Fluoride concentration in dentin of exfoliated primary teeth as a biomarker for cumulative fluoride exposure.

A biomarker for lifetime fluoride exposure would facilitate population-based research and policy making but currently does not exist. This study examined the suitability of primary tooth dentin as a biomarker by comparing dentin fluoride concentration and fluoride exposures. Ninety-nine children's exfoliated primary teeth were collected from 2 fluoridated and 2 fluoride-deficient communities in North Carolina. Coronal dentin was isolated by microdissection and fluoride concentration assayed using the microdiffusion, ion-specific electrode technique. Information on children's fluoride exposures since birth from drinking water, toothpaste, supplements, rinses, food and beverages was collected by a self-reported questionnaire administered to caregivers. Only a small portion of the variance (10%) in incisor dentin fluoride (mean 792, SD 402 mg/kg) was accounted for by the best linear regression model as evaluated by the adjusted R(2). A moderate portion of the variance (60%) of molar dentin fluoride (mean 768, SD 489 mg/kg) was predicted by dietary fluoride supplement exposures, community of residence, and frequent tea consumption. Results for molars suggest that primary tooth dentin concentration may prove to be a satisfactory biomarker for fluoride exposure.

The immunohistochemical localization of Bax and Bcl-2 and their relation to apoptosis during amelogenesis in developing rat molars.

Bax and Bcl-2 are members of a family of intracellular, membrane-associated proteins that regulate programmed cell death. It has been suggested that, when Bax predominates, programmed cell death is accelerated and the apoptosis inhibitory activity of Bcl-2 is suppressed. The present study was undertaken to immunohistochemically (IHC) localize Bax and Bcl-2 in the cells of the enamel organ during amelogenesis in rat molars. Also, apoptotic cells were detected by TUNEL staining. The IHC intense localization of Bcl-2 and light staining for Bax in the pre-ameloblasts suggest that apoptosis is inhibited in the proliferating pre-ameloblasts. This is consistent with an absence of TUNEL staining for apoptosis in these cells. However, in the late secretory and transition ameloblasts, and adjacent stratum intermedium, evidence of apoptosis of the ameloblasts was observed. Bax and Bcl-2 were co-localized in the proximal ends of late secretory, transition and early maturation-stage ameloblasts, but immunoreactivity for Bax markedly increased in the proximal ends of late secretory and transition ameloblasts, while the Bcl-2 staining appeared to be lighter. This suggests that Bax antagonized Bcl-2 function, limiting the ability of Bcl-2 to prolong cell survival. In the early maturation stage, Bax staining faded while the immunoreactivity for Bcl-2 increased. Evidence of distinct apoptosis was reduced in the early maturation stage ameloblasts. When related to the occurrence of apoptosis during amelogenesis, the relative intensity of expression of Bax and Bcl-2 changed in a pattern consistent with that observed in other cell lines. This indicates that these proteins play essential roles in the process of amelogenesis, as predicted by their proposed mechanisms of action in the control of apoptosis.

Immunohistochemical localization of Galphaq, PLCbeta, Galphai1-2, PKA, and the endothelin B and extracellular Ca2+-sensing receptors during early amelogenesis.

Antibodies specific to Galphaq, PLCbeta, Galphai 1-2, and PKA were immunohistochemically (IHC) localized in the pre-ameloblasts up to initial dentin matrix deposition and continued in the distal ends of the pre-secretory ameloblasts to the beginning of enamel matrix secretion. It was hypothesized that the endothelin B receptor (ETBR) and/or the extracellular Ca2+-sensing receptor (CaR) would localize in the same locations as their known downstream signal transduction pathway (STP) effectors during events related to early amelogenesis. Localization was similar for the 4 signal transduction pathway elements and the CaR. The ETBR was not localized in any of the cells of the enamel organ. These findings indicate that the CaR and its related STPs are expressed in the pre-ameloblasts and pre-secretory ameloblasts in positions where they may be able to detect increases in extracellular Ca2+ concentrations observed in the pre-dentin matrix in a previous study. These observations are consistent with the hypothesis that increased levels of free Ca2+ in the pre-dentin matrix serve as a primary signal for modification of gene expression important to amelogenesis.

Fluoride release from restorative materials and its effects on dentin demineralization.

As the use of adhesive restorative materials has increased during the last several years, interest in adhesive materials that release fluoride has also grown. The purpose of this study was to measure fluoride release from several adhesive restorative materials and to evaluate its effect on dentin resistance to demineralization and on bacterial metabolism in a modified in vitro system. Standardized cavities (1.8 mm in diameter) were prepared in bovine teeth that had been ground to dentin. One cavity in each tooth was restored with one of the following restorative systems: (a) Single Bond and Z100; (b) Single Bond and Tetric Ceram; (c) Fuji Bond LC and Z100; (d) Fuji Bond LC and Tetric Ceram; (e) Fuji II LC; or (f) Fuji IX GP. The other cavity in each tooth was "restored" with wax as a control. For each restorative system, 12 specimens were evaluated for fluoride release during the first 24 hrs after restoration placement. Dentin adjacent to the restored sites was subjected to lactic acid challenge (pH 4.3) for 3 hrs, after which calcium release was measured. Another 12 specimens in each group were stored for 24 hrs in de-ionized water, and were exposed to an S. mutans suspension (1:1 THB/de-ionized water and 50 mM glucose, A660 = 0.2) for 6 hrs, followed by calcium release and pH measurement. Bulk specimens of each material were also made and stored in water. Fluoride released from Fuji Bond LC, Fuji IX GP, and Fuji II LC in bulk was significantly greater than from the other materials. In the restored dentin specimens, increased resistance to demineralization from a lactic acid challenge was directly related to fluoride release. The same effects were seen as a result of the S. mutans challenge. While fluoride release from restorative materials increased the resistance of dentin to demineralization in this system, the clinical relevance of the findings is not known.

The immunohistochemical localization of phospholipase Cgamma and the epidermal growth-factor, platelet-derived growth-factor and fibroblast growth-factor receptors in the cells of the rat molar enamel organ during early amelogenesis.

Findings on the localization and possible roles of the major growth factors, epidermal (EGF), platelet-derived (PDGF) and fibroblast (FGF) in early amelogenesis are contradictory and inconclusive. This study sought to localize immunohistochemically phospholipase (PLCgamma) and the EGF, PDGF and FGF receptors in the cells of the enamel organ during the events leading directly to early enamel formation in rat molars. PLCgamma is an immediate, downstream, signal-transduction pathway effector unique to the three receptors. A whole-head, freeze-dried sectioning method was used to reduce the possibilities of false-negative staining. A modification of the avidin/biotin complex method of immunohistochemical localization was used. Anti-PLCgamma and antibodies to each of EGF, PDGF and FGF receptors colocalized in the preameloblasts of the cervical loop, adjacent to the undifferentiated mesenchymal cells of the dental pulp. This staining disappeared shortly after the beginning of dentine mineralization. Staining for all four antibodies appeared on the proximal ends of the differentiating presecretory ameloblasts at the level of the beginning of predentine matrix deposition and continued in the secretory ameloblasts. It appears that EGF, PDGF and FGF have roles in the differentiation of ameloblasts and in control of cellular functions in presecretory and secretory ameloblasts. Their roles may represent redundancy of the kind seen in highly conserved tissues.

The immunohistochemical localization of the interferon-gamma and granulocyte colony-stimulating factor receptors during early amelogenesis in rat molars.

Previous studies, in which the known janus kinase and signal transducer and activator of transcription (STAT) isoforms were immunohistochemically mapped in developing rat molars, implicated a sizeable list of cytokine superfamily receptor (CSR)/signal-transduction pathway (STP) linkages in the cells of the enamel organ involved in the events leading directly to early amelogenesis. Various combinations of upregulated janus kinases and STATs are known to be linked to single or small groups of CSRs. On the basis of the previous observations it was hypothesized that the interferon-gamma receptor (IFNgamma r) and the granulocyte colony-stimulating factor receptor (G-CSF receptor) would be localized in specific sites in the cells of the enamel organ during early amelogenesis. To verify this, whole-head, freeze-dried sections were here obtained at the level of the mandibular first and second molar from newborn and 5-day-old rats. These sections were not demineralized or fixed, reducing the possibility of false-negative results. Antibodies to the IFNgamma r and the G-CSF receptor were localized using a modification of the avidin-biotin complex method. In the newborn rats, IFNgamma r was localized in the preameloblasts in the cervical loop, the proximal and distal ends of presecretory ameloblasts, the outer enamel epithelium, the dental lamina, and in bone. In 5-day-old rats, it was confined to the proximal ends of the presecretory and secretory ameloblasts. The G-CSF receptor was observed in the molars of newborn rats in the preameloblasts, the proximal and distal ends of the presecretory ameloblasts, outer enamel epithelium, and in bone. In 5-day-old rats, G-CSF receptor was localized in the preameloblasts, the proximal ends of presecretory and secretory ameloblasts, the stellate reticulum, the outer enamel epithelium, and in bone. These findings indicate that the IFNgamma r and the G-CSF receptor, and their downstream STP linkages, are upregulated in the cells of the enamel organ and may be involved in the events leading directly to early enamel formation.

A survey of private pediatric dental practices in North Carolina.

PURPOSE: In response to concerns about current and future demands for specialized pediatric dental care in North Carolina, a survey of private pediatric dental practices was conducted. METHODS: Data were collected on the demographics and other practice variables. Information was also collected on the ages, caries activity, Medicaid status, estimated treatment needs, fluoridation status, and location of residence (urban/rural) of all new patients seen in each practice during three designated, consecutive days in November 1996. RESULTS: The survey response rate was 76%. The data indicated that most pediatric dentists in North Carolina are quite busy. A total of 519 new patients were seen during the three-day survey period. The mean age was 4.7 years and 22% had advanced caries. Forty seven percent were caries free. Most of the disease was found in the primary dentitions of young children. CONCLUSIONS: The findings indicate that the specialized pediatric dental care system in North Carolina is operating close to its capacity and is overtaxed in many areas of the state.

Mercury exposure due to environmental factors and amalgam restorations in a sample of North Carolina children.

PURPOSE: Dental amalgam restorations provide a potential source for mercury (Hg) exposure in children. This study explored the possibility that Hg levels in dentin of exfoliated primary maxillary canines could detect cumulative Hg exposure from amalgam restorations in a sample of North Carolina children. METHODS: Twenty-seven exfoliated maxillary canines from 3.3 children, without restorations or caries, were assayed for dentin Hg concentration ([Hg]). Urine samples were obtained from 21 subjects and assayed for [Hg] and diet surveys for seafood ingestion were completed for 26 subjects. A surface/month exposure index (SMEI) was compiled from dental records to quantify each child's cumulative exposure to amalgam restorations. RESULTS: Results showed that dentin [Hg] ranged from undetectable levels to 15.7 ppm with a mean of 3.7 ppm. The SMEI scores ranged from 0-638 with a mean of 95. Ten subjects had low SMEI scores of 0-3, nine had scores 4-100, and eight had scores higher than 100. No statistical correlation was found for SMEI scores and dentin [Hg]. Urine Hg levels were found to be negligible and no relationship was found between urine [Hg] and reported ingestion of seafood or SMEI scores. CONCLUSIONS: Hg exposure in this sample of children was low and additional exposure from amalgam restorations could not be detected by the methods used in this study.

Dental wear and growth performance in steers fed sweetpotato cannery waste.

OBJECTIVE: To determine whether feeding sweetpotato cannery waste (SPCW) to cattle had adverse effects on dental wear, growth performance, or ruminal tissues. DESIGN: Clinical trial. ANIMALS: 36 Holstein steers. PROCEDURE: Steers were assigned to 1 of 3 groups. All steers received ryegrass hay ad libitum. In addition, steers in group 1 were fed 3.2 kg of corn and soybean meal/steer/d, steers in group 2 were fed 0.45 kg of soybean meal/steer/d and SPCW ad libitum, and steers in group 3 were fed a mixture of SPCW and broiler litter ad libitum. Samples of rumen fluid were collected on day 56. Steers were slaughtered on day 84, and samples of rumen were submitted for histologic examination. Teeth from control steers were removed, and calcium ion loss in response to etching with 2.28% lactic acid solutions buffered to pH of 3.75, 4.0, 4.25, 4.5, and 4.75 was determined. RESULTS: Average daily gain was lower for steers fed SPCW than for steers in the other 2 groups. Steers fed the SPCW-broiler litter mixture had only mild increases in tooth wear and tooth color scores, compared with control steers, whereas steers fed unbuffered SPCW had substantial increases in tooth wear and tooth color scores. Histologic abnormalities were detected in rumens from steers fed diets containing SPCW. Calcium ion loss decreased as pH of the etching solution increased. CLINICAL IMPLICATIONS: Results indicate that feeding cattle unbuffered SPCW can cause dental erosion, ruminal epithelial changes, and poor growth; however, SPCW buffered with broiler litter can be used as a cattle feed.

Fluoride varnish: a useful new tool for public health dentistry.

Since first shown to be effective by Bibby in 1942, professionally applied topical fluorides have been used successfully as a caries-preventive intervention. In the United States, the acidulated phosphate fluoride (APF) gels have been the most widely used agent for over two decades. While effective, APF has several practical disadvantages, including unpleasant taste and the risk of fluoride overingestion. The use of APF on infants and very young children is not practical or safe. Recently, fluoride varnish has become available in the United States. It has been used widely in Europe and Scandinavia for 25 years. Its effectiveness and safety are documented in over 50 clinical trials. Fluoride varnish is easy to apply, is well accepted by children, and eliminates the risk of overingestion of fluoride. Fluoride varnish is approved by the FDA as a "device" and must be used "off label" for the prevention of caries. Because of the large body of published data documenting its effectiveness and safety, there is no legal risk in using fluoride varnish off label. In fact, the American Dental Association has granted its seal of approval to Duraphat, one of the varnish products. Varnish offers considerable advantages in the dental public health setting. Of particular note, it is practical and safe to apply to the teeth of infants and very young children.

In vitro effects of an acidic by-product feed on bovine teeth.

OBJECTIVE: To evaluate in vitro erosive effects of sweet potato cannery waste (SPCW) on bovine incisor enamel. SAMPLE POPULATION: 20 bovine mandibles. PROCEDURE: Mandibles were collected and incisors were classified into 3 categories: lacking observable wear, advanced normal wear, or abnormal wear associated with feeding SPCW. Intact mandibles were radiographed. Contralateral normal teeth from the same jaw were used to compare Ca2+ loss (etching) with SPCW, lactic acid (pH 3.2), or SPCW neutralized with NaOH to pH 5.0 or 5.5. Scanning electron microscopy was performed to compare etched and unetched specimens. Two abnormally worn teeth were evaluated histologically. Knoop hardness testing was conducted on unexposed areas of surface enamel and enamel exposed to SPCW. RESULTS: Radiography revealed large periapical abscesses in the mandibles exposed to SPCW. Nearly identical amounts of Ca2+ were removed by SPCW and lactic acid solution at the same pH. Scanning electron microscopy did not indicate consistent differences between etch patterns resulting from exposure to SPCW or lactic acid. Mean rate of calcium removal was 56% higher in deciduous than permanent teeth. Knoop hardness data suggested that softening occurred in enamel exposed to SPCW. Neutralizing SPCW to pH 5.5 eliminated calcium removal. Histologic examination of sections indicated that SPCW degraded and removed some dentin matrix proteins. CONCLUSIONS: Exposure to SPCW results in enamel erosion in vitro; low pH is the most likely cause of erosion. Neutralizing SPCW to pH 5.5 eliminated erosive effects. CLINICAL RELEVANCE: Confirmation of SPCW's erosive effects on enamel in vitro supported the field diagnosis.

Protein characterization of fluorosed human enamel.

Despite extensive investigation, the development mechanism or mechanisms resulting in dental fluorosis are unknown. Several hypotheses suggest abnormal matrix synthesis, secretion, and delayed and/or defective matrix degradation with retention of enamel protein. The purpose of this study was to characterize the protein composition of fluorosed human enamel. Nine permanent moderately fluorosed (developed in a 3.2 ppm H2O area) and ten permanent normal control teeth (from individuals with < 0.2 ppm F in their drinking water) were evaluated. The enamel fluoride concentration, protein content, and amino acid composition were determined for each tooth. The enamel proteins were further characterized by gel electrophoresis and by Western blot analysis by means of polyclonal antibodies raised against recombinant amelogenin protein. Fluorotic enamel had significantly elevated (p = 0.0001) F levels compared with normal enamel (mean [F-] fluorosed = 431 ppm; mean [F-] control = 62 ppm). While there was a significantly greater protein content by weight in fluorosed enamel compared with normal enamel (mean fluorosed = 0.27%; mean control = 0.11%), the amino acid profiles were similar for fluorosed and normal enamel. Gel electrophoresis showed fluorosed enamel to have a greater diversity of primarily low-molecular-weight proteins compared with normal enamel. Western blot analysis did not indicate retention of amelogenin in either fluorosed or normal enamel. This investigation showed that the protein content of fluorosed enamel was greater than that of normal enamel; however, the amino acid compositions were similar for fluorosed and normal enamel. Furthermore, there does not appear to be retention of significant amounts of amelogenin in fully mature, moderately fluorosed human enamel. Although delayed removal of the enamel matrix proteins may play a role in the hypomineralization defects seen in fluorosed enamel, the majority of these proteins are absent in the mature tissue of these moderately fluorosed teeth.

The immunohistochemical localization of stat-2, -3, -4 and -5 during early enamel and dentine formation in rat molars.

STATs (signal transduction and activators of transcription) are key components of the signal transduction pathways in the cytokine receptor superfamily-linked pathway. STATs are activated directly by members of the Jak (Janus kinase) family and, when activated, migrate to the nucleus to modify gene expression to produce a variety of cellular responses. Individual cytokines activate specific combinations of the Jak/STAT isoforms. A previous study localized the known Jak isoforms and STAT-1 in 5-day-old rat molars during the early stages of enamel and dentine formation. The present study was undertaken to localize immunohistochemically STAT isoforms STAT-2. -3, -4 and -5 in association with events involved in early dentine and enamel formation in 5-day-old rat molars. Each of the isoform localization patterns was different from the others. Combining the results of the previous study with the present findings, it appears that all of the known Jaks and STATs-1, -2, -3, -4 and -5 are located in the cells directly involved in early enamel or dentine formation. Using colocalization patterns of the individual Jaks and STATs, individual receptor locations may be predicted. In the proximal ends of differentiated ameloblasts, several cytokine receptors [interleukin (IL) -5, -6, -7, -9, -10, -12, growth hormone granulocyte colony-stimulating factor interferon-alpha/beta. -gamma] are predicted. In other areas of the early odontogenic cells, the proximal ends of differentiating ameloblasts are predicted to have IL-7 receptors, inner enamel epithelium IL-6 and IL-10 receptors, and stratum intermedium cells IL-6 receptors. In the early developing dentine, differentiating odontoblasts are predicted to have IL-6 and IL-10 receptors, and differentiated odontoblasts no cytokine receptors identified by known Jak/STAT combinations. Mapping of the Jak and STAT isoforms in the cells involved in early enamel and dentine formation indicates that a sizeable list of ligands and their respective cytokine receptor/pathway complexes are involved in the regulation of these processes.

Immunohistochemical localization of signal transduction pathways during amelogenesis: an initial exploration.

This study was undertaken to map signal transduction pathway (STP) components uniquely associated with the four major receptor groups and their related STPs in association with the events involved in amelogenesis in the rat. Whole-head, freeze-dried sagittal sections were obtained at the level of the maxillary first molars and picked up on transparent adhesive tape. The sections were not decalcified or fixed, providing optimum conditions for immunohistochemical (IHC) localization. Antibodies to pathway components Gs alpha, Gi alpha, Gq alpha, Sos-1, Grb-2, p125Fak, Jak2, and Vav were localized. The respective patterns of localization indicate that the Gq alpha-linked, the receptor tyrosine kinase-initiated, and the integrin receptor-initiated pathways are involved in the proliferating pre-ameloblast cells. In the differentiating and differentiated ameloblasts, the Gs alpha-linked cAMP pathway is involved, apparently reading a factor(s) released by the dentin matrix. The Gq alpha-linked, the receptor tyrosine kinase-initiated, the integrin receptor-initiated, and the cytokine receptor-initiated pathways are also up-regulated in the proximal ends of the ameloblasts. These observations indicate that all four of the major receptor groups are involved in amelogenesis and that the role of classes of ligands not previously implicated in enamel formation must now be considered. It seems that the cells of the enamel organ respond to the appearance and disappearance of autocrine and paracrine growth factors, but they also up-regulate specific STPs to enable them to respond to circulating hormones and growth factors whose concentrations in the extracellular fluids remain relatively constant.

The immunohistochemical localization of signal-transduction pathway components Jak1, Jak2, Jak3, Tyk2 and STAT-1 during early enamel and dentine formation in rat molars.

This study sought to localize immunohistochemically Janus kinase (Jak) and Tyk isoforms and STAT-1 in association with events involved in early dentine and enamel formation in the rat molar. The Jaks and STATs (signal transducers and activators of transcription) are key signal-transduction pathway components in the cytokine receptor-linked pathway. The histological sections were not demineralized or fixed, providing optimum conditions for immunohistochemical localization. It appears that all of the Jak isoforms and STAT-1 are involved in enamel formation. Jak2 and STAT-1 colocalized in the proximal ends of presecretory and secretory-stage ameloblasts, supporting work by others that growth hormone receptor is located at that site. The colocalization of Jak1, Jak2 and STAT-1 along the proximal ends of presecretory and secretory ameloblasts suggests that the interferon receptor is up-regulated in these cells as well. Also, colocalization of Jak3 and STAT-1 in the proximal ends of the ameloblasts and the cells of the stratum intermedium predicts the location of the interleukin-7 receptor in those locations. Jak1, Tyk2 and STAT-1, but not Jak2 or Jak3, stain was seen in the odontoblasts.

Expression of amelogenin mRNA sequences during development of rat molars.

The expression of amelogenin mRNA in growing rat molars was studied. Northern blotting and the analysis of cDNA isolates revealed two predoninant variants. One group of cDNA inserts contained sequences of a long mRNA version and the other group contained mRNA sequences of the shorter leucin-rich amelogenin polypeptide (LRAP). The LRAP group was deficient in an internal stretch which coded for a peptide with a high potential for beta turns. Northern blot experiments showed that most amelogenin RNA in rat teeth was represented by two bands of 1.1 and 0.8 kb. Two oligonucleotide probes were designed that were specific for the long version and for the LRAP variant. The probes were used for in situ hybridization experiments on sections of developing maxillar teeth of rats between day 2 and day 15 after birth. Both RNA species were accumulated concomitantly and exclusively in cells of the inner enamel epithelium. Expression was first observed at the mesial cusp sides and finally involved the whole ameloblast layer except for the cells adjacent to the enamel-free region at the tip of the cusps. The early amelogenin RNA expression occurred adjacent to the initial deposition of the dentin matrix. Low amounts of amelogenin RNA persisted after the differentiation of ameloblasts into the maturative stage. The sequence of events was similar in all three molars.

Problems associated with estimation of net calcium uptake during enamel formation using 45Ca.

45Ca uptake in mineralizing tissues may occur by net Ca uptake or by isotopic exchange. It is rarely possible to differentiate between these effects, making interpretation of the findings difficult. Unfortunately, this problem is not often considered, and 45Ca uptake is usually regarded as representative of only net calcium uptake. The study reported here was undertaken to estimate the extent to which 45Ca uptake in mineralizing enamel is due to net Ca deposition or to isotopic exchange, and to consider the implications. The enamel surfaces of the lower incisors of adult rats were notched at the gingival line, and the eruption distance over 16 hours was measured. This distance was used to establish the position of a 0.3-mm-wide increment of enamel at the beginning and end of the 16-hour period, during which it passed through the early-maturation stage of enamel formation. The rate of Ca uptake was determined by chemical assay. Other rats were injected with 45Ca, mean plasma specific activity values for the experimental period determined, and the rate of Ca uptake through the same area of enamel formation was estimated. The estimates were from two- to nearly ten-fold greater than those established by chemical assay, indicating that from 50 to 90% of the 45Ca uptake occurred by isotopic exchange. 45Ca uptake may indicate more about the labile state of Ca in mineralizing enamel than about the rate of mineral deposition.

Distribution of protein kinase C alpha and accumulation of extracellular Ca2+ during early dentin and enamel formation.

Activation of the protein kinase C (PKC)-related signal transduction system has been associated with phenotypic expression in a wide variety of cell types. In in vitro studies, it has often been activated by relatively small increases in the Ca2+ concentration ([Ca2+]) in the medium. The studies reported here explored the hypothesis that localized increases in the extracellular [Ca2+] and activation of the PKC-related pathway may be involved in early dentin and enamel formation. Whole-head, freeze-dried sections through the developing molars of 5-day-old rats were evaluated by methods that localized non-crystalline Ca2+. Immunohistochemical methods were adapted for use with the freeze-dried sections, and two monoclonal antibodies were used to localize PKC alpha in the formative cells of the developing teeth. Low concentrations of extracellular Ca2+ were observed in the early, unmineralized dentin in the area of ameloblast differentiation. Increased concentrations occurred at the point of initial dentin mineralization, immediately before the beginning of enamel matrix deposition. PKC alpha was localized in the differentiating odontoblasts, at the beginning of dentin matrix deposition. It was intensely localized in the distal borders of the pre-ameloblasts, and appeared to redistribute in the cells during ameloblast differentiation. These observations suggest that local increases in the extracellular [Ca2+] and the PKC signal transduction pathway may be involved in key inductions in the early stages of dentin and enamel formation.