Longitudinal bond strength of a universal adhesive and chemical dentin characterization under different acid etching protocols.

OBJECTIVE: This study aimed to analyze the longitudinal bond strength of a universal adhesive and chemically characterize the dentin substrate under different acid etching protocols. METHODOLOGY: Dentin samples were etched with polyacrylic acid 25% (PAA) for 10 seconds (n=3) and phosphoric acid 32% (PA) for 15 seconds (n=3) and analyzed by Fourier transform infrared spectroscopy - attenuated total reflectance (FTIR-ATR) before and after treatment. For collagen degradation, samples (n=12) were divided into 3 groups: PAA, PA, and Deionized water (control), and analyzed by the quantity of solubilized type I collagen C-terminal cross-linked telopeptides and solubilized C-terminal peptide in relation to total protein concentration (ICTPtp and CTXtp) and by their ultimate tensile strength (UTS). For the adhesive interface analysis, dentin samples (n=72) were divided into 3 groups: PAA, PA, and Self-etch (SE), and subdivided into 2 groups: 24 h (baseline) and 1 year. The following tests were performed: microtensile bond strength (µTBS) (n=48), scanning electron microscopy (SEM) (n=12), and nanoleakage (n=12). RESULTS: The FTIR of PAA showed lower reduction of the peaks in the phosphate group when compared to PA. For ICTPtp, PA showed a significantly higher value. For CTXtp, PA and PAA groups failed to statically differ from each other. UTS was significantly lower for PA. For µTBS, storage time significantly affected bond strength. The results were unaffected by the etching protocol. For SEM, after 1 year, PA had little evidence of degradation in the upper third of the adhesive interface in comparison to the other groups. Nanoleakage showed no considerable silver impregnation after 1 year in the SE group. CONCLUSION: The use of PAA prior to a universal adhesive (when compared to PA) represents a less aggressive type of etching to dentin. However, self-etching still seems to be the best option for universal adhesive systems that have functional monomers in their composition.

Longitudinal bond strength of a universal adhesive and chemical dentin characterization under different acid etching protocols

Abstract Objective This study aimed to analyze the longitudinal bond strength of a universal adhesive and chemically characterize the dentin substrate under different acid etching protocols. Methodology Dentin samples were etched with polyacrylic acid 25% (PAA) for 10 seconds (n=3) and phosphoric acid 32% (PA) for 15 seconds (n=3) and analyzed by Fourier transform infrared spectroscopy - attenuated total reflectance (FTIR-ATR) before and after treatment. For collagen degradation, samples (n=12) were divided into 3 groups: PAA, PA, and Deionized water (control), and analyzed by the quantity of solubilized type I collagen C-terminal cross-linked telopeptides and solubilized C-terminal peptide in relation to total protein concentration (ICTPtp and CTXtp) and by their ultimate tensile strength (UTS). For the adhesive interface analysis, dentin samples (n=72) were divided into 3 groups: PAA, PA, and Self-etch (SE), and subdivided into 2 groups: 24 h (baseline) and 1 year. The following tests were performed: microtensile bond strength (μTBS) (n=48), scanning electron microscopy (SEM) (n=12), and nanoleakage (n=12). Results The FTIR of PAA showed lower reduction of the peaks in the phosphate group when compared to PA. For ICTPtp, PA showed a significantly higher value. For CTXtp, PA and PAA groups failed to statically differ from each other. UTS was significantly lower for PA. For μTBS, storage time significantly affected bond strength. The results were unaffected by the etching protocol. For SEM, after 1 year, PA had little evidence of degradation in the upper third of the adhesive interface in comparison to the other groups. Nanoleakage showed no considerable silver impregnation after 1 year in the SE group. Conclusion The use of PAA prior to a universal adhesive (when compared to PA) represents a less aggressive type of etching to dentin. However, self-etching still seems to be the best option for universal adhesive systems that have functional monomers in their composition.

Interaction of epigallocatechin-gallate and chlorhexidine with Streptococcus mutans stimulated odontoblast-like cells: Cytotoxicity, Interleukin-1ß and co-species proteomic analyses.

OBJECTIVES: The dentin therapeutic agent chlorhexidine has inflammatory and cytotoxic characteristics urging investigation of alternatives like the natural compound epigallocatechin-gallate. The aim is to verify the effect of epigallocatechin-gallate and chlorhexidine on viability, interleukin-1ß (IL-1ß) and differential protein expression of MDPC-23 odontoblast-like cells stimulated by Streptococcus mutans. DESIGN: Cells were stimulated with heat-killed S. mutans at multiplicity of infection (MOI) of 100-1000 and subsequently treated with 100-1 µM of epigallocatechin-gallate. Cells with no treatment or chlorhexidine were controls. Combined stimulated/treated cells were tested for cytotoxicity (Alamar-Blue, N = 3, n = 3), total protein (N = 3, n = 3), IL-1ß (ELISA, N = 3, n = 3), and differential protein expression by liquid chromatography-tandem mass spectrometry (LC-MS/MS, n = 2). RESULTS: Cells stimulated at MOI 100/1000 and treated with 10 µM epigallocatechin-gallate and chlorhexidine did not present cytotoxicity. IL-1ß significantly increased in both un-stimulated and stimulated chlorhexidine 10 µM groups when compared to un-treated control (p < 0.05). MOI 100 chlorhexidine 10 µM group significantly increased IL-1ß compared to un-stimulated chlorhexidine 10 µM and epigallocatechin-gallate 10 µM groups, as well as to MOI 100 epigallocatechin-gallate 10 µM group (p < 0.05). LC-MS/MS revealed S. mutans and mammalian proteins, with tooth-specific proteins exhibiting different abundance levels, depending on the tested condition. CONCLUSIONS: Odontoblast-like cells stimulated with S. mutans at different MOI combined with epigallocatechin-gallate treatment did not cause cytotoxicity. S. mutans stimulation combined with chlorhexidine 100 µM treatment decreased cell viability, while treatment with chlorhexidine 10 µM concentration significantly increased IL-1ß. S. mutans stimulation and treatment of cells resulted in varied protein expression.

Analysis of permeability and biological properties of dentin treated with experimental bioactive glasses.

OBJECTIVES: To evaluate obliterating capability and biological performance of desensitizing agents. METHODS: 50 dentin blocks were distributed according to the desensitizing agent used (n = 10): Control (Artificial saliva); Ultra EZ (Ultradent); Desensibilize Nano P (FGM); T5-OH Bioactive Glass (Experimental solution); F18 Bioactive Glass (Experimental solution). Desensitizing treatments were performed for 15 days. In addition, specimens were subjected to acid challenge to simulate oral environment demineralizing conditions. Samples were subjected to permeability analysis before and after desensitizing procedures and acid challenge. Cytotoxicity analysis was performed by using Alamar Blue assay and complemented by total protein quantification by Pierce Bicinchoninic Acid assay at 15 min, 24-h and 48-h time points. Scanning electron microscopy and energy dispersion X-ray spectroscopy were performed for qualitative analysis. Data of dentin permeability was analyzed by two-way repeated measures ANOVA and Tukey's test. For cytotoxicity, Kruskal-Wallis and Newman-Keuls tests. RESULTS: for dentin permeability there was no significant difference among desensitizing agents after treatment, but control group presented highest values (0.131 ± 0.076 Lp). After acid challenge, control group maintained highest values (0.044 ± 0.014 Lp) with significant difference to other groups, except for Desensibilize Nano P (0.037 ± 0.019 Lp). For cytotoxicity, there were no significant differences among groups. CONCLUSION: Bioglass-based desensitizers caused similar effects to commercially available products, regarding permeability and dentin biological properties. CLINICAL SIGNIFICANCE: There is no gold standard protocol for dentin sensitivity. The study of novel desensitizing agents that can obliterate dentinal tubules in a faster-acting and long-lasting way may help meet this clinical need.

Functionalized epigallocatechin gallate copolymer inhibit dentin matrices degradation: Mechanical, solubilized telopeptide and proteomic assays.

OBJECTIVES: We investigated the biostability of dentin organic matrices treated with epigallocatechin gallate (EGCG) in comparison to chlorhexidine (CHX), both extracted from functionalized copolymers. METHODS: Copolymers were prepared with bis-GMA:TEGDMA and incorporated with 1% of EGCG or CHX (w/w). Blank copolymers were used as control. Copolymer samples were individually stored in 1mL deionized water to produce copolymer extracts. Dentin matrices were obtained by demineralization of dentin disks in 10% phosphoric acid solution. Matrices were individually treated with 1mL of the copolymer extracts or distilled water for 48h. Collected extracts were analyzed by high-performance liquid chromatography (HPLC) for the presence and quantification of EGCG, CHX, and copolymer by-products. Treated dentin matrices were tested for ultimate tensile strength, gravimetric changes, and swelling ratio. The treatment media were tested for total protein concentration, and dentin protease activity through solubilized telopeptide (ICTP- and CTX-ELISA) assays. The treatment media were also submitted to proteomic analysis. RESULTS: HPLC identified released unreacted copolymer species and showed higher release of CHX compared to EGCG from respective copolymer extracts. EGCG extract inhibited activity of dentin proteolytic enzymes and promoted collagen biomodification observed by the telopeptide assays and in the changes to dentin matrix properties. The proteomic results showed less collagenous peptide hits in the EGCG extract media compared to CHX, and suggest compound-specific dentin protein binding interactions. SIGNIFICANCE: This study demonstrates specific antiproteolytic effect and protein interactions of EGCG copolymer extract directly on dentin. This represents an advancement in dental materials which can impact the clinical procedures.

Profiling cytokine levels in chlorhexidine and EGCG-treated odontoblast-like cells.

OBJECTIVE: To screen the effect of two compounds, chlorhexidine diacetate (CHX) and epigallocatechin-gallate (EGCG), on the levels of cytokines produced by odontoblast-like cells (MDPC-23). METHODS: Cells were seeded at 24h and 48h with serial dilution of the compounds to determine cell metabolic activity by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (n=3). Cells with no compound treatment were used as control (Ctr). For the highest equal non-cytotoxic compound dilution tested at 48h cell treatment, total protein concentration was measured using a Pierce bicinchoninic acid (BCA) assay (n=3), and expression of 23 cytokines was analyzed using the Bio-Plex cytokine assay (n=2). Data were analyzed by one-way ANOVA and Tukey's test (α=5%). RESULTS: The MTT assay revealed that at 24h and 48h, CHX and EGCG did not reduce cell metabolic activity at concentrations of 2.5-20µM (CHX) and 2.5-160µM (EGCG), respectively (p>0.05). At 48h, total protein levels were consistent across all groups for 20µM compound dilution (Ctr: 1.04mg/mL; CHX: 0.98mg/mL; and EGCG: 1.06mg/mL). At 20µM dilution, both CHX and EGCG significantly increased the secretion of IL-1ß, IL-10, IL-12, KC, MIP-1α, IFN-γ and IL-6 (p<0.05). Treatment with CHX significantly increased secretion of IL-4 and RANTES (p<0.05). TREATMENT: with EGCG significantly increased Eotaxin secretion (p<0.05). Both CHX and EGCG significantly decreased secretion of IL-17 (p<0.05). GM-CSF and TNF-α did not present significant change in secretion after treatment with either CHX or EGCG (p>0.05). SIGNIFICANCE: Both CHX and EGCG modulate secretion of various inflammatory and anti-inflammatory mediators in odontoblastic cells.

Cytotoxicity and potential anti-inflammatory activity of velutin on RAW 264.7 cell line differentiation: Implications in periodontal bone loss.

OBJECTIVES: Hypoxia-inducible factor-1α (HIF-1α) has been implicated in periodontal tissue inflammation and possibly in osteoclast differentiation, while polyphenols are known to be anti-inflammatory natural compounds that are capable of regulating the NF-κB protein complex pathway. The objective of this study was to investigate cytotoxicity and HIF-1α expression through the NF-κB pathway by polyphenol velutin (Euterpe oleracea Mart.), found in the pulp of acai fruit, during inflammatory RAW 264.7 differentiation. DESIGN: RAW 264.7 mouse monocyte macrophage cells were stimulated with RANKL (30ng/mL) and Porphyromonas gingivalis lipopolysaccharide (1µg/mL). Cells were treated with various concentrations of velutin (0.5-2µM) to check for viability, morphology, osteoclast differentiation, and HIF-1α expression (Western blot). RESULTS: Alamar blue cell viability assay showed no toxicity to RAW cells with the use of velutin in all concentrations tested (p>0.05). Velutin did not induce cell apoptosis based on caspase 3/7 assay (p>0.05). Fluorescence images stained by DAPI showed no alteration in the morphology of RAW cell nuclei (p>0.05) treated with velutin. TRAP assays demonstrated a dose-dependent reduction in osteoclast formation by velutin when compared with control (p<0.05). Velutin showed a reduction in HIF-1α expression related to IκB phosphorylation when compared with control (p<0.001). CONCLUSIONS: At the tested concentrations, velutin was not cytotoxic to RAW 264.7 and differentiated cells. Velutin reduced osteoclast differentiation and downregulated HIF-1α through the NF-κB pathway.

Allium cepa L. and Quercetin Inhibit RANKL/Porphyromonas gingivalis LPS-Induced Osteoclastogenesis by Downregulating NF-κB Signaling Pathway.

Objectives. We evaluated the in vitro modulatory effects of Allium cepa L. extract (AcE) and quercetin (Qt) on osteoclastogenesis under inflammatory conditions (LPS-induced). Methods. RAW 264.7 cells were differentiated with 30 ng/mL of RANKL, costimulated with PgLPS (1 µg/mL), and treated with AcE (50-1000 µg/mL) or Qt (1.25, 2.5, or 5 µM). Cell viability was determined by alamarBlue and protein assays. Nuclei morphology was analysed by DAPI staining. TRAP assays were performed as follows: p-nitrophenyl phosphate was used to determine the acid phosphatase activity of the osteoclasts and TRAP staining was used to evaluate the number and size of TRAP-positive multinucleated osteoclast cells. Von Kossa staining was used to measure osteoclast resorptive activity. Cytokine levels were measured on osteoclast precursor cell culture supernatants. Using western blot analysis, p-IκBα and IκBα degradation, inhibitor of NF-kappaB, were evaluated. Results. Both AcE and Qt did not affect cell viability and significantly reduced osteoclastogenesis compared to control. We observed lower production of IL-6 and IL-1α and an increased production of IL-3 and IL-4. AcE and Qt downregulated NF-κB pathway. Conclusion. AcE and Qt may be inhibitors of osteoclastogenesis under inflammatory conditions (LPS-induced) via attenuation of RANKL/PgLPS-induced NF-κB activation.

Dental implant surface treatments may modulate cytokine secretion in Porphyromonas gingivalis-stimulated human gingival fibroblasts: a comparative study.

Peri-implantitis is an inflammation that affects dental implants and can lead to implant loss. The aim of this study was to analyze the in vitro effect of different implant surface treatments on cytokine production by human gingival fibroblasts (HGFs) stimulated or not with Porphyromonas gingivalis lipopolysaccharide (PgLPS). Six different titanium implants were tested: turned, sandblasted, anodized, acid-etched, TiO2-blasted/acid-etched, and grit-blasted/acid-etched. HGFs were seeded with each implant in a 6-well plate and assayed before LPS treatment (-LPS) or after 36 h of LPS (+LPS) treatment. Protein concentrations were measured using a Pierce bicinchoninic acid (BCA) assay and cytokine secretions were analyzed using a multiplex cytokine array. Scanning electron microscopy was performed for sterile implants and after cell attachment. Protein levels were consistent across all implants indicating that cell growth was uniform (p > 0.05). Sandblasted and turned surfaces significantly increased the secretion of interleukin (IL)-6, -8, -10, MCP-1 and VEGF (p < 0.05) when compared with the other surfaces. PgLPS stimulus increased cytokine secretion in all tested surfaces. In conclusion, different implant surfaces had various effects on HGFs' cytokine secretion. The findings may provide insights into the progression of peri-implantitis.

Effect of Allium cepa L. on Lipopolysaccharide-Stimulated Osteoclast Precursor Cell Viability, Count, and Morphology Using 4',6-Diamidino-2-phenylindole-Staining.

Allium cepa L. is known to possess numerous pharmacological properties. Our aim was to examine the in vitro effects of Allium cepa L. extract (AcE) on Porphyromonas gingivalis LPS and Escherichia coli LPS-stimulated osteoclast precursor cells to determine cell viability to other future cell-based assays. Osteoclast precursor cells (RAW 264.7) were stimulated by Pg LPS (1 µg/mL) and E. coli LPS (1 µg/mL) in the presence or absence of different concentrations of AcE (10-1000 µg/mL) for 5 days at 37°C/5% CO2. Resazurin reduction and total protein content assays were used to detect cell viability. AcE did not affect cell viability. Resazurin reduction assay showed that AcE, at up to 1000 µg/mL, did not significantly affect cell viability and cellular protein levels. Additionally a caspase 3/7 luminescence assay was used to disclose apoptosis and there was no difference in apoptotic activity between tested groups and control group. Fluorescence images stained by DAPI showed no alteration on the morphology and cell counts of LPS-stimulated osteoclast precursor cells with the use of AcE in all tested concentrations when compared to control. These findings suggest that Allium cepa L. extract could be used for in vitro studies on Porphyromonas gingivalis LPS and Escherichia coli LPS-stimulated osteoclast precursor cells.