Osteointegration of titanium implant is sensitive to specific nanostructure morphology

Acta Biomaterialia 8 (2012) 1976–1989

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Osteointegration of titanium implant is sensitive to specific nanostructure morphology V.V. Divya Rani, Lakshmanan Vinoth-Kumar, V.C. Anitha, Koyakutty Manzoor, Menon Deepthy ⇑, V. Nair Shantikumar ⇑ Amrita Institute of Medical Sciences & Research Centre, Amrita Centre for Nanosciences & Molecular Medicine, Amrita Lane, Ponekkara P O, Kochi, Kerala 682041, India

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Article history: Received 23 September 2011 Received in revised form 17 January 2012 Accepted 18 January 2012 Available online 28 January 2012 Keywords: Nanosurface modification Titanium Hydrothermal Implant In vivo osteointegration

a b s t r a c t An important aspect of orthopedic implant integration is the enhancement of functional activity of osteoblasts at the tissue–implant interface without any fibrous tissue intervention. Nanostructured implant surfaces are known to enhance osteoblast activity. Previously, we have reported a simple hydrothermal method for the fabrication of non-periodic nanostructures (nanoscaffold, nanoleaves and nanoneedles) on titanium implants showing good biocompatibility and a distinct osteoblast response in vitro in terms of osteoblast adhesion to the surface. In the present work, these nanostructures have been evaluated for their detailed in vitro cellular response as well as in vivo osteointegration. Our studies showed that a specific surface nanomorphology, viz. nanoleaves, which is a network of vertically aligned, non-periodic, leaf-like structures with thickness in the nanoscale, provided a distinct increase in osteoblast cell proliferation, alkaline phosphatase (ALP) activity and collagen synthesis compared to several other types of nanomorphology, such as nanotubes, nanoscaffold and nanoneedles (rods). Gene expression analysis of ALP, osteocalcin, collagen, decorin and Runx2 showed 20- to 40-fold up-regulation on the leaf-like topography. Cytoskeletal arrangement studies on this substrate again revealed a unique response with favorable intracellular protein expressions of vinculin, FAK and src. In vivo osteointegration study over 12 weeks on rat model (Sprague–Dawley) showed early-stage bone formation (60% bone contact by week 2 and 85% by week 8, p < 0.01) in the leaf-like nanopattern, without any inflammatory cytokine production. Ó 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction The success of any orthopedic or dental implantation procedure is based on the formation of an effective interface between the surface of the implant material and the bone tissue, without any fibrous tissue intervention [1]. Current orthopedic implants are limited by the lack of appropriate cell adhesion and osteointegration, leading to reduced implant lifespan. Improvements in implant surface topography as well as surface chemistry have been established as ways to improve bone bonding [2–4]. Recent efforts in this field have highlighted the importance of nanotechnology in altering the surface topography of materials such as metals, ceramics, polymers and composites to better mimic the surface roughness features of natural bone [5–10]. The principle behind surface structural modifications on implants at the nanoscale is that such surfaces would mimic the extracellular matrix with which cells normally interact and hence would favor positive interaction with cells [11]. Indeed, ⇑ Corresponding authors. Tel.: +91 484 4008750; fax: +91 484 2802030. E-mail addresses: [email protected], [email protected] (M. Deepthy).

this principle has been verified in several cases. Various studies have shown that surface energy and nanotopography influence the type, quantity and conformation of adsorbed protein, and control cellular adhesion to the surface [12–17]. Specifically, the active site of vitronectin (RGD sequence) has been found to be more exposed on nanophase ceramics than on conventional ceramics [7,18]. Studies on lithographically patterned nanofeatures have shown that a periodic pattern of 400 nm dots enhanced osteoblast differentiation of human mesenchymal stem cells (hMSCs), but not when cultured on 150 and 600 nm dot patterns [19]. Similarly, periodic, vertically arranged nanotube arrays, with less than 30 nm spacing and varying pore diameters (<100 nm), enhanced osteoblast cell functions [20], as well as endothelial cell proliferation without vascular smooth muscle proliferation [21]. Dalby et al. [5], in a detailed investigation on patterned polymeric surfaces with disordered arrangement of dots in square arrays, having a displacement of 50 nm between dots, showed enhanced osteoblast differentiation of hMSCs compared to an ordered substrate. All these reports highlight the significance of nanoscale substrate topographies in controlling cellular response. In our previous study [22], we reported a simple, scalable, inexpensive and one-step wet chemical (hydrothermal) method for the

1742-7061/$ - see front matter Ó 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2012.01.021

V.V. Divya Rani et al. / Acta Biomaterialia 8 (2012) 1976–1989

generation of non-periodic, homogeneous nanostructured morphologies, having similar surface chemistry, on metallic titanium surface. In that study, initial cell adhesion characterization revealed an enhanced, mature focal adhesion formation of cells on non-periodic, nanoleaf-like morphology over all other nanotopographies. A more in-depth understanding of the biological activity of such a specific nanomorphology would have considerable industrial and scientific importance. Accordingly, in the present study, a detailed investigation of osteoblast cell-surface interaction on these hydrothermally modified, disordered nanomorphologies was conducted both in vitro and in vivo. The results are compared with the corresponding results on ordered, vertically oriented nanotube structures, with nanopolished Ti as a control. The investigation included cytoskeletal arrangement and early signal transduction events, as well as gene expression changes, which offered clues on the nature of osteoblast cell response obtained on the non-periodic nanomorphologies. 2. Materials and methods 2.1. Hydrothermal synthesis and characterization of nanostructures Three different non-periodic nanostructures were used in this work, viz. nanoscaffold (NS), nanoleaf (NL) and nanoneedles (NN), which were synthesized on commercially pure titanium plates (Grade II, ASTMF-67) by a hydrothermal processing technique using 0.5 N NaOH established by our group previously [22]. The detailed protocol is given as supplementary information. TiO2 nanotubes (NT) were prepared by electrochemical anodization of Ti in 0.5% HF for 1 h [23]. Nanomodified as well as control polished Ti plates were used for all the in vitro experiments carried out in this investigation. For in vivo experiments, commercially pure Ti screws of 4 mm length and 2 mm diameter were used. As before, the screws were first cleaned thoroughly to remove any surface contaminants. They were then subjected to hydrothermal modification as detailed above. The morphology of the as-synthesized TiO2 nanostructures on the commercial Ti screws was characterized by scanning electron microscopy (SEM), using a JEOL JSM-6490L analytical scanning electron microscope. Surface characteristics such as surface area, contact angle, surface roughness and mechanical strength were analyzed (Supplementary information). All the samples were subjected to ultraviolet sterilization under identical conditions before the in vitro and in vivo studies. 2.2. Cell culture Human hipbone primary osteoblastic cells (pHOB), purchased from Promocell, Germany, were used in this study. The cells were maintained in specified Promocell osteoblast growth medium (without antibiotic) supplemented with recommended Promocell osteoblast supplement mix for their normal growth. The cells were incubated in a CO2 incubator with 5% CO2. After reaching confluency, the cells were detached from the flask with trypsin–ethylenediaminetetraacetic acid (EDTA) (Sigma–Aldrich, USA) and then neutralized with trypsin-neutralizing solution. The cell suspension was centrifuged at 3000 rpm for 3 min and then resuspended in growth medium for further studies. 2.3. Cell morphology and cellular proliferation studies For cell morphology studies using SEM, cells (15,000 cells cm2) were seeded on titanium plates and incubated for 24 and 96 h. After incubation, titanium plates were washed with phosphate-buffered saline (PBS), and cells were fixed with 2.5%

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gluteraldehyde solution (Sigma–Aldrich, USA) for 1 h. Following three rinses with PBS, the samples were dehydrated in an ethanol series (75, 80, 90 and 100%) twice. Samples were dried overnight and mounted on aluminium stubs, sputter-coated with platinum and observed under SEM. The cell proliferation of primary osteoblast cells on surfacemodified Ti samples was assessed and compared with that of control polished Ti using an alamarBlue assay. Briefly, cells were seeded at a density of 15,000 cells cm2 onto the Ti samples placed in a 12-well plate and cultured for up to 7 days in osteoblast-specific medium with supplement mix under standard culture conditions. At different time intervals, viz. days 3, 5 and 7 of incubation, the cells were incubated with 10% alamarBlue (Invitrogen, USA) in complete medium for 4 h. After incubation, the medium was pipetted into 96-well plates, and the optical density was recorded using a microplate spectrophotometer at 570 nm, with 600 nm set as the reference wavelength. Results are presented as the mean ± standard deviation of three independent analysis performed in triplicates. 2.4. Total intracellular protein, alkaline phosphatase (ALP) activity and intracellular collagen content Osteoblast cells (15,000 cells cm2) were seeded on all the titanium plates and incubated for 7 and 14 days. The medium was replaced every other day. At the end of the prescribed time period, the plates were rinsed three times with tris(hydroxymethyl)aminomethane (Tris)-buffered saline and the cells were lysed using 1% Triton X-100, followed by sonication. The amount of total protein content in the cell lysates was determined spectrophotometrically using a bicinchoninic acid (BCA) protein assay method. For this, 25 ll of each sample and 200 ll of BCA reagents A and B (Sigma Aldrich, USA) at a 50:1 ratio were incubated at 37 °C for 30 min. The absorbance of the samples was then recorded using a spectrophotometer at 562 nm. The total intracellular protein synthesized by osteoblasts cultured on the substrates was determined from a standard curve of absorbance vs. known concentrations of albumin run in parallel with experimental samples. To assess the total intracellular collagen content, 50 ll of osteoblast cell lysate prepared as described earlier was added to each well of a 96-well plate. The collagen was allowed to dry on the plate through incubation at 37 °C for 16 h and was incubated at 37 °C for 24 h in the presence of silica (desiccant). The 96-well plate was subsequently rinsed three times with distilled water before 100 ll of 0.1% Sirius red stain (Sirius red powder in picric acid, Sigma, USA) was dispensed into each well and incubated for 1 h at room temperature. Each well was then washed five times with 200 ll of 0.01 M HCl for 10 s per wash. About 200 ll of 0.1 M NaOH was added to each well for 5 min. Finally the solution in each well was mixed, transferred to a second plate and the optical absorbance was recorded at 540 nm in a spectrophotometer. The total intracellular collagen synthesized by osteoblasts cultured on the substrates was determined from a standard curve of absorbance vs. known concentration of collagen run in parallel with experimental samples. The concentration of collagen was normalized with the total protein content (per lg). A 25 ll aliquot of the prepared cell lysate was transferred to a 96-well plate and 100 ll of paranitrophenylphosphate substrate (Sigma-Aldrich, USA) substrate was added and incubated at 37 °C for 30 min. Optical absorbance at 650 nm was recorded spectrophotometrically and the ALP synthesized by osteoblasts cultured on substrates was determined from a standard curve of absorbance vs. known standards of ALP enzyme run in parallel. The ALP concentration was normalized with the total protein content (per lg). Results are presented as the mean ± standard deviation of three independent analysis performed in triplicates.

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2.5. Focal adhesion and cytoskeletal arrangement Focal adhesion points and cytoskeletal arrangement of pHOBs cultured on modified Ti samples were analyzed by immunocytochemistry. Samples were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS for 15 min at room temperature and then rinsed three times with PBS. Non-specific binding sites were blocked by incubating the samples with 3% bovine serum albumin (BSA) in PBS for 30 min at room temperature. The cells were stained with mouse monoclonal anti-human vinculin antibody (Sigma-Aldrich, USA) for 60 min at room temperature followed by three rinses with 1% BSA in PBS. The cells were then incubated with fluorescein isothiocyanate-conjugated anti-mouse IgG antibody (Sigma-Aldrich, USA). To detect polymerized actin, the cells grown on titanium plates were incubated with tetramethyl rhodamine isothiocyanate (TRITC)-conjugated phalloidin (SigmaAldrich, USA) for 25 min and the nucleus was stained with 40 ,6-diamidino-2-phenylindole (Sigma-Aldrich, USA). The samples then were rinsed with PBS and mounted using glycerol. The cells were examined using an Olympus BX-51 epifluorescent microscope equipped a cooled color CCD camera (Model DP71) and 63 and 100 oil immersion objectives. High-magnification immunofluorescence images were used to study the focal adhesion points and actin arrangement using band-pass excitation and emission filters (BP 480–550 and 460–490 nm excitation respectively). 2.6. Gene expression For gene expression analysis, total RNA was extracted from the cells and reverse transcribed to cDNA. Quantitative real-time reverse transcription–polymerase chain reaction (RT-PCR) experiments were performed using an Applied Biosystems RT-PCR system (7300-RT-PCR) according to the manufacturer’s instruction. The detailed protocol for RNA isolation, reverse transcription and RT-PCR is detailed in the supplementary data. The levels of expression of each sample were normalized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression and the results were obtained by the comparative method of relative quantification. Statistical analysis (n = 9, 3 groups repeated 3 times) of 2DDCT was performed by one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test to a confidence level of p < 0.05.

Institute of Medical sciences and Research Centre, Kochi. Thirty 4-week-old male Sprague-Dawley rats were used for the study. The animals were divided into three groups, viz. weeks 2, 8 and 12, with 10 animals in each group. Each of the surface-modified implants was used in duplicate for each time point mentioned above. Animals were anaesthetized with 0.5 ml of xylazine (20 mg/ml, 0.2 ml) and ketamine (50 mg/ml, 0.3 ml) injection, and their legs were shaved and scrubbed with betadine. The implants were uniformly placed in the outer lateral side of the left femur condyle of all animals. Animals were sacrificed after 2, 8 and 12 weeks of implantation respectively. Euthanasia was done by administering an overdose of xylazine and ketamine. The condyle region with the implant was then removed and fixed in neutral buffered formalin for 7 days, then sectioned for histopathological analysis to be carried out. The surgical implantation procedure and histomorphometrical analysis are given as supplementary information. 2.9. In vivo inflammation studies To understand whether the implanted nanosurface-modified Ti induced any inflammation in the animals, the serum cytokine level was analyzed using BD™ Cytometric Bead Array rat inflammation flex sets (CBA,BD Biosciences, USA) in FACS Aria (BD Biosciences, USA). Before sacrificing the animals at 12 weeks, 1 ml of blood was collected in vacutainers by retro-orbital bleeding. The tubes were centrifuged at 10,000 rpm for 3 min to separate the serum, which was stored at 20 °C for analysis. 2.10. Statistical analysis All of the above-mentioned in vitro and in vivo analyses were performed in triplicate in three independent experiments. Student’s t-test was used to evaluate the statistical significance for in vitro experiments and one-way ANOVA and Tukey’s multiple comparison test were used for in vivo experiments. A probability of p < 0.05 was considered statistically significant. 3. Results and discussion 3.1. Preparation of nanostructured Ti screws and their characterization

2.7. Western blot For analysis of focal adhesion components, whole cell lysate was collected. Cells were washed with PBS and lysed with ice-cold modified RIPA buffer (10 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate, protease inhibitors and phosphatase inhibitors). For this, 20 lg of total cell lysate was separated by denaturing sodium dodecyl sulphate–ppolyacrylamide gel electrophoresis, electroblotted onto PVDF membrane and blocked with 5% non-fat milk. The membrane was incubated with specific primary antibodies (mouse monoclonal anti-vinculin antibody (Sigma-Aldrich, USA), rabbit polyclonal FAK, rabbit polyclonal p-FAK, rabbit polyclonal src, rabbit polyclonal p-src (Cell Signaling Technology, Inc, Danvers, MA) and mouse monoclonal GAPDH antibody (SigmaAldrich, USA)). Specific protein bands were detected using horseradish peroxidase-conjugated secondary antibodies (Santacruz biotechnology, USA) and Immobilin western chemiluminiscent substrate (Millipore, USA). GAPDH was used as loading control. 2.8. In vivo osteointegration study All the animal experiments were carried out after Ethical Committee approval from the Animal Ethical Committee at Amrita

A range of unique nanostructures were fabricated on clinically used titanium implant screws by a single-step hydrothermal technique, similar to our earlier work on Ti plates [22]. We obtained identical structures, which included dense, non-periodic, but homogeneous morphologies (NL, NN, NS), all having similar surface chemistry. One set of nanotubular titania samples were also prepared by the process of anodization of Ti in hydrogen fluoride [23]. Physicochemical characterization using SEM, X-ray diffraction and energy-dispersive spectroscopy carried out on the modified Ti samples confirmed that all hydrothermally prepared substrates were a mixed phase of rutile and anatase titania, and exhibited excellent structural stability in physiological medium [22]. Titania NT with a pore size (diameter) of 60–80 nm and length 100 ± 5 nm, created by applying an anodization voltage of 20 V for 1 h in an aqueous electrolyte of 0.5 wt.% HF, were used for our experiments [23]. Energy-dispersive analysis of these NT showed that the elements present on the anodized Ti were titanium and oxygen, chemically as TiO2 (data not shown), and the SEM image (Fig. 1b) reveals its well ordered structure. Fig. 1 represents the scanning electron micrograph images of hydrothermally modified nanostructures (NS, NN, NL) fabricated on Ti screws as well as the anodized TiO2 NT selected for the present in vivo work. Similar structures fabricated on titanium plates as reported earlier

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Fig. 1. Representative SEM images of (a) nanopolished Ti screw: (i) low magnification image and (ii) featureless surface morphology of nanopolished Ti. (b) Nanostructural features generated on surface modified Ti screws: (i) NT, (ii) NS (iii) NL and (iv) NN.

Table 1 Surface area, contact angle, surface energy, surface roughness and critical load for adhesive failure values of different nanomodified Ti and control Ti plates. Samples

Surface area of samples (cm2)

Contact angle (degrees)

NP NT NS NL NN

*

1.96 ± 0.130 3.08 ± 0.112 2.84 ± 0.156 * 4.01 ± 0.099 * 6.41 ± 1.140

79.2 ± 0.14 75.0 ± 0.71 76.9 ± 1.40 43.2 ± 0.98 9.30 ± 1.20

*

Surface roughness/lm

Critical load (N)

0.05 ± 0.01 0.44 ± 0.02 0.16 ± 0.01 * 0.28 ± 0.03 * 0.94 ± 0.05

– 0.26 ± 0.01 0.33 ± 0.02 0.44 ± 0.02 0.14 ± 0.01

* *

Statistical significance was assessed relative to control nanopolished Ti for each nanostructures. p < 0.05.

*

Table 2 Concentration of fibronectin and vitronectin protein adsorbed as well as cellular proliferation of different nanomodified Ti and control Ti plates. Samples

Protein adsorption (lg/ml)

Cellular proliferation (OD at 570 nm)

Fibronectin

NP NT NS NL NN

Vitronectin

1h

6h

12 h

1.47 ± 0.31 1.57 ± 0.06 1.44 ± 0.04 * 2.41 ± 0.11 1.29 ± 0.21

*

2.41 ± 0.09 2.70 ± 0.10 3.66 ± 0.11 * 3.97 ± 0.06 3.80 ± 0.20

2.50 ± 0.27 3.05 ± 0.49 3.65 ± 0.13 3.43 ± 0.15 2.94 ± 0.67

*

1h

6h

12 h

3rd day

5th day

7th day

1.47 ± 0.01 2.15 ± 0.01 1.61 ± 0.03 * 2.80 ± 0.02 * 2.62 ± 0.01

3.51 ± 0.07 3.68 ± 0.09 3.07 ± 0.07 3.70 ± 0.19 3.98 ± 0.04

3.45 ± 0.05 3.61 ± 0.15 2.96 ± 0.22 3.67 ± 0.31 3.81 ± 0.12

0.18 ± 0.02 0.16 ± 0.01 0.18 ± 0.01 0.19 ± 0.02 0.16 ± 0.01

0.34 ± 0.02 0.43 ± 0.01 0.42 ± 0.03 0.48 ± 0.01 0.22 ± 0.02

0.48 ± 0.02 0.49 ± 0.03 0.50 ± 0.02 * 0.65 ± 0.02 0.31 ± 0.02

*

Statistical significance was assessed relative to control nanopolished Ti for each nanostructures. p < 0.05.

*

[22] were used for all the in vitro studies presented here. A nanopolished Ti plate or screw served as the control substrate in all our experiments. Low-magnification SEM images did not reveal any differences between the modified and unmodified Ti screws (Fig. 1). It has been proven that this hydrothermal modification technique can be extended to other metallic surfaces, such as that of the titanium alloy TAV, stainless steel and nitinol, implying the utility of this technique for generating nanostructured metallic implants for biomedical applications [24]. 3.2. Surface area, contact angle, surface roughness and mechanical strength To further characterize the surface property of our developed nanomodified Ti, a dye adsorption test was carried out to find

out the surface area (Supplementary data provided). The results of the dye adsorption analysis are summarized in Table 1. It should be noted that all the nanosurface-modified samples have a statistically significant increase in surface area in comparison to the nanopolished Ti surface, implying that the surface modification alters the surface area. The amount of dye adsorbed as well as the surface area calculated were observed to be the highest for NN amongst all the tested samples, perhaps owing to its vertically arrayed onedimensional nature, which projects various surfaces to the outside. Surprisingly, anodized nanotubular Ti displayed a lower surface area in comparison to the hydrothermally modified NL-like structure, possibly due to the short length of the periodically arrayed NT used in the study (<100 nm). However, the surface area of nanotubular Ti was measured to be higher than that of NS, perhaps due to its tubular geometry.

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Contact angles were measured on the different nanomodified surfaces along with the control Ti surface using water as the medium [25], and are depicted in Table 1. It was noted that different nanomodified surfaces exhibited significantly different contact angles. The Ti control sample showed an average value of 79° with water as the contacting medium, while NT and NS patterned surfaces showed values in the range of 75–76°. A considerably different contact angle of 44° was measured on the nanoleafy patterned surface. In contrast to all these, NN Ti showed the lowest contact angle of 9°. Further, the average surface roughness measured using optical profilometry (Table 1) revealed that nanomodified surfaces showed a statistically significant difference in surface roughness in comparison to the control polished Ti. Amongst the various nanostructures, NN showed the highest average roughness value (0.94 ± 0.05 lm). The surface roughnesses of the nanosurfaces used in the present study were in the order: NN > NT > NL > NS > nanopolish. Hence, it can inferred that the size of the nanoscale features on the Ti nanostructures altered their water contact angles and thereby their hydrophilicity, mainly due to the changes in surface area and surface roughness. It has been reported that superhydrophobicity or superhydrophilicity provided by the nanostructures can alter the protein conformation [26]. Kasemo and Lousmaa [27,28] observed that surfaces which do not recruit proteins or adsorb proteins strongly with altered conformation do not permit good cell adhesion. The results of our study enable us to conclude that variations in nanotopography of metallic Ti influence its wettability and are thereby expected to significantly alter its biological functions. The critical load (Lc) in the scratch adhesion test is a measure of the adhesive strength in the absence of complete delamination of the film from the substrate [29]. No statistically significant difference in critical load to adhesive failure was observed on the different nanomodified titania samples studied. However, amongst all the modified surfaces tested, it was found that the critical load to adhesive failure was higher in hydrothermally modified NS and nanoleafy structures, with values 0.33 ± 0.0246 and 0.44 ± 0.023 N respectively (Table S1). In the case of anodized nanotubular titania, the critical load to adhesive failure was 0.26 ± 0.019 N. Nevertheless, the hydrothermally modified NN Ti surface showed a lower critical load of 0.14 ± 0.014 N, implying a reduced adhesion strength, which can be attributed to the greater thickness and roughness of the NN surfaces. A recent report from Krishna et al. [30] showed that a rough Ti surface (0.92 lm thick) prepared on stainless steel can act as a stress raiser, which deteriorates the anti-scratch performance. This may be due to the presence of a mechanically non-adherent layer on the metallic substrate, as in our case with the NN surface. Thus, it can be concluded that the non-periodic nanoleafy patterned Ti presents a stable oxide layer which adheres well to the metallic substrate. The lowest mechanical adhesion was measured on the NN topography, possibly due to the comparatively greater thickness of the oxide layer. This implies that, amongst the various nanostructured Ti substrates, nanoleafy Ti has the optimal mechanical adhesion, suggesting it to be an ideal biomaterial for implant applications. 3.3. Fibronectin and vitronectin adsorption The biocompatibility of an implant material depends, in part, upon the capacity of the material’s surface to adsorb the endogenous proteins that regulate cell behavior. A time-lapse protein adsorption study at different time intervals, viz. 1, 6 and 12 h, was therefore carried out. The adsorption of specific cell adhesion extracellular matrix proteins from fetal bovine serum, such as fibronectin and vitronectin, on titanium plates was found to increase with incubation time, as is evident from Table 2. It was observed that both fibronectin and vitronectin adsorption reached

a plateau at 6 h of incubation on modified as well as control titanium plates, implying a saturation effect at longer incubation times. Pro-adhesive proteins such as fibronectin and vitronectin are abundant in blood and may play an important role in mediating cell/biomaterial interactions by providing integrin binding sites for cell adhesion [31,32]. An accepted theory is that any surface adsorbs proteins with the highest arrival rate, but these are gradually displaced by molecules with a higher affinity for the surface [27]. Amongst the nanotopographies studied, all the hydrothermally modified nanostructures showed a nearly 1.5-fold increase in fibronectin adsorption than on the control polished surface at 6 h. Likewise, vitronectin adsorption was found to be significantly higher (2-fold) on nanoleafy as well as NN structures even after 1 h, with respect to the control. This can be attributed mainly to the increased surface area to volume ratio of these nanostructures in comparison to all the other samples. Dolatshahi-Pirouz et al. [33] recently evaluated the adsorption and availability of fibronectin cell-binding domains on nanostructured tantalum surfaces using ellipsometry and showed that differences in structural dimensions at the nanoscale influence fibronectin mass uptake and the availability of cell-binding domains. Woo and colleagues [34] reported that three-dimensional nanofibrous scaffolds selectively adsorbed more proteins, including fibronectin and vitronectin, compared with a solid pore wall consisting of the same material. Similarly, an increased unfolding of vitronectin was reported by Webster et al. [18,35]. Likewise, the differences in adsorption of fibronectin and vitronectin on different periodic as well as non-periodic nanostructured surfaces in the present work can be attributed to the subtle changes in surface topography, which in turn alter the affinity of proteins on these substrates. 3.4. Cellular proliferation Hydrothermally modified Ti surfaces were observed to be cytocompatible when tested using primary osteoblast cells in our early investigation [22]. In the present work, we analyzed the influence of non-periodic arrays of nanostructures on cellular proliferation in comparison to the periodic nature of NT as well as nanopolished Ti after 3, 5 and 7 days of incubation using alamarBlue assay. Statistically significant (p < 0.05), enhanced proliferation was observed on nanoleafy surface Ti in comparison to the control (Table 2). No significant differences in cell number were observed until day 3 of incubation. However on days 5 and 7, the proliferation rate was measured to be higher on the nanoleafy surface amongst all substrates and the control (Table 2). These results correlate well with the enhanced protein adsorption measured on nanoleafy samples. However, cells grown on NN surfaces showed a significant reduction in proliferation, despite high protein adsorption. The SEM images in Fig. 2a and b clearly reveal the differences in cell density on different surfaces at different time points. Our results are consistent with previous studies reporting that nanostructuring substantially alters the proliferation of fibroblast and osteoblast cells in comparison to flat substrates [36–41]. However, it has also been pointed out by several researchers that the size, shape, spacing and randomness (disorder) of nanofeatures have a tremendous influence on cellular behavior [5,42]. The important parameters that influence the interaction of cells with nanostructured surfaces include surface topography, surface energy and protein adsorption, as well as its conformation and formation of focal adhesion points [11–13,17,18]. It has been reported that an increase in surface area as observed for nanostructured surfaces enhances the amount of proteins adsorbed onto the surface, which in turn influences cellular adhesion and proliferation [11–13,17,18]. In the present study, although the amount of vitronectin and fibronectin adsorbed were significantly higher on all

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Fig. 2. SEM images of cellular adhesion of primary osteoblast cells cultured on nanomodified Ti (a) after 24 h and (b) after 96 h. (a) Nanopolish, (b) NT, (c) NS, (d) NL and (e) NN. Lane 1, 2 and 3 represents magnifications of 30,1000 and 10,000, respectively.

nanomodified surfaces (Table 1), the cellular response was measured to be different for the different surface topographies. Specifically, the cellular response (proliferation) was relatively less on the NN surface, possibly due to the differences in the wettability (superhydrophilicity) of the surface, which alters its protein adhesion characteristics [27,28]. In addition, cell spreading is also regulated to a great extent by the clustering of integrins, which in turn is dictated by the spacing as well as arrangement of surface nanofeatures, as observed by Kunzler et al. [42]. It is possible that the combined effect of surface topography, surface roughness, surface area and hydrophilicity is responsible for the enhanced cellular behavior observed on nanostructured samples. Our results provide supporting evidence for this observation, with the non-periodic nature of the NL-like patterns, together with its varying surface characteristics, promoting a rapid proliferation of osteoblast cells in comparison to other nanostructures. Even though the influence of nanofeatures on cell behavior can be explained by several different possible mechanisms, it is speculated that osteoblast functions could be enhanced by unique nanometer surface features and/or their disorder [4,43], as in our case with nanoleafy architecture. 3.5. Osteoblast long-term functions For a detailed understanding of the cell–nanostructure interaction, it is important to analyze osteoblast long-term functions such as ALP activity and collagen production. ALP is an enzyme whose synthesis indicates the differentiation of osteoblasts from non-calcium-depositing to calcium-depositing cells [44]. In the present study, we observed a significantly higher level of ALP (p < 0.01) production by the cells grown on nanoleafy structures compared to other samples after 14 days of growth (Fig. 3a). Similar effects were observed for the intracellular collagen production. After 7 and 14 days of culture, osteoblast cells grown on nanostructured surfaces produced a significantly higher (p < 0.05 and p < 0.01 respectively) amount of collagen (Fig. 3b). Our results are consistent with the observations of Webster et al. [45–47] that nanophase titania enhances alkaline phosphatase activity and extracellular calcium deposition over conventional titania. Several previous in vitro cell biology reports show that cells can respond to the shape of their environment [5,48].

The modulation of adsorbed extracellular matrix (ECM) protein conformation by the underlying substrates can alter the integrin binding and signaling, which in turn directs the function/activity of cells [49]. A recent report by Binulal et al. [50] revealed the influence of in vivo regulators in an in vitro setting with hMSCs for bone tissue engineering on polycaprolactone (PCL) nanofibrous matrices. They reported that more cells on nanofibrous PCL were found to differentiate into the osteoblast lineage and subsequently mineralize upon addition of in vivo osteogenic regulators than on microfibrous scaffold. Thus, it is possible that the unique nanotopography of any surface primarily modulates the adsorption of ECM proteins in its proper conformation and thereby enhances cellular adhesion and its subsequent function. Thus, our results on the remarkable differences in protein adsorption and thereby osteoblast proliferation on nanoleafy surfaces in comparison to other nanostructures is justifiable. 3.6. Gene expression analysis Although the interaction between cells and biomaterials is observed to be regulated by nanoscale features [51], the mechanism behind enhanced osteoblast functions due to these nanofeatures is still poorly understood. To confirm the enhanced osteoblast function obtained in our study, gene expression analysis was carried out using RT-PCR. The pattern of five osteoblast specific genes, viz. ALP, osteocalcin (OCN), collagen type 1 (COL), decorin (DCN) and RunX2 transcription factor, in primary osteoblast cells cultured on various nanostructured surfaces were compared with nanopolished Ti. ALP, COL, OCN and DCN are matrix-mineralizing proteins, and their expression has been proved to be significant for bone matrix assembly [52]. At 7 days of culture, the cells on nanoleafy surfaces expressed 2-fold higher ALP mRNA than control titanium, which increased nearly 10-fold after 14 days of incubation (Fig. 4a). However, the ALP gene expression was more or less reduced in cells cultured on all other nanofeatured surfaces. OCN and collagen are two extracellular matrix proteins secreted by osteoblast cells, whose expressions are enhanced during bone formation [53]. In this study, both collagen and OCN genes showed a significant upregulation when compared to cells cultured on the control surface.

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Fig. 3. (a) ALP and (b) COL production of primary osteoblast cells cultured on nanomodified titanium in comparison to control polished titanium. Statistical significance was assessed relative to control nanopolished Ti for each nanostructures. ⁄p < 0.05.

Fig. 4. Gene expression analysis of primary osteoblast cells cultured on different nanomodified Ti surfaces after 7 and 14 days of growth using RT-PCR: (a) ALP, (b) OCN, (c) COL, (d) DCN and (e) Runx2 transcription factor. Statistical significance was assessed relative to control nanopolished Ti for each nanostructures. ⁄p < 0.05.

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Fig. 5. Immunofluorescent staining images of vinculin focal adhesion points (FAPs) after 30 min, 1 h and 5 h. Scale bar = 5 lm.

The OCN mRNA level was 35.4 ± 8.03-fold higher for cells cultured on nanoleafy surface, whereas only a 2- to 3-fold higher expression was measured for the cells cultured on nanotubular surface after 14 days. In contrast, this gene was not observed to be up-regulated on NS surface after 7 or 14 days of culture. Likewise, the gene expression on NN surfaces did not reveal considerable up-regulation after 14 days, even though an initial increase was noted (Fig. 4b). A similar trend was observed for the collagen mRNA expression on all the nanostructured surfaces in comparison to control. Collagen levels for cells cultured on nanoleafy surfaces exhibited a sharp increase, from 2.3 ± 1.4 to 31.1 ± 4.5-fold (p < 0.05), after 14 days. In contrast, no change in gene expression was revealed on nanotubular surfaces after 7 or 14 days of culture (Fig. 4c). It was surprising to observe that for NS as well as NN surfaces collagen mRNA expression was reduced on day 7, with the levels reaching that the same as the control Ti after 14 days. DCN is one of the proteoglycans synthesized by osteoblast cells and is responsible for collagen assembly during bone matrix formation [54]. We noted a statistically significant 37 ± 9.7-fold (p < 0.05) up-regulation of DCN mRNA on nanoleafy substrates after 14 days of incubation (Fig. 4d). However, the gene expression on nanotubular and NN surfaces exhibited only a 4.6 ± 1.7-fold increase after 7 days of culture, with a downward trend for longer incubation periods (14 days). RunX2 expression was also significantly higher (17.8 ± 1.4-fold, p < 0.05) on nanoleafy surfaces after 7 and 14 days of growth (Fig. 4e). Nevertheless, cells grown on nanotubular Ti showed an initial increase of mRNA level on day 7, with a subsequent decline in its expression level. Various researchers have demonstrated the beneficial effects of osteoblast differentiation and bone formation around nanostructured Ti implants, both in vitro and in vivo [55–59]. The role of

nanoscale structures specifically on osteoblast gene expression has been investigated by a group of researchers on a variety of materials [56,57]. The influence of nanopatterns studied by Dalby et al. [5] on lithographically modified polymeric substrates showed increased expression of OCN and ALP (10- and 5-fold respectively) above the flat control. A recent report by Hori et al. [57] revealed an enhanced expression of OCN and ALP genes on hybrid micro–nanostructured TiO2. Likewise, the changes in mRNA expression observed in our study on osteoblast differentiation can be ascribed to the influence of the nanotopography presented by the non-periodic nanoleafy architecture. It is the 17.8 ± 1.4-fold enhancements in RunX2 transcription factor measured on the nanoleafy surface that has subsequently resulted in the increased expression of ALP, COL, OCN and DCN. Such an obvious augmentation in gene expression levels was not apparent in any other nanostructures and hence the nanoleafy Ti substrate can be considered as the ideal biomaterial surface for implant applications. 3.7. Cytoskeleton and focal adhesion arrangement Fundamental cellular processes such as focal adhesion, stress fiber formation and spreading are shown to be dependent on the spatial distribution of integrin clusters at the nanoscale [42]. In the present study, we investigated in detail the early stages of focal adhesion point (FAP) formation as well as cytoskeletal arrangement of primary osteoblast cells on various Ti surfaces. The cytoplasmic structural proteins at the FAP that directly bind to integrins include talin and a-actinin, which bind to other structural proteins such as vinculin, paxillin and tensin [60]. Herein, we evaluated the initial FAP formation and its distribution on all experimental samples by immunoflourescent staining of vinculin at 30 min, 1 h and 5 h of incubation (Fig. 5). As is clearly evident from

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Fig. 6. Actin cytoskeletal arrangement at different time points using TRITC-conjugated phalloidin dye after 30 min, 1 h and 5 h. Scale bar = 5 lm.

the fluorescent microscopic images, a higher density of FAPs was observed on nanoleafy surface at both 1 and 5 h of incubation compared to control Ti. In the case of NS and nanotubular surfaces, the number of mature focal points was far less than on nanoleafy surface. However, in NN structures, even after 5 h of incubation, the cells failed to form mature focal contacts. The enlarged images shown in Fig. 5 (lane d) clearly reveal distinct differences in FAPs on the various substrates. The formation of mature FAPs ultimately leads to the recruitment of actin filaments, which occurs through integrin-mediated phosphorylation of several cytoskeletal proteins. The spatial organization of actin cytoskeleton plays an important role in maintaining cell mechanics. Fig. 6 represents the actin cytoskeletal arrangement on various substrates during the early phase of cell attachment (30 min, 1 h and 5 h). The images reveal that, from 30 min to 1 h, the actin fibers appeared more concentrated at the periphery of cells, allowing them to adhere to the substratum. At 5 h, actin monomers rearranged to form specific stress fibers. Fig. 6 shows that stress fiber formation was more prominent in the nanoleafy surface than in the control or other nanopatterned surfaces. In the case of NS, actin fibers present a wavy arrangement along the margins, with the cell maintaining its typical morphology. In contrast, for the cells on the NN surface, actin fibers were

Fig. 7. The effect of different nanopatterns on the expression of vinculin FAK, src, pFAK and p-src. Immunoblots were carried out with specific antibodies against vinculin, FAK, p-FAK, src, p-src and GAPDH. GAPDH was used as the loading control.

observed to be more concentrated at the cellular periphery as concentric layers, leading to a distorted morphology. It is reported that extracellular stimulus can pass through the cytoskeletal components to the nucleoskeleton, where the

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Fig. 8. Cartoon depicting signal transduction and mechanotransduction event of adherent cell on the surface of material.

interphase chromosome position inside the nucleus will be altered, resulting in changes in the gene expression [61,62]. The characterization of focal adhesion and cytoskeleton arrangements would hence provide evidence of how topography affects the mechanotransduction of the cell. In our study, we noted distinct differences in FAP distribution as well as stress fiber formation on the periodic as well as non-periodic arrays of nanostructures fabricated on the surface of metallic Ti. In the control Ti sample, FAPs are less numerous and more focused towards the ends of the filopodia, while the non-periodicity of nanoleafy surfaces might have induced mature FAP formation concentrated at the filopodial ends as well as in the lamellopodia. Accompanying this, the formation of actin stress fiber was seen to be more pronounced on nanoleafy surface than the control, resulting in a well spread morphology of the cells. In contrast, on the NS surface, cells showed a wavy actin fiber assembly, while on the NN surface, despite its disordered geometry, the spacing and arrangement of nanofeatures did not allow the cells to form mature focal adhesion formation. Similarly, cells grown on the periodically arrayed nanotubular array also failed to form good focal adhesion formation. This altered stress fiber formation observed on various nanotopographies might reduce the mechanical forces that activate connections between cytoskeleton and focal adhesion signaling, which in turn can affect the ability of the cells to sense and respond to extracellular stimuli. This is a probable reason for the reduction in ALP and collagen synthesis as well as gene expression on these surfaces. It is thus clearly evident that vertically arrayed, non-periodic, nanoleafy surface provides a topographic induction of changes in cytoskeletal organization, which in turns alters the gene expression profile. 3.8. Intracellular protein expression We further analyzed the early signaling events, such as expression of FAK and src proteins, since they are the root of subsequent signal transduction events, leading to altered cellular functions.

Integrin-mediated adhesion generally induces the autophosphorylation of FAK, generating a binding site for the src homology 2 domain of the src protein, which further phosphorylates other tyrosine residues of FAK [63]. It was previously reported that there is extensive cross-talk between integrins, src family kinases and Rho-GTPase [63]. In this study, we have analyzed the influence of nanopatterned surfaces on the activation of FAK and src proteins. A general trend towards the enhanced expression of phosphorylated FAK and src was observed on the nanoleafy surface in comparison to all other substrates, as is evident from Fig. 7. Moreover, an elevated level of vinculin was also observed on the nanoleafy surface. Surprisingly, the NN surface did not reveal any expression of the activated src. This supports our observations on the altered cytoskeletal arrangement and stress fiber formation on NN Ti surfaces. Hence, collectively, our results on protein expression and enhanced ECM matrix protein production (gene expression) enable us to conclude that nanoleafy surfaces can activate the FAK–src complex, which triggers primary osteoblast cells in a unique way, resulting in increased gene expression and cellular function. The tyrosine phosphorylation at Tyr 576/577 positions maximizes the catalytic activity of FAK [56]. This active FAK–src complex can activate multiple signaling cascades, including MAPKinase, RhoGTPase, Rac 1 and Cdc42, and augment the cellular response to growth factors [64]. Furthermore, it is reported that several of the pathways described above have an important role in mechanosensing and mechanotransduction, and cells can respond to mechanical tension created by the ECM through the activation of integrins, Rho-GTPase and SFKs [65,66]. A cartoon depicting the above signal transduction and mechanotransduction events due to the changes in surface nanotopography is illustrated in Fig. 8. In addition to the activation of FAK–src complex, which regulates GEFs and GAPs for Rho-GTPases, there are several alternative routes linking integrins to src family kinase activity, which will comprise a future aspect of the present study.

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Fig. 9. Representative histological images of bone growth on nanomodified titanium implants using Stevenel’s blue staining in 250 and 50 lm magnifications. Histological images at (a) 2 weeks, (b) 8 weeks and (c) 12 weeks after implantation are shown, with the representative quantitative results of percentage bone contact around the implant as insets.

3.9. In vivo osteointegration study It has been reported that nanofeatures can enhance bone formation in vivo [67–70]. The in vitro findings of the present work were further reinforced through the in vivo studies using a rat model. Nanosurface modified Ti screws bearing structural features identical to those used for the in vitro studies were employed for the in vivo experiments. Different sets of animals were euthanized after 2, 8 and 12 weeks of implantation and histology sections of

the samples analyzed. From the histology images shown in Fig. 9a–c, it is apparent that there was no necrosis or inflammation around any of the implants studied. In all the histology sections, Ti screws were identified as a circular, black region bearing a regular margin, and were observed near the epiphyseal plate, surrounded by the trabeculae of new woven bone. Fig. 9a shows that, after 2 weeks of implantation, a thin layer of woven bone formation was observed around the control Ti screw, although not as a continuous layer. There was no cellular infiltration

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Fig. 9 (continued)

of fibroblasts or fibrocytes around the implant. However, in the case of NT and NS screws, despite the new bone formation, there was clear indication of fibrous tissue formation near the implant surface. In the case of implants with NN topography, although fibrous tissue intervention was reduced, the formation of new bone was insignificant. In contrast, nanoleafy screws exhibited a comparatively thick and well-defined continuous layer of new bone formation without any fibrous tissue. The results of the quantitative analysis of percentage of bone contact around the implant (Fig. 9a, inset) revealed that, at 2 weeks of implantation, a significantly higher percentage of bone contact (53.34 ± 5.16%, p < 0.01) was observed around the nanoleafy implant surface. In contrast, all other nanomodified surfaces showed only about 30–40% bone contact, which is very similar to that observed around the unmodified control titanium screws. After week 8 of implantation (Fig. 9b), scanty new bone formation was seen around control Ti screws, with infiltration of fibrous tissue, fibrocytes, fibroblasts and occasional macrophages. Similar observations were noted around the NS and NN topographies, where the implant was partially in contact with fibrous tissues and fibrocytes. In the case of NT, there was a significant reduction in fibrous tissue formation, but the bone growth was not robust. In contrast, a continuous thick layer of lamellar bone, with new blood vessel formation near the bone–implant interface, was noticed on nanoleafy Ti screws. Quantitative analyses also confirmed these results (Fig. 9b, inset). It was found that the percentage of bone contact showed a significant increase (p < 0.001) to 80% for the nanoleafy implant, in comparison to all the other experimental samples, which showed only about 40–55%. At week 12 of implantation, although control Ti and nanotube implants were surrounded with directly deposited bone, the coverage was not complete. In the case of NS and NN samples, a partial encapsulation of thick fibrous tissue layer was observed around the implant. However, around the nanoleafy implants we consistently observed a thick mature lamellar bone with abundant new blood vessels in close proximity to the implant surface (Fig. 9c). Quantitative analyses (Fig. 9c inset)

Fig. 10. In vivo serum inflammatory cytokine analyses after 12 weeks of implantation using FACS. Serum from normal animals without any implant was taken as control.

revealed that the percentage of bone contact around the NT, NS, NN and control screws reached only up to 60% after week 12 of implantation. The most notable observation was the enhanced percentage of bone contact on nanoleafy implants for all study periods, with a value of 90% at week 12. This is significantly higher (p < 0.05) than all other nanomodified samples as well as control screws. Hence it is evident that bone bonding is critically influenced by the specific nanoscale topography generated on implant surfaces. 3.10. Inflammatory cytokine analysis Inflammation is mediated by a variety of soluble factors, including a group of secreted polypeptides known as cytokines, resulting in acute or chronic inflammation. The inflammatory response around Ti implants has been reported to be transient and lower in magnitude than on several other implant materials [71,72]. In this study, we have analyzed how nanosurface modification of Ti implants alters the inflammatory response in vivo. Analysis of both acute inflammatory cytokines such as IL-6 and TNF-a and chronic cytokines such as IFN-c, IL-10 and IL-4 were monitored using flow

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cytometry. Since the animals were administered with anti-inflammatory drug (0.01 ml/100 g ketoprofen) during the first week following surgery, cytokine analysis was carried out only after 12 weeks of implantation. Serum levels of acute and chronic inflammatory cytokines after week 12 of implantation revealed no significant increase due to the presence of nanoscale features on Ti in comparison to the control (Fig. 10). Hence, we conclude that nanosurface modification of implants by a hydrothermal processing technique does not impart any kind of inflammatory response in vivo.

[2]

[3] [4]

[5]

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4. Conclusions In summary, using a hydrothermal technique, tunable, nonperiodic nanostructures were developed to surface modify metallic titanium implants, and these helped to improve bone integration. It was observed that not all non-periodic nanomorphologies on implants imparted a similar response in vitro. Among the nanomorphologies, the nanoleafy pattern had the strongest influence on protein adsorption, in vitro osteoblast cell proliferation and differentiation. Osteoblast-specific gene expression analysis on this substrate using primary osteoblast cells revealed enhanced expression of collagen, OCN, DCN and RunX2. Further analysis also showed that early signal transduction events were altered by the nanoleafy structure. A comparative in vivo study of all the nanostructured implants in Sprague-Dawley rats demonstrated a higher percentage of bone contact without any inflammatory response on NL-patterned Ti screws. These results point to the importance of specific nanomorphologies in controlling tissue integration. Also, the fact that a hydrothermal processing technique for generating tunable nanostructures can be extended to other metallic surfaces (e.g. nitinol, TAV, stainless steel, Co–Cr) holds great promise for biomedical applications, especially in the field of metal implant therapies.

[7]

[8] [9] [10] [11]

[12]

[13] [14] [15] [16] [17]

[18]

[19]

Acknowledgements The authors are grateful for the support of this work by the DBT, Government of India under the Bioengineering Program and DST, Government of India, under the Nanomission Program. V.V.D. acknowledges the CSIR India, for financial support through a Senior Research Fellowship. We gratefully acknowledge Dr. Mira Mohanty and Mr. Muralidharan CV (Sree Chitra Tirunal Institute of Medical Sciences, Kerala) for their help with histopathology and materials testing studies. Mr. Sajin P. Ravi is acknowledged for his help in SEM analysis; Dr. Amrita Suresh for her help with RT-PCR experiments; and Ms. Roshni Ramachandran for her help during the in vivo work. The authors are also grateful to Amrita Vishwa Vidyapeetham, for providing substantial infrastructure support for basic research in nanobiomedical sciences.

[20] [21]

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[25]

[26]

Appendix A. Figures with essential colour discrimination Certain figures in this article, particularly Figures 1, 5–10, are difficult to interpret in black and white. The full colour images can be found in the on-line version, at doi:10.1016/j.actbio.2012.01.021.

[27] [28] [29] [30]

Appendix B. Supplementary data [31]

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.actbio.2012.01.021. References [1] Varioloa F, Vetrone F, Richert, L, Jedrzejowski P, Yi J, Zalzal S, et al. Improving biocompatibility of implantable metals by nanoscale modification of surface.

[32]

[33]

An overview of strategies, fabrication methods and challenges. Small 2009;5:996–1006. Bowers KT, Keller JC, Randolph BA, Wick DG, Michaels CM. Optimization of surface micromorphology for enhanced osteoblast responses in vitro. Int. J Oral Maxillofac Implants 1992;7:302–10. Brunette DM. The effects of implant surface topography on the behavior of cells. Int. J Oral Maxillofac Implants 1988;3:231–46. Groessner-Schreiber B, Tuan RS. Enhanced extracellular matrix production and mineralization by osteoblasts cultured on titanium surfaces in vitro. J Cell Sci 1992;101(Pt 1):209–17. Dalby MJ, Gadegaard N, Tare R, Andar A, Riehle MO, Herzyk P, et al. Control of human mesenchymal cell differentiation using nanoscale symmetry and disorder. Nat Mater 2007;6:997–1003. Popat KC, Leoni L, Grimes CA, Desai TA. Influence of engineered titania nanotubular surfaces on bone cells. Biomaterials 2007;28:3788–97. Iwasa F, Hori N, Ueno T, Minamikawa H, Yamada M, Ogawa T. Enhancement of osteoblast adhesion to UV-photofunctionalized titanium via an electrostatic mechanism. Biomaterials 2010;31:2717–27. Balasundaram G, Webster TJ. An overview of nanopolymers for orthopedic applications. Macromol Biosci 2007;7:635–42. Webster TJ. Nanotechnology for the regeneration of hard and soft tissues. Singapore: World Scientific Publishers; 2007. Zhang L, Webster TJ. Nanotechnolgy and nanomaterials: promises for improved tissue integration. Nano Today 2009;4:66–80. Engel E, Michiardi A, Navarro M, Lacroix D, Planell JA. Nanotechnology in regenerative medicine: the material side. Trends Biotechnol 2007;26(1). doi:10.1016/j.tibtech.2007.10.005. Puckett S, Taylor E, Raimondo T, Webster TJ. The relationship between the nanostructure of titanium surfaces and bacterial attachment. Biomaterials 2010;31(4):706–13. Taylor E, Webster TJ. The use of superparamagnetic nanoparticles for biofilm prevention. Int J Nanomed 2009;4:145–52. Gillani R, Ercan B, Qiao A, Webster TJ. Nanofunctionalized zirconia and barium sulfate particles as bone cement additives. Int J Nanomed 2010;5:1–11. Tran N, Webster TJ. Nanotechnology for bone materials. Wiley Interdiscip Rev Nanomed Nanobiotechnol (WIREs) 2009;1(3):336–51. Yang L, Webster TJ. Nanotechnology controlled drug delivery for treating bone diseases. Expert Opin Drug Deliv 2009;6(8):851–64. Raimondo T, Puckett S, Webster TJ. Greater osteoblast and endothelial cell adhesion on nanostructured polyethylene and titanium. Int J Nanomedicine 2010;5:647–52. Webster TJ, Schadler LS, Siegel RW, Bizios R. Mechanisms of enhanced osteoblast adhesion on nanophase alumina involve vitronectin. Tissue Eng 2001;7:291–301. You MH, Kwak MK, Kim DH, Kim K, Levchenko A, Kim DY, et al. Synergistically enhanced osteogenic differentiation of human mesenchymal stem cells by culture on nanostructures surfaces with induction mediua. Biomacromolecules 2010;11(7):1856–62. Park J, Bauer S, von der Mark K, Schmuki P. Nanosize and vitality: TiO2 diameter directs cell fate. Nano Lett 2007;7:1686–91. Pengt L, Barczak AJ, Barbeau RA, Xiaos Y, LaTempa TJ, Grimes CA, et al. Whole genome expression analysis revealed differential effects of TiO2 nanotubes on vascular cells. Nano Lett 2010;10(1):143–8. Rani DVV, Manzoor K, Deepthy M, Selvamurugan N, Nair SV. The design of novel nanostructures on titanium by solution chemistry for an improved osteoblast response. Nanotechnology 2009;20:195101–11. Anitha VC, Deepthy M, Nair SV, Prasanth R. Electrochemical tuning of titania nanotube morphology in inhibitor electrolytes. Electrochimica Acta 2010;55:3703–13. Mohan CC, Sreerekha PR, Divyarani VV, Nair S, Chennazhi K, Menon D. Influence of titania nanotopography on human vascular cell functionality and its proliferation in vitro. J Mater Chem 2012. doi:10.1039/c1jm13726c. Liu X, Lim JY, Donahue HJ, Dhurjati R, Mastro AM, Vogler EA. Influence of substratum surface chemistry/energy and topography on the human fetal osteoblastic cell line hFOB 1.19: phenotypic and genotypic responses observed in vitro. Biomaterials 2007;28:4535–50. Martin GR, Timpl R, Muller PK, Kuhn K. The genetically distinct collagens. Trends Biochem Res 1985;251:285–7. Kasemo B, Lousmaa J. Biomaterial and implant surfaces: surface science approach. Int J Oral madxillofac Implants 1988;3:247–59. Kasemo B, Lousmaa J. Material tissue interfaces: the role of surface properties and processes. Environ Health Perspect 1994;103(5):41–54. Bull SJ. Failure mode maps in the thin film scratch adhesion test. Trib Int 1997;30(7):491. Krishna DSR, Sun Y, Chen Z. Magnetron sputtered TiO2 films on a stainless steel substrate: selective rutile phase formation and its tribological and anticorrosion performance. Thin Solid Films 2011. doi:10.1016/j.tsf.2011.01.042. Siebers MC, Brugge TPJ, Walboomers XF, Jansen JA. Integrins as linker proteins between osteoblasts and bone replacing materials. A critical review. Biomaterials 2005;26(2):137–46. Clem WC et al. Mesenchymal stem cell interaction with ultra smooth nanostructured diamond for wear resistant orthopaedic implants. Biomaterials 2008;29:3461–8. Dolatshahi-Pirouz A et al. Fibronectin adsorption, cell adhesion, and proliferation on nanostructured tantalum surfaces. ACS Nano 2010;4: 2874–82.

V.V. Divya Rani et al. / Acta Biomaterialia 8 (2012) 1976–1989 [34] Woo KM, Chen VJ, Ma PX. Nano-fibrous scaffolding architecture selectively enhances protein adsorption contributing to cell attachment. J Biomed Mater Res A 2003;67:531. [35] Webster TJ, Ergun C, Doremus RH, Siegel RW, Bizios R. Specific proteins mediate enhanced osteoblast adhesion on nanophase ceramics. J Biomed Mater Res 2000;51(3):475–83. [36] Curtis ASG, Gadegaard N, Dalby MJ, Riehle M, Wilkinson C, Aitchinson G. Cells react to nanoscale order and symmetry in their surroundings. IEEE Trans Nanobiosci 2004;3:61–5. [37] Gittens RA, McLachlan T, Olivares-Navarrete R, Cai Y, Berner S, Tannenbaum R, et al. The effects of combine micron-/submicron scale surface roughness and nanoscale features on cell proliferation and differentiation. Biomaterials 2011;32:3395–403. [38] Yang W-E, Huang H-H. Improving the biocompatibility of titanium surface through formation of a TiO2 nanomesh layer. Thin solid films 2010;518:7545–50. [39] Zhao C, Cao P, Ji W, Zhang J, Zhang F, Jiang Y, et al. Hierarchical titanium surface textures affect osteoblastic functions. J Biomed Mater Res Part A 2011;99:666–75. [40] Jimbo R, Coelho PG, Vandeweghe S, Schwartz-Filho HO, Hayashi M, Ono D, et al. Histological and three-dimensional evaluation of osseointegration to nanostructured calcium phosphate coated implants. Acta biomaterialia 2011;7:4229–34. [41] Annunziata M, Oliva A, Buosciolo A, Giordano M, Guida A, Guida L. Bone marrow mesenchymal stem cell response to nano-structured oxidized and turned titanium surfaces. Clin Oral Implants Res 2011. doi:10.1111/j.16000501.2011.02194.x. [42] Kunzler TP, Huwiler C, Drobek T, Voros J, Spencer ND. Systematic study of osteoblast response to nanotopography by means of nanoparticle-density gradients. Biomaterials 2007;28:5000–6. [43] Yao C, Slamovich EB, Webster TJ. Enhanced osteoblast functions on anodized titanium with nanotube like structures. J Biomed Mater Res Part A 2007;1:157–66. [44] Kaplan FS, Hayes WC, Keaveny TM, Boskey A, Einhorn TA, Iannotti JP. Form and function of bone. In: Simon SP, editor. Orthopedic basic science. Columbus, OH: American Academy of Orthopedic Surgeons; 1994. p. 127–85. [45] Elias KL, Price RL, Webster TJ. Enhanced functions of osteoblasts on nanometer diameter carbon fibers. Biomaterials 2002;23:3279–87. [46] Sato M, Sambito MA, Aslani A, Kalkhoran NM, Slamovich E, Webster TJ. Increased osteoblast functions on undoped and yttrium-doped nanocrystalline hydroxyapatite coatings on titanium. Biomaterials 2006;27:2358–69. [47] Webster TJ, Massa-Schlueter EA, Smith JL, Slamovich EB. Osteoblast response to hydroxyapatite doped with divalent and trivalent cations. Biomaterials 2004;25:2111–21. [48] Curtis ASG, Varde M. Control of cell behavior: topological factors. J Natl Cancer Res Inst 1964;33:15–26. [49] Stephansson SN, Byers BA, García AJ. Enhanced expression of the osteoblastic phenotype on substrates that modulate fibronectin conformation and integrin receptor binding. Biomaterials 2002;23:2527–34. [50] Binulal NS, Deepthy M, Selvamurugan N, Shalumon KT, Suja S, Mony U, et al. Role of nanofibrous poly(caprolactone) scaffolds in human mesenchymal stem cell attachment and spreading for in vitro bone tissue engineering response to osteogenic regulators. Tissue Eng Part A 2010;16:393–404. [51] Whitesides GM. The ‘right’ size in nanobiotechnology. Nat Biotechnol 2003;21:1161–5. [52] Stevens MM, George JH. Exploring and engineering the cell surface interface. Science 2005;310:1135–8.

1989

[53] Lian JB, Stein GS. Osteoblast biology. Osteoporosis 2008;3–13. [54] Wiberg C, Heinegard D, Wenglen C, Timpl R, Morgelin M. Biglycan organizes collagen VI into hexagonal-like networks resembling tissue structures. J Biol Chem 2002;277(51):49120–6. [55] Nebe JB, Muller L, Luthen F, Ewald A, Bergemann C, Conforto E, et al. Osteoblast response to biomimetically altered titanium surfaces. Acta Biomaterialia 2008;4:1985–95. [56] Schneider GB, Perinpanayagam H, Clegg M, Zaharias R, Seabold D, Keller J, et al. Implant surface roughness affects osteoblast gene expression. J Dent Res 2003;82:372–6. [57] Hori N et al. Selective cell affinity of biomimetic micro–nano-hybrid structured TiO2 overcomes the biological dilemma of osteoblasts. Dent Mater 2010;26:275–87. [58] Mendonca G, Mendonca DBS, Simoes LGP, Araujo AL, Leite ER, Golin AL, et al. Nanostructured implant surface effect on osteoblast gene expression and bone to implant contact in vivo. Mater Sci Eng C 2011;31:1809–18. [59] Wang N, Li H, Lü W, Wang J, Zhang Z, Liu Y. Effects of TiO2 nanotubes with different diameters on gene expression and osseointegration of implants in minipigs. Biomaterials 2011;32:6900–11. [60] Aplin AE, Howe A, Alahari SK, Juliano RL. Signal transduction and signal modulation by cell adhesion receptors: the role of integrins, cadherins, immunoglobulin-cell adhesion molecules and selectins. Pharmacol Rev 1998;50(2):197–263. [61] Dalby MJ, Biggs MJ, Gadegaard N, Kalna G, Wilkinson CD, Curtis ASG. Nanotopographical stimulation of mechanotransduction and changes in interphase centromere positioning. J Cell Biochem 2007(a);100:326–38. [62] Dalby MJ, Gadegaard N, Herzyk P, Sutherland D, Agheli H, Wilkinson CD, et al. Nanomechanotransduction and interphase nuclear organization influence on genomic control. J Cell Biochem 2007;102:1234–44. [63] Huveneers Danen. Adhesion signaling – cross talk between integrins src and Rho. J Cell Sci 2009;122:1059–69. [64] Mitra SK, Schlaepfer DD. Integrin-regulated FAK–Src signaling in normal and cancer cells. Curr Opin Cell Biol 2006;18:516–23. [65] Schober M et al. Focal adhesion kinase modulates tension signaling to control actin and focal adhesion dynamics. J Cell Biol 2007;176:667–80. [66] Matthews BD, Overby DR, Mannix R, Ingber DE. Cellular adaptation to mechanical stress: role of integrins, Rho, cytoskeletal tension and mechanosensitive ion channels. J Cell Sci 2006;119:508–18. [67] Facca S, Lahiri D, Fioretti F, Messadeq N, Mainard D, Benkirane-Jessel N, et al. In vivo osseointegration of nanodesigned composite coatings on titanium implants. ACS Nano 2011;5:4790–9. [68] Ueno T, Tsukimura N, Yamada M, Ogawa T. Enhanced bone integration capability of alkali and heat treated nanopolymorphic titanium in micro to nanoscale hierarchy. Biomaterials 2011;32:7297–308. [69] Aita H, Hori N, Takeuchi M, Suzuki T, Yamada M, Anpo M, et al. The effect of ultraviolet functionalization of titanium on integration with bone. Biomaterials 2009;30(6):1015–25. [70] Chiang C, Chiou S, Yang W, Hsu M, Yung M, Tsai M, et al. Formation of TiO2 nanonetwork on titanium surface increases the human cell growth. Dent Mater 2009;25(8):1022–9. [71] Holgers KM, Esposito M, Kalltorp M, Thomsen P. Titanium in soft tissues. In: Brunette D, Tengvall P, Textor P, Thomsen P, editors. Titanium in medicine. Heidelberg: Springer; 2001. p. 513–60. [72] Kou PM, Schwartz Z, Boyan BD, Babensee JE. Dendritic cell responses to surface properties of clinical titanium surfaces. Acta Biomaterialia 2011;7:1354–63.