Research Article

Cite This: ACS Appl. Mater. Interfaces 2018, 10, 16977−16991 www.acsami.org

Silk Sponges Ornamented with a Placenta-Derived Extracellular

Matrix Augment Full-Thickness Cutaneous Wound Healing by
Stimulating Neovascularization and Cellular Migration
Arun Prabhu Rameshbabu,† Kamakshi Bankoti,† Sayanti Datta,† Elavarasan Subramani,‡
Anupam Apoorva,§ Paulomi Ghosh,† Priti Prasanna Maity,† Padmavati Manchikanti,∥ Koel Chaudhury,‡
and Santanu Dhara*,†
†
Biomaterials and Tissue Engineering Laboratory, School of Medical Science and Technology, ‡School of Medical Science and
Technology, §School of Bio Science, and ∥School of Energy Science & Engineering, Indian Institute of Technology Kharagpur,
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Kharagpur 721302, India

*
S Supporting Information

ABSTRACT: Regeneration of full-thickness wounds without scar formation is a multifaceted

process, which depends on in situ dynamic interactions between the tissue-engineered skin
substitutes and a newly formed reparative tissue. However, the majority of the tissue-
engineered skin substitutes used so far in full-thickness wound healing cannot mimic the natural
extracellular matrix (ECM) complexity and thus are incapable of providing a suitable niche for
endogenous tissue repair. Herein, we demonstrated a simple approach to fabricate porous
hybrid ECM sponges (HEMS) using a placental ECM and silk fibroin for full-thickness wound
healing. HEMS with retained cytokines/growth factors provided a noncytotoxic environment in
vitro for human foreskin fibroblasts (HFFs), human epidermal keratinocytes (HEKs), and
human amniotic membrane-derived stem cells to adhere, infiltrate, and proliferate.
Interestingly, HEMS-conditioned media accelerated the migration of HFFs and HEKs owing
to the presence of cytokines/growth factors. Also, the ex vivo chick chorioallantoic membrane
assay of HEMS demonstrated its excellent vascularization potential by inducing and supporting
blood vessels. Additionally, HEMS when subcutaneously implanted demonstrated no severe
immune response to the host. Furthermore, HEMS implanted in full-thickness wounds in a rat model showed augmented healing
progression with well-organized epidermal−dermal junctions via pronounced angiogenesis, accelerated migration of HFFs/
HEKs, enhanced granulation tissue formation, and early re-epithelialization. Taken together, these findings show that porous
HEMS ornamented with cytokines/growth factors having superior physicomechanical properties may be an appropriate skin
substitute for full-thickness cutaneous wounds.
KEYWORDS: extracellular matrix, silk fibroin, cytokine/growth factor, neovascularization, cellular migration,
full-thickness wound healing

1. INTRODUCTION thickness skin injuries is the autologous skin grafting.5

Skin is the largest organ of the human body that serves the
onerous function of providing a physical shield against
microorganisms, thermal regulation for average hydration
retention, sensory information about the external environment,
and other vital functions.1 Skin injuries can be caused by
genetic disorders, acute trauma, chronic wounds, or by complex
surgeries.2 In complex full-thickness wounds, epidermal layer
along with dermal layer, sweat glands, hair follicles as well as the
underlying subcutaneous fat tissue is damaged. Specifically,
wounds of critical size hamper the crucial functions of the skin
and can lead to complications such as microbial infection, a
water−electrolyte imbalance in the body, and severe cases that
can be life-threatening.3 Therefore, skin injuries of critical size
(greater than 1 cm in diameter) require bioactive support with
clinical intervention to accelerate healing.4 Also, scarless wound
healing is desirable for regaining tissue functionality as well as
improved aesthetics. The gold standard treatment for full-
However, the limitations such as donor site morbidity and
limited donor site availability have resulted in the need for the
development of skin substitutes. In this context, bioengineered
skin substitutes (hydrogels,6 sponges,7 and electrospun mats8)
from natural polymers and proteins can solve the problem of
autologous donor graft shortage, provide protection from fluid
loss and contamination, as well as can deliver bioactive factors
such as cytokines, dermal matrix components, and growth
factors to the wound bed for enhancing the host wound healing
responses.4,9
Silk fibroin (SF) from Bombyx mori, a versatile natural fibrous
protein, has been extensively explored as a skin substitute
material in tissue engineering applications owing to its
Received: December 14, 2017
Accepted: May 2, 2018
Published: May 2, 2018

© 2018 American Chemical Society 16977 DOI: 10.1021/acsami.7b19007

ACS Appl. Mater. Interfaces 2018, 10, 16977−16991
ACS Applied Materials & Interfaces
biocompatibility, slow degradability, low immunogenicity,
remarkable mechanical properties, and hemostatic action.10−12
However, SF alone is inadequate for dermal tissue regeneration
because it lacks adequate cell-specific binding sites and limited
growth factor adsorbing capacity.13 Thus, to enhance the
properties of SF, it has been blended with various natural
macromolecules such as gelatin,14 hyaluronic acid,15 collagen,16
chitosan,17 and so forth. For instance, a scaffold fabricated from
SF/elastin facilitated accelerated re-epithelialization in burn
wounds.18 In another study, the blend scaffold of SF/keratin
significantly enhanced the cell adhesion/proliferation of L929
fibroblasts and increased the extracellular matrix (ECM)
deposition of collagen type I.19 Though the scaffolds
mentioned above displayed superior cellular activities and
ECM deposition, yet they lack a plethora of structural proteins,
growth factors, and cytokines required for skin regeneration.
In recent years, decellularized ECM has emerged as an
attractive biomaterial in regenerative medicine, which provides
abundant biological cues for cell migration and proliferation
and further directs/promotes differentiation.20 The human
placenta is a unique, complex organ that serves multiple
functions to the developing fetus; also, it is a rich reservoir for a
variety of growth factors and cytokines such as epidermal
growth factor (EGF), transforming growth factor-β (TGF-β),
fibroblast growth factor (FGF), platelet-derived growth factor
(PDGF), and vascular endothelial growth factor (VEGF).21
These growth factors/cytokines play an essential role in
migration/proliferation of fibroblasts/keratinocytes, homing of
mesenchymal stem cells, re-epithelialization, and neovasculari-
zation that are considered essential for tissue repair and
regeneration in the early stage of wound healing. Direct
administration of the human placental extract in the wound
margin of full-thickness wounds was found to accelerate the
wound healing mechanism associated with an increase in TGF-
β levels during the initial phase, resulting in increased
inflammatory cell infiltration, and increased VEGF levels during
the later stages, leading to increased new blood vessel
formation.22 Furthermore, the properties of the placenta, such
as low immunogenicity, anti-inflammatory, and anti-scarring
make it an ideal choice to treat full-thickness skin wounds.23
In the present work, a hybrid ECM sponge (HEMS) was
fabricated for skin tissue engineering by incorporating a
placenta-derived ECM (pECM) with SF, thus bringing together
the inherent advantages of both SF and pECM. Collagen (the
major ECM component of native skin) is considered to be the
promising material of choice for wound healing applications
owing to its biodegradability, superior biocompatibility, low
antigenicity, nontoxicity upon exogenous application, and high
tensile strength. Therefore, collagen was selected as a
representative control to demonstrate the superiority of
pECM containing intrinsic cytokines/growth factors for
wound healing. Hence, a collagen-incorporated SF matrix
sponge (CIMS) was also fabricated for comparative studies.
The fabricated HEMS and CIMS were characterized for their
physicomechanical properties and evaluated for their bio-
compatibility using the primary fibroblasts, keratinocytes, and
human amniotic membrane-derived stem cells (HAMSCs). In
vitro wound healing/migration potential was evaluated by the
scratch assay, and the ability to induce vascularization was
assessed using the chick chorioallantoic membrane (CAM)
model. Also, HEMS and CIMS were implanted subcutaneously
in rats to study the host immune reaction/toxicity in vivo.
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Further, HEMS and CIMS were assessed for their potential in
accelerating full-thickness wound healing using a rat model.
2. MATERIALS AND METHODS
2.1. Decellularization of Human Placenta and Processing of
Soluble ECM (pECM). All the experimental procedures were
approved by the Institutional Ethical Committee of Indian Institute
of Technology, Kharagpur, India. Decellularization of human placenta
was performed according to the protocol reported in our previous
work.24 Briefly, the collected placentas were repeatedly washed with
Dulbecco’s phosphate-buffered saline (DPBS) until the blood was
removed and then minced into small fragments by using a sterile
scalpel. The minced placenta was decellularized using 0.5% sodium
dodecyl sulfate (SDS; Sigma-Aldrich, USA) with 0.2% DNase (2000
U; Sigma-Aldrich, USA), 200 mg/mL RNase (Sigma-Aldrich, USA),
0.05% trypsin/ethylenediaminetetraacetic acid (EDTA; Gibco, USA),
100 U/mL penicillin, 100 mg/mL streptomycin (Gibco, USA), and 1
mM phenyl methyl sulfonyl fluoride (Sigma-Aldrich, USA) in a sealed
rotating vessel. The procedure was conducted under strictly sterile
conditions, and the solution was changed every 24 h to prevent tissue
degradation and contamination. The decellularized placentas were
washed with PBS and stored at −80 °C for further processing.
Solubilization of ECM was performed as described elsewhere.25
Briefly, the decellularized human placenta was pulverized to a powder
using a tissue homogenizer in the presence of liquid nitrogen. The
resulting powder was immersed and stirred in a 4 M urea buffer (240 g
urea, 9 g NaCl, and 6 g Tris base in 1 L distilled water) containing a
protease inhibitor cocktail for 24 h. Subsequently, the samples were
subjected to ultrasonic homogenization (Branson Ultrasonics, USA) in
an ice bath. After centrifuging the samples at 14 000 rpm for 20 min at
4 °C, the supernatant was collected and then dialyzed using an 8000
molecular weight cutoff (MWCO) dialysis tubing against Tris-buffered
saline (TBS), that is, 9 g NaCl and 6 g Tris base in 1 L distilled water
to remove urea. The contents of the dialysis tubes were centrifuged
again to remove protein aggregates, and then the supernatant was
frozen at −80 °C, freeze-dried for 42 h, and pulverized to obtain
pECM.
2.2. Quantification of Collagen and Glucosaminoglycans
(GAGs). The total collagen content was assessed by the hydroxypro-
line assay, and the sulfated GAG (sGAG) content was assessed by the
alcian blue assay26 in native placenta (NP) and pECM, as detailed in
the Supporting Information.
2.3. Cytokine Array for Detection of Growth Factors. Growth
factors in NP and pECM were analyzed using a human cytokine
antibody array (RayBio C-Series C1000, USA) following the
manufacturer’s protocol. Briefly, the samples were dissolved at 4 °C
for 36 h in the buffer containing 0.1× protease inhibitor, 2 M urea, and
50 mM Tris-HCl. The supernatant was collected by centrifugation at 4
°C. The obtained supernatant was incubated onto an array chip
containing 120 different human cytokine antibodies after blocking.
The chip was then washed and incubated with biotinylated antibody
cocktail. Subsequently, the signals were detected using a chemilumi-
nescence detection system after horseradish peroxidase (HRP)−
streptavidin incubation and washing.
2.4. SDS-Polyacrylamide Gel Electrophoresis (PAGE) and
Western Blotting. The finely powdered NP and pECM were
obtained by homogenizing in a buffer containing 65 mM dithiothreitol,
4 M guanidine HCl, protease inhibitor cocktail, 10 mM EDTA, and 50
mM sodium acetate (all Sigma-Aldrich, USA). The resulting mixture
was exposed to ultrasonic homogenization, and the supernatant was
collected after centrifugation (13 000 rpm for 20 min at 4 °C).
Bicinchoninic acid assay kit (Thermo Scientific, Rockford, USA) was
used to quantify the total protein concentration of the homogenates
according to the manufacturer’s instruction. The extracted proteins
from NP and pECM were run in 10% PAGE and stained with
Coomassie blue after fixing.
The extracted proteins (40 μg) from NP and pECM after resolving
in SDS-PAGE were transferred onto the nitrocellulose membrane
(Millipore, USA) at 90 V for 2 h in the presence of a Tris-glycine
buffer. The nitrocellulose membranes containing the transferred
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proteins were treated with a primary antibody [hepatocyte growth
factor (HGF; Santa Cruz, USA), TGF-β1 (Abcam, USA), VEGFA
(Abcam, USA), EGF (Santa Cruz, USA), insulin-like growth factor-1
(IGF-1; Santa Cruz, USA), and PDGF-B (Santa Cruz, USA)] at 4 °C
overnight after blocking. The blots were subsequently incubated with
the HRP-conjugated secondary antibody for 1 h. Pierce ECL western
blotting substrate kit (Thermo Scientific, USA) was used for
visualizing the immunoreactive proteins according to the manufac-
turer’s instructions. Further, an automatic X-ray film processor was
used to develop the images, which were analyzed by ImageJ software
(Rasband WS; NIH).
2.5. Preparation of SF Solution. SF solution was prepared
according to the procedure described elsewhere.27 Briefly, B. mori
cocoons were chopped into small pieces and boiled (98 °C) for 30
min in an alkaline bath containing 0.02 M sodium carbonate (Sigma-
Aldrich, USA) to obtain fibroin and to remove the sericin/
glycoproteins. The degummed silk was rinsed thoroughly with
double-distilled water and allowed to dry at room temperature
overnight. The extracted fibroin was dissolved in 9.3 M LiBr (Sigma-
Aldrich, USA) solution at 60 °C for 4 h followed by dialysis for 72 h
against double-distilled water using a dialysis membrane (3500
MWCO) to remove LiBr. The obtained fibroin solution was
centrifuged (9000 rpm at 4 °C for 20 min) to remove large
aggregates, and the supernatant was concentrated to a final
concentration of 10% (w/v) by dialysis against poly(ethylene glycol)
(10 000 MW, Sigma-Aldrich, USA). The concentrated fibroin solution
was passed through a 5 μm syringe filter (Millipore) to eliminate
minor impurities.
2.6. Fabrication of CIMS and HEMS. Collagen type I was
extracted from fish scales according to the procedure described
elsewhere28 and was grounded to powder in the presence of liquid
nitrogen. Fibroin solution and pECM were obtained as described
above. For fabrication of CIMS/HEMS, 0.25 g of pulverized collagen
type I/pECM, respectively, was dispersed in 5 mL of 10% (w/v) SF
solution under constant stirring for 24 h at 4 °C. The homogeneously
mixed viscous solution was subjected to sonication in an ice bath for 2
min, gently poured into sterile molds, frozen at −80 °C, and
subsequently lyophilized for 72 h to obtain CIMS/HEMS. The
lyophilized CIMS/HEMS was soaked in 70% (v/v) ethanol for 6 h to
induce formation of β-sheets in the SF protein. Homogeneous
distribution of pECM or collagen in the SF matrix was assessed by
Masson’s trichrome (MT) staining as described in the Supporting
Information.
2.7. Morphological Analysis by Scanning Electron Micros-
copy. The microstructure of CIMS/HEMS was examined under a
scanning electron microscope (EVO ZEISS, Carl Zeiss SMT AG,
Oberkochen, Germany) at an accelerating voltage of 10−20 kV after
fixing the samples with 4% paraformaldehyde. Briefly, the dried
samples were placed on a sample holder using double-sided adhesive
tapes and gold-coated for 90 s using a plasma coater under high
vacuum to avoid the charging effect.
2.8. Porosity Measurement by Micro-Computed Tomog-
raphy (Micro-CT). CIMS/HEMS was scanned (1000 scan slices/
sample) using a micro-CT (GE Phoenix v|tom|ex s, Germany) at a
voltage of 155 kV and a current of 45 mA, with a voxel of 7.2 μm.
When analyzing the porosity of the samples, the threshold to be used
was obtained for each specimen by the threshold histogram offered by
the VG Studio Max software (Volume Graphics Germany).
2.9. Swelling Behavior and Degradation Kinetics. The
swelling behavior of CIMS and HEMS was examined for 64 h at 37
°C in sterile PBS (pH 7.4). At predetermined time intervals, the excess
PBS in samples was removed by gently wiping with a filter paper and
then instantly weighed in an electronic balance (Mettler-Toledo
International Inc., USA) to calculate the PBS content in swollen
CIMS/HEMS. The swelling ratio was calculated using the formula
below
Ts − Td
Swelling ratio (%) = × 100
Td
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where Td and Ts are the dry and wet weight of CIMS/HEMS,
respectively. The experiments were conducted in triplicate and
averaged.
In vitro degradation of CIMS/HEMS (n = 5) was performed in PBS
containing 1 U/mL of lysozyme (hen egg-white, Sigma-Aldrich), pH
7.4 at 37 °C for a specified period according to the protocol described
in our previous publication.29
2.10. Fourier Transform Infrared (FTIR) Spectroscopy. The
infrared spectra of CIMS and HEMS were obtained using a Thermo
Nicolet spectrophotometer (model NEXUS-870; Thermo Nicolet
Corporation, Madison, WI) in attenuated total reflection mode. The
absorbance of spectra was recorded in the range of 4000−500 cm−1.
2.11. Mechanical Testing. The mechanical properties of the
samples (n = 10) were determined by tensile testing using the
universal testing machine (25 K, Hounsfield, UK) at room
temperature with a 20 N load cell. The samples were cut into a
rectangular strip (∼5 mm width, ∼20 mm length, ∼1 mm thickness)
and soaked in PBS for 48 h. Uniaxial tensile testing was performed
under tension at a crosshead speed of 2 mm/min.
2.12. Isolation of HAMSCs/HFFs/HEKs. HAMSCs were isolated
and characterized according to the protocol reported in our previous
publication.24 Briefly, the human placentas collected under stringent
sterile conditions were transported to the laboratory at 4 °C. The
blood components were removed by rinsing with Hank’s balanced salt
solution (Gibco, USA) containing 200 mg/mL streptomycin and 200
U/mL penicillin. The amniotic membrane was detached from chorion
by a blunt dissection and was incubated with 0.05% trypsin−EDTA
solution (Gibco, USA) for two cycles of 30 min each, followed by
discarding the supernatant. Subsequently, the tissue was digested with
Earle’s balanced salt solution (Gibco, USA) containing 2 mg/mL
collagenase Type IV (Gibco, USA) and 10 U/mL DNase I (Sigma-
Aldrich, USA) for 60 min. After the end of the digestion, the
suspension was centrifuged at 1500 rpm to collect the cell pellet. The
cell pellet was seeded in flasks (Nunc, USA) after suspending it in the
Dulbecco’s modified Eagle’s medium (DMEM; containing low
glucose, 10% fetal bovine serum (FBS), 1% antibiotic−antimycotic)
and transferred to an incubator. TrypLE Express Enzyme (Gibco,
USA) was used to subpassage the cells, and cells before passage
number five were used for experiments.
Human foreskin fibroblasts (HFFs) and human epidermal
keratinocytes (HEKs) were isolated as described elsewhere from
circumcised human foreskin.30 Briefly, skin samples were collected in
sterile containers and rinsed in PBS containing 200 mg/mL
streptomycin and 200 U/mL penicillin. Later, the samples were
chopped into small pieces and incubated in Dispase II (Sigma-Aldrich,
USA) for overnight at 4 °C. Subsequently, epidermal and dermal
layers were separated and incubated in TrypLE Express Enzyme/
collagenase I (Gibco, USA), respectively, for further digestion. The
corresponding cell suspension was strained, and the cell pellet was
obtained by centrifugation. Defined keratinocyte-serum-free media
(SFM) (Gibco, USA)/DMEM high glucose media (Gibco, USA) were
used for culturing HEK/HFF, respectively. Cells after reaching 80%
confluency were passaged with TrypLE Express, and cells before
passage five were used for further experiments.
2.13. Indirect Cytotoxicity Testing. 2.13.1. Preparation of
HEMS-/CIMS-Conditioned Medium. HEMS/CIMS in the presence of
liquid nitrogen was grounded to a fine powder with a sterile porcelain
mortar and pestle. The resulting HEMS/CIMS powder (25 mg/mL)
was added to a complete DMEM/defined keratinocyte-SFM medium
with 1% antibiotic−antimycotic in a sterile container and stirred inside
an incubator at 37 °C and 5% CO2 for 24 h. The conditioning material
was removed from the media by filtering the medium through a 0.22
μm syringe filter (Millipore, USA).
2.13.2. Morphological and Apoptosis Assessment. Primary HFFs
and HEKs were used for investigating the effect of the CIMS-/HEMS-
conditioning medium on their morphological and functional attributes.
Briefly, HFFs/HEKs were cultured either with CIMS-/HEMS-
conditioned or normal medium on lysine-coated coverslips placed in
a 12-well plate. The coverslips were removed after 96 h, stained with
rhodamine phalloidin (Invitrogen, USA) and DAPI (Invitrogen, USA)
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according to the manufacturer’s instructions. Apoptosis of HEKs in the
presence of CIMS-/HEMS-conditioned media was also investigated
after 7 days of cultivation using the DeadEnd Fluorometric TUNEL
system (Promega, USA) according to the manufacturer’s guidelines.
2.13.3. Scratch Assay. The scratch assay for wound healing was
performed according to the protocol described elsewhere.31 HFFs/
HEKs were cultured in 35 mm Petri dishes (Tarsons, India) in
DMEM/defined keratinocyte-SFM medium at 37 °C in a 5% CO2 in
an air atmosphere to produce confluent monolayers. To mimic
wounds, a vertical scratch was made in the center of the monolayer
HFFs/HEKs using a sterile 200 μL pipette tip, and subsequently, the
Petri dishes were washed with sterile PBS to remove the detached
HFFs/HEKs. Later, the Petri dishes were filled with either CIMS-/
HEMS-conditioned medium or control medium, and the Petri dishes
were incubated at 37 °C in a 5% CO2 for 72 h. The migration of
HFFs/HEKs in the denuded path was monitored by taking optical
images using an inverted phase contrast microscope (Carl Zeiss
AxioObserver) at the different period, and three independent
observers counted the number of cells that had migrated into the
initially cell-free scratch area.
2.14. Analysis of HEMS-Conditioned Medium by ELISA. The
quantitative direct enzyme immunoassay was performed to estimate
the levels of interleukin-6 (IL-6), interleukin-8 (IL-8), monocyte
chemotactic protein-1 (MCP-1), TGF-β1, and RANTES in HEMS-
conditioned medium. The protein concentration of 20 μg/mL was
used for antigen binding in poly-L-lysine-coated 96-well plates.
Following blocking with 3% bovine serum albumin (BSA), the wells
were incubated with antibodies against IL-6, IL-8, MCP-1, TGF-β1,
and RANTES (Santa Cruz Biotechnology, Inc., USA). All wells were
washed with (Phosphate Buffered Saline with Tween 20) (PBST) and
incubated with a suitable secondary antibody conjugated with HRP
(Santa Cruz Biotechnology, Inc. CA, USA). After washing with PBST,
100 μL of Femto-ELISA-HRP substrate (G-Biosciences, USA) was
added to each well and incubated for 10−15 min. The reaction was
stopped with 1 N HCl, and the absorbance was measured at 450 nm.
2.15. Direct Cytotoxicity Testing. The cytotoxicity was tested by
the direct cultivation of HAMSCs/HEKs/HFFs on TCP/CIMS/
HEMS. Briefly, 5 × 104 HAMSCs/HFFs/HEKs were seeded on
samples (n = 5) and cultivated in a 24-well tissue culture plate for 72 h.
The number of viable cells was calculated using Vybrants MTT Cell
Proliferation Assay Kit (Invitrogen, USA) according to the
manufacturer’s instructions.
The viability of HEKs seeded on CIMS and HEMS was assessed
using Live/Dead Assay Kit (Life Technologies, NY) according to the
manufacturer’s protocol. Briefly, cell-seeded CIMS/HEMS after 7 days
was incubated for 60 min at 37 °C in a solution containing 2 μM
calceinacetomethoxy (AM) and 4 μM ethidium homodimer. The
samples were repeatedly washed with DPBS to avoid background
staining and visualized under an inverted fluorescence microscope
(AxioVision, Zeiss, Germany).
2.16. CAM Assay. The CAM assay was used as an ex vivo model to
assess the potential of CIMS/HEMS to induce vascularization. In this
study, fertilized white Leghorn chicken eggs were used for the CAM
assay by following the procedure described elsewhere.32 Briefly, the
eggs were incubated at 37 °C, 65% relative humidity egg incubator. On
day 5, disks (5 mm diameter; n = 5) of CIMS and HEMS were
punched, sterilized, and hydrated with sterile PBS overnight. The
sterile disk of CIMS and HEMS was placed over CAM, respectively;
later, the eggshell was sealed. On day 8, the window was carefully
dissected, and CIMS/HEMS was photographed in situ. Three blind
observers counted the number of blood vessels approaching toward
the scaffolds. Further, the CIMS/HEMS along with the surrounding
membrane was retrieved carefully with forceps without getting tore off,
fixed in 4% paraformaldehyde, dehydrated, and subsequently
embedded in paraffin blocks. The paraffin blocks were sectioned and
stained with hematoxylin and eosin (H&E; Sigma-Aldrich, USA), MT
(Sigma-Aldrich, USA), and DAPI.
2.17. In Vivo CIMS/HEMS Cellular Response and Organ
Toxicity Analysis. The in vivo cellular response of CIMS/HEMS was
studied by subcutaneous implantation in albino Wistar rats (250 ± 10
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g; males; n = 5). The experimental protocol followed was permitted by
the Institutional Ethical Committee of Indian Institute of Technology,
Kharagpur, and all surgery was performed under anesthesia. Extreme
caution was taken to minimize the suffering of the animals. Cylindrical
CIMS/HEMS (diameter = ∼1 cm; height = ∼0.5 cm) was implanted
on the dorsal side of rats after dissection and sutured using chromic
catgut under aseptic condition. After 28 days of implantation, the rats
were euthanized, and the site of implantation along with the adjacent
tissue was retrieved for further analysis. The harvested samples were
fixed, dehydrated, and subsequently embedded in paraffin blocks. The
paraffin blocks containing the sample were microtomed, and the
sections were then stained with H&E, MT, toluidine blue (TB; Sigma-
Aldrich, USA), and anti-CD31 antibody (Abcam, USA) staining
according to the manufacturer’s instructions. All the sections were
observed under a microscope to understand the cellular events. For
organ toxicity analyses, the major organs (heart, kidney, lung, and
liver) were harvested (n = 3 per group) after 28 days of the treatment
period of different groups and processed for the histological
assessment of the tissue.
2.18. Full-Thickness Cutaneous Wound Healing Study. The
in vivo experimental protocols were approved by the Institutional
Ethical Committee of Indian Institute of Technology, Kharagpur.
Albino Wistar rats (2 months old; 200 ± 10 g) were used to evaluate
the full-thickness skin wound healing capacity of the different groups.
Randomly, the rats were divided into three groups (n = 9 each group)
according to the treatments received on each rat. Three treatment
groups were as follows: (1) SHAM: wounds that were left untreated
and (2) CIMS and (3) HEMS were applied into the wound exclusively
within the wound area. Before surgery, 2 cm diameter wounds were
marked using a template, and standardized full-thickness skin wounds
were made on the dorsal side by excising to the level of the fascia, by
using sterile forceps and scissors under general anesthesia. Petrolatum
gauze pressure dressing was applied to all the groups, which prevented
the samples from moving away from the wound bed. Care was taken
to ensure that the pressure dressing was not detached during the 21
day treatment period. The rats were individually housed in
temperature-controlled cabins and fed with a standard protein diet.
The cutaneous wounds were photographed after 0, 7, 14, and 21 days,
and the unhealed area was measured using ImageJ (Rasband WS;
NIH) to assess the wound healing kinetics. The percentage of wound
reduction was calculated according to the following formula
A0 − At
Rate of wound closure = × 100
A0
where A0 and At are designated as the initial wound area and wound
area at the designated time, respectively.
The rats were sacrificed after 7, 14, and 21 days post-implantation,
and the wound site with the surrounding muscle and skin was
retrieved. They were then immediately fixed with 4% formaldehyde,
dehydrated, and embedded in a paraffin block. Sections of 3 μm
thickness were cut and stained with H&E and MT. Further, the
sections were also immune-stained with anti-CD31 and anti-
cytokeratin 10 (CK-10; Abcam, USA) according to the manufacturer’s
instruction. The photomicrograph of different stains was captured and
examined to document cardinal features such an inflammatory
response, necrosis, neovascularization, collagen deposition, granula-
tion, and re-epithelialization to understand healing progression at
different periods.
2.19. Reverse Transcriptase-PCR (RT-PCR) Analyses of the
Wound Area. RT-PCR was performed on a newly formed tissue in
the wound area from different treatment groups, 21 days post-surgery.
The regenerated tissues (n = 5 per group) from different study groups
were excised from the animal after sacrificing and ground to a powder
using liquid nitrogen. The total RNA was extracted using the TRIzol
reagent (Invitrogen, USA) according to the manufacturer’s instruc-
tions. An equal quantity of isolated RNA was transcribed into cDNA
using a cDNA synthesis kit (Thermo Scientific, USA) according to the
manufacturer’s protocol. PCR amplification of the gene-specific
primers (Supporting Information Table S1) was performed in a
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Figure 1. Profiling of cytokines/growth factors in NP and pECM. Y-error bars represent standard deviation.

Figure 2. (A) Total protein extracted from NP and pECM was separated by SDS-PAGE; (B) immunoblotting using different antibodies (anti-EGF,
anti-PDGF-B, anti-TGF-β1, anti-VEGFA, anti-IGF-1, and anti-HGF) and (C) their respective relative band intensities. Y-error bars represent
standard deviation, and single asterisks signify P < 0.05.

thermal cycler (Eppendorf Mastercycler, USA), and the PCR product
was imaged in UV gel doc (Bio-Rad, USA) after running it in 1%
agarose gels. The band intensity was analyzed using ImageJ (Rasband
WS; NIH).
2.20. Statistical Analysis. The data were analyzed using
GraphPad Prism software (version 5.02, La Jolla, CA, USA) by one-
way analysis of variance and Tukey’s multiple comparison tests. The
level of significance was determined as P < 0.05 significance.
Experiments were repeated in triplicates, and data were represented
as mean ± standard deviation (SD) for n = 3 unless mentioned.
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3. RESULTS
3.1. Biochemical Characterization of Soluble Placenta
ECM (pECM). The NP was subjected to decellularization using
a combinatorial treatment involving homogenization, centrifu-
gation, SDS, and nucleases. For the preparation of soluble
placenta ECM proteins (pECM), the ECM from the
decellularized placenta was solubilized using urea. To estimate
the biochemical preservation in pECM after the decellulariza-
tion and the solubilization process, pECM was quantified for
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Figure 3. SEM micrographs of (A) CIMS and (C) HEMS; micro-CT analysis of (B) CIMS and (D) HEMS.

Figure 4. (A) Swelling kinetics of CIMS and HEMS at 37 °C in PBS; (B) degradation profile of CIMS and HEMS incubated in PBS containing
lysozyme; (C) FTIR analysis of CIMS and HEMS; and (D) tensile testing of CIMS and HEMS incubated in PBS for 48 h (P > 0.05). Y-error bars
represent standard deviation.

major ECM components such as collagen and sGAG. As
observed in Supporting Information (Figure S1), retention of
collagen (409.8 ± 45.44 μg mg−1) and sGAG (38.45 ± 7.42 μg
mg−1) in pECM after the decellularization and solubilization
process was not significantly (P > 0.05) different from the
collagen (460.4 ± 18.622 μg mg−1) and sGAG (53.59 ± 14.41
μg mg−1) content of NP. These results demonstrated that the
major ECM components were sufficiently retained in pECM
after the decellularization and solubilization process, which will
make it an ideal matrix for tissue engineering applications.
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3.2. Cytokine Array for Detection of Growth Factors.
pECM and NP were arrayed on a glass chip containing 120
different cytokine antibodies for detection of the retained
endogenous cytokines. As shown in Figure 1, 84 cytokines were
readily detected in pECM, and the remaining 36 cytokines were
not detected, which may be due to the sensitivity limitations of
the array. Among the 84 detected bioactive molecules,
cytokines [EGF, IGF-1, PDGF-B, HGF, VEGFA, TGF-β1,
and FGF] which are considered critical for regulating the
angiogenesis and skin wound healing were detected at high
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levels. Also, growth factors such as IL-6, IL-8, MCP-1, and HEKs were cultivated on lysine-coated coverslips with either
RANTES, which are reported to accelerate fibroblast and CIMS-/HEMS-conditioning medium or control medium for 96
keratinocyte migration and proliferation, were also detected in h. As shown in Figure 5A, HFFs/HEKs grown in HEMS-
pECM.
3.3. SDS-PAGE and Western Blotting of NP and pECM.
SDS-PAGE was used to investigate the proteins retained in
pECM. Figure 2A reveals some bands at lower molecular
weights signifying the presence of various proteins/peptides in
the pECM, which demonstrates the complexity of the pECM.
Western blotting was performed to confirm the presence of
endogenous bioactive molecules that are known to regulate
blood vessel formation and skin regeneration. Figure 2B reveals
the presence of retained growth factors such as EGF, IGF-1,
PDGF-B, HGF, VEGFA, and TGF-β1 after the decellulariza-
tion and solubilization process in pECM. From Figure 2C, no
significant difference (P > 0.05) was observed in the levels of
EGF, PDGF-B, TGF-β1, and VEGFA in pECM compared to
that in NP. However, a significant decrease (P < 0.05) was
observed in the levels of IGF-1 and HGF in pECM compared
to that in NP (Figure 2C).
3.4. Physicochemical Characterization of Scaffolds. In
this study, collagen I/pECM was blended with SF in aqueous
environment and successfully fabricated into CIMS/HEMS,
respectively, by molding and freeze-drying process. The
scanning electron microscopy (SEM) microstructures of
CIMS and HEMS are shown in Figure 3A,C, which revealed
a high degree of interconnected heterogeneous porous
structures within the matrices. The porosity of the samples
analyzed by micro-CT was approximately similar for CIMS
(85.27%) and HEMS (90.2%) as shown in Figure 3B,D. The
swelling behavior of the scaffolds is critical not only for
arbitrating the rate of nutrition and waste transport within the
scaffolds in situ, but it also helps in biological fixation to the Figure 5. (A) Rhodamine phalloidin−DAPI images of HFFs and
wound bed. As observed in Figure 4A, CIMS and HEMS HEKs cultivated in CIMS- and HEMS-conditioned medium (scale bar
demonstrated 55 and 64% swelling, respectively, when exposed represents 50 μm, red represents nucleus staining DAPI and green
to PBS for 0.5 h because of the rapid uptake of water. After this depicts cytoskeleton expression); (B) TUNEL assay images of HEKs
time, it attained a plateau, and no further increase in mass was cultivated in CIMS- and HEMS-conditioned medium (scale bar
observed until 64 h. Extended long-term swelling observations represents 50 μm).
are essential for more critical assessment. As shown in Figure
4B, CIMS (mass loss 97%) exhibited faster degradation as conditioned medium proliferated rapidly and were able to make
compared to HEMS (mass loss 90%) when incubated in intermittent contact via cellular protrusions and extensions with
lysozyme at 37 °C for 16 days. Also, CIMS and HEMS when well-spread cytoskeletons compared to those of HFFs/HEKs
incubated in PBS (pH 7.4) without lysozyme for 48 h showed a cultured in CIMS-conditioned/control medium. Also, there was
negligible mass loss (data not shown) and displayed a wet no difference in the morphology of HFFs/HEKs cultured in the
tensile strength of 0.065 ± 0.004 and 0.076 ± 0.003 MPa (P > CIMS-conditioned medium compared with the cells cultured in
0.05; Figure 4D). control medium. These observations demonstrated that CIMS/
Infrared absorption spectra (Figure 4C) of CIMS and HEMS HEMS did not release cytotoxic substances.
showed characteristic absorption peaks assigned to the peptide The TUNEL assay was performed to detect apoptosis after 7
bonds (−CONH−) that give rise to amide I (1600−1700 cm), days of HEKs’ cultivation either in control or in CIMS-/
amide II (1520−1540 cm), and amide III (1220−1300 cm) HEMS-conditioned medium. As shown in Figure 5B, no
signature peaks. Pure silk scaffolds showed amide I, amide II, significant apoptotic HEKs were observed in control or CIMS-/
and amide III bands at 1617, 1511, and 1226 cm−1; CIMS HEMS-conditioned medium treated groups, indicating that the
scaffolds showed amide I, amide II, and amide III bands at CIMS-/HEMS-conditioned medium had no detrimental effect
1636, 1528, and 1226 cm−1, whereas HEMS scaffolds showed on cells.
the characteristic peaks of amide I, amide II, and amide III 3.5.2. Scratch Assay. HFFs started to migrate from the
bands at 1623, 1533, and 1222 cm−1. Also, in CIMS and edges of the denuded/wounded areas within 6 h, whereas HEK
HEMS, the peak at 2869−1 corresponds to C−H symmetric migration was witnessed after 12 h in both the treatment
stretching. Notably, in CIMS, the 1636 cm−1 band is (CIMS-/HEMS-conditioned medium) and control groups
characteristic of the triple helix of native collagen and thus (Figure 6A,B). As determined by three independent observers,
suggests the existence of collagen in the native triple helical the numbers of HFFs that migrated into the scratch areas for
structures in the CIMS scaffold. HEMS-conditioned, CIMS-conditioned, and control group
3.5. Indirect Cytotoxicity Testing for CIMS and HEMS. were found to be (6 h): 134 ± 06, 86 ± 12, and 50 ± 05
3.5.1. Morphological and Apoptosis Assessment. HFFs and and (12 h): 295 ± 16, 160 ± 14, and 110 ± 06, respectively
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Figure 6. Scratch assay images of (A) HFFs and (B) HEKs cultivated in CIMS- and HEMS-conditioned medium at different time durations (scale
bar represents 100 μm); cell migration quantification of (C) HFFs and (D) HEKs. Y-error bars represent standard deviation, and triple asterisks
signify P < 0.001.

Figure 7. (A) Growth factors and cytokines detected in HEMS-conditioned medium by ELISA multiarray; (B) cellular metabolic activity of HFFs,
HEKs, and HAMSCs cultured in CIMS/HEMS according to the MTT assay; live−dead cell staining of HEKs cultivated in (C) CIMS and (D)
HEMS. Y-error bars represent standard deviation, and scale bar represents 50 μm.

(Figure 6C). Similarly, the numbers of HEKs that migrated into
the scratch areas for HEMS-conditioned, CIMS-conditioned,
and control group were (12 h): 313 ± 11, 208 ± 12, and 81 ±
06 and (24 h): 654 ± 16, 355 ± 9, and 268 ± 08, respectively
(Figure 6D). It can be interpreted from Figure 6C,D that the
number of migrated HFFs/HEKs was more significant (p <
0.05) in the HEMS-conditioned medium treated group
compared to that in other groups.
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3.6. Analysis of HEMS-Conditioned Medium by ELISA.
The HEMS-conditioned media were assayed by ELISA to
evaluate the presence of growth factors (IL-6, IL-8, MCP-1,
TGF-β1, and RANTES; known to accelerate migration and
proliferation of HFFs and HEKs) released from HEMS during
the conditioning process. From Figure 7A, it can be observed
that detectable levels of MCP-1, TGF-β1, RANTES, IL-6, and
IL-8 were present in the HEMS-conditioned media.
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Figure 8. Proangiogenic property analysis of CIMS and HEMS by the CAM assay; (A) macroscopic view, (B) quantification of vessels converging
toward the scaffolds, and (C) histological analysis of the retrieved CIMS and HEMS after 72 h of incubation (scale bar represents 100 μm). *
represents the implanted scaffold; Y-error bars represent standard deviation, and triple asterisks signify P < 0.001.

Figure 9. H&E, MT, TB, and anti-CD31 of the explanted CIMS/HEMS on day 21 after subcutaneous implantation (scale bar represents 50 μm;
blue represents nucleus staining DAPI, and green depicts antibody expression).

3.7. Direct Cytotoxicity Testing for CIMS and HEMS.
The 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bro-
mide (MTT) cytotoxicity assay was performed to assess the
remnant toxic chemical residues that may be present in CIMS
and HEMS during the process of extraction/decellularization
and solubilization. As shown in Figure 7B, it was evident that
CIMS and HEMS supported the proliferation and viability of
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HFFs, HEKs, and HAMSCs, which indicates the potential of
CIMS/HEMS in skin tissue engineering applications. Interest-
ingly, the cells cultivated in HEMS displayed increased cellular
metabolic activity compared to that in CIMS and TCP,
demonstrating its cytocompatibility. This increased cellular
metabolic activity in HEMS may be attributed to the presence
of cytokines/growth factors. Also, CIMS displayed higher cell
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Figure 10. Healing progression of full-thickness cutaneous wounds treated with SHAM, CIMS, and HEMS; (A) photographs of wounds and (B)
rate of wound closure on days 0, 7, 14, and 21; (C-D) RT-PCR analysis of skin wounds treated with SHAM, CIMS, and HEMS at 21 day
postwounding. Y-error bars represent standard deviation, single asterisks signify P < 0.05, and triple asterisks signify P < 0.001.
viability compared to TCP, owing to the 3D architecture of the
scaffold.
The Live/Dead assay was used to determine the adhesion
and viability of HEKs within CIMS/HEMS. On day 7, the
majority of cells cultivated in CIMS and HEMS expressed green
fluorescence (live cells), and dead cells were rarely detected
(Figure 7C,D). These results suggest that CIMS and HEMS
provided the necessary environment for adequate cell function
and did not induce significant cytotoxicity.
3.8. CAM Assay. The potential of CIMS/HEMS to induce
angiogenesis was examined by implanting CIMS/HEMS on the
CAM. After 3 days of implantation, HEMS was completely
enveloped by the CAM and facilitated superior integration with
the host, whereas this feature was not visualized in CIMS. Also,
a number of allantoic vessels converged approaching toward the
HEMS, while the CIMS group showed the random progression
of the allantoic vessels (Figure 8A,B). The quantitative analysis
confirmed that the numbers of allantoic vessels converging
toward the HEMS group (28 ± 3) were significantly (p <
0.001) higher than the CIMS group (12 ± 2). Also, H&E, MT,
and DAPI-stained sections (Figure 8C) revealed that HEMS
promoted cell infiltration with homogeneous distribution, while
CIMS supported moderate cell infiltration.
3.9. In Vivo CIMS/HEMS Cellular Response and Organ
Toxicity Analysis. The CIMS/HEMS subcutaneously im-
planted groups appeared healthy, and no mortality was
recorded throughout the study period. After 28 days of
implantation, CIMS/HEMS was retrieved with the surrounding
tissues and histologically stained as shown in Figure 9. H&E
staining revealed tissue integration/in-growth between the host
tissue and the implanted CIMS/HEMS. Interestingly, CD31
staining revealed considerably more number of blood vessels
(neo-capillary formation) in the HEMS-implanted group
compared to that in the CIMS-implanted group. From TB
staining, few mast cells were visualized in the host region
surrounding the CIMS/HEMS, but no mast cells were found
infiltrated into CIMS/HEMS. This minor inflammatory
response after CIMS/HEMS implantation is attributed to the
early inflammatory response, which is similar to the regular
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wound healing process. From MT staining, no capsular layer/
fibrosis was found, and dense collagen fiber deposition was
visualized in the interfacial layer between the host and CIMS/
HEMS, demonstrating stimulated cell infiltration. These results
suggest that CIMS/HEMS does not trigger adverse host
immune inflammatory response in vivo.
The rat’s body mass of different groups was regularly
monitored, and after 28 days of implantation, there was no
significant difference in the weight of animals before and after
treatment (CIMS-/HEMS-implanted group). Histological
analysis of the organs retrieved after 28 days of post-
implantation showed no necrosis or significant pathological
changes in the anatomical structure of the liver, heart, lung, and
kidney of the treated group (Supporting Information, Figure
S2). Further, the cardiac muscle of both the CIMS and HEMS
groups showed no indication of fibrosis or inflammatory
response. Also, while observing the liver, the distribution of
hepatocytes in the treatment group was found to be similar to
the typical anatomy. Likewise, no pulmonary fibrosis was
detected in the lungs of the treatment groups. Besides, the
glomerulus structure in the kidney section was observed
without difficulty in both the groups. These results indicate that
CIMS and HEMS did not cause any organ toxicity.
3.10. Full-Thickness Cutaneous Wound Healing
Study. The efficacy of CIMS/HEMS on accelerating the skin
wound healing process was evaluated by creating a full-
thickness skin wound in a rat model. Full-thickness cutaneous
wounds treated with CIMS, HEMS, and SHAM groups were
photographed at different time durations (0, 7, 14, and 21 days)
to visualize the reduction of wound size and to understand the
post-operative healing process (Figure 10A). On day 0, CIMS
and HEMS were firmly attached to the wound bed and readily
absorbed the wound exudates, thus leading to the biological
fixation of dressing, and also prevented the exposure of wounds
to the external environment. Also, there was no indication of
infection or inflammation in both the CIMS- and HEMS-
treated group throughout the study period. On day 7 post-
operation, wound sizes were reduced in all groups (Figure
10B), but notably, the wound covered with HEMS-treated
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Figure 11. Histological micrographs of wound sections implanted with CIMS and HEMS at days 7, 14, and 21 after dermal excision by H&E staining
(black arrow indicates the initial wound boundary created; scale bar represents 250 μm; inset scale bar represents 50 μm).

Figure 12. Representative images of (A) MT and (B) anti-CD31 of wounds treated with SHAM, CIMS, and HEMS (scale bar for MT represents 50
μm; scale bar for anti-CD31 represents 100 μm; red represents nucleus staining DAPI, and green depicts antibody expression).

group (77 ± 1.8%) showed significantly (p < 0.05) higher
wound contraction compared to the CIMS (65 ± 3.4%) and
SHAM (47 ± 2.09%) groups. Subsequently, after 14 days of
treatment, there was 83 ± 3.3% reduction (P < 0.05) in the
wound area covered by HEMS, whereas wound size reductions
were 71 ± 4.3 and 61 ± 5.3% for the wounds covered by the
CIMS and SHAM groups, respectively. Interestingly, 21 days
post-treatment, there were 81 ± 4.9 and 90 ± 5.7% reductions
in wound area covered by the SHAM and CIMS groups,
respectively, while complete closure of wounds was observed in
the HEMS (99 ± 1%) group.
After 7 days post-treatment, H&E staining (Figure 11)
revealed eschar formation in all the groups. Considerably, a
high number of fibroblast infiltration and pronounced neo-
vascularization was witnessed in the wounds treated with
HEMS. On day 14 in wounds treated with HEMS, partial
development of the epidermis was observed, while CIMS and
SHAM groups exhibited poorly developed epidermis. Remark-
ably, a small number of epidermal appendages such as
sebaceous glands and hair follicles were also observed in the
regenerated skin treated with HEMS, while, in contrast, it was
absent in the CIMS and SHAM groups. Further, 21 days post-
treatment, the SHAM group displayed incomplete healing
associated with partially developed epithelium, whereas CIMS
displayed discontinuous epithelium. Interestingly, in the
wounds treated with HEMS, well-developed epidermis
associated with differentiated epidermal cells, closely arranged
basal cells, and layers of keratinocytes were evident, whereas
these features were not prominent in CIMS and SHAM.
Moreover, the increased number of secondary structures such
as sebaceous glands and hair follicles was witnessed in the
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HEMS group compared to that in the CIMS and SHAM
groups.
MT staining of different groups was performed to
demonstrate the collagen (major ECM component) deposition
in the wounds (Figure 12A). After 7 days post-treatment, low
level of regenerative collagen deposition was observed in the
SHAM group, whereas comparatively more collagen fibers with
granulation tissue formation were visualized in the CIMS- and
HEMS-treated groups. Interestingly, on day 14, dermal collagen
fibers of the SHAM group were disorganized and sparse,
whereas they were bundled and arranged in a parallel fashion
for the CIMS and HEMS groups. On day 21, more pronounced
basket weave type arrangement revealing higher maturation
level of collagen was demonstrated particularly for the HEMS-
treated group, while it was not distinctly visualized for CIMS
and SHAM.
Immunofluorescence staining for CD31 for the CIMS/
HEMS group was further performed at 7 and 14 days to
demonstrate neovascularization (Figure 12B). After 7 days
post-treatment, a large number of neo-microvessels were seen
in the HEMS-treated group compared to that in the CIMS and
SHAM groups. This increased vascularization in the HEMS
group may provide more blood supply to the wound area and
thus may accelerate the wound healing cascade. At day 14, neo-
microvessels were still developing in the SHAM group,
demonstrating the slow healing progression therein, while in
contrast, the number of neo-microvessels reduced in CIMS and
HEMS revealing superior healing. Further, immunofluores-
cence staining for CK-10 was performed at 14 and 21 days
post-wounding to investigate the re-epithelialization and
epidermal differentiation (Figure 13). After 14 days, no
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Figure 13. Representative immunohistochemistry images of CK-10-stained histological sections on days 14 and 21 of SHAM, CIMS, and HEMS
(scale bar represents 50 μm; red represents nucleus staining DAPI, and green depicts antibody expression).
expression of CK-10 was observed in the SHAM group because
of the lack of new epithelium formation. Similarly, the CIMS-
treated group also showed no significant expression of CK-10 at
the suprabasal epithelium layer because the epithelialization and
its stratification were not distinct. Interestingly, HEMS
supported the development of a new stratified thick epithelial
layer, which resulted in the enhanced expression of CK-10. On
day 21, the HEMS-treated group underwent further remodeling
and exhibited thinner epithelium revealing complete re-
epithelialization, hence mimicking native epidermal tissue
architecture, whereas comparatively thicker epithelium was
observed in the CIMS and SHAM groups.
3.11. Reverse Transcriptase-PCR (RT-PCR) Analyses of
the Wound Area. In addition to histological and immuno-
fluorescence staining to evaluate overall wound healing, RT-
PCR was further utilized to assess the expression profiles of
genes associated with skin wound healing (COL I, COL III,
KRT 10, KRT 14). As shown in Figure 10C,D, COL I
expression is upregulated for the HEMS-treated group
compared to that for the CIMS and SHAM groups. This
increased expression of COL I (predominant type of collagen
present in skin) may be attributed to the enhanced remodeling
in the HEMS group. However, on the other hand, the
expression of COL III, KRT 10, and KRT 14 was down-
regulated in the HEMS-treated group when compared to that
in CIMS and SHAM (Figure 10C,D), which confirms that the
wound healing process is more complete in the HEMS-treated
group compared to that in the CIMS and SHAM groups.
4. DISCUSSION
To stimulate accelerated regeneration of full-thickness skin
injuries, porous, non-immunogenic, bioactive matrix are
scaffolds of choice into which HFFs and HEKs can infiltrate,
proliferate, and form reparative tissues. In the present study, we
demonstrated a simple and efficient technique to fabricate
HEMS by blending pECM containing cytokines/growth factors
with SF, thus bringing together the inherent advantages of both
SF and pECM for skin tissue engineering. Because collagen I is
widely reported for its efficacy in skin tissue engineering,33,34 SF
was also blended with collagen I to fabricate CIMS and thus
used for comparison studies with HEMS. pECM was
successfully prepared by decellularizing human placenta using
the protocol described in our previous study24 and solubilized
using urea.25 ECM, a significant noncellular component of the
cell, is considered as a direct modulator in regulating many
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cellular functions such as adhesion, migration, proliferation, and
differentiation. Also, it provides the structural support and
biochemical cues that are required for tissue development as
well as rejuvenation.35 It is of prime importance to retain these
biological entities in pECM during the decellularization and
solubilization process. The collagen and GAGs which are the
primary ECM components were well-preserved in pECM
(Supporting Information Figure S1). Further, cytokine growth
factor array (Figure 1) and western blot analysis (Figure 2B,C)
of pECM established the presence of cytokines/growth factors
such as EGF, IGF-1, PDGF-B, HGF, VEGFA, TGF-β1, IL-6,
IL-8, MCP-1, and RANTES at high levels. Thus, the HEMS
containing pECM with the relevant cytokines/growth factors
required for wound healing will be advantageous for accelerated
wound healing when compared to CIMS.36
Porosity and mechanical attributes of the scaffold are
essential criteria for skin repair and regeneration because it
provides a personalized environment for the cells to initiate the
neotissue formation.37 Less porosity of the scaffold can lead to
insufficient swelling affecting nutrient/gas transport, while
ultrahigh-porosity of the scaffold can result in excess swelling
and structural deformation. The SEM (Figure 3A,C) and
micro-CT (Figure 3B,D) images of CIMS and HEMS revealed
interconnected fibrous morphology with an adequate degree of
porosity and swelling (Figure 4A), which will be beneficial for
cell adhesion, proliferation, and migration into the 3D
architecture of the scaffold. The tensile strength values (Figure
4D) of CIMS and HEMS were in the range of the values
reported for the naturally derived porous scaffold (heart
muscles of humans/rats0.003−0.07 MPa; collagen gels of
calfskin0.001−0.009 MPa) proposed for soft tissue engineer-
ing applications.38,39 Further, HFFs and HEKs were cultivated
in the conditioning medium extracted from CIMS/HEMS to
study the effect of leachates from the scaffolds. Rhodamine
phalloidin and DAPI staining (Figure 5A) of HFFs/HEKs
grown in CIMS-/HEMS-conditioned media did not show any
morphological aberrations, but interestingly HFFs/HEKs
grown in HEMS-conditioned media demonstrated more well-
spread actin filaments compared to that in CIMS-conditioned
media. The well-spread actin filaments are beneficial for
establishing enhanced cell−cell contact and interaction with
the environment.40 Also, the tunnel assay (Figure 5B) revealed
that the CIMS-/HEMS-conditioned media did not cause any
apoptosis-related deleterious effect to the HEKs. The results of
the scratch assay (Figure 6) showed that the HEMS-
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conditioned medium significantly accelerated the migration of
HFFs/HEKs in vitro compared to other groups. The presence
of cytokines/growth factors such as TGF-β1, IL-6, IL-8, MCP-
1, and RANTES in the HEMS-conditioned medium was
detected by ELISA (Figure 7A). TGF-β1 and IL-6 are reported
to stimulate increased cell migration of dermal fibroblasts and
keratinocytes.41−45 Likewise, IL-8 is also reported to promote
skin re-epithelialization by increasing keratinocyte migration.45
Also, MCP-1 and RANTES are known to promote dermal
wound healing by acting as a chemoattractant for the cells.46
Thus, it may be concluded that the presence of cytokines/
growth factors (TGF-β1, IL-6, IL-8, MCP-1, and RANTES) in
the HEMS-conditioned medium is responsible for the
accelerated migration of HFFs/HEKs in vitro. Thus, in the
course of wound healing, augmented fibroblast infiltration into
the wound site during the initial days after injury has been
reported to stimulate the wound healing process, and
accelerated migration of basal keratinocytes from the wound
edges to the lesion leads to closure of the wound area, which in
turn triggers the differentiation of basal keratinocyte to
keratinizing epidermal cells because of contact inhibition.47
The MTT assay (Figure 7B) demonstrated that HAMSCs/
HEKs/HFFs cultivated directly in HEMS had a higher rate of
proliferation compared to that in CIMS and TCP. Also, live−
dead staining (Figure 7C,D) revealed that HEKs successfully
adhered, infiltrated, and remained viable in the HEMS
microenvironment. Thus, taken together, these findings
strongly suggest that HEMS containing cytokines/growth
factors with desirable physical attributes supported the
adhesion, infiltration, migration, and proliferation of
HAMSCs/HEKs/HFFs and therefore proved to be nontoxic
and functionally competent for skin tissue engineering in vitro.
Vascularization is considered to be a critical aspect for the
survival of engineered scaffolds; insufficient vasculature can lead
to necrosis or volume reduction after implantation and inhibits
efficient regeneration of the skin defect.48 Herein, ex vivo CAM
assay (Figure 8) using scaffolds revealed significantly more
number of allantoic vessels approaching toward the HEMS
compared to the CIMS group. This enhanced angiogenic
response of the HEMS may be attributed to the preservation of
VEGFA (the most potent growth factor reported to promote
the formation of new blood vessels49) in the HEMS. Further, it
is of crucial importance to assess the host immune response in
vivo by subcutaneous grafting before investigating the healing
ability of CIMS/HEMS in full-thickness skin wounds.
Histological evaluation (Figure 9) of the subcutaneously
implanted CIMS/HEMS after 28 days displayed the absence
of tissue necrosis, granulomatous inflammation, or fibrous layer
development surrounding the CIMS/HEMS. Furthermore, the
implanted HEMS exhibited its outstanding biocompatibility in
vivo by supporting considerably more number of blood vessel
formation compared to CIMS. Also, from H&E staining of
organs (Supporting Information, Figure S2) retrieved from the
animals treated with CIMS/HEMS, no organ toxicity was
visualized in the major organs (heart, liver, lung, and kidney).
Overall, the absence of any adverse immune reaction in the
host animals suggests that these engineered scaffolds are
nonimmunogenic in vivo.
The efficacy of CIMS/HEMS in accelerating wound healing
was evaluated in the full-thickness cutaneous wound in a rat
model. ECM components such as collagen, laminin,
fibronectin, elastin, and GAG are essential at every phase
(hemostasis to remodeling) of the wound healing cascade via
16989
Research Article
dynamic cell−cell or cell−matrix interactions and modulate
various cellular responses.50 Also, different growth factors [IGF-
1, PDGF-B, TGF-β1, EGF, and VEGFA] stimulate angio-
genesis, collagen deposition, and re-epithelialization by
recruiting fibroblasts, keratinocytes, endothelial cells, stem
cells, and macrophages into the wound site.51 Histological
(Figure 11) and immunofluorescence (Figures 12B and 13)
investigation of the wounds covered with HEMS demonstrated
rapid healing with pronounced neovascularization and re-
epithelialization compared to the CIMS and SHAM groups.
Further, MT staining (Figure 12A) of the wounds treated with
HEMS revealed the higher turnover rate of bundled and
arranged collagen fibers, which lead to early wound stabilization
suggesting its role in supporting skin remodeling. Predom-
inantly during the proliferation and remodeling phases, HGF is
a vital growth factor in scar formation by controlling the
equilibrium between synthesis and degradation of ECM via
stimulating the secretion of matrix metalloproteinase in
fibroblasts.52 Also, bFGF inhibits the phenotypic conversion
of fibroblasts to myofibroblasts, thus preventing scarring.53
Further, the complete wound closure without scar formation
was witnessed only in the HEMS group. Thus, our results
indicate that the release of endogenous growth factors (IGF-1,
PDGF-B, TGF-β1, EGF, VEGFA, HGF, and bFGF) from
HEMS has contributed to superior neovascularization and
accelerated re-epithelialization without scar formation. Also,
expression profiles of genes (COL I, COL III, KRT 10, KRT
14) associated with wound healing were studied in wound area
retrieved from different treatment groups after 21 days by RT-
PCR (Figure 10C,D). The results demonstrated that SHAM
expressed higher expression of KRT 10 and KRT 14 compared
to other groups because of the presence of more proliferating
or differentiating keratinocytes, which is indicative of
incomplete healing and infers that the re-epithelialization
process is still ongoing.54 On the other hand, the HEMS
expressed lower expression of KRT 10 and KRT 14 as the
epithelialization process is more complete. Also, downregulated
expression of COL III and upregulation of COL I in HEMS-
treated wounds relative to CIMS/SHAM are suggestive of early
remodeling because COL III is produced during early phases of
wound healing and subsequently replaced by COL I in the final
stages of wound healing.55 Therefore, HEMS is evidenced to be
a promising matrix that delivered the required cytokines/
growth factors and desirable microenvironment to augment
cutaneous wound healing.
5. CONCLUSIONS
We successfully fabricated HEMS by a combinatorial approach
for skin tissue engineering applications. The fabricated HEMS
retained the natural ECM composition and the cytokines/
growth factors which are known to accelerate the migration of
HFFs/HEKs, blood vessel induction/development, and re-
epithelialization. Also, the 3D architecture of HEMS with
adequate porosity and mechanical stability supported excellent
viability and proliferation of HFFs/HEKs/HAMSCs in vitro.
Further, HEMS demonstrated its superior vascularization
potential by the CAM assay, and also no immune rejection
or organ toxicity was observed in the host after subcutaneous
implantation. We also evidenced the ability of HEMS to
mediate early skin repair in full-thickness wound models.
Therefore, this study provides an approach wherein human
pECM is incorporated with SF to produce a bioactive hybrid
DOI: 10.1021/acsami.7b19007
ACS Appl. Mater. Interfaces 2018, 10, 16977−16991
ACS Applied Materials & Interfaces
scaffold that has enormous potential for skin tissue engineering
applications.
■
ASSOCIATED CONTENT
*
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsami.7b19007.
Collagen and GAG quantification; H&E staining of
heart, liver, lung, and kidney harvested from different
treatment groups; MT staining of hybrid scaffolds; and
gene-specific primers used for RT-PCR (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: sdhara@smst.iitkgp.ernet.in.
ORCID
Koel Chaudhury: 0000-0002-9390-1179
Santanu Dhara: 0000-0003-4443-7610
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
The authors A.P.R. and S.D. acknowledge the fellowship
support received from the Ministry of Human Resource
Development (MHRD, Govt. of India), K.B. from the
Department of Science & Technology (DST, Govt. of India)
INSPIRE, and P.P.M. from the Indian Council of Medical
Research (ICMR, Govt. of India). The funding of the project
(BT/PR7818/MED/32/279/2013) from the Department of
Biotechnology (DBT, Govt. of India) is greatly acknowledged.
■
REFERENCES
(1) Yildirimer, L.; Thanh, N. T. K.; Seifalian, A. M. Skin
Regeneration Scaffolds: A Multimodal Bottom-up approach. Trends
Biotechnol. 2012, 30, 638−648.
(2) Shevchenko, R. V.; James, S. L.; James, S. E. A Review of Tissue-
Engineered Skin Bioconstructs Available for Skin Reconstruction. J. R.
Soc., Interface 2010, 7, 229−258.
(3) Chen, X.; Zhang, M.; Wang, X.; Chen, Y.; Yan, Y.; Zhang, L.;
Zhang, L. Peptide-Modified Chitosan Hydrogels Promote Skin
Wound Healing by Enhancing Wound Angiogenesis and Inhibiting
Inflammation. Am. J. Transl. Res. 2017, 9, 2352−2362.
(4) Choi, J. S.; Kim, J. D.; Yoon, H. S.; Cho, Y. W. Full-Thickness
Skin Wound Healing Using Human Placenta-Derived Extracellular
Matrix Containing Bioactive Molecules. Tissue Eng., Part A 2013, 19,
329−339.
(5) Simman, R.; Phavixay, L. Split-Thickness Skin Grafts Remain the
Gold Standard for the Closure of Large Acute and Chronic Wounds. J.
Am. Col. Certif. Wound Spec. 2011, 3, 55−59.
(6) Lotz, C.; Schmid, F. F.; Oechsle, E.; Monaghan, M. G.; Walles,
H.; Becker, F. G. Cross-Linked Collagen Hydrogel Matrix Resisting
Contraction to Facilitate Full-Thickness Skin Equivalents. ACS Appl.
Mater. Interfaces 2017, 9, 20417−20425.
(7) Li, Q.; Niu, Y.; Diao, H.; Wang, L.; Chen, X.; Wang, Y.; Dong, L.;
Wang, C. In Situ Sequestration of Endogenous PDGF-BB with an
ECM-Mimetic Sponge for Accelerated Wound Healing. Biomaterials
2017, 148, 54−68.
(8) Selvaraj, S.; Fathima, N. N. Fenugreek Incorporated Silk Fibroin
NanofibersA Potential Antioxidant Scaffold for Enhanced Wound
Healing. ACS Appl. Mater. Interfaces 2017, 9, 5916−5926.
(9) Devalliere, J.; Dooley, K.; Hu, Y.; Kelangi, S. S.; Uygun, B. E.;
Yarmush, M. L. Co-Delivery of a Growth Factor and a Tissue-
Protective Molecule Using Elastin Biopolymers Accelerates Wound
Healing in Diabetic Mice. Biomaterials 2017, 141, 149−160.
16990
Research Article
(10) Zhang, W.; Chen, L.; Chen, J.; Wang, L.; Gui, X.; Ran, J.; Xu,
G.; Zhao, H.; Zeng, M.; Ji, J.; Qian, L.; Zhou, J.; Ouyang, H.; Zou, X.
Silk Fibroin Biomaterial Shows Safe and Effective Wound Healing in
Animal Models and a Randomized Controlled Clinical Trial. Adv.
Healthcare Mater. 2017, 6, 1700121.
(11) Guan, G.; Bai, L.; Zuo, B.; Li, M.; Wu, Z.; Li, Y.; Wang, L.
Promoted Dermis Healing From Full-Thickness Skin Defect by
Porous Silk Fibroin Scaffolds (PSFSs). Biomed. Mater. Eng. 2010, 20,
295−308.
(12) Gil, E. S.; Panilaitis, B.; Bellas, E.; Kaplan, D. L. Functionalized
Silk Biomaterials for Wound Healing. Adv. Healthcare Mater. 2013, 2,
206−217.
(13) Yan, S.; Zhang, Q.; Wang, J.; Liu, Y.; Lu, S.; Li, M.; Kaplan, D. L.
Silk Fibroin/Chondroitin Sulfate/Hyaluronic Acid Ternary Scaffolds
for Dermal Tissue Reconstruction. Acta Biomater. 2013, 9, 6771−
6782.
(14) Xiong, S.; Zhang, X.; Lu, P.; Wu, Y.; Wang, Q.; Sun, H.; Heng,
B. C.; Bunpetch, V.; Zhang, S.; Ouyang, H. A Gelatin-Sulfonated Silk
Composite Scaffold Based on 3D Printing Technology Enhances Skin
Regeneration by Stimulating Epidermal Growth and Dermal Neo-
vascularisation. Sci. Rep. 2017, 7, 4288.
(15) Fuentes, M. G.; Meinel, A. J.; Hilbe, M.; Meinel, L.; Merkle, H.
P. Silk Fibroin/Hyaluronan Scaffolds for Human Mesenchymal Stem
Cell Culture in Tissue Engineering. Biomaterials 2009, 30, 5068−5076.
(16) Lv, Q.; Hu, K.; Feng, Q.; Cui, F. Fibroin/Collagen Hybrid
Hydrogels with Crosslinking Method: Preparation, Properties, and
Cytocompatibility. J. Biomed. Mater. Res., Part A 2008, 84A, 198−207.
(17) Bhardwaj, N.; Kundu, S. C. Silk Fibroin Protein and Chitosan
Polyelectrolyte Complex Porous Scaffolds for Tissue Engineering
Applications. Carbohydr. Polym. 2011, 85, 325−333.
(18) Vasconcelos, A.; Gomes, A. C.; Paulo, A. C. Novel Silk Fibroin/
Elastin Wound Dressings. Acta Biomater. 2012, 8, 3049−3060.
(19) Bhardwaj, N.; Sow, W. T.; Devi, D.; Ng, K. W.; Mandal, B. B.;
Cho, N.-J. Silk Fibroin−Keratin Based 3D Scaffolds as a Dermal
Substitute for Skin Tissue Engineering. Integr. Biol. 2015, 7, 53−63.
(20) Aamodt, J. M.; Grainger, D. W. Extracellular Matrix-Based
Biomaterial Scaffolds and the Host Response. Biomaterials 2016, 86,
68−82.
(21) Wildman, D. E. Review: Toward an Integrated Evolutionary
Understanding of the Mammalian Placenta. Placenta 2011, 32, S142−
S145.
(22) Hong, J. W.; Lee, W. J.; Hahn, S. B.; Kim, B. J.; Lew, D. H. The
Effect of Human Placenta Extract in a Wound Healing Model. Ann.
Plast. Surg. 2010, 65, 96−100.
(23) De, D.; Chakraborty, P. D.; Bhattacharyya, D. Regulation of
Trypsin Activity by Peptide Fraction of an Aqueous Extract of Human
Placenta Used as Wound Healer. J. Cell. Physiol. 2011, 226, 2033−
2040.
(24) Rameshbabu, A. P.; Ghosh, P.; Subramani, E.; Bankoti, K.;
Kapat, K.; Datta, S.; Maity, P. P.; Subramanian, B.; Roy, S.; Chaudhury,
K.; Dhara, S. Investigating the Potential of Human Placenta-Derived
Extracellular Matrix Sponges Coupled with Amniotic Membrane-
Derived Stem Cells for Osteochondral Tissue Engineering. J. Mater.
Chem. B 2016, 4, 613−625.
(25) Moore, M. C.; Pandolfi, V.; Mcfetridge, P. S. Novel Human-
Derived Extracellular Matrix Induces In Vitro and In Vivo
Vascularization and Inhibits Fibrosis. Biomaterials 2015, 49, 37−46.
(26) Frazier, S. B.; Roodhouse, K. A.; Hourcade, D. E.; Zhang, L. The
Quantification of Glycosaminoglycans: A Comparison of HPLC,
Carbazole, and Alcian Blue Methods. Open Glycosci. 2008, 1, 31−39.
(27) Rockwood, D. N.; Preda, R. C.; Yücel, T.; Wang, X.; Lovett, M.
L.; Kaplan, D. L. Materials Fabrication from Bombyx Mori Silk
Fibroin. Nat. Protoc. 2011, 6, 1612−1631.
(28) Pati, F.; Adhikari, B.; Dhara, S. Isolation and Characterization of
Fish Scale Collagen of Higher Thermal Stability. Bioresour. Technol.
2010, 101, 3737−3742.
(29) Ghosh, P.; Rameshbabu, A. P.; Dogra, N.; Dhara, S. 2,5-
Dimethoxy 2,5-Dihydrofuran Crosslinked Chitosan Fibers Enhance
DOI: 10.1021/acsami.7b19007
ACS Appl. Mater. Interfaces 2018, 10, 16977−16991
ACS Applied Materials & Interfaces Research Article

Bone Regeneration in Rabbit Femur Defects. RSC Adv. 2014, 4, (53) Desmouliere, A.; Darby, I. A.; Laverdet, B.; Bonté, F. Fibroblasts
19516−19524. and Myofibroblasts in Wound Healing. Clin., Cosmet. Invest. Dermatol.
(30) Aasen, T.; Izpisúa Belmonte, J. C. Isolation and Cultivation of 2014, 7, 301−311.
Human Keratinocytes from Skin or Plucked Hair for the Generation of (54) Levengood, S. L.; Erickson, A. E.; Chang, F.-c.; Zhang, M.
Induced Pluripotent Stem Cells. Nat. Protoc. 2010, 5, 371−382. Chitosan−Poly(Caprolactone) Nanofibers For Skin Repair. J. Mater.
(31) Liang, C.-C.; Park, A. Y.; Guan, J.-L. In Vitro Scratch Assay: A Chem. B 2017, 5, 1822−1833.
Convenient and Inexpensive Method for Analysis of Cell Migration in (55) Volk, S. W.; Wang, Y.; Mauldin, E. A.; Liechty, K. W.; Adams, S.
vitro. Nat. Protoc. 2007, 2, 329−333. L. Diminished Type III Collagen Promotes Myofibroblast Differ-
(32) Ribatti, D.; Nico, B.; Vacca, A.; Presta, M. The Gelatin Sponge- entiation and Increases Scar Deposition in Cutaneous Wound Healing.
Chorioallantoic Membrane Assay. Nat. Protoc. 2006, 1, 85−91. Cells Tissues Organs 2011, 194, 25−37.
(33) Brett, D. A Review of Collagen and Collagen-Based Wound
Dressings. Wounds 2008, 20, 347−356.
(34) Chattopadhyay, S.; Raines, R. T. Collagen-Based Biomaterials
for Wound Healing. Biopolymers 2014, 101, 821−833.
(35) Frantz, C.; Stewart, K. M.; Weaver, V. M. The Extracellular
Matrix at a Glance. J. Cell Sci. 2010, 123, 4195−4200.
(36) Kim, E. J.; Choi, J. S.; Kim, J. S.; Choi, Y. C.; Cho, Y. W.
Injectable and Thermosensitive Soluble Extracellular Matrix and
Methylcellulose Hydrogels for Stem Cell Delivery in Skin Wounds.
Biomacromolecules 2016, 17, 4−11.
(37) O’brien, F. J. Biomaterials & Scaffolds for Tissue Engineering.
Mater. Today 2011, 14, 88−95.
(38) Chen, Q.-Z.; Harding, S. E.; Ali, N. N.; Lyon, A. R.; Boccaccini,
A. R. Biomaterials in Cardiac Tissue Engineering: Ten Years of
Research Survey. Mater. Sci. Eng. 2008, 59, 1−37.
(39) Ö zyazgan, I.; Liman, N.; Dursun, N.; Güneş, I. The Effects of
Ovariectomy on the Mechanical Properties of Skin in Rats. Maturitas
2002, 43, 65−74.
(40) Revenu, C.; Athman, R.; Robine, S.; Louvard, D. The Co-
Workers of Actin Filaments: From Cell Structures to Signals. Nat. Rev.
Mol. Cell Biol. 2004, 5, 635−646.
(41) Postlethwaite, A. E.; Keski-Oja, J.; Moses, H. L.; Kang, A. H.
Stimulation of the Chemotactic Migration of Human Fibroblasts by
Transforming Growth Factor Beta. J. Exp. Med. 1987, 165, 251−256.
(42) Li, Y.; Fan, J.; Chen, M.; Li, W.; Woodley, D. T. Transforming
Growth Factor-Alpha: A Major Human Serum Factor that Promotes
Human Keratinocyte Migration. J. Invest. Dermatol. 2006, 126, 2096−
2105.
(43) Luckett, L. R.; Gallucci, R. M. Interleukin-6 (IL-6) Modulates
Migration and Matrix Metalloproteinase Function in Dermal
Fibroblasts from IL-6KO Mice. Br. J. Dermatol. 2007, 156, 1163−1171.
(44) Gallucci, R. M.; Sloan, D. K.; Heck, J. M.; Murray, A. R.; O’dell,
S. J. Interleukin 6 Indirectly Induces Keratinocyte Migration. J. Invest.
Dermatol. 2004, 122, 764−772.
(45) Michel, G.; Kemény, L.; Peter, R. U.; Beetz, A.; Ried, C.;
Arenberger, P.; Ruzicka, T. Interleukin-8 Receptor-Mediated Chemo-
taxis of Normal Human Epidermal Cells. FEBS Lett. 1992, 305, 241−
243.
(46) Dipietro, L. A.; Reintjes, M. G.; Low, Q. E. H.; Levi, B.; Gamelli,
R. L. Modulation of Macrophage Recruitment into Wounds by
Monocyte Chemoattractant Protein-1. Wound Repair Regen. 2001, 9,
28−33.
(47) Werner, S.; Grose, R. Regulation Healing by Growth of Wound
Factors and Cytokines. Physiol. Rev. 2003, 83, 835−870.
(48) Rouwkema, J.; Khademhosseini, A. Vascularization and
Angiogenesis in Tissue Engineering: Beyond Creating Static Net-
works. Trends Biotechnol. 2016, 34, 733−745.
(49) Yancopoulos, G. D.; Davis, S.; Gale, N. W.; Rudge, J. S.;
Wiegand, S. J.; Holash, J. Vascular-Specific Growth Factors and Blood
Vessel Formation. Nature 2000, 407, 242−248.
(50) Schultz, G. S.; Wysocki, A. Interactions between Extracellular
Matrix and Growth Factors in Wound Healing. Wound Repair Regen.
2009, 17, 153−162.
(51) Barrientos, S.; Brem, H.; Stojadinovic, O.; Tomic-Canic, M.
Clinical Application of Growth Factors and Cytokines in Wound
Healing. Wound Repair Regen. 2014, 22, 569−578.
(52) Jackson, W. M.; Nesti, L. J.; Tuan, R. S. Mesenchymal Stem Cell
Therapy for Attenuation of Scar Formation during Wound Healing.
Stem Cell Res. Ther. 2012, 3, 20.

16991 DOI: 10.1021/acsami.7b19007

ACS Appl. Mater. Interfaces 2018, 10, 16977−16991