International Journal of Biological Macromolecules 76 (2015) 10–24

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International Journal of Biological Macromolecules

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

Bone formation of a porous Gelatin-Pectin-biphasic calcium

phosphate composite in presence of BMP-2 and VEGF
Jhaleh Amirian a , Nguyen Thuy Ba Linh a,c , Young Ki Min b,c , Byong-Taek Lee a,c,∗
a
Department of regenerative medicine, College of Medicine, Soonchunhyang University366-1, Ssangyong-dong, Cheonan-City,
ChungCheongNam-Do 330-090, Republic of Korea
b
Department of Physiology, College of Medicine, Soonchunhyang University366-1, Ssangyong-dong, Cheonan-City, ChungCheongNam-Do 330-090,
Republic of Korea
c
Institute of Tissue Regeneration, Soonchunhyang University366-1, Ssangyong-dong, Cheonan-City, ChungCheongNam-Do 330-090, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history: A composite scaffold of gelatin (Gel)-pectin (Pec)-biphasic calcium phosphate (BCP) was fabricated for the
Received 21 August 2014 successful delivery of growth factors. Bone morphogenetic protein-2 (BMP-2) and vascular endothelial
Received in revised form 9 February 2015 growth factor (VEGF) were coated on the Gel-Pec-BCP surface to investigate of effect of them on bone
Accepted 9 February 2015
healing. Surface morphology was investigated by scanning electron microscopy, and BCP dispersion in
Available online 20 February 2015
the hydrogel scaffolds was measured by energy dispersive X-ray spectroscopy. The results obtained from
Fourier transform infrared spectroscopy showed that BMP-2 and VEGF were successfully coated on Gel-
Keywords:
Pec-BCP hydrogel scaffolds. MC3T3-E1 preosteoblasts were cultivated on the scaffolds to investigate the
Freeze-drying
Pectin
effect of BMP-2 and VEGF on cell viability and proliferation. VEGF and BMP-2 loaded on Gel-Pec-BCP
Biphase Calcium Phosphate (BCP) scaffold facilitated increased cell spreading and proliferation compared to Gel-Pec-BCP scaffolds. In vivo,
Gelatin bone formation was examined using rat models. Bone formation was observed in Gel-Pec-BCP/BMP-2 and
VEGF Gel-Pec-BCP/VEGF scaffolds within 4 weeks, and was greatest with Gel-Pec-BCP/BMP-2 scaffolds. In vitro
BMP-2 and in vivo results suggest that Gel-Pec-BCP/BMP-2 and Gel-Pec-BCP/VEGF scaffolds could enhance bone
regeneration.
© 2015 Elsevier B.V. All rights reserved.

1. Introduction a carrier for growth factors that include bone morphogenetic pro-
tein (BMP), vascular endothelial growth factor (VEGF), and other
Recent strategies for bone regeneration applied to bone tissue growth factors [14]. Moreover, adding ceramics, such as calcium
engineering have provided promising alternatives to use of allo- phosphate, biphase calcium phosphate (BCP), ␤-tricalcium phos-
grafts and autografts, which are limited by complications including phate (␤-TCP) [12,15], and hydroxyapatite (HAp) [12,16] to the
donor site morbidity, disease transmission, and immunological polymer can increase the osteoconductivity and bioactivity in the
rejection [1–6]. In bone tissue engineering, the main purpose is composite [7]. Composite scaffolds have attracted more atten-
designing a biocompatible porous scaffold with high interconnec- tion because of the specific advantages conferred by both the
tivity that can provide an appropriate environment for nutrient polymer and ceramic properties, such as flexibility, biocompati-
delivery and waste removal. In addition, pore sizes >300 ␮m along bility, osteoconductivity, and capacity as effective drug reservoirs
with interconnectivity are essential for the attachment of cells with [5,17–19].
sufficient separation to permit nutrient delivery, with the aim of Gel as a denatured collagen has been extensively utilized for
creating new bone tissue [4,5,7]. pharmaceutical and medical applications, given that its biosafety
Different types of polymer matrix have been used for bone tissue has been proven in long-term clinical applications [13,20,21].
engineering. These include gelatin (Gel), collagen [8], hyaluronic Furthermore, the ease of chemical modification of Gel and its
acid (HA)[9], and pectin [10,11] as natural polymers, and polylac- commercial availability have fueled the extensive application as
tic acid (PLA), polyglycolic acid (PGA), and their copolymer (PLGA) a biomaterial in tissue engineering and drug delivery [13,20,21].
[12] as synthetic polymers [5,12,13]. In addition, they can act as Pectin (Pec) is also an excellent carbohydrate polymer derived
mainly from the structural component of the plant cell wall. Pec
facilitates homogenous immobilization of cells, genes, and proteins,
∗ Corresponding author. Tel.: +82 41 5702427; fax: +82 5772415. which makes Pec a suitable carrier for drug delivery applications
E-mail address: [email protected] (B.-T. Lee). [10,22–24].

http://dx.doi.org/10.1016/j.ijbiomac.2015.02.021
0141-8130/© 2015 Elsevier B.V. All rights reserved.
 J. Amirian et al. / International Journal of Biological Macromolecules 76 (2015) 10–24 11

Furthermore, growth factors are also vital in bone tissue engi- polyethylene mold with a diameter of 12 mm and a height of 2 cm.
neering. Among growth factors, BMPs have gained more attention The slurry was frozen at −20 ◦ C for 10 h and then placed in a freeze-
due to their simulation of new bone formation and osteoin- dryer for 2 days. The porous scaffolds were cross-linked using EDC
ductive properties [4,6,14,25,26]. VEGF is another growth factor; (N-(3-dimethylaminopropyl) N -ethylcarbodimide hydrochloride;
its osteogenic and angiogenic properties are valuable in bone Sigma-Aldrich) and NHS (N-hydroxysuccinimide; Sigma–Aldrich)
regeneration. In vivo, neovascularization is necessary for new at a 5:2 molar ratio in 80% ethanol overnight at 4 ◦ C. This step was
bone formation and blood supply [4,6,14,26]. Previous studies repeated to stabilize the scaffolds so they would not degrade dur-
have focused on enhancing the angiogenic potential in bioma- ing cell culture. The scaffolds were washed five times with distilled
terials by incorporating of growth factors, such as VEGF, into water for 15 min to remove residual EDC, frozen at −20 ◦ C for 3 h,
the carrier matrix. Generally, ideal scaffold matrices in cell/drug and then dried [29].
delivery should fulfill requirements that include biocompatibil-
ity [5], biodegradability, and appropriate mechanical properties 2.3. Coating of scaffolds with BMP-2 and VEGF
depending on the application. Such properties include high surface-
to-volume ratio, interface adherence, interconnected porosity rhBMP-2 and rhVEGF were individually coated on scaffolds by
[4,5,7] and appropriate pore size for delivery of nutrients and export drop-wise addition of 10 ␮g/ml solutions in 1% BSA to the surface of
of wastes, capability to mimic the native extracellular matrix, ease Gel-Pec-BCP freeze-dried scaffolds for 24 h at 4 ◦ C and 50 rpm. Then
of processing, avid binding of drug to scaffold, controlled release scaffolds were stored at −20 ◦ C for 12 h and freeze-dried for 24 h.
of drugs from scaffold, and physiological stability [27]. Scaffolds Fig. 1(a) and (b) was drawn according to experiments and reactions
constructed of Gel-Pec-BCP and a combination of growth factors, done in this study.
such as BMP-2 and VEGF, with Gel-Pec-BCP, Gel-Pec-BCP/BMP-2,
and Gel-Pec-BCP/VEGF may be ideal for bone tissue engineering. 2.4. Characterization
This study was conducted with the purpose of developing a
freeze-dried Gel-Pec-BCP system for delivery of BMP-2 and VEGF, 2.4.1. Scaffold morphology
for preparation of Gel-Pec-BCP/BMP-2 and Gel-Pec-BCP/VEGF. The morphology of the scaffold was characterized using scan-
BMP-2 and VEGF were separately coated on the surface of Gel-Pec- ning electron microscopy (SEM) using a JSM-6701F microscope
BCP and the effect on the physical properties in vitro and in vivo (JEOL, Japan) equipped for (EDS). A small part of scaffold was placed
were examined. A range of functional and histological analyses was on the SEM sample holder and sputter coated with platinum. An
applied for evaluation of the in vivo effects of BMP-2 and VEGF accelerating voltage of 10 kV was used.
coating in a rat cranial defect model.
The hypothesis driving the study was that coating of Gel-Pec- 2.4.2. Mercury intrusion porosimetry
BCP scaffolds with BMP-2 and VEGF as a unique system will A mercury porosimeter (PoremasterTM ; Quantachrome Instru-
significantly enhance and accelerate bone repair within a critical- ments, FL, USA) was used to analyze the porosity and pore size
size defect during 4 weeks. The scaffolds were prepared to improve distribution of the scaffolds. Low pressure intrusion porosime-
vascularization and bone formation observed in vitro and in vivo. try was used to obtain the interconnected macro pore diameter
and porosity, as well the width × length × height (5 × 6 × 6 mm)
2. Material and methods dimensions. For each different pore diameter scaffold, three mea-
surements were performed.
2.1. Materials
2.4.3. Fourier Transform Infrared (FTIR) Spectroscopy
Gelatin (Sigma-Aldrich, USA), pectin from citrus (Pec; The chemical compositions of the samples were determined by
Sigma–Aldrich, Denmark), and BCP were used as starting mate- FTIR using a C1000 Thermal Cycler.
rials for fabrication of the Gel-Pec-BCP composite scaffold. BCP
powder was synthesized using a microwave assisted process 2.4.4. Biodegradation and swelling
with a particle size of around 90–100 nm [28]. Recombinant Samples were immersed in PBS for 1, 3, 5, 10, 15, 20, 25, 30, 40,
human BMP-2(rhBMP-2), recombinant human VEGF (rhVEGF) and 50 days. Three samples were considered for each type of scaf-
and enzyme-linked immunosorbent assay (ELISA) kits for rhBMP- fold. At the end of each immersion period, samples were washed
2 and rhVEGF were purchased from R&D Systems (USA). For with deionized water. Weight loss was determined by drying the
in vitro study, bovine serum albumin (BSA; Sigma–Aldrich, samples to a constant weight and comparing to their initial weight
USA), dimethylsulfoxide 99.0% (DMSO; Samchun Pure Chemical, according to the following equation:
Korea), ethanol (Merck, Germany), fetal bovine serum (FBS), peni- (W0 − Wt )
cillinstreptomycin antibiotic (PS), 3-[4,5-dimethylthiazol-2-yl]-2,5 Pt = × 100% (1)
W0
diphenyltetrazolium bromide (MTT) solution, trypsin-EDTA (all
from Gibco, CA), phosphate buffered saline tablets (PBS; Amresco, Where Wt is the weight of each sample at time t (1, 3, 5, 10, 15, 20,
Korea), and Minimum Essential Medium (MEM; Gibco, USA) were 25, 30, 40, and 50 days) and W0 is the initial weight of each sample.
used. MC3T3-E1 cells were derived from mouse preosteoblast cells The percent weight loss, Pt , was measured with Eq. (1).
obtained from the American Type Culture Collection (ATCC, USA). To consider of swelling behavior of the three types of scaffold,
dried samples were weighed and then immersed in PBS for 1, 2, and
3 days. The samples were removed at the specific intervals, gently
2.2. Preparation of Gel-Pec-BCP scaffolds blotted with tissue paper to remove the excess surface water, and
the weight was recorded. This process was repeated at several time
Gelatin (5 wt.%) was dissolved in distilled water at 40 ◦ C. The intervals. At the end of the test period, the samples were weighed
same amount of Pec (5 wt.%) was dissolved in water, added to and compared against the original sample weight according to the
the gel solution, and mixed for 3 h. BCP powder (5 wt.%) was following equation:
added to deionized water and added to the Gel-Pec solution until
it became a slurry. The slurry was mixed with a magnetic stir- (Wt − W0 )
Pa = × 100% (2)
rer for 3 h. After mixing, 2 ml of the slurry was poured into a W0
12 J. Amirian et al. / International Journal of Biological Macromolecules 76 (2015) 10–24

Fig. 1. Schematic overview the (a) current manufacturing process of Gel-Pec-BCP, Gel-Pec-BCP/BMP-2 and Gel-Pec-BCP/VEGF scaffolds and in vitro and in vivo design, and
(b) crosslinking method were used for Gel-Pect cross-linking by EDS/NHS.
 J. Amirian et al. / International Journal of Biological Macromolecules 76 (2015) 10–24 13

Fig. 2. Scanning electron micrographs of the surface of the scaffolds: (a) and (b) low and high magnification of Gel-Pec-BCP scaffolds porous structure, (c) high resolution
image of Gel-Pec-BCP inserted EDS profile, (d) pore size distribution histogram of the Gel-Pec-BCP composite, and (e1), (e2), (e3), and (f) interconnectivity in three X, Y and
Z direction and porosity percent value graph of Gel-Pec-BCP.
14 J. Amirian et al. / International Journal of Biological Macromolecules 76 (2015) 10–24

Fig. 3. Fourier transform infrared analysis of Gel-Pec-BCP, Gel-Pec-BCP coated BMP-2 loaded VEGF scaffolds.

Where Wt is the weight of each sample at time t (1, 2, and 3 days) at 37 ◦ C under 5% CO2 for 1, 3, and 7 days. After the incubation, MTT
and W0 is the initial weight of each sample. The percent of PBS solution (100 ␮l of 5 mg/ml) was added to each well, and the plate
uptake, Pa , was measured with Eq. (2). was incubated at 37 ◦ C for 4 h. Finally, the solution was removed
from each well, 1000 ␮l of DMSO was added to each well, and incu-
2.5. In vitro BMP-2 and VEGF release bated for 1 h to allow formation of the purple formazan crystals.
Absorbance of the solution was determined at 595 nm using an
The levels of BMP-2 and VEGF coated on Gel-Pec-BCP hydro- ELISA reader. Samples were tested in quadruplicate.
gel were determined by BMP-2 ELISA and VEGF ELISA kits (R&D
Systems, USA), respectively. To evaluate the amount of BMP-2 and 2.6.2. Live–dead assay
VEGF released from Gel-Pec-BCP/BMP-2 and Gel-Pec-BCP/VEGF Cells (1 × 105 ) were seeded on the scaffolds in 24 well plates.
hydrogel scaffolds, specimens were soaked in 1 ml PBS at 37 ◦ C. After 1, 3, and 7 days of culture in a humidified CO2 incubator
Supernatant was collected and replaced with fresh PBS at 1, 2, providing 5% CO2 at 37 ◦ C, scaffolds were washed twice with PBS
3, 5, 7, 10, and 15 days. The absorbance of BMP-2 and VEGF and stained with 0.4 mM ethidium homodimer-1 (Invitrogen, USA)
were determined with the aforementioned ELISA kits according to and 0.1 mM Calcein AM (Invitrogen) for 5 min at room temper-
the manufacture’s instruction using an ELISA EL 312 Biokinetics ature. After washing with PBS, the scaffolds were visualized by
microplate reader (Bio-Tek Instruments, USA) at a wavelength of confocal fluorescence microscopy using a model FV10i-W micro-
450 nm. scope (Olympus, USA). Images were analyzed using FV10i-ASW 2.0
Viewer software.
2.6. Cell culture and seeding
2.6.3. Cell proliferation
MCT3T3-E1 preosteoblasts were passaged in a primary medium Cells (1 × 105 ) were seeded on each scaffold in 24 well
consisting of MEM, 10% FBS, and 1% antibiotics (penicillin 100 U/ml, plates. After 1, 3, and 7 days of CO2 culture as described
streptomycin 100 U/ml). Cells recovered after passage three were above, scaffolds were washed three times with PBS and
seeded onto the three types of hydrogel scaffold. The medium was then fixed by 4% paraformaldehyde (Sigma–Aldrich) for 10 min
replaced 3 times in a week. at room temperature. After washing with PBS, the cells
were permeabilized with 0.5% Triton X-100 (Sigma–Aldrich)
2.6.1. Cell viability assay for 10 min. The procedure used 2.5% of BSA for 60 min as
Viability of cells on Gel-Pec-BCP, Gel-Pec-BCP-BMP-2, and Gel- blocking reagent. Cells were immunostained using fluores-
Pec-BCP-VEGF scaffolds was determined using the MTT assay and cein isothiocyanate (FITC) conjugated phalloidin (25 ␮m/ml;
a standard testing protocol [ISO 10993-5:2009(E)]. The specimens Sigma–Aldrich) overnight in 4 ◦ C in the dark. Nuclei were
were placed in a 24-well culture plate. In brief, the samples were stained with 1 ␮g/ml Hoechst 33342 (2’-[4-ethoxyphenyl]-5-
immersed in 70% ethanol for 15 min to sterilize them. The sam- [4-methyl-1-piperazinyl]-2,5’-bi-1H-benzimidazole trihydrochlo-
ples were washed in PBS to eliminate the ethanol. One milliliter of ridetrihydrate; Sigma–Aldrich). The scaffolds were visualized
the MC3T3-E1 preosteoblast suspension (about 1 × 104 cells) was using confocal fluorescence microscopy and images analyzed as
placed on the samples. The cell-seeded scaffolds were maintained described above.
 J. Amirian et al. / International Journal of Biological Macromolecules 76 (2015) 10–24 15

Fig. 4. (a) In vitro degradation behavior of scaffold in PBS solution at 37 ◦ C for period time, and PBS uptake of (b) porous Gel-Pec-BCP, Gel-Pec-BCP coated BMP-2 and
Gel-Pec-BCP coated VEGF at 37 ◦ C.

2.7. In vivo study the skin was closed with a 4-0 sutures and antibiotics were injected.
Rats were euthanized 2 weeks and 4 weeks post-operatively. After
Eighteen Rattus norvegicus male rats (9 weeks old, 300–350 g) euthanasia, rat skulls were harvested and the surfaces of the cranial
were used as animal experimental model (three rats for each group defect were analyzed and described macroscopically. The protocol
for 2 and 4 weeks). Animals were housed individually in standard was approved by the Animal Ethical Committee of Soonchunhyang
rat cages with an ambient temperature of 24 ◦ C to 26 ◦ C, and a University for care and use of laboratory animals.
12/12 h light/dark cycle. The animals had free access to drinking
water and standard laboratory pellets. The rats were anesthetized
by diethyl ether (Daejung, South Korea). The dorsal area of each 2.7.1. Micro-computed tomography (micro-CT)
rat’s cranium was shaved before the surgery, and the surgical field The samples and the surrounding bone tissue were fixed in 10%
was prepared with povidone iodine. After exposure of the partial formalin solution for micro-CT M analysis and histopathology study
skull, defects on both side of rat skull were made using a trephine for 2 weeks and 4 weeks after implantation using a model 1076
drill Critical size defect were 5 mm diameter for 1 month [30,31]. apparatus (Skyscan, Belgium). Micro-CT was used to observe the
This defect diameter cannot be used any longer than 30 days in rats new bone formation in defected skull sites. Each sample was fixed
[30]. Right defect was grafted with a hydrogel scaffolds (samples) on the object stage, and imaging was performed on the sample
and the left side without sample as a negative control. At the end, for 360◦ of rotation with an exposure time of 20 min. Micro-CT
16 J. Amirian et al. / International Journal of Biological Macromolecules 76 (2015) 10–24

Fig. 5. Cumulative release profiles of BMP-2 and VEGF from (a) Gel-Pec-BCP/BMP-2 and(b) Gel-Pec-BCP/VEGF scaffolds. Error bars represent mean ± standard deviation.

were reconstructed over the region of interest (ROI) using CTAn Where g is the graft remains each sample at 2 and 4 weeks and W is
(SKYscan) and CTVol (SKyscan) to make three-dimensional (3D) whole area of defect. The graft remains percent, GR, was measured
images. The bone volume (BV/TV, %) was calculated from the total with Eq. (4).
bone (TV) and bone volume (BV) to evaluate the new bone quantity,
and the bone surface density (BS/TV, mm−1 ) was calculated from 2.7.3. Immunohistochemistry study
the bone surface (BS) and as a measure of bone surface density. Each section was cut at a thickness of 5 ␮m. Sections were
deparaffinized in xylene, hydrated in descending alcohol, and
2.7.2. Histomorphometry rinsed in running water for 10 min. For staining of osteopontin
After the micro-CT analysis, implanted samples including the (OPN), osteocalcin (OCN), and collagen I (COL I) sections were boiled
surrounding bone and the bone without implanted samples were in 0.1 M citrate buffer (pH 6.0) in a microwave oven (580 W) for
decalcification with 5% nitric acid for 2 days. After washing with 20 min to retrieve antigenicity. The endogenous peroxidase activ-
deionized water for 1 h and dehydration in 70%, 80%, 90%, and ity was blocked by immersing the sections in peroxidase-blocking
100% ethanol solutions, xylene solutions were used to remove the in methanol for 5 min at room temperature. After washing in
alcohol prior to embedding in paraffin wax. The sample was cut wash buffer, sections were incubated with a protein blocker for
into sections 5 ± 2 ␮m in thickness using a microtome (Thermo- 10 min. Primary antibody of OCN (5 ␮g/ml; Abcam), OPN (1:100;
scientific, USA), deparaffinzed, and stained using hematoxylin and Santa Cruz Biotechnology, USA), and COL I (1:300, Abcam) were
eosin (H&E) and Masson Trichrome. Tissue sections were viewed added to sections for 1 h. After washing with wash buffer, the sec-
with a BX53 light microscope (Olympus) and photographed with ondary antibody (Envision/horseradish peroxidase) was added for
an Olympus DP72 camera. Images were analyzed using the accom- 30 min. After washing in wash buffer, sections were treated with
panying Cellsens software. substrate-chromogen solution for 10 min and subsequently coun-
After microscope examination of each section, computer- terstained with hematoxylin for 30 s. Following dehydration, slides
assisted histometric measurements were made using an automated were mounted and coverslipped for visualizing using an optical
image-analysis system equipped with Cellsens software. Two microscope.
parameters were measured: the percentage of new bone and per-
centage of remaining scaffold (graft). The amount of new bone was 3. Results and discussion
measured by calculating the amount of newly formed mineralized
bone (including lamellar and woven bone) in each defect as a per- 3.1. Characterization of microstructure and material properties of
centage of the whole area of the defect, which was measured as all Gel-Pec-BCP, Gel-Pec-BCP/BMP-2, and Gel-Pec-BCP/VEGF
tissue within the boundaries of newly formed bone. The amount composites
of remaining scaffold was measured by calculating the amount of
remaining graft material as percentage of whole area defect. The 3.1.1. SEM and porosity
parameters for measurements and the calculation methods are Fig. 2(a)–(c) displays low and high magnification SEM micro-
defined according to the following equation: graphs of Gel-Pec-BCP composite scaffold, respectively. The highly
n interconnected pores of the scaffold were visible. The pores in
NB = × 100% (3) the hydrogel system were interconnected with a pore size ran-
w
ging from100 to 400 ␮m. In addition to, high magnification of
Where n is new bone at 2 and 4 weeks and W is whole area of defect.
SEM image was shown in Fig. 2(c) shows that BCP particles
The percent of new bone, NB, was measured with Eq. (3).
properly were embedded in the hydrogel. The porosity and micro-
g CT data shown in Fig. 2(d), (e1), (e2), and (e3) confirmed that
GR = × 100% (4)
w the scaffolds were interconnected. Fig. 2(f) shows the open and
 J. Amirian et al. / International Journal of Biological Macromolecules 76 (2015) 10–24 17

Fig. 6. Cell viability on Gel-Pec-BCP, Gel-Pec-BCP/BMP-2 and Gel-Pec-BCP/VEGF crosslinked scaffold after 1, 3 and 7 days observed by (a) Live- Dead cell assay and b) MTT
assay.

total porosity percent of 92.88% and 92.98% derived from the the accompanying SEM image in Fig. 2c, it was confirmed that the
micro-CT data. As mentioned in previous studies, interconnec- hydrogel scaffold contained C, O, Ca, and P due to the composi-
tivity of dependence on both typical porosity more than 90% tion of Gel, Pec, and BCP powder. This data showed that the surface
as well as a pore size more than 100 ␮m that cell can pen- microstructure of scaffold was composed of BCP powder. BCP pow-
etrate inside scaffolds, so that proper vascularization happen der was dispersed throughout the surface of the scaffolds, apparent
[7,32]. as a white contrast region on the surface. The EDS profile of the Gel-
EDS analysis revealed many BCP particles dispersed on the sur- Pec-BCP composite scaffold shown in Fig. 2(c) indicated the atomic
face of Gel-Pec-BCP scaffolds. From the EDS graph inserted in Fig. 2c percentage of Ca and P were 5.74% and 3.92%, respectively (Ca/P
taken from the point marked with a blue rectangle and shown on ratio = 1.46).
18 J. Amirian et al. / International Journal of Biological Macromolecules 76 (2015) 10–24

Fig. 7. Cell proliferation on the Gel-Pec-BCP, Gel-Pec-BCP/BMP-2 and Gel-Pec-BCP/VEGF crosslinked scaffolds after 1, 3 and 7 days as observed by confocal microscopic
images.

All scaffolds had a pore size between 4 and 1000 ␮m; the mean Gel-Pec-BCP and Gel-Pec-BCP samples coated with BMP-2 and
porosity of samples was 100-300 ␮m, as confirmed by mercury VEGF. Fig. 3 shows the presence of a PO4 3− band, the strongest band
porosimetry (Fig. 2(d)). Also, low magnification SEM examinations of the phosphates, which appears at 1050 cm−1 [36]. The appear-
of the prepared scaffolds (Fig. 2(a) and (b)) revealed that the fab- ance of gelatin at 3443 cm−1 indicates that, due to N-H stretching
ricated scaffolds had a porosity ranging from 100-300 ␮m, which amine bond, C O stretching at 1640 cm−1 is apparent [37]. More-
is good for bone regeneration [33]. Pore sizes exceeding 300 ␮m over, the spectrum of Pectin displayed a peak at 3400 cm−1 due
had a greater penetration of mineralized tissue in comparison with to stretching of–OH groups. The peaks at 2913 cm−1 indicate C H
smaller pores. In addition, increasing in bone formation and vas- stretching vibration. The peak at 1742 cm−1 indicate C O stretch-
cularization have been reported for scaffolds with pore sizes larger ing vibrations due to the presence of COOCH3 group. The peaks
than 300 ␮m [33–35]. at 1441 cm−1 and 1342 cm−1 could be attributed to CH2 scissoring
and OH bending vibrations, respectively. The peak at 1150 cm−1
3.1.2. FT-IR spectra suggested the presence of CH-OH group [37,38]. Furthermore, FT-IR
To further illustrate the chemical interaction between BCP was used to confirm protein coating on surface of Gel-Pec-BCP scaf-
and the Gel-Pec-BCP network matrix, FT-IR analysis was carried folds. The spectra of Gel-Pec-BCP/BMP-2 and Gel-Pec-BCP/VEGF
out. Fig. 3 shows the FT-IR spectra of three types of sample: showed peaks at 1650 and 1540 cm−1 that are also observed in
 J. Amirian et al. / International Journal of Biological Macromolecules 76 (2015) 10–24 19

Fig. 8. (a) Photograph of implanted scaffolds, (b) 3D micro-CT images of implanted scaffolds of Gel-Pec-BCP, Gel-Pec-BCP/BMP-2 and Gel-Pec-BCP/VEGF and without scaffold,
(c) bone volume (BV/TV) and (d) surface density (BS/TV) for each rat in 2 and 4 weeks after implantation.

Gel-Pec-BCP, but the intensity after coating with BMP-2 and VEGF 3.1.4. Swelling
increased dramatically. These peaks are consistent with the pres- The ability to absorb and retain a large amount of water is
ence of amide bonds found in proteins [39], our finding also confirm a key feature for 3D polymeric networks, such as hydrogel sys-
that the success of coating between Gel-Pec-BCP and BMP-2 and tems. Swelling is thus the first point to investigate [42]. Drug
VEGF. release rate according to Higuchi-type release is inversely related
with the swelling rate [43]. Consequently, drug loaded in a poly-
mer with lower PBS uptake may result in a burst release of
3.1.3. Biodegradation drug. A model proposed by Ju et al. described the relationship of
As a gold standard for scaffold degradation measurement, we swelling/dissolution mechanism and drug release from hydropho-
performed a gravimetric assay that is commonly used in tissue bic matrices [43]. Quintanar-Guerrero et al. [40] reported factors
engineering to provide the absolute value of mass changes in the affecting drug release, such as matrix swelling (surface expansion),
scaffolds [40]. Quantitative consideration of hydrogel degradation polymer dissolution, and thickness of the gel layer that supported
was performed as a function of weight loss during incubation time the prior study. Fig. 4(b) shows the swelling kinetics of Gel-Pec-BCP
in PBS at 37 ◦ C, as shown in Fig. 4(a). After coating of material alone and coated with BMP-2 or VEGF. All three scaffolds exhibited
with growth factor that encapsulated in BSA, Gel-Pec-BCP hydro- high swelling kinetics. The coated scaffolds absorbed less PBS than
gel scaffolds were less stable and degradation occurred faster. At the Gel-Pec-BCP scaffold at 1, 2, and 3 days. On day 1, Gel-Pec-
day 10, degradation of Gel-Pec-BCP/BMP-2 and Gel-Pec-BCP/VEGF BCP reached 360.08% compared to scaffolds coated with BMP-2
scaffold was 14.27% and 17.02%, respectively, compared with scaf- and VEGF, representing an approximately 297.27% and 254.84%
fold without coating (10.80%). In addition, this continued; at day increase, respectively. However, all scaffolds displayed a decreased
40, the decrease reached 59.60% and 50.36%, respectively. These after 3 days and reached equilibrium. In addition, PBS uptake by
results confirmed that the scaffold degradation was facilitated pro- Gel-Pec-BCP/VEGF scaffolds was lower than for scaffolds coated
portionally with coating of BMP-2 and VEGF. Micro porosities that with BMP-2, with both scaffolds displaying lower uptake than
were created may affect increase of the biodegradation rate of the Gel-Pec-BCP scaffolds. It was obvious that the decrease was more
scaffold, as well as increasing surface area available for attack [41]. for VEGF-coated scaffolds than BMP-2 coated on hydrogel. Conse-
Previous studies also confirmed that freeze-dried scaffolds exhibit quently, the burst release of VEGF from scaffold would occur, rather
higher porosity [41]. than from BMP-2 coated scaffold. That should be considered that
20 J. Amirian et al. / International Journal of Biological Macromolecules 76 (2015) 10–24

Fig. 9. Histological section of rat skull defects (10 and 40x magnification, H&E staining) without implantation, with Gel-Pec-BCP, Gel-Pec-BCP/BMP-2 and Gel-Pec-BCP/VEGF
scaffold (a) 2 weeks, and (b) 4 weeks after implantation. (LB: lamellar bone, and WB: woven bone).

drug release in swellable matrixes depends on diffusion of drug into In research done by Shen et al. in 2010, they shows improv-
the scaffold and on swelling of the scaffold after penetration of PBS ing in hydophilicity with loading of rhBMP-2 in PLGA/HA(50/50)
[44]. composite microspheres[48]. The authors described a moderate
burst release during first day of about 21.99% of the loaded rhBMP-
3.2. In vitro BMP-2 and VEGF release 2, with a continuous release observed thereafter until 21 days.
This sustained release could be related to the avid bonding of
BMP-2 and VEGF release measurements were conducted over HA and rhBMP-2, as well as the polar interaction and hydrogen
15 days using ELISA kits. Fig. 5 shows the results from experiment bonding between PLGA and rhBMP-2 [48]. Another study that
designed to monitor the release of VEGF and BMP-2, the two most examined the effect of BMP-2 and VEGF delivery in bone regen-
commonly studied growth factors [45]. Differences in the release eration used glycidyl methacrylate modified hyaluronic acid (HA)
profiles were evident. In vitro, an initial VEGF burst release over the as the matrix for BMP-2 and VEGF delivery reported higher release
first 3 days subsequently tapered over 1 week to assume an almost of VEGF [47]. This might be related to the different interaction of
linear sustained release up to 15 days. The in vitro release profile VEGF and BMP-2 with the matrix, with BMP-2 having a stronger
of BMP-2 showed a minimal burst of 4.27 (±1.75)% the first day interaction. Our findings substantiate these earlier results. Exam-
and 20.38 (±1.32)% on day 3. The release of VEGF during the first 3 ination of the effect of the cross-linking amount (concentration;
days was 51.45(±2.57)%, representing 2.5 times more that of BMP- 0.5% to 2% glutaraldehyde) and time of cross-linking (1 to 24 h) [45]
2. After 7 days the release profiles of VEGF and BMP-2 changed, revealed that increases in both parameters were associated with
with VEGF having leveled off with BMP-2 release doubling from increased cross-linking of the gelatin microspheres, and decreases
34.90% to 70.61%. Additionally, a burst release was observed, with in the burst release and release amount of clonidine hydrochlo-
total release near 15 days being 74.15% of total release amount. The ride. This behavior was confirmed [49]. Composite scaffolds depend
means by which growth factors (VEGF and BMP-2) are coated on on different type of polymer and ceramics in terms of their value
scaffolds affects the release profile. Release occurred sooner com- in drug delivery for bone regeneration [50]. Scaffolds used for
pared with the previous method and release profile was measured biomedical applications need to be capable of generating the best
more quickly, in consistent with another study [46]. After 15 days a environment for cell growth and tissue formation, and should be
slow and steady protein release followed until completion of study. able to deliver the essential growth factor for regeneration [51].
It seems reasonable to suppose that biodegradation of the hydro- Designing the system that possesses these ideal properties is cru-
gel scaffolds affected release of growth factor [47]. Comparison of cial.
three types of scaffolds (Fig. 4(a)) shows that degradation of Gel- The activities of the presently-examined scaffolds after 1, 3, and
Pec-BCP/VEGF scaffold was faster than for scaffolds coated with 7 days of in vitro release are shown in Fig. 6(a) and (b). Both growth
BMP-2. The fast degrading hydrogels would be expected to display factors showed acceptable bioactivity after 1 and 3 days. Bioactivity
a high burst release of VEGF followed by steady release until 15 of the VEGF decreased after 1 week and was related to a 66.55%
days. deterioration of scaffolds. Overall, VEGF release from scaffolds was
 J. Amirian et al. / International Journal of Biological Macromolecules 76 (2015) 10–24 21

Fig. 10. Histological section of rat skull defects (40x magnification, Masson trichrome staining) without implantation, with Gel-Pec-BCP, Gel-Pec-BCP/BMP-2 and Gel-Pec-
BCP/VEGF scaffold (a) 2 weeks, and (b) 4 weeks after implantation(COL: Collagen tissue, OC: Osteocyte, NB: New Bone tissue, NV: New Vessel formation).

faster than BMP-2 release. But, BMP-2 release was more sustained number, viability, and proliferation on Gel-Pec-BCP and VEGF and
over the 15 day experiment. BMP-2 coated on Gel-Pec-BCP surfaces were monitored on days
1, 3, and 7 day after cell seeding. Cultured MC3T3-E1 cells in all
3.3. In vitro evaluation the groups increased throughout the incubation period for up to
7 days. In the case of Gel-Pec-BCP scaffolds coated with VEGF, the
3.3.1. Cell viability proliferation rate increased in a similar manner to that of the scaf-
The change in surface properties of Gel-Pec-BCP scaffolds by folds coated with BMP-2 over 7 days. The proliferation rate of the
BMP-2 coating may affect cell proliferation and viability. The coat- cells increased on both coated hydrogel scaffolds. A F-actin staining
ing of BMP-2 and VEGF increased the hydrophilicity Gel-Pec-BCP assay of the MC3T3-E1 cells showed similar results to those of live-
substrates, which may be able to provide topographical simu- dead and MTT assay. The number of cells increased with increased
lation that can facilitate focal adhesion formation in the cells BMP-2 and VEGF coating. The MTT assay of this material confirmed
[52]. In addition, with examine proliferation of cells on the sub- a high percentage of viable cells in scaffolds loaded with VEGF and
strates, the coating of BMP-2 and VEGF on surface of scaffold may BMP-2 than in the absence of the growth factors and the control
promote cell proliferation and viability because the topographi- group. It can be concluded that the release of growth factors affects
cal simulation induced by increased hydrophilicity may activate cell viability and proliferation.
intracellular signal transductions for cell proliferation [52]. The
increased hydrophilic surface may provide increased wettability 3.3.2. Cell proliferation
for cell attachment and proliferation [52]. Biomaterial properties significantly influenced cellular behavior
The ability of cells to adhere to porous scaffolds that mimic when cells were seeded on the scaffolds. As mentioned above, coat-
the structure of the extracellular matrix is an important property ing BMP-2 and VEGF on Gel-Pec-BCP affect the hydrophilicity of the
for tissue engineering applications. We present here preliminary surface of scaffolds. These change in surface properties of scaffolds
results of the effect of BMP-2 and VEGF coating on Gel-Pec-BCP after coating and also growth factor effect confirmed that coat-
hydrogel matrix on cell viability. Fig. 6(a) and (b) show repre- ing procedure may affect cell proliferation and viability, because
sentative laser confocal scanning microscopy (LCSM) images of increasing hydrophilicity may activate intercellular signal trans-
MC3T3-E1 preosteoblast cells cultured on surfaces of Gel-Pec-BCP, duction for cell proliferation via enhance focal adhesion formation
Gel-Pec-BCP/BMP-2, and Gel-Pec-BCP/VEGF scaffolds. In the exam- [52]. Fig. 7 shows the morphology of MC3T3-E1 preosteoblast cells
ination system, live cells stain green and dead cells are red. Cell (1 × 105 cell/well) that had adhered and spread to the hydrogels.
22 J. Amirian et al. / International Journal of Biological Macromolecules 76 (2015) 10–24

The staining results showed varying cell response to the hydrogel Table 1
Histomorphometry demonstrating percentage of remain graft and bone formation
and hydrogels loaded BMP-2 and VEGF. Compared to Gel-Pec-BCP,
in Gel-Pec-BCP, Gel-Pec-BCP/BMP-2, Gel-Pec-BCP/VEGF scaffolds, and control dur-
both Gel-Pec-BCP/BMP-2 and Gel-Pec-BCP/VEGF scaffolds showed ing (a) 2weeks, and (b) 4weeks.
a rapid cell spreading after 1, 3, and 7 days. More cells adhered and
(a) 2 Weeks
spread to the Gel-Pec-BCP/BMP-2 and Gel-Pec-BCP/VEGF scaffolds
than Gel-Pec-BCP (Fig. 7). Therefore, it can be concluded that the Sample type Remain graft (%) New bone (%)
hydrogels containing BMP-2 or VEGF growth factor effectively pro- Control – 2.13
mote cell adhesion and that all hydrogels are biocompatible. VEGF Gel-Pec-BCP 45.72 4.25
is the main angiogenic factor; it contributes to postnatal neovas- Gel-Pec-BCP/BMP-2 20.13 7.51
cularization [53]. The effects of rhBMP-2 and rhVEGF on osteoblast Gel-Pec-BCP/VEGF 15.34 6.74

proliferation are not clear [54]. Kim et al. reported that cell prolif-
(b) 4 Weeks
eration on rhBMP-2 immobilized on surface of Ti disc compared to
that on pristine Ti did not show a significant increase [54,55]. In con- Sample type Remain graft (%) New bone (%)
trast, Park et al. reported the positive effect of BMP-2 on osteoblast Control – 3.75
proliferation in the chitosan membranes with immobilized BMP-2 Gel-Pec-BCP 42.32 8.63
compared to chitosan membranes [54]. Gel-Pec-BCP/BMP-2 10.81 20.75
Gel-Pec-BCP/VEGF 8.95 8.95

3.4. In vivo evaluation

3.4.1. Micro-CT bone formed and remaining graft from histology section for 2 and 4
Mineralized bone formation was evaluated using micro-CT with weeks in Gel-Pec-BCP, Gel-Pec-BCP/BMP2, and Gel-Pec-BCP/VEGF
the BV/TV ratio indicating the regeneration of bone skull. However, scaffolds. The percentage of scaffold remaining in case of Gel-Pec-
the main problem of micro-CT examination of polymeric scaffolds BCP was greater when scaffold were coated with BMP-2 and VEGF,
is that the technique cannot differentiate mineralized tissue (new which was confirmed by an in vitro degradation assessment. Faster
bone formed) from polymeric blend graft material [56]. As shown degradation of scaffold can provide a favorable environment for
in Fig. 8, the degree of bone regeneration was higher in Gel-Pec- bone growth in biomaterial designing. But, faster scaffold degrada-
BCP/BMP-2 and Gel-Pec-BCP/VEGF at 2 and 4 weeks than that in tion does not mean that new bone formation will definitely happen.
and Gel-Pec-BCP, while there was no significant difference either In case of Gel-Pec-BCP/VEGF, the percentage of new bone formed
between the Gel-Pec-BCP and control. The percent bone volume was not greater at 4 weeks compared with Gel-Pec-BCP/BMP-2.
(BV/TV) and bone surface density (BS/TV) are shown in Fig. 8(c) and In addition, the type of bone formed in vivo in the presence of
(d). The BV/TV and BS/TV values are larger for scaffold with growth different growth factor (BMP-2 and VEGF) was different. Obser-
factors (BMP-2 and VEGF) than the without growth factors, and vation of woven and lamellar bone formation within 2 and 4 weeks
also more than the control. BV/TV values after 2 weeks of implan- (Fig. 9(a) and (b)) revealed that VEGF-containing scaffolds aided
tation were 2.32 ± 0.35 4.83 ± 0.37, 5.38 ± 0.89, and 6.39 ± 1.39, woven bone formation. In contrast, Gel-Pec-BCP scaffolds contain-
respectively. The respective values after 4 weeks were 3.26 ± 2.19, ing BMP-2 better supported lamellar bone formation compared
6.49 ± 0.43, 12.26 ± 0.48, 6.91 ± 0.82. The micro-CT results clearly with VEGF-coated scaffolds. Woven bone is dependent on angio-
demonstrate the effect of VEGF and BMP-2 coated on surface of genesis and growth factors like VEGF help to woven bon formation
scaffolds. [56]. In contrast, scaffold Gel-Pec-BCP with BMP-2 aided lamellar
bone formation in this group at 2 and 4 weeks after implantation
3.4.2. Histology (Fig. 9(a) and (b)). In a prior study, in BMP-2 containing scaffolds,
Fig. 9(a) and (b) displays representative histological sections of new bone remodeled into lamellar bone, which provided good
rat skull defect implantation with and without scaffold 2 weeks mechanical strength [57].
and 4 weeks after implantation. Histological analysis of retrieved Because the delivery of VEGF increased blood formation and
samples at 2 weeks after implantation demonstrated the ben- function in the compromised osseous defects, we sought to deter-
eficial effect of Gel-Pec-BCP mediated by BMP-2 and VEGF on mine whether this boost in the angiogenic response correlated
bone formation in defects. The Gel-Pec-BCP/BMP-2 scaffold showed with wound healing of the defect measured by bone regenera-
more evidence of new bone tissue between hydrogel scaffold, with tion. The presence of early bone regeneration within these defects
new bone formation occurring along the scaffold. The scaffold- was performed through histological analysis 2 and 4 weeks after
implanted area did not show foreign body reaction apparent in implantation of composite scaffolds. Gross qualitative histologi-
Fig. 9(a) and (b). Thus, the Gel-Pec-BCP composite scaffold with cal analysis of whole sections suggested that implantation of VEGF
BMP-2 and VEGF enhanced bone formation. Masson’s trichrome- scaffolds resulted in formation of more bone tissue than implanta-
stained tissue slides were used to identify new bone formation in tion of scaffold without any growth factors. In a previous study by
the defect healing area. Collagen fiber was stained blue and new Patterson et al., BMP-2 and VEGF growth factor were loaded in gly-
bone, residual scaffold and cytoplasm were stained red. Masson’s cidyl methacrylate modified hyaluronic acid (HA-GMA) separately
trichrome staining indicated scar tissue formation in the defect of and also in a mix of BMP-2 and VEGF in the gel system. The authors
control group (without treatment) in Fig. 9(a, b). After implanta- found that VEGF delivered alone did not have an osteoinductive
tion of the three types of scaffold, Gel-Pec-BCP, Gel-Pec-BCP/BMP-2 effect in in vivo [47]. In addition, it can be supposed that regenera-
and Gel-Pec-BCP/VEGF, hydrogels coated with BMP-2 exhibited the tion in calvaria occurs through interamembranous ossification and,
greatest collagen formation in defects sites. In case of VEGF-coated therefore, that VEGF may not have a strong osteogenic activity [47].
hydrogel, new blood vessels with red blood cells were formed and Ninety percent of the organic bone matrix is collagen and the
observed to perfuse the host vasculature red arrow as shown in rest consists of non-collagenous proteins. OPN and OCN account
Fig. 10(a) and (b). for remaining 10% of the non-collagenous proteins [58]. Appropri-
Histomorphometry analyses were done to measure the per- ately, evaluation the role of BMP-2 and VEGF coated on Gel-Pec-BCP
cent bone formation and remaining graft after implantation. We hydrogels involved immunohistochemical staining of sections
assumed that bone growth could be accelerated further in the pres- OPN, OCN, and collagen I. During the initial period of prolifera-
ence of BMP-2 and VEGF. Table 1 presents data of the percent of new tion and extracellular matrix biosynthesis, COL I was expressed.
 J. Amirian et al. / International Journal of Biological Macromolecules 76 (2015) 10–24 23

Fig. 11. Histological section of rat skull defects (40x magnification, immunohistochemical staining) without implantation, with Gel-Pec-BCP, Gel-Pec-BCP/BMP-2 and Gel-
Pec-BCP/VEGF scaffold (a) 2 weeks, and (b) 4 weeks after implantation (OPN: Osteopontin, OCN: Osteocalcin, and COL I: Collagen I).
24 J. Amirian et al. / International Journal of Biological Macromolecules 76 (2015) 10–24

Expression of OPN and OCN occurred later during the period of [6] C.-H. Lu, et al., Biotechnol. Adv. 31 (8) (2013) 1695–1706.
extracellular-matrix mineralization [59]. Therefore, the maturation [7] S.-I. Roohani-Esfahani, et al., Mater. Lett. 107 (0) (2013) 378–381.
[8] A.M. Ferreira, et al., Acta Biomaterial. 8 (9) (2012) 3191–3200.
of osteoblasts can be determined to some extent by their expres- [9] J. Kim, et al., Biomaterials 28 (10) (2007) 1830–1837.
sions of OPN and OCN, because bone matrix proteins depend on it [10] N. Ninan, et al., Carbohydr. Polym. 98 (1) (2013) 877–885.
[58]. To make further confirmation, immunohistochemical staining [11] S.H. Jahromi, et al., J. Mech. Behav. Biomed. Mater. 4 (7) (2011) 1157–1166.
[12] H. Cao, N. Kuboyama, Bone 46 (2) (2010) 386–395.
was applied to detect the level of OCN, which is one bone-specific [13] S.C.C.C. Miranda, et al., Three-dimensional culture of rat BMMSCs in a porous
extracellular matrix protein produced by the osteoblast cells in new chitosan-gelatin scaffold: A promising association for bone tissue engineering
bone formation. Scaffolds coated with BMP-2 and VEGF showed in oral reconstruction, Arch. Oral Biol. 56 (1) (2011) 1–15.
[14] E.A. Abou Neel, et al., Journal of Dentistry 40 (8) (2014) 915–925.
the highest level of OCN 2 weeks post-surgery, suggesting that the
[15] G. Hannink, J.J.C. Arts, Injury 42 (Suppl 2) (2011) S22–S25.
scaffold induces the highest activity of new extracellular matrix [16] S. Uma Maheshwari, V.K. Samuel, N. Nagiah, Ceram. Int. 40 (6) (2014)
secretion at that time in synergizing with the applied growth fac- 8469–8477.
[17] S.E. Lobo, T. Livingston Arinzeh, Materials 3 (2) (2010) 815–826.
tors. As expected, our data revealed a strong relationship between
[18] A. Dubnika, D. Loca, L. Berzina-Cimdina, P EST ACAD SCI 61 (3) (2012) 193–199.
cell density and accumulation of bone matrix proteins, including [19] M.I. Tadros, R.H. Fahmy, Int. J. Pharm. 472 (1–2) (2014) 27–39.
OPN, OCN, and COL I. The outer region of scaffolds, populated with [20] K.-H. Chang, H.-T. Liao, J.-P. Chen, Acta Biomater. 9 (11) (2013) 9012–9026.
a higher number of cells, showed the strongest presence of all mark- [21] K. Siimon, et al., Mater. Sci. Eng.C 42 (0) (2014) 538–545.
[22] B. Gupta, et al., Carbohydr. Polym. 106 (0) (2014) 312–318.
ers (Fig. 11(a) and (b)). The expression of OPN, OCN, and COL I [23] L.N.M. Ribeiro, et al., Int. J. Pharm. 463 (1) (2014) 1–9.
reached a peak at 4 week (Fig. 11(b)). At week 2, the expression [24] P.T.S. Kumar, et al., Colloids Surf. B: Biointerfaces 106 (0) (2013) 109–116.
in the Gel-Pec-BCP/BMP-2 and Gel-Pec-BCP/VEGF was higher than [25] J. Zhang, et al., Biomaterials 34 (37) (2013) 9381–9392.
[26] S.-H. Lee, H. Shin, Adv. Drug Delivery Rev. 59 (4–5) (2007) 339–359.
the Gel-Pec-BCP. From Fig. 11(b) we can see that the expression of [27] G.H. Billström, et al., Injury 44 (Suppl 1) (2013) S28–S33.
OCN and OPN at 4 weeks in scaffold coated with BMP-2 and VEGF [28] M.-H.Y. Byong-Taek Lee, R.K. Paul, K.-H. Lee, H.-Y. Song, Mater. Chem. Phys. 104
was higher than without growth factors. (2007) 249–253.
[29] N.T.B. Linh, Y. Min, B.-T. Lee, Tissue Engineering Part A (2014).
[30] G.G. Porto, et al., Acta Cir. Bras. 27 (11) (2012) 757–760.
4. Conclusions [31] S. Zhao, et al., J. Mater. Chem. B 2 (36) (2014) 6106–6118.
[32] K. Rezwan, et al., Biomaterials 27 (18) (2006) 3413–3431.
[33] A.C. Jones, et al., Biomaterials 25 (20) (2004) 4947–4954.
Gel-Pec-BCP scaffolds were fabricated using a freeze-drying [34] S. Teixeira, M. Ferraz, F. Monteiro, J. Mater. Sci. 19 (2) (2008) 855–859.
method. BMP-2 and VEGF growth factors were coated on surface [35] S. Bose, S. Vahabzadeh, A. Bandyopadhyay, Mater. Today 16 (12) (2013)
of scaffolds. SEM micrographs showed interconnected pores with 496–504.
[36] C.R. Yang, et al., Mater. Lett. 59 (28) (2005) 3635–3640.
almost 300 ␮m pore size also confirmed with porosity measuring.
[37] S. Gautam, A.K. Dinda, N.C. Mishra, Mater. Sci. Eng.C 33 (3) (2013) 1228–1235.
In addition, the release result shows that the scaffolds were con- [38] U. Kalapathy, A. Proctor, Food Chem. 73 (4) (2001) 393–396.
trolled carriers for BMP-2 and VEGF and were confirmed to have [39] L. Moore, et al., J. Dental Res. 92 (11) (2013) 976–981.
[40] S.H. Kim, et al., Sci Rep. 3 (2013).
good sustained release properties. In vitro results also showed that
[41] B. Zuki Abu, F.H. Bahaa, M. Noordin Mohamed, Shell-Based Biocomposite Scaf-
scaffold with BMP-2 and VEGF showed high cell viability and pro- fold for Bone Tissue Engineering, in: D. Eberli (Ed.), Regenerative Medicine and
liferation compared to those without growth factors. In vivo study Tissue Engineering-Cells and Biomaterial, InTech., China, 2011, pp. 365–390.
shows that among the three types of scaffolds, those coated with [42] R. Filippo, et al., Int. J. Mol. Sci. 12 (6) (2011).
[43] D. Quintanar-Guerrero, et al., Drug Dev. Ind. Pharm. 25 (2) (1999) 169–174.
BMP-2 and VEGF are suitable for new bone formation. This study [44] P. Colombo, et al., Int. J. Pharm. 88 (1–3) (1992) 99–109.
suggests that Gel-Pec-BCP/BMP-2 and Gel-Pec-BCP/VEGF hydrogel [45] L. Platt, L. Kelly, S. Rimmer, J. Mater. Chem. B 2 (5) (2014) 494–501.
scaffold can be used for biopolymer composite scaffolds in bone [46] J. Su, et al., Int. J. Mol. Sci. 14 (6) (2013) 12714–12728.
[47] J. Patterson, et al., Biomaterials 31 (26) (2010) 6772–6781.
tissue engineering. [48] H. Shen, et al., Acta Biomater. 6 (2) (2010) 455–465.
[49] V. Mouriño, et al., Composite polymer-bioceramic scaffolds with drug delivery
capability for bone tissue engineering, Expert Opin. Drug Deliv. 10 (10) (2013)
Acknowledgments
1353–1365.
[50] C. Romagnoli, F. D’Asta, M.L. Brandi, Drug delivery using composite scaffolds in
This work was supported by- Mid-carrer Researcher program the context of bone tissue engineering, Clin. Cases Miner. Bone Metab. 10 (3)
through National Research Foundation of Korea (NRF) grant funded (2013) 6.
[51] A. Zieris, et al., Biomaterials 31 (31) (2010) 7985–7994.
by Ministry of Education, Science and Technology (MEST), and par- [52] E. Ko, et al., Biomacromolecules 14 (9) (2013) 3202–3213.
tially supported by Soonchunhyang university research fund. [53] W. zhang, et al., Eur. Cell. Mater. 27 (2014) 1–12.
[54] S.E. Kim, et al., Biomaterials 32 (2) (2011) 366–373.
[55] Z. Shi, et al., Biomacromolecules 10 (6) (2009) 1603–1611.
References [56] M.H. Mardirosian, Effects of Bone Morphogenetic Protein (rhBMP-2) and
Platelet Derived Growth Factor (rhPDGF-BB) on Ectopic Bone Formation In Rats,
[1] A.I. Rodrigues, et al., Acta Biomater. 8 (10) (2012) 3765–3776. University of California, Los Angeles, 2012, pp. 1–29.
[2] S.C.C.C. Miranda, et al., J. Biomed. Mater. Res. A 100A (10) (2012) 2775–2786. [57] T.T. Tang, et al., J. of Bone Joint Surg. Br. 89–B (1) (2007) 127–132.
[3] Y. Liu, J. Lim, S.-H. Teoh, Biotechnol. Adv. 31 (5) (2013) 688–705. [58] A. Ono, et al., Oral Oncol. 43 (4) (2007) 339–344.
[4] R.R. Rao, J.P. Stegemann, Cytotherapy 15 (11) (2013) 1309–1322. [59] F. Barrere, C.A.v. Blitterswijk, K.d. groot, Int. J. Nanomed. 1 (3) (2006)
[5] J. Qian, et al., Mater. Sci. Eng. C 33 (8) (2013) 4587–4593. 317–332.