Enamel Organ Epithelial Cells Research Articles

Bmp2 deletion causes an amelogenesis imperfecta phenotype via regulating enamel gene expression.

Although Bmp2 is essential for tooth formation, the role of Bmp2 during enamel formation remains unknown in vivo. In this study, the role of Bmp2 in regulation of enamel formation was investigated by the Bmp2 conditional knock out (Bmp2 cKO) mice. Teeth of Bmp2 cKO mice displayed severe and profound phenotypes with asymmetric and misshaped incisors as well as abrasion of incisors and molars. Scanning electron microscopy analysis showed that the enamel layer was hypoplastic and enamel lacked a typical prismatic pattern. Teeth from null mice were much more brittle as tested by shear and compressive moduli. Expression of enamel matrix protein genes, amelogenin, enamelin, and enamel-processing proteases, Mmp-20 and Klk4 was reduced in the Bmp2 cKO teeth as reflected in a reduced enamel formation. Exogenous Bmp2 up-regulated those gene expressions in mouse enamel organ epithelial cells. This result for the first time indicates Bmp2 signaling is essential for proper enamel development and mineralization in vivo.

Binding of amelogenin to MMP-9 and their co-expression in developing mouse teeth

Amelogenin is the most abundant matrix protein in enamel. Proper amelogenin processing by proteinases is necessary for its biological functions during amelogenesis. Matrix metalloproteinase 9 (MMP-9) is responsible for the turnover of matrix components. The relationship between MMP-9 and amelogenin during tooth development remains unknown. We tested the hypothesis that MMP-9 binds to amelogenin and they are co-expressed in ameloblasts during amelogenesis. We evaluated the distribution of both proteins in the mouse teeth using immunohistochemistry and confocal microscopy. At postnatal day 2, the spatial distribution of amelogenin and MMP-9 was co-localized in preameloblasts, secretory ameloblasts, enamel matrix and odontoblasts. At the late stages of mouse tooth development, expression patterns of amelogenin and MMP-9 were similar to that seen in postnatal day 2. Their co-expression was further confirmed by RT-PCR, Western blot and enzymatic zymography analyses in enamel organ epithelial and odontoblast-like cells. Immunoprecipitation assay revealed that MMP-9 binds to amelogenin. The MMP-9 cleavage sites in amelogenin proteins across species were found using bio-informative software program. Analyses of these data suggest that MMP-9 may be involved in controlling amelogenin processing and enamel formation.

Gene‐expression analysis of early‐ and late‐maturation‐stage rat enamel organ

Enamel maturation is a dynamic process that involves high rates of mineral acquisition, associated fluctuations in extracellular pH, and resorption of extracellular enamel proteins. During maturation, ameloblasts change from having a tall, thin, and highly polarized organization, characteristic of the secretory stage, to having a low columnar and widened morphology in the maturation stage. To identify potential differences in gene expression throughout maturation, we obtained enamel organ epithelial cells derived from the early- and late-maturation stages of rat incisor and analyzed the global gene-expression profiles at each stage. Sixty-three candidate genes were identified as having potential roles in the maturation process. Quantitative PCR was used to confirm the results of this genome-wide analysis in a subset of genes. Transcripts enriched during late maturation (n = 38) included those associated with lysosomal activity, solute carrier transport, and calcium signaling. Also up-regulated were transcripts involved in cellular responses to oxidative stress, proton transport, cell death, and the immune system. Transcripts down-regulated during the late maturation stage (n =25) included those with functions related to cell adhesion, cell signaling, and T-cell activation. These results indicate that ameloblasts undergo widespread molecular changes during the maturation stage of amelogenesis and hence provide a basis for future functional investigations into the mechanistic basis of enamel mineralization.

Bioactive Nanofibers Instruct Cells to Proliferate and Differentiate During Enamel Regeneration

During tooth development, ectoderm-derived ameloblast cells create enamel by synthesizing a complex protein mixture serving to control cell to matrix interactions and the habit of hydroxyapatite crystallites. Using an in vitro cell and organ culture system, we studied the effect of artificial bioactive nanostructures on ameloblasts with the long-term goal of developing cell-based strategies for tooth regeneration. We used branched peptide amphiphile molecules containing the peptide motif Arg-Gly-Asp, or "RGD" (abbreviated BRGD-PA), known to self-assemble in physiologic environments into nanofibers that display on their surfaces high densities of this biological signal. Ameloblast-like cells (line LS8) and primary enamel organ epithelial (EOE) cells were cultured within PA hydrogels, and the PA was injected into the enamel organ epithelia of mouse embryonic incisors. The expression of amelogenin, ameloblastin, integrin alpha 5, and integrin alpha 6 was detected by quantitative real-time PCR and immunodetection techniques. We performed cell proliferation assay using BrdU labeling and a biomineralization assay using Alizarin red S staining with quantitative Ca(2+) measurements. In the cell culture model, ameloblast-like cells (LS8) and primary EOE cells responded to the BRGD-PA nanostructures with enhanced proliferation and greater amelogenin, ameloblastin, and integrin expression levels. At the site of injection of the BRGD-PA in the organ culture model, we observed EOE cell proliferation with differentiation into ameloblasts as evidenced by their expression of enamel specific proteins. Ultrastructural analysis showed the nanofibers within the forming extracellular matrix, in contact with the EOE cells engaged in enamel formation and regeneration. This study shows that BRGD-PA nanofibers present with enamel proteins participate in integrin-mediated cell binding to the matrix with delivery of instructive signals for enamel formation.

Enamel Tissue Engineering Using Subcultured Enamel Organ Epithelial Cells in Combination with Dental Pulp Cells

We describe a strategy for the in vitro engineering of enamel tissue using a novel technique for culturing enamel organ epithelial (EOE) cells isolated from the enamel organ using 3T3-J2 cells as a feeder layer. These subcultured EOE cells retain the capacity to produce enamel structures over a period of extended culture. In brief, enamel organs from 6-month-old porcine third molars were dissociated into single cells and subcultured on 3T3-J2 feeder cell layers. These subcultured EOE cells were then seeded onto a collagen sponge in combination with primary dental pulp cells isolated at an early stage of crown formation, and these constructs were transplanted into athymic rats. After 4 weeks, complex enamel-dentin structures were detected in the implants. These results show that our culture technique maintained ameloblast lineage cells that were able to produce enamel in vivo. This novel subculture technique provides an important tool for tooth tissue engineering.

Quiescent epithelial cell rests of Malassez can differentiate into ameloblast‐like cells

Epithelial cell rests of Malassez (ERM) are quiescent epithelial remnants of Hertwig's epithelial root sheath (HERS) that are involved in the formation of tooth roots. After completion of crown formation, HERS are converted from cervical loop cells, which have the potential to generate enamel for tooth crown formation. Cervical loop cells have the potential to differentiate into ameloblasts. Generally, no new ameloblasts can be generated from HERS, however this study demonstrated that subcultured ERM can differentiate into ameloblast-like cells and generate enamel-like tissues in combination with dental pulp cells at the crown formation stage. Porcine ERM were obtained from periodontal ligament tissue by explant culture and were subcultured with non-serum medium. Thereafter, subcultured ERM were expanded on 3T3-J2 feeder cell layers until the tenth passage. The in vitro mRNA expression pattern of the subcultured ERM after four passages was found to be different from that of enamel organ epithelial cells and oral gingival epithelial cells after the fourth passage using the same expansion technique. When subcultured ERM were combined with subcultured dental pulp cells, ERM expressed cytokeratin14 and amelogenin proteins in vitro. In addition, subcultured ERM combined with primary dental pulp cells seeded onto scaffolds showed enamel-like tissues at 8 weeks post-transplantation. Moreover, positive staining for amelogenin was observed in the enamel-like tissues, indicating the presence of well-developed ameloblasts in the implants. These results suggest that ERM can differentiate into ameloblast-like cells.

The Effect of LRAP on Enamel Organ Epithelial Cell Differentiation

Leucine-rich amelogenin peptide (LRAP) is an alternatively spliced amelogenin found in the developing enamel organ. LRAP functions to regulate the development of mesenchymal-derived cells; however, its effect on cells of the enamel organ remains unclear. The hypothesis tested in this study is that LRAP also regulates human enamel organ epithelial cells. Recombinant human LRAP (rH58) was synthesized in E. coli, purified, and exogenously added to cultures of human primary enamel epithelial cells, which were analyzed for changes in cell proliferation and differentiation. rH58 had no effect on cell proliferation, but altered enamel epithelial cell morphology, resulting in larger, more rounded cells. Immunofluorescence showed that rH58 treatment increased amelogenin synthesis, but down-regulated Notch1 expression in enamel epithelial cells. LAMP-1, a membrane receptor for LRAP in mesenchymal cells, was identified and was up-regulated in the presence of rH58. These results suggest that rH58 promotes differentiation of human enamel organ epithelial cells.

Growth of ameloblast‐lineage cells in a three‐dimensional Matrigel environment

Enamel organ epithelial cells grow in culture as two distinct cell populations--either stellate-shaped or polygonal-shaped cells. The polygonal cells have an ameloblast cell phenotype and are difficult to grow in culture beyond two passages. This study was designed to determine the effects of a Matrigel three-dimensional (3D) environment on polygonal cells, as compared with stellate cells, derived from porcine tooth enamel organ. Enamel organs were dissected free from the unerupted molars of 30-kg pigs and then grown in LCH-8e media, either with or without serum. Cells grown in serum-free media were primarily polygonal shaped, whereas cells grown in media containing serum were stellate shaped. Both types of cells were grown in a 3D Matrigel matrix. In addition, polygonal-shaped cells were mixed with hydroxyapatite powder and transplanted subcutaneously into nude mice. Polygonal-shaped epithelial cells formed cell groups, similar to epithelial pearls, both in vitro and in vivo. The stellate-shaped cells, in contrast, did not form similar structures, but remained suspended in the Matrigel and gradually disappeared from the culture. These results suggest that a Matrigel environment, rich in basement membrane and matrix proteins, selects for polygonal-shaped ameloblast-lineage cells and induces the formation of epithelial pearls.

Characterization of human primary enamel organ epithelial cells in vitro

Tooth enamel is formed by ameloblasts, which are derived from the epithelial cells of the enamel organ. The purpose of this study was to grow human ameloblast-like epithelial cells in culture. Human fetal tooth organs were isolated, and the cells were separated by digestion in collagenase/dispase. The cells were cultured in KGM-2 media with and without serum and at different calcium concentrations. The expression of enamel matrix proteins was analyzed by RT-PCR and cytokeratin 14 was detected by immunohistochemistry. The cells were further characterized by osteogenesis/odontogenesis-related DNA array. Cells isolated from the tooth organs grown in KGM-2 media containing 2-10% serum, were mixture of cobblestone and spindle shaped cells. Culturing these cells in KGM-2 with 0.05 mM calcium was selective for cobblestone ameloblasts-like cells (CAB), which were immunopositive for cytokeratin 14. Amelogenin, ameloblastin, enamelin, MMP-20 and KLK-4 were detected in CAB cells by RT-PCR. Osteogenesis SuperArray analyses could not detect the presence of typical molecules related to mesenchymal odontoblast or osteoblast lineage cells in these cultures. These studies showed that cobblestone-shaped ameloblast-like cells are selected from the tooth organ cells, by culture in KGM-2 media with 0.05 mM calcium.

Antagonistic effects of Smad2 versus Smad7 are sensitive to their expression level during tooth development.

Members of the transforming growth factor-beta (TGF-beta) superfamily regulate cell proliferation, differentiation, and apoptosis, controlling the development and maintenance of most tissues. TGF-beta signal is transmitted through the phosphorylation of Smad proteins by TGF-beta receptor serine/threonine kinase. During early tooth development, TGF-beta inhibits proliferation of enamel organ epithelial cells but the underlying molecular mechanisms are largely unknown. Here we tested the hypothesis that antagonistic effects between Smad2 and Smad7 regulate TGF-beta signaling during tooth development. Attenuation of Smad2 gene expression resulted in significant advancement of embryonic tooth development with increased proliferation of enamel organ epithelial cells, while attenuation of Smad7 resulted in significant inhibition of embryonic tooth development with increased apoptotic activity within enamel organ epithelium. These findings suggest that different Smads may have differential activities in regulating TGF-beta-mediated cell proliferation and death. Furthermore, functional haploinsufficiency of Smad2, but not Smad3, altered TGF-beta-mediated tooth development. The results indicate that Smads are critical factors in orchestrating TGF-beta-mediated gene regulation during embryonic tooth development. The effectiveness of TGF-beta signaling is highly sensitive to the level of Smad gene expression.

Development and characterization of an SV40 immortalized porcine ameloblast-like cell line.

Enamel is secreted as a protein matrix by the ameloblasts. These same cells then control the maturation of the enamel matrix, secreting proteinases that hydrolyze proteins as mineralization progresses, until mature enamel containing less than 1% protein by weight remains. Further understanding of the factors that control ameloblast function and differentiation requires an in vitro cell culture system. In this study, we report immortalization of enamel organ epithelial cells and the selection of a cell line with characteristics of ameloblasts. Porcine enamel organ cells were dissected from unerupted porcine molars, cultured in serum-free medium, and passaged twice. These cells were transfected with an origin-of-replication defective SV40 plasmid by calcium phosphate precipitation, and a cell line with mRNA expression characteristic of ameloblasts was cloned. This cell line (PABSo-E) expressed mRNA for amelogenin, matrix metalloproteinase-20 (enamelysin), and enamel matrix serine proteinase 1 (EMSP1), but not ameloblastin. PABSo-E cells have been passaged more than 55 times, while continuing to maintain characteristics of ameloblasts. These cells will be useful for future studies of ameloblast function.

Primary Culture and Characterization of Enamel Organ Epithelial Cells

The cells of the enamel organ are programmed by signals such as growth factors and extracellular matrix components to differentiate and form dental enamel. To study how the enamel organ epithelial cells control enamel development, we have begun to characterize a primary porcine enamel organ epithelial cell culture system. The unerupted molars of 3 month old pigs were isolated, the cells were digested into a single cell suspension and grown in media either with or without serum. Expression of amelogenin and ameloblastin mRNA was monitored by RT PCR, and protein secretion was identified by immunohistochemistry. Cells grown in MEM formed a mixed cell population of epithelial- and fibroblast-like cells which grew past confluence, formed nodules, mineralized, and expressed low levels of amelogenin and ameloblastin protein. In LHC-9 media, which is selective for epithelial cells, the cells did not grow past confluence but secreted amelogenin and ameloblastin proteins more strongly. Cell viability was maintained in both serum-free and serum-containing media. However, in the serum-free media, cell proliferation proceeded slowly. Although cells grown in MEM mineralized, the mixed cell population may make studies of specific ameloblast-like cells more difficult. However, cells grown in a culture media selective for epithelial cells will require modifications such as cell immortalization to allow long term studies of cell regulation and interaction. In summary, we have established an enamel organ epithelial cell culture system which will enable us to study the role of ameloblasts in enamel matrix formation, ameloblast regulation, as well as cell-matrix interactions. Selection of specific culture conditions will depend on the questions being addressed in individual studies.

Co-distribution of Enamel- and Cementum-Related Proteins in Association with Enamel Organ Epithelial Cells

Co-distribution of Enamel- and Cementum-Related Proteins in Association with Enamel Organ Epithelial Cells

Antisense inhibition of AMEL translation demonstrates supramolecular controls for enamel HAP crystal growth during embryonic mouse molar development

During tooth development, enamel organ epithelial cells express a tissue-specific gene product (amelogenin) which presumably functions to control calcium hydroxyapatite crystal growth patterns during enamel biomineralization. The present studies were designed to test the hypothesis that amelogenin as a supramolecular aggregate regulates crystal growth during enamel biomineralization. Antisense oligodeoxynucleotide strategy was used in a simple organ culture system to inhibit amelogenin translation. Under these experimental conditions, antisense treatment prior to and during amelogenin expression resulted in inhibition of amelogenin translation products within immunoprecipitated [35S]methionine metabolically labeled proteins. To determine the efficiency of antisense treatment in this model system, digoxigenin-labeled oligodeoxynucleotides were observed to diffuse throughout the tooth explants including the target ameloblast cells within 24 hours. Ultrastructural analyses of amelogenin supramolecular assembly as electron-dense stippled materials in antisense treated cultures demonstrated dysmorphology of the extracellular enamel matrix with a significant reduction in crystal length and width. We conclude that secreted extracellular proteins form a supramolecular aggregate, which controls both the orientation and dimensions of enamel crystal formation during tooth development.