Wear 266 (2009) 349355

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Wear
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Nanometer size wear debris generated from ultra high molecular weight polyethylene in vivo
Monika Lapcikova a , Miroslav Slouf a, , Jiri Dybal a , Eva Zolotarevova b , Gustav Entlicher b , David Pokorny c , Jiri Gallo d , Antonin Sosna c
a

Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, v.v.i., Heyrovskeho namesti 2, 162 06 Prague 6, Czech Republic Faculty of Sciences, Charles University, Hlavova 8, 128 40 Prague 2, Czech Republic Orthopaedics Clinic, Faculty Hospital Motol, V Uvalu 84, 156 06 Prague 5, Czech Republic d Department of Orthopaedics, Faculty of Medicine and Dentistry, Palacky University, I.P. Pavlova 6, 775 20 Olomouc, Czech Republic
b c

a r t i c l e

i n f o

a b s t r a c t
Ultra high molecular weight polyethylene (UHMWPE) wear debris is a major cause of long-term failure of total hip replacements. UHMWPE wear particle sizes range from submicron to several millimeters, but the particles below 10 m exhibit the highest biological activity. Some in vitro wear particles, produced in joint simulators, were shown to be smaller than 0.2 m and recently even in vitro particles as small as several tens of nanometers have been detected. This study brings the rst evidence that nano-sized wear particles with sizes below 0.05 m are produced in vivo. UHMWPE wear nanoparticles were revealed by high-resolution, eld emission gun scanning electron microscopy (FEGSEM) in the periprosthetic tissues of two different patients. Purity of the isolated wear nanoparticles was conrmed by energy-dispersive analysis of X-rays (EDS) and infrared spectroscopy (IR). Morphology of wear nanoparticles was determined by image analysis of FEGSEM micrographs. The average equivalent diameters of wear particles in the rst and the second patient were 18.5 and 21.2 nm, respectively. Nanoscale wear debris could only be reliably detected if the isolation protocol included intensive sonication and if higher-than-usual magnications were employed during FEGSEM analysis. 2008 Elsevier B.V. All rights reserved.

Article history: Received 20 December 2007 Received in revised form 21 May 2008 Accepted 9 June 2008 Available online 17 July 2008 Keywords: Ultra high molecular weight polyethylene Nanometer size wear debris Morphology of wear particles Total hip replacement

1. Introduction Ultra high molecular weight polyethylene (UHMWPE) has been used as a bearing material in total joint replacements (TJR) for more than four decades. The success of UHMWPE in TJRs is due to its excellent bulk biocompatibility, very good friction properties, sufcient mechanical performance and high wear resistance [1]. Therefore, UHMWPE is regarded as the gold standard for given application [2,3]. Nevertheless, articulation between polymer and metallic/ceramic components of TJR leads to generation of tiny UHMWPE wear particles, which are released from the joint space and cause complex inammatory reactions leading to osteolysis and aseptic loosening [4]. Since UHMWPE wear particles were recognized as one of the major causes of TJR failures, a number of studies describing their isolation [5], characterization [6] and biological activity [7] have been published. The particle sizes range from submicron to several millimeters. It was shown that the most biologically active and

thus the most dangerous wear particles have sizes below 10 m [8]. A few years ago in vitro experiments on wear testing machines proved that a signicant fraction of wear particles can be smaller than 0.2 m [9]. Recently in vitro wear particles of nanometer sizes below 0.1 m were observed [10]. The present study proves, for the rst time, that nanometer-sized wear particles are also generated in vivo. In our previous work, we focused on development of a reliable technique for sampling, isolation and quantication of in vivo UHMWPE wear particles in periprosthetic tissues [1115]. More than 100 samples of periprosthetic tissues have been processed and both total amount and morphology of wear particles were determined. In two cases we found that the periprosthetic tissues contained mostly nanometer-sized wear debris. 2. Materials and methods 2.1. Isolation of wear particles 2.1.1. Sampling UHMWPE wear particles were detected in periprosthetic (granuloma) tissues obtained during revisions of TJRs in Orthopaedics

Corresponding author. Tel.: +420 296809310; fax: +420 296809410. E-mail address: slouf@imc.cas.cz (M. Slouf). 0043-1648/$ see front matter 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.wear.2008.06.005

350 Table 1 Summary of periprosthetic tissues studied in this work Patient H1 H2 Sex M M Birth 1937 1957 Weight (kg) 82 70

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Particle separation on low-concentration membranes was further improved by sonication as documented below.
Implant type Balgrist/CF 30 ABG

Implant duration (years) 8.5 8.8

2.2. Characterization of wear particles 2.2.1. Field-emission gun scanning electron microscopy (FEGSEM) High-resolution scanning electron microscope Quanta 200 FEG (FEI), equipped with a eld-emission gun, was used to visualize wear debris. Polycarbonate (PC) membranes with isolated polyethylene (PE) wear particles were xed onto metallic support with a conductive silver paste, and the specimen was sputter coated with a 10-nm thick platinum layer using a vacuum sputter coater SCD 050 (Balzers). This xation was found necessary to eliminate sample drift and sample damage under the electron beam at very high magnications, which were necessary to detect polyethylene nanoparticles. The micrographs were obtained with a secondary electron detector at an accelerating voltage of 10 kV and the smallest possible spot size. In the rst step, homogeneity of PE wear particles on each PC membrane was investigated at low magnications (500 to 5000). In the second step, selected representative locations on the lters were analyzed at three different magnications: medium magnication (7500), high magnication (12,000) and very high magnication (50,000). In this paper we use exclusively the above three magnications (medium, high and very high) for sake of simplicity and easy comparison of all samples. 2.2.2. Energy-dispersive analysis of X-rays (EDS) The same samples as those used for SEM were observed in the microscope Quanta 200 FEG as described in the previous section, using an EDS detector (EDAX). EDS spectra were taken at 30 kV. The signal was collected both from the whole areas of SEM micrographs and from selected places containing large agglomerates of wear particles. 2.2.3. Fourier-transform infrared spectroscopy (FTIR) Infrared-spectra were recorded with an IFS-55 spectrometer (Bruker, Germany) equipped with MCT detector (256 scan/spectrum, resolution 4 cm1 ). IR spectra of selected PC membranes with PE particles were measured before sputtering with platinum and investigation by SEM and EDS. The measurement was performed in two ways: (i) several locations on each membrane were measured by the attenuated total reection (ATR) technique using a Golden Gate TM Heated Diamond ATR Top-Plate (Specac Ltd.) and (ii) the whole membrane was also measured in the transmission mode. 2.2.4. Image analysis of FEGSEM micrographs Image analysis of FEGSEM micrographs was performed using the Lucia program (Laboratory Imaging, Czech Republic). FEGSEM micrographs with magnications of 50,000 were used to distinguish and measure individual wear nanoparticles. Lower magnication micrographs were not used because the two analyzed samples contained only nanoparticles, as demonstrated below. Both image analyses were based on more than 500 particles. The particles were described by means of equivalent circle diameter (D), circularity (C) and elongation (E). Equivalent diameter of an object is equal to the diameter of a circle with the same area as the corresponding object: D = (4 Area/ )1/2 . Circularity is calculated from the object area and perimeter: C = 4 Area/Perimeter2 ; C = 1 for circles and C < 1 for all other shapes. Elongation is dened as the ratio of maximum and minimum diameter of the particle: E = MaxDiameter/MinDiameter; E = 1 for circles and E > 1 for all other shapes.

Clinic, Faculty Hospital Motol, Prague, Czech Republic and in the Orthopaedic Clinic of Faculty Hospital Olomouc, Czech Republic. A summary of periprosthetic tissues analyzed in this study is given in Table 1. 2.1.2. Purication of chemicals Distilled water and isopropyl alcohol (iPrOH) were puried before use by successive ltration through 10 and 0.1 m polycarbonate (PC) Isopore membranes (Millipore, Ireland). More aggressive chemicals, such as 65% HNO3 and 12 mol/l KOH were ltered through 10 and 0.1 m Teon (PTFE) Omnipore membranes (Millipore, Ireland). 2.1.3. Delipidation of samples Typically, 0.3 g of a freeze-dried tissue sample was cut into small pieces and extracted twice with 10 ml of a chloroform/methanol mixture (2:1, v/v) for 12 h. The solvents were decanted. After two decantations the tissue samples were dried in a stream of ltered air and by heating for 2 h at 60 C. 2.1.4. Acid hydrolysis and washing The delipidated samples of tissue (obtained from 0.3 g of freezedried sample) were hydrolyzed with 5 ml of 65% HNO3 at room temperature for 24 h. Only the upper 2 ml layer of the suspension was used for further isolation. The remaining lower part was removed by aspiration and discarded. The upper layer was washed twice with 5 ml of 65% HNO3 and twice with 5 ml of distilled water: 5 ml of solvent (HNO3 or H2 O) were added, the sample was centrifuged (500 g, 1 min) and the lower 5 ml were removed by aspiration. The resulting washed 2 ml layer was neutralized with 12 mol/l KOH and washed twice with water in the same way as described above. The nal 2 ml of the sample was used for further purication. 2.1.5. Separation of wear debris by ltration The wear debris suspension obtained by acid hydrolysis was mixed with 2 ml of iPrOH and ltered through a 10-m Teon membrane Omnipore (Millipore, USA). The ltrate was lled up to 6 ml with iPrOH. Then these 6 ml of the suspension were divided into four parts: (i) 4.5 ml of the suspension was ltered through a 0.1m polycarbonate membrane Isopore (Millipore, USA), (ii) 0.2 ml of the suspension was ltered through another 0.1-m polycarbonate membrane Isopore (Millipore, USA), (iii) another 0.2 ml of the suspension was sonicated (5 min, 30 kHz, amplitude 112 m, energy density 125 W/cm2 ) just before the ltration through the 0.1 m membrane Isopore and (iv) the remaining 1.5 ml of the suspension was left as a reserve. 2.1.6. Usage of lter membranes containing wear particles All membranes were investigated by all the methods described below, however, the image analysis results were based exclusively on membranes with lower concentrations of particles (0.2 ml of the suspension), whereas infrared spectroscopy and microanalysis were performed on all the membranes (both 0.2 and 4.5 ml). The low-concentration membranes showed better particle separation on electron micrographs, while the high-concentration membranes gave higher signals in infrared spectroscopy and microanalysis.

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particles (Al, Zr), were found; a typical EDS spectrum is shown in Fig. 2. 3.2. Separation of nanoparticles by sonication Fig. 3 shows PC membranes with PE particles isolated from periprosthetic tissue of the rst patient H1 (Table 1). On the membranes, nanometer-sized wear particles tended to form aggregates (Fig. 3a, c and e), which could be disintegrated by sonication of the ltered suspension (Fig. 3b, d and f). The aggregates of non-sonicated nanoparticles at medium magnications (Fig. 3a; magnication 7500) and high magnications (Fig. 3c; magnication 12,000) looked as if they were microparticles. Single nanoparticles within the aggregates were only revealed at very high magnications (Fig. 3e; magnication 50,000). Sonication resulted in nanoparticle separation, which could be observed even at medium magnications (Fig. 3b). At high magnications (Fig. 3d), the single nanoparticles could be clearly resolved. The micrographs of sonicated nanoparticles at very high magnications (Fig. 3f) were suitable for image analysis. 3.3. Image analysis of separated nanoparticles Fig. 4 shows nal FEGSEM micrographs of PC membranes with in vivo PE wear particles, which were isolated from periprosthetic tissues of both patients (Table 1). Both suspensions of the isolated particles were sonicated immediately before ltration through the membrane to separate particles. It is worth noting that FEGSEM micrographs displayed neither bigger particles (>0.1 m) nor impurities (such as bone fragments with their characteristic texture or dust particles with sharp edges). Image analysis of the wear particles of each patient was based on nine FEGSEM micrographs from different parts of the membrane. All nine micrographs in each set have the same magnication (50,000; the same as in Fig. 4e and f) and each set of micrographs contained more than 700 well-separated particles for analysis. The wear particles from patients H1 and H2 exhibited average sizes 18.5 and 21.2 nm, respectively. Complete histograms (Fig. 5) proved that in both cases the wear particles were quite small, with relatively narrow size distributions. Complete numerical results of image analysis (Table 2) re-conrmed the small particle size, conrmed narrow size distributions and indicated that all particles were spherical in shape. 3.4. Nanoparticles in other samples In the previous sections we have demonstrated that 2 samples out of 100 contained mostly nanoparticles. Detailed microscopic analysis of the two samples suggested that they contained almost no microparticles. At higher magnications, practically all microparticles were identied as the agglomerates of nanoparticles as documented by FEGSEM (Figs. 3 and 4) and conrmed by image analysis (Fig. 5 and Table 2). The other samples exhibited no exceptional features and contained mostly microparticles, in agreement with numerous previous studies (e.g. [1120]). Particles coming from one of our typical samples of periprosthetic tissue are shown in Fig. 6. The difference between the two samples containing mostly nanoparticles (Figs. 3 and 4) and the typical samples containing mostly microparticles (Fig. 6) was visible even at medium magnication FEGSEM micrographs (cf. Figs. 3a and b, 4a and b and 6a). In the two samples containing nanoparticles there were no particles >0.1 m, whereas a typical sample contained a number of particles with dimensions >1 m. This was the main reason why we focused our attention on the two exceptional samples and revealed wear

Fig. 1. Comparison of IR spectra of pure PE (from a catalog), blank PC membrane and PC membrane with isolated PE particles. The spectra were measured in ATR mode (PE = polyethylene, PC = polycarbonate, ATR = attenuated total reection infrared spectroscopy).

3. Results 3.1. Purity of isolated particles Three facts indicated that observed nanoparticles were not artifacts, but pure UHMWPE. Firstly, we used a well-established isolation technique, which had already been employed and perfected in our previous studies [1215]. Secondly, the purity of the isolated PE particles on PC membranes was investigated by means of FTIR: comparison of IR spectra of pure PE, unused PC membrane and PC membrane with PE nanoparticles showed that all samples exhibited only peaks of PE and/or PC (Fig. 1). Thirdly, we performed EDS analysis of all membranes, which were investigated by FEGSEM. In all cases, we found only peaks of C (mostly from PC membrane and partially from PE particles), O (from PC membrane), Pt (10 nm thick platinum layer from vacuum sputter coater), Ag and Cl (from conductive silver paste that xed the membranes to the support). No traces of other elements, which would indicate bone fragments (Ca, P), dust (Si), organic impurities (N, S), metallic wear particles (Cr, Mo, Ti) or ceramic wear

Fig. 2. EDS spectrum of UHMWPE wear particles on a PC membrane. The peaks of C and O come from the PC membrane and/or UHMWPE particles. The peaks of Pt, Ag and Cl result from the fact that the membrane was sputter coated with platinum and xed with a conductive silver paste containing chlorine before investigation in an electron microscope (PC = polycarbonate, EDS = energy-dispersive analysis of X-rays).

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Fig. 3. FEGSEM micrographs of UHMWPE wear particles on 0.1 m PC membrane. All particles come from patient H1 in Table 1. Left column (a, c and e): particles without sonication before ltration; right column (b, d and f): particles with sonication before ltration. The selected micrographs were taken at medium (a and b), high (c and d) and very high (e and f) magnication.

nanoparticles, which become clearly visible at higher magnications (Figs. 3cf and 4cf). Nevertheless, further analysis of the other samples, which contained mostly microparticles, showed certain small amounts of wear nanoparticles as well (Fig. 6b and c). Although FEGSEM micrographs of typical samples re-conrmed clear predominance of wear microparticles, small amounts of wear nanoparticles could be detected (Fig. 6c), nanoparticles marked with white arrows. This indicated that a certain number of wear nanoparticles might be present in all periprosthetic tissues. Accurate determination of volume fraction of wear nanoparticles in typical samples of periprosthetic tissues is a subject of our subsequent study.

4. Discussion In the previous section it has been demonstrated for the rst time that UHMWPE wear nanoparticles are generated in vivo. We should re-emphasize that in this study the term wear nanoparticles denotes UHMWPE particles with sizes below 0.05 m, i.e. the particles whose size is even smaller than the lowest pore size in commonly used lters [9]. Three independent methods, FEGSEM, EDS and IR suggested that the observed nanoparticles are a polyethylene and not an artifact. Moreover, the nanoparticles with average sizes 18.5 and 21.2 nm were observed in two independent samples. On the other hand, many previous studies reported much larger average sizes of in vivo UHMWPE wear nanoparticles.

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Fig. 4. Final FEGSEM micrographs of PC membranes with UHMWPE wear particles of both studied patients, H1 and H2 (Table 1). Left column (a, c and e): particles from H1; right column (b, d and f): particles from H2. The selected micrographs were taken at medium (a and b), high (c and d) and very high (e and f) magnications.

Why were similar wear nanoparticles not found in previous works? The rst reason why wear nanoparticles were not reported before might consist in the fact that we investigated more than 100 samples and detected signicant volumes of wear nanoparticles just in 2 of them. In the other samples the microparticles predominated and the amounts of nanoparticles were either small or negligible. Elck et al. [16] summarized the average sizes of in vivo wear particles from various studies; their sizes varied around 0.5 m. Also in vitro wear particles from joint simulators showed average sizes of several tenths of micrometers [17]. Thus it seems that standard wear particles are microparticles, whereas wear nanoparticles might be an exception.

The second reason why wear nanoparticles escaped the attention of previous investigators could be sonication. Wear particles are usually isolated from periprosthetic tissues by means of acid/base digestion method. Then the suspension of the particles is ltered through a membrane and the particles are analyzed by means of scanning electron microscopy. Our results showed that the nanoparticles on the membrane tend to form agglomerates looking like single microparticles (Fig. 3). Intensive sonication of the suspension just before its ltration through a membrane is necessary to achieve nanoparticle separation. As this sonication was not always used in other studies, the agglomerates of nanoparticles could have been misinterpreted as single microparticles in particular at low and medium magnication micrographs. In this study the nanoparticles on the membranes occurred as loose agglomer-

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The fourth reason why wear nanoparticles were not found on the membranes after isolation might consist in the isolation and characterization methods themselves. Over the course of time, the methods of isolation of wear particles from periprosthetic tissues have been gradually improved. Some of the isolation protocols were far from perfect, as discussed in our previous work [13].

Fig. 5. Histogram showing size distributions of UHMWPE wear particles isolated from patient H1 (full columns) and patient H2 (striped columns). Size of the particles was described by means of equivalent diameter. Number of particles used for the analysis was >700 in both cases.

ates (Figs. 3 and 4). If the sonication was even more intensive, the nanoparticles might be completely separated and, consequently, they might go through the membrane without being detected. It seems that just sufcient sonication is needed to catch the nanoparticles. The third reason why wear nanoparticles were not observed might be associated with a combination of sonication, high magnications and the use of FEGSEM instead of standard SEM microscopy. The effect of sonication has already been discussed in the previous paragraph. The effect of high magnication is as follows: an ISO standard for quantitative image analysis of wear nanoparticles [18] recommends using SEM micrographs with magnications of at least 5000. Such medium magnications (5000 to 10,000) were used in most of the previous studies, whereas here we have demonstrated that non-sonicated wear nanoparticles were observed only at very high magnications (50,000). In a standard SEM microscope, it is possible to achieve such a high magnication with a good signal and resolution only if an intensive electron beam and acceleration voltage around 30 kV are used. We have veried that under such conditions the PC membranes with PE particles were burnt by the electron beam and, as a result, the nanoparticles could not be observed. In a FEGSEM microscope, the magnication 50,000 with good resolution could be achieved at 10 kV and a small probe size, which did not destroy the specimens. Therefore, somewhat unusual combination of sonication, very high magnication and FEGSEM microscopy seems to be necessary for detection of wear nanoparticles. At the highest magnications the nanoparticles could be observed even without sonication, but they were not so well separated from each other.
Table 2 Results of the image analysis of UHMWPE nanoparticles Patient Eq. diameter (D) (nm) H1 H2 18.47 21.20 (nm) 5.29 8.01 Circularity (C) () 0.97 0.93 () 0.07 0.09 Elongation (E) () 1.29 1.35 () 0.13 0.29

Each numerical value in this table was calculated from a distribution obtained by image analysis of nine FEGSEM micrographs with magnication 50,000 and real width 1.26 m. The mean of the distribution, , was calculated as the arithmetic xi ) and the width of the distribution, , was calculated as estimean ( = 1/n
i

mated standard deviation ( =

(1/n

of the distribution, n is the number of all elements in the distribution and summation runs through all elements.

xi ) ), where xi is the ith element

Fig. 6. FEGSEM micrographs of PC membranes with UHMWPE wear particles of a typical patient, whose periprosthetic tissue contained mostly microparticles. The micrographs were taken at medium (a), high (b) and very high (c) magnications. A few nanoparticles are visible at very high magnications (marked with white arrows).

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In some other studies, the authors used techniques, which were not able to detect the smallest particles. For example, some particle counters detected only particles with sizes above 0.3 m [19], in transmission electron microscopy the nanoparticles might be hardly observable due to insufcient contrast as their size may be lower than the thickness of investigated ultrathin section (typically around 60 nm) [20] and in the methods based on light scattering the signal from bigger particles may entirely predominate [11,12]. Our results suggest that a combination of isolation method (veried in previous studies) and good detection method (sonication + FEGSEM) are necessary to catch wear nanoparticles. In future studies the in vivo UHMWPE wear nanoparticles might be observed more and more frequently as the sampling, isolation and detection methods are further improved. 5. Conclusion Methods for isolation and visualization of UHMWPE wear particles are still being improved. As a result, it has been possible to isolate and detect smaller and smaller wear particles. A few years ago Scott et al. [9] proved that a signicant fraction of in vitro UHMWPE wear particles can be smaller than 0.2 m. Recently Galvin et al. [10] found in vitro UHMWPE wear nanoparticles with sizes below 0.1 m. In the present work we found, for the rst time, in vivo UHMWPE wear nanoparticles with sizes below 0.05 m. These nanoparticles were isolated from periprosthetic tissues of two different patients and had average particle size of 18.5 and 21.2 nm in the rst and the second case, respectively. Acknowledgement Financial support through grant 2B06096 (Ministry of Education, Youth and Sports of the Czech Republic) is gratefully acknowledged. References
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