| Cell adhesion receptors play a key role in sensing and integrating signals provided by cellular environment, such as the physicochemical property of substrate surface and the stiffness of substrate. The chemical signals and force signal will be transformed into the corresponding biological signals and regulate the cell adhesion, spreading, proliferation and differentiation. It is very important to quantitative study cell-substrate interaction for revealing the essence of cell physiological process changes, which provides an important experimental data for biomaterials design.In recent years, the development of atomic force microscopy(AFM) provides a powerful technology on researches of cell-biomaterials interaction at single cell level, which exhibit many important micro information and experimental basis for the design of biomaterials and the change of cell physiological process. Hence, this paper carried out the research of the following three aspects based on AFM single cell force spectrum. 1. At present, it is not very mature for AFM technology to detected cell Young’s modulus. In order to get a precise result, many operating parameter need further test. Firstly, a 5 μm(in diameter) silicon dioxide spheres was modified to a tip-less AFM cantilever by AFM nanomanipulation technology. This nanomanipulation technology is the base of AFM single cell force spectrum. Simultaneously, a systematic study was performed to research the effect of physical cues on living MC3T3-E1 cell mechanical property based on AFM detection. The physical cues include indentation force, probe loading rate and the shape of the AFM tips. All of these cues show a significant effect on the cell Young’s modulus assessment. Moreover, each sort of cells exhibit specific Young’s modulus value when they are cultured in cell culture dish. It suggests that AFM can be used as a new method for disease diagnosis and cell physiological status characterization. 2. A new method for real-time and in-situ detection of the effect of chemical composition on MC3T3-E1 cell adhesion, morphology and biomechanical property was developed based on AFM. The changes of cell adhesion force between different substrate were probed by AFM single cell force spectrum. The cell adhesion force increased from 0.76±017 n N to 1.70±0.19 n N, and cell Young’s modulus reduced from 11.94±3.19 k Pa to 1.81±0.52 k Pa with the increase of collagen content. In addition, with the collagen content increased, the area of cell spreading and cell proliferation increased, cell height decreased and pseudopod fusion. Those methods and quantitative results have guiding significance for investigating the mechanism of chitosan based cell-targeting drug carrier and the preparation of chitosan-collagen composite biomaterials. 3. The effect of substrate stiffness on MC3T3-E1 cell adhesion and spreading was studied based on AFM force spectrum technology. The quantitative adhesion force between cell and different stiffness chitosan substrates was detected by living cell adhered AFM tip-less probe. The results showed cell adhesion force was 0.53±0.08 n N on the soft chitosan film, while the adhesion force was 1.19±0.14 n N on stiffer chitosan film. Simultaneously, cell Young’s modulus was probed by AFM. The results of cell area and cell morphology indicated that with the increase of substrate stiffness cell area and adhesion force increased respectively, and cell Young’s modulus reduced from 12.6±2.3 k Pa to 3.6±1.5 k Pa. Moreover, the changes of cell proliferation and adhesion rate were interpreted by CCK-8 assay. Our results indicate that the change of cell adhesion force and Young’s modulus plays a key role in cell adhere and spreading on single cell level. The stiffness of substrate has a significant influence on the cytocompatibility of biomaterial. The research provided new important experimental evidence and method for the design of chitosan-based biomaterials. |
PDF Full Text Request