Novel grooved substrata stimulate macrophage fusion, CCL2 and MMP-9 secretion.

Rough surface topographies on implants attract macrophages but the influence of topography on macrophage fusion to produce multinucleated giant cells (MGC) and foreign body giant cells (FBGC) is unclear. Two rough novel grooved substrata, G1 and G2, fabricated by anisotropic etching of Silicon <110> crystals without the use of photolithographic patterning, and a control smooth surface (Pol) were produced and replicated in epoxy. The surfaces were compared for their effects on RAW264.7 macrophage morphology, gene expression, cyto/chemokine secretion, and fusion for one and five days. Macrophages on grooved surfaces exhibited an elongated morphology similar to M2 macrophages and increased cell alignment with surface directionality, roughness and cell culture time. Up-regulated expression of macrophage chemoattractants at gene and protein level was observed on both grooved surfaces relative to Pol. Grooved surfaces showed time-dependent increase in soluble mediators involved in cell fusion, CCL2 and MMP-9, and an increased proportion of multinucleated cells at Day 5. Collectively, this study demonstrated that a rough surface with surface directionality produced changes in macrophage shape and macrophage attractant chemokines and soluble mediators involved in cell fusion. These in vitro results suggest a possible explanation for the observed accumulation of macrophages and MGCs on rough surfaced implants in vivo. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2243-2254, 2016.

The effect of surface roughness on RAW 264.7 macrophage phenotype.

Monocyte-derived cells, including macrophages and foreign body giant cells, can determine the performance of implanted devices. Upon contact with biomaterials, macrophages can be activated into a classic inflammatory (M1) or wound-healing (M2) phenotype. Previously, we showed that high macrophage density on rough SLA implants was associated with early bone formation. This study examined a possible mechanism, namely, surface roughness activation of macrophages to the M2 phenotype to enhance bone formation on the SLA surface. RAW 264.7 macrophages were seeded on SLA or smooth (Po) epoxy substrates and the expression of the M1 and M2 specific markers, NOS2 and Arg-1 measured by qPCR on days 1, 3, and 5. Additionally, secretion of inflammation-associated cytokines and chemokines was studied by antibody arrays and ELISAs. Controls included RAW 264.7 macrophages primed into the M1 or M2 phenotypes by LPS/IFN-γ and IL-4, respectively. Rough SLA surfaces did not activate Arg-1 and NOS2 expression, but relative to Po surfaces MCP-1 and MIP-1α were upregulated after 5 days, whereas the secretion of the M1-associated chemokine IP-10 was lowered. RAW 264.7 macrophages on the SLA surface thus adopted elements of an M2-like phenotype, suggesting that when implanted the SLA surfaces may enhance wound repair.

Infection control in digital intraoral radiography: evaluation of microbiological contamination of photostimulable phosphor plates in barrier envelopes.

BACKGROUND AND OBJECTIVE: The detectors (both solid-state sensors and photostimulable phosphor [PSP] plates) used for digital intraoral radiography cannot be autoclaved, and barriers are typically used to prevent the spread of infection. The aim of this study was to determine the effectiveness of a barrier envelope system for PSP plates. METHODS: Disinfected PSP plates were aseptically inserted into barrier envelopes and placed in a periapical location. One PSP plate was placed in each of 28 patients, and 12 plates in each of 2 volunteers (D.S.M., J.D.W.). After retrieval, each PSP plate was removed from its barrier envelope, immersed in trypticase soy broth and aliquots were plated on trypticase soy agar. Bacterial colonies were counted 2 days later. RESULTS: Fifty-two PSP plates in barrier envelopes were evaluated for contamination. Quality assurance of the PSP plates before clinical placement revealed defects in the integrity of 4 barrier envelopes, caused by forceps-related damage or failure to achieve a uniform seal. These defects allowed substantial contamination. Contamination also occurred as a result of failure to extract the PSP plate from the barrier envelope cleanly. Of the 44 barriers with no obvious defects that were placed by either final-year dental students or a radiologist, only 3 allowed bacterial contamination of the PSP plate. CONCLUSION: Detectors contained in barrier envelopes remain a potential source of contamination. PSP plates must be disinfected between removal from a contaminated barrier envelope and placement in a new barrier envelope. In addition, placement into the barrier envelope should ideally be carried out under aseptic conditions. Finally, the integrity of each sealed barrier envelope must be verified visually before release to the clinic.

The effect of surface topography on early NFκB signaling in macrophages.

Surface topography modulates macrophage expression of pro-inflammatory cytokines through triggering of a number of different signaling pathways. In this article, we investigated the early activation of the NFκB pathway in RAW 264.7 macrophages in response to four surface topographies: mechanically polished (PO), coarse sand blasted (CB), acid etched (AE), and sandblasted and acid etched (SLA). We found that activation of the NFκB pathway was topography dependent. The PO and CB surfaces showed the highest level of activation, followed by the AE, then the SLA. Addition of suboptimal stimulatory concentrations of lipopolysaccharide (LPS) enhanced the response. Second, we determined that topography dependent cell signaling occurred in the absence of fetal bovine sera in the media. Third, we demonstrated that disruption of the lipid rafts by removal of cholesterol from cells in suspension using methyl ß cyclodextrin (MßCD) affected signaling through the NFκB pathway and transcription of the pro-inflammatory cytokine IL-1 ß, but did not affect cell adhesion, spreading or morphology. The number of macrophages adhered to the surfaces after 30 min followed the order PO, CB, AE, and SLA. In conclusion, our study suggests that one mechanism by which surface topography modulates activation of the NFκB pathway is through cholesterol-enriched raft-associated adhesive/signaling structures.

The CCL3 family of chemokines and innate immunity cooperate in vivo in the eradication of an established lymphoma xenograft by rituximab.

The therapeutic mAb rituximab induced the expression of the CCL3 and CCL4 chemokines in the human lymphoma line BJAB following binding to the CD20 Ag. Induction of CCL3/4 in vitro was specific, was observed in several cell lines and freshly isolated lymphoma samples and also took place at the protein level in vitro and in vivo. To investigate the role of these beta-chemokines in the mechanism of action of rituximab, we synthesized a N-terminally truncated CCL3 molecule CCL3(11-70), which had antagonist activity on chemotaxis mediated by either CCL3 or BJAB supernatant. We also set up an established s.c. BJAB tumor model in athymic mice. Rituximab, given weekly after tumors had reached 250 mm2, led to complete disappearance of the lymphoma within 2-3 wk. Treatment of mice with cobra venom factor showed that complement was required for rituximab therapeutic activity. Treatment of BJAB tumor bearing mice every 2 days with the CCL3(11-70) antagonist, starting 1 wk before rituximab treatment, had no effect on tumor growth by itself, but completely inhibited the therapeutic activity of the Ab. To determine whether CCL3 acts through recruitment/activation of immune cells, we specifically depleted NK cells, polymorphonuclear cells, and macrophages using mAbs, clodronate treatment, or Rag2-/-cgamma-/- mice. The data demonstrated that these different cell populations are involved in BJAB tumor eradication. We propose that rituximab rapidly activates complement and induces beta-chemokines in vivo, which in turn activate the innate immunity network required for efficient eradication of the bulky BJAB tumor.

Effect of titanium surface topography on macrophage activation and secretion of proinflammatory cytokines and chemokines.

The macrophage has a major role in normal wound healing and the reparative process around implants. Murine macrophage-like cells RAW 264.7 were used to investigate the effect of titanium surfaces on macrophage activation and secretion of proinflammatory cytokines [interleukin (IL)-1 beta, IL-6, and tumor necrosis factor (TNF)-alpha] and chemokines (monocyte chemoattractant protein-1 and macrophage inflammatory protein-1 alpha). Four topographies were used: those produced by mechanically polishing, coarse sand blasting, acid etching, and sandblasting and acid etching (SLA). Macrophages were plated on the four titanium surfaces at a population density of 5 x 10(5) cells/mL/well. Tissue culture plastic and tissue culture plastic plus lipopolysaccharide (LPS) served as negative and positive control, respectively. In addition, all surfaces were tested for their effects on macrophages in the presence of LPS. Supernatants were collected for assays after 6, 24, and 48 h and the numbers of macrophages attached to the surfaces were quantified using the DAPI (4,6-di-amidino-2-phenylindole) assay. Cytokine and chemokine levels were measured with sandwich enzyme-linked immunosorbent assays. Statistical comparison between the surfaces and the controls was determined by using the two-way analysis of variance including interaction effect (two tailed and p < or = 0.05). Unstimulated macrophages increased their secretion of the proinflammatory cytokine (TNF-alpha) when attached to rough surfaces (acid etching and SLA, p < or = 0.05). In macrophages stimulated with LPS, the roughest surface SLA produced higher levels of IL-1 beta, IL-6, and TNF-alpha at 24 and 48 h than all other surfaces (p < or = 0.05). Surface topography also modulated the secretion of the chemokines monocyte chemoattractant protein-1 and macrophage inflammatory protein-1 alpha by macrophages. Unstimulated macrophages attached to the SLA surface down-regulated their production of chemokines (p < or = 0.05) whereas LPS-stimulated macrophages attached to the SLA surface up-regulated their production (p < or = 0.05). Moreover, the SLA surface was found to act synergistically with LPS as well as the combination of blasting and etching features of the SLA surface resulted in significant release of proinflammatory cytokines and chemokines by stimulated macrophages at 24 and 48 h (p < or = 0.05). This in vitro study has demonstrated that surface topography, in particular the SLA surface, modulated expression of proinflammatory cytokines and chemokines by macrophages in a time-dependent manner.

Proinflammatory cytokine profiles in pulp fibroblasts stimulated with lipopolysaccharide and methyl mercaptan.

Pulpal disease is intimately associated with the immune system's response to bacteria products. Clinical pathology is mediated in part by the production of pyrogenic cytokines, especially interleukin (IL)-1, tumor necrosis factor (TNF)-alpha, and IL-6. Methyl mercaptan (CH3SH), a volatile sulfur compound produced by anaerobic Gram-negative bacteria, has been shown to contribute to the production of IL-1 by human mononuclear cells. In this report, we investigated the production of IL-1, TNF-alpha, and IL-6 by human pulp fibroblasts when stimulated for various periods of time with lipopolysaccharide (LPS) with or without the presence of CH3SH. We found that LPS and CH3SH had no effect on the production of IL-1 or TNF-alpha. However, LPS stimulated IL-6 production, and this production was augmented when CH3SH was present. We conclude that the volatile sulfur compound CH3SH plays a role in activation and modulation of the immune response through its role in production of IL-6.