表面功能化聚乳酸膜的构建及其细胞相容性评价
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摘要
组织工程的核心内容之一是构建细胞与支架材料的复合体,而细胞在材料上的粘附、增殖、分化等行为主要依赖于材料的表面物理化学性质,因此不同表面性质材料的制备及其与细胞之间的相互作用就成为目前国内外研究的热点。由于材料制备方法及表面改性技术的限制,目前大多数关于材料不同表面性质对细胞行为影响的研究仍以相对较易实现功能化,但在组织工程领域应用并不广泛的材料为基底,如聚苯乙烯(PS)、石英等,而以常用的生物材料例如聚乳酸(PLA)为基底进行的研究则较少报道,而后者恰恰对组织工程的研究与开发更具有实际意义。为了探讨常用组织工程支架材料表面与细胞的相互作用,本研究以目前组织工程领域常用的PLA为基质,考察了表面具有不同物理化学性质的PLA膜对细胞生物学性能的影响,为今后研制生物相容性良好的PLA材料与细胞的复合支架探索一些理论基础和实际技能。
为了较系统的研究材料表面与细胞之间的相互作用,本研究构建了一系列表面形态和化学组成功能化的PLA膜。首先是在单模微波有机合成仪中,采用有别于传统减压封管热聚合的微波聚合方式,在常压氮气保护下合成了分子量约为4.86×10~4和10.47×10~4的PLA。其次,以此为原料,采用SNS(Solvent-Non-Solvent)技术构建了微孔孔径介于1μm—20μm的PLA膜和PS膜,并以多孔PS膜为模板,制备了表面具有微岛结构的PLA膜,微孔孔径的大小以及孔密度可通过改变溶液的浓度、非溶剂组成、环境温度和溶剂的挥发速率等方法进行控制。最后采用光氧化接枝法,以过氧化氢为光氧化剂,在紫外光辐照下成功的将丙烯酸(AA)、丙烯酰胺(AAm)、丙烯酸羟乙酯(HEA)等亲水性单体接枝到PLA膜表面,从而在PLA膜表面引入了羧基、酰胺基以及羟基等基团,接触角测定显示这些极性基团的引入改善了PLA膜的亲水性,提高了材料的表面自由能。
作为骨组织工程支架材料,不仅材料与骨细胞的相互作用非常重要,而且材料表面性质对矿化性能及胶原蛋白吸附的影响也非常关键,因此本论文首先系统的研究了材料表面性质对生物矿化及胶原蛋白吸附性能的影响。对PLA膜的诱导矿化性能的研究结果显示纯PLA膜表面由于缺少成核位点而不利于矿化产物的形成,小分子量的PLA由于其表面羧基密度大于高分子量的PLA而矿化沉积的产物相对较多;对于物理形貌功能化的表面,具有1μm微岛结构的PLA膜上有大量的结晶性矿化物产生,显示此结构的表面有利于促进矿化产物的沉积,而另外三种形貌的表面均未见到有明显的结晶性矿化产物形成,其中又以20μm微孔结构的表面沉积的钙磷盐最少;对比不同基团功能化的表面,所引入的三种极性基团由于带有负电荷,因此能够诱导大量的结晶性钙磷产物形成,X射线衍射(XRD)显示此产物为具有一定结晶性的羟基磷灰石(HA)。采用石英晶体微天平(QCM)技术探讨了PLA膜的分子量及表面基团对胶原蛋白吸附的影响。研究结果显示胶原蛋白的吸附基本符合Langmuir等温吸附方程,胶原蛋白在引入功能化基团的表面能够更快的达到吸附平衡,并有较强的吸附力;对比三种功能化基团表面,胶原蛋白在羧基化表面的PLA膜上的吸附量最大;原子力显微镜(AFM)观察显示在小分子量PLA上吸附的胶原蛋白较少,呈弯曲状,而在较大分子量PLA上的胶原吸附量则相对较多,部分胶原蛋白形态呈直线形;在基团功能化表面,胶原蛋白在羧基化表面呈密集的网状分布,显示羧基化的表面有利于胶原蛋白的吸附。
对成骨细胞在不同PLA膜表面的相容性评价显示,低分子量的PLA比高分子量PLA更有利于细胞粘附,但因前者的降解速率过快,对细胞的长期生长不利;表面具有微孔及微岛结构的PLA膜有利于细胞的粘附与铺展,但对细胞的增殖性能贡献不大。虽然细胞有贴壁的特性,但对直径小于20μm的微孔及微岛结构,细胞伪足均不会沿着孔壁及岛壁贴附生长,而是在微孔及微岛上方铺展;而对于不同表面基团的PLA膜,不论是细胞的粘附与铺展还是细胞的增殖性能都得到一定程度的改善,其中又以引入酰胺基的PLA膜的细胞相容性最好。
最后,本研究还创新性的探讨了不同性质PLA膜对骨髓间充质干细胞(BMSCs)的诱导分化性能的影响。结果显示在诱导剂的存在下,BMSCs在纯PLA膜上均能够正常的向成骨细胞分化,而对于不同形貌的PLA膜,1μm微岛结构的表面对BMSCs分化性能的影响与纯PLA膜相似,而另外三种形貌则不同程度的对BMSCs朝成骨细胞分化产生抑制作用;更值得注意的是,在PLA膜表面引入酰胺基不仅能够促进BMSCs的诱导分化,甚至在无诱导剂的作用下,其自身也具有一定的诱导BMSCs分化为成骨细胞的功能。
One of the central contents of tissue engineering is to fabricate a composite containing cell and scaffold.The adhesion,proliferation and differentiation functions of cells depend mainly on the physical and chemical characters of materials surface,hence the preparation of different characters surface and the interaction between surfaces and cells become the research hotspot at present.Due to the restriction of preparation method and surface modification technique,most researches of the effect of surfaces characters on cells behavior use the easily functionlized materials which aren't applied widely in tissue engineering as substrate such as polystyrene(PS) and quartz,and few report utilizes frequently-used biomaterials like polylactide acid(PLA) as substrate,however the latter has more actual significant to the investigation and exploiture of tissue engineering.In order to discuss the interaction between the surface of popular scaffolds and cells,the PLA film is used as substrate to review the effects of different surfaces on the capability of cell in this study to offer some academic basics and actual skills in tissue engineering.
In order to study the interactions between surface and cells systemically,the PLA films with different morphology and chemical compositive functionlized surfaces were prepared in this study.Firstly,the PLA with molecular weight of 4.86×10~4 and 10.47×10~4 were synthesized by microwave irradiation at natural pressure with nitrogen protection,which differed from traditional thermal polymerization.Afterwards the SNS(Solvent-Non-Solvent) technique was used to fabricate micro-pits surfaces of PLA film and PS film with different diameters between 1μm and 20μm,which could be controlled by changing the solution concentration,non-solvent, temperature and volatilization speed of solvent.The micro-pits PS film was used as template ulteriorly to fabricate micro-islands patterned PLA film.Finally the hydrophilic monomers like acrylic(AA),acrylamide(AAm) and hydroxyethyl acrylate(HEA) were grafted onto PLA surface to introduce carboxyl,amide and hydroxyl group respectively.The contact angel measurements appeared that the introduction of polar groups improved its hydrophilicity and increased its surface energy.
As the bone tissue engineering scaffold,it is important to understand not only the interaction between materials and osteocyte but also the effect of surface characters on the biomineralization and collagen adsorption abilities.The results of induced mineralization showed that pure PLA film made against mineral formed on its surface because it lacked nuclear site,correspondingly the lower molecular weight PLA appeared better mineralization ability than higher molecular weight due to its higher carboxyl density.For the different patterned surfaces,a great number of crystallization mineral was aggraded on PLA film with 1μm micro-islands,proved that this structure would stimulate mineralization,however the other three kinds of patterned surfaces had no obvious crystallization mineral formed and the least mineral was aggraded on the patterned surface with 20μm micro-pits.Compared the group functionlized surfaces,plentiful of crystallization mineral,which was proved to be hydroxyapatite(HA) by X-ray diffraction(XRD), was formed on all of the surfaces because of their negative charges.Collagen adsorption on the surfaces of different molecular weight and group functionlized PLA films was measured by quartz crystal microbalance(QCM).The results showed that the collagen adsorption on these surfaces accorded with Langmuir isothermal adsorption equation.Collagen could achieve adsorption balance faster and the adsorption force was stronger on group functionlized PLA film than pure PLA film.Furthermore the most quality of collagen was adsorbed on the carboxyl surface among three kinds of group functionlized films.The images of atomic force microscope (AFM) appeared that the quantity of adsorbed collagen was less and the conformation was curly on lower molecular weight PLA surface,however,the adsorption quantity on higher molecular weight PLA surface was more and some of them were straight.The collagen adsorbed on the carboxyl functionlized surface appeared a compact dicyto-structure,which meant that carboxyl functionlized surface benefited collagen adsorption.
The cytocompatibility of different PLA film was evaluated by osteoblast incubation in vitro. Although lower molecular weight PLA showed better adhesion for osteobalst than higher molecular weight,it restrained osteoblast extended growth because of its faster degradation.The PLA film with micro-patterned surface offered a better substrate for cells adhesion and spread, however,it contributed less for cells proliferation.In spite of having "adherence" ability,the pseudopodium of cells only spread on the top surface of PLA film and couldn't grow along the curvature walls of pits and islands.Both cells adhesion and proliferation abilities were improved on the group functionlized surfaces,and the best cytocompatibility surface was the amide functionlized film.
Lastly,the effect of surface characters on the differentiation of bone mesenchymal stem cells (BMSCs) was investigated innovatively in this study.BMSCs could differentiate normally towards osteoblast direction on pure PLA film in existence of revulsant.The PLA film with 1μm micro-islands showed a same trend with pure PLA film to affect BMSCs differentiation,whereas the other morphologies restrained its differentiation;The introduction of amide could accelerate BMSCs differentiating to wards osteoblast on PLA film,it also had a weak ability to induce BMSCs differentiation in a certain extend without revulsant.
为了较系统的研究材料表面与细胞之间的相互作用,本研究构建了一系列表面形态和化学组成功能化的PLA膜。首先是在单模微波有机合成仪中,采用有别于传统减压封管热聚合的微波聚合方式,在常压氮气保护下合成了分子量约为4.86×10~4和10.47×10~4的PLA。其次,以此为原料,采用SNS(Solvent-Non-Solvent)技术构建了微孔孔径介于1μm—20μm的PLA膜和PS膜,并以多孔PS膜为模板,制备了表面具有微岛结构的PLA膜,微孔孔径的大小以及孔密度可通过改变溶液的浓度、非溶剂组成、环境温度和溶剂的挥发速率等方法进行控制。最后采用光氧化接枝法,以过氧化氢为光氧化剂,在紫外光辐照下成功的将丙烯酸(AA)、丙烯酰胺(AAm)、丙烯酸羟乙酯(HEA)等亲水性单体接枝到PLA膜表面,从而在PLA膜表面引入了羧基、酰胺基以及羟基等基团,接触角测定显示这些极性基团的引入改善了PLA膜的亲水性,提高了材料的表面自由能。
作为骨组织工程支架材料,不仅材料与骨细胞的相互作用非常重要,而且材料表面性质对矿化性能及胶原蛋白吸附的影响也非常关键,因此本论文首先系统的研究了材料表面性质对生物矿化及胶原蛋白吸附性能的影响。对PLA膜的诱导矿化性能的研究结果显示纯PLA膜表面由于缺少成核位点而不利于矿化产物的形成,小分子量的PLA由于其表面羧基密度大于高分子量的PLA而矿化沉积的产物相对较多;对于物理形貌功能化的表面,具有1μm微岛结构的PLA膜上有大量的结晶性矿化物产生,显示此结构的表面有利于促进矿化产物的沉积,而另外三种形貌的表面均未见到有明显的结晶性矿化产物形成,其中又以20μm微孔结构的表面沉积的钙磷盐最少;对比不同基团功能化的表面,所引入的三种极性基团由于带有负电荷,因此能够诱导大量的结晶性钙磷产物形成,X射线衍射(XRD)显示此产物为具有一定结晶性的羟基磷灰石(HA)。采用石英晶体微天平(QCM)技术探讨了PLA膜的分子量及表面基团对胶原蛋白吸附的影响。研究结果显示胶原蛋白的吸附基本符合Langmuir等温吸附方程,胶原蛋白在引入功能化基团的表面能够更快的达到吸附平衡,并有较强的吸附力;对比三种功能化基团表面,胶原蛋白在羧基化表面的PLA膜上的吸附量最大;原子力显微镜(AFM)观察显示在小分子量PLA上吸附的胶原蛋白较少,呈弯曲状,而在较大分子量PLA上的胶原吸附量则相对较多,部分胶原蛋白形态呈直线形;在基团功能化表面,胶原蛋白在羧基化表面呈密集的网状分布,显示羧基化的表面有利于胶原蛋白的吸附。
对成骨细胞在不同PLA膜表面的相容性评价显示,低分子量的PLA比高分子量PLA更有利于细胞粘附,但因前者的降解速率过快,对细胞的长期生长不利;表面具有微孔及微岛结构的PLA膜有利于细胞的粘附与铺展,但对细胞的增殖性能贡献不大。虽然细胞有贴壁的特性,但对直径小于20μm的微孔及微岛结构,细胞伪足均不会沿着孔壁及岛壁贴附生长,而是在微孔及微岛上方铺展;而对于不同表面基团的PLA膜,不论是细胞的粘附与铺展还是细胞的增殖性能都得到一定程度的改善,其中又以引入酰胺基的PLA膜的细胞相容性最好。
最后,本研究还创新性的探讨了不同性质PLA膜对骨髓间充质干细胞(BMSCs)的诱导分化性能的影响。结果显示在诱导剂的存在下,BMSCs在纯PLA膜上均能够正常的向成骨细胞分化,而对于不同形貌的PLA膜,1μm微岛结构的表面对BMSCs分化性能的影响与纯PLA膜相似,而另外三种形貌则不同程度的对BMSCs朝成骨细胞分化产生抑制作用;更值得注意的是,在PLA膜表面引入酰胺基不仅能够促进BMSCs的诱导分化,甚至在无诱导剂的作用下,其自身也具有一定的诱导BMSCs分化为成骨细胞的功能。
One of the central contents of tissue engineering is to fabricate a composite containing cell and scaffold.The adhesion,proliferation and differentiation functions of cells depend mainly on the physical and chemical characters of materials surface,hence the preparation of different characters surface and the interaction between surfaces and cells become the research hotspot at present.Due to the restriction of preparation method and surface modification technique,most researches of the effect of surfaces characters on cells behavior use the easily functionlized materials which aren't applied widely in tissue engineering as substrate such as polystyrene(PS) and quartz,and few report utilizes frequently-used biomaterials like polylactide acid(PLA) as substrate,however the latter has more actual significant to the investigation and exploiture of tissue engineering.In order to discuss the interaction between the surface of popular scaffolds and cells,the PLA film is used as substrate to review the effects of different surfaces on the capability of cell in this study to offer some academic basics and actual skills in tissue engineering.
In order to study the interactions between surface and cells systemically,the PLA films with different morphology and chemical compositive functionlized surfaces were prepared in this study.Firstly,the PLA with molecular weight of 4.86×10~4 and 10.47×10~4 were synthesized by microwave irradiation at natural pressure with nitrogen protection,which differed from traditional thermal polymerization.Afterwards the SNS(Solvent-Non-Solvent) technique was used to fabricate micro-pits surfaces of PLA film and PS film with different diameters between 1μm and 20μm,which could be controlled by changing the solution concentration,non-solvent, temperature and volatilization speed of solvent.The micro-pits PS film was used as template ulteriorly to fabricate micro-islands patterned PLA film.Finally the hydrophilic monomers like acrylic(AA),acrylamide(AAm) and hydroxyethyl acrylate(HEA) were grafted onto PLA surface to introduce carboxyl,amide and hydroxyl group respectively.The contact angel measurements appeared that the introduction of polar groups improved its hydrophilicity and increased its surface energy.
As the bone tissue engineering scaffold,it is important to understand not only the interaction between materials and osteocyte but also the effect of surface characters on the biomineralization and collagen adsorption abilities.The results of induced mineralization showed that pure PLA film made against mineral formed on its surface because it lacked nuclear site,correspondingly the lower molecular weight PLA appeared better mineralization ability than higher molecular weight due to its higher carboxyl density.For the different patterned surfaces,a great number of crystallization mineral was aggraded on PLA film with 1μm micro-islands,proved that this structure would stimulate mineralization,however the other three kinds of patterned surfaces had no obvious crystallization mineral formed and the least mineral was aggraded on the patterned surface with 20μm micro-pits.Compared the group functionlized surfaces,plentiful of crystallization mineral,which was proved to be hydroxyapatite(HA) by X-ray diffraction(XRD), was formed on all of the surfaces because of their negative charges.Collagen adsorption on the surfaces of different molecular weight and group functionlized PLA films was measured by quartz crystal microbalance(QCM).The results showed that the collagen adsorption on these surfaces accorded with Langmuir isothermal adsorption equation.Collagen could achieve adsorption balance faster and the adsorption force was stronger on group functionlized PLA film than pure PLA film.Furthermore the most quality of collagen was adsorbed on the carboxyl surface among three kinds of group functionlized films.The images of atomic force microscope (AFM) appeared that the quantity of adsorbed collagen was less and the conformation was curly on lower molecular weight PLA surface,however,the adsorption quantity on higher molecular weight PLA surface was more and some of them were straight.The collagen adsorbed on the carboxyl functionlized surface appeared a compact dicyto-structure,which meant that carboxyl functionlized surface benefited collagen adsorption.
The cytocompatibility of different PLA film was evaluated by osteoblast incubation in vitro. Although lower molecular weight PLA showed better adhesion for osteobalst than higher molecular weight,it restrained osteoblast extended growth because of its faster degradation.The PLA film with micro-patterned surface offered a better substrate for cells adhesion and spread, however,it contributed less for cells proliferation.In spite of having "adherence" ability,the pseudopodium of cells only spread on the top surface of PLA film and couldn't grow along the curvature walls of pits and islands.Both cells adhesion and proliferation abilities were improved on the group functionlized surfaces,and the best cytocompatibility surface was the amide functionlized film.
Lastly,the effect of surface characters on the differentiation of bone mesenchymal stem cells (BMSCs) was investigated innovatively in this study.BMSCs could differentiate normally towards osteoblast direction on pure PLA film in existence of revulsant.The PLA film with 1μm micro-islands showed a same trend with pure PLA film to affect BMSCs differentiation,whereas the other morphologies restrained its differentiation;The introduction of amide could accelerate BMSCs differentiating to wards osteoblast on PLA film,it also had a weak ability to induce BMSCs differentiation in a certain extend without revulsant.
引文
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