Page 272 - Prevention and Treatment of Incisional Hernia- New Techniques and Materials

Chapter 13
ELISA (R&D Systems, Minneapolis, Minnesota, USA) was used to analyse CCL18 in 100 μl cell culture supernatant according to the manufacturer’s instructions. These nine proteins were selected based on previous experiments, where the read-out parameters were chosen after stimulation of macrophages towards either the M1 or M2 phenotype16. To correct for the numbers of macrophages on the di erent biomaterials, the cells were lysed in 0.1 per cent Triton in PBS (Sigma-Aldrich) and samples were frozen at −80°C before being analysed with CyQUANT® cell proliferation assay kit (Invitrogen, Carlsbad, California, USA). DNA content was measured according to the manufacturer’s recommendation.
Statistical analysis
The in vitro experiments were performed in triplicate with four di erent monocyte donors. All data are presented as scatterdot plots, with each dot representing an individual sample. The mean of the four donors is indicated by a line in the graphs. When evaluating the e ect of an in ammatory environment, the data are presented as the ratio of the LPS/IFN-γ-stimulated condition versus the non-stimulated condition for each biomaterial. To calculate the ratio between LPS/IFN-γ-stimulated samples and non-stimulated samples, the stimulated samples were divided by the mean of the non-stimulated samples per donor. To compare the e ect of the four biomaterials on the macrophage phenotype in an in ammatory environment, a relative M1/M2 index for each material was determined by calculating for each cytokine the percentage of production relative to the mean production on the four materials. This was followed by taking the mean of the percentages of the M1 cytokines (MIP-1α, TNF-α, MCP-3, IL-1β, IL-6) divided by the mean percentages of the M2 cytokines (MDC, RANTES, IL-1RA and CCL18) per sample. Groups were compared in SPSS® for Windows® version 20.0 (IBM, Armonk, New York, USA) using the Kruskal– Wallis test (independent samples median test) and Mann–Whitney U test, because the data were not normally distributed. Correlation between proteins was analysed by Spearman correlation. The Bonferroni correction was used. Di erences were considered statistically signi cant when P < 0.050.
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