Journal of Biomaterials and Nanobiotechnology, 2011, 2, 28-35
doi:10.4236/jbnb.2011.21004 Published Online January 2011 (http://www.SciRP.org/journal/jbnb)
Controlling Bovine Serum Albumin Release from
Biomimetic Calcium Phosphate Coatings
Xiaohua Yu, Mei Wei*
Department of Chemical, Materials & Biomolecular Engineering, University of Connecticut, Storrs, CT, 06269,USA
Received November 30th, 2010; revised December 20th, 2010; accepted December 30th, 2010
ABSTRACT
Biomimetic calcium phosphate (CaP) coating has been used successfully for protein delivery, but the release of protein
from CaP coating is mainly dependent on the limited dissolution of the CaP coating and the passive diffusion of the
protein in the CaP coating. In the present work, our aim is to improve the release behavior of protein from CaP coating
and make it more controllable. By using bovine serum albumin (BSA) as a model protein, our strategy is to tailor BSA
release profiles by controlling the distribution of BSA in CaP coatings. To achieve this aim, BSA was added to a modi-
fied simulated body flu id (m-SBF) at different stages of coating fo rmation to obtain tailored BSA release profiles. Su s-
tained BSA releas e was obtained when BSA was added to m-SBF at the initial stag e of the coatin g where the BSA was
incorporated into the lattice structure of the coating. In contrast, a relatively faster release was observed when BSA
was added during the later stage of coating formation where BSA was mainly adsorbed to the coating surface. As a
result, the BSA release efficiency could be tailored by adding BSA into m-SBF at different coating formation stages.
More importantly, the coatin g composition was not altered with the ch ange of BSA adding times and all the beneficial
properties of the biomimetic coating were reserved. Therefore, the BSA release from CaP coatings can be tailored by
adjusting its distribution in the coating to achieve a more satisfactory release profile.
Keywords: Biomimetic Coating, Apatite, Controlled Release, Release Efficiency
1. Introduction
Calcium phosphate (CaP) coating has been applied widely
onto titanium implants in orthopedic and dental applica-
tions to enhance bone formation around implants and
improve clinical success at an early stage after implanta-
tion [1,2]. Many methods have been used to produce CaP
coatings on titanium substrate, such as plasma splaying,
sputtering deposition, sol-gel coating and biomimetic
coating [3-6]. Among these coating techniques, biomi-
metic coating has attracted more and more attention in
the past two decades [7,8]. Generally, calcium phosphate
is deposited onto a substrate in a simulated body fluid
(SBF) which is maintained at a mild temperature and
physiological pH [9,10]. The main advantage of using
this method is that bioactive agents such as growth fac-
tors, enzymes and DNAs, can be co-deposited with CaP
crystals onto substrates without losing their bioactivity
[11,12]. It has b een reported tha t the biomolecule-containing
CaP coating not only improves bone integration with
implants, but also induces more new bone formation
[13,14].
More recently, researchers have focused on the appli-
cation of biomimetic CaP coating as a protein carrier
system. Although CaP has been used for protein delivery
in the past, most of the therapeutic agents are simply ad-
sorbed onto CaP surfaces [15]. These agents then disso-
ciate from CaP in a “burst release” fashion. The desirable
sustained release is not achieved. In comparison, when
proteins are co-deposited with CaP crystals in the bio-
mimetic coating process, a sustained protein release is
resulted as the coating slowly degrades in vivo [16,17].
However, it was found that only a very small portion of
the protein incorporated into the coating released out due
to the slow degradation rate of most of the CaP coatings
at physiological conditions [18-20]. More importantly,
the release profile with this approach is not controllable
as the release simply relies on the dissolution of the
coating.
In the present work, bovine serum albumin (BSA), a
protein with similar size of growth factors, was used as a
model protein. The distribution of BSA in the biomimetic
CaP coating was carefully tailored by varying the BSA
adding time. The BSA release profile and its release effi-

Controlling Bovine Serum Albumin Release from Biomimetic Calcium Phosphate Coatings 29
ciency at different BSA adding times were investigated
systemically.
2. Materials and Methods
2.1. Materials
Commercially available pure titanium strips (18 mm × 2
mm × 0.5 mm) were used in the cu rr ent study. They wer e
roughened using 800# sandpaper, followed by alkaline
treatment in 5 M NaOH at 60˚C for 24 h. The treated
strips were then rinsed thoroughly with de-ionized water
and dried at room temperature.
Modified SBF was prepared based on the procedures
described by Qu [21], but the ion concentrations were
adjusted according to Table 1. Alexa Fluor 488 conju-
gate BSA (Alexa488-BSA, Invitrogen, USA) was pur-
chased and used as a model protein in this study.
2.2. Pre par ati on of Biom imet ic C oat ings with
Different BSA Adding Times
To avoid protein absorption onto the wall of container,
all the tubes used in this study were pre-coated with 1%
non-fluorescent labeled BSA solution by filling the tub es
with the BSA solution for 60 minutes, and then washed
with PBS for three times. To better tailor the release of
BSA, BSA was added to m-SBF at different time periods
after the coating process started to control the BSA dis-
tribution within the coating. Basically, each of the six-
teen pretreated titanium strips was vertically placed in a
1.5 mL tube containing 1.0 mL m-SBF. All the tubes
were then incubated in a water bath at 42˚C for 24 h.
BSA was added to each tube after the strip was immersed
in m-SBF for 0, 4, 6 and 8 h, respectively. At each inter-
val, four samples were chos en and 26 µL Alexa488-BSA
at a concentration of 2.0 mg/mL was added into each
tube, resulting in a final BSA con centration of 50 µg/mL.
After 24 h incubation in m-SBF, all the strips were taken
out, washed thoroughly with de-ionized water and dried
Table 1. Inorganic composition of human blood plasma and
m-SBF.
Concentration/mM
Ion Blood plasma m-SBF
K+ 5.0 6.0
Na+ 142.0 110.0
Ca2+ 2.5 8.0
Mg2+ 1.5 1.5
Cl- 103.0 110.0
HCO3- 27.0 18
SO42- 0.5 0
HPO42- 1.0 3.0
at room temperature.
2.3. Characterization of Biomimetic Coatings
Prepared with Different BSA Adding Time
The morphology of the coatings prepared at different
BSA adding times was observed using field emission
scanning electron microscopy (FESEM, JEOL 6335F,
Japan). The coatings were also examined using X-ray
diffractometer (Bruker AXS D5005) with a copper targ et.
The voltage and current setup were 40 kV and 40 mA,
respectively. A step size of 0.02˚ and a scan speed of
0.5˚/min were used. Infrared spectra of the coatings were
recorded by Fourier transform infrared (FTIR) spectros-
copy (Nicolet XS60). The coatings were removed gently
from substrate using a knife and ground into powder for
FTIR examination. FTIR spectra were obtained between
wave numbers 4000-400 cm-1 at a resolution of 2 cm-1
and an average scan of 128.
2.4. Incorporation of BSA, Calcium, and
Phosphate into Bbiomimetic Coatings
The BSA concentration in the remained m-SBF was
measured using a microplate reader (Molecular Devices
M2 plate reader, USA) at a fluorescence absorbance
mode with an excitation wavelength of 497 nm and
emission wavelength of 520 nm. The incorporation effi-
ciency (in percentage) of BSA incorporated into the
coating was calculated using the following equation:
initial remained
incorporation initial
(CC )
E (%)100%
C

 (1)
where Eincorporation: BSA incorporation efficiency in the
coating;
Cinitial: Initial BSA concentration in m-SBF (amount
pre-determined);
Cremained: BSA concentration remained in m-SBF after
the coating.
The amount of calcium and phosphate in m-SBF par-
ticipating in the coating formation was also calculated
using a similar equation. The calcium concentration in
the remaining m-SBF was measured using an atomic
absorbance spectro meter (AAS, Perkin Elmer-5000 , USA ),
and the phosphate concentration was determined using a
molybdenum blue chemistry method. Briefly, pure water,
2.5% ammonium molybdate, and 10 wt% ascorbic acid
(v:v:v = 5:1:1) were added in the above order to form a
working solution. The working solution was further
mixed with the testing solution at a volume ratio of
4:1(v/v), and then incubated at 60˚C for 15 min. The
mixed solution was subsequently examined using a mi-
croplate reader (Biotek, MQX200) at a wavelength of
830 nm.

Controlling Bovine Serum Albumin Release from Biomimetic Calcium Phosphate Coatings 30
2.5. BSA Release Behavior in Vitro from
Biomimetic Coatings Prepared with
Different BSA Adding Time
To study the effect of BSA adding time on BSA release
behavior, the biomimetic apatite coated titanium strips
were further employed for the release study. The strips
with different Alexa488-BSA adding times (n = 4) were
placed in a sealed 1.5 mL tube containing 1.0 mL phos-
phate buffered saline ( PBS, pH = 7.4) and kep t at 37˚C in
a dynamic water bath. All soaking media were removed
from the tube and saved for Alexa488-BSA concentra-
tion determination at 1, 2, 4, 8, 12 and 24 h for the first
24 hours. Fresh PBS of 1.0 mL was refilled to the tube to
maintain a constant volu me of the soaking medium. After
that, the soaking medium was assessed every 24 h up to
240 h. The concentration of Alexa488-BSA was meas-
ured as stated in Section 2.4.
2.6. Degradation Study of the Coatings Prepared
with Different BSA Adding Times
After the release study, the strips were collected and
rinsed with de-ionized water thoroughly and dried at
room temperature overnight. The morphology of the re-
maining coatings was observed using a FESEM. To quan-
tify the degradation rate, the coatings after the release
study were dissolved in 1.0 mL HCl (1.0 M) and the su-
pernatants were collected for calcium and phosphate
quantifications. Calcium and phosphate amounts remained
in the coating after the release study were measured using
the same met hod descri bed in Section 2.4.
3. Results
3.1. Characterization of Biomimetic Coatings
Prepared with Different BSA Adding Times
FESEM observation indicates that the surface morphol-
ogy of the coatings changed significantly with different
BSA adding times. Homogenous, porous coatings were
deposited on all titanium strip surfaces regardless of the
BSA adding time. These coatings demonstrated mor-
phology of long sharp interconnected crystal plates cov-
ered by a leaf-like material. The later the BSA was added
to the coating, the more leaf-like material observed on
the surface of the coating (Figure 1). When BSA was
added at the initial stage of the coating (e.g. 0 h adding
time), a porous uniform coating was observed with the
crystal plates slightly bended (Figures 1(a), (b)). No
leaf-like material was observed for this grou p of samples.
The leaf-like material started to appear as BSA was add-
ed 4 h after the coating process (Figures 1(c), (d)). When
BSA was added to m-SBF 6 h after soaking, nearly half
of the coating was covered by the leaf-like material
(Figures 1(e), (f)). Much more leaf-like materials was ob-
Figure 1. SEM micrographs of CaP coatings soaked in m-
SBF containing 50 µg/mL BSA: BSA was added into m-SBF
after the coating process proceeded for 0, 4, 6 and 8 h, re-
spectively. (a) 0 h, × 2000 (b) 0 h, × 10000 (c) 4 h, × 2000 (d)
4 h, × 10000 (e) 6 h, × 2000 (f) 6 h, × 10000 (g) 8 h, × 2000 (h)
8 h, × 10000.
served when BSA was added 8 h after soaking (Figures
1(g), (h)).
Under a higher magnification, it was noted that the
plate-like mineral layer had been formed under the leaf-
like material.
The biomimetic coatings were also characterized by
FTIR spectroscopy and XRD analysis. As shown in Fig-
ure 2(a), the XRD patterns indicated that the biomimetic
coating is a typical apatite phase. The peaks around
31-33˚ is an overlap of three major peaks of apatite: (21 1),
(112) and (3 00). The sharp peak at 26˚ is assigned to the
CaP coating formed with a strong preferential crystallo-
graphic direction of (002). No considerable differences in
those characteristic diffraction peaks were discerned
from the coatings prepared by adding BSA at different
time periods. The FTIR spectra of all CaP coatings with
different BSA adding times show phosphate absorption
bands at 1031, 600, and 561 cm-1 (Figure 2(b)). An am-
ide peak is shown in all four coatings at 1652 cm-1 while
another low amide peak was only detected at 1539 cm-1

Controlling Bovine Serum Albumin Release from Biomimetic Calcium Phosphate Coatings 31
(a)
(b)
Figure 2. Chemical and crystalline structure of CaP coat-
ings soaked in m-SBF containing 50 µg/mL BSA versus
soaking time. (a) XRD patterns of CaP coatings with dif-
ferent BSA adding times: (A) 0 h, (B) 4 h, (C) 6 h, (D) 8 h;
and (b) FTIR spectra of CaP coatings with different BSA
adding times: (A) 0 h, (B) 4 h, (C) 6 h, (D) 8 h.
in the coatings with earlier BSA adding times, such as 0
h and 4 h. The absence of the 1539 cm-1 peak in the
coatings with a BSA adding time of 6 h and 8 h might
due to their lower BSA incorporation efficiencies com-
paring to those with a BSA adding time of 0 h and 4 h.
3.2. The Effect of BSA Adding Time on the
Incorporation Efficiency of BSA, Calcium
and Phosphate
Figure 3 shows the incorporation efficiency of BSA,
calcium and phosphate at different BSA adding times. It
was found that the BSA incorporation efficiency de-
creased dramatically as the BSA adding time was de-
layed. When BSA was added to m-SBF after 4 h of
soaking, its incorporation efficiency was hardly affected.
An incorporation efficiency of approximately 75% was
achieved. However, BSA incorporation efficiency
dropped sharply to 55% when the adding time was de-
layed from 4 h to 6 h of soaking. When BSA was added
at 8 h after soaking, only 26% BSA in the m-SBF was
incorporated into the coating. In contrast, the incorpo-
Figure 3. Incorporation efficiencies of BSA, calcium and
phosphate versus BSA adding times: BSA was added into
m-SBF after 0 h, 4 h, 6 h and 8 h of samples soaked in
m-SBF.
ration of calcium and phosphate in m-SBF was not af-
fected by the change of the BSA adding time. About 40%
of calcium and 70% of phosphate in m-SBF were par-
ticipated the calcium phosphate coating formation re-
gardless of the change of BSA adding time.
3.3. BSA Release Behavior from Biomimetic
Coatings Prepared with Different BSA
Adding Time in Vitro
The BSA release profiles at different BSA adding times
are shown in Figure 4. Although a “bursting release” is
shown at the initial stage, a sustained release was subse-
quently observed, suggesting a typical two-stage release
mechanism when the BSA adding times were 0, 4, and 6
h. However, when BSA was added 8 h after soaking, a
more pronounced “burst release” at the initial stage was
observed, which resulted in the release of more than 80%
of the BSA incorporated into the coating. The total
amount of BSA released from the coatings with BSA
adding times at 0, 4, and 6 h are similar, approximately
7.5 µg, but a dr amatic drop wa s observed for th e coating
with the BSA adding time at 8 h (Figure 4(a)). The per-
centage of the incorporated BSA released with time was
also calculated (Figure 4(b)). When BSA was added to
m-SBF at the very beginning of the soaking, the per-
centage of the BSA released was very low. Only 22% of
the BSA incorporated into the coating was released after
10 days of soaking. In comparison, the percentage of
BSA release was much higher for the coating with BSA
added at 4 and 6 h after soaking, 28% and 32% respec-
tively.
3.4. Degradation Study of the Coatings
Prepared with Different BSA Adding Time
To study the coatings degradation, calcium and phos-
phate amounts of the remaining coating after 10 day of
(a)
(b)

Controlling Bovine Serum Albumin Release from Biomimetic Calcium Phosphate Coatings 32
Figure 4. BSA release profiles from CaP coatings prepared
with different BSA adding time: (a) The amount of BSA
released from CaP coatings. (b) The percentage of BSA
released from CaP coatings. BSA was added to m-SBF after
the coating process proceeded for 0, 4, 6 and 8 h, respec-
tively.
soaking were measured and the results are demonstrated
in Figure 5. It was found that the BSA adding time had a
distinct influence on the coating degradation rate. When
BSA was added at the beginning of the coating (adding
time at 0 h), about 28% calcium and 17% phosphate were
dissolved during the 10-day soaking. The amount of de-
graded calcium increased to approximately 35% and
phosphate to 25% for both the adding time of 4 and 6 h.
It is worth to notice that the coating prepared with BSA
adding time at 8 h had the slowest degradation rate, 20%
for calcium and 14% for phosphate.
The morphology of the coatings changed dramatically
after the 10-day of BSA release study, as shown in Fig-
ure 6. The leaf-like material presented on the surface of
most of the coatings disappeared completely after the 10-
day release study. In turn, porous, homogenous coatings
were observed. It was also discovered that the large
plate-like mineral layer, which was found beneath the
leaf-like material after 24 h of soaking in m-SBF, also
disappeared. Instead, a coating with small pores was ma-
Figure 5. Degradation of CaP coatings after 10 days immer-
sion in PBS. The degradation of both calcium and phos-
phate was calculated based on the amount of calcium and
phosphate in coatings before and after the release study.
BSA was added to m-SBF after the coating process pro-
ceeded for 0, 4, 6 and 8 h, respectively.
Figure 6. SEM micrographs of CaP coatings after 10 d in
vitro BSA release study: BSA was added to m-SBF after the
coating process proceeded for 0, 4, 6 and 8 h, respectively.
(a) 0 h, × 2000 (b) 0 h, × 5000 (c) 4 h, × 2000 (d) 4 h, × 5000
(e) 6 h, × 2000 (f) 6 h, × 5000 (g) 8 h, × 2000 (h) 8 h, × 5000.
(a)
(b)
(a) (b)
(c) (d)
(e) (f)
(g) (h)

Controlling Bovine Serum Albumin Release from Biomimetic Calcium Phosphate Coatings 33
nifested (Figures 6(a)-(f)), which was similar to that of
the coating formed after 6 h soaking in m-SBF (data not
shown). In addition, the morphology of the coating with
BSA adding time at 8 h was slightly different from those
with BSA added at an earlier stage (Figure 6(g), (h)). No
small pores were observed.
4. Discussion
Biomimetic calcium phosphate coatings have been
proved to be a suitable biomolecule carrier [19,22-24].
Different biomolecules, such as growth factors, antibiot-
ics, enzymes and DNAs, have been successfully incor-
porated into CaP coatings in recent years [12,14,25].
However, most of these applications suffer from both
low bimolecular incorporation efficien cy and low release
efficiency. In addition, the release behavior of these
biomolecules from CaP coatings is far from satisfaction
[18,19-20]. In our previous study, we managed to im-
prove the BSA incorporation efficiency up to 90% by
closely controlling the ratio of m-SBF solutio n volume to
sample surface area [26]. In the present study, we tai-
lored the release behavior of BSA by simply delaying the
BSA adding time to the m-SBF. Our results demon-
strated that the carrier system has a high protein incorpo-
ration rate, improved release efficiency and sustained
release profile when the BSA adding time is 6 h.
To control the release profile of BSA from CaP coat-
ing, it is crucial to develop a better understanding of the
mechanism of BSA incorporation into the CaP coating.
Our results (data not shown) indicated that most of the
BSA incorporated in to the CaP coating in the in itial 4-12
h soaking in m-SBF. Moreover, the BSA incorporation
profile perfectly matched with the incorporation profiles
of calcium and phosphate during the initial 4-12 h. As a
result, it is believed that BSA is attached to the CaP and
co-deposited onto the strip. When it is added to the
m-SBF at the beginning of the coating, BSA is homoge-
nously distributed within the entire depth of the coating,
as illustrated in Figure 7(a). However, when BSA is
added after a certain time period after the coating starts,
it only presents in the top section of the coating (Figure
7(b)). Such incorporated BSA would be easier to release
compared to those embedded in the bottom coating adja-
cent to the substrate. To test this hypothesis, we have
managed to retain all the BSA to the top section of the
coating by delaying the BSA adding time. One advantage
of this method is that we can easily control the BSA dis-
tribution within the coating, which in turn controls the
protein release from the coatings. Another advantage of
the method is that the amount of BSA released from the
coating is substantially increased as less BSA is trapped
in the coating co mparing to the conventional approach.
By adjusting the BSA adding time, variable release
curves with different release efficiency was attained,
which indicated that varying BSA adding time can be an
efficient approach to control the release of biomolecules
from the biomimetic CaP coating (Figure 4). It was also
found that the BSA release amount was inversely propor-
tional to BSA adding time, but the release efficiency was
proportional to BSA adding time (Figure 4(b)). This
might be explained as follows: Since BSA is co-precipi-
tated with CaP coating on the substrate, it is distributed
evenly through the entire depth of the coating when it is
added at 0 h (Figure 7). However, the bottom section of
the coating hardly degrades, which results in a relatively
large amount of protein is still trapped in the coating after
the 10-day release test [27,28]. As a result, low release
efficiency was attained for the coating with an adding
time of 0 h. When the adding time is increased, the BSA
is only distributed in the top section of the coating which
makes it easier for BSA to release from the coating via
degradation. In the case of adding time at 8 h, the major-
ity of BSA was simply adsorbed onto the surface of the
coating, so an stronger initial bursting release was ob-
served due to the desorption of BSA from the coating.
The results of coating degradation study are also consis-
tent with this explanation. It is known that biomimetic
CaP coating can be gradually dissolved under physio-
logical conditions [29]. The dissolution rate of CaP is
affected by its crystallinity, composition, and porosity
[27]. It was observed that only the top layer of the coat-
ings was dissolved after 10 d release study, which sug-
gests only the BSA in this part of the coating be released.
It is worth to note that high release efficiency is achieved,
Figure 7. Schematic illustration of BSA distribution in CaP coatings (a) BSA is added to m-SBF from the beginning of the
coating process. (b)BSA is added to m-SBF after the coating process proceeded for certain time period.
(a) (b)

Controlling Bovine Serum Albumin Release from Biomimetic Calcium Phosphate Coatings 34
but it is accompanied with lower protein incorporation
efficiency. According to the release data, adding BSA to
m-SBF at 6 h after soaking is an optimal time to achieve
both high incorporation rate and release efficiency.
BSA adding time also significantly affects BSA in-
corporation efficiency into/onto the biomimetic coating.
It was observed that the BSA incorporation efficiency
decreased nearly linearly as a function of BSA adding
time (Figure 3). It is known that the BSA incorporation
efficiency is closely associated with calcium and phos-
phate precipitation rate. Since the incorporation effi-
ciency of calcium and phosphate remains around 40%
and 70% respectively regardless of the BSA adding time,
the BSA incorporation efficiency is mainly determined
by the time when BSA is added to the system. The later
the BSA is added, the less the Ca2+ ions are available to
bind to BSA and co-precipitate onto the substrate surface,
and the lower the BSA incorporation efficiency.
Especially, in the case of the adding time at 8 h, the
BSA incorporation relied mainly on pseudo-Langmuir
adsorption as the majority of the coating had been
formed [30]. In contrast to BSA incorporation efficiency,
the incorporation of calcium and phosphate was not af-
fected by BSA adding time (Figure 3). Besides, the XRD
and FTIR results also suggest that there is no significant
change in the coating composition by varying the BSA
adding time. Thus, changing BSA adding time during
coating formation not only provides better control over
release profiles, but also reserves all the properties of the
biomimetic CaP coating, such as coating thickness, com-
position, and crystallinity.
5. Conclusion
In this study, we have successfully optimized the release
profile of BSA from CaP coating by adjusting its adding
time. At a BSA adding time of 6 h, its release efficiency
was improved to 26% while its incorporation efficiency
was maintained as high as 55%. In this study, BSA was
employed as a model protein. The method established
here, however, can be applied for loading other drugs or
proteins using biomimetic CaP coatings as a carrier.
6. Acknowledgements
The authors would like to thank the supports from Na-
tional Science Foundation (DMI 0500269 and BES 0503-
315) and Connecticut Innovations under the Yankee In-
genuity Technology Competition. The authors would
also like to thank Prof. Yong Wang in the Department of
Chemical, Material and Biomolecular Engineering, Uni-
versity of Connecticut for helpful discussion.
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