Journal of Industrial and Engineering Chemistry 67 (2018) 244–254

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Journal of Industrial and Engineering Chemistry

journal homepage: www.elsevier.com/locate/jiec

Polydopamine-mediated surface modifications of poly L-lactic acid

with hydroxyapatite, heparin and bone morphogenetic protein-2 and
their effects on osseointegration
Young Jin Yuna , Han-Jun Kimb , Deok-Won Leec , Sewook Umd , Heung Jae Chuna,*
a
Institute of Cell & Tissue Engineering, Department of Biomedical Sciences, College of Medicine, The Catholic University of Korea, Seoul 06591, Republic of
Korea
b
Department of Clinical Pathology, College of Veterinary Medicine, Konkuk University, Seoul 05029, Republic of Korea
c
Department of Oral and Maxillofacial Surgery, Kyung Hee University Dental Hospital at Gangdong, Kyung Hee University, Seoul 05278, Republic of Korea
d
Department of Veterinary Surgery, College of Veterinary Medicine, Seoul National University, Seoul 08826, 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 23 May 2018 Surface modified poly L-lactic acid (PLLA) samples with hydroxyapatite (HA), heparin and bone
Received in revised form 18 June 2018 morphogenetic protein-2 (BMP-2) mediated by polydopamine (pDA) coating (PLLA/pDA/HA/Hep/BMP-2)
Accepted 27 June 2018 were prepared, and their effects on the enhancements of bone formation and osseointegration were
Available online 11 July 2018 evaluated in vitro and in vivo as compared to PLLA, PLLA/pDA/HA, and PLLA/pDA/Hep/BMP-2. The changes
in surface chemical compositions, morphologies and wettabilities were observed by X-ray photoelectron
Keywords: spectroscopy (XPS), field-emission scanning electron microscopy (FE-SEM), atomic force microscopy
Polydopamine (AFM) and water contact angle measurements. Pre-coating of HA particles with pDA provided uniform
Poly L-lactic acid
and homogeneous anchoring of particles to PLLA surface. In addition, the strong ionic interaction
Hydroxyapatite
between heparin and pDA led PLLA surface readily heparinized for loading of BMP-2. In vitro experiments
Bone morphogenetic protein-2
Bone formation revealed that the levels of alkaline phosphatase (ALP) activity, calcium deposition, and osteocalcin (OCN)
gene expression were higher in MG-63 human osteosarcoma cell lines grown on PLLA/pDA/HA/Hep/
BMP-2 than on control PLLA, PLLA/pDA/HA, and PLLA/pDA/Hep/BMP-2. In vivo studies using micro-
computed tomography (micro-CT) also showed that PLLA/pDA/HA/Hep/BMP-2 screw exhibited greatest
value of bone volume (BV) and bone volume/tissue volume (BV/TV) among samples. Histological
evaluations with H&E and Von Kossa staining demonstrated that a combination of HA and BMP-2
contributed to the strong osseointegration.
© 2018 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights
reserved.

Introduction because HA has proved to be a successful substitute for calcium

Poly L-lactic acid (PLLA), as the biodegradable aliphatic
polyester, has gained the great attention for potential use in
orthopedic applications because it is hydrolytically degraded over
time in vivo with minimal inflammatory response [1–3]. These
benefits of PLLA may avoid the necessity of second surgery for
removal of metallic internal fixation devices [4]. However, PLLA
implants also have drawbacks including hydrophobicity and lack of
bioactivity that eventually delay the new bone formation [5–7]. For
decades, numerous attempts have been made to fabricate ceramic/
polymer composites made of hydroxyapatite (HA) and PLLA,
* Corresponding author.
E-mail address: chunhj@catholic.ac.kr (H.J. Chun).
phosphate in terms of unique osteoconductive properties [8,9].
Several research groups have shown the possibilities of HA/PLLA
composites for suitable clinical applications, but the failure rate
derived from the poor bonding ability between ceramic and
polymer phases, the insufficient mechanical properties and the
biodegradation behavior still remain a challenge to overcome
[10,11].
In this regard, some efforts have been put into the surface
modification of PLLA with HA in order to improve the bone
formation and osseointegration. Typically, various types of
plasma deposition methods have been employed to modify PLLA
surface with HA [12,13]. However, the temperature in the range of
800–1000
C for sputtering or sintering of HA cannot be used
because PLLA has low glass transition and melting point.
Although there exist some reports that utilize non-thermal

https://doi.org/10.1016/j.jiec.2018.06.035
1226-086X/© 2018 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
 Y.J. Yun et al. / Journal of Industrial and Engineering Chemistry 67 (2018) 244–254
plasma surface treatment, the coated HA layer formed at low
temperature are not strongly attached to PLLA surface results in
easy separation from the interface [14].
A decade ago, simple and versatile surface modification
methods based on the self-polymerization and deposition of
dopamine (DA) was proposed [15]. DA creates a spontaneous
thin coating on solid surfaces through polymerization at pH 8.5,
which does not rely on specific surface chemistries. Further-
more, polydopamine (pDA) may provide binding sites for the
introduction of bioactive factors. A precise mechanism of the
coating with pDA, indeed, has not been fully established yet,
however, the application of pDA to the introduction of HA and/
or bioactive factors such as bone morphogenetic proteins
(BMPs) on the surface of PLLA is of great significance to be
explored [16–18].
In this work, surface modified PLLA samples with HA, heparin
and bone morphogenetic protein-2 (BMP-2) mediated by pDA
coating (PLLA/pDA/HA/Hep/BMP-2) were prepared. HA particles
were pre-coated with pDA to avoid the aggregation due to the
electrostatic interactions between Ca and P ions, and plastered to
the surface of PLLA for uniform and homogeneous anchoring. In
addition, the strong ionic interaction between heparin and pDA led
PLLA surface readily heparinized for loading of BMP-2. The effects
the surface modifications on the enhancements of bone formation
and osseointegration were evaluated in vitro and examined for
efficacy in vivo using micro-computed tomography (micro-CT)
together with histologic analysis.
Experimental
Materials
Poly L-lactic acid (PLLA) discs (diameter: 15.0 mm; thickness:
1.9 mm; made of Samyang PLLA 1604) were supplied by Samyang
Biopharmaceuticals Corporation (Seongnam, Republic of Korea).
PLLA screws (FXS 150204; f 2.0  5.0 mm; made of RESOMER
L210S) and recombinant human bone morphogenetic protein 2
(rhBMP-2, BM-100) were gifted from Cellumed Ltd. (Seoul,
Republic of Korea). Alizarin red S (ARS), cetylpyridinium chloride
(CPC), dopaminehydrochloride (DAHCl), hydroxyapatite nano-
powder (HA, diameter: <200 nm) and heparin sodium salt (Hep)
were purchased from Sigma-Aldrich (St. Louis, USA). Tri(hydrox-
ymethyl) aminomethane (Tris) buffer was purchased from
Dynebio Inc. (Seongnam, Republic of Korea). Quantikine1 BMP-2
Immunoassay was bought from R&D Systems (Minneapolis, USA).
MG-63 human osteosarcoma (MG-63) cell line was provided by
Korean Cell Line Bank (Seoul, Republic of Korea). Dulbecco’s
minimum essential medium high glucose (DMEM), fetal bovine
serum (FBS), and phosphate buffered saline (PBS) were bought
from Biowest (Riverside, USA). StemPro osteogenesis supplement
and StemPro osteocyte differentiation basal medium were
purchased from Gibco (Grand Island, USA). Alkaline phosphatase
(ALP) assay kit was purchased from Abcam (Cambridge, USA). Cell
counting kit-8 (CCK-8) was provided by Dojindo (Kumamoto,
Japan). ReliaPrepTM RNA Cell MiniPrep System was supplied by
Promega (Madison, USA). SuperScript III first-strand synthesis
system for reverse transcription polymerase chain reaction (RT-
PCR) was purchased from Invitrogen (Carlsbad, USA). AccuPower1
RT PreMix was purchased from Bioneer (Daejeon, Republic of
Korea). iQTM SYBR1 Green Supermix was bought from Bio-Rad
(Hercules, USA). Zoletil1 (Virbac Laboratories, Carros, France),
Alfaxan1 (Jurox Pty Ltd., Rutherford, Australia), Rumpun1 (Bayer
Korea Ltd., Seoul, Republic of Korea), Gentamicin sulfate (Kukje
Pharm, Seongnam, Republic of Korea), Ketoprofen (SCD Pharm,
Seoul, Republic of Korea), Betadine1 (Purdue Pharma L.P.,
245
Stamford, USA), Ethicon Vicryl1 and Ethilon1 (Johnson & Johnson
Services Inc., Somerville, USA) were used for animal studies.
Preparation of pDA pre-coated HA, pDA/Hep/BMP-2 and pDA/HA/Hep/
BMP-2 surface-modified PLLA samples (PLLA/pDA/HA, PLLA/pDA/Hep/
BMP-2, and PLLA/pDA/HA/Hep/BMP-2) of disc and screw types
pDA-coated HA
DAHCl (0.11 mmol, 20 mg) was dissolved in 10 mM Tris buffer
(10 mL, pH 8.5). HA (10 mg) was suspended in DA solution (20 mL)
and ultra-sonicated for 30 min to minimize aggregation. The
mixture was incubated at room temperature (RT) for 24 h with
shaking at 80 rpm. pDA-coated HA was precipitated by centrifuga-
tion at 4000 rpm for 10 min and supernatant was discarded. The
resulting pDA-coated HA was washed by ultrasonication with fresh
Tris buffer for 30 min and re-precipitated. This procedure was
repeated three times to obtain pDA-coated HA slurry without
unreacted DA and HA (Fig. 1a) [19].
PLLA/pDA/HA
The pDA-coated HA slurry (1 mg) was plastered onto PLLA. The
HA plastered PLLA was immersed into 1 mL DA solution (2 mg/mL
in 10 mM Tris-HCl, pH 8.5) and kept at RT for 24 h for coating by
Michael addition and Schiff’s base reaction (Fig. 1b). Resulting HA-
coated PLLA was ultra-sonicated five times for 15 min to eliminate
loosely coated HA, then lyophilized at 90
C and stored in vacuum
desiccator before use.
PLLA/pDA/Hep/BMP-2 and PLLA/pDA/HA/Hep/BMP-2
PLLA was immersed into a solution of Hep (0.1 wt%) and DA
(2 mg) in 10 mM Tris buffer (1 mL, pH 8.5) and kept at RT for 24 h to
incorporate Hep into pDA chains through ionic interactions and/or
hydrogen bonding (Fig. 1e) [20–22], then the sample was washed
with an excess of distilled water several times and lyophilized at
90
C (PLLA/pDA/Hep). The specific interaction between Hep and
BMP-2 was achieved by incubation of PLLA/pDA/Hep in a solution
of BMP-2 in PBS (50 ng/mL PBS solution, pH 7.4) at 4
C for 24 h
with shaking at 80 rpm (Fig. 1f). PLLA/pDA/HA/Hep/BMP-2 was
prepared according to the process used for PLLA/pDA/Hep/BMP-2
using PLLA/pDA/HA as shown in Fig. 1c and d. The samples
containing BMP-2 (PLLA/pDA/Hep/BMP-2 and PLLA/pDA/HA/Hep/
BMP-2) were washed with an excess of PBS several times,
lyophilized at 90
C under 5 mTorr and stored at 20
C.
Surface characterizations
X-ray photoelectron spectroscopy (XPS)
Atomic compositions of PLLA discs after the surface modifica-
tions were analyzed by X-ray photoelectron spectroscopy (XPS)
using PHI 5000 VersaProbe (ULVAC PHI Inc., Chigasaki, Japan) at
source 15 kV with 24.5 W monochromated X-ray beam (photo-
electron energy = 1486.6 eV) and an aluminum anode. Pass energy
of wide scan was recorded at 117.4 eV under a gazing angle of 45
at
a high vacuum (2.0  107 Pa). Atomic percentages of PLLA, PLLA/
pDA/HA, PLLA/pDA/Hep/BMP-2, and PLLA/pDA/HA/Hep/BMP-2
discs were determined by fitting curves with symmetrical
Gauss-Lorentz functions. The spectra were fitted using MultiPak
software (ULVAC PHI Inc., Chigasaki, Japan) and Shirley type
background subtraction.
Scanning electron microscopy (SEM)
Surface morphologies of PLLA discs were investigated by field-
emission scanning electron microscopy (FE-SEM) using Inspect F
(FEI Company, Hillsboro, USA). Samples on metal mounts were
platinum-coated using Eiko IB-3 ion coater (Eiko Engineering Co.
246 Y.J. Yun et al. / Journal of Industrial and Engineering Chemistry 67 (2018) 244–254
Fig. 1. Schematic illustration of PLLA, PLLA/pDA/HA, PLLA/pDA/Hep/BMP-2, and PLLA/pDA/HA/Hep/BMP-2 samples of discs (diameter: 15.0 mm; thickness: 1.9 mm) and
screws (f 2.0  5.0 mm). These disc and screw types of PLLA were surface-modified by the same process.
Ltd., Hitachinaka, Japan). SEM images of these samples were
obtained at magnification of 30.0 k.
Atomic force microscopy (AFM)
XE-100 (Park Systems, Suwon, Republic of Korea) atomic force
microscopy (AFM) was used to observe surface morphologies of
PLLA discs. An area of 20  20 mm2 was scanned using a tapping
mode. Arithmetic mean of surface roughness (Ra) was calculated
from the roughness profile determined by AFM.
Contact angle measurement
Wettabilities of PLLA discs were determined by measuring
water contact angles. After dropping 10 mL of a water droplet, the
angle of the droplet on the surface was captured using OCA 40
(DataPhysics Instruments GmbH, Filderstadt, Germany).
Loading efficiencies and release behaviors of BMP-2
Loading efficiencies of BMP-2 on PLLA/pDA/Hep/BMP-2 and
PLLA/pDA/HA/Hep/BMP-2 were determined by measuring the
remaining BMP-2 after the preparation of samples. PLLA/pDA/Hep/
BMP-2 and PLLA/pDA/HA/Hep/BMP-2 disc was immersed in 3 mL
PBS (pH 7.4) and incubated at 37
C in a shaking water bath at
100 rpm. At set time intervals (1, 2, 8 h, and 1, 3, 5, 7, 14, 21, 28 days),
the supernatant was collected and replaced with fresh PBS. The
amount of BMP-2 in the collected solution was measured with a
Quantikine1 BMP-2 Immunoassay according to the manufacturer’s
protocol. Briefly, collected supernatants were transferred into an
antibody coated 96-well plate (50 mL/well) and then incubated at
RT on a shaker at 100 rpm for 2 h. Following the reaction, 200 mL of
BMP-2 conjugate solution protected from light was added to each
well and incubated at RT on the shaker at 100 rpm for 2 h. The
optical density (OD) of the solution in each well was measured at
wavelength of 450 nm with a microplate reader PowerWave XS
(BIO-TEK, Winooski, USA).
MG-63 cell line culture
To assess cellular responses of MG-63 cells cultured onto PLLA
discs, cell proliferation, ALP activity, amount of calcium deposition,
and level of OCN gene expression were investigated. MG-63 cells
were cultured with DMEM supplemented with 10% FBS and 1%
penicillin-streptomycin. These cells were sub-cultured at 80%
confluence. The culture medium was changed every 3 days. The
cells between passage 4 and 6 were used for in vitro tests.
Cell proliferation assay
To determine proliferation of MG-63 cells grown on PLLA discs,
cells were seeded on discs in 24-well plate (1.0  104 cells/well)
and incubated at 37
C with 5% CO2 for 3, 5, and 7 days. Culture
media were changed every 3 days. Cell proliferation was measured
using CCK-8 reagent. Briefly, CCK-8 (100 mL) was added to each
well and incubated at 37
C for 2 h. OD values of wells were
measured at wavelength of 450 nm using a microplate reader.
Measurement of ALP activity
ALP activities of MG-63 cells on PLLA discs were measured by
enzyme-linked immunosorbent assay. Briefly, cells were seeded
 Y.J. Yun et al. / Journal of Industrial and Engineering Chemistry 67 (2018) 244–254
onto PLLA discs in 24-well plate at density of 1.0  104 cells per
well. After 1 day of culture, the growth medium was replaced with
osteogenic differentiation medium and cells were further cultured
for 7, 14, and 21 days. Following differentiation for predetermined
time duration, ALP activity was determined in cultured media.
Supernatants were collected and mixed with 5 mM p-nitrophenyl
phosphate solution. These mixtures protected from light were then
incubated at 25
C for 60 min. Subsequently, the absorbance was
measured at 450 nm using a microplate reader.
Amount of calcium deposition
To evaluate the amount of calcium deposition, MG-63 cells were
seeded onto control PLLA and surface-modified PLLA discs and
cultured for 7,14, and 21 days using osteogenic culture medium. Cells
grown on these discs were washed with PBS three times and fixed for
10 min using 1 mL of 4% formaldehyde. Fixed cells were washed with
PBS again and stained with 1 mL of ARS solution (40 mM, pH 4.2) for
30 min. After removing the staining solution, ARS-stained cells were
washed three times with distilled water. For quantitative analysis,
stained ARS was extracted with 10% CPC solution. The OD was then
measured at 540 nm using a microplate reader.
OCN gene expression analysis
OCN gene expression was analyzed by real-time polymerase
chain reaction (real-time PCR). After 7, 14, and 21 days of
osteogenic differentiation, total RNA was extracted from MG-63
cells grown on control PLLA and surface-modified PLLA discs using
ReliaPrepTM RNA Cell Miniprep System. Then cDNA was synthe-
sized using SuperScript III first-strand synthesis system for RT-PCR
with AccuPower1 RT PreMix. PCR amplification was carried out for
Fig. 2. Implantation process of PLLA screw to a proximal tibia using rabbit animal model. Steps are as follows: (I) anesthetize the surgical site and shave; (II) open the proximal
tibia by incision; (III) drill a tibial head to insert a PLLA screw; (IV) install the PLLA screw in the drill hole; (V) suture the fascia and skin; (VI) disinfect and finish the surgery.
247
human OCN gene using the following primers: OCN forward (F) 50 -
GCA GGG TCA GGA GAA TC-30 , and OCN reverse (R) 50 -TTC CCT GTG
TCT TAG CAG TC-30 , product size 100 bp, annealing temperature
59
C. Real-time PCR amplification and detection were carried out
using a CFX96TM Real-Time PCR system (Bio-Rad, Hercules, USA).
Relative levels of OCN were normalized with GAPDH, forward (F)
50 - GAT TCC ACC CAT GGC AAA TT-30 , reverse (R) 50 - AAG ATG GTG
ATG GGA TTT CCA TT-30 , product size 83 bp, annealing temperature
54
C.
In vivo animal test
Six New Zealand white rabbits (2.5–3.5 kg, n = 3) were obtained
from DooYeol Biotech (Seoul, Republic of Korea) and used for
animal test (Fig. 2). Prior to surgery, these rabbits were
anesthetized with Zoletil1, Alfaxan1, and Rumpun1 at recom-
mended dosages. Their legs were depilated and disinfected with
Betadine1. Gentamicin sulfate and Ketoprofen were then injected
for anti-inflammatory and pain relief, respectively. The proximal
tibial metaphysis was exposed by incisions through the skin, fascia,
and periosteum. After inserting screw types of PLLA, PLLA/pDA/HA,
PLLA/pDA/Hep/BMP-2, PLLA/pDA/HA/Hep/BMP-2, fascia was su-
tured with Vicryl1 and skin incisions were closed with Ethilon1. At
4 weeks after the operation, rabbits were sacrificed by CO2 gas. The
implanted areas were harvested and fixed with 4% formaldehyde
for micro-computer tomography (micro-CT) and histological
analysis.
Micro-CT images
Screw implanted-tibia was visualized with a SkyScan 1173
micro-CT system (Bruker-CT, Kontich, Belgium). The tube voltage
248 Y.J. Yun et al. / Journal of Industrial and Engineering Chemistry 67 (2018) 244–254
and current were 70 kVp and 114 mA, respectively. The exposure
time was 500 ms. Images were taken at 0.3
increments over 180
.
Cross sections were reconstructed using NRecon (Ver 1.6.10.1).
Scanned images were aligned using a data viewer (Ver. 1.5.1.2).
Bone volume (BV) and bone volume to tissue volume (BV/TV) were
analyzed with a CT Analyzer (Ver. 1.14.4.1). A cylindrical volume of
interest (VOI) (diameter: 3.0 mm; height: 1.0 mm) was selected for
tissue around the PLLA screw. Bone volume was measured at
distance of 200 mm from the PLLA screw.
Histological evaluations
Harvested tissues were fixed with 4% formaldehyde solution for
2 weeks. Specimens were dehydrated in ethanol solutions (80, 90,
and 100%). Acrylic resin blocks were made with Technovit 7200
(Heraeus KULZER, Wehrheim, Germany). After polishing to
40  5 mm using a micro grinder EXAKT 400 CS (EXAKT Advanced
Technologies GmbH, Norderstedt, Germany), these blocks were
stained with hematoxylin and eosin (H&E) and Von Kossa. Stained
tissue slides were then visualized with an AX70 Fluorescence
Attached Microscope (Olympus, Tokyo, Japan).
Statistical analysis
All experiments were conducted at least three times. All
quantitative data were expressed as mean  standard deviation
(SD). One-way analysis of variance (ANOVA) was performed and
*p < 0.05 was considered statistically significant.
Institutional Review Board
Animal experimental protocols were approved by the Institu-
tional Animal Care and Use Committee (IACUC) in College of
Medicine, the Catholic University of Korea (CUMC-2017-0088-01).
Results
Surface chemical composition
XPS survey scan spectra of PLLA, surface treated PLLA discs
(PLLA/pDA/HA, PLLA/pDA/Hep/BMP-2, and PLLA/pDA/HA/Hep/
BMP-2) and their corresponding elemental atomic percentages
are shown in Fig. 3 and Table 1, respectively. PLLA showed C1s and
O1s spectra at 286 and 533 eV due to carbon and oxygen atoms in
its backbone chain. The survey scan of PLLA/pDA/HA revealed the
additional N1s, Ca2p, and P2p spectra, indicating the successful
Fig. 3. Wide-scan XPS spectra of PLLA, PLLA/pDA/HA, PLLA/pDA/Hep/BMP-2, and
PLLA/pDA/HA/Hep/BMP-2 discs.
Table 1
Chemical compositions of PLLA, PLLA/pDA/HA, PLLA/pDA/Hep/BMP-2, and PLLA/
pDA/HA/Hep/BMP-2. Calculated from XPS spectra of samples.
Samples Atomic percentage (%)
C1s O1s N1s Ca2p P2p S2p
PLLA 67.18 32.82 0 0 0 0
PLLA/pDA/HA 49.38 36.76 1.68 7.18 5 0
PLLA/pDA/Hep/BMP-2 67.08 24.78 6.78 0 0 1.37
PLLA/pDA/HA/Hep/BMP-2 60.79 31.19 2.79 2.16 1.41 1.67
coating of pDA with HA. The incorporation of Hep to pDA also
results in the S2p spectra at 168 eV. The immobilization of BMP-2
to Hep via specific interaction on the surface of PLLA/pDA/Hep/
BMP-2 and PLLA/pDA/HA/Hep/BMP-2 was confirmed by a decrease
in the C1s and an increase in the N1s compared to PLLA/pDA/HA
(Table 1).
Surface morphology, roughness, and wettability
Surface morphologies of control PLLA and surface-modified
PLLA discs were observed by SEM (Fig. 4). Control PLLA showed
smoother surface than surface-modified PLLA discs (Fig. 4a). In
SEM image of PLLA/pDA/Hep/BMP-2, thread-like texture was
observed due to coating of Hep/BMP-2 (Fig. 4b). Fig. 4c and d shows
the surface of PLLA/pDA/HA and PLLA/pDA/HA/Hep/BMP-2 pre-
pared by conventional dipping method. The surfaces are very
coarse and sparse, therefore, it is doubtful whether the deposited
HA show osteoconductivity. The surfaces prepared by using pDA
pre-coated HA slurry, on the other hand, exhibit dense and uniform
coating of HA (Fig. 4e and f). The HA particles on the surface of
PLLA/pDA/HA/Hep/BMP-2 seem to be larger than those on PLLA/
pDA/HA, probably because of the additional coating Hep/BMP-2
with pDA on the sample surface. Fig. 5 shows surface roughness of
control PLLA and surface-modified PLLA discs. Surface-modified
PLLA had larger surface roughness than PLLA. The surface
roughness parameters (Ra) of the samples, PLLA, PLLA/pDA/Hep/
BMP-2, PLLA/pDA/HA and PLLA/pDA/HA/Hep/BMP-2 were
2.97 nm, 13.29 nm, 38.71 nm and 50.60 nm, respectively. Surface
wettabilities of control PLLA and surface-modified PLLA discs were
examined by water contact angle measurement (Fig. 6). Surface
modification of HA and/or BMP-2 decreased the contact angle of
PLLA. Contact angles on PLLA, PLLA/pDA/HA, PLLA/pDA/Hep/BMP-
2, and PLLA/pDA/HA/Hep/BMP-2 were 85.3
, 52.2
, 47.0
, and 28.9
,
respectively. These results indicated that surface modifications
improved the surface wettability of PLLA. In particular, surface
modification with both HA and BMP-2 resulted in improved
surface wettability.
Release behaviors of BMP-2 and loading efficiencies
The loading efficiencies and release behaviors of BMP-2 onto
PLLA/pDA/Hep/BMP-2 and PLLA/pDA/HA/Hep/BMP-2 discs are
shown in Fig. 7. The loading efficiencies of the samples were 86.4%
(43.2 ng) and 85.4% (42.7 ng), respectively (Fig. 7a). This
demonstrates that the loading of BMP-2 is made through the
specific interaction between BMP-2 and heparin as described in
discussion section. In Fig. 7b, an initial burst release of BMP-2
from PLLA/pDA/Hep/BMP-2 and PLLA/pDA/HA/Hep/BMP-2 was
observed during the 1 day. After that, release behaviors of BMP-2
showed controlled release throughout 28 days in a sustained
manner. At day 1, cumulative release percentages of BMP-2 from
PLLA/pDA/Hep/BMP-2 and PLLA/pDA/HA/Hep/BMP-2 were
30.6  5.2% (13.2  1.7 ng) and 29.4  8.0% (12.6  4.0 ng), respec-
tively. Cumulative release percentages of BMP-2 from PLLA/pDA/
Hep/BMP-2 and PLLA/pDA/HA/Hep/BMP-2 for 28 days were
 Y.J. Yun et al. / Journal of Industrial and Engineering Chemistry 67 (2018) 244–254 249

Fig. 4. SEM images of surface-modified PLLAs. (a) PLLA, (b) PLLA/pDA/Hep/BMP-2, (c, d) controls of PLLA/pDA/HA and PLLA/pDA/HA/Hep/BMP-2 which conventionally
dipping to DA solution with HA and (e, f) PLLA/pDA/HA and PLLA/pDA/HA/Hep/BMP-2 which plastering pDA-coated HA, observed at 30.0k.

Fig. 5. AFM images of (a) PLLA, (b) PLLA/pDA/Hep/BMP-2, (c) PLLA/pDA/HA, and (d) PLLA/pDA/HA/Hep/BMP-2 discs.

72.9  1.9% (31.5  3.5 ng) and 69.9  5.6% (30.0  5.5 ng), respec-
tively.
In vitrocell proliferation, ALP activity, calcium deposition, and OCN
gene expression level
Results of in vitro cell proliferation, ALP activity, calcium
deposition, and OCN gene expression level for MG-63 cells
cultured on control PLLA and surface-modified PLLA discs are
shown in Fig. 8. Cell proliferation rate was increased gradually
throughout the culture period (Fig. 8a). At 5 and 7 days, surface-
modified PLLAs exhibited the increased cell proliferation rates
compared to control PLLA. Among them, PLLA/pDA/HA/Hep/BMP-
2 sample showed the highest cell proliferation rate. ALP activities
of MG-63 cells on the samples surfaces were increased until 14
days and decreased thereafter (Fig. 8b). In particular, cells on PLLA/
pDA/HA/Hep/BMP-2 showed the highest ALP activities among
samples, that is, ALP activities for cells on PLLA/pDA/HA/Hep/BMP-
2 at 14 days were 1.63-, 1.47-, and 1.14-fold higher than those for
cells on PLLA, PLLA/pDA/HA, and PLLA/pDA/Hep/BMP-2, respec-
tively. Gradual increases in the amount of calcium deposition were
observed in control PLLA and surface-modified samples through-
out the culture period (Fig. 8c). Until 14 days, similar amounts of
calcium were deposited, and the priority between the samples
250 Y.J. Yun et al. / Journal of Industrial and Engineering Chemistry 67 (2018) 244–254
Fig. 6. Water contact angles on PLLA, PLLA/pDA/HA, PLLA/pDA/Hep/BMP-2, and
PLLA/pDA/HA/Hep/BMP-2 discs.
could not be figured out. At 21 days, however, surface-modified
PLLAs showed the sharp increase in calcium accumulation
compared to control PLLA. The amount of calcium deposited on
PLLA/pDA/HA, PLLA/pDA/Hep/BMP-2, and PLLA/pDA/HA/Hep/
BMP-2 were 1.93-, 2.07-, and 2.57-fold greater than on control
PLLA. In the case of OCN gene expression level, a similar pattern to
calcium deposition was observed (Fig. 8d). At 14 and 21 days, OCN
gene expression levels of surface-modified PLLAs were higher
than control PLLA. At 14 days, the levels of OCN gene of PLLA,
PLLA/pDA/HA, PLLA/pDA/Hep/BMP-2 and PLLA/pDA/HA/Hep/
BMP-2 were slightly increased. At 21 days, the OCN gene levels
of PLLA/pDA/HA, PLLA/pDA/Hep/BMP-2, and PLLA/pDA/HA/Hep/
BMP-2 were 1.89-, 3.08-, and 5.15-fold higher than control PLLA.
Eventually, PLLA/pDA/HA/Hep/BMP-2 sample showed the best
performance for all in vitro tests regarding proliferation and
differentiation.
Fig. 7. (a) BMP-2 loading efficiencies to PLLA/pDA/Hep and PLLA/pDA/HA/Hep after 24 h. (b) Release behaviors of BMP-2 from PLLA/pDA/Hep/BMP-2 and PLLA/pDA/HA/Hep/
BMP-2 samples in PBS (pH 7.4).
Micro-CT images
Newly formed bones were observed at the interfaces between
host bones and PLLA screws (Fig. 9). Surface-modified PLLA screws
produced larger amounts of new bone at the interfaces between
host bone and screw than control PLLA. To determine the amount
of bone formation, BV and BV/TV were measured (Fig. 10). Surface-
modified PLLA exhibited greater BV and BV/TV than control PLLA.
The BV of PLLA, PLLA/pDA/HA, PLLA/pDA/Hep/BMP-2, and PLLA/
pDA/HA/Hep/BMP-2 was 0.22  0.08, 0.44  0.02, 0.54  0.07, and
0.69  0.04 mm3, respectively. The BV/TV ratio of PLLA, PLLA/pDA/
HA, PLLA/pDA/Hep/BMP-2, and PLLA/pDA/HA/Hep/BMP-2 was
23.75  8.20, 46.07 4.59, 55.99  6.19, 68.73  3.07%, respectively.
In particular, PLLA/pDA/HA/Hep/BMP-2 showed the most im-
proved bone formation at the interface. The BV and BV/TV ratio of
PLLA/pDA/HA/Hep/BMP-2 was 3.18- and 3.21-fold higher than
PLLA, respectively.
Histological evaluations
H&E and Von Kossa stained images showed new bone
formation of tissues around control PLLA and surface-modified
PLLA screws implanted in tibia head for 4 weeks. Osteoblasts,
osteoid, osteocytes, and lacunae were observed on the implant-
bone interfaces (Fig. 11a–d ). Empty and osteocyte-occupied
lacunae were found at the host bone. Few inflammatory cells were
observed, and bone lining cells were mainly covered the clear bone
surface (yellow arrow). Osteoblasts formed osteoid, known as
immature bone matrix, across the interfaces between host bone
and PLLAs (green arrow). The Mineralized bone matrix was
distinguished by Von Kossa staining in black (Fig. 11a0 –d0 ).
Compared to control PLLA, surface-modified PLLA screws exhibited
large amounts of bone mineralization at interfaces. In particular,
PLLA/pDA/HA/Hep/BMP-2 had a superior bone-implant contact at
the interface.
Discussion
The main benefit of the use of the absorbable PLLA orthopedic
device is to avoid the necessity of second surgery for removal.
However, PLLA has to persist for years in vivo after its surgical role
has ended. Therefore, an early bone fixation through osseointe-
gration at the implant-host bone interface is necessary during the
 Y.J. Yun et al. / Journal of Industrial and Engineering Chemistry 67 (2018) 244–254 251

Fig. 8. (a) In vitro cell proliferation rates of MG-63 cells cultured on PLLA, PLLA/pDA/HA, PLLA/pDA/Hep/BMP-2, and PLLA/pDA/HA/Hep/BMP-2 discs for 3, 5, and 7 days, (b)
levels of ALP activity, (c) amounts of calcium deposition, and (d) levels of OCN gene expression on PLLA, PLLA/pDA/HA, PLLA/pDA/Hep/BMP-2, and PLLA/pDA/HA/Hep/BMP-2
discs cultured with MG-63 cells for 7, 14, and 21 days.

initial tissue reaction period [23]. The introduction of HA to the
implant may take the priority over the candidates, because the ion
exchange with host tissue leads to the formation of a chemical
bond along the interface, so called bonding osteogenesis [24]. The
resulting CaP-rich layers at the interface are osteoconductive and
thus act as scaffolds allowing bone growth [25–27]. In addition to
HA, BMP-2 can be a key factor for neo bone formation at the
interface as osteoinductive candidate. BMP-2 has stem cell-
recruiting ability, and is demonstrated to potently induce
osteoblast differentiation in a variety of cell types [28]. In the
present study, in order to provide osteoinductive and osteocon-
ductive capability to PLLA device, the surface of PLLA samples were
modified with BMP-2 and HA using DA. The pDA can be a good
material due to its excellent coating ability for biomolecules
[21,29]. The coating of pDA can be made by the instant
polymerization of DA on the materials surface at pH 8.5 [30].
During coating process, biomolecules are anchored to pDA layer
through Michael addition and Schiff’s base reaction with catechol
and/or diketone groups in the layer [15,31]. By virtue of pDA, PLLA
surface could be readily heparinized and ionically coupled with
BMP-2 resulted in smooth surface (Figs. 4b and 5b). In the case of
HA, however, no dramatic coating could be made through
conventional dipping method (Fig. 4c and d). There is no
uniformity in morphology and arrangement of HA on the PLLA
surface. Moreover, the bonding strength between HA and PLLA
does not seem to be tight enough to be used as implantable
devices. This phenomenon is probably because HA has no
nucleophilic group to the pDA layer, therefore, uniformly anchor-
ing HA to pDA is hardly achieved. The electrostatic interactions
between Ca and P can be another factor that hinders HA coating to
PLLA surface because (surface) HA powder was dissociated into
calcium cation and phosphate anion in aqueous solution, in turn
tend to aggregate rather than disperse [32–34]. In this work, HA
particles were pre-coated with pDA to overcome the inherent
drawbacks of HA for coating. The coating with pDA provide the
amine groups exposed to the surface of HA that not only interfere
the aggregation of calcium and phosphate ions but also allow
Michael addition and Schiff’s base reaction with catechol or
diketone groups for uniform and homogeneous anchoring (Fig. 4e
and f) [29].
Surface properties of modified PLLA discs including morpholo-
gy, roughness, and wettability were confirmed by SEM, AFM, and
contact angle measurement, respectively (Figs. 4–6). The results
showed that surface modification using HA and BMP-2 improved
surface roughness and hydrophilicity. It is known that surface
properties are closely related to adhesion and proliferation of
osteoblasts, thus affecting the improvement of bone formation
[35]. The hydrophilic property as well as surface roughness of solid
252 Y.J. Yun et al. / Journal of Industrial and Engineering Chemistry 67 (2018) 244–254

Fig. 9. Scheme of rabbit tibia model, micro-CT, and reconstructed 3D images. Volume of interest (VOI) (3.0 mm diameter and 1.0 mm height) was selected for host bone. Bone
volume was measured at a distance of 200 mm around the PLLA screw. Sagittal, axial and coronal views of micro-CT images are shown. Reconstructed 3D images for (a–d) host
bone and new bone with screws, (a0 –d0 ) host bone and new bone without screws, and (a00 –d00 ) new bone (gray, screws; orange, host bone; red, new bone) are shown at 4 weeks
after implantation into the tibia head. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 10. (a) Amounts of BV on PLLA, PLLA/pDA/HA, PLLA/pDA/Hep/BMP-2, and PLLA/pDA/HA/Hep/BMP-2 screws at 4 weeks after implantation. (b) Percentages of BV/TV on
PLLA, PLLA/pDA/HA, PLLA/pDA/Hep/BMP-2, and PLLA/pDA/HA/Hep/BMP-2 screws at 4 weeks after implantation.

substrates provides suitable topographical environments to
improve bone formation because various proteins and growth
factors are attached to these substrates, thereby enhancing
adhesion and proliferation of osteoblasts [36].
The loading efficiency of BMP-2 on PLLA samples is important.
Many materials such as gels, scaffolds, and composite have been
used for BMP-2 delivery. However, they often fail to effective
delivery of proteins due to their low loading efficiency [37]. This
problem can be solved by modifying the BMP-2 delivery system
using Hep. It is well known that BMP-2 can bind to Hep with high
affinity and maintain its biological activity [38,39]. Results of
loading efficiency of BMP-2 suggest that PLLA/pDA/Hep/BMP-2
and PLLA/pDA/HA/Hep/BMP-2 has an effective BMP-2 delivery
system. Controlled release of BMP-2 in a sustained manner with
initial burst plays a critical role in improving bone formation at the
interface with host bone. Initial burst stimulates the initiation of
bone formation while the sustained manner continuously main-
tains bone formation [40]. However, the use of BMP-2 alone has
some problems due to its short half-life, low bioactivity in solution,
and the need for high dose of BMP-2 for clinical treatment [41].
Conjugation of Hep on PLLA can overcome such problems of BMP-
2. Heparin, as a highly sulfated linear polysaccharide, has strong
binding affinity to peptide 1-17 representing the N-terminal part of
BMP-2, therefore, it not only provides excellent loading capacity of
 Y.J. Yun et al. / Journal of Industrial and Engineering Chemistry 67 (2018) 244–254
Fig. 11. (a–d) H&E stained and (a0 –d0 ) Von Kossa stained images for surrounding tissues around (a, a0 ) PLLA; (b, b0 ) PLLA/pDA/HA; (c, c0 ), PLLA/pDA/Hep/BMP-2; and (d, d0 )
PLLA/pDA/HA/Hep/BMP-2 screws at 4 weeks after implantation into the tibia head.
Scale bar = 200 mm.
BMP-2 but also allows initial burst as well as sustained controlled
release of BMP-2 [39]. The results turned out as expected (Fig. 7);
both PLLA/Hep/BMP-2 and PLLA/pDA/HA/Hep/BMP-2 samples
showed the BMP-2 loading efficiencies more than 85% and the
initial burst of BMP-2 within one day followed by controlled
release in a sustained manner. In addition, there were only a little
difference in BMP-2 loading efficiencies and release behaviors
between PLLA/pDA/Hep/BMP-2 and PLLA/pDA/HA/Hep/BMP-2,
convincingly demonstrating the superiority of heparin in the
delivery of BMP-2 in the samples used in this study.
To evaluate cellular response on surface-modified PLLAs, in vitro
tests, including cell proliferation rate, ALP activity, calcium
deposition, and OCN gene expression were carried out using
MG-63 cell line. The surface-modification of HA promoted cell
growth (Fig. 8a) which could be attributed that the enhanced
osteoblasts adhesion on HA surfaces induced activation of
proliferative genes such as c-fos and c-jun [42–44]. The samples
containing BMP-2, a proven strong osteoinductive factor, also
increased the levels of osteogenic differentiation markers (ALP and
OCN) because the BMP-2 binds BMP receptors, in turn, activate
Smad proteins that stimulate osteogenic differentiation directly or
via Runx-2 (Fig. 8b–d) [45,46]. In particular, PLLA/pDA/HA/Hep/
BMP-2 sample showed the best performance for all in vitro tests
among samples. This can be probably because of the cocktail effect
of collaboration between the inductive and conductive factors. As
demonstrated in our previous report [28], the enhanced adhesion
by HA cannot cover the whole process of osteogenesis, however, it
is a primary factor for osseointegration [43,47–50].
In vivo animal test was conducted using a rabbit tibia defect
model to investigate the effect of surface modifications using HA
and/or BMP-2 on the improvement of bone formation. In rabbit
tibial defect model, bone formation occurs strong enough to
support implant at 4 weeks [51,52]. Hence, PLLA screws were
implanted in the tibial head of rabbit for 4 weeks to examine bone
formation. Quantitative micro-CT analysis exhibited that the HA
and BMP-2 induced a noticeable bone formation at the interface
(Figs. 9 and 10). These results were consistent with previous
reports that osteoconductive HA and osteoinductive BMP-2 act
synergistically to induce bone regeneration [28,53]. H&E stained
images showed no evidence of inflammatory cells at the host bones
(Fig. 11) [54,55]. In particular, the osteoblasts and bone lining cells
were presented on the smooth bone surface [56]. Osteoblast begin
253
osteoblast, osteoid, osteoblast lining cell, osteocyte and 4 mineralized bone.
the process of forming bone tissue by secreting unmineralized
bone matrix (osteoid). When the osteoid becomes mineralized,
adjacent osteoblasts have developed into new bone tissue [57]. The
Von Kossa stained images provided information on bone minerali-
zation at these interfaces indicated as black color. In the PLLA/pDA/
HA samples, mineralization was observed to progress toward the
screw due to the osteoconductivity of HA (Fig. 11b0 ). In the PLLA/
pDA/Hep/BMP-2 samples, the gap between the screw and host
bone seemed to be wide, but the bone mineralization was further
proceeded than PLLA/pDA/HA due to the osteoinductivity of BMP-2
(Fig. 11c0 ). In addition, the bone mineralization close to the PLLA/
pDA/HA/Hep/BMP-2 screw surface suggested a combination of HA
and BMP-2 contributed to the strong osseointegration (Fig. 11d0 ).
Conclusion
In this study, surface modified poly L-lactic acid (PLLA) samples
with hydroxyapatite (HA), heparin and bone morphogenetic
protein-2 (BMP-2) mediated by polydopamine (pDA) coating
(PLLA/pDA/HA/Hep/BMP-2) were successfully prepared. Pre-coat-
ing of HA particles with pDA provided uniform and homogeneous
anchoring of particles to PLLA surface. In addition, the strong ionic
interaction between heparin and pDA led PLLA surface readily
heparinized for loading of BMP-2. In vitro test revealed that the
proliferation rates as well as the osteogenic differentiation markers
of MG-63 cells were remarkably increased in PLLA/pDA/HA/Hep/
BMP-2 than other groups because of the synergistic effect of HA on
the adhesion and proliferation, with BMP-2 on differentiation. In
vivo animal tests further demonstrated that a combination of HA
and BMP-2 contributed to the strong osseointegration.
Acknowledgment
This research was supported by the grant of Ministry of Trade,
Industry and Energy (MOTIE) (Grant no. 10047811).
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