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

本文将探索一种基于可再生资源的纤维素和壳聚糖衍生物作为生物源性、生物相容性和环境友好型生物聚合物形成热响应性表面涂层的新方法，用于制作哺乳动物细胞培养基质，使细胞和细胞片在温度变化下非酶释放。三种不同的热敏元件连同硫酸盐作为阴离子基团将被引入这些生物聚合物的主链。所得到的聚电解质将系统地表征其化学结构、分子量、zeta电位和较低的临界溶液温度（LCST），以获得具有足够电荷密度和37°C左右转变温度的热响应化合物。在选择合适的LCST和电荷密度的候选衍生物后，这些聚电解质将通过层接层（LbL）技术用于多层材料的形成，并将通过表面等离子体共振、石英微天平、椭偏仪和接触角测量进行研究。在5°C-37°C的温度范围内，通过多层涂层的含水量和润湿性能的变化来分析热响应效应。蛋白质吸附是细胞粘附的先决条件，并且已被证明是细胞在热响应表面涂层上粘附的决定因素，本项目将研究纤维连接蛋白和血清蛋白的结合。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粘附和生长。温度从37°C降至5°C时，细胞形状的变化和单个细胞的脱离，以及响应温度变化的完整细胞片的释放，将研究热响应效应。总的来说，新系统的开发比现有的完全合成的热反应性聚合物（如聚n -异丙基丙烯酰胺）具有重要的优势，因为硫酸盐纤维素/壳聚糖对有丝分裂和形态发生生长因子的固有生物活性，出色的生物相容性以及潜在的长期可降解性。这些优点使得这些系统不仅可以在体外应用于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，而且还可以用于各种体内应用，其中需要热响应性释放蛋白质和/或细胞。

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.