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doi:10.1016/j.bi

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Biomaterials 27 (2006) 5199–5211

www.elsevier.com/locate/biomaterials

Differential inflammatory macrophage response to rutileand titanium particles

Gema Vallesa, Pablo Gonzalez-Melendib, Jose L. Gonzalez-Carrascoc, Laura Saldanaa,Elena Sanchez-Sabatea, Luis Munuerad, Nuria Vilaboaa,�

aUnidad de Investigacion, Hospital Universitario La Paz, Paseo de la Castellana 261, 28046 Madrid, SpainbCentro de Investigaciones Biologicas, CIB-CSIC, C/Ramiro de Maeztu 9, 28040 Madrid, Spain

cCentro Nacional de Investigaciones Metalurgicas, CENIM-CSIC, Avda. Gregorio del Amo 8, 28040 Madrid, SpaindDepartamento de Traumatologıa y Cirugıa Ortopedica, Hospital Universitario La Paz, Paseo de la Castellana 261, 28046 Madrid, Spain

Received 8 March 2006; accepted 29 May 2006

Available online 21 June 2006

Abstract

Titanium and its alloys are widely used as implant materials for dental and orthopaedic applications due to their advantageous bulk

mechanical properties and biocompatibility, compared to other metallic biomaterials. In order to improve their wear and corrosion

resistance, several surface modifications that give rise to an outer ceramic layer of rutile have been developed. The ability of rutile wear

debris to stimulate the release of inflammatory cytokines from macrophages has not been addressed to date. We have compared the in

vitro biocompatibility of sub-cytotoxic doses of rutile and titanium particles in THP-1 cells driven to the monocyte/macrophage

differentiation pathway as well as in primary cultures of human macrophages. Confocal microscopy experiments indicated that

differentiated THP-1 cells and primary macrophages efficiently internalised rutile and titanium particles. Treatment of THP-1 cells with

rutile particles stimulated the release of TNF-a, IL-6 and IL-1b to a lesser extent than titanium. The influence of osteoblasts on the

particle-induced stimulation of TNF-a and IL-1b was analysed by co-culturing differentiated THP-1 cells with human primary

osteoblasts. Under these conditions, secretion levels of both cytokines after treatment of THP-1 cells with rutile particles were lower than

after exposure to titanium. Finally, we observed that primary macrophages released higher amounts of TNF-a, IL-6 and IL-1b after

incubation with titanium particles than with rutile. Taken together, these data indicate that rutile particles are less bioreactive than

titanium particles and, therefore, a higher biocompatibility of titanium-based implants modified with an outer surface layer of rutile is

expected.

r 2006 Elsevier Ltd. All rights reserved.

Keywords: Osteolysis; Wear debris; Macrophage; Cytokine; Titanium; Titanium oxide

1. Introduction

The biological response to wear particles at the bone–implant interface is considered the main cause of asepticloosening and osteolysis [1,2]. Currently, the only clinicalintervention for aseptic loosening is implant revisionsurgery, and bone loss associated with this procedurelimits the number of times that it can be performed.Histological analysis of periprosthetic tissues retrievedfrom loose implants reveals the existence of a granuloma-

e front matter r 2006 Elsevier Ltd. All rights reserved.

omaterials.2006.05.045

ing author. Tel./fax: +34 912071034.

ess: nvilaboa.hulp@salud.madrid.org (N. Vilaboa).

tous membrane composed of numerous macrophages,fibroblasts, lymphocytes, giant cells, and wear particles[2–5]. Enlarged macrophages, often showing internalisedwear particles, were detected in these tissues [5–7].Biochemical and immunohistochemical analysis of peri-implant tissues from patients at revision indicated highlevels of cytokines known to stimulate bone resorption,such as interleukin-1b (IL-1b), interleukin-6 (IL-6) ortumor necrosis factor-a (TNF-a) [7–10]. Moreover, a highpercentage of macrophages in these tissues showedpositive staining for those cytokines [8,10]. In vivo and invitro experiments indicate that particulate debris withdiverse characteristics are phagocytosed by macrophages,

ARTICLE IN PRESSG. Valles et al. / Biomaterials 27 (2006) 5199–52115200

stimulating the secretion of the inflammatory cytokinesTNF-a or IL-1b [2]. These soluble factors act onosteoblasts inducing the expression of intercellular adhe-sion molecule-1 (ICAM-1), which mediates the recruitmentof osteoclast precursors, and receptor activator of NF-kBligand (RANKL), which stimulates the maturation andactivation of osteoclasts [11,12]. TNF-a and IL-1b alsostimulate osteoblastic IL-6 secretion, a potent activator ofbone resorption, which mediates their effects by stimulat-ing the secretion of RANKL [12]. In addition, TNF-a andIL-1b directly stimulate osteoclast differentiation andactivity through specific receptors expressed by cells ofthe osteoclast lineage [12]. Particles can also be internalisedby osteoblasts, stimulating the secretion of pro-resorptivefactors such as IL-6 and RANKL [13,14].

Titanium and Ti-based alloys are widely used for dentaland orthopaedic implants due to their good corrosionbehaviour and advantageous bulk mechanical properties,compared to other metallic biomaterials. The reduced ionrelease and excellent biocompatibility is largely attributedto the spontaneous formation of an inert surface passivefilm of non-stoichiometric TiO2, typically 4–6 nm thick,which is amorphous or poorly crystallized. However, metaldevices fail to integrate completely with the surroundingbone, whereby a thin fibrous layer forms at the bone–tissueinterface. Such a soft layer, under continuous mechanicalstress, is believed to initiate micromotions that give rise towear debris and favour the migration of macrophages tothe bone–implant interface. In fact, a significant number oftitanium particles have been detected in retrieval studiesof tissues surrounding failed implants [15,16]. The ability ofthese particles to stimulate focal bone resorption has beenwell established in vivo [14,17]. In vitro studies indicatethat titanium particles in the phagocytosable range are ableto induce macrophages to secrete osteolytic cytokines andinitiate an inflammatory response [18,19]. Increased thick-ness and crystallinity of the outside oxide layer of Ti-basedimplants can be obtained by treatments such as micro-arcoxidation or thermal oxidation [20–22]. These surfacemodifications not only improve corrosion resistance, butalso reduce the friction coefficient in rubbing contact. Theresulting rutile layer enhances osteoblast adhesion in vitro[23,24] and improves bone fixation in vivo [25]. Thus, thesemodified surfaces should limit the access of macrophagesand wear debris to the bone–implant interface. None-theless, functional loading for a long service period willlead to release of titanium oxide particles. In addition,rutile particles are increasingly proposed as reinforcementagents in the fabrication of composite materials [26], whichalso can suffer from particle loss.

To our knowledge, there is no information availablerelated to the inflammatory behaviour of macrophages inthe presence of rutile particles. In this work we haveevaluated the in vitro biocompatibility of these particles inTHP-1 cells differentiated to the monocyte–macrophagelineage as well as in human primary macrophages. To thisend, secretions of TNF-a, IL-6 and IL-1b were assessed in

cultures of both cell types after treatment with sub-cytotoxic doses of particles. For comparative purposes,titanium particles were used.

2. Materials and methods

2.1. Characterisation and preparation of particles

Rutile (TiO2) of 0.9–1.6mm-diameter and commercially pure titanium

(Ti) particles of o20 mm-diameter were obtained from Johnson Matthey

(Ward Hill, MA, USA). In order to characterise the morphology of the

materials, powders of the investigated particles were deposited on a

conductive and adhesive tape and then studied with scanning electron

microscopy (SEM) using a JEOL JSM 6500F (Jeol, Peabody, MA, USA).

Chemical composition of the particles was confirmed by electron

dispersive spectroscopy (EDS) using a Rontec EDR288 Software (Rontec,

Berlın, Germany). The equivalent circle diameter (ECD) was determined

by quantitative analysis of representative SEM images obtained from a

minimum of five different fields of view using the same magnifications.

Particle size analysis was performed with an Optimas Image Analyzer

equipped with SigmaScan Pro 4.0 Image Analysis Software (Jandel

Scientic Co., San Rafael, CA, USA). Particle characterisation was

completed by X-ray diffraction, confirming that TiO2 and Ti particles

were composed of rutile and pure titanium structures, respectively (data

not shown).

TiO2 and Ti particles were weighed and sterilised by incubation in

isopropanol at room temperature and dried under UV light in a laminar

flow hood. Prior to the addition to the cells, particles were re-suspended in

the appropriate culture medium (20mg/ml) and sonicated at maximum

power for 10min in a bath sonicator (Bransonic 12, Branson Ultrasonidos

S. A. E., Barcelona, Spain).

2.2. Cell culture and treatments

THP-1 cells (ECACC, Salisbury, Wiltshire, UK) were grown in RPMI-

1640 medium supplemented with 10% (v/v) heat-inactivated fetal bovine

serum (FBS), 500UI/ml of penicillin and 0.1mg/ml of streptomycin, in a

humidified 5% CO2 atmosphere at 37 1C. Cells were maintained in

continuous logarithmic growth by passage every 3 days. To induce

macrophage differentiation, cells were seeded into six-well plates at a

density of 2� 105 cells/well and continuously treated with 10 ng/ml 12-O-

tetradecanoyl phorbol 13-acetate (TPA) (Sigma, Madrid, Spain) for 36 h,

or treated only for 12 h and thoroughly washed with phosphate-buffered

saline (PBS) and incubated in fresh medium for a further 24 h. Then, cells

were washed with PBS and supplemented with 2ml of fresh medium.

Appropriate volumes of particle suspensions were added to cells to achieve

doses of 0.5, 5 and 50 ng/cell and cells were cultured for a further 24 h. As

controls, TPA-differentiated THP-1 cells were incubated in the absence of

particles.

Peripheral blood mononuclear cells (PBMC) were isolated from the

blood of healthy donors by differential centrifugation on Ficoll-Paque

Plus (Amersham Bioscience, Uppsala, Sweden). PBMC were seeded at a

density of 2� 106 cells/ml and cultured for 2 h in RPMI supplemented

with 2% heat-inactivated FBS and 500UI/ml of penicillin and 0.1mg/ml

of streptomycin. The medium was removed and adherent cells were

cultured in the same medium supplemented with 10% heat-inactivated

FBS. After 7 days, cells were harvested with 5mM EDTA in PBS and

plated in six-well plates at a density of 2� 105 cells/well for 24 h. PBMC

were washed with PBS and supplemented with 2ml of fresh medium.

Appropriate volumes of particle suspensions were added to cells to achieve

doses of 0.5, 5 and 50 ng/cell and cells were cultured for further 24 h. As

controls, PBMC were incubated in the absence of particles.

Human osteoblastic cells (OB) were derived from fresh trabecular bone

explants removed during total knee arthroplasty, as previously described

[23]. Bone samples were obtained from patients aged 6975 years old.

Patients enrolled in this research signed an Informed Consent form and all

ARTICLE IN PRESSG. Valles et al. / Biomaterials 27 (2006) 5199–5211 5201

procedures using human tissue designated ‘‘surgical waste’’ were approved

by the Human Research Committee of Hospital La Paz (Date of

Approval: 15 March 2001). Each bone sample was processed in a separate

primary culture and experiments were performed using cultures obtained

from independent patients. OB were cultured in Dulbecco0s modified

Eagle’s medium (DMEM) containing 15% (v/v) FBS, 100UI/ml penicillin

and 0.1mg/ml streptomycin in a humidified 5% CO2 atmosphere at 37 1C.

Culture medium was changed every 3 days until confluence was reached.

THP-1 and OB were co-cultured using a transwell insert system

(Corning, Life Sciences, MA, USA) that allows humoral contact of both

cell types, through a microporous membrane with a pore size of 0.4 mm,

without direct cell contact. 2� 105 THP-1 cells were seeded in six-well

plates and treated with 10 ng/ml TPA for 12 h, thoroughly washed with

PBS and cultured in fresh medium for 24 h. Parallel groups of 2� 105 OB

were seeded in inserts and cultured for 24 h. Then, THP-1 cells were

washed with PBS and supplemented with 3ml of a mixture of 50%

RPMI and 50% DMEM, containing 12.5% (v/v) heat-inactivated FBS,

500UI/ml of penicillin and 0.1mg/ml of streptomycin. Inserts containing

OB were washed with PBS and placed into the wells containing THP-1

cells. Appropriate volumes of particle suspensions were added to THP-1

cells to achieve doses of 50 ng/cell and co-cultures were incubated for

further 24 h. As controls, co-cultures of THP-1 and OB were subjected to

the same manipulations but incubated in the absence of particles.

2.3. Endotoxin test

Particles and culture media were endotoxin-free as demonstrated by the

Sigma E-TOXATE assay for detection and semi-quantification of

endotoxins (Sigma). Particles and culture media used in this study

contained levels of endotoxins below 0.015EU/ml.

2.4. Cytotoxicity assays

Lactate dehydrogenase (LDH) release was assayed with the Cyto Tox

96 (Promega, Madison, WI, USA), following the manufacturer’s

instructions.

2.5. Immunoenzymatic assays

Culture media were collected, filtered and centrifuged at 1200g for

10min, supplemented with a mixture of proteases inhibitors (17.5mg/ml

phenylmethylsulfonyl fluoride, 1mg/ml pepstatin A, 2 mg/ml aprotinin,

50 mg/ml bacitracin, all from Sigma) and frozen at �80 1C. Human-specific

ELISA kits were used to measure TNF-a (CLB, Amsterdam, Holland),

IL-6 and IL-1b (Biosource International Inc., Camarillo, CA, USA). The

detection limits of the kits were 1 pg/ml for TNF-a and IL-1b and 2 pg/ml

for IL-6. All procedures were performed following the manufacturer’s

instructions. The secreted levels were normalised to the total protein

amount, measured by the BCA protein assay (Pierce, Rockford, IL, USA),

using bovine serum albumin as standard.

2.6. Flow cytometry assays

Cell surface immuno-fluorescence staining was performed by incubat-

ing 1� 105 cells with fluorescein isothiocyanate (FITC)-conjugated anti-

human CD14 (Cymbus Biotechnology Ltd, Chandlers Ford, UK) for

30min at 4 1C in the dark. Cells incubated with FITC-conjugated mouse

inmunoglobulin G (Cymbus Biotechnology Ltd) were used as negative

controls. Cells were washed with PBS and fluorescence was measured by

flow cytometry using a FACSCalibur analyzer and CELLQUEST

software (both from Becton Dickinson Biosciences, San Jose, CA,

USA). For determination of CD11b antigen expression, 1� 105 cells were

incubated with 0.1mg/ml of human g-globulin in PBS for 15min at 4 1C in

the dark. Cells were washed with PBS and incubated with a mouse anti-

human CD11b (Becton Dickinson Biosciences) for 30min at 4 1C. Cells

incubated in the absence of CD11b were used as negative controls. Cells

were washed with PBS and incubated with FITC-conjugated anti-mouse

immunoglobulin G (Sigma) in PBS. Cells were washed with PBS and

fluorescence was measured by flow cytometry using the same equipment.

2.7. Confocal microscopy

Cells were seeded in eight-well chambers (Nunc, Wiesbaden, Germany)

(2� 104 cells/well) and cultured for 24 h in the presence or in the absence

of particles. Cells were extensively washed with PBS and fixed for 45min

with 2.5% glutaraldehyde in PBS, in darkness. Cells were washed with

PBS and mounted with a 1:1 mixture of glycerol/PBS; then a cover slip

was placed onto the specimen and sealed with nail varnish without

pressure. The specimens were observed in a confocal microscope (LEICA

TCS-SP2-AOBS, Leica microsystems, Heidelberg GMBH, Germany)

under a He/Ne laser (excitation lines 543 and 633 nm). The autofluorescent

signal of the biological samples due to glutaraldehyde fixation was

collected in the emission ranges 553–625nm and 675–753nm. The

presence of particles within the cells can be detected as non-fluorescent,

dark areas in an autofluorescent background, which were absent in the

untreated cells. Stacks of 0.5 mm optical sections spanning completed cells

were recorded and the confocal images were analysed using the LEICA

software LCS, version 2.5 Build 1227, which was also used for

morphometric analysis and measures of areas. Once we determined that

we had detected internalised rutile and Ti particles, we measured their

sizes. The areas of intracellular dark regions of different sizes were

measured using the same software, by manually outlining a region of

interest (ROI) on the projected maximum view of the confocal stack. The

software is calibrated with the original data from the confocal stack

collection in order to measure the real ROI area and therefore, calculate

the corresponding ECD. For each experimental condition, individual cells

were examined in order to determine the number of internalised particles/

cell. A significant number of measures were collected for each specimen.

2.8. Statistical analysis

The data are presented as mean7S.D. of several independent

experiments, each performed with an individual primary culture. Secretion

differences between isolated cultured and co-cultured cells as well as

characterisation of particles internalised into cells were analysed using

one-way analysis of variance (ANOVA) with Bonferroni0s correction for

post hoc comparisons. Two-way ANOVA for repeated measures were

performed for the rest of experiments. Post hoc comparisons were

analysed by 95% confidence interval adjusted by the Bonferroni’s method.

In all cases we confirmed results by non-parametric pair-wise Friedman’s

test with the appropriate post hoc comparisons. The p values o0.05 were

considered to be statistically significant. All statistical analyses were

performed using personal computer-based statistical software (SPSS

version 10.0.1; SPSS, Chicago, IL, USA).

3. Results

3.1. Characterisation of the particles

Morphological assessment of TiO2 particles showedthem to be globular with a grainy surface structure(Fig. 1(A)). Most of Ti particles were round with a smoothsurface, although the larger Ti particles were usuallyelongated and rough (Fig. 1(B)). The mean sizes of parti-cles were 0.4570.26 mm for TiO2 (range was 0.1–1.5 mm,92% were lower than 0.9 mm) and 3.3272.39 mm forTi (range was 1–15 mm, 89% were lower than 7 mm)(Figs. 1(A and B), respectively).

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Fig. 1. Scanning electron microscopy images and particle size distribution of TiO2 (A) and Ti particles (B) (n ¼ 588).

G. Valles et al. / Biomaterials 27 (2006) 5199–52115202

3.2. Response of TPA-differentiated THP-1 cells to Ti or

TiO2 particles

TNF-a released from THP-1 cells at levels below thedetection limits of immunoenzymatic assays and treatmentof THP-1 cells with 50 ng/cell of Ti or TiO2 particles for24 h did not raise the secretion rates to detectable levels(Fig. 2(A)). THP-1 cells were incubated with 10 ng/ml TPAfor 36 h and then thoroughly washed and cultured in theabsence or presence of 50 ng/cell of particles for further24 h. TPA treatment of cells resulted in measurable levelsof TNF-a but incubation of TPA treated cells with Ti orTiO2 particles did not enhance the release of TNF-a to themedia (Fig. 2(A)). Parallel groups of THP-1 cells weretreated with TPA for only 12 h, washed and cultured infresh medium for 24 h, and then incubated in the absenceor in the presence of 50 ng/cell of particles for a further24 h. THP-1 cells treated with TPA for only 12 h secretedsubstantially lower amounts of TNF-a than the grouptreated continuously with the phorbol ester for 36 h andincubation with Ti or TiO2 particles increased TNF-a levels(Fig. 2(A)). Expression levels of the surface antigens CD14and CD11b, characteristics of the monocyte/macrophagelineage, increased to same extent in cells transiently treatedwith TPA for only 12 h or continuously for 36 h (Fig. 2(B)).Since treatment with TPA for only 12 h increased theexpression of differentiation markers and cells respondedto particles by releasing substantial amounts of TNF-a,subsequent experiments along this work were performedwith THP-1 cells differentiated according to this procedure.

3.3. Secretion of LDH after treatment of TPA-

differentiated THP-1 cells with Ti or TiO2 particles

Treatment of TPA-differentiated THP-1 cells with Ti orTiO2 particles for 24 h, at doses of 0.5, 5 or 50 ng/cell, didnot result in a significant increase of LDH release, ascompared to the levels detected in media from untreatedTPA-differentiated cells (Fig. 3).

3.4. Internalisation of Ti and TiO2 particles

In order to assess if the particles were internalised intoTPA-differentiated THP-1 cells, we used confocal micro-scopy. The cells were fixed in a solution of glutaraldehyde,which provides an autofluorescence background to theoverall cell structure where the presence of the particlescan be inferred by the absence of staining. We first noticeda dose-dependent effect of the particles in the cells. At0.5 ng/cell of TiO2 or Ti, some dark areas of differentsizes were observed on projections of 3D confocal stacks(Figs. 4(A and D)); the dark areas were larger and moreevident at a concentration of 5 ng/cell (Figs. 4(B and E)).At the highest tested dose, an increasing number of largedark areas were observed (Figs. 4(C and F)). The 3Dposition of these dark areas was determined by analysingorthogonal sections along the planes XZ and YZ acrossparticular, small, dark areas visualised in the confocalstack. When the stack was virtually sectioned along the XZ

plane (bottom panel) and the YZ plane (right panel), weobserved that the dark area penetrated inside the cell and

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Fig. 2. Response of TPA-differentiated THP-1 cells to Ti or TiO2 particles. THP-1 cells were cultured in the absence of TPA (�), with TPA for 36 h, or for

12 h and then cultured for 24 h in fresh medium (12 h, rec 24 h). (A) TNF-a secretion from THP-1 cells after treatment with Ti or TiO2 particles. Untreated

or TPA-treated cells were incubated in the absence (�) or in the presence (+) of 50 ng/cell of Ti or TiO2 particles for 24 h. The data are relative to the level

of TNF-a measured in media from THP-1 cells treated with TPA for 12 h, cultured in fresh medium and not exposed to particles (0.5170.06 pg TNF-a/mgtotal proteins), which was given the arbitrary value of 1. Each value represents the mean7S.D. of six independent experiments. *po0:05 compared to

TPA (12 h, rec 24 h) not exposed to particles; #po0:05 compared to TPA (36 h); &po0:05 compared to treatment with TiO2 particles in TPA (12 h, rec

24 h). N.D.: Not detected. (B) Flow cytometric determination of the expression of the surface markers CD14 and CD11b in THP-1 cells untreated or

treated with TPA. The grey dashed lines indicate the boundary for the positive staining.

G. Valles et al. / Biomaterials 27 (2006) 5199–5211 5203

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with particles. Cells were untreated or treated with the indicated doses of

TiO2 ( ) or Ti ( ) particles for 24 h. The data are relative to the

absorbance measured in untreated cells, which was given the arbitrary

value of 1. Each value represents the mean7S.D. of six independent

experiments.

G. Valles et al. / Biomaterials 27 (2006) 5199–52115204

showed a three-dimensional arrangement. Images corre-sponding to cells treated with the intermediate doses(5 ng/cell) of TiO2 (Fig. 4(H)) and Ti (Fig. 4(I)) are shown.Fig. 4(G) shows orthogonal sections of untreated culturesof TPA-differentiated THP-1 cells, without any dark areasinside. The size of the dark areas inside the cells wasmeasured by manually outlining their contour in the 3Dstacks, using the image processing software of the confocalmicroscope. The distribution of areas vs. their relativefrequencies was represented in histograms as shown inFigs. 4(J and K) for TiO2 and Ti particles, respectively.Average size of particles was smaller in cells exposed toTiO2 than to Ti particles. However, the number ofparticles/cell was higher for TiO2 than for Ti particles(Table 1).

3.5. Secretion of TNF-a, IL-6 and IL-1b from TPA-

differentiated THP-1 cells treated with Ti or TiO2 particles

Treatment of TPA-differentiated THP-1 cells with Tiparticles at any tested dose for 24 h stimulated the secretionof TNF-a and IL-6 (po0:05) (Figs. 5(A and B)). Levels ofboth cytokines in media from cells treated with 5 ng/cell ofparticles were higher than those measured in media fromcells treated with 0.5 ng/cell (po0:05). The highest testeddose did not enhance the released amounts of thesecytokines above levels detected in media from cells treatedwith 5 ng/cell of Ti particles. Incubation with TiO2 particlesresulted in a dose-dependent significant increase of TNF-aand IL-6 secretion (po0:05). TiO2 particles at 0.5 and5 ng/cells resulted in lower TNF-a and IL-6 levels thanthose measured after treatment with the corresponding

doses of Ti. At the highest dose, TiO2 particles stimulatedsecretion of TNF-a at lower extent than Ti although nosignificant differences were observed in levels of IL-6.Finally, levels of IL-1b also increased in a dose-dependentmanner in the media of cells treated with Ti or TiO2

particles (po0:05), but the levels were substantially lowerin cells treated with TiO2 particles at any tested dose(Fig. 5(C)).

3.6. Secretion of TNF-a and IL-1b from co-cultures of OB

and TPA-differentiated THP-1 cells treated with Ti or TiO2

particles

We wondered about the ability of OB to modulate,through secretion of soluble factors, the TiO2 and Tiparticles-induced TNF-a and IL-1b secretion in TPA-differentiated THP-1 cells. To this end, these cells were co-cultured with OB without direct cell contact and incubatedfor 24 h in the absence or presence of 50 ng/cell of Ti orTiO2 particles. For comparative purposes, media fromisolated cultured OB or THP-1 cells, incubated in theabsence of particles, were also evaluated. TNF-a and IL-1bwere not detected in the media from isolated cultured OB(Figs. 6(A and B)). Compared to the amounts released byisolated cultured THP-1 cells, co-culturing of THP-1 cellswith OB diminished or abolished the secretion of TNF-aand IL-1b, respectively. Treatment of co-cultured THP-1cells with Ti particles increased the levels of both cytokines.TiO2 particles only increased IL-1b release and comparedto incubation with Ti, secretion levels were significantlylower.

3.7. Secretion of LDH and internalisation assays after

treatment of human primary macrophages with Ti and TiO2

particles

The human primary macrophages cultures containedmore than 85% CD14-positive cells as determined by flowcytometry (Fig. 7(A)). Treatment of macrophages with Tior TiO2 particles for 24 h, at doses of 0.5, 5 or 50 ng/cell,did not result in significant increase of LDH release, ascompared to the levels detected in media from untreatedmacrophages (Fig. 7(B)).Internalisation of the particles into the cells was assessed

by confocal microscopy. As observed in THP-1 cells, wenoticed a dose-dependent effect of the particles (datanot shown). After exposure to 5 ng particles/cell of TiO2

(Fig. 7(D)) and Ti (Fig. 7(E)) for 24 h, we observed somesmall, dark areas of different sizes in the cells in 3D stacks,which were not detected in untreated cells (Fig. 7(C)). The3D position of these unstained regions in the cells wasdetermined by virtual sectioning of the stack along the XZ

(bottom panels in Figs. 7(C, D and E)) and YZ planes(right panels in Figs. 7(C, D and E)) at the central point ofthese areas (arrows in central panels). Observation of theseorthogonal sections showed that the particles are locatedinside the cells (arrows in side-panels of Figs. 7(D and E)).

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20

25

30

35

ECD (µm)

(A) (B) (C)

(F)(E)(D)

(G)

(J) (K)

(H) (I)

Fig. 4. Internalisation of particles into TPA-differentiated THP-1 cells. (A–F) Cells were treated for 24 h with TiO2 or Ti particles at doses of 0.5 ng/cell (A

or D), 5 ng/cell (B or E) and 50 ng/cell (C or F). (G–I) Orthogonal projections along the XZ (bottom panels) and YZ (right panels) at a random position of

untreated cells (G) or at a central position of unstained areas of cells treated with 5 ng/cell of TiO2 (H) or Ti particles (I). Bars ¼ 40 mm. (J,K) Size

distribution of internalised particles in cells treated with 5 ng/cell of TiO2 (J) or Ti (K) particles (n ¼ 63).

Table 1

Characterization of particles internalized in TPA-differentiated THP-1 cells and human primary macrophages

TPA-differentiated THP-1 cells Human primary macrophages

TiO2 Ti TiO2 Ti

ECD (mm) 2.2670.86* 5.0472.55 1.9070.96 3.9271.99

Particles/cell 1975* 973 1474** 1272

ECD and number of particles/cell into cells treated with TiO2 or Ti particles. *po0:05 compared to Ti particles in TPA-differentiated THP-1 cells;

**po0:05 compared to TiO2 particles in TPA-differentiated THP-1 cells.

G. Valles et al. / Biomaterials 27 (2006) 5199–5211 5205

No dark areas inside the cells were observed on orthogonalprojections of the untreated controls (Fig. 7(C)). The sizeof these dark areas inside the cells was measured by

manually outlining their contour in the 3D stacks and thedistribution of areas vs. their relative frequencies wasrepresented in histograms as shown in Figs. 7(F and G) for

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0

1

2

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-α s

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ng /cell

ng /cell

*

*

*

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-6 s

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*

*

150

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024

152535

0.5 5 50

Rel

ativ

e IL

-1β

secr

etio

n

*

*

*

(A) (B)

(C)

Fig. 5. TNF-a (A), IL-6 (B) and IL-1b (C) secretion in TPA-differentiated THP-1 cells treated with particles. Cells were untreated or treated with the

indicated doses of TiO2 ( ) or Ti ( ) particles for 24 h. The data are relative to cytokines levels measured in untreated cells, which were given the arbitrary

value of 1. Untreated cells released 0.5170.06 pg TNF-a/mg total proteins, 1.5570.13 pg IL-6/mg total proteins and 0.3470.07 pg IL-1b/mg total proteins.

Each value represents the mean7S.D. of six independent experiments. *po0:05 compared to treatment with Ti particles, at the corresponding tested dose.

G. Valles et al. / Biomaterials 27 (2006) 5199–52115206

TiO2 and Ti particles, respectively. Average sizes andnumber of particles/cell were similar in cells treated withTiO2 or Ti (Table 1).

3.8. Secretion of TNF-a, IL-6 and IL-1b from human

primary macrophages treated with Ti or TiO2 particles

Treatment of macrophages with Ti particles at 0.5, 5 or50 ng/cell for 24 h resulted in a dose-dependent inductionof TNF-a (po0:05). While secreted levels of TNF-aincreased by about 30 fold after treatment with Ti particlesat 0.5 ng/cell and to 250 fold after treatment with thehighest tested dose, TiO2 particles at 0.5 or 5 ng/cell onlystimulated the secretion of this cytokine by about 2-fold(Fig. 8(A)). After treatment with the highest tested dose ofTiO2 particles, macrophages secreted similar levels ofTNF-a to untreated cells. Similar behaviour was observedafter evaluation of released IL-6 after treatments with bothtypes of particles. Treatment with Ti particles resulted in adose-dependent induction of IL-6 (po0:05), which in-creased by 200 fold after treatment with 50 ng/cell of thistype of particle, while TiO2 treatment marginally affectedthe released levels of IL-6 (Fig. 8(B)). Untreated humanprimary macrophages released IL-1b at levels below thedetection limit of the immunoenzymatic assay and we only

could detect this cytokine after treatment with Ti or TiO2

particles at 50 ng/cell. At this dose, TiO2 particlesstimulated macrophages to release significantly lower levelsof IL-1b than Ti particles (Fig. 8(C)).

4. Discussion

The THP-1 cell line is a useful in vitro model forstudying the wear response to particles in macrophages. Todrive these cells along the monocytic/macrophage differ-entiation pathway, several inducers such as the active formof vitamin D3 (1a, 25 dihydroxyvitamin D3) or the phorbolester TPA have been employed [27,28]. Examination ofphagocytosis ability and expression of the surface markersCD14 and CD11b indicate that TPA treatment results in amore differentiated phenotype than treatment with 1a, 25dihydroxyvitamin D3 [29]. However, TPA differentiationalso stimulates the release of substantial amounts ofinflammatory cytokines such as TNF-a, IL-1b, IL-6 orprostaglandin E2 (PGE2) [29–31]. Results from this workindicate that neither Ti particles, which are recognised tostimulate the release of inflammatory cytokines in macro-phages [18,19], nor rutile particles enhanced the secretionof TNF-a when applied to continuously TPA-treatedTHP-1 cells. Transient application of the phorbol ester

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elat

ive

TN

F-α

sec

reti

on

Rel

ativ

e IL

-1β

secr

etio

n

-

THP-1 THP-1

OB

THP-1

OB

2.0

0.0

1.0

3.0

* #

N.D.

OB

-

2

10

8

6

4

0

-

THP-1 THP-1

OB

THP-1

OBOB

-

N.D.N.D.

* &

&

+

+

*

(A)

(B)

Fig. 6. TNF-a (A) or IL-1b (B) secretion in TPA-differentiated THP-1

cells co-cultured with OB. Levels of both cytokines were determined in

cultures of TPA-differentiated THP-1 cells ( ), OB, or co-cultures of OB

and TPA-differentiated THP-1 cells untreated ( ) or treated with

50 ng/cell of TiO2 ( ) or Ti ( ) particles for 24 h. The data are relative

to cytokines levels measured in TPA-differentiated THP-1 cells, which

were given the arbitrary value of 1. Untreated cells released 0.5170.06 pg

TNF-a/mg total proteins and 0.3470.07 pg IL-1b/mg total proteins. Each

value represents the mean7S.D. of five independent experiments.

*po0:05 compared to TPA-differentiated THP-1 cells; #po0:05 compared

to co-cultures treated with Ti particles; &po0:05 compared to treatment

with TiO2 particles. (�) indicates the absence of the corresponding cell

type; (+) indicates that co-cultured TPA-differentiated THP-1 cells were

treated with 50 ng/cell of TiO2 or Ti particles. N.D.: Not detected.

G. Valles et al. / Biomaterials 27 (2006) 5199–5211 5207

drove cells to an equivalent maturation stage to thatachieved by continuous treatment, according to theexpression levels of CD14 and CD11b antigens. Ti andrutile particles stimulated the secretion of TNF-a, IL-6 andIL-1b by cells transiently treated with TPA, indicating thatthis is a suitable method for studying the inflammatoryresponse to particles. Moreover, the behaviour of thesecells after treatment with Ti or rutile particles followedsimilar trends to those observed in human primarymacrophages under the same experimental conditions.

Internalisation of wear particles into cultured cells hasbeen examined using several microscopy techniques.Among these, confocal and transmission electron micro-scopy are more sensitive than the visualisation of particlesunder phase contrast on a light microscope. Only confocalor physical sectioning of the specimens permits theidentification of particles internalised into cells. Althoughobservation of ultra-thin sections on the electron micro-scope allows a direct visualisation of mineral particlesbecause they are dense to the electrons [32], this procedureis more time-consuming and requires higher technical skillsthan confocal microscopy, which in turn allows definitionof the intracellular location where the particles arecontained as the absence of intracellular staining in afluorescence background. To this aim, confocal microscopymakes use of specific dyes such as phalloidin staining toreveal the actin network [33], or general dyes, such asacridine orange [34]. In this work, we have developed aone-step technical advance to visualise rutile and Tiparticles by taking advantage of the autofluorescenceproperties of glutaraldehyde, one of the most commonlyused fixatives for microscopy studies. Together withoptimal preservation of the cell’s structure, this offerssatisfactory and reproducible results while avoiding addi-tional staining, washing and further manipulation of thespecimens. Phalloidin staining leaves non-stained spacescorresponding to the position of the cell’s organelles, whichmakes the identification of internalised particles difficult,while treatment with glutaraldehyde extensively crosslinkscellular proteins and provides an autofluorescence signal tothe overall cell structure. Thus, the non-stained areascorrespond to a non-biological composition. On the otherhand, the resolution of the confocal microscopy, 0.2 mm inthe XY plane, was enough to detect the size of particlesused in this study. The location of rutile and Ti particleswas inferred by the presence of small, dark areas ofdifferent sizes, which correspond to a non-biologicalnature, in the autofluorescent cells. The 3D position ofthe particles was determined by virtually sectioning thestack across the XZ and YZ planes at a given position,finding that the particles were unequivocally internalisedinto the cells. A dose-dependent effect was observed: thehigher the dose, the more and larger the dark areas. 3Dconfocal stacks are amenable to morphometric studies bymeasuring the areas of manually outlined ROIs usingsuitable software, such as the one we employed in this work[35]. Mean sizes of Ti and rutile particles internalised byboth THP-1 cells and primary macrophages were higherthan the mean sizes of the powders of particles used in thisstudy, indicating the existence of aggregates of particlesinside the cells. Together with the work of others reportingthe agglomeration of alumina and titania particles inculture media [36], our results suggest that cells mayinternalise aggregates rather than individual particles. Indespite that the mean size of dry rutile particles was about7 fold lower than that of dry Ti, intracellular aggregates ofrutile and Ti presented similar dimensions. Interestingly,

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100

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101 102 103 104

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l nu

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87.50 %CONTROL

CD14

ECD (µm) ECD (µm)

(A) (B)

(C)

(F) (G)

(D) (E)

Fig. 7. Response of human primary macrophages to particles. (A) Flow cytometric determination of the surface marker CD14 expression. The grey

dashed lines indicate the boundary for the positive staining. (B) LDH release in cultures of macrophages after treatment with particles. Cells were

untreated or treated with the indicated doses of TiO2 ( ) or Ti ( ) particles for 24 h. The data are relative to the absorbance measured in untreated cells,

which was given the arbitrary value of 1. Each value represents the mean7S.D. of eight independent experiments. (C–E) Orthogonal projections along the

XZ (bottom panels) and YZ (right panels) at a random position in untreated cells (C) or at a central position of unstained areas of cells treated with

5 ng/cell of TiO2 (D) or Ti particles (E). Bars ¼ 40mm. (F,G) Size distribution of internalised particles in cells treated with 5 ng/cell of TiO2 (F) or Ti (G)

particles (n ¼ 63).

G. Valles et al. / Biomaterials 27 (2006) 5199–52115208

titania particles of 4 mm diameter also aggregated in theculture media to the same extent as alumina particles of0.17 mm [36].

Chemistry of the particles influences their ability tostimulate an inflammatory response in macrophages[37,38]. In agreement with several reports addressing theinflammatory potential of Ti particles [18,19], levels ofTNF-a, IL-6 and IL-1b raised in culture media of THP-1cells treated with this type of particle. Rutile particles alsostimulated their release, but to a lesser extent than Tiparticles, suggesting the better biocompatibility of rutile. Inthis regard, it has been shown that other ceramic particles,such as Al2O3 or ZrO2, induce a lower release of TNF-athan Ti particles in THP-1 cells differentiated with 1a, 25dihydroxyvitamin D3 and granulocyte-macrophage colony-

stimulating factor (GM-CSF) [39]. Particles of sub-micro-meter size are more effective inducers of macrophageactivation than those of larger size [40,41]. Consequently,levels of secreted cytokines after treatment with rutileparticles should be expected to be higher than afterexposure to Ti particles. The opposite effect we havedetected supports the higher biocompatibility of rutile,compared with Ti debris.Co-cultures of osteoblasts and macrophages have proven

to be a valuable tool for studying interactions betweenthese types of cells in the presence of wear particles [42–45].Co-cultures of differentiated THP-1 cells and humanprimary osteoblasts, in the absence of particles, decreasedthe levels of secreted TNF-a or IL-1b by THP-1 cells. Theinfluence of osteoblasts to modulate TNF-a secretion by

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pg

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0.5 5 50 ng /cell ng /cell

ng /cell

210

3

30

40

250

450

50

* **

(A) (B)

(C)

Fig. 8. TNF-a (A), IL-6 (B) and IL-1b (C) secretion in human primary macrophages treated with particles. Cells were untreated or treated with the

indicated doses of TiO2 ( ) or Ti ( ) particles for 24 h. The data corresponding to TNF-a and IL-6 are relative to cytokines levels measured in untreated

cells, which were given the arbitrary value of 1. Untreated cells released 0.1270.30 pg TNF-a/mg total proteins and 3.3570.53 pg IL-6/mg total proteins.

Each value represents the mean7S.D. of six independent experiments. *po0:05 compared to treatment with Ti particles in C and *po0:001 in A and B, at

the corresponding tested dose. N.D.: Not detected.

G. Valles et al. / Biomaterials 27 (2006) 5199–5211 5209

macrophages has been previously detected in other co-culture models [42,44] and in this work we have detectedthat IL-1b secretion is also regulated. Particles modulatethe secretion of cytokines typically involved in boneturnover in co-cultures of osteoblasts and macrophages[42–45]. In contrast to Ti, in our co-culture experiments,rutile particles were not able to influence the secretion ofTNF-a and stimulated IL-1b release to a lower extent. Tiparticles enhanced the release of TNF-a in a co-culturemodel of J774.A1 and MC3T3-E1 cells, but polymethyl-methacrylate particles failed to regulate its production [43],proving that the chemistry of the particles stronglyinfluences the interactions of macrophages and osteoblasts.Compared to Ti, attenuation in the levels of TNF-a andIL-1b after treatment of co-cultured macrophage-like cellswith rutile particles predicts their lower bioreactivity, asboth cytokines play a relevant role in the initiation of theinflammatory response to wear debris [2,19].

Finally, we studied the behaviour of human primarymacrophages treated with Ti and rutile particles. As inTHP-1 cultures, both types of particles were efficientlyinternalised by these cells without eliciting a harmfulresponse, as determined by release of LDH to the media.Inductions of TNF-a, IL-6 and IL-1b after treatment withTi particles were much higher in human primary macro-

phages than in differentiated THP-1 cells. These resultsconcur with earlier observations indicating that Ti particlesmore efficiently stimulate the levels of IL-1 in mouseprimary macrophages than in murine cell lines P388D1 orIC-21 [46]. Interestingly, treatment of human primarymacrophages with Ti particles released much higher levelsof TNF-a, IL-6 and IL-1b than rutile, which only margin-ally stimulated the levels of these cytokines.In summary, the data in this work indicate that,

compared with Ti, rutile particles induce a lower responsein vitro, defined as their ability to induce TNF-a, IL-6 andIL-1b secretion in macrophages cultures of differentsources. Ti-based surfaces modified to create an outerlayer of rutile show improved corrosion [21] and wearresistance [22]. We provide now evidence that rutile debrisare less prone to initiate a inflammatory response than Tiparticles, accounting for the better biocompatibility of thiskind of surfaces.

5. Conclusions

In this study we have comparatively analysed theinflammatory potential of rutile and titanium particles ofphagocytosable size. Confocal microscopy experimentsrevealed that both types of particles were efficiently

ARTICLE IN PRESSG. Valles et al. / Biomaterials 27 (2006) 5199–52115210

internalised in cultures of TPA-differentiated THP-1 cellsas well as in primary cultures of human macrophages.Rutile particles stimulated the release of TNF-a, IL-6 andIL-1b to a lesser extent than titanium particles in thesecellular models. Compared to treatment with titaniumparticles, TNF-a and IL-1b were also released to a lesserextent in co-cultures of human osteoblasts and TPA-differentiated cells treated with rutile particles. Theseresults support the higher biocompatibility of titanium-based implants modified to create an outer layer of rutileon their surfaces.

Acknowledgements

This work was supported by grants from CICYTMAT2001/0019/CO2/O1-O2 from the Spanish Ministryof Science and Technology, FIS PI03/0036 from Fondo deInvestigaciones Sanitarias and Fundacion Mutua Madri-lena. NV is supported by Grant award FIS 01/3027 fromthe Fondo de Investigaciones Sanitarias. P G-M is fundedby the programme ‘‘Ramon y Cajal’’ of the SpanishMinistry of Education and Science. We thank MarıaAngeles Ollacarizqueta and Marıa Teresa Seisdedos, of theconfocal microscopy service of the Centro de Investiga-ciones Biologicas (CIB, CSIC) for excellent technicalsupport. The authors thank the medical staff of the Ortho-paedic Department and Haematology Service (HospitalLa Paz, Madrid, Spain) for providing us with bone andblood samples.

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