Cytochemical localization of Ca2+-Mg2+ adenosine triphosphatase in rat incisor ameloblasts during enamel secretion and maturation.

A modified Wachstein-Meisel medium containing lead or cerium as capturing ions was used to localize Ca2+-Mg2+ adenosine triphosphatase (ATPase; EC 3.6.1.3) in rat incisor ameloblasts during enamel formation. Sections representing different developmental stages were processed for electron microscopic cytochemistry. Distribution and intensity of the observed reaction product, which was almost exclusively associated with cell membranes, varied according to the stage of enamel formation. During the secretory stage, intense reaction product was evident along the entire plasma membrane of ameloblasts and papillary cells. The early transitional ameloblasts showed reaction product on their proximal and lateral cell membranes, but not distally. In late transitional (pre-absorptive) ameloblasts, distal cell membranes exhibited intense reaction product. During enamel maturation, smooth-ended ameloblasts showed reaction product proximally and laterally, but not distally. Ruffle-ended maturative ameloblasts exhibited intense reaction product along their lateral and distal membranes. The intensity of the latter was decreased but not eliminated by levamisole. In the transition from smooth-ended to ruffle-ended cells, the reaction product became evident distally, concomitant with the appearance of cell membrane invaginations. These data are consistent with a possible role for Ca2+-Mg2+ ATPase in controlling calcium availability at the enamel mineralization front.

Tubular lysosomes in ruffle-ended ameloblasts associated with enamel maturation in rat incisor.

Trimetaphosphatase (TMPase) and cytidine-5'-monophosphatase (CMPase) were localized to investigate the lysosomal system, particularly tubular lysosomes, in ruffle-ended ameloblasts associated with maturation of enamel in rat incisor. Demineralized specimens were incubated for TMPase and for CMPase in a modified medium where cerium was used as the capture ion. Ruffle-ended ameloblasts showed distal invaginations and membrane-bound bodies filled with fine granular material, some of which displayed CMPase reaction product. Elongated tubular configurations 80-140 nm wide were distributed throughout the cytoplasm and were reactive with both TMPase and CMPase, thus characterizing these structures as lysosomes. They often contained fine granular material morphologically similar to that present in multivesicular bodies. During late enamel maturation, fewer tubular lysosomes were observed when compared to early maturation. These cytochemical results demonstrate the presence of tubular lysosomes in ruffle-ended ameloblasts, and it is suggested that they are elements of the endosomal system in these cells. These findings are also consistent with a resorptive function for ruffle-ended ameloblasts during enamel maturation.

Expression of plasma membrane Ca++ pump epitopes parallels the progression of enamel and dentin mineralization in rat incisor.

We investigated the expression of Ca++ pump epitopes during enamel and dentin mineralization in the rat incisor. Secretory and maturation ameloblasts were studied as well as odontoblasts, using a monoclonal antibody (5F10) against human erythrocyte plasma membrane Ca++, Mg(++)-ATPase. A progressive increase in staining intensity in ameloblasts and the odontoblasts was observed beginning with the onset of mineralization. The mainly membrane-related labeling of ameloblasts showed variable intensity depending on the stage of enamel formation, whereas that of the odontoblasts showed even intensity during continued dentinogenesis. Staining of papillary cells was evident only during enamel maturation. Western blot analysis of freeze-dried ameloblasts was also used to determine the molecular weight of the Ca++ pump epitopes as well as the distribution and relative concentration of epitopes at each stage. An immunoreactive band of MW 140 KD and lower molecular weight bands that are more intense in late than in early maturation were demonstrated. Our studies suggest that the expression of plasma membrane Ca++ pump parallels the progression of mineralization in rat incisor enamel and dentin.

Scanning and transmission electron microscopy of tubular structures presumed to be human odontoblast processes.

Tubular structures interpreted as being odontoblast processes can be observed with the scanning electron microscope (SEM) on fractured dentin surfaces which have been demineralized and treated with collagenase. To confirm the nature of these structures, SEM preparations exhibiting similar tubular structures were subsequently examined with the transmission electron microscope (TEM). Newly-erupted human third molars were fractured buccolingually with heavy-gauge industrial nippers or sectioned mesiodistally with a Leitz saw microtome and fixed in glutaraldehyde. The exposed dentin surfaces were decalcified to a depth of approximately 500 microns and then treated with bacterial collagenase. Half of the specimens were critical-point-dried and coated for SEM. The other half were post-fixed and processed for TEM. After examination by SEM, the specimens were embedded and thin-sectioned for TEM. SEM observations of both the fractured and cut surfaces of dentin showed tubular structures running from the surface of the pulp to the dentino-enamel junction. When the SEM preparations were examined with TEM, the tubular structures were seen to be the inner sheath of the peritubular matrix, not odontoblast processes. In the specimens directly processed for TEM, the structures lying inside the sheath could be visualized clearly. In the outer two-thirds of the dentin, the tubules were essentially empty. Well-defined odontoblast processes were seen lying inside the sheath only in the inner dentin.

Multi-method analysis of calcium localization in the secretory ameloblast.

X-ray micro-analysis and electron energy loss analysis were used to confirm cytochemical localization by potassium pyro-antimonate of calcium in secretory ameloblasts. Neither intercellular calcium concentration between ameloblasts nor over-all calcium levels of the enamel organ were lowered during a period of inhibition of enamel mineralization by injected fluoride. Calcium concentrations in mitochondria and secretory granules were reduced.

Fine structure of the secretory and non-secretory ameloblasts in the frog. II. Fine structure of the non-secretory ameloblast.

The non-secretory ameloblasts present at the enamel-free surfaces of maxillary teeth in the frog Rana pipiens were examined by electron microscopy at different stages of tooth development. Their main fine structural features seem to reflect a transport function. During early tooth development, the non-secretory ameloblasts adjacent to odontoblasts and predentin exhibit extensive lateral surface specializations and numerous cytoplasmic vesicles. During late tooth development, the non-secretory ameloblasts adjacent to mineralizing dentin show numerous cellular junctions, well-developed intercellular channels with numerous interdigitating processes and labyrinthine configurations at their distal surfaces. An intact basal lamina is present between the non-secretory ameloblasts and the dentin surface until the dentin becomes fully mineralized. At this stage the adjacent cells no longer exhibit surface specializations. It is suggested that the non-secretory ameloblasts may participate in the mineralization of adjacent dentin at the enamel-free surfaces. This surface dentin becomes fully mineralized at a later stage of development than the underlying dentin.

Comparison of scanning and transmission electron microscopy of the epithelial pocket wall in juvenile and adult periodontitis.

Using scanning (SEM) and transmission (TEM) electron microscopy, this study compared fine structural features of the pocket walls in both juvenile and adult periodontitis (JP and AP, respectively) in 40 cases. Gingiva was also obtained from a control group consisting of periodontally noninvolved teeth. Clinical parameters were assessed in both JP and AP patients as well as in controls. Clinical findings showed low plaque accumulation, marked periodontal tissue destruction and less gingival inflammation in JP. Bone destruction and attachment loss were more marked in JP than in AP. AP had a higher plaque index and more evident gingival inflammation. SEM observations of JP as compared to AP showed gross distortions in pocket walls, an increased beaded appearance of microridges, and separation between pocket epithelial cells. TEM showed partially desquamated and separated superficial epithelial cells, but only in JP were fine granular precipitates observed in the intercellular spaces. The observations demonstrated structural features indicative of more prominent degenerative changes in JP than in AP. Also, these features were coincidental with a higher plaque index in AP than in JP, where clinical features (including a low plaque index) were not proportional to the epithelial destructive changes present.

Histology and lysosomal cytochemistry of the postsurgically inflamed dental pulp after topical application of steroids. I. Histological study.

The purpose of this study was to histologically investigate steroid effects on the dental pulp. Three steroid preparations, hydrocortisone, betamethasone and triamcinolone, were locally applied to the exposed pulp tissue in rat incisor after pulpectomy. After 24 h, the effects on the tissues were assessed by light microscopy. The results showed that topical application of corticosteroids as an intracanal medicament reduced inflammatory changes in the pulp as compared with controls. Furthermore, triamcinolone and betamethasone demonstrated more potent anti-inflammatory effects than did hydrocortisone.

Morphology and cytidine-5'-monophosphatase cytochemistry of odontogenic epithelium at the enamel-free areas of mouse molars.

This epithelium in developing first and second molars of 1-15 days old balb/c mice was examined by light and electron microscopy. The lysosomal content of these epithelial cells was assessed using CMPase as a marker. At the early developmental stages (4-7 days), the epithelial cells at the enamel-free areas had morphological features of secretory cells. A distinct afibrillar layer was identified covering the enamel-free dentine surfaces. This layer was morphologically different from both the underlying dentine and the enamel at its periphery. At a later stage (9 days), the epithelial cells had extensive infoldings of their distal membranes. Also, the secretory organelles were markedly reduced and mostly replaced by abundant mitochondria concentrated in the distal cytoplasm. Numerous CMPase-reactive lysosomal structures were demonstrated in the 9- and 11-day-old specimens. These cytochemical and morphological features are consistent with a resorptive activity for the enamel-free cells during this developmental stage (9-11 days). Subsequently, at 15 days, these cells had regressed into a flattened squamous epithelium that remained in contact with the tooth surface until eruption.

Morphometry and autoradiography of altered rat enamel protein processing due to chronic exposure to fluoride.

Female Sprague-Dawley rats had 6 weeks of 0 (control), 75 or 100 parts/10(6) sodium fluoride in their drinking water. Whole mandibular incisors were removed, fixed, demineralized and sections prepared for light-microscopic morphometric analysis of dose-related alterations in enamel protein retention. Other rats given 0 and 75 parts/10(6) only (control and experimental groups) were used for autoradiographic evaluation of alterations in enamel protein removal 35S-methionine was applied directly over secretory ameloblasts at the end of the fifth week of fluoride exposure. Incisors were removed either 5 or 7 days later and processed for autoradiographic analysis. The results indicated: (1) extended retention of enamel proteins in fluoride-exposed maturation enamel as well as reduced enamel protein synthesis and/or secretion in the secretory stage; (2) negative linear correlation between extended enamel protein retention and reduced enamel protein secretion among groups; and (3) repression of enamel protein removal. The data are also consistent with the concept that the fluoride effect is multifactorial.

Ameloblast cycling patterns as measured by fluorochrome infiltration of rat incisor enamel.

Rapid modulation of maturation ameloblasts between smooth-ended and ruffle-ended forms may play an important part in the development of normal dental enamel. Previous studies of modulation rates relied upon measurements of stained or fluorescing bands on the enamel surface of whole incisors along with separate histological sections for cell-band dimensions. The present study utilized direct measurement of maturation-ameloblast bands and fluorescing regions of underlying enamel in the same histological sections, which increased the accuracy and ease with which modulation rates could be determined. Rats were injected with calcein at various times before killing and preparation of survey midsagittal sections of the lower incisors. The lengths of bands of smooth-ended ameloblasts and underlying fluorescing regions of enamel were measured throughout the maturation zone. Modulation rates were found to range from 238 microns/h (early maturation) to 91 microns/h (late maturation). Calcein diffused into enamel to varying degrees depending upon the location within the maturation stage. This new approach of direct measurement greatly facilitates the investigation of ameloblast modulation and provides additional insights into progressive structural changes in enamel during maturation.

Comparisons of processing with and without potassium pyroantimonate in quantitative autoradiography of calcium in developing teeth of the frog Rana pipiens.

To assess the effect of potassium pyroantimonate (PPA) on the retention and localization of 45Ca in developing teeth, six frogs were injected with 45CaCl2, and pairs decapitated after 5 min, 1 h and 24 h; blocks of developing teeth were dissected and processed with and without PPA. Sections from 36 teeth in similar stages of development were selected from the PPA-processed blocks and similar sections from 36 teeth without PPA treatment served as controls; all were similarly processed for autoradiographic examination. Each tooth was drawn to scale; the areas of enamel, dentine and predentine were measured and the net number of silver grains in each tissue determined after subtraction of background counts. Counts in the PPA specimens were consistently higher for all tissues and all time intervals and the differences between experimental and control mean counts were statistically significant. No differences in the distribution pattern of 45Ca were seen in any of the three tissues. The unaltered distribution but increased concentration of 45Ca with PPA suggest the formation of PPA complexes with loosely-bound calcium, which will enhance its retention during processing for autoradiography.

The use of autoradiography and cycloheximide to determine the origin of enamel proteins in the maturation ameloblasts of the rat incisor.

Morphological and cytochemical studies have suggested that maturation ameloblasts participate in protein loss by absorbing and degrading enamel proteins as the enamel matures. Several immunocytochemical and autoradiographic studies have suggested other possible explanations for the presence of enamel matrix proteins in maturation ameloblasts. The weakness of these autoradiographic studies is the uncontrolled distribution of systemically injected radioactive amino acids, making it impossible to trace the source of the visualized intracellular isotope. This study used a localized technique to control the targets of the applied isotope and to identify enamel matrix proteins in the maturation ameloblasts with more confidence about their origin. The amount of labelled enamel protein was higher in maturation ameloblasts than transitional ameloblasts. When cycloheximide, a protein-synthesis inhibitor, was applied, there was no effect on the amount of labelled protein in the maturation ameloblasts. These findings support the hypothesis that maturation ameloblasts actively resorb and degrade enamel matrix proteins during enamel formation in the mandibular incisor of the rat.

Tubular lysosomes in papillary cells during maturation of enamel in the rat.

TMPase and CMPase were used as lysosomal markers. Demineralized-incisor specimens were incubated for TMPase in a lead-acetate based medium and for CMPase in a modified medium where cerium was used as the capture ion. Papillary cells contained an extensive network of branched and interconnected tubular structures reactive for both TMPase and CMPase; there were some of these structures also in the endothelium of adjacent capillaries. Fine granular material was observed in these tubular structures, as well as in coated vesicles and multivesicular bodies in the papillary cells. These cytochemical results demonstrate the presence of tubular lysosomes in papillary cells, as have already been found in adjacent maturation ameloblasts. These structures may be elements of an extensive endosomal system involved in the degradation of enamel protein. These findings also support the concept that the various cells of the enamel organ constitute a functional unit.

Quantitative cytochemistry of lysosomal structures in rat incisor maturation enamel organ.

Trimetaphosphatase was used as a lysosomal marker in the ruffle-ended maturation ameloblasts and associated papillary cells. Morphometric analysis was carried out of the percentage area of these cells (density) occupied by the various enzyme-reactive lysosomal structures. The density of total TMPase-positive lysosomal structures, tubular lysosomes and multivesicular bodies in ruffle-ended ameloblasts were all significantly greater (p less than or equal to 0.05) in early than in late maturation enamel formation. In papillary cells the same was true of tubular lysosomes, whereas the greater density of enzyme-positive total structures in early maturation was not statistically significant when compared to late maturation. These findings demonstrate a corresponding pattern between enamel-organ lysosomal activity and the period of early enamel maturation when most enamel protein is lost. They support the likely involvement by ruffle-ended ameloblasts and papillary cells in absorption and degradation of exogenous enamel proteins.

Dentin matrix protein 1 induces cytodifferentiation of dental pulp stem cells into odontoblasts.

Odontoblasts are postmitotic cells that differentiate from the dental papilla. These cells are responsible for producing the calcified dentin matrix. The pulp-odontoblast interphase contains undifferentiated mesenchymal stem cells, which have the ability to cytodifferentiate into odontoblast-like cells in response to specific signaling molecules. Dentin matrix protein 1 (DMP1) is one of the dentin noncollagenous extracellular matrix proteins that has been implicated in regulation of mineralization. In this study, we have examined the potential role of DMP1 in inducing cytodifferentiation of dental pulp stem cells into odontoblast-like cells and formation of reparative dentin in a rat model. Cavities were drilled and pulps exposed in maxillary first molars. Collagen matrix impregnated with recombinant DMP1 was implanted directly in Group 1, while calcium hydroxide, a commonly used pulp-capping agent was implanted in group 2, collagen matrix that was not impregnated with rDMP1 was implanted directly in group 3, which served as control. Each of these three groups was subdivided into two subgroups, A for 2 weeks time duration and B for 4 weeks duration. At the end of the time period the maxillae were excised, tissues were processed for histological and immunohistochemical evaluations. The results showed that DMP1 could act as a morphogen on undifferentiated mesenchymal cells present in the dentin-pulp complex. These differentiated cells had the potential to regenerate dentin-like tissue, which was confirmed by the presence of collagenous matrix, odontoblast specific markers and calcified deposits.

Fine structure of the secretory and nonsecretory ameloblasts in the frog. I. Fine structure of the secretory ameloblasts.

Amelogenesis in the tooth germs of the frog Rana pipiens was examined by electron microscopy at different stages of tooth development. Cellular changes in secretory ameloblasts during this process showed many basic similarities to those in mammalian amelogenesis. Amelogenesis can be divided into three stages based on histological criteria such as thickness of enamel and the relative position of the tooth germ within the continuous succession of teeth. These stages are early, transitional and late. The fine structure of the enamel-secreting cells reflects the functional role of these ameloblasts as primarily secretory in the early stage, possibly transporting in the late stage and reorganizing between the two functions in the transitional stage. In early amelogenesis the cell exhibits well-developed granular endoplasmic reticulum, Golgi complex, microtubules, dense granules, smooth and coated vesicles, lysosome-like bodies in supranuclear and distal portions of the cell and mitochondria initially concentrated in the basal part of the cell. Numerous autophagic vacuoles are observed concomitant with the loss of some cell organelles at the transitional stage. During late amelogenesis the ameloblasts exhibit numerous vesicles, granules, convoluted cell membranes, junctional complexes and widely distributed mitochondria. Toward the end of amelogenesis, cells become oriented parallel to the enamel surface and the number of organelles is reduced. Amelogenesis in the frog is an extracellular process and mineralization seems to occur simultaneously with matrix formation.