Hsien-Chung Chiu

Three‐dimensional analysis of the root morphology of mandibular first molars with distolingual roots

AIM To determine the prevalence of distolingual roots in mandibular first molar teeth in Taiwanese Han Chinese, and its impact on root morphology. METHODOLOGY The presence of distolingual roots in 375 subjects (521 molars) were assessed from 624 patients attending the dental clinics of medical centres around Taiwan island from August 2004 to April 2007 using computed tomography. The following observations were made: (i) numbers of roots and canals, (ii) mesial and distal root types and (iii) levels of furca in the molars presence or absence of distolingual root. RESULTS The mean age of the subject was 45; 43% were women. Among all the examined molars, 56%, 27% and 18% were two-, three- and four-rooted, respectively. Two per cent, 72% and 26% of molars had two, three and four canals, respectively. All of the four-rooted molars had four canals, but all of the molars with four canals varied in the number of roots. All molars with distolingual roots had two mesial canals. Bilateral consistency in terms of distolingual root, root canal number, root number and root type was observed in subjects with bilateral molars. In molars with distolingual roots, a higher prevalence of two mesial roots and a shorter mesial root trunk were observed than in teeth without distolingual roots. CONCLUSIONS A distolingual root was found in 22% of molars and in 24% of the subjects examined. Most subjects with a distolingual root had them bilaterally. The presence of a distolingual root was associated with variation in the root morphology, including the furcation level, the root type and the number of roots and canals.

Cyclosporine A inhibits the expression of membrane type-I matrix metalloproteinase in gingiva.

BACKGROUND AND OBJECTIVE Membrane type-I matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase-2 (TIMP-2) regulate the activation of MMP-2; however, their roles in the activation of MMP-2 in gingiva during treatment with cyclosporine A are still unknown. Therefore, the expressions of membrane type-I MMP and TIMP-2, as well as MMP-2, in gingivae upon treatment with cyclosporine A were examined in vivo and in vitro. MATERIAL AND METHODS Thirty-four rats were divided into two groups after edentulous ridges were established. The experimental group received 30 mg/kg/d of cyclosporine A and the control group received vehicle. At the end of the experimental period, the rats were killed, the gingivae were obtained and the expression of mRNA and protein of membrane type-I MMP, TIMP-2 and MMP-2 in gingiva were examined using real-time polymerase chain reaction and immunohistochemistry. In human gingival fibroblasts, the activity of MMP-2 and the expression of MMP-2, membrane type-I MMP and TIMP-2 mRNAs were examined (using zymography and reverse transcription-polymerase chain reaction, respectively) after treatment with cyclosporine A. RESULTS In gingivae of rats, cyclosporine A significantly decreased the expression of mRNA and protein of membrane type-I MMP, but not of TIMP-2. The expression of MMP-2 mRNA was unaffected but the expression of MMP-2 protein showed a significant decrease upon treatment with cyclosporine A. In fibroblast culture medium, the presence of cyclosporine A induced a decrease in MMP-2 activity in a dose-dependent manner. The expression of MMP-2, membrane type-I MMP and TIMP-2 mRNAs in fibroblasts was not significantly affected by cyclosporine A; however, in fibroblasts the ratio of mRNA expression of membrane type-I MMP to that of TIMP-2 decreased as the cyclosporine A dose was increased. CONCLUSION Cyclosporine A inhibits the expression of membrane type-I MMP in gingiva and it may further reduce the activation of MMP-2.

Effect of high glucose, Porphyromonas gingivalis lipopolysaccharide and advanced glycation end-products on production of interleukin-6/-8 by gingival fibroblasts.

BACKGROUND AND OBJECTIVE It is known that chronic periodontal infection can magnify the cytokine responses in patients with diabetes. Hyperglycemia increases the proinflammatory status, including the levels of advanced glycation end-products (AGEs), in patients with periodontitis. However, whether AGEs have additional effects on the production of those proinflammatory cytokines in diabetic patients with periodontitis is still unknown. To examine in vitro the effect of hyperglycemia and AGEs on the amounts of interleukin (IL)-6 and IL-8 produced in periodontally infected gingiva, human gingival fibroblasts (HGFs) were stimulated with glucose, AGE-modified bovine serum albumin (AGE-BSA) and Porphyromonas gingivalis LPS in the present study. MATERIAL AND METHODS Primary culture of HGFs was incubated with various concentrations of AGE-BSA (0, 50, 100 and 200 μg/mL) and LPS (0, 10, 100 or 1000 ng/mL) at two different glucose concentrations - normal glucose (5 mm) and high glucose (25 mm). The amounts of IL-6 and IL-8 produced by HGFs were evaluated using ELISA. Expression of the AGE receptor on HGFs was determined by flow cytometry. RESULTS High glucose stimulated a significant increase in the production of IL-6 and IL-8 by HGFs compared with normal glucose. This enhanced production of IL-6 and IL-8 could also be observed in the presence of LPS and/or AGE-BSA. When both LPS and AGE-BSA were present, especially at high concentrations (≥ 500 μg/mL of LPS and ≥ 25 μg/mL of AGE-BSA), a synergistic effect on IL-8 production was found in the high-glucose condition. CONCLUSIONS A synergistic effect of the production of IL-8 could be induced in HGFs with the combination of high glucose, LPS and AGEs.

The effects of diallyl sulfide upon Porphyromonas gingivalis lipopolysaccharide stimulated proinflammatory cytokine expressions and nuclear factor-kappa B activation in human gingival fibroblasts.

BACKGROUND AND OBJECTIVE Diallyl sulfide (DAS), a flavor compound from garlic, has varied potential therapeutic activities. Periodontitis is a disease that develops because of host-mediated inflammation to periodontal pathogens. In this study, the effects of DAS on the common proinflammatory cytokines and nuclear factor-kappa B (NF-κB) in human gingival fibroblasts (HGFs) being stimulated with lipopolysaccharide from Porphyromonas gingivalis, a potent periodontal pathogen, were evaluated. MATERIAL AND METHODS Cytotoxicities of DAS and lipopolysaccharide on HGFs were measured with MTS assay. The mRNA and protein expressions of proinflammatory cytokines, including interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α, from the HGFs treated with lipopolysaccharide with and without DAS were examined with reverse transcription-polymerase chain reaction and immunocytochemistry, respectively. In addition, the activation and nuclear translocation of NF-κB with and without DAS were compared. RESULTS DAS and lipopolysaccharide treatments within 3 mm and 10 μg/mL, respectively, did not affect the survival rate of HGFs. Lipopolysaccharide (1 μg/mL) significantly increased the mRNA expressions of IL-1β, IL-6 and TNF-α; however, DAS (1 mm) inhibited these expressions. The protein expressions of TNF-α, IL-1β, as well as the NF-κB nuclear translocation were increased after lipopolysaccharide treatment, but decreased when there was a DAS pretreatment. CONCLUSION DAS diminished P. gingivalis lipopolysaccharide-stimulated cytokine expression and NF-κB activation in HGFs; we therefore suggest DAS may be beneficial on periodontal inflammation.

Association of CCL5 and CCR5 Gene Polymorphisms With Periodontitis in Taiwanese

BACKGROUND It has been suggested that genetic factors may predispose individuals to periodontal diseases. The present case-control study aims to test whether the -403 single nucleotide polymorphism of chemokine ligand 5 (CCL5-403) and the 32-bp deletion of CCR5 (CCR5Δ32) polymorphisms are associated with susceptibility to chronic and aggressive periodontitis. METHODS Taiwanese participants (N = 213) were grouped into control group (CG), generalized aggressive periodontitis (GAgP), or chronic periodontitis (CP) groups. DNA samples were obtained from peripheral blood. CCL5-403, evaluated by polymerase chain reaction-restriction fragment length polymorphism, and CCR5Δ32, evaluated by polymerase chain reaction, were compared among the three groups. RESULTS There was a significant association between type of periodontitis and having allele A or G in the CCL5-403 polymorphism. GAgP patients were 3.7 times more likely than CP patients and 2.0 times more likely than CG patients to have allele A, instead of allele G, in CCL5-403. GAgP patients were 3.1 times more likely than CG patients to have AG versus GG genotype. GAgP patients were also 5.0 and 19.8 times more likely than CP patients to have AG and AA genotypes, respectively, compared to GG. For the CCR5Δ32 polymorphism, no association was found between the type of periodontitis and having different genotype or allele distributions among GAgP, CP, or CG patients. CONCLUSION The single nucleotide polymorphism of CCL5-403 G substitution by A may play a role in AgP; however, the CCR5Δ32 polymorphism may not.

Association between History of Dental Amalgam Fillings and Risk of Parkinson's Disease: A Population-Based Retrospective Cohort Study in Taiwan.

The impact of dental amalgam on the development of Parkinson’s disease (PD) is still uncertain, although a positive association between dental amalgam and PD has been found in a few case-control studies. The patients with amalgam fillings restored between 2000 and 2008 were identified by using the National Health Insurance Research Database (NHIRD) in Taiwan. The same number of patients who had no new amalgam filling restored was matched by sex, age, and treatment date. Both cohorts were followed up from the treatment date until the date of diagnosis of PD, death, or the end of the year 2008. The individuals who received amalgam fillings had a significantly higher risk of PD afterward (adjusted hazard ratio [HR]=1.583, 95% confidence interval [CI]=1.122–2.234, p=0.0089) than those who did not. In the individuals who received amalgam fillings, being diagnosed with diabetes or hyperlipidemia demonstrated a significantly lower HR of PD occurrence than in the patients without diabetes or hyperlipidemia (HR=0.449, 95% CI=0.254–0.794, p=0.0059; HR=0.445, 95% CI=0.260–0.763, p=0.0032) after adjusting for comorbidities and Charlson-Deyo Comorbidity Index (CCI) scores. Meanwhile, hypertension increased the hazard risk of PD (HR=1.645, 95% CI=1.098–2.464, p=0.0159). The patients exposed to dental amalgam fillings were 1.583 times more likely to have PD afterward compared to their non-exposed counterparts after adjusting for comorbidities and CCI scores.

Gelatinases and Extracellular Matrix Metalloproteinase Inducer Are Associated With Cyclosporin-A-Induced Attenuation of Periodontal Degradation in Rats

BACKGROUND The present study aims to examine the inhibitory effect of cyclosporin-A (CsA) on periodontal breakdown and to further explore the correlations of CsA-induced attenuation of periodontal bone loss with the expressions of gelatinases (i.e., matrix metalloproteinase [MMP]-2 and MMP-9) and extracellular matrix metalloproteinase inducer (EMMPRIN). METHODS Forty Sprague-Dawley rats were randomly divided into four groups: 1) control; 2) CsA; 3) ligature (Lig); and 4) ligature plus CsA (Lig + CsA). The CsA group received 10 mg ⋅ Kg(-1) ⋅ d(-1) CsA for 8 days. The Lig group received silk ligature on selected molars. The Lig + CsA group received silk ligature and CsA treatment. The inhibitory effects of CsA on the ligature-induced periodontal breakdown was examined with microcomputed tomography (micro-CT) and histometric analyses to analyze the amount of attachment loss, crestal bone loss, connective tissue attachment, and the surface area with inflammatory cell infiltration. The effects of CsA on ligature-induced expressions of gelatinases and EMMPRIN in gingival tissues were examined with Western blotting and zymography, respectively. RESULTS By micro-CT and histology, the Lig + CsA group had significantly more periodontal breakdown than the control and CsA groups but less periodontal breakdown than the Lig group. Consistent results were found for the expressions of gelatinases and EMMPRIN among the groups demonstrating that the Lig + CsA group had significantly less gingival protein expression of gelatinases and EMMPRIN than the Lig group. CONCLUSIONS CsA inhibited the expressions of gelatinase MMPs and EMMPRIN and partially prevented the periodontal breakdown in ligature-induced experimental periodontitis. The CsA-induced attenuation of periodontal bone loss was strongly correlated positively with the expressions of MMP-2, MMP-9, and EMMPRIN in gingiva.

Up‐regulation of retinoblastoma protein phosphorylation in gingiva after cyclosporine A treatment: an in vivo and in vitro study

BACKGROUND AND OBJECTIVE Cyclosporine A can induce gingival cell proliferation; however, the precise molecular regulation of the proliferation is uncertain. Therefore, this study was carried out to examine, in vivo and in vitro, the expression of genes and proteins associated with gingival cell proliferation after treatment with cyclosporine A. MATERIAL AND METHODS Forty Sprague Dawley rats with right maxillary posterior edentulous gingivae were assigned to a cyclosporine A group (30 mg/kg daily of cyclosporine A, administered orally) or a control group (administered mineral oil only). The animals were killed 4 wk after treatment. The edentulous gingivae were dissected out and analyzed for the expression of proliferating cell nuclear antigen (PCNA), cyclin D1, cyclin-dependent kinase 4 (CDK4) and retinoblastoma protein (Rb1) mRNA and/or protein, and phosphorylated Rb1 (pRb1), by real-time RT-PCR or immunohistochemistry. In human gingival fibroblast (HGF) cultures, the expression of PCNA, CDK4, cyclin D1 and Rb1 proteins and Rb1 phosphorylation were determined by western blotting after cyclosporine A treatment (0-10(4) ng/mL). RESULTS Proliferating cell nuclear antigen and cyclin D1 mRNAs (Pcna and Ccnd1, respectively) were expressed more strongly in the gingivae of cyclosporine A-treated animals than in the gingivae of the controls. Immunohistochemical analyses showed that a greater number of gingival cells stained positive for cyclin D1, CDK4 and pRb1 in the cyclosporine A group than in the control group. Increased expression of cyclin D1, CDK4 and PCNA proteins was observed in HGFs after cyclosporine A treatment. The phosphorylation of Rb1 was enhanced in HGFs after treatment with cyclosporine A at concentrations of 10(2)-10(3) ng/mL. CONCLUSION The increases in cyclin D1, PCNA and CDK4, together with the enhanced phosphorylation of Rb1, suggest that cyclosporine A promotes cell-cycle progression through the G(1)/S transition in the gingiva.

Role of Shh and TGF in cyclosporine-enhanced expression of collagen and α-SMA by gingival fibroblast.

OBJECTIVE Cyclosporine-A (CsA)-induced gingival overgrowth may arise from an alteration in stoma matrix homeostasis. Sonic hedgehog (Shh) plays a key role during embryogenic development and fibrotic progression, and may be involved in CsA-altered gingival matrix homeostasis. METHODS Using the reverse transcription-polymerase chain reaction and Western blot analysis, we investigated the mRNA and protein expressions of Shh, type 1 collagen (COL1), alpha-smooth muscle actin (α-SMA) and transforming growth factor-beta (TGF-β) in human gingival fibroblasts after CsA treatments. The effect of Shh on CsA-induced alterations was further evaluated by the extra-supplement or inhibition of Shh or TGF-β. RESULTS Cyclosporine-A enhanced COL1, α-SMA, Shh and TGF-β expressions in human gingival fibroblasts. The exogenous Shh/TGF-β augmented the expression of COL1 and α-SMA, and the Shh/TGF-β inhibition suppressed the CsA-enhanced COL1 and α-SMA expressions. Moreover, Shh mRNA and protein expressions increased if extra-supplementing the exogenous TGF-β, whereas the CsA-upregulated Shh was mitigated by the TGF-β pathway inhibitor. However, neither exogenous Shh nor the Shh pathway inhibitor alters TGF-β expression or CsA-up-regulated TGF-β expression. CONCLUSIONS Shh, regulated by TGF-β, mediates CsA-altered gingival matrix homeostasis.

Role of Transforming Growth Factor-beta1 in Cyclosporine-Induced Epithelial-to-Mesenchymal Transition in Gingival Epithelium

BACKGROUND It has been proposed that cyclosporin A (CsA) may induce epithelial-to-mesenchymal transition (EMT) in gingiva. The aims of the present study are to confirm the notion that EMT occurs in human gingival epithelial (hGE) cells after CsA treatment and to investigate the role of transforming growth factor beta1 (TGF-β1) on this CsA-induced EMT. METHODS The effects of CsA, with and without TGF-β1 inhibitor, on the morphologic changes of primary culture of hGE cells were examined in vitro. The changes of protein and messenger RNA (mRNA) expressions of two EMT markers (E-cadherin and alpha-smooth muscle actin) in the hGE cells after CsA treatment with and without TGF-β1 inhibitor were evaluated with immunocytochemistry and real-time polymerase chain reaction. RESULTS The epithelial cells became spindle-like, elongated, and disassociated from neighboring cells and lost their original cobblestone monolayer pattern when CsA was added. However, the epithelial cells stayed in their original cobblestone morphology with treatment of TGF-β1 inhibitor on top of the CsA treatment. When CsA was given, the protein and mRNA expressions of E-cadherin and α-SMA were significantly altered, and these alterations were significantly reversed with pretreatment of TGF-β1 inhibitor. CONCLUSIONS CsA could induce Type 2 EMT in gingiva by changing the morphology of epithelial cells and altering the EMT markers/effectors. The CsA-induced gingival EMT is dependent or at least partially dependent on TGF-β1.