The John Charnley Award: an accurate and sensitive method to separate, display, and characterize wear debris: part 1: polyethylene particles

Abstract

Background: Numerous studies indicate highly crosslinked polyethylenes reduce the wear debris volume generated by hip arthroplasty acetabular liners. This, in turns, requires new methods to isolate and characterize them.

Questions/purposes: We describe a method for extracting polyethylene wear particles from bovine serum typically used in wear tests and for characterizing their size, distribution, and morphology.

Methods: Serum proteins were completely digested using an optimized enzymatic digestion method that prevented the loss of the smallest particles and minimized their clumping. Density-gradient ultracentrifugation was designed to remove contaminants and recover the particles without filtration, depositing them directly onto a silicon wafer. This provided uniform distribution of the particles and high contrast against the background, facilitating accurate, automated, morphometric image analysis. The accuracy and precision of the new protocol were assessed by recovering and characterizing particles from wear tests of three types of polyethylene acetabular cups (no crosslinking and 5 Mrads and 7.5 Mrads of gamma irradiation crosslinking).

Results: The new method demonstrated important differences in the particle size distributions and morphologic parameters among the three types of polyethylene that could not be detected using prior isolation methods.

Conclusion: The new protocol overcomes a number of limitations, such as loss of nanometer-sized particles and artifactual clumping, among others.

Clinical relevance: The analysis of polyethylene wear particles produced in joint simulator wear tests of prosthetic joints is a key tool to identify the wear mechanisms that produce the particles and predict and evaluate their effects on periprosthetic tissues.

Fig. 1

A flowchart shows an outline of the experiment.

Fig. 2

A schematic diagram shows the SWD protocol, highlighting the digestion, purification, and display phases.

Fig. 3A

FE-SEM secondary electron image (accelerating voltage, 15 kV; spot size, 1 nm) shows noncrosslinked PE particles recovered with the SWD protocol and displayed on a silicon wafer.

Fig. 4

A FE-SEM secondary electron image (accelerating voltage, 15 kV; spot size, 1 nm) shows noncrosslinked PE particles recovered with the NaOH protocol and displayed on a PC filter membrane (0.01 μm).

Fig. 5A–B

FE-SEM images show particles isolated by the SWD protocol and displayed on a silicon wafer. (A) This image was selected from among those showing the highest presence of agglomerates, likely formed during the wear process. (B) In the inset, fibril-like particles appear to be a complex network of particles of different shape and size “fused” together. These structures formed during the wear process suggest a specific wear mechanism that produced irresolvable agglomerates, confirming what has already been suggested by others. The high power and sensitivity of the SWD protocol allow separation and characterization of particles as small as 20 to 80 nm (arrows).

Fig. 6A–C

Graphs show the size distribution according to (A) maximum Feret’s diameter (d_max), (B) as percentage of total particles, and (C) as relative number of particles per million cycles. NXL-poly = noncrosslinked PE; 5XL-poly = 5-Mrad crosslinked PE; 7.5XL-poly = 7.5-Mrad crosslinked PE.

Fig. 7

A graph illustrates the results of the particle recovery experiments. The size distribution of 5-Mrad crosslinked PE (5XL-poly) particles after isolation by the SWD method is shown (blue line). Particles on these wafers were the source of particles for all other curves: after reisolation from serum by SWD digestion (R-SWD) and display on a silicon wafer (red line); after reisolation from serum by SWD digestion (R-SWD) and display on a filter membrane (green line); and after reisolation from serum by NaOH digestion (R-NaOH) and display on a silicon wafer (dotted purple line).