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Enamel matrix derivative (EMD) has demonstrated the potential to stimulate periodontal regeneration with mineralized tissue formation. Molecular regulators of bone metabolism include osteoprotegrin (OPG), receptor activator of nuclear factor kappa B ligand (RANKL), cyclooxygenase 2 (COX2), and core binding factor alpha 1 (Cbfa1). The role of these regulatory molecules within the context of EMD stimulation of mineralized tissue formation is unknown. Therefore, the purpose of this investigation was to explore the effects of EMD on these bone-related molecules in human periodontal ligament (PDL) cells.Human PDL-cell cultures were treated with EMD (5 to 100 mg/ml) for 24 hours. Total RNA was isolated using phenolchloroform, and reverse transcription-polymerase chain reaction (RT-PCR) was performed using primers specific for OPG, RANKL, COX2, Cbfa1, and aldolase, with amplification in the exponential range for each molecule studied.The results of this study show that there is a significant (P <0.05) increase in COX2 mRNA levels with EMD treatment, and no effects were noted on mRNA levels for Cbfa1. RANKL mRNA levels were significantly decreased (P <0.01) up to 50% with EMD treatment ≥25 μg/ml. OPG levels showed minimal effects with EMD treatment. However, the RANKL/ OPGratio showed a 40%to 55%reductionwith EMD ≥ 25 μg/ml.This study supports a role for EMD stimulation of mineralized tissue formation consistent with periodontal regeneration by modulating regulatory molecules critical to bone metabolism at the RNA level. J Periodontol 2006;77: 891-898.