| [页数] 54 [字数] 27434 [目录] 第1章 引言 1 第2章 NHAC/PLA骨组织工程框架材料的细胞相容性研究 14 第3章 复合BMP的骨框架材料诱导成骨细胞分化的初步研究 27 第4章 磷酸化壳聚糖的细胞生物相容性和骨诱导性研究 32 第5章 结论 40 参考文献 41 致谢、声明 46 个人简历、攻读硕士学位期间发表的SCI和待发表的学术论文 47 [原文] 第1章 引 言 1.1 课题目的和意义 基于近年来组织工程学科的迅速发展，组织工程骨修复材料有望成为最早实现的组织工程产品[1,2]。骨组织工程的基本出发点是以“诱导成骨”的方式而不是单纯以“爬行替代”的方式实现骨的修复和再生。骨组织工程的三个关键要素为信号分子（骨生长因子、骨诱导因子）、框架材料、靶细胞。通过研究骨系细胞与骨修复材料的相互作用，可以为实现组织工程骨提供切实可靠的实现方法和机理研究模型。骨材料，无机非金属骨修复材料以及羟基磷灰石/胶原复合材料的生物相容性都已有了较全面的报道，目前纳米－羟基磷灰石/胶原复合物(nHAC composite)因从成分和结构上比一般骨修复材料更接近天然骨，近年来颇受众人关注[3,4]。本实验室在此方面也有一定的研究基础[6-8]。纳米相羟基磷灰石/胶原复合物的复合框架材料是当前骨组织工程学中新型的骨修复材料。羟基磷灰石，胶原，高分子聚合物如PLA和壳聚糖等都已先后被证明具有良好的生物相容性。进一步实验验证纳米—羟基磷灰石/胶原/聚乳酸的骨修复框架材料具有良好的细胞生物相容性，对于指导外科骨缺损的修复具有重要的临床意义。并进一步研究复合细胞生长因子的框架材料是否具有诱导成骨细胞分化的能力，对于指导框架材料的进一步的临床应用，包括细胞分子水平的治疗都具有指导意义。 甲壳素是自然界中仅次于纤维素的天然多糖，广泛存在于昆虫、甲壳类动物外壳及真菌细胞壁中。经脱乙酰化反应变成甲壳胺，即壳聚糖。壳聚糖一般不溶于水、碱和常规有机溶剂中。只溶于盐酸等无机酸及甲醇、乙醇等。这类天然多糖具有明显碱性、良好的生物相容性和生物可降解性。壳聚糖类分子中有许多胺基和羟基，容易进行化学修饰和改性。壳聚糖的生物相容性一般认为是良好的，但这些评价大都是将壳聚糖制成膜材料、线材料和药物载体的研究观察到的...... [摘要] 矿化的胶原和壳聚糖类等一些天然的高分子物质近年来越来越多地被用于骨缺损的研究，其中纳米-羟基磷灰石/胶原/聚乳酸框架材料 (nano-Hydroxyapatite/collagen scaffold, nHAC/PLA scaffold) 和磷酸化的壳聚糖是近年来骨缺损修复材料领域中的热点。在此基础之上，本研究进行了这两类材料的细胞相容性的研究和部分动物实验。通过用体外细胞培养的方法，建立了成骨细胞与nHAC/PLA框架材料的三维复合体，并将其植入大块骨缺损的动物模型中，结果表明，材料具有很好的生物相容性，细胞在材料上正常的贴壁、增殖和迁移生长，并能够长入框架材料的空隙内部。同时还有实验表明nHAC/PLA复合材料具有优异的骨传导性，当植入缺损后骨可在这种支架上进行爬行替代性生长，一定程度上起到缩小缺损尺寸的作用。 nHAC/PLA与rhBMP-2复合后又具有了高效的骨诱导性，在本研究中又关于材料和细胞生长因子对成骨细胞的诱导分化作用的进行了初步探讨。良好的细胞相容性和骨传导性以及高效的骨诱导性使这类材料具有较大的临床应用潜力。可以为临床治疗骨缺损提供高效的备选材料。 壳聚糖被磷酸根基团修饰后，可以得到具有水溶性的磷酸化壳聚糖，由于改性以后具备了水溶性，因此这一类材料就可以被广泛地应用于生物材料领域。通过研究磷酸化壳聚糖与成骨细胞的相互作用，我们得知经过改性的磷酸化的壳聚糖仍然具有良好的生物相容性，同时对成骨细胞具有骨诱导性。因此，也为临床骨修复材料的研究提供了另一种可行的途径。 [参考文献] [1] Langer R and Vacanti J P. Tissue engineering. Science. 1993, 260: 920-926. [2] Suchanek W and Yoshimura M. Processing and properties of hydroxyapatite-based biomaterials for use as hard tissue replacement implants. J Mater Res. 1998, 13: 94-117. [3] Stock UA and Vacanti JP. Tissue Engneering: Current State and Prospects. Annu.Rev.Med.2001.52: 443-451. [4] Paula K, Orla S, Colin N. Bone health in the balance. Science. 2000, 289: 1497. [5] Robert F. Tissue Engineers Build New Bone. Science. 2000, 289: 1498-1500. [6] Wang RZ, Cui FZ, Lu HB, Wen HB, Ma CL, Li HD. Synthesis of Nanophase Hydroxyapatite collagen composite. Journal of Materials Science Letters. 1995, 14: 490-492. [7] Du C, Cui FZ, Zhang W, Feng QL, Zhu XD, K. de Groot. Formation of calcium phosphate/collagen composites through mineralization of collagen matrix. J Biomed Mater Res. 2000, 50: 518-527. [8] Du C, Cui FZ, Feng QL, et al. Tissue response to nano-hydroxyapatite/ collagen composite implants in marrow cavity. J Biomed Mater Res. 1998, 42: 540-548. [9] Helga Ertesvag, Svein Valla, Biosynthesis and application of alginates. Polymer degradation and stability. 1998, 59: 85-91. [10] Wang XH, Ma JB, Wang YN, et al. Reinforcement of calcium phosphate cements with phosphorylated chitin. CHINESE J POLYM SCI 20 (4): 325-332 JUL 2002 [11] Park J B and Lakes R S. Biomaterials: An Introduction. Plenum Press, New York, 1992 [12] 崔福斋, 冯庆玲. 生物材料学. 科学出版社, 1996. [13] 顾汉卿, 徐国风. 生物医学材料学. 天津科技翻译出版公司, 1993. [14] Recum AF, Lucas LC. Trans. of the Sixth World Biomaterials Congress in Kamuela Hawaii, USA, May 15-20, 2000. [15] 刘子君主编. 骨关节病理学. 人民卫生出版社, 1992. [16] Marks S C and Popoff S N. Bone cell biology: the regulation of development, structure, and function in the skeleton. Amer J Anat. 1988, 183: 1-44. [17] Gay C V. Osteoclast ultrastructure and enzyme histochemistry: Functional implications. In: Biology and Physiology of the osteoclast. Rifkin B R, Gay C V, eds. CRC Press, Boca Raton, Florida, 1992, pp129-150. [18] Nijweide P J and de Grooth R. Ontogeny of the osteoclast. In: Biology and Physiology of the osteoclast. Rifkin B R, Gay C V, eds. CRC Press, Boca Raton, Florida, 1992, pp81-104. [19] Buckwalter J A, Glimcher M J, Cooper R R, et al. Bone biology. J Bone Joint Surg. 1995, 77A: 1276-1289. [20] Pechak D G, Kujawa M J, Caplan A I. Morphological and histochemical events during first bone formation in embryonic chick limbs. Bone. 1986, 7: 441-458. [21] Frost H M. The biology of fracture healing. Clin Orthop. 1989, 248: 283-309. [22] Tang X M, Chai B F. Light microscopic and electron microscopic observation on experimental fracture healing. Chine Med J. 1982, 95(10): 721-730. [23] Cui FZ, ZhangY, Wen HB, Zhu XD. Microstructure evolution in external callus of human long bone. Materials Science & Engineering C. 2000, 11:27-33. [24] Buckwalter J A, Glimcher M J, Cooper R R, et al. Bone biology. J Bone Joint Surg. 1995, 77A(8): 1276-1289. [25] Vicente V, Meseguer L, Martinez F, et al., Ultrastructural study of the osteointegration of bioceramics (whitlockite and composite beta-TCP plus collagen) in rabbit bone. Ultrastruct Pathol. 1996, 20(2): 179-188. [26] Urist MR. Bone: Formation by autoinduction. Science. 1965, 150: 893-899. [27] Reddi AH and Huggins CB. Biochemical sequences in the transformation of normal fibroblasts in adolescent rats. Proc Natl Acad Sci USA. 1972, 69: 1601-1605. [28] Sampath TK and Reddi AH. Dissociative extraction and reconstitution of extracellular matrix components involved in local bone differentiation. Proc Natl Acad Sci USA. 1981, 78: 7599-7602. [29] Urist MR, Lietze A, Dawson E. ?-tricalcium phosphate delivery system for bone morphogenetic protein. Clin Orthop Rel Res. 1984, 187: 277-280. [30] Suh DY, Boden SD, Louis-Ugbo J, et al. Delivery of recombinant human bone morphogenetic protein-2 using a compression-resistant matrix in posterolateral spine fusion in the rabbit and in the non-human primate. Spine 2002, 27(4): 353-360. [31] Moursi AM; Winnard AV; Winnard PL; Lannutti JJ; Seghi RR. Enhanced osteoblast response to a polymethylmethacrylate–hydroxyapatite composite. Biomaterials. 2002, 23: 133-144. [32] Buttery LDK, Bourne S, Xynons JD, et al. Differentiation of osteoblasts and in vitro bone formation from murine embryonic stem cells. Tissue Engineering. 2001,7:89-99 [33] Lowry OH. The Evolution OF Analytical Biochemistry (1933-1983)-A Biased 50-Year Review. Faseb Journal. 1994, 8 (2): 262-264. [34] Lowry OH, Rosebrough NJ, Farr Al, Rand RJ. Proteion measurement with folin phenol reagent. J Biol Chem. 1951, 193: 265-275. [35] Ishaug SL, Crane GM, Miller MJ, et al. Bone formation by three-dimensional stromal osteoblast culture in biodegrable polymer scaffolds. J Biomed Mater Res. 1997, 37: 17-28. [36] Fu YH, Chueh SC, Wang YJ. Micropheres of hydroxyapatite/reconstituted collagen as supports for osteoblast cell growth. Biomaterials. 1999,20:1931-1936. [37] Folkman J, Mascona A. Role of cell shape in growth control. Nature. 1978, 273: 345-349. [38] Archer CW, Rooney P, Wolpert L. Cell-shape and cartilage differentiation of early chick limb bud cells in culture. Cell Differentiation. 1982, 11: 245-251. [39] Hunter A, Archer CW, Walker PS, Blunn GW. Attachment and proliferation of osteoblasts and fibroblasts on biomaterials for orthopedic use. Biomaterials. 1995, 16: 287-295. [40] 廖素三.矿化胶原基组织工程骨材料的研究.清华大学博士生论文. 北京：清华大学材料科学与工程系，2003 pp 35-53,70-88. [41] Boyan BD, Hummert TW, Dean DD, Schwartz Z. The role of material surfaces in regulating bone and cartilage cell response. Biomaterials. 1996, 17: 137-146. [42] Vacanti CA. The Impact of Biomaterials Research on Tissue Engineering. MRS Bull. 2001, 26: 798-799. [43] Reddi AH. Morphogenesis and Tissue Engineering of Bone and Cartilage: Inductive Signals, Stem Cells, and Biomimetic Biomaterials. Tissue Engineering. 2000, 6: 351-359. [44] Shea LD, Wang D, Franceschi RT, Mooney DJ. Engineering Bone Development from a Pre-Osteoblast Cell Line on Three-Dimensional Scaffolds. Tissue Engineering. 2000, 6: 605-617. [45] Younger EM, Chapman MW. Morbidity at bone graft donor sites. J Orthop Trauma. 1989, 3: 192-195. [46] Boden SD, Schimandle JH, Hutton WC, Chen MI. 1995 Volvo Award in Basic Science: the use of an osteoinductive growth factor for lumbar spinal fusion. Part I: biology of spinal fusion. Spine. 1995, 20: 2626-2632. [47] Boden SD, Schimandle JH, Hutton WC. 1995 Volvo Award in Basic Science: the use of an osteoinductive growth factor for lumbar spinal fusion. Part II: study of dose, carrier, and species. Spine. 1995, 20: 2633-2644. [48] 孙靖，遇秀玲. 实验动物学讲义. 北京市实验动物管理办公室. 1999，pp133-149. [49] Nilsson E S, Urist M R, Dawson E G, et al. Bone repair induced by bone morphogenetic protein in ulnar defects in dogs. J Bone Joint Surg. 1986, 68B: 635-642. [50] Bolander M E and Balian G. The use of demineralized bone matrix in the repair of segmental defects. J Bone Joint Surg. 1986, 68A(8): 1264-1273. [51] Spiro RC, Thompson AY, Poser JW. Spinal fusion with recombinant human growth and differentiation factor-5 combined with a mineralized collagen matrix. The Anatomical Record. 2001, 263: 388-395. [52] Emery SE, Fuller DA, Stevenson S. Ceramic anterior spinal fusion biologic and biomechanical comparison. Spine. 1996, 21: 2713-2719. [53] Thalgott JS, Fritts K, Giffre JM, et al. Anterior interbody fusion of cervical spine with coralline hydroxyapatite. Spine. 1999, 24: 1295-1299. [54] Chaudhari A, Ron Eyal, Rethman MP. Recombinant human morphogenetic protein-2 stimalates differentiation in primary culture of fetal rat calvarial. Molecular and Cellular Biochemistry. 1997, 167: 31-39. [55] Buckwalter J A, Glimcher M J, Cooper R R, et al. Bone biology. J Bone Joint Surg. 1995, 77A: 1276-1289. [56] Buckwalter J A, Glimcher M J, Cooper R R, et al. Bone biology. J Bone Joint Surg. 1995, 77A(8): 1276-1289. [57] Wang XH, Ma JB, Feng QL, et al. Skeletal repair in rabbits with calcium phosphate cements incorporated phosphorylated chitin. BIOMATERIALS 23 (23): 4591-4600 DEC 2002 [58] Wang XH, Ma JB, Wang YN, et al. Bone repair in radii and tibias of rabbits with phosphorylated chitosan reinforced calcium phosphate cements. BIOMATERIALS 23 (21): 4167-4176 NOV 2002 [59] Bronwyn MK, Fiona JM, John HM. Serum –free cultue of rat post-natal and fetal brainstem neurons. Development Brain Research 2000;120:199-210. [60] Cai K, Yao K, Lin S, Yang Z, Li X, Xie H, Qing T, Gao L. Poly(D, L-lactic acid) surfaces modified by silk fibroin: effects on the culture of osteoblast in vitro. Biomaterials 2002;23:1153-1160. [61] Zund G, Ye Q, Hoerstrup S P, Shoeberlein A, Schmid AC, Grunenfelder J, Vogt P, Turina M. Tissue engineering in cardiovascular surgery: MTT, a rapid and reliable quantitative method to assess the optimal human cell seeding on polymeric meshes. Eur J Cardio-thoracic Surg 1999;15:519–24 [62] Ciapetti G, Cenni E, Pratelli L, Pizzoferrato A. In vitroevaluation of cell/biomaterial interaction by MTT assay. Biomaterials1993;14:359–364. [63] Heide Siggelkow, Dirk Hilmes, Katja Rebenstorff, Wiebke Kurre, Iris Engel, Michael Hüfner. Analysis of human primary bone cells by florescence activated cell scanning: methodological problems and preliminary results. Clinica Chimica Acta 272 (1998) 111-125. [64] Webster TJ, Siegel RW, Bizios R. Osteoblast adhesion on nanophase ceramics. Biomaterials. 1999, 20: 1221-1227. [65] C. Du, F. Z. Cui, X. D. Zhu, K. de Groot. Three-dimensional nano-HAp/collagen matrix loading with osteogenic cells in organ culture. J Biomed Mat Res 1999; 44: 4: 407-415 [66] Gerstenfld LC, Chipman SD, Kelly CM, Hodgnes KJ, Lee DD, Landis WJ. Collagen expression, ultrastructure assembly and mineralization in cultures of chicken embryo osteoblasts. J Cell Biol 1998;106:979-89. [67] Mansur Ahmad, MaryBeth McCarthy, Gloria Gronowicz. An in vitro model for mineralization of human osteoblast-like cells on implant materials. Biomaterials 20(1999)211-220 [原文截取] 矿化胶原和磷酸化壳聚糖骨修复材料的细胞生物相容性研究 The Study on Cytocompatibility of Mineralized Collagen and P-Chitosan Bone Repair Materials 院（系、所） ： 材料系 工程领域 ： 材料工程 申请人 ： 指导教师 ： 教 授 联合指导教师 ： 副主任医师 二零零 年 月 其复合框架材料的制备及性能研究 张 曙 明 关于论文使用授权的说明 本人完全了解 大学有关保留、使用学位论文的规定，即：学校有权保留送交论文的复印件，允许论文被查阅和借阅；学校可以公布论文的全部或部分内容，可以采用影印、缩印或其他复制手段保存论文。 (保密的论文在解密后应遵守此规定) 签 名： 导师签名： 日 期： 中文摘要 矿化的胶原和壳聚糖类等一些天然的高分子物质近年来越来越多地被用于骨缺损的研究，其中纳米-羟基磷灰石/胶原/聚乳酸框架材料 (nano-Hydroxyapatite/collagen scaffold, nHAC/PLA scaffold) 和磷酸化的壳聚糖是近年来骨缺损修复材料领域中的热点。在此基础..... |
矿化胶原和磷酸化壳聚糖骨修复材料的细胞生物相容性研究
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