Lysyl oxidase-mediated VEGF-induced differentiation and angiogenesis in human dental pulp cells.

AIM: To investigate the effects of recombinant human vascular endothelial growth factor (rhVEGF) on odontoblastic differentiation, in vitro angiogenesis, and expression and activity of lysyl oxidase (LOX) in human dental pulp cells (HDPCs), compared with rhFGF-2. To identify the underlying molecular mechanisms, the study focused on whether LOX was responsible for the actions of rhVEGF. METHODOLOGY: Recombinant human vascular endothelial growth factor (rhVEGF) was constructed using the pBAD-HisA plasmid in Escherichia coli. HDPCs were treated with 1-50 µg mL-1 rhVEGF for 14 days. Alkaline phosphatase (ALP) activity was measured, and the formation of calcified nodules was assessed using alizarin red staining after the induction of odontogenic differentiation of HDPCs. The expression level of the odontogenic differentiation markers was detected by reverse transcription polymerase chain reaction. Signal pathways were assessed by Western blot and immunocytochemistry. The data were analysed by anova with Bonferroni's test (α = 0.05). RESULTS: Recombinant human vascular endothelial growth factor significantly increased cell growth (P < 0.05), ALP activity (P < 0.05) and mineralization nodule formation and upregulated the mRNA expression levels of the osteogenic/odontogenic markers that were lower with rhFGF-2. rhVEGF significantly increased amine oxidase activity (P < 0.05) and upregulated LOX and LOXL mRNA expression in HDPCs. Additionally, rhVEGF dose-dependently upregulated angiogenic gene mRNAs and capillary tube formation to a greater degree than rhFGF-2. Inhibition of LOX using ß-aminopropionitrile (BAPN) and LOX or LOXL gene silencing by RNA interference attenuated rhVEGF-induced growth, ALP activity, mineralization, the expression of marker mRNAs and in vitro angiogenesis. Furthermore, treatment with rhVEGF resulted in phosphorylation of Akt, ERK, JNK and p38, and activation of NF-κB, which was inhibited by LOX or LOXL silencing and BAPN. CONCLUSION: Recombinant human vascular endothelial growth factor promoted cell growth, odontogenic potential and in vitro angiogenesis via modulation of LOX expression. These results support the concept that rhVEGF may offer therapeutic benefits in regenerative endodontics.

Role of PIN1 on in vivo periodontal tissue and in vitro cells.

BACKGROUND: Although expression of peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (PIN1) was reported in bone tissue, the precise role of PIN1 in periodontal tissue and cells remain unclear. MATERIAL & METHODS: To elucidate the roles of PIN1 in periodontal tissue, its expression in periodontal tissue and cells, and effects on in vitro 4 osteoblast differentiation and the underlying signaling mechanisms were evaluated. RESULTS: PIN1 was expressed in mouse periodontal tissues including periodontal ligament cells (PDLCs), cementoblasts and osteoblasts at the developing root formation stage (postnatal, PN14) and functional stage of tooth (PN28). Treatment of PIN1 inhibitor juglone, and gene silencing by RNA interference promoted osteoblast differentiation in PDLCs and cementoblasts, whereas the overexpression of PIN1 inhibited. Moreover, osteogenic medium-induced activation of AMPK, mTOR, Akt, ERK, p38 and NF-jB pathways were enhanced by PIN1 siRNA, but attenuated by PIN1 overexpression. Runx2 expressions were induced by PIN1 siRNA, but downregulated by PIN1 overexpression. CONCLUSION: In summary, this study is the first to demonstrate that PIN1 is expressed in developing periodontal tissue, and in vitro PDLCs and cementoblasts. PIN1 inhibition stimulates osteoblast differentiation, and thus may play an important role in periodontal regeneration.

PIN1 Suppresses the Hepatic Differentiation of Pulp Stem Cells via Wnt3a.

This study aimed to investigate the role of PIN1 on the hepatic differentiation of human dental pulp stem cells (hDPSCs) and its signaling pathway, as well as the potential therapeutic effects of hDPSC transplantation and PIN1 inhibition on CCl4 (carbon tetrachloride)-induced liver fibrosis in mice. The in vitro results showed that hepatic differentiation was suppressed by infection with adenovirus-PIN1 and promoted by PIN1 inhibitor juglone via the downregulation of Wnt3a and ß-catenin. Compared with treatment with either hDPSC transplantation or juglone alone, the combination of hDPSCs and juglone into CCl4-injured mice significantly suppressed liver fibrosis and restored serum levels of alanine transaminase, aspartate transaminase, and ammonia. Collectively, the present study shows for the first time that PIN1 inhibition promotes hepatic differentiation of hDPSCs through the Wnt/ß-catenin pathway. Furthermore, juglone in combination with hDPSC transplantation effectively treats liver fibrosis, suggesting that hDPSC transplantation with PIN1 inhibition may be a novel therapeutic candidate for the treatment of liver injury.

Anti-inflammatory and anti-osteoclastogenic effects of zinc finger protein A20 overexpression in human periodontal ligament cells.

BACKGROUND AND OBJECTIVE: Although overexpression of the nuclear factor κB inhibitory and ubiquitin-editing enzyme A20 is thought to be involved in the pathogenesis of inflammatory diseases, its function in periodontal disease remains unknown. The aims of the present study were to evaluate A20 expression in patients with periodontitis and to study the effects of A20 overexpression, using a recombinant adenovirus encoding A20 (Ad-A20), on the inflammatory response and on osteoclastic differentiation in lipopolysaccharide (LPS)- and nicotine-stimulated human periodontal ligament cells (hPDLCs). MATERIAL AND METHODS: The concentration of prostaglandin E2 was measured by radioimmunoassay. Reverse transcription-polymerase chain reactions and western blot analyses were used to measure mRNA and protein levels, respectively. Osteoclastic differentiation was assessed in mouse bone marrow-derived macrophages using conditioned medium from LPS- and nicotine-treated hPDLCs. RESULTS: A20 was upregulated in the gingival tissues and neutrophils from patients with periodontitis and in LPS- and nicotine-exposed hPDLCs. Pretreatment with A20 overexpression by Ad-A20 markedly attenuated LPS- and nicotine-induced production of prostaglandin E2 , as well as expression of cyclooxygenase-2 and proinflammatory cytokines. Moreover, A20 overexpression inhibited the number and size of tartrate-resistant acid phosphatase-stained osteoclasts, and downregulated osteoclast-specific gene expression. LPS- and nicotine-induced p38 phosphorylation and nuclear factor κB activation were blocked by Ad-A20. Ad-A20 inhibited the effects of nicotine and LPS on the activation of pan-protein kinase C, Akt, GSK-3ß and protein kinase Cα. CONCLUSIONS: This study is the first to demonstrate that A20 overexpression has anti-inflammatory effects and blocks osteoclastic differentiation in a nicotine- and LPS-stimulated hPDLC model. Thus, A20 overexpression may be a potential therapeutic target in inflammatory bone loss diseases, such as periodontal disease.

Dosimetric planning study for the prevention of anal complications after post-operative whole pelvic radiotherapy in cervical cancer patients with hemorrhoids.

OBJECTIVE: Radiation-induced anal toxicity can be induced by low radiation doses in patients with haemorrhoids. The object of this study was to determine the dosimetric benefits of different whole pelvic radiotherapy (WPRT) techniques in terms of dose delivered to the anal canal in post-operative patients with cervical cancer. METHODS: The planning CT images of 10 patients with cervical cancer undergoing postoperative radiotherapy were used for comparison of three different plans. All patients had been treated using the conventional box technique WPRT (CV-WPRT), and we tried low-margin-modified WPRT (LM-WPRT), three-dimensional conformal techniques WPRT (CF-WPRT) and intensity-modulated WPRT (IM-WPRT) planning for dosimetric comparison of the anal canal, retrospectively. RESULTS: Mean anal canal doses of the IM-WPRT were significantly lower (p < 0.05) than those of CV-WPRT, LM-WPRT and CF-WPRT, and V10, V20, V30 and V40 to the anal canal were also significantly lower for IM-WPRT (p < 0.05). The proportion of planning target volumes (PTVs) that received ≥98% of the prescribed dose for all plans was >99%, and the proportion that received ≥108% of the prescribed dose for IM-WPRT was <2%. Volumes of bladders and rectums that received ≥30 or ≥40 Gy were significantly lower for IM-WPRT than for three of the four-field WPRT plans (p = 0.000). CONCLUSION: IM-WPRT can significantly reduce radiation dose delivered to the anal canal and does not compromise PTV coverage. In patients with haemorrhoids, IM-WPRT may be of value for the prevention of anal complications. ADVANCES IN KNOWLEDGE: Although tolerance of the anal canal tends to be ignored in patients undergoing post-operative WPRT, patients with haemorrhoids may suffer complications at low radiation doses. The present study shows IM-WPRT can be meaningful in these patients.

Educational intervention as an effective step for reducing blood culture contamination: a prospective cohort study.

BACKGROUND: Contaminated blood cultures lead to diagnostic challenges and place a burden on healthcare services. AIM: To determine the impact of introducing a clinical skills test (CST) as part of the medical licensing examination and an institutional education programme on the contamination rates of blood cultures. METHODS: A prospective cohort study was conducted from 2009 through 2013 in all wards of a tertiary-care teaching hospital. We evaluated the effects of the CST, which was added to the National Medical Licensing Examination in Korea (KMLE) in 2010 and our institutional education programme, which began in 2013. The medical interns in charge of collection of blood for culture were divided in three groups with presence or absence of CST and the institutional education programme. The primary outcome was the percentage of blood cultures contaminated in each group, which were compared using the Poisson regression model. Participants' self-rated scores for the blood draw procedure were also analysed. FINDINGS: Although introduction of the CST in the KMLE failed to reduce blood culture contamination rate (1.36% vs 1.35%; P = 0.734), the institutional education programme significantly reduced the contamination rate (1.35% vs 1.00%; P < 0.0001). Most participants answered that they always followed each step correctly except for waiting the recommended contact time after applying the antiseptic. CONCLUSION: The educational intervention, not the introduction of CST in the KMLE, was effective in reducing overall contamination rates.

2,4,5-Trimethoxyldalbergiquinol promotes osteoblastic differentiation and mineralization via the BMP and Wnt/ß-catenin pathway.

Dalbergia odorifera has been traditionally used as a medicine to treat many diseases. However, the role of 2,4,5-trimethoxyldalbergiquinol (TMDQ) isolated and extracted from D. odorifera in osteoblast function and the underlying molecular mechanisms remain poorly understood. The aim of this study was to investigate the effects and possible underlying mechanisms of TMDQ on osteoblastic differentiation of primary cultures of mouse osteoblasts as an in vitro assay system. TMDQ stimulated osteoblastic differentiation, as assessed by the alkaline phosphatase (ALP) activity, ALP staining, mineralized nodule formation, and the levels of mRNAs encoding the bone differentiation markers, including ALP, bone sialoprotein (BSP), osteopontin, and osteocalcin. TMDQ upregulated the expression of Bmp2 and Bmp4 genes, and increased the protein level of phospho-Smad1/5/8. Furthermore, TMDQ treatment showed the increased mRNA expression of Wnt ligands, phosphorylation of GSK3, and the expression of ß-catenin protein. The TMDQ-induced osteogenic effects were abolished by Wnt inhibitor, Dickkopf-1 (DKK1), and bone morphogenetic protein (BMP) antagonist, noggin. TMDQ-induced runt-related transcription factor 2 (Runx2) expression was attenuatted by noggin and DKK1. These data suggest that TMDQ acts through the activation of BMP, Wnt/ß-catenin, and Runx2 signaling to promote osteoblast differentiation, and we demonstrate that TMDQ could be a potential agent for the treatment of bone loss-associated diseases such as osteoporosis.

TNF-α and LPS activate angiogenesis via VEGF and SIRT1 signalling in human dental pulp cells.

AIM: To assess whether SIRT1 and VEGF are responsible for tumour necrosis factor-α (TNF-α) and lipopolysaccharide (LPS)-induced angiogenesis and to examine the molecular mechanism(s) of action in human dental pulp cells (HDPCs). METHODOLOGY: Immortalized HDPCs obtained from Prof. Takashi Takata (Hiroshima University, Japan) were treated with LPS (1 µg mL(-1) ) and TNF-α (10 ng mL(-1) ) for 24 h. mRNA and protein levels were examined by RT-PCR and Western blotting, respectively. Migration and tube formation were examined in human umbilical vein endothelial cells (HUVECs). The data were analysed by one-way anova. Statistical analysis was performed at α = 0.05. RESULTS: LPS and TNF-α upregulated VEGF and SIRT1 mRNA and protein levels. Inhibition of SIRT1 activity by sirtinol and SIRT1 siRNA or inhibition of the VEGF receptor by CBO-P11 significantly attenuated LPS + TNF-α-stimulated MMPs production in HDPCs, as well as migration and tube formation in HUVECs (P < 0.05). Furthermore, sirtinol, SIRT1 siRNA and CBO-P11 attenuated phosphorylation of Akt, extracellular signal-regulated kinase (ERK), p38 and c-Jun N-terminal kinase (JNK) and the nuclear translocation of NF-κB p65. Pre-treatment with inhibitors of p38, ERK, JNK, PI3K and NF-κB decreased LPS + TNF-α-induced VEGF and SIRT1 expression, MMPs activity in HDPCs and angiogenesis (P < 0.05) in HUVECs. CONCLUSIONS: TNF-α and LPS led to upregulation of VEGF and SIRT1, and subsequent upregulation of MMP-2 and MMP-9 production, and promote angiogenesis via pathways involving PI3K, p38, ERK, JNK and NF-κB. The results suggest that inhibition of SIRT1 and VEGF might attenuate pro-inflammatory mediator-induced pulpal disease.

Role of resistin in the inflammatory response induced by nicotine plus lipopolysaccharide in human periodontal ligament cells in vitro.

BACKGROUND AND OBJECTIVE: Resistin was recently reported to play a role in inflammation-related diseases such as arthritis. However, the precise role of resistin in chronic inflammatory diseases, such as periodontal disease, remains unclear. The aim of this study was to investigate the combined effects of nicotine and lipopolysaccharide (LPS) on the expression of resistin and to assess whether resistin expression influences the levels of inflammatory cytokines, extracellular matrix (ECM) molecules and MMPs in human periodontal ligament cells (PDLCs) stimulated with both nicotine and LPS. MATERIAL AND METHODS: PDLCs were pretreated with isoproterenol or resistin-specific small interfering RNA (siRNA), stimulated with LPS plus nicotine for 24 h, and then monitored for the production of inflammatory mediators. The concentrations of prostaglandin E2 (PGE2) and nitric oxide (NO) were measured by radioimmunoassay and the Griess method, respectively. RT-PCR and western blot analysis were used to measure the levels of mRNA and protein, respectively. Western blot analysis was also used to assess the activation of various signal-transduction pathways. RESULTS: Treatment with nicotine plus LPS up-regulated the expression of resistin mRNA and the production of resistin protein in PDLCs in a time- and concentration-dependent manner. Isoproterenol-mediated interference with the function of resistin, or siRNA-mediated knockdown of resistin expression, markedly attenuated the LPS plus nicotine-mediated stimulation of PGE2 and NO production, the production of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase proteins and the expression of proinflammatory cytokines [tumor necrosis factor-α, interleukin (IL)-1ß, IL-6 and IL-12] and MMPs (MMP-1, MMP-2 and MMP-9); however, these treatments restored the expression of ECM molecules. Furthermore, pretreatment with isoproterenol or resistin-specific siRNA blocked nicotine plus LPS-induced activation of phosphoinositide-3-kinase, glycogen synthase kinase-3 beta, ß-catenin, p38, ERK, JNK and nuclear factor-κB. CONCLUSION: This is the first study to show that the inhibition of resistin, by either a pharmacological or a genetic silencing approach, has anti-inflammatory effects. These effects include decreased levels of inflammatory cytokines and the prevention of ECM breakdown in a nicotine plus LPS-stimulated PDLC model.

Accuracy of the Xpert® MTB/RIF assay for the diagnosis of extra-pulmonary tuberculosis in South Korea.

SETTING: The Xpert(®) MTB/RIF assay has been endorsed by the World Health Organization for the detection of pulmonary and extra-pulmonary tuberculosis (EPTB). OBJECTIVE: To determine the accuracy of the Xpert assay in diagnosing EPTB in South Korea, a country with an intermediate TB burden. DESIGN: We retrospectively reviewed the medical records of 1429 patients in whom the Xpert assay using EPTB specimens was requested between 1 January 2011 and 31 October 2013 in a tertiary referral hospital in South Korea. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for the diagnosis of EPTB and detection of rifampicin (RMP) resistance were calculated. RESULTS: Using culture as gold standard, the sensitivity, specificity, PPV and NPV of the assay were respectively 67.7%, 98.1%, 60% and 98.6%. Using a composite reference standard, the sensitivity, specificity, PPV and NPV were respectively 49.3%, 100%, 100% and 95.1%. The sensitivity, specificity, PPV and NPV for the detection of RMP resistance among specimens with positive results for Mycobacterium tuberculosis were respectively 80%, 100%, 100% and 97.7%. CONCLUSION: The Xpert assay showed acceptable sensitivity in certain groups and excellent specificity in diagnosing EPTB and detecting RMP resistance in an intermediate TB burden country.

Anti-inflammatory effects of glutamine on LPS-stimulated human dental pulp cells correlate with activation of MKP-1 and attenuation of the MAPK and NF-κB pathways.

AIM: To evaluate the anti-inflammatory effects of glutamine and the underlying signal pathway mechanisms in lipopolysaccharide (LPS)-stimulated human dental pulp cells (HDPCs). METHODS: Human dental pulp cells were exposed to 10 µg mL(-1) LPS and various concentrations of glutamine for 24 h. The production of PGE2 and nitric oxide was determined by enzyme-linked immunosorbent assay (ELISA) and Griess reagent kit, respectively. Cytokines were examined by ELISA, reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time PCR. iNOS and COX protein expression as well as signal pathways were accessed by Western blot. The data were analysed by anova with Bonferroni's test (α = 0.05). RESULTS: Glutamine reduced LPS-induced iNOS and COX-2 protein expression as well as production of NO and PGE2 in a dose-dependent fashion. Additionally, glutamine suppressed the production and mRNA expression of inflammatory cytokines including interleukin-1ß (IL-1ß), TNF-α, and IL-8. Furthermore, glutamine attenuated phosphorylation of extracellular signal-regulated kinase (ERK), p38, c-Jun N-terminal kinase (JNK) and IκB-α, and nuclear translocation of NF-κB p65, but enhanced mitogen-activated protein kinase phosphatase-1 (MKP-1) expression in LPS-treated HDPCs. CONCLUSION: Glutamine exerted an anti-inflammatory effect via activation of MKP-1 and inhibition of the NF-κB and MAPK pathways in LPS-treated HDPCs.

PIN1 inhibition suppresses osteoclast differentiation and inflammatory responses.

Inflammatory responses and osteoclast differentiation play pivotal roles in the pathogenesis of osteolytic bone diseases such as periodontitis. Although overexpression or inhibition of peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (PIN1) offers a possible therapeutic strategy for chronic inflammatory diseases, the role of PIN1 in periodontal disease is unclear. The aim of the present study was to evaluate PIN1 expression in periodontitis patients as well as the effects of PIN1 inhibition by juglone or PIN1 small-interfering RNA (siRNA) and of PIN1 overexpression using a recombinant adenovirus encoding PIN1 (Ad-PIN1) on the inflammatory response and osteoclastic differentiation in lipopolysaccharide (LPS)- and nicotine-stimulated human periodontal ligament cells (PDLCs). PIN1 was up-regulated in chronically inflamed PDLCs from periodontitis patients and in LPS- and nicotine-exposed PDLCs. Inhibition of PIN1 by juglone or knockdown of PIN1 gene expression by siRNA markedly attenuated LPS- and nicotine-stimulated prostaglandin E2 (PGE2) and nitric oxide (NO) production, as well as cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression, whereas PIN1 overexpression by Ad-PIN1 increased it. LPS- and nicotine-induced nuclear factor (NF)-κB activation was blocked by juglone and PIN1 siRNA but increased by Ad-PIN1. Conditioned medium prepared from LPS- and nicotine-treated PDLCs increased the number of tartrate-resistant acid phosphatase-stained osteoclasts and osteoclast-specific gene expression. These responses were blocked by PIN1 inhibition and silencing but stimulated by Ad-PIN1. Furthermore, juglone and PIN1 siRNA inhibited LPS- and nicotine-induced osteoclastogenic cytokine expression in PDLCs. This study is the first to demonstrate that PIN1 inhibition exhibits anti-inflammatory effects and blocks osteoclastic differentiation in LPS- and nicotine-treated PDLCs. PIN1 inhibition may be a therapeutic strategy for inflammatory osteolysis in periodontal disease.

NOD2 Mediates Odontoblast Differentiation and RANKL Expression.

The precise regulation of odontoblast differentiation and osteoclastogenic cytokine expression in human dental pulp cells (HDPCs) is crucial for the pathology of bacteria-related pulpitis. Although the up-regulation of nucleotide-binding oligomerization domain-containing protein 2 (NOD2) has been reported in inflamed human dental pulps, the role of NOD2 in the differentiation of HDPCs remains unclear. Here, we show the involvement of NOD2 in odontoblast differentiation together with osteoclastogenic cytokine expression in HDPCs. Treatment with muramyl dipeptide (MDP), a known NOD2-agonist, significantly inhibited odontoblast differentiation of HDPCs, as revealed by reduced ALP activity, osteoblast/odontoblast marker expression, and mineralized nodule formation. Importantly, the forced down-regulation of NOD2 by small interfering RNA (siRNA) recovered MDP-down-regulated odontoblast differentiation. MDP-elicited suppression of odontoblast differentiation resulted from the increased expression of MKP-1 protein and the subsequent decline of MAPKs phosphorylation, which is a prerequisite for odontoblast differentiation. Furthermore, we found that MDP treatment elevated the expression of osteoclastogenic cytokines in HDPCs, which was also reversed by NOD2 silencing. Analysis of these data, taken together, suggests that the regulation of NOD2 expression upon MDP challenge might serve as an intrinsic mechanism that underlies the hindered dentin formation and accelerated dentin resorption in bacterial infection-mediated pulpitis.

Spontaneous fungal peritonitis: a severe complication in patients with advanced liver cirrhosis.

Treatment of cirrhotic patients with spontaneous peritonitis using antibiotics occasionally fails. Fungal infections may be one of the causes of antibiotic treatment failure in such patients. In this study we evaluated the clinical significance and characteristics of spontaneous fungal peritonitis (SFP). Consecutive cirrhotic patients with spontaneous peritonitis treated between 2000 and 2005 at a tertiary care center in Seoul, Korea, were included. We analyzed the clinical characteristics and the prognosis of SFP patients compared with patients with spontaneous bacterial peritonitis (SBP). During the study period 416 patients developed spontaneous peritonitis and 15 (3.6 %) had SFP. Compared with patients with SBP, nosocomial peritonitis (peritonitis that developed after hospitalization for >72 h) was more common and the Child-Pugh score was higher in SFP patients (both, P < 0.01). Ten patients were infected with Candida spp. (C. albicans, 8; C. tropicalis, 1; C. glabrata, 1), and 5 with Cryptococcus neoformans. Eleven patients were co-infected with bacteria that were susceptible to the antibiotics administered. Only 5 patients were treated using appropriate anti-fungal agents. The 1-month mortality rate for SFP patients was 73.3 % (11 out of 15; median time to death, 2 days [range, 0-22]), which was significantly higher than patients with SBP alone (28.7 %, P = 0.0007). SFP is severe complication related to high mortality in cirrhotic patients. A longer admission and a higher Child-Pugh score may be risk factors. Immediate anti-fungal treatment is warranted in patients with spontaneous peritonitis, once fungus is found in the ascitic fluid.

Involvement of SDF-1 and monocyte chemoattractant protein-1 in hydrogen peroxide-induced extracellular matrix degradation in human dental pulp cells.

AIM: To determine whether chemokines such as SDF-1 and monocyte chemoattractant protein-1 (MCP-1) are responsible for hydrogen peroxide (H2 O2 )-induced extracellular matrix (ECM) degradation and to identify the underlying mechanism in human dental pulp cells (HDPCs). METHOD: Human dental pulp cells were exposed to 0.4 mmol H2 O2 for 48 h. mRNA expression and protein expression were examined by RT-PCR and Western blot analysis, respectively. The mRNA expression of chemokine (SDF-1 and MCP-1), their receptors (CXCR4 and CXCR2) and extracellular matrix proteins was evaluated by reverse transcriptase-polymerase chain reaction. The production of SDF-1, MCP-1, CXCR4 and CCR2 in the culture medium was determined by enzyme-linked immunosorbent assay. Signal transduction pathway was examined by Western blotting. RESULTS: Hydrogen peroxide provoked the activation of MCP-1 and SDF-1 mRNA and their respective receptors, CXCR4 and CXCR2. H2 O2 treatment concomitantly downregulated the expression of ECM molecules, such as type I collagen, elastin and fibronectin, and upregulated the mRNA expression of matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-8 and MMP-9. Hydrogen peroxide-induced ECM degradation and MMP upregulation were blocked by neutralizing antibodies and siRNAs directed against SDF-1 and MCP-1. Inhibition of SDF-1 and MCP-1 blocked the H2 O2 -induced activation of Akt, p38, ERK and NF-kB. CONCLUSION: Inhibition of SDF and MCP-1 is a potent component of reducing release reactive oxygen species-induced ECM degradation in HDPCs and may play an important role in pulpal and periapical inflammation.

The role of SDF-1 and CXCR4 on odontoblastic differentiation in human dental pulp cells.

AIM: To examine the role of stromal cell-derived factor 1 (SDF-1) signalling during odontogenic differentiation in human dental pulp cells (HDPCs). METHODOLOGY: Human dental pulp cells were treated with differentiation medium, recombinant human SDF-1, neutralizing antibody for SDF-1 or CXCR4, pertussis toxin (PTX) and AMD3100. The expression of SDF-1 and its receptor chemokine receptor type 4 (CXCR4) was measured by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. Odontoblastic differentiation was determined using alkaline phosphatase (ALP) activity assay, mineralized nodule formation and marker mRNAs by RT-PCR. RESULTS: Marked upregulation of SDF-1 and CXCR4 mRNA and protein was observed in cells grown 7 days in osteogenic induction medium. The addition of recombinant human SDF-1 to HDPCs significantly (P < 0.05) increased ALP activity, mineralized nodule formation and odontoblast marker mRNAs in a dose-dependent manner. Blocking SDF-1 signalling using antibodies against SDF-1 or CXCR4, or the G-protein-coupled receptor inhibitor PTX, and CXCR4 inhibitor AMD3100, strongly suppressed induction of odontogenic differentiation in HDPCs. CONCLUSIONS: Odontoblastic differentiation was stimulated by SDF-1 activation and repressed by SDF-1/CXCR4 inhibition. Thus, SDF-1/CXCR4 signalling may be a new therapeutic target and strategy to promote repair and regeneration in endodontics.

Trace metal contents of three tricalcium silicate materials: MTA Angelus, Micro Mega MTA and Bioaggregate.

AIM: To investigate the levels of nine metals [aluminium (Al), antimony (Sb), arsenic (As), beryllium (Be), cadmium (Cd), chromium (Cr), iron (Fe), lead (Pb) and molybdenum (Mo)] in MTA Angelus, Micro Mega MTA and Bioaggregate using inductively coupled plasma-optical emission spectrometry (ICP-OES). METHODOLOGY: Each material (0.2 g) was digested using a mixture of hydrochloric and nitric acids and then filtered. The levels of nine metals in the resulting filtrates were measured by ICP-OES. The results were statistically analysed using one-way anova and the Bonferroni test. RESULTS: MTA Angelus contained more aluminium, beryllium and chromium than Micro Mega MTA (P < 0.05), whilst their levels of arsenic, cadmium and iron were similar. Antimony, lead and molybdenum were not detected in any of the three tested cements. Bioaggregate contained trace amounts of aluminium. CONCLUSIONS: MTA Angelus and Micro Mega MTA contained small amounts of seven tested metal oxides. Bioaggregate only contained trace amounts of aluminium.

Deferoxamine promotes osteoblastic differentiation in human periodontal ligament cells via the nuclear factor erythroid 2-related factor-mediated antioxidant signaling pathway.

BACKGROUND AND OBJECTIVE: Recently it was reported that deferoxamine (DFO), an iron chelator, stimulates bone formation from MG63 and mesenchymal stem cells, but inhibits differentiation in rat calvarial cells; however, the effect of DFO on osteoblastic differentiation in human periodontal ligament cells (hPDLCs) has not been reported. The aim of this study was to investigate the effects and the possible underlying mechanism of DFO on osteoblastic differentiation of hPDLCs. MATERIAL AND METHODS: The effect of DFO on osteoblast differentiation was determined by the staining intensity of calcium deposits with Alizarin red and by RT-PCR analysis of the expression of osteoblastic markers. Signal transduction pathways were analyzed by western blotting. RESULTS: DFO increased osteogenic differentiation in a concentration-dependent manner by expression of the mRNA for differentiation markers and calcium nodule formation. Exposure of hPDLCs to DFO resulted in increases in the production of reactive oxygen species and in the levels of nuclear factor erythroid 2-related factor (Nrf2) protein in nuclear extractions, as well as a dose-dependent increase in the expression of Nrf2 target genes, including glutathione (GSH), glutathione S-transferase, γ-glutamylcysteine lygase, glutathione reductase and glutathione peroxidase. Pretreatment with Nrf2 small interfering RNA, GSH depletion by buthionine sulfoximine and diethyl maleate, and with antioxidants by N-acetylcysteine and vitamin E, blocked DFO-stimulated osteoblastic differentiation. Furthermore, pretreatment with GSH depletion and antioxidants blocked DFO-induced p38 MAPK, ERK, JNK and nuclear factor-kappaB pathways. CONCLUSION: These data indicate, for the first time, that nontoxic DFO promotes osteoblastic differentiation of hPDLCs via modulation of the Nrf2-mediated antioxidant pathway.

Cryopreservation induces macrophage colony stimulating factor from human periodontal ligament cells in vitro.

Cryopreservation is used to protect vital periodontal ligaments during the transplantation of teeth. We investigated which gene products implicated in root resorption are upregulated in human periodontal ligament cells by cryopreservation, and whether cryopreservation affects the expression of macrophage-colony stimulating factor (M-CSF) in human periodontal ligament cells. We used customized microarrays to compare gene expression in human periodontal ligament cells cultured from teeth immediately after extraction and from cryopreserved teeth. Based on the result of these assays, we examined M-CSF expression in periodontal ligament cells from the immediately extracted tooth and cryopreserved teeth by real-time PCR, enzyme-linked immunosorbent assay (ELISA), Western blot analysis, and immunofluorescence. We also investigated whether human bone marrow cells differentiate into tartrate-resistant acid phosphatase (TRAP) positive osteoclasts when stimulated with RANKL (Receptor Activator for Nuclear Factor κ B Ligand) together with any secreted M-CSF present in the supernatants of the periodontal ligament cells cultured from the various groups of teeth. M-CSF was twofold higher in the periodontal ligament cells from the rapid freezing teeth than in those from the immediately extracted group (p < 0.05). Cryopreservation increased M-CSF expression in the periodontal ligament cells when analyzed by real time PCR, ELISA, Western blotting, and immunofluorescence (p < 0.05). TRAP positive osteoclasts were formed in response to RANKL and the secreted M-CSF present in the supernatants of all the experimental groups except negative control. These results demonstrate that cryopreservation promotes the production of M-CSF, which plays an important role in root resorption by periodontal ligament cells.

Transverse stability of the proximal segment after bilateral sagittal split ramus osteotomy for mandibular setback surgery.

This study aimed to evaluate the correlation between the transverse displacement of the proximal segment after bilateral sagittal osteotomy for mandibular setback and the amount and design of the mandibular setback. Patients who underwent either bilateral sagittal split ramus osteotomy (BSSRO) alone or two-jaw surgery were selected, and cephalographic postero-anterior (PA) measurements were taken pre-operatively (T1), immediately post-operatively (T2), and at follow-up (T3). The inter-gonal (IG) and inter-ramal (IR) width increased immediately after surgery, but decreased to the initial value during follow-up (P=0.002; IR, P=0.046). Only the immediate IG changes after surgery correlated with the amount of mandibular setback (P=0.009). The IG changes were significant in the symmetric group, but not in the asymmetric group. There was no difference in the IG and IR changes between the symmetric group and the asymmetric group. The immediate IG change in two-jaw patients with symmetric setback showed correlation with the setback amount. The gonial width of the deviated group showed more significant changes than that of the non-deviated group. There was no difference in the unilateral gonial width between the deviated and the non-deviated group, but the difference was significant for the unilateral ramal angle between the two groups. These correlations will be helpful in predicting post-surgical results for patients.