Fibronectin

This article is protected by copyright. All rights reserved fibrinogen depleted of fibronectin, plasminogen, and von Willebrand factor (Enzyme Research Laboratories, South Bend, IN, USA) at 8 mg/ml and supplemented with or not (w/o APR) 17 µg/ml aprotinin (Sigma-Aldrich) was cross-linked with 2 U/ml human thrombin (Sigma-Aldrich) and 8 U/ml factor XIIIa (Fibrogammin; Behring, Marburg, Germany) in 2.5 mM calcium chloride in HEPES buffer. FN III9-10/12-14 was produced as described to comprise a factor XIIIa substrate at the N terminus, consisting of residues 1-8 of the protein alpha 2 plasmin inhibitor (a 2 PI 1-8 , NQEQVSPL): a 2 PI 1-8 -FN III9-10/12-14 (Martino et al. 2011) . a 2 PI 1-8 -FN III9-10/12-14 was covalently incorporated in 80 µl (SC grafts) or 20 µl (EFP grafts) fibrin gels at 2 µM (ISL+FIB). For experimental conditions containing GFs ('ISL+FIB+GFs'), hPDGF-BB (2500 ng/ml; 50 ng in the EFP site or 200 ng in the SC site, carrier-free from R&D Systems) and hVEGF-A165 (5000 ng/ml; 100 ng in the EFP site or 400 ng in the SC site, carrier-free from R&D Systems, Minneapolis, MN) were also incorporated.

Ninety-six-well ELISA plates (Greiner Bio-One) were coated with recombinant human ECM proteins (10 mg/ml), fibronectin (Sigma-Aldrich), fibrinogen (von Willebrand factor-and fibronectin-depleted; Enzyme Research Laboratories), osteopontin (Sigma-Aldrich), vitronectin (Sigma-Aldrich), collagen I (EMD Millipore), collagen II (EMD Millipore), collagen III (EMD Millipore), or collagen IV (EMD Millipore) in PBS for 1 hour at 37°C, followed by blocking with 2% BSA in PBS with 0.05% Tween 20 (PBS-T) for 1 hour at room temperature. Then, wells were washed with PBS-T and further incubated with PlGF-2 123-144 -Abs or unmodified Abs (10 mg/ml each) for 1 hour at room temperature. After three washes with PBS-T, wells were incubated for 1 hour at room temperature with horseradish peroxidase (HRP)-conjugated Abs against rat IgG or hamster IgG (Jackson ImmunoResearch). After washes, bound Abs were detected with tetramethylbenzidine sub-strate by measurement of the absorbance at 450 nm with subtraction of the absorbance at 570 nm.