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.

Defining critical inflammatory parameters for endotoxin impurity in manufactured alginate microcapsules.

Since current purification methods cannot completely remove all traces of endotoxin in biomaterials intended for use in implantable or blood-contacting devices, acceptable levels of endotoxin contamination that will not cause a significant inflammatory reaction need to be defined. Inflammatory reactions to biomaterials may include production of high concentrations of potentially harmful nitric oxide (NO) generated by macrophages. Nitrite accumulation was measured from RAW264.7 cells treated with either lipopolysaccharide (LPS) free in solution or defined quantities of LPS incorporated into alginate in the absence or presence of murine interferon-gamma (mrIFN-gamma). In the absence of IFN-gamma, significant NO production by RAW 264.7 cells occurred for LPS levels down to 0.018 EU/mL. In the presence of mrIFN-gamma, the lowest concentration of LPS tested in solution (0.006 EU/mL) elicited a significant increase in NO production. In the absence or presence of mrIFN-gamma, five times the concentration of LPS incorporated into alginate as compared to LPS free in solution was necessary to elicit a similar NO response by RAW264.7. These results demonstrate that very low concentrations of endotoxin can elicit significant NO responses from macrophages, particularly when inflammatory cytokines are present. Biomaterials may sequester endotoxin, resulting in lower inflammatory reactions that otherwise might be expected.

Sensitivity of insulin production from encapsulated islets to endotoxin-stimulated macrophage inflammatory mediators.

Chronic inflammation may compromise function of implanted encapsulated islets. Increased purity of alginate used for encapsulation prolongs encapsulated graft function, correlating with decreased presence of impurities like bacterial endotoxin. Limits for endotoxin contamination in biomaterials based on indirect inhibition of function of embedded cells have yet to be established. In a coculture system with RAW 264.7 monocyte/macrophage cells in the presence of 50 ng/mL murine recombinant gamma-interferon (mrIFN-gamma), the insulin response to glucose challenge of both rat and pig unencapsulated islets was prevented by endotoxin (LPS) in the medium down to 0.3 EU/mL (LOEL), but not 0.06 EU/mL (NOEL). Evaluation of nitrite concentrations in supernatants revealed that pig islets were more resistant to LPS-stimulated macrophage mediators than rat islets. Encapsulation in highly purified alginate produced little change in observed inhibitory effects of macrophage-generated nitric oxide (NO) toward islet function. Chemically released NO was much less effective in inhibiting insulin responsiveness to glucose challenge than was coculture of islets with LPS and mrIFN-gamma-stimulated RAW 264.7. These results taken together with other data suggest that an upper limit of 0.3 EU/mL LPS within the encapsulating alginate will not impair the function of implanted encapsulated islets by toxic concentrations of macrophage-mediated inflammatory agents.

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.