Int J Artif Organs 2013; 36 (7): 506-517 DOI: 10.5301/ijao.5000215
ORIGINAL ARTICLE
Immobilization of BMP-2 on a nano-hydroxyapatite-
coated titanium surface using a chitosan calcium
chelating agent
Sung-Hyun Kim1, Jung-Keug Park1, Kug-Sun Hong2, Hyun-Suk Jung3, Young-Kwon Seo1
1
Department of Medical Biotechnology, Dongguk University, Seoul - Korea
2
Department of Materials Science and Engineering, Seoul National University, Shillim-dong, Kwanak-gu, Seoul - Korea
3
Department of Medical Biotechnology Dongguk University Seoul - - University Shillim-dong, Kwanak-gu, Seoul -School of Advanced Materials Science and Engineering, Sungkyunkwan University, Suwon - Korea
Department of Medical Biotechnology , Dongguk University , Seoul - Kor ea , Suwon - Kor ea Kor ea
Department of Materials Science and Engineering, Seoul National Kor ea University ,
School of Advanced Materials Science , and Engineering, Sungkyunkwan
Department of Medical Biotechnology Dongguk University Seoul Kor ea
, , ,
We conducted experiments to determine the most effective calcium chelating agents for use in en-
hancing adhesion of human bone marrow mesenchymal stem cells (BM-MSCs) on nano-hydroxyap-
atite (nHAp)-coated titanium substrates by covalently immobilizing bone morphogenetic protein-2
(BMP-2). The quantity of amine groups on the chitosan chelated surface was 7 µg/surface area, and
it was 1.4 µg/surface area on the alendronate chelated surface. The quantity of BMP-2 on the BMP-2
immobilized surface chelated with chitosan (4 ng/surface area) was higher than that on BMP-2 immo-
bilized surface chelated with alendronate (2.2 ng/surface area). Contact angles of the nHAp-coated
titanium, alendronate chelated, chitosan chelated, and BMP-2 immobilized surfaces chelated with
alendronate were 68.8 Ä… 3.6°, 78.2 Ä… 1.9°, 74.8 Ä… 5.2°, and 76.0 Ä… 2.5°, respectively. The contact
angle of the BMP-2 immobilized surface chelated with chitosan was significantly lower (56.2 Ä… 2.0°)
than that of any of the other groups. BM-MSCs on the chitosan surface and BMP-2 immobilized on
the surface chelated with chitosan appeared to be healthy and showed a spindle-like fibroblastic
morphology. In addition, BM-MSCs on these surfaces appeared to have the ability to differentiate
into bone-forming cells. We suggest that chitosan can be used as an effective calcium chelating
agent for implants.
Keywords: Titanium, Nano-hydroxyapatite, Bone morphogenetic protein-2, Chitosan, Osseointegration
Accepted: February 20, 2013
Interest in surface modification methods to stimulate cell
INTRODUCTION
function at the bone-implant interface has increased (4).
It is essential to maintain a stable bone-biomaterial inter- The key part of a clinical implant application is to immo-
face to ensure long-term success of an endosseous den- bilize biomolecules on biomaterials, and this has been a
tal implant. Early osseointegration and biocompatibility in widely researched approach to modify metal surfaces to
the location of the implant are associated with long-term control cell and tissue responses (5). Hence, delivering
success (1). Metal prostheses have excellent mechanical growth factors to the bone-implant interface using implant
properties (2); however, osseointegration and biocompat- coating techniques has recently become a popular method
ibility are dependent on biomolecules that enhance bone to control healing and fixation of implants (6). Adsorption
regeneration. In addition, it has been revealed that the of bone morphogenetic protein-2 (BMP-2) on the surface
use of bone cement for permanent bone implantation of titanium or hydroxyapatite (HAp) ceramics results in
presents several problems (3). intense acceleration of implant osseointegration (7-9).
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Kim et al
Osteoconductive calcium phosphate, which is mainly com-
posed of HAp, is an attractive and typical biocompatible
ceramic material used in prosthetic devices and has re-
ceived much attention for its application as an endosse-
ous dental implant (10-12). HAp, Ca10(PO4)6(OH)2, was first
established as the mineral component of bone in 1926 by
DeJong, and synthetic HAp was approved as a biomate- Fig. 1 - 8 mm and 14 mm nano-hydroxyapatite (nHAp)-coated
titanium discs. Sample (1) is the 14 mm cont nHAp group disc,
rial for use in orthopedics, bone grafts, and dentistry about
(2) is the same size as that of the alen group disc, and (3) is the same
35 years ago (13). HAp can enhance osseointegration,
size as that of the chito group discs, respectively. Sample (4) is the
but it is brittle, which restricts its use in endosseous den- 8 mm cont nHAp group disc, (5) is the same size as that of the alen
group discs, and (6) is the same size as that of the chito group discs,
tal applications (14-15). The technique of HAp coating by
respectively.
methods such as plasma spray, sputtering, electrolysis,
and sol-gel have been studied to overcome this defect.
Effective coating of a load-bearing substrate with HAp can human or mouse fibroblasts in tissue cell culture (20).
overcome the physical weakness of HAp (13). Chitosan (C6H13NO5) has chelating ability toward metal
However, despite the enormous benefit of biomimetic and calcium ions, since the calcium chelating effect oc-
coatings on a titanium surface, proteins such as BMP-2, curs with the amine group of the chitosan molecule (21).
which is pre-adsorbed onto the material surface, may be In our study, we evaluated the possibility of using chito-
insufficiently immobilized and release may be uncontrolled san as a novel calcium chelating agent for immobilizing
due to a lack of functional groups. Covalent immobiliza- BMP-2 by culturing bone marrow mesenchymal stem
tion on the material surface achieves prolonged retention cells (BM-MSCs) and Jurkat cells on a modified titanium
of BMP-2 and growth factors at their site of action. A two- surface to test biocompatibility.
step, zero length, cross-linking strategy can be applied
to covalently immobilize BMP-2 by exposing the amino-
MATERIALS AND METHODS
functionalized ceramic discs to a cross-linker (16). A solu-
tion to this problem may be the use of calcium chelating
Materials
techniques that provide amine groups needed for covalent
attachment of proteins with the cross-linker (17).
Many calcium chelating agents have been researched for Grade 4 titanium discs were used. The triethyl phosphate
calcium chelating technique applications. Pamidronate (P(OC2H5)3) solution, propionic acid, and calcium ethox-
(C3H11NO7P2) and alendronate (C4H18NNaO10P2) have been ide (Ca(OC2H5)2) powder were obtained from the Elec-
used widely by researchers to evaluate the suitability of tronic Functional Materials Laboratory of Seoul National
this approach (16). In addition, EDTA (C10H16N2O8) is an ef- University. 4-amino-1-hydroxy-1-phosphonobutyl phos-
fective calcium chelating agent as it is a single hexadentate phonic acid monosodium (alendronate sodium trihydrate)
chelon. Due to the lone pairs of electrons, the four oxygen was purchased from Sigma-Aldrich Chemical (St Louis,
atoms in the four carboxyl groups and two nitrogen atoms MO, USA). Chitosan and Protosan UP CL 213 were pur-
chelate with the metal and calcium ions (18). However, chased from Novametrix (Brakeroya, Drammen, Norway).
due to their cytotoxic properties, bisphosphonates such Human BMP-2 was a gift from Daewoong Research and
as pamidronate and alendronate can cause apoptosis in a Development, Daewoong Pharmaceutical Co., Ltd (Seoul,
variety of cell types in vitro (19). Korea).
In this study, we investigated the effectiveness of calcium
chelating agents for providing amine groups to immobilize
Preparation of titanium discs
protein on nano-hydroxyapatite (nHAp) surfaces. We ad-
opted natural chelating material that is non-cytotoxic, has The diameters of the titanium discs used in this experiment
excellent biocompatibility, and immobilizes BMP-2. Chito- were 8 and 14 mm (Fig. 1), and the size of titanium foil
san, which is widely used in various forms of biomaterials, was 5 cm × 5 cm. The titanium discs and foil were coated
shows good biocompatibility and no cytotoxicity in either with nHAp using a spin-coater. The nHAp solution was
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Immobilization of BMP-2 using a chitosan calcium chelating agent
prepared inside a glove box that was purged with argon coated titanium surface (cont nHAp group), the alendro-
gas (99.999%). P(OC2H5)3 liquid was diluted with propionic nate chelated cont nHAp group (alen group), the BMP-2
acid to produce the nHAp solution, then Ca(OC2H5)2 pow- immobilized alen group (alen + BMP group), the chitosan
der was dissolved with propionic acid as a solvent. Two chelated cont nHAp group (chito group), and the BMP-2
bottles of each solution were stored separately in the glove immobilized chito group (chito + BMP group).
box and stirred until absolutely dissolved. The two bottles
of each solution were then mixed in a 1:1 ratio in the glove
Amine assay
box for 10 min. Afterwards, the mixed solution was stirred
at a temperature of 60°C in a water bath for 6 h. The nHAp The surface density of the amine groups introduced onto
solution was spin-coated onto the titanium discs with a the 8 mm calcium chelated titanium discs was quantified
spinning velocity of 4000 rpm and a 20 s spin coating time. by reaction with 2,4,6-trinitrobenzenesulfonic acid (TNBS),
nHAp spin-coated titanium discs were then sintered in a which interacts with primary amine groups to form trinitro-
sintering oven at a temperature of 500°C for 2 h. phenyl derivatives (22). The amine assay was performed ac-
cording to the method of Puleo (5). Titanium samples were
incubated with 0.1% TNBS in 3% sodium borate at 70°C for
Calcium chelating agent treatment
5 min, followed by washing with triple distilled water, then
The nHAp coated 8 mm and 14 mm discs and the 5 cm × hydrolyzed with 1 N NaOH at 70°C for 10 min. These reac-
5 cm titanium foil were placed in 12-well plates containing tions provided a yellow color that was proportional to the
2 mL of a 1 mg/ml alendronate or chitosan aqueous solu- number of trinitrophenyl groups, which was proportional to
tion. The titanium discs were then totally immersed in solu- the number of amine groups. Absorbance of hydrolysate
tion. The well plates were shaken at room temperature for was detected at 410 nm. Standard curves were prepared
4 h on an orbital shaker set at 20 rpm in the dark. The tita- by adding L-DOPA (Sigma-Aldrich) in 0.1% TNBS in 3% so-
nium samples were washed three times with distilled water, dium borate, followed by a serial dilution. The standard was
dried, and stored under vacuum until further use. then hydrolyzed with 1 N NaOH at 70°C for 10 min.
Immobilization of BMP-2 BMP-2 assay
The concentration of human BMP-2 stock solution was The BMP-2 assay was performed using the Human BMP-2
1 µg/ml in phosphate-buffered saline (PBS). BMP-2 was Super X-ELISA kit (Antigenix America, Huntington Station,
dispersed on the nHAp coated titanium surfaces, which NY, USA). BMP-2 immobilized on the 8 mm nHAp-coated
had been treated with each of the calcium chelating titanium discs and other titanium discs were placed in
agents. Before BMP-2 treatment, human BMP-2 stock 48-well plates. These titanium discs were then immersed
solution and cross-linking solution were mixed for 5 h in 1 mL of 0.1 mg/ml bovine serum albumin (BSA) solu-
at a 1:1 ratio to achieve efficient covalent immobilization tion and incubated at room temperature for 2 h in the dark.
of BMP-2 on the calcium chelated nHAp coating sur- Then, the samples were washed four times with the wash
face. The cross-linking solution was composed of 20 mL buffer provided in the kit. A 20 µL aliquot of the 0.5 µg/ml
40% (v/v) ethanol and 50 mM MES (pH 5.5), 24 mM 1-eth- biotin-labeled tracer (tracer antibody) stock solution was
yl-3-(3-dimethyl aminopropyl)carbodiimide (Fluka Chemic loaded onto each titanium disc surface, and the titanium
AG, Milwaukee, WI, USA) and 5 mM N-hydroxysuccin- discs were incubated at room temperature for 2 h in the
imide (Fluka Chemic AG). The diluted human BMP-2 dark. Samples were then washed four times again using
solution was 0.5 µg/ml. A 10 µL aliquot of the diluted hu- the wash buffer. The streptavidin-HRP conjugate solu-
man BMP-2 solution was loaded onto the 8 mm titanium tion was diluted with diluent, 0.05% Tween-20 (Uniqema,
disc surfaces, 30 µL was loaded onto the 14 mm titanium Wilmington, DE, USA), and 0.1% BSA in PBS, at a ratio of
disc surfaces, and 500 µL was added to the 5 cm × 5 cm 1:500. A 20 µL aliquot of the diluted streptavidin-HRP con-
titanium foil surfaces. All samples were placed overnight jugate solution was loaded onto each titanium disc surface,
at room temperature, washed five times with distilled water, and the titanium discs were incubated at room tempera-
and dried at room temperature. We compared the nHAp- ture for 30 min in the dark. The samples were then washed
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Kim et al
four times again using the wash buffer provided in the kit. were fixed in 2% OsO4:0.2 M phosphate buffer (1:1) re-
Titanium discs were placed upside down in another 48-well agent for 2 h in the dark. The samples were then rinsed
plate. TMB substrate solutions A and B were mixed at a 1:1 with PBS twice for 10 min. The BM-MSC samples were
ratio. A 100 µL aliquot of TMB substrate mixed solution was washed with an ethanol: water mixture (30%, 50%, 70%,
added to the 48-well plates, followed by a 30-min incuba- 90%, and 100% ethanol) twice for 10 min, in sequence.
tion at room temperature in the dark. After completing the Samples were then treated with 98% 1,1,1,3,3,3-hexa-
incubation, 100 µL of stop solution (2 N sulphuric acid) was methyldisilazane (HMDS):100% ethanol (1:1) solution for
added, and absorbance was measured at 450 nm. 5 min. They were then treated with 98% HMDS twice for
10 min and allowed to dry overnight.
The morphology of the BM-MSCs on nHAp-coated titanium
Contact angle measurement
disc surfaces was examined under SEM (Model HITACHI
A contact angle measurement device (Tensiometer/Pen- S-3000N; Hitachi Instruments, Tokyo, Japan).
dant Drop, Model DSA100; KRUSS Advancing Surface
Science, Hamburg, Germany) was used to measure wa-
MTT assay of BM-MSCs
ter contact angles. The Drop Shape Analysis software was
used to estimate the contact angle of various nHAp-coat- Cell number was determined using a 3-(4,5-dimethylthiazol-
ed titanium surfaces. Water contact angles were measured 2-yl)-2,5-diphenyl tetrazolium bromide (MTT; Sigma-Aldrich)
on dry surfaces. assay (18). This assay identifies metabolically active cells
through the action of a mitochondrial dehydrogenase that
is changed into an insoluble formazan pigment. BM-MSCs
BM-MSC culture on surface-modified titanium
in 24-well plates were incubated for the designated times in
discs
0.33 mg/ml MTT supplemented cell culture medium at 37°C
BM-MSCs were purchased from Lonza (Basel, Switzer- and 5% CO2 for 2 h. The intense purple-colored formazan
land). Human BM-MSCs were cultured on 14 mm nHAp derivative formed during active cell metabolism was eluted
coated titanium discs and 5 cm × 5 cm titanium foil surfaces and dissolved in 1 mL dimethyl sulfoxide and absorbance
in high glucose DMEM medium (Invitrogen, Carlsbad, CA, was measured at 540 nm.
USA) containing 10% foetal bovine serum (FBS; Cambrex,
East Rutherford, NJ, USA), 1% penicillin streptomycin
T-lymphocyte culture
(10,000 units/ml penicillin, 10,000 µg/ml streptomycin,
WelGENE Inc., Daejeon, Korea), and 25 µM ascorbic acid T-cells were cultured for the biocompatibility analysis. The
(Sigma-Aldrich) at a density of 2.5 × 104 cells/disc and Jurkat cell line (T-lymphocyte, human leukemia, suspen-
1 × 105 cells/foil. The 14 mm nHAp-coated titanium discs sion cell line) was purchased from the ATCC (Manassas, VA,
were placed in 24-well plates with the medium, and the USA). Jurkat cells were cultured in 12-well plates containing
cells were incubated at 37°C in a humidified atmosphere 8 mm nHAp-coated titanium discs with 1 mL of RPMI1640
of 5% CO2 for 3 days. The 5 cm × 5 cm titanium foil sheets (Invitrogen) containing 10% FBS and 1% penicillin strepto-
were placed in 100 mm diameter Petri dishes with medium, mycin (10,000 units/ml of penicillin, 10,000 µg/ml streptomy-
and the cells were incubated at 37°C in a humidified atmo- cin) at a density of 5 × 105 cells/well. Cells were incubated
sphere of 5% CO2 for 10 days. Then, the culture medium, at 37°C in a humidified atmosphere of 5% CO2 for 3 days.
which was low glucose DMEM medium containing 10% The BrdU assay was performed using the Cell Prolifera-
FBS, 1% penicillin streptomycin, 50 µM ascorbic acid, tion ELISA Assay, BrdU (colorimetric) kit (Roche Diagnos-
10 mM ²-glycerophosphate (TCI, Seoul, Korea), and 10-7 M tics, Mannheim, Germany) to evaluate proliferation of the
dexamethasone (Sigma-Aldrich) was used during 2 weeks. Jurkat cell line. All 8 mm nHAp-coated titanium discs were
removed during this assay. One microliter of the BrdU label-
ing solution provided in the kit was added to the Jurkat cell
Scanning electron microscopy (SEM)
line cultured in 12-well plates, followed by a 2-h incubation
SEM samples were fixed overnight at room temperature at 37°C in the dark. The final concentration of BrdU label-
using a fixing reagent (2.5% glutaraldehyde in PBS); they ing solution was 10 µM. The solution was centrifuged at
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Immobilization of BMP-2 using a chitosan calcium chelating agent
300 × g for 10 minutes using a 1.5 mL tube and the labeling cellular RNA was isolated using 1 mL of Trizol reagent (In-
medium was removed. The 1.2 mL of FixDenat provided in vitrogen). cDNA was synthesised by reverse transcription
the kit was then added to the cells, followed by a 30-min using 1 µg of total RNA. PCR was conducted by subject-
room temperature incubation. The solution was centrifuged ing the samples to 23 to 35 cycles (within the linear range
again at 300 × g for 10 minutes, followed by removal of the of amplification) of denaturation (94°C, 1 min), annealing
FixDenat solution. Then, 600 µL of anti-BrdU-POD working (53-57°C, 1 min), and extension (72°C, 1 min). The prod-
solution (antibody conjugate), which was diluted 1:100 with ucts were then analyzed on 2% agarose gels and visualized
antibody dilution solution, was added to the cells, followed by SYBR Safe DNA Gel Staining (Invitrogen). The relative
by a 90-min room temperature incubation. The antibody abundance of type I collagen, type III collagen, osteonec-
conjugate was removed by aspiration of the centrifuged tin, osteopontin, vimentin, BMP-2, bone sialoprotein (BSP),
samples, and the wells were rinsed three times with 1.2 mL and GAPDH (an internal control) transcripts was measured
PBS. A 600 µL aliquot of substrate solution was added to using RT-PCR. Primers used for RT-PCR were purchased
the cells, followed by 15-min room temperature incubation. from Bioneer, and their sequences, reaction conditions,
Absorbance was measured immediately at 370 nm. and product size (bp) are listed in Table I. Image J software
(Wayne Rasband, National Institutes of Health, Bethesda,
MD, USA) was used to quantitatively analyze the RT-PCR
Tumor necrosis factor-Ä… (TNF-Ä…) assay
amplicons on digitized gel images.
The quantity of secreted TNF-Ä…, an inflammatory cytokine,
was estimated after the T-cell culture. The TNF-Ä… assay was
Statistical analysis
performed using the TNF-Ä… ELISA kit (Biosource, Nivelles,
Belgium). Two hundred microliters of standards and sam- Student s t-test was used to evaluate the artificial titanium
ples were added to anti-TNF-Ä…-coated wells of the microti- sample data. Data are given as means Ä… standard devia-
ter plate with 50 µL of incubation buffer provided in the kit. tions. A p<0.05 was considered significant.
The 96-well microtiter plates were incubated for 2 h at room
temperature on a horizontal shaker set at 700 Ä… 100 rpm.
RESULTS
The liquid was aspirated from each well, and the plates were
washed three times by dispensing 0.4 mL of wash solution
Quantification of amine groups
into each well, and aspirating the contents. One hundred mi-
croliters of standard 0 and 50 µL of anti-TNF-Ä… conjugate,
which were diluted 1:10 with conjugate buffer, were added to Amine groups were quantified using the amine assay with
all wells. The 96-well plates were incubated for 2 h at room the TNBS reaction. The quantity of amine groups on the
temperature on a horizontal shaker set at 700 Ä… 100 rpm. The chito group surface was approximately 7 µg/surface area
liquid was then aspirated from each well. The plates were (Fig. 2). However, the alen group surface contained ap-
washed three times by dispensing of 0.4 mL of wash so- proximately 1.4 µg/surface area of amine groups. Thus, the
lution into each well and aspirating the contents. Following chitosan chelated surface appeared to have a large num-
the washing step, 200 µL of freshly prepared chromogenic ber of amine groups, compared with the alendronate che-
solution was added to each well within 15 min and the plates lated surface. As expected, the cont nHAp group surface
were incubated for 30 min at room temperature on a hori- had scarcely any amine groups on its surface.
zontal shaker set at 700 Ä… 100 rpm, avoiding direct sunlight.
Finally, 50 µL of Stop Solution was added to each well, and
BMP-2 estimation
absorbance was read at 450 nm within 3 h.
The BMP-2 assay was performed when all samples were
dried perfectly. We compared the quantity of BMP-2 im-
Reverse transcription polymerase chain reaction
mobilized on the cont nHAp, alen, alen + BMP, chito, and
(RT-PCR) of BM-MSCs
chito + BMP groups (Fig. 3). The quantity of BMP-2 on
The RT-PCR analysis was performed to compare the ex- the chito + BMP (about 4 ng/surface area) group was
pression of bone-inducing markers in BM-MSCs. Total higher than that on the alen + BMP (about 2.2 ng/surface
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Kim et al
TABLE I - PRIMER SEQUENCES, REACTION CONDITIONS, AND PRODUCT SIZE FOR THE REVERSE TRANSCRIPTION-
POLYMERASE CHAIN REACTION (RT-PCR) ANALYSIS
Gene Sequence Product Size (bp) Annealing (°C) Cycles
GAPDH F : ACC ACA GTC CAT GCC ATC AC 450 55 25
R : TTC ACC ACC CTG TTG CTG TA
Collagen 1 F : GAA AAC ATC CCA GCC AAG AA 270 57 23
R : CAG GTT GCC AGT CTC CTC AT
Collagen 3 F : CAG GTG AAC GTG GAG CTG C 661 57 23
R : TGC CAC ACG TGT TTC CGT GG 849
Osteonectin F : CCA GAA CCA CCA CTG CAA AC 161 57 23
R : GGC AGG AAG AGT CGA AGG TC
Osteopontin F : TCG CAG ACC TGA CAT CCA GT 267 57 32
R : TCG GAA TGC TCA TTG CTC TC
Vimentin F : GGA ACA GCA TGT CCA AAT CG 214 55 25
R : TCA GTG GAC TCC TGC TTT GC
BMP-2 F : GTC CAG CTG TAA GAG ACA CC 316 54 31
R : GTA CTA GCG ACA CCC ACA AC
BSP F : AAC CTA CAA CCC CAC CAC AA 147 57 36
R : GTT CCC CGT TCT CAC TTT CA
Fig. 2 - Quantification of amine groups on the calcium chelated
Fig. 3 - Estimation of bone morphogenetic protein-2 (BMP-2) on the
surface. The nano-hydroxyapatite (nHAp)-coated titanium surface
calcium chelated surface. The Cont nHAp graph indicates quanti-
(cont nHAp) graph indicates quantification of amine groups on the
fication of BMP-2 on the nano-hydroxyapatite (nHAp) coated tita-
nHAp-coated titanium surface. The Alen graph indicates quantifi-
nium surface. The Alen graph indicates quantification of BMP-2 on
cation of amine groups on the alendronate chelated surface. The
the alendronate chelated surface. The Alen + BMP graph indicates
Chito graph indicates quantification of amine groups on the chitosan
quantification of BMP-2 on the BMP-2 immobilized surface chelated
chelated surface.
with alendronate. The Chito graph indicates quantification of BMP-2
on the chitosan chelated surface. The Chito + BMP graph indicates
quantification of BMP-2 on the BMP-2 immobilized surface chelated
with chitosan. Results are expressed as means Ä… standard deviation
area). The chitosan agent appeared to hold many BMP-2
(n = 3) (*p<0.0001).
growth factors due to the large quantity of amine functional
groups. We also detected some BMP-2 on the surface of
Contact angle goniometry
the sample groups that were not immobilized with BMP-2,
which may have been caused by a reaction with the cal- Contact angles of the alen and chito groups were signifi-
cium chelating agent. cantly higher (more hydrophobic) than those of the cont
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Immobilization of BMP-2 using a chitosan calcium chelating agent
Fig. 4 - Results for the calcium chelated surface contact angles. (A): Fig. 5 - Scanning electron microscopy (SEM) images of bone mar-
The contact angle of the alendronate chelated surface (alen group) row mesenchymal stem cells (BM-MSCs) on a calcium chelated
was approximately 78.2 Ä… 1.9°. (B): The contact angle of the bone surface (magnification: ×2.0 k). (A): SEM of BM-MSCs on an alendro-
morphogenetic protein-2 (BMP-2) immobilized surface chelated nate chelated surface (alen group). (B): SEM of BM-MSCs on a bone
with alendronate (alen + BMP group) was approximately 76.0 Ä… 2.5°. morphogenetic protein-2 (BMP-2) immobilized surface chelated
(C): The contact angle of the chitosan chelated surface (chito group) with alendronate (alen + BMP group). (C): SEM of BM-MSCs on a
was approximately 74.8 Ä… 5.2°. (D): The contact angle of the BMP-2 chitosan chelated surface (chito group). (D): SEM of BM-MSCs on
immobilized surface chelated with chitosan (chito + BMP group) was a BMP-2 immobilized surface chelated with chitosan ( chito + BMP
approximately 56.2 Ä… 2.0°. (E): The contact angle of the nano-hy- group). (E): SEM of BM-MSCs on a nano-hydroxyapatite (nHAp)-
droxyapatite (nHAp)-coated titanium surface (cont nHAp group) was coated titanium surface (cont nHAp group).
approximately 68.8 Ä… 3.6°.
nHAp group. In addition, the contact angle of the alen + were more adherent on the alen + BMP (Fig. 5B) group than
BMP group was also significantly higher than that of the those on the cont nHAp and alen groups (Figs. 5A and E).
Cont nHAp group. Angles of the cont nHAp, alen, chito, No differences were observed between the cont nHAp and
and alen + BMP groups were 68.8 Ä… 3.6°, 78.2 Ä… 1.9°, alen groups. BM-MSCs on the chito and chito + BMP (Figs.
74.8 Ä… 5.2°, and 76.0 Ä… 2.5°, respectively. However, the 5C and D) groups were more adherent than those on the
contact angle of the chito + BMP group was significantly cont nHAp group.
lower (56.2 Ä… 2.0°) than that of any other group (Fig. 4). In particular, adhesion of the chito + BMP group was higher
Consequently, the chitosan chelated surface was modified than that of the chito group, which may have occurred due
by immobilizing BMP-2 to improve the hydrophilicity of its to the presence of abundant amine functional groups, sug-
surface. gesting that chitosan has the ability to increase adhesion
of BM-MSCs. Thus, chitosan is an effective biomolecule
with an adhesion role in cells. In addition, BMP-2 on chito-
BM-MSC morphology and spreading
san increased cell adhesion.
The morphology of BM-MSCs on the nHAp-coated titanium Normally, cells attached on the surface, and the adhesion
discs was assessed by SEM. High-magnification images area was not large. But if the cells spread their cytoplasm
(× 1.0 K) of these samples show cell adhesion. BM-MSCs on the ECM or growth factor-coated surface, then the
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Kim et al
Fig. 7 - Proliferation of bone marrow mesenchymal stem cells
Fig. 6 - Analysis of spreading cell area measured by Image J software.
(BM-MSCs) on the calcium chelated surface. The Cont nHAp
Group area was divided by the nHAp-coated titanium surface (cont
graph represents cell proliferation on the nano-hydroxyapatite
nHAp) area. (Group area = spreading cell area for each experiment
(nHAp) coated titanium surface. The Alen graph represents cell
group; cont nHAp = spreading cell area of the control group).
proliferation on the alendronate chelated surface. The Alen + BMP
graph represents cell proliferation on the bone morphogenetic
protein-2 (BMP-2) immobilized surface chelated with alendronate.
The Chito graph represents cell proliferation on the chitosan che-
surface increased adhesion and the spreading area in-
lated surface. The Chito + BMP graph represents cell proliferation
on the BMP-2 immobilized surface chelated with chitosan. Each
creased. In Figure 6, when the cells adhered on the HAp-
cell proliferation graph is divided into 12 and 72 h after cell seed-
coated surface, the adhesion area of the cytoplasm was
ing. Results are expressed as means Ä… standard deviations (n = 3)
not large. But, when MSCs attached to the BMP linked
(*p<0.5).
surface, the spreading area increased because BMP in-
creased cytoplasmic spreading. Thus, even though the
cell number was similar, the spreading area was wider. The Ä… 0.07 × 104 cells/disc and 2.06 Ä… 0.12 × 103 cells/disc.
spreading cell area was measured by Image J software The numbers on the chito + BMP group surface after 12 h
and is shown in Figure 6. and 72 h were approximately 2.04 Ä… 0.21 × 104 cells/disc
and 2.10 Ä… 0.17 × 103 cells/disc, respectively. We found no
differences in cell proliferation. Despite what appeared to
BM-MSC proliferation
be enhanced cell attachment on the alen group, no differ-
BM-MSC proliferation on nHAp coated titanium discs ences were observed among the samples.
was evaluated by the MTT assay (Fig. 7) 3 days after cell
seeding. The number of BM-MSCs was measured with a
Number of Jurkat cells
standard MTT assay.
The number of BM-MSCs on the cont nHAp group sur- The BrdU assay was performed to measure the number of
face after 12 h and 72 h was approximately 2.01 Ä… 0.33 × Jurkat cells 3 days after cell seeding (Fig. 8). The number
104 cells/disc and 2.27 Ä… 0.23 × 103 cells/disc, respectively. of Jurkat cells on the alen + BMP group (approximately
The cell number appeared to increase slightly over 3 days. 33.3 Ä… 4.94 × 105 cells/well) was higher than that of cells
However, the other groups did not show an outstanding on the alen group (approximately 15 Ä… 5.06 × 105 cells/
growth effect. The numbers of BM-MSCs on the alen group well). We also confirmed that the number of Jurkat cells
surface after 12 h and 72 h were approximately 2.32 Ä… on the chito + BMP group (approximately 41.4 Ä… 4.23 ×
0.20 × 104 cells disc and 2.43 Ä… 0.54 × 103 cells/disc. The 105 cells/well) was higher than that of cells on the chito
numbers on the alen + BMP group surface after 12 h group (approximately 34.7 Ä… 2.04 × 105 cells/well). In other
and 72 h were approximately 2.18 Ä… 0.09 × 104 cells/disc reported research, exposure to toxic molecules results in
and 2.25 Ä… 0.13 × 103 cells/disc. The numbers on the chito a concentration-dependent decrease in Jurkat T-cell pro-
group surface after 12 h and 72 h were approximately 2.02 liferation (23). Our results suggest that alendronate is more
© 2013 Wichtig Editore - ISSN 0391-3988
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Immobilization of BMP-2 using a chitosan calcium chelating agent
Fig. 9 - Tumor necrosis factor-Ä… (TNF-Ä…) assay results for Jurkat
Fig. 8 - The number of Jurkat cells was measured by the BrdU
cells on the calcium chelated surface. The Con nHAp graph repre-
assay. The Con nHAp graph represents Jurkat cell number on the
sents the quantity of TNF-Ä… on the nHAp coated titanium surface.
nano-hydroxyapatite (nHAp) coated titanium surface. The Alen graph
The Alen graph represents the quantity of TNF-Ä… on the alendronate
represents Jurkat cell number on the alendronate chelated surface.
chelated surface. The Alen + BMP graph represents the quantity
The Alen + BMP graph represents Jurkat cell number on the bone
of TNF-Ä… on the bone morphogenetic protein-2 (BMP-2) immobi-
morphogenetic protein-2 (BMP-2) immobilized surface chelated with
lized surface chelated with alendronate. The Chito graph represents
alendronate. The Chito graph represents Jurkat cell number on the
the quantity of TNF-Ä… on the chitosan chelated surface. The Chito +
chitosan chelated surface. The Chito + BMP graph represents Jurkat
BMP graph represents the quantity of TNF-Ä… on the BMP-2 immobi-
cell number on the BMP-2 immobilized surface chelated with chi-
lized surface chelated with chitosan. Results are means Ä… standard
tosan. Results are means Ä… standard deviations (n = 3) (*p<0.0001).
deviations (n = 3) (*p<0.005).
cytotoxic than chitosan; however, BMP-2 had the ability to
reduce cytotoxicity of alendronate by increasing the num-
ber of Jurkat cells.
Quantification of TNF-Ä…
The TNF-Ä… assay was performed with RPMI1640 me-
dium from Jurkat cells, which was obtained 3 days after
cell seeding (Fig. 9). This result showed a large amount of
TNF-Ä… secreted in the alen group. TNF-Ä… decreased sig-
nificantly in the alen + BMP group, when compared with
that of the alen group due to immobilization of BMP-2 on
the surface. In addition, the chito and chito + BMP groups
showed decreased TNF-Ä…. Thus, chitosan was less cyto-
toxic than alendronate in Jurkat cells.
RT-PCR analysis
We investigated whether preconditioning BM-MSCs re-
sults in an increase in their bone-inducing activity us-
ing RT-PCR analysis (Fig. 10). Although type I collagen
Fig. 10 - Reverse transcription polymerase chain reaction (RT-PCR)
mRNA expression appeared to increase in the chito group
analysis of GAPDH, collagen type I, collagen type III, osteonectin,
when compared with that in the control group, no signifi-
osteopontin, vimentin, bone morphogenetic protein (BMP), and
cant difference was observed among any of the groups. bone sialoprotein (BSP).
© 2013 Wichtig Editore - ISSN 0391-3988
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Kim et al
In contrast, type III collagen expression increased in the
chito + BMP group when compared with that in the other
groups. Levels of osteonectin and osteopontin expression
were higher in the chito and chito + BMP groups, com-
pared with the other groups. Osteonectin expression in the
chito + BMP group increased more than that in the chito
group. In addition, the chito + BMP group showed great-
er BMP expression than that in any of the other groups.
These combined results suggest that chitosan may have
a significant autonomous, osteoconductive effect. Thus,
bone-inducing activity was more enhanced when BMP-2
was immobilized on the chitosan chelated surface. Ad-
ditionally, we analyzed BMP release from the BMP-2 im-
mobilized surface and found that 10% of the BMP was
released into the media every 24 h during the 4 days. As a
result, this osteogenic effect was due to immobilized BMP
and BMP released into the media. Alendronate may also
have an osteoconductive effect. However, the levels of
type III collagen, osteopontin, vimentin, and BSP mRNA
expression decreased when BMP-2 was immobilized on
the alendronate chelated surface. The quantitative analysis
of the RT-PCR amplicons on digitized gel images is shown
in Figure 11. We suggest that chitosan can be used as a
ligand to bind BMP-2 and can work as a bioactive agent
for biomaterials that promote osteogenic differentiation.
DISCUSSION
The key point of our study was immobilization of BMP bio-
molecules on biomaterials, which is a widely researched
Fig. 11 - Quantitative analysis of reverse transcription-polymerase
chain reaction (RT-PCR) amplicons on digitized gel images mea-
approach to modify metal surfaces to control cell and
sured by Image J software (*p<0.1, **p<0.05).
tissue responses (5). We used alendronate and chitosan
as calcium chelating agents to improve immobilization of
BMP-2 on an nHAp-coated surface. We hoped to find the We found that the chitosan chelated surface had a larger
most effective calcium chelating agent to enhance adhe- number of amine groups compared to that of the alendro-
sion of BM-MSCs on nHAp-coated titanium substrates by nate chelated surface. Alendronate has a primary amine on
covalently immobilizing BMP-2. The results suggest that its R2 side chain (25), whereas chitosan has many amine
chitosan, a biomolecule that contains amine functional groups that have a chelating effect (26). BMP-2 tends to
groups, increased the ability to retain BMP-2, increased adhere to cross-linker activated surfaces. Other research-
the adhesion of BM-MSCs, and reduced cytotoxicity. In ers have revealed that titanium surface hydroxyl groups
addition, BMP-2 immobilized on a chitosan chelated sur- can be activated with carbonyldiimidazole and that the
face was increasingly hydrophilic, which can enhance carboxyl groups are activated with N-hydroxysuccinimide
differentiation to bone compared with a chitosan-only che- to bind amine-containing molecules. In addition, BMP-2
lated surface. Chitosan is a non-toxic, non-immunogenic, covalently attaches to activated titanium surfaces (27).
and biodegradable natural biopolymer that enhances bone Therefore, chitosan has a large number of amine functional
healing in various animal models (24). groups that enhanced immobilization of BMP-2 with the
© 2013 Wichtig Editore - ISSN 0391-3988
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Immobilization of BMP-2 using a chitosan calcium chelating agent
cross-linker. We also found that the quantity of BMP-2 on Chitosan enhanced binding ability and cell adhesion of
the chito + BMP group was higher than that on the alen + BMP-2 and also reduced cytotoxicity. We also demon-
BMP group. Thus, we demonstrated an association be- strated that chitosan had an excellent autonomous osseo-
tween the number of amine groups on the surface and that integration effect. In addition, BMP-2 immobilization on a
of BMP-2 immobilized onto an amine-grafted surface. chitosan chelated surface increased hydrophilicity of the
We found that the hydrophilicity of the BMP-2 immobilized surface. BM-MSC differentiation to bone on a BMP-2 im-
surface chelated with chitosan could be improved. Bone- mobilized surface chelated with chitosan was better than
inducing activity was enhanced when BMP-2 was immo- that on a chitosan chelated surface.
bilized on the chitosan chelated surface. The correlation
between surface hydrophilicity and osteogenic activity of
CONCLUSIONS
cells has been demonstrated in other studies. Our results
suggest that hydrophilic titanium can lead to an alterations
in osteogenic activity (28). However, we found no significant More amine groups were found on the chitosan chelated
difference in cell proliferation among the samples. Other re- surface than on the alendronate chelated surface. The
searchers have already shown that cell proliferation on both quantity of BMP-2 on the BMP-2 immobilized surface che-
titanium grafted with carboxymethyl chitosan and BMP-2 lated with chitosan was higher than that on the BMP-2 im-
functionalized substrates does not increase compared with mobilized surface chelated with alendronate. Hydrophilicity
that on pristine titanium (29). We propose that although we of the BMP-2 immobilized surface chelated with chitosan
did not detect a significant increase in cell proliferation, en- showed a significantly greater increase than that of any
hanced cell adhesion due to the presence of chitosan and other group. BM-MSCs on a BMP-2 immobilized surface
BMP-2 would lead to more complete osseointegration. chelated with chitosan appeared to have a significant abil-
Research has shown that chitosan has a wide range of ap- ity to differentiate into bone-forming cells. Based on these
plications, including antibacterial activity (30, 31). Analogous results, we suggest that chitosan is an effective calcium
studies of immobilizing BMP-2 to enhance the osteocon- chelating agent for implants. Our future work will focus on
ductive effect of a titanium surface using chitosan have also methods to immobilize various bioactive molecules on sur-
been reported. Shi et al immobilized BMP-2 on a titanium faces grafted with bioinert material. In addition, we will in-
surface, which was functionalized by covalent grafting with vestigate various analytical tests.
carboxymethyl chitosan, and showed an antibacterial effect
Financial Support: This study was supported by the R & D Program
(29). Shi et al grafted chitosan on titanium by immobilizing
of the Ministry of Knowledge and Economy/Korea Evaluation Institute
L-DOPA on its surface. Their results showed that bacterial ad-
of Industrial Technology (MKE/KEIT 10033290, Development of sur-
hesion on both the carboxymethyl chitosan-grafted (CMCS)
face activation technology for best function modification of implants
surface and BMP-2 immobilized CMSC surfaces decreased containing bioactive materials).
significantly, compared with that on pristine substrates. In
Conflict of Interest: The authors declare no conflict of interest.
addition, BMP-2 immobilized CMSC surfaces promote
significant attachment, alkaline phosphatase activity, and
Address for correspondence:
calcium mineral deposition in both osteoblasts and human
Young-Kwon Seo
BM-MSCs. However, we immobilized BMP-2 on chitosan, Department of Medical Biotechnology
Dongguk University
which was chelated on an nHAp-coated titanium surface.
3-26, Pil-dong, Chung-gu
We adopted an nHAp-coated titanium surface, as it contains
Seoul 100-715, Korea
a large number of calcium ions for chelation with chitosan. [e-mail]
2. Fujishiro Y, Sato T, Okuwaki A. Coating of hydroxy-
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