A relation between osteoarthritis (OA) and increased cholesterol levels is apparent. In the present study we investigate OA pathology in apolipoprotein E (ApoE)−/− mice with and without a cholesterol-rich diet, a model for high systemic low density lipoprotein (LDL) cholesterol levels independent of weight.Wild type (WT), Apoe−/−, S100a9−/− and Apoe−/−S100a9−/− mice (C57BL/6 background) received a standard or cholesterol-rich diet. Experimental OA was induced by intra-articular injection of collagenase and animals were sacrificed at day 10 and day 36.Although minimal differences in cartilage damage were found between the WT and ApoE−/− mice, increased synovial thickening was found in the latter. Thirty-six days after OA-induction, ApoE−/− mice on a standard diet showed increased ectopic bone formation, particularly at the medial collateral ligament, compared with OA in WT mice. Furthermore, a significant increase in synovial gene expression of both S100a8 and S100a9 and S100A8/S100A9 protein levels was found in ApoE−/− mice, suggesting an activated inflammatory status of synovial cells.In both ApoE−/− and WT mice, addition of a cholesterol-rich diet resulted in excessive bone formation in the medial collateral ligament at late-time-point OA. Interestingly, at the early time point, proteoglycan deposition was already significantly increased in ApoE−/− mice compared with WT mice. Mice deficient for both ApoE and S100a9 also showed increased ectopic bone formation, but not synovial activation, suggesting a role for S100-proteins in cholesterol-mediated synovial activation.Increased cholesterol levels strongly elevate synovial activation and ectopic bone formation in early-stage collagenase-induced OA.