Fluoride reduces the expression of enamel proteins and cytokines in an ameloblast-derived cell line

Objective

To investigate the effects of two different fluoride concentrations on the expression of enamel proteins, alkaline phosphatase (ALP), cytokines and interleukins by an ameloblast-derived cell line.

Methods

Murine ameloblast-derived cells (LS-8), mouse odontogenic epithelia, were exposed to 1 or 5 ppm sodium fluoride (NaF) (0.46 and 2.25 ppm F, respectively) for 1, 3 and 7 days. The effect of NaF on the mRNA expression of enamel proteins was quantified; the secretion of cytokines, and interleukins, and the alkaline phosphatase (ALP) activity, into the cell culture medium was measured and compared to untreated controls. The effect on cell growth after 1- and 3-days in culture was measured using BrdU incorporation.

Results

Fluoride at 2.25 ppm reduced mRNA expression of the structural enamel matrix proteins amelogenin (amel), ameloblastin (ambn), enamelin (enam), and the enamel protease matrix metallopeptidase-20 (MMP-20). Similarly several vascularisation factors (vascular endothelial growth factor (VEGF), monocyte chemoattractant proteins (MCP-1) and interferon inducible protein 10 (IP-10), was also reduced by 2.25 ppm fluoride. ALP activity and proliferation were stimulated by 0.46 ppm fluoride but inhibited by 2.25 ppm fluoride.

Conclusions

These results indicate that fluoride may impact on the expression of structural enamel proteins and the protease responsible for processing these proteins during the secretory stage of amelogenesis and go some way to explaining the mineralization defect that characterises fluorotic enamel.     

High-fluoride acitivates the FasL signalling pathway and leads to damage of ameloblast ultrastructure

ObjectiveHigh fluoride can induce stress-mediated apoptosis and degradation of ameloblasts. Fas ligand (FasL) has been regarded as a key regulator in intracellular responses for stress-induced apoptosis in reproductive or cancerous cell lineages. The objective of this study is to explore the role of FasL in the regulation of ameloblast ultrastructure damage.DesignPrimary ameloblasts were isolated from the molar tooth germ of 4-day-old SD rats. The ameloblasts were incubated with 3.2 mM NaF or nothing. After incubation for different time arranging from 12 h to 72 h, ELISA was used to detected the secretion levels of FasL in the medium. Then at 48 h post treatment, the ameloblast ultrastructure was detected with Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM), and expression of apoptotic proteins and peroxidative enzymes/products were examined. Finally, a specific FasL inhibitor was applied to co-treat the ameloblasts with NaF, and the ameloblast ultrastructure was detected with TEM and SEM.ResultsThe secretion of FasL was notably increased by 3.2 mM NaF treatment, and the increase reached to the peak after incubation for 48 h. High fluoride incubation damaged the ameloblast untrastructure manifesting a series of intracelluar stress responsing cell organelle destruction, and a marked increase in expression of apoptotic genes and oxidative stress. The FasL inhibitor treatment partially mitigated the untrastructure damage caused by high dose NaF.ConlusionHigh-fluoride leads to damage of the ameloblast ultrastructure through paritially acitivating the FasL signalling pathway.     

Indirect caries‐preventive effect of silver diamine fluoride on adjacent dental substrate: A single‐section demineralization study

This study assessed the indirect effect of 38% silver diamine fluoride (SDF) on demineralization of adjacent untreated sound and pre‐demineralized enamel and dentine using a single‐section model for digital transverse microradiography (TMR‐D). Forty‐eight bovine dentine single sections were demineralized, stratified (n = 12) according to integrated mineral loss (ΔZ), and treated with SDF or deionized water (DIW). Each “treated dentine” section was attached between untreated sound and pre‐demineralized enamel or dentine and then subjected to demineralization. ΔZ and lesion depths (LD) of all specimens at baseline, 24 and 48 h demineralization, and after treatment of “treated dentine” were quantified using TMR‐D. Fluoride in the demineralization solution of SDF clusters was determined using an ion‐selective electrode. ΔZ and LD of sound and ΔZ of pre‐demineralized enamel adjacent to SDF‐treated dentine did not increase over time. All untreated dentine demineralized significantly; however, ΔZ of sound dentine adjacent to SDF‐treated specimen was still significantly lower than control. SDF‐treated dentine remineralized and released fluoride even after 48 h. Consistent with clinical findings, when applied only to demineralized teeth in this chemical model, 38% SDF completely inhibited demineralization in adjacent untreated sound enamel. Demineralization prevention was observed to a lesser extent in adjacent pre‐demineralized enamel but not in dentine.     

Genetic variation may explain why females are less susceptible to dental erosion

Not all individuals at risk for dental erosion (DE) display erosive lesions. The prevalence of DE is higher among male subjects. The occurrence of DE may depend on more than just acidic challenge, with genetics possibly playing a role. The aim of this study was to investigate the association of enamel‐formation genes with DE. One premolar and a saliva sample were collected from 90 individuals. Prepared teeth were immersed in 0.01 M HCl (pH 2.2), and enamel loss (μm) was measured using white light interferometry. DNA was extracted from saliva, and 15 single‐nucleotide polymorphisms were analysed. Allele and genotype frequencies were related to the enamel loss of the specimens. Single‐marker and haplotype analyses were performed using sex as a covariate. Mean enamel loss was higher for male donors than for female donors (P = 0.047). Significant associations were found between enamel loss and amelogenin, X‐linked (AMELX), tuftelin 1 (TUFT1), and tuftelin‐interacting protein 11 (TFIP11). Analyses showed significant associations between variation in enamel‐formation genes and a lower susceptibility to DE in female subjects. The results indicate that susceptibility to DE is influenced by genetic variation, and may, in part, explain why some individuals are more susceptible than others to DE, including differences between female subjects and male subjects.     

Aspects of the final phase of enamel formation as evidenced by observations of superficial enamel of human third molars using scanning electron microscopy

ObjectiveEnamel structure reflects ameloblast function. By studying the structure of the superficial enamel, information about ameloblast function toward the end of the secretory stage may be obtained.DesignThe superficial enamel in midcoronal areas of acid-etched facio-lingual sections from human third molars was studied in the scanning electron microscope (SEM).ResultsA great variation was observed in occurrence of prism-free enamel. Prism-free enamel dominated in 40% (mandibular) and 47% (maxillary) of observed areas and had a mean thickness of about 30 μm. Striations in the prism-free enamel had an interstriae distance of about 3.3–3.8 μm. The angle between prisms and enamel surface was about 60°, between prisms and Retzius lines about 45° and between Retzius lines and enamel surface about 15°. The distances between regularly occurring Retzius lines and between striations in the prism-free enamel both tended to decrease toward the enamel surface. Prisms could change direction as they approached the enamel surface, mostly in cervical direction. Where Retzius lines curved and converged occlusally, prisms tended to deviate in an occlusal direction.ConclusionsJudged from the incremental lines and occurrence of prism-free enamel, ameloblasts slow down and tend to lose their Tomes’ process as they approach the end of secretion. The crystals of prism-free enamel belong to the same system as the interprism crystals of prismatic enamel. A method, based on the disposition of fine incremental lines, is suggested for evaluation of ameloblast dynamics in the last stage of enamel secretion.     

低氧对人牙周膜细胞骨向分化能力的影响

目的 研究低氧对人牙周膜细胞（PDLC）的碱性磷酸酶（ALP）活性及成骨相关基因表达的影响.方法 采用组织块法体外分离培养人PDLC;取第3 ～ 5 代细胞分别在常氧和低氧条件下培养后检测其ALP 活性;并通过荧光定量聚合酶链反应（PCR）观察低氧处理后人PDLC 中成骨相关基因的表达变化.结果 低氧12、24、36、48 h 组ALP 活性均比常氧组高;其中低氧48 h 组与常氧组相比明显升高,差异有统计学意义（P ＜ 0.05）;低氧48 h 组ALP mRNA 表达比常氧组高,差异具有统计学意义（P ＜ 0.05）;4 个时间点低氧组骨钙蛋白（OCN）mRNA 表达均比常氧组高,且差异有统计学意义（12、36 h：P ＜ 0.05;24、48 h：P ＜ 0.01）.结论 低氧增强人PDLC 的ALP 活性,上调ALP mRNA 和OCN mRNA 的表达,促进人PDLC 向成骨细胞分化,同时促进成骨细胞的矿化,提示低氧环境对人PDLC 的骨向分化功能产生一定的影响.     

Buffering of protons released by mineral formation during amelogenesis in mice

Regulation of pH by ameloblasts during amelogenesis is critical for enamel mineralization. We examined the effects of reduced bicarbonate secretion and the presence or absence of amelogenins on ameloblast modulation and enamel mineralization. To that end, the composition of fluorotic and non‐fluorotic enamel of several different mouse mutants, including enamel of cystic fibrosis transmembrane conductance regulator‐deficient (Cftr null), anion exchanger‐2‐deficient (Ae2a,b null), and amelogenin‐deficient (Amelx null) mice, was determined by quantitative X‐ray microanalysis. Correlation analysis was carried out to compare the effects of changes in the levels of sulfated‐matrix (S) and chlorine (Cl; for bicarbonate secretion) on mineralization and modulation. The chloride (Cl^?) levels in forming enamel determined the ability of ameloblasts to modulate, remove matrix, and mineralize enamel. In general, the lower the Cl^? content, the stronger the negative effects. In Amelx‐null mice, modulation was essentially normal and the calcium content was reduced least. Retention of amelogenins in enamel of kallikrein‐4‐deficient (Klk4‐null) mice resulted in decreased mineralization and reduced the length of the first acid modulation band without changing the total length of all acidic bands. These data suggest that buffering by bicarbonates is critical for modulation, matrix removal and enamel mineralization. Amelogenins also act as a buffer but are not critical for modulation.     

Protective effect of alpha‐lipoic acid against apical periodontitis‐induced cardiac injury in rats

This study was designed to assess the possible protective effect of alpha‐lipoic acid (ALA) on apical periodontitis (AP)‐induced cardiac injury. Wistar albino rats were randomized into four groups: control; ALA; AP; and ALA + AP. Rats of the control and ALA groups were not endodontically treated, but saline and ALA (100 mg kg^?1) were administered. In rats of the AP and ALA + AP groups, the pulp chambers of mandibular first molar teeth were exposed and left open for 30 d to induce AP. Saline and ALA (100 mg kg^?1) were administered intraperitoneally every 24 h during the experiment. At the end of the experiment, the rats were killed. Establishment of AP was verified by radiographic and histopathological evaluation. Serum alkaline phosphatase (ALP), lactate dehydrogenase (LDH), creatine kinase (CK), and superoxide dismutase (SOD) activities were determined using an automated biochemical analyzer, and the structural cardiac injury was assessed pathologically. Serum ALP, LDH, and CK activities were elevated, and SOD activities were decreased, in the AP group. The changed enzyme activities were significantly normalized by treatment with ALA. We conclude that ALA administration alleviated the AP‐induced heart injury and improved cardiac structure and function, and therefore this agent may be of potential therapeutic value in protecting cardiac tissue from systemic injury caused by AP.     

Sheath proteins: synthesis,secretion, degradation and fate in forming enamel

We investigated expression of ameloblastin and sheathlin, recently cloned enamel matrix proteins from the rat and pig, in forming enamel immunocytochemically and immunochemically, using region-specific antibodies. The results obtained from the rat and pig were essentially the same. Antibodies which recognize the N-terminal region stained the secretory machinery of the secretory ameloblast and the entire thickness of the enamel matrix, especially the peripheral region of the enamel rod. Immunostained protein bands were observed near 65 or 70 kDa and below 20 kDa. C-terminal-specific antibodies stained the secretory machinery of the ameloblast and the immature enamel adjacent to the secretion sites. Immunostained protein bands were found ranging from 25 to 70 kDa. Antibodies which recognize a region in the protein just prior to the C-terminal region stained the cis-side of the Golgi apparatus but not the enamel matrix. Immunostained protein bands were observed of about 55 kDa. These results suggest that post-translational and post-secretory modifications of ameloblastin and sheathlin are similar to each other, and further showed that their cleaved N-terminal polypeptides concentrate in the prism sheath. We propose that sheathlin and ameloblastin share the same role in amelogenesis and should be classified as sheath proteins.     

Short‐term antimicrobial properties of mineral trioxide aggregate with incorporated silver‐zeolite

Abstract – The purpose of this in vitro study was to determine whether adding silver‐zeolite (SZ) to mineral trioxide aggregate (MTA) would enhance the antimicrobial activity of MTA against Staphylococcus aureus (ATCC #25923), Enterococcus faecalis (ATCC #29212), Escherichia coli (ATCC#25922), Pseudomonas aeruginosa (ATCC #27853), Candida albicans (ATCC #90028), Porphyromonas gingivalis (ATCC #33277), Actinomyces israelii (ATCC #12102), and Prevotella intermedia (ATCC# 15032). SZ was added at 0.2% and 2% mass fraction concentration to MTA powder. The control group was MTA powder with no SZ. The antimicrobial effect test was accomplished by placing freshly mixed MTA specimens on agar plates inoculated with microorganisms and comparing the zones of inhibition at 24, 48, and 72 h. The amounts of silver ion release from MTA specimens were measured with atomic absorption spectrophotometry at 10‐min, 24‐, 48‐, and 72‐h periods. The pH of MTA specimens was measured with a pH meter at 10‐min, 24‐, 48‐, and 72‐h periods. MTA with 2% and 0.2% SZ specimens showed inhibitory effects on some microorganisms at all time periods, whereas no antimicrobial activity showed for P. intermedia and A. israelii. MTA without SZ inhibited C. albicans, E. Coli, and P. intermedia. The highest silver release was detected in 2% SZ MTA at 24 h. The incorporation of SZ may enhance the antimicrobial activity of MTA.     

Apoptotic gene expression by human periodontal ligament cells following cyclic stretch

Xu C, Hao Y, Wei B, Ma J, Li J, Huang Q, Zhang F. Apoptotic gene expression by human periodontal ligament cells following cyclic stretch. J Periodont Res 2011; 46: 742–748. © 2011 John Wiley & Sons A/S Background and Objective: Periodontal ligament cells play an important role in maintaining homeostasis of periodontal tissue upon mechanical force loading caused by mastication or orthodontic force. Previous studies revealed force‐driven periodontal ligament cell death via apoptosis, but the force‐sensing genes assigned to the apoptotic pathway have not been fully characterized. The present study aimed to identify force‐sensing genes implicated in the apoptotic pathway in periodontal ligament cells. Material and Methods: Human periodontal ligament cells were exposed to 20% stretch strain for 6 or 24 h, and the differential expression of 84 genes implicated in the apoptotic pathway were quantified by real‐time PCR array technology. Results: Ten and 11 genes showed upregulated expression after 6 and 24 h stretches, respectively, and there were two downregulated genes in response to both 6 and 24 h stretches. These genes included those encoding the tumor necrosis factor ligand family (TNFSF8), tumor necrosis factor receptor family (FAS, TNFRSF10B, TNFRSF11B, TNFRSF25 and CD27), the Bcl‐2 family (BAG3, BAK1, BCL2L11 and BCLAF1), the caspase family (CASP5 and CASP7), the inhibitor of apoptosis proteins family (BIRC3, BIRC6 and NAIP), the caspase recruitment domain family (RIPK2 and PYCARD) and the death domain family (DAPK1), as well as an oncogene (BRAF). Conclusion: This study identified several force‐sensing genes implicated in the apoptotic pathway in periodontal ligament cells and should facilitate future studies on force‐driven apoptosis by providing putative target genes.     

Hypoxia inhibits mineralization ability of human dental pulp cells treated with TEGDMA but increases cell survival in accordance with the culture time

ObjectiveTo evaluate the cytotoxicity and mineralization effects of TEGDMA in human dental pulp cells (hDPCs) under hypoxic and normoxic culture conditions.DesignCell viability was evaluated using XTT assay after incubation periods of 24, 48, or 72 h. The expression of mineralization-related genes (osteonectin, osteopontin, dentin sialophosphoprotein, collagen type 1) and heme oxygenase 1 (HO-1) was assessed by quantitative real-time polymerase chain reaction at 24 and 72 h.ResultsIn XTT assay, viability was higher in 0.3, 1, 2, 4, and 5 mM groups in the presence of 21% O₂ after 24 h (p < 0.05). Additionally, while 0.3, 1, 2 mM groups had higher cell viability in the presence of 21% O₂ after 48 h (p < 0.05), in 3 mM groups cell viability was higher under 3% O₂ than 21% O₂ after both 24 and 48 h (p < 0.05). 1–3 mM groups had higher cell viability under 3% O₂ after 72 h (p < 0.05). There was no difference between 4 and 5 mM groups with regards to cell viability after 48 or 72 h (p > 0.05). In the gene expression study, TEGDMA-treated hDPCs showed lower mineralization potential in the presence of 3% than with 21% O₂ (p < 0.05). hDPCs revealed higher HO 1 expression in 0.3 and 1 mM groups under hypoxic than under normoxic conditions after a 72-h time period (p < 0.001).ConclusionsHypoxic conditions increased cell survival in accordance with the culture period but inhibited the odontoblastic differentiation of hDPCs treated with TEGDMA.     

The fate of Treponema denticola within human gingival epithelial cells

Treponema denticola is one of the major pathogens associated with chronic periodontitis. Bacterial invasion into gingival tissues is a critical process in the pathogenesis of periodontal disease. We recently reported that T. denticola can invade gingival epithelial cells. The aim of this study is to determine the fate of internalized T. denticola in gingival epithelial cells. Immortalized human gingival epithelial HOK‐16B cells were infected with 5‐ (and 6‐) carboxy‐fluorescein diacetate succinimidyl ester (CFSE)‐labeled live or heat‐killed T. denticola for 24 h, and the presence of bacteria inside the cells was confirmed by confocal microscopy. Live T. denticola, but not heat‐killed bacteria, invaded HOK‐16B cells. Confocal microscopy also revealed that internalized T. denticola rarely colocalized with either endosomes or lysosomes. Transmission electron microscopy of infected cells showed that intracellular T. denticola was localized inside endosome‐like structures. Although a culture‐based antibiotics protection assay could not detect viable intracellular T. denticola 12 h after infection, a substantial number of bacteria were observed by confocal microscopy and weak expression of bacterial 16S ribosomal RNA was detected 48 h after infection. In addition, flow cytometric analysis of HOK‐16B cells infected with CFSE‐labeled T. denticola showed no loss of fluorescence over 48 h. Collectively, T. denticola invades gingival epithelial cells and remains within the host cells for many hours by resisting endolysosomal degradation. These findings may provide new insight into the role of T. denticola in the pathogenesis of periodontitis.