TiO2 nanotube heterostructures on microporous titanium

Accepted Manuscript Characterization and osteogenic activity of SrTiO3/TiO2 nanotube heterostructures on microporous titanium

Lu Yin, Jie Zhou, Lili Gao, Chanjuan Zhao, Junhong Chen, Xiong Lu, Jianxin Wang, Jie Weng, Bo Feng PII: DOI: Reference:

S0257-8972(17)31011-3 doi:10.1016/j.surfcoat.2017.09.075 SCT 22752

To appear in:

Surface & Coatings Technology

Received date: Revised date: Accepted date:

10 January 2017 8 May 2017 26 September 2017

Please cite this article as: Lu Yin, Jie Zhou, Lili Gao, Chanjuan Zhao, Junhong Chen, Xiong Lu, Jianxin Wang, Jie Weng, Bo Feng , Characterization and osteogenic activity of SrTiO3/TiO2 nanotube heterostructures on microporous titanium. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Sct(2017), doi:10.1016/j.surfcoat.2017.09.075

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Characterization and osteogenic activity of SrTiO3/TiO2 nanotube heterostructures on microporous titanium

Lu Yina, Jie Zhoua, Lili Gaoa, Chanjuan Zhaob, Junhong Chena, Xiong Lua, Jianxin

Key Laboratory of Advanced Technologies of Materials, Ministry of Education, School

SC

a

RI

PT

Wanga, Jie Wenga, Bo Fenga*

of Materials Science and Engineering, Southwest Jiaotong University, Chengdu,

CE

PT E

D

MA

West China Second University Hospital, Sichuan University

AC

b

NU

610031,Sichuan

Author information *Corresponding author: Bo Feng Tel: +86 028 87634023; Fax: +86 28 87601371. E-mail addresses: fengbo@ swjtu.edu.cn (Bo Feng).

1

ACCEPTED MANUSCRIPT Abstract

Surface properties such as physicochemical characteristics and topographical parameters are important considerations in the design of implant materials, as they

PT

determine interactions with living cells and tissues. Micro-/nanostructurization of Ti surfaces can enhance osteointegration, and strontium (Sr) is able to decrease

RI

osteoresorption. Sr loaded micro/-nanotubular structures that allow controlled and

SC

long-term Sr release are expected to yield favorable osteogenic effects. In this work, we

NU

constructed SrTiO3/TiO2 nanoparticle-nanotube heterostructures on a microporous

MA

titanium (Ti) surface and characterized their properties. Ti plates were etched with acid to create a micro-rough surface (M) and then anodized to generate a surface layer of

D

TiO2 nanotubes (MN). Strontium (Sr) was loaded onto MN by hydrothermal treatment

PT E

in Sr(OH)2 solution (MN-Sr) for 1 or 3 h to obtain SrTiO3/TiO2 nanotube heterostructures with different Sr contents. The in vitro biocompatibility of MN-Sr was

CE

investigated by evaluating protein adsorption, using osteoblast and osteoclast (RAW

AC

264.7 cell) cultures. The micro-/nanostructured porous samples (MN and MN-Sr) promoted protein adsorption owing to their large specific surface area and high reactivity; the amount of protein adsorbed onto MN-Sr was independent of Sr content. Sr in SrTiO3/TiO2 heterostructures exhibited controllable and sustained Sr2+ ion release in phosphate-buffered saline. Moreover, heterostructures with an appropriate SrTiO3 content promoted osteoblast adhesion, proliferation and differentiation, and inhibited 2

ACCEPTED MANUSCRIPT osteoclast proliferation and differentiation. These results indicate that the micro-/nano heterostructure with an appropriate content of Sr has excellent osteogenic activity and anti-bone resorption ability. Keywords: Micro-/nano structuralization; SrTiO3/TiO2 heterostructures; Protein

AC

CE

PT E

D

MA

NU

SC

RI

PT

adsorption; Osteoblasts; Osteoclasts

3

ACCEPTED MANUSCRIPT 1. Introduction

Titanium (Ti) and its alloys are widely used in orthopedics and dentistry owing to their excellent biocompatibility [1], but the metallic Ti surfaces do not satisfy the

PT

demands of the rapid osseointegration in clinical use. The biological activity of Ti can be improved by modifying its surface morphology and incorporating drugs [2-4]; for

RI

instance, anodic TiO2 nanotubular layers with outstanding biocompatibility and

SC

bioactivity have been shown to stimulate osteoblast adhesion and proliferation [5, 6].

NU

Micro-rough Ti surfaces are more suitable for growing osteoblasts and exhibit less bone

MA

resorption than a smooth surface [7], whereas micro-/nanostructured Ti surfaces can promote osteogenesis and enhance bonding between Ti implants and bone tissue owing

D

to their dimensional similarity to bone collagen fibrils and elasticity resembling that of

PT E

bone [8, 9]. In addition, TiO2 nanotubes also act as a reservoir for drug delivery, particularly for inorganic, bioactive elements such as silver, zinc, and strontium [10-12].

CE

Some studies have investigated the use of TiO2 nanotubes as a delivery platform

AC

decorated with SrTiO3 for biomedical applications [12-14]. Strontium (Sr) is an essential trace element in the human body that plays an important role in bone rebuilding by promoting bone formation and reducing bone resorption[15, 16]. Sr has attracted extensive clinical interest especially after the development of the anti-osteoporosis drug strontium ranelate (SR), which decreases the bone fracture risk in osteoporotic patients [17, 18]. Oral administration of SR has been 4

ACCEPTED MANUSCRIPT reported to effectively enhance bone implant fixation in osteoporotic and intact rats [19]. However, constant in situ Sr release at the implant-tissue interface gives rise to better results while avoiding the potential deleterious effects associated with oral administration at high concentrations [20]. In vivo and in vitro studies have shown that

PT

Sr incorporation into biomaterials such as calcium phosphates and bioactive glasses can

RI

stimulate osteoblast proliferation and reduce osteoclast generation [21, 22]. Nonetheless,

SC

because the solubility of calcium phosphates and bioglasses changes appreciably with the amount of Sr [23], it is difficult to achieve long-lasting Sr release at a reasonably

NU

constant rate. In this respect, a more efficient delivery platform with controlled Sr

MA

release is urgently needed.

In addition, it has been reported that high Sr content loaded on implants has

D

adverse effects on cell behaviors such as adhesion, proliferation, and differentiation [19,

PT E

24]. However, some studies have demonstrated that calcium phosphates with higher than a particular content of Sr can suppress osteoclast differentiation [7, 25]. Thus, an

AC

performance.

CE

appropriate Sr content in implants is also a major determinant of biological

We hypothesized that micro-/nano structuralization and loading an appropriate amount of Sr on Ti can improve osseointegration and prevent implant loosening, especially over the long term. To investigate this possibility, we fabricated SrTiO 3 nanoparticle-TiO2 nanotube heterostructures on microporous Ti (MN-Sr) by acid etching, anodization, and hydrothermal processing. The effect of the heterostructures 5

ACCEPTED MANUSCRIPT and Sr content on osteogenic induction capacity were also evaluated by cultures of osteoblasts and osteoclasts.

2. Material and methods

PT

2.1 Preparation of TiO2 nanotubes on microporous Ti substrate

RI

Titanium plates (Ф10 mm x 2 mm, purity > 99.7%) were first polished using SiC

SC

sandpaper and then ultrasonically cleaned with acetone, ethanol, and deionized water for 10 min sequentially. Micropores on titanium plates were fabricated by acid etching

NU

according to our previous report [26]. Briefly, the Ti plate was immersed in an aqueous

MA

solution of HCl and CaCl2 with a 7:1 molar ratio at 60°C for 24 h, and then ultrasonically rinsed in deionized water. The resultant sample (M) was anodized in an

D

ethylene glycol solution with 0.3 wt % NH4F and 5 vol % H2O at 40 V for 2 h. TiO2

PT E

nanotubes were formed on the as-prepared M after anodization (denoted as MN). All

CE

samples were then annealed at 450 °C for 3 h in air at a heating rate of 2°C/min and then cooled in a muffle furnace. As a control, TiO2 nanotubes on flat Ti plates (N) were

AC

fabricated by anodization and annealing.

2.2 Hydrothermal synthesis of SrTiO3/TiO2 heterostructures on MN

A hydrothermal method was applied to synthesize the strontium titanate nanoparticle-titania nanotube heterostructures. The loaded Sr content in the heterostructures was controlled with different hydrothermal treatment time. Annealed 6

ACCEPTED MANUSCRIPT MN samples were placed in 80 mL of 0.025 M Sr(OH)2 solution in a teflon autoclave. Then, the autoclave was heated at 180°C for 1 h and 3 h to obtain MN-Sr1h and MN-Sr3h, respectively. Finally, the samples were ultrasonically cleaned with distilled water for 2 min to remove surface deposits and dried in air. The samples were

PT

characterized by X-ray diffraction (XRD, Philips X’Pert PRO ) with Cu K radiation,

RI

field emission scanning electron microscopy (FE-SEM, JSM-7001F, JEOL Inc., Japan),

SC

and transmission electron microscopy (TEM, JEM-2100, JEOL) with an accelerating

NU

voltage of 200 kV.

MA

2.3 Sr release determination

The MN-Sr1h and MN-Sr3h were immersed in 5 mL of PBS at 37°C and the

D

solution was refreshed every 2 d. This process was repeated for a total of 65 d. The

PT E

solutions at designated time points were used to determine the Sr release time profile by atomic absorption spectrometry (AAS, Agilent 55B). The Sr-containing layers were

CE

completely dissolved in the HF and HNO3 mixture for detection of the total amounts of

AC

Sr2+ ions by AAS.

2.4 Protein adsorption assay

The protein adsorption assay was conducted in minimum essential medium alpha (α-MEM, Corning cellgro@, USA) containing 10% fetal calf serum (FCS). After incubation in the medium at 37°C for 3 h, the amounts of proteins adsorbed onto the 7

ACCEPTED MANUSCRIPT samples were tested using a protein assay kit (Beyotime). The functional groups of the proteins were analyzed by Fourier transform infrared spectroscopy (FT-IR, Nicolet 5700, Thermo, USA). The working reagent (150 μL) was pipetted onto all samples in a 24-well plate (BD Falcon). After incubation at 60°C for 30 min, 100 μL of working

PT

reagent was collected and analyzed at 562 nm using a microplate reader (uQuant,

RI

Biotech, USA). The topography of the sample surfaces after incubation for 2 d in the

SC

medium was inspected by FE-SEM.

NU

2.5 Osteoblast culture and evaluation

MA

Primary mouse osteoblasts were obtained from mouse calvaria following standard tissue culture protocols. The Institutional Animal Ethics Committee of Southwest

D

Jiaotong University approved all animal experimental protocols and the experiments

PT E

were conducted in accordance with the guidelines of Committee on the Use of Live Animals in Teaching & Research, Government of China, for animal care and

CE

experimentation. The cells were cultured in α-MEM supplemented with 10% FCS at

AC

37°C in a 5 % CO2 humidified atmosphere and the medium was changed every 2 d. The samples for cell culture were sterilized by ultraviolet (UV) irradiation for 2 h. For cell culture experiments, osteoblasts were seeded on the surfaces of samples with a density of 2 x 104 cells per well in 24-well culture plates. The number of adherent cells after 1 and 2 h of culture and cell proliferation after 1, 3, and 7 d of culture were assessed by Alamer Blue assay according to the manufacturer’s instructions. After 3 and 7 d of 8

ACCEPTED MANUSCRIPT culture, the samples with attached osteoblast cells were rinsed twice with PBS, fixed in 2.5% glutaraldehyde in PBS at room temperature, dehydrated in a graded ethanol series, dried in air, sputter-coated with gold, and observed by FE-SEM.

PT

2.6 Quantitative reverse transcription polymerase chain reaction (qRT-PCR )

Table 1

Sequences

SC

Target gene

RI

Nucleotide sequences for each primer couple used for RT-PCR.

Forward primer (5’→3’)

NU

Reverse primer (5’→3’)

TACACGGACACTGAGATGCGCT

BMP-2

GCTAAACTTGACGACGCTCGT

CTGCTCGGTTCCCGTTA

Col-I

CTAAAGAATGACTACAGCTA

TCCACCCCTAGTGCGTTGCT

OCN

CTGTTTAGGGTGTGTGTCGC

OPN

TCTGGAGAGGAGTGAAGCTC

AGGGAAGCGTCAATCTTAAG

RUNX2

CTGTCCCTCCCTCACCCCGT

GACGCCTGCTGTCTTCTGTAG

CCTCCAGGAACTCTCCTCAG

GTATGAGAGCGCAGCCAACGG

CE

TCGTGCATTATCTGATAGGTGA

TAGCGAGGCAACGAGATCAA

AC

GAPDH (IC)

PT E

D

MA

ALP

Osteogenic differentiation was assessed. The expression levels of genes implicated in bone matrix synthesis and degradation were measured by qRT-PCR. The osteoblasts were seeded at a density of 2 x 104 cells/well, cultured for 14 d, and harvested using TRIzol (Invitrogen, Milan, Italy). Total mRNA was extracted and cleaned using an RNeasy Micro Kit. Residual genomic DNA was removed by an on-column DNase 9

ACCEPTED MANUSCRIPT digestion step using an RNase-Free DNase Set (Qiagen). RNA was quantified on a Qubit quantitation platform (Invitrogen, Cergy Pontoise, France). RNA quality was checked by 1% agarose gel electrophoresis and optical density measurement. Reverse transcription followed immediately using a high-capacity reverse transcription kit

PT

(Applied Biosystems) for real-time PCR experiments. RNA was frozen and stored at

RI

−80°C and cDNA at −20°C. Primers were designed and tested for specificity and

SC

efficiency for amplifying alkaline phosphatase (ALP), type I collagen (Col-I), osteocalcin (OCN), osteopontin (OPN), bone morphogenetic protein-2 (BMP-2), and

NU

runt related transcription factor 2 (RUNX2) (Table 1). Glyceraldehyde-3-phosphate

MA

dehydrogenase (GAPDH) was used as an internal control (IC). The relative expression level of each mRNA was calculated by the 2-ΔΔCt method.

PT E

D

2.7 Osteoclast culture and evaluation

The mouse monocyte cell line, RAW264.7, which differentiates to osteoclasts

CE

under appropriate conditions [25], was purchased from the American Type Culture

AC

Collection (ATCC, Manassas, VA). RAW 264.7 cells (1 x 105/well) were cultured on samples in Dulbecco's modified Eagle’s medium (DMEM, high glucose formulation) containing 10 % FCS and 20 ng/mL receptor activator of nuclear factor kappa-B ligand (RANKL, PeproTech EC, London, UK), which induced the differentiation of RAW 264.7 cells into osteoclasts, at 37°C in a 5% CO2 humidified atmosphere. The medium was changed every 2 d. All samples were the same as those in osteoblast culture. The 10

ACCEPTED MANUSCRIPT osteoclast proliferation was assessed by the Alamer Blue assay.

2.8 Tartrate resistant acid phosphatase (TRAP) activity

TRAP activity, a marker of osteoclast differentiation and resorbing activity [27],

PT

was measured by enzyme-linked immunosorbent assays (ELIAS, BIOSS) following the manufacturer’s protocol. After 1, 3, 5, and 7 d of culture, the cells were lysed by

RI

freezing and thawing three times in PBS with 1% Triton X-100. The supernatants of the

SC

osteoclasts cultured were collected at designed time points and frozen at −20°C until

NU

analysis. TRAP was detected using a TRAP assay kit (Beyotime) according to the

MA

manufacturer’s instructions.

D

2.9 Statistical analysis

PT E

The data were collected from three separate experiments and expressed as the mean ± standard deviation. One-way analysis of variance (ANOVA) and

CE

Student-Newman-Keuls post hoc test were performed to determine the level of

AC

significance, and p < 0.05 and 0.01 were considered to be significant and highly significant, respectively.

3. Results

3.1 Characterization of SrTiO3/TiO2 heterostructures

Ti plates were etched with acid to create a micro-rough surface (M) and then 11

ACCEPTED MANUSCRIPT anodized to generate a surface layer of TiO2 nanotubes (MN); the fabricated material was analyzed by X-ray diffraction (XRD; Fig. 1). The crystal phases of micropores (M) (Fig. 1a) included only Ti (Joint Committee on Powder Diffraction Standards [JCPDS] no. 03-065-9622). The crystal phases of nanotubes (N) included Ti and the anatase TiO2

PT

(JCPDS no. 75-1537). The XRD pattern of MN-Sr (Fig. 1d, e) showed peaks

RI

corresponding to SrTiO3 (JCPDS no. 35-0734) that were not observed for N and MN

SC

(Fig. 1b, c). SrTiO3 peaks in MN-Sr3h (Fig. 1e) were stronger, whereas anatase TiO2 peaks were reduced as compared to those in MN-Sr1h (Fig. 1d).The intensity SrTiO3

NU

peaks increased with longer hydrothermal reaction times, indicating gradual

AC

CE

PT E

D

MA

transformation of TiO2 into SrTiO3.

Fig.1. XRD patterns of (a) M, (b) N, (c) MN, (d) MN-Sr1h, and (e) MN-Sr3h. The three-dimensional microporous structure of M was produced by acid etching (Fig. 2a). The diameter of micropores on the Ti surface was 10-20 μm and the pores were interconnected. N consisted of a dense array of highly ordered nanotubes on 12

ACCEPTED MANUSCRIPT the Ti substrate with an average outer diameter of 100 nm and length of 8 μm. MN retained the original micropores of M but also had new nanotubes in the walls and at the bottom of the micropores (Fig. 2c, c1). These nanotube arrays formed a micro/nanostructural TiO2 layer on the Ti substrate. The diameter of the nanotubes was

CE

PT E

D

MA

NU

SC

RI

PT

similar to that of N (Fig. 2c2).

AC

Fig. 2. SEM micrographs of M (a) and N (b). Insets show corresponding sectional views. (c) MN; (c1) low- and (c2) high-magnification views of MN. We investigated the morphology of MN-Sr1h and MN-Sr3h (Fig. 3) and found that nanotubes on the micropores showed distinct surface topographies after hydrothermal reaction with Sr(OH)2. SrTiO3 nanoparticles grew on the TiO2 nanotubes; the MN-Sr1h sample retained micropores and nanotubes, but the nanotube diameter decreased from 13

ACCEPTED MANUSCRIPT 100 nm to approximately 70 nm as a result of hydrothermal treatment, which caused TiO2 nanotubes to be transformed into SrTiO3, with a concomitant increase in nanotube wall thickness. SrTiO3 nanoparticles almost covered the MN-Sr3h surface, and the SrTiO3/TiO2 nanotube diameter was approximately 60 nm. These results indicate that

PT

more SrTiO3 nanoparticles were generated with a longer reaction time. The crystal

RI

structure of MN-Sr1h was confirmed by transmission electron microscopy, which

SC

revealed that SrTiO3 nanoparticles were closely associated with the walls of TiO2 nanotubes (Fig. 3c, d), providing direct evidence for the formation of SrTiO3/TiO2

AC

CE

PT E

D

MA

NU

heterostructures.

Figure 3. SEM micrographs of MN-Sr1h (a) and MN-Sr3h (b). Insets show corresponding high-magnification SEM images and (c, d) TEM micrographs of

14

ACCEPTED MANUSCRIPT MN-Sr1h at (c) low- and (d) high- resolution.

3.2 Sr2+ release from SrTiO3/TiO2 heterostructures

We determined the fraction of Sr2+ released from MN-Sr after immersion in

PT

phosphate-buffered saline (PBS) for 65 d (Fig. 4). The Sr2+ concentration increased rapidly from 0 to 10 d and more slowly from 10 to 65 d. The total amount of loaded Sr2+

RI

was 123.35 ± 2.68 μg/cm2 and 204.18 ± 2.24 μg/cm2 for MN-Sr1h and MN-Sr3h,

AC

CE

PT E

D

MA

NU

MN-Sr1h at a given time point (17% vs. 10%).

SC

respectively. That is, the fraction of released Sr2+ was higher for MN-Sr3h than for

Fig. 4. Proportion of Sr2+ released from MN-Sr after immersion in PBS for 65 d.*, **, p < 0.05 or 0.01 vs. MN-Sr1h.

15

ACCEPTED MANUSCRIPT 3.3 Protein adsorption

FT-IR spectra of the samples were obtained after protein adsorption following immersion for 3 h in α-Minimal Essential Medium (α-MEM) containing 10% fetal calf serum (FCS; Fig. 5a). The characteristic absorption peaks at 1655 cm−1 and 1549 cm−1

PT

were attributed to the amide I band corresponding to C=O stretching vibration and the

RI

amide II band corresponding to N-H formation vibration, respectively. The

SC

characteristic absorption peaks of protein on M surfaces indicated that a small amount

NU

of protein was adsorbed; stronger characteristic absorption peaks were observed on N surfaces. Band intensity was stronger for MN and MN-Sr than for M and N.

MA

Characteristic protein absorption peaks whose intensities showed no differences were detected on MN and MN-Sr surfaces, indicating that more protein was adsorbed onto

PT E

D

the surfaces of micro-/nanostructures than onto micro- and nanostructures. We examined protein adsorption on the samples after 3 h of immersion in α-MEM

CE

containing 10 % FCS (Fig. 5b). N adsorbed more protein than M and the micro-/nanostructure surfaces adsorbed even greater amounts. The amounts of protein

AC

adsorbed on MN, MN-Sr1h, and MN-Sr3h did not differ significantly.

16

PT

ACCEPTED MANUSCRIPT

RI

Fig. 5. (a) FT-IR spectra of samples. (b) Proportion of protein adsorbed to samples in

SC

α-MEM containing 10 % FCS after 3 h of culture. *,**p < 0.05 or 0.01 vs M, #,## p <

NU

0.05 or 0.01 vs MN, and %,%% p < 0.05 or 0.01 vs MN-Sr1h. The amount and distribution of adsorbed proteins are known to influence

MA

cell/biomaterial interactions. We therefore examined the topography of each sample after protein adsorption by scanning electron microscopy (Fig. 6). M absorbed a few

PT E

D

proteins that were not seen in SEM (micrograph not shown). Proteins accumulated on the top end of the nanotubes on the N surface. Micro-/nanostructure surfaces (MN,

CE

MN-Sr1h, and MN-Sr3h) aggregated more nanoparticles than N. However, it was difficult to distinguish proteins from SrTiO3 nanoparticles because they both appeared

AC

white (Fig. 6c, d). We determined that the proteins on the N and MN surfaces were evenly distributed. The amounts of protein adsorbed on MN, MN-Sr1h, and MN-Sr3h did not differ significantly. These observations imply that micro/nanotopographies promote protein adsorption, which was unaffected by Sr2+ ions in the samples. It was assumed that proteins on the MN-Sr1h and MN-Sr3h surfaces were similarly

17

ACCEPTED MANUSCRIPT

MA

NU

SC

RI

PT

distributed.

PT E

D

Fig. 6. SEM micrographs of samples after 3 h of incubation in α-MEM containing 10% FCS. (a) N, (b) MN, (c) MN-Sr1h, and (d) MN-Sr3h.

CE

3.4 Osteoblast adhesion and proliferation

AC

Cell adhesion is critical for the proliferation and differentiation of cells on biomaterials. The initial number of adherent cells did not differ between samples after 1 h of culture (Fig. 7a). However, there were more adherent cells on MN than on M or N after 2 h, indicating that nanotubes on microporous Ti stimulated initial cell adhesion. There were more adherent cells on MN-Sr1h than on MN or MN-Sr3h after 2 h of culture following Sr incorporation, demonstrating that an appropriate amount of Sr 18

ACCEPTED MANUSCRIPT promoted early cell adhesion, as previously suggested [28]. However, it was also previously reported that the presence of Sr did not affect initial cell adhesion to Ti-6Al-4V [29]. These conflicting results may be explained by differences in the amount of Sr that was loaded. Cell proliferation on the five different surfaces was analyzed after

PT

1, 3, and 7 d of culture (Fig. 7b). There were no differences in proliferation after 1 d of

RI

culture, with the number of cells on the samples increasing over time. There were more

SC

osteoblasts on micro/nanostructured surfaces (MN and MN-Sr) than on N and M, with the latter showing the fewest cells. A higher proliferation rate was observed for cells

NU

grown on MN-Sr1h than for those grown on MN-Sr3h. These results indicate that

CE

PT E

D

MA

MN-Sr1h is most effective for inducing cell adhesion and proliferation.

AC

Fig. 7. Osteoblast (a) adhesion and (b) proliferation on N, M, MN, MN-Sr1h, and MN-Sr3h. *,**p < 0.05 or 0.01 vs M, #,## p < 0.05 or 0.01 vs MN, and %,%% p < 0.05 or 0.01 vs MN-Sr1h.

3.5 Osteoblast morphology

Osteoblast morphology was examined by scanning electron microscopy after 3 and 19

ACCEPTED MANUSCRIPT 7 d of culture (Fig. 8). Osteoblasts on the N surface spread and extended filopodia after 3 and 7 d of culture (Fig. 8a), whereas those on the M surface exhibited a fusiform and shrunken morphology with a few filopodia after 3 and 7 d of culture (Fig. 8b), suggesting that they were unhealthy. Cells grown on MN and MN-Sr1h surfaces showed

PT

more filopodia after 7 d of culture (Fig. 8c, d) than those on the MN-Sr3h surface (Fig.

AC

CE

PT E

D

MA

NU

SC

RI

8e). These results suggest that excess Sr was cytotoxic to osteoblasts.

Fig. 8. SEM micrographs of osteoblasts after 3 and 7 d of culture on each sample. (a) N, 20

ACCEPTED MANUSCRIPT (b) M, (c) MN, (d) MN-Sr1h, and (e) MN-Sr3h.

3.6 Osteogenic differentiation

To investigate the potential for each type of surface to induce osteogenic

PT

differentiation, we evaluated gene expression levels of osteoblasts grown on the samples (Fig. 9). MN and MN-Sr1h promoted the expression of osteogenesis-related genes,

RI

including ALP (an early marker of osteogenic differentiation), type 1 collagen (Col-1,

SC

the main component of bone extracellular matrix), bone morphogenetic protein 2,

NU

runt-related transcription factor (RUNX)2, and osteocalcin (OCN) and osteopontin

MA

(OPN) (late markers of osteogenic differentiation). The MN and MN-Sr1h samples had excellent osteogenic activity. In contrast, the expression levels of all of these genes were

D

lower in cells grown on MN-Sr3h, N, and M. Although both MN-Sr1h and MN-Sr3h

PT E

were composed of SrTiO3 nanoparticles and had similar topographies, osteoblasts grown on MN-Sr1h had higher expression of osteogenesis-related genes than those

CE

grown on MN-Sr3h. In addition, osteogenesis-related gene expression was lower in

AC

cells grown on MN-Sr3h than in those grown on MN. Thus, an appropriate Sr content stimulates initial osteogenic differentiation.

21

SC

RI

PT

ACCEPTED MANUSCRIPT

Figure 9. Relative expression levels of ALP, type 1 collagen (Col-1), bone

NU

morphogenetic protein (BMP-2), RUNX2, osteocalcin (OCN), and osteopontin (OPN)

MA

in osteoblasts cultured on each sample for 14 d. Values were normalized to the expression level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). *,**p < 0.05

PT E

D

or 0.01 vs M, #,## p < 0.05 or 0.01 vs MN, and %,%% p < 0.05 or 0.01 vs MN-Sr1h.

3.7 Osteoclast proliferation

CE

We assessed the proliferation of osteoclasts grown on the five surface types (Fig.

AC

10a). After 1 d of culture, the number of osteoclasts did not differ among samples. From 3 to 7 d, the number of osteoclasts on M did not change obviously, and was the lowest among the samples. The number of osteoclasts on N and MN increased from 1 to 5 d and declined slightly from 5 to 7 d, with more cells on MN than on N. The number of osteoclasts on MN-Sr1h and MN-Sr3h increased from 1 to 5 d but decreased from 5 to 7 d, with fewer cells than on the MN surface at these time points. After 7 d of culture, 22

ACCEPTED MANUSCRIPT there were fewer osteoclasts on MN-Sr1h and MN-Sr3h than on M. These results

SC

RI

PT

indicated that Sr loading on MN inhibits osteoclast proliferation.

NU

Fig. 10. (a) Osteoclast proliferation and (b) TRAP expression in cells grown on each sample. *,**p < 0.05 or 0.01 vs M, #,## p < 0.05 or 0.01 vs MN, and %,%% p < 0.05 or

MA

0.01 vs MN-Sr1h.

PT E

D

3.8 Osteoclast differentiation

RAW264.7 cells differentiate into osteoclasts in the presence of receptor activator

CE

of nuclear factor kappa-B ligand. Under normal conditions, activated macrophages and osteoclasts express tartrate-resistant acid phosphatase (TRAP), which is considered as a

AC

marker of osteoclast differentiation, at high levels [27]. We measured TRAP activity in osteoclasts grown on each type of sample and found similar levels after 1 d of culture (Fig. 10b). TRAP activity was lowest and highest in cells grown on M and MN after 3, 5, and 7 d of culture, respectively. TRAP activity in cells grown on N increased with culture time, but remained lower than the level in cells grown on MN. Although cells on MN-Sr1h and MN-Sr3h showed relatively high TRAP activity from 3 to 7 d, a marked 23

ACCEPTED MANUSCRIPT decline was observed after 7 d of culture. Moreover, TRAP activity in cells on MN-Sr1h was lower than that in cells on MN-Sr3h during the culture period. Sr loading on micro/nanostructured Ti suppressed osteoclast differentiation, and this trend became more pronounced over time, especially after 7 d of culture.

PT

4. Discussion

SC

RI

4.1 Mechanism of Sr2+ loading and release in SrTiO3/TiO2 heterostructures

The SrTiO3/TiO2 composite was a heterostructure with SrTiO3 nanoparticles

NU

grafted on TiO2 nanotubes. We previously described similar heterostructures with high

MA

photoelectrochemical performance [30]. SrTiO3 nanoparticles were formed on TiO2 nanotube arrays via a dissolution-precipitation process [31] according to the following

D

chemical reactions.

(1)

TiO2 + 2 OH− + 2 H2O → [Ti(OH)6]2−

(2)

Sr2+ + [Ti(OH)6]2− → SrTiO3 + 3 H2O

(3)

CE

PT E

Sr(OH)2 → Sr2+ + 2 OH−

AC

The total reaction is

TiO2 + Sr(OH)2 → SrTiO3 + H2O

(4)

SrTiO3 was precipitated and nucleated at the nanotube surface due to the presence of free [Ti(OH)6]2−. The initial SrTiO3 layer was loosely packed or porous and did not completely block the flow of Sr2+ and OH− ions. With a longer hydrothermal reaction, transport or diffusion of Sr2+ and OH− ions to the reaction site became more difficult 24

ACCEPTED MANUSCRIPT owing to the smaller diameter of the nanotubes, which slowed reactions (1) and (2) and inhibited SrTiO3 synthesis, as this was controlled by in situ hydrolysis of TiO2. The process of SrTiO3 hydrolysis was similar to that of other titanates such as BaTiO3 [6]. The release of Sr2+ ions from SrTiO3/TiO2 heterostructure nanotubes in

PT

neutral solution (pH = 7.4) can be described as follows. (5)

RI

SrTiO3 + H2O → Sr2+ + TiO2 + 2 OH−

SC

The high SrTiO3 content on the MN-Sr3h surface resulted in a higher concentration of Sr2+ in PBS as compared to that on MN-Sr1h. After 65 d of immersion in PBS, the

NU

cumulative proportion of Sr2+ released from MN-Sr1h and MN-Sr3h was 10% and 17%,

MA

respectively. The release rate decreased with longer culture times. We predict that Sr release from MN-Sr can persist for longer than 1 year.

PT E

D

4.2 Protein adsorption on SrTiO3/MN

Protein adsorption on implants was related to surface electricity, chemical

CE

composition, and topography [32, 33]. According to a previous report [34] and reaction

AC

(5) above, hydrated TiO2 and SrTiO3 in the culture medium (α-MEM, pH = 7.4) have negatively charged surfaces. α-MEM contains many positively charged amino acids, such as arginine, aspartic acid, histidine, and lysine. Thus, all of the samples readily adsorbed proteins in α-MEM via electrostatic interactions. However, the amounts of proteins adsorbed on the sample surface differed, which was attributed to variations in surface topography. The lowest amount adsorbed was on M, which had only micropores; 25

ACCEPTED MANUSCRIPT moreover, M had the smallest specific surface area among the samples and consequently the fewest adsorption sites and lowest surface energy. The micro/nanostructured MN and MN-Sr surfaces exhibited protuberant and more curved topography and adsorbed more proteins than N or M. The micro/nanostructure of Ti surfaces generates more

PT

anatase and nanotube arrays, which increases specific surface area and the number of

RI

surface active sites available for protein adsorption [35, 36]. MN, MN-Sr1h, and

SC

MN-Sr3h had similar micro/nanostructure morphology and adsorbed near-equivalent amounts of protein (Fig. 5b). Sr loaded on MN had no effect on protein adsorption

NU

capacity despite alterations in surface chemistry, which may be due to the small amount

MA

of Sr that was loaded. Thus, the main factor affecting protein adsorption was surface topography, which is related to specific surface area and energy.

PT E

D

4.3 Behavior of osteoblasts on SrTiO3/MN

There was less osteoblast adhesion after 2 h of culture and proliferation after 3 and

CE

7 d of culture on M surface than that on N and MN surface (Fig. 7a, b), which was

AC

consistent with the results of osteoblast differentiation after 14 d of culture (Fig. 9). This corresponded to the amount of protein adsorbed; that is, more protein was absorbed on implants that were favorable for osteoblast adhesion, proliferation, and differentiation. A previous study showed that micropitted nanotube surfaces promote cell adhesion and proliferation and induce osteogenesis-related gene expression [37]. The Sr content of SrTiO3-TiO2 heterostructures was also previously shown to influence osteoblast 26

ACCEPTED MANUSCRIPT adhesion, proliferation, and differentiation [38], whereas other studies have also shown that Sr in implants stimulates osteoblast growth [15, 25, 39]. In the present study, MN-Sr1h was superior to MN, whereas MN-Sr3h was inferior to MN in the terms of ability to promote osteoblast adhesion, proliferation, and differentiation. MN-Sr1h

PT

expressed osteogenesis-related genes at higher levels than NM-Sr3h and MN,

RI

suggesting that an appropriate amount of Sr loaded on the heterostructure would

SC

enhance these behaviors but that an excess amount would have an inhibitory effect. Sr is known to induce osteogenic differentiation via Wnt/β-catenin and mitogen-activated

NU

protein kinase signaling pathways and the downstream transcription factor RUNX2 [40,

MA

41]. However, a high concentration of Sr in biphasic calcium phosphate and strontium ranelate inhibited the expression of osteogenesis-related genes by suppressing the

D

synthesis of 1,25-dihydroxyvitamin D3 [42, 43]. Thus, in addition to protein adsorption

PT E

capacity, Sr content can influence osteoblast adhesion, proliferation, and differentiation. The topography of implant surfaces determined the spreading of osteoblasts [12,

CE

44]. Osteoblasts on M displayed a fusiform and shrunken shape with few filopodia,

AC

whereas those on N were spread out and had many filopodia after 3 and 7 d of culture. M lacks the nanostructured topography that induces the generation of extracellular matrix fibrils and filopodia [45]. The edges and vertical walls of TiO2 nanotubes has more lateral points available for filopodia to bind with implants via integrins. In addition, cells on MN had a greater number of filopodia as compared to those on M and N, demonstrating that MN is more effective in promoting cell growth. This is due to not 27

ACCEPTED MANUSCRIPT only the presence of nanotubes, but also the co-existence of nanotubes and micropores, resulting in larger and more numerous focal adhesions. Osteoblasts on MN and MN-Sr1h showed no obvious differences in morphology, but those on MN-Sr3h had comparatively fewer filopodia. A previous study showed that osteoblast adhesion and

PT

filopodial spreading on the surface were inhibited at high Sr concentrations [38],

RI

implying that only heterostructures containing an appropriate amount of Sr have the

SC

ability to promote osteoblast growth. An excess of SrTiO3 nanoparticles covered the top edges and vertical walls of TiO2 nanotubes in MN-Sr3h, there by inhibiting filopodial

NU

spreading.

MA

4.4 Behavior of osteoclasts

D

In this study, osteoclasts on N and MN showed higher proliferative and

PT E

differentiation potential than those on M, which was positively correlated with the amount of protein adsorbed. Nano- and micro/nanostructures on Ti surfaces both

CE

showed excellent cytocompatibility with osteoblasts and osteoclasts. MN and MN-Sr

AC

had similar topography but differed in terms of Sr content. The proliferation and differentiation rates of osteoclasts grown on MN-Sr were lower than those on MN, and decreased over time. Previous reports have shown that Sr inhibits bone resorption by negatively regulating osteoclast matrix metalloproteinase production and induces osteoclast apoptosis via a calcium-sensing receptor-dependent mechanism [29, 43, 46]. MN-Sr3h inhibited osteoclast proliferation and differentiation, but the high Sr content of 28

ACCEPTED MANUSCRIPT MN-Sr3h had a deleterious effect on osteoblast growth. Ideally, implants should promote the growth of osteoblasts while inhibiting that of osteoclasts, achieving a coordinated balance between osteoclasts and osteoblasts. Thus, appropriate Sr loading on MN (MN-Sr1h) had a positive effect on bone formation.

PT

5. Conclusion

RI

We fabricated SrTiO3/TiO2 nanotube heterostructures on microporous Ti via acid

SC

etching and anodization combined with hydrothermal treatment. The Sr content of

NU

heterostructures was controlled with different hydrothermal reaction time. Long-lasting

MA

and controllable Sr release from SrTiO3/TiO2 heterostructures (MN-Sr) might occur for longer than 1 year. Our results indicate that SrTiO3/TiO2 heterostructures with

D

appropriate Sr content (MN-Sr1h) showed excellent capacity for inducing osteogenic

PT E

differentiation and promoting osteoblast spreading while inhibiting osteoclast differentiation. In addition, MN-Sr1h had good osteogenic activity without the need for

CE

exogenous biomolecules. These results indicate that SrTiO3/TiO2 heterostructures on

AC

microporous titanium with appropriate Sr content have the ability to enhance osseointegration and prevent osteoresorption. This novel biomaterial is promising for bone implants in clinical applications.

Acknowledgements

This work was supported by the Natural Science Foundation of China (31570955), 29

ACCEPTED MANUSCRIPT National Key Research and Development Program of China (2017YFGX090007-02)

AC

CE

PT E

D

MA

NU

SC

RI

PT

and Applied Basic Research Programs of Sichuan Province, China (2015JY0036).

30

ACCEPTED MANUSCRIPT References

AC

CE

PT E

D

MA

NU

SC

RI

PT

[1] M. Niinomi, Recent metallic materials for biomedical applications, Metall. Mater. Tran. A, 33 (2002) 477-486. [2] J.E. Davies, Understanding peri-implant endosseous healing, J. Dent. Educ., 67 (2003) 932-949. [3] D.O. Costa, P.D. Prowse, T. Chrones, S.M. Sims, D.W. Hamilton, A.S. Rizkalla, S.J. Dixon, The differential regulation of osteoblast and osteoclast activity by surface topography of hydroxyapatite coatings, Biomaterials, 34 (2013) 7215-7226. [4] D.F. Williams, On the mechanisms of biocompatibility, Biomaterials, 29 (2008) 2941-2953. [5] N.K. Shrestha, J.M. Macak, F. Schmidt-Stein, R. Hahn, C.T. Mierke, B. Fabry, P. Schmuki, Magnetically guided titania nanotubes for site-selective photocatalysis and drug release, Angew. Chem. Int. Ed. Engl., 48 (2009) 969-972. [6] S. Su, R. Zuo, D. Lv, J. Fu, Synthesis and characterization of (001) oriented BaTiO3 platelets through a topochemical conversion, Powder Technol., 217 (2012) 11-15. [7] C. Capuccini, P. Torricelli, F. Sima, E. Boanini, C. Ristoscu, B. Bracci, G. Socol, M. Fini, I.N. Mihailescu, A. Bigi, Strontium-substituted hydroxyapatite coatings synthesized by pulsed-laser deposition: in vitro osteoblast and osteoclast response, Acta Biomater., 4 (2008) 1885-1893. [8] G.A. Crawford, N. Chawla, K. Das, S. Bose, A. Bandyopadhyay, Microstructure and deformation behavior of biocompatible TiO2 nanotubes on titanium substrate, Acta Biomater., 3 (2007) 359. [9] M. Tzaphlidou, The role of collagen in bone structure: an image processing approach, Micron, 36 (2005) 593-601. [10] Y. Xin, J. Jiang, K. Huo, T. Hu, P.K. Chu, Bioactive SrTiO3 nanotube arrays: strontium delivery platform on Ti-based osteoporotic bone implants, ACS Nano, 3 (2009) 3228-3234. [11] L. Zhao, H. Wang, K. Huo, L. Cui, W. Zhang, H. Ni, Y. Zhang, Z. Wu, P.K. Chu, Antibacterial nano-structured titania coating incorporated with silver nanoparticles, Biomaterials, 32 (2011) 5706-5716. [12] L. Zhao, H. Wang, K. Huo, X. Zhang, W. Wang, Y. Zhang, Z. Wu, P.K. Chu, The osteogenic activity of strontium loaded titania nanotube arrays on titanium substrates, Biomaterials, 34 (2013) 19-29. [13] J. Zhang, J.H. Bang, C. Tang, P.V. Kamat, Tailored TiO2-SrTiO3 heterostructure nanotube arrays for improved photoelectrochemical performance, ACS Nano, 4 (2010) 387-395. [14] Z. Jiao, Y. Zhang, T. Chen, Q. Dong, G. Lu, Y. Bi, TiO2 nanotube arrays modified with Cr-doped SrTiO3 nanocubes for highly efficient hydrogen evolution under visible light, Chemistry (Easton), 20 (2014) 2654-2662. 31

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

SC

RI

PT

[15] P.J. Meunier, C. Roux, E. Seeman, S. Ortolani, J.E. Badurski, T.D. Spector, J. Cannata, A. Balogh, E.M. Lemmel, S. Porsnielsen, The effects of strontium ranelate on the risk of vertebral fracture in women with postmenopausal osteoporosis, N. Engl. J. Med., 350 (2004) 459-468. [16] N.S. Pors, The biological role of strontium, Bone, 35 (2004) 583-588. [17] J.L. Saffar, The effects of strontium ranelate on the risk of vertebral fracture in women with postmenopausal osteoporosis, N. Engl. J. Med., 350 (2004) 459-468. [18] J.Y. Reginster, E. Seeman, M.C.D. Vernejoul, S. Adami, J. Compston, C. Phenekos, J.P. Devogelaer, M.D. Curiel, A. Sawicki, S. Goemaere, Strontium ranelate reduces the risk of nonvertebral fractures in postmenopausal women with osteoporosis: treatment of peripheral osteoporosis (TROPOS) study, J. Clin. Endocrinol. Metab., 90 (2005) 2816-2822. [19] L. Maïmoun, T.C. Brennan, I. Badoud, V. Dubois-Ferriere, R. Rizzoli, P. Ammann, Strontium ranelate improves implant osseointegration , Bone, 46 (2010) 1436. [20] Y. Xin, J. Jiang, K. Huo, T. Hu, P.K. Chu, Bioactive SrTiO3 nanotube arrays: strontium delivery platform on Ti-based osteoporotic bone implants, ACS Nano, 3 (2009) 3228-3234. [21] Y. Li, Q. Li, S. Zhu, E. Luo, J. Li, G. Feng, Y. Liao, J. Hu, The effect of strontium-substituted hydroxyapatite coating on implant fixation in ovariectomized rats, Biomaterials, 31 (2010) 9006-9014. [22] E. Gentleman, Y.C. Fredholm, G. Jell, N. Lotfibakhshaiesh, M.D. O'Donnell, R.G. Hill, M.M. Stevens, The effects of strontium-substituted bioactive glasses on osteoblasts and osteoclasts in vitro, Biomaterials, 31 (2010) 3949-3956. [23] W. Zhang, Y. Shen, H. Pan, K. Lin, X. Liu, B.W. Darvell, W.W. Lu, J. Chang, L. Deng, D. Wang, W. Huang, Effects of strontium in modified biomaterials, Acta Biomater., 7 (2011) 800-808. [24] Y. Li, G. Feng, Y. Gao, E. Luo, X. Liu, J. Hu, Strontium ranelate treatment enhances hydroxyapatite-coated titanium screws fixation in osteoporotic rats, J. Orthop. Res., 28 (2010) 578-582. [25] C.J. Chung, H.Y. Long, Systematic strontium substitution in hydroxyapatite coatings on titanium via micro-arc treatment and their osteoblast/osteoclast responses, Acta Biomater., 7 (2011) 4081-4087. [26] J. Zhou, L. Yin, H. Li, Z. Liu, J. Wang, K. Duan, S. Qu, J. Weng, B. Feng, Heterojunction of SrTiO3/TiO2 nanotubes with dominant (001) facets: Synthesis, formation mechanism and photoelectrochemical properties, Mater. Sci. Semicond. Process., 40 (2015) 107-116. [27] S. Peng, X.S. Liu, S. Huang, Z. Li, H. Pan, W. Zhen, K.D. Luk, X.E. Guo, W.W. Lu, The cross-talk between osteoclasts and osteoblasts in response to strontium treatment: involvement of osteoprotegerin, Bone, 49 (2011) 1290-1298. [28] S. Panzavolta, P. Torricelli, L. Sturba, B. Bracci, R. Giardino, A. Bigi, Setting properties and in vitro bioactivity of strontium-enriched gelatin-calcium phosphate bone cements, J. Biomed. Mater. Res. A, 84 (2008) 965-972. 32

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

SC

RI

PT

[29] J.W. Park, H.K. Kim, Y.J. Kim, J.H. Jang, H. Song, T. Hanawa, Osteoblast response and osseointegration of a Ti-6Al-4V alloy implant incorporating strontium, Acta Biomater., 6 (2010) 2843-2851. [30] J. Zhou, L. Yin, K. Zha, H. Li, Z. Liu, J. Wang, K. Duan, B. Feng, Hierarchical fabrication of heterojunctioned SrTiO3/TiO2 nanotubes on 3D microporous Ti substrate with enhanced photocatalytic activity and adhesive strength, Appl. Surf. Sci, 367 (2016) 118-125. [31] C.W. Kim, S.P. Suh, M.J. Choi, Y.S. Kang, Y.S. Kang, Fabrication of SrTiO3-TiO2 heterojunction photoanode with enlarged pore diameter for dye-sensitized solar cells, J. Mater. Chem. A, 1 (2013) 11820. [32] B. Feng, J. Weng, B.C. Yang, J.Y. Chen, J.Z. Zhao, L. He, S.K. Qi, X.D. Zhang, Surface characterization of titanium and adsorption of bovine serum albumin, Mater. Charact., 49 (2002) 129-137. [33] D.T. Wassell, G. Embery, Adsorption of bovine serum albumin on to titanium powder, Biomaterials, 17 (1996) 859-864. [34] G.A. Parks, The isoelectric points of solid oxides, solid hydroxides, and aqueous hydroxo complex systems, Chem. Rev., 65 (1965) 177-198. [35] J. Shi, B. Feng, X. Lu, J. Weng, Adsorption of bovine serum albumin onto titanium dioxide nanotube arrays, Int. J. Mater. Res., 103 (2012) 889-896. [36] L. Gao, B. Feng, J. Wang, X. Lu, D. Liu, S. Qu, J. Weng, Micro/nanostructural porous surface on titanium and bioactivity, J. Biomed. Mater. Res. A Appl. Biomater., 89B (2009) 335-341. [37] L. Zhao, L. Liu, Z. Wu, Y. Zhang, P.K. Chu, Effects of micropitted/nanotubular titania topographies on bone mesenchymal stem cell osteogenic differentiation, Biomaterials, 33 (2012) 2629-2641. [38] Y. Wang, D. Zhang, C. Wen, Y. Li, Processing and characterization of SrTiO 3-TiO2 nanoparticle-nanotube heterostructures on titanium for biomedical applications, ACS Appl. Mater. Interfaces, 7 (2015) 16018-16026. [39] G.X. Ni, B. Shu, G. Huang, W.W. Lu, H.B. Pan, The effect of strontium incorporation into hydroxyapatites on their physical and biological properties, J. Biomed. Mater. Res. B Appl. Biomater., 100 (2012) 562-568. [40] S. Peng, G. Zhou, K.D. Luk, K.M. Cheung, Z. Li, W.M. Lam, Z. Zhou, W.W. Lu, Strontium promotes osteogenic differentiation of mesenchymal stem cells through the Ras/MAPK signaling pathway, Cell. Physiol. Biochem., 23 (2009) 165-174. [41] C. Fournier, A. Perrier, M. Thomas, N. Laroche, V. Dumas, A. Rattner, L. Vico, A. Guignandon, Reduction by strontium of the bone marrow adiposity in mice and repression of the adipogenic commitment of multipotent C3H10T1/2 cells, Bone, 50 (2012) 499-509. [42] J. Braux, F. Velard, C. Guillaume, S. Bouthors, E. Jallot, J.M. Nedelec, D. Laurent-Maquin, P. Laquerriere, A new insight into the dissociating effect of strontium on bone resorption and formation, Acta Biomater., 7 (2011) 2593-2603. [43] G.T. Huang, W.G. Liu, G.X. Ni, Effect of strontium on the biological characteristics 33

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

SC

RI

PT

of osteoblasts and osteoclasts, J. Clin. Reh. Tissue Engin. Res., 12 (2008) 9113-9116. [44] X. Liu, P.K. Chu, C. Ding, Surface modification of titanium, titanium alloys, and related materials for biomedical applications, Mater. Sci. Engin., R, 47 (2003) 49-121. [45] K.S. Brammer, S. Oh, C.J. Frandsen, S. Varghese, S. Jin, Nanotube surface triggers increased chondrocyte extracellular matrix production, Mater. Sci. Engin., C, 30 (2010) 518-525. [46] F. Yang, D. Yang, J. Tu, Q. Zheng, L. Cai, L. Wang, Strontium enhances osteogenic differentiation of mesenchymal stem cells and in vivo bone formation by activating Wnt/catenin signaling, Stem Cells, 29 (2011) 981-991.

34

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

SC

RI

PT

Graphical abstract

35

ACCEPTED MANUSCRIPT Highlights

AC

CE

PT E

D

MA

NU

SC

RI

PT

1. The fabrication of SrTiO3/TiO2 nanoparticle-nanotube heterostructures on a microporous titanium (Ti) surface 2. SrTiO3/TiO2 heterostructures exhibited controllable and sustained Sr2+ ion release . 3. SrTiO3/TiO2 heterostructures exhibited excellent Osteogenesis.

36