On the pattern of ameloblast migration

Abstract The kinetics of ameloblast cells in continuously growing guinea pig molars were studied using autoradiography. The results showed that there was no direct relationship between ameloblast migration rate and ameloblast production rate, which indicated that ameloblasts actively migrated coronally. It was found that ameloblast migration rate was maximal at the root apex, and then reduced to a minimum value as the ameloblasts left their proliferative compartment and migrated coronally. A multiple regression model was found to be the most suitable one to represent the ameloblast migration pattern.     

Carbonic anhydrase II regulates differentiation of ameloblasts via intracellular pH‐dependent JNK signaling pathway

Differentiation of ameloblasts from undifferentiated epithelial cells is controlled by diverse growth factors, as well as interactions between epithelium and mesenchyme. However, there is a considerable lack of knowledge regarding the precise mechanisms that control ameloblast differentiation and enamel biomineralization. We found that the expression level of carbonic anhydrase II (CAII) is strongly up‐regulated in parallel with differentiation of enamel epithelium tissues, while the enzyme activity of CA was also increased along with differentiation in ameloblast primary cultures. The expression level of amelogenin, a marker of secretory‐stage ameloblasts, was enhanced by ethoxzolamide (EZA), a CA inhibitor, as well as CAII antisense (CAIIAS), whereas the expression of enamel matrix serine proteinase‐1 (EMSP‐1), a marker for maturation‐stage ameloblasts, was suppressed by both. These agents also promoted ameloblast proliferation. In addition, inhibition of ameloblast differentiation by EZA and CAIIAS was confirmed using tooth germ organ cultures. Furthermore, EZA and CAIIAS elevated intracellular pH in ameloblasts, while experimental decreases in intracellular pH abolished the effect of CAIIAS on ameloblasts and triggered the activation of c‐Jun N‐terminal kinase (JNK). SP600125, a JNK inhibitor, abrogated the response of ameloblasts to an experimental decrease in intracellular pH, while the inhibition of JNK also impaired ameloblast differentiation. These results suggest a novel role for CAII during amelogenesis, that is, controlling the differentiation of ameloblasts. Regulation of intracellular pH, followed by activation of the JNK signaling pathway, may be responsible for the effects of CAII on ameloblasts. J. Cell. Physiol. 225: 709–719, 2010. © 2010 Wiley‐Liss, Inc.     

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.     

Localization of transcription factor AP-1 family proteins in ameloblast nuclei of the rat incisor.

We examined by immunocytochemistry the localization of the AP-1 family proteins c-Jun, JunB, JunD, c-Fos, FosB, Fra-1, and Fra-2 in rat incisor ameloblasts. Most of the antibodies against AP-1 family proteins, except for c-Fos-specific antibody, labeled ameloblast nuclei. The labeling intensity of the c-Jun, JunD, and Fra-2 antibodies was stronger than that of JunB, FosB, and Fra-1. Antibody reactivities of c-Jun, JunD, and Fra-2 were greatly enhanced during or after the transition zone. Furthermore, c-Jun antibodies labeled maturation ameloblasts in a cyclic pattern, which was correlated with ameloblast modulation. Disruption of ameloblast modulation by colchicine injection resulted in greatly decreased reactivity of the c-Jun antibody in the ameloblast nuclei of the maturation zone. Phospho-specific antibodies to c-Jun labeled ameloblast nuclei only weakly throughout the secretion, transition, and maturation zones. These results suggest that the stage-specific localization of AP-1 in ameloblasts is closely related to tooth enamel formation.     

Effect of Vinblastine On the Cell Cycle and Migration of Ameloblasts of Mouse Incisors As Shown By Autoradiography Using 3H-Thymidine

Abstract. 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 μg/g of body weight vinblastine intraperitoneally and 6 hr after these animals and those of a control group were injected with 1μCi/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 μ/hr respectively) as compared with the control (3.21 cell positions/hr and 18.88 μm/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.     

Ameloblast differentiation in the human developing tooth: Effects of extracellular matrices

Tooth enamel is formed by epithelially-derived cells called ameloblasts, while the pulp dentin complex is formed by the dental mesenchyme. These tissues differentiate with reciprocal signaling interactions to form a mature tooth. In this study we have characterized ameloblast differentiation in human developing incisors, and have further investigated the role of extracellular matrix proteins on ameloblast differentiation. Histological and immunohistochemical analyses showed that in the human tooth, the basement membrane separating the early developing dental epithelium and mesenchyme was lost shortly before dentin deposition was initiated, prior to enamel matrix secretion. Presecretary ameloblasts elongated as they came into contact with the dentin matrix, and then shortened to become secretory ameloblasts. In situ hybridization showed that the presecretory stage of odontoblasts started to express type I collagen mRNA, and also briefly expressed amelogenin mRNA. This was followed by upregulation of amelogenin mRNA expression in secretory ameloblasts. In vitro, amelogenin expression was upregulated in ameloblast lineage cells cultured in Matrigel, and was further up-regulated when these cells/Matrigel were co-cultured with dental pulp cells. Co-culture also up-regulated type I collagen expression by the dental pulp cells. Type I collagen coated culture dishes promoted a more elongated ameloblast lineage cell morphology and enhanced cell adhesion via integrin α2β1. Taken together, these results suggest that the basement membrane proteins and signals from underlying mesenchymal cells coordinate to initiate differentiation of preameloblasts and regulate type I collagen expression by odontoblasts. Type I collagen in the dentin matrix then anchors the presecretary ameloblasts as they further differentiate to secretory cells. These studies show the critical roles of the extracellular matrix proteins in ameloblast differentiation.     

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.     

Distribution of calcium-ATPase in developing teeth of embryonic American alligators (Alligator mississippiensis)

Light microscopic and ultrastructural observations were carried out to evaluate the cell morphology and histochemistry (calcium-ATPase activity) of developing teeth in embryonic American alligators (Alligator mississippiensis). Ca-ATPase activity was observed in the distal and lateral cell membranes, rough endoplasmic reticulum (rER), mitochondria, vacuoles, and other organelles of the ameloblast, but only in the distal cell membrane and process of the odontoblast. Enzyme activity in the ameloblasts increased gradually during development. These sites of enzyme activity are related to mineralization of the enamel layer, similar to that in mammalian tooth development. Alligator teeth are heavily mineralized like mammalian teeth; however, alligator ameloblasts have undeveloped distal processes during mineralization in contrast to mammalian ameloblasts in which Tomes' processes are found near the distal portion of ameloblasts at maturation stage. The localization of intense enzyme activity in the distal and lateral ameloblast cell membrane suggests that these regions are the site of accumulation of calcium as enamel differentiates in the developing tooth. © 1993 Wiley-Liss, Inc.     

Fine structure of differentiating ameloblasts in the kitten.

The fine structure of differentiating ameloblasts was studied in the lower second molar of 1-week-old kittens after perfusion fixation with and without subsequent decalcification. The differentiation zone was divided into three phases. In Differentiation 1, ameloblasts are about 27 mum long and face an uninterrupted basal lamina. The predentin adjacent to the basal lamina contains a few collagen fibrils oriented mainly at right angles to the ameloblast surface. The specialized predentin forms a well-defined layer, up to 1.5 mum thick, referred to as the junctional layer. In Differentiation 2, ameloblast processes extend through the basal lamina and the thickness of the junctional layer. The processes consist of cytoplasmic sheets forming a honeycomb-like network. Dentin starts to calcify after process-formation is underway. Two distinct types of odontoblast processes, having different shapes and contents, come in contact with the ameloblasts and push into the ameloblastic layer. In Differentiation 3, stippled material appears in the extracellular spaces between ameloblasts. Later, stippled material-like substances appear in the predentin close to the ameloblast apex and close to odontoblast processes within the dentin. Ameloblasts now are up to 40 mum high. Enamel secretion starts in small circumscribed areas which gradually enlarge, leading to the disappearance of the ameloblast processes. These findings are compared with results obtained in other species, including man, and their possible functional significance is discussed.     

CHIR-99021与FGF8协同诱导人表皮干细胞牙向分化

人表皮干细胞(human keratinocyte stem cells, hKSCs)可作为上皮源性的成体干细胞应用于牙齿再生,但是其诱导效率较低.本研究利用小分子化合物CHIR-99021提高hKSCs的Wnt/β-catenin信号活性,再与具有诱导成牙潜能的小鼠牙胚间充质重组,构建嵌合体,并移植裸鼠肾囊膜下培养20 d. 将嵌合体组织切片,并利用组织染色和免疫组化等方法鉴定牙齿结构. 结果显示,经FGF8诱导处理的hKSCs与小鼠牙胚间充质构成的嵌合体的成牙率为27.80%,其中成釉率仅为40.00%;经CHIR 99021诱导处理的hKSCs与小鼠牙胚间充质构成的嵌合体的成牙率仅为18.20%,其中成釉率高达100%;而CHIR 99021与FGF8协同作用,则进一步提高嵌合体成牙率至40.00%,其中成釉率也达75.00%. 进一步的研究发现,经CHIR-99021处理后,hKSCs的Wnt/β-catenin信号活性明显提高,同时FGF8的表达水平也显著上调. 以上结果表明,CHIR-99021可通过上调Wnt/β-catenin信号活性水平,同时促进FGF8表达,与FGF8协同,高效诱导hKSCs分化为具有分泌釉质功能的成釉质细胞. 研究结果对利用hKSCs作为上皮来源的成体细胞应用于人类牙齿再生的研究具有重要意义.     

Ameloblast apoptosis and IGF-1 receptor expression in the continuously erupting rat incisor model

Enamel-producing cells (ameloblasts) pass through several phenotypic and functional stages during enamel formation. In the transition between secretory and maturation stages, about one quarter of the ameloblasts suddenly undergo apoptosis. We have studied this phenomenon using the continuously erupting rat incisor model. A special feature of this model is that all stages of ameloblast differentiation are presented within a single longitudinal section of the developing tooth. This permits investigation of the temporal sequence of gene and growth factor receptor expression during ameloblast differentiation and apoptosis. We describe the light and electron microscopic morphology of ameloblast apoptosis and the pattern of insulin-like growth factor-1 receptor expression by ameloblasts in the continuously erupting rat incisor model. In the developing rat incisor, ameloblast apoptosis is associated with downregulated expression of the insulin-like growth factor-1 receptor. These data are consistent with the hypothesis that ameloblasts are hard wired for apoptosis and that insulin-like growth factor-1 receptor expression is required to block the default apoptotic pathway. Possible mechanisms of insulin-like growth factor-1 inhibition of ameloblast apoptosis are presented. The rat incisor model may be useful in studies of physiological apoptosis as it presents apoptosis in a predictable pattern in adult tissues.     

Odontogenic ameloblasts-associated protein (ODAM), via phosphorylation by bone morphogenetic protein receptor type IB (BMPR-IB), is implicated in ameloblast differentiation

To elucidate the function of the odontogenic ameloblast-associated protein (ODAM) in ameloblasts, we identified more than 74 proteins that interact with ODAM using protoarray. Of the identified proteins, bone morphogenetic protein receptor type-IB (BMPR-IB) was physiologically relevant in differentiating ameloblasts. ODAM and BMPR-IB exhibited similar patterns of expression in vitro, during ameloblast differentiation. ODAM and BMPR-IB interacted through the C-terminus of ODAM, which resulted in increased ODAM phosphorylation in the presence of bone morphogenetic protein 2 (BMP-2). Immunoprecipitation assays using Ser-Xaa-Glu (SXE) mutants of ODAM demonstrated that the phosphorylation of ODAM by BMPR-IB occurs at this motif, and this phosphorylation is required for the activation of MAPKs. ODAM phosphorylation was detected in ameloblasts during ameloblast differentiation and enamel mineralization in vitro and involved in the activation of downstream factors of MAPKs. Therefore, the BMP-2-BMPR-IB-ODAM-MAPK signaling cascade has important roles in ameloblast differentiation and enamel mineralization. Our data suggest that ODAM facilitates the progression of tooth development in cooperation with BMPR-IB through distinct domains of ODAM.     

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.     

Maturation ameloblasts of the porcine tooth germ do not express amelogenin

 Amelogenins are the most abundant constituent in the enamel matrix of developing teeth. Recent investigations of rodent incisors and molar tooth germs revealed that amelogenins are expressed not only in secretory ameloblasts but also in maturation ameloblasts, although in relatively low levels. In this study, we investigated expression of amelogenin in the maturation stage of porcine tooth germs by in situ hybridization and immunocytochemistry. Amelogenin mRNA was intensely expressed in ameloblasts from the differentiation to the transition stages, but was not detected in maturation stage ameloblasts. C-terminal specific anti-amelogenin antiserum, which only reacts with nascent amelogenin molecules, stained ameloblasts from the differentiation to the transition stages. This antiserum also stained the surface layer of immature enamel at the same stages. At the maturation stage, no immunoreactivity was found within the ameloblasts or the immature enamel. These results indicate that, in porcine tooth germs, maturation ameloblasts do not express amelogenins, suggesting that newly secreted enamel matrix proteins from the maturation ameloblast are not essential to enamel maturation occurring at the maturation stage. Accepted: 14 January 1999     

Freeze-fracture replicas of rat ameloblasts reveal unusual membrane domains around gap junctions

Specimens of aldehyde-fixed and glycerol-impregnated tooth germs obtained from 1-2 day old rats were prepared for ultrathin section studies and for freeze-fracture, with the purpose of studying the structural organization of membranes of developing ameloblasts. In this report we describe unusual membrane domains which were found surrounding several ameloblast gap junctions. Developing ameloblasts - when examined in ultrathin sections - exhibit gap junctions which appear straight, curved or invaginated. In freeze-fracture replicas, in addition to their typical appearance, several gap junctions were found to be surrounded by a membrane margin which was undulating and devoid of intramembrane particles (IMP's). We believe that these hitherto unreported particle-free membrane margins are associated with the formation of curved or invaginated gap junctions. It is possible that these membrane margins are particle-free because plasma membrane proteins (presumably IMP's) become transiently detached from the cytoskeleton and move laterally. It is therefore likely that these margins are pure lipid domains which are more flexible, thus providing a transient hinge-like mechanism which facilitates the movement required for the formation of the curved or invaginated ameloblast gap junctions.     

A survey of carbonic anhydrase mRNA expression in enamel cells

Enamel formation requires rigid control of pH homeostasis during all stages of development to prevent disruptions to crystal growth. The acceleration of the generation of bicarbonate by carbonic anhydrases (CA) has been suggested as one of the pathways used by ameloblasts cells to regulate extracellular pH yet only two isozymes (CA II and CA VI) have been reported to date during enamel formation. The mammalian CA family contains 16 different isoforms of which 13 are enzymatically active. We have conducted a systematic screening by RT-PCR on the expression of all known CA isoforms in mouse enamel organ epithelium (EOE) cells dissected from new born, in secretory ameloblasts derived from 7-day-old animals, and in the LS8 ameloblast cell line. Results show that all CA isoforms are expressed by EOE/ameloblast cells in vivo. The most highly expressed are the catalytic isozymes CA II, VI, IX, and XIII, and the acatalytic CA XI isoform. Only minor differences were found in CA expression levels between 1-day EOE cells and 7-day-old secretory-stage ameloblasts, whereas LS8 cells expressed fewer CA isoforms than both of these. The broad expression of CAs by ameloblasts reported here contributes to our understanding of pH homeostasis during enamel development and demonstrates its complexity. Our results also highlight the critical role that regulation of pH plays during the development of enamel.     

Mitochondrial migration and Ca-ATPase modulation in secretory ameloblasts of fasted and calcium-loaded rats

Migration of mitochondria and modulation of Ca-ATPase activity in secretory ameloblasts were investigated ultrastructurally and ultracytochemically by using lower incisors taken from normally fed, 30-hr-fasted, and calcium (Ca)-loaded rats. In normally fed rats, almost all mitochondria were localized in a narrow infranuclear compartment between the nucleus and proximal cell webs of secretory ameloblasts. In 30-hr-fasted rats, a prominent migration of many mitochondria into the supranuclear region of the cells was noted. Mitochondria returned to the infranuclear compartment and seldom appeared in the supranuclear region when fasted rats were Ca-loaded by transcardiac perfusion with physiological Ca solution. Normally, the mitochondria of secretory ameloblast exhibited moderate Ca-ATPase activity along their inner membranes. This mitochondrial Ca-ATPase was decreased by a 30-hr fast and became prominent again after Ca loading. Plasma-membrane Ca-ATPase was demonstrated in the entire cell surface of secretory ameloblasts. An especially abundant reaction was found along the invaginated cell surface of the Tomes process. This Ca-ATPase also became very weak and was almost abolished from the Tomes process after fasting, but Ca loading caused reappearance of an intense Ca-ATPase activity on the entire cell surface, including along Tomes's processes. These results suggest that 1) mitochondrial localization in secretory ameloblasts is influenced by the Ca concentration of the extracellular milieu, and 2) the level of mitochondrial and cell-membrane ATPase activity is responsive to the concentration of extracellular calcium.     

Follistatin regulates enamel patterning in mouse incisors by asymmetrically inhibiting BMP signaling and ameloblast differentiation

Rodent incisors are covered by enamel only on their labial side. This asymmetric distribution of enamel is instrumental to making the cutting edge sharp. Enamel matrix is secreted by ameloblasts derived from dental epithelium. Here we show that overexpression of follistatin in the dental epithelium inhibits ameloblast differentiation in transgenic mouse incisors, whereas in follistatin knockout mice, ameloblasts differentiate ectopically on the lingual enamel-free surface. Consistent with this, in wild-type mice, follistatin was continuously expressed in the lingual dental epithelium but downregulated in the labial epithelium. Experiments on cultured tooth explants indicated that follistatin inhibits the ameloblast-inducing activity of BMP4 from the underlying mesenchymal odontoblasts and that follistatin expression is induced by activin from the surrounding dental follicle. Hence, ameloblast differentiation is regulated by antagonistic actions of BMP4 and activin A from two mesenchymal cell layers flanking the dental epithelium, and asymmetrically expressed follistatin regulates the labial-lingual patterning of enamel formation.     

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.     

Establishment of primary cultures for mouse ameloblasts as a model of their lifetime

To understand how the properties of ameloblasts are spatiotemporally regulated during amelogenesis, two primary cultures of ameloblasts in different stages of differentiation were established from mouse enamel epithelium. Mouse primary ameloblasts (MPAs) prepared from immature enamel epithelium (MPA-I) could proliferate, whereas those from mature enamel epithelium (MPA-M) could not. MPA-M but not MPA-I caused apoptosis during culture. The mRNA expression of amelogenin, a marker of immature ameloblasts, was down-regulated, and that of enamel matrix serine proteiase-1, a marker of mature ameloblasts, was induced in MPA-I during culture. Using green fluorescence protein as a reporter, a visualized reporter system was established to analyze the promoter activity of the amelogenin gene. The region between -1102bp and -261bp was required for the reporter expression in MPA-I. These results suggest that MPAs are valuable in vitro models for investigation of ameloblast biology, and that the visualized system is useful for promoter analysis in MPAs.