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 was 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 24hr 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.

Mesenchymal Stromal/Stem Cell and Minocycline-Loaded Hydrogels Inhibit the Growth of Staphylococcus aureus that Evades Immunomodulation of Blood-Derived Leukocytes

“…Venipuncture was executed on healthy, medication-refrained donors after obtaining written consent approved by the University of Wisconsin Hospital and Clinics Regulatory Committee. Monocytes and PMNs were isolated from whole blood using established density gradient methods (23). The collected monocytes were examined for purity using CD14-PE (AbD Serotec, Raleigh, NC) and CD45-FITC (BD Biosciences) monoclonal antibodies with appropriate fluorescence-minus-one and negative controls, which resulted in~80-90% monocyte purity with the contaminant being lymphocytes using flow cytometry characterization (24,25).…”

“…The collected monocytes were examined for purity using CD14-PE (AbD Serotec, Raleigh, NC) and CD45-FITC (BD Biosciences) monoclonal antibodies with appropriate fluorescence-minus-one and negative controls, which resulted in~80-90% monocyte purity with the contaminant being lymphocytes using flow cytometry characterization (24,25). Autologous human serum (AHS) was obtained from whole blood without sodium citrate as previously described (23,26).…”

Cell Encapsulating Biomaterial Regulates Mesenchymal Stromal/Stem Cell Differentiation and Macrophage Immunophenotype

“…The cell pellet was then resuspended in Iscove's modified Dulbecco's medium (IMDM) (Cellgro Mediatech) (without phenol red) at approximately 1-2 ϫ 10 6 Mø per milliliter, and then 25 ml of 46% Percoll/IMDM solution (with phenol red) was slowly underlayered using a spinal needle, which formed a bilayer solution that was then centrifuged at 550g for 30 minutes. The Mø band was collected in 15-ml volumes diluted with 35 ml of DPBSE, centrifuged at 400g for 10 minutes, and then resuspended for the coculture studies [20,21]. The Møs were assessed for purity using CD14-PE (AbD Serotec, Raleigh, NC, http://www.abdserotec.com) and CD45-FITC (BD Biosciences) monoclonal antibodies with appropriate monoculture controls, resulting in ϳ80%-90% monocyte purity, with the primary contaminant being lymphocytes using flow cytometry analysis.…”

Biomaterials differentially regulate Src kinases and phosphoinositide 3-kinase-γ in polymorphonuclear leukocyte primary and tertiary granule release

“…PMNs were isolated as previously described using a density gradient method [28]. Unless otherwise indicated, PMNs were adjusted to 1 × 10 6 cells/ml in RPMI-1640 medium supplemented with 10% autologous human serum (AHS) and were statically seeded onto the biomaterial substrates in 48-well plates (0.5 ml/well for a density of approximately 5.3 × 10 3 PMNs/mm 2 ).…”