Biomaterials 24 (2003) 737–748

Comparison of the response of human peripheral blood mononuclearcells to challenge with particles of three bone cements in vitro

Wendy Mitchella,*, J. Bridget Matthewsa, Martin H. Stoneb, John Fisherc, Eileen Inghama

aDivision of Microbiology, The University of Leeds, Leeds LS2 9JT, West Yorkshire, UKbDepartment of Orthopaedic Surgery, Leeds General Infirmary, Leeds LS1 3EX, West Yorkshire, UKcDepartment of Mechanical Engineering, The University of Leeds, Leeds LS2 9JT, West Yorkshire, UK

Received 11 October 2001; accepted 21 August 2002

Abstract

This study compared the effects of different sizes of three clinically relevant endotoxin free bone cement particles on primary

human macrophage TNF-a production in vitro. The bone cements used were CMW original, CMW1RO and Palacos R. The cementwear debris was generated aseptically and then sequentially filtered to produce the size ranges 0.1–1 mm; 0.1–10 mm; 1–10 mm and> 10 mm: The debris was cultured with human peripheral blood mononuclear cells at particle volume (mm3) per cell ratios of 100:1,10:1 and 1:1. TNF-a production was determined by ELISA and cell viability by MTT conversion. CMW1RO particles inducedincreased TNF-a production by PBMNCs when tested in the size range 0.1–1 mm; and also to a lesser degree in the sizes 0.1–10 mmand 1–10 mm at the particle volume (mm3) to cell number ratios of 100:1 and 10:1.The increase in TNF-a production induced by Palacos R debris was only observed with the particle size ranges less than 10 mm at

the ratio of 100:1. This study demonstrated that bone cement particles are capable of inducing raised TNF-a production in vitro.This is dependent upon cement particle size, volume and cement particle type, with cement particles containing radio-opaque

additives being the most active.

r 2002 Elsevier Science Ltd. All rights reserved.

Keywords: Bone cement; Wear debris; THR; Cytokine

1. Introduction

Currently, the primary cause of revision of ultra highmolecular weight polyethylene (UHMWPE) on metaltotal hip prostheses is aseptic loosening due to osteolysisof the femoral bone. It is widely accepted thatUHMWPE particles produced as a consequence ofarticulation are engulfed by macrophages within theperiprosthetic tissue [1]. In vitro studies have clearlydemonstrated the capacity of UHMWPE wear particlesto activate mononuclear phagocytes [2] and have shownthe importance of particle size, number and volumetricconcentration [2–4]. These studies have indicated thatthe most biologically active UHMWPE particles aresubmicrometre in size. Following activation by wearparticles, macrophages have been shown to produce

the inflammatory mediators TNF-a; IL-6, PGE2 andIL-1b amongst others, and these factors have beenimplicated in osteoclast activation and bone resorption[3,5–8].In addition to UHMWPE debris generation, produc-

tion of wear debris from the femoral stem and cementmantle may also occur in vivo. The purpose ofthe cement mantle is to provide fixation of the stem intothe femoral bone and to evenly distribute stressalong the length of the cement mantle [9]. Littleattention has been paid to the biological effects of thebone cement wear particles which are produced follow-ing fretting wear or motion at the polymethylmethacry-late (PMMA) cement/prosthesis and PMMA/boneinterfaces.To date, studies utilising various established cell lines

and primary peripheral blood mononuclear cells(PBMNC) cultured with bone cements have highlightedthe capacity of bone cement particles to activatemononuclear phagocytes [10–12]. Sabokbar et al. [13]

*Corresponding author. Tel.: +44-113-233-5693; fax: +44-113-233-

5638.

E-mail address: micwm@bmb.leeds.ac.uk (W. Mitchell).

0142-9612/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved.

PII: S 0 1 4 2 - 9 6 1 2 ( 0 2 ) 0 0 4 0 5 - 2

demonstrated that cement particles containing theradio-opaque additive barium sulphate stimulatedmore bone resorption than those containing zirconiumoxide. Ingham et al. [10] compared the capacity ofclinically relevant wear debris particles produced fromseven different bone cements to induce IL-1b; IL-6 andTNF-a production in the U937 human histiocytic cellline. This study not only demonstrated the capacity ofclinically relevant cement wear debris particles toactivate macrophages but also showed that cementparticles containing radio-opaque additives were themost biologically active. However, no difference wasobserved in the level of cytokine produced by cellsin response to stimulation with cement particles, whichcontained either barium sulphate or zirconium oxide.Moreover, Quinn et al. [11] demonstrated the capacityof PMMA stimulated macrophages to resorb bonein vitro.Granchi et al. [12] investigated the reaction of primary

peripheral blood mononuclear cells (PBMNC) presti-mulated with phytohemoagglutinin-P (PHA) or unsti-mulated, to extracts of 10 different bone cements thatcontained radio-opaque additives. This study concludedthat bone cement extracts were capable of modifyingthe profile of proinflammatory cytokines secreted bythe PBMNCs, particularly when prestimulated withPHA.In contrast with previous research which in-

dicated that PMMA in its bulk form appeared to bebiologically inert [14,15], Herman et al. [16] showedthat PMMA coated onto plastics was able to activatemonocyte enriched cells extracted from PBMNCsand unfractionated mononuclear cells. However, thiscould have been explained by the presence of contam-inating endotoxin in the PMMA since levels were notdetermined. Endotoxin, adherent to the PMMA, maycause cellular activation not attributable to the PMMAalone.The potential of bone cements to induce apoptosis or

necrosis, was investigated by Ciapetti et al. [17] using theHL60 cell line (leukemic cells sensitive to apoptoticsignals). Three of the 10 bone cements tested werecapable of inducing cell death after 24 h: Both necrosisand apoptosis of the cells was observed.Iwaki et al. [18] characterised the PMMA particles

extracted from retrieved tissues and indicated that99.5% of the particles were less than 10 mm and 82%were submicrometre in size (in the phagocytosable sizerange). This research therefore indicated that PMMAwear debris may contribute to osteolysis and loosening.In order to further investigate the osteolytic potential

of PMMA particles, the aim of this study was todetermine the effect of different sizes and volumes ofthree types of clinically relevant, endotoxin free, bonecement particles on the activation of primary humanmononuclear phagocytes in vitro.

2. Materials and methods

2.1. Generation of bone cement debris

Three polymethylmethacrylate (PMMA) bone ce-ments were tested: CMW original (PMMA only);CMW1 RO (PMMA plus 9.2% (w/v) BaSO4 of meangranule diameter 1 mm) and Palacos R (PMMA plus15.2% (w/v) ZrO2 of mean granule diameter 10 mm).Bone cements were moulded at a constant pressure intocylinders to ensure uniform density, and were suppliedin rod-form by DePuy International (a Johnson &Johnson Company). The cement rods were thenmachined into pins 12 mm in length, with a truncatedcontact surface area of 5 mm diameter. Sterile pyrogenfree water (Baxter Healthcare Ltd., Newbury, UK) wasused as the lubricant and all tests were performed in aclass I laminar flow cabinet (Heraeus, Germany) usingaseptic technique throughout. Stainless-steel counter-faces were manufactured with a ‘vaquasheened’ finish tomimic the commercial Charnley stem (Proprietarytechnique; DePuy; Leeds). After thorough cleaning ina sonicating water bath, all wear test rig componentsand stainless steel counterfaces were rinsed with sterilepyrogen-free water and then dry heat sterilised at 1801Cfor 4 h to destroy any adherent endotoxin.Prior to use, pins were soaked in sterile, pyrogen-free

water for 24 h: Cement wear debris was generated in apin-on-plate tribometer, using a unidirectional motionof 0:0071 m s�1 with a constant load of 40 N and afrequency of 0:1 Hz:

2.2. Isolation and characterisation of particulate

cement debris

Lubricants containing the particulate wear debriswere filtered through preweighed 10 mm pore sizepolycarbonate Cycloporetm

s

membrane filters (What-man International Ltd., Maidstone, England). Thefiltrate was then shaken vigorously and split into twoequal volumes. One half was sequentially filteredthrough preweighed 1 mm then 0:1 mm pore sizeCycloporetm

s

membranes. The remaining half of thefiltrate was filtered directly onto a preweighed 0:1 mmpore size membrane. Thus four test cement debrisfractions were produced: > 10; 1–10 mm; 0.1–1 and0.1–10 mm: The filters were then dried under infraredlamps, reweighed and the mass of debris on each filterdetermined. The total mass of debris generated wasconfirmed by calculating the loss of volume from eachpin. This was achieved by measuring the changein diameter of the contact surface area (Profile projectorV-16, Nixon Atlanta USA) and the length of the pin. Inorder to determine the effect of cement on the metalcounterface, the surface roughness (Ra) of wear plateswas calculated using a laser monitor dialus talysurf

W. Mitchell et al. / Biomaterials 24 (2003) 737–748738

(6 mm range 120L; Taylor Hobson) both before and aftereach test. Samples of each particle fraction were thenmounted, gold coated and viewed by scanning electronmicroscopy (SEM; Hitachi 5700; Japan). The particle sizedistributions were then determined by image analysis(Image Pro Plus). For each cement type, the particle area,equivalent circle diameter, aspect ratio, roundness, formfactor, elongation and length were characterised for 100particles according to the ASTM, standard F1887.

2.3. Quantification of endotoxin contamination in cement

wear particle preparations using the limulus amoebocyte

lysate assay

Endotoxin contamination of the cement wear debriswas measured using the kinetic turbidimetric limulusamoebocyte lysate (LAL) method (Associates of CapeCod Inc., Liverpool, UK). Cement wear particles fromthree separate wear tests were resuspended at0:05 mg ml�1 in endotoxin-free LAL reagent water bysonication for 2 h: The suspension was then incubatedwith continuous shaking at 371C for 1 h; and thenfurther sonicated for 1 h to remove attached endotoxin.The debris suspension was then centrifuged at 1000g for10 min to pellet the particles and the supernatant wasremoved.The rate of increase in turbidity over time of the

‘Pyrotell-T’tm LAL solution was determined using theLAL 5000 Automatic Endotoxin Detection Systemtm

and Pyrostm software. Positive sample controls weretested in duplicate, spiked with 0:01 EU ml�1 or0:0125 EU ml�1: A standard curve was constructed witheach assay using standard endotoxin (CSE) from 0.1 to0:00156 EU ml�1: In order to calculate the equivalentlipopolysaccharide (LPS) concentration from EU ml�1 astandard curve of LPS (E. coli 055: B5) was constructedby serial 10 fold dilution of a 1 mg ml�1 sample.

2.4. Culture of bone cement particles with primary

peripheral blood mononuclear cells

Peripheral blood mononuclear cells (PBMNC) wereisolated from 30 ml of heparinised whole blood usingdensity gradient centrifugation over Lymphopreps

(Gibco Life Technologies Ltd., Paisley, Scotland). Cellsfrom three donors were evaluated in this study. Cellswere then resuspended in RPMI 1640 culture medium(Gibco Life Technologies Ltd., Paisley, Scotland)supplemented with 10% (v/v) foetal calf serum (FCS;Gibco), 20 mm l-glutamine (Gibco), 60 mg l�1 penicil-lin (Britannia Pharmaceuticals Ltd., Redhill, Surrey,UK) and 100 mg l�1 streptomycin (ICN Pharmaceuti-cals Ltd., Basingstoke, Hampshire, UK). The percen-tage of monocytes was then calculated using a latex beadingestion assay and haematoxylin and eosin staining.Cells (100 ml) were incubated with 50 ml of 1% (w/v)

1 mm latex bead suspension (Sigma Chemical Co., Ltd.,Poole, UK) for 30 min at 371C: The cells were thenlayered over 100 ml FCS, then centrifuged at 42g for1 min; after which the supernatant containing unin-gested latex beads was removed and the cell pelletresuspended in 100 ml of RPMI 1640 culture medium.The proportion of latex containing mononuclear pha-gocytic cells was then quantified by light microscopy.Further evaluation of the cells using a trypan blue dyeexclusion assay enabled calculation of the total numberand percentage viability of the cells. Cells (1� 104

mononuclear phagocytes in 200 ml well�1) were thenseeded into U-bottomed 96-well tissue culture plates andincubated at 371C in an atmosphere of 5% (v/v) CO2 inair. After overnight incubation, 150 ml of culture super-natant was removed from each well and replaced with50 ml of fresh culture medium.Cement particles were resuspended at 0:01 mg ml�1 in

supplemented RPMI 1640 culture medium by sonicationin a sonicating water bath for 2 h; and then sterilised bygamma irradiation. Cement particle fractions were thenadded to the wells containing cells (100 ml well�1) at threeparticle volumes (mm3) to mononuclear phagocytic cellnumber ratios: 1:1; 10:1; and 100:1. Cells and particleswere cultured together for 24 h prior to harvesting culturesupernatants for cytokine quantification. The viability ofthe cells was determined by the MTT conversion assay.All tests were performed in sextuplet and positive controls(cells plus LPS at 100 pg ml�1; E. coli strain 055.B5;Sigma Chemicals Co. Ltd., Poole, UK) and negativecontrols (cells plus culture medium only) were included.The optimal concentration of this particular LPS wasdetermined in preliminary experiments.

2.5. Assessment of PBMNC viability using the MTT

conversion assay

Cell viability was determined using a 3-[4,5-di-methylthiazol-2-yl]-2;5-diphenyltetrazolium bromide(MTT) conversion assay (Sigma Chemicals Co. Ltd.,Poole, England). Following removal of 100 ml of super-natant from the cocultures for cytokine analysis, 10 ml ofMTT was added to each well (5 mg ml�1 in PBS). Cellswere then incubated for 4 h at 371C in an atmosphere of5% (v/v) CO2 in air, after which, 100 ml of 10% (w/v)sodium dodecyl sulphate in 0:1 m HCl was added. Cellswere then incubated overnight in the dark to allowsolubilisation of the formazan crystals. The opticaldensity (OD) was then determined at 570 nm (reference630 nm) using a Dynex MRX plate reader (DynexTechnologies UK Ltd., Sussex, UK).

2.6. TNF-a quantification

The levels of TNF-a present in culture supernatantswere quantified using a double antibody capture and

W. Mitchell et al. / Biomaterials 24 (2003) 737–748 739

detection sandwich ELISA with antibodies produced‘‘in house’’ by Professor E. Ingham and purified by Dr.R. Banks (St. James University Hospital, Leeds, UK)[19]. Between each step the plates were washed 3 timeswith 200 ml well�1 of wash buffer (PBS, Tween; Sigma0.05% v/v adjusted to pH 7.4) then blotted dry byinversion onto tissue paper. Each incubation was at371C in a humidified atmosphere.Maxisorb s ELISA plates (Gibco Life Technologies

Ltd., Paisley, Scotland) were coated with polyclonalrabbit antihuman TNF-a antibody (100 ml well�1)diluted 1 in 1000 in coating buffer (0.16% w/vNa2CO3; 0.29% w/v NaHCO3 in distilled wateradjusted to pH 9.6). Plates were incubated for 2 h andthen washed. The plates were then blocked with100 ml well�1 blocking buffer (coating buffer, bovineserum albumin; Sigma 1% w/v) and incubated over-night, then washed again. A standard curve of humanTNF-a was constructed by serial 2-fold dilution ofhuman TNF-a (National Institute for biological Stan-dards and Controls NIBSC Potters bar Hertfordshire,UK) in RPMI 1640 supplemented culture medium. Testsupernatant in triplicate and standard in duplicate(100 ml) were added, and then incubated for 2 h:Following washing of the plate, a monoclonal mouseantihuman TNF-a antibody was diluted 1 in 3000in wash buffer supplemented with 0.5% (w/v) BSAand added to the wells (100 ml well�1). The plateswere incubated for 1 h: The plates were then washedas before and 100 ml well�1 of biotinylated rabbitantimouse antibodies diluted 1 in 2000 in dilution buffer(wash buffer supplemented with 0.5% w/v BSA, 2% v/vrabbit serum; Sigma) was added. The plates were thenincubated for 1 h and then washed. A horse radishperoxidase (HRP) conjugated with avidin D was added(diluted 1 in 6000 in dilution buffer). The plates wereincubated for 1 h: Following removal of the HRP-avidinD, 100 ml well�1 of o-phenylenediamine dihydrochloride(OPD; Sigma) 0.04% w/v in phosphate citrate buffer(0:0243 m citric acid, 0:0514 m Na2HPO4 adjusted to pH5.0) was added and incubated for 20–30 min until yellowcolouration occurred. The reaction was stopped byaddition of 50 ml well�1 of 2:5 m H2SO4: The absor-bance at 490 nm was then determined using an ELISAplate reader.

2.7. Statistical analysis of data

Levels of TNF-a in particle stimulated cultures wereconverted to specific activities (ng ml�1TNF-a=OD 570 nm) and compared with the negative controls(cells cultured without particles) by one way analysis ofvariance and calculation of the minimum significantdifference using the T-method at the Po0:05 and 0.01level. All results are expressed as the mean 795%confidence limits.

3. Results

3.1. Generation and characterisation of bone cement

wear debris

Sample SEM images of the wear debris producedfrom the three bone cements are displayed in Fig. 1. Thethree bone cements appeared globular with an irregularsurface. The frequency of the particle length (0.1–0.5,0.5–1.0, 1–10 and > 10 mm) within each of the fourfractions was determined by image analysis.The particle mass distribution for CMW original in

the size ranges (a) 0.1–1, (b) 0.1–10, (c) 1–10 and (d)> 10 mm is shown in Fig. 2. Wear debris particlesfractionated between the pore sizes 0.1 and 1 mm had84.3% of the mass less than 1 mm in length (Fig. 2a).The fraction obtained between the pore sizes 0.1 and10 mm displayed 78% of the mass which was submicro-metre in size (Fig. 2b). The remaining 22% of the masswas between 1 and 5 mm in length. The debrisfractionated between the pore sizes 1 and 10 mm; had96% of the mass between 1 and 5 mm (Fig. 2c). Thedebris filtered onto 10 mm filter had 43% of the mass> 10 mm in length, and 47% of the mass between 5 and10 mm in length (Fig. 2d).The particle mass distribution for CMW 1RO debris

fractionated in the size ranges (a) 0.1–1, (b) 0.1–10, (c)1–10 and (d) > 10 mm is shown in Fig. 3. Debrisfractionated between the pore sizes 0.1 and 1 mm had100% of the mass which was submicrometre in size(Fig. 3a). The fraction between the pore sizes 0.1 and10 mm contained 67% of the mass which was > 1 mm inlength (Fig. 3b). The debris fractionated between thepore sizes 1 and 10 mm contained 74% of the massbetween 1 and 5 mm in length (Fig. 3c). The debrisfiltered onto the 10 mm filter contained 87% of the mass,which was > 10 mm in length (Fig. 3d).The particle mass distribution for Palacos R

cement debris fractionated in the size ranges (a) 0.1–1,(b) 0.1–10, (c) 1–10 and (d) > 10 mm is shown in Fig. 4.Debris fractionated between the pore sizes 0.1 and 1 mmcontained 62.1% of the mass less than 1 mm in length(Fig. 4a). The remaining 38% of the mass was between 1and 5 mm: The fraction filtered between the pore sizes0.1 and 10 mm contained 75.7% of the mass between 1and 5 mm in length (Fig. 4b). The debris fractionatedbetween the pore sizes 1 and 10 mm contained 78% ofthe mass between the sizes 1 and 10 mm (Fig. 4c). Of theoverall mass, 70% was between the size ranges 1 and5 mm: The debris fractionated onto the 10 mm filtercontained 66.3% of the mass, which was > 10 mm inlength (Fig. 4d).The three cement debris types were characterised

according to the ASTM standard F1877 by theirequivalent circle diameter (ECD), elongation, round-ness, form factor and aspect ratio. The results displayed

W. Mitchell et al. / Biomaterials 24 (2003) 737–748740

in Table 1 indicated that there were small differences inthe particles between the three cements. CMW 1ROparticles had the greatest aspect ratio (mean of 2.86),Palacos R had the smallest aspect ratio (mean of 1.72).

3.2. Quantification of adherent endotoxin contamination

in cement debris

The level of adherent endotoxin contamination in thecement debris is shown in Table 2. The bone cementCMW original contained 0:0007570:00078 EU ml�1

(mean7SD). This is equivalent to 0:03 pg ml�1 LPS(E. coli 055: B5) 70:032 pg ml�1: The bone cementPalacos R contained 0:0001171:45� 10�5 EU ml�1

(mean7SD), which is equivalent to 0:004 pg ml�1 LPS(E. coli 055: B5) 70:00058 pg ml�1: The bonecement CMW1RO contained 0:008770:011 EU ml�1

(mean7SD). This is equivalent to 0:36 pg ml�1

LPS70:46 pg ml�1:

3.3. TNF-a production by PBMNC stimulated with bone

cement wear debris

The PBMNCs were cultured with debris fractionatedinto the four pore size ranges: 0.1–1 mm; 0.1–10 mm;1–10 mm and > 10 mm in length at particle volumes (mm3)per mononuclear phagocyte of 100, 10 and 1 for 24 h:MTT conversion was used to determine any cytotoxiceffect caused by the bone cement wear debris particles.No significant cell death caused by the presence of weardebris particles was observed with any of the particle sizeranges or particle volume ratios (data not shown).Mononuclear phagocyte activation by the cement

debris was determined by measuring the TNF-a

Fig. 1. SEM images of cement wear particles fractionated using polycarbonate membrane filters in the size ranges (a) CMW 1RO 1–10 mm; (b)CMW 1RO > 10 mm; (c) CMW 1RO 0.1–1 mm; (d) CMW 1RO 0.1–10 mm; (e) CMW original 0.1–1 mm; (f) CMW original 0.1–10 mm (g) CMWoriginal 1–10 mm (h) Palacos R 1–10 mm and (i) Palacos R > 10 mm:

W. Mitchell et al. / Biomaterials 24 (2003) 737–748 741

production using ELISA. The data was convertedto specific activity (SA; TNF-a ng ml�1=OD570 nm).The level of TNF-a produced by cells stimulated

with debris was compared to the level of TNF-aproduced by the cells without stimulation andstatistical significance was determined by analysis of

% m

ass

size range µm

40

60

80

0.1 - 0.5 0.5 - 1 1- 5 5 - 10 >10

0.1 - 0.5 0.5 - 1 1 - 5 5 - 10 >10

80

60

40

20

00

20

40

60

80

0.1 - 0.5 0.5 - 1 1 - 5 5 - 10 >10

(A)

0

40

0.1 - 0.5 0.5 - 1 1- 5 5 - 10 >10

(C) (D)

- - - -

- - 1 1 - 5 5 -- - 1 - 5 -

- 1- -0

- - - -

- - 1 - 5 -

(B)

- 1 - -

80

- 1 -

20

- -

- - - -- --

20

60

100

- -

63.1

21.216.7

30.4

47.4

22.2

3.2

95.7

10.2

47.2

42.6

1.0

Fig. 2. Particle mass distribution as a function of size for CMW original fractionated in the size ranges (a) 0.1–1 (b) 0.1–10 (c) 1–10 and (d) > 10 mm:

0

20

40

60

80

0.1 - 0.5 0.5 - 1 1 - 5 5 - 10 >10

0

20

40

60

80

0.1 - 0.5 0.5 - 1 1 - 5 5 - 10 >100

80

60

40

20

100

80

60

40

20

0

0.1 – 0.5 0.5 - 1 1 - 5 5 - 10 >10

0.1 – 0.5 0.5 - 1 1 - 5 5 - 10 >10

(A) (B)

(C) (D)

Size range µm

% m

ass

- - - -

- - - - – - - -

– - -

75

25

10.7

22

67.3

7

19.1

73.9

2.70.4

10.1

86.7

Fig. 3. Particle mass distribution as a function of size for CMW 1RO fractionated in the size ranges (a) 0.1–1 (b) 0.1–10 (c) 1–10 and (d) > 10 mm:

W. Mitchell et al. / Biomaterials 24 (2003) 737–748742

variance and calculation of the minimum significantdifference.PBMNC from three donors were cultured with CMW

original. The SA of the negative controls ranged from0.07 to 10 pg ml�1=OD570 nm: The SA of the positivecontrols (LPS 100 pg ml�1) ranged from 1170 to2580 pg ml�1=OD570 nm: No significant TNF-a pro-duction was observed with any particle size range orparticle volume ratio (SA range from 0.07 to38 pg ml�1=OD570 nm).

The results for PBMNC from three donors followingculture with CMW 1RO are shown in Fig. 5. The SA ofthe negative controls ranged from 0.05 to200 pg ml�1=OD570 nm: The SA of the positive con-trols (LPS 100 pg=ml) ranged from 1700 to5170 pg ml�1=OD570 nm: A statistically significant re-sponse was observed with cement debris in the sizeranges 0.1–1 mm; at a ratio of 100:1 and 10:1 (rangefrom 3090 to 17,900 and 2100 to 4700 pg ml�1=OD570 nm; respectively), and 0.1–10 mm; at a ratio of

0

20

40

60

80

0.1 - 0.5 0.5 - 1 1 - 5 5 - 10 >100

20

40

60

80

8080

60

40

20

0

60

40

20

0

0.1 - 0.5

0.1 - 0.50.1 - 0.5

0.5 - 1

0.5 - 10.5 - 1

1 - 5

1 - 51 - 5

5 - 10

5 - 105 - 10

>10

>10>10

Size range µm - - - -

-

- - -

-

% m

ass

8.5

53.6

37.9

0.76.9

75.7

16.7

6.315.9

69.8

8.05.1 0.7

25.7

2.2

66.3

(A) (B)

(C) (D)

Fig. 4. Particle mass distribution as a function of size for Palacos R fractionated in the size ranges (a) 0.1–1 (b) 0.1–10 (c) 1–10 and (d) > 10 mm:

Table 1

Characterisation of wear debris particles of three bone cements

Equivalent circle Aspect ratio Roundness Elongation Form factor

diameter mm (mean7SD) (mean7SD) (mean7SD) (mean7SD) (mean7SD)

CMW 1RO 0.2570.24 2.8672.52 0.5770.19 1.870.77 0.6670.25CMWoriginal 0.2570.23 1.9571.05 0.6970.15 1.670.85 0.8270.2Palacos R 0.9070.58 1.7270.61 0.6870.14 1.4970.37 0.8670.16

Values expressed as Mean7SD:

Table 2

Quantification of endotoxin in cement particles by kinetic turbidimetric limulus amoebocyte lysate LAL assay

EU ml�1 in 0:01 mg ml�1 % recovery of spike Equivalent LPS pg ml�1

debris (mean7SD) (mean7SD) (mean7SD)

CMW original 0.0007570.000785 95.774.07 0.0370.032Palacos R 0.0001170.0000145 75.7724.3 0.00470.000586CMW 1RO 0.0087870.011375 76.3712.33 0.3670.46

Results expressed as mean endotoxin EU ml�1 in cement particles ð0:01 mg ml�1Þ7SD: Results are also converted to equivalent LPS (pg ml�1).

W. Mitchell et al. / Biomaterials 24 (2003) 737–748 743

100:1 and 10:1 (range from 2030 to 8530 and 1290 to5050 pg ml�1=OD570 nm; respectively). Additionallythe particle size 1–10 mm at the ratios of 100:1 and10:1 induced significantly greater TNF-a productioncells alone (range from 2080 to 9130 and 910 to6720 pg ml�1=OD570 nm; respectively). No responsewas observed with particles > 10 mm in size or anyparticle size range at the ratio of 1:1.The SA of PBMNCs from three donors following

culture with Palacos R wear particles is shown in Fig. 6.The SA of the negative controls ranged from 6 to

167 pg ml�1=OD570 nm: The positive control (LPS100 pg ml�1) SA ranged from 460 to 3550 pg ml�1=OD570 nm: A statistically significant response wasobserved with the particle size range 0.1–1 mm at theratio of 100:1 with cells from two of the three donors(range from 33 to 740 pg ml�1=OD570 nm). No in-crease in TNF-a production was observed with the sizerange 0.1–1 mm at the ratios of 10:1 and 1:1 (range0.005–190 pg ml�1=OD570 nm).When the PBMNC were stimulated with Palacos

R particles ranging from 0.1 to 10 mm; an increase in

Fig. 5. Levels of TNF-a produced by PBMNC following 24 h culture with clinically relevant CMW 1RO bone cement particles of the size ranges0.1–1, 0.1–10, 1–10 and > 10 mm at particle volume (mm3) to cell ratio of 100:1, 10:1 and 1:1. Results are expressed as the mean specific activity ofTNF-a ðng TNF-a=OD570 nmÞ795% confidence limits. LPS (lipopolysaccharide 100 pg ml�1; positive control). Control: (PBMNC in culturemedium only; C; negative control). Data were analysed by one-way analysis of variance. The minimum significant difference (MSD Po0:05;Po0:01) was then determined. Means which are significantly greater than the negative control are indicated (* ; Po0:05; * * ; Po0:01).

W. Mitchell et al. / Biomaterials 24 (2003) 737–748744

TNF-a production was observed in comparison to thenegative control at the ratio of 100:1 of the donorsPBMNCs (range from 280 to 1200 pg ml�1=OD570 nm). At the ratio of 10:1, an increase in TNF-a production relative to the negative control wasobserved with 1 of the donors PBMNCs (mean569 pg ml�1=OD570 nm). Culture of PBMNC withPalacos R debris at the size range 1–10 mm resulted inan increased TNF-a production at the ratio of 100:1 forPBMNCs from all three donors (range from 440 to790 pg ml�1=OD570 nm). At the ratio of 10:1 and 1:1within the size range 1–10 mm; no increase in TNF-a

production was observed (range 0.006–189 pg ml�1=OD570 nm). Stimulation of the PBMNC with Palacos Rdebris particles > 10 mm in size failed to stimulate anincrease in TNF-a production (range from 0.006 to240 pg ml�1=OD570 nm).

4. Discussion

The effect of particle size of clinically relevant cementdebris on the activation of human monocytes hasreceived little attention. Since the size of UHMWPE

Fig. 6. Levels of TNF-a produced by PBMNC following 24 h culture with clinically relevant Palacos R bone cement particles of the size ranges0.1–1, 0.1–10, 1–10 and > 10 mm at particle volume (mm3) to cell ratio of 100:1, 10:1 and 1:1. Results are expressed as the mean specific activity ofTNF-a ðng TNF-a=OD570 nmÞ795% confidence limits. LPS (lipopolysaccharide 100 pg ml�1; positive control). Control: (PBMNC in culturemedium only; C; negative control). Data were analysed by one-way analysis of variance. The minimum significant difference (MSD Po0:05;Po0:01) was then determined. Means which are significantly greater than the negative control are indicated (* ; Po0:05; * * ; Po0:01).

W. Mitchell et al. / Biomaterials 24 (2003) 737–748 745

wear debris is a major factor in macrophage activation,the aim of this study was to determine the effect ofcement wear particle size on macrophage stimulationwhen cultured with PBMNCs.Wear debris generation was carried out against a

vaquasheened stainless-steel plate under aseptic condi-tions. The particle size ranges used were 0.1–1 mm;0.1–10 mm 1–10 mm and > 10 mm in length. These sizeranges were chosen since previous studies of UHMWPEwear particles indicated that the submicrometre particleswere the most biologically active when cultured withmononuclear phagocytic cells in vitro [2–4]. Followingparticle generation, the LAL assay was used todetermine that the debris was endotoxin free. The levelof endotoxin present in the wear particle preparationswas less than the equivalent of 1 pg ml�1 in all of theparticle preparations. This level of endotoxin contam-ination was shown in preliminary experiments to bebelow that which is required to cause activation of thePBMNCs in vitro. Therefore, the debris used for culturewas clinically relevant and contained negligible contam-inating endotoxin. Endotoxin contamination may causeactivation of macrophages and therefore may signifi-cantly affect the reliability of assays.It has been shown that TNF-a was responsible for

30% of the bone resorbing activity of conditionedsupernatant from UHMWPE stimulated macrophages[20]. It is therefore considered to be an appropriatemarker for phagocyte activation in this context.Three donors were used for this study since genetic

polymorphism in the TNF-a promoter region has beenshown to be responsible for variations in the level ofproduction of TNF-a by PBMNC’s within the popula-tion. Since considerable variation in the level of responseby the PBMNCs to the cement particles was observedbetween the three donors, it was considered appropriateto analyse the data for TNF-a production by thePBMNCs from each donor separately.Interestingly none of the three cements, at the size

range greater than 10 mm stimulated the PBMNC toproduce TNF-a above the negative controls. This wasconsistent with studies using UHMWPE particles,which demonstrated that the most biologically activesize range was 0.2–0:8 mm in size [2–4]. It is thought thatparticles larger than 10 mm may cause a giant cellresponse in vivo, whereby macrophages may fusetogether to wall off the foreign material. This responsemay be less pernicious than the response to smallerparticles, particularly submicrometre in size, which isthought to result in activation of the macrophages toproduce osteolytic cytokines.CMW original bone cement (no radio opaque

additive) particles failed to stimulate an increase inTNF-a production by PBMNCs from 3 donors atany size range or ratio tested. This was in agreementwith previous work carried out by Ingham et al. [10]

which demonstrated little stimulatory activity of CMWoriginal particles on U937 activation following culture[10].The response of PBMNCs from all 3 donors to the

0.1–1 mm fraction of CMW 1RO wear debris wasconsiderably greater than the response to the PalacosR, wear debris in the same size range. This may havebeen due to the fact that a greater proportion (100%) ofthe mass of the CMW 1RO debris was achieved in thissize range compared to the Palacos R (62%) and doesnot necessarily indicate that the CMW 1RO debris wasmore biologically active. Both the CMW1RO and thePalacos R debris in the 0.1–10 and 1–10 mm size rangesstimulated TNF-a production by the PBMNCs from all3 donors at a ratio of 100:1. However, the CMW 1ROdebris in the size ranges 0.1–10 and 1–10 mm alsostimulated the PBMNCs to produce TNF-a at a ratio of10:1 whereas the Palacos R debris did not. AlthoughPalacos R contains 15.2% w/v ZrO2 whilst CMW 1ROcontains 9.2% w/v BaSO4; the smaller mean granulediameter size of the BaSO4 (1 mm) as opposed to that ofPalacos R (10 mm) may play some part in this difference.The submicrometre particles of CMW 1RO stimulatedthe greatest levels of TNF-a production by PBMNCs atparticle volume (mm3) per cell number ratios of 100:1and 10:1. The response to the fractions of 0.1–10 and1–10 mm was lower and the levels of TNF-a producedsimilar response to these size ranges. This was undoubt-edly due to the similar mass distribution exhibited bythese fractions. In both fractions, the majority of themass was between 1 and 5 mm in size. The lesser capacityof these fractions to induce elevated levels of TNF-aproduction indicated that the most biologically activesize range of CMW 1RO cement particles in this studywas submicrometre. In comparison to the TNF-aresponse to LPS, the TNF-a response to Palacos Rwas small. No fraction appeared consistently optimalfor PBMNC activation. The finding that wear debrisgenerated from Palacos R bone cement had a lessercapacity to induce elevated levels of TNF-a secretion isin agreement with previous research. Sabokbar et al. [13]demonstrated that particles containing the radio opaqueadditive barium sulphate induced 50% more boneresorbtion than those containing zirconium oxide. Thismay perhaps be due to the larger surface area of theradio opaque additive in the bone cement CMW 1RO.In addition to the difference in the radio-opaqueadditive, other small differences exist between PalacosR compared with CMW1RO and CMW original. BothCMW 1RO and original CMW contain prepolymerisedpolymethylmethacrylate. Palacos R however, containsprepolymerised polymethyl methacrylate and poly-methyl acrylate. CMW1RO and CMW original bothcontain hydroquinone as a stabiliser. AdditionallyPalacos R contains chlorophyllin, which providesits distinctive green colour. These differences may

W. Mitchell et al. / Biomaterials 24 (2003) 737–748746

contribute to the difference in the biological responsemade by the PBMNCs to the bone cement particles.Iwaki et al. [18] calculated that 1 g of periprosthetic

tissue contained between 2:4� 107 and 6:8� 109 parti-cles with an average equivalent circle diameter of1:07 mm: From this it may be assumed that 1 g of tissuecontained 1:24� 107–3:5� 109 mm3 of cement debris. If1 g of tissue was composed of 100% macrophages and 1macrophage was 1000 mm3 then it may be inferred thatthe tissue may contain between 0.0125 and 3:536 mm3

cement debris particles per macrophage. This alsoassumes an even distribution of cement debris withinthe tissue. It is unlikely that an even distribution ofcement debris would be observed in vivo, or thatperiprosthetic tissue would comprise 100% macro-phages. Therefore, it is not unreasonable to assumethat a particle volume mm3 per macrophage ratio of 10may be reached or even exceeded in areas of peripros-thetic tissue in the proximity of cement mantle failure ordefects.Characterisation of cement wear debris particles by

Iwaki et al. [18], demonstrated that 82% of cementparticles were submicrometre in size. This study hasdemonstrated that wear debris particles of bone cementscontaining radio opaque additives within the size rangefound present in periprosthetic tissue have the capacityto induce significantly elevated TNF-a production byhuman PBMNCs in vitro.Previous studies have demonstrated that comparable

volumes of clinically relevant UHMWPE wear particlesare required in order to stimulate an increase in TNF-aproduction by PBMNCs following culture in vitro[4,21,22]. These studies indicate that both cementparticles and UHMWPE wear debris may contributeequally to osteolysis and late aseptic loosening ofcemented total hip replacements.

Acknowledgements

This work was funded by the Arthritis ResearchCampaign (ARC). Technical assistance with the scan-ning electron microscopy was given by Dr. JohnHarrington. Surface preparation of wear materials wereprepared by Mr. H. Devon Derby. Surface roughnessmeasurements were carried out by Mr. Alan Heald.

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