Sze-Kwan Lin

Cytotoxicity of sodium fluoride on human oral mucosal fibroblasts and its mechanisms

Because sodium fluoride (NaF) is widely used for prevention of dental caries, pathobiological effects of NaF were investigated on human oral mucosal fibroblasts. The results showed that NaF was cytotoxic to oral mucosal fibroblasts at concentrations of 4 mmol/L or higher. Exposure of cells to NaF for 2 h also inhibited protein synthesis, cellular ATP level and functional mitochondrial activities in a dose-dependent manner. However, incubation of cells with NaF up to 12 mmol/L for 2 h depleted only 13% of cellular glutathione level. The IC50 of NaF on cellular ATP level was about 5.75 mmol/L. Preincubation of the cells with pyruvate and succinate did not protect cells from NaF-induced ATP depletion. At concentrations of 4 mmol/L, 8 mmol/L and 12 mmol/L, NaF inhibited 31%, 56% and 57% of mitochondrial functions, respectively, after 2 h incubation. No significant inhibition for NaF was found at concentrations lower than 2 mmol/L (40 ppm). These results indicate that NaF can be toxic to oral mucosal fibroblasts in vitro by its inhibition of protein synthesis, mitochondrial function and depletion of cellular ATP. Because of repeated and long-term usage of NaF, more detailed studies should be undertaken to understand its toxic effects in vitro and in vivo.

Collagen gene expression in human dental pulp cell cultures

Pulp cells from human permanent molars were isolated and established in culture; 40% showed positive alkaline phosphatase staining. When incubated with 50 micrograms/ml of ascorbic acid and 10 mM of beta-glycerophosphate, the cells formed a mineralized extracellular matrix; they could thus have the potential to differentiate into odontoblast-like cells in vitro. Collagen synthesis was analysed by SDS interrupted gel electrophoresis, Northern blot and slot blot: the cells produced predominantly (approximately 99%) type I collagen and only trace amount of type III collagen. The ratio of alpha 1 (I) to alpha 2(I) procollagen chains was about 68:32, indicating that no significant amount of collagen type I trimer was synthesized in this system. The ratios of alpha 1(I), alpha 2(I) and alpha 1(III) procollagen mRNAs were about 61:25:1; these were compatible with the ratios of corresponding procollagen alpha chains. In addition, a novel 5.8 kb pro alpha 1(III) mRNA was detected. These observations indicate that collagen synthesis in these cultured pulp cells was regulated at the transcriptional level.

Induction of Dental Pulp Fibroblast Matrix Metalloproteinase–1 and Tissue Inhibitor of Metalloproteinase–1 Gene Expression by Interleukin–1α and Tumor Necrosis Factor–α Through a Prostaglandin–Dependent Pathway

Matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) are involved in the degradation of extracellular matrix in many inflammatory diseases. Little is known regarding the expression of these mediators in dental pulp fibroblasts. The effects of proinflammatory cytokines (interleukin (IL)-1alpha and tumor necrosis factor-alpha (TNF-alpha)) and prostaglandin E2 (PGE2) on pulp fibroblast MMP-1 and TIMP-1 gene expression were investigated. Northern hybridization showed that IL-1alpha and TNF-alpha induced significant MMP-1 gene expression, with only little effect on TIMP-1 gene. Exogenous PGE2, however, upregulated TIMP-1 mRNA synthesis but not MMP-1. Concomitant addition of IL-1alpha and PGE2 or TNF-alpha and PGE2 suppressed MMP-1 mRNA production, compared with the groups treated with IL-1alpha or TNF-alpha alone. In contrast, PGE2 enhanced the upregulatory effects of TIMP-1 mRNA by IL-1alpha or TNF-alpha. Furthermore, cytokine stimulation of MMP-1 and TIMP-1 gene expressions can be enhanced or blocked by indomethacin, respectively, and reversed by exogenous PGE2. These results suggested that cytokine-stimulated MMP-1 and TIMP-1 gene expression in dental pulp fibroblasts was mediated, at least in part, by a prostaglandin-dependent pathway. The differential regulation of IL-1alpha or TNF-alpha-induced MMP-1 and TIMP-1 mRNA synthesis, as well as the direct upregulation of TIMP-1 gene expression by PGE2, also implied that prostaglandin may serve as a protective mechanism from excessive tissue breakdown during pulpitis.

Epigallocatechin‐3‐gallate diminishes CCL2 expression in human osteoblastic cells via up‐regulation of phosphatidylinositol 3‐Kinase/Akt/Raf‐1 interaction: A potential therapeutic benefit for arthritis

OBJECTIVE To assess the effects of epigallocatechin-3-gallate (EGCG) on oncostatin M (OSM)-induced CCL2 synthesis and the associated signaling pathways in human osteoblastic cells. The therapeutic effect of EGCG on collagen-induced arthritis (CIA) in rats was also studied. METHODS CCL2 and c-Fos messenger RNA expression was analyzed by Northern blotting. The modulating effects of EGCG on the activation of Raf-1, Akt, and phosphatidylinositol 3-kinase (PI 3-kinase) were examined by coimmunoprecipitation, Western blotting, and PI 3-kinase activity assay. Interactions between c-Fos and CCL2 promoter were evaluated by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay. The effect of EGCG on CIA in rats was examined clinically and immunohistochemically. RESULTS EGCG inhibited OSM-stimulated CCL2 expression in primary human osteoblasts and MG-63 cells. In MG-63 cells, EGCG alleviated the OSM-induced phosphorylation of Raf-1 at Ser338 but restored the dephosphorylation of Raf-1 at Ser259. EGCG increased the activity of PI 3-kinase, the level of phosphorylated Akt (Ser473), and binding between Raf-1 and active Akt. EMSA and ChIP assay revealed that EGCG attenuated activator protein 1 (AP-1)-CCL2 promoter interaction, possibly by reducing c-Fos synthesis. Codistribution of CD68+ macrophages and CCL2+ osteoblasts in osteolytic areas was obvious in the CIA model. Administration of EGCG markedly diminished the severity of CIA, macrophage infiltration, and the amount of CCL2-synthesizing osteoblasts. CONCLUSION By stimulating PI 3-kinase activity, EGCG promoted Akt/Raf-1 crosstalk, resulting in decreased AP-1 binding to CCL2 promoter, and finally reduced CCL2 production in osteoblasts. EGCG alleviated the severity of CIA, probably by suppressing CCL2 synthesis in osteoblasts to diminish macrophage infiltration. Our data support the therapeutic potential of EGCG on arthritis.

Nitric oxide promotes infectious bone resorption by enhancing cytokine-stimulated interstitial collagenase synthesis in osteoblasts

This experiment was undertaken to determine the role of macrophage‐derived nitric oxide (NO) in mediating lipopolysaccharide (LPS)‐induced bone resorption by using an in vitro co‐culture system and an in vivo model of infectious bone resorption. Our results demonstrated that LPS stimulated the expression of inducible nitric oxide synthase (iNOS) and tumor necrosis factor (TNF)‐α mRNAs and nitrite synthesis in the J774 mouse macrophage cell line but not in the UMR‐106 (rat) and MC3T3‐E1 (mouse) osteoblast cell lines. Conditioned media (CM) from LPS‐stimulated J774 triggered only low to moderate levels of iNOS mRNAs in MC3T3‐E1 and a trivial effect in UMR‐106. On the other hand, CM induced matrix metalloproteinase‐1 (MMP‐1) gene expression in both osteoblast cell lines. The NOS inhibitor NG‐monomethyl‐L‐arginine (L‐NMMA) did not alter this effect in MC3T3‐E1 and UMR‐106, whereas TNF‐α antibody diminished the CM‐induced MMP‐1 gene expression in both cell lines. Interestingly, SNAP, a NO donor, although by itself is not a MMP‐1 stimulator for UMR‐106, augmented the TNF‐α‐stimulated MMP‐1 mRNA production in UMR‐106. In a J774/UMR‐106 co‐culture system, LPS stimulated significant MMP‐1 gene expression in UMR‐106, and this upregulation was abolished by L‐NMMA and TNF‐α antibodies. Immunohistochemical analysis in a rat model of infectious bone resorption (periapical lesion) showed co‐distributions of iNOS+ macrophages and MMP‐1+ osteoblasts around the osteolytic areas. Administration of L‐NMMA markedly reduced the extent of bone loss and the percentage of MMP‐1‐synthesizing osteoblasts. These data suggest that NO derived from macrophages after LPS stimulation may enhance bone loss by augmenting the cytokine‐induced MMP‐1 production in osteoblasts.

Comparisons of norcantharidin cytotoxic effects on oral cancer cells and normal buccal keratinocytes

Norcantharidin (NCTD) is the demethylated analogue of cantharidin. In this study, multi-parameter assessments of morphological alterations, clonogenic efficiency, cell growth curves, DNA synthesis, and DNA strand break were employed to determine and compare the cytotoxic effects of NCTD on oral cancer KB cell line and normal buccal keratinocytes. The results showed NCTD induced significant cytotoxicity in KB cells after 24 h of exposure. Normal buccal keratinocytes were more resistant to NCTD induced cytotoxicity. The IC(50) of 24 h NCTD treatment for KB and keratinocytes were 15.06 and 216.29 microg/ml, respectively with a keratinocyte/KB selective index of 14.36. Anoikis and membrane blebbing, morphological characterization of apoptosis, were observed in about 90% of KB cells after exposure to 100 microg/ml of NCTD for 24 h compared to about 30% in keratinocytes. In addition, inhibition of colony formation was noted in KB cells even when exposed to low concentration of drug (5 microg/ml) for a short period of time (6 h). NCTD inhibited subsequent cell proliferation in KB but growth of normal keratinocytes was retarded only temporarily. NCTD inhibited DNA synthesis in both KB and normal keratinocytes. However, keratinocytes were more sensitive to DNA synthesis inhibition by low dose of NCTD. Significant DNA strand break was noted in KB cells only after cell viability was reduced to less than 60% of the control. In comparison, normal keratinocytes were resistant to NCTD induced DNA strand break. These results indicated KB cells were more sensitive to NCTD induced cytotoxicity compared to normal keratinocytes. NCTD may be of value in treating oral cancers. The underlying mechanisms of the differential actions of NCTD on these two cell types are worthy of further investigations.

A comparison of the morphological changes after Nd-YAG and CO2 laser irradiation of dentin surfaces.

The purpose of this study was to compare the morphological changes after Nd-YAG and CO2 laser irradiation on dentin surfaces with or without the smear layer. Eighty-one 3-mm-thick dentin specimens collected from the middle third of molar crowns were used. The dentin surfaces were ground to #320, #400, and #600 grit in series to create a smear layer. Half of the specimens were treated with 14% EDTA for 2 min to remove the smear layers. The lasers were applied on each specimen perpendicularly with 1-mm focus distance to the dentin surface for 4 s. The parameters for the Nd-YAG laser were 50 mJ, 100 mJ, and 150 mJ at 10 pps, 20 pps, and 30 pps, and for the CO2 laser were 2 W, 3 W, and 4 W at 5 ms x 20 pps, 10 ms x 10 pps, 20 ms x 20 pps, 50 ms x 2 pps, 100 ms x 2 pps, and 200 ms x 2 pps. The results showed that the Nd-YAG laser caused crater and melting of the dentin surface, especially in dentin specimens with smear layers. The CO2 laser produced extensive cracking lines on dentin surfaces with a smear layer, whereas surface erosion and crater formation were found on specimens without a smear layer. In conclusion, both the laser types and smear layer have a significant influence on the morphological changes of dentin surfaces irradiated by lasers.

Immunolocalization of Macrophages and Transforming Growth Factor-β1 in Induced Rat Periapical Lesions

Apical periodontitis was induced in Wistar rats by exposing the pulp chamber of right mandibular first molars to the oral environment. Animals were killed 0, 5, 10, 15, 20, 30, 60, and 80 days after lesion induction. Microradiographic and automated image analysis showed that the lesions expanded significantly in a time-dependent manner from day 0 to day 20 (0.039 mm2/day, p < 0.05, active phase) and stabilized thereafter (chronic phase). A linear regression test revealed a positive correlation between the numbers of ED-1 positive macrophage per microscopic high power field and the periapical lesion size during the active phase (r = 0.98, p < 0.01). Immunohistochemical studies showed that transforming growth factor-beta 1 positive macrophages distributed around the root apex and areas showing bone resorption during active lesion phase, whereas TGF-beta 1-positive osteoblasts were detected during the chronic stage (days 30, 60, and 80 after pulp exposure). Histologically TGF-beta 1 positive osteoblasts possessed a large, round nucleus as well as an abundant cytoplasm and located in close vicinity to areas exhibiting reparative bone formation. These results suggest that macrophages may play important role(s) in the initiation and development of periapical lesions and TGF-beta 1 may play dual roles in both bone resorption and deposition in induced rat periapical lesions.

Simvastatin alleviates the progression of periapical lesions by modulating autophagy and apoptosis in osteoblasts.

INTRODUCTION Autophagy is a process for recycling intracellular organelles as a survival mechanism. Apoptosis has important biological roles in the pathogenesis of many diseases. This study elucidated the effect of simvastatin on autophagy/apoptosis in MC3T3E1 murine osteoblastic cells and also the significance of this action on the progression of induced rat apical periodontitis. METHODS We examined the H2O2-stimulated expression of LC3-II (an autophagy marker) and poly (adenosine phosphate ribose) polymerase (PARP) fragmentation (an apoptosis marker) in MC3T3E1 by Western analysis. In a rat model of induced apical periodontitis, the relation between disease progression and osteoblastic expression of Beclin-1 (an autophagy marker) and terminal deoxyuridine triphosphate nick end-labeling (an apoptosis marker) was studied by radiographic and immunohistochemistry analyses. RESULTS Western blot showed elevated levels of LC3-II and PARP cleavage after H2O2 treatment. An autophagy inhibitor 3-methyladenine promoted whereas rapamycin (an autophagy enhancer) diminished H2O2-induced PARP cleavage. Simvastatin enhanced H2O2-induced LC3-II formation and simultaneously decreased PARP fragmentation. Radiography and immunohistopathology demonstrated that simvastatin reduced the number of apoptotic osteoblasts and the extension of periapical lesions in rats. The number of Beclin-1-synthesizing osteoblasts also increased markedly after simvastatin treatment. CONCLUSIONS We found a negative relation between autophagy and apoptosis in osteoblastic cells. In addition, simvastatin suppressed apoptosis and enhanced autophagy both in vitro and in vivo. Our data implied that simvastain might alleviate the progression of apical periodontitis by promoting autophagy to protect osteoblasts from turning apoptotic.

Simvastatin as a Novel Strategy To Alleviate Periapical Lesions

Hydroxymethylglutaryl-coenzyme A reductase inhibitors (statins) are widely used cholesterol-lowering agents that also possess anti-inflammatory activities. Cysteine-rich 61 (Cyr61) and CCL2 are potential osteolytic mediators in inflammatory bone diseases. The study assessed the effect of simvastatin on tumor necrosis factor alpha (TNF- alpha)-induced synthesis of Cyr61 and CCL2 in MG-63 human osteoblastic cells. The therapeutic effect of simvastatin on rat apical periodontitis was also examined. The synthesis of Cyr61 in MG-63 was assessed by Western analysis. Expression of CCL2 was examined by an enzyme-linked immunosorbent assay. The effect of simvastatin on induced rat periapical lesion was examined radiographically and immunohistochemically. Western blot showed that TNF-alpha stimulated Cyr61 synthesis in MG-63, whereas simvastatin attenuated this effect in a dose-dependent manner. Simvastatin also reduced the levels of TNF-alpha-induced CCL2, and exogenous Cyr61 restored the inhibitory effects. Radiography and histopathology revealed that the administration of simvastatin markedly diminished the severity of induced rat periapical lesions. The numbers of Cyr61-synthesizing osteoblasts and CD-68-positive macrophages were also decreased. Simvastatin suppresses the progression of apical periodontitis, possibly by diminishing Cyr61 expression in osteoblasts and, subsequently, macrophage chemotaxis into the lesions.