温敏性微凝胶用于可注射三维细胞支架材料的研究
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摘要
本文制备了两种温敏性水凝胶细胞支架材料：
1. P(NIPAM-HEMA)微凝胶细胞支架的制备及表征以往的研究者多采用线性或枝型高分子来合成凝胶支架材料,在本文中我们尝试用高分子微球作为构建支架结构的单元。我们以N-异丙基丙烯酰胺(N-isopropyl-acrylamide/ NIPAM)、甲基丙烯酸-2-羟基乙酯(2-hydroxyethyl methacrylate/HEMA)为单体,N,N’-亚甲基双丙烯酰胺(N,N'-methylene-bis-acrylamide/BIS)为交联剂,制备了温敏性P (NIPAM-HEMA)微凝胶。该微凝胶具有良好的温度敏感性和生物相容性。粒度仪实验表明,该P(NIPAM-HEMA)微凝胶的相转变温度为29℃。然而流变实验表明,该凝胶分散液在温度高于其VPTT实仍保持稳定,说明疏水作用不能单独引发凝胶化。在加入氯化钙,即引入了离子键后,凝胶乳液在37℃下凝胶迅速凝胶化,而且增加CaCl2的浓度,凝胶水溶液的VPTT逐渐降低。SEM实验表明,该凝胶呈互穿的多孔结构,该多孔结构有利于培养物质的传递及细胞代谢物质的排泄。我们以293T细胞为模型,将单细胞分散液与浓缩后的凝胶水溶液在室温下混合,在37℃下加热形成凝胶支架,并置于培养箱中长期培养。MTT实验及荧光倒置显微镜实验表明,293T细胞在该支架材料中能正常生长增殖,但长期培养的293T细胞会变小,这是由于PNIPAM水凝胶的收缩作用导致的。
2.丙烯酸对PNIPAM微凝胶收缩作用的影响在第一个工作中,P(NIPAM-HEMA)水凝胶仍然存在不足。水凝胶中的293T细胞会变小,这是P(NIPAM--HEMA)水凝胶的收缩作用导致的,有可能会改变293T细胞的生理功能。因此,在第二个工作中,我们尝试在PNIPAM长链中引入丙烯酸基团(Acrylic acid/AAc),丙烯酸基团具有良好的亲水性及良好的生物相容性,通过丙烯酸的亲水作用使水凝胶部分亲水,从而减弱微凝胶的收缩作用。我们仍然以N-异丙基丙烯酰胺、丙烯酸(AAc)为单体,N,N’-亚甲基双(丙烯酰胺)为交联剂,合成系列不同丙烯酸含量的P(NIPAM-AAc)共聚微凝胶。在37℃下原位形成大块凝胶,并研究该系列凝胶的原位收缩现象。实验表明,AAc能有效改变PNIPAM微凝胶的相转变行为,系列P(NIPAM-AAc)大块凝胶在37℃下的收缩现象呈现出一定的规律性。我们选取了其中几个较低AAc含量的P(NIPAM-AAc)共聚微凝胶样品用于制备可注射细胞支架。将HepG2细胞与P(NIPAM-AAc)系列共聚微凝胶混合,在37℃下加热原位形成细胞支架,长期培养以观察细胞的生长情况。经系统观察,我们发现不同AAc含量的凝胶支架的细胞形态、数量明显不同,其中含量为AAc%=1%的P(NIIPAM-AAc)的细胞生长增殖现象最为明显,说明AAc%=1%的P(NIPAM-AAc)共聚微凝胶形成的细胞支架最为适合细胞生长。
In present work,two kinds of thermosensitive hydrogel scaffold materials have been faricated:
1. Injectable 3D cell scaffold built with thermal microgels In this work we try to develop a new thermal gelling injectable scaffold for 3D cell culture. Instead of using linear, branched, or grafted macromolecules, thermosensitive microgel particles or microspheres are used as building blocks for the construction of the macroscopic hydrogel scaffold. As a proof of concept, thermosensitive poly(N-isopropylacrylamide-co-2-hydroxyethylmethacrylate) (P(NIPAM-HEMA)) microgel particles were synthesized, which present a volume phase transition temperature (VPTT) at about 29 C. Rheological test shows that the concentrated P(NIPAM-HEMA) microgel dispersion is colloidally stable when heated above its VPTT, indicating hydrophobic interaction alone can not induce thermal gelation of the dispersion. In the presence of a low concentration of CaC12, however, with the introduction of additional ionic cross-linking, the microgel dispersion gelates and forms macroscopic hydrogel. Gelation temperature of the microgel dispersion decreases with increasing ionic strength. SEM observation reveals that the resultant bulky gel has an interconnected porous microstructure.293T cells, a human cell line, were encapsulated inside the hydrogel by simple mixing with the microgel dispersion at room temperature and heating to 37℃. MTT assays reveal that the cells are viable and proliferate inside the 3D scaffold.
2. Syneresis effect of thermal gelable injectable 3D cell caffold shrinkage phenomonen is popular in PNIPAM hydrogels.in this work,we try to fabricate a scaffold which would not shrink or less shrinkage.we propose to introduce acrylic acid group (AAc) into the PNIPAM chains, acrylic acid group is hydrophilic and biocompatible.the hydrophilic property of AAc can weaken the hydrophobic effect of the thermal microgels,thereby leads to the weakening of syneresis effect of the micro-gel. To study shrinkage behavior of the formed bulk gel,a serie of thermal sensitive P (NIPAM-AAc) copolymeric microgels were prepaerd with various content of acrylic acid,the microgels were then heated at 37 C to formed physical network in situ. Rheologic experiments indicate that, the induced AAc group can change the phase transition behavior of P(NIPAM-AAc) microgels. P (NIPAM-AAc) copolymeric microspheres with different AAc content were then used to form injectable 3D cell scaffold with HepG2 cells by mixed together and heated at 37℃. Through systematic observation, we found that morphology and number of the cells in the cell scaffold with different AAc content are different,the cell number in the scaffold with a content AAc%=1% is the most one,indicating that P(NIPAM-AAc) with a AAc content of 1% is most suitable one for cell proliferation.
1. P(NIPAM-HEMA)微凝胶细胞支架的制备及表征以往的研究者多采用线性或枝型高分子来合成凝胶支架材料,在本文中我们尝试用高分子微球作为构建支架结构的单元。我们以N-异丙基丙烯酰胺(N-isopropyl-acrylamide/ NIPAM)、甲基丙烯酸-2-羟基乙酯(2-hydroxyethyl methacrylate/HEMA)为单体,N,N’-亚甲基双丙烯酰胺(N,N'-methylene-bis-acrylamide/BIS)为交联剂,制备了温敏性P (NIPAM-HEMA)微凝胶。该微凝胶具有良好的温度敏感性和生物相容性。粒度仪实验表明,该P(NIPAM-HEMA)微凝胶的相转变温度为29℃。然而流变实验表明,该凝胶分散液在温度高于其VPTT实仍保持稳定,说明疏水作用不能单独引发凝胶化。在加入氯化钙,即引入了离子键后,凝胶乳液在37℃下凝胶迅速凝胶化,而且增加CaCl2的浓度,凝胶水溶液的VPTT逐渐降低。SEM实验表明,该凝胶呈互穿的多孔结构,该多孔结构有利于培养物质的传递及细胞代谢物质的排泄。我们以293T细胞为模型,将单细胞分散液与浓缩后的凝胶水溶液在室温下混合,在37℃下加热形成凝胶支架,并置于培养箱中长期培养。MTT实验及荧光倒置显微镜实验表明,293T细胞在该支架材料中能正常生长增殖,但长期培养的293T细胞会变小,这是由于PNIPAM水凝胶的收缩作用导致的。
2.丙烯酸对PNIPAM微凝胶收缩作用的影响在第一个工作中,P(NIPAM-HEMA)水凝胶仍然存在不足。水凝胶中的293T细胞会变小,这是P(NIPAM--HEMA)水凝胶的收缩作用导致的,有可能会改变293T细胞的生理功能。因此,在第二个工作中,我们尝试在PNIPAM长链中引入丙烯酸基团(Acrylic acid/AAc),丙烯酸基团具有良好的亲水性及良好的生物相容性,通过丙烯酸的亲水作用使水凝胶部分亲水,从而减弱微凝胶的收缩作用。我们仍然以N-异丙基丙烯酰胺、丙烯酸(AAc)为单体,N,N’-亚甲基双(丙烯酰胺)为交联剂,合成系列不同丙烯酸含量的P(NIPAM-AAc)共聚微凝胶。在37℃下原位形成大块凝胶,并研究该系列凝胶的原位收缩现象。实验表明,AAc能有效改变PNIPAM微凝胶的相转变行为,系列P(NIPAM-AAc)大块凝胶在37℃下的收缩现象呈现出一定的规律性。我们选取了其中几个较低AAc含量的P(NIPAM-AAc)共聚微凝胶样品用于制备可注射细胞支架。将HepG2细胞与P(NIPAM-AAc)系列共聚微凝胶混合,在37℃下加热原位形成细胞支架,长期培养以观察细胞的生长情况。经系统观察,我们发现不同AAc含量的凝胶支架的细胞形态、数量明显不同,其中含量为AAc%=1%的P(NIIPAM-AAc)的细胞生长增殖现象最为明显,说明AAc%=1%的P(NIPAM-AAc)共聚微凝胶形成的细胞支架最为适合细胞生长。
In present work,two kinds of thermosensitive hydrogel scaffold materials have been faricated:
1. Injectable 3D cell scaffold built with thermal microgels In this work we try to develop a new thermal gelling injectable scaffold for 3D cell culture. Instead of using linear, branched, or grafted macromolecules, thermosensitive microgel particles or microspheres are used as building blocks for the construction of the macroscopic hydrogel scaffold. As a proof of concept, thermosensitive poly(N-isopropylacrylamide-co-2-hydroxyethylmethacrylate) (P(NIPAM-HEMA)) microgel particles were synthesized, which present a volume phase transition temperature (VPTT) at about 29 C. Rheological test shows that the concentrated P(NIPAM-HEMA) microgel dispersion is colloidally stable when heated above its VPTT, indicating hydrophobic interaction alone can not induce thermal gelation of the dispersion. In the presence of a low concentration of CaC12, however, with the introduction of additional ionic cross-linking, the microgel dispersion gelates and forms macroscopic hydrogel. Gelation temperature of the microgel dispersion decreases with increasing ionic strength. SEM observation reveals that the resultant bulky gel has an interconnected porous microstructure.293T cells, a human cell line, were encapsulated inside the hydrogel by simple mixing with the microgel dispersion at room temperature and heating to 37℃. MTT assays reveal that the cells are viable and proliferate inside the 3D scaffold.
2. Syneresis effect of thermal gelable injectable 3D cell caffold shrinkage phenomonen is popular in PNIPAM hydrogels.in this work,we try to fabricate a scaffold which would not shrink or less shrinkage.we propose to introduce acrylic acid group (AAc) into the PNIPAM chains, acrylic acid group is hydrophilic and biocompatible.the hydrophilic property of AAc can weaken the hydrophobic effect of the thermal microgels,thereby leads to the weakening of syneresis effect of the micro-gel. To study shrinkage behavior of the formed bulk gel,a serie of thermal sensitive P (NIPAM-AAc) copolymeric microgels were prepaerd with various content of acrylic acid,the microgels were then heated at 37 C to formed physical network in situ. Rheologic experiments indicate that, the induced AAc group can change the phase transition behavior of P(NIPAM-AAc) microgels. P (NIPAM-AAc) copolymeric microspheres with different AAc content were then used to form injectable 3D cell scaffold with HepG2 cells by mixed together and heated at 37℃. Through systematic observation, we found that morphology and number of the cells in the cell scaffold with different AAc content are different,the cell number in the scaffold with a content AAc%=1% is the most one,indicating that P(NIPAM-AAc) with a AAc content of 1% is most suitable one for cell proliferation.
引文
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