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1.
Eur J Prosthodont Restor Dent ; 27(4): 145-153, 2019 Nov 28.
Article in English | MEDLINE | ID: mdl-31609100

ABSTRACT

Titanium has been used for biomedical devices due to its excellent biocompatibility, which is based partly on its 2-8 nm thick titanium oxide layer. However, the relatively poor surface hardness, wear resistance and metal release of these layers may cause some problems in clinical application. In this study, titanium surfaces were modified using a TiO2 sol-gel coating, in order to improve surface properties and osteoblast function. No significant difference in surface roughness was observed between titanium and TiO2 sol-gel discs. The surface of TiO2 sol-gel discs possessed more wettability than titanium discs. The X-ray diffraction results showed amorphous TiO2 phase on titanium discs, whereas TiO2 sol-gel surfaces presented TiO2 rutile and anatase phase. After 4 hours, the number of osteoblasts seeded on TiO2 surface was significantly higher than those on titanium discs. The mRNA expression of bone sialoprotein and osteocalcin were also higher on day 5 and 7, respectively. Enzyme-linked immunosorbent assay(ELISA) analysis confirmed the increase of osteocalcin protein synthesis in osteoblasts grown on the TiO2 sol-gel surface. Alizarin red-S staining showed higher amount of calcium deposition from osteoblasts cultured on TiO2 surface than those on titanium discs at day 20. In conclusion, TiO2 sol-gel coated-titanium could enhance osteoblasts differentiation and promote mineralization, indicating its potential in improving osseointegration for clinical application.


Subject(s)
Osteoblasts , Titanium , Coated Materials, Biocompatible , Osseointegration , Surface Properties
2.
Oral Dis ; 24(4): 619-627, 2018 May.
Article in English | MEDLINE | ID: mdl-29117466

ABSTRACT

OBJECTIVE: To investigate physical characteristics and behaviours of dental pulp cells of teeth isolated from a dentinogenesis imperfecta (DGI) patient with a novel dentin sialophosphoprotein (DSPP) mutation. SUBJECTS AND METHODS: Whole exome and Sanger sequencing were employed to identify mutations. Physical characteristics of the teeth were examined. Pulp cells' behaviours including cell proliferation, colony-forming unit, osteogenic differentiation, pluripotent markers, and mesenchymal stem cell markers were investigated. RESULTS: The proband had opalescent brown primary teeth with extensive loss of enamel. Mutation analysis revealed a novel heterozygous 4-bp deletion, c.1915_1918delAAGT (p.K639QfsX674), in exon 5 of the DSPP associated with DGI. Analysis of the extracted primary incisor demonstrated a decrease in brightness but an increase in yellow and red chroma. The dentin showed reduced mineral density. The dentinal tubules were present in the predentin, but progressively collapsed in the dentin. The pulp cells exhibited markedly reduced CD105 expression, decreased cell proliferation, and smaller colony-forming units. CONCLUSIONS: We identified a novel mutation in the DSPP gene which disturbed dentin characteristics and pulp cells' behaviours. Our study expands the mutation spectrum and understanding of pathologic dentin phenotypes related to the frameshift deletion in the dentin phosphoprotein (DPP) region of the DSPP gene.


Subject(s)
Base Sequence , Dental Pulp/pathology , Dental Pulp/physiopathology , Dentinogenesis Imperfecta/genetics , Extracellular Matrix Proteins/genetics , Phosphoproteins/genetics , Sequence Deletion , Sialoglycoproteins/genetics , Cell Differentiation/genetics , Cell Proliferation/genetics , Cells, Cultured , Child, Preschool , Colony-Forming Units Assay , Dental Pulp/ultrastructure , Dentin/physiopathology , Dentin/ultrastructure , Endoglin/metabolism , Humans , Male , Pedigree , Phenotype
3.
J Periodontal Res ; 52(3): 546-555, 2017 Jun.
Article in English | MEDLINE | ID: mdl-27663623

ABSTRACT

BACKGROUND AND OBJECTIVE: The cytokine interleukin 12 (IL-12) has been implicated as a potent stimulator of tissue degradation in the pathogenesis of several inflammatory diseases, including periodontitis. In patients with periodontitis, an increased level of IL-12 is found in serum and gingival crevicular fluid. As inflammatory cytokines have been demonstrated to induce activation of the immunomodulatory properties of mesenchymal stem cells (MSCs), this study aimed to investigate the influence of IL-12 on these properties in human periodontal ligament (hPDL) cells. MATERIAL AND METHODS: Human PDL cells were isolated from periodontal tissue and incubated with 0-10 ng/mL of IL-12 for 24 h. The levels of expression of interferon gamma (IFN-γ), indoleamine 2,3-dioxygenase (IDO) and human leukocyte antigen G (HLA-G), as well as of the stem cell markers, CD73, CD90 and CD105, were assessed by quantitative PCR. The level of IFN-γ protein was measured by ELISA, and IDO activity was measured by activity assay. The participation of IFN-γ in the expression of IDO and HLA-G was analyzed using neutralizing antibody against IFN-γ. RESULTS: IL-12 upregulated the expression of IFN-γ in a dose-dependent manner. Moreover, IL-12 induced the expression of the immunomodulatory proteins IDO and HLA-G via an IFN-γ-dependent pathway, as indicated by experiments using an IFN-γ neutralizing antibody. Addition of exogenous IFN-γ upregulated the expression of HLA-G and IDO. Expression of the stem cell markers CD73, CD90 and CD105, as well as the pluripotent markers Nanog homeobox, octamer-binding transcription factor 4 and SRY-box 2, were also upregulated in IL-12-treated hPDL cells. Finally, IL-12 inhibited osteogenic differentiation of the hPDL cells and preserved the self-clonal expansion property of these cells, as assessed by Alizarin Red S staining and the colony-forming unit assay. CONCLUSION: Expression of IL-12 during periodontitis may play an important role in the control of the inflammatory response via the induction of immunosuppressive molecules by hPDL cells. We hypothesize that this immunomodulatory property of IL-12 will serve as a protective mechanism to preserve a population of stem cells under inflammatory conditions.


Subject(s)
Interleukin-12/pharmacology , Periodontal Ligament/drug effects , 5'-Nucleotidase/metabolism , Cells, Cultured , Endoglin/metabolism , Enzyme-Linked Immunosorbent Assay , HLA-G Antigens/metabolism , Humans , Indoleamine-Pyrrole 2,3,-Dioxygenase/metabolism , Interferon-gamma/metabolism , Periodontal Ligament/cytology , Periodontal Ligament/immunology , Real-Time Polymerase Chain Reaction , Thy-1 Antigens/metabolism
4.
J Periodontal Res ; 51(6): 789-799, 2016 Dec.
Article in English | MEDLINE | ID: mdl-26751719

ABSTRACT

BACKGROUND AND OBJECTIVE: Notch signaling regulates bone homeostasis. The present study investigated the effect of Jagged1 on osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) expression in human periodontal ligament stromal (hPDL) cells. MATERIAL AND METHODS: hPDL cells were seeded on to indirect immobilized Jagged1 surfaces. OPG expression was determined using real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Lentiviral small hairpin RNA particles against NOTCH2 were employed to inhibit NOTCH2 expression. Osteoclast formation was evaluated using RAW264.7 cells. An influence of exogenous OPG on osteogenic differentiation was determined by real-time polymerase chain reaction and Alizarin Red S staining. RESULTS: Jagged1 significantly enhanced HES1 and HEY1mRNA expression in a dose-dependent manner. Furthermore, OPG mRNA and protein levels dramatically decreased upon exposing hPDL cells to Jagged1. However, RANKL mRNA levels were not significantly different. There was also no difference in M-CSF and MCP-1mRNA expression. A γ-secretase inhibitor and cycloheximide treatment rescued Jagged1-attenuated OPG expression. Furthermore, shNOTCH2 overexpressing hPDL cells did not exhibit a decrease in OPG expression upon exposure to Jagged1, implying the involvement of NOTCH2 in the regulatory mechanism. Culturing RAW264.7 cells with conditioned medium from Jagged1-treated hPDL cells enhanced osteoclast formation compared with those cultured with conditioned medium of the control group. Lastly, OPG treatment did not influence osteogenic differentiation by hPDL cells. CONCLUSION: These results suggest that Jagged1 activates Notch signaling in hPDL cells, leading to decreased OPG expression. This may imply an indirect role of Jagged1 on the regulation of osteoclast differentiation via hPDL cells.


Subject(s)
Jagged-1 Protein/physiology , Osteoprotegerin/metabolism , Periodontal Ligament/metabolism , Blotting, Western , Cell Differentiation/physiology , Enzyme-Linked Immunosorbent Assay , Humans , Jagged-1 Protein/pharmacology , Osteoprotegerin/antagonists & inhibitors , Periodontal Ligament/cytology , RANK Ligand/metabolism , Real-Time Polymerase Chain Reaction , Signal Transduction/physiology
5.
J Dent Res ; 94(7): 983-9, 2015 Jul.
Article in English | MEDLINE | ID: mdl-25870205

ABSTRACT

The aim of this study was to investigate the response of human periodontal ligament (hPDL) fibroblasts to an intermittent compressive force and its effect on the expression of SOST, POSTN, and TGFB1. A computerized cell compressive force loading apparatus was introduced, and hPDL cells were subjected to intermittent compressive force. The changes in messenger RNA (mRNA) and protein expression were monitored by real-time polymerase chain reaction and Western blot analysis, respectively. An increased expression of SOST, POSTN, and TGFB1 was observed in a time-dependent fashion. Addition of cycloheximide, a transforming growth factor (TGF)-ß inhibitor (SB431542), or a neutralizing antibody against TGF-ß1 attenuated the force-induced expression of SOST and POSTN as well as sclerostin and periostin, indicating a role of TGF-ß1 in the pressure-induced expression of these proteins. Enzyme-linked immunosorbent assay analysis revealed an increased level of TGF-ß1 in the cell extracts but not in the medium, suggesting that intermittent compressive force promoted the accumulation of TGF-ß1 in the cells or their surrounding matrix. In conclusion, an intermittent compressive force regulates SOST/POSTN expression by hPDL cells via the TGF-ß1 signaling pathway. Since these proteins play important roles in the homeostasis of the periodontal tissue, our results indicate the importance of masticatory forces in this process.


Subject(s)
Bone Morphogenetic Proteins/analysis , Cell Adhesion Molecules/analysis , Fibroblasts/metabolism , Periodontal Ligament/metabolism , Transforming Growth Factor beta1/physiology , Adaptor Proteins, Signal Transducing , Adolescent , Adult , Benzamides/pharmacology , Biomechanical Phenomena , Blotting, Western , Cell Culture Techniques , Cells, Cultured , Coloring Agents , Dioxoles/pharmacology , Extracellular Matrix/chemistry , Fibroblasts/drug effects , Gene Expression Regulation/genetics , Genetic Markers , Humans , Mechanotransduction, Cellular/physiology , Periodontal Ligament/cytology , Periodontal Ligament/drug effects , RNA, Messenger/analysis , Real-Time Polymerase Chain Reaction , Stress, Mechanical , Tetrazolium Salts , Thiazoles , Time Factors , Transforming Growth Factor beta1/analysis , Transforming Growth Factor beta1/antagonists & inhibitors , Young Adult
6.
J Periodontal Res ; 49(6): 777-84, 2014 Dec.
Article in English | MEDLINE | ID: mdl-24410709

ABSTRACT

BACKGROUND AND OBJECTIVE: Prostaglandin E2 (PGE2) has been shown to be able to influence both bone formation and resorption. The purpose of this study was to investigate the effect of PGE2 on the osteogenic differentiation of human periodontal ligament (HPDL) cells. MATERIAL AND METHODS: HPDL cells were cultured with 0.001-1 µm PGE2 in osteogenic medium. In-vitro mineral deposition was determined by Alizarin Red S staining, and gene expression was determined by real-time PCR. RESULTS: PGE2 inhibited in-vitro mineral deposition by HPDL cells in a dose-dependent manner. PCR analyses showed that PGE2 upregulated the expression of Runt-related transcription factor 2 (RUNX2), but had no effect on osteocalcin expression. Upregulation of TWIST-related protein1 (TWIST1), a functional antagonist of RUNX2, was also observed. In addition, increased levels of RUNX2 and TWIST1 proteins, induced by PGE2, were detected by western blot analysis. Using a chemical activator of E prostanoid (EP) receptors as well as small interfering RNA against an EP receptor, it was shown that PGE2 regulated RUNX2 and TWIST1 via the EP2 receptor. The role of protein kinase A in the inductive effect of PGE2 was also demonstrated. CONCLUSION: The results of this study revealed that PGE2 modulates the osteogenic differentiation of HPDL cells via regulating the expression of RUNX2 and TWIST1. The results suggest a possible role for PGE2 in regulating the homeostasis of periodontal ligament tissue.


Subject(s)
Core Binding Factor Alpha 1 Subunit/drug effects , Dinoprostone/pharmacology , Nuclear Proteins/drug effects , Osteogenesis/drug effects , Periodontal Ligament/drug effects , Twist-Related Protein 1/drug effects , Anthraquinones , Cell Culture Techniques , Cell Differentiation/drug effects , Cells, Cultured , Coloring Agents , Cyclic AMP-Dependent Protein Kinases/drug effects , Dinoprostone/administration & dosage , Dose-Response Relationship, Drug , Gene Expression Regulation/drug effects , Homeostasis/drug effects , Humans , Osteocalcin/drug effects , Periodontal Ligament/cytology , Polymerase Chain Reaction/methods , RNA, Small Interfering/genetics , Receptors, Prostaglandin E, EP2 Subtype/agonists , Receptors, Prostaglandin E, EP2 Subtype/drug effects , Receptors, Prostaglandin E, EP2 Subtype/genetics , Up-Regulation
7.
J Periodontal Res ; 49(5): 595-602, 2014 Oct.
Article in English | MEDLINE | ID: mdl-24219423

ABSTRACT

BACKGROUND AND OBJECTIVE: Pannexin 1 (Panx1) has been found to form nonjunctional hemichannels. It is also proposed to combine with the P2X7 receptor, forming a complex involved in adenosine triphosphate (ATP)-induced interleukin-1beta (IL-1ß) release in macrophages. Previously, we reported that mechanical stress induced IL-1ß expression via the ATP/P2X7 receptor-dependent pathway in human periodontal ligament (HPDL) cells and that ATP was released through the connexin 43 (Cx43) hemichannel. In the present work, we examined the role of Panx1 in stress-induced IL-1ß induction in HPDL cells. MATERIAL AND METHODS: Cultured HPDL cells were treated with compressive loading or ATP to stimulate IL-1ß expression. Inhibitors, antagonists and the small interfering RNA technique were used to investigate the involvement of Panx1 in IL-1ß induction. Co-immunoprecipitation (Co-IP) and immunostaining were used to determine the association of Panx1 with the P2X7 receptor. The IL-1ß release mechanism was analyzed using inhibitors. RESULTS: Blocking Panx1 significantly decreased ATP release, as well as IL-1ß up-regulation, upon stimulation with stress or ATP. Co-IP revealed the association of Panx1 and the P2X7 receptor in HPDL cells, which was increased in response to mechanical loading. Pretreatment with vesicular trafficking inhibitors significantly reduced the amount of IL-1ß released from stimulated cells, suggesting that IL-1ß might be released through vesicles. CONCLUSION: We clearly illustrated the contribution of Panx1 in ATP release, as well as in IL-1ß induction in HPDL cells. The association of Panx1 and the P2X7 receptor might be required for IL-1ß induction, and their possible novel role in IL-1ß vesicular release was indicated.


Subject(s)
Connexins/immunology , Interleukin-1beta/immunology , Nerve Tissue Proteins/immunology , Periodontal Ligament/cytology , Receptors, Purinergic P2X7/immunology , Adenosine Triphosphate/analysis , Adenosine Triphosphate/pharmacology , Biomechanical Phenomena , Carbenoxolone/pharmacology , Cell Culture Techniques , Cells, Cultured , Connexin 43/antagonists & inhibitors , Connexin 43/immunology , Connexins/antagonists & inhibitors , Connexins/genetics , Humans , Interleukin-1beta/antagonists & inhibitors , Meclofenamic Acid/pharmacology , Nerve Tissue Proteins/antagonists & inhibitors , Nerve Tissue Proteins/genetics , Periodontal Ligament/immunology , Probenecid/pharmacology , Quinine/pharmacology , RNA, Small Interfering/genetics , Spermine/pharmacology , Stress, Mechanical
8.
Oral Dis ; 20(4): 352-8, 2014 May.
Article in English | MEDLINE | ID: mdl-23651465

ABSTRACT

OBJECTIVE: An investigation on neuronal differentiation capacity of human dental pulp stem cells (DPSCs) was still lacking. In this study, two different neuronal induction protocols were investigated and compared. METHODS: The neuronal differentiation was induced using chemical or growth factor induction protocol. The differentiation was confirmed by the neurogenic mRNA and protein expression using polymerase chain reaction and immunocytochemistry, respectively. RESULTS: Chemical-induced neuronal differentiation protocol promoted morphological change and ß3-TUBULIN protein expression. Though, SOX2, SOX9, and ß3-TUBULIN mRNA levels were not different compared with the control, indicating a defective differentiation. For growth factor induction protocol, the cells were exhibited neurite-like cellular process and positively stained with ß3-TUBULIN. In addition, the increase in intracellular calcium was noted upon NMDA stimulation, implying the neuronal function. A dramatic increased mRNA expression of neurogenic markers [SOX2, SOX9, ß3-TUBULIN, and gamma-aminobutyric acid (GABA receptors)] was noted as compared to the control. In addition, a remarkable increased expression of Notch signaling target gene, HEY1, was observed in growth factor-induced DPSCs derived neuronal-like cells compared with the control. CONCLUSION: These data indicate that growth factor induction method is a preferable protocol for neuronal differentiation by DPSCs.


Subject(s)
Cell Differentiation , Dental Pulp/cytology , Neurons/cytology , Stem Cells/cytology , Cell Differentiation/drug effects , Cells, Cultured , Cytological Techniques/methods , Humans
9.
Oral Dis ; 19(7): 673-82, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23279369

ABSTRACT

OBJECTIVE: To investigate the effect of mechanical stress on stem cells from human exfoliated deciduous teeth (SHED). METHODS: Cells were stimulated with mechanical stress ranging from 0 to 2.5 g cm⁻² for 2 h in serum-free condition. The expression of stem cell markers was examined by polymerase chain reaction and immunocytochemistry. The molecular mechanisms of these effects were investigated by means of inhibitors and siRNA. RESULTS: Mechanical stress induced Rex-1 expression in a force-dependent manner. The upregulation of interleukin-6 (IL-6) was also observed. Application of neutralizing antibody against IL-6 and IL-6 siRNA could attenuate the mechanical stress-induced Rex-1 expression. In addition, the increase in mRNA levels of Rex-1 was observed after treating SHED with an exogenous IL-6. This inductive effect was attenuated by JAK inhibitor, suggesting the involvement of JAK signaling pathway. CONCLUSION: These results suggest the role of mechanical stress in the regulation of stemness via IL-6-Rex-1 interaction.


Subject(s)
Interleukin-6/physiology , Kruppel-Like Transcription Factors/physiology , Mechanotransduction, Cellular/physiology , Mesenchymal Stem Cells/physiology , Tooth, Deciduous/cytology , Zinc Fingers/physiology , Adipogenesis/physiology , Biomechanical Phenomena , Calcification, Physiologic/physiology , Cell Culture Techniques , Cell Differentiation/physiology , Gene Silencing , Humans , Interleukin-6/genetics , Janus Kinases/physiology , Neurogenesis/physiology , Osteogenesis/physiology , RNA, Small Interfering/genetics , Stress, Mechanical , Up-Regulation
10.
J Periodontal Res ; 48(2): 169-76, 2013 Apr.
Article in English | MEDLINE | ID: mdl-22881405

ABSTRACT

BACKGROUND AND OBJECTIVE: Mechanical stress is an important factor in maintaining homeostasis of the periodontium. Interleukin-1beta (IL-1ß) and adenosine triphosphate (ATP) are considered potent inflammatory mediators. In macrophages, ATP-activated P2X7 receptor is involved in IL-1ß processing and release. Our previous works demonstrated mechanical stress-induced expression of osteopontin and RANKL through the ATP/P2Y1 receptor in human periodontal ligament (HPDL) cells. This study was designed to examine the effect of mechanical stress on IL-1ß expression in HPDL cells, as well as the mechanism and involvement of ATP and the P2 purinergic receptor. MATERIAL AND METHODS: Cultured HPDL cells were treated with continuous compressive loading. IL-1ß expression was analyzed at both mRNA and protein levels, using RT-PCR and ELISA, respectively. Cell viability was examined using the MTT assay. ATP was also used to stimulate HPDL cells. Inhibitors, antagonists and the small interfering RNA (siRNA) technique were used to investigate the role of ATP and the specific P2 subtypes responsible for IL-1ß induction along with the intracellular mechanism. RESULTS: Mechanical stress could up-regulate IL-1ß expression through the release of ATP in HPDL cells. ATP alone was also capable of increasing IL-1ß expression. The induction of IL-1ß was markedly inhibited by inhibitors and by siRNA targeting the P2X7 receptor. ATP-stimulated IL-1ß expression was also diminished by intracellular calcium inhibitors. CONCLUSION: Our work clearly indicates the capability of HPDL cells to respond directly to mechanical stimulation. The results signified the important roles of ATP/P2 purinergic receptors, as well as intracellular calcium signaling, in mechanical stress-induced inflammation via up-regulation of the proinflammatory cytokine, IL-1ß, in HPDL cells.


Subject(s)
Adenosine Triphosphate/physiology , Interleukin-1beta/physiology , Periodontal Ligament/cytology , Receptors, Purinergic P2X7/physiology , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/analogs & derivatives , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/pharmacology , Adenosine Diphosphate/analogs & derivatives , Adenosine Diphosphate/pharmacology , Adenosine Triphosphate/antagonists & inhibitors , Adenosine Triphosphate/pharmacology , Apyrase/pharmacology , Benzenesulfonates/pharmacology , Calcium Channel Blockers/pharmacology , Calcium Signaling/physiology , Cell Culture Techniques , Cell Survival/physiology , Cells, Cultured , Coloring Agents , Dactinomycin/pharmacology , Homeostasis/physiology , Humans , Interleukin-1beta/analysis , Interleukin-1beta/drug effects , Luminescent Measurements , Purinergic P2X Receptor Antagonists/pharmacology , RNA, Small Interfering/genetics , Receptors, Purinergic P2X7/genetics , Signal Transduction/physiology , Stress, Mechanical , Suramin/analogs & derivatives , Suramin/pharmacology , Tetrazolium Salts , Thiazoles , Time Factors , Up-Regulation
11.
J Periodontal Res ; 48(1): 22-9, 2013 Feb.
Article in English | MEDLINE | ID: mdl-22587561

ABSTRACT

BACKGROUND AND OBJECTIVE: Increasing evidence has shown the presence of transient receptor potential vanilloid-1 (TRPV1) in a variety of nonneuronal tissues; however, the function of TRPV1 in these cells is not well understood. In this study, we aimed to investigate the expression and function of TRPV1 in human periodontal ligament (HPDL) cells. As HPDL cells are known to play an important role in the bone-remodeling process, we hypothesized that TRPV1 might be implicated in the regulation of osteoprotegerin (OPG) and RANKL expression. MATERIAL AND METHODS: TRPV1 expression was examined by western blot analysis. The function of TRPV1 was studied using capsaicin, a well-known TRPV1 agonist. RT-PCR was performed to study the expression of OPG and RANKL mRNAs. The expression of OPG and RANKL proteins was analyzed by ELISA and western blotting, respectively. The mechanisms of capsaicin-induced OPG expression in HPDL cells were studied using inhibitors. RESULTS: In this study we found that TRPV1 was present in HPDL cells. Treatment with capsaicin induced OPG expression in a dose-dependent manner but did not affect the expression of RANKL. The increase of the OPG/RANKL ratio was also found in human osteoblasts, but not in MC3T3-E1 cells, a mouse osteoblastic cell line, suggesting species specificity. Capsazepine, the competitive TRPV1 antagonist, significantly abolished the effect of capsaicin on OPG expression in HPDL cells. In addition, studies investigating the effects of a calcium chelator and a phospholipase C inhibitor indicated that calcium ions and phospholipase C were required for the induction. Interestingly, capsaicin was able to increase the OPG/RANKL ratio, even in the presence of prostaglandin E2, a potent inducer of RANKL. CONCLUSION: Our study demonstrates that activation of TRPV1 leads to an increase of the OPG/RANKL ratio in HPDL cells. These findings suggest the novel function of TRPV1 in periodontal tissues, at least, as the regulator of the OPG/RANKL axis.


Subject(s)
Homeostasis/physiology , Osteoprotegerin/physiology , Periodontal Ligament/physiology , RANK Ligand/physiology , TRPV Cation Channels/physiology , 3T3 Cells , Animals , Bone Remodeling/physiology , Calcium/physiology , Capsaicin/analogs & derivatives , Capsaicin/pharmacology , Cell Culture Techniques , Chelating Agents/pharmacology , Dinoprostone/pharmacology , Dose-Response Relationship, Drug , Egtazic Acid/analogs & derivatives , Egtazic Acid/pharmacology , Estrenes/pharmacology , Humans , Mice , Osteoblasts/drug effects , Osteoblasts/physiology , Osteoprotegerin/analysis , Osteoprotegerin/drug effects , Periodontal Ligament/cytology , Phosphodiesterase Inhibitors/pharmacology , Pyrrolidinones/pharmacology , RANK Ligand/analysis , RANK Ligand/drug effects , TRPV Cation Channels/agonists , TRPV Cation Channels/analysis , TRPV Cation Channels/antagonists & inhibitors , Type C Phospholipases/antagonists & inhibitors , Type C Phospholipases/physiology
12.
J Periodontal Res ; 46(5): 607-15, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21615411

ABSTRACT

BACKGROUND AND OBJECTIVE: Our previous studies showed that mechanical stress could induce ATP release in human periodontal ligament (HPDL) cells. By signaling through P2 purinergic receptors, ATP increased the expression and the synthesis of osteopontin and RANKL. In this study, the mechanism of stress-induced ATP release was investigated. MATERIAL AND METHODS: Continuous compressive forces were applied on cultured HPDL cells. The ATP released was measured using luciferin-luciferase bioluminescence. The expression of gap-junction proteins was examined using RT-PCR and western blot analysis. The opening of hemichannels was demonstrated by cellular uptake of a fluorescent dye, 5(6)-carboxyfluorescein, which is known to penetrate hemichannels. Intracellular signal transduction was investigated using inhibitors and antagonists. RESULTS: Mechanical stress induced the release of ATP into the culture medium, which was attenuated by carbenoxolone, a nonspecific gap-junction inhibitor. Addition of meclofenamic acid sodium salt, a connexin43 inhibitor, inhibited ATP release by mechanical stress. Knockdown of connexin43 expression by small interfering RNA reduced the amount of ATP released by mechanical stress, suggesting the role of connexin43 hemichannels. In addition, intracellular Ca(2+) blockers could also inhibit mechanical stress-induced ATP release and the opening of the gap junction. CONCLUSION: Our study demonstrated the involvement of gap-junction hemichannels, especially connexin43, in the stress-induced ATP-release mechanism. Furthermore, this mechanism may be regulated by the intracellular Ca(2+) signaling pathway. These results suggest an important role of gap-junction hemichannels in the function and behavior of HPDL cells.


Subject(s)
Adenosine Triphosphate/metabolism , Calcium Signaling , Connexin 43/physiology , Dental Stress Analysis , Periodontal Ligament/metabolism , Analysis of Variance , Calcium Channel Blockers/pharmacology , Calcium Signaling/physiology , Cells, Cultured , Compressive Strength , Connexins/biosynthesis , Connexins/genetics , Female , Gene Knockdown Techniques , Humans , Luminescent Measurements/methods , Male , Periodontal Ligament/cytology , Purinergic P2 Receptor Antagonists/pharmacology , Receptors, Purinergic P2/physiology , Stress, Mechanical , Young Adult
13.
J Cell Physiol ; 224(3): 743-7, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20578243

ABSTRACT

Cbl-b is a member of Cbl family of E3 ubiquitin (Ub) ligase. Besides the important role in ubiquitination process, other members of Cbl family have been suggested to show non-ubiquitination-related function in regulation of osteoblastic differentiation. However, the role of Cbl-b in regulation of osteoblastic function has not been known yet. To elucidate the role of Cbl-b in regulation of osteoblastic function, we examined its effects on Runx2, a master gene of osteoblastic differentiation. We co-expressed Cbl-b and Runx2 in osteoblastic cell lines and tested their effects on osteocalcin promoter activity together with the expression of Runx2 and its downstream genes. Luciferase assay demonstrated that Cbl-b synergistically enhances osteocalcin promoter activity in conjunction with the effect on Runx2. Co-transfection of Cbl-b and Runx2 further upregulated Runx2 protein levels without any alteration in Runx2 mRNA expression. The upregulation of Runx2 protein by Cbl-b was inhibited by the treatment with lactacystin, a specific inhibitor of the 26S proteasome. These results indicated that Cbl-b would control Runx2 protein levels at the post-translational event. Moreover, the upregulation of downstream genes of Runx2 such as osteocalcin and alkaline phosphatase mRNA was also observed. These data propose the involvement of Cbl-b in the regulation of osteoblast-related genes expression.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Core Binding Factor Alpha 1 Subunit/metabolism , Osteoblasts/physiology , Osteocalcin/genetics , Promoter Regions, Genetic , Proto-Oncogene Proteins c-cbl/metabolism , Adaptor Proteins, Signal Transducing/genetics , Animals , Cell Line , Core Binding Factor Alpha 1 Subunit/genetics , Gene Expression Regulation , Genes, Reporter , Mice , Osteoblasts/cytology , Osteocalcin/metabolism , Protein Stability , Proto-Oncogene Proteins c-cbl/genetics
14.
J Periodontal Res ; 45(3): 404-11, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20337886

ABSTRACT

BACKGROUND AND OBJECTIVE: Our previous study showed that human periodontal ligament cells responded to mechanical stress by increasing adenosine triphosphate (ATP) release, accompanied by the increased expression of RANKL and osteopontin. We found that the signaling pathway of mechanical stress-induced osteopontin was mediated through ATP/P2Y(1) receptor and Rho kinase activation but that of mechanical stress-induced RANKL was different. In this study, we further investigated the effect of extracellular ATP on the expression of RANKL and the mechanism involved. MATERIAL AND METHODS: Human periodontal ligament cells were treated with ATP (10-40 microm). The expressions of RANKL and cyclo-oxygenase 2 (COX-2) were examined by RT-PCR and western blot analysis. The level of prostaglandin E(2) was determined using ELISA. Signaling pathways were investigated by using inhibitors and antagonist. RESULTS: Adenosine triphosphate induced the expression of RANKL. Indomethacin, an inhibitor of COX, could abolish the induction of RANKL expression, suggesting a COX-dependent mechanism. A cAMP-dependent protein kinase inhibitor, H89, and a nuclear factor kappaB (NF kappaB) inhibitor, pyrrolidine dithiocarbamate, inhibited RANKL expression, prostaglandin E(2) production and NF kappaB translocation. In addition, a specific P2Y(1) receptor antagonist, MRS2179, and P2Y(1) small interfering RNA diminished the effect of ATP. CONCLUSION: Extracellular ATP stimulates RANKL expression in human periodontal ligament cells through a pathway dependent on the P2Y(1) receptor, cAMP-dependent protein kinase, NF kappaB and COX. Our results suggest that, among the molecules responsible for the effect of mechanical stress, ATP participates in bone resorption or bone homeostasis by mediating its signal through the P2Y(1) receptor and the NF kappaB-COX-RANKL axis in periodontal tissue.


Subject(s)
Adenosine Triphosphate/pharmacology , Cyclooxygenase 2/drug effects , Periodontal Ligament/drug effects , RANK Ligand/drug effects , Receptors, Purinergic P2/drug effects , Adenosine Diphosphate/analogs & derivatives , Adenosine Diphosphate/pharmacology , Biomechanical Phenomena , Bone Remodeling/physiology , Bone Resorption/physiopathology , Cells, Cultured , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclooxygenase 2 Inhibitors/pharmacology , Dinoprostone/antagonists & inhibitors , Dinoprostone/metabolism , Enzyme Inhibitors/pharmacology , Estrenes/pharmacology , Humans , Indomethacin/pharmacology , Isoquinolines/pharmacology , NF-kappa B/antagonists & inhibitors , Periodontal Ligament/cytology , Periodontal Ligament/enzymology , Phosphodiesterase Inhibitors/pharmacology , Purinergic P2 Receptor Antagonists , Pyrrolidines/pharmacology , Pyrrolidinones/pharmacology , RANK Ligand/antagonists & inhibitors , RNA, Small Interfering/pharmacology , Receptors, Purinergic P2/genetics , Receptors, Purinergic P2Y1 , Sulfonamides/pharmacology , Thiocarbamates/pharmacology , Type C Phospholipases/antagonists & inhibitors
15.
J Periodontal Res ; 44(6): 776-83, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19602124

ABSTRACT

BACKGROUND AND OBJECTIVE: Our previous study found that thrombin induced osteoprotegerin synthesis in human periodontal ligament cells. As elevated levels of osteoprotegerin can exert biological effects on various cell types, in the present study we investigated the effect of osteoprotegerin on the expression of osteopontin in human periodontal ligament cells. MATERIAL AND METHODS: Cultured human periodontal ligament cells were treated with recombinant human osteoprotegerin (0-100 ng/mL) for 24-48 h. The expression of osteopontin mRNA and protein was analyzed using reverse transcription-polymerase chain reaction and western blot analyses, respectively. Phosphoinositol 3-kinase inhibitor, Akt inhibitor, heparinase, neutralizing antibody against receptor activator of nuclear factor-kappaB ligand (RANKL) and syndecan-1, and small interfering RNA against syndecan-1, were used to determine the mechanism involved. RESULTS: Osteoprotegerin up-regulated the mRNA and protein expression of osteopontin in human periodontal ligament cells in a dose-dependent manner. Addition of neutralizing antibody against RANKL attenuated the inductive effect of osteoprotegerin on osteopontin expression. In addition, the inductive effect of osteoprotegerin was abolished by phosphoinositol 3-kinase and Akt inhibitors, as well as by syndecan-1 antibody or syndecan-1 small interfering RNA. None of the inhibitors had any effect on the background level of osteopontin expression. CONCLUSION: An increased level of osteoprotegerin can generate signals via a RANKL/syndecan-1/phosphoinositol 3-kinase/Akt pathway. The results also suggest that osteopontin is one of the downstream targets of the pathway mediated by osteoprotegerin in human periodontal ligament cells. Thus, in addition to counteracting RANKL in the RANKL-osteoprotegerin system, osteoprotegerin may play a role in periodontal tissue remodeling through modulation of the extracellular matrix.


Subject(s)
Osteopontin/drug effects , Osteoprotegerin/pharmacology , Periodontal Ligament/drug effects , Phosphatidylinositol 3-Kinases/drug effects , Proto-Oncogene Proteins c-akt/drug effects , Syndecan-1/analysis , Antibodies, Neutralizing/pharmacology , Cells, Cultured , Chromones/pharmacology , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Humans , Immunoglobulin G/pharmacology , Morpholines/pharmacology , Osteoprotegerin/administration & dosage , Periodontal Ligament/cytology , Phosphoinositide-3 Kinase Inhibitors , Polysaccharide-Lyases/pharmacology , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , RANK Ligand/antagonists & inhibitors , RNA, Messenger/drug effects , RNA, Small Interfering/pharmacology , Recombinant Proteins , Syndecan-1/antagonists & inhibitors , Time Factors , Up-Regulation
16.
J Periodontal Res ; 43(5): 537-43, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18565131

ABSTRACT

BACKGROUND AND OBJECTIVE: Thrombin influences the biological behavior of periodontal ligament cells and plays multiple roles in the early stages of bone healing. Osteoprotegerin (OPG) is one of the key molecules that regulate bone homeostasis and prevent osteoclastogenesis. The purpose of this study was to evaluate the biological effects of thrombin on OPG synthesis in human periodontal ligament (HPDL) cells in vitro. MATERIAL AND METHODS: Cells were treated with various concentrations (0.001, 0.01 and 0.1 U/mL) of thrombin. The mRNA expression and protein synthesis of OPG, as well as of receptor activator of nuclear factor kappaB ligand (RANKL), were determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. The influence of thrombin on OPG synthesis and its signaling pathway were investigated using inhibitors. RESULTS: Thrombin profoundly induces protein synthesis of OPG at 0.1 U/mL. The inductive effect was inhibited by cycloheximide, but not by indomethacin. The phosphatidylinositol 3'-kinase (PI3K) inhibitor, LY294002, and the mammalian target of rapamycin (mTOR) inhibitor, rapamycin, exerted an inhibitory effect on the thrombin-induced OPG synthesis. In addition, the effect was inhibited by protease-activated receptor (PAR)-1 antagonist. Activation of phospho-Akt (p-Akt) was observed and the effect was abolished by LY294002. CONCLUSION: Thrombin induces OPG synthesis in HPDL cells post-transcriptionally, possibly through PAR-1. The regulation was through the PI3K/Akt and mTOR pathway. This finding suggests that thrombin may play a significant role in alveolar bone repair and homeostasis of periodontal tissue, partly through the OPG/RANKL system.


Subject(s)
Anti-Bacterial Agents/pharmacology , Osteoprotegerin/biosynthesis , Periodontal Ligament/cytology , Phosphatidylinositol 3-Kinases/metabolism , Sirolimus/pharmacology , Thrombin/physiology , Blotting, Western , Cells, Cultured , Cycloheximide/pharmacology , Gene Expression Regulation , Humans , Osteoprotegerin/antagonists & inhibitors , Periodontal Ligament/metabolism , Phosphoinositide-3 Kinase Inhibitors , Protein Kinases/metabolism , Protein Synthesis Inhibitors/pharmacology , Proto-Oncogene Proteins c-akt/metabolism , RANK Ligand/biosynthesis , Receptor, PAR-1/antagonists & inhibitors , Receptor, PAR-1/metabolism , Reverse Transcriptase Polymerase Chain Reaction , TOR Serine-Threonine Kinases , Thrombin/pharmacology
17.
J Dent Res ; 87(6): 564-8, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18502966

ABSTRACT

Our previous study showed that mechanical stress induced the expression of osteopontin (OPN) in human periodontal ligament (HPDL) cells through the Rho kinase pathway. The increase of OPN expression via Rho kinase has been demonstrated to be triggered by nucleotide. Therefore, we hypothesized that nucleotides, particularly adenosine triphosphate (ATP), participated in the stress-induced OPN expression in HPDL cells. In the present study, the roles of ATP and P2Y1 purinoceptor were examined. Reverse-transcription polymerase chain-reaction and Western blot analysis revealed that the stress-induced ATP exerted its stimulatory effect on OPN expression. The inductive effect was attenuated by apyrase and completely inhibited by the Rho kinase inhibitor, as well as by the P2Y1 antagonist. We here propose that stress induces release of ATP, which in turn mediates Rho kinase activation through the P2Y1 receptor, resulting in the up-regulation of OPN. Stress-induced ATP could play a significant role in alveolar bone resorption.


Subject(s)
Adenosine Triphosphate/metabolism , Osteopontin/biosynthesis , Periodontal Ligament/metabolism , Receptors, Purinergic P2/metabolism , Stress, Mechanical , Alveolar Bone Loss/metabolism , Blotting, Western , Cells, Cultured , Humans , Periodontal Ligament/cytology , Receptors, Purinergic P2Y1 , Reverse Transcriptase Polymerase Chain Reaction , Up-Regulation , rho-Associated Kinases/metabolism
18.
Oral Dis ; 14(4): 335-40, 2008 May.
Article in English | MEDLINE | ID: mdl-18410576

ABSTRACT

AIM: The aim of this study was to investigate the influence of secreted protein acidic, rich in cysteine (SPARC) on the migration of human dental pulp (HDP) cells. METHODS: Secreted protein acidic, rich in cysteine was applied in the lower chamber of the chemotaxis apparatus and migration was determined by counting the cells that migrated through the membrane. To determine the signaling pathway involved, cells were incubated with inhibitors for 30 min prior to the migration assay. RESULTS: The results indicated that SPARC induced HDP cell migration in a dose-dependent manner via extracellular signal-regulated kinase (ERK). The migration could be inhibited both by the anti-alphavbeta3 integrin antibody and by suramin, a non-selective growth factor receptor and G-protein coupled receptor antagonists. The anti-alphavbeta3 integrin antibody could also inhibit ERK activation, suggesting the possible role of alphavbeta3 integrin on the regulation of ERK and cell migration. Interestingly, both suramin and SB225002, another G-protein coupled receptor antagonist, suppressed ERK activation. CONCLUSIONS: Secreted protein acidic, rich in cysteine could act as a chemotactic factor and facilitate migration, possibly through the G-protein coupled receptor, alphavbeta3 integrin and ERK. The data support that SPARC could play a crucial role in dental pulp tissue repair by inducing dental pulp cell migration.


Subject(s)
Chemotaxis/physiology , Dental Pulp/physiology , Extracellular Signal-Regulated MAP Kinases/physiology , Integrin alphaVbeta3/physiology , Osteonectin/physiology , Cells, Cultured , Chemotaxis/drug effects , Dental Pulp/cytology , Enzyme Inhibitors/pharmacology , Extracellular Signal-Regulated MAP Kinases/analysis , Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors , Humans , MAP Kinase Signaling System , Phenylurea Compounds/pharmacology , Receptors, G-Protein-Coupled/antagonists & inhibitors , Receptors, G-Protein-Coupled/physiology , Suramin/pharmacology
19.
Oral Dis ; 10(6): 383-8, 2004 Nov.
Article in English | MEDLINE | ID: mdl-15533216

ABSTRACT

OBJECTIVE: Matrix metalloproteinase 2 (MMP-2) has been implicated to play a role in pathogenesis of periodontal disease. We recently reported that Porphyromonas gingivalis supernatant could activate MMP-2 in human periodontal ligament (HPDL) cells. In this study, activation of MMP-2 by Actinobacillus actinomycetemcomitans supernatant and the mechanism was investigated. METHODS: HPDL cells were treated with either A. actinomycetemcomitans or P. gingivalis supernatant for 48 h. To verify the mechanism, pretreated inhibitors were used. Gelatin zymography, RT-PCR and Western blot analysis were used to detect the activation of MMP-2, expression of MT1-MMP and TIMP-2 mRNA and the proteins, respectively. RESULTS: The supernatant from A. actinomycetemcomitans could activate MMP-2 in HPDL cells similar to that from P. gingivalis but by a different mechanism. Activation by A. actinomycetemcomitans supernatant was correlated with a reduction of TIMP-2 secretion without any alteration of MT1-MMP, while activation by P. gingivalis increased MT1-MMP but no change of TIMP-2 was found. CONCLUSION: The supernatant from A. actinomycetemcomitans and P. gingivalis could induce the activation of MMP-2 possibly through the imbalance of MT1-MMP and TIMP-2 in HPDL cells but by different mechanisms. The imbalance of MT1-MMP and TIMP-2 may be another factor that is involved in the severity of periodontal disease.


Subject(s)
Aggregatibacter actinomycetemcomitans/metabolism , Matrix Metalloproteinase 2/metabolism , Tissue Inhibitor of Metalloproteinase-2/metabolism , Analysis of Variance , Blotting, Western , Cell Line , Enzyme Activation , Humans , Periodontal Ligament/enzymology , Polymerase Chain Reaction
20.
Matrix Biol ; 15(6): 423-32, 1996 Dec.
Article in English | MEDLINE | ID: mdl-9049980

ABSTRACT

In a previous study, we presented evidence that the synthesis of hyaluronan by hypertrophic chondrocytes is one of the principal factors driving the interstitial expansion of the growth plate (Pavasant et al., J. Cell Sci. 109: 327-334, 1996). To test this possibility further, we used two different approaches to examine the effects of hormones on the production of hyaluronan in the growth plate. In the first approach, we examined the growth plate of the lit/lit mouse that lacks growth hormone and found that its hypertrophic lacunae were smaller and contained less hyaluronan than those of wild type mice. Moreover, the ratios of hyaluronan staining density to total area of the lacunae were similar for the lit/lit and the wt/wt mice, indicating that the amount of hyaluronan is directly related to lacuna size. In the second approach, we examined the effects of hormones on segments of the epiphysial growth plate placed in organ culture. Under normal culture conditions, a band of hyaluronan staining progressed across the length of the growth plate, reflecting the maturation of chondrocytes into the hypertrophic stage. When insulin-like growth factor-1, a factor known to promote chondrocyte maturation, was added to the culture medium, the production of hyaluronan and the enlargement of the lacunae were stimulated. In contrast, when either parathyroid hormone or transforming growth factor-beta 1, both of which inhibit chondrocyte differentiation, was added to the medium of cultured segments, new pericellular hyaluronan was not detected and the lacunae did not enlarge. Taken together, these results indicate that factors that either stimulate or inhibit the maturation of epiphysial chondrocytes have a corresponding effect on the production of hyaluronan. This, in turn, further supports the importance of hyaluronan in the process of lacuna enlargement.


Subject(s)
Cartilage, Articular/metabolism , Growth Hormone/pharmacology , Growth Plate/metabolism , Hyaluronic Acid/biosynthesis , Insulin-Like Growth Factor I/pharmacology , Parathyroid Hormone/pharmacology , Transforming Growth Factor beta/pharmacology , Animals , Cartilage, Articular/drug effects , Growth Hormone/deficiency , Growth Plate/cytology , Growth Plate/drug effects , Humans , Hyaluronic Acid/analysis , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Organ Culture Techniques , Recombinant Proteins/pharmacology
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