【作者】 胡小红；

【导师】 高长有；

【作者基本信息】 浙江大学 ， 材料学， 2009， 博士

【摘要】 在软骨修复中，支架为细胞和组织生长提供适宜的环境。水凝胶的结构和天然软骨的细胞外基质相似，并可通过微创注射等方法来实现组织修复，因此是用于软骨修复和再生的一类非常重要的材料。我们利用光引发聚合的方法制备了可注射型水凝胶并在水凝胶中引入微载体、生物大分子、生长因子等，研究了水凝胶的结构与性能。针对软骨组织的特点，进一步设计了仿生化的水凝胶。这些工作对软骨的修复与再生用水凝胶支架的设计和制备有重要意义。在本课题组以前的工作基础上，水溶性可交联聚合的壳聚糖(CML)通过在壳聚糖上依次接枝甲基丙烯酸(MA)和乳酸(LA)制得。光引发剂(Irgacure2959)在特定波长(365 nm)的紫外光作用下引发CML在水溶液中聚合，制备了可注射型壳聚糖水凝胶。水凝胶的凝胶时间从分钟到几十分钟，且当引发剂浓度低于0.05％时，对凝胶时间影响较大，因此用0.05％引发剂来制备水凝胶。干态的水凝胶吸收水分后可以溶胀到原重量几十到一百多倍。当交联密度较高、pH较高、溶液中有二价阴离子时，水凝胶的平衡溶胀比较低。水凝胶的粘弹性测试表明水凝胶的储能模量在0.8-7 kPa，损耗模量在10-100 Pa。交联密度较高的水凝胶，储能模量和损耗模量也相对较高，在含溶菌酶的PBS中降解相对较慢。体外软骨细胞的培养表明由Irgacure2959引发形成的水凝胶的细胞毒性小于由过硫酸铵／四甲基乙二胺(APS／TEMED)引发形成的水凝胶的细胞毒性。为了促进水凝胶中软骨细胞的生长，将明胶引入CML水凝胶中，用光聚合的方法制备了含明胶的CML水凝胶。以明胶分子为模板研究了生物大分子在CML水凝胶中的释放行为。随着明胶的交联程度增大、含量增加，水溶液的pH增大，从CML水凝胶中释放出的明胶相对含量减少。在20℃下从CML水凝胶中释放出的明胶相对含量比37℃下少。另外，溶液中的离子和氨基酸等也会影响明胶的释放。明胶的包埋也会影响CML水凝胶的结构与性能。如随着明胶含量的增大，水凝胶的网孔尺寸变小，平衡溶胀比降低，对水凝胶的粘弹性和水凝胶的降解性能也有一定的影响。体外软骨细胞培养发现CML水凝胶中的明胶一定程度上可以促进软骨细胞的生长和细胞外基质的分泌；随着培养时间的延长，软骨细胞在CML水凝胶中逐渐向下迁移。通过MA的接枝成功地合成了可共价交联的明胶(GM)，并通过光引发聚合的方法制备了可注射型明胶水凝胶。明胶接枝MA后分子量分布变宽，zeta电位升高。光照20 min后，固体核磁的结果显示GM化学凝胶中不存在未反应的双键。通过差示扫描量(?)仪(DSC)表征了明胶接枝前后的物理溶胶-凝胶转变点，MA的接枝、聚合物浓度等会影响其熔融温度和熔融焓。圆二色谱(CD)证实了明胶中存在三螺旋结构，改性后三螺旋结构的含量减少。随着聚合物浓度增加，水凝胶的凝胶时间缩短、平衡溶胀比降低、储能模量和损耗模量增加、平均网孔尺寸减小。我们还将TGF-β1包埋在明胶水凝胶中，研究了软骨细胞在水凝胶中的生长。体外软骨细胞的培养表明GM水凝胶可以支持软骨细胞生长和保持软骨细胞的表型；TGF-β1能促进软骨细胞生长并保持软骨细胞的表型。结合可注射型水凝胶支架和可注射型微载体支架两者的优点，我们将GM固定在聚乳酸／乙醇酸(PLGA)粒子表面。然后把改性后的粒子分散在CML的溶液中，在Irgacure2959的引发下，PLGA粒子表面的双键和CML的双键一起聚合，形成复合的可注射型水凝胶支架。这种复合型水凝胶支架具有更好的力学性能和生物活性，有着更广泛的应用前景。最后，针对软骨组织的特点，利用点击化学制备了透明质酸／明胶／硫酸软骨素的仿生水凝胶。首先合成了含有叠氮基团的透明质酸(HA-AA)、硫酸软骨素(CS-AA)和含有炔基的明胶(G-PA)。在Cu(I)催化下，叠氮基团和炔基反应使含HA-AA、CS-AA、G-PA的水溶液凝胶化。水溶液的凝胶时间随氯化亚铜的浓度的增加而缩短，直到1 mg／mL。水凝胶在PBS中的溶胀比为18.8±2.4，比单纯的明胶水凝胶的平衡溶胀比大。动态粘弹性的结果显示了水凝胶为弹性体。水凝胶在PBS中4周的失重为50％左右。两周内大约有20％的明胶和10％的CS从水凝胶中释放出来。原子力显微镜显示了水凝胶表面较为平整。体外软骨细胞的培养表明软骨细胞可以在水凝胶表面生长，并保持软骨细胞的表型。更多还原

【Abstract】 In cartilage regeneration, scaffolds can provide suitable environment for cellgrowth and tissue formation. Hydrogel is a kind of hydrated polymer networks, whichmimics the native extracellular matrix （ECM） of cartilage. The hydrogel can betransplanted into damaged site to realize tissue repair through injection in a minimallyinvasive way. Therefore, the hydrogel is a kind of very important materials incartilage repair and regeneration. In this dissertation, injectable hydrogels wereprepared by photoinitiating polymerization, into which microcarriers,biomacromolecules and growth factors were introduced. The structure and propertiesof the hydrogels were studied. A biomimic hydrogel was designed at last. Theseworks had great significance in designing ideal biomaterials for cartilage repair andregeneration.Based on the previous research work in our laboratory, water-soluble andcrosslinkable chitosan was synthesized by sequentially grafting of methacrylic acid（MA） and lactic acid （LA）. Irradiated by 365 nm UV light, Photoinitiator（Irgacure2959） was used to initiate CML polymerization in water solution and therebyobtain chitosan hydrogel. The gelation time was controlled from several minutes totens minutes. When the photoinitiator concentration was lower than 0.05％, thegelation time was longer and influenced greatly by the photoinitiator concentration.Therefore, in this work, the hydrogel was obtained using 0.05％photoinitiator. Thedry hydrogels could adsorb water by a factor of tens to hundred. The swelling ratiowas smaller at larger crosslinking density, higher pH value, and in salt solutions,especially in Na₂SO₄ and Na₂HPO₄ solution. Rheological measurement recorded thestorage modulus and loss modulus of 0.8-7 kPa and 10-100 Pa, respectively. With ahigher degree of MA substitution, the resultant hydrogel had a larger modulus anddegraded slowly too. In vitro chondrocyte culture showed that the cytotoxicity of thehydrogel made by Irgacure2959 was much smaller than that of the hydrogel made byammonium persulfate （APS）/ N,N,N’,N’-tetramethylethylenediamine （TEMED）. In order to promote the chondrocytes growth in hydrogel, gelatin was introducedinto the CML hydrogel. The release behaviors of gelatin as a biomacromoleculartemplate in CML hydrogel were studied. The released gelatin ratio was smaller at alarger crosslinking extent of gelatin, original gelatin content in hydrogel and higherpH value. The released gelatin ratio was smaller at 20℃than that at 37℃. Moreover,the released gelatin ratio was influenced by ions and amino acids in solution. Thestructure and properties of the CML hydrogel were also influenced by the embeddedgelatin. When the embedded gelatin increased, mesh size of the hydrogel was smallerand swelling ratio of the hydrogel decreased. The rheological properties anddegradation properties were also influenced by the embedded gelatin. In vitrochondrocyte culture showed that the gelatin containing hydrogel could promotechondrocyte growth and secrete ECM. Chondrocytes in the CML hydrogel couldmove downward along with the culture time.We modified gelatin with MA and obtained crosslinkable gelatin （GM）, whichwas used to form injectable gelatin hydrogel by photoinitiating polymerization. Thepolydispersity of molecular weight broadened and zeta potential increased too aftermodification with MA. There were no double carbon bonds in hydrrogel detected byhydrogen spectrum of high resolution magic angle spinning nuclear magneticresonance （¹H HR-MAS NMR） after irradiation for 20 min. The sol-gel transitionpoint of gelatin before and after modification was characterized by differentialscanning calorimeter （DSC）, revealing that it is influenced by MA modification andpolymer concentration. Triple helix structure existed in the gelatin gel and wasconfirmed by circular dichroism （CD）, whose content decreased after modification.With polymer concentration increasing, the gelation time decreased, swelling ratiodecreased, storage modulus and loss modulus increased and mesh size decreased.Transform growth factor-β1 （TGF-β1） also combined into the GM hydrogel topromote the chondrocyte growth. In vitro chondrocyte culture showed that the GMhydrogel both with and without TGF-β1 could support the chondrocyte growth andmaintain the chondrocyte phenotype. TGF-β1 could promote chondrocyte growth andmaintaining chondrocytic phenotype. Combination of advantages of the injectable hydrogels and the injectablemicrocarriers, a composite hydrogel was prepared. First, the surface of poly（lactic-co-glycolic acid） （PLGA） microparticles were modified with GM. Themodified particles were then dispersed in CML solution. Under UV irradiation,particles were polymerized with CML to obtain a composite hydrogel, which showedbetter mechanical property and cytoviability than that of the CML hydrogel. Asinjectable materials, the composite hydrogel might have wider potential applications.At last, hyaluronic acid （HA）/gelatin/chondroitin sulfate hydrogel was formed byclick chemistry to mimic the compositions of natural cartilage matrix. HA and CSwere modified with 11-azido-3,6,9-trioxaundecan-1-amine （AA）, which contain -N₃group. Gelatin was modified by propargylamine （PA）, which contain acetylene group.Catalyzed by Cu （Ⅰ）, -N₃ group was reacted with acetylene group to obtain thehydrogel. As Cu （Ⅰ） concentration increasing, the gelation time was shortened until1 mg/mL. The swelling ratio of this hydrogel was 18.8±2.4, larger than that of thegelatin hydrogel. The rheological result showed that this hydrogel was elastomeric.The weight loss of this hydrogel in 4 weeks reached to 50％. About 20％gelatin and10％CS was released from this hydrogel in 2 weeks. The surface of hydrogel filmwas smooth characterized by atomic force microscopy （AFM）. In vitro chondrocyteculture showed that the chondrocytes could grow on the surface of the hydrogel andmaintain its phenotype.更多还原