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A physical vapor deposition method for controlled
evaluation of biological response to biomaterial
chemistry and topography
S.A. Hacking,
1,2,3
M. Zuraw,
4
E.J. Harvey,
1,2,5
M. Tanzer,
1,2,5
J.J. Krygier,
1
J.D. Bobyn
1,2,3,5
1
Jo Miller Orthopaedic Research Laboratory, McGill University, Montreal, Canada
2
Division of Orthopaedics, McGill University, Montreal, Canada
3
Department of Biomedical Engineering, McGill University, Montreal, Canada
4
Fused Metals Inc., Georgetown, Canada
5
Department of Surgery, McGill University, Montreal, Canada
Received 13 October 2005; revised 1 September 2006; accepted 28 September 2006
Published online 31 January 2007 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.31131
Abstract: The purpose of this study was to characterize a
technique to effectively mask surface chemistry without
modifying surface topography. A thin layer of titanium was
deposited by physical vapor deposition (PVD) onto differ-
ent biomaterial surfaces. Commercially pure titanium disks
were equally divided into three groups. Disks were either
polished to a mirror finish, grit blasted with alumina par-
ticles, or grit blasted and subsequently plasma sprayed with
a commercial grade of hydroxyapatite (HA). A subgroup of
each of these treatment types was further treated by mask-
ing the entire disk surface with a thin layer of commercially
pure titanium deposited by PVD. A comparison of surface
topography and chemical composition was carried out
between disks within each treatment group. Canine marrow
cells were seeded on all disk surfaces to determine the sta-
bility of the PVD Ti mask under culture conditions. The
PVD process did not significantly alter the surface topogra-
phy of any samples. The thin titanium layer completely
masked the underlying chemistry of the plasma sprayed
HA surface and the chemistry of the plasma vapor depos-
ited titanium layer did not differ from that of the commer-
cially pure titanium disks. Aliquots obtained from the
media during culture did not indicate any significant differ-
ences in Ti concentration amongst the Ti and Ti-masked
surfaces. The PVD application of a Ti layer on HA coatings
formed a stable, durable, and homogenous layer that effec-
tively masked the underlying surface chemistry without
altering the surface topography. Ó2007 Wiley Periodicals,
Inc. J Biomed Mater Res 82A: 179–187, 2007
Key words: hydroxyapatite; surface chemistry; physical
vapor deposition; biomaterial; surface topography
INTRODUCTION
Biomaterial evaluation is becoming increasingly more
complex as new manufacturing techniques, materials,
coatings, methods of analysis, and devices are devel-
oped. The long-term success of any implantable device
depends in part upon its interaction with the host tissue.
In many cases, a better understanding of the tissue–
implant interaction is fundamental to improving implant
function or performance. As a result, the relationship
between the implant substrate and the peri-implant
environment is a subject of great interest.
In such investigations, it is important to recognize
that subtle changes in biomaterial surface morphol-
ogy can have profound effects on peri-implant tissue
formation
1–11
or cellular
7,12–17
response. These responses
to surface topography, or more precisely, surface
morphology, need to be dissociated from other factors
such as local surface chemistry in order to determine
the exact causes for tissue response. As a result, ex-
perimental controls must not just approximate sur-
face morphology, but match it exactly, since subtle
changes in surface morphology have the potential to
confound experimental findings.
However, it may not be practical or even possible
to produce an exact morphological control from a dif-
ferent biomaterial. This approach can be particularly
problematic in cases where the surface morphology is
complex
5,18–20
or is a consequence of the manufactur-
ing process.
5,18–22
One solution is to produce a control
by coating the surface of interest with a thin film that
is dense, durable, homogenous, and does not alter the
surface morphology. To be truly useful, the film must
also be practical to apply and the ideal result of the
application of the thin film would be a change only in
the surface chemistry of the coated component.
Correspondence to: S.A. Hacking; e-mail: ahacking@yahoo.
com
'2007 Wiley Periodicals, Inc.
There are a variety of thin film deposition tech-
niques collectively referred to as physical vapor depo-
sition (PVD): DC and RF magnetron sputtering,
23–25
electron beam (E-Beam) evaporation,
26–28
cathodic arc
and thermal evaporation. Medical applications of PVD
coatings include wear resistant coatings
29–31
or bio-
compatible coatings
29,32–35
for medical devices. While
other coating methods exist, PVD has the advantage
of being a viable process at lower temperatures in a
relatively inert environment.
36
In the PVD process, an
ionized gas (e.g., Ar
þ
) strikes a target (cathode) and
releases the source material which travels across a
vacuum and condenses on the surface to be coated. E-
Beam coatings are typically produced at about
5008C.
36
Cathodic arc coatings are typically deposited
at temperatures much lower than 4008C, and in some
cases near room temperature.
36,37
The hypothesis of this study was that a thin, com-
mercially pure titanium film applied by PVD could
effectively mask the surface chemistry of a textured
biomaterial without substantially altering its surface
topography.
MATERIALS AND METHODS
PVD process (PVD-mask)
A modified PVD process was developed to coat (mask)
a variety of specimens described later with commercially
pure titanium. All samples were placed directly into the
PVD chamber. The base pressure before processing was
not greater than 6 10
2
Pa. Radiant heaters maintained a
temperature of 1408C, as measured in the center of the ves-
sel. The chamber was backfilled with prepurified argon
(99.99% pure) to a pressure of 2.0 Pa. Titanium was evapo-
rated from a commercially pure source (99.99%) with an
arc current of 125 A. A bias voltage of 250 V pulsing at a
frequency of 20 kHz was maintained on the specimens.
Initial experiments with this technique had shown that
small particles of Ti condensed on the specimen surface
[Fig. 1(A)]. To reduce this contamination, a shield with a
floating bias with respect to ground was placed 10 cm in
front of the titanium source [Fig. 1(B)].
Test specimens
One hundred and twenty cell culture disks, 22 mm in
diameter and 3 mm thick, were divided equally into three
groups. One group was polished (Pol), one group was grit
blasted (GB) with Al
2
O
3
particles, and another group was
grit blasted then plasma sprayed with a layer of hydroxy-
apatite (HA) using industry standard techniques (Implex
Corp, Allendale, NJ) (Fig. 2). The plasma spray process
resulted in a 60-mm thick HA coating that was 98% HA
and 64% crystalline with a density of 99% and a calcium:
phosphate ratio of 1.67. Twenty of the Pol, GB, and HA
disks were left untreated and 20 of each group were PVD-
masked, as described earlier. Representative disks from
each group were randomly selected for surface characteri-
zation.
Characterization of the disk surfaces
Implant topography
Scanning electron micrographs of all disk surfaces were
obtained to provide a qualitative impression of surface
morphology. Surface topography was quantified using a
Wyko NT 2000 (Veeco, Rochester, NY) noncontact optical
profiler. The profiler was calibrated before use and the
operational parameters were: VSI mode, 52Mag, VSI fil-
ter, and tilt correction. Three random regions from three
disks of each group were analyzed yielding nine measure-
ments per surface.
Surface chemistry
Surface chemical analysis was performed using X-ray
photoelectron spectroscopy (XPS). All measurements were
carried out using a dual-anode source in a VG Escalab
MKII instrument (Thermo VG Scientific, Beverly, MA) with
nonmonochromatized Mg Karadiation (hn¼1253.6 eV)
operated at 20 mA and 15 kV. Survey spectra were
Figure 1. (A) First generation PVD Ti surface showing
particle contamination and (B) second generation PVD Ti
surface with a gross reduction in particle contamination.
180 HACKING ET AL.
Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a
obtained at 908from the sample surface using a pass
energy of 100 eV, 1.0 eV steps, and a 15 mm 6 mm slit-
width, which result in an analyzed surface area of 3 mm
2 mm. When present, specimen-charging effects were com-
pensated by adjusting the binding energy of the survey
spectra to fix the binding energy of the hydrocarbon peak
at 285.0 eV. The concentration of each element was deter-
mined from the XPS signal area and the corresponding
XPS atomic sensitivity factor relative to Fluorine 1s elec-
tron. The sensitivity of the technique was 0.1 at 100%.
With this technique, measurements below 0.2% are consid-
ered contaminant levels. Three cell culture disks from each
group were selected for analysis. Five locations were ran-
domly analyzed on each disk.
XPS was selected for the chemical analysis of the disk
surfaces since it has an effective penetration depth of
25 nm. This was especially necessary for the PVD-mask
specimens to limit analysis to the thin PVD Ti film without
influence from the substrate below.
The Pol and GB disks were scanned with XPS and com-
pared with the PVD-mask, Pol, and GB disks. Scans of the
PVD-mask HA-coated disks were also included to deter-
mine if the underlying HA Ca and P chemistry could be
detected. Three disks were randomly selected from each
group and scanned five times each.
Stability of the PVD coating in an in vitro
cell culture model
An in vitro cell culture model was utilized to determine
the effect of tissue growth and culture media on the control
and PVD-mask surfaces (n¼4). Canine marrow cells
obtained from the iliac crest were harvested in a sterile envi-
ronment, maintained in a-MEM medium supplemented
with 10% FBS, 100 U/mL penicillin, and 100 mg/mL strepto-
mycinandgrowninprimarycultureforaperiodof7days.
Cells were seeded either on disks or on tissue culture plastic
(TCP) at a plating density of 3 10
5
cells/cm
2
(100,000 cells
per disk) in 12-well plates and maintained in 2.0 mL stand-
ard medium (a-MEM containing 10% FBS, 100 U/mL peni-
cillin, and 100 mg/mL streptomycin and 50 mg/mL ascorbate
and 5 mMb-glycerophosphate) in a 5% CO
2
air-balanced in-
cubator at 378Cuptoday12.
Media was changed every 2 days and aspirates were
obtained prior to media change at days 0, 2, 5, 7, 10, and
12 days. The resistance to dissolution of the PVD-mask
film used in this experiment was determined by analysis
of the Ti concentration in aliquots of media from cell cul-
ture. Aliquots from the HA-coated and PVD-mask HA-
coated disks were also analyzed for Ca concentration. Ali-
quots were analyzed by a sequential inductively coupled
plasma spectrometer (Trace Scan, Jarrell-Ash Corp, Frank-
lin, MA). Certified commercial standards (1000 ppm) were
used and subsequently diluted with deionized water.
Standard concentrations bracketed the test samples. Two-
level standardization was used and titanium compounds
were used in the standard preparation. The detection lim-
its for titanium and calcium by this system were 5 ppb
and 5 ppm, respectively.
Assessment of cell proliferation
A parallel set of disks (n¼4 at each time period) was
harvested at days 2, 5, 7, and 12 to demonstrate cell
Figure 2. Cell culture disks used in the study. (A) Polished Ti, (B) polished Ti þPVD mask, (C) grit blasted Ti, (D) grit
blasted Ti þPVD mask, (E) HA coated, and (F) HA coated þPVD mask. Small slot in disk facilitates media change with-
out disturbing culture surface.
PVD METHOD FOR MASKING SURFACE CHEMISTRY 181
Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a
growth and proliferation. Cell proliferation was deter-
mined by quantification of total DNA according to the
method of Labarca and Paigen.
38
Briefly, culture media
was aspirated from the wells and disks were washed three
times in PBS, following which cells were harvested in a so-
lution of 2MPBS. In cases where a cell layer existed it was
removed in its entirety, otherwise cells were removed by a
combination of cell scraping and vigorous pipetting. Previ-
ous experiments determined that this method provided
more consistent results than using trypsin or collagenase.
Cells were kept on ice and lysed by sonification (2 30 s).
DNA content was determined by Hoescht dye.
39
The sample solutions were diluted as follows: 1 day –
no dilution, 3 days – 5 times dilution, 6 days – 10 times
dilution, 12 days – 20 times dilution. Each sample received
35 mL of Hoechst dye and if the sample was diluted the
balance to 965 mL was made up with 2MPBS.
Where appropriate data were analyzed with the Stu-
dent’s ttest or with ANOVA and various post hoc tests. All
analysis was performed at the 95% confidence level.
RESULTS
Surface topography
Qualitative SEM images of the surfaces are pre-
sented in Figure 3. Three dimensional scans of the
test surfaces are presented in Figure 4. There was no
significant difference in surface topography between
the Pol (R
a
¼0.01 60.02 mm) and Pol PVD-mask
(R
a
¼0.01 60.04 mm) disks, between the GB (R
a
¼
3.26 60.95 mm) and GB PVD-mask (R
a
¼3.32 61.38
mm) disks or between the HA (R
a
¼2.98 60.81 mm)
and HA PVD-mask (R
a
¼3.01 60.37 mm) disks (Table I).
The preservation of surface topography is further
illustrated in Figure 1(B), where one half of a polished
disk was PVD-masked. The continuity of small pol-
ishing artifacts is visible on both the polished and pol-
ished PVD-mask regions.
Surface chemistry
Chemical analyses of the specimen surfaces are pre-
sented in Figure 5. The chemical profiles for the Pol,
Pol PVD-mask, GB, GB PVD-mask, and HA PVD-
mask surfaces were nearly identical. This indicated
that on all test surfaces the PVD deposited Ti film
possessed a surface chemistry that was the same as
the Ti substrate. As anticipated, no Ca or P was
detected on the Pol, Pol PVD-mask, GB, or GB PVD-
mask disks. Ca and P were detected in a ratio of *2:1
on the HA-coated disks. Importantly, there was no Ca
or P detected in any of the 15 scans of the PVD-mask
HA disks, indicating that the applied Ti film had pro-
duced a homogeneous mask that effectively occluded
the chemistry of the underlying HA surface. This fur-
ther indicated that the thickness of the PVD Ti coating
was at least 25 nm, the maximum penetration depth
of XPS.
In vitro stability of the PVD titanium mask
Cell growth and proliferation occurred on all disks
except the HA-coated disks (Fig. 6). There was no sig-
nificant difference in the rate of cell growth between
Figure 3. Scanning electron micrographs of cell culture disks with (A) polished Ti, (B) polished Ti þPVD Ti mask, (C)
grit blasted Ti, (D) grit blasted Ti þPVD Ti mask, (E) HA coated, (F) HA coated þPVD Ti mask surface. SEM, 1000.
182 HACKING ET AL.
Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a
Pol Ti and Pol Ti þPVD mask and GB Ti and GB
Ti þPVD mask surfaces.
There was a slight increase in Ti levels (40–50 ppb)
in the culture media of each sample (Table II). These
Ti levels were extremely low and near the detection
limits of the instrument involved (5 ppb). A paired
Student’s ttest showed that there was no significant
difference in Ti levels detected between Pol and Pol
PVD-mask, GB and GB PVD-mask, and HA PVD-
mask disks. The rougher disks (GB and GB PVD-
mask, HA PVD-mask disks) had a slightly higher
level of Ti in the media when compared with the
smooth disks (Pol and Pol PVD-mask), however, this
difference was not significant.
There was a significant difference in Ca level from
aspirates of the HA and HA PVD-mask disks (Ta-
ble III). Calcium aspirate concentration for the HA-
coated disks was maximal at day 2 at 2452 642 ppm
and gradually diminished to 714 636 ppm at day 12.
In contrast, the calcium aspirate concentration for the
HA PVD-mask disks was 62.5 62.1 ppm at day 2
and 58.4 61.9 ppm at day 12. This compared well
with the blank value of 69.8 60.71 ppm.
DISCUSSION
Subtle changes in surface morphology have signifi-
cant and predictable effects on cellular behavior and
peri-implant tissue formation. In the context of bio-
medical investigations, it is imperative that surface
morphology be strictly controlled as an experimental
variable to avoid confounding experimental results.
In this study, a 100-nm Ti film applied by PVD effec-
tively masked the surface chemistry of an HA-coated
implant without altering its surface topography.
Other researchers have used similar techniques
such as RF sputtering to successfully produce thin Ti
films for biomedical evaluation.
40–46
Thin TiO, TiN,
and C films have also been successfully applied to
mask the underlying chemistry to prevent corrosion
or ion release from CoCr alloy surfaces.
27,47–49
How-
ever, as far as the authors are aware, this is the first
study to use a thin Ti film to specifically produce a
morphological control with a different surface chem-
istry.
A comparison of the surface topographies indicates
that the application of the PVD coating did not signif-
icantly alter the surface roughness of any of the cul-
ture surfaces. Chemical analysis of the Pol and GB
disks with and without the PVD mask demonstrated
that the application of the PVD Ti mask did not pro-
duce a surface coating that was significantly different
from that of the uncoated Ti specimens. There were
trace levels of Al present in the POL samples, which
Figure 4. Non contact optical profile of cell culture disks with (A) polished Ti, (B) polished Ti þPVD Ti mask, (C) grit
blasted Ti, (D) grit blasted Ti þPVD Ti mask, (E) HA coated, (F) HA coated þPVD Ti mask surfaces. 102vertical reso-
lution not to scale amongst images.
TABLE I
Surface Roughness of Cell Culture Surfaces (n=9)
Surface
Roughness Parameter (mm)
R
a
R
q
TCP <0.01 <0.01
Pol Ti Disk 0.01 60.02 0.02 60.02
Pol Ti Disk þPVD Ti Mask 0.01 60.03 0.02 60.03
GB Ti Disk 3.26 60.95 4.04 61.04
GB Ti Disk þPVD Ti Mask 3.32 61.38 4.01 61.53
HA-coated Disk 2.98 60.81 3.72 61.03
HA-coated Disk þPVD Ti Mask 3.01 60.37 3.87 60.41
PVD METHOD FOR MASKING SURFACE CHEMISTRY 183
Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a
were most likely a residue from the Al
2
O
3
polishing
process. Analysis of the HA and PVD-mask HA sam-
ples demonstrated that the PVD Ti mask produced a
uniform and dense barrier that effectively masked the
underlying surface chemistry. Ca and P were detected
in a *2:1 ratio in the HA-coated disk, but were not
detected on the PVD-mask HA-coated disks.
For all samples where cell growth occurred, there
was slight elevation of Ti in the media. The Ti levels
presented here are higher than those of the PVD-
mask HA in the previous soak study (40 ppb vs.
5 ppb), but still in the order of parts per billion. It is
interesting to note that the rougher surfaces (GB Ti,
PVD GB Ti, and PVD HA) had a slightly elevated
level of Ti in the media, compared with the Pol surfa-
ces; this perhaps was due to their greater surface
area. At some time periods, aspirations from the HA-
coated disks indicated trace amounts of Ti, near the
detection limits of the instrument. This Ti artifact may
be a result of the media being exposed to Ti from the
non-HA-coated sides and bottom of the disks (only
the top of the disk was coated with HA).
Figure 5. Chemical analysis of the culture disk surfaces used in this study. There is no Ca or P present in any of the
PVD Ti mask HA samples. In addition, the chemical profile of the Ti and PVD mask Ti samples are not significantly dif-
ferent.
Figure 6. Growth of canine marrow culture on Pol Ti, PVD-masked Pol Ti, GB Ti, PVD-masked GB Ti, HA, PVD-masked
HA, and TCP surfaces. Cells proliferated on all surfaces except the HA surface.
184 HACKING ET AL.
Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a
A previous soak study also confirmed the stability
of the PVD coating for long periods of time in a modi-
fied saline environment.
50
The aim of the present
study was to test the stability of the PVD coating
under tissue culture conditions. With the exception
the HA-coated disks, all samples exhibited cell
growth and proliferation. In the current study, media
aspirated from the HA-coated disks presented signifi-
cantly increased Ca levels. To determine if cellular ac-
tivity was affecting the Ca levels, a subsequent test
was conducted by immersing the same HA-coated
disks in the same media and incubating for 12 days
without cells. In this soak test, elevated levels of Ca
were observed that were not significantly different to
those reported in this study. This finding suggests
that there may be an interaction between the HA and
culture media. While HA is generally not soluble, the
HA coating used in this study was amorphous (36%
noncrystalline, amorphous HA).
The elevated levels of Ca are believed to arise from
a dissolution of the HA coating. Since the aim of this
study was to determine if the PVD Ti mask effectively
concealed the HA chemistry under culture conditions,
the dramatically elevated Ca levels from aspirates of
the HA-coated surfaces are a fortunate finding. The
levels of Ca in the PVD Ti-masked samples were
lower than the measured media Ca concentration at
day 0 (aspirates were obtained after 2 days of cul-
ture). If the HA coating from the PVD Ti mask HA
disks had been exposed to the media, a much higher
concentration of Ca would have been expected, indi-
cating once again that the PVD-mask effectively con-
cealed the underlying HA surface chemistry.
It is important to note that Ti was selected as the
coating material for this study because of its resist-
ance to degradation in a physiologic environ-
ment.
40,51,52
Other investigators have coated implant
surfaces with thin films of CaP by sputtering techni-
ques.
53–57
However, in this context, the degradation
of these thin CaP coatings with subsequent change in
morphology upon exposure to physiologic fluids or
media mitigates their utility as an effective coating for
long term morphological controls. A plasma spray
HA surface was selected as a substrate material since
it was chemically dissimilar to the commercially pure
Ti mask, but like Ti it is commonly associated with
orthopaedic implants. The plasma spray HA surface
also presented a complex geometry that tested the ef-
ficacy of the PVD Ti to mask irregular biomaterial
substrates.
For nonporous metallic materials such as Ti in non-
abrasive conditions like these, only the first 10 nm of
the substrate surface actually participates in any
chemical interaction with the surrounding environ-
ment.
52
Since the PVD Ti coating used in this study
was 100 nm thick, it is reasonable to assume that the
tissue reaction to the PVD mask Ti-coated implant
was no different from that of the same implant made
entirely of Ti.
In conclusion, the PVD Ti coating effectively
masked the underlying surface chemistry of the HA-
coated disk without altering its surface topography.
In addition, the PVD process produced a thin, dense,
and homogenous Ti film possessing the same surface
chemistry as commercially pure Ti. The PVD of thin
Ti films has utility for the investigation of the effects
TABLE II
Dissolved Titanium in Media Aspirates
Disk Surface
Dissolved Titanium (ppb)
0
a
2 5 7 10 12
Pol Ti 0 38.2 65.6 41.0 65.8 37.3 64.6 39.0 62.0 40.1 63.8
Pol Ti þPVD 0 39.8 67.5 43.8 67.3 41.8 62.8 40.3 63.5 40.3 62.8
GB Ti 0 44.9 63.7 46.9 63.8 47.7 61.9 49.3 62.2 48.1 63.5
GB Ti þPVD 0 46.3 64.4 51.2 65.6 50.5 62.6 48.9 64.6 50.6 62.5
HA 0 2.5 62.9 1.3 62.5 0.0 60 1.8 63.5 1.5 63
HA þPVD 0 45.5 64.4 50.8 61.9 47.1 63.6 46.8 64.9 49.9 64.8
TCP 000000
a
0, 2, 5, 7, 10, and 12 are time periods (days).
TABLE III
Dissolved Calcium in Media Aspirates
Disk Surface
Dissolved Calcium (ppm)
0
a
2571012
HA þPVD 69.8 60.71 62.5 62.1 59.8 61.8 58.1 62.0 58.5 61.8 58.4 61.9
HA 70.2 60.66 2452 642 1913 645 1030 638 844 641 714 636
a
0, 2, 5, 7, 10, and 12 are time periods (days).
PVD METHOD FOR MASKING SURFACE CHEMISTRY 185
Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a
of surface topography and surface chemistry on the cel-
lular and/or tissue response to various biomaterials.
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