Enamel matrix derivative promotes osteoclast cell formation by RANKL production in mouse marrow cultures

Abstract

Enamel matrix derivative (EMD) is used clinically to promote periodontal tissue regeneration, however, there are few reports regarding effects of EMD on bone metabolism. We evaluated the influence of EMD on osteoclast formation using in vitro bone marrow culture. Bioactive fractions were purified from EMD by reverse-phase HPLC on a C18 hydrophobic support, then mouse bone marrow cells were cultured with EMD or its purified fractions for 8 days. Following tartrate resistant acid phosphatase (TRAP) staining, TRAP-positive multinucleated cells were counted. The expression of receptor activator of NF-kappaB ligand (RANKL) in osteoblastic cells was detected using immunoblotting. EMD was dissolved in 0.1% (vol/vol) trifluoroacetic acid and applied to a C18 column for HPLC. Two major peaks were obtained of which the second (fraction numbers 21-25) was found to induce the formation of osteoclasts in mouse marrow cultures. Further, osteoprotegerin completely inhibited osteoclast formation in mouse marrow cultures with or without osteoblastic stromal cells, when being cultured with EMD or its purified fractions. In addition, Western blot analysis revealed the presence of RANKL in mouse osteoblastic cells stimulated with EMD or its purified fractions. Our results indicate that EMD induces the formation of osteoclasts through RANKL expressed by osteoblastic cells, and suggest that EMD may regulate both bone formation and bone resorption during periodontal tissue regeneration.