April 2011
Volume 52, Issue 14
ARVO Annual Meeting Abstract  |   April 2011
Inhibition of RANKL-Induced Osteoclast Formation in RAW264.7 Cells by HC•HA Complex Purified from Human Amniotic Membrane
Author Affiliations & Notes
  • Hua He
    Research & Development, TissueTech, Inc., Miami, Florida
  • Shunsuke Sakurai
    Research & Development, TissueTech, Inc., Miami, Florida
  • Scheffer C. Tseng
    Research & Development, TissueTech, Inc., Ocular Surface Center, and Ocular Surface Research Education Foundation, Miami, Florida
  • Footnotes
    Commercial Relationships  Hua He, TissueTech, Inc. (E); Shunsuke Sakurai, TissueTech, Inc. (E); Scheffer C. Tseng, TissueTech, Inc. (I, P)
  • Footnotes
    Support  NIH Grant R44-EY7497
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 2060. doi:
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      Hua He, Shunsuke Sakurai, Scheffer C. Tseng; Inhibition of RANKL-Induced Osteoclast Formation in RAW264.7 Cells by HC•HA Complex Purified from Human Amniotic Membrane. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2060.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : HC•HA, a complex formed by a covalent linkage between heavy chains (HC) of inter-α-inhibitor and hyaluronic acid (HA) and its tightly bound proteins, has recently been purified from human amniotic membrane extract and had effects in inhibiting inflammation, scarring, and angiogenesis. Herein, we investigated HC•HA complex’s inhibitory activity on osteoclast formation.

Methods: : Macrophage RAW264.7 cells were cultivated in DMEM /10% FBS, harvested with enzyme-free dissociation buffer, and seeded at 1 x 105/ml simultaneously with HMW HA or HC•HA (both at 25 µg/ml HA). After 2 h, cells were further treated with 25 ng/ml RANKL. During a period of 5 days, the multi-nucleated cell morphology was monitored. At Day 5, cells were either stained with acid phosphatase leukocyte kit for osteoclasts or extracted for total RNAs, which were subsequently used for quantitative measurement of mRNA expression of RANK, β3 integrin, and TRAP. Cell death/apoptosis were determined by Live & Dead and Cell Death ELISA.

Results: : Consistent with published reports, the treatment of RAW264.7 cells with only RANKL for 5 days induced the multi-nucleated osteoclast differentiation which was confirmed by TRAP staining. The mRNA levels of both RANK and β3 integrin were significantly higher while that of TRAP was lower. Treatment of HA did not inhibit or promote the osteoclast formation. However, treatment of HC•HA significantly down-regulated the mRNA expression of both RANK and β3 integrin, increased the cell death, and abolished the osteoclast formation.

Conclusions: : HC•HA complex can abolish RANKL-induced osteoclast formation in RAW264.7 cells. The abolishment was probably resulted from the down-regulation of both RANK and β3 integrin and subsequently leading to the cell death under RANKL stimulation.

Keywords: differentiation • gene/expression • signal transduction 

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