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) derived from rat incisor dental pulp. Mineralized nodules in extracellular matrix were formed by RPC-C2A cells cultured in the presence of conditioned medium (CM) from confluent osteoblastic MC3T3-E1 cells. These nodules were stained 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 medium. The CM-treated RPC-C2A cells showed high alkaline phosphatase activity, a high mRNA level of osteocalcin and decreases in the mRNA levels of osteopontin and osteonectin, but undetectable levels of mRNA of dentin sialophosphoprotein by Northern blot analyses. A pan-specific anti-transforming growth factor (TGF)-beta antibody and a soluble form of receptor for bone morphogenetic protein (BMP)-2/-4 did not neutralize the CM-induced mineralization. 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.
Ueno
A
A
Department of Biochemistry, School of Dentistry, The University of Tokushima, 3-18-15 Kuramoto-cho, Tokushima 770-8504, Japan. akemichi@dent.tokushima-u.ac.jp
Kitase
Y
Y
Moriyama
K
K
Inoue
H
H
D000469:Alkaline Phosphatase / Q000187:drug effects*; D000818:Animals; D019485:Bone Morphogenetic Proteins / Q000378:metabolism; D002460:Cell Line / Q000166:cytology; D002999:Clone Cells; D018920:Coculture Techniques; D017077:Culture Media, Conditioned / Q000378:metabolism / Q000494:pharmacology*; D003782:Dental Pulp / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D003784:Dental Pulp Calcification / Q000378:metabolism*; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D051379:Mice; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism; D012333:RNA, Messenger / Q000187:drug effects* / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D016212:Transforming Growth Factor beta / Q000378:metabolism
D019485:Bone Morphogenetic Proteins; D017077:Culture Media, Conditioned; D016326:Extracellular Matrix Proteins; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; D000469:Alkaline Phosphatase