丝素蛋白对PP和PET的表面改性及改性后材料表面矿化研究

发布时间：2018-06-11 20:27

  本文选题：PP + PET　； 参考：《复旦大学》2012年硕士论文

【摘要】：作为物理化学性能优异、原料来源丰富且价格低廉的塑料,聚丙烯(Polypropylene, PP)和聚对苯二甲酸乙二醇酯(Poly(ethylene terephthalate), PET)已在汽车、家电、日用品及家具、包装等国民经济和日常生活中各个领域得到广泛应用。但与大多数聚合物材料一样,PP和PET存在着大量的表面和界面问题,如表面硬度低、阻隔性差、难印刷、难粘接,特别是生物相容性差,这大大限制了其在生物医药领域的应用。目前,生物材料的研究捷径之一是对已有的物理机械性能俱佳的合成高分子材料进行表面改性,因此,若能对PP、PET的惰性表面进行生物相容性改进,我们将有望开发出以此为基体的生物材料,极大地拓展其应用空间。 蚕丝素蛋白(Silk Fibroin, SF)作为一种来源广,价格低的生物大分子,由于具备优异的生物相容性、可生物降解性、透气透氧性以及较低的炎症反应而被广泛用于生物医药领域,其在酶固定化电极、药物缓释载体和组织工程支架等的研究中表现出了优异的非免疫原性和生物相容性,能够很好地促进细胞的粘附、生长和分化。与此同时,作为脊椎动物骨骼的主要组成部分,磷酸钙(Calcium Phosphate, CP),特别是其代表羟基磷灰石(Hydroxyapatite, HAP)由于良好的生物相容性和诱导成骨能力被广泛应用于组织工程支架研究。这其中,利用交替矿化法、模拟体液矿化法和共沉淀等方法,大量的丝素蛋白／羟基磷灰石支架材料已被相继报道。 基于此,本文希望从丝素蛋白固定和羟基磷灰石矿化两方面进行PP和PET的生物相容性改进研究。丝素蛋白的固定主要采用了共价结合和溶液浸涂两种方法,并以对PP的改性为基础进行了实验方法的初步探索。PET的改性中我们对两种丝素蛋白固定方法进行了深入的探讨,全面分析了二者改性效果的异同及其产生原因。文章最后,我们在表面改性的PET上进行了羟基磷灰石的矿化,讨论了不同改性对矿化的影响及矿化后材料的生物相容性。 丝素蛋白与材料的共价结合通过其与接枝于材料表面的聚丙烯酸(Poly(acrylic acid), PAA)的相互作用来实现。为此,我们首先进行了PP表面接枝PAA的试验,分别讨论了聚合反应时间、单体浓度、聚合反应温度、等离子体处理功率和时间等对PP表面PAA接枝率的影响,并初步得到PAA接枝反应的较适宜条件：单体浓度40%,反应温度80℃,等离子体处理功率75W,等离子体处理时间90s。 我们以等离子体功率100W、处理时间120s,丙烯酸(Acrylic Acid, AA)单体浓度40%(v/v),聚合反应温度800C、时间4h的条件在PP膜表面接枝PAA(标记为PP-PAA,其中PAA接枝率GD=0.33±0.18μg/cm2),并借助偶联剂在其表面固定丝素蛋白(所得材料为PP-PAA-SF);同时利用等离子体处理后直接浸涂丝素蛋白的方法制得PP-SF材料。大鼠间骨髓间充质干细胞(Mesenchymal stem cells, MSCs)的培养实验结果表明,PP-PAA-SF由于PAA的存在引入了细胞毒性,对MSCs的生长有抑制作用,而PP-SF则促进了MSCs的生长。 为降低PAA的接枝率,在PET表面接枝PAA的实验中我们将AA浓度降至20%(v/v),得到接枝率GD=0.19±0.05μg/cm2的PET-PAA,并采取与PP表面固定SF相同的方法制取PET-PAA-SF和1PET-SF两种材料。我们利用表面衰减全反射红外光谱(ATR-FTIR)、X射线光电子能谱(XPS)、原子力显微镜(AFM)和水接触角(WCA)等对经过不同表面改性处理的PET膜进行了表征。XPS结果表明,PET-PAA-SF膜上确实建立了PAA与SF的共价连接,PET-SF由于等离子体处理在PET表面引入了羧基等极性基团,除了疏水、静电力相互作用外,SF还通过部分共价相互作用固定在PET表面。两者相比较,PET-SF表面丝蛋白的固定量多于PET-PAA-SF,这样的差异可能是由于固定SF前两者表面粗糙度(对应于比表面积)的不同造成的：AFM表征显示,等离子体处理对PET产生了明显的刻蚀,极大提高了材料表面粗糙度,而接枝PAA后由于PAA对材料表面的填平效应,得到的PET-PAA其表面粗糙度大大降低至原始PET的水平。固定SF后PET-PAA-SF与PET-SF的表面粗糙度相近,但是后者的水接触角小于前者。 在MSCs的培养中,与PP-SF和PP-PAA-SF的实验结果类似,PET-SF再次表现出优异的细胞亲和力,而PET-PAA-SF则抑制了细胞的生长。这表明相对于将丝素蛋白共价结合于接枝PAA的聚合物材料表面,等离子体处理后直接浸涂丝素蛋白是更简便易行且能显著提高材料生物相容性的改性方法。 对于经不同改性的PET无纺布于CaCl2和Na2HPO4溶液中交替矿化后的样品,扫描电子显微镜(SEM)和热失重分析(TGA)结果表明,PAA接枝和丝素蛋白固定由于引入了可以螯合钙离子的羧基等极性基团,极大提高了材料表面磷酸钙盐的沉积量,这种影响在矿化次数较少时表现明显,并随着矿化次数的增多,无机物逐渐布满基底后减弱。XRD表征结果证明这种交替矿化方法在材料表面产生的磷酸钙盐为羟基磷灰石。随后,我们对PET-SF和PET-PAA-SF膜材料进行1O个矿化循环的磷酸钙沉积制备PET-SF-10(HAP)和PET-PAA-SF-10(HAP)。MSCs培养结果表明,相对于有较好MSCs亲和力的PET-SF, PET-SF-10(HAP)由于HAP的引入生物相容性进一步提高。PET-PAA-SF-10(HAP)相对于PET-PAA-SF虽然对MSCs的粘附生长有所促进,但由于HAP引入的量大部分用于抵消PAA的细胞毒性,所以改善幅度不是很大。 至此,我们的实验为韧带组织工程的材料改性提供了很好的参考依据,即在构建两端具有成骨能力而中部有良好生物相容性的人工韧带时,可以采用等离子体处理后浸涂丝素蛋白的方法先将韧带材料表面固定一层具有良好生物相容性的丝素蛋白,而后材料两端可以利用在CaCl2和Na2HPO4溶液中交替矿化的方法引入羟基磷灰石,使其在生物体内更好的促进成骨。
[Abstract]:As a plastic with excellent physical and chemical properties, rich in raw materials and low price, polypropylene (Polypropylene, PP) and polyethylene terephthalate (Poly (ethylene terephthalate), PET) have been widely used in various fields such as automobiles, household appliances, daily necessities and furniture, packaging and other fields of daily life. PP and PET have a large number of surface and interface problems, such as low surface hardness, poor barrier property, difficult printing, difficult adhesion, especially poor biocompatibility, which greatly restricts its application in the field of biological medicine. At present, one of the shortcuts for biological materials is synthetic polymer material with good physical and Mechanical properties. The material is surface modified, so if we can improve the biocompatibility of the inert surface of PP and PET, we will be expected to develop the biomaterial based on it and greatly expand its application space.
Silk Fibroin (SF) is widely used in biological medicine because of its excellent biocompatibility, biodegradability, permeability, oxygen permeability and low inflammatory response. It has been used in the research of enzyme immobilized electric pole, drug sustained-release carrier and tissue engineering scaffold, for its excellent biocompatibility, biodegradability, permeability and low inflammatory reaction. It shows excellent non immunogenicity and biocompatibility, and can promote cell adhesion, growth and differentiation. At the same time, as the main component of vertebrate skeleton, Calcium Phosphate (CP), especially it represents hydroxyapatite (Hydroxyapatite, HAP) due to good biocompatibility and induction of osteogenesis. Capacity has been widely used in the study of tissue engineering scaffolds. Among them, a large number of silk fibroin / hydroxyapatite scaffolds have been reported in succession by means of alternation mineralization, simulated humoral mineralization and coprecipitation.
Based on this, this paper hopes to study the biocompatibility improvement of PP and PET from two aspects of silk fibroin fixation and hydroxyapatite mineralization. The immobilization of silk fibroin mainly adopts covalent binding and solution leaching two methods, and the experimental method based on the modification of PP is a preliminary exploration of the modification of the two kinds of silk fibroin in the modification of the silk fibroin. The method of protein fixation was deeply discussed, and the differences and similarities between the two modified effects were analyzed and the causes were analyzed. At the end of this article, we carried out the mineralization of hydroxyapatite on the surface modified PET, and discussed the effect of different modification on mineralization and the biocompatibility of the materials after mineralization.
The covalent binding of silk fibroin to the material is realized by its interaction with polyacrylic acid (Poly (acrylic acid) and PAA) grafted on the surface of the material. To this end, we first carried out an experiment on the grafting of PAA on the PP surface. The polymerization reaction time, monomer concentration, polymerization temperature, plasma treatment power and time were discussed respectively on the PP table. The effect of the grafting ratio of PAA on the surface was obtained, and the suitable conditions for the graft reaction of PAA were obtained: the monomer concentration was 40%, the reaction temperature was 80, the plasma treatment power was 75W, and the plasma treatment time was 90s.
We use plasma power 100W, treatment time 120s, acrylic acid (Acrylic Acid, AA) monomer concentration 40% (v/v), polymerization reaction temperature 800C, time 4H conditions on the PP membrane surface grafting PAA (labeled PP-PAA, PAA graft ratio is + 0.18 mu), and with the aid of coupling agent on the surface of silk fibroin fixed (obtained material is); PP-SF material was prepared by direct dip coating of silk fibroin after plasma treatment. The experimental results of Mesenchymal stem cells (MSCs) in rat bone marrow mesenchymal stem cells (MSCs) showed that PP-PAA-SF caused cytotoxicity in the presence of PAA and inhibited the growth of MSCs, while PP-SF promoted the growth of MSCs.
In order to reduce the grafting rate of PAA, the concentration of AA was reduced to 20% (v/v) in the experiment on the surface of PET, and the graft ratio was GD=0.19 + 0.05 g/cm2 PET-PAA, and two kinds of PET-PAA-SF and 1PET-SF materials were made with the same method as PP surface fixed SF. S), atomic force microscopy (AFM) and water contact angle (WCA) were used to characterize the PET films treated with different surface modification, and the.XPS results showed that the covalent connection between PAA and SF was established on the PET-PAA-SF film, PET-SF because the plasma treatment introduced the carboxyl group and other polar groups on the PET surface, except for hydrophobicity and static electricity interaction. A partial covalent interaction is fixed on the PET surface. Compared to the two, the fixed amount of the PET-SF surface silk protein is more than that of PET-PAA-SF. This difference may be caused by the difference of surface roughness (corresponding to the surface area) before the fixed SF. The AFM characterization shows that the plasma treatment has produced a significant etching of the PET, which greatly improved. The surface roughness of the material, and the surface roughness of the PET-PAA obtained by the grafting of PAA on the surface of the material after the grafting of PAA, is greatly reduced to the level of the original PET. After fixed SF, the surface roughness of PET-PAA-SF is similar to that of the PET-SF, but the water contact angle of the latter is less than that of the former.
In the culture of MSCs, similar to the experimental results of PP-SF and PP-PAA-SF, PET-SF showed excellent cell affinity again, while PET-PAA-SF inhibited the growth of cells. This indicates that it is easier and easier to directly dip silk fibroin in plasma at the surface of the polymer material grafted with fibroin protein. A modified method that can significantly improve the biocompatibility of materials.
The results of scanning electron microscopy (SEM) and thermal weight loss analysis (TGA) for samples of different modified PET non-woven fabrics in CaCl2 and Na2HPO4 solutions show that PAA graft and fibroin protein immobilization have greatly improved the deposition of calcium phosphate on the surface of the material by introducing the carboxyl group and other polar groups that chelate calcium ions. The effect is obvious when the number of mineralization is less, and with the increase of the number of mineralization, the.XRD characterization results show that the calcium phosphate produced by this alternating mineralization method is hydroxyapatite with the increase of the number of mineralization. Then, the calcium phosphate deposition of the 1O mineralization cycle of the PET-SF and PET-PAA-SF membrane materials is then made. The results of PET-SF-10 (HAP) and PET-PAA-SF-10 (HAP).MSCs culture showed that, as compared with PET-SF with better MSCs affinity, PET-SF-10 (HAP) was further enhanced by the introduction of HAP to the biocompatibility of HAP. Cytotoxicity, so the improvement is not very large.
At this point, our experiment provides a good reference for the material modification of the ligament tissue engineering, that is, when constructing the artificial ligaments with good biocompatibility in the middle of the two ends, we can use plasma treatment to fix the surface of the ligament material on the surface of the ligament with good biocompatibility first. The silk fibroin, which can be used in CaCl2 and Na2HPO4 solutions alternately mineralized, is introduced into the hydroxyapatite, which makes it better to promote osteogenesis in the organism.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.08

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