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Stem cells

TiO2 Nanotube Surfaces: 15 nm—An Optimal LengthScale of Surface Topography for Cell Adhesion andDifferentiation**

Jung Park, Sebastian Bauer, Karl Andreas Schlegel, Friedrich W. Neukam,

Klaus von der Mark, and Patrik Schmuki*

Studies of biomimetic surfaces in medicine and biomaterial

fields have explored extensively how the micrometer-scale

topography of a surface controls cell behavior, but only

recently has the nanoscale environment received attention as a

critical factor for cell behavior. Several investigations of cell

interactions have been performed using surface protrusion

topographies at the nanoscale; such topographies are typically

based on polymer demixing, ordered gold cluster arrays, or

islands of adhesive ligands at distinct length scales.[1–3] Recent

work has indicated that the fabrication of ordered TiO2

nanotube layers with controlled diameters can be achieved by

anodization of titanium in adequate electrolytes.[4–6] Such

surfaces can almost ideally be used as nanoscale spacing

models for size-dependent cellular response. This is particu-

larly important as these studies are carried out on titanium

surfaces—a material used for clinical titanium implantations

for the purpose of bone, joint, or tooth replacements.

Therefore, principles elucidated from this work can guide

implant surface modifications toward an optimized surface

geometry and profile to best fit and cell interactions for

adequate bone growth.[7,8]

[�] Prof. P. Schmuki, S. Bauer

Department of Materials Science,

Institute for Surface Science and Corrosion (LKO)

University of Erlangen-Nuremberg,

Martensstrasse 7, 91058 Erlangen (Germany)

E-mail: schmuki@ww.uni-erlangen.de

Dr. J. Park, Prof. K. von der Mark

Department of Experimental Medicine I,

Nikolaus-Fiebiger-Center of Molecular Medicine

Friedrich-Alexander-University of Erlangen-Nuremberg,

91054 Erlangen (Germany)

Dr. K. A. Schlegel, Prof. F. W. Neukam

Oral and Maxillofacial Surgery

Friedrich-Alexander-University of Erlangen-Nuremberg,

91054 Erlangen (Germany)

[��] J. P. and S. B. contributed equally to the presented work. Wegratefully thank Mrs. Friedrich for SEM investigations, the Depart-ment of Materials Science, and Mrs. Rummelt, Eye-Hospital,University of Erlangen-Nuremberg. This work was supported bythe Deutsche Forschungsgemeinschaft (SCHM1597/9-1 andMA534/20-1).

: Supporting Information is available on the WWW under http://www.small-journal.com or from the author.

DOI: 10.1002/smll.200801476

� 2009 Wiley-VCH Verl

Previously we showed that vitality, proliferation, and

motility of mesenchymal stem cells (MSCs) and their differ-

entiation to bone-forming cells is critically influenced by

nanoscale TiO2 surface topography with a specific response to

nanotubes with diameters between 15 and 100nm.[9,10] We

demonstrated that adhesion, proliferation, migration, and

differentiation of MSCs was maximally induced on 15-nm

nanotubes, but prevented on 100-nm nanotubes, which induced

cell death. It remained unclear, however, whether this high

sensitivity of cell response—detecting minute differences of

pore size from15nmup to100nm—isa specificphenomenonof

stem cells or reflects a universal cell behavior. Therefore, in the

present work, we explore the nanoscale response of two main

bone cells: osteoblasts and osteoclasts.

Formaintaining bone homeostasis, the balance between the

bone-forming activity of osteoblasts and the bone-resorbing

activity of osteoclasts is finely regulated by a complex

mechanism involving paracrine and autocrine signals as well

as cellular interactions between these cells and their extra-

cellular matrix. Osteoclasts are originally derived from

hematopoietic stem cells (HSCs) capable of differentiating into

monocytes/macrophages and activated monocytes/macro-

phages, while osteoblasts are derived from mesenchymal

stem/progenitor cells.[11–16] Their differentiation canbe induced

by cytokines such as m-CSF (macrophage colony-stimulating

factor) and by interaction with osteoblasts through the RANK/

RANKL (receptor activator of nuclear factor-kB ligand)

system. Bone-resorbing cells play an important role not only

for daily bone remodeling but also for bone regeneration as

occurring in osseous integration of implant materials.[17,18]

Therefore, we address here the interaction of osteoclasts

with TiO2 nanoscale environments in order to explore i) if the

nanotopography of cell interactions as observed previously

with MSCs[10] is of a universal nature, and ii) if the balance

between bone-forming and bone-resorbing cells can be

affected by nanoscale topography. We show that the cell

response is sensitive to nanoscale surface topography in bone-

forming/resorbing cells as well as stem cells. Our present data

show that this nanoscale surface topography largely affects

bone cell differentiations involving osteoclastic activation and

bone-forming activity, indicating that 15 nm is a universal

geometric constant of surface-topography-supporting cell

adhesion and differentiation.

For this study we used surfaces of vertically aligned TiO2

nanotubes with six different diameters between 15 and 100 nm

ag GmbH & Co. KGaA, Weinheim small 2009, 5, No. 6, 666–671

as described previously.[9,10] Synthesis of these self-assembled

TiO2 nanotube layers on titanium in a highly regular

arrangement was achieved by anodizing Ti sheets in a

phosphate-fluoride electrolyte at different voltages ranging

from 1 to 20V, thus precisely controlling tube diameter[9,19]

(Figure 1). On these surfaces we seeded HSCs from human

umbilical cord blood and induced differentiation into multi-

nucleated osteoclast-like cells using standard m-CSF and

RANKL procedures.

As shown in Figure 2, the response of freshly isolated

HSCs from human umbilical cord blood with respect to

differentiation to multinucleated osteoclasts showed the same

size-dependent response to TiO2 nanotubes as described

previously for osteoblastic differentiation,[10] that is, a highly

distinct reaction to the nanoscale spacing distance below

100 nm. On nanotubes below 30 nm differentiation to multi-

nucleated (Figure 2a) and tartrate-resistant acid phosphatase

(TRAP)-positive (Figure 2b) osteoclasts was significantly

stimulated compared to smooth TiO2 surfaces. In contrast,

osteoclast differentiation was severely inhibited on larger pore

sized nanotubes (Figure 2a–c). Differentiation of HSCs

(Figure 2c, left panel) to osteoclasts on 15-nm nanotubes

was evident by the appearance of large, multinucleated cells

(Figure 2c, middle panel, and 2d), which were not observed on

100-nm nanotubes (Figure 2c, right panel). Furthermore,

under the fluorescence microscope, differentiated osteoclasts

on 15-nm nanotubes showed a typical cortical actin ring

(arrows in Figure 2d, left upper panel) commonly observed in

active osteoclasts, and also showed more clearly enhanced

aVb3-integrin expression on 15-nm TiO2 nanotubes as

compared to 100-nm nanotubes (Figure 2d, lower panel).

Scanning electron microscopy (SEM) (Figure 2e) revealed the

Figure 1. Top-view SEM images of self-assembled layers of vertically

oriented TiO2 nanotubes of six different diameters ranging between 15

and 100nm formed in 1 M H3PO4þ0.3 wt% HF at potentials between 1

and 20V for 1 h. Scale bars: 200 nm.

small 2009, 5, No. 6, 666–671 � 2009 Wiley-VCH Verlag Gmb

protrusion of extensive filipodia on 15-nm but not on 100-nm

nanotubes, confirming that HSCs can be actively stimulated to

differentiate into bone-resorbing cells on a TiO2 nanoporous

surface microenvironment with a diameter less than 30 nm.

In bone marrow and circulating blood, activated mono-

cytes/macrophages as well as HSCs are also sources for

osteoclast differentiation. Using blood monocytes we con-

firmed that not only stem cells but also activated monocytes/

macrophages can be induced to osteoclast differentiation

depending on the nanotube diameter (see Supporting

Information, Figure S1).

The possibility existed that the insufficient response of

HSCs to 100-nm nanotubes was due to cellular degeneration

or irreversible loss of differentiation capacity. In Figure 2f we

show, however, that HSCs retain their osteoclastic differ-

entiation potential even though they failed to differentiate into

osteoclasts on 100-nm nanotubes. By harvesting cells from

100-nm nanotubes after culture and replating on tissue culture

dishes, cells differentiated into multinucleated osteoclasts

(OCLs) within 10 days (Figure 2f, left panel). These cells were

TRAP-positive (Figure 2f, middle panel) and showed

osteoclastic resorption pits on bone disks (Figure 2f, right

panel). These findings indicate that osteoclastic differentiation

on large nanoporous structures is only temporarily impaired

on 100-nm nanotubes, but reversible by changing the

nanoscale microenvironment.

These findings show that—apart from this reversible

differentiation block of HSCs on 100-nm nanotubes—the

size-dependent response of HSCs to TiO2 nanotubes with

respect to osteoclast differentiation is very similar to the cellular

response ofMSCs reported previously.[10] MSCs did not spread

properly on nanotube surfaces of pore size larger than 30nm,

and showed unstable filopodia extensions.[10] Further motility

was reduced as shown in a gap-filling cell migration experiment

(see Supporting Information, Figure S2). Interestingly, HSCs

also showedreducedmotilityon100-nmnanotubesas compared

to 15-nm nanotubes as visualized by video microscopy (see

Supporting Information, Movies S1 and S2).

These findings imply that the nanoscale spacing of 15 nm,

which corresponds approximately the diameter of an intergrin

extracellular domain,[10] may represent a universal spacing

constant supporting a maximum of cellular responses to

surfaces. In order to investigate whether differentiation of

osteoblasts (a cell of mesenchymal origin in contrast to the

hematopoietic origin of osteoclasts) also follows a similar size

response to TiO2 nanotubes, we plated primary human

osteoblast-like cells (hOBs) from human iliac bone marrow

on six different sizes of nanotubes and on smooth TiO2

surfaces as a control. As shown in Figure 3, cell proliferation of

hOBs was again highest on 15-nm nanotubes (Figure 3a).

Similar to MSCs,[10] immunofluorescence analysis of hOBs

with antibodies to paxillin and fibronectin indicated a strongly

enhanced formation of focal contacts and a remarkably

stronger deposition of fibronectin fibers on the cell surface on

15-nm nanotubes as compared to 100-nm nanotubes

(Figure 3b). SEM revealed that cells spread out normally

on smaller size nanotubes (15 nm), forming lamellopodia and

wide, thick filopodia, while cell adhesion and spreading was

impaired without stable extension of filopodia on 100-nm

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Figure 2. HSCs can be actively differentiated to multinucleated osteoclasts on nanotubes of

diameter less than 30nm. a) Osteoclast differentiation measured by counting multinucleated

cells; the 100nm value was set as 1. b) Enzymatic assay for TRAP. c) Cell morphology reveals

large osteoclasts only on 15-nm nanotubes. Scale bars: 50, 200, and 200mm. d) Immu-

nofluorescence staining reveals the typical ring of actin cortex (arrows, left upper panel)

shown only in active osteoclasts on 15-nm nanotubes, and enhanced aVb3-integrin

expression on 15-nm nanotubes (lower panels). Scale bars: 100mm. e) SEM analysis of

filopodia formation. Scale bars: 3mm. f) Replating of HSCs that had remained undifferen-

tiated after 10 days on 100-nm nanotube surfaces retained their ability to differentiate toward

bone-resorbing cells on conventional culture dishes. Scale bars: 200, 50, and 50mm.

668

nanotubes (Figure 3b). Mineralization of hOBs as measured

by Alizarin red staining (Figure 4a) and expression of

osteogenic marker proteins such as osteocalcin (Figure 4b)

also reaches a maximum on 15-nm nanotubes as compared to

larger size nanotubes or smooth surfaces. Interestingly,

osteogenic differentiation including mineralization was not

hampered by coculture with osteoclasts on 15-nm nanotubes,

while mineralization was not stimulated in coculture on 100-

nm nanotubes (Figure 4c–e). This indicates that bone cell

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differentiation was largely controlled by the

nanoscale microenvironment even under

the influence through the interaction

between osteoblast and osteoclast via

cell–cell contacts and soluble factors from

each cell type during coculture in vitro.

For both cell types of MSCs and hOBs,

differentiation was severely impaired on

tube diameters larger than 70nm, and cell

proliferation and migration were dramati-

cally reduced, indicating that bone-forming

cells and their progenitor cells have less

osteogenic differentiation potential in that

range of nanoscale spacing. These findings

confirm that both cell types of MSCs and

hOBs react similarly to lateral nanospacing.

This result is consistent with a previous

report on (RGDfK)-coated cell-adhesive

gold nanodots spotted in distinct spacings

of 28, 58, 73, and 85nmdistance, usingblock-

copolymer micelle nanolithography.[20] In

that study a separation of >73nm between

dots resulted in limited cell attachment and

spreading. Our findings support recent

reports on cells reacting sensitively to

nanoscale roughness on silicon or silica

substrates,[21,22] indicating that cell res-

ponses to biomimetic surfaces do not only

depend on the chemistry of the biomaterial

but also on the geometry of the nanoscale

microenvironment. Cell interactions with

extracellular surfaces are mediated by clus-

tering of integrins into focal adhesion

complexes and activation of intracellular

signaling cascades into thenucleus and to the

cytoskeleton.[23] The results presented here

are consistent with our hypothesis that a

spacing of 15–30 nm may be a result of

compact clustering of integrin receptor

molecules with an actual size of the extra-

cellular domain of about 10 nm into focal

contacts by the 15nm spacing of the

nanotubes.[10,24] This may explain why focal

contact formation, cell proliferation, migra-

tion, and differentiation occur at a higher

rate on 15-nm nanotubes than on polished

TiO2 or non-nanoporous surfaces. The

almost identical response of MSCs, HSCs,

andosteoclasts to the15-nmspacing suggests

that this nanoscale spacing may be a

universal scaffold for several cell types, or at least for bone-

remodeling-associated cells.

Bone marrow and surrounding bone are the major

supports for dental implants and total hip replacement

implants.[25] The maintenance of an appropriate balance of

bone resorption and bone remodeling during and after wound

healing, as well as stable integration of the implants have been

a long-standing challenge in the biomaterial implantology

field. Two rather different counteracting cell types of different

im small 2009, 5, No. 6, 666–671

Figure 3. Size-dependent response of primary human osteoblasts to

TiO2 nanotubes. a) Cell proliferation measured by cell counting (100 nm

values were set as 1). b) SEM images show that development of focal

contacts measured by paxillin staining (upper panel) (scale bars:

100mm), extracellular deposition of fibronectin matrix (middle panel)

(scale bars: 50mm), and filopodia formation (lower panel), were highest

on 15 nm nanotubes (scale bars: 10mm).

origin, osteoblasts and osteoclasts, are responsible for the bone

healing process. In most aspects, osteoblasts and osteoclasts

behave different in vitro and in vivo and underlie different

regulatory mechanisms. Thus, the present findings that the

activities of both bone-forming cells and bone-resorbing cells

on biomimetic surfaces responded almost identically to the

same spacing topography within a narrow range between 15

and 100 nm are quite surprising. Since MSCs also showed the

same size-dependence in their responses to TiO2 nanotubes,

we propose that a surface geometry with a lateral spacing of

approximately 15 nm that corresponds to the dimension of

integrin heads will be preferentially recognized by many more

(at least bone remodeling cells (osteoblast/osteoclast) as well

as MSCs) if not all cell types. Although several facts, such as

the formation of focal contacts, the induction of paxillin,

phosphorylation, and the formation of stress fibers, indicate

the key role of the integrin cluster size in the recognition of

nanoscale surface topography, further work is needed to

ultimately confirm this point. It is, however, noteworthy that

our pilot in vivo experiments also have shown that bone

regeneration on Ti implants with 30-nm nanotubes are

small 2009, 5, No. 6, 666–671 � 2009 Wiley-VCH Verlag Gmb

significantly different from that on rough Ti implants.[26] It

is still elusive how the balance between osteoblastic and

osteoclastic activity can be modulated by controlling the

surface geometry. However, this implies a general change in

our concepts in the design of nanoscale surfaces and

biomaterials used for implantation and other biotechnology

developments.

Experimental Section

Nanotube formation: Titanium foils (99.6% purity, Advent Ltd.)

were used for producing nanoporous surfaces. Coatings consisting

of highly ordered self-assembled TiO2 nanotubes of different

diameters were applied on the foils using an electrochemical cell

with a three-electrode configuration. Platinum gauze served as

a counter electrode and a Haber–Luggin capillary with a Ag/AgCl

(1 M KCl) electrode was used as a reference electrode. Electro-

chemical treatments were carried out according to previous

reported work[9] in 1 M H3PO4 (Merck) with addition of 0.3wt%

HF (Merck) with applied potentials from 1V up to 20 V for 1 h at

room temperature. For smooth, polished surfaces, titanium sheets

(99.99% purity, Alfa Aesar) were mechanically ground, lapped,

and finally polished (New Lam System) followed by anodization in

fluoride free 1 M H3PO4 at 20 V. All electrolytes were prepared from

reagent grade chemicals and deionized water. The samples were

sterilized using an autoclave at 121 8C prior to cell seeding. High

resolution X-ray diffraction (XRD) of the nanotube layers after

autoclaving showed the tubes to be of an amorphous nature. X-ray

photoelectron spectroscopy (XPS) investigations of the tubes

revealed the composition to be approximately 58 at% Ti, 37 at%

O, and 5 at% F prior to the wash with distilled water.[6] For

morphological characterization of sample surfaces, a field

emission SEM (FESEM, S-4800, Hitachi) was used.

Cell culture: For the isolation of HSCs and mononuclear cells

(MNCs), fresh umbilical cord blood samples were collected from

healthy, full-term placentas. The MNC fraction was isolated by

density gradient centrifugation using Ficoll-Paque Plus (Amersham

Biosciences) according to the manufacturer’s protocol. CD133(þ)

cells were obtained by incubating the cells with an anti-CD133-

phycoerythrin antibody (Miltenyi Biotech) followed by separation

on a high-performance flow cytometry cell sorter (MoFlo, DAKO)

with forward-side scatter gating. The resulting fraction of

CD133(þ) cells was sorted again to increase its purity to >98%.

Primary human osteoblast-like cells were isolated from

remaining bone chips of iliac bone marrow obtained after

autologous bone graft with patient consent. After two passages

of primary cell culture with alpha medium (Invitrogen) containing

10% fetal calf serum (FCS), cells were harvested at their

confluency and stored frozen until usage. Rat MSCs were isolated

and expanded from fresh bone marrow from femurs of 4-week-old

wistar rats. Selected clonal cells were further expanded as

described previously.[10,27] For migration assay the cells were

infected with retroviruses containing a green fluorescence protein

(GFP) cDNA. For all studies, stably GFP-expressing cell clones were

used.

For osteoclastic differentiation, HSCs at a cell density of

10 000 cm�2 or MNCs at a cell density of 500 000 cm�2 were

cultivated on 15- and 100-nm nanotubes in alpha medium

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Figure 4. a) Mineralization (alizarin red staining) and osteogenic differentiation measured by

osteocalcin expression (b) of primary osteoblasts was highest on 15-nm tube diameters, but

severely impaired on nanotubes with diameters larger than 70-nm. Scale bars: 100mm. c,d)

Mineralization assay of a 2-week coculture (c) and of primary osteoblasts and osteoclasts (d),

showing that overall mineralization in spite of osteoclast differentiation was much enhanced

on 15-nm nanotubes compared to 100-nm nanotubes. d) Alizarin red staining showing

mineralization was consistent with quantitative analysis of mineralization in (c), confirming

that the activity of osteoblastic differentiation dramatically was stimulated on 15-nm

nanotubes. Scale bar: 1 cm. Osteoblastic differentiation in cocultures on 15-nm nanotubes

was further supported by immunofluorescence staining for e) osteocalcin at a similar level as

osteoblasts in the absence of osteoclasts (b). Osteocalcin in red, nuclear stain in blue, scale

bars: 400 and 100mm.

670

containing 10% FCS, 50 ng mL�1 human recombinant RANKL, and

20 ng mL�1 m-CSF. The culture medium was replaced every 2 days

in all experiments.

For osteogenic differentiation cells were plated at a cell

density of 50 000 cm�2 in alpha medium (Invitrogen) containing

10% FCS. Five days after cell plating, the culture medium was

changed into a differentiation medium containing 10% FCS,

dexamethasone (100 nM), b-glycerophosphate (10mM), and as-

corbic acid (50mg mL�1). The cells were cultivated for 2 weeks

with differentiation medium and analyzed by immunocytochem-

istry and quantitative mineralization assay.

For the coculture experiment, human primary osteoblasts at a

cell density of 50000 cm�2 and primary MNCs at a cell density of

500000 cm�2 were plated on 15- and 100-nm nanotubes in alpha

medium containing 10% FCS, 50 ng mL�1 human recombinant

RANKL, and 20ng mL�1 m-CSF. From the 5th day of coculture, the

cocultured cells were stimulated for osteoblastic/osteoclastic

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differentiation using differentiation medium

containing dexamethasone (100nM), b-glycer-

ophosphate (10mM), ascorbic acid (50mg

mL�1), 50ng mL�1 human recombinant RANKL,

20ngmL�1m-CSF, and 10% FCS. The cells were

cultivated further for 2 weeks with differentia-

tion medium and analyzed by immunocyto-

chemistry and quantitative mineralization

assay.

TRAP staining and solution assays: To

analyze osteoclastic differentiation, HSCs or

MNCs were cultivated on nanotubes in differ-

entiation medium, and fixed and immunos-

tained after 10 days with 40,6-diamidino-2-

phenylindole (DAPI) and phalloidin as de-

scribed previously.[12] Multinucleated cells

containing more than three nuclei were

considered differentiated osteoclast-like cells,

and 100–300 cells in at least three fields were

counted under the fluorescence microscope

(Zeiss Axiophot).

To quantify TRAP activity, cells after 10 days

culture in differentiation medium were washed

once with phosphate buffer saline (PBS) and

lyzed in 80mL of cold lysis buffer (90mM citrate

buffer, pH 4.8, 0.1% Triton X-100 containing

80mM sodium tartrate) for 10min. After lysis,

80mL of substrate solution (20mM p-nitrophe-

nyl phosphate in the above lysis buffer) was

added and incubated for an additional 3–

5min, and the reaction was stopped by adding

40mL of 0.5 M NaOH. The optical density was

read at a 405-nm wavelength.

To verify the differentiation potential of

HSCs on TiO2 nanotubes, cultivated HSCs on

100-nm tubes were trypsinized and replated on

24-well plastic culture plates for TRAP staining

or BD BioCoatTM OsteologicTM disks for osteo-

clastic resorption tests. Replated cells were

cultivated with the same differentiation med-

ium. Then cells were washed once with PBS and

fixed in 10% formalin for 10min. After washing with PBS, cells were

permeabilized with 0.1% Triton X-100 for 1min, washed once with

PBS, and incubated with substrate solution napthol AS-BI phos-

phate (Sigma) in the presence of 50mM sodium tartrate at 37 8C for

10min. Resulting red-stained TRAP activity Osteoclast resorption

pits were visualized by light microscopy.

Detection and quantification of mineralization: For detection of

mineralization, alizarin red staining was performed on three

samples each with 15- and 100-nm nanotubes after 2week

differentiation culture as previously described.[28] Briefly, cocul-

tured cells were fixed in 4% paraformaldehyde (PFA) and treated

with 40mM alizarin red S (pH 4.1, Sigma) for 20min at room

temperature with gentle shaking. After aspiration of the unin-

corporated dye, the samples were washed four times with d-H2O

while shaking for 5min. Stained mineralized nodules were

visualized. For quantification of staining, 10% v/v acetic acid

was added to each sample and incubated for 30min with shaking.

im small 2009, 5, No. 6, 666–671

The monolayer on nanotubes was then scraped from the sample

surfaces with 10% v/v acetic acid and transferred to a

microcentrifuge tube. After vortexing for 30 s, the tube was

heated to 85 8C for 10min and centrifuged at 20 000 � g for

15min. The supernatant was transferred to a new microcentrifuge

tube and 10% v/v ammonium hydroxide was added to neutralize

the acid. Aliquots of the supernatant were read in triplicate at

405 nm in a 96-well format enzyme-linked immunosorbent assay

(ELISA) reader.

Immunocytochemistry: For immunocytochemistry, cells grown

on nanotubes were rinsed in PBS and fixed with 2% PFA in PBS at

room temperature for 10min. After fixation, cells were permeabi-

lized with 0.2% Triton X-100 in PBS for 2min, washed with PBS,

and incubated with antibodies of mouse monoclonal anti-paxillin

(Signal Transduction), anti-aVb3-integrin (Chemicon), and mouse

monoclonal anti-osteocalcin (Takara) for 1 h. The F-actin was

visualized with Alexa488-labeled phalloidin (Biosource). Second-

ary antibodies labeled with Cy5 (Biosource) were used. Cell nuclei

were stained blue with DAPI (Roth). Cell images were taken using

an Axiophot 2000 ApoTome microscope with AxioCam digital

camera and AxioVision software (Zeiss).

For SEM observation cells were fixed with 2.5% glutaraldehyde

solution (Merck) overnight at 4 8C. Samples were rinsed in PBS

solution, dehydrated in a series of acetone solutions (60, 70, 80,

90, and 100%) and critical point dried with a Critical Point Dryer

(CPD 030, Balzers).

Cell proliferation and migration assay: Primary human

osteoblast-like cells and GFP-labeled MSCs were plated on a

titanium surface at a cell density of 5 000 cm�2. Cell proliferation

was analyzed by a cell count 3 days after cell plating. For cell

counting of primary osteoblast-like cells cell nuclei were stained

with DAPI before counting. Adherent cells were counted at three

different areas using 1280�1024 pixels resolution, where each

sample was depicted under a fluorescent microscope (50�magnification).

To analyze cell migration, GFP-labeled MSCs were plated at a

cell density of 50 000 cm�2 and 3 h later cells were removed in a

streak (3.4mm in width) in the center of the field. Thirty-six hours

later the immigration of cells into the cleft was analyzed using

Openlab software (Improvision).

To analyze HSC motility on different sizes of nanotubes, HSCs

were labeled using CM-DiI fluorescence cell tracker (Invitrogen)

before cell plating on nanotubes as previously described.[29] One

day after cell plating, cell migration of HSCs was monitored by

time-lapse video microscopy every 2min during 4 h and analyzed

using Openlab software.

Keywords:biomimetics . nanotubes . stem cells . surface topography .

titanium dioxide

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Received: October 6, 2008Revised: November 7, 2008Published online: February 20, 2009

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