Fabrication and characterization of silk fibroin–gelatin/chondroitin

Materials Letters 126 (2014) 207–210
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Materials Letters
journal homepage: www.elsevier.com/locate/matlet
Fabrication and characterization of silk ﬁbroin–gelatin/chondroitin
sulfate/hyaluronic acid scaffold for biomedical applications
Nopporn Sawatjui a, Teerasak Damrongrungruang b, Wilairat Leeanansaksiri c,d,
Patcharee Jearanaikoon e, Temduang Limpaiboon e,n
a
Biomedical Sciences, Graduate School, Khon Kaen University, Khon Kaen 40002, Thailand
Department of Oral Diagnosis, Faculty of Dentistry, Khon Kaen University, Khon Kaen 40002, Thailand
c
Stem Cell Therapy and Transplantation Research Group, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand
d
School of Microbiology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand
e
Centre for Research and Development of Medical Diagnostic Laboratories(CMDL), Faculty of Associated Medical Sciences, Khon Kaen University,
Khon Kaen 40002, Thailand
b
art ic l e i nf o
a b s t r a c t
Article history:
Received 12 December 2013
Accepted 5 April 2014
Available online 13 April 2014
We fabricated silk ﬁbroin–gelatin/chondroitin sulfate/hyaluronic acid (SF–GCH) scaffolds with different
blending ratios using freeze-drying technique. The properties of the scaffolds in term of structural
morphology, porosity, water uptake, compressive strength, degradability and cell viability were
investigated. The blended scaffolds showed porous structure with pore diameters ranging 211–
272 mm, and high porosity and water uptake. The addition of SF reduced degradation rate of scaffold
in collagenase solution and increased mechanical strength by 168% in comparison with GCH scaffold.
in vitro cytocompatibility demonstrated that the blended SF–GCH scaffold supported cell viability and
proliferation, which may be of value for tissue engineering applications.
& 2014 Elsevier B.V. All rights reserved.
Keywords:
Biomaterials
Poly-blended composites
Fabricated scaffold
Freeze drying technique
1. Introduction
Many natural biomaterials such as collagen, chondroitin sulfate
(C) and hyaluronic acid (H) are essential components in extracellular matrix (ECM). Gelatin (G) is a partial derivative of collagen
but less immunogenic and retains the Arg–Gly–Asp motif that can
promote cell adhesion and proliferation [1]. Chondroitin sulfate
comprises repeating disaccharide units containing sulfate ester
and carboxylic groups to which the positively charged growth
factors can be attached [2,3]. Hyaluronic acid is a water-soluble
polysaccharide widely distributed in ECM. The speciﬁc interaction
of hyaluronic acid with CD44 cell surface receptor plays an
important role in cell migration [4]. The GCH scaffold is capable
of supporting keratinocytes and dermal ﬁbroblasts to proliferate
and secrete their ECM [5]. However, the lower mechanical strength
and rapid degradation are the critical limitation of these materials
leading to the easy loss of scaffold integrity prior to complete cell
proliferation, differentiation and development of the cultured cells
for reconstitution of tissue and organ. The gelatin/chitosan/hyaluronan scaffold showed low compressive modulus and loss of
integrity after 3 weeks in PBS solution [6].
n
Corresponding author. Tel./fax: þ 66 43202088.
E-mail address: [email protected] (T. Limpaiboon).
http://dx.doi.org/10.1016/j.matlet.2014.04.018
0167-577X/& 2014 Elsevier B.V. All rights reserved.
Silk ﬁbroin (SF), originated from B. mori silk, is an attractive
biomaterial because of its excellent mechanical properties, nontoxicity, biodegradability, biocompatibility and supporting incorporation of many molecules such as polymer, peptide and hydrogel making
SF utilizable in biomedical applications [7,8]. Moreover, enriched SF
content in silk ﬁbroin–chitosan scaffold has signiﬁcantly higher
compressive modulus and slower degradation in lysozyme than poor
SF content [9]. Regarding its impressive properties, the 3-dimensional
SF scaffolds were fabricated by blending with the GCH tri-copolymer
in different ratios and evaluated for their physical and biological
properties.
2. Experimental
Material preparation: Bombyx mori silk cocoons (Nangnoi-Srisaket)
were obtained from the Queen Sirikit Sericulture Center, Khon
Kaen, Thailand. Type A gelatin, chondroitin sulfate, hyaluronic
acid, N-(3-Dimethylaminopropyl)-N0 -ethylcarbodiimide hydrochloride
(EDC), N-Hydroxysuccinimide (NHS), MTT (Methylthiazolyldiphenyltetrazolium bromide) and collagenase from Clostridium histolyticum
were purchased from Sigma-Aldrich, St. Louis, MO.
SF solution was prepared as described previously [10] and
ﬁnally adjusted with distilled water to 3% (w/v). The mixture of
gelatin, chondroitin sulfate and hyaluronic acid (3:1:0.05 w/w)
was used to prepare 3% (w/v) GCH solution at 40 1C for 30 min.
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N. Sawatjui et al. / Materials Letters 126 (2014) 207–210
Scaffold fabrication: The 3-dimensional SF and SF-GCH scaffolds were fabricated by freeze-drying technique. SF and GCH
solutions were blended with ratios 2:1, 1:1 and 1:2 (v/v) before
1% EDC/0.5% NHS was added for cross-linking at room temperature for 15 min. Subsequently, the mixed solutions were injected
into the cylindrical Teﬂon molds, frozen at 20 1C for 4 h, at
80 1C for 4 h and lyophilized for 48 h. The freeze-dried scaffolds were immersed in methanol for 1 h, then repeatedly crosslinked in 0.2% EDC/0.1% NHS and lyophilized under the same
condition.
Physical characterization of scaffolds: The cross sectional scaffold morphology was observed by a Hitachi S-3000N scanning
electron microscope (SEM). The pore diameter of scaffolds was
determined from SEM images using NIS elements BR 3.0 imaging
software. The porosity of scaffolds was measured using a liquid
displacement method [11]. The mechanical compression of the
scaffolds was evaluated using a universal testing machine
(LR30K, LLOYD Instrument) with 100 N load cells at room
temperature and the loading rate was 1 mm/min. The compressive strength was assessed by drawing a parallel line starting at
1%. The point at which this line crossed the stress–strain curve
was deﬁned as the compressive strength. The percentage of
water uptake was determined by weighing the scaffolds before
and after incubation in PBS at 37 1C for 24 h. The degradation of
scaffolds was evaluated by mass remaining of scaffolds after
incubation in PBS containing 10 mg/mL collagenase at 37 1C for 7,
14 and 21 days.
Cell culture: The human bone marrow mesenchymal stem
cells (BM-MSCs) [12] were expanded to passage 3 in DMEM-low
glucose containing 10% fetal bovine serum, 100 U penicillin and
100 mg/mL streptomycin. The sterile scaffolds were soaked in
culture medium for 4 h, seeded with 3 104 BM-MSCs and left
for 3 h at 37 1C with 5% CO2 before culture medium being added.
The cell-seeded scaffolds were incubated at 37 1C with 5% CO2
for 1, 3 and 5 days, and subsequently determined for cell
viability using MTT assay. The absorbance was measured at
570 nm by microplate reader (Sunrise, TECAN). Cellular distribution was histologically examined on the hematoxylin-eosin
(H&E) stained sections of scaffolds after being cultured for
5 days.
Data are presented as mean 7SD. One-way ANOVA and Tukey's
post hoc tests were used to determine signiﬁcant differences
among groups. Student's t-test was used to compare cell viability
between SF and SF–GCH scaffold. P o0.05 was considered statistically signiﬁcant.
3. Results and discussion
The SEM images of SF, GCH and SF–GCH scaffolds with different
blending ratios are shown in Fig. 1a–e. The porous structures were
distributed throughout the scaffolds. The pore diameter of SF and
GCH scaffolds was 101.8719.0 mm and 561.2740.1 mm, respectively while that of SF–GCH was 211.2730.0–272.0 734.5 mm
which was related to the increased GCH content (Fig. 2a), suggesting the effect of GCH content on porous structure of the scaffolds.
The optimal scaffold pore size required for tissue engineering
varies widely depending on biological applications in which a
minimum pore size of 200–250 mm is proposed for the growth of
soft tissue and 150 mm for hard tissue [12,13].
The percent porosity of scaffolds was decreased when SF
content was reduced as shown in Fig. 2b suggesting that the
structural morphology is dependent on the ratio of the SF added.
Water retention is an important property of scaffold that inﬂuences its efﬁcacy in tissue engineering. A high water uptake was
beneﬁcial to maintain the shape of scaffold and transfer nutrients
and wastes. The water uptake was increased when GCH was
blended into the SF scaffolds (Fig. 2c). The gelatin, chondroitin
sulfate and hyaluronic acid have a large number of hydrophilic
groups that can be easily hydrated [14]. The swelling ratio of silk
ﬁbroin/chondroitin sulfate/hyaluronic acid was increased signiﬁcantly with an increase in hyaluronic acid content [10]. The pure SF
scaffold showed signiﬁcantly higher compressive strength than
GCH scaffold. The compressive strength of SF–GCH scaffolds was
increased from 108.5 kPa to 252.3 kPa when SF content was
increased (Fig. 2d). The methanol treatment of silk ﬁbroin could
induce β-sheet formation which increased compressive strength of
the blended scaffolds [15]. The GCH scaffold was degraded at
signiﬁcantly faster rate than SF scaffold and its mass remaining
was 36.7% after 3 weeks (Fig. 2e). The larger pore facilitates
enzyme diffusion leading to quick degradation as seen in the
GCH scaffold [16]. The addition of SF could impede GCH composite
degradability in collagenase as observed in SF–GCH scaffold
(Fig. 2e).
Since the SF–GCH 2:1 showed high compressive strength and
slow degradation which enable to retain the integrity of scaffold
Fig. 1. SEM images of the SF, GCH and SF–GCH scaffolds with different blending ratios. (a) SF scaffold, (b) SF–GCH 2:1, (c) SF–GCH 1:1, (d) SF–GCH 1:2 and (e) GCH scaffold.
N. Sawatjui et al. / Materials Letters 126 (2014) 207–210
209
Fig. 2. Effect of SF and GCH blending ratios on physical properties. (a) Pore diameter, (b) Percent porosity, (c) Percent water uptake, (d) Compressive strength and (e) Scaffold
degradation rate in collagenase. (S1) SF, (S2) SF–GCH 2:1, (S3) SF–GCH 1:1, (S4) SF–GCH 1:2, (S5) GCH. Groups without the same Roman numeral are statistically different
(Po 0.05).
Fig. 3. The cell viability of BM-MSCs cultured on SF and SF–GCH 2:1 scaffolds (a) and H&E staining of BM-MSCs on SF (b) and SF–GCH (c) after cultured for 5 days.
and protect seeded cells from being damaged, we selected this
scaffold for determining cell viability and proliferation, which are
indicators of cellular biocompatibility and suitability for tissue
engineering. BM-MSCs grown in SF–GCH 2:1 showed 1.48-, 1.55and 1.86-fold higher proliferation rate than in pure SF after day 1,
3 and 5, respectively, which statistically signiﬁcant difference was
observed in day 3 and 5 (Po0.05) (Fig. 3a). It was shown that SF
blended with gelatin enhanced ﬁbroblast proliferation compared to
pure SF [16]. Histological sections showed homogenous distribution
of typically spindle-shaped characteristic of BM-MSCs grown in SF
and SF–GCH scaffolds of which higher cell distribution was found in
SF–GCH than in SF (Fig. 3b, c) indicating that the fabricated SF–GCH
scaffolds are non-toxic and suitable for cell proliferation.
4. Conclusions
The three-dimensional SF–GCH scaffold was fabricated successfully using freeze-drying technique and the effect of SF and GCH
blending ratios was investigated. The SF–GCH scaffolds show
different pore size, porosity and water uptake depending on the
ratios of SF to GCH. Moreover, the blended SF–GCH has several
physical and biological advantages over SF or GCH alone such as
increased scaffold integrity, slower degradation rate, improved
mechanical strength, non-cytotoxicity and enhanced cell proliferation. With these impressive properties, our fabricated SF–GCH
scaffold is an ideal biomaterial which may be suitable for tissue
engineering applications.
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N. Sawatjui et al. / Materials Letters 126 (2014) 207–210
Acknowledgments
We appreciate Professor Suradej Hongeng for kindly supplying
BM-MSCs. This work was supported by the Royal Golden Jubilee Ph.D.
Program (grant no. PHD/0149/2550 to N. Sawatjui); Division of
Research Administration, Khon Kaen University; and the Centre for
Research and Development of Medical Diagnostic Laboratories
(CMDL), Faculty of Associated Medical Sciences, Khon Kaen University,
Khon Kaen, Thailand.
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