Silk Sponges Ornamented with a Placenta-Derived Extracellular

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Biological and Medical Applications of Materials and Interfaces

Silk Sponges Ornamented with Placenta-Derived Extracellular Matrix Augments 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 ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b19007 • Publication Date (Web): 02 May 2018 Downloaded from http://pubs.acs.org on May 2, 2018

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ACS Applied Materials & Interfaces

1

Silk Sponges Ornamented with Placenta-Derived Extracellular Matrix

2

Augments Full-thickness Cutaneous Wound Healing by Stimulating

3

Neovascularization and Cellular Migration

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Arun Prabhu Rameshbabu¥, Kamakshi Bankoti¥, Sayanti Datta¥, Elavarasan Subramani$,

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Anupam Apoorva*, Paulomi Ghosh¥, Priti Prasanna Maity¥, Padmavati Manchikantiα, Koel

7

Chaudhury$ and Santanu Dhara¥#

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¥

Biomaterials and Tissue Engineering Laboratory

10

School of Medical Science and Technology

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Indian Institute of Technology Kharagpur

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Kharagpur–721302, India

13 14

$

15


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School of Medical Science and Technology

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*School of Bio Science

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21 22

α

23


24


School of Energy Science & Engineering

25 26

#corresponding author

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Dr. Santanu Dhara

28

E-mail: [email protected]

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ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract

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Regeneration of full-thickness wound without scar formation is a multifaceted process which

32

depends on in situ dynamic interactions between the tissue-engineered skin substitutes and

33

newly formed reparative tissue. However, the majority of the tissue-engineered skin

34

substitutes used so far in full-thickness wound healing cannot mimic the natural extracellular

35

matrix (ECM) complexity and thus are incapable of providing a suitable niche for

36

endogenous tissue repair. Herein, we demonstrated a simple approach to fabricate porous

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Hybrid Extracellular Matrix Sponges (HEMS) using Placental Extracellular Matrix (pECM)

38

and Silk Fibroin (SF) for full-thickness wound healing. HEMS with retained

39

cytokines/growth factors provided a non-cytotoxic environment in vitro for Human Foreskin

40

Fibroblasts (HFFs), Human Epidermal Keratinocytes (HEKs) and Human Amniotic

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Membrane-derived Stem Cells (HAMSCs) to adhere, infiltrate and proliferate. Interestingly,

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HEMS-conditioned media accelerated the migration of HFFs & HEKs owing to the presence

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of cytokines/growth factors. Also, HEMS ex-vivo Chick Chorioallantoic Membrane (CAM)

44

assay demonstrated its excellent vascularization potential by inducing and supporting blood

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vessels. Additionally, HEMS when subcutaneously implanted demonstrated no severe

46

immune response to the host. Furthermore, HEMS implanted in full-thickness wounds in a rat

47

model showed augmented healing progression with well-organized epidermal-dermal

48

junctions via pronounced angiogenesis, accelerated HFFs/HEKs migration, enhanced

49

granulation tissue formation and early re-epithelialization. Taken together, these findings

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show that porous HEMS ornamented with cytokines/growth factors having superior

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physicomechanical properties may be an appropriate skin substitute for full-thickness

52

cutaneous wounds.

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Keywords: Extracellular Matrix; Silk Fibroin; Cytokine/Growth Factor; Neovascularization;

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Cellular Migration; Full-thickness Wound Healing.

56

1. Introduction

57

Skin is the largest organ of the human body that serves the onerous function of providing

58

a physical shield against microorganisms, thermal regulation for average hydration retention,

59

sensory information about the external environment, and other vital functions.1 Skin injuries

60

can be caused by genetic disorders, acute trauma, chronic wounds, or by complex surgeries.2

61

In complex full-thickness wounds, epidermal layer along with dermal layer, sweat glands,

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hair follicles as well as the underlying subcutaneous fat tissue is damaged. Specifically,

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wounds of critical size hamper the crucial functions of the skin and can lead to complications

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such as microbial infection, a water-electrolyte imbalance in the body and severe cases that

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can be life-threatening.3 Therefore, skin injuries of critical size (greater than 1 cm in

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diameter) require bioactive support with clinical intervention to accelerate healing.4 Also,

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scarless wound healing is desirable for regaining tissue functionality as well as improved

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aesthetics. The gold standard treatment for full-thickness skin injuries is the autologous skin

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grafting.5 However, the limitations such as donor site morbidity and limited donor site

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availability have resulted in the need for the development of skin substitutes. In this context,

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bioengineered skin substitutes (hydrogels,6 sponges,7 and electrospun mats8) from natural

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polymers and proteins can solve the problem of autologous donor graft shortage, provide

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protection from fluid loss and contamination as well as can deliver bioactive factors such as

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cytokines, dermal matrix components, and growth factors to the wound bed for enhancing the

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host wound healing responses.4,9

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Silk fibroin (SF) from Bombyx mori, a versatile natural fibrous protein has been

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extensively explored as a skin substitute material in tissue engineering application owing to

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its biocompatibility, slow degradability, low immunogenicity, remarkable mechanical



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properties, and hemostatic action.10-12 However, SF alone is inadequate for dermal tissue

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regeneration since it lacks adequate cell-specific binding sites and limited growth factor

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adsorbing capacity.13 Thus, to enhance the properties of SF, it has been blended with various

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natural macromolecules such as gelatin,14 hyaluronic acid,15 collagen,16 chitosan,17 etc. For

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instance, scaffold fabricated from SF/elastin facilitated accelerated re-epithelialization in burn

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wounds.18 In another study, blend scaffold of SF/keratin significantly enhanced the cell

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adhesion/proliferation of L929 fibroblasts and increased extracellular matrix (ECM)

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deposition of collagen type I.19 Though the scaffolds mentioned above displayed superior

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cellular activities and ECM deposition, yet they lack a plethora of structural proteins, growth

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factors, and cytokines required for skin regeneration.

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In recent years, decellularized Extracellular Matrix (dECM) has emerged as an attractive

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biomaterial in regenerative medicine which provides abundant biological cues for cell

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migration, proliferation and further directing/promoting differentiation.20 The human placenta

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is a unique, complex organ that serves multiple functions to the developing fetus; also, it is a

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rich reservoir for a variety of growth factors and cytokines such as epidermal growth factor

94

(EGF), transforming growth factor-β (TGF- β), fibroblast growth factor (FGF), platelet-

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derived growth factor (PDGF), and vascular endothelial growth factor (VEGF), etc.21 These

96

growth

97

migration/proliferation,

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neovascularization that are considered essential for tissue repair and regeneration in the early

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stage of wound healing. Direct administration of human placental extract in the wound

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margin of full thickness wounds was found to accelerate the wound healing mechanism

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associated with an increase in TGF- β levels during the initial phase resulting in increased

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inflammatory cell infiltration, and increased VEGF levels during the later stages leading to

103

increased new blood vessel formation.22 Furthermore, the properties of the placenta, such as

factors/cytokines

play

an

mesenchymal

essential stem

cells

role

in

homing,


fibroblasts/keratinocytes re-epithelialization,

and

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low immunogenicity, anti-inflammatory, anti-scarring make it an ideal choice to treat full-

105

thickness skin wound.23

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In the present work, a Hybrid Extracellular Matrix Sponges (HEMS) was fabricated for

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skin tissue engineering by incorporating placenta-derived Extracellular Matrix (pECM) with

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SF; thus bringing together the inherent advantages of both SF and pECM. Collagen (major

109

ECM component of native skin) is considered to be the promising material of choice for

110

wound healing application owing to its biodegradability, superior biocompatibility, low

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antigenicity, non-toxic upon exogenous application, and high tensile strength. Therefore,

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collagen was selected as a representative control to demonstrate the superiority of pECM

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containing intrinsic cytokines/growth factors for wound healing. Hence, collagen

114

incorporated SF Matrix sponges (CIMS) was also fabricated for comparative studies. The

115

fabricated HEMS and CIMS were characterized for their physicomechanical properties and

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evaluated for their biocompatibility using the primary fibroblasts, keratinocytes, and

117

HAMSCs. In vitro wound healing/migration potential was evaluated by scratch assay and the

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ability to induce vascularization was assessed using Chick Chorioallantoic Membrane (CAM)

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model. Also, HEMS and CIMS were implanted subcutaneously in rats to study the host

120

immune reaction/toxicity in vivo. Further, HEMS and CIMS were assessed for their potential

121

in accelerating full-thickness wound healing using a rat model.

122

2. Materials and Methods

123 124

2.1. Decellularization of Human Placenta and Processing of Soluble Extracellular Matrix (pECM)

125

All the experimental procedures were approved by the Institutional Ethical Committee

126

of Indian Institute of Technology, Kharagpur, India. Decellularization of human placenta was

127

performed according to the protocol reported in our previous work.24 Briefly, the collected

128

placentas were repeatedly washed with Dulbecco's phosphate-buffered saline (DPBS) until

129

the blood was removed and then minced into small fragments by using a sterile scalpel. The



130

minced placenta was decellularized using 0.5% sodium dodecyl sulfate (SDS; Sigma-Aldrich,

131

USA) with 0.2% DNase (2000 U; Sigma-Aldrich, USA), 200mg/ml RNase (Sigma-Aldrich,

132

USA), 0.05% trypsin/EDTA (Gibco, USA), 100 U/ml penicillin, 100 mg/ml streptomycin

133

(Gibco, USA) and 1 mM phenyl methyl sulfonyl fluoride (Sigma-Aldrich, USA) in a sealed

134

rotating vessel. The procedure was conducted under strictly sterile conditions, and the

135

solution was changed every 24 h to prevent tissue degradation and contamination. The

136

decellularized placentas were washed with PBS and stored at -80 °C for further processing.

137

Solubilisation of extracellular matrix was performed as described elsewhere.25

138

Briefly, the decellularized human placenta was pulverized to a powder using tissue

139

homogenizer in the presence of liquid nitrogen. The resulting powder was immersed and

140

stirred in a 4 M urea buffer (240 g urea, 9 g NaCl, and 6 g Tris base in 1 L distilled water)

141

containing protease inhibitor cocktail for 24 h. Subsequently, the samples were subjected to

142

ultrasonic homogenization (Branson Ultrasonics, USA) in an ice bath. After centrifuging the

143

samples at 14000 rpm for 20 min at 4 °C, the supernatant was collected and then dialyzed

144

using 8000 MWCO dialysis tubing against Tris-buffered saline (TBS), i.e., 9 g NaCl and 6 g

145

Tris base in 1 L distilled water to remove urea. The contents of the dialysis tubes were

146

centrifuged again to remove protein aggregates, and then the supernatant was frozen at -80 °C

147

followed by freeze-drying for 42 h and pulverized to obtain pECM.

148

2.2. Collagen and GAGs Quantification

149

The total collagen content by hydroxyproline assay and sGAGs content was assessed

150

by alcian blue assay26 in native placenta (NP) and pECM as detailed in Supporting

151

Information S1.

152

2.3. Cytokine Array for Detection of Growth Factors


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Growth factors in NP and pECM were analyzed using a human cytokine antibody

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array (RayBio® C-Series C1000, USA) following the manufacturer’s protocol. Briefly, the

155

samples were dissolved at 4 °C for 36 h in the buffer containing 0.1X protease inhibitor, 2 M

156

urea, and 50 mM Tris-HCl. The supernatant was collected by centrifugation at 4 °C. The

157

obtained supernatant was incubated onto an array chip containing 120 different human

158

cytokine antibodies after blocking. The chip was then washed and incubated with biotinylated

159

antibody cocktail. Subsequently, the signals were detected using a chemiluminescence

160

detection system after HRP-Streptavidin incubation and washing.

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2.4. SDS-PAGE & Western Blotting

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The finely powdered NP and pECM were obtained by homogenizing in a buffer

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containing 65 mM DTT, 4 M guanidine HCl, protease inhibitor cocktail, 10 mM EDTA and

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50 mM sodium acetate (all Sigma-Aldrich, USA). The resulting mixture was exposed to

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ultrasonic homogenization, and the supernatant was collected after centrifugation (13000 rpm

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for 20 min at 4 °C). Bicinchoninic acid assay kit (Thermo Scientific, Rockford, USA) was

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used to quantify the total protein concentration of the homogenates according to the

168

manufacturer’s instruction. The extracted proteins from NP and pECM were run in 10 %

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PAGE and stained with Coomassie blue after fixing.

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The extracted proteins (40 µg) from NP and pECM after resolving in SDS-PAGE was

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transferred onto the nitrocellulose membrane (Millipore, USA) at 90 V for 2 h in the presence

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of a Tris-glycine buffer. The nitrocellulose membranes containing the transferred proteins

173

were treated with a primary antibody [Hepatocyte growth factor (HGF; Santa Cruz, USA),

174

TGF-β1 (Abcam, USA), VEGFA (Abcam, USA), EGF (Santa Cruz, USA), Insulin-like

175

growth factor-1 (IGF-1; Santa Cruz, USA) and PDGF-B (Santa Cruz, USA)] at 4 °C

176

overnight after blocking. The blots were subsequently incubated with HRP-conjugated



177

secondary antibody for 1 h. Pierce ECL-western blotting substrate kit (Thermo Scientific,

178

USA) was used for visualizing the immunoreactive proteins according to the manufacturer’s

179

instructions. Further, an automatic x-ray film processor was used to develop the images,

180

which were analyzed by ImageJ software (Rasband WS; NIH).

181

2.5. Preparation of Silk Fibroin (SF) Solution

182

Silk fibroin solution was prepared according to the procedure described elsewhere.27

183

Briefly, Bombyx mori cocoons were chopped into small pieces and boiled (98 °C) for 30 min

184

in an alkaline bath containing 0.02 M sodium carbonate (Sigma-Aldrich, USA) to obtain

185

fibroin and to remove the sericin/glycoproteins. The degummed silk was rinsed thoroughly

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with double distilled water and allowed to dry at room temperature overnight. The extracted

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fibroin was dissolved in 9.3 M LiBr (Sigma-Aldrich, USA) solution at 60 °C for 4 h followed

188

by dialysis for 72 hours against double distilled water using a dialysis membrane (3500

189

MWCO) to remove LiBr. The obtained fibroin solution was centrifuged (9,000 rpm at 4 °C

190

for 20 min) to remove large aggregates and the supernatant concentrated to a final

191

concentration of 10 % w/v by dialysis against polyethylene glycol (10,000 MW, Sigma-

192

Aldrich, USA). The concentrated fibroin solution was filtrated through a 5 µm syringe filter

193

(Millipore) to eliminate minor impurities.

194

2.6. Fabrication of Collagen Incorporated SF Matrix Sponges (CIMS) and Hybrid

195

Extracellular Matrix Sponges (HEMS)

196

Collagen type I was extracted from fish scales according to the procedure described

197

elsewhere28 and was grounded to powder in the presence of liquid nitrogen. Fibroin solution

198

and pECM was obtained as described above. For fabrication of CIMS/HEMS, 0.25 g of

199

pulverized collagen type I/pECM respectively was dispersed in 5 ml of 10 % w/v SF solution

200

under constant stirring for 24 h at 4 °C. The homogeneously mixed viscous solution was


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subjected to sonication in an ice bath for 2 min, gently poured into sterile moulds, frozen at -

202

80 °C and subsequently lyophilized for 72 h to obtain CIMS/HEMS. The lyophilized

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CIMS/HEMS were soaked in 70% (v/v) ethanol for 6 h to induce β-sheets formation in SF

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protein. Homogeneous distribution of pECM or collagen in SF matrix was assessed by

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Masson’s Trichrome (MT) staining as described in Supporting Information S3.

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2.7. Morphological Analysis by Scanning Electron Microscopy

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The microstructure of CIMS/HEMS was examined under scanning electron

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microscope (EVO ZEISS, Carl Zeiss SMT AG, Oberkochen, Germany) at an accelerating

209

voltage of 10-20 kV after fixing the samples with 4% paraformaldehyde. Briefly, the dried

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samples were placed on a sample holder using double-sided adhesive tapes and gold coated

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for 90 s using plasma coater under high vacuum to avoid charging effect.

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2.8. Porosity Measurement by Micro-Computed Tomography (Micro-CT)

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CIMS/HEMS were scanned (1000 scan slices/sample) using a micro-CT (GE Phoenix

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v|tom|ex s, Germany) at a voltage of 155 kV, current 45 mA with a voxel of 7.2 microns.

215

When analyzing the porosity of the samples, the threshold to be used was obtained for each

216

specimen by the threshold histogram offered by the VG Studio Max software (Volume

217

Graphics Germany).

218

2.9. Swelling Behaviour and Degradation Kinetics

219

The swelling behavior of CIMS and HEMS was examined for 64 h at 37 °C in sterile

220

PBS (pH 7.4). At predetermined time intervals, the excess PBS in samples was removed by

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gently wiping with filter paper and then instantly weighed in electronic balance (Mettler-

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Toledo International Inc., USA) to calculate the PBS content in swollen CIMS/HEMS.

223

Swelling ratio was calculated using the formula below:



Swelling ratio (%) =

224

× 100

225

Where Td and Ts are the dry and wet weight of CIMS/HEMS respectively. The experiments

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were conducted in triplicate and averaged.

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In vitro degradation of CIMS/HEMS (n=5) was performed in PBS containing 1U/ml

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of lysozyme (hen egg-white, Sigma-Aldrich), pH 7.4 at 37 °C for a specified period

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according to the protocol described in our previous publication.29

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2.10. Fourier Transform Infrared (FTIR) Spectroscopy

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Infrared spectra of CIMS and HEMS were obtained using a Thermo Nicolet

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Spectrophotometer (Model NEXUS-870; Thermo Nicolet Corporation, Madison, WI) in ATR

233

mode. The absorbance of spectra was recorded in the range of 4000 to 500 cm-1.

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2.11. Mechanical Testing

235

The mechanical properties of the samples (n=10) were determined by tensile testing

236

using the universal testing machine (25 K, Hounsfield, UK) at room temperature with 20 N

237

load cell. The samples were cut into rectangular strip (~ 5 mm width, ~ 20 mm length,

238

~ 1 mm thickness) and soaked in PBS for 48 h. Uniaxial tensile testing was performed under

239

tension at a crosshead speed of 2 mm/min.

240

2.12. Isolation of Human Amniotic Mesenchymal Stem Cells (HAMSCs)/Human

241

Foreskin Fibroblast (HFF)/ Human Epidermal Keratinocytes (HEK)

242

HAMSCs were isolated and characterized according to the protocol reported in our

243

previous publication.24 Briefly, the human placentas collected under stringent sterile

244

conditions were transported to the laboratory at 4 °C. The blood components were removed

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by rinsing with Hank's Balanced Salt Solution (Gibco, USA) containing 200 mg/ml

246

streptomycin and 200 U/ml penicillin. The amniotic membrane was detached from chorion


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247

by a blunt dissection and was incubated with 0.05 % trypsin–EDTA solution (Gibco, USA)

248

for two cycles of 30 min each, followed by discarding the supernatant. Subsequently, the

249

tissue was digested with Earle's Balanced Salt Solution (Gibco, USA) containing 2 mg/ml

250

collagenase Type IV (Gibco, USA) and 10 U/ml DNase I (Sigma-Aldrich, USA) for 60 min.

251

After the end of the digestion, the suspension was centrifuged at 1500 rpm to collect the cell

252

pellet. The cell pellet was seeded in flasks (Nunc, USA) after suspending it in DMEM

253

medium (containing low glucose, 10% FBS, 1% Antibiotic-Antimycotic) and transferred to

254

an incubator. TrypLE™ Express Enzyme (Gibco, USA) was used to sub-passage the cells

255

and cells before passage number five were used for experiments.

256

HFFs and HEKs were isolated as described elsewhere from circumcised human

257

foreskin.30 Briefly, skin samples were collected in sterile containers and rinsed in PBS

258

containing 200 mg/ml streptomycin and 200 U/ml penicillin. Later, the samples were

259

chopped into small pieces and incubated in Dispase II (Sigma-Aldrich, USA) for overnight at

260

4 °C. Subsequently, epidermal and dermal layers were separated and incubated in TrypLE™

261

Express Enzyme/collagenase I (Gibco, USA), respectively for further digestion. The

262

corresponding cell suspension was strained and the cell pellet was obtained by centrifugation.

263

Defined Keratinocyte-SFM media (Gibco, USA)/DMEM high glucose media (Gibco, USA)

264

were used for culturing HEK/HFF respectively. Cells after reaching 80% confluency were

265

passaged with TrypLE™ Express and cells before passage five were used for further

266

experiments.

267

2.13. Indirect Cytotoxicity Testing

268

2.13.1. Preparation of HEMS/CIMS Conditioned Medium

269

HEMS/CIMS in the presence of liquid nitrogen was grounded to a fine powder with a

270

sterile porcelain mortar and pestle. The resulting HEMS/CIMS powder (25 mg/ml) was



271

added to a complete DMEM/Defined Keratinocyte-SFM medium with 1% Antibiotic-

272

Antimycotic in a sterile container and stirred inside an incubator at 37 °C and 5 % CO2 for 24

273

h. Conditioning material was removed from the media by filtering the medium through a 0.22

274

µm syringe filter (Millipore, USA).

275

276

2.13.2. Morphological and Apoptosis Assessment

277

Primary HFFs and HEKs were used for investigating the effect of CIMS/HEMS

278

conditioning medium on their morphological and functional attributes. Briefly, HFFs/HEKs

279

were cultured either with CIMS/HEMS-conditioned or normal medium on lysine coated

280

coverslips placed in a 12-well plate. The coverslips were removed after 96 h, stained with

281

Rhodamine phalloidin (Invitrogen, USA) and DAPI (Invitrogen, USA) according to the

282

manufacturer’s instructions. Apoptosis of HEKs in the presence of CIMS/HEMS-conditioned

283

media was also investigated after 7 days of cultivation using the DeadEnd Fluorometric

284

TUNEL system (Promega, USA) according to the manufacturer’s guidelines.

285

2.13.3. Scratch Assay

286

Scratch assay for wound healing was performed according to the protocol described

287

elsewhere.31 HFFs/HEKs were cultured in 35 mm Petri dishes (Tarsons, India) in

288

DMEM/Defined Keratinocyte-SFM medium at 37 °C in a 5% CO2 in an air atmosphere to

289

produce confluent monolayers. To mimic wounds, a vertical scratch was made in the center

290

of the monolayer HFFs/HEKs using a sterile 200 µl pipette tip, and subsequently, the Petri

291

dishes were washed with sterile PBS to remove the detached HFFs/HEKs. Later, the Petri

292

dishes were filled with either CIMS/HEMS-conditioned medium or control medium, and the

293

Petri dishes were incubated at 37 °C in a 5 % CO2 for 72 h. The migration of HFFs/HEKs in

294

the denuded path was monitored by taking optical images using an inverted phase contrast

295

microscope (Carl Zeiss AxioObserver) at the different period, and three independent


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296

observers counted the number of cells that had migrated into the initially cell-free scratch

297

area.

298

2.14. Analysis of HEMS Conditioned Medium by ELISA

299

The quantitative direct enzyme immunoassay was performed to estimate the levels of

300

Interleukin-6 (IL-6), Interleukin-8 (IL-8), monocyte chemotactic protein-1 (MCP-1), TGF-β1

301

and RANTES in HEMS conditioned medium. The protein concentration of 20 µg/ml was

302

used for antigen binding in poly-L-lysine coated 96-well plates. Following blocking with 3%

303

BSA, wells were incubated with antibodies against IL-6, IL-8, MCP-1, TGF-β1 and

304

RANTES (Santa Cruz Biotechnology, Inc., USA). All wells were washed with PBST and

305

incubated with a suitable secondary antibody conjugated with HRP (Santa Cruz

306

Biotechnology, Inc. CA, USA). After washing with PBST, 100 µl of Femto-ELISA-HRP

307

substrate (G-Biosciences, USA) was added to each well and incubated for 10-15 min. The

308

reaction was stopped with 1N HCl and absorbance was measured at 450 nm.

309

2.15. Direct Cytotoxicity Testing

310

Cytotoxicity was tested by the direct cultivation of HAMSCs/HEKs/HFFs on TCP/

311

CIMS/HEMS. Briefly, 5 x 104 HAMSCs/HFFs/HEKs were seeded on samples (n=5) and

312

cultivated in a 24-well tissue culture plates for 72 h. The number of viable cells was

313

calculated using Vybrants MTT Cell Proliferation Assay Kit (Invitrogen, USA) according to

314

the manufacturer’s instructions.

315

The viability of HEKs seeded on CIMS and HEMS were assessed using Live/Dead

316

Assay Kit (Life Technologies, NY) according to the manufacturer’s protocol. Brieﬂy, cell-

317

seeded CIMS/HEMS after 7 days were incubated for 60 min at 37 °C in a solution containing

318

2 µM calceinacetomethoxy (AM) and 4 µM ethidium homodimer. The samples were



319

repeatedly washed with DPBS to avoid background staining and visualized under an inverted

320

ﬂuorescence microscope (AxioVision, Zeiss, Germany).

321

2.16. Chick Chorioallantoic Membrane (CAM) Assay

322

CAM assay was used as an ex vivo model to assess the potential of CIMS/HEMS to

323

induce vascularization. In this study, fertilized white Leghorn chicken eggs were used for

324

CAM assay by following the procedure described elsewhere.32 Briefly, the eggs were

325

incubated at 37 °C, 65% relative humidity egg incubator. On day 5, disks (5 mm diameter;

326

n=5) of CIMS and HEMS were punched, sterilized and hydrated with sterile PBS overnight.

327

The sterile disk of CIMS and HEMS was placed over CAM respectively; later the eggshell

328

was sealed. On day 8, the window was carefully dissected, and CIMS/HEMS were

329

photographed in situ. Three blind observers counted the number of blood vessels approaching

330

towards the scaffolds. Further, the CIMS/HEMS along with surrounding membrane were

331

retrieved carefully with forceps without getting tore off, fixed in 4 % paraformaldehyde,

332

dehydrated and subsequently embedded in paraffin blocks. The paraffin blocks were

333

sectioned and stained with hematoxylin and eosin (H & E; Sigma-Aldrich, USA), Masson's

334

trichrome (MT; Sigma-Aldrich, USA) and DAPI.

335

2.17. In vivo CIMS/HEMS Cellular Response and Organ Toxicity Analysis

336

In vivo cellular response of CIMS/HEMS was studied by subcutaneous implantation

337

in albino Wistar rats (250 ± 10 g; Males; n=5). The experimental protocol followed was

338

permitted by the Institutional Ethical Committee of Indian Institute of Technology

339

Kharagpur, and all surgery was performed under anesthesia. Extreme caution was taken to

340

minimize suffering to the animals. Cylindrical CIMS/HEMS (diameter = ~1 cm; height =

341

~0.5 cm) were implanted on the dorsal side of rats after dissection and sutured using chromic

342

catgut under aseptic condition. After 28 days of implantation, rats were euthanized, and the


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343

site of implantation along with the adjacent tissue was retrieved for further analysis. The

344

harvested samples were fixed, dehydrated and subsequently embedded in paraffin blocks.

345

The paraffin blocks containing sample were microtomed, and the sections were then stained

346

with H&E, MT, toluidine blue (TB; Sigma-Aldrich, USA) and anti-CD31 antibody (Abcam)

347

staining according to the manufacturer’s instructions. All the sections were observed under a

348

microscope to understand the cellular events. For organ toxicity analyses, the major organs

349

(Heart, Kidney, Lung, and Liver) were harvested (n=3 per group) after 28 days of the

350

treatment period of different groups and processed for histological assessment of the tissue.

351

352

2.18. Full-thickness Cutaneous Wound Healing Study

353

The in vivo experimental protocols were approved by the Institutional Ethical

354

Committee of Indian Institute of Technology Kharagpur. Albino Wistar rats (2 months old;

355

200 ± 10 g) were used to evaluate the full-thickness skin wound healing capacity of the

356

different groups. Randomly, the rats were divided into three groups (n=9 each group)

357

according to the treatments received on each rat. Three treatment groups were as follows (1)

358

SHAM: wounds that were left untreated; (2) CIMS and (3) HEMS were applied into the

359

wound exclusively within the wound area. Before surgery, 2 cm diameter wounds were

360

marked using a template, and standardized full-thickness skin wounds were made on dorsal

361

side by excising to the level of the fascia, by using sterile forceps and scissors under general

362

anesthesia. Petrolatum gauze pressure dressing was applied to all the groups, which prevented

363

the samples from moving away from the wound bed. Care was taken to ensure that the

364

pressure dressing was not detached during the twenty-one day treatment period. The rats were

365

individually housed in temperature-controlled cabins and fed with a standard protein diet.

366

The cutaneous wounds were photographed after 0, 7, 14, and 21 days and the unhealed area



Page 16 of 52

367

was measured using ImageJ (Rasband WS; NIH) to assess the wound healing kinetics. The

368

percentage of wound reduction was calculated according to the following formula: Rate of Wound Closure =

369

370 371

× 100

Where, A0 and At are designated to initial wound area and wound area at the designated time, respectively.

372

Rats were sacrificed after 7, 14, 21 days post-implantation, and the wound site with

373

the surrounding muscle & skin was retrieved. They were then immediately fixed with 4%

374

formaldehyde, dehydrated and embedded in a paraffin block. Sections of 3 µm thickness

375

were cut and stained with H & E and MT. Further, the sections were also immune-stained

376

with anti-CD31 and anti-cytokeratin 10 (CK-10; Abcam, USA) according to the

377

manufacturer’s instruction. Photomicrograph of different stains was captured and examined

378

to document cardinal features like an inflammatory response, necrosis, neovascularization,

379

collagen deposition, granulation, and re-epithelialization to understand healing progression at

380

different periods.

381

2.19. Reverse Transcriptase-PCR (RT-PCR) Analyses of the Wound Area

382

RT-PCR was performed on newly formed tissue in the wound area from different

383

treatment groups, 21 days post-surgery. The regenerated tissues (n=5 per group) from

384

different study groups were excised from the animal after sacrificing and ground to a powder

385

using liquid nitrogen. The total RNA was extracted using TRIzol reagent (Invitrogen, USA)

386

according to the manufacturer’s instructions. An equal quantity of isolated RNA was

387

transcribed into cDNA using a cDNA synthesis kit (Thermo Scientific, USA) according to

388

the manufacturer’s protocol. PCR amplification of the gene-specific primers (Supporting

389

Information Table S1) was performed in a thermal cycler (Eppendorf Mastercycler, USA)


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390

and the PCR product was imaged in UV gel doc (Bio-Rad, USA) after running it in 1%

391

agarose gels. The band intensity was analyzed using ImageJ (Rasband WS; NIH).

392

2.20. Statistical Analysis

393

The data were analyzed using GraphPad Prism software (version 5.02, La Jolla, CA,

394

USA) by one-way ANOVA, Tukey's multiple comparison tests. The level of significance was

395

determined as P < 0.05 significance. Experiments were repeated in triplicates and data were

396

represented as mean ± standard deviation (SD) for n=3 unless mentioned.

397

398

399

3. Results

400

3.1. Biochemical Characterization of Soluble Placenta Extracellular Matrix (pECM)

401

The native placenta (NP) was subjected to decellularization using a combinatorial

402

treatment involving homogenization, centrifugation, SDS, and nucleases. For the preparation

403

of soluble placenta extracellular matrix proteins (pECM), the ECM from the decellularized

404

placenta was solubilized using urea. To estimate the biochemical preservation in pECM after

405

the decellularization and the solubilization process, pECM was quantified for major ECM

406

components such as collagen and sGAG. As observed in Supporting Information (Figure S1),

407

retention of collagen (409.8 ± 45.44 µg mg-1) and sGAG (38.45 ± 7.42 µg mg-1) in pECM

408

after the decellularization and solubilization process, was not significantly (P > 0.05)

409

different from the collagen (460.4 ± 18.622 µg mg-1) and sGAG ( 53.59 ± 14.41 µg mg-1)

410

content of NP. These results demonstrated that the major ECM components were sufficiently

411

retained in pECM after the decellularization and solubilization process which will make it an

412

ideal matrix for tissue engineering application.



413

3.2. Cytokine Array for Detection of Growth Factors

414

pECM and NP were arrayed on a glass chip containing 120 different cytokine

415

antibodies for detection of the retained endogenous cytokines. As shown in Figure 1, 84

416

cytokines were readily detected in pECM, and the remaining 36 cytokines were not detected,

417

which may be due to the sensitivity limitations of the array. Among the 84 detected bioactive

418

molecules, cytokines [EGF, IGF-1, PDGF-B, HGF, VEGFA, TGF-β1, and FGF] which are

419

considered critical for regulating the angiogenesis and skin wound healing were detected at

420

high levels. Also, growth factors such as IL-6, IL-8, MCP-1, and RANTES; which are

421

reported to accelerate fibroblast and keratinocyte migration and proliferation was also

422

detected in pECM.

423

3.3. SDS PAGE and Western Blotting of NP and pECM

424

SDS-PAGE was used to investigate the proteins retained in pECM. Figure 2A reveals

425

some bands at lower molecular weights signifying the presence of various proteins/peptides

426

in the pECM, which demonstrates the complexity of the pECM. Western blotting was

427

performed to confirm the presence of endogenous bioactive molecules which are known to

428

regulate blood vessel formation and skin regeneration. Figure 2B reveals the presence of

429

retained growth factors such as EGF, IGF-1, PDGF-B, HGF, VEGFA, and TGF-β1 after the

430

decellularization and solubilization process in pECM. From Figure 2C, no significant

431

difference (P > 0.05) was observed in the levels of EGF, PDGF-B, TGF-β1, and VEGFA in

432

pECM compared to the NP. However, a significant decrease (P < 0.05) was observed in the

433

levels of IGF-1 and HGF in pECM compared to NP (Figure 2C).

434

3.4. Physicochemical Characterization of Scaffolds

435

In this study, collagen I/pECM was blended with SF in aqueous environment and

436

successfully fabricated into CIMS/HEMS respectively by molding and freeze-drying process.


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437

The SEM microstructure of CIMS and HEMS are shown in Figure 3A and C which revealed

438

a high degree of interconnected heterogeneous porous structures within the matrices. The

439

porosity of the samples analyzed by Micro-CT was approximately similar for CIMS (85.27

440

%) and HEMS (90.2%) as shown in Figure 3B and D. The swelling behavior of the scaffolds

441

is critical not only for arbitrating the rate of nutrition and waste transport within the scaffolds

442

in situ but it also helps in biological fixation to the wound bed. As observed in Figure 4A,

443

CIMS and HEMS demonstrated 55% and 64% swelling respectively when exposed to PBS

444

for 0.5 h due to the rapid uptake of water. After this time; it attained a plateau and no further

445

increase in mass was observed until 64 h. Extended long-term swelling observations are

446

essential for more critical assessment. As shown in Figure 4B, CIMS (mass loss 97%)

447

exhibited faster degradation as compared to HEMS (mass loss 90%) when incubated in

448

lysozyme at 37 °C for 16 days. Also, CIMS and HEMS when incubated in PBS (pH 7.4)

449

without lysozyme for 48 h showed a negligible mass loss (data not shown) and displayed a

450

wet tensile strength of 0.065 ± 0.004 MPa and 0.076 ± 0.003 MPa (P > 0.05; Figure 4D).

451

Infrared absorption spectra (Figure 4C) of CIMS and HEMS showed characteristic

452

absorption peaks assigned to the peptide bonds (–CONH–) that give rise to amide I (1600–

453

1700 cm1), amide II (1520–1540 cm1), and amide III (1220–1300 cm1) signature peaks. Pure

454

silk scaffolds showed amide I, amide II and amide III bands at 1617 cm-1, 1511 cm-1, and

455

1226 cm-1; CIMS scaffolds showed amide I, amide II and amide III bands at 1636 cm-1, 1528

456

cm-1, and 1226 cm-1 whereas HEMS scaffolds showed the characteristic peaks of amide I,

457

amide II and amide III bands at 1623 cm-1, 1533 cm-1, and 1222 cm-1. Also in CIMS and

458

HEMS peak at 2869-1 corresponds to C-H symmetric stretching. Notably, in CIMS the 1636

459

cm–1 band is characteristic of the triple helix of native collagen, and thus suggests the

460

existence of collagen in the native triple helical structures in the CIMS scaffold.

461

3.5. Indirect Cytotoxicity Testing for CIMS and HEMS



462

3.5.1. Morphological and Apoptosis Assessment

463

HFFs and HEKs were cultivated on lysine coated coverslips with either CIMS/HEMS

464

conditioning medium or control medium for 96 h. As shown in Figure 5A, HFFs/HEKs

465

grown in HEMS-conditioned medium proliferated rapidly and were able to make intermittent

466

contact via cellular protrusions and extensions with well-spread cytoskeletons compared to

467

those of HFFs/HEKs cultured in CIMS-conditioned/control medium. Also, there was no

468

difference in the morphology of HFFs/HEKs cultured in the CIMS-conditioned medium

469

compared with the cells cultured in control medium. These observations demonstrated that

470

CIMS/HEMS did not release cytotoxic substances.

471

TUNEL assay was performed to detect apoptosis after 7 days of HEKs cultivation

472

either in control or CIMS/HEMS-conditioned medium. As shown in Figure 5B, no significant

473

apoptotic HEKs was observed in control or CIMS/HEMS-conditioned medium treated

474

groups; indicating that the CIMS/HEMS-conditioned medium had no detrimental effect on

475

cells.

476

3.5.2. Scratch Assay HFFs started to migrate from the edges of the denuded/wounded areas within 6 h

477 478

while

HEKs migration was witnessed after 12 h in both the treatment (CIMS/HEMS-

479

conditioned medium) and control groups (Figure 6A and B). As determined by three

480

independent observers, the number of HFFs that migrated into the scratch areas for HEMS-

481

conditioned, CIMS-conditioned, and control group were found to be (6 h): 134 ± 06, 86 ± 12,

482

50 ± 05 and (12 h): 295 ± 16, 160 ± 14, 110 ± 06 respectively (Figure 6C). Similarly, the

483

number of HEKs that migrated into the scratch areas for HEMS-conditioned, CIMS-

484

conditioned, and control group were (12 h): 313 ± 11, 208 ± 12, 81 ± 06 and (24 h): 654 ± 16,

485

355 ± 9, 268 ± 08, respectively (Figure 6D). It can be interpreted from Figure 6C and D, the


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486

number of migrated HFFs/HEKs was more significant (p 0.05). Y-error bars represent standard deviation. 126x94mm (300 x 300 DPI)


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Figure 5: (A) Rhodamine Phalloidin-DAPI images of HFFs and HEKs cultivated in CIMS and HEMS-conditioned medium (scale bar represents 50 µm, Red represents nucleus staining DAPI and green depicts cytoskeleton expression); (B) TUNEL assays images of HEKs cultivated in CIMS and HEMS conditioned medium (scale bar represents 50 µm). 213x268mm (300 x 300 DPI)



Figure 6: Scratch assay images of (A) HFFs and (B) HEKs cultivated in CIMS and HEMS-conditioned medium at different time duration (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. 119x84mm (300 x 300 DPI)


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Figure 7: (A) Growth factors and cytokines detected in HEMS-conditioned medium by ELISA multi-array; (B) Cellular metabolic activity of HFFs, HEKs and HAMSCs cultured in CIMS/HEMS according to MTT assay; Livedead cell staining of HEKs cultivated in (C) CIMS and (D) HEMS. Y-error bars represent standard deviation and scale bar represents 50 µm. 125x92mm (300 x 300 DPI)



Figure 8: Pro-angiogenic property analysis of CIMS and HEMS by CAM assay; (A) Macroscopic view, (B) Quantification of vessels converging towards 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. 182x197mm (300 x 300 DPI)


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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). 114x77mm (300 x 300 DPI)



Figure 10: Healing progression of full-thickness cutaneous wounds treated with SHAM, CIMS and HEMS; (A) Photographs of wounds and (B) The rate of wound closure on days 0, 7, 14, and 21; (C) RT-PCR analysis of skin wounds treated with SHAM, CIMS and HEMS 21 days post-wounding. Y-error bars represent standard deviation, single asterisks signify P < 0.05, and triple asterisks signify P < 0.001. 99x47mm (300 x 300 DPI)


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Figure 11: Histological micrographs of wound sections implanted with CIMS and HEMS at day 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). 84x33mm (300 x 300 DPI)



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). 104x51mm (300 x 300 DPI)


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Figure 13: Representative Immunohistochemistry images of CK-10 stained histological sections on day 14 and 21 of SHAM, CIMS and HEMS (scale bar represents 50 µm; Red represents nucleus staining DAPI and Green depicts antibody expression). 86x44mm (300 x 300 DPI)



Graphical Abstract 192x218mm (300 x 300 DPI)


Page 52 of 52

Silk Sponges Ornamented with a Placenta-Derived Extracellular

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