Direct Gingival Fibroblast/Osteoblast Transdifferentiation via Epigenetics

Cho, Y.,Kim, B.,Bae, H.,Kim, W.,Baek, J.,Woo, K.,Lee, G.,Seol, Y.,Lee, Y.,Ku, Y. Journal of Dental Research, Inc 2017 Journal of dental research Vol.96 No.5

<P>Alveolar bone resorption caused by trauma or periodontal diseases has represented a challenge for both dental clinicians and researchers. In this study, we evaluate the osteogenic potential of human gingival fibroblasts (HGFs) through a direct transdifferentiation from HGFs to functional osteoblasts via epigenetic modification and osteogenic signaling with bone morphogenetic protein 2 (BMP2) in vitro and in vivo. HGF treatment with 5-aza-2'-deoxycytidine (5-aza-dC) induced demethylation in the hypermethylated CpG islands of the osteogenic lineage marker genes RUNX2 and ALP, and subsequent BMP2 treatment successfully drove the fibroblasts to the osteoblasts' lineage. Cell morphological changes viewed under microscopy and alkaline phosphatase (ALP) and alizarin red S (ARS) staining confirmed the osteoblastic change mediated by epigenetic modification as did real-time polymerase chain reaction (PCR), methylation-specific PCR (MSP), and chromatin immunoprecipitation (ChIP) assay, which demonstrated the altered methylation patterns in the RUNX2 and ALP promoter regions and their effect on gene expression. Furthermore, micro-computed tomography (CT) analysis of in vivo mouse cell transplantation experiments showed high-density signal in the epigenetically modified HGF group; in addition, a significant amount of bone formation was observed in the transplanted material using hematoxylin and eosin (H&E) staining as well. Collectively, our results indicate that epigenetic modification permits the direct programming of HGFs into functional osteoblasts, suggesting that this approach might open a novel therapeutic avenue in alveolar bone regeneration.</P>

COUP-TFII Stimulates Dentin Sialophosphoprotein Expression and Mineralization in Odontoblasts

Hur, S.-W.,Oh, S.-H.,Jeong, B.-C.,Choi, H.,Kim, J.-W.,Lee, K.-N.,Hwang, Y.-C.,Ryu, J.-H.,Kim, S.-H.,Koh, J.-T. Journal of Dental Research, Inc 2015 Journal of dental research Vol.94 No.8

<P>Chicken ovalbumin upstream promoter transcription factor 2 (COUP-TFII), an orphan nuclear receptor belonging to the steroid-thyroid hormone receptor superfamily, plays an important role in cell fate determination of various tissues. However, the specific role of COUP-TFII in tooth development has not yet been elucidated. In the present study, we aimed to explore the role of COUP-TFII in <I>dentin sialophosphoprotein</I> (<I>DSPP</I>) expression and matrix mineralization in odontoblast-lineage cells. In primary human dental pulp cells (HDPCs) and murine dental papilla-derived cells (MDPC-23) cultured in a mineralizing medium, the expression of <I>COUP-TFII</I> was induced along with the increased odontoblast-specific <I>dentin matrix protein-1</I> (<I>DMP-1</I>) and <I>DSPP</I> expression. Endogenous expression of <I>COUP-TFII</I> in maxillary second molar germs of rats showed an increasing tendency as development of the tooth progressed. Also, COUP-TFII protein was detected in greater quantity in the odontoblastic layer of second molar germs than in that of third molar germs of rats. Overexpression of <I>COUP-TFII</I> using an adenoviral system upregulated the expression of odontoblast-specific genes with increased alkaline phosphatase activity and matrix mineralization in odontoblast-lineage cells. In contrast, knockdown of <I>COUP-TFII</I> using small interfering RNA decreased the expression of odontoblast-specific genes, which reduced matrix mineralization. Mechanistic studies revealed that COUP-TFII increased <I>DSPP</I> transcription by direct binding on the <I>DSPP</I> promoter. In addition, COUP-TFII physically interacted with the homeodomain transcription factor Msx2 and antagonistically regulated the Msx2 effect on <I>DSPP</I> promoter activity. Taken together, these results suggest that COUP-TFII has a stimulatory role in <I>DSPP</I> expression and matrix mineralization in odontoblast-lineage cells.</P>

The Grooved Rodent Incisor Recapitulates Rudimentary Teeth Characteristics of Ancestral Mammals

Li, L.,Tang, Q.,Jung, H.-S. Journal of Dental Research, Inc 2016 Journal of dental research Vol.95 No.8

<P>It is known from the paleontology studies of eutherian mammals that incisor numbers were reduced during evolution. The evolutionary lost incisors may remain as vestigial structures at embryonic stages. The recapitulation of the incisor patterns among mammalian species will potentially uncover the mechanisms underlying the phenotypic transition of incisors during evolution. Here, we showed that a minute tooth formed in the presumptive groove region of the gerbil upper incisor at the early developmental stages, during which multiple epithelial swellings and Shh transcription domains spatiotemporally appeared in the dental epithelium, suggests the existence of vestigial dental primordia. Interestingly, when we trimmed the surrounding mesenchyme from incisor tooth germs at or before the bud stage prior to ex vivo culture, the explants developed different incisor phenotypes ranging from triplicated incisors, duplicated incisors, to Lagomorpha-like incisors, corresponding to the incisor patterns in the eutherian mammals. These results imply that the phenotypic transition of incisors during evolution, as well as the achievement of ultimate incisors in adults, arose from differential integrations of primordia. However, when the incisor tooth germ was trimmed at the cap stage, a grooved incisor developed similar to the normal condition. Furthermore, the incisor tooth germ developed a small but smooth incisor after the additional removal of the minute tooth and a lateral rudiment. These results suggest that multiple dental primordia integrated before the cap stage, with the labial primordia contributing to the labial face of the functional incisor. The minute tooth that occupied the boundary of the 2 labial primordia might be implicated in the groove formation. This study sheds light on how rudiments incorporate into functional organs and aids the understanding of incisor evolution.</P>

Deletion of <i>Osr2</i> Partially Rescues Tooth Development in <i>Runx2</i> Mutant Mice

Kwon, H.J.E.,Park, E.K.,Jia, S.,Liu, H.,Lan, Y.,Jiang, R. Journal of Dental Research, Inc 2015 Journal of dental research Vol.94 No.8

<P>Tooth organogenesis depends on genetically programmed sequential and reciprocal inductive interactions between the dental epithelium and neural crest–derived mesenchyme. Previous studies showed that the Msx1 and Runx2 transcription factors are required for activation of odontogenic signals, including Bmp4 and Fgf3, in the early tooth mesenchyme to drive tooth morphogenesis through the bud-to-cap transition and that Runx2 acts downstream of Msx1 to activate <I>Fgf3</I> expression. Recent studies identified Osr2 as a repressor of tooth development and showed that inactivation of <I>Osr2</I> rescued molar tooth morphogenesis in the <I>Msx1</I><SUP>-/-</SUP> mutant mice as well as in mice with neural crest–specific inactivation of <I>Bmp4</I>. Here we show that <I>Runx2</I> expression is expanded in the tooth bud mesenchyme in <I>Osr2</I><SUP>-/-</SUP> mutant mouse embryos and is partially restored in the tooth mesenchyme in <I>Msx1<SUP>-/-</SUP>Osr2<SUP>-/-</SUP></I> mutants in comparison with <I>Msx1<SUP>-/-</SUP></I> and wild-type embryos. Whereas mandibular molar development arrested at the bud stage and maxillary molar development arrested at the bud-to-cap transition in <I>Runx2<SUP>-/-</SUP></I> mutant mice, both mandibular and maxillary molar tooth germs progressed to the early bell stage, with rescued expression of <I>Msx1</I> and <I>Bmp4</I> in the dental papilla as well as expression of <I>Bmp4, p21</I>, and <I>Shh</I> in the primary enamel knot in the <I>Osr2<SUP>-/-</SUP>Runx2<SUP>-/-</SUP></I> compound mutants. In contrast to the <I>Msx1<SUP>-/-</SUP>Osr2<SUP>-/-</SUP></I> compound mutants, which exhibit nearly normal first molar morphogenesis, the <I>Osr2<SUP>-/-</SUP>Runx2<SUP>-/-</SUP></I> compound mutant embryos failed to activate the expression of <I>Fgf3</I> and <I>Fgf10</I> in the dental papilla and exhibited significant deficit in cell proliferation in both the dental epithelium and mesenchyme in comparison with the control embryos. These data indicate that Runx2 synergizes with Msx1 to drive tooth morphogenesis through the bud-to-cap transition and that Runx2 controls continued tooth growth and morphogenesis beyond the cap stage through activation of <I>Fgf3</I> and <I>Fgf10</I> expression in the dental papilla.</P>

Hypotonic Stress Induces RANKL via Transient Receptor Potential Melastatin 3 (TRPM3) and Vaniloid 4 (TRPV4) in Human PDL Cells

Son, G.Y.,Yang, Y.M.,Park, W.S.,Chang, I.,Shin, D.M. Journal of Dental Research, Inc 2015 Journal of dental research Vol.94 No.3

<P>Bone remodeling occurs in response to various types of mechanical stress. The periodontal ligament (PDL) plays an important role in mechanical stress–mediated alveolar bone remodeling. However, the underlying mechanism at the cellular level has not been extensively studied. In this study, we investigated the effect of shear stress on the expression of bone remodeling factors, including receptor activator of nuclear factor–kappa B (NF-κB) ligand (RANKL) and osteoprotegerin (OPG), as well as its upstream signaling pathway in primary human PDL cells. We applied hypotonic stress to reproduce shear stress to PDL cells. Hypotonic stress induced the messenger RNA (mRNA) and protein expression of RANKL but not OPG. It also increased intracellular Ca<SUP>2+</SUP> concentration ([Ca<SUP>2+</SUP>]<SUB>i</SUB>). Extracellular Ca<SUP>2+</SUP> depletion and nonspecific plasma membrane Ca<SUP>2+</SUP> channel blockers completely inhibited the increase in both [Ca<SUP>2+</SUP>]<SUB>i</SUB> and RANKL mRNA expression. We identified the expression and activation of transient receptor potential melastatin 3 (TRPM3) and vaniloid 4 (TRPV4) channels in PDL cells. Pregnenolone sulfate (PS) and 4α-phorbol 12, 13-didecanoate (4α-PDD), which are agonists of TRPM3 and TRPV4, augmented Ca<SUP>2+</SUP> influx and RANKL mRNA expression. Both pharmacological (2-aminoethoxydiphenyl borate [2-APB], ruthenium red [RR], ononetin [Ono], and HC 067047 [HC]) and genetic (small interfering RNA [siRNA]) inhibitors of TRPM3 and TRPV4 reduced the hypotonic stress–mediated increase in [Ca<SUP>2+</SUP>]<SUB>i</SUB> and RANKL mRNA expression. Our study shows that hypotonic stress induced RANKL mRNA expression via TRPM3- and TRPV4-mediated extracellular Ca<SUP>2+</SUP> influx and RANKL expression. This signaling pathway in PDL cells may play a critical role in mechanical stress–mediated alveolar bone remodeling.</P>

Pyrosequencing Analysis of Subgingival Microbiota in Distinct Periodontal Conditions

Park, O.-J.,Yi, H.,Jeon, J.H.,Kang, S.-S.,Koo, K.-T.,Kum, K.-Y.,Chun, J.,Yun, C.-H.,Han, S.H. Journal of Dental Research, Inc 2015 Journal of dental research Vol.94 No.7

<P>Subgingival microorganisms are potentially associated with periodontal diseases. However, changes in the subgingival microbiota during the progress of periodontal diseases are poorly understood. In this study, we analyzed bacterial communities in the subgingival paper point samples from 32 Korean individuals with no sign of disease, gingivitis, or periodontitis using 454 FLX Titanium pyrosequencing. A total of 256,113 reads representing 26 phyla, 433 genera, and 1,016 species were detected. Bacteroidetes, Fusobacteria, Synergistetes, and Spirochaetes were the abundant phyla in periodontitis subjects, whereas Firmicutes and Proteobacteria were identified as the dominant phyla in the gingivitis and healthy subjects, respectively. Although high levels of <I>Porphyromonas, Fusobacterium, Fretibacterium, Rothia, Filifactor</I>, and <I>Treponema</I> genera were observed in the periodontitis subjects, <I>Streptococcus, Capnocytophaga, Leptotrichia</I>, and <I>Haemophilus</I> genera were found at high frequency in the gingivitis subjects. Species including <I>Porphyromonas gingivalis, Fusobacterium nucleatum</I>, and <I>Fretibacterium fastidiosum</I> were significantly increased in periodontitis subjects. On the other hand, <I>Streptococcus pseudopneumoniae, Haemophilus parainfluenzae</I>, and <I>Leptotrichia hongkongensis</I> were preferentially observed in the gingivitis subjects. Intriguingly, the halophile <I>Halomonas hamiltonii</I> was revealed as a predominant species in the healthy subjects. Based on Fast UniFrac analysis, distinctive bacterial clusters were classified for the healthy, gingivitis, and periodontitis state. The current findings might be useful for understanding the pathogenesis, diagnosis, and treatment of periodontal diseases.</P>

Piezo2 Expression in Mechanosensitive Dental Primary Afferent Neurons

Won, J.,Vang, H.,Lee, P. R.,Kim, Y. H.,Kim, H. W.,Kang, Y.,Oh, S. B. Journal of Dental Research, Inc 2017 Journal of dental research Vol.96 No.8

<P>Mechanosensitive ion channels have been suggested to be expressed in dental primary afferent (DPA) neurons to transduce the movement of dentinal fluid since the proposal of hydrodynamic theory. Piezo2, a mechanosensitive, rapidly inactivating (RI) ion channel, has been recently identified in dorsal root ganglion (DRG) neurons to mediate tactile transduction. Here, we examined the expression of Piezo2 in DPA neurons by in situ hybridization, single-cell reverse transcriptase polymerase chain reaction, and whole-cell patch-clamp recordings. DPA neurons with Piezo2 messenger RNA (mRNA) or Piezo2-like currents were further characterized based on their neurochemical and electrophysiological properties. Piezo2 mRNA was found mostly in medium- to large-sized DPA neurons, with the majority of these neurons also positive for Nav1.8, CGRP, and NF200, whereas only a minor population was positive for IB4 and peripherin. Whole-cell patch-clamp recordings revealed Piezo2-like, RI currents evoked by mechanical stimulation in a subpopulation of DPA neurons. RI currents were pharmacologically blocked by ruthenium red, a compound known to block Piezo2, and were also reduced by small interfering RNA-mediated Piezo2 knockdown. Piezo2-like currents were observed almost exclusively in IB4-negative DPA neurons, with the current amplitude larger in capsaicin-insensitive DPA neurons than the capsaicin-sensitive population. Our findings show that subpopulation of DPA neurons is indeed mechanically sensitive. Within this subpopulation of mechanosensitive DPA neurons, we have identified the Piezo2 ion channel as a potential transducer for mechanical stimuli, contributing to RI inward currents. Piezo2-positive DPA neurons were characterized as medium- to large-sized neurons with myelinated A-fibers, containing nociceptive peptidergic neurotransmitters.</P>

Spatiotemporally Controlled Microchannels of Periodontal Mimic Scaffolds

Park, C.H.,Kim, K.H.,Rios, H.F.,Lee, Y.M.,Giannobile, W.V.,Seol, Y.J. Journal of Dental Research, Inc 2014 Journal of dental research Vol.93 No.12

<P>Physiologic bioengineering of the oral, dental, and craniofacial complex requires optimized geometric organizations of fibrous connective tissues. A computer-designed, fiber-guiding scaffold has been developed to promote tooth-supporting periodontal tissue regeneration and functional restoration despite limited printing resolution for the manufacture of submicron-scaled features. Here, we demonstrate the use of directional freeze-casting techniques to control pore directional angulations and create mimicked topographies to alveolar crest, horizontal, oblique, and apical fibers of natural periodontal ligaments. For the differing anatomic positions, the gelatin displayed varying patterns of ice growth, determined <I>via</I> internal pore architectures. Regardless of the freezing coordinates, the longitudinal pore arrangements resulted in submicron-scaled diameters (~50 µm), along with corresponding high biomaterial porosity (~90%). Furthermore, the horizontal + coronal ([Formula]) freezing orientation facilitated the creation of similar structures to major fibers in the periodontal ligament interface. This periodontal tissue-mimicking microenvironment is a potential tissue platform for the generation of naturally oriented ligamentous tissues consistent with periodontal ligament neogenesis.</P>

<i>Glechoma hederacea</i> Suppresses RANKL-mediated Osteoclastogenesis

Hwang, J.K.,Erkhembaatar, M.,Gu, D.R.,Lee, S.H.,Lee, C.H.,Shin, D.M.,Lee, Y.R.,Kim, M.S. Journal of Dental Research, Inc 2014 Journal of dental research Vol.93 No.7

<P> <I>Glechoma hederacea</I> (GH), commonly known as ground-ivy or gill-over-the-ground, has been extensively used in folk remedies for relieving symptoms of inflammatory disorders. However, the molecular mechanisms underlying the therapeutic action of GH are poorly understood. Here, we demonstrate that GH constituents inhibit osteoclastogenesis by abrogating receptor activator of nuclear κ-B ligand (RANKL)-induced free cytosolic Ca<SUP>2+</SUP> ([Ca<SUP>2+</SUP>]<SUB>i</SUB>) oscillations. To evaluate the effect of GH on osteoclastogenesis, we assessed the formation of multi-nucleated cells (MNCs), enzymatic activity of tartrate-resistant acidic phosphatase (TRAP), expression of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), and [Ca<SUP>2+</SUP>]<SUB>i</SUB> alterations in response to treatment with GH ethanol extract (GHE) in primarily cultured bone marrow–derived macrophages (BMMs). Treatment of RANKL-stimulated or non-stimulated BMMs with GHE markedly suppressed MNC formation, TRAP activity, and NFATc1 expression in a dose-dependent manner. Additionally, GHE treatment induced a large transient elevation in [Ca<SUP>2+</SUP>]<SUB>i</SUB> while suppressing RANKL-induced [Ca<SUP>2+</SUP>]<SUB>i</SUB> oscillations, which are essential for NFATc1 activation. GHE-evoked increase in [Ca<SUP>2+</SUP>]<SUB>i</SUB> was dependent on extracellular Ca<SUP>2+</SUP> and was inhibited by 1,4-dihydropyridine (DHP), inhibitor of voltage-gated Ca<SUP>2+</SUP> channels (VGCCs), but was independent of store-operated Ca<SUP>2+</SUP> channels. Notably, after transient [Ca<SUP>2+</SUP>] elevation, treatment with GHE desensitized the VGCCs, resulting in an abrogation of RANKL-induced [Ca<SUP>2+</SUP>]<SUB>i</SUB> oscillations and MNC formation. These findings demonstrate that treatment of BMMs with GHE suppresses RANKL-mediated osteoclastogenesis by activating and then desensitizing DHP-sensitive VGCCs, suggesting potential applications of GH in the treatment of bone disorders, such as periodontitis, osteoporosis, and rheumatoid arthritis.</P>

Wntless Regulates Dentin Apposition and Root Elongation in the Mandibular Molar

Bae, C.H.,Kim, T.H.,Ko, S.O.,Lee, J.C.,Yang, X.,Cho, E.S. Journal of Dental Research, Inc 2015 Journal of dental research Vol.94 No.3

<P>Wnt signaling plays an essential role in the dental epithelium and mesenchyme during tooth morphogenesis. However, it remains unclear if Wnt ligands, produced from dental mesenchyme, are necessary for odontoblast differentiation and dentin formation. Here, we show that odontoblast-specific disruption of Wntless (Wls), a chaperon protein that regulates Wnt sorting and secretion, leads to severe defects in dentin formation and root elongation. Dentin thickness decreased remarkably and pulp chambers enlarged in the mandibular molars of <I>OC-Cre;Wls<SUP>CO/CO</SUP> </I> mice. Although the initial odontoblast differentiation was normal in the mutant crown, odontoblasts became cuboidal and dentin thickness was reduced. In immunohistochemistry, Wnt10a, β-catenin, type I collagen, and dentin sialoprotein were significantly down-regulated in the odontoblasts of mutant crown. In addition, roots were short and root canals were widened. Cell proliferation was reduced in the developing root apex of mutant molars. Furthermore, Wnt10a and Axin2 expression was remarkably decreased in the odontoblasts of mutant roots. Deletion of the <I>Wls</I> gene in odontoblasts appears to reduce canonical Wnt activity, leading to inhibition of odontoblast maturation and root elongation.</P>