摘要
组织工程支架材料是当前的热点研究方向,尤其是采用静电纺丝技术制备的纤维膜在组织工程领域得到广泛的应用。本学位论文以可生物降解的L-聚乳酸(PLLA)为研究对象,采用共聚、共混、复合的方法对PLLA进行改性,研究其静电纺丝工艺及其纤维膜的结构与性能,并在纤维膜表面进行硫酸软骨素、壳聚糖的静电自组装以提高纤维膜的亲水性、扩大其在生物医学等领域的应用。论文的研究内容和主要结果如下：
(1)通过偶联反应,以羟基封端的L-聚乳酸(PLLA-diols)为硬段、聚己内酯(PCL)或聚乙二醇(PEG)为软段合成了PLLA-b-PCL、PLLA-b-PEG嵌段共聚物,采用静电纺丝技术制备了共聚物纤维膜。研究了共聚组分对PLLA结晶性能、电纺纤维的形貌、纤维膜力学性能的影响。结果表明在共聚物中结晶相主要是PLLA相,且共聚物中PLLA的结晶度随着PLLA含量增加而增加；共聚物电纺纤维的直径随着PCL、PEG组分含量的增加及溶液浓度的降低而减小；在保持较高强度和杨氏模量的同时,PCL和PEG提高了共聚物纤维膜的断裂伸长率。
(2)采用溶液静电纺丝工艺制备了PLLA/PCL共混物纤维膜,考察了影响纤维形貌的因素,研究了纤维膜的孔隙率、结晶性、力学性能和生物相容性。在PLLA/PCL质量比1/1、溶液浓度为10wt%、电压为20kV、接收距离为15cm的条件下得到最佳形貌的纤维；随着PCL含量增加,共混纤维膜的模量和拉伸强度逐渐降低、而断裂伸长率增加,高的孔隙率和良好的力学性能使其具备成为组织工程支架的条件。倒置相差显微镜(IPCM)和SEM结果表明脂肪源干细胞(ADSCs)在PLLA/PCL纤维膜支架上生长的数量随着种植时间的增加而增加,形成沿着支架三维网络方向生长的结构。MTT实验结果证明共混纤维膜支架有利于ADSCs的粘附和生长,且ADSCs在支架上分别向成骨细胞和脂肪细胞诱导分化成功,进一步说明PLLA/PCL纤维膜支架可以在组织工程中替代原生ECM。
(3)采用静电纺丝工艺制备了高度取向的PLLA/PCL/F-MWNT复合纤维膜。研究了F-MWNT对纤维的形貌、孔隙率、热稳定性、结晶性、降解速率、力学性能和生物相容性的影响。结果表明F-MWNTs在纤维中沿着轴向分布,随着F-MWNTs含量增加,电纺纤维直径由于电纺溶液的电导率增加而变得更细；F-MWNTs显著提高了复合纤维膜的热稳定性和降解速率。虽然在电纺过程中,F-MWNTs妨碍了PLLA/PCL的结晶,但在冷结晶过程中,F-MWNTs起到晶核的作用促进PLLA相的结晶。1.25wt%含量的F-MWNTs对取向复合纤维同时起到增强增韧的作用。MTT实验证明PLLA/PCL/F-MWNT复合纤维膜支架有利于ADSCs的粘附,具有良好的细胞相容性。
(4)采用静电自组装技术在PLLA/PCL、PLLA/PCL/F-MWNT复合纤维膜表面组装硫酸软骨素(CS)/壳聚糖(CHS)复合膜。研究纤维膜表面修饰前后的形貌、结构和亲水性能。实验结果证实1,6-己二胺与PLLA发生胺化反应,且CS和CHS通过静电作用力吸附到纤维膜表面。随着CS/CHS组装层数的增加,纤维膜表面的亲水性也逐步得到改善。
Tissue engineering scaffolds material is the current research hotspot, especially the electrospun fibrous membranes are expected to have wide applications in tissue engineering field. In this dissertation, the biodegradable poly(L-lactide) (PLLA)was used as the research object, and it was modified with copolymerization, blending and composite. The electrospinning process, the structure and properties of the fibrous membranes were investigated. In order to improve the hydrophilic of the fibrous membranes, chondroitin sulfate (CS) and chitosan (CHS) were selected as the polyelectrolyte to deposit on the fibrous membranes substrate via the electrostatic assemble technique, which will expand their application in biomedical fields. The main contents and resuluts of this dissertation are as follows:
(1)PLLA/PCL, PLLA/PEG multiblock copolymers were synthesized from diol-terminated PLLA (PLLA-diols), as hard segments and PCL, as soft segments. The PLLA/PCL fibrous membranes were fabricated by electrospinning. The crystalline of PLLA, the morphology of the fibers and mechanical properties of the fibrous membranes were investigated. The results showed that the crystalline phase is mainly PLLA phase in these block copolymers, and high crystallinity in all copolymers with the higher PLLA ratios. The fiber diameter of copolymers decreased with decreasing solution concentration and increasing PCL, PEG content. Comparing with the electrospun PLLA membrane, the electrospun fibrous membranes of the copolymers demonstrated an enhanced elongation with still high tensile strength and Young's modulus.
(2) The electrospun PLLA/PCL blend fibers could be optimally prepared with a 1/1 ratio of PLLA/PCL blend under a solution concentration of 10 wt%, an applied voltage of 20 kV and a TCD of 15cm. The porosity, crystalline, mechanical properties and biocompatibility of the fibrous membranes were investigated. The results showed that with increasing the content of PCL, the tensile strength and Young's modulus decreased gradually but the elongation at break increased. The high porosity and large ductility characteristics of the electrospun PLLA/PCL (1/1) blend fibrous membrane have made it a strong candidate for use as tissue scaffolds. The morphology and interactions between ADSCs and PLLA/PCL blend fibrous scaffolds were examined by using IPCM and SEM. With increasing seeding time, an increasing number of cells grow along the fiber orientation direction, forming a three-dimensional network with a fibrous pattern. The results of MTT assay demonstrated that the electrospun PLLA/PCL blend fibrous scaffolds provide ADSCs with a supportive interface to survive. Furthermore, the multi-lineage differentiation of ADSCs on such scaffold indicated the prospect to replace the native ECM for tissue engineering.
(3) Aligned PLLA/PCL/F-MWNT composite fibrous membranes were fabricated with strong-acid oxidized MWNTs by electrospinning. The morphology, structure, porosity, crystalline, thermal stability, bio-degradation, mechanical properties and biocompatibility of the fibrous membranes were investigated. The results showed that F-MWNTs are oriented along the fiber axis. With the incorporation of F-MWNTs, the PLLA/PCL/F-MWNT composite fibers become thinner due to the increment in conductivity. PLLA/PCL/F-MWNT composite fibrous membranes show good thermal stability and bio-degradation. During electrospinning, F-MWNTs hinder the crystallization of PLLA/PCL blend, but during cold crystallization, the F-MWNTs act as nucleation sites to enhance the crystallization of PLLA. Moreover, the 1.25 wt% F-MWNTs toughen the electrospun PLLA/PCL blend fibrous membrane. MTT assay indicates that the electrospun PLLA/PCL/F-MWNT composite fibrous scaffolds have no toxic effect on proliferation of ADSCs.
(4) CS/CHS multiplayer were selected as the polyelectrolyte to deposit on the PLLA/PCL, PLLA/PCL/F-MWNT fibrous membranes via the electrostatic assemble technique. The results verified the chemical reaction between 1,6-diaminohexane and PLLA, and more CS/CHS adsorption on the fibrous membranes. As CS/CHS multiplayers increase, the hydrophilic of the membranes increase gradually.
引文
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