表面微纳米图案化技术及细胞黏附临界面积等问题的研究
摘要
随着生物材料从生物惰性向生物活性的不断发展,细胞与材料之间的相互作用研究也越来越受到人们的关注和重视,并成为将生物材料应用于再生医学和组织工程等环节中的一个重要课题。表面图案化材料可以将复杂的生物学问题进行简化,对其进行抽丝剥茧地研究,并因其规则、可控、可调、类型多变等特点,因而被大量用来进行材料表面细胞行为及其相互作用机理的研究,同时也成为调控细胞行为的一种重要手段。生物体系是一个复杂的多尺度体系,图案化技术在微米、纳米及微纳米不同尺度或跨尺度水平的不断发展,使得图案化材料可以方便地对细胞外基质进行细胞水平和分子水平的各种体外仿生与模拟。微米图案可以实现对细胞位置、尺寸、形状等方面的精确控制,因而被率先用于细胞黏附及凋亡行为的研究,并逐渐扩展到细胞增殖、取向、迁移、分化等各个方面；纳米图案可以在纳米尺度对细胞与材料相互作用中的黏附结构进行精细控制,因此也成为调控细胞行为的一种重要手段,并在揭示一些分子或超分子水平的相互作用机理方面具有独特的优势；微纳米图案可以实现对细胞位置、尺寸、形状与细胞黏附精细结构的同时控制,对于全面、系统地研究细胞—材料相互作用问题具有独到之处。
本博士论文旨在运用表面图案化技术研究细胞黏附的一些基础科学问题,并且对现有的图案化表面技术作出改进,以制备新型的微纳米复合图案、拓展图案化技术在细胞黏附和干细胞分化等问题上的运用空间。
本文的主要创新性工作包括以下几个方面：
(1)首次提出了微米图案化表面细胞黏附具有多个临界面积的概念,并给出了获得这些基本参数的实验方法。
本文定义了七种细胞在微米图案上黏附的特征面积：细胞凋亡—黏附之间的临界面积A*或Acl、单细胞黏附到多细胞黏附的临界面积Ac2、后期每增加一个黏附细胞所需增加的面积A△、细胞黏附数为1和2时对应分布几率最大的黏附面积Apeak(1)和Apeak(2)、平均细胞黏附数为1和2所对应的黏附面积AN(1)和AN(2)。通过在具有黏附反差和黏附面积精细变化的聚乙二醇(PEG)水凝胶基微米图案化材料表面进行细胞培养,证实了所定义特征面积的存在。然后对此尺寸依赖性进行了半定量的描述,并提出了实际可行的确定方法。进而通过另两种细胞类型的培养证实了特征面积的普适性,并分析了其在细胞类型间的差异性,最后揭示了这些特征面积的普遍性规律。此部分工作对具有特定黏附数量的细胞定位研究,提供了理论分析方法和基础数据,对生物材料的设计具有指导意义,于某些相关领域如细胞芯片或生物模拟等也有积极作用。
(2)改进适于细胞研究的微纳米复合图案技术,不仅发展了nano-then-micro和micro-then-nano策略下的若干具体方法,还拓展到nanol-then-micro-then-nano2的综合策略、首次设计并成功制备了微米尺度嵌套的双间距纳米图案。
论文发展了适于细胞研究的微纳米复合图案技术,对现有微纳米复合图案中的一种micro-on-nano图案的制备技术进行了方法改进,同时提出“先微米后纳米”的制备路线,并将该类型图案应用于解决微米图案化表面细胞定位黏附与自由铺展之间的矛盾,初步实现了单细胞的铺展定位(定位+铺展,在定位的同时保持铺展的形态),丰富了微纳米复合图案在细胞—材料相互作用研究中的应用,有望提高基于单细胞黏附的研究中对细胞外基质的仿生程度。论文对于nano-then-micro和micro-then-nano的策略进行了总结。
论文进一步提出一种新的微米尺度嵌套的双纳米图案(我们称之为双间距纳米图案),这是一种nano1-then-micro-then-nano2的策略,其两种纳米阵列的纳米颗粒大小、间距、规整度等参数,具有独立可调性和可反转性,设计了“二次自组装”的技术路线,并成功制备出可反转的双间距图案；对现有的均一阵列两种颗粒尺寸的双纳米图案(简称为双点大小纳米图案)制备技术,引入巯基试剂溶液超声处理法,发展了一种纳米颗粒“减小”的制备路线,成功制备出可反转的双点大小纳米图案。这两种双纳米图案对进一步综合不同空间尺度研究细胞行为提供了重要的技术平台。
(3)首次开展了纳米图案及微纳米图案化表面的干细胞共诱导分化行为研究。
首次在纳米图案化表面进行了间充质干细胞的成骨成脂共诱导,研究了纳米间距对不同分化方向比例分配的影响作用,对于纳米图案化表面调控干细胞共分化行为具有指导意义。
首次在我们所制备的新型微纳米复合图案表面进行了间充质干细胞的共诱导。提出“利用基底微纳米特征调控成骨成脂共分化空间分布”的设想,通过共诱导证实了其可行性,并指出此中纳米间距的重要性。本工作进一步扩展了微纳米复合图案的应用,为主动调控干细胞行为提供了新的研究工具和方法,对某些与不同分化方向间比例、分布等有关的疾病问题也有一定启迪。
总之,本论文改进和拓展了现有的微纳米复合图案的制备技术,为考察和调控细胞在生物材料表面的行为提供了新工具,并丰富了微米、纳米及微纳米复合图案在细胞—材料相互作用中的基础知识和应用,对生物医用材料的设计具有一定的指导意义。
Cell-material interactions have been paid m(?)re attention with the development of biomaterials from the bioinert generation to the (?)oactive generation. The patterned surfaces have been applied to control and study c(?)llular responses to biomaterials and could draw deterministic conclusions for c mplex biological problems. Appropriate patterns are capable to mimic the extracellular matrix in vitro on cellular and molecular levels thanks for the progress of microscale and nanoscale patterning techniques. Micropatterns provide a unique tool for a precise control of the localization, spreading size and shape of cells, and nanopatterns allow a relatively fine control of adhesion complex that mediates the cell-biomaterial interactions. Micro/nano-composite patterns, which combine the features of micro-and nano-patterns, can achieve better control of cells, and yet the patterning technique needs to be developed.
Besides exploring some basic cell behaviors such as adhesion areas on micropatterned surfaces, this Ph. D thesis is aimed to improve the techniques of micro/nano composite patterns and extend the space to use patterned surfaces to study cell behaviors including differentiation of stem cells.
The main creative achievements in this thesis are as follows:
(1) We first introduced the concept of a few critical areas of cell adhesion on micropatterned surfaces and put forward the approaches to determine these critical values experimentally.
We defined seven characteristic areas of cell adhesion on micropatterned surfaces (A*, also named Ac1is the critical area from apoptosis to survival; Ac2, the critical area from single-to multi-cell adhesion; A△, the area for one more cell to adhere;Apeak(1), the area with respect to the maximum population among all microislands occupied exactly by a single cell;Apeak(2), the area with respect to the maximum population among all microislands occupied exactly by two cells; AN(1), the area of the microisland occupied by one cell on average; AN(2), the area of the microisland occupied by two cells on average), and justified their existence via cell culture on a micropatterned surface. On the surface, cell-adhesive RGD microislands were covalently linked to the non-fouling PEG hydrogels. We described semi-quantitatively the basic size-dependent behaviors of cell adhesion, and put forward the practical approaches of their determination. The concepts were justified for all of three cell lines we examined. The general relationships between these critical values were also revealed. This work provides a model surface with a preferred given number of cells localized on adhesive microislands, and seems also stimulating for design of cell chips with appropriate dimensions.
(2) We extended the techniques of fabricating patterned surfaces appropriate for studies on cells. Not only the nano-then-micro and micro-then-nano strategies were summarized with inputs of some new approaches, but also a nano1-then-micro-then-nano2strategy was suggested to prepare microscale-nested dual nanopatterns.
We improved the fabrication technique of micro-on-nano-pattern, a kind of micro/nano composite pattern, and meanwhile put forward a new route named as "micro-then-nano" method. We also applied the micro-on-nano-patterns for resolving the conflict between the controlled localization and free spreading of cells on micropatterned surface, and achieved the "spreading-localization" of single cells. This work extended the application of micro/nano composite patterns in the investigation on cell-biomaterial interactions.
We suggested a nano1-then-micro-then-nano2strategy and prepared a new type of micro/nano composite patterns named as "microscale-nested dual-spacing nanopatterns". The dotsize, spacing and regularity of the two nanoarrays could be independently tuned and conveniently reversed. We designed a fabrication route named as "double self-assembly", and successfully obtained such reversible dual-spacing nanopattern. What's more, we also introduced thiol-reagent-assisted sonication method for fabricating another nested dual nanopattern with uniform lattice but two kinks of sizes of nanodots, denoted as "dual-dotsize nanopattern", and developed a new route via a selective diminishing method, and also successfully fabricated reversible dual-dotsize nanopatterns.
(3) We first carried out the investigation on co-differentiation of mesenchymal stem cells on nanopatterned and micro/nano composite patterns.
We first co-induced mesenchymal stem cells (MSCs) towards osteogenesis and adipogenesis on nanopatterned surfaces. The effect of nanospacing on the partition ratio between two directions was found.
We also examined co-induction of MSCs on micro-on-nano-patterned surfaces. We proposed an assumption to modulate the spatial distribution of osteogenesis and adipogenesis which can be driven by micro/nano features of underlying material surface, and then verified its feasibility via co-induction of MSCs, which also revealed the importance of nano-spacing. This work further broadens the application of micro/nano composite pattern, suggests a new tool to regulate the co-differentiation behaviors of MSCs, and may provide cues for some diseases related to the balance and spatial distribution between different lineage commitments.
In summary, this Ph. D thesis has improved the fabrication technique of micro/nano composite patterns, affords new tools for modulating cell behaviors on biomaterials, shed new insights upon cell-biomaterial interactions and basic cell behaviors such as cell adhesion and differentiation on micropatterns, nanopatterns, and micro-nano composite patterns. The fundamental study might be meaningful for design of biomaterials of the new generation.
本博士论文旨在运用表面图案化技术研究细胞黏附的一些基础科学问题,并且对现有的图案化表面技术作出改进,以制备新型的微纳米复合图案、拓展图案化技术在细胞黏附和干细胞分化等问题上的运用空间。
本文的主要创新性工作包括以下几个方面：
(1)首次提出了微米图案化表面细胞黏附具有多个临界面积的概念,并给出了获得这些基本参数的实验方法。
本文定义了七种细胞在微米图案上黏附的特征面积：细胞凋亡—黏附之间的临界面积A*或Acl、单细胞黏附到多细胞黏附的临界面积Ac2、后期每增加一个黏附细胞所需增加的面积A△、细胞黏附数为1和2时对应分布几率最大的黏附面积Apeak(1)和Apeak(2)、平均细胞黏附数为1和2所对应的黏附面积AN(1)和AN(2)。通过在具有黏附反差和黏附面积精细变化的聚乙二醇(PEG)水凝胶基微米图案化材料表面进行细胞培养,证实了所定义特征面积的存在。然后对此尺寸依赖性进行了半定量的描述,并提出了实际可行的确定方法。进而通过另两种细胞类型的培养证实了特征面积的普适性,并分析了其在细胞类型间的差异性,最后揭示了这些特征面积的普遍性规律。此部分工作对具有特定黏附数量的细胞定位研究,提供了理论分析方法和基础数据,对生物材料的设计具有指导意义,于某些相关领域如细胞芯片或生物模拟等也有积极作用。
(2)改进适于细胞研究的微纳米复合图案技术,不仅发展了nano-then-micro和micro-then-nano策略下的若干具体方法,还拓展到nanol-then-micro-then-nano2的综合策略、首次设计并成功制备了微米尺度嵌套的双间距纳米图案。
论文发展了适于细胞研究的微纳米复合图案技术,对现有微纳米复合图案中的一种micro-on-nano图案的制备技术进行了方法改进,同时提出“先微米后纳米”的制备路线,并将该类型图案应用于解决微米图案化表面细胞定位黏附与自由铺展之间的矛盾,初步实现了单细胞的铺展定位(定位+铺展,在定位的同时保持铺展的形态),丰富了微纳米复合图案在细胞—材料相互作用研究中的应用,有望提高基于单细胞黏附的研究中对细胞外基质的仿生程度。论文对于nano-then-micro和micro-then-nano的策略进行了总结。
论文进一步提出一种新的微米尺度嵌套的双纳米图案(我们称之为双间距纳米图案),这是一种nano1-then-micro-then-nano2的策略,其两种纳米阵列的纳米颗粒大小、间距、规整度等参数,具有独立可调性和可反转性,设计了“二次自组装”的技术路线,并成功制备出可反转的双间距图案；对现有的均一阵列两种颗粒尺寸的双纳米图案(简称为双点大小纳米图案)制备技术,引入巯基试剂溶液超声处理法,发展了一种纳米颗粒“减小”的制备路线,成功制备出可反转的双点大小纳米图案。这两种双纳米图案对进一步综合不同空间尺度研究细胞行为提供了重要的技术平台。
(3)首次开展了纳米图案及微纳米图案化表面的干细胞共诱导分化行为研究。
首次在纳米图案化表面进行了间充质干细胞的成骨成脂共诱导,研究了纳米间距对不同分化方向比例分配的影响作用,对于纳米图案化表面调控干细胞共分化行为具有指导意义。
首次在我们所制备的新型微纳米复合图案表面进行了间充质干细胞的共诱导。提出“利用基底微纳米特征调控成骨成脂共分化空间分布”的设想,通过共诱导证实了其可行性,并指出此中纳米间距的重要性。本工作进一步扩展了微纳米复合图案的应用,为主动调控干细胞行为提供了新的研究工具和方法,对某些与不同分化方向间比例、分布等有关的疾病问题也有一定启迪。
总之,本论文改进和拓展了现有的微纳米复合图案的制备技术,为考察和调控细胞在生物材料表面的行为提供了新工具,并丰富了微米、纳米及微纳米复合图案在细胞—材料相互作用中的基础知识和应用,对生物医用材料的设计具有一定的指导意义。
Cell-material interactions have been paid m(?)re attention with the development of biomaterials from the bioinert generation to the (?)oactive generation. The patterned surfaces have been applied to control and study c(?)llular responses to biomaterials and could draw deterministic conclusions for c mplex biological problems. Appropriate patterns are capable to mimic the extracellular matrix in vitro on cellular and molecular levels thanks for the progress of microscale and nanoscale patterning techniques. Micropatterns provide a unique tool for a precise control of the localization, spreading size and shape of cells, and nanopatterns allow a relatively fine control of adhesion complex that mediates the cell-biomaterial interactions. Micro/nano-composite patterns, which combine the features of micro-and nano-patterns, can achieve better control of cells, and yet the patterning technique needs to be developed.
Besides exploring some basic cell behaviors such as adhesion areas on micropatterned surfaces, this Ph. D thesis is aimed to improve the techniques of micro/nano composite patterns and extend the space to use patterned surfaces to study cell behaviors including differentiation of stem cells.
The main creative achievements in this thesis are as follows:
(1) We first introduced the concept of a few critical areas of cell adhesion on micropatterned surfaces and put forward the approaches to determine these critical values experimentally.
We defined seven characteristic areas of cell adhesion on micropatterned surfaces (A*, also named Ac1is the critical area from apoptosis to survival; Ac2, the critical area from single-to multi-cell adhesion; A△, the area for one more cell to adhere;Apeak(1), the area with respect to the maximum population among all microislands occupied exactly by a single cell;Apeak(2), the area with respect to the maximum population among all microislands occupied exactly by two cells; AN(1), the area of the microisland occupied by one cell on average; AN(2), the area of the microisland occupied by two cells on average), and justified their existence via cell culture on a micropatterned surface. On the surface, cell-adhesive RGD microislands were covalently linked to the non-fouling PEG hydrogels. We described semi-quantitatively the basic size-dependent behaviors of cell adhesion, and put forward the practical approaches of their determination. The concepts were justified for all of three cell lines we examined. The general relationships between these critical values were also revealed. This work provides a model surface with a preferred given number of cells localized on adhesive microislands, and seems also stimulating for design of cell chips with appropriate dimensions.
(2) We extended the techniques of fabricating patterned surfaces appropriate for studies on cells. Not only the nano-then-micro and micro-then-nano strategies were summarized with inputs of some new approaches, but also a nano1-then-micro-then-nano2strategy was suggested to prepare microscale-nested dual nanopatterns.
We improved the fabrication technique of micro-on-nano-pattern, a kind of micro/nano composite pattern, and meanwhile put forward a new route named as "micro-then-nano" method. We also applied the micro-on-nano-patterns for resolving the conflict between the controlled localization and free spreading of cells on micropatterned surface, and achieved the "spreading-localization" of single cells. This work extended the application of micro/nano composite patterns in the investigation on cell-biomaterial interactions.
We suggested a nano1-then-micro-then-nano2strategy and prepared a new type of micro/nano composite patterns named as "microscale-nested dual-spacing nanopatterns". The dotsize, spacing and regularity of the two nanoarrays could be independently tuned and conveniently reversed. We designed a fabrication route named as "double self-assembly", and successfully obtained such reversible dual-spacing nanopattern. What's more, we also introduced thiol-reagent-assisted sonication method for fabricating another nested dual nanopattern with uniform lattice but two kinks of sizes of nanodots, denoted as "dual-dotsize nanopattern", and developed a new route via a selective diminishing method, and also successfully fabricated reversible dual-dotsize nanopatterns.
(3) We first carried out the investigation on co-differentiation of mesenchymal stem cells on nanopatterned and micro/nano composite patterns.
We first co-induced mesenchymal stem cells (MSCs) towards osteogenesis and adipogenesis on nanopatterned surfaces. The effect of nanospacing on the partition ratio between two directions was found.
We also examined co-induction of MSCs on micro-on-nano-patterned surfaces. We proposed an assumption to modulate the spatial distribution of osteogenesis and adipogenesis which can be driven by micro/nano features of underlying material surface, and then verified its feasibility via co-induction of MSCs, which also revealed the importance of nano-spacing. This work further broadens the application of micro/nano composite pattern, suggests a new tool to regulate the co-differentiation behaviors of MSCs, and may provide cues for some diseases related to the balance and spatial distribution between different lineage commitments.
In summary, this Ph. D thesis has improved the fabrication technique of micro/nano composite patterns, affords new tools for modulating cell behaviors on biomaterials, shed new insights upon cell-biomaterial interactions and basic cell behaviors such as cell adhesion and differentiation on micropatterns, nanopatterns, and micro-nano composite patterns. The fundamental study might be meaningful for design of biomaterials of the new generation.
引文
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