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Nanoscience and
Nanotechnology Letters
Vol. 5, 162–166, 2013
Study of Bone Morphogenetic Protein-2 Delivery with
Different TiO2 Nanotube Structures
Kun Liang∗ , Xiao Cheng Li, and Beng Kang Tay
School of Electrical and Electronic Engineering, Nanyang Technological University,
Block S1(S1-B3a-01), 50 Nanyang Avenue, Singapore 639798
A release controlled and localized carrier is important for bone morphogenetic protein delivery in
order to enhance the effectiveness in bone repair. In this work, various TiO2 nanotubes with tunable
morphologies are fabricated by electrochemical anodization in different organic electrolytes. Their
effect on delivery of bone morphogenetic protein-2 (BMP-2) was investigated. During the protein
delivery test, it was found that the nanotube structures show superhydrophilic property and tight
interaction with protein, compared with the Ti ﬂat surface. The elution proﬁles of TiO2 nanotube
structures have shown slowed down release rate, suppressed initial burst and reduced total amount
of BMP-2. The protein retention amount is over 90% for all nanotube structures. The release rate
and protein retention are highly related to surface wettability: the more hydrophilic the surface, the
slower the release rate and the higher the protein retention. Furthermore, a larger initial burst could
be caused by a smaller nanotube density in protein release process.
Keywords: TiO2 , Nanotube, Electrochemical, Anodication, BMP-2, Protein Delivery.
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Bone morphogenetic proteins (BMP) have the ability to
interspace and length, could be easily controlled by adjustinduce bone formation and are used widely on bone surging the electrolyte compositions, anodization voltage and
eries such as spinal fusion and implant surface treatment.1
experimental duration.7 8 Until now, nanotubes with diamParticularly, BMP-2 has the ability to direct human meseter ranging from 55 to 300 nm and length up to 260 m
enchymal stem cells to differentiate into mature osteoblasts
can be easily produced.9 10 TiO2 nanotube has proven its
2
and enhance bone formation. After administration, BMPs
advantage on cell adhesion in a variety of biological appliface the presence of ﬂuid, enzymatic activity and other
cations including implant coating with BMP treatment.11 12
3
factors that can affect their bioactivity. All these factors
It was hypothesized that the tube structure provides more
can cause great lose of protein at the injected site and
storage space for protein and might improve protein delivmay introduce denatured bone growth.4 In order to localery with slower release rate. Nanotube structures were
ize BMP and have a proper elution process, a carrier or
found having strong interaction with proteins and released
implant surface modiﬁcation is normally demanded.
less total amount of protein. The study of BMP-2 delivTitanium itself and its alloys have been used as implant
ery with different TiO2 nanotube structures might help on
material in orthopedic and dental implant surgeries. Northe development of a localized and release controlled carmally a thin oxide layer is formed on the surface and it
rier. However, there is a lack of dynamic study on BMP
is actually this thin layer that has direct interaction with
delivery with different TiO2 nanotube structures.
cell environment. The good biocompatibility and mechanIn this study, various TiO2 nanotube structures are
ical properties make Ti/TiO2 excellent implant material.5
fabricated via an electrochemical anodization method in
Nowadays, introducing of nanotechnology is expected to
different types of organic electrolytes. Effects of their
give development of new therapeutic tools. Implant surmorphologies on the BMP delivery are also investigated.
face could be modiﬁed with nanostructured coatings with
BMP-2 is used as the model protein.
different surface roughness and surface energy to inﬂuTiO2 nanotube was prepared by an electrochemical
ence protein loading and release.6 TiO2 nanotube can be
anodization within a two-electrode polytetraﬂuoroethylene
easily fabricated on Ti surface by electrochemical anodiza(PTFE) cell at room temperature. The distance between
tion method. The nanotube parameters, such as diameter,
electrodes is 2 cm. Pure Ti foil (Sigma-Aldrich; 250 m
thick, 99.7% purity) was cut into 2 cm × 2 cm size
∗
Author to whom correspondence should be addressed.
and used as anode. Before usage, it was cleaned by
162
Nanosci. Nanotechnol. Lett. 2013, Vol. 5, No. 2
1941-4900/2013/5/162/005
doi:10.1166/nnl.2013.1492
Liang et al.
Study of Bone Morphogenetic Protein-2 Delivery with Different TiO2 Nanotube Structures
ultrasonication in a mixture of isopropyl alcohol (IPA) and
To compare the protein delivery function between differacetone solution, followed by thoroughly rinsing with DI
ent structures, six samples were tested for BMP-2 delivery
water and dried under N2 gas. A Pt wire (Alfa Aesar,
test: Ti substrate (with nanotubes fully removed), rough
1.0 mm diameter, 5 cm length, 99.95% purity) was used
surface sample (DEG-HF electrolyte under 20 V), EGas cathode. The anodization process was carried under
NH4 F tube (EG-NH4 F electrolyte under 60V), DMSO-HF
DC voltages. Three types of solutions were used as electube (DMSO-HF electrolyte under 60 V), DEG-HF 60 V
trolytes and the total amount was ﬁxed at 30 ml. Ethytube (DEG-HF electrolyte under 60 V) and DEG-HF TiO2
lene glycol (EG, 99%, Sigma Aldrich) with 2% water and
100 V tube (DEG-HF electrolyte under 100 V). The mor0.3% ammonia ﬂuoride (NH4 F, 40%, Sigma Aldrich) was
phologies of TiO2 nanotubes vary with the types of the
used under 60 V for 40 min to fabricated alumina-like
employed electrolytes. Figure 1 shows the SEM images of
nantoube (EG-NH4 F tube). Dimethyl sulfoxide (DMSO,
all samples. The Ti substrate (Fig. 1(a)) is smooth with
99.5%, Sinopharm Chemical Reagent) with 2% hydrogen
nanotubes completely removed. The sample (Fig. 1(b)),
ﬂuoride (HF, 48% aqueous solution, Sinopharm Chemfabricated under 20 V electrochemical treatment in DEGical Reagent) was used under 60 V for 24 h for disHF electrolyte, has a rough surface but no nanotubes covordered nanotube (DMSO-HF tube). Diethylene glycol
ered on its surface. On the other hand, all the nanotube
(DEG, 99%, Sigma Aldrich) with 1% HF was used to prostructures have surface more than rough. TiO2 nanotubes
duce sample with rough surface under 20 V voltage for
fabricated from EG-NH4 F electrolyte (Figs. 1(c), (d)) has a
24 h (rough surface), ordered nanotube sample under 60 V
alumina-like nanotube membrane morphology. This nanosupply for 72 h (DEG-HF 60 V tube). The DEG-HF electube structure is more like a porous ﬁlm with tubes
trolyte was reused with a new Ti substrate under 100 V
for 72 h to fabricate ordered large diameter nanotube fabrication (DEG-HF 100 V tube). After fabrication, the TiO2
samples were rinsed with IPA and DI water for several
times to remove residual chemical and then dried under
N2 gas. For comparison, a ﬂat reference sample (Ti substrate) was made by placing the fabricated TiO2 nanotube
samples into
a high power
ultrasonicTechnology
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remove
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Scanning electron microscope (SEM,
JEOL 5910)
with Scientific Publishers
15 kV accelerating voltage and 15 mm working distance
was used to examine the TiO2 nanotube microstructure.
Mechanically broken nanotube arrays were attached to carbon conductive tapes and observed for cross section and
bottom view. Surface wettability of TiO2 samples was
examined using water on contact angle equipment (OCA
20, Datalhysics) with sessile drop method.
All the samples were cut into the same 1 cm × 1 cm
size, sterilized under UV light in a bio safety cabinet (BSC) for 30 min. They were then put into a six
well culture plate and loaded with 200 ng BMP-2 (RnD
Systems, Minneapolis) which was reconstituted in sterile 4 mM hydrogen chloride solution with 0.1% bovine
serum albumin (BSA). The plate was put under BSC
and dried naturally overnight. After drying, 1 ml of 1×
phosphate buffer solution (PBS) with 0.1% BSA were
put into wells with samples and incubated at 37 C.
The buffer solutions were replaced at time intervals of
1, 2, 4, 8 h and 1, 3, 7 days. The collected solutions
were kept in 1.5 ml vials and stored at −20 C until
next usage. An enzyme-linked immunosorbent assay kit
(ELISA kit, RnD Systems, DBMP 200) was used to determine the protein amount in solution.13 The amount of
protein were read with microplate reader (Tecan Genios)
Fig. 1. SEM images of fabricated samples. Ti substrate (a), rough surat 450 nm. Three readings were made for one solution
face sample (b), top and side view of EG-NH4 F TiO2 nanotube (c), (d),
sample and an average was taken to reduce unnecessary
DMSO-HF TiO2 nanotube (e), (f), DEG-HF TiO2 nanotube fabricated at
60 V (g), (h) and DEG-HF TiO2 nanotube fabricated at 100 V (i), (j).
errors.
Nanosci. Nanotechnol. Lett. 5, 162–166, 2013
163
Study of Bone Morphogenetic Protein-2 Delivery with Different TiO2 Nanotube Structures
Liang et al.
highly bundled together. The diameter is 100–150 nm.
small areas (as marked with red circle). Naturally, the TiO2
Figures 1(e), (f) displays the top view and cross sectional
nanotubes fabricated under 100 V have smaller tube denview of nanotubes fabricated in DMSO-HF electrolyte.
sity compared to that fabricated under 60 V.
TiO2 nanotubes are separated but still bundled together
Surface wettabilities of the samples were evaluated
in small areas. The nanotubes have larger diameter at
using water contact angle. A hydrophilic surface (lower
200–250 nm. In DEG-HF electrolyte, the produced TiO2
contact angle) is normally preferred in biocompatibility.
nanotubes (Figs. 1(g), (h)) are well separated and higher
Figure 3 demonstrates the water contact angles of all samordered. The average tube diameter is 300 nm. By comples. The contact angle of Ti substrate and rough surparing EG-NH4 F tube, DMSO-HF tube and DEG-HF tube,
face samples are both around 60–70 (Figs. 3(a), (b)). The
we can see that the morphologies of TiO2 nanotubes fabEG-NH4 F nanotube has smaller contact around 20–30
ricated under same DC supply have big difference with
(Fig. 3(c)). The contact angle of DMSO-HF nanotube and
composition of employed electrolytes. The electrolyte contwo DEG-HF samples are almost 0 which makes their
ductivity, pH value and other properties maybe the other
surface superhydrophilic. It is quite obvious that contact
factors that affect the tube morphologies, such as tube
angles of nanotube surfaces are lower than that from tube
diameter, tube interspace and growth rate.14
free surfaces, indicating the higher hydrophilic property
After one process at 60 V, the DEG-HF electrolyte
of nanotube structures. The order of surface hydrophilic
was reused with new anode material under 100 V DC
property is as follows: DEG-HF tube ≈ DMSO-HF tube >
supply. TiO2 nanotubes with similar structure but much
EG-NH4 F tube > sample with rough surface ≈ Ti reference
larger diameter were successfully fabricated. From Figsubstrate. The difference in surface wettablility of the samures 1(i), (j), the average diameter is 650 nm which is
ples is contributed to the different microstructure, compomuch larger then that fabricated from 60 V. With same
sition and physical chemistry properties of their surfaces.17
electrolyte, tube diameter is linearly related to anodizaThe Ti substrate and rough sample have smaller roughness
tion voltage within certain voltage limit.9 15 Too low
compared to nanotube structures. The two samples allowed
voltage cannot initiate tube growth and too high voltwater to sit on their surface. On the other hand, the tubular
age make tubes clogged because of fast dissolution rate.
structures let water droplet disperse into the gaps easily by
The upper limit voltage for HF contained electrolyte is
capillary action.18
around 70 Delivered
V and corresponding
TiOTechnology
During
protein
process,
proteinNTU
solutions were
by Publishing
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formed
on Ti surface
up to 350 nm.15 Tubes are hardlyIP:
quickly
dispersed
and penetrated into DMSO-HF nanotube
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for fresh electrolyte under 100 V. However,
withAmerican
reused Scientific
and DEG-HF
nanotube samples. For EG-NH4 F nanotube
electrolyte, large diameter TiO2 nanotubes were successsample, the protein solution gives a small contact angle.
fully formed on Ti surface. The electrolyte composition
Similarly, protein solution sat on Ti substrate and rough
was changed after ﬁrst usage hence established new tube
formation balance. The reused electrolyte can initialize
tube growth faster due to higher conductivity. Meanwhile
tubes produced with re-used electrolyte have thicker walls
and rougher surface.16 However, the higher voltage means
faster etching rate, and consequently, some of the tubes
were etched away. Figure 2 exhibits the over-view SEM
images for TiO2 nanotubes fabricated under 60 and 100 V.
As we can notice, for the sample fabricated under 100 V,
tubes were etches away and substrate was exposed in some
Fig. 2. Overview of TiO2 nanotubes fabricated in DEG-HF electrolyte
under 60 V in fresh electrolyte (a) and under 100 V in reused electrolyte
(b). Red circles show area with tubes etched away.
164
Fig. 3. Contact angle of (a) Ti substrate, (b) rough surface, (c) EGNH4 F TiO2 nanotube, (d) DMSO-HF TiO2 nanotube, (e) DEG-HF TiO2
nanotube fabricated at 60 V and (f) DEG-HF TiO2 nanotube fabricated
at 100 V.
Nanosci. Nanotechnol. Lett. 5, 162–166, 2013
Liang et al.
Study of Bone Morphogenetic Protein-2 Delivery with Different TiO2 Nanotube Structures
surface sample. These phenomenons are well ﬁtted with
the surface hydrophilic property. Naturally, the drying process was faster for nanotube structures than ﬂat samples
(Ti substrate and rough surface sample).
Figure 4(a) displays the cumulative release proﬁle of
BMP-2 on all samples. It can be noticed that both ﬂat
samples eluted almost 100% protein loaded. Furthermore,
more than 95% is eluted in the ﬁrst few hours. Expectedly, nanotube samples only released a few percent of
the total amount; this indicates that majority of protein
loaded were stored in sample. This could be mainly due to
the enlarged storage space and strong interaction between
nanotube and protein. The nanotube samples have a tubular structure which provides much larger surface area and
more storage space for protein. Furthermore, due to their
superhydrophilic property, protein solution can wet entire
surface and sit between or into nanotubes. Proteins could
bind tightly with nanotube through van der Waals and electrostatic forces.19 Flat samples have less surface area hence
less binding sites for protein.
Figure 4(b) indicates the protein release for Ti substrate
and rough surface sample for the ﬁrst few hours. Rough
surface sample has smaller slope which suggests slower
release rate. This could be contributed by the larger surface
roughness. By comparing the release proﬁle of different
TiO2 nanotube structures in Figure 4(c), we can ﬁnd that
the order of released protein amount can be sequenced
as: EG-NH4 F tube > DMSO-HF tube > DEG-HF 60 V
tube. While the order of release rate is opposite. These
sequences are well ﬁtted with the surface hydrophilic property and degree of order for nanotubes. TiO2 sample with
most highly ordered tubes and smallest contact angle has
elution School
proﬁle of
with
the least total
amount
and slowest
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Studies,
NTU
release
rate among
all the samples. This implies that, the
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more hydrophilic
the surface, the tighter the binding with
protein. In Figure 4(c), DEG-HF nanotubes fabricated at
100 V releases the largest amount of protein even though it
has same surface wettability and tube structure with tubes
fabricated at 60 V. This is due to tube density. For the TiO2
nanotube sample fabricated at 100 V, it could not hold
protein tightly because the nanotube structure are etched
away in some area, correspondingly, it eluted more protein
(Fig. 3(b)).
Figure 5 exhibits the release amount of BMP-2 at different time intervals. Flat samples have elution with fast
bust (Fig. 5(a)). Both Ti substrate and rough surface sample have initial burst over 80% in the ﬁrst 10 min. At the
following time intervals, the release protein amount has
a steady decline trend. By comparing the two ﬂat samples only, the rough surface sample has a smaller initial
burst (80%) than Ti substrate (90%). Furthermore, at the
following time intervals, the released protein amounts are
more than that from Ti substrate. This indicates that rough
surface has a better performance on protein delivery with
suppressed burst and sustained elution.
As expected, nanotube samples have relatively constant
amount of protein released over the time with small ﬂuctuation, except for the DEG-HF 100 V nanotube (Fig. 5(b)).
The DEG-HF 100 V sample has a small initial burst of
2.2% followed by comparable protein eluted amount. The
fast initial burst, which is a big obstacle for drug delivery,20
Fig. 4. BMP-2 cumulative release proﬁle of (a) all samples, (b) Ti subis signiﬁcantly suppressed by using nanotube structure.
strate and rough surface sample (zoom in), (c) TiO2 nanotube (zoom in).
Nanosci. Nanotechnol. Lett. 5, 162–166, 2013
165
Study of Bone Morphogenetic Protein-2 Delivery with Different TiO2 Nanotube Structures
Liang et al.
adhesion. With the superhydrophilic TiO2 nanotube structure, the big initial burst from ﬂat surface is signiﬁcantly
suppressed. The nanotube surfaces showed a more sustained release proﬁle but reduced total release amount. The
order of released amount and elution rate are tightly related
to the surface hydrophilic property. The more hydrophilic
the surface, the less released amount and slower elution
rate. Furthermore, density of nanotube has obvious effect
on both initial burst and release amount. Smaller density
shows higher initial burst.
In summary, this study suggests that the TiO2 nanotube structures can be used as a suitable carrier for BMP2 release for enhanced bone repair. Fabrication of TiO2
nanostructure by anodization can be used for implant
surface modiﬁcation. The BMP-2 elution process could
be controlled by TiO2 nanotube structures with different
morphologies. Further studies will be needed to test on
BMP-2’s delivery efﬁciency and bioactivity using nanotube structures.
Fig. 5. Separate release amount of BMP-2 at different time intervals of
(a) Ti substrate and rough surface sample; (b) nanotube samples fabricated in different electrolyte.
References and Notes
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Delivered by Publishing Technology to: S. Rajaratnam School of International Studies, NTU
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Copyright:
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13. Datasheet of Quantikine BMP-2 Immunoassay, RnD Systems,
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Received: 1 December 2011. Accepted: 28 February 2012.
166
Nanosci. Nanotechnol. Lett. 5, 162–166, 2013