Odontogenic ameloblast-associated protein nature of the amyloid found in calcifying epithelial odontogenic tumors and unerupted tooth follicles.

We have previously reported that the amyloid found in three patients with calcifying epithelial odontogenic tumors (CEOT) was composed of N-terminal fragments of a putative 153-residue protein specified by a gene designated FLJ20513 now known to represent exons 5 through 10 of the odontogenic ameloblast-associated protein (ODAM) locus that encodes a 279-residue polypeptide. Confirmation of the amyloidogenic potential of ODAM has resulted from analyses of four other cases where we found, in addition, a 74-residue segment specified by exon 4. Through preparation of ODAM-related synthetic peptides, it was possible to localize the fibril-forming region of this molecule, as well as generate a monoclonal antibody that reacted specifically with the amyloid associated with CEOT. Notably, we also detected green birefringent congophilic material in unerupted tooth follicles - a precursor of CEOT - and demonstrated through immunologic and chemical analyses the ODAM nature of the deposits. Our studies have provided further evidence for this unique form of odontogenic amyloid that we provisionally designate "AODAM".

MIB-1 expression in odontogenic epithelial rests, epithelium of healthy oral mucosa and epithelium of selected odontogenic cysts. An immunohistochemical study.

The aim of this study is to investigate the proliferative potential of rests of odontogenic epithelium found in follicles of unerupted teeth, epithelium of oral mucosa and epithelial linings of various odontogenic cysts. MIB-1 expression was studied in the rests of odontogenic epithelium (n=10), healthy oral mucosa (n=10), odontogenic keratocysts (n=10) and other odontogenic cysts (n=10) using an avidin-biotin peroxidase technique on paraffin sections. The number of positively stained cells was counted on 10 representative areas of epithelium using a x40 objective. The average number of MIB-1 positive cells in each group was calculated. No MIB-1 positive cells were seen in the rests of odontogenic epithelium. The mean numbers of MIB-1 positive cells detected within the epithelium of oral mucosa, and of radicular and dentigerous cysts were similar. The number of MIB-1 positive cells was found to be increased in the presence of marked inflammatory cell infiltration. The highest number of MIB-1 positive cells was seen in the keratocysts. These findings suggest that removal of an unerupted tooth to prevent the possibility of neoplastic transformation of rests of odontogenic epithelium is not a justifiable rationale.

Is fluoride concentration in dentin and enamel a good indicator of dental fluorosis?

Despite some studies correlating dental fluorosis (DF) and fluoride (F) concentration in dental enamel, no information is available about DF and dentin F concentration. Our objective was to determine the correlation between teeth F concentration and DF severity in unerupted human 3rd molars, and the correlation between dentin and enamel F concentrations in the same tooth. Ninety-nine 3rd molars were studied-53 from Fortaleza, Brazil (F water, 0.7 ppm), 22 from Toronto (1.0 ppm), and 24 from Montreal (0.2 ppm). DF severity was evaluated according to the Thylstrup-Fejerskov Index, while F concentration was analyzed by Instrumental Neutron Activation Analysis. DF severity varied between TF0 and TF4, while F concentration ranged between 39 and 550 ppm in enamel and 101 and 860 ppm in dentin. Our results showed correlation between dentin F concentration and DF (r(S) = 0.316, p = 0.001), but no correlation between enamel F concentration and DF (r(S) = 0.154, p = 0.133). No correlation was observed between dentin and enamel F concentrations in the same tooth (r(S) = 0.064, p = 0.536).

Development of a novel mouse osteoclast culture system including cells of mandibular body and erupting teeth.

Osteoclasts are multinucleated cells with the specialized function of resorbing calcified tissues. These cells develop from hemopoietic cells of the monocyte-macrophage lineage with the support of osteoblasts/stromal cells. Tooth eruption is a vertical movement of teeth via creation of an eruption pathway in and through the alveolar bone. The precise cellular and molecular determinants of tooth eruption are not yet clear, and a cell culture system that can reproduce the activity of osteoclast formation during tooth eruption is expected to be a useful tool to clarify the mechanism of eruption pathway formation. To this end, mandibular bodies, including incisors and molars, were isolated from 9- to 11-day-old mice undergoing active tooth eruption. Primary cells were obtained from mandibular bodies by enzymatic digestion and cultured in alphaMEM containing 15% FBS without any cytokine or growth factor or hormone in the culture (AFT culture, for alveolar bone, dental follicle, and tooth). A progressive increase in the number of tartrate-resistant acid phosphatase-positive multinucleated osteoclastic cells was observed in AFT culture. The osteoclastic cells generated were immunopositive for cathepsin K and calcitonin receptor, and formed resorption pits when cultured on dentine slices. Parathyroid hormone-related protein (PTHrP), expressed by the enamel organ of tooth, is reported to be an essential factor in creation of the eruption pathway. To verify this point, cells were isolated from mandibular bodies from which all teeth and dental follicles had been removed and cultured similarly (A culture, for alveolar bone). Osteoclastic cells were not formed and PTHrP production was hardly detected in the medium of A culture, in contrast to the high level of PTHrP in AFT culture. Since our previous study demonstrated that neonatal homozygous PTHrP-knockout mice show impaired osteoclastogenesis around tooth germs, AFT culture was performed by using this sample to examine whether this culture system can reproduce the status of osteoclastogenesis observed in vivo. The result showed that none of the osteoclastic cells were generated from cells of homozygous mice. We here report a novel mouse osteoclast culture system that reproduces the activity of osteoclast formation around erupting teeth without addition of any cytokine or growth factor or hormone to the medium. Histological examination of various transgenic and mutant mice now offers valuable findings on studies of tooth eruption and the present culture system using these animals would be a powerful tool in clarifying the cellular and molecular mechanisms of eruption pathway formation.

Study of the maturation of the organic (type I collagen) and mineral (nonstoichiometric apatite) constituents of a calcified tissue (dentin) as a function of location: a Fourier transform infrared microspectroscopic investigation.

Fourier transform infrared microspectroscopy (FTIRM) was used to investigate the organic and mineral phases of a calcified tissue (dentin) as a function of its location from predentin toward enamel. Thin dentin slices (decalcified or not) were fixed in formaldehyde and embedded in glycolmethylmethacrylate (GMA). Fixation did not denature collagen, and GMA did not interact with organic or mineral constituents of dentin. The v1v3 PO4 domain was studied in particular in order to estimate mineral maturity and amide I, II, A, and B to obtain data on protein conformation. The results showed that dentin apatite became increasingly mature (stoichiometric) from the mineralization front toward the enamel, especially through loss of HPO4(2-) groups and vacancies. Moreover, collagen fibrils became less and less hydrated, suggesting that intrafibrillar mineralization partially dehydrated the collagen. Combined study of the organic and mineral fractions of calcified tissues may help clarify their relationships in physiological and pathological tissues.

Alkali-soluble and insoluble fluoride in erupted and unerupted human enamel from a high fluoride area with a low fluorosis score.

The amounts of fluoroapatite and 'CaF2-like' fluoride (F) were determined in enamel of unerupted and erupted teeth that had been exposed in vivo to 1.8-2.6 parts/10(6) F in the drinking water and to brushing with F dentifrice at least once a day, and occasionally to a F mouth-rinse (0.022% F). Enamel was sampled by acid-etching and the F levels were measured with an adapted F ion-selective electrode. More F was built into the deeper enamel in the high-F area than in a similar low-F area. Unerupted enamel did not etch significantly (p > 0.05) deeper than erupted enamel. No significant differences (p > 0.05) were found in the F concentrations amongst the following: alkali-washed erupted, unwashed erupted, alkali-washed unerupted and unwashed unerupted at the outer most enamel (approx. 6 microns). However the erupted enamel (alkali-washed or not) showed higher F levels than unerupted enamel (alkali-washed or not) between approx. 6 microns and greater than 100 microns. The increase of F for this high-F area was about 100% in the deeper enamel while for a low-F area it was approx. 78% in the most outer enamel with no increase after a depth of about 20 microns. In contrast to a similar low-F area (water F < 0.10 parts/10(6)), no significant 'CaF2-like' F could be detected in erupted or unerupted enamel for the high-F area.

Alkali-soluble and insoluble fluoride in erupted and unerupted sound enamel of human third molars in vivo.

The amounts of firmly and loosely bound fluoride were determined in sound enamel of unerupted and erupted teeth which had been exposed in vivo for 1-16 yr to brushing at least once a day, and occasionally to mouth rinsing and the application of sealers. Enamel was sampled by an acid-etch procedure, and the fluoride levels were measured with an adapted fluoride ion-selective electrode. Unerupted enamel was etched significantly (p less than 0.05) deeper than erupted enamel up to a depth of at least 8 microns. Significant differences (p less than 0.05) were found between the mean enamel fluoride concentrations of unwashed and alkali-washed, erupted teeth up to a depth of at least 3 microns and also between unwashed or washed, erupted versus unwashed or washed, unerupted teeth. At a depth of 3 microns, the fluoride treatments of enamel had increased the total amount of fluoride by approx. 78% of which approx. 53% was loosely bound fluoride (like CaF2) and 47% firmly bound (like fluorapatite). No increase in sound enamel fluoride as a result of topical treatments over a period of up to 16 yr could be found at a level deeper than 20 microns.

Measurement of the permeability of dental enamel and its variation with depth using an electrochemical method.

To investigate the permeability of the dental enamel of erupted and unerupted human pre-molars and its variation with depth, we determined the resistivity of successive 100-micron-thick layers. The electrical resistance of halved tooth crowns at zero Hertz, which according to Scholberg et al. (1982, 1984) is inversely related to the permeability, was measured before and after removal of successive enamel layers from the outer surface toward the dentino-enamel junction (D.E.J.). The resistivity of the successive enamel layers increased from the D.E.J. toward the outer surface in approximately the same way in erupted as well as in unerupted pre-molars, except within the outermost layer of 100 to 200 microns thickness. The resistivity of this layer in erupted pre-molars was considerably higher than that in unerupted pre-molars, which may be due to post-eruptive mineralization.

Selenium uptake into teeth determined by fluorimetry.

Selenium concentrations in successive layers of bovine incisor enamel showed no concentration gradient and no increase with age. Selenium was taken upt into the continuously-growing rat incisor in proportion to the amount of selenium in the diet. A sensitive (approximately 1 ng Se) and relatively simple fluorimetric method for determining selenium in small amounts of hard tissues is presented.

Dental phosphoprotein-induced formation of hydroxylapatite during in vitro synthesis of amorphous calcium phosphate.

(Ethylenedinitrilo)tetraacetic acid soluble phosphoproteins were isolated from rat incisor and bovine unerupted teeth. This material was examined for its effect on the stability of amorphous calcium phosphate in vitro. When the precipitation of amorphous calcium phosphate was attempted in the presence of small amounts of these phospho-proteins, an apatite-like mineral was observed to form, which was approximately 60% crystalline, as determined by infrared measurements. This apatite phase could not be induced by addition of phosphoprotein after the precipitation reaction. The organic phosphate bound to these phosphoproteins was shown to be directly responsible for the formation of the apatite phase, since removal of 60% of the covalently bound phosphate with alkaline phosphatase destroyed the protein's ability to induce hydroxylapatite formation. The properties of the dental phosphoproteins appear to be consistent with their possible involvement in the development of the mineral phase of dentine.