物理-化学双重梯度纳米复合水凝胶的可控构筑及对体外三维细胞培养机制研究

It has been challenging to fabricate bionic cell and organ microenvironment with similar structure, mechanical properties and gradient biological function to extracellular matrix. Hydrogels are regarded as excellent candidates due to similar structure to tissues and high biocompatibility. However, traditional non-gradient hydrogels cannot imitate cell microenvironment, while the present gradient hydrogels have obvious disadvantages of complex preparation methods, uncontrollable performance and toxic residue caused by chemical crosslinkers. So this proposal aims to design mechanically strong nanocomposite hydrogels with physical-chemical dual gradients and set up a facile and controllable preparation method under the induction of electric field based on the colloidal property of Laponite and interaction between Laponite and functional monomers. Furthermore, interaction between Laponite and monomers, and their migration behaviors under electric field are discussed. The gradient distribution of Laponite and functional monomers are adjusted in attempted to precisely control mechanical and functional gradients. Relationship between gradient structure and functions is investigated when external stimuli are applied. The effect of physical and chemical gradients on cell behaviors is investigated and mechanism of cell culture is explored. The proposal will provide a new insight on development of functionally gradient hydrogels, and open new opportunity for regenerative medicine and biomaterials.

如何仿生构筑具有类细胞外基质的结构、力学特性和梯度生物学功能的微环境一直是再生医学领域的难点。水凝胶因具有与生命组织类似的结构和生物相容性，在体外仿生构筑细胞生长微环境中具有巨大的应用潜力。针对均质水凝胶无法真实模拟细胞微环境，而现有梯度水凝胶存在合成方法复杂、性能调控不便以及化学交联导致的残留危害等缺点，本项目拟利用锂藻土的胶体性质及其与功能单体的相互作用，发展一种电场诱导下，简便、通用和可控的合成方法，构建新型高强度物理-化学双重梯度纳米复合水凝胶。通过系统研究电场中锂藻土和功能单体的相互作用和迁移规律，精确调控水凝胶中锂藻土和功能单体的梯度分布，实现力学性质和功能梯度可控；探明外部刺激对梯度结构与梯度功能的影响；考察物理和化学梯度与细胞反应间的关系，建立相关细胞培养机制；为发展功能梯度水凝胶的新方法和新结构提供思路与参考依据，并为构建真实模拟细胞微环境的新型医学再生材料提供新的借鉴。

仿生构筑类细胞外基质的梯度水凝胶对细胞培养等生物医学领域的研究具有重要意义。本项目利用锂藻土特有的带电性质和交联作用，通过电泳法合成了系列交联密度梯度和聚合物分布梯度的粘土基纳米复合水凝胶，用荧光、SEM、FTIR、TG和AFM等手段验证了锂藻土和聚合物的分布梯度，建立了梯度结构定性和定量表征方法。考察了凝胶硬度、含水量、温敏性及吸附性等梯度性质与电场条件的关系规律，实现了梯度结构的简单可控：通过电压调节，可改变凝胶硬度梯度跨度，当电压从2V增至4V时，平均硬度梯度可从0.18 kPa/mm增至1.28 kPa/mm，而对于离子型梯度凝胶，胺基质子化也会进一步增加梯度跨度；凝胶LCST呈梯度变化，表现出热致收缩和变形行为，通过引入亲水性单体，可在温敏响应过程中保持凝胶尺寸的稳定性，此时水在凝胶中存在动态平衡，总含水量变化不大，但自由水量在LCST之上时有所增加；锂藻土和聚合物的梯度分布导致其具有梯度吸附性能。发展了酸热法制备梯度纳米复合水凝胶的新方法，利用酸从水凝胶外层到内层的不同扩散速率，以及凝胶中酰胺基团在热处理下的酰亚胺化反应，成功构建了酰亚胺基团的梯度分布结构；同时凝胶的亲水性和交联度均发生改变，故可通过调节酰亚胺化程度灵活调节凝胶的溶胀度和机械性能，凝胶从外部到内部模量变化范围为2.09-0.9 GPa。研究了梯度纳米复合水凝胶在肿瘤细胞体外培养中的应用，凝胶表面的粘附及增殖行为除有明显的趋硬性外，还与凝胶的电荷，亲水性等密切相关。研究成果为发展功能梯度水凝胶的新方法和新结构提供思路与参考依据，并为构建真实模拟细胞微环境的新型医用材料提供新的借鉴。在ACS Appl. Mater. Interfaces, J. Mater. Chem. B, Chem. Eng. J., Macromol. Rapid Comm. 等国际重要期刊上发表论文8篇，申请并授权中国发明专利1项，培养研究生4名。