The Effects of PMMA Surface Chemistry and Extracellular Traps on Macrophages, Lens Epithelial Cells and the Wound Healing Response
Posterior capsule opacification (PCO) is a condition that affects 20-40% of patients that undergo cataract lens replacement surgery. The design of the replacement intraocular lens (IOL) (for instance material, shape, and surface chemistry) is thought to be one of the main contributing factors to its development. Following the implantation of a biomaterial, there is a response by the immune system, activating cells and the release of various factors. The neutrophil is one of the first immune cells to respond and consequently has an effect on local cells and downstream immune cells. Also, neutrophils release a meshwork of DNA and proteins termed neutrophil extracellular traps (NETs). NETs are present during the inflammatory response and can affect downstream immune cells (i.e. macrophages) and the local cells. Therefore, it is hypothesized that the lens surface chemistry and the immune response of the neutrophils affect the behaviour of lens epithelial cells (LECs) and macrophages in the wound healing response. In this research, NETs were isolated from an HL60 cell line and used to investigate their effect on macrophages (activated THP1 cells) and LECs in vitro. Cell viability and behaviour of these cell types were assessed to determine the effects that NETs have on local and immune cells involved in PCO and the wound healing response. Poly(methyl methacrylate) (PMMA) disks were functionalized with amine or carboxyl surface groups to explore how the surface chemistry can affect LEC behaviour and viability in vitro. Lastly, in vivo analysis of functionalized PMMA beads investigated the healing response and neutrophil presence to different surface chemistries. When THP1 cells were incubated on tissue cultured polystyrene (TCPS) pre-treated with NETs, the NETs increased monocyte differentiation and TNF-α production when paired with iii activation with phorbol 13-myrstate 12-acetate (PMA) but there was no significant increase in THP1 cells differentiation when they were incubated on NETs only. It was also found that there were paracrine effects on the activated macrophages, leading to increased cell activation when THP1 cells were seeded onto TCPS at a higher cell density. When LEC were incubated on TCPS with pre-adsorbed NETs, the NETs decreased LEC viability but, interestingly, increased the production of alpha smooth muscle actin (α-SMA), a marker of epithelial to mesenchymal transition. When LEC were incubated on PMMA, the PMMA surface chemistry altered the viability of LECs and α-SMA production, with increased viability and α-SMA expression on aminated PMMA. When PMMA beads were injected subcutaneously into C57BL/6J mice, there was increased neutrophil presence and vessel formation 7 days after injection as well as differences in these two measures surrounding the aminated and carboxylated PMMA. Neutrophil presence was increased surrounding PMMA-COOH and vessel structure formation was increased surrounding PMMA-NH2 at 1 day and 7 days after injection.