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ORIGINAL ARTICLE
A BMSCs-laden quercetin/duck's feet collagen/hydroxyapatite
sponge for enhanced bone regeneration
Jeong Eun Song
1
| Jingwen Tian
2
| Yeon Ji Kook
1
| Muthukumar Thangavelu
1
|
Joo Hee Choi
1
| Gilson Khang
1
1
Department of BIN Convergence Technology,
Department of Polymer Nano Science &
Technology and polymer Materials Fusion
Research Center, Chonbuk National
University, Jeonju-si, Jeollabuk-do, Republic of
Korea
2
Department of Nuclear Medicine, Molecular
Imaging and Therapeutic Medicine Research
Center, Cyclotron Research Center, Institute
for Medical Science, Biomedical Research
Institute, Chonbuk National University Medical
School and Hospital, Jeonju-si, Jeollabuk-do,
Republic of Korea
Correspondence
Gilson Khang, Department of BIN
Convergence Technology, Department of
Polymer Nano Science & Technology and
polymer Materials Fusion Research Center,
Chonbuk National University, 567 Baekje-
daero, Deokjin-gu, Jeonju-si, Jeollabuk-do
54896, Republic of Korea.
Email: gskhang@chonbuk.ac.kr
Funding information
Korea Health Industry Development Institute,
Grant/Award Number: HI15C2996; Ministry
of Science, ICT and Future Planning, Grant/
Award Number: 2017K1A3A7A03089427
Abstract
Treating critical-sized bone defects is an important issue in the field of tissue engi-
neering and bone regeneration. From the various biomaterials for bone regeneration,
collagen is an important and widely used biomaterial in biomedical applications,
hence, it has numerous attractive properties including biocompatibility, hyper elastic
behavior, prominent mechanical properties, support cell adhesion, proliferation, and
biodegradability. In the present study, collagen was extracted from duck's feet
(DC) as a new collagen source and combined with quercetin (Qtn), a type of flavo-
noids found in apple and onions and has been reported to affect the bone metabo-
lism, for increasing osteogenic differentiation. Further, improving osteoconductive
properties of the scaffold hydroxyapatite (HAp) a biodegradable material was used.
We prepared 0, 25, 50, and 100 μM Qtn/DC/HAp sponges using Qtn, DC, and
HAp. Their physiochemical characteristics were evaluated using scanning electron
microscopy, compressive strength, porosity, and Fourier transform infrared spectros-
copy. To assess the effect of Qtn on osteogenic differentiation, we cultured bone
marrow mesenchymal stem cells on the sponges and evaluated by alkaline phospha-
tase, 3-4-2, 5-diphenyl tetrazolium bromide assay, and real-time polymerase chain
reaction. Additionally, they were studied implanting in rat, analyzed through Micro-
CT and histological staining. From our in vitro and in vivo results, we found that Qtn
has an effect on bone regeneration. Among the different experimental groups,
25 μM Qtn/DC/HAp sponge was found to be highly increased in cell proliferation
and osteogenic differentiation compared with other groups. Therefore, 25 μM
Qtn/DC/HAp sponge can be used as an alternative biomaterial for bone regeneration
in critical situations.
KEYWORDS
bone marrow mesenchymal stem cells, bone regeneration, duck's feet collagen,
hydroxyapatite, quercetin
1|INTRODUCTION
One among the primary goals of tissue engineering and regeneration
is to develop a variety of techniques to reconstruct defected bone
(Amini, Laurencin, & Nukavarapu, 2012; Bose, Roy, & Bandyopadhyay,
2012; Henkel et al., 2013; Polo-Corrales, Latorre-Esteves, & Ramirez-
Vick, 2014; Ranganathan, Ferrari, & Decuzzi, 2013; Vaquette et al.,
2012). Skeletal development and fracture repair include the coordina-
tion of migration, differentiation, and activation of multicell types and
tissues (Dirckx, Van Hul, & Maes, 2013; Ivkovic et al., 2003; Kanczler &
Received: 7 June 2019 Revised: 27 November 2019 Accepted: 29 November 2019
DOI: 10.1002/jbm.a.36857
J Biomed Mater Res. 2019;1–11. wileyonlinelibrary.com/journal/jbma © 2019 Wiley Periodicals, Inc. 1
Oreffo, 2008; Ozasa et al., 2014). Currently, autografts are widely
used to treat defected bone in clinics. However, it has some draw-
backs, like limited availability and the risk of second surgery (Rabkin,
Reid, & Doty, 2015; Temponi et al., 2017; Yanamadala et al., 2017).
Therefore, scaffolds arise as a viable alternative for bone tissue engi-
neering to treat bone-related diseases (Hollister, 2005; Murphy,
O'Brien, Little, & Schindeler, 2013; Saranya, Saravanan, Moorthi,
Ramyakrishna, & Selvamurugan, 2011; Wang et al., 2007).
Currently, numerous tissue engineering techniques have been
used to produce three-dimensional (3D) scaffolds for the defective
bone (Jones et al., 2009). The application of 3D scaffolds offers tem-
porary supports for cell growth and new tissue development. When
we choose bone graft scaffolds to treat fractures in human body, the
desired physical and chemical characteristic of scaffolds must be con-
sidered such as biocompatibility, biodegradability, and nontoxicity
(Chen et al., 2013; Hoornaert, d'Arros, Heymann, & Layrolle, 2016;
Mistry, Mikos, & Jansen, 2007; Thi Hiep et al., 2017). Collagen is the
most renowned material used in biomaterial research (Behring, Junker,
Walboomers, Chessnut, & Jansen, 2008; Dong-Soo et al., 2015;
Elango et al., 2016; Inzana et al., 2014). Collagen comprises the mate-
rial of bone, teeth, and connected tissue and can be extracted from
porcine, duck's feet and human tissues (Kim et al., 2016; Song et al.,
2016). The unique collagen properties include prominent mechanical
properties. Hyper-elastic behavior similar to natural human tissue,
facilitate and encourage cell adhesion, nontoxic, biocompatible, and
biodegradable. Collagen is considered as one of the best useful bioma-
terials. Collagen can be prepared in sponge's foams, nanofibrous pow-
ders, viscous solution, and dispersions (Kook et al., 2017), in this
study, collagen is extracted from duck's feet and sponges were pre-
pared. Usually, duck's feet are discarded as byproducts, and they are
very cheap when used commercially. When comparing the character-
istics of collagen extracted from commercially available animals, there
is no significant difference (El-Jawhari, Sanjurjo-Rodríguez, Jones, &
Giannoudis, 2016; Sultan et al., 2018).
HAp is one of the main inorganic components of bone, with a lot
of application in the field of bone tissue engineering owing to its excel-
lent biocompatibility, osteoconductivity, and biointegration properties.
HAp has attracted considerable attention as a bone substitute (Ambre,
Katti, & Katti, 2015; Fu, Rahaman, Dogan, & Bal, 2008; Michel, Penna,
Kochen, & Cheung, 2015). HAp helps cell adhesion, proliferation due
to its surface roughness, and has been known to enhance osteoblast
adhesion, proliferation, and differentiation (Chen et al., 2013).
Quercetin (Qtn) is flavonoid materials, and it induced mineral depo-
sition and also enhances osteoblast response of calcium phosphate
(Forte et al., 2016; Gupta, Kumar, & Mishra, 2017; Wong & Rabie,
2008a; Zhou et al., 2015). Qtn has been reported to favor adhesion
and proliferation of osteoblast by positively interacting with them and
effectively slowing-down or inhibiting the osteoclast activity (Kanter,
Altan, Donmez, Ocakci, & Kartal, 2007; Liang et al., 2011; Napimoga
et al., 2013; Siddiqui et al., 2011; Wong, 2011; Wong & Rabie, 2008b).
Therefore, if Qtn and Hap are used in bone tissue engineering applica-
tions, it could activate the metabolism of bone cells and it will promote
osteoblast proliferation, differentiation, and mineralization.
Bone marrow mesenchymal stem cells (BMSCs) have shown great
potential in the clinical application upon activation by pharmacological or
biological means, result in improved bone healing by modulating their dif-
ferentiation into osteoblasts. In this study, unique collagen isolated from
DC which has superior mechanical properties compared with fish and
chicken collagen (Theng, Huda, Wariyah, Hashim, & Nik Muhammad,
2018), that makes it highly suitable for biomedical application and the col-
lagen fibers were used to create biocomposite material. In addition, they
were coated with Hap, an osteoinductive and osteoconductive material
that facilitates cell adhesion and proliferation, they are combined differ-
ent ratio of Qtn in our developed DC/HAp sponges. They prepared spon-
ges were physio chemically characterized for its mechanical behavior,
biocompatibility in an in vitro model by seeding BMSCs and further eval-
uated in in vivo rat as an animal model for the bone regeneration.
2|MATERIALS AND METHODS
2.1 |Materials
Duck's feet were purchased from Korean local market. Qtn, acetone,
NaOH, methanol, citric acid, chloroform, pepsin, and acetic acid were
obtained from Sigma-Aldrich. All other reagents used in this study
were of high-performance liquid chromatography grade.
2.2 |Collagen isolation from ducks feet
DC was extracted using our previously reported protocol (Kook et al.,
2017; Song et al., 2016). The percentage of DC to be used in this
study was selected from our earlier study (Kook et al., 2017; Song
et al., 2016), where we have found that 2% DC have good mechanical
and cell viability properties. Therefore, we fabricated 2% of DC. First,
iced duck's feet were washed with distilled water several times and
their feet were cut using a blade and then immersed in 70% alcohol to
disinfect for 3 hr. Followed by, immersing them in 0.5M NaOH solu-
tion for 24 hr to remove fat from the tissue by placing in stirring at
60 rpm. Later, it was washed for a moment with methanol:chloroform
in 3:1 ratio. For collagen extraction, the feet were submerged again in
5% citric acid for 24 hr. Later, they were treated with 3 g of pepsin for
48 hr. Following the acid treatment, the supernatant was centrifuged
at 12,000 rpm for 15 min. The precipitated collagen was treated with
alcohol and centrifuged again at 3500 rpm for 5 min. Subsequently,
precipitated collagens were stored at −80C for 24 hr. Then, the
extracted collagen was freeze-dried for 5 days.
2.3 |Fabrication of Qtn/DC/HAp sponges
We dissolved collagen in 0.5M acetic acid to make 2% collagen solu-
tion followed by stirring for 72 hr at room temperature in 100 rpm.
Qtn was prepared in dimethyl sulfoxide (DMSO) under stirring at
60 rpm for 5 min at room temperature. To make a different ratio
2SONG ET AL.
(0, 25, 50, and 100 μM), Qtn solution was mixed into 2% collagen
solution. The mixture was then poured into 48 well plates (1 ml/well),
freeze at −80C, later freeze-dried. Afterward, the Qtn/DC/HAp
sponges were prepared following the methods: briefly, 2% DC or
Qtn/DC sponges were immersed in 1X stimulated body fluid (SBF) for
24 hr at room temperature, then washed with distilled water and
lyophilized for 48 hr. In this study, DC/HAp sponges and a different
ratio of Qtn/DC/HAp were referred as 0 μM Qtn/DC/HAp, 25 μM
Qtn/DC/HAp, 50 μM Qtn/DC/HAp, and 100 μM Qtn/DC/HAp. All
these characterization were carried out using the dry samples.
2.4 |Sponge characterizations
The surface morphology and pore size of sponges were examined by
scanning electron microscopy (SEM; Bio-LV SEM, Hitachi, S-2250N,
Japan). The chemical properties of the sponges were analyzed by Fou-
rier transform infrared spectroscopy (FTIR; Spectrum GX, Perkin Elmer).
The compressive mechanical strength of each sponge was measured
using TMS-Pro instrument (Food Technology Corporation, Sterling,
VA), by moving the samples down at a target distance to specimen of
1.5 mm with a speed of 10 mm/s at a force of 0.5N for calculating the
strength and height of the sponges. Porosity was evaluated by Archi-
medes principles via fluid displacement measurement techniques.
Sponges were immersed in a known volume of water (V
1
) for 10 min.
Then the total water volume along with the immersed sponge was
referred to as V
2
. Then the water impregnated sponges were removed,
and the remaining volume of water was recorded as V
3
. The porosity of
sponges was estimated by using the following equation.
Porosity %ðÞ=V1−V3
V2−V3
×100
2.5 |Cell culture
BMSCs were isolated from the femurs and tibias of 6 weeks old
female New Zealand white rabbits. Isolated femurs and tibias moved
into the clean bench and washed three times with phosphate buffered
saline (PBS). After cutting both ends of the bone, the bone marrow
was isolated using a 20-G needle. Then bone marrow was incubated
in culture plate containing Alpha-MEM (Lonza, Walkersville, MD) sup-
plemented with 20% fetal bovine serum, and 1% penicillin/streptomy-
cin (Invitrogen, Carlsbad, CA). After 3 hr, bone marrow was
resuspended using 100 μm cell strainer in the medium. Isolated
BMSCs were plated in the culture medium at 37Cin5%CO
2
. The
culture medium was replaced once in every 3 days.
2.6 |In vitro cell adhesion and proliferation
The surface morphology and the initial attachment of BMSCs on the
sponges were observed by SEM (S-2250N, Hitachi, Tokyo, Japan).
BMSCs were seeded on the sponges at a concentration of
5×10
4
cells/sponge (n= 3). After 21 days of culture, the cultured
sponges were washed with PBS and fixed using 2.5% glutaraldehyde
(Sigma-Aldrich, Saint Louis) at room temperature for 24 hr. Fixed sam-
ples were dehydrated in ethanol graded series (50, 60, 70, 80, 90, and
100%) for 30 min each. Dried samples were coated with gold using a
plasma sputter under argon gas. The cell morphology on sponge was
observed by SEM.
The cell viability and proliferation on the sponges was measured
using MTT (3-4-2, 5-diphenyl tetrazolium bromide, Sigma-Aldrich)
assay. The density of BMSC per sponge was seeded at 5 ×10
4
. After
1, 7, 14, and 21 days of culture, 100 μl of MTT solution was added on
the sponges. Then it was incubated for 4 hr at 37C in 0.5% CO
2
.
After the formation of purple crystals, the solution was removed,
sponges were carefully washed with PBS followed by addition of 1 ml
DMSO and incubated for 24 hr. Then 100 μl of the solution was
transferred into 96-well plate, and the absorbance was measured at
570 nm using Synergy Mx monochromator-based multimode micro-
plate reader (Biotek Instuments, Inc.).
2.7 |Alkaline phosphatase activity
The osteogenic differentiation of BMSCs on the sponges was ana-
lyzed using alkaline phosphatase (ALP) Assay Kit (Takara Bio Inc.,
Tokyo, Japan) according to the manufacturer's instruction. BMSCs
were seeded on the sponges at the concentration of 5 ×10
4
cells/
sponge (n= 3) and cultured for 1, 7, 14, and 21 days. At each time
point, the samples were retrieved and rinsed using PBS. The protein
from each BMSCs seeded on the sponges were extracted using 5%
ALP extraction solution and para-nitrophenyl phosphate (pNPP) solu-
tion was added. The reaction solution incubated at 37Cin5%CO
2
for 1 hr. The reaction was stopped by adding 0.9N NaOH. The absor-
bance was measured using a microplate reader at 405 nm.
2.8 |Gene expression using real-time polymerase
chain reaction
Osteogenic gene expression was evaluated using real-time polymer-
ase chain reaction (RT-PCR). On 1, 7, 14, and 21 days of BMSCs
(5 ×10
4
) cultured on sponges (n= 3), the total RNA was extracted
using a Trizol reagent (Takara Bio, Inc., Tokyo, Japan) and 0.2 ml chlo-
roform. The supernatants were precipitated using 0.5 ml of isopropanol
(Sigma-Aldrich). Each of primers; glyceraldehyde 3-phosphate dehydro-
genase (GAPDH), type-I collagen (COLI), runt-related transcription fac-
tor 2 (Runx2), and osteocalcin (OCN) were purchased from Genotec
(Daejeon, Korea) and extended using TOPscript™One-step RT PCR
DryMIX (Enzynomics, Korea) by Authorized Thermal Cycler (TP 600,
Takara Bio, Inc., Japan). PCR products were examined by electrophore-
sis on 1.2% (wt/vol) agarose gel containing ethidium bromide and visu-
alized under UV light (FluorChem HD2 Gel Imaging System, Alpha
Innotech) at 300 nm.
SONG ET AL.3
2.9 |In vivo bone regeneration-micro-CT
The sponges were cut into 0.5 cm thick and 4 mm diameter of
round shapes. Two pieces of 0.5 cm ×4 mm calvarial bone defects
were carved off from each sprague–dawley rats (SD rat, 6 weeks,
female, Hanil laboratory animal center, Wanju, Korea). Scaffolds
were implanted and fixed with silicone caps (Summit medical;
n= 2). Then, the wounds were sutured (Ethicon) to protect the
implanted sites from external contamination. The in vivo minerali-
zation and compatibility of 0 μM Qtn DC/HAp and 25 μM
Qtn/DC/HAp sponges were evaluated by micro-CT (SkyScan 1173,
SkyScan, Belgium) after 2 and 8 weeks of implantation. The 3D
images of calvarial defects implanted sponges were built from cor-
onal images to evaluate bone formation in the defects. We ana-
lyzed bone volume (BV), and bone mineral density (BMD), through
μ-CT analysis.
2.10 |Histological evaluation
To evaluate the bone regeneration of 0 μM Qtn DC/HAp and 25 μM
Qtn/DC/HAp, sponges were seeded with BMSCs at 2 ×10
5
(cells/
sponge), and sponges were implanted at the calvarial defected site of
SD rat with 4 mm biopsy punch (n= 2). Histological evaluation of SD
rat cranial section was performed after 8 weeks postsurgery. Sponges
along with the surrounding tissues were collected and fixed using
10% formaldehyde (Sigma-Aldrich), and dehydrated with a series of
graded ethanol, and embedded in paraffin blocks, sectioned into
8 mm. Then samples were deparaffinized and stained with Hematoxy-
lin and Eosin (H&E) and Masson's trichrome stain (MTS). All the animal
experimental procedures were performed with the approval from the
Chonbuk National University Animal Care Committee, Jeonju, South
Korea.
2.11 |Statistical analysis
The data were expressed as means ± standard deviation (SD) of exper-
iments performed in triplicate (n= 3). Statistical significance was ana-
lyzed with one-way analysis of variance using GraphPad Prism 5.0
(San Diego, CA). Probability (p) values of <.05(*), <.01(**), and <.001
(***) were considered as statistically significant.
3|RESULTS
3.1 |Morphology of Qtn/DC/HAp sponges
SEM analysis was used to observe the surface morphology of the
scaffold and the pore size that is shown in Figure 1, showing the gross
images of 0, 25, 50, and 100 μM Qtn/DC/HAp sponges (photographic
images of the scaffolds are presented as an insert in Figure 1). All the
sponges presented smooth, soft surface, and sponge-like morphology
with the 2 mm thickness and 6 mm wide length. Also, all sponge's
pore shape showed uniform elliptical shapes in the cross-sectional
images. All the scaffold had a pore structure with well-oriented pores
that are enclosed by a thin wall from the sponge surface to inside,
the majority of the pores observed are of uniform size. This special
orientation of the scaffolds structure helps cells moving inside the
pores of the scaffold and form adhesion and proliferation. For an ideal
biomaterial scaffold must possess a suitable 3D structure and pore
size for the nutrients transport and prevent cell loss.
3.2 |Physiochemical properties
Figure 2a FTIR spectra illustrated the change in the chemical structure
of Qtn, hydroxyapatite, and different concentration of Qtn/DC/HAp
FIGURE 1 Scanning electron microscopy images of 0, 25, 50, and 100 μM Qtn/DC/HAp sponges showing in ×100 and their imagined images
at ×200 (Insert shows the gross images of sponges)
4SONG ET AL.
sponges. DC spectrum displays characteristic peaks at 1645 cm
−1
(amide-I; C O stretch), 1561 cm
−1
(amide-II; N H bend), 1469 cm
−1
(C H bond), and 1249 cm
−1
(amide-III; C N stretch), respectively.
The peaks at 1740 and 1680 cm
−1
corresponded to the stretch vibra-
tions of C O and C C groups. Qtn spectrum at ~3422 cm
−1
cor-
responded to the stretch vibration of the O H bond. Also, at 1663,
1609, 1574, and 1523 cm
−1
corresponding to its benzene ring and
CO group. Hydroxyapatite spectrum showed PO
4
−3
at 1033 and
571 cm
−1
. The peaks at 3400 cm
−1
corresponded to O H. In the
spectra of the different ratios of Qtn/DC/HAp sponges, the 100 μM
Qtn/DC/HAp sponge exhibited comparatively higher intensity at the
same absorption bands that were observed in Qtn spectrum. More-
over, the absorption peaks at 3325, 1642 cm
−1
were observed in the
Qtn/DC/HAp sponge due to O H and C O stretch by the interac-
tion of Qtn and collagen, and the effect of hydroxyapatite was rela-
tively small. This sponge was coated with SBF solution by immersing
the sponge for 1 day instead of direct addition of hydroxyapatite and
was expected to have low absorption. We confirmed Qtn, HAp, and
DC peaks in the fabricated Qtn/DC/HAp. And the Qtn peak intensity
was increased at higher concentration of Qtn. So it is considered that
all sponges have unique properties of Qtn, DC, and HAp during the
production process. Mechanical strength of the scaffold plays a vital
role in bone tissue engineering. Figure 2b shows the compressive
strength of the fabricated sponges. The compressive strength was
found to 0.178 ± 0.02, 0.173 ± 0.02, 0.150 ± 0.02, and 0.122
± 0.01 MPa for 0, 25, 50, and 100 μM Qtn/DC/HAp sponges, respec-
tively. The compressive strength of the scaffolds is observed to be
decreasing with increasing concentration of Qtn in the scaffold. Scaf-
fold containing 0% Qtn had the highest compressive strength 0.178
± 0.02 MPa compared with other scaffolds but they have the lowest
porosity, pore size. Figure 2c presents the porosity of the 0, 25,
50, and 100 μM Qtn/DC/HAp sponges. We observed that the porosi-
ties of all groups of Qtn/DC/HAp sponges were over 70%. The poros-
ities of 0, 25, 50, and 100 μM Qtn/DC/HAp sponges were 71.36
± 1.93, 73.46 ± 1.28, 76.36 ± 5.14, and 80.90 ± 4.89%, respectively.
Most of the pores were of uniform size.
3.3 |Cell proliferation and adhesion of BMSCs on
Qtn/DC/HAp sponges
To further study the biocompatibility, cell adhesion, proliferation, and
cytotoxicity of the sponges, SEM analysis was carried out to confirm
the cell adherence and morphology of BMSC in Qtn/DC/HAp spon-
ges. As a result, BMSCs were attached to the surface of all sponges in
a round shape, and the extracellular matrix was observed to extend
like a spider web (Figure 3). The images clearly showed cells randomly
distributed on the surface and inside the walls of the sponge pores.
In order to evaluate the effect of Qtn on cell growth and their
toxicity, we performed MTT assay during 21 days in different time
point (1, 7, 14, and 21 days) by culturing BMSCs on 0, 25, 50, and
100 μM Qtn/DC/HAp sponges. OD values of all the three different
groups showed not much statistically significant difference (Figure 4a)
over time compared with the control groups without Qtn, the prolifer-
ation of BMSCs seeded on the Qtn/DC/HAp sponges gradually
increased. All of the experimental groups showed similar proliferation
on day 1.
3.4 |Osteogenic differentiation on Qtn/DC/HAp
sponges
The previous experimental results confirmed that the prepared scaf-
fold possessed good physiochemical properties with good biocompati-
bility. Further, study to evaluate its ability to promote osteogenic
differentiation of BMSCs in vitro model was carried out. The osteo-
genic differentiation of BMSCs on Qtn/DC/HAp sponges was evalu-
ated by culturing BMSCs on the sponges for 21 days and they were
analyzed on different time point (1, 7, 14, and 21 days). BMSCs grown
in osteoblast-like environments undergo osteoblast differentiation
into osteoblasts cells. ALP activity is an important analysis carried out
for evaluating osteoblast differentiation. In general, unlike BMSCs, dif-
ferentiated osteoblasts have ALP that hydrolyzes other substances to
produce phosphoric acid. The BMSCs present in the Qtn/DC/HAp
FIGURE 2 (a) Fourier transform infrared spectroscopy spectra, (b) Compressive strength, and (c) Porosity of 0, 25, 50, and 100 μM Qtn/DC/
HAp sponges (n=3,**p< .005)
SONG ET AL.5
sponge was evaluated by measuring ALP activity using the fact that
the number of BMSCs differentiated into osteoblasts was increased
thereby increasing ALP activity with the same trend. Figure 4b shows
that BMSCs in the Qtn/DC/HAp sponge gradually increased osteo-
blast differentiation. A total of 25 μM Qtn/DC/HAp sponge had the
highest ALP value compared with other sponges studied at all-time
point.
3.5 |mRNA expression of BMSCs on Qtn/DC/
HAp sponges
To analyze the ability of osteogenic differentiation of BMSCs on
0, 25, 50, and 100 μM Qtn/DC/HAp sponges, RT-PCR was performed
(Figure 5). To study, whether the sponges affected the osteogenic dif-
ferentiation of rBMSCs, the expressions of bone-specific genes such
as OCN, COL1, and RUNX-2 were examined on 21 days of cell culture
(GAPDH was used as a housekeeping gene). Runx2 and COL1 are
considered as early markers of osteogenic differentiation, whereas
OCN is middle and late markers (Komori, 2010). Comparing the gene
expression on the different concentration of the sponges analyzed,
25 μM Qtn/DC/HAp sponges real-time quantitative values for COL1
(1.05-fold), OCN (1.22-fold), and Runx2 (1.11-fold) showed higher
expression. These results suggested that 25 μM Qtn/DC/HAp possess
stronger stimulating effects on osteogenic differentiation than 0 μM
Qtn /DC/HAp as a control.
3.6 |Sponge scaffold improved bone regeneration
in vivo
To investigate osteoconduction ability of the Qtn/DC/HAp sponges,
sponges were implanted in a full thickness rat calvarial bone defect.
All rats survived the surgery and recovered well postoperatively,
FIGURE 3 SEM images of BMSCs cultured on the surface of 0, 25, 50, and 100 μM Qtn/DC/HAp sponges over a period of 21 days, showing
enhanced cell attachment and proliferation (magnifications with ×500 and ×1000). BMSCs, bone marrow mesenchymal stem cells; SEM, scanning
electron microscopy
FIGURE 4 Biocompatibility and osteogenic differentiation of BMSCs cultured on 0, 25, 50, and 100 μM Qtn/DC/HAp sponges for 1, 7,
14, and 21 days studied by (a) MTT and (b) ALP assay (*p< .05, **p< .005, ***p< .001). ALP, alkaline phosphatase; BMSCs, bone marrow
mesenchymal stem cells; MTT, 3-4-2, 5-diphenyl tetrazolium bromide
6SONG ET AL.
without infection. All the animals were sacrificed on 8 weeks of post-
surgery for further examination. Micro-CT images were performed to
analyze the new bone formation in the defected region (Figure 6). In
the control group, the newly formed bones appeared a little around
the hole. At 8 weeks postsurgery, the BMD of the rate on the
25 μM Qtn/DC/HAp group was almost 3.65 fold higher than con-
trol groups and 2.5 fold higher than 0 μM Qtn/DC/HAp group
showing the significantly largest area of new bone regeneration. BV
(mm
3
) on the 0 μM Qtn/DC/HAp group showed a slight increase
(2 weeks: 0.12 ± 2.1 mm
3
, 8 weeks: 0.76 ± 0.32 mm
3
). While BV on
the 25 μM Qtn/DC/HAp sponge showed a significant increase
(2 weeks: 2.92 ± 0.99 mm
3
, 8 weeks: 3.92 ± 1.63 mm
3
). These
results indicate the 25 μM Qtn/DC/HAp sponge could stimulate
new bone formation.
3.7 |Histological analysis
To evaluate bone regeneration in the rat calvarial defect model,
12 SD-rats were sacrificed at 8 weeks postsurgery. H&E staining rev-
eled significantly more new calcified bone formation in implanted
defected models and inside the porous scaffold. In the H&E staining,
the blank group with no sponge implanted in the defective part no vis-
ible newly produced bone were seen, and the group with 0 μM
Qtn/DC/HAp inserted into the defective part and the group with
25 μM Qtn/DC/HAp, it was observed and confirmed that the sponge
pores were filled with a pale red color. In particular, this new bone for-
mation was found to be regenerated significantly higher at 25 μM
Qtn/DC/HAp sponge compared with 0 μM Qtn/DC/HAp sponge.
MTS staining showed new bone (red), collagen sponge (blue), and
FIGURE 5 mRNA expression of BMSCs bone-specific markers analyzed from the BMSCs cultured on 0, 25, 50, and 100 μM Qtn/DC/HAp
sponges for 21 days. (a) RT-PCR results of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), type-I collagen (COLI), osteocalcin (OCN), and
runt-related transcription factor 2 (Runx2), (b) quantitative analysis of COL1, OCN, and Runx2 were normalized using GAPDH (**p< .005,
***p< .001). BMSCs, bone marrow mesenchymal stem cells; RT-PCR, real-time polymerase chain reaction
FIGURE 6 Micro-CT images of SD-Rat cranial new bone regeneration after implantation at 2 and 8 weeks: (a) Micro-CT images and (b) Bone
mineral density (BMD) and (c) Bone volume (BV; ***p< .001)
SONG ET AL.7
tissue (blue) in part of the implanted area in 0 μM Qtn/DC/HAp and
25 μM Qtn/DC/HAp sponges (Figure 7). When comparing blank with
0μM Qtn/DC/ HAp and 25 μM Qtn/DC/HAp in MT staining, it was
observed that only the tissue was filled with the bone defects. The
0μM Qtn/DC/HAp sponge confirmed that the new bone tissue
appeared as a red color between the collagen sponge pores and that
the 25 μM Qtn/DC/HAp sponge had more new bones between the
sponge pores and the red color part was more distributed than
DC/HAp sponges.
4|DISCUSSION
In bone tissue engineering, cells cultured with a specific scaffold/
sponge/biomaterial to construct composite materials to repair bone
defects have achieved initial success. There is an urgent need for
developing suitable scaffolds with good physiochemical, nontoxic, and
biocompatible properties for bone tissue engineering applications. An
ideal bone scaffold/sponge should possess biocompatibility, optimal
mechanical properties, and good morphology for cell adhesion, prolif-
eration, and ensuring the integration of the scaffold with bone tissue
(Ho-Shui-Ling et al., 2018). For tissue bone engineering sponge/scaf-
fold, minimal porosity, appropriate pore size, and well interconnecting
pores are very important for ingrowth, cell adhesion, proliferation, and
metabolic transport of the cells (Gomes, Bossano, Johnston, Reis, &
Mikos, 2006; Przekora, 2019). In our present study, 25 μM Qtn/DC/
HAp sponge had well-interconnected pores and a porosity (73.46
± 1.28%), compared with cancellous bone having a porosity of
50–90%. Considering that the porosity of the cancellous bone was
reported to be >75% (Polo-Corrales, Latorre-Esteves, & Ramirez-Vick,
2014; Uth, Mueller, Smucker, & Yousefi, 2017), our finding was so
close to the cancellous bone supporting that it can be used for tissue
engineering application. This uniform pore shape was formed through
the 3 hr refrigeration, freeze-drying method. The pore size of each
sponge was increased by the increasing content of Qtn. The reason
for the different pore size of each sponge is due to DMSO, the sol-
vent used to dissolve Qtn. It affects the ice particle size during
freeze-dry.
The change in functional groups was confirmed by the FTIR spec-
trum. The spectrum clearly shows the respective peaks of Qtn, DC,
and HAp, and their intensity was observed to be increasing with the
increasing concentration of Qtn in the sponge materials. We found
the addition of Qtn slightly decreased the compressive strength of the
sponges. It is known that DMSO enhanced the hydrogen bonding of
water to strengthen the binding force between the molecules of colla-
gen (Ho-Shui-Ling et al., 2018; Parimaladevi & Srinivasan, 2016) nev-
ertheless, as the concentration of Qtn increased, the compressive
strength of the sponge decreased slightly. This indicates that the pore
size has a larger influence on the compressive strength than the
above-mentioned bond between collagen molecules. The porosity
was increased with the increasing concentration of Qtn in the sponge.
These results are also owing to the influence of the pore size.
The MTT assay was performed to evaluate the proliferation and
biocompatibility of the sponges by seeding BMSCs on the surface of
the materials in vitro. The activity of the cells (live cells) presented
through observance reading of the different groups on several time
points. On day 1, all the groups showed similar proliferative activities
with no significant difference. On day 7, there was a significant differ-
ence in proliferative activities between the groups. We observed a
decrease in cell proliferative activities on increasing in Qtn concentra-
tions. But over time, among the four groups, 25 μM Qtn/DC/HAp
sponges exhibited the highest proliferation rate. Compared with
BMSCs cultured without sponges in cell culture plates, the cell prolif-
erative activities of BMSCs cultured on the sponges was not
FIGURE 7 Histological evaluation of 0 and 25 μM Qtn/DC/HAp sponges implanted calvarial defected SD-Rat for 8 weeks (Red arrow
indicates the edge of the defect, magnification ×40)
8SONG ET AL.
decreased and after day 7 compared with control, the cell proliferative
activities was observed to be increasing in 25 μM Qtn/DC/HAp sam-
ples, the same trend was followed till day 21. However, cell prolifera-
tion was inhibited with the increase of Qtn concentration. Since Qtn
inhibits cell proliferation at higher concentrations, different mecha-
nisms have been proposed, including cell cycle arrest, DNA strand
breakage, induction of apoptosis, and possibly influencing the activity
of various kinases including phosphatidylinositol-3-kinase, tyrosine
protein kinase, and protein kinase C (van der Woude et al., 2005).
Thus, it is considered that the higher amount of Qtn is suppressed cell
proliferation in the DC/HAp sponges. SEM images showed BMSCs to
be well adhered and proliferated and integrated within the pores
sponge.
In vitro ALP activities of the 25 μM, Qtn/DC/HAp sponge con-
firmed a stimulatory effect on the differentiation of osteoblasts. Also,
when the content of Qtn was increased in the sponge, the degree of
bone differentiation gradually decreased. These results suggest that
the Qtn/DC/HAp sponge with Qtn tends to inhibit BMSC prolifera-
tion by Qtn, but the present BMSCs further promote bone differentia-
tion into osteoblasts. Stem cells were optimized in 25 μM Qtn/DC/
HAp sponge. This suggests that Qtn reduced osteoclastic differentia-
tion by RANKL, and osteoblastic differentiation is indirectly induced
for that reason, thus promoting bone morphogenesis of bone
marrow-derived stem cells in the sponge (Wattel et al., 2004). The
gene expression results revealed among the sponge analyzed 25 μM
Qtn/DC/HAp sponge showed better results. These results indicated
that Qtn could enhance osteogenic differentiation at various stages,
additionally, the concentration of 25 μM Qtn/DC/HAp was found to
be the optimum concentration for COL1, Runx2, and OCN expression.
Micro-CT results revealed that the implanted 0 μM Qtn /DC/HAp and
25 μM Qtn/DC/HAp sponges with rBMSCs promotes more bone
regeneration than the blank group. These results indicated that the
density and area of tissue regenerated in the 25 μM Qtn/DC/HAp
sponge were the highest, and their osteogenic activity was strong.
The results confirm the integration between the surrounding tissues
and implanted sponges. Histological analysis results confirmed that
compared with control the 25 μM Qtn/DC/HAp sponge treated
enhanced defect closure and mineralization and the analysis comple-
mented the Micro-CT results, this suggests that Qtn has an effect on
the osteogenic differentiation of BMSC, resulted in an increase in
bone regeneration.
5|CONCLUSION
In this study, we fabricated porous composite Qtn/DC/HAp sponges
by a freeze-drying process with the aim of biomaterial for bone regen-
eration. We found that 25 μM Qtn/DC/HAp sponge is the most effi-
cient promoter of BMSCs osteogenic differentiation with
osteoinductive and osteoconductive features as evidenced by the
in vitro and in vivo outcomes without any negative effects on rats. In
spite of fulfilling all the desired properties of an ideal tissue engineer-
ing scaffold, Qtn/DC/HAp sponges has, 3D structure with pore size
for the nutrients transport, preventing cell loss, biocompatible, non-
toxic, good morphology for cell adhesion, proliferation, and ensuring
the integration of the scaffold with bone tissue. Thus, this bio-
engineered Qtn/DC/HAp sponge might be applicable for bone regen-
eration in larger animals and future clinical trials. In conclusion, the
proper amount of the Qtn incorporated with collagen sponges may
price clinical support for the patients with various bone defects and
might be applied in several tissue engineering fields.
ACKNOWLEDGMENTS
This research was supported by a grant of the Korea Health Technol-
ogy R&D Project (HI15C2996) through the Korea Health Industry
Development Institute (KHIDI), the International Research & Develop-
ment Program of the National Research Foundation of Korea (NRF)
funded by the Ministry of Science, ICT & Future Planning (NRF-
2017K1A3A7A03089427).
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How to cite this article: Song JE, Tian J, Kook YJ,
Thangavelu M, Choi JH, Khang G. A BMSCs-laden quercetin/
duck's feet collagen/hydroxyapatite sponge for enhanced
bone regeneration. J Biomed Mater Res. 2019;1–11. https://
doi.org/10.1002/jbm.a.36857
SONG ET AL.11
