International Journal of Pharmaceutics 479 (2015) 291–301

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International Journal of Pharmaceutics

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

Pharmaceutical nanotechnology

Silk fibroin/gelatin electrospun nanofibrous dressing functionalized

with astragaloside IV induces healing and anti-scar effects on burn
wound
Ying-Hui Shan a,1, Li-Hua Peng a,1, Xin Liu b , Xi Chen a , Jie Xiong b , Jian-Qing Gao a,c, *
a
Institute of Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, PR China
b
College of Materials and Textiles, Zhejiang Sci-Tech University, Hangzhou 310018, PR China
c
Jiangsu Engineering Research Center for New-Type External and Transdermal Preparations, PR China

A R T I C L E I N F O A B S T R A C T

Article history: Functional wound dressing has provided new challenges for researchers who focus on burn to improve
Received 27 September 2014 skin graft quality, reduce scarring, and develop a pluristratified dermal or epidermal construct of a burn
Received in revised form 23 December 2014 wound. This study aimed to investigate the effect of a silk fibroin/gelatin (SF/GT) electrospun nanofibrous
Accepted 27 December 2014
dressing loaded with astragaloside IV (AS) on deep partial-thickness burn wound. AS-loaded SF/GT-
Available online 30 December 2014
blended nanofibrous dressing was prepared by electrospinning nanotechnology. The optimal ratio
(25:75) of silk fibroin to gelatin was further optimized by evaluating ATR-FTIR characteristics, mechanical
Keywords:
properties, porosity, swelling rate, degradation, and release profile of the AS-loaded SF/GT nanofibrous
Burn
Nanofibrous dressing
dressing. In contrast to the blank control, the AS-loaded SF/GT nanofibrous dressing promoted cell
Astragaloside IV adhesion and proliferation with good biocompatibility in vitro (p < 0.01). This dressing also accelerated
Wound healing wound healing and inhibited scar formation in vivo by stimulating wound closure (p < 0.05), increasing
Scar inhibition angiogenesis, regulating newly formed types of collagen, and improving collagen organization. These
results showed that SF/GT nanofibrous dressing is a promising topical drug delivery system. Furthermore,
AS-functionalized SF/GT nanofibrous dressing is an excellent topical therapeutic that could be applied to
promote healing and elicit anti-scar effects on partial-thickness burn wound.
ã 2015 Elsevier B.V. All rights reserved.

1. Introduction The risk of serious systemic infection originating from burn

Burn, one of the worst forms of trauma, is accounted for an
increased worldwide incidence of approximately two million cases
annually (Brigham and McLoughlin, 1996). This condition is treated
by implementing effective burn care that covers four major areas of
advances: (1) control of infection; (2) fluid resuscitation and early
patient management; (3) modulation of hypermetabolic response;
and (4) surgery and wound care (Janzekovic, 1970; Merrell et al.,
1989). Different methods have been used to treat varying degrees
of burn wounds. For instance, superficial partial-thickness burns
often spontaneously re-epithelialize in 7–28 days. Full-thickness
burn (third-degree burn) is effectively managed by conducting an
immediate full excision and applying autologous skin graft.
* Corresponding author at: Institute of Pharmaceutics, College of Pharmaceutical
Sciences, Zhejiang University, PR China. Tel.: +86 571 88208437;
fax: +86 571 88208437.
E-mail address: gaojianqing@zju.edu.cn (J.-Q. Gao).
1
These authors contributed equally to this work.
wound has been reduced because of the routine incorporation of
early burn wound excision and coverage (Janzekovic, 1970). Deep
dermal burns are dry and less sensate and characterized by poor or
no capillary refill. These burns exhibit a pale and/or mottled
appearance involving the reticular dermis; furthermore, re-
epithelialization occurs in cells residing in hair follicles, often
resulting in severe scarring (Jeschke et al., 2013). Hence, deep
dermal burns are considered as a valuable direction in external
preparation studies. In several cases, the size and/or depth of
wounds may actually increase because of improper burn treat-
ments, such as lack of proper wound care, resuscitation, and edema
formation. In burn wound care, topical agents with relevant
qualities, such as broad-spectrum antibacterial activities, low
development of resistance, limited adverse effects, and decreased
risk of systemic toxicity, are used. Various agents, including
creams, ointments, and biological or non-biological dressings, are
also used to treat burn wounds.
Burn wound healing is an interactively dynamic process and
intercellular stimulating response; this process involves tempo-
rary skin replacement, wound debridement, cytokine and growth

http://dx.doi.org/10.1016/j.ijpharm.2014.12.067
0378-5173/ ã 2015 Elsevier B.V. All rights reserved.
292 Y.-H. Shan et al. / International Journal of Pharmaceutics 479 (2015) 291–301
factor secretion, and collagen synthesis. Burn wound is finally
replaced with new extracellular matrix; new epithelial cells then
resurface and cover the injured skin from wound edges
(Rhett et al., 2008). In a previous study, astragaloside IV (AS)
can significantly promote healing and inhibit full-thickness acute
wound (Chen et al., 2012). As a natural product, AS has been
considered as a promising agent to promote burn wound healing. A
preliminary experiment has demonstrated the effect of AS on deep
partial-thickness burn wound; results show that the AS group
exhibits improved healing compared with the control group.
Hence, the treatment effect of AS is dependent on drug
concentration and interval dosage time.
Burn wounds require topical treatments to prevent infection,
debride wound eschar, and promote healing. Furthermore, wound
dressings provide temporary coverage for burn wounds, maintain
temperature homeostasis, prevent desiccation, and promote
healing. Electrospun nanofibers have been investigated for tissue
engineering applications because these materials are structural
mimics of extracellular matrix (ECM) (Stitzel et al., 2001; Yim and
Leong, 2005). Drugs releasing scaffolds can promote cellular and
biological activities; as such, wound healing and tissue regenera-
tion are facilitated. Therefore, the application of electrospun fibers
in drug delivery has been extensively studied.
Silk fibroin (SF) produced by silk worm has been recognized as a
promising scaffold material because of biocompatibility, biode-
gradability, high mechanical strength, and flexibility (Altman et al.,
2003; Meinel et al., 2005). SF scaffolds can support cell adhesion,
proliferation, and differentiation in vitro, as well as promote tissue
repair in vivo (Altman et al., 2003; Lu et al., 2009; Tonsomboon
et al., 2013). However, slow degradation is not advantageous for
clinical applications. Gelatin, a biocompatible and degradable
substance, has been widely used to develop wound dressings
(Peng et al., 2012a). This substance is derived from the partial
hydrolysis of collagen (Elzoghby et al., 2012). Therefore, SF with
gelatin may be advantageous to prepare scaffolds that can mimic
the ECM in terms of topography and chemical composition
(Altman et al., 2003; Lu et al., 2009; Tonsomboon et al., 2013).
Manunya demonstrated that SF degradation is accelerated and
cell proliferation is improved by adding gelatin to SF scaffolds
(Okhawilai et al., 2010). However, the optimal ratio of silk and
gelatin remains unknown.
This study aimed to develop a nanofibrous wound dressing
prepared from optimized SF/gelatin material incorporated with AS
by electrospinning. The prepared functionalized nanofibrous
dressing was then applied topically on burn wounds; the effects
on burn wound healing and scar formation, skin angiogenesis, and
collagen deposition were investigated in vitro and in vivo.
2. Materials and methods
2.1. Materials
Astragaloside IV (purity > 98%, HPLC) was purchased from
Shanghai Sunny Biotech Co., Ltd., China. Dulbecco’s modified Eagle’s
medium (DMEM) and fetal bovine serum (FBS) were purchased from
Gibco BRL (Gaithersburg, MD, USA). Proteinase K was purchased
from Aladdin, Inc. (Shanghai, China). Purified mouse anti-rat
CD31 and VEGF monoclonal antibody was purchased from Abcom
(UK). Masson’s trichrome staining kit was purchased from Nanjing
Keygen, Inc. (Nanjing, China). Immortalized keratinocyte line was
obtained from Jiangsu Biomics, Inc. (Jiangsu, China). Buffered
paraformaldehyde (4%) was purchased from Boster, Inc. (Wuhan,
China). Gelatin was purchased from Aladdin, Inc. (Shanghai, China).
Silk fibroin was made from Bombyx mori silk fibers by Key Laboratory
of Advanced Textile Materials and Manufacturing Technology of
Zhejiang Sci-Tech University.
2.2. Animals
Eight-week-old Sprague–Dawley rats (180–200 g) were pro-
vided by Zhejiang University Experimental Animal Center, China.
All of the experimental procedures were in accordance with
Zhejiang University guidelines for the welfare of experimental
animals. All of the rats were provided free access to standard
diet and water and raised under constant conditions (tempera-
ture 26
1  C). Animal experimentation ethics approval nos.
Zju2012-0052.
2.3. Optimization and preparation of the SF/GT nanofibrous dressing
by electrospinning technology
The homogeneous solution of a certain amount of SF/gelatin
and AS was filled up in a 5 ml syringe connected with a needle. A
high voltage of 15 kV was applied to the droplet of injected
solution. A grounded rotating collector with aluminum foil
attached to the drum was placed 13 cm away from the needle
tip. The collector was rotated slowly to minimize any preferential
alignment of the fiber. Solution flow rate was fixed at 0.01 ml/min.
The suitable blending weight ratios [SF/GT (100/0) (SF), SF/GT (75/
25), SF/GT (25/75), SF/GT (0/100) (GT)] were chosen and electro-
spun for 30 h to obtain nanofibrous dressing. The collected
nanofibrous dressing was left overnight in a vacuum dryer to
ensure that the solvent was completely removed.
2.4. Characterization of the SF/GT nanofibrous dressing
2.4.1. Morphological observation by scanning electron microscopy
(SEM)
The surface of the nanofibrous dressing specimens was viewed
using a SEM (Hitachi 3000, Japan) at 15 kV. Before observing, we
sputtered the samples with an ultrathin layer of gold in a coating
system. The diameters of the obtained nanofibrous dressing were
determined using image analysis software (ImageJ, NIH). The
average diameter of nanofibers in the dressing was determined by
measuring the diameters of 100 randomly selected fibers.
2.4.2. ATR-FTIR
Nanofibrous dressing was analyzed by attenuated total reflec-
tion Fourier transform infrared (ATR-FTIR) spectroscopy in a
Nicolet spectrometer (Thermo Scientific, USA) at a spectral range of
4000–600 cm1 (Jankovic et al., 2013).
2.4.3. Mechanical properties
The tensile properties of the nanofibrous dressing were
evaluated using a multifunctional tensile tester (KES-G1). These
tests were performed using rectangular specimens with a size of
5 mm  50 mm, a clamping length of 40 mm, and a constant speed
of 0.2 mm/s.
2.4.4. Porosity
The structural pore size of the nanofibrous dressing was
performed using a capillary flow porometer (AutoPore IV 9510,
USA). The capillary flow porometer was used to measure scaffold
pore size and distribution. All of the necessary calculations were
performed using the software from Porous Materials (9500SETUP,
USA).
2.4.5. Swelling rate
The expansion rate of the nanofibrous dressing was measured
to determine the ability of organized exudate absorption. In brief,
the samples were treated with ethyl alcohol for 10 min, dried at
37  C in an oven, and exposed to deionized water. Swelling rate
was in a dry state according to the following equation:
 Y.-H. Shan et al. / International Journal of Pharmaceutics 479 (2015) 291–301
ðM1  M0 Þ
Swelling rate ð%Þ ¼  100%
M0
where M1 represents the average height of the swollen fiber and M0
represents the average height of the dry fiber. Data were expressed
as mean
standard deviation (n = 3) (Jankovic et al., 2013).
2.4.6. Degradation
The in vitro biodegradation of the nanofibrous dressing was
observed in a solution of phosphate-buffered saline (PBS, pH
7.2–7.6) and 0.01 U/ml of proteinase K solution (1 mg = 35.8 units,
Sigma, St. Louis, MO) at pH 7.4 and 37  C. After SF/GT nanofibrous
dressing were socked in each degradation solution for 2, 4, 8, 12, 24,
36, and 48 h, they were removed and washed with deionized
water; afterward, the degraded nanofibrous dressing was dried at
37  C in a constant-temperature oven and weighed. The result of in
vitro biodegradation was calculated as follows:
ðD1  D2 Þ
Degradation rate ð%Þ ¼  100%
D1
where D1 represents the initial weight of the nanofibrous dressing
(g) and D2 represents the final weight of the same nanofibrous
dressing after this dressing was exposed to the degradation
solution. Data were expressed as mean
standard deviation (n = 3)
(Okhawilai et al., 2010).
2.4.7. Drug release
Drug release was investigated by placing the AS-loaded SF/GT
nanofibrous dressing in a release medium with PBS (pH 7.2–7.6)
containing 0.5% sodium dodecyl sulfate. The AS-loaded SF/GT
nanofibrous dressing (0.5 g) was placed in a beaker containing
20 ml of the release medium. The beaker was capped and placed on
a magnetic stirrer at 37  C and 50 rpm. At predetermined time
points, 1 ml of the release medium (sample) was withdrawn from a
beaker and replaced with 1 ml of fresh release medium to maintain
the sink condition. The samples were analyzed by HPLC.
2.5. Biocompatibility evaluation
2.5.1. Cell culture
Sterilized nanofibrous dressing membranes were inserted in
96-well culture plates (Nunc, Denmark) to evaluate the biocompati-
bility of the nanofibrous dressing in vitro. Immortalized human
fibroblast cells (Institute of Biochemistry and Cell Biology, Shanghai
Institutes for Biological Sciences, Chinese Academy of Sciences) were
seeded onto the nanofibrous dressing at a density of 104 cells/cm2
and incubated in DMEM with 10% FBS (Gaithersburg, MD, U.S.A) and
1% penicillin and streptomycin. To maintain the culture environment
at 37  C and 5% CO2, we changed the media every 2 days.
2.5.2. Cell proliferation and attachment in SF/GT nanofibrous dressing
The attachment and proliferation of cells on nanofibrous dressing
were observed using the standard MTT assay. At 1, 3, and 5 h and 1, 4,
and 7 days, the media were removed; non-adherent and loosely
attached cells were also removed by tapping the plates gently and
washing the wells with PBS (pH 7.2–7.6). Afterward, the nanofibrous
dressing with attached cells were stained with 5 mg/ml thiazolyl
blue (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bro-
mide, MTT) for 4 h at 37  C and 5% CO2. A purple-colored formazan
derivative was formed and then dissolved thoroughly with 200 ml of
dimethyl sulfoxide (Pierce) for 20 min; approximately 100 ml of
supernatant of the well was transferred to a 96-well plate. The
cultured cells on the blank plate were used as a control sample.
Absorbance was determined at 490 nm (MULTISKAN MK3, Thermo
Electron Corporation). Cell adhesion and proliferation were
expressed as mean
standard deviation (n = 4).
293
The morphological characteristics of adherent cells on the
nanofibrous dressing were investigated by SEM. At the end of 7
days, the nanofibrous dressings were washed thoroughly with PBS
(pH 7.2–7.6) thrice and fixed with 2% glutaraldehyde solution for
4 h. In SEM analysis, the samples were prepared by washing them
twice with PBS (pH 7.2–7.6); these samples were then fixed by
immersing in 2.5% osmic acid solution for 24 h at 4  C. The sample
sheet was prepared by acetone dehydration with critical point
drying, sputter-coated with gold, and viewed under a SEM at an
accelerating voltage of 15 kV.
2.6. Animal study
2.6.1. Establishment of partial-thickness burn wound in rats
A partial-thickness burn wound model was used to assess the
wound healing efficacy of nanofibrous dressing functionalized
with AS by utilizing an instrument at constant temperature and
pressure. The rats were anesthetized with 10% chloral hydrate
(4 ml/kg) intraperitoneally; afterward, all of the rats were
transferred to a warm environment to obtain a constant body
temperature. Hair over the dorsum was clipped, and the skin was
scrubbed with 75% ethanol solution to prepare for wounding. A
circular burn wound with an area of approximately 4.0 cm2 was
made using the instrument with constant temperature and
pressure. The contact surface of the electrode was subjected to a
constant temperature that could be adjusted from 0 to 400  C
depending on the depth and degree of injury that would be
inflicted. The rats were scalded for 6, 8, and 20 s by using an
instrument at constant temperature (80  C) and pressure (12 KPa).
This procedure was conducted to create a uniform wound with a
sharp margin. The depth of injury was determined by a pathologist
who examined tissue sections at 2 days after injury in a blinded
manner. The total skin injury area was approximately 4% of the
whole skin surface.
A total of 32 eight-week-old Sprague–Dawley rats were used in
this study. These rats were distributed into four groups: blank
control; blank nanofibrous dressing; AS solution; and AS-loaded
SF/GT nanofibrous dressing. Normal saline, AS solution, blank
wound dressing, and AS-loaded SF/GT nanofibrous dressing were
locally delivered at the wound site every 2 days. The dosage of AS in
nanofibrous dressing and AS solution groups was 0.5 mg every 2
days. The wounds were copied with filter papers and the weight
percentage of filter papers was calculated at different post-
wounding time points; wound closure percentage was then
obtained as follows:
Wound closure percentageð%Þ
 
Area on day0  Open area on dayn
¼  100%
Area on day0
2.6.2. Masson’s trichrome staining, HE staining, and Sirius red staining
At 31 days post-wounding, the wound samples, including
full-thickness skin layers (epidermis, dermis, and hypodermis),
were fixed in 4% buffered paraformaldehyde. The tissues were
sequentially dehydrated in 75, 85, 95, and 100% ethanol and
dimethylbenzene. Afterward, the processed tissues were embed-
ded in paraffin. Tissue sections (thickness of 8 mm) were analyzed
by Masson’s trichrome, HE, and Sirius red staining (Peng et al.,
2013a). The processed specimens were examined and then
photographed using a Leica image analyzing system. Tissue
sections with Sirius red staining were photographed using a
polarizing microscope (Nikon, Japan) (Peng et al., 2013b).
294 Y.-H. Shan et al. / International Journal of Pharmaceutics 479 (2015) 291–301

[(Fig._1)TD$IG]

Fig. 1. SEM images of the nanofibrous dressing. (A) SF; (B) SF/GT 75/25; (C) SF/GT 25/75; (D) GT; and (E) AS-loaded SF/GT 25/75, and the corresponding fiber diameter
distribution histograms.
 Y.-H. Shan et al. / International Journal of Pharmaceutics 479 (2015) 291–301 295

2.6.3. VEGF and CD31 immunohistological staining
The sample slices (4 mm) were dewaxed in xylol and then
soaked in graded alcohol series to rehydrate. The sections were
washed with distilled water and soaked in 0.01 M citrate buffer (pH
6.0) to retrieve epitope; these sections were then soaked in 3%
H2O2 in methanol for 10 min to inhibit endogenous peroxidase. The
slices were washed with 1 M PBS (pH 7.2–7.6), incubated with
diluted VEGF and CD31 primary antibody (1:100) at 4  C overnight,
and washed thrice with 1 M PBS (pH 7.2–7.6) for 5 min in each
wash. The slices were subsequently incubated with PV-6001 and
PV-6002 secondary antibodies at 37  C for 30 min. The tissue
sections were washed again thrice with 1 M PBS (pH 7.2–7.6) for
5 min in each wash and stained with 3,30 -diaminobenzidine
chromogenic kit and hematoxylin. The stained sections were
washed with distilled water, stored in resin, and observed using a
Leica image analyzing system.
2.6.4. SEM of collagen deposition
The hypodermis of the prepared skin tissues was removed;
these tissues were fixed in 4% buffered paraformaldehyde for 24 h
at 4  C and then thoroughly washed with PBS (pH 7.2–7.6) to
remove excess fixatives. The tissues were sequentially dehydrated
in 75, 85, 95, and 100% acetone and dried in a critical point dryer.
The specimen was subsequently coated with a thin layer of gold
film. Afterward, collagen deposition was imaged and observed
using SEM (Hitachi 3000, Japan) (Kwan et al., 2011).
2.7. Statistical analysis
Data were expressed as mean
standard error (SD). Signifi-
cance was calculated using Student’s t-test. p < 0.05 was consid-
ered statistically significant.
3. Results
3.1. SEM and appearance of the prepared nanofibrous dressing
Electrospun nanofibrous dressing was fabricated by electro-
spinning under optimized controlled conditions to obtain porous,
nanoscale structures. The SEM images presented in Fig. 1 show the
morphological characteristics of random SF/GT (75/25), SF/GT (25/
75), AS-loaded SF/GT (25/75), SF, and GT nanofibrous dressings.
The average diameters of random fibers were 300 (
34), 403
(
87), 480 (
79), 521 (
97), and 476 (
89) nm for SF, SF/GT (75/

[(Fig._2)TD$IG]

Fig. 2. (A) FTIR spectra of nanofibrous dressings. (B) Swelling degrees in water of various nanofibrous dressings. (C) Stress-strain curves of nanofibrous dressings. (D) Porosity
factors of nanofibrous dressings. (E) Degradation rates of SF, SF/GT (75/25), and SF/GT (25/75) nanofibrous dressing after exposure in 0.01 U/ml of proteinase K solution at
37  C. (F) Cumulative AS release profiles of AS-loaded SF/GT nanofibrous dressing.
296 Y.-H. Shan et al. / International Journal of Pharmaceutics 479 (2015) 291–301

25), SF/GT (25/75), and AS-loaded SF/GT (25/75), respectively. No
significant differences between the diameters of SF/GT (25/75) and
ASG-loaded SF/GT (25/75) nanofibrous dressing were observed.
3.2. Characterization of the SF/GT nanofibrous dressing
In Fig. 2(A), the ATR-FTIR spectra indicated that AS was
successfully loaded in the nanofibers. The structural integrity of
nanofibrous dressing should be studied depending on application,
in which the surrounding environment of the nanofibrous dressing
once placed in an in vivo system is considered. In general, wounds
of a burn patient contain organized exudate. Fig. 2(B) indicates that
the swelling rate of the nanofibrous dressing increased as gelatin
content increased. The swelling rate of AS-loaded SF/GT nano-
fibrous dressing was thrice higher than that of silk alone. Fig. 2(C)
illustrates the stress-strain curves of nanofibrous dressings. As
gelatin content increased, the break strength of nanofibrous
dressing decreased compared with that of pure silk nanofibrous
dressing (Acharya et al., 2009); the maximum and minimum break
strengths were 2.28 and 1.49 MPa, respectively. By contrast,
fracture strain increased. The fracture strain of gelatin was 112%;
this result demonstrated that gelatin with a preferable flexibility
should be used. This result could be accounted for the viscosity of
the spinning solution that increased as gelatin increased; the
diameter of the obtained nanofiber was also increased, thereby
decreasing the entanglement point of the nanofibrous dressing.
The entanglement acting force was weaker during stretching; as a
result, break strength decreased. With an external force, gelatin
exhibited excellent viscoelasticity and delivery energy; gelatin also
functions as a flexibilizer. Thus, elongation was increased at the
break of nanofibers that could be suitable for a wound dressing.
Porosity is also an important influencing factor of cell-dressing
interactions, thereby affecting cell attachment and proliferation. In
general, pores should be sufficiently wide to allow gas transfer and
maintain cell homeostasis. Fig. 2(D) shows that the porosity factors
of SF, SF/GT (75/25), SF/GT (25/75), GT and AS-loaded SF/GT (25/75)
were 88.6, 88.1, 87.9, 87.3, and 89.1%, respectively, which are
suitable for a wound dressing (Yin et al., 2009; Freyman et al.,
2001; Li et al., 2003). Fig. 2(E) illustrates the degradation rate of the
SF/GT nanofibrous dressing at various weight blending ratios after
this dressing was degraded in a collagenase solution at 37  C for
48 h. After 2 h of incubation, the weight of the SF nanofibrous
dressing was decreased by 10%; by contrast, the blending ratios of
the two other nanofibrous dressings, particularly 75/25 and 25/75,
were decreased rapidly by 30 and 50%, respectively. The results
revealed that the SF/GT nanofibrous dressing with higher gelatin
content was degraded faster than the dressings with lower gelatin
content. After 48 h of incubation, the weights of SF, SF/GT(75/25)
and SF/GT(25/75) nanofibrous dressing decreased to 79.2, 44.2, and
18.1% of their initial weights. Therefore, the weight loss of
nanofibrous dressing, after this dressing was immersed in
collagenase for 48 h, was approximately the amount of gelatin
contained in nanofibrous dressing. Fig. 2(F) shows that the
AS-loaded SF/GT nanofibrous dressing (25/75) released AS in a
complicated manner. At 12 and 48 h, the accumulative release
percentages of the nanofibrous dressing were approximately 82
and 96%, respectively. At 2 h, the accumulative release percentage
increased by 58%, that is, more than half of the release content was

[(Fig._3)TD$IG]

Fig. 3. Cell attachment and proliferation on nanofibrous dressing. (A) Cell attachment when cultured on each fiber composite at 1, 3, and 5 h. (B) Cell proliferation when
cultured on each nanofibrous dressing composite at 1, 4, and 7 days. MTT assay was performed to determine the metabolic activities of the cells attached to each surface, each
value was normalized to that of cultured cells without AAV infection. Statistically significant differences at *p < 0.05, **p < 0.01, and ***p < 0.001. (C) SEM images of
keratinocyte cells that adhered on each nanofibrous dressing at 7 days. (I) SF; (II) SF/GT 75/25; (III) SF/GT 25/75; and (IV) AS-loaded SF/GT 25/75.
 Y.-H. Shan et al. / International Journal of Pharmaceutics 479 (2015) 291–301 297

obtained. This result indicated that AS was rapidly released from
the AS-loaded SF/GT nanofibrous dressing (25/75) in 12 h;
furthermore, AS exhibited a sustained release from 12 to 24 h.
3.3. Biocompatibility evaluation (cell attachment and proliferation)
Keratinocytes were seeded onto the meshes and MTT assays
were performed to observe cell attachment and growth on
nanofibrous dressings (Fig. 3(A) and (B)). In this study, the
obtained nanofibrous dressings were cyto-compatible, and cell
growth was observed at a higher extent than that of the control
sample without the nanofibrous dressing. Cell proliferation was
significantly increased in the samples at 4 and 7 days on all of the
nanofibrous dressing types compared with the blank control
sample. No significant difference between SF/GT (25/75) and
AS-loaded SF/GT (25/75) nanofibrous dressings was observed in all

[(Fig._4)TD$IG]

Fig. 4. (A) Photo of the constant temperature and pressure instrument (YLS-5Q). (B-a) Images of skin wounds obtained post-wounding; (B-b) Masson’s trichrome staining at
24 h post-wounding; (B-c) HE staining at 24 h post-wounding; (I) normal skin; (II) superficial partial-thickness burn wound; (III) deep partial-thickness burn wound; and (IV)
third-degree burn wound (200 mm). (C) Wound closure percentage post-wounding in vivo (*p < 0.05, versus blank control; #p < 0.05, versus AS solution; &p < 0.05, versus
blank nanofibrous dressing). (D) Graphical process of the experiment. (E) Images of skin wounds at 31 days post-wounding; (I) blank control group; (II) blank nanofibrous
dressing group; (III) AS solution group; and (IV) AS-loaded SF/GT nanofibrous dressing group.
298 Y.-H. Shan et al. / International Journal of Pharmaceutics 479 (2015) 291–301
of the time points. This result suggested that AS did not induce any
observable toxicity or inhibit cell proliferation. In Fig. 3(C), the
morphological characteristic of the nanofibrous dressing was very
similar to that of cellular pseudopodia and junctions between cells.
Cells proliferated significantly, as culturing was continued. The
cells that tethered on the surface of the nanofibrous dressing with
polygonal and flattening extensions became confluent gradually
with typical keratinocyte morphology.
3.4. In vivo investigation
3.4.1. Established rat model and wound closure rate
Fig. 4(B) shows the sections of skin at 24 h post-wounding. In
the superficial partial-thickness burn wound group, the epidermis
was necrotic. Collagenous fibers in the superficial partial dermis
were swollen slightly and blood vessels were dilated in HE-stained
samples. Collagenous fibers in the superficial partial dermis also
appeared red in the Masson’s trichrome staining. In the deep
partial-thickness burn wound group, the epithelial cells in the
follicular sheath were disrupted, the deep partial dermis was
swollen, and blood vessels were intensely dilated. Numerous
lymphocytes and monocytes were infiltrative, and collagenous
fibers in the deep partial dermis appeared red in the Masson’s
trichrome staining. Skin appendages, such as hair follicles,
sebaceous glands, and sweat glands, were also disrupted in
third-degree burn wounds.
The wound healing of the burned skin was determined from the
percentage of the wound surface covered by regenerating
epidermis (Chen et al., 2012). In our previous study, 0.5% AS
solution locally delivered daily on wounded sites can accelerate
wound healing and inhibit scar formation. In the present study,
blank nanofibers, AS solution, and AS-loaded SF/GT nanofibrous
dressing were administrated every 2 days (Fig. 4(C)). Fig. 4(D)
shows that the AS-loaded SF/GT nanofibrous dressing significantly
contributed to wound healing compared with the blank control
group, the AS solution group, and the blank nanofibrous dressing
group at 7 and 15 days post-wounding (p < 0.05). Fig. 4(E) shows
that the appearance of the recovered burn wound of the AS-loaded
SF/GT
Nanofibrous dressing was slightly better than the blank control
group. Although these four groups did not exhibit remarkable
differences, the microstructure was subsequently observed.
3.4.2. Masson’s trichrome, HE, Sirius, VEGF, and
CD31 immunohistological staining and SEM of collagen deposition
Fig. 5 shows that AS solution and AS-loaded SF/GT nanofibrous
dressing contributed to angiogenetic effects and regular deposition
of newly formed collagen at 31 days post-wounding compared
with the blank control group and the blank nanofibrous dressing
group. Masson’s trichrome staining on the newly formed skin
tissues at 31 days post-wounding (Fig. 5(A)) revealed that the
wounds in the blank control group and the blank nanofibrous
dressing group exhibited wide areas of a dense dermis devoid of
skin appendages, showing the characteristics of scars. By
comparison, many blood vessels were found in the regenerated
skin of the wounds treated with AS solution and AS-loaded SF/GT
nanofibrous dressing. In these two groups, the collagen deposition
of the regenerated skin was similar to that of the normal skin. The
characteristics of scar in the newly formed skin in the blank control
group and the blank nanofibrous dressing group demonstrated the
angiogenetic effect of AS on wound repair; regenerated skin with
blood vessels in the AS solution group and the AS-loaded SF/GT
nanofibrous dressing group also exhibited the same effect of AS on
wound repair. These results further indicated the inhibitory
efficacy of AS on scar complication accompanied by the healing
process in adult tissues. Fig. 5(C) shows that the expressions of
VEGF increased in the AS-loaded SF/GT nanofibrous dressing group
and the AS solution group compared with those in the control
group and the blank nanofibrous dressing group. In Fig. 5(D),
CD31 immunohistological staining results showed no newly
formed blood vessels in the blank control group. In the blank
nanofibrous dressing group, small blood vessels (indicated by black
arrows) were observed. The AS solution and the ASG-loaded SF/GT
nanofibrous dressing could drastically contribute to angiogenetic
effect by enhancing the amount and dimension of blood vessels.
SEM imaging revealed the arrangement of collagen fibrils in the
normal skin. The collagen fibrils of the AS solution group, and the
AS-loaded SF/GT nanofibrous dressing group exhibited regular
characteristics. Both groups were organized and close to the
normal skin (Fig. 5(E)). The collagen fibrils were arranged in a
relatively random and loose aligned fibril matrix in the blank
control group and the blank nanofibrous dressing group. AS
solution and AS-loaded SF/GT nanofibrous dressing could adjust
collagen deposition in wound healing. This finding also explains
why the AS solution and the AS-loaded SF/GT nanofibrous dressing
promoted wound healing rather than scar formation.
In normal skin, the majority of collagen is type I. Fig. 5(F) reveals
that the collagen composition of the AS-loaded SF/GT nanofibrous
dressing group was similar to that of the normal skin, which
contained mainly type I collagen. However, only type III collagen
was observed in the blank control group and the blank nanofibrous
dressing group. This result indicated that the AS-loaded SF/GT
nanofibrous dressing significantly contributed to wound healing
compared with blank control, AS solution, and blank nanofibrous
dressing groups.
4. Discussion
An ideal burn wound dressing should exhibit the following
characteristics: (1) allowing gaseous exchange and preventing
bacterial penetration; (2) maintaining high humidity at the
dressing/wound interface; (3) absorbing toxic components and
exudates from wound surfaces; and (4) stimulating healing
cascade, among others (Meng et al., 2009).
The well porous structure of the prepared wound dressing
almost reached 90%; high specific surface areas of nanofibrous
dressings allowed gaseous and fluid exchange and provided a
barrier for bacteria and blood components in an advantageous
humid microenvironment (Zhong et al., 2012). A flexible nanofiber,
suitable as wound dressing could be obtained by mixing SF and
gelatin. After burn injury occurred, blood components and
subcutaneous fluid overflowed. As such, the ability of rapid
swelling in wound dressing was necessary to absorb excess
exudates and wound odor. Gelatin could be added to promote the
swelling rate of a nanofiber remarkably. These properties indicated
that silk and gelatin could be suitably used as wound dressing
materials when closely attached to the wound surface in a
particular characterization field (Hromadka et al., 2008).
Gelatin was directly hydrolyzed by collagenase. By contrast, SF
may resist collagenase hydrolysis; this result could be attributed to
the b-sheet conformation and hydrophobicity of SF. This result is
also similar to that of Jeeratawatchai. Fig. 2(E) shows that silk
degradation is very slow. This process could be accelerated by
increasing the percentage of gelatin component. Considering that
the dosing frequency in this experiment was 2 days, we found that
the optimal condition necessary to promote the degradation of
nanofibrous dressing was 48 h; in 48 h, patients’ pain induced by
changing the dressing could be decreased. In addition, the quick
release of AS in the early stage is beneficial for wound healing, as
revealed in our pre-experiment; combining this quick release with
swelling rate and strain stress, we chose the SF/GT (25/75) as the
optimal ratio of nanofibrous dressing used for wound healing. The
 Y.-H. Shan et al. / International Journal of Pharmaceutics 479 (2015) 291–301 299
[(Fig._5)TD$IG]

Fig. 5. (A) Masson’s trichrome staining at 31 days post-wounding (black arrows indicate newly formed blood vessels). (B) HE staining at 31 days post-wounding. (C) VEGF
immunohistological staining at 31 days post-wounding (black arrows indicate positive VEGF factor). (D) CD31 immunohistological staining at 31 days post-wounding (black
arrows indicate newly formed blood vessels). (E) Collagen deposition of regenerated skin observed through SEM at 31 days post-wounding. (F) Sirius red staining at 31 days
post-wounding (pink arrows indicate type I collagen and green arrows indicate type III collagen (magnification = 40)). (I) Blank control group; (II) blank nanofibrous dressing
group; (III) AS solution group; (IV) AS-loaded SF/GT nanofibrous dressing group; and (V) normal skin (200 mm). (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of this article.)

cumulative AS release from the SF/GT (25/75) nanofibrous dressing
is shown in Fig. 2(F). The main influencing factor of drug release is
the degradation rate due to the property of nanofibrous dressing
which was prepared by homogeneous solution of a certain amount
of SF/gelatin and AS, and with the influencing about porosity,
swelling rate and mechanical properties. At 12 h, the degradation
rate and the drug release content were approximately 80%; this
result indicated that the drug was rapidly dissolved and released
from the degraded gelatin component. The drug was diffused from
SF at 12–48 h because of the final cumulative AS release of 95%;
however, the degraded nanofibrous dressing was <80%. These
results suggested that 80% of the drug was rapidly released from
the formulation, particularly from gelatin in the early stage. The
remaining 15% in the silk component exhibited a sustained release
from 12 to 48 h; this result is advantageous for the rapid
acceleration of wound closure.
Materials used as drug delivery vehicles and tissue engineering
scaffolds should be biocompatible and biodegradable. Moreover,
the degradation products of these materials should be non-toxic
and be easily eliminated from the system. For instance, the
degradation products of silk and gelatin are mainly amino acids
and other products, which are readily absorbed by the skin and
implicated in nutrition and tissue repair. The degradation products
of silk and gelatin are also acidic, which is beneficial for wound
healing.
In a deep burn wound, as presented in this study, the basement
membrane was breached and the skin appendages (hair follicles,
sebaceous glands, and sweat glands) were disrupted. Thus,
300 Y.-H. Shan et al. / International Journal of Pharmaceutics 479 (2015) 291–301
epithelial cells could only migrate from wound edges on the basis
of granulation tissues that fill the wound bed (Falanga, 2004).
Rapid migration corresponds to a low extent of scar formation. The
blended nanofibrous dressing as a substitute for collagen could
improve cell adhesion and proliferation. Furthermore, nanofibrous
dressing with a high surface-to-volume ratio could promote cell
adhesion and proliferation (Sethuraman et al., 2010; Wen and
Tresco, 2006). SEM confirmed the adhesion of cells on nanofibrous
dressing and demonstrated the desirable cell-substrate interaction
between cells and the SF/GT nanofibrous dressing. In conclusion,
the composition and structure of SF/GT nanofibrous dressing were
similar to those of the native ECM; this dressing could also provide
a favorable environment for cell attachment and proliferation.
Therefore, the SF/GT composite is a promising candidate for tissue
engineering scaffolds with fairly good biocompatibility.
Angiogenesis is imperative in wound healing to maintain
cellular activity for tissue repair (Stojadinovic et al., 2008). The
precursor cells present in the blood should migrate to sites of
injury via a high density of blood vessels to adjust skin
regeneration (Peng et al., 2011, 2012b). In Fig. 5(C), the expression
of VEGF increased in the AS-loaded SF/GT nanofibrous dressing
group, compared with the control group. Therefore, AS likely
increased the expression of VEGF. We also investigated the extent
of new blood vessel formation by analyzing the expression of
CD31-positive endothelial cells (Chen et al., 2013). Our result
showed that neovascularization was significantly different be-
tween the AS-loaded SF/GT nanofibrous dressing group and the
control groups. We did not evaluate whether VEGF is produced by
cells or is derived from AS; nevertheless, we found that AS
influenced wound healing by increasing VEGF levels. We further
speculated that these differences were partially due to the
different conditions of hypoxic environment and acid degradation
products of nanofibrous dressing (Kondo and Ishida, 2010). In
general, cell growth and proliferation result in high metabolic rates
and low oxygen contents in regenerated tissues, likely promoting
vasculogenesis (Kondo and Ishida, 2010). Hence, the number of
microvessels in the histological sections of the AS-loaded SF/GT
nanofibrous dressing group increased; these microvessels were
also closely similar to those of the normal skin with a sustained
supply of AS, a hypoxic environment, and acid degradation
products.
Hypertrophic scarring usually occurs after deep injury of the
skin and is mainly caused by excessive collagen production by
fibroblasts (Xi-Qiao et al., 2009). Type III collagen was the first to
emerge among the collagen types and functions as a bridge in
wounds during wound healing. As tissues are regenerated to build
a solid bracket and facilitate wound healing, type I collagen
appears in conjunction with type III (Friedman et al., 1993). To
evaluate wound healing effect, we performed Sirius staining,
which is an effective method. In Fig. 5(F), the collagen composition
of the AS-loaded SF/GT nanofibrous dressing group was the most
similar to that of the normal skin. Moreover, the collagen
deposition of the AS solution group and the AS-loaded SF/GT
nanofibrous dressing group exhibited organized and regular
characteristics, as shown in SEM results. In the blank control
group and the blank nanofibrous dressing group, the collagen fibril
was arranged in a relatively loose and randomly aligned fibril
matrix.
In summary, the optimal ratio of SF/GT electrospun nanofibrous
dressing functionalized with AS was chosen and obtained
successfully with excellent biological properties; these properties
showed a remarkable effect when the functionalized AS was
applied on partial-thickness burn wound. Compared with the AS
solution, the AS-loaded SF/GT nanofibrous dressing was more
beneficial for wound healing because this dressing involves a
combination of diffusion and dissolution mechanisms. This
combination of mechanisms allowed a quick drug release in early
stage, prolonged drug residency on/in the skin, exhibited good
compatibility, and provided an efficient barrier for bacteria and
blood constituents with an optimal humid microenvironment.
5. Conclusions
AS, a poorly soluble drug, was successfully carried by the SF/GT
nanofibrous dressing via electrospinning. The AS-loaded SF/GT
nanofibrous dressing evidently enhanced cell adhesion and
proliferation with good biocompatibility in vitro. This dressing
also promoted rapid healing, induced pro-angiogenesis of partial-
thickness burn wound, and inhibited scar complications in
wounds.
Acknowledgements
The study was supported by National Natural Science Founda-
tion of China (81102393,11272289) and the Zhejiang Provincial
Program for the Cultivation of High-Level Innovative Health
Talents.
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