Abstract
Aim: To assess whether Osteoclast-associated receptor (OSCAR) is produced by human peripheral blood derived osteoclast cells in culture and regulated by RANKL +/-TNF alpha.
Methods: Human peripheral blood mononuclear cells were pre-cultured with MCSF for 7 days. Adherent cells were then differentiated into osteoclasts with 10 or 50 ng/ml RANKL and 10 ng/ml MCSF over 10 days. TNF alpha was added to duplicate 10 ng/ml RANKL treated cultures. Media was changed every 3–4 days. After 7 days differentiation supernatant was collected during media change and cells were fixed for TRAP staining (to identify pre-osteoclasts) or RNA extracted for RT QPCR. Levels of soluble OSCAR were determined by commercial ELISA. After 10 days differentiation, dentine slices were imaged by Scanning Electron Microscopy (SEM) for resorption pit analysis to verify the presence of active osteoclasts.
Results: OSCAR was produced by PBMC derived osteoclast cells in response to RANKL at 10 ng/ml as well as 50 ng/ml. Addition of TNFα to 10 ng/ml RANKL treated cells raised OSCAR levels to those treated with 50 ng/ml RANKL alone (although not significant). TRAP positive cells, osteoclast gene expression and resorption pits were present with all treatments confirming the presence of active osteoclasts.
Conclusions: OSCAR expression and production by osteoclast cells is mediated by RANKL and TNFα, supporting a role for OSCAR in the pathophysiology of RA.
Methods: Human peripheral blood mononuclear cells were pre-cultured with MCSF for 7 days. Adherent cells were then differentiated into osteoclasts with 10 or 50 ng/ml RANKL and 10 ng/ml MCSF over 10 days. TNF alpha was added to duplicate 10 ng/ml RANKL treated cultures. Media was changed every 3–4 days. After 7 days differentiation supernatant was collected during media change and cells were fixed for TRAP staining (to identify pre-osteoclasts) or RNA extracted for RT QPCR. Levels of soluble OSCAR were determined by commercial ELISA. After 10 days differentiation, dentine slices were imaged by Scanning Electron Microscopy (SEM) for resorption pit analysis to verify the presence of active osteoclasts.
Results: OSCAR was produced by PBMC derived osteoclast cells in response to RANKL at 10 ng/ml as well as 50 ng/ml. Addition of TNFα to 10 ng/ml RANKL treated cells raised OSCAR levels to those treated with 50 ng/ml RANKL alone (although not significant). TRAP positive cells, osteoclast gene expression and resorption pits were present with all treatments confirming the presence of active osteoclasts.
Conclusions: OSCAR expression and production by osteoclast cells is mediated by RANKL and TNFα, supporting a role for OSCAR in the pathophysiology of RA.
| Original language | English |
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| Publication status | Published - 2014 |
| Event | Australian Rheumatology Association 55th Annual Scientific Meeting - Hobart, Australia Duration: 17 May 2014 → 20 May 2014 |
Conference
| Conference | Australian Rheumatology Association 55th Annual Scientific Meeting |
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| Country/Territory | Australia |
| City | Hobart |
| Period | 17/05/14 → 20/05/14 |