Nano-hydroxyapatite crystal formation based on calcified TiO2 nanotube arrays

Accepted Manuscript Nano-hydroxyapatite crystal formation based on calcified TiO2 nanotube arrays

Qiaoxia Lin, Di Huang, Jingjing Du, Yan Wei, Yinchun Hu, Xiaojie Lian, Xin Xie, Weiyi Chen, Yu Shrike Zhang PII: DOI: Reference:

S0169-4332(19)30257-0 https://doi.org/10.1016/j.apsusc.2019.01.226 APSUSC 41625

To appear in:

Applied Surface Science

Received date: Revised date: Accepted date:

29 September 2018 7 January 2019 25 January 2019

Please cite this article as: Q. Lin, D. Huang, J. Du, et al., Nano-hydroxyapatite crystal formation based on calcified TiO2 nanotube arrays, Applied Surface Science, https://doi.org/10.1016/j.apsusc.2019.01.226

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

Nano-Hydroxyapatite Crystal Formation Based on Calcified TiO2 Nanotube Arrays Qiaoxia Lina, Di Huanga,b,c,*, Jingjing Dua, Yan Weia, Yinchun Hua, Xiaojie Liana,

Department of Biomedical Engineering, Research Center for Nano-biomaterials & Regenerative

SC

a

RI

PT

Xin Xiec, Weiyi Chenb, Yu Shrike Zhangc,*

Medicine, College of Biomedical Engineering, Taiyuan University of Technology, Taiyuan 03002

b

NU

4, PR China

Shanxi Key Laboratory of Material Strength & Structural Impact, Institute of Biomedical Engin

c

MA

eering, Taiyuan University of Technology, Taiyuan 030024, PR China Division of Engineering in Medicine, Department of Medicine, Brigham and Women’s Hospital

, Harvard Medical School, Cambridge, MA 02139, USA

PT E

D

* Corresponding authors. Tel: +86-351-3176651; Fax: +86-351-3176658. E-mail addresses: [email protected] (D. Huang); [email protected] (Y.S.

AC

CE

Zhang)

ACCEPTED MANUSCRIPT ABSTRACT The lack of biological activity of pure titanium as a dental implant has attracted increasing attention. This condition can be improved by preparing a layer of nano-hydroxyapatite (nano-HA) coating that is stable and does not easily fall off, which however, remains a challenge. Here, we report the preparation of nano-HA crystals on calcified titanium oxide (TiO2) nanotube arrays by means

PT

of anodization, heat treatment, calcification in vacuum and hydrothermal phosphorylation, achieving the chemical bonding between the nano-HA coating and TiO2 substrate. The mechanism of

RI

coating formation was also investigated. The results confirmed that annealing at 450 oC could

SC

convert the amorphous nanotubes to the anatase structure. Calcium titanate (CaTiO3) was formed on the surface of nanotubes by heat treatment after calcification, which could provide nucleation

NU

sites for the formation of nano-HA. Needle-like nano-HA crystals were subsequently formed on the surface of the TiO2 nanotube arrays. The scratch test showed that the adhesion strength be-

MA

tween the formed nano-HA coating and the TiO2 nanotube arrays was higher than 29.17 ± 1.07 N. In vitro cell proliferation and adhesion experiments showed that the nano-HA coating had good

D

biocompatibility. Alkaline phosphatase (ALP) activity and osteocalcin (OCN) expressions high-

PT E

lighted the higher osteoconductivity of the nano-HA coating on the surface of TiO2 nanotube arrays compared to the pure Ti.

AC

CE

Keywards: Nano-hydroxyapatite; Titanium; Anodization; Titania nanotube arrays; Calcification

ACCEPTED MANUSCRIPT 1. Introduction Owing to its excellent mechanical properties, corrosion resistance, and biocompatibility, the pure titanium (Ti) metal is extensively applied in load-bearing dental implant prosthesis [1]. However, due to the intrinsic bioinertness of the material, the bonding of Ti surfaces with surrounding bone is difficult at the early stage following implantation. In fact, it always results in the eventual

PT

failure of the implants [2, 3]. To achieve rapid osseointegration and necessary mechanical strength of implants, numerous research has been devoted to forming hydroxyapatite (HA) coatings on the

RI

surface of Ti substrates. However, the HA coating easily flakes away from the Ti substrate due to

SC

the mismatch in thermal and mechanical properties between HA and Ti, which may lead to surgical failures [4]. To date, various attempts have been made to address this challenge.

NU

A widely adopted solution is to prepare a porous titanium oxide (TiO2) layer on the Ti substrate prior to HA coating, which obviously can increase the bonding strength and reduce mis-

MA

match in material properties at the interface [5, 6]. TiO2 nanotube arrays have attracted increasing attention due to their high surface-to-volume ratio and good bioactivity[7]. Anodization in a elec-

D

trolyte containing fluorinated ammonia (NH4F) is the most suitable strategy for obtaining highly

PT E

ordered TiO2 nanotube arrays [8]. On the other hand, a series of techniques have been developed to fabricate HA coatings on Ti substrates such as plasma spraying [9], electrophoretic deposition [10], biomineralization methods [11], and radio-frequency magnetron sputtering [12], among oth-

CE

ers. While the most widely used method for its controllability, plasma-spraying has several problems that can easily cause inferior coating including poor bonding strength between the coating

AC

and the substrate, residual stress in the sprayed coatings, and non-uniformity in the coating density [13]. Electrophoretic deposition has high HA growth rate using lower reaction temperatures, but the bonding strength is weak as well [14]. Consequently, it is still a challenge to achieve a high quality of the HA coatings, a fast speed of crystal formation, and a good bonding strength at the same time, because the formation process of HA is complicated. It has been demonstrated in a number of studies that TiO2 nanotubes can accelerate rates of apatite formation on the surface of the Ti metal [15]. Various types of surface modification methods for improving the bioactivity of TiO2 layers have thus been studied, such as heat treatment [16] and alkali-heat treatment [17]. It has become increasingly evident that the anatase form of TiO2

ACCEPTED MANUSCRIPT after an appropriate heat treatment can effectively improve the bioactivity of the TiO2 layers [18]. For example, Wen et al. have reported the effect of hydrothermal treatment in calcium hydroxide (Ca(OH)2) solution on the formation of apatite [19], which was evaluated by soaking in a modified simulated body fluids (m-SBF). It was proven that this treatment could accelerate apatite formation. In the present study, we first prepared a layer of TiO2 nanotube arrays on top of Ti as the

PT

transition layer by anodic oxidation, and then formed a thin apatite coating by calcification prior to

RI

hydrothermal treatment, achieving a stable coating of nano-HA crystals. Scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS), X-ray photoelectron spectroscopy

SC

(XPS), attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy, atomic

NU

force microscopy (AFM), and X-ray diffraction (XRD) were used to explore the surface morphology, crystal structure, composition, roughness and formation mechanism of these coatings. In ad-

MA

dition, the adhesion strength between the coating and the substrate was measured with an automatic scratch tester. Finally, pre-osteoblast behaviors were investigated to demonstrate the biocompat-

D

ibility and osteogenic performance of the nano-HA crystal coating.

PT E

2. Materials and methods 2.1. Preparation of TiO2 layers

CE

The surfaces of commercially pure Ti sheets (99.7%, 10×10×0.1 mm3) were sequentially abraded and polished smooth with 400-grit, 800-grit, and 1000-grit carborundum papers. Prior to

AC

anodization, Ti substrates were ultrasonically cleaned in ethanol and distilled water for 10 min each to remove stains and grease, followed by drying at room temperature. Next, electrochemical anodization was carried out with a two-electrode electrolytic cell that used Ti as the anode and platinum (Pt) as the cathode. The electrolyte solution consisted of 50 wt% propanetriol, 50 wt% deionized water, and 0.9 wt% NH4F. The anodizing voltage was kept constant at 30 V by using a DC power supply (Maynuo, China) during the course. The anodization was conducted at room temperature for 2 h. After anodizing, the samples were rinsed with distilled water and dried at room temperature, followed by annealing at 450 oC for 2 h and then cooling to room temperature.

ACCEPTED MANUSCRIPT 2.2. Preparation of nano-HA coatings The anodized samples were subjected to calcification treatment by soaking in a CaCl2 solution (1 M) at 37 oC for 30 min in vacuum. Then, the samples were taken out and dried at ambient temperature, followed by annealing at 450 oC for 2 h with a heating rate of 5 oC/min. Next, the annealed samples were rinsed with distilled water. The treated samples above were placed vertically in sodium phosphate aqueous solution (Na3PO4·12H2O, 0.1 M, pH = 12) and reacted at 90 o

RI

water. Finally, the samples were dried at room temperature in air.

PT

C for 1 h, 3 h, or 5 h. Then, the as-formed specimens were retrieved and rinsed with deionized

2.3. Characterizations

SC

The morphology of the samples was observed using SEM (JSM-7100F, Japan). EDS (Oxford MaxN, UK) equipped on the SEM was used to detect the elemental composition on the surface of

NU

the samples. The crystal structure was examined by XRD (X’Pert PRO, The Netherlands) in a

MA

step-scan mode with a step size of 0.02° ranging from 20° to 80°. The samples were characterized by XPS (XSAM800, UK) and ATR-FTIR (Bruker Alpha, Germany) to determine their chemical compositions. The surface morphology and roughness of specimens were analyzed using an AFM

D

(SPA-300HV, Japan) in the tapping mode. Automatic scratch tester (HT-3002, China) with a dia-

PT E

mond stylus (120° cone with a 200-μm-radius tip) was used to measure the bonding strength between the coating and substrate, following ASTM C1624-05, i.e., Standard Test Method for Adhe-

CE

sion Strength and Mechanical Failure Modes of Ceramic Coating by Quantitative Single Point Scratch Testing [20]. The loading rate was 10 N/min. Each sample was scratched three times to

AC

ensure the reliability of the results. 2.4. Cell proliferation and adhesion Mouse pre-osteoblasts (MC3T3-E1) were employed to evaluate the cell proliferation and adhesion on different samples. The samples were disinfected by high-pressure steam sterilization and then placed in 24-well plates. Afterwards, the cells were seeded on the surfaces of the samples at a density of 2×104/mL and incubated at 37 oC in an incubator containing 5% CO2. The cells were cultured for 1, 4, and 7 days for the proliferation assay or 5 days for the attachment assay. The Cell Counting Kit (CCK)-8 (Beyotime, China) was used to analyze cell proliferation. At each time point of measurement, 1 mL of medium was added to each well with 100 μL CCK-8 reagent

ACCEPTED MANUSCRIPT according to the manufacturer’s instructions. After 1 h incubation at 37 oC, the absorbance was read with a microplate reader (Biorad iMark, USA) at 450 nm. Each group included three parallel samples. The samples cultured for 5 days were rinsed with phosphate-buffered saline (PBS), fixed with 2.5% glutaraldehyde (Aladdin, China), and dehydrated by sequential soaking in 30%, 50%, 75%, 80%, 95%, and 100% ethanol for 15 min each, followed by dealcoholization with isoamyl acetate for 30 min. Then the dried samples were observed under SEM at 15kV.

PT

2.5. Alkaline phosphatase (ALP) activity

RI

The ALP activity was determined by quantifying the amount of p-nitrophenol phosphate (pNPP), the product generated by the reaction of alkaline phosphatase, using an ALP Kit (Be-

SC

yotime) according to the assay protocol. A 1-mL cell suspension was seeded on each sample in 24-well plates at a density of 2×104 cells/mL. After culturing for 1, 4, and 7 days, the media were

NU

removed and the samples were washed with PBS. Then, 100 μL of cell lysis solution (Beyotime) was added to each well for 4 min. The lysates were collected and centrifuged, and the supernatant

MA

was taken. 50 μL of the supernatant was added to the wells of a 96-well plate and incubated with 50 μL of the substrate solution (pNPP) at 37 oC for 10 min. Finally, 100 μL of stop solution was

D

added and the optical density (OD) values were measured with a microplate reader set to 405 nm.

PT E

A calibration curve was obtained using the p-nitrophenol solution of known concentrations. The diethanolamine (DEA) enzyme activity unit was calculated according to the definition of the ALP

CE

Kit instruction.

2.6. Osteocalcin (OCN) enzyme-linked immunosorbent assay (ELISA)

AC

The contents of OCN in cell culture media were evaluated using a Mouse Osteocalcin ELISA Kit (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer’s instructions. Briefly, media were harvested at day 1, 4, and 7. To each well 50 μL of sample and 50 μL of biotinylated antigen working solution were added. After 30 min of incubation at 37 oC and washing, 50 μL of horseradish peroxidase (HRP) working solution were added, followed by 30 min of incubation and further washing. Then, 50 μL of chromogenic solution A and 50 μL chromogenic solution B were added to each well. After 10 min of incubation at 37 oC away from light, 50 μL of stop solution was added to stop the reaction and OD was determined using a microplate reader at 450-nm wavelength.

ACCEPTED MANUSCRIPT 3. Results and discussions Fig. 1a shows the surface morphology of pure Ti. Fig. 1b, c shows SEM images of anodized and annealed TiO2 nanotube arrays on the pure Ti substrate, in which the nanotubes were vertically aligned. These nanotubes presented a highly ordered hollow structure with an average diameter of 129.34 ± 3.62 nm and an average length of 560.15 ± 20.02 nm. This morphology may facilitate

PT

the growth of nano-HA for its large surface roughness and area. From Fig. 1d, it could be observed that after calcification, sheet-like products appeared on the surface of TiO2 nanotubes. EDS

AC

CE

PT E

D

MA

NU

SC

RI

spectrum (the inset in Fig. 1d) shows the existence of Ca element in the sheet-like products.

Fig. 1. SEM images of pure Ti (a); TiO2 nanotubes: top-view and distribution of nanotube diameters (b); and TiO2 nanotubes: side-view and distribution of nanotube lengths (c), and the SEM image with EDS mapping and EDS spectrum of calcified TiO2 nanotubes (d).

After calcification, surface analysis was carried out using XPS. The chemical states of Ti and O were investigated and the spectra of coatings of calcified samples are shown in Fig. 2. The Ti 2p XPS spectrum of the annealed sample exhibited two contributions, 2p3/2 and 2p1/2, located respectively at 458.86 and 464.57 eV, and the peak of O 1s was located at 530.10 eV,

ACCEPTED MANUSCRIPT which could be assigned to TiO2 [21]. Compared with the annealed sample, the chemical state of O changed after calcification. The Ti 2p spectrum of the calcified sample showed two main peaks. From curve fitting, the double peak could be deconvoluted into 4 subpeaks at 457.30, 458.39, 462.60, and 464.09 eV, respectively, which clearly evidenced the presence of two chemical environments for Ti atoms. The first predominant doublet, present at 458.39 and 464.09 eV, is characteristic of TiO2. The second one, with a lower intensity, was located at lower binding energies of

PT E

D

MA

NU

SC

RI

PT

457.30 and 462.60 eV, probably attributing to CaTiO3 [22, 23].

CE

Fig. 2. XPS spectrum and Ti 2p, O 1s peaks of annealed and calcified TiO2 nanotube samples.

Fig. 3 shows the XRD patterns of all samples. It was confirmed from the XRD analysis that

AC

an anatase structure of TiO2 was formed after annealing at 450 °C for 2 h (Fig. 3b). It has been reported that an anatase or a rutile structure can be more favorable to apatite formation than an amorphous structure [24]. Hence, the as-anodized samples were annealed at 450 °C. In Fig. 3c, a new peak at 33.2° was detected, which is corresponding to the diffraction peak of the (121) plane of CaTiO3 (JCPDS no. 42-0423). Other peaks of CaTiO3 were not measured, probably because the products were too thin to be detected by XRD. The vacuum effectively removed the air from the nanotubes and then drove CaCl2 solution into the TiO2 nanotubes, leading to a homogeneous adsorption of calcium ions on the surface of nanotubes. In the process of annealing, CaTiO3 was

ACCEPTED MANUSCRIPT possibly formed by solid-phase reaction, which could provide nucleation sites for the formation of apatite. The presence of CaTiO3 further possibly introduced a chemical bonding for the formation of nano-HA crystals on the TiO2 nanotube layers. The crystal phase of the formed apatite was further investigated by XRD. As shown in Fig. 3d, the pattern for phosphorylated sample had several

PT E

D

MA

NU

SC

RI

PT

peaks (2θ = 25.9°, 31.8°, 32.9°) that might be well-indexed to HA (JCPDS no.09-0432) [25, 26].

Fig. 3. XRD patterns of samples: pure Ti (a), annealed TiO2 nanotubes (b), calcified samples (c),

CE

and samples phosphorylated for 3h (d), respectively.

AC

ATR-FTIR analysis was further carried out to verify the conversion of layer composition in the reaction process. As shown in Fig. 4, a peak in the range of 450-650 cm-1 appeared in the spectra b and c, which could be ascribed to the stretching vibration of Ti-O bond of the oxide layer [27]. Upon treatment with CaCl2 solution, the new peak at 420 cm-1 could be assigned to the stretching vibration of the Ti-O (from TiO32- groups) [28]. The new band at 877 cm-1 is related to the stretching vibration along Ca-O-Ca bonds [29]. In addition, the characteristic peaks of HA (PO43-) appeared at 1025 cm-1 and 930 cm-1, which demonstrated successful formation of nanoHA crystals [30, 31].

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

Fig. 4. ATR-FTIR spectra of samples: pure Ti (a), annealed TiO2 nanotubes (b), calcified samples (c),

AC

CE

PT E

D

MA

and samples phosphorylated for 3h (d), respectively.

Fig. 5. The SEM images, EDS spectra and nano-HA crystal diameter distributions of phosphorylated samples in different time: 1 h (a), 3 h (b) and 5 h (c).

The morphology and composition of the nano-HA crystal coatings varied with time for which the samples were soaked in the phosphate solution. The results characterized by SEM and EDS are shown in Fig. 5. The crystals of phosphorylated samples exhibited a needle-like morphology. Nano-HA crystals formed by hydrothermal treatment for 1 h were relative sparse (Fig. 5a). The

ACCEPTED MANUSCRIPT average diameter was 88.81 ± 15.78 nm. The TiO2 nanotubes could still be seen at the bottom of the formed nano-HA crystals. The density of nano-HA crystals increased with soaking time and the coatings became more intensive and covered the entire surface of TiO2 nanotubes at 3 h (Fig. 5b). The average diameter of the crystals was 96.24 ± 15.19 nm. After immersion for 5 h, it could be seen that the crystals continued to grow and some crystals bundled together (Fig. 5c). The average diameter increased up to 101.33 ± 12.23 nm. EDS analysis was used to quantify Ca and P el-

PT

ements of nano-HA crystals. The Ca/P ratios of the products soaked for 1, 3, and 5 h were approx-

RI

imately 1.92, 1.67, and 1.44, respectively. At the early stage (1 h), the products were rich in calcium. With increase in the immersion time, the calcium content of products was reduced. The de-

SC

crease of calcium might be due to the formation of new nano-HA crystals. The nano-HA crystals

NU

produced by soaking in phosphate for 3 h had a Ca/P ratio of roughly 1.67, which is stoichiometrically consistent with the natural HA. The Ca/P ratio of the crystals reacted for 1 h and 5 h was

MA

1.92 and 1.44, respectively, which indicated that the formed nano-HA crystals were deficient in

AC

CE

PT E

D

composition with the natural HA.

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

D

Fig. 6. XPS spectrum and O 1s, Ca 2p, and P 2p peaks of samples phosphorylated for 3 h.

PT E

XPS analysis was performed on the phosphorylated samples reacted for 3 h to further confirm the chemical compositions of the coatings. The results in Fig. 6 revealed that the spectra on

CE

the coating surface consisted of O 1s, Ca 2p, and P 2p. Five characteristic peaks of O, Ca, and P could be observed. The binding energies of O 1s peaks were 530.59 eV and 531.65 eV, corre-

AC

sponding to PO43- and OH-, respectively, of HA. The measured binding energies of 350.50 eV for the Ca 2p1/2 peak, 346.97 eV for the Ca 2p3/2 peak, and 133.01 eV for the P 2p peak are in agreement with previously published results relating to HA crystal structure[6, 32]. These findings showed that the apatite formed by treatment for 3 h corresponding to the typical HA coating, and were thus used for the following cell evaluations.

RI

PT

ACCEPTED MANUSCRIPT

SC

Fig. 7. The mechanism diagram of nano-HA crystals formation based on calcified TiO2 nanotube arrays.

NU

Fig. 7 shows the mechanism diagram of nano-HA crystals formation based on calcified TiO2

MA

nanotube surface. TiO2 nanotube arrays were formed under the etching action of F- contained in electrolyte and converted to anatase phase by annealing. By immersing in the calcium solution under vacuum, large amount of Ca2+ were adsorbed on the surface and inside of the TiO2 nano-

D

tubes. After annealing, a thin layer of CaTiO3 was formed by the reaction between adsorbed Ca2+

PT E

and TiO2, providing nucleation sites for the formation of nano-HA crystals. By immersing in the phosphate solution, needle-like nano-HA crystals were finally formed on the surface of calcified

CE

TiO2 nanotube arrays.

The surface morphology and roughness of pure Ti, TiO2 nanotube arrays, and nano-HA were

AC

characterized using AFM and the 2D and 3D morphologies are showed in Fig.8. The centerline average roughness (Ra) was 1.316 nm for pure Ti, 21.53 nm for TiO2 nanotubes, and 19.79 nm for nano-HA based on a 2×2-μm2 scan area. The TiO2 nanotube layer and the HA layer had greater surface roughness than the pure Ti, which may facilitate cell adhesion on the surface. Meanwhile, the rough surface of the TiO2 nanotubes produced high specific surface area, which could be beneficial to improving the bonding between the nanotube layer and the HA layer.

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

Fig. 8. AFM images of the surface topographies: pure Ti (a, d), TiO2 nanotubes (b, e), and nano-HA

AC

CE

PT E

D

phosphorylated for 3 h (c, f), respectively.

Fig. 9. AE signal and tangential friction force curves: TiO2 nanotube samples (a) and nano-HA crystal samples phosphorylated for 3 h (b).

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

PT E

ples phosphorylated for 3 h (b, d, f).

D

Fig. 10. SEM micrographs of the scratches: TiO2 nanotube samples (a, c, e) and nano-HA crystal sam-

Bonding strength between the coating and substrate was analyzed with a scratch tester and

CE

the morphology of the scratch track was observed by SEM. Fig. 9a shows the acoustic emission (AE) signal and tangential friction force curves of the coatings formed on pure Ti substrates. Gen-

AC

erally, the load at which the coating is totally peeled off from the substrate is designated as the critical load (Lc). From the inflection point of friction and the flex points of AE signal, the average Lc of TiO2 nanotube coating was calculated to be 28.78 ± 2.71 N. From the SEM micrographs (Fig. 10a, c, and e), the TiO2 nanotubes were brittlely peeled off from the substrate, which is consistent with results of AE and friction tests. For the nano-HA crystal samples, the tangential friction force curve had an inflection point at normal load of 28.5 N. Moreover, the fluctuations in the AE signal curves (Fig. 9b) also indicated that the coatings hardly underwent brittle spalling. The SEM images (Fig. 10b, d, and f) of the scratch of nano-HA crystal samples showed that the HA crystals still adhered to the exfoliated TiO2 layers. The average Lc calculated from tangential fric-

ACCEPTED MANUSCRIPT tion force curves of the nano-HA crystal samples was 29.17 ± 1.07 N. It means that the adhesion strength between th formed nano-HA coating and the TiO2 nanotube arrays was higher than 29.17 ± 1.07 N. Meanwhile, the bonding strength between the nano-HA layer and TiO2 nanotube layer is strong enough compared with that of other coatings on Ti by biomimetic deposition [33] and electrochemical [34] results. The high bonding strength and the stable bonding could be reasonably attributed to the chemical bonding between the nano-HA layer and the TiO2 nanotube

AC

CE

PT E

D

MA

NU

SC

RI

PT

layer.

Fig. 11. The SEM images and high-magnification images inserted of MC3T3 cells cultured for 5 days on pure Ti samples (a) TiO2 nanotube samples (b) nano-HA crystal samples (c); CCK-8 assay for proliferation (d), ALP activity (e) and osteocalcin (OCN) enzyme-linked immunosorbent assay of MC3T3E1 cells cultured on different samples for 1, 4 and 7 days. The data are represented as mean ± standard deviation, n = 3 (*P < 0.05, **P < 0.01, ***P < 0.001)

ACCEPTED MANUSCRIPT Surface contact and interactions between cell and biomaterials play critical roles in the success of implantation. Thus, the morphology of MC3T3-E1 preosteoblasts on various substrates after culturing for 5 days was evaluated by SEM (Fig. 11a-c). The large amounts of cells attached well on the three samples, which suggested that all samples had good biocompatibility. On the nano-HA crystal-coated surfaces, the cells exhibited superior attachment as compared to the pure Ti and TiO2-coated surfaces, and high-magnification images showed that the cells had extending

PT

lamellipodia and filopodia to adhere to the surface. After 1, 4, and 7 days of culture, proliferation

RI

of the cells on the different samples was characterized by using the CCK-8 proliferation assay. The results in Fig. 11d demonstrated that the number of cells on the nano-HA crystal-coated sam-

SC

ples was slightly higher than that on pure Ti at 4 days and 7 days (P < 0.001). After culturing for 4

NU

days, the number of cells on the nano-HA crystal-coated samples was more than that on the TiO2 nanotube-coated samples (P < 0.05). For proliferation of the cells on each type of samples, the

MA

number of cells was shown to monotonically increase with prolonged culture time from 1 day to 7 days. Therefore, as compared with the pure Ti and TiO2 coatings, the nano-HA crystal-coated substrates exhibited better biocompatibility to promote the proliferation of preosteoblasts.

D

As biocoatings on the surface of implants, they should have excellent osteogenic ability. ALP

PT E

activity and the expression of OCN are commonly used to reflect the osteoblast differentiation at the early and late stages, respectively. To evaluate the osteogenic differentiation of the MC3T3-E1

CE

preosteoblasts on the different substrates, ALP activity and OCN levels were measured. Fig. 11e shows the ALP activity results for the different samples at 1, 4, and 7 days of culture. At day 1,

AC

ALP activity for the cells on the pure Ti was significantly lower than that of cells on the TiO2 nanotube- and nano-HA crystal-coated samples. Moreover, the quantification results showed that the ALP activities for cells cultured on all samples except the pure Ti substrate increased with time throughout the measurement period. At 7 days, the highest ALP activity was found in the lysates of the cells cultivated on the nano-HA crystal-coated samples. The formation of nano-HA crystals, therefore, obviously improved the ALP activity of the cells. ELISA was further used to evaluate the OCN secretions by the cells (Fig. 11f). The results showed that the levels of OCN continued to increase as the culture time was prolonged, which was consistent with the trend of ALP. Additionally, the OCN levels of the cells on nano-HA crystal-coated samples were higher

ACCEPTED MANUSCRIPT than those of the cells cultured on pure Ti and TiO2 nanotube-coated samples at all time points (P < 0.05).

4. Conclusions In the present work, the anodization, vacuum calcification, and hydrothermal phosphorylation were used to prepare nano-HA crystals based on TiO2 nanotube arrays. CaTiO3 was formed

PT

on the surface of nanotubes by heat treatment after calcification, which provided nucleation sites for later phosphorylation to form HA. The chemical bonding between nano-HA crystals and the

RI

substrate was achieved, which may present great potential applications in dental prosthesis. In

SC

vitro cytological test results indicated that the MC3T3-E1 preosteoblasts on the nano-HA crystalcoated samples had better cell adhesion, spreading, proliferation, and osteogenic differentiation

NU

than on the pure Ti and TiO2 nanotube-coated substrates.

MA

Acknowledgements

This work was supported by the Natural Science Foundation of China (Grant no.: 11502158, 11632013,

51503140,

11802197,

51502192)

and

China

Scholarship

Council

(CSC,

D

201706935057). The support of the International Cooperation Project Foundation of Shanxi Prov-

PT E

ince (Grant no: 201803D421060) and the Natural Science Foundation for Young Scientist of

References

CE

Shanxi Province, China (201801D221439) is also acknowledged with gratitude.

[1] X. Liu, P.K. Chu, C. Ding, Surface nano-functionalization of biomaterials, Materials Sci-

AC

ence and Engineering: R: Reports, 70 (2010) 275-302. [2] Q. Liu, J. Ding, F.K. Mante, S.L. Wunder, G.R. Baran, The role of surface functional groups in calcium phosphate nucleation on titanium foil: a self-assembled monolayer technique, Biomaterials, 23 (2002) 3103-3111. [3] H. Cheng, W. Xiong, Z. Fang, H. Guan, W. Wu, Y. Li, Y. Zhang, M.M. Alvarez, B. Gao, K. Huo, J. Xu, N. Xu, C. Zhang, J. Fu, A. Khademhosseini, F. Li, Strontium (Sr) and silver (Ag) loaded nanotubular structures with combined osteoinductive and antimicrobial activities, Acta Biomaterialia, 31 (2016) 388-400. [4] S.D. Cook, K.A. Thomas, J.F. Kay, Experimental coating defects in hydroxylapatitecoated implants, Clinical orthopaedics and related research, (1991) 280-290.

ACCEPTED MANUSCRIPT [5] S. Ahmadi, I. Mohammadi, S.K. Sadrnezhaad, Hydroxyapatite based and anodic Titania nanotube biocomposite coatings: Fabrication, characterization and electrochemical behavior, Surface & Coatings Technology, 287 (2016) 67-75. [6] R.V. Chernozem, M.A. Surmeneva, B. Krause, T. Baumbach, V.P. Ignatov, A.I. Tyurin, K. Loza, M. Epple, R.A. Surmenev, Hybrid biocomposites based on titania nanotubes and a hydroxyapatite coating deposited by RF-magnetron sputtering: Surface topography, structure, and mechanical properties, Applied Surface Science, 426 (2017) 229-237.

PT

[7] A.W. Tan, B. Pingguan-Murphy, R. Ahmad, S.A. Akbar, Review of titania nanotubes: Fabrication and cellular response, Ceramics International, 38 (2012) 4421-4435.

RI

[8] A. Ghicov, P. Schmuki, Self-ordering electrochemistry: a review on growth and functionality of TiO2 nanotubes and other self-aligned MOx structures, Chemical Communications,

SC

(2009) 2791-2808.

[9] R. Palanivelu, S. Kalainathan, A.R. Kumar, Characterization studies on plasma sprayed

NU

(AT/HA) bi-layered nano ceramics coating on biomedical commercially pure titanium dental implant, Ceramics International, 40 (2014) 7745-7751.

[10] B.K. Zhang, C.T. Kwok, Hydroxyapatite-anatase-carbon nanotube nanocomposite coat-

MA

ings fabricated by electrophoretic codeposition for biomedical applications, Journal of Materials Science-Materials in Medicine, 22 (2011) 2249-2259. [11] F. Barrère, C.M. van der Valk, G. Meijer, R.A.J. Dalmeijer, K. de Groot, P. Layrolle,

D

Osteointegration of biomimetic apatite coating applied onto dense and porous metal implants

67B (2003) 655-665.

PT E

in femurs of goats, Journal of Biomedical Materials Research Part B: Applied Biomaterials,

[12] M.A. Surmeneva, R.A. Surmenev, Y.A. Nikonova, I.I. Selezneva, A.A. Ivanova, V.I. Putlyaev, O. Prymak, M. Epple, Fabrication, ultra-structure characterization and in vitro stud-

CE

ies of RF magnetron sputter deposited nano-hydroxyapatite thin films for biomedical applications, Applied Surface Science, 317 (2014) 172-180.

AC

[13] V. Jokanović, M. Vilotijević, B. Čolović, M. Jenko, I. Anžel, R. Rudolf, Enhanced Adhesion Properties, Structure and Sintering Mechanism of Hydroxyapatite Coatings Obtained by Plasma Jet Deposition, Plasma Chemistry and Plasma Processing, 35 (2015) 1-19. [14] Y.-q. Wang, J. Tao, L. Wang, P.-t. He, T. Wang, HA coating on titanium with nanotubular anodized TiO(2) intermediate layer via electrochemical deposition, Transactions of Nonferrous Metals Society of China, 18 (2008) 631-635. [15] A.F. Cipriano, C. Miller, H. Liu, Anodic Growth and Biomedical Applications of TiO2 Nanotubes, Journal of Biomedical Nanotechnology, 10 (2014) 2977-3003. [16] B. Yang, M. Uchida, H.-M. Kim, X. Zhang, T. Kokubo, Preparation of bioactive titanium metal via anodic oxidation treatment, Biomaterials, 25 (2004) 1003-1010.

ACCEPTED MANUSCRIPT [17] S.Y. Kim, Y.K. Kim, I.S. Park, G.C. Jin, T.S. Bae, M.H. Lee, Effect of alkali and heat treatments for bioactivity of TiO2 nanotubes, Applied Surface Science, 321 (2014) 412-419. [18] J. He, W. Zhou, X. Zhou, X. Zhong, X. Zhang, P. Wan, B. Zhu, W. Chen, The anatase phase of nanotopography titania plays an important role on osteoblast cell morphology and proliferation, Journal of Materials Science-Materials in Medicine, 19 (2008) 3465-3472. [19] X.-f. Xiao, T. Tian, R.-f. Liu, H.-d. She, Influence of titania nanotube arrays on biomimetic deposition apatite on titanium by alkali treatment, Materials Chemistry and Physics,

PT

106 (2007) 27-32. [20] S.T. Gonczy, N. Randall, An ASTM standard for quantitative scratch adhesion testing of

RI

thin, hard ceramic coatings, International Journal of Applied Ceramic Technology, 2 (2005) 422-428.

SC

[21] R. Rahal, T. Pigot, D. Foix, S. Lacombe, Photocatalytic efficiency and self-cleaning properties under visible light of cotton fabrics coated with sensitized TiO2, Applied Catalysis

NU

B: Environmental, 104 (2011) 361-372.

[22] L.P. Zhang, M. Li, U. Diebold, Characterization of Ca impurity segregation on the TiO2(110) surface, Surface Science, 412-413 (1998) 242-251.

MA

[23] S.-W. Lee, L.M. Lozano-Sánchez, V. Rodríguez-González, Green tide deactivation with layered-structure cuboids of Ag/CaTiO3 under UV light, Journal of Hazardous Materials, 263 (2013) 20-27.

D

[24] Z. Zhang, H. Liu, Q. Shi, X. Liu, L. Wan, Calcium ion modification of TiO2 nanotube

PT E

arrays to enhance apatite formation, Materials Technology, 31 (2016) 791-798. [25] Y. Ha, J. Yang, F. Tao, Q. Wu, Y.J. Song, H.R. Wang, X. Zhang, P. Yang, PhaseTransited Lysozyme as a Universal Route to Bioactive Hydroxyapatite Crystalline Film, Advanced Functional Materials, 28 (2018).

CE

[26] J. Shen, Y. Qi, B. Jin, X. Wang, Y. Hu, Q. Jiang, Control of hydroxyapatite coating by self-assembled monolayers on titanium and improvement of osteoblast adhesion, Journal of

AC

Biomedical Materials Research Part B-Applied Biomaterials, 105 (2017) 124-135. [27] P. Agilan, N. Rajendran, In-vitro bioactivity and electrochemical behavior of polyaniline encapsulated titania nanotube arrays for biomedical applications, Applied Surface Science, 439 (2018) 66-74. [28] C. Peng, Z. Hou, C. Zhang, G. Li, H. Lian, Z. Cheng, J. Lin, Synthesis and luminescent properties of CaTiO3: Pr3+ microfibers prepared by electrospinning method, Opt. Express, 18 (2010) 7543-7553. [29] L.M. Lozano-Sanchez, S.-W. Lee, T. Sekino, V. Rodriguez-Gonzalez, Practical microwave-induced hydrothermal synthesis of rectangular prism-like CaTiO3, Crystengcomm, 15 (2013) 2359-2362.

ACCEPTED MANUSCRIPT [30] S. Durdu, O.F. Deniz, I. Kutbay, M. Usta, Characterization and formation of hydroxyapatite on Ti6Al4V coated by plasma electrolytic oxidation, Journal of Alloys and Compounds, 551 (2013) 422-429. [31] J. Esguerra Arce, A. Esguerra Arce, Y. Aguilar, L. Yate, S. Moya, C. Rincon, O. Gutierrez, Calcium phosphate-calcium titanate composite coatings for orthopedic applications, Ceramics International, 42 (2016) 10322-10331. [32] S. Kaciulis, G. Mattogno, L. Pandolfi, M. Cavalli, G. Gnappi, A. Montenero, XPS study

PT

of apatite-based coatings prepared by sol-gel technique, Applied Surface Science, 151 (1999) 1-5.

RI

[33] N.-b. Li, G.-y. Xiao, B. Liu, Z. Wang, R.-f. Zhu, Y.-p. Lu, Rapid deposition of spherical apatite on alkali-heat treated titanium in modified simulated body fluid at high temperature,

SC

Surface & Coatings Technology, 301 (2016) 121-125.

[34] M.C. Kuo, S.K. Yen, The process of electrochemical deposited hydroxyapatite coatings

NU

on biomedical titanium at room temperature, Materials Science & Engineering C-Biomimetic

AC

CE

PT E

D

MA

and Supramolecular Systems, 20 (2002) 153-160.

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

SC

RI

PT

Graphical Abstract

ACCEPTED MANUSCRIPT Highlights 

Calcium titanate (CaTiO3) was formed on the surface of TiO2 nanotubes by calcification, which could provide nucleation sites for the formation of nano-HA.



Needle-like nano-HA crystals were subsequently formed on the surface of the calcified TiO2 nanotube arrays.

CE

PT E

D

MA

NU

SC

RI

PT

The nano-HA coating modified substrates showed good cytocompatibility.

AC

