Silk Sponges Ornamented with a Placenta-Derived Extracellular

May 2, 2018 - Eng. Data, J. Chem. Educ. .... In the present work, a hybrid ECM sponge (HEMS) was fabricated for skin tissue engineering by incorporati...
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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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Silk Sponges Ornamented with Placenta-Derived Extracellular Matrix

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Augments Full-thickness Cutaneous Wound Healing by Stimulating

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Neovascularization and Cellular Migration

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

6

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

7

Chaudhury$ and Santanu Dhara¥#

8 9

¥

Biomaterials and Tissue Engineering Laboratory

10

School of Medical Science and Technology

11

Indian Institute of Technology Kharagpur

12

Kharagpur–721302, India

13 14

$

15

Indian Institute of Technology Kharagpur

16

Kharagpur–721302, India

School of Medical Science and Technology

17 18

*School of Bio Science

19

Indian Institute of Technology Kharagpur

20

Kharagpur–721302, India

21 22

α

23

Indian Institute of Technology Kharagpur

24

Kharagpur–721302, India

School of Energy Science & Engineering

25 26

#corresponding author

27

Dr. Santanu Dhara

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E-mail: [email protected]

29

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Abstract

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

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

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

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

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

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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)

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

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

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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)

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assay demonstrated its excellent vascularization potential by inducing and supporting blood

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vessels. Additionally, HEMS when subcutaneously implanted demonstrated no severe

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

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

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

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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

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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.

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1. Introduction

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

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

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

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

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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

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(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

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growth

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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

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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,

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fibroblasts/keratinocytes re-epithelialization,

and

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

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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

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

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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

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

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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

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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

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

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

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2. Materials and Methods

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

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All the experimental procedures were approved by the Institutional Ethical Committee

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

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

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

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

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

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

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

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

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

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

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

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Solubilisation of extracellular matrix was performed as described elsewhere.25

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

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

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

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

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

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

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

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

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

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

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2.2. Collagen and GAGs Quantification

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

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

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Information S1.

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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

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

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

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

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

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

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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

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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

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

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

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

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

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

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

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

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

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2.5. Preparation of Silk Fibroin (SF) Solution

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

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

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

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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

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

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

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

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

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

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(Millipore) to eliminate minor impurities.

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

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Extracellular Matrix Sponges (HEMS)

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

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

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

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

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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 -

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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

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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.

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

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

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Graphics Germany).

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2.9. Swelling Behaviour and Degradation Kinetics

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

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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.

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Swelling ratio was calculated using the formula below:

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× 100

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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

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

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2.11. Mechanical Testing

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

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

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

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

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tension at a crosshead speed of 2 mm/min.

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

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

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

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

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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

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

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

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

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

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

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

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

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

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

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and cells before passage number five were used for experiments.

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HFFs and HEKs were isolated as described elsewhere from circumcised human

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

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

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

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

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

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

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

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

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

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experiments.

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2.13. Indirect Cytotoxicity Testing

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2.13.1. Preparation of HEMS/CIMS Conditioned Medium

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

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

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

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

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

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µm syringe filter (Millipore, USA).

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2.13.2. Morphological and Apoptosis Assessment

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

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

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

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

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

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

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

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

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2.13.3. Scratch Assay

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

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

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

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

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

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

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

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

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

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

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

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area.

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2.14. Analysis of HEMS Conditioned Medium by ELISA

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

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

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

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

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

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

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

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

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

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

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2.15. Direct Cytotoxicity Testing

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

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

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

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

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the manufacturer’s instructions.

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

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

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

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

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

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fluorescence microscope (AxioVision, Zeiss, Germany).

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2.16. Chick Chorioallantoic Membrane (CAM) Assay

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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permitted by the Institutional Ethical Committee of Indian Institute of Technology

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

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

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

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

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

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

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

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

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

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

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

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

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2.18. Full-thickness Cutaneous Wound Healing Study

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The in vivo experimental protocols were approved by the Institutional Ethical

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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× 100

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

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

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

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

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

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

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

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

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

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different periods.

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

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

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

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

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

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

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

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

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

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

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

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2.20. Statistical Analysis

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

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

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

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

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3. Results

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

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

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

403

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

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

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

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

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

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

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

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

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

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ideal matrix for tissue engineering application.

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3.2. Cytokine Array for Detection of Growth Factors

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

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

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

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

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

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

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

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reported to accelerate fibroblast and keratinocyte migration and proliferation was also

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detected in pECM.

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3.3. SDS PAGE and Western Blotting of NP and pECM

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

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

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

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

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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

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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

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

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3.4. Physicochemical Characterization of Scaffolds

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

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

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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

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

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

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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

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

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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)

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

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

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

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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

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

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

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

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

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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.

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3.5. Indirect Cytotoxicity Testing for CIMS and HEMS

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3.5.1. Morphological and Apoptosis Assessment

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

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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

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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

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

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

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

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cells.

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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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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)

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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 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)

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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)

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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) 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)

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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)

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Graphical Abstract 192x218mm (300 x 300 DPI)

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