mTOR plays a pivotal role in multiple processes of enamel organ development principally through the mTORC1 pathway and in part via regulating cytoskeleton dynamics.

We herein report that deletion of mTOR in dental epithelia caused defective development of multiple cell layers of the enamel organ, which culminated in tooth malformation and cystogenesis. Specifically, cells of the stellate reticulum and stratum intermedium were poorly formed, resulting in cystic changes. The pre-ameloblasts failed to elongate along the apical-basal axis and persisted vigorous expression of Sox2 and P63, which are normally downregulated during cytodifferentiation. Expression of amelogenic markers was also attenuated in mutants. Cell proliferation and cell sizes in mutants were significantly reduced over time. Importantly, we found reduced amounts and aberrant aggregations of cytoskeletal components in mutants, along with attenuated expression of cytoskeleton regulator Cdc42, whose epithelial deletion causes a similar phenotype. Moreover, disruption of actin assembly in an organ culture system affected cell proliferation and cytodifferentiation of tooth germs, supporting a causative role of mTOR-regulated cytoskeleton dynamics for the observed phenotype of mTOR mutant mice. In further support of this view, we showed that mTOR overactivation caused increased cytoskeletal component synthesis and assembly, along with accelerated cytodifferentiation in the enamel organ. Finally, we demonstrated that mTOR regulated enamel organ development principally through the mTORC1 pathway.

Fluoride exposure alters Ca2+ signaling and mitochondrial function in enamel cells.

Fluoride ions are highly reactive, and their incorporation in forming dental enamel at low concentrations promotes mineralization. In contrast, excessive fluoride intake causes dental fluorosis, visually recognizable enamel defects that can increase the risk of caries. To investigate the molecular bases of dental fluorosis, we analyzed the effects of fluoride exposure in enamel cells to assess its impact on Ca2+ signaling. Primary enamel cells and an enamel cell line (LS8) exposed to fluoride showed decreased internal Ca2+ stores and store-operated Ca2+ entry (SOCE). RNA-sequencing analysis revealed changes in gene expression suggestive of endoplasmic reticulum (ER) stress in fluoride-treated LS8 cells. Fluoride exposure did not alter Ca2+ homeostasis or increase the expression of ER stress-associated genes in HEK-293 cells. In enamel cells, fluoride exposure affected the functioning of the ER-localized Ca2+ channel IP3R and the activity of the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) pump during Ca2+ refilling of the ER. Fluoride negatively affected mitochondrial respiration, elicited mitochondrial membrane depolarization, and disrupted mitochondrial morphology. Together, these data provide a potential mechanism underlying dental fluorosis.

A Regenerative Endodontic Approach in Mature Ferret Teeth Using Rodent Preameloblast-conditioned Medium.

BACKGROUND: This study evaluated the effectiveness of a regenerative endodontic approach to regenerate the pulp tissue in mature teeth of ferret. The presence of odontoblast-like cells in the newly-formed tissue of teeth treated with or without preameloblast-conditioned medium was evaluated based on morphological criteria. MATERIALS AND METHODS: Twenty-four canines from six ferrets were treated. The pulp was removed, and the apical foramen was enlarged. After inducing the formation of a blood clot, a collagen sponge with or without preameloblast-conditioned medium was placed underneath the cementoenamel junction. The samples were analyzed at the eighth week of follow-up. RESULTS: Vascularized connective tissue was observed in 50% of teeth, without differences between groups. The tissue occupied the apical third of the root canals. Odontoblast-like cells were not observed in any group. CONCLUSION: Revitalization of mature teeth is possible, at least in the apical third of the root canal. Further experimental research is needed to produce more reliable outcomes.

Loss of transforming growth factor-ß1 in epithelium cells affects enamel formation in mice.

OBJECTIVES: In order to understand the specific in vivo function of transforming growth factor-beta1 (TGF-ß1), we successfully established aTGF-ß1 deficient mouse model using a conditional knockout method. In the present study, we aimed to further understand the potential role of TGF-ß1 in enamel formation. DESIGN: Transgenic mice withoutTGF-ß1 in epithelial cells were generated. Scanning electron microscopy and micro-computed tomography analysis were used to detect the dental appearance, enamel microstructure and tooth density. Histological analysis was used to examine the residual organic matrix of enamel. Quantitative real-time polymerase chain reaction was used to analyze the expressions of enamel matrix proteins at the mRNA level. RESULTS: The enamel of mandibular molars and incisors inTGF-ß1 conditional knockout mice displayed severe attrition and lower density compared with the wild-type littermates. A slender microstructure of enamel rod was observed, and enamel matrix proteins were retained in the enamel space at the maturation stage in conditional knockout mice. Moreover, the expressions of enamel matrix protein-encoding genes, such as amelogenin (Amelx), ameloblastin (Ambn), Enamelin (Enam) and matrix metalloproteinase-20 (Mmp-20), were increased in enamel organs of conditional knockout mice. On the other hand, the expressions of Amelotin (Amtn), kallikrein-related peptidase-4 (Klk4), C4orf26 and WD repeat-containing protein 72 (Wdr72) were dramatically decreased at the transition and maturation stages. CONCLUSIONS: TGF-ß1 played an important role in enamel mineralization through decreasing synthesis ofAmelx, Ambn and Enam and increasing synthesis of Klk4, Amtn, Corf26 and Wdr72.

Are Hertwig's epithelial root sheath cells necessary for periodontal formation by dental follicle cells?

OBJECTIVE: The role of Hertwig's epithelial root sheath (HERS) cells in periodontal formation has been controversial. This study aimed to further clarify whether HERS cells participate in formation of the periodontium, and the necessity of HERS cells in differentiation of dental follicle cells (DFCs) for periodontal regeneration. DESIGN: HERS cells and DFCs were isolated and identified from post-natal 7-day Sprauge-Dawley rats. In vitro, direct co-culture of HERS cells and DFCs as well as the individual culture of HERS and DFCs were performed and followed by alizarin red staining and the quantitative real-time polymerase chain reaction analysis. For in vivo evaluation, the inactivated dentin matrix (iTDM) was fabricated. HERS cells and DFCs were seeded in combination or alone on iTDM and then transplanted into the rat omentum. Scanning electron microscope and further histological analysis were carried out. RESULTS: In vitro, mineral-like nodules were found in the culture of HERS cells alone or HERS + DFCs either by alizarin red staining or scanning electronic microscope. The mineralization and fiber-forming relevant mRNA expressions, such as bone sialoprotein, osteopontin, collagen I and collagen III in HERS + DFCs were significantly higher than that of the HERS or DFCs alone group. After transplantation in vivo, cementum and periodontal ligament-like tissues were formed in groups of HERS + DFCs and HERS alone, while no evident hard tissues and attached fibers were found in DFCs alone. CONCLUSIONS: Hertwig's epithelial root sheath cells directly participate in the formation of the periodontium, and they are essential for the differentiation of dental follicle cells to form periodontal structures. The combination use of Hertwig's epithelial root sheath cells and dental follicle cells is a promising approach for periodontal regeneration.

Comparative study on differentiation of cervical-loop cells and Hertwig's epithelial root sheath cells under the induction of dental follicle cells in rat.

Cervical loop cells (CLC) and Hertwig's epithelial root sheath (HERS) cells are believed to play critical roles in distinct developmental patterns between rodent incisors and molars, respectively. However, the differences in differentiation between CLC and HERS cells, and their response to inductions from dental follicle cells, remain largely unknown. In present study, CLC and HERS cells, as well as incisor dental follicle (IF) cells and molar dental follicle (MF) cells were isolated from post-natal 7-day rats. IF and MF cell derived conditioned medium (CM) was obtained for induction of CLC and HERS cells. In vitro experiments, we found that, under the induction of dental follicle cell derived CM, CLC cells maintained the epithelial polygonal-shapes and formed massive minerals, while part of HERS cells underwent shape transformation and generated granular minerals. CLC cells expressed higher enamel-forming and mineralization related genes, while HERS cells showed opposite expression patterns of BMP2, BMP4, AMBN and AMGN. In vivo, CLC cells generated enamel-like tissues while HERS cells formed cementum-periodontal ligament-like structures. Taken together, CLC and HERS cells present distinct differentiation patterns under the inductions from dental follicle cells.

Laminin γ2 knockout mice rescued with the human protein exhibit enamel maturation defects.

The epithelial ameloblasts are separated from the maturing enamel by an atypical basement membrane (BM) that is enriched in laminin 332 (LM-332). This heterotrimeric protein (α3, ß3 and γ2 chains) provides structural integrity to BMs and influences various epithelial cell processes including cell adhesion and differentiation. Mouse models that lack expression of individual LM-332 chains die shortly after birth. The lethal phenotype of laminin γ2 knockout mice can be rescued by human laminin γ2 (LAMC2) expressed using a doxycycline-inducible (Tet-on) cytokeratin 14 promoter-rtTA. These otherwise normal-looking rescued mice exhibit white spot lesions on incisors. We therefore investigated the effect of rescue with human LAMC2 on enamel maturation and structuring of the atypical BM. The maturation stage enamel organ in transgenic mice was severely altered as compared to wild type controls, a structured BM was no longer discernible, dystrophic matrix appeared in the maturing enamel layer, and there was residual enamel matrix late into the maturation stage. Microtomographic scans revealed excessive wear of occlusal surfaces on molars, chipping of enamel on incisor tips, and hypomineralization of the enamel layer. No structural alterations were observed at other epithelial sites, such as skin, palate and tongue. These results indicate that while this humanized mouse model is capable of rescue in various epithelial tissues, it is unable to sustain structuring of a proper BM at the interface between ameloblasts and maturing enamel. This failure may be related to the atypical composition of the BM in the maturation stage and reaffirms that the atypical BM is essential for enamel maturation.

Appropriate real-time PCR reference genes for fluoride treatment studies performed in vitro or in vivo.

OBJECTIVE: Quantitative real-time PCR (qPCR) is routinely performed for experiments designed to identify the molecular mechanisms involved in the pathogenesis of dental fluorosis. Expression of reference gene(s) is expected to remain unchanged in fluoride-treated cells or in rodents relative to the corresponding untreated controls. The aim of this study was to select optimal reference genes for fluoride experiments performed in vitro and in vivo. DESIGN: Five candidate genes were evaluated: B2m, Eef1a1, Gapdh, Hprt and Tbp. For in vitro experiments, LS8 cells derived from mouse enamel organ were treated with 0, 1, 3 and/or 5mM sodium fluoride (NaF) for 6 or 18 h followed by RNA isolation. For in vivo experiments, six-week old rats were treated with 0 or 100 ppm fluoride as NaF for six weeks at which time RNA was isolated from enamel organs. RNA from cells and enamel organs were reverse-transcribed and stability of gene expression for the candidate reference genes was evaluated by qPCR in treated versus non-treated samples. RESULTS: The most stably expressed genes in vitro according to geNorm were B2m and Tbp, and according to Normfinder were Hprt and Gapdh. The most stable genes in vivo were Eef1a1 and Gapdh. Expression of Ddit3, a gene previously shown to be induced by fluoride, was demonstrated to be accurately calculated only when using an optimal reference gene. CONCLUSIONS: This study identifies suitable reference genes for relative quantification of gene expression by qPCR after fluoride treatment both in cultured cells and in the rodent enamel organ.

Effects of FGFR Signaling on Cell Proliferation and Differentiation of Apert Dental Cells.

The Apert syndrome is a rare congenital disorder most often arising from S252W or P253R mutations in fibroblast growth factor receptor (FGFR2). Numerous studies have focused on the regulatory role of Apert FGFR2 signaling in bone formation, whereas its functional role in tooth development is largely unknown. To investigate the role of FGFR signaling in cell proliferation and odontogenic differentiation of human dental cells in vitro, we isolated dental pulp and enamel organ epithelia (EOE) tissues from an Apert patient carrying the S252W FGFR2 mutation. Apert primary pulp and EOE cells were established and shown to exhibit normal morphology and express alkaline phosphatase under differentiation conditions. Similar to control cells, Apert dental pulp and EOE cells expressed all FGFRs, with highest levels of FGFR1 followed by FGFR2 and low levels of FGFR3 and FGFR4. However, Apert cells had increased cell growth compared with control cells. Distinct from previous findings in osteoblast cells, gain-of-function S252W FGFR2 mutation did not upregulate the expression of epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFRα), but elevated extracellular signal-regulated kinase (ERK) signaling in cells after EGF stimulation. Unexpectedly, there was little effect of the S252W mutation on odontogenic gene expression in dental pulp and EOE cells. However, after inhibition of total FGFR signaling or ERK signaling, the expression of odontogenic genes was upregulated in both dental cell types, indicating the negative effect of whole FGFR signaling on odontogenic differentiation. This study provides novel insights on FGFR signaling and a common Apert FGFR2 mutation in the regulation of odontogenic differentiation of dental mesenchymal and epithelial cells.

Analysis of expression patterns of IGF-1, caspase-3 and HSP-70 in developing human tooth germs.

AIMS: To analyze expression patterns of IGF-1, caspase-3 and HSP-70 in human incisor and canine tooth germs during the late bud, cap and bell stages of odontogenesis. MATERIALS AND METHODS: Head areas or parts of jaw containing teeth from 10 human fetuses aged between 9th and 20th developmental weeks were immunohistochemically analyzed using IGF-1, active caspase-3 and HSP-70 markers. Semi-quantitative analysis of each marker's expression pattern was also performed. RESULTS: During the analyzed period, IGF-1 and HSP-70 were mostly expressed in enamel organ. As development progressed, expression of IGF-1 and HSP-70 became more confined to differentiating tissues in the future cusp tip area, as well as in highly proliferating cervical loops. Few apoptotic bodies highly positive to active caspase-3 were observed in enamel organ and dental papilla from the cap stage onward. However, both enamel epithelia moderately expressed active caspase-3 throughout the investigated period. CONCLUSIONS: Expression patterns of IGF-1, active caspase-3 and HSP-70 imply importance of these factors for early human tooth development. IGF-1 and HSP-70 have versatile functions in control of proliferation, differentiation and anti-apoptotic protection of epithelial parts of human enamel organ. Active caspase-3 is partially involved in formation and apoptotic removal of primary enamel knot, although present findings might reflect its ability to perform other non-death functions such as differentiation of hard dental tissues secreting cells and guidance of ingrowth of proliferating cervical loops.

Immunohistochemical expression of CD56 in dog (Canis familiaris) odontogenesis.

AIM: To investigate the expression of CD56 in dog odontogenesis in order to elucidate the expression found in ameloblastomas. MATERIALS AND METHODS: Immunohistochemical analysis of CD56 expression of developing dog teeth in the bud, cap and bell stages including the remnants of the dental lamina. RESULTS: Weak CD56 expression was observed in the dental epithelium during the bud stage with intense staining of certain peripheral epithelial cells. Positive staining of epithelial cells was also observed in the cap stage with intense staining of the inner enamel epithelium at this stage. During the bell stage the staining was concentrated on the cervical loop areas. The dental papilla revealed positive staining throughout the cap and bell stages while the dental follicle stained intensely positive throughout all the phases examined. The dental lamina and Serres rests also stained positive for CD56. CONCLUSIONS: The expression of CD56 in dog odontogenic tissue varies according to the stage of tooth development. There is a positive correlation between the positive staining observed in ameloblastomas and their odontogenic cells of origin.

Cell dynamics in Hertwig's epithelial root sheath and surrounding mesenchyme in mice irradiated to the head.

OBJECTIVE: To investigate the mechanisms that cause damage to root formation as a result of irradiation to the mouse head, morphological changes in molar dental roots and cell dynamics in Hertwig's epithelial root sheath (HERS), and surrounding mesenchymal tissue were examined. MATERIALS AND METHODS: To perform the experiments, 5-day-old C57BL/6 mice were randomly divided into three groups: the control group (0 Gy) and irradiated groups (10 and 20 Gy). Micro-CT analysis, HE staining, immunohistochemistry analysis, and TUNEL assay were then performed. RESULTS: Roots in irradiated mice were dose-dependently shorter than those of control mice. Cells located outside the root dentin, with abnormal morphology in irradiated mice, were positive for an odontoblast marker. HERS fragmentation occurred earlier in irradiated mice than in control mice, and HERS was trapped by the calcified apical tissue. A dose-dependent reduction in the number of proliferating cells within the apical dental pulp and periapical periodontal ligament surrounding HERS was observed in irradiated mice. Apoptotic cells in the dental pulp and periodontal ligament surrounding HERS were hardly seen. CONCLUSIONS: These results indicate that the early disappearance of HERS and the proliferative suppression of the surrounding mesenchymal cells, which was induced by irradiation, caused dental root malformation.

E-cadherin can replace N-cadherin during secretory-stage enamel development.

BACKGROUND: N-cadherin is a cell-cell adhesion molecule and deletion of N-cadherin in mice is embryonic lethal. During the secretory stage of enamel development, E-cadherin is down-regulated and N-cadherin is specifically up-regulated in ameloblasts when groups of ameloblasts slide by one another to form the rodent decussating enamel rod pattern. Since N-cadherin promotes cell migration, we asked if N-cadherin is essential for ameloblast cell movement during enamel development. METHODOLOGY/PRINCIPAL FINDINGS: The enamel organ, including its ameloblasts, is an epithelial tissue and for this study a mouse strain with N-cadherin ablated from epithelium was generated. Enamel from wild-type (WT) and N-cadherin conditional knockout (cKO) mice was analyzed. µCT and scanning electron microscopy showed that thickness, surface structure, and prism pattern of the cKO enamel looked identical to WT. No significant difference in hardness was observed between WT and cKO enamel. Interestingly, immunohistochemistry revealed the WT and N-cadherin cKO secretory stage ameloblasts expressed approximately equal amounts of total cadherins. Strikingly, E-cadherin was not normally down-regulated during the secretory stage in the cKO mice suggesting that E-cadherin can compensate for the loss of N-cadherin. Previously it was demonstrated that bone morphogenetic protein-2 (BMP2) induces E- and N-cadherin expression in human calvaria osteoblasts and we show that the N-cadherin cKO enamel organ expressed significantly more BMP2 and significantly less of the BMP antagonist Noggin than did WT enamel organ. CONCLUSIONS/SIGNIFICANCE: The E- to N-cadherin switch at the secretory stage is not essential for enamel development or for forming the decussating enamel rod pattern. E-cadherin can substitute for N-cadherin during these developmental processes. Bmp2 expression may compensate for the loss of N-cadherin by inducing or maintaining E-cadherin expression when E-cadherin is normally down-regulated. Notably, this is the first demonstration of a natural endogenous increase in E-cadherin expression due to N-cadherin ablation in a healthy developing tissue.

NBCe1 (SLC4A4) a potential pH regulator in enamel organ cells during enamel development in the mouse.

During the formation of dental enamel, maturation-stage ameloblasts express ion-transporting transmembrane proteins. The SLC4 family of ion-transporters regulates intra- and extracellular pH in eukaryotic cells by cotransporting HCO3 (-) with Na(+). Mutation in SLC4A4 (coding for the sodium-bicarbonate cotransporter NBCe1) induces developmental defects in human and murine enamel. We have hypothesized that NBCe1 in dental epithelium is engaged in neutralizing protons released during crystal formation in the enamel space. We immunolocalized NBCe1 protein in wild-type dental epithelium and examined the effect of the NBCe1-null mutation on enamel formation in mice. Ameloblasts expressed gene transcripts for NBCe1 isoforms B/D/C/E. In wild-type mice, weak to moderate immunostaining for NBCe1 with antibodies that recognized isoforms A/B/D/E and isoform C was seen in ameloblasts at the secretory stage, with no or low staining in the early maturation stage but moderate to high staining in the late maturation stage. The papillary layer showed the opposite pattern being immunostained prominently at the early maturation stage but with gradually less staining at the mid- and late maturation stages. In NBCe1 (-/-) mice, the ameloblasts were disorganized, the enamel being thin and severely hypomineralized. Enamel organs of CFTR (-/-) and AE2a,b (-/-) mice (CFTR and AE2 are believed to be pH regulators in ameloblasts) contained higher levels of NBCe1 protein than wild-type mice. Thus, the expression of NBCe1 in ameloblasts and the papillary layer cell depends on the developmental stage and possibly responds to pH changes.

Ionizing radiation effects on the secretory-stage ameloblasts and enamel organic extracellular matrix.

This study assessed the effects of high doses of ionizing radiation on eruption rate, odontogenic region morphology, secretory-stage ameloblasts, and enamel organic extracellular matrix (EOECM) of rat maxillary incisors. For the study, 30 male rats were divided into three experimental groups: control (non-irradiated), irradiated by 15 Gy, and irradiated by 25 Gy. Irradiated groups received a single dose of 15 or 25 Gy of X-rays in the head and neck region. The maxillary incisor eruption rate was measured. Sections of 5-µm thickness of the maxillary incisor odontogenic regions were evaluated using bright field light microscopy. Ultrathin sections of secretory ameloblasts and their EOECM were analyzed by transmission electron microscopy (TEM). Irradiated groups showed significantly diminished eruption rate values at the 4th and at the 6th day after irradiation. Reduced optical retardation values were observed in the irradiated groups. The odontogenic region of maxillary incisors from irradiated rats exhibited altered and poorly organized preameloblasts. TEM showed degeneration areas in the secretory-stage EOECM and several autophagosomes in the secretory ameloblasts from irradiated animals. In conclusion, high radiation doses delay eruption and induce disturbances in secretory ameloblasts and EOECM of rat maxillary incisors. These findings may be associated with structural defects of mature enamel.

Contribution of mesenchymal proliferation in tooth root morphogenesis.

In mouse tooth development, the roots of the first lower molar develop after crown formation to form 2 cylindrical roots by post-natal day 5. This study compared the morphogenesis and cellular events of the mesial-root-forming (MRF) and bifurcation-forming (BF) regions, located in the mesial and center of the first lower molar, to better define the developmental mechanisms involved in multi-rooted tooth formation. We found that the mesenchyme in the MRF showed relatively higher proliferation than the bifurcation region. This suggested that spatially regulated mesenchymal proliferation is required for creating cylindrical root structure. The mechanism may involve the mesenchyme forming a physical barrier to epithelial invagination of Hertwig's epithelial root sheath. To test these ideas, we cultured roots in the presence of pharmacological inhibitors of microtubule and actin polymerization, nocodazole and cytochalasin-D. Cytochalasin D also inhibits proliferation in epithelium and mesenchyme. Both drugs resulted in altered morphological changes in the tooth root structures. In particular, the nocodazole- and cytochalasin-D-treated specimens showed a loss of root diameter and formation of a single-root, respectively. Immunolocalization and three-dimensional reconstruction results confirmed these mesenchymal cellular events, with higher proliferation in MRF in multi-rooted tooth formation.

Analysis of the proliferative potential of odontogenic epithelial cells of pericoronal follicles.

AIM: To evaluate the proliferative potential and the cell proliferation rate of odontogenic epithelial cells. MATERIALS AND METHODS: Forty-two cases of pericoronal follicles of impacted third molars were submitted to silver impregnation technique for quantification of argyrophilic nucleolar organizer regions (AgNOR) and immunohistochemical staining for EGFR and Ki-67. For AgNOR quantification, the mean number of active nucleolar organizer regions per nucleus (mAgNOR) and the percentage of cells with 1, 2, 3 and 4 or more AgNORs per nucleus (pAgNOR) were quantified. Ki-67 immunolabeling was quantified, whereas for EGFR, a descriptive analysis of staining patterns (membrane, cytoplasm or membrane + cytoplasm positivity) was performed. We evaluated the reduced epithelium of the enamel organ and/or islands of odontogenic epithelium present in the entire connective tissue. RESULTS: mAgNOR were 1.43 (1.0-2.42) and were significantly different among pericoronary follicles from upper and lower teeth (p = 0.041). Immunostaining of Ki-67 was negative in all cases. EGFR immunolabeling was found mainly in the cytoplasm and was more intense in islands and cords when compared to reduced epithelium of the enamel organ. CONCLUSION: Odontogenic epithelial cells of some pericoronal follicles have proliferative potential, suggesting their association with the development of odontogenic lesions. CLINICAL SIGNIFICANCE: The authors suggest that nonerupted, especially of the lower teeth, should be monitored and if necessary removed.

MMP20 modulates cadherin expression in ameloblasts as enamel develops.

Matrix metalloproteinase-20 (enamelysin, MMP20) is essential for dental enamel development. Seven different MMP20 mutations in humans cause non-syndromic enamel malformations, termed amelogenesis imperfecta, and ablation of Mmp20 in mice results in thin brittle enamel with a dysplastic rod pattern. Healthy enamel formation requires the sliding movement of ameloblasts in rows during the secretory stage of development. This is essential for formation of the characteristic decussating enamel rod pattern observed in rodents, and this is also when MMP20 is secreted into the enamel matrix. Therefore, we propose that MMP20 facilitates ameloblast movement by cleaving ameloblast cell-cell contacts. Here we show that MMP20 cleaves the extracellular domains of the E- and N-cadherin adherens junction proteins, that both E- and N-cadherin transcripts are expressed at significantly higher levels in Mmp20 null vs. wild-type (WT) mice, and that in Mmp20 ablated mice, high-level ameloblast N-cadherin expression persists during the maturation stage of development. Furthermore, we show that E-cadherin gene expression is down-regulated from the pre-secretory to the secretory stage, while N-cadherin levels are up-regulated. This E- to N-cadherin switch supports epithelial migration in other tissues and may be an important event necessary for the ameloblasts to start moving in rows that slide by one another.

Reciprocal expressions between α-dystroglycan and integrin ß1, perlecan receptors, in the murine enamel organ development.

Signals of perlecan, an extracellular matrix molecule, which accumulates within the intercellular spaces of the stellate reticulum of the enamel organ, are mediated by at least two receptors, dystroglycan (DG) and integrin ß1, in a case-dependent manner in various events in embryogenesis and pathogenesis. This study aims to understand the expression profiles of these two perlecan receptors at both protein and gene levels in murine enamel organ development. Before birth, α-DG was immunolocalized in stellate reticulum cells, in which perlecan was colocalized, while integrin ß1 was mainly distributed in the peripheral enamel organ cells as well as the dental mesenchymal cells. On and after postnatal Day 1, the expression of α-DG was dramatically decreased in the stellate reticulum, while integrin ß1 was enhanced around blood vessels within the enamel organ. Furthermore, biosyntheses of α-DG and integrin ß1 by dental epithelial and pulp mesenchymal cells were confirmed in vitro by using immunofluorescence and reverse-transcriptase polymerase chain reaction. The results suggest that DG is a perlecan receptor that specifically functions in the stellate reticulum of the embryonic stage, but that dental epithelial and mesenchymal cells are maturated by capturing perlecan signals differentially through integrin ß1.

Isolation and enhancement of a homogenous in vitro human Hertwig's epithelial root sheath cell population.

Hertwig's epithelial root sheath (HERS) cells play a pivotal role during root formation of the tooth and are able to form cementum-like tissue. The aim of the present study was to establish a HERS cell line for molecular and biochemical studies using a selective digestion method. Selective digestion was performed by the application of trypsin-EDTA for 2 min, which led to the detachment of fibroblast-like-cells, with the rounded cells attached to the culture plate. The HERS cells displayed a typical cuboidal/squamous-shaped appearance. Characterization of the HERS cells using immunofluorescence staining and flow cytometry analysis showed that these cells expressed pan-cytokeratin, E-cadherin, and p63 as epithelial markers. Moreover, RT-PCR confirmed that these cells expressed epithelial-related genes, such as cytokeratin 14, E-cadherin, and ΔNp63. Additionally, HERS cells showed low expression of CD44 and CD105 with absence of CD34 and amelogenin expressions. In conclusion, HERS cells have been successfully isolated using a selective digestion method, thus enabling future studies on the roles of these cells in the formation of cementum-like tissue in vitro.