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SAS Journal 3 (2009) 133–142

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Metal wear particles: What we know, what we do not know, and whyFabrizio Billi, PhD a,*, Paul Benya, PhD a, Edward Ebramzadeh, PhD a,

Pat Campbell, PhD a, Frank Chan, PhD b, Harry A. McKellop, PhD a

a Orthopaedic Hospital – UCLA, Los Angeles, CAb Medtronic, Memphis, TN

bstract

The importance of wear particle characterization for orthopaedic implants has long been established in the hip and knee arthroplastyiterature. With the increasing use of motion preservation implants in the spine, the characterization of wear debris, particularly metallicature, is gaining importance. An accurate morphological analysis of wear particles provides for both a complete characterization of theiocompatibility of the implant material and its wear products, and an in-depth understanding of the wear mechanisms, ion release, andssociated corrosive activity related to the wear particles. In this paper, we present an overview of the most commonly-used publishedrotocols for the isolation and characterization of metal wear particles, and highlight the limitations and uncertainties inherent to metalarticle analysis.

2009 SAS - The International Society for the Advancement of Spine Surgery. Published by Elsevier Inc. All rights reserved.

eywords: Wear particles; Morphology; Characterization; Metal-on-metal; Digestion protocol

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In the past decade, there has been growing acceptance ofhe use of motion-preservation implants in the spine. Priorxperience with orthopaedic devices, such as hip and kneemplants, has shown that particulate wear debris can bessociated with postoperative complications such as peri-mplant osteolysis.1–7 In the last 20 years, new materialsave been developed for use in total hip and knee arthro-lasty that effectively reduce the total amount of wearenerated in situ. These materials include cross-linked poly-thylenes, as well as combinations of metal-on-metal anderamic-on-ceramic materials for articulating surfaces.

The introduction of modern metal-on-metal bearing sur-aces for hip replacement components has resulted in re-ewed interest in the analysis of metal particles due tooncerns for the potential long-term effects of metal wearroducts in vivo. Despite their increasing popularity, it isidely recognized that modern metal-on-metal bearings for

otal hips can generate extremely small sized metal parti-les.8–10 For a given period of usage, the total volume ofetal wear debris from a metal-on-metal articulation may

e much smaller compared to the wear volume of polyeth-

* Corresponding author: Fabrizio Billi, PhD, Orthopaedic Hospital –CLA, 2400 S. Flower Street -5th Floor, Los Angeles, CA 90007-2629.

mE-mail address: [email protected]

935-9810 © 2009 SAS - The International Society for the Advancement of Spinoi:10.1016/j.esas.2009.11.006

lene from a polyethylene-on-metal articulation.9 However,ue to the smaller metal particle size, it is estimated that theumber of metal particles can be up to 100 times greaterhan the number of polyethylene particles.8,10,11 The smallize of metal particles vastly increases the total surface areaf the metal exposed to the aggressively corrosive bodynvironment, increasing the propensity to release metalons. Moreover, concerns have been raised regarding theoxicity, corrosion, and ion release associated with metalear debris; although the long-term implications related to

arcinogenicity remain unknown to date.12–16

As motion preservation gains traction as a treatmentaradigm for spinal disorders, the need for accurate char-cterization of the wear particles generated at articulatingurfaces in the spine is growing in importance. An accurateorphological analysis of wear particles can provide an

nderstanding of the specific mechanisms of wear, and alsollows for a more complete characterization of the biocom-atibility of the implant materials employed. Previous ex-erience in particle characterization for large joint ortho-aedic implants has shown that these techniques areomplex and on the leading edge of scientific development.owever, we do stand to gain significantly from this bodyf knowledge as it relates to particle characterization forpinal implants. In this paper, we present an overview of the
ost commonly used published protocols to isolate, display,
e Surgery. Published by Elsevier Inc. All rights reserved.

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134 F. Billi et al. / SAS Journal 3 (2009) 133–142

nd morphologically and chemically characterize metalear particles, and highlight the advantages and limitations

n each.

he necessity and importance of particleharacterization

In the literature, the most widely used particle character-stic is particle size which is evaluated in different ways,ormally estimating the Feret’s diameter (the longest dis-ance between any 2 points along the particle boundary) orhe equivalent circle diameter (the diameter of the circle thatould have the equivalent area as the particle). Shape, if

valuated, is normally assessed qualitatively or semi-quali-atively. Most of the previous particle analysis work inrthopaedic biomaterials has focused on polyethylene wearebris. It has been suggested that small (less than 1 �m)olyethylene particles can be phagocytosed more easilyhan larger particles and that elongated particles may inducestronger cellular reaction than rounded particles.17,18 More

ecently, the importance of metallic wear particle analysisas gained interest in the community. As a result, it is nowecognized that particles interact differently with the bio-ogical environment depending on a multitude of particleharacteristics including size, shape, surface topography,nd chemistry.6,19–24

The tribological properties of materials strongly affecthe characteristics of wear particles produced during im-lant use. Conversely, particle characteristics can often pro-ide information regarding not only the magnitude of wearut also the types of underlying wear mechanisms in action.or example, under normal conditions of use, the continu-us generation and removal of the protective layer of aetal surface may produce extremely small particles in

ound or oval shapes. This generally indicates mild abrasiveear. In contrast, more severe service conditions will pro-uce greater number of irregularly shaped and larger parti-les whose chemistry is closer to that of the bulk material,ndicating severe abrasion. It has been recently hypothe-ized that microstructural changes at and below the surfacere responsible for the generation of nanometer-sized wearebris with metal-on-metal implants.25

There has also been increasing interest in the evaluationf biological response to particles, and, in particular, toetal wear particles. It is important to note, therefore, that

valuation of host bioreactivity to wear debris requires firstnd foremost a full morphological and chemical character-zation of the debris. Clearly, a well-established and vali-ated method to characterize wear debris is necessary toacilitate rigorous bioreactivity testing, identify the under-ying wear mechanisms, and understand the material deg-adation phenomena.

Laboratory wear simulations provide an accurate andeliable prediction of an implant’s wear performance. Inddition to providing the amount of wear, it is important for
aboratory wear tests to also analyze the morphology, dis- u
ribution, and chemical composition of the wear particlesenerated. Furthermore, the validation of wear simulationtudies requires an accurate and direct comparison of thearticles from the wear simulator to particles extracted fromeri-implant tissues. Therefore, the isolation protocolhould ideally be designed so that it can be used for thesolation of particles from both laboratory wear simulationtudies and from explanted tissues. Unfortunately, metalarticles obtained from tissues have already gone throughome extent of degradation in vivo, complicating suchomparisons.

In addition to wear simulations, laboratory studies ofioreactivity also have the potential to provide vital infor-ation in predicting clinical performance of new materials.or such studies ideally, the same method used to isolatend characterize particles from wear simulations should alsoe used to isolate and characterize particles for prediction ofioreactivity. This would ensure that the particles providedor cellular bioreactivity testing have the same morphology,ize distribution, and chemical composition as those produceduring the wear process. This fundamental observationeads to an important consequence: the isolation processust be designed so that particles can be fully characterized

nd virtually free of any contaminant or residue.A synopsis of the information that can typically be ob-

ained from analysis of wear particles is illustrated in Fig. 1.In recent years, numerous novel implants have been

ntroduced for the spine. Typically, FDA requirements forpproval of a new implant include, among others, results ofatigue and wear testing.26 Increased recognition of themportance of wear particle analysis has resulted in regula-ory organizations requiring particle analysis as a routineomponent of implant fatigue and wear testing, generalrocedures of which are described in various ASTM andSO documents.27,28 So far, these documents provide gen-ral guidelines but do not outline the details of the meth-dology required to conduct reliable and reproducible par-icle isolation and characterization. Specifically, an FDAuidance document26 specifies, “You should . . . extract theear debris from the test solution using a filter with a pore

ize that allows collection of sub-micron particles,” and that. . . you should characterize the wear debris.” As discussedelow, these requirements are not well-defined and over-ook the complexities of the process of particle analysis.ubstantial challenges remain towards the goal of establish-

ng a single, accurate and repeatable method for the analysisf metal particles generated by implant wear for universalpplication in the orthopaedic and spine communities.

ast accomplishments

Many different techniques and approaches have beensed over the years in order to establish a reliable andepeatable methodology for the isolation and characteriza-ion of metal wear particles from tissues or from lubricants
sed for wear simulations.8,9,29–47 A thorough review of

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135F. Billi et al. / SAS Journal 3 (2009) 133–142

hese methods is beyond the scope of this article. However,n this section, we briefly summarize the most significantast accomplishments of key investigators towards particlesolation and analysis.

Any particle analysis protocol essentially consists of 4arts: digestion and isolation, display and image acquisition,orphological and chemical characterization, and statistical

nalysis.

igestion and isolation

The first step in the analysis of particles is their isolationrom serum lubricant solutions, tissues, or synovial fluid. Eachf these is characterized by the presence of different proteins,ipids, and other constituents such as hyaluronic acid. Particlesan bind to these macromolecules or be trapped along withther residues. In order to free the particles from any substancehat eventually might interfere with their characterization, re-earchers have used different approaches.

Selection of each particular approach also depends on the

Fig. 1. Interrelations among wear particle char

able 1rotocols for metal particle isolation/characterization

uthors Digestion Display

chmiedberg Papain � pK (NaOH wash) Drop Evaporationoorn Papain � pK Nebulizationatelas Papain � pK Resin Embeddingrown Papain � p-K � yeast lytic Sequential Filtration

enzyme � Zymolyase

ype of particles and type of media in which the particles areispersed. Particles from the wear of metal alloys are of 2ypes: ceramic particles, which are oxides or carbides andhus fairly inert, and metal alloy particles coming from theulk materials, thus having all the characteristics of theetal constituents including susceptibility to corrosion and

lution processes. For example, it has been established thathe use of an aggressive alkaline solution, such as sodiumydroxide which is typically used for the isolation of poly-thylene particles,30 has a detrimental effect on metal par-icles, affecting both their shape and chemical composi-ion.37,48 Various (and widely different) protocols involvinghe use of enzymes and detergents have been developedith the goal of digesting the proteinaceous content of the

olution, while at the same time preserving the characteris-ics of the particles as closely as possible.8,9,37,46,47

For the digestion and isolation of metal particles, variousethods have been published over the years, but 4 areost commonly recognized (Table 1). Chronologically,

ics, wear mechanisms and biological activity.

ingSizeanalysis

Shapeanalysis

Size/Shapedistribution

Evaluation of sampledistribution

Y N N NY N N NY Y Y YY N Y N

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chmiedberg et al46 applied a double enzymatic protocolsing papain and proteinase-K followed by a final washith NaOH. Doorn et al8 applied an enzymatic protocol to

solate particles from tissue and used a combination ofeagents to first defat and denature the samples. Enzymaticigestion with papain and then proteinase-K in Tris-HClas adopted for the digestion step. Catelas et al37 applied an

nzymatic protocol similar to the one developed bychmiedberg et al. Sodium dodecyl sulfate (SDS) was useds a detergent. Digestion was performed using papain androteinase-K in Tris-HCL at 65°C and 55°C, respectively.ore recently, Brown et al47 applied a multi-step protocol

nvolving the use of 4 different enzymes (papain, protein-se-K, yeast lytic enzyme, and Zymolyase) and variousther reagents including SDS, MOPS, TCEP, Tris-HCl,EPES buffer, Sodium Phosphate buffer, �-mercaptoetha-ol and Elmaclean 65 solution (Table 2).

isplay and image acquisition

Scanning electron microscopy (SEM) and transmissionlectron microscopy (TEM) are the 2 main tools usedor imaging particles. Both instruments, if equipped withnergy-dispersive x-ray spectroscopy (EDS), can give in-ormation on the chemical composition of the particles,iscriminating those of interest from contaminants and res-dues. Furthermore, SEM and TEM can collect other infor-ation that can be used to better understand the wearechanism that created the particles. It should be noted thatEM and TEM can provide complementary data. SEM cane used to obtain information about the surface topographyf the particles, and TEM can be used to understand therystallinity and thus the phase composition of the constit-ent alloy. Both instruments provide images containing sizend shape information.

SEM and TEM function in fundamentally different ways,nd, therefore, have different limitations with regard toarticle analysis. The TEM beam, in fact, passes through theample, and the image obtained is the projection of thebject in the path of the beam. Depending on the orientationf the particle to the beam, the error associated with itsimensions and any following estimation of its shape andize may not be trivial. Therefore, great caution is requiredn analyzing TEM images for size and shape estimations.

able 2teps involved in enzymatic protocols for metal particle isolation

TEPS Schmiedberg40 Doorn15 Catelas10 Brown4

ashing 6 7 9 12entrifugation 7 4 6 2ilution 2 4 3 8igestion 2 2 2 12oiling - 4 3 5eating - - - 1ransfer 3 - -upernatant removal 7 4 6

n the other hand, until recently, TEM provided much n

reater magnifications than SEM, allowing visualizationnd chemical characterization of extremely small particles.

The greatest consideration when performing TEM anal-sis for imaging isolated wear particles is sample prepara-ion, which is more difficult for TEM than it is for SEM andtrongly dependent on operator skills. The quality of parti-le sample preparation determines in large part whether orot the micrograph will be clear and representative of thentire particle distribution. Vastly different methods haveeen adopted during the years to prepare particles for char-cterization either by SEM or TEM. Filtering for SEMreparation has been the most commonly used technique,lthough resin-embedding, nebulization, and drop evapora-ion have been also employed. Similarly, Schmiedberg etl46 evaporated 5 �L of solution onto a carbon planchet forEM analysis. In contrast, Doorn et al8 used a nebulizer toisplay the particles on a copper grid for TEM analysis.atelas et al37 developed an original procedure to embedarticles in resin and then obtain slices of about 90-nmhickness for TEM analysis. Brown et al47 used vacuumltration to collect particles onto filters for SEM analysis.

orphological and chemical characterization

All investigators who have conducted particle analysisave provided an estimation of particle size. Some have alsoncluded size distribution as a part of their analysis. Inontrast to determining size distributions, analysis of parti-le shapes typically requires subjective assessments by thebserver. For example, in determining size distribution,chmiedberg et al46 defined length and width as the longestxis through the particle and the shorter line segment thatpans the length at a 90° angle. However, the shape infor-ation was not quantified; rather, the authors observed that

fragments appeared to be predominantly spherical inhape, but some ovoid fragments were also observed.”oorn et al8 reported the median size of particles regardlessf their shape; however, those authors reported most of thearticles to be round in shape.

Unlike previous authors, Catelas et al37 reported quanti-cation of shape as well as size distributions. For sizestimation, the length, defined as the maximum dimension,nd the width, defined as the maximum orthogonal dimen-ion, were used. For shape and morphology estimation, theatio of length to width was used to categorize particles intoound, oval, and needle-shaped.

More recently, Brown et al47 evaluated a maximum di-meter to calculate the size distribution of particles andenerically categorized them as round or irregular shapearticles.

tatistical analysis

Statistical analysis is used to estimate characteristics ofhe general population using sample observations. It followshat microscopic images (micrographs) of each samplehould be representative of the overall sample. It may be
ecessary to obtain several different micrographs of the

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ame sample to determine the morphological distribution ofhe whole sample.

There is currently no consensus among researchers onhe best procedure to follow to evaluate the size distributionf particles in a sample. Schmiedberg et al46 evaluated aotal of 20 particles and, assuming a normal distribution,eported mean and standard deviation of length and width.y comparison, Doorn et al8 reported the widest diameter of

he particle on a much larger population. A different numberf particles were characterized for each sample (from 1 to80) and no indication about the accuracy of the distributionas given. Catelas et al37 reported the mean maximumimension (length), the mean maximum orthogonal dimen-ion (width), and shape of an average of 300 particlesvaluated on different micrographs. In a more recent article,p to 1300 particles from up to 10 micrographs were ana-yzed.49 It must be acknowledged that Doorn et al analyzedarticles from tissues, explaining probably why the authorsad a different number of particles for each sample, as wearreatly varies depending on the patients.

iscussion

The particle analysis methods discussed above have con-ributed significantly towards our understanding of the na-ure of particulate wear debris. However, a consensus stilloes not exist as to which method should become thetandard for routine use. The absence of a universally ac-epted method for particle analysis and the use of differentechniques to isolate and characterize particles make it dif-cult to compare results from different studies. Despite the

ack of consensus, attempts have been made to establishtandards for a universally accepted method.

As discussed below, accurate analysis of wear particles isimited by multiple problems. The lubricants used in labo-atory wear simulations are typically serum solutions con-aining high concentrations of protein. Digestion of lubri-ant protein is necessary to retrieve the debris efficiently.owever, inefficient and/or incomplete digestion of lubri-

ant proteins can lead to agglomeration or loss of particlesFig. 2). In particular, nanoparticles can easily bind to pro-eins or be lost by adsorption to surfaces and/or adhesion toonpelleting debris. That is, particles, especially in theanometer size, can bind to centrifuge tubes surfaces or toipids and proteins or fragments of them. Furthermore, evenn the presence of complete digestion, separation of thearticles from lubricant digests can lead to particle lossnd/or particle agglomeration; thus the choice of the tech-ique to retrieve particles from the digest lubricant canffect the final distribution. Moreover, information on par-icle morphology, particle size, and particle size distributions as critical a component as designing and conducting theigestion and isolation methods that are used to generate thearticle images. Because of these difficulties, the errors
ssociated with particle characterization can be very high. t
igestion and isolation

As indicated above, the process of digestion involveseveral steps to obtain particles that are as clean as possibley the removal of any proteinaceous content and residue.nitially, researchers used fewer steps in their digestionrotocol; but over time, in the interest of obtaining purer andetter-separated particles, more steps were added to theigestion protocols. All of the particle isolation protocolsiscussed here have involved a large number of steps, ashown in Table 2, and all of them involved several centrif-gation steps to purify and collect particles. However, theres an optimum balance between loss of particles and puri-cation. In fact, every purification step introduced into arotocol can potentially lead to loss of particles or beggressive enough to distort their physical and chemicalharacteristics.

A discussion of specific side effects of each reagent inhe published digestion protocols is beyond the scope of thiseview. However, it is worth mentioning that if centrifuga-ion is involved in isolating nanoparticles, any substancehat can affect their buoyant density can be a source of lossf particles.

In consideration of these issues, any enzymatic digestionrotocol needs to be optimized and fine-tuned so that theumber of steps and enzymes can be reduced to a minimum.imilarly, reagents need to be selected so that their interac-

ion with particles is minimal.In the final analysis, the reasoning behind digestion and

solation protocols to obtain purified particles is 2-fold: theeed to obtain particles that can be easily identified andharacterized, and the need to access contaminant-free par-icles that can subsequently be fed to cells in bioreactivityests. However, the images obtained using protocols estab-ished so far frequently depict apparent residues and ag-lomerates, indicating that these needs have only been par-

ig. 2. Metal wear particle and organic residue after double enzymaticigestion.

ially addressed.

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isplay and image acquisition

The ultimate goal of an efficient display techniquehould be clear and sharp images of well separated particleshat allow for the use of an image analysis software toutomatically or semi-automatically measure and calculatehe features associated with each single particle. Thus theontrast between the background and the observed particlehould be as high as possible so that the error associatedith image analysis is minimized. Moreover, the back-round should be chosen for absence of features that couldnterfere with the recognition of particles. Ideally, the back-round should be as smooth and as uniform as possible. Ifhe particles are not well separated, their chemical compo-ition cannot be accurately identified through EDS. Conse-uently, it may not be possible to discriminate particles ofnterest from contaminants.

Most researchers have used filtration in order to separatearticles.9,47 Filtering is the easiest way of collecting parti-les on a support that can then be used for SEM analysis.owever, filter membranes normally present a distributionf pores that induce agglomeration by a funnel-effect. Thisffect occurs because the washing solution (and thus thearticles) is forced to follow a path that is not straight,oncentrating the wear particles around the pores (see Fig.). Furthermore, some of the pores can easily get clogged byarticles bigger than the pore size or by residue, thus re-ucing the overall filtering area and increasing the possibil-ty of further agglomeration. Lastly, the use of membranesf specific pore size introduces an artifactual threshold inhe size of recovered particles, since most of the particlesmaller than the pore size are lost. The number of particlesost through pores may be significant; consequently, theesulting size distribution might not represent the actualistribution of particles in the sample. Furthermore, display-ng on filter membranes may be unacceptable in terms ofontrast between the background and the particles, or be-ause the pores/features on the filter can incorrectly be

Fig. 3. Metal wear particles on a polycarbonate filter.

ounted as particles or may be difficult to distinguish. s

Although they are quite different, both nebulization androp evaporation may very likely lead to agglomeration ofarticles. This is due to natural formation of droplets thatay hold several particles together, eventually forming ag-

lomerates once the liquid has evaporated. If the evapora-ion is carried out at high temperature, the agglomerates cane extremely compacted, destroying the resolution of indi-idual particles. Furthermore, during nebulization, it isrobable that particles of different sizes are unevenly neb-lized and distributed, remain trapped, or fall away from thearget.

Embedding in resin for TEM analysis has gained popu-arity in the particle analysis community. Unfortunately,mbedding, if not done accurately, can also be a source ofarticle agglomeration. Specifically, the particles are pel-eted at a bottom of a centrifuge tube and thus initiallyorced together into potential artifactual agglomerates. Theesin is then poured into the tube and allowed to cure on topf the pellet, leaving no possibility for particle separation.urthermore, some of the particles can remain attached at

he bottom of the tube or be lost due to partial embedding.lices of 90-100 nm are then cut once the resin is cured. Ascknowledged by some authors, slices may contain particleshose orientation could lead to erroneous evaluation of therojected dimension obtained through TEM. Consequently,here may be an artificial skewing of particle size, becausearger particles may not be sectioned and the resin willimply tear around them, leaving only the smaller particlesn the successful sections. Finally, particles may be stackedoo close on top of each other within the slice to beiscriminated.

ize and morphological distribution

Analysis of wear particle size and morphological distribu-ions has often been limited to the evaluation of few parame-ers; eg, length and width, precluding a detailed characteriza-ion and discrimination. This is typically compounded by theact that image analysis is normally performed in the presencef significant amounts of agglomerates and, in general, usingoorly contrasted images. Consequently, the operator is forcedo select the particles manually and then extract the parametersf interest either manually or via dedicated software, makinghe entire process tedious and heavily dependent on the oper-tor’s skills and judgment.

For estimation of size distribution, a few simple descrip-ors are often sufficient and established in the literature. Onhe other hand, description of morphology has to be donesing a much more elaborate analysis. Morphological anal-sis should give an accurate description of the shape andexture of the observed object. Typically, this cannot bextrapolated by applying simple algorithms to dimensionaleasurements. The difficulty of proper morphological anal-

sis is often underestimated by researchers in the orthopae-ic field.

A more accurate description of particle size and shape
hould involve the use of multiple parameters and morpho-

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ogical descriptors calculated via image analysis software.STM F1877-98 standard specifies different parameters to

valuate the size of each particle and to further defineorphological descriptors.28 Commonly measured charac-

eristics are width (W), height (H), dmax or length, dmin orreadth, fiber length (FL), fiber breadth (FB), perimeter (P),nd area (A). These data can be used to calculate the 5orphological parameters specified in the standard, ie,

quivalent circle diameter (ECD), aspect ratio (AR), elon-ation (E), roundness (R), and form factor (FF). Figure 4hows an example of the particle length, breadth, height andidth for different types of particles. These shapes areutlined from SEM particle images and grouped togetheror demonstration purposes. Table 3 shows the shape andorphological descriptors obtained for each of the objects

n Figure 4.A simple example demonstrates how each of these pa-

ameters alone is hardly sufficient to fully characterize aiven particle. In Figure 4, particles 6 and 4 have the same

ig. 4. Example of particles outlined from SEM micrographs for morpho-ogical characterization.

eight and length but are very different in shape. Most of the1

articles shown have the same dmax but very differenthapes. A combination of parameters might be useful for aore precise separation. Orientation of the particles in 3-di-ensional space also has an influence on the evaluation of

he size when only 1 parameter is chosen to evaluate sizeistribution. The use of artificial intelligence software couldelp in automatically discriminating particles of differenthapes. These difficulties indicate that the complicationsssociated with morphological analysis are far from resolved.

It is not clear whether, and if so how, differences in shapend morphology as described above may affect bioreactiv-ty. However, in the absence of characterization, it is virtu-lly impossible to detect such clinical importance.

tatistics

Appropriate statistical analysis is crucial in describingnd comparing distributions of particle size and shape de-criptors. Different micrographs of the same sample shoulde compared to ensure that they provide the same distribu-ion estimates; otherwise, the previous steps in the methodhould be questioned. This necessary step has not beeneported in many of the commonly cited publications onetal particle analysis. Another critical point is to analyze

nd average a large enough number of particles from dif-erent micrographs, after having classified them by shape.ikewise, comparison of the results of one study to the nextmong different investigators or using different methodsan only be made using proper statistical estimates of theistributions.

It is well established (and it stands to reason) that theistribution of particles is far from normal; however, com-arison of particle distributions is often conducted usingnalyses intended only for normal distributions. Specifi-ally, the most common error in statistical representation ofarticle distributions is the use of means, standard devia-ions, and other parametric analysis such as t tests andnalysis of variance, all of which are valid only if distribu-ions are normal in the general population. Fortunately,ppropriate statistical methods are available for describingnd comparing any general type of distribution (eg, P and Qlots). However, the literature on metal particle analysis hasot taken advantage of these methods so far.

able 3orphological characterization of particles in Fig. 4

bject # SHAPE ECD AR E R FF

1 Fibril 0.31 1.96 48.0 0.06 0.062 Rod 0.37 8.33 16.6 0.09 0.173 Rod 0.35 5.80 11.4 0.11 0.234 Oval 0.84 1.97 1.7 0.48 0.735 Rod 0.32 7.30 13.4 0.09 0.206 Oval 0.79 1.54 3.0 0.46 0.597 Fibril 0.51 1.23 29.5 0.16 0.108 Fibril 0.28 3.61 33.6 0.06 0.099 Round 1.10 1.02 1.0 0.89 0.83
0 Oval 0.64 2.90 2.7 0.34 0.62

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The next most common source of error in statisticalepresentation of particles of an image is disproportionatelyounting a greater fraction of large or small particles, givingise to bias towards that fraction compared to what is actu-lly present in the whole sample. Figure 5 shows the dmax

alculated for the same image, but counting a differentumber of particles. In the first image, only 30% of thearticles are taken into consideration, while in the secondne, 75% of the particles are included in the distribution.learly, including a greater fraction of the particles in this

ample results in a substantially more accurate estimate ofhe peak dmax.

Another potential source of error in estimation of sizeistribution is the image magnification. For example, anmage at a given magnification can give a good estimate ofhe bigger particles, but may not be sufficiently detailed tollow a good estimation of the smaller particles. Severalmages taken at different magnification should thus be con-idered. In this case, the contribution of each image to thenal distribution must be accurately taken into account.

It is important to accurately evaluate the distribution ofarticles across different images. The minimum number ofarticles to be analyzed to ensure statistical accuracy instimating the general distribution of size and morphologys often overlooked by researchers. More often than not, theample simply does not contain enough particles, perhapsue to processing time and expense.

Comparison of distribution of the particles on each singleicrograph is a strong indicator of the uniform dispersion of

articles and the accuracy of the measurement. However, soar, the state of dispersion of isolated particles is a charac-eristic that has not been taken into account. The relativeumber of primary (single) particles in comparison to ag-lomerates could produce useful parameters that could helphe researcher to better understand the results obtained with

ig. 5. Effect of number of particles on apparent size distribution: (A) 30%articles visible on the micrograph are taken into account.

specific protocol. p

onclusion

Metal particle analysis consists of multiple, complexrocesses that span several disciplines in science and tech-ology. Each of the steps (digestion and isolation, displaynd image acquisition, morphological and chemical charac-erization, statistical analysis) requires taking advantage ofhe state of the art in technology and knowledge in theespective field. In part, due to the technical difficulties andhe many challenges involved in each step, investigatorsave established significantly different techniques to ad-ress each of the problems. Consequently, each of theseifferent techniques may produce entirely different resultsor a given sample. Therefore, the results obtained usingifferent techniques cannot be compared without taking intoonsideration the differences among these complex, multi-tep processes. The lack of agreement in particle isolationnd analysis methodology also poses tremendous challengesn studies of the host biological response to particulate wearebris. While there is a need to better characterize andontrol the particle size, shape, and distribution for cellulture or animal studies, there is no consensus in theethods needed to accomplish this.Today, the machinery is in place to isolate, visualize and

hemically characterize the metallic wear particles frommplants with articulating surfaces. In spite of the varioustandards and guidance documents, there is currently noonsensus in the community on the methods used to gen-rate these data. Equally important is that there is also noonsensus on the methodology for the analysis and inter-retation of the results. Therefore, data on wear particleharacterization must be critically assessed by the reader iniew of the limitations of each specific set of techniquesmployed. In general, these data do provide valuable infor-ation on the potential characteristics of the wear particles.owever, caution is still recommended in extrapolating

particles visible on the micrograph are taken into account; (B) 75% of the
of the
article analysis results to a predictive statement of clinical

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ost response to a particular implant design. Further re-earch must be conducted in order to develop and validateefined techniques within these interrelated areas of wearnd biology to arrive at a reasonable set of specificationsnd requirements for the design and material selection ofmplants for motion preservation implants for the spine and,ore generally, for arthroplasty devices.

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metal wear particles: what we know, what we do not know, and why

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