Neutrophils exhibit distinct phenotypes toward chitosans with different degrees of deacetylation: implications for cartilage repair.

INTRODUCTION

Osteoarthritis is characterized by the progressive destruction of cartilage in the articular joints. Novel therapies that promote resurfacing of exposed bone in focal areas are of interest in osteoarthritis because they may delay the progression of this disabling disease in patients who develop focal lesions. Recently, the addition of 80% deacetylated chitosan to cartilage microfractures was shown to promote the regeneration of hyaline cartilage. The molecular mechanisms by which chitosan promotes cartilage regeneration remain unknown. Because neutrophils are transiently recruited to the microfracture site, the effect of 80% deacetylated chitosan on the function of neutrophils was investigated. Most studies on neutrophils use preparations of chitosan with an uncertain degree of deacetylation. For therapeutic purposes, it is of interest to determine whether the degree of deacetylation influences the response of neutrophils to chitosan. The effect of 95% deacetylated chitosan on the function of neutrophils was therefore also investigated and compared with that of 80% deacetylated chitosan.

METHODS

Human blood neutrophils from healthy donors were isolated by centrifugation on Ficoll-Paque. Chemotaxis was performed using the chemoTX system. Production of superoxide anions was evaluated using the cytochrome c reduction assay. Degranulation was determined by evaluating the release of myeloperoxidase and lactoferrin. The internalization of fluorescently labelled 80% deacetylated chitosan by neutrophils was studied by confocal microscopy.

RESULTS

Neutrophils were dose dependently attracted to 80% deacetylated chitosan. In contrast, 95% deacetylated chitosan was not chemotactic for neutrophils. Moreover, the majority of the chemotactic effect of 80% deacetylated chitosan was mediated by phospholipase-A2-derived bioactive lipids. Contrary to the induction of chemotaxis, neither 80% nor 95% deacetylated chitosan activated the release of granule enzymes or the generation of active oxygen species. Despite the distinct response of neutrophils toward 80% and 95% deacetylated chitosan, both chitosans were internalized by neutrophils.

CONCLUSIONS

Eighty per cent deacetylated chitosan induces a phenotype in neutrophils that is distinct from the classical phenotype induced by pro-inflammatory agents. Our observations also indicate that the degree of deacetylation is an important factor to consider in the use of chitosan as an accelerator of repair because neutrophils do not respond to 95% deacetylated chitosan.