用于生物人工肝反应器的半乳糖化壳聚糖/聚已内酯复合材料的制备与性能研究
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摘要
肝组织工程主要研究内容和应用包括两大方面:即体外生物人工肝支持系统中的应用和可植入体内的细胞支架的应用。无论是体外还是体内的应用,都需要制备能长时间维持肝细胞的增殖和肝特异性功能水平的胞外基质材料。
本文首先利用水溶性碳二亚胺(EDC))和N-羟基琥珀酰亚胺(NHS)为活化剂,将乳糖酸共价连接到壳聚糖分子上,得到了含有β-半乳糖基结构的半乳糖化壳聚糖(GC)。利用碱解反应处理聚己内酯(PCL)膜,利用碱解反应引入的羧基与GC上剩余氨基的反应,将GC固定到PCL膜上,得到了可用于肝细胞培养的GC/PCL复合材料。结果表明碱解可以增加PCL膜表面的羧基密度,并且羧基密度受溶液浓度、反应时间和反应温度的影响。而碱解带来材料表面化学和物理性质的改变也引起了材料亲水性的变化,碱解PCL膜的亲水性得到了一定程度的改善。固定在膜上的GC的量也随着碱解处理时间的改变而发生变化
在此基础上,用碱解涂层的方法制备了GC/PCL复合多孔支架。首先用无规明胶粒子致孔剂法制备了PCL多孔支架,然后用碱解涂层的办法将GC固定在支架内外表面得到了GC/PCL复合支架。并对支架的微观形貌,羧基密度和GC含量变化,以及力学性能等进行了研究。
在用复合支架材料对人肝癌细胞HepG2进行体外培养的过程中,复合支架表现出了良好的细胞相容性。并在对肝细胞白蛋白分泌功能的维持上有着显著的优越性。该GC/PCL复合支架能够作为一种合适的支架材料用于生物人工肝支持系统的应用中。
Tissue engineering for liver treatment has two major applications.One is the application in extracorporeal bioartificial liver(BLA) assist system,and the other one is for liver regeneration with implantable cell-scaffold constructs.A suitable extracellular matrix which can sustain long-term hepatocytes proliferation and maintenance of liver-specific functions is essential for both applications.
In this work,a kind of galatosylated chitosan(GC) was prepared by coupling Lactobionic Acid(LA) to chitosan under the catalyzation of 1-Ethyl-3-(3-dimethyl aminopropyl) carbodiimide(EDC) and N-hydroxysuccinimide(NHS).The obtained GC having galatose moieties was characterized by infrared spectroscopy(IR), nuclear magnetic resonance(NMR) and elemental analysis,confirming the resultant molecular structure.Polycaprolactone(PCL) membrane was hydrolyzed in alkaline solution to introduce carboxyl groups which can react with GC to form a GC/PCL hybrid materials for hepatocytes culture.The results showed that hydrolysis could increase the density of carboxyl on the PCL membrane,which was controled by the concentration of solution,reaction time and reaction temperature.The hydrolysis could also change hydrophilicity of the PCL membrane due to the change of these surface chemical and physical properties.The amount of immobilized GC on the membrane could be altered by the change of hydrolysis time.
Next,a hybrid porous three-dimensional scaffold was fabricated by using the same method.The polycaprolactone(PCL) scaffold was firstly prepared by a particle leaching method,which was then integrated with galactosylated chitosan(GC) via a method of hydrolysis to obtain a hybrid GC/PCL scaffold.The micro morphology, the density of carboxyl,the GC content and mechanical property of the hybrid scaffold were analyzed as well.
In vitro culture of HepG2 cells on the hybrid scaffold demonstrated a good cell viability and long-term maintenance of liver-specific functions.All the results suggest that the GC/PCL scaffold could be a potential candidate applied for scaffolds bioreactor in bioartificial liver assist system.
本文首先利用水溶性碳二亚胺(EDC))和N-羟基琥珀酰亚胺(NHS)为活化剂,将乳糖酸共价连接到壳聚糖分子上,得到了含有β-半乳糖基结构的半乳糖化壳聚糖(GC)。利用碱解反应处理聚己内酯(PCL)膜,利用碱解反应引入的羧基与GC上剩余氨基的反应,将GC固定到PCL膜上,得到了可用于肝细胞培养的GC/PCL复合材料。结果表明碱解可以增加PCL膜表面的羧基密度,并且羧基密度受溶液浓度、反应时间和反应温度的影响。而碱解带来材料表面化学和物理性质的改变也引起了材料亲水性的变化,碱解PCL膜的亲水性得到了一定程度的改善。固定在膜上的GC的量也随着碱解处理时间的改变而发生变化
在此基础上,用碱解涂层的方法制备了GC/PCL复合多孔支架。首先用无规明胶粒子致孔剂法制备了PCL多孔支架,然后用碱解涂层的办法将GC固定在支架内外表面得到了GC/PCL复合支架。并对支架的微观形貌,羧基密度和GC含量变化,以及力学性能等进行了研究。
在用复合支架材料对人肝癌细胞HepG2进行体外培养的过程中,复合支架表现出了良好的细胞相容性。并在对肝细胞白蛋白分泌功能的维持上有着显著的优越性。该GC/PCL复合支架能够作为一种合适的支架材料用于生物人工肝支持系统的应用中。
Tissue engineering for liver treatment has two major applications.One is the application in extracorporeal bioartificial liver(BLA) assist system,and the other one is for liver regeneration with implantable cell-scaffold constructs.A suitable extracellular matrix which can sustain long-term hepatocytes proliferation and maintenance of liver-specific functions is essential for both applications.
In this work,a kind of galatosylated chitosan(GC) was prepared by coupling Lactobionic Acid(LA) to chitosan under the catalyzation of 1-Ethyl-3-(3-dimethyl aminopropyl) carbodiimide(EDC) and N-hydroxysuccinimide(NHS).The obtained GC having galatose moieties was characterized by infrared spectroscopy(IR), nuclear magnetic resonance(NMR) and elemental analysis,confirming the resultant molecular structure.Polycaprolactone(PCL) membrane was hydrolyzed in alkaline solution to introduce carboxyl groups which can react with GC to form a GC/PCL hybrid materials for hepatocytes culture.The results showed that hydrolysis could increase the density of carboxyl on the PCL membrane,which was controled by the concentration of solution,reaction time and reaction temperature.The hydrolysis could also change hydrophilicity of the PCL membrane due to the change of these surface chemical and physical properties.The amount of immobilized GC on the membrane could be altered by the change of hydrolysis time.
Next,a hybrid porous three-dimensional scaffold was fabricated by using the same method.The polycaprolactone(PCL) scaffold was firstly prepared by a particle leaching method,which was then integrated with galactosylated chitosan(GC) via a method of hydrolysis to obtain a hybrid GC/PCL scaffold.The micro morphology, the density of carboxyl,the GC content and mechanical property of the hybrid scaffold were analyzed as well.
In vitro culture of HepG2 cells on the hybrid scaffold demonstrated a good cell viability and long-term maintenance of liver-specific functions.All the results suggest that the GC/PCL scaffold could be a potential candidate applied for scaffolds bioreactor in bioartificial liver assist system.
引文
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