Wear 265 (2008) 19141917

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Wear

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Short communication

Can centrifugation affect the morphology of polyethylene wear debris?

E. Zolotarevova
a
, Z. Fejfarkova
a
, G. Entlicher
a
, M. Lapcikova
b
, M. Slouf
b,
, D. Pokorny
c
, A. Sosna
c


a
Faculty of Science, Charles University, Hlavova 8, 12840 Praha 2, Czech Republic
b
Institute of Macromolecular Chemistry AS CR, v.v.i., Heyrovskeho namesti 2, 162 06 Praha 6, Czech Republic
c
Orthopedic Clinic, Faculty Hospital Motol, V Uvalu 84, 156 06 Praha 5, Czech Republic

a r t i c l e i n f o a b s t r a c t

Article history: It was suggested that centrifugation may affect the morphology of ultra high molecular weight polyethy-
Received 23 July 2007
lene wear particles isolated from periprosthetic tissues. In order to investigate this problem, several sets
Received in revised form 20 March 2008
Accepted 11 April 2008
Available online 3 June 2008
of in vivo polyethylene wear particles were isolated in the same way, using four different centrifugation
speeds in the nal isolation step. The rst set of particles was separated only by otation, the second
set was centrifuged at 500 g, the third at 16, 000 g and the fourth at 105,000 g. The morphology
Keywords:
Polyethylene wear particles
Total joint replacements
Centrifugation
Morphology
of the wear particles was assessed by image analysis of SEM micrographs. Centrifugation speeds up to
105, 000 g had no effect on the morphology: quantitative image analysis parameters, such as equiva-
lent diameter, circularity and elongation, remained the same as for particles obtained by spontaneous
otati on.
2008 Elsevier B.V. All rights reserved.


1. Introduction 2. Materials and methods

Although the majority of the total joint replacements (TJR) UHMWPE wear particles were isolated from periprosthetic
containing ultra-high molecular weight polyethylene (UHMWPE) (granuloma) tissues obtained during revisions of TJR in the Ortho-
components often perform well for many years, particulate pedic Clinic, Faculty Hospital Motol, Prague, Czech Republic and in
UHMWPE wear debris formed during their use causes osteoag- the Orthopedic Clinic of Faculty Hospital Olomouc, Czech Republic.
gressive granuloma formation, osteolysis and aseptic loosening of Revised TJRs, from which the wear particles were isolated, are given
joint parts [13]. The amount, size and possibly also composition in Table 1. The isolation method was essentially the same as that
and shape of UHMWPE wear particles may inuence macrophage described earlier [7], the only difference consisted in that all cen-
response and subsequent complications [4,5]. As a result, many trifugations were substituted by spontaneous otation for at least
authors paid attention to the isolation, purication and charac- 24 h. The result of the isolation procedure was a set of four aqueous
terization of in vivo, native, biologically active UHMWPE wear suspensions of UHMWPE wear particles (size 0.110 m), coming
particles [6,7] from periprosthetic tissues. The existing isolation from periprosthetic tissues of four different revised TJRs (Table 1).
methods are being improved [7,8] and new methods appear [9]. Each of the UHMWPE wear particle suspensions was divided
Most of the methods rely on tissue digestion, washing, ltrati on into four parts: (i) the rst part was left as it was, (ii) the second
and/or centrifugation [7,1012]. In one of the studies [9] it was part was centrifuged for 2 min at 500 g, (iii) the third for 5 min
suggested that ultracentrifugation or centrifugation may change at 16,000 g, and (iv) the fourth for 30 min at 105,000 g. Then
particle morphology. However, this assumption has been presented the suspensions were ltered through 0.1- m polycarbonate mem-
without any proof. Therefore, we decided to analyze systemati- branes (Cyclopore, Whatman, UK). The purity of isolated particles
cally the possible effect of centrifugation on morphology of in vivo was veried by scanning electron microscopy (SEM), energy dis-
UHMWPE wear particles isolated from periprosthetic tissues. persive analysis of X-rays (EDS) and infrared spectroscopy (IR) as
described in our previous study [7]. SEM micrographs and EDS spec-
tra were obtained by means of a high-resolution, eld-emission gun
SEM microscope Quanta 200 FEG (FEI). IR spectra were recorded
with an IFS-55 spectrometer (Bruker, Germany) equipped with a
MCT detector (256 scan/spectrum, resolution 4 cm
1
).
The morphology of isolated UHMWPE wear particles on poly-
Corresponding author. Tel.: +420 296 809 291; fax: +420 296 809 410.
E-mail address: slouf@imc.cas.cz (M. Slouf).
carbonate membranes was assessed by image analysis of SEM
micrographs. The total number of samples was 16 (4 cases 4

0043-1648/$ see front matter 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.wear.2008.04.005
bution,

,

was

calculated

as

the

arithmetic

mean

(

=1/n

i
i





E. Zolotarevova et al. / Wear 265 (2008) 19141917 1915

Table 1
Summary of studied patients and total joint replacements
Case Sex Birth Implant type Implant duration (years)
H1 M 1940 ABG (Howmedica) 8.5
H2 F 1955 ABG (Howmedica) 9
H3 F 1933 Poldi/Ultima (Poldi) 8
K1 M 1927 PFC (J + J) 8
Case: three cases of total hip replacement (H1H3), one case of total knee replace-
ment (K1).

different centrifugations; Table 2) and the total number of inves-
tigated micrographs was 320 (20 micrographs per each sample).
Image analysis of all the micrographs was performed with pro-
gram Lucia (Laboratory Imaging). Three morphological parameters
were determined: Equivalent diameter (D), Circularity (C) and Elon-
gation (E). Equivalent diameter is a size feature derived from the
object area, giving the diameter of a circle with the same area as
the corresponding object: D=(4 Area/ )
1/2
. Circularity is calcu-
lated from the object area and perimeter: C =4 Area/Perimeter
2
;
C =1 for circles and C <1 for all other shapes. Elongation is cal-
culated 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.
Distributions of all morphology parameters (C, D and E) were
characterized by their mean values and widths. Mean of the distri-
x )
and width of the distribution, , was calculated as estimated
standard

deviation

(

=

(1/(n

1)

i
(

x
i
)
2
),

where

x
i

is

the ith element of a distribution, n is the number of all ele-
ments in a distribution and summation runs through all elements.
The elements in the above summations were either morphol-
ogy parameters of single wear particles (Table 2) or their mean
values calculated for different centrifugation conditions/patients
(Figs. 2 and 3). Condence intervals from the values of were
not calculated, because we characterized widths of the distribu-
tions (Table 2) or variances of the data (Figs. 2 and 3) rather than
probabilities that morphological parameters are within a particular
range.






































Fig. 1. SEM micrographs showing UHMWPE wear particles on 0.1- m polycarbonate membranes. Particles were separated from solution only by otation (a) and by
centrifugation at 500 g (b), 16,000 g (c) and 105,000 g (d). Particles were isolated from the rst patient (case H1 in Table 1).




1916 E. Zolotarevova et al. / Wear 265 (2008) 19141917

Table 2
patients with similar particle size distributions (Fig. 2) so that it
UHMWPE wear particle sizes and shapes obtained by image analysis of SEM micro-
graphs for all patients and centrifugation conditions
was readily possible to compare and average all results (Fig. 3).
After centrifugation, both average morphological parameters
Sample Equivalent diameter (D) Circularity (C) Elongation (E)
(Table 2) and histograms (Fig. 3) demonstrated that particle sizes
( m) ( m) () () () () (given by D) and shapes (given by C and E) remained more-or-less
H1/F 0.32 0.24 0.66 0.24 1.78 0.71
H1/C1 0.32 0.25 0.67 0.25 1.75 0.53
constant and did not change with centrifugation in any systematic
way. This suggested that centrifugation had negligible effect on
H1/C2 0.26 0.16 0.68 0.23 1.71 0.51 particle morphology.
H1/C3 0.30 0.22 0.67 0.26 1.75 0.62
Selection of the patients for this study was made as follows:
H2/F 0.30 0.36 0.66 0.26 1.92 0.83
H2/C1 0.24 0.21 0.70 0.23 1.70 0.49
H2/C2 0.29 0.26 0.65 0.26 1.79 0.77
H2/C3 0.23 0.17 0.70 0.24 1.76 0.64
Our isolation procedure based on HNO3 digestion [7] was applied
to samples from more than 100 patients. In 25 cases we corre-
lated total amounts of wear particles with osteolysis in various
H3/F 0.35 0.32 0.70 0.28 1.76 0.78 zones around TJR [13], while the remaining results are being pro-
H3/C1 0.36 0.27 0.62 0.25 1.82 0.67
H3/C2 0.31 0.22 0.64 0.25 1.74 0.66
H3/C3 0.30 0.26 0.67 0.25 1.75 0.67
K1/F 0.26 0.23 0.73 0.23 1.69 0.57
K1/C1 0.34 0.30 0.67 0.26 1.76 0.72
K1/C2 0.29 0.23 0.67 0.26 1.72 0.73
K1/C3 0.36 0.32 0.64 0.27 1.86 0.72

Samples are denoted as Case/Centrifugation, where Cases are TJRs described in
Table 1 and Centrifugations are (F or C1 or C2 or C3), where F = otation and
C1/C2/C3 = centrifugation at 500/16,000/105,000 g. Each numerical value in the
table was calculated from a distribution obtained by image analysis of exactly
twenty SEM micrographs with magnication 10,000 and real width 24 m. Mean
of the distribution, , was calculated as the arithmetic mean ( = 1/n i xi)
and width of the distribution, , was calculated as estimated standard deviation
(

=

(1/(n

1)

i
(

xi
)
2
),

where

x
i

is

the

ith

element

of

a

distribution,

n

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

3. Results

Illustrative SEM micrographs of in vivo UHMWPE wear particles
isolated from the rst patient are shown in Fig. 1. No qualitative dif-
ference was found between the particles separated by spontaneous
otation (Fig. 1a) and centrifugation at 500, 16,000 or 105,000 g
(Fig. 1bd). All micrographs showed both spherical and elongated
particles indicating that centrifugation did not impact on particle
morphology signicantly.
Quantitative information from SEM micrographs was conrmed
by image analysis, whose results are summarized in Table 2. Particle
morphology was described by equivalent diameter D, circularity C
and elongation E. After otation, the morphology of wear particles
was quite similar for all four cases (H1/F, H2/F, H3/F and K1/F in
Table 2). This resulted from the fact that we intentionally selected














Fig. 2. Particle size distributions in all four studied patients. Each column represents Fig. 3. Distributions of particle sizes (a) and shapes (b and c). Each column rep-
the average through all centrifugation conditions (which are denoted as F, C1, C2 and resents the average through all four patients (which are denoted as H1, H2, H3
C3 in Table 2); error bars were calculated as estimated standard deviations of the and K1 in Table 2); error bars were calculated as estimated standard deviations
corresponding average. The columns are given in quadruplets; the columns in each of the corresponding average. The columns are given in quadruplets; the columns in
quadruplet, from left to right, correspond to case H1, H2, H3 and K1 in Table 1, each quadruplet, from left to right, correspond to otation, centrifugation at 500 g,
respectively. 16,000 g and 105,000 g, respectively.




E. Zolotarevova et al. / Wear 265 (2008) 19141917 1917

cessed [14]. In 10 cases we compared morphology of the particles
isolated with or without centrifugation and found no qualitative dif-
ferences in SEM micrographs. Detailed quantitative morphological
study was performed in four cases listed in Table 1; this means that
the presented results are based on image analysis of 320 SEM micro-
graphs (4 patients 4 centrifugation speeds 20 micrographs per
sample).

4. Discussion and conclusion

From the physico-chemical point of view there is little chance
that centrifugation forces would change the morphology of
UHMWPE wear particles. Centrifugation at 105,000 g is generally
used for separation of cell organelles without any signicant dam-
age to them [15]. However, when such a possibility was suggested
for polyethylene wear particles [9], it was desirable to investi-
gate the problem and verify the correctness of both our own [7]
and other isolation techniques [1012], which all employ centrifu-
gation. There are two theoretical possibilities how centrifugation
could have inuenced wear particle morphology: (i) elongated par-
ticles might have been changed to spherical due to mutual collisions
and collisions with vessel edges at very high centrifugation speeds
and (ii) very small particles might have been isolated in higher
amounts at higher centrifugation rates. Nevertheless, neither of the
above-mentioned effects was observed and even ultracentrifuga-
tion at 105,000 g did not change the morphology of UHMWPE
wear debris.

Acknowledgement

This work was supported by grant of the Ministry of Education,
Youth and Sports of the Czech Republic (MSMT 2B06096).

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