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Ek Kia Tan, Hua He, Scheffer C. Tseng; Inhibition of Osteoclasts Differentiation from RAW264.7 Cells by Amniotic Membrane and Its Derivatives. Invest. Ophthalmol. Vis. Sci. 2011;52(14):440.
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© ARVO (1962-2015); The Authors (2016-present)
Previous studies have shown that osteoclast differentiation could be induced from macrophage RAW264.7 cells by adding Receptor Activator for Nuclear Factor Kβ Ligand (RANKL). Clinically and experimentally, amniotic membrane (AM), AM extract (AME), and HC•HA purified from AME have been shown to inhibit inflammation, scarring, and angiogenesis. We would like to determine whether AM and its derivatives affect such osteolclast differentiation from macrophage RAW264.7 cells.
Macrophage RAW264.7 cells were cultivated in DMEM /10% FBS, harvested with enzyme-free dissociation buffer, and seeded at 320 cells/mm2 simultaneously with either PBS (CTL), AME (25 µg/ml HA), HC•HA (25 µg/ml HA), or on either side of intact AM (iAM), denuded AM (dAM), and lyophilized AM (lAM). After incubation for 2 h, cells were further treated with 25 ng/ml RANKL and subsequently monitored for cell death and formation of multi-nucleated cell (osteoclast) for a period of 7 days. After termination, cells were stained with acid phosphatase leukocyte kit for osteoclast cells or extracted for total RNAs, which were subsequently used for quantitative (qPCR) measurement of RANK, β3 integrin, and tartrate-resistant acid phosphatase (TRAP) transcript levels.
Phase contrast microscopy showed formation of multi-nucleated cells on Day 6 in the CTL and AME group but not in the HC•HA, iAM, dAM, and lAM group. Positive TRAP staining on single-nucleated cells was noted on all culture conditions except HC•HA, which also uniquely displayed cell death. The mRNA expression of RANK and β3 Integrin were significantly down-regulated for both HC•HA (p=0.04 and p=0.02m respectively) and stromal side of dAM stroma (p=0.03 and p=0.005, respectively). TRAP mRNA level was downregulated by stromal side of dAM (p=0.04), but upregulated by the epithelial side of iAM (p=0.005) and by either side of lAM (p=0.08 and p=0.01 for epithelial and stroma side respectively).
Osteoclast differentiation from macrophage RAW264.7 cells by RANKL treatment is inhibited when cells were in contact with stromal side of dAM and HC•HA purified from AME, suggesting that HC•HA is the prime candidate in AM stroma to exert this unique action. The negative finding exerted by lyophilization or presence of amniotic epithelial cells deserves further investigation.
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