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Toll-like receptors (TLRs) make a crucial contribution to the innate immune response. TLR5 was expressed in embryoid body derived from mouse embryonic stem cells (mESCs) and βIII-tubulin-positive cells under all-trans retinoic acid-treated condition. TLR5 was upregulated during neural differentiation from mESCs and augmented the neural differentiation of mESCs via nuclear factor-κB and interleukin 6/CREB pathways. Besides, TLR5 was expressed in SOX2- or doublecortin-positive cells in the subgranular zone of the hippocampal dentate gyrus where adult neurogenesis occurs. TLR5 inhibited the proliferation of adult hippocampal neural stem cells (NSCs) by regulating the cell cycle and facilitated the neural differentiation from the adult hippocampal NSCs via JNK pathway. Also, TLR5 deficiency impaired fear memory performance in mice. Our data suggest that TLR5 is a crucial modulator of neurogenesis from mESCs and adult hippocampal NSCs in mice and represents a new therapeutic target in neurological disorders related to cognitive function.

Background/purpose Autophagy is involved in controlling differentiation of various cell types. The present study aimed to investigate the mechanism related to autophagy in regulating odontogenic differentiation of dental pulp cells. Materials and methods Human dental pulp cells (HDPCs) were cultured in differentiation inductive medium (DM) and odontoblastic differentiation and mineralization were evaluated by alkaline phosphatase (ALP) staining and Alizarin red S staining, respectively. Tooth cavity preparation was made on the mesial surface of lower first molars in rat. The expression of autophagy-related signal molecules was detected using Western blot analysis and Immunohistochemistry. Results HDPCs cultured in DM showed increased autophagic flux and declined phosphorylation of phosphoinositide 3-kinases (PI3K), protein kinase B (Akt), and mTOR. Dentin matrix protein-1 (DMP-1) and dentin sialoprotein (DSP), markers of odontoblastic differentiation, were upregulated and autophagic activation showing increased LC3-II and decreased p62 levels was observed during odontogenic differentiation of HDPCs. However, PI3K blocker 3-methyladenine (3MA), lentiviral shLC3 and Akt activator SC79 attenuated the expression of LC3II as well as DMP-1, ALP activity and mineralization enhanced in HDPCs under DM condition. In addition, 3MA, shLC3 and SC79 recovered the expression of pluripotency factor CD146, Oct4 and Nanog downregulated in DM condition. In rat tooth cavity preparation model, the expression of LC3B and DMP-1 was elevated near odontoblast-dentin layer during reparative dentin formation, whereas 3MA significantly reduced the expression of LC3B and DMP-1. Conclusion These findings indicated autophagy promotes the odontogenic differentiation of dental pulp cells modulating stemness via PI3K/Akt inactivation and the repair of pulp.

To study the role of sclerostin in periodontal ligament (PDL) as a homeostatic regulator in biophysical-force-induced tooth movement (BFTM).BFTM was performed in rats, followed by microarray, immunofluorescence, in situ hybridization, and real-time polymerase chain reaction for the detection and identification of the molecules. The periodontal space was analysed via micro-computed tomography. Effects on osteoclastogenesis and bone resorption were evaluated in the bone-marrow-derived cells in mice. In vitro human PDL cells were subjected to biophysical forces.In the absence of BFTM, sclerostin was hardly detected in the periodontium except in the PDL and alveolar bone in the furcation region and apex of the molar roots. However, sclerostin was up-regulated in the PDL in vivo by adaptable force, which induced typical transfiguration without changes in periodontal space as well as in vitro PDL cells under compression and tension. In contrast, the sclerostin level was unaffected by heavy force, which caused severe degeneration of the PDL and narrowed periodontal space. Sclerostin inhibited osteoclastogenesis and bone resorption, which corroborates the accelerated tooth movement by the heavy force.Sclerostin in PDL may be a key homeostatic molecule in the periodontium and a biological target for the therapeutic modulation of BFTM.

Aim We aimed to identify a key molecule that maintains periodontal tissue homeostasis during biophysical force-induced tooth movement (BTM) by orchestrating alveolar bone (AB) remodelling. Materials and methods Differential display-PCR was performed to identify key molecules for BTM in rats. To investigate the localization and expression of the identified molecules, immunofluorescence, real-time RT-PCR and Western blotting were performed in rats and human periodontal ligament (PDL) cells. Functional test and micro-CT analysis were performed to examine the in vivo effects of the identified molecules on BTM. Results Secretory leucocyte peptidase inhibitor (SLPI) in the PDL was revealed as a key molecule for BTM-induced AB remodelling. SLPI was enhanced in the PDL under both compression and tension, and downregulated by an adenyl cyclases inhibitor. SLPI induced osteoblastogenic genes including runt-related transcription factor 2 (Runx2) and synergistically augmented tension-induced Runx2 expression. SLPI augmented mineralization in PDL cells. SLPI induced osteoclastogenic genes including receptor activator of nuclear factor kappa-Β ligand (RANKL) and synergistically augmented the compression-induced RANKL and macrophage colony-stimulating factor (MCSF) expression. Finally, the in vivo SLPI application into the AB significantly augmented BTM. Conclusions SLPI or its inhibitors might serve as a biological target molecule for therapeutic interventions to modulate BTM.

Fusion and apoptosis share a breakdown of the membrane phospholipids asymmetry, modes of which are largely unknown in osteoclastogenesis. Here, we investigated the externalization of phosphatidylserine (PS) and its receptors, and their biological functions in osteoclastogenesis. Strong immunoreactivities in vivo for the PS receptors TIM4, BAI1, and STAB2 were observed in the TRAP-positive multinucleated cells in the alveolar bone that was being remodeled around the developing dental follicles in rats. These receptors were significantly upregulated during M-CSF/RANKL-induced in vitro osteoclastogenesis using mouse bone marrow-derived cells. PS externalization in preosteoclasts was increased by the M-CSF/RANKL treatment. Multinucleation of preosteoclasts was markedly inhibited by antibodies against PS and its receptors. Among the investigated lipid transporter proteins, floppases (Abcb4, Abcc5, and Abcg1) were upregulated, whereas flippases (Atp11c and Atp8a1) downregulated during osteoclastogenesis. Preosteoclast fusion was markedly blocked by the ATPase inhibitor Na3VO4 and siRNAs against Abcc5 and Abcg1, revealing the importance of these lipid transporters in PS externalization. Further, the levels of Cd47 and Cd31, don’t-eat-me signal inducers, were increased or sustained in the early phase of osteoclastogenesis, whereas those of AnnexinI and Mfg-e8, eat-me signals inducers, were increased in the late apoptotic phase. In addition, Z-VAD-FMK, a pan caspase inhibitor, had no effect on preosteoclast fusion in the early phase of osteoclastogenesis, whereas Abs against PS, TIM4, and BAI1 decreased osteoclast apoptosis during the late phase. These results suggest that PS externalization is essential for the whole process of osteoclastogenesis and share PS receptors and transporters in the early stage fusion and late stage apoptosis. Therefore, modulation of PS and its receptors could be a useful strategy to develop anti-bone resorptive agents.

Much information is currently available for molecules in early odontogenesis, but there is limited knowledge regarding terminal cytodifferentiation of ameloblasts and odontoblasts for the determination of normal crown morphology. The present differential display PCR (DD-PCR) revealed that insulin-like growth factor-binding protein 5 (IGFBP5) was differentially expressed in molar tooth germs between the cap (before crown mineralization) and root formation (after crown mineralization) stages. Real-time PCR confirmed that the expression levels of IGFBP1-4 were not significantly changed but those of IGFBP5-7 were upregulated in a time-dependent manner. Immunoreactivities for IGFBP5-7 were hardly seen in molar germs at the cap/early bell stage and protective-stage ameloblasts at the root formation stage. However, the reactivity was strong in odontoblasts and maturation-stage ameloblasts, which are morphologically and functionally characterized by wide intercellular space and active enamel matrix mineralization. The localization of each IGFBP was temporospatial. IGFBP5 was localized in the nuclei of fully differentiated odontoblasts and ameloblasts, while IGFBP6 was localized in the apical cytoplasm of ameloblasts and odontoblasts with dentinal tubules, and IGFBP7 was mainly found in the whole cytoplasm of odontoblasts and the intercellular space of ameloblasts. IGFBP silencing using specific siRNAs upregulated representative genes for dentinogenesis and amelogenesis, such as DMP1 and amelogenin, respectively, and augmented the differentiation media-induced mineralization, which was confirmed by alizarin red s and alkaline phosphatase staining. These results suggest that IGFBP5-7 may play independent and redundant regulatory roles in late-stage odontogenesis by modulating the functional differentiation of ameloblasts and odontoblasts.

Direct reprogramming, also known as a trans-differentiation, is a technique to allow mature cells to be converted into other types of cells without inducing a pluripotent stage. It has been suggested as a major strategy to acquire the desired type of cells in cell-based therapies to repair damaged tissues. Studies related to switching the fate of cells through epigenetic modification have been progressing and they can bypass safety issues raised by the virus-based transfection methods. In this study, a protocol was established to directly convert fully differentiated fibroblasts into diverse mesenchymal-lineage cells, such as osteoblasts, adipocytes, chondrocytes, and ectodermal cells, including neurons, by means of DNA demethylation, immediately followed by culturing in various differentiating media. First, 24 h exposure of 5-azacytidine (5-aza-CN), a well-characterized DNA methyl transferase inhibitor, to NIH-3T3 murine fibroblast cells induced the expression of stem-cell markers, that is, increasing cell plasticity. Next, 5-aza-CN treated fibroblasts were cultured in osteogenic, adipogenic, chondrogenic, and neurogenic media with or without bone morphogenetic protein 2 for a designated period. Differentiation of each desired type of cell was verified by quantitative reverse transcriptase-polymerase chain reaction/ western blot assays for appropriate marker expression and by various staining methods, such as alkaline phosphatase/alizarin red S/oil red O/alcian blue. These proposed procedures allowed easier acquisition of the desired cells without any transgenic modification, using direct reprogramming technology, and thus may help make it more available in the clinical fields of regenerative medicine.

BACKGROUND: The delivery of recombinant human bone morphogenetic protein 2 (rhBMP2) by using various carriers has been used to successfully induce bone formation in many animal models. However, the effect of multiple administration of rhBMP2 on bone formation and BMP2 antibody production has not been determined. Our aim was to examine the bone formation activity of rhBMP2 and serum levels of anti-BMP2 antibodies following the repeated administration of rhBMP2 in mice. METHODS: Absorbable collagen sponges or polyphosphazene hydrogels containing rhBMP2 were subcutaneously implanted or injected into one side on the back of six-week-old C57BL/6 mice. Three or 4 weeks later, the same amount of rhBMP2 was administered again with the same carrier into the subcutaneous regions on the other side of the back or into calvarial defects. The effects of a single administration of rhBMP2 on the osteoinductive ability in the ectopic model were compared with those of repeated administrations. In vivo ectopic or orthotopic bone formation was evaluated using microradiography and histological analyses. Serum concentrations of anti-rhBMP2 antibodies were measured by ELISAs. RESULTS: Re-administration of the same amount of rhBMP2 into the subcutaneous area showed a comparable production of ectopic bone as after the first administration. The bone forming ability of repeated rhBMP2 administrations was equal to that of single rhBMP2 administration. The administration of rhBMP2 into calvarial defects, following the first subcutaneous administration of rhBMP2 on the back, completely recovered the defect area with newly regenerated bone within 3 weeks. Repeated administration of rhBMP2 at 4-week intervals did not significantly alter the serum levels of anti-BMP2 antibodies and did not induce any inflammatory response. The serum obtained from rhBMP2-exposed mice had no effect on the ability of rhBMP2 to induce osteogenic gene expressions in MC3T3-E1. CONCLUSION: We suggest that the osteoinductive ability of rhBMP2 is not compromised by repeated administrations. Thus, rhBMP2 can be repeatedly used for bone regeneration at various sites within a short duration. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13770-020-00290-4) contains supplementary material, which is available to authorized users.

Introduction Few studies have reported direct pulp capping in inflamed pulp conditions. The purpose of this study was to investigate the in vitro and in vivo responses of dental pulp during direct pulp capping using various pulp capping materials in inflamed conditions. Methods Human dental pulp cells were treated with lipopolysaccharide (LPS) and cultured with Dycal (Dentsply Caulk, Milford, DE), ProRoot MTA (Dentsply Maillefer, Ballaigues, Switzerland), and Endocem MTA (Maruchi, Wonju, South Korea). The expressions of interleukin (IL)-1β, IL-6, dentin matrix protein 1, and dentin sialophosphoprotein were analyzed through real-time polymerase chain reaction. The maxillary molars of Sprague-Dawley rats were exposed for 2 days. The exposed pulps were capped with Dycal, ProRoot MTA, and Endocem MTA and sealed with resin-modified glass ionomer followed by histologic and immunohistochemical analyses. Results The expression of IL-1β and IL-6 was increased with LPS and decreased by Dycal, ProRoot MTA, and Endocem MTA. Dentin matrix protein 1 and dentin sialophosphoprotein levels were decreased with LPS and increased after treatment with pulp capping materials.In the in vivo study, inflammation associated with Dycal was higher than that associated with ProRoot MTA and Endocem MTA at week 1, without any significant difference between the 2. At 4 weeks, inflammation was decreased, and mineralization was increased compared with week 1 in all 3 of the materials. At week 1, IL-6 immunoreactivity was strongly expressed. Dycal exhibited stronger immunoreactivity than ProRoot MTA and Endocem MTA. However, the immunoreactivity was decreased in all groups at week 4. Conclusions Successful direct pulp capping requires more effective pulp capping materials for the treatment of inflamed pulps.

Objective It has been well known that Hedgehog (Hh) signaling plays an important role in bone development, however, its function in cementogenesis has not yet been reported. This study was intended to elucidate the role of Hh signaling in cementoblast differentiation. Design Expression changes of various Hh signaling components and levels of skeletogenic markers (alkaline phosphatase, osteocalcin, osteopontin) and osteogenic transcription factors (RUNX2, Osterix) by Hh signaling modulators during OCCM-30 cementoblast differentiation were determined by quantitative real-time reverse transcriptase polymerase chain reaction. To investigate effects of Hh signaling modulators on the mineralization of cementoblast, alkaline phosphatase and alizarin red S staining were used. Then, the interaction between Hh and BMP signaling during cementoblast differentiation was evaluated using co-treatment of BMP7 and Hh signaling modulators. Results We observed the consistent expression of Hh signaling molecules in the OCCM-30, which were up-regulated during cementoblast differentiation. We also found that the treatment of cells with Purmo, an Hh activator, enhanced cementoblast differentiation by increasing the mRNA expression of skeletogenic markers and osteogenic transcription factors, as well as increasing alkaline phosphate activity and mineralization capability. On the contrary, an Hh antagonist, like Cyclo, effectively inhibited cementoblast differentiation. Furthermore, BMP7 promoted cementoblast differentiation through crosstalk with the Hh signaling. Conclusion These results suggest that Hh signaling is involved in cementoblast differentiation, and Hh signaling molecules may therefore represent new therapeutic targets in periodontal treatment and regeneration.

Recent progress in chemotherapy has significantly increased its efficacy, yet the development of chemoresistance remains a major drawback. In this study, we show that GFRA1/GFRα1 (GDNF family receptor α 1), contributes to cisplatin-induced chemoresistance by regulating autophagy in osteosarcoma. We demonstrate that cisplatin treatment induced GFRA1 expression in human osteosarcoma cells. Induction of GFRA1 expression reduced cisplatin-induced apoptotic cell death and it significantly increased osteosarcoma cell survival via autophagy. GFRA1 regulates AMPK-dependent autophagy by promoting SRC phosphorylation independent of proto-oncogene RET kinase. Cisplatin-resistant osteosarcoma cells showed NFKB1/NFκB-mediated GFRA1 expression. GFRA1 expression promoted tumor formation and growth in mouse xenograft models and inhibition of autophagy in a GFRA1-expressing xenograft mouse model during cisplatin treatment effectively reduced tumor growth and increased survival. In cisplatin-treated patients, treatment period and metastatic status were associated with GFRA1-mediated autophagy. These findings suggest that GFRA1-mediated autophagy is a promising novel target for overcoming cisplatin resistance in osteosarcoma.

Ascorbic acid induces apoptosis, autophagy, and necrotic cell death in cancer cells. We investigated the mechanisms by which ascorbic acid induces death in laryngeal squamous cell carcinoma Hep2 cells. Ascorbic acid markedly reduced cell viability and induced death without caspase activation and an increase in cytochrome c. Hep2 cells exposed to ascorbic acid exhibited membrane rupture and swelling, the morphological characteristics of necrotic cell death. The generation of reactive oxygen species (ROS) was increased in Hep2 cells treated with ascorbic acid, and pretreatment with N-acetylcysteine blocked ascorbic acid-induced cell death. Ascorbic acid also stimulated protein kinase C (PKC) signaling, especially PKC α/β activation, and subsequently increased cytosolic calcium levels. However, ascorbic acid-induced necrotic cell death was inhibited by Ro-31-8425 (PKC inhibitor) and BAPTA-AM (cytosolic calcium-selective chelator). ROS scavenger NAC inhibited PKC activation induced by ascorbic acid and Ro-31-8425 suppressed the level of cytosolic calcium increased by ascorbic acid, indicating that ROS is represented as an upstream signal of PKC pathway and PKC activation leads to the release of calcium into the cytosol, which ultimately regulates the induction of necrosis in ascorbic acid-treated Hep2 cells. These data demonstrate that ascorbic acid induces necrotic cell death through ROS generation, PKC activation, and cytosolic calcium signaling in Hep2 cells. J. Cell. Physiol. 232: 417-425, 2017. © 2016 Wiley Periodicals, Inc.

Introduction Recent reports have shown that the combined use of platelet-rich fibrin (PRF), an autologous fibrin matrix, and mineral trioxide aggregate (MTA) as root filling material is beneficial for the endodontic management of an open apex. However, the potential of the combination of MTA and PRF as an odontogenic inducer in human dental pulp cells (HDPCs) in vitro has not yet been studied. The purpose of this study was to evaluate the effect of the combination of MTA and PRF on odontoblastic maturation in HDPCs. Methods HDPCs extracted from third molars were directly cultured with MTA and PRF extract (PRFe). Odontoblastic differentiation of HDPCs was evaluated by measuring the alkaline phosphatase (ALP) activity, and the expression of odontogenesis-related genes was detected using reverse-transcription polymerase chain reaction or Western blot. Mineralization formation was assessed by alizarin red staining. Results HDPCs treated with MTA and PRFe significantly up-regulated the expression of dentin sialoprotein and dentin matrix protein-1 and enhanced ALP activity and mineralization compared with those with MTA or PRFe treatment alone. In addition, the combination of MTA and PRFe induced the activation of bone morphogenic proteins (BMP)/Smad, whereas LDN193189, the bone morphogenic protein inhibitor, attenuated dentin sialophosphoprotein and dentin matrix protein-1 expression, ALP activity, and mineralization enhanced by MTA and PRFe treatment. Conclusions This study shows that the combination of MTA and PRF has a synergistic effect on the stimulation of odontoblastic differentiation of HDPCs via the modulation of the BMP/Smad signaling pathway.

The present study is aimed at investigating the effects of the exogenous estrogen 17β-estradiol (E2) on odontoblastic differentiation in human dental pulp cells (HDPCs) immotalized with hTERT gene and their molecular mechanism. Proliferation was detected by BrdU assay, and odontoblast differentiation induction was evaluated by the expression of dentin sialophosphoprotein (DSPP), dentin sialoprotein (DSP) and dentin matrix protein1 (DMP1), and alkaline phosphatase (ALP) activity and mineralization. Estrogen receptor-α (ER-α), c-Src, and mitogen-activated protein kinases (MAPKs) were examined and their inhibitors were used to determine the roles on odontogenic induction. E2 significantly promoted the HDPC proliferation, which was mediated by extracellular signal-related kinase 1/2. E2 upregulated DSPP, DSP, and DMP1 as the odontogenic differentiation markers and enhanced ALP activity and mineralization. E2 increased phosphorylation of ER-α and fulvestrant, an ER downregulator, significantly downregulated DSPP, DMP1, and DSP induced by E2. Moreover, E2 treatment activated c-Src and MAPKs upon odontogenic induction, whereas chemical inhibition of c-Src and MAPKs decreased expression of DSPP, DMP1, and DSP and mineralization augmented by E2. Moreover, fulvestrant reduced E2-induced phosphorylation of c-Src and MAPK and inhibition of c-Src by PP2 attenuated activation of MAPKs during E2-induced odontoblastic differentiation. Taken together, these results indicated that E2 stimulates odontoblastic differentiation of HDPCs via coordinated regulation of ER-α, c-Src, and MAPK signaling pathways, which may play a key role in the regeneration of dentin.

Objective This study aimed to determine the expression of chemokine (C-X-C motif) ligand 14 (CXCL14) in pulpal and periodontal cells in vivo and in vitro, and investigate function of CXCL14 and its underlying mechanism in the proliferation and osteogenic differentiation of human periodontal ligament (hPDL) cells. Methods To determine the expression level of CXCL14 in adult rat oral tissues and in hPDL cells after application of biophysical forces, RT-PCR, western blot, and histological analyses were performed. The role of CXCL14 in proliferation and osteogenic differentiation of PDL cells was evaluated by measuring dehydrogenase activity and Alizarin red S staining. Results Strong immunoreactivity against CXCL14 was observed in the PDL tissues and pulpal cells of rat molar, and attenuated apparently by orthodontic biophysical forces. As seen in rat molar, highly expressed CXCL14 was observed in human dental pulp and hPDL cells, and attenuated obviously by biophysical tensile force. CXCL14 expression in hPDL cells was increased in incubation time-dependent manner. Proliferation of hPDL cells was inhibited dramatically by small interfering (si) RNA against CXCL14. Furthermore, dexamethasone-induced osteogenic mineralization was inhibited by recombinant human (rh) CXCL14, and augmented by CXCL14 siRNA. rhCXCL14 increased transforming growth factor-beta1 (TGF- β1) in hPDL cells. Inhibition of the cell proliferation and osteogenic differentiation of hPDL cells by CXCL14 siRNA and rhCXCL14 were restored by rhTGF-β1 and SB431542, respectively. Conclusion These results suggest that CXCL14 may play roles as a growth factor and a negative regulator of osteogenic differentiation by increasing TGF-β1 expression in hPDL cells.

Background The association between diabetes mellitus (DM) and bone diseases is acknowledged. However, the mechanistic pathways leading to the alveolar bone (AB) destruction remain unclear. This study aims to elucidate the mechanical forces (MF)-induced AB destruction in DM and its underlying mechanism. Methods In vivo periodontal tissue responses to MF were evaluated in rats with diabetes. In vitro human periodontal ligament (PDL) cells were either treated with advanced glycation end products (AGEs) alone or with AGEs and MF. Results In vivo, the transcription of VEGF-A, colony stimulating factor-1 (CSF-1), and Ager was upregulated in diabetes, whereas changes in DDOST and Glo1 mRNAs were negligible. DM induced VEGF-A protein in the vascular cells of the PDL and subsequent angiogenesis, but DM itself did not induce osteoclastogenesis. MF-induced AB resorption was augmented in DM, and such augmentation was morphologically substantiated by the occasional undermining resorption as well as the frontal resorption of the AB by osteoclasts. The mRNA levels of CSF-1 and vascular endothelial growth factor (VEGF) during MF application were highly elevated in diabetes, compared with those of the normal counterparts. In vitro, AGEs treatment elevated Glut-1 and CSF-1 mRNA levels via the p38 and JNK pathways, whereas OGT and VEGF levels remained unchanged. Compressive MF especially caused upregulation of VEGF, CSF-1, and Glut-1 levels, and such upregulation was further enhanced by AGEs treatment. Conclusions Overloaded MF and AGEs metabolites may synergistically aggravate AB destruction by upregulating CSF-1 and VEGF. Therefore, regulating the compressive overloading of teeth, as well as the levels of diabetic AGEs, may prove to be an effective therapeutic modality for managing DM-induced AB destruction.

Amelogenin, an enamel matrix protein has been considered to be exclusively expressed by ameloblasts during odontogenesis. However, burgeoning evidence indicates that amelogenin is also expressed in non‐mineralizing tissues. Under the hypothesis that amelogenin may be a functional molecule in developing hair follicles which share developmental features with odontogenesis, this study for the first time elucidated the presence and functional changes of amelogenin and its receptors during rat hair follicle development. Amelogenin was specifically localized in the outer epithelial root sheath of hair follicles. Its expression appeared in the deeper portion of hair follicles, i.e. the bulbar and suprabulbar regions rather than the superficial region. Lamp‐1, an amelogenin receptor, was localized in either follicular cells or outer epithelial sheath cells, reflecting functional changes during development. The expression of amelogenin splicing variants increased in a time‐dependent manner during postnatal development of hair follicles. Amelogenin expression was increased by treatment with cyclosporin A, which is an inducer of anagen in the hair follicle, whereas the level of Lamp‐1 and ‐2 was decreased by cyclosporin A treatment. These results suggest that amelogenin may be a functional molecule involved in the development of the hair follicle rather than an inert hair shaft matrix protein.

Human mesenchymal stem cells (hMSCs), characterized by rapid in vitro expandability and multi-differentiation potential, have been widely used in the clinical field of tissue engineering. Recent studies have shown that various signaling networks are involved in the growth and differentiation of hMSCs. Although Wnts and their downstream signaling components have been implicated in the regulation of hMSCs, the role of Wnt signaling in hMSC self-renewal is still controversial. Here, it was observed that activation of endogenous canonical Wnt signaling with LiCl, which decreased β-catenin phosphorylation, leads to a decrease in hMSC proliferation. The fact that this growth arrest is not linked to apoptosis was verified by annexin V-FITC/propidium iodide assay. It was associated with sealing off of the cells in the G1 phase of the cell cycle accompanied by changes in expression of cell cycle-associated genes such as cyclin A and D. In addition, activation of Wnt signaling during hMSC proliferation seemed to reduce their clonogenic potential. On the contrary, Wnt signaling activation during hMSC proliferation had little effect on the osteogenic differentiation capability of cells. These findings show that canonical Wnt signaling is a critical regulator of hMSC proliferation and clonogenicity.

Differentiated ameloblasts secret enamel matrix proteins such as amelogenin, ameloblastin, and enamelin. Expression levels of these proteins are regulated by various factors. To find a new regulatory factor for ameloblast differentiation, we performed 2D-PAGE analysis using mouse ameloblast lineage cell line (mALCs) cultured with mineralizing medium. Of identified proteins, family with sequence similarity 50 member A (Fam50a) was significantly increased during differentiation of mALCs. Fam50a protein was also highly expressed in secretory ameloblasts of mouse tooth germs. In mALCs cultures, forced expression of Fam50a up-regulated the expression of enamel matrix protein genes such as amelogenin, ameloblastin, and enamelin. In addition, up-regulation of Fam50a also increased ALP activity and mineralized nodule formation in a dose-dependent manner. In contrast, knockdown of Fam50a decreased expression levels of enamel matrix protein genes, ALP activity, and mineralized nodule formation. By fluorescence microscopy, endogenous Fam50a protein was found to be localized to the nucleus of ameloblasts. In addition, Fam50a synergistically increased Ambn transactivation by Runx2. Moreover, Fam50a increased binding affinity of Runx2 to Ambn promoter by physically interacting with Runx2. Taken together, these results suggest Fam50a might be a new positive regulator of ameloblast differentiation.

The transcription factor Twist1 is known to be closely associated with the formation of bone by mesenchymal stem cells and osteoblasts, however, the role of Twist1 in cementogenesis has not yet been determined. This study was undertaken to elucidate the roles of Twist1 in cementoblast differentiation by means of the gain- or loss-of-function method. We used alkaline phosphatase (ALP) and alizarin red S staining and quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) to determine whether the forced transient expression or knock-down of Twist1 in a mouse cementoblast cell line, OCCM-30, could affect cementogenic differentiation. Silencing Twist1 with small interference RNA (siRNA) enhanced the formation of mineralized tissue. The expression of several cementogenesis markers, such as bone sialoprotein (BSP), osteopontin (OPN), dentin matrix protein1 (DMP1), and dentin sialophosphoprotein (DSPP) mRNA, were upregulated. Transient Twist1 overexpression in OCCM-30 consistently suppressed mineralization capacity and downregulated the differentiation markers. These results suggest that the Twist1 transcription factor may play a role in regulating cementoblast differentiation.

Ascorbic acid induces apoptosis, autophagy, and necrotic cell death in cancer cells. We investigated the mechanisms by which ascorbic acid induces death in laryngeal squamous cell carcinoma Hep2 cells. Ascorbic acid markedly reduced cell viability and induced death without caspase activation and an increase in cytochrome c. Hep2 cells exposed to ascorbic acid exhibited membrane rupture and swelling, the morphological characteristics of necrotic cell death. The generation of reactive oxygen species (ROS) was increased in Hep2 cells treated with ascorbic acid, and pretreatment with N-acetylcysteine blocked ascorbic acid-induced cell death. Ascorbic acid also stimulated protein kinase C (PKC) signaling, especially PKC α/β activation, and subsequently increased cytosolic calcium levels. However, ascorbic acid-induced necrotic cell death was inhibited by Ro-31-8425 (PKC inhibitor) and BAPTA-AM (cytosolic calcium-selective chelator). ROS scavenger NAC inhibited PKC activation induced by ascorbic acid and Ro-31-8425 suppressed the level of cytosolic calcium increased by ascorbic acid, indicating that ROS is represented as an upstream signal of PKC pathway and PKC activation leads to the release of calcium into the cytosol, which ultimately regulates the induction of necrosis in ascorbic acid-treated Hep2 cells. These data demonstrate that ascorbic acid induces necrotic cell death through ROS generation, PKC activation, and cytosolic calcium signaling in Hep2 cells. J. Cell. Physiol. 232: 417-425, 2017. © 2016 Wiley Periodicals, Inc.

Objective To determine the diameter of the crown, total crown area, individual cusp area, and occlusal table area in Korean maxillary permanent molars, as well as dental characteristics relevant to the hypocone reduction trait. Materials and methods Subjects included 121 dental school students in Korea (81 men and 40 women). A digital image analysis system was used for measurements and we relied on visual scoring to assign categories of hypocone expression. Results The mean crown dimension was larger in the first molar (M1) than in the second molar (M2). Regarding differences according to gender, the crown diameter, total crown area, and individual cusp area were significantly larger in the men than in the women. The mean occlusal table area ratios were 61% for M1 and 57% for M2, and these ratios increased in proportion to the total crown area. With respect to the hypocone and other features of the maxillary molars, differences between the men and women were more prominent for M1 than for M2. The M2 hypocone tended to be smaller than the M1 hypocone, and hypocone reduction was inversely related to the cusp area of the protocone. The protocone area may be considered a reliable parameter for comparing race and/or gender differences in maxillary molars. Conclusion This study concerning characteristics of the maxillary molar occlusal surface in Koreans provides useful information for comparative studies among human races.

Periodontal disease is one of the most prevalent chronic disorders worldwide. It is accompanied by inflammation of the gingiva and destruction of periodontal tissues, leading to alveolar bone loss. Here, we focused on the role of adipokines, which are locally expressed by periodontal tissues, in the regulation of catabolic gene expression leading to periodontal inflammation. The expression of the nicotinamide phosphoribosyltransferase (NAMPT) adipokine was dramatically increased in inflamed human and mouse gingival tissues. NAMPT expression was also increased in lipopolysaccharide- and proinflammatory cytokine-stimulated primary cultured human gingival fibroblasts (GF). Adenovirus-mediated NAMPT (Ad-Nampt) overexpression upregulated the expression and activity of COX-2, MMP1 and MMP3 in human GF. The upregulation of IL-1β- or Ad-Nampt-induced catabolic factors was significantly abrogated by the intracellular NAMPT (iNAMPT) inhibitor, FK866 or by the sirtuin (SIRT) inhibitor, nicotinamide (NIC). Recombinant NAMPT protein or extracellular NAMPT (eNAMPT) inhibition using a blocking antibody did not alter NAMPT target gene expression levels. Moreover, intragingival Ad-Nampt injection mediated periodontitis-like phenotypes including alveolar bone loss in mice. SIRT2, a part of the SIRT family, was positively associated with NAMPT actions in human GF. Furthermore, in vivo inhibition of the NAMPT-NAD+-SIRT axis by NIC injection in mice ameliorated the periodontal inflammation and alveolar bone erosion caused by intragingival injection of Ad-Nampt. Our findings indicate that NAMPT is highly upregulated in human GF, while its enzymatic activity acts as a crucial mediator of periodontal inflammation and alveolar bone destruction via regulation of COX-2, MMP1, and MMP3 levels.

Objectives The purpose of this study was to assess the ability of two new calcium silicate-based pulp-capping materials (Biodentine and BioAggregate) to induce healing in a rat pulp injury model and to compare them with mineral trioxide aggregate (MTA). Materials and Methods Eighteen rats were anesthetized, cavities were prepared and the pulp was capped with either of ProRoot MTA, Biodentine, or BioAggregate. The specimens were scanned using a high-resolution micro-computed tomography (micro-CT) system and were prepared and evaluated histologically and immunohistochemically using dentin sialoprotein (DSP). Results On micro-CT analysis, the ProRoot MTA and Biodentine groups showed significantly thicker hard tissue formation (p < 0.05). On H&E staining, ProRoot MTA showed complete dentin bridge formation with normal pulpal histology. In the Biodentine and BioAggregate groups, a thick, homogeneous hard tissue barrier was observed. The ProRoot MTA specimens showed strong immunopositive reaction for DSP. Conclusions Our results suggest that calcium silicate-based pulp-capping materials induce favorable effects on reparative processes during vital pulp therapy and that both Biodentine and BioAggregate could be considered as alternatives to ProRoot MTA.

Bisphosphonates have been reported to have chondroprotective activities in addition to its original functions. However, mechanisms for these just began to be elucidated. Under the hypothesis that bisphosphonates may regulate expression and activities of matrix enzymes during degradation of cartilage for bone formation, we administrated an alendronate (1 mg/kg) to newborn rats subcutaneously once a day for 4, 7, and 10 days. To identify the effects of alendronate on cartilage, thickness of cartilage layer was measured by histomorphometry on the proximal epiphysis of tibia. Immunofluorescence staining and RT-PCR were performed to investigate the expressions of matrix enzymes in both in vitro and in vivo. MTS assay revealed that at 10 -3 M in concentration, alendronate significantly reduced viability of chondrocytes. The mRNA expressions of MMP-1, MMP-9, EMMPRIN, and TIMP-3 in primary chondrocytes were decreased by the alendronate treatment. Interestingly, TIMP-1 mRNA expression was significantly increased, whereas a constitutive form, TIMP-2 was relatively unchanged by the treatment. The thickness of proliferating layer at postnatal day 7 was not significantly different, whereas thickness of hypertrophied layer was significantly thicker in the alendronate group than in the control (p These results suggest that the alendronate have chondroprotective properties by down-regulation of MMPs and up-regulation of TIMPs during endochondral ossification.

The aim of this study was to investigate the characteristics of mandibular premolars regarding size and morphology in Koreans. Moreover, comparisons of gender difference in mandibular premolars were examined to expand anatomical database in Koreans. Data was obtained from students in School of Dentistry, Chonnam National University, Gwangju, Korea. The total number of participants was 66 (33 men and 33 women) and dental casts were fabricated. A total of nine items was investigated using a digital measuring software. Five measurements were performed including intercuspal distance (ID), buccolingual diameter (BL), mesiodistal diameter (MD), total crown area, and each cusp area. One item as each cusp area ratio was calculated, and three items were observed including the number of lingual cusp, occlusal groove patterns, and mesiolingual developmental groove. Comparison measurements were analyzed using paired t-tests, independent t-tests and Pearson correlation tests. Average values in mandibular second premolars were larger than first premolars in most of measurements with a significance with the exception of mesiodistal diameter (p=0.223). Overall average values were significantly higher in male than in female except intercuspal distance (p=0.607) and lingual cusp area (p=0.070) in mandibular premolars. The presence of mesiolingual developmental grooves in the first premolars was 59.1% (male 51.5%, female 66.7%). The most common occlusal groove patterns of the second premolar were a Y pattern, followed in order by H and U patterns. These results provide valuable morphological characteristics of mandibular premolars in Koreans.