【作者】 洪奕；

【导师】 沈家骢； 高长有；

【作者基本信息】 浙江大学 ， 材料学， 2005， 博士

【摘要】 本文构建了组织工程中可注射性聚乳酸细胞微载体/壳聚糖凝胶复合支架。首先,制备了聚乳酸微球,并对其表面进行修饰,得到具良好细胞相容性的细胞微载体;其次,制备了可聚合的水溶性壳聚糖,可在温和条件下交联形成壳聚糖水凝胶;最后,将细胞微载体与壳聚糖凝胶复合形成新型的可注射性支架。 采用乳化溶剂挥发法制各了聚乳酸微球。通过调节搅拌速度、聚乳酸浓度和分散剂浓度,获得了不同粒径及粒径分布的聚乳酸微球。在此基础上,采用乳化溶剂挥发法和液致相分离相结合制备了聚乳酸多孔微球。它的表面存在大量的小孔,内部为不规则的多孔结构。通过调节搅拌速度、不良溶剂/良溶剂比、聚乳酸浓度和分散剂浓度,不仅能控制多孔微球的粒径及粒径分布,还能控制多孔微球内部孔径的大小。其内部平均孔径最大可达30μm。 以180～280μm的聚乳酸微球为模板,采用胺解和接枝涂层技术相结合的方法,在聚乳酸微球表面固定生物大分子,如胶原和壳聚糖,以提高细胞相容性。胺解过程中,微球重量随胺解时间的延长而线性下降,而微球表面的自由氨基随胺解时间的延长,先增加后恒定在一稳定值。采用戊二醛为偶联剂,将胶原和壳聚糖接枝涂层在聚乳酸微球表面。胶原和壳聚糖的固定量均随自由氨基含量的增加而增大。体外软骨细胞实验表明,与未改性的聚乳酸微球相比较,胶原涂层和壳聚糖涂层聚乳酸微球的细胞相容性都得到提高,且前者优于后者。 以碳化二亚胺为缩合剂,将甲基丙烯酸和乳酸依次接枝在壳聚糖分子链上,获得了可聚合的水溶性壳聚糖(CML)。采用FTIR、~1HNMR以及元素分析证明了甲基丙烯酸(MA)和乳酸(LA)在壳聚糖分子链上的接枝。且二者的接枝量可由碳化二亚胺的加入量进行控制。壳聚糖衍生物(含23%MA和52%LA)在pH值为9时才发生沉淀。衍生物水溶液的粘度也较低,1%时,粘度只有～40cp(50rpm)。 在氧化还原引发体系过硫酸铵与四甲基乙二胺作用下,壳聚糖衍生物交联形成壳聚糖水凝胶。荧光探针芘检测结果表明交联反应速率主要依赖于引发剂的浓度,而对大分子单体浓度依赖性较小。其凝胶时间随着引发剂浓度的增加而下降,随温度的升高而减少。当引发剂浓度为5mM时,1%CML在25℃和37℃下的凝胶时间分别为～20min和5.5min。平衡溶胀比随引发剂浓度的增加先下降后不变,对离子浓度的变化非常敏感,在培养基和PBS中的平衡溶胀比只有在水中的1/3左右。壳聚糖水凝胶的降解与交联度有关,且对溶菌酶比较敏感。在1mg/ml的溶菌酶/PBS溶液中,水凝胶(CML-15)8天内就完全降解。但凝胶的弹性模量远低于正常软骨的弹性模量。更多还原

【Abstract】 A novel injectable scaffold consisting of polylactide cell microcarriers and chitosan hydrogel was fabricated. Polylactide (PLA) microspheres were fabricated by emulsion-solvent evaporation. Their surfaces were modified to improve cytocompatibility. Crosslinkable and water-soluble chitosan was synthesized, which could be crosslinked to form chitosan hydrogel under mild conditions. The microcarriers and the chitosan hydrogel were finally compounded to form the novel injectable scaffold.PLA microspheres were fabricated by emulsion-solvent evaporation. Their particle size and size distribution could be controlled by stirring rate, PLA concentration and dispersant concentration. Moreover, PLA porous microspheres were fabricated by solution induced phase separation based on emulsion-solvent evaporation. There were lots of small pores on the surfaces of the porous microspheres, and irregular pores throughout the whole inner structure. The particle size, size distribution and inner pore size all could be controlled by stirring rate, PLA concentration, dispersant concentration and non-solvent/solvent ratio.Biomacromolecules, such as collagen type I and chitosan, were immobilized on the surfaces of the PLA microspheres with a diameter of 180~280μm via the combination of aminolysis and grafting-coating to improve the cytocompatibility. The weight of microspheres decreased with the prolongation of aminolyzed time, while the ammo content increased initially, then reached a constant. Using glutaraldehyde as a couple reagent, collagen type I and chitosan were grafted and coated on the surfaces of the PLA microspheres. In vitro chondrocytes culture showed that compared with the unmodified PLA microspheres, collagen and chitosan coated PLA microspheres possessed better cytocompatibility. Chondrocytes could adhesion, spread and proliferate on the surfaces, in particular on those coated with collagen.Using water-soluble carbodiimide (EDAC) as condensation reagent, methyl acrylic acid (MA) and lactic acid (LA) were grafted on the chitosan chain to obtain a crosslinkable and water-soluble chitosan derivative (CML). The molecular structure of CML was confirmed by FTIR, ~1HNMR and elemental analysis. The grafting ratios of MA and LA increased with the increase of EDAC content. CML (23%MA and 52%LA) was readily soluble in pure water and did not precipitate till pH 9. And the viscosity of its solution was very low, only ~40cp (1% CML and 50rpm).Gelation of the CML was realized by thermal treatment at body temperature under the initiation of a redox system, ammonium persulfate (APS)/N,N,N’,N’-tetramethylethylenediamine(TMEDA). Dynamic investigation of hydrogel formation showed that the reaction rate mainly depended on the initiator concentration. The gelation time could be mediated in a wide range, e.g. from 6min to 20min, by reaction temperature and/or initiator’s concentration. In vitro lysozyme degradation of chitosan hydrogel was related to crosslinking degree. At lower crosslinking degree, the hydrogel has been degraded completely at day 8. But the elastic modulus of the hydrogel was smaller than that of the normal human cartilage.3T3 fibroblast culture showed that the cytotoxicity of the hydrogel extractant was attributed to the existence of the initiators. The cytotoxicity of the hydrogel extractant was dependent on the cell seeding number and the initiator’s concentration. With large enough number of cells (>2.5xlO4) and low initiator’ concentration (5mM), the cytotoxicity introduced by the initiator is very minimal and neglectable. 3T3 and chondrocytes encapsulated in the hydrogel could survive but could not proliferate. The cell viability increased initially to a highest value with the prolongation of culture time, then decreased. Although the hydrogel could cause acute inflammation and foreign body reaction, no tissue necrosis and malignancy were evidenced in vivo, demonstrating that the material has better histocompatibility. These features have endowed the chitosan with great opportunity as injectable biomaterials, which may find wide applications in the rapid developed fields such as tissue engineering and orthopaedics.To ensure that PLA microspheres can be suspended in 1% CML solution and can be injected easily, konjac glucomannan (KGM) as thickening agent was added into 1 % CML solution to increase its viscosity. According to the suspension test, gelation time and elastic modulus, when the KGM content was 0.6%, the mixture met with the requirement of PLA microspheres’ suspension and also was found no side effect on the gelation time. The addition of KGM could increase the elastic modulus of the hydrogel.PLA microcarriers were mixed with KGM/CML solution firstly, and then under the initiation of APS/TMEDA, PLA microcarriers/chitosan hydrogel scaffold was formed. The elastic modulus of the scaffold increased with the increase of microspheres’ content. When the microcarriers’ content was 5%, the elastic modulus of the scaffold was 0.12~1.15MPa, a value closed to that normal human cartilage(0.25~2.5MPa). In vitro chondrocyte culture showed that cells could survive, grow and proliferate in this scaffold. Cell viability increased with the prolongation of culture time, and then reached a constant after 9d. The morphology of cells in hydrogel was round, but on the microcarriers surface spread cells showed pebble-like shape. CLSM and SEM observation indicated that chondrocytes in hydrogel moved to the surfaces of the PLA microcarriers, and then attached, spread and proliferated. And the increase of cell viability in this case was attributed to the cell proliferation on the PLA microcarriers. The combination of the PLA microcarriers and the chitosan hydrogel possessed synergistic effect. Liquid chitosan hydrogel precursor could be used as the carrier to inject PLA microcarriers into body. After gelation, hydrogel could help PLA microcarriers to shape and to prevent microcarriers from moving in vivo. On the other hand, addition of the PLA microcarriers could improve the mechanical strength of chitosan hydrogel. These features indicate that this novel PLA microcarriers/chitosan hydrogel scaffold has potential application for injectable scaffold in tissue engineering.更多还原