ELABORATION OF THE MATRIX GLYCOPROTEIN OF ENAMEL BY THE SECRETORY AMELOBLASTS OF THE RAT INCISOR AS REVEALED BY RADIOAUTOGRAPHY AFTER GALACTOSE-3H INJECTION

The elaboration of enamel matrix glycoprotein was investigated in secretory ameloblasts of incisor teeth in 30–40-g rats. To this end, the distribution of glycoprotein was examined histochemically by the use of phosphotungstic acid at low pH, while the formation of glycoprotein was traced radioautographically in animals sacrificed 2.5–30 min after galactose-³H injection. Histochemically, the presence of glycoprotein is observed in ameloblasts as well as in the enamel matrix; in ameloblasts glycoprotein occurs within the Golgi apparatus in amounts increasing from the outer to the inner face of the stacks of saccules, and is concentrated in condensing vacuoles and secretory granules; in the enamel matrix, glycoprotein is observed within linear subunits. Radioautographs at 2.5 min after injection demonstrate the uptake of galactose-³H label by Golgi saccules, indicating that galactose-³H is incorporated into glycoprotein within this organelle. After 5–10 min, the label collects in the condensing vacuoles and secretory granules of the Golgi region. By 20–30 min, the label appears in the secretory granules of the apical (Tomes') processes, as well as in the enamel matrix (next to the distal end of the apical processes, and at the tips of matrix prongs). In conclusion, galactose contributes to the formation of glycoprotein within the Golgi apparatus. The innermost saccules then distribute the completed glycoprotein to condensing vacuoles, which later evolve into secretory granules. These granules rapidly migrate to the apical processes, where they discharge their glycoprotein content to the developing enamel.     

Enamel protein biosynthesis and secretion in mouse incisor secretory ameloblasts as revealed by high-resolution immunocytochemistry

Mouse secretory ameloblasts express a number of enamel proteins, which have been divided into amelogenin and enamelin subfamilies. We have used polyclonal antibodies to murine amelogenins to reveal enamel proteins in mouse ameloblasts using the protein A-gold immunocytochemical technique. Specific immunolabeling was detected over the extracellular enamel matrix and over the rough endoplasmic reticulum, the saccules of the Golgi apparatus, and the secretory granules of the ameloblasts. In addition, some lysosome-like granules were also labeled. Only background labeling was obtained over mitochondria, nuclei, cytosol, adjacent odontoblasts, and dentin. Quantitation of the intensity of labeling showed the presence of an increasing gradient along the secretory pathway, which may correspond to the concentration or the maturation of these proteins as they are processed by the cell. These findings indicate that the ameloblast displays an intracellular distribution of its secretory products similar to that of other merocrine secreting cells. The presence of enamel proteins in lysosomes suggests that crinophagy and/or resorption occurs in these cells.     

A correlated scanning and transmission electron microscopic study of maturation ameloblasts in developing molar teeth of rats

Summary Maturation ameloblasts of developing molar teeth of the rat were studied by both scanning and transmission electron microscopy. After fixation, teeth were frozen and split. One face of the fractured tooth was used for SEM, the other for TEM.It was found that in some regions proximal junctional complexes separate the interameloblast space from the intercellular space of the papillary layer. Thereby an intercellular ameloblastic compartment is delineated which in some specimens contains a substance interpreted to be colloidal. Elsewhere the proximal junctions of ameloblasts are not present and free communication between the extracellular spaces is evident. The apical pole of ameloblasts varies in structure. Over some areas there is a distinct distal border zone with membranous infoldings which in some regions resembles a striated or ruffled border, but in other regions the membranes show whorl configurations. The distal border zone also contains granules with flocculent material. Elsewhere the ameloblasts display no distal border zone and the cells show a smooth membrane (except for pinocytotic vesicles and hemidesmosomes) facing the enamel surface. The lateral surface of ameloblasts exhibits a variety of surface configurations similar to but not as pronounced as those reported previously in rat incisor maturation ameloblasts.The authors wish to thank Pauletta Sanders and Helen Ruane for technical assistance. This project was supported in part by USPHS NIH Grant DE04059-03 and by the Medical Research Council of Great Britain     

Localization of actin during differentiation of the ameloblast, its related epithelial cells and odontoblasts in the rat incisor using NBD-phallacidin

Using NBD-phallacidin, which specifically binds to F-actin, we investigated changes in the localization of actin during the differentiation of ameloblasts, related epithelial cells and odontoblasts in rat incisors. In cryosections treated with NBD-phallacidin, intense fluorescence was observed in undifferentiated epithelial cells in the apical loop and at the proximal extremity of undifferentiated inner enamel epithelial cells. During differentiation, the distal extremity began to exhibit strong fluorescence. In cross-sections of secretory ameloblasts, the fluorescence took the form of polygons of uniform intensity at the proximal end, and of rectangles of non-uniform intensity at the distal end. At the distal end, the fluorescence was more intense at right angles to the long axis of the incisor. At the distal end, this pattern was established just before the appearance of the enamel layer. These patterns were maintained during the secretory stage of ameloblasts. The location, pattern and time of appearance of these sites were identical to those of the terminal webs in ameloblasts. NBD-phallacidin weakly labelled the peripheral cytoplasm of the cell body of ameloblasts, and also labelled Tomes' process. The cells forming the stratum intermedium were mainly labelled at their periphery (i.e. forming larger polygons), while the overlying epithelial cells exhibited labelling throughout their cytoplasm. Except for the terminal webs, the cell bodies of odontoblasts were weakly labelled throughout the period of differentiation. Young odontoblasts secreting predentin were first labelled on the terminal web, with the fluorescence becoming gradually more intense as the thickness of the dentin increased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fine structural changes and lysosomal phosphatase cytochemistry of ameloblasts associated with the transitional stage of enamel formation in the rat incisor.

Trimetaphosphatase (TMPase) and cytidine-5'-monophosphatase (CMPase) were used as lysosomal markers in the transitional ameloblasts (TA) to investigate the distribution of lysosomal structures and to correlate the cytochemical findings with the ultrastructural features of these cells. Of particular interest were the cytochemical and morphological changes which occur as the ameloblasts approach the maturation stage of enamel formation. The sequence of changes observed provides a basis for designation of three regions of the transitional zone (early and late TA and modulating ameloblasts). In the early TA region, the cells decreased in height and contained phagic vacuoles as well as numerous TMPase and CMPase reactive structures. Late transitional ameloblasts had invaginations at their distal ends as well as membrane-bound structures, both filled with fine granular material. Dense bodies, phagic vacuoles, and other elements of the lysosomal system were enzyme reactive. Modulating ameloblasts lacked the phagic vacuoles but exhibited large numbers of multivesicular bodies, vesicles, and secretory granules. Their distal ends were morphologically altered indicating a change towards ruffle- or smooth-ended varieties of maturation ameloblast. In the former, increased granular material was observed within cell membrane invaginations and associated membrane-bound structures. In the latter, intercellular spaces widened and were filled with granular material. The present cytochemical findings of an extensive lyosomal system in transitional ameloblasts confirm the function of those cells in reducing the secretory ameloblast population and in the selective elimination of their protein-synthesizing organelles. Furthermore, this extensive lysosmal system and the present morphological findings are consistent with a potential role for transitional ameloblasts in contributing to the marked loss of enamel protein known to occur during maturation.     

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.     

Fine structural and immunohistochemical detection of collar enamel in the teeth of <Emphasis Type="Italic">Polypterus senegalus</Emphasis>, an actinopterygian fish

This is the first detailed report about the collar enamel of the teeth of Polypterus senegalus. We have examined the fine structure of the collar enamel and enamel organ of Polypterus during amelogenesis by light and transmission electron microscopy. An immunohistochemical analysis with an antibody against bovine amelogenin, an antiserum against porcine amelogenin and region-specific antibodies or antiserum against the C-terminus, middle region and N-terminus of porcine amelogenin has also been performed to examine the collar enamel matrix present in these teeth. Their ameloblasts contain fully developed Golgi apparatus, rough endoplasmic reticulum and secretory granules. During collar enamel formation, an amorphous fine enamel matrix containing no collagen fibrils is found between the dentin and ameloblast layers. In non-demineralized sections, the collar enamel (500 nm to 1 μm thick) is distinguishable from dentin, because of its higher density and differences in the arrangement of its crystals. The fine structural features of collar enamel in Polypterus are similar to those of tooth enamel in Lepisosteus (gars), coelacanths, lungfish and amphibians. The enamel matrix shows intense immunoreactivity to the antibody and antiserum against mammalian amelogenins and to the middle-region- and C-terminal-specific anti-amelogenin antibodies. These findings suggest that the proteins in the enamel of Polypterus contain domains that closely resemble those of bovine and porcine amelogenins. The enamel matrix, which exhibits positive immunoreactivity to mammalian amelogenins, extends to the cap enameloid surface, implying that amelogenin-like proteins are secreted by ameloblasts as a thin matrix layer that covers the cap enameloid after enameloid maturation.     

THE CYTOLOGY OF THE NORMAL PARATHYROID GLANDS OF MAN AND VIRGINIA DEER : A Light and Electron Microscopic Study with Morphologic Evidence of Secretory Activity

The normal parathyroids of six humans and a Virginia deer were studied by light and electron microscopy. The parenchyma of the deer parathyroid is composed of uniform chief cells, which contained 100 to 400 mµ electron-opaque, membrane-limited granules, presumed to be secretory granules, in addition to the usual cytoplasmic organelles. Desmosomes are present between adjacent cells, and rare cilia are observed protruding from the chief cells into the intercellular space. The human parathyroids contain chief cells in two phases—active and inactive—as well as oxyphil cells. Active chief cells have a large Golgi apparatus, sparse glycogen, numerous secretory granules, and rare cilia. Inactive chief cells contain a small Golgi apparatus, abundant glycogen, and few secretory granules. Both forms have the usual cytoplasmic organelles and, between adjacent cells, desmosomes. Oxyphil cell cytoplasm is composed of tightly packed mitochondria and glycogen granules, with rare secretory granules. Cells with cytoplasmic characteristics intermediate between chief and oxyphil cells, possibly representing transitional cells, have been observed. Secretory granules of both man and deer are composed of 100 to 200 A particles and short rods, and the granules develop from prosecretory granules in the Golgi region of the cell. The human secretory granules are smaller and more variable in shape than those of the deer. The granules are iron and chrome alum hematoxylin-positive, argyrophilic, and aldehyde fuchsin-positive, permitting light microscopic identification. They are also found in the capillary endothelial cells of the parathyroid and in its surrounding connective tissue. The secretory granules of the parathyroid cells can thus be followed from their formation in the Golgi apparatus almost to their extrusion into the blood stream.     

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.     

Fluorescence histochemical observations on catecholamine-containing cell bodies in Auerbach's plexus

Summary The nature and distribution of cell contacts have been examined in the human enamel organ in bell stage. The lateral cell surfaces of secretory ameloblasts are linked at their distal (apical) and proximal (basal) parts by junctional complexes consisting of tight junctions, large intermediate junctions (zonulae adherentes), occasional gap junctions and one or more series of desmosomes. Scattered desmosomes and large gap junctions link epithelial cells of the external enamel epithelium, stellate reticulum, stratum intermedium and internal enamel epithelium including secretory ameloblasts. Furthermore the above-mentioned layers are also linked together by desmosomes and gap junctions.With increasing maturation of the enamel organ an increase in size and number of gap junctions is observed. Some possible implications of the role of the different junctions are considered. The gap junctions probably participate in cell differentiation in the normal morphogenesis of the teeth as well as in metabolic and ionic coupling of the cells of the enamel organ. By means of tight junctions, adjacent secretory ameloblasts cooperate to form a physical barrier which might prevent the diffusion of some types of molecules or substances (e.g. secretory material distally and acid mucopolysaccharides proximally) through the interspaces between the cells. Adhering junctions might assist in regulation of the mechanical properties of the enamel organ as a whole.This work was supported by grants from Statens almindelige Videnskabsfond, Copenhagen, and the Association for the Aid of the Crippled Children, New York.     

Ultrastructural study of tracer permeability through the cat and ferret enamel organ

The access of exogenous materials to the developing enamel surface has been intensively studied in rodents, but not in other mammalian species. This ultrastructural study investigates the permeability of injected horseradish peroxidase (HRP) and lanthanum tracers in cat and ferret tooth buds. In cat enamel organs fixed by immersion, lanthanum did not escape the capillaries overlying secretory stage tooth buds, but it did permeate up to the distal junctions of ruffle-ended (RA) and the proximal junctions of smooth-ended (SA) ameloblasts. Perfusion fixation with lanthanum compromised junctional integrity of cat ameloblasts at all stages of development. Similarly, HRP rarely escaped the capillaries associated with cat secretory stage enamel organs. However, unlike lanthanum, HRP was mostly confined to the vasculature of maturation stage enamel organs in immersion fixed cats at all time intervals examined. In ferrets, HRP penetrated up to, but not beyond, the distal junctional complexes of secretory ameloblasts. In maturation stage enamel organs, HRP coated the papillary and RA cells, but did not penetrate the RA distal cell junctions. HRP did permeate the extracellular spaces of SA to reach the underlying enamel surface. Ameloblasts in transitional phases of SA and RA endocytosed HRP at the distal cell surface. This data leads to several conclusions. First, HRP localization in the ferret paralleled that observed in rodents. Second, the results of cat enamel organs substantiate previous studies showing perfusion fixation can increase vascular and intercellular permeability to lanthanum. However, in cats fixed by immersion, both lanthanum and HRP were restricted to capillaries associated with the secretory stage enamel organ, and only lanthanum escaped maturation stage capillaries. It is suggested that variations in the fenestrations and distribution of capillaries associated with the cat enamel organ may differentially retain some materials and permit other materials to escape with relative ease.     

Ultrastructure of secretory ameloblasts in the house musk shrew, Suncus murinus, Insectivora

Tooth germs from neonatal house musk shrews, Suncus murinus, were used for the study. The tooth morphogenesis was compared electron microscopically to that of Primates. In the tooth germ at the bell stage, the ameloblast was 3 x 50 microns in size, columnar in shape and had several tubular-type Golgi apparatus which were at the distal end of the cell. Most mitochondria were noted at the proximal end of ameloblasts. Tomes' processes were 1 micron in width, protruded 10 microns from the ameloblast and had many dense bodies and two kinds of vesicles. They were morphologically different from human ameloblasts and enamel rods.     

Cytochemical calcium distribution in secretory ameloblasts of the rat in relation to enamel mineralization

Calcium distribution in secretory ameloblasts was studied in rat incisor enamel in which mineralization was temporarily disturbed by injection of either fluoride or cobalt. Pyroantimonate precipitates of calcium were analysed morphometrically in regions of the cell membranes, mitochondria and secretory granules. The disturbances in mineralization were characterized by accumulations of unmineralized enamel matrix at the secretory regions of Tomes' process within 1 h after injection. Fluoride-induced disturbances in mineralization were not accompanied by marked changes in calcium concentration and distribution. It may be that fluoride causes alterations in the synthesis and secretion of the organic matrix which affects its ability to mineralize. Secretory ameloblasts treated with cobalt showed a broad basis for interference with calcium, in particular that which is associated with cell membranes and secretory granules. Secretory ameloblasts may be actively controlling the availability of calcium to enamel by mechanisms involving the cell membrane as well as the secretory granules.     

FURTHER STUDIES ON THE THETA CELL OF THE MOUSE ANTERIOR PITUITARY AS REVEALED BY ELECTRON MICROSCOPY, WITH SPECIAL REFERENCE TO THE MODE OF SECRETION

Theta cells reported previously as a new cell type in the anterior pituitary of the mouse were examined with the electron microscope. This type of cell is distinguished by the presence of pleomorphic secretory granules, a characteristic arrangement of the rough surfaced variety of endoplasmic reticulum, a well developed Golgi complex, and an eccentrically located nucleus. The secretory granules are seen at first as small granules of low density within the Golgi vesicles. While they are within the Golgi vesicles they become larger and denser. Simultaneously they move from the proximal to the distal part of the Golgi region and finally emerge from the Golgi area as mature granules in the cytoplasm. Thus, secretory granules are always enveloped by a limiting membrane which originates from the wall of the Golgi vesicle. At the stage of granule-extrusion, the cell membrane fuses with the limiting membrane of the granules and openings in the cell membrane appear at the place of extrusion. The granules then appear to lie within inpocketings of the cell membrane. They lose their density within these inpocketings or within the cytoplasm and occasionally show fragmentation. After complete loss of density, the granules are extruded as amorphous materials to the territory outside of the cell.     

Urokinase-type Plasminogen Activator mRNA is Expressed in Normal Developing Teeth and Leads to Abnormal Incisor Enamel in αMUPA Transgenic Mice

The urokinase-type plasminogen activator (uPA) is a secreted, inducible serine protease implicated in extracellular proteolysis and tissue remodeling. Here we detected uPA mRNA through in situ hybridization in developing molar and incisor teeth of normal mice at multiple sites of the cap and bell developmental stages. The mRNA was confined to epithelial cells, however, was undetectable in ameloblasts or their progenitor preameloblasts and the inner enamel epithelium. Furthermore, mice of five lines of previously described αMUPA transgenic mice, carrying a transgene consisting of the uPA cDNA linked downstream from the αA-crystallin promoter, overexpressed uPA mRNA in the same epithelial sites. In addition, αMUPA mice showed remarkably high levels of uPA mRNA in ameloblasts, however, exclusively in two specific sites late in incisor development. First, at the late secretory stage, but only on sides of the ameloblast layer. Second, in a limited zone of ameloblasts near the incisal end, coinciding with a striking morphological change of the ameloblast layer and the enamel matrix. In adult αMUPA mice, the incisor teeth displayed discoloration and tip fragility, and reduction of the outer enamel as determined by scanning electron microscopy. These results suggest that balanced uPA activity could play a role in normal tooth development. The αMUPA tooth phenotype demonstrates a remarkable sensitivity to excessive extracellular proteolysis at the incisor maturation stage of amelogenesis.     

Collagen biogenesis and assembly into fibrils as shown by ultrastructural and 3H-proline radioautographic studies on the fibroblasts of the rat food pad

To examine whether collagen is assembled into fibrils within or outside fibroblasts, the connective tissue of the rat foot pad was investigated by electron microscopy and by radioautography at times varying from 4 min to 3 days after an intravenous injection of 3H-proline. The fibroblasts of the rat food pad are long polarized cells with the nucleus at one end, the Golgi apparatus in the center, and a region with long processes at the other end. This region contains secretory granules and is considered to be the secretory pole of the cell. In the Golgi apparatus the stacks of saccules are separated from rough endoplasmic reticulum (rER) by groups of "intermediate vesicles" including similarly structured tubules which may be over 300 nm long and are referred to as "intermediate tubules." The Golgi saccules exhibit distended portions which differ at the various levels of the stack. On the cis side, the distentions tend to be spherical and contain fine looping threads; in the middle of the stack, they are cylindrical and present distinct straight threads; whereas on the trans side, they are again cylindrical, but the straight threads are grouped in parallel aggregates. Between these cylindrical distentions and the secretory granules, there are transitional forms within which thread aggregates are packaged more and more tightly. Finally, the fibroblasts are associated with two types of collagen fibrils: extracellular ones arranged into large groups between the cells and intracellular ones located within long intracytoplasmic channels. Quantitative radioautography after 3H-proline injection reveals that the number of silver grains per unit area reaches a peak over the rER at 4-10 min, Golgi apparatus at 40 min, secretory granules at 60 min, and extracellular collagen fibrils at 3 h. At no time are intracellular collagen fibrils labeled. Qualitative observations further indicate that spherical Golgi distentions are mainly labeled at 40 min, and cylindrical distentions, at 60 min. In addition, from 20 min to 3 hr, some lysosomal elements are labeled. The biogenetic pathway leading to the formation of collagen fibrils is interpreted as follows.(ABSTRACT TRUNCATED AT 400 WORDS)     

Expression of connexin 43 and ZO-1 in differentiating ameloblasts and odontoblasts from rat molar tooth germs

We studied the distribution of connexin (Cx) 43 and ZO-1 by confocal laser scanning microscopy at early stages of dentinogenesis and amelogenesis. Labeling for Cx43 was observed at early stages of differentiation in both the epithelial cells and differentiating odontoblasts. Immunolabeling was detected at the distal and medial regions of undifferentiated ameloblasts and between cells from stratum intermedium and stellate reticulum. Differentiating odontoblasts exhibited immunoreaction for this antibody at their distal end. Immunoreactivity for ZO-1 was observed at regions that correspond to the proximal and distal junctional complexes of differentiating ameloblasts. Staining for ZO-1 was observed at apical regions of odontoblasts with a punctate appearance. In more advanced stages, expression of Cx43 was more evident on ameloblasts, especially at the junctional complexes. Punctate immunolabeling for Cx43 was observed at the lateral sides of differentiating ameloblasts and between the other cells of the enamel organ. Immunoreaction for ZO-1 in ameloblasts was more evident than at the previous stage. It was also observed at the distal end of differentiated odontoblasts. The present study showed that differentiating ameloblasts and odontoblasts express Cx43 and ZO-1 as early as the start of the differentiation process. In addition, the expression of these junctional proteins increases as differentiation of cells continues.     

Stromelysin-1 (MMP-3) in forming enamel and predentine in rat incisor-coordinated distribution with proteoglycans suggests a functional role

Stromelysin-1 (matrix metalloproteinase-3) or proteoglycanase was visualized by light and electron microscopy immunolabelling in the forming zone of rat incisors. In predentine, labelling was more dense at the transition zone between the inner proximal third and the two outer thirds. Odontoblast processes were also positively stained, mostly in predentine and to a lesser degree in dentine. The dentine–enamel junction was intensely labelled, whereas dentine and forming enamel were only faintly stained. Gold–antibodies complexes were seen inside secretory ameloblasts and odontoblasts in cytosolic locations. The distribution of stromelysin-1 was compared with the distribution of 2-B-6 epitope, an antibody recognizing chondroitin-4-sulphate/dermatan sulphate and which showed a decreasing gradient from the proximal zone to the distal part of predentine. In contrast, both 5-D-4, an anti-keratan sulphate antibody and an anti-lumican antibody displayed a reversed distribution, with an increase seen from the proximal and central thirds to the distal part of predentine. This coordinated distribution suggests that stromelysin-1 may have a functional role, being implicated in predentine in the degradation of chondroitin-4-sulphate/dermatan sulphate-containing proteoglycans, and consequently allowing keratan sulphate proteoglycan concentration to increase near the border where mineralization is initiated.     

Stromelysin-1 (MMP-3) in Forming Enamel and Predentine in Rat Incisor – Coordinated Distribution with Proteoglycans Suggests a Functional Role

Stromelysin-1 (matrix metalloproteinase-3) or proteoglycanase was visualized by light and electron microscopy immunolabelling in the forming zone of rat incisors. In predentine, labelling was more dense at the transition zone between the inner proximal third and the two outer thirds. Odontoblast processes were also positively stained, mostly in predentine and to a lesser degree in dentine. The dentine–enamel junction was intensely labelled, whereas dentine and forming enamel were only faintly stained. Gold–antibodies complexes were seen inside secretory ameloblasts and odontoblasts in cytosolic locations. The distribution of stromelysin-1 was compared with the distribution of 2-B-6 epitope, an antibody recognizing chondroitin-4-sulphate/dermatan sulphate and which showed a decreasing gradient from the proximal zone to the distal part of predentine. In contrast, both 5-D-4, an anti-keratan sulphate antibody and an anti-lumican antibody displayed a reversed distribution, with an increase seen from the proximal and central thirds to the distal part of predentine. This coordinated distribution suggests that stromelysin-1 may have a functional role, being implicated in predentine in the degradation of chondroitin-4-sulphate/dermatan sulphate-containing proteoglycans, and consequently allowing keratan sulphate proteoglycan concentration to increase near the border where mineralization is initiated.     

Immunocytochemical and immunochemical study of enamelins, using antibodies against porcine 89-kDa enamelin and its N-terminal synthetic peptide, in porcine tooth germs

Enamelins comprise an important family of the enamel matrix proteins. Porcine tooth germs were investigated immunochemically and immunocytochemically using two antibodies: a polyclonal antibody raised against the porcine 89-kDa enamelin (89 E) and an affinity purified anti-peptide antibody against the porcine enamelin amino-terminus (EN). Immunochemical analysis of layers of immature enamel from the matrix formation stage detected immunopositive protein bands ranging from 10 kDa to 155 kDa in the outer layer enamel sample irrespective of the antibodies used. In contrast, the middle and inner enamel layer mainly contained lower molecular weight enamelins. In immunocytochemical analyses of the differentiation stage, 89 E stained enamel matrix islands around mineralized collagen fibrils of dentin, while EN stained both enamel matrix islands and stippled material. At the matrix formation stage, both antibodies intensely stained enamel prisms located in the outer layer. In the inner layer, 89 E moderately stained enamel matrix homogeneously, while EN primarily stained the prism sheath. The intense immunoreaction over the surface layer of enamel matrix at the matrix formation stage, following staining with 89 E and EN, disappeared by the end of the transition stage and the early maturation stage, respectively. The Golgi apparatus and secretory granules in the ameloblasts from the late differentiation stage to the transition stage were immunostained by both antibodies. These results suggest that expression of enamelin continues from late differentiation to the transition stage and the cleavage of N-terminal region of enamelin occurs soon after secretion. Some enamelin degradation products, which apparently have no affinity for hydroxyapatite crystals, concentrate in the prism sheaths during enamel maturation.