In vitro penetration of 125I-amelogenin into the enamel and enamel organ of rat incisors.

This work attempted to introduce pre-labelled intact amelogenins into developing rat enamel in vitro. The intention was ultimately to trace the fate of such proteins in the developing enamel matrix and to examine the effect of these and other molecules on ameloblast behavior. The penetration of 125I-amelogenin (25 KDa) directly into the young enamel of rat incisors, in vitro, keeping the enamel organ intact is described. The enamel (an extension approximately 8 mm from the apical end of the tooth) together with the enamel organ, was separated from the underlying dentine and placed over a strip of filter paper covering a well of micro-culture slide filled with Eagle's medium containing 125I labelled amelogenin and incubated at 37 degrees C. The enamel faced the strip of filter paper and so was adjacent to the medium. After 1 h pieces were either washed in cold medium, fixed and embedded in Epoxy-resin, or incubated in cold medium for another 1 to 10 h at 37 degrees C before embedding. One-micron thick sections were processed for autoradiography and the results showed a decreasing gradient of silver grain concentration from the dentino-enamel junction towards the enamel organ. It is expected that by determining the nature of the labelled material incorporated into the enamel and enamel organ the fate of amelogenins could be better understood.

Effect of vinblastine on the cell cycle and migration of ameloblasts of mouse incisors as shown by autoradiography using 3H-thymidine.

The effects of vinblastine on the cell cycle and the migration of ameloblasts were studied in the lower incisors of mice by labelling the cells with 3H-thymidine ([3H]TdR) and radioautography. A group of mice received 2 micrograms/g of body weight vinblastine intraperitoneally and 6 hr after these animals and those of a control group were injected with 1 microCi/g body weight of [3H]TdR, and sacrificed at time intervals from 0.75 hr to 15 days. The generation time of ameloblasts in the progenitor compartment was 14.8 hr in animals treated with vinblastine and 17 hr in the controls, using the FLM curve method; with the grain dilution method the duration was respectively 29.25 hr and 25.96 hr. The thymidine labelling index of the treated animals was 50% higher than the controls. The velocity of ameloblast migration, determined either by the displacement of the most incisally labelled cell or by the grain dilution method, was lower in the experimental group (2.48 cell positions/hr and 9.18 microns/hr respectively) as compared with the control (3.21 cell positions/hr and 18.88 microns/hr respectively). The results on the ameloblast production rate are contradictory but the slowing down in the velocity of cell migration is compatible with a decrease of the rate of cell production in the progenitor compartment as a vinblastine effect.

The effect of colchicine and vinblastine on the secretory activity of ameloblasts in the mouse lower incisor as revealed by autoradiography after injection of 35S-sodium sulphate.

The effect of colchicine (0.1 mg/100 g of body weight) and vinblastine (0.2 mg/100 g of body weight) on the secretory activity of ameloblasts in the mouse lower incisor was investigated using autoradiography, 10, 30, 120 min after a single injection of 25S-sodium sulphate. colchicine did not inhibit the synthesis of glycosaminoglycans but delayed the exocytosis of the labeled products into the enamel. Vinblastine inhibited both the biosynthetic activity of ameloblasts and the exocytosis, the latter in an higher degree than colchicine.

New evidences for the role of secretory ameloblasts in the removal of proline labelled proteins from young enamel as visualized by autoradiography.

Eight mice (M. musculus, albinus), two hours old, were sacrificed from 1 to 168 hours following the administration of a single dose of 1 microCi/g body weight of 3H-Thymidine. An other group of six mice received a single dose of 5 microCi/g body weight of 3H-Proline and the animals were killed from 20 min. to 120 h after the injection. The unfixed hemi-jaws containing one incisor tooth, were processed to obtain frozen cross serial sections. With time, as the tooth grows, the 3H-Thymidine labelled ameloblasts move away from the apical end of the incisor at a daily rate of 550 micrometer. By determining the silver grains content over ameloblasts and related enamel matrix in animals which received 3H-Proline, using serial sections separated from each other by 550 micrometers, it was shown that : 1) The silver grains concentration over secretory ameloblasts, which was maximum 20 min. after 3H-Proline administration, showed a decrease after 24 h and a new increase between 48 and 72 h and a further decrease at later time intervals ; 2) such second reactive peak was not observed in post-secretory ameloblasts; 3) the radioactive reaction over the enamel matrix after reaching a maximum 24 h after the injection, decreased at 48 h and another increase in radioactivity was detected at 96 h when the matrix moved incisally but was still related to secretory ameloblasts. The results were interpreted as indicative of the participation of the secretory ameloblasts in the removal of proline-containing protein, which can be considered either as one of the steps of enamel maturation, or also as evidence of a distinct process by which there would be a partial turnover of the organic matter in young enamel.

Histochemical methods for proteins in tissues submitted to decalcification. A histophotometric study.

4 histochemical methods for demonstration of protein acid groups: Nihydrin-Schiff, Millon, DDD and DMAB-nitrite were applied in sections of rat pancreas fixed in the following fixatives: 10% formalin, 10% formalin in saline, ZENKER and GENDRE, and treated with the following decalcifying solutions: 5% nitric acid, 5% trichloroacetic acid, 33% formic acid, 5% EDTA pH=7.0 JENKINS, GREEP and citrate buffer pH=4.5. The colour intensity of each reaction was measured photometrically over the zymogen granules region of the acinar cells, using a Zeiss histophotometer, and compared with controls, i.e., fixed tissue which have not been treated with decalcifying solutions. The results have shown that for each method there is more than one combination fixative-decalcifying solution which gives results similar to those of the control.