Secretory Enamel Research Articles

Effects of Fluoride on Matrix Proteins and Their Properties in Rat Secretory Enamel

This publication concerns the selective adsorption of rat enamel proteins onto hydroxyapatite, their solubility in aqueous solutions, and the effect that systemic fluoride has on these properties. The enamel proteins used as adsorbates were extracted in 0.5 mol/L acetic acid from the secretory enamel of the upper and lower incisors of SD rats (females, 200-220 g body weight). Equilibration of the proteins with hydroxyapatite was performed in two solutions: (i) 50 mmol/L acetate buffer at pH 6.0 and 0 degrees C, and (ii) 50 mmol/L Tris buffer containing 4 mol/L guanidine at pH 7.4 and room temperature. Enamel was dissected from animals, which were given either de-ionized water (control group) or water containing 25, 50, 75, or 100 ppm fluoride as NaF for four weeks. From these enamel samples, the proteins were extracted in sequence with 160 mmol/L NaCl and 3 mmol/L phosphate (pH 7.3), 50 mmol/L carbonate buffer (pH 10.8), and finally, with 0.5 mol/L acetic acid for dissolution of the enamel mineral. The F, Ca, and P contents of the various enamel samples were determined.(ABSTRACT TRUNCATED AT 250 WORDS)

Properties of phosphorylated 32 kd nonamelogenin proteins isolated from porcine secretory enamel

Enamel proteins were isolated from specific locations of permanent porcine incisors at various developmental stages, namely, the outer (young) and inner (old) secretory, and maturing (chalk-like in appearance) enamel. The selective adsorption of these matrix proteins onto hydroxyapatite (HA) crystals was investigated in the presence of dissociative agents. The results showed that the proteins with the highest adsorption affinity were present at the highest concentration in the vicinity of the ameloblasts, i.e., in the outer enamel layer; a substantial reduction of these proteins was observed in the older (inner) secretory enamel and in the tissue in the maturing stage. An interesting finding was that a group of proteins having molecular masses of 32 kd present only in the inner secretory enamel, adsorbed strongly onto the HA crystals and were potent inhibitors of HA crystal growth. This 32 kd group contains phosphorylated glycoproteins; they are rich in Pro, Glu, Gly, and Asp and the N-terminal sequence was LXQVPGRIPPGYGRPPTP-, having no resemblance to the reported sequences of amelogenins. It was also found that the 32 kd moieties remained only as trace constituents in the maturing enamel, suggesting that most of them were removed as soluble constituents in the tissue fluid or further degraded by enzymatic activity during the late secretory stage. The results obtained support the view that amelogenetic mineralization is regulated by the presence of various organic matter and, importantly, that their efficacy as inhibitors of mineralization may be modulated through their degradation.

Nature and Mechanisms of Dental Fluorosis in Animals

Recent studies of dental fluorosis in animals have been reviewed. The aim was to describe studies which have provided information which may be relevant to explaining the mechanisms involved in human dental fluorosis. Studies on rats, sheep, and pigs have provided details of dosage regimens which produce lesions which resemble those described in human fluorosis. In the pig and rat, the plasma fluoride concentrations associated with these dental lesions are of the same order of magnitude as those which may occur in man. Three different kinds of studies in different species have shown that fluoride affects processes occurring during enamel maturation. One study on rats has indicated that fluoride may reduce matrix removal during maturation by an effect on enamel proteases. Many studies have demonstrated accumulation of fluoride in secretory enamel and that fluoride concentrations in maturation enamel are lower than in secretory enamel. This phenomenon had previously been explained by the binding of fluoride to enamel proteins, but recent studies indicate that such binding does not occur. The hypothesis that enamel fluorosis might be caused by general effects of fluoride on calcium metabolism has not been supported by more recent studies. It was concluded that, although the mechanisms involved in dental fluorosis remain obscure, recent animal studies do seem to have provided new information which may prove to be important for our understanding of mechanisms whereby fluoride causes dental fluorosis in man.

Ultrastructural study of tracer permeability through the cat and ferret enamel organ

The access of exogenous materials to the developing enamel surface has been intensively studied in rodents, but not in other mammalian species. This ultrastructural study investigates the permeability of injected horseradish peroxidase (HRP) and lanthanum tracers in cat and ferret tooth buds. In cat enamel organs fixed by immersion, lanthanum did not escape the capillaries overlying secretory stage tooth buds, but it did permeate up to the distal junctions of ruffle-ended (RA) and the proximal junctions of smooth-ended (SA) ameloblasts. Perfusion fixation with lanthanum compromised junctional integrity of cat ameloblasts at all stages of development. Similarly, HRP rarely escaped the capillaries associated with cat secretory stage enamel organs. However, unlike lanthanum, HRP was mostly confined to the vasculature of maturation stage enamel organs in immersion fixed cats at all time intervals examined. In ferrets, HRP penetrated up to, but not beyond, the distal junctional complexes of secretory ameloblasts. In maturation stage enamel organs, HRP coated the papillary and RA cells, but did not penetrate the RA distal cell junctions. HRP did permeate the extracellular spaces of SA to reach the underlying enamel surface. Ameloblasts in transitional phases of SA and RA endocytosed HRP at the distal cell surface. This data leads to several conclusions. First, HRP localization in the ferret paralleled that observed in rodents. Second, the results of cat enamel organs substantiate previous studies showing perfusion fixation can increase vascular and intercellular permeability to lanthanum. However, in cats fixed by immersion, both lanthanum and HRP were restricted to capillaries associated with the secretory stage enamel organ, and only lanthanum escaped maturation stage capillaries. It is suggested that variations in the fenestrations and distribution of capillaries associated with the cat enamel organ may differentially retain some materials and permit other materials to escape with relative ease.

A simple, disposable, and improved organ culture system for maintaining three-dimensional development of mouse embryonic molars

Mandibular first molars from 17-d-old mouse embryos were cultured in vitro for 2 to 4 d by a simple, disposable, improved floatation method. This method consisted of using a 24-well multidish and a plastic culture chamber with a membrane filter. The improved floatation method, as well as our previous method, was capable of the three-dimensional development of tooth germs. Cytodifferentiation of odontoblasts and ameloblasts and formation of extracellular matrices were accelerated by the present culture system, in comparison with our previous method. All the molars cultivated by this method were very similar in morphology to in vivo. On Day 2 of culture the terminal cytodifferentiation of odontoblasts and the formation of predentin were ascertained in the bucco-lingual sections of the cultured molars. A thick layer of predentin was formed at the tip of the cusp and gradually decreased toward the cervical loop and the fissure between the buccal and lingual cusps. On Day 4 in vitro, secretory ameloblasts produced enamel matrix, and the mineralized enamel showed showed prismatic structure very similar to that in vivo. Dentin and predentin also were normal in ultrastructure. The extracellular matrices (enamel, dentine, and predentin) were formed in line with the pattern of the cusp and the formation of matrices normally started at the tip of the cusp. We conclude that the three-dimensional development of whole tooth germs in vitro may be very important for normal expression of the developmental program intrinsic to mouse embryonic molars.

Effect of prenatal and postnatal fluoride on the human deciduous dentition. A literature review.

Fluoride passes from the mother to fetal teeth. Much of the fluoride is taken up in secretory enamel, probably by the forming mineral apatite crystals. Some is retained with residual proteins. The low concentration of fluoride in the inner enamel is incorporated mainly during the secretory stage, while the enhanced concentration in the surface enamel is produced during the much longer maturation stage. Mature, hard enamel is generally absent during fetal life. The clinical question is whether prenatal fluoride imparts an additional benefit to the universally accepted effect of postnatal fluoride. In general, surface enamel fluoride levels of deciduous teeth increase with increasing pre- and postnatal fluoride administration. A consistent level of caries protection has been reported with pre- and postnatal administration of fluoride unrelated to the acquisition of fluoride in the surface enamel. Many children develop enamel opacities in their deciduous dentition related by various factors to enamel mineralization disturbances in drinking water areas even low in fluoride. Accumulation of fluoride due to an increased fluoride intake is a feature of fluorosed enamel in the deciduous as well as permanent dentition. The resulting mature fluorosed enamel retains a relatively high proportion of immature matrix proteins onto the crystal surface. The degree of fluorosis of the deciduous dentition is less compared with that of the permanent dentition, due probably to a partial protection afforded by the maternal loss of fluoride, formerly known as the "placental barrier".

Possible roles of partial sequences at N- and C-termini of amelogenin in protein-enamel mineral interaction.

The purpose of this study was to assess the functional significance of homologous sequences of mammalian amelogenins at their N- and C-termini. A porcine 5-kDa fragment corresponding to the N-terminal 45 residues of amelogenins was purified from the secretory enamel. The decapeptide TDKTKREEVD corresponding to the C-terminal 10 residues of amelogenins was synthesized according to conventional solid-phase procedures. The inhibitory activity of both moieties on apatite crystal growth was determined in a supersaturated solution having an ionic composition similar to that of the fluid phase separated from porcine secretory enamel. The 5-kDa amelogenin fragment was sparingly soluble in neutral solutions and (in condensed forms because of aggregation) showed no significant inhibition of crystal growth, whereas the fragment molecules pre-adsorbed onto the seed crystals yielded modest inhibition of hydroxyapatite precipitation. However, their inhibitory activity was significantly lower than that of parent porcine amelogenin (25-kDa molecular mass). The high solubility of synthesized decapeptide allowed us to determine the adsorption isotherm onto hydroxyapatite at 37 degrees C, at an ionic strength similar to that of the enamel fluid. The obtained adsorption isotherm was described by a Langmuir model; the adsorption affinity and the maximum adsorption sites were 6.2 mL/mumol and 0.53 mumol/m2, respectively. As expected from the low adsorption affinity, the peptide showed a much weaker inhibition of apatite crystal growth than the parent amelogenin.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro Effects of Calcitonin and/or Parathyroid Hormone on Odontogenesis of Mouse Embryonic Molars

Mandibular first molars of mouse embryos were cultivated for examination of the effects of calcitonin (CT) and/or parathyroid hormone (PTH) on the odontogenesis of the molars, and for determination of whether and how CT, which is a PTH antagonist, has an influence on the effect of PTH on odontogenesis. On the second day, the inner enamel epithelium in the control group had already differentiated into pre-ameloblasts. Typical odontoblasts had secreted a layer of predentin. On the fourth day of culture, the pre-ameloblasts achieved terminal differentiation into secretory ameloblasts, and enamel and dentin had already been deposited. PTH (1 unit/mL) inhibited the odontogenesis of the cultured molars during the designated culture periods (two and four days), while CT (0.5 unit/mL) stimulated odontogenesis. On the second day, the development of the molars in the CT + PTH group showed an intermediate stage between the control and PTH-treated explants, but on day 4 it corresponded to that of the controls. Moreover, when the molars exposed to PTH for two days were untreated and treated with CT for an additional two days, the former produced a small quantity of enamel matrix, while the latter formed a large amount of the matrix. These histological findings were also supported by a morphometric analysis of the enamel matrix in the cultured molars. The present results suggest that CT stimulates, but PTH suppresses, the odontogenesis of the mouse embryonic molars, and that CT is an antagonist to the inhibitory effect of PTH on odontogenesis.

Possible Function of Matrix Proteins in Fluoride Incorporation into Enamel Mineral during Porcine Amelogenesis

The present study was undertaken to elucidate the mechanism of fluoride incorporation into secretory enamel mineral, with porcine enamel used as a model. Although the fluoride content in the enamel varied greatly among the animals, we observed that the fluoride-to-calcium ratio in the enamel tissue was maximal at the beginning of the secretory stage; the F/Ca ratio decreased (and leveled off) with the advancement of mineralization. In vitro work showed that some of the fluoride in the secretory enamel tissue was removed with the extraction of organic matter, mostly amelogenins. Furthermore, coating hydroxyapatite crystals with enamel matrix proteins resulted in a retardation of fluoride incorporation into the crystals when exposed to fluoride solutions, as a result of an inhibition of apatite reprecipitation. We also confirmed that the growth kinetics of fluoridated apatite onto HA seeds decreased with increasing coverage of the seed surface with the enamel proteins. All the results of the present study strongly suggest that the fluoride incorporation into enamel mineral during the secretory stage may be regulated by the kinetics of mineralization, which is highly dependent on the driving force for precipitation and the presence of proteinaceous inhibitors, mainly amelogenins.

Separation by polyacrylamide gel electrophoresis of multiple proteases in rat and bovine enamel

At least seven protein bands of protease activity were found in rat secretory enamel and six bands in bovine secretory enamel. The bovine proteases had molecular weights similar to those found in rat enamel. Two bands were found to be active in gels containing enamel proteins. All proteases were acid-resistant and calcium-dependent. In vitro digestion of secretory enamel resulted in a changing profile of proteases. Rat maturation enamel contained a 23,000 and a 33,000 molecular-weight protease which were not present in the secretory enamel. The 33,000 molecular weight protease was also active against enamel proteins. Thus multiple proteases are present in rat and bovine secretory enamel and in rat maturation enamel; some of these may be important in hydrolysis of enamel proteins.

Effect of fluoride in the apatitic lattice on adsorption of enamel proteins onto calcium apatites.

The selective adsorption of enamel proteins onto crystalline calcium apatites having different specific surface areas and various degrees of fluoride substitution was investigated. The proteins were obtained from the outer (close to the ameloblast) layer of secretory enamel of porcine permanent incisors. The adsorption of the enamel proteins was not affected markedly by the variation of specific surface area of the hydroxyapatites used as adsorbents, but it was enhanced substantially with increasing fluoride content in the crystalline lattice. Through the use of SDS- and two-dimensional polyacrylamide gel electrophoresis, it was shown that the originally secreted amelogenin (25 kd) as well as 60-90-kd and 5-6-kd molecules adsorbed most selectively onto the hydroxyapatites and that additional moieties having 21-23-kd and 14-18-kd molecular masses commenced to adsorb onto the apatitic surfaces with increasing degrees of fluoride substitution in the lattice. In contrast, the 20-kd amelogenin, a product partially degraded from the 25-kd amelogenin, showed no significant adsorption, even onto the fluoridated apatites. These results suggest that the retention of proteinaceous matrix in the developing enamel might be affected by the nature of the forming crystals.

The in vitro Uptake of Fluoride by Secretory and Maturation Stage Bovine Enamel

The objectives of this study were to determine the specific surface area of secretory-stage and of maturation-stage enamel, to compare the fluoride uptake by isolated enamel at these two stages on a surface-area basis, and to examine the effect of the organic matrix on the fluoride uptake by whole enamel. Fetal bovine secretory and maturation stage enamel samples were collected, and a portion of the enamel at each developmental stage was treated with hydrazine for removal of the organic matrix. The specific surface areas of the enamel mineral, as determined by the multi-point BET method, were 59.3 m2/g in the secretory stage and 37.9 m2/g in the maturation stage. Whole and deproteinated enamel samples were equilibrated in buffered solutions containing 10(-5) to 10(-3) mol/L fluoride, and the uptake was measured with a fluoride specific electrode. The results indicate that the in vitro fluoride uptake was controlled solely by the surface area of the apatitic mineral and that the organic matrix did not contribute to the fluoride uptake.

Temperature-related ultrastructural changes in early stage amelogenesis in vascularly perfused rat incisors.

Vascularly perfused rat incisors were investigated by electron microscopy in order to achieve further definition of the origin and nature of stippled material in developing enamel. A prolonged (20-minute) pre-wash perfusion of the rat with a physiological solution at 37°C prior to perfusion fixation retained good morphology of the secretory ameloblasts and adjacent enamel, showing virtually no extracellular accumulation of granular material. Granular material appeared throughout the developing enamel after pre-wash perfusion at low temperatures (0-2°C), and accumulated in the extensively dilated extracellular spaces between the proximal portions of Tomes' processes, where numerous coated pits and coated vesicles were located. Vascular perfusion at 37°C, preceded by a cold perfusion, abolished the granular material in the developing enamel, whereas the granular material in the extracellular spaces between Tomes' processes remained as huge droplets of dense material. These results indicate that at least a part of the matrix protein of rat incisor developing enamel has the physico-chemical property to dissociate at low temperature during pre-wash perfusion and move toward the enamel surface. Thus, in perfusion-fixed specimens, an intimate relationship between the artifactual formation of stippled material and the temperature of the pre-wash perfusate is suggested.

Effects of fluoride on protein secretion and removal during enamel development in the rat.

Enamel maturation consists of a loss of the early secreted matrix proteins and an increase in mineralization. This study investigated the changes in enamel proteins of the rat incisor, caused by the ingestion of fluoride at various stages of enamel formation. Rats were given 0, 10, 25, 50, or 100 ppm fluoride in drinking water for five weeks. Changes in the protein composition of the secretory, early-maturation, and late-maturation enamel were investigated by means of gel filtration chromatography and polyacrylamide gel electrophoresis. No differences were found between fluorosed and control enamel proteins in secretory enamel. In fluorosed early-maturation enamel, amelogenins were retained in larger quantities than in control enamel in animals ingesting 25 ppm fluoride or greater. At the late-maturation stage of enamel formation, only enamel from animals ingesting 100 ppm fluoride in drinking water contained more protein, when compared with control enamel. This study suggests that fluoride ingestion levels resulting in enamel fluorosis inhibit the mechanisms involved in the removal of proteins during enamel maturation.

Cyclical uptake pattern of tetracycline in post-secretory maturation phase enamel demonstrated in rooted teeth

Uptake of tetracycline by enamel in the short-term was studied at an advanced stage of crown formation and after completion of crown formation in deciduous molars in the cat. Both secretory phase enamel and bands of postsecretory, maturation phase enamel labeled rapidly. The pattern of labeling mimicked that seen in the continuously growing, rootless incisor teeth of the rat, with narrow doublets fusing to form narrow bands with wide unlabeled intervals in the short term. This is a physiological demonstration which indicates that cyclical activity and changes may occur in vivo during the maturation phase of amelogenesis in rooted teeth. It is also noted that dentine did not, and that some circumscribed patches of bone did label in the same animals in the same time interval. Short-term tetracycline labels are lost following conventional histological processing, but are retained after freeze-drying or air-drying.

Multiple forms of acid phosphatase in rat secretory enamel organ.

Acid phosphatase activity was studied biochemically in homogenates of secretory enamel organs from the rat. Incubations with crude homogenate failed to show distinct pH optima or kinetics characteristic for single enzymes. Crude homogenate activity was strongly inhibited by concentrations higher than 1 mM of NaF and Na-tartrate, and higher than 10 mM of ZnSO4 and para-bromotetramisole oxalate. 10 mM MgCl2 gave a slight stimulation. CaCl2, KCl and EDTA were uneffective. Electrophoretic separation of the crude homogenate acid phosphatase on Triton X-100 containing polyacrylamide gel demonstrated the presence of at least three multiple forms of the enzyme. Two of them showed distinct pH optima at pH 4.4. The third one showed a broad pH plateau in the acid pH range. Kinetic studies of the three forms indicated single enzyme reactions. Two forms had electrophoretic mobilities similar to alkaline phosphatase. One form could be solubilized only after Triton X-100 treatment. All forms were strongly sensitive to 10 mM NaF when added to the reaction mixture. The sensibility to 10 mM ZnSO4, CuSO4, Na-tartrate and para-bromotetramisole oxalate differed between the different forms.

Calcium-stimulated ATPase activity in homogenates of the secretory enamel organ in the rat.

The activity of calcium-stimulated ATPase (E.C. 3.6.1.3) in homogenates of the secretory enamel organ of rat incisors was studied biochemically. ATP hydrolysis was estimated from the amount of inorganic phosphate liberated. An analysis of the total degradation of ATP was initially performed to ensure that the enzyme assays pertained to the original substrate, ATP, and were not influenced by reaction products formed. Standard incubations were run in tris-maleate buffer, pH 8.2, with 3 mM ATP, 3 mM Ca2+ and 0.5 mM R 8231 at 37 degrees C. The presence of R 8231 was necessary to inhibit nonspecific alkaline phosphatase. The calcium-stimulated ATPase was completely inhibited when heated at 55-60 degrees C for 5 min. The pH optimum was found to be 8.2. The hydrolysis was substantially dependent on Ca2+ and was fastest when the ATP:Ca2+ ratio was 1:1. High substrate concentrations inhibited the hydrolysis. The addition of 1 mM Zn2+ and Ni2+ to the incubation medium markedly inhibited the hydrolysis as did, though less strongly, p-hydroxymercuribenzoate, oligomycin, EDTA and ruthenium red. l-Cysteine, mercaptoethanol, iodoacetic acid and sodium azide were without effect. F- was without effect unless added to a final concentration above 15 mM to media where Ca2+ had first been allowed to react with ATP.

Histochemical and electron probe analysis of secretory ameloblasts of developing rat molar teeth.

Calcium was not found in secretory ameloblasts and stratum intermedium cells when treated with OsO4-pyroantimonate or when surfaces prepared by fracturing fresh, rapidly frozen, developing molar tooth germs were subject to electron probe X-ray analysis. Pyroantimonate reaction product, considered to be calcium, was found in mitochondria of enamel organ cells which were first placed in a bath containing calcium and potassium. The plasma membrane was disrupted in cells ehich showed mitochondrial localization of reaction product. The results provide no data which indicates that enamel organ cells have a direct, active role in the movement of calcium into the enamel. Rather, it is suggested that the secretory enamel organ might serve as a selective barrier in regulating the initial mineralization of enamel.

Adenosine triphosphate catabolism in homogenates of rat secretory enamel organs incubated in histochemical lead media.

To investigate how lead, when used as trapping agent, influences the ATP hydrolysis and to study how ATP is catalyzed in histochemical systems, homogenized secretory enamel organs were incubated in histochemical [3H]-ATP media. Aliquots from the media were taken after 3, 10, 20 and 30 min, the ATP and formed metabolites were separated by electrophoresis and radiometrically quantitated. In media lacking both lead and homogenate 2% of the ATP was spontaneously hydrolyzed during 30 min incubation at room temperature. The presence of lead caused an additional 8% hydrolysis at pH 7.2 and an additional 20% hydrolysis at pH 9.4. In the presence of homogenate, however, lead caused a net decrease of the hydrolysis of ATP as well as of ADP and AMP. This enzyme inhibition varied from around zero to some 80%, depending on pH and substrated involved. In homogenate-containing lead media, at both pH 7.2 AND 9.4, ATP was rapidly hydrolyzed primarily to ADP and subsequently to AMP and adenosine and/or inosine. After 5--10 min ADP constituted the predominant substrate at both pH:s. At pH 7.2 ADP remained so for the rest of the incubation, whereas at pH 9.4 AMP was predominant substrate at the end of the incubation. AMP was the finan catabolic product in experiments at pH 7.2, and adenosine and/or inosine at pH 9.4. Inorganic phosphate was liberated almost linearly during the whole incubation period. The results indicate that histochemical studies of substrate specific ATP-ases should be performed with short incubation times and, when high specific activities are present, in large quantities of incubation media to reduce interference by ADP and AMP hydrolyzing enzymes.