Page 270 - Prevention and Treatment of Incisional Hernia- New Techniques and Materials

Chapter 13
slides were analysed with an Olympus BX50 light microscope (Olympus, Tokyo, Japan).
Monocyte isolation
Ficoll density gradient (Ficoll-PaqueTM PLUS; GE Healthcare, St Giles, UK) was used to isolate monocytes from the bu y coat of four healthy donors (men and women aged 25–65 years). All bu y coats were obtained from the blood bank (Sanquin, Rotterdam, The Netherlands). Some 30 ml of 1 : 5 diluted bu y coat with 0.1 per cent bovine serum albumin (BSA) in phosphate-bu ered saline (PBS) was layered on 15 ml Ficoll. After 15 min centrifugation at 1000g with no brake, the interphase band containing peripheral blood mononuclear cells was aspirated and washed in PBS/BSA 0.5 per cent 2 mmol/l EDTA and labelled with 100 μl anti- CD14+ magnetic beads (CD14 microbeads human, MACS Separation columns LS and MidiMACSTM Separator; all from Miltenyi Biotec, Bergisch Gladbach, Germany), and isolated according to the manufacturer’s guidelines. This positive selection of monocytes will not activate the cells(16). Purity of the isolation was assessed by uorescence-activated cell sorting (FACS) analysis, in which 1 × 106 monocytes were incubated for 15 min at room temperature with the following antibodies: FITC-conjugated CD14 and peridinin chlorophyll protein complex (PerCP)-conjugated CD45 (all BD Biosciences, Franklin Lakes, New Jersey, USA). After incubation, cells were washed in PBS/BSA 0.1 per cent and FACS analysis was performed with CellQuestTM Pro on a FACSCaliburTM (both BD Biosciences); the purity of the freshly isolated CD14+ monocytes was above 95 per cent (data not shown). In the case of donors 1, 2 and 4, the yield of monocytes was not su cient to allow for testing of all four biomaterials in the experiments.
Culturing macrophages on biomaterials
Four di erent biomaterials were chosen to study macrophage response in relation to the biomaterial (all from Covidien – Sofradim Production): a multi lament PP biomaterial (ParieteneTM), hydrophobic with a contact angle of 95°; a collagen-based material (COL) (PermacolTM), processed from porcine skin and cross-linked with hexamethylene di-isocyanate; a multi lament polyethylene terephthalate (PET) biomaterial, hydrophilic with a contact angle of 80.9°; and a multi lament PET biomaterial with an absorbable, continuous and hydrophilic collagen lm on one of its sides (PET+COL) (ParietexTM Composite). The PET and PP biomaterials have a similar weave (Figure 1).
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