Bilayer Cryogel Wound Dressing and Skin Regeneration Grafts for the

Subscriber access provided by UNIV OF NEBRASKA - LINCOLN

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 43

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

ACS Applied Materials & Interfaces

1

Bilayer Cryogel Wound Dressing and Skin Regeneration Grafts

2

for the Treatment of Acute Skin Wounds

3

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

4

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

5

2, 3

and Ashok Kumar1, 2,*

6

1

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

7

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

8 9

3

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

10

11

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)

23

#

24

Department of Biomedical Engineering; Saint Louis University;

25

Saint Louis, MO, USA, 63103

26

$

27

Biochemical Sciences Division, CSIR-National Chemical Laboratories,

28

Pune-411008, Maharashtra, India

Present address:

Present address:

29

1

ACS Paragon Plus Environment


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

Page 2 of 43

30

Abstract

31

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

32

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

33

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

34

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

35

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

36

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

37

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

38

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

39

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

40

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

41

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

42

proliferation of fibroblasts and keratinocytes. Microparticles loaded with bioactive molecules

43

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

44

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

45

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

46

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

47

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

48

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

49

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

50

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

51

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

52

bilayer incorporated with human fibrinogen microparticles showed early signs of skin

53

regeneration; epidermis formation occurred at two weeks of implantation.

54

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

55

fibrinogen 2


Page 3 of 43

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


56

Introduction

57

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

58

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

59

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

60

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

61

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

62

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

63

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

64

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

65

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

66

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

67

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

68

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

69

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

70

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

71

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

72

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

73

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

74

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

75

dressings5–14.

76

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

77

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

78

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

79

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

80

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

81

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



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

Page 4 of 43

82

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

83

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

84

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

85

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

86

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

87

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

88

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

89

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

90

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

91

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

92

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

93

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

94

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

95

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

96

wound healing24,25.

97

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

98

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

99

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

100

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

101

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

102

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

103

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

104

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

105

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

106

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

107

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


Page 5 of 43

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


108

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

109

scaffolds were also incorporated with gelatin microparticles containing bioactive molecules to

110

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

111

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

112

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

113

Material and methods

114

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

115

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

116

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

117

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

118

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

119

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

120

(25%),

121

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

122

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

123

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

124

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

125

and reagents used were of analytical grade.

126

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

127

Synthesis of polyvinylpyrrolidone cryogel sheet

128

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

129

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

130

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

131

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

132

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

recrystallized

iodine,

ammonium

persulfate

(APS),

N,N,N′,N′-

5



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

Page 6 of 43

133

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

134

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

135

deionized (DI) water.

136

Coupling and estimation of iodine onto PVP cryogel sheets

137

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

138

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

139

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

140

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

141

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

142

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

143

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

144

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

145

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

146

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

147

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

148

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

149

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

150

calculated.

151

Preparation of gelatin cryogel regenerative layer

152

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

153

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

154

Synthesis of gelatin microparticles incorporated with M6P and human fibrinogen

155

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

156

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

6


Page 7 of 43

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


157

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

158

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

159

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

160

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

161

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

162

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

163

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

164

and were incorporated in gelatin cryogel as follows.

165

Synthesis of gelatin cryogel scaffold with and without synthesized microparticles

166

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

167

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

168

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

169

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

170

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

171

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

172

stored for further use.

173

Physicochemical characterizations

174

Iodine release from PVP-I cryogel sheets

175

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

176

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

177

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

178

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

179

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

180

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

181

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

182

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



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

Page 8 of 43

183

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

184

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

185

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

186

by titration with 0.01 N sodium thiosulphate32.

187

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

188

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

189

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

190

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

191

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

192

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

193

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

194

following formula

195

=

196

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

197

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

198

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

199

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

200

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

201

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

202

microparticles were then resuspended in fresh buffer.

203

Swelling kinetics

204

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

205

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

206

equilibrium was reached.

207

The water uptake capacity was determined as:

8


Page 9 of 43

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


208

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

209

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

210

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

211

Microstructure analysis

212

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

213

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

214

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

215

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

216

reconstruction of the scanned images using CT-volume software.

217

Flow rate and mercury porosimetry

218

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

219

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

220

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

221

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

222

any back pressure34.

223

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

224

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

225

pore diameter, D, was determined as per Wasburn equation

226

D = (4g cos θ)/ P

227

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

228

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

229

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

230

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

231

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

232

determined34.

233

Mechanical testing: compression and tensile strength 9



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

Page 10 of 43

234

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

235

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

236

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

237

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

238

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

239

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

240

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

241

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

242

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

243

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

244

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

245

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

246

(%) curve.

247

Degradation rate of gelatin cryogel scaffold

248

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

249

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

250

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

251

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

252

final dry weight of the samples35.

253

DD = (M1-M2)/M1x100

254

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

255

M2 - Dry weight of the samples after incubation.

256

In vitro biocompatibility of gelatin cryogel scaffolds

257

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

258

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


Page 11 of 43

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


259

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

260

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

261

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

262

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

263

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

264

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

265

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

266

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

267

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

268

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

269

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

270

were carried out individually on fibroblasts L929 and keratinocytes A431.

271

Microscopic analysis

272

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

273

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

274

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

275

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

276

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

277

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

278

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

279

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

280

Peel test on porcine skin

281

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

282

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

283

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

284

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



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

Page 12 of 43

285

Antibacterial activity of the bilayer cryogel sheets

286

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

287

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

288

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

289

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

290

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

291

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

292

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

293

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

294

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

295

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

296

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

297

showed no turbidity.

298

Cytotoxicity testing of bilayer cryogel sheets

299

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

300

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

12


Page 13 of 43

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


309

(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

13



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

Page 14 of 43

328

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



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

Page 34 of 43

757 758

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


Page 35 of 43

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


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

References

788

(1)

Shevchenko, R. V; James, S. L.; James, S. E. A Review of Tissue-Engineered Skin Bioconstructs Available for Skin Reconstruction. J. R. Soc. Interface 2010, 7, 229–258.

789 790

(2)

Papini, R. Management of Burn Injuries of Various Depths. BMJ 2004, 329, 158–160.

791

(3)

Ulubayram, K. EGF Containing Gelatin-Based Wound Dressings. Biomaterials 2001, 22, 1345–1356.

792 793

(4)

Ru, C.; Wang, F.; Pang, M.; Sun, L.; Chen, R.; Sun, Y. Suspended, Shrinkage-Free,

794

Electrospun PLGA Nanofibrous Scaffold for Skin Tissue Engineering. ACS Appl.

795

Mater. Interfaces 2015, 7, 10872–10877.

796

(5)

Mi, F. L.; Wu, Y. B.; Shyu, S. S.; Schoung, J. Y.; Huang, Y. B.; Tsai, Y. H.; Hao, J. Y.

797

Control of Wound Infections Using a Bilayer Chitosan Wound Dressing with

798

Sustainable Antibiotic Delivery. J. Biomed. Mater. Res. 2002, 59, 438–449.

799

(6)

Fox, C. L.; Rappole, B. W.; Stanford, W. Control of Pseudomonas Infection in Burns by

35



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

Silver Sulfadiazine. Surg. Gynecol. Obstet. 1969, 128, 1021–1026.

800 801

Page 36 of 43

(7)

Saymen, D. G.; Nathan, P.; Holder, I. A.; Hill, E. O.; Macmillan, B. G. Control of

802

Surface Wound Infection: Skin versus Synthetic Grafts. Appl. Microbiol. 1973, 25, 921–

803

934.

804

(8)

Robb, E. C.; Nathan, P. Control of Experimental Burn Wound Infections: Comparative

805

Delivery of the Antimicrobial Agent (silver Sulfadiazine) Either from a Cream Base or

806

from a Solid Synthetic Dressing. J. Trauma 1981, 21, 889–893.

807

(9)

Bianco, C.; Adami, G.; Crosera, M.; Larese, F.; Casarin, S.; Castagnoli, C.; Stella, M.;

808

Maina, G. Silver Percutaneous Absorption after Exposure to Silver Nanoparticles: A

809

Comparison Study of Three Human Skin Graft Samples Used for Clinical Applications.

810

Burns 2014, 40, 1390–1396.

811

(10)

Sarhan, W. A.; Azzazy, H. M. E.; El-Sherbiny, I. M. Honey/Chitosan Nanofiber Wound

812

Dressing Enriched with Allium Sativum and Cleome Droserifolia: Enhanced

813

Antimicrobial and Wound Healing Activity. ACS Appl. Mater. Interfaces 2016, 8, 6379–

814

6390.

815

(11)

Rigo, C.; Ferroni, L.; Tocco, I.; Roman, M.; Munivrana, I.; Gardin, C.; Cairns, W. R. L.;

816

Vindigni, V.; Azzena, B.; Barbante, C.; Zavan, B. Active Silver Nanoparticles for

817

Wound Healing. Int. J. Mol. Sci. 2013, 14, 4817–4840.

818

(12)

Kuroyanagi, Y.; Kim, E.; Kenmochi, M.; Ui, K.; Kageyama, H.; Nakamura, M.; Takeda,

819

A.; Shioya, N. A Silver-Sulfadiazine-Impregnated Synthetic Wound Dressing

820

Composed of Poly-L-Leucine Spongy Matrix: An Evaluation of Clinical Cases. J. Appl.

821

Biomater. 1992, 3, 153–161.

822

(13)

Collagen Dressing Containing Antibiotics. J. Biomed. Mater. Res. 1997, 36, 163–166.

823 824 825

Grzybowski, J.; Kołodziej, W.; Trafny, E. A.; Struzyna, J. A New Anti-Infective

(14)

Loke, W. K.; Lau, S. K.; Yong, L. L.; Khor, E.; Sum, C. K. Wound Dressing with Sustained Anti-Microbial Capability. J. Biomed. Mater. Res. 2000, 53, 8–17. 36


Page 37 of 43

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

826


(15)

Biomaterials 1993, 14, 1030–1035.

827 828

Matsuda, K.; Suzuki, S.; Isshiki, N.; Ikada, Y. Re-Freeze Dried Bilayer Artificial Skin.

(16)

Hinrichs, W. L.; Lommen, E. J.; Wildevuur, C. R.; Feijen, J. Fabrication and

829

Characterization of an Asymmetric Polyurethane Membrane for Use as a Wound

830

Dressing. J. Appl. Biomater. 1992, 3, 287–303.

831

(17)

1999, 17, 17–23.

832 833

Kramer, S. A. Effect of Povidone-Iodine on Wound Healing: A Review. J. Vasc. Nurs.

(18)

Nakao, H.; Yamazaki, M.; Tsuboi, R.; Ogawa, H. Mixture of Sugar and Povidone--

834

Iodine Stimulates Wound Healing by Activating Keratinocytes and Fibroblast

835

Functions. Arch. Dermatol. Res. 2006, 298, 175–182.

836

(19)

Regeneration. Tissue Eng. Part B. Rev. 2008, 14, 105–118.

837 838

Priya, S. G.; Jungvid, H.; Kumar, A. Skin Tissue Engineering for Tissue Repair and

(20)

Kössi, J.; Vähä-Kreula, M.; Peltonen, J.; Risteli, J.; Laato, M. Hexose Sugars

839

Differentially Alter Collagen Gene Expression and Synthesis in Fibroblasts Derived

840

from Granulation Tissue, Hypertrophic Scar and Keloid. Arch. Dermatol. Res. 2004,

841

295, 521–526.

842

(21)

Viera, M. H.; Amini, S.; Konda, S.; Berman, B. Do Postsurgical Interventions Optimize

843

Ultimate Scar Cosmesis. G. Ital. di dermatologia e Venereol. organo Uff. Soc. Ital. di

844

dermatologia e Sifilogr. 2009, 144, 243–257.

845

(22)

of Keloids and Hypertrophic Scars. J. Clin. Aesthet. Dermatol. 2010, 3, 20–26.

846 847

Viera, M. H.; Amini, S.; Valins, W.; Berman, B. Innovative Therapies in the Treatment

(23)

Li, B.; Clemons, T. D.; Agarwal, V.; Kretzmann, J.; Bradshaw, M.; Toshniwal, P.;

848

Smith, N. M.; Li, S.; Fear, M.; Wood, F. M.; Swaminathan Iyer, K. Regulation of

849

Collagen Expression Using Nanoparticle Mediated Inhibition of TGF-β Activation. New

850

J. Chem. 2016, 40, 1091–1095.

851

(24)

Donaldson, D. J.; Mahan, J. T. Fibrinogen and Fibronectin as Substrates for Epidermal 37



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

Cell Migration during Wound Closure. J. Cell Sci. 1983, 62, 117–127.

852 853

(25)

Geer, D. J.; Swartz, D. D.; Andreadis, S. T. Fibrin Promotes Migration in a ThreeDimensional in Vitro Model of Wound Regeneration. Tissue Eng. 2002, 8, 787–798.

854 855

Page 38 of 43

(26)

Singh, D.; Vishnoi, T.; Kumar, A. Effect of Alpha-Ketoglutarate on Growth and

856

Metabolism of Cells Cultured on Three-Dimensional Cryogel Matrix. Int. J. Biol. Sci.

857

2013, 9, 521–530.

858

(27)

Gupta, A.; Bhat, S.; Jagdale, P. R.; Chaudhari, B. P.; Lidgren, L.; Gupta, K. C.; Kumar,

859

A. Evaluation of Three-Dimensional Chitosan-Agarose-Gelatin Cryogel Scaffold for the

860

Repair of Subchondral Cartilage Defects: An in Vivo Study in a Rabbit Model. Tissue

861

Eng. Part A 2014, 20, 3101–3111.

862

(28)

Mishra, R.; Goel, S. K.; Gupta, K. C.; Kumar, A. Biocomposite Cryogels as Tissue-

863

Engineered Biomaterials for Regeneration of Critical-Sized Cranial Bone Defects.

864

Tissue Eng. Part A 2014, 20, 751–762.

865

(29)

Lu, H.; Oh, H. H.; Kawazoe, N.; Yamagishi, K.; Chen, G. PLLA–collagen and PLLA–

866

gelatin Hybrid Scaffolds with Funnel-like Porous Structure for Skin Tissue Engineering.

867

Sci. Technol. Adv. Mater. 2016, 13, 064210.

868

(30)

Sudhakar, P.; Bhagyamma, S. N.; Siraj, S.; Sekharnath, K. V.; Rao, K. C.; Subha M. C.

869

S. Preparation and Characterization of Microspheres for Controlled Release of Anti HIV

870

Drug. J. Appl. Pharm. Sci. 2015, 5, 051–057.

871

(31)

Pharm. Sci. 2011, 73, 355–366.

872 873

(32)

Gradle, C.D.; Dee, A.O. Iodine-Propylene Glycol Teat Dip. US7,153,527 B2, December 26, 2006.

874 875

Mitra, A.; Dey, B. Chitosan Microspheres in Novel Drug Delivery Systems. Indian J.

(33)

Negrulescu, A.; Patrulea, V.; Mincea, M. M.; Ionascu, C.; Vlad-Oros, B. A.; Ostafe, V.

876

Adapting the Reducing Sugars Method with Dinitrosalicylic Acid to Microtiter Plates

877

and Microwave Heating. J. Braz. Chem. Soc. 2012, 23, 2176–2182. 38


Page 39 of 43

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

878


(34)

Jain, E.; Kumar, A. Designing Supermacroporous Cryogels Based on Polyacrylonitrile

879

and a Polyacrylamide-Chitosan Semi-Interpenetrating Network. J. Biomater. Sci. Polym.

880

Ed. 2009, 20, 877–902.

881

(35)

Deschamps, A. A.; van Apeldoorn, A. A.; Hayen, H.; de Bruijn, J. D.; Karst, U.;

882

Grijpma, D. W.; Feijen, J. In Vivo and in Vitro Degradation of Poly(ether Ester) Block

883

Copolymers Based on Poly(ethylene Glycol) and Poly(butylene Terephthalate).

884

Biomaterials 2004, 25, 247–258.

885

(36)

Aramwit, P.; Ratanavaraporn, J.; Ekgasit, S.; Tongsakul, D.; Bang, N. A Green Salt-

886

Leaching Technique to Produce sericin/PVA/glycerin Scaffolds with Distinguished

887

Characteristics for Wound-Dressing Applications. J. Biomed. Mater. Res. B. Appl.

888

Biomater. 2015, 103, 915–924.

889

(37)

Aramwit, P.; Bang, N.; Ratanavaraporn, J.; Ekgasit, S. Green Synthesis of Silk Sericin-

890

Capped Silver Nanoparticles and Their Potent Anti-Bacterial Activity. Nanoscale Res.

891

Lett. 2014, 9, 79.

892

(38)

2011.

893 894

(39)

(40)

Kumar,

A.

Supermacroporous

Cryogels:

Biomedical

and

Biotechnological

Applications.; CRC Press: Boca Raton, 2016.

897 898

Kumar, A.; Mishra, R.; Reinwald, Y.; Bhat, S. Cryogels: Freezing Unveiled by Thawing. Mater. Today 2010, 13, 42–44.

895 896

Jain, E.; Kumar, A. Wound Dressing Polymer Matrix. WO2011055388 A3, June 30,

(41)

Lamme, E. N.; Gustafsson, T. O.; Middelkoop, E. Cadexomer-Iodine Ointment Shows

899

Stimulation of Epidermal Regeneration in Experimental Full-Thickness Wounds. Arch.

900

Dermatol. Res. 290, 18–24.

901

(42)

Vogt, P. M.; Hauser, J.; Rossbach, O.; Bosse, B.; Fleischer, W.; Steinau, H.-U.; Reimer,

902

K. Polyvinyl Pyrrolidone-Iodine Liposome Hydrogel Improves Epithelialization by

903

Combining Moisture and Antisepis. A New Concept In Wound Therapy. Wound Repair 39



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

Regen. 2001, 9 (2), 116–122.

904 905

Page 40 of 43

(43)

Selvaggi, G.; Monstrey, S.; Van Landuyt, K.; Hamdi, M.; Blondeel, P. The Role of

906

Iodine in Antisepsis and Wound Management: A Reappraisal. Acta Chir. Belg. 2003,

907

103, 241–247.

908

(44)

Shih, J. S.; Merianos, J. J. Aqueous Stable Complex of a Strongly Swellable,

909

Moderately Crosslinked Polyvinylpyrrolidone and Iodine. US5242985 A, September 7,

910

1993.

911

(45)

Atemnkeng, M. A.; Plaizier-Vercammen, J.; Schuermans, A. Comparison of Free and

912

Bound Iodine and Iodide Species as a Function of the Dilution of Three Commercial

913

Povidone-Iodine Formulations and Their Microbicidal Activity. Int. J. Pharm. 2006,

914

317, 161–166.

915

(46)

Characterization and Uses. Polym. J. 1985, 17, 143–152.

916 917

(47)

(48)

(49)

dward, Starch-Iodine-Peroxide Preservation of Foods. WO1994009635 A1, May 11, 1994.

922 923

Rackur, H. New Aspects of Mechanism of Action of Povidone-Iodine. J. Hosp. Infect. 1985, 6 Suppl A, 13–23.

920 921

William, P.; Oliver, I. Iodine-Containing Germicidal Preparations and Method of Controlling Germicidal Activity. US4946673 A, August 7, 1990.

918 919

Haaf, F.; Sanner, A.; Straub, F. Polymers of N-Vinylpyrrolidone: Synthesis,

(50)

Bigi, A.; Cojazzi, G.; Panzavolta, S.; Rubini, K.; Roveri, N. Mechanical and Thermal

924

Properties of Gelatin Films at Different Degrees of Glutaraldehyde Crosslinking.

925

Biomaterials 2001, 22, 763–768.

926

(51)

Nguyen, T.-H.; Lee, B.-T. Fabrication and Characterization of Cross-Linked Gelatin Electro-Spun Nano-Fibers. J. Biomed. Sci. Eng. 2010, 03, 1117–1124.

927 928

(52)

Ikada, Y. Challenges in Tissue Engineering. J. R. Soc. Interface 2006, 3, 589–601.

929

(53)

Oh, S. H.; Kang, S. G.; Lee, J. H. Degradation Behavior of Hydrophilized PLGA 40


Page 41 of 43

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


930

Scaffolds Prepared by Melt-Molding Particulate-Leaching Method: Comparison with

931

Control Hydrophobic One. J. Mater. Sci. Mater. Med. 2006, 17, 131–137.

932

(54)

Raina, D. B.; Koul, R.; Bangroo, A.; Kumar, A. Fabrication Temperature Modulates

933

Bulk Properties of Polymeric Gels Synthesized by Different Crosslinking Methods. RSC

934

Adv. 2014, 4, 31855.

935

(55)

and Polymeric Biomaterials; Taylor & Francis: Boca Raton, 2016.

936 937

(56)

(57)

Rasmussen, C.; Thomas-Virnig, C.; Allen-Hoffmann, B. L. Classical Human Epidermal Keratinocyte Cell Culture. Methods Mol. Biol. 2013, 945, 161–175.

940 941

Su, K.; Wang, C. Recent Advances in the Use of Gelatin in Biomedical Research. Biotechnol. Lett. 2015, 37, 2139–2145.

938 939

Rahman, M. M. Gelatin: Tissue Engineering - Encyclopedia of Biomedical Polymers

(58)

C Kinikoglu, B. In Cell and Material Interface: Advances in Tissue Engineering,

942

Biosensor, Implant, and Imaging Technologies; Vrana, N. E., Ed.; CRC Press: Boca

943

Raton, 2015; Chapter 6, pp 147–162.

944

(59)

Dinarvand, R.; Mahmoodi, S.; Farboud, E.; Salehi, M.; Atyabi, F. Preparation of Gelatin

945

Microspheres Containing Lactic Acid--Effect of Cross-Linking on Drug Release. Acta

946

Pharm. 2005, 55, 57–67.

947

(60)

Mak, W. C.; Olesen, K.; Sivlér, P.; Lee, C. J.; Moreno-Jimenez, I.; Edin, J.; Courtman,

948

D.; Skog, M.; Griffith, M. Controlled Delivery of Human Cells by Temperature

949

Responsive Microcapsules. J. Funct. Biomater. 2015, 6, 439–453.

950

(61)

Mladenovska, K.; Kumbaradzi, E.; Dodov, G.; Makraduli, L.; Goracinova, K.

951

Biodegradation and Drug Release Studies of BSA Loaded Gelatin Microspheres. Int. J.

952

Pharm. 2002, 242, 247–249.

953

(62)

Eds.; Taylor and Francis: Boca Raton, 2012; Chapter 8.

954 955

Vibrational Spectroscopy for Tissue Analysis; Rehman, I.; Movasaghi, I.; Rehman, S;

(63)

McDonnell, G.; Russell, A. D. Antiseptics and Disinfectants: Activity, Action, and 41



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

Resistance. Clin. Microbiol. Rev. 1999, 12, 147–179.

956 957

Page 42 of 43

(64)

Damour, O.; Zhi Hua, S.; Lasne, F.; Villain, M.; Rousselle, P.; Collombel, C.

958

Cytotoxicity Evaluation of Antiseptics and Antibiotics on Cultured Human Fibroblasts

959

and Keratinocytes. Burns 1992, 18, 479–485.

960

(65)

Wilson, J. R.; Mills, J. G.; Prather, I. D.; Dimitrijevich, S. D. A Toxicity Index of Skin

961

and Wound Cleansers Used on in Vitro Fibroblasts and Keratinocytes. Adv. Skin Wound

962

Care 2005, 18, 373–378.

963

(66)

Müller, G.; Kramer, A. Biocompatibility Index of Antiseptic Agents by Parallel

964

Assessment of Antimicrobial Activity and Cellular Cytotoxicity. J. Antimicrob.

965

Chemother. 2008, 61, 1281–1287.

966

(67)

Horch, R. E.; Stark, G. B. Comparison of the Effect of a Collagen Dressing and a

967

Polyurethane Dressing on the Healing of Split Thickness Skin Graft (STSG) Donor

968

Sites. Scand. J. Plast. Reconstr. Surg. Hand Surg. 1998, 32, 407–413.

969 970 971 972 973 974 975 976 977 978 979 980 981 42


Page 43 of 43

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


982

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


Bilayer Cryogel Wound Dressing and Skin Regeneration Grafts for the

Recommend Documents