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Rampant caries and periodontal diseases occur in patients treated with antidepressants such as serotonin reuptake inhibitors (SRIs; e.g., Prozac) which target the serotonin transporter (SERT). As the serotonin 2B receptor (5HT2BR) regulates SERT functionality and capacity to recognize SRIs, we investigated the potential role of 5HT2BR on dental tissues by exploiting 5HT2BR knockout (KO) mice. Compared to wild-type (WT) mice, several structural differences were identified in the teeth of KO mice. In the molar of KO mice, rod curvatures and twisting were altered compared to WT mice, suggesting involvement of 5HT2BR at early stages of enamel formation. The volume of the KO enamel layer was also reduced, and larger porosities were observed in the prismatic enamel, with smaller crystallite thickness. Crystallite pattern disorganization and occlusal abrasion were enhanced in female KO mice, indicating a sexual dimorphism. In the incisor, no difference was detected in the width of the enamel layer between KO and WT mice; however, enamel maturation differed in absence of 5HT2BR. Specifically, the outer aprismatic enamel border was 1.5- to 2-fold larger in KO compared to WT mice, together with a decreased etching pattern. Finally, although no noticeable difference was observed in dentin, the micro-CT three-dimensional pulp reconstruction evidenced a decrease in both length and width of dentin formation in the root canals of the KO versus WT mice. These data provide evidence that 5HT2BR-mediated signaling pathways are involved in enamel formation and dentinogenesis.

Transforming growth factor-ß (TGF-ß) signaling plays an important role in regulating crucial biological processes such as cell proliferation, differentiation, apoptosis, and extracellular matrix remodeling. Many of these processes are also an integral part of amelogenesis. In order to delineate a precise role of TGF-ß signaling during amelogenesis, we developed a transgenic mouse line that harbors bovine amelogenin promoter-driven Cre recombinase, and bred this line with TGF-ß receptor II floxed mice to generate ameloblast-specific TGF-ß receptor II conditional knockout (cKO) mice. Histological analysis of the teeth at postnatal day 7 (P7) showed altered enamel matrix composition in the cKO mice as compared to the floxed mice that had enamel similar to the wild-type mice. The µCT and SEM analyses revealed decreased mineral content in the cKO enamel concomitant with increased attrition and thinner enamel crystallites. Although the mRNA levels remained unaltered, immunostaining revealed increased amelogenin, ameloblastin, and enamelin localization in the cKO enamel at the maturation stage. Interestingly, KLK4 mRNA levels were significantly reduced in the cKO teeth along with a slight increase in MMP-20 levels, suggesting that normal enamel maturation is regulated by TGF-ß signaling through the expression of KLK4. Thus, our study indicates that TGF-ß signaling plays an important role in ameloblast functions and enamel maturation.

The role of the serotonin 2B receptor (5-HT(2B) R) in enamel formation and mineralization was explored in adult 5HT(2B) R knockout (KO) mice compared with wild-type (WT) mice. In the molar, quantitative data obtained by micro-computed tomography imaging showed that the overall volume of the enamel layer was firmly reduced in KO mice. Defective mineralization was ascertained by energy-dispersive X-ray microanalysis. We also observed, using scanning electron microscopy, that parazones in the KO mice included two or three helicoidally twisted rods within Hunter-Schreger bands, instead of a single rod, as found in the WT mice. Minor disturbances were also detected in the incisors of KO mice. Structural modifications, thinner enamel crystallites, and porosities observed in KO mice indicate that the 5-HT(2B) R-mediated signaling pathways as part of the enamel formation process. These data provide a basis for evaluating the role of 5-HT(2B) R in ameloblast functions. Defects observed in the mineralization and structure of enamel in KO mice highlight that the 5-HT(2B) R interferes with the mechanisms directing amelogenesis., (© 2011 Eur J Oral Sci.)

Previously, it was shown that the volume of forming enamel of molar teeth in biglycan-null mice was greater than that in genetically matched wild-type mice. This phenotypic change appeared to result from an increase in amelogenin expression, implying that biglycan directly influences amelogenin synthesis. To determine whether biglycan overexpression resulted in decreased amelogenin expression, we engineered transgenic mice to overexpress biglycan in the enamel organ epithelium. Biglycan overexpression did not significantly affect the amelogenin expression in incisor and molar teeth in 3-day postnatal transgenic mice. In the transgenic animals, we observed that the immature and mature enamel appeared normal. These results suggested that increasing the biglycan expression, in the cells that synthesize the precursor protein matrix for enamel, has a negligible influence on amelogenesis.

To determine the functions of fibromodulin (Fmod), a small leucine-rich keratan sulfate proteoglycan in tooth formation, we investigated the distribution of Fmod in dental tissues by immunohistochemistry and characterized the dental phenotype of 1-day-old Fmod-deficient mice using light and transmission electron microscopy. Immunohistochemistry was also used to compare the relative protein expression of dentin sialoprotein (DSP), dentin matrix protein-1 (DMP 1), bone sialoprotein (BSP), and osteopontin (OPN) between Fmod-deficient mice and wild-type mice. In normal mice and rats, Fmod immunostaining was mostly detected in the distal cell bodies of odontoblasts and in the stratum intermedium and was weaker in odontoblast processes and predentin. The absence of Fmod impaired dentin mineralization, increased the diameter of the collagen fibrils throughout the whole predentin, and delayed enamel formation. Immunohistochemistry provides evidence for compensatory mechanisms in Fmod-deficient mice. Staining for DSP and OPN was decreased in molars, whereas DMP 1 and BSP were enhanced. In the incisors, labeling for DSP, DMP 1, and BSP was strongly increased in the pulp and odontoblasts, whereas OPN staining was decreased. Positive staining was also seen for DMP 1 and BSP in secretory ameloblasts. Together these studies indicate that Fmod restricts collagen fibrillogenesis in predentin while promoting dentin mineralization and the early stages of enamel formation.

Transforming growth factor-beta1 (TGF-beta1) is a key regulator of many cellular processes, including cell adhesion, the immune response and synthesis of extracellular matrix proteins. In the present study, we report the characterization of enamel defects in a transgenic mouse model overexpressing TGF-beta1 in odontoblasts and ameloblasts, its expression being driven by the promoter sequences of the dentin sialophosphoprotein gene. As reported earlier, these mice develop distinct dentin defects similar to those seen in human dentin dysplasia and dentinogenesis imperfecta. A further detailed examination of enamel in these mice revealed that from the early secretory stage, ameloblasts began to detach from dentin to form cyst-like structures. A soft X-ray analysis revealed that this cyst-like structure had a disorganized and partially mineralized matrix with an abnormal mineralization pattern and a globular appearance. In the molars, the enamel was not only pitted and hypoplastic, but enamel rods were completely lost. Thus, altered TGF-beta1 expression in the tooth seems to trigger detachment of ameloblasts and abnormal secretion and deposition of minerals in the cyst-like structures adjoining the dentin. We speculate that the altered expression of TGF-beta1 in teeth impacts the adhesion process of ameloblasts to dentin.

Extracellular matrix proteins control the formation of the inorganic component of hard tissues including bone, dentin, and enamel. The structural proteins expressed primarily in the enamel matrix are amelogenin, ameloblastin, enamelin, and amelotin. Other proteins, like biglycan, are also present in the enamel matrix as well as in other mineralizing and nonmineralizing tissues of mammals. In addition, the presence of sulfated enamel proteins, and "tuft" proteins has been examined and discussed in relation to enamel formation. The structural proteins of the enamel matrix must have specific protein-protein interactions to produce a matrix capable of directing the highly ordered structure of the enamel crystallites. Protein-protein interactions are also likely to occur between the secreted enamel proteins and the plasma membrane of the enamel producing cells, the ameloblasts. Such protein-protein interactions are hypothesized to influence the secretion of enamel proteins, establish short-term order of the forming matrix, and to mediate feedback signals to the transcriptional machinery of these cells. Membrane-bound proteins identified in ameloblasts, and which interact with the structural enamel proteins, include Cd63 (cluster of differentiation 63 antigen), annexin A2 (Anxa2), and lysosomal-associated glycoprotein 1 (Lamp1). These and related data help explain the molecular and cellular mechanisms responsible for the removal of the organic enamel matrix during the events of enamel mineralization, and how the enamel matrix influences its own fate through signaling initiated at the cell surface. The knowledge gained from enamel developmental studies may lead to better dental and nondental materials, or materials inspired by Nature. These data will be critical to scientists, engineers, and dentists in their pursuits to regenerate an entire tooth. For tooth regeneration to become a reality, the protein-protein interactions involving the key dental proteins must be identified and understood. The scope of this review is to discuss the current understanding of protein-protein interactions of the developing enamel matrix, and relate this knowledge to enamel biomineralization.

Small leucine-rich proteoglycans have been suggested to affect mineralization of dental hard tissues. To determine the functions of two of these small proteoglycans during the early stages of tooth formation, we characterized the dental phenotypes of biglycan (BGN KO) and decorin deficient (DCN KO) mice and compared them to that of wild type mice. Each targeted gene disruption resulted in specific effects on dentin and enamel formation. Dentin was hypomineralized in both knock out mice, although the effect was more prominent in the absence of decorin. Enamel formation was dramatically increased in newborn biglycan knockout mice but delayed in absence of decorin. Increased enamel formation in the former case resulted from an upregulation of amelogenin synthesis whereas delayed enamel formation in the later case was most probably an indirect consequence of the high porosity of the underlying dentin. Enamelin expression was unchanged in BGN KO, and reduced in DCN KO. Dentin sialoprotein (DSP), a member of the family of phosphorylated extracellular matrix proteins that play a role in dentinogenesis, was overexpressed in BGN-KO odontoblasts and in the sub-odontoblastic layer. In contrast, a decreased expression of DSP was detected in DCN KO. Dentin matrix protein-1 (DMP-1), bone sialoprotein (BSP) and osteopontin (OPN) were upregulated in BGN KO and downregulated in the DCN KO. Despite the strong effects induced by these deficiencies in newborn mice, no significant difference was detected between the three genotypes in adult mice, suggesting that the effects reported here in newborn mice are transient and subjected to self-repair.

Tooth enamel is formed in the extracellular space within an organic matrix enriched in amelogenin proteins. Amelogenin nanosphere assembly is a key factor in controlling the oriented and organized growth of enamel apatite crystals. Recently, we have reported the formation of higher ordered structures resulting from organized association and self-orientation of amelogenin nanospheres in vitro. This remarkable hierarchical organization includes self-assembly of amelogenin molecules into subunits of 4-6 nm in diameter followed by their assembly to form nanospheres of 15-25 nm in radii. Chains of >100 nm length are then formed as the result of nanosphere association. These linear arrays of nanospheres assemble to form the microribbons that are hundreds of microns in length, tens of microns in width, and a few microns in thickness. Here, we review the step by step process of amelogenin self-assembly during the formation of microribbon structures in vitro. Assembly properties of selected amelogenins lacking the hydrophilic C terminus will then be reviewed. We will consider amelogenin as a template for the organized growth of crystals in vitro. Finally, we will compare the structures formed in vitro with globular and periodic structures observed earlier, in vivo, by different sample preparation conditions. We propose that the alignment of amelogenin nanospheres into long chains is evident in vivo, and is an important indication for the function of this protein in controlling the oriented and elongated growth of apatite crystals during enamel biomineralization., (2005 S. Karger AG, Basel)

The mouse X-chromosomal amelogenin gene promoter was used to drive the expression of mutated amelogenin proteins in vivo. Two different transgenic mouse lines based on deletions to either the amino-terminal (A-domain deletions) or to the carboxyl-region (B-domain deletions) were bred. In the molars of newborn A-domain deleted transgenic mice the formation of the initial layer of aprismatic enamel was delayed. There were severe structural alterations in the enamel of incisors of newborn mice bearing the A-domain deletion which were not apparent in animals bearing the B-domain deletion. In the A-domain-deleted animals, stippled material accumulated throughout the entire thickness of the forming enamel apparently causing a disruption of the normal rod-to-inter-rod relationship. This stippled material was likened to and interpreted as being groupings of amelogenin nanospheres. In the B-domain-deleted animals the stippled material was detected only in minute defects of the forming enamel. These data suggest significant differences in nanosphere assembly properties for animals bearing either the A-domain or the B-domain-deleted transgene. The present in vivo experimental approach suggests that at early stages of enamel formation, the A-domain plays a greater role than does the B-domain in amelogenin self-assembly, and consequently in enamel architecture and structure.

The structure of rat incisor enamel is established at the topographically complex interface between secretory ameloblasts and forming enamel. The aim of this study was to gain additional information on this interface by sectioning parallel with the rows and the long axis of Tomes' processes and prisms. Rats were sacrificed and fixed by glutaraldehyde/paraformaldehyde perfusion. After dissection, demineralization and embedding transverse jaw/incisor segments were cut, reembedded, and reoriented. Sections were prepared for and observed in the transmission electron microscopy (TEM). The intraenamel part of Tomes' process was about 18 microns long. The forming prism occupied a longitudinally grooved invagination on its apical aspect. The parts of Tomes' process forming the side walls of the groove were attenuated and showed variation in extent and outline. Prism growth occurred over the whole grooved area. An estimation of Tomes' process secretory area in rat compared with data from humans suggests that there may be a relationship between secretory area and rate of prism formation. Prism crystals were oriented obliquely or parallel to the secretory surface of Tomes' process. At interprism growth sites matrix deposition was irregular and required some redistribution to conform to the pattern of interprism sheets.

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.

Enamel formation is a powerful model for the study of biomineralization. A key feature common to all biomineralizing systems is their dependency upon the biosynthesis of an extracellular organic matrix that is competent to direct the formation of the subsequent mineral phase. The major organic component of forming mouse enamel is the 180-amino-acid amelogenin protein (M180), whose ability to undergo self-assembly is believed to contribute to biomineralization of vertebrate enamel. Two recently defined domains (A and B) within amelogenin appear essential for this self-assembly. The significance of these two domains has been demonstrated previously by the yeast two-hybrid system, atomic force microscopy, and dynamic light scattering. Transgenic animals were used to test the hypothesis that the self-assembly domains identified with in vitro model systems also operate in vivo. Transgenic animals bearing either a domain-A-deleted or domain-B-deleted amelogenin transgene expressed the altered amelogenin exclusively in ameloblasts. This altered amelogenin participates in the formation an organic enamel extracellular matrix and, in turn, this matrix is defective in its ability to direct enamel mineralization. At the nanoscale level, the forming matrix adjacent to the secretory face of the ameloblast shows alteration in the size of the amelogenin nanospheres for either transgenic animal line. At the mesoscale level of enamel structural hierarchy, 6-week-old enamel exhibits defects in enamel rod organization due to perturbed organization of the precursor organic matrix. These studies reflect the critical dependency of amelogenin self-assembly in forming a competent enamel organic matrix and that alterations to the matrix are reflected as defects in the structural organization of enamel., (Copyright 2000 Academic Press.)

Stromelysin-1 (matrix metalloproteinase-3) or proteoglycanase was visualized by light and electron microscopy immunolabelling in the forming zone of rat incisors. In predentine, labelling was more dense at the transition zone between the inner proximal third and the two outer thirds. Odontoblast processes were also positively stained, mostly in predentine and to a lesser degree in dentine. The dentine-enamel junction was intensely labelled, whereas dentine and forming enamel were only faintly stained. Gold-antibodies complexes were seen inside secretory ameloblasts and odontoblasts in cytosolic locations. The distribution of stromelysin-1 was compared with the distribution of 2-B-6 epitope, an antibody recognizing chondroitin-4-sulphate/dermatan sulphate and which showed a decreasing gradient from the proximal zone to the distal part of predentine. In contrast, both 5-D-4, an anti-keratan sulphate antibody and an anti-lumican antibody displayed a reversed distribution, with an increase seen from the proximal and central thirds to the distal part of predentine. This coordinated distribution suggests that stromelysin-1 may have a functional role, being implicated in predentine in the degradation of chondroitin-4-sulphate/dermatan sulphate-containing proteoglycans, and consequently allowing keratan sulphate proteoglycan concentration to increase near the border where mineralization is initiated.

In order to study the uptake and fate of [3H]choline into cellular and extracellular phospholipids in the forming part of mandibular rat incisors, radioautography was carried out after treatment with the iodoplatinate reaction which retains phospholipids. Thirty minutes and 1 hour after the intravenous injection of the radiolabeled precursor, grain density in secretory odontoblasts and ameloblasts was not significantly above background labeling whereas dentin was actually labeled. Therefore, at this early period, odontoblasts cannot be responsible for the secretion of phospholipids incorporated into dentin, and intercellular diffusion of components originating from blood could explain this early dentin labeling. After 2 hours, odontoblasts and ameloblasts were labeled. In cells, grain density reached a maximum at 4 hours, reduced at 24 hours, and strongly decreased at 4 days. In predentin and enamel, grain density peaked at 24 hours and diminished at 4 days. However, in the forming enamel 4 days after the injection, labeling was twice as high as in any other compartment. Altogether, the results highlighted two distinct pathways for phospholipids in dental mineralized dental tissues: a first one shows evidence of early incorporation of [3H]choline into dentin resulting from intercellular diffusion independently from odontoblasts secretion, whereas inside the forming enamel, higher labeling and longer retention of choline-containing membrane components were detected between 4 hours and 4 days. This suggests an accumulation of membranes that are not subjected to rapid turnover in contrast with other dental compartments.

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.

(3H)-choline, a precursor for phosphatidylcholine (PC) and sphingomyelin (SM), was injected into rats killed after 4, 24, 48, and 96 hrs. Radioautography carried out on malachite-green/aldehyde-fixed tissues demonstrated that labeled choline was incorporated into cells and further released into the extracellular matrix. In predentin, labeling decreased rapidly, whereas in dentin, silver grains formed a stable band. In contrast, labeling was still high at 48 and 96 hrs in secretory ameloblasts as well as in the forming enamel. This indicates that ameloblasts are actively involved in the synthesis of membranes. Membrane remnants of the ameloblasts could be released into the forming enamel. In rats fed with an essential fatty-acid-deficient (EFAD) diet for 42 days, (3H)-choline uptake was delayed and reduced in pulp cells and odontoblasts, and consequently the migration of labeled phospholipids into dentin. The influence of the EFAD diet on secretory ameloblasts was limited. No difference was detected between normally fed and EFAD-fed rats in the forming enamel.

The present investigation studies the effects of persistently cutting one lower rat incisor out of occlusion. Within four days, the rate of eruption of the cut (unimpeded) incisor increased to 216% and that of the uncut (impeded) contralateral to 136% of the baseline rate. While the former remained high, the latter decreased gradually to about 90% within three weeks. The rate of attrition of the impeded incisor increased to 233% of the baseline rate within two days, then fell abruptly, and remained at a slightly lower level than the rate of eruption. Accordingly, the length of the erupted part of the impeded incisor decreased initially, but increased gradually after about four days. Measurements made on SEM micrographs of the series of transverse tooth segments obtained when cutting the incisor out of occlusion, showed that growth-related increase in mesiodistal tooth width was arrested from the 10th segment, dentin thickness decreased gradually to about 50% in the 12th segment, and enamel thickness, after an initial increase, decreased to about 80% in the 11th segment. The present study provides experimental evidence that accelerated eruption affects morphogenesis and histogenesis of the rat lower incisor. An impeded incisor, especially the contralateral, may not serve as an ideal control.

The forming enamel of rat incisor was either treated before fixation with one of these five treatments: ultrasonication, NaOCl, chloroform/methanol, NaOH or tween 20; or the incisors were first fixed and then submitted to one of these treatments. The structure of the forming enamel, compared with sections fixed with aldehyde and submitted to osmic maceration, depends on the way the samples were treated and on the fixative used. An amorphous material, removed by osmium tetroxide but not by aldehyde solution was seen to be located between fiber-like structures. In the region of inner enamel secretion the thickness of interrod septa was fairly constant, whereas in the region of outer enamel secretion, transverse septa of interrod enamel gradually became thinner and finally disappeared. The longitudinal septa did not display such changes. Longitudinal grooves and ridges were seen at the end of enamel completion. These longitudinal septa finally merged and formed a flat surface.

This paper review the effects of orthophosphoric acid and other acidic agents on dental enamel. The effects of other treatments such as proteolytic agents or of mixture able to remove the lipids are also described. Specific problems are related to the dentine treatment. Acids and chelators should be avoided, because of the dentine tubules, the odontoblasts and pulp. We review also the problem of the smear layer, the surface of the carious dentine and the consequences for dental treatment. Researches which are still in progress are reported including the use of enzymes and alcali gels submitted to feeble electrolysis, this latter being able to prepare dentine, cementum and bone surfaces in order to increase the adhesion of biomaterials.

Enamel cylinders, submitted to the action of NaHCO3 solutions or kept in water, were immersed in a remineralizing solution containing radioactive calcium and phosphate. The calcium uptake was increased in NaHCO3 treated samples, but not the phosphate uptake. Less calcium adsorption was found in 72 h--NaHCO3 treated samples, than after 1 h and 24 h respectively.

Preparation of the cavo-surface margin for fillings with "composites resins" is an important operative step. The good peripheral fit of the restoration and the stability of the bond between tooth and material depend on that preparation. How should the "bevel" be shaped? Scan electron microscopy of long bevels and rounded bevels show that the former seem more favorable to the fit of the filling. The section of enamel prisms is clean. The aprismatic surface area disappears. This enables to etch the cristallites in a more favorable axis as opposed to the rounded bevel.

Planes of longitudinal fractures of human dental enamel observed in scanning electron microscopy showed morphological variations related to age. This late maturation was observed along the enamel sheaths which disappeared in the subsurface and decreased in the rest of the structure. The destructions produced by demineralizing substances (phosphoric acid, citric acid, EDTA) were always more severe in enamel of older teeth when compared to younger samples. A pretreatment of the fragments with a urea solution increased the acid effects only in young enamel. It is suggested that the late maturation of enamel consists in a reduction of matrix components.

Almost twice as much fatty acid was extracted without demineralization from the mature enamel of erupted teeth treated with 2:1 chloroform-methanol as from the enamel of unerupted teeth (32.64 micrograms/g versus 15.60 micrograms/g). Palmitic C16:0, stearic C18:0, oleic C18:1 omega 9 and linoleic acids C18:2 omega 6 were the chief fatty acids detected by gas-liquid chromatography. The percentage of free fatty acids and diglycerides was larger in unerupted than erupted teeth, whereas that of triglycerides, phosphatidylethanolamine and phosphatidylcholine was larger in erupted teeth. These changes may be due to bacteria and other contaminants (saliva and pellicle). The large amounts of free fatty acids, diglycerides, cholesterol and phospholipids as intrinsic components were probably due to the persistence of membrane remnants entombed during enamel formation, as indicated by the visualization of holes and by the increase in the size and number of focal holes after lipid-solvent interaction with the enamel surface. These defects always occurred in rods and never in inter-rod material. Other minute defects, including empty-widened intercrystal spaces, were also observed inside some rods.

In the surfaces zones of artificial carious lesions, tiny microchannels, about 0.5 - 1.5 micrometers in diameter and roughly 100 micrometers in length, have been observed. The mineral components seem to be concentrated at the lumen borders, whereas the organic components have disappeared or are disorganized. These observations suggest that the channels mentioned are involved in the lesion development and play an important role in diffusion (transport) processes taking place between the sub-surface lesion and the liquid phase outside the lesion. The microchannels originate in prisms in the perikymata of the surface enamel. They extend in the direction of the Hunter-Schreger bands, mainly in connection with diazones. No relationship could be found between the microchannels and the direction of the prism sheaths. This indicates that in initial artificial lesion formation the enamel structure plays an important role.

The ultrastructural distribution of complex carbohydrates in an early formation stage of rat incisor enamel was investigated by staining with the periodic acid-thiocarbohydrazide-silver proteinate reaction (PA-TCH-SP) for vicinal glycol-containing glycoconjugates, the phosphotungstic acid-chromic acid mixture (PTA) for glycoproteins, and the cationic dyes alcian blue or bismuth nitrate for sulfated glycoconjugates. In order to remove selectively sulfated complex carbohydrates, half of the serial sections obtained were digested with a bovine testicular hyaluronidase prior to staining. Far fewer electron-dense deposits were observed with the PA-TCH-SP method on hyaluronidase-treated sections, especially those subsequently treated for 48 hours with TCH. On the other hand, the minimal staining obtained with PTA was much more intense on sections treated with hyaluronidase where linear fiberlike structures were observed. With cationic dyes, staining of dotlike alignment structures and ground substance was obtained but was completely abolished by hyaluronidase treatment. Cuprolinic blue in a critical electrolyte concentration, ruthenium hexamine trichloride used with aldehyde during fixation, as well as rapid-freezing followed by freeze-substitution validate that this dotlike distribution is not an artefact of processing. The staining results demonstrated that the glycoproteins and sulfated complex carbohydrates in developing rat incisor enamel each display a specific distribution pattern. The glycoproteins were present as fiberlike structures and the sulfated carbohydrates appeared as dotlike formations located close to the surface of the fiberlike structures, and/or in the spaces between them.

Morphologic examination of the developing enamel of rat incisors showed the presence of cell processes and remnants. Histochemical investigation of rapid-frozen freeze-substituted samples, using p-phenylenediamine or a phosphotungstic acid chromic acid mixture, revealed osmiophilic components which were extractable in chloroform-methanol solution and were located inside the tubule-like structures of the extracellular matrix. The presecretory cell-rich and developing enamel zones underwent quantitative and qualitative lipid analysis. Comparison of the biochemical data as well as of the morphological observations, suggests a cellular origin for enamel lipids randomly adsorbed by extracellular matrix components during enamel processing. The nature of the material which appeared as an osmiophilic intra-tubular filling is still unresolved.

The distribution pattern of the protein-polysaccharides in the rat incisor enamel was similar whether stained for glycoproteins (phosphotungstic acid) or for proteoglycans (alcian blue, bismith nitrate). Except for the holes at the dentino-enamel junction, where these components seem to exist as amorphous gel, protein-polysaccharides are closely associated to the proteinic fibrous matrix in rods and interrods structures as in prism sheaths where they are particularly abundant.

The amount of free water in 50 samples of air-dried enamel from permanent and deciduous teeth was measured by the Karl Fischer method. Samples included both contemporary and burial specimens. The mean values obtained showed that free water constituted about 1% of enamel mass. However, the range of individual values varied considerably, from 0.56 to 1.48%. The proportion of free water did not seem to depend on the patient's age, the type of tooth, or the relationship between the tooth and its oral environment. On the other hand, the deciduous enamel tested displayed a mean free water content that was three times the mean for the permanent teeth (3.01 vs. 1.00%) and the five burial teeth, a mean content of 1.68%.

A morphological study of the cells of the outer epithelium layer, of the stellate reticulum and of the stratum intermedium of the rat incisor on thin sections and after freeze-etching allowed the differentiation of an intracellular and an intercellular compartment. Both were implicated in transport and diffusion phenomenons. The structure of these cells suggested that they synthetize non exportable proteins. Nevertheless they could be implicated in some none precised activities of biosynthesis and secretion.

A new microsampling method is described for measuring variations in calcium and phosphorus distribution in dental enamel. Ten successive layers, 10 micron thick, from five human enamel posts with previously abraded flat upper surfaces were ground and analyzed. Mean Ca and P values were, respectively, 1116 +/- 110 and 527 +/- 47 micrograms/mm3, and mean Ca/P w/w and molar ratios, 2.11 +/- 0.10 and 1.63 +/- 0.08, respectively. Despite its limitations, the present method allows for accurate investigation of the mineral content in sound, experimentally-treated enamel.