BIOLOGICAL ACTIVITY OF NANOMETRESIZED POLYMER PARTICLES

Abstract

Introduction: Nanometre sized UHMWPE particles have recently been isolated from periprosthetic tissues and hip simulator lubricants [1,2]. The biological response to UHMWPE particles of 0.1 μm and above has been well characterised, with particles in the 0.1–1.0 μm size range having the highest biological activity [3]. The purpose of the study was to determine the biological activity of nanometre-sized particles in terms of osteolytic cytokine release from primary human monocytes.

Methods: Monocytes were isolated from peripheral blood from 5 healthy donors by density gradient centrifugation over Lymphoprep. Cells were cultured using the agarose gel technique [3] at particle volume (μm3):cell number ratios of 10:1 and 100:1. The particles used were:

• 1 Polystyrene FITC-conjugated FluoSpheres (FS; Invitrogen) in 20 nm, 40 nm, 0.2 μm and 1.0 μm sizes.

• 2a Complete Ceridust® 3615 (CD), a low MW polyethylene powder (size range 15 nm – 53 μm).

• 2b Nanometresized Ceridust® (fractionated by filtration using 10, 1, 0.1, 0.05 & 0.015 μm filters).

• 3 Clinically relevant GUR 1120 UHMWPE debris produced aseptically using a multidirectional wear rig.

All particles were tested for the presence of endotoxin prior to culture with cells. Cells without particles were used as a negative control and 200 ng/ml LPS was used as a positive control. Cell viability was assessed using the ATP Lite assay (Perkin Elmer) and ELISA was used to determine TNF-alpha, IL-1beta, IL-6 and IL-8 release at 3, 6, 12 and 24 h.

Results: FluoSpheres and CD had no effect on cell viability at 10 or 100:1. Clinically relevant UHMWPE particles had no effect on cell viability at 10:1, however, at 100:1 significant differences (P< 0.05) were seen at 3, 12 and 24 h for Donors 1 and 3. The 40 nm, 0.2μm and 1.0 μm FS caused significant elevation of TNF-α release at the 12 and 24 h time points at 100:1. There was no significant increase in TNF-α release for the 20 nm FS (3/5 donors). Particle volume and particle size showed correlation with cellular response, with the 20 nm FS showing the lowest biological activity. Clinically relevant UHMWPE particles and nanometre sized CD produced significantly higher quantities of TNF-alpha at 100:1. Release of interleukins IL-1beta, IL-6 and IL-8 followed a similar trend to TNF-alpha release.

Discussion: This study found that all nanometre-sized particles had the potential to provoke inflammatory cytokine release from macrophages. Particle volume and particle size played critical roles in initiating cellular responses. There was a lower particle size limit, with the 20 nm FS showing the lowest activity. Nanometre-sized polyethylene particles (CD) caused elevated TNF-α release, and since it has been shown that nanometre-sized UHMWPE particles are produced in large numbers in vivo [2], the relative contribution of these particles to osteolysis should be considered. The biological response to nanometre-sized clinically relevant UHMWPE particles is currently under investigation.