Satoshi Monnouchi

Mineral Trioxide Aggregate Induces Bone Morphogenetic Protein-2 Expression and Calcification in Human Periodontal Ligament Cells

INTRODUCTION Mineral trioxide aggregate (MTA) is a therapeutic, endodontic repair material that is reported to exhibit calcified tissue-conductive activity although the mechanisms remain unclear. We hypothesize that the dissolution of calcium from MTA into the surrounding environment may play an important role in the osteoblastic/cementoblastic differentiation of human periodontal ligament cells (HPLCs). METHODS Two populations of HPLCs were obtained from two patients, respectively, and were cultured in the presence or absence of MTA discs and/or CaCl(2) in order to investigate calcium release, calcification activity, calcium-sensing receptor (CaSR) gene expression and bone morphogenetic protein-2 (BMP-2), and BMP-2 receptor protein and gene expression. RESULTS MTA released a substantial accumulation of calcium (4 mmol/L) within 14 days into culture media. After 4 weeks, the two populations of HPLCs independently exhibited calcification as well as BMP-2 distribution in the vicinity of MTA. HPLCs inherently expressed genes encoding for the CaSR and BMP-2 receptors. Exogenous CaCl(2) media supplementation induced CaSR gene expression in HPLCs and calcification and BMP-2 synthesis throughout the entire HPLC cultures, whereas MgCl(2) had no effect. Both MTA and CaCl(2) stimulated BMP-2 gene expression above that of baseline levels. CONCLUSION Here we show the first report showing that HPLCs cocultured directly with MTA up-regulated BMP2 expression and calcification. These results may be through CaSR interactions that were potentially activated by the release of calcium from MTA into the culture environment.

Effects of TGF-β1 on the proliferation and differentiation of human periodontal ligament cells and a human periodontal ligament stem/progenitor cell line

Periodontal ligament (PDL) is a specialized connective tissue that influences the lifespan of the tooth. Transforming growth factor-β1 (TGF-β1) is a multifunctional cytokine, but little is known about the effects of TGF-β1 on PDL cells. Our aim has been to demonstrate the expression of TGF-β1 in rat PDL tissues and to evaluate its effects on the proliferation and gene expression in human PDL cells (HPLCs) and a human PDL stem/progenitor cell line, line 1-11, that we have recently developed. The expression of TGF-β1 in the entire PDL tissue was confirmed immunohistochemically, and both HPLCs and cell line 1-11 expressed mRNA from the TGF-β1, TGF-β type I receptor, and TGF-β type II receptor genes. Although exogenous TGF-β1 stimulated the proliferation of HPLCs, it did not upregulate the expression of alpha-smooth muscle actin (α-SMA), type I collagen (Col I), or fibrillin-1 (FBN1) mRNA or of α-SMA protein in HPLCs, whereas expression for these genes was attenuated by an anti-TGF-β1 neutralizing antibody. In contrast, exogenous TGF-β1 reduced the proliferation of cell line 1-11, although it upregulated the expression of α-SMA, Col I, and FBN1 mRNA and of α-SMA protein in this cell line. In addition, interleukin-1 beta stimulation significantly reduced the expression of TGF-β1 mRNA and protein in HPLCs. Thus, TGF-β1 seems to play an important role in inducing fibroblastic differentiation of PDL stem/progenitor cells and in maintaining the PDL apparatus under physiological conditions.

A multipotent clonal human periodontal ligament cell line with neural crest cell phenotypes promotes neurocytic differentiation, migration, and survival

Repair of injured peripheral nerve is thought to play important roles in tissue homeostasis and regeneration. Recent experiments have demonstrated enhanced functional recovery of damaged neurons by some types of somatic stem cells. It remains unclear, however, if periodontal ligament (PDL) stem cells possess such functions. We recently developed a multipotent clonal human PDL cell line, termed cell line 1‐17. Here, we investigated the effects of this cell line on neurocytic differentiation, migration, and survival. This cell line expressed the neural crest cell marker genes Slug, SOX10, Nestin, p75NTR, and CD49d and mesenchymal stem cell‐related markers CD13, CD29, CD44, CD71, CD90, CD105, and CD166. Rat adrenal pheochromocytoma cells (PC12 cells) underwent neurocytic differentiation when co‐cultured with cell line 1‐17 or in conditioned medium from cell line 1‐17 (1‐17CM). ELISA analysis revealed that 1‐17CM contained approximately 50 pg/ml nerve growth factor (NGF). Cell line 1‐17‐induced migration of PC12 cells, which was inhibited by a neutralizing antibody against NGF. Furthermore, 1‐17CM exerted antiapoptotic effects on differentiated PC12 cells as evidenced by inhibition of neurite retraction, reduction in annexin V and caspase‐3/7 staining, and induction of Bcl‐2 and Bcl‐xL mRNA expression. Thus, cell line 1‐17 promoted neurocytic differentiation, migration, and survival through secretion of NGF and possibly synergistic factors. PDL stem cells may play a role in peripheral nerve reinnervation during PDL regeneration. J. Cell. Physiol. 227: 2040–2050, 2012.

The Roles of Angiotensin II in Stretched Periodontal Ligament Cells

The loading caused by occlusion and mastication plays an important role in maintaining periodontal ligament (PDL) tissues. We hypothesized that a loading magnitude would be involved in the production of biological factors that function in the maintenance of PDL tissues. Here, we identified up-regulated gene expressions of transforming growth factor-β1 (TGF-β1), alkaline phosphatase (ALP), and angiotensinogen in human PDL fibroblastic cells (HPLFs) that were exposed to 8% stretch loading. Immunolocalization of angiotensin I/II (Ang I/II), which was converted from angiotensinogen, was detected in rat PDL tissues. HPLFs that were stimulated by Ang II also increased their gene expressions of TGF-β1 and ALP. Furthermore, the antagonist for Ang II type 2 receptor, rather than for type 1, significantly inhibited gene expressions induced by the stretch loading. Analysis of these data suggests that Ang II mediates the loading signal in stretched HPLFs to induce expressions of TGF-β1 and ALP.

Prospective potency of TGF-β1 on maintenance and regeneration of periodontal tissue

Periodontal ligament (PDL) tissue, central in the periodontium, plays crucial roles in sustaining tooth in the bone socket. Irreparable damages of this tissue provoke tooth loss, causing a decreased quality of life. The question arises as to how PDL tissue is maintained or how the lost PDL tissue can be regenerated. Stem cells included in PDL tissue (PDLSCs) are widely accepted to have the potential to maintain or regenerate the periodontium, but PDLSCs are very few in number. In recent studies, undifferentiated clonal human PDL cell lines were developed to elucidate the applicable potentials of PDLSCs for the periodontal regenerative medicine based on cell-based tissue engineering. In addition, it has been suggested that transforming growth factor-beta 1 is an eligible factor for the maintenance and regeneration of PDL tissue.

Semaphorin 3A Induces Mesenchymal-Stem-Like Properties in Human Periodontal Ligament Cells

Periodontal ligament stem cells (PDLSCs) have recently been proposed as a novel option in periodontal regenerative therapy. However, one of the issues is the difficulty of stably generating PDLSCs because of the variation of stem cell potential between donors. Here, we show that Semaphorin 3A (Sema3A) can induce mesenchymal-stem-like properties in human periodontal ligament (PDL) cells. Sema3A expression was specifically observed in the dental follicle during tooth development and in parts of mature PDL tissue in rodent tooth and periodontal tissue. Sema3A expression levels were found to be higher in multipotential human PDL cell clones compared with low-differentiation potential clones. Sema3A-overexpressing PDL cells exhibited an enhanced capacity to differentiate into both functional osteoblasts and adipocytes. Moreover, PDL cells treated with Sema3A only at the initiation of culture stimulated osteogenesis, while Sema3A treatment throughout the culture had no effect on osteogenic differentiation. Finally, Sema3A-overexpressing PDL cells upregulated the expression of embryonic stem cell markers (NANOG, OCT4, and E-cadherin) and mesenchymal stem cell markers (CD73, CD90, CD105, CD146, and CD166), and Sema3A promoted cell division activity of PDL cells. These results suggest that Sema3A may possess the function to convert PDL cells into mesenchymal-stem-like cells.

Wnt5a Induces Collagen Production by Human Periodontal Ligament Cells Through TGFβ1-Mediated Upregulation of Periostin Expression.

Wnt5a, a member of the noncanonical Wnt proteins, is known to play important roles in the development of various organs and in postnatal cell functions. However, little is known about the effects of Wnt5a on human periodontal ligament (PDL) cells. In this study, we examined the localization and potential function of Wnt5a in PDL tissue. Immunohistochemical analysis revealed that Wnt5a was expressed predominantly in rat PDL tissue. Semi‐quantitative reverse‐transcription polymerase chain reaction and Western blotting analysis demonstrated that human PDL cells (HPDLCs) expressed Wnt5a and its receptors (Ror2, Fzd2, Fzd4, and Fzd5). Removal of occlusal pressure by extraction of opposing teeth decreased Wnt5a expression in rat PDL tissue, and the expression of Wnt5a and its receptors in HPDLCs was upregulated by exposure to mechanical stress. Stimulation with Wnt5a significantly enhanced the proliferation and migration of HPDLCs. Furthermore, Wnt5a suppressed osteoblastic differentiation of HPDLCs cultivated in osteogenic induction medium, while it significantly enhanced the expression of PDL‐related genes, such as periostin, type‐I collagen, and fibrillin‐1 genes, and the production of collagen in HPDLCs cultivated in normal medium. Both knockdown of periostin gene expression by siRNA and inhibition of TGFβ1 function by neutralizing antibody suppressed the Wnt5a‐induced PDL‐related gene expression and collagen production in HPDLCs. Interestingly, in HPDLCs cultured with Wnt5a, TGFβ1 neutralizing antibody significantly suppressed periostin expression, while periostin siRNA had no effect on TGFβ1 expression. These results suggest that Wnt5a expressed in PDL tissue plays specific roles in inducing collagen production by PDL cells through TGFβ1‐mediated upregulation of periostin expression. J. Cell. Physiol. 9999: 2647–2660, 2015.

Effect of CTGF/CCN2 on Osteo/Cementoblastic and Fibroblastic Differentiation of a Human Periodontal Ligament Stem/Progenitor Cell Line

Appropriate mechanical loading during occlusion and mastication play an important role in maintaining the homeostasis of periodontal ligament (PDL) tissue. Connective tissue growth factor (CTGF/CCN2), a matricellular protein, is known to upregulate extracellular matrix production, including collagen in PDL tissue. However, the underlying mechanisms of CTGF/CCN2 in regulation of PDL tissue integrity remain unclear. In this study, we investigated the effect of CTGF/CCN2 on osteo/cementoblastic and fibroblastic differentiation of human PDL stem cells using the cell line 1–11. CTGF/CCN2 expression in rat PDL tissue and human PDL cells (HPDLCs) was confirmed immunohisto/cytochemically. Mechanical loading was found to increase gene expression and secretion of CTGF/CCN2 in HPDLCs. CTGF/CCN2 upregulated the proliferation and migration of 1–11 cells. Furthermore, increased bone/cementum‐related gene expression in this cell line led to mineralization. In addition, combined treatment of 1–11 cells with CTGF/CCN2 and transforming growth factor‐β1 (TGF‐β1) significantly promoted type I collagen and fibronectin expression compared with that of TGF‐β1 treatment alone. Thus, these data suggest the underlying biphasic effects of CTGF/CCN2 in 1–11 cells, inducible osteo/cementoblastic, and fibroblastic differentiation dependent on the environmental condition. CTGF/CCN2 may contribute to preservation of the structural integrity of PDL tissue, implying its potential use as a therapeutic agent for PDL regeneration. J. Cell. Physiol. 230: 150–159, 2015.

Expression and effects of glial cell line-derived neurotrophic factor on periodontal ligament cells

AIM To investigate Glial cell line-derived neurotrophic factor (GDNF) expression in normal and wounded rat periodontal ligament (PDL) and the effects of GDNF on human PDL cells (HPDLCs) migration and extracellular matrix expression in HPDLCs. MATERIAL AND METHODS The expression of GDNF and GDNF receptors was examined by immunocyto/histochemical analyses. Gene expression in HPDLCs treated with GDNF, interleukin-1 beta (IL-1β), or tumour necrosis factor-alpha (TNF-α) was quantified by quantitative RT-PCR (qRT-PCR). In addition, we examined the migratory effect of GDNF on HPDLCs. RESULTS GDNF was expressed in normal rat PDL and cultured HPDLCs. HPDLCs also expressed GDNF receptors. In wounded rat PDL, GDNF expression was up-regulated. QRT-PCR analysis revealed that IL-1β and TNF-α significantly increased the expression of GDNF in HPDLCs. Furthermore, GDNF induced migration of HPDLCs, which was blocked by pre-treatment with the peptide including Arg-Gly-Asp (RGD) sequence, or neutralizing antibodies against integrin αVβ3 or GDNF. Also, GDNF up-regulated expression of bone sialoprotein (BSP) and fibronectin in HPDLCs. CONCLUSIONS GDNF expression is increased in rat wounded PDL tissue and HPDLCs treated with pro-inflammatory cytokines. GDNF enhances the expression of BSP and fibronectin, and migration in an RGD-dependent manner via the integrin αVβ3. These findings suggest that GDNF may contribute to wound healing in PDL tissue.

Alternation of extracellular matrix remodeling and apoptosis by activation of the aryl hydrocarbon receptor pathway in human periodontal ligament cells.

It is well known that the aryl hydrocarbon receptor (AhR) is involved in the toxicity of halogenated aromatic hydrocarbons (HAH) and polycyclic aromatic hydrocarbons (PAH). Recent experiments have shown the induction of impaired tooth and hard‐tissue formation by AhR pathway activation, however, the effect on periodontal ligament (PDL) tissue remains unclear. Here, we investigated the effects of benzo(a)pyrene (BaP), a member of PAH, on the extracellular matrix (ECM) remodeling‐related molecules, collagen type I (COL‐I), matrix metalloproteinase‐1 (MMP‐1), alpha‐smooth muscle actin (α‐SMA) expression, and apoptosis in two different human periodontal ligament cells (HPDLCs). The transduction of AhR from the cytoplasm to the nucleus and the increase of AhR‐responsive genes; that is, cytochrome P450 1A1 (CYP1A1), cytochrome P450 1B1 (CYP1B1), and aryl‐hydrocarbon receptor repressor (AhRR), expression was induced by BaP exposure in both HPDLCs. BaP treatment significantly enhanced MMP‐1 mRNA expression and MMP‐1 protein production, while markedly suppressing COL‐I and a‐SMA mRNA expression in both HPDLCs. Furthermore, these BaP‐treated HPDLCs fell into apoptotic cell death as evidenced by induction in annexin V and caspase‐3/7 staining and reduction of total cell number and Bcl‐2 mRNA expression. Thus, BaP exposure altered the expression of ECM‐related molecules and induced apoptosis in HPDLCs through activation of the AhR pathway. Overactivity of the AhR pathway may induce an inappropriate turnover of PDL tissue via disordered ECM remodeling and apoptosis in PDL cells. J. Cell. Biochem. 113: 3093–3103, 2012.