可注射型智能凝胶类的医用植入材料研究适应了微创伤外科技术发展的要求，具有微创伤修复组织缺损或畸形、组织损伤小、不破坏修复区血供、操作简便易行等优点，在药物缓释和组织工程领域日益引人注目。可注射型智能凝胶在注射前保持溶液状态，注射后可在人体环境下形成凝胶。溶剂、紫外辐射、离子交联剂、pH和温度等因素都能够导致可注射型凝胶的原位形成。其中，通过温度改变实行溶液-凝胶转变可原位形成凝胶的温敏型智能材料，不需要有机溶剂，共聚剂和引发剂等化学试剂，可降低水凝胶的生物毒性，是生物医用材料应用发展的重要方向之一。 壳聚糖(CS)是自然界唯一大量存在的碱性多糖，生物毒性小，具有良好的组织相容性、生物可降解性和粘附性，在医学、生物学领域得到了深入的研究和广泛的应用，是一种理想的医用植入材料。但在一般情况下，壳聚糖只溶于酸性水溶液中，壳聚糖的酸性溶液被中和到一定pH值(>6.2)后，可形成类似凝胶的沉淀，该过程与温度关系不大。近年来，有些研究通过对壳聚糖进行共混、衍生化和接枝反应，可得到具有温度敏感性的壳聚糖基物理凝胶。溶液在生理pH(7.0)及室温下保持溶液状态，而在温度升至体温(37℃)时迅速凝胶化。 本论文概述了可注射型温度敏感性智能水凝胶的性质及其在药物缓释和组织工程中的研究进展；从分子设计的角度出发，制备了一系列壳聚糖基可注射型温度敏感性水凝胶及其与纳米粒子的复合凝胶，用于药物缓释与软骨组织细胞培养的三维支架。采用傅立叶红外光谱(FTIR)、共聚焦激光显微镜(CLSM)、X —衍射(XRD)、扫描电镜(SEM)、透射电镜(TEM)、动态光散射(DLS)和动态流变仪(ARES)等分析手段表征了凝胶的物理化学特征。考察了不同多羟基聚合物与壳聚糖共混凝胶的制备条件及其温度响应性，研究了多糖的分子参数如分子量和脱乙酰度等对凝胶形成的影响，讨论了不同盐类在凝胶形成中的作用，评价其对蛋白质和小分子药物释放的可行性，以及作为组织工程中的三维支架培养细胞的性能。 主要研究内容和结论如下： 1.制备了壳聚糖-甘油的可注射型温敏凝胶体系。考察碱、甘油、壳聚糖浓度和分子量对凝胶化温度及凝胶结构和性能的影响。实验结果表明：碱、甘油和壳聚糖的混合比例影响溶液凝胶化的温度和速度。壳聚糖浓度越高，形成凝胶所需时间越短，凝胶强度越大；壳聚糖分子量越低越难以形成凝胶，分子量低于1.0×10⁴时，不能形成凝胶。采用牛血清白蛋白作为模型药物，研究不同制备条件和缓释体系中凝胶的缓释情况。当直接添加牛血清白蛋白于浓度为2wt%、分子量为2.7×10⁵的壳聚糖溶液中，并用海藻酸钠溶液处理形成后的凝胶时，药物缓释时间最长。不同pH、离子强度等缓释体系也影响药物缓释效果。由于凝胶制备过程避免高温、有机化学试剂及其它有害的条件，药物缓释时间较长，壳聚糖基凝胶作为蛋白类药物的包埋剂和口服制剂有良好的应用前景。 2.制备了壳聚糖-聚乙烯醇(PVA)的可注射型温敏凝胶体系。CS/PVA溶液的凝胶行为是一个具有低临界溶液温度(LSCT)的复杂热致凝胶体系。当CS浓度为1wt%时，PVA浓度为1、2.5和5wt%时，凝胶化温度分别为21、26和34℃，即凝胶化温度随着PVA浓度的增加而提高。CS脱乙酰度的降低也能导致凝胶化温度的提高。CS/PVA溶液的凝胶化时间随温度升高和PVA溶液浓度的减少而减少。凝胶强度也随CS溶液浓度的增加而提高，相反PVA溶液浓度的增加导致凝胶强度的减少。CS分子量和脱乙酰度的降低也减少了凝胶的强度。采用CLSM和SEM观察了不同浓度CS和PVA对凝胶结构的影响，并根据动态流变分析的结果，表明凝胶形成机理主要取决于两个作用：一是低温下，聚合物分子链以及水分子之间的氢键作用，主要起到分离聚合物的链段，避免其发生相互缠绕；二是升温后，氢键作用被破坏，聚合物分子链之间的疏水作用，促使凝胶形成。因此高温下，CS链(pH>6.2)之间的疏水作用被认为是促使凝胶形成的主要驱动力。CS浓度越高，PVA浓度越高，凝胶内部网络孔径越小。 3.制备了壳聚糖-甲基纤维素的可注射型温敏凝胶体系。考察了不同盐类(NaCl， Na₃PO₄，NaHCO₃和甘油基磷酸钠(GP)对凝胶形成的影响。含不同盐类的CS/MC凝胶具有不同的凝胶温度、速度和强度，可满足不同的应用需求。含NaCl的CS/MC水凝胶，在50℃以上，MC的疏水作用导致强凝胶态的形成，并且凝胶是可逆的，CS对凝胶的形成没有影响。对于含Na₃PO₄和NaHCO₃的CS/MC水凝胶，甚至在40℃，远高于它们的凝胶温度27℃和24℃，凝胶强度依然很弱。主要由于40℃，MC凝胶不能够形成，凝胶的支架主要由疏水的壳聚糖链构成，MC仅依靠弱的氢键作用被包裹于CS链中。对于含GP的CS/MC水凝胶，凝胶能够在40℃形成强凝胶态，主要是由于依靠疏水的作用，CS和MC凝胶能够同时形成，凝胶强度最大。因此，在同样的温度下，凝胶形成速度为GP>NaHCO₃>Na₃PO₄，凝胶强度为GP>NaHCO₃>Na₃PO₄。 4.采用直接添加羟基磷灰石(HA)于凝胶溶液和原位生成HA于凝胶溶液两种方法，制备了HA纳米粒子与CS/PVA的复合凝胶。HA的生物复合提高了可注射型温敏性CS/PVA凝胶的力学强度和生物活性。40℃时，纯CS/PVA凝胶、直接添加HA的CS/PVA凝胶和原位生成HA的CS/PVA凝胶的储能模量G'分别为30，130和550Pa。可见原位形成HA的CS/PVA凝胶强度有了很大提高。并且不同含量HA的CS/PVA凝胶强度也不同。对于直接添加HA的凝胶，当HAS 0.1mM，凝胶强度最大；HA含量提高到0.2mM时，凝胶强度没有提高，过多的粒子对凝胶化起了阻碍作用。对于原位形成HA的CS/PVA凝胶，凝胶强度随着HA含量的提高而增强，主要由于部分PO₄³⁻与CS发生静电吸引而导致凝胶强度的提高。含HA的CS/PVA纳米复合凝胶用作药物缓释载体时，克服了单纯CS/PVA凝胶容易发生的药物突释现象。纯CS/PVA凝胶，药物在开始的5h内出现突释，缓释量达60%，并由于凝胶支架的崩解，24h内缓释完毕。对于直接添加HA的CS/PVA凝胶，药物在开始的5h内仍出现突释，缓释量为50%，但缓释时间延长。原位形成的CS/PVA凝胶，在开始的5h内缓释仅为15%。可见药物缓释速率与凝胶强度有关，凝胶强度越大，缓释速率越慢。 5.通过-NH₃⁺和-COO⁻的静电交联作用，制备了羧甲基-季铵盐壳聚糖纳米粒子。调控-NH₃⁺和-COO⁻的摩尔比例，能够制备表面带不同电荷的纳米粒子，适宜于相反电荷药物的负载。采用普奈洛尔和双氯芬酸钠两种不同电荷的小分子作为模型药物，将纳米粒子与可注射型温敏性CS/PVA凝胶原位复合用作药物缓释载体。对于两种不同电荷的药物，空白凝胶药物缓释速度都是最快的；而对于负电荷的药物，含正电荷纳米粒子凝胶的缓释速度最慢；同样对于正电荷的药物，含负电荷纳米粒子凝胶的缓释速度最慢。说明凝胶纳米粒子的添加有利于减慢药物缓释速度。由于纳米粒子的添加并没有提高凝胶的强度，药物缓释速度的减少是因为不同电荷的纳米粒子能够和相反电荷的药物产生静电引力，阻碍了药物的突释；纳米粒子填充在凝胶的孔径内，也增加了药物渗出的难度，延长药物缓释时间。 6.制备了 NaHCO₃-CS/PVA、Na₃PO₄-CS/MC 和 NaHCO₃-CS/MC 三种凝胶支架，用于软骨细胞的三维培养。细胞主要以球形于支架表面或合适的凝胶孔洞里粘附生长。Na₃PO₄-CS/MC凝胶支架具有最合适的凝胶孔径，大孔孔径约为300um左右，小孔孔径约在20-100um之间，有利于细胞的长入；孔与孔之间相互贯通，孔隙率达到90%以上，有利于营养物质的运输和废物的排出，因此软骨细胞在支架上大量增殖，适宜于用作组织工程中细胞培养的三维支架。 关键词：可注射；温度敏感；水凝胶；壳聚糖；纳米粒子；药物释放；组织工程

 Hydrogels that are hydrophilic three-dimensional polymeric networks capable of absorbing large quantities of water have become increasingly important in the biomedical field. The intelligent gels exhibit phase transitions (i.e. volume change) in response to changes in external conditions such as pH, ionic strength, temperature and electric currents. One of the recent trends in intelligent gels research is injectable in situ-forming systems that can be introduced into the body in a minimally invasive manner for various biomedical applications, including drug delivery and tissue engineering. The injectable systems do not require a surgical procedure for placement (and withdrawal if not biodegradable), and various therapeutic agents can be incorporated by simple mixing. When they are used to fill a cavity or a defect, their flowing nature enables a good fit. The injectable systems are liquid aqueous solutions before administration, but gel under physiological conditions. There are several possible mechanisms that lead to in situ gel formation: solvent exchange, UV-irradiation, ionic cross-linkage, pH change, and temperature modulation. The temperature responsive hydrogels are triggered by changes in environmental temperature thus resulting in in situ hydrogel formation. They do not require organic solvents, copolymerization agents, or an externally applied trigger for gelation, which have caused the interest of many investigators for biomedical applications. Chitosan, derived from the second greatest biomass resource, has special mechanical, processable and chemical reactive activities, and is applied widely in chemical engineering, environment, agriculture et al. In recent years, chitosan receives great attention in the field of biology and medicine owing to its good biocompatibility, biodegradability, nontoxicity and bioactivities, such as antibacterial, antitumor. Developing all kinds of advanced functional materials and biomaterials based on chitosan, is the necessary direction of chitosan study and application in future. In general, chitosan is a pH-dependent cationic polymer, which is soluble in water up to pH 6.2. Basification of chitosan aqueous solutions above this pH leads to the formation of a hydrated gel-like precipitate. Phase separation ensues from the neutralization of chitosan amine groups and the consequent elimination of repulsive interchain electrostatic forces, which subsequently allow for extensive hydrogen bonding and hydrophobic interactions between chains. The above progress has no relation to the temperature. However, pH-gelling, cationic chitosan solutions have been transformed into thermally sensitive, pH dependent, gel-forming systems by the complexes、derivations and grafting in recent years. These formulations possess a neutral pH, remain liquid at or below room temperature, and form monolithic gels at body temperature. In this dissertation, the biochemistry properties and application of the injectable and thermosensitive hydrogels based on biodegradable chitosan are reviewed. A serious of chitosan-based hydrogles has been prepared according to molecular design. The prepared complexes hydrogels and nanocomposite gels, which are thermosensitive, have been used as drugs sustain release carriers and three-dimensional scaffolds for chondrocytes culture. The physical chemistry properties of hydrogels were characterized by FT-IR, CLSM, DLS, TEM, SEM, XRD and ARES. We investigated a series of factors affecting gel formation and thermosensitive properties. Effect of molecular structure of chitosan on hydrogels properties was investigated. The drug release and scaffolds behavior of the hydrogels were studied. It provides scientific data for modulating the application of chitosan-based hydrogels. The main contents and conclusions are summarized as below: 1.The injectable and thermosensitive chitosan/glycerin hydrogel was prepared. The contents of basic salts, glycerin and chitosan had an important effect on the gel formation and gel structure. The different content of basic salts and glycerin used in the system resulted in the difference of gelation temperature and time. With an increase of chitosan content, the gel time decreased and gel intensity was enhanced. But it didn't obviously change the size of gel pores. The higher molecular weight of chitosan was, the greater the gel pores was. When the molecular weight of chitosan was reduced, it was necessary to improve the concentration of chitosan solution in order to form the gel. At the same time, the gelation time was increased and the size of gel pores was reduced. When the molecular weight was lower to 1×10⁴, the gel didn't form. When bovine serum albumin(BSA) was directly added to chitosan solution(200㎎/10mlHCl, M〓=2.7×10⁵) and the formed gel was treated by alginate solution, the delivery time of BSA was the longest(about 30d) in phosphate buffer solution(PBS, PH 7.4). Additionally, the different conditions of delivery systems also made great effect on the properties of protein delivery, such as pH and ionic strength. In general, the formation and treatment progress of gels didn't destroy the activities of protein delivery and increased drug release, which can be used as protein drug delivery. 2.Through the mixture of chitosan (CS), poly (vinyl alcohol) (PVA) and sodium bicarbonate, the thermosensitive complex hydrogel was obtained. The mixture was liquid aqueous solutions at low temperature (about 4℃), but a gel under physiological conditions. The effect of hydrogel composition and temperature on both the gel process and the gel strength was investigated by rheological analysis. The increase of PVA content resulted in the increase of the gelation temperature. The gelation temperatures were 21, 26 and 34℃ for the hydrogels containing 1, 2.5 and 5wt% PVA, respectively. The higher the temperatures, the faster the formation speed of the gel. With an increase of CS content, the gel time decreased and gel intensity was enhanced. However, for an increase of PVA content, the phenomenon was the reverse. The lower molecular weight or DA of chitosan also result the decrease of gel intensity. The hydrogel interior morphology as well as porosity of structure was evaluated by SEM and CLSM, from which possible hydrogel formation mechanisms were inferred. The hydrophobic interactions are assumed to be the main driving force to form the gel among chitosan chains (pH ＞ 6.2) at high temperature. In addition, the potential of the hydrogels as vehicles for delivering bovine serum albumin (BSA) were also examined. In this study, the physically crosslinked chitosan/PVA gel was prepared under mild conditions without organic solvent, high temperature or harsh pH, which was very suitable for local and sustained delivery of proteins, cell encapsulation and tissue engineering. 3.Under mild conditions without organic solvent, high temperature or harsh pH, the thermosensitive hydrogels were firstly prepared through the mixture of chitosan (CS), methylcellulose (MC) and different salts. The mixtures were liquid at low temperature (about 4℃), but gels under physiological conditions (37℃). The effect of different salts including NaCl, Na₃PO₄, NaHCO₃ and glycerophosphate (GP) on the CS/MC gel process was firstly investigated by rheological analysis from which possible gelation mechanisms were inferred. The viscoelastic properties, as investigated rheologically, indicated that the gels formed with different salts had different gel temperature, strength and speed. For NaCl, the strong gel was mainly formed by the hydrophobic effects of MC chains above 50℃ and it was reversible. For Na₃PO₄ and NaHCO₃, the gel structure was mainly formed by the hydrophobic effects of CS chains and the MC chains only entangled with CS chains possibly by the weak hydrogen-bonding interactions because the MC solution couldn't form gel singly at 37℃, For GP, the CS and MC chains could form gel by the hydrophobic effects simultaneity at 37℃. The gel formation speed was GP＞NaHCO₃＞Na₃PO₄ and the gel strength was GP＞NaHCO₃＞Na₃PO₄ at 37℃. The hydrogels were also characterized by IR, SEM, XRD and swelling. The results showed the gels with different salts had different gel structures, which were from nonporous to microporous. 4.The synthesis and characterization of a thermosensitive chitosan (CS) /poly(vinyl alcohol) (PVA) hydrogel containing hydroxyapatite (HA) for drug delivery were firstly reported. Two synthetic processes were introduced, i.e. in situ and ex situ routes. The gel formation decreased the crustallinity of HA crystal. FTIR results showed that the HA crystals were partially substituted by carbonate. It was supposed the structural features of HA were close to those of biological apatites. The thermosensitive property of the gels was evaluated by rheological ananlysis, which indicated that the strength of HA-CS/PVA composite gels was notably enhanced in comparison with that of pure CS/PVA gel, especially for HA-CS/PVA composite gels synthesized through in situ process. The performances of composite gels containing different amounts of HA were further studied. For the HA-CS/PVA composite gels containing 0.1 mM HA synthesized through in situ process, the swelling ratio was the lowest and the speed of drug release was the slowest. 5.Through the electrostatic effect of -NH₃⁺ and -COO⁻, the nanoparticles of carboxymethyl- quatemized chitosan were prepared. The nanoparticles with different charges were obtained by the different ratio of -NH₃⁺ and -COO⁻, which was suitable for drug delivery with opposite charges. The composites of nanoparticels and CS/PVA hydrgels were used to model drug release (propranolol and diclofenac sodium). For two drugs, the releases of control hydrogels were both the fastest; for the positive drug, the release of hydrogels containing negative nanoparticles was the slowest; for the negative drug, the release of hydrogels containing positive nanoparticles was the slowest It indicated the addition of nanoparticles was helpful to slow the release. Though the nanoparticles didn't reinforce the gel strength, the electrostatic effect between nanoparticles and drugs reduced the obvious release. 6.The properties of three scaffolds used as chondrocytes culture (NaHCO₃-CS/PVA、Na₃PO₄-CS/MC and NaHCO₃-CS/MC) were investigated. The cells were adhered on the scaffolds surface and gel pores by sphere. The Na₃PO₄-CS/MC scaffolds had the suitable gel pores, which were helpful to cell growth, the nutrition transport and the waste eduction. Therefore, the chondrocytes were largely proliferated on the scaffolds. The main creative ideas are listed as following: 1.The injectable and thermosensitive hydrogels are normally prepared by synthetical polymers. The synthetical hydrogels are easy to control the gel structures and properties, but they are non-biodegradable and toxic. For natural polymers, they are abundant, biocompatible and biodegradable. However, the strength of natural hydrogels is too weak to satisfy the biomaterial applications. In this dissertation, the complexes of chitosan and hydroxyl polymers were firstly synthesized. The novel hydrogels improve the gel strength and are suitable to be used as biomaterials, which avoid the toxic, non-biodegradable and high gel temperature of other thermosensitive materials. 2.A series of chitosan-based injectable and thermosensitive hydrogels were prepared under neutral conditions without organic solvent, high temperature or harsh pH. The systems will be a solution that is a injectable liquid at ambient temperature and gel at body temperature. Moreover, loading with drugs or cells should be achieved by simple mixing. When administered parenterally, the system should exhibit a pH close to neutrality and should be biodegradable. 3.The effect of hydroxyl polymers on the gel structure and properties was investigated from which possible hydrogel formation mechanisms were inferred. The high temperature could reduce the intermolecular hydrogen bonding interactions between hydroxyl polymers and chitosan. It also accelerated the mobility of chitosan molecules. So the energized water molecules surrounding the chitosan chains were removed. The dewatered hydrophobic chitosan chains associated with each other. As a result, a gel is formed. The increase of hydroxyl polymers content resulted in the increase of the gelation temperature, which is corresponding to the gelation mechanism. The higher hydroxyl polymers content could produce the more hydrogen-bonding interactions, which needed the higher gelation temperature to reduce the hydrogen bonds and promote the gel formation. 4.The novel HA-CS/PVA composite gels with complicated porous structure were firstly synthesized. The two composite methods were introduced, i.e. ex situ and in situ routs. The composites were liquid aqueous solutions at low temperature (about 4℃), but gels under physiological conditions. The rheological analysis indicated that the gel strength was notably enhanced by the introduction of HA particles, especially HA synthesized via in situ process. It was because that these CS chains could be associated weakly by the PO₄³⁻, which resulted in increasing the gelation speed and enhancing the gel strength. The swelling ratio of HA/CS-PVA composite gels in situ containing about 0.1 mM HA was the lowest and the speed of drug release was the slowest. The burst drug release from it was avoided in comparison with the pure CS/PVA gel. Therefore, the novel HA-CS/PVA composite gels are attractive for applications as drug delivery, artificial bones and scaffolds for tissue engineering. Keywords: Injectable; Thermosensitivity; Hydrogel; Chitosan; Nanoparticles; Drug Delivery; Tissue Engineering