壳聚糖基角膜内皮组织工程载体支架的制备及性能评价
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摘要
目的：在我国,角膜病仅次于白内障是第二大致盲眼病,我国的单眼和双眼角膜盲患者共有约400万人,其中80%可以通过角膜移植手术复明,但由于角膜供体来源短缺,目前我国每年能完成的角膜移植手术仅约5000例。随着组织工程技术的迅猛发展以及角膜细胞体外培养方法的建立,利用组织工程技术构建人工角膜为治疗角膜盲提供了新的途径和方法,也成为当前研究的热点之一。壳聚糖是甲壳素脱乙酰后得到的聚阳离子生物多糖,安全、无毒、具有良好的组织相容性和生物降解性、降解产物可被人体吸收。近年来,壳聚糖在组织工程领域的研究也越来越受到人们的重视,以其为主要成分制备载体支架用于皮肤组织工程、骨骼组织工程、神经组织工程、血管组织工程等的实验研究已有报道；另外,以壳聚糖复合胶原、透明质酸或硫酸软骨素等制备的共混膜在构建人工角膜的研究中也得到应用。本研究以克服高脱乙酰度壳聚糖共混膜硬度高、植入体内降解速度慢等缺点为切入点,首次选择脱乙酰度50%的水溶性壳聚糖为基本材料,采用共混的方法旨在制备出理化性质优异、组织相容性良好、适合角膜内皮细胞贴附生长的共混膜,为该种共混膜作为角膜内皮组织工程载体支架的可行性提供理论依据。
方法：1、参照组织工程医疗产品行业标准对实验室制备的水溶性壳聚糖(Cts)的水分、灰分、重金属含量、粒度、pH、细胞毒性等性质进行测定；2、以水溶性壳聚糖为主体,按照体积比9:1:0.1、8:2:0.1、7:3:0.1的比例与明胶(Gel)、硫酸软骨素(CS)进行共混制备出三组共混膜；测定共混膜的含水量、通透性、透光率、细胞毒性等性质,评价兔角膜基质细胞在共混膜上贴附生长性能,筛选出一组共混膜作为进一步研究的对象；3、从体外酶解-皮下、肌肉植入-眼前房植入三个层面评价共混膜的生物降解性和组织相容性；4、在体外以共混膜为载体支架构建人工角膜内皮,考察兔角膜内皮细胞在共混膜上的贴附牢固程度及生长情况,研究共混膜与角膜内皮细胞的相容性,并制备曲率共混膜。
结果：1、实验室制备的水溶性壳聚糖符合组织工程和生物医用材料对植入体内应用的材料的要求。细胞毒性试验显示水溶性壳聚糖对L929细胞完全没有毒性,而且对其生长具有一定的促进作用,具有良好的细胞相容性。2、通过对三组共混膜性质的比对,发现体积比Cts:Gel:CS=9:1:0.1的共混膜含水量合适、透光率优异、通透性良好、无细胞毒性、适合兔角膜基质细胞生长,可作为角膜载体支架材料。3、Cts-Gel-CS共混膜比单纯壳聚糖膜体外降解速度快,而且组织中炎症反应较轻,具有良好的生物降解性和组织相容性。其中,共混膜在大鼠背部皮下和股部肌肉内60天左右基本被降解吸收；在兔眼前房内,观察期内无明显的炎症反应,植入后2个月共混膜基本被吸收。4、Cts-Gel-CS共混膜与角膜内皮细胞相容性良好,曲率共混膜也具有良好的透明性和一定机械强度,为后续动物移植实验奠定了基础。
结论：实验室自制的脱乙酰度50%的水溶性壳聚糖符合组织工程和生物医用材料对植入体内应用的材料的要求；以该种水溶性壳聚糖为主要材料,按体积比Cts:Gel:CS=9:1:0.1,与明胶、硫酸软骨素进行共混制备的膜片含水量合适、透光率优异、通透性良好、细胞毒性为0级、适合兔角膜基质细胞和内皮细胞生长,在大鼠背部皮下、股部肌肉和兔眼前房内组织反应比较温和,炎症反应较轻,具有良好的组织相容性。总之,该共混膜具备作为组织工程角膜内皮载体支架的基本条件,通过对该共混膜的进一步研究,有望在构建组织工程活性人工角膜方面发挥积极作用。
Objective:As we all known, inferior to cataract, keratinous is the second illness which leads to blindness in our country. There are 4 million patients suffered blindness caused by keratinous, and only 5 thousand patients can be treated by transplantation of donor corneas every year bacause donor corneas are insufficient.-With the development of tissue engineering and the methodological establishment of corneal cells culture, it would be advantageous by the way of corneal tissue engineering for the treatment of diseases caused by corneal endothelial disorders. Chitosan (Cts) is a partially deacetylated derivative of chitin, which shows a lot of interesting biological properties such as low immunogenicity, low cytotoxicity, biodegradability and biocompatibility. Recently, Cts is being taken more seriously in tissue engineering field, and it is reported that carriers made of Cts have been used in skin tissue engineering, bone tissue engineering, nerve tissue engineering, vessel tissue engineering and so on. In addition, blend membranes composed of Cts, gelatin (Gel), chondroitin sulfate (CS) or hyaluronic acid (HA) have been used for reconstructing artificial cornea. Aim to accelerate biodegradation and bring down the hardness of Cts(high deacetylation degree) based blend membrane, in this research the soluble Cts (D.D.≈50%) as main ingredient is crosslinked with Gel and CS, in order to obtain a kind of blend membrane which has excellent physicochemical property, good tissue compatibility, and is suitable for corneal endothelial cells.
Methods:1.To improve the solubility of Cts, Cts with deacetylation degree at 50% was pepared in our lab. Strictly according to biomedical material and tissue engineering standard, aquiferous capacity, ash content, heavy metal content, pH, particle size, cytotoxicity of the soluble Cts were determined.2. The soluble Cts as main ingredient was crosslinked with Gel and CS to obtain three kinds of blend membranes at different proportions. Then transparency, water content, cytotoxicity, NaCl permeability, cytocompatibility with the rabbit keratocytes were tested, and Cts-Gel-CS blend membrane with the volume ratio at 9:1:0.1 was selected out.3. Biodegradability and biocompatibility of Cts-Gel-CS blend membrane were studied in vitro and in vivo respectively. The in vitro degradation was carried out by immersing the blend membrane in pH 7.4 buffered lysozyme solution at 37℃. While the in vivo biocompatibility and biodegradability were studied by implanting the blend membrane in the subcutaneous tissue, leg muscle of rats and in atria of rabbits.4. New Zealand white rabbit corneal endothelial cells were seeded on Cts-Gel-CS blend membrane to construct artificial cornea endothelium. And concave blend membrane was prepared through special equipment in our lab.
Results:1. The soluble Cts prepared in our lab is in agreement with standard of biomedical material and tissue engineering. Cytotoxicity experiments showed that the soluble Cts has no cytotoxicity. The Cts solutions with different concentrations can promote L929 proliferation, significantly at 250μg/mL.2. Through Comparing the properties of three kinds of blend membranes, the blend membrane with the volume ratio at 9:1:0.1 of Cts-Gel-CS is selected out as the corneal tissue engineering carrier which has good optical transmittance, suitable water content, no cytotoxicity and better cytocompatibility with the rabbit keratocytes.3. Cts-Gel-CS blend membrane prepared not only degrades faster than Cts film in vitro, but also has good tissue compatibility. It is found that Cts-Gel-CS blend membrane can be degraded in less than 60 days in the subcutaneous tissue and leg muscle of rats and 2 months in rabbits' atria. HE staining results showed that the tissue reaction towards the blend membrane was very mild.4. Cts-Gel-CS blend membrane has good cytocompatibility with the rabbit corneal endothelial cells. In addition, the concave blend membrane has certain mechanical strength and good transparency.
Conclusions:As we hoped, the soluble Cts prepared in our lab is in agreement with standard of biomedical material and tissue engineering. The blend membrane with the volume ratio at 9:1:0.1 of Cts-Gel-CS has good optical transmittance, suitable water content, no cytotoxicity and better cytocompatibility with the rabbit keratocytes and corneal endothelial cells. Animal tissue compatibility experiments showed that no remarkable inflammation appeared and the tissue reaction towards the blend membrane was very mild. The concave blend membrane prepared through special equipment has certain mechanical strength and good transparency. In summary, this blend membrane is a promising carrier of corneal endothelial cells to reconstruct tissue engineering cornea.
方法：1、参照组织工程医疗产品行业标准对实验室制备的水溶性壳聚糖(Cts)的水分、灰分、重金属含量、粒度、pH、细胞毒性等性质进行测定；2、以水溶性壳聚糖为主体,按照体积比9:1:0.1、8:2:0.1、7:3:0.1的比例与明胶(Gel)、硫酸软骨素(CS)进行共混制备出三组共混膜；测定共混膜的含水量、通透性、透光率、细胞毒性等性质,评价兔角膜基质细胞在共混膜上贴附生长性能,筛选出一组共混膜作为进一步研究的对象；3、从体外酶解-皮下、肌肉植入-眼前房植入三个层面评价共混膜的生物降解性和组织相容性；4、在体外以共混膜为载体支架构建人工角膜内皮,考察兔角膜内皮细胞在共混膜上的贴附牢固程度及生长情况,研究共混膜与角膜内皮细胞的相容性,并制备曲率共混膜。
结果：1、实验室制备的水溶性壳聚糖符合组织工程和生物医用材料对植入体内应用的材料的要求。细胞毒性试验显示水溶性壳聚糖对L929细胞完全没有毒性,而且对其生长具有一定的促进作用,具有良好的细胞相容性。2、通过对三组共混膜性质的比对,发现体积比Cts:Gel:CS=9:1:0.1的共混膜含水量合适、透光率优异、通透性良好、无细胞毒性、适合兔角膜基质细胞生长,可作为角膜载体支架材料。3、Cts-Gel-CS共混膜比单纯壳聚糖膜体外降解速度快,而且组织中炎症反应较轻,具有良好的生物降解性和组织相容性。其中,共混膜在大鼠背部皮下和股部肌肉内60天左右基本被降解吸收；在兔眼前房内,观察期内无明显的炎症反应,植入后2个月共混膜基本被吸收。4、Cts-Gel-CS共混膜与角膜内皮细胞相容性良好,曲率共混膜也具有良好的透明性和一定机械强度,为后续动物移植实验奠定了基础。
结论：实验室自制的脱乙酰度50%的水溶性壳聚糖符合组织工程和生物医用材料对植入体内应用的材料的要求；以该种水溶性壳聚糖为主要材料,按体积比Cts:Gel:CS=9:1:0.1,与明胶、硫酸软骨素进行共混制备的膜片含水量合适、透光率优异、通透性良好、细胞毒性为0级、适合兔角膜基质细胞和内皮细胞生长,在大鼠背部皮下、股部肌肉和兔眼前房内组织反应比较温和,炎症反应较轻,具有良好的组织相容性。总之,该共混膜具备作为组织工程角膜内皮载体支架的基本条件,通过对该共混膜的进一步研究,有望在构建组织工程活性人工角膜方面发挥积极作用。
Objective:As we all known, inferior to cataract, keratinous is the second illness which leads to blindness in our country. There are 4 million patients suffered blindness caused by keratinous, and only 5 thousand patients can be treated by transplantation of donor corneas every year bacause donor corneas are insufficient.-With the development of tissue engineering and the methodological establishment of corneal cells culture, it would be advantageous by the way of corneal tissue engineering for the treatment of diseases caused by corneal endothelial disorders. Chitosan (Cts) is a partially deacetylated derivative of chitin, which shows a lot of interesting biological properties such as low immunogenicity, low cytotoxicity, biodegradability and biocompatibility. Recently, Cts is being taken more seriously in tissue engineering field, and it is reported that carriers made of Cts have been used in skin tissue engineering, bone tissue engineering, nerve tissue engineering, vessel tissue engineering and so on. In addition, blend membranes composed of Cts, gelatin (Gel), chondroitin sulfate (CS) or hyaluronic acid (HA) have been used for reconstructing artificial cornea. Aim to accelerate biodegradation and bring down the hardness of Cts(high deacetylation degree) based blend membrane, in this research the soluble Cts (D.D.≈50%) as main ingredient is crosslinked with Gel and CS, in order to obtain a kind of blend membrane which has excellent physicochemical property, good tissue compatibility, and is suitable for corneal endothelial cells.
Methods:1.To improve the solubility of Cts, Cts with deacetylation degree at 50% was pepared in our lab. Strictly according to biomedical material and tissue engineering standard, aquiferous capacity, ash content, heavy metal content, pH, particle size, cytotoxicity of the soluble Cts were determined.2. The soluble Cts as main ingredient was crosslinked with Gel and CS to obtain three kinds of blend membranes at different proportions. Then transparency, water content, cytotoxicity, NaCl permeability, cytocompatibility with the rabbit keratocytes were tested, and Cts-Gel-CS blend membrane with the volume ratio at 9:1:0.1 was selected out.3. Biodegradability and biocompatibility of Cts-Gel-CS blend membrane were studied in vitro and in vivo respectively. The in vitro degradation was carried out by immersing the blend membrane in pH 7.4 buffered lysozyme solution at 37℃. While the in vivo biocompatibility and biodegradability were studied by implanting the blend membrane in the subcutaneous tissue, leg muscle of rats and in atria of rabbits.4. New Zealand white rabbit corneal endothelial cells were seeded on Cts-Gel-CS blend membrane to construct artificial cornea endothelium. And concave blend membrane was prepared through special equipment in our lab.
Results:1. The soluble Cts prepared in our lab is in agreement with standard of biomedical material and tissue engineering. Cytotoxicity experiments showed that the soluble Cts has no cytotoxicity. The Cts solutions with different concentrations can promote L929 proliferation, significantly at 250μg/mL.2. Through Comparing the properties of three kinds of blend membranes, the blend membrane with the volume ratio at 9:1:0.1 of Cts-Gel-CS is selected out as the corneal tissue engineering carrier which has good optical transmittance, suitable water content, no cytotoxicity and better cytocompatibility with the rabbit keratocytes.3. Cts-Gel-CS blend membrane prepared not only degrades faster than Cts film in vitro, but also has good tissue compatibility. It is found that Cts-Gel-CS blend membrane can be degraded in less than 60 days in the subcutaneous tissue and leg muscle of rats and 2 months in rabbits' atria. HE staining results showed that the tissue reaction towards the blend membrane was very mild.4. Cts-Gel-CS blend membrane has good cytocompatibility with the rabbit corneal endothelial cells. In addition, the concave blend membrane has certain mechanical strength and good transparency.
Conclusions:As we hoped, the soluble Cts prepared in our lab is in agreement with standard of biomedical material and tissue engineering. The blend membrane with the volume ratio at 9:1:0.1 of Cts-Gel-CS has good optical transmittance, suitable water content, no cytotoxicity and better cytocompatibility with the rabbit keratocytes and corneal endothelial cells. Animal tissue compatibility experiments showed that no remarkable inflammation appeared and the tissue reaction towards the blend membrane was very mild. The concave blend membrane prepared through special equipment has certain mechanical strength and good transparency. In summary, this blend membrane is a promising carrier of corneal endothelial cells to reconstruct tissue engineering cornea.
引文
[1]曹谊林.组织工程学研究进展.上海交通大学学报(医学版),2008,28(7)：763-766
[2]Langer R, Vacanti JP. Tissue engineering. Science,1993,260:920-926
[3]曹谊林.组织工程的基本问题.生命科学,17(2)：106-111
[4]Walgenbach K J, Voigt M, Riabikhin A W, et al.Tissue engineering in plastic reconstructive surgery.Anat Rec.,2001,263:372-378
[5]Ren T, Ren Jl, Jia X, et al. The bone formation in vitro and mandibular defect repair using PLGA porous scaffolds. J Biomed Mater Res A,2005,74(4):562-569
[6]Ouyang H W, Goh J C, Thambyah A, et al. Knitted polylactid ecogtycolide scaffold loaded with bone m arrow stromal cells in repair and regeneration of rabbit Achilles tendon. Tissue En, 2003,9(3):431-439
[7]Pelled G, Tai K, Sheyn D, et al. Structural and nanoindentation studies of stem cell-based tissue-engineered bone. Biomech 2007,40(2):399-411
[8]王明科.干细胞与组织工程.重庆医学,2006,35(21)：1990-1992
[9]C.Liu, Z.Xia, J.T.Czernuszka, Design and Development of Three-Dimensional Scaffolds for Tissue Engineering.Trans IChemE, PartA, Chemical Engineering Research and Design,2007, 85(A7):1051-1064
[10]王身国,贝建中.组织工程细胞支架及其相关技术的研究.中国创伤骨科杂志,2000,2(4)：277-279
[11]凌生林.组织工程支架材料研究进展.泸州医学院学报,2008,31(1)：95-97
[12]王海林.眼科解剖学图谱.沈阳：辽宁科学技术出版社,2002,85-86
[13]谢立信.中国眼科现状.中国工程科学,2003,5(1)：1-5
[14]Stocker F W, Eiring A, Georging, et al. A tissue culture technique for growing corneal epithelial stromal,and endothelial tissues separately. Am J Ophthalmol,1958,46(5):294-298.
[15]谢立信,Gebh BM兔角膜组织细胞培养方法的技术改进.中华眼科杂志,1990,26：237-239
[16]屈雷,王馨,杨学义,等.家兔角膜内皮细胞快速培养.国际眼科杂志,2004,4(2)：243-246
[17]Joyce NC, Joyce SJ, Powell SM, et al. EGF and PGE2:effects on corneal endothelial cell migration and monolayer spreading during wound repairin vitro. CurrEye Res,1995,14(7): 601-609
[18]EngelmannK, DrexlerD, Bohnke M. Transplantation of adulthuman or porcine corneal endothelial cells onto human recipientsin vitro. Part Ⅰ:Cell culturing and transplantation procedure. Cornea,1999,18(2):199-206
[19]邵应峰,胡诞宁,陈家祺β-FGF.EGF和NGF对人角膜内皮细胞生长调控的实验研究.眼科学报,2008,24(1)：9-12
[20]葛健,项行,等.不同浓度牛磺酸对兔角膜内皮细胞的影响.上海交通大学学报(医学版),2009,29(7)：825-827
[21]王丽娅,李辰,曾耀英,等.角膜内皮细胞培养方法的改进.眼科研究,1995,13(3)：215-216
[22]胡晓琴,袁进,陈家祺,等.角膜内皮细胞改良培养技术的初步探讨.眼科研究,2008,26(5)：321-325
[23]陆雯娟,傅瑶,邵春益,范先群.培养猫角膜内皮细胞的新方法—联合酶消化法.眼科新进展,2008,28(2)：81-84
[24]杨鹏,刘瑶,等.兔角膜内皮细胞的新法培养.东南大学学报(医学版),2001,20(3)：170-172
[25]Bednarz J, TeifelM, Friedl P, Engelmann K. Immortalization ofhuman corneal endothelial cells using electroporation protocol optimized for human corneal entothelial and human retinal pigment epithelial cell. Acta Ophthalm ol Scand,2000,78(2):130-136
[26]Shin J S, Jang I K, Kim C W, Kim J C. Development and characterization of a rabbit corneal endothelial cell line. Jpn J Ophthalm ol,2004,48(5):454-459
[27]樊廷俊,赵君,付永锋,从日山,郭瑞超,刘万顺,等.家兔(Oryctolagus curiculus)角膜内皮细胞系的建立.中国科学C辑：生命科学,2007,37(1)：20-27.
[28]刘小伟.兔角膜内皮细胞移植和自体骨耽间充质干细胞替代移植治疗角膜内皮损伤的初步研究：[博士学位论文].北京：中国医学科学院眼科研究中心,北京协和医院眼科,1999
[29]Gospodarowicz D, Greenburg G. The coating ofbovine and rabbit corneas denuded of their entothelium with bovine corneal endothelial cells. Exp Eye Res.,1979,28(3):249-265
[30]易伟斌,胡竹林,徐岩泽,王廷华,黄桂琴,陈娟,等.兔血管内皮细胞对角膜后弹力层代谢的影响.国际眼科杂志,2007,7(4)：956-959
[31]Ishino Y, Sano Y, Nakamura T, et al. Amniotic membrane as a carrier for cultivated human corneal endothelial cell transplantation. Invest Ophthalmol. Vis. Sci.,2004,45 (3):800-806
[32]傅瑶,范先群,等.羊膜载体培养标记兔角膜内皮细胞移植的研究.中华眼科杂志,2006,42(10)：925-929.
[33]林旭初,惠延年,等.猪脱细胞角膜基质的制备及组织学特征观察.国际眼科杂志,2007,7(1)：49-51
[34]林旭初,惠延年,等.脱细胞猪角膜基质体外支持角膜上皮和基质细胞的生长.国际眼科杂志,2008,8(7)：1293-1295
[35]吴连胜,陈建苏,徐锦堂,丁勇,等.脱细胞角膜材料的生物相容性研究.眼科研究,2009,27(5)：359-363
[36]张姝江,杨志明,等.脱细胞异种角膜基质的制备及细胞相容性.中国医学科学院学报,2005,27(6)：670-673
[37]张超,金岩,聂鑫,等.组织工程角膜生物材料载体制备的比较性研究.中国修复重建外科杂志,2006,20(2)：185-188
[38]罗小玲,徐锦堂,黄菊天,赵松滨,等.以异种后弹力膜基质为载体培养角膜内皮细胞的研究.眼科研究,2004,22(5)：449-452
[39]吴静,李姝燕,郝念,等.脱水猪角膜基质为载体体外构建猫角膜内皮组织.中国病理生理杂志,2004,20(6)：1017-1020
[40]范先群,陈苹,傅瑶,等.以异种角膜脱细胞基质为载体构建角膜上皮-载体-内皮复合物的实验研究.中华眼科杂志,2007,43(5)：437-441
[41]Amano S. Transplantation of cultured human corneal endothelial cells. Cornea,2003,22 (7): 66-74
[42]朱梅湘,穆畅道,等.胶原作为生物医学材料的优势与应用.化学世界,2003,3：161-164
[43]Y Minami, H Sugihara, S Oono. Reconstruction of cornea in three-dimensional collagen gel matrix culture. Investigative Ophthalmology & Visual Science,1993,34:2316-2324
[44]陈家祺,张胜,郭琳洁,等.应用三维培养技术体外重建角膜组织的初步研究.中华眼科杂志,2001,37(4)：244-247
[45]Han B, Schwab IR, Madsen TK, et al. A fibrin-based bioengineered ocular surfacewith human corneal epithelial stem cells. Cornea,2002,21 (5):505-510.
[46]顾国贞,王芳,谷树严,等.以纤维蛋白为基质网架的组织工程角膜上皮的构建.吉林大学学报(医学版),2008,34(5)：814-816
[47]梁晓东,陈建苏,等.纤维蛋白胶与兔角膜3种细胞构建组织工程细胞片研究.中国病理生理杂志,2008,24(4)：797-801
[48]Shilu Wang, Wanshun Liu, Baoqin Han,et al. Study on a hydroxypropylchitosan-gelatin based scaffold for cornealstrom tissue engineering. Applied Surface Science,2009, 255:8701-8705.
[49]陈列欢,刘万顺,韩宝芹,位晓娟,杨朝忠.三种角膜内皮细胞载体膜片的性质研究.中国生物医学工程学报,2005,24(6)：755-762
[50]位晓娟,刘万顺,韩宝芹,等.兔角膜内皮细胞载体的体外培养及移植的研究.中国生物医学工程学报,2006,25(5)：590-595
[51]Hadlock T, Singh S, Vacanti JP, et al. Ocular cell monolayers cultured on biodegradable substrates. Tissue Eng.,1999,5 (3):187-190
[52]胡晓洁,王敏,柴岗,张艳,等.组织工程技术构建兔角膜基质组织的实验研究.中华眼科杂志,2004,40(8)：517-521
[53]蒋挺大.甲壳素.第一版.北京：中国环境科学出版社,1996：99,301-328
[54]Majeti NV, Kumar R. A review of chitin and chitosan applications. React. Funct. Polym., 2000,46(1):1-27
[55]M. Thanou, J. C. Verhoef, H. E. Junginge. Oral drug absorption enhancement by chitosan and its derivatives. Adv.Drug Delivery Rev.,2001,52 (2):117-126.
[56]张建湘,汤健,徐斌,等.壳聚糖棒材的组织相容性和安全性评价.生物医学工程学杂志,1996,13(4)：293-297
[57]贾文英,景毅,程琳,等.壳聚糖的遗传毒性研究.透析与人工器官,2001,12(2)：44-46
[58]Y.M. Yang, W. Hu, X.D. Wang, X.S. Gu. The controlling biodegradation of chitosan bersbynacetylation in vitro and in vivo. J.Mater.Sci.Mater.Med.,2007,18:2117-2121
[59]付辰炜.氨基葡萄糖及壳寡糖对小鼠免疫功能的影响：[硕士学位论文].青岛：中国海洋大学,2006
[60]卢凤奇,曹宗顺,庄昭霞,等.壳聚糖膜的降解与生物相容性研究.生物医学工程学杂志,1998,15(2)：183-185
[61]李伟,肖传国.壳聚糖膜在大鼠脊髓组织中的降解及其生物相容性.中国组织工程研究与临床康复,2008,12(36)：7131-7134
[62]Park J H, Cha B J, Lee Y N. Antibacterial activity of chitosan acetate on food-borne enteropathogenic bacteria. Food Science Biotechnol,2003,12(1):100-103
[63]Liu X F, Guan Y L, Yang D Z, et al. Antibacterial action of chitosan and carboxymethylated chitosan. Appl Polym Sci.,2000,79(7):1324-1335
[64]No H K, Park N Y, Lee S H, et al. Antibacterial activity of chitosans and chitosanoligomers with different molecular weights. Int J Food Microbiol,2002, (74):65-72
[65]王鸿,沈月新.不同脱乙酰度壳聚糖的抑菌性.上海水产大学学报,2001,10(4)：380-382.
[66]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
[67]蒋霞云.p-壳聚糖及其脱乙酰衍生物的特性.上海水产大学学报,2002,11(4)：348-352.
[68]Devlieghere F, Vermeulen A, Debevere J. Chitosan:antimicrobial activity, interactions with food components and applicability as a coating on fruit and vegetables. Food Microbiol.,2004, 21(6):703-714.
[69]陈威,吴清平,张菊梅,吴慧清.壳聚糖抑菌机制的初步研究.微生物学报,2008,48(2)：164-168
[70]张惠珍,马国芳,钱洪军,等.不同种类壳聚糖抗菌性能的比较.河北师范大学学报(自然科学版),2009,33(1)：74-77
[71]吴小勇,曾庆孝,莫少芳,肖仔君.几种壳聚糖的抑菌性能.食品与发酵工业,2005,31(2)：18-21
[72]彭翠珍.壳聚糖对几种农作物病害的抑制作用研究.云南农业科技,2009,4：12-13
[73]于广利,李八方,梁平方,万忠良.水溶性甲壳胺制备及其对铅离子的螯合作用.中国海洋药物,1996,4：13-17
[74]Sannan K, Kurita K, Iwakura Y. Studies on chitin. Makromol Chem.,1976,177:3589-3600
[75]Sannan K, Kurita K, Iwakura Y. Kinetics of deacetylation reaction. Polym.J.,1977,9(6): 649-651
[76]席敏,王子飞.水溶性壳聚糖的制备及结构表征.安徽农业科学,2001,29(5)：694-696
[77]陈鲁生.完全水溶性壳聚糖制备条件的研究.化学通报,1998,(8)：48-50
[78]陈天,严俊,徐荣南,夏炎.部分N-酰化脱乙酰壳多糖的水溶性.应用化学,1989,6(3)： 90-92
[79]高兴爽,刘万顺,韩宝芹,梁晔.羟丙基壳聚糖的制备、表征及其对角膜细胞生长的影响.中国海洋大学学报(自然科学版),2007,37(6 sup)：131-134.
[80]Yanfei Peng, Baoqin Han, Wanshun Liu, XiaojuanXu. Preparation and antimicrobial activity of hydroxypropyl chitosan. Carbohydrate Research,2005,340:1846-1851
[81]万荣欣.羧甲基壳聚糖的制备及其生物学评价：[硕士学位论文].天津：天津医科大学,2006
[82]黄攀,韩宝芹,刘万顺,等.N-羧甲基壳聚糖的制备及其生物相容性评价.功能材料,2009,40(7)：1198-1203
[83]Hao Wu, Aiping Zhu, LanhuaYuan. Interactions between O-carboxymethyl chitosan and bovine serum albumin. Materials Chemistry and Physics,2008(112):41-46
[84]Changzheng Wei, Chunlin Hou, Qisheng Gu, et al.A thermosensitive chitosan-based hydrogel barrier for post-operative adhesions' prevention. Biomaterials,2009 (30):5534-5540
[85]刘双利,侯春林,魏长征,等.羟丁基壳聚糖预防大鼠腹腔粘连的实验研究.中国修复重建外科杂志,2009,23(6)：718-722
[86]程金伟,魏锐利,李由,顾其胜.壳聚糖在眼科疾病治疗中的应用.上海生物医学工程,2001,22(4)：44-47
[87]De Campos AM, Diebold Y, Carvalho EL, et al. Chitosan nanoparticles as new ocular drug delivery systems:in vitro stability, in vivo fate, and cellular toxicity. Pharm Res., 2004:(5):803-810
[88]游向东,金中秋,吴建国,宋艳萍.壳聚糖缓释给药系统植入脉络膜上腔治疗兔眼穿通伤.国际眼科杂志,2005,5(1)：74-76
[89]李平,张建林,李家仁,常威,等.氧氟沙星壳聚糖滴眼液的研制.中国生化药物杂志,1999,20(2)：68-69
[90]黄虹,康琦文,樊丽蓉,等.医用几丁糖用作氯霉素滴眼液增稠剂的稳定性考察.中国医院药学杂志,1999,19(11)：689-690
[91]高玉英.治疗眼科疾病用可降解复合膜的制备及性能研究：[硕士学位论文].武汉：武汉理工大学,2005
[92]Howling GI, Dettmar PW, Goddard PA, et al.The effect of chitin and chitosan on the proliferation of human skin fibroblasts and keratinocytes in vitro. Biomaterials,2001,22(22): 2959-2966
[93]郭晟,魏锐利,王文斌,蒋丽霞,金玲.壳聚糖预防兔眼眶手术术后组织粘连.眼科新进展,2006(3)：191-193
[94]李平,王爱勤,谭干祖,张建林.壳聚糖人工泪液的制备及应用.中国医院药学杂志,1996,16(12)：569-570
[95]Felt O, Carrel A, Baehni P, et al. Chitosan as tear substitute:a wetting agent endowed with antimicrobial efficacy. J Ocul Pharmaco Ther,2000,16(3):261-270
[96]李由,周韵秋,侯春林,等.壳聚糖眼内应用的实验研究.第二军医大学学报,1997,18(5)：455-457.
[97]Li Y, Cheng JW, Wei RL, et al. Intraocular pressure and endothelium cell counts after cataract surgery with chitosan and sodium hyaluronate (Healon GV):3-year follow-up results of a randomised clinical trial. Adv Ther,2008,25(5):422-429
[98]吴刚,盛祖力.壳聚糖在牙周组织工程中的应用进展.国外医学生物医学工程分册,2004,27(6)：380-383
[99]向珊珊,高秀秋.壳聚糖-胶原支架与人牙周膜细胞复合培养的实验研究.锦州医学院学报,2006,27(5)：1-4
[100]Feng Z, Yuji Y, William WL, et al. Preparation and histological evaluation of biomimetic three-dimensional hydroxyapatite/chitosan-gelatin network composite scaffolds. Biomaterials,2002,23 (15):3227-3234
[101]Gregory RD, Evans MD, et al. Challenges to nerve regeneration. Seminars in Surgical Oncology,2000,19:312-318.
[102]敖强,王爱军,孙志刚,张秀芳.一种壳聚糖神经组织工程支架的制备及表征.中国组织工程研究与临床康复,2008,12(1)：47-50
[103]张建英,雷德林,李建虎,张圃,等.壳聚糖复合胶原蛋白周围神经支架材料的制备研究.中国美容医学,2006,15(3)：241-244
[104]Lim SS, Gaziano TA, Gakidou E, et al. Prevention of cardiovascular disease in high-risk individuals in low-income and middle-income countries:health effects and costs. Lancet, 2007,370(9604):2054-2062.
[105]Niklason LE. Replacement arteries made to order. Science,1999,286:1493-1494
[106]张鲁生,杨飞,公衍道,等.壳聚糖和明胶材料对血管平滑肌细胞的作用.生物物理学 报,2004,20(1)：19-25
[107]徐迎新,尹太,李瑞新,吴仕和,等.壳聚糖-生物蛋白胶用于工程化血管构建的初步研究.中国康复理论与实践,2005,11(8)：623-625
[108]Suh JK, MatthewHW. Application of chitosan-based polysaccharide biomaterials in cartilage tissue engineering. Biomaterials,2000,21(24):2589-2598.
[109]高海玲,管英俊,申志新,姜俊怡,等.软骨细胞与壳聚糖复合物体内构建软骨组织的研究.潍坊医学院学报,2006,28(4)：266-267
[110]Chenite A, Chaput C, Wang D, et al. Novel injectable neutral solutions of chitosan form biodegradable gels in situ. Biomaterials,2000,21 (21):2155-2161
[111]Tangsadthakun C, Kanokpanont S, Sanchavanakit N, et al. The influence of molecular weight of chitosan on the physical and biological properties of collagen/chitosan scaffolds. J Biomater SciPolym Ed.,2007,18(2):147-163
[112]Mao J, Zhao L, De Yao K, et al. Study of novel chitosan-gelatin artificial skin in vitro. J Biomed Mater Res A.,2003,64(2):301-308
[113]贾之慎,李齐彪.双突跃电位滴定法测定壳聚糖脱乙酰度.化学世界,2001,5：240-241
[114]蒋挺大.壳聚糖.北京.化学工业出版社,2001：26-32
[115]GB/T16886.5-2003,体外细胞毒性试验[S]//郝和平,医疗器械生物学评价标准实验指南.第1版.北京：中国标准出版社,2000：80-82
[116]王昕,施嬿萍,朱雪涛,等.MTT法评价医用高分子材料的细胞毒性.山东生物医学工程,2003,1：46-47
[117]Kawada K, Yonei T, Ueoka H, et al. comparison of the mosensitivity test:clonogenic assay versus MTT assay. Acta Medokayama,2002,56(3):129-134.
[118]郑晓燕.国产碳纤维桩材料的生物学评价：[硕士学位论文].北京：首都医科大学口腔临床医学,2005
[119]张青.黏多糖在眼科中的应用.食品与药品,2005,7(8A)：57-59
[120]牛晓霞,洪晶,段辉,刘蕾.不同保存方法对角膜内皮细胞活性的影响.国际眼科杂志,2006,6(5)：1015-1017
[121]Dawlee S, Sugandhi A, Balakrishnan B, et al. Oxidized chondroitin sulfate-cross-linked gelatin matrixes:a new class of hydrogels. Biomacromolecules,2005,6(4):2040-2048
[122]Huang Y, Onyeri S, Siewe M, et al. In vitro characterization of chitosan-gelatin scaffolds for tissue engineering. Biomaterials,2005,26 (36):7616-7627
[123]Mehrdad Rafat, Fengfu Li, Per Fagerholm, et al. PEG-stabilized carbodiimide crosslinked collagen-chitosan hydrogels for corneal tissue engineering. Biomaterials,2008,29: 3960-3972
[124]余家会,杜予民,等.壳聚糖-明胶共混膜.武汉大学学报(自然科学版),1999,45(4)：440-444
[125]陈丽娟,盛瑞兰,等.AO/EB荧光染色法测定HHT诱导的HL-60细胞凋亡.南京医科大学学报(中文版),1997,17(5)：514-516
[126]周贵,奚廷斐,郑裕东,汤京龙,等.三种羧甲基壳聚糖防粘连膜的降解及其生物相容性.中国组织工程研究与临床康复,2009,13(8)：1411-1418
[127]卢凤琦,曹宗顺.壳聚糖-明胶混合膜的制备及其生物降解性研究.生物医学工程学杂志,1998,15(4)：323-324
[128]Hiraku O, Yoshiharu M. Biodegradation and distribution of water-soluble chitosan in mice. Biomaterials,1999,20(2):175-182
[129]马如,黄明智.壳聚糖的溶菌酶降解.北京化工大学学报,2002,29(6)：40-43
[130]王征,刘万顺,韩宝芹,魏长征.低分子量壳聚糖及其衍生物的制备与溶菌酶对其降解的实验研究.海洋科学,2007,31(10)：36-40
[131]陈列欢.组织工程人工角膜内皮载体膜片的制备,性质及对角膜内皮损伤修复的实验研究.[博士学位论文].青岛：中国海洋大学,2006
[132]杨晓芳,奚廷斐.生物材料生物相容性评价研究进展.生物医学工程学杂志,2001,18(1)：123-128
[133]梁卫东,石应康.细胞培养法评价生物材料生物相容性研究进展.生物医学工程学杂志,1999,16(1)：86-90
[134]戴建国.生物材料生物相容性的分子生物学研究进展.国外医学生物医学工程分册,2004,27(6)：360-364
[135]刘小亮,张彩霞,闻学雷.评价生物材料的体内外实验相关性研究-体外细胞培养的MTT法与体内皮下埋植实验.口腔材料器械杂志,1998,8(1)：10-13
[136]Lobner D. Comparison of the LDH and MTT assays for quantifying cell death:validity for neuronal apoptosis. Journal of Neuroscience Methods,2000,96:147-152.
[137]刘立,王友联,张枢,等.环孢霉素A对局部脑缺血再灌流损伤的改善作用.中风与神 经疾病杂志,2002,19(2)：106-107
[138]邵京山,薛敏,冯玉兰,等.复方丹参注射液对脑出血CT及脑脊液LDH的影响.实用中西医结合临床,2002,2(5)：1-2
[139]陈晓东,吴伯瑜,江琼,林新华,等.芦荟粗多糖对体外培养人表皮细胞增殖作用的研究.中国药理学通报,2005,21(11)：1363-1366
[140]张永红,胡伟,Stark GB,等.乳酸脱氢酶实验检测细胞新陈代谢的实验研究.山西医科大学学报,2003,34(4)：308-310
[2]Langer R, Vacanti JP. Tissue engineering. Science,1993,260:920-926
[3]曹谊林.组织工程的基本问题.生命科学,17(2)：106-111
[4]Walgenbach K J, Voigt M, Riabikhin A W, et al.Tissue engineering in plastic reconstructive surgery.Anat Rec.,2001,263:372-378
[5]Ren T, Ren Jl, Jia X, et al. The bone formation in vitro and mandibular defect repair using PLGA porous scaffolds. J Biomed Mater Res A,2005,74(4):562-569
[6]Ouyang H W, Goh J C, Thambyah A, et al. Knitted polylactid ecogtycolide scaffold loaded with bone m arrow stromal cells in repair and regeneration of rabbit Achilles tendon. Tissue En, 2003,9(3):431-439
[7]Pelled G, Tai K, Sheyn D, et al. Structural and nanoindentation studies of stem cell-based tissue-engineered bone. Biomech 2007,40(2):399-411
[8]王明科.干细胞与组织工程.重庆医学,2006,35(21)：1990-1992
[9]C.Liu, Z.Xia, J.T.Czernuszka, Design and Development of Three-Dimensional Scaffolds for Tissue Engineering.Trans IChemE, PartA, Chemical Engineering Research and Design,2007, 85(A7):1051-1064
[10]王身国,贝建中.组织工程细胞支架及其相关技术的研究.中国创伤骨科杂志,2000,2(4)：277-279
[11]凌生林.组织工程支架材料研究进展.泸州医学院学报,2008,31(1)：95-97
[12]王海林.眼科解剖学图谱.沈阳：辽宁科学技术出版社,2002,85-86
[13]谢立信.中国眼科现状.中国工程科学,2003,5(1)：1-5
[14]Stocker F W, Eiring A, Georging, et al. A tissue culture technique for growing corneal epithelial stromal,and endothelial tissues separately. Am J Ophthalmol,1958,46(5):294-298.
[15]谢立信,Gebh BM兔角膜组织细胞培养方法的技术改进.中华眼科杂志,1990,26：237-239
[16]屈雷,王馨,杨学义,等.家兔角膜内皮细胞快速培养.国际眼科杂志,2004,4(2)：243-246
[17]Joyce NC, Joyce SJ, Powell SM, et al. EGF and PGE2:effects on corneal endothelial cell migration and monolayer spreading during wound repairin vitro. CurrEye Res,1995,14(7): 601-609
[18]EngelmannK, DrexlerD, Bohnke M. Transplantation of adulthuman or porcine corneal endothelial cells onto human recipientsin vitro. Part Ⅰ:Cell culturing and transplantation procedure. Cornea,1999,18(2):199-206
[19]邵应峰,胡诞宁,陈家祺β-FGF.EGF和NGF对人角膜内皮细胞生长调控的实验研究.眼科学报,2008,24(1)：9-12
[20]葛健,项行,等.不同浓度牛磺酸对兔角膜内皮细胞的影响.上海交通大学学报(医学版),2009,29(7)：825-827
[21]王丽娅,李辰,曾耀英,等.角膜内皮细胞培养方法的改进.眼科研究,1995,13(3)：215-216
[22]胡晓琴,袁进,陈家祺,等.角膜内皮细胞改良培养技术的初步探讨.眼科研究,2008,26(5)：321-325
[23]陆雯娟,傅瑶,邵春益,范先群.培养猫角膜内皮细胞的新方法—联合酶消化法.眼科新进展,2008,28(2)：81-84
[24]杨鹏,刘瑶,等.兔角膜内皮细胞的新法培养.东南大学学报(医学版),2001,20(3)：170-172
[25]Bednarz J, TeifelM, Friedl P, Engelmann K. Immortalization ofhuman corneal endothelial cells using electroporation protocol optimized for human corneal entothelial and human retinal pigment epithelial cell. Acta Ophthalm ol Scand,2000,78(2):130-136
[26]Shin J S, Jang I K, Kim C W, Kim J C. Development and characterization of a rabbit corneal endothelial cell line. Jpn J Ophthalm ol,2004,48(5):454-459
[27]樊廷俊,赵君,付永锋,从日山,郭瑞超,刘万顺,等.家兔(Oryctolagus curiculus)角膜内皮细胞系的建立.中国科学C辑：生命科学,2007,37(1)：20-27.
[28]刘小伟.兔角膜内皮细胞移植和自体骨耽间充质干细胞替代移植治疗角膜内皮损伤的初步研究：[博士学位论文].北京：中国医学科学院眼科研究中心,北京协和医院眼科,1999
[29]Gospodarowicz D, Greenburg G. The coating ofbovine and rabbit corneas denuded of their entothelium with bovine corneal endothelial cells. Exp Eye Res.,1979,28(3):249-265
[30]易伟斌,胡竹林,徐岩泽,王廷华,黄桂琴,陈娟,等.兔血管内皮细胞对角膜后弹力层代谢的影响.国际眼科杂志,2007,7(4)：956-959
[31]Ishino Y, Sano Y, Nakamura T, et al. Amniotic membrane as a carrier for cultivated human corneal endothelial cell transplantation. Invest Ophthalmol. Vis. Sci.,2004,45 (3):800-806
[32]傅瑶,范先群,等.羊膜载体培养标记兔角膜内皮细胞移植的研究.中华眼科杂志,2006,42(10)：925-929.
[33]林旭初,惠延年,等.猪脱细胞角膜基质的制备及组织学特征观察.国际眼科杂志,2007,7(1)：49-51
[34]林旭初,惠延年,等.脱细胞猪角膜基质体外支持角膜上皮和基质细胞的生长.国际眼科杂志,2008,8(7)：1293-1295
[35]吴连胜,陈建苏,徐锦堂,丁勇,等.脱细胞角膜材料的生物相容性研究.眼科研究,2009,27(5)：359-363
[36]张姝江,杨志明,等.脱细胞异种角膜基质的制备及细胞相容性.中国医学科学院学报,2005,27(6)：670-673
[37]张超,金岩,聂鑫,等.组织工程角膜生物材料载体制备的比较性研究.中国修复重建外科杂志,2006,20(2)：185-188
[38]罗小玲,徐锦堂,黄菊天,赵松滨,等.以异种后弹力膜基质为载体培养角膜内皮细胞的研究.眼科研究,2004,22(5)：449-452
[39]吴静,李姝燕,郝念,等.脱水猪角膜基质为载体体外构建猫角膜内皮组织.中国病理生理杂志,2004,20(6)：1017-1020
[40]范先群,陈苹,傅瑶,等.以异种角膜脱细胞基质为载体构建角膜上皮-载体-内皮复合物的实验研究.中华眼科杂志,2007,43(5)：437-441
[41]Amano S. Transplantation of cultured human corneal endothelial cells. Cornea,2003,22 (7): 66-74
[42]朱梅湘,穆畅道,等.胶原作为生物医学材料的优势与应用.化学世界,2003,3：161-164
[43]Y Minami, H Sugihara, S Oono. Reconstruction of cornea in three-dimensional collagen gel matrix culture. Investigative Ophthalmology & Visual Science,1993,34:2316-2324
[44]陈家祺,张胜,郭琳洁,等.应用三维培养技术体外重建角膜组织的初步研究.中华眼科杂志,2001,37(4)：244-247
[45]Han B, Schwab IR, Madsen TK, et al. A fibrin-based bioengineered ocular surfacewith human corneal epithelial stem cells. Cornea,2002,21 (5):505-510.
[46]顾国贞,王芳,谷树严,等.以纤维蛋白为基质网架的组织工程角膜上皮的构建.吉林大学学报(医学版),2008,34(5)：814-816
[47]梁晓东,陈建苏,等.纤维蛋白胶与兔角膜3种细胞构建组织工程细胞片研究.中国病理生理杂志,2008,24(4)：797-801
[48]Shilu Wang, Wanshun Liu, Baoqin Han,et al. Study on a hydroxypropylchitosan-gelatin based scaffold for cornealstrom tissue engineering. Applied Surface Science,2009, 255:8701-8705.
[49]陈列欢,刘万顺,韩宝芹,位晓娟,杨朝忠.三种角膜内皮细胞载体膜片的性质研究.中国生物医学工程学报,2005,24(6)：755-762
[50]位晓娟,刘万顺,韩宝芹,等.兔角膜内皮细胞载体的体外培养及移植的研究.中国生物医学工程学报,2006,25(5)：590-595
[51]Hadlock T, Singh S, Vacanti JP, et al. Ocular cell monolayers cultured on biodegradable substrates. Tissue Eng.,1999,5 (3):187-190
[52]胡晓洁,王敏,柴岗,张艳,等.组织工程技术构建兔角膜基质组织的实验研究.中华眼科杂志,2004,40(8)：517-521
[53]蒋挺大.甲壳素.第一版.北京：中国环境科学出版社,1996：99,301-328
[54]Majeti NV, Kumar R. A review of chitin and chitosan applications. React. Funct. Polym., 2000,46(1):1-27
[55]M. Thanou, J. C. Verhoef, H. E. Junginge. Oral drug absorption enhancement by chitosan and its derivatives. Adv.Drug Delivery Rev.,2001,52 (2):117-126.
[56]张建湘,汤健,徐斌,等.壳聚糖棒材的组织相容性和安全性评价.生物医学工程学杂志,1996,13(4)：293-297
[57]贾文英,景毅,程琳,等.壳聚糖的遗传毒性研究.透析与人工器官,2001,12(2)：44-46
[58]Y.M. Yang, W. Hu, X.D. Wang, X.S. Gu. The controlling biodegradation of chitosan bersbynacetylation in vitro and in vivo. J.Mater.Sci.Mater.Med.,2007,18:2117-2121
[59]付辰炜.氨基葡萄糖及壳寡糖对小鼠免疫功能的影响：[硕士学位论文].青岛：中国海洋大学,2006
[60]卢凤奇,曹宗顺,庄昭霞,等.壳聚糖膜的降解与生物相容性研究.生物医学工程学杂志,1998,15(2)：183-185
[61]李伟,肖传国.壳聚糖膜在大鼠脊髓组织中的降解及其生物相容性.中国组织工程研究与临床康复,2008,12(36)：7131-7134
[62]Park J H, Cha B J, Lee Y N. Antibacterial activity of chitosan acetate on food-borne enteropathogenic bacteria. Food Science Biotechnol,2003,12(1):100-103
[63]Liu X F, Guan Y L, Yang D Z, et al. Antibacterial action of chitosan and carboxymethylated chitosan. Appl Polym Sci.,2000,79(7):1324-1335
[64]No H K, Park N Y, Lee S H, et al. Antibacterial activity of chitosans and chitosanoligomers with different molecular weights. Int J Food Microbiol,2002, (74):65-72
[65]王鸿,沈月新.不同脱乙酰度壳聚糖的抑菌性.上海水产大学学报,2001,10(4)：380-382.
[66]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
[67]蒋霞云.p-壳聚糖及其脱乙酰衍生物的特性.上海水产大学学报,2002,11(4)：348-352.
[68]Devlieghere F, Vermeulen A, Debevere J. Chitosan:antimicrobial activity, interactions with food components and applicability as a coating on fruit and vegetables. Food Microbiol.,2004, 21(6):703-714.
[69]陈威,吴清平,张菊梅,吴慧清.壳聚糖抑菌机制的初步研究.微生物学报,2008,48(2)：164-168
[70]张惠珍,马国芳,钱洪军,等.不同种类壳聚糖抗菌性能的比较.河北师范大学学报(自然科学版),2009,33(1)：74-77
[71]吴小勇,曾庆孝,莫少芳,肖仔君.几种壳聚糖的抑菌性能.食品与发酵工业,2005,31(2)：18-21
[72]彭翠珍.壳聚糖对几种农作物病害的抑制作用研究.云南农业科技,2009,4：12-13
[73]于广利,李八方,梁平方,万忠良.水溶性甲壳胺制备及其对铅离子的螯合作用.中国海洋药物,1996,4：13-17
[74]Sannan K, Kurita K, Iwakura Y. Studies on chitin. Makromol Chem.,1976,177:3589-3600
[75]Sannan K, Kurita K, Iwakura Y. Kinetics of deacetylation reaction. Polym.J.,1977,9(6): 649-651
[76]席敏,王子飞.水溶性壳聚糖的制备及结构表征.安徽农业科学,2001,29(5)：694-696
[77]陈鲁生.完全水溶性壳聚糖制备条件的研究.化学通报,1998,(8)：48-50
[78]陈天,严俊,徐荣南,夏炎.部分N-酰化脱乙酰壳多糖的水溶性.应用化学,1989,6(3)： 90-92
[79]高兴爽,刘万顺,韩宝芹,梁晔.羟丙基壳聚糖的制备、表征及其对角膜细胞生长的影响.中国海洋大学学报(自然科学版),2007,37(6 sup)：131-134.
[80]Yanfei Peng, Baoqin Han, Wanshun Liu, XiaojuanXu. Preparation and antimicrobial activity of hydroxypropyl chitosan. Carbohydrate Research,2005,340:1846-1851
[81]万荣欣.羧甲基壳聚糖的制备及其生物学评价：[硕士学位论文].天津：天津医科大学,2006
[82]黄攀,韩宝芹,刘万顺,等.N-羧甲基壳聚糖的制备及其生物相容性评价.功能材料,2009,40(7)：1198-1203
[83]Hao Wu, Aiping Zhu, LanhuaYuan. Interactions between O-carboxymethyl chitosan and bovine serum albumin. Materials Chemistry and Physics,2008(112):41-46
[84]Changzheng Wei, Chunlin Hou, Qisheng Gu, et al.A thermosensitive chitosan-based hydrogel barrier for post-operative adhesions' prevention. Biomaterials,2009 (30):5534-5540
[85]刘双利,侯春林,魏长征,等.羟丁基壳聚糖预防大鼠腹腔粘连的实验研究.中国修复重建外科杂志,2009,23(6)：718-722
[86]程金伟,魏锐利,李由,顾其胜.壳聚糖在眼科疾病治疗中的应用.上海生物医学工程,2001,22(4)：44-47
[87]De Campos AM, Diebold Y, Carvalho EL, et al. Chitosan nanoparticles as new ocular drug delivery systems:in vitro stability, in vivo fate, and cellular toxicity. Pharm Res., 2004:(5):803-810
[88]游向东,金中秋,吴建国,宋艳萍.壳聚糖缓释给药系统植入脉络膜上腔治疗兔眼穿通伤.国际眼科杂志,2005,5(1)：74-76
[89]李平,张建林,李家仁,常威,等.氧氟沙星壳聚糖滴眼液的研制.中国生化药物杂志,1999,20(2)：68-69
[90]黄虹,康琦文,樊丽蓉,等.医用几丁糖用作氯霉素滴眼液增稠剂的稳定性考察.中国医院药学杂志,1999,19(11)：689-690
[91]高玉英.治疗眼科疾病用可降解复合膜的制备及性能研究：[硕士学位论文].武汉：武汉理工大学,2005
[92]Howling GI, Dettmar PW, Goddard PA, et al.The effect of chitin and chitosan on the proliferation of human skin fibroblasts and keratinocytes in vitro. Biomaterials,2001,22(22): 2959-2966
[93]郭晟,魏锐利,王文斌,蒋丽霞,金玲.壳聚糖预防兔眼眶手术术后组织粘连.眼科新进展,2006(3)：191-193
[94]李平,王爱勤,谭干祖,张建林.壳聚糖人工泪液的制备及应用.中国医院药学杂志,1996,16(12)：569-570
[95]Felt O, Carrel A, Baehni P, et al. Chitosan as tear substitute:a wetting agent endowed with antimicrobial efficacy. J Ocul Pharmaco Ther,2000,16(3):261-270
[96]李由,周韵秋,侯春林,等.壳聚糖眼内应用的实验研究.第二军医大学学报,1997,18(5)：455-457.
[97]Li Y, Cheng JW, Wei RL, et al. Intraocular pressure and endothelium cell counts after cataract surgery with chitosan and sodium hyaluronate (Healon GV):3-year follow-up results of a randomised clinical trial. Adv Ther,2008,25(5):422-429
[98]吴刚,盛祖力.壳聚糖在牙周组织工程中的应用进展.国外医学生物医学工程分册,2004,27(6)：380-383
[99]向珊珊,高秀秋.壳聚糖-胶原支架与人牙周膜细胞复合培养的实验研究.锦州医学院学报,2006,27(5)：1-4
[100]Feng Z, Yuji Y, William WL, et al. Preparation and histological evaluation of biomimetic three-dimensional hydroxyapatite/chitosan-gelatin network composite scaffolds. Biomaterials,2002,23 (15):3227-3234
[101]Gregory RD, Evans MD, et al. Challenges to nerve regeneration. Seminars in Surgical Oncology,2000,19:312-318.
[102]敖强,王爱军,孙志刚,张秀芳.一种壳聚糖神经组织工程支架的制备及表征.中国组织工程研究与临床康复,2008,12(1)：47-50
[103]张建英,雷德林,李建虎,张圃,等.壳聚糖复合胶原蛋白周围神经支架材料的制备研究.中国美容医学,2006,15(3)：241-244
[104]Lim SS, Gaziano TA, Gakidou E, et al. Prevention of cardiovascular disease in high-risk individuals in low-income and middle-income countries:health effects and costs. Lancet, 2007,370(9604):2054-2062.
[105]Niklason LE. Replacement arteries made to order. Science,1999,286:1493-1494
[106]张鲁生,杨飞,公衍道,等.壳聚糖和明胶材料对血管平滑肌细胞的作用.生物物理学 报,2004,20(1)：19-25
[107]徐迎新,尹太,李瑞新,吴仕和,等.壳聚糖-生物蛋白胶用于工程化血管构建的初步研究.中国康复理论与实践,2005,11(8)：623-625
[108]Suh JK, MatthewHW. Application of chitosan-based polysaccharide biomaterials in cartilage tissue engineering. Biomaterials,2000,21(24):2589-2598.
[109]高海玲,管英俊,申志新,姜俊怡,等.软骨细胞与壳聚糖复合物体内构建软骨组织的研究.潍坊医学院学报,2006,28(4)：266-267
[110]Chenite A, Chaput C, Wang D, et al. Novel injectable neutral solutions of chitosan form biodegradable gels in situ. Biomaterials,2000,21 (21):2155-2161
[111]Tangsadthakun C, Kanokpanont S, Sanchavanakit N, et al. The influence of molecular weight of chitosan on the physical and biological properties of collagen/chitosan scaffolds. J Biomater SciPolym Ed.,2007,18(2):147-163
[112]Mao J, Zhao L, De Yao K, et al. Study of novel chitosan-gelatin artificial skin in vitro. J Biomed Mater Res A.,2003,64(2):301-308
[113]贾之慎,李齐彪.双突跃电位滴定法测定壳聚糖脱乙酰度.化学世界,2001,5：240-241
[114]蒋挺大.壳聚糖.北京.化学工业出版社,2001：26-32
[115]GB/T16886.5-2003,体外细胞毒性试验[S]//郝和平,医疗器械生物学评价标准实验指南.第1版.北京：中国标准出版社,2000：80-82
[116]王昕,施嬿萍,朱雪涛,等.MTT法评价医用高分子材料的细胞毒性.山东生物医学工程,2003,1：46-47
[117]Kawada K, Yonei T, Ueoka H, et al. comparison of the mosensitivity test:clonogenic assay versus MTT assay. Acta Medokayama,2002,56(3):129-134.
[118]郑晓燕.国产碳纤维桩材料的生物学评价：[硕士学位论文].北京：首都医科大学口腔临床医学,2005
[119]张青.黏多糖在眼科中的应用.食品与药品,2005,7(8A)：57-59
[120]牛晓霞,洪晶,段辉,刘蕾.不同保存方法对角膜内皮细胞活性的影响.国际眼科杂志,2006,6(5)：1015-1017
[121]Dawlee S, Sugandhi A, Balakrishnan B, et al. Oxidized chondroitin sulfate-cross-linked gelatin matrixes:a new class of hydrogels. Biomacromolecules,2005,6(4):2040-2048
[122]Huang Y, Onyeri S, Siewe M, et al. In vitro characterization of chitosan-gelatin scaffolds for tissue engineering. Biomaterials,2005,26 (36):7616-7627
[123]Mehrdad Rafat, Fengfu Li, Per Fagerholm, et al. PEG-stabilized carbodiimide crosslinked collagen-chitosan hydrogels for corneal tissue engineering. Biomaterials,2008,29: 3960-3972
[124]余家会,杜予民,等.壳聚糖-明胶共混膜.武汉大学学报(自然科学版),1999,45(4)：440-444
[125]陈丽娟,盛瑞兰,等.AO/EB荧光染色法测定HHT诱导的HL-60细胞凋亡.南京医科大学学报(中文版),1997,17(5)：514-516
[126]周贵,奚廷斐,郑裕东,汤京龙,等.三种羧甲基壳聚糖防粘连膜的降解及其生物相容性.中国组织工程研究与临床康复,2009,13(8)：1411-1418
[127]卢凤琦,曹宗顺.壳聚糖-明胶混合膜的制备及其生物降解性研究.生物医学工程学杂志,1998,15(4)：323-324
[128]Hiraku O, Yoshiharu M. Biodegradation and distribution of water-soluble chitosan in mice. Biomaterials,1999,20(2):175-182
[129]马如,黄明智.壳聚糖的溶菌酶降解.北京化工大学学报,2002,29(6)：40-43
[130]王征,刘万顺,韩宝芹,魏长征.低分子量壳聚糖及其衍生物的制备与溶菌酶对其降解的实验研究.海洋科学,2007,31(10)：36-40
[131]陈列欢.组织工程人工角膜内皮载体膜片的制备,性质及对角膜内皮损伤修复的实验研究.[博士学位论文].青岛：中国海洋大学,2006
[132]杨晓芳,奚廷斐.生物材料生物相容性评价研究进展.生物医学工程学杂志,2001,18(1)：123-128
[133]梁卫东,石应康.细胞培养法评价生物材料生物相容性研究进展.生物医学工程学杂志,1999,16(1)：86-90
[134]戴建国.生物材料生物相容性的分子生物学研究进展.国外医学生物医学工程分册,2004,27(6)：360-364
[135]刘小亮,张彩霞,闻学雷.评价生物材料的体内外实验相关性研究-体外细胞培养的MTT法与体内皮下埋植实验.口腔材料器械杂志,1998,8(1)：10-13
[136]Lobner D. Comparison of the LDH and MTT assays for quantifying cell death:validity for neuronal apoptosis. Journal of Neuroscience Methods,2000,96:147-152.
[137]刘立,王友联,张枢,等.环孢霉素A对局部脑缺血再灌流损伤的改善作用.中风与神 经疾病杂志,2002,19(2)：106-107
[138]邵京山,薛敏,冯玉兰,等.复方丹参注射液对脑出血CT及脑脊液LDH的影响.实用中西医结合临床,2002,2(5)：1-2
[139]陈晓东,吴伯瑜,江琼,林新华,等.芦荟粗多糖对体外培养人表皮细胞增殖作用的研究.中国药理学通报,2005,21(11)：1363-1366
[140]张永红,胡伟,Stark GB,等.乳酸脱氢酶实验检测细胞新陈代谢的实验研究.山西医科大学学报,2003,34(4)：308-310