Effects of a functional agar surface on in vitro dentinogenesis induced in proteolytically isolated, agar-coated dental papillae in rat mandibular incisors

In an attempt to study the effects of a three-dimensional agar surface on in vitro dentinogenesis both in the growing end and in incisally cross-cut pulp, the possible expression of odontoblast phenotype was investigated morphologically, autoradiographically and immunohistochemically. Explants were incubated for 8 days. In the growing end, duringt the last 4 days, mitotic cells differentiated into [3H]-thymidine-labelled, tubular matrix-forming cells. In cross-cut pulp, however, during the first 4 days, mitotic cells differentiated into [3H]-thymidine-labelled, tubular matrix-forming cells. Electron microscopy demonstrated that, in both regions, tubular matrix-forming cells had characteristics similar to those of primary odontoblasts. When agar was incubated alone, exogenous fibronectin was deposited on it rapidly. After 12 h, endogenous fibronectin appeared on explant peripheral cells. Collagen and materials reacting positively to periodic acid-Schiff (PAS) were first interposed between agar and explant after 4 days. After 8 days, an inner immunonegative layer corresponding to materials reacting positively to PAS or toluidine blue and an outer immunopositive layer of fibronectin or collagen were visible adjacent to the rows of elongated columnar cells. In the presence of GlyArgGlyAspSerPro (GRGDSP), a competitive inhibitor of attachment of cells to fibronectin, explants became detached from the agar surface, and no dentinogenesis occurred. These results indicate that, when in contact with an agar surface that becomes modified by fibronectin and/or by a complex of fibronectin with deposited matrix, dental mesenchymal cells progressively differentiate into tubular matrix-forming cells. Possibly the functional agar surface has the important role of providing a foothold for cell attachment, which is the first step towards in vitro odontoblast differentiation. This system of inducing tubular matrix-forming cells constitutes a useful model for the study of in vitro dentinogenesis.