Effect of Cold Atmospheric Plasma (CAP) on Osteogenic Differentiation Potential of Human Osteoblasts.

Bone regeneration after oral and maxillofacial surgery is a long-term process, which involves various mechanisms. Recently, cold atmospheric plasma (CAP) has become known to accelerate wound healing and have an antimicrobial effect. Since the use of CAP in dentistry is not yet established, the aim of the present study was to investigate the effect of CAP on human calvaria osteoblasts (HCO). HCO were treated with CAP for different durations of time and distances to the cells. Cell proliferation was determined by MTT assay and cell toxicity by LDH assay. Additionally, RT-qPCR was used to investigate effects on osteogenic markers, such as alkaline phosphatase (ALP), bone morphogenic protein (BMP)2, collagen (COL)1A1, osteonectin (SPARC), osteoprotegerin (OPG), osterix (OSX), receptor activator of NF-κB (RANK), RANK Ligand (RANKL), and Runt-related transcription factor (RUNX)2. There were small differences in cell proliferation and LDH release regarding treatment duration and distance to the cells. However, an increase in the expression of RANK and RANKL was observed at longer treatment times. Additionally, CAP caused a significant increase in mRNA expression of genes relevant to osteogenesis. In conclusion, CAP has a stimulating effect on osteoblasts and may thus represent a potential therapeutic approach in the regeneration of hard tissue defects.

Influence of Implant Material and Surface on Mode and Strength of Cell/Matrix Attachment of Human Adipose Derived Stromal Cell.

A fundamental step for cell growth and differentiation is the cell adhesion. The purpose of this study was to determine the adhesion of different cell lineages, adipose derived stromal cells, osteoblasts, and gingival fibroblast to titanium and zirconia dental implants with different surface treatments. Primary cells were cultured on smooth/polished surfaces (titanium with a smooth surface texture (Ti-PT) and machined zirconia (ZrO2-M)) and on rough surfaces (titanium with a rough surface texture (Ti-SLA) and zirconia material (ZrO2-ZLA)). Alterations in cell morphology (f-actin staining and SEM) and in expression of the focal adhesion marker were analysed after 1, 7, and 14 days. Statistical analysis was performed by one-way ANOVA with a statistical significance at p = 0.05. Cell morphology and cytoskeleton were strongly affected by surface texture. Actin beta and vimentin expressions were higher on rough surfaces (p < 0.01). Vinculin and FAK expressions were significant (p < 0.05) and increased over time. Fibronectin and laminin expressions were significant (p < 0.01) and did not alter over time. Strength of cell/material binding is influenced by surface structure and not by material. Meanwhile, the kind of cell/material binding is regulated by cell type and implant material.

Evaluation of the biocompatibility of root canal sealers on human periodontal ligament cells ex vivo.

The aim of this study was to evaluate the biocompatibility of two comparatively new calcium silicate containing sealers (MTA-Fillapex and BioRoot-RCS) with that of two established sealers (AH-Plus, epoxy resin-based; Pulp-Canal-Sealer, zinc oxide eugenol containing). Human periodontal ligament cells (PDL-cells) were brought in contact with eluates from freshly mixed and set sealer. The sealers were mixed strictly according to the manufacturers' instructions and identically samples were produced. 1:1, 1:2, and 1:10 dilutions of sealers extract were used. Extracts from freshly mixed sealer were added to the PDL-cells on day one to simulate a clinical scenario. Subsequently, at 24 h, 7, 14, and 21 days extracts form set sealers were used for PDL-cell culturing. PDL-cell viability was analyzed by living-cell-count, MTT-assay, and living/dead-staining, cytotoxicity by LDH-assay, and changes by Richardson-staining. All data were statistically evaluated by one way ANOVA and a posthoc analysis with Bonferroni-Holm testing (p < 0.05). In contact with BioRoot-RCS a regeneration of the PDL-cells were observed over time. This sealer showed the lowest toxicity in a freshly mixed and set state (p < 0.05). MTA-Fillapex and Pulp-Canal-Sealer were cytotoxic in a fresh as well as in a set state, whereas AH-Plus was cytotoxic in a freshly mixed state, but not when the sealer was set. BioRoot-RCS is biocompatible and bioactive because it seems to have a positive influence on the PDL-cell metabolism. Pulp Canal Sealer and MTA-Fillapex showed no biocompatibility in contact with PDL-cells at all. Freshly mixed AH Plus is less biocompatible on PDL than in a set state.

Influence of Implant Material and Surface on Differentiation and Proliferation of Human Adipose-Derived Stromal Cells.

For the guided regeneration of periimplant hard and soft tissues, human adipose-derived stromal cells (hADSC) seem to be a promising source for mesenchymal stromal cells. For this, the proliferation and differentiation of hADSC were evaluated on titanium and zirconia dental implants with different surface treatments. Results were compared to edaphic cells as human osteoblasts (hOB) and human gingival fibroblasts (HGF). Primary cells were cultured on (1) titanium implants with a polished surface (Ti-PT), (2) sandblasted and acid-etched titanium (Ti-SLA), (3) sandblasted and alkaline etched zirconia (ZrO2-ZLA) and (4) machined zirconia (ZrO2-M). The cell proliferation and differentiation on osteogenic lineage were assessed after 1, 7 and 14 days. Statistical analysis was performed by one-way ANOVA and a modified Levene test with a statistical significance at p = 0.05. PostHoc tests were performed by Bonferroni-Holm. Zirconia dental implants with rough surface (ZrO2-ZLA) showed the highest proliferation rates (p = 0.048). The osteogenic differentiation occurred early for zirconia and later for titanium implants, and it was enhanced for rough surfaces in comparison to polished/machined surfaces. Zirconia was more effective to promote the proliferation and differentiation of hADSCs in comparison to titanium. Rough surfaces were able to improve the biological response for both zirconia and titanium.

Simvastatin Induces In Vitro Mineralization Effects of Primary Human Odontoblast-Like Cells.

Simvastatin (SV) is an often prescribed statin reducing the LDL-concentration in circulating blood. The aim of this study was to evaluate the pleiotropic effects of SV to primary human odontoblast-like cells. Twenty four wisdom teeth of different subjects were extracted and the pulp tissue was removed and minced under sterile conditions. After mincing, the requested cells were passaged according to established protocols. Osteoblastic marker (ALP conversion), viability and mineralization were determined at days 14, 17 and 21 after simvastatin exposition (0.01 µM, 0.1 µM, 1.0 µM, 2.0 µM). The sample size per group was 24 cultures with three replicates per culture for ALP-conversion and mineralization and 6 replicates for viability. A Kruskal-Wallis test was used for statistical analysis. After adding SV, viability was significantly (p < 0.01) decreased in a time- and dose-dependent manner, whereas after 21 days, mineralization was significant (p < 0.01). ALP-conversion in groups with SV concentrations of 1 and 2 µM SV was significantly (p < 0.01) increased. Pleiotropic effects regarding mineralization in higher SV concentrations were possibly induced via alternative mineralization pathways as almost equal elevations of ALP conversion were not evident in the control and experimental groups.

The Biological Effect of Particulate Titanium Contaminants of Dental Implants on Human Osteoblasts and Gingival Fibroblasts.

PURPOSE: To evaluate the effects of different titanium particle concentrations on viability of human calvarial osteoblasts and human gingival fibroblasts. MATERIALS AND METHODS: Primary human calvarial osteoblasts (HCO, 3H Biomedical) and human gingival fibroblasts (HGF-1, ATCC) were cultivated and allowed to adhere for 24 hours. Titanium powder concentrations (0.01 to 1.0 mg/mL) were added, and samples were analyzed at three time points (24 hours, 7 days, 21 days). Cell viability was analyzed using living cell count, proliferation (MTT) assay, and a live/dead staining. Cytotoxic effects were evaluated using lactated dehydrogenase assay. Qualitative analysis of cell viability was performed. In addition, scanning electron microscopy (SEM) analysis was performed. Release of interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-±) was estimated with Human IL-6 / Human TNF-± ELISA. RESULTS: Titanium concentrations of 0.1 mg/mL and 1.0 mg/mL showed medium- and long-term effects on cell growth and proliferation rates. Cytotoxic effects by release of lactate dehydrogenase were observable during the first 24 hours. Human gingival fibroblast cells showed a release factor between 2.6 to 3.4. Titanium powder seemed to be more cytotoxic to human gingival fibroblast cells than to human calvarial osteoblast cells. For human calvarial osteoblasts, only the highest concentration showed cytotoxic effects with a release factor of 2.7. Human calvarial osteoblasts secreted IL-6 only during the first 24 hours and only in the highest titanium concentration, whereas human gingival fibroblasts secreted IL-6 during the entire period. The lowest titanium concentration showed stronger secretion of IL-6 compared to control. Incorporation of smaller and single titanium particles by cells was identified under SEM analysis. CONCLUSION: Cell viability is negatively correlated with titanium concentration. Further, titanium debris might lead to an inflammatory biologic response of dental peri-implant tissue. Also, cells interact with the debris, eg, with incorporation of particles.

Self-Assessment of Oral Health-Related Quality of Life in People with Ectodermal Dysplasia in Germany.

Background: Ectodermal dysplasia describes a heterogeneous group of hereditary, congenital malformations involving developmental dystrophies of ectodermal structures. The aim of this study was to analyse the oral health-related quality of life (OHRQoL) in people with ectodermal dysplasia and to evaluate the influence of different variables. Methods: The study was designed as an anonymous epidemiological survey study among people with ectodermal dysplasia to evaluate oral symptoms, satisfaction with the health system and their respective OHRQoL using the validated German version of the OHIP-14 (Oral Health Impact Profile) questionnaire. Results: When asked about oral symptoms, 110 of the participants provided responses, of which 109 (99.09%) described oral symptoms. The average age of the female participants at the time of diagnosis was 17.02 years (range: 0 to 48 years), the average age of men was 5.19 years (range: 0 to 43 years). The average OHIP-14 overall score for female participants was 12.23 points (SD: 12.39), for male participants an average OHIP score of 11.79 points was recorded (SD: 11.08 points). Difficulty in finding a dentist (p = 0.001), and the dissatisfaction with the health system (p = 0.007) showed a negative impact on the OHRQoL. Conclusion: People with ectodermal dysplasia rate their OHRQoL worse than is usually prevalent in the normal German population (4.09 points); women are diagnosed with "ectodermal dysplasia" later than men. Participants who reported difficulties in finding a dentist for treatment exhibited higher OHIP values. Likewise, dissatisfaction with the health system demonstrated a negative impact on the oral health-related quality of life.

The Biological Effect of Particulate Titanium Contaminants of Dental Implants on Human Osteoblasts and Gingival Fibroblasts.

PURPOSE: To evaluate the effects of different titanium particle concentrations on viability of human calvarial osteoblasts and human gingival fibroblasts. MATERIALS AND METHODS: Primary human calvarial osteoblasts (HCO, 3H Biomedical) and human gingival fibroblasts (HGF-1, ATCC) were cultivated and allowed to adhere for 24 hours. Titanium powder concentrations (0.01 to 1.0 mg/mL) were added, and samples were analyzed at three time points (24 hours, 7 days, 21 days). Cell viability was analyzed using living cell count, proliferation (MTT) assay, and a live/dead staining. Cytotoxic effects were evaluated using lactated dehydrogenase assay. Qualitative analysis of cell viability was performed. In addition, scanning electron microscopy (SEM) analysis was performed. Release of interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-±) was estimated with Human IL-6 / Human TNF-± ELISA. RESULTS: Titanium concentrations of 0.1 mg/mL and 1.0 mg/mL showed medium- and long-term effects on cell growth and proliferation rates. Cytotoxic effects by release of lactate dehydrogenase were observable during the first 24 hours. Human gingival fibroblast cells showed a release factor between 2.6 to 3.4. Titanium powder seemed to be more cytotoxic to human gingival fibroblast cells than to human calvarial osteoblast cells. For human calvarial osteoblasts, only the highest concentration showed cytotoxic effects with a release factor of 2.7. Human calvarial osteoblasts secreted IL-6 only during the first 24 hours and only in the highest titanium concentration, whereas human gingival fibroblasts secreted IL-6 during the entire period. The lowest titanium concentration showed stronger secretion of IL-6 compared to control. Incorporation of smaller and single titanium particles by cells was identified under SEM analysis. CONCLUSION: Cell viability is negatively correlated with titanium concentration. Further, titanium debris might lead to an inflammatory biologic response of dental peri-implant tissue. Also, cells interact with the debris, eg, with incorporation of particles.

Cytotoxic effects of four different root canal sealers on human osteoblasts.

The aim of this study was to evaluate the effect of an epoxy resin-based (AH-Plus), a zinc oxide eugenol containing (Pulp-Canal-Sealer) and two calcium silicate containing (MTA-Fillapex and BioRoot-RCS) sealers on primary human osteoblasts (hOB) in freshly mixed and set state. All sealers were mixed strictly according to the manufacturers´ instructions and identically samples were produced. In a pretest cytotoxic sealer concentrations were determined. Thus, for the main cell culture study, dilutions of sealer extract 1:1, 1:2, and 1:10 were used. To simulate a clinical scenario, extracts from freshly mixed sealer were added to the cells on day one. Extracts form set sealers were used for subsequent culturing for 24h, 7d, 14d, and 21d. Cell viability was analyzed by living-cell-count, MTT-assay, and living/dead-staining, cytotoxicity by LDH-assay, and changes by Richardson-staining. All data were statistically evaluated by one way ANOVA and a posthoc analysis with Bonferroni-Holm testing (p<0.05). AH-Plus was cytotoxic in a freshly mixed state, but not when the sealer was set. MTA-Fillapex and Pulp-Canal-Sealer were cytotoxic in a fresh as well as in a set state. BioRoot-RCS showed the lowest toxicity in both states; where as a regeneration of the cells could be observed over time (p<0.05). Contact of freshly mixed AH-Plus to osteoblasts should be avoided. Pulp Canal Sealer and MTA-Fillapex showed no biocompatibility in contact with osteoblasts at all. BioRoot-RCS had a positive influence on the cell metabolism (bioactivity) and is biocompatible.