Which cells form enamel

It seems likely that some of these genes are critically important for normal enamel formation and will be implicated in hereditary defects of enamel in the future. In addition to the many genetic defects of enamel formation there are known to be a similar number of environmental etiologies about producing enamel phenotypes.

Collectively, the environmental and genetic etiologies of enamel defects result in a high prevalence. Enamel hypoplasia is known to be a predisposing factor for the development of dental caries and conditions such as molar incisor hypomineralization contribute to the high morbidity of first permanent molars. The third talk in this session explored stemness, lineage commitment and developmental potential in the enamel organ.

To address the commitment and lineage differentiation potential of the four cell layers of the enamel organ, 41 Tom Diekwisch and his team conducted studies to explain their fate and differentiation potential. The stratum intermedium transiently expresses the epithelial stem cell marker p63, supportive of a potential role as an enamel organ stem cell layer that may provide a reservoir for ameloblast renewal.

The stellate reticulum is linked to the papillary layer of the eruption stage tooth organ via keratin immunostaining. The papillary layer provides an important cell layer to facilitate infection-free tooth eruption. Finally, the outer enamel epithelium gives rise to the outer layer of Hertwig's Epithelial Root Sheath in mammals, while in reptiles, the outer enamel layer directly continues with the general lamina responsible for continuous successional tooth organ growth.

Diekwisch therefore highlighted that the developing enamel organ is a multifunctional, complex cell assembly, in which different cell layers co-develop and synergize to serve various functions during enamel development, tooth organ succession, and tooth eruption.

Beta-catenin forms distinct transcriptional complexes in differentiating versus proliferating cells, enabling it to activate different sets of target genes. The third session was opened by Pamela DenBesten University of California, San Francisco, CA, USA , who explored how ameloblast differentiation requires stage specific, and yet to be identified, factors in the dental mesenchyme.

Tooth loss due to genetic causes, trauma, caries or periodontal disease continues to be major health issue for both adults and children. Although dental implants have resulted in improved strategies to replace missing teeth, implants are a less optimal solution as compared to the ultimate goal of tooth regeneration.

Key strategies for tooth regeneration were identified in the classic studies in mice conducted by Kollar and co-workers, who showed that either the early dental epithelium combined with non-dental mesenchyme, or the later differentiating bud-stage dental mesenchyme combined with non-dental epithelium, could result in tooth formation. DenBesten et al. They found that similar to the previously reported mouse studies, co-culture of the human epithelial cells with the human bud-stage dental mesenchyme resulted in the formation of tooth like structures.

Co-culture of human epithelial cells with mature dental pulp from erupted human teeth formed acinar type epithelial structures, similar to those found when ameloblast lineage cells were cultured alone.

From this perspective, good model systems recapitulate human enamel performance for example, protection of dentin from wear over the lifetime of a human, formation of structurally similar caries lesions 59 , properties such as wear resistance, toughness, and resistance to acid dissolution , hierarchical structure including 3D weave of rods, arrangement of crystallites, presence of an amorphous intergranular phase and ultimately the crystal growth process.

While some data are available, a comprehensive view of how different model systems murine, porcine, canine, bovine and human differ in these interrelated areas is lacking, which makes comparisons difficult.

In addition, there is also a lack of data on the heterogeneity of enamel within one tooth, between different teeth in the same individual, and between equivalent teeth in one species. While it is often held that continuously growing incisors are poor model systems for human teeth, Joester suggested that understanding developmental and functional differences between continuously erupting teeth and those that erupt just once remains an important topic of fundamental research.

Joester further discussed how imaging at the atomic scale, using atom probe tomography, reveals clues to amelogenesis and enamel function. Specifically, the existence of a relatively more soluble Mg-rich amorphous intergranular phase AIGP at the boundaries between enamel crystallites in rodent 60 and human enamel 61 appears to be linked to the susceptibility of enamel to biofilm-derived and ingested acids.

Even within this AIGP, organics, carbonate and possibly water show distinct distribution patterns with important implications for the resistance to acid corrosion, mechanical properties, and the mechanism by which enamel crystals grow during amelogenesis. At least in the rodent model, Mg is excluded from the growing crystal, which means that one would expect there to be an elevated concentration of it right at the interface between the mineral and the surrounding aqueous phase.

The AIGP is likely dynamic in structure and composition, however, these dynamics are an underexplored field at this time. Finally, knowledge of the AIGP and its biogenesis may help engineer teeth that are chemically and or mechanically more robust.

From an engineering point of view, the ability to accelerate tooth growth, and the impact of high growth rates on enamel defects will become of central importance in the future. Janet Oldak University of Southern California, Los Angeles, CA, USA presented recent advances in understanding molecular interactions in the enamel matrix, and how these can move us towards the development of biomimetic strategies for synthetic enamel. Because dental enamel does not regenerate itself, efforts to develop improved biomaterials with mechanical and esthetic attributes close to those of natural enamel are timely and justified.

The dominant proteins in the developing enamel matrix are amelogenin, ameloblastin, amelotin and enamelin, and while all have a significant impact on the resulting enamel, 69 amelogenin and splice variants with HAP are the most studied. Overall it is a beautifully balanced system that is easily disrupted. Continued challenges include understanding structure-function relationships which is exacerbated by the intrinsically disordered nature and the prevalence to self-assemble, and understanding the timing and importance of different protein-protein interactions.

Also a challenge is that quantitative structural and interfacial studies with atomic resolution can only be down ex situ , but over-simplifies the complexity of the developing enamel environment. Understanding the role of proteins in ion movement, and measuring transient structures of the proteins are also future challenges. The most commonly used mouse line for conditional gene deletion in the enamel organ is the Kcre line. The limitation of this line is that it deletes genes in all layers of the enamel organ: ameloblasts, stratum intermedium and papillary layer.

In order to understand the cellular complexity of the organ that produces enamel, it would be essential to target gene deletion in subpopulations of cells in the enamel organ.

The AMELX- cre line is readily available and can be used to target gene deletion specifically in ameloblasts from the secretory stage onwards.

However, the restricted expression of Alpl in the stratum intermedium 87 gives potential for the development of Alpl-creERT mice that could be used for inducible deletion of genes in this compartment of the enamel organ. Characterizing the transcriptome of each layer of the enamel organ would help identify novel markers that are restricted to subcompartments of the enamel organ and develop genetic tools to dissect the function of each cell type in the process of enamel formation.

Duverger also discussed how enamel scientists can learn from other organs. Amelogenesis imperfecta is a rare monogenic disorder that can be found in non-syndromic and syndromic forms. The other organs affected in syndromic forms of AI include the skin, the kidney, the lungs, the eyes, the brain, and the immune system, among others. When studying genes involved in the pathogenicity of these forms of AI , understanding their function in the other affected organs can help elucidate their role in amelogenesis.

The day ended with a group discussion focused on how to move the enamel field forward and more efficiently translate research findings to clinical applications. Even though tremendous progress has been made in the last century in our understanding of the genetic, physiologic and developmental mechanisms that drive the secretion and maturation of enamel, many crucial questions remain unanswered. What makes ameloblasts migrate in a coordinated way that leads to enamel rod decussation?

What is the involvement of the other cells that make up the enamel organ? What are the key determinants in the ion transport function of ameloblasts at each stage? Addressing these questions is an essential prerequisite to regenerating enamel in an ex vivo setting. Workshop participants were in general agreement that a major hurdle to progress is the need for heightened interaction among biologists and materials scientists.

Enamel research is a multidisciplinary field, and one idea that was discussed was a platform where data and resources could be shared in a more efficient way. Participants also agreed that additional animal models would be useful to deepen our understanding of enamel formation in vivo. In addition, current ameloblast-like cell lines are limited in their replication cycles in culture, their ability to consistently generate typical enamel-like matrices, and their origin from different cell types of the enamel organ.

Thus, the generation of improved ameloblast-like cell lines will enable scientists to exploit the ability of tissue-specific cells to form enamel-like extracellular matrices and facilitate faithful enamel crystal growth. Finally, the potential for organoid and organ on a chip technology in the future is quite exciting. Thus, there is enormous potential for progress in the field of enamel biology through increased interdisciplinary collaboration and improved approaches.

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Isl1 controls patterning and mineralization of enamel in the continuously renewing mouse incisor. J Bone Miner Res ; epub ahead of print 26 June ; doi: Ras signaling regulates stem cells and amelogenesis in the mouse incisor. It is worthy to note that often several claudin family members are co-expressed and interact with each other to regulate the tight junction structure in epithelial cells.

These stem cells were maintained as previously reported, and their differentiation into epithelial cells ES-ECs was induced and characterized according to the established protocols [ 68 , 69 ]. These cells served as controls for microarray analyses. Fetal buccal mucosa and maxillae were isolated and embedded in O.

M Laser Dissecting Microscope Zeiss. Oral buccal mucosal epithelial cells OE were collected from the cheek tissues. Presecretory ameloblasts PABs were identified as the single layer of columnar epithelial cells directly bordering with the dentin matrix.

Secretory ameloblasts SABs were identified as the elongated and polarized epithelial cells adjoining to the organic enamel matrix shown in Fig. The robust multichip average RMA method was used for background adjustment, quantile normalization, and median polish summarization using Affymetrix Expression Console Software. To determine the relative expression level of the target gene, the comparative CT threshold cycle method was used [ 74 ].

Kohwi-Shigematsu et al. The hemimandibles were then processed, embedded, and sectioned sagittally. To detect the biotin-labeled antibody, the sections were next incubated with alkaline phosphatase-conjugated streptavidin Vector Laboratories, Inc. Counter-staining was performed with methyl green Dako. The apical tight junction is an important indicator for ameloblast polarity and can be measured by the functional selectivity of the paracellular apical barrier.

Sulfo-NHS-Biotin was used as a tracer molecule to evaluate the effects of SATB1 on the ameloblast layer barrier function as previously described [ 78 ]. Ten minutes after receiving Sulfo-NHS-Biotin injection, the pups were sacrificed, and the hemimandibles were dissected, processed, and paraffin-embedded for histological sectioning as previously mentioned.

Ameloblasts were removed from the first molars, which have the enamel primarily at the secretory stage [ 79 ] were microdissected from the surface of the teeth.

Human ameloblast lineage cells hALCs were harvested from the tooth buds as mentioned above. We followed the protocol previously established in our laboratory to maintain these cells [ 80 , 81 , 82 ].

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Required enhancer-matrin-3 network interactions for a homeodomain transcription program. Genome organizing function of SATB1 in tumor progression. Semin Cancer Biol. Cancer Lett. Rab30 is required for the morphological integrity of the Golgi apparatus. Biol Cell. Rho protein regulates tight junctions and perijunctional actin organization in polarized epithelia. Eps8 controls actin-based motility by capping the barbed ends of actin filaments. Nat Cell Biol. Mol Biol Cell. Loss of the actin remodeler Eps8 causes intestinal defects and improved metabolic status in mice.

Eps8 is increased in pancreatic cancer and required for dynamic actin-based cell protrusions and intercellular cytoskeletal organization. Shaping the intestinal brush border. These data provide greater insight as to the suitability of both cell lines to contribute to biological studies on enamel formation and biomineralization, and highlight some of the strengths and weaknesses when relying on enamel epithelial organ-derived cell lines to study molecular activities of amelogenesis.

Dental enamel, the most highly mineralized and hardest tissue in vertebrates, is comprised of highly organized hydroxyapatite Hap crystallites formed by ameloblast cells Smith, Dental enamel formation is tightly controlled by ameloblast cells whose differential activities toward enamel formation can be broadly divided into the initial secretory-stage, followed by a maturation-stage Smith, ; Lacruz et al. The enamel organ transcriptome for secretory-stage vs. There is a lack of available immortalized ameloblast-derived cell lines and this limits the possibilities to explore in detail the cellular events characteristic of ameloblasts through the different stages of amelogenesis, thus increasing the immediate need for animal experimentation.

The few available ameloblast-like cell lines that are under investigation by the research community include mouse LS8 Chen et al. LS8 Lacruz et al. PABSo-E cells have been used to study calcium-sensing receptor activities in enamel formation Mathias et al. In this study we compare the molecular profiles of selected enamel-specific genes, and the ability to form calcified nodules in the two mouse-derived ameloblast-like cells: the Swiss-Webster derived LS8 cells Chen et al. This comparative investigation better characterizes both cell lines, and identifies some of the advantages of experimenting with one cell line in preference to the other, to focus studies to ameloblast-specific cellular phenomena such as matrix formation vs.

Swiss-Webster mice were treated in accordance with Institutional and Federal guidelines. For quantitative real-time PCR and Western blot analysis, the first molars from mice at postnatal PN days 3, 6, and 9 were extracted and total RNA and protein recovered for analysis. These three PN time points were chosen to demonstrate the shift in ameloblast gene expression from primarily secretory-stage activities PN3 to a stage where maturation-stage activities predominate PN9 ; and a point midway PN6 where there is a mixed population of secretory and maturation ameloblasts Simmer et al.

For the purpose of this study isolated first molar teeth at the three developmental stages serve as a suitable control, however RNA and protein isolated from these teeth are not only attributed by enamel organ epithelial cells but also non-epithelial cells including odontoblast and pulp cells Jacques et al.

The cDNA was analyzed using mouse specific Actb a. All samples were run in triplicate. All data points were normalized to Actb. For protein extraction from mouse molars, the samples were homogenized manually with a pestle for 40 s. Samples were then cleared by centrifugation and protein concentration quantified using the Bicinchoninic Acid BCA assay Pierce Biotechnology.

Secondary antibodies used were: horseradish peroxidase labeled goat-derived anti-mouse Cat , Pierce Biotechnology ; goat-derived anti-rabbit Cat , Pierce Biotechnology ; and rabbit-derived anti-chicken Cat , Pierce Biotechnology.

The cells were evaluated for Alizarin Red stain at 1-, 3-, 7-, , , and days post inoculation. Cells were recovered by mechanically scrapping off the well, and the cells recovered into a microcentrifuge tube and vortexed vigorously. One hundred and fifty microliter of this supernatant was transferred into an opaque walled transparent bottom well plate, and the optical density of the suspension was measured by absorption at nm.

Alizarin Red stain incorporated into the cells was quantified using a standard curve generated as per the protocol provided by the manufacturer. OD was applied to quantify the unknown samples. Expression of Amtn in both cell lines was negligible.

This in vivo derived molar data clearly demonstrated a shift from a more dominate secretory function, with high levels of expression for Amelx, Ambn, Enam , and Mmp20 at PN3, to a more dominant maturation-stage gene expression profile, with the highest levels of Amtn, Odam , and Klk4 seen at PN9. The data may indicate that ALC gene expression profiles represent a later stage of gene expression associated with enamel maturation, when compared to the gene expression profiles of LS8 cells that may represent better a secretory function.

Figure 1. Expression levels relative to Actb expression are graphed. B The expression of each gene with the enamel genes is compared from three stages of development; PN3, 6, and 9, and normalized to Actb levels. Gapdh was applied as a normalizing control. Suitable antibodies for Western blot analysis of Enam, Mmp20, and Klk4 protein were not immediately available thus their expression profiles were not examined.

No protein gene expression associated with enamel matrix was detected in LS8 cell lysates, or by the NIH3T3 cells used as a negative control. In the case of the in vivo derived molar PN3, 6, and 9 samples, the selected protein expression profiles reflected the levels observed for the mRNA expression profile, with the highest expression levels for Amelx and Ambn apparent at PN3, and the highest levels of Amtn and Odam expression apparent at PN9. Figure 2. LS8 cells did not appear to express the enamel proteins examined.

No Amtn expression was noted in either enamel organ cell line. NIH3T3 fibroblasts cells are used here as a negative control. All cells cultured for 7 days. Groupings to right: Protein from PN3, 6, and 9 mouse first molars were examined to confirm the suitability of each antibody to assess mouse-derived enamel proteins; and also to illustrate the decreasing levels of Amelx and Amtn from PN3 to PN9, and the increasing levels of Amtn and Odam over the same time course.

B A second series of Western blots were completed with ALC cells isolated at days 3 and 7 after passage, and also from PN9 mouse molars to directly compare protein levels for Amelx, Ambn, and Odam in ALC cells when compared to in vivo derived tooth tissues.

Glyceraldehydephosphate dehydrogenase Gapdh was used as a loading normalizing control. A second series of Western blots were performed using protein extracts from ALC cells cultured for 3- and 7-days after passage, alongside protein extracted from PN9 first molars Figure 2B with Gapdh applied as the normalizing factor.

ALC cells showed positive Alizarin Red S staining, which became apparent at day-3 and increased over the days duration of culture Figure 3A. Alizarin Red S staining was quantified by extrapolating from a standard curve of concentrations measured at nm. Graphical representation of the solubilized Alizarin Red S stain from each cell culture indicated a progressive, and highly significant, increase in calcified nodule formation for ALC cells only, reacting a maximum staining at day 21 Figure 3B.