Validation of a novel particle isolation procedure using particle doped tissue samples

Abstract

A novel particle isolation method for tissue samples was developed and tested using particle-doped peri-articular tissues from ovine cadavers. This enabled sensitivity of the isolation technique to be established by doping tissue samples of 0.25 g with very low particle volumes of 2.5 µm3 per sample. Image analysis was used to verify that the method caused no changes to particle size or morphologies.

Recovery of low volumes of wear debris from rat stifle joint tissues using a novel particle isolation method

“…The size and shape of CoCrMo and Ti-6Al-4V particles showed slight variation between the validation study [34] and the rat study. In particular, CoCrMo particles were slightly smaller in the rat study and Ti-6Al-4V particles had a higher average circularity.…”

“…Particle amounts could therefore be extremely low, especially in well-functioning ceramic-on-ceramic bearings which may produce less than 0.5 mm 3 of wear per year in vivo [38]. The current method was therefore tested in particledoped tissue samples at starting volumes as low as 0.0025 mm 3 per sample of tissue [34]. The in vivo exposure time of seven days was the maximum time point that could enable protein binding to particles before a large volume of particles would be lost through dissemination.…”

“…Particles were first located within histological tissue sections with a previously used technique [40] to enable accurate tissue harvesting. Due to the small amount of tissue available from a rat stifle joint, initial tissue masses (wet weight) were 150 mg; however, since larger quantities of tissue are sometimes available from human explant samples, the particle isolation method was also tested on initial tissue masses of 250 mg [34].…”

“…A novel tissue digestion method was developed and optimised as described in [33] to produce an isolation protocol for tissue samples. The protocol was validated in each material by recovering particles from particle-doped tissue samples as detailed in [34] prior to commencing with particle isolation from the injected rat stifle joints.…”

“…Tissue digestion optimisation experiments showed that the use of papain, glycine in the buffer, and frequent proteinase K enzyme replenishment were required to efficiently digest tissue proteins [33]. Validation of the method in tissue samples doped with 0.0025 mm 3 of particles per sample demonstrated that the protocol used had no effect on particle size or morphologies for any of the materials, and was effective at very low particle doses [34]. Digested rat tissue samples were visibly homogeneous, containing no large pieces of undigested tissue, and during the isolation process the proteins in each sample formed a faint band within the upper density gradient layers, enabling separation from particles.…”