| Smart materials are widely researched because of their sensitive, controllable and reversible response to environmental changes. Poly(N-isopropylacrylamide) is a kind of temperature sensitive polymer which has been widely used for harvesting cell sheet and has great potential in scaffold-free tissue engineering technology and regenerative medicine. However, using this temperature responsive polymer may cause problems such as reduction in cell viability at lower temperatures, thus there is an urgent need to develop novel smart materials. Phenylboronic acid and its derivatives such as 4-carboxy-3-fluorophenylboronic acid(CFPBA) and 3-acrylamidophenylboronic acid(AAPBA) could combine with glucose or fructose and some other small biological molecules with cis-diol structure to form stable and reversible covalent bonds. These compounds demonstrate saccharide sensitivity, and as a result, they hold great promise in biomedicine. The purpose of this thesis is to prepare temperature or saccharide responsive polymer, and study the effect of temperature and sugar content on the polymer substrates. Then the polymer is used as a substrate for cell culture in order to control cell and cell sheet adhesion or detachment behavior according to the change in temperature or sugar concentration of the surroundings, thereby improving the technology for harvesting cell sheets.Firstly, we modified the glass substrate with the saccharide responsive polymer brush by the AGET-ATRP method and post-modification. Through reversible saccharide sensitive covalent interactions we introduced a linear polymer(RGD-PGAPMA) with bioactive adhesion peptide RGD in its ends and glycosyl groups in its side chains. Glass surfaces modified with PBA and RGD-PGAPMA were used for cell culture. Obvious cell detachment was observed after adding glucose or fructose. After replacing high saccharide concentrion culture medium with fresh low glucose medium, we found that almost all of the released cells re-adhered on the substrate. From the above, a reversible muticovalent PBA/RGD-PGAPMA interface could be applied to fabricate stable and dynamical biointerface material. The bioactive surface can be turned on and off by changing the sugar concentration of the medium, and ultimately achieve within the organism a stimuli-responsive regulated(via in vivo glucose concentration changes) cell attachment and desorption behavior.Next, we prepared temperature and saccharide dual-responsive hydrogel layers by redox polymerization. Swelling ratio and surface hydrophilic/hydrophobic properties were examinated, and the results showed that by lowering temperature or adding sugar the hydrogel swelled and became more hydrophilic, so the hydrogel showed dual-sensitivity. Those hydrogel layers were then used for cell culture. Cell sheet was detached after reducing the temperature or replacing the medium with the higher sugar concentration medium, and then the intact cell sheet was harvested. Moreover, cell sheets were detached most efficiently and completely from substrate surfaces in the case of simultaneously reducing the temperature and adding sugar to the medium. The harvested cell sheet was cultured in regular fresh medium and the cells proliferated with good morphology. In conclusion, we rapidly harvested intact or low-injury cell sheets by controlling temperature and sugar content, and improved the efficiency of harvesting cell sheets.In conclusion, based on the research of intelligent materials on cell sheet engineering, we fabricated novel saccharide responsive or temperature and saccharide dual-responsive substrates for controlling cell adhesive behavior and havesting cell sheets, therefore, this research has greatly improved cell sheet technology. |
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