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Stem cells

TiO2 Nanotube Surfaces: 15 nmAn Optimal Length Scale of Surface Topography for Cell Adhesion and Differentiation**
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 elds 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.[13] 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.[46] Such surfaces can almost ideally be used as nanoscale spacing models for size-dependent cellular response. This is particularly important as these studies are carried out on titanium surfacesa 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 modications toward an optimized surface geometry and prole to best t and cell interactions for adequate bone growth.[7,8] Previously we showed that vitality, proliferation, and motility of mesenchymal stem cells (MSCs) and their differentiation to bone-forming cells is critically inuenced by nanoscale TiO2 surface topography with a specic response to nanotubes with diameters between 15 and 100 nm.[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 responsedetecting minute differences of pore size from 15 nm up to 100 nmis a specic phenomenon of stem cells or reects a universal cell behavior. Therefore, in the present work, we explore the nanoscale response of two main bone cells: osteoblasts and osteoclasts. For maintaining bone homeostasis, the balance between the bone-forming activity of osteoblasts and the bone-resorbing activity of osteoclasts is nely regulated by a complex mechanism involving paracrine and autocrine signals as well as cellular interactions between these cells and their extracellular matrix. Osteoclasts are originally derived from hematopoietic stem cells (HSCs) capable of differentiating into monocytes/macrophages and activated monocytes/macrophages, while osteoblasts are derived from mesenchymal stem/progenitor cells.[1116] Their differentiation can be 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 boneforming/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
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[] 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. We gratefully thank Mrs. Friedrich for SEM investigations, the Department of Materials Science, and Mrs. Rummelt, Eye-Hospital, University of Erlangen-Nuremberg. This work was supported by the Deutsche Forschungsgemeinschaft (SCHM1597/9-1 and MA534/20-1). : Supporting Information is available on the WWW under http:// www.small-journal.com or from the author. DOI: 10.1002/smll.200801476

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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-uoride electrolyte at different voltages ranging from 1 to 20 V, thus precisely controlling tube diameter[9,19] (Figure 1). On these surfaces we seeded HSCs from human umbilical cord blood and induced differentiation into multinucleated 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 multinucleated (Figure 2a) and tartrate-resistant acid phosphatase (TRAP)-positive (Figure 2b) osteoclasts was signicantly stimulated compared to smooth TiO2 surfaces. In contrast, osteoclast differentiation was severely inhibited on larger pore sized nanotubes (Figure 2ac). 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 uorescence 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 100 nm formed in 1 M H3PO4 0.3 wt% HF at potentials between 1 and 20 V for 1 h. Scale bars: 200 nm.
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protrusion of extensive lipodia on 15-nm but not on 100-nm nanotubes, conrming 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 monocytes/macrophages as well as HSCs are also sources for osteoclast differentiation. Using blood monocytes we conrmed 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 insufcient 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 differentiation 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 ndings indicate that osteoclastic differentiation on large nanoporous structures is only temporarily impaired on 100-nm nanotubes, but reversible by changing the nanoscale microenvironment. These ndings show thatapart from this reversible differentiation block of HSCs on 100-nm nanotubesthe size-dependent response of HSCs to TiO2 nanotubes with respect to osteoclast differentiation is very similar to the cellular response of MSCs reported previously.[10] MSCs did not spread properly on nanotube surfaces of pore size larger than 30 nm, and showed unstable lopodia extensions.[10] Further motility was reduced as shown in a gap-lling cell migration experiment (see Supporting Information, Figure S2). Interestingly, HSCs also showed reduced motility on 100-nm nanotubes as compared to 15-nm nanotubes as visualized by video microscopy (see Supporting Information, Movies S1 and S2). These ndings 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] immunouorescence analysis of hOBs with antibodies to paxillin and bronectin indicated a strongly enhanced formation of focal contacts and a remarkably stronger deposition of bronectin bers 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 lopodia, while cell adhesion and spreading was impaired without stable extension of lopodia on 100-nm

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differentiation was largely controlled by the nanoscale microenvironment even under the inuence through the interaction between osteoblast and osteoclast via cellcell 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 70 nm, and cell proliferation and migration were dramatically reduced, indicating that bone-forming cells and their progenitor cells have less osteogenic differentiation potential in that range of nanoscale spacing. These ndings conrm 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 85 nm distance, using blockcopolymer micelle nanolithography.[20] In that study a separation of >73 nm between dots resulted in limited cell attachment and spreading. Our ndings support recent reports on cells reacting sensitively to nanoscale roughness on silicon or silica substrates,[21,22] indicating that cell responses 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 clustering of integrins into focal adhesion complexes and activation of intracellular signaling cascades into the nucleus and to the cytoskeleton.[23] The results presented here are consistent with our hypothesis that a spacing of 1530 nm may be a result of compact clustering of integrin receptor molecules with an actual size of the extraFigure 2. HSCs can be actively differentiated to multinucleated osteoclasts on nanotubes of cellular domain of about 10 nm into focal diameter less than 30 nm. a) Osteoclast differentiation measured by counting multinucleated contacts by the 15 nm spacing of the cells; the 100 nm value was set as 1. b) Enzymatic assay for TRAP. c) Cell morphology reveals nanotubes.[10,24] This may explain why focal large osteoclasts only on 15-nm nanotubes. Scale bars: 50, 200, and 200 mm. d) Immucontact formation, cell proliferation, migranouorescence staining reveals the typical ring of actin cortex (arrows, left upper panel) tion, and differentiation occur at a higher shown only in active osteoclasts on 15-nm nanotubes, and enhanced aVb3-integrin rate on 15-nm nanotubes than on polished expression on 15-nm nanotubes (lower panels). Scale bars: 100 mm. e) SEM analysis of TiO2 or non-nanoporous surfaces. The lopodia formation. Scale bars: 3 mm. f) Replating of HSCs that had remained undifferentiated after 10 days on 100-nm nanotube surfaces retained their ability to differentiate toward almost identical response of MSCs, HSCs, bone-resorbing cells on conventional culture dishes. Scale bars: 200, 50, and 50 mm. and osteoclasts to the 15-nm spacing suggests that this nanoscale spacing may be a nanotubes (Figure 3b). Mineralization of hOBs as measured universal scaffold for several cell types, or at least for boneby Alizarin red staining (Figure 4a) and expression of remodeling-associated cells. Bone marrow and surrounding bone are the major osteogenic marker proteins such as osteocalcin (Figure 4b) also reaches a maximum on 15-nm nanotubes as compared to supports for dental implants and total hip replacement larger size nanotubes or smooth surfaces. Interestingly, implants.[25] The maintenance of an appropriate balance of osteogenic differentiation including mineralization was not bone resorption and bone remodeling during and after wound hampered by coculture with osteoclasts on 15-nm nanotubes, healing, as well as stable integration of the implants have been while mineralization was not stimulated in coculture on 100- a long-standing challenge in the biomaterial implantology nm nanotubes (Figure 4ce). This indicates that bone cell eld. Two rather different counteracting cell types of different

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signicantly 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 conguration. Platinum gauze served as a counter electrode and a HaberLuggin capillary with a Ag/AgCl (1 M KCl) electrode was used as a reference electrode. Electrochemical treatments were carried out according to previous reported work[9] in 1 M H3PO4 (Merck) with addition of 0.3 wt% HF (Merck) with applied potentials from 1 V 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 nally polished (New Lam System) followed by anodization in uoride 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 eld 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 manufacturers protocol. CD133() cells were obtained by incubating the cells with an anti-CD133phycoerythrin antibody (Miltenyi Biotech) followed by separation on a high-performance ow cytometry cell sorter (MoFlo, DAKO) with forward-side scatter gating. The resulting fraction of CD133() 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 cm2 or MNCs at a cell density of 500 000 cm2 were cultivated on 15- and 100-nm nanotubes in alpha medium

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: 100 mm), extracellular deposition of bronectin matrix (middle panel) (scale bars: 50 mm), and lopodia formation (lower panel), were highest on 15 nm nanotubes (scale bars: 10 mm).

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 ndings 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 bers, indicate the key role of the integrin cluster size in the recognition of nanoscale surface topography, further work is needed to ultimately conrm 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
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differentiation using differentiation medium containing dexamethasone (100 nM), b-glycerophosphate (10 mM), ascorbic acid (50 mg mL1), 50 ng mL1 human recombinant RANKL, 20 ng mL1 m-CSF, and 10% FCS. The cells were cultivated further for 2 weeks with differentiation medium and analyzed by immunocytochemistry and quantitative mineralization assay. TRAP staining and solution assays: To analyze osteoclastic differentiation, HSCs or MNCs were cultivated on nanotubes in differentiation medium, and xed and immunostained after 10 days with 40 ,6-diamidino-2phenylindole (DAPI) and phalloidin as described previously.[12] Multinucleated cells containing more than three nuclei were considered differentiated osteoclast-like cells, and 100300 cells in at least three elds were counted under the uorescence 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 80 mL of cold lysis buffer (90 mM citrate buffer, pH 4.8, 0.1% Triton X-100 containing 80 mM sodium tartrate) for 10 min. After lysis, 80 mL of substrate solution (20 mM p-nitrophenyl phosphate in the above lysis buffer) was added and incubated for an additional 3 Figure 4. a) Mineralization (alizarin red staining) and osteogenic differentiation measured by 5 min, and the reaction was stopped by adding osteocalcin expression (b) of primary osteoblasts was highest on 15-nm tube diameters, but 40 mL of 0.5 M NaOH. The optical density was severely impaired on nanotubes with diameters larger than 70-nm. Scale bars: 100 mm. c,d) read at a 405-nm wavelength. Mineralization assay of a 2-week coculture (c) and of primary osteoblasts and osteoclasts (d), To verify the differentiation potential of showing that overall mineralization in spite of osteoclast differentiation was much enhanced HSCs on TiO2 nanotubes, cultivated HSCs on on 15-nm nanotubes compared to 100-nm nanotubes. d) Alizarin red staining showing mineralization was consistent with quantitative analysis of mineralization in (c), conrming 100-nm tubes were trypsinized and replated on 24-well plastic culture plates for TRAP staining 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 or BD BioCoatTM OsteologicTM disks for osteowas further supported by immunouorescence staining for e) osteocalcin at a similar level as clastic resorption tests. Replated cells were osteoblasts in the absence of osteoclasts (b). Osteocalcin in red, nuclear stain in blue, scale cultivated with the same differentiation medbars: 400 and 100 mm. ium. Then cells were washed once with PBS and containing 10% FCS, 50 ng mL1 human recombinant RANKL, and fixed in 10% formalin for 10 min. After washing with PBS, cells were 20 ng mL1 m-CSF. The culture medium was replaced every 2 days permeabilized with 0.1% Triton X-100 for 1 min, washed once with PBS, and incubated with substrate solution napthol AS-BI phosin all experiments. For osteogenic differentiation cells were plated at a cell phate (Sigma) in the presence of 50 mM sodium tartrate at 37 8C for density of 50 000 cm2 in alpha medium (Invitrogen) containing 10 min. Resulting red-stained TRAP activity Osteoclast resorption 10% FCS. Five days after cell plating, the culture medium was pits were visualized by light microscopy. Detection and quantication of mineralization: For detection of changed into a differentiation medium containing 10% FCS, dexamethasone (100 nM), b-glycerophosphate (10 mM), and as- mineralization, alizarin red staining was performed on three corbic acid (50 mg mL1). The cells were cultivated for 2 weeks samples each with 15- and 100-nm nanotubes after 2 week [28] Briey, coculwith differentiation medium and analyzed by immunocytochem- differentiation culture as previously described. tured cells were xed in 4% paraformaldehyde (PFA) and treated istry and quantitative mineralization assay. For the coculture experiment, human primary osteoblasts at a with 40 mM alizarin red S (pH 4.1, Sigma) for 20 min at room cell density of 50 000 cm2 and primary MNCs at a cell density of temperature with gentle shaking. After aspiration of the unin500 000 cm2 were plated on 15- and 100-nm nanotubes in alpha corporated dye, the samples were washed four times with d-H2O medium containing 10% FCS, 50 ng mL1 human recombinant while shaking for 5 min. Stained mineralized nodules were RANKL, and 20 ng mL1 m-CSF. From the 5th day of coculture, the visualized. For quantication of staining, 10% v/v acetic acid cocultured cells were stimulated for osteoblastic/osteoclastic was added to each sample and incubated for 30 min with shaking.

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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 10 min and centrifuged at 20 000 g for 15 min. 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 xed with 2% PFA in PBS at room temperature for 10 min. After xation, cells were permeabilized with 0.2% Triton X-100 in PBS for 2 min, 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). Secondary 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 cm2. 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 uorescent microscope (50 magnication). To analyze cell migration, GFP-labeled MSCs were plated at a cell density of 50 000 cm2 and 3 h later cells were removed in a streak (3.4 mm 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 2 min during 4 h and analyzed using Openlab software.

Keywords:

biomimetics . nanotubes . stem cells . surface topography . titanium dioxide

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

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