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Journal of Dental Research Aug 2023Tooth enamel is generated by ameloblasts. Any failure in amelogenesis results in defects in the enamel, a condition known as amelogenesis imperfecta. Here, we report...
Tooth enamel is generated by ameloblasts. Any failure in amelogenesis results in defects in the enamel, a condition known as amelogenesis imperfecta. Here, we report that mice with deficient autophagy in epithelial-derived tissues ( and conditional knockout mice) exhibit amelogenesis imperfecta. Micro-computed tomography imaging confirmed that enamel density and thickness were significantly reduced in the teeth of these mice. At the molecular level, ameloblast differentiation was compromised through ectopic accumulation and activation of NRF2, a specific substrate of autophagy. Through bioinformatic analyses, we identified , , , , , and as candidate genes related to amelogenesis imperfecta and the NRF2-mediated pathway. To investigate the effects of the ectopic NRF2 pathway activation caused by the autophagy deficiency, we analyzed target gene expression and NRF2 binding to the promoter region of candidate target genes and found suppressed gene expression of , , , and but not of and . Taken together, our findings indicate that autophagy plays a crucial role in ameloblast differentiation and that its failure results in amelogenesis imperfecta through ectopic NRF2 activation.
Topics: Mice; Animals; Ameloblasts; Amelogenesis Imperfecta; X-Ray Microtomography; NF-E2-Related Factor 2; Amelogenesis; Mice, Knockout; Tumor Suppressor Proteins; Repressor Proteins
PubMed: 37249312
DOI: 10.1177/00220345231169220 -
Journal of Dental Research Oct 2023Amelogenin plays a crucial role in tooth enamel formation, and mutations on X-chromosomal amelogenin cause X-linked amelogenesis imperfecta (AI). Amelogenin...
Amelogenin plays a crucial role in tooth enamel formation, and mutations on X-chromosomal amelogenin cause X-linked amelogenesis imperfecta (AI). Amelogenin pre-messenger RNA (mRNA) is highly alternatively spliced, and during alternative splicing, exon4 is mostly skipped, leading to the formation of a microRNA (miR-exon4) that has been suggested to function in enamel and bone formation. While delivering the functional variation of amelogenin proteins, alternative splicing of exon4 is the decisive first step to producing miR-exon4. However, the factors that regulate the splicing of exon4 are not well understood. This study aimed to investigate the association between known mutations in exon4 and exon5 of X chromosome amelogenin that causes X-linked AI, the splicing of exon4, and miR-exon4 formation. Our results showed mutations in exon4 and exon5 of the amelogenin gene, including c.120T>C, c.152C>T, c.155C>G, and c.155delC, significantly affected the splicing of exon4 and subsequent miR-exon4 production. Using an amelogenin minigene transfected in HEK-293 cells, we observed increased inclusion of exon4 in amelogenin mRNA and reduced miR-exon4 production with these mutations. In silico analysis predicted that Ser/Arg-rich RNA splicing factor (SRSF) 2 and SRSF5 were the regulatory factors for exon4 and exon5 splicing, respectively. Electrophoretic mobility shift assay confirmed that SRSF2 binds to exon4 and SRSF5 binds to exon5, and mutations in each exon can alter SRSF binding. Transfection of the amelogenin minigene to LS8 ameloblastic cells suppressed expression of the known miR-exon4 direct targets, and , related to multiple pathways. Given the mutations on the minigene, the expression of has been significantly upregulated with c.155C>G and c.155delC mutations. Together, we confirmed that exon4 splicing is critical for miR-exon4 production, and mutations causing X-linked AI in exon4 and exon5 significantly affect exon4 splicing and following miR-exon4 production. The change in miR-exon4 would be an additional etiology of enamel defects seen in some X-linked AI.
Topics: Humans; Amelogenin; Amelogenesis Imperfecta; HEK293 Cells; Mutation; Dental Enamel Proteins; MicroRNAs; RNA, Messenger
PubMed: 37563801
DOI: 10.1177/00220345231180572 -
Frontiers in Cell and Developmental... 2023Tooth formation relies on two types of dental cell populations, namely, the dental epithelium and dental mesenchyme, and the interactions between these cell populations...
Tooth formation relies on two types of dental cell populations, namely, the dental epithelium and dental mesenchyme, and the interactions between these cell populations are important during tooth development. Although human-induced pluripotent stem cells (hiPSCs) can differentiate into dental epithelial and mesenchymal cells, organoid research on tooth development has not been established yet. This study focused on the hiPSC-derived human ameloblast organoid (hAO) using a three-dimensional (3D) culture system. hAOs had similar properties to ameloblasts, forming enamel in response to calcium and mineralization by interaction with the dental mesenchyme. hAOs simultaneously had osteogenic and odontogenic differentiation potential. Furthermore, hAOs demonstrated tooth regenerative potential upon interaction with the mouse dental mesenchyme. Our findings provide new insights into a suitable hiPSC-derived dental source and demonstrate that hAOs can be beneficial not only for tooth regeneration but also for the study of various dental diseases for which treatment has not been developed yet.
PubMed: 37457296
DOI: 10.3389/fcell.2023.1164811 -
Frontiers in Physiology 2023Mouse and human genetic studies indicate key roles of the ligand in odontogenesis. Previous studies have identified effectors and regulators of the Wnt signaling...
Mouse and human genetic studies indicate key roles of the ligand in odontogenesis. Previous studies have identified effectors and regulators of the Wnt signaling pathway actively expressed during key stages of tooth morphogenesis. However, limitations in multiplexing and spatial resolution hindered a more comprehensive analysis of these signaling molecules. Here, profiling of transcriptomes using fluorescent multiplex hybridization and single-cell RNA-sequencing (scRNA-seq) provide robust insight into the synchronized expression patterns of , , and simultaneously during tooth development. First, we identified transcripts restricted to the epithelium at the stage of tooth bud morphogenesis, contrasting that of and localization to the dental mesenchyme. By embryonic day 15.5 (E15.5), a marked shift of expression from dental epithelium to mesenchyme was noted, while and expression remained enriched in the mesenchyme. By postnatal day 0 (P0), co-localization patterns of , , and were observed in both terminally differentiating and secreting odontoblasts of molars and incisors. Interestingly, exhibited robust expression in fully differentiated ameloblasts at the developing cusp tip of both molars and incisors, an observation not previously noted in prior studies. At P7 and 14, after the mineralization of dentin and enamel, expression was limited to odontoblasts. Meanwhile, Wnt modulators showed reduced or absent signals in molars. In contrast, strong signals persisted in ameloblasts (for ) and odontoblasts (for , , and ) towards the proximal end of incisors, near the cervical loop. Our scRNA-seq analysis used CellChat to further contextualize Wnt pathway-mediated communication between cells by examining ligand-receptor interactions among different clusters. The co-localization pattern of , , and in both terminally differentiating and secreting odontoblasts of molars and incisors potentially signifies the crucial ligand-modulator interaction along the gradient of cytodifferentiation starting from each cusp tip towards the apical region. These data provide cell type-specific insight into the role of Wnt ligands and mediators during epithelial-mesenchymal interactions in odontogenesis.
PubMed: 38274045
DOI: 10.3389/fphys.2023.1316635 -
International Journal of Oral Science Dec 2023Ameloblasts are specialized cells derived from the dental epithelium that produce enamel, a hierarchically structured tissue comprised of highly elongated...
Ameloblasts are specialized cells derived from the dental epithelium that produce enamel, a hierarchically structured tissue comprised of highly elongated hydroxylapatite (OHAp) crystallites. The unique function of the epithelial cells synthesizing crystallites and assembling them in a mechanically robust structure is not fully elucidated yet, partly due to limitations with in vitro experimental models. Herein, we demonstrate the ability to generate mineralizing dental epithelial organoids (DEOs) from adult dental epithelial stem cells (aDESCs) isolated from mouse incisor tissues. DEOs expressed ameloblast markers, could be maintained for more than five months (11 passages) in vitro in media containing modulators of Wnt, Egf, Bmp, Fgf and Notch signaling pathways, and were amenable to cryostorage. When transplanted underneath murine kidney capsules, organoids produced OHAp crystallites similar in composition, size, and shape to mineralized dental tissues, including some enamel-like elongated crystals. DEOs are thus a powerful in vitro model to study mineralization process by dental epithelium, which can pave the way to understanding amelogenesis and developing regenerative therapy of enamel.
Topics: Mice; Animals; Durapatite; Dental Enamel; Ameloblasts; Amelogenesis; Stem Cells; Organoids
PubMed: 38062012
DOI: 10.1038/s41368-023-00257-w -
Regenerative Therapy Dec 2024Understanding the composition and function of cells constituting tissues and organs is vital for unraveling biological processes. Single-cell analysis has allowed us to... (Review)
Review
Understanding the composition and function of cells constituting tissues and organs is vital for unraveling biological processes. Single-cell analysis has allowed us to move beyond traditional methods of categorizing cell types. This innovative technology allows the transcriptional and epigenetic profiling of numerous individual cells, leading to significant insights into the development, homeostasis, and pathology of various organs and tissues in both animal models and human samples. In this review, we delve into the outcomes of major investigations using single-cell transcriptomics to decipher the cellular composition of mammalian teeth and periodontal tissues. The recent single-cell transcriptome-based studies have traced in detail the dental epithelium-ameloblast lineage and dental mesenchyme lineages in the mouse incisors and the tooth germ of both mice and humans; unraveled the microenvironment, the identity of niche cells, and cellular intricacies in the dental pulp; shed light on the molecular mechanisms orchestrating root formation; and characterized cellular dynamics of the periodontal ligament. Additionally, cellular components in dental pulps were compared between healthy and carious teeth at a single-cell level. Each section of this review contributes to a comprehensive understanding of tooth biology, offering valuable insights into developmental processes, niche cell identification, and the molecular secrets of the dental environment.
PubMed: 38596822
DOI: 10.1016/j.reth.2024.03.023 -
Ecotoxicology and Environmental Safety Jul 2023Perfluorooctanoic acid (PFOA) is an artificial fluorinated organic compound that has generated increased public attention due to its potential health hazards. Unsafe...
Perfluorooctanoic acid (PFOA) is an artificial fluorinated organic compound that has generated increased public attention due to its potential health hazards. Unsafe levels of PFOA exposure can affect reproduction, growth and development. During tooth enamel development (amelogenesis), environmental factors including fluoride can cause enamel hypoplasia. However, the effects of PFOA on ameloblasts and tooth enamel formation remain largely unknown. In the present study we demonstrate several PFOA-mediated cell death pathways (necrosis/necroptosis, and apoptosis) and assess the roles of ROS-MAPK/ERK signaling in PFOA-mediated cell death in mouse ameloblast-lineage cells (ALC). ALC cells were treated with PFOA. Cell proliferation and viability were analyzed by MTT assays and colony formation assays, respectively. PFOA suppressed cell proliferation and viability in a dose dependent manner. PFOA induced both necrosis (PI-positive cells) and apoptosis (cleaved-caspase-3, γH2AX and TUNEL-positive cells). PFOA significantly increased ROS production and up-regulated phosphor-(p)-ERK. Addition of ROS inhibitor N-acetyl cysteine (NAC) suppressed p-ERK and decreased necrosis, and increased cell viability compared to PFOA alone, whereas NAC did not change apoptosis. This suggests that PFOA-mediated necrosis was induced by ROS-MAPK/ERK signaling, but apoptosis was not associated with ROS. Addition of MAPK/ERK inhibitor PD98059 suppressed necrosis and increased cell viability compared to PFOA alone. Intriguingly, PD98059 augmented PFOA-mediated apoptosis. This suggests that p-ERK promoted necrosis but suppressed apoptosis. Addition of the necroptosis inhibitor Necrostatin-1 restored cell viability compared to PFOA alone, while pan-caspase inhibitor Z-VAD did not mitigate PFOA-mediated cell death. These results suggest that 1) PFOA-mediated cell death was mainly caused by necrosis/necroptosis by ROS-MAPK/ERK signaling rather than apoptosis, 2) MAPK/ERK signaling plays the dual roles (promoting necrosis and suppressing apoptosis) under PFOA treatment. This is the initial report to indicate that PFOA could be considered as a possible causative factor for cryptogenic enamel malformation. Further studies are required to elucidate the mechanisms of PFOA-mediated adverse effects on amelogenesis.
Topics: Mice; Animals; Reactive Oxygen Species; Ameloblasts; Cell Death; Necrosis
PubMed: 37271104
DOI: 10.1016/j.ecoenv.2023.115089 -
Heliyon Mar 2024The junctional epithelium (JE) serves a crucial protective role in the periodontium. High glucose-related aging results in accelerated barrier dysfunction of the...
The junctional epithelium (JE) serves a crucial protective role in the periodontium. High glucose-related aging results in accelerated barrier dysfunction of the gingival epithelium, which may be associated with diabetic periodontitis. Metformin, an oral hypoglycemic therapeutic, has been proposed as a anti-aging agent. This study aimed to clarify the effect of metformin on diabetic periodontitis and explore its mechanism in ameliorating senescence of JE during hyperglycemia. The db/db mice was used as a diabetic model mice and alterations in the periodontium were observed by hematoxylin-eosin staining and immunohistochemistry. An ameloblast-like cell line (ALC) was cultured with high glucose to induce senescence. Cellular senescence and oxidative stress were evaluated by SA-β-gal staining and Intracellular reactive oxygen species (ROS) levels. Senescence biomarkers, P21 and P53, and autophagy markers, LC3-II/LC3-I, were measured by western blotting and quantitative real-time PCR. To construct a stable SIRT1 (Sirtuin 1) overexpression cell line, we transfected ALCs with lentiviral vectors overexpressing the mouse SIRT1 gene. Cellular senescence was increased in the JE of db/db mice and the periodontium was destroyed, which could be alleviated by metformin. Moreover, oxidative stress and cellular senescence in a high glucose environment were reduced by metformin in assays. The autophagy inhibitor 3-MA and SIRT1 inhibitor EX-527 could dampen the effects of metformin. Overexpression of SIRT1 resulted in increased autophagy and decreased oxidative stress and cellular senescence. Meanwhile, AMPK (AMP-activated protein kinase) inhibition reversed the anti-senescence effects of metformin. Overall, these results suggest that metformin alleviates periodontal damage in db/db mice and cellular senescence in ALCs under high glucose conditions via the AMPK/SIRT1/autophagy pathway.
PubMed: 38496895
DOI: 10.1016/j.heliyon.2024.e27478 -
Scientific Reports Nov 2023Enamel forming ameloblasts move away from the dentino-enamel junction and also move relative to each other to establish enamel shape during the secretory stage of enamel...
Enamel forming ameloblasts move away from the dentino-enamel junction and also move relative to each other to establish enamel shape during the secretory stage of enamel development. Matrix metalloproteinase-20 (MMP20) is a tooth specific proteinase essential for proper enamel formation. We previously reported that MMP20 cleaves cadherins and may regulate ameloblast movement. Here, we used an Amelx promoter driven tdTomato reporter to label mouse ameloblasts. With these transgenic mice, we assessed ameloblast mobility group dynamics and gene expression. Three-dimensional imaging of mouse ameloblasts were observed in hemi-mandibles by using a tissue clearing technique. The three-dimensional ameloblast layer in Tg(Amelx-Mmp20) mice that overexpress MMP20 was uneven and the ameloblasts migrated away from this layer. Mouse ameloblast movement toward incisal tips was monitored by ex vivo time-lapse imaging. Gene expression related to cell migration and adhesion was analyzed in ameloblasts from wild-type mice, Mmp20 mice with no functional MMP20 and from Tg(Amelx-Mmp20) overexpressing mice. Gene expression was altered in Mmp20 and Tg(Amelx-Mmp20) mice compared to wild type. Among the genes assessed, those encoding laminins and a gap junction protein were upregulated in Mmp20 mice. New techniques and findings described in this study may lead to an improved understanding of ameloblast movement during enamel formation.
Topics: Mice; Animals; Ameloblasts; Matrix Metalloproteinase 20; Mice, Transgenic; Cadherins; Gene Expression
PubMed: 37914726
DOI: 10.1038/s41598-023-44627-0 -
Archives of Razi Institute Aug 2023Amoxicillin is one of the most commonly prescribed antibiotics in children. As a result, it is prescribed as the first line of defence against cutaneous,...
Amoxicillin is one of the most commonly prescribed antibiotics in children. As a result, it is prescribed as the first line of defence against cutaneous, gastrointestinal, and respiratory infections. The objective of this study was to evaluate the effects of Amoxicillin on the formation of dentin and enamel during the secretory and early phases of mineralization. Regarding the materials and methods used to perform this study, 16 pregnant adult Wistar rats were equally divided into two groups. The first group did not receive the drug and was prescribed a saline solution (control group), and the other group received 250 mg/kg/day of Amoxicillin (experimental group). From the 13 gestational day until delivery, the treatment was given every day by oral gavage. After birth, the newborns also received the same treatment as their mothers from the first day until 7 or 12 days after birth. The newborns were sacrificed at 7 and 12 days postnatally, the jaws were dissected, the maxilla was taken, the samples were fixed in 10% formaldehyde solution, and the upper first molars were analyzed histologically by H & E stain and histomorphometrically by image J to examine the enamel, dentin, ameloblast and odontoblast mean thickness in both groups and each healing periods. The study's results showed that the mean enamel, as well as ameloblastic and odontoblastic layer thickness, were significantly different in the Amoxicillin 250 mg/kg group, compared to the control group. The result also revealed a non-significant group difference in the dentin thickness in both durations (P-value at day 7=0.147 and the P-value at day 12=0.054). Vacuolization of the ameloblastic and odontoblastic layers was observed in the Amoxicillin-treated group in both durations.
Topics: Infant, Newborn; Humans; Pregnancy; Female; Child; Rats; Animals; Amoxicillin; Rats, Wistar; Anti-Bacterial Agents; Wound Healing
PubMed: 38226389
DOI: 10.32592/ARI.2023.78.4.1333