acetylation. Carbohydrate Research. 2005,340(15):2403-2410.
[63]Rao SB, Sharma CP. Use of chitosan as a biomaterial:studies on its safety and hemostatic potential. Journal of Biomedial Materials Research.1997,34(1):21-28.
[64]Usami Y, Okamoto Y, Takayama T, Shigemasa Y, Minami S. Chitin and chitosan stimulate canine polymorphonuclear cells to release leukotriene B4 and prostaglandin E2. Journal of Biomedial Materials Research.1998,42(4):517-522.
[65]Ueno H, Yamada H, Tanaka I, Matsuura M, Okumura M, Kadosawa T, et al. Accelerating effects of chitosan for healing at early phase of experimental open wound in dogs. Biomaterials.1999,20(15):1407-1414.
[66]马莉华,马小军.壳聚糖抗菌支架的制备及体外释药性能.中国组织工程研究与临床康复.2007,11(26)：5144-5146.
[67]胡瑛,杜予民,刘慧.壳聚糖抗菌性与分子量和环境介质相关性研究.分析科学学报.2003,19(4):305-308.
[68]张俊英,葛琦,杨春,王天民.蓄能光催化材料对大肠杆菌的抗菌性能研究.稀有金属材料与工程.2007,36(6)：1037-1040.
[69]Dutta PK, Tripathi S, Mehrotra GK, Dutta J. Perspectives for chitosan based antimicrobial films in food applications. Food Chemistry.2009,114(4):1173-1182.
[70]Seiichi T, Yoshiaki M, Masayoshi J, Norio N, Shin-Ichiro N. Induction of drug specific antibody and the controlled release of drug by 6-O-carboxymethyl-chitin. Journal of Controlled Release.1994,28(1-3):235-241.
[71]洪咏龙,魏建华,隋文,刘宝林.牙骨质基质提取物促成骨细胞在钛表面上粘附与移行的实验研究.稀有金属材料与工程.2003,32(7)：527-529.
[72]杨志明.组织工程基础与临床.成都：四川科学技术出版社,2000：3-4.
[73]杨志明,余希杰,解慧琪,陈旭,屈艺.不同来源成骨细胞生物学特性的比较研究.中华创伤杂志.2001,17(1)：10-13.
[74]Diduch DR, Coe MR, Joyner C. Two cell lines from bone marrow that differ in terms of collagen synthesis, osteogenic characteristics, and matrix mineralization. Journal of Bone and Joint Surgery.1993,75A:92-105.
[75]陈琨.细胞电化学及细胞相容性材料研究.湖南大学硕士论文.2007：6-14.
[76]Dunn GA, Brown AF. Alignment of fibroblasts on grooved surfaces described by a simple geometric transformation. Journal of Cell Science.1986,83(1):313-340.
[77]Chou LS, Firth JD, Uitto VJ, Brunette DM. Effects of titanium substratum and grooved surface topography on metalloproteinase-2 expression in human fibroblasts. Journal of Biomedial Materials Research.1998,39(3):437-445.
[78]Matsuzaka K, Ruijter JE, Jansen JA, Walboomers XF. The effect of poly-L-lactic acid with parallel surface micro-groove on osteoblast-like cells in vitro. Biomaterials. 1999,20(14):1293-1301.
[79]Ann SA, Petra O. The effects of continuous and discontinuous groove edges on cell shape and alignment. Experimental Cell Research.2003,288(1):177-188.
[80]Lu X, Leng Y. Comparison of the osteoblast and myoblast behaviors on hydroxyapatite microgrooves. Journal of Biomedial Materials Research B Applications Biomater. 2009,90B:438-445.
[81]范能全,彭兰.Alamar Blue法测定细胞毒性.华西药学杂志.2007,22(4)：472-474.
[82]O'Brien J. Investigation of the alamar blue (resazurin) fluorescent dye for the assessment of mammalian cell cytoxicity. European Journal of Biochemistry. 2000,267(17):5421-5426.
[83]Liu YR, Qu SX, Maitz MF, Tan R, Weng J. The effect of the major components of salvia miltiorrhiza bunge on bone marrow cells. Journal of Ethnopharmacology. 2007,111(3):573-583.
[84]Sabokbar A, Millett PJ, Myer B, Rushton N. A rapid, quantitative assay for measuring alkaline phosphatase activity in osteoblastic cells in vitro. Bone Mineral. 1994,27(1):57-67.
[85]Ramires PA, Romito A. The influence of titania/hydroxyapatite composite coatings on in vitro osteoblasts behaviour. Biomatirials.2001,22(12):1467-1474.
[86]Peter K, Thomas EM, Thu-Trang T, Tracy LL, Herbert MG, Matthew RL. Length-scale mediated adhesion and directed growth of neural cells by surface-patterned poly(ethylene glycol) hydrogels. Biomaterials.2009,30(5):721-729.
[87]Recknor JB, Recknor JC, Sakaguchi DS, Mallapragada SK. Oriented astroglial cell growth on micropatterned polystyrene substrates. Biomaterials.2004,25(14):2753-2767.
[88]吴峰.壳聚糖复合膜的制备及表面形貌的原子力显微镜观察.暨南大学硕士学位论文.2006：14-21.
[2]俞耀庭,王连永,王深琪.生物医用材料发展状况与对策.北京：科学出版社,2003：13-19.
[3]袁佐平.生物医用材料的研究进展.新经济导刊.2003,(17)：56-57.
[4]刘敬肖,杨大智,王伟强,陈吉华,蔡英骥.表面改性在生物医用材料研究中的应用.材料研究学报.2000,14(3)：225.
[5]曾晟宇,赵乃勤,崔振铎,朱胜利,杨贤金.金属生物材料表面改性研究的进展.材料保护.2000,33(1)：5-8.
[6]张丽,成炜,李铁军.钛种植体表面改性常用方法及其研究进展.社区医学杂志.2010,8(6)：11-13.
[7]Toner M, Irimia D. Blood-on-a-chip. Annual Review of Biomedical Engineering. 2005,7:77-103.
[8]Bransky A, Korin N, Nemirovski Y, Dinnar U. Correlation between erythrocytes deformability and size:a study using a microchannel based cell analyzer. Microvascular Research.2007,73(1):7-13.
[9]姜海,郑小林,彭承琳,翁璇.基于MEMS技术的红细胞变形性测量系统的研究.中国医疗器械信息.2006,12(12)：28-38.
[10]王小章,王朝晖,蒋庄德,叶鹏.硅微通道内血细胞流动特性测试.西安交通大学学报.2009,43(5)：128-132.
[11]Nguyen NT, Wereley S. Fundamentals and applications of microfluidics. USA:Artech House Press,2003:27-45.
[12]Wang FG, Song SY, Zhang JY. Surface texturing of porous silicon with capillary stress and its superhydrophobicity. Chemical Communications.2009,(28):4239-4241.
[13]谈国强,博海洋,苗鸿雁,夏傲,贺中亮.硅基图案化Hf02表面微结构的制备和表征.功能材料.2009,40(10)：1660-1662.
[14]陈长琦,王艳,王旭迪,汪力.软刻蚀技术及其应用.真空.2003,(6)：11-14.
[15]Xia Y, Whitesides GM. Soft lithography. Annual Review of Materials Research. 1998,28:153-184.
[16]Xia Y, Whitesides GM. Soft lithography. Angewandte Chemie International Edition. 1998,37(5):550-575
[17]张冬梅.基于全息光刻及软刻蚀技术制备二维光子晶体模板.浙江大学硕士学位论文.2007：17-19.
[18]赵小力.基于PDMS的自组装及转移印刷装备微结构的研究.哈尔滨工业大学博士学位论文.2007：17-21.
[19]Sotomayor TCM. Alternative lithography. USA:Springer Press,2003:169-204.
[20]石锦霞.软刻蚀技术在高分子科学中的应用.中国科学技术大学博士学位论文.2008：3-5.
[21]罗运军,桂红军.有机硅树脂及其应用.北京：化学工业出版社,2001：6-17.
[22]Mark JE. Polymer data handbook. USA:Oxford University Press,1999:411-435.
[23]潘力佳,何平笙.纳米器件制备的新方法：微接触印刷术.化学通报.2000,(12)：12-17.
[24]Kumar A, Whitesides GM. Features of gold having micrometer to centimeter dimensions can be formed through a combination of stamping with an elastomeric stamp and an alkanethiol "ink" followed by chemical etching. Applied Physics Letters. 1993,63(14):2002-2004.
[25]崔婧怡,马莒生,王广龙.微接触印刷术：制备纳米器件的新技术.电子元件与材料.2005,24(8)：56-69.
[26]洪吉,刘伟庭,陈裕泉.软光刻技术.国外医学(生物医学工程分册).2001,34(3)：134-137.
[27]周明,郑傲然,杨加宏.复制模塑法制备超疏水表面及其应用.物理化学学报.2007,23(8)：1296-1300.
[28]王哲,邢汝博,韩艳春,李滨耀.聚合物三维微图案加工的转移微模塑新方法.高等学校化学学报.2003,24(5)：946-948.
[29]吴福贵,杨海洋,王政,何平笙.毛细微模塑和转移微模塑制备PVP/Ru(dpphen)3Cl2微图形.高分子材料科学与工程.2006,22(6)：188-196.
[30]陈默.微全分析系统中微传递成模软光刻技术研究.重庆大学硕士论文.2004：18.
[31]Rogers JA, Paul KE, Jackman RJ. Using an elastomeric phase mask for sub-100 nm photolithography in the optical near field. Applied Physics Letters. 1997,70(20):2658-2660.
[32]蒋稼欢.生物医学微系统技术及应用.北京：化学工业出版社,2006：80-81,375.
[33]Krsko P, McCann TE, Thach TT, Laabs TL, Geller HM, Libera MR. Length-scale mediated adhesion and directed growth of neural cells by surface-patterned poly(ethylene glycol) hydrogels. Biomaterials.2009,30(5):721-729.
[34]Charest JL, Garci'a AJ, King WP. Myoblast alignment and differentiation on cell culture substrates with microscale topography and model chemistries. Biomaterials. 2007,28(13):2202-2210.
[35]Recknora JB, Recknorb JC, Sakaguchi DS, Mallapragada SK. Oriented astroglial cell growth on micropatterned polystyrene substrates. Biomaterials.2004,25(14):2753-2767.
[36]巩沅鑫,赵先英.成骨细胞生物矿化过程中的调控机制.重庆医学.2009,38(5)：596-599.
[37]Chen W, Oh S, Ong AP, Oh N, Liu Y, Courtney HS, et al. Antibacterial and osteogenic properties of silver-containing hydroxyapatite coatings produced using a sol gel process. Journal of biomedical materials research. Part A.2007,82(4):899-906.
[38]Ravi Kumar MNV. A review of chitin and chitosan applications. Reactive and Functional Polymers.2000,46(1):1-27.
[39]Sorlier P, Denuziere A, Viton C, Domard A. Relation between the degree of acetylation and the electrostatic properties of chitin and chitosan. Biomacromolecules. 2001,2(3):765-772.
[40]Constantine CA, Mello SV, Dupont A, Cao XH, Santos D, Oliveira ON, et al. Layer-by-Layer self-assembled chitosan/poly(thiophene-3-acetic acid) and organophosphorus hydrolase multilayers. Journal of the American Chemical Society. 2003,125 (7):1805-1809.
[41]Camilo CS, Santos DS, Rodrigues JJ, Jr, Vega ML, Campana Filho SP, et al. Surface-relief gratings and photoinduced birefringence in layer-by-layer films of chitosan and an azopolymer. Biomacromolecules.2003,4(6):1583-1588.
[42]付寅,姚桂君,董宇平,曹宇静,段恩奎.重氮树脂与牛血清白蛋白共价结合自组装膜及其表面细胞培养.高等学校化学学报.2003,24(1)：181-183.
[43]Feng J, Gao CY, Wang B, Shen JC. Co-patterning chitosan and bovine serum albumin on an aldehyde-enriched glass substrate by microcontact printing. Thin Solid Films. 2004,460(1-2):286-290.
[44]Xia YN, Rogers JA, Paul KE, Whitesides G. Unconventional methods for fabricating and patterning nanostructures. Chemical Reviews.1999,99(7):1823-1848.
[45]Franssila S. Introduction to microfabrication. Chen D, Liu JQ, Zhu J. Beijing:Electronics Industry Press,2006:154-163
[46]Tavana H, Simon F, Grundke K, Kwok DY, Hair ML, Neumann AW. Interpretation of contact angle measurements on two different fluoropolymers for the determination of solid surface tension. Journal of Colloid and Interface Science.2005,291(2):497-506.
[47]丁晓峰,陈沛智,管蓉.接触角测量技术的应用.分析试验室.2008,27：72-75.
[48]Kim CJ, Muller RS, Pisano AP. Residual strain measurement of thin films using microfabricated vernier gauges. Ensors and Materials.1993,4(4):291-304.
[49]薛晓康.电化学表面增强红外光谱的应用与发展.复旦大学博士学位论文.2008：17-35.
[50]谢佳,鲁雄,张红平,周先礼,屈树新,冯波,等.硅表面CS/BSA复合微图形的制备及表征.高等学校化学学报.2009,30(10)：2319-2325.
[51]陈瑜,吴俊徐,郭平生,王连卫,Zwan M, Sarro PM.一种用于硅基MEMS加工的深刻蚀技术.微细加工技术.2005,4：37-41.
[52]张莉,卓馨,王红艳,王聪.层层静电自组装构筑壳聚糖/磷钨酸复合膜的研究.无机化学学报.2007,23(11)：1988-1993.
[53]Fukuzaki S, Urano H, Nagata KJ. Adsorption of bovine serum albumin onto metal oxide surfaces. Journal of Fermentation and Bioengineering.1996,81(2):163-167.
[54]Kopac T, Bozgeyik K, Yener J. Effect of pH and temperature on the adsorption of bovine serum albumin onto titanium dioxide. Colloids and Surfaces A. 2008,322(1-3):19-28.
[55]Kurrat R, Prenosil JE, Ramsden JJ. Kinetics of human and bovine serum albumin adsorption at silica-titania surfaces. Journal of Colloid and Interface Science. 1997,185(1):1-8.
[56]沈玉华,杨展澜,吴瑾光.BSA-羟基磷灰石可溶性复合物的FTIR光谱.北京大学学报自然科学版.1999,35(4)：431-436.
[57]包志明,潘继伦,吴晨,俞耀庭.半乳糖修饰大孔壳聚糖支架的制备及表征.功能材料.2004,35：2454-2456.
[58]季君晖,史维明.抗菌材料.北京：化学工业出版社,2003.
[59]徐孝华.微生物世界.北京：农业大学出版社,1989.
[60]余贺,龙振洲.医学微生物学.北京：人民卫生出版社,1986.
[61]Onishi H, Machida Y. Biodegradation and distribution of water-soluble chitosan in mice. Biomaterials.1999,20(2):175-182.
[62]Dong WR, Hong FY, Ma XJ, Wei W.The enzymatic degradation and swelling properties of chitosan matrices with different degree of N-acetylation. Carbohydrate Research. 2005,340(15):2403-2410.
[63]Rao SB, Sharma CP. Use of chitosan as a biomaterial:studies on its safety and hemostatic potential. Journal of Biomedial Materials Research.1997,34(1):21-28.
[64]Usami Y, Okamoto Y, Takayama T, Shigemasa Y, Minami S. Chitin and chitosan stimulate canine polymorphonuclear cells to release leukotriene B4 and prostaglandin E2. Journal of Biomedial Materials Research.1998,42(4):517-522.
[65]Ueno H, Yamada H, Tanaka I, Matsuura M, Okumura M, Kadosawa T, et al. Accelerating effects of chitosan for healing at early phase of experimental open wound in dogs. Biomaterials.1999,20(15):1407-1414.
[66]马莉华,马小军.壳聚糖抗菌支架的制备及体外释药性能.中国组织工程研究与临床康复.2007,11(26)：5144-5146.
[67]胡瑛,杜予民,刘慧.壳聚糖抗菌性与分子量和环境介质相关性研究.分析科学学报.2003,19(4):305-308.
[68]张俊英,葛琦,杨春,王天民.蓄能光催化材料对大肠杆菌的抗菌性能研究.稀有金属材料与工程.2007,36(6)：1037-1040.
[69]Dutta PK, Tripathi S, Mehrotra GK, Dutta J. Perspectives for chitosan based antimicrobial films in food applications. Food Chemistry.2009,114(4):1173-1182.
[70]Seiichi T, Yoshiaki M, Masayoshi J, Norio N, Shin-Ichiro N. Induction of drug specific antibody and the controlled release of drug by 6-O-carboxymethyl-chitin. Journal of Controlled Release.1994,28(1-3):235-241.
[71]洪咏龙,魏建华,隋文,刘宝林.牙骨质基质提取物促成骨细胞在钛表面上粘附与移行的实验研究.稀有金属材料与工程.2003,32(7)：527-529.
[72]杨志明.组织工程基础与临床.成都：四川科学技术出版社,2000：3-4.
[73]杨志明,余希杰,解慧琪,陈旭,屈艺.不同来源成骨细胞生物学特性的比较研究.中华创伤杂志.2001,17(1)：10-13.
[74]Diduch DR, Coe MR, Joyner C. Two cell lines from bone marrow that differ in terms of collagen synthesis, osteogenic characteristics, and matrix mineralization. Journal of Bone and Joint Surgery.1993,75A:92-105.
[75]陈琨.细胞电化学及细胞相容性材料研究.湖南大学硕士论文.2007：6-14.
[76]Dunn GA, Brown AF. Alignment of fibroblasts on grooved surfaces described by a simple geometric transformation. Journal of Cell Science.1986,83(1):313-340.
[77]Chou LS, Firth JD, Uitto VJ, Brunette DM. Effects of titanium substratum and grooved surface topography on metalloproteinase-2 expression in human fibroblasts. Journal of Biomedial Materials Research.1998,39(3):437-445.
[78]Matsuzaka K, Ruijter JE, Jansen JA, Walboomers XF. The effect of poly-L-lactic acid with parallel surface micro-groove on osteoblast-like cells in vitro. Biomaterials. 1999,20(14):1293-1301.
[79]Ann SA, Petra O. The effects of continuous and discontinuous groove edges on cell shape and alignment. Experimental Cell Research.2003,288(1):177-188.
[80]Lu X, Leng Y. Comparison of the osteoblast and myoblast behaviors on hydroxyapatite microgrooves. Journal of Biomedial Materials Research B Applications Biomater. 2009,90B:438-445.
[81]范能全,彭兰.Alamar Blue法测定细胞毒性.华西药学杂志.2007,22(4)：472-474.
[82]O'Brien J. Investigation of the alamar blue (resazurin) fluorescent dye for the assessment of mammalian cell cytoxicity. European Journal of Biochemistry. 2000,267(17):5421-5426.
[83]Liu YR, Qu SX, Maitz MF, Tan R, Weng J. The effect of the major components of salvia miltiorrhiza bunge on bone marrow cells. Journal of Ethnopharmacology. 2007,111(3):573-583.
[84]Sabokbar A, Millett PJ, Myer B, Rushton N. A rapid, quantitative assay for measuring alkaline phosphatase activity in osteoblastic cells in vitro. Bone Mineral. 1994,27(1):57-67.
[85]Ramires PA, Romito A. The influence of titania/hydroxyapatite composite coatings on in vitro osteoblasts behaviour. Biomatirials.2001,22(12):1467-1474.
[86]Peter K, Thomas EM, Thu-Trang T, Tracy LL, Herbert MG, Matthew RL. Length-scale mediated adhesion and directed growth of neural cells by surface-patterned poly(ethylene glycol) hydrogels. Biomaterials.2009,30(5):721-729.
[87]Recknor JB, Recknor JC, Sakaguchi DS, Mallapragada SK. Oriented astroglial cell growth on micropatterned polystyrene substrates. Biomaterials.2004,25(14):2753-2767.
[88]吴峰.壳聚糖复合膜的制备及表面形貌的原子力显微镜观察.暨南大学硕士学位论文.2006：14-21.