摘要
近年来随着组织工程等方面的再生医学发展,有关材料表面改性和细胞粘附等方面的问题越来越引起人们的关注。而人体组织可被视为是细胞与细胞外基质的复合体,如果能够利用仿细胞外基质的新型生物材料构筑一定的类生命环境,并在其表面或内部培植细胞,这样我们就可以在体外利用仿生手段来研究机体内部重要的生命活动。要在体外构造细胞与生物材料的复合体,就必须了解细胞与生物材料相互作用方面的诸多基本科学问题,而其中最关键的问题之一就是细胞在生物材料表面的粘附机制。在研究细胞黏附机制方面,生物材料表面的图案化处理使原本复杂的问题简单化,并为更深入地研究细胞粘附、迁移等活动机制提供了契机。原有的图案化技术大都针对硬基质或者硅橡胶等少数疏水性聚合物,而在力学性质接近人体大部分组织的水凝胶表面构筑合适的微图案则存在技术困难;另一方面,有关水凝胶的研究木身已经成为生物材料界一个活跃的领域。因此,拓展具有细胞黏附反差的水凝胶表面微图案化制备于段、建立相应技术平台就具有重要意义,也是一个富有挑战性的研究课题。
本论文发展了一种可用于水凝胶材料表面图案化修饰的光刻转移技术,以及由此技术制备出的具有良好生物学性质的Au-PEG复合图案化材料,此水凝胶材料能够区域选择性的抵抗抗细胞黏附,可用于考察细胞与生物材料相互作用的基础研究,揭示细胞与生物材料相互作用的部分基本规律,为新型生物材料的表面设计提供理论依据。
本博士论文的主要贡献可以分为以下几个部分:
1.拓展了聚合物表面制备稳定微图案的转移制备策略。本论文利用光刻技术和转移技术在高分子水凝胶表面制备微米级Au图案,成功地解决了软基底材料表面的图案化修饰问题,并彻底保证了材料的湿态稳定性。在Au图案化修饰的PEG水凝胶表面培植细胞,考察了材料的生物相容性和抗细胞黏附反差性。首先利用光刻技术在硬质基底表面制备出Au微图案,然后利用自组装技术在Au表面接上带巯基的转移试剂,进而在PEG水凝胶的聚合过程中将微图案转移到水凝胶表面,实现PEG水凝胶的Au微图案化修饰。所获得的图案即使长期浸泡在水中也具有稳定性。
2.设计了一个具有异种端基的大分子转移试剂,并克服了两个端基本身易于反应的困难成功地予以了合成。本文设计和合成一种大分子量的具有异种官能端基的转移试剂ACRL-PEG5K-SH。以ACRL-PEG5K-NHS为基础合成了大分子量的转移试剂ACRL-PEG5K-SH,并进行了GPC,IR,~1H NMR等表征。在合成反应中,采用双硫模型以拟制不希望发生的Michael-加成反应和一系列副反应,从而得到一端巯基一端丙烯酰基的功能化大分子转移试剂。进而,将其成功用于水凝胶材料的表面图案化修饰,细胞实验证明了用该转移试剂合成的图案化修饰的PEG水凝胶材料的生物相容性和抗细胞黏附差异。在具有抗细胞黏附反差和不同直径的Au点阵表面的细胞实验表明,细胞能够选择性黏附,并且随着点尺度的变化呈现出一定的规律。
3.研究了条纹状微图案表面诱导的细胞取向,并提出了系列统计参量。本论文制备了条纹状Au-PEG水凝胶复合图案化表面,用此材料表面研究了3T3成纤维细胞在金条纹表面的取向黏附行为,定义了一系列统计参数用于细胞黏附行为的研究。发现随着条纹间距的增加,细胞的取向度、长短轴比以及金条纹上的细胞占有率均逐渐增大,但单细胞的铺展面积总体呈减小趋势,并且它们随条纹间距增大而发生突然变化的区域并不完全一致;同时黏附细胞在金条纹上的有效黏附比呈现非单调变化。5个统计参数组合在一起,良好地描述了细胞在条纹表面粘附后的取向。条纹状Au-PEG水凝胶复合图案表面的利用以及相应的参量统计和比较为今后研究细胞在生物材料表面的取向行为提供了一种有效的模型化方法。
Recent development of regenerative medicine including tissue engineering triggers research of surface modification of biomaterials and cell adhesion on modified substrate. Considering that tissues could be thought as a composite of cells and extracellular matrix (ECM), ideal biomaterials for tissue regeneration are desired to mimic ECM. Development of the new-generation biomaterials requires extensive understanding of cell-material interaction. Surface-patterning techniques shed light on simplifying the understanding of fundamental investigation in the cost of complicated material fabrication. The surface patterning techniques so far are, however, limited to hard substrates or some hydrophobic rubber such as silicone; on the other hand, hydrogels as a soft matter have emerged as a hot issue in material studies. Hence, a surface-patterning technique platform for generating patterns with cell-adhesion contrast on hydrogels is called for.
This paper introduces a photolithography transfer strategy which can modify poly(ethylene glycol) (PEG) hydrogel by Au microarrays. The micropatterned biomaterial has cell-adhesion contrast, which can regulate cell adhesion and be used to study cell-biomaterial interactions. The fundamental studies might be helpful for the development of novel biomaterials.
The main achievements are summarized as follows:
1. To suggest a transfer strategy to fabricate stable micropatterns on hydrogels.
The coupling of photolithography and microtransfer techniques is used to fabricate Au microarrays on PEG hydrogel surface, which resolves a problem of patterned modification of soft materials. Firstly, gold microarray is prepared on a hard inorganic substrate by the conventionally photolithography technique. Then, a thiol-end macromolecular linker is linked to gold micropattern and transferred to a polymeric substrate in photopolymerization of PEG-DA macromonomers. The resultant patterns are confirmed to keep stability even in water.
2. To design a hetero-bifunctional macromolecular linker, and perform successfully the synthesis after overcoming an "inherent" difficulty of reaction between the two end groups. A well-designed hetero-bifunctional macromonomer linker, ACRL-PEG5K-SH, is synthesized and successful used to prepare gold microarrays onto a PEG hydrogel surface. The disulfide model is used to prevent the Michael addition between -SH and ACRL groups and other side reactions. Cell experiments confirm the biocompatibility and cell-adhesion contrast of the resulting patterned hydrogel. The adhesion of cells exhibits dependence of microdot sizes.
3. To put forward a series of parameter to semi-quantified cell orientation on stripe-patterned surfaces. We further prepared, by the photolithography transfer technique, stable gold (Au) micropattems on PEG hydrogel surfaces with defined cell-resistant (PEG hydrogel) and cell-adhesive (gold microstripes) properties. 3T3 fibroblasts were cultured on Au-microstripe surfaces to observe cell adhesion and orientation. Five statistical parameters were defined and used to describe cell orientation on micropattems. The abrupt changes of these parameters did not happen at the same inter-distance. The combination of the 5 statistical parameters represented well the cell orientation behaviors semi-quantitatively.
引文
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