Nasal-type extranodal natural killer/T-cell lymphoma with oral ulcer as the earliest clinical manifestation.

Nasal-type extranodal natural killer/T-cell lymphoma (ENKTL-NT) is a special subtype of non-Hodgkin's lymphoma derived from natural killer cells or cytotoxic T cells. Oral ulcers as the first symptom makes its diagnosis challenging because of its rarity and lack of understanding. We report a case of ENKTL-NT in this paper. We analyzed the clinicopathological features, differential diagnosis, treatment, prognosis and the causes of misdiagnosis to provide a diagnostic basis for dentists to make better clinical diagnosis and treatment.

Salivary chromogranin A and myeloid-related protein-8/14 from periodontitis patients with type 2 diabetes.

OBJECTIVES: The bidirectional relationship between diabetes mellitus and chronic periodontitis is well known from clinical trials. Periodontitis in diabetic patients is characterized by severe inflammation and tissue destruction. The purpose of this study was to investigate the levels of chromogranin A (CgA), a stress marker, and myeloid-related protein (MRP)-8/14, an inflammatory marker, in saliva from patients with periodontitis and diabetes mellitus, and to investigate the relationship between CgA and MRP-8/14 in all individuals and in the three groups separately. METHOD AND MATERIALS: Stimulated saliva was collected from 20 diabetic patients with chronic periodontitis, 16 patients with chronic periodontitis, and 21 healthy individuals. Salivary CgA and MRP-8/14 were determined with enzyme-linked immunosorbent assay. Salivary CgA and MRP-8/14 levels were assessed in the saliva of diabetic periodontitis and periodontitis patients, and the relationship with periodontal disease severity was investigated. RESULTS: CgA values in saliva samples from chronic periodontitis patients and diabetic patients with chronic periodontitis were significantly higher than those of the control group. MRP-8/14 values in saliva from chronic periodontitis patients and diabetic patients with chronic periodontitis was significantly higher than that in the control group. Salivary CgA level was positively correlated to MRP-8/14 in all individuals, but there was no significant correlation within the chronic periodontitis patient group, diabetic patients with chronic periodontitis group, and the healthy patient group. No significant correlation between salivary CgA/MRP-8/14 and clinical parameters of periodontitis was found in the three groups. CONCLUSIONS: The results suggest that salivary CgA and MRP-8/14 could be related to the pathogenesis of periodontitis and diabetes. CgA concentration in saliva was positively associated with increased MRP-8/14 in all individuals.

Strontium ion attenuates lipopolysaccharide-stimulated proinflammatory cytokine expression and lipopolysaccharide-inhibited early osteogenic differentiation of human periodontal ligament cells.

BACKGROUND AND OBJECTIVE: Periodontitis is a chronic inflammatory disease characterized by the destruction of the periodontium. The strontium ion (Sr2+ ) can prevent the bone loss associated with periodontitis and promote the regeneration of the bone. The mechanisms by which the Sr2+ works remain poorly understood. We aim to investigate the effects of the Sr2+ ion on cell proliferation, inflammatory regulation and osteogenic differentiation of human periodontal ligament cells (hPDLCs) in pathological conditions. MATERIAL AND METHODS: hPDLCs were obtained from premolars that came from the orthodontic extraction. The hPDLCs were treated with Sr2+ and/or lipopolysaccharide (LPS), which was applied as the pathological condition of periodontitis. The effect of the dose of Sr2+ on cell proliferation was analyzed using a Cell Counting Kit-8 assay. The gene and protein expression of proinflammatory cytokines were detected by the real-time polymerase chain reaction and enzyme-linked immunosorbent assay. The osteogenic differentiation and mineralization were assessed by the real-time polymerase chain reaction, alkaline phosphatase activity assay and alizarin red staining. RESULTS: Results demonstrated that Sr2+ in a range of concentrations from 0.02 to 2.5 mmol/L significantly improved the proliferation of hPDLCs. Sr2+ reversed LPS-stimulated proinflammatory cytokine expressions such as tumor necrosis factor alpha, interleukin (IL)-1ß, IL-6 and IL-8. Moreover, Sr2+ rescued the LPS-inhibited gene expression of osteogenic differentiation. Although it appeared to suppress the late mineralization, Sr2+ can reverse the LPS-inhibited early osteogenic differentiation of hPDLCs. CONCLUSION: These results indicated that Sr2+ could attenuate the LPS-stimulated proinflammatory molecule expression and inhibit early osteogenic differentiation of hPDLCs.

Alpha-Lipoic Acid Alleviates High-Glucose Suppressed Osteogenic Differentiation of MC3T3-E1 Cells via Antioxidant Effect and PI3K/Akt Signaling Pathway.

BACKGROUND/AIMS: Patients with diabetes mellitus have a higher risk of dental implant failure. One major cause is high-glucose induced oxidative stress. Alpha-lipoic acid (ALA), a naturally occurring compound and dietary supplement, has been established as a potent antioxidant that is a strong scavenger of free radicals. However, few studies have yet investigated the effect of ALA on osteogenic differentiation of osteoblasts cultured with high glucose medium. The aim of this study is to investigate the effects of ALA on the osteoblastic differentiation in MC3T3-E1 cells under high glucose condition. METHODS: MC3T3-E1 cells were divided into 4 groups including normal glucose (5.5 mM) group (control), high glucose (25.5 mM) group, high glucose + 0.1 mM ALA group, and high glucose + 0.2 mM ALA group. The proliferation, osteogenic differentiation and mineralization of cells were evaluated by MTT assay, alkaline phosphatase (ALP) activity assay, alizarin red staining and real time-polymerase chain reaction. High-glucose induced oxidative damage was also assessed by the production of reactive oxygen species (ROS) and superoxide dismutase (SOD). Western blots were performed to examine the role of PI3K/Akt pathway. RESULTS: The proliferation, osteogenic differentiation and mineralization of MC3T3-E1 cells were significantly decreased by the ROS induced by high-glucose. All observed oxidative damage and osteogenic dysfunction induced were inhibited by ALA. Moreover, the PI3K/Akt pathway was activated by ALA. CONCLUSIONS: We demonstrate that ALA may attenuate high-glucose mediated MC3T3-E1 cells dysfunction through antioxidant effect and modulation of PI3K/Akt pathway.

The Effects of Hierarchical Micro/Nano-Structured Titanium Surface on Osteoblast Proliferation and Differentiation Under Diabetic Conditions.

PURPOSE: The aim of the present study was to mimic the hierarchical structure of bone tissues by simple sandblasting/acid-etching and anodization to investigate the effects of such surface characteristics on proliferation and differentiation of osteoblasts in high glucose concentrations. By the way, the effects of high glucose levels on osteoblast functions were tested. METHODS: MC3T3-E1 cells cultured on sand-blasted and acid-etched (SLA) surface and nano-modified SLA (NMSLA) surface were subjected to normal serum (NS) and diabetic serum (DS), respectively. The surface characteristics were evaluated by scanning electron microscopy. Cell proliferation was assessed using MTT assay. The levels of alkaline phosphatase (ALP) activity and mineralization were measured and compared. Real-time polymerase chain reaction was applied to detect the expression levels of osteogenic genes. RESULTS: NMSLA significantly increased cell proliferation at time points ranging from 3 to 7 days under both serums. Cells cultured on NMSLA surfaces displayed significantly higher ALP activities and mineralization. The expression levels of Runx2 (indicates runt-related protein 2), collagen I (COL1), and osteocalcin (OCN) were notably increased on NMSLA surface compared with SLA surface. Moreover, we found that high glucose increased osteoblast proliferation but decreased differentiation of osteoblast slightly. CONCLUSION: The hierarchical micro/nano-structured titanium surface has a favorable biocompatibility on simultaneously improving osteoblast proliferation and differentiation in diabetic serum.

A rare case of juvenile localised scleroderma with intra-oral and dental involvement.

Juvenile localised scleroderma is a rare childhood disorder of the immune system and connective tissue with unknown etiology. There are different types of localised scleroderma, including linear scleroderma (where the lesion appears as a line or streak) and plaque or circumscribed morphea (where the lesion appears as a roundish lesion). The present report describes the case of a 10-year-old girl, who presented with a history of linear scleroderma with nose, oral and dental involvement, and outlines the diagnosis of the case based on the clinical presentation, pathology, X-ray and cone beam computed tomography images. The treatment strategy that was selected for the patient is additionally discussed.

[Comparative analysis of 6 kinds of bacteria in the subgingival plaque in different types of patients with periodontal diseases].

PURPOSE: To detect the existence of Aa,Pg,Tf,Cr,Ec and Pn in the subgingival plaque, and determine their relationships among different types of periodontal diseases. METHODS: Dental plaques from 120 subjects were sampled, including 40 volunteers with health periodontal status(Group A) , forty patients with dental plaque-induced gingival diseases(Group B) and 40 patients with moderate or severe chronic periodontitis (Group C) . These samples were detected based on bacterial composition using the terminal restriction fragment length polymorphism of 16S rRNA genes by multiple-polymerase chain reaction. The data was analysed with SPSS 13.0 software package for Chi-square test. RESULTS: The detection rate of Pn, Cr and Pg had significant differences between group A and B. The detection rate of Ec, Cr, Pg, Aa and Tf had significant differences between group C and B. The detection rate of Ec, Pn, Cr, Pg, Aa and Tf had significant differences between group A and C. CONCLUSIONS: The rate of Ec, Pn, Cr, Pg and Tf detected in moderate or patients with moderate or severe chronic periodontitis are significantly higher than that in healthy subjects, indicating that these bacteria have certain correlation with chronic periodontitis. The rate of Ec, Cr, Pg and Tf detected in severe chronic periodontitis are significantly higher than that in dental-induced gingivitis, suggesting their close relationship with the progress of periodontal disease.

Potential mechanism for osseointegration of dental implants in Zucker diabetic fatty rats.

Our aim was to investigate the impact of diabetes mellitus and different durations of glycaemic control on early osseointegration of dental implants, and to explore possible mechanisms by measuring the expression of integrin α5ß1 and fibronectin in bone around the implant. We divided 33 male Zucker diabetic fatty (ZDF) rats aged 3 months into 3 groups. The first group comprised diabetic rats with dental implants (controls); the second group was treated with insulin and implants were placed simultaneously (exenatide alone group); and the third group was treated with insulin until the serum glucose was at a constant concentration (< 16 mmol/L), and implants were then inserted (exenatide+normal glucose group). Rats were killed 7, 14, 30, and 60 days after implants had been inserted. The expression of integrin α5ß1 and fibronectin in bone around the implants was detected by immunohistochemical analysis in each group. The expression in the exenatide+normal glucose group was stronger than in the other 2 groups. Fourteen days after implantation, expression of integrin α5ß1 in the exenatide alone group was significantly stronger than that in the control group (p=0.027), and 60 days after implantation the expression of fibronectin in the exenatide alone group was also significantly stronger than that among the controls (p=0.001). Both fibronectin and integrin α5ß1 participate in the adhesion of osteoblasts and act as signals at the bone/implant interface. Diabetes interferes with the osseointegration of implants by deferring expression of fibronectin and integrin α5ß1.

Different concentrations of glucose regulate proliferation and osteogenic differentiation of osteoblasts via the PI3 kinase/Akt pathway.

INTRODUCTION: The aim of this study was to investigate the effect of high glucose levels on proliferation and osteogenic ability in MC3T3-E1 osteoblastic cell line and to explore the regulatory mechanism of PI3 kinase (PI3K)/Akt signaling pathway. METHODS: The cultures were divided into 8 treatment groups: 4 concentrations of glucose (5.5, 15.5, 25.5, and 35.5 mM) with or without LY294002. Cell proliferation, alkaline phosphatase (ALP) assay, alizarin red staining of mineralized nodule, osteogenic genes, and P-AKT expression were analyzed. RESULTS: Cell proliferation, ALP activity, mineralization, osteogenic genes (RUNX2, OSX, OPN, OCN) and P-AKT expression in MC3T3-E1 cells were increased, whereas the glucose concentration changed from 5.5 to 15.5 mM. However, when the glucose concentrations continue to increase from 25.5 to 35.5 mM, the proliferation and osteogenic ability in MC3T3-E1 cells were gradually declined. Furthermore, these effects were significantly inhibited by PI3K/Akt inhibitor LY294002 at a glucose concentration of 15.5 mM, which was the optimum. CONCLUSIONS: Appropriate high glucose concentration (15.5 mM) can increase osteogenic differentiation by activating PI3K/Akt pathway in MC3T3-E1 cells, but exorbitant high glucose concentrations (25.5 and 35.5 mM) inhibited the biomineralization process. Findings indicated that PI3K/Akt pathway plays an important role in the physiological process of MC3T3-E1 cells.

Different effects of Porphyromonas gingivalis lipopolysaccharide and TLR2 agonist Pam3CSK4 on the adhesion molecules expression in endothelial cells.

Recent researches suggest an association between periodontitis and cardiovascular disease. Periodontopathic bacteria and/or their component might play a role in the development of atherosclerotic lesions. In the present study, we investigated in vitro the effect of Porphyromonas gingivalis lipopolysaccharide (LPS) on the expression of adhesion molecules in human umbilical vein endothelial cells (HUVECs) as well as its effect on Escherichia coli LPS-induced response. The effect of P. gingivalis LPS was compared with that of toll-like receptor 2 agonist synthetic triacylated lipoprotein Pam3-Cys-Ser-(Lys)4 (Pam3CSK4). Gene and protein expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin were measured using RT-PCR and flow cytometry, respectively. P. gingivalis LPS stimulated the expression levels of all adhesion molecules in a dose-dependent manner. However, the response of HUVECs to P. gingivalis LPS was markedly lower than that to E. coli LPS. Moreover, P. gingivalis LPS attenuated E. coli LPS-induced responses when HUVECs were simultaneously stimulated with both kinds of LPS. Treatment with Pam3CSK4 resulted in a minor increase of adhesion molecule expression and did not diminish E. coli LPS-induced responses. Our data suggest that P. gingivalis LPS induces in vitro the expression of adhesion molecules in endothelial cells, which might promote atherogenesis. Qualitatively different responses of HUVECs to P. gingivalis LPS and Pam3CSK4 suggest that besides TLR2 other signaling pathways might be involved in the effects of P. gingivalis LPS.

Total Antioxidant Capacity and Total Oxidant Status in Saliva of Periodontitis Patients in Relation to Bacterial Load.

The detection of salivary biomarkers has a potential application in early diagnosis and monitoring of periodontal inflammation. However, searching sensitive salivary biomarkers for periodontitis is still ongoing. Oxidative stress is supposed to play an important role in periodontitis progression and tissue destruction. In this cross-sectional study, we investigated total antioxidant capacity (TAC) and total oxidant status (TOS) in saliva of periodontitis patients compared to healthy controls and their relationship with periodontopathic bacteria and periodontal disease severity. Unstimulated saliva was collected from 45 patients with generalized severe periodontitis and 37 healthy individuals and the TAC/TOS were measured. In addition, salivary levels of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, and Fusobacterium nucleatum in saliva were measured. Salivary TAC was lower in periodontitis patients compared to healthy controls. Moreover, a significant negative correlation of salivary TAC with clinical attachment loss was observed in periodontitis patients. No significant difference in the salivary TOS was observed between periodontitis patients and healthy controls. Bacterial load was enhanced in periodontitis patients and exhibited correlation with periodontal disease severity but not with salivary TAC/TOS. Our data suggest that changes in antioxidant capacity in periodontitis patients are not associated with increased bacterial load and are probably due to a dysregulated immune response.

[Role of extracellular signal-regulated kinase in osteoblast differentiation on roughened titanium surfaces].

PURPOSE: The purpose of this study was to evaluate the effect of sand-blasted and acid-etched titanium surface on MC3T3-E1 murine pre-osteoblast cell differentiation, and investigate the pathway of regulating osteogenic differentiation of MC3T3-E1 cells on sand-blasted and acid-etched titanium surface in order to elucidate the regulatory mechanisms of surface roughness on osteoblastic differentiation. METHODS: The characteristic of PT polished titanium (PT), sand-blasted and acid-etched (SLA) titanium surface were examined by scanning electron microscopy (SEM). Real-time PCR was applied to detect the expression of osteogenic genes including Runx2, OSX, OCN and OPN of the MC3T3-E1 cells cultured on the 2 groups of substrates.ERK1/2 activities in MC3T3-T1 cells were measured by Western blot on SLA surface. The data was analyzed using SAS9.0 software package. RESULTS: The result of SEM observation showed that the PT surface was turned titanium surfaces with the mean peak to valley roughness (Ra) of 0.2 µm and the corresponding Ra value of SLA was 3.2 µm. The expression levels of Runx2, OSX, OPN and OCN were significantly higher and the cell proliferation was significantly lower on SLA surfaces than on PT surfaces (P<0.05). The expression levels of Runx2, OSX, OPN and OCN were up-regulated by the effect of SLA surface with PD98095. Compared with PT surface, ERK1/2 phosphorylation was continuously inhibited by SLA. Moreover, PT surfaces treated with PD98095 and SLA surfaces without PD98095 both demonstrated reduced ERK1/2 phosphorylation of the cells and the inhibitive effect of SLA surfaces was milder than that of PD98095. CONCLUSIONS: Surface roughness is an important factor that determines osteoblast behaviors. Surface roughness of titanium substrates seems to enhance the osteoblastic differentiation of MC3T3-E1 cells and the enhancing effect of surface roughness on cell differentiation may be mediated by suppressing the activity of ERK1/2 pathway.

Differentiation of rabbit bone mesenchymal stem cells into endothelial cells in vitro and promotion of defective bone regeneration in vivo.

Tissue engineering strategies often fail to regenerate bones because of inadequate vascularization, especially in the reconstruction of large segmental bone defects. Large volumes of vascular endothelial cells (ECs) that functionally interact with osteoblasts during osteogenesis are difficult to obtain. In this study, we simulated bone healing by co-culturing differentiated ECs and mesenchymal stem cells (MSCs) either on a culture plate or on a polylactide glycolic acid (PLGA) scaffold in vitro. We also evaluated the effect of osteogenesis in repairing rabbit mandible defects in vivo. In this study, MSCs were separated from rabbit as the seed cells. After passage, the MSCs were cultured in an EC-conditioned medium to differentiate into ECs. Immunohistochemical staining analysis with CD34 showed that the induced cells had the characteristics of ECs and MSC. The induced ECs were co-cultured in vitro, and the induction of MSCs to osteoblast served as the control. Alkaline phosphatase (ALP) and alizarin red (AZR) staining experiments were performed, and the Coomassie brilliant blue total protein and ALP activity were measured. The MSCs proliferated and differentiated into osteoblast-like cells through direct contact between the derived ECs and MSCs. The co-cultured cells were seeded on PLGA scaffold to repair 1 cm mandible defects in the rabbit. The effectiveness of the repairs was assessed through soft X-ray and histological analyses. The main findings indicated that MSCs survived well on the scaffold and that the scaffold is biocompatible and noncytotoxic. The results demonstrated that the co-cultured MSC-derived ECs improved MSC osteogenesis and promoted new bone formation. This study may serve as a basis for the use of in vitro co-culturing techniques as an improvisation to bone tissue engineering for the repair of large bone defects.

Escin inhibits lipopolysaccharide-induced inflammation in human periodontal ligament cells.

Periodontitis is a chronic inflammatory disease associated with gram-negative subgingival microflora infection. Accumulating experimental evidence suggests that escin exerts anti-inflammatory and anti-edematous effects. This study was designed to investigate the in vitro effects of escin on the inflammatory reaction of human periodontal ligament cells (hPDLs). hPDLs were stimulated with lipopolysaccharide (LPS). The cells were treated with various concentrations of escin. The viability of hPDLs was evaluated using the MTT method. The expression of Toll-like receptor 2 (TLR2) in hPDLs and the levels of IL-1ß, TNF-α and IL-6 in the supernatant were measured. Escin significantly attenuated LPS-induced cytotoxicity in a concentration-dependent manner in hPDLs. Treatment with escin partly blocked the expression of TLR2. Escin also lowered the increase of pro-inflammatory cytokines (IL-1ß, TNF-α and IL-6) induced by LPS. The present findings show that escin exerts a protective effect against LPS-induced inflammation in hPDLs. It was also shown that escin is a promising medicine for the treatment of periodontitis.

Effect of simvastatin on the osteogenetic behavior of alveolar osteoblasts and periodontal ligament cells.

Statins are routinely used in the clinic as cholesterol lowering drugs, but recently they were reported to also have anabolic effects on bone tissue. Since regeneration of alveolar bone is one of the primary aims of periodontal treatment, in the present study we investigated the effects of simvastatin, a lipophilic statin, on primary alveolar osteoblasts (AOBs) and periodontal ligament cells (PDLs) in vitro. The effect of simvastatin (1-100 nM) on the cells proliferation/viability after 24, 48, and 72 h stimulation was measured using 3,4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT)-assay. The alkaline phosphatase (ALP) activity was measured after stimulation with simvastatin using specific colorimetric assay. Finally, the mRNA expression levels of osteocalcin (OC), receptor activator of NF-κB ligand (RANKL), and osteoprotegerin (OPG) were measured by real-time PCR. The proliferation/viability of AOBs was significantly decreased by all simvastatin concentrations after 72 h stimulation. The proliferation/viability of PDLs was not influenced by simvastatin. ALP activity of AOBs and PDLs was increased by 1 and 100 nM simvastatin, respectively. Simvastatin induced a dose-dependent increase in OC mRNA expression of AOBs and did not influence that in PDLs. RANKL expression of AOBs was increased at all tested simvastatin concentrations and that in PDLs was increased by higher simvastatin concentrations (10-100 nM). Finally, the expression of OPG in AOBs and PDLs was stimulated by 1-10 and 100 nM simvastatin, respectively. Simvastatin seems to slightly increase the expression of osteogenic markers in AOBs and PDLs, indicating its ability to influence alveolar bone formation and periodontal regeneration.

[Effect of simvastatin on the function of MG63 cell line].

PURPOSE: To investigate the effect of simvastatin on the function of MG63 cell line. METHODS: The function of proliferation, osteoprotegerin (OPG) and osteocalcin (OC) gene expression, and migration of MG63 cells were detected with MTT, fluorescent real-time PCR and micro chemotaxis, respectively. The data was analyzed using ANOVA followed by Tukey test with SPSS14.0 software package. RESULTS: 10-9mol/L and 10-8mol/L concentrations of simvastatin slightly promoted MG63 cell proliferation; 10-7mol/L and 10-6mol/L concentrations of simvastatin greatly enhanced the OPG and OC mRNA expression; All concentrations of simvastatin had an inhibitory effect on MG63 cell migration. CONCLUSION: Simvastatin could promote bone defect regeneration by enhancing the bone-related genes expression.

[Effect of simvastatin on residual ridge resorption after tooth extraction].

OBJECTIVE: To observe the effect of simvastatin carried by poly (lactide-co-glycolide) (PLGA) on residual ridge resorption following tooth extraction. METHODS: Sixty male Wistar rats were divided into experimental groups and control groups (30 rats/group). PLGA was immediately implanted with or without simvastatin into extraction sockets of the mandibular incisors. Soft X-ray photography, bone mineral density (BMD) and histopathologic study were conducted at 7, 14, 28, 56, and 84 days after implantation. RESULTS: The relative length values of residual alveolar ridge of the experimental groups were greater than those of the controls at 14, 28, 56, and 84 days after implantation, and there was a significant difference between the experimental and control groups (P < 0.05). The BMD of the specific region was higher in the experimental groups [(7.101 +/- 0.025), (7.178 +/- 0.039), and (7.162 +/- 0.052) g/cm(2)] than that in the control groups [(7.074 +/- 0.014), (7.117 +/- 0.012), and (7.059 +/- 0.037) g/cm(2)] (P < 0.05) after 28, 56, and 84 days. Light microscopy showed that bone formation rate and quality of the experimental group were better than those in the control group at the same time. CONCLUSIONS: Simvastatin carried by PLGA could induce bone formation of tooth socket. Local application of simvastatin would be potential to preserve the length and bone volume of alveolar ridge after tooth extraction.