Stages Of Enamel Formation Research Articles

ENAM Mutations Can Cause Hypomaturation Amelogenesis Imperfecta

Amelogenesis imperfecta (AI) is a diverse group of inherited diseases featured by various presentations of enamel malformations that are caused by disturbances at different stages of enamel formation. While hypoplastic AI suggests a thickness defect of enamel resulting from aberrations during the secretory stage of amelogenesis, hypomaturation AI indicates a deficiency of enamel mineralization and hardness established at the maturation stage. Mutations in ENAM, which encodes the largest enamel matrix protein, enamelin, have been demonstrated to cause generalized or local hypoplastic AI. Here, we characterized 2 AI families with disparate hypoplastic and hypomaturation enamel defects and identified 2 distinct indel mutations at the same location of ENAM, c588+1del and c.588+1dup. Minigene splicing assays demonstrated that they caused frameshifts and truncation of ENAM proteins, p.Asn197Ilefs*81 and p.Asn197Glufs*25, respectively. In situ hybridization of Enam on mouse mandibular incisors confirmed its restricted expression in secretory stage ameloblasts and suggested an indirect pathogenic mechanism underlying hypomaturation AI. In silico analyses indicated that these 2 truncated ENAMs might form amyloid structures and cause protein aggregation with themselves and with wild-type protein through the added aberrant region at their C-termini. Consistently, protein secretion assays demonstrated that the truncated proteins cannot be properly secreted and impede secretion of wild-type ENAM. Moreover, compared to the wild-type, overexpression of the mutant proteins significantly increased endoplasmic reticulum stress and upregulated the expression of unfolded protein response (UPR)–related genes and TNFRSF10B, a UPR-controlled proapoptotic gene. Caspase, terminal deoxynucleotidyl transferase UTP nick-end labeling (TUNEL), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays further revealed that both truncated proteins, especially p.Asn197Ilefs*81, induced cell apoptosis and decreased cell survival, suggesting that the 2 ENAM mutations cause AI through ameloblast cell pathology and death rather than through a simple loss of function. This study demonstrates that an ENAM mutation can lead to generalized hypomaturation enamel defects and suggests proteinopathy as a potential pathogenesis for ENAM-associated AI.

Mapping the Tooth Enamel Proteome and Amelogenin Phosphorylation Onto Mineralizing Porcine Tooth Crowns.

Tooth enamel forms in an ephemeral protein matrix where changes in protein abundance, composition and posttranslational modifications are critical to achieve healthy enamel properties. Amelogenin (AMELX) with its splice variants is the most abundant enamel matrix protein, with only one known phosphorylation site at serine 16 shown in vitro to be critical for regulating mineralization. The phosphorylated form of AMELX stabilizes amorphous calcium phosphate, while crystalline hydroxyapatite forms in the presence of the unphosphorylated protein. While AMELX regulates mineral transitions over space and time, it is unknown whether and when un-phosphorylated amelogenin occurs during enamel mineralization. This study aims to reveal the spatiotemporal distribution of the cleavage products of the most abundant AMLEX splice variants including the full length P173, the shorter leucine-rich amelogenin protein (LRAP), and the exon 4-containing P190 in forming enamel, all within the context of the changing enamel matrix proteome during mineralization. We microsampled permanent pig molars, capturing known stages of enamel formation from both crown surface and inner enamel. Nano-LC-MS/MS proteomic analyses after tryptic digestion rendered more than 500 unique protein identifications in enamel, dentin, and bone. We mapped collagens, keratins, and proteolytic enzymes (CTSL, MMP2, MMP10) and determined distributions of P173, LRAP, and P190 products, the enamel proteins enamelin (ENAM) and ameloblastin (AMBN), and matrix-metalloprotease-20 (MMP20) and kallikrein-4 (KLK4). All enamel proteins and KLK4 were near-exclusive to enamel and in excellent agreement with published abundance levels. Phosphorylated P173 and LRAP products decreased in abundance from recently deposited matrix toward older enamel, mirrored by increasing abundances of testicular acid phosphatase (ACPT). Our results showed that hierarchical clustering analysis of secretory enamel links closely matching distributions of unphosphorylated P173 and LRAP products with ACPT and non-traditional amelogenesis proteins, many associated with enamel defects. We report higher protein diversity than previously published and Gene Ontology (GO)-defined protein functions related to the regulation of mineral formation in secretory enamel (e.g., casein α-S1, CSN1S1), immune response in erupted enamel (e.g., peptidoglycan recognition protein, PGRP), and phosphorylation. This study presents a novel approach to characterize and study functional relationships through spatiotemporal mapping of the ephemeral extracellular matrix proteome.

Expression patterns of the Stim1 protein during different stages of mouse tooth development

Objectives: Stromal interaction molecule 1 (STIM1), a calcium sensor protein, was shown to be significantly up-regulated during the maturation stage of amelogenesis. This result highlighted a potentially important role for STIM1 in calcium ion handling during the latter stages of enamel formation. Our work aimed to study in detail the expression patterns of STIM1 during all stages of mouse tooth development.Material and Methods: C57BL mice were used for all the experiments in this study. The embryos were obtained from time mated pregnant mice. Embryonic day 0 (E0) was designated as the day on which a vaginal plug was confirmed. Embryos at E12.5, E14.5, E15.5 and pups at PNO, PN4, PN8 and PN14 were used to describe different developmental stages of incisors (E12.5 – E15.5) and molars (PN0 – PN14).Results: Stim1 signals start to be detectable in the cells of the enamel organ at the beginning of the cap stage. Its expression was similar between the incisor and molar tooth germs. Odontoblasts appeared completely negative, while there was some strong expression in the middle of the dental papilla. Conclusion: In our study, we have shown that Stim1 was most highly expressed in the ameloblasts during the maturation stage. This highly expression of Stim-1 at the maturation stages of enamel development supports the contention that stored operated calcium channels are central to Ca2+ transport/extrusion mechanism during the final phase of enamel crystal growth.

Towards unraveling the human tooth transcriptome: the dentome.

The goal of the study was to characterize the transcriptome profiles of human ameloblasts and odontoblasts, evaluate molecular pathways and advance our knowledge of the human “dentome”. Laser capture microdissection was used to isolate odontoblasts and ameloblasts from human tooth buds (15-20week gestational age) from 4 fetuses. RNA was examined using Agilent 41k whole genome arrays at 2 different stages of enamel formation, presecretory and secretory. Probe detection was considered against the array negative control to control for background noise. Differential expression was examined using Significance Analysis of Microarrays (SAM) 4.0 between different cell types and developmental stages with a false discovery rate of 20%. Pathway analysis was conducted using Ingenuity Pathway Analysis software. We found that during primary tooth formation, odontoblasts expressed 14,802 genes, presecretory ameloblasts 15,179 genes and secretory ameloblasts 14,526 genes. Genes known to be active during tooth development for each cell type (eg COL1A1, AMELX) were shown to be expressed by our approach. Exploring further into the list of differentially expressed genes between the motile odontoblasts and non-motile presecretory ameloblasts we found several genes of interest that could be involved in cell movement (FN1, LUM, ASTN1). Furthermore, our analysis indicated that the Phospholipase C and ERK5 pathways, that are important for cell movement, were activated in the motile odontoblasts. In addition our pathway analysis identified WNT3A and TGFB1 as important upstream contributors. Recent studies implicate these genes in the development of Schimke immuno-osseous dysplasia. The utility of laser capture microdissection can be a valuable tool in the examination of specific tissues or cell populations present in human tooth buds. Advancing our knowledge of the human dentome and related molecular pathways provides new insights into the complex mechanisms regulating odontogenesis and biomineralization. This knowledge could prove useful in future studies of odontogenic related pathologies.

The Molecular Basis of Hereditary Enamel Defects in Humans

The formation of human enamel is highly regulated at the molecular level and involves thousands of genes. Requisites for development of this highly mineralized tissue include cell differentiation; production of a unique extracellular matrix; processing of the extracellular matrix; altering of cell function during different stages of enamel formation; cell movement and attachment; regulation of ion and protein movement; and regulation of hydration, pH, and other conditions of the microenvironment, to name just a few. Not surprising, there is a plethora of hereditary conditions with an enamel phenotype. The objective of this review was to identify the hereditary conditions listed on Online Mendelian Inheritance in Man (OMIM) that have an associated enamel phenotype and whether a causative gene has been identified. The OMIM database was searched with the terms amelogenesis, enamel, dental, and tooth, and all results were screened by 2 individuals to determine if an enamel phenotype was identified. Gene and gene product function was reviewed on OMIM and from publications identified in PubMed. The search strategy revealed 91 conditions listed in OMIM as having an enamel phenotype, and of those, 71 have a known molecular etiology or linked genetic loci. The purported protein function of those conditions with a known genetic basis included enzymes, regulatory proteins, extracellular matrix proteins, transcription factors, and transmembrane proteins. The most common enamel phenotype was a deficient amount of enamel, or enamel hypoplasia, with hypomineralization defects being reported less frequently. Knowing these molecular defects allows an initial cataloging of molecular pathways that lead to hereditary enamel defects in humans. This knowledge provides insight into the diverse molecular pathways involved in enamel formation and can be useful when searching for the genetic etiology of hereditary conditions that involve enamel.

Transcriptional regulation of amelotin gene by proinflammatory cytokines in gingival fibroblasts

Amelotin (AMTN) is a secreted protein expressed during the late stages of enamel formation and in the junctional epithelium. Among many differentially expressed genes, we found significantly increased AMTN mRNA level in inflamed gingiva by DNA microarray. The inductions of AMTN mRNA expressions in inflamed gingiva and human gingival fibroblasts (HGF) were confirmed by real-time polymerase chain reaction. To determine the molecular basis of the expression of AMTN and its regulation by proinflammatory cytokines, we have isolated and characterized the promoter region of mouse AMTN gene. Transient transfection assays were performed using luciferase constructs including mouse AMTN gene promoter. Interleukin-1β, Interleukin-6 and tumor necrosis factor-α induced AMTN mRNA levels in HGF. These cytokines increased the luciferase activities of the AMTN promoter constructs in HGF. The results suggest that proinflammatory cytokines induce AMTN gene transcription and a role for AMTN in gingival inflammation.

WDR72 models of structure and function: A stage-specific regulator of enamel mineralization

Amelogenesis Imperfecta (AI) is a clinical diagnosis that encompasses a group of genetic mutations, each affecting processes involved in tooth enamel formation and thus, result in various enamel defects. The hypomaturation enamel phenotype has been described for mutations involved in the later stage of enamel formation, including Klk4, Mmp20, C4orf26, and Wdr72. Using a candidate gene approach we discovered a novel Wdr72 human mutation in association with AI to be a 5-base pair deletion (c.806_810delGGCAG; p.G255VfsX294). To gain insight into the function of WDR72, we used computer modeling of the full-length human WDR72 protein structure and found that the predicted N-terminal sequence forms two beta-propeller folds with an alpha-solenoid tail at the C-terminus. This domain iteration is characteristic of vesicle coat proteins, such as beta′-COP, suggesting a role for WDR72 in the formation of membrane deformation complexes to regulate intracellular trafficking. Our Wdr72 knockout mouse model (Wdr72−/−), containing a LacZ reporter knock-in, exhibited hypomineralized enamel similar to the AI phenotype observed in humans with Wdr72 mutations. MicroCT scans of Wdr72−/− mandibles affirmed the hypomineralized enamel phenotype occurring at the onset of the maturation stage. H&E staining revealed a shortened height phenotype in the Wdr72−/− ameloblasts with retained proteins in the enamel matrix during maturation stage. H+/Cl− exchange transporter 5 (CLC5), an early endosome acidifier, was co-localized with WDR72 in maturation-stage ameloblasts and decreased in Wdr72−/− maturation-stage ameloblasts. There were no obvious differences in RAB4A and LAMP1 immunostaining of Wdr72−/− mice as compared to wildtype controls. Moreover, Wdr72−/− ameloblasts had reduced amelogenin immunoreactivity, suggesting defects in amelogenin fragment resorption from the matrix. These data demonstrate that WDR72 has a major role in enamel mineralization, most notably during the maturation stage, and suggest a function involving endocytic vesicle trafficking, possibly in the removal of amelogenin proteins.

Amelogenesis imperfecta: A clinician′s challenge

Defective enamel formation can be explained as defects occurring at the stages of enamel formation. Quantitative defects in matrix formation leads to hypoplastic form of amelogenesis imperfecta. Inadequate mineralization of matrix leads to hypocalcification and hypomaturation variants. The demarcation of matrix formation and mineralization is not so distinct. This paper describes a case of a 7-year-old boy with amelogenesis imperfecta - Type IA i.e., hypoplastic pitted autosomal dominant.

The Sodium Bicarbonate Cotransporter (NBCe1) Is Essential for Normal Development of Mouse Dentition

Proximal renal tubular acidosis (pRTA) is a syndrome caused by abnormal proximal tubule reabsorption of bicarbonate resulting in metabolic acidosis. Patients with mutations to the SLC4A4 gene (coding for the sodium bicarbonate cotransporter NBCe1), have pRTA, growth delay, ocular defects, and enamel abnormalities. In an earlier report, we provided the first evidence that enamel cells, the ameloblasts, express NBCe1 in a polarized fashion, thereby contributing to trans-cellular bicarbonate transport. To determine whether NBCe1 plays a critical role in enamel development, we studied the expression of NBCe1 at various stages of enamel formation in wild-type mice and characterized the biophysical properties of enamel in NBCe1(-/-) animals. The enamel of NBCe1(-/-) animals was extremely hypomineralized and weak with an abnormal prismatic architecture. The expression profile of amelogenin, a known enamel-specific gene, was not altered in NBCe1(-/-) animals. Our results show for the first time that NBCe1 expression is required for the development of normal enamel. This study provides a mechanistic model to account for enamel abnormalities in certain patients with pRTA.

Type XVII Collagen is a Key Player in Tooth Enamel Formation

Inherited tooth enamel hypoplasia occurs due to mutations in genes that encode major enamel components. Enamel hypoplasia also has been reported in junctional epidermolysis bullosa, caused by mutations in the genes that encode type XVII collagen (COL17), a component of the epithelial-mesenchymal junction. To elucidate the pathological mechanisms of the enamel hypoplasia that arise from the deficiency of epithelial-mesenchymal junction molecules, such as COL17, we investigated tooth formation in our recently established Col17(-/-) and Col17 rescued mice. Compared with wild-type mice, the incisors of the Col17(-/-) mice exhibited reduced yellow pigmentation, diminished iron deposition, delayed calcification, and markedly irregular enamel prisms, indicating the presence of enamel hypoplasia. The molars of the Col17(-/-) mice demonstrated advanced occlusal wear. These abnormalities were corrected in the Col17 rescued humanized mice. Thus, the Col17(-/-) mice clearly reproduced the enamel hypoplasia in human patients with junctional epidermolysis bullosa. We were able to investigate tooth formation in the Col17(-/-) mice because the Col17(-/-) genotype is not lethal. Col17(-/-) mouse incisors had poorly differentiated ameloblasts that lacked enamel protein-secreting Tomes' processes and reduced mRNA expression of amelogenin, ameloblastin, and of other enamel genes. These findings indicated that COL17 regulates ameloblast differentiation and is essential for normal formation of Tomes' processes. In conclusion, COL17 deficiency disrupts the epithelial-mesenchymal interactions, leading to both defective ameloblast differentiation and enamel malformation.

Scanning electron microscopy and energy-dispersive X-ray microanalysis studies of afibrillar cementum and cementicle-like structures in human teeth.

We examined afibrillar cementum (AFC) and cementicle-like structures (CLS) in human teeth by scanning electron microscopy and energy-dispersive X-ray microanalysis. The AFC showed a spur- or island-, plate- and mass-like structure with appositional laminations, while large masses in the enamel fissures enclosed CLS showing concentric appositional rings. Such AFC was observed in enamel fissures, an abnormal enamel pit, dens invaginatus and root furcations with enamel droplets, as well as on the cervical enamel surfaces, where ameloblasts are differentiated at the later or last stage of enamel formation. Cementicle-like structures were occasionally found independent from AFC and some CLS contained epithelial cell-like or ameloblast-like remnants in the core, surrounded by a few or many concentric rings. In addition, cementicles (CEC) in the root furcations also contained the remnants of Malassez's epithelial-rest cells surrounded by a few concentric rings. In some areas, AFC was mixed with enamel structures. These results show that the organic material in some parts of AFC and CLS may be derived from epithelial cells similar to that of CEC. Calcification values of AFC and CLS were significantly higher than that of fibrillar cementum, and the minute crystals are probably apatite.

The Dentino-Enamel Junction Revisited

The dentino-enamel junction is not an simple inert interface between two mineralized structures. A less simplistic view suggests that the dentino-enamel junctional complex should also include the inner aprismatic enamel and the mantle dentin. At early stages of enamel formation, fibroblast growth factor (FGF)-2 is stored in and released from the inner aprismatic enamel, possibly under the control of matrix metalloproteinase (MMP)-3. The concentration peak for MMP-2 and -9 observed in the mantle dentin coincided with a very low labeling for TIMP-1 and -2, favoring the cross-talk between mineralizing epithelial and connective structures, and as a consequence the translocation of enamel proteins toward odontoblasts and pulp cells, and vice versa, the translocation of dentin proteins toward secretory ameloblasts and cells of the enamel organ. Finally, in X-linked hypophosphatemic rickets, large interglobular spaces in the circumpulpal dentin were the major defect induced by the gene alteration, whereas the mantle dentin was constantly unaffected. Altogether, these data plead for the recognition of the dentino-enamel junctional complex as a specific entity bearing its own biological characteristics.

Inositol Hexasulphate, a Casein Kinase Inhibitor, Alters Enamel Formation in Cultured Embryonic Mouse Tooth Germs

Post-translational modification of enamel proteins is regulated by casein kinases (CK) and results in binding sites for calcium ions that subsequently play a key role during the initial stages of mineralization. Phosphorylation may also influence the secretion and extracellular organization of enamel proteins. Previous studies indicated that inositol hexasulphate inhibited the activity of CK-I and/or CK-II in mouse tooth germs (Torres-Quintana et al., 1998). We hypothesized that inositol hexasulphate would also inhibit the activity of the specific casein kinase(s) identified in secretory ameloblasts, and would prove useful for determination of the extent to which phosphorylation might influence the organization of enamel proteins at early stages of enamel formation. To test this hypothesis, we dissected mandibular first molars from 18-day-old mouse embryos and cultured them for 11 days in the presence of 0-0.1 mM inositol hexasulphate. Ultastructural analysis revealed that the formation of enamel was largely impaired at an inhibitor concentration > or = 0.08 mM. Quantitative radioautographic analysis of [33P]phosphate incorporation indicated that radiolabeled phosphate normally secreted into forming enamel was retained within ameloblasts. In contrast, no significant difference was observed between control and inositol-hexasulphate-treated tooth germs when cultures were labeled with [3H]serine and [3H]proline. SDS-PAGE and Western blot analysis confirmed that while inositol hexasulphate inhibited CK-mediated phosphorylation, it did not significantly alter protein synthesis. We conclude that impairment of phosphorylation leads to intracellular accumulation of [3H]phosphate-containing material by ameloblasts. We also conclude that when non-phosphorylated enamel matrix proteins are secreted, they are either unable to form an enamel matrix that supports mineralization, or they diffuse throughout a poorly mineralized dentin.

Effects of accelerated eruption on the enamel of the rat lower incisor.

The effect of accelerated eruption of the rat lower incisor on enamel was studied in a series of segments obtained when the incisor was cut repeatedly out of occlusion over a five-week period. The segments were ground, cleaned, acid-etched, observed with SEM, and analyzed with EDX. Pigmentation was lost within 11 days. Pigmented superficial enamel was more acid-resistant than the rest of the enamel, but this quality decreased with decreasing iron content. Hypomineralized enamel first appeared in a restricted area at the mesio-labial angle of the tooth in the 6th-7th segment obtained after 11-14 days. Later, hypomineralization became more generalized. All enamel zones were retained throughout the experiment. The geometry of the prism pattern was affected. The angle between prism rows and the enamel-dentin junction increased from 44 degrees to 48 degrees, while the angle of decussation increased from 60 degrees to 70 degrees. The angle between the enamel surface and prisms in the outer enamel was more difficult to assess, but tended to increase from about 25 degrees to 29 degrees. However, the prisms retained their incisal direction. The connection between enamel and dentin was partly disrupted from about the 9th segment onward. The depth of the mesial concavity of the enamel-dentin junction decreased from about the 10th segment onward. Accelerated eruption affects all stages of enamel formation and is a suitable and predictable model for studying regulatory mechanisms in amelogenesis.

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.

Expression of plasma membrane Ca++ pump epitopes parallels the progression of enamel and dentin mineralization in rat incisor.

We investigated the expression of Ca++ pump epitopes during enamel and dentin mineralization in the rat incisor. Secretory and maturation ameloblasts were studied as well as odontoblasts, using a monoclonal antibody (5F10) against human erythrocyte plasma membrane Ca++, Mg(++)-ATPase. A progressive increase in staining intensity in ameloblasts and the odontoblasts was observed beginning with the onset of mineralization. The mainly membrane-related labeling of ameloblasts showed variable intensity depending on the stage of enamel formation, whereas that of the odontoblasts showed even intensity during continued dentinogenesis. Staining of papillary cells was evident only during enamel maturation. Western blot analysis of freeze-dried ameloblasts was also used to determine the molecular weight of the Ca++ pump epitopes as well as the distribution and relative concentration of epitopes at each stage. An immunoreactive band of MW 140 KD and lower molecular weight bands that are more intense in late than in early maturation were demonstrated. Our studies suggest that the expression of plasma membrane Ca++ pump parallels the progression of mineralization in rat incisor enamel and dentin.

Biological mechanisms of fluorosis and level and timing of systemic exposure to fluoride with respect to fluorosis.

Enamel fluorosis can occur following either an acute or chronic exposure to fluoride during tooth formation. Fluorosed enamel is characterized by a retention of amelogenins in the early-maturation stage, and by the formation of a more porous enamel with a subsurface hypomineralization. The mechanisms by which fluoride affects enamel development include specific effects on both the ameloblasts and on the developing enamel matrix. Maturation-stage ameloblast modulation is more rapid in fluorosed enamel as compared with control enamel, and proteolytic activity in fluorosed early-maturation enamel is reduced as compared with controls. Secretory enamel appears to be more susceptible to the effects of fluoride following acute fluoride exposure, such as may occur with the use of fluoride supplements. However, both human and animal studies show that the transition/early-maturation stage of enamel formation is most susceptible to the effects of chronic fluoride ingestion at above-optimal levels of fluoride in drinking water.

Effects of single doses of 1-hydroxyethylidene-1,1-bisphosphonate on the mineralizing front of rat incisor enamel: A microradiographic and scanning electron microscopic study

The effects of a single dose of HEBP were examined by injecting rats subcutaneously with various doses (0.5, 1, 2.5, 5 and 10 mg P/kg body wt) and killing them either 2 or 9 days after injection. The maxillary incisors were processed for microradiography and the mandibular incisors for scanning electron microscopy after enzymatic digestion of the enamel organ remains. All doses resulted in a hypomineralized incremental band corresponding to the position of the mineralizing front at the time of injection. At 5 and 10 mg P/kg body wt, a hypomineralized lesion was found throughout the whole thickness of the enamel in an area which corresponded to the stage of late enamel secretion at the time of injection. The surface layer covering this lesion was elevated or disrupted. By scanning electron microscopy, there were three different types of lesions along the enamel surface: a “demarcation groove” corresponding to the initial enamel formation at the time of injection; an “area containing mineral globules” and a “bright band” corresponding to the stages of late secretory and final enamel formation, respectively, at the time of injection. A single injection of HEBP thus interferes with different stages of enamel formation. The findings may be explained as of the physico-chemical effects of HEBP on the mineral phase alone, but a direct effect of the drug on ameloblast function cannot be excluded.

EFFECT OF DRUGS ON DEVELOPING ENAMEL IN RAT INCISORS

The basic structure of rat incisors and the process of enamel formation are reviewed, and the drug-induced lesions of rat incisors are discribed. The rodent incisors grow, calcify and erupt continuously throughout the life of the animal, and show in one longitudinal section the complete life cycle of tooth development from inception to maturity. Therefore it is a valuable biologic indicator which reflects and records, during its development, the metabolic status of the animal. Drug-induced lesions in developing enamel of incisors are white discoloration of enamel surface, degeneration, necrosis, and atrophy of ameloblasts and/or papillary cells, disturbance of pigmentation, and hypocalcification of enamel. These changes correlate with the stage of enamel formation. The advantage of using rat incisors for toxicity studies is stressed.

Calcium transport during mineralization.

The translocation of calcium from the extracellular fluid compartment into the mineralizing matrix during hard tissue formation is not well understood. There are two general means by which such calcium movement may occur: 1) diffusion through the pericellular space, or 2) transcellular transport. Cementum and bone are difficult tissues in which to study the system and little is known about the mechanisms involved. Dentin offers certain advantages for study and it appears that calcium movement into the mineralizing matrix is by transcellular transport. Information concerning the transport mechanism is meager. Enamel is more easily explored. The apparent existence of intercellular junctions tight to calcium in the ameloblast layer at all stages of enamel formation indicates that calcium movement occurs by transcellular transport. Based on published findings, a hypothesis concerning mechanisms of transcellular transport may be advanced. It is proposed that the relatively low level of calcium transport through secretory ameloblasts occurs without direct involvement of a calcium binding protein. During the maturation stage, when calcium influx to the matrix is greatly increased, a calcium binding protein (9 kd) appears and facilitates transport while preventing unphysiologic increases in the cytosolic free calcium ion concentration. Differences in the calcium ion concentrations of extracellular fluid and enamel matrix fluid appear to be critical to the influx of calcium across the proximal cell membrane and the efflux of calcium across the distal cell membrane.