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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.

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.

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.

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.

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.

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.

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.

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.

Rat incisor tissue sections were fixed by a modified version of the malachite green-aldehyde method (MGA) composed of rapid-freezing, malachite green-acrolein staining, and osmium tetroxide freeze-substitution (Fr.MGAO). In the pre-dentin, a thick, dense network of branched fibrous structures was observed. Cryotechniques allowed visualization of complexes about twice as thick and dense as the aggregates visualized on MGA-treated sections. Pretreatment of rapid-frozen samples with methanol before freeze-substitution fixation and staining prevented staining of the complexes otherwise revealed by the Fr.MGAO method. Electron-dense material stained by this procedure resisted de-mineralization with EDTA, while intramitochondrial granules and dentin crystallites were dissolved. EDTA treatment demonstrated unequal distribution of Fr.MGAO staining in dentin in the form of tiny dots underlining the collagen fibers. These results support the concept that rapid-freezing, followed by staining and freeze-substitution fixation, improves preservation of the phospholipids visualized as extracellular matrix components in pre-dentin and dentin of rat incisors.

A comparative study of the organic matrix in crown dentin and root dentin of the bovine incisor has been performed utilizing biochemical analyses and histochemical stainings. Root dentin contained only half the amount of dentin phosphophoryn present in crown dentin. Composition of noncollagenous matrix other than phosphophoryn was also suggested to be different between crown and root dentin. These data indicate that odontoblasts forming root dentin may secrete a noncollagenous matrix having a composition different from that of crown dentin, and that crown and root dentin may be separate in nature. The distinct compositions of noncollagenous matrix may reflect the difference in the mineralization process between crown and root dentin.

A topographic description of the concentration profiles of Ca, P, Mg, Zn, F, S and K in coronal dentin of red deer is given. The concentrations reported should be regarded as semiquantitative values. An area along the mesiodistal axis of the crowns showed lower concentrations of Ca and P and higher concentrations of Mg than the surrounding bulk of dentin. The inner dentin revealed downward slopes of the Ca, P and Mg profiles toward the pulpal cavity, whereas the Zn, F and usually also the S profiles showed elevations. Incidental findings were elevations of S and K profiles near the worn incisal edges. The walls of giant tubules, which are frequent in red deer dentin, showed in many cases modest elevations of the Ca, P, Mg and Zn profiles. The lumina of these tubules revealed in a few instances elevation of the S profiles.

Phosphophoryn, the most abundant of the dentin non-collagenous proteins, has been considered to be related in function to the mineralization process. In the present study, identification of dentin phosphophoryn localization was attempted using newly developed, precautionary histological methods by which phosphophoryn was retained in the sections during the specimen preparation and stained selectively in situ. Phosphophoryn was found to be present widely in all of the calcified dentin except the mantle dentin, the external, first-formed portion of dentin, but was not found in the predentin, the inner, uncalcified layer of dentin. These results indicate that phosphophoryn is apparently related to the mineral phase of calcified dentin and that the mineralization process of mantle dentin, which is formed before the odontoblasts are fully differentiated, may be different from that of circumpulpal dentin.

Dentinogenesis offers a unique system for the study of changes in collagen structure occurring simultaneously with mineralization. Bovine dentin was found to contain about one reducible crosslink per collagen molecule; rat dentin contained twice this amount. In contrast, bovine dentin contained twice as much pyridinium crosslink as did rat dentin collagen. These results indicate that the collagen in rat teeth is less mature and again emphasize the difference in composition between the organic matrices of rat and bovine dentin. In dissected bovine predentin, the unmineralized precursor of dentin, the content of reducible crosslinks was almost double that of dentin. Only minute amounts of non-reducible crosslinks were found in predentin, whereas both pyridinoline and deoxy-pyridinoline were present in collagen from mineralized dentin. The observed differences in crosslinking between predentin and dentin of the same teeth may indicate some alterations within the area of mineralization.