Informed healthy blood donors (n=10) from both genders and in the age range of 21-50 were recruited following written consent. The study was approved by the University of Brighton local research ethical committee. Heparinized venous peripheral blood samples (20 ml) were collected on different days and mononuclear cells were freshly isolated within 2 h from vein puncturing using a Histopaque-1077 medium (Sigma, UK) by the Boyum method as previously described (Santin et al.2004). The cells were washed three times in phosphate-buffered saline pH 7.2 (PBS, Oxoid, UK) and re-suspended in DMEM enriched with 10% (v/v) heat-inactivated foetal calf serum (FCS). Cell viability was assessed using trypan blue staining and always found to be higher than 95%. The cell isolation step also provided a fraction of platelet-rich plasma (PRP) which was collected and stored at room temperature in sterile conditions until use (approximately 30 min).  PRP was used for preconditioning both the NP and the 24-well tissue culture plate (TCP) for 30 min. Mononuclear cells (1×105 cells/ml) were seeded onto a PRP pre-conditioned TCP and incubated for 3 h at 37oC, in 5% CO2 and 95% air. Meanwhile, 5 mg of each NP [Table 1] were re-suspended in 10% (v/v) FCS-supplemented DMEM and sonicated for 5 min. The medium was removed from the adhering mononuclear cells and the samples were washed 3 times by 10% (v/v) FCS-supplemented DMEM and finally incubated in 1 ml of the same medium containing 0.05 mg/ml of the NP. NP-spiked samples were incubated overnight (20 h) at 37oC, in 5% CO2 and 95% air in static conditions. MM adhering on PRP pre-conditioned TCP (Nunc, UK) were used as controls.

Adhering cell SEM analysis, staining and immunocytochemistry.

After 20 h incubation, adhering cells were washed three times in PBS and then fixed in 3.7% (v/v) formaldehyde (Sigma,UK) in PBS. Following fixing, cell samples (n=2) were either prepared for SEM or processed for epifluorescence microscopy. Specimens for SEM were prepared by dehydration in increasing ethanol concentrations (25, 50, 75 and 90% by volume), air-dried, sputter-coated by palladium and finally analysed by SEM at 5Kv, at different magnifications. SEM analysis focussed on MM and platelet morphology as well as on their interactions with the NP. In particular, the degree of plasmalemma roughness and cell spreading were evaluated in the case of MM, while aggregation and fragmentation were assessed in platelets. Where detectable, platelet analysis was associated with observations of NP-induced fibrin polymerization as a measure of the NP potential to induce clotting.

 

Phalloidin staining was performed by making the cell membrane permeable to the dye by treatment in 0.1% (w/v) Triton X-100 in PBS followed by incubation with phalloidin-tetremethylrhodamine isothiocyanate (Sigma, UK) for 30 m, at room temperature, in dark conditions. The samples were washed twice in PBS prior to final analysis at 40x magnification by a fluorescent inverted microscope (Eclipse TE2000-U, Nikon, UK) connected to a digital camera (D1x, Nikon, UK).

To confirm the presence of MM cells, the fixed samples were subjected to immunolabelling by CD68 antibody, a marker for both monocytes and macrophages. The samples were blocked in 4% (w/v) bovine serum albumin  for 30 min, incubated in 1:1000-diluted mouse monoclonal anti CD68 (Vector Labs, UK) solution overnight, room temperature, and finally washed with an excess of PBS and incubated in anti-mouse AlexaFluor 548 tagged antibody (Invitrogen, UK) for 1h, room temperature. The samples (n=3) were analysed by inverted fluorescent microscope as described above. The morphology of MM when incubated with the different NP and their marker expression were compared to that of control MM adhering on plasma preconditioned TCP without any NP challenge.

Evaluation of NP-induced biochemical signalling and cytotoxicity in monocytes/macrophages.

The supernatants from each experiment were collected and centrifuged at 1,000 x g to eliminate any unbound cells and stored at -70oC until tested by ELISA kits for PDGF-BB (PeproTech EC, UK, catalogue no. 900-K04), VEGF (R&D Systems, UK, catalogue no. DVE00) and TNFα (Amersham, UK, catalogue no. RPN2781). TNFalpha secretion was evaluated as a marker of MM pro-inflammatory phenotype, while PDGF-BB and VEGF were tested to assess the cell potential to stimulate blood vessel formation (VEGF) and fibroblast proliferation (PDGF-BB) and, therefore, as markers of granulation and fibrous tissue formation. Standard curves with a linear coefficient no lower than 0.99 were obtained in triplicate by the kit pure growth factor standards. Absorbance reading from the blank (10% FCS-enriched tissue culture medium) were subtracted from the sample values and data were expressed as pg/ml mean ± standard deviation from n>5.

Cytotoxicity was evaluated by lactate dehydrogenease (LDH) activity assay of the supernatants using the Cytotox 96 kit (Promega, UK) (Levinsky et al. 2008). The assay measures cell LDH activity via the oxidation of NADH to NAD+ in the presence of lactate and pyruvate. The enzyme activity was measured by reading the absorbance at 490nm by spectrophotometer (ELx800 BioTek, UK). Positive controls were obtained from the measurement of the LDH activity liberated from the cytoplasm of Triton-lysed cells (1×105 cells/ml). Data were expressed as mean ± standard deviation of the enzymatic arbitrary units from n=6.

Statistical Analysis

Data were statistically analysed by pair t tests and by ANOVA (Tukey’s test) to allow comparative analysis across the samples. Data analysis was performed by Minitab Release 15 software (Minitab, YK) and samples were considered significantly different at either p ≤ 0.01 or p ≤ 0.05.

 

 

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