Kengo Iwasaki

Twist negatively regulates osteoblastic differentiation in human periodontal ligament cells

Periodontal ligament (PDL) is a thin fibrous connective tissue located between two mineralized tissues, alveolar bone and cementum, which maintains a constant width physiologically. The mechanisms by which PDL resists mineralization are not well understood. Twist is a basic helix loop helix protein that plays a central role in regulation of early osteogenesis. We investigated the localization of Twist in PDL and compared the expression of Twist and osteoblast‐related genes in PDL cells with those in osteoblast‐like cells in the presence or absence of recombinant human bone morphogenetic protein (BMP)‐2. Histochemical analysis showed that Twist was expressed along alveolar bone surface in PDL. PDL cells constitutively expressed Twist gene and the expression level was higher than that in osteoblast‐like cells. In osteoblast‐like cell culture, BMP‐2 enhanced osteoblast‐related gene expression, while Twist expression was slightly decreased. In contrast, BMP‐2 increased runt‐related transcription factor (Runx)‐2, but failed to enhance alkaline phosphatase (ALP) and osteocalcin (OCN) gene expression in PDL cells. Interestingly, unlike in osteoblast‐like cells, Twist expression was upregulated by BMP‐2 in PDL cells. We transiently knocked down Twist gene in PDL cells using a short interference RNA expression vector (siTwist) and found that ALP, osteopontin (OPN), bone sialoprotein (BSP) genes expression and basal level of ALP activity were slightly increased, whereas Runx2 and OCN genes were not affected. Collectively, these results suggest that Twist may act as a negative regulator of osteoblastic differentiation in PDL cells. J. Cell. Biochem. 100: 303–314, 2007.

The effect of chemical and/or mechanical conditioning on the Er:YAG laser-treated root cementum: analysis of surface morphology and periodontal ligament fibroblast attachment.

This study compared the surface morphology as well as the biocompatibility of dental root cementum treated with Er:YAG laser irradiation alone and with the laser irradiation followed by chemical and/or mechanical conditioning.

Cementum protein 1 (CEMP1) induces a cementoblastic phenotype and reduces osteoblastic differentiation in periodontal ligament cells

Cementum is a calcified tissue covering the tooth root surface, which functions as rigid tooth‐anchoring structure. Periodontal ligament is a unique non‐mineralized connective tissue, and is a source of mineralized tissue forming cells such as cementoblasts and osteoblasts. The CEMP1 is a novel cementum component the presence of which appears to be limited to cementoblasts and their progenitors. In order to understand the function of CEMP1, we investigated CEMP1 expression during the differentiation of human periodontal ligament cells. Immunomagnetically enriched alkaline phosphatase (ALP)‐positive periodontal ligament cells preferentially expressed CEMP1. CEMP1 expression was reduced when periodontal ligament cells differentiated to osteoblasts in vitro. Over‐expression of CEMP1 in periodontal ligament cells enhanced cementoblast differentiation and attenuated periodontal and osteoblastic phenotypes. Our data demonstrate for the first time that the CEMP1 is not only a marker protein for cementoblast‐related cells, but it also regulates cementoblast commitment in periodontal ligament cells. J. Cell. Physiol. 227: 649–657, 2012.

Periodontal Ligament Stem Cells Possess the Characteristics of Pericytes

BACKGROUND Periodontal ligament (PDL) contributes to maintaining homeostasis in periodontal tissues by supplying stem/progenitor cells. It has long been suggested that PDL stem cells/progenitors are located around blood vessels. Recently mesenchymal stem cells (MSCs) have been isolated and cultured from PDL in vitro, although the location of the stem cells in PDL is unclear. The purpose of this study is to test the characteristics of human PDL stem cells (PDLSCs) and examine their similarity to related vascular cell types, such as pericytes and endothelial cells. METHODS PDLSCs were obtained from healthy extracted teeth using the collagenase/dispase enzyme digestion method. MSC and pericyte characteristics of PDLSCs were examined by cell surface marker expression using flow cytometry. The expression of pericyte markers was tested using immunohistochemistry. Pericyte-like functions of PDLSCs were examined in co-culture of PDLSCs and umbilical vein endothelial cells on a gel matrix. RESULTS Cultured PDLSCs were positive for both MSC markers and pericyte markers, including cluster of differentiation 146 (CD146), neural/glial antigen 2 (NG2), and CD140b. When pericyte marker expression was explored in rat periodontal tissue sections, CD146- and NG2-positive signals were observed in the perivascular area of the PDL. Further, when the cells were cultured with human umbilical cord endothelial cells under conditions for forming capillary-like structures in vitro, PDLSCs localized adjacent to endothelial cells and contributed to the stability of the capillary-like structure. CONCLUSIONS PDLSCs possess pericyte-like characteristics and may localize as pericytes in the PDL. These data provide useful information for stem cell biology in periodontal research and stem cell-based periodontal therapy.

Human gingival fibroblasts release high-mobility group box-1 protein through active and passive pathways

INTRODUCTION The nuclear protein high-mobility group box-1 (HMGB1) acts as a late mediator of inflammation when secreted in the extracellular milieu. In this study, we examined the effect of lipopolysaccharides from periodontal pathogens and apoptotic and necrotic cell death on HMGB1 production in human gingival fibroblasts (HGF). METHODS HGF from healthy periodontal tissue were cultured and stimulated with lipopolysaccharides (LPS) from Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Escherichia coli. We also initiated apoptotic and necrotic cell deaths in HGF. The HMGB1 released in the supernatants from stimulated or dying cells was measured. Immunocytochemical staining against HMGB1 was performed in LPS-stimulated HGF. RESULTS A significantly higher amount of HMGB1 was detected from necrotic and apoptotic HGF. LPS from A. actinomycetemcomitans, P. gingivalis, and E. coli significantly induced the production of HMGB1 in a time-dependent manner. After 6 h of LPS stimulation, HMGB1 was present in the cytoplasm of cells whereas its location was mainly nuclear after 24 h. CONCLUSIONS LPS from two major periodontal pathogens, A. actinomycetemcomitans and P. gingivalis, induced HMGB1 secretion from HGF. Apoptotic and necrotic cell deaths resulted in the enhancement of HMGB1. Our results suggest that HGF can be a source of HMGB1 by both active secretion and passive release, and that HMGB1 from HGF may contribute to periodontal tissue destruction.

Comparison of characteristics of periodontal ligament cells obtained from outgrowth and enzyme-digested culture methods

OBJECTIVE Periodontal ligament (PDL) cells have an important role in periodontal regeneration. The unique characteristics of PDL cells, mainly outgrown cells derived from PDL tissue, have been investigated. Recently, mesenchymal stem cells have been obtained from PDL tissue using enzyme digestion. The differences in properties of those PDL cells cultured by the two methods (outgrowth and enzyme digestion) are unclear. The objective of this study was to investigate the characteristics of PDL cells obtained by these methods. METHODS PDL cells from extracted tooth were cultured using outgrowth and enzyme digest methods. Cell proliferation, colony-forming activity and differentiation capacity to osteoblast, adipocyte and chondrocyte were compared. Gene expressions for PDL cells, mesenchymal stem cells and fibroblasts were also investigated by reverse transcription polymerase chain reaction. Procollagen type I c-peptide (PIP) production was measured using an enzyme-linked immunosorbent assay (ELISA) kit. RESULTS PDL cells cultured by enzyme digest methods showed a higher proliferation rate, colony-forming activity and differentiation capacity into osteoblast, adipocyte and chondrocyte than those in PDL cells by outgrowth method. CD166, one of the mesenchymal stem cell markers, was slightly higher in enzyme-digested PDL than in outgrowth PDL, whilst gene expressions for type 1 collagen alpha 1 and type 3 collagen were higher in outgrown PDL cells. Moreover, outgrowth PDL exhibited higher PIP production than enzyme-digested PDL cells. CONCLUSION PDL cells obtained by outgrowth and enzyme digestion showed different characteristics. The enzyme digestion method yielded cells with higher proliferation rate and mesenchymal stem cell-like properties, whereas cells with fibroblast-like properties were collected in the outgrowth method. PDL cell properties by different culture methods may provide information for inventing new therapeutic uses of PDL cells.

The Enamel Protein Amelotin Is a Promoter of Hydroxyapatite Mineralization

Amelotin (AMTN) is a recently discovered protein that is specifically expressed during the maturation stage of dental enamel formation. It is localized at the interface between the enamel surface and the apical surface of ameloblasts. AMTN knock‐out mice have hypomineralized enamel, whereas transgenic mice overexpressing AMTN have a compact but disorganized enamel hydroxyapatite (HA) microstructure, indicating a possible involvement of AMTN in regulating HA mineralization directly. In this study, we demonstrated that recombinant human (rh) AMTN dissolved in a metastable buffer system, based on light scattering measurements, promotes HA precipitation. The mineral precipitates were characterized by scanning and transmission electron microscopy and electron diffraction. Colloidal gold immunolabeling of AMTN in the mineral deposits showed that protein molecules were associated with HA crystals. The binding affinity of rh‐AMTN to HA was found to be comparable to that of amelogenin, the major protein of the forming enamel matrix. Overexpression of AMTN in mouse calvaria cells also increased the formation of calcium deposits in the culture medium. Overexpression of AMTN during the secretory stage of enamel formation in vivo resulted in rapid and uncontrolled enamel mineralization. Site‐specific mutagenesis of the potential serine phosphorylation motif SSEEL reduced the in vitro mineral precipitation to less than 25%, revealing that this motif is important for the HA mineralizing function of the protein. A synthetic short peptide containing the SSEEL motif was only able to facilitate mineralization in its phosphorylated form (PSPSEEL), indicating that this motif is necessary but not sufficient for the mineralizing properties of AMTN. These findings demonstrate that AMTN has a direct influence on biomineralization by promoting HA mineralization and suggest a critical role for AMTN in the formation of the compact aprismatic enamel surface layer during the maturation stage of amelogenesis.

Cyclooxygenase-2-dependent Prostaglandin E2 Down-regulates Intercellular Adhesion Molecule-1 Expression via EP2/EP4 Receptors in Interleukin-1ß-stimulated Human Gingival Fibroblasts

Prostaglandin E2 (PGE2), which exerts its actions via EP receptors (EP,, EP2, EP 3, and EP4), is a bioactive metabolite of arachidonic acid produced by cyclooxygenase (COX)-l and/or COX-2. We have previously demonstrated that PGE, downregulates intercellular adhesion molecule-1 (ICAM-1) expression in interleukin-1β (IL-1β)-stimulated human gingival fibroblasts (HGF). In the present study, we investigated which COX was involved in down-regulation of ICAM-1 expression by PGE2 in IL-1β-stimulated HGF and which subtypes of EP receptors modulated the ICAM-1 expression. NS-398, a specific COX-2 inhibitor, completely inhibited PGE2 production by IL-1β-stimulated HGF, as did indomethacin, a COX-1/COX-2 inhibitor. Northern blot analysis and immunocytochemical staining showed that mRNA and protein of COX-2 were expressed in IL-1β-challenged HGF, but not in unstimulated HGF, and that the expression of mRNA and protein of COX-1 was similar both in unstimulated and in stimulated cells. NS-398 and indomethacin enhanced ICAM-1 expression in IL-1β-challenged HGF. EP1, EP 2, and EP4 receptor mRNA was expressed in HGF according to reverse-transcription/polymerase chain-reaction. PGE2, 11-deoxy-PGE 1 (a selective EP2/EP4 agonist), and Butaprost (a selective EP2 agonist) attenuated IL-1β-elicited ICAM-1 expression, although Butaprost was less potent than PGE2 and 11-deoxy-PGE1. AH-23848B, an EP4 antagonist, antagonized the inhibitory effect of IL-1β-elicited ICAM-1 expression by PGE2. Sulprostone, an EP1/EP3 agonist, had no effect on IL-1β-elicited ICAM-1 expression. Analysis of these data suggests that COX-2-derived PGE 2 downregulates ICAM-1 expression via EP2/EP 4 receptors in IL-1β-stimulated HGF.

IL-6 Induces Osteoblastic Differentiation of Periodontal Ligament Cells

Interleukin (IL)-6 has been considered as an osteolytic factor involved in periodontal disease. However, the function of IL-6 in osteoblastic differentiation of periodontal ligament cells is not clear. We examined the effects of IL-6 and its soluble receptor (sIL-6R) on osteoblastic differentiation of periodontal ligament cells. Osteoblastic differentiation was induced by ascorbic acid. Osteoblast markers, including alkaline phosphatase activity and Runx2 gene expression, were examined. The mechanism of action of IL-6 on osteoblastic differentiation was evaluated by insulin-like growth factor (IGF)-I production and specific inhibitors for the IL-6-signaling molecule. IL-6/sIL-6R enhanced alkaline phosphatase activity and Runx2. Alkaline phosphatase activity was reduced by anti-IGF-I antibody. Mitogen-activated protein kinase and Janus protein tyrosine kinase inhibitors diminished alkaline phosphatase induced by IL-6/sIL-6R. We conclude that IL-6/sIL-6R increases ascorbic-acid-induced alkaline phosphatase activity through IGF-I production, implying that IL-6 acts not only as an osteolytic factor, but also as a mediator of osteoblastic differentiation in periodontal ligament cells.

Downregulation of Lipopolysaccharide-Induced Intercellular Adhesion Molecule-1 Expression via EP2/EP4 Receptors by Prostaglandin E2 in Human Fibroblasts

In the present study, the effect of prostaglandin E2 (PGE2) on intercellular adhesion molecule-1 (ICAM-1) expression in human gingival fibroblasts (HGF) stimulated with lipopolysaccharides (LPS) was investigated. LPS were isolated from periodontopathic bacteria, Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans) and Porphyromonas gingivalis (P. gingivalis), by the phenol-water method and Escherichia coli (E. coli) LPS was used as a control. PGE2 significantly inhibited A. actinomycetemcomitans-, P. gingivalis- and E. coli-LPS-induced ICAM-1 expression. Next, of four PGE2 receptor subtypes (EP1, EP2, EP3 and EP4), we examined which subtype(s) was involved in inhibition of LPS-elicited ICAM-1 expression by PGE2. Eleven-deoxy-PGE1, a selective EP2/EP4 agonist, and butaprost, a selective EP2 agonist, attenuated A. actinomycetemcomitans-, P. gingivalis- and E. coli-LPS-elicited ICAM-1 expression, although butaprost was less potent than PGE2 and 11-deoxy-PGE1. Sulprostone, an EP1/EP3 agonist, and ONO-AP-324, an EP3 agonist, was inert to the LPS-elicited ICAM-1 expression. Furthermore, dibutyryl cAMP, a cAMP analogue, and forskolin, an adenylate cyclase activator, downregulated A. actinomycetemcomitans-, P. gingivalis- and E. coli-LPS-elicited ICAM-1 expression in HGF. Our data suggest that PGE2 downregulates A. actinomycetemcomitans- and P. gingivalis-LPS-induced ICAM-1 expression in HGF, via EP2/EP4 receptors by cAMP-dependent signaling pathways. The cAMP-elevating agents such as EP2/EP4 receptor activators may serve to control inflammatory and immune responses in periodontal disease.