Chemical Engineering Journal 347 (2018) 711–720

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Chemical Engineering Journal

journal homepage: www.elsevier.com/locate/cej

In situ modulation of crystallinity and nano-structures to enhance the T

stability and osseointegration of hydroxyapatite coatings on Ti-6Al-4V
implants
⁎ ⁎
Lunguo Xiaa,1, Youtao Xieb,1, Bing Fanga, Xiuhui Wangb, , Kaili Linc,
a
Department of Orthodontics, Collage of Stomatology, Ninth People’s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200011, China
b
Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, China
c
Department of Oral & Cranio-Maxillofacial Surgery, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of
Stomatology, Shanghai Research Institute of Stomatology, Shanghai 200011, China

H I GH L IG H T S G R A P H I C A L A B S T R A C T

• Incoatings
situ modulation of hydroxyapatite
with high crystallinity and
nano-structure on Ti-6Al-4V implants
is developed.
• The high crystallinity realizes long-
term service life span of the hydro-
xyapatite coatings.
• The constructed nano-structured sur-
face enhances cell responses and os-
seointegration.

A R T I C LE I N FO A B S T R A C T

Keywords: Maintaining the high crystallinity and tailoring the nano-structured surface of hydroxyapatite (HA) coatings on
Bioactive hydroxyapatite coatings Ti-6Al-4V implants play critical roles in realizing their long-term service life span and fast osseointegration for
In situ modulation biomedical implants. Herein, a nanorod structured HA (nHA) coating with a pure phase and high crystallinity
Nano-structure was successfully developed by combining atmospheric plasma spraying (APS) with hydrothermal treatment
Stability
(HT). The crystallinity degree of the plasma-sprayed HA coating was increased from 51.3% to over 85.3% by
Osteogenic differentiation
hydrothermal treatment, which meets the medical device industrial standard of having a crystallinity no less
Osseointegration
than 62% and significantly decreased the degradation rate of the HA coating. In addition, the construction of a
nanorod structured surface promoted the attachment, proliferation and differentiation of rat bone mesenchymal
stem cell (rBMSCs), thus further improving the osseointegration between implants and the surrounding bone
tissue. Hence, the stable and bioactive nHA-coated Ti-6Al-4V implant has considerable potential in dental and
orthopedic applications.

⁎
Corresponding authors.
E-mail addresses: WXH200801@163.com (X. Wang), lklecnu@aliyun.com (K. Lin).
1
These authors contributed equally.

https://doi.org/10.1016/j.cej.2018.04.045
Received 2 February 2018; Received in revised form 3 April 2018; Accepted 7 April 2018
Available online 25 April 2018
1385-8947/ © 2018 Elsevier B.V. All rights reserved.
L. Xia et al.
1. Introduction
Titanium and its alloys (such as Ti-6Al-4V), as the preferred metallic
materials for orthopedic implants, have been successfully applied in all
kinds of clinical applications, such as surgical implements and artificial
joints due to their high mechanical strength, low elastic modulus close
to that of bone and biocompatibility [1,2]. However, the release of toxic
ions (such as Al and V) in vivo might cause undesirable long-term ef-
fects, such as Alzheimer’s disease and cytotoxic reactions [3]. Fur-
thermore, it is well known that the titanium surface is usually un-
suitable for sufficient osseointegration because of its bio-inert quality
and poor osteoconductivity [4]. Long-term clinical applications have
revealed that there is usually no direct contact between bone and a
titanium implant; instead, a layer of fiber tissue formed between the
implant and bone, leading to a loosening of the implant–native bone
interface and to eventual implant failure [2]. Hence, the surface mod-
ification of titanium and its alloys is necessary for the implants to in-
tegrate tightly with the surrounding host bone [5]. The surface che-
mical stability and topography play vital roles in the development of
optimal osseointegration properties [6,7].
When considering beneficial surface modifications for titanium and
its alloys, a hydroxyapatite [Ca10(PO4)6(OH)2, HA] coating on the Ti-
6Al-4V implant has attracted considerable attention because of its ex-
cellent biocompatibility, bioactivity and osteoconductivity properties
compared with an uncoated implant [8]. As is well known, HA is one of
the main inorganic components in native bone and supports bone in-
growth and osseointegration [9,10]. Furthermore, it has been demon-
strated that HA coatings on Ti-alloy substrates significantly enhance
osteoblast functions and bone formation and shorten the curative time
of metal based implants [11–14].
To date, various kinds of surface modification techniques, including
plasma spraying, ion beam deposition, ion implantation and micro-arc
oxidation, have been developed to fabricate HA coatings on the metal
and its alloy substrates [5,15,16]. Among these methods, plasma
spraying is considered a prospective method for modifying Ti-alloy
implant surfaces due to its high efficiency and easily controlled coating
thickness [17,18]. However, plasma-sprayed HA coatings still exhibit
some problems, including low purity, low crystallinity and a micro-
rough surface [19,20]. It is well known that the plasma-spraying pro-
cess is composed of sharp heating under a super-high temperature,
quick cooling and solidifying, which leads to the phase decomposition
of HA and amorphous phase formation, further resulting in the low
purity and low crystallinity [21]. Unfortunately, the low crystallinity
usually accelerates the degradation rate of the HA coating, thus further
shortening the service life span of implants [22]. In addition, plasma-
sprayed HA coatings presented a micro-scale rough surface, which has a
suboptimal biological response, including poor osteoconductivity
[20,23]. To overcome these problems, various post-deposition heat
treatments have been proposed, such as vapor-flame treatment, laser
treatment and furnace heating treatment. These traditional post-de-
position heat treatments increase the purity and crystallinity under
suitable conditions by removing non-HA compounds [24], while it is
still difficult to obtain a nano-structured coating using these post-
treatment methods. However, constructing a nano-structure on the
implant surface is considered an important method for improving the
osseointegration of the Ti-alloys implants [25]. Previous studies have
showed that a nano-structural surface both enhanced functional protein
adsorption and promoted cell attachment, proliferation and differ-
entiation [1,26]. Furthermore, a nano-structured topography is bene-
ficial to osseointegration between Ti-alloy implants and host bone
tissue [27]. Recent studies have also reported that a nano-structural
surface modulates some key steps of the osseointegration processes
[28,29].
Currently, it is still a challenge to create nano-structured HA coat-
ings with high purity and crystallinity on Ti-6Al-4V implants. The at-
mosphere plasma spraying (APS) technology is advantageous because it
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Chemical Engineering Journal 347 (2018) 711–720
can control the coating thickness on a Ti-6Al-4V substrate, but it in-
evitably generates an amorphous phase and simultaneously drastically
decreases the crystallinity of the HA coating [22]. The reversion of the
decomposed and amorphous phases might be an effective approach for
obtaining highly pure and crystalline HA coatings with a nano-struc-
tured surface on Ti-6Al-4V implants.
In the present study, we combined APS and hydrothermal treatment
(HT) technology to overcome the aforementioned challenges. In this
method, the hydrothermal treatment realizes the reversion of the de-
composed and amorphous phases into a nanorod structured HA (nHA)
crystal phase to increase the anti-degradation performance and induce
an osseointegration capacity. The biological responses of rat bone me-
senchymal stem cells (rBMSCs) in vitro and osseointegration in vivo of
the nHA-coated Ti-6Al-4V implants were also evaluated.
2. Materials and methods
2.1. Preparation and characterization of HA coatings with nano-structured
surface on Ti-6Al-4V substrate
Commercially available Ti-6Al-4V slices (dimensions:
10 × 10 × 2 mm) and rods (diameter: 2 mm; length: 10 mm) (Shanghai
Yantai Metallic Material Co., Ltd, China) were prepared for use as
substrates by machining for the in vitro and in vivo studies, respectively.
All Ti-6Al-4V substrate surfaces were roughened by grit blasting. In
addition, HA powders with a particle size ranging from 40 to 80 μm
were obtained from Sulzer Metco (Europe GmbH). Subsequently, HA
powders were coated on the rough Ti-6Al-4V substrate using atmo-
spheric plasma spraying (APS, F4-MB, Sulzer Metco, Switzerland). The
optimal spraying parameters are listed in Table 1.
After plasma spraying, the HA-coated Ti-6Al-4V samples received an
additional hydrothermal treatment (HT). Some of them were hydro-
thermally treated in Milli-Q water at 180 °C for 24 h and labeled T2.
Some of them were hydrothermally treated in 0.2 mol/L Na3PO4 solu-
tion at 180 °C for 24 h and labeled T3. Next, T2 and T3 were soaked in
Milli-Q water for several days to remove the redundant salt ions. The
plasma-sprayed HA coatings without hydrothermal treatment were
used as controls and labeled T1.
The surface morphology of the HA coatings was observed by field
emission scanning electron microscopy (FESEM: SU8220, Hitachi,
Japan). The crystal phases of the Ti-6Al-4V substrate and the fabricated
HA coating were detected by using X-ray diffraction with Cu Kα ra-
diation [30]. The crystallinity degree, Xc, of the HA coatings before HT
and after HT was evaluated according to the International Standard
Organization (ISO 13779-3: 2008).
2.2. Evaluation the wettability and degradability of the HA coatings before
and after HT
The surface wettability of the HA coatings was evaluated by char-
acterizing the contact angle using Milli-Q water as the medium on an
Automatic Contact Angle Meter Model (SL200B, Solon Information
Technology Co., Ltd, China). The contact angle value was calculated by
the Young-Laplace drop profile fitting method [31]. In addition, the
average value and error bar were taken from five measurements
Table 1
Atmospheric plasma-spraying parameters.
Main gas auxiliary gas Arc Arc Spraying Power
Ar (slpm) H2 (slpm*) current voltage distance feeding rate
(A) (V) (mm) (rpm**)
40 8 550 60 100 25
* slpm = standard liter per minute.
** rpm = revolutions per minute.
L. Xia et al.
performed at different locations on the coating surface.
The degradation rate of the HA coatings with and without hydro-
thermal treatment was evaluated by immersing in Tris-HCl buffer so-
lution for 14 days, during which the pH value was adjusted to 7.25. The
Tris-HCl buffer solution was changed every two days during this ex-
periment. The Ca ionic concentration released from the HA coating at 2,
4, 8 and 14 days was measured by inductively coupled plasma atomic
emission spectroscopy (ICP-AES, Varian 715ES, USA).
2.3. Characterization of protein adsorption capacity of the HA coatings
before and after HT
T1, T2 and T3 were soaked in fluorescent-labeled bovine serum
albumin (FITC-labeled BSA, Sigma) solution (0.25 mg/mL, in PBS) for
3 h to observe the protein adsorption ability by using confocal laser
scanning microscopy (CLSM, Leica, Germany).
2.4. Cell cultures on the HA coatings before and after HT
2.4.1. Culture of rat bone mesenchymal stem cell (rBMSCs)
rBMSCs were isolated from male rats as described in a previous
study [32]. The obtained cell suspension was centrifuged to remove the
supernatant. Then, the cells were cultured in an incubator at 37 °C with
an atmosphere of 5% CO2 and 95% relative humidity. The culture
medium was removed to discard the non-adherent cells after 1 day and
was then renewed every two days. In addition, rBMSCs of passages 2–4
were used in the following cell biological experiments. No os-
teoinductive factors were applied in the present study.
2.4.2. Early morphology and adhesion of the seeded rBMSCs
The adhesion morphology of rBMSCs cultured on the HA coating
surface was visually observed using confocal laser scanning microscopy
(CLSM, Leica, Germany). After 6 h of culturing, the samples were re-
moved from the culture medium, and the non-adherent cells were re-
moved using PBS. Then, the adherent cells were fixed with 4% paraf-
ormaldehyde. Before staining, the samples were treated by 0.1% Triton
X-100 and 1% BSA to block the nonspecific binding sites. Then, the
actin cytoskeleton was stained by TRITC-Phalloidin (Sigma), and the
cell nuclei were stained with 4′,6-Diamidino-2-phenylindole dihy-
drochloride (DAPI, Sigma) [33]. Finally, the cell adhesion morphology
was imaged’ by confocal laser scanning microscopy.
2.4.3. Cell proliferation of the seeded rBMSCs
rBMSCs were seeded on the surface of HA coatings (TI, T2 and T3)
and cultured for 4 days. Then, the cell proliferation was evaluated by
MTT assay after culturing for 1 and 4 days. Briefly, at each time point,
three samples of each group were immersed in 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyl tetrazolium bromide (MTT: Amresco, USA) solution
to form formazan; and then, the solution was replaced with dimethyl
sulfoxide (DMSO, Sigma) to dissolve the formazan. Finally, the ELX
Ultra Microplate Reader (BioTek, USA) was used to measure the ab-
sorbance value at λ = 590 nm.
2.4.4. Alkaline phosphatase (ALP) activity assays of the seeded rBMSCs
To evaluate the ALP activity, rBMSCs were cultured on HA coating
surfaces for 4, 7 and 10 days. After culturing the rBMSCs for 10 days,
ALP staining was performed according to the manufacturer’s instruc-
tions. The ALP activity at days 4, 7 and 10 was quantified with an ALP
Assay Kit. Briefly, the rBMSCs were lysed from the HA coating surfaces
using lysis buffer. Then, the cell lysates were centrifuged to obtain the
supernatant, and the supernatant was reacted with 1 mg/mL pNPP so-
lution. Finally, the OD value was detected using a microplate reader at
405 nm. In addition, the data were normalized to the total cellular
proteins as measured by the Bradford method [33].
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2.4.5. Quantitative real-time PCR assay of osteogenic differentiation
analysis
The osteogenic differentiation of the rBMSCs after culturing on HA
coating surfaces for 4, 7 and 10 days was evaluated by qRT-PCR.
Briefly, total RNA was extracted from cells culturing on HA coating by
Trizol reagent (Invitrogen, USA). Next, the obtained RNA was reverse-
transcribed to cDNA using a Prime-Script RT reagent kit (Takara Bio,
Japan) according to the manufacturer’s instructions. Finally, the
quantitative expression of osteogenic markers, including bone sialo-
protein (BSP), bone morphogenetic protein 2 (BMP-2), type I collagen
(Col I), osteocalcin (OCN), runt-related transcription factor 2 (Runx2)
and osteopontin (OPN), was detected using Bio-Rad MyiQ single color
Real-time PCR system [34].
2.5. In vivo osseointegration evaluation
2.5.1. Surgical procedures
Four-week-old male SD rats were used in the animal experiment.
First, general anesthesia was induced by an intravenous injection of
pentobarbital. Then, the bone defects with a 2 mm diameter and 10 mm
depth were made in the femur of the rat using a KaVo drill. Finally, T1,
T2 and T3 with a size of 2 × 10 mm (diameter × length) were ran-
domly inserted into the bone defects sites, and the surgical wounds
were carefully closed. All surgical procedures were approved by the
Animal Research Committee of 9th People’s Hospital, Shanghai Jiao
Tong University School of Medicine.
Twelve weeks after the implant surgery, all rats were sacrificed by
euthanasia. The specimens were harvested for histological analysis.
2.5.2. Histological and histomorphometric observation
The specimens were fixed by 10% formaldehyde solution, dehy-
drated by a graded series of ethanol from 75% to 100% and embedded
in polymethylmethacrylate (PMMA). Then, the slices of the embedded
specimens were obtained by a saw microtome (SP1600, Leica,
Germany). Finally, the slices were stained by Van Gieson’s picrofuchsin.
In addition, the percentage of newly formed areas was quantified using
a computer-based image analysis system (Image Pro. 5.0, Media
Cybernetic, USA). The bone-implant contact ratio (BCR) was calculated
as the ratio of the bone-contacting region length (Lb) to the total cir-
cumference length (Lf).
2.6. Statistical analysis
All data were expressed as the mean ± standard deviation with
each group prepared in triplicate (n = 3). The statistical analysis was
employed an ANOVA and an equal variance assumption test. The dif-
ferences were considered statistically significant when the p-value was
less than .05.
3. Results
3.1. Characterization of HA coatings before and after hydrothermal
treatment
The surface morphology of HA coatings before and after hydro-
thermal treatment (HT) is presented in Fig. 1. Before hydrothermal
treatment, the original plasma-sprayed coating T1 had a micro-rough
surface that was formed via the random deposition of fully and partially
melted HA particles. The corresponding higher-magnification images
showed that the fully melted coating surface was relatively smooth and
presented a caky structure. In contrast, the distinctly different surface
morphologies of the HA coating were obtained after hydrothermal
treatment in appropriate reaction media. The HA coating T2 presented
an erosion surface and sedimentary crystal boundary with different
sizes between 150 nm and 740 nm following hydrothermal treatment in
Milli-Q water at 180 °C for 24 h. The coatings on T3 showed a nanorod-
L. Xia et al. Chemical Engineering Journal 347 (2018) 711–720

Fig. 1. SEM images of the HA coatings before and after hydrothermal treatment. T1: the plasma-sprayed HA coatings without hydrothermal treatment; T2: the
samples obtained by hydrothermal treatment of T1 in Milli-Q water at 180 °C for 24 h; T3: the samples obtained by hydrothermal treatment of T1 in 0.2 mol/L
Na3PO4 solution at 180 °C for 24 h.

structured surface with a mean diameter of 35 nm following hydro-
thermal treatment in 0.2 mol/L Na3PO4 solution at 180 °C for 24 h.
The XRD patterns presented in Fig. 2A suggest that before plasma
spraying HA coatings, the substrates showed the characteristic peaks of
Ti-6Al-4V, while all the Ti-6Al-4V characteristic peaks disappeared
after plasma-spraying HA coatings on the substrates. In addition, the
HA powers could be identified as standard HA crystal phase (JCPDS
card: No. 09-0432) without any line-broadening effect and served as the
standard of 100% crystallinity but that the plasma-sprayed HA coating
still presented characteristic HA structural peaks accompanied by an
obvious glassy phase. After HT, the amorphous phase almost dis-
appeared (shown in Fig. 2B). Furthermore, the crystallinity degree Xc of
the HA coating was calculated in terms of ISO 13779-3: 2008. The re-
sults of Fig. 2C show that the crystallinity degree, Xc, of the original

Fig. 2. A. XRD patterns of Ti-6Al-4V substrates, HA powders and the plasma-sprayed HA coatings, B. XRD patterns of the HA coating before and after hydrothermal
treatment, C. The crystallinity degree (Xc) before and after hydrothermal treatment.

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L. Xia et al.
Fig. 3. Surface contact angle and water drop profile of HA coating before and
after hydrothermal treatment.
Fig. 4. Ca ion concentration by soaking the T1, T2 and T3 in Tris-HCl buffer
solution for 2, 4, 8 and 14 days.
plasma-sprayed coating (T1) was approximately 51.3% and that after
hydrothermal treatment, the crystallinity degree of HA coatings T2 and
T3 increased significantly to 87.7% and 85.3%, respectively.
3.2. Effect of HT on wettability and degradability of the HA coatings
Fig. 3 presented that the contact angle of the plasma-sprayed HA
Fig. 5. CLSM images of protein absorption on T1, T2 and T3 after soaking in PBS solution containing FITC-BSA for 3 h.
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Chemical Engineering Journal 347 (2018) 711–720
coating (T1) surface before hydrothermal treatment was approximately
38.1° and that the contact angle of T2 decreased to 32.3° after hydro-
thermal treatment in Milli-Q water. Furthermore, T3 with nanorod-
structured surfaces possessed a super-hydrophilic property with contact
angle of 0° by HT in 0.2 mol/L Na3PO4 solution.
Fig. 4 shows the amount of Ca ion released from the HA coatings
(T1, T2 and T3) after soaking in Tris-HCl buffer solution for 2, 4, 8 and
14 days. The results illustrated the released amount of Ca ion from T2
and T3 was much lower than that of T1. In addition, the released
concentration of Ca ion from T3 was the lowest throughout the de-
gradation experiment.
3.3. The effect of hydrothermal treatment on the protein adsorption
capacity of the HA coatings
The protein absorption results showed that only a few proteins at-
tached to the original plasma-sprayed HA coating (T1) surface after
being immersed in FITC-BSA solution (0.25 mg/mL, in PBS) for 3 h,
whereas more proteins absorbed on the surface of T2 after HT.
Furthermore, T3 with the nano-structured surface showed the highest
level of protein absorption (Fig. 5).
3.4. The effect of HT on the adhesion, proliferation and osteogenic
differentiation of rBMSCs on HA coating
3.4.1. The stimulation of rBMSCs adhesion by the HA coating with high-
crystallinity and nano-structured surfaces
The CLSM results (Fig. 6) showed that after culturing for 6 h, the
rBMSCs attached on the original plasma-sprayed HA coating T1 spread
only slightly, whereas the rBMSCs adhered on T2 and T3 after HT
showed much better cell adhesion. Compared with the rBMSCs on T1,
the cells on T2 and T3 spread significantly with more typical filopodia
attachment. More importantly, the enhanced effect on cell attachment
was more obvious on T3 which had the nanorod-structured surface.
3.4.2. The stimulation of rBMSCs proliferation by the HA coatings with
high-crystallinity and nanorod-structured surfaces
The rBMSCs on all HA coatings surface showed a significant pro-
liferation throughout the cell culture period (Fig. 7). Moreover, the
rBMSCs cultured on T2 and T3 proliferated significantly faster than did
those on T1 at 4 days of culture.
3.4.3. The stimulation of ALP expression by HA coatings with high-
crystallinity and nanorod-structured surfaces
Fig. 8A shows ALP staining intensity on T1, T2 and T3 increased in
sequence. Moreover, on T3, the ALP staining was the most intense
compared with those of T1 and T2. Furthermore, the quantitative ALP
expression result suggested that the rBMSCs on T2 and T3 had higher
ALP expression than did those on T1 throughout the culture period. In
addition, the highest expression amount of ALP was observed on T3
L. Xia et al.
Fig. 6. CLSM images of rBMSCs attachment after culturing on different HA coating samples for 6 h. A: actin stained with red, B: cell nuclei stained with blue, C:
merged A with B. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 7. MTT assay of rBMSCs cultured on different HA coatings at days 1 and 4
for cell proliferation.
throughout the culture period, with a significant difference (p < .05).
3.4.4. Stimulation of osteogenic differentiation by the HA coatings with
high-crystallinity and nanorod-structured surfaces
The gene expression of osteogenic markers was further evaluated by
qRT-PCR assay after culturing the rBMSCs on T1, T2 and T3 for up to
10 days (Fig. 9). The result indicated that the osteogenic marker ex-
pression of rBMSCs cultured on T2 and T3 was significantly enhanced
compared with that of T1 in a particular period. Specifically, at 4 days
of culture, the gene expression levels of BSP, BMP-2, COL1, OCN,
RUNX2 and OPN in rBMSCs cultured on T3 were significantly up-
regulated compared with those of T1, and enhanced expression of BSP,
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Chemical Engineering Journal 347 (2018) 711–720
COL1 and OPN was observed for rBMSCs cultured on T2 compared to
that of T1. In addition, the osteogenic markers, including BSP, BMP-2
and OCN, of rBMSCs maintained the higher expression on T3 compared
with that of T1 when the culture time increased to 10 days. Moreover,
the gene expression level of BSP and OCN was significantly up-regu-
lated in rBMSCs cultured on T3 than those of T2 throughout the cell
culture period.
3.5. In vivo osseointegration of the stable nHA-coated Ti-6Al-4V implants in
rat model
Micro-CT images (Fig. 10A) visually showed the new bone forma-
tion morphology around the bone defect site. The results suggested that
new bone was formed around the HA-coated Ti-6Al-4V implants. More
importantly, more new bone was formed around the T2 implant com-
pared with T1 implant, whereas much more new bone was formed
around the T3 implant than around the T1 and T2 implants. In addition,
Van Gieson’s staining (Fig. 10B) showed that little new bone formed
around the cancellous bone region on the T1 implant surface. More
newly formed bone was observed around the cancellous bone region on
the T2 implant surface compared with that of the T1 implant, while the
T3 implant surface showed the most newly formed bone around the
cancellous bone region compared with the T1 and T2 implants. The
quantitative analysis of histomorphometry (Fig. 10B1 and B2) further
showed that the new bone area percentage around the T3 implant was
approximately 43.27 ± 2.98%, which was significantly higher than
that around the T1 (20.38 ± 2.68%) and T2 (28.59 ± 3.33%) im-
plants. It also showed a significant difference between the T1 and T2
implants. Moreover, the trend of bone-implant contact ratio is con-
sistent with the new bone area percentage. The bone-T3 implant con-
tact ratio percentage was approximately 65.04 ± 4.73%, which was
significantly higher than those of the T1 (36.82 ± 4.16%) and T2
(46.3 ± 5.15%) implants.
L. Xia et al. Chemical Engineering Journal 347 (2018) 711–720

Fig. 8. A. ALP staining for rBMSCs cultured on different HA coatings at day 10, B. Quantitative ALP expression after culturing rBMSCs on different HA coatings for 4,
7 and 10 days.

Fig. 9. Osteogenic factor expression of rBMSCs cultured on different HA coating samples for 4, 7 and 10 days.

4. Discussion implants, due to the high mechanical strength of Ti-6Al-4V and the
good bioactivity of the HA coating [18,35]. Among the various pre-
Ti-6Al-4V implants with HA coating have been widely used in the paration methods for HA coatings, plasma spraying is considered a
clinic, including surgical implements, artificial joints and dental prospective method due to its high efficiency and easily controlled

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Fig. 10. A. Histological images of newly formed bone on the HA-coated Ti-6Al-4V implants surface after implanted in rat femur for 12 weeks, B. The percentage of
new bone areas, C. The percentage of bone-implant contact ratio.

Fig. 11. Schematic illustration for the formation mechanism of nHA coatings on Ti6Al4V substrate via combination of APS and HT technology.

coating thickness [36,37]. However, there are still several problems
with plasma-sprayed HA coatings, including low purity, low crystal-
linity and a micro-rough surface [20]. Additionally, the low crystal-
linity usually accelerates the degradation rate of the HA coating, thus
further shortening the service life span of the implants [22]. Moreover,
the micro-rough surface lacks sufficient osteoconductivity at the early
stage of new bone formation compared with the nano-structure of
human bones [20]. Recent papers have also reported that having a
nano-structured surface on an implant enhanced the adsorption of
bioactive proteins, thus promoting cellular responses and ultimately
improving osseointegration [38]. Hence, it is significant to modulate
the nano-structured surface of HA coatings for better osseointegration
[25].
In the current study, we developed an effective approach for mod-
ulating HA coatings with high crystallinity and nano-structured sur-
faces by combining the plasma-spraying technology with hydrothermal
treatment in an appropriate reaction medium [39]. Under hydro-
thermal treatment, the amorphous phase and decomposition products

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L. Xia et al.
(tricalcium phosphate and calcium oxide) generated by plasma
spraying were successfully transformed into a crystalline HA phase.
These phase transformation increased the crystallinity degree of the HA
coating from 51.3% to over 85%, which meets the requirement for a
crystallinity of no less than 62% in the medical devices industrial
standard (YY0304-1998).
Most importantly, a nanorod structured surface on the HA coating
was successfully achieved via hydrothermal treatment in 0.2 mol/L
Na3PO4 solution. Furthermore, the formation mechanism of the na-
norod HA surface was deduced as follows (Fig. 11): First, hydrothermal
treatment is a dissolution–recrystallization process. At a hydrothermal
temperature of 180 °C, the tricalcium phosphate and amorphous phase
dissolved into calcium and phosphate ions. At the same time, the re-
crystallization process occurred. The trisodium phosphate reaction
medium played an important role in this process and offered a super-
saturated degree of phosphate ions, further resulting in a nano-struc-
tured crystalline HA phase. Moreover, the nanorod structure growth
mechanism was based on “Posner’s cluster growth model” [40]. The
calcium and phosphate ions gradually dissolved into solution to form
Posner’s clusters (molecular formula: Ca9(PO4)6). Posner’s clusters were
considered the growth units of HA crystals and presented a positive
charge on the surface. In contrast, the HA crystal surface has two dif-
ferent charged types: the a-surface with a positive charge and the c-
surface with a negative charge [41]. The Posner’s clusters were posi-
tively charged prior to adsorbing on the negatively charged c-surface.
Moreover, the excess PO43- group prevented the Posner’s clusters from
absorbing on the a-surface. Hence, HA crystals grew along the c-axis,
which increased its aspect ratio, and finally formed a nano-rod HA
crystal [42].
The nanorod structured surface of the HA coating offered a super-
hydrophilic environment (as shown in Fig. 3), which is beneficial for
protein adsorption and cell adhesion and further promotes cell pro-
liferation and differentiation [34,43]. Moreover, cell-material interac-
tions plays vital roles in further osseointegration in vivo [44]. As soon as
they were implanted in vivo, the stabile nHA-coated Ti-6Al-4V implants
induced a cascade of biological responses at different stages. At the
beginning, the adsorption of water molecules and protein was induced,
and the cells then attached on the nano-structured HA surface. In ad-
dition, this super-hydrophilic surface on nHA-coated Ti-6Al-4V im-
plants (T3) is beneficial for the interaction of the implants with body
fluid.
Moreover, the nanorod structured surface presented a high surface
area, which was good for surface protein (such as integrin) enrichment
and cell attachment (as shown in Figs. 5 and 6). Next, cell proliferation
and differentiation occurred on the implant surface. Cell adhesion is a
key regulator of cellular activation, migration, proliferation and dif-
ferentiation, and it influences the biological interfacial interactions
with the implants [45]. The rBMSCs attached well on the implant
surface with a regular cytoskeleton is beneficial for the proliferation
and differentiation of rBMSCs (as shown in Figs. 7–9). As an early
marker for osteogenic differentiation, ALP regulates the inorganic
phosphate metabolism and acts as a plasma membrane transporter for
phosphates [46]. The current studies suggested that ALP activity was
significantly promoted for rBMSCs by introducing a nano-structured
surface. More importantly, the expression of bone-specific markers for
osteogenic differentiation, including BSP, BMP-2, COL-1, OCN, RUNX2
and OPN, was also enhanced in a particular period due to the combi-
nation of high crystallinity with a nano-structured surface.
Finally, the formation of new bone on the implant interface induced
tight interlocking between the surrounding bone tissue and the im-
plants, which resulted in excellent osseointegration [47]. The me-
chanical stability became higher, and the probability of the implant
loosening decreased as the osseointegration degree increased. Thus, the
required implant interface for excellent osseointegration should con-
sider not only the surface chemical composition but also the construc-
tion of the nano-structured surface [25]. In this study the chemically
719
Chemical Engineering Journal 347 (2018) 711–720
stable HA coating with high crystallinity was prepared, and a nanorod
structured surface was constructed on the Ti-6Al-4V substrate through a
combination of atmosphere plasma spraying (APS) with hydrothermal
treatment. After being implanted into healthy rat femurs for 12 weeks,
the stable nHA-coated Ti-6Al-4V implants demonstrated superior os-
seointegration (as shown in Fig. 10). These results indicated that the
design of the nanorod-structured HA coating with high crystallinity is
beneficial for a long-term service life span and excellent osseointegra-
tion of Ti-6Al-4V implants.
5. Conclusion
A stable nHA-coated Ti-6Al-4V implant that was produced by
combining atmospheric plasma spraying (APS) with hydrothermal
treatment (HT) was developed in this study via the in situ reversion of
the decomposed and amorphous phases of the HA coatings in trisodium
phosphate aqueous solution. The fabricated coatings possessed high
crystallinity, which decreased the degradation rate and improved the
stability, thus potentially improving their long-term service life span.
Moreover, the features of the constructed nanorod surface promoted the
adhesion, proliferation, differentiation of rBMSCs and further improved
the osseointegration between the implants and the surrounding bone
tissue. Hence, the stable nHA-coated Ti-6Al-4V implants developed by
our strategy have the potential to improve osseointegration and prolong
the service life span in dental and orthopedic applications.
Conflict of interest
Without competing financial interest declared by the authors.
Acknowledgments
The authors gratefully acknowledge the support of Natural Science
Foundation of China (81672134, 81771115, 81400554), Science and
Technology Commission of Shanghai Municipality (17510710800), the
Fund of Shanghai Municipal Commission of Health and Family
Planning (201540369), Municipal Human Resources Development
Program for Outstanding Young Talents in Medical and Health Sciences
in Shanghai (2017YQ058), Excellent Youth Program of Ninth People’s
Hospital Affiliated to Shanghai Jiao Tong University School of Medicine
(jyyq08201621).
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