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Background Severe inflammation with necrosis and fibrosis of the gallbladder in acute cholecystitis increases operative difficulty during laparoscopic cholecystectomy. This study aimed to assess the use of preoperative MRI in predicting pathological changes of the gallbladder associated with surgical difficulty. Methods Patients who underwent both preoperative MRI and early cholecystectomy for acute cholecystitis between 2012 and 2018 were identified retrospectively. On the basis of the layered pattern of the gallbladder wall on MRI, patients were classified into three groups: high signal intensity (HSI), intermediate signal intensity (ISI), and low signal intensity (LSI). The endpoint was the presence of pathological changes of the gallbladder associated with surgical difficulty, such as necrosis, abscess formation and fibrosis. Results Of 229 eligible patients, pathological changes associated with surgical difficulty were found in 17 (27 per cent) of 62 patients in the HSI group, 84 (85 per cent) of 99 patients in the ISI group, and 66 (97 per cent) of 68 patients in the LSI group (P, Variations in the layered pattern of the gallbladder wall on MRI were significantly associated with fibrosis and necrosis of the gallbladder wall in acute cholecystitis. Intermediate to low signal intensity of the gallbladder wall on MRI had a sensitivity of 90 per cent and a specificity of 73 per cent for predicting pathological changes in the gallbladder wall associated with surgical difficulty during laparoscopic cholecystectomy for acute cholecystitis. The results indicate that MRI could be a novel method of predicting surgical difficulty in laparoscopic cholecystectomy. MRI could possilby predict surgical difficulty of cholecystectomy.

Background Few studies have been conducted on patterns of recurrence after resection for distal cholangiocarcinoma (DCC). The aim of this study was to investigate the incidence and pattern of recurrence after resection of DCC, and to evaluate prognostic factors for time to recurrence and recurrence-free survival (RFS). Methods Patients who underwent pancreatoduodenectomy with curative intent for DCC between 2001 and 2010 at one of 30 hospitals in Japan were reviewed retrospectively, with special attention to recurrence patterns. The Cox proportional hazards model was used for multivariable analysis. Results In the study interval, 389 patients underwent pancreatoduodenectomy for DCC with R0/M0 status. Recurrence developed in 213 patients (54·8 per cent). The estimated cumulative probability of recurrence was 54·3 per cent at 5 years. An initial locoregional recurrence occurred in 55 patients (14·1 per cent) and initial distant recurrence in 168 (43·2 per cent), most commonly in the liver. Isolated initial locoregional recurrence occurred in 45 patients (11·6 per cent). Independent prognostic factors for time to recurrence and RFS were perineural invasion (P = 0·001 and P = 0·009 respectively), pancreatic invasion (both P < 0·001) and lymph node metastasis (both P < 0·001). RFS worsened as the number of risk factors increased: the 5-year RFS rate was 70·6 per cent for patients without any risk factors, 50·3 per cent for patients with one factor, 31·8 per cent for those with two factors, and 13·4 per cent when three factors were present. Conclusion More than half of patients with DCC experienced recurrence after R0 resection, usually within 5 years. Perineural invasion, pancreatic invasion and positive nodal involvement are risk factors for recurrence.

Background Little is known about the value of portal vein (PV) resection in distal cholangiocarcinoma. The aim of this study was to evaluate the clinical significance of PV resection in distal cholangiocarcinoma. Methods Patients who underwent pancreatoduodenectomy (PD) for distal cholangiocarcinoma between 2001 and 2010 at one of 31 hospitals in Japan were reviewed retrospectively with special attention to PV resection. Short- and long-term outcomes were evaluated. Results In the study interval, 453 consecutive patients with distal cholangiocarcinoma underwent PD, of whom 31 (6·8 per cent) had combined PV resection. The duration of surgery (510 versus 427 min; P = 0·005) and incidence of blood transfusion (48 versus 30·7 per cent; P = 0·042) were greater in patients who had PV resection than in those who did not. Postoperative morbidity and mortality were no different in the two groups. Several indices of tumour progression, including high T classification, lymphatic invasion, perineural invasion, pancreatic invasion and lymph node metastasis, were more common in patients who had PV resection. Consequently, the incidence of R1/2 resection was higher in this group (32 versus 11·8 per cent; P = 0·004). Survival among the 31 patients with PV resection was worse than that for the 422 patients without PV resection (15 versus 42·4 per cent at 5 years; P < 0·001). Multivariable analyses revealed that age, blood loss, histological grade, perineural invasion, pancreatic invasion, lymph node metastasis and surgical margin were independent risk factors for overall survival. PV resection was not an independent risk factor. Conclusion PV invasion in distal cholangiocarcinoma is associated with locally advanced disease and several negative prognostic factors. Survival for patients who have PV resection is poor even after curative resection.

Background The aim of the study was to investigate the prognostic impact of lymph node metastasis in cholangiocarcinoma using three different classifications. Methods Patients who underwent pancreaticoduodenectomy for distal cholangiocarcinoma in 24 hospitals in Japan between 2001 and 2010 were included. Survival was calculated by means of the Kaplan–Meier method and differences between subgroups were assessed with the log rank test. The Cox proportional hazards model was used to identify independent predictors of survival. χ2 scores were calculated to determine the cut-off value of the number of involved nodes, lymph node ratio (LNR) and total lymph node count (TLNC) for discriminating survival. Results Some 370 patients were included. The median (range) TLNC was 19 (3–59). Nodal metastasis occurred in 157 patients (42·4 per cent); the median (range) number of involved nodes and LNR were 2 (1–19) and 0·11 (0·02–0·80) respectively. Four or more involved nodes was associated with a significantly shorter median survival (1·3 versus 2·2 years; P = 0·001), as was a LNR of at least 0·17 (1·4 versus 2·3 years; P = 0·002). Involvement of nodes along the common hepatic artery, present in 21 patients (13·4 per cent), was also associated with a shorter survival (median 1·3 versus 2·1 years; P = 0·046). Multivariable analysis among 157 node-positive patients identified the number of involved nodes as an independent prognostic factor (risk ratio 1·87; P = 0·002). Conclusion The number of involved nodes was a strong predictor of survival in patients with distal cholangiocarcinoma.

Background Enamel mineralization taking place during amelogenesis is a unique model to investigate carbonatoapatite formation in vivo. The abundance of proteinaceous crystal growth inhibitors, in particular amelogenins, contributes significantly to the mineralization process. Their putative roles are to prevent random proliferation of crystal nuclei and to regulate the growth kinetics and orientation of the formed enamel crystals. Methods The enamel fluid surrounding the forming enamel crystals contains high concentrations of carbonate and magnesium ions, both of which seem to modulate the mineralization process. Particularly, Mg ions can adsorb onto enamel crystal surfaces in a manner to compete with Ca ions. Enamel mineral formed during amelogenesis is featured as calcium-deficient, acid phosphate-rich carbonatoapatites. Currently the most putative stoichiometry model for enamel mineral is (Ca)5-x(HPO4)v(CO3)w(PO4)3-x(OH)1-x. Results Very significant changes in the morphology, stoichiometry, and solubility of enamel crystals occur during the various stages of amelogenesis. The early enamel mineralization comprises two events: the initial precipitation of the well-documented thin ribbons and the subsequent over-growth of apatite crystals on those templates. The thin ribbons precipitated in the vicinity of the secretory ameloblasts have the highest contents of acid phosphate, particularly in the form of exchangeable species, whereas both the exchangeable and unexchangeable acid phosphate decrease concomitantly with the progress of the apatite overgrowth and the appearance of elongated hexagonal crystals in the late secretory stages. Conclusions Those morphological and compositional features seem to be consistent with the formation of precursors, such as octacalcium phosphate. © 1996 Wiley-Liss, Inc.

In order to learn more about how the microenvironment for enamel mineralization is modified by fluoride at low concentrations (0 through 1 ppm) and how excess fluoride retards the degradation and removal of amelogenins, we studied precipitation reactions in an in vitro model utilizing a dialysis chamber. The results showed that, with the limited supply of Ca ions through the ultrafiltration membrane, the solution composition surrounding the seed crystals showed a proximity to the steady-state condition after 12-24 h equilibration. Major findings were that (a) fluoride overcame partially the inhibition of precipitation and growth reactions by enamel proteins and (b), with this accelerating effect of fluoride, the steady-state Ca concentrations in the media surrounding the seed crystals decreased substantially as a function of fluoride concentration. The overall results support the concept that the presence of fluoride in the mineralizing milieu can modify markedly the steady-state concentrations of mineral lattice ions, particularly decreasing free Ca2+ concentrations, which in turn may modulate protease activities in situ.

In order to assess fluoride accumulation and effects in developing dental tissues, one must determine the concentration profile of fluoride in the tissue and to assess separately the labile (i.e., free ions in fluid and ions associated with organic matter) and stable (i.e., incorporated into apatite lattice) pools of fluoride. Free fluoride ions in the mineralizing milieu markedly affect the driving force for precipitation and, as a result, the nature of precipitating crystals. The fluoride incorporated into the crystalline lattice increases the stability of the formed mineral. Improvement in the understanding of the mechanism of dental fluorosis requires more comprehensive information about the effects of fluoride on the ionic composition of the fluid phase, the nature of the initially precipitating mineral(s), the interactions between crystals and matrix proteins, and the enzymatic degradation of the proteins. Recent observations relevant to the role of fluoride in enamel formation include: (1) that there are threshold concentrations of fluoride below which the precipitation and hydrolysis of thin-platy octacalcium phosphate is facilitated but beyond which de novo apatite precipitation prevails; (2) that the presence of fluoride in the mineralizing milieu most likely affects the steady-state concentrations of mineral lattice ions; (3) that incorporation of fluoride into the stable pool is retarded by the presence of matrix proteins, particularly amelogenins, which inhibit the growth of apatite crystals; (4) that increasing the degree of fluoridation of apatite crystals enhances the adsorption of amelogenins onto the crystal surface, and (5) that amelogenins pre-adsorbed onto apatite crystals are more resistant to enzymatic cleavages by trypsin (used as a prototype of amelogeninases).

The aim of the present work was to investigate changes in cross-sectional morphologies of enamel crystallites as a function of location in secretory porcine enamel. Enamel tissues were obtained from 5- to 6-month-old slaughtered piglets. For examination by electron microscopy, a portion of the secretory enamel was embedded in resin and ultrathin sections were prepared with a diamond knife. In parallel studies, compositional and structural changes of enamel mineral were assessed by chemical analysis and Fourier transform infrared (FTIR) spectroscopy. For this purpose, two consecutive layers of the outer secretory enamel, each approximately 30 μm thick, were separated from the labial side of permanent incisors. Using high-resolution electron microscopy, early events of enamel crystal growth were characterized as the epitaxial growth of small apatite units on the lateral surfaces of the initially precipitated thin ribbon. These apatite units had regular triangle or trapezoid cross-sections. After fusions of those isolated trapezoids on both lateral sides of the platy template, the resulting enamel crystallites had the well-documented flattened-hexagonal shapes in cross-sections. The initially precipitated thin plate was buried inside the overgrown apatite lamella and then retained as a central dark line. Similar morphological evidence for the epitaxial nucleation and overgrowth of carbonatoapatite on the platy template was obtainedin vitro. Chemical and FTIR analyses of the enamel layer samples showed that the characteristics of the youngest enamel mineral were distinct from those of enamel crystals found in older secretory enamel. The overall results support the concept that initial enamel mineralization comprises two events: the initial precipitation of thin ribbons and the subsequent epitaxial growth of apatite crystals on the two-dimensional octacalcium phosphate-like precursor.

The present study was undertaken to assess the labile or surface pools of Mg, Na, and K ions in porcine enamel tissues at various developmental stages. The enamel samples, corresponding to the outer and the inner secretory, the early maturing, and the mature hard enamel, were dissected from the labial sides of permanent incisors of 6- to 8-month-old piglets. Each enamel sample was extracted successively with solutions of de-ionized water and 50 mmol/L Tris-4 mol/L guanidine buffer (for removal of organic matrix proteins, mainly amelogenins). The labile (free or organically bound) pools of Mg, Na, and K were assessed by the total amounts of these ionic species extracted by the water and Tris-guanidine buffer. The surface (adsorbed onto enamel mineral) pool of Mg was assessed directly by determination of the adsorption of Mg onto enamel mineral at various developmental stages. The results showed that: (i) 30-40% of the Mg in the secretory and early maturation enamel was in the surface pool (adsorbed onto the enamel mineral); (ii) 25 to 40% of the total sodium in the enamel samples was in labile forms; and (iii) most (around 70-80%) of the total potassium was readily extracted in water and appeared to originate from the enamel fluid; only marginal portions remained in the solids. The present adsorption studies also indicated that the maximum uptake of magnesium in the early maturation enamel was due mostly to an increase of the occupancy by Mg ions of adsorption sites on the crystal surfaces, which become accessible with a massive removal of enamel matrix proteins. The observation that the surface pool of Mg (and the total Mg content) decreased markedly with the advancement of mineralization is explained by the displacement of the adsorbed Mg from the crystal surfaces by Ca2+ having a higher adsorption affinity for the same adsorption sites and by changes in the properties of the mineral surfaces. The overall results support the contention that incorporation of Mg into developing enamel upon resorption of the organic matrix may play a key role in regulating enamel crystal growth.

Magnesium (Mg) is a conspicuous constituent of hard tissues but its possible role in biomineralization is poorly understood. It is possible that Mg2+ adsorbed onto bioapatites may contribute to the modulation of crystal growth as such inhibitory activity has been reported for synthetic apatites. The present study was undertaken to determine the adsorption isotherms of Mg ions onto synthetic apatites and biominerals in tooth and bone tissues in the presence of other ions of natural occurrence. Synthetic crystals used as adsorbents were hydroxyapatite and, as a better prototype for the biomineral, Mg-containing carbonatoapatite. Human enamel and dentin materials were obtained from extracted, caries-free, permanent teeth. Porcine dentin materials at two developmental stages were obtained from erupted deciduous and unerupted permanent teeth of a 6-month-old slaughtered piglet. Porcine bone was obtained from the cortical portion of the mandible of the same animal. All biomineral samples were pulverized and then treated by plasma ashing (deproteination) at about 60°C. Each of the powdered samples was equilibrated in solutions containing various initial concentrations of Mg2+, Ca2+, and Na+ (or K+) as nitrate salts. Following equilibration, concentrations (and activities) of magnesium and calcium ions in the experimental solution were determined. The pH values of the equilibrium solutions were in the range of 6.2–6.5. Experimental data of the Mg adsorption onto hydroxyapatite were interpreted on the basis of a Langmuir-type model for binary systems assuming competition of Mg2+ and Ca2+ for the same adsorption sites on the crystal surfaces of the apatites. According to this model, the adsorbed Mg is expressed as a function of the ionic activity ratio (Mg2+)/(Ca2+) in the equilibrium solution. The model contains two parameters, the adsorption selectivity constant Ks and the maximum number of adsorption sites N (μmol/g). The numerical values of Ks were similar for all adsorbents used (synthetic and biological) and indicated the preferential adsorption of Ca2+ probably due to spacial restrictions extending to the very surface of the crystals. The initial level of Mg2+ in the surface pool was different in the various biominerals, probably reflecting the composition of fluid in which the biominerals were formed. Whereas the surface pool of Mg of human enamel was marginal, only 5% of the total Mg, significant fractions of the total Mg in human and porcine dentins (about 20–30%), and porcine bone (about 40%) existed on the crystal surfaces. There were significant differences in the total Mg and the value of the parameter N between young (unerupted) and mature (erupted) dentin minerals. It was ascertained that the occupancy of adsorption sites by Mg ions became greater with maturation of the dentin tissues. The overall results suggest that the Mg-mineral interaction in tooth and bone tissues may be a highly tissue-specific process, presumably reflecting differences in fluid composition (particularly Ca and Mg activities) responsible for biomineralization.

The composition and morphology of crystals formed in fish enameloid were investigated at various developmental stages. Species studied were shark, skate, red seabream, puffer, and carp. For comparative purposes, mammalian enamel samples were obtained from developing porcine teeth and erupted human teeth. Chemical and physical analyses (FTIR, X-ray diffraction, and electron microprobe) indicated that the mineral phase of enameloid in elasmobranch and teleost fish was most adequately characterized as fluoridated carbonatoapatites but that the degree of fluoridation and carbonation of the apatite latice varied among species and, within species, with the developmental stage. High resolution electron microscopy demonstrated differences in the nature and morphology of the initially precipitating crystallites of the enameloids of elasmobranch as compared with those of teleost fish. The elasmobranch enameloid contained high levels of fluoride (2.5% wt or more) at the beginning of precipitation and its mineralization was characterized by the initial formation and subsequent growth of prismatic apatite crystals having hexagonal (frequently equilateral) cross-sectional areas. In contrast, the initially precipitating crystallites in the teleost fish appeared as thin ribbons, like those commonly reported in mammalian enamel. The crystal morphology of the teleost fish enameloid may be related to the low fluoride contents maintained in the early stages of enameloid formation. However, the growth process of enameloid crystallites varied within the teleostei depending on the fluoride accretion during the mineralization stages. In the seabream enameloid (the highfluoride group), the growth on the side planes of the apatitic prisms was accelerated with increasing fluoride concentration in the tissue; the resulting crystallites had equilateral hexagonal cross sections. The morphology and growth of enameloid crystallites in puffer and carp (the low-fluoride group) were similar to those reported in mammalian enamel. However, appreciable differences existed between the enameloid of this fish group and the mammalian enamel with respect to carbonation, fluoridation, and central defects of their crystallites. The overall results support the contention that fluoride significantly affects the morphology and structure of enameloid crystals, most probably by increasing the driving force for carbonatoapatite formation and by accelerating hydrolysis of possible acidic precursors.

The solubility properties of hydroxyapatite (HA) are compared with those of human dental enamel and dentin. The apatites used in this study were equilibrated with dilute phosphoric acid solutions in CO2-containing atmospheres. The experimental results are interpreted in terms of solubility models which consider the biological materials as either HA or carbonatoapatites. Both in the HA and the dental mineral systems, the results are consistent with the precipitation of another carbonate-containing apatitic phase during equilibration. However, although the chemical behavior of the HA systems is in very good agreement with predictions based on the solubility models, the results with the bioapatities are not; this inconsistency is more marked for dentin than for enamel but in both cases the results clearly indicate the inadequacy of assuming for these dental apatites the stoichiometry of HA. The models and the experimental results show that, in principle, it is possible to define the two dental minerals in terms of respective solubility product constants, if independent information is attained on the stoichiometry of these bioapatites.

The composition of enamel mineral corresponds to that of a calcium carbonato-apatite. For insight to be gained into the precipitation of carbonato-apatites having specific properties (crystal size, morphology, and carbonate incorporation into the crystal lattice), apatites were prepared at 80°C in aqueous systems having various CO3 concentrations and pH values of around 7.5 or 10.5 (± 0.5). The various preparations had a wide range (0.005 to 0.19) of CO3/Ca molar ratios that bracket the ratios found in porcine enamel mineral at various developmental stages. Fourier transform infrared spectroscopy (FTIR) and x-ray diffraction analyses showed that the calcium apatites precipitating at neutral pH incorporated the carbonate into both the hydroxyl and phosphate ion sites in their lattices (A,B-types), whereas the preparations made at the alkaline pH (high OH-CO32- competition) or in the presence of fluoride (F--CO32- competition) yielded only the B-type carbonato-apatite. It was also ascertained that the size and morphology of the carbonato-apatites, assessed by specific surface area determination and high-resolution electron microscopy, were highly dependent on the driving force for precipitation and the presence of regulators (CO 32- and F-) in solution. In neutral media, early precipitates were thin-ribbon in appearance, but grew into crystals having flattened-hexagonal cross-sections. In the presence of fluoride or in alkaline media, acicular apatite crystals, precipitated initially, grew into large rod-like carbonato-apatites having a symmetric-hexagonal cross-section. In both neutral and alkaline solutions, carbonate inhibited the growth of apatite crystals along their c axis, leading to the formation of bulkier crystals. The formation of carbonato-apatites at the neutral pH and their properties are consistent with observations made on enamel minerals formed in the early developmental stages.

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.

Ameloblastic fibro-odontoma is the unique entity of epithelial-ectomesenchymal odontogenic tumors, which is characterized by enamel formation in addition to dentine. We examined immunohistochemically a case of this tumor in which enamel having prism structures was developed in the absence of odontoblast differentiation but was in contact with mesenchymal matrices. Histological examination showed diverse morphological features of epithelial tumor cells, e.g., cuboidal cells comprising tooth bud-like projections, ameloblast- and stellate reticulum-like cells, and residual cells in forms of extended cords or islands of odontogenic epithelium. Immunostaining with anti-amelogenin sera proved that the intracellular production of amelogenins was initiated at the tooth bud-like stage. The secreted amelogenins were detected almost exclusively in the induced enamel and dentinoid areas, as well as in the core region of cementicle-like spheres deposited in the encapsulating stroma. The results obtained indicate that the odontogenic tumor epithelia and its products, i.e., amelogenins, participate in multifaceted aspects of dental hard tissue formation that takes place during oncogenesis.

Fluoride participates in many aspects of calcium phosphate formation in vivo and has enormous effects on the process and on the nature and properties of formed mineral. The most well-documented effect of fluoride is that this ion substitutes for a column hydroxyl in the apatite structure, giving rise to a reduction of crystal volume and a concomitant increase in structural stability. In the process of enamel mineralization during amelogenesis (a unique model for the cell-mediated formation of well-crystallized carbonatoapatite), free fluoride ions in the fluid phase are supposed to accelerate the hydrolysis of acidic precursor(s) and increase the driving force for the growth of apatitic mineral. Once fluoride is incorporated into the enamel mineral, the ion likely affects the subsequent mineralization process by reducing the solubility of the mineral and thereby modulating the ionic composition in the fluid surrounding the mineral, and enhancing the matrix protein-mineral interaction. But excess fluoride leads to anomalous enamel formation by retarding tissue maturation. It is worth noting that enameloid/enamel minerals found in vertebrate teeth have a wide range of CO3 and fluoride substitutions. In the evolutionary process from elasmobranch through teleost enameloid to mammalian enamel, the biosystems appear to develop regulatory functions for limiting the fluoridation of the formed mineral, but this development is accompanied by an increase of carbonate substitution or defects in the mineral. In research on the cariostatic effect of fluoride, considerable emphasis is placed on the roles of free fluoride ions (i.e., preventing the dissolution and accelerating the kinetics of remineralization) in the oral fluid bathing tooth mineral. Fluoride also has been used for the treatment of osteoporosis, but much still remains to be learned about maximizing the benefit and minimizing the risk of fluoride when used as a public health measure.

Possession of full-thickness hard enamel appears to be one of the indispensable life-saving characteristics of rats. Previous studies by Suga and his colleagues and by others demonstrated that various types of malformation are evoked in continuously erupting rat incisors. In the current report, we directed our effort to oversee various types of enamel malformation caused experimentally in rat incisors. We surveyed the specimens collected by Suga and his colleagues, as well as specimens we obtained. From the results, it is conceivable that perturbation of the programmed sequential events during enamel development is a major factor in the establishment of enamel malformation. Animal studies with either 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) or a multidentate phosphonic acid (EDTPO) confirmed that dentin mineralization provides a certain inductive effect on the secretion of enamel matrix and subsequent enamel crystallization. Our recent studies using anti-microtubular agents led to the conclusion that the acceleration of mineralization in outer enamel is a type of enamel malformation, most likely due to disruption of the cellular regulation of calcium transport under severe toxic regimens. In future work, experimental approaches combining measurements of kinetic factors with static observation of enamel lesions are required before we can gain a comprehensive understanding of the pathogenesis of disturbed enamel mineralization. The kinetic factors to be considered include the rates of tissue apposition and tooth eruption which determine the total volume of tooth substance formed, and the rate of mineral accretion. Furthermore, information as to the composition, crystallinity, solubility, and mechanical properties of enamel defects is needed before we can assess the susceptibility of teeth having those lesions to caries and other physico-chemical attacks in the oral environment.

Acid phosphate is one of the major impurities incorporated into bioapatites, and its quantity and environment in forming mineral have been used as diagnostic probes to pursue acidic precursor(s). Currently, little is known about the structural feature of non-stoichiometric octacalcium phosphate (OCP), which has been advocated to be, most plausibly, mineral salt initially formed during amelogenesis. In the present report, we attempt to define the state of acid phosphate in OCP crystals which were Ca-deficient and contained 40% total phosphate as acid phosphate. We assessed fractions of acid phosphate in discrete environments by extracting the crystals in either deionized water, 10 mmol/L NaOH solution (initial pH 11), or 150 mmol/L Tris buffer at pH 7.4. Solid samples before and after the treatments were examined by chemical analyses and x-ray diffraction. The results indicated that successive extractions with use of the alkaline solution brought about a reversible change (not hydrolysis) in the interior structure of OCP, which accompanied a marked decrease in acid phosphate. A substantial part of the lost acid phosphate was restored during subsequent treatments at neutral pH, and, intriguingly, this restoration accompanied a re-ordering of OCP structure. The data suggested that the acid phosphate in OCP is separated into three pools: (a) a stable pool corresponding to roughly 50 to 60% of the total acid phosphate, (b) a reversibly exchangeable pool corresponding to 25 to 30% of the acid phosphate which may exist either in the water layer or on crystal surfaces, and (c) an unstable (or irreversibly lost) pool corresponding to 15 to 20% of the acid phosphate, a part of which might be explained by the presence of excess hydrogen in OCP. The present work supports the concept that protons and, to a lesser magnitude, phosphate species can diffuse into and out of the OCP lattice prior to initiation of its hydrolytic transition into apatite.

Alveolar bone reconstruction in growing dog during the retention period following orthodontic tooth movement was studied. Three beagle dogs (8-10 kg body weight, about one-year-old) were used and two of the animals were subjected to histological observation. The upper 2nd and lower 3rd premolars on both sides were extracted prior to the orthodontic treatments. After a healing period of one month, the upper 3rd premolar and the lower 4th premolar on the right side were moved mesially with a conventional orthodontic force for 8 weeks, and then retained in their new position for 4 weeks. The contralateral corresponding premolars were used as control. The alveolar bone was double-labeled with tetracycline (TC) during the movement and calcein (Cal) during the retention period. Alveolar bone structure and labeling patterns were examined by contact microradiography, conventional fluorescence microscopy, and confocal laser scanning microscopy (CLSM). Optimizing the separation of TC and Cal labelings in the alveolar bone was attained by the simultaneous use of ultraviolet (364 nm) and argon (488 nm) laser sources for excitation of TC and Cal, respectively. Cal labeling, indicative of new bone deposition showed two distinct patterns: lamination at the periodontal surface and rings circumscribing the vascular canal. The cementum surface also exhibited active deposition during the experimental period. Bone formation was affected by slight changes in magnitude and direction of orthodontic or occlusal forces. CLSM is valuable in deciphering the process of alveolar bone remodeling.

A mouse cDNA encoding a 180 amino acid amelogenin was subcloned into the pET expression plasmid (Novagen, Madison, WI) for production in Escherichia coli. A simple growth and purification protocol yields 20–50 mg of 95–99% pure recombinant amelogenin from a 4.5-liter culture. This is the first heterologous expression of an enamel protein. The expressed protein was characterized by partial Edman sequencing, amino acid composition analysis, SDS-PAGE, Western blotting, laser desorption mass spectrometry, and hydroxyapatite binding. The recombinant amelogenin is 179 amino acids in length, has a molecular weight of 20,162 daltons, and hydroxyapatite binding properties similar to the porcine 173 residue amelogenin. Solubility analyses showed that the bacterially expressed protein is only sparingly soluble in the pH range of 6.4–8.0 or in solutions 20% saturated with ammonium sulfate. The purified protein was used to generate rabbit polyclonal anti-amelogenin antibodies which show specific reaction to amelogenins in both Western blot analyses of enamel extracts and in immunostaining of developing mouse molars.

Circular dichroism (CD) studies were conducted to gain a better insight into the conformation of amelogenins, which were isolated from developing enamel of piglets. The intact porcine amelogenin and its degraded products were purified chromatographically. The 25-residue peptide corresponding to the segment at the C-terminus was synthesized. CD spectra of these samples were measured at pH 5.0-5.3 in the temperature range between 4 and 90 degrees C. The most remarkable finding was that the CD spectrum of the intact amelogenin was accounted for by the sum of the spectra of the three fragments at the N-terminal, central, and C-terminal regions, supporting the hypothesis that the structure of the whole protein consists of discrete folding units. Furthermore, low-angle laser light scattering analysis provided evidence that the 20 kDa amelogenin, the most abundant extracellular matrix protein in forming enamel tissue, exists in a monomeric form at pH 5.3 and 25 degrees C. It was tentatively concluded that the N-terminal region contains beta-sheet structures, while the spectral characteristics of the C-terminal region are similar to those of a random coil conformation. The conformation of the central region was characterized by a strong negative ellipticity at 203 nm, although its nature remains to be defined.

The purpose of this research was to clarify the influence of high speed swimsuits on the lactate curve test among competitive swimmers. Method: 8 Japanese collegiate student championship participants were evaluated. These students were divided randomly into two groups. Four of the subjects wore a high-speed swimsuits (LZRR) and the four remaining subjects wore conventional swimwear, all the members performed the lactate curve test on the same day. Lactic acid was measured using Lactate ProR (Arclay, Japan) with CCD enzyme electrode method. The Lactate curve was described using analysis program MEQNET Lactate ManagerR from the acquired value, and the velocity of lactic acid was computed with values of 2, 4, 6, 8 mmol/l. In the lactate curve test all members swam 200 m free style swimming 4 times (+40 seconds of best time, +30 seconds, +20 seconds, maximal effort). The rest time for each trial was 15 min, immediately after swimming lactic acid and HR was measured and velocity was computed from the time required. All the subjects were monitored with a video camera (SK-2130, SONY, Japan) with a sampling frequency of 60Hz in the sagittal plane to measure the angular displacement of their different joints during the periods of the griding motion. A motion analysis system (Frame-DIAS4, DKH, Japan) was used to digitize ten body landmarks. After 3 days, each one wore a different swimsuit, and performed a 2nd lactate curve test and motion analyzing. All the members performed the same training programs for all the periods. Result and conclusions: The subjects wore LZRR, in all cases, shifted their lactate curve to the right. The mean of the velocity equivalent to eight persons at lactic value 2, 4 mmol/l (LZRR: 1.27± 0.03, 1.33± 0.03 conventional swimsuit: 1.25± 0.04, 1.31± 0.04 m/s), a difference was not shown depending on the swimsuits although at the lactic value 6,8mmol/l (LZRR: 1.39± 0.03, 1.44± 0.04 conventional swimsuit: 1.36± 0.04, 1.41± 0.04 m/s) a significant difference (p< 0.01) was shown between the LZRR and conventional ones. The required to swim 200 m at maximal speed in the LZRR was 1.6± 0.1 m/s and that of the conventional swimsuit was 1.54± 0.1 m/s. It showed a significant difference (p< 0.01). Moreover, the subjects wore LZRR maintained the highest speed during the gliding motion longer than that of the conventional swimsuit. It was thought that the high-speed swimsuits (LZRR) obviously had a different functionality to the conventional swimsuits.

The present study was undertaken to investigate glycosylation of porcine enamel proteins secreted in the secretory stage of amelogenesis and to gain insight into functional roles of glycosylated proteins in enamel mineralization. Enamel proteins, isolated from various zones of the secretory enamel, were separated by SDS-PAGE and then transferred on to a nitrocellulose membrane. The transblotted proteins were visualized with either antibodies against porcine amelogenins or various biotin-conjugated lectins. The lectins used were Con-A, GS-II, STA, WGA, s-WGA, GS-I, MPA, VVA, PNA, RCA-I, DBA, SJA, UEA-I, Lotus-A and LPA. The results of the immuno- and lectin blottings revealed that most of the lectins did not bind to porcine amelogenins, while a large number of non-amelogenins having various molecular masses were stained strongly with the conjugated WGA, Con A and MPA lectins. On the basis of the binding specificity with the lectins, porcine non-amelogenins were classified into two groups: WGA (and Con A)-binding moieties at 60–90 kDa (WGA-HMW); and MPA-binding moieties at 13–17 kDa (MPA-LMW). These two groups of non-amelogenins differed distinctly in terms of their localization and stability in the secretory tissue and their adsorption properties onto hydroxyapatite. The WGA-HMW were concentrated in the outer region adjacent to the ameloblasts and disappeared (due to degradation) in the underlying inner secretory enamel. In contrast, the MPA-LMW were found in all zones of the secretory enamel and their quantity remained relatively constant. Histochemical studies using FITC-conjugated WGA and MPA showed that the fluorescence-labelling of WGA was localized in the core region of prism rods, while the fluorescence-labelling of MPA was locally limited at the rim of prism rods or at the prism sheath. In separate adsorption studies, it was found that the WGA-HMW, as well as the intact amelogenins, displayed a high adsorption affinity on to apatite crystals, whereas the MPA-LMW showed only marginal adsorption on to apatitic surfaces. The overall results indicate that part of the heterogeneity found in porcine enamel proteins can be ascribed to variations of carbohydrate moieties attached to non-amelogenins. As to possible functional roles of porcine enamel proteins, the results strongly suggest that the WGA-HMW non-amelogenins and the intact amelogenins participate directly in regulation of the precipitation and growth of enamel crystals taking place in the vicinity of the ameloblasts, while the MPA-LMW non-amelogenins may contribute to development of a well-organized enamel structure by supporting a framework of prism rods.

The present studies were undertaken to investigate the presence of common epitopes of mammalian amelogenins at the C-terminus and the possible functional importance of the conserved C-terminal domain in enamel mineralization during mammalian amelogenesis. Enamel proteins, including the intact amelogenins and their degraded polypeptides, were isolated from the secretory enamel of pig, cow, rat, and rabbit incisors. Rabbit and rat antipeptide sera, as well as rat anti-25 kD and 20 kD pig amelogenin sera, were used to identify the amelogenins among the isolated matrix proteins of each of the animal species. The antipeptide sera were developed previously (Aoba et al. [19]) using as immunogens the two synthetic peptides, C13 and C25, which correspond to the last 12 (plus Cys for KLH-conjugation) and 25 amino acid residues of pig intact amelogenin, respectively. Reactivity of the enamel proteins with each antiserum was examined by Western blot analysis. The results of immunoblotting showed that a few enamel matrix proteins in each of the mammalian species were recognized by the anti-C13 serum, specifically, pig amelogenin at 25 kD (and trace components at 27, 22, and 18 kD), cow amelogenin at 28 kD (trace components at 26, 22, 19, and 14 kD), rat amelogenins at 28 and 26 kD (and a trace component at 20 kD), and rabbit amelogenins at 24 and 21 kD (and a trace at 13 kD). The anti-C25 serum reacted additionally with pig amelogenin at 23 kD, cow amelogenin at 27 kD (a major matrix constituent), and rabbit protein at 19 kD. The anti-pig 20 kD amelogenin (lacking the last 25 amino acid residues at the C-terminus) serum reacted with a large number of pig, cow, and rat amelogenins but, interestingly, with none of the rabbit enamel proteins. Probing of rat enamel proteins with Maclura pomifera lectin showed the heterogeneity of glycosylation of rat amelogenins, particularly between the 28 and 26 kD intact amelogenins. In parallel adsorption studies, part of the enamel protein samples isolated from each of the species was used as adsorbates to investigate the selective adsorption of amelogenins onto hydroxyapatite. Immunoblot analysis of the proteins adsorbed onto the crystals revealed that the mammalian amelogenins having the common epitopes at the C-terminus, in general, adsorb preferentially onto hydroxyapatite. The adsorption affinity of the degraded amelogenins decreased significantly with the loss of reactivity toward the anti-C13 serum. The overall results support the contention that the intact mammalian amelogenins, including rat and rabbit amelogenins, share common epitopes at the C-terminus and that the conserved C-terminal domain plays an important role in setting the molecular structures of the intact amelogenins so as to facilitate the protein-enamel mineral interaction.

This was an immunological investigation of the processing of porcine amelogenins in situ. Rabbit and rat anti-peptide sera reacted specifically with the hydrophilic segment of the intact amelogenins at the C-terminus. The immunogens used were the synthetic peptides: (a) C13 composed of PATDKTKREEVDC and (b) C25 composed of MQSLLPDLPLEAWPATDKTKREEVD. These peptides correspond to the C-terminal 12- and 25-residue segments of porcine amelogenin, respectively. Cystine was introduced at the C-terminus of C12 for KLH-binding (C13). Western blot analysis disclosed that: (i) both rabbit and rat anti-C13 sera reacted selectively with the 25-kDa porcine amelogenin and three other minor components (27, 22 and 18 kDa); (ii) anti-C25 peptide sera, additionally, reacted with the 23-kDa amelogenins (a degradation derivative of the 25-kDa protein, lacking the 12-residue segment at the C-terminus) and as trace components, 20-, 16- and 14-kDa moieties. Importantly, all the proteins reactive with the anti-C13 serum were concentrated in the outer secretory enamel adjacent to the ameloblasts, decreasing significantly in the underlying inner secretory enamel. Immunohistochemical studies applying the anti-peptide sera to the developing tooth germs of a minipig also confirmed the localization of reactivity in the outer secretory region. Neither anti-peptide serum reacted with porcine non-amelogenins, serum proteins nor dentine matrix proteins at the dilutions tested. however, it was found that both the anti-C13 and C25 sera reacted with human keratin.(ABSTRACT TRUNCATED AT 250 WORDS)

The solubility of enamel mineral (a carbonated apatite) formed at various stages of porcine amelogenesis was investigated at controlled partial pressures of CO2. Enamel samples were obtained from the outer (young) secretory, inner (old) secretory, early (soft) and late (hard) mature enamel of the permanent dentition of slaughtered piglets. The dissected enamel was pulverized and subjected to a plasma ashing at low temperature to remove organic matter. The composition (Ca, total P, HPO4, and CO3) of the enamel mineral was determined chemically. The enamel mineral contained significant amounts of carbonate and acid phosphate; the model adopted for its stoichiometry was [Ca]5-x [HPO4]v[CO3]w[PO4]3-x[OH]1-x. Each enamel sample was equilibrated in dilute phosphoric acid solutions (0.01-1.2 mM) under Pco2 = 1.86 and 1.75%. Equilibration of the enamel samples usually took 20-25 days; the solution composition (pH, concentrations of Ca, P, Mg, Na, and K, and activity of Ca2+) was determined periodically. The composition of the solution at equilibrium showed that (1) the outer (younger) secretory mineral was the most soluble and the solubility of enamel mineral decreased with advancing developmental stages; (2) the mean activity product in the saturated solutions for the outer secretory enamel was the same as that calculated on the basis of the reported composition of the enamel fluid; and (3) the solubility data obtained with most of the enamel samples were consistent with a model in which the equilibration includes two processes: dissolution of the original enamel mineral and precipitation of a new carbonatoapatite. Analyses of the equilibrated samples, particularly the mature enamel, by electron microscopy, supported the precipitation of carbonatoapatite.(ABSTRACT TRUNCATED AT 250 WORDS)

Biominerals in tooth and bone are most adequately described as carbonatoapatites. In order to elucidate the mechanism of carbonated apatite formation in vivo, our experimental strategies have been to (1) determine changes in the nature and properties of the biominerals with advancement of mineralization or tissue development, (2) assess the driving force for precipitation of the crystals in extracellular fluids, and (3) investigate temporal and spacial modifications of mineralization kinetics caused by various native regulators. Enamel mineralization taking place during amelogenesis is a unique and most useful model to investigate carbonatoapatite formation in vivo and the mineral changes at various stages of development. It was established that (1) substantial amounts of carbonate substitute both the PO4 3− and OH− ions in enamel crystals, (2) early enamel mineralization occurs in an environment isolated from circulating blood plasma, under a partial pressure of CO2 of around 0.018 atm, and (3) the precipitation and growth of the crystals proceed steadily, without interference due to remodeling of the formed tissue, yielding the largest crystallites in the body. In the past few years, our effort was focused on the assessment of the labile and stable pools of the crystal constituents (i.e., carbonate, acid phosphate and fluoride) in developing enamel tissues, the development of a new solubility model taking into account most of the lattice constituents, and the determination of the solubility of enamel mineral under controlled partial pressures of CO2. Although our knowledge is still limited and further refinement of the adopted stoichiometry and solubility models is required, the information obtained is valuable to predict the driving force for carbonatoapatite precipitation, as well as the stability of enamel mineral, in normal and pathologic environments.

The present study was undertaken to investigate changes in the acid-phosphate content of porcine enamel mineral during its development and to assess separately the HPO42-pools in labile and stable forms. Enamel samples at the secretory and maturing stages of amelogenesis were obtained from the permanent incisors of five- to six-month-old slaughtered piglets. Human enamel from erupted, extracted teeth, synthetic hydroxyapatite, and carbonatoapatite containing acid phosphate were included as references. The acid-phosphate content of each sample was determined chemically through its pyrolytic conversion to pyrophosphate. The assessment of HPO42-in labile forms was made by analysis of samples pre-equilibrated with solutions containing 3 mmol/L phosphate at pH 11 1 (to de-protonate the HPO42-species on crystal surfaces). The analytical results of porcine enamel samples showed that: (a) the outermost secretory (youngest) enamel contained the highest HPO42-, corresponding to about 16% of the total phosphate; (b) the acid-phosphate content decreased gradually to 10% in the inner (older) secretory and to 6% in the maturing tissue; (c) a substantial part of the HPO42-in developing enamel tissue (50-60% of the HPO42-for the secretory enamel) was in labile forms; and (d) the pool of the labile HPO42-decreased with the growth of enamel mineral. In parallel studies with mature human enamel, it was ascertained that the total acid phosphate was only about 3% of the total phosphate, much lower than in developing porcine enamel, and that the labile pool of HPO42-was also small, corresponding to about 15% of the total acid phosphate determined. The overall results indicate that acid phosphate is one of the major constituents of the early enamel crystals formed during amelogenesis and that the surfaces of the growing enamel crystallites are rich in HPO42-.

In recent years, the dentition of the pig has been increasingly used as a model for the study of amelogenesis. Indeed, much of our current knowledge on enamel formation derives from biochemical and physicochemical analyses of the organic and inorganic components, respectively, of porcine enamel. As an extension of this previous work, and as the first step in our attempt to correlate known enamel matrix and mineral changes with adjacent enamel organ morphology, the present study was undertaken to provide a description of the morphological events occurring in the enamel organ during porcine amelogenesis. Two-week-old miniature swine (minipigs) were fixed by vascular perfusion with glutaraldehyde, the deciduous teeth present at this age were embedded in Epon resin and sectioned, and the cells of the enamel organ at each of the various developmental stages of amelogenesis were examined by light and transmission electron microscopy. In many respects, the morphology of the porcine enamel organ was similar to that previously described in other mammalian species. On the other hand, several particularities were noted and these are discussed in the context of available data correlating cell ultrastructure with putative function during enamel formation.

The understanding of the basic mechanisms by which biological systems exert a precise control over the nature and properties (size, morphology, structural defects, and orientation) of the crystals in hard tissues, is greatly enhanced by comparative studies of mineralization processes in various species and their similarities in the evolutionary scale. It is known that carbonate and fluoride are common constituents of enameloid and enamel in vertebrate teeth [1–3] and the fluoride incorporation into fish enameloid does not reflect the fluoride concentration in environmental water and feeding habits but is related to the phylogeny of fish species [4,5]. However, little is known about the mechanism of species-dependent carbonation and fluoridation of the forming tooth mineral. The objectives of the present study were to investigate the nucleation and crystal growth of carbonated calcium apatites in various animal species and to gain an insight into the regulatory mechanism of in vivo precipitation processes, particularly in relation to fluoride.

The carbonate ion is consistently found in the crystalline tooth minerals in quantities that vary across species, from higher than 6%wt in secretory porcine enamel [1] and mature human dentin [2] to about 0.6%wt for mature shark enameloid [3]. Although part of the carbonate may not be occupying sites in the apatitic crystal lattice of the minerals, there is little doubt that a significant amount [4] resides in lattice sites. Thus, the dental apatites should be considered as carbonatoapatites similar to the mineralogical group denominated dahllites [5], i.e., carbonated apatites having a fluoride content of less than 1%wt. The chemical and biological importance of these compounds, recognized for a long time, is reflected by the numerous articles [6–9] on the structure and chemical properties of these compounds. However, investigators [10–14] studying the chemical properties of dental minerals, particularly their solubility and stability, invariably consider them as hydroxyapatite, Ca5OH(PO4)3, HA. Thus, the solubility product constant for dental enamel has been calculated [12–14] as the ionic activity product for stoichiometric HA in solutions equilibrated with enamel powder; the enamel is then assimilated to an HA displaying a higher solubility than synthetic preparations. Such a simplification facilitates experimental designs and interpretation of results, but it introduces undesirable inconsistences, particularly when comparing the chemical properties of different tissues, e.g., enamel and dentin demineralization in organic buffers. In such a case, it is highly unlikely that a buffer having a given degree of undersaturation with respect to HA will exhibit the same driving force for the dissolution of both tooth minerals. However, without knowing the actual composition of the minerals and their respective solubilities, it is impossible to define the proper driving forces for de- or remineralization.

The present study was undertaken to investigate changes in the crystalline structure and composition of procine enamel mineral during amelogenesis. Special attention was given to the carbonate location in the forming apatite crystal. Enamel samples were obtained from the outer (young) secretory, inner (old) secretory, maturation, and mature (hard) enamel of the permanent incisors of slaughtered piglets. The crystalline structure and composition of these enamel samples were studied using Fourier transform infrared spectroscopy, X-ray diffraction, and chemical analyses. The initial enamel mineral in the outer secretory enamel was rich in acid phosphate and carbonate. The carbonate was mainly substituted for phosphate groups in the apatite crystals of the early (outer) secretory enamel. Developmental advancement from the outer secretory to the inner secretory (as well as early maturation) stages brought about significant changes in crystal parameters, namely, shrinkage and expansion of thec anda unit cell dimensions, respectively, and the shift of av3 PO4 band to higher wavenumbers in the FTIR spectrum. X-ray diffraction patterns indicated that mineralization during the tissue maturation was characterized by a gradual growth of enamel crystals parallel to thea-axis direction. A most prominent finding was that, with developmental advancement, a decrease in CO3 per unit mass of P (or Ca) in the tissue, and a concomitant increase in the CO3 occupying OH sites in the crystalline lattice became apparent. The overall results may reflect (1) changes in the composition of the medium in which precipitation of enamel carbonatoapatite occurs, (2) initial formation of an acid phosphate such as octacalcium phosphate-like mineral, or (3) modifications of the precipitating phase induced by changes in the kinetics of the mineral formation.

The present 1H-nmr study was undertaken to investigate the molecular structure of porcine amelogenins in solution using photo-CIDNP (chemically induced dynamic nuclear polarization). The proteins of interest were the parent 25 kD amelogenin consisting of 173 amino acid residues and its degraded products having molecular masses of 23 kD, 20 kD, 13 kD and 5 kD on SDS-PAGE. From the recorded 1H-nmr and photo-CIDNP spectra, it was found that: 1) the Trp161 at the C-terminus of the 25 kD protein showed a stronger photo-CIDNP effect than the other two Trp25,45 at the N-terminus; 2) Tyr residues at the N-terminus of the 25 kD and 20 kD amelogenins gave rise to the strong peak around 6.8 ppm, indicating that at least some of the six Tyr residues are surface residues; and 3) the accessibility of His residues was quite different between the 13 kD fragment and the 25 kD and 20 kD proteins. These results suggest that the hydrophilic segment at the C-terminus is most likely exposed on the molecular surface and that the molecular structure of the amelogenin in solution may change substantially by the cleavage of the segments at the N- and C-termini.

The solubility of human enamel was investigated in dilute phosphoric acid solutions (0.07 mMols/L to 1.54 mMols/L) under controlled partial pressures of CO2 (0.005% to 3.33% in mixtures with N2) at 25 degrees C. The enamel mineral was considered as carbonatoapatite containing acid phosphate having the stoichiometry [Ca]5-x-y[HPO4]v[PO4]3-x[CO3]w[OH]l-x-y. During equilibration, a second apatitic phase precipitated, as observed under the electronmicroscope. Solubility models are advanced consistent with the adapted stoichiometry and the precipitation of the second phase. The latter appears to have a carbonate content similar to that of the enamel mineral even when precipitated under the lowest partial pressure of CO2, suggesting that a molar content of about 0.3 of carbonate stabilizes the apatitic lattice. Based on the adopted stoichiometry of enamel mineral, Ca4.34 (HPO4).08 (CO3).29 (PO4)2.63 (OH).05, its solubility product constant was calculated as Ksp = 1.74 (+/- 0.081)x10(-6), expressed as the mean activity product.

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°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 NaCI 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. The results showed that: (i) The amelogenin having a 26-kD molecular mass on SDS-PAGE displayed the highest adsorption affinity onto the apatite crystals among the amelogenins; (ii) the same selective adsorption onto the crystals was also observed for the amelogenins separated from the teeth of the animals ingesting fluoride; (iii) the amelogenins can be classified into two groups, neutral-soluble moieties (14-15 kD) and alkaline-soluble moieties (19-28 kD), by the use of solutions having ionic strengths of about 160 mmol/L; and (iv) with regard to the electrophoretic patterns, quantities, and amino acid composition of these two groups of amelogenins, no major differences were obtained between the control group and any groups of animals ingesting fluoride, although the fluoride substitution into enamel mineral increased proportionally with an increase in fluoride concentration in the drinking water, and the incisors from animals in the 75 and 100 ppm F treatment were distinctively brittle. All the foregoing results, as well as those previously reported, indicate that possible functional roles of the amelogenins in early enamel mineralization are common to rat and porcine models, and that the fluoride administration at the doses tested, or the resulting incorporation of fluoride in the forming enamel mineral, caused only a marginal effect, if any, on the properties and degradation processes of amelogenins at the secretory stage of rat amelogenesis.