Background Resolvin-D1 (RvD1) is a derivative of omega-3 fatty acids and a potent anti-inflammatory agent synthesized during the resolution phase of inflammation. In human cartilage, we recently reported that RvD1 strongly inhibited a number of factors involved in inflammation, catabolism, oxidative stress, and apoptosis.
Objectives The overall objective of this study is to further investigate RvD1 effects on bone metabolism in vitro.
Methods First, murine macrophages RAW267.4 were used to assess osteoclasts (OC) recruitment and bone resorption. RAW264.7 cells were incubated with 50 ng/ml LPS with or without RvD1 (0 - 10 μM) for 48 hours. Cell viability was verified with the MTS test. OC phenotype markers, namely TRAP and cathepsin-K, were assessed by western blot, enzymatic staining and immunocytochemistry. Levels of TNF-α, IL-1β, IL-6, IL-10, were measured by ELISA, and PGE2 levels by EIA. NO release was measured by Greiss reaction.
To investigate bone resorption, RAW264.7 cells were seeded in hydroxyapatite plates, then treated with 50 ng/ml LPS with or without RvD1 (0.5 and 1 μ M) for 48 hours. Plot formation was assessed by Von Kossa staining.
Second, human osteoblasts (Ob) were obtained from post-surgery discarded trabecular bone of osteoarthritic (OA) patients who underwent total knee arthroplasty. First passage human OA Ob were treated either with RvD1 (0.1 - 1 μM) alone, or with 20 nM VitD3 with or without RvD1 (0.1 - 1 μ M), for 48 hours. Cell viability was evaluated with the MTS test. Alkaline phosphatase (PAL) activity and osteocalcin (OCN) release was determined by colorimetric reaction and ELISA, respectively.
Results In RAW264.7 cells, our results clearly show that RvD1 strongly reduces OC recruitment and activation as indicated by the inhibition of TRAP and cathepsin K expression as well as TNF-α, IL-1β, IL-6, PGE2 and NO release, as well as the concurrent enhancement of IL-10 levels. Besides, RvD1 decreases bone resorption through the inhibition of plots formation in hydroxyapatite matrix. In human OA Ob, RvD1 partially decreases VitD3-induced PAL activity, while it maintains OCN expression at control levels.
Conclusions Our in vitro results clearly show that RvD1 may play an important role in the regulation of bone metabolism. Additionally to our previous data, our findings suggest that RvD1 may offer a novel and original perspective to make a real contribution to musculoskeletal and bone diseases therapy.
Acknowledgements This study was supported by Canadian Institute of Health Research (CIHR) grant (#IMH 131570) and by the Center of Excellence for Arthroplasty Research.
Disclosure of Interest None declared