Production of recombinant human ameloblastin by a fully native purification pathway.

Ameloblastin (Ambn) is an intrinsically disordered protein (IDP) with a specific function of forming heterogenous homooligomers. The oligomeric function is led through a specific sequence encoded by exon 5 of Ambn. Due to the IDP character of Ambn to form oligomers, protein purification is subject to many challenges. Human ameloblastin (AMBN) and its two isoforms, I and II have already been purified as a recombinant protein in a bacterial expression system and functionally characterized in vitro. However, here we present a new purification protocol for the production of native AMBN in its original formation as a homooligomeric heterogeneous IDP. The purification process consists of three chromatographic steps utilizing His-tag and Twin Strep-tag affinity chromatography, along with size exclusion and reverse affinity chromatography. The presented workflow offers the production of AMBN in sufficient yield for in vitro protein characterizations and can be used to produce both AMBN isoforms I and II.

FAM20C plays an essential role in the formation of murine teeth.

FAM20C is highly expressed in bone and tooth. Previously, we showed that Fam20C conditional knock-out (KO) mice manifest hypophosphatemic rickets, which highlights the crucial roles of this molecule in promoting bone formation and mediating phosphate homeostasis. In this study, we characterized the dentin, enamel, and cementum of Sox2-Cre-mediated Fam20C KO mice. The KO mice exhibited small malformed teeth, severe enamel defects, very thin dentin, less cementum than normal, and overall hypomineralization in the dental mineralized tissues. In situ hybridization and immunohistochemistry analyses revealed remarkable down-regulation of dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein in odontoblasts, along with a sharply reduced expression of ameloblastin and amelotin in ameloblasts. Collectively, these data indicate that FAM20C is essential to the differentiation and mineralization of dental tissues through the regulation of molecules critical to the differentiation of tooth-formative cells.

Characterization of rat apical tissues in different root development stage.

In this study, we try to compare the histological characteristics and the odontogenic capability of apical tissues (AT) at different root development stages of rat molar teeth. AT of mandibular first molars from 8-day-old, 21-day-old, and 35-day-old Sprague-Dawley rats were selected as being representative of root-initiating, root-forming, and root-completing stages, respectively. Cell counting, flow cytometry assays, alkaline phosphatase activity, alizarin red staining, and reverse transcription polymerase chain reaction were performed to assess the proliferation and mineralization potential of apical tissue cells at different stages of root development in vitro. In vivo transplantation of apical tissue cells combined with ceramic bovine bone was used to characterize the differentiation capacity. It was shown that there was a structurally and functionally dynamic change in the apical tissue of developing tooth root of rats, of which the unique developmental potential will reduce gradually with the ending up of root development. The AT of root-initiating and root-forming stage exhibited much higher proliferation and tissue-regenerative capacity than those of root-completing stage. Our present results indicate that the apical tissue, with the sustainable developmental ability throughout almost the whole process of tooth development, can yet be regarded as a competent candidate source for root/periodontal tissues regeneration.

Biphasic influence of hypoxia on tuftelin expression in mouse mesenchymal C3H10T1/2 stem cells.

Tuftelin, an acidic protein, thought to play a role in the initial stages of ectodermal enamel mineralization, has since been detected in mesenchymal-derived tissues. During bone/cartilage development and regeneration, mesenchymal stem cells (MSCs) undergo an avascular period in a hypoxic environment, involving induction of hypoxia-inducible factor 1-alpha (HIF-1-alpha), a key component in this process. In the present study we investigated, in a mouse mesenchymal C3H10T1/2 stem cell model, the hypothesis that oxygen stress modulates tuftelin 1 expression in relation to HIF-1-alpha (Hif1a), in a mouse mesenchymal C3H10T1/2 stem cell model. The results of the present study showed a biphasic induction of tuftelin, similar to the pattern of HIF-1-alpha expression, in MSCs subjected to a hypoxic insult of 1% O(2) through a period of 2-24 h. Immunocytochemistry analysis of the cells exposed to hypoxic insult for 2-24 h revealed the same biphasic pattern of tuftelin protein expression. Tuftelin localization appears to be mainly in the cytoplasm, and concentrated at the perinuclear region of the cells by 24 h of hypoxic insult. Based on our previous studies using the neuronal PC12 cell model, in which tuftelin induction was mediated by Hif1a, we propose that tuftelin is a member of oxygen-sensitive genes and implicated in the adaptive mechanisms regulating MSC function.

Epithelial-specific knockout of the Rac1 gene leads to enamel defects.

The Ras-related C3 botulinum toxin substrate 1 (Rac1) gene encodes a 21-kDa GTP-binding protein belonging to the RAS superfamily. RAS members play important roles in controlling focal adhesion complex formation and cytoskeleton contraction, activities with consequences for cell growth, adhesion, migration, and differentiation. To examine the role(s) played by RAC1 protein in cell-matrix interactions and enamel matrix biomineralization, we used the Cre/loxP binary recombination system to characterize the expression of enamel matrix proteins and enamel formation in Rac1 knockout mice (Rac1(-/-)). Mating between mice bearing the floxed Rac1 allele and mice bearing a cytokeratin 14-Cre transgene generated mice in which Rac1 was absent from epithelial organs. Enamel of the Rac1 conditional knockout mouse was characterized by light microscopy, backscattered electron imaging in the scanning electron microscope, microcomputed tomography, and histochemistry. Enamel matrix protein expression was analyzed by western blotting. Major findings showed that the Tomes' processes of Rac1(-/-) ameloblasts lose contact with the forming enamel matrix in unerupted teeth, the amounts of amelogenin and ameloblastin are reduced in Rac1(-/-) ameloblasts, and after eruption, the enamel from Rac1(-/-) mice displays severe structural defects with a complete loss of enamel. These results support an essential role for RAC1 in the dental epithelium involving cell-matrix interactions and matrix biomineralization.

Molecular and circadian controls of ameloblasts.

Stage-specific expression of ameloblast-specific genes is controlled by differential expression of transcription factors. In addition, ameloblasts follow daily rhythms in their main activities (i.e. enamel protein secretion and enamel mineralization). This time-related control is orchestrated by oscillations of clock proteins involved in the regulation of circadian rhythms. Our aim was to identify the potential links between daily rhythms and developmental controls of ameloblast differentiation. The effects of the transcription factors distal-less homeobox 3 (Dlx3) and runt-related transcription factor 2 (Runx2), and the clock gene nuclear receptor subfamily 1, group D, member 1 (Nr1d1), on secretory and maturation ameloblasts [using stage-specific markers amelogenin (Amelx), enamelin (Enam), and kallikrein-related peptidase 4 (Klk4)] were evaluated in the HAT-7 ameloblast cell line. Amelx and Enam steady-state mRNA expression levels were down-regulated in Runx2 over-expressing cells and up-regulated in Dlx3 over-expressing cells. In contrast, Klk4 mRNA was up-regulated by both Dlx3 and Runx2. Furthermore, a temporal and spatial relationship between clock genes and ameloblast differentiation markers was detected. Of interest, clock genes not only affected rhythmic expression of ameloblast-specific genes but also influenced the expression of Runx2. Multiscale mathematical modeling is being explored to further understand the temporal and developmental controls of ameloblast differentiation. Our study provides novel insights into the regulatory mechanisms sustaining ameloblast differentiation.

Differentiation of mouse embryonic stem cells into dental epithelial-like cells induced by ameloblasts serum-free conditioned medium.

Embryonic stem cells (ESCs) possess an intrinsic self-renewal ability and can differentiate into numerous types of functional tissue cells; however, whether ESCs can differentiate toward the odontogenic lineage is still unknown. In this study, we developed an efficient culture strategy to induce the differentiation of murine ESCs (mESCs) into dental epithelial cells. By culturing mESCs in ameloblasts serum-free conditioned medium (ASF-CM), we could induce their differentiation toward dental epithelial cell lineages; however, similar experiments with the tooth germ cell-conditioned medium (TGC-CM) did not yield effective results. After culturing the cells for 14days in the differentiation-inducing media, the expression of ameloblast-specific proteins such as cytokeratin (CK)14, ameloblastin (AMBN), and amelogenin (AMGN) was markedly higher in mESCs obtained with embryoid body (EB) formation than in mESCs obtained without EB formation. We observed that immunocompromised mice implanted with induced murine EBs (mEBs) showed tissue regenerative capacity and produced odontogenic epithelial-like structures, whereas those implanted with mSCE monolayer cells mainly formed connective tissues. Thus, for the first time, we report that ASF-CM provides a suitable microenvironment for inducing mESC differentiation along the odontogenic epithelial cell lineage. This result has important implications for tooth tissue engineering.

Expression pattern of odontogenic ameloblast-associated and amelotin during formation and regeneration of the junctional epithelium.

The junctional epithelium (JE) adheres to the tooth surface, and seals off periodontal tissues from the oral environment. This incompletely differentiated epithelium is formed initially by the fusion of the reduced enamel organ with the oral epithelium (OE). Two proteins, odontogenic ameloblast-associated (ODAM) and amelotin (AMTN), have been identified in the JE. The objective of this study was to evaluate their expression pattern during formation and regeneration of the JE. Cytokeratin 14 was used as a differentiation marker for oral epithelial cells, and Ki67 for cell proliferation. Immunohistochemistry was carried out on erupting rat molars, and in regenerating JE following gingivectomy. In the reducing enamel organ and in established JE, ODAM and AMTN were present at the cell-tooth interface while only ODAM and CK14 were found throughout the JE. Both were also conspicuously present in cell clusters situated between the erupting tooth and OE. During JE regeneration, ODAM was detected first at the leading wound edge and then in the regenerating JE. Some cell clusters in the subjacent connective tissue were also positive for ODAM. AMTN appeared later and both AMTN and ODAM accumulated at the interface with the tooth. Cytokeratin 14 gradually appeared in the regenerating JE but the cell clusters showed variable labeling. Cells associated with JE formation and regeneration exhibited higher division activity than adjacent epithelial cells. These findings suggest that ODAM and AMTN have a role at the cell-tooth interface, and that ODAM is likely also implicated in cellular events during formation and regeneration of the JE.

Cytomegalovirus induces stage-dependent enamel defects and misexpression of amelogenin, enamelin and dentin sialophosphoprotein in developing mouse molars.

Of the approximately 8,400 children born each year in the US with cytomegalovirus (CMV)-induced birth defects, more than one third exhibit hypoplasia and hypocalcification of tooth enamel. Our prior studies indicated that CMV severely delayed, but did not completely interrupt, early mouse mandibular first molar morphogenesis in vitro. The aim of the present study was to examine the effects of CMV infection on progressive tooth differentiation and amelogenesis. Since initial CMV infection in human fetuses can occur at different developmental times, we varied the stage of initial viral infection (that is, Cap stage, Early Bell stage and Bell stage), as well as the duration of infection. CMV infection of embryonic mouse mandibular first molars in vitro induces tooth dysmorphogenesis and enamel defects in a developmental stage- and duration-dependent manner. Cap stage- and Early Bell stage-infected molars exhibit enamel agenesis and Bell stage-infected molars exhibit enamel hypoplasia. This viral-induced pathology is coincident with stage-dependent changes in Amelx, Enam and Dspp gene expression, distribution of amelogenin, enamelin and DSP proteins, cell proliferation localization and dedifferentiation of secretory ameloblasts. Importantly, our data indicate that specific levels of Amelx and Dspp gene expression define whether mouse CMV induces enamel agenesis or hypoplasia.

Human cementoblasts express enamel-associated molecules in vitro and in vivo.

BACKGROUND AND OBJECTIVE: Cementum is a mineralized tissue that facilitates the attachment of periodontal ligament to the root and surrounding alveolar bone and plays a key role in the regeneration of periodontal tissues. The molecular mechanisms that regulate the proliferation and differentiation of cementoblasts, however, have not been elucidated to date. Enamel molecules are believed to regulate cementoblast differentiation and to initiate the formation of acellular extrinsic fiber cementum. The purpose of this study was therefore to isolate and culture human root-derived cells (HRDC) in order to determine whether they are able to express both cementum and specific enamel proteins and subsequently to confirm these findings in vivo. MATERIAL AND METHODS: Human root-derived cells were isolated and expanded in vitro. Cells were characterized using RT-PCR, immunostaining, western blotting and by examination of total mRNA to determine the expression of cementum and enamel markers. Human periodontal tissues were also examined for the expression of enamel-related proteins by immunostaining. RESULTS: We showed that HRDC express mRNA corresponding to ameloblastin (AMBN), amelogenin (AMEL), enamelin (ENAM), tuftelin (TUFT) and cementum-associated molecules such as cementum protein 1 (CEMP1) and cementum attachment protein (CAP). Western blotting revealed that HRDC express both AMEL and AMBN gene products, as well as the cementum markers CEMP1 and CAP. In vivo, we have showed that AMBN and AMEL are expressed by cementoblasts lining cementum, paravascular cells and periodontal ligament cells. CONCLUSION: These results suggest that enamel-associated and cementum-associated proteins could act synergistically in regulating cementoblast differentiation and cementum deposition and offer new approaches on how the cementogenesis process is regulated.

Disruption of periodontal integrity induces expression of apin by epithelial cell rests of Malassez.

BACKGROUND AND OBJECTIVE: It has been suggested that epithelial cell rests of Malassez (ERM) may express enamel matrix proteins and play an important role in periodontal regeneration. Two novel proteins, apin (APIN) and amelotin (AMTN), produced by maturation-stage ameloblasts and junctional epithelium, have recently been identified. The objective of this study was to evaluate whether the ERM express APIN and AMTN under normal conditions and after periodontal challenge. MATERIAL AND METHODS: Gingivectomy and orthodontic tooth movement were carried out on the left side of the maxillae of rats. The control group included the untreated contralateral side of these animals and the maxillae of normal, untreated rats. Animals were sacrificed by intracardiac perfusion on days 3 and 5 after the experimental procedures and maxillary molars were decalcified and processed for paraffin embedding. Immunohistochemistry was used to evaluate the expression of various ameloblast products, including APIN, AMTN, ameloblastin (AMBN) and amelogenin (AMEL). RESULTS: At 3 and 5 days after periodontal challenge, ERM were more evident in the periodontal ligament along the root surface and in the root furcations. Immunodetection of APIN, but not of the other three proteins, was observed in the ERM following the disruption of periodontal integrity. No immunolabeling for APIN, AMTN, AMBN and AMEL was detected in the ERM under normal conditions. CONCLUSION: The expression of APIN at an early time-point following disruption of periodontal integrity suggests that this protein may be part of the cascade of events leading to the activation of ERM during periodontal healing and regeneration.

Tooth induction in chick epithelium: expression of quiescent genes for enamel synthesis.

Intraocular grafts of chick epithelium combined with mouse molar mesenchyme produced a variety of dental structures including perfectly formed crowns with differentiated ameloblasts depositing enamel matrix. The results suggest that the loss of teeth in Aves did not result from a loss of genetic coding for enamel synthesis in the oral epithelium but from an alteration in the tissue interactions requisite for odontogenesis.

Distal cis-regulatory elements are required for tissue-specific expression of enamelin (Enam).

Enamel formation is orchestrated by the sequential expression of genes encoding enamel matrix proteins; however, the mechanisms sustaining the spatio-temporal order of gene transcription during amelogenesis are poorly understood. The aim of this study was to characterize the cis-regulatory sequences necessary for normal expression of enamelin (Enam). Several enamelin transcription regulatory regions, showing high sequence homology among species, were identified. DNA constructs containing 5.2 or 3.9 kb regions upstream of the enamelin translation initiation site were linked to a LacZ reporter and used to generate transgenic mice. Only the 5.2-Enam-LacZ construct was sufficient to recapitulate the endogenous pattern of enamelin tooth-specific expression. The 3.9-Enam-LacZ transgenic lines showed no expression in dental cells, but ectopic beta-galactosidase activity was detected in osteoblasts. Potential transcription factor-binding sites were identified that may be important in controlling enamelin basal promoter activity and in conferring enamelin tissue-specific expression. Our study provides new insights into regulatory mechanisms governing enamelin expression.

[Construction of the stable cell line expressing the mouse recombinant enamelin gene].

OBJECTIVE: To construct a mouse recombinant enamelin eukaryocyte expression system, and establish the stable cell line which can produce the protein continuously. METHODS: The mRNA transcript from the 3-day mouse jaw was extracted. and the enamelin gene fragment amplified with RT-PCR techniques. Then the PCR product was cat with two restriction enzymes, and subcloned into the eukaryotic gene expression vector pcDNA3.1TM/mycj His(-)B.The recombinant plasmid was transformed into E.coli DH5alpha bacterial cells, and harvested with plasmid midi kit. The recombinant expression plasmid was transferred to the HEK 293A eukaryocyte cells, cultured selectively with 800 mg/L G418, and examined with SDS-PAGE and Western Blot at the protein level. RESULTS: The mouse enamelin gene was cloned to the eukaryotic expression plasmid successfully by sequence measuring. After the recombinant plasmid was transferred into the HEK 293A cells, about 32,000 enamelin protein was checked out by SDS-PAGE and Western Blot. CONCLUSION: The recombinant eukaryocyte expression plasmid and the stable cell line were established. This is a basic research to obtain high-yeild biologically active enamelin protein, which may facilitate further investigation of its function.

Enamel dysplasia with hamartomatous atypical follicular hyperplasia (EDHFH) syndrome: suggested pathogenic mechanisms.

The syndrome of enamel dysplasia with hamartomatous atypical follicular hyperplasia (EDHFH) is an unusual syndrome and is unique to black South Africans. Major criteria for the syndrome are enamel dysplasia with generalized amelogenesis imperfecta-like features and atypical hyperplastic dental follicles with microscopic features of central odontogenic fibroma WHO-type (follicle analogue) attached to the crowns of multiple impacted teeth. Minor features of some cases are anterior open-bite malocclusion, supernumerary teeth, pulpal calcification, aberrant roots with hypercementosis, and hypodontia. The pathogenic mechanisms that lead to the development of EDHFH are unknown. We speculate that faulty synthesis of enamel matrix proteins may interfere with enamel formation and play a role in the generalized enamel hypoplasia described in this syndrome. Alterations in inductive signalling by the odontogenic epithelium mediated by enamel matrix proteins may explain the development of the follicle analogues, the root hypercementosis and the presence of dysplastic cementum deposition juxtaposed to odontogenic epithelium in the gingival overgrowth. Thus, alterations in the function of enamel matrix protein function, may be the common denominator responsible for the development of the EDHFH phenotype.

The potential use of enamel matrix derivative for in situ anterior cruciate ligament tissue engineering: a translational in vitro investigation.

Polyester scaffolds have been used as an alternative to autogenous tissues for the reconstruction of the anterior cruciate ligament (ACL). They are biocompatible and encourage tissue infiltration, leading to neoligament formation. However, rupture can occur, caused by abrasion of the scaffold against the bone tunnels through which it is implanted. Good early tissue induction is therefore considered essential to protect the scaffold from this abrasion. Enamel matrix derivative (EMD) is used clinically in the treatment of periodontal disease. It is a complex mix of proteins with growth factor-like activity, which enhances periodontal ligament fibroblast attachment, proliferation, and differentiation, leading to the regeneration of periodontal bone and ligament tissues. We hypothesized that EMD might, in a similar manner, enhance tissue induction around scaffolds used in ACL reconstruction. This preliminary investigation adopted a translational approach, modelling in vitro 3 possible clinical modes of EMD administration, to ascertain the suitability of each protocol for application in an animal model or clinically. Preliminary investigations in monolayer culture indicated that EMD had a significant dose-dependent stimulatory effect (p < 0.05, n = 6) on the proliferation of bovine primary synovial cells. However, pre-treating culture plates with EMD significantly inhibited cell attachment (p < 0.01, n = 6). EMD's effects on synovial cells, seeded onto ligament scaffolds, were then investigated in several in vitro experiments modelling 3 possible modes for clinical EMD administration (pre-, intra-, and post-operative). In the pre-operative model, EMD was adsorbed onto scaffolds before the addition of cells. In the intra-operative model, EMD and cells were added simultaneously to scaffolds in the culture medium. In the post-operative model, cells were pre-seeded onto scaffolds before EMD was administered. EMD significantly inhibited cell adhesion in the pre-operative model (p < 0.05, n = 6) and had no significant benefit in the intra-operative model. In the post-operative model, the addition of EMD to previously cell-seeded scaffolds significantly increased their total deoxyribonucleic acid content (p < 0.01, n = 5). EMD's stimulative effect on cell proliferation in vitro suggests that it may accelerate scaffold colonization by cells (and in turn tissue induction) in situ. However, its inhibitory effect on synovial cell attachment in vitro implies that it may only be suited to post-operative administration.

[Immunohistochemical localization of enamelin in developing rat tooth germ].

OBJECTIVE: To observe the immunohistochemical localization of enamelin in enamel formationand mineralization. METHODS: Tissue sections of the first mandibular molar tooth germ from 1, 3, 7, 10, 14 days rats after birth were prepared, expression of the enamelin protein was identified by immunohistochemical technique. RESULTS: Enamelin was found in the cytoplasm of ameloblasts in 1-10 days old rat postnatal first mandibular molar tooth germs. Enamelin appeared weakly in the tooth germs of 1 day rats. From 3 to 10 days, enamelin localized both in the cytoplasm of ameloblasts and the uncalcified enamel from the dentino-enamel junction to surfaces of the tooth. Enamelin protein was negative in the tooth germs of 14 days rats postnatally. CONCLUSION: Enamelin protein is synthesised and secreted by ameloblasts, specially localized in enamel from DEJ to surfaces of the tooth, suggesting that enamelin has important roles in enamel formation.

Fibromodulin-deficient mice display impaired collagen fibrillogenesis in predentin as well as altered dentin mineralization and enamel formation.

To determine the functions of fibromodulin (Fmod), a small leucine-rich keratan sulfate proteoglycan in tooth formation, we investigated the distribution of Fmod in dental tissues by immunohistochemistry and characterized the dental phenotype of 1-day-old Fmod-deficient mice using light and transmission electron microscopy. Immunohistochemistry was also used to compare the relative protein expression of dentin sialoprotein (DSP), dentin matrix protein-1 (DMP 1), bone sialoprotein (BSP), and osteopontin (OPN) between Fmod-deficient mice and wild-type mice. In normal mice and rats, Fmod immunostaining was mostly detected in the distal cell bodies of odontoblasts and in the stratum intermedium and was weaker in odontoblast processes and predentin. The absence of Fmod impaired dentin mineralization, increased the diameter of the collagen fibrils throughout the whole predentin, and delayed enamel formation. Immunohistochemistry provides evidence for compensatory mechanisms in Fmod-deficient mice. Staining for DSP and OPN was decreased in molars, whereas DMP 1 and BSP were enhanced. In the incisors, labeling for DSP, DMP 1, and BSP was strongly increased in the pulp and odontoblasts, whereas OPN staining was decreased. Positive staining was also seen for DMP 1 and BSP in secretory ameloblasts. Together these studies indicate that Fmod restricts collagen fibrillogenesis in predentin while promoting dentin mineralization and the early stages of enamel formation.

Cloning, sequencing, and expression of the amelogenin gene in two scincid lizards.

Our knowledge of the gene coding for amelogenin, the major enamel protein, is mainly based on mammalian sequences. Only two sequences are available in reptiles. To know whether the snake sequence is representative of the amelogenin condition in squamates, we have studied amelogenin in two scincid lizards. Lizard amelogenin possesses numerous conserved residues in the N- and C-terminal regions, but its central region is highly variable, even when compared with the snake sequence. This rapid evolution rate indicates that a single squamate sequence was not representative, and that comparative studies of reptilian amelogenins might be useful to detect the residues which are really important for amelogenin structure and function. Reptilian and mammalian enamel structure is roughly similar, but no data support amelogenin being similarly expressed during amelogenesis. By performing in situ hybridization using a specific probe, we showed that lizard ameloblasts express amelogenin as described during mammalian amelogenesis. However, we have not found amelogenin transcripts in odontoblasts. This indicates that full-length amelogenin is specific to enamel matrix, at least in this lizard.

Ameloblastin and amelogenin expression in posnatal developing mouse molars.

Ameloblastin and amelogenin are structural proteins present in the enamel matrix of developing teeth. Here we report the results of in situ hybridization analyses with DNA probes of ameloblastin and amelogenin expression in the mandibular first molars of ICR/Jcl mice from postnatal day 1 to day 15. Ameloblastin mRNA expression was observed in ameloblasts at day 2 while amelogenin mRNA was detected in secretory ameloblasts at day 3. Significant expression of both molecules was observed at days 4, 5 and 6, after which the levels decreased. Amelogenin expression ended on day 10, while ameloblastin mRNA was only weakly detected on day 12. Neither amelogenin nor ameloblastin expression was observed in day 15 mouse molars. Amelogenin and ameloblastin mRNAs were restricted to ameloblasts. We conclude that amelogenin and ameloblastin expression is enamel-specific, and suggest that these genes might be involved in the mineralization of enamel. It is possible that ameloblastin could participate in the attachment of ameloblasts to the enamel surface. In this case, the downregulation of expression may indicate the beginning of the maturation stage in which the ameloblasts tend to detach from the enamel layer.