Phosphorylated extracellular proteins are important in the early phases of biomineralization through such as binding Ca²⁺ ions, forming calcium phosphate clusters and affecting the conversion of amorphous calcium phosphate to hydroxyapatite, thus promoting the formation of initial nuclei and at later stages inhibiting crystal growth. In dentine, a group of phosphorylated extracellular-matrix proteins have been identified. These include osteopontin (bone sialoprotein I), bone sialoprotein II, osteonectin, dentine matrix protein 1, dentine phosphoproteins and probably dentine sialoprotein, which is coexpressed with dentine phosphoproteins. The phosphoproteins constitute the major group of dentine non-collagenous proteins. Depending on the species, low, intermediate and highly phosphorylated molecules have been extracted. The highly phosphorylated dentine phosphoproteins—also called phosphophoryns—are specifically found in dentine, not in bone or any other mineralized tissue. They are synthesized exclusively by odontoblasts and seem to be important in dentine mineralization. Some abnormal mineralizations of dentine, such as dentinogenesis imperfecta and reparative dentine, display alterations at the phosphorylated protein level, due either to a post-transductional alteration in phosphorylation, or to the fact that dentine phosphoproteins are not genetically expressed and therefore are lacking in the dentine matrix.

Dentine phosphorylated proteins possess several sequences which act as substrates for the kinases associated with intracellular membranes, but independent of the intracellular messengers, casein kinases I and II are capable of phosphorylating the different dentine phosphoproteins. In addition, Zeichner-David et al. (1992 and Zeichner-David et al. (1995) have identified a dentine phosphoprotein-specific kinase, which has not yet been fully characterized.

Casein kinases are ubiquitous protein kinases belonging to the family that catalyses the phosphorylation of serines and threonines. They act independently from cyclic nucleotides and phosphorylate casein and phosvitin, but not histones or protamine. Their activity is not modified by the heat-stable inhibiting protein and they are independent of Ca²⁺ ions, calmodulin and phospholipids. Two distinct types of casein kinase, type I and II, have been recognized; they have different structures and specificity for the phosphate donor nucleotide, and are specific to a different phosphorylation site within the substrate.

A low concentration of heparin and 2,3 diphosphoglycerate inhibits the activity casein kinase II; moreover, haemin and inositol hexasulphate inhibit both casein kinases by competing with the substrate. Their inhibiting action has been tested only in vitro on subcellular fractions containing either or both enzymes. It is still not known whether or not the inhibitors are also active at the cellular level.

We report here the effects of inositol hexasulphate on organ cultures of 18-day-old mouse embryonic molars, cultured for 11 days. In order to investigate the suitability of the model, we examined the dose response of embryonic tooth germs cultured in the presence of increasing concentrations of the inhibitor. Casein kinases are known to phosphorylate over 100 cytosolic and nuclear proteins, suggesting that their effects are not restricted to extracellular phosphorylated proteins. Therefore our purpose now was firstly to establish the border between the cytotoxic and inhibitory effect of the inhibitor on matrix-protein phosphorylation in odontogenesis, and secondly to discriminate between the specific effects of the inhibitor on extracellular-matrix protein phosphorylation, and non-specific effects on cells. To this end a quantitative radioautographic study was carried out on germs cultured with or without 0.1 mM inositol hexasulphate (at which concentration, staining of phosphorylated protein was eliminated), using [³³P]phosphate as a phosphorylation tracer, [³H]serine as an amino acid mostly incorporated into the protein skeleton of dentine phosphoproteins and [³H]proline as a precursor for collagen synthesis.

Mandibular first molar germs were dissected from embryos of Swiss mice killed at the 18th day of gestation (M18) (vaginal plug=day 0). The isolated molars were dissected in Hank’s balanced salt solution. The tooth germs were first cultured for 1 day in a chemically defined semisolid medium, BGJb (Gibco, Cergy-Pontoise, France) supplemented with 15% fetal bovine serum (Gibco), 20 mg/ml -glutamine, 275 ?g/ml sodium ascorbate (Merck, Darmstadt, Germany), and an antibiotic solution (Gibco) containing penicillin (10,000 IU/ml) and streptomycin (10,000 ?g/ml) diluted to a final concentration of 1%. The medium was gelled with 0.5% agar (Bacto Agar; Difco).

On the second day of culture, myo-inositol hexasulphate (Sigma, St Quentin Fallavier, France) was added to the culture medium at the following six final concentrations, 0.04, 0.08, 0.1, 0.2, 0.4 and 0.9 mM, obtained from a stock solution prepared in culture medium at a concentration of 10 mg/ml. These concentrations were chosen according to published data about their efficiency in inhibiting the action of casein kinase in subcellular fractions. An equal volume of culture medium was included in the control culture.

All the germs were cultured for 11 days, the time needed to synthesize and secrete a thin layer of dentine, 40–60 ?m thick at the top of the highest cusp, easy to distinguish from the predentine. The medium was renewed every other day. The culture dishes were kept in an incubator at 37°C in an atmosphere rich in oxygen (50% concentration).

For light microscopy the M18 and the cultured tooth germs (M18+11 days) were rinsed in Hank’s solution and immersed for 4 h at 4°C in a fixative solution containing 4% paraformaldehyde, buffered with 0.1 M sodium cacodylate at pH 7.4. The samples were rinsed in the same buffer, dehydrated in graded ethanols and embedded in paraffin. Thick sections (7 ?m) were stained either with haematoxylin and eosin or with Stains all (Sigma) following the method of Gruber and Mekikian (1991), or with the von Kossa method.

For electron microscopy the M18 and the cultured tooth germs were rinsed in Hank’s medium, immersed for 4 h at 4°C in a fixative solution containing 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4 and postfixed in 2% of OsO₄ for 30 min at room temperature. The samples were rinsed in the same buffer, dehydrated in graded ethanols and embedded in epoxy resin (Epon 812). Ultrathin sections stained with uranyl acetate and lead citrate were examined with a JEOL 100 B transmission electron microscope operating at 80 kV. In addition, about 1 ?m-thick semithin sections were stained with either toludine Blue or Stains all and examined by light microscopy.

Tooth-germ buds were cultured in the presence or absence of the inhibitor. As the Stains all disappeared from the sections only at a 0.1 mM concentration of inositol hexasulphate, this dose was chosen even though some toxic reactions became detectable inside cell bodies. On day 9 of culture, one of each of the three radiolabelled precursors was added for 30 min to the culture medium, either 1.85 MBq/ml [³³P]phos phate (The Radiochemical Centre, Amersham, England; spec. act. 1.85 TBq/mmol), or 740 KBq -[3-³H]serine (Radiochemical Centre; spec. act. 0.74 TBq/mmol), or 740 KBq -[5-³H]proline (Radio chemical Centre; spec. act. 0.74 TBq/mmol). The germs were then rinsed three times for 10 min in the culture medium containing an excess of cold precursor sodium phosphate, or serine, or proline, respectively. They were further cultured for 1, 2, 4, and 24 h, fixed in 2.5% glutaraldehyde buffered with 0.1 M sodium cacodylate, pH 7.4, for 4 h at 4°C, postfixed in 2% OsO₄ for 30 min at room temperature and further processed for embedding in Epon 812. Five germs were used for each period of time per precursor.

After embedding in Epon, semithin sections were coated with Ilford K5 emulsion for radioautography at half dilution, at 40°C and stored at 4°C for 11 days for [³³P]phosphate, and 20 days for [³H]serine and [³H]proline. They were developed with D19 (Kodak) for 20 min at 15°C and fixed with Hypam (Ilford) for 10 min. The sections were stained for 30 sec with 0.1% aqueous basic fuchsin.