| Summary: | 碩士 === 國立嘉義大學 === 生化科技學系研究所 === 104 === Chitosan, the N-deacetylated derivative of chitin found in nature resources, is used in bone tissue engineering because its biocompatible, biodegradable, non-toxic characteristics and the structure is similar to the glycosaminoglycans (GAGs) in the bone extracellular matrix. Besides, chitosan has been used in biomaterials in against gram-positive and gram-negative bacteria. In this study, chitosan/poly(vinyl alcohol)/gelatin composite matrices were prepared by glutaraldehyde crosslinking with two chitosan concentrations (2.5% and 3%) and three glutaraldehyde concentrations (0.5%, 0.75% and 1%). The physical properties of chitosan/PVA/gelatin composite matrices were characterized by swelling measurement and the capacity of protein absorption. Surface roughness of chitosan composite matrices also measured by atomic force microscope (AFM). The antibacterial ability test and bacterial invasion study were investigated against the bacteria species: Staphylococcus epidermidis (S. epidermidis) and Escherichia coli (E. coli). For cell study, 7F2 osteoblasts and SW1353 chondrocytes were studied in vitro for cell viability and proliferation on chitosan composite matrices and anti-inflammatory effect of chitosan composite matrices were investigated by RAW 264.7 murine macrophage-like cells.
The results showed that chitosan/PVA/gelatin composite matrices prepared using higher chitosan concentration (3%) could improve the capacity of protein absorption. In chitosan/PVA/gelatin composite matrices prepared using 2.5% chitosan concentration, chitosan composite matrices prepared using 1% glutaraldehyde had better protein absorption than others. But in the measurement of swelling ratio, all matrices prepared using 3% chitosan concentration showed lower swelling ratio than ones prepared using 2.5% chitosan concentration. In the analysis of surface roughness, the roughness of chitosan composite matrices was reduced with the increase of glutaraldehyde concentration but increased after 1 day immersion in 10% FBS medium. Therefore, the increase of surface roughness also proved protein absorption on the surface of chitosan composite matrices.
Although 7F2 and SW1353 cells showed only 20% ~ 30% cell attachment on chitosan composite matrices at day 1, chitosan composite matrices C2.5-G0.5 and C3-G0.5 after 1 day immersion in 10% FBS containing medium showed high cell proliferation rate at day 3 . In bacterial inhibition test, chitosan composite matrices C2.5-G0.5 and C3-G0.5 showed the best inhibitory effects on S. epidermidis, however, all chitosan composite matrices were unable to inhibit the growth of E. coli. Furthermore, all chitosan composite matrices had good effect on bacterial invasion because chitosan composite matrices could prevent S. epidermidis invading to 7F2 and SW1353 cells no matter at a multiplicity of infection (MOI) of 40 or 100. Chitosan composite matrices could significantly inhibit LPS-induced nitrite production in RAW 264.7 macrophages and also suppressed the inflammatory responses induced by S. epidermidis except chitosan matrices C2.5-G1. All together, chitosan composite matrices may have the potential to assist bone repair and prevent bacterial invasion.
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