Inhibition of chondroitin sulfate glycosaminoglycans incorporation affected odontoblast differentiation in cultured embryonic mouse molars.

Chondroitin sulfate proteoglycan (CSPG) is an important component of extracellular matrix (ECM), it is composed of a core protein and one or more chondroitin sulfate glycosaminoglycan side chains (CS-GAGs). To investigate the roles of its CS-GAGs in dentinogenesis, the mouse mandibular first molar tooth germs at early bell stage were cultivated with or without ß-xyloside. As expected, the CS-GAGs were inhibited on their incorporation to CSPGs by ß-xyloside, accompanied by the change of morphology of the cultured tooth germs. The histological results and the transmission electron microscopy (TEM) investigation indicated that ß-xyloside exhibited obvious inhibiting effects on odontoblasts differentiation compared with the control group. Meanwhile the results of immunohistochemistry, in situ hybridization and quantitative RT-PCR for type I collagen, dentin matrix acidic phosphoprotein 1 and dentin sialophosphoprotein, the products of differentiated odontoblasts, further proved that odontoblasts differentiation was inhibited. Collagen fibers detected in TEM decreased and arranged in disorder as well. Thus we conclude that the inhibition of CS-GAGs incorporation to CSPGs can affect odontoblast differentiation in cultured embryonic mouse molars.

[Structure of teeth in mature ovarian teratoma].

MATERIAL AND METHODS: The authors investigated three mature ovarian dermoid cysts that were found to contain atypically developed teeth (one tooth in one cyst; 2 teeth in another cyst, and a set of 8 teeth with jawbone fragments in the third one). Their structure was examined using the epoxy resin plastination technique to obtain plastinated sections. RESULTS: Some of these teratomous teeth have obvious signs of alteration in both dentin and enamel, which may be referred to as fluorosis or macular enamel hypoplasia in one case whereas other sections indicate a caries lesion. CONCLUSION: These facts are contradictory to the concept of the exogenous nature of dental caries, which is accepted in dentistry.

Autophagy appears during the development of the mouse lower first molar.

Odontogenesis consists of a series of consecutive tooth morphogenesis stages, in which apoptosis is involved to eliminate the unnecessary cells. Autophagy, a lysosome or endosome-mediated self-degradation process, is indicated to participate in embryogenesis and tissue morphogenesis associated with apoptosis. This study hypothesized that autophagy may be involved and associated with apoptosis in odontogenesis. The transcripts of autophagy-related genes (Atg5, Atg7, and Atg12) were positively detected in tooth germs at embryonic day (E) 14.5 and postnatal day (P) 5.5 by quantitative real-time PCR. The protein expression of Atg5-Atg12 conjugate and lipidation of LC3 (microtubule-associated protein 1 light chain 3, autophagic marker) were revealed in the developing tooth germs by western blot. Meanwhile, LC3 was immunolocalized in the enamel organ and dental papilla at embryonic stages (E13.5-E18.5), especially stage E14.5 cervical loop and the PEK that facing the mesenchyme. At postnatal stages (P1.5-P15.5), besides the dental epithelium cells, LC3 was detected in the differentiating and differentiated odontoblasts, dental follicle cells, and Hertwig's epithelium root sheath cells. Moreover, double-immunofluorescence analysis revealed the partial colocalization of LC3 and TUNEL signal in the E14.5 PEK that facing the mesenchyme, the E16.5 stratum intermedium and outer enamel epithelium, the P5.5 stratum intermedium and stellate reticulum. Nevertheless, LC3 was also found in non-apoptotic cells. Furthermore, the transmission electron microscopic images revealed the presence of autophagy, as well as the partial colocalization of autophagic vacuoles and apoptotic nuclei during tooth development. Our findings imply the developmental appearance of autophagy and its partial colocalization with apoptosis during odontogenesis.

[Histological analysis of dental follicles after unerupted mandibular third molar extraction]. / Analyse histologique des follicules dentaires après germectomies des troisièmes molaires mandibulaires.

INTRODUCTION: The extraction of third mandibular tooth germ (M3) is often prophylactic to avoid orthodontic treatment relapse and to prevent infectious or tumoral diseases developing from the dental sac. The purpose of this study was to screen for early histopathological modification of dental follicles (inflammatory, infiltration, or epithelial metaplasia) after extraction of third mandibular tooth germ (M3) on asymptomatic patients. The secondary objective was to study the proliferative activity of the epithelium by dosing the anti Ki-67 antibody. PATIENTS AND METHOD: Twenty dental follicles extracted from 12 boys and eight girls between 14 and 18 years of age were examined under phototonic microscopy. The proliferative activity of the epithelium was assessed by immuno-histochemistry. RESULTS: Three dental follicles presented with focal epidermoid metaplasia of the epithelium, without odontogenic tumoral proliferation. In all other cases, the cylindrical epithelial cell structure was normal. A mild chronic inflammatory infiltrate was present in 30% of the cases. Immuno-histochemical analysis revealed labeling of very rare epithelial lining cells, slightly more in cases presenting with metaplasia. DISCUSSION: The prevalence of early morphological changes of dental sac is low. This histo-morphological study does not support the systematic extraction of asymptomatic mandibular tooth germs (M3).

[Effect of intrauterine hypoxia upon newborn albino rat tooth germ cells anabolic activity].

The aim of this study was to examine the intrauterine hypoxia influence on dental hard tissue development. Pregnant rats were exposed in hypoxic environments between day 14 and 19 of pregnancy. The study was performed on 36 newborn albino rats. Analysis of nucleolar organizator parameters were performed in enameloblasts, odontoblasts and saliva gland epitheliocytes. Data obtained demonstrated that intrauterine hypoxia decreased nucleolar organizator quantity in enameloblasts of tooth germ.

Gene expression and dental enamel structure in developing mouse incisor.

At the mouse incisor tip the initially differentiated ameloblasts produce a thin, prism-free enamel, while further apically, in the immediate adjacent segment, the enamel thickness increases and the four-layered enamel of mouse incisor is formed. Comparative gene-expression profiling was carried out on RNA isolated from these two segments of incisor tooth germs at embryonic day (E)17.5 and at postnatal days (P)0, 1, 2, and 10 using microarrays to measure messenger RNA (mRNA) and microRNA (miRNA) species present in the segments. Validation of expression data was achieved using real-time reverse transcription-polymerase chain reaction (RT-PCR) and western blotting. Bioinformatic data suggested enhanced cellular apoptosis in the incisal tip segment, which, together with diminished expression of the Amelx and Enam genes, may contribute to the production of the thin enamel seen in this tooth segment. For genes exhibiting higher levels of expression in the adjacent segment where complex enamel is being formed, bioinformatic analysis suggested significant associations with cellular functions involving the actin cytoskeleton, cellular development, morphology, and movement. This is suggested to reflect that ameloblasts with Tomes' process are being organized in transverse rows, facilitating the transverse movement that results in prism decussation in the inner enamel of the adjacent segment. Bioinformatic analysis of miRNA expression data lends support to these suggestions.

Differential gene expression profiling of the molar tooth germ in peroxisome proliferator-activated receptor-alpha (PPAR-alpha) knockout mouse and in wild-type mouse: molar tooth phenotype of PPAR-alpha knockout mouse.

Gene expression profiling of the first molar tooth germ at embryonic days (E)17.5 and 18.5, and at postnatal days (P)0, 2, and 6 from peroxisome proliferator-activated receptor-alpha (PPAR-alpha) knockout mouse and from wild-type mouse was carried out using microarrays and validated using real-time reverse transcription-polymerase chain reaction (RT-PCR) and western blotting. When comparing expression profiles at each time-point, a total of 1,235 genes showed significantly different expression, 772 of which exhibited significantly decreased expression in tooth germ from knockout mouse. With genes exhibiting significantly decreased levels of expression in tooth germ from PPAR-alpha knockout mouse, bioinformatic analysis using ingenuity pathway analysis yielded significant associations to cellular functions related to cellular growth/proliferation and to networks related to regulation of calcium homeostasis. Using scanning electron microscopy to investigate molars from adult PPAR-alpha knockout mouse, the molar size was found to be slightly reduced, the enamel structure was found to be normal, but cervical molar enamel exhibited evidence suggesting hypomineralization. Although the PPAR-alpha knockout had no significant effect on molar morphology, the results suggest that active PPAR-alpha signaling is required to achieve normal mineralization of molar enamel, most probably through regulation of calcium homeostasis and metabolism of vitamin D. Cyp27b1 was expressed in tooth germ, suggesting that tooth germ can synthesize active vitamin D. Expression of Cyp27b1 was significantly enhanced in postnatal PPAR-alpha knockout tooth germ.

Neonatal lines in the enamel of primary teeth--a morphological and scanning electron microscopic investigation.

OBJECTIVE: The neonatal line (NNL) is in principle found in all primary teeth and the line represents the time of birth. Earlier findings of the appearance of the NNL in light microscope and in microradiographs have shown not only changes in the prism direction of the enamel, but that the NNL has a hypomineralized character. METHODS: The neonatal line was analyzed in un-decalcified sections of primary lower and central incisors, collected from individuals of different ages utilizing polarized light microscopy, microradiography, scanning electron microscopy (SEM) and X-ray analysis (XRMA). RESULTS: In polarized light the NNL appeared to have a more porous structure than the enamel in general. The appearance of the NNL as a dark line in microradiographs is interpreted as the NNL being less mineralized than neighbouring enamel. Analysis with ImageJ visualized the reduction of the amount of grey value, indicating that the NNL is less mineralized. Analysis of the NNL in SEM showed a reduction of the diameter of enamel prisms, the more narrow diameters continued through the postnatal enamel. A change of the growth direction of the prisms was also observed at the NNL. In a three-dimensional image the NNL appeared as a grove, however, in non-etched enamel no grove was seen. The elemental analyses with XRMA showed no marked changes in the content of C, Ca, P, N, O or S in the area around the NNL. CONCLUSIONS: The NNL is an optical phenomenon due to alterations in height, and degree of mineralization of the enamel prisms.

Morphological analysis of human and bovine dentine by scanning electron microscope investigation.

OBJECTIVE: The objective of this study was to compare the superficial morphology of bovine and human sclerotic dentine. DESIGN: For the morphological analysis, bovine (n = 3) and human (n = 3) incisors exhibiting exposed dentine were used. Dentine presented characteristics of sclerosis: brownish, smooth and shiny--the vitreous appearance. The teeth were prepared for assessment on a scanning electron microscope (SEM). Three pre-determined areas of each sample were submitted to SEM. The number of open tubules per area was obtained from the electron micrographs (n = 9 per group) for comparison purposes. RESULTS: The number of open tubules in both species compared were similar (p > 0.05). Human dentine presented 31.89 +/- 23.94 open tubules per area, whereas bovine dentine showed 30.33 +/- 18.14 open tubules per area. CONCLUSION: Based on the results, we concluded that dentine exposed at the incisal surface of human and bovine teeth presented similar clinical and micro-morphological aspects, represented by surfaces with equivalent numbers of open dentinal tubules.

Immunocytochemical and biochemical detection of EMMPRIN in the rat tooth germ: differentiation-dependent co-expression with MMPs and co-localization with caveolin-1 in membrane rafts of dental epithelial cells.

In tooth development matrix metalloproteinases (MMPs) are under the control of several regulatory mechanisms including the upregulation of expression by inducers and downregulation by inhibitors. The aim of the present study was to monitor the occurrence and distribution pattern of the extracellular matrix metalloproteinase inducer (EMMPRIN), the metalloproteinases MMP-2 and MT1-MMP and caveolin-1 during the cap and bell stage of rat molar tooth germs by means of immunocytochemistry. Strong EMMPRIN immunoreactivity was detected on the cell membranes of ameloblasts and cells of the stratum intermedium in the bell stage of the enamel organ. Differentiating odontoblasts exhibited intense EMMPRIN immunoreactivity, especially at their distal ends. Caveolin-1 immunoreactivity was evident in cells of the internal enamel epithelium and in ameloblasts. Double immunofluorescence studies revealed a focal co-localization between caveolin-1 and EMMPRIN in ameloblastic cells. Finally, western blotting experiments demonstrated the expression of EMMPRIN and caveolin-1 in dental epithelial cells (HAT-7 cells). A substantial part of EMMPRIN was detected in the detergent-insoluble caveolin-1-containing low-density raft membrane fraction of HAT-7 cells suggesting a partial localization within lipid rafts. The differentiation-dependent co-expression of MMPs with EMMPRIN in the enamel organ and in odontoblasts indicates that EMMPRIN takes part in the induction of proteolytic enzymes in the rat tooth germ. The localization of EMMPRIN in membrane rafts provides a basis for further investigations on the role of caveolin-1 in EMMPRIN-mediated signal transduction cascades in ameloblasts.

Diverse effects of c-src deficiency on molar tooth development and eruption in mice.

C-src deficiency is characterized by osteopetrosis due to impaired bone resorption by hypofunctional osteoclasts and the resultant failure of tooth eruption. In preliminary observations, we frequently encountered erupted molars in c-src deficient mice unlike in other osteopetrotic animals. Here we examine the effects of c-src deficiency on the development of molar teeth with an emphasis on the spatial relation of growing teeth with the surrounding bones. In c-src deficient mice, the magnitude of tooth impaction differed considerably among the types of molars; all maxillary 1st molars were totally impacted deep in the alveolar sockets, whereas most mandibular 1st molars fully erupted into oral cavity. Distribution of osteoclasts in the alveolar bone was identical among all types of molars, and electron microscopy revealed signs of bone resorbing activity in these osteoclasts despite the absence of a ruffled border. From early development, the alveolar space was much narrower in the upper molar tooth germs than in the lower ones in both wild type and homozygous animals, and particularly so in the upper 1st molars. Current observations thus indicate a significant contribution of "hypofunctional osteoclasts" in c-src deficient mice in molar tooth development except for the upper 1st molars, which appear to require highly functional osteoclasts to gain sufficient space for them to grow normally. Taken together, these findings on the seemingly tooth-type specific effects of c-src deficiency on the development and eruption of molar teeth in c-src deficient mice can be attributed to the given differential spatial relation of the respective tooth germs with the surrounding bones in the presence of hypofunctional osteoclasts.

Formation of a successional dental lamina in the zebrafish (Danio rerio): support for a local control of replacement tooth initiation.

In order to test whether the formation of a replacement tooth bud in a continuously replacing dentition is linked to the functional state of the tooth predecessor, I examined the timing of development of replacement teeth with respect to their functional predecessors in the pharyngeal dentition of the zebrafish. Observations based on serial semithin sections of ten specimens, ranging in age from four week old juveniles to adults, indicate that (i) a replacement tooth germ develops at the distal end of an epithelial structure, called the successional dental lamina, budding off from the crypt epithelium surrounding the erupted part of a functional tooth; (ii) there appears to be a developmental link between the eruption of a tooth and the formation of a successional dental lamina and (iii) there can be a time difference between successional lamina formation and initiation of the new tooth germ, i.e., the successional dental lamina can remain quiescent for some time. The data suggest that the formation of a successional lamina and the differentiation of a replacement tooth germ from this lamina, are two distinct phases of a process and possibly under a different control. The strong spatio-temporal coincidence of eruption of a tooth and development of a successional dental lamina is seen as evidence for a local control over tooth replacement.

Anisotropic properties of the enamel organic extracellular matrix.

Enamel biosynthesis is initiated by the secretion, processing, and self-assembly of a complex mixture of proteins. This supramolecular ensemble controls the nucleation of the crystalline mineral phase. The detection of anisotropic properties by polarizing microscopy has been extensively used to detect macromolecular organizations in ordinary histological sections. The aim of this work was to study the birefringence of enamel organic matrix during the development of rat molar and incisor teeth. Incisor and molar teeth of rats were fixed in 2% paraformaldehyde/0.5% glutaraldehyde in 0.2 M phosphate-buffered saline (PBS), pH 7.2, and decalcified in 5% nitric acid/4% formaldehyde. After paraffin embedding, 5-microm-thick sections were obtained, treated with xylene, and hydrated. Form birefringence curves were obtained after measuring optical retardations in imbibing media, with different refractive indices. Our observations showed that enamel organic matrix of rat incisor and molar teeth is strongly birefringent, presenting an ordered supramolecular structure. The birefringence starts during the early secretion phase and disappears at the maturation phase. The analysis of enamel organic matrix birefringence may be used to detect the effects of genetic and environmental factors on the supramolecular orientation of enamel matrix and their effects on the structure of mature enamel.

Developmental movements of the inner enamel epithelium as derived from micromorphological features.

It was the purpose of this article to analyze the (micro) morphological structure of enamel at different stages of development in order to deduce movement patterns of ameloblasts during formation of the human dental primordium. Developing enamel and overlying ameloblasts were dried and fractured for scanning electron microscopy (SEM) and sectioned for transmission electron microscopy (TEM). Specimens of human permanent enamel were either fractured and/or ground and etched to visualize the enamel rods. All specimens were viewed by SEM. Moreover, three-dimensional reconstructions were made from serial ground sections of enamel blocks to follow the enamel rods for a longer distance. In addition, the outline of the dentino-enamel junction was analyzed under the SEM after removal (using nitric acid) of the enamel cap, and in serial histological sections. Two basic movements of the inner enamel epithelium can be derived from the micromorphological features: (i) the scalloped dentino-enamel junction may be a consequence of a bulged inner enamel epithelium owing to initial spatial impediment; and (ii) the undulating path of the enamel rods may be a consequence of unequal growth of the cells in the cervical loop.

Immunocytochemical study of amelogenin deposition during the early odontogenesis of molars in alendronate-treated newborn rats.

Newborn rats were treated with sodium alendronate to study how enamel is formed and the effect of alendronate during early odontogenesis. Ultrastructural analysis combined with high-resolution immunocytochemistry for amelogenin was carried out. Twelve rats were subjected to daily SC injections of sodium alendronate (2.5 mg/kg/day) for 3 days on their dorsal region, whereas three rats were daily injected with saline solution as a control. Molar tooth germs from 3-day-old rats were fixed under microwave irradiation in 0.1% glutaraldehyde + 4% formaldehyde buffered at pH 7.2 with 0.1 M sodium cacodylate. The specimens were left undecalcified, postfixed with osmium tetroxide, dehydrated, and embedded in LR White resin. Ultrathin sections were incubated with a chicken anti-24-kDa rat amelogenin antibody, a secondary antibody, and finally with a protein A-gold complex. Large patches of amelogenin were present over the unmineralized mantle dentin and at early secretory ameloblasts. At more advanced stages, they were also detected at the enamel matrix, as well as in the mineralized dentin, at the periodontoblastic space of the dentinal tubules, and at the predentin. It is likely that the main effect of alendronate at early stages of odontogenesis is the increase of synthesis/secretion of amelogenin, promoting its deposition within the forming dentin and enamel.

Collagen analysis in human tooth germ papillae.

The extracellular matrix (ECM) performs a very important role in growth regulation and tissue differentiation and organization. In view of this, the purpose of this study was to analyze the collagen, the major organic component of dental pulp ECM, in papillae of human tooth germs in different developmental phases. The maxillas and mandibles of 9 human fetuses ranging from 10 to 22 weeks of intrauterine life were removed and 16 tooth germs (1 in the cap stage, 8 in the early bell stage and 7 in the late bell stage) were obtained. The pieces were processed for histological analysis and stained with hematoxylin-eosin, Masson's Trichrome and picrosirius staining technique. Both types of collagen in the dental papilla were only detected by the picrosirius staining technique under polarized light microscopy. Type III collagen was detected in all specimens. Type I collagen was present in focal areas of the dental papilla only in some specimens. In conclusion, the findings of this study showed that type III collagen is a regular component of the papillae of human tooth germs whereas type I collagen is present in a significantly lesser amount.

Collagen analysis in human tooth germ papillae

The extracellular matrix (ECM) performs a very important role in growth regulation and tissue differentiation and organization. In view of this, the purpose of this study was to analyze the collagen, the major organic component of dental pulp ECM, in papillae of human tooth germs in different developmental phases. The maxillas and mandibles of 9 human fetuses ranging from 10 to 22 weeks of intrauterine life were removed and 16 tooth germs (1 in the cap stage, 8 in the early bell stage and 7 in the late bell stage) were obtained. The pieces were processed for histological analysis and stained with hematoxylin-eosin, Masson's Trichrome and picrosirius staining technique. Both types of collagen in the dental papilla were only detected by the picrosirius staining technique under polarized light microscopy. Type III collagen was detected in all specimens. Type I collagen was present in focal areas of the dental papilla only in some specimens. In conclusion, the findings of this study showed that type III collagen is a regular component of the papillae of human tooth germs whereas type I collagen is present in a significantly lesser amount.

A matriz extracelular (MEC) tem um papel importante na regulação do crescimento e na diferenciação e organização dos tecidos. Com base nestes aspectos o objetivo do deste estudo foi analisar o colágeno, maior componente orgânico da MEC da polpa dentária, na papila de germes dentários humanos, em diferentes fases do desenvolvimento. Foram obtidos fragmentos de maxilas e mandíbulas de 9 fetos humanos com 10 a 22 semanas de vida intra-uterina, dos quais foram analisados 16 germes dentários (1 em estágio de capuz, 8 em estágio de campânula precoce e 7 em estágio de campânula tardia). Secções histológicas seriadas foram coradas com hematoxilina e eosina, tricrômico de Masson e técnica de coloração do picrosirius. Ambos os tipos de colágeno na papila dentária foram somente detectados pela técnica de coloração do picrosirius usando microscopia de luz polarizada. Colágeno tipo III foi detectado em todas as amostras. Colágeno tipo I estava presente em áreas focais da papila dental em algumas amostras. Concluiu-se que o colágeno tipo III mostrou-se um componente regular da papila de germes dentários humanos, enquanto o colágeno tipo I esteve presente em quantidade significativamente menor.

Tooth development in vitro in two teleost fish, the cichlid Hemichromis bimaculatus and the cyprinid Danio rerio.

A technique for organotypic in vitro culture with serum-free medium was tested for its appropriateness to mimic normal odontogenesis in the cichlid fish Hemichromis bimaculatus and the zebrafish Danio rerio. Serial semithin sections were observed by light microscopy to collect data on tooth patterning and transmission electron microscopy was used to compare cellular and extracellular features of tooth germs developing in vitro with the situation in vivo. Head explants of H. bimaculatus from 120 h post-fertilization (hPF) to 8.5 days post-fertilization (dPF) and of zebrafish from 45 hPF to 79 hPF and adults kept in culture for 3, 4 or 7 days revealed that tooth germs developed in vitro from explants in which the buccal or pharyngeal epithelium was apparently undifferentiated and, when present at the time of explantation, they continued their development up to a stage of attachment. In addition, the medium allowed the morphogenesis and cytodifferentiation of the tooth germs similar to that observed in vivo and the establishment of a dental pattern (place and order of tooth appearance and of attachment) that mimicked that in vivo. Organotypic culture in serum-free conditions thus provides us with the means of studying epithelial-mesenchymal interactions during tooth development in teleost fish and of analysing the genetic control of either mandibular or pharyngeal tooth development and replacement in these polyphyodont species. Importantly, it allows heads from embryonically lethal (zebrafish) mutants or from early lethal knockdown experiments to develop beyond the point at which the embryos normally die. Such organotypic culture in serum-free conditions could therefore become a powerful tool in developmental studies and open new perspectives for craniofacial research.

Rat enamel contains RP59: a new context for a protein from osteogenic and haematopoietic precursor cells.

We have recently identified a protein, RP59, in bone marrow cells and young osteoblasts, in cells involved in bone repair and in young erythroblasts and megakaryocytes. Here, we report immunohistochemical data at the light- and electron-microscope level indicating that RP59 is also present in newly secreted tooth enamel of the rat and in ameloblasts, the formative cells. In enamel matrix, RP59 was located proximal to secretory ameloblasts only, i.e. in newly secreted material. Distal enamel and enamel in association with maturation stage ameloblasts were unlabelled. Secretory ameloblasts contained RP59 in the matrix-proximal region including Tomes' processes, post-secretory ameloblasts in the cell-matrix interface. Western blotting of proteins from tooth germs identified RP59 as a band at 90 kD, co-migrating with RP59 from bone marrow and spleen. Antisera versus a chemically synthesised RP59 peptide and versus a bacteria-synthesised protein fragment reacted in the same manner. In situ hybridisation of tooth tissue revealed RP59 RNA specifically in ameloblasts. The reverse transcription/polymerase chain reaction method identified tooth RNA coding for RP59. Sequence analysis indicated that RP59 RNA from tooth and marrow had the same sequence. An internal sequence motif was found in rat RP59 resembling a signal implicated in secretion of the chicken "engrailed" gene product. The findings indicate that RP59 is a genuine product of ameloblasts and that it is secreted in the course of enamel formation together with other matrix components.

Interactions of amelogenins with octacalcium phosphate crystal faces are dose dependent.

Amelogenins, the major protein components of the enamel extracellular matrix, are postulated to be involved in controlling the elongated and oriented growth of enamel carbonated apatite crystals. In order to clarify the functional role of amelogenin during the early stage of enamel biomineralization, octacalcium phosphate (OCP) crystals, known to be potent precursors of hydroxyapatite, were grown in 1-10% (w/w) native bovine and two recombinant murine amelogenins. Amelogenins were solution-like at 1% and formed gel at 10%, while 5% amelogenins became gel after reaction and it was inhomogeneous and porous. Morphological changes of OCP crystals were evaluated as the function of amelogenin concentration by analyzing the mean values of length, width, thickness, their reduction ratios (L/Lc, W/Wc, T/Tc) as well as L/W and W/T ratios. Length, width, and thickness decreased in a does-dependent manner. Length decreased almost linearly in 1%-10%, whereas width decreased drastically in 1%-5% while the decrease from 5% to 10% was small. As a result, elongated morphology of OCP crystal was most emphasized in 5% bovine amelogenins and rM166 and 2%-5% rM179. The size reduction was in the order of W/Wc < L/Lc < T/Tc. We therefore concluded that amelogenin interaction with crystal faces was in the order (010) > (001) > (100). At all concentrations, W/ Wc was significantly the smallest. This indicated that the primary role of amelogenin was to decrease the width of OCP by blocking the hydrophobic (010) faces. We suggest that the drastic decrease of crystal width is the result of interaction of the densely packed nanospheres in 5%-10% amelogenin.