Accepted Manuscript

Title: Fabrication of Nano-structured Calcium SilicateCoatings with Enhanced Stability, Bioactivity and Osteogenicand Angiogenic Activity

Author: Xiuhui Wang Yuning Zhou Lunguo Xia Cancan ZhaoLei Chen Deliang Yi Jiang Chang Liping Huang XuebinZheng Huiying Zhu Youtao Xie Yuanjin Xu Kaili Lin

PII: S0927-7765(14)00670-5DOI: http://dx.doi.org/doi:10.1016/j.colsurfb.2014.11.044Reference: COLSUB 6769

To appear in: Colloids and Surfaces B: Biointerfaces

Received date: 5-10-2014Revised date: 13-11-2014Accepted date: 26-11-2014

Please cite this article as: X. Wang, Y. Zhou, L. Xia, C. Zhao, L. Chen, D. Yi,J. Chang, L. Huang, X. Zheng, H. Zhu, Y. Xie, Y. Xu, K. Lin, Fabrication ofNano-structured Calcium Silicate Coatings with Enhanced Stability, Bioactivity andOsteogenic and Angiogenic Activity, Colloids and Surfaces B: Biointerfaces (2014),http://dx.doi.org/10.1016/j.colsurfb.2014.11.044

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 proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.

Page 1 of 40

Accep

ted

Man

uscr

ipt

1

Fabrication of Nano-structured Calcium Silicate Coatings with Enhanced

Stability, Bioactivity and Osteogenic and Angiogenic Activity

Xiuhui Wanga, Yuning Zhoub, Lunguo Xiab, Cancan Zhaoa, Lei Chena, Deliang Yia,

Jiang Changa, Liping Huangc, Xuebin Zhengc, Huiying Zhua, Youtao Xiec,*, Yuanjin

Xub,* , Kaili Lina,*

aState Key Laboratory of High Performance Ceramics and Superfine Microstructure,

Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050,

China

bDepartment of Oral and Maxillofacial Surgery, College of Stomatology, 9th People’s

Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200011,

China

cLaboratory of Inorganic Coating Materials, Shanghai Institute of Ceramics, Chinese

Academy of Sciences, Shanghai 200050, China

Corresponding Author: Tel.: 86-21-52412264. *E-mail: lklsic@mail.sic.ac.cn (K.

Lin); xieyoutao@mail.sic.ac.cn (Y. Xie); xuyuanjin@hotmail.com (Y. Xu).

Page 2 of 40

Accep

ted

Man

uscr

ipt

2

ABSTRACT:

The bioactivity and stability of coatings on alloy implants play critical roles in the

fast osseointegration and maintenance of a long-term life span of the implants,

respectively. Herein, nano-sheet surface on bioactive calcium silicate (CaSiO3, CS)

coatings on metal substrates was fabricated by combining atmosphere plasma

spraying (APS) and hydrothermal technology (HT). The glassy phase in CS coatings

generated by APS was converted into crystalline sheet-like nano-structures after HT

treatment. Compared with the original CS coating samples, HT treatment decreased

the degradation rate of the CS coatings. Moreover, the fabricated nano-structured

topography of CS coatings increased the apatite mineralization ability and

significantly enhanced the cell attachment, proliferation, differentiation, alkaline

phosphatase (ALP) activity and expression of osteogenic genes and angiogenic factors

of rat bone marrow stromal cells (bMSCs). Our results suggest that the

nano-structured CS coatings have immense potential in improving the clinical

performance of medical implants.

Keywords: Calcium silicate coating; Nano-structured surface; Stability; Osteogenic

activity; Angiogenic activity

Page 3 of 40

Accep

ted

Man

uscr

ipt

3

1. Introduction

Orthopedic implants, such as artificial joints, intravascular stents and dental

implants, play a critical role in improving the life quality of aged people and injured

individuals. Clinical applications reveal that the successful integration of orthopedic

implants with host bone tissue not only requires initial stability supported by enough

bone stock but also the tight fixation of the implants to the bone tissue, which is called

rapid osseointegration [1]. Titanium (Ti)-alloys have been widely used in the

preparation of orthopedic and dental implants because of their superior mechanical

properties, corrosion resistance and biocompatibility [2]. However, Ti-alloys are

usually unable to sufficiently integrate chemically with the bone at the early stage

after implantation due to their suboptimal osteoconductivity, which is deemed as an

important reason that leads to mechanical loosening and the subsequent premature

failure of the implants [3, 4]. To improve the physiochemical osseointegration and

increase the success rate of Ti-alloy implants, various surface modification methods

including plasma spraying, micro-arc oxidation and sol-gel method have been utilized

[5-7]. Plasma spraying a bioactive coating on the implants is considered a prospective

method for enhancing osseointegration between the implant and the surrounding bone

because the bioactive coating can produce lasting physiochemical osseointegration,

reduce implant loosening and ameliorate other adverse reactions such as the toxic

effects of metal ions released from the prosthesis [8].

Meanwhile, modifying the surface topographic design is another important

approach to improve the biological responses and osseointegration of the implants.

Page 4 of 40

Accep

ted

Man

uscr

ipt

4

The existing studies on metal implants show that peculiar nano-structured surfaces

can enhance the enrichment of functional proteins and growth factors from host

tissues, accelerate cell adhesion, proliferation, differentiation and, subsequently,

successful osseointegration of the implants [9-11]. Moreover, recent studies have

shown that the nano-structured surfaces on metal implants could stimulate the

secretion of angiogenic factors, which might facilitate angiogenesis in vivo, thereby

ensuring nutrient transport, metabolism and cell migration [12, 13]. Considering the

significant importance of nano-topographical features, the acceleration of

osseointegration may be obtained by fabricating coatings with nano-structured

surfaces on alloy implants.

Calcium silicate (CaSiO3, CS) has been developed as a bioactive coating on

Ti-alloy implants [14-17]. Previous studies have shown that CS coatings possess

excellent bioactivity and biocompatibility, which could quickly induce the formation

of a bone-like apatite layer on their surface after soaking in simulated body fluid

(SBF), both in cell culture and after implantation in vivo [17-19]. This type of apatite

layer plays an essential role in the formation of tight bone bonding between the

bioactive coatings and the adjacent tissues [20]. Furthermore, the silicon (Si) ions

released from the CS coatings or implants can significantly enhance the proliferation

and osteogenic differentiation of osteoblasts and bone marrow stromal cells (bMSCs)

and thereby promote bone formation in vivo [21, 22]. In addition, the thermal

expansion coefficient of CS is close to that of Ti-alloys, which brings the benefit of

tight bonding between the coating and the metal substrate compared with traditional

Page 5 of 40

Accep

ted

Man

uscr

ipt

5

hydroxyapatite (HA) coatings [17, 23]. However, plasma spraying consists of rapid

heating in an ultra-high temperature environment, rapid cooling and a solidification

process, which ultimately results in the formation of a smooth glassy phase. The

formation of the glassy phase apparently leads to a fast degradation rate of the

inorganic coating, which might result in the coatings falling off and subsequent

premature failure of the implant [24]. Moreover, the high degradation rate of the

glassy phase may lead to a high pH value in the surrounding environment and

disturbances in cell vitality, which may limit the clinical applications of CS-coated

bone implants [25, 26].

Therefore, the present study hypothesized that the construction of a nano-structured

surface on a CS coating could not only reduce the glassy phase of the CS coating and

decrease the coating’s degradability but also improve the osseointegration and

biological response of the CS coating. To prove our hypothesis, hydrothermal

technology (HT) was applied to convert the glassy phase generated from the

atmosphere plasma spraying (APS) process into a nano-structured calcium silicate

phase with high crystallinity. Moreover, the effect of the hydrothermal treatment on

the stability and bioactivity of the coating and the in vitro biological responses of rat

bMSCs was also investigated.

2. Materials and methods

2.1 Fabrication and characterization of nano-structured CS coatings on Ti-6Al-4V

substrates

Page 6 of 40

Accep

ted

Man

uscr

ipt

6

The CaSiO3 (CS) powders were synthesized by the chemical precipitation method

using Ca(NO3)2 and Na2SiO3 solutions as raw materials (for details, see the

supplementary information). Atmospheric plasma spraying (APS, F4-MB, Sulzer

Metco, Switzerland) was applied to deposit the CS coating onto the commercial

Ti-alloy Ti-6Al-4V substrate (Shanghai Yantai Metallic Material Co., Ltd, China) with

dimensions of 10×10×2 mm. The substrates were grit blasted, ultrasonically washed

with ethanol and dried at 60°C before plasma spraying. The following parameters: an

argon plasma gas flow rate of 40 slpm, a hydrogen plasma gas flow rate of 10 slpm, a

spray distance of 120 mm, a current of 600 Å and a voltage of 67 V were used in the

plasma processing [14].

The nano-structured CS coatings on Ti-6Al-4V substrates were fabricated via

hydrothermal treatment of the generated CS coatings in aqueous solutions with pH=7,

pH=9 and pH=11, respectively, at 180 °C for 1-24 h. The pH values of the aqueous

solutions were adjusted with 1 mol/L nitric acid (HNO3) and sodium hydroxide

(NaOH). After hydrothermal treatment, the samples were washed by soaking in

distilled water several times. The samples obtained by reaction in the aqueous

solutions with pH=7, pH=9 and pH=11 were denoted as S1, S2 and S3, respectively,

and the original CS coating without hydrothermal treatment was denoted as S0 and

used as a control sample for comparison.

The crystal phase composition of the fabricated CS coatings and the

nano-structured CS coatings was characterized by X-ray diffraction (XRD: D/Max

2550V, Rigaku, Japan) using Cu Kα radiation. The surface microstructure of the

Page 7 of 40

Accep

ted

Man

uscr

ipt

7

samples was observed by scanning electron microscopy (FESEM: JSM-6700F, JEOL,

Japan). The cross-sections of the fabricated samples before and after hydrothermal

treatment were cut using a microtome (SP1600, Leica, Germany), and then observed

by SEM.

The tensile bonding strength between the coatings and Ti-6Al-4V substrates was

measured by mechanical tester (Instron-5592, SATEC, USA) in accordance with

ASTM C-633. The contact angle of the fabricated samples was evaluated with

Automatic Contact Angle Meter Model (SL200B, Solon information technology Co.,

Ltd, China). The detailed methods are shown in the supplementary information.

2.2 Degradability and the apatite-mineralization ability of the coatings

To investigate the effect of the hydrothermal treatment on the bio-dissolution of the

coatings in vitro test, the fabricated samples were immersed in 50 mL of a Tris-HCl

buffer solution. The Tris-HCl buffer solution was refreshed every other day in this

process till the end. After soaking for 1, 3, 7, 14 and 21 days, the Ca ionic

concentrations released from the CS coatings were tested by inductively coupled

plasma atomic emission spectroscopy (ICP-AES, Varian 715ES, USA) [27]. In

addition, the apatite-mineralization ability of the coatings was evaluated by soaking

the fabricated samples in 15 mL simulated body fluid (SBF) at 37 °C for 1, 3 and 7

days with a constant shaking rate of 90 rpm. The XRD, Fourier transformed infrared

spectroscopy (FTIR, Nicolet Co., USA) and FESEM were used to identify the

apatite-mineralization ability of the coatings. The detailed methods are presented in

the supplementary information.

Page 8 of 40

Accep

ted

Man

uscr

ipt

8

2.3 Cell cultures on CS and nano-structured CS coatings

In this study, bMSCs were isolated from a 4-week-old male SD rat for use in the

cell experiments. Briefly, the effect of nano-structured surface on bMSC adhesion was

assessed by visualizing the actin cytoskeletons with confocal laser scanning

microscopy (CLSM, Leica, Wetzlar, Germany). For the cell proliferation assay, the

bMSCs were cultured on coating surfaces in 24-well tissue culture plates for up to 7

days, and the absorbance was measured at λ=590 nm using an ELX Ultra Microplate

Reader (BioTek, USA) [28]. Moreover, the Si ionic concentration in the cell culture

medium after culturing the bMSCs on samples for 1 day were tested by ICP-AES.

ALP staining was performed according to the manufacturer’s instructions (Beyotime,

China) after bMSCs were cultured for 10 days. Moreover, a quantitative ALP activity

assay was performed according to previous studies [28, 29]. In addition, the effect of

the nano-structured CS coating on the osteogenic and angiogenic differentiation of

bMSCs was assessed with qRT-PCR to measure the mRNA expression of type I

collagen (Col I), bone morphogenetic protein 2 (BMP-2), bone sialoprotein (BSP),

osteopontin (OPN), vascular endothelial growth factor (VEGF) and angiopoietin-1

(ANG-1). All experiments were performed in triplicate. The detailed methods are

shown in the supplementary information.

2.4 Statistical analysis

The mean and the standard deviation of the data were calculated. Differences

between groups were analyzed using a paired T-test. The statistical analysis was

Page 9 of 40

Accep

ted

Man

uscr

ipt

9

performed using SAS 9.0 software (SAS Institute, Cary, NC, USA). A p value of less

than 0.05 was considered statistically significant.

3. Results

3.1 Characterization of the nano-structured CS coatings on Ti-6Al-4V substrates

The surface morphologies of the fabricated samples S0-S3 are presented in Fig. 1.

The original coating (S0) has a rough surface and a few of micro-cracks built by

random stacking of fully and partially melted CS particles. Moreover, these

micro-cracks also presented within the cross-sections (Fig. S6c), and the

cross-sectional observation results showed that the thickness of coatings was around

120-150 μm (Fig. S6c, d). After hydrothermal treatment (HT) of the samples in

aqueous solutions with different pH values at 180 °C for 24 h, sheet-like topographic

surfaces with a thickness of approximately 65 nm and a length and width up to 7 μm

were obtained (S1-S3). It is interesting to find that the micro-cracks on coating

surfaces disappeared after HT due to the newly formed sheet-like nano-structures

covering on the top surface of the coatings (Fig. S6b), which might be beneficial to

inhibit the leaching of toxic ions from Ti-6Al-4V alloy substrates. However, the

micro-cracks still presented within the cross-section image (Fig. S6d). The XRD

patterns presented in Fig. 2 suggest that the original CS coating (sample S0) could be

identified as a wollastonite phase (JCPDS card: No. 75-1396). In addition, the sharp

peaks of characteristic of a wollastonite phase coexisted with an obvious glass bulge.

Though the glassy phase almost disappeared after hydrothermal treatment, new

Page 10 of 40

Accep

ted

Man

uscr

ipt

10

diffraction peaks of a tobermorite phase (JCPDS card: No. 89-6458) appeared

(samples S1-S3). In addition, the intensity of the diffraction peaks for wollastonite

phase became less after hydrothermal treatment, which was due to the formation of

the tobermorite layers on the top surface of the coatings. These results suggested the

facilely hydrothermal transformation of the glassy phase into a crystalline tobermorite

phase with nano-sheet structure, which was further confirmed by comparing the S0

and S2 samples with small angle XRD scanning (Fig. 2B). The S2 sample was

selected for further characterization and property studies because it had the lowest

diffraction intensity of the tobermorite phase.

To further investigate the transformation mechanism of the glass phase into the

sheet-like nano-structures on the coating surfaces, the topographies of the samples

after hydrothermal treatment at 180 °C for 1, 3, 6 and 24 h were observed (Fig. 3).

Compared with the morphologies of the original coating (S0) (Fig. 1), the erosion-like

topography appeared after hydrothermal treatment of the sample for 1 h (the early

stage). With an increase in the hydrothermal time to 3 h, small sheet-like crystals with

a thickness of approximately 5 nm and a length and width of approximately 1 μm

completely covered the coating surface. Prolonging the hydrothermal time to 6 h

caused the crystal size to increase. When the hydrothermal time was further increased

to 24 h (final product), the nano-sheet-like topography with a thickness of

approximately 65 nm and a length and width up to 7 μm was obtained.

The bonding strength results showed that the mean bond strength of the samples

before and after hydrothermal treatment was 43.83 ± 3.67 and 17.55 ± 8.69 MPa,

respectively (Fig. S5). The wettability characterization results (Fig. S4) showed that

the contact angle of the original coating surface was approximately 85 ± 4o. It is

Page 11 of 40

Accep

ted

Man

uscr

ipt

11

interesting to find that the contact angle of the sample S2 decreased to 0o, which

suggested that the coatings with nano-structured surfaces possessed excellent

super-hydrophilic property.

3.2 Degradability and apatite-mineralization ability of the coatings

Fig. 4 illustrates that the concentration of Ca ions released from the hydrothermally

treated samples (S2) was significantly lower than that of the control sample (S0) after

immersion in a Tris-HCl buffer solution for up to 21 days. The results suggested that

the hydrothermal treatment could significantly reduce the degradation rate of the CS

coatings.

After soaking in SBF for 7 days, the newly formed clusters deposited on the surface

of the coatings (Fig. S1 a, b). Higher magnification FESEM images showed that the

newly formed clusters consist of smaller lath-like microcrystals with a thickness of

approximately 6 nm and a length and width of approximately 160 nm. The EDS

results revealed that the Ca/P (molar ratio) of the deposited clusters reached 1.87-1.89,

which was close to that of stoichiometric HA (Ca/P=1.67) (Fig. S1 c, d). The XRD

results indicated that the newly deposited clusters were a HA phase (JCPDS card: No.

09-0432) (Fig. S2). The broad diffraction peaks (2θ = 26° and 32°) of HA were

observed in the XRD patterns after soaking the CS coating sample (S0) in SBF for 3

days, and the intensity of the HA diffraction peaks increased with an increase in

soaking time. As for the nano-structured CS coating sample (S2), the HA diffraction

could be detected earlier, after 1 day of soaking, and the strong diffraction peaks of

the wollastonite and tobermorite phases between 25° and 35° almost disappeared after

Page 12 of 40

Accep

ted

Man

uscr

ipt

12

7 days of soaking. However, the diffraction peaks for the wollastonite phase were still

present in sample S0 after 7 days of soaking. FTIR was used to further elucidate the

newly formed deposits (Fig. S3). As observed in the spectra generated before soaking,

the adsorption bands of the silicate group were evident. After soaking in SBF for 7

days, bands at 602 and 563 cm-1 were present because of the bending vibration modes

of the PO4 group. A weak water absorption band at approximately 1650 cm-1 and a

broad OH- absorption band from 2500 to 3700 cm-1 also appeared in the spectra. The

band between 1400 cm-1 and 1550 cm-1 was carbonate (CO32-) IR absorption v3. The

peak at approximately 870 cm-1 was the joint contribution of HPO42- and CO3

2-�ions.

The FTIR spectra further confirmed that a bone-like hydroxycarbonate apatite (HCA)

layer deposited on the coatings after soaking in SBF. In addition, the broad and low

weak diffraction peaks of XRD patterns suggested that the newly deposited clusters

were amorphous apatite.

3.3 The effect of nano-structured coatings on the adhesion, growth and osteogenic and

angiogenic differentiation of bMSCs

3.3.1 The effect of nano-structured coatings on the adhesion and growth of seeded

bMSCs

The CLSM results showed that the cells attached on the control sample (S0) only

spread slightly, whereas the cells attached on sample S2 with a nano-structured

surface exhibited almost full adhesion with apparent cytoplasmic extensions and

typical filopodial attachments (Fig. 5). Moreover, the adherent cells on sample S2

Page 13 of 40

Accep

ted

Man

uscr

ipt

13

presented a long-strip shape and actin filaments with regular direction were well

defined by the actin microfilament system, ranging parallel to the long axis of the

cells.

3.3.2 The effect of nano-structured coatings on proliferation of bMSCs

According to the results of the MTT assay (Fig. 6), continuous proliferation of

bMSCs was detected on coating surfaces throughout the entire culture period.

Furthermore, the proliferation of bMSCs was significantly higher on the sample S2

with a nano-structured surface than on the control sample S0. Furthermore, the Si

ionic concentration in cell culture medium after culture of the bMSCs on samples S0

and S2 for 1 day reached 23.64 ± 0.85 ppm and 24.85 ± 1.04 ppm, respectively. In

addition, without significant difference between them (p = 0.19 ) was observed.

3.3.3 The effect of nano-structured coatings on the ALP activity of bMSCs

As shown in Fig. 7, a more intense ALP staining was observed in bMSCs cultured

on the nano-structured coating sample (S2) compared with the original CS coating

sample (S0). Furthermore, quantitative analysis showed that the ALP activity of

bMSCs cultured on the coating surfaces continuously increased throughout the assay

period, and the ALP activity of the bMSCs seeded on sample S2 was significantly

higher than that on sample S0 throughout the culture period.

3.3.4 The effect of nano-structured coatings on osteogenic and angiogenic

Page 14 of 40

Accep

ted

Man

uscr

ipt

14

differentiation of bMSCs

qRT-PCR for markers of osteogenic and angiogenic differentiation was performed

on bMSCs cultured on samples S0 and S2 at 4, 7 and 10 days (Fig. 8). The results

showed that the nano-structured sample S2 significantly enhanced the osteogenic

differentiation of bMSCs to various degrees compared with the control sample S0.

Specifically, at day 4, the cells cultured on sample S2 showed increased expression of

Col I and OPN compared with sample S0. With the increase of culture time to 7 and

10 days, the expression of BSP, BMP-2, Col 1 and OPN was higher in bMSCs

cultured on sample S2 than in cells cultured on the control sample S0, except for BSP

at day 7 and OPN at day 10. More importantly, a higher expression of the angiogenic

factor VEGF throughout the entire culture period and ANG-1 at day 7 was observed

on sample S2 compared with sample S0.

4. Discussion

Plasma-sprayed bioactive inorganic coatings on Ti-6Al-4V substrate possess good

bioactivity and mechanical properties, which allow them to be widely used for

orthopedic implant applications [30]. However, plasma spraying consists of rapid

heating in an ultra-high temperature environment, rapid cooling and a solidification

process, which results in the formation of a smooth glassy phase and a high

degradation rate of the coatings [24]. The fast degradation rate might lead to the

exfoliation of the CS coatings and the subsequent premature failure of the implants. In

addition, recent studies have suggested that the nano-topographical surface on metal

Page 15 of 40

Accep

ted

Man

uscr

ipt

15

implants can improve the osseointegration and even stimulate the secretion of

angiogenic factors [10, 12, 13]. For example, titanium dioxide (TiO2) nanotubes

fabricated via anodization on titanium cylinder implants strongly enhanced

osseointegration at the molecular level [10], and micro/nano-structured topography on

Ti surfaces fabricated by an acid-etching method could modulate the secretion of

angiogenic growth factors by osteoblasts [13]. Hence, it will be an important task to

maintain and even improve the biological activity of the coatings while also reducing

the degradation rate of the coatings generated from the plasma spraying process. In

the present study, the nano-structured CS coatings on Ti-6Al-4V substrates were

successfully fabricated by combining atmosphere plasma spraying (APS) and

hydrothermal technology (HT). The glassy phase generated from the APS process

could be converted into a nano-structured calcium silicate phase with high

crystallinity via HT.

Under hydrothermal treatment, the erosion occurred on the coating surface in an

aqueous solution, followed by the release of Ca2+ and SiO32- ions into the solution. In

addition, some of the SiO32- ions hydrolyzed into HSiO3

- ions. With the continual ion

release and hydrolysis, the concentration of Ca2+, SiO32- and HSiO3

- in the solution

reached oversaturation and the tobermorite nucleated on the eroded CS coating

surface via the following chemical reaction equation: 5Ca2+ + 4SiO32- + 2HSiO3

- +

4H2O → Ca5Si6O16(OH)2·4H2O (Fig. 2). During this process, the CS coating itself

offered Ca2+ and SiO32- ion sources. In addition, the pH value of the aqueous solution

influenced the existing form of silicon oxygen tetrahedron ([SiO4]4-), which further

Page 16 of 40

Accep

ted

Man

uscr

ipt

16

influenced the morphology of the formed tobermorite crystals. Previous studies have

shown that the silicon oxygen tetrahedron ([SiO4]4-) arranged in a layer shape in an

aqueous solution with a pH < 13.0 and finally resulted in sheet-like tobermorite

nano-crystals (Fig. 3) [31]. At the same time, the glassy phase observed in the original

coatings was successfully converted into a crystalline tobermorite phase after

hydrothermal treatment, which reduced the degradation rate of the CS coatings (Fig.

4). Clinical applications suggest that successful coatings on metal implants should be

relatively stable to avoid implant loosening and maintain a long-term life span [30,

32]. The increase in crystallinity of the hydroxyapatite (HA) coatings can apparently

increase the stability of HA-coated implants [33, 34]. Therefore, the fabricated

nano-structured CS coating via hydrothermal treatment might possess better stability

in clinical applications compared with the original CS coating. However, the bonding

strength characterization results suggested that the hydrothermal treatment decreased

the bond strength of the coatings due to thermal stress generated during the

hydrothermal process, which has been widely observed in the second heat treatment

of the plasma spraying coatings [35]. However, bond strength of sample S2 with

nano-structured surfaces after hydrothermal treatment was still higher than that of the

traditional hydroxyapatite coatings in biomedical fields [36].

It is well known that the apatite-mineralization ability of the implants plays an

important role in the proliferation and differentiation of osteoblasts and bMSCs [37].

Moreover, the newly formed bone-like apatite can stimulate the formation of tight

bone bonding between the material and the adjacent tissues [18, 20]. After soaking

Page 17 of 40

Accep

ted

Man

uscr

ipt

17

sample S2 in SBF for 7 days, the sample was completely covered by HCA, whereas

the phase of the substrate completely disappeared according to the XRD

characterization. However, the original CS phase was maintained in sample S0,

suggesting that the fabricated nano-structured CS coating (S2) possessed a better

apatite mineralization ability (Fig. S3). It is well known that bone-like apatite

formation ability depends on the chemical composition and surface topography of the

material [38]. The better HCA formation ability of sample S2 might be due to the

specific sheet-like nano-structure. The nano-structured surface exhibits higher

porosity and specific surface area, which promote a higher hydroxylation degree and

allows the formation of SiOH on the coating surface, and provides more sites for the

nucleation of HCA [38, 39].

Based on the cell adhesion results (Fig. 5), it was obvious that the actin filaments of

cells on sample S2 with nano-structured surface were well defined and had a regular

direction, and the cells exhibited full attachment with apparent cytoplasmic extensions

and presented a long-strip shape. Comparing with sample S0, sample S2 with

nano-structured surface possessed excellent super-hydrophilic property, which is

beneficial to cell adhesion [28, 40]. Moreover, the samples with nano-structured

surfaces possess higher specific surface area, which can enhance the enrichment of

functional proteins, activate surface integrin, help the cells to anchor themselves to

biomaterial surfaces, and then further accelerate the cell adhesion, migration,

proliferation and differentiation, etc. [11, 41].

It is well known that the cell adhesion and spreading not only initiate interactions

Page 18 of 40

Accep

ted

Man

uscr

ipt

18

between the cells and the material but also further regulate cellular functions such as

migration, proliferation and differentiation [42, 43]. According to the results of the

MTT and ALP activity assays in the present study (Fig. 6, 7), it is clear that the

proliferation and differentiation of bMSCs was significantly higher on sample S2 with

nano-structured surface than on the control sample S0 throughout the assay period,

which is consistent with the above deduction.

BSP and BMP-2 are important markers of osteogenic differentiation of bone

forming cells. The binding of BSP to collagen is thought to be important for initiating

bone mineralization and bone cell adhesion to the mineralized matrix, whereas

BMP-2 plays an important role in stimulating differentiation of mesenchymal stem

cells and is relevant to osteogenic commitment and differentiation [44, 45].

Furthermore, as another bone-specific marker in osteogenic differentiation, Col I is

considered to be the most crucial fibrillar type of ECM collagen and serves as

initiation sites for bone apatite deposition [45]. OPN is relevant to the maturation

stage of osteoblasts during matrix synthesis and attachment before mineralization,

which is considered to be a relatively early or intermediate marker of osteogenic

differentiation [46]. Hence, qRT-PCR analysis of osteogenic markers BSP, BMP-2,

Col I and OPN was performed as an additional evaluation of the osteogenic

differentiation promoted by the nano-structured surface topography (Fig. 8). These

results showed that the expression levels of osteoblastic markers, such as BSP, BMP-2,

Col I and OPN, were significantly higher in cells on sample S2 with a nano-structured

surface than in the original CS coating sample S0 at particular culture time points.

Page 19 of 40

Accep

ted

Man

uscr

ipt

19

Specifically, Col I expression was significantly up-regulated and maintained on cells

on sample S2 compared with cells on sample S0 at all culture time points. BSP and

BMP-2 expression in cells on sample S2 was markedly higher compared with that in

cells on control samples (S0) at day 7. With the increase in the culture time to day 10,

the increased expression level for BMP-2 in cells on sample S2 was maintained. The

OPN expression in cells cultured on sample S2 was significantly higher than that on

sample S0 at days 4 and 7. The increased expression levels of these osteogenic factors

suggested that the nano-structured CS coating surface could stimulate osteogenic

differentiation of bMSCs.

In the present study, it was very interesting to find that the nano-structured surface

topography of the fabricated CS coating sample S2 could promote the expression of

angiogenic factors such as VEGF and ANG-1 (Fig. 8). After 4 and 7 days of culture, a

significant increase in VEGF expression in cells on sample S2 was observed, and an

increased expression of ANG-1 was also detected in cells on sample S2 at day 7. It is

well knowm that angiogenesis plays an extraordinarily important role in

osseointegration during bone regeneration [47]. The induction of angiogenesis is

beneficial for cell survival, integration and functionality of newly formed bone tissue

[48]. As a key angiogenic factor, VEGF has been widely considered to be the most

significant biological factor with the strongest activity in improving blood formation.

Recently, it was revealed that Ti surfaces with micro/nano-topography could promote

the secretion of VEGF from osteoblasts [13]. Likewise, the present study showed that

the fabricated CS coatings with a nano-structured topographical surface could also

Page 20 of 40

Accep

ted

Man

uscr

ipt

20

enhance ANG-1 expression, which might play an essential role in the later stages of

blood vessel formation, such as the stabilization of endothelial sprouting and the

interaction with pericytes [40]. Furthermore, it could also reduce VEGF-mediated

vascular permeability [49]. Therefore, it is an effective approach to design

nano-structured topography on coating surfaces for promoting vascularization.

In present study, without significant difference of Si ionic concentration in cell

culture medium was observed. Therefore, it could be concluded that nano-structures

on the fabricated sample S2 played the critical role on better cell biological responses.

Taken together with the results of cell adhesion, proliferation, and osteogenic and

angiogenic differentiation of bMSCs in this study, an implant with a coating surface

with a nano-structured topography might enhance the osteointegration by promoting

bone formation in cells in contact with the surface directly and in the adjacent tissue,

which might also provide insight into the future development of implants with

bioactive coatings. However, further in vivo implantation investigations are required

to confirm the promotion of osseointegration by calcium silicate (CS) coatings with

nano-structured topographical surfaces.

5. Conclusion

In this study, calcium silicate (CS) coatings with a sheet-like nano-structured

surface on Ti-6Al-4V substrates were successfully fabricated by a combination of

atmosphere plasma spraying (APS) and hydrothermal technology (HT). The HT could

facilely convert the glassy phase generated from the APS into a nano-structured

Page 21 of 40

Accep

ted

Man

uscr

ipt

21

calcium silicate phase with high crystallinity, which significantly reduced the

degradation rate of the coatings. Moreover, the nano-structured CS coatings possessed

a better apatite-mineralization ability compared with the original CS coatings.

Additionally, the fabricated nano-structured surface on the CS coatings promoted cell

attachment, cell proliferation, the osteogenic differentiation of bMSCs and the

increased expression of angiogenic factors. The present study suggests that the

nano-structured CS coatings fabricated by combining APS and HT methods show

potential for improving the bioactivity and osteointegration of implants and might

provide implants with the long-term life span that is necessary for clinical

applications.

Conflict of interest

The authors declare no competing financial interest.

Acknowledgments

The authors gratefully acknowledge the support of Natural Science Foundation of

China (Grant 81171458, 81190132), Science and Technology Commission of

Shanghai Municipality (Grant 13NM1402102, 14140904100), and the Funds of Key

Laboratory of Inorganic Coating Materials, Chinese Academy of Sciences.

Appendix A. Supplementary data

Supplementary information associated with this article can be found in the Appendix

Page 22 of 40

Accep

ted

Man

uscr

ipt

22

A. Supplementary data.

References

[1] P.I. Brinemark, R.A.T. Albrektsson, U. Lekholm, S. Lundkvist, B. Rockier,

Osseointegrated titanium fixtures in the treatment of edentulousness,

Biomaterials 4 (1983) 25-28.

[2] M. Geetha, A.K. Singh, R. Asokamani, A.K. Gogia, Ti based biomaterials, the

ultimate choice for orthopaedic implants – A review, Prog. Mater. Sci. 54 (2009)

397-425.

[3] D.L. DuQuesnary, A.K. Lynn, Hydroxyapatite-coated Ti–6Al–4V Part 2: the

effects of post-deposition heat treatment at low temperatures, Biomaterials 23

(2002) 1947–1953.

[4] M. Wen, C. Wen, P. Hodgson, Y. Li, Improvement of the biomedical properties of

titanium using SMAT and thermal oxidation, Colloid Surface B 116 (2014)

658-665.

[5] Y.T. Sul, B.S. Kang, C. Johansson, H.S. Um, C.J. Park, T. Albrektsson, The roles

of surface chemistry and topography in the strength and rate of osseointegration

of titanium implants in bone, J. Biomed. Mater. Res. A 89 (2009) 942-950.

[6] G. Mendonça, D. Mendonca, F.J. Aragao, L.F. Cooper, Advancing dental implant

surface technology–from micron-to nanotopography, Biomaterials 29 (2008)

3822-3835.

[7] X.B. Zheng, Y.T. Xie, Progress on biomedical ceramic coatings prepared by

thermal spraying, J. Inorg. Mater. 28 (2013) 12-20.

[8] S. Vercaigne, J.G.C. Wolke, I. Naer, J.A. Jansen, The effect of titanium

plasma-sprayed implants on trabecular bone healing in the goat, Biomaterials 19

Page 23 of 40

Accep

ted

Man

uscr

ipt

23

(1998) 1093-1099.

[9] N. Tran, T.J. Webster, Nanotechnology for bone materials, Wires. Nanomed.

Nanobi. 1 (2009) 336-351.

[10] N. Wang, H. Li, W. Lu, J. Li, J. Wang, Z. Zhang, Y. Liu, Effects of TiO2

nanotubes with different diameters on gene expression and osseointegration of

implants in minipigs, Biomaterials 32 (2011) 6900-6911.

[11] J. Park, S. Bauer, K.V.D. Mark, P. Schmuki, Nanosize and vitality: TiO2

nanotube diameter directs cell fate, Nano Lett. 7 (2007) 1686-1691.

[12] M. Adam, C. Ganz, W. Xu, H.R. Sarajian, W. Gotz, T. Gerber, In vivo and in

vitro investigations of a nanostructured coating material - a preclinical study, Int.

J. Nanomed. 9 (2014) 975-984.

[13] A.L. Raines, R. Olivares-Navarrete, M. Wieland, D.L. Cochran, Z. Schwartz,

B.D. Boyan, Regulation of angiogenesis during osseointegration by titanium

surface microstructure and energy, Biomaterials 31 (2010) 4909-4917.

[14] X.Y. Liu, C.X. Ding, Phase compositions and microstructure of plasma sprayed

wollastonite coating, Surf. Coat. Tech. 141 (2001) 269-274.

[15] B.Q. Lu, Y.J. Zhu, G.F. Cheng, Y.J. Ruan, Synthesis and application in drug

delivery of hollow-core-double-shell magnetic iron oxide/silica/calcium silicate

nanocomposites, Mater. Lett. 104 (2013) 53-56.

[16] H. Hu, Y. Qiao, F. Meng, X. Liu, C. Ding, Enhanced apatite-forming ability and

cytocompatibility of porous and nanostructured TiO2/CaSiO3 coating on

titanium, Colloid Surface B 101 (2013) 83-90.

[17] X.Y. Liu, Z.Y. Wang, Apatite formed on the surface of plasma-sprayed

wollastonite coating immersed in simulated body fluid, Biomaterials 22 (2001)

2007-2012.

Page 24 of 40

Accep

ted

Man

uscr

ipt

24

[18] K.L. Lin, M.L. Zhang, W. Zhai, H. Qu, J. Chang, Fabrication and

characterization of hydroxyapatite/wollastonite composite bioceramics with

controllable properties for hard tissue repair, J. Am. Ceram. Soc. 94 (2011)

99-105.

[19] S. Xu, K. Lin, Z. Wang, J. Chang, L. Wang, J. Lu, C. Ning, Reconstruction of

calvarial defect of rabbits using porous calcium silicate bioactive ceramics,

Biomaterials 29 (2008) 2588-2596.

[20] T. Kokubo, H. Takadama, How useful is SBF in predicting in vivo bone

bioactivity?, Biomaterials 27 (2006) 2907-2915.

[21] I.D. Xynos, A.J. Edgar, L.D.K. Buttery, L.L. Hench, J.M. Polak,

Gene-expression profiling of human osteoblasts following treatment with the

ionic products of Bioglass 45S5 dissolution, J. Biomed. Mater. Res. A 55 (2001)

151-157.

[22] I.D. Xynos, A.J. Edgar, L.D.K. Buttery, L.L. Hench, J.M. Polak, Ionic products

of bioactive glass dissolution increase proliferation of human osteoblasts and

induce insulin-like growth factor II mRNA expression and protein synthesis,

Biochem. Bioph. Res. Co. 276 (2000) 461-465.

[23] K.A. Khor, Y.W. Gu, D. Pan, P. Cheang, Microstructure and mechanical

properties of plasma sprayed HA/YSZ/Ti–6Al–4V composite coatings,

Biomaterials 25 (2004) 4009-4017.

[24] X.Y. Liu, C.X. Ding, Phase compositions and microstructure of plasma sprayed

wollastonite coating, Surf. Coat. Tech. 141 (2001) 269-274.

[25] Y. Ramaswamy, C. Wu, H. Zhou, H. Zreiqat, Biological response of human bone

cells to zinc-modified Ca-Si-based ceramics, Acta Biomater. 4 (2008)

1487-1497.

Page 25 of 40

Accep

ted

Man

uscr

ipt

25

[26] H. Zreiqat, Y. Ramaswamy, C. Wu, A. Paschalidis, Z. Lu, B. James, O. Birke,

M. McDonald, D. Little, C.R. Dunstan, The incorporation of strontium and zinc

into a calcium-silicon ceramic for bone tissue engineering, Biomaterials 31

(2010) 3175-3184.

[27] K. Li, J. Yu, Y. Xie, L. Huang, X. Ye, X. Zheng, Effects of Zn content on crystal

structure, cytocompatibility, antibacterial activity, and chemical stability in

Zn-modified calcium silicate coatings, J. Therm. Spray. Techn. 22 (2013)

965-973.

[28] K.L. Lin, L.G. Xia, J. Gan, Z. Zhang, H. Chen, X. Jiang, J. Chang, Tailoring the

nanostructured surfaces of hydroxyapatite bioceramics to promote protein

adsorption, osteoblast growth, and osteogenic differentiation, ACS Appl. Mater.

Inter. 5 (2013) 8008-8017.

[29] U. Stucki, J. Schmid, C.F. Hammerle, N.P. Lang, Temporal and local appearance

of alkaline phosphatase activity in early stages of guided bone regeneration. A

descriptive histochemical study in humans, Clin. Oral implan. Res. 12 (2001)

121-127.

[30] W. Zhang, G. Wang, Y. Liu, X. Zhao, D. Zou, C. Zhu, Y. Jin, Q. Huang, J. Sun,

X. Liu, X. Jiang, H. Zreiqat, The synergistic effect of hierarchical

micro/nano-topography and bioactive ions for enhanced osseointegration,

Biomaterials. 34 (2013) 3184-3195.

[31] X. Huang, D.L. Jiang, S.H. Tan, The chelating agent method, hydrothermal

synthesis of bioactive calcium silicate whisker, J. Inorg. Mater. 18 (2003)

143-148.

[32] X.Y. Liu, Progress in research on the surface/interface of materials for hard

tissue implant, J. Inorg. Mater. 26 (2011) 1-11.

Page 26 of 40

Accep

ted

Man

uscr

ipt

26

[33] M.A. Faghihi-Sani, A. Arbabi, A. Mehdinezhad-Roshan, Crystallization of

hydroxyapatite during hydrothermal treatment on amorphous calcium phosphate

layer coated by PEO technique, Ceram. Int. 39 (2013) 1793-1798.

[34] R.A. Surmenev, M.A. Surmeneva, A.A. Ivanova, Significance of calcium

phosphate coatings for the enhancement of new bone osteogenesis--a review,

Acta Biomater. 10 (2014) 557-579.

[35] Y.C. Tsui, C. Doyle, T.W. Clyne, Plasma sprayed hydroxyapatite coatings on

titanium substrates Part 2: optimisation of coating properties, Biomaterials 19

(1998) 2031-2043.

[36] M.H. Huang, X.B. Zheng, C.X. Ding, Bond strength of plasma-sprayed

hydroxyapatite_Ti composite coatings, Biomaterials 21 (2000) 841-849.

[37] Y.F. Chou, W. Huang, J.C. Dunn, T.A. Miller, B.M. Wu, The effect of

biomimetic apatite structure on osteoblast viability, proliferation, and gene

expression, Biomaterials 26 (2005) 285-295.

[38] P. Sepulveda, J.R. Jones, L.L. Hench, Characterization of melt-derived 45S5 and

sol-gel-derived 58S bioactive glasses, J. Biomed. Mater. Res. (2001) 734-740.

[39] Z. Wu, T. Tang, H. Guo, S. Tang, Y. Niu, J. Zhang, W. Zhang, R. Ma, J. Su, C.

Liu, J. Wei, In vitro degradability, bioactivity and cell responses to mesoporous

magnesium silicate for the induction of bone regeneration, Colloid Surface B 120

(2014) 38-46.

[40] L. Xia, K. Lin, X. Jiang, B. Fang, Y. Xu, J. Liu, D. Zeng, M. Zhang, X. Zhang, J.

Chang, Z. Zhang, Effect of nano-structured bioceramic surface on osteogenic

differentiation of adipose derived stem cells, Biomaterials 35 (2014) 8514-8527.

[41] M. Razavi, M. Fathi, O. Savabi, B. Hashemi Beni, D. Vashaee, L. Tayebi,

Surface microstructure and in vitro analysis of nanostructured akermanite

Page 27 of 40

Accep

ted

Man

uscr

ipt

27

(Ca2MgSi2O7) coating on biodegradable magnesium alloy for biomedical

applications, Colloid Surface B 117 (2014) 432-440.

[42] D.H. Kim, C.H. Seo, K. Han, K.W. Kwon, A. Levchenko, K.Y. Suh, Guided cell

migration on microtextured substrates with variable local density and anisotropy,

Adv. Funct. Mater. 19 (2009) 1579-1586.

[43] H.Y. Chang, J.T. Chi, S. Dudoit, C. Bondre, M.V.D. Rijn, D. Botstein, P.O.

Brown, Diversity, topographic differentiation, and positional memory in human

fibroblasts, P. Natl. Acad. Sci. USA 99 (2002) 12877-12882.

[44] P.J. Marie, F. Debiais, E. Hay, Regulation of human cranial osteoblast phenotype

by FGF-2, FGFR-2 and BMP-2 signaling, Histol. Histopathol. 17 (2002)

877-885.

[45] L. Xia, K. Lin, X. Jiang, Y. Xu, M. Zhang, J. Chang, Z. Zhang, Enhanced

osteogenesis through nano-structured surface design of macroporous

hydroxyapatite bioceramic scaffolds via activation of ERK and p38 MAPK

signaling pathways, J. Mater. Chem. B 1 (2013) 5403.

[46] A. Scherberich, A.M. Muller, D.J. Schafer, A. Banfi, I. Martin, Adipose

tissue-derived progenitors for engineering osteogenic and vasculogenic grafts, J.

Cell. Physiol. 225 (2010) 348-353.

[47] S.K. Levengood, M.J. Poellmann, S.G. Clark, D.A. Ingram, M.C. Yoder, A.J.

Johnson, Human endothelial colony forming cells undergo vasculogenesis within

biphasic calcium phosphate bone tissue engineering constructs, Acta Biomater. 7

(2011) 4222-4228.

[48] C. Wang, K. Lin, J. Chang, J. Sun, Osteogenesis and angiogenesis induced by

porous beta-CaSiO(3)/PDLGA composite scaffold via activation of

AMPK/ERK1/2 and PI3K/Akt pathways, Biomaterials 34 (2013) 64-77.

Page 28 of 40

Accep

ted

Man

uscr

ipt

28

[49] S. Fukuhara, K. Sako, K. Noda, J. Zhang, M. Minami, N. Mochizuki,

Angiopoietin-1/Tie2 receptor signaling in vascular quiescence and angiogenesis,

Histol. Histopathol. 25 (2010) 387-396.

Page 29 of 40

Accep

ted

Man

uscr

ipt

29

Figure captions:

Fig. 1. FESEM images of the original CS coating sample S0 and the samples after

hydrothermal treatment in aqueous solutions with pH=7 (S1), pH=9 (S2) and pH=11

(S3), respectively.

Fig. 2. XRD patterns (A) of the original sample S0 and the samples after

hydrothermal treatment in aqueous solutions with pH=7 (S1), pH=9 (S2) and pH=11

(S3), respectively, and the small angle XRD scanning (B) for S0 and S2.

Fig. 3. FESEM images of the samples after hydrothermal treatment in aqueous

solution with pH=9 at 180 oC for 1, 3, 6 and 24 h.

Fig. 4. The Ca ion concentration of sample S0 and S2 after soaking in Tris-HCl buffer

solution up to 21 days.

Fig. 5. CLSM images for the attachment of bMSCs on original CS coating sample (S0)

and the nano-structured sample (S2) after 6 h of seeding. Actin filament (cytoskeleton)

stained red (a, d), while the cell nuclei stained blue (b, e), and (c, f) represented the

merged images of the two fluorochromes for the samples.

Fig. 6. MTT assay of bMSCs seeded on the original CS coating sample (S0) and the

nano-structured sample (S2) at days 1, 4 and 7 for cell viability and proliferation. *p <

0.05.

Fig. 7. (A) ALP staining for bMSCs cultured on original CS coating sample (S0) and

the nano-structured sample (S2) at days 10; (B) The quantitative results of ALP

activity after culture bMSCs on samples S0 and S2 for 4, 7 and 10 days. *p < 0.05.

Fig. 8. Osteogenic and angiogenic factor expression of bMSCs cultured on original

Page 30 of 40

Accep

ted

Man

uscr

ipt

30

CS coating sample (S0) and the nano-structured sample (S2) for 4, 7 and 10 days. *p

< 0.05.

Page 31 of 40

Accep

ted

Man

uscr

ipt

31

Highlights:

1. Nanostructured CaSiO3 coating was fabricated by combining the APS and HT.

2. HT treatment apparently decreased the degradation rate of CaSiO3 coating.

3. Nanostructured topography increased the apatite mineralization ability.

4. Nanostructured topography enhanced osteogenic and angiogenic activity.

Page 32 of 40

Accep

ted

Man

uscr

ipt

*Graphical Abstract (for review)

Page 33 of 40

Accep

ted

Man

uscr

ipt

Figure(1)

Page 34 of 40

Accep

ted

Man

uscr

ipt

Figure(2)

Page 35 of 40

Accep

ted

Man

uscr

ipt

Figure(3)

Page 36 of 40

Accep

ted

Man

uscr

ipt

Figure(4)

Page 37 of 40

Accep

ted

Man

uscr

ipt

Figure(5)

Page 38 of 40

Accep

ted

Man

uscr

ipt

Figure(6)

Page 39 of 40

Accep

ted

Man

uscr

ipt

Figure(7)

Page 40 of 40

Accep

ted

Man

uscr

ipt

Figure(8)