MC3T3-E1-conditioned medium-induced mineralization by clonal rat dental pulp cells

Dental pulp is thought to participate in supplementary mineralization, such as reparative dentin and pulp stones, but no direct proof of this has been reported. To study this process at a molecular level, we investigated the matrix mineralization of dental pulp using a clonal cell line (RPC-C2A) der ived from rat incisor dental pulp. Mineralized nodules in extracellular mat rix were formed by RPC-C2A cells cultured in the presence of conditioned me dium (CM) from confluent osteoblastic MC3T3-E1 cells. These nodules were st ained by the von Kossa method and with alizarin red S and quantified by the measurement of acid-soluble calcium deposition. This CM was most effective when collected 3-6 days after confluency and added at 50% to the culture m edium. The CM-treated RPC-C2A cells showed high alkaline phosphatase activi ty, a high mRNA level of osteocalcin and decreases in the mRNA levels of os teopontin and osteonectin, but undetectable levels of mRNA of dentin sialop hosphoprotein by Northern blot analyses. A pan-specific anti-transforming g rowth factor (TGF)-beta antibody and a soluble form of receptor for bone mo rphogenetic protein (BMP)-2/-4 did not neutralize the CM-induced mineraliza tion. These results suggest that some soluble factor(s) other than TGF-beta or BMP-2/-4 in the CM from MC3T3-E1 cells cause differentiation of RPC-C2A cells to osteoblast-like cells. (C) 2001 Elsevier Science B.V. /Internatio nal Society of Matrix Biology. All rights reserved.