Objective: Insulin-like growth factor-binding protein 3 (IGFBP3) is a multifunctional protein involved in various cellular functions. However, the function of IGFBP3 in periodontal ligament (PDL) tissue remains unclear. In this study, we investigated the localization and function of IGFBP3 in PDL tissues and human PDL-derived cell line. Design: Small interfering RNA (siRNA) and recombinant protein of IGFBP3 were used to examine the effect of IGFBP3 in human PDL-derived cell line. mRNA expression was determined by quantitative reverse transcriptionpolymerase chain reaction. Protein expression was determined using immunohistochemical staining, immunofluorescence staining, and Western blotting analyses. WST-1 and migration assays were used to analyze the effects on proliferation and migration. Collagen production was examined using picrosirius red staining. Calcium nodule formation was examined using Alizarin Red S staining. Results: IGFBP3 was expressed in both mouse PDL tissues and human PDL-derived cell line (2-23 cells). Mechanical stretch and Transforming growth factor-beta 1 (TGF-(31) stimulation upregulated IGFBP3 expression in 2-23 cells. Knockdown of IGFBP3 using siRNA significantly suppressed PDL-related gene expression, collagen production, and inhibited Smad2/3 phosphorylation induced by TGF-(31, while IGFBP3 knockdown enhanced calcium chloride-induced osteogenic differentiation in 2-23 cells and activated Akt phosphorylation. Furthermore, treatment with exogenous rhIGFBP3 showed the opposite trend. Conclusions: IGFBP3 plays a dual role in PDL homeostasis, by promoting collagen production and inhibiting osteogenic differentiation. IGFBP3 is involved in TGF-(3-Smad2/3 pathway and related with phosphorylation of Akt, highlighting IGFBP3 as a potential therapeutic target for periodontal regeneration.

Background/purpose:Medication-related osteonecrosis of the jaw (MRONJ), a serious adverse effect of antiresorptive agents, remains difficult to manage. Our previous studies showed that statins, widely prescribed for dyslipidemia, promote healing in MRONJ-like lesions, however, the mechanisms underlying their therapeutic effects remain unclear. This study investigated the efficacy of the therapeutic effect of fluvastatin (FS) in a rat MRONJ-like model, with particular attention to macrophage polarization. Materials and methods:Four-week-old female rats were allocated to three groups. Two groups received zoledronic acid (ZOL) and dexamethasone (DEX) for two weeks prior to extraction of maxillary first molars. After extraction, one group was treated with a single local injection of FS near the extraction site, whereas the other received saline. A control group underwent identical procedures, but received saline instead of ZOL, DEX, and FS. All animals were evaluated two weeks after treatment. Results:The control (saline) group showed complete socket healing. In contrast, ZOL/DEX-treated rats exhibited incomplete epithelial closure and exposed necrotic bone. However, local FS administration in ZOL/DEX-treated rats extensively promoted healing, resulting in smaller epithelial gaps and reduced bone exposure, and decreased areas of necrotic bone. Total macrophage numbers were significantly higher in both experimental groups compared with the control group. The ZOL/DEX group showed a predominance of pro-inflammatory M1 macrophages, whereas the FS-treated group exhibited relatively increased proportions of tissue-reparative M2 macrophages. Conclusion:Local FS administration enhances bone healing of MRONJ-like lesions by promoting macrophage polarization toward M2 phenotype.

Background and objective Oral metastases are rare. We experienced a case of oral metastasis of renal cell carcinoma (RCC) and discussed its characteristics with a systematic review to survey the literature for useful features for the diagnosis of oral metastatic RCC. Methods A systematic review was conducted according to the PRISMA 2020 statement. An electronic search was performed using three databases and the literature of cases of renal cancer metastasis to the jawbone and gingiva. Results Thirty studies were identified (male-to-female ratio, 2.75:1). The site of metastasis was the jawbone in 66.7% (mandible, 60%; maxilla, 6.7%) and the gingiva in 30%. Clinical symptoms were a mass in 86.7% and hypoesthesia in 23.3%. A total of 76.7% of oral metastases were clear cell type. In our case, the male patient showed a mass in the maxillary gingiva without hypoesthesia. Metastatic clear cell RCC was suspected by an oral biopsy, which was confirmed on clinical examinations, including a renal biopsy. Conclusion Oral metastases are rare, and the clinical and imaging findings for oral metastases of RCC are wide-ranged with low specificity, depending on the metastatic site. It is necessary to fully understand the key points in histopathological differentiation. Because the clinician plays a fundamental role in the early diagnosis of oral cancer and the identification of metastatic lesions, awareness of the possibility of rare tumors as in this study can facilitate an accurate preoperative diagnosis and the planning of appropriate treatment.

Cellular responses to mechanical stimulation are involved in tissue development and the maintenance of biological functions. Teeth function as receptors for mastication and occlusal pressure. During tooth development, the tooth germ begins with an invagination of the epithelium, and its morphology matures through dynamic interactions between epithelial cells and mesenchymal cells, suggesting that mechanosensors may play an important role in this process. We analyzed the expression and function of Piezo1, a mechanically activated ion channel, during tooth development and clarified the involvement of Piezo1 in tooth morphogenesis. The expression of Piezo1 was observed in both the enamel organ and the surrounding mesenchymal cells at the early stage and in the ameloblasts and odontoblasts during enamel and dentin matrix formation. Yoda1, a Piezo1 activator, inhibited cell proliferation in mouse dental epithelial (mDE6) cells and E15 tooth germs, and suppressed cell migration in mDE6 cells. Meanwhile, GsMTx4, a Piezo1 inactivator, showed opposite results. Furthermore, in the organ culture of E15 tooth germs, the activation and inactivation of Piezo1 were found to affect the expression of ameloblast differentiation marker genes and control the arrangement of ameloblasts. Interestingly, the expression of E-cadherin was reduced in the cell membrane of ameloblasts at the cusp in the GsMTx4-treated tooth germs of organ culture, and enamel formation was significantly decreased. Yoda1-treated mDE6 cells showed upregulated E-cadherin expression, which was downregulated by calpain inhibitor. These findings suggest that Piezo1 may be involved in tooth morphogenesis during ameloblast development by playing an essential role in cell proliferation, migration, arrangement, differentiation, and mineralization.

OBJECTIVE:Insulin-like growth factor-binding protein 3 (IGFBP3) is a multifunctional protein involved in various cellular functions. However, the function of IGFBP3 in periodontal ligament (PDL) tissue remains unclear. In this study, we investigated the localization and function of IGFBP3 in PDL tissues and human PDL-derived cell line. DESIGN:Small interfering RNA (siRNA) and recombinant protein of IGFBP3 were used to examine the effect of IGFBP3 in human PDL-derived cell line. mRNA expression was determined by quantitative reverse transcription-polymerase chain reaction. Protein expression was determined using immunohistochemical staining, immunofluorescence staining, and Western blotting analyses. WST-1 and migration assays were used to analyze the effects on proliferation and migration. Collagen production was examined using picrosirius red staining. Calcium nodule formation was examined using Alizarin Red S staining. RESULTS:IGFBP3 was expressed in both mouse PDL tissues and human PDL-derived cell line (2-23 cells). Mechanical stretch and Transforming growth factor-beta 1 (TGF-β1) stimulation upregulated IGFBP3 expression in 2-23 cells. Knockdown of IGFBP3 using siRNA significantly suppressed PDL-related gene expression, collagen production, and inhibited Smad2/3 phosphorylation induced by TGF-β1, while IGFBP3 knockdown enhanced calcium chloride-induced osteogenic differentiation in 2-23 cells and activated Akt phosphorylation. Furthermore, treatment with exogenous rhIGFBP3 showed the opposite trend. CONCLUSIONS:IGFBP3 plays a dual role in PDL homeostasis, by promoting collagen production and inhibiting osteogenic differentiation. IGFBP3 is involved in TGF-β-Smad2/3 pathway and related with phosphorylation of Akt, highlighting IGFBP3 as a potential therapeutic target for periodontal regeneration.

Manuka oil and tea tree oil are essential oils with known antibacterial properties that are believed to be caused by one main component: terpinen-4-ol. Terpinen-4-ol has potent antibacterial activity against caries-related microorganisms. However, few studies have investigated the antimicrobial effects of terpinen-4-ol on bacteria in apical periodontitis. Thus, the objective of the present study was to evaluate the antibacterial and antibiofilm potential of terpinen-4-ol against Enterococcus faecalis, Porphyromonas gingivalis, Prevotella intermedia, and Fusobacterium nucleatum, which have all been detected in apical periodontitis. The minimum inhibitory and minimum bactericidal concentrations of terpinen-4-ol were determined to assess its activity against biofilms. The minimum inhibitory concentration of terpinen-4-ol was 0.25% against E. faecalis and F. nucleatum, 0.05% against P. gingivalis, and 0.1% against P. intermedia. The minimum bactericidal concentration of terpinen-4-ol was 1.0% against E. faecalis, 0.2% against P. gingivalis and P. intermedia, and 0.5% against F. nucleatum. In the biofilm evaluations, all terpinen-4-ol-treated bacteria had significant reductions in biofilm viability compared with controls in experiments assessing attachment inhibitory activity. Furthermore, structural alterations and decreased bacterial cell clumping were observed under scanning electron microscopy, and significantly decreased cell survival was noted using fluorescence microscopy. Together, these results suggest that terpinen-4-ol is a potential antibacterial agent with bactericidal properties, and can also act on established biofilms.

Background/purpose: Actin alpha 2, smooth muscle (ACTA2) is an actin isoform that forms the cytoskeleton. Actin plays a crucial role in numerous cellular functions. ACTA2 is a marker of functional periodontal ligament (PDL) fibroblasts and is upregulated by transforming growth factor-beta 1 (TGF-beta 1); however, the underlying function of ACTA2 in PDL tissue is unknown. We aimed to examine the localization and potential function of ACTA2 in PDL tissues and cells. Materials and methods: RNA expression was determined using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and quantitative RT-PCR. Protein expression was determined using immunofluorescence staining and Western blot analysis. Soluble and insoluble collagen production was examined using the Sircol collagen assay and picrosirius red staining, respectively. Small interfering RNA (siRNA) was used for knockdown assay to examine the effect of ACTA2 in human PDL cells. Results: ACTA2 expression was observed in human primary PDL cells and PDL cell line (2-23 cells). TGF-81 upregulated ACTA2, collagen type I alpha1 chain (COL1A1), periostin (POSTN), and fibrillin-I(FBN1) expression and soluble and insoluble collagen production in 2-23 cells. However, ACTA2 depletion by siRNA strongly suppressed PDL-related gene expression and collagen production compared with those of TGF-beta 1-stimulated control cells. Furthermore, ACTA2 knockdown significantly suppressed the phosphorylation of Smad2 and Conclusion: ACTA2 plays a crucial role in PDL-related marker expression and collagen production via Smad2/3 phosphorylation. Our findings might contribute to the development of novel and effective periodontal therapies. (c) 2022 Association for Dental Sciences of the Republic of China. Publishing services by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Medication-related osteonecrosis of the jaw (MRONJ) is an intractable disease that is typically observed in patients with osteoporosis or tumors that have been treated with either bisphosphonate (BP) or antiangiogenic medicine. The mechanism of MRONJ pathogenesis remains unclear, and no effective definitive treatment modalities have been reported to date. Previous reports have indicated that a single injection of benidipine, an antihypertensive calcium channel blocker, in the vicinity of a tooth extraction socket promotes wound healing in healthy rats. The present study was conducted to elucidate the possibility of using benidipine to promote the healing of MRONJ-like lesions. In this study, benidipine was administered near the site of MRONJ symptom onset in a model rat, which was then sacrificed two weeks after benidipine injection, and analyzed using histological sections and CT images. The analysis showed that in the benidipine groups, necrotic bone was reduced, and soft tissue continuity was recovered. Furthermore, the distance between epithelial edges, length of necrotic bone exposed in the oral cavity, necrotic bone area, and necrotic bone ratio were significantly smaller in the benidipine group. These results suggest that a single injection of benidipine in the vicinity of MRONJ-like lesions can promote osteonecrotic extraction socket healing.

Introduction Dental caries and apical periodontitis are ones of the most prevalent chronic diseases and involve infection by cariogenic and endodontic bacteria. It can be said that the most required to cure is disinfection. We hypothesized that NB-UVB could be used for intraoral disinfection without affecting cells, and that it could be used in combination with TiO 2 to disinfect complex root canals. The objectives were to investigate the effects of UV on dental pulp cells and oral bacteria and to evaluate the enhancement effect of a photocatalyst on bactericidal effects of UV irradiation. Methods UV irradiation devices of UVB (310, 285 nm) and UVC (265 nm) were prepared. Cell proliferation and cytotoxicity assays after UV irradiation were performed using human dental pulp cells. The antibacterial activity of UV irradiation was investigated in Streptococcus mutans , Staphylococcus aureus , Enterococcus faecalis , Actinomyces naeslundii , Porphyromonas gingivalis , and Fusobacterium nucleatum. A curved simulated root canal with plastic training block was used to evaluate the effect of combined UV and TiO 2 treatment. Data were analyzed by one-way ANOVA ( p < 0.05) followed by Tukey’s post hoc tests ( p < 0.05). Results Human dental pulp cell proliferation was decreased by 265 nm, 285 nm, and 310 nm UV irradiation, although 310 nm UV irradiation did not show cytotoxic effects on these cells. Oral bacterial growth was suppressed following UV irradiation at 285 nm and 265 nm. Viability of all bacteria significantly decreased with UV irradiation. In the curved simulated root canal, viability of E. faecalis in UV irradiation at 285 nm with long taper fibers was significantly decreased in the 300 s irradiation group. E. faecalis proliferation was inhibited by combined UV irradiation and TiO 2 application with long taper fibers in the curved simulated root canal. Conclusions The wavelength of UVB and UVC showed bactericidal effects on oral bacteria including caries-related bacteria and apical periodontitis–related bacteria while NB-UVB did not. UVB with longer fibers was more effective in disinfection on E. faecalis in curved simulated root canal, and the combined use of photocatalyst further improved the disinfection effect.

We recently demonstrated that Semaphorin 3 A (Sema3A), the expression of which is negatively regulated by Wnt/β-catenin signaling, promotes odontogenic epithelial cell proliferation, suggesting the involvement of Sema3A in tooth germ development. Salivary glands have a similar developmental process to tooth germ development, in which reciprocal interactions between the oral epithelium and adjacent mesenchyme proceeds via stimulation with several growth factors; however, the role of Sema3A in the development of salivary glands is unknown. There may thus be a common mechanism between epithelial morphogenesis and pathogenesis; however, the role of Sema3A in salivary gland tumors is also unclear. The current study investigated the involvement of Sema3A in submandibular gland (SMG) development and its expression in adenoid cystic carcinoma (ACC) specimens. Quantitative RT-PCR and immunohistochemical analyses revealed that Sema3A was expressed both in epithelium and in mesenchyme in the initial developmental stages of SMG and their expressions were decreased during the developmental processes. Loss-of-function experiments using an inhibitor revealed that Sema3A was required for AKT activation-mediated cellular growth and formation of cleft and bud in SMG rudiment culture. In addition, Wnt/β-catenin signaling decreased the Sema3A expression in the rudiment culture. ACC arising from salivary glands frequently exhibits malignant potential. Immunohistochemical analyses of tissue specimens obtained from 10 ACC patients showed that Sema3A was hardly observed in non-tumor regions but was strongly expressed in tumor lesions, especially in myoepithelial neoplastic cells, at high frequencies where phosphorylated AKT expression was frequently detected. These results suggest that the Sema3A-AKT axis promotes cell growth, thereby contributing to morphogenesis and pathogenesis, at least in ACC, of salivary glands.

Most human malignant tumor cells arise from epithelial tissues, which show distinctive characteristics, such as polarization, cell-to-cell contact between neighboring cells, and anchoring to a basement membrane. When tumor cells invaginate into the stroma, the cells are exposed to extracellular environments, including the extracellular matrix (ECM). Increased ECM stiffness has been reported to promote cellular biological activities, such as excessive cellular growth and enhanced migration capability. Therefore, tumorous ECM stiffness is not only an important clinical tumor feature but also plays a pivotal role in tumor cell behavior. Transient receptor potential vanilloid 4 (TRPV4), a Ca 2+ -permeable nonselective cation channel, has been reported to be mechano-sensitive and to regulate tumorigenesis, but the underlying molecular mechanism in tumorigenesis remains unclear. The function of TRPV4 in oral squamous cell carcinoma (OSCC) is also unknown. The current study was conducted to investigate whether or not TRPV4 might be involved in OSCC tumorigenesis. TRPV4 mRNA levels were elevated in OSCC cell lines compared with normal oral epithelial cells, and its expression was required for TRPV4 agonist-dependent Ca 2+ entry. TRPV4-depleted tumor cells exhibited decreased proliferation capabilities in three-dimensional culture but not in a low-attachment plastic dish. A xenograft tumor model demonstrated that TRPV4 expression was involved in cancer cell proliferation in vivo. Furthermore, loss-of-function experiments using siRNA or an inhibitor revealed that the TRPV4 expression was required for CaMKII-mediated AKT activation. Immunohistochemical analyses of tissue specimens obtained from 36 OSCC patients showed that TRPV4 was weakly observed in non-tumor regions but was strongly expressed in tumor lesions at high frequencies where phosphorylated AKT expression was frequently detected. These results suggest that the TRPV4/CaMKII/AKT axis, which might be activated by extracellular environments, promotes OSCC tumor cell growth.

Fibro-osseous and osteochondromatous lesions are non-neoplastic lesions containing metaplastic bone and/or cementum. The World Health Organization classification (4th edition, 2017) has categorized these lesions into subgroups: ossifying fibroma, familial gigantiform cementoma, fibrous dysplasia, cemento-osseous dysplasia and osteochondroma. We report a highly unusual case of cemento-osseous dysplasia in a 32-year-old male complaining of discomfort of the left cheek. The radiographic examination revealed that a spherical radiopaque lesion, which involved a tooth, was located in the left maxillary sinus. A definitive diagnosis could not be made based only on the radiographic and clinical findings. An excisional biopsy was performed by opening the left maxillary sinus under the general anaesthesia. Histopathological examination revealed that the lesion consists of fibrous tissue, woven bone and masses of cementum-like material associated with a dislocated tooth. Together with the radiographic, clinical and histopathological findings, the lesion was diagnosed as cemento-osseous dysplasia. Differential diagnosis between fibro-osseous lesions and tumor is critical; the present case highlights the importance of correlating clinical, radiological and histopathological findings in order to correctly diagnose and manage oral and maxillofacial lesions.

Undifferentiated odontogenic epithelium and dental papilla cells differentiate into ameloblasts and odontoblasts, respectively, both of which are essential for tooth development. These differentiation processes involve dramatic functional and morphological changes of the cells. For these changes to occur, activation of mitochondrial functions, including ATP production, is extremely important. In addition, these changes are closely related to mitochondrial fission and fusion, known as mitochondrial dynamics. However, few studies have focused on the role of mitochondrial dynamics in tooth development. The purpose of this study was to clarify this role. We used mouse tooth germ organ cultures and a mouse dental papilla cell line with the ability to differentiate into odontoblasts, in combination with knockdown of the mitochondrial fission factor, dynamin related protein (DRP)1. In organ cultures of the mouse first molar, tooth germ developed to the early bell stage. The amount of dentin formed under DRP1 inhibition was significantly larger than that of the control. In experiments using a mouse dental papilla cell line, differentiation into odontoblasts was enhanced by inhibiting DRP1. This was associated with increased mitochondrial elongation and ATP production compared to the control. These results suggest that DRP1 inhibition accelerates dentin formation through mitochondrial elongation and activation. This raises the possibility that DRP1 might be a therapeutic target for developmental disorders of teeth.

Purpose Keratin 17 (KRT17) has been suggested as a potential diagnostic marker of squamous cell carcinoma including oral squamous cell carcinoma (OSCC). The current study was conducted to clarify the function of KRT17 and its expression mechanism in OSCC.Methods Immunohistochemical analyses were carried out to examine the expression of KRT17, GLI family zinc finger (GLI)-1, GLI-2, or cleaved caspase-3 in OSCCs. The expression of KRT17, GLI-1, or GLI-2 was investigated among OSCC cell lines, and the effects of loss-of-function of KRT17 or GLI, using siRNA or inhibitor, on the cell growth of the OSCC cell line HSC-2 particularly with respect to apoptosis were examined.Results Immunohistochemical analyses of tissue specimens obtained from 78 OSCC patients revealed that KRT17 was not observed in non-tumor regions but was strongly expressed at high frequencies in tumor regions. Knockdown of KRT17 increased the number of cleaved caspase-3-positive cells, leading to the reduction of cell number. Loss-of-function of GLI-1 or GLI-2 also increased the cell numbers of apoptotic cells positive for staining of Annexin-V and propidium iodide (PI) and the terminal deoxynucleotidyl transferase dUTP-biotin nick-end labeling (TUNEL) method, and induced DNA fragmentation. This inhibitory effect on cell growth was partially rescued by exogenous KRT17 expression. In the KRT17-positive regions in OSCCs, GLI-1 or GLI-2 was frequently detected, and the number of cells with cleaved caspase-3 positive was decreased.Conclusions KRT17 promotes tumor cell growth, at least partially, through its anti-apoptotic effect as a result of the KRT17 overexpression by GLIs in OSCC.

To evaluate the feasibility of using ultrasound-obtained tumor depth (TD) and margin shape (MS) to predict the prognosis of tongue cancer.

Abnormal expression of Facioscapulohumeral muscular dystrophy (FSHD) region gene 1 (FRG1) is involved in the pathogenesis of FSHD. FRG1 is also important for the normal muscular and vascular development. Our previous study showed that FRG1 is one of the highly expressed genes in the mandible on embryonic day 10.5 (E10.5) than on E12.0. In this study, we investigated the temporospatial expression pattern of FRG1 mRNA and protein during the development of the mouse lower first molar, and also evaluated the subcellular localization of the FRG1 protein in mouse dental epithelial (mDE6) cells. The FRG1 expression was identified in the dental epithelial and mesenchymal cells at the initiation and bud stages. It was detected in the inner enamel epithelium at the cap and early bell stages. At the late bell and root formation stages, these signals were detected in ameloblasts and odontoblasts during the formation of enamel and dentin matrices, respectively. The FRG1 protein was localized in the cytoplasm in the mouse tooth germ in vivo, while FRG1 was detected predominantly in the nucleus and faintly in the cytoplasm in mDE6 cells in vitro. In mDE6 cells treated with bone morphogenetic protein 4 (BMP4), the protein expression of FRG1 increased in cytoplasm, suggesting that FRG1 may translocate to the cytoplasm. These findings suggest that FRG1 is involved in the morphogenesis of the tooth germ, as well as in the formation of enamel and dentin matrices and that FRG1 may play a role in the odontogenesis in the mouse following BMP4 stimulation.

In previous studies by our group, we reported that thymosin beta 4 (Tb4) is closely associated with the initiation and development of the tooth germ, and can induce the expression of runt-related transcription factor 2 (RUNX2) during the development of the tooth germ. RUNX2 regulates the expression of odontogenesis-related genes, such as amelogenin, X-linked (Amelx), ameloblastin (Ambn) and enamelin (Enam), as well as the differentiation of osteoblasts during bone formation. However, the mechanisms through which Tb4 induces the expression of RUNX2 remain unknown. In the present study, we employed a mouse dental epithelial cell line, mDE6, with the aim to elucidate these mechanisms. The mDE6 cells expressed odontogenesis-related genes, such as Runx2, Amelx, Ambn and Enam, and formed calcified matrices upon the induction of calcification, thus showing characteristics of odontogenic epithelial cells. The expression of odontogenesis-related genes, and the calcification of the mDE6 cells were reduced by the inhibition of phosphorylated Smad1/5 (p-Smad1/5) and phosphorylated Akt (p-Akt) proteins. Furthermore, we used siRNA against Tb4 to determine whether RUNX2 expression and calcification are associated with Tb4 expression in the mDE6 cells. The protein expression of p-Smad1/5 and p-Akt in the mDE6 cells was reduced by treatment with Tb4-siRNA. These results suggest that Tb4 is associated with RUNX2 expression through the Smad and PI3K-Akt signaling pathways, and with calcification through RUNX2 expression in the mDE6 cells. This study provides putative information concerning the signaling pathway through which Tb4 induces RUNX2 expression, which may help to understand the regulation of tooth development and tooth regeneration.

Previous studies have shown that the recombination of cells liberated from developing tooth germs develop into teeth. However, it is difficult to use human developing tooth germ as a source of cells because of ethical issues. Previous studies have reported that thymosin beta 4 (Tmsb4x) is closely related to the initiation and development of the tooth germ. We herein attempted to establish odontogenic epithelial cells from non-odontogenic HaCaT cells by transfection with TMSB4X. TMSB4X-transfected cells formed nodules that were positive for Alizarin-red S (ALZ) and von Kossa staining (calcium phosphate deposits) when cultured in calcification-inducing medium. Three selected clones showing larger amounts of calcium deposits than the other clones, expressed PITX2, Cytokeratin 14, and Sonic Hedgehog. The upregulation of odontogenesis-related genes, such as runt-related transcription factor 2 (RUNX2), Amelogenin (AMELX), Ameloblastin (AMBN) and Enamelin (ENAM) was also detected. These proteins were immunohistochemically observed in nodules positive for the ALZ and von Kossa staining. RUNX2-positive selected TMSB4X-transfected cells implanted into the dorsal subcutaneous tissue of nude mice formed matrix deposits. Immunohistochemically, AMELX, AMBN and ENAM were observed in the matrix deposits. This study demonstrated the possibility of induction of dental epithelial cell differentiation marker gene expression in non-odontogenic HaCaT cells by TMSB4X.

Itm2a is a type II transmembrane protein with a BRICHOS domain. We investigated the temporospatial mRNA and protein expression patterns of Itm2a in the developing lower first molar, and examined the subcellular localization of Itm2a in murine dental epithelial (mDE6) cells. From the initiation to the bud stage, the in situ and protein signals of Itm2a were not detected in either the dental epithelial or mesenchymal cells surrounding the tooth bud. However, at the bell stage, these signals of Itm2a were primarily observed in the inner enamel epithelium of the enamel organ. After the initiation of the matrix formation, strong signals were detected in ameloblasts and odontoblasts. Itm2a showed a punctate pattern in the cytoplasm of the mDE6 cells. The perinuclear-localized Itm2a displayed a frequent overlap with the Golgi apparatus marker, GM130. A tiny amount of Itm2a was colocalized with lysosomes and endoplasmic reticulum. Minimal or no overlap between the Itm2a-EGFP signals with the other organelle markers for endoplasmic reticulum, lysosome and mitochondria used in this study noted in the cytoplasm. These findings suggest that Itm2a may play a role in cell differentiation during odontogenesis, rather than during the initiation of tooth germ formation, and may be related to the targeting of proteins associated with enamel and dentin matrices in the secretory pathway.

This study presents the expression pattern and functions of thymosin beta 10 (Tbeta10), a Tbeta4 homologue during the development of mouse lower first molars. An in situ signal of Tbeta10 was detected on embryonic day 10.5 (E10.5)-E15.5 mainly in dental mesenchymal cells as well as in dental epithelial cells, while Tbeta4 was expressed in dental epithelial cells. In the late bell stage, preodontoblasts with strong Tbeta10 expression and preameloblasts with strong Tbeta4 expression exhibited face-to-face localization, suggesting that an intimate cell-cell interaction might exist between preodontoblasts and preameloblasts to form dentin and enamel matrices. A strong Tbeta10 signal was found in odontoblasts in the lateral side of the dental pulp and in Hertwig’s epithelial root sheath, thus suggesting that Tbeta10 participates in the formation of the outline of the tooth root. An inhibition assay using Tbeta10-siRNA in E11.0 mandibles showed significant growth inhibition in the tooth germ. The Tbeta10-siRNA-treated E15.0 tooth germ also showed significant developmental arrest. The number of Ki67-positive cells significantly decreased in the Tbeta10-siRNA-treated mandibles. The cellular proliferative activity was also significantly suppressed in Tb10-siRNA-treated cultured mouse dental pulpal and epithelial cells. These results indicate that developmental arrest of the tooth germ might be caused by a reduction in cell proliferative activity. The stage-specific temporal and spatial expression pattern of Tbeta10 in the developing tooth germ is indicative of multiple functions of Tbeta10 in the developmental course from initiation to root formation of the tooth germ.