Bilayer Cryogel Wound Dressing and Skin Regeneration Grafts for the

May 25, 2016 - CSIR−Indian Institute of Toxicology Research, Lucknow-226 001, Uttar Pradesh, India. ABSTRACT: In this study, the potential of cryoge...
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Bilayer Cryogel Wound Dressing and Skin Regeneration Grafts for the Treatment of Acute Skin Wounds S. Geetha Priya, Ankur Gupta, Era Jain, Joyita Sarkar, Apeksha Damania, Pankaj R. Jagdale, Bhushan P. Chaudhari, Kailash C. Gupta, and Ashok Kumar ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b04711 • Publication Date (Web): 25 May 2016 Downloaded from http://pubs.acs.org on May 30, 2016

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Bilayer Cryogel Wound Dressing and Skin Regeneration Grafts

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for the Treatment of Acute Skin Wounds

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S. Geetha Priya1, Ankur Gupta1, Era Jain1,#, Joyita Sarkar1, Apeksha

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Damania1, Pankaj R. Jagdale3, Bhushan P. Chaudhari3,$, Kailash C. Gupta1,

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2, 3

and Ashok Kumar1, 2,*

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1

Department of Biological Sciences and Bioengineering; 2Centre for Environmental Sciences

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and Engineering, Indian Institute of Technology Kanpur, Kanpur- 208016, Uttar Pradesh, India

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CSIR- Indian Institute of Toxicology Research, Lucknow - 226 001, Uttar Pradesh, India

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12 13 14 15 16 17 18 19 20 21 22

*

Correspondence should be addressed to:

Ashok Kumar Tel: +91 512 2594051 Fax: +91 512 2594010 E-mail: [email protected] (A. Kumar)

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#

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Department of Biomedical Engineering; Saint Louis University;

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Saint Louis, MO, USA, 63103

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$

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Biochemical Sciences Division, CSIR-National Chemical Laboratories,

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Pune-411008, Maharashtra, India

Present address:

Present address:

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1

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Abstract

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In this study, potential of cryogel bilayer wound dressing and skin regenerating graft for the

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treatment of surgically created full thickness wound was evaluated. The top layer was

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composed of polyvinylpyrrolidone-iodine (PVP-I) cryogel and served as the antiseptic layer

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while the bottom regenerative layer was made using gelatin cryogel. Both components of the

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bilayer showed typical features of cryogel interconnected macropore network, rapid swelling,

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high water uptake capacity of about 90%. Both PVP and gelatin cryogel showed high tensile

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strength of 45 kPa and 10 kPa, respectively. Gelatin cryogel sheets were essentially elastic and

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could be stretched without any visible deformation. The antiseptic PVP-I layer cryogel sheet

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showed sustained iodine release and suppressed microbial growth when tested with skin

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pathogens (zone of inhibition ~2 cm for sheet of 0.9 cm diameter). The gelatin cryogel sheet

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degraded in vitro in weeks. The gelatin cryogel sheet supported cell infiltration, attachment and

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proliferation of fibroblasts and keratinocytes. Microparticles loaded with bioactive molecules

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(mannose-6-phosphate and human fibrinogen) were also incorporated in the gelatin cryogel

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sheets for their role in enhancing skin regeneration and scar free wound healing. In vivo

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evaluation of healing capacity of the bilayer cryogel was checked in rabbits by creating full

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thickness wound defect (diameter 2 cm). Macroscopic and microscopic observation at regular

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time intervals for 4 weeks demonstrated better and faster skin regeneration in the wound

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treated with cryogel bilayer as compared to untreated defect and the repair was comparable to

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commercial skin regeneration scaffold Neuskin-F®. Complete skin regeneration was observed

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after 4 weeks of implantation with no sign of inflammatory response. Defects implanted with

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cryogel having mannose-6-phosphate showed no scar formation, while the wound treated with

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bilayer incorporated with human fibrinogen microparticles showed early signs of skin

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regeneration; epidermis formation occurred at two weeks of implantation.

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Keywords: Cryogel, bilayer wound dressing, skin graft, mannose-6-phosphate, human

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fibrinogen 2

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Introduction

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There are several reasons where considerable loss of skin occurs like chronic wounds,

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traumatic accidents, burns, etc. In majority of such cases, possibility of skin regeneration is

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lost. Extensive and deep wounds which cannot be cured with common techniques may lead to

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death of the patient1. Most serious type is full thickness injuries in which all the regenerative

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elements are destroyed and healing occur from the edges with considerable contraction1,2. In

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case of injuries where large amount of skin is lost, immediate coverage of wound with dressing

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is required. It protects loss of fluids and proteins from wound area and prevents any bacterial

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invasion and subsequent damage of tissue. Additionally, it also shows regenerative capacity by

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promoting healing by providing support for proliferation of cells3,4. Severe problem occurs

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when wounds get infected by microorganisms, during the treatment. These organisms target

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beneath the surface of dressing at the wound site which leads to frequent change of the wound

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dressing causing damage to the healed area. Thus, it is necessary to prevent bacterial invasion

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and multiplication at the wound site. In past, antimicrobial creams were applied over the

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injured area which causes discomfort to the patients. Additionally, grafts, either coated or

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incorporated with germicidals such as antimicrobial agents and silver nanoparticles, have also

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been developed. But, major limitation associated with such grafts is that the antimicrobial

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activity diminishes as the graft is degraded, thereby impairing long-term protection from

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infection. Along with this, lots of nursing efforts are also required for the change of wound

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dressings5–14.

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To overcome these drawbacks, in recent past, researchers have focused on developing

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bilayer wound dressing. These types of wound dressings constitute top elastic external layer

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and lower sheet of soft layer. The external layer controls bacterial invasion and prevents wound

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surface dehydration. The soft underlying layer acts as scaffold which allows cell infiltration for

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tissue regeneration3,5,15,16. These bilayer wound dressings not only decrease chances of

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infection but also decrease the medical care required to change dressing and prevent damage 3

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caused to the newly formed epithelium during the change of dressing5. A commonly used anti-

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microbial agent is Povidone/Betadine, composed of iodine and polyvinylpyrrolidone (a

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synthetic polymer)17–19. Povidone has often been used as a skin cleanser.

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In addition to this, active research is being pursued to study the role of the bioactive

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molecules in the wound healing process. Several studies have shown that the presence of

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bioactive molecules like mannose-6-phosphate (M6P) accelerates wound healing and inhibits

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scar formation20. Previous studies have shown the role of M6P surface receptors in the

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proteolytic activation of TGF β. Thus, when M6P is injected into the wounds it competes for

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M6P receptors with latent M6P, therefore inhibiting the activation of TGF β 1 and TGF β 2

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that leads to reduction in fibrosis. Results of a phase I dose-escalation trial reveals M6P to be

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safe and also presence of M6P enhance epithelialization significantly21–23. Other bioactive

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molecule which is extensively studied for its role in wound healing is human fibrinogen. The

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presence of human fibrinogen considerably enhances the rate of epithelial cells migration over

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the wound surface thereby reducing the time of re-epithelialization and increasing rate of

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wound healing24,25.

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In this study, a bilayer wound dressing was developed using cryogelation technology.

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Cryogel matrix has large and interconnected pores which helps in cell infiltration in the

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matrices, resulting in homogenous distribution of cells and tissue formation26,27. They have

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high liquid absorptive capacity which would help in fluid retention and prevent accumulation

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of fluid in the wounds. The in vivo potential of cryogel as a scaffold for the repair and

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regeneration of different tissues like cartilage and bone have already been reported27,28. In the

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developed bilayer wound dressing, top external antiseptic layer was synthesized using

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polyvinylpyrrolidone polymer along with cotton as a support matrix, this cryogel matrix was

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further coupled with iodine; giving it antimicrobial property. And the soft underlying

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regenerative layer was synthesized using gelatin. Gelatin has some inherent advantage that it

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promotes cell adhesion and proliferation and has low antigenicity3,29. Moreover, gelatin cryogel 4

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scaffolds are biodegradable which could be degraded and replaced by regenerated tissue. These

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scaffolds were also incorporated with gelatin microparticles containing bioactive molecules to

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further enhance the healing process. The bilayer wound dressing was implanted on the

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surgically created full thickness wound on the rabbit and were monitored at regular time

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intervals to evaluate the healing potential of the cryogel bilayer dressing.

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Material and methods

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Gelatin from cold water fish skin, MW: ~ 60,000 (pI 6.0), Dulbecco’s Modified Eagles

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Medium (DMEM), Trypsin-EDTA, Penicillin-Streptomycin antibiotic, Polyethyleneglycol

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diacrylate (PEGda), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and

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fibrinogen from human plasma were purchased from Sigma-Aldrich (St. Louis, MO, USA).

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Fetal Bovine Serum (FBS) was purchased from Invitrogen (Carlsbad, CA, USA). N-vinyl

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pyrrolidone (NVP) was brought from Acros (New Jersey, USA). Glutaraldehyde solution

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(25%),

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tetramethylethylenediamine (TEMED), dimethyl sulfoxide (DMSO) and mannose-6-phosphate

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was obtained from Merck chemicals (Mumbai, India). Cell lines L929 and A431 were obtained

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from National Centre for Cell Sciences, Pune. Commercial skin graft, Neuskin-F®, has been

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obtained from Eucare Pharmaceuticals Private Limited, Chennai, India. All other chemicals

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and reagents used were of analytical grade.

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Preparation of polyvinylpyrrolidone–iodine (PVP-I) cryogel antiseptic layer

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Synthesis of polyvinylpyrrolidone cryogel sheet

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A 5% v/v NVP and 2% v/v PEGDa was prepared in degassed water. The solution was pre-

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cooled for 20 min at 4 °C and underwent three freeze thaw cycle of 20 min each. To this

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solution, 0.2% w/v of APS and 0.25% v/v of TEMED was added and mixed quickly and

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thoroughly. Thereafter, it was quickly transferred into pre-cooled plastic petri-plates with or

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without cotton as the adsorbent matrix. These petri-plates were then incubated in a methanol

recrystallized

iodine,

ammonium

persulfate

(APS),

N,N,N′,N′-

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bath maintained at -20 °C, for 12-16 h. The polyvinlylpyrrilodone (PVP) cryogel sheets (~2

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mm thick) were obtained by thawing the polymerized matrix and washing extensively with

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deionized (DI) water.

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Coupling and estimation of iodine onto PVP cryogel sheets

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Dried sheets of PVP cryogels were weighed and placed in a glass container. The sheets were

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swollen with DI water and then iodine equivalent to 1/5th quantity of the total dried weight of

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PVP cryogel sheet was added. The container was closed and placed in a dry air oven at 75 °C

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for 24 h. The iodine vapours formed in the process got complexed with the PVP cryogel sheets.

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The iodine complexed PVP cryogel sheets were then removed and washed with hexane till

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excess of uncomplexed iodine was removed. The iodine coupled PVP (PVP-I) cryogel sheets

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were lyophilized for future use.The available iodine was estimated by titration with 0.01N

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sodium thiosulphate. Dried and weighed iodine coupled cryogel sheets were taken in a glass

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test tube or beaker and 0.01N sodium thiosulphate was added dropwise. After the color of the

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sheet became pale yellow, 200 µl of 0.1% starch solution was added which turned the color of

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the solution violet. Again 0.01N sodium thiosulphate was added until the sheet becomes

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colorless. The volume of sodium thiosulphate solution consumed was recorded. From the

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volume of sodium thiosulphate consumed, amount of iodine complexed with the cryogels was

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calculated.

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Preparation of gelatin cryogel regenerative layer

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The regenerative layer was made using gelatin cryogel with or without the gelatin

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microspheres containing mannose-6-phosphate (M6P) or human fibrinogen (HF).

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Synthesis of gelatin microparticles incorporated with M6P and human fibrinogen

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Gelatin microparticles containing the M6P or HF were synthesized by emulsion polymerization

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method30,31. Briefly, aqueous solution of gelatin polymer (25% w/v) was mixed with equal

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volume of either 0.1 mg/ml of M6P or 3 mg/ml of HF. Ten ml of this solution was then added

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gradually to 30 ml of pre-homogenized mixture of heavy and light paraffin oils containing

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Span 80 (1% w/v) as stabilizer. The emulsion was allowed to form by high speed mixing at

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1200 rpm using three blade propeller for 5 to 10 min. Glutaraldehyde (2.5 ml of 25% v/v

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solution) was then added to above mix to give a final concentration of 6.25% v/v of gelatin.

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The mixture was stored for 2h at an increased speed of 1500 rpm. Finally, the microparticles

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were obtained by decanting the supernatant and washing with n-hexane to remove residual oil

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and were incorporated in gelatin cryogel as follows.

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Synthesis of gelatin cryogel scaffold with and without synthesized microparticles

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Briefly, the gelatin cryogel scaffold was synthesized by mixing 5% w/v aqueous gelatin (with

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or without the 0.1% w/v gelatin microparticles) with 0.125% v/v of glutaraldehyde. About 12

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ml of this solution was swiftly transferred into the pre-cooled 90 mm plastic petri-dishes. It

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was quickly transferred to methanol bath maintained at -12 °C and incubated for 16 h. Post

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incubation the cryogel scaffold with and without microparticles (~2 mm thick) were obtained

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by thawing in DI water at room temperature. Gelatin cryogel sheets were lyophilized and

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stored for further use.

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Physicochemical characterizations

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Iodine release from PVP-I cryogel sheets

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For determination of free iodine the pre-weighed and dried gels were incubated in hexane for 2

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h and the absorbance of the hexane solution was measured at 535 nm using fresh hexane as

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blank. The amount of iodine released was measured by a standard curve obtained by dissolving

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known concentration of molecular iodine in hexane and measuring its absorbance. PVP-I

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cryogel sheets (~2 x 2 cm, 175 mg) was submerged in 10 ml PBS. At regular time intervals of

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2, 4, 6, 24, 48, 72 and 96 h, 1 ml of the sample were removed and its absorbance was measured

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at 595 nm. The rate of release of iodine from PVP-I was determined in aqueous system using

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water. Pre-dried and pre-weighed sheets were placed in 10 ml of water in separate 7

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compartments. Water sample (1 ml) was removed every hour and the released amount of

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iodine was estimated. For the initial period of 4 h samples were collected every hour, thereafter

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the samples were collected at an interval of 24 h. The amount of iodine released was estimated

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by titration with 0.01 N sodium thiosulphate32.

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Incorporation and in vitro release of M6P and HF from gelatin microparticles

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M6P microparticles (250 mg) and HF microparticles (100 mg) were suspended in 0.1 M PBS,

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pH 7.4, sonicated for 1 min to break open the particles and then centrifuged at 3000 rpm for 10

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min. The supernatant was collected and assayed. M6P was estimated by dinitrosalicylic acid

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method for reducing sugars as reported elsewhere33. HF was estimated by Pierce® BCA

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protein assay kit according to the manufacturer’s instructions. Blank microparticles, without

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any active ingredients, were taken as control. The loading efficiency was calculated by

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following formula

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   =

   

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M6P microparticles (250 mg) and HF microparticles (100 mg) was added to 500 µl and

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200 µl of 5% gelatin solution, respectively with 0.125% glutaradehyde. The mixture was then

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frozen at -12 °C for 16 h to frm cryogel. M6P, HF and blank microparticles alone or

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incorporated in gelatin cryogel scaffolds were immersed in 0.1 M PBS, pH 7.4 and incubated

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at 37 °C. At each time point the samples were centrifuged at 3000 rpm for 5 min, the

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supernatant was collected and assayed for the presence of either M6P or HF. The

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microparticles were then resuspended in fresh buffer.

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Swelling kinetics

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Lyophilized cryogel sheets were immersed in de-ionized water and removed at regular time

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intervals. The excess water on the surface was wiped off and the gels were weighed until

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equilibrium was reached.

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The water uptake capacity was determined as:

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Wu = [(Mt-Mg)/Me] x 100

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where Wu is the water uptake capacity, Mt is the weight at regular time intervals, Mg is the

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weight of the xerogel and Me is the weight of swollen cryogel at equilibrium34.

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Microstructure analysis

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All microsphere and cryogel lyophilized samples were gold coated and analyzed using Zeiss

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EVO-18 scanning electron microscope, Germany. Gelatin cryogel scaffold (2 mm thick) were

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also analyzed using Bruker micro-CT Skyscan 1172 high resolution, Belgium, at a resolution

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of 11.5 µm. The overall architecture and average pore size and porosity was determined by 3D

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reconstruction of the scanned images using CT-volume software.

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Flow rate and mercury porosimetry

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The resistance to flow in gelatin and PVP cryogel (diameter 13 mm x thickness 20 mm) was

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measured by inserting either monolith into the plastic syringe that was connected to a

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peristaltic pump. Maximum rate at which the aqueous solvent can pass through the cryogel was

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examined by circulating water at a controlled speed up to a rate at which cryogel does not show

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any back pressure34.

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The porosity and pore size distribution of lyophilized gelatin cryogel sheets (1 x 1 cm) was

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determined using a mercury porosimeter (AMP-60K-L-A, Porous Materials Inc, USA). The

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pore diameter, D, was determined as per Wasburn equation

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D = (4g cos θ)/ P

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where P is the applied pressure; θ, contact angle of mercury on the surface (commonly

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accepted as 140°) was determined by mercury intrusion porosimeter and g the surface tension

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of mercury (484 dyn cm-1). The pore distribution and pore structure of the cryogel was

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determined by running the machine in a hysteresis mode at a maximum pressure of 10,000 psi.

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The critical pressure Pc, i.e. the minimum pressure required to intrude the largest pore was

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determined34.

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Mechanical testing: compression and tensile strength 9

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Gelatin cryogel monoliths were sliced into uniform parallel discs of diameter and thickness of

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13 mm. Samples were equilibrated in 0.1 M PBS (pH 7.4). The mechanical stability of

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cryogels was investigated by applying uniaxial compression using Zwick/Roell Z010 machine

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(Germany) by applying uniaxial compression (strain applied). Samples were compressed up to

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90% of their original length under a load cell of 10 kN at the displacement rate of 1 mm min-1.

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The compressive modulus of the gelatin cryogels was calculated from the slope of the graph

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obtained by stress (kPa) versus strain (%).

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For tensile strength measurement, gelatin and PVP-I cryogel sheets were cut into total

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length of 5.5 cm, breadth of 1.5 cm and for the gauge length of 2.5 cm. The samples were

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equilibrated with 0.1PBS (pH 7.4) and the elasticity was investigated by applying a load of 200

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g and at a cross-head speed of 0.5 mm/min with a chart speed of 20 mm/min (INSTRON 1195).

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The ultimate tensile strength of the material was determined from the stress (kPa) versus strain

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(%) curve.

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Degradation rate of gelatin cryogel scaffold

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Gelatin cryogels dried by lyophillization (1x1 cm) were weighed and sterilized by incubating

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in ethanol gradients (20 to 100 %). Each sample was incubated in sterile 15 ml 1X PBS. At

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regular time of 5 days, samples were removed, washed with DI water, lyophilized and the dry

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weight was measured. The degree of degradation was determined by the change in initial and

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final dry weight of the samples35.

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DD = (M1-M2)/M1x100

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where, DD - Degree of Degradation, M1 – Dry weight of the samples before incubation, and

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M2 - Dry weight of the samples after incubation.

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In vitro biocompatibility of gelatin cryogel scaffolds

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Fibroblast (L929) and human keratinocyte (A431) cell lines were cultured in DMEM media

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supplemented with 1% penicillin-streptomycin and 10% FBS in an incubator at 37 °C, 5% 10

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CO2. Gelatin cryogels (2-3 mm thickness and 1 mm diameter) were sterilized by treatment with

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70% ethanol. The cryogel scaffolds were washed 2-3 times with 1X PBS with 10-15 min

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incubation for each wash. Finally the cryogels were incubated in cell culture media for 12-20 h.

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To test the biocompatibility of the gelatin cryogel, 1 X 105 cells were seeded on each cryogel

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sample. The media was refreshed every 2nd day. Cell viability was measured at regular time

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intervals by MTT assay. Briefly, at each time point cryogel scaffolds containing cells were

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incubated with 1.5 ml basal media containing MTT (0.5 mg/ml) for 4 h at 37 °C in an

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incubator. Post incubation media containing MTT was removed and the purple formazan

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crystals formed were dissolved by incubating the cryogel in 1.5 ml of dimethyl sulfoxide

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(DMSO) for 15-20 min at RT with constant shaking. The DMSO solution was then collected

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and the absorbance of violet colored solution so obtained was read at 570 nm. Experiments

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were carried out individually on fibroblasts L929 and keratinocytes A431.

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Microscopic analysis

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The cells were allowed to grow for a period of 7 days in gelatin cryogel scaffolds. These were

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analyzed by SEM and fluorescent staining. The cell seeded scaffolds were washed with PBS

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and then fixed with 2.5% glutaraldehyde for 4 h. For SEM, the scaffolds were dehydrated in

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increasing gradient of ethanol, dried under vacuum, sputter coated with gold and observed

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under SEM. For fluorescent analysis 100 µm thick sections of cryogel scaffold were used for

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the study, the scaffolds were fixed in 2.5% glutaraldehyde as mentioned above, then

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permeabilized with 0.1% Triton X-100, stained with 100 µl of propidium iodide (1 µg/ml) for

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30 min and observed under confocal laser scanning microscope (CLSM).

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Peel test on porcine skin

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The peel test was done by a method described elsewhere with some modifications36. Briefly,

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wounds of depth 1 cm were created on the porcine skin and dressed with gelatin cryogel sheets.

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The scaffolds were either removed immediately (0 h) or after 24 h. The cell attachment to the

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sheets was analysed by MTT assay and fluorescent staining by DAPI as mentioned above. 11

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Antibacterial activity of the bilayer cryogel sheets

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PVP-I, PVP-I + gelatin (bilayer) sheets of 1 x 1 cm were equilibrated in 1X PBS. It was then

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placed in agar plates with lawn cultures of pathogenic strains Staphylococcus aureus (S.

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aureus) and Staphylococcus epidermidis (S. epidermidis). Further the plates were incubated at

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37 ºC for 24 h. After incubation the zone of inhibition (Z.I) i.e., the clear region surrounding

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the PVP-I sheets and PVP-I + gelatin sheets were measured. To further substantiate the

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antibacterial activity, the minimum amount of PVP-I required to inhibit bacterial growth was

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also analysed by a method described elsewhere with some modifications37. The PVP-I sheets

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were crushed in liquid nitrogen and different amounts (0.17-175 mg) obtained powder were

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suspended in 2 ml of Luria Bertani (LB) broth. To each of the PVP-I suspension, 1 ml of

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bacterial suspension (either S. aureus or S. epidermidis) was added with a count of 105

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CFU/ml. After incubation for 24 h at 37 °C, the MIC was recorded as the lowest amount that

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showed no turbidity.

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Cytotoxicity testing of bilayer cryogel sheets

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The toxicity of PVP-I (single layer) and PVP-I + gelatin (bilayer) to fibroblast cell lines was

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assessed by direct contact assay. The single layer and bilayer sheets were kept in direct contact

301

with an almost confluent layer of L929 cells. After 12 h of contact, the viability of the cells was

302

measured by MTT assay as mentioned above. The viability of the cells was compared to a

303

positive control which contained only cells and did not come in contact with the material. A

304

macroporous hydrogel of gelatin was also taken as a control to evaluate the effect of pressure

305

exerted by the weight of the cryogels on the growing cells.

306

In vivo studies in rabbit animal model

307

All in vivo experiments were conducted according to the guidelines of Institute Animal Ethical

308

Committee (IAEC) of Indian Institute of Toxicological Research (IITR), Lucknow, India

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(Reference number: ITRC/IAEC/13/2012). Thirty six, 3-month old healthy male white New

310

Zealand rabbits weighing 2-3 kg were used for experiments.

311

Surgical procedure

312

Before starting the surgery, rabbits were anaesthetized by giving intramuscular injection

313

consisting of a mixture having xylazine (8 mg/kg) and ketamine hydrochloride (40 mg/kg).

314

Thereafter, electric shaver was used to remove hairs from the dorsal area and skin was

315

sterilized using 70% ethanol. Two full thickness circular defects were created (diameter 2 cm)

316

on one rabbit. The wounds were treated with gelatin cryogel layer, gelatin cryogel layer

317

containing microparticles with either M6P or human fibrinogen. Each of these scaffolds was

318

implanted on four wounds for each time point (1st, 2nd, 3rd and 4th week after implantation). For

319

comparison, six wounds were treated with commercially available scaffold Neuskin-F®, a

320

Type I collagen film, and six wounds were left untreated (control) and were analysed on 1st and

321

4th week after implantation (Figure 1). After scaffold implantation, wound area was covered

322

completely with synthesized antiseptic layer, thus creating the bilayer system. Finally, the

323

wound area was covered with cotton gauze. After surgery, rabbits were housed individually in

324

the cages. To evaluate repair, rabbits were sacrificed by giving intracardial injection of

325

thiopentone, after 1st, 2nd, 3rd and 4th week of implantation. Specimens having whole wound

326

area were collected for further analysis.

327

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Figure 1. Flow chart showing experimental design of in vivo studies on rabbit.

329

Macroscopic and histological evaluation

330

After sacrificing the rabbits, the skin samples of wound site were collected and fixed in 10%

331

formalin saline. Further, samples were dehydrated by treating them with ethanol gradient.

332

Thereafter, they were embedded in paraffin. Sections of 5 µm were cut and mounted on glass

333

slide. For histological analysis, sections prepared were stained with hematoxylin and eosin.

334

Histological analysis was performed by experienced veterinary pathologist who did not had a

335

prior knowledge of the sample identity or experiment setup to avoid bias. Samples of all the

336

animals were analyzed for the wound healing. There 2 wounds per rabbit and 2 rabbits per

337

condition making it 4 per time point per condition. For each parameter entire histological

338

samples were analyzed with 10 fields taken under consideration. For comparison, histology of

339

normal rabbit skin was also performed.

340

Hematological analysis

341

To determine if the different treatments given generate any inflammatory response in rabbits,

342

hematological analysis was done. For this, blood was collected by puncturing ear vein of the

343

rabbits before surgery and just before sacrifice. Blood samples collected were analyzed for the

344

change in the level of inflammatory cells in response to different treatments.

345

Statistical analysis

346

All the experiments were carried out in triplicate and the results are represented as average ±

347

SD of 3-6 samples used per experiment. Single factor analysis of variance (ANOVA) mean ±

348

standard deviation. Two tailed student t-test was used to compare differences between two

349

groups. A value of difference and p 0.05).

33

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Figure 10. Hematological analysis of rabbits treated with (A) gelatin cryogels containing

759

microparticles incorporated with mannose-6-phosphate, (B) gelatin cryogels containing

760

microparticles incorporated with fibrinogen and (C) commercial scaffold (Neuskin-F®).

761

Conclusion and future direction

762

The bilayer cryogel wound dressing and skin regeneration graft showed potential for the

763

treatment of full thickness skin defect without any infection. The incorporation of iodine in

764

separate PVP cryogel in a separate layer allowed for sustained release of iodine. Also, the

765

antiseptic layer can be removed and easily replaced as per the requirement for long term

766

prevention from infection. Incorporation of microparticles having bioactive molecules like

767

M6P and human fibrinogen shows improvement in the process of repair. The gelatin cryogel

768

matrix alone or along with bioactive molecules does not generate any inflammatory response in

769

the rabbits. These cryogel scaffolds are biocompatible as well as biodegradable. Along with it,

770

its properties like high fluid absorption capacity also helps in faster and better healing of the

771

wound. These results were comparable to commercially available product (Neuskin-F®) used

772

for the repair of wound. In future, optimization of the dosage of the bioactive molecules used

773

for the repair of the wounds is desired. After the optimization, these cryogel scaffolds shows

774

the possibility to be used as a wound dressing.

34

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775

Conflict of Interests

776

The authors disclose no conflict of interests.

777

Acknowledgement

778

The authors would like to acknowledge Life Science Research Board, Defense Research and

779

Development Organisation, Govt. of India for funding this research project (Project No.

780

DLS/81/48222/LSRB-226/SHDD/2010). SGP would like to thank both IIT Kanpur and

781

Council for Scientific and Industrial Research (CSIR), India for providing research fellowship.

782

AG and EJ would like to acknowledge IIT Kanpur, India and JS would like to thank CSIR,

783

India for providing research fellowship. KCG acknowledges Indian Council of Medical

784

Research, New Delhi, India for awarding a distinguished scientist chair at CSIR-IGIB, Delhi-

785

110007. AK acknowledges TATA Innovation Fellowship from Department of Biotechnology,

786

Ministry of Science and Technology, Govt. of India.

787

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For Table of contents only

983

Bilayer Cryogel Wound Dressing and Skin Regeneration Grafts for the Treatment of

984

Acute Skin Wounds

985

Subramanian Geetha Priya1, Ankur Gupta1, Era Jain1,#, Joyita Sarkar1, Apeksha Damania1,

986

Pankaj R. Jagdale3, Bhusan P. Chaudhari3,$, Kailash C. Gupta1, 2, 3 and Ashok Kumar1, 2,*

987

43

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