新型PEG亚胺衍生物共聚物改性聚乳酸材料的细胞相容性评价

发布时间：2018-05-31 14:00

本文选题：细胞相容性 + 改性聚乳酸　；参考：《重庆大学》2012年硕士论文

【摘要】：改性聚乳酸材料与单纯的PLA材料相比在理化性能及生物应用等方面具有明显的优势，因此成为组织工程材料研究的热点。本研究是将具有良好生物性能的聚乙二醇(PEG)单体引入到聚乳酸中，结合均苯四甲酸酐共聚形成了PAP单体，进一步缩合反应生成了PAP-PLA共聚物。首先，采用静态水接触角及吸水率对材料的亲/疏水性进行了研究，其次对降解产物PAP片段及PMA的细胞相容性进行了考察，同时对PAP-PLA材料膜的细胞相容性进行了评价，主要检测了细胞的粘附及铺展形态、细胞的增殖、细胞的分化及矿化等，其中细胞的粘附及铺展状态以显微镜及激光共聚焦观察，细胞增殖通过CCK-8试剂盒检测，细胞分化通过对碱性磷酸酶(ALP)的检测来考察，而细胞矿化通过茜素红法及胶原分泌量来检测。实验得出以下主要结论： ①材料的静态水接触角及吸水率的实验结果表明，PAP-PLA共聚物材料的亲水性相较于PLA材料有明显的改善，亲/疏水性能合适，而且其亲疏水性能可以通过调节嵌段的不同比例进行调解，这为材料在组织工程领域的应用奠定了基础。同时，由于PAP-PLA共聚物材料中所具有的多数结构及基团较为常见，具有良好的细胞相容性，，而聚酰亚胺由于研究较少，包括其降解产物PMA在内的细胞相容性问题都还有待于检测。 ②原代成骨细胞的培养与鉴定实验表明，获取的原代成骨细胞形态良好，细胞增殖分化活性高，且具备了矿化能力，适合进行相关细胞实验。 ③采用浸提液法直接考察了合成材料中新型嵌段PAP的细胞相容性。结果表明，使用PAP浸提不同天数的浸提液培养的细胞与对照组相比，细胞的形态、增殖、分化矿化等均表现正常，该材料表现出良好的细胞相容性。 ④同时采用浸提液法考察了合成材料的降解产物PMA的细胞相容性。即将PAP和两组不同PAP-PLA样品完全降解后可能产生的最大量降解产物PMA浸提于培养液中用于培养成骨细胞，结果表明，PAP降解后产生的PMA与对照组相比其分化活力略有下降，但增长趋势相同，而PAP-PLA样品组完全降解产生的PMA与对照组不论是形态、增殖和分化都没有明显差异。这些结果表明PAP-PLA材料降解产物中的PAP片段及PMA没有明显的细胞毒性，具有良好的细胞相容性。 ⑤对PAP-PLA材料膜上的细胞粘附及铺展形态的检测表明，过于亲水或疏水的表面都不利于细胞的粘附，其中PAPD4/20与PAPD4/25两种材料的亲疏水性适中，细胞的粘附及铺展状态较好，而PAPD4/15与PLA材料则相对较差。 ⑥对PAP-PLA材料膜上的细胞的增殖检测表明，细胞在PLA上的增殖速率低于PAPD4/20和PAPD4/25，细胞在PAP-PLA材料的增殖状况良好，其在各组材料表面上的增殖速率均呈上升趋势。 ⑦对PAP-PLA材料膜上的细胞分化和矿化实验检测表明，细胞在PAP-PLA共聚物材料上的分化、矿化及胶原分泌等都较PLA材料要好。通过以上的检测表明，PAP-PLA共聚物材料的细胞相容性良好，这为本改性聚乳酸材料及其他潜在的PAP改性材料的生物相容性评价提供了良好的借鉴。
[Abstract]:The modified poly (lactic acid) materials have obvious advantages in physical and chemical properties and biological applications compared with simple PLA materials. Therefore, it has become a hot spot in the research of tissue engineering materials. This study is to introduce PEG monomers with good biological properties into polylactic acid, and to form PAP monomers by copolymerization of benzylic anhydride. PAP-PLA copolymer was formed by step condensation reaction. First, the parent / hydrophobicity of the material was studied by the static water contact angle and water absorption. Secondly, the cell compatibility of the degradation product PAP fragment and PMA was investigated, and the cell compatibility of the PAP-PLA membrane was evaluated, and the cell adhesion and spreading morphology were mainly detected. Cell proliferation, cell differentiation and mineralization, in which cell adhesion and spreading state are observed by microscope and laser confocal, cell proliferation is detected by CCK-8 kit, cell differentiation is detected by the detection of alkaline phosphatase (ALP), and cell mineralization is detected by alizarin red method and collagen secretion. To conclude:
The experimental results of the static water contact angle and water absorption of the material show that the hydrophilic property of the PAP-PLA copolymer is obviously improved compared with the PLA material, and the hydrophobicity and hydrophobicity are suitable, and the hydrophobicity can be mediated by adjusting the different proportion of the block. This lays the foundation for the application of the material in the field of tissue engineering. Because most of the structures and groups in PAP-PLA copolymer are more common and have good cellular compatibility, the biocompatibility problems of polyimides, including their degradation product PMA, are still to be detected because of less research.
2. The culture and identification of primary osteoblasts showed that the acquired osteoblasts had good morphology, high proliferation and differentiation activity, and had the ability to mineralogate, which was suitable for the related cell experiments.
(3) the cell compatibility of the new block PAP in the synthetic material was investigated by the extraction method. The results showed that the cells cultured with PAP in different days were compared with the control group, and the cell morphology, proliferation and differentiation mineralization were all normal, and the material showed good cytocompatibility.
At the same time, the biocompatibility of the degradation product PMA of the synthetic material was examined by the leaching solution. The maximum degradation product of the PAP and two groups of different PAP-PLA samples, which may be produced after the complete degradation of the PAP-PLA samples, was extracted in the culture medium for the culture of osteoblasts. The results showed that the PMA produced after the degradation of PAP had a slight differentiation activity compared with the control group. The growth trend was the same, but the total degradation of PMA in the PAP-PLA group was not significantly different from the control group, no matter its morphology, proliferation and differentiation. These results showed that the PAP fragments and PMA in the degradation products of the PAP-PLA material had no obvious cytotoxicity, and had good cytocompatibility.
(5) the detection of cell adhesion and spreading morphology on the PAP-PLA material membrane showed that the overly hydrophilic or hydrophobic surfaces were not conducive to cell adhesion, of which the two materials of PAPD4/20 and PAPD4/25 were moderately hydrophobic, the cell adhesion and spreading state were better, while PAPD4/15 and PLA materials were relatively poor.
6. The proliferation of cells on the PAP-PLA membrane showed that the proliferation rate of the cells on PLA was lower than that of PAPD4/20 and PAPD4/25, and the proliferation of the cells in PAP-PLA material was good, and the proliferation rate of the cells on the surface of each group showed an upward trend.
The test of cell differentiation and mineralization on the PAP-PLA material membrane showed that the differentiation, mineralization and collagen secretion on the PAP-PLA copolymer were better than that of the PLA material.
The results of the above tests show that the PAP-PLA copolymer has good cytocompatibility, which provides a good reference for the biocompatibility evaluation of the modified polylactic acid materials and other potential PAP modified materials.
【学位授予单位】：重庆大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.08

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