Acta Biomaterialia 7 (2011) 2337–2344

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Acta Biomaterialia

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Analysis on migration and activation of live macrophages on transparentflat and nanostructured titanium

Soyoung Lee a,1, Jungil Choi b,1, Sangwon Shin c, Yeon-Min Im d, Jonghan Song c, Sang Soo Kang b,Tae-Hyun Nam d, Thomas J. Webster e, Sang-Hyun Kim a,⇑, Dongwoo Khang d,⇑a Department of Pharmacology, Kyungpook National University Medical School, Daegu, 700-422, South Koreab Department of Anatomy, Institute of Health Science and School of Medicine, Gyeongsang National University, Jinju, 660-701, South Koreac Nanomaterials Analysis Center, Korea Institute of Science and Technology, Seoul, 136-791, South Koread Center for NMBE and School of Nano and Advanced Materials Science and Engineering, Gyeongsang National University, Jinju, 660-701, South Koreae School of Engineering and Department of Orthopaedics, Brown University, Providence, RI 02912, USA

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

Article history:Received 14 September 2010Received in revised form 24 December 2010Accepted 6 January 2011Available online 11 January 2011

Keywords:ImmunotoxicityImplantMacrophageTitaniumLive cell migration

1742-7061/$ - see front matter � 2011 Acta Materialdoi:10.1016/j.actbio.2011.01.006

⇑ Corresponding authors. Fax: +82 53 420 4838(D. Khang).

E-mail addresses: shkim72@knu.ac.kr (S. Kim), dkh1 These authors contributed equally.

The immunotoxicity of implanted nanostructured titanium is a paramount issue for vascular, dental andorthopedic applications. However, it has been unclear whether implanted surface nanostructures caninhibit or aggrevate inflammatory responses. Herein, macrophage activation, as evidence of migration,on transparent flat and nanostructured titanium correlated with pro-inflammatory protein synthesisand cytokine release. Through the real-time monitoring of initial cytoskeleton variations, this study iden-tified that macrophage movement was restricted on nanostructured titanium compared to flat titaniumsurfaces. Furthermore, nanostructured titanium elicited secretion of fewer pro-inflammatory enzymemolecules and cytokines, as well as reduced nitric oxide production. All results collectively indicated thatinitial macrophage activation can be mitigated by nanoscale surface topography alone, without modifi-cation of surface chemistry or stiffness.

� 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction

Nanotechnology has engendered the development of noveltreatments and approaches to medicine [1]. Its applications have,in particular, facilitated the improvement of biomedical implants[2]. Studies conducted within the past decade have observed thatthe nanoscale surface treatment of biomaterials plays a critical rolein accelerating tissue formation in vitro and in vivo [3–6]. Rapidtissue growth at the implant site, however, requires moderateimmunological activity [7]. Few studies have elucidated the immu-notoxic effects of modulating immune cells on nanostructured tita-nium, though the majority of studies have focused on tissue cellresponses on nanofeatured titanium surfaces [8–11]. In this light,understanding the interactions of immune cells on nanomaterialsis critical to the design of the next generation of implant coatings.

Macrophages are a type of immune cells that mediate inflam-mation by inducing a common wound healing response from thebody to the implant [7]. Macrophage migration, or its movementat the implant site, is an integrated mechano-molecular process

ia Inc. Published by Elsevier Ltd. A

(S. Kim), +82 55 759 1745

ang@gnu.ac.kr (D. Khang).

that involves dynamic, coordinated changes in cell adhesion andcytoskeletal organization, and signal transduction often triggersan immunological response [4,12–14]. During migration, cells con-stantly break and form new integrin contacts. Depending on thecomposition of the extracellular matrix (ECM), integrins may acti-vate one or more intracellular signaling pathways. These pathwaystypically involve phosphorylation of focal adhesion kinase (FAK),recruitment of adaptor proteins, activation of small GTPase andsubsequent activation of downstream effecter molecules [15].FAK is a cytoplasmic protein kinase that co-localizes with integrinsto form cell focal adhesion sites, and its activation has been shownto promote cell survival and migration [16]. Cells respond to thesesignals through the clustering of integrin receptors and recruit-ment of focal adhesions cytoskeleton proteins by regulating cyto-skeletal tension [17,18]. These signals, in concert with signalsderived from growth factors, regulate cell behavior in the complexmicroenvironment of tissues [19].

Murine macrophages J774A.1 was selected for this study be-cause they do not require the combination of interferon (IFN)-rwith tumor necrosis factor (TNF)-a/interleukin (IL)-4/etc. toproduce NO, though Raw264.7 do require such combinations toproduce NO. Ideally, human primary macrophages or the humanmacrophage cell line would be an ideal cell to examine the roleof nanotitanium on the migration and activation of macrophages.

ll rights reserved.

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However, there are two realistic problems: (i) so far there is no hu-man macrophage cell line, only monocyte/macrophage-like cells(e.g. THP-1); and (ii) human primary macrophages are not strongenough for transfection/immunostaining/long-term culture.

Cell migration can be altered by the chemical and physicalproperties of the biomaterial substrate, such as its surface rough-ness and wettability [20,21]. Previous studies from other groupshave shown that nanocomposites induced activation of FAK andactin stress fibers in endothelial cells [20]. Furthermore, nanoscaletopography was shown to regulate integrin-mediated focal adhe-sion by increasing levels of actin stress fibers and phosphorylatedFAK [22]. In addition, physical stimulants, such as ECM modifica-tion and nanoscale surface features, may regulate macrophage pro-duction of nitric oxide synthase (iNOS), a catalyst of NOproduction, in a manner similar to chemical stimulants (e.g. lipo-polysaccharide (LPS), chemokines and cytokines). NO, in turn,influences the tissue microenvironment, vascular regulation, andthe immune system [23–25]. Moreover, examining macrophageactivation on nanomaterials could ameliorate chronic and acuteinflammatory diseases by providing a means of assessing immuneresponses (e.g. cytokine expression of TNF-a and IL-1b) to nanom-aterials [22,26]. Importantly, most of the suggested immune re-sponses to an implanted biomaterial primarily develop within24 h after implantation, thus direct monitoring of macrophagebehavior on nanometer-scale surface roughness (defined as nano-rough) and subnanometer-scale surface roughness (defined as flat)samples can provide critical insight into the initial immune cellinteractions on titanium nanomaterials.

Despite the importance of such studies, it has been difficult todirectly observe the dynamics of live immunological cell interac-tions on nanomaterial surfaces because most nanoscaled biomate-rials (such as ceramics, metal alloys and polymers) are opticallyopaque due to their thickness and are thus incompatible with livecell tracking analysis. Research has hitherto focused on macro-phage cytokine expression in biomaterials and direct monitoringof live macrophage behavior, such as cell morphology and migra-tion, which can provide more conclusive evidence of immune acti-vation by implanted nanomaterials. The transparent nanoroughtitanium thin films (35 nm height thickness) used in this study en-abled the monitoring of macrophage cytoskeleton morphology,migration speed and migration distance in real time. Importantly,the surface structures developed in this study elucidated the initialcytoskeleton dynamics and confirmed that nanoscaled topographyalone (i.e. without mechanical and chemical stimuli) can dictatemacrophage migration.

2. Materials and methods

2.1. Sample preparation

A 35 nm coating of pure titanium (99.8% of purity: T-2069, Cer-ac Inc.) was deposited on glass coverslips (12-546-1, Fisher-Scien-tific) using an e-beam evaporator (vacuum state of 10�8 torr) toproduce flat (defined as subnanometer dimension in height) andnanorough (defined as nanometer dimension in height) titaniumsurfaces. Both sample types were of the same thickness(35 ± 2 nm) to enable identical optical contrast during live macro-phage recording. The e-beam deposition rate and e-beam currentdensity were 2 Å s–1 and 60–70 mA cm–2 for the flat titanium sur-faces and 40 Å s–1 and 170 mA cm–2 for the nanorough titaniumsurfaces, respectively. The e-beam energy remained constant at7.9 keV throughout the experiment. Cover glass was used as a con-trol for all experiments and was prepared by cleaning with acetonethree times, rinsing with DI water and finally drying in a vacuumoven (at a temperature of 80 �C) overnight.

2.2. Surface characterizations

Rutherford backscattering spectroscopy (RBS/channeling) wasconducted using 2 MeV He and 3.05 MeV He oxygen resonancemodes to measure the surface chemistry and thickness of titaniumthin film. The surface roughness of both the flat and the nanoroughtitanium thin films was measured using a non-contact mode atom-ic force microscope (AFM; XE-100, Parks System). The dimension ofthe scan field of view was 3 lm � 3 lm for both sample types.Commercially available AFM tips (radius of curvature less than10 nm, PPP-NCHR, Parks System) were used in non-contact modeat a scan rate of 0.5 Hz, a tip height of 125 lm and a constant forceof 42 N m–1. Water surface contact angles were measured using adrop shape analysis system (Easy Drop Contact Angle System,Kruss, Germany) and corresponding software (DSA1, Kruss) underambient conditions. Distilled water (3 ll) was used as the contactsolvent. All data were obtained 5s after placing the droplet on thesurface.

2.3. Cell culture and viability (MTT)

The BALB/c macrophage cell line, J774A.1 (TIB-67, ATCC), wascultivated in Dulbecco’s modified Eagle’s medium supplementedwith 2 mM glutamine, 100 units ml–1 penicillin, 100 lg ml–1 strep-tomycin (1% antibiotics) and 10% non-heat-inactivated fetal bovineserum (16000-044, Gibco) under standard cell culture conditions(5% CO2 at 37 �C). Upon reaching confluence, cells were resus-pended, seeded at a density of 5 � 104 cells cm–2 onto each sampleand cultured for 24 h. Cell viability was determined using a 3(4,5-dimethylthiazolyl-2)2 and 5-diphenyl tetrazolium bromide assay(MTT; M-2128, Sigma). Specifically, MTT (10 mg ml–1) was addedto each well that contained a sample and incubated for 4 h. Isopro-panol (in 0.04 N-hydrochloric acid) was added to dissolve the for-mazan crystals. Absorbance was read at 570 nm using aspectrophotometer (Anthos 2010, Biochrom). Cell viability, definedas the relative absorbance of each sample compared to that of thecontrol, was calculated and expressed as a percentage.

2.4. Fluorescence imaging

Macrophages were seeded at 5 � 103 cells cm–2 onto samplesurfaces and incubated for 12 and 24 h to stain for actin and iNOS,respectively. The cells were then washed with phosphate-bufferedsaline (PBS), fixed with 3.7% paraformaldehyde for 15 min and per-meabilized with 0.5% Triton X-100 in PBS for 10 min prior to stain-ing. Next, cells were blocked using 3% bovine serum albumin for30 min. The cells were stained with either rhodamine phalloidin(50 lg ml–1, P-1951, Sigma) or iNOS antibody (1:200 dilution,651, Santa Cruz Biotech) for 40 min and washed three times withPBS. Coverslips were mounted on microscope slides and sealedwith a synthetic mount. Images were obtained using a fluorescenceconfocal microscope (LSM 5 exciter, Carl Zeiss) and fluorescencemicroscope (BX51, Olympus).

2.5. Live cell tracking analysis

Cell Observer (Carl Zeiss) was used to analyze real-time macro-phage migration on transparent flat and nanorough titanium thinfilms. For this experiment, macrophages were seeded at5 � 103 cells cm–2 onto the flat and nanorough titanium surfacesand incubated under standard cell culture conditions during theadhesion process. After 4 h, culture plates were transferred to theCell Observer and maintained at 37 �C in 5% CO2 and 95% air.Images were captured at 5 min intervals for a total duration of24 h. The recorded images were analyzed with additional software(Axio Vision Rel. 4.8, Carl Zeiss) to determine average cell

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migration distance and migration velocity for each sample type.Mean value is based on the averaged cell migration of selected tra-jectories (n = 60) on each sample (n = 3 for both flat and nanotita-nium surfaces). Activated cells, identified based upon theirpolarized cell morphology, were indicative of cell migration atthe surface. By the same token, cells embodying a round shapewere not activated and exhibited no movement on the surface.The ratio of activated macrophages, defined as the fraction of acti-vated cells to all detected cells counted in the images(800 lm � 800 lm; 800–1000 cells for each sample type (nano-rough and conventional)), was calculated after 5, 12 and 24 h.

2.6. NO assay

The amount of stable nitrite, which is the final NO product gener-ated by activated macrophages, was determined with a colorimetricassay after 48 h of cell seeding (5 � 104 cells cm–2). Briefly, 50 ll ofculture supernatant was mixed with an equal volume of Griess re-agent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochol-oride, 2.5% H3PO4) and incubated at room temperature for 10 min.The absorbance at 540 nm was read using a spectrophotometer

Fig. 1. Surface topography, chemistry, thickness and wettability of transparent flat andAFM scanning of (b) flat and (c) nanorough titanium thin films. (d) 2 MeV RBS measuraluminum foil. The red curve represents the fitting of the obtained spectrum for the calcuAFM line scan of the flat and nanorough titanium (b, c) exhibiting a different order of ve(nanorough).

(Anthos 2010). Nitrite concentration was determined by extrapola-tion from a sodium nitrite standard curve.

2.7. Western blot

Macrophages were seeded on sample surfaces at a cell densityof 1 � 105 cells cm–2 and incubated for the indicated times: 24 hfor iNOS and 4 h for FAK. Samples were then rinsed twice withice-cold PBS and total cell lysates were gathered in 200 ll of lysisbuffer (20 mM Tris–HCl, 120 mM NaCl, 50 mM HEPES, 1%Triton-X, 1 mM ethylenediaminetetraacetic acid, 2 mM sodiumorthovanadate, 1 mM dithiothreitol, 10% glycerol, 0.02 mM phen-ylmethylsulfonyl fluoride, 1 mg ml–1 leupeptin, 1 mg ml–1 aproti-nin). The lysates were spun in a microcentrifuge for 20 min at4 �C and the supernatant was collected. Proteins were electropho-resed using 8–12% sodium dodecyl sulfate (SDS)–polyacrylamidegel electrophoresis, and then transferred to nitrocellulose mem-branes. The membranes were stained with reversible Ponceau Sto ascertain equal loading of samples in the gel. iNOS was assayedusing anti-iNOS antibody (651, Santa Cruz Biotech). FAK activationwas determined using anti-phospho-FAK antibody (3281, Cell

nanorough titanium. (a) Optical transparency of glass, flat and nanorough titanium.ement for the 35 nm thick titanium layer (both flat and nanorough) deposited onlation of deposited thickness and surface chemistry of titanium using RUMP code. (e)rtical dimensions of subnanometer- (flat) and nanometer-scaled surface roughness

Fig. 2. Surface roughness, altered wettability and protein adsorption on flat andnanotitanium surfaces. (a) RMS values of nanoscale surface roughness for glass, flatand nanostructured titanium were 0.43 ± 0.02, 0.37 ± 0.005 and 4.76 ± 0.01 nm,respectively (mean ± SD). (b) Reduced contact angle on nanorough surface com-pared to flat titanium. Decrease in water contact angle due to nanoscaled surfaceroughness; wettability (and hydrophilicity) increased on the randomly distributednanostructured islands (height of 10 nm). (c) Serum protein (10% FBS in PBS)adsorption on glass, flat and nanorough titanium surfaces. Due to the increasedsurface wettability, protein adsorption amount on the nanorough titanium washigher than flat titanium. Significantly different at ⁄⁄p < 0.01 and ⁄⁄⁄p < 0.001,respectively; data analysis was based on mean ± SEM for n = 3.

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Signaling). Immunodetection was performed using an enhancedchemiluminescence detection kit (34080, Thermo scientific).

2.8. Real-time polymerase chain reaction (PCR)

Real-time PCR was used to analyze the expression of mRNA forvarious cytokines. Cells were cultured on sample surfaces(5 � 104 cells cm–2) for 12 h, after isolation of RNA from cells usinga RNAiso Plus solution (TakaRa Bio Inc., Japan). First-strand com-plementary DNA (cDNA) was synthesized using a Maxime RT-Pre-Mix Kit (iNtRON Biotechnology, Korea). The respective primerswere chosen by the Primer3 program (Whitehead Institute) andall primers were obtained from Genotech (Daejeon, Korea). PCRwas carried out with the following primers for: TNF-a (s 50-AAGCCT GTA GCC CAC GTC GTA-30; as 50-GGC ACC ACT AGT TGG TTGTCT TTG-30) and IL-1b (s 50-ATA ACC TGC TGG TGT GTG AC-30; as50-AGG TGC TGA TGT ACC AGT TG-30). D-actin (s 50-TAG ACT TCGAGC AGG AGA TG-30; as 50-TTG ATC TTC ATG GTG CTA GG-30)was used to verify that equal amounts of RNA were used foramplification. mRNA level was quantified using a real-time PCRtechnique supplied by Thermal Cycler Dice Real Time System(TP-800; TakaRa Bio Inc.). LPS served as a positive control. Productswere separated by electrophoresis on a 1.5% agarose gel andvisualized by staining with ethidium bromide. The gels werecertificated using a Kodak DC 290 digital camera (Eastman Kodak).

2.9. Protein assay

Serum proteins (10% FBS in PBS) were adsorbed on flat andnanostructured titanium surfaces and kept for4 h in a cell cultureincubator (to ensure identical environments for cell adhesion).After 4 h, the adsorbed proteins were completely removed with2% SDS (L3771, Sigma). The protein concentration was measuredwith a protein assay kit (Coomassie Plus 23236, Thermo). Theabsorbance at 595 nm was read using a spectrophotometer (AsysUVM 340, Biochrom). The protein concentration was determinedby extrapolation from a standard albumin linear slope and wasnormalized to glass as a final step.

2.10. Statistical analysis

The statistical differences amongst several sample types wereanalyzed by an analysis of variance followed by the Student–New-man–Keuls multiple comparison test. A Student’s t-test was usedto compare two groups. Significance was indicated at p values ofless than 0.05, 0.01 and 0.001. At least three samples were usedfor three independent experiments. All results are presented asmean ± SEM with n = 3.

3. Results and discussion

3.1. Surface properties and protein adsorption on nanorough and flattitanium thin films

Nanoscale surface topography was generated under opticallytransparent conditions (Fig. 1a). Total deposition thickness wasset to 35 nm for both the flat (defined as subnanometer dimensionin height) and nanorough (defined as nm dimension in height andwidth) titanium (Fig. 1b and c) for direct comparison during livecell recording. RBS verified that the pure titanium coating depos-ited onto the cover glass was indeed 35 nm thick (Fig. 1d). Oxideresonant signals from RBS analysis further showed that 3–4 nmof oxide thickness was deposited onto the titanium surfaces(Supplementary Fig. 1). This is, however, the same oxide thicknessthat coats bare aluminum foil in ambient air and, hence, it was

further confirmed that the 35 nm (in 10�8 torr) coating predomi-nantly comprised pure titanium. Altogether, these results estab-lished that the surface chemistry was identical for both the flatand nanorough titanium. The averaged nanoscale surface rough-ness (RMS) for glass, flat and nanorough titanium surfaces were0.43 (±0.02), 0.37 (±0.005) and 4.76 (±0.01) nm, respectively(Fig. 2a). Nanoscaled surface features reduced water contact angleson titanium from 80� (on flat) to 70� (on nanorough) (Fig. 2b). Inaddition, enhancing the surface roughness (i.e. from flat to nano-rough) increased the surface hydrophilicity [6]. Enhanced proteinadsorption (10% FBS in PBS) was observed on the nanostructuredtitanium compared to the flat titanium (Fig. 2c).

3.2. Macrophage activation and morphology on nanostructuredsurface features

Tissue cells, such as bone and vascular cells, selectively respondto their surrounding environment and to the extracellular signals of

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biomaterials (e.g. surface chemistry, nanoscale topography, charge,surface energy and wettability) through altered protein adsorptionon nanometer surface features [6,27–29]. In addition, nanotopogra-phy induces changes in the focal adhesion, cytoskeleton organiza-tion and mechanical properties of fibroblasts [13] and humanmesenchymal stem cells [30]. These studies demonstrated a strongcorrelation between nanoscale surface features and the cytoskele-ton, migration and surface interactions of cells. Results obtainedfrom this study corroborated this relationship, establishing it forthe first time for altered macrophages dynamics on flat and nano-rough titanium thin films using a real-time monitoring system.

It is worthwhile pondering how the nanoscale surface featurestriggered different orders of macrophage activation. Differentamounts or configurations of serum protein adsorption may influ-ence the immune response of macrophages through the stimula-tion of Toll-like receptors to release pro-inflammatory cytokines(TNF-a, IL-6) via NF-jB during macrophage activation [31] orthrough other signal pathways [32]. However, it is still unclearwhich specific proteins in the serum stimulated the macrophageimmune signal cascades. Further investigation is required to eluci-date the underlying mechanism.

In this study, the migration of immune cells (i.e. macrophages)on flat and nanotitanium surfaces within 24 h was measured bymeans of live cell tracking analysis; Fig. 3a provides images docu-menting the macrophage activation. For calculating the numbers ofmacrophages activated, polarized cells (as evidence of migration)were considered as activated whereas static macrophages were

Fig. 3. Macrophage activation and morphology on flat and nanotitanium surfaces. (a) Inactivation. Analysis distinguished activated (migrating: red circle) and static (inactivatetotal cells in the images (in terms of migration) was calculated for the indicated times (i.e.indicated that flat surfaces elicited greater macrophage activation than nanorough surfananorough and flat titanium surfaces because of nearby activated cells (see Supplementaron flat titanium and (d) inactivation of macrophages on nanotitanium surfaces. The polaCells were seeded at 5 � 103 cells cm–2 and monitored for 21 h after adhesion (4 h). Sig

considered as inactivated cells (Fig. 3a, c and d). The number ofactivated cells on each sample (flat and nanorough) was deter-mined at 5, 12 and 24 h (Fig. 3b). The percentage of activated mac-rophages (defined as the number of activated cells per totalnumber of cells) differed between the nanorough and flat titaniumsurfaces during the initial 12 h (Fig. 3b). Magnified confocal imagesshow the evidence of activation of macrophages on the flat tita-nium (c) and nanotitanium (d) surfaces. The polarized actin fila-ment in (c) shows evidence of migration (see also SupplementaryMovie).

3.3. Real-time macrophage migration and FAK activation onnanostructured surface feature

Analysis of the activated (live) macrophages on the nanosurfac-es exhibited a clear difference in migration trajectories: shortermigration distances and a lower migration velocity were observedon the nanorough than on the flat titanium surfaces (Fig. 4a and band Supplementary Movie). The results obtained provide clear evi-dence that macrophage interactions significantly differed on thenanorough (reduced migration distance and circular cytoskeleton)and flat (greater migration distance with polarized cytoskeleton)titanium surfaces within the initial 12 h, after which macrophagesmay be activated by neighboring activated cells (specifically be-tween 12–24 h; see Supplementary Movie).

Live cell tracking analysis revealed that macrophages migratedover greater distances on the flat (mean of 850 lm) than on the

stantaneous image of live macrophages on titanium surfaces depicting macrophaged: yellow) cells on transparent titanium surfaces. (b) The ratio of activated cells to5, 12 and 24 h). The percentage of activated macrophages (based on total cell count)ces after 12 h. After 24 h, however, the majority of cells are activated on both the

y Movie). Magnified confocal images show evidence of (c) activation of macrophagesrized actin filament in (c) shows evidence of migration (see Supplementary Movie).nificantly different at ⁄⁄p < 0.05; data analysis was based on mean ± SEM for n = 3.

Fig. 4. Real-time migration dynamics, cytoskeleton morphology and FAK activation on flat and nanotitanium surfaces. (a) The trajectory of each macrophage suggested thatmore macrophages were active on flat than on nanorough titanium surfaces. Macrophage movement was restricted (i.e. less migration) on nanorough surfaces (see alsoSupplementary Movie). (b) Live cell tracking analysis revealed that macrophages migrated over greater distances on flat (mean of 850 lm) than on nanorough (mean of720 lm) titanium surfaces. Accelerated mean migration speed was also greater on flat (1.3 lm min–1) than on nanorough (1 lm min–1) titanium surfaces. Cells were seededat 5 � 103 cells cm–2 and monitored for 12 h via a real-time live cell system. (c) Fluorescence images illustrating that flat surfaces contained more polarized actin filamentsthan nanorough titanium and glass (control) surfaces. (d) Magnified images of macrophages obtained by confocal microscopy. Cells were seeded at 5 � 103 cells cm–2 andincubated for 12 h prior to fixing. (e) Strong signals of FAK phosphorylation, compared to normalized FAK activation, on flat surfaces suggested cell migration. Cells wereseeded at 1 � 105 cells cm–2 and incubated for 4 h prior to Western blot. All results represented mean ± SEM and ⁄⁄⁄p < 0.001.

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nanorough (mean of 720 lm) titanium surfaces (Fig. 4b). Acceler-ated mean migration speed was also greater on flat (1.3 lm min–

1) than on nanorough (1 lm min–1) titanium surfaces. In addition,the macrophage morphology on the nanomaterials was observedby fluorescence microscopy (Fig. 4c and d).

Adherent macrophages, identified by their circular morphology,were more abundant on the nanorough compared to the flat sur-faces. Actin filaments on the flat surfaces were either stretchedor polarized and indicative of migration, whereas actin stress fiberswere reduced on the nanostructured surface features (Fig. 4d).Thus, the two types of titanium surfaces featured clear differencesin cytoskeleton structures and cell migration.

The cytoskeleton arrangement also provides an important in-sight into the intracellular signaling pathways of a cell throughPI3 K/Akt, MAPK and FAK [13,16,22,33]. Transmembrane integrinsserve as linker proteins that facilitate focal adhesion by connectingextracellular substrates to actin stress fibers extended from withincells [34]. To monitor this signaling pathway for cell migration, FAKactivation in macrophages was analyzed on flat and nanoroughtitanium surfaces since it is the transduction of integrin-mediatedintracellular signaling molecules that promotes cell migration. At4 h, FAK phosphorylation (i.e. FAK activation) declined on thenanorough surfaces and reflected restricted macrophage migrationon the nanorough (as compared to flat) surfaces (Fig. 4e).

3.4. NO production and pro-inflammatory cytokine expression onnanoscaled surface structures

To characterize the inflammatory response elicited in macro-phages, this study examined NO production and pro-inflammatorycytokine expression on the nanorough and flat titanium surfaces.Although MTT assays detected no meaningful differences in celldensity or cell viability after 24 h (Supplementary Fig. 2), the flatsurface structures induced the highest production of NO comparedto control and nanorough titanium after 48 h (Fig. 5a). This can beinterpreted as the initial difference in macrophage activation onthe nanoscaled and flat titanium surfaces until 12 h leading to al-tered NO secretion at 24 h.

Although Raw264.7 has been highly investigated, J774A.1 isalso widely used in the field of immunology and considered to bea good macrophage cell line. Furthermore, it is well known thatRaw264.7 needs the combination of IFN-r with TNF-a/IL-4/etc. toproduce NO but J774A.1 does not require these cytokines. To con-firm the observed NO trend, iNOS expression was also analyzedusing Western blot and immunofluorescence. The results revealedlower iNOS expression on the nanorough than on the flat surfacesat 24 h (Fig. 5b and c). Furthermore, real-time PCR revealedthat macrophage expression of pro-inflammatory cytokines(e.g. TNF-a and IL-1b) were lower on the nanorough than on the

Fig. 5. Decreased NO, iNOS and pro-inflammatory cytokines on nanorough titanium surfaces. (a) Macrophages produced higher levels of NO on flat titanium than on glass andon nanorough titanium surfaces. Cells were seeded at 5 � 104 cells cm–2 and incubated for 48 h prior to testing for NO production. ⁄p < 0.05 compared to glass. (b) Flattitanium shows a stronger intensity corresponding to iNOS (inflammatory protein synthesis) than glass and nanotitanium. Cells were seeded at 1 � 105 cells cm–2 andincubated for 24 h before measuring for iNOS. Western blot was used to determine iNOS expression based upon normalized b-actin. (c) Macrophages were also stained with afluorescence probe for iNOS and visualized using fluorescence microscopy. The results show that mRNA levels of TNF-a (d) and IL-1b (e) were higher on flat (conventional)substrates. All obtained mRNA levels were normalized to b-actin levels and LPS was used as a positive control. Cells were seeded at 5 � 104 cells cm–2 and incubated for 12 hprior to determining gene expression of cytokines. Products were separated by electrophoresis on a 1.5% agarose gel and visualized by staining with ethidium bromide. Allresults represented mean ± SEM with n = 3. ⁄p < 0.05 and ⁄⁄p < 0.01 compared to flat (d and e), respectively.

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flat titanium surfaces (LPS was used as a positive control; Fig. 5dand e). These results confirmed that the nanorough surface struc-tures mitigated the inflammatory response by down-regulatinginflammatory protein synthesis (Fig. 5b–d).

4. Conclusions

This study investigated the effect of nanostructured titaniumsurface features on macrophage activation by analyzing real-timechanges in the macrophage cytoskeleton and migration. Live mac-rophage functions were analyzed on transparent nanostructuredtitanium. Although it has been argued that nanometer surface fea-tures on titanium may stimulate pro-inflammatory responses, thisstudy clearly suggested that initial macrophage migration and acti-

vation were reduced on nanostructured compared to flat titanium.Dynamic cell morphology on transparent titanium surfaces viareal-time monitoring of macrophages correlated to NO signalingpathways and pro-inflammatory cytokine expression (i.e. TNF-aand IL-1b). Inhibition of iNOS, NO and pro-inflammatory cytokinesfrom macrophages corresponded to macrophage inactivation, orrestricted cytoskeleton movement, on nanoscaled titanium surfacefeatures.

Acknowledgements

This research was supported by the Pioneer Research CenterProgram (2010-0002178), Basic Science Research Programs(2010-0016245 and 2010-0003359) and by the Regional Core

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Research Program/Anti-aging and Well-being Research Center Pro-gram through Ministry of Education, Science and Technology(MEST) in South Korea.

Appendix A. Figures with essential colour discrimination

Certain figures in this article, particularly Figures 1, 3–5, are dif-ficult to interpret in black and white. The full colour images can befound in the on-line version, at doi:10.1016/j.actbio.2011.01.006.

Appendix B. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.actbio.2011.01.006.

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