Materials Science and Engineering C 78 (2017) 443–451
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
A dual-layer macro/mesoporous structured TiO2 surface improves the initial adhesion of osteoblast-like cells Ranran Zhang a,c, Tarek A. Elkhooly a,e, Qianli Huang a,c, Xujie Liu c,d, Xing Yang a,c, Hao Yan a,c, Zhiyuan Xiong c, Jing Ma a, Qingling Feng a,c,⁎, Zhijian Shen a,b,⁎ a
State Key Laboratory of New Ceramics and Fine Processing, School of Materials Science and Engineering, Tsinghua University, Beijing 100084, China Department of Materials and Environmental Chemistry, Arrhenius Laboratory, Stockholm University, S-106 91 Stockholm, Sweden Key Laboratory of Advanced Materials of Ministry of Education of China, Tsinghua University, Beijing 100084, China d Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, China e Department of Ceramics, Inorganic Chemical Industries Division, National Research Centre, Dokki, 12622 Cairo, Egypt b c
a r t i c l e
i n f o
Article history: Received 17 March 2017 Received in revised form 13 April 2017 Accepted 15 April 2017 Available online 17 April 2017 Keywords: Titanium Macro/mesoporous TiO2 Cell adhesion RhoA/ROCK
a b s t r a c t A dual-layer TiO2 surface with hierarchical macro and mesoporous structure was prepared by a combinational approach of micro-arc oxidation followed by evaporation-induced self-assembly of nano-crystallites. The mesoporous layer contains pores with an average size of b 10 nm and consists of anatase TiO2 nanocrystallites. The dual-layer hierarchical macro/mesoporous structured TiO2 surface improves the hydrophilicity and fibronectin adsorption ability in comparison with the sole macroporous or smooth TiO2 surface. With the formation of an additional mesoporous layer on macroporous TiO2 surface, the attached number of human osteogenic sarcoma cells (SaOS-2) increases in the initial incubation of 4 h but it does not show significant difference after 24 h compared to that attached on the macroporous or smooth surfaces. Whereas, it was noticed that SaOS-2 cells have larger spread area and more stress fibers on the macro/mesoporous structured surface than those on the other surfaces. To understand the intracellular mechanism of the initial cell adhesion on the macro/mesoporous surface, the Rho/ROCK pathway was investigated to reveal the topography-induced biological functions by introducing the ROCK inhibitor Y-27632 during cell culture. In the presence of Y-27632, cells on the macroporous surface and macro/mesoporous surface both show stellate appearance, with poor assembly stress fibers and long cell membrane protrusions. Cells on the smooth surface have larger spread areas compared to the former two surfaces. And the attached cells significantly reduced but there are no differences among the three surfaces. It reveals that the ROCK inhibitor invalidates the promotion of initial cell adhesion on the macro/mesoporous structure. This study may shed light on the mechanism behind the enhancement effect of macro/mesoporous structure for initial cell adhesion. © 2017 Published by Elsevier B.V.
1. Introduction Titanium and its alloys are the most widely used load-bearing implants for substituting loss in bone tissue. These metals have several advantages such as high specific strength, excellent corrosion resistance and biocompatibility, but their surface bioactivity interacting with the bone tissue still requires enhancement [1]. Since the proposal of “osseointegration” by Branemark, great efforts have been made to improve the integration between the implant and bone structures [2]. The chemical, physical or topological properties of the alloy surface ⁎ Corresponding authors at: School of Materials Science and Engineering, Tsinghua University, Beijing 100084, China. E-mail addresses:
[email protected] (Q. Feng),
[email protected] (Z. Shen).
http://dx.doi.org/10.1016/j.msec.2017.04.082 0928-4931/© 2017 Published by Elsevier B.V.
rule the success of the osseointegration at the contact zones with bone tissue. For this purpose, different surface modifications of titanium and its alloys have been extensively studied during the last decade. Titanium alloys are spontaneously oxidized in air and this process accelerates greatly when heated to elevated temperatures. This smooth oxide layer can improve the biocompatibility and corrosion resistance, but it is fairly thin and has low wear resistance. There exists a consensus that the implant surface oxide chemistry and structural topography can influence both the bone cell behavior and the healing [3]. Synergistic effects of surface micro/nanostructures on their in vitro and in vivo performances have been summarized recently [4]. The influences vary with the structural sizes, so that microscale features can indirectly promote cellular activity [5], whereas nanoscale topographies interact directly with bone tissue [6].
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A combination of sandblasting and acid etching (SLA) is a simple and commonly used method that creates microtopography on the metal implant surface and clinical studies have shown a success rate around 99% [7]. However, a superimposed film with nanoscale topography on this microscale structure was found to be even more favorable to improve the osteoblast cell responses. In general, two or more steps are needed to prepare hierarchical oxide structures on an implant surface. For instance, Huang et al. created TiO2 coatings with hierarchical micro/nanostructures by micro-arc oxidation (MAO) for the microsized porosity followed by hydrothermal treatment for achieving a simultaneous nanotopography [8–10]. These micro/nanostructures showed improved in vitro mineralization and osteogenic properties. Li et al. fabricated hierarchical micro/nanostructures by a novel duplex coating process, including MAO and a subsequent electrochemical reduction in alkaline solution [11]. The latter coating induced higher in vitro alkaline phosphatase (ALP) activity and mineralization capacity. After insertion of such implants with modified surface into canine femurs for 6 weeks, the speed of bone formation and bone-implant contact ratio were significantly promoted. Han et al. used MAO and anodic oxidation to prepare a macro/nanoporous TiO2 coating structure, which showed considerably enhanced hydrophilicity and in vitro biocompatibility compared to the smooth alloy implant surface [12]. As seen above, the MAO technique is considered very suited to create a pore structure [13]. It is a general electrochemical method that generates an oxide coating on a metal surface. The MAO-fabricated coatings prepared on Ti metal or its alloys have very strong binding to the underlying metal and excellent wear and corrosion resistance. This process, used in (NaPO3)6-NaF-NaAlO2 solution, by Wang et al. gave a ceramic TiO2 coating with measured adhesion strength of 40 MPa [14]. In addition, the MAO process enables addition of bioactive elements in the electrolyte solution to be incorporated into the coating. Furthermore, it is very convenient and effective to use the MAO technique for components with complex geometry giving a well-covered and controllable macro/microtopography. In the latter case, however, an additional surface modification process is needed for achieving a superimposed TiO2 film with nanostructures for promotion of bone cell interactions. Porous biomaterials at the nanoscale level have also got much attention in the past for other applications, e.g. selective tissue reactions or drug adsorption and delivery. Studies on mesoporous biomaterials are mainly concerned with mesoporous particles or coatings on implant materials [15]. It was demonstrated that titanium implant directly with a thin mesoporous TiO2 film promoted osseointegration and had a comparable biomechanical stability similar to a nonporous oxide film [16]. Several methods have been employed to generate the mesoporous structures. Evaporation-induced self-assembly (EISA) is excellent for obtaining a controlled mesoporous topography. It can be used to superimpose a uniform mesoporous TiO2 film on previous oxide coatings, e.g. on a macroporous MAO coating. In the EISA method surfactants with hydrophilic blocks and hydrophobic groups are used as templates for controlling the mesoporosity. Inorganic oxide species aggregate around these templates to form the skeleton of the mesopores and after the removal of the template (by heat treatment), a mesoporous oxide structure remains. The EISA method has many advantages such as controlling pore numbers and sizes; it results in a narrow pore size distribution with a large surface area. Samples with complex geometry can be coated using this method. Thus, a mesoporous film created on anodic oxidized titanium components was prepared by Han et al. using EISA [17]. This hierarchically structured TiO2 layer promoted cell adhesion (cultured for 3 days), proliferation and mineralization compared with the original topography of anodic oxidized titanium. The first step of bone cell/material interaction is the cell adhesion to the implant material. The quality of this cell attachment will influence bone cell functions like proliferation and differentiation. Therefore, the interaction between surface topography of the implant and the cells has been studied intensively and is still not fully understood in detail.
There are several possible pathways for controlling shape and focal adhesion strength of the bone cells on a biomaterial surface. Among them, RhoA/ROCK is known to have effects on cell adhesion and reorganization of F-actin cytoskeleton, which in turn will affect the cell behavior. Chang et al. created a micropattern topography (2 mm lattice pattern with 3 mm intervals) and found that the RhoA/ROCK pathway can regulate the focal adhesion formation and cytoskeleton organization of bone cells on the substrate microtopography [18]. Thus, RhoA/ROCK seems to play a major role in regulating cell morphology, F-actin microfilament polymerization and stability, as well as cell contractility. Therefore, a crucial question is how the RhoA/ROCK pathway affects the cell adhesion on the macro/mesoporous structure. The study reported below has two major objects. The first one is the preparation method used to obtain a biomimetic and hierarchical macro/mesoporous surface by a combination of MAO and EISA. That is, the feasibility of using two uncomplicated methods to enhance the adhesion of osteoblast-like cells (SaOS-2). The second purpose is to provide some insight on the effect of surface structure on the initial cell adhesion and functions, especially, whether the RhoA/ROCK pathway is an essential route for cell adhesion. 2. Materials and methods 2.1. Preparation Ti-6Al-4V discs (diameter: 9 mm, thickness: 2 mm) were grinded with sequentially used 240, 800 and 1500 grits SiC abrasive sandpapers. Then, the samples were cleaned ultrasonically in acetone, ethanol and deionized water, each for 5 min. The micro-arc oxidation (MAO) treatment of the Ti-6Al-4V discs was carried out in an aqueous electrolyte containing 0.13 M calcium acetate monohydrate (Ca(CH3COO)2·H2O) and 0.12 M sodium dihydrogen phosphate (NaH2PO4·2H2O). A pulsed AC field was used (WHD-20, Haerbin, China) for power supply. The voltage, frequency, duty and time of the pulsed AC power were 400 V in positive and 5 V in negative, 50 Hz, 50% and 5 min, respectively. Circulating water was used for cooling. After the process, samples were ultrasonically cleaned as above in acetone, ethanol and deionized water. Mesoporous TiO2 thin films were formed using evaporation-induced self-assembly (EISA) method where 2.1 g of titanium (IV) tetraethoxide (TEOT, 95% Aldrich, Aladdin) were dissolved in 1.35 ml of concentrated hydrochloric acid under stirring. Another solution was made of 0.55 g of poly-(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) block copolymer EO20PO70EO20 (Pluronic P123, Sigma) dissolved in 7.5 ml of ethanol (99.5% EtOH, Aldrich) under vigorous stirring. The two solutions were mixed and stirred at room temperature for 3 h to get a homogeneous solution. Subsequently, films were deposited on the macroporous TiO2 coatings by dip coating (1.5 mm/s) and aged at room temperature overnight. Finally, the films were calcined at 400 °C for 4 h to remove the block copolymer to create the mesoporous titania layer. The heating and cooling rates of the furnace were adjusted at 1 °C/ min. 2.2. Physicochemical properties Morphology of the surfaces was examined using a field emission scanning electron microscopy (FESEM, Merlin Compact, Zeiss, Germany) operating at an accelerating voltage of 15 kV. The cross-sections of the macro/mesoporous coating were prepared by an argon ion cross section polisher and were inspected by SEM. The surface morphology and roughness were examined with interferometer (MicroXAM instrument, ADE Co., USA).The crystalline structure of the coatings were analyzed using a Bruker D 8 Advance X-ray diffractometer, collecting a 2θ scanning range of 20°–70°, with a step size of 0.02° and using monochromatic CuKα1 X-ray radiation (λ = 1.5405 Å). Mesoporous film flakes were carefully scratched from the surface of the macro/mesoporous TiO2 coating and investigated using a transmission electron microscopy (TEM,
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JEM-2100, Japan). Selected area diffraction was also applied to analyze the crystallinity of the film. The static contact angles were measured using an Attension Theta Optical Tensiometer (JC2000C1, Powereach, China).
evaluate differences between these groups. Values of p b 0.05 and 0.01 were considered statistically significant and high significant.
2.3. Protein adsorption
The morphologies observed by SEM of the grinded and untreated Ti6Al-4V alloy surface are shown in Fig. 1a–c. Parallel weak and shallow scratches aligned along the grinding direction can be distinguished in the micrographs exposed at low-magnification (Fig. 1a). Higher SEM magnification reveals, however, that the surface is relatively smooth (Fig. 1b and c). After the MAO process, a macroporous TiO2 surface is created on the surface of the Ti-6Al-4V alloy (MAT group), as shown in Fig. 1d and e. The macropores have a size ranging from 0.5 to 3 μm, while higher magnification SEM images show that no nanostructure is present, as seen in Fig. 1f. After the EISA process and dip-coating (MA/ MET group), a typical hierarchical micro/nanostructure was observed by SEM. A SEM overview at low magnification is shown in Fig. 1g, a typical macropore in Fig. 1h, as well as the mesoporosity at higher magnification in Fig. 1i. The cross-section of the MA/MET coating is shown by SEM in Fig. 2a. The total thickness of the TiO2 oxidation layer varies between 2 and 5 μm along the Ti-6Al-4V alloy substrate. The thin mesoporous film follows the contour of the underlying macroporous coating, indicated by the white arrows in Fig. 2a. The film thickness ranges from tens to several hundreds of nanometers. The XRD patterns of the uncoated Ti alloy surface and the oxidized MAT and MA/MET surfaces are shown in Fig. 2b. It is found that anatase is the predominant TiO2 phase for both coatings. TEM was used to give a further look on the shape and phase content of the thin titania top layer. A flake from the top layer was gently scratched of the oxide coating. The flake consisted of the mesoporous film deposited on the thicker macroporous oxide layer. The TEM image shows that the mesoporous film has a random porous structure (Fig. 2c) and the presence of electron diffraction rings confirms the presence of nanosized crystals (Fig. 2d). Indexing of the diffraction rings proved that the mesoporous film was composed of anatase nanocrystals in agreement with the XRD patterns. It is known that nanosized anatase, with a large exposed surface area, has relatively more Ti-OH bindings than the much larger micron sized anatase crystals. The exposed hydroxide groups are prone to form strong chemical bonds with ions like Ca2+ [20]. Therefore, the anatase nanocrystallites may exhibit a better ability to react and form hydroxyapatite at bone contact zones. The white light interferometry images of the three investigated groups indicated that the surface roughness values of the oxide coatings fall in the sub- or micrometric range, see Fig. 3a–c. The recorded values are found printed at the top of the images. The uncoated Ti alloy surface is regarded as smooth, despite the shallow grinding scrapes mentioned above. As expected, the MAO formed oxide coating, with coarse sized open porosity, gives a roughness of micron size. Further deposition of the thin mesoporous film layer partly smooths this roughness giving a slightly lower value. The MAT surface (Ra = 1.036 μm) has much larger roughness value than the substrate surface (Ra = 122 nm). A surface roughness around 1–2 μm has been suggested to be suitable for improving the mechanical interlocking between the implant and bone [21]. This is of the same order of the roughness as observed for both our modified surfaces MAT and MA/MAT. Contact angle measurements indicated that all the three experimental groups are fairly hydrophilic, as shown in Fig. 4. However, the uncoated alloy substrate had the highest contact angle and was somewhat less hydrophilic than the two oxide layers. Among the oxide layers the MA/MET coating showed slightly higher hydrophilicity and this behavior might be due to the nanostructure of the covering film. In addition, the fluorescence intensities of rhodamine-conjugated fibronectin reveal that more fibronectin was adsorbed on MA/MET group, as shown in Fig. 5. In fact, the proteins adsorption on a material surface occurs immediately when biomaterials exposes to the biological
Specimens were soaked in 20 μg/mL of rhodamine-conjugated fibronectin (Cytoskeleton, USA) dissolved in a solution of Dulbecco's phosphate buffered saline (DPBS, WELGENE Inc., Korea) at 37 °C for 4 h. After washing with pure DPBS for three times, samples were photographed by a fluorescence microscope (Leica, Germany).The amounts of adsorbed protein on the surface were semi-quantitatively determined by an Image J2X analysis software [8]. 2.4. Cell culture SaOS-2 cells (human osteogenic sarcoma cells) were seeded on 48 sample plates with a density of 5.0 × 104 cells/cm2. The culture medium was prepared by McCoy's 5A (Gibco®, USA) medium supplemented with 15% fetal bovine serum (FBS, Gibco®, USA) and 1% penicillin-streptomycin (Gibco®, USA). The cells were incubated in a 5% CO2 incubator at 37 °C. 2.5. Initial cell attachment 2.5.1. SEM observations The cell morphology of SaOS-2 was visualized by SEM after 4 h or 24 h after the initial cell seeding. Three material groups were investigated: a titanium alloy substrate without any modification (samples referenced as substrate), macroporous oxide surface after MAO (samples referenced as MAT) and macro/mesoporous modified surface after a MAO and subsequent EISA-film process (samples referenced as MA/ MET). The cells were fixed with 2.5% glutaraldehyde solution in DPBS overnight at 4 °C. Then, the samples were washed three times with pure DPBS to remove excess of glutaraldehyde solution. Specimens were dehydrated in sequentially increased concentrations of ethanol (from 30, 50, 75, 80, 90, 95 to 100%) followed by increased concentrations of tertiary butanol (from 25, 50, 75 to 100%) for 10 min each. Finally, samples were freeze dried before being sputter-coated with platinum and observed by SEM. 2.5.2. Cytoskeleton and nucleus staining in the absence or presence of ROCK inhibitor (Y-27632) The cells were cultured in the absence or presence of 10 μM Y-27632 (AbMole, USA) dissolved in dimetylsulfoxide (DMSO, Sigma-Aldrich, Biodee Biotechnology Co. Ltd, China). After 4 h and 24 h, the attached cells were fixed with 4% paraformaldehyde for 30 min. Then washed by pure DPBS for three times and permeabilized with 0.1% Trion X100 (Sigma-Aldrich, Sigma, USA) in DPBS solution for 5 min and washed by pure DPBS again. F-actin of the cells were stained with phalloidinTRITC (Sigma, USA) and nuclear DNA were stained with 4', 6diamidino-2-phenylindole (DAPI, Sigma-Aldrich, Sigma, USA). The morphologies of the cells attached to each specimen were observed using confocal laser scanning microscope (Zeiss 710, Germany). Attached cells were determined by measuring three randomly selected images of the DAPI staining. The attached cells were counted by the Image J2X software [19]. 2.6. Statistical analysis Means ± standard deviations (SD) of three independent replicates for each sample group were used for the calculated statistical analysis. One-way analysis of variance (ANOVA) followed by post hoc comparisons with least significant difference (LSD) method was performed to
3. Results
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Fig. 1. SEM images showing the surface morphologies at three different magnifications. The Ti-6Al-4V alloy substrate surface (a, b, c), the MAT surface (d, e, f) and the MA/MET surface (g, h, i).
environment. This layer of extracellular proteins offers cell recognition sites to promote cell adhesion by interacting with cell membrane integrins. SaOS-2 cells were cultured in DMEM solution in the absence
or presence of FCS (Fetal calf serum) by Degasne et al. [22]. They found that cells displayed a globular appearance and were piled and packed in the absence of FCS, while in the presence of FCS absorbed
Fig. 2. Cross-section of a MA/MET coating as seen by SEM (a); the three XRD patterns of Ti alloy substrate, MAT and MA/MET groups are compared (b); A TEM images of a flake of the mesoporous film showing randomly oriented mesopores (c) and the TEM diffraction rings of this film showing nanocrystallinity (d).
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Fig. 3. Surface roughness of the Ti alloy substrate surface (a), MAT surface (b) and MA/MET surface (c).
proteins can support formation of focal adhesion points resulting in good cell adhesion. Further, Huang et al. found that MG63 cells exhibit larger spreading area on micron/nano-topography surface in the presence of FBS compared to a surface without FBS [8]. These reports indicate that amounts and type of adsorbed proteins are important determining factors for cell behavior. Protein adsorption depends on surface properties such as roughness, microstructure, wettability and chemical properties. In the current work, the two oxide coatings have much larger surface roughness and area plus better wettability than the substrate. The MA/MET group with a mesoporous film has the largest specific surface area, which also leads to the high protein adsorption, cf. Fig. 5d. Cells will attach to the biomaterials surface within minutes, but it will take several hours to establish a stronger binding. Studies by Rajaraman divided the process of cell attachment and spreading into four phases: attachment, filopodial growth, cytoplasmic spreading and flattening of the central mass [23]. Different substrates lead to differences in the duration of these four phases and degree of overlapping. In this study, cells showed different developments and morphologies by time after the initial cell seeding, as demonstrated by the SEM micrographs in Fig. 6a–f. After 4 h, some of the cells on the substrate surface were still in a spherical appearance, but most cells were elongated in a spindle like shape and had long cell membrane protrusions. Cells on the MAT and MA/MET oxide surfaces were flattened with larger spreading areas compared to the substrate surface. However, the cells on the substrate surface would spread more by time, as seen after 24 h, cf. Fig. 6a and d. The substrate cells, however, had still less spreading area than those found for the MAT and MA/MET groups after prolonged time. Especially, the cells on the MA/MET surface had the largest and thinnest cytoplasmic layer (Fig. 6c and f). As seen above the initial cell attachment was enhanced on the MA/MET coating and so is the ability of rapid spreading and flattening following the suggested scheme by
Fig. 4. Contact angles of the substrate surface, MAT surface and MA/MET surface. * depicts statistical differences. Values are mean ± SD (n = 3); *p b 0.05; **p b 0.01.
Rajaraman [23]. It is generally believed that well spread cells with larger areas are more disposed to bone lineage [24]. Hence, the ability to promote cell spreading will have a positive impact on osteogenic inducing ability. In order to explore the underlying molecular mechanism of cell adhesion on the three different surfaces a specific inhibitor for the Rho-associated protein kinases (ROCKs) was used. It was added to the growth medium before the seeding of SaOS-2 cells and tested on the substrate and the two different oxide topographies. A synthetic chemical compound (abbreviated as Y-27632) was used to mark the conserved kinase domains of the ROCK's isoforms (ROCK-I and ROCK-2) [25]. It is known that the stability of cell adhesion and cell migration is highly dependent on the activation of RhoA [18]. Therefore, the downstream pathways of RhoA are of special interest in this study. The attached cells on the three investigated surfaces in the presence or absence (control) of Y-27632 after 4 h and 24 h were summarized in Fig. 7. Used as an internal reference, the numbers of cells found on the substrate after 4 h was considered to be 100%. In the absence of Y-27632, the MA/MET group showed the largest attached cell number after 4 h, cf. the staple diagram of Fig. 7. However, after prolonged time (24 h) there was no significant difference among the three groups. In the presence of Y-27632, the amount of attached cell was significantly reduced for all, but there were no significant differences among the three groups either at 4 h or 24 h. The observations clearly showed that the presence of the ROCK inhibitor impaired the cell attachment and, especially, offset the observed positive effect of the macro/mesoporous structure. Cell attachment and spreading are regulated by assembly and disassembly of the cell skeleton. Direct after the initial cell attachment and after 4 h or 24 h the F-actin cytoskeletal arrangement for all studied groups were illustrated in Fig. 8a-l. The cells on the relatively smoothed substrate surface formed only actin stress fibers after 4 h in the absence of a Rock inhibitor; indicated by green arrows in Fig. 8a. This shows that the cells are static and immobile on the surface. Whereas, the cells on the oxide coated samples behaved differently and were seen in Fig. 8e and Fig. 8i, respectively. Protrusions (white arrows), were observed in the cell membranes and these would direct the formation of lamellipods and filopodia. Stress fibers consist of large bundles of F-actin which would traverse the cells and anchor them at both ends to the substrate. Consequently, this hindered their migration through excessive formation of focal adhesion complexes [26]. After inhibition of ROCK by Y27632, an expected formation of finger-like filopodia was seen for the cells of all groups after 4 h, as shown by white arrows in Fig. 8b, f and j. This observation proved that the cells sensed the surface influence prior to migration. On the other hand, the cells seen on the two oxide surfaces had reduced sizes; as seen in Fig. 8f and j, respectively. This behavior indicated that the formation of cytoskeleton fibers was significantly influenced by the ROCK inhibition. On the other hand, cells on the substrate surface (still with ROCK inhibition) both spread and increased in area without well defined development of stress fiber bundles after 24 h (see in Fig. 8d). In contrast, the cells without ROCK inhibition had the same morphology but larger spreading areas as the one observed after 4 h (Fig. 8c). Compared to the smooth substrate
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Fig. 5. Fibronectin adsorption (red color) at the substrate surface (a), MAT surface (b) and MA/MET surface (c). A summary is given in (d). * depicts statistical differences. Values are mean ± SD (n = 3); ** p b 0.01.
surface (Fig. 8g) and MAT surface (Fig. 8g), the cells on the rougher MA/ MET (Fig. 8k) surface had larger spreading areas and several membrane protrusions. Stress fibers were observed on the MAT and MA/MET surfaces after 24 h (green arrows). When cells were treated with ROCK inhibitor for 24 h, they still remained attached to the surface, but loss their cytoskeleton completely for the MAT surface (Fig. 8h) and they shrunk drastically in sizes for the MA/MET surface (Fig. 8l). Based on cell morphologies (exposed by F-actin immunostaining) before and after ROCK inhibition, we can now conclude that the initial adhesion and spreading of SaOS-2 cells on either microtopography or micro/ nanotopography surfaces are highly dependent on the RhoA/ROCK pathway. However, the spreading of cells on the relatively smooth
substrate surface was not affected by ROCK inhibition, but the formation of stress fiber was suppressed. 4. Discussion Cell attachment and spreading depend on the formation of focal adhesion, which involves extracellular proteins adsorbed onto the surfaces, specific membrane receptors, and cytoskeletal proteins [27]. Proteins in an extracellular matrix, such as fibronectin, vitronectin and laminin, contain the specific amino acid sequence Arg–Gly–Asp or the RGD sequence and can be recognized by the cell membrane receptors. Establishment of mature and stable integrin adhesions is regulated by
Fig. 6. SEM micrographs illustrating the morphologies and spreading of SaOS-2 cells on the substrate surface at 4 h (a) and 24 h (d); the MAT surface at 4 h (b) and 24 h (e); and the MA/ MET surface at 4 h (c) and 24 h (f).
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Fig. 7. Statistical analysis of attached cells on the three surfaces, where attached cells on the substrate surface at 4 h is assigned as 100%. * depicts statistical differences. Values are mean ± SD (n = 3); *p b 0.05; **p b 0.01.
the RGD density. Surface properties significantly influence the concentration, conformation and bioactivity of adsorbed proteins contained in serum, which is reported responsible for enhanced cell functions [28]. An increase in adsorbed proteins provides more integrin anchorage sites for filopodia and lamellipodia extension and lead to larger cell spreading area. As mentioned and stressed above, the two oxide surfaces are much rougher than the smooth alloy substrate surface. It has been confirmed in literature that osteoblastic cell adhesion is correlated to surface roughness [29]. The macro/mesoporous structure has the highest specific surface area due to the presence of the mesoporous titania film. The greater surface area provided by pores can be synergistically exploited to link more efficiently bioactive molecules to metals [30]. The protein adsorption experiment (Fig. 5) showed that fibronectin adsorbed more on the macro/mesoporous coating compared to other
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topographies. The diameter of fibronectin is about 2 nm in its folded configuration and its length is 140 nm when it is unfolded due to its high molecular weight, 440 KDa [31]. Therefore, this important protein for initial cell adhesion has smaller diameter than the pore size of mesoporous titanium film which will support the early cell adhesion with the substrate, as revealed by increasing the number of attached cell to the mesoporous film 4 h after cell seeding (Fig. 7). It is worth mentioning that the adhesion of α5β1 and αVβ3 integrins to fibronectin activates RhoA effectors that regulate cell adhesion. This is done through downstream signaling by either activating ROCK or mDia [26,32]. The former one increases the stability of the actin filaments by inhibiting cofilin and enhances contractility of the cells by the activation of myosin II and, the later one, increases actin polymerization [24]. On the other hand, hydrophilicity of a titanium or titanium alloy surface has been widely identified as a key parameter influencing early cell adhesion and more hydrophilic surfaces are known to adsorb more extracellular matrix proteins [30]. This is correlated with our results showing that the macro/mesoporous structure has the highest hydrophilicity value and adsorbed more fibronectin than the macroporous structure or the substrate. Inhibition of ROCK activity reduces the number of adherent cells to a certain extent on all hydrophilic surfaces; however, it does not inhibit cell adhesion completely, as can be seen in Fig. 7. Extracellular stimulus can pass through the cytoskeleton and cause changes in gene expression related to cell adhesion. Mesoporous film of nanoparticles on an implant surface is indeed favorable to deliver small biological molecules and drugs to the site of implantation. Mechanotransduction signaling is generated within the cells when they sense different physical or chemical properties on the implant surface on direct contact. Different pathways act simultaneously to regulate the morphology of the cells by affecting cytoskeleton organization, cell contractility, cell organelles polarization, cell membrane protrusions, focal adhesion and stress fiber stability [33]. The shape of the cells, especially bone related cells, is essential in enhancing their biological function including proliferation and mineralization ability. The morphology of osteoblast can be altered by changing the shape and dimensions of nanoscale features superimposed on
Fig. 8. F-actin staining to visualize the attached cells on the smooth alloy substrate (a: 4 h without Y-27632, b: 4 h with Y-27632, c: 24 h without Y-27632 and d: 24 h with Y-27632). Similar on the MAT oxide surface (e: 4 h without Y-27632, f: 4 h with Y-27632, g: 24 h without Y-27632, and h: 24 h with Y-27632) and the MA/MET oxide surface (i: 4 h without Y-27632, j: 4 h with Y-27632, k: 24 h without Y-27632 and l: 24 h with Y-27632). Green arrow: stress fibers, white arrow: protrusions.
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microscale topography which consequently will alter the differentiation capacity of osteoblast and the formation of bone minerals [10]. Although there are many studies focusing on the extracellular process, the mechanism of how the topography induces intracellular changes has not been fully understood. In this work, we focused on the fabrication of mesoporous titanium film on a macroporous titanium oxide topography as well as the influence of the RhoA/ROCK pathways on the initial osteoblast-like cell adhesion. RhoA, Rac1 and Cdc42 are three small GTP phases belonging to Rho family which is well known to regulate cell adhesion and migration. RhoA and its downstream effector ROCK promote the stability of focal adhesions, cell contractility and the formation of stress fiber in the middle of the cells. The Rac1 and Cdc42 exert their function at cell periphery leading to the formation of nascent focal adhesion and development of filopodia and lamellipodia [34–36]. On the polished titanium alloy surface (Fig. 8a), it is clearly shown that stress fiber is formed after 4 h of cell seeding which indicates strong adhesion at an early point. After inhibiting ROCK, the formation of stress fiber is greatly affected. However, the protrusion of the cell membrane observed indicates that other pathways such as Rac and Cdc42 are still active [37]. The cells adhere and spread on the substrate after 24 h by the formation of filopodia and lamellipodia. Furthermore, it is also clear that the orientation of SaOS-2 cells on the smooth substrate surface is significantly affected by inhibiting ROCK (Fig. 8b and d). Based on many previous reports, the initial cell adhesion through RhoA/ROCK pathway is highly sensitive to the change in the scale of surface topography and roughness, which are consistent with our results. Since the quality of initial cell adhesion is crucial factor that positively influence bone mineralization capacity of osteoblasts, fabrication of dual layer of oxidized macroporous titania overlaid with a thin layer of mesoporous titania has shown an improvement in initial cell attachment. Therefore, this surface modification with hierarchical multiscale topography is believed to be promising to enhance the integration between titanium based implant and the host bone, which will be beneficial for dental and orthopedic applications. Additionally, mesoporous structure possesses an ability to serve as a drug carrier which can also endow this hierarchical structure an additional useful function such as antibacterial property by delivering bactericidal drugs. 5. Conclusions A dual-layer macro/mesoporous structured TiO2 surface has been successfully prepared through a combinational approach of micro-arc oxidation followed by the evaporation-induced self-assembly of nanocrystallites. The mesoporous layer contains pores with an average size of b10 nm and consists of anatase TiO2 nanocrystallites. The duallayer macro/mesoporous structured TiO2 surface, having a roughness very closed to that of macroporous TiO2 surface, demonstrates improved hydrophilicity and fibronectin adsorption than the macroporous surface. Initial cell attachment and spreading are enhanced on the duallayer macro/mesoporous structured surface. ROCK plays a major role in regulating morphology, attachment and spread of human osteogenic sarcoma cells (SaOS-2). Cell skeleton alterations are associated with the RhoA/ROCK pathway inhibitor Y-27632. Acknowledgement The authors are grateful for the financial support from National Natural Science Foundation of China (51361130032, 51472139), and International S&T Cooperation of the Ministry of Science and Technology of China (2015DFG52490). References [1] X. Liu, P.K. Chu, C. Ding, Surface modification of titanium, titanium alloys, and related materials for biomedical applications, Mater. Sci. Eng. R 47 (2004) 49–121.
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