Analysis of cell-material interforce by using AFM

• Main Authors: Sheng-wen Hsiao, 蕭盛文
• Other Authors: Ming-hua Ho
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/pp4sau

博士 === 國立臺灣科技大學 === 化學工程系 === 100 === Chitosan, a biocompatible material which has been widely used in the bone tissue engineering, is believed to be with high affinity to osteoblastic cells. This hypothesis was first proved in this research. By using AFM with the chitosan-modified cantilever, the quantitative evaluation of the interforce between chitosan and osteoblastic cells can be carried out. In the last part of this research, the cell attachment and spreading on chitosan substrates were analyzed to further clarify the interactions between cells and chitosan. The results showed that the force between chitosan and osteoblasts or osteoblastic cells was specifically high. Comparatively, the smallest adhesion force appeared between chitosan and muscle fibroblasts, the cell without any osteogenic properties. This result proved that there was a significant interaction existing between chitosan and bone cells, which agreed with the tendency in cell attachment and spreading. The technique developed in this research revealed the adhesion force between chitosan and cells directly and quantitatively. The specific interaction exists between chitosan and osteoblasts which was also first testified in this study. Chitosan substrates were also treated by simulated body fluid (SBF) for different time to fabricate scaffolds with different osteoconductivity. XRD and EDS were used to identify crystallinity and Ca/P ratios of the biomimetic layer from SBF deposition. The properties of deposition have been proved to be controlled by the time for SBF treatment. In vitro and in vivo tests revealed that with the SBF treatment longer than 14 days, scaffold showed excellent biocompatibility and osteoconductivity. AFM was used in liquid system to quantify the interforce between the biomimetic layer on scaffolds and cells. The results supported that the interforce between cells and scaffolds dominant in the osteoconductivity of scaffolds. To understand how the topographical cues influence cell behaviors, the microgrooved surfaces were used to culture osteoblasts and osteogenic cells in this study. Osteoblasts were cultured on the grooved surfaces , and then cell alignment, elongation, attachment, spreading, cytoskeleton distribution, penetration depth, stiffness and differentiation were analyzed. The cell alignment and elongation were significantly guided by microgroove patterns and enhanced with the decrease in the groove width. In addition, the organization of actin filaments was visualized by the cytoskeleton immunostaining. On the grooved surface, cells adopted bipolar morphology with few lamellipodia and expressed highly orientated actin filaments along the groove direction. The density as well as orientation degree of actin filaments would be increased with the narrowing of groove width. On the other hand, cells exhibited meshwork of peripheral actin filament with many lamellipodia when they were cultured on flat surface. The attachment, spreading and proliferation of osteoblasts were retarded by the small groove patterns (1-6 ?慆) despite this structure could offer a larger specific area. In contrast, topography with wide grooves promoted the attachment, spreading, and proliferation of osteoblasts. The outcomes were different for osteosarcoma cells where the cell attachment, spreading and proliferation were all promoted by narrow grooves. According to the analysis of cell stiffness and penetration depth, the influences of grooves on cell behaviors would be determined by the mechanical properties of cells. Furthermore, ALPase expression of osteoblasts was notably enhanced by the reduction of groove width. The narrow groove patterns are able to promote osteoblasts differentiation. The results suggested that the cell stiffness would greatly induce the osteogenic differentiation.