Effect of polar, hydrophobic, and ionic polyurethane character on adsorbed IgG-mediated monocyte activation

Kyle Battiston (University of Toronto)
Ben Ouyang (University of Toronto)
Eilyad Honarparvar (University of Toronto)
Jenny Qian (University of Toronto)
Rosalind Labow (University of Ottawa Heart Institute)
Craig Simmons (University of Toronto)
Paul Santerre (University of Toronto)

Introduction

One of the first cell types present following biomaterial implantation is the monocyte. These cells can play an important role in supporting tissue integration and wound healing, or conversely contribute to an adverse foreign body response and implant rejection, depending on the activation state induced by the cell’s interaction with the particular biomaterial [1]. A degradable polar hydrophobic ionic polyurethane (D-PHI) has previously been investigated with regards to its interactions with human monocytes. Monocytes adherent to D-PHI release less of the pro-inflammatory cytokine TNF-α and more of the anti-inflammatory cytokine IL-10 relative to the ubiquitous cell culture substrate tissue culture polystyrene (TCPS), as well as the commonly used degradable polyester poly(lactide-co-glycolide) (PLGA) [2,3]. One of the most common factors associated with biomaterial-mediated monocyte activation is the nature of the adsorbed protein layer [1]. Both the type and conformation of protein present, along with the exposure of critical binding sites important for interacting with monocytes, have been linked with the monocyte’s response to the biomaterial [4]. Immunoglobulin G (IgG) is known to be a particularly potent substrate for activating monocytes/macrophages [4]. The objective of the present study was to determine the role of IgG interactions with D-PHI and TCPS on monocyte activation observed for these surfaces, as well as to elucidate how D-PHI chemistry elicits this differential response.

Materials and Methods

D-PHI films were fabricated according to previously established methods [5]. Briefly, a divinyl oligomer (DVO), methacrylic acid (MAA), and methyl methacrylate (MMA) were mixed together in a 1:5:15 molar ratio, along with the initiator benzoyl peroxide (0.032 mol/mol vinyl group). 50 µl of the resulting solution was pipetted into a polypropylene 96-well plate and cured at 110°C for 24 hr. Human IgG (Jackson ImmunoResearch) was pre-adsorbed to D-PHI and TCPS surfaces for 24 hr at a concentration of 200 µg/ml (100 µl per film). To assess total IgG content, films were washed 3x with PBS and then incubated with 2% SDS for 24 hr, after which a micro BCA assay was performed (Thermo Scientific). To quantify the exposure of the Fab region of IgG after surfaces were coated with IgG as described above, surfaces were rinsed 3x with PBS, incubated with an HRP-conjugated Fab-specific IgG (Sigma) for 1 hr, rinsed again, and subsequently incubated with a tetramethylbenzidine substrate solution (eBioscience) for 25 min, after which the absorbance was read at 450 nm. To assess the role of IgG in supporting monocyte activation on D-PHI and TCPS, monocytes were isolated from peripheral blood obtained from healthy volunteers (University of Toronto ethics approval %2322203) and seeded at a density of 200,000 per well (96-well plate) on IgG pre-adsorbed and non-coated D-PHI and TCPS surfaces. Monocytes were cultured for up to 72 hr in RPMI-1640 medium containing 10% autologous human serum (AHS), at which point assays were performed to assess the release of pro- and anti-inflammatory cytokines (ELISAs for TNF-α, IL-10; eBioscience), cell number (DNA mass quantification), and cell morphology (scanning electron microscopy). To block monocyte interactions with the Fab region of adsorbed IgG, medium was supplemented with a Fab-specific IgG (Sigma) that was supplemented with every medium change.

Results

While monocytes cultured on IgG-coated TCPS had significantly more DNA than non-coated TCPS after 72 hr (4.3 ± 0.7 times, p<0.05), IgG did not affect monocyte adherence to D-PHI (1.0 ± 0.1 times for IgG-coated/non-coated). SEM analysis indicated that IgG coating had a noticeable effect on cell spreading on TCPS, whereas no effect was observed for D-PHI (Figure 1). The role of IgG conformation in these differential responses was evaluated, and it was determined by ELISA that there was significantly more exposure of the Fab region on TCPS vs. D-PHI (1.27 ± 0.01 A.U. vs. 0.14 ± 0.01 A.U. for TCPS vs. D-PHI, respectively, p<0.05). To confirm the role of the Fab region in promoting monocyte survival and spreading, the medium was supplemented with a Fab-specific IgG to block exposed IgG from interacting with adherent monocytes. Blocking exposed Fab acted to reverse the effect of IgG on monocytes for TCPS, and had no effect on D-PHI (Figure 1).

Discussion and Conclusion

Results indicate that D-PHI attenuates monocyte activation induced by adsorbed IgG by limiting the exposure of the Fab region of the protein. Future work will involve evaluating the importance of D-PHI chemistry in the observed favourable material-protein and material-cell interactions, which will help provide insight into the use of tailored polyurethane chemistry as a means to develop new materials with favourable interactions with proteins and immune cells.

Figure 1 (A) DNA mass quantification and (B) representative SEM images at day 3 post-seeding for monocytes seeded on non-coated (NC) or IgG-coated D-PHI and TCPS surfaces with or without exposure to a Fab-specific antibody. Scale bars = 30 um. Data are the mean /- S.E. *p<0.05 vs IgG coating. n=9

Acknowledgements

CIHR grant %23230762, Cell Signals training fellowship (Battiston), Ontario Graduate Scholarship (Battiston).

References

[1] Anderson JM et al. Semin Immunol 2008;20(2):86-100. [2] McBane JE et al. Biomaterials 2011;32(14):3584-95. [3] McBane JE et al. Biomaterials 2009;30(29):5497-504. [4] Jenney CR and Anderson JM. J Biomed Mater Res 2000;50(3):281-90. [5] Sharifpoor S et al. Biomacromolecules 2009;10(10):2729-39.

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