In order to elucidate the mechanisms involved in human dentin formation, we developed a cell culture system to promote differentiation of dental pulp cells into odontoblasts. Explants from human teeth were cultured in Eagle's basal medium supplemented with 10% or 15% fetal calf serum, with or without beta-glycerophosphate (beta GP). Addition of beta GP to the culture medium induced odontoblast features in the cultured pulp cells. Cells polarized and some of them exhibited a typical cellular extension. In some cases, cells aligned with their processes oriented in the same direction and developed junctional complexes similar to the terminal web linking odontoblasts in vivo. Fine structural analyses showed the presence of typical intracellular organelles of the odontoblast body, whereas the process contained only cytoskeleton elements and secretory vesicles. Polarized cells deposited onto the plastic dishes an abundant and organized type I collagen-rich matrix with areas of mineralization appearing thereafter. X-ray microanalysis showed the presence of calcium and phosphorus and the electron diffraction pattern confirmed the apatitic crystal structure of the mineral. High expression of alpha 1 (1) collagen mRNAs was detected in all polarized cells whereas dentin sialoprotein gene was mainly expressed in mineralizing areas. This cell culture system allowed for the differentiation of pulp cells into odontoblasts, at both the morphological and functional level. Moreover, these cells presented a spatial organization similar to the odontoblastic layer.

Gram-positive bacteria entering the dentinal tissue during the carious process are suspected to influence the immune response in human dental pulp. Odontoblasts situated at the pulp/dentin interface are the first cells encountered by these bacteria and therefore could play a crucial role in this response. In the present study, we found that in vitro-differentiated odontoblasts constitutively expressed the pattern recognition receptor TLR1–6 and 9 genes but not TLR7, 8, and 10. Furthermore, lipoteichoic acid (LTA), a wall component of Gram-positive bacteria, triggered the activation of the odontoblasts. LTA up-regulated the expression of its own receptor TLR2, as well as the production of several chemokines. In particular, an increased amount of CCL2 and CXCL10 was detected in supernatants from LTA-stimulated odontoblasts, and those supernatants augmented the migration of immature dendritic cells in vitro compared with controls. Clinical relevance of these observations came from immunohistochemical analysis showing that CCL2 was expressed in vivo by odontoblasts and blood vessels present under active carious lesions but not in healthy dental pulps. In contrast with this inflammatory response, gene expression of major dentin matrix components (type I collagen, dentin sialophosphoprotein) and TGF-β1 was sharply down-regulated in odontoblasts by LTA. Taken together, these data suggest that odontoblasts activated through TLR2 by Gram-positive bacteria LTA are able to initiate an innate immune response by secreting chemokines that recruit immature dendritic cells while down-regulating their specialized functions of dentin matrix synthesis and mineralization.

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Transforming growth factor-beta1 (TGF beta1) is a potent modulator of tissue repair in various tissues. To analyze its role during human dental repair, we used thick-sliced teeth cultured as described previously (Magloire et al., 1996). The supply of TGF beta1 to the pulp tissue was accomplished by means of a small tube glued onto the dentin. We show that this device allowed the growth factor to diffuse locally through dentinal tubules and to bind to the cells present in the coronal pulp opposite the TGF beta1-delivery tube. The tube was filled with 20 ng/mL TGF beta1, and slices were cultured for 4 days. Results show a preferential accumulation of cells in the odontoblastic and subodontoblastic layers in the vicinity of the tube. Cell proliferation increased in the subodontoblastic layer and in the underlying pulp, and BrdU-positive cells were abundant around the blood vessels. TGF beta1 induced type I collagen production by the odontoblastic/subodontoblastic/pulp cells in the stimulated zone, as demonstrated by in situ hybridization. These results suggest that TGF beta1 could be directly involved in the regulation of cell proliferation, migration, and extracellular matrix production in the human dental pulp and eventually in the repair process occurring after tooth injury.

Odontoblasts are responsible for the dentin formation. They are suspected to play a role in tooth pain transmission as sensor cells because of their close relationship with nerve, but this role has never been evidenced. We demonstrate here that human odontoblasts in vitro produce voltage-gated tetrodotoxin-sensitive Na ؉ currents in response to depolarization under voltage clamp conditions and are able to generate action potentials. Odontoblasts express neuronal isoforms of ␣2 and ␤2 subunits of sodium channels. Co-cultures of odontoblasts with trigeminal neurons indicate a clustering of ␣2 and ␤2 sodium channel subunits and, at the sites of cell-cell contact, a co-localization of odontoblasts ␤2 subunits with peripherin. In vivo, sodium channels are expressed in odontoblasts. Ankyrin G and ␤2 co-localize, suggesting a link for signal transduction between axons and odontoblasts. Evidence for excitable properties of odontoblasts and clustering of key molecules at the site of odontoblast-nerve contact strongly suggest that odontoblasts may operate as sensor cells that initiate tooth pain transmission.

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Abstract. Pulp tissue responds to dentin injury by laying down reactionary dentin secreted by existing odontoblasts or reparative dentin elaborated by odontoblast-like cells that differentiated from precursor cells in the absence of inner dental epithelium and basement membrane. Furthermore, growth factors or active dentin matrix components are fundamental signals involved in odontoblast differentiation. In vitro, dental pulp cells cultured under various conditions are able to express typical markers of differentiation, but no culture system can re-create pulp response to dentin drilling. This paper reports the behavior of thick slices from human teeth drilled immediately after extraction and cultured from 3 days to 1 month. Results show that the damaged pulp beneath the cavity is able to develop, in vitro, some typical aspects correlated to tissue healing, evidenced by cell proliferation (BrdU-positive cells), neovascularization (positive with antitype-IV collagen antibodies), and the presence of functional (3H proline-positive) cuboidal cells close to the injured area.After 30 days of culture, elongated spindle-shaped cells can be seen aligned along the edges of the relevant dentin walls, whereas sound functional odontoblasts are well-preserved beneath healthy areas. This tissue recovery leads us to believe that such a culture model will be a useful system for testing factors regulating pulp repair.