Dual Functionalized Injectable Hybrid Extracellular Matrix Hydrogel for Burn Wounds

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Dual Functionalized Injectable Hybrid Extracellular Matrix Hydrogel

for Burn Wounds

Kamakshi Bankoti, Arun Prabhu Rameshbabu, Sayanti Datta, Piyali Goswami, Madhurima Roy,

Dipankar Das, Sudip Kumar Ghosh, Amit Kumar Das, Analava Mitra, Sagar Pal, Dhrubajyoti Maulik,

Bo Su, Paulomi Ghosh, Bikramajit Basu, and Santanu Dhara *

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ABSTRACT: Low strength and rapid biodegradability of acellular dermal matrix

(ADM) restrict its wider clinical application as a rapid cell delivery platform in situ

for management of burn wounds. Herein, the extracted ADM was modiﬁed by a dual

cross-linking approach with ionic crosslinking using chitosan and covalent cross-

linking using an iodine-modiﬁed 2,5-dihydro-2,5-dimethoxy-furan cross-linker,

termed as CsADM-Cl. In addition, inherent growth factors and cytokines were

found to be preserved in CsADM-Cl, irrespective of ionic/covalent crosslinking.

CsADM-Cl demonstrated improvement in post crosslinking stiﬀness with a

decreased biodegradation rate. This hybrid crosslinked hydrogel supported adhesion,

proliferation, and migration of human foreskin-derived ﬁbroblasts and keratinocytes.

Also, the angiogenic potential of CsADM-Cl was manifested by chick chorioallantoic

membrane assay. CsADM-Cl showed excellent antibacterial activity against

Escherichia coli and Staphylococcus aureus. Moreover, CsADM-Cl treated full

thickness burn wounds and demonstrated rapid healing marked with superior angiogenesis, well-deﬁned dermal−epidermal

junctions, mature basket weave collagen deposition, and development of more pronounced secondary appendages. Altogether, the

bioactive CsADM-Cl hydrogel established signiﬁcant clinical potential to support wound healing as an apt injectable antibacterial

matrix to encounter unmet challenges concerning critical burn wounds.

application.^6,7Also, hydrogels with antibacterial activity are

1. INTRODUCTION

widely explored, either by encapsulating antibacterial mole-

Burn injuries, a multifaceted acute wound, represent complex

cules into the hydrogel or self-assembling alternate Lys−Val

inﬂammatory progression, that is, intense local inﬂammation

peptides. However, the former causes burst release of

marked by a pronounced propensity to infections. Especially,

antibiotics, whereas in case of latter, the high cost of

full thickness (FT) wounds of critical size (diameter >1 cm),

production, complex preparation protocol, and poor tenability

induced by burn, result in rapid loss of liquid and also curtail

limit their application.^8,9Iodine is reported to exhibit broad-

various vital functions of the skin, leading to the development

spectrum antimicrobial properties and hence is being used

of chronic wounds. Hence, these critical wounds are prone to

frequently in clinics as an antiseptic and antimicrobial agent.¹⁰

complications, such as secondary necrosis.¹Further, healing

Notably, the potential of bacterial resistance is signiﬁcantly

progression is impeded by restricted blood ﬂow and complex

reduced due to the alternative mechanism of antimicrobial

inﬂammatory reactions, which are stimulated by an initial burn

activity of iodine.¹¹For instance, iodine conjugated with

injury.²To add up to this slowed down regeneration, these

polyvinylpyrrolidone known as povidone-iodine has been

wounds are susceptible to microbial infections and lead to

demonstrated to exhibit antimicrobial property, even against

scarring upon healing, thus complicating the treatment

methicillin-resistant strains.¹²Altogether, there is an urgent

modalities.³

clinical need to develop simple techniques to tackle the

Hydrogels with in situ gelation property are preferred as a

stringent pathophysiological requirements of chronic injuries

skin substitute for burn wound management due to their

minimal invasiveness and ability to ﬁll the irregular tissue

defects. Hydrogels are capable of lending a moist environment

that accelerates cell proliferation and collagen deposition in the

Received: September 28, 2020

Revised: November 20, 2020

Published: December 8, 2020

wound bed along with the delivery of bioactive target

molecules.^4,5Bioactive molecules like hormones, cytokines,

growth factors, and peptides are reported to promote healing

progression. However, their high cost of production limits their

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to induce rapid wound closure and to minimize scar

development.

physio-mechanical attributes and subsequently assessed for

cytocompatibility using human foreskin-derived ﬁbroblasts

(HFCs) and keratinocytes (HKC). Wound healing and

angiogenic potential of hydrogels were evaluated in vitro

using scratch and chick chorioallantoic membrane (CAM)

assay. Wound regeneration was analyzed through histological

observations, marker protein expressions, and immunostaining

of the wound tissue across days of recovery. Furthermore, neo-

blood vessel formation, collagen organization, and skin

appendage formation were determined to evaluate the eﬀect

of bioactive cross-linked hybrid hydrogel treatment in the

wound repair process.

Chitosan (CTS) dressings have been used widely for wound

healing purposes owing to their excellent biocompatibility,

biodegradability, antibacterial property, hemostatic nature, and

also for drug/biomolecule delivery.^13,14CTS resembles

glycoaminoglycan (GAGs) in structure; however, CTS lacks

bioactive signals equivalent to those existing in the extracellular

matrix (ECM) for cell attachment, growth, and diﬀerentiation.

The decoration of cytokines, growth factors, signaling

molecules, proteins, or small cell-binding peptides onto the

polymer surface is used as a strategy to enhance as well as to

direct cell responses on biomaterial surfaces.¹⁵⁻²⁰In

comparison to synthetic matrices that fail to mimic the

complexity of the ECM in vivo, biological scaﬀolds composed

of the decellularized ECM (dECM) embody structures akin to

the host tissue with superior properties such as a natural 3D

morphology, RGDs promoting cell adhesion and proliferation,

and inherent biodegradability. The dECM has been reported

to have potential to guide cell diﬀerentiation into appropriate

tissue type structures and phenotype.²¹⁻²³Further, the dECM

supports in vivo tissue remodeling and regeneration. Also,

ECM degradation products act as a chemoattractant for the

recruitment of endogenous stem and progenitor cells and

modulates innate immune response.^24,25dECM scaﬀolds from

various species and tissue have been approved by FDA and are

being used clinically for soft tissue repair, wound management,

or heart valve replacement.^26,27However, decellularized

scaﬀolds may restrict its application due to irregular contours

of a wound bed. Thus, in situ gelling hydrogels of the pepsin-

digested dECM will be of great importance. pH-sensitive in

vivo gelling hydrogels of the ECM have been characterized for

the preservation of biomolecules in an intact form.^28,29Also,

the hybrid dECM has been explored for preparing scaﬀolds,

hydrogels, nanoﬁbers, and microspheres.³⁰⁻³³However, the

dECM has weak mechanical strength and poor biostability and

is prone to microbial contamination. Therefore, its use for cell

encapsulation and wound healing is questionable. To this end,

crosslinking of the ECM is being explored in this work. A

cross-linking reaction can be performed using chemical

reagents (like glutaraldehydes, carbodiimide, and genipin),

UV light, and enzyme catalysis (such as transglutaminase).³⁴

Developing bioactive hydrogel with inherent antibacterial

activity is a fascinating challenge for the clinical management

of burn wounds.

To address this challenge, we have developed injectable

soluble acellular dermal matrix (sADM)-based hybrid hydrogel

platforms based on double crosslinksforming ionic and

covalent bonds for facilitating the recovery of critical FT

thermal burn injury in a rat model. A process to dissolve ADM

was developed, and hybrid crosslinked hydrogel (CsADM-Cl)

was formed by blending with CTS, a natural linear

biocompatible polysaccharide, and iodine-modiﬁed 2,5-dihy-

dro-2,5-dimethoxy-furan (DHF-I), a covalent crosslinker. To

the best of our knowledge, there is no report of crosslinking

ADM using DHF-I, which not only preserves the ECM

biological composition and ultrastructure but also enhances its

mechanical strength and control the biodegradation rate as

well as provides the unique antibacterial and antioxidant

property to the gel. Collagen (Col), being the major protein in

the dermal matrix, has been explored for its eﬃcacy in wound

healing models, and therefore, it was selected as a control for

the study. Fabricated hydrogels were characterized for their

2. EXPERIMENTAL SECTION

2.1. Decellularization of Skin Tissue. Decellularization of the

skin was carried out using a modiﬁed protocol described else-

where.^35,36FT skin was harvested from adult rats (weighing ∼150−

200 g) and preserved in cold phosphate buﬀer saline (PBS)

containing 0.1% gentamycin, followed by washing several times

using PBS to remove the adhered blood. Samples were processed

within 4 h from harvesting. Dermal hair and epidermis were removed

using a hypertonic solution (4 g of sodium chloride, 605 mg of tris

base, and 202.5 mg of EDTA in 100 mL PBS) for 2−4 h in an orbital

shaker (Thermocon Instruments Private Limited, Bangalore, India)

and maintained at 37 °C. The isolated dermal layer was denuded

using trypsin (0.25%, Thermo Fisher Scientiﬁc, USA) and Triton X-

100 (Sigma-Aldrich, USA, 0.5% v/v) treatments and washed several

times using PBS. The ADM was further treated with a fat digestion

solution (chloroform/methanol1:1 v/v) for 2 h and subsequently

washed with sterile PBS thoroughly and stored at −80 °C. Frozen

ADM was lyophilized and pulverized mechanically at 4 °C. A

pulverized ADM was solubilized enzymatically (acidic pepsin solution

1 mg/mL) to form a sADM and stored at −80 °C until required. The

ADM was characterized by staining with hematoxylin and eosin

(H&E), anti-Col I antibody (Col I), alcian blue (AB), DAPI, and

DNA quantiﬁcation, following the protocol described in the

Supporting Information. To investigate the retained proteins after

decellularization, the native skin (NS) and ADM were assessed by

sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-

PAGE) as detailed in the Supporting Information. Further, sGAGs

and Col were also quantiﬁed using colorimetric assays (detailed in

Supporting Information). The tissue homogenate prepared aforesaid

was analyzed using ELISA Kits (Invitrogen, USA) to investigate the

presence of the growth factors [BMP-2, vascular endothelial growth

factor (VEGF), and transforming growth factor-β (TGF-β)]. Samples

were run in triplicate; averaged and standard curve was used to

measure the concentrations of growth factors in the NS and ADM.

For Col isolation, rat tail was harvested from freshly euthanized

adult albino rats (average weight 150−200 g) using a protocol

described elsewhere.³⁷Brieﬂy, the tail was washed with PBS to

remove the adherent dirt, and the epidermis was removed

mechanically using a surgical blade and tweezers. Col ﬁbers from

rat tail tendon were cut to smaller fragments and washed successively

with acetone and 70% v/v isopropanol. Rinsed ﬁbers were dissolved

in acetic acid (0.02 N) for 48 h at 4 °C and dialyzed against 1%

chloroform water for 1 h and then changed to water for 2 days to

remove acetic acid. Col solution was lyophilized after freezing and

further stored at −80 °C, until use. Col was characterized using SDS

PAGE (details in Supporting Information).

2.2. Synthesis of Iodine-Modiﬁed DHF-I. DHF-I was

synthesized as described elsewhere.³⁸Iodine was dissolved in 20%

v/v ethanol and mixed with DHF (15%) at 25 °C. The reaction was

allowed to complete in an acidic environment for 12 h until reaction

color changes from reddish brown to yellow. Subsequently, pH 7 was

maintained using sodium hydroxide (NaOH) solution. The product

was ﬁltered to remove the precipitate, if any. The iodination of DHF

was conﬁrmed by proton nuclear magnetic resonance spectroscopy

(400 MHz, Advance DAX-400 Bruker, Sweden) using deuterium

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oxide (Sigma-Aldrich, USA) as a solvent, and the spectrum was

compared with the spectrum of pristine DHF.

measuring their potential to scavenge the stable 1,1-diphenyl-2-

picrylhydrazyl (DPPH, Sigma-Aldrich, USA) free radical, following

the protocol described elsewhere with minor modiﬁcations.³⁹The

uncrosslinked/crosslinked hydrogels were homogenized to powder

using liquid nitrogen. Brieﬂy, DPPH (3.0 mL, 100 μM) and

dispersion of samples (containing 5 mg) in methanol were stirred

and incubated in a dark place for 30 min. Then, DPPH scavenging

was assessed by measuring the absorbance using a UV−vis

spectrophotometer (Multiskan GO Microplate Spectrophotometer,

Thermo Fisher Scientiﬁc, USA) at 517 nm and calculated using the

following equation

2.3. Preparation of Hybrid Hydrogel. CCol-Cl and CsADM-Cl

hydrogels were prepared by blending CTS (molecular weight

50,000−190,000 Da; 75−85% deacetylated, Sigma-Aldrich, USA)

and Col/sADM using DHF-I as a crosslinker. In brief, the blend was

neutralized using 0.1 M NaOH in 10× PBS buﬀer in ice (to maintain

temperature 0−10 °C), such that the ﬁnal concentration of the

polymer and protein in the gel would be 8 mg/mL each and PBS will

become 1×. Further, pregel solution was crosslinked using 0.5% DHF-

I, forming a rapid hydrogel. Crosslinked hydrogels were denoted as

CCol-Cl and CsADM-Cl, while uncrosslinked hydrogels (without

DHF-I) were designated as CCol and CsADM. Tube inversion

method was used for measuring gelation time. Also, the pregel, not

ﬂowing for more than 30 s, was taken as a hydrogel. Surface topology

of the hydrogels was observed using SEM, and ﬁber networks were

analyzed using ImageJ (version 6). Further iodine distribution in the

crosslinked matrix were conﬁrmed by EDAX mapping of iodine on

CCol-Cl/CsADM-Cl hydrogel. In brief, the hydrogels were coated on

coverslips and kept at 37 °C for 15 min to facilitate gelation. Coated

coverslips were dried in a desiccator and stored therein until SEM

analysis was carried out after gold coating of the sample to avoid

moisture.

2.4. Immunohistochemistry of Hydrogel. To assess the

retention of important matrix proteins and biomolecules after

crosslinking, immunohistochemistry (IHC) was performed on NS,

ADM, CsADM, and CsADM-Cl. The primary antibodies against Col-

I (Abcam), ﬁbronectin, VEGF, TGF-β, interleukin-8 (IL-8), and

monocyte chemoattractant protein-1 (MCP-1) (all Santa Cruz

Biotechnology, Inc.) were used. The stained samples were imaged

under an inverted ﬂuorescent microscope (AxioVision, Zeiss,

Germany).

A_B− A_H

DPPH scavenging % =

× 100

A_B

where A_Bis the absorption of the blank (DPPH in methanol) and A_H

the absorption of the hydrogel (hydrogel with DPPH in methanol).

Samples were run in triplicate and averaged.

2.9. Hemocompatibility. The hemocompatible characteristics of

the hydrogels was evaluated using the heparinized blood, following a

protocol described elsewhere.⁴⁰Heparinized blood was centrifuged to

obtain RBC pellets, and the pellet was subsequently washed thrice

with (4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid) (5 mM)

buﬀer containing sodium chloride (150 mM). To hydrogels/normal

saline (negative control)/1% Triton X-100 (positive control), 200 μL

of the suspension (5% diluted anticoagulant blood solution in 0.9%

NaCl solution) was added and further incubated for 30 min at 37 °C.

The supernatant was collected post-centrifugation (1000g, 10 min),

and the absorbance was recorded at 540 nm (n = 3).

The % hemolysis was evaluated using the following equation

A_s− A_NC

A_PC− A_NC

Hemolysis (%) =

× 100

2.5. Rheological Characterization. CCol, CsADM, CCol-Cl,

and CsADM-Cl were further characterized for gelation kinetics using

a dynamic shear rheometer (DSR+, Malvern, U.K.). Pregel solution

was formed by blending CTS with Col/sADM. Gelation kinetics was

assessed by a rheometer using parallel plate geometry; a gap of 200

μm and parallel plate of 25 mm were maintained in all the

experiments. Pregel solution was neutralized in ice and loaded

immediately to a pre-cooled rheology plate at 15 °C. Mineral oil was

used to reduce evaporation from the edges, and the temperature was

raised to 37 °C to induce gelation. Gelation kinetics was studied by

applying constant frequency (1 Hz) and strain (0.1%). Further, the

hydrogels were subjected to small oscillatory frequency sweep at

strain 0.5% and frequency 0.5−20 Hz.

2.6. Fourier Transformer Infrared. Fourier transformer infrared

(FTIR) spectroscopy of the hydrogels was performed in ATR mode

in the wavelength range of 500−4000 cm⁻¹on a Thermo Nicolet

Spectrophotometer (model NEXUS-870; Thermo Nicolet Corpo-

ration, Madison, WI). Vacuum-dried hydrogels (CCol, CsADM,

CCol-Cl and CsADM-Cl) were stored carefully in desiccators to

avoid moisture, before FTIR spectra were recorded.

where A_sis the absorption of the sample, A_PCis absorbance of positive

control, and A_NCis the absorption of negative control.

2.10. Bacterial Inhibition. The hydrogels were prepared in sterile

24-well plates and washed with sterile PBS to ensure sterility of the

samples before analyzing antibacterial eﬃcacy.⁴¹Staphylococcus aureus

(N315) and Escherichia coli (DH5α) strains were taken for the

present study. 20 μL of bacterial suspensions (5 × 10⁷cfu mL⁻¹) was

spread onto each hydrogel (CCol, CsADM, CCol-Cl, and CsADM-

Cl) in a tissue culture plate (TCP) and incubated for 2 h at 37 °C,

relative humidity >90%. To collect bacterial survivors, post 2 h

treatment, sterile PBS (1 mL) was added, and a series of 10-fold

dilution was prepared, followed by plating out in Luria Bertani agar.

Survivor bacteria were allowed to grow for 16−18 h by incubating at

35 °C and counted for colony-forming units (cfu). The TCP served as

a control and was processed in a similar fashion like other samples.

The results are expressed as

C_c− C_H

Log Reduction (%) =

× 100

C_c

2.7. Iodine Dynamic Release Kinetics. To study the iodine

dynamic release from cross-linked variant CsADM-Cl, hydrogel was

incubated in PBS and buﬀered enzymatic solution (Collagenase I,

Sigma, USA) to imitate in vivo conditions. The supernatant was

collected at a predetermined period, that is, 2, 4, 6, 12, 24, 48, 72, and

96 h. In brief, 1 mL of pregel solution was gelled for 24 h in 37 °C,

and the hydrogel was incubated with 10 mL of PBS/collagenase I

(125 U/mL) in 0.1 M Tris base and 0.25 M CaCl₂with pH

maintained at 7.4 in 37 °C in an incubator and stirred using a

mechanical stirrer at 100 rpm (schematic representation in Figure

S4). At a predetermined period, 400 μL of supernatant was extracted

from the sample vial and replenished with 400 μL of fresh PBS/

buﬀered enzymatic solution to maintain a constant volume. Samples

were centrifuged, and the collected supernatant was ﬁltered through

0.2 μm syringe ﬁlter and stored at −80 °C until analyzed using Dionex

ICS 2100 (Thermo Scientiﬁc, USA).

where C_cis log (cell count of control), and C_His survivor count on

hydrogel.

For Live/Dead assay, the chosen bacterial strains were seeded on

hydrogels as aforesaid, and post-incubation, the nutrient broth was

removed. Subsequently, the hydrogels were washed with PBS, Live/

Dead solution (3 μg/mL ethidium bromide and 5 μg/mL acridine

orange in PBS) was added to each hydrogel, and the plates were

incubated at 37 °C for 15 min in the dark. After this, the samples were

washed with PBS and imaged using a Carl Zeiss ﬂuorescence

microscope. The experiment was repeated twice in triplicate, and the

data represent average with standard deviation (SD).

2.11. CAM Assay. The angiogenic potential of the hydrogel

(CCol-Cl and CsADM-Cl) was investigated by CAM assay, following

a protocol reported earlier.³³Fertilized chicken eggs procured

(regional poultry farm, Midnapore, West Bengal, India) were cleaned

with warm sterile saline, wiped, and incubated at 37 °C, 60%

humidity. After 3 days, sterilized hydrogels (CCol-Cl and CsADM-

Cl) were implanted onto the CAM of the eggs with careful sterile

2.8. Antioxidant Eﬃciency of Hydrogels. The ex vivo

antioxidant eﬃcacy of CCol-Cl and CsADM-Cl was assessed by

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dissection, making a small window away from the embryo (n = 3).

The window was resealed through adhesive tape, and the eggs were

further incubated until day 8 of embryo development. Post-incubation

period, the hydrogel-treated CAM membrane was photographed and

analyzed for angiogenesis. The vessels approaching toward the

scaﬀold was counted by three independent observers, and results

were reported by taking the average.

seeded (15,000 cells/coverslip) and cultivated for 5 days. Post 5 days

of cultivation, samples were stained after processing, and images were

documented using a ZEISS inverted ﬂuorescence microscope.

2.12.3. Cells Encapsulation in Pre-Gel. For encapsulating HFCs in

hydrogel, CCol-Cl and CsADM-Cl were processed in a sterile parallel

blinded fashion. Gels were fabricated in 24-well plates containing 2

mL of pregel solution of each group with 1,00,000 HFCs/mL. After

gelling, media was supplied to gels, and media was changed every day

to avoid cell death. The viability of HFCs in hydrogels was

determined post 72 h using the Live/Dead kit (Invitrogen, USA),

following manufacturer’s protocol. Labeled cells were then micro-

graphed under an inverted microscope, and images were captured

using the Zen software. Viable/Live cells were stained green (tagged

with calcein AM), while dead cells were stained red (tagged with

EthD-1).

2.12.4. Quantiﬁcation of Hydrogel Contraction. CCol, CsADM,

CCOl-Cl, and CsADM-Cl contraction after cells seeding/unseeded

was analyzed using macroscopic image analysis (from two

independent batches of ADM isolation). Free-ﬂoating crosslinked

and uncrosslinked hydrogels were imaged after 12 h, 1, 3, and 7 days

in culture.

2.13. Host Response of CCol-Cl and CsADM-Cl In Vivo. All in

vivo experiments were executed under compliance of the Institutional

Animal Ethical Committee guidelines of the Indian Institute of

Technology, Kharagpur, India. To assess the host response in vivo, the

subcutaneous injection of CCol-Cl and CsADM-Cl in the dorsal

region was performed in albino Wistar rats (150−200 kg; n = 3 per

group). Prior to the experiment, peritoneal injection of ketamine was

performed to anesthetize the rats. Food and water supply were

provided freely to the rats post-surgery 12 h, and the light/dark cycle

of 12 h was maintained during the study. On day 14, the rats were

sacriﬁced; CCol-Cl and CsADM-Cl along with surrounding tissues

were retrieved, followed by ﬁxation in 4% PFA and processed for

H&E, TB, and Masson’s trichome (MT) staining. Major organs were

also harvested from rat on day 14 to assess organ toxicity, and H&E

was performed to establish the biocompatibility of the hydrogels.

2.14. FT Wound Healing in Burn Model. 2.14.1. In Vivo Burn

Model Creation. For burn model, Wistar rats (150−200 g each) were

taken, and all in vivo experiments were performed as per guidelines of

the Institutional Animal Ethical Committee, Indian Institute of

Technology, Kharagpur, India. Rats were anesthetized by intra-

peritoneal injection of ketamine hydrochloride; then their dorsum was

shaved to remove hair. The burn model was created as previously

reported.⁴³Brieﬂy, a custom-made 220 g aluminum rod with a copper

template of 1.5 cm diameter was heated in a 100 °C water bath for 5

min and placed on the posterior-dorsum of each rat for 8 s.

Subsequently, the rat was resuscitated by intraperitoneal injection of

saline within 1 h post burning. To follow the current clinically

accepted treatment, burn wound excisions were executed 48 h post

burn injury. The FT skin was removed to generate a 2 cm diameter

circular wound; CCol-Cl/CsADM-Cl pregel was injected at the

wound site, and Tegaderm dressing (3M Science Applied to Life,

USA) was applied to hydrogels on the wound bed. SHAM wounds

were only covered with Tegaderm dressing. For the study, three rats

were taken for each group and for each period.

2.12. Cell Culture Study. 2.12.1. Conditioning Media and

Related Assays. The matricryptic peptides or cytokines, a major

portion of eluted components, may aﬀect scaﬀold cellularization and

remodeling. All materials were processed under sterile conditions in a

blinded fashion. Each hydrogel was weighed equally and minced using

a sterile razor; subsequently, minced hydrogels were placed in six-well

plates. Dulbecco’s minimum essential medium high glucose (Life

Technologies, USA) was added to minced hydrogels (ratio: 50 mg

hydrogel/mL medium) and incubated at 37 °C, 5% CO₂. Untreated

medium was processed simultaneously and acted as a control for the

study. 72 h post incubation, the media was centrifuged (16,000g for

10 min) for removing hydrogel particles, and the untreated medium

was added to attain a ﬁnal concentration of 25 mg crushed hydrogel/

mL medium. The morphological changes of HFCs/HKCs was

documented using rhodamine and DAPI (Invitrogen, Thermo

Scientiﬁc, USA) staining.⁴²15,000 cells/coverslip were seeded and

cultivated for a predetermined period, that is, 72 h. Post the preset

period, cells were ﬁxed using 4% paraformaldehyde (PFA) and stained

for rhodamine & DAPI, and images were recorded using a

ﬂuorescence microscope (Carl Zeiss) for evaluating cytocompatibility

of diﬀerent hydrogel’s conditioned media, where the complete media

acted as a control for the study.

The conditioning media was also subjected to scratch assay using

HFCs/HKCs to study the role of conditioning media in supporting

the migration potential of HFCs/HKCs. Scratch assay was performed

to evaluate in vitro wound healing potential of the conditioning media

of the hydrogel. In brief, HFCs were grown until the formation of the

monolayer; then wound/scratch was created using a sterile 200 μm

tip. The plates were washed carefully twice with sterile PBS to remove

any unattached cells on the plate and wound area. The complete

media was replaced with the conditioning media, and micrographs

were obtained after a predetermined period (i.e. HFC6 and 12 h;

HKC12 and 24 h). The number of migrated cells in denuded area

was also calculated for both HFCs and HKCs by three independent

observers, and the results were demonstrated as mean SD.

2.12.2. Direct Cytotoxicity Testing. 2.12.2.1. MTT Assay. The

cytotoxicity of hydrogels was evaluated using 3-(4,5-dimethyl-2-

thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay by

directly cultivating HFCs/HKCs on CCol, CsADM, CCol-Cl, and

CsADM-Cl. Viable cells were calculated at a predetermined (24, 72,

and 120 h) period by incubating the hydrogel-coated coverslips with

an MTT (Merk Millipore, Germany) solution and subsequently

dissolving the formazan crystal by adding an equivalent amount of

dimethyl sulfoxide. Subsequently, absorbance was taken at 570 nm,

experiments were repeated thrice, and mean was taken.

2.12.2.2. Proliferation Assay. Proliferating cell nuclear antigen

(PCNA) antibody (BioLegend, USA) was used for the immuno-

ﬂuorescent study (IF) of PCNA (green) in proliferating HFCs on

hydrogels. Hydrogel-coated coverslips were seeded with HFCs

(15,000 cells/coverslip) and incubated for 5 days. Post incubation,

coverslips were ﬁxed and permeabilized with ice-cold 70% ethanol for

15 min, followed by washing with PBS and blocking in BSA (1% in

PBS) for 15 min at room temperature. Cells seeded with hydrogels

were stained with PCNA monoclonal antibody tagged with secondary

for 1 h at room temperature and subsequently washed with PBS to

remove background and imaged using an inverted ﬂuorescence

microscope. The experiment was repeated thrice to conﬁrm the

reproducibility of data.

2.14.2. Histomorphometric Analysis. Hydrogel explants were

collected at days 7, 14, and 21 and ﬁxed using 4% PFA. Following

ﬁxation, tissues were dehydrated in ethanol graded series (70−100%),

embedded in paraﬃn to form a block, sectioned using a microtome (3

μm thickness), and stained with H&E. Section images were processed

using ImageJ (version 6) for quantiﬁcation of re-epithelialization,

wound distance, granulation area, newly formed appendages, and

thickness of the newly formed epithelium. MT staining was performed

for the analysis of collagen morphology and intensity. Sections were

also stained with anti-CD31 and anti-CK10 for conﬁrmation of blood

vessels and re-epithelialization in diﬀerent groups.

2.12.2.3. Apoptosis Assay. Apoptosis assay was executed using a

DeadEnd Fluorometric Terminal deoxynucleotidyl transferase dUTP

nick end labeling system (Promega, USA) following the manufac-

turer’s guidelines. Brieﬂy, hydrogel-coated coverslips were taken as the

sample, and lysine-coated coverslips served as the control. HFCs were

2.15. Gene Expression (RT-PCR Analysis). Regenerated tissues

were retrieved on day 21 from each group, and TRIzol Reagent

(Thermo Scientiﬁc, USA) was used for isolation of total RNA

following the manufacturer’s instructions. RNA quality was

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a

Scheme 1. Synthesis Strategy of Crosslinked Hybrid Hydrogel

a

Schematic representation of physical cross-linking between CTS and ADM at speciﬁc conditions to form hydrogel CsADM (a); further, CsADM

was cross-linked using DHF-I to form CsADM-Cl (b).

Figure 1. Decellularized skin was used as an ADM in developing a crosslinked hydrogel. Histological and immunohistochemical analysis of NS and

ADM (a), total protein extracted from NS and ADM separated by SDS-PAGE (b), DNA isolation and quantiﬁcation from NS and ADM (c,d), and

quantiﬁcation of GAGs and collagen from NS and ADM (e). Scale bar represents 50 μm, Y-error bars symbolize SD, and *** signiﬁes p < 0.001.

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Figure 2. Crosslinking enhances viscoelastic properties, while retaining nanoﬁbrillar hydrogel architecture. Optical images, surface topology, and

ﬁber network analysis of CCol (a,e,i), CCol-Cl (b,f,j), CsADM (c,g,k), and CsADM-Cl (d,h,l); inset (g & i) shows magniﬁed images. EDX

mapping of iodine on the ﬁbrillar network of CCol-Cl (m,n) and CsADM-Cl (o,p). Gelation kinetics of hydrogel (q) without a crosslinker (CCol

and CsADM) and (r) with a crosslinker (CCol-Cl and CsADM-Cl); (s) maximum elastic modulus (G′); frequency sweep analysis post gelation for

(t) uncrosslinked (CCol and CsADM); and (u) crosslinked (CCol-Cl and CsADM-Cl) samples.

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determined using NanoDrop (NanoDrop 2000/2000c, Thermo,

USA) (high quality RNA has OD 260/280 nm ratio between 1.8

and 2.0), and equivalent RNA of each group was reverse transcribed

to cDNA using the cDNA synthesis kit (Thermo Scientiﬁc, USA),

following the manufacturer’s protocol. The primer sequences used for

the study are given in the Supporting Information.

2.16. Statistics. GraphPad Prism software (version 5.02, La Jolla,

CA, USA) was used to execute statistical evaluations of data obtained

from diﬀerent groups. All experiments were repeated thrice unless

methoxy proton (H1) of cis and trans isomers. The chemical

shifts between δ = 6.27−6.29 ppm are because of H2 protons.

The chemical shifts at δ = 5.78 and 6.07 ppm are due to the

presence of unsaturated protons (H3). The disappearance of

the chemical shift at δ = 5.78 ppm and the appearance of a new

chemical shift between δ = 3.78−3.82 ppm in the NMR

spectrum of an iodinated product reveal the successful

formation of DHF-I from DHF through iodination reaction.

3.3. Macroscopic Appearance of Hydrogel. Uncros-

slinked hydrogels (CCol/CsADM) were softer with round

edges in comparison to crosslinked hydrogels (CCol-Cl/

CsADM-Cl), which were observed to be rigid structures with

deﬁned edges and easy to handle with the help of tweezers

(Figure 2a−d). SEM analysis of the hydrogel microstructure

revealed a nano-ﬁbrillar morphology in both groups, that is,

CCol (Figure 2e) and CsADM (Figure 2g). Further,

crosslinking of hydrogels did not aﬀect adversely the intrinsic

property of the self-assembly into a nano-ﬁbrillar structure, and

a similar structure was observed in CCol-Cl (Figure 2f) and

CsADM-Cl (Figure 2h). After crosslinking, the ﬁber diameter

was reduced marginally; the ﬁber diameters of CCol, CCol-Cl,

CsADM and CsADM-Cl hydrogel were ∼110, ∼105, ∼135,

and ∼95 nm, respectively (Figure 2i−l). The presence of

iodine in the crosslinked matrix was conﬁrmed by EDAX

scanning; both crosslinked hydrogel variants CCol-Cl (Figure

2m,n) and CsADM-Cl (Figure 2o,p) showed uniform iodine

distribution.

3.4. Hydrogel ICC. For evaluating the presence of

characteristic ECM biomolecules such as Col I and ﬁbronectin,

ICC was performed against speciﬁc antibody (Figure S3). The

distribution of ECM active biomolecules was similar in NS and

ADM. Also, cytokines and growth factors that are vital for

dermal neovascularization and regeneration, like TGF-β,

VEGFA, MCP-1, and IL-8, were also evaluated in the NS,

ADM, and CsADM and CsADM-Cl hydrogels. The ICC

results revealed the fact that although intensity of the ICC

stains diminished, yet part of each growth factor was preserved

in the crosslinked hydrogel (Figure S3). Altogether, the native

nanoﬁbrillar structure and bioactive components were well

preserved after crosslinking with CTS and DHF.

3.5. Rheological Characterization of Hydrogel. The

rheological properties of hybrid hydrogel were studied using a

parallel plate rheometer (Figure 2q−u). The average viscosity

at 25 °C of pregel solution was 1.5 and 1.9 Pa s for CCol and

CsADM, respectively. Storage modulus (G′) and loss modulus

(G″) increase with an increase in temperature from 15 to 37

°C (Figure 2q,r). The G′ value was higher than G″ for both the

variants by a factor of ∼5, which denotes the solid-like

behavior of pregel solutions after gelation. The steady-state G′

after complete gelation varied non-linearly with temperature/

time, and when compared to a uncrosslinked variant, the

crosslinked variant showed higher G′ (Figure 2s). After

gelation, frequency response was studied, and the value of G′

was independent of frequency (Figure 2t,u), indicating the

frequency-independent gelation behavior. At all frequencies,

crosslinked hydrogel showed higher G′ when compared to the

uncrosslinked variant, representing superior stability and

strength.

mentioned, and the data signiﬁes mean

signiﬁcance was determined as P < 0.05.

SD. The level of

3. RESULTS

Hybrid hydrogels (CCol, CCol-Cl, CsADM, and CsADM-Cl)

were fabricated successfully at physiological pH and temper-

ature using CTS, DHF-I, and ADM/Col. The possible

mechanism of hydrogel formation with ADM/Col and cross-

linking reaction by CTS and DHF-I is shown in Scheme 1.

Interestingly, both CCol-Cl and CsADM-Cl were observed to

be injectable using a syringe with 18 G needle. Uncrosslinked

hydrogels took 15

2 min for gelation, while crosslinked

hydrogels gelled within 5 1 min at 37 °C.

3.1. Extraction of sADM and Col. The ADM was

successfully prepared from rat dermis by a combinatorial

treatment of trypsin and Triton X-100. After decellularization,

the NS turned milky white from pinkish red, which represents

the removal of cells. The eﬃciency of the decellularization

process was conﬁrmed using H&E, Col I, AB, and DAPI

staining. The ADM, in contrast to the NS, showed the absence

of any cell nucleus as observed by H&E and DAPI staining,

whereas major protein Col and GAGs had similar intensity in

both NS and ADM sections as shown by Col I and AB

staining, respectively (Figure 1a). SDS-PAGE (Figure 1b)

shows the existence of a mixture of proteins/peptides in the

ADM as evident from the presence of diﬀerent molecular

weights bands, especially bands at a lower molecular weight.

Such an observation signiﬁes the preservation of bioactive

molecules in the ADM post decellularization. The absence of

the DNA fragment in the ADM was demonstrated by the

GelDoc image (Figure 1c), which corroborates with the

ﬁndings of H&E and DAPI staining. The NS and ADM were

processed to isolate and to quantify DNA. In comparison to

the NS (1121 210 ng/mg dry weight), the ADM showed

signiﬁcantly reduced (p < 0.001) DNA content (30.14 12.34

ng/mg dry weight), as observed in Figure 1d. Further, Col

content of the NS and ADM did not show any signiﬁcant

diﬀerence; the Col content in the ADM was 60.12 5.31% of

dried tissue, whereas in the NS, it was 52.31 4.61% of dried

tissue (Figure 1d). Also, sGAG content was not observed to

vary signiﬁcantly in the ADM (0.44 0.06% dry weight) when

compared to NS (0.512 0.07%) (Figure 1d). The presence

of essential growth factors (BMP-2, VEGF, and TGF-β) was

revealed through ELISA assay in both the NS and ADM

(Figure S1a).

The isolated rat tail Col was characterized using SDS-PAGE.

Rat tail Col demonstrated a classic band pattern for type I Col,

that is, a doublet at molecular weights ∼215 and ∼235 kDa;

also, an additional doublet at ∼115 and ∼130 kDa was

observed in SDS-PAGE (Figure S1b).

3.6. FTIR. FTIR spectra of CCol, CCol-Cl, CsADM, and

CsADM-Cl are shown in Figure S2b. The CCol hydrogel

(Figure S2b(A)) showed peaks at 3357, 2982, 1640, 1578, and

1414 cm⁻¹which are responsible for frequencies of N−H bond

stretching (amide A), C−H stretching, amide-I stretching,

3.2. DHF-I Synthesis. Iodine reacts with the double bond

of DHF via electrophilic addition reaction and formed DHF-I

1

which was conﬁrmed through H NMR analysis of pristine

1

DHF and DHF-I. In the H NMR (Figure S2a) spectrum of

DHF, the chemical shifts between δ = 3.49−3.52 ppm signify

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Figure 3. Antimicrobial and angiogenic potential, both are important in wound healing applications. Microbes (E. coli and S. aureus) colony grown

on an agar plate after contact with control and hydrogel (a); log reduction in microbial count (b); Live/Dead staining post 2 h of incubation with

hydrogel (c); and culture plates of control-treated E. coli and S. aureus (10−5 dilution) (d). CAM assay macroscopic view (e) and quantiﬁcation of

vessels (f) growing toward scaﬀolds. Y-error bars represent SD, ** signiﬁes p < 0.01, and scale bar represent 50 μm.

amide-II stretching, and amide III stretching, respectively. The

crosslinked hydrogel (Figure S2b(B)) CCol−Cl exhibited

peaks for amide A, C−H stretching, amide I, amide II and

amide III at 3340, 2972, 1634, 1556, and 1405 cm⁻¹,

respectively, while the CsADM hydrogel (Figure S2b(C))

demonstrated peaks of N−H stretching, C−H stretching,

amide I, amide II, and amide III at 3366, 2982, 1633, 1567,

and 1414 cm⁻¹, respectively. The crosslinked hydrogel (Figure

S2b(D)) CsADM-Cl exhibited peaks for amide A, C−H

stretching, amide I, amide II, and amide III stretching

frequencies at 3325, 2982, 1634, 1556, and 1409 cm⁻¹,

respectively. Further, the increase of peak intensity at 1634

cm⁻¹for both crosslinked hydrogels (Figure S2b(B,D))

implies the formation of the Schiﬀ base by chemical

crosslinking between CTS, collagen/sADM, and the iodinated

DHF via imine (CN) bond.³⁸

mechanical and biological properties.⁴⁴This further will dictate

the hydrogel behavior in vivo. The ratio of O.D. 1235 cm⁻¹and

O.D. 1450 cm⁻¹can assess Col’s triple helix integrity. Also,

values around 0.5 have been reported for denatured, while

those around 1 indicate native triple helix structures.^45,46In

case of the blend samples (CCol and CsADM), the values

obtained were 1.01 and 1.00, respectively. Similarly, the values

obtained were 1.03 and 1.04 for crosslinked samples CCol-Cl

and CsADM-Cl, respectively. Such observations indicate that

the crosslinking did not destabilize the triple helix structure.

Absence of band at 1706 cm⁻¹(corresponding to free acetic

acid)⁴⁵in any of the groups evidences complete neutralization

of the hydrogels, which is essential for imparting the bioactive

biocompatible surface in situ for neighboring cell inﬁltration

and proliferation.

3.7. Iodine Release Kinetics. Figure S4 demonstrates the

iodine release curves of the CsADM-Cl hybrid hydrogel in PBS

and buﬀered enzymatic solution at a diﬀerent predetermined

The triple helix integrity of Col ﬁbers in both isolated Col

and ADM is a critical factor attributing to good combination of

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Figure 4. Crosslinked hybrid hydrogels exhibit better cytocompatibility and cell migration behavior of skin tissue cells. Rhodamine phalloidin−

DAPI images of HFCs and HKCs cultivated in CCol-Cl and CsADM-Cl-conditioned medium at diﬀerent time durations; cell migration

quantiﬁcation of (d) HFCs and (e) HKCs. (Scale bar represents 50 μm, red represents nucleus staining DAPI; and green depicts cytoskeleton

expression). Y-error bars represent SD, ** represent p < 0.01, and*** signiﬁes p < 0.001.

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Figure 5. Quantitative and qualitative cell inﬁltration and proliferation of HFC-encapsulated hybrid hydrogels. PCNA staining images (a) and

quantiﬁcation of PCNA positive cells (b) of HFCs cultivated in CCol-Cl and CsADM-Cl. Live/Dead staining images of CCol-Cl (c) and CsADM-

Cl (d) encapsulated with HFCs post 72 h of incubation; images of contraction of HFCs seeded hybrid hydrogels at diﬀerent time periods (e) and

quantiﬁcation of area remaining (f). Scale bar represents 50 μm; Y-error bars represent SD; and *** signify p < 0.001.

period. The iodine release rate from hydrogel was slower in

PBS when compared to the enzymatic solution. There was an

initial rapid release (in PBS: 1919.84 105.9 ppm; in enzyme:

3.8. Antioxidant Assay. Antioxidant molecules have been

reported to exert a positive eﬀect on wound healing

progression and pace by regulating burden of excess reactive

oxygen species (ROS) in the wound site.⁴⁷Thus, iodine

introduction in the hydrogel matrix plays another vital role

since iodine has an antioxidant activity.⁴⁸Antioxidant potential

was evaluated by measuring the eﬃciency of hydrogels to

3771.51

163.4 ppm) at 6 h of incubation. Further, the

CsADM-Cl hydrogel showed a prolonged release behavior of

iodine (in PBS: 2190.67 106.5 ppm; in enzyme: 3959.35

191.23 ppm) till 24 h, after which it attained the plateau phase.

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scavenge DPPH. All hydrogels showed diﬀerential reduction in

the DPPH peak, which was attributed to hydrogen atom

donation or electron transfer from the parent molecule to

DPPH free radical. The DPPH scavenging eﬃciency of CCol,

CsADM, CCol-Cl, and CsADM-Cl was 19.54 0.9, 24.14

2.12, 58.60 5.21, and 63.20 6.21%, respectively (Figure

S2c). These results demonstrated that crosslinked variants had

signiﬁcantly (p < 0.001) superior antioxidant potential when

compared to uncrosslinked variants. Also, hybrid hydrogels can

prevent cell damage and enhance viability by reducing excess

ROS in vitro.

3.9. Hemocompatibility. Blood compatibility of the skin

substitute is important because it would stimulate the blood

defense systems (i.e., coagulation or ﬁbrinolysis) which can

further worsen the situation.⁴⁹Hemocompatibility is especially

essential for FT/critical burn wounds with signiﬁcant loss of

skin components (either epidermis or epidermis dermis both)

and hence bringing the skin substitute in direct contact to

diﬀerent body ﬂuids. The percentage hemolysis is a direct

indicator of the extent of erythrocyte damage when exposed to

sample of interest. CCol-Cl, CsADM-Cl, positive control

(Triton X-100), and negative control (saline) on direct contact

with RBCs showed an absorbance of 0.047 0.02, 0.048

CsADM-Cl hydrogel (50 3) when compared to CCol-Cl (21

2) hydrogel on treated CAM. Moreover, CsADM-Cl-treated

CAM led to a higher number of branch points when compared

to CCol-Cl-treated CAM.

3.12. In Vitro Assays. 3.12.1. Indirect Assay. To evaluate

the toxicity of the eluted remnant material hydrogels

conditioning media, treated cells were stained using rhodamine

phalloidin/DAPI. HFCs and HKCs showed a similar

morphology in a conditioned medium like control after 72 h

of incubation (Figure 4a). HFCs treated with the CsADM-Cl

conditioning media displayed a well-spread cytoskeleton and

extended F-actin ﬁlaments, revealing a higher cell-to-cell

contact.

In vitro, wound healing or scratch assay was also performed

to evaluate the eﬀect of conditioning media on the

proliferation potential of HFCs. Interestingly, none of the

group showed an adverse eﬀect on the proliferation rate of

HFCs when treated for 24 h. Wounds/scratches treated with

CCol-Cl/CsADM-Cl conditioning media showed a signiﬁ-

cantly (p < 0.01) higher rate of migration of HFC in the

denuded path after 6 h of treatment (Figure 4b,d). After 12 h

of treatment, the CsADM-Cl-treated group showed complete

wound coverage (100%) in comparison to control (68.675

5.61%) and CCol-Cl (80.52 6.21%). Similarly, in the case of

HKCs, they were observed to follow an increasing trend of

migration with time. A signiﬁcantly higher migration rate (p <

0.001) of HKCs was supported by the conditioning media of

CCol-Cl/CsADM-Cl, as observed in Figure 4c,e. After 24 h of

treatment, the CsADM-Cl conditioning media-treated group

showed a wound closure of 93.12 2.11% in comparison to

the control of 33.56 1.24% and the CCol-Cl conditioning

media-treated group of 64.00 3.44% (p < 0.001).

3.12.2. Direct Assay. HFCs were directly plated on

hydrogel-coated coverslips and subjected to MTT assay to

assess the metabolic activity, proliferation assay (PCNA) to

identify actively proliferating cells, and apoptosis staining to

identify apoptotic cells. Both HFCs and HKCs showed an

increasing growth trend of metabolically active cells with time,

revealing the non-toxic behavior of CCol-Cl/CsADM-Cl. The

proliferation rate of cells was signiﬁcantly (p > 0.05) higher in

the CsADM-Cl treated group when compared to CCol-Cl

(Figure S5a,b). After 5 days of cultivation, there was a

signiﬁcantly (p > 0.001) higher number of PCNA positive cells

(Figure 5a,b) in CsADM-Cl (210 15) when compared to

CCol-Cl (142 8). Also, cells cultivated on CCol-Cl showed

signiﬁcantly (p > 0.001) higher number of PCNA positive cells

when compared to that on control. Further, there were no

apoptotic cells in any of the group (Figure S5c), demonstrating

the excellent cellular compatibility of the hydrogel.

0.01, 0.6835

0.02 and 0.038

0.01, respectively. The

percentage hemolysis due to CCol-Cl/CsADM-Cl was 1.39

0.12 and 1.55 0.13%. Generally, hemolysis <5% is acceptable

for a suitable biomaterial to be used in vivo. Higher percentage

of hemolysis reveals biomaterial’s poor hemocompatibility. All

materials used clinically in current practice, including PVC,

meet these guidelines.⁵⁰The data suggested that CCol-Cl and

CsADM-Cl can exhibit clinically desired hemocompatibility.

3.10. Antibacterial Assay. Burn wounds are prone to

bacterial infection.⁵¹Therefore, dressings with the inherent

antibacterial property will be beneﬁcial to remove the bacterial

load and inﬂammatory response from the wounds. Iodine is

reported to exhibit a signiﬁcant antibacterial property even at 1

ppm.^51,52For demonstrating antibacterial activity, hydrogels

were challenged with two pathogens (E. coli and S. aureus) at a

concentration of about 10⁶cfu, and the cell-count reductions

after 2 h treatment were recorded post incubation for 12 h in

agar gel. Figure 3a−d shows the antibacterial activity of the

hydrogel. CCol and CsADM exhibited good activity against

Gram positive and Gram-negative bacteria. CCol and CsADM

showed a log reduction of 7.72 4.2 and 7.89 3.5 against E.

coli, respectively, whereas 8.39 5.2 and 8.55 3.4 against S.

aureus, respectively (Figure 3b). The control group displayed

development of lawn post direct plating (Figure 3a). For

calculating log reduction, cfu from 10⁵dilution (Figure 3d) in

control was utilized. On other hand, all DHF-I crosslinked

hydrogel, that is, CCol-Cl and CsADM-Cl demonstrated

outstanding antibacterial property (Figure 3a). No survival

colony was observed after incubation for 12 h in agar gel.

Further, Live/Dead assay also revealed the presence of red

stained cells denoting non-viable bacterial cells on the hydrogel

surface (Figure 3c).

3.12.3. HFC Encapsulation in Hydrogel. Figures 5c,d and

S6 represent the cells encapsulated into hydrogels. It is

noticeably demonstrated that the majority of cells were alive,

depicting green ﬂuorescence post 72 h of incubation in

hydrogels, suggesting that the dual crosslinked nanoﬁbrillar

microenvironment were non-toxic to the HFCs.

3.11. CAM Assay. Capillary formation on CAM was

observed to investigate the angiogenesis potential of CCol-Cl

and CsADM-Cl hydrogels (Figure 3e,f). After incubation with

the CCol-Cl/CsADM-Cl disc for 3 days, the embryo was alive,

indicating the non-toxic nature of hydrogel. Further, the blood

vessels were also found to be branched therein. Interestingly,

after 72 h of incubation, there were signiﬁcantly (p < 0.001)

higher number of blood vessels approaching toward the

3.12.4. ECM Hydrogel Contraction. Hydrogels showed an

increased contraction, when incubated from 12 h to 7 days in

culture. Also the contraction rate relied on the crosslinking

status of the hydrogel (Figure 5e,f). The unseeded hydrogel

area remained the same regardless of being crosslinked/

uncrosslinked/left for diﬀerent time points (data not shown).

Hydrogel contraction (Figure 5f) in CsADM-Cl (81.88

3.1%) and CCol-Cl (76.35

6.5%) hydrogel groups was

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Figure 6. Pre-clinical study in a rat model. Biocompatibility of subcutaneously injected hybrid hydrogels (CCol-Cl and CsADM-Cl) was evaluated

in dorsal skin in a rat model (a) and histological study post 10 days of injection (b). Healing progression of full-thickness cutaneous wounds treated

with SHAM, CCol-Cl, and CsADM-Cl; optical images of wounds (c) and wound closure rate (d) on days 0, 7, 14, and 21; (e and f) RT-PCR

analysis at 21 day post wounding. Yellow star represents the blood vessel in MT staining (b), red star in TB staining represents TB-positive stained

cells (b), yellow dotted line represent adjoining area between the host and implant (b), and yellow circle area of interest (b). Scale bar represents

50 μm; Y-error bars represent SD, yellow dotted circle represent wound area (c),* signiﬁes p < 0.05, ** signiﬁes p < 0.01, and *** signiﬁes p <

0.001.

signiﬁcantly lesser when compared to CCol (42.35 5.89%)

and CsADM (47.35 3.2%) groups. Crosslinking imparted

stability to hydrogel networks. Hence, crosslinked hydrogels

were observed to contract lesser when compared to

uncrosslinked hydrogels.

3.13. Host Response to the Hydrogel. To evaluate the

host response to hydrogel, CCol-Cl/CsADM-Cl was injected

subcutaneously in the rat. The treated groups were observed to

be healthy, and there were no mortality during the study. The

rat weight did not vary abruptly during the study period (data

not shown). After 2 weeks of injection, CCol-Cl/CsADM-Cl

was retrieved along with the surrounding tissue and was

subjected to histological staining for studying the interaction

between the hydrogel and the host tissue, as shown in Figure

6a,b. H&E staining demonstrated host tissue integration with

CCol-Cl/CsADM-Cl and also host cell inﬁltration in hydrogel

was evident. Interestingly, as displayed in Figure 6b, CsADM-

Cl showed more inﬁltration and proliferation of cells, as

compared to CCol-Cl.

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Figure 7. Histomorphometric analysis provides quantitative wound healing eﬃcacy of injectable hybrid hydrogels. Histological micrographs of

wound sections implanted with CCol-Cl and CsADM-Cl at days 7, 14, and 21 after dermal excision by H&E staining (a), quantiﬁcation of length of

wound area (b), granulation area (c), length of reepithelization (d), thickness of epithelial layer (e), and no. of newly formed skin appendages at

predetermined period (f). Scale bar represents 50 μm; black dotted line represent healed area; ** signiﬁes p < 0.01, and *** signiﬁes p < 0.001.

MT staining of the retrieved hydrogels showed absence of

capsular layer/ﬁbrosis and the presence of blood vessels in the

interface area. Further, the interfacial layer was also

characterized by the presence of dense collagen ﬁber

deposition, demonstrating stimulated cell inﬁltration in

CCol-Cl/CsADM-Cl. To assess inﬂammatory response to

hydrogel, TB staining was performed. TB staining revealed

very few mast cells near the implanted zone, and also, there

were no mast cell inﬁltration inside CCol-Cl/CsADM-Cl. This

minimal inﬂammatory response after CCol-Cl/CsADM-Cl

injection is similar to the normal wound healing cascade. These

ﬁndings establish the biocompatible nature of CCol-Cl and

CsADM-Cl and also demonstrate that neither of hydrogels

triggered an adverse immune response in vivo.

The plausible toxicity of CCol-Cl/CsADM-Cl to vital organs

was also determined by retrieving major organs after two-week

post-injection. Major organs (liver, kidney, lung, and heart)

retrieved were analyzed via histological staining, and there

were no signiﬁcant necrosis or pathological changes in the

anatomy of the organs of the CCol-Cl/CsADM-Cl-treated

group in comparison to the untreated group (Figure S7).

Cardiac muscles in the heart had the similar anatomy for all

groups, and also there was the absence of any sign of ﬁbrosis or

inﬂammation. Hepatocyte distribution in the liver of treatment

groups showed the normal anatomy. Also, lungs had no signs

of ﬁbrosis in treatment groups. Moreover, in kidney sections,

glomerulus structure was distinctly visible for all groups. These

results signify that CCol-Cl and CsADM-Cl did not exert any

toxic eﬀect on major organs in the rat model.

crosslinked hybrid hydrogels CCol-Cl/CsADM-Cl, whereas

Tegaderm covered wounds were marked as the SHAM group,

which served as a control for the study. Tegaderm was also

placed on top of hydrogels to secure their position on the

wound bed. Animals were sacriﬁced after a predetermined

period, that is, 7, 14, and 21 days post-treatment for

histological analysis. Optical images revealing the morpho-

logical changes in wound closure for various groups are

provided in Figure 6c,d. Wound treated with CsADM-Cl

showed signiﬁcantly enhanced wound closure (∼89.23%

healing) 7 days post-surgery when compared to groups treated

with CCol-Cl (∼78.21% healing)/SHAM (∼65.11% healing)

(Figure 6d).

By day 14, ∼90.21 and ∼95.40% of the wounded area were

re-epithelialized in wounds treated with CCol-Cl and CsADM-

Cl, respectively. This was signiﬁcantly higher than the SHAM

group (∼75.52%). 21 days post-surgery CsADM-Cl-treated

groups showed complete wound closure, that is, ∼99.12%,

while CCol-Cl/SHAM groups were still in the healing phase.

However, CCol-Cl (∼97.254%)-treated groups showed

signiﬁcantly higher % healing when compared to the SHAM

group (∼88.62) on day 21 (Figure 6d). All these ﬁndings

suggest that the wound treated with CsADM-Cl showed

enhanced wound closure and hence better-wound healing as

compared to CCol-Cl/SHAM groups.

3.15. Histomorphometric Analysis. Wound healing is a

multifaceted repair process involving recruitment of cells to the

wound area by releasing chemoattractants, followed by cell

proliferation and diﬀerentiation. These pathobiological pro-

cesses lead to the formation of new tissue and hence wound

closure. To further analyze the healing progression in diﬀerent

groups, H&E staining was performed to detail the morpho-

3.14. Hydrogel Treatment of Dorsal Burn Injury

Model. Burn model was prepared as demonstrated in Figure

S8; FT wounds created post burn injury were treated with

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Figure 8. Collagen staining and immunohistochemical analysis demonstrated the potential of the hybrid hydrogels to heal chronic wounds in a rat

model. MT staining (a) and quantiﬁcation of collagen index (b) 21 days post treatment with CCol-Cl and CsADM-Cl. Representative IHC images

of anti CD-31 (c) and anti CK-10-(e) stained histological sections on determined periods. Quantiﬁcation of blood vessels on day 7 and day 14 of

wounds treated with SHAM, CCol-Cl, and CsADM-Cl (d). Scale bar represents 50 μm; red represents nucleus staining DAPI, and green depicts

antibody expression; Y error bar represent SD; ** signiﬁes p < 0.01, and *** signiﬁes p < 0.001.

logical changes in diﬀerent layers of skin (Figure 7). At day 7

post surgery, the length of the wound reduced signiﬁcantly in

wounds treated with CsADM-Cl (0.56 0.09 cm) and CCol-

organized granulation tissue when compared to the CCol-Cl

(re-epithelialization 9.74 0.85 cm and Granulation area

42.69 2.87 mm²)/SHAM treated group (re-epithelialization

7.46 0.65 cm and Granulation area 74.97

5.21 mm²)

Cl (1.08

0.12 cm) when compared to the SHAM group

(1.36

0.14 cm). In addition, CsADM-Cl-treated wounds

(Figure 7b−d). Granulation tissue formation is an essential

aspect of wound repair and regeneration. The granular tissue is

composed of synthesized ECM, ﬁbroblasts, and bioactive

molecules, dictating the diﬀerent phases of healing.⁵²⁻⁵⁶By

demonstrated signiﬁcantly enhanced re-epithelialization (13.49

1.2 cm) accompanied with lower area of granulation (31.26

2.05 mm²). This was characterized with a thicker and well-

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day 14, the length of wounds (control: 0.9307

0.11 cm,

7. Further, the newly formed vessels were distinctly visible on a

more organized granulation tissue of the CsADM-Cl-treated

group. The angiogenesis induced was signiﬁcantly higher (p <

0.001) when compared to both SHAM and CCol-Cl-treated

groups post 7-days of surgery. The higher angiogenic activity of

CsADM-Cl may be attributed to the release of bioactive

molecules from the hydrogel in situ. At day 14, blood vessels

were observed to follow an increasing trend in the control-

treated group. In contrast, both CCol-Cl- and CsADM-Cl-

treated groups showed a decrease in number of vessels. The

reason may be the rapid closure of wounds in CCol-Cl- and

CsADM-Cl-treated groups. Hence, requirements of blood

vessels for the transport of nutrients and oxygen is less,

whereas in the SHAM group, healing is still in progress.

Further, to demonstrate re-epithelialization and epidermal

diﬀerentiation, CK 10 antibody was used for staining 14- and

21-days post-surgery sections (Figure 8e). By day 14, no

distinct expression of CK 10 was observed in both SHAM and

CCol-Cl-treated groups, revealing the absence of neo-

epithelium in the wound area. Surprisingly, the CsADM-Cl-

treated group revealed little CK-10 expression in the thick

epithelial layer, developing at the interface of the wound. At 21

day post-wounding, the CsADM-Cl-treated group showed a

clear, intense expression of CK-10 in the epithelial layer,

revealing complete regeneration and resembling of the NS

tissue architecture. However, the CCol-Cl-treated group

showed that the thick epithelium and expression of CK-10

was mild comparatively, while the in control-treated group, the

expression of CK-10 was non-speciﬁc.

3.17. RT-PCR Analysis. To evaluate the molecular aspect

of healing in diﬀerent groups, the expression of genes

associated with skin wound healing (COL I, COL III, KRT

10, and KRT 14) was assessed, 21 days post-wounding. As

shown in Figure 6e,f, there was upregulation in the Col I

expression in the CsADM-Cl-treated group when compared to

the SHAM/CCol-Cl-treated group. This enhanced expression

can be credited to accelerated wound healing observed in the

CsADM-Cl-treated group. Interestingly, the downregulation in

the expression of Col III, KRT 10, and KRT 14 in the

CsADM-Cl-treated group when compared to other group

reveals that the re-epithelialization process was more complete

and the remodeling phase has started to regain the tissue native

strength.

CCol-Cl: 0.7026 0.1 cm, CsADM-Cl: 0.3187 0.05 cm)

and granulation area (control: 38.447 4.21 mm², CCol-Cl:

28.293

1.92 mm², CsADM-Cl: 18.368 mm²) further

decreased, whereas the length of reepithelization (control:

11.578 0.98 cm, CCol-Cl: 14.189 1.18 cm, CsADM-Cl:

16.749 1.48 cm) increased, indicating healing progression in

all the groups (Figure 7b−d).

At 21 days post-surgery, no wound was observed in groups

treated with CsADM-Cl, whereas CCol-Cl and control-treated

group showed a wound length of 0.514 0.09 and 0.319

0.08 cm, respectively (Figure 7b). The CsADM-Cl-treated

group exhibited complete re-epithelization with a deﬁned

epidermal−dermal junction, when compared to the CCol-Cl/

control group. The epidermal−dermal junction forms a surging

interdigitating interface (inset of Figure 7), which increases the

surface area and hence strengthening the contact between the

dermis and epidermis. Also, the CsADM-Cl-treated group

exhibited a signiﬁcantly higher number of newly formed

appendages (40 5) when compared to the CCol-Cl (12

2)/control (4

2)-treated group (Figure 7f). The newly

formed appendages in the wound area signify a better quality

of wound healing. Additionally, we also measured the thickness

of the epithelium post 21 days of surgery. The epidermis

thickness of the control, CCol-Cl, and CsADM-Cl-treated

groups was 97

8.21, 84

10.1, and 59

3.24 μm,

respectively (Figure 7e). The epidermal thickness of the

CsADM-treated group was much closer to the reported values

of the epidermal thickness of NS, that is, 42.0 7.1 μm.⁵⁷

As shown in Figure 8a, collagen deposition and arrangement

were visualized and analyzed using MT staining 21 days post-

wounding. Augmented deposition of collagen was observed in

CCol-Cl- and CsADM-Cl-treated groups when compared to

the SHAM group. However, collagen deposition in the dermis

was more organized with a basket weave pattern in the

CsADM-Cl-treated group. Also, densely packed random

collagen ﬁbers were observed in the CCol-Cl-treated group

and SHAM group exhibited partial dysplastic collagen ﬁbers.

Quantitative image analysis of MT-stained sections revealed

acollagen index (intensity of collagen deposition) of 0.42

0.02, 0.74 0.06, and 0.639 0.04 for SHAM, CCol-Cl, and

CsADM-Cl, respectively (Figure 8a,b). The collagen index of

CsADM-Cl was closest to the collagen index of NS, as reported

earlier (i.e. 0.601 0.038).⁵⁷

In wound healing, dermal regeneration, re-epithelialization,

and formation of the secondary structure play a pivotal role to

restore tissue functionality, strength, and aesthetic view.⁵⁸It

has been widely reported that cytokines and growth factors are

essential for accelerating wound healing, regarding enhanced

wound closure and re-epithelialization.^59,60Therefore, accel-

erated healing in the CsADM-Cl-treated group can be

attributed to embedded native proteins, GAGs, cytokines,

growth factors, and also to the unique antibacterial and ROS

scavenging activity of the hydrogel variant.

3.16. Immuno Histochemical Analysis. Angiogenesis is

a fundamental necessity for rapid wound closure, especially in

the case of chronic wounds as blood vessels increase the inﬂow

of oxygen and nutrients to the wound area and henceforth

promote healing therein.^61,63In the present study, angiogenesis

through the in vivo model was assessed via CD31

immunoﬂuorescence staining, as shown in Figure 8c,d.

Compared to SHAM, the CCol-Cl treated group showed

signiﬁcantly (p < 0.05) a higher number of blood vessels at day

4. DISCUSSION

Bioactive matrices with appropriate physiochemical and

biological properties are required to induce rapid wound

closure to support cell inﬁltration, adhesion, and diﬀerentiation

to regain tissue morphology and function. It has been

established in the literature that burn wounds, if healed within

21 days, exhibit minimal scar formation.⁶⁴In the present work,

a rapid injectable bioactive hydrogel was developed by dual

cross-linking of the sADM. The compositional training allows

the hydrogels to preserve their inherent biochemical

composition and also lending the gel a unique antioxidant

and antibacterial property. Although sADMs from varied

origins have been utilized either alone or as a hybrid for tissue

engineering, minimal reports are available for an antibacterial

sADM-based hydrogel with retained bioactive cues. Our study

has utilized DHF-I as a crosslinker for the development of an

injectable sADM-based hydrogel with inherent antibacterial

property for application in critical burn wounds. Perhaps, this

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is the ﬁrst report to utilize DHF-I crosslinker for wound

healing study.

is required. Herein, we have taken low molecular-weight CTS

for the study since it has been reported for its antibacterial

property. Further, DHF-I crosslinking synergizes with the

polymer’s inherent property, and the hybrid is observed to

have excellent antibacterial property against E. coli and S.

aureus taken for the study. The antimicrobial activity of iodine

has been reported not only against bacteria but also fungi,

tubercle bacilli, and viruses; additionally, bacteria does not

develop resistance against iodine unlike antibiotics. Iodine is a

vital component of the thyroid hormone and hence is

biologically safe and can be excreted by the kidneys.¹¹The

antibacterial property therefore can be attributed to the release

of iodine from the crosslinked hybrid hydrogel. It has also been

reported in the literature that iodine has antibacterial property

owing to its irreversible reaction to tyrosine residues/sites of

unsaturation in lipids, oxidation of the sulfhydryl group, and

interference in the H-bonding of certain amino acid/nucleic

acid.⁷⁴The release of iodine may be due to the instability of

the di-iodinated compound in room temperature/light leading

to the tendency to revert to iodine and oleﬁn. Iodine, being an

acceptor, is able to form complexes with donor compounds.

The complex is reported to be weak when formed with ethers,

esters, benzene, and ketones using polar solvents, while it

forms a strong complex with the amino group-containing

compounds like triethylamine/pyridine.⁷⁵However, the exact

mechanism of the release of elemental iodine from the hybrid

is not established. Thus, antibacterial CsADM-Cl-containing

peptides, GAGs, cytokines, and growth factors can act as an apt

matrix for facilitating rapid wound closure when compared to

CCol-Cl.

Skin mainly consists of ﬁbroblasts and keratinocytes, while

stem cells and melanocytes are present in a small number.

Fibroblasts or keratinocytes or both have been utilized for the

assessment of graft potential as a skin substitute in vitro. Hence,

both ﬁbroblast and keratinocytes were selected for in vitro

biological evaluation of the hydrogel. In the present study,

conditioning media of the hydrogels revealed the absence of

any toxic remnant chemicals, while promoted cell adhesion,

migration, and proliferation of cells (HFCs and HKCs). All

these cell fate processes are conﬁrmed using a spectrum of

assays, including MTT assay, scratch assay, and rhodamine &

DAPI staining. The hybrid hydrogel showed uniform viable

cellularization on blending the pregel solution and ﬁbroblast

cells, that is, as demonstrated by Live/Dead staining post 3

days of encapsulation. Further, no apoptotic cell could be

observed in any of the hydrogel posts 7 days seeding, revealing

the excellent cytocompatibility of the hydrogels. In addition,

cells were observed to be actively proliferating on hydrogel-

coated coverslips when stained with PCNA staining.

Interestingly, statistically higher (p < 0.001) PCNA positive

cells were found on CsADM-Cl-coated coverslips. This may be

attributed to bioactive molecules in CsADM-Cl.

In our processing approach, ADM was decellularized using a

combinatorial approach, and to lessen surgical trauma due to

scaﬀold implantation in particular, an injectable hydrogel of a

decellularized tissue was developed using enzymatic (pepsin)

digestion, as reported in the literature.²⁹The biggest obstacle

for decellularization is to get rid of cellular components

(including RNA and DNA) and hence minimizing the immune

response, while retaining the inherent bioactive molecules.

DAPI and H&E staining of NS and ADM reveal the absence of

cells in the ADM; further, DNA quantiﬁcation supported

successful decellularization of the ADM. The DNA content of

the ADM (30.14

12.34 ng/mg dry wt) is less than the

minimal criterion (50 ng/mg dry wt) required for complete

decellularization.⁶⁵To emulate the intricacy of the NS, the

ADM must retain multifarious structural and functional

proteins, GAGs, glycoproteins, and bioactive cues (growth

factors and cytokines), which are the unique features of the

NS. Both NS and ADM were observed to have non-signiﬁcant

diﬀerence in collagen and GAG contents, and these results

were consistent with the literature.³⁶Further, SDS-PAGE of

NS and ADM revealed the presence of several similar bands of

similar intensities revealing the complexity of the ECM which

was maintained during the decellularization process. Moreover,

ELISA assay showed the presence of TGF-β, VEGF, and BMP-

2 in both NS and ADM. TGF-β plays an essential role in cell

proliferation and diﬀerentiation, immune response, angio-

genesis, and tissue repair. It also assists in the production of a

new matrix in the graft and promotes graft acceptance.^66,67

BMP2 serves as a chemotactic guide to graft for host cells,

induces adipogenesis in soft tissue spaces, and promotes stem

cell diﬀerentiation.⁶⁸VEGF is reported to induce cell

migration and neovascularization in the newly formed tissue.⁶⁹

An ideal skin substitute should mimic the native ECM

proteins structurally that provides mechanical support and also

biologically active that helps in regulating cellular activities. In

native tissue ECM, collagen ﬁbrils exist in a 3D network

structure composed of multi-layered nano ﬁbrils (10−500

nm).⁷⁰Our fabricated hybrid hydrogels are 3D networks which

are ﬁbrillar owing to phase separation induced by physiological

pH and temperature, as demonstrated by SEM (ﬁbril size: 90−

120 nm). Interestingly, DHF-I cross-linking did not aﬀect the

self-assembly of ﬁber formation in the hydrogel although the

ﬁber diameter reduced after crosslinking, yet it was not

signiﬁcantly diﬀerent from the uncrosslinked variant. Also, the

triple helix structure of collagen was intact in both CCol-Cl

and CsADM-Cl, as demonstrated by FTIR data. CsADM-Cl

immunostaining revealed the presence Col I, ﬁbronectin, Col

III, VEGF, IL-8, and MCP-1. IL-8 is reported to promote skin

re-epithelialization by increasing keratinocyte migration, and

MCP-1 has been utilized in the literature to promote dermal

wound healing by acting as a chemo attractant for the

cells.⁷¹⁻⁷³DHF-I crosslinking enhanced the mechanical

strength of the hydrogel; crosslinked hydrogels were

approximately double in strength when compared to the

uncrosslinked variant (as revealed by rheological study).

Wound healing may be hindered by the bacterial load

present on the chronic wounds since the high bacterial load

present on the wound site delays the wound healing by

upregulating the MMP production, which in turn adversely

aﬀects collagen deposition and remodeling. To reduce the

wound region’s bacterial load, the antibacterial skin substitute

Angiogenesis is a vital aspect in the wound healing cascade,

which governs the initial granulation tissue formation and

tissue remodeling.^61,62Moreover, insuﬃcient vasculature can

cause necrosis, ultimately leading to the rejection of the graft.

In this context, the angiogenic potential of CsADM-Cl was

demonstrated by ex vivo CAM assay. The CsADM-Cl hydrogel

implant was observed to attract signiﬁcantly a higher number

of allantoic vessels when compared to CCol-Cl hydrogel-

implanted CAM. The angiogenic property may be assigned to

growth factors (TGF-β and VEGF), preserved during the

decellularization and fabrication process. The good injectability

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of the crosslinked hydrogel was established by injecting the

pregel solution into the rat subcutaneous tissue. The qualitative

and quantitative outcome of in vivo experiments clearly

indicated that the new hybrid hydrogel is suitable as an

injectable material for minimally invasive surgery. Histological

assessment of the hydrogels revealed in vivo degradation of the

hydrogel and also good attachment of the remnant hydrogel to

the host tissue was observed. The degradation of the hydrogel

would release many bioactive components, such as peptides,

which are useful for recruitment of cell and tissue remodeling,

although many components are still unknown. The CsADM-

Cl-injected group showed more inﬁltration of cells when

compared to the CCol-Cl-injected group. The cell inﬁltration

into the scaﬀolds is especially useful for tissue remodeling and

regeneration. In addition, no immune cell or capsular protein

layer encapsulating the hydrogel was observed in any of the

group. Iodine is known for its toxicity, so to ensure non-toxic

degradation of the hydrogel, vital organs were harvested and

histological evaluation was performed. All the organs showed a

similar histological morphology to the control/untreated

group, and characteristic morphological features can be easily

identiﬁed revealing the non-toxic nature of CCol-Cl and

CsADM-Cl upon degradation.

tion, pepsin digestion, and crosslinking. The newly fabricated

CsADM-Cl hydrogel successfully retained cytokines and

growth factors, which have the potential to guide the tissue

regeneration even in chronic conditions. The CsADM-Cl

hydrogel exhibited a ﬁbrous morphology and also, gel strength

can be tailored by altering crosslinker’s concentration. The

CsADM-Cl exhibited excellent antibacterial activity against E.

coli and S. aureus and good cytocompatibility with HFCs and

HKCs in vitro. HFCs were easily encapsulated into the

CsADM-Cl hydrogel and proliferated well inside. Importantly,

the CsADM-Cl hydrogel allowed a rapid cell inﬁltration in vitro

and in vivo. It possessed superior angiogenic potential and was

observed to be nonimmunogenic without toxicity to major

organs studied. Moreover, the CsADM-Cl hydrogel-treated

wounds were completely healed with well-deﬁned DEJ and

more pronounced secondary appendages. Altogether, our

study clearly demonstrates that the CsADM-Cl hydrogel

with its nanoﬁbrillar topography, physiochemical composition,

antibacterial activity, and injectability can serve as an apt graft

for rapid wound healing in chronic wounds.

ASSOCIATED CONTENT

■

sı

The biomedical potential of CCol-Cl and CsADM-Cl to

treat chronic wounds was assessed in thermal burn wounds

created in a rat model. The ECM from the tissue of origin is

the best alternative for regeneration as it contains all proteins,

proteoglycans, glycosaminoglycans, cytokines, and growth

factors required for the normal functioning of a tissue.⁷⁰

Also, the presence of iodine in the hydrogel protected the

wound bed from bacterial infection and free-radical load,

which otherwise can delay the healing progression therein.^76,77

CsADM-Cl-treated wounds were observed to be rapidly

closing with enhanced re-epithelialization when compared to

the control- or CCol-Cl-treated group. Moreover, the CsADM-

Cl-treated wounds showed the development of more organ-

ized/structured collagen deposition, which is much closer to

the distribution of collagen in the native tissue. Further, the

collagen index of CsADM-Cl-treated wounds was close to the

NS as reported in the literature. The presence of proteins,

GAGs, cytokines, and growth factors (CK-8, MCP-1, TGF-β,

VEGF, and others not quantiﬁed) in CsADM-Cl along with a

nanoscale ﬁbrillar topology provided ambient biochemical and

physiological cues for rapid tissue regeneration. Gene proﬁling

post 21 days for the healing tissue also supported our data.

COL I was upregulated in all the group revealing the

progression of the healing process, that is, conversion of

COL III to COL I, with the expression being strongest in

CsADM-Cl. In addition, COL III, KRT 10, and KRT 14 were

down-regulated in CsADM-CL, demonstrating more complete

tissue regeneration when compared to SHAM/CCol-Cl. Taken

together, our study demonstrates that CsADM-Cl hydrogel

promoted rapid wound closure, enhanced neovascularization,

and complete skin regeneration post 21 days of wounding with

superior well-deﬁned dermal−epidermal junctions (DEJ),

collagen index native to NS, and skin appendages. Thus,

CsADM-Cl hydrogel holds signiﬁcant potential to serve as a

unique graft for superior treatment of dermal wounds in

clinical applications.

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.biomac.0c01400.

Histological studies, DNA quantiﬁcation, SDS PAGE,

sGAG assay, collagen quantiﬁcation, and primer

sequences (PDF)

AUTHOR INFORMATION

■

Corresponding Author

Santanu Dhara − Biomaterials and Tissue Engineering

Laboratory, School of Medical Science and Technology,

Indian Institute of Technology Kharagpur, Kharagpur

721302, India; orcid.org/0000-0003-4443-7610;

Email: sdhara@smst.iitkgp.ernet.in

Authors

Kamakshi Bankoti − Biomaterials and Tissue Engineering

Laboratory, School of Medical Science and Technology,

Indian Institute of Technology Kharagpur, Kharagpur

721302, India

Arun Prabhu Rameshbabu − Biomaterials and Tissue

Engineering Laboratory, School of Medical Science and

Technology, Indian Institute of Technology Kharagpur,

Kharagpur 721302, India

Sayanti Datta − Biomaterials and Tissue Engineering

Laboratory, School of Medical Science and Technology,

Indian Institute of Technology Kharagpur, Kharagpur

721302, India

Piyali Goswami − Department of Biotechnology, Indian

Institute of Technology Kharagpur, Kharagpur 721302,

India

Madhurima Roy − Department of Biotechnology, Indian

Institute of Technology Kharagpur, Kharagpur 721302,

India

Dipankar Das − Polymer Chemistry Laboratory, Department

of Applied Chemistry, Indian Institute of Technology (Indian

School of Mines), Dhanbad 826004, India; orcid.org/

0000-0001-9298-6871

5. CONCLUSIONS

An injectable hybrid hydrogel CsADM-Cl derived from the rat

dermal tissue was developed through modiﬁed decellulariza-

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Sudip Kumar Ghosh − Department of Biotechnology, Indian

Institute of Technology Kharagpur, Kharagpur 721302,

India

Amit Kumar Das − Department of Biotechnology, Indian

Institute of Technology Kharagpur, Kharagpur 721302,

India

Analava Mitra − Natural Products Research Laboratory,

School of Medical Science and Technology, Indian Institute of

Technology Kharagpur, Kharagpur 721302, India;

orcid.org/0000-0003-2597-0794

Sagar Pal − Polymer Chemistry Laboratory, Department of

Applied Chemistry, Indian Institute of Technology (Indian

School of Mines), Dhanbad 826004, India; orcid.org/

0000-0002-3425-7010

Dhrubajyoti Maulik − Department of Surgery, Bankura

Sammilani Medical College, Bankura 722102, India

Bo Su − Bristol Dental School, University of Bristol, Bristol

BS1 2LY, U.K.

Paulomi Ghosh − Structural Biology and Bioinformatics,

CSIR-Indian Institute of Chemical Biology (CSIR-IICB),

Kolkata 700032, India

Bikramajit Basu − Materials Research Center, Indian Institute

of Science, Bangalore 560012, India

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Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

Authors acknowledge fellowship received from Department of

Science and Technology (DST, Govt. of India) for K.B. and

Ministry of Human Resource Development (MHRD, Govt. of

India) for S.D. and A.P.R. Nantu Dogra is acknowledged for

helping in laboratory experiments. Project (BT/PR7818/

MED/32/279/2013) funding from the Department of

Biotechnology is acknowledged.

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Dual Functionalized Injectable Hybrid Extracellular Matrix Hydrogel for Burn Wounds

DOI: 10.1021/acs.biomac.0c01400

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