OBJECTIVETo determine the effect of aspirin on cell proliferation, alkaline phosphatase (ALP) activity, cell cycle and apoptosis in murine bone marrow stromal cells, so as to explore an appropriate dose range to improve bone regeneration in periodontal treatment.

METHODSST2 cells were stimulated with aspirin (concentrations of 1, 10, 100 and 1 000 μmol/L) for 1, 2, 3, 5 and 7 d. Cell proliferation was measured by methyl thiazolyl tetrazolium (MTT) assay. After ST2 cells were treated for 1, 3 and 7 d, ALP activity was measured by ALP kit, cell cycle and apoptosis were measured by flow cytometry (FCM) after treated for 48 h.

RESULTSMTT assays showed that various doses of aspirin have different effects on the cell growth. Briefly, lower concentrations (1, 10 μmol/L) of aspirin promoted the cell growth, the A value of 0, 1 and 10 μmol/L aspirin 7-day-treated cells were 0.313±0.012, 0.413±0.010 and 0.387±0.017 respectively (P <0.01 vs control), and so did the ALP level ([4.3±0.9], [6.0±0.3] and [7.7±0.4] μmol·min(-1)·g(-1), P <0.05 vs control), while higher concentrations, especially 1000 μmol/L of aspirin might inhibit the cell growth with time going, A value and ALP level were 0.267±0.016, (4.3±1.3) μmol·min(-1)·g(-1) respectively (P <0.05 vs control). Cell cycle analysis revealed no changes in comparison to control cells after treatment with 1 or 10 μmol/L aspirin, but it was observed that cell mitosis from S phase to G2/M phase proceeded at higher concentrations of 100 μmol/L aspirin, and the cell cycle in phase G0/G1 arrested at 1000 μmol/L. Parallel apoptosis/necrosis studies showed that the percentage of cells in apoptosis decreased dramatically at all doses of aspirin, the apoptosis rates of ST2 cells responded to 0, 1, 10, 100 and 1000 μmol/L aspirin were (11.50±0.90)%, (5.30±0.10)%, (5.50±0.10)%, (4.90±0.90)% and (7.95±0.25)% respectively (P<0.05 vs control).

CONCLUSIONSThis study demonstrated that lower dosage of aspirin can promote ST2 cells growth, osteogenic activity and inhibit its apoptosis. Aspirin maybe used for the bone reconstruction with a proper concentration.

Alkaline Phosphatase ; metabolism ; Animals ; Apoptosis ; drug effects ; Aspirin ; administration & dosage ; pharmacology ; Bone Regeneration ; Cell Cycle ; drug effects ; Cell Division ; Cell Line, Tumor ; Cell Proliferation ; Flow Cytometry ; Formazans ; Mesenchymal Stromal Cells ; cytology ; drug effects ; enzymology ; Mice ; Periodontics ; Tetrazolium Salts ; Time Factors

Bisphosphonate-Associated Osteonecrosis of the Jaw ; complications ; Humans ; Periodontal Abscess ; complications

OBJECTIVETo investigate the expression of chemokine stromal cell-derived factor-1 (SDF-1) receptor CXCR4 in human gingival mesenchymal stem cells (GMSCs) and the migration potential of GMSCs stimulated with SDF-1.

METHODSHuman GMSCs were isolated by single-cell cloning method. Their cell surface markers were characterized by flow cytometry, and the rate of colony formation was evaluated. Differentiation assay was used to detect the differentiation potential of GMSCs. The expression of chemokine SDF-l receptor CXCR4 in GMSCs was detected by immunocytochemical staining. The chemotactic effect of SDF-1 on GMSCs was detected using a 24-multiwell Transwell cell culture chamber. The number of net migrated cells was counted in different microscope fields.

RESULTSHuman GMSCs possessed high self-renewal potential and formed single-cell colonies cultured in vitro. GMSCs expressed mesenchymal stem cells-associated markers CD44, CD73, CD90, CD105, and CD166, and the expression of hemopoietic stem cell surface markers CD14, CD34, and CD45 was negative. GMSCs differentiated into osteoblasts and adipocytes under defined culture conditions. The colony forming unit-fibroblastic for GMSCs was 21.4%/±2.8%. Immunocytochemical staining demonstrated that GMSCs expressed chemokine SDF-1 receptor CXCR4. The number of GMSCs migrating at concentrations of 100 ng.mL-1 and 200 ng.mL-1 of SDF-l in the Transwell cell culture chamber was significantly higher than that of the negative control (189.3±4.4, 164.6±4.9 cells/field vs. 47.8±2.5 cells/field, P<0.01). Treatment with the CXCR4 neutralizing antibody, an antagonist for CXCR4, significantly reduced the migratory effect compared with the negative controls (29.0±2.4 cells/field vs. 47.8±2.5 cells/field, P<0.01).

CONCLUSIONHuman GMSCs express chemokine SDF-l receptor CXCR4. SDF-1 may participate in regulating chemotaxis of human GMSCs. Results suggest that the migration induced by SDF-1 is mediated by CXCR4.

Adipocytes ; Cell Culture Techniques ; Cell Differentiation ; Chemokine CXCL12 ; metabolism ; Chemotaxis ; Flow Cytometry ; Gingiva ; physiology ; Humans ; Mesenchymal Stromal Cells ; physiology ; Osteoblasts ; Receptors, CXCR4 ; Signal Transduction

ObjectiveTo?investigate?the?expression?of?chemokine?stromal?cell-derived?factor-1?(SDF-1)?receptor?CXCR4?in?human?gingival?mesenchymal?stem?cells?(GMSCs)?and?the?migration?potential?of?GMSCs?stimulated?with?SDF-1.?Methods?Human?GMSCs?were?isolated?by?single-cell?cloning?method.?Their?cell?surface?markers?were?characterized?by?flow?cytometry,?and?the?rate?of?colony?formation?was?evaluated.?Differentiation?assay?was?used?to?detect?the?differentiation?potential?of?GMSCs.?The?expression?of?chemokine?SDF-1?receptor?CXCR4?in?GMSCs?was?detected?by?immunocytochemical?staining.?The?che-motactic?effect?of?SDF-1?on?GMSCs?was?detected?using?a?24-multiwell?Transwell?cell?culture?chamber.?The?number?of?net?migrated?cells?was?counted?in?different?microscope?fields.?Results???Human?GMSCs?possessed?high?self-renewal?potential?and?formed?single-cell?colonies?cultured?in vitro.?GMSCs?expressed?mesenchymal?stem?cells-associated?markers?CD44,?CD73,?CD90,?CD105,?and?CD166,?and?the?expression?of?hemopoietic?stem?cell?surface?markers?CD14,?CD34,?and?CD45?was?negative.?GMSCs?differentiated?into?osteoblasts?and?adipocytes?under?defined?culture?conditions.?The?colony?forming?unit-fibroblastic?for?GMSCs?was?21.4%±2.8%.?Immunocytochemical?staining?demonstrated?that?GMSCs?expressed?chemokine?SDF-1?receptor?CXCR4.?The?number?of?GMSCs?migrating?at?concentrations?of?100?ng·mL-1?and?200?ng·mL-1?of?SDF-1?in?the?Transwell?cell?culture?chamber?was?significantly?higher?than?that?of?the?negative?control?(189.3±4.4,?164.6±4.9?cells/field?vs.?47.8±2.5?cells/field,?P<0.01).?Treatment?with?the?CXCR4?neutralizing?antibody,?an?antagonist?for?CXCR4,?significantly?reduced?the?migratory?effect?compared?with?the?negative?con-trols?(29.0±2.4?cells/field?vs.?47.8±2.5?cells/field,?P<0.01).?Conclusion???Human?GMSCs?express?chemokine?SDF-1?receptor?CXCR4.?SDF-1?may?participate?in?regulating?chemotaxis?of?human?GMSCs.?Results?suggest?that?the?migration?induced?by?SDF-1?is?mediated?by?CXCR4.

OBJECTIVETo investigate the expression of high mobility group box 1 (HMGB1) in gingival tissues of chronic periodontitis.

METHODSHuman peripheral blood mononuclear cells(PBMC) were stimulated with 1 microg x mL(-1) lipopolysaccharide (LPS) for 24 h or 48 h. Expression and release of HMGB1 were checked by immunofluorescence and enzyme-linked immunosorbent assay (ELISA), respectively. PBMC were stimulated with 100 ng x mL(-1) HMGB1 or 50 ng x mL(-1) tumor necrosis factor-alpha (TNF-alpha), the expressions of TNF-alpha and HMGB1 in the supernatant were studied by ELISA. Gingival tissues and gingival crevicular fluids (GCF) were collected from patients and healthy people. Expression of HMGB1 in gingival tissues and GCF was studied using immunofluorescence and ELISA, respectively.

RESULTSHMGB1 was translocated from nucleus to cytosol in PBMC after LPS stimulation for 24 h. The content of HMGB1 in the supernatant from stimulated cells was significantly higher than that from unstimulated cells after 48 h (P < 0.01). HMGB1 was released by PBMC in response to TNF-alpha stimulation, it also stimulated PBMC to release TNF-alpha (P < 0.01). Translocation of HMGB1 from nucleus to cytosol was also found in infiltrated cells in gingival tissues from patients, and HMGB1 in GCF from patients was significantly higher than that from healthy people P < 0.01).

CONCLUSIONThe results suggest that HMGB1 may play an important role in the pathological progress of chronic periodontitis.

Chronic Periodontitis ; Gingiva ; HMGB1 Protein ; Humans ; Leukocytes, Mononuclear ; Male ; Tumor Necrosis Factor-alpha

OBJECTIVETo evaluate the therapeutic effects of 2% minocycline hydrochloride liposome controlled-release gel on the periodontitis in an established rat periodontitis model.

METHODSBiocompatibility was tested by oral perfusion sample solution for long-term observation. Minocycline hydrochloride liposome controlled-release gel was utilized to treat the established rat periodontitis model. The rats were selected randomly and divided into three groups: group A (PERIO-treated group), group B (minocycline hydrochloride liposome controlled-release gel treated group), and group C (negative control group). The gingival index (GI) and probing depth (PD) were detected, and the number of mononuclear and broken bone cells were examined after 7, 14, 28, and 56 d.

RESULTSThe minocycline hydrochloride liposome controlled-release gel exhibited excellent biocompatibility based on weight measure and tissue section evaluation. The rats with periodontitis demonstrated that GI, PD, and the number of mononuclear and broken bone cells of group B decreased in 14, 28, and 56 d. Pathological observation showed that new bones and fibers were formed in group B.

CONCLUSIONMinocycline hydrochloride liposome controlled-release gel improves rat periodontitis, thereby providing valuable evidence for clinical application.

Animals ; Anti-Bacterial Agents ; Delayed-Action Preparations ; Dental Scaling ; Liposomes ; Minocycline ; Periodontal Index ; Periodontitis ; Rats

OBJECTIVETo investigate the mechanism by which carbon monoxide inhibits the expression of adhesion molecules on human gingival fibroblasts (HGF) stimulated with inflammatory cytokines.

METHODSHGF were cultured in vitro, and stimulated with 50 ng x mL tumor necrosis factor-alpha (TNF-alpha) and 10 ng x mL(-1) interleukin-1beta (IL-1beta) concurrently in the presence or absence of carbon monoxide releasing molecule-3 (CORM-3) at 500 micromol x L-1. Expression of phosphorylated extracellular regulated protein kinase (ERK), phosphorylated c-Jun N-terminal kinase (NK) and phosphorylated p38 in mitogen-activated protein kinase(MAPK) pathway was studied by Western blot at 10 min and 20 min, respectively. Nuclear expression of nuclear factor-kappaB (NF-kappaB) was checked by Western blot after 4 h stimulation. In some experiments, cells were prestimulated by 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ) for 8 h before cytokine stimulation and the expression of intercellular adhesion molecule-1 (ICAM-1) was checked by Western blot after 24 h.

RESULTSCORM-3 significantly inhibited the phosphorylation of MAPK p38 after 10 min stimulation with cytokines, but had no signifi-cant effect on the phosphorylation of ERK and JNK. CORM-3 significantly inhibited the nuclear expression of NF-KB-p65 on HGF after 4 h stimulation by inflammatory cytokines. The inhibitory effect of CORM-3 on the expression of ICAM-1 was not influenced by guanylate cyclase inhibitor ODQ.

CONCLUSIONThe inhibitory effect of carbon monoxide on the expression of adhesion molecules might be exerted by its inhibitory effect on the NF-kappaB activity and MAPK p38 phosphorylation.

Carbon Monoxide ; Cytokines ; Fibroblasts ; Gingiva ; Humans ; Intercellular Adhesion Molecule-1 ; JNK Mitogen-Activated Protein Kinases ; Mitogen-Activated Protein Kinases ; NF-kappa B ; Phosphorylation ; Tumor Necrosis Factor-alpha

OBJECTIVETo investigate the chemotactic response of human periodontal ligament stem cells (PDLSCs) to bone morphogenetic protein-2 (BMP-2).

METHODSHuman PDLSCs were obtained from clinically healthy premolars extracted for orthodontic reasons and used to isolate PDLSCs by limited dilution method. The expression of Vimentin and stem cell marker STRO-1 on PDLSCs were demonstrated with immunocytochemical staining. Differentiation assay was used to detect the differentiation potential of PDLSCs. Cloning formation experiment and 5-bromo-2-deoxyuridine (BrdU) incorporation assay were used to determine the stem cell characteristics of PDLSCs. The chemotactic effect of BMP-2 on PDLSCs was detected by using a 24-multiwell Transwell cell culture chamber. The number of net migrated cells was counted in different microscope fields.

RESULTSHuman PDLSCs displayed positive staining for Vimentin and expressed the stem cell marker STRO-1. These cells differentiated into osteoblasts and adipocytes under defined culture conditions, possessed high self-renewal potential and formed single-cell colonies in vitro. The number of cells migrating at concentrations of 100, 200 ng mL(-1) of BMP-2 in Transwell cell culture chamber was significantly higher than that of negative control (P<0.01).

CONCLUSIONBMP-2 may participate in regulating chemotaxis of human PDLSCs.

Adipocytes ; Bone Morphogenetic Protein 2 ; Cell Culture Techniques ; Cell Differentiation ; Humans ; Osteoblasts ; Periodontal Ligament ; Stem Cells

OBJECTIVETo investigate the influence of carbon monoxide on the expression of adhesion molecules stimulated by inflammatory cytokines on human gingival fibroblasts.

METHODSHuman gingival fibroblasts were stimulated with 50 ng x mL(-1) tumor necrosis factor (TNF)-alpha and 10 ng x mL(-1) interleukin (IL)-1beta concurrently in the presence or absence of 500 micromol x L(-1) carbon monoxide releasing molecule (CORM). Expression of intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1 at protein and mRNA level was examined by Western blot and reverse transcription polymerase chain reaction (RT-PCR), respectively. Activity of transcription factor NF-kappaB was evaluated by reporter gene assay.

RESULTSExpression of ICAM-1 and VCAM-1 on human gingival fibroblasts increased dramatically after concurrent stimulation of TNF-alpha and IL-1beta, while CORM inhibited the upregulation of ICAM-1 and VCAM-1. CORM decreased the activity of NF-KB stimulated by TNF-alpha and IL-1beta.

CONCLUSIONCarbon monoxide could be a promising way in treating of periodontitis.

Carbon Monoxide ; Cells, Cultured ; Fibroblasts ; Gingiva ; Humans ; Intercellular Adhesion Molecule-1 ; Interleukin-1beta ; NF-kappa B ; RNA, Messenger ; Tumor Necrosis Factor-alpha ; Vascular Cell Adhesion Molecule-1

OBJECTIVETo investigate the expression pattern of FHL2, which is an intracellular signaling transcription molecule during mineralization in cultured human periodontal ligament cells (hPDLCs) in vitro.

METHODShPDLCs were cultured in vitro. Test group was cultured with mineral induction media while control group without induction media. 0, 14, 28 days after culture, alizarin red staining was used to measure the mineral nodules formation. Immunocytochemistry was used to examine the expression of FHL2 protein 0 day and 14 days after mineral induction. Meanwhile, mRNA expression level of FHL2 was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) on the 0, 14, 28 days after induction.

RESULTS14 and 28 days after cultivation, mineral nodules formed and were stained positively with alizarin red staining in test group while no mineral nodule formed in control group. Immunocytochemical results indicated that hPDLCs in test group expressed FHL2 positively. According to RT-PCR results, 14 and 28 days after mineral induction, the expression levels of FHL2 both increased significantly when compared with 0 day (P<0.01), and the expression level at 14 days was 1.4 folds of 0 day.

CONCLUSIONFHL2 protein is found to be involved in the in vitro mineralization of hPDLCs. FHL2 protein may play a role in the differentiation and mineralization of hPDLCs.

Cell Differentiation ; Cells, Cultured ; Humans ; In Vitro Techniques ; LIM-Homeodomain Proteins ; biosynthesis ; Muscle Proteins ; biosynthesis ; Periodontal Ligament ; RNA, Messenger ; Transcription Factors ; biosynthesis