AbstractThe cell population of peripheral blood monocytes/macrophages (MO) is heterogeneous: The majority of the MO are CD14++ CD16- and named “classical” (= MO1). Furthermore two other subpopulations were described: CD14++ CD16+ (“intermediate” = MO2) and CD14+ CD16++ (“non-classical” = MO3). It is reported that MO2 posses anti-inflammatory properties and express the MO lineage marker CD163. On a hydrophilic neutrally charged acrylamide-based hydrogel human intermediate (CD14++ CD16+) CD163++ monocytes/macrophages (aMO2) angiogenically stimulated maintained a pro-angiogenic and non-inflammatory status for at least 14 days. Here we explored whether this aMO2 subset adhered to hydrophobic poly(n-butyl acrylate) networks (cPnBA) and also remained in its pro-angiogenic and non-inflammatory status. Because substrate elasticity can impact adherence, morphology and function of cells, cPnBAs with different Young’s modulus (250 and 1100 kPa) were investigated, whereby their elasticity was tailored by variation of the crosslinker content and matched to the elasticity of human arteries.
The cPnBAs exhibited similar surface properties (e.g. surface roughness), which were maintained after ethylene oxide sterilization and exposure in serum-free cell culture medium for 18 h at 37 °C.
aMO2 were seeded on cPnBA samples (1.7 × 105 cells / 1.33 cm2) in a cell culture medium (DMEM) supplemented with VEGF-A165 (10 ng/ml) and fetal calf serum (10 vol%) for 3 and 72 hrs. On both polymeric samples (n=3 each) the numbers of adherent cells per unit area were significantly higher (p<0.01; cPnBA0250: 3 hrs 13±5 cells/mm2, 72 hrs 234±106 cells/mm2; cPnBA1100: 3 hrs 14±3 cells/mm2, 72 hrs 198±113 cells/mm2) compared to control cultures (glass, 3 hrs: 6±3 cells/mm2, 72 hrs: 130±83 cells/mm2) and showed a typically spread morphology.
The mRNA expression profile of the aMO2 was not influenced by the substrate elasticity. In the supernatant of aMO2 on cPnBA0250 significantly less VEGF-A165 product was found than expected based on the mRNA level measured (p<0.01). Tests with recombinant VEGF-A165 then demonstrated that significantly more VEGF-A165 was adhered on cPnBA0250 than on cPnBA1100 (p<0.01).
Seeded on cPnBA, aMO2 – unaffected by the elastic moduli of both substrates – seemed to remain in their subset status and secreted VEGF-A165 without release of pro-inflammatory cytokines. These in vitro results might indicate that this MO subset can be used as cellular delivery system for pro-angiogenic and non-inflammatory mediators to support the endothelialization of cPnBA.