Rat Incisor Enamel Organ Research Articles

Lectin cytochemistry on the stratum intermedium and the papillary layer in the rat incisor enamel organ.

Lectin cytochemistry was carried out to elucidate the role of stratum intermedium cells and papillary layer cells in amelogenesis, especially in the process of sugar incorporation and on membrane characteristics according to their cytodifferentiation. Regarding the lectin-reaction on the plasma membrane, little or at best a weak reaction of Con A, UEA-I, PNA, MPA and WGA was seen in stratum intermedium cells from the late differentiation stage to the early secretory stage of ameloblasts. Lectin-stainability in the stratum intermedium increased in accordance with the cytodifferentiation of ameloblasts. At the active secretory stage of ameloblasts, lectins intensely stained the plasma membranes of stratum intermedium cells. The plasma membranes of papillary layer cells at both stages of ruffle-ended and smooth-ended ameloblasts were stained by same lectins as well. The results therefore suggest that: 1) stratum intermedium cells bring about changes in the glycolipids and glycoproteins of their plasma membranes in accordance with the cytodifferentiation of ameloblasts; 2) they regulate the transport of mineral and/or organic materials between ameloblasts and extracellular fluid via highly charged plasma membranes generated by glycocalyx; 3) the cell-cell interaction of stratum intermedium cells with ameloblasts, in which carbohydrate-protein (endogenous lectin) interaction plays a significant role, is important for the cytodifferentiation of these cells. Regarding the papillary layer cells, the results suggest that they also regulate the transport of minerals by their charged plasma membranes and participate in the removal of the enamel matrix.

Histochemical localization of calcium in the enamel organ of rat incisors in early-stage amelogenesis.

The localization of calcium in the enamel organ of rapidly-frozen, freeze-substituted rat incisors in early-stage amelogenesis was examined by a histochemical calcium-staining method. In secretory ameloblasts, glyoxal bis(2-hydroxyanil) (GBHA) staining revealed intense red reactions in mitochondria and tubulovesicular structures located throughout the cytoplasm, while no reaction was seen in the nucleus and cytosol, nor along the plasma membranes of the respective cells. No significant GBHA reaction was observed in the intercellular compartment and other cells of the enamel organ. Some granular reactions were localized in the cells of the adjacent connective tissue. Control tests confirmed the specificity of GBHA reactions for calcium. Thus, the present observations provide histochemical evidence indicating an exclusive localization of calcium in mitochondria and tubulovesicular structures of the secretory ameloblast, and support their contributions to the translocation of calcium from the proximal to the distal pole of the cytoplasm.

Calmodulin immunocytochemistry in rat incisor enamel organ through its life cycle.

The enamel organ of the growing rat incisor was perfusion-fixed with a mixture of formaldehyde and glutaraldehyde and processed for indirect immunogold labeling of calmodulin on post-embedded ultrathin sections. Throughout the zones of presecretion, secretion, and maturation of enamel, specific protein A-immunogold labeling was localized on polyribosomes and those attached to endoplasmic reticulum, mitochondria, nuclear chromatin, phagolysosomes, and cytoplasm adjacent to the plasma membrane, and tonofilaments associated with desmosomes of ameloblasts and cells of outer layer of enamel organ. Golgi membranes, condensing vacuoles, secretion granules, primary lysosomes, and micropinocytotic coated vesicles were hardly labeled. In the presecretion zone, the basal lamina of the preameloblasts and the matrix vesicles and collagen fibrils of the predentin matrix were not immunoreactive. Tomes' process of secretory ameloblast and adjacent enamel crystals were labeled. In addition to the above immunoreactive structures, some phagolysosomes, ferritin granules, and the cytoplasm of the ruffled border zone of maturation ameloblast contained immunogold particles. In control sections incubated with either protein A-gold complex alone, or antiserum preabsorbed with an excess of calmodulin and protein A-gold complex, only a few gold particles were observed to be randomly associated with the tissues. These results indicate that calmodulin is present in the cells of the enamel organ through all stages of amelogenesis. Its wide distribution is consistent with its involvement in various cytoplasmic functions.

H+-K+-ATPase activity in the rat incisor enamel organ during enamel formation.

The enamel organ of growing rat incisors was perfusion-fixed with a mixture of formaldehyde and glutaraldehyde and processed for ultracytochemical demonstration of ouabain-resistant, K+-stimulated p-nitrophenylphosphatase representing the second dephosphorylative step of H-K-ATPase by use of the one-step lead method. Throughout the stages of amelogenesis, the enzymatic activity was found in the plasma membranes, mitochondrial membranes, and lysosomal structures of the cells of stratum intermedium, papillary layer, and ameloblast layer. Gap junctions and desmosomes between these cells were, however, free of reaction product or showed slight precipitates of reaction. The stellate reticulum and the outer enamel epithelium at the stage of enamel secretion were usually negative for reaction. Although secretory, transition, and ruffle-ended maturation ameloblasts showed enzymatic activity at their basolateral cell surfaces, their distal cell surfaces facing the enamel were always free of reaction product. On the other hand, the smooth-ended maturation ameloblasts seldom showed a positive reaction, except in lysosomes and along their basal cell surfaces. An energy-dispersive X-ray microanalysis of reaction products of H-K-ATPase in unosmicated tissue sections demonstrated that they were composed of lead and phosphorus, which had been released during the dephosphorylation of substrate. In cytochemical controls, the enzymatic activity was completely dependent on substrate and potassium ion, resistant to ouabain and levamisole, and inhibited by nolinium bromide, a specific inhibitor of H-K-ATPase. In addition, inorganic trimetaphosphatase as enzymatic marker of lysosome was localized in dark and pale lysosomes, phagosomes, multivesicular bodies, and ferritin-containing vesicles of the ameloblasts and the cells of stratum intermedium and papillary layer. These membrane-bound structures were also positive for H-K-ATPase reaction. These results suggest that: 1) H-K-ATPase functions to maintain an acidic internal pH of lysosomes in the enamel organ cells; and 2) H-K-ATPase localization in the plasma membranes of enamel organ cells is concerned with efflux of protons derived from cytoplasmic water.

A histochemical demonstration of calcium in the maturation stage enamel organ of rat incisors.

The location of calcium in a rapid-frozen and freeze-substituted maturation stage enamel organ of the rat incisors was demonstrated by means of the glyoxal bis(2-hydroxyanil) (GBHA) staining method, which formed insoluble red precipitates of calcium-GBHA complex. In the ameloblast layer, highly GBHA-reactive tubulo-vesicular structures corresponding to mitochondria and some other membrane-bound structures were localized in both ruffle-ended and smooth-ended ameloblasts, although no significant GBHA reaction was localized in the nucleus, Golgi region, nor along the plasma membrane of these cells. In addition, numerous granular GBHA reactions appeared exclusively in association with the ruffled border of ruffle-ended ameloblasts. GBHA reactions were positive, but were considerably weaker in papillary cells than in the ameloblast. These observations provide a first published histochemical mapping of calcium in the maturation stage enamel organ, and suggest the active participation of mitochondria in maturation stage ameloblasts in calcium regulation.

Na-K-ATPase in the enamel organ: localization and possible roles in enamel formation.

Ouabain-sensitive, K-dependent p-nitrophenyl phosphatase (p-NPPase) activity was localized ultra-Ocytochemically in the lateral plasma membranes of secretory ameloblasts and the stratum intermedium and principally in the papillary layer cells of aldehyde-fixed rat incisor enamel organs by the one-step lead method. Daily intraperitoneal injection of ouabain (250 μg, 500 μg, and 1 mg/100 g body weight) for two weeks reduced p-NPPase activity in the enamel organ cells. However, the degree to which this activity was reduced appeared to vary among the experimental animals. Addition of ouabain to the cytochemical incubation medium completely inhibited p-NPPase activity in the tissues. Although long-term ouabain injection did not result in any morphological alterations of the enamel organ cells, it caused, in part, an appearance of electron-dense, homogeneous matrix-like substances (MS) in the extracellular spaces of the ameloblast layers at both the secretion and maturation stages. In addition, long-term ouabain injection appeared to have resulted in delayed maturation of enamel as measured by energy-dispersive x-ray analysis of Ca and P in surface enamel. These results suggest that Na-K-ATPase of enamel organ cells may participate in the net flow (removal) of organic matrix components and water from the enamel during the maturation stage of enamel formation. It is suggested that this flow is maintained by local osmotic gradients generated by Na-K-ATPase within the papillary layer.

Calcium Transport by a Calmodulin-Regulated Ca-ATPase in the Enamel Organ

Using aldehyde-fixed rat incisor enamel organ, we localized Ca-ATPase activity ultracytochemically in the plasma membranes, the mitochondrial inner membranes, and the Golgi membranes of secretory ameloblasts and the cells of stratum intermedium at the secretory stage and papillary layer cells at the maturation stage, but not in maturation ameloblasts. This Ca-ATPase activity was totally dependent on substrate ATP, the enzyme activator CaCl2, and also sensitive to the specific calmodulin blocker trifluoperazine (TFP) in the incubation media. Specific antigenic sites of endogenous calmodulin were demonstrated in polyribosomes, the nucleus, mitochondria, and the cytoplasmic matrix along the plasma membranes of secretory ameloblasts, by the protein A-immunogold technique using sheep antiserum against bovine testis calmodulin. All other enamel organ cells-such as stratum intermedium, papillary layer cells, and maturation ameloblasts-were also weakly immunoreactive. In control sections incubated with antiserum pre-absorbed with an excess of calmodulin and protein A-gold complex, only a few gold particles were observed to be randomly associated with the tissues. Daily intraperitoneal injection of TFP (1 and 5 mg per 100 g body weight) for one week resulted in prominent migration of mitochondria from the infranuclear to supranuclear regions of secretory ameloblasts but caused no other morphological alterations in the enamel organ cells. EDX analysis of ultrathin sections revealed significantly lower peaks of Ca and P in the forming enamel of TFP-injected rats than those in controls. However, little reduction in the Ca and P levels in the maturing enamel was observed in TFP-injected rats. When growing enamel surfaces were exposed with NaOCl and examined with SEM, a remarkable defect in the enamel matrix was observed in the forming enamel but not in the maturing enamel. These results suggest that early enamel mineralization is dependent upon an intact calmodulin-regulated Ca-transporting ATPase in secretory ameloblasts and that enamel maturation is controlled by different mechanism(s).

Calmodulin in rat incisor secretory ameloblasts as revealed by protein A-gold immunocytochemistry.

Thin sections of aldehyde-fixed, undecalcified, embedded rat incisor enamel organ were incubated with sheep antiserum to bovine testes calmodulin to reveal the sites of antigen-antibody reaction at the ultrastructural level in secretory ameloblasts using the protein A-gold immunocytochemical technique. Specific immunolabelling was localized intensely on free polyribosomes and those attached to rough-surfaced endoplasmic reticulum but only rarely observed in the cisternal space. The nuclei, mitochondria, cytosol, and plasma membranes were also immunoreactive. The Golgi membranes and related vesicles, secretion granules, and lysosomes were unlabelled. The proximal and distal cell web junctional complex systems were not immunoreactive. These findings suggest that calmodulin location reflects its synthetic site and multifunctional roles in the immunolabelled cytoplasmic components of secretory ameloblasts.

Subcellular fractionation of rat incisor enamel organs by differential and rate zonal microcentrifugation: [formula omitted][formula omitted][formula omitted] Depts. of Anatomy 1 & Oral Biology 2, McGill University & Dépts. de stomatologie et d'anatomie 3, Université de Montréal, Montreal, Canada

Subcellular fractionation of rat incisor enamel organs by differential and rate zonal microcentrifugation: [formula omitted][formula omitted][formula omitted] Depts. of Anatomy 1 & Oral Biology 2, McGill University & Dépts. de stomatologie et d'anatomie 3, Université de Montréal, Montreal, Canada

Ultracytochemistry of ouabain-sensitive K+-dependent p-nitrophenyl phosphatase in rat incisor enamel organ.

Sprague-Dawley strain rats of 4-5 weeks old were perfusion-fixed with either a mixture containing 0.1 or 0.25% glutaraldehyde and 2% formaldehyde, or a 2% formaldehyde in 0.1 M sodium cacodylate buffer for 10 minutes. Non-decalcified 30-50-micron sections of the enamel organ taken from lower incisors were then processed for ultracytochemical demonstration of ouabain-sensitive, K+-dependent, p-nitrophenyl phosphatase, by use of the one-step lead method, representing the second dephosphorylative step of Na+-K+-ATPase. Throughout the secretory, transition, and maturation stages of amelogenesis, the enzymatic activity was demonstrated along the cytoplasmic side of the plasma membranes of the stratum intermedium and the papillary layer cells, especially along their numerous microvilli. The plasma membranes forming gap junctions and desmosomes were free of reaction or showed slight focal precipitates of reaction products. The stellate reticulum and the outer enamel epithelium exhibited either a weak reaction or were reaction negative. Secretory ameloblasts showed a weak trace-like reaction along the basal and lateral cell surfaces; however, the latter surfaces were sometimes completely free of reaction. Tomes' processes were usually reaction negative. Ameloblasts in the transition and maturation stages were devoid of enzymatic activity, except for a slight reaction along the plasma membranes of the basal cell surfaces of transition ameloblasts facing the papillary layer. The enzymatic activity described above was completely dependent on the presence of potassium and substrate in the incubation media and was almost completely inhibited by an addition of 10 mM ouabain to the incubation media.

Ultracytochemical demonstration of ATP-dependent calcium pump in ameloblasts of rat incisor enamel organ.

The enamel organ of the growing rat incisor was fixed with a mixture of formaldehyde and glutaraldehyde and processed for ultracytochemical demonstration of Ca- and Mg-activated membrane ATPase by a one-step lead technique at alkaline pH. To inhibit nonspecific alkaline phosphatase, 5 mM levamisole was added to the incubation media. Intense Ca- and Mg-ATPase activity was demonstrated in the cell surfaces of the secretory ameloblasts, except at the proximal and distal junctional complexes and the gap junctions in the lateral and basal cell surfaces. Deep plasma membrane invaginations at the proximal and distal parts of Tomes processes facing interrod- and rod-enamel growth regions exhibited the strongest enzymatic reaction. Mg-ATPase activity was also shown to be present in the plasma membranes of secretory ameloblasts but it was less intense than Ca-ATPase. Except for a slight reaction in the Golgi membranes, all other cell organelles of the secretory ameloblasts and the adjacent enamel matrix were free of enzymatic reaction. However, when the tissues were incubated in media lacking levamisole, a prominent enzymatic reaction was observed in the newly secreted enamel matrix of the rod and interrod growth regions as well as on the plasma membranes of the cells. In maturation ameloblasts of both ruffle-ended and smooth-ended types, a weak reaction for Ca- and Mg-ATPase was restricted to basal cell surfaces facing the papillary cell layer. In tissues incubated in media lacking levamisole, a variable deposition of reaction products was observed in the Golgi membranes, mitochondrial membranes, tubular elements of smooth endoplasmic reticulum in the ruffled border zone, and along the plasma membranes of the ruffled border. Throughout the secretory and maturation stages, a moderate and/or weak enzymatic reaction for both Ca- and Mg-ATPase was seen in the plasma membranes of the cells of the stratum intermedium and the papillary layer when incubated in media with levamisole. Omission of substrate ATP and/or the enzyme activator CaCl2 from the incubation media for Ca-ATPase produced a negative reaction in the tissues examined. When the calmodulin blocker trifluoperazine was administered to the rats intravenously, Ca-ATPase activity was almost completely abolished from the plasma membranes of secretory ameloblasts, but not of other cell types.

Correlation of apical and lateral membrane modulations of maturation ameloblasts.

Maturation ameloblasts of rat incisor teeth have smooth-ended and ruffle-ended apical membrane configurations. It has also been reported that maturation ameloblasts have several lateral membrane configurations. The purpose of this study was to determine the correlation between the modulations of lateral and apical cell membranes of murine incisor ameloblasts in the maturation stage of amelogenesis. Maxillary and mandibular incisors were dissected, demineralized, embedded in paraffin, sectioned and then de-paraffinized, and the enamel organs were prepared for scanning electron microscopy. Additional mouse and rat incisor enamel organs were fixed and teased apart during dehydration, then observed in the SEM. The lengths of smooth- and ruffle-ended ameloblast segments were measured, and the site, length, and frequency of each lateral membrane configuration were determined within each segment. The lateral membrane configuration with folds forming from 12 to 14 channels around the periphery of the cells was most predominant in both smooth- and ruffle-ended cells. Cells surrounded by from six to eight channels were the only other lateral membrane configuration observed in ruffle-ended ameloblasts. Smooth-ended ameloblasts had lateral membrane configurations with either dense or sparse microvillous projections in addition to both types of channel cells. The observation that channelled extracellular spaces are always associated with ruffle-ended cells suggests that channels somehow function in conjunction with the ruffled apical membrane in resorption and removal of enamel matrix proteins. The smooth-ended ameloblasts lack tight apical junctions, and their microvillous lateral membranes permit the passage of plasma fluids around cells to the maturing enamel surface.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo experimentation on rat incisor enamel organs through a surgical window.

Experimental agents administered systemically are costly and often toxic to animals. An in vivo technique has been developed whereby a surgical window in the alveolar bone allows selected areas of the rat incisor enamel organ and underlying enamel to be exposed to various drugs, radiolabeled molecules, and molecular weight markers. Sherman rats weighing 100 gm were anesthetized and the inferior surface of each hemimandible was surgically exposed. A slow-speed dental hand drill was used to drill a small hole through the alveolar bone overlying the secretion or maturation zones of the enamel organ. The wound was closed and during recovery the mechanical trauma to the underlying tissue moved away from the hole due to the continuous eruption of the tooth. Two to 5 days later the hole was reexposed and microinjections of 3H-proline, 125I-salmon calcitonin, vinblastine sulphate, and normal saline (as control) were administered through the hole with a micromanipulator and a microliter syringe. Radioautographic detection of 3H-proline incorporation in secretory ameloblasts and enamel at 10 minutes, 30 minutes, 1 hour, 4 hours, 1 day, and 2 days after microinjection was identical to that obtained previously by systemic injection. Two hours after microinjection of vinblastine sulphate the cellular response was again identical to that following systemic injection; 125I-salmon calcitonin (M.W. approximately 3,600D) was used as a molecular weight marker and was seen to diffuse into the enamel of the maturation zone at 10 minutes after microinjection. This study has demonstrated the feasibility of this new technique for experimentation on rat incisor enamel organs.

A freeze-fracture study of ruffle-ended post-secretory ameloblasts.

The post-secretory portion of the rat incisor enamel organ was prepared for routine transmission electron microscopy and freeze-fracture replication in order to define further the structural surface features of the ruffle-ended ameloblasts. Surface views of the distal plasma membrane of the ruffle-ended ameloblasts revealed a well-developed zonula occludens junction with from six to ten rows of tight junctional strands. Gap junctions were also observed just proximal to the tight junctional strands. The membranes of the ruffled border contained a rich supply of intramembrane particles (IMP). The IMPs were approximately 7 to 8 nm in diameter and preferentially located on the P-face profiles of the membrane. The density of IMPs on the membranes of the ruffled border was higher than that on the lateral borders of the cell. It is suggested that the IMPs of the ruffled border may represent enzymatic proteins in the basal cell membrane of absorptive ameloblasts. In addition, the large, highly-developed zonula occludens appeared structurally capable of sealing the intercellular spaces between the ruffle-ended ameloblasts.

Ultrastructure of granular materials in rat incisor enamel organ at early maturation stage

With scanning electron microscopy, globules were seen in inter-cellular spaces between ameloblasts with ruffled borders and papillary cells at early maturation stage. Numerous globules were located on longitudinal folds between parallel ridges on the sides of ameloblasts, but only a few in the papillary layer. In previously-scanned specimens sectioned for transmission electron microscopy, the globules were composed of finely granular material. Vacuoles containing material identical to extra-cellular granules were observed in intra-cellular portions of the ameloblasts and papillary cells.

A freeze-fracture study of the papillary layer of the rat incisor enamel organ

After tooth enamel has been secreted it undergoes maturation or hardening. This process is mediated by ruffled and smooth-ended ameloblasts and associated papillary layer cells. The cells of the papillary layer are characterized by large numbers of mitochondria, coated vesicles, microvilli, and gap junctions. These features have led numerous investigators to speculate that the papillary layer is an ion-transporting epithelium. We have conducted freeze-fracture studies of the rat papillary layer in order to better characterize the surface features of these cells. The cell membranes of the papillary cells contained large numbers of intramembrane particles of various sizes ranging from 4 to 9 nm in diameter. Gap junctions were present at the cell surface and in the cytoplasm in the form of annular gap junctions. The intramembrane particles or connexons of both types of gap junctions were about 8–9 nm wide and were either packed randomly or present in the so-called ‘crystallized’ state. At the interface between smooth-ended ameloblasts and papillary layer cells, a well-developed zonula occludens was present along the basal surfaces of the ameloblasts and several large gap junctions were formed between the two cell types. The capillary network associated with the papillary layer was characterized by a thin endothelium containing large numbers of fenestrations.

Microvascular architecture of the enamel organ in the rat-incisor maturation zone. Scanning and transmission electron microscopic studies.

In order to clarify the microvascular architecture and ultrastructural features of the capillary vessels related to transendothelial transport of metabolites, scanning electron microscopy of tissues digested by HCl-collagenase and of vascular corrosion casts as well as thin-section, tracer, and freeze-fracture replications were employed to study the maturation zone of rat-incisor enamel organ. The enamel-organ maturation zone was shown to have a well-developed, dense capillary plexus. The capillary vessels were distributed along furrows formed by the enamel-organ papillary ridges. In central regions they formed a regular, blindlike network; in the peripheral regions, however, they formed an irregular, circular network. Everywhere except in the nuclear and perinuclear regions, the very thin capillary-vessel endothelial walls were pierced with numerous fenestrations. Such fenestrations were evident in endothelial walls facing the ameloblast-layer site. In tracer experiments, intravenously injected horseradish peroxidase passed through the fenestrations in the endothelial walls to diffuse throughout the enamel-organ extracellular spaces. It did not, however, pass through intercellular spaces or transendothelial channels. The dense, regular distribution of highly fenestrated capillaries in the enamel organ is thought to make possible the rapid transcapillary exchange of various metabolites between the vascular system and the ameloblast and papillary layers that is necessary for enamel maturation.

A morphological, tracer and cytochemical study of the role of the papillary layer of the rat-incisor enamel organ during enamel maturation

Thin-section, freeze-fracture, tracer and cytochemical methods showed that papillary cells were characterized by the presence of numerous mitochondria and microvilli. The cytoplasms contained Golgi apparatus, multivesicular bodies (MVB), tubular structures and coated vesicles. In freeze-fracture replicas, some tubular structures and vesicles were continuous with the cell surfaces. Intravenously-injected horseradish peroxidase (HRP) was rapidly incorporated into the papillary cells via coated vesicles and tubular structures. Horseradish peroxidase-labelled coated vesicles and tubular structures then fused with endocytic vacuoles and MVB. These endocytic vacuoles and MVB showed HRP-labelling of various degrees. Acid-phosphatase activity was demonstrated in the Golgi apparatus, endocytic vacuoles and MVB of papillary cells. Enzymic activity sometimes took place in tubular structures but never occurred in coated vesicles. These results indicate that absorptive and digestive activities of papillary cells are closely related to loss of organic materials from the enamel matrix during enamel maturation.

Ultrastructure of the Papillary Layer and the Blood Capillaries in the Rat Incisor Enamel Organ

The ultrastructure of the papillary cells and capillaries in the papillary layer of the enamel organ during enamel maturation were studied by means of thin sectioning and freezefracturing. Thirty rats were perfused with Karnovsky's fixative, tannic acid-glutaraldehyde fixative and ruthenium red-glutaraldehyde fixative.The papillary cells of the proximal part possessed numerous mitochondria, coated vesicles and microvilli. The papillary cells were connected with each other by desmosomes and gap junctions, and also firmly connected with the proximal ends of the ameloblasts by means of desmosomes, gap junctions and interdigitations. Wide intercellular spaces in the papillary layer were continuous to the basement membrane of the capillaries and to the intercellular spaces of the ameloblast layer. In these cell layers, complete sealing junctions could not be observed. The endothelial wall of the capillaries except for the nuclear portions became very thin and the fenestrations of 50-70 nm in diameter were clearly observed. Ruthenium red positive diaphragms were frequently recognized in the fenestrations. In replica images the fenestrated areas of endothelia were seen near the papillary cells, and the pinocytotic vesicles were mainly distributed in the nonfenestrated areas. Cell to cell attachments of the endothelia were performed by tight junctions. From these results the ameloblasts, the papillary cells and neighboring capillaries were considered to form a functional unit in relation to enamel maturation. They may be concerned in the transport of calcium and phosphate ions from the capillaries to the enamel matrix and in the resorption of organic substances and water from the enamel matrix.

Access of horseradish peroxidase (HRP) to the extracellular spaces of the maturation zone of the rat incisor enamel organ

Adult rats received a single dose of HRP intravenously and were killed from 10 min to 6 hr after injection. Following fixation with glutaraldehyde, the enamel organs were treated with a Graham-Karnovsky-type procedure for peroxidase activity, post-osmicated, and embedded in plastic. Sections were studied with light and electron microscopes. Ten minutes after injection, reaction product was found in all extra-cellular spaces of the enamel organ, at the enamel-ameloblast interface over smooth-ended and intermediate ameloblasts, and in apical surface invaginations and vesicles of the latter cell types. The enamel-ameloblast interface over the ruffle-ended aemlo-blasts and the extracellular spaces within the ruffled border were free of reaction product and remained so for up to 6 hr. The apical terminal bars of the ruffle-ended ameloblasts functioned as a barrier to HRP. The basal terminal bars of the smooth-ended ameloblasts likewise seemed to prevent the passage of the HRP. Possibly, HRP flows in a lateral direction from groups of ruffle-ended into groups of smooth-ended ameloblasts. Between 10 min and 6 hr, HRP was cleared more rapidly from the extra-cellular spaces of the papillary layer than from those of the ameloblast layer, and there was little backflow of tracer from the ameloblast into the papillary layer. Eventually, tracer was cleared also from the extracellular spaces of the ameloblast layer, probably mainly through micropinocytosis by the ameloblasts. A working model is proposed regarding the handling of large molecules by the enamel organ in the maturation zone.