Neutrophil NETosis, viability, and gene or protein expression in response to surface chemistry using isodecyl acrylate copolymer coatings
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Undesirable host responses to implants commonly lead to impaired device function. Biomaterial-based strategies focused on neutrophil behavior to improve host responses to implants have not been significantly researched. As the first immune cell to respond to inflammation, neutrophil activation resulting in the release of antimicrobials and neutrophil extracellular traps primes this microenvironment for macrophages and other infiltrating cells. Methacrylic acid (MAA) and methyl methacrylate (MM) based polymers modulate healing responses through altered macrophage behavior and vascularization. This project investigates the neutrophil response to MAA and MM copolymerized with isodecyl acrylate (IDA) (MAAcoIDA and MMcoIDA) and hexamethylenediamine-functionalized MMcoIDA coatings (HMD-MMcoIDA). In this research, MAAcoIDA and MMcoIDA were coated onto glass coverslips and MMcoIDA. MAAcoIDA coatings had 4975 ± 580 nmol/cm2 of carboxyl groups at the surfaces while MMcoIDA was further functionalized to add 58 ± 18 nmol/cm2 of amine groups to the surfaces via aminolysis with HMD. The HL60 cell line and murine bone marrow derived neutrophils (BMDN) were then incubated with the coatings to study neutrophil-material interactions. HL60 gene expression and viability were assessed to determine inflammatory and anti-inflammatory characteristics induced by the different surface chemistries. NETosis, indicated by citrullinated histone H3 (citH3), elicited by the coatings was visualized for both HL60 cells and murine BMDN, and protein synthesis was additionally quantified for BMDN. HL60 cells demonstrated increased viability when incubated with MAAcoIDA relative to MMcoIDA. HL60 viability on HMD-MMcoIDA decreased after 48 hours, coinciding with increased citH3. HL60 cells exhibited increased expression of ICAM-1 and TNF-α when incubated on aminated coatings compared to those with MMcoIDA or MAAcoIDA after 48 hours. Murine BMDN displayed lower ratios of viable cells on the coatings than uncoated glass, and cells with MAAcoIDA coatings underwent less histone citrullination while HMD-MMcoIDA elicited increased histone citrullination, relative to MMcoIDA. BMDN secreted varied concentrations of MIP-1α and MIP-2 in response to coatings with no added stimulus after 2 hours and increased over 16 hours, although no significant differences were noted between surface chemistries. Future work looks to characterize these coatings and examine protein-material interactions, as well as follow up studies with these materials in vivo.