低温常压微波辐射下CS-g-PLLA的制备与性能研究
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摘要
壳聚糖(CS)和聚乳酸(PLA)是目前国内外应用最广泛的生物材料。为改善壳聚糖的降解速率和聚乳酸的生物相容性，合成同时具备天然材料和合成材料优势的壳聚糖接枝聚乳酸材料(CS-g-PLLA)，本研究首先在低温常压下利用微波辐射快速高效地合成了具有较高分子量和纯度，良好结晶性、热稳定性和力学性能的PLLA材料。以此为基础，首次采用生物酶为活性催化剂，以含羟基和氨基的壳聚糖为共引发剂，在常压低温微波辐射下引发L-LA开环共聚，合成了CS-g-PLLA，避免了金属催化剂的使用，提高了反应效率。系统研究了微波反应条件、催化剂种类和用量、投料比对产物接枝率和性能的影响。结果表明共聚物接枝率可控，反应时间为30min，50℃时，在1.0wt％的猪胰酶催化作用下可得接枝率最大为178.75％的共聚物；FTIR和~(13)C-NMR分析结果证明产物为符合预期分子结构的CS-g-PLLA共聚物；DSC、TG和X-RD分析结果表明随着L-LA和CS投料比的增大，共聚物的接枝率增大，结晶性降低。
为指导该接枝共聚物在组织工程及其它生物医学领域的实际应用，本文还从体外降解、细胞毒性和溶血试验三个方面初步考察了材料的生物学性能，结果表明所得共聚物的降解速率比单纯CS快且可通过调节产物的接枝率进行调控；体外细胞培养和溶血试验结果表明，使用猪胰酶为催化剂制备的CS-g-PLLA能有效地避免金属催化剂残留物带来的细胞毒性，并显著改善材料的血液相容性。
Nowadays, chitosan (CS) and polylactide (PLA) are widely used in the field of biomaterials. To improve the slow degradation rate of CS and biocompatibility of PLA, make up for native material and synthetic material's deficiencies, we synthesized the chitosan-graft-poly (L-lactide) copolymer (CS-g-PLLA). Under microwave irradiation we prepared PLLA which exhibited high purity, good crystallinity and satisfactory mechanical performance, then to make the biomaterials' biocompatibility more better, we firstly used porcine pancreatic lipase (PPL) as the catalyst, which completily asepsis, to get the CS-g-PLLA. We can operate with much lower temperature than ever before and under ordinary pressure. The copolymer was synthesized under different reaction time, temperature, reagent mole ratio and different kinds and dosages of catalyst. The copolymers were characterized by FTIR, ~(13)C-NMR, DSC, TG and X-RD, and used weight method to get the grafting percentage. The copolymers' grafting percentage can be controled by change of the feeding molar ratio of L-lactide and chitosan. The results of these spectra showed that the product was CS-g-PLLA copolymer; the crystallinity of raw CS was destroyed and the products' thermal stability descented after the copolymerization. And the grafting percentage of the products can be controled.
The results of vitro degradation test by lysozyme in PBS indicated that the copolymers had a higher weight loss in PBS than that of pure CS, and degradation rate increased with the increasing the feeding molar of L-LA, so we can adjust the degradation velocity of the products and PH value by change of the feeding molar ratio of L-lactide and chitosan, which can reduce acidity of the degradation product of the pure PLLA. Cell culture of vitro was assessed using the MTT assay, the results show that in vitro the copolymers' biocompatibility is more better than PLLA, and the PPL catalysis system's cell viabilitys was much better than the tin octoate catalysis systerm. So using the PPL as the catalyst maybe a really good method to synthesis the biomaterials because of it is non-cytotoxicity and totally harmless to human. The results of hemolysis test indicated that the copolymers consistent with the standard of the hemolysis test's requirement, and the PPL system is the best.
为指导该接枝共聚物在组织工程及其它生物医学领域的实际应用，本文还从体外降解、细胞毒性和溶血试验三个方面初步考察了材料的生物学性能，结果表明所得共聚物的降解速率比单纯CS快且可通过调节产物的接枝率进行调控；体外细胞培养和溶血试验结果表明，使用猪胰酶为催化剂制备的CS-g-PLLA能有效地避免金属催化剂残留物带来的细胞毒性，并显著改善材料的血液相容性。
Nowadays, chitosan (CS) and polylactide (PLA) are widely used in the field of biomaterials. To improve the slow degradation rate of CS and biocompatibility of PLA, make up for native material and synthetic material's deficiencies, we synthesized the chitosan-graft-poly (L-lactide) copolymer (CS-g-PLLA). Under microwave irradiation we prepared PLLA which exhibited high purity, good crystallinity and satisfactory mechanical performance, then to make the biomaterials' biocompatibility more better, we firstly used porcine pancreatic lipase (PPL) as the catalyst, which completily asepsis, to get the CS-g-PLLA. We can operate with much lower temperature than ever before and under ordinary pressure. The copolymer was synthesized under different reaction time, temperature, reagent mole ratio and different kinds and dosages of catalyst. The copolymers were characterized by FTIR, ~(13)C-NMR, DSC, TG and X-RD, and used weight method to get the grafting percentage. The copolymers' grafting percentage can be controled by change of the feeding molar ratio of L-lactide and chitosan. The results of these spectra showed that the product was CS-g-PLLA copolymer; the crystallinity of raw CS was destroyed and the products' thermal stability descented after the copolymerization. And the grafting percentage of the products can be controled.
The results of vitro degradation test by lysozyme in PBS indicated that the copolymers had a higher weight loss in PBS than that of pure CS, and degradation rate increased with the increasing the feeding molar of L-LA, so we can adjust the degradation velocity of the products and PH value by change of the feeding molar ratio of L-lactide and chitosan, which can reduce acidity of the degradation product of the pure PLLA. Cell culture of vitro was assessed using the MTT assay, the results show that in vitro the copolymers' biocompatibility is more better than PLLA, and the PPL catalysis system's cell viabilitys was much better than the tin octoate catalysis systerm. So using the PPL as the catalyst maybe a really good method to synthesis the biomaterials because of it is non-cytotoxicity and totally harmless to human. The results of hemolysis test indicated that the copolymers consistent with the standard of the hemolysis test's requirement, and the PPL system is the best.
引文
[1] 周长忍．生物材料学．中国医药科技出版社，2004，第1版，288
[2] 杨志明，组织工程．化学工业出版社，2002，第1版，1
[3] Skalak R, Fox C F, Fung B. Preface. In: Tissue Engineering. New York: Alan R Liss Inc;1988:1
[4] Langer R, Vacanti JP Tissue engineering. Science. 1993; 260: 920～926.
[5] 王常勇，赵强，范明．组织工程研究进展．第一届全国组织工程会议论文集，1999：199
[6] 张涤生，组织工程学简介．中华整形烧伤外科杂志，1998；14：218～224
[7] JosepH Vacanti. The 8th annual meeting of tissue engineering society international 2005
[8] 商庆新，曹谊林，涤生，翁雨来．生物工程领域的崭新前沿组织工程．实用美容整形外科杂志，2000年4月第11卷第2期，57～58
[9] Mooney D. J, Langer R. The Biomedical Engineering Handbook. CRC Press 1995: 1609～1618
[10] Slukinami Y., Okuno M.. Bioresorrable devices made of forged composites of hydroxyapatite particles and poly-L-lactide. Biomaterials, 1999, 20:859
[11] Freed C. E., Vunjak, Novakovic G., Iron K. J., Biodegradable polymer scaffold for tissue engineering. Biotechnology N. Y., 1994, 12(7): 689-693
[12] Kim B. S., Mooney D. J., Develepment of biocompatible synthetic extracellar matrices for tiddue engineering. Trends biotecnol, 1998: 16(5): 224～230
[13] Kulkarni R. K., Moore E. G., Hegyeli A. F. and Leonard E, J. Biomed. Mater. Res., 1971,5(15): 169
[14] Schmitt E. A., Flanagan D. R. and Linhardt R. J., Macromolecules, 1994, 27:743
[15] Li S., Garreau H. and Vert M., J. Mater. Sci.: Mater. Med., 1990, 1:198
[16] Grizzi, I., Garreau, H., Li, S. and Vert, M.. Biomaterials, 1995, 16:305
[17] Bostman O., Hirvensalo E., MaE kinen, J. and Rokkanen, P., J. Bone Joint Surg., 1990, 72B,592
[18] Suganama J., Alexander H., Traub J. and Ricci J. L., Tissue-Inducing Biomaterials, ed. L. G.Cima. Materials Research Society, 1992:339-343
[19] Migamae J. A., Biotechnology, 1986, 13:565～576
[20] Allock H. R. Poly (organophosphazenes)--unusual new high polymers. Angewandte Chemie(International ed. in English), 1977, 16(3):147-156
[21] Laurencin C. T., EIAmim S. E, Ibim S. E., et al., A highly porous 3-dimensional polypHospHazene polymer mareix for skeletal tissue regeneration, J. Biomed. Mater. Res., 1996,30(2): 133
[22] Attwia M. A., Uhrich K. E., Botchwey E., et al., Cytotoxicity testing of poly(anhydride-co-imides) for orthopedic applications, J. Biomed. Mater. Res., 1995, 29(10): 1233
[23] 蒋挺大．壳聚糖．化学工业出版社2001年第1版：9
[24] Yamane S, Iwasaki N, Majima T, Funakoshi T, Masuko T, Harada K, Minami A, Monde K, Nishimura SI. Feasibility of chitosan-based hyaluronic acid hybrid biomaterial for a novel scaffold in cartilage tissue engineering. Biomaterials, 2005; 26:611～19
[25] Chen J S, Li Q H, Xu J T, Huang Y X, Ding Y, Deng H W, Zhao S B, Chen R. Study on biocompatibility of complexes of collagen-chitosan-sodium hyaluronate and cornea. Artif Organs,2005, 29:104-113
[26] Mao J S, Zhao L G, Yao K D, Shang Q X, Yang G H, Cao Y L. Study of novel chitosan-gelatin artificial skin in vitro. J Biomed Mater Res 2003; 64A: 301～308
[27] Ma Lie, Gao Changyou, Mao Zhengwei, Zhou Jie, Shen Jiacong, Hu Xueqing, Han Chunmao. Collagen/chitosan porous scaffolds with improved biostability for skin tissue engineering. Biomaterials 2003; 24:4833-4841
[28] Constantia E, Wolf ram Frick, Udo Losert, et al. Chitosan-thioglycolic acid conjugate: a new scaffold material for tissue engineering. Inter J pHarm 2003; 256:183-189
[29] 丁珊，李立华，周长忍．新型组织工程支架材料．生物医学工程学杂志，2002；19：122～126
[30] 李立华，李红，罗丙红，周长忍．壳聚糖及其衍生物在组织工程中的应用．功能高分子学报，2005，18：347～351
[31] Putnam A J, Mooney D J. Tissue engineering using synthetic extra cellular matrices. Nature Med, 1996, (2): 824
[32] Giguere R J, Bray T L, Duncan S M. et al. Application of commercial microwave ovens to organic synthesis. Tetrahedron Letters, 1986, 27(41): 4945～4948
[33] Sandor K, Ildiko B, Gyorgy D, et al. Fast microwave-mediated bulk polymerization of D,L-lactic acid. Macromolecular Rapid Commun, 2001, 22(13): 1063～1605
[34] 杨秀英，张德庆．微波法合成低分子量聚L-乳酸研究．齐齐哈尔大学学报，2005，24(4)：17～20
[35] Liu L J, Zhang C. Microwave-assisted polymerization of D,L-lactide with stannous octanoate as catalyst. Chin. Chem. Lett., 2001, 12(8): 663～664
[36]刘立建，廖立琼，宋英等．己内酯和丙交酯的微波辅助开环聚合反应．高分子通报，2003，5：1～6
[37] Zhang C, Liao L Q, Liu L J. Rapid ring-opening polymerization of D,L-lactide by microwaves. Macromolecular Rapid Communications, 2004, 25(15): 1402～1405
[38] 张科，王鹏，李文科等．聚乳酸的微波辐射合成方法研究．高分子材料科学与工程，2004，20(3)：46～48
[39] 舒静，王鹏，张科等．聚D,L-丙交酯的常压微波辐射合成．高校化学工程学报，2005，19(4)：498～502
[40] Shu J, Wang P , Zheng T, et al. Microwave-irradiated ring-opening polymerization of D,L-lactide under atmospHere. Appl. Polym. Sci., 2006, 100(3): 2244～2247
[41] 王鹏，舒静．微波下PLLA的熔融缩聚反应．发明专利公开说明书，2005
[42] Pandey A, Aswath P B. Microwave synthesis of Poly L-Lactic Acid and Polyglycolic Acid blends for medical application. 60th Southwest Regional Meeting of the American Chemical Society, University of Texas at Arlington, USA, 2004
[43] Imbronito A V, Scarano A, Orsini G, Ultrastructure of bone healing in defects grafted with a copolymer of polylactic/polyglycolic acids . Journal of biomedical materials research, 2005,74(2): 215～221
[44] Albert P, Warth H, Muelhaupt R. et al. Comparison of thermal and microwave-activated polymerization of e-caprolactone with titanium tetrabutylate as catalyst. Macromol. Chem.pHys., 1996, 197(5): 1633～1641
[45] Liao L Q, Liu L J, Zhang C, et al. Microwave-assisted ring opening polymerization of e-caprolactone. Polym. Sci. Polym. Chem., 2002, 40:1749-1755
[46] Barbier-Baudry D, Brachais C H, Cretu A, et al. An easy way toward e-caprolactone macromonomers by microwave irradiation using early lanthanide halides as catalysts. Macromol.Rapid. Commun., 2002, 23(3): 200～204
[47] Barbier-Baudry, D.; Brachais, L.; Cretu, A.; et al. Synthesis of polycaprolactone by microwave irradiation - an interesting route to synthesize this polymer via green chemistry.Environmental Chemistry Letters, 2003, 1(1): 19～23
[48] 余兆菊．酸、醇引发的e-己内酯微波开环聚合．武汉大学，博士学位论文，2004
[49] Z J Yu, L J Liu. Effect of microwave energy on chain propagation of poly(e-caprolactone) in benzoic acid-initiated ring opening polymerization of e-caprolactone. European Polymer Journal, 2004, 40:2213～2220
[50] 宋英．2，2-二甲基三亚甲基破酸酣和e-己内酯的微波开环聚合．武汉大学，博士学位论 文，2004
[51] Song Y, Liu L J, Weng X C, et al. Acid-initiated polymerization ofe-caprolactone under microwave irradiation and its application in the preparation of drug controlled release system. Journal of Biomaterials Science, Polymer Edition, 2003, 14(3): 241～253
[52] Yu Z J, Liu L J. Microwave-assisted synthesis of poly(e-caprolactone)-poly (ethylene glycol)-poly-(caprolactone) tri-block co-polymers and use as matrices for sustained delivery of ibuprofen taken as model drug. Journal of biomaterials science, Polymer edition, 2005, 16(8):957～971
[53] Zhang C, Liu L J, Liao L Q, et al. Microwave-assisted polymerization of trimethylene carbonate. Polym. Prepr., 2003, 44(1): 874-875
[54] 丁盈红，黄小文，张红．微波辐射制备壳聚糖．化学试剂，2003，25(1)：41～42．
[55] 居红芳．微波新技术制备壳聚糖的研究．常熟高专学报，2004，18(4)：56～59
[56] 张立彦，曾庆孝，李作为等．密闭条件下微波场中甲壳素脱乙酰反应的条件研究．化工进展．2005，24(3)：295～298
[57] 丁盈红，李若琦，伍锟贤等．微波辐射快速制备水溶性壳聚糖．中国生化药物杂志，2002，23(3)：132～133
[58] Shao J, Yang Y M, Zhong Q Q. Studies on preparation of oligoglucosamine by oxidative degradation under microwave irradiation. Polym. Deg. Stab., 2003, 82(3): 395～398
[59] 胡思前．微波条件下制备水溶性壳聚糖的研究．高等函授学报(自然科学版)，2004，17(2)：10～12
[60] Xing R G, Liu S, Yu H H, et al. Salt-assisted acid hydrolysis of chitosan to oligomers under microwave irradiation. Carbohydrate Research, 2005, 340:2150～2153
[61] 郭国瑞，钟海山，王科军等．微波作用下甲壳素的脱乙酰化和壳聚糖的羧甲基化．赣南师范学院学报，1997，3：57～59
[62] 徐云龙，冯屏，钱秀珍等．微波合成羧甲基壳聚糖．华东理工大学学报，2003，29(4)：380～383
[63] 关丽，王燕妹，李国明等．微波辐射制各羧甲基壳聚糖研究初探．广东药学院学报，2003，19(4)：307～309
[64] 罗登科，葛华才．微波辐射水溶液体系壳聚糖羧甲基化反应的研究．广州化学，2004，29(3)：14～17
[65] 郝红英，徐文国，邵自强．微波法制备羧甲基壳聚糖及其在水处理中的应用．高分子材料科学与工程，2006，22(2)：223～226
[66] 来水利，王青玲，胡益平．微波作用下磺化羟乙基壳聚糖制备的初步研究．陕西科技大学学报，2005，23(5)：62～64
[67] 马宁，汪琴，孙胜玲等．甲壳素和壳聚糖化学改性研究进展．化学进展，2004，16(4)：643～653
[68] Xing R G, Liu S, Yu H H, et al. Preparation of low-molecular-weight and high-sulfate-content antioxidant activity in vitro. Carbohydrate Research, 2004, 339:2515～2519
[69] 李鹏程，刘松．磺化壳聚糖的金属混合物及其制备方法．发明专利公开说明书，2006
[70] 董奇志，郭振楚，谭凤娇．微波作用下壳聚糖的O-苯甲基化反应．南京化工大学学报，2001，23(1)：72～74
[71] 董奇志，郭振楚，谭凤娇．微波作用下壳聚糖的O-苄基化反应．合成化学，2001，9(6)：560～562
[72] 董奇志，郭振楚．微波作用下壳聚糖的O．烷基化反应．湘潭大学自然科学学报，2001，23(2)：57～60
[73] Liu L, Li Y P, Li Y, et al. Rapid N-pHthaloylation of chitosan by microwave irradiation. Carbohydrate Polymers., 2004, 57:97～100
[74] Vandana S, Devendra N T, Ashutosh T, et al. Microwave promoted synthesis of chitosan-graft-poly(acrylonitrile). Appl. Polym. Sci., 2005, 95(4): 820～825
[75] Vandana S, Ashutosh T, Devendra N T, et al. Microwave enhanced synthesis of chitosan-graft-polyacrylamide. Polymer, 2006, 47(1): 254～260
[76] Liu L, Li Y, Fang Y, et al. Microwave-assisted graft copolymerization ofe-caprolactone onto chitosan via the pHthaloyl protection method. Carbohydrate Polymers, 2005, 60(3):351～356
[77] Aime S, Gianolio E, Uggeri F, et al. New paramagnetic supramolecular adducts for MRI applications based on non-covalent interactions between Gd(Ⅲ)-complexes and-cyclodextrin units anchored to chitosan. Journal of inorganic biochemistry, 2006, 100(56): 931-938
[78] 蒋挺大．壳聚糖．北京：化学工业出版社，2001：63，17
[79] 曹佐英，赖声礼，葛华才．微波辐射下甲醛交联壳聚糖的制备及其吸附性能的研究．现代化工，1999，19(6)：24
[80] 曹佐英，赖声礼，葛华才．微波辐射下乙二醛交联壳聚糖的制备及其吸附性能的研究．微波学报，2000，16(1)：96～99
[81] 曹佐英，葛华才，赖声礼．微波辐射下壳聚糖的化学改性及其应用研究(Ⅱ)．华南理工大学学报(自然科学版)，2000，28(10)：15
[82] Cao Z Y, Ge H C, Lai S L. Studies on synthesis and adsorption properties of chitosan cross-linked by glutaraldehyde and Cu(Ⅱ) as template under microwave irradiation . Europ.Polym., 2001, 37(10): 2141～2143
[83] 曹佐英，张启修，魏琦峰．微波辐射下交联壳聚糖树脂固载化B-环糊精．高分子材料科学与工程，2003，19(3)：204
[84] 郝红英，徐文国，邵自强．模板法微波辐射下交联壳聚糖的合成及其吸附性能．环境污染与防治．2005，27(8)：568～570
[85] 周悦，龙来寿．微波辐射下交联壳聚糖树脂的制备及其吸附性能．韶关学院学报(自然科学版)，2005，26(6)：71～74
[86] 曹佐英，葛华才，赖声礼．微波能促进壳聚糖钙离子配合物的制备研究．食品工业科技，2000，21(2)：11～13
[87] 曹佐英，赖声礼，曹珍年．微波辐射下壳聚糖zn(Ⅱ)配合物的合成．现代化工，1999，19(11)：24～27
[88] 朱惠光，计剑，高长有等，聚乳酸组织工程支架材料，功能高分子学报，2001，14(4)：488～492
[89] Inoue Y., Kamei S., Jmicromol Sci Pure Appl Chem, 1995, A322:787
[90] 周贤爵，李进，邵惠丽，胡学超．高分子材料科学与工程，2005，5，21(3)：66～69
[91] 滕新荣，任杰，易辉．合成纤维，2005．1：3～5
[92] Liu L J, Zhang C. Chinese Chemical Letters, 2001, 12(8): 663～664
[93] 张科，王鹏，李文科．高分子材料科学与工程,2004,20(3)：46～48
[94] Shu J, Wang P, Zheng T. Appl. Polym. Sci., 2006, 100(3): 2244～2247
[95] Wang P, Shu J, Tian L Y. Faming Zhuanli Shenqing Gongkai Shuomingshu, 2005
[96] Elisseeff J H. Embryonic stem cells: potential for more impact. Trends in Biotechnology,2004; 22:155～156
[97] Szilvassy S J. The biology of hematopoietic stem cells. Archives of Medical Research, 2003,34:446～460
[98] Peng H, Huard J. Muscle-derived stem cells for musculoskeletal tissue regeneration and repair. Transplant Immunology, 2004, 12:311～319
[99] 李姝丹，L-丙交酯的合成研究，硕士论文，哈尔滨理工大学，2004年：8～9
[100] Schindler A, Harper D. Journal of Polymer Science, Polymer Chemistry Edition, 1979,17(8): 2593～2599
[101] Stolt M, Sodergard A. Macromolecules, 1999, 32:6412～6417
[102] Wang N, Wu X S, Mater H. Sci. Eng. 1997, 76:373～374
[103] Witzke D R, Narayan R. Macromolecules, 1997, 30:7075～7085
[104] Hideto T, Shinya M. Polymer Degradation and Stability. 2001, 71(3): 415～424
[105] 王伟超，L-乳酸熔融缩聚及聚乳酸的结晶和熔融行为，浙江大学硕士学位论文，2006．6：41～42
[106] 郝红，梁国正．聚乳酸的溶液结晶行为．高分子材料科学与工程，2005，21(5)：185～188
[107] 刘莹，阮建明，张海坡，申雄军，周忠诚．聚L-乳酸(PLLA)的热稳定性．粉末冶金材料科学与工程．2006年12月，11(6)：365～369
[108] Zhang X C, Macdonald D A, Goosen F A, et al.. Polym.Sci. PartA: Polym.Chem., 1994,32(15): 2965～2970
[109] Cai Q, Yang J, Bei J Z. Biomaterials, 2002, 23:4483～4492
[110] Luo B H, Zhou C R. The world Engineers' Convention 2004 (WEC2004), November 2-6,2004, Shanghai, China; Zhu Y B, Gao C Y. Tissue engineering, 2004, 10:53～61
[111] 赵小东，刘文广，姚康德．离子交换与吸附，2003，19(4)：304～310
[112] Cui Y L, Qi A D, Yao K D. J. Biomed. Mater. Res., 2002, 60(3): 398～404
[113] Kim J Y, Ha C S, Jo N J. Polym. Int, 2002, 51:1123～1128
[114] Yan Wu, Yongli Zheng, Wuli Yang, Changchun Wang, Jianhua Hu, Shoukuan Fu. Synthesis and characterization of a novel ampHipHilic chitosan-polylactide graft copolymer. Carbohydrate Polymers, 2005 (59): 165～171
[115] Y. Liu, E Tian, K. A. Hu. Systhesis and characterization of a brush-like copolymer of a polylactide grafted onto chitosan. Carbohydrate Research, 2004 (339): 845～851
[116] Fujioka M, Okada H, Kusala Y. Macromol. Rapid Commun., 2004, 25:1776～1780
[117] 仰振球，李巧霞，樊红雷，宋宝珍，NMR，FTIR及电位滴定法测定壳聚糖脱乙酰度．功能高分子学报，2003．9：313-318
[118] X.Ou,A.Wirsen,A.-C.Albertsson，Effect of lactic/glycolic acid side chains on the thermal degradationkinetics of chitosan derivatives.Polymer 41(2000):4841～4847
[119] 朱爱萍，吴钧，张娜，沈健．壳聚糖一种具有应用潜力的组织工程支架材料．化学通报，2002(65)：w003，1-5
[120] Ariela V Paula, Daniele Urioste, Julio C Santos and Heizir F de Castro.. Porcine pancreatic lipase immobilized on polysiloxane-polyvinyl alcohol hybrid matrix: catalytic properties and feasibility to mediate synthesis of surfactants and biodiesel. Journal of Chemical Technology and Biotechnology. 2007(82): 281～288
[121] Witzke D R, Narayan R.. Macromolecules, 1997, 30:7075～7085
[122] 陈百万．生物医学工程学．北京：科学出版社，1997
[123] 邓倩莹，缓释溶菌酶纳米微球/壳聚糖复合支架材料的制备及性能，学位论文，暨南大学，2006
[124] 陈华，张伯勋，张巨松，梁雨田，胡成栋，黄靖香，张莉，高飞．柠檬酸化半水硫酸钙作为骨移植替代材料的安全性及生物相容性评价．中国临床康复，第9卷第34期，2005．9：56～59
[125] 赵建华，廖维宏，王远亮，潘君，柳峰．消旋聚乳酸羟基磷灰石脱钙骨基质的制备及其体外降解特性研究．中国修复重建外科杂志，2003年，第17卷第1期：61～64
[126] 司徒镇强，吴军正．细胞培养．西安：世界图书出版西安公司，2004
[2] 杨志明，组织工程．化学工业出版社，2002，第1版，1
[3] Skalak R, Fox C F, Fung B. Preface. In: Tissue Engineering. New York: Alan R Liss Inc;1988:1
[4] Langer R, Vacanti JP Tissue engineering. Science. 1993; 260: 920～926.
[5] 王常勇，赵强，范明．组织工程研究进展．第一届全国组织工程会议论文集，1999：199
[6] 张涤生，组织工程学简介．中华整形烧伤外科杂志，1998；14：218～224
[7] JosepH Vacanti. The 8th annual meeting of tissue engineering society international 2005
[8] 商庆新，曹谊林，涤生，翁雨来．生物工程领域的崭新前沿组织工程．实用美容整形外科杂志，2000年4月第11卷第2期，57～58
[9] Mooney D. J, Langer R. The Biomedical Engineering Handbook. CRC Press 1995: 1609～1618
[10] Slukinami Y., Okuno M.. Bioresorrable devices made of forged composites of hydroxyapatite particles and poly-L-lactide. Biomaterials, 1999, 20:859
[11] Freed C. E., Vunjak, Novakovic G., Iron K. J., Biodegradable polymer scaffold for tissue engineering. Biotechnology N. Y., 1994, 12(7): 689-693
[12] Kim B. S., Mooney D. J., Develepment of biocompatible synthetic extracellar matrices for tiddue engineering. Trends biotecnol, 1998: 16(5): 224～230
[13] Kulkarni R. K., Moore E. G., Hegyeli A. F. and Leonard E, J. Biomed. Mater. Res., 1971,5(15): 169
[14] Schmitt E. A., Flanagan D. R. and Linhardt R. J., Macromolecules, 1994, 27:743
[15] Li S., Garreau H. and Vert M., J. Mater. Sci.: Mater. Med., 1990, 1:198
[16] Grizzi, I., Garreau, H., Li, S. and Vert, M.. Biomaterials, 1995, 16:305
[17] Bostman O., Hirvensalo E., MaE kinen, J. and Rokkanen, P., J. Bone Joint Surg., 1990, 72B,592
[18] Suganama J., Alexander H., Traub J. and Ricci J. L., Tissue-Inducing Biomaterials, ed. L. G.Cima. Materials Research Society, 1992:339-343
[19] Migamae J. A., Biotechnology, 1986, 13:565～576
[20] Allock H. R. Poly (organophosphazenes)--unusual new high polymers. Angewandte Chemie(International ed. in English), 1977, 16(3):147-156
[21] Laurencin C. T., EIAmim S. E, Ibim S. E., et al., A highly porous 3-dimensional polypHospHazene polymer mareix for skeletal tissue regeneration, J. Biomed. Mater. Res., 1996,30(2): 133
[22] Attwia M. A., Uhrich K. E., Botchwey E., et al., Cytotoxicity testing of poly(anhydride-co-imides) for orthopedic applications, J. Biomed. Mater. Res., 1995, 29(10): 1233
[23] 蒋挺大．壳聚糖．化学工业出版社2001年第1版：9
[24] Yamane S, Iwasaki N, Majima T, Funakoshi T, Masuko T, Harada K, Minami A, Monde K, Nishimura SI. Feasibility of chitosan-based hyaluronic acid hybrid biomaterial for a novel scaffold in cartilage tissue engineering. Biomaterials, 2005; 26:611～19
[25] Chen J S, Li Q H, Xu J T, Huang Y X, Ding Y, Deng H W, Zhao S B, Chen R. Study on biocompatibility of complexes of collagen-chitosan-sodium hyaluronate and cornea. Artif Organs,2005, 29:104-113
[26] Mao J S, Zhao L G, Yao K D, Shang Q X, Yang G H, Cao Y L. Study of novel chitosan-gelatin artificial skin in vitro. J Biomed Mater Res 2003; 64A: 301～308
[27] Ma Lie, Gao Changyou, Mao Zhengwei, Zhou Jie, Shen Jiacong, Hu Xueqing, Han Chunmao. Collagen/chitosan porous scaffolds with improved biostability for skin tissue engineering. Biomaterials 2003; 24:4833-4841
[28] Constantia E, Wolf ram Frick, Udo Losert, et al. Chitosan-thioglycolic acid conjugate: a new scaffold material for tissue engineering. Inter J pHarm 2003; 256:183-189
[29] 丁珊，李立华，周长忍．新型组织工程支架材料．生物医学工程学杂志，2002；19：122～126
[30] 李立华，李红，罗丙红，周长忍．壳聚糖及其衍生物在组织工程中的应用．功能高分子学报，2005，18：347～351
[31] Putnam A J, Mooney D J. Tissue engineering using synthetic extra cellular matrices. Nature Med, 1996, (2): 824
[32] Giguere R J, Bray T L, Duncan S M. et al. Application of commercial microwave ovens to organic synthesis. Tetrahedron Letters, 1986, 27(41): 4945～4948
[33] Sandor K, Ildiko B, Gyorgy D, et al. Fast microwave-mediated bulk polymerization of D,L-lactic acid. Macromolecular Rapid Commun, 2001, 22(13): 1063～1605
[34] 杨秀英，张德庆．微波法合成低分子量聚L-乳酸研究．齐齐哈尔大学学报，2005，24(4)：17～20
[35] Liu L J, Zhang C. Microwave-assisted polymerization of D,L-lactide with stannous octanoate as catalyst. Chin. Chem. Lett., 2001, 12(8): 663～664
[36]刘立建，廖立琼，宋英等．己内酯和丙交酯的微波辅助开环聚合反应．高分子通报，2003，5：1～6
[37] Zhang C, Liao L Q, Liu L J. Rapid ring-opening polymerization of D,L-lactide by microwaves. Macromolecular Rapid Communications, 2004, 25(15): 1402～1405
[38] 张科，王鹏，李文科等．聚乳酸的微波辐射合成方法研究．高分子材料科学与工程，2004，20(3)：46～48
[39] 舒静，王鹏，张科等．聚D,L-丙交酯的常压微波辐射合成．高校化学工程学报，2005，19(4)：498～502
[40] Shu J, Wang P , Zheng T, et al. Microwave-irradiated ring-opening polymerization of D,L-lactide under atmospHere. Appl. Polym. Sci., 2006, 100(3): 2244～2247
[41] 王鹏，舒静．微波下PLLA的熔融缩聚反应．发明专利公开说明书，2005
[42] Pandey A, Aswath P B. Microwave synthesis of Poly L-Lactic Acid and Polyglycolic Acid blends for medical application. 60th Southwest Regional Meeting of the American Chemical Society, University of Texas at Arlington, USA, 2004
[43] Imbronito A V, Scarano A, Orsini G, Ultrastructure of bone healing in defects grafted with a copolymer of polylactic/polyglycolic acids . Journal of biomedical materials research, 2005,74(2): 215～221
[44] Albert P, Warth H, Muelhaupt R. et al. Comparison of thermal and microwave-activated polymerization of e-caprolactone with titanium tetrabutylate as catalyst. Macromol. Chem.pHys., 1996, 197(5): 1633～1641
[45] Liao L Q, Liu L J, Zhang C, et al. Microwave-assisted ring opening polymerization of e-caprolactone. Polym. Sci. Polym. Chem., 2002, 40:1749-1755
[46] Barbier-Baudry D, Brachais C H, Cretu A, et al. An easy way toward e-caprolactone macromonomers by microwave irradiation using early lanthanide halides as catalysts. Macromol.Rapid. Commun., 2002, 23(3): 200～204
[47] Barbier-Baudry, D.; Brachais, L.; Cretu, A.; et al. Synthesis of polycaprolactone by microwave irradiation - an interesting route to synthesize this polymer via green chemistry.Environmental Chemistry Letters, 2003, 1(1): 19～23
[48] 余兆菊．酸、醇引发的e-己内酯微波开环聚合．武汉大学，博士学位论文，2004
[49] Z J Yu, L J Liu. Effect of microwave energy on chain propagation of poly(e-caprolactone) in benzoic acid-initiated ring opening polymerization of e-caprolactone. European Polymer Journal, 2004, 40:2213～2220
[50] 宋英．2，2-二甲基三亚甲基破酸酣和e-己内酯的微波开环聚合．武汉大学，博士学位论 文，2004
[51] Song Y, Liu L J, Weng X C, et al. Acid-initiated polymerization ofe-caprolactone under microwave irradiation and its application in the preparation of drug controlled release system. Journal of Biomaterials Science, Polymer Edition, 2003, 14(3): 241～253
[52] Yu Z J, Liu L J. Microwave-assisted synthesis of poly(e-caprolactone)-poly (ethylene glycol)-poly-(caprolactone) tri-block co-polymers and use as matrices for sustained delivery of ibuprofen taken as model drug. Journal of biomaterials science, Polymer edition, 2005, 16(8):957～971
[53] Zhang C, Liu L J, Liao L Q, et al. Microwave-assisted polymerization of trimethylene carbonate. Polym. Prepr., 2003, 44(1): 874-875
[54] 丁盈红，黄小文，张红．微波辐射制备壳聚糖．化学试剂，2003，25(1)：41～42．
[55] 居红芳．微波新技术制备壳聚糖的研究．常熟高专学报，2004，18(4)：56～59
[56] 张立彦，曾庆孝，李作为等．密闭条件下微波场中甲壳素脱乙酰反应的条件研究．化工进展．2005，24(3)：295～298
[57] 丁盈红，李若琦，伍锟贤等．微波辐射快速制备水溶性壳聚糖．中国生化药物杂志，2002，23(3)：132～133
[58] Shao J, Yang Y M, Zhong Q Q. Studies on preparation of oligoglucosamine by oxidative degradation under microwave irradiation. Polym. Deg. Stab., 2003, 82(3): 395～398
[59] 胡思前．微波条件下制备水溶性壳聚糖的研究．高等函授学报(自然科学版)，2004，17(2)：10～12
[60] Xing R G, Liu S, Yu H H, et al. Salt-assisted acid hydrolysis of chitosan to oligomers under microwave irradiation. Carbohydrate Research, 2005, 340:2150～2153
[61] 郭国瑞，钟海山，王科军等．微波作用下甲壳素的脱乙酰化和壳聚糖的羧甲基化．赣南师范学院学报，1997，3：57～59
[62] 徐云龙，冯屏，钱秀珍等．微波合成羧甲基壳聚糖．华东理工大学学报，2003，29(4)：380～383
[63] 关丽，王燕妹，李国明等．微波辐射制各羧甲基壳聚糖研究初探．广东药学院学报，2003，19(4)：307～309
[64] 罗登科，葛华才．微波辐射水溶液体系壳聚糖羧甲基化反应的研究．广州化学，2004，29(3)：14～17
[65] 郝红英，徐文国，邵自强．微波法制备羧甲基壳聚糖及其在水处理中的应用．高分子材料科学与工程，2006，22(2)：223～226
[66] 来水利，王青玲，胡益平．微波作用下磺化羟乙基壳聚糖制备的初步研究．陕西科技大学学报，2005，23(5)：62～64
[67] 马宁，汪琴，孙胜玲等．甲壳素和壳聚糖化学改性研究进展．化学进展，2004，16(4)：643～653
[68] Xing R G, Liu S, Yu H H, et al. Preparation of low-molecular-weight and high-sulfate-content antioxidant activity in vitro. Carbohydrate Research, 2004, 339:2515～2519
[69] 李鹏程，刘松．磺化壳聚糖的金属混合物及其制备方法．发明专利公开说明书，2006
[70] 董奇志，郭振楚，谭凤娇．微波作用下壳聚糖的O-苯甲基化反应．南京化工大学学报，2001，23(1)：72～74
[71] 董奇志，郭振楚，谭凤娇．微波作用下壳聚糖的O-苄基化反应．合成化学，2001，9(6)：560～562
[72] 董奇志，郭振楚．微波作用下壳聚糖的O．烷基化反应．湘潭大学自然科学学报，2001，23(2)：57～60
[73] Liu L, Li Y P, Li Y, et al. Rapid N-pHthaloylation of chitosan by microwave irradiation. Carbohydrate Polymers., 2004, 57:97～100
[74] Vandana S, Devendra N T, Ashutosh T, et al. Microwave promoted synthesis of chitosan-graft-poly(acrylonitrile). Appl. Polym. Sci., 2005, 95(4): 820～825
[75] Vandana S, Ashutosh T, Devendra N T, et al. Microwave enhanced synthesis of chitosan-graft-polyacrylamide. Polymer, 2006, 47(1): 254～260
[76] Liu L, Li Y, Fang Y, et al. Microwave-assisted graft copolymerization ofe-caprolactone onto chitosan via the pHthaloyl protection method. Carbohydrate Polymers, 2005, 60(3):351～356
[77] Aime S, Gianolio E, Uggeri F, et al. New paramagnetic supramolecular adducts for MRI applications based on non-covalent interactions between Gd(Ⅲ)-complexes and-cyclodextrin units anchored to chitosan. Journal of inorganic biochemistry, 2006, 100(56): 931-938
[78] 蒋挺大．壳聚糖．北京：化学工业出版社，2001：63，17
[79] 曹佐英，赖声礼，葛华才．微波辐射下甲醛交联壳聚糖的制备及其吸附性能的研究．现代化工，1999，19(6)：24
[80] 曹佐英，赖声礼，葛华才．微波辐射下乙二醛交联壳聚糖的制备及其吸附性能的研究．微波学报，2000，16(1)：96～99
[81] 曹佐英，葛华才，赖声礼．微波辐射下壳聚糖的化学改性及其应用研究(Ⅱ)．华南理工大学学报(自然科学版)，2000，28(10)：15
[82] Cao Z Y, Ge H C, Lai S L. Studies on synthesis and adsorption properties of chitosan cross-linked by glutaraldehyde and Cu(Ⅱ) as template under microwave irradiation . Europ.Polym., 2001, 37(10): 2141～2143
[83] 曹佐英，张启修，魏琦峰．微波辐射下交联壳聚糖树脂固载化B-环糊精．高分子材料科学与工程，2003，19(3)：204
[84] 郝红英，徐文国，邵自强．模板法微波辐射下交联壳聚糖的合成及其吸附性能．环境污染与防治．2005，27(8)：568～570
[85] 周悦，龙来寿．微波辐射下交联壳聚糖树脂的制备及其吸附性能．韶关学院学报(自然科学版)，2005，26(6)：71～74
[86] 曹佐英，葛华才，赖声礼．微波能促进壳聚糖钙离子配合物的制备研究．食品工业科技，2000，21(2)：11～13
[87] 曹佐英，赖声礼，曹珍年．微波辐射下壳聚糖zn(Ⅱ)配合物的合成．现代化工，1999，19(11)：24～27
[88] 朱惠光，计剑，高长有等，聚乳酸组织工程支架材料，功能高分子学报，2001，14(4)：488～492
[89] Inoue Y., Kamei S., Jmicromol Sci Pure Appl Chem, 1995, A322:787
[90] 周贤爵，李进，邵惠丽，胡学超．高分子材料科学与工程，2005，5，21(3)：66～69
[91] 滕新荣，任杰，易辉．合成纤维，2005．1：3～5
[92] Liu L J, Zhang C. Chinese Chemical Letters, 2001, 12(8): 663～664
[93] 张科，王鹏，李文科．高分子材料科学与工程,2004,20(3)：46～48
[94] Shu J, Wang P, Zheng T. Appl. Polym. Sci., 2006, 100(3): 2244～2247
[95] Wang P, Shu J, Tian L Y. Faming Zhuanli Shenqing Gongkai Shuomingshu, 2005
[96] Elisseeff J H. Embryonic stem cells: potential for more impact. Trends in Biotechnology,2004; 22:155～156
[97] Szilvassy S J. The biology of hematopoietic stem cells. Archives of Medical Research, 2003,34:446～460
[98] Peng H, Huard J. Muscle-derived stem cells for musculoskeletal tissue regeneration and repair. Transplant Immunology, 2004, 12:311～319
[99] 李姝丹，L-丙交酯的合成研究，硕士论文，哈尔滨理工大学，2004年：8～9
[100] Schindler A, Harper D. Journal of Polymer Science, Polymer Chemistry Edition, 1979,17(8): 2593～2599
[101] Stolt M, Sodergard A. Macromolecules, 1999, 32:6412～6417
[102] Wang N, Wu X S, Mater H. Sci. Eng. 1997, 76:373～374
[103] Witzke D R, Narayan R. Macromolecules, 1997, 30:7075～7085
[104] Hideto T, Shinya M. Polymer Degradation and Stability. 2001, 71(3): 415～424
[105] 王伟超，L-乳酸熔融缩聚及聚乳酸的结晶和熔融行为，浙江大学硕士学位论文，2006．6：41～42
[106] 郝红，梁国正．聚乳酸的溶液结晶行为．高分子材料科学与工程，2005，21(5)：185～188
[107] 刘莹，阮建明，张海坡，申雄军，周忠诚．聚L-乳酸(PLLA)的热稳定性．粉末冶金材料科学与工程．2006年12月，11(6)：365～369
[108] Zhang X C, Macdonald D A, Goosen F A, et al.. Polym.Sci. PartA: Polym.Chem., 1994,32(15): 2965～2970
[109] Cai Q, Yang J, Bei J Z. Biomaterials, 2002, 23:4483～4492
[110] Luo B H, Zhou C R. The world Engineers' Convention 2004 (WEC2004), November 2-6,2004, Shanghai, China; Zhu Y B, Gao C Y. Tissue engineering, 2004, 10:53～61
[111] 赵小东，刘文广，姚康德．离子交换与吸附，2003，19(4)：304～310
[112] Cui Y L, Qi A D, Yao K D. J. Biomed. Mater. Res., 2002, 60(3): 398～404
[113] Kim J Y, Ha C S, Jo N J. Polym. Int, 2002, 51:1123～1128
[114] Yan Wu, Yongli Zheng, Wuli Yang, Changchun Wang, Jianhua Hu, Shoukuan Fu. Synthesis and characterization of a novel ampHipHilic chitosan-polylactide graft copolymer. Carbohydrate Polymers, 2005 (59): 165～171
[115] Y. Liu, E Tian, K. A. Hu. Systhesis and characterization of a brush-like copolymer of a polylactide grafted onto chitosan. Carbohydrate Research, 2004 (339): 845～851
[116] Fujioka M, Okada H, Kusala Y. Macromol. Rapid Commun., 2004, 25:1776～1780
[117] 仰振球，李巧霞，樊红雷，宋宝珍，NMR，FTIR及电位滴定法测定壳聚糖脱乙酰度．功能高分子学报，2003．9：313-318
[118] X.Ou,A.Wirsen,A.-C.Albertsson，Effect of lactic/glycolic acid side chains on the thermal degradationkinetics of chitosan derivatives.Polymer 41(2000):4841～4847
[119] 朱爱萍，吴钧，张娜，沈健．壳聚糖一种具有应用潜力的组织工程支架材料．化学通报，2002(65)：w003，1-5
[120] Ariela V Paula, Daniele Urioste, Julio C Santos and Heizir F de Castro.. Porcine pancreatic lipase immobilized on polysiloxane-polyvinyl alcohol hybrid matrix: catalytic properties and feasibility to mediate synthesis of surfactants and biodiesel. Journal of Chemical Technology and Biotechnology. 2007(82): 281～288
[121] Witzke D R, Narayan R.. Macromolecules, 1997, 30:7075～7085
[122] 陈百万．生物医学工程学．北京：科学出版社，1997
[123] 邓倩莹，缓释溶菌酶纳米微球/壳聚糖复合支架材料的制备及性能，学位论文，暨南大学，2006
[124] 陈华，张伯勋，张巨松，梁雨田，胡成栋，黄靖香，张莉，高飞．柠檬酸化半水硫酸钙作为骨移植替代材料的安全性及生物相容性评价．中国临床康复，第9卷第34期，2005．9：56～59
[125] 赵建华，廖维宏，王远亮，潘君，柳峰．消旋聚乳酸羟基磷灰石脱钙骨基质的制备及其体外降解特性研究．中国修复重建外科杂志，2003年，第17卷第1期：61～64
[126] 司徒镇强，吴军正．细胞培养．西安：世界图书出版西安公司，2004