HL-60 cell response to PMMA surfaces of different chemistry and roughness with a focus on extracellular traps

Abstract

Event Abstract Back to Event HL-60 cell response to PMMA surfaces of different chemistry and roughness with a focus on extracellular traps Fairuz Hoque1*, Martin Kwok1 and Laura A. Wells1 1 Queen's University, Chemical Engineering, Canada Introduction: Shortly after a material is implanted, neutrophil cells infiltrate the implantation site. Neutrophils have recently been identified to reside at injury sites for up to 5 days, to polarize to different phenotypes, and to release DNA structures known as neutrophil extracellular traps (NETs) [1]. NETs are associated with inflammation, but little is known on how they influence host responses to biomaterials. We hypothesize that the surface properties of materials will influence NET generation, which will contribute to the host response. This work investigates how poly methyl methacrylate (PMMA) disks of different roughness and surface chemistry influence the behaviour and generation of NETs from “neutrophil-like” HL60 cells. Methods: PMMA disks (d=18 mm) were modified with carboxyl groups by reacting them in a 1:1 methanol and 1 M sodium hydroxide solution for 2 hours at 60°C [2] or with amine groups by reacting them in 10% hexamethylene diamine in 100 mM sodium borate buffer (pH 11.5) solution for 48 hours at 21°C [3]. The reactions were monitored by ATR-FTIR and quantified by back titration of sodium hydroxide for carboxyl groups and the ninhydrin assay for amine groups. Angiogenic methacrylic acid (MAA) and control methyl methacrylate (MM) copolymerized with isodecylacrylate [4] were also compared. HL60 were activated with the different polymers using 50 mM phorbol 12-myristate 13-actetate (PMA) for 48 hours and then evaluated using the alamarBlue assay for cell viability and nucBlue and sytox green stains to visualize DNA. Results: HL60 were activated onto rough PMMA (maximum roughness peak, Rp=460 nm) and smooth PMMA (Rp= 163 nm) disks with 28 ± 6 μmol carboxyl groups/cm2 or 13 ± 3 μmol amine groups/cm2. As shown in Figure 1, HL60 incubated with PMMA and modified PMMA adhered to the surfaces in the absence of PMA (in comparison to polystyrene controls, p<0.05), suggesting that the materials themselves are activating HL60. HL60 treated with PMA resulted in similar absorbance onto the different PMMA disks, but it remains to be determined if this is due to differences in cell number or cell viability. Studies with the copolymers showed a 1.6 fold increase in viable cells on MAA copolymers in comparison to controls, further illustrating that chemistry affects HL60 activation. Figure 1: The alamarBlue assay on HL-60 activated with or without 50 nM PMA onto PMMA with different surface groups and topography. (*=p<0.05) Sytox green was used to evaluate the generation of NETs by HL60 activated with rough PMMA of different chemistry. In comparison to PMMA, there was an increase in NETs from HL60 cells activated onto carboxyl-modified PMMA and to lower degrees on amine-modified surfaces (Figure 2), suggesting that carboxyl-modified PMMA results in increased NETosis. Figure 2: HL-60 cells activated onto PMMA and modified PMMA (Green=sytox green, Blue=nucBlue). Conclusions: Viable HL60 cells adhered to PMMA and modified PMMA in the absence of the activator PMA, suggesting that the surface chemistry is able to activate the cells. Interestingly, preliminary DNA stains suggest that NETosis is increased on carboxyl-modified PMMA. This could relate to previously observed differences in the healing surrounding polymers of different chemistry [5]. Future work will evaluate these observations by immunohistochemistry and real-time assays, and also determine the influence of NETs on downstream cells, such as macrophages. This work was supported by a Queen's University Research Initiation Grant and Senate Advisory Research Grant