Uptake of Nanotitania by Gingival Epithelial Cells Promotes Inflammatory Response and Is Accelerated by Porphyromonas gingivalis Lipopolysaccharide: An In Vitro Study.

Titanium is often used in the medical field and in dental implants due to its biocompatibility, but it has a high rate of leading to peri-implantitis, which progresses faster than periodontitis. Therefore, in the present study, the expression of cytokines from gingival epithelial cells by nanotitania was investigated, which is derived from titanium in the oral cavity, and the additional effect of Porphyromonasgingivalis (periodontopathic bacteria) lipopolysaccharide (PgLPS) was investigated. Ca9-22 cells were used as a gingival epithelial cell model and were cultured with nanotitania alone or with PgLPS. Cytokine expression was examined by reverse transcription-quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. In addition, cellular uptake of nanotitania was observed in scanning electron microscopy images. The expression of interleukin (IL)-6 and IL-8 significantly increased in Ca9-22 cells by nanotitania treatment alone, and the expression was further increased by the presence of PgLPS. Nanotitania was observed to phagocytose Ca9-22 cells in a dose- and time-dependent manner. Furthermore, when the expression of IL-11, related to bone resorption, was investigated, a significant increase was confirmed by stimulation with nanotitania alone. Therefore, nanotitania could be associated with the onset and exacerbation of peri-implantitis, and the presence of periodontal pathogens may worsen the condition. Further clinical reports are needed to confirm these preliminary results.

Bone marrow-derived mesenchymal stem cells propagate immunosuppressive/anti-inflammatory macrophages in cell-to-cell contact-independent and -dependent manners under hypoxic culture.

Immunosuppressive/anti-inflammatory macrophage (Mφ), M2-Mφ that expressed the typical M2-Mφs marker, CD206, and anti-inflammatory cytokine, interleukin (IL)-10, is beneficial and expected tool for the cytotherapy against inflammatory diseases. Here, we demonstrated that bone marrow-derived lineage-positive (Lin+) blood cells proliferated and differentiated into M2-Mφs by cooperation with the bone marrow-derived mesenchymal stem cells (MSCs) under hypoxic condition: MSCs not only promoted proliferation of undifferentiated M2-Mφs, pre-M2-Mφs, in the Lin+ fraction via a proliferative effect of the MSCs-secreted macrophage colony-stimulating factor, but also promoted M2-Mφ polarization of the pre-M2-Mφs through cell-to-cell contact with the pre-M2-Mφs. Intriguingly, an inhibitor for intercellular adhesion molecule (ICAM)-1 receptor/lymphocyte function-associated antigen (LFA)-1, Rwj50271, partially suppressed expression of CD206 in the Lin+ blood cells but an inhibitor for VCAM-1 receptor/VLA-4, BIO5192, did not, suggesting that the cell-to-cell adhesion through LFA-1 on pre-M2-Mφs and ICAM-1 on MSCs was supposed to promoted the M2-Mφ polarization. Thus, the co-culture system consisting of bone marrow-derived Lin+ blood cells and MSCs under hypoxic condition was a beneficial supplier of a number of M2-Mφs, which could be clinically applicable to inflammatory diseases.

Novel SCRG1/BST1 axis regulates self-renewal, migration, and osteogenic differentiation potential in mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) remodel or regenerate various tissues through several mechanisms. Here, we identified the hMSC-secreted protein SCRG1 and its receptor BST1 as a positive regulator of self-renewal, migration, and osteogenic differentiation. SCRG1 and BST1 gene expression decreased during osteogenic differentiation of hMSCs. Intriguingly, SCRG1 maintained stem cell marker expression (Oct-4 and CD271/LNGFR) and the potentials of self-renewal, migration, and osteogenic differentiation, even at high passage numbers. Thus, the novel SCRG1/BST1 axis determines the fate of hMSCs by regulating their kinetic and differentiation potentials. Our findings provide a new perspective on methods for ex vivo expansion of hMSCs that maintain native stem cell potentials for bone-forming cell therapy.

Proteome analyses of human macrophages exposed to low cytotoxic IC90 copper (2+) ions.

Dental noble alloys often contain copper (Cu). Eluted metal ions sometimes irritate oral tissues. The most eluted ions are Cu ions. The purpose of this study was to investigate the effects of low cytotoxic (IC90, 100 µmol/L) Cu ions on macrophages by proteome analyses consisting of two-dimensional (2D) electrophoresis and matrix-assisted laser desorption/ionization -time of flight (MALDI-TOF) mass spectrometry. The analyses revealed that stimulation with IC90 Cu ions for 1 day caused the macrophage to significantly increase five specific protein spots. Mascot peptide mass finger-print matching suggested that four of them were attributed to 70 kDa heat shock protein 1A/1B (HSP70). HSP70 expression was verified by expressions of corresponding HSPA1A and HSPA1B mRNAs of the macrophage in quantitative real-time PCR analyses. It was concluded that by producing abundant HSP70, the macrophage protected itself against intracellularly intruding cytotoxic Cu ions that might un-fold and crosslink cellular proteins.

Microscopic observations and inflammatory cytokine productions of human macrophage phagocytising submicron titanium particles.

This study was performed to microscopically observe and measure inflammatory cytokine production by human macrophages phagocytosing submicron titanium (Ti) particles. Observations with secondary electron microscopy (SEM), SEM/electron probe microanalysis (EPMA) and transmission electron microscopy (TEM) indicated that macrophages [phorbol-12-myristate-13-acetate (PMA)-differentiated THP-1 cells] at 24 h in culture actively phagocytosed and accumulated submicron Ti particles in intracellular phagosomes, in which refinement of Ti particles occurred. The macrophages were also cultured for 24 h in four media with and without submicron Ti particles and lipopolysaccharide (LPS; components of bacteria). Whilst neither stimulus reduced cell viability, submicron Ti particles and LPS activation independently and synergistically caused the macrophages to produce three inflammatory cytokines (TNF-alpha, IL-1beta and IL-6) at high levels in the culture supernatants. The inflammatory and osteolysis conditions caused by macrophages phagocytosing submicron Ti particles would be worsened by challenge with LPS in patients wearing Ti prostheses.

Gene expression analyses of human macrophage phagocytizing sub-micro titanium particles by allergy DNA chip (Genopal).

The purpose of this study was to examine gene expressions of macrophage phagocytizing sub-micro Ti particles by a DNA chip. Human monocytic cell line THP-1 was differentiated into macrophages by culturing for two days in medium supplemented with 200 nM phorbol ester (PMA). The macrophages were then cultured in four media: medium without PMA (control); medium with suspended sub-micro Ti particles (0.5 wt%); medium with 1.0 microg/ml lipopolysaccharide (LPS); and medium with LPS and Ti particles. After 6 hours' culture, total RNA were extracted and gene expressions were evaluated by DNA allergy chip with 205 allergy and inflammation related gene spots. We found that phagocytosis of sub-micro Ti particles and LPS independently and synergistically up-regulated 17 inflammation-related genes more than two-fold. The extensive expressions of four genes (CCL1, IL1B, IL6 and IL8) were further confirmed by real-time quantitative PCR. It turned out that dual stimulation of LPS and Ti particles most up-regulated three genes (IL1B, IL6 and IL8), followed by LPS while Ti particles moderately but least increased, suggesting that phagocytosis of sub-micro Ti particles induces moderate inflammation with its degree less than LPS, but phagocytosis of sub-micro Ti particles has the potential to worsen inflammation caused by LPS-stimulated macrophages.

Dose-dependent effects of Ni (II) ions on production of three inflammatory cytokines (TNF-alpha, IL-1beta and IL-6), superoxide dismutase (SOD) and free radical NO by murine macrophage-like RAW264 cells with or without LPS-stimulation.

The effect of Ni (II) ions on macrophages is not well understood. The purpose of this study was to examine the dose-dependent effects of Ni (II) ions up to 1,000 micromol/L on production of three inflammatory cytokines (TNF-alpha, IL-1beta and IL-6), superoxide dismutase (SOD) and nitric oxide (NO) by murine macrophage-like RAW264 cells with (+) or without (-) lipopolysaccharide (LPS) -stimulation. Ni (II) ions caused LPS (-) RAW264 cells to slightly increase production of TNF-alpha and IL-6, proportionally to the Ni (II) ion concentration while IL-1beta was not produced, and to slightly increase production of SOD and NO. It can be concluded that Ni (II) ions dose-dependently increased the inflammatory and oxidative stress conditions of LPS (-) RAW264 cells. LPS-stimulation caused RAW264 cells to produce in abundance the three inflammatory cytokines, SOD and NO. Ni (II) ions dose-dependently reduced the three cytokine quantities and NO amounts in LPS (+) RAW264 cells, while dose-independently increasing SOD amounts. It was noted that Ni (II) ions dose-dependently reduce the resistance power against bacteria of LPS (+) macrophages, because the production of volatile NO--bacteria killer is diminished proportionally to the Ni (II) ion concentration.

Inhibitory effect of triamcinolone acetonide on corneal neovascularization.

BACKGROUND: Corneal neovascularization (NV) plays an important role in the pathogenesis of corneal disorders. Recently, triamcinolone acetonide (TA) has been reported as a potential treatment for ocular angiogenesis. However, there are no reports on the inhibitory effect of TA on the corneal NV. METHODS: Triamcinolone acetonide (2 mg) was administered to four rabbits' eyes by a subconjunctival injection immediately after a basic fibroblast growth factor (bFGF)-pellet was placed into the cornea. As a control, four eyes received an injection of distilled water. Four weeks later, the inhibition of corneal NV was evaluated as the percentage ratio of the vessel invasion area to the area that was sandwiched between the pellet and the limbus cornea. To identify the characteristic appearance of new corneal vessels, the control cornea was examined by using the antibody of vascular endothelial growth factor (VEGF). To confirm TA concentration in TA-treated corneas, the TA level was measured using high-performance liquid chromatography. RESULTS: Neovascularization from the limbus to the pellet was detected in control eyes 4 weeks after the bFGF pellet implantation. TA-treated eyes demonstrated the inhibition of the neovascular response to the pellet. The severity of NV as compared between control and TA-treated eyes was statistically significant (P<0.05). Morphologically, new vessel growth was shown in the control cornea, and endothelial cells of new vessels were positively stained with the antibody of VEGF. TA concentration in TA-treated corneas at 2 weeks showed 63.5+/-42.8 microg/g (n=4, mean +/- SD), while TA was not detected in control and TA-treated corneas at 4 weeks. The level of TA was effectively maintained for at least 2 weeks after the subconjunctival injection. CONCLUSION: We have demonstrated that subconjunctival TA administration inhibited rabbit corneal NV. This agent may prove useful in the treatment of corneal angiogenic disorders.

Accumulation of element Ti in macrophage-like RAW264 cells cultured in medium with 1 ppm Ti and effects on cell viability, SOD production and TNF-alpha secretion.

The adverse effect of Ti on body-defense macrophage is not well understood. The aims of this study were twofold: (1) to examine the intracellular accumulation of Ti element; and (2) to measure the cell viability, superoxide dismutase (SOD) production, and TNF-alpha secretion of macrophage-like RAW264 cells cultured for two days in medium with 1 ppm Ti prepared from acidic ICP Ti standard solution. PIXE analysis showed that element Ti was accumulated up to 7.3 ppm in RAW264 cells when cultured in the medium with 1 ppm Ti. Further, RAW264 cells cultured in the medium with 1 ppm Ti exhibited cell viability of about 60%, SOD production of about 180%, and TNF-alpha secretion of about 170% relative to those of control cells cultured in the medium without Ti. It was speculated that phagocytosis of minute Ti-containing complex (mostly TiO2) by macrophage caused oxidative stress and inflammatory reaction, leading to cell proliferation arrest and increased production of SOD and TNF-alpha.

Effects of Ni2+ ions on cell viability and NO production of murine peritoneal exudate cells (macrophages) with and without lipopolysaccharide stimulation.

The purpose of this study was to clarify the cytotoxicity of Ni2+ ions against murine peritoneal exudate cells (PEC) (macrophages). First, we examined the cell viability of PEC with and without lipopolysaccharide (LPS) stimulation in culture media containing Ni2+ ions up to 1000 micromol/L. Results showed that the cytotoxicity of Ni2+ ions against PEC was dose-dependent and accelerated by LPS stimulation, especially in media with Ni2+ ions exceeding 100 micromol/L. Second, we measured the production of nitric oxide (NO) from PEC and found that LPS caused the PEC to produce abundant NO. However, high dose of Ni2+ ions at concentration more than 200 micromol/L hindered and inhibited NO production. These results pointed out that the cytotoxicity of Ni2+ ions against macrophages depended on both the Ni2+ ion concentration and the presence of bacteria with LPS. Further, NO--a killer of bacteria--was lost when LPS-stimulated macrophages were exposed to high dose of Ni2+ ions.