Inflammatory Response to Wear Particles: Comparisons Between Zirconia and Titanium In Vitro.

PURPOSE: To compare the inflammatory responses of peripheral blood mononuclear cells (PBMCs) subjected to titanium (Ti) and/or zirconia (Zr) particles while growing on Ti or Zr discs. MATERIALS AND METHODS: In total, 240 discs were fabricated at a size of 2 mm in height and 5 mm in diameter. Of the 240 discs, 120 discs were made of Ti (commercially pure [CP] grade 4), and 120 discs were made of Zr (3 mol% yttria-stabilized zirconia polycrystals [3Y-TZP]). The PBMCs were cultured on the two-disc materials, and particles with a size up to 20 mm Ti (99.5% Ti) and 0.1 to 0.2 mm Zr (3Y-TZP) were added to the cultures. The concentration levels of inflammatory cytokines in culture supernatants were measured through Bio-Plex assay (Bio-Rad Laboratories), and light microscopic analysis was performed to detect cell attachment and characterize particle shape and cell-particle interaction. RESULTS: The inflammatory responses of PBMCs were generally higher when cells were cultured on a Ti surface compared to a Zr surface. In addition, higher cytokine levels were seen when cells were cultured in the presence of Ti particles compared to Zr particles when no discs were used. However, there were only significantly increased levels for three cytokines (MCP-1, IFN-γ, and TNF-α) when particles were added to Ti discs. Higher release of neutrophil extracellular traps (NETs) from neutrophils were seen in presence of Zr particles compared to Ti particles. A reduction in cell death was observed in the presence of Zr particles compared to Ti particles and unstimulated control samples. CONCLUSIONS: The type of growth material and presence of particle affects PBMCs in vitro. Cells seeded on Ti discs and together with Ti particles generated higher levels of inflammatory cytokines compared to the Zr counterparts.

The effect of a non-tobacco-based nicotine pouch on mucosal lesions caused by Swedish smokeless tobacco (snus).

Oral mucosal lesions are commonly found in Swedish smokeless tobacco (snus) users where the pouch is placed. These lesions are reversible, that is, clinical and histological tissue changes return to normal following cessation. However, the exact mechanisms behind these changes are unknown. The main aim of this study was to investigate how snus-like non-tobacco-based nicotine pouches affect the oral mucosa and the severity of pre-existing lesions. Sixty regular users of Swedish smokeless tobacco were encouraged to substitute their snus with non-tobacco-based nicotine pouch products during a 6-week period. Meanwhile, oral mucosal lesions were assessed using a four-degree scale. Over time, a reduction of pre-existing mucosal lesions was observed between baseline and the final visit. In a second part, the effect of exposure to regular snus on the production of 48 different cytokines in peripheral blood mononuclear cells was compared in vitro with that resulting from exposure to the non-tobacco-based nicotine products. Results showed significantly increased production of proinflammatory cytokines in cells exposed to regular snus compared to untreated or cells exposed to the non-tobacco-based nicotine products. This may be related to the improved clinical appearance of the oral mucosa in the participants that used the non-tobacco-based nicotine test pouches.

Immunological response of human leucocytes after exposure to lipopolysaccharides from Porphyromonas gingivalis.

Porphyromonas gingivalis (P. gingivalis) is a gram-negative bacterium and an important etiologic agent of periodontitis. P. gingivalis releases outer membrane vesicles containing lipopolysaccharides (LPS), which can penetrate periodontal tissues. Once in the periodontal tissues and in contact with immune cells, it may participate in the destructive innate host response associated with the disease. The exact mechanism of P. gingivalis LPS in the disease process is not clear, but it is known to affect a variety of immune responses. OBJECTIVES: To investigate how LPS from P. gingivalis affect neutrophil extracellular trap (NET) formation, cell death and production of cytokines from human neutrophils and peripheral mononuclear blood mononuclear cells (PBMCs). MATERIALS AND METHODS: Isolated neutrophils and PBMCs were cultured with LPS from P. gingivalis or Escherichia coli (E. coli) (control). The NET formation was measured using Sytox green stain. Cell death of neutrophils and PBMCs was analyzed using flow cytometry or Sytox green stain. Cytokine production was measured using enzyme-linked immunosorbent assay (ELISA) kit or Bio-Plex assay. RESULTS: Exposure to LPS from P. gingivalis and E. coli caused significantly lower cell death in neutrophils. NETs were formed after exposure to the two different LPS. In PBMCs, exposure to P. gingivalis and E. coli LPS caused increased levels of IL-1ß and IL-6 compared to unstimulated controls. Increased cell death in PBMCs after exposure to LPS from E. coli in comparison to LPS from P. gingivalis and unstimulated controls was also observed. CONCLUSIONS: LPS from P. gingivalis has the ability to affect both human neutrophils and PBMCs with regard to cytokine production, cell death and production of NETs. LPS from P. gingivalis could be involved in the pathogenesis of periodontitis, and our results may contribute information regarding possible markers for diagnosis and targets for treatment of periodontal disease.

Inflammatory Response to Cobalt-Chromium Alloys Fabricated With Different Techniques.

OBJECTIVES: To explore the in vitro cytokine expression of human peripheral blood mononuclear cells exposed to cobalt-chromium alloys, manufactured with different techniques, in comparison with commercially pure titanium grade 4 and titanium alloy grade 23. MATERIAL AND METHODS: Peripheral blood mononuclear cells (PBMC) were collected from 10 healthy blood donors and exposed to machine-ground coin-shaped: (a) cobalt-chromium (Co-Cr) specimens (n = 5) manufactured by four techniques, i.e. cast, milled, laser melted and presintered milled; (b) commercially pure titanium grade 4; and (c) titanium alloy grade 23. The cells were cultured for 4, 24 and 72 hours followed by investigations of pro- and anti-inflammatory cytokine release using Bio-Plex Pro™. RESULTS: In general, the PBMC produced significantly more cytokines when exposed to the cast and presintered milled Co-Cr materials compared to laser melted, milled Co-Cr and titanium materials. CONCLUSIONS: Within the limitation of the present study, it may be suggested that cast and presintered milled cobalt-chromium alloys provoke a stronger inflammatory response compared to milled and laser melted cobalt-chromium alloys and titanium materials.

Oral Lactobacillus strains reduce cytotoxicity and cytokine release from peripheral blood mononuclear cells exposed to Aggregatibacter actinomycetemcomitans subtypes in vitro.

BACKGROUND: This study evaluated the effect of oral lactobacilli on the cytotoxicity and cytokine release from peripheral blood mononuclear cells (PBMCs) when exposed to Aggregatibacter actinomycetemcomitans subtypes in vitro. The supernatants and cell wall extracts (CWEs) of eight A. actinomycetemcomitans strains, representing different subtypes, and three Lactobacillus strains were used. The PBMCs from six blood donors were exposed to supernatants and CWEs of A. actinomycetemcomitans or Lactobacillus strains alone or combinations and untreated cells as control. The cytotoxicity was determined by trypan blue exclusion method and IL-1ß secretion by ELISA. TNF-α, IL-6, and IL-8 secretions were measured using Bioplex Multiplex Immunoassay. RESULTS: Supernatants or CWEs from all bacterial strains showed cytotoxicity and IL-1ß secretion and the subtypes of A. actinomycetemcomitans showed generally a significantly higher effect on PBMCs than that of the Lactobacillus strains. Two highly toxic A. actinomycetemcomitans strains (JP2 and JP2-like) induced a higher response than all other strains. When combined, Lactobacillus significantly reduced the toxicity and the IL-1ß secretion induced by A. acinomycetemcomitans. The effect varied between the subtypes and the reduction was highest for the JP2 and JP2-like strains. The Lactobacillus paracasei strain SD1 had a higher reducing effect than the other Lactobacillus strains. This strain had a consistent reducing effect on all subtypes of A. actinomycetemcomitans cytotoxicity, and release of IL-1ß, IL-6, IL-8, and TNF-α from PBMCs of the blood donors. A strong and significant variation in cytokine release between the six blood donors was noticed. CONCLUSIONS: Lactobacillus spp. and L. paracasei SD1 in particular, showed a limited but statistically significant reducing interaction with A. actinomycetemcomitans toxicity and release of cytokines in vitro.

Inflammatory cytokine release from human peripheral blood mononuclear cells exposed to polyetheretherketone and titanium-6 aluminum-4 vanadium in vitro.

Objective To investigate the cytokine expression profiles of blood cells exposed to polyetheretherketone and titanium-6 aluminum-4 vanadium materials in vitro. Materials and methods Coin-shaped samples composed of titanium-6 aluminum-4 vanadium, polyetheretherketone, and blasted polyetheretherketone were manufactured. The surfaces of the coins were characterized using optical interferometry, scanning electron microscopy, and contact angle measurements. Peripheral blood mononuclear cells collected from 10 blood donors were cultured for one, three, and six days in the presence or absence of the coins, and then assayed for cytokine production. Quantification of the peripheral blood mononuclear cells attached to the coins was performed using confocal microscopy after immunofluorescence staining. Results The machined titanium-6 aluminum-4 vanadium coins had a smoother surface topography compared to the machined polyetheretherketone and blasted polyetheretherketone. The highest mean contact angle was noted for the blasted polyetheretherketone, followed by the machined polyetheretherketone and titanium-6 aluminum-4 vanadium. The peripheral blood mononuclear cells produced significantly more proinflammatory cytokines when exposed to the polyetheretherketone surface compared to the titanium-6 aluminum-4 vanadium surface, while the blasted polyetheretherketone induced the highest level of proinflammatory cytokine release from the peripheral blood mononuclear cells. Significantly more cells attached to both polyetheretherketone surfaces, as compared to the titanium-6 aluminum-4 vanadium surface. Conclusion Polyetheretherketone induces a stronger inflammatory response from peripheral blood mononuclear cells than does titanium-6 aluminum-4 vanadium. Surface topography has an impact on cytokine release from peripheral blood mononuclear cells.

The Immunomodulatory Properties of 2-Hydroxyethyl Methacrylate are Mediated by the NLRP3 Inflammasome.

PURPOSE: The methacrylate monomer 2-hydroxyethyl methacrylate (HEMA), commonly used in dentistry, has multiple effects on the immune system. This study examined whether HEMA affects the immune system by inducing formation of the NLRP3 inflammasome. MATERIALS AND METHODS: Human peripheral blood mononuclear cells (PBMCs) and the human monocyte cell line THP1 were cultured with or without 1000 µM HEMA. To block NLRP3 inflammasome activation, 130 mM KCl was also added to some of the cultures. For the in vivo studies, two different experimental setups were used. In the first experimental setup, mice were injected subcutaneously at the base of the tail with 20 µmol HEMA with or without 100 mM KCl. After 3 weeks, the animals were given an identical booster injection. Two weeks after the last injection, the mice were sacrificed and splenectomized. In the second experimental setup, HEMA (20 µmol), with or without 100 mM KCl, was injected subcutaneously into the tails of BALB/c mice. The mice were given two similar injections at 3-week intervals to allow evaluation of the local inflammation induced by HEMA. After the last inoculation, the injection site was examined daily for 4 days, after which the mice were sacrificed. RESULTS: Cultures of PBMCs and THP1 cells exposed to HEMA in vitro produced more IL-1ß and IL-18 than did control cells. Increased extracellular concentration of KCl inhibited the secretion of IL-1ß. HEMA exposure did not induce cytokine production in variants of the THP1 cell line unable to form the NLRP3 inflammasome. For the first experimental setup, the level of unstimulated basic splenocyte proliferation in vitro was significantly higher in cultures from mice exposed in vivo to HEMA only than in cultures from mice injected with HEMA plus KCl. In the second experimental setup of the in vivo studies, the HEMA-treated mice developed more pronounced inflammation at the site of injection compared to the group of mice given HEMA plus KCl. CONCLUSION: HEMA affects the immune system by inducing formation of the NLRP3 inflammasome.

Sublingual administration of 2-hydroxyethyl methacrylate enhances antibody responses to co-administered ovalbumin and Streptococcus mutans.

OBJECTIVE: The oral mucosa of patients undergoing dental procedures is often exposed to residual monomers leaking from incompletely cured acrylic resins. We investigated whether 2-hydroxyethyl methacrylate (HEMA) monomers applied to the sublingual mucosa in mice modulate the antibody responses towards co-administered ovalbumin (OVA) or live oral bacteria. MATERIAL AND METHODS: OVA, live mouse oral commensal Lactobacillus murinus or live human oral commensal Streptococcus mutans were administered sublingually with or without HEMA to BALB/c mice on four weekly occasions. One week after the last administration, the experiment was terminated and serum antibody levels were analyzed using ELISA. RESULTS: Significantly increased IgG and IgE anti-OVA antibody activity was found in the sera from mice immunized with OVA together with HEMA, as compared to mice immunized with OVA alone. Likewise, S. mutans together with HEMA induced an IgG anti-S. mutans antibody response that was significantly higher than the antibody response detected after application of S. mutans alone. No IgG anti-L. murinus antibody response was detected in mice immunized with L. murinus together with HEMA, as compared to the background activity. CONCLUSIONS: We report that HEMA monomers have adjuvant properties when sublingually administered in combination with OVA or S. mutans.

Triethylene glycol dimethacrylate: adjuvant properties and effect on cytokine production.

Objective: Leakage of monomers from dental fillings due to incomplete curing is very common. The objective of the present study was to examine the cytokine profile in cells exposed to triethyleneglycol dimethacrylate (TEGDMA) and the adjuvant properties of TEGDMA. Materials and methods: Human peripheral blood mononuclear cells were exposed to TEGDMA (500 and 1000 µM) for 24 h in vitro. Bio-Plex Pro™ assays were used for analysis and detection of cytokines. In vivo, BALB/c mice were immunized subcutaneously in the base of the tail with TEGDMA in combination with ovalbumin (OVA). Results: The cytokine levels of IL-8, IL-18, GRO-α and MCP-1 were significantly increased for both concentrations. IL-1ß, IL-6 and TNF-α was only significantly increased in cultures exposed to 500 µM TEGDMA. The concentration of TNF-α was significantly decreased in cultures exposed to 1000 µM TEGDMA. Animals immunized with OVA co-administrated with TEGDMA had a significantly higher IgE and IgG anti-OVA antibody levels in blood than animals immunized with OVA only. Conclusions: TEGDMA affects production of proinflammatory cytokines IL-1ß, IL-6, IL-8, IL-18 and TNF-α. This inflammatogenic capacity renders TEGDMAs adjuvant properties, which may interfere with the homeostasis between the immune system and the indigenous microflora in the oral cavity.

Effects of the methacrylate/acrylate monomers HEMA, TEGDMA, DEGDA, and EMA on the immune system.

Incomplete curing of dental fillings may lead to leakage of methacrylate/acrylate monomers, which may come in contact with different cells of the immune system in oral tissues. Very little is known about the different immunologic effects caused by these methacrylates/acrylates. The objective of the present study was to study if and how the methacrylate/acrylate monomers ethyl methacrylate (EMA) and diethylene glycol diacrylate (DEGDA) affect the immune system in vivo and in vitro in comparison to 2-hydroxyethyl methacrylate (HEMA) and triethylene glycol dimethacrylate (TEGDMA). Human peripheral blood mononuclear cells were exposed to the different monomers (500 and 1000 µM) for 24 hr in vitro. BioPlex Pro™ assays were used for cytokine analysis. In vivo, BALB/c mice were immunized subcutaneously at the base of the tail with HEMA, TEGDMA, EMA, or DEGDA in combination with ovalbumin (OVA) in order to study adjuvant properties of the 4 monomers. Peripheral blood mononuclear cells exposed to DEGDA had viability less than 50% of the cells. A pattern was observed where the levels of most cytokines were elevated after exposure to HEMA or TEGDMA. Since that, many cells died after DEGDA-exposure, the only observed cytokine secretion was a significantly increased production of interleukin-18. In the in vivo experiments, all mice immunized with DEGDA died after the booster injection. Mice receiving OVA in combination with HEMA, TEGDMA, or EMA developed a higher immunoglobulin G anti-OVA antibody levels compared to the group immunized with OVA alone. We could not demonstrate any significant difference in antibody levels among the mice receiving the various methacrylate/acrylate monomers. The different monomers affected the production, increase and decrease, of different cytokines in vitro but resulted also in vivo in increased antibody production and T-cell activity.

The engagement of oral-associated lymphoid tissues during oral versus gastric antigen administration.

The role of oral-associated lymphoid tissues during induction of oral tolerance still remains elusive. Therefore, the aim was to compare T-cell activation and induction of tolerance to ovalbumin (OVA) presented through either of two routes; deposited into the oral cavity, or the stomach, thereby bypassing the oral cavity. OVA was administered by the oral or gastric route to BALB/c mice that had received OVA-specific DO11.10+ CD4(+) T cells, stained with CellTrace(™) Violet dye, through intravenous injection. Proliferating OVA-specific T cells were detected in the nose-associated lymphoid tissues (NALT) and the cervical, mesenteric and peripheral lymph nodes at different time-points following OVA exposure. OVA-specific T-cell proliferation was initially observed in the NALT 1 hr after oral, but not gastric, administration. However, at day 1, proliferation at this site was also detected after gastric administration and profound proliferation was observed at all sites by day 4. For the oral route the degree of proliferation observed was lower in the peripheral lymph nodes by day 4 compared with the other sites. These results demonstrate a similar activation pattern achieved by the two routes. However, the NALT distinguishes itself as a site of rapid T-cell activation towards fed antigens irrespective of feeding regimen. To evaluate induction of tolerance a semi-effective OVA dose was used, to detect differences in the degree of tolerance achieved. This was performed in a model of OVA-induced airway hypersensitivity. No differences in tolerance induction were observed between the two administration routes.

Inflammatory cytokine release is affected by surface morphology and chemistry of titanium implants.

To investigate in vitro cellular cytokine expression in relation to commercially pure titanium discs, comparing a native surface to a fluorinated oxide nanotube surface. Control samples pure titanium discs with a homogenous wave of the margins and grooves and an often smeared-out surface structure. Test samples pure titanium discs with a fluorinated titanium oxide chemistry and surface morphology with nanopore/tube geometry characterized by ordered structures of nanotubes with a diameter of ≈ 120 nm, a spacing of ≈ 30 nm, and a wall thickness of ≈ 10 nm. Cross-section view showed vertically aligned nanotubes with similar lengths of ≈ 700 nm. Peripheral blood mononuclear leucocytes were cultured for 1, 3, and 6 days according to standard procedures. BioPlex Pro™ assays were used for analysis and detection of cytokines. Selected inflammatory cytokines are reported. A pronounced difference in production of the inflammatogenic cytokines was observed. Leucocytes exposed to control coins produced significantly more TNF-α, IL-1ß, and IL-6 than the test nanotube coins. The effect on the TH2 cytokine IL-4 was less pronounced at day 6 compared to days 1 and 3, and slightly higher expressed on the control coins. The morphology and surface chemistry of the titanium surface have a profound impact on basic cytokine production in vitro. Within the limitations of the present study, it seems that the fluorinated oxide nanotube surface results in a lower inflammatory response compared to a rather flat surface that seems to favour inflammation.

Proinflammatory cytokine gene expression in the stomach correlates with vaccine-induced protection against Helicobacter pylori infection in mice: an important role for interleukin-17 during the effector phase.

CD4(+) T cells have been shown to be essential for vaccine-induced protection against Helicobacter pylori infection in mice. Less is known about the relative contributions of individual cell subpopulations, such as T(h)1 and T(h)17 cells, and their associated cytokines. The aim of the present study was to find immune correlates to vaccine-induced protection and further study their role in protection against H. pylori infection. Immunized and unimmunized mice were challenged with H. pylori, and immune responses were compared. Vaccine-induced protection was assessed by measuring H. pylori colonization in the stomach. Gastric gene expression of T(h)1- and/or T(h)17-associated cytokines was analyzed by quantitative PCR, and contributions of individual cytokines to protection were evaluated by antibody-mediated in vivo neutralization. By analyzing immunized and unimmunized mice, a significant inverse correlation between the levels of interleukin-12p40 (IL-12p40), tumor necrosis factor alpha (TNF), gamma interferon (IFN-γ), and IL-17 gene expression and the number of H. pylori bacteria in the stomachs of individual animals after challenge could be demonstrated. In a kinetic study, upregulation of TNF, IFN-γ, and IL-17 coincided with vaccine-induced protection at 7 days after H. pylori challenge and was sustained for at least 21 days. In vivo neutralization of these cytokines during the effector phase of the immune response revealed a significant role for IL-17, but not for IFN-γ or TNF, in vaccine-induced protection. In conclusion, although both T(h)1- and T(h)17-associated gene expression in the stomach correlate with vaccine-induced protection against H. pylori infection, our study indicates that mainly T(h)17 effector mechanisms are of critical importance to protection.

Sublingual immunization protects against Helicobacter pylori infection and induces T and B cell responses in the stomach.

Sublingual (SL) immunization has been described as an effective novel way to induce mucosal immune responses in the respiratory and genital tracts. We examined the potential of SL immunization against Helicobacter pylori to stimulate immune responses in the gastrointestinal mucosa and protect against H. pylori infection. Mice received two SL immunizations with H. pylori lysate antigens and cholera toxin as an adjuvant, and after challenge with live H. pylori bacteria, their immune responses and protection were evaluated, as were immune responses prior to challenge. SL immunization induced enhanced proliferative responses to H. pylori antigens in cervicomandibular lymph nodes and provided at least the same level of immune responses and protection as corresponding intragastric immunization. Protection in SL-immunized mice was associated with strong H. pylori-specific serum IgG and IgA antibody responses in the stomach and intestine, with strong proliferation and gamma interferon (IFN-γ) and interleukin-17 (IL-17) production by spleen and mesenteric lymph node T cells stimulated with H. pylori antigens in vitro, and with increased IFN-γ and IL-17 gene expression in the stomach compared to levels in infected unimmunized mice. Immunohistochemical studies showed enhanced infiltration of CD4(+) T cells and CD19(+) B cells into the H. pylori-infected stomach mucosa of SL-immunized but not unimmunized H. pylori-infected mice, which coincided with increased expression of the mucosal addressin cell adhesion molecule (MAdCAM-1) and T and B cell-attracting chemokines CXCL10 and CCL28. We conclude that, in mice, SL immunization can effectively induce protection against H. pylori infection in association with strong T and B cell infiltration into the stomach.