Low molecular weight hyaluronic acid effects on murine macrophage nitric oxide production.

Hyaluronic acid (HA) is increasingly used for a number of medical device applications. Since the chemical structure of HA is identical no matter its bacterial or animal origin, it should be the ideal biomaterial. However, short term transient inflammatory reactions are common, while rare long-term adverse events may correlate with subclinical chronic inflammation. Concern has been raised that low molecular weight components or degradation fragments from implanted HA may directly stimulate inflammatory reactions. This study examined a panel of HA molecular weights from the unitary disaccharide up to 1.7 x 10(6) Dalton lengths, in which endotoxin was assayed at a very low level (less than 0.03 EU/mg). The murine cell line RAW 264.7, rat splenocytes, and rat adherent differentiated primary macrophages were assayed for nitric oxide production under a variety of inflammatory conditions plus or minus HA. Under the highest inflammatory states, nitric oxide production was mildly suppressed by HMW-HA while slightly augmented by LMW-HA at mg/mL concentrations. However, at micromolar concentrations fragments below 5000 Daltons, thought to have drug-like qualities, were without effect. These data support the hypothesis that if endotoxin is reduced to an extremely low level, LMW-HA may not directly provoke normal tissue macrophage-mediated inflammatory reactions.

Screening biomaterials for stimulation of nitric oxide-mediated inflammation.

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.