Properties of dissociatively extracted fetal tooth matrix proteins. I. Principal molecular species in developing bovine enamel.

A sequential dissociative extraction scheme is described in which tooth matrix proteins are extracted first in 4 M guanidine HCl, pH 7.4, and then in 4 M guanidine HCl, 0.5 M EDTA, pH 7.4, both with protease inhibitors present. The latter step dissolves the mineralized portion of the tissue and extracts noncollagenous proteins closely associated with hydroxyapatite crystallites in the mineralized matrix. In fetal bovine enamel, the initial dissociative extraction step completely removes proline-rich amelogenins from the tissue without dissolving the enamel apatite. The amelogenin proteins consist of several species on polyacrylamide gel electrophoresis with sodium dodecyl sulfate, but display anomalous migration behavior relative to conventional marker proteins in this technique. Subsequent extraction of fetal bovine enamel with guanidine HCl/EDTA removes matrix enamelins, acidic glycoproteins that are tightly bound to the enamel hydroxyapatite. This latter fetal protein type has not been isolated previously. The enamelins are adsorbed strongly by DEAE-cellulose in 7 M urea and totally adsorb to synthetic apatite, even in 4 M guanidine HCl. The enamelins display normal behavior on polyacrylamide gels and stain positively for sialic acid/phosphate and carbohydrate. With advancing tooth maturation, amelogenins disappear while enamelins are conserved. Gel filtration chromatography in 4 M guanidine HCl showed amelogenin components at apparent molecular weights of approximately 25,000, 15,000, 9,500, 7,500, and 6,000, while the enamelins eluted at Mr positions of approximately 72,000, 56,000, 42,000, 30,000, 21,000, 13,000, and 8,000. The gel filtration data showed a clear shift in molecular size population from higher to lower components for both amelogenins and enamelins with progressive enamel maturation.

Calcium fluoride containing granules produced in vitro in rat bones.

Femurs and tibias of normal rats were fixed in 4% unbuffered solutions of glutaraldehyde or formaldehyde containing 1% NaF. Following washing, radiographically controlled demineralization was performed in 3.25% HNO3. Light microscopy and transmission electron microscopy revealed the presence of so-called fluoride granules in bone trabeculae, cartilage, cartilage remnants, bone cells, chondrocytes and in the bone marrow. Selected area diffraction indicated that the fluoride granules contained CaF2. X-ray diffraction analysis of non-demineralized NaF-fixed bones were negative for CaF2. The results are discussed and related to precipitation artifacts reported in demineralized hard tissues and to similar CaF2-containing granules observed in demineralized bones and incisors of rats given high doses of NaF in diet or drinking water.

Matrix-mineral relationships--a morphologist's viewpoint.

The literature on the ultrastructural morphology of the enamel matrix and its relationship to the crystals is reviewed. Two morphological entities of the matrix are discussed. One is the so-called stippled material which may be the initial cell product; the other, variously described as fibrillar, lamellar, tubular or helical, is thought by many to play a crucial role in nucleation and orientation of the crystals. A number of observations, however, suggest that the latter structures form secondarily to the crystals and that in reality they represent organic material adsorbed to the crystal surface and maintained as independent structures upon removal of the mineral. The need for additional studies is stressed including systematic studies of interactions between constituents of the organic matrix and the apatite crystals.

Dose and age dependent variations in effect of tetracycline on enamel formation in rat.

Incidence of rats with gross enamel lesions in response to tetracycline hydrochloride (TC) was studied in relationship to age, tooth type, and concentration of antibiotic in the injection fluid. Incidence was defined as percentage of animals with defects in at least one incisor or one molar. Serum levels were measured at various intervals in 4- and 75-day-old rats, which received a single i.p. injection with 130 mg/kg bw in concentrations of either 13 or 25 mg/ml saline. The study confirmed that the effect of TC on enamel varied with age of the animal and that the molar responded more readily than the incisor. Furthermore, the incidence was higher the higher the concentration of antibiotic. This effect appeared tied to a direct relationship between concentration and serum level. Serum levels declined more slowly in the younger than in the older rats, resulting in significantly higher levels 6, 24 and 48 h after dosing, which correlated with a more extensive labeling of the dentin in the young rats. The possible roles of serum levels, capillary supply and cell susceptibility are discussed in relation to differences in response with animal age, between tooth types, tooth surfaces and with age of the secretory cells.