Effects of ofloxacin on chondrogenesis in murine cartilage organoid culture

The fluoroquinolone, ofloxacin, a widely used antimicrobial agent, has been shown to cause athropathogenic syndromes in juvenile animals. In the present study, the effect of ofloxacin on chondrogenesis in cartilage organoid cultures of limb-bud mesenchymal cells obtained from day-12 mouse embryos was investigated. Cultures treated with increasing concentrations of ofloxacin (10, 30 and 100 mug/ml medium) for 6 days showed no significant changes in overall protein content and dry weight. Collagen type II, as a specific marker for cartilage, and collagen type I, as a marker protein in the internodular loose connective tissue in this culture system, were estimated by an inhibition-ELISA after cyanogen-bromide digestion. The collagen type I content of treated cultures remained constant, but the collagen type II level decreased in a dose-dependent manner to about 40% of that of the controls. There was no detectable increase in the concentration of collagen type II in the medium suggesting that ofloxacin inhibits synthesis rather than stimulate degradation of collagen type II. Cultures treated with 100 mug ofloxacin/ml were further investigated by indirect immunofluorescence and electron microscopy. Anticollagen type II antibodies demonstrated irregularities, several defects in the cartilage nodules, and much weaker staining in the treated cultures compared with controls. Similar results were obtained with antibodies directed against the core protein of large chondroitin sulphate proteoglycan monomers. Using monoclonal antibodies specific for unsulphated, 4-sulphated and 6-sulphated disaccharide "stubs" that remain attached to the core protein after chondroitinase ABC digestion of proteoglycans, different changes in these glycosaminoglycans were observed. While unsulphated chondroitin seemed to disappear nearly completely from the cartilage matrix, the level of chondroitin 4-sulphate remained unchanged and in the case of chondroitin 6-sulphate a slight increase in staining intensity was observable in the ofloxacin-treated cultures. Ultrastructurally, there was a reduction in the number of collagenous fibrils in the cartilage matrix of treated cultures and necrotic chondroblasts could be demonstrated. The present results resemble, in some aspects, observations that have been made in vivo after ofloxacin treatment, indicating that this in vitro model may provide a suitable system for examining the mechanism of quinolone-induced athropathia.