Barbara Cvikl

Prolyl Hydroxylase Inhibitors Increase the Production of Vascular Endothelial Growth Factor in Dental Pulp–derived Cells

INTRODUCTION Prolyl hydroxylase (PHD) inhibitors can induce a proangiogenic response that stimulates regeneration in soft and hard tissues. However, the effect of PHD inhibitors on the dental pulp is unclear. The purpose of this study was to evaluate the effects of PHD inhibitors on the proangiogenic capacity of human dental pulp-derived cells. METHODS To test the response of dental pulp-derived cells to PHD inhibitors, the cells were exposed to dimethyloxalylglycine, desferrioxamine, L-mimosine, and cobalt chloride. To assess the response of dental pulp cells to a capping material supplemented with PHD inhibitors, the cells were treated with supernatants from calcium hydroxide. Viability, proliferation, and protein synthesis were assessed by formazan formation, (3)[H]thymidine, and (3)[H]leucine incorporation assays. The effect on the proangiogenic capacity was measured by immunoassays for vascular endothelial growth factor (VEGF). RESULTS We found that all 4 PHD inhibitors can reduce viability, proliferation, and protein synthesis at high concentrations. At nontoxic concentrations and in the presence of supernatants from calcium hydroxide, PHD inhibitors stimulated the production of VEGF in dental pulp-derived cells. When calcium hydroxide was supplemented with the PHD inhibitors, the supernatants from these preparations did not significantly elevate VEGF levels. CONCLUSIONS These results show that PHD inhibitors can stimulate VEGF production of dental pulp-derived cells, suggesting a corresponding increase in their proangiogenic capacity. Further studies will be required to understand the impact that this might have on pulp regeneration.

Toothbrushing after an erosive attack: will waiting avoid tooth wear?

The purpose of this study was to determine if storage for up to 4 h in human saliva results in a decrease of erosive tooth wear (ETW) and in an increase of surface microhardness (SMH) of enamel samples after an erosive attack with subsequent abrasion. Furthermore, we determined the impact of individual salivary parameters on ETW and SMH. Enamel samples were distributed into five groups: group 1 had neither erosion nor saliva treatment; groups 2-5 were treated with erosion, then group 2 was placed in a humid chamber and groups 3-5 were incubated in saliva for 30 min, 2 h, and 4 h, respectively. After erosion and saliva treatments, all groups were treated with abrasion. Surface microhardness and ETW were measured before and after erosion, incubation in saliva, and abrasion. Surface microhardness and ETW showed significant changes throughout the experiment: SMH decreased and ETW increased in groups 2-5, regardless of the length of incubation in saliva. The results of groups 3-5 (exposed to saliva) were not significantly different from those of group 2 (not exposed to saliva). Exposure of eroded enamel to saliva for up to 4 h was not able to increase SMH or reduce ETW. However, additional experiments with artificial saliva without proteins showed protection from erosive tooth wear. The recommendation to postpone toothbrushing of enamel after an erosive attack should be reconsidered.

Effects of Prolyl Hydroxylase Inhibitor L-mimosine on Dental Pulp in the Presence of Advanced Glycation End Products.

INTRODUCTION Proangiogenic prolyl hydroxylase (PHD) inhibitors represent a novel approach to stimulate tissue regeneration. Diabetes mellitus involves the accumulation of advanced glycation end products (AGEs). Here we evaluated the impact of AGEs on the response of human pulp tissue to the PHD inhibitor L-mimosine (L-MIM) in monolayer cultures of dental pulp-derived cells (DPCs) and tooth slice organ cultures. METHODS In monolayer cultures, DPCs were incubated with L-MIM and AGEs. Viability was assessed based on formazan formation, live-dead staining, annexin V/propidium iodide, and trypan blue exclusion assay. Vascular endothelial growth factor (VEGF), interleukin (IL)-6, and IL-8 production was evaluated by quantitative polymerase chain reaction and immunoassays. Furthermore, expression levels of odontoblast markers were assessed, and alizarin red staining was performed. Tooth slice organ cultures were performed, and VEGF, IL-6, and IL8 levels in their supernatants were measured by immunoassays. Pulp tissue vitality and morphology were assessed by MTT assay and histology. RESULTS In monolayer cultures of DPCs, L-MIM at nontoxic concentrations increased the production of VEGF and IL-8 in the presence of AGEs. Stimulation with L-MIM decreased alkaline phosphatase levels and matrix mineralization also in the presence of AGEs, whereas no significant changes in dentin matrix protein 1 and dentin sialophosphoprotein expression were observed. In tooth slice organ cultures, L-MIM increased VEGF but not IL-6 and IL-8 production in the presence of AGEs. The pulp tissue was vital, and no signs of apoptosis or necrosis were observed. CONCLUSIONS Overall, in the presence of AGEs, L-MIM increases the proangiogenic capacity, but decreases alkaline phosphatase expression and matrix mineralization.

The response of dental pulp-derived cells to zoledronate depends on the experimental model.

AIM To investigate whether zoledronate (ZOL) can cause a cytotoxic response in dental pulp-derived cells (DPCs) in vitro. METHODOLOGY Cell activity was assessed utilizing MTT tests, (3) [H]thymidine, and (3) [H]leucine incorporation assays in human DPCs in response to ZOL. Cell activity assays were also preformed on calcium phosphate-coated plates. Cell death was analysed with annexin V/propidium iodide, trypan blue staining and Western blot analysis. RESULTS Micromolar concentrations of ZOL were required to decrease the activity of DPCs. The decreased activity of DPCs was associated with the occurrence of apoptosis and necrosis. No adverse effects were observed when DPCs were cultured on calcium phosphate-coated plates with ZOL. CONCLUSION High concentrations of soluble ZOL were required to cause adverse effects in vitro. These adverse effects are abolished when the bisphosphonate was bound to a mineralized surface. However, the clinical relevance of these results remains to be determined.

Saliva Suppresses Osteoclastogenesis in Murine Bone Marrow Cultures

Saliva can reach mineralized surfaces in the oral cavity; however, the relationship between saliva and bone resorption is unclear. Herein, we examined whether saliva affects the process of osteoclastogenesis in vitro. We used murine bone marrow cultures to study osteoclast formation. The addition of fresh sterile saliva eliminated the formation of multinucleated cells that stained positive for tartrate-resistant acid phosphatase (TRAP). In line with the histochemical staining, saliva substantially reduced gene expression of cathepsin K, calcitonin receptor, and TRAP. Addition of saliva led to considerably decreased gene expression of receptor activator of nuclear factor kappa-B (RANK) and, to a lesser extent, that of c-fms. The respective master regulators of osteoclastogenesis (c-fos and NFATc1) and the downstream cell fusion genes (DC-STAMP and Atp6v0d2) showed decreased expression after the addition of saliva. Among the costimulatory molecules for osteoclastogenesis, only OSCAR showed decreased expression. In contrast, CD40, CD80, and CD86—all costimulatory molecules of phagocytic cells—were increasingly expressed with saliva. The phagocytic capacity of the cells was confirmed by latex bead ingestion. Based on these in vitro results, it can be concluded that saliva suppresses osteoclastogenesis and leads to the development of a phagocytic cell phenotype.

Migration background is associated with caries in Viennese school children, even if parents have received a higher education

BackgroundA low level of education and the migration background of parents are associated with the development of caries in children. The aim of this study was to evaluate whether a higher educational level of parents can overcome risks for the development of caries in immigrants in Vienna, Austria.MethodsThe educational level of the parents, the school type, and the caries status of 736 randomly selected twelve-year-old children with and without migration background was determined in this cross sectional study. In children attending school in Vienna the decayed, missing, and filled teeth (DMFT) index was determined. For statistical analysis, a mixed negative-binomial-model was used.ResultsThe caries status of the children with migration background was significantly worse compared to that of the native Viennese population. A significant interaction was found between migration background and the educational level of the parents (p = 0.045). No interaction was found between the school type and either the migration background (p = 0.220) or the education level of the parents (p = 0.08). In parents with a higher scholarly education level, migration background (p < 0.01) and school type (p = 0.018) showed an association with DMFT values. In parents with a low education level, however, migration background and school type had no significant association with DMFT values.ConclusionThese data indicate that children with a migration background are at higher risk to acquire caries than other Viennese children, even when the parents have received a higher education.

The in vitro impact of toothpaste extracts on cell viability

Toothpastes contain three main components: detergents, abrasives, and fluoride. Detergents, particularly sodium lauryl sulfate, have been proposed as components that enable toothpastes to produce cytotoxic effects in vitro. However, not all toothpastes contain sodium lauryl sulfate, and almost no studies have found an association between detergents and the in vitro cytotoxicity of toothpastes. The present study examined the in vitro cytotoxicity of nine commercially available toothpastes containing four different detergents. Toothpastes were diluted in serum-free medium, centrifuged, and filter sterilized. The half-lethal concentration of the toothpaste-conditioned medium (TCM) was calculated based on the formation of formazan by gingival fibroblasts, oral squamous cell carcinoma HSC-2 cells, and L929 cells. Cell proliferation was analyzed, and live-dead staining was performed, after exposure of cells to conditioned medium prepared with 1% toothpaste (1% TCM). It was found that toothpastes containing sodium lauryl sulfate and amine fluoride strongly inhibited cell viability with the half-lethal concentration being obtained with conditioned medium prepared with approximately 1% toothpaste (1% TCM). Toothpastes containing cocamidopropyl betaine and Steareth-20 showed higher half-lethal concentration values, with the half-lethal concentration being obtained with conditioned medium prepared with 10% (10% TCM) and 70% (70% TCM) toothpaste, respectively. Proliferation and live-dead data were consistent with the cell-viability analyses. These results demonstrate that the type of detergent in toothpastes can be associated with changes in in vitro cell toxicity.

Salivary pellets induce a pro-inflammatory response involving the TLR4-NF-kB pathway in gingival fibroblasts.

BackgroundWhole saliva provokes a substantial pro-inflammatory response in gingival fibroblasts. This raises the question whether the salivary pellet, which is used for diagnostic purposes, also has a pro-inflammatory capacity and, if yes, what the underlying mechanisms at the molecular level are.MethodsWe examined the ability of extensively washed salivary pellets to provoke the expression of chemokines in gingival fibroblasts by real-time polymerase chain reaction and immunoassays. Protein composition was determined with proteomic analysis. Endotoxins were analyzed by a Limulus assay and removed by affinity chromatography. The inhibitors TAK-242 and BAY11-7082 were used to determine the involvement of the TLR4 and NF-kB signaling, respectively. Western blot was performed to detect phosphorylated p65.ResultsThe experiments show that salivary pellets and the corresponding washing solution contain pro-inflammatory activity without impairing cell viability. Proteomic analysis revealed proteins with a binding capacity for lipopolysaccharides, and the Limulus assay indicated the presence of endotoxin in the salivary pellets. Blocking TLR4 with TAK-242 and depletion of endotoxins both lowered the capacity of salivary pellets to increase chemokine expression and phosphorylation of p65. BAY11-7082 suppressed chemokine expression in response to the salivary pellets. Autoclaving salivary pellets also reduced their pro-inflammatory activity.ConclusionsThe data support the molecular mechanism of a TLR4-NF-kB-dependent pro-inflammatory response of the gingival fibroblasts exposed to preparations of washed salivary pellets. Together, the data indicate that the salivary pellet is rich in endotoxin but it is mainly a heat labile fraction that accounts for the chemokine expression in the bioassay.

Enamel Surface Changes After Exposure to Bleaching Gels Containing Carbamide Peroxide or Hydrogen Peroxide.

OBJECTIVE This study evaluated the differences in enamel color change, surface hardness, elastic modulus, and surface roughness between treatments with four bleaching gels containing carbamide peroxide (two at 10% and one each at 35%, and 45%) and two bleaching gels containing hydrogen peroxide (two at 40%). METHODS Enamel specimens were bleached and color changes were measured. Color change was calculated using either ΔE or the Bleaching Index (BI). Then, surface hardness, elastic modulus, and surface roughness of the enamel specimens were evaluated. All measurements were performed at baseline and directly after the first bleaching treatment for all carbamide peroxide- and hydrogen peroxide-containing bleaching gels. In addition, final measurements were made 24 hours after each of a total of 10 bleaching treatments for carbamide peroxide bleaching gels, and 1 week after each of a total of three bleaching treatments for hydrogen peroxide bleaching gels. RESULTS After the last bleaching treatment, respective ΔE scores were 17.6 and 8.2 for the two 10% carbamide peroxide gels, 12.9 and 5.6 for the 45% and 35% carbamide peroxide gels, and 9.6 and 13.9 for the two 40% hydrogen peroxide gels. The respective BI scores were -2.0 and -2.0 for the two 10% carbamide peroxide gels, -3.5 and -1.5 for the 45% and 35% carbamide peroxide gels, and -2.0 and -3.0 for the two 40% hydrogen peroxide gels. Each bleaching gel treatment resulted in significant whitening; however, no significant difference was found among the gels after the last bleaching. Whitening occurred within the first bleaching treatments and did not increase significantly during the remaining treatments. Surface hardness significantly decreased after the last bleaching treatment, when 10% carbamide peroxide was used. Furthermore, significant changes in the elastic modulus or surface roughness occurred only after treatment with 10% carbamide peroxide. CONCLUSION All six bleaching gels effectively bleached the enamel specimens independent of their concentration of peroxide. Gels with low peroxide concentration and longer contact time negatively affected the enamel surface.

Chemokine expression of oral fibroblasts and epithelial cells in response to artificial saliva.

ObjectivesArtificial saliva is widely used to overcome reduced natural salivary flow. Natural saliva provokes the expression of chemokines in oral fibroblasts in vitro. However, if artificial saliva changes the expression of chemokines remains unknown.Materials and methodsHere, we investigated the ability of Saliva Orthana®, Aldiamed®, Glandosane®, and Saliva Natura® to change the expression of chemokines in human oral fibroblasts and the human oral epithelial cell line HSC-2 by means of reverse transcription polymerase chain reaction and immunoassays. Mucins isolated from bovine submaxillary glands and recombinant human mucin 1 were included in the bioassay. Formazan formation and LIVE/DEAD® staining determined the impact of artificial saliva on cell viability. The involvement of signaling pathways was determined by pharmacologic inhibitors and Western blotting.ResultsIn gingival fibroblasts, Saliva Orthana®-containing mucins provoked a significantly increased expression of CXC ligand 8 (CXCL8, or interleukin 8), CXCL1, and CXCL2. Immunoassays for CXCL8 and CXCL1 confirmed the translation at the protein level. The respective dilution of artificial saliva had no impact on formazan formation and LIVE/DEAD® staining. Mucins isolated from bovine submaxillary glands also increased the panel of chemokine expression in gingival fibroblasts. BAY 11–7082, a nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) inhibitor, but also TAK-242, an inhibitor of toll-like receptor 4 signaling, blocked chemokine expression of Saliva Orthana® and bovine mucins. In HSC-2 cells, Glandosane® significantly increased CXCL8 expression.ConclusionsSaliva Orthana® stimulated chemokine expression in gingival fibroblasts. Mammalian mucins, but also possible contaminations with endotoxins, might contribute to the respective changes in gene expression. Epithelial cells have a differential response to artificial saliva with Glandosane® changing CXCL8 expression.Clinical relevanceArtificial saliva can incite a cellular response, if however the changing expression of chemokines by isolated fibroblasts and epithelial cells in vitro translates into a clinical condition, is not clear.