Acta Biomaterialia 6 (2010) 2264–2273

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Acta Biomaterialia
journal homepage: www.elsevier.com/locate/actabiomat

Functionally graded hydroxyapatite coatings doped with antibacterial components

Xiao Bai a, Karren More b, Christopher M. Rouleau c, Afsaneh Rabiei a,*
a
Mechanical and Aerospace Engineering, North Carolina State University, Raleigh, NC 27695, USA
b
Materials Science and Technology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA
c
Center for Nanophase Materials Sciences, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA

a r t i c l e i n f o a b s t r a c t

Article history: A series of functionally graded hydroxyapatite (FGHA) coatings incorporated with various percentages of
Received 13 July 2009 silver were deposited on titanium substrates using ion beam-assisted deposition. The analysis of the coat-
Received in revised form 26 November 2009 ing’s cross-section using transmission electron microscopy (TEM) and scanning transmission electron
Accepted 1 December 2009
microscopy equipped with energy dispersive X-ray spectroscopy has shown a decreased crystallinity
Available online 5 December 2009
as well as a distribution of nanoscale (10–50 nm) silver particles from the coating/substrate interface
to top surface. Both X-ray diffraction and fast Fourier transforms on high-resolution TEM images revealed
Keywords:
the presence of hydroxyapatite within the coatings. The amount of Ag (wt.%) on the outer surface of the
Ion beam processing
Hydroxyapatite coating
FGHA, as determined from X-ray photoelectron spectroscopy, ranged from 1.09 to 6.59, which was about
Adhesion strength half of the average Ag wt.% incorporated in the entire coating. Average adhesion strengths evaluated by
Antimicrobial pull-off tests were in the range of 83 ± 6 to 88 ± 3 MPa, which is comparable to 85 MPa for FGHA without
silver. Further optical observations of failed areas illustrated that the dominant failure mechanism was
epoxy failure, and FGHA coating delamination was not observed.
Ó 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction Among those, Ag incorporation has gained more attention due to

Infection, which results in morbidity and implantation failure, is
a serious problem associated with prosthetic implantation, and
usually leads to subsequent implant removal and debridement
[1–3]. The infection mechanism has been postulated as bacterial
colonization of the prosthesis at the time of implantation by direct
inoculation during manipulation of tissue and implantation, or as a
result of contaminated sterile implants [3]. In 1033 cases of total
hip and total knee prosthetic arthroplasty infections, the major
ones have been found to be caused by aerobic cocci, most com-
monly Staphylococcus aureus (23%), and coagulase-negative staph-
ylococci (25%) [3]. The overall rate of prosthetic joint infection is
highest in the first 6 months postoperatively, and declines contin-
uously thereafter [3]. It has also been observed that the combined
incidence of rates of total hip and knee arthroplasty infections dur-
ing the first and second postoperative years, and after 2 years, were
approximately 6.5, 3.2 and 1.4 per 1000 joint-years, respectively
[3].
Hydroxyapatite (HA)-coated implants incorporated with anti-
microbial agents are able to prevent or cure infections by releasing
directly the antimicrobial agents to local regions. HA incorporated
with Ag/Ag+ [2,4–22], Cu2+/Zn2+ [18,21], ampicillin [23] and doxy-
cycline [12] have been studied to evaluate their antibacterial effect.
* Corresponding author. Tel.: +1 919 513 2674; fax: +1 919 515 7968.
E-mail address: arabiei@eos.ncsu.edu (A. Rabiei).
its broad-spectrum antibacterial properties [4,8]. The most com-
mon technique to incorporate Ag into HA coatings is via an ion ex-
change method, in which the Ca ions in HA are replaced by Ag ions
while dipping the HA coatings into AgNO3 for a period of time
[2,4,24]. The limitation of the ion exchange method is that Ag will
reside mostly on the outer surface of the coating and will be
quickly depleted in vivo/in vitro without long-term antibacterial
effect. In order to achieve the continuous release of Ag, HA coatings
doped with Ag through the entire thickness has been developed
using sol–gel [10,18], co-sputtering [5,9], and thermal or cold
spraying [25,26]. Although Ag in small percentages can have an
antibacterial effect, larger amounts can be toxic [9], and therefore
optimization of the Ag concentration in the coating is critical to
guarantee an optimum antibacterial effect without cytotoxicity.
It has been reported that the presence of 2 wt.% Ag in HA coating
processed by co-sputtering can have significant antibacterial effect
to Staphylococcus aureus and Staphylococcus epidermidis without
osteoblast-precursor cell cytotoxicity [9].
In this study, we investigated processing and characterization of
functionally graded hydroxyapatite (FGHA) coatings incorporated
with Ag as an antibacterial component. The processing technique
was ion beam-assisted deposition (IBAD) with in situ heat treat-
ment. While the amorphous top layer of the coating allows a high-
er release rate of silver due to its higher dissolution immediately
after implantation, the crystalline layer will maintain the silver
as a reservoir, resulting in long term infection protection. It is

1742-7061/$ - see front matter Ó 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.actbio.2009.12.002
 X. Bai et al. / Acta Biomaterialia 6 (2010) 2264–2273
anticipated that this technique will be more efficient in infection
control than conventional oral/intravenous antibiotic intake with
fewer side effects. In this study, the processing of FGHA coatings
with three different silver contents is reported along with their
mechanical and microstructural properties. The biological re-
sponses of these coatings will be reported in a future article.
2. Materials and methods
2.1. Material processing
Titanium disc substrates of 7.62 mm thickness were machined
from a 12.7 mm diameter, 99.5% commercially pure titanium rod
(Alfa Asear). Prior to deposition, the Ti substrate surfaces were pre-
pared by wet grinding with 240-, 400- and 600-grit silicon carbide
paper (Buehler) and subsequent polishing with 9, 3 and 1 lm poly-
crystalline diamond suspension (Buehler). In between each of the
grinding and polishing steps the discs were washed and ultrason-
ically cleaned to prevent cross contamination of abrasive particles.
After polishing and grinding the discs were ultrasonically cleaned
in acetone and isopropyl alcohol for 10 min each and passivated
by exposing the discs to a 40% (by volume) nitric acid solution at
room temperature for 30 min. The discs were then rinsed with
water and dried by compressed air (ASTM F86-91).
Two sintered HA targets (Cerac Inc.) of 152.4 mm diameter,
6.6 mm thickness and 99% purity were used to deposit either pure
FGHA or FGHA-Ag coatings in order to avoid cross contamination.
5 N purity silver wires with various diameters (ESPI) were affixed
to the top of the target to dope the FGHA coatings with various
concentrations of silver.
The IBAD system (Ion Tech.) employed to synthesize the coat-
ings consisted of a primary 5 cm Kaufman ion source for sputtering
the target and a secondary 15 cm Kaufman ion source for bom-
barding the substrate/coating surface; both primary and secondary
beams were aligned to focus on the center of target and substrate
holder. The incident angle from the target and secondary beam was
45°. Substrates were heated by a radiant heater (Momentative Per-
formance Mater.) located behind the substrate holder, but without
direct contact. A base pressure of 5  10 7 Torr was reached before
starting each deposition run.
The FGHA coatings were deposited over a total period of 6 h; the
substrate temperature was 550 °C for the first 4 h and 450 °C for
the last 2 h in order to grow the functionally graded microstruc-
ture. Beam voltage and current density of the primary source were
1200 V and 8.92 mA cm 2, respectively, during deposition. Beam
voltage and current density of the secondary ion source, however,
were gradually decreased in order to facilitate a well-bonded inter-
face and appropriate composition at the top surface of the coatings.
Manual rotation by 90° of the substrate holder was performed
every 5 min in the first hour of deposition and every 15 min during
the remainder of the deposition in order to improve the uniformity
of the coating. Water vapor was introduced into the chamber by
passing a 20 sccm argon gas flow through a water bubbler to pro-
vide more OH at the surface of the substrate and prevent dehydr-
oxylation of HA. For the duration of the depositions, 10 and
20 sccm of argon gas flow was provided to the primary and second-
ary ion source, respectively, and 5 sccm of argon gas flow was pro-
vided to the plasma bridge neutralizer of each ion source. Using the
parameters above, three sets of Ag-containing FGHA coatings
(FGHA-Ag1, FGHA-Ag2 and FGHA-Ag3) were deposited onto a total
of 21 Ti substrates secured into a custom-made substrate holder
for each run. One line of Ag wire with diameter 1 mm, two lines
of diameter 1 mm and two lines of diameter 1.5 mm were secured
on the surface of the target to provide an average Ag content of
2.34 ± 0.37, 6.29 ± 0.24 and 13.53 ± 0.22 wt.% silver (measured by
2265
SEM–EDS) in FGHA-Ag1, FGHA-Ag2 and FGHA-Ag3, respectively.
For comparison of coating properties, FGHA coatings without silver
were deposited using the second HA target using the same deposi-
tion parameters.
2.2. Materials characterization
2.2.1. Transmission electron microscopy and scanning transmission
electron microscopy
A combined transmission electron microscope/scanning trans-
mission electron microscope (TEM/STEM, Hitachi model HF3300)
equipped with an energy dispersive X-ray spectrometer (EDS,
Thermo Scientific) and operated at 300 kV was used to examine
the cross-sectional microstructure of each of the coatings. The
cross-section samples for TEM and STEM analysis were prepared
in a dual-beam focused ion beam (FIB) instrument (Hitachi
NB5000) using an in situ lift-out technique.
2.2.2. Scanning electron microscopy–energy dispersive spectroscopy
A scanning electron microscope (SEM, Hitachi S3200) equipped
with an EDS was used to examine the composition of the different
coatings from the top surface down to the FGHA/Ti interface. A
minimum of three random locations on the coating surface were
selected for analysis. The instrument was calibrated by measuring
and comparing a pure Mn standard to the ideal for Mn.
2.2.3. X-ray diffraction
X-ray diffraction (XRD, Xpert-Pro Powder Diffractometer) was
used to identify various phases in the FGHA coatings along with
the blank Ti and HA target. The coatings were analyzed with CuKa1
(k = 0.1540598 nm) radiation with 2h between 10° and 70° (with a
step size of 0.017°). Results were then compared to JCPDS powder
diffraction standards to confirm the presence of crystalline HA, Ag
or any other calcium phosphate compounds that might be present
in the coating.
2.2.4. X-ray photoelectron spectroscopy
X-ray photoelectron spectroscopy (XPS, Thermo Scientific K-Al-
pha) Ar+ depth-profiling was employed to quantify the chemical
composition of the coating throughout the thickness. Prior to the
XPS measurements, the XPS was calibrated with the pure HA target
having a Ca/P ratio of 1.67.
2.2.5. Fourier transform infrared spectroscopy
Fourier transform infrared spectroscopy (FTIR, Nicolet Nexus
470) with a smart orbit diamond attenuated total reflectance
(ATR) insert was used to evaluate the structural and molecular
composition of the coatings on the titanium substrates as well as
the HA target in order to identify the presence of the chemical
groups OH and PO43 . Each sample was scanned with a resolution
of 2 cm 1 with a scan number of 64.
2.2.6. Adhesion strength
The adhesion strength of the coating to the titanium substrate
was evaluated using a Romulus materials tester (Pull off tester,
Quad Group). Epoxy coated studs with a cured adhesive strength
of 70–100 MPa were secured perpendicular to the coating surface.
The samples were cured for 1 h at 150 °C in an oven, then removed
and allowed to cool to room temperature in an ambient atmo-
sphere. Using the Romulus Materials Tester IV and Romulus auto-
mated program for coating adhesion strength, each sample was
loaded at a rate of 26.7 N s–1 until failure, with a maximum load
of 445 N. The adhesion strength was averaged from a minimum
of four measurements for each set of coatings. Failure areas under
the stud were observed via an optical microscope (Zeiss) and fail-
2266 X. Bai et al. / Acta Biomaterialia 6 (2010) 2264–2273
ure modes were then classified as epoxy failure (EF), coating cohe-
sive failure (CF) or coating delamination (CD).
2.2.7. Nanoindentation test
Nanohardness and Young’s modulus values for all the coatings
were determined using an MTS Nanoindenter IIÒ instrument (MTS
Instruments). The samples were indented with a Standard Berkovich
indenter, with a strain rate set to 0.05 s 1 and an allowable drift rate
of 0.05 nm s 1. The maximum penetration depth was 1200 nm. The
tip was calibrated with fused silica (SiO2) and was cleaned with iso-
propanol between indentations. The Young’s modulus and hardness,
measured as a function of indentation depth (only 100–200 nm in
order to exclude the substrate effect), were determined using the
Oliver–Pharr model [27] with the Poisson’s ratio set to 0.3 [28]. A
minimum of 10 measurements from each coating were used to eval-
uate the average nanohardness and Young’s modulus.
3. Results
Fig. 1 shows TEM images of the cross-sections of samples FGHA-
Ag1, FGHA-Ag2 and FGHA-Ag3 and Fig. 2 shows the high-resolu-
tion TEM image of FGHA-Ag2 and its fast Fourier transform. All
the coatings consist of three distinct FGHA layers, as labeled in
images (a-1), (b-1) and (c-1) shown in Fig. 1: an equiaxed polycrys-
talline interface layer adjacent to the Ti, a crystalline bottom layer
and a highly-amorphous top layer. Higher magnification images of
the coating interface and bottom layers are shown in images (a-2),
(b-2) and (c-2) of Fig. 1. The thicknesses of the coatings are consis-
tently in the range of 700 to 800 nm. Both the interface and crys-
Fig. 1. TEM images of cross-section of coatings FGHA-Ag1, FGHA-Ag2 and FGHA-Ag3. (a-1, b-1 and c-1) Showing the entire thickness of coatings; (a-2, b-2 and c-2) close-up
of the interface areas; (a-3, b-3 and c-3) close-up of the top layer.
talline bottom layers in all three coatings, with their sum
thickness around 2/3 of the total coating thickness, were formed
at a substrate temperature of 550 °C during the first 4 h of deposi-
tion; the initial interface layer was formed during the first hour of
deposition with a beam voltage and current of 200 V and
0.28 mA cm 2, respectively, as well as a substrate rotation of 90°/
5 min. The top (surface) amorphous layer as shown in a-3, b-3
and c-3, on the other hand, was formed during the final 2 h of
deposition with a substrate temperature of 450 °C and secondary
beam parameters of 50 V/0.17 mA cm 2. Fast Fourier transform
(FFT) on a high-resolution image of coating FGHA-Ag2 shows three
rings, with d-spacing of 2.8, 2.4 and 1.7Å, corresponding to (1 1 2),
(2 2 0) and (0 0 4) of HA. The selective area diffraction was used to
verify the transmission electron microscopy observation of the
mostly amorphous top layer and crystalline bottom layer in the
coatings. However, the high density of crystalline silver particles
in the coating precludes us from having a meaningful selected area
diffraction (SAD) result. In addition, converging the beam (CBED) to
collect data only from the areas between Ag particles resulted in
rapid degradation of the sample. Therefore, a set of samples depos-
ited under the same exact substrate temperatures, but without
including Ag, was used to identify the crystalline and amorphous
regions in the coatings. TEM, SAD and XRD observations confirmed
the crystalline nature of the coating deposited at 550 °C and mostly
amorphous nature of coating deposited at 450 °C (Figs. 3 and 4).
More detailed information on such experiments can be found else-
where [29].
The isolated dark particles shown in Fig. 1 were identified as
metallic Ag by STEM-EDS. Ag particles were found throughout
the coating thickness in all three layers of the coating, for
 X. Bai et al. / Acta Biomaterialia 6 (2010) 2264–2273
Fig. 2. High-resolution TEM image of coating FGHA-Ag2 (left) and its FFT (right). The FFT shows three rings labeled by 1, 2 and 3 at the top; their measured d-spacing and
planes are also labeled.
Fig. 3. TEM and SAD analysis of coating without silver deposited at 450 and 550 °C.
FGHA-Ag1, FGHA-Ag2 and FGHA-Ag3. There were, however, clear
variations in their distribution and size. In coating FGHA-Ag1, the
Ag was mostly distributed in the transitional region between the
top amorphous HA layer and the bottom crystalline HA layer. On
the other hand, coating FGHA-Ag1, which had the lowest percent-
age of Ag, exhibited a columnar grain structure within the bottom
layer. Coatings FGHA-Ag2 and FGHA-Ag3, however, with higher
percentage of Ag, had a more equiaxed grain structure in their
crystalline layer. This can be attributed to the fact that silver can
act as nucleation sites for smaller, equiaxed HA grains in samples
with higher percentages of Ag at higher substrate temperatures
(550 °C) during the first 4 h of deposition. An equiaxed grain struc-
ture will accommodate more silver atoms/particles compared to a
columnar grain structure because the silver can be pushed away by
the tip of the growing columnar grains. In the amorphous layer,
however, the Ag particle size is much smaller due to the lower dif-
Fig. 4. XRD analysis of coating without silver deposited at 450 and 550 °C.
2267
fusion resulted from a lower substrate temperature during the final
stage of deposition. It can be concluded that, in order to achieve a
more uniform distribution of silver throughout the thickness of the
coating, a minimum amount of silver (more than the amount of sil-
ver in FGHA-Ag1) is needed to facilitate the formation of an equi-
axed grain structure in the crystalline layer.
Fig. 5 shows the XRD data from the HA target, blank Ti, FGHA-
Ag1, FGHA-Ag2, FGHA-Ag3 and FGHA coatings. As can be seen in
Fig. 5, the most intense peaks are HA(2 1 1), HA(3 0 0), HA(1 1 2)
and HA(0 0 2) in the HA target. The HA peak (2 1 1) at 32.11°, ob-
served in all coatings, is broad enough to cover both HA(2 1 1)
and HA(1 1 2), which are the two most intense peaks in the target.
Broad peaks of HA(3 2 3), CaO (2 2 0) and (2 2 2) were also ob-
served in all coatings. In general, all coatings show broader peaks,
which is due to the nanoscale structure of the coating. An Ag dif-
fraction peak was not clearly observed in coating FGHA-Ag1, which
is likely due to the low concentration of Ag in this coating. How-
ever, a broad Ag peak (2 0 0) has been clearly observed at 44.13°
in coatings FGHA-Ag2 and FGHA-Ag3. In general, the higher the
concentration of Ag in the coating, the stronger the diffraction
peaks in the XRD data.
Fig. 6 shows the average Ag wt.% evaluated from the coating top
surface by SEM–EDS and XPS. The average Ag wt.% for the entire
thickness of coatings FGHA-Ag1, FGHA-Ag2 and FGHA-Ag3 mea-
sured by SEM–EDS were 2.34 ± 0.37, 6.29 ± 0.24, and 13.53 ± 0.22,
respectively, whereas the Ag wt.% measured on the coating top sur-
face by XPS were 1.09, 3.16 and 6.59, respectively. The average Ag
concentration for the entire coating is about twice that of the out-
ermost coating surface, which confirmed that Ag was not uni-
2268 X. Bai et al. / Acta Biomaterialia 6 (2010) 2264–2273
Fig. 5. XRD results of samples FGHA-Ag1, FGHA-Ag2, FGHA-Ag3 and FGHA.
formly distributed within the coatings, as shown in Fig. 1. In addi-
tion to the variation of Ag distributions on the cross section of coat-
ings, the high sputtering rate of Ag compared to HA could lower the
Ag concentrations at the coating surface because of ion beam etch-
ing during XPS characterization.
Fig. 7 shows the Ca/P measured from the coating top surfaces by
SEM–EDS and XPS. The average Ca/P ratios of coating FGHA, FGHA-
Ag1, FGHA-Ag2 and FGHA-Ag3 measured by SEM–EDS were
2.33 ± 0.01, 2.95 ± 0.03, 2.19 ± 0.10 and 3.09 ± 0.03, respectively,
whereas the Ca/P ratios measured by XPS were 3.25, 3.33, 3.75
and 2.96, respectively. The higher Ca/P ratio measured by XPS
may also be related to the higher sputtering rate of P compared
to Ca, which may sputter more P from the sample during XPS
characterization.
In order to evaluate the variation of the Ca/P ratio and the Ag
concentration throughout the coating thickness, XPS depth-profil-
ing was performed on coatings FGHA, FGHA-Ag1, FGHA-Ag2 and
FGHA-Ag3. These results are presented in Fig. 8. Fig. 8a, b–1, c-1
and d-1 showing the depth-profile for the primary elements ob-
served in each coating (P, Ca, O), while Fig. 8b–2, c-2 and d-2 show
the corresponding observation of the Ag concentration throughout
the coating thickness. The profiles are segmented and labeled in
Fig. 8 as interface, bottom and top layer, consistent with layer
thicknesses measured from images in Fig. 1. Ca, P and O were the
primary elements in coating FGHA (Fig. 8a), and Ag, Ca, P and O
were detected in coatings FGHA-Ag1, FGHA-Ag2 and FGHA-Ag3.
Measured by XPS Measured by SEM-EDS
16
14
12
Ag wt %
10
8
6
4
2
0 ings fall within the range of 83–88 MPa. The fracture surfaces after
FGHA-Ag1 FGHA-Ag2 FGHA-Ag3
Fig. 6. Ag wt.% evaluated by XPS and SEM–EDS on the top surface of coatings.
Measured by XPS Measured by SEM-EDS
4.0
3.5
Atomic Ca/P ratio
3.0
2.5
2.0
1.5
1.0
0.5
0.0
FGHA FGHA-Ag1 FGHA-Ag2 FGHA-Ag3
Fig. 7. Atomic Ca/P ratio evaluated by XPS and SEM–EDS on the top surface of
coatings.
For the FGHA coating, the Ca/P is 3.5 within the amorphous re-
gion and 4 within the crystalline region. Similar Ca/P trends were
observed in the coatings FGHA-Ag1, FGHA-Ag2 and FGHA-Ag3.
However, atomic Ca/P ratios in the top layer of coatings FGHA-
Ag1, FGHA-Ag2 and FGHA-Ag3 were 3.5, 3.5 and 3.7, respectively,
whereas Ca/P was 5, 4.5 and 5 in the crystalline region, respec-
tively. The Ag concentration steadily increased from the coating
top surface inward, and approached a maximum concentration at
the transition region between the top layer and bottom layer for
all coatings. The Ag concentration steadily decreased within the
bottom layer towards the interface with the Ti substrate. Another
high concentration of Ag was observed at the interface region with
the substrate, and this could be formed during the first half hour of
deposition. It is well known that Ag has a higher sputtering rate
when compared to HA [30,31]. As a result, the density of Ag parti-
cles sputtered from the target and deposited on the substrate are
higher than HA. However, the re-sputtering from the heated sub-
strate surface is greater for Ag particles than Ca and P during the
first few minutes, lowering the concentration of Ag in the coating.
Fig. 9 shows the FTIR spectra of the coatings and the HA target.
The peaks are labeled with their corresponding structures. As can
be seen in the HA target spectrum in Fig. 9, OH peaks were ob-
served in stretching mode and OH liberation mode, while the sharp
peaks at 564 and 599 belong to v4 of PO43 . The tiny peak at
879 cm 1 is v2 of CO32 whereas the peak at 963 cm 1 is v1 of
PO43 . The peaks at 1027 and 1091 cm 1 are v3 of PO43 . The peaks
within 1300–1650 cm 1 are due to v3 vibration mode of surface
carbonate ions instead of carbonate ions in the lattice of the mate-
rial, which have been observed in commercial pure HA [33]. The
peaks within 1700–2500 cm 1 are noise of ATR–FTIR. Compared
to the target material, all the coatings show the presence of OH-
at 3640 cm 1 [32]. However, the OH- peaks of stretching and libra-
tional modes were not observed at 3570 and 633 cm 1, which is
the same as the HA coating processed by ion beam mixing deposi-
tion [34]. In addition, all the coatings show a broad peak of PO43
v4 mode at 564 cm 1 instead of the two sharp peaks of PO43 v4
mode at 564 cm 1 and PO43 v3 mode at 599 cm 1 presented in
the target material. Moreover, a broad peak of PO43 v3 mode
was observed at 1004 cm 1 for all the coatings. It is concluded that
all coatings include OH and PO43 . These results, in combination
with the XRD and SAD results presented earlier, confirm the pres-
ence of functionally graded hydroxyapatite in the coating. It is
notable that the presence of broader peaks in this study is attrib-
uted to the small thickness of the coating together with its partially
amorphous structure.
Table 1 lists the average adhesion strength results from pull-off
testing of the coatings. The average adhesion strengths for all coat-
pull-off test were also observed using optical microscopy in order
to further evaluate the failure modes. The optical images are
 X. Bai et al. / Acta Biomaterialia 6 (2010) 2264–2273 2269

Fig. 8. Results of depth-profiling measured by XPS: (a) FGHA, (b) coating FGHA-Ag1, (c) FGHA-Ag2 and (d) FGHA-Ag3.

shown in Fig. 10. The associated failure modes are classified as
coating cohesive failure, coating delamination and epoxy failure,
and are also summarized in Table 1. The epoxy failures given in Ta-
ble 1 are the sum of both epoxy failures (EF) that occurred within
the epoxy and the epoxy delaminations (ED), as shown in Fig. 10.
There were no coating delaminations (CD) observed in any of coat-
ings, which indicates a well-bonded interface between the coating
and the substrate.
Table 2 summarizes the results of the Young’s modulus and
hardness measured by nanoindentation. The coatings FGHA and
FGHA-Ag1 have similar elastic moduli in the range of 77 to
78 GPa, respectively, and hardness values ranging from 2.59 to
2.97 GPa. However, the elastic modulus and hardness of coatings
FGHA-Ag2 and FGHA-Ag3 are higher than those of FGHA and
FGHA-Ag1, ranging from 101.85 to 106.92 GPa and from 4.17 to
4.59 GPa.
2270 X. Bai et al. / Acta Biomaterialia 6 (2010) 2264–2273
Fig. 9. FTIR results of FGHA, FGHA-Ag1, FGHA-Ag2 and FGHA-Ag3 coatings along with the HA target.
Table 1
The adhesion strength measured by the pull-off test.
Coatings Average adhesion Failure modes
strength (MPa)
Coating Cohesive
delamination (%) failure (%)
FGHA 85 ± 6 0 40
FGHA-Ag1 83 ± 6 0 18
FGHA-Ag2 86 ± 3 0 33
FGHA-Ag3 88 ± 3 0 35
4. Discussion
The coatings’ microstructures, as well as their mechanical prop-
erties, resulted from manipulating deposition parameters and sub-
strate temperature during deposition. The well-bonded interfaces
shown in Fig. 1 as well as the compositional transition interface
shown in Fig. 8 illustrate that higher values for secondary beam
parameters of 200 V/50 mA at the beginning of deposition facili-
tated the formation of a dense and compositionally graded struc-
ture. The high bond strength (greater than 83 MPa) without any
coating delamination for all the coatings verified the formation of
this well-bonded interface. In addition, standard deviations less
than 6.42 MPa (7.6%) of the bonding strength resulted from a high
reproducibility of coatings due to continuous substrate rotation of
90o/5 min at the beginning of deposition. Although the in situ sub-
strate temperature has been found to be the dominant factor con-
trolling the degree of crystallinity of HA coatings [29,35–39], a
difference in grain structures in coatings deposited at the same
temperature has been observed. The columnar grain structure in
the bottom layer of FGHA-Ag1 and the equiaxed grain structure
in bottom layer of both FGHA-Ag2 and FGHA-Ag3, which were all
deposited at 550 °C, suggest that Ag concentrations exceeding
6.29 wt.% may enhance the crystallization of the calcium phos-
phate coating. In addition, dense coating structures throughout
the coating thickness for all the coatings were resulted from ion
bombardment by the secondary beam.
Similarly, the variation of coating composition throughout the
coating thickness (as shown in Fig. 8) was a direct result of varying
the deposition parameters of the secondary beam and the sub-
strate temperature. The highest Ca/P ratio near the interface re-
sulted from the highest secondary beam parameters of 200 V/
50 mA at a substrate temperature of 550 °C during the first hours
of deposition; with decreasing values of the secondary beam
parameters to 100 V/50 mA in the second hour and 60 V/30 mA
in the third and fourth hours of deposition, the Ca/P ratio steadily
deceased. During the first 4 h of deposition, the decreasing Ca/P
Epoxy was controlled primarily by the gradual reduction of beam volt-
failure (%) age/current because the re-sputtering rate reduction for Ca is sig-
60 nificant when compared to that of P, although Ca and P both
82 have reduced re-sputtering rates at a reduced beam voltage/cur-
67 rent. In the final 2 h, the voltage was decreased to 50 V and the
65
temperature decreased to 450 °C, which resulted in decreased Ca/
P ratios of 3.25, 3.33, 3.75 and 2.96 for the FGHA, FGHA-Ag1,
FGHA-Ag2 and FGHA-Ag3, respectively. The other primary factor
controlling the Ca/P ratio for this stage was the substrate temper-
ature that is significantly affecting the re-sputtering rate of Ca
when compared to that of P.
The Ag concentration in the coating is critical to its antibacterial
effects and cytotoxicity. In particular, the concentration of Ag in
the top amorphous region of the coating has more severe impact
on its antibacterial effect and cytotoxicity than the average amount
of Ag within the entire coating. This is due to the fact that the top
amorphous region is directly in contact with the body tissue, and is
the first layer to be gradually dissolved and thus enhance new bone
growth by providing Ca and P, as well as inhibiting possible infec-
tions by releasing Ag. The amorphous top surface, with the lowest
Ag concentration and highest dissolution rate, is able to quench
infection immediately after implantation. Ag release then reaches
a steady-state level since the high concentration of the Ag in the
crystalline layer will compensate for its lower release rate. Based
on the current observations, a silver concentration between the
measured in coating FGHA-Ag2 and that in coating FGHA-Ag3
should provide the best cell attachment/cell growth as well as anti-
bacterial effect. Further studies are needed to pinpoint the exact
optimum Ag concentration in FGHA coatings to achieve the best
biological response.
XRD revealed the broad peaks of HA along with calcium oxide
and silver in the coatings; particularly, the HA peaks (2 1 1) at
32.11° in all the coatings is broad enough to cover the most intense
peaks of HA(211) at 31.74° and HA(112) at 32.16° that appeared in
both the HA target and JCPDS card (9–432). The broad peaks to-
gether with the absence of some other HA peaks in these coatings
compared to the HA target or JCPDS card (9–432) are due to the
nature of the coating, which is partially amorphous, with some
nanoscale crystals much smaller than 100 nm in size. In a study
of size effect on X-ray diffraction patterns of HA crystals ranging
 X. Bai et al. / Acta Biomaterialia 6 (2010) 2264–2273 2271

Fig. 10. Optical images of the pull-off area for coatings FGHA, FGHA-Ag1, FGHA-Ag2 and FGHA-Ag3.

Table 2
32.11° and HA(3 2 3) at 64.10° combined with FFT results indicated
Nanoindentation results.
the presence of HA phase. Compared to the fact that various cal-
Coatings Elastic modulus (GPa) Hardness (GPa) cium phosphate compounds instead of HA were observed in func-
FGHA 78.01 ± 8.47 2.97 ± 0.57 tionally graded coatings processed without the presence of water
FGHA-Ag1 77.04 ± 8.05 2.59 ± 0.45 vapor during deposition in our previous work [39], it can be con-
FGHA-Ag2 106.92 ± 9.47 4.59 ± 0.73 cluded that the presence of water vapor with a pressure of
FGHA-Ag3 101.85 ± 9.90 4.17 ± 0.55
2  10 4 Torr during deposition was favorable to the formation of
HA according to both XRD and FFT data. A significantly higher
from 0.5 lm to 50 nm, the well-defined characteristic HA peaks water vapor pressure of 0.25 Torr has been observed to better en-
were observed in HA crystals with an average length of about hance the formation of HA using pulsed laser deposition with
0.5 lm, while only two broad peaks ranging from 25° to 35° and water vapor [41]. It is suggested that a higher pressure of water va-
from 45° to 55° were observed in HA crystals with an average size por might be better to incorporate OH into coatings during future
of 50 nm [40]. In our coatings, both broad peaks of HA(2 1 1) at depositions.
2272 X. Bai et al. / Acta Biomaterialia 6 (2010) 2264–2273
No significant difference of adhesion strength was observed be-
tween the coatings with or without silver, suggesting that incorpo-
ration of Ag (2.34–13.53 wt.%) into the coating does not have any
significant impact on the coating’s adhesion strength. In addition,
the high adhesion strength (greater than 83 MPa) for all the coat-
ings illustrates a well-bonded interface and overall good mechan-
ical properties of the coatings. The optical observations showed
that epoxy failure was the dominant failure mode during the
pull-off test, with 60–80% of the failed area falling under this mode,
indicating that the bond strength of the coating is superior to that
of the epoxy. The superior bond strength of the coating can be
attributed to the presence of the intermixed interface layer and
dense coatings produced by IBAD.
Nanoindentation results from the coatings suggest that incor-
poration of Ag into the FGHA coatings generally resulted in an in-
crease in both the elastic modulus and nanohardness, with the
exception being coating FGHA-Ag1 as compared with coating
FGHA. Apparently, the very low Ag content (2.34 ± 0.37 wt.%) in
coating FGHA-Ag1 rendered it similar to coating FGHA, whereas
for coatings having higher Ag concentrations, of 6.29 ± 0.24 and
13.53 ± 0.22 wt.% (less than 5 vol.%) for FGHA-Ag2 and FGHA-
Ag3, the Young’s modulus increased sharply by 30% compared
to coating FGHA-Ag1, indicating that the Ag acts as a reinforcement
in the HA matrix [42].
5. Conclusions
A series of functionally graded hydroxyapatite coatings incor-
porated with different concentrations of Ag were deposited using
IBAD. TEM/STEM observations on FIB-prepared coating cross-sec-
tions show three distinct layers within the coatings: a ‘‘mixed”
crystalline interface layer adjacent to the Ti substrate, a crystal-
line bottom layer and a mostly amorphous layer on the top sur-
face. TEM/STEM-EDS confirmed the presence of 10–50 nm silver
particles distributed throughout the coating thickness, with lar-
ger particles observed in the crystalline layer(s) and smaller Ag
particles in the amorphous layer. XRD results showed that the
coatings are primarily HA phase, with small amounts of CaO.
SEM–EDS analysis on the coating top surfaces indicated that
the average Ag wt.% in coatings FGHA-Ag1, FGHA-Ag2 and
FGHA-Ag3 were 2.34, 6.29 and 13.53, respectively, whereas
XPS analysis of the top surfaces of the coatings indicated an
average Ag wt.% of 1.09, 3.16 and 6.59, respectively. Adhesion
strength, evaluated by pull-off tests, showed that the average
bonding strengths were in the range of 83–88 MPa, which is
comparable to 85 MPa for an FGHA coating without incorporated
silver. The optical observation of failure areas indicated that the
dominant failure mode was epoxy failure and no coating delam-
inations were observed. The biological responses of these coat-
ings are being prepared for future publication.
Acknowledgements
The authors would like to acknowledge the financial support
from the National Science Foundation (Grant No. 0600596) and ac-
cess to the research facilities at the Center for Nanophase Materials
Sciences (CNMS) and the Shared Research Equipment (SHaRE) User
Facilities at Oak Ridge National Laboratory, both of which are sup-
ported by the Scientific User Facilities Division, Office of Basic En-
ergy Sciences, the U.S. Department of Energy (Award No.
CNMS2005-070). The authors would also like to acknowledge Mr.
Stefan Sandukas from NCSU for his assistance with SEM–EDS,
and Drs. Harry M. Meyer, Sang Hoon Shim, Adam J. Rondinone
and Andrew Payzant from ORNL for their training and assistance
with XPS, nanoindentation and X-ray diffraction analysis.
Appendix A. Figures with essential color discrimination
Certain figures in this article, particularly Figures 4–10, are dif-
ficult to interpret in black and white. The full color images can be
found in the on-line version, at doi: 10.1016/j.actbio.2009.12.002.
References
[1] Vaudaux PE, Daniel PL, Waldvgel FA. Host factors predisposing to and
influencing therapy of foreign body infections. In: Bisno AL, Waldvogel FA,
editors. Infections associated with indwelling medical devices. Washington,
DC: ASM Press; 1994. p. 1.
[2] Shirkhanzadeh M, Azadegan M, Liu GQ. Bioactive delivery systems for the
slow-release of antibiotics – incorporation of Ag+ ions into micro-porous
hydroxyapatite coatings. Mater Lett 1995;24:7.
[3] Steckelberg JM, Osmon DR. Prothetic joint infections. In: Bisno AL, Waldvogel
FA, editors. Infections associated with indwelling medical
devices. Washington, DC: ASM Press; 1994. p. 259.
[4] Feng QL, Kim TN, Wu J, Park ES, Kim JO, Lim DY, et al. Antibacterial effects of
Ag–HAp thin films on alumina substrates. Thin Solid Films 1998;335:214.
[5] Park SS, Lee HJ, Oh IH, Lee BT. Effects of Ag-doping on microstructure and
mechanical properties of hydroxyapatite films. Key Eng Mat 2005;277–
279:113.
[6] Alt V, Bechert T, Steinrucke P, Wagener M, Seidel P, Dingeldein E, et al. An
in vitro assessment of the antibacterial properties and cytotoxicity of
nanoparticulate silver bone cement. Biomaterials 2004;25:4383.
[7] Rameshbabu N, Kumar TSS, Prabhakar TG, Sastry VS, Murty KVGK, Rao KP.
Antibacterial nanosized silver substituted hydroxyapatite: synthesis and
characterization. J Biomed Mater Res A 2007;80A:581.
[8] Feng QL, Wu J, Chen GQ, Cui FZ, Kim TN, Kim JO. A mechanistic study of the
antibacterial effect of silver ions on Escherichia coli and Staphylococcus aureus. J
Biomed Mater Res 2000;52:662.
[9] Chen W, Liu Y, Courtney HS, Bettenga M, Agrawal CM, Bumgardner JD, et al. In
vitro anti-bacterial and biological properties of magnetron co-sputtered silver-
containing hydroxyapatite coating. Biomaterials 2006;27:5512.
[10] Chen W, Oh S, Ong AP, Oh N, Liu Y, Courtney HS, et al. Antibacterial and
osteogenic properties hydroxyapatite coatings produced using of silver-
containing a sol gel process. J Biomed Mater Res A 2007;82A:899.
[11] Alt V, Bechert T, Steinrucke P, Wagener M, Seidel P, Dingeldein E, et al. In vitro
testing of antimicrobial activity of bone cement. Antimicrob Agents Ch
2004;48:4084.
[12] Ramesh N, Kumar KR, Kumar TSS, Sunder M, Victor SP. Biphasic calcium
phosphates for antibiotic release. Trends Biomater Artif Organs 2005;18:6.
[13] Feng QL, Cui FZ, Kim TN, Kim JW. Ag-substituted hydroxyapatite coatings with
both antimicrobial effects and biocompatibility. J Mater Sci Lett 1999;18:559.
[14] Badrour L, Sadel A, Zahir M, Kimakh L, El Hajbi A. Synthesis and physical and
chemical characterization of Ca10 xAgx(PO4)(6)(OH)(2 x)square(x)
apatites. Ann Chim-Sci Mat 1998;23:61.
[15] Aimanova OJ, LeGeros RZ, Sinyayev VA. Antimicrobiologic property hydrated
amorphous calcium phosphates containing silver. Key Eng Mat 2005;17:439.
[16] Karlov AV, Shakhov VP, Kolobov JR. Definition of silver concentration in
calcium phosphate coatings on titanium implants ensuring balancing of
bactericidity and cytotoxicity. Key Eng Mat 2000;192-1:207.
[17] Oh KS, Kim KJ, Jeong YK, Park EK, Kim SY, Kwon JH, et al. Cytotoxicity and
antimicrobial effect of Ag doped hydroxyapatite. Key Eng Mat 2004;264–
268:2107.
[18] Chung RJ, Hsieh MF, Huang KC, Perng LH, Chou FI, Chin TS. Anti-microbial
hydroxyapatite particles synthesized by a sol–gel route. J Sol-Gel Sci Techn
2005;33:229.
[19] Lee BT, Shin NY, Han JK, Song HY. Microstructures and fracture characteristics
of spark plasma-sintered HAp–5 vol.% Ag composites. Mat Sci Eng A-Struct
2006;429:348.
[20] Montali A. Antibacterial coating systems. Injury 2006;37:81.
[21] Kim TN, Feng QL, Kim JO, Wu J, Wang H, Chen GC, et al. Antimicrobial effects of
metal ions (Ag+, Cu2+, Zn2+) in hydroxyapatite. J Mater Sci-Mater M
1998;9:129.
[22] Choi JW, Cho HM, Kwak EK, Kwon TG, Ryoo HM, Jeong YK, et al. Effect of Ag-
doped hydroxyapatite as a bone filler for inflamed bone defects. Key Eng Mat
2004;254-2:47.
[23] Paul W, Sharma CP. Antibiotic loaded hydroxyapatite osteoconductive implant
material – in vitro release studies. J Mater Sci Lett 1995;14:1792.
[24] Lee IS, Whang CN, Oh KS, Park JC, Lee KY, Lee GH, et al. Formation of silver
incorporated calcium phosphate film for medical applications. Nucl Instrum
Meth B 2006;242:45.
[25] Noda I, Miyaji F, Ando Y, Miyamoto H, Shimazaki T, Yonekura Y, et al.
Development of novel thermal sprayed antibacterial coating and evaluation of
release properties of silver ions. J Biomed Mater Res B: Appl Biomater
2008;89B.
[26] Sanpo N, Tan ML, Cheang P, Khor KA. Antibacterial property of cold-sprayed
HA–Ag/PEEK coating. J Therm Spray Techn 2009;18:10.
[27] Oliver WC, Pharr GM. Measurement of hardness and elastic modulus by
instrumented indentation: advances in understanding and refinements to
methodology. J Mater Res 2004;19:3.
 X. Bai et al. / Acta Biomaterialia 6 (2010) 2264–2273
[28] Cofino B, Fogarassy P, Millet P, Lodini A. Thermal residual stresses near the
interface between plasma-sprayed hydroxyapatite coating and titanium
substrate: finite element analysis and synchrotron radiation measurements.
J Biomed Mater Res A 2004;70A:20.
[29] Blalock T, Bai X, Rabiei A. A study on microstructure and properties of calcium
phosphate coatings processed using ion beam assisted deposition on heated
substrates. Surf Coat Tech 2007;201:5850.
[30] Ding SJ, Ju CP, Lin JHC. Characterization of hydroxyapatite and titanium
coatings sputtered on Ti–6Al–4V substrate. J Biomed Mater Res 1999;44:
266.
[31] Ohring M, NetLibrary Inc.. Materials science of thin films: deposition and
structure. San Diego, CA: Academic Press; 2002.
[32] Hou XH, Choy KL, Leach SE. Processing and in vitro behavior of hydroxyapatite
coatings prepared by electrostatic spray assisted vapor deposition method. J
Biomed Mater Res A 2007;83A:683.
[33] Rehman I, Bonfield W. Characterization of hydroxyapatite and carbonated
apatite by photo acoustic FTIR spectroscopy. J Mater Sci-Mater M
1997;8:1.
[34] Wang CX, Chen ZQ, Wang M, Liu ZY, Wang PL. Ion-beam-sputtering/mixing
deposition of calcium phosphate coatings. I. Effects of ion-mixing beams. J
Biomed Mater Res 2001;55:587.
2273
[35] Blalock TL, Bai X, Narayan R, Rabiei A. Effect of substrate temperature on
mechanical properties of calcium phosphate coatings. J Biomed Mater Res B
2008;85B:60.
[36] Rabiei A, Thomas B, Neville B, Lee JW, Cuomo J. A novel technique for
processing functionally graded HA coatings. Mat Sci Eng C-Bio S 2007;27:523.
[37] Rabiei A, Thomas B, Jin C, Narayan R, Cuomo J, Yang Y, et al. A study on
functionally graded HA coatings processed using ion beam assisted deposition
with in situ heat treatment. Surf Coat Tech 2006;200:6111.
[38] Rabiei A, Blalock B, Thomas B, Cuomo J, Yang Y, Ong J. Microstructure,
mechanical properties, and biological response to functionally graded HA
coatings. Mat Sci Eng C-Bio S 2007;27:529.
[39] Xiao Bai, Stefan Sandukas, Appleford MR, Ong Joo L, Rabiei A. Deposition and
investigation of functionally graded calcium phosphate coatings on titanium.
Acta Biomater 2009;5:3563.
[40] Suvorova EI, Buffat PA. Size effect in X-ray and electron diffraction patterns
from hydroxyapatite particles. Crystallogr Rep 2001;46:722.
[41] Lee WJ, Lee SW, Kim HL, Kim DJ, Han JS. Characteristics of calcium phosphate
films prepared by pulsed laser deposition under various water vapor
pressures. J Korean Phys Soc 2005;47:152.
[42] Zhang X, Gubbels GHM, Terpstra RA, Metselaar R. Toughening of calcium
hydroxyapatite with silver particles. J Mater Sci 1997;32:235.