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1.
Arch Oral Biol ; 117: 104775, 2020 Sep.
Article in English | MEDLINE | ID: mdl-32512258

ABSTRACT

OBJECTIVE: The aim of the study is to investigate the apoptotic mechanism in salivary glands in the rat experimental periodontitis model. DESIGN: A rat periodontitis model was prepared by using a ligature around the second upper molar. In the salivary (parotid and submandibular) glands and blood samples, putative apoptotic factors and pathway molecules were investigated in vivo and in vitro. RESULTS: Four weeks of ligation (chronic periodontitis) demonstrated significant apoptotic atrophy of the salivary gland, but one week of ligation (initial periodontitis) did not. In the blood plasma, tumor necrosis factor-α (TNF-α) was increased in the periodontitis model, but interleukin-1ß and -6 were not. TNF-α receptor type 1, which has an intracellular apoptotic pathway, was expressed in the salivary glands of rats. Western blot analysis of cultured rat primary salivary gland cells demonstrated that TNF-α induced cleavage of poly (ADP-ribose) polymerase (PARP) and caspase-3 in a dose-dependent manner, indicating apoptosis induction. Additionally, we found increment of circulating lymphocytes in the model. Expression of mRNA and immunoreactive cells for the B lymphocyte marker CD19 were increased in the salivary gland in the model. Western blotting showed that coculture with extracted B cells from the periodontitis model increased cleaved PARP in salivary gland cells. CONCLUSIONS: Chronic periodontitis status leads to an increase in circulating TNF-α and B lymphocyte infiltration, resulting in apoptotic atrophy of the salivary gland as a periodontitis-induced systemic response.


Subject(s)
Apoptosis , Chronic Periodontitis/pathology , Salivary Glands/pathology , Animals , B-Lymphocytes/cytology , Rats , Tumor Necrosis Factor-alpha/blood
2.
J Periodontal Res ; 53(5): 870-882, 2018 Oct.
Article in English | MEDLINE | ID: mdl-29900548

ABSTRACT

OBJECTIVE AND BACKGROUND: Human periodontal ligament mesenchymal stem cells (hPDLMSCs) are reported to be responsible for homeostasis and regeneration of periodontal tissue. Although hPDLMSCs are commonly cultured in monolayers, monolayer cultures have been reported as inferior to 3-dimensional cultures such as spheroids, which are spherical clusters of cells formed by self-assembly. The aim of this study was to examine the osteogenic phenotype of spheroids of hPDLMSCs, compared with monolayer cultures of hPDLMSC, in vitro and in vivo. MATERIAL AND METHODS: Spheroids were formed using microwell chips that were tagged with polyethylene glycol. Mesenchymal stem cell (MSC) markers in hPDLMSC spheroids were examined by flow cytometer. Real-time polymerase chain reaction analysis was examined to measure the expressions of stemness markers and osteogenesis-related genes in monolayer and spheroid-cultured hPDLMSCs. Immunofluorescence analysis was performed to confirm protein expressions of stemness markers in PDLMSC spheroids. Nodule formation assay, alkaline phosphatase (ALP) activity assay and transplantation assay in a mouse calvarial defect model were performed to confirm the osteogenic potential of hPDLMSC spheroids. To elucidate the mechanism of spheroid culture enhanced osteogenesis in hPDLMSCs with osteoinductive medium (OIM), a small interfering RNA (siRNA) assay targeted with secreted frizzled-related protein 3 (SFRP3) was examined. The levels of SFRP3 expression in monolayer and spheroid-cultured hPDLMSCs with OIM were measured by real-time polymerase chain reaction and western blotting analysis. ALP gene expression and ALP activity were examined in SFRP3-deficient hPDLMSC spheroids. RESULTS: The hPDLMSC spheroids expressed MSC markers, which were similar to hPDLMSCs grown in monolayer cultures. Intriguingly, the protein and mRNA expressions of transcription factors that regulate "stemness" were significantly increased in hPDLMSC spheroids, compared with hPDLMSCs in monolayer cultures. Nodule formation by hPDLMSCs was significantly increased in spheroid cultures grown with OIM, compared with monolayer-cultured hPDLMSCs. ALP activity and expression of osteogenesis-related genes were also significantly enhanced in hPDLMSC spheroids, compared with monolayer cultures. Treatment with hPDLMSC spheroids significantly enhanced new bone formation in a murine calvarial defect model, compared with hPDLMSCs in monolayer culture. Finally, to elucidate mechanisms by which spheroid culture enhances ALP activation in hPDLMSCs grown with OIM, an siRNA assay was used to manipulate expression of SFRP3, a Wnt signaling antagonist. Knockdown of SFRP3 suppressed ALP gene expression in hPDLMSCs grown in OIM; further, it suppressed ALP activity in spheroid culture. These data suggest that the enhancement of osteogenic potential in hPDLMSC spheroids is regulated through SFRP3-mediated ALP activation. CONCLUSION: Spheroid cultures of hPDLMSCs may be a novel and useful tool in regenerative medicine.


Subject(s)
Cell Culture Techniques/methods , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/physiology , Osteogenesis/physiology , Periodontal Ligament/cytology , Spheroids, Cellular , Alkaline Phosphatase/genetics , Alkaline Phosphatase/metabolism , Animals , Cells, Cultured , Culture Media , Gene Expression , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Mice , Osteogenesis/genetics , Periodontal Ligament/metabolism , Signal Transduction/physiology
3.
Int Endod J ; 49(3): 271-8, 2016 Mar.
Article in English | MEDLINE | ID: mdl-25702817

ABSTRACT

AIM: To investigate the effects of PRP on odontoblastic differentiation using dental pulp progenitor cells derived from the dental papilla of rat incisors. METHODOLOGY: Monolayer cultures of odontoblastic lineage KN-3 cells were incubated with PRP for various time periods. The expression of dentine sialophosphoprotein (DSPP) and dentine matrix protein-1 (DMP-1) was determined using real-time reverse transcription-polymerase chain reaction and Western blot analyses. To further clarify the role of PRP in odontogenesis, KN-3 cells were stimulated with PRP in the presence of ascorbic acid and ß-glycerophosphate. The cells were stained for alkaline phosphatase (ALP), and ALP activity was quantified in cell lysates. The formation of mineralized nodules was assessed by alizarin red staining. Statistical analysis was performed by one-way analysis of variance. RESULTS: PRP increased the mRNA and protein expressions of odontoblastic markers, such as DSPP and DMP-1. Furthermore, PRP stimulated the ALP activity and mineralized nodule formation induced by ascorbic acid and ß-glycerophosphate in a time-dependent manner. CONCLUSIONS: PRP enhances odontoblastic differentiation of KN-3 cells. These results indicate that PRP could be a potential candidate for use in the regeneration of dentine-pulp complex.


Subject(s)
Dental Pulp/cytology , Odontoblasts/drug effects , Platelet-Rich Plasma , Alkaline Phosphatase/metabolism , Animals , Ascorbic Acid/pharmacology , Blotting, Western , Calcium/metabolism , Cell Differentiation , Cells, Cultured , Extracellular Matrix Proteins/metabolism , Glycerophosphates/metabolism , Humans , Phosphoproteins/metabolism , Rats , Real-Time Polymerase Chain Reaction , Sialoglycoproteins/metabolism
4.
Osteoarthritis Cartilage ; 22(1): 111-20, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24185105

ABSTRACT

OBJECTIVE: To determine the effects of high molecular weight hyaluronic acid (HMW-HA) on osteoclast differentiation by monocytes co-cultured with stromal cells. METHODS: Mouse bone marrow stromal cell line ST2 cells were incubated with HMW-HA or 4-methylunbeliferone (4-MU) for various times. In some experiments, cells were pre-treated with the anti-CD44 monoclonal antibody (CD44 mAb) or Rho kinase pathway inhibitors (simvastatin or Y27632), then treated with HMW-HA. The expression of receptor activator of NF-κB ligand (RANKL) was determined using real-time reverse transcription polymerase chain reaction (RT-PCR), western blotting, and immunofluorescence microscopy, while the amount of active RhoA was measured by a pull-down assay. To further clarify the role of HMW-HA in osteoclastogenesis, mouse monocyte RAW 264.7 cells were co-cultured with ST2 cells pre-stimulated with 1,25(OH)2D3. Osteoclast-like cells were detected by staining with tartrate-resistant acid phosphatase (TRAP). RESULTS: HMW-HA decreased RANKL mRNA and protein expressions, whereas inhibition of hyaluronic acid (HA) synthesis by 4-MU enhanced RANKL expression. Blockage of HA-CD44 binding by CD44 mAb suppressed HMW-HA-mediated inhibition of RANKL. Pull-down assay findings also revealed that HMW-HA transiently activated RhoA in ST2 cells and pre-treatment with CD44 mAb inhibited the activation of RhoA protein mediated by HMW-HA. Moreover pre-treatment with Rho kinase pathway inhibitors also blocked the inhibition of RANKL by HMW-HA. Co-culture system results showed that HMW-HA down-regulated differentiation into osteoclast-like cells by RAW 264.7 cells induced by 1,25(OH)2D3-stimulated ST2 cells. CONCLUSIONS: These results indicated that HA-CD44 interactions down-regulate RANKL expression and osteoclastogenesis via activation of the Rho kinase pathway.


Subject(s)
Hyaluronic Acid/pharmacology , Osteoclasts/drug effects , RANK Ligand/antagonists & inhibitors , rho-Associated Kinases/physiology , Animals , Antibodies, Monoclonal/immunology , Cell Differentiation/drug effects , Cell Differentiation/physiology , Cells, Cultured , Coculture Techniques , Dose-Response Relationship, Drug , Down-Regulation/drug effects , Down-Regulation/physiology , Enzyme Activation/drug effects , Enzyme Activation/physiology , Hyaluronan Receptors/immunology , Hyaluronan Receptors/metabolism , Hyaluronic Acid/administration & dosage , Hyaluronic Acid/chemistry , Hyaluronic Acid/metabolism , Mice , Molecular Weight , Monocytes/cytology , Monocytes/drug effects , Osteoclasts/cytology , RANK Ligand/genetics , RANK Ligand/metabolism , RANK Ligand/physiology , RNA, Messenger/genetics , Stromal Cells/cytology , Stromal Cells/drug effects , Stromal Cells/metabolism
5.
J Dent Res ; 92(3): 241-6, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23318766

ABSTRACT

The objective of this study was to examine whether native low-density lipoprotein (LDL) induces foam cell formation by macrophages and to examine the effect of lipopolysaccharide (LPS) on native LDL-induced foam cell formation by macrophages in vitro. RAW 264.7 cells were cultured with LDL or high-density lipoprotein (HDL) in the presence of LPS derived from Aggregatibacter actinomycetemcomitans. Foam cell formation was determined by staining with Oil-red-O to visualize cytoplasmic lipid droplet accumulation. The expression of LDL-receptor and the degree of internalization of FITC-conjugated LDL in RAW 264.7 cells were examined by immunofluorescence microscopy. The images were digitally recorded and analyzed with Image J software. Statistical analysis was performed by JMP software. Foam cell formation was induced by the addition of native LDL in dose- and time-dependent manners, whereas HDL showed no effect. LPS enhanced the foam cell formation induced by native LDL. In addition, LPS stimulated the expression of LDL-receptor protein on RAW 264.7 cells and enhanced the internalization of LDL. The enhancement of foam cell formation induced by LPS and LDL was inhibited by the depolymerizing agent nocodazole and amiloride analog 5-(N-ethyl-N-isoprophyl) amiloride (EIPA). Our findings indicate that LPS plays an important role in foam cell formation by LDL-stimulated macrophages.


Subject(s)
Aggregatibacter actinomycetemcomitans/physiology , Foam Cells/metabolism , Lipoproteins, LDL/pharmacology , Pinocytosis/drug effects , Receptors, LDL/biosynthesis , Tubulin Modulators/pharmacology , Acid Sensing Ion Channel Blockers/pharmacology , Aggregatibacter actinomycetemcomitans/chemistry , Amiloride/analogs & derivatives , Amiloride/pharmacology , Animals , Cell Line , Drug Synergism , Foam Cells/cytology , Foam Cells/drug effects , Lipopolysaccharides/antagonists & inhibitors , Lipopolysaccharides/pharmacology , Lipoproteins, LDL/physiology , Macrophages/cytology , Macrophages/drug effects , Macrophages/physiology , Mice , Microtubules/drug effects , Nocodazole/pharmacology , Periodontitis/microbiology , Sodium-Hydrogen Exchangers/antagonists & inhibitors
6.
Osteoarthritis Cartilage ; 18(3): 447-54, 2010 Mar.
Article in English | MEDLINE | ID: mdl-19874928

ABSTRACT

OBJECTIVE: Small hyaluronan (HA) oligosaccharides displace HA from the cell surface and induce cell signaling events. In articular chondrocytes this cell signaling is mediated by the HA receptor CD44 and includes stimulation of genes involved in matrix degradation such as matrix metalloproteinases (MMPs) as well as matrix repair genes including collagen type II, aggrecan and HA synthase-2 (HAS-2). The objective of this study was to determine whether stimulation of HAS-2 and MMP-3 by HA oligosaccharides is due to the activation of a single, cascading pathway or multiple signaling pathways. METHOD: Bovine articular chondrocytes were pre-treated with a variety of inhibitors of major signaling pathways prior to the addition of HA oligosaccharides. Changes in HA were monitored by real time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of HAS-2 mRNA, HA ELISA and HA accumulation at the cell surface. A 1900 base pair sequence containing the proximal promoter of HAS-2 was inserted into a luciferase reporter construct, transfected into human immortalized chondrocytes and assayed in a similar fashion. RESULTS: While our previous studies demonstrated that HA oligosaccharides stimulate MMP-13 activity via activation of p38 MAP kinase and NF-kappaB, inhibitors of these pathways did not affect the stimulation of HAS-2 mRNA expression. However, inhibiting the phosphatidylinositol-3-kinase pathway blocked HA oligosaccharide-mediated stimulation of HAS-2 yet had no effect on MMP-3. Wortmannin and LY294002 also blocked HA oligosaccharide-induced serine and threonine Akt phosphorylation. Treatment of transfected immortalized chondrocytes with HA oligosaccharides resulted in stimulation of HAS-2 mRNA, activation of Akt and enhanced luciferase activity-activity that was blocked by inhibitors of Akt phosphorylation. CONCLUSIONS: Changes in chondrocyte-matrix interactions by HA oligosaccharides induce altered matrix metabolism by the activation of least two distinct signaling pathways.


Subject(s)
Cartilage, Articular/metabolism , Chondrocytes/drug effects , Hyaluronan Receptors/metabolism , Hyaluronic Acid/metabolism , Matrix Metalloproteinase 13/metabolism , Oligosaccharides/metabolism , Animals , Cattle , Cells, Cultured , Humans , Hyaluronan Receptors/genetics , Hyaluronic Acid/genetics , Male , Matrix Metalloproteinase 13/genetics , Oligosaccharides/genetics , Signal Transduction/drug effects , Signal Transduction/genetics , Time Factors
7.
Osteoarthritis Cartilage ; 15(3): 291-9, 2007 Mar.
Article in English | MEDLINE | ID: mdl-16979913

ABSTRACT

OBJECTIVE: Although biochemical studies have examined the synovial fluid (SF) of patients with temporomandibular joint (TMJ) disorders (TMDs), the details of the molecular mechanism of bone destruction and remodeling remain unknown. In this study, we induced and characterized osteoclast-like cells from the SF of patients with TMD and investigated the participation of these cells in the pathogenesis of TMD. METHODS: We collected SF cells from patients with TMD after a pumping procedure, cultured osteoclast-like cells, and examined their characteristics, including osteoclast markers and bone resorption activities. In addition, we obtained fibroblastic cells from the SF of TMD patients by continuous sub-culturing. Using these fibroblastic cells, we examined fibroblast markers using immunocytochemical staining and analyzed the receptor activator of nuclear-factor-kappaB ligand (RANKL) mRNA levels. Detection of soluble form of RANKL (sRANKL) in the SF was measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Osteoclast-like cells were induced from the SF cells of patients with TMD by adding recombinant human (rh) macrophage colony stimulating factor (M-CSF) and either 1,25-dihydroxy vitamin D3 [1,25(OH)2D3] or prostaglandin E2 (PGE2). These multinucleated giant cells were positive for tartrate-resistant acid phosphatase (TRAP) and had the ability to absorb bone. The fibroblastic cells from the SF of TMD patients were positive for fibroblast markers and RANKL mRNA was up-regulated. Detection of sRANKL in SF of patient group was significantly higher than control group. CONCLUSION: The results suggest that the joint-infiltrating SF cells from TMD patients play important roles in the pathogenesis of these disorders, which is characterized by progressive bone destruction or remodeling.


Subject(s)
Bone Resorption/pathology , Fibroblasts/metabolism , Osteoclasts/metabolism , RANK Ligand/metabolism , Receptor Activator of Nuclear Factor-kappa B/metabolism , Synovial Fluid/cytology , Temporomandibular Joint Disorders/metabolism , Acid Phosphatase/analysis , Adolescent , Adult , Biomarkers/analysis , Calcitriol/pharmacology , Cell Differentiation/drug effects , Cells, Cultured , Colony-Stimulating Factors/pharmacology , Dinoprostone/pharmacology , Female , Giant Cells/enzymology , Giant Cells/metabolism , Humans , Isoenzymes/analysis , Male , Middle Aged , Osteoclasts/enzymology , RNA, Messenger/metabolism , Tartrate-Resistant Acid Phosphatase , Temporomandibular Joint Disorders/pathology
8.
J Periodontal Res ; 41(4): 273-9, 2006 Aug.
Article in English | MEDLINE | ID: mdl-16827720

ABSTRACT

BACKGROUND AND OBJECTIVE: Enamel matrix derivative (EMD) is used clinically to promote periodontal tissue regeneration, and it has been reported that EMD can induce the formation of osteoclasts in mouse marrow cultures. In the present study, we investigated the mechanisms of EMD-induced osteoclast formation using a mouse monocytic cell line, RAW 264.7. MATERIAL AND METHODS: Bioactive fractions were purified from EMD by reverse-phase HPLC using a C18 hydrophobic support, following which RAW 264.7 cells were cultured with EMD or its purified fractions in the presence of receptor activator of nuclear factor-kappaB ligand (RANKL) for 8 d. Following staining with tartrate-resistant acid phosphatase (TRAP), TRAP-positive multinucleated cells were counted. The expression of receptor activator of nuclear factor-kappaB (RANK), as well as phosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinase, in RAW 264.7 cells were detected using immunoblotting. To determine whether EMD has an effect on osteoclast function, differentiated RAW 264.7 cells were cultured on Osteologic Multitest slides with RANKL in the presence of EMD. RESULTS: Purified EMD fractions (fraction numbers 21-25; EMD peak 2) were found to enhance the formation and function of RAW 264.7 cells induced by RANKL. Moreover, EMD peak 2 enhanced the levels of phosphorylation of ERK p38 and RANK in RAW 264.7 cells stimulated with RANKL. CONCLUSION: Our results indicate that EMD induces the formation of osteoclasts through interaction with RANKL, while ERK and p38 MAPK may play a critical role in the enhancement of osteoclast formation in RAW 264.7 cells.


Subject(s)
Bone Regeneration/drug effects , Dental Enamel Proteins/pharmacology , Osteoclasts/drug effects , Animals , Blotting, Western , Carrier Proteins/pharmacology , Cell Differentiation/drug effects , Cell Line , Cell Proliferation/drug effects , Extracellular Signal-Regulated MAP Kinases/metabolism , Membrane Glycoproteins/pharmacology , Mice , Mice, Inbred Strains , Monocytes/drug effects , Monocytes/metabolism , Osteoclasts/cytology , RANK Ligand , Receptor Activator of Nuclear Factor-kappa B , p38 Mitogen-Activated Protein Kinases/metabolism
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