Active leukocyte detachment and apoptosis/necrosis on PEG hydrogels and the implication in the host inflammatory response

Abstract

Monocytes/Macrophages have long been recognized as key players in inflammation and wound healing and are often employed in vitro to gain an understanding of the inflammatory response to biomaterials. Previous work has demonstrated a drastic decrease in primary monocyte adherent density on biomaterial surfaces coupled with a change in monocyte behavior over time. However, the mechanism responsible for this decrease remains unclear. In this study, we explored active detachment and cellular death as possible regulating factors. Specifically, extracellular TNF-α and ROS production were analyzed as potential endogenous stimulators of cell death. MMPs, but not calpains, were found to play a key role in active monocyte detachment. Monocyte death was found to peak at 24 h and occur by both apoptosis and necrosis as opposed to polymorphonuclear leukocyte death which mainly occurred through apoptosis. Finally, TNF-α and ROS production were not found to have a causal relationship with monocyte death on TCPS or PEG surfaces. The occurrence of primary monocyte apoptosis/necrosis as well as active detachment from a material surface has implications not only in in vitro study, but also in the translation of the in vitro inflammatory response of these cells to in vivo applications.

Figure 1

The number of adherent monocytes on TCPS cultured with various concentrations of (A) PD150606 and (B) GM6001 for 2 hours. Cells without treatment of PD150606 or GM6001 served as the control. §: significantly different compared to the control at 2 hours, p<0.05.

Figure 2

The number of adherent monocytes on TCPS (A) and PEG hydrogel (B) surfaces after 2 hours incubation with10µM GM6001 () or 50µM PD150606 () before each time point. Cells cultured without GM6001 or PD150606 served as control (). §: significantly different compared to the control without additive at the same time point, p<0.05;

Figure 3

Viable () and non-viable () monocytes in the cell culture supernatant after treatment with 10µM GM6001 (B) or 50µM PD150606 (C) for 2 hours before each time point on TCPS and PEG hydrogel substrates. The fluorescence of live and dead cells was measured for each sample simultaneously (MultiTox-Fluor™, Promega). Cells cultured on TCPS without GM6001 or PD150606 served as control (A).

Figure 4

TNF-α concentration in monocyte culture supernatant on TCPS ()and PEG hydrogel () substrates at 2, 24, 96 and 168 hours. §: significantly different compared to TCPS at the same time point, p<0.05;

Figure 5

The adherent monocyte density on TCPS after incubation for 2 () or 12 () hours in media supplemented 0.01, 0.05, 0.5, 5 or 50 ng/mL human recombinant TNF-α. Media without exogenous TNF-α served as the control. §: significantly different compared to the control without cells at 2 hours, p<0.05; ‡: significantly different compared to the cell number at the same time point treated with 0.01 ng/mL TNF-α, p<0.05.

Figure 6

Extracellular ROS production by monocytes (A) and PMNs (B) adhered to TCPS and PEG hydrogels at 2(), 24() and 96() hr. ROS levels were determined using a cell membrane impermeable probe which fluoresces upon oxidation (485_ex/520_em).

Figure 7

The level of monocyte (A) and PMN (B) viability (), apoptosis (), and necrosis () at 2, 24, 48, 96, and 168hr after exposure to TCPS and PEG hydrogel substrates. Viability was assessed by measuring the cleavage of a fluorogenic, cell-permeant, peptide substrate by cells with intact membranes. Primary and secondary necrosis is assessed by measuring the cleavage of fluorogenic, cell-impermeant, peptide substrate, by a “dead-cell protease” which is released from cells with permeable membranes. Mean fluorescent intensity (MFI) of the samples were compared to a fluorescein standard curve to allow for comparison between time points. Finally, cleavage of a luminogenic caspase-3/7 substrate was used to assess apoptosis.

Figure 8

Levels of secondary necrosis present in monocyte and PMN cultures as determined by measuring the activity of caspase-3 in the supernatant. Supernatant from cells exposed to TCPS () and PEG hydrogel () substrates for 2, 24, 48, 96, and 168hr were exposed to fluorogenic caspase-3 substrate and the mean fluorescence intensity from the cleaved probe was assessed.

Figure 9

Photomicrographs of apoptotic and necrotic monocytes on TCPS (A) and PEG hydrogel (B) surfaces at 24hr. Apoptosis detection was based on the translocation of phosphatidylserine from the inner to outer membrane of the plasma membrane in apoptotic cells (Blue). Primary and secondary necrosis was detected using the nucleic acid-binding propidium iodide dye which cannot penetrate the membranes of live or early apoptotic cells (Red). Live cells demonstrated no or a very low level of fluorescence. The PEG hydrogel created blue background fluorescence. The white reference line represents 75µm.