Application of MS-Based Proteomics to Study Serum Protein Adsorption/Absorption and Complement C3 Activation on Poly(ethylene glycol) Hydrogels

Abstract

Although the interaction between cells and poly(ethylene glycol) (PEG) hydrogels is well documented, there lacks a thorough investigation into the adsorption of blood proteins on these surfaces which dictates the observed cellular and in vivo host response. Thus, a clear understanding of how surface-bound proteins mediate the unique biological property of PEG hydrogels is fundamentally important. The information obtained will also provide insights into future biomaterial design. In this study, several mass-spectrometry-based proteomic tools coupled with complementary immunoassays were employed to survey the complex surface-bound serum proteome. The adsorption of vitronectin, thrombin, fibrinogen and complement component C3 was significantly lower on PEG hydrogels than on tissue culture polystyrene (TCPS). Although PEG hydrogels mediated lower C3 adsorption than TCPS, the extent of C3 activation between the two surfaces was comparable. Adherent monocyte density was also significantly lower on PEG hydrogels as compared to TCPS. Taken together, these results support the critical role of the complement C3 in mediating monocyte adhesion on biomaterials. Thus we conclude that the biocompatibility of PEG hydrogels both in vitro and in vivo can be partly contributed to their limited C3 interaction and monocyte activity.

Cell Encapsulating Biomaterial Regulates Mesenchymal Stromal/Stem Cell Differentiation and Macrophage Immunophenotype

“…This trend was consistent with previous observations in our laboratory using a semi-interpenetrating network composed of physically entangled polyethylene glycol and gelatin macromolecules [25]. For the Mø monoculture gelatin/polyethylene glycol condition, temporal variations in the type, composition, and concentration of adsorbed protein can contribute to changes in macrophage phenotypic behavior causing lowered TNF-␣ expression observed at 4 days [26,27]. MSCs entrapped within gelatin/polyethylene glycol respond to potentially lethal TNF-␣ levels via a nuclear factor ␤ (NF-B) mechanism that causes several anti-inflammatory factors to be secreted (TNF-␣ stimulated gene-6 protein, interleukin-1 receptor antagonist, prostaglandin E2, and stanniocalcin-1) that downregulate TNF-␣ expression and direct adherent macrophages toward an M2 alternatively activated state [28 -30].…”

Fetal bovine serum xenoproteins modulate human monocyte adhesion and protein release on biomaterials in vitro

“…For example, PEG hydrogels are well known as resistant to protein adsorption; however, a study on the PEG-like surface tetraglyme indicated that complement protein C3 is the primary mediator of monocyte adhesion to tetraglyme [38]. Additionally, C3 activity on PEG hydrogels is comparable to TCPS, and monocyte adhesion to PEG hydrogels is lowered when using C3-depleted serum [24]. C3 is present only at very low, nearly undetectable levels in FBS, while adult humans have a C3 concentration of approximately 75-135 mg/dl [39,40].…”

“…Depending on the surface properties, biomaterials promote differential adsorption of certain serum proteins, and thus we hypothesized that the effect of serum type may vary by surface [1]. For example, polyethylene glycol (PEG) hydrogels limit adsorption of common serum proteins but may sustain significant complement component 3 (C3) activity comparable to other surfaces, potentially allowing this protein to take on a more influential role in mediating monocyte/macrophage responses [24]. FBS high abundance proteins include: bovine serum albumin (BSA), alpha-1-antiproteinase, plasminogen, lactoperoxidas, kniogen (LMW II), alpha-2-HS-glycoprotein, hemiferrin, prothrombin, apolipoprotein A-I, integrin beta-1, IGFBP2, IGF II, TGF-beta1 [25].…”

“…TCPS and PDMS were selected because they are both commonly used reference materials with differing surface properties, and PEG hydrogels were selected because they have been shown to support monocyte adhesion while limiting protein adsorption along with having substantially differing surface properties from TCPS and PDMS [1,16,24, 27,28]. PEG hydrogels are highly hydrophilic swollen polymer networks, while the PDMS discs are highly hydrophobic films, and TCPS is a rigid, slightly hydrophobic surface.…”

Mesenchymal Stromal/Stem Cell and Minocycline-Loaded Hydrogels Inhibit the Growth of Staphylococcus aureus that Evades Immunomodulation of Blood-Derived Leukocytes

“…The collected monocytes were examined for purity using CD14-PE (AbD Serotec, Raleigh, NC) and CD45-FITC (BD Biosciences) monoclonal antibodies with appropriate fluorescence-minus-one and negative controls, which resulted in~80-90% monocyte purity with the contaminant being lymphocytes using flow cytometry characterization (24,25). Autologous human serum (AHS) was obtained from whole blood without sodium citrate as previously described (23,26).…”