Interactions between human whole blood and modified TiO2-surfaces: influence of surface topography...

* Corresponding author. Tel.: #46-31-773-33-88; fax: #46-31-773-33-30.E-mail address: [email protected] (C. Eriksson).

Biomaterials 22 (2001) 1987}1996

Interactions between human whole blood and modi"ed TiO�-surfaces:

In#uence of surface topography and oxide thicknesson leukocyte adhesion and activation

Cecilia Eriksson��*, Jukka Lausmaa�, Ha� kan Nygren�

�Applied Cell Biology, Department of Anatomy and Cell Biology, University of Go( teborg, Box 420, 405 30 Go( teborg, Sweden�Department of Chemistry and Materials Technology, SP Swedish National Testing and Research Institute, Bora� s, Sweden

Received 2 May 2000; accepted 6 November 2000

Abstract

An in vitro model (Nygren et al., J Lab Clin Med 129 (1997) 35}46) was used to investigate interactions between leukocytes and fourmodi"ed TiO

�-surfaces. Surface topography was measured using scanning electron microscopy and optical pro"lometry while Auger

electron spectroscopy was used to determine surface composition and oxide thickness. The surfaces were either smooth or rough witheither thin or thick oxides. All surfaces consisted of TiO

�covered by a carbonaceous layer. The surfaces were incubated with capillary

blood for time periods of between 8 min and 32 h. Immuno#uorescence techniques together with computer aided image analysis andchemiluminescence technique were used to detect cell adhesion, expression of adhesion receptors and the zymosan-stimulatedrespiratory burst response. Leukocyte adhesion to the surfaces increased during the "rst hours of blood}material contact and thendecreased. Polymorphonuclear granulocytes were the dominating leukocytes on all surfaces followed by monocytes. Cells adhering torough surfaces had higher normalized expression of adhesive receptors than cells on smooth surfaces. Maximum respiratory burstresponse occurred earlier on the smooth than on the rough surfaces. In conclusion, topography had a greater impact than oxidethickness on most cellular reactions investigated, but the latter often had a dampening e!ect on the responses. � 2001 ElsevierScience Ltd. All rights reserved.

Keywords: Leukocytes; CD11b; Respiratory burst; TiO�-surfaces; Surface roughness; Surface composition

1. Introduction

Titanium implant systems have been exploited as boneanchored implants for many years [1], and while tita-nium is well tolerated by the body the reason for thesuccessful use of this metal is not clear. Bene"cial e!ectsdue to the oxide that spontaneously forms on titaniumhave been implicated [2]. The success is not due totitanium being passively incorporated into the body,since the body does recognize titanium as foreign andtitanium elicits an in#ammatory response. The response,however, resembles normal wound healing regarding cellrecruitment and persistence [3,4]. Attempts have beenmade to improve integration of titanium into bone by

varying parameters like surface roughness and oxidethickness of the implant [5,6]. Studies made have gener-ally investigated healing of titanium implants in terms ofweeks and months. Less is known about events takingplace between the implant surface and the biologicalenvironment in terms of hours, when the conditions areset out for healing or rejection.

During the surgical procedure of implantation thebiomaterial most likely will encounter blood. Almostinstantly following contact with blood the implant sur-face will be covered with plasma proteins that becomeadsorbed to the surface. Titanium in contact with serumor plasma is known to adsorb high molecular weightkininogen (HMWK), Factor XII, "brinogen, IgG,prekallikrein and C1q [7,8]. After 5 s platelets are foundon titanium surfaces exposed to whole blood whileit takes around 10 min before polymorphonuclear(PMN) granulocytes adhere [9]. Protein-platelet [10],leukocyte-platelet [11], and protein-leukocyte [12] are

0142-9612/01/$ - see front matter � 2001 Elsevier Science Ltd. All rights reserved.PII: S 0 1 4 2 - 9 6 1 2 ( 0 0 ) 0 0 3 8 2 - 3

possible interactions at the surface. Furthermore, releaseof in#ammatory mediators at the surface may in#uenceboth recruitment and activation of cells [10].

The PMN granulocytes are, as already mentioned, the"rst leukocytes to be recruited to a titanium surfaceexposed to blood [9]. Adhesion and activation are twoprocesses PMN granulocytes may go through upon ma-terial contact. Adhesion involves receptor occupationwith appropriate surface ligands [12,13] and when PMNgranulocytes are challenged with di!erent stimuli theyactivate their NADPH oxidase. By using oxygen theNADPH oxidase produce reactive oxygen species (ROS)that are used in the cells defense against microorganisms[10,14].

In a previous study [9] we have shown that dependingon surface preparation signi"cant di!erences in proteinadsorption and cellular reactions occur at titanium surfa-ces exposed to human blood. The TiO

�-surfaces investi-

gated had either a smooth surface with thick oxide ora rough surface with thin oxide. It was not possible todistinguish whether the observed di!erences in biologicalresponse were due to di!erences in surface topography oroxide thickness. We have therefore added two types oftitanium surfaces in this study: (i) smooth surface withthin oxide, and (ii) rough surface with thick oxide.

Using a previously developed in vitro model [9] wehave exposed titanium surfaces to blood. The focus hasbeen on interactions between human leukocytes and thefour titanium surfaces with special emphasis on PMNgranulocytes, since they were shown to be the domina-ting leukocytes on all the TiO

�-surfaces and are the "rst

blood cells recruited to in#ammatory sites in vivo. Wefound that expression of receptors (CD11b, CD16 andCD29), production of ROS and distribution ofleukocytes (monocytes, PMN granulocytes and lympho-cytes) di!ered between the four TiO

�-surfaces. In general,

the rough surfaces elicited a stronger biological responsethan the smooth and the surfaces with thick oxides hada dampening e!ect on most of the cellular reactionsinvestigated. To conclude, the adhering leukocytes weresusceptible to both changes in topography and composi-tion of the TiO

�-surfaces.

2. Materials and methods

2.1. Surface modixcation of titanium

Commercially pure (99.6 at%) polished (P) titaniumsheets were cut in squares of 64 or 16 mm�. The 64 mm�

pieces were used in immuno#uorescence experimentswhile the 16 mm� pieces were used when measuringchemiluminescence. All pieces were cleaned by boiling for5 min in a solution of 5 parts H

�O, 1 part H

�O

�and

1 part NH�OH followed by rinsing in distilled water. To

grow a thick oxide on titanium, the metal was annealed

(A) over an open #ame at approximately 7003C. Etching(E) in 10% HF for 3 min resulted in a rough (facetted)titanium surface. To grow a novel thin oxide on therough surface titanium was immersed in concentratedHNO

�for 20 min. After the surface treatments all pieces

were rinsed and stored in distilled water. The followingsurfaces were used: P"smooth surface with thin TiO

�;

P/A"smooth surface with thick TiO�; E"rough sur-

face with thin TiO�; E/A"rough surface with thick

TiO�. The wettability of the surfaces after modi"cations

was estimated by placing 10 �l water on the surfaces andmeasuring the spreading of the liquid. The water spreadat least 7 mm (1 mm edge e!ect) and the surfaces wereconsidered hydrophilic.

2.2. Surface characterization

The surface topography of one sample from eachgroup was analyzed by scanning electron microscopy(SEM) (JEOL JSM-5800) and optical pro"lometry (Topscan 3D). Four SEM measurements micrographs weretaken at di!erent magni"cation in the secondary electronmode with a beam voltage of 20 keV. Pro"lometry datawas acquired from areas of 245�245�m at a resolutionof 1 �m. Surface roughness (R

��), mean lateral distance

between surface asperities (S��

) and surface enlargement(S

��) were measured.

One sample from each group was analyzed by Augerelectron spectroscopy (AES) in a scanning Auger micro-probe (SAM) (Physical Electronics, PHI 660, EdenPrairie, MN). Survey spectra (30}1630 eV) were acquiredfrom two points of 200 �m diameter on each sample inorder to detect the elements present in the outermost fewnanometers of the surface. The oxide thickness was mea-sured by depth pro"ling, using 1.5 keV Ar� ions foretching at a rate of 3.8 nm/min (as calibrated for TiO

�).

The thickness of the oxide was de"ned as the depth atwhich the oxygen signal had decreased to half its max-imum value. A primary electron beam energy of 5.0 keVand a primary beam current of around 200 nA were usedin all AES analyses.

Relative concentrations of the detected elements wereobtained by taking the percentages of their peak-to-peakheights in di!erentiated survey spectra, after correctionby atomic sensitivity factors [15]. This quanti"cationprocedure does not take into account depth distributionsor chemically induced variations in the atomic sensitivityfactors, so the stated concentrations should not be seenas absolute concentrations, but are instead given for thepurpose of comparing the composition of the di!erentsamples.

2.3. Exposing titanium to whole blood

Pieces of titanium were exposed to human capil-lary blood, obtained from healthy individuals not on

1988 C. Eriksson et al. / Biomaterials 22 (2001) 1987}1996

medication. No anticoagulant agents were used so bloodcoagulated on the surfaces. The surfaces were exposed toblood in a humid environment at 373C for times rangingbetween 8 min and 32 h. After blood exposure the clotwas removed and the surface gently rinsed in Dulbeccos'phosphate bu!ered saline (DPBS), pH 7.3, or Hanks'balanced salt solution (HBSS), pH 7.4.

2.4. Fluorescent cell staining techniques

2.4.1. Staining with acridine orangePieces of titanium were incubated with blood between

8 and 64 min. The pieces were rinsed in 0.06 M phosphatebu!er, pH 6.0, and stained with 0.01% acridine orangefor 3 min at room temperature. Acridine orange was usedto detect nucleated cells. After rinsing the pieces wereincubated with 1% CaCl

�for 30 s followed by a "nal

rinse and mounting in the phosphate bu!er on a glassslide.

2.4.2. Viability stainingAfter incubation with blood from 30 min to 32 h the

adhering cells were stained for viability according toJones and Senft [16]. In short, the surface was stained for3 min in room temperature with a stain containing #uor-escein diacetate (FDA), 18 �g/ml, and propidium iodide(PI), 5.4 �g/ml. The titanium pieces were then rinsed inDPBS and dried.

2.4.3. ImmunocytochemistryPieces of titanium were incubated with blood from

8 min to 24 h and washed in DPBS. The pieces wereplaced on a cooling plate (Histolab, Sweden) at 43C.Speci"c antibodies were used to detect cell markers.Mouse antihuman CDllb, CD29, CD66b, CD99R andCD14 antibodies were from Serotec Ltd, England.FITC-conjugated rabbit antimouse immunoglobulinsand mouse antihuman CD16 were from DAKO A/S,Denmark. The pieces were stained with primary antibod-ies for 20 min, rinsed in DPBS and stained with FITC-conjugated antibodies for another 20 min. After rinsingthe pieces were placed on a glass slide and 1,4-dia-zabicyclo [2,2,2] octane was used as mounting mediumto prevent #uorescence fading.

2.5. Opsonization of zymosan and chemiluminiscencedetection of ROS

Zymosan (Sigma Chemical Co., St Louis, MO) wasmixed with human serum and incubated for 30 min at373C. The zymosan was then spun for 2�30 min andwashed with HBSS. After the last centrifugation thezymosan was dissolved in HBSS to a "nal concentrationof 12 mg/ml and stored at !203C until use.

Pieces of titanium were exposed to capillary blood for0.5, 1, 2, 4 and 24 h. After rinsing in HBSS, the pieces wereplaced in a 96 well microtiter plate. 100 �l 1 mM luminol(5-amino-2,3-dihydro-1,4-phthalazinedione; Sigma Che-mical Co., St Louis, MO) was added to all wells followedby 100 �l of opsonized zymosan (12 mg/ml). Productionof ROS was measured for approximately 42 min in aVictor Multilabel Counter luminometer (Wallac,Sweden) and detected as counts per second (CPS).

2.6. Photography and computer aided image analysis

All #uorescent specimens were examined and photo-graphed, using Elite chrome 400 "lms, in a #uorescencemicroscope (Zeiss, Germany), where exposure time andmagni"cation were kept constant. After development pic-tures were scanned into and analyzed in a computer usingAdobe Photoshop 3.0. The area and resolution scannedwere always constant. The #uorescent surface coverageabove background was used to quantify amount of posi-tive staining cells on the surface. When measuring surfacecoverage the di!erence in surface area between the roughand smooth surfaces was not accounted for.

2.7. Statistical evaluation

Experiments were repeated at least 3 times. Studentst-test was used to determine the statistical signi"cance ofdi!erences between two sets of data. Signi"cance wasde"ned at p(0.05.

3. Results

3.1. Surface topography

Fig. 1 shows SEM images of the four di!erent surfaces.The surfaces of the two polished samples (P and P/A) hada very similar appearance (Fig. 1a and b). They wererelatively smooth, with polishing grooves and ridges inthe micron range as the main features and some pits ofsize 1}10 �m. The surface topography measurements re-sulted in very similar values for both polished surfaces.The surface topography of the two etched samples (E andE/A) were very similar to each other but quite di!erentfrom the two mechanically polished ones (Fig. 1c and d).The etched surfaces had a rough appearance and thegrain structure of the material was clearly visible.

Optical pro"lometry con"rmed the di!erences be-tween the polished and etched surfaces observed withSEM. R

��was 0.45�m for the polished and 1.90 �m for

the etched surfaces. The polished surfaces had a S��

valueof 10.7 �m and a S

��value of 1.17, while for the etched

surfaces corresponding values were 14.0 �m and 1.79,respectively.

C. Eriksson et al. / Biomaterials 22 (2001) 1987}1996 1989

Fig. 1. SEM images of the modi"ed TiO�-surfaces P (a), P/A (b), E (c) and E/A (d).

Table 1Relative concentrations (in at%) of elements detected in AES surveyspectra

Sample(point)

Ti O C Ca Cl Fe Si P Sn� S Dioxide(nm)�

P (1) 14.4 57.3 23.8 0.8 0.2 3.5 * * * * 9P (2) 15.4 53.8 25.4 0.8 0.8 3.8 * * * * 10P/A (1) 8.7 37.1 47.4 2.5 0.5 2.6 0.5 0.3 * 0.4 38P/A (2) 8.4 34.9 52.8 0.4 0.5 1.8 * 0.7 * 0.6 34E (1) 12.1 44.1 37.9 * * * 3.7 2.2 * * 5E (2) 13.8 46.6 38.8 * * * 0.3 0.4 * 0.1 5E/A (1) 13.5 47.2 36.7 * * * * 0.2 2.4 * 27E/A (2) 15.0 48.6 33.7 0.5 * * * * 2.3 * 30

�Uncertain values due to overlap with TiLMM signal.�Oxide thickness measured by depth pro"ling.

3.2. Surface composition and oxide thickness

Table 1 shows the relative concentrations of the detec-ted elements and the measured oxide thickness for the

four analyzed samples. All spectra taken from the surfa-ces were dominated by Ti, O and C signals. Upon ionetching to a depth of 1}2 nm, most of the C signalsdisappeared, while those of Ti and O increased. Afterfurther ion etching, also the O signals disappeared atdi!erent depths depending on sample preparation. Theseobservations were consistent with surfaces consisting ofa Ti surface oxide covered by an adsorbed layer ofcarbonaceous molecules [17].

The P samples had the lowest carbon levels (24}25%),and the P/A samples the highest (47}53%). The twoetched samples, E and E/A, had similar and intermediatecarbon levels (34}39%). The variations in C levels in-dicated di!erent coverage of adsorbed carbonaceousmolecules, which in turn in#uenced the measured relativeconcentrations of Ti and O. In addition to these ele-ments, smaller amounts (a few % or less) of Ca, Cl, Fe, Si,P, Sn and S were occasionally detected. The Ca, Fe andCl signals were almost exclusively observed on the twopolished samples, while Si, P and Sn appeared mainly on


Fig. 2. (a}c) Fluorescent surface coverage of (a) anti-CD14 (monocytes), (b) anti-CD66b (granulocytes) and (c) anti-CD99R (lymphocytes) on thetitanium surfaces P, P/A, E and E/A during 24 h of surface exposure to blood. Mean surface coverage and standard error of mean (SE) are shown in thegraphs.

the etched samples. The oxides of the two non-annealedsamples, P and E, were 5}10 nm thick, while the twoannealed samples, P/A and E/A, had considerablythicker oxides, 27}38 nm.

3.3. Viability of adherent cells

The viability of adherent cells was investigated usingFDA in combination with PI. PI stains the cell nucleusand was used to detect cells with leaky membranes. FDAdetects an intact intracellular pool of esterases found inviable cells. Cell viability was investigated after surfaceexposure from 30 min up to 32 h. Injured or dead cellswere absent on all surfaces at all times investigated.Surface coverage of FDI/PI stained cells increased on allsurfaces during the "rst hours of blood}material interac-tion. This was followed by a slow decrease in surfacecoverage with increased blood}surface contact time (datanot shown).

3.4. Adhesion of leukocytes on the titanium surfaces

The four surfaces were stained with speci"c FITC-conjugated antibodies against monocytes (CD14),granulocytes (CD66b) and lymphocytes (CD99R). Sur-face coverage of #uorescence was measured after 0.5, 1, 2,4 and 24 h of blood}surface interaction (Fig. 2a}c).

3.4.1. MonocytesMonocytes were present on all four surfaces during the

observed time (Fig. 2a). During the "rst 4 h in contactwith blood the surfaces with thick oxides had clearly lesssurface coverage of cells (0.01}0.06%) than the surfaceswith thin oxides (P: 0.2}0.4%; E: 0.3}0.8%). On P, P/Aand E/A surface coverage of monocytes was rather con-stant during the "rst 4 h while on E coverage increasedwith time. The surfaces with thin oxides were di!erent inthe respect that the rough surface always had higher cellcoverage than the smooth one. At 24 h, however, the tworough surfaces had higher surface coverage than thesmooth ones and the smooth surface with thick oxidehad strikingly low surface coverage compared to theother three surfaces.

3.4.2. PMN granulocytesPMN granulocytes were the dominant leukocytes on

all four surfaces (Fig. 2b). On P and E surface coverage ofcells increased during the "rst hour and then decreased.P/A and E/A had a much more homogeneous distribu-tion of granulocytes during the "rst 4 h of blood contact.The two rough surfaces had higher surface coverage ofcells, 18}26%, than the smooth surfaces, 9}13%, duringthe "rst 2 h. On all surfaces only low surface coverage,0.8}1.5%, of cells was detected after 24 h of blood en-counter.


Table 2Distribution (%) of monocytes, granulocytes and lymphocytes on thetitanium surfaces P, E, P/A and E/A during 24 h of blood}surfaceinteraction

Cell type Surface 0.5 h 1 h 2 h 4 h 24 h

Monocyte P 2 2 4 5 31E 2 2 3 4 32P/A * * * * 5E/A * * * * 32

Granulocyte P 98 98 96 95 54E 98 98 97 96 55P/A 100 100 100 100 82E/A 100 100 100 100 56

Lymphocyte P * * * * 15E * * * * 13P/A * * * * 13E/A * * * * 12

Fig. 3. Normalized surface coverage of anti-CD11b stained leukocyteson the surfaces P, P/A, E and E/A during 64 min of surface exposure toblood. Mean and SE are shown in the graph.

3.4.3. LymphocytesCompared to the other leukocytes lymphocytes were

present in only minute amounts, less than 0.1%, on allsurfaces during the "rst 4 h investigated (Fig. 2c). After24 h, however, lymphocytes were detected on all surfacesin still small but measurable amounts. At this time thetwo rough surfaces had a surface coverage of around0.35%. Less surface coverage was found on the twosmooth surfaces where P, 0.2$0.01%, had signi"cantlyhigher coverage of cells than P/A, 0.15$0.01%.

3.5. Distribution of leukocytes on the surfaces

Table 2 shows how many percentages of the totalleukocyte population each cell type represented on eachsurface at di!erent incubation times. The dominance ofPMN granulocytes, 95}99%, on all surfaces during the"rst 4 h of blood}surface interaction was clear. On P andE monocytes contributed to the total population ofleukocytes during this time with 2}5% while on P/A andE/A the monocyte contribution was non-existent. Thelymphocyte population was negligible on all four surfa-ces during the "rst 4 h. After 24 h the distribution of cellsdi!ered from earlier time points. The distribution wasvery similar on P, E and E/A with around 30% mono-cytes, 55% granulocytes and 15% lymphocytes. P/A,however, di!ered from the other surfaces in the respectthat while the lymphocyte distribution was similar tothat seen on the other surfaces, 15%, less than 5% of thecells were monocytes and as high as 80% granulocytes.The surfaces with thin oxides showed a slight increase inmonocyte population at the expense of a slight decreasein granulocyte population. The thick oxide surfaces hada more pure granulocyte population during the "rst 4 hof blood encounter, and in the case of P/A even after 24 h.

3.6. CD11b expression of PMN granulocytes during thexrst hour

Expression of CD11b (�2 integrin subunit) on the cellsurface of leukocytes was detected with speci"c antibod-ies during the "rst hour of blood}surface interaction,and normalized to surface coverage of acridine orange(Fig. 3). On all surfaces a transient expression of CD11bwas found, where maximum expression of CD11b per celloccurred after approximately 0.5 h of blood contact.PMN granulocytes on the two smooth surfaces had verysimilar CD11b expression during the whole time mea-sured, with maximum normalized CD11b values ofaround 0.3. The PMN granulocytes on the two roughsurfaces had signi"cantly higher expression of CD11bthan cells on the smooth surfaces. At 0.5 h normalizedvalues of CD11b on E and E/A were 1.6 and 0.6, respec-tively. The surface structure and oxide thickness bothin#uenced the expression of CD11b on the rough surfa-ces, while only the surface structure appeared to in#uencethe CD11b expression on the two polished surfaces.

3.7. Spontaneous and zymosan stimulated respiratory burstof PMN granulocytes

Both spontaneous and induced respiratory burst ofadhering PMN granulocytes was measured after 0.5, 1, 2,4 and 24 h of incubation with blood on the titaniumsurfaces. ROS production was measured as chemilumin-escence normalized to surface coverage of FDA/PIstained cells and expressed as CPS per % surface cover-age (s.c.).

The spontaneous ROS production by the adheringcells did not change signi"cantly from background levelsregardless of blood}surface incubation time and TiO

�-

surface (data not shown).Opsonized zymosan was added to induce a respiratory

burst of the PMN granulocytes on titanium (Fig. 4a}d).After 0.5 h of blood exposure ROS was produced without


Fig. 4. (a}d) Normalized production of ROS expressed as CPS/% s.c. on the four titanium surfaces P (a), P/A (b), E (c) and E/A (d) during 4 hours ofblood}material interaction. Mean values are shown in the graphs (n"6).

Table 3Zymosan stimulated ROS production during 4 h of blood surface inter-actions. Normalized values, expressed as CPS/% s.c., after 39 min ofchemiluminescence measuring are shown together with SE

Surface 0.5 h 1 h 2 h 4 h

P 218$18 119$12 85$4 84$8P/A 102$10 81$9 61$4 44$5E 89$2 88$4 104$6 239$30E/A 97$6 73$5 45$4 168$10

delay. At longer incubation times an induction of ROSwas needed and the induction time increased with in-creasing time of blood exposure. A general trend seen onall surfaces was that approximately 25 min after addedstimuli the ROS production reached its maximum andremained constant during the rest of the assay. No respir-atory burst was detected on the surfaces after 24 h ofblood contact (data not shown).

In order to compare ROS production between surfacesand di!erent exposure times a speci"c time point, at39 min, during the assay was chosen (Table 3). On P andP/A a trend towards decreasing ROS production withincreasing exposure time was observed. E and E/A elici-ted a quite di!erent response. On these surfaces thehighest production of ROS was detected after 4 h of

blood}surface interaction. After 0.5 and 1 h cells on P pro-duced signi"cantly more ROS than cells on the othersurfaces. No signi"cant di!erence in production was ob-served between the other three surfaces. At 4 h granulo-cytes on the two rough surfaces produced more ROS thangranulocytes on the smooth surfaces. Cells on E producedsigni"cantly more ROS than cells on E/A and cells onP produced signi"cantly more ROS than cells on P/A.

3.8. Expression of adhesion receptors on the surfacesafter 4 h

After 4 h of blood exposure adherent cells were stainedwith speci"c antibodies against CD11b (�2 integrin),CD16 (Fc�III receptor) and CD29 (�1 integrin). Cover-age of antibodies was normalized to FDA/PI coverage(Fig. 5). The reason for using these antibodies at this timepoint was to see whether the receptor expression di!eredbetween the surfaces and whether possible di!erencescould explain the results obtained with opsonizedzymosan after 4 h. Leukocytes on E, 0.3$0.03, and E/A,0.2$0.02, had the highest normalized surface coverageof CD11b while coverage on P and P/A was around 0.05.Using CD16 a similar trend was found between thesurfaces with the exception that signi"cantly lessCD16/cell was found on P/A compared to on P. Approx-imately, 2 times higher values of CD11b than of CD16


Fig. 5. Normalized #uorescent surface coverage of anti-CD11b, anti-CD16 and anti-CD29 after 4 h of blood}titanium interaction on thesurfaces P, P/A, E and E/A. Mean and SE are shown in the graph.

were found on the surfaces. CD29 was detected on allsurfaces. The rough surfaces had the signi"cant highestnormalized coverage, 0.05}0.07, while on P/A only min-ute amounts were detected.

4. Discussion

All four surfaces consisted of a titanium oxide coveredby a carbon-dominated overlayer and minor amounts ofother impurities. Based on our previous X-ray photo-emission spectroscopy (XPS) analysis on similarly pre-pared titanium samples [9] it can be assumed that thesurface oxide was TiO

�. The four surfaces, however,

showed major di!erences in surface topography and ox-ide thickness. The two polished surfaces were relativelysmooth and featureless on the scale of cellular dimension(10 �m) in contrast to the two etched surfaces. The ap-pearance of the latter indicates that the etching processremoved the amorphous surface layer, which normallyresults from mechanical machining or polishing. Thefacetted surface structure is most likely a result of prefer-ential etching, exposing predominantly low-index crystalfaces.

The oxide thickness for the two non-annealed sampleswere in the range 5}10 nm, which was similar to the oxidethickness found on clinically prepared titanium materials[17]. The two annealed samples had considerably thickeroxide layers. The di!erences in oxide thickness betweenthe latter two samples, despite similar oxidation temper-atures and times used, most likely re#ects di!erences inoxidation rate due to the di!erent surface pre-treatments.Apparently, the thicker oxide layer of the polished(amorphous) surfaces indicates that they have a loweroxidation rate than the etched (crystalline) ones. Inaddition to di!erences in oxide thickness and surfaceroughness the samples also showed less systematic dif-ferences with respect to the elements found in the surfacecontamination layer. Most of the observed impurities,

except Fe and Sn, have frequently been detected in pre-vious analyses of titanium surfaces used in biomaterialsresearch [17].

The normalized expression of CD11b was transient onall surfaces during the "rst hour of blood}metal contact,with a peak in expression after 0.5 h. This peak coincidedwith rapid onset of ROS production following stimula-tion with opsonized zymosan. Up-regulation ofCD11b/CD18 is seen after PMN granulocyte stimula-tion, and priming of cells has been shown to reducethe lag phase before onset of the respiratory burst[18]. Furthermore, the phenomenon of transient primingof PMN granulocytes has been reported by others[19,20].

The higher normalized expression of CD11b on therough than on the smooth surfaces found during the "rsthour might also re#ect adhesive events since it has beenshown that the CD11b/CD18 receptor is responsible for"rm adhesion of PMN granulocytes to surfaces withimmune complex deposition [21]. The rough surfacescould then have cells attached with stronger adhesiveforces, which might explain the higher surface coverageof PMN granulocytes seen on the rough surfaces com-pared to the smooth during this time, even after takingthe larger surface area (52%) into consideration.

Our results showed that no clear correlation existedbetween multitude of CD11b/CD18 receptors on the cellsurface and magnitude of ROS produced. After 0.5 h ofblood}titanium contact, for instance, PMN granulocyteson P produced more ROS than cells on E and E/A, whilethe opposite was true for the CD11b expression. Follow-ing stimulation CD11b/CD18 is rapidly mobilized fromintracellular stores to the cell membrane. Although anincrease in receptor expression of CD11b/CD18 can re-sult in increased adhesion and priming of cells not allnewly recruited receptors are believed to be functional[22]. Instead further modi"cations are needed in order torender the receptors functionally competent and sub-sequently the expression of CD11b/CD18 cannot fullypredict degree of cell activation [23,24]. This "nding mayexplain the results obtained by us.

We found that PMN granulocytes on titanium did notproduce ROS spontaneously but were able to elicit a re-sponse when exposed to opsonized zymosan. Cells adher-ent to titanium thus retained their ability to respond tomicroorganisms when challenged but were not active inabsence of stimuli. Others have shown in vitro that PMNgranulocytes adhering to coatings of the extra cellularmatrix (ECM) protein "bronectin or to polystyrene andpolyurethane in the presence of serum do not produceROS unless exposed to stimuli [25,26]. It has been sug-gested that this behavior might be necessary in vivo inorder to protect tissues from ROS during leukocytemigration through ECM [27]. Maybe the proper com-position of proteins at a surface renders the in#am-matory cells non-reactive towards the surface but still


functionally responsive. The reason for lack of ROSproduction in this study could also derive from the pres-ence of erythrocytes. Erythrocytes are known to have theability to scavenge ROS [28] and chemokines [29] andalso to bind immune complex through the CR1 receptor[30]. A consequence of this would be a less stimulatingenvironment for the cells.

CD29 positive cells were found after 4 h of incubationwith blood. Both PMN granulocytes and monocytesexpress CD29 [31,32]. Normally, PMN granulocyteshave a very low expression of CD29, but it can beinduced under certain conditions like during extra-vascular migration and when cells adhere to "bronectinunder static conditions [33,34]. The presence ofCD29 might indicate that ECM proteins like laminin or"bronectin were present on the surfaces, since theyare ligands for CD29 [31]. These ECM proteins havenot been reported to adsorb initially on titaniumexposed to serum or plasma [8], so the surface com-position of proteins may have been subjected to alter-ations with time, exposing new ligands for adheringleukocytes.

We found that adhesion of monocytes was sensitive tochanges in oxide thickness while PMN granulocytes re-sponded more to surface roughness. Others have shownpreferences of certain cell types for certain surface prop-erties and also that surface roughness in#uence the stateof cell maturation [35}38]. Macrophages, for instance,have been shown to prefer rough-to-smooth surfaces,which is in agreement with the results obtained hereregarding PMN granulocytes and lymphocytes and tosome degree monocytes [39]. Looking at ROS produc-tion and receptor expression of cells there were di!er-ences between rough and smooth surfaces, but the thickoxides also in#uenced the responses by having a passivat-ing or dampening e!ect.

The overall picture that emerges from the presentstudy is therefore that the leukocyte reactions investi-gated were susceptible to both changes in topographyand oxide thickness. It cannot, however, be excludedthat surface properties other than those of primaryinterest here also play a decisive role in cell responsesto the surfaces. It would, for example, be of interestto systematically investigate the role of surface con-tamination (species and concentrations) or the micro-structure of the oxides (crystal structure andmorphology), which both can be expected to be ofimportance.

Acknowledgements

This study was supported by grants from the SwedishResearch Council of Engineering Sciences. We thankAnn Wennerberg for performing the optical pro"lometryanalysis.

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