Abstract
Introduction
Periprosthetic joint infection (PJI) and particle-induced osteolysis are closely related to peri-implant local immunity and macrophage function. We previously demonstrated that titanium particles attenuate the immune response of macrophages caused by chronic inflammation [1]. In a separate study, we have determined that UHMWPE wear particles containing vitamin E (VE) induce less osteolysis compared to HXL UHMWPE wear particles in a murine calvarium model [2]. For this study we hypothesized that macrophages exposed to HXL UHMWPE particles containing VE would better maintain their ability to respond to S. aureus compared to HXL UHMWPE without VE.
Methods
A gamma-sterilized, HXL UHMWPE tibial bearing containing VE (E1, Biomet, “VE-PE”) and 100kGy irradiated and melted UHMWPE (“CISM 100”) were cryomilled to particles by Bioengineering Solutions (Oak Park, IL). In the first in vitro study, RAW 264.7 mouse macrophages were exposed (inverted co-culture) to either VE-PE particles or CISM100 particles and lipopolysaccharide (LPS) for 1–7 days. Macrophage viability was measured using a cell counting kit (CCK-8). Control group with no particles and a LPS group were also included. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed to determine macrophage apoptosis rate in response to particle exposure over time. In the second study, macrophages were exposed to VE-PE or CISM100 particles for 48h, then exposed to LPS for 30 min. Subsequently, reactive oxygen species (ROS) generation and extracellular regulated protein kinase (ERK) phosphorylation were measured. In a third study, after exposure to particles for 48h, fatigued macrophages were co-cultured with bioluminescent S. aureus strain Xen29 for 3h and 6h. Bioluminescence signal was determined to measure the total amount of bacteria. Bacterial live/dead staining and optical density at 600 nm (OD 600) were also performed to determine S. aureus viability. Statistical analysis was performed using one-way or two-way ANOVA with a post hoc examination. *indicates p<0.05.
Results
CISM100 particles significantly decreased macrophage viability at day 5 and day 7 (p<0.05, Fig. 1A), while the viability of macrophages exposed to VE-PE particles was similar to controls (macrophages not exposed to particles). After 48h, macrophages exposed to VE-PE particles showed a lower TUNEL-positive rate (less apoptosis) compared to CISM100 particles (Fig. 1B, C). 48h-exposure to VE-PE particles increased ROS generation and ERK phosphorylation in 30 min-LPS-activated macrophages when compared to CISM100 particles (Fig. 2). This immune response caused by VE-PE particles resembles that of macrophages without particles. Furthermore, 48h exposure to E1 particles showed less S. aureus at 6h (Fig. 3).
Conclusions
These results suggest that VE-PE particles cause reduced macrophage apoptosis and protect the macrophages' immune response. VE-PE particles also preserved the innate immunity of macrophages, unlike CISM100, as evidenced by the S. aureus co-culture study. Thus, patients with vitamin-E containing implants may be less likely to develop PJI.
