【作者】 马祖伟；

【导师】 沈家骢； 高长有；

【作者基本信息】 浙江大学 ， 高分子化学与物理， 2003， 博士

【摘要】 研究了聚-L-乳酸（PLLA）软骨组织工程支架的制备、改性及细胞相容性。首先以PLLA平面膜为研究模型，研究了亲水性单体在PLLA表面的接枝聚合、生物大分子在PLLA平面膜表面的化学接枝和涂层、生长因子在PLLA平面膜表面的引入。以致孔剂法和热致相分离法制备了PLLA多孔支架。最后将PLLA平面膜的表面改性技术应用于PLLA多孔支架的改性，制备了具有良好细胞相容性的软骨组织工程用可降解多孔支架。 采用过氧化氢溶液中紫外光氧化的方法在PLLA膜表面引入了大分子过氧化氢基团。采用碘量法测定了PLLA膜表面大分子过氧化氢基团的浓度，并考察了氧化时间和氧化温度的影响。随氧化时间延长，大分子过氧化氢基团的浓度先升后降。氧化温度升高能够加快氧化过程的进行。 利用光氧化后PLLA膜表面的大分子过氧化氢基团作为自由基接枝聚合的引发剂，采用紫外光引发或Fe²⁺辅助引发的方式，引发了亲水性单体甲基丙烯酸羟乙酯（HEMA）、丙烯酰胺（AAm）或甲基丙烯酸（MAA）在PLLA膜表面的接枝聚合，从而将羟基、酰氨基或羧基引入材料表面。ATR-IR和XPS测试结果证明了接枝聚合的发生。接触角测试结果表明改性后PLLA膜表面的亲水性明显提高。采用ATR-IR、XPS和比色法表征了PLLA膜表面亲水性聚合物的接枝量，结果表明接枝量随单体浓度的提高和接枝时间的延长而增大。采用SEM和SFM考察了改性前后材料表面形貌的变化，结果表明接枝改性使得材料表面的粗糙度增大。与紫外光引发接枝聚合所得到的PLLA改性膜相比，由Fe²⁺辅助引发接枝聚合所得到的PLLA改性膜表面的亲水性聚合物的分布更为均匀，表面粗糙度较低。 采用软骨细胞培养考察了接枝改性对材料细胞相容性的影响。材料表面亲水性官能团的引入没有明显提高细胞的粘附率和增殖率，但却对细胞的铺展性能有重要影响。通过改变单体浓度和接枝时间，制备了一系列不同接枝量的PLLA-g-PAAm和PLLA-g-PMAA平面膜，并考察了材料表面不同的官能团以及材料表面的亲水性对细胞铺展性能的影响。结果表明细胞只有在适度亲水性的表面才有良好的铺展性能，呈多角状；而在较高疏水性或较高亲水性的表面细胞主要呈球形并团聚成细胞团。官能团的种类以及表面电荷对细胞的铺展性也有重要影响。 为了将生物大分子化学接枝于PLLA膜表面，首先在PLLA膜表面接枝PMAA，将羧基引入PLLA表面，利用水溶性碳化二亚胺EDAC作为缩合剂，与含有氨基的生物大分子反应，可将生物大分子如明胶、胶原和壳聚糖化学接枝在PLLA膜表面。ATR-IR、XPS和茚三酮测试结果均证明了接枝的发生，但也表明一生物大分子的接枝量较小(q卜g／cm勺。为了提高材料表面生物大分子的含量，在化学接枝生物大分子后的PLLA表面进一步涂层生物大分子溶液。采用PBS中浸洗的方法研究了生物大分子涂层的稳定性，结果表明胶原涂层和壳聚糖涂层具有良好的稳定性。软骨细胞培养结果表明明胶涂层或胶原涂层可明显提高细胞在PLLA膜表面的粘附率、增殖率、细胞活性和铺展性能。进一步利用“接枝－涂层”技术，将含有生长因子BMP或bFGF的胶原溶液涂层在PLLA膜表面，制备了含有生长因子的活性PLLA材料。软骨细胞培养结果表明BMP可明显提高单个细胞的MTT活性。bFGF虽然能够大大提高单个细胞的MTT活性，但不利于细胞的增殖。 分别利用腊球滤出法和热致相分离法制备了具有良好孔连通性的PLLA多孔支架材料。对于腊球滤出法，研究了腊球直径、腊球用量和PLLA溶液浓度对多孔支架结构的影响。对于热致相分离法，研究了PLLA溶液浓度、溶剂组成、粗化时间、粗化温度对多孔支架结构的影响。通过控制适当的制备条件，可得到符合组织工程所要求的孔径在150上50仰，孔隙率大于90％的多孔支架。 利用抽真空然后恢复常压的办法将液体导入多孔支架内部，利用离心的办法将液体从支架内部去除，上述简单技术解抉了对多孔支架改性时的物质扩散难的问题，使得将PLLA平面膜的改性技术应用于PLLA多孔支架的改性成为可能。研究了采用h”辅助引发的方式在PLLA多孔支架内部表面接枝PAin和PMAA，与空白PLLA多孔支架相比，细胞在改性后的支架内部铺展良好、分布均匀。研究了明胶和胶原在多孔支架内部的接枝和涂层，利用苟三酮法考察了多孔支架中的胶原或明胶的含量及其稳定性，结果表明多孔支架中的明胶和胶原涂层具有良好的稳定性。细胞在涂层明胶或胶原的支架内部有优良的铺展性能。利用“接枝－涂层”技术将含有BMP或bFGF的胶原涂层在多孔支架内部，制备了含有生长因子的活性多孔支架，活性支架中的细胞铺展良好，分布均匀，并有较高的MTT活性。利用激光共聚焦显微镜，研究了软骨细胞在多孔支架中的分布情况，在热致相分离法所制备的支架中，软骨细胞主要存在于孔的内壁表面，而在腊球法所制备的多孔支架中，软骨细胞不仅存在于孔壁表面，还大量存在于孔壁内部的微孔之中。更多还原

【Abstract】 The preparation, modification, and cytocompatibility of poly-L-lactic acid (PLLA) scaffold for cartilage tissue engineering were studied. First, hydrophilic polymers were grafted on the PLLA membranes. Biomacromolecules were then grafted and coated on the PLLA membrane. Growth factors containing PLLA membranes were also prepared. Finally, these technologies were used to modify the 3-D PLLA scaffolds, which were prepared either by paraffin sphere leaching method or thermal induced phase separation method, to provide 3-D scaffolds with good cytocompatibility for cartilage tissue engineering.The PLLA membrane was immersed in hydrogen peroxide solution under UV-light to introduce the macromolecular hydroperoxide groups on the membrane surface. lodometry method was used to measure the content of the hydroperoxide groups on the membrane surface. The content of the hydroperoxide groups on the photooxidized PLLA membrane surface increased initially and then decreased with the photooxidization time. Under a high temperature, the process of photooxidization can be accelerated.Using the macromolecular hydroperoxide groups on the photooxidized membrane surface as initiators, three kinds of hydrophilic monomers, i.e. hydroxyethyl methacrylate (HEMA), methacrylic acid (MAA) or acrylamide (AAm) were polymerized on the PLLA membranes, under induction by either UV irradiation or Fe2+ reduction. ATR-IR and XPS spectra verified the occurrence of the grafting. Water contact angle measurement of the control and the modified PLLA membranes indicated that the hydrophilicity of the membrane was improved obviously after the grafting modification. ATR-IR, XPS and colorimetric method were used to characterize the grafting density of the hydrophilic polymers on the PLLA membranes, which increased with the monomer concentration and the grafting time. SEM and SFM were used to observe the surface morphology of the membranes. After grafting modification, the surface of the PLLA membrane became rougher than the control membrane. For the membranes modified by Fe2+/OOH initiation, the grafted hydrophilic polymers on the membranes surface has a more evenly distribution and a smoother surface morphology than that of the membranes modified by UV initiation.Chondrocyte culturing was performed to evaluate the cytocompatibility of the control and the modified PLLA membranes. The results showed that the improvement of surface hydrophilicity did not improve the cell adhesion and cell proliferation effectively, but had great influence on the cell spreading properties. Only on the surfaces with moderate hydrophilicity could the cell have a polygonal shape and spread well. On the control PLLA and the grafted PLLA membranes with high density of hydrophilic polymers the cells were round shape and aggregated with each other to form cell aggregates. However, the hydrophilicity is not the only factors affecting the cell spreading properties. Other factors such as the kind of functional groups and surface charge must be considered.Using water-soluble carbodiimide as condensation agent, biomacromolecules suchas gelatin, collagen and chitosan were covalently grafted on the PLLA-g-PMAA membranes. Solution of the biomacromolecules was further coated on the above membranes to increase the surface density of the biomacromolecules. This technology was designated "grafting and coating" method. The biomacromolecular layers on the PLLA membrane surfaces had good stability and good cytocompatibiliry. Cells spread very well on the gelatin or collagen coated membranes and had obviously improved adhesion rate, proliferation rate and MTT activity. By coating the collagen solution containing bone morphogenic protein (BMP) or basic fibroblast growth factors (bFGF) on the PLLA membrane surface via the "grafting and coating" method, bioactive PLLA membranes were prepared. Both BMP and bFGF can improve the MTT activity per cell obviously, but the bFGF inhibit the proliferation of the chondrocytes.3-D PLLA scaffolds with good interconnectivity were prepared using更多还原