可注射酶交联水凝胶的制备及其在软骨组织工程中的应用
文献类型：学位论文
| 作者 | 任凯旋 |
| 学位类别 | 博士 |
| 答辩日期 | 2015-04 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 中国科学院长春应用化学研究所 |
| 导师 | 李杲 ; 陈学思 |
| 关键词 | 酶交联 可注射水凝胶 软骨组织工程 仿生支架 软骨细胞 间充质干细胞 |
| 中文摘要 | 软骨组织工程的发展为关节软骨缺损的修复提供了新的思路和方法。利用软骨组织工程修复关节软骨缺损时，需要将体外培养扩增后的种子细胞，如干细胞、软骨细胞等，种植于支架材料中，从而在相关的生长因子作用下实现软骨组织的修复和重建。可注射水凝胶用作软骨组织工程支架时，不仅需要为种子细胞的生长提供支撑作用，还需要具备能够促进种子细胞增殖和分化的功能。其中，可注射酶交联水凝胶由于其形成条件温和、反应速率容易控制、具有良好的生物相容性而受到了广泛关注。酶交联水凝胶采用天然的酶作为催化剂，安全性高，不会引入有机溶剂，具有良好的生物相容性，便于包载药物、细胞和生物活性分子，不会对细胞造成损伤，且能够避免药物及生物活性分子的失活。酶交联水凝胶的反应速率容易控制，可以通过调节酶的催化活性进行有效调控，可控的反应速率能够有效地防止水凝胶的前驱体溶液的扩散。酶交联水凝胶可以进行原位注射，适用于修复形状复杂的组织缺损部位，不仅避免了预塑型支架材料难以与修复部位吻合的缺陷，还避免了植入型支架材料带来的手术创伤及并发炎症等，实现微创治疗。 本论文制备了多种可注射酶交联水凝胶，侧重研究酶交联水凝胶的性能调控及其对软骨细胞的表型、骨髓间充质干细胞的软骨向分化的影响，评价了可注射酶交联水凝胶作为3D细胞载体和软骨组织工程支架的可行性。具体研究内容和主要结论如下： （1）从酶催化苯酚类化合物的聚合出发，设计并合成了一种两端为苯酚基团的亲水性的线形聚合物TA-SA-PEG-SA-TA，对该线形聚合物的成凝胶行为和水凝胶性能进行了考察，通过改变聚合物、HRP的浓度和H2O2与苯酚基团的摩尔比，实现了对水凝胶的成凝胶时间和强度的有效调控。进一步合成了单端为苯酚基团的亲水性的线形聚合物mPEG-SA-TA，深入研究了其在HRP和H2O2的存在下的酶催化反应，结果表明，在HPR和H2O2的存在下，mPEG-SA-TA发生酶催化反应，其末端的苯酚基团能够相互反应形成二聚体或多聚体。我们推断，线形聚合物TA-SA-PEG-SA-TA采用同样的机理制备得到酶交联水凝胶，当H2O2与TA的摩尔比大于0.5时，TA-SA-PEG-SA-TA相互反应生成较多的苯酚多聚体，能够形成足够多的交联点，从而制备得到水凝胶，而当H2O2与TA的摩尔比小于0.5时，TA-SA-PEG-SA-TA上的苯酚基团虽然也能够相互反应生成部分多聚体，但产物中仍存在较多的单体，交联点数量较少，无法形成水凝胶。成功论证了两端为苯酚基团的线形聚合物的酶催化成凝胶机理，拓宽了酶交联水凝胶的材料选择范围。 （2）将酶催化的交联方式引入聚氨基酸领域，制备了一种新型的基于聚谷氨酸-g-酪胺/聚乙二醇的可注射酶交联水凝胶，水凝胶的理化性能，如机械强度、微孔结构、溶胀率等能够通过改变HRP、H2O2的浓度进行有效调节。进一步评价了水凝胶在体内外的生物相容性。细胞实验表明，该水凝胶及其浸提液具有良好的细胞相容性。用作3D细胞载体时，低浓度的H2O2制备的水凝胶具有更大的多孔结构，便于营养物质的渗透和细胞代谢产物的扩散，能够提供较大的空间以供细胞生长，更有利于细胞增殖。将水凝胶的前驱体溶液皮下注射到大鼠背部，水凝胶能够原位形成，14周后完全降解。水凝胶在降解过程中不会引起组织的病变，具有良好的生物相容性。 （3）制备了一种基于聚谷氨酸-g-酪胺的酶交联水凝胶，水凝胶的理化性能，如强度、微孔结构及体外降解性能等能够通过改变聚合物浓度进行有效调控。聚合物材料及水凝胶具有良好的细胞相容性。水凝胶能够在大鼠皮下完全降解，且降解过程中不会引起组织的病变，具有良好的生物相容性。通过改变聚合物浓度构建了不同的水凝胶微环境，选择兔的骨髓间充质干细胞（BMSCs）作为种子细胞，考察不同的水凝胶微环境对BMSCs的3D细胞形貌、增殖以及软骨向分化等的影响。结果表明，低浓度的水凝胶具有更大的多孔结构，便于营养物质的渗透和细胞代谢产物的扩散，能够提供较大的空间，促进BMSCs的铺展及增殖。在诱导培养基中培养4 周后，BMSCs在低浓度的水凝胶中形成结节，能够表达和分泌II型胶原、糖胺聚糖等软骨特异性细胞外基质，促进BMSCs向软骨分化。 （4）从模拟天然软骨细胞外基质中蛋白多糖的结构出发，设计并制备了基于类糖肽的可注射酶交联水凝胶，通过改变聚合物、HRP、H2O2浓度能够有效调控水凝胶的理化性能。聚合物材料及水凝胶具有良好的细胞相容性。水凝胶能够在大鼠皮下完全降解，且降解过程中不会引起组织的病变，具有良好的生物相容性。以类糖肽水凝胶作为仿生软骨组织工程支架材料，选用软骨细胞为种子细胞，评价了该凝胶作为软骨组织工程支架材料的可行性。结果表明，在体外3D培养和裸鼠皮下模型中，类糖肽水凝胶有利于软骨细胞的存活和增殖，促进去分化的软骨细胞在类糖肽水凝胶中3D环境中再分化，表达并分泌软骨特异性细胞外基质，在软骨组织工程中具有一定的应用潜力。 |
| 英文摘要 | Cartilage tissue engineering provides a promising approach for the regeneration of cartilage defects. This approach involves the combination of chondrocytes or progenitor cells, growth factors with three-dimensional scaffolds. Injectable hydrogels may be suitable platforms for supporting the survival, proliferation and differentiation of incorporated chondrocytes or progenitor cells and serve as scaffolds for cartilage tissue engineering, attributed to their physical properties that resemble the native ECM. Recently, increasing attention has been paid to enzymatically crosslinked hydrogels for biomedical applications due to their excellent biocompatibility, fast gelation process and tunable mechanical properties. Enzymatically crosslinked hydrogels can be formed in situ under physiological conditions with the presence of natural enzymes. Meanwhile, cells and bioactive molecules can be homogeneously incorporated in the hydrogels, avoiding the damage to cells and the inactivation of bioactive molecules. The gelation time could be adjusted effectively by varying the concentrations of the enzymes, preventing the diffusion of precursor solutions. The precursor solutions of enzymatically crosslinked hydrogels can be administrated via a minimally invasive procedure, and are able to appropriately ?ll irregular-shaped defects. In this dissertation, we have prepared several injectable enzymatically crosslinked hydrogels with the presence of horse horseradish peroxidase (HRP) and hydrogen peroxide (H2O2) and focused on the regulation of the properties of hydrogels, chondrocyte phenotype and differentiation of bone marrow mesenchymal stem cells (BMSCs). The feasibility of injectable enzymatically crosslinked hydrogels as three dimensional cell carriers and scaffolds for cartilage tissue engineering has been evaluated. The main contents and conclusions of this dissertation are summarized as follows: (1) Motivated by enzyme-catalyzed polymerization of phenol compounds, a hydrophilic linear polymer containing terminal phenol groups, TA-SA-PEG-SA-TA was designed. When the molar ratio of H2O2 to TA was greater than 0.5, the hydrogels based on the linear polymer were rapidly formed under physiological conditions with the presence of HRP and H2O2. The gelation time and storage modulus of the hydrogels were adjusted by changing the concentrations of polymer, HRP and the molar ratio of H2O2 to TA. Another hydrophilic linear polymer containing single terminal phenol group, mPEG-SA-TA, was then synthesized to clarify the enzyme-catalyzed polymerization reaction and gelation mechanism. The results demonstrated that mPEG-SA-TA formed dimers or oligomers with the presence of HRP and H2O2. Therefore, we inferred that the gelation process of TA-SA-PEG-SA-TA occurred in the similar manner. When the molar ratio of H2O2 to TA was great than 0.5, the oxidative coupling reaction of phenol moieties in TA-SA-PEG-SA-TA generated enough oligomers, resulting in the formation of intermolecular crosslinks and the formation of hydrogels. While the molar ratio of H2O2 to TA was less than 0.5, the precursor solutions didn’t undergo sol-gel transition, owing to the formation of less oligomers. (2) A kind of enzyme-mediated injectable hydrogel based on poly(L-glutamic acid) grafted with tyramine and poly(ethyleneglycol) (PLG-g-TA/PEG) was prepared. The hydrogels were rapidly formed under physiological conditions with the presence of HRP and H2O2. The gelation time could be adjusted by varying the concentrations of HRP and H2O2. The physicochemical properties of the hydrogels, including mechanical strength, swelling ratio and porous structure, were dependent on the concentrations of HRP and H2O2. The live-dead staining and cell counting kit-8 revealed that the PLG-g-TA/PEG hydrogels exhibited good cytocompatibility in vitro. The in situ formed hydrogels in the subcutaneous layer of rats persisted for up to 14 weeks and displayed acceptable biocompatibility in vivo. There |
| 语种 | 中文 |
| 公开日期 | 2016-05-03 |
| 源URL | [http://ir.ciac.jl.cn/handle/322003/64494]  |
| 专题 | 长春应用化学研究所_长春应用化学研究所知识产出_学位论文 |
| 推荐引用方式 GB/T 7714 | 任凯旋. 可注射酶交联水凝胶的制备及其在软骨组织工程中的应用[D]. 中国科学院长春应用化学研究所. 中国科学院研究生院. 2015. |
入库方式： OAI收割
来源：长春应用化学研究所
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