Page 271 - Prevention and Treatment of Incisional Hernia- New Techniques and Materials

Macrophages in a contaminated environment
Figure 1. Detailed picture of wave pattern of polypropylene (A) and polyethylene terephthalate multi laments (B)
The materials were cut into 1.5 × 1.5-cm pieces with a sterile scalpel. Before cell seeding, materials were incubated in 100 per cent non-heat-inactivated fetal calf serum (FCS) (Lonza, Verviers, Belgium) for 2 h to provide protein attachment. Freshly isolated monocytes were adjusted to a concentration of 0.7 × 106 cells/ ml in a total volume of 25 ml in a 50-ml PP tube (FalconTM; Becton, Dickinson, Franklin Lakes, New Jersey, USA). Twelve samples were incubated per 25 ml for 2 h at 37°C. Subsequently, samples were placed in a 24-well non-adherent plate (NUNCTM, non-treated multiplate; Thermo Scienti c, Rochester, New York, USA) and cultured for 3 days in serum-free X-VIVOTM 15 medium with 20 per cent FCS (Lonza). To simulate an in ammatory environment caused by bacterial infection, macrophages on biomaterials were cultured with 10 ng/ml LPS (Sigma-Aldrich, St Louis, Missouri, USA) and 1 ng/ml recombinant human IFN-γ (PeproTech, Rocky Hill, New Jersey, USA), and compared with macrophages on the same materials without simulation. The medium was refreshed after 48 h of culturing, and after a further 24 h in culture the supernatant was harvested for protein analysis.
Analysis of the production of in ammatory and anti-in ammatory cytokines
Proteins were measured in 25 μl cell culture supernatant using a multiplex system (Millipore, Billerica, Massachusetts, USA)(17). IL-1β, IL-6, TNF-α, monocyte chemotactic protein (MCP) 3 and macrophage in ammatory protein (MIP) 1α, IL-1RA, RANTES (regulated on activation, normal T cell expressed and secreted, or CCL5), and macrophage-derived chemokine (MDC, or CCL22) were measured according to manufacturer recommendations. The CCL18 DuoSet®
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