Abstract
Inflammatory reactions to biomaterials may include macrophage-mediated generation of nitric oxide (NO), which may harm patient tissue or potentially interfere with proper function of an implanted device. RAW 264.7 cells were grown in culture and treated at various times with lipopolysaccharide (LPS, endotoxin), murine recombinant gamma-interferon (mrIFN-gamma), and different preparations of hyaluronic acid (HA). Increase in fluorescence of 2,3-diaminonaphthalene (DAN) allowed for detection of initial (24 h or less) NO inflammatory responses of RAW 264.7 to LPS from E. coli O26:B6. By looking at early time points, mrIFN-gamma augmentation of the LPS effect was observed, simulating a complex immune reaction. Activation through nuclear factor-kappaB (NF-kappaB), was confirmed in this system by parthenolide inhibition of LPS stimulation. Stimulation of RAW 264.7 by different HA preparations resulted in NO responses that correlated with the amount of LPS present. In the presence of mrIFN-gamma, a significant inflammatory reaction to HA was observed when the concentration of contaminating LPS was as low as 0.15 EU/mL. NO production in the presence of mrIFN-gamma by RAW 264.7 may serve as a convenient in vitro system to routinely screen biomaterials for potentially harmful macrophage-mediated inflammation whereby the safety of implanted medical devices might be compromised.
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