Simple colorimetric methods for determination of sub-milligram amounts of ultra-high molecular weight polyethylene wear particles.

New colorimetric methods are described for determination of sub-milligram amounts of ultra-high molecular weight polyethylene (UHMWPE) wear particles. These methods are based on the irreversible binding of the fluorescein-conjugated bovine serum albumin or the hydrophobic dye Oil Red O to wear particles. UHMWPE particles bind both substances from their solutions and thus decrease the absorbance of these solutions. The decrease is linearly dependent on the amount of added wear particles in the sub-milligram range suitable for practical use. The newly developed method offers improved accuracy and precision compared to Fourier transformed infrared spectroscopy (Slouf M, et al. Quantification of UHMWPE wear in periprosthetic tissues of hip arthoplasty: description of a new method based on IR and comparison with radiographic appearance. Wear 2008;265:674-684.).

[MORF method for assessment of the size and shape of UHMWPE wear microparticles and nanoparticles in periprosthetic tissues]. / Metoda MORF pro sledování velikostí a tvaru oterových mikro- a nanocástic UHMWPE v periprotetických tkáních.

PURPOSE OF STUDY: Aseptic loosening of total joint replacement (TJR) due to wear of ultra-high molecular weight polyethylene (UHMWPE) is regarded as one of the major problems in the field of arthroplasty. This work describes a newly developed method, called MORF, which completely describes the morphology of UHMWPE wear particles. The differences in wear particle morphology may help to elucidate individual differences in TJR failures. MATERIAL AND METHODS: During the years 2002-2010, a set of 47 typical damaged periprosthetic tissues, coming from 16 TJR revisions, was collected. Isolated on polycarbonate (PC) filters were quantified. Quantification of the particles consisted in determination of their concentration and description of their morphology by means of the newly developed MORF method. Firstly, the micrographs of isolated UHMWPE particles were obtained with a scanning electron microscope (Quanta 200 FEG; FEI) at two magnifications: x1200 and x6000. Secondly, both high- and low-magnification micrographs were processed by a standard image analysis software (program NIS Elements; Laboratory Imaging) in order to obtain basic morphological descriptors. Finally, the results from image analysis of high- and low- magnification micrographs were combined by means of our own program MDISTR in order to obtain correct particle sizes and shapes. RESULTS: In the first stage, the method was applied to 25 samples and yielded an average particle size of 0.51 µm. In the second stage, the method was further improved in order to calculate not only the size of particles but also the shape of descriptors. The improved method was applied to eight samples and gave an average size of particles (equivalent diameter, D) in the range of 0.27 - 0.60 µm, circularity (C) of 0.66-0.85 and elongation (E) of 1.75-1.79, suggesting that the great majority of particles were approximately spherical. Finally, in the third stage, the MORF method was applied to two exceptional samples which contained extremely small wear particles (D = 18.5 nm and 21.2 nm). The shape of these small wear nano- particles (C = 0.97 and 0.93; E = 1.29 and 1.35) was even more spherical than that of wear microparticles described abo- ve. This was one of the first two studies which proved the presence of UHMWPE wear nanoparticles in vivo. DISCUSSION: Our newly developed MORF method described in this contribution yields both size and shape descriptors of UHMWPE wear particles, with sizes from 0.1 to 10 µm, which are regarded as most biologically active. The main objective of the method is to yield the highest accuracy. This is achieved by parallel analyses of high- and low-magnification micrographs taken of the same sample. In the end, the two analyses are combined together in order to obtain the correct and complete size and shape description of all particles in the sample. The morphology of UHMWPE wear particles influences TJR lifetime both directly (size and shape of the particles is related to their biological activity) and indirectly (for the given total volumetric wear, size and shape of the particles influence their concentration, which is associated with the biological respon- se of the organism). CONCLUSION: The authors have developed a new method which yields a complete description of the size and shape of UHMWPE wear particles in periprosthetic tissues. The method, which was called MORF, can be applied to studies of TJR failures and also used to evaluate the quality of different UHMWPE components of TJR. The method is quite universal and therefore can be used not only for analyses of wear particles, but also for other types of particles, such as microparticles in polymer blends or inorganic/metallic nanoparticles.

Binding of proteins to ultra high molecular weight polyethylene wear particles as a possible mechanism of macrophage and lymphocyte activation.

Binding of five human plasma proteins (IgG, serum albumin, α(1)-acid glycoprotein, holo-transferrin, α(1)-antitrypsin) to ultra high molecular weight polyethylene wear particles (0.1-10 µm) isolated from hip periprosthetic tissues was studied in vitro. All tested plasma proteins were bound to wear particles in a similar way indicating irreversible binding. Analogous interaction was found also between GUR 4120 particles (diameter ∼250 µm) and two tested plasma proteins (human serum albumin and α(1)-acid glycoprotein). The binding was not affected by pH of a buffer or the isoelectric point of bound proteins; thus it was apparently of clearly hydrophobic nature. We hypothesize that the binding causes some unfolding of the bound proteins, thus exposing new determinants with which sensitive cells may react. This could be a mechanism by which polyethylene wear particles trigger, for example, macrophages activity and thence initiate aseptic inflammation and cause the failure of total joint replacements. Results can contribute to the choice of a convenient construction type of prostheses.

Distribution of polyethylene wear particles and bone fragments in periprosthetic tissue around total hip joint replacements.

Ultra-high molecular weight polyethylene (UHMWPE) wear particles play a significant role in failures of total joint replacements (TJRs). In this work, we investigated the distribution of these wear particles in periprosthetic tissues obtained from nine revisions of hip TJR. In the first step, all periprosthetic tissues were combined and mechanically separated into granuloma tissue (containing hard granules visible to the naked eye) and surrounding tissue (without visible granules). In the second step, the tissues were hydrolyzed by protease from Streptomyces griseus and granules were separated by filtration; this divided the sample into four groups: (i) lyzate and (ii) non-degraded large granules from the granuloma tissue plus (iii) lyzate and (iv) non-degraded small granules from the surrounding tissue. In the third step, the large as well as small granules were hydrolyzed by collagenase from Clostridium histolyticum. In the last step, the UHMWPE wear particles from all four groups were purified by HNO3 digestion and weighed. The purity of the isolated particles was verified by scanning electron microscopy, infrared spectroscopy and energy-dispersive X-ray analysis. Of the total amount of polyethylene particles in the whole granuloma tissue, 72% of particles in the size range 0.1-10 microm and 68% of those larger than 10 microm were found in granules. Therefore, the formation of granules significantly lowers the effective amount of wear particles available for interaction with reactive cells and seems to be a natural defense mechanism.

[New metod for quantification of UHMWPE wear particles around joint replacements]. / Nová metoda kvantifikace oterových cástic UHMWPE v okolí kloubních náhrad.

PURPOSE OF THE STUDY: The orthopaedic community has unanimously adopted the view that ultra high molecular weight polyethylene (UHMWPE) wear particles are a very frequent cause of aseptic implant loosening. Some studies have tried to provide objective evidence for this.We have found descriptions of particle distribution or morphology, but no report that would objectively cor- relate the number of particles in zones surrounding an implant with the extent of damage to these zones. The aim of this study was to develop a method allowing us to evaluate a number of samples with polyethylene abrasive wear large enough to find association between the extent of damage around a THA and the number of biologically active UHMWPE wear particles, 0.1 to 10 microm in size. MATERIAL AND METHODS: In 28 patients undergoing revision total hip arthroplasty (THA) at the 1st Orthopaedic Clinic, 1st Faculty of Medicine, Char- les University, we took samples of typical osteoaggressive granuloma from defined zones around the implant; the zones corresponded to those described by Gruen and DeLee. The extent of tissue damage in each zone was evaluated on the basis of pre-operative radiographs and by the extent of osteolysis and damage to soft tissues actually observed during revision THA. The volume of wear particles in each zone was assessed by the IRc method developed by us; this is based on a quantitative evaluation of infrared spectra. To verify the methodology, a comparison between tissue damage and the number of particles in each zone was made in three randomly selected patients. RESULTS: We introduced a method of detailed orthopaedic evaluation which enabled us to categorize zones around a revised THA according to the extent of damaged tissue. As a result, a series of zones ranked by the extent of damaged tissue, or an "orthopaedist's statement" (OS), was obtained. At the same time we adopted a method, based on infrared spectroscopy and termed IRc, by which the number of particles in the samples of damaged tissues and osteoaggressive granulomas collected from the area around a revised THA was determined.The results of evaluation were presented as numerical data that, in a defined way, were converted into a series of zones ranked according to the number of wear particles, i.e., the "result of measurement" (RM). In this study we verified the methods described above and made a comparison of OSs and RMs for three randomly selected patients. The very good agreement found confirmed the reliability of both methods which will soon be used to evaluate a group of patients large enough to provide statistically significant results. DISCUSSION: The IRc method determines a total volume of UHMWPE wear particles, 0.1 to 10 microm in size, which are generally considered to be most biologically active. This study suggests that the distribution of particles around a THA is uneven and that relation between tissue damage and the number of wear particles in individual zones surrounding a THA does exist. The major conclusion from the orthopaedic point of view is a confirmation of the assumption that UHMWPE wear particles are one of the chief causes of THA failure. Although this fact is generally accepted, studies correlating the number of particles with tissue damage and osteolysis in individual zones are very scarce. CONCLUSIONS: The quick and simple IRc method offers a possibility to quantify polyethylene wear particles in soft tissues. The number of 0.1 to 10 microm wear polyethylene particles correlated with pre-operative radiographic findings and orthopaedic evaluation of revision THAs in three randomly selected patients. The confirmed correlation between the extent of tissue damage in individual zones surrounding a THA and the volume of wear particles detected in these zones supports the view that UHMWPE wear particles are one of the main causes of THA failure.