The Suppressive Effect of Enamel Matrix Derivative on Osteocalcin Gene Expression of Osteoblasts Is Neutralized by an Antibody Against TGF‐β

Abstract

Enamel matrix derivative (EMD) plays a crucial role in periodontal tissue regeneration. However, the precise mechanism of tissue regeneration by EMD remains obscure. The purpose of this study was to clarify what factors present in EMD show bioactivity. We first examined the effect of EMD on MC3T3-E1 osteoblastic cells. To evaluate the differentiation, the expression of osteoblast-related genes was measured by reverse transcription-polymerase chain reaction, and the osteocalcin (OCN) content was measured by enzyme-linked immunosorbent assay. Alkaline phosphatase activity and the mineralization were examined histologically. EMD (intact EMD) was filtrated to separate the soluble fraction (soluble EMD), and the effects of soluble and intact EMD were examined. Neutralization of the bioactivity of EMD was performed using a polyclonal antibody against porcine transforming growth factor-beta (TGF-beta). EMD inhibited the expression of osteoblastic phenotypes, and we used the inhibitory effect of EMD on osteoblastic differentiation as a benchmark of activity of EMD. The soluble fraction separated from EMD inhibited osteoblast-related gene expression and OCN synthesis. Soluble EMD suppressed the OCN gene level within 24 hours, and the effect of soluble EMD mimicked that of TGF-beta (10 ng/ml). The antibody against TGF-beta diminished the inhibitory effect of soluble EMD on OCN gene expression. The inhibitory effect of EMD on OCN gene expression of osteoblastic cells is neutralized by the antibody against TGF-beta in it. This result might indicate that EMD contains TGF-beta and that it participates in the bioactivity of EMD.