Biogenic thermoresponsive polyelectrolyte multilayers as potential substrata for the generation of cell sheets for tissue engineering

探索一种基于纤维素和壳聚糖衍生物的热响应性表面涂层的新方法，这些聚合物是来自可再生资源的生物来源、生物相容和环境友好的生物聚合物，用于制作哺乳动物细胞的培养基质，允许通过温度变化非酶释放细胞和细胞膜。三种不同的热响应元素和硫酸盐作为阴离子基团将被引入到这些生物聚合物的主干中。为了获得具有足够电荷密度和约37°C转变温度的热响应性化合物，将系统地表征所得到的聚电解质的化学结构、相对分子质量、Zeta电位和较低的临界溶液温度(LCST)。在选择了具有合适的LCST和电荷密度的候选衍生物后，这些聚电解质将用于通过逐层(LBL)技术形成多层膜，这将通过表面等离子激元共振、石英微天平、椭偏仪和接触角测量进行研究。热响应效应应通过在5°C-37°C温度范围内多层涂层的水分含量和润湿性能的变化来分析。蛋白质吸附是细胞黏附的先决条件，已被证明是细胞在热敏表面涂层上黏附的决定因素，这将在本项目中关于纤维连接蛋白和血清蛋白质的结合进行研究。具有热响应特性的多层膜允许蛋白质的可逆吸附，将进一步用于研究来自三个不同胚层的细胞的黏附和生长。热响应效应将通过温度从37°C降至5°C时细胞形状和单个细胞分离的变化以及整个细胞膜对温度变化的响应来研究。总而言之，新体系的开发提供了比现有的完全合成的热响应性聚合物(如聚N-异丙基丙烯酰胺)的基本优势，因为硫化纤维素/壳聚糖对有丝分裂和形态形成生长因子具有固有的生物活性，良好的生物兼容性以及潜在的长期降解性。这些优点使这些系统不仅可用于体外应用，以生成用于移植的各种组织工程的细胞片，包括皮肤、角膜、心肌等，而且还潜在地用于各种体内应用，其中需要蛋白质和/或细胞的热响应释放。

A novel approach for the formation of thermoresponsive surface coatings based on derivatives from cellulose and chitosan as biogenic, biocompatible and environmentally-friendly biopolymers from renewable resources will be explored for making culture substrata for mammalian cells to permit non-enzymatic release of cells and cell sheets by temperature change. Three different thermoresponsive elements together with sulfate as anionic groups will be introduced into the backbone of these biopolymers. The resulting polyelectrolytes will be systematically characterized regarding their chemical structures, molecular weights, zeta potentials and lower critical solution temperatures (LCST), in order to achieve thermoresponsive compounds with a sufficient charge density as well as a transition temperature around 37°C. After the selection of candidate derivatives with suitable LCST and charge density, these polyelectrolytes will be used for the formation of multilayers via the layer-by-layer (LbL) technique, which will be studied by surface plasmon resonance, quartz micro balance, ellipsometry and contact angle measurements. The thermoresponsive effects shall be analyzed by changes of water content and wetting properties of the multilayer coatings in the temperature range from 5°C-37°C. Protein adsorption is a prerequisite for the adhesion of cells and has been shown to be a determinant of cell adhesion on thermoresponsive surface coatings, which will be studied in this project regarding the binding of fibronectin and serum proteins. Multilayers with thermoresponsive properties that permit reversible adsorption of proteins will be further used to study the adhesion and growth of cells from three different germ layers. The thermoresponsive effect will be studied by changes in cell shape and detachment of single cells during decrease of temperature from 37°C to 5°C as well as by the release of complete cell sheet in response to temperature changes. Altogether, the development of the new system provides essential advantages over existing fully synthetic thermoresponsive polymers like poly(N-isopropylacrylamide) because of the inherent bioactivity of sulfated cellulose/chitosan towards mitogenic and morphogenic growth factors, the excellent biocompatibility as well as the potential long-term degradability. These advantages allow these systems not only to be feasible for in vitro applications to generate cell sheets for engineering various tissues for transplantation including skin, cornea, myocardium, etc., but also to be potentially useful for diverse in vivo applications, where the thermoresponsive release of proteins and/or cells is desirable.