the cyto-genotoxicity evaluation of biomedical grade...
TRANSCRIPT
THE CYTO-GENOTOXICITY EVALUATION OF BIOMEDICAL GRADE CHITOSAN ON PRIMARY KERATINOCYTE CULTURES AND ITS ROLE IN
SCAR FORMATION PATHWAYS USING SKIN COCULTURE MODEL
by
LIM CHIN KEONG
Thesis submitted in fulfillment of the requirements
for the degree of Doctor of Philosophy
July 2010
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ACKNOWLEDGEMENT
I would like to express my sincere appreciation and gratitude to my supervisor
Professor Dr. Ahmad Sukari Halim and Co-supervisor Associate Professor Dr. Nik
Soriani Yaacob for their invaluable support, guidance, motivation and constructive
suggestions throughout the entire period of my research work.
My sincere thanks are also extended to all the staffs at National Tissue Bank Unit
of Universiti Sains Malaysia (USM), Burn unit of hospital Universiti Sains Malaysia
(HUSM), Reconstructive Sciences unit, Centre Research Laboratory (CRL) and
Immunology Laboratory of School of Medical Sciences, Craniofacial Science
Laboratory of School of Dental Sciences and Molecular Biology Laboratory of School
of Health Sciences in USM, for their immeasurable assistance and support in providing
me the facilities to accomplish my experiments during period of my studies.
My special acknowledgement is also dedicated to my fellow lab-mates, Syaiful
Bahari, Nor Asiah, Siti Mahirah Yusof, Siti Nazmin Saifuddin for their help and co-
operation throughout my research. Also, my greatest appreciation goes to the Ministry of
Science Technology and Innovation (MOSTI), Malaysia, for funding me with the
National Science Fellowship (NSF) scholarship and a research grant, namely
Intensification of Research in Priority Area Program (IRPA) (No.: 03-03-01-0000-
PR0071/ 05).
Last but not least, my deepest and warmest gratitude to my beloved wife,
parents and brothers for their moral supports, encouragements, love, concern and never-
ending patience throughout the period of my studies.
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TABLE OF CONTENTS
Page
ACKNOWLEDGEMENT ii
TABLE OF CONTENTS iii
LIST OF TABLES xvi
LIST OF FIGURES xvii
LIST OF SYMBOLS AND ABBREVIATIONS xx
ABSTRAK xxvii
ABSTRACT xxix
CHAPTER 1 INTRODUCTION 1
1.1 Research background 1
1.2 Literature review 6
1.2.1 Structure and function of human skin 6
1.2.2 Cutaneous wound healing 9
1.2.2.1 Inflammation phase 9
1.2.2.2 Proliferation phase 10
1.2.2.3 Tissue remodeling 12
1.2.3 Chitosan as wound dressing 14
1.2.4 Biocompatibility in vitro of biomedical-grade chitosan 17
1.2.4.1 Cytocompatibility assessment in vitro 21
1.2.4.2 Genotoxicity assessment in vitro 23
1.2.4.3 Assessment of human skin pro-inflammatory
cytokines in vitro 26
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1.2.5 Human keloid disease 30
1.2.5.1 Histopathology and pathogenesis of keloid 30
1.2.5.2 Epithelial-mesenchymal interactions in keloid disease 34
1.2.5.3 Transforming growth factor beta (TGF-β) and
SMAD proteins in keloid pathogenesis pathway 38
1.3 Rationale and objectives of the study 44
CHAPTER 2 MATERIALS AND METHODS 46
2.1 Materials 46
2.1.1 Human skin samples 46
2.1.1.1 Primary human epidermal keratinocytes (pHEK) 46
2.1.1.1 (a) Primary normal human
epidermal keratinocytes (pNHEK) 46
2.1.1.1 (b) Primary keloid human
epidermal keratinocytes (pKHEK) 46
2.1.1.2 Primary human dermal fibroblasts (pHDF) 46
2.1.1.2 (a) Primary normal human dermal
fibroblasts (pNHDF) 46
2.1.1.2 (b) Primary keloid human dermal
fibroblasts (pKHDF) 46
2.1.1.3 Commercial available skin cultures 46
2.1.1.3 (a) Commercial normal human skin
epidermal keratinocytes (cNHEK) 46
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2.1.1.3 (b) Commercial normal human skin
dermal fibroblasts (cNHDF) 46
2.1.2 Chitosan porous skin regenerating templates (CPSRTs) 48
2.1.2 (a) CPSRT-NC-etha 48
2.1.2 (b) CPSRT-lactic-PVA 48
2.1.2 (c) CPSRT-NC-bicarbonate 48
2.1.2 (d) CPSRT-NC-drytech 49
2.1.3 Laboratory apparatus and equipments 50
2.1.3.1 Apparatus and equipments for primary cell culture 50
2.1.3.2 Primary culture and coculture vessels 50
2.1.3.3 Apparatus and equipments for immunosorbent assay 50
2.1.3.4 Apparatus and equipment for western blotting 51
2.1.3.5 Apparatus and equipment for real-time PCR 51
2.1.4 Bioreagents and biochemicals 52
2.1.4.1 Defined keratinocyte serum free medium (DKSFM) 52
2.1.4.2 Dulbecco’s modified eagle medium (DMEM) (1X) 52
2.1.4.3 Dulbecco’s phosphate-buffered saline (DPBS) 52
2.1.4.4 Penicillin-Streptomycin, liquid 52
2.1.4.5 Gentamicin, liquid 53
2.1.4.6 Dispase 53
2.1.4.7 Collagenase type-I 53
2.1.4.8 0.25% trypsin-ethylenediaminetetraacetic
acid (EDTA) 53
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2.1.4.9 Accutase™ 53
2.1.4.10 Versene (1:5000) 53
2.1.4.11 Fetal bovine serum (FBS) 54
2.1.4.12 EpiLife® medium 54
2.1.4.13 Human keratinocyte growth supplement-V2
(HKGS-V2) 54
2.1.4.14 Gentamicin-Amphotericin B solution 54
2.1.4.15 Coating matrix (collagen type-I) 54
2.1.4.16 Dimethyl sulphoxide (DMSO) 55
2.1.4.17 Thiazolyl blue tetrazolium blue (MTT) 55
2.1.4.18 Sodium hydroxide (NaOH) 55
2.1.4.19 Hydrochloric acid (HCl) 55
2.1.4.20 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid (HEPES) 55
2.1.4.21 Ethanol absolute 55
2.1.4.22 Phosphoric acid 56
2.1.4.23 Acetic acid 56
2.1.4.24 Sodium chloride (NaCl) 56
2.1.4.25 Trizma® hydrochloride 56
2.1.4.26 Tergitol® solution 56
2.1.4.27 Sodium deoxycholate (Na-deoxycholate) 56
2.1.4.28 Sodium dodecyl sulfate (SDS) 56
2.1.4.29 Phenylmethanesulfonyl fluoride (PMSF) 57
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2.1.4.30 Radio-Immuno Precipitation Assay (RIPA) buffer 57
2.1.4.31 Bovine serum albumin (BSA) 57
2.1.4.32 Protein assay dye reagent concentrate 57
2.1.4.33 Ammonium persulphate (APS) 57
2.1.4.34 N,N,N',N'-Tetramethylethylenediamine (TEMED) 58 2.1.4.35 Bromophenol blue solution 58 2.1.4.36 Glycerol 58 2.1.4.37 2-Mercaptoethanol 58
2.1.4.38 3-cyclohexylamino-1-propanesulfonic acid
(CAPS) buffer 58
2.1.4.39 Methanol 59
2.1.4.40 Tween-20 59
2.1.4.41 30% Acrylamide/Bis solution, 37.5:1 59
2.1.4.42 Trizma® base solution 59
2.1.4.43 Tris 59
2.1.4.44 Glycine 59
2.1.4.45 4X running gel buffer 59
2.1.4.46 4X stacking gel buffer 60
2.1.4.47 Water-saturated butanol 60
2.1.4.48 2X protein sample loading buffer (Laemmli buffer) 60
2.1.4.49 1X Tris-buffered saline (TBS)-Tween-20 60
2.1.4.50 Coomasie® dye binding protein reagent
(Bradford reagent) 60
2.1.4.51 Protein standard bovine serum albumin (BSA) 60
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2.1.4.52 Blocking solution 61
2.1.4.53 Western blot electrophoresis tank buffer 61
2.1.4.54 Stripping solution 61
2.1.4.55 2X RNA loading dye 61
2.1.4.56 10X 3-morpholinopropanesulfonic acid
(MOPS) buffer 61
2.1.4.57 37% formaldehyde solution 61
2.1.5 Experimental kits 62
2.1.5.1 IHC Select® secondary goat anti-mouse IgG,
anti-rabbit IgG, biotinylated 62
2.1.5.2 CometAssay® for DNA damage 62
2.1.5.3 CometAssay® silver staining 62
2.1.5.4 IL-1α ELISA 62
2.1.5.5 TNF-α ELISA 62
2.1.5.6 IL-8 ELISA 62
2.1.5.7 TGF-β1 ELISA 62
2.1.5.8 PageRuler™ plus prestained protein ladder 62
2.1.5.9 Immobilon™ Western chemiluminescent
horseradish peroxidase (HRP) substrate 62
2.1.5.10 RNeasy® Plus mini kit 62
2.1.5.11 QIAshredder™ homogenizer 62
2.1.5.12 Brilliant® II QRT-PCR master mix kit, 1-step 62
2.1.5.13 RiboRuler™ RNA ladder, high range 62
2.1.6 Primary and secondary antibodies 63
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2.1.6.1 Primary monoclonal mouse anti-human
Cytokeratin-6 63
2.1.6.2 Primary monoclonal mouse anti-human
Involucrin 63
2.1.6.3 Primary monoclonal mouse anti-human
Heat Shock Protein (HSP47) 63
2.1.6.4 Primary monoclonal mouse anti-human Fibroblast
Surface Protein (FSP), [1B10] 63
2.1.6.5 Primary monoclonal mouse anti-human Collagen
Type-I (COL-1) 63
2.1.6.6 Primary monoclonal mouse anti-human Collagen
Type-III (FH-7A) 64
2.1.6.7 Primary monoclonal mouse anti-human Collagen
Type-V (MC5-HA) 64
2.1.6.8 Primary mouse monoclonal anti-human
Alpha-tubulin (DM1A) 64
2.1.6.9 Secondary goat anti-mouse IgG antibody 64
2.1.6.10 Secondary goat anti-mouse IgM antibody 64
2.1.7 Primers and probes 65
2.1.7.1 Primers and probes for SMAD 2 65
2.1.7.2 Primers and probes for SMAD 3 65
2.1.7.3 Primers and probes for SMAD 4 65
2.1.7.4 Primers and probes for SMAD 7 65
2.1.7.5 Primers and probes for GAPDH 66
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2.2 Methodology 67
2.2.1 Establishment of primary human epidermal keratinocyte (pHEK)
and primary human dermal fibroblasts (pHDF) cultures 67
2.2.1.1 Establishment of pHEK cultures 67
2.2.1.2 Establishment of pHDF cultures 68
2.2.1.3 Maintenance and subculturing of pHEK and
pHDF cultures 68
2.2.1.4 Cryopreservation of pHEK and pHDF cultures and
the recovery of cryopreserved cells 69
2.2.1.5 Verification of pHEK and pHDF cultures
via immunocytostaining 70
2.2.2 Biocompatibility evaluation of chitosan porous skin regenerating
templates (CPSRTs) in primary normal human epidermal
keratinocyte (pNHEK) cultures 72
2.2.2.1 Treatment of CPSRTs on adherent pNHEK via
direct contact method 72
2.2.2.2 Cytotoxicity in vitro: Cell viability assay using the
colorimetric tetrazolium salt (MTT) 73
2.2.2.3 Genotoxicity in vitro: A detection of DNA damage
by Comet assay 74
2.2.2.3.1 Single cell gel electrophoresis (SCGE)
of pNHEK 74
2.2.2.3.2 Silver staining and scoring of
comet assay 75
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2.2.2.4 Human epidermal pro-inflammatory cytokine
(IL-1, TNF- and IL-8) expression in vitro 76
2.2.3 Effect of CPSRT on human collagen type I, III, V expression and
TGF-β1/SMADs signaling pathway in human keloid by using
skin epithelial-mesenchymal coculture model 78
2.2.3.1 Establishment of primary human skin
keratinocyte-fibroblast coculture models 78
2.2.3.1.1 Primary human skin coculture
medium mixtures 78
2.2.3.1.2 Experimental CPSRT treatment in
pHEK-pHDF coculture models 80
2.2.3.2 Western blot analysis for human collagen type I,
III and V in pHEK-pHDF cocultures 83
2.2.3.2.1 Protein extraction from
pHEK-pHDF cocultures 83
2.2.3.2.2 Protein quantification 84
2.2.3.2.3 Sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE) 84
2.2.3.2.4 Protein blotting 85
2.2.3.2.5 Immuno-detection and
chemiluminescent detection 85
2.2.3.3 Analysis of human TGF-β1 expression in
pHEK-pHDF cocultures 87
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2.2.3.4 Quantitative real-time polymerase chain reaction
(QRT-PCR) for SMAD 2, 3, 4 and 7 89
2.2.3.4.1 Total RNA extraction and
Quantification 89
2.2.3.4.2 RNA integrity by denaturing agarose
gel electrophoresis 90
2.2.3.4.3 QRT-PCR on SMAD 2, 3, 4 and 7
gene expression 91
2.2.3.4.4 Relative quantification of SMADs
mRNAs expression in QRT-PCR 93
2.2.4 Statistical analysis 94
CHAPTER 3 ESTABLISHMENT OF pHEK AND pHDF
CULTURES 95
3.1 Introduction 95
3.2 Results 97
3.2.1 Morphological characterization of the established pHEK
cultures under inverted light microscopy system 97
3.2.2 Morphological characterization of the established pHDF
cultures under inverted light microscopy system 103
3.2.3 Immunocytochemical verification of the established
pHEK cultures 106
3.2.4 Immunocytochemical determination of the established
pHDF cultures 109
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3.3 Discussion 112
CHAPTER 4 IN VITRO CYTOTOXICITY EVALUATION
OF CPSRTs 118
4.1 Introduction 118
4.2 Results 119
4.3 Discussion 123
CHAPTER 5 IN VITRO GENOTOXICITY EVALUATION OF
CPSRTs VIA COMET ASSAY 129
5.1 Introduction 129
5.2 Results 130
5.3 Discussion 134
CHAPTER 6 INFLAMMATORY CYTOKINE (IL-1, TNF-
AND IL-8) EXPRESSION IN pNHEK CULTURES
TREATED WITH CPSRTs 138
6.1 Introduction 138
6.2 Results 139
6.3 Discussion 147
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CHAPTER 7 ESTABLISHMENT OF HUMAN EPITHELIAL
MESENCHYMAL INTERACTION VIA HUMAN
SKINKERATINOCYTE-FIBROBLAST
COCULTURES 152
7.1 Introduction 152
7.2 Results 154
7.3 Discussion 157
CHAPTER 8 HUMAN COLLAGEN TYPE-I, III AND V EXPRESSION
IN HUMAN SKIN KERATINOCYTE-FIBROBLAST
COCULTURES TREATED WITH CPSRT-NC-
BICARBONATE 160
8.1 Introduction 160
8.2 Results 162
8.3 Discussion 173
CHAPTER 9 TGF-β1 EXPRESSION IN HUMAN SKIN
KERATINOCYTE-FIBROBLAST COCULTURES
TREATED WITH CPSRT-NC-BICARBONATE 178
9.1 Introduction 178
9.2 Results 180
9.3 Discussion 182
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CHAPTER 10 EXPRESSION OF SMAD 2, 3, 4 AND 7 IN HUMAN
SKIN KERATINOCYTE-FIBROBLAST
COCULTURES TREATED WITH CPSRT-NC-
BICARBONATE 186
10.1 Introduction 186
10.2 Results 187
10.3 Discussion 197
CHAPTER 11 SUMMARY AND CONCLUSION 202
11.1 Summary 202
11.2 Conclusion 207
11.3 Recommendations for future research 209
REFERENCES 211
APPENDICES
LIST OF PUBLICATIONS
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LIST OF TABLES
Table Page
2.1 Serum-free medium mixtures for human skin primary keratinocyte-fibroblast coculture 79
2.2 pHEK and pHDF coculture pairing 82
2.3 Volume and concentrations of each probe and primer with respect to each target gene and endogenous control per reaction 92
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LIST OF FIGURES
Figure Page
2.1 Schematic presentation of a keratinocyte-fibroblast coculture model 81 3.1 Morphologically rounded cells suspending in the culture medium observed
at the second hour post-seeding for (A) pNHEK and (B) pKHEK 98 3.2 The attached, elongating cells of (A) cultured pNHEK and (B) pKHEK
observed at the twelfth hour after primary seeding 99 3.3 Morphological observation of pHEK cultures at 96 hours post-seeding 100 3.4 Morphological observation of pHEK cultures at 120 hours post-seeding 101 3.5 The morphology of confluent pHEK cultures at 168 hours post-seeding 102 3.6 The pHDF cultures initialized from human skin dermal at 2-hour and
12-hour post-seeding 104 3.7 The pHDF cultures initialized from human skin dermal at 96-hour and
168-hour post-seeding 105 3.8 Immunocytochemical determination of pHEK using monoclonal mouse
anti-human CK6 107 3.9 Immunocytochemical determination of pHEK using monoclonal mouse
anti-human involucrin 108 3.10 Immunocytochemical determination of pHDF using monoclonal mouse
anti-human HSP47 110 3.11 Immunocytochemical determination of pHDF using monoclonal mouse
anti-human FSP 111 4.1 Average percentages (mean % ± S.D.) of cell viability treated with
CPSRTs in comparison with negative control 120 4.2 Average percentages (mean % ± S.D.) of cell viability treated with
CPSRTs in comparison with treatment-free-control 121 5.1 DNA-strand breaking potency of cultured pNHEK at 72 hours
post-treatment with CPSRTs 131 5.2 Assessment of DNA damage via comet assay in human pNHEK
cultures after treatment with CPSRTs 132
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6.1 Concentrations (mean pg/ml ± S.D.) of IL-1 induced by CPSRT treatments of pNHEK culture at 24, 48 and 72 hours 140
6.2 Fold change concentration (mean ± S.D) of IL-1α in pNHEK cultures
treated with CPSRTs compared with (A) negative control and (B) treatment-free-control 141
6.3 Concentrations (mean pg/ml ± S.D.) of TNF- induced by CPSRT
treatments of pNHEK culture at 24, 48 and 72 hours 142 6.4 Fold change concentration (mean ± S.D) of TNF-α in pNHEK cultures
treated with CPSRTs compared with (A) negative control and (B) treatment-free-control 144
6.5 Concentrations (mean pg/ml ± S.D.) of IL-8 induced by CPSRT
treatments of pNHEK culture at 24, 48 and 72 hours 145
6.6 Fold change concentration (mean ± S.D) of IL-8 in pNHEK cultures treated with CPSRTs compared with (A) negative control and (B) treatment-free-control 146
7.1 Average cell viability (mean % of all ratios per medium type ± S.D.) of each type of culture medium used in pHEK and pHDF cultures
at 72 hours 155
8.1 Expression of collagen type-I in cocultures at 24 hours determined via western blotting 163
8.2 Expression of collagen type-I in cocultures at 72 hours determined
via western blotting 164 8.3 Effect of CPSRT-NC-bicarbonate on human collagen type-I
expression in vitro 165 8.4 Expression of collagen type-III in cocultures at 24 hours determined
via western blotting 166 8.5 Expression of collagen type-III in cocultures at 72 hours determined
via western blotting 167 8.6 Effect of CPSRT-NC-bicarbonate on human collagen type-III
expression in vitro 169 8.7 Expression of collagen type-V in cocultures at 24 hours determined
via western blotting 170
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8.8 Expression of collagen type-V in cocultures at 72 hours determined via western blotting 171
8.9 Effect of CPSRT-NC-bicarbonate on human collagen type-V
expression in vitro 172 9.1 Effect of CPSRT-NC-bicarbonate on TGF-β1 expression in
the cocultures 181 10.1 RNA integrity examination by denaturing gel electrophoresis for
coculture sets at 24 hours 188 10.2 RNA integrity examination by denaturing gel electrophoresis for
coculture sets at 72 hours 189 10.3 Real-time PCR efficiency (E) curve for SMAD 2, 3, 4, 7 and GAPDH 190 10.4 Effect of CPSRT-NC-bicarbonate on SMAD 2 mRNA expression in vitro 191 10.5 Effect of CPSRT-NC-bicarbonate on SMAD 3 mRNA expression in vitro 192 10.6 Effect of CPSRT-NC-bicarbonate on SMAD 4 mRNA expression in vitro 193 10.7 Effect of CPSRT-NC-bicarbonate on SMAD 7 mRNA expression in vitro 194
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LIST OF SYMBOLS AND ABBREVIATIONS
α Alpha
& And
~ Approximately
β Beta
oC Degree Celsius
≥ Equal or more than
< Less than
% Percent
± Plus or minus
® Registered
™ Trademark
ACS American Chemical Society
AIU Arbitrary intensity unit
ALS Alkali-labile sites
AMREC Advanced Materials Research Centre
APS Ammonium persulphate
BCG Bacillus Calmette-Guerin
BHQ1 Black hole quencher-1
bp Base pair
BPE Bovine pituitary extract
BSA Bovine serum albumin
BSE Bovine spongiform encephalopathy
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Ca2+ Ion calcium
CaCl2 Calcium chloride
CAPS 3-cyclohexylamino-1-propanesulfonic acid
cDNA Complementary deoxyribonucleic acid
cNHDF Commercial normal human dermal fibroblasts
cNHEK Commercial normal human epidermal keratinocytes
CO2 Carbon dioxide
CPSRTs Chitosan porous skin regenerating templates
CTGF Connective tissue growth factor
cu Caseinolytic unit
Ct Cycle threshold
ddH2O Double distilled water
dH2O Distilled water
DKSFM Defined keratinocyte serum free medium
DMEM Dulbecco’s modified eagle medium
DMSO Dimethylsulphoxide
DNA Deoxyribonucleic acid
DPBS Dulbecco’s phosphate-buffered saline
DPX Di-n-butylPhthalate in Xylene
DSB Double-stranded breaks
DTBP Dimethyl 3-3,dithio-bis’propionimidate
E Real-time polymerase chain reaction efficiency
ECM Extracellular matrix/ extracellular matrices
EGF Epidermal growth factor
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ECVAM European Center for the Validation of Alternative Methods
e.g. exempli gratia (Example)
EDTA Ethylenediaminetetraacetic acid
ELISA Enzyme-linked immunosorbent assay
et al. Et alii
6-FAM 6-carboxyfluorescein
FBS Fetal bovine serum
FDA United States Food and Drug Administration
g Earth’s gravitational acceleration
g Gram
gDNA Genomic deoxyribonucleic acid
GAGs Glycosaminoglycans
GAPDH Glyceraldehyde 3-phosphate dehydrogenase
H2O Water
HCl Hydrochloric acid
HDF Human dermal fibroblasts
HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
HKGS-V2 Human keratinocytes growth supplements
HMWC High molecular weight chitosan
HRP Horseradish peroxidase
HSP47 Heat shock protein 47
IgG Immunoglobulin G
IgM Immunoglobulin M
IL Interleukin
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ISO International Standards Organization
JMHW Japanese Ministry of Health and Welfare
kb Kilobase
KCl Potassium chloride
kDa Kilodalton
KH2PO4 Potassium phosphate monobasic
L Liter
LDPE Low density polyethylene
LMAgarose Low melting point agarose
LPS Lipopolysaccharide
M Molar
mA Milliampere
MAPK Mitogen-activated protein kinase
mg Milligram
Mg2+ Ion magnesium
MgCl2 Magnesium chloride
mL Milliliter
mM Millimolar
mm2 Millimeter square
MTT 3-[4, 5-dimethyl-2-thiazolyl]-2, 5-diphenyl tetrazolium bromide
N Normality
n Number of samples/ replicates
NaCl Sodium chloride
NaHCO3 Sodium bicarbonate
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Na2HPO4-7H2O Sodium phosphate dibasic
NaOH Sodium hydroxide
NC-bicarbonate Neutralized chitosan by sodium bicarbonate
NC-drytech Neutralized chitosan by drying technique
NC-etha Neutralized chitosan solution by ethanol
ng Nanogram
nm Nanometer
nM Nanomolar
Na-deoxycholate Sodium deoxycholate
No. Number
NO-CMC N, O- carboxymethyl-chitosan
NP-40 Nonyl Phenoxylpolyethoxylethanol-40
NTC No-template-control
O-C Oligo-chitosan
OD Optical density
PAI-1 Tissue plasminogen activator inhibitor
PBS Phosphate buffered saline
PDGF Platelet derived growth factor
PDLC Periodontal ligament cells
PMSF Phenylmethanesulphonyl fluoride
PEG Polyethylene glycol
PET Polyethylene terephthalate
pg Picogram
pH Power of hydrogen
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pHDF Primary human dermal fibroblasts
pHEK Primary human epidermal keratinocytes
pKHDF Primary keloid human dermal fibroblasts
pKHEK Primary keloid human epidermal keratinocytes
PMSF Phenylmethanesulphonyl fluoride
pNHDF Primary normal human dermal fibroblasts
pNHEK Primary normal human epidermal keratinocytes
PSA Penicillin, Streptomycin and Amphotericin
PVA Polyvinyl alcohol
PVC Polyvinyl chloride
PVDF Polyvinylidene fluoride
R2 Linear regression
Real-time PCR Real-time polymerase chain reaction
QRT-PCR Quantitative real-time polymerase chain reaction
rEGF Recombinant epidermal growth factor
RFU Relative fluorescence unit
RIPA Radio-immuno precipitation assay
RNA Ribonucleic acid
rpm Round per minute
SCGE Single cell gel electrophoresis
S.D. Standard deviation
SDS Sodium dodecyl sulfate
SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis
SEM Scanning electron microscopy
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SIRIM Standards and Industrial Research Institute of Malaysia
SMAD Similar to mothers against Decapentaplegic Drosophilia
SSB Single-stranded breaks
TBS Tris-buffered saline
TEMED N,N,N',N'-Tetramethylethylenediamine
TGF Transforming growth factor
TMB 3.3’, 5.5’-tetramethylbenzidine
TNF Tumor necrosis factor
Tris-Cl Tris base added with concentrated hydrochloric acid
USA United States of America
µg Microgram
µL Microliter
µm Micrometer
µM Micromolar
V Volt
VEGF Vascular endothelial growth factor
v/v Volume by volume
WSC Water-soluble chitosan
w/v Weight by volume
YIGSR Tyr-Ile-Gly-Ser-Arg, (Tyrosine-Isoleucine-Glycine-Serine-Arginine)
xxvii
PENILAIAN KETOKSIKAN SECARA SITO-GENO DENGAN MENGGUNAKAN KITOSAN BERGRED BIOPERUBATAN PADA KULTUR
KERATINOSIT ASAS DAN PERANANNYA DALAM LITAR PEMBENTUKAN PARUT DENGAN MENGGUNAKAN MODEL KO-KULTUR KULIT
ABSTRAK
Kitosan (-1, 4—D-glukosamin) merupakan kitin yang kumpulan asetilnya telah
disingkirkan dan ia mempunyai ciri-ciri biologi yang unggul dan berguna dalam rawatan
luka. Tetapi, pengubahsuaian kitosan secara fizikal dan kimia akan menjejaskan bio-
keserasiannya. Selain daripada itu, dilaporkan juga bahawa kitosan mempunyai
kegunaan dalam pencegahan parut. Oleh itu dalam kajian ini, dengan menggunakan
kultur normal keratinosit asas daripada epidermis manusia (pNHEK), bio-keserasian in
vitro pada templat perbentukan kulit berpori kitosan (CPSRT) yang diubahsuai telah
dinilai melalui analisis ketoksikan secara sito melalui ujian 3-(4, 5-dimetilthiazol-2-yl)-
2, 5-difenil tetrazolium bromida (MTT), analisis ketoksikan secara geno dengan
menggunakan ujian komet serta analisis rembesan sitokin keradangan keratinosit kulit
[interleukin-1 (IL-1), faktor tumor nekrosis- (TNF-) dan interleukin-8 (IL-8)].
Peranan CPSRT yang bio-serasi dalam pencegahan keloid telah diselidik dengan
menggunakan ko-kultur keratinosit-fibroblast. Pengekspresan protein kolagen manusia
berbentuk-I, III dan V telah diselidik dengan menggunakan analisis Western blot.
Sementara itu, rembesan faktor pertumbuhan-β1 (TGF- β1) telah dianalisis dengan
menggunakan ujian enzim-linked immunosorbent (ELISA) dan pengekspresan mRNA
melalui molekul intraselular (SMAD 2, 3, 4 dan 7) juga telah dihitung dengan
menggunakan analisis real-time PCR. Kultur keratinosit asas daripada epidermis
manusia (pHEK) yang selnya berbentuk skuamosa telah disahkan dengan sitokeratin-6
xxviii
(CK6) dan involukrin, manakala kultur fibroblast asas daripada dermis manusia (pHDF)
telah dibuktikan dengan kewujudan heat shock protein 47 (HSP47) dan protein
permukaan fibroblast (FSP). CPSRT-NC-bikarbonat telah meningkatkan kadar
pertumbuhan sel dalam kultur pNHEK. Ia tidak mencetuskan kemusnahan DNA dan
juga tidak meningkatkan respon sitokin keradangan secara in vitro. Ko-kultur
keratinosit-fibroblast telah dipelihara dalam DKSFM:DMEM:F12 (2: 2: 1) sebagai satu
medium yang dikongsi dalam ko-kultur. Kolagen bentuk-I adalah dominan dalam kultur
normal fibroblast asas daripada dermis manusia (pNHDF) manakala kolagen bentuk-III
pula banyak didapati dalam ko-kultur fibroblast daripada keloid dermis manusia
(pKHDF). Kolagen bentuk-V berkemungkinan wujud dengan lebih banyak di dalam
dermis yang mempunyai kolagen bentuk-I yang banyak. SMAD 2, SMAD 4 dan TGF-
β1 telah didapati lebih banyak di dalam ko-kultur pKHDF berbanding dengan pNHDF.
Ko-kultur yang wujud dengan keratinosit normal telah mengurangkan pengekspresan
kolagen bentuk-III, SMAD 2, SMAD 4 dan TGF-β1. Rawatan dengan CPSRT-NC-
bikarbonat meningkatkan lagi kesan itu. Kesimpulannya, CPSRT-NC-bikarbonat yang
bio-serasi berhubungan dengan kultur normal keratinosit mempunyai bio-fungsi untuk
mengurangkan fosforilasi TGF-β1 dalam kultur fibroblast yang berasal keloid. Ianya
mungkin melalui peningkatan dalam pengekspresan SMAD 7 dan faktor-faktor larut
CPSRT-NC-bikarbonat yang melembapkan reseptor TGF-β, lalu mengurangkan
transkripsi ekstrasellular matriks (ECM) terutamanya kolagen bentuk-III, di mana ia
berguna dalam pencegahan keloid.
xxix
THE CYTO-GENOTOXICITY EVALUATION OF BIOMEDICAL GRADE CHITOSAN ON PRIMARY KERATINOCYTE CULTURES AND ITS ROLE IN
SCAR FORMATION PATHWAYS USING SKIN COCULTURE MODEL
ABSTRACT
Chitosan (-1, 4—D-glucosamine) is a deacetylated form of chitin with excellent
biological properties useful in wound management. However, improvement of the
physical and chemical properties of the chitosan will alter its biocompatibility.
Additionally, chitosan has been reported to be useful in scar intervention. Therefore in
this study, the in vitro biocompatibility of the modified chitosan porous skin
regenerating templates (CPSRTs) has been evaluated based on cytotoxicity analysis by
3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay,
genotoxicity by comet assay, and skin keratinocyte pro-inflammatory cytokine
secretions [interleukin-1 (IL-1), tumor necrosis factor- (TNF-) and interleukin-8
(IL-8)], using primary normal human epidermal keratinocyte (pNHEK) cultures. The
most biocompatible CPSRT was then investigated for its role in keloid intervention
using keratinocyte-fibroblast cocultures. The human collagen type-I, III and V protein
expressions were assayed in Western blot analysis. Meanwhile, the secreted
transforming growth factor-β1 (TGF-β1) was analyzed by enzyme-linked
immunosorbent assay (ELISA), and the mRNA expressions of its intracellular signaling
molecules (SMAD 2, 3, 4 and 7) were quantified using real-time PCR analysis. The
established squamous-shaped primary human epidermal keratinocyte (pHEK) cultures
have been verified using cytokeratin-6 (CK6) and involucrin, whereas spindle-shaped
primary human dermal fibroblast (pHDF) cultures were tested for the presence of heat
xxx
shock protein 47 (HSP47) and fibroblast surface protein (FSP). Treatment with CPSRT-
NC-bicarbonate enhanced the cell growth of pNHEK cultures. It did not induce DNA
damage and did not provoke pro-inflammatory cytokine responses in vitro.
Keratinocyte-fibroblast cocultures were maintained in DKSFM: DMEM: F12 (2: 2: 1) as
a shared serum-free coculture medium. Collagen type-I is the dominant form in primary
normal human dermal fibroblast (pNHDF) whereas collagen type-III is more abundant
in primary keloid-derived human dermal fibroblast (pKHDF) cocultures. Collagen type-
V may be present more in the skin dermal that is rich in collagen type-I. SMAD 2 and
SMAD 4, as well as the TGF-β1 were expressed more in the pKHDF than pNHDF
cocultures. Cocultures present with normal keratinocytes were observed to suppress
collagen type-III, SMAD 2, SMAD 4 and TGF-β1 expressions. Treatment with CPSRT-
NC-bicarbonate further enhanced this effect. In conclusion, the biocompatible CPSRT-
NC-bicarbonate which was in association with normal-derived keratinocyte cultures
exerted bio-functionality to decelerate TGF-β1 phosphorylation in the keloid-derived
fibroblast culture, perhaps through the increased expressions of SMAD 7 and the soluble
factors of CPSRT-NC-bicarbonate in deactivating the TGF-β receptors, thus reducing
transcription of extracellular matrix (ECM) particularly collagen type-III, which can be
useful in keloid intervention.
1
CHAPTER 1
INTRODUCTION
1.1 Research background
Wound healing is a process of restoration that is necessary for tissue repair,
typically comprising a continuous sequence of inflammation and repair in which
epithelial, endothelial, inflammatory cells, platelets and fibroblasts briefly interact to
resume their normal functions. This healing process consists of four different and
overlapping phases, namely: inflammation, granulation tissue formation, matrix
remodeling and reepithelialization (Christine et al., 1999). The orderly sequence of
healing events is regulated by various interplayed cytokines and growth factors (Ono
et al., 1995). One of the ultimate goals in wound healing research involves finding
ways for humans to heal via the regeneration of similar tissues. This could be
accomplished via the invention and modification of a variety of dressing materials to
facilitate proper wound management.
Chitosan is a partially deacetylated derivative of natural chitin, a primary
structural polymer in arthropod exoskeletons. One of the present trends in biomedical
research requires materials that are derived from nature as natural materials have
been shown to exhibit better biocompatibility with humans. For example, chitosan’s
monomeric unit, N-acetylglucosamine, occurs in hyaluronic acid, an extracellular
macromolecule that is important in wound repair. Studies on chitosan have been
intensified since 1990 due to its low cytotoxicity, antimicrobial activity and excellent
biodegradable properties in the human body, where these have contributed to the
better biocompatibility of chitosan (Sashiwa et al., 1990; Shigemasa et al., 1994;
Van et al., 2006; Prabharan and Mano, 2006). The potential of chitosan stems from
2
its cationic nature and high charge density in solution. An effective approach for
developing a clinically applicable chitosan is to modify the surface of the material
that already has excellent biofunctionality and bulk properties. However, blending
technologies with various additives may cause cytotoxicity. Hence, the modified
biomedical-grade of chitosan with various derivatives should be submitted for
biocompatibility testing to promote the engineering of high-quality, biocompatible
products for human wound management.
In the pre-commercialization steps of a biomedical-grade chitosan derivative
as wound dressing, it is of great importance that the dressing be submitted to a
number of in vitro pre-toxicity assays. In vitro toxicity examinations are preferable
to in vivo tests since the newly developed dressing materials can be examined outside
the body and the results are more reproducible, facilitating clinical evaluation of a
medicinal product that can be replicated and estimated (Soheili et al., 2003; Cheong
et al., 2008). Biocompatibility in vitro is useful to characterize cytotoxic effects of
leachable materials, such as residual monomers, catalysts as well as polymer erosion
and related factors (e.g. composition, molecular weight and polydispersity) which are
suitable to monitor the quality and purity of the biodegradable biomedical-grade
chitosan. Cytotoxicity assays measure only finite effects on cells during the first 12
to 72 hours after exposure to toxic substances. The host cells either recover from or
succumb to their chemical injury. However, many biological reactions in vivo are not
simply cytotoxic and are propagated beyond 24 hours. Examples are improper cells
programmed apoptosis and necrosis due to deoxyribonucleic acid (DNA) damage;
inflammatory and immune reactions that all resulted from non-biocompatible
chitosan derivatives. Therefore, in vitro biocompatibility assessment for wound
3
management must consider cytocompatibility, genotoxicity and related pro-
inflammatory cytokines expression for better in vitro biocompatibility evaluation.
Abnormal wound healing is related to improper fibroblasts activities in the
wound bed that have commonly encountered in multiple pathologies ranging from
diabetic ulcers, to fibrosis and scarring. Keloids render a form of abnormal wound
healing affecting only human which involve persistent itchy pseudotumoral skin
lesions expanding beyond edges of the original wounds and frequently recurring
following surgical excision. The exuberant scarring of keloid leads to disfiguring and
dysfunctional scars without any satisfactory treatment until today. Hence, by
understanding the keloid formation pathway may serve as a corner stone for the
treatment. Unluckily, there are still no keloid studies using established animal model
(Xia et al., 2004). Alternatively, keloid studies are mostly performed using an in
vitro model (Uitto et al., 1985; Smith et al., 1999; Higham et al., 2002; Xia et al.,
2004; Naitoh et al., 2005).
Wound healing is a complex physiological response that involves various
interactions between epithelial and mesenchymal. Many earlier in vitro studies on
keloid pathogenesis were largely on the use of keloid fibroblasts simply because it
mainly produces collagen and extracellular matrix (ECM) that form the bulk of
keloid tissue. Recently, evidence has indicated that autocrine, paracrine and
endocrine epithelial-mesenchymal interactions may be involved in normal skin
homeostasis, growth and differentiation (Maas-Szabowski et al., 1999; Higham et
al., 2005; Amjad et al., 2007). Therefore, the application of in vitro coculture system
may serve as a useful tool to elucidate the pathogenesis of keloid. Besides,
biochemical and ultrastructural studies of the engineered skin via cultures or
cocultures had also shown the similarities in metabolic and differentiation program
4
compared with human skin (Asbil et al., 2000; Robinson et al., 2000; Wagner et al.,
2001). Human skin tissue culture may provide a controlled yet realistic model to
study the molecular mechanisms of barrier disruption and identify the involvement of
genes in a disease development such as scar formation. By using a specifically made
coculture inserts, that is a membrane with certain pore sizes used to culture one type
of cell line while the other cell line on another plastic dishes. The combination of
both inserts will imitate the in vivo-like interactions in which the skin keratinocytes
are separated from dermal mesenchymal cells by a complex ECM, the basement
membrane, thus rendering cell-cell contact-mediated mechanisms less probable.
Fibroblasts are the primary cell type that functions in the organization of the
ECM following of skin injuries. To restore the barrier function of the skin as quickly
as possible, closure of skin wounds and replacement of missing skin tissues occur
rapidly in adult skin by some mechanisms that lead to scarring in which
overproduction of collagen bundles renders significant keloid pathology. Collagen
type-I, III and V constitutes a large portion in the keloid ECM (Ala-Kokko et al.,
1987). In accordance with the clinical observation that delayed reepithelialization is
associated with the scar formation, in vitro investigations have demonstrated that
keratinocytes decrease fibroblasts expression of collagen and pro-fibrotic factors
(Garner, 1998; Le Poole and Boyce, 1999). Some have reported that the chitosan
may be potentially used in scar prevention (Wang et al., 1998; Paul and Sharma,
2004; Yang et al., 2006; Delorino and Cresidio, 2009). Among the TGF-β isoforms,
TGF-β1 is one of the most reported and important stimulators of collagen synthesis.
It affects the ECM not only by stimulating collagen synthesis but also by preventing
its breakdown (Yang et al., 1997; Szulgit et al., 2002; Quan et al., 2004). TGF-β1 is
a pro-fibrotic factor that increases the formation of granulation tissue and stimulates
5
wound contraction besides being a negative regulator of inflammation and
responsible in the switching of the inflammatory phase to the proliferation phase in
wound healing. Similar to mothers against Decapentaplegic Drosophilia (SMAD)
proteins function as intracellular signaling mediators of TGF-β family members and
can be subdivided into receptor-regulated SMADs (R-SMADs), common mediator
SMADs, and inhibitory SMADs (Ten and Hill, 2004). Kopp et al. (2005) proposed
the relationship of TGF-β/ SMADs in human fibroproliferative disorders and
elucidated that autocrine TGF-β/ SMADs signaling is involved in contractility and
matrix gene expression of the fibroproliferative diseases.
Therefore, in this study, primary normal human epidermal keratinocytes
(pNHEK) was implemented in the cytotoxicity pre-evaluation of newly developed
chitosan porous skin regenerating templates (CPSRTs). Those contributed more than
70% of cell viability were then chosen for genotoxicity assay using alkaline single
cell gel electrophoresis (SCGE) or comet assay. Additionally, their potential to
induce skin irritation or inflammatory responses was assessed by quantitatively
examining the secretion of pro-inflammatory cytokines (IL-1, TNF- and IL-8) in
the pNHEK cultures. Keratinocyte-fibroblast coculture model was also constructed
to study the epithelial-mesenchymal interactions of normal and keloid-derived
human skin tissues, with regard to the effectiveness of CPSRTs on the keloid
intervention, by elucidating the expressions of type-I, III and V collagen and its
response in TGF-β1/ SMADs signaling pathway.
6
1.2 Literature review 1.2.1 Structure and function of human skin
Skin is the largest organ in the human body besides commonly known as a
vital outpost of the nervous system and is the closest contact with the outside world.
It naturally provides a protective covering layer for the other tissues in the body,
acting as a barrier to micro-organisms, water and chemicals, regulating body
temperature while still maintaining the water content of the body, removing waste
products from the body and providing sensory information. In addition, the skin
produces Vitamin D in response to sunlight (Brad Spellberg, 2000). Generally,
human skin is composed of three structurally and functionally integrated layers
namely the outer epidermis, the dermis and the inner hypodermis (Haake and
Holbrook, 2001).
The human skin epidermis consists mainly of keratinocytes that form a
stratified squamous epithelium (Eckert et al., 1997). Its thickness is about 0.2 mm on
average and this thickness varies depending on the location on the body.
Furthermore, the thickness also varies according to the volume of water that the
epidermis holds. Epidermal layer itself is further divided under skin cell
differentiation process into four main layers that are stratum basal, stratum spinous,
stratum granular and stratum corneum. These layers render to different stages of cell
maturation. Keratinocytes at the basal layer progressively differentiate upward into
cells with morphologically bigger and flatter-shaped meanwhile loosing proliferative
ability (Jetten and Harvat, 1997). Epidermal population of stem cells that are
confined to the basal cell layer of the epidermis will constitutively provide the
epidermis with its regenerative capacity. Cells from the basal layer migrate and
replicate during reepithelialization of wound healing (Haake and Holbrook, 2001).
7
Basal layer keratinocytes are columnar in shape and adhere with the underlying
basement membrane through the hemidesmosomes. In the outermost layer which is
stratum corneum, cells are cornified, lacking nuclei and organelles besides composed
primarily of keratins. The cornified cells desquamate in order to balance the
proliferation in the basal layer at the turnover rate of approximately 21 days. There
are also small portions of cells residing in the human skin epidermal namely
pigment-producing melanocytes, antigen-presenting Langerhans cells and
neuroendocrine Merkel cells. The epidermal appendages (hair follicles, apocrine
glands, sweat glands and sebaceous glands) extend into the human skin dermis. In
superficial wounds where most of the dermis is intact and viable, the cells in the
epidermal appendages proliferate and regenerate the epidermis (Fairley, 2001).
The dermis is the second layer of human skin, beneath the epidermal layer.
This layer is much thicker than the epidermis ranging from 1 to 4 mm. The dermis is
structurally organized into the uppermost papillary and the innermost reticular
dermis. At the dermal-epidermal junction, the dermal papillae interact with
extensions of the epidermis called rete pegs. The sub-papillary plexus which is lying
as horizontally vessels constitutes the boundary between the papillary and reticular
regions besides extending capillaries toward the skin epidermis (Haake and
Holbrook, 2001). The skin dermis has few cells and is mainly composed of
connective tissue and extracellular matrix (ECM) (Lee et al., 2006). The essential
dermal cell type is fibroblast which is responsible for the production and
maintenance of the structural elements of skin. These elements are collagen and
elastin which are the major fibrous components in the reticular dermis, combining
with non-fibrous substances such as glycosaminoglycans (GAGs) to form ECM
(Butler et al., 1999). Fibroblasts are more abundant in the papillary dermis and have
8
higher proliferative and metabolic activity at this location. Collagen is the major
constituent accounting approximately 75% of the dry weight of human skin. There
are several types of collagen present in mammalian skins. Collagen type-I, III and V
commonly are found in human in which normal skin dermal consists approximately
80% of collagen type-I. The connective tissues of the dermis give the skin its
pliability, elasticity, tensile strength and protect the body from mechanical injuries
(Brown et al., 1994). During deep wound healing, skin dermal fibroblasts are
responsible for the formation of scar tissue accompanied with activated immune
response and reparative processes.
Hypodermis is a layer underneath the dermis which is an abrupt transition
from the reticular dermis. This layer is rich in adipose tissue. However, skin dermis
and hypodermis are integrated through nerve and vascular networks as well as
epidermal appendages. The adipocytes are organized into lobules separated by septa
of connective tissues that contain nerves, vessels and lymphatics (Wajchenberg,
2002). This hypodermis insulates the human body, provides energy and allows for
mobility of the skin on underlying structures. Adipose tissue that is underlying the
hypodermis serves as an energy storage, in which is virtually limitless via lipogenesis
over lipolysis and pre-adipocyte replication and differentiation (Prins and O’Rahilly,
1997).
9
1.2.2 Cutaneous wound healing
Wound is defined as the disruption of the anatomic structures and their
functions in any body parts. This can be due to a simple cut or burns. Wounds can be
generally classified as wounds without tissue loss (e.g. in surgery) and wounds with
tissue loss such as burn wounds, wounds due to trauma, abrasions or as secondary
events in chronic ailments (e.g. venous stasis, diabetic ulcers and iatrogenic wounds
such as skin graft donor sites and derma-brasions) (Willi and Chandra, 2004). In
contrast, wound healing is a process of restoration by which tissue repair takes place
and usually comprises of a continuous sequence of inflammation and reparation in
which epithelial, endothelial, inflammatory cells, platelets and fibroblasts briefly
interact to restore a semblance of their usual discipline and resume their normal
functions. The orderly sequence of healing events are accomplished and regulated by
cytokines and growth factors (Ono et al., 1995). Wound healing is a process
involving generally three phases namely inflammation, proliferation and tissue
remodeling. Soon after the elimination of macrophage that appears during the
inflammatory phase, wound healing is then impeded and the tensile strength of the
scar is diminished (Leibovich and Ross, 1975).
1.2.2.1 Inflammation phase
Cutaneous injury disrupts primarily the blood vessels and lymphatics. An
initial five to ten minutes period of vasoconstriction is then followed by more
persistent vasodilatation. This presents itself as erythema, swelling, warmth and is
often accompanied with pain. Blood components are recruited into wound cavity.
Endothelial cells retract and loose their attachments with adjoining cells, exposing
subendothelial factor VII related Von Willebrand factor and fibrillar collagen in the
10
injured tissue. Platelets adhere to these surfaces to form platelet plug, with
association of fibrin clot provides a protective wound matrix. The provisional matrix
composed of fibrin and small amounts of vitronectin, fibronectin and
thrombospondin. Platelets release inflammatory mediators that together with
bacterial peptides and products of matrix degradation attract leukocytes to the
wound. Neutrophil migrations engulf debris and microorganisms providing the first
line of defense against infection for a few days. If acute inflammation phase persists
due to wound hypoxia, infection, nutritional deficiencies or other factors related to
one’s immune response, it can interfere with the late inflammatory phase
(Stadelmann et al., 1998). Macrophages replace neutrophils towards the end of the
inflammatory phase. Macrophages release several growth factors that stimulate the
migration of proliferation of fibroblasts as well as the production of ECM.
Thrombocytopenia decreases inflammatory activity during wound healing but it does
not impede cutaneous tissue repair (Szpaderska et al., 2003).
1.2.2.2 Proliferation phase
The subsequent proliferation phase is dominated by the formation of
granulation tissue and reepithelialization. Chemotactic and growth factors released
from platelets and macrophages stimulate the migration and activation of wound
fibroblasts that produce a variety of substances such as glycosaminoglycans (mainly
hyaluronic acid, chondroitin-4-sulfate, dermatan sulfate and heparin sulfate) and
collagen (Stadelmann et al., 1998). This amorphous and gel-like connective tissue
matrix is necessary for cell migration. Reepithelialization begins where the basal
keratinocytes flatten and detach from the basement membrane and migrate over the
wound matrix. There is also evidence that suprabasal keratinocytes take part in the
11
reepithelialization (Laplante et al., 2001). Fibronectin, laminin 5 and collagen type I
that reside in the ECM are known to assist the keratinocytes migration while collagen
type IV, thrombospondin and laminin may inhibit the migration (Frank and Carter,
2004; Woodley et al., 1991). Antimicrobial peptide LL37 has also been regarded as
one of the factors involved in the reepithelialization (Heilborn et al., 2003). Matrix
degrading enzymes are highly increased in wound healing and facilitate the
migration of keratinocytes (Pilcher et al., 1999).
During the end of migratory phase of keratinocytes and where the
components of basement membrane begin to aggregate, the migration is terminated
leading subsequently to the proliferation and differentiation of keratinocytes to
reconstitute the epidermis. Epidermal reconstitution requires migration, proliferation,
differentiation and stratification of keratinocytes in cutaneous wound healing
(Laplante et al., 2001). Interactions between skin keratinocytes and fibroblasts in
regulating basement membrane formation, cell proliferation and differentiation in
vitro, may probably occur in vivo (Smola et al., 1998). Dermal fibroblasts may
promote reepithelialization by secreting keratinocyte growth factor (KGF), insulin
growth factor-1 (IGF-1) and the epidermal growth factor family (Haase et al., 2003;
Werner and Smola, 2001). There is accumulating evidence for a possible role of
interleukin-1 in reepithelialization (Werner and Smola, 2001; Maas-Szabowski et al.,
2000). Thus, hypertrophic scar formation in wounds due to delayed
reepithelialization suggests a role for keratinocytes in diminishing the production of
ECM by fibroblasts.
The dermal component of the wound heals through matrix synthesis
(fibroplasia) and wound contraction, which is a centripetal movement of the edges of
a full thickness wound in order to facilitate closure of the defect (Grillo, 1964).
12
Blood vessels are formed in the process of angiogenesis. Collagen synthesis and
cross-linkage is responsible for vascular integrity and strength of new capillary beds.
Improper cross-linkage of collagen fibers has been reported in nonspecific post-
operative bleeding in patients with normal coagulation parameters (Blee et al., 2002).
Fibroblasts and endothelial cells proliferate and start to migrate into the wound about
48 hours after injury in which fibroblasts form the new connective tissue while
endothelial cells give rise to new vessels. Some fibroblasts differentiate into
myofibroblasts that are characteristically similar to both smooth muscle cells and
fibroblasts. Singer (1979) has described such a specialized structure as the
‘fibronexus’ which associates across the cytoplasmic membrane of the
myofibroblasts of intracellular actin microfilaments and extracellular fibronectin
fibers. Therefore, contraction of the actin filaments within the myofibroblasts will
lead to the fibronexus to transmit this force to the surrounding matrix and mediate
the clinical phenomenon of wound contraction that aids in closing the wound.
1.2.2.3 Tissue remodeling
Tissue remodeling is the final stage of cutaneous wound healing which
continues for months after injury. The events in remodeling are responsible for the
increase in tensile strength, decrease in erythema and formation of scar tissue bulk
with the final appearance of the healed scar. As collagen is laid down, fibronectin
gradually disappears. The nonsulfated glycosaminoglycans and hyaluronic acid are
replaced by more resilient proteoglycans such as chondroitin-4-sulfate. Additionally,
re-absorption of water from the scar allows the collagen fibers and other matrix
components to lie closer together leading to an increase in the tensile strength. A
13
matrix rich in collagen type-I replaces the provisional matrix and granulation tissue
which are rich in fibronectin and collagen type-III (Guo et al., 1990).
14
1.2.3 Chitosan as wound dressing
Wound dressings are generally classified based on its nature of action as
passive products, interactive products and bioactive products. Traditional dressings
such as gauze and tulle dressings that account for the largest market segment are
known as passive products. Polymeric films and forms which are mostly transparent,
permeable to water vapor and oxygen but impermeable to bacteria are commonly
recognized as interactive products. Bioactive dressings are important in delivering
substances in wound healing by which the delivery of bioactive compounds or
dressings is constructed from material having endogenous activity. These materials
include proteoglycans, collagen, non-collagenous proteins, alginates or chitosan
(Willi and Chandra, 2004).
Chitosan is an N-deacetylated derivative bio-product of chitin and is found in
the cell walls of some fungi (e.g. Basidiomycetes spp.), arthropods and marine
invertebrates. Chitosan is usually produced from chitin by alkali treatment (Roller
and Covill, 1999). Commercial chitosan is commonly deacetylated greater than 70%
of deacetylation with molecular weights ranging between 100 000 to 1.2 million
dalton (Onsoyen and Skaugrud, 1990). Chitosan is polycationic at pH < 6 and
interacts readily with negatively charged substances such as protein, anionic
polysaccharides (e.g. alginate, carragenan), fatty acids, bile acids and phospholipids
due to the high density of amino groups present in the polymer (Muzzarelli, 1996).
Their unique properties include polyoxysalt formation, the ability to form films,
chelate metal ions and optical structural characteristics (Larry, 1998). Most of the
current polymers are synthetic materials. Their biocompatibility and biodegradability
are much more limited than chitosan and their derivatives. However, these naturally
abundant materials also exhibit a limited reactivity and processing ability (Mass et
15
al., 1998). Chitosan’s positive surface charge enables it to effectively support cell
growth (Zileinski and Acbischer, 1994).
Chitosan is well known for its haemostatic properties to prevent excessive
bleeding, while its biological activities can mediate macrophage function to secrete
numerous enzymes (e.g. collagenase) and cytokines [e.g. interleukins, tumor necrosis
factor (TNF)] in wound healing processes. Its biological properties include
bacteriostatic and fungistatic which are particularly useful for wound treatment
(Majeti and Ravi, 2000). It appears that chitosan which has structural characteristics
similar to glycosamino glycans, could be considered for developing such substratum
for skin replacement. Yannas et al. (1982) proposed a design of artificial skin for
long term chronic use, focusing on a non-antigenic membrane in which can perform
as a biodegradable template for the synthesis of neodermal tissue. According to Yao
et al. (1994), chitosan can be used as an artificial skin in accelerating wound and
ulcer healing besides serving as a biocompatible vehicle for sustained release of
drug. Recently, application of chitosan has been given serious research attention due
to its potentially important renewable resource that is non-toxic and biodegradable.
Malette et al. (1986) studied the effect of treatment with chitosan and saline solution
on healing and fibroplasia of wounds made by scalpel insertions in skin and
subcutaneous tissue in the abdominal surface of dogs. It has been suggested that
chitosan may be used to inhibit fibroplasia in wound healing and to promote tissue
growth and differentiation in tissue culture (Muzzarelli et al., 1997). Some have
reported that the chitosan may be potentially used in scar prevention (Wang et al.,
1998; Paul and Sharma, 2004; Yang et al., 2006). Delorino and Cresidio (2009)
concluded that by using 10% lactic acid for decalcification and 1M NaOH for
deproteination chitosan is effective as active ingredient in scar remover cream.
16
Schmidt et al. (1993) have also reported the properties of chitosan in preventing
overgrowth of fibroblasts besides being cytocompatible (Risbud et al., 2000) and
blood biocompatible (Risbud and Bhat, 2001). The excellent biological properties of
chitosan can be potentially improved with a variety of additional chemicals such as
polyethylene glycol and carboxymethyl, N-acyl groups in order to produce
biocompatible chitosan derivatives for use as wound dressing (Zhang et al., 2002).
Chitosan-gelatin sponge wound dressing had shown better antibacterial effect by
vacuum dryness on Escherichia coli K88 than that of penicillin besides having the
superior healing effect to that by 0.2% (v/v) ethacridine. Additionally, chitosan-
gelatin sponge assisted the wound contraction and shortened the wound healing time
compared with sterile Vaseline gauze (Deng et al., 2007). Clinical assessment was
performed using a Hyphecan cap (1-4,2-acetamide-deoxy-β-D-glucan), as a
biological fingertip dressing and was proven to be effective in achieving a good
functional and cosmetic result within a relatively short time, aside from being
comfortable during conventional dressing changes (Halim et al., 1996).
17
1.2.4 Biocompatibility in vitro of biomedical-grade chitosan
Biocompatibility of a biomedical-grade chitosan refers to the quality of the
material that does not have toxic or injurious effects on biological systems.
Biocompatibility of any biomaterials is necessary to be tested for their strength,
esthetics and clinical manipulation. Current regulations in accordance with the
United States Food and Drug Administration (FDA), the International Organization
for Standardization (ISO) and the Japanese Ministry of Health and Welfare (JMHW)
require that manufacturers conduct adequate safety testing of their finished devices
through pre-clinical and clinical phases as part of the regulatory clearance process.
Toxicity induced by a chemical substance or drug can exert limitation on dose and
duration of treatment besides adversely affecting patients’ quality of life and may be
life threatening. Side effects together with tissue toxicity will lead to long term
effects such as alteration to the immune system and development of malignancies
probably due to genetic damage induced by a certain kind of drug treatment (Paola et
al., 2002). In the pre-commercialization steps of any biomaterial, it is of importance
to undergo a number of pre-toxicity tests in vitro or in vivo. In vitro toxicity
examinations are more favorable over the in vivo tests since the newly developed
biomaterials can be examined outside the body and the results are more reproducible.
Thus, preliminary in vitro tests are always being carried out to pre-screen and
characterize the potentially harmful effects of a newly synthesized dressing material
before it is clinically used on humans (Zhang et al., 2002). However, in vitro tests
may not necessarily fully represent the in vivo test whereby differences in
sensitivities had been observed between primary and established cell lines (Geurtsen
et al., 1998; Lovschall et al., 2002). Various in vitro cell culture systems have been
applied to investigate and evaluate the cellular processes, such as fibroblast and
18
keratinocyte proliferation and migration response to the growth factors that are
present in a wound (Schreier et al., 1993; Cha et al., 1996; Kawada et al., 1997).
Such in vitro models simplify and enable chemicals and biomaterials to be assessed
for their potential, at least in a preliminary way, to promote wound repair by
stimulating cell proliferation and for their biocompatibility.
Chitosan is a cationic polymer having an amino group in its chemical
structure. Its anti-tumor, anti-microbial and anti-inflammatory properties can be
largely attributed to the cationic structure. The N-acetylglucosamine moiety in
chitosan is structurally similar to glycosaminoglycans (GAGs), heparin, chondroitin
sulphate and hyaluronic acid in which they are biocompatible, and have specific
interactions with various growth factors, receptors and adhesion proteins besides
being the biologically important mucopolysaccharides in all mammals. Therefore,
the analogous structure in chitosan may also exert similar bioactivity and
biocompatibility (Li et al., 1992; Suh and Matthew, 2000). Additionally, chitosan
can function as a bridge to improve the mechanical strength of collagen-based
scaffolds owing to the large number of amino groups in its molecular chain (Taravel
and Domard, 1996). Lee et al. (2002) performed trypan blue dye exclusion
cytotoxicity assay on tumor cell lines, after increased positive charge density of
chitosan by dialkylaminoalkylation and reductive amination followed by
quaternization. Amino groups of chitosan attached with biologically active moieties
such as vanillin, p-hydroxybenzaldehyde, p-chlorobenzaldehyde, anisaldehyde,
methyl 4-hydroxybenzoate, methyl 2,4-dihydroxybenzoate, propyl 3,4,5-
trihydroxybenzoate and 2-hydroxymethylbenzoate can promote anti-microbial ability
(Kenaway et al., 2005).
19
A thorough understanding of cell and protein interactions with artificial
surfaces is of importance to design suitable implant surfaces and substrates. The
surface properties of newly synthesized biomedical-grade chitosan derivatives,
including surface composition, wettability, domain composition, surface oxidation,
surface charge and morphology, may influence protein adsorption and subsequently,
the cellular responses to biomaterial implants. Though it has once been suggested
that surfaces modified with polyethylene glycol (PEG) may limit protein adsorption
and cell adhesion, the results for PEG blended with chitosan increased both
adsorption and cell adhesion (Saneinejad and Shoichet, 1998). Peng et al. (2006)
demonstrated that chitosan-collagen composites appeared as a biocompatible
biomaterial for periodontal tissue engineering. Zhang et al. (2002) examined the
biocompatibility of newly synthesized chitosan-PEG by growing and observing the
morphological changes of 3T3 embryonic mouse fibroblasts and osteogenesis nodule
cell suspension in culture. Cytotoxicity of chitosan in three dimensional fiber meshes
was well determined with scanning electron microscopy (SEM) and cell
morphological observation on the use of mouse lung fibroblast (L929) (Tuzlakoglu et
al., 2004). Recent studies have been focused on the development of antibacterial
surfaces of biomedical-grade chitosan derivatives to attain high functionality and
high-value products. Huh et al. (2001) prepared chitosan-grafted poly(ethylene
terepthalate) (PET) and quaternized chitosan-grafted PET, both showed high growth
inhibition in the range of 75% to 86% and still retained 48% to 58% bacterial growth
inhibition after laundering. But, higher antibacterial activity of a wound dressing can
retard cell growth in culture. Laminin is known to be involved in metastasis of tumor
cells. A peptide containing the Tyrosine-Isoleucine-Glycine-Serine-Arginine [Tyr-
Ile-Gly-Ser-Arg (YIGSR)] sequence, corresponding to a partial sequence of laminin,
20
inhibited angiogenesis and therefore depressed tumor growth. Nishiyama et al.
(2000) prepared YIGSR-chitosan conjugate which proved to have higher inhibitory
activity against experimental lung metastasis of B16BL6 melanoma cells in mice
than did the parent peptide. According to Koo et al. (2002), water-soluble chitosan
(WSC) significantly protected the cultured cells from serum starvation-induced
cellular rounding up and from serum starvation-induced cell death through p53
activation as tested by flow-cytometry and western blot. From these results, it
appears that WSC may prevent serum starvation-induced apoptosis of human
astrocytes via p53 inactivation. Cross-linking will increase the toxicity of chitosan
scaffold leachate to fibroblast cells. However, the chitosan crosslinked with dimethyl
3-3,dithio-bis’propionimidate (DTBP) permitted cell proliferation after three days in
culture (Adekogbe and Ghanem, 2005).
Denuziere et al. (1998) examined the cytocompatibility of films of chitosan
and chitosan associated with GAGs in human skin keratinocytes and found that the
cell growth was consistently to approximate 60% of that of controls. Highly
deacetylated chitosans can modulate human skin cell mitogenesis in vitro. Cell
compatibility of N-sulfoalkylchitosan derivatives with human dermal fibroblasts
(HDFs) was dependent on the concentration of sulfonate moieties in which higher
concentration led to decrease in cell compatibility (Jung et al., 2007). Additionally,
Zainol et al. (2008) described the usefulness of chitosan to be developed into porous
bilayer membrane as a skin regenerating template for treatment of third degree burn,
and was also proven to be cytocompatible for skin fibroblasts as the cultured cells
were noticed to proliferate on the chitosan membrane. Analysis of their effects on
cells in culture may be useful as a screening tool for their potential activity in vivo as
21
wound healing agents. But, it is important to select appropriate strains of cells for use
in the in vitro biocompatibility screening (Howling et al., 2001).
1.2.4.1 Cytocompatibility assessment in vitro
One of the most basic and important parameters in the in vitro measurement
of a medicinal product’s biocompatibility is cytocompatibility in vitro, which
measures the qualitative and quantitative aspects of the impact of a medicinal
product with regard to the viability of target cultured cells. There are a number of
assays which are capable of evaluating the activation of biological processes (e.g.
inflammation, immune reactions and mutagenesis) which require longer periods of
tissue reaction to materials compared with cytotoxic reactions. Traditional assays
have measured cytotoxicity in vitro by means of either an end-stage event (e.g.
permeability of cytoplasmic membranes of dead and dying cells, or some metabolic
parameters such as cell division or an enzymatic reaction) (Hanks et al., 1996).
Cytotoxicity assays are commonly used to indicate the only finite effects on cells
during the exposure to toxic substances at multiple concentrations and time course
parameters. The colorimetric 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium
bromide (MTT) assay is a simple assay which is selected to determine detrimental
intracellular effects on mitochondria and metabolic activity based on the selective
ability of viable cells to reduce tetrazolium bromide into purple formazan crystals
which are only soluble in organic solvents. The quantity of formazan crystal formed
depends on intact metabolic activity and is frequently used for screening of
cytotoxicity (Mossman, 1983). In vitro biocompatibility evaluation of biomedical-
grade oligo-chitosan (O-C) and N, O- carboxymethyl-chitosan (NO-CMC)
derivatives (O-C 1%, O-C 5%, NO-CMC 1% and NO-CMC 5%) correlated well
22
with in vivo study in which O-C 1% being the most cytocompatible using MTT cell
viability assay. The experiment was carried out as described by the International
Standards Organization (ISO), with some modifications (Lim et al., 2007). The MTT
assessment of cytocompatibility was adequately good and the results were easily
interpreted, besides being cost effective (Mori et al., 1997; Chellat et al., 2000;
Janvikul et al., 2005; Mei et al., 2005; Neamnark et al., 2007).
Appropriate target cell to be cultured should be in accordance with the end
usage of a newly synthesized biomaterial. Je et al. (2006) suggested that chitosan
derivatives in their studies were specific to tumor cell lines [HeLa (cervix cancer),
HT108 (human fibrosarcoma) and A549 (lung cancer)]. Several transformed
keratinocyte culture models have been used to study carcinogenesis, differentiation,
apoptosis and cell cycle regulation (Yuspa et al., 1994). L929 mouse fibroblast cell
line was often applied to evaluate cytotoxicity in vitro of various chitosan derivatives
(Mori et al., 1997; Janvikul et al., 2005). Zange et al. (1998) implemented the MTT
assay to pre-screen the cytotoxicity of poly(L-lactic-co-glycolic acid) attached with
poly(ethylene oxide), namely ABA triblocks by using L929 mouse fibroblast cell
lines and proved the better sensitivity of in vitro model for the cytotoxicity
evaluation than studies in experimental animal model. Peng et al. (2006) observed
the cell viability of human periodontal ligament cells (PDLCs) seeded within the
chitosan-collagen scaffolds by using MTT assay and proved that chitosan-collagen
composites are promising scaffold materials for periodontal tissue engineering.
Others such as F1000 embryonic skin and muscle fibroblasts (Chung et al., 1994)
were also implemented for the cytotoxicity evaluation. Cytotoxicity of chitosan with
various degrees of N-deacetylation to form aminoethyl-chitosan, dimetylaminoethy-
chitosan and diethylaminoethyl-chitosan, was examined with MTT on tumor cell
23
lines and concluded that cytotoxic effect may be caused by cationic charge of the
chitosan derivatives (Je et al., 2006). Howling et al. (2001) had successfully
demonstrated the use of human skin dermal fibroblasts and epidermal keratinocytes
to evaluate the effect of chitosan with various deacetylation levels and polymer
lengths in vitro. Jung et al. (2007) explored the cytocompatibility of newly
synthesized N-sulfopropylchitosan derivatives by using MTT assay on human dermal
fibroblasts (HDF) culture and suggested the usefulness of MTT assay together with
the use of other cell types in assessing the cytocompatibility of chitosan derivatives.
HDFs cultures were also implemented along with MTT cell viability assay to
examine the cell proliferation within the newly developed chitosan-based porous
scaffold (Lim et al., 2007).
1.2.4.2 Genotoxicity assessment in vitro
Although measures of a medical product's biocompatibility have largely been
reported in terms of irritation, sensitization, and systemic toxicity, there is a growing
concern that devices or biomaterial extracts may exert some genotoxic effects. Thus,
any attempts to assess the safety of a device intended for intimate body contact or
permanent implantation would be incomplete without testing for the presence of
toxins that might exert an effect on the genetic material of cells. In its set of
harmonized standards for the biological evaluation of medical devices, the ISO has
outlined the need for such genotoxicity testing in ISO 10993-3: "Tests for
Genotoxicity, Carcinogenicity, and Reproductive Toxicity."
A number of techniques for detecting deoxyribonucleic acid (DNA) damage
as opposed to the biological effects (e.g. Ames test, micronuclei, mutations,
structural chromosomal aberrations) (Ames et al., 1973; Savage, 1976; Ishidate,
24
1979; Maron and Ames, 1983) that result from DNA damage have been used to
identify substances with genotoxic activity. According to Muramatsu et al. (2004),
they did not detect any genotoxicity via Ames test and chromosome aberration assay,
performed on Chinese hamster lung fibroblasts with 5mg/ml (maximum
concentration) of beta-tricalcium phosphate/carboxymethyl-chitin. Abou Sereih et al.
(2007) reported that their chitosan with concentrations of 3.00 and 4.50 mg/ml have
induced mutation on Trichoderma. However, those techniques have some
shortcomings (e.g. time consuming, requirement for proliferating cell populations)
(Fernandez and Ferrao, 1999; Giri et al., 1999; Ikken et al., 1999; Umbuzeiro et al.,
1999).
Until recently, a more useful approach for assessing DNA damage is the
single-cell gel electrophoresis (SCGE) or comet assay. The terms “Comet” is used to
identify the individual cell DNA migration patterns produced. According to Ostling
and Johanson (1984), they were the first to develop a microgel electrophoresis
technique for detecting DNA damage at the single cell level. In their technique, cells
were embedded in agarose and placed on a microscope slide. The cells were lysed by
detergents and high salt concentration to liberate the DNA. The liberated DNA was
then electrophoresed under neutral conditions. Therefore, cells with an increased
frequency of DNA double strand breaks (DSB) displayed increased migration of
DNA toward the anode. The migrating DNA was then measured quantitatively by
staining with ethidium bromide using a microscope photometer. The neutral
condition used in the comet assay detects only DSB of a cell. Subsequently, Singh et
al. (1988) introduced a microgel technique involving electrophoresis under alkaline
(pH >13) conditions for detecting DNA single strand breaks (SSB) and alkali-labile
sites (ALS) in single cells. Singh (2000) mentioned that the alkaline comet assay can