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A low molecular weight protein fraction isolated under dissociative conditions during the demineralization of rat incisor dentine has the ability to modulate, in culture, the expression of fibroblast-like cells explanted from neonatal rat muscle. The protein fraction enhances the incorporation of 35S-sulphate into a proteoglycan larger in weight than that produced by the uninduced cells; furthermore it induces the production of type II collagen. These changes take place in the absence of cell proliferation as measured by 3H-thymidine incorporation. The altered fibroblast-like cells form nodules and secrete an abundant extracellular matrix which stains for proteoglycan after 7-9 days in culture. These data show that the dentine matrix does contain a factor which can initiate a mitogenesis-independent alteration in the expression of the muscle-explant outgrowth cells. Those changes are consistent with a shift to a chondrogenic mode.

Shed deciduous teeth (incisors only) were collected from children (n = 199) living in Duisburg and Gummersbach (F.R.G.) in 1976 and 1988. The teeth were analysed for lead and cadmium. Considering all teeth, there was a significant of tooth lead and tooth cadmium from 1976 to 1988. The reduction was between -40 and -50% for tooth lead and -45% for tooth cadmium. Regarding tooth lead, the reduction was more pronounced in children from Duisburg, an area heavily polluted by lead and other heavy metals due to the presence of large iron and steel plants and a large lead-/zinc smelter. Teeth from the upper jaw were found to have higher lead and calcium concentrations than teeth from the lower jaw. Moreover, it was found that central incisors had higher lead and cadmium concentrations than lateral incisors. Even after the effects of jaw and tooth type had been allowed for, the reduction of tooth lead and tooth cadmium could be demonstrated. Children living in pre-war houses were found to have higher tooth lead and tooth cadmium levels than children living in post-war houses. The higher lead and cadmium burden of children living in old houses seems to be related to a higher degree of exposure to lead and cadmium via drinking water resulting from the release of these metals from old zinc-plated steel water pipes. In total, our results indicate that there has been a significant decrease of lead and cadmium exposure in West-German children since the mid-70s. The reduction of tooth lead levels parallels the decrease of blood and bone lead levels, which has been observed in previous studies.

Enamel proteins from fully mineralized human molars and from bovine tooth germs were separated by electrophoresis. The gels were stained for detection of glycoproteins, lipoproteins, and phosphoproteins. Glycoproteins were shown by periodic acid-Schiff staining and lectin blotting. In mature human enamel a number of high molecular weight proteins could be demonstrated after ethylenediaminetetra-acetic acid demineralization and subsequent Triton X-100 extraction. These proteins are suggested to be lipoproteins. Phosphoproteins could only be visualized in enamel matrix from the tooth germs.

A double-isotope technique that does not necessitate urine and fecal collections but requires only the extraction of the incisor teeth for isotopic analysis has been devised. A precalibrated dose of 45Ca in solution with stable carrier calcium is administered to the rat orally. An intraperitoneal injection delivers a precalibrated dose of 47Ca in isotonic saline. The ratio of the percentage uptake of the two radionuclides in the incisor tooth is equal to the fraction of the 45Ca and, therefore, the calcium absorbed by the gut. The fraction of calcium absorbed by 5-mo-old rats, as determined by collection and measurement of excreta, was found to be 39.1%. The ratio of uptake of the two calcium radionuclides in the incisor teeth yields an absorption measurement of 38.8%, nonsignificantly different from the value obtained from the excretion data. The measurement of radiocalcium uptake in the incisor tooth affords one an accurate in vivo determination of intestinal calcium absorption without the collection of excreta or multiple blood sampling.

The phosphoprotein of rat incisors has been purified by successive gel and ion-exchange chromatography. The product gave a single band on polyacrylamide gel electrophoresis and contained approximately 34% phosphoserine and 32% aspartic acid. Alkaline elimination experiments showed all the phosphate to be present as phosphoserine. Ultraviolet spectra in the presence or absence of ATP showed that the phosphoprotein did not contain an nucleotide moiety as suggested by Veis, A., Spector, A. R. and Zamoscianyk, H. ((1972) Biochim. Biophys. Acta 257, 404-413) for bovine dentin phosphoprotein.

Phosphoprotein was obtained from rat incisor dentin either by extraction at elevated ionic strength after acetic acid demineralization, or by extraction simultaneous with demineralization in neutral EDTA solution. Purification of solubilized proteins was achieved by Sepharose 4B and DEAE-cellulose chromatography. Polyacrylamide gel electrophoresis of the material from the two preparations resulted in one single band. Except for the amino acid analyses, no evidence for a difference between the two phosphoprotein preparations could be found. After additional purification by iso-electric focusing the amino acid analyses demonstrated a similar composition. It is concluded that the two methods for phosphoprotein extraction yield the same product when purified properly. The study did not give any unequivocal answer as to if any phosphoprotein component exists in rat incisor dentin which is covalently linked to the collagen matrix.

The molecular weight of highly phosphorylated phosphoprotein from rat incisor dentin was estimated by three different methods: sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, gel chromatography in 6 M guanidinium chloride (GdnHCl) and analytical ultracentrifugation at high salt concentration. SDS-polyacrylamide gel electrophoresis revealed an abnormal migration of the phosphoprotein and its enzymatically dephosphorylated derivative in comparison to standard proteins. Depending on the acrylamide concentration in the gel (7.5-20%), the apparent molecular weight of the phosphoprotein varied from 90,000 to 27,000. Similar results were obtained for the dephosphorylated protein. In gel chromatography using 6 M GdnHCl as eluent the phosphoprotein had an elution volume corresponding to that of a standard protein with a molecular weight of 67,000, while the enzymatically dephosphorylated phosphoprotein showed an apparent molecular weight of 30,000. The phosphoprotein behaved non-ideally in equilibrium sedimentation runs. The apparent molecular weight was strongly concentration-dependent and the extrapolation to zero concentration was uncertain. However, after enzymatic dephosphorylation the concentration dependence disappeared and a molecular weight of 28,000 could be calculated. Since the phosphate groups represent 26% of the phosphoprotein by weight, the true molecular weight of the highly phosphorylated phosphoprotein components from rat incisor dentin should be 38,000. The study shows that it is not possible directly to estimate the molecular weight of this type of protein by standard methods elaborated for normal globular proteins.

Freshly excised rat incisors were immediately cleaned and demineralized in 0.5 M ethylene diaminetetracetic acid at pH 7.5. The extracts were freed of calcium, diffusible phosphate and low molecular weight polypeptide components by dialysis in membranes with cut-off of 3500 molecular weight. The extract was resolved into at least 7 protein components by chromatography on DEAE-cellulose at pH 8.2. The composition of each protein component was determined. Two proteins, rich in serine, phosphorous and aspartic acid were unlike any proteins attributed to enamel, and hence were considered to be components of incisor dentin. These were the principal non-collagenous components of the teeth. Further purification was carried out under dissociative conditions on Sepharose CL-6B gel filtration columns in 3.0 M guanidine hydrochloride. The two phosphoproteins have mol wts, by this method, of 71,000 and 65,000, respectively, and differ in content of apolar amino acids, although both contain greater than 70 residue % of seryl (or phosphoseryl) and aspartyl residues. The name "phosphophoryns" is proposed to describe these dentinal proteins. The insoluble collagenous matrix remaining after the original demineralizing extraction was degraded with cyanogen bromide. Several non-collagenous protein components were released as well as the typical collagen derived peptides. Two collagen phosphoprotein complex peptides were also isolated, demonstrating as in bovine dentin, the probable direct covalent interaction of a dentin phosphoprotein with hte collagen of the mineralized matrix.

Electron-dense particles, approximately 4 nm (40 A) in diameter were present. Evidence is offered that these particles represent iron probably in the form of amorphous hydrous ferric hydroxide. From X-ray probe analytical data, it is suggested that this iron component is accommodated through the process of adsorption rather than by direct substitution of calcium and phosphorus. The iron-rich surface layer was more resistant to acid etching than subsurface enamel. Reported areas of reduced mineral density of enamel at the enamel-dentine junction appear to be preparation artifacts.

The non-collagenous proteins of rat dentin that remain firmly bound to the matrix after demineralization were studied in order to ascertain if they are covalently linked to insoluble dentin collagen. After solubilization with CNBr or with bacterial collagenase, unusually small amounts of dentin phosphoprotein were detected in the matrix. The phosphoprotein obtained by CNBr digestion of the matrix was separated from collagen peptides using two chromatographic steps. Thus even this small quantity of phosphoprotein found in decalcified rat dentin matrix was not covalently bound to collagen.

Sixteen female, Sprague-Dawley rats were divided into four equal groups. Two groups served as controls receiving low or regular concentrations of fluoride (F); animals in the other two groups were given drinking water, containing 100 parts/10(6) F, for 3 weeks either during or immediately before pregnancy. Thirteen days after delivery, the pups and dams were killed and various tissues analysed for F content. Prenatal F supplementation increased F concentrations in plasma, mandibular incisors and femoral epiphyses of pups by 25, 36 and 38% respectively, when given during pregnancy. Only a slight increase of 9 and 11% in the respective F concentrations of incisors and epiphyses occurred when the supplement was given before pregnancy. The fluoride level of milk was consistently higher than that of the maternal plasma. These results suggest the need for further study of prenatal F supplementation.

The appearance and chemical composition of a number of developing human deciduous incisors indicated that the enamel passes through the following four developmental stages: 1. Partially mineralized matrix is secreted and some extracellular breakdown occurs. 2. Selective replacement of matrix proteins by tissue fluid begins. 3. Almost all of the matrix protein is replaced by tissue fluid and an influx of calcium phosphate occurs. 4. The enamel becomes almost fully mineralized, mature and hard. These stages of development are similar to those described in rat and bovine tissue. the number of stages simultaneously present in a single tooth differed from rat and bovine enamel, however, as did the rate of change in amino acid composition from developing to mature tissue.

Enamel-matrix components from rat incisor enamel were extracted from tissue at different stages of development on single teeth. Separations of proteins using urea and SDS acrylamide gel electrophoresis were compared. The bulk of the matrix exhibited SDS mol. wt of 25-30,000 with smaller amounts at approximately 18,000 and about 10-12,000. Trace amounts of material at -50,000 and 70,000 were detected. These were presumably associated with the mineral phase as their yield increased after demineralization. The proportion of small molecular weight components increased with tissue age. Using urea, many more proteins were separated (up to 20) into fast, intermediate and slowly-migrating components. Disappearance of small bands of intermediate mobility at the end of matrix secretion suggested that they were early ameloblast products which were rapidly degraded after secretion. Both slowly- and rapidly-migrating components increased with tissue age indicating progressive degradation of parent molecules of intermediate mobility into highly charged and relatively uncharged molecules.

Bovine incisors were classified into three developmental stages and non-collagenous proteins extracted from them. Sodium dodecyl sulphate gel electrophoresis of the extracts showed a reduction in osteonectin with the various stages. The reduction was confirmed by enzyme immunoassay using antiserum against bone osteonectin. This change is in contrast to dentine phosphoprotein, indicating functional differences between these two proteins.

Antiserum to the 28-kilodalton vitamin D-dependent calcium-binding protein (CaBP) was used to localize CaBP in histologic sections of the continuously erupting incisor in mandibles obtained from normal rats. With the peroxidase--anti-peroxidase technique, no CaBP was detected in undifferentiated ameloblasts or in those which had become columnar and were facing pulp. Calcium-binding protein was first noted in the cytoplasm of random ameloblasts facing dentin in the presecretion zone. As the ameloblasts became more mature in the zone of enamel secretion, CaBP was uniformly present in their cytoplasm. Ameloblasts with Tome's processes clearly contained CaBP in these processes as well as in the cell-body cytoplasm. Near the later developmental stages of the zone of enamel secretion, some of the adjacent underlying cells of the stratum intermedium also contained CaBP in their cytoplasm. In some stratum intermedium cells and papillary cells, CaBP extended into the zone of enamel maturation, but not to the end of that zone. Cytoplasmic CaBP continued to be present in ameloblasts as they progressed through the zone of enamel maturation to the final, shortened cells at the gingival margin of the erupting incisor. No CaBP was detected in odontoblasts, pulpal cells, the stellate reticulum, or the outer dental epithelium.

The mineralization levels of erupted buccal enamel from 24 deciduous teeth were compared to those of 28 permanent teeth. Sections were prepared in a defined plane using a lapping machine which gave plano-parallel sections. Mineralization levels were recorded by quantitative microradiography at 25 equivalent anatomical sites in each section. Deciduous incisors and canines were compared with their homologous successors: overall mineralization levels were lower in the deciduous dentition, with no significant differences being found close to the amelo-dentinal junction, but highly significant differences being found in the outermost sites. Deciduous molars were compared with premolars, and were also relatively less mineralized. However, deciduous molars did not show the consistent diminishing occlusocervical gradient observed in all other tooth types tested; on the contrary, they showed a cervical reversal with higher values than permanent premolar enamel. These results confirm the generally lower mineral levels in deciduous enamel, and provide quantitative information on site-specific mineralization levels.

Selenium concentrations in successive layers of bovine incisor enamel showed no concentration gradient and no increase with age. Selenium was taken upt into the continuously-growing rat incisor in proportion to the amount of selenium in the diet. A sensitive (approximately 1 ng Se) and relatively simple fluorimetric method for determining selenium in small amounts of hard tissues is presented.

The absolute concentrations of Mn, Fe, Ni, Cu, Zn, Sr and Pb in whole enamel, and in labial and lingual surface enamel of deciduous incisors were determined with proton-induced X-ray emission calibrated with the International Atomic Energy Agency animal-bone standard. The differences between the mean concentration values of the 19 samples from the urban area and those of the nine samples from the rural region were not significant. The concentrations of the elements measured on the surface, depth 0-20 microns, were higher than the corresponding average values of the whole enamel by factors of 1.1-35. The lowest factor, 1.1, was for Sr and the highest, 35, for Pb. There were, however, large variations in concentration between the lingual and labial surfaces of individual teeth. Nevertheless, there were no significant differences between the mean concentrations for the labial and lingual surface, except for lead.

Pregnant rats were dosed with 0, 3 or 10 parts/10(6) lead (as acetate) in their drinking water, during pregnancy and during lactation until 21 days post partum. The litters were killed at the age of 21 days and the incisors, first, second and third molars analysed for lead by atomic absorption spectrophotometry. The findings confirmed that even at low concentrations lead has an affinity for hard tissues. The mean lead levels of the teeth of the three groups differed at the 1 per cent level. Pb levels of the first and second molars and the incisors also differed at the 1 per cent level. No significant differences between the four types of teeth within a group could be demonstrated. The no-effect level of Pb administered in drinking water, to the mother during lactation, and during pregnancy, was lower than 3 parts/10(6).

Immunohistochemical methods were used to determine whether type IV collagen, laminin, fibronectin, and heparan sulfate proteoglycan were present in diverse basement membranes. Antisera or antibodies against each substance were prepared, tested by enzyme-linked immunosorbent assay, and exposed to frozen sections of duodenum, trachea, kidney, spinal cord, cerebrum, and incisor tooth from rats aged 20 days to 34 months. Bound antibodies were then localized by indirect or direct peroxidase methods for examination in the light microscope. Immunostaining for type IV collagen, laminin, fibronectin, and heparan sulfate proteoglycan was observed in all of the basement membranes encountered. Fibronectin was also found in connective tissue. In general, the intensity of immunostaining was strong for type IV collagen and laminin, moderate for heparan sulfate proteoglycan, and weak for fibronectin. The pattern was similar in the age groups under study. Very recently the sulfated glycoprotein, entactin, was also detected in the basement membranes of the listed tissues in 20-day-old rats. It is accordingly proposed that, at least in the organs examined, type IV collagen, laminin, fibronectin, heparan sulfate proteoglycan, and entactin are present together in basement membranes.

The lead content of teeth of children in five different environments has been measured to determine the relative contribution of different sources of lead. The importance of lead paint in children living in dilapidated housing is clearly observed, but no effect attributable to automobile exhaust or industrial emissions is apparent. High lead levels were found among children living in new public housing projects within the high lead area of the city, even though a lead paint problem presumably should not exist in the projects themselves. The data are analyzed to determine the frequency with which excessive lead contents occur in the problem area in comparison to a low risk area.

Using electron probe microanalysis, the authors determined the calcium and magnesium contents in the incisors of rats treated with different doses of tetracycline and oxytetracycline, and in those of untreated rats. The contents of these elements were significantly lower in the dentine of the treated animals than in that of the control animals, whereas the enamel samples showed no statistically significant differences. The concentration tendency in different tooth sections, from the periphery to the pulp canal, i.e., decrease in calcium and increase in magnesium, was not influenced.

In order to elucidate the cytochemical properties of the membranous structure between enamel and ameloblasts of the rat incisor at the maturation stage, chromic phosphotungstic acid (Cr-PTA) and periodic acid-silver methenamine (PA-silver) techniques for electron microscopy were employed in combination with a digestion test with hyaluronidase, neuraminidase, collagenase or trypsin. Also, acid phosphatase activity of ameloblasts at the maturation stage was examined with a modified GOMORI's metal salt method. An intensely Cr-PTA reactive band approximately 0.1 micron thick appeared along the surface layer of enamel at the transitional stage, and at the very beginning of the maturation stage another intensely Cr-PTA reactive band which was seen by uran-lead stain to be a delicate electron-dense membranous structure appeared as well between enamel and ameloblasts. A lot of cytoplasmic small vesicles or tubular structures, both intensely reactive to Cr-PTA, were observed near the apical membranes of the overlying ameloblasts indicating that those organelles must have been responsible for the secretion of the latter band. Acid phosphatase activity was clearly demonstrated at Cr-PTA reactive large vesicles in the cytoplasm of those cells. The PA-silver staining technique manifested a band heavily deposited with silver grains along the surface layer of enamel, i.e., where the former band existed, but showed no particular reaction at the latter, the band-like layer between enamel and ameloblasts. Hyaluronidase or neuraminidase treatment remarkably decreased the Cr-PTA reaction of the latter band. Trypsin or collagenase treatment, on the other hand, not only eliminated the Cr-PTA reaction but digested the band itself. These results suggest that the membranous structure between enamel and ameloblasts of a rat incisor is not so-called enamel cuticle but a basal lamina produced by overlying ameloblasts and that the basal lamina contains collagenous components even though it lies on enamel.

Noncollagenous proteins (NCPs) were obtained rrom rat dentin using several precautionary measures to prevent artifactual degradation and losses of the proteins. Prior to demineralization, rat incisor dentin was extracted with 4 M guanidine hydrochloride (GdmCl) containing enzyme inhibitors. The only major component extracted with GdmCl was a proteoglycan fraction. Most of the NCPs were extracted when the incisors were decalcified with an EDTA solution containing protease inhibitors. The EDTA extract contained four types of macromolecules: acidic glycoproteins, gamma-carboxyglutamic acid (Gla)-containing proteins, phosphoproteins, and proteoglycans. With two exceptions, the apparent molecular weights of these NCPs were greater than 50,000. Our observations contrast sharply with the results obtained by others for human dentin NCPs and suggest that artifactual degradation and losses of some NCPs occurred in these previous studies. The organic phosphate-containing fraction was biphasic when the EDTA extract was chromatographed on DEAE-cellulose. Rechromatography of this fraction by two different procedures separated the material into a complex glycoprotein-containing fraction and, a single rat incisor phosphoprotein peak (RIP). Thus, the earlier interpretation by others that the biphasic nature of the RIP-containing fraction represents two widely differing phosphoprotein species was premature. Highly purified RIP, prepared by passage through a sulfonated polystyrene column, contained no cysteine, valine, methionine, leucine, phenylalanine, or arginine, and the level of phosphoseryl residues was higher than for any previous report. When this preparation of phosphoprotein was dephosphorylated (dP-RIP) and rechromatographed on DEAE-cellulose, a partial separation into two fractions was observed. Automated Edman degradation of fraction dP-RIP suggested the presence of two NH2-terminal sequences: Asp-Asp-Asp-Asn and Asp-Asp-Pro-Asn. However, this material displayed a single protein band when applied to 7.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue. The apparent molecular weight of dP-RIP, compared to standard globular proteins, was about 72,000. The data suggest that rat dentin contains at least two major molecular species of RIP that are closely related in size and-structure. In addition, preliminary evidence suggested that other minor forms of related phosphoproteins may exist.

The degree of microcrystal alignment in enamel of clinically sound upper and lower central incisors, canines and premolars was determined by means of electron paramagnetic resonance. The results show that the degree of alignment depends on the individual properties of the enamel as determined by biological variations, tooth morphology and tooth position. Upper incisors and canines have a much higher degree of crystallite alignment as compared to all other teeth investigated. These results indicate that the caries susceptibility of tooth enamel is not defined only by the degree of microcrystal alignment.

Demineralized dentin matrix has the capacity to induce bone formation via a chondrogenic pathway when implanted into muscle, in a fashion entirely analogous to bone matrix implants. In this work we have attempted to isolate, from rat incisor dentin, the matrix factor responsible for initiating osteogenesis. Rat incisor dentin was demineralized with EDTA plus 4.0 M guanidine. HCl. The proteins in the extracts were collected and, after a CaCl2 precipitation step, fractionated on Sephacryl S-200 in 6.0 M guanidine. HCl. The primary assay for activity was the incorporation of 35S-sulfate into proteoglycan in cultures of the fibroblast-like outgrowth cells from explants of neonatal rat muscle. Two Sephacryl S-200 fractions showed enhanced sulfate incorporating activity, but only one showed enhanced incorporation without a concomitant increase in cell number. In the presence of this fraction, the cell cultures produced a larger amount of a new small proteoglycan, as compared to controls, and a significant amount of a much larger proteoglycan. The active fraction had proteins in the Mr range from 8,000 to 15,000 as the major components. These data suggest that the fraction identified may contain the factors responsible for initiating the osteogenic response to dentin matrix upon its implantation in muscle in vivo.

Dentin phosphophoryn was quantified on bovine and rabbit dentin at three developmental stages. Phosphophoryn was extracted from teeth with 0.6M HCl, and quantified as optical density on DEAE-cellulose chromatogram or as phosphoserine content. Bovine phosphophoryn showed progressive increase with formation of dentin. Matrix-associated phosphophoryn was also quantified as phosphoserine content in insoluble dentin residue which was extracted with 6 M urea after decalcification. This fraction increased with formation of dentin both in bovine and rabbit dentin. Phosphophoryn is thought to be related to the later stage of dentin formation.

The effect of parathyroid hormone (PTH) on mineralization and matrix formation was studied on the incisal dentin of immature rats by using a time marker as well as the histochemical method. 1. When the hypersecretion of PTH was caused by a low calcium diet, mineralization and matrix formation were both accelerated. 2. Both mineralization and matrix formation were clearly inhibited by parathyroidectomy or thyro-parathyroidectomy which brought about a widening of the predentin (dentinoid) as a result of the conspicuous inhibition of mineralized matrix formation. The maturation of the matrix seemed to be inhibited also. 3. Demineralization was lower and slower in the labial than in the lingual dentin. 4. The inhibitory effect disappeared totally by the PTH injection, but the restoration of matrix formation was faster compared with that of mineralization. 5. The increase or decrease in mineralization did not necessarily occur in parallel with that in matrix formation. 6. Acid mucopolysaccharide formation in the dentin depended clearly on the quantity of PTH.

Cattle were fed forage containing fluoride at a yearly average of 40 mg of fluoride/kg of forage for 5 or 6 years from the time they were 4 months of age. A significant (P < 0.05) negative correlation was observed between the average microhardness of the 3rd premolar and 3rd molar enamel and the fluorosis score of the 2nd incisor. The microhardness of fluorotic outer molar enamel was only 41% of the microhardness values of enamel from control teeth not exposed to long-term fluoride, and electron probe microanalysis indicated increased fluoride concentrations in the molar coronal cementum, enamel, and dentin. Increased incisor fluorosis scores were diagnostic of softer molar enamel. When severe, such changes may have a detrimental effect on the proper mastication and subsequent nutrition of cattle with fluorosis.

Whole teeth obtained from handicapped children and from controls have been analysed for lead by anodic stripping voltammetry. No significant differences were found between the two groups. Teeth originating from the same patient showed systematic variations in lead content that are dependant on anatomical tooth type. The nature of this association is discussed. Analyses of sectioned teeth revealed, for cadmium and copper, unexpectedly large variations. High concentrations of cadmium found in the tips of deciduous incisor teeth indicate a probable contribution from maternal blood.