Quantifying Protein Adsorption to Physically Crosslinked Gelatin-Based Networks
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Quantifying Protein Adsorption to Physically Crosslinked Gelatin-Based Networks Axel T. Neffe1,2, Benjamin F. Pierce1, Joanna Blaszkiewicz1,2, and Andreas Lendlein1,2 1 Center for Biomaterial Development and Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Helmholtz-Zentrum Geesthacht, Kantstr. 55, 14513 Teltow, Germany 2 Institute of Chemistry, University of Potsdam, 14476 Potsdam-Golm, Germany ABSTRACT Physically crosslinked hydrogels based on gelatin functionalized with desaminotyrosine (DAT) (giving Gel-DAT) or desaminotyrosyl tyrosine (DATT) (resulting in Gel-DATT) have shown high potential as biomaterials. Here, protein adsorption to the functionalized gelatins in comparison to gelatin was quantified to see if the functionalization and chain organization of gelatins has an influence on the amount of proteins being adsorbed. For this purpose, gelatin, Gel-DAT, and Gel-DATT were incubated with water or aq. solutions of bovine serum albumin (BSA), fibrinogen, or fibronectin, respectively, at physiological concentrations. Protein concentrations in the supernatant were determined with the bicinchoninic acid (BCA) assay before and after the contact. BSA adsorption to the materials was influenced as well by the hydrophobicity of the material as the degree of swelling, with the observation that higher protein concentrations led to lower protein adsorption. The highest amount of fibronectin was adsorbed to Gel-DAT, followed by gelatin and Gel-DATT, with only small differences for different initial protein concentrations. Fibrinogen adsorption increased with increasing concentration. In the future, adsorption studies based on specific antibody-based techniques might enable quantification of the proteins also in competition assays and direct quantification of adsorbed material. INTRODUCTION Biomaterial-induced autoregeneration is a concept of regenerative medicine that envisions the implantation of a biomaterial, which acts temporarily as biological and structural substitute of a damaged tissue and is replaced over time by functional neo-tissue [1]. After implantation, contact with blood unavoidably leads to protein adsorption to biomaterials [2]. The adhering dynamic protein layer directs cell adhesion and therefore affects endogenous regenerative processes [3]. Therefore, the quantification of protein adsorption to biomaterials is an essential in vitro study in the biomaterial evaluation process [4]. Protein concentrations in solution can be easily determined with the bicinchoninic acid (BCA) assay [5]. This assay uses the coordination of Cu2+ ions by proteins and reduction to Cu+ under basic conditions (Biuret reaction), followed by specific complexation of Cu+ with BCA, which can be quantified by UV spectroscopy. Protein adsorption can be calculated by determination of protein concentrations in solution before and after contact to the target material. In earlier studies we have shown that gelatin functionalized with desaminotyrosine (DAT) (giving Gel-DAT) or desaminotyrosyl tyrosine (DATT) (giving Gel-DAT
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