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A. Sharma, R.R. Mohan, S. Sinha, S.E. Wilson; Corneal Fibroblasts Support Osteoclast–Like Cell Differentiation of Macrophages . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2605.
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Purpose: Osteoblasts and corneal fibroblasts both express membrane–bound receptor activator of NFkB (RANK) ligand and macrophage colony stimulating factor (mCSF), which confer on osteoblasts the capacity to modulate macrophages to differentiate into osteoclasts. The aim of the present study was to investigate the capacity of corneal fibroblasts to support osteoclast–like differentiation from macrophages in vitro. Methods: Corneal fibroblasts were isolated from C57BL/6 mice by dispase and collagenase digestion. For macrophage cell culture, mouse bone marrow obtained from tibia and femur was dispersed in alpha MEM medium and incubated at 37°C for 24 hours. Non–adherent cells were collected, layered over Ficoll and centrifuged. The interphase thus obtained was collected, washed, and diluted with alpha MEM containing 10 ng/ml of mCSF and plated at 50,000 cell/well in 4–chamber culture plates. Three days later, 50,000 first passage corneal fibroblasts were added to the macrophage culture. These co–cultured cells were incubated together. Cells were stained for osteoclast marker tartrate–resistant acid phosphatase (TRAP) using a commercially available kit (SIGMA). The effects of osteoprotegerin (soluble decoy RANKL receptor antagonist) (100 ng/ml) and/or dexamethasone (100 nM) in these co–cultures were also evaluated. Dexamethasone has been shown to down regulate OPG expression in fibroblast cells. Soluble RANKL added to macrophage cultures served as positive controls. Results: Cells staining positive for TRAP were generated after 15 days of co–culture of corneal fibroblasts and macrophages, suggesting formation of osteoclast–like cells. No TRAP staining was noted in corneal fibroblasts or macrophages that were cultured separately to serve as negative controls. Addition of OPG, to the fibroblast–macrophage co–culture significantly decreased the number of TRAP–positive cells. Supplementing co–cultures with dexamethasone increased TRAP–positive cells several fold. Conclusions: Corneal fibroblasts are competent to induce the formation of osteoclast–like cells from macrophages in vitro. This interaction is likely mediated by RANKL, since formation of the osteoclast–like cells is blocked by OPG and augmented by dexamethasone.
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