Enhanced osteogenic differentiation of MC3T3-E1 on rhBMP-2-immobilized titanium via click reaction

Article abstract of DOI:10.1016/j.carbpol.2013.12.023

In the present study, we report about the efficacy of titanium surface-immobilized with bone morphogenetic protein-2 (BMP-2) via click reaction on enhanced osteogenic differentiation of MC3T3-E1 cells. The surface was characterized by static contact angles and XPS measurements, which indicated that pristine titanium (Ti-1) was successfully surface-modified via click chemistry (aminated titanium, Ti-4). By quantitative analysis of heparin immobilized on aminated titanium (Ti-4), we found that the Ti-4 can be used as a good candidate to immobilize biomolecules such as heparin. BMP-2 from titanium immobilized with BMP-2 (Ti-6) was released for a period of 28 days in a sustained manner. The highest proliferation rate of MC3T3-E1 cells was observed on Ti-6. Through in vitro tests including alkaline phosphatase (ALP) activity, calcium deposition and real-time polymerase chain reaction (real-time PCR), we found that Ti-6 can be used as a good implant to enhance the osteogenic differentiation of MC3T3-E1 cells.

Full text of DOI:10.1016/j.carbpol.2013.12.023

Carbohydrate Polymers 103 (2014) 170–178
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Enhanced osteogenic differentiation of MC3T3-E1 on
rhBMP-2-immobilized titanium via click reaction
Eun-Cheol Kima, Tae-Hee Kimb, Jae-Hoon Jungb, Sung Ok Hongb, Deok-Won Leea,b,∗
a Department of Maxillofacial Tissue Regeneration and Research Center for Tooth & Periodontal Regeneration (MRC), School of Dentistry, Kyung Hee
University, 1 Heogi-dong, Dongdaemun-gu, Seoul 130-701, Republic of Korea
b Department of Oral and Maxillofacial Surgery, Kyung Hee University Dental Hospital at Gangdong, Kyung Hee University, Seoul 134-727, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 14 August 2013
Received in revised form 4 November 2013
Accepted 9 December 2013
Available online 16 December 2013
In the present study, we report about the efficacy of titanium surface-immobilized with bone morpho-
genetic protein-2 (BMP-2) via click reaction on enhanced osteogenic differentiation of MC3T3-E1 cells.
The surface was characterized by static contact angles and XPS measurements, which indicated that pris-
tine titanium (Ti-1) was successfully surface-modified via click chemistry (aminated titanium, Ti-4). By
quantitative analysis of heparin immobilized on aminated titanium (Ti-4), we found that the Ti-4 can be
used as a good candidate to immobilize biomolecules such as heparin. BMP-2 from titanium immobilized
with BMP-2 (Ti-6) was released for a period of 28 days in a sustained manner. The highest proliferation
rate of MC3T3-E1 cells was observed on Ti-6. Through in vitro tests including alkaline phosphatase (ALP)
activity, calcium deposition and real-time polymerase chain reaction (real-time PCR), we found that Ti-6
can be used as a good implant to enhance the osteogenic differentiation of MC3T3-E1 cells.
© 2013 Elsevier Ltd. All rights reserved.
Keywords:
Titanium surface
BMP-2
MC3T3-E1 cell
Osteogenic differentiation
1. Introduction
Kapoor, Bhaumik, Inagaki, Kuraoka, & Yazawa, 2002; Lee, Kim, Kim,
Choi, & Lee, 2004; Xiao, Textor, & Spencer, 1998; Xue, Deng, Lai, &
The surface properties of materials contacting with biological
systems play significant roles in determining the outcome at the
interface between them (Mager, LaPointe, & Stevens, 2011; Prime &
Whitesides, 1991). In dental and orthopedic surgeries, the surfaces
of implant materials are often modified to improve the rates of
osseointegration and biomechanical fixation.
Titanium (Ti) and titanium alloy have been widely used in hard
tissue replacement as well as cardiac and cardiovascular applica-
tions (Liu, Chu, & Ding, 2004). There have been numerous reports
about surface modification methods for the enhanced osteogenic
activity of these materials; mechanical methods (Buser et al., 1999;
Sutherland, Forshaw, Allen, Brown, & Williams, 1993; Wennerberg,
Albrektsson, Johansson, & Andersson, 1996) including machin-
ing, grinding, polishing and blasting as well as chemical methods,
such as acid-etching, H2O2-etching, alkali-etching, sol–gel coat-
ing, anodic oxidation, chemical vapor deposition and biochemical
modification have been studied to enhance the osteogenic activity
(Andreazza, De Barros, Andreazza-Vignolle, Rats, & Vandenbulcke,
1998; Domenichini et al., 1999; Hua, Shi, Zhang, Ruan, & Zhao, 2001;
Chen, 1998).
The formation of hydroxyl, carboxyl, amine, azide and alkyne
groups on the surface of Ti can be effective ways to conjugate var-
ious biomolecules by these functional groups. Among these, click
reaction, based on the reaction between azide and alkyne groups,
is highly efficient due to both high conversion and selectivity under
mild conditions, reactive process occurring in an aqueous medium
and high yield (Kolb, Finn, & Sharpless, 2001; Rostovtsev, Green,
Fokin, & Sharpless, 2002), as compared to another reactions such as
amide reaction, disulfide reaction, silylanization and polydopami-
nation. Moreover, click reaction has been developed as a versatile
method for conjugating a variety of organic, polymeric and biologi-
cal molecules (van Dijk, Rijkers, Liskamp, van Nostrum, & Hennink,
2009).
To examine the feasibility of aminated Ti surface via click
reaction for immobilizing biological molecules, heparin was
surface-immobilized, followed by immobilization of recombinant
human bone morphogenetic protein-2 (rhBMP-2) via physical
interaction with heparin (Ruppert, Hoffmann, & Sebald, 1996).
rhBMP-2, an effective growth factor, plays a significant role in the
formations of bone and cartilage.
In the present study, we designed and prepared aminated
Ti (Ti-4), which was conjugated via click reaction between the
azide group of 2-azidoethanamine and the propargyl group of
propargylated Ti (Ti-3). Heparin was conjugated to Ti-4 via amide
bond by condensation reaction (heparinized Ti, Ti-5), followed by
∗
Corresponding author at: Department of Oral and Maxillofacial Surgery, Kyung
Hee University Dental Hospital at Gangdong, #892 Dongnam-ro, Gangdong-gu,
Seoul 134-727, Republic of Korea. Tel.: +82 2 440 6172; fax: +82 2 440 7525.
E-mail addresses: verycutebear@hanmail.net, verycutebear@khu.ac.kr
(D.-W. Lee).
0144-8617/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carbpol.2013.12.023

E.-C. Kim et al. / Carbohydrate Polymers 103 (2014) 170–178
171
Fig. 1. (A) Synthetic route of 2-azidoethanamine and (B) schematic illustration of preparing Ti-1–6.
immobilization of rhBMP-2 onto Ti-5 (rhBMP-2-immobilized Ti,
Ti-6). In vitro tests, including MC3T3 cell proliferation, alkaline
phosphatase (ALP) activity, calcium deposition and bone-related
mRNA gene expressions, were carried out.
room temperature and reacted at 70 ◦C for 24 h. After washing with
distilled water three times, the samples were dried with nitrogen
gas, and then stored at room temperature (Ti-4). In order to escape
the immobilization of heparin at the one-side and the circumfer-
ence of Ti-4, the points were scrubbed with sandpaper. Heparin
(92 mg) and DMT-MM (1 g, 3.61 mmol) were dissolved in distilled
water (40 mL). This solution was continuously stirred at room tem-
perature for 1 h, and then Ti-4 was added. The mixture was reacted
at room temperature for 48 h to induce amide bond by condensa-
tion reaction. After washing the samples with distilled water three
times, they were dried with nitrogen gas, and then stored at −20 ◦C
(Ti-5) prior to use. Ti-5 and rhBMP-2 (10 ng) were mixed in PBS
(1 mL, pH 7.4) and incubated at 37 ◦C for 24 h with orbital agitation
at 80 rpm. After washing the samples with PBS three times, they
were dried with nitrogen gas. Prior to use, they were stored in a
freezer (−20 ◦C) (Ti-6) (Fig. 1).
2. Materials and methods
2.1. Materials
Titanium dioxide disks (Ti-1, diameter: 10 mm, height: 3 mm)
were kindly obtained from DIO Co. (Busan, Republic of Korea).
Propargyl bromide, potassium carbonate (K2CO3), sodium ascor-
bate and copper (II) sulphate pentahydrate (CuSO4·5H2O) were
supplied from Sigma–Aldrich (St. Louis, MO, USA). Heparin sodium
(molecular weight: 12,000–15,000 g/mol) was purchased from
Acrose Organics (NJ, USA). rhBMP-2 was purchased from Peprotech
(NJ, USA). Fetal bovine serum (FBS), penicillin–streptomycin (PS)
and phosphate buffered saline (×1 PBS, pH 7.4) were supplied from
Gibco BRL (Gaithersburg, MD). (4-(4,6-dimethoxy-1,3,5-triazin-2-
yl)-4-methylmorpholinium chloride) (DMT-MM) was purchased
from Wako (Osaka, Japan). All organic solvents were used as
received without further purification.
2.4. Scanning electron microscope observation
The surface morphologies of Ti-1–6 were observed by scanning
electron microscopy (SEM, S-2300, Hitachi, Japan). First, Ti-1–6
fixed on metal mounts were gold-coated by a sputter-coater (Eiko
IB, Japan) with an accelerating voltage of 15 kV, and then observed
at a magnification of ×1.5k.
2.2. Surface propargylation (Ti-3)
2.5. Static contact angles measurement
Ti-1 was treated using 2.5 N NaOH aqueous solution (500 mL) at
80 ◦C for 48 h. After sonicating, the surface-treated samples were
washed with distilled water for 5 min three times, dried with nitro-
gen gas, and then stored at room temperature (Ti-2). Ti-2 and
K2CO3 (6.91 g, 0.05 mol) were mixed together in anhydrous ace-
tone (500 mL). Propargyl bromide (4.31 mL, 0.05 mol) was added to
this mixture at room temperature and refluxed for 24 h to induce
click reaction. After taking out the samples, they were repeatedly
washed with acetone and distilled water three times, dried with
nitrogen gas, and then stored at room temperature (Ti-3) (Fig. 1).
Static contact angles measurement was carried out using a ses-
sile drop method at 20 ◦C with 3 ␮L of distilled water droplet under
a relative humidity of 60%, in advance adjusted to 71.8 mN/m of
surface tension, as measured by a telescopic goniometer (Pheonix
300, Seoul, Republic of Korea).
2.6. Surface chemical compositions measurement
The surface chemical compositions of Ti-1–6 were examined by
X-ray photoelectron spectroscopy (XPS) measurement. XPS mea-
surement was carried out using a Thermo Electron (U.K) unit
under a grazing angle of 90◦ and in a high vacuum (less than
3.1 × 10−9 Torr). A monochromatic aluminum K␣ X-ray radiation
source (photoelectron energy = 1486.6 eV) was used. Wide scanned
2.3. Surface heparinization (Ti-6)
Ti-3 and 2-azidoethanamine (1.41 g, 0.02 mol) were mixed in
distilled water (50 mL). Sodium ascorbate (3.96 g, 0.02 mol) and
CuSO4·5H2O (0.89 g, 3.57 mmol) were added to this mixture at

172
E.-C. Kim et al. / Carbohydrate Polymers 103 (2014) 170–178
Table 1
XPS was obtained at a pass of 187.7 eV and measured from 0 to
1400 eV.
Primers of bone-related mRNA genes; OSX, Runx2, COL1, OP, OC and BSP.
Genes
OSX
Primers
2.7. Quantitative analysis of heparin
Sense
Antisense
Sense
Antisense
Sense
Antisense
Sense
Antisense
Sense
Antisense
Sense
5ꢀ-TTG AGG AAG AAG CTC ACT ATG GCT CCA G-3ꢀ
5ꢀ-GCT GAA AGG TCA GCG TAT GGC T-3ꢀ
5ꢀ-TCG TCA GCA TCC TAT CAG TTC CCA-3ꢀ
5ꢀ-CCA TCA GCG TCA ACA CCA TCA TTC TGG TTA G-3ꢀ
5ꢀ-CGT GGC GAC CAA GGT CCA GT-3ꢀ
Runx2
COL1
OP
Quantitative analysis of heparin immobilized on Ti-5 was car-
ried out using toluidine blue method (Kim et al., 2011). Briefly,
500 ␮L toluidine blue solution (0.005%) was added to a mix-
ture of Ti-5 in 2 mL aqueous NaCl solution (0.2%). After gentle
shaking for 30 min, hexane (3 mL) was added. The Ti-5 was
removed from the solution, and then the absorbance of the
aqueous layer was measured at a wavelength of 620 nm. The
quantitative amount of heparin was calculated using a calibra-
tion curve determined from known concentrations of heparin.
The calibration curve was prepared using seven concentrations of
heparin (0.1, 0.5, 2, 3, 4, 5 and 10 ␮g/mL) in aqueous NaCl solu-
tion.
5ꢀ-AGG GAG ACC CAG AAT ACC GGG AG-3ꢀ
5ꢀ-TGT GTC CTC TGA AGA AAC CA-3ꢀ
5ꢀ-TGG CTT TCG TTG GAC TTA CT-3ꢀ
OC
5ꢀ-AGC AAA GGT GCA GCC TTT GT-3ꢀ
5ꢀ-GCG CCT GGG TCT CTT CAC T-3ꢀ
BSP
5ꢀ-CAG CGG CCC TGA GTC TGA CAA A-3ꢀ
5ꢀ-TCA CAA GCA GGG TTA AGC TCA CAC TG-3ꢀ
Antisense
and 0.1 ␮M dexamethasone) was added to each well and the plates
were incubated for 7, 14 and 21 days. At each predetermined time
point, all of the cell-seeded samples were washed with PBS (pH
7.4), and then diluted in 1× RIPA buffer (50 mM Tris–HCl (pH 7.4),
150 mM NaCl, 0.25% deoxycholic acid, 1% NP-40 and 1 mM EDTA).
A protease inhibitor cocktail tablet (Roche, Mannheim, Germany)
was added. The cells in RIPA buffer were lysed for 20 min in ice. Each
lysate was centrifuged at 4 ◦C for 10 min to remove the cell debris.
The supernatant was then incubated with p-nitrophenyl phosphate
(PNPP). The reaction with PNPP was terminated by adding 50 ␮L of
1 N NaOH. ALP activity was determined by measuring the conver-
sion of PNPP to p-nitrophenol. The optical density was measured
by a microplate reader at 405 nm.
2.8. Practical immobilizing amount of rhBMP-2 on Ti-6
A fixed amount of rhBMP-2 (10 ng) was immobilized on Ti-
5 as mentioned in Section 2.3. After the immobilizing process,
100 ␮g of the remaining solution was analyzed by a microplate
reader (Bio-Rad, Hercules, CA, USA) using an enzyme-linked
immunosorbent assay (ELISA) (Mundy et al., 1999) to examine
free rhBMP-2. The practical amount of rhBMP-2 immobilized was
calculated by the comparison with standard curve obtained from
several concentration of rhBMP-2. Optical density was measured
at 495 nm.
2.12. Calcium deposition assay
2.9. Release kinetics of rhBMP-2 from Ti-6
MC3T3-E1 cells (a density of 5 × 103 cells/well) were seeded on
Ti-1, 2, 5 and 6 as well as control (48-well plate), and then incu-
bated for 7, 14 and 21 days. At each predetermined time point,
the cell-seeded samples were washed twice with PBS (pH 7.4),
fixed for 20 min using 3.7% formaldehyde and washed with PBS
again. All of the samples were stained using 40 mM alizarin S-
red staining solution (pH 4.2) and incubated at 37 ◦C under 5%
CO2 for 1 h. The staining solution was removed after 1 h, the
cells were washed with distilled water three times, and then
observed by confocal laser scanning microscopy (CLSM, Eclipse
E600W, Nikon, Tokyo, Japan). For a quantitative analysis, the
stained cells were desorbed with 10% 1-hexadecylpyridinium chlo-
ride and the absorbance was measured by a microplate reader at
540 nm.
Ti-6 was added to a vial filled with 3 mL PBS (pH 7.4) and then
incubated at 37 ◦C at 100 rpm. At each predetermined time point (1,
2, and 8 h, 1, 3, 5, 7, 14, 21 and 28 days), 1 mL of the supernatant was
collected, and then same volume of fresh PBS was added to the vial.
Prior to the measurement, the collected supernatant was stored
in a refrigerator (−20 ◦C). The release of rhBMP-2 was determined
using the ELISA kit according to manufacturer’s instructions men-
tioned in Section 2.7. Optical density of the collected supernatant
was measured at 495 nm.
2.10. MC3T3-E1 cell proliferation assay
MC3T3-E1 cells (density: 5 × 103 cells/well) were seeded on
Ti-1, 2, 5 and 6 in a 48-well culture plate as well as con-
trol (48-well plate). 1 mL of Dulbecco’s modified eagle medium
(DMEM) containing 10% FBS and 1% PS was added to these cells
and the plate was incubated for 1, 3 and 7 days. The medium
was changed once in every three days. Nuclei and cytoskele-
tons of the cells cultured on all samples were stained using
by 4ꢀ,6-diamidino-2-phenylindole (DAPI) and F-actin methods, as
observed by fluorescence microscope (OLYMPUS DP2-BSW, NY,
USA) at ×200. At each predetermined time point, each sample
was rinsed with PBS (pH 7.4) and then analyzed using a cell
counting kit (CCK-8). The reagent was added to the samples and
carefully transferred to a 96-well culture plate after 2 h of incuba-
tion. The optical density was measured by a microplate reader at
450 nm.
2.13. Real-time polymerase chain reaction (real-time PCR)
The total RNA of MC3T3-E1 cells (a density of 5 × 103 cells/mL)
cultured on Ti-1, 2, 5 and 6 as well as control (48-well plate) for 7,
14 and 21 days were isolated using RNeasy Plus Mini Kit (Qiagen,
CA, USA). According to manufacturer’s instructions, 1 ␮g of total
RNA was extracted from all samples and transcribed into cDNA
with AccuPower CycleScript RT Premix (Bioneer, Daejeon, Republic
of Korea). Real-time PCR amplifications on all samples were car-
ried out by AccuPower PCR PreMix (Bioneer, Daejeon, Republic of
Korea). The primers of the measured mRNA genes were as shown
in Table 1: osterix (OSX) – 5ꢀ-TTG AGG AAG AAG CTC ACT ATG GCT
CCA G-3ꢀ (sense) and 5ꢀ-GCT GAA AGG TCA GCG TAT GGC T-3ꢀ (anti-
sense), runt-related transcription factor 2 (Runx2) – 5ꢀ-TCG TCA
GCA TCC TAT CAG TTC CCA-3ꢀ (sense) and 5ꢀ-CCA TCA GCG TCA ACA
CCA TCA TTC TGG TTA G-3ꢀ, type I collagen (COL1) – 5ꢀ-CGT GGC GAC
CAA GGT CCA GT-3ꢀ (sense) and 5ꢀ-AGG GAG ACC CAG AAT ACC GGG
AG-3ꢀ (antisense), osteopontin (OP) – 5ꢀ-TGT GTC CTC TGA AGA AAC
CA-3ꢀ (sense) and 5ꢀ-TGG CTT TCG TTG GAC TTA CT-3ꢀ (antisense),
osteocalcin (OC) – 5ꢀ-AGC AAA GGT GCA GCC TTT GT-3ꢀ (sense) and
2.11. Alkaline phosphatase (ALP) activity assay
MC3T3-E1 cells (a density of 5 × 103 cells/well) were seeded on
Ti-1, 2, 5 and 6 in a 48-well culture plate as well as control (48-well
plate). Osteogenic media (DMEM containing 10% FBS, 1% PS, 10 mM
␤-glycero phosphate disodium salt hydrate, 300 ␮M ascorbic acid

E.-C. Kim et al. / Carbohydrate Polymers 103 (2014) 170–178
173
Fig. 2. SEM images of Ti-1–6, as observed at ×1500.
5ꢀ-GCG CCT GGG TCT CTT CAC T-3ꢀ (antisense), and bone sialopro-
3. Results
tein (BSP) – 5ꢀ-CAG CGG CCC TGA GTC TGA CAA A-3ꢀ (sense) and
5ꢀ-TCA CAA GCA GGG TTA AGC TCA CAC TG-3ꢀ (antisense). Real-
time PCR was analyzed using iQ SYBR Green supermix (Bio-Rad,
Hercules, CA, USA). Threshold cycle values were calculated by a
comparative CT method. The fold change of control (48-well plate)
at 7 days of culture was set at 1-fold and the ratio of the normalized
fold change was then calculated. The real-time PCR amplifications
were carried out under a cycle of 10 s at 95 ◦C, 30 s at 57–62 ◦C
(OSX: 59 ◦C, Runx2: 62 ◦C, COL1: 60 ◦C, OP: 56 ◦C, OC: 57 ◦C, BSP:
61 ◦C) and 30 s at 72 ◦C for 45 cycles after the initial denaturation
step for 10 min at 95 ◦C.
3.1. Surface characterizations
Fig. 2 shows the surface morphologies of Ti-1–6, as observed
by SEM. Significant differences in the surface morphologies were
observed between unmodified and surface-modified samples. The
smooth surface of Ti-1 was cracked by the treatment of 2.5 NaOH.
The crack still remained after the surface treatment of propargyl
bromide, whereas it was disappeared on the surfaces of Ti-4–6
due to the surface treatments of 2-azidoethanamine, heparin
and rhBMP-2. The static contact angles of Ti-1–6 were shown
in Table 2. The static contact angle of Ti-1 was 71.55◦, whereas
Ti-2 showed a more hydrophilic surface (37.91◦), indicating that
hydroxyl groups were produced on the surface of Ti-2. Moreover,
the static contact angles of Ti-3–6 were different due to the sur-
face modifications using different components including propargyl,
2-azidoethanamine as the linker, heparin and rhBMP-2. By wide-
scanned XPS spectra measurement (Table 2), the C1s percentage of
2.14. Statistical analysis
All experiments were carried out three times. Student’s t-test
was used to confirm the statistical significance of each data, as com-
pared to control designated in each experiment. A value of *p < 0.05
was considered statistically significant.
Table 2
Changes in the S2p, C1s, N1s and O1s percentages of Ti-1–6 and their static contact angles.
C1s (283.7 eV, %)
N1s (399.2 eV, %)
O1s (528.8 eV, %)
S2p (163.8 eV, %)
Static contact angle (◦)
Ti-1
Ti-2
Ti-3
Ti-4
Ti-5
Ti-6
29.21
14.11
32.09
28.50
30.05
29.06
0.32
0.14
0.47
3.12
1.28
0.66
51.27
62.24
52.12
46.15
45.04
54.82
0.25
0.31
0.32
0.21
5.89
3.26
71.55 2.95
37.91 2.80
64.10 2.53
42.45 1.75
40.80 1.08
29.19 2.37

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E.-C. Kim et al. / Carbohydrate Polymers 103 (2014) 170–178
1, as compared to that cultured on Ti-1 as well as the control. On
the other hand, the cell proliferation rates of MC3T3-E1 cultured
on Ti-5 and 6 were similar to that cultured on Ti-1 as well as the
control at 3 and 7 days of culture. Moreover, Ti-6 showed the high-
est cell proliferation rate even though the cell proliferation rate
was not statistically significant, as compared that of the control.
But, the cell proliferation rate showed a statistical significance, as
compared to that of Ti-1. The results indicated that Ti-6 can be
used as a good implant to enhance the cell proliferation rate of
MC3T3-E1.
3.5. Level of ALP activity
Fig. 3. Release kinetic of BMP-2 from Ti-6 for 28 days of incubation.
We examined the ALP activity level of MC3T3-E1 cells cultured
on Ti-6 for 7, 14 and 21 days, as compared to that on the control (48-
well plate), Ti-1, 2 and 5 (Fig. 5). Generally, ALP activity is expressed
at the initial stage of bone formation (Takagishi et al., 2006), which
was well in agreement with our result. Although ALP activity lev-
els on Ti-1, 2, 5 and 6 statistically showed significant differences
compared to that on control, the ALP activity levels were not sub-
stantial at 7 days of culture. However, significant increases in the
ALP activity levels were observed on Ti-2, 5 and 6 at 14 and 21
days of culture. Moreover, Ti-6 showed the highest ALP activity
level, indicating that the sample can be used as a good implant to
accelerate the osteogenic differentiation of MC3T3-E1 cells.
Ti-1 was 29.33%, which might be due to contamination. This fact
made it difficult to quantitatively analyze the XPS spectra of Ti-
1–6. However, Ti-1 showed higher O1s percentage than Ti-1 and
3, which was attributed to the production of OH group. A signif-
icant increase in the N1s percentage was observed on Ti-3 due
to the existence of propargyl group. Decreased N1s percentage
was observed on Ti-4–6 due to the immobilization of heparin and
rhBMP-2. Moreover, a significant increase in the S2p percentage
was observed on Ti-5 and 6, mainly attributed to the immobiliza-
tion of heparin. However, S2p percentage was decreased on Ti-6
due to the immobilization of BMP-2.
3.6. Amount of calcium deposition
3.2. Quantitative analysis of heparin
We examined the amount of calcium deposited on Ti-6 cultured
with MC3T3-E1 cells for 14 and 21 days as compared to that on the
control (48-well plate), Ti-1, 2, and 5 (Fig. 6). Calcium was visually
found to be heavily deposited on Ti-2, 5 and 6 at 21 days of cul-
ture, as compared to the control and Ti-1 (Fig. 6A). Moreover, Ti-6
showed the largest amount of calcium deposition (Fig. 6B), indicat-
ing that the sample can be used as a good implant to accelerate the
osteogenic differentiation of MC3T3-E1 cells.
The amount of heparin immobilized on Ti-5 was
4.1 0.27 ␮g/sample, which was bound to the titanium sur-
face more than 68-fold, as compared to the previous study (Kim
et al., 2011). 3-Aminopropyltriethoxysilane (APTES) was surface-
treated on titanium to immobilize heparin in the previous study
(Shimasaki et al., 1999). On the other hand, propargyl groups
were produced onto the surface of Ti-2 in the present study. As a
result, it was judged that propargyl group onto titanium surface
can be used as an intermediate tool to effectively immobilize
biomolecules such as heparin.
3.7. Real-time PCR
3.3. Release kinetics of rhBMP-2 from Ti-6
The expressions of osteoblast mRNA gene markers, including
OXS, Runx2, COL1, OC, OP and BSP, was examined on Ti-2, 5 and
6 cultured with MC3T3-E1 cell, as compared to the control (48-
well plate) and Ti-1 (Fig. 7). As osteogenic transcription factors,
a gradual increase in the OXS mRNA expression was observed on
all samples over time (Fig. 7A). On the other hand, Runx2 mRNA
expression increased for 14 days of culture, and then decreased
thereafter (Fig. 7B). Among the samples, Ti-6 showed the highest
expressions of OSX and Runx2 mRNA genes. Fig. 7C shows mRNA
expression of COL1 synthesis on all samples for 21 days of culture.
COL1 is generally regarded as an early marker during the prolifera-
tion of osteoblast cells (Lian, Stein, & Aubin, 2003), which is well in
agreement with our results. A gradual decrease in the COL1 synthe-
sis was observed on all samples over time. At each predetermined
time point, COL1 synthesis significantly increased in the order of Ti-
1, 2, 5 and 6. Fig. 7D shows the OC mRNA expression on all samples
for 21 days of culture. Gradual increases and decreases in the OC and
the OP mRNA expressions were observed on all samples over time
(Fig. 7D and E). The mRNA genes showed increased expressions in
the order of Ti-1, 2, 5, and 6. In particular, significant increases of
OC and OP mRNA expressions were observed on Ti-6 at 14 and 21
days of culture. The mRNA expression of BSP on all samples showed
a similar behavior, as compared to that of OC (Fig. 7F). Among the
samples, a significant increase in the BSP mRNA expression was
observed on Ti-6.
The practical amount of rhBMP-2 immobilized was
9.55 0.01 ng, indicating that the loading efficiency was 99.5%.
Fig. 3 shows the release kinetic of rhBMP-2 from Ti-6 for a period of
28 days. The initial burst release happened within 8 h of incubation
and was below 10% (0.78 0.1 ng). Afterward, rhBMP-2 showed
a sustained release kinetic for up to 28 days of incubation and
the cumulative release percentage was below 29% (2.71 0.3 ng),
which indicated that rhBMP-2 may be released in a sustained
manner during a prolonged period (more than 28 days).
3.4. MC3T3-E1 cell proliferation
The proliferation and morphology of MC3T3-E1 cells on Ti-6
for 1, 3 and 7 days was visually observed by fluorescence micro-
scope, as compared to those on the control (48-well plate), Ti-1,
2 and 5 (Fig. 4A). Gradual increases in the cell proliferations were
observed on all samples over time. In addition, the cells cultured on
all samples were stretched in all directions over time. In particu-
lar, Ti-6 showed the highest cell proliferation rate and the quickest
stretching velocity in all samples. We quantitatively examined the
proliferation rates of MC3T3-E1 cells cultured on all samples for
1, 3 and 7 days (Fig. 4B). The cell proliferation rates of MC3T3-E1
cultured on Ti-2, 5 and 6 only showed a marginal difference at day

E.-C. Kim et al. / Carbohydrate Polymers 103 (2014) 170–178
175
Fig. 4. Proliferation of MC3T3-E1 cells cultured on control (48-well plate), Ti-1–2, and Ti-5–6 for 1, 3 and 7 days, examined by (A) fluorescence microscope and (B) CCK-8
assays.
4. Discussion
present study, we modified the surface of titanium by the concept
of click reaction. Pristine and surface-modified Ti-1–6 were char-
acterized by static contact angle and XPS measurements (Table 2),
resulting in the successful surface modifications.
We examined the release kinetics of rhBMP-2 from Ti-6 for
28 days of incubation (Fig. 3). Generally, BMP family comprises
significant growth factors for the regeneration of bone and car-
tilage. Among these, rhBMP-2 has been particularly well known
as a growth factor for very strong osteoinductive activity. How-
ever, BMP-2 has several problems including high cost, the need of
high dose and short half-life (Sellers et al., 2010). Hence, effective
Click reaction was first introduced by Kolb et al. (2001), which
is not a specific reaction but one that mimics nature. Click reaction
has been used in several applications including the bioconjuga-
tion of synthetic labels to biomolecules, materials science such as
synthesis of dendrimers, surface modification of single walled car-
bon nanotubes (SWNTs) for molecular electronics, sensors and field
emission devices, engineering techniques for self assembled mono-
layer (SAM) formation on metal surfaces, the synthesis of rotaxanes
and drug discovery (Kolb et al., 2001; Rostovtsev et al., 2002). In the

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E.-C. Kim et al. / Carbohydrate Polymers 103 (2014) 170–178
good binding with heparin (Ruppert et al., 1996). We found that
rhBMP-2 using heparin-based system was released for 28 days in a
sustained manner.
The proliferation rate of MC3T3-E1 cell was examined on all
samples, resulting that the cells cultured on Ti-6 had the highest cell
proliferation rate in all samples (Fig. 4), which may be attributed to
the effect of rhBMP-2.
ALP activity and calcium deposition are early and late markers
of osteoblastic cells, respectively. Enhanced ALP activity level and
calcium deposition amount were observed on Ti-6, as compared to
the control, Ti-1, 2 and 5 (Figs. 5 and 6), suggesting that rhBMP-2
accelerated new bone formation.rhBMP-2 has also known as one
of the most potent stimulators of osteoblastic cell differentiation.
Osteoblastic differentiation can be confirmed by the assay of
bone-related mRNA gene expressions. We examined the mRNA
expressions of OSX as a significant transcriptional factor involved
in the differentiation step from preosteoblasts to fully functioning
osteoblast and Runx2 as a significant regulator of osteoblastic
differentiation (Nakashima & de Crombrugghe, 2003). A zinc finger
transcriptional factor OSX acts downstream from Runx2, which is
predictive of bone mass formation (Nakashima & de Crombrugghe,
2003). As shown in Fig. 7A, an increase in the OSX mRNA
Fig. 5. ALP activity level of MC3T3-E1 cells cultured on control (48-well plate), Ti-1,
2, 5 and 6 for 7, 14, and 21 days.
systems for the controlled release of rhBMP-2 have to be consid-
ered to stimulate bone regeneration. In the present study, we used
heparin-binding system to release rhBMP-2 in a sustained manner
because rhBMP-2 contains a strong heparin-binding site allowing
Fig. 6. (A) CLSM images of calcium deposited on control (48-well plate), Ti-1–2, 5 and 6 at 21 days of culture and (B) their quantitative calcium degree after 14 and 21 days
of cultures, respectively.

E.-C. Kim et al. / Carbohydrate Polymers 103 (2014) 170–178
177
Fig. 7. Gene expressions of (A) OSX, (B) Runx2, (C) COL1, (D) OC, (E) OP and (F) BSP on control (48-well plate), Ti-1–2, 5 and 6 at 7, 14 and 21 days of cultures.
expression was observed on Ti-6, which may lead to the acceler-
ation of new bone formation. Runx2 mRNA expression is induced
by a Smad-dependent pathway (Nakashima & de Crombrugghe,
2003). Ti-6 gradually showed over-expressed Runx2 mRNA
expression, as compared to the control, Ti-1, 2 and 5 (Fig. 7B). A
gradual decrease in the Runx2 mRNA expression was observed on
all samples after 14 days of culture (Fig. 7B). Although Runx2 is
not essential for the expression of main osteogenic mRNA genes
in mature osteoblastic cells because the overexpression produces
osteopenia by decreasing osteoblasts and increasing osteoclasts,
the gene inhibits osteoblastic proliferation by acting on p85 PIK3
and GADD45␤ (Gordeladze, Reseland, Duroux-Richard, Apparailly,
& Jorgensen, 2010). In the present study, we suggested that a
decrease in the Runx2 mRNA expression after 14 days might accel-
erate osteoblasts. Also, bone is formed by complicated biological
processes and controlled by expressions of the mRNA genes includ-
ing COL1, ALP, OC, BSP and OP. COL1 is an early marker during the
proliferation of osteoblast cells (Lamolle et al., 2009). As shown in
Fig. 7C, Ti-6 showed more significant mRNA expression of COL1, as
compared to the control and Ti-1, mainly attributed to the effect
of rhBMP-2. ALP mRNA gene expresses in the matrix maturation.
In addition, OC and BSP are closely related to calcium deposition
in the extracellular matrix during matrix mineralization (Lamolle
et al., 2009; Stein, Lian, Stein, Van Wijnen, & Montecino, 1996).
Aronow et al. (1990) have reported that OC mRNA gene harvested
from the cranial bone of rat was more expressed later than ALP.
Among all samples, the highest level of ALP activity and expression
of OC mRNA gene were observed on Ti-6, mainly attributed to
the effect of rhBMP-2. Unlike OC mRNA gene, OP mRNA gene
expresses in the initial stage of osteoblastic differentiation and
matrix mineralization (Owen et al., 1990). However, the result of
Fig. 7E was different from the above-mentioned fact, which may
be due to the influences of the culture condition and media. BSP
is generally a late marker expressed during matrix mineralization.
Ti-6 showed the highest expression of BSP mRNA gene over
time. Consequently, BMP-2 immobilized on titanium surface may
induce the enhanced osteogenic differentiation of MC3T3-E1
cells.

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5. Conclusions
on nanostructural and chemical changes and the growth of MC3T3-E1 cells.
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We designed and prepared surface-propargylated Ti-3 via click
reaction and rhBMP-2-immobilized Ti-6 to evaluate the osteogenic
differentiation of MC3T3-E1 cells. The surfaces of Ti-1–6 were char-
acterized by SEM, XPS and contact angle measurements, resulting
in the successful surface modifications. Ti-3 was found to be used
as a good intermediate tool to immobilize heparin. BMP-2 from Ti-
6 was released for 28 days in a sustained manner. Ti-6 showed the
highest cell proliferation rate and bone-related mRNA gene expres-
sion including OSX, Runx2, COL1, OC, OP and BSP. It is concluded
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Acknowledgments
osteoblast differentiation and bone formation. Trends in Genetics, 19(8),
458–466.
This work was supported by grants from the Kyung Hee
University in 2012 (KHU-20121678) and the National Research
Foundation of Korea (NRF) Grant funded by the Korean Government
(MSIP) (no. 2012R1A5A2051384).
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