Fabrication, characterisation and in vitro biological activities of a sulfuretin-supplemented nanofibrous composite scaffold for tissue engineering

Article abstract of DOI:10.1039/c5ra06648d

Electrospun micro/nanofibrous scaffolds are widely used in various tissue regeneration applications because they have a similar structure to the extracellular matrix and can induce high attachment, proliferation and even differentiation of cultured cells. Here, we designed a new composite scaffold consisting of poly(ε-caprolactone) (PCL), bone morphogenetic protein (BMP-2) and sulfuretin fabricated using a combined process, i.e. electrospinning/plasma-treatment/coating. In the composite, we introduced a new bioactive component, sulfuretin, which was used as a cell stimulant to regenerate bone tissue. Sulfuretin release from the composite was controlled by coating of a fixed concentration of alginate. The in vitro biocompatibilities of the fibrous composites were examined using preosteoblasts (MC3T3-E1s), and the composite showed high cell adhesion and differentiation for a limited range of sulfuretin compared to the control, which lacked sulfuretin. These results suggest sulfuretin to be an effective supplemental bioactive agent for enhancing bone tissue growth on fibrous composite scaffolds.

Full text of DOI:10.1039/c5ra06648d

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PAPER
Fabrication, characterisation and in vitro biological
activities of a sulfuretin-supplemented nanofibrous
composite scaffold for tissue engineering
Cite this: RSC Adv., 2015, 5, 44943
YoungWon Koo,a Hyeongjin Lee,a Suji Kim,b No-Joon Song,b Jin-Mo Ku,c
b
a
b
a
*
JaeHwan Lee, Chang Hyun Choi, Kye Won Park and GeunHyung Kim
Electrospun micro/nanofibrous scaffolds are widely used in various tissue regeneration applications
because they have a similar structure to the extracellular matrix and can induce high attachment,
proliferation and even differentiation of cultured cells. Here, we designed a new composite scaffold
consisting of poly(3-caprolactone) (PCL), bone morphogenetic protein (BMP-2) and sulfuretin fabricated
using
a combined process, i.e. electrospinning/plasma-treatment/coating. In the composite, we
introduced a new bioactive component, sulfuretin, which was used as a cell stimulant to regenerate
bone tissue. Sulfuretin release from the composite was controlled by coating of a fixed concentration of
alginate. The in vitro biocompatibilities of the fibrous composites were examined using preosteoblasts
(MC3T3-E1s), and the composite showed high cell adhesion and differentiation for a limited range of
sulfuretin compared to the control, which lacked sulfuretin. These results suggest sulfuretin to be an
effective supplemental bioactive agent for enhancing bone tissue growth on fibrous composite scaffolds.
Received 14th April 2015
Accepted 13th May 2015
DOI: 10.1039/c5ra06648d
www.rsc.org/advances
peptide—to induce high cell adhesion, growth and differentia-
tion.11 To overcome the low cell-activating or -stimulating
Introduction
functions of the synthetic polymer, we designed a new bio-
composite system supplemented with bone morphogenetic
protein (BMP-2) and sulfuretin. BMPs belong to the trans-
forming growth factor-b (TGF-b) superfamily, which can control
cell migration, proliferation, and even differentiation and
angiogenesis.12,13 Since BMP-2 of the BMPs is one of the signif-
icantly important signalling molecules in bone formation, it has
been widely used as an imperative osteoinductive material.14–17
Sulfuretin can be extracted from Rhus verniciua Stokes (RVS;
the lacquer tree native to East Asia) and has been subjected to
evaluation of medical properties, such as antihuman cancer,18–20
anti-inammatory,21,22 antileukaemia,19 antimutagenic,23 anti-
platelet,24 and anti-rheumatoid arthritis25 effects. Although
sulfuretin has shown exceedingly positive effects as a medical
assisting and/or treating component, it has not been used in
tissue regenerative material, in particular as a bioactive
component for bone regeneration. Moreover, sulfuretin has not
been combined in any composite system as a dispersed bioac-
tive material supplemented to the nanobrous structure.
In this study, we designed a new composite system that used
various concentrations of sulfuretin/alginate coated onto
PCL/BMP-2 electrospun bres. The function of BMP-2 powder,
which was composed of rhBMP-2 coated on b-TCP (b-tricalcium
phosphate) and hydroxyapatite (HA) granules, in the composite
was to enhance the mechanical properties and osteogenesis.26
To effectively control its function, sulfuretin release from the
composite scaffolds was controlled by coating with a xed
The ultimate object of tissue engineering, an interdisciplinary
eld of study that combines engineering, material science and
cell biology, is to regenerate injured tissues and organs by using
biocompatible porous materials that mimic the original native
tissue.1,2 For successful regeneration of tissues, various fabri-
cation methods have been used—including e.g. electro-
spinning,3 gas foaming,4 solid free-form fabrications,5 and salt
leaching.6,7 Of these methods, electrospinning is widely used to
produce micro/nanobres, which have a similar physical
structure to the extracellular matrix (ECM) and a high surface-
to-volume ratio. These physical structures of the micro/
nanobres can provide biological cues to the cells, resulting
in successful regeneration of various tissues.8
The synthetic polymer poly(3-caprolactone) (PCL) has been
extensively used in fabricating bioscaffolds because it shows
easily controllable degradability; moreover, it is nontoxic and
exhibits easy and versatile processability and possesses
considerable mechanical properties compared to natural
biopolymers.9,10 However, like the most synthetic biopolymers,
the PCL absences cell adhesive motifs—proteins and peptides
such as bronectin binding to integrin and Arg-Gly-Asp (RGD)
aDepartment of Biomechatronic Eng., Sungkyunkwan University, Suwon 440-746,
Korea. E-mail: gkimbme@skku.edu; Tel: +82 31 2907828
bDepartment of Food Science and Biotechnology, Sungkyunkwan University, Suwon
440-746, Korea
cGyeonggi Bio-Center, Suwon 443-270, Korea
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concentration (20 wt%) of alginate. In general, alginate, an mixture of methylene chloride (Junsei, Tokyo, Japan) and
anionic polysaccharide commonly present in the cell walls of dimethylformamide (Junsei) in a 4 : 1 ratio). BMP-2 powder
brown algae, has been used in drug-delivery systems as a (mixture of rhBMP-2, b-TCP, and HA, Cowellmedi, Busan, South
controllable drug-releasing material due to its rapid gelation Korea) was mixed with the PCL solution at 0.5 wt% to fabricate
upon addition of divalent cations (Ca2+).27,28 In addition, because nanobres. Alginate solution for coating on the electrospun
the alginate has outstanding physiochemical properties such as PCL/BMP-2 bres was prepared by dissolving 20 wt% G-content
being nontoxic and nonallergic, it has been applied extensively nonmedical grade LF10/60 alginate (FMC BioPolymer, Dram-
as a bioink for cell printing, in wound dressings and as a tissue men, Norway) in triple-distilled water. To cross-link the alginate
regenerative material.29 Recently, Liu et al. fabricated alginate solution, CaCl2 (Sigma-Aldrich) was used. Sulfuretin (Mw
¼
struts (diameter z 20 mm) that incorporated nanocapsules, 270.241 g molꢀ1), kindly provided by Dr Ku of Gyeonggi Bio-
which are drug-encapsulated alginate beads. The alginate-based Center (Suwon, South Korea), was obtained by extraction from
nanocapsules enabled manipulation of drug release.30
RVS using ethanol. Fig. 1(a–d) shows optical and scanning
By using a combined processing method ((1) electrospinning electron microscopy (SEM) images and the chemical structure
of PCL/BMP-2, (2) plasma-treatment of the electrospun bres of sulfuretin and BMP-2 powder, respectively. We used 70% (v/v)
and (3) coating of sulfuretin and alginate), we generated a novel ethanol to dissolve the sulfuretin powder; the sulfuretin
composite material. MC3T3-E1 cells were used to assess the in concentrations used were 0.5, 1, 5 and 10 mM.
vitro responses of cells on the composite scaffold, including
proliferation determined by MTT assay, live/dead cell staining,
ALP activity, Runx2 expression and Alizarin Red S (ARS) stain-
ing. Furthermore, sulfuretin release over time and the physical
properties, water contact angle (WCA), biodegradation and
mechanical properties of the composite scaffolds were
determined.
Preparation and chemical identication of sulfuretin
To a mixture of 3.74 g (24.9 mmol) of methyl 6-hydroxy-
benzofuran-3(2H)-one and 3.44 g (24.9 mmol) of 3,4-dihydrox-
ybenzaldehyde in ethanol (124 mL), 12 N HCl (37.4 mL) was
added drop-wise at 0 ꢁC. Then the reaction mixture was reacted
ꢁ
at 60 C until the reaction was nished from TLC monitoring.
The mixture was poured into water and the resulting precipitate
was ltered and dried in vacuum. The crude product was
recrystallized by MeOH/H2O to yield 5.38 g (80.0%) of sulfuretin
as a yellow powder. The synthesized sulfuretin was identied by
LC/MS (Fig. 1(e and f)). LC was performed on WATERS
Materials and methods
Materials
A PCL solution was generated by dissolving 10 wt% PCL (Mw
¼
80 000 g molꢀ1; Sigma-Aldrich, St. Louis, MO, USA) in methy- ACQUITY UPLC BEH C18 column (2.1 ꢂ 100 mm; Milford, MA,
lene chloride/dimethyl formamide (MC/DMF) solvent (a USA). The mobile phase A was 0.1% formic acid in water and
Fig. 1 (a) Optical and (b) SEM images of sulfuretin powder. (c) The molecular structure of sulfuretin. (d) A SEM image of BMP-2 powder, which
was composed of rhBMP-2 coated on b-TCP/HA granules. (e) Liquid chromatography was performed on C18 column (2.1 ꢂ 100 mm) (mobile
phase: phase A: 0.1% formic acid in water, phase B: 0.1% formic acid in acetonitrile). (f) Positive ion mass spectrum was generated using a
Micromass Quattro Micro API mass spectrometer. Protonated sulfuretin [M + H]+ was detected at a retention time of 2.64 min.
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phase B was 0.1% formic acid in acetonitrile. Mass spectrum was dried and re-coated with 20 wt% alginate solution by
was performed with Micromass Quattro Micro API mass spec- dipping. Then, the coated alginate was cross-linked in 10 wt%
trometer (Milford, MA, USA). The monoisotopic mass of CaCl2 solution for 10 min and washed with phosphate-buffered
protonated sulfuretin [M + H]+ was detected at a retention time saline (PBS) three times to eliminate CaCl2 from the brous
of 2.64 min.
composites. The amount of coated alginate on the brous mat
was determined by weighing ber mats before and aer the
coating process. 0.6 ꢃ 0.01 mg of alginate was coated per
brous mat (diameter ¼ 5 mm and thickness ¼ 0.13 mm) (n ¼
10). The composite scaffold fabrication procedure is shown in
Fig. 2(a–e).
Fabrication of PCL/BMP-2/sulfuretin/alginate brous
composites
To obtain nanobres of PCL/BMP-2, the mixed PCL/BMP-2
solution was placed in a 10 mL syringe with a 16 G needle.
The ow rate of the solution (0.5 mL hꢀ1) was controlled by a
syringe pump system (KDS 230, KD Scientic, Ansan, South
Characterisations of the composite scaffolds
Korea). A high-voltage power supply (HVDC, SHV300RD-50K, The WCAs for ve independent samples were averaged and are
Convertech, Suwon, South Korea) was used and the applied presented as the means ꢃ SD. Water (10 mL) was placed on the
electric eld was set at 1.2 kV cmꢀ1 to obtain nanobres.
surface of the composite scaffolds, and aer 1 min the WCA was
Because the electrospun PCL/BMP-2 nanobres were measured under an atmospheric temperature of 26 ꢁC and
hydrophobic, the brous mats were treated with oxygen plasma humidity of 37%.
using low-frequency plasma (CUTE-MP/R, Femto-Science, Inc.,
To assess the degradation of the composite scaffolds, the
Busan, South Korea) for 5 min. A frequency of 50 kHz, a power initial weight (Wi) was measured. The composite scaffolds were
of 50 W and a pressure of 5.3 ꢂ 10ꢀ1 Torr were used.
then immersed in PBS (pH 7.4) containing lysozyme (10 000 U
The plasma-treated brous mats were dipped in 0.5, 1, 5 and mLꢀ1) at 37 C. Aer incubation for various time periods, the
10 mM sulfuretin solutions for 2 h, and each coated brous mat samples were rinsed with deionised water and freeze-dried,
ꢁ
Fig. 2 Schematic of fabrication of the PCL/BMP-2/sulfuretin/alginate composite scaffolds. (a and b) The PCL/BMP-2 fibres were deposited on a
collecting plate via electrospinning process and cut into 5 mm diameter round pieces. (c and d) To enhance their hydrophilicity, the composite of
the PCL/BMP-2 fibres was treated with oxygen plasma for 5 minutes and the plasma-treated composites were coated with 0.5, 1, 5 or 10 mM
sulfuretin for 2 h. (d and e) The sulfuretin-coated fibrous composite was coated with 20 wt% alginate, and the coated alginate was cross-linked
with CaCl2 solution (10 wt%). (f) A schematic of the effect of alginate coating for the release of sulfuretin from the composite scaffolds.
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aer which their weight (Wf) was measured. The per cent weight density of 1 ꢂ 105 per sample and incubated at 37 ꢁC in an
loss was calculated with the following equation: weight loss atmosphere of 5% CO2.
(%) ¼ [(Wi ꢀ Wf)/Wi] ꢂ 100. Weight loss is shown as the mean ꢃ
Cell proliferation was examined by the MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay
SD of ve samples.
The mechanical properties of samples were determined (Cell Proliferation Kit I, Boehringer Mannheim, Mannheim,
using a micro-tensile tester (Toptech 2000, Chemilab, Seoul, Germany) aer cell culture for 1, 3 and 7 days. Cells/scꢁaffolds
South Korea) in tensile mode. Samples 5 ꢂ 20 mm (width ꢂ were incubated with MTT (0.5 mg mLꢀ1) for 4 h at 37 C and
length) in size were prepared. The thickness of the samples was the absorbance at 570 nm was measured using a microplate
measured at three points under an optical microscope and the reader (EL800, Bio-Tek Instruments, Winooski, VT, U.S.A.). Five
values were averaged. The tensile test was performed in a “dry” samples were used for each incubation period and each test was
state. The samples were stretched to failure at a stretching performed in triplicate.
speed of 1 mm sꢀ1 at room temperature. All values are
expressed as means ꢃ standard deviation (n ¼ 5).
Live/dead cell staining
The scaffolds were exposed to 0.15 mM calcein AM and 2 mM
ethidium homodimer-1 for 45 min in an incubator (aer culture
Sulfuretin release test
Sulfuretin released from the composites was monitored by for 1, 3 and 7 days) to observe live and dead cells. The analysis
UV-spectrophotometry (Optizen 2120UVPlus, Mecasys Co., was performed under a microscope (TE2000-S, Nikon, Tokyo,
Daejeon, South Korea) in PBS (pH 7.4) at a wavelength of Japan) equipped with an epiuorescence attachment and a
405 nm. The sulfuretin release test was carried out by incu- SPOT RT digital camera (SPOT Imaging Solutions, Sterling
bating the scaffold in 15 mL of PBS solution at 37 ꢁC. The Heights, MI, U.S.A.). We captured images and processed the
scaffold was incubated in PBS solution for various periods. To area of green spots using the ImageJ soware (NIH, Bethesda,
measure sulfuretin release, 1 mL of the 15 mL of release solu- MD, U.S.A.) to assess the percentage of live cells.
tion was extracted and tested for the drug; the aliquot was then
returned to the release solution. To avoid contamination of
release solution, the pipette tip and cuvette of the spectropho-
tometer were sterilised with 70% ethanol. The cumulative
amount of sulfuretin released was calculated as a function of
time.
The total amount of sulfuretin coated in the brous mats,
which were not dipped in alginate solution, was measured
using a standard curve using the spectrophotometry. To obtain
the standard curve, we measured the optical density for 20, 40,
60, 80, and 100 mM sulfuretin. A good linearity (correlation
coefficient of 0.999) for the standard plot of the optical density
versus sulfuretin concentrations ranging from 20 to 100 mM was
DAPI/phalloidin staining
Aer 7 and 14 days of cell culture, the composite scaffolds were
subjected to diamidino-2-phenylindole (DAPI) uorescent
staining to detect cell nuclei. Phalloidin (Invitrogen, Carlsbad,
CA, U.S.A.) staining was performed to visualise the actin cyto-
skeleton of proliferated cells. The stained scaffolds were
visualised under a microscope (TE2000-S, Nikon, Tokyo, Japan)
equipped with an epiuorescence attachment and a SPOT RT
digital camera (SPOT Imaging Solutions).
Alizarin Red S (ARS) staining
obtained. The sulfuretin concentrations released from the Mineralisation levels were determined by ARS staining in
brous mats, which were dipped in 0.5, 1, 5 and 10 mM sul- 24-well plates. MC3T3-E1 cells were cultured in a-MEM and
furetin
solutions,
were
determined
using
the then washed three times with PBS, xed in 70% (v/v) cold
UV-spectrophotometer at 405 nm and evaluated in triplicate. ethanol (4 ꢁC) for 1 h and air-dried. Ethanol-xed specimens
The released solution was taken at 2 days, and PBS at 37 ꢁC was were stained with 40 mM ARS (pH 4.2) for 1 h and washed three
used as the dissolution medium.
times with puried water. Specimens were then distained with
10% cetylpyridinium chloride in 10 mM sodium phosphate
buffer (pH 7.0) for 15 min. The optical density (OD) at 562 nm
was measured using a Spectra III UV microplate reader (Spectra
III, SLT-Lab Instruments, Salzburg, Austria).
In vitro cell culture
The composite scaffolds were sterilised with 70% ethanol and
ultraviolet (UV) light, and then placed in culture medium
overnight. Mouse preosteoblast cells (MC3T3-E1; ATCC, Mana-
ssas, VA, U.S.A.) were used to evaluate the behaviour of cells
Real-time PCR analysis
cultured on the scaffolds. The cells were cultured for up to four Total RNAs from MC3T3-E1 cells cultured on scaffolds were
passages in 24-well plates containing a-minimum essential extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, U.S.A.)
medium (a-MEM, Life Technologies, Carlsbad, CA, U.S.A.) with as described previously.31 Briey, total RNAs were extracted and
10% foetal bovine serum (Gemini Bio-Products, West Sacra- 0.5 mg was subjected to reverse transcription to synthesise
mento, CA, U.S.A.) and 1% penicillin/streptomycin (Cellgro, complementary DNAs (cDNAs) using RTase M-MLV (2640A,
Mediatech, Manassas, VA, U.S.A.) for a concentration of 100 IU TaKaRa, Ohtsu, Japan). Expression of alkaline phosphatase
mLꢀ1 penicillin and 100 mg mLꢀ1 streptomycin.
(ALP) and Runx2 was measured using Power SYBR Premix Ex
The cells were collected by trypsin–ethylenediaminetetra- Taq (RP041A, TaKaRa) with gene-specic primer sets in a
acetic acid (EDTA) treatment, seeded onto the scaffolds at a thermal cycler (TaKaRa). The gene-specic forward and reverse
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oligonucleotide primer sets were synthesised by Integrated DNA actions of sulfuretin. Expression of genes linked to osteoblast
Technologies (San Diego, CA, U.S.A.). The following gene- differentiation was further measured to conrm the actions of
specic primers were used for real-time PCR: ALP F (accession sulfuretin in osteogenesis (Fig. 3(b)). Consistent with increased
#: NM_007431), 50-AAACCCAGAACACAAGCATTCC-30, ALP R, 50- ALP activity, real time PCR analysis showed that sulfuretin at
ACCAGCAAGAAGAAGCC-30; 36B4 F (accession #: NM_007475), 20 mM stimulated the expression of genes encoding osteocalcin
50-AGATGCAGCAGATCCGCAT-30, 36B4 R, 50-GTTCTTGCCCAT (OCN), colagen I (Col I), osterix, and Runx2 compared to control
CAGCACC-30; Runx2
F
(accession #: NM_001145920), 50- treatment. Therefore, ALP activity and gene expression analyses
GCCCAGGCGTATTTCAGA-30, Runx2 R, 50-TGCCTGGCTCTTC show the sulfuretin derived from natural herbal products can
TTACTGAG-30. The relative expression levels (fold change) were act as an osteoinductive factor.
obtained using the 2ꢀDCT method and normalised to the levels
of 36B4.
Preparation of PCL/BMP-2/sulfuretin/alginate brous
composite using electrospun PCL/BMP-2 bres
Statistical analysis
In the electrospinning process, we used an auxiliary cylindrical
electrode connected to the spinning nozzle to stabilise the
initial jet zone by concentrating the electric eld in the spinning
axis.32 Fig. 4(a) shows the initial spun jet and whipped/stretched
All data are presented as means ꢃ SD. Statistical analyses were
performed using the SPSS soware (ver. 20.0, SPSS, Inc., Chi-
cago, IL, U.S.A.) and consisted of single-factor analyses of vari-
ance (ANOVAs). In all analyses, p < 0.05 was considered to
indicate statistical signicance.
micro/nanobres using PCL/BMP-2 solution (electrical
conductivity ¼ 6.47 mS cmꢀ1; surface tension ¼ 0.034 N mꢀ1).
Fig. 4(b) shows an SEM image of PCL/BMP-2 bres with diverse
Result and discussion
diameters. In Fig. 4(c), the diameters of PCL/BMP-2 nano/
microbres were found to be distributed in two diameter
ranges (0.5–2.5 mm and 3.5–6.5 mm), which is termed a bimodal
distribution. Deitzel et al. rst investigated a bimodal brous
Stimulatory effects of sulfuretin on osteoblastic
differentiation in MC3T3-E1 cells
To test the pro-osteogenic effects of the sulfuretin, MC3T3-E1
cells were exposed to sulfuretin for 10 days and its effects on
osteoblast differentiation were assessed. An osteoblast marker,
alkaline phosphatase (ALP) activity was measured to examine
the effects on osteoblast differentiation (Fig. 3(a)). Treatments
of sulfuretin at 10–20 mM strongly increased ALP activity with a
modest induction at 30 mM suggesting the pro-osteogenic
Fig. 3 (a) MC3T3-E1 cells were treated with sulfuretin for 10 days and Fig. 4 (a) Whipped/stretched fibres in PCL/BMP-2 solution during the
ALP-positive cells were stained. (b) MC3T3-E1 cells were treated with electrospinning process, and (b) SEM image and (c) size distribution of
sulfuretin at the indicated doses for 10 days and relative expression of electrospun PCL/BMP-2 fibres. SEM images of (d) sulfuretin-coated
osteocalcin (OCN), collagen I (Col I), osterix (OSX), and Runx2 mRNA PCL/BMP-2 fibres and (e) an alginate-coated PCL/BMP-2/sulfuretin
were determined by real-time polymerase chain reaction. Data composite scaffold. (f) Weight loss of PCL/BMP-2/sulfuretin/alginate
are means standard deviations from three independent experiments scaffolds for the indicated time periods. (g) Stress–strain curves for the
(*p < 0.05, **p < 0.005).
PCL, PCL/BMP-2 and PCL/BMP-2/sulfuretin/alginate scaffolds.
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structure using an electrospinning process.33 Although this similar because degradation principally involves the coated
bimodal structure is still under investigation in terms of the alginate (not PCL); therefore, only the PBSA-4 curve is shown
exact relationship between the bimodal form and cell prolifer- (Fig. 4(f)). As revealed in the gure, the degradation of the
ation,34 bimodal bres with a nano- and microscale hierarchical composite increased linearly with time. The result is directly
structure can induce various cellular activities.35–38 For this correlated with sulfuretin release from the composite scaffolds.
reason, we can estimate that the bimodal topological structure Based on the degradation curve, sulfuretin was released
of the PCL/BMP-2 bres would enhance cellular activities constantly owing to continuous degradation of the coated
compared to homogeneous structural bres. Following their alginate.
fabrication sulfuretin was coated on the PCL/BMP-2 bres.
The mechanical properties of biomedical scaffolds are
However, because the bres are hydrophobic (WCA ¼ 115 ꢃ important parameters for successful regeneration of various
2.6ꢁ), they were treated with an oxygen plasma to increase their tissues because appropriate mechanical properties can main-
hydrophilicity. Aer 5 min of plasma treatment, the WCA of the tain the predesigned tissue structure and inuence cellular
PCL/BMP-2 bres was 10 ꢃ 1.4ꢁ. Four sulfuretin concentrations activities.40 Fig. 4(g) shows the stress–strain curves of the PCL,
of 500 mM, 1 mM, 5 mM and 10 mM were used to coat the PCL/BMP-2 and PBSA-4 scaffolds determined using a tensile
brous PCL/BMP-2 composite. Finally, to control sulfuretin tester under a 1 mm sꢀ1 crosshead speed. The initial slope of
release, 20 wt% alginate was used, based on our previous the curves was used to calculate the Young's modulus; the
work.27 In that study, rhodamine was used as the releasing moduli of the PCL, PCL/BMP-2 and PBSA-4 were 7.73 ꢃ
agent and 5, 10 and 20 wt% alginate were coated onto porous 1.31 MPa, 12.61 ꢃ 2.37 MPa and 51.16 ꢃ 4.26 MPa, respectively.
collagen scaffolds. The results showed that coating with 20 wt% Therefore, the PBSA scaffold showed greater stiffness compared
alginate showed the most appropriate controllability of rhoda- to the PCL and PCL/BMP-2 scaffolds due to its reinforced algi-
mine release from the scaffold. Fig. 4(d and e) shows SEM nate component.
images of PCL/BMP-2/sulfuretin (10 mM) bres (PBA) and a
PCL/BMP-2/sulfuretin (10 mM)/alginate composite scaffold
(PBSA). The amount of sulfuretin coated was measured using a
standard curve. The amount of sulfuretin coated on the
composite scaffolds, which were dipped in 0.5, 1, 5 and 10 mM
sulfuretin solutions, was 12.66 ꢃ 0.65, 20.24 ꢃ 2.15, 59.54 ꢃ
7.83 and 103.51 ꢃ 1.67 mM, respectively. In this work, the
scaffolds coated using 0.5, 1.0, 5.0 and 10.0 mM sulfuretin were
labelled PBSA-1, PBSA-2, PBSA-3 and PBSA-4, respectively.
Sulfuretin release from the PBSA scaffold
In this study, to interrupt the initial burst of sulfuretin release
from the composite scaffolds, we coated the sulfuretin-treated
brous structure with alginate. Fig. 5(a and b) shows the
cumulative percent release for composite scaffolds with (PBSA
scaffold) and without (PCL/BMP-2/sulfuretin (PBS) scaffold) an
alginate coating, respectively. Sulfuretin release from PBSA
scaffolds was inhibited by the alginate coating. In the initial
bursts (0–6 h), the sulfuretin release percentage was 0.91% hꢀ1
in PBSA scaffolds and 15.70% hꢀ1 in PBS scaffolds, which
Characterisation of PBSA brous composite
In general, the hydrophilic property of biomedical substitutes is suggests that the initial burst was disturbed by the alginate
related to cell growth, migration and physiological responses.39 coating of PBSA composite scaffolds. Moreover, the second
The hydrophilicity of the materials was assessed by measuring burst occurred between days 1 and 5 in PBSA following a time
the WCA. The PBSA-1, -2, -3 and -4 brous composite scaffolds interval of 0.5 days (grey-coloured), which was postulated to be a
showed similar hydrophilic property (WCA ¼ 6.7 ꢃ 1.2ꢁ aer release-induction phase by Batycky et al.41 This process can be
1 min).
interpreted according to Batycky et al. who formulated the
To assess the biodegradability of the PBSA composites, the process of drug release to determine the amount of released
weight change was tested for 7 days in lysozyme solution at 37 ꢃ drug over time. According to that study, the mass of the released
ꢁ
2 C. The biodegradation of each PBSA composite scaffold was drug can be formulated using the following equation:
Fig. 5 Cumulative percentage release of sulfuretin from (a) PCL/BMP-2/sulfuretin (PBS) composite scaffolds and from (b) PCL/BMP-2/sulfuretin/
alginate (PBSA). The numbers, 2, 3, and 4 indicate the sulfuretin concentrations, 1, 5, and 10 mM, respectively.
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mr(t) ¼ mi4(1 ꢀ eꢀk t
)
(1)
d
À
Á
mdðtÞ
¼ 1 ꢀ 4 1 ꢀ eꢀk t ꢀ ð1 ꢀ 4Þð1 ꢀ dðt ꢀ tiÞÞ
(2)
d
mi
where mi is the initial mass of drug, 4 is the mass fraction of the
drug involved in the burst relative to the initial drug and kd is
the drug desorption rate constant. The second burst (aer
induction) can be formulated according to the following
equation:41
where d is directly proportionate to the effective drug diffusivity
and inversely to the square of the micro-geometry of the poly-
mer. Based on the result, we can conrm that the sulfuretin
release was interrupted effectively by the alginate coating.
Fig. 6 (a) MTT results and (b) rate of increase in viable cell numbers (slope of optical density of MTT vs. culture day) on the composite scaffolds.
PBA indicates non-sulfuretin-coated scaffolds and PBSA-1, -2, -3, and -4 indicate the scaffolds with sulfuretin coating concentrations of 0.5, 1, 5,
and 10 mM, respectively. (c) Live (green)/dead (red) cells on the indicated composite scaffolds at 1, 3 and 7 days of culture. (d) Magnified image of
live cells on the composite scaffolds at 7 days and (e) percentage of spread cells. Asterisks and NS indicate significant and non-significant
difference, respectively.
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In vitro cell responses
To evaluate the relationship between in vitro cell activities and
the composite scaffolds with different concentrations of sul-
furetin, preosteoblast cells (MC3T3-E1) were cultured on the
PBA, PBSA-1, PBSA-2, PBSA-3 and PBSA-4 composite scaffolds.
Cell viability is one of the most important parameters for
assessing biomedical substitutes as viability can directly affect
the nal cell responses, such as proliferation and differentia-
tion. In Fig. 6(a), the MTT assay results indicate that the
proliferation of viable cells increased on the brous composites
with increasing culture time and increasing weight fraction of
sulfuretin, with the exception of only PBSA-4. In addition, a
signicant increase in the cell viability rate of the composite
scaffolds was observed in the PBSA-3 scaffold for all culture
times (Fig. 6(b)). Therefore, the sulfuretin released from the
composites facilitates preosteoblast proliferation, but only over
a limited range of sulfuretin concentrations.
To qualitatively evaluate live and dead cells on the composite
scaffolds, preosteoblasts were stained with calcein AM and
ethidium homodimer-1. Fig. 6(c) shows uorescence images of
live (green) and dead (red) cells on composite scaffolds aer
culturing for 1, 3 and 7 days. As expected from the MTT result,
PBSA-3 showed a signicantly higher number of viable cells
compared to the PBA, PBSA-1 and PBSA-4 scaffolds. Aer 7 days
in culture, the per cent cell spreading was considerably greater
on the PBSA-2 and PBSA-3 composite scaffolds than on the PBA
and PBSA-4 scaffolds (Fig. 6(d and e)). However, as evidenced by
MTT result, the effect of sulfuretin concentration in terms of
enhancing cell proliferation was limited.
Fig. 7 (a) DAPI (nuclei)/phalloidin (F-actin) images of composite
scaffolds after cell culture for 7 and 14 days. (b) Cell number per mil-
limetre squared and (c) F-actin area per cent (%) calculated using
images of the composite scaffolds. Asterisks and NS indicate signifi-
cant and non-significant difference, respectively.
In Fig. 7(a), the nuclei (blue) and F-actin (red) of preosteo-
blasts on the PBA, PBSA-1, PBSA-2, PBSA-3 and PBSA-4 scaffolds
were examined using uorescence imaging aer culturing for
7 and 14 days. The number of nuclei and cytoskeleton devel-
opment (actin) were more highly developed on the PBSA-2 and
PBSA-3 scaffolds compared to the PBA and PBSA-4 scaffolds
(Fig. 7(b and c)). The results indicate that the composite scaf-
folds supplemented with sulfuretin can induced signicant
development of the cytoskeleton, but only over a limited range
of sulfuretin concentrations.
Generally, ARS is used to evaluate the level of calcium
mineral. To assess the effect of sulfuretin, we conducted
experiments in which the composite scaffolds were incubated
in medium for 7 and 14 days. Fig. 8(a) displays optical images of
ARS staining of the composite scaffolds aer 7 and 14 days of
cell culture. The red colour in the images indicates the area of
calcium mineral. As the sulfuretin in the composite scaffolds
increased, the density of the red colour rose, indicating an
increase in calcium mineralisation, while the PBSA-4 scaffold
Fig. 8 Calcium mineralisation in MC3T3-E1 cells cultured for 7 and 14
days. (a) Optical images of ARS staining of the composite scaffolds. (b)
Optical density of the composites. Asterisks indicate significant
differences.
These results are consistent with the optical images of calcium
mineralisation. These results propose that conjugation of sul-
furetin to nanobres promoted the osteogenic differentiation of
the cells, but only over a limited range of sulfuretin concen-
trations. These results suggest sulfuretin to be a promising
bioactive factor for bone tissue regeneration.
(highest sulfuretin concentration) showed
a signicantly
reduced area of calcium mineralisation. The optical density
(OD) of composite scaffolds extracted from the stained
composite scaffolds is shown in Fig. 8(b). The OD value at
14 days was considerably higher than that at 7 days. In addition,
as the sulfuretin concentration in the brous composites
increased, the OD value rose. In particular, the OD value of
PBSA-3 at 14 days was almost 2.5-fold that of the control (PBA).
Finally, to conrm the effect of the released sulfuretin from
the composite scaffolds on osteogenic differentiation, we
measured the expression levels of two osteoblastic markers—
ALP and Runx2—using real-time PCR. MC3T3-E1 cells were
cultured on PBA (the control scaffold lacking sulfuretin) and
PBSA-2 scaffolds for 10 days. As shown in Fig. 9(a), the mRNA
expression of ALP increased 2.5-fold in cells grown on the PBSA-
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Fig. 9 Effects of sulfuretin scaffolds on osteoblast differentiation. MC3T3-E1 cells were cultured on sulfuretin scaffolds for 10 days, and relative
levels of (a) alkaline phosphatase (ALP) and (b) Runx2 mRNA were determined by real-time PCR. Asterisks indicate significant differences.
2 scaffold compared to the control. In addition, mRNA expres- (PJT200673), by the National Research Foundation of Korea
sion of Runx2 was induced approximately 2-fold in cells on the (NRF) grant funded by the Korea government (MSIP) (no.
sulfuretin-coated scaffold (Fig. 9(b)). These observations 2014R1A2A1A11050047), and also by the Basic Science Research
conrm that the released sulfuretin promotes osteoblast Programme through the National Research Foundation of
differentiation in MC3T3-E1 cells. However, although we Korea (NRF) funded by the Ministry of Education, Science and
showed that the sulfuretin-containing composite scaffolds Technology (NRF-2013R1A1A2060447).
induce osteoblast proliferation and differentiation, the mech-
anisms underlying the effect of sulfuretin on preosteoblast cells
are unknown. Various signalling pathways modulate the
proliferation and differentiation of osteoblasts, including the
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