Wnt5a Induces Collagen Production by Human Periodontal Ligament Cells Through TGFß1-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 TGFß1 function by neutralizing antibody suppressed the Wnt5a-induced PDL-related gene expression and collagen production in HPDLCs. Interestingly, in HPDLCs cultured with Wnt5a, TGFß1 neutralizing antibody significantly suppressed periostin expression, while periostin siRNA had no effect on TGFß1 expression. These results suggest that Wnt5a expressed in PDL tissue plays specific roles in inducing collagen production by PDL cells through TGFß1-mediated upregulation of periostin expression.

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-ß1 (TGF-ß1) significantly promoted type I collagen and fibronectin expression compared with that of TGF-ß1 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.

Identification of the microbiota in carious dentin lesions using 16S rRNA gene sequencing.

While mutans streptococci have long been assumed to be the specific pathogen responsible for human dental caries, the concept of a complex dental caries-associated microbiota has received significant attention in recent years. Molecular analyses revealed the complexity of the microbiota with the predominance of Lactobacillus and Prevotella in carious dentine lesions. However, characterization of the dentin caries-associated microbiota has not been extensively explored in different ethnicities and races. In the present study, the bacterial communities in the carious dentin of Japanese subjects were analyzed comprehensively with molecular approaches using the16S rRNA gene. Carious dentin lesion samples were collected from 32 subjects aged 4-76 years, and the 16S rRNA genes, amplified from the extracted DNA with universal primers, were sequenced with a pyrosequencer. The bacterial composition was classified into clusters I, II, and III according to the relative abundance (high, middle, low) of Lactobacillus. The bacterial composition in cluster II was composed of relatively high proportions of Olsenella and Propionibacterium or subdominated by heterogeneous genera. The bacterial communities in cluster III were characterized by the predominance of Atopobium, Prevotella, or Propionibacterium with Streptococcus or Actinomyces. Some samples in clusters II and III, mainly related to Atopobium and Propionibacterium, were novel combinations of microbiota in carious dentin lesions and may be characteristic of the Japanese population. Clone library analysis revealed that Atopobium sp. HOT-416 and P. acidifaciens were specific species associated with dentinal caries among these genera in a Japanese population. We summarized the bacterial composition of dentinal carious lesions in a Japanese population using next-generation sequencing and found typical Japanese types with Atopobium or Propionibacterium predominating.

Effects of Activin A on the phenotypic properties of human periodontal ligament cells.

Periodontal ligament (PDL) tissue plays an important role in tooth preservation by structurally maintaining the connection between the tooth root and the bone. The mechanisms involved in the healing and regeneration of damaged PDL tissue, caused by bacterial infection, caries and trauma, have been explored. Accumulating evidence suggests that Activin A, a member of the transforming growth factor-ß (TGF-ß) superfamily and a dimer of inhibinßa, contributes to tissue healing through cell proliferation, migration, and differentiation of various target cells. In bone, Activin A has been shown to exert an inhibitory effect on osteoblast maturation and mineralization. However, there have been no reports examining the expression and function of Activin A in human PDL cells (HPDLCs). Thus, we aimed to investigate the biological effects of Activin A on HPDLCs. Activin A was observed to be localized in HPDLCs and rat PDL tissue. When PDL tissue was surgically damaged, Activin A and IL-1ß expression increased and the two proteins were shown to be co-localized around the lesion. HPDLCs treated with IL-1ß or TNF-α also up-regulated the expression of the gene encoding inhibinßa. Activin A promoted chemotaxis, migration and proliferation of HPDLCs, and caused an increase in fibroblastic differentiation of these cells while down-regulating their osteoblastic differentiation. These osteoblastic inhibitory effects of Activin A, however, were only noted during the early phase of HPDLC osteoblastic differentiation, with later exposures having no effect on differentiation. Collectively, our results suggest that Activin A could be used as a therapeutic agent for healing and regenerating PDL tissue in response to disease, trauma or surgical reconstruction.

Expression and effects of epidermal growth factor on human periodontal ligament cells.

Repair of damaged periodontal ligament (PDL) tissue is an essential challenge in tooth preservation. Various researchers have attempted to develop efficient therapies for healing and regenerating PDL tissue based on tissue engineering methods focused on targeting signaling molecules in PDL stem cells and other mesenchymal stem cells. In this context, we investigated the expression of epidermal growth factor (EGF) in normal and surgically wounded PDL tissues and its effect on chemotaxis and expression of osteoinductive and angiogenic factors in human PDL cells (HPDLCs). EGF as well as EGF receptor (EGFR) expression was observed in HPDLCs and entire PDL tissue. In a PDL tissue-injured model of rat, EGF and IL-1ß were found to be upregulated in a perilesional pattern. Interleukin-1ß induced EGF expression in HPDLCs but not EGFR. It also increased transforming growth factor-α (TGF-α) and heparin-binding EGF-like growth factor (HB-EGF) expression. Transwell assays demonstrated the chemotactic activity of EGF on HPDLCs. In addition, EGF treatment significantly induced secretion of bone morphogenetic protein 2 and vascular endothelial growth factor, and gene expression of interleukin-8 (IL-8), and early growth response-1 and -2 (EGR-1/2). Human umbilical vein endothelial cells developed well-formed tube networks when cultured with the supernatant of EGF-treated HPDLCs. These results indicated that EGF upregulated under inflammatory conditions plays roles in the repair of wounded PDL tissue, suggesting its function as a prospective agent to allow the healing and regeneration of this tissue.

The roles of calcium-sensing receptor and calcium channel in osteogenic differentiation of undifferentiated periodontal ligament cells.

Elevated extracellular calcium has been shown to promote the differentiation of osteoblasts. However, the way that calcium affects the osteogenic differentiation of human periodontal ligament stem/progenitor cells (PDLSCs) remains unclear. Our aim has been to investigate the proliferation and osteogenic differentiation of a calcium-exposed human PDLSC line (cell line 1-17) that we have recently established and to elucidate the roles of the calcium-sensing receptor (CaSR) and L-type voltage-dependent calcium channel (L-VDCC) in this process. Proliferation activity was investigated by WST-1 assay, and gene and protein expression was examined by quantitative reverse transcriptase plus the polymerase chain reaction and immunostaining, respectively. Calcification assay was performed by von Kossa and Alizarin red staining. Treatment with 5 mM CaCl2 significantly induced proliferation, bone-related gene expression, and calcification in cell line 1-17. During culture with 5 mM CaCl2, this cell line up-regulated the gene expression of CaSR, which was reduced after 7 days. Simultaneous treatment with NPS2143, a CaSR inhibitor, and calcium significantly further increased bone-related gene expression and calcification as compared with CaCl2 exposure alone. The L-VDCC inhibitor, nifedipine, significantly suppressed osteogenic differentiation of cell line 1-17 treated with 5 mM CaCl2 and promoted the expression of CaSR, as compared with calcium treatment alone. Thus, elevated extracellular calcium promotes the proliferation and osteogenic differentiation of a PDLSC line. Antagonizing CaSR further enhances the effect of calcium on osteogenic differentiation, with CaSR expression being regulated by L-VDCC under extracellular calcium. Extracellular calcium might therefore modulate the osteogenic differentiation of PDLSCs through reciprocal adjustments of CaSR and L-VDCC.

Regeneration of the periodontium for preservation of the damaged tooth.

The population of the world grows every year, and life expectancy tends to increase. Thus, long-term preservation of teeth in aged individuals is an urgent issue. The main causes of tooth loss are well known to be periodontitis, caries, fractures, and orthodontic conditions. Although implant placement is a widely accepted treatment for tooth loss, most patients desire to preserve their own teeth. Many clinicians and researchers are therefore challenged to treat and preserve teeth that are irreversibly affected by deep caries, periodontitis, fractures, and trauma. Tissue engineering techniques are beneficial in addressing this issue; stem cells, signal molecules, and scaffolds are the main elements of such techniques. In this review, we describe these three elements with respect to their validation for regeneration of the periodontium and focus particularly on the potency of diverse scaffolds. In addition, we provide a short overview of the ongoing studies of 4-methacryloxyethyl trimellitate anhydride/methyl methacrylate-tri-n-butyl-borane resin including calcium chloride or hydroxyapatite for periodontium regeneration.

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.

Periodontal tissue engineering: defining the triad.

The idea that somatic stem cells are localized in periodontal ligament (PDL) tissues as PDL stem cells (PDLSCs) responsible for construction and reconstruction of the periodontium has been widely accepted. Many dental scientists have attempted to clarify the identity of these PDLSCs, but the number of PDLSCs localized in PDL tissues is too small to be routinely and conveniently analyzed. Therefore, researchers have been attempting to develop undifferentiated PDL cell lines by transducing them with genes that are suitable for immortalization. The present authors were the first to succeed in establishing two clonal human PDL stem/progenitor cell lines that possessed multipotency derived from PDL tissues and that expressed PDL-related molecules as well as neural crest- and embryonic stem-related markers. The differentiation stages of these cell lines appeared to vary based on their potential to differentiate into other lineage cells, their response to tissue regeneration-related cytokines, and their behavior when transplanted into immunodeficient rats. This review describes the phenotypes of these cell lines compared with reported PDLSCs or other MSCs and discusses contemporary circumstances related to PDL regenerative medicine. Differential analyses between these two clones will reveal the mechanism of differentiation of PDLSCs as well as their phenotypes. The results will also allow for the acquisition of a mass population of PDLSCs or other stem cells directed toward PDL-lineage cells and to develop an unmet treatment needed for construction and reconstruction of PDL tissues based on tissue engineering techniques.

Prospective potency of TGF-ß1 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.

Exposure to transforming growth factor-ß1 after basic fibroblast growth factor promotes the fibroblastic differentiation of human periodontal ligament stem/progenitor cell lines.

Basic fibroblast growth factor (bFGF) is a cytokine that promotes the regeneration of the periodontium, the specialized tissues supporting the teeth. bFGF, does not, however, induce the synthesis of smooth muscle actin alpha 2 (ACTA2), type I collagen (COL1), or COL3, which are principal molecules in periodontal ligament (PDL) tissue, a component of the periodontium. We have suggested the feasibility of using transforming growth factor-ß1 (TGFß1) to induce fibroblastic differentiation of PDL stem/progenitor cells (PDLSCs). Here, we investigated the effect of the subsequent application of TGFß1 after bFGF (bFGF/TGFß1) on the differentiation of PDLSCs into fibroblastic cells. We first confirmed the expression of bFGF and TGFß1 in rat PDL tissue and primary human PDL cells. Receptors for both bFGF and TGFß1 were expressed in the human PDLSC lines 1-11 and 1-17. Exposure to bFGF for 2 days promoted vascular endothelial growth factor gene and protein expression in both cell lines and down-regulated the expression of ACTA2, COL1, and COL3 mRNA in both cell lines and the gene fibrillin 1 (FBN1) in cell line 1-11 alone. Furthermore, bFGF stimulated cell proliferation of these cell lines and significantly increased the number of cells in phase G2/M in the cell lines. Exposure to TGFß1 for 2 days induced gene expression of ACTA2 and COL1 in both cell lines and FBN1 in cell line 1-11 alone. BFGF/TGFß1 treatment significantly up-regulated ACTA2, COL1, and FBN1 expression as compared with the group treated with bFGF alone or the untreated control. This method might thus be useful for accelerating the generation and regeneration of functional periodontium.

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.

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-1ß), 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-1ß 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 αVß3 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 αVß3. These findings suggest that GDNF may contribute to wound healing in PDL tissue.

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.

Promise of periodontal ligament stem cells in regeneration of periodontium.

A great number of patients around the world experience tooth loss that is attributed to irretrievable damage of the periodontium caused by deep caries, severe periodontal diseases or irreversible trauma. The periodontium is a complex tissue composed mainly of two soft tissues and two hard tissues; the former includes the periodontal ligament (PDL) tissue and gingival tissue, and the latter includes alveolar bone and cementum covering the tooth root. Tissue engineering techniques are therefore required for regeneration of these tissues. In particular, PDL is a dynamic connective tissue that is subjected to continual adaptation to maintain tissue size and width, as well as structural integrity, including ligament fibers and bone modeling. PDL tissue is central in the periodontium to retain the tooth in the bone socket, and is currently recognized to include somatic mesenchymal stem cells that could reconstruct the periodontium. However, successful treatment using these stem cells to regenerate the periodontium efficiently has not yet been developed. In the present article, we discuss the contemporary standpoints and approaches for these stem cells in the field of regenerative medicine in dentistry.

Visualization of irrigant flow and cavitation induced by Er:YAG laser within a root canal model.

INTRODUCTION: Laser-activated irrigation (LAI) has recently been introduced as an innovative method for root canal irrigation. However, there is limited information about the cleaning mechanism of an Er:YAG laser. In this study, we visualized the action of laser-induced bubbles and fluid flow in vitro to better understand the physical mechanisms underlying LAI. METHODS: An Er:YAG laser was equipped with a novel cone-shaped tip with a lateral emission rate of approximately 80%. Laser light was emitted at a pulse energy of 30, 50, or 70 mJ (output energy: 11, 18, or 26 mJ) and a repetition rate of 1 or 20 pulses per second, without air or water spray. Fluid flow dynamics in a root canal model were observed by using glass-bead tracers under a high-speed camera. Moreover, laser-induced bubble patterns were visualized in both free water and the root canal model. RESULTS: Tracers revealed high-speed motion of the fluid. A full cycle of expansion and implosion of vapor and secondary cavitation bubbles were clearly observed. In free water, the vapor bubble expanded for 220 microseconds, and its shape resembled that of an apple. In the root canal model, the vapor bubble expanded in a vertical direction along the canal wall, and bubble expansion continued for ≥700 microseconds. Furthermore, cavitation bubbles were created much more frequently in the canal model than in free water. CONCLUSIONS: These results suggest that the cleaning mechanism of an Er:YAG laser within the root canal might depend on rapid fluid motion caused by expansion and implosion of laser-induced bubbles.

The usefulness of three-dimensional imaging in the diagnosis and treatment of clinically ambiguous gingival swelling.

We evaluated and treated a 54-year-old woman with gingival swelling. Conventional intraoral and panoramic radiography did not provide sufficient information for either determining the cause of gingival swelling or planning treatment of clinical symptoms. The 3D Accuitomo XYZ Slice View Tomograph (3DX) is a compact dental computed tomography device that allowed for accurate identification and optimal treatment of the causes of gingival swelling. At four years after treatment, 3DX radiographs showed no abnormalities in treated teeth or healing of surrounding structures. We conclude that high-resolution 3D images obtained with 3DX promise to be very effective for diagnosing oral diseases and determining effective treatment.

[An epidemiologic examination on the prevalence of the periodontal diseases and oral pigmentation in Yusho patients in 2010].

An epidemiologic examination was carried out to reveal the prevalence of the periodontal diseases and oral pigmentation in patients with Yusho in 2010. The results obtained were as follows. 1) Yusho patients complained of tooth pain and periodontal diseases such as gingival swelling, but not of oral pigmentation. 2) 104 patients out of 117 patients with Yusho, who were measured periodontal pocket depth according to Ramfjord' methods, had at least one tooth with periodontal pocket deeper than 3 mm. Similarly, 314 teeth out of a total 551 examined teeth showed a periodontal pocket with more than 3 mm in depth. However, it was determined that 57 teeth had a periodontal pocket deeper than 4 mm. 3) Oral pigmentation was observed in 63 patients out of 122 patients with Yusho. In this study, gingival pigmentation was most predominant among oral pigmentation. The prevalence of oral pigmentation in male patients seemed to be somewhat higher than that in female patients. In addition, the prevalence of oral pigmentation tended to be higher in patients under seventy years old than patients beyond the age of seventy. These results indicated that PCB-related compounds may be responsible for the higher prevalence of both periodontal diseases and oral pigmentation.

Effects of TGF-ß1 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-ß1 (TGF-ß1) is a multifunctional cytokine, but little is known about the effects of TGF-ß1 on PDL cells. Our aim has been to demonstrate the expression of TGF-ß1 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-ß1 in the entire PDL tissue was confirmed immunohistochemically, and both HPLCs and cell line 1-11 expressed mRNA from the TGF-ß1, TGF-ß type I receptor, and TGF-ß type II receptor genes. Although exogenous TGF-ß1 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-ß1 neutralizing antibody. In contrast, exogenous TGF-ß1 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-ß1 mRNA and protein in HPLCs. Thus, TGF-ß1 seems to play an important role in inducing fibroblastic differentiation of PDL stem/progenitor cells and in maintaining the PDL apparatus under physiological conditions.

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.