Experimental design
Gel fabrication
Gel functionalization with fibronectin
Gel functionalization with TAMRA-RGD peptide
Silicone breast implant–derived materials
Mechanical characterization
Interfacial rheology measurements
Contact angle measurements
Steel ball indentation measurements
Gel preparation with Span85 surfactant treatment for cellular assays
Cell lines and culture
Antibodies and reagents
Cell morphology and spreading assays
Cell proliferation assays
Gene expression analysis
Preparation of a collagen master mix for 3D collagen I gels
PBMC-silicone interaction assay
PBMC—silicone droplets interaction assay
Immunofluorescence imaging
Hertz contact model with surface tension
Statistics
PBMCs were isolated from three thoroughbred horses: an 8-year-old gelding, a 12-year-old gelding, and an 18-year-old mare. The Institutional Animal Care and Use Committee (IACUC) of Cornell University approved the use of horses in these studies (IACUC no. 2018-0024). Blood (50 ml) was collected from the jugular vein using an 18-gauge needle and 60-ml syringe containing 5 ml of heparin (1000 U/ml; Sagent Pharmaceuticals), resulting in a final concentration of 100 U/ml of heparin. The blood was immediately stored on ice and transported to the laboratory for processing within 1 hour using Ficoll density gradient centrifugation (50). Briefly, freshly collected blood was diluted with equal volume of PBS containing 2% FBS (VWR Life Science Seradigm). A total of 20 ml of diluted blood was overlaid on 20-ml Ficoll-Paque PLUS (GE Healthcare, Chicago, IL) in a 50-ml conical tube. The Ficoll gradient centrifugation was performed at 400g for 30 min with low acceleration and no deceleration at room temperature. The PBMCs were collected from the plasma-Ficoll interface with a sterile Pasteur pipette and diluted 2:1 in PBS. After centrifugation at 300g for 5 min, the PBMCs pellet was resuspended in 10 ml of PBS and centrifuged at 300g for 5 min to wash the cells. The PBMCs were then cultured in RPMI supplemented with 1% GlutaMAX and 10% FBS.