Introduction of a three-dimensional and layered (TDL) culture, a novel primary co-culture method for ameloblasts and pulp-derived cells.

The enamel organ engaged in enamel matrix formation in tooth germs comprises four different cell types: the ameloblasts, the cells of the stratum intermedium, stellate reticulum, and the outer enamel epithelium, each characterized by distinct structural features. In ordinary primary cultures of tooth-derived cells, these cells generally become flat in profile and hardly regain their original profiles comparable to those in vivo, even under conditions that can induce the expression of functional markers from these cells. To overcome this limitation inherent to the cell culture of tooth-derived cells, we introduced a novel co-culture method, a "three-dimensional and layered (TDL) culture", a three-dimensional (3D) culture of dental pulp-derived cells dispersed in type I collagen gel combined with a layered culture of enamel epithelial cells seeded on top of the gel to establish thereby a culture condition where the functional tooth-derived cells regain their original structures and spatial arrangements. We subjected the TDL gels thus prepared to floating cultures and found that, in the layered epithelial cells, those facing the 3D gel became cuboidal/short columnar in shape, showed cell polarity and well-developed intercellular junctions, had PAS positive material in their cytoplasm, and expressed a distinct immunoreactivity for cyotokeratin 14 and amelogenins. Pulpal cells in the gel displayed a strong ALP activity throughout the 3D gel. The current observations have clearly shown that the structural and functional features reminiscent of early secretory ameloblasts could be restored in the enamel organ-derived cells in a TDL culture.

Development and terminal differentiation of pulp and periodontal nerve elements in subcutaneous transplants of molar tooth germs and incisors of the rat.

Ectopic tooth transplants are known to receive rich innervation of local neurons, but the precise location and structural features of neurites in the pulp and periodontal ligament (PDL) of such transplants are unclear. In this experiment, the molar tooth germs of rat embryos and incisors of young rats were subcutaneously transplanted into the dorsal regions of rats and processed, at various time intervals, for immunohistochemical demonstration of neural elements. Teeth with periodontal tissue elements developed in most of the molar transplants in 6 or 8 wk and received rich innervation, including some autonomic fibres, in the pulp. Nerve elements were also confirmed to be present in the PDL of these transplants, including specialized nerve ending-like structures reminiscent of the periodontal Ruffini endings. Mechanoreceptor-like structures were also induced in the regenerated PDL of similarly transplanted incisors, although the success rate was low. We conclude that rich and highly ordered innervation of the pulp, and occasional development of mechanoreceptors in the regenerated PDL of ectopic dental transplants, imply a high probability of successful induction of teeth with both nociceptive and mechanical sensations in the ectopic tooth and/or tooth germ transplant systems, although differentiation of mechanoreceptor-like nerve endings occurred in only a few rare cases.

Phosphatase actions at the site of appositional mineralization in bisphosphonate-affected bones of the rat.

Tissue-nonspecific alkaline phosphatase (TNSALP) and Ca-ATPase are known to play roles in bone mineralization, but how these enzymes contribute to appositional mineralization has been illusive. Here we examined the active sites of these enzymes in appositional mineralization using the bones of young rats being administered with 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) for 5 days. The doses of HEBP totally abolished mineralization of newly formed bone matrix except in matrix vesicles (MVs), and hence allowed precise localization of MVs and phosphatase reactions within non-mineralized extracellular matrix. Intense TNSALP and ATPase reactions were confirmed along the limited portions of osteoblast membranes where intimate cell-cell contacts were maintained. Diffuse reactions of these enzymes were throughout the osteoid implicating efflux of TNSALP and ATPase molecules into extracellular matrix from the osteoblast membranes. Phosphatase reactions associated with MVs varied both in intensity and location among the individual vesicles; newly formed MVs were almost free of reactions but appeared to gain those activities later in the osteoid. These data suggest that TNSALP and ATPase are released from the osteoblast membrane and later integrated into MVs within the osteoid. The osteoblasts may thus regulate appositional mineralization of bone from a distance at least in part by providing phosphatases via MVs.