Doc1

Molecular Biological Studies to Evaluate
the Treatment Role of Irradiated Scaffolds
in Ulcers and Wounds in Rat Skin
A Thesis
Submitted In Partial Fulfillment of the Requirements for
the Master Degree of Science in
(BIOCHEMISTRY)
Presented by
Amir Mohammed Mohammed Ali Abdo
B.Sc
Chemistry and Biochemistry
2009
Helwan University
Faculty of Sciences
Chemistry Department
2014

Molecular Biological Studies to Evaluate the Treatment Role
of Irradiated Scaffolds in Ulcers and Wounds in Rat Skin
By
Amir Mohammed Mohammed Ali Abdo
B.Sc in Chemistry and Biochemistry, 2009
In the partial fulfillment of the requirement of the
Master Degree in Science (BIOCHEMISTRY)
Under the supervision of
Prof. Dr. Elsayed Mahdy Prof.Dr. Eglal Eldegheidy
Professor of Biochemistry& Emeritus professor of Biochemistry
Dean of Faculty of Sciences Radiobiology department
Helwan University National Center for Radiation Research
and Technology (NCRRT)
Egyptian Atomic Energy Authority
Prof.Dr. Tarek Khaled Dr. Hatem Abdel Monem El-
Elmaghraby mezayen
Professor of Molecular Biology Assistant professor of Biochemistry
Radiobiology department Faculty of sciences
National Center for Radiation Helwan University
Research and Technology
Egyptian Atomic Energy Authority
Chemistry Department
Faculty of Science-Helwan University
2014

I hear you say "Why?" Always "Why?"
You see things; and you say "Why?"
But I dream things that never were; and I say
"Why not?"
(George Bernard Shaw)

Acknowledgments
i
ACKNOWLEDGMENTS
With the pen in hand, I am proud to think that no words do justice to express my thanks to
ALMIGHTY ALLAH (The Omnipotent, The Omniscient, The Most Merciful and The Most
Powerful) who is the entire source of all knowledge and wisdom to mankind and everything
is submitted to his will.
This work would not be performed without the support of the Academy of Scientific Research
and Technology grant (Scientists for Next Generation (SNG)). I wish to thank each one
working with this great project.
I would like to heartily thank my supervisor Professor Tarek Almaghraby, the
head of the molecular biology lab/ Radiobiology department/ National Center for Radiation
Research and Technology (NCRRT)/ The Egyptian Atomic energy Authority (EAEA), without
whom, this study would not have been possibly completed. Especially, recognition must be
given for offering me with guidance during the research and providing me with the scientific
expertise to complete my thesis.
I would like to express my sincere gratitude to Professor Eglal Eldegheidy, the
emeritus professor of biochemistry/Radiobiology department/ NCRRT/ EAEA for her ever-
inspiring guidance and constructive suggestions throughout the course of this effort.
To professor Elsayed Mahdy, the Dean of Faculty of Science/ Helwan university. I
am very proud to have a supervisor like you. No words can express my feelings and respect
for you.
I wish also to thank Dr. Hatem Abdel-Moneim El-mezayen, the assistant
professor of Biochemistry/ Faculty of Science/ Helwan university for kindly supervising the
present work, reading and scholarly criticizing the manuscript.
I wouldn`t have been able to complete this research without the generosity of Professor
Waleed Nazmy, the head of the Innovation Development Unit at VACSERA, Egypt. He
has provided me with extraordinary mentorship during my work in his lab. His meticulous
concern and patience were the ways to complete this research.

Acknowledgments
ii
The work presented in this study was impossible to be accomplished without the sympathetic
attitude and utmost care of my teacher, Dr. Sanaa Abdel-Hamid.
I wish to express my deepest feeling of gratitude to Dr. Mohammed Abdel-Baseer,
Dr. Saad Attiya and Dr.Wael Abul-Noor. Without their observant pursuit,
cheering perspective and the enlightened supervision, this work would not be completed.
Thanks to Prof. Renee Georgy, NCRRT, prof. Kawkab Abdel-aziz, Cairo Univ. and
Mr. Moussa Hussein, the National Cancer Institute (NCI) for conducting the
hisological staining and examinations.
Great thanks to Prof. Doaa Mekawy and Dr. Wael Hossam, the National
Research Center (NRC) for their help during my study.
I am grateful to prof. Hisham Attiya, prof. Ahmed Shafik, prof. Magdy Senna
,Dr. Mohammed Almohmady and Dr. Ahmed Elbarbary, NCRRT for their kind
advice and support during the work.
I can`t forget the kind support from Dr. Mohammed Hamdy, so I wish him to receive
my great thanks.
Words are lacking to express my humble obligation to my late father and my loving mother
who always longed for my successful and happy life. Their endless efforts and best wishes
sustained me at all stages of my life and their hands always remain raised in prayer for my
success. It’s my ever pray that may Allah bless my mother with long, happy and healthy life.
If the pearls were words and flowers feelings, it would be easier to express my deepest heart
gratitude and indebtedness to the all the peoples and friends who have helped me during this
research. May Allah Almighty infuses me with the energy to fulfill their noble inspiration and
expectations and further modify my competence. May Allah bless us all with long, happy and
peaceful lives.
Amier Mohammed

iii
List of Contents
Section Contents Page
Acknowledgments i
List of Contents Iii
List of Figures v
List of Tables and Appendices vii
List of Abbreviations viii
Abstract xi
1 Introduction 1
Aim of Work 3
2 Review of literature 4
Chapter (1) Skin and Wound Healing 4
2.1.1 Skin Structure 4
2.1.2 Skin Anatomy 4
2.1.3 Functions of Skin 10
2.1.4 Skin Wounds 12
2.1.5 Skin Ulcers 13
2.1.6 Wound Healing 14
2.1.7 Classification of Wound Dressing Products 17
Chapter (2) Review on Alginates 27
2.2.1 Chemical Structure of Alginate 27
2.2.2 Sources of Alginates 28
2.2.3 Properties of Alginate 29
2.2.4 Alginate Gelation 30
2.2.5 Modification of Alginate 35
2.2.6 Purification of Alginate 43
Chapter (3) Review on Chitosans 46
2.3.1 Chemical Structure of Chitosan 46
2.3.2 Sources of Chitosans 47
2.3.3 Properties of Chitosan 49
2.3.4 Production of Chitosan from Chitin of Shrimp Shells 52
Chapter (4) Review on PolyElectrolyte Complexes (PECs) 53
2.4.1 Properties of the Alginate/Chitosan PECs 53
2.4.2 Principle of Formation of Alginate/Chitosan PECs 55
Chapter (5) Biological effects of alginates & chitosans-based dressings 58
2.5.1 Alginates as Wound Dressings 58
2.5.2 Chitosans as Wound Dressings 62
2.5.3 Alginate/ Chitosan-Based Wound Dressings 63
2.5.4 Angiogenesis and Angiogenesis-Controlling Genes 66
2.5.4.1 Vascular Endothelial Growth Factor (VEGF) 64
2.5.4.2 von Willebrand Factor (vWF) 72
3 Materials and Methods 80
3.1 Materials 80
3.2 Preparation of reagents 80

iv
Section Contents Page
3.3 Modification of Alginate 81
3.3.1 Irradiation of Sodium Alginate 81
3.3.2 Oxidation of Sodium Alginate 82
3.3.3 Characterization of the Different Modified Alginates 83
3.4 Purification Protocol of Sodium Alginate 86
3.4.1 Acid-washing of Activated Charcoal 86
3.4.2 Method of Alginate Purification 86
3.4.3 Testing the Effects of Purification in Alginate 87
3.5 Preparation of Chitosan 90
3.5.1 The Extraction and Deacetylation Steps 93
3.5.2 Characterization of the Prepared Chitosans Products 93
3.6 Method of Preparing the Alginate-Chitosan PECs hydrogels 93
3.7 Major Steps for Choosing the Best Type of Hydrogels 94
3.7.1 In vitro Swelling of Hydrogels in Simulated Wound Fluids 94
3.7.2 Stability Characterization Studies 94
3.7.3 Blood Compatibility Tests 95
3.7.4 Rate of Evaporation of Water from Gel 96
3.7.5 In vitro Degradation of the Prepared Hydrogel Films 96
3.7.6 Primary Skin Irritation Test for the Hydrogels 96
3.7.7 Testing the Optimum Composition of Hydrogel 97
3.7.7.1 Detection of the Best Concentration for the used(CaCl2) 97
3.7.7.2 Characterizing the Effects of (γ-irradiation) on (F-20) 97
3.7.7.3 Choosing the Best Working Film Structure 99
3.8 Statistical Analyses 100
3.9 Wounding and Wound Healing Assessment 102
3.9.1 Animals 102
3.9.2 Wounding Procedures 102
3.9.3 Wound and Skin Assessment 103
3.9.3.1 Monitoring the Visible Changes in Wounds During Healing 103
3.9.3.2 Measurement of Residual Wound Area 103
3.9.3.3 Histological Studies 104-105
3.9.3.4 Quantification of RNA corresponding to(VEGF and vWF) 105-110
3.9.3.5 Screening of Kidney Functions 110
4 Results 113
5 Discussion 142
Recommendations for future work 188
Summary and Conclusion 189
References 191
Appendices 235

v
List of figures
No. Title Page
1 Cross-sections of Skin and the Epidermis layer 5
2 Summary for phases of Wound Healing 15
3 Summary for the overlapping periods of healing stages 15
4 Schematically drawn alginate block structure with a segment
showing structure of the molecules
27
5 The binding of a divalent cation to contiguous dimers of
guluronate residues
32
6 Proposed mechanism for Alginate degradation in the solid state 39
7 Suggested reaction scheme describing periodate oxidation of a
mannuronan residue within the alginate chain
41
8 The chemical structures of Chitin and Chitosan 46
9 Schematic diagram of counter-ions release upon PEC formation 56
10 Schematic Interpretation of the (Alginate-Chitosan physical
complex and Semi-IPN complex)
57
11 Genomic location of human VEGFA Gene on chromosome (6) 66
12 Exon structure and function of rat VEGFA 67
13 Different VEGF Receptors&the corresponding binding cytokines 68
14 Genomic location of human vWF gene on chromosome (12) 73
15 Structure of vWF Protein 74
16 FT-IR spectra for the irradiated and oxidized sodium alginates 114
17 FT-IR Spectra showing the changes within the activated charcoal
after washing with different acids
115
18 Quantitative evaluation of the major Alginate contaminants
before /after purification
116&
117
19 FT-IR spectra for (Na-Alginate) before and after purification 117
20 (FT-IR) spectra for the chitosans (1, 2) 119
21 Swelling kinetics for different formulae immersed in PBS
medium (pH 7.4) for 24 hours
121
22 Time dependence of water loss from the 3 formulae (F-20,18,5) 124
23 Rate of degradation for the different formulae (F-20,18,5) 125
24 The influence of cross-linking agent (CaCl2) concentration on the
swelling degree for the formulae (F-18 and 20)
127
25 Comparison of the swelling kinetics for the unirradiated and
irradiated formulae (F-20 and 5)
127
26 FTIR spectra for physical mixture of alginates, alginate with
chitosan, the unirradiated and irradiated PECs (F-20)
128
27 Scanning Electron Micrographs for the surfaces of different
formulae based on (Alginate/Chitosan PECs).
129

vi
No. Title Page
29 Time dependence of water loss from the 2 hydrogel forms (F-20,
and F-20/I)
131
30 Rate of degradation for the unirradiated and irradiated forms (F-
20 and F-20/I)
131
31 Representative digital photographs assessment of healing
progression during the first 2 post-operative weeks
132
32 Rate of closure of wounds in large rats, treated with the prepared
dressing, fusidin cream or the untreated wounds.
134
33 Comparison of healing models between dressed and undressed
wounds in small rats
134
34 Histology of wound sections stained with Hematoxylin (H),
Eosin (E) and Masson`s Trichome (MT) under polarized light
after 1 and 3 days of wounding.
135
35 Representative images of (H, E and MT) histological stained
wound sections (Day:7)
135
36 Representative images of (H, E and MT) stained wound sections
(Days: 11& 15)
136
37 Representative images of (H, E and MT) stained wound sections
(Day: 16)
137
38 VEGF mRNA quantification by r.t-PCR during the healing days
of both dressed (D)& non-treated control wounds (C)
138
39 Amplification curves for the Quantitative real time PCR of VEGF
and β-actin cDNAs from both the 2 wounding groups
138
40 Melting curves for PCR products of VEGF cDNA amplification
from wounds of both groups
139
41 vWF mRNA quantification by r.t-PCR during the healing days of
both dressed (D)& non-treated control wounds (C)
140
42 Amplification curves for the Quantitative real time PCR of vWF
and β-actin cDNAs from both the 2 wounding groups
140
43 Melting curves for PCR products of vWF cDNA amplification
from both wounded groups
141
44 Schematic interpretation of chitin backbone structure 147
45 Deacetylation Mechanism for chitin into chitosan 147

vii
List of Tables
No. Title Page
1 Antiangiogenic agents, approved by FDA 22
2 Summary for commercial Alginate and Chitosan-based
dressings
61
3 Primers sequences, expected product length and PCR program
for amplification of (β-actin, VEGF and vWF genes)
109
4 Aldehyde analyses for the different alginates (Formyls/ mol. of
alginate)
114
5 Average Molecular Weights for the different alginates 114
6 Properties of the prepared chitosans 118
7 General overview for the swelling and stability results of the
different formulae composing of [chitosan fraction plus
alginate fraction whose composition is only shown]
122&
123
8 Blood compatibility parameters for different hydrogels 124
9 PDI test results for the Non-irradiated Hydrogel 125
10 PDI test results for the irradiated Hydrogel 125
11 Levels of BUN (mg/dl) and Creatinine (mg/dl) in plasma 141
List of Appendices
No. Appendix Page
Appendix (A) The different groups frequency wave-numbers (cm-1
)
for the Raw charcoal & different washed charcoals
235
Appendix (B) The groups frequency wave-numbers for sodium
Alginate
236
Appendix (C) The different groups frequency wave-numbers (cm-1
)
for the two Prepared chitosans (Ch-1 and Ch-2)
237

List of Abbreviations
viii
Abbreviation Name
AFU Arbitrary Fluorescence Unit
Alg Alginate
APS Ammonium persulphate
A.T Adipose Tissues
BCs Basal Cells
BCC Basal Cell Carcinoma
BSA Bovine Serum Albumin
CFU Colony Forming Units
Ch Chitosan
Con Control
CP Carrier Proteins
CXCR-4 Chemokine Receptor type 4
DD Degree of Deacetylation
DDS Drug Delivery System
D.S Degree of Swelling
DFUs Diabetic Feet Ulcers
Dre Dressing
DSwG Duct of Sweat Gland
ECs Endothelial Cells
ECM Extra-Cellular Matrix
EDC 1-Ethyl-3 (-3-Dimethylaminopropyl) Carbodiimide. HCl
EGF Epidermal Growth Factor
EGFR Endothelial Growth Factor Receptor
EGT Early Granulation Tissues
EGTA Sodium Ethylene Glycol Tetra Acetic acid
EPCs Endothelial Progenitor Cells
FBG Fasting Blood Glucose
FBS Fetal Bovine Serum
FDA Food and Drug Administration
FFE Free Flow Electrophoresis
FGF-2 (bFGF) Basic Fibroblast Growth Factor
Flk-1 Fetal liver kinase-1
Flt fms Related Tyrosine Kinase
FTIR Fourier Transform Infrared
Fus Fusidin
GRAS Generally Recognized As Safe
G α-L-guluronate
GGG Polyguluronates

ix
Abbreviation Name
GLcN 2-amino-2-deoxy-β-glucopyranose (glucosamine)
GlcNAc 2-acetamido-2-deoxy-β-D-glucopyranose (N-
acetylglucosamine)
GP GlycoProtein
Gy Gray
H-bonding Hydrogen bonding
HCB Human Citrated Blood
H&E Haematoxylin and Eosin
HF Hair Follicle
HMW High molecular Weight
Il Interleukin
IMC Inter-Macromolecular Complexes
IPN Inter Penetrating Network
KDR Kinase insert Domain Receptor
LCD Linear Charge Density
LCST Lower Critical Solution Temperature
M ß-D-mannuronate
MMM Polymannuronates
MASA Multi Aldehyde Sodium Alginate
MHC Major Histocompatibility Complexes
MHS Mark–Houwink–Sakurada equation
MMP Matrix Metallo-Proteinase
MT Masson`s Trichrome
MWD Molecular Weight Distribution
Mn Number Average Molecular Weight
Mv Viscosity Average Molecular Weight
Mw Weight Average Molecular Weight
Na-Alg Sodium Alginate
NMF Natural Moisturizing Factors
NO Nitric Oxide
PBS Phosphate Buffered Saline
PC Polyphenol-like Compounds
PDA Parenteral Drug Association
PDGFR Platelet-Derived Growth Factor Receptor
PEC PolyElectrolyte Complex
PG12 Prostacyclin (Prostaglandin 12)
PLC-γ phospholipase C
PlGF placental Growth Factor
ROS Reactive Oxygen Species

x
Abbreviation Name
R.T Room Temperature (25o
C)
SAL Sterility Assurance Level
SCCs Stratum Corneum Cells
SDF-1 Stromal Cell -Derived Factor-1
SEC Size Exclusion Chromatography
SGCs Stratum Granulosum Cells
SGl Sebaceous Gland
SMCs Smooth Muscle Cells
SSCs Stratum Spinosum Cells
SSD Silver Sulphadiazine
STZ Streptozotocin
SwG Sweat Gland
TEMPO 2,2,6,6-tetramethylpiperidine-1-oxy radical
TGF- β Transforming Growth Factor-β
TS Tensile strength
U.V Ultra-Violet
VEGF Vascular Endothelial Growth Factor
VPF Vascular Permeability Factor
VWD von Willebrand Disease
vWF von Willebrand Factor
WPBs Weibel-Palade bodies

Abstract
xi
Molecular Biological Studies to Evaluate the Treatment Role
of Irradiated Scaffolds in Ulcers and Wounds of Rat skin
ABSTRACT
Skin is the first line of defense in the body and can be easily injured with
either external object or with internal blunt force trauma. There are many
types of wound dressings with different properties and mechanism of action
for accelerating healing. They may activate the wound repair, help in the skin
regeneration process, provide the moisture environment for wound or help in
its drying. Biomaterials, the non-drug biologically-derived materials have
become very important means to treat, enhance or replace any tissue, organ
or function in an organism based on their structural rather than biological
properties. For viable translational outcomes, we considered that a hydrogel
made of the 2 polymeric biomaterials; alginate and chitosan alone, with no
additional growth factors, cytokines or cells would prove sufficiency to treat
wound injuries and can act as a scaffold for activating cells migration and
proliferation as well as promoting the angiogenesis. The present study aimed
at preparing a new type of Alginate/ Chitosan PolyElectrolye Complex
(PEC) hydrogel and testing the required wound healing properties of the
hydrogel in vitro which were then tested in vivo with excisional acute wound
models in rats and compared with those of a commercial cream dressing and
non-treated wounded rats. The healing promoting effects were assessed using
different methods including the quantification of expression of two
angiogenesis-controlling genes (VEGF and vWF) and measurement of the
wound closure rate % with histological examinations for skin and wounds
beds. In addition, the effect of gel degradation in the body was monitored by
routine measuring of kidney functions.

Abstract
xii
The dressed wounds showed maintained suitable levels of the angiogenic
genes for activating hemostasis and accelerating the angiogenic cascades for
maintaining the blood supply to the newly formed skin tissue in the wound
area. Accelerated rebuilding for the layers of wound area was observed
proving efficiency of the hydrogel in the treatment of acute wounds and its
role in the regeneration of the damaged skin tissues. The wound closure rate
was faster with wounds treated with the chosen hydrogel than those treated
with the cream and the non-treated wounds.
Key words: Wound, Wound Healing, Biomaterial, Alginate, Chitosan,
PolyElectrolyte Complex, Angiogenesis, VEGF, vWF.

Introduction and Aim of the work
1
INTRODUCTION
Skin is the largest organ of the integumentary system consisting of
multiple layers of ectodermal tissues which guard the underlying muscles,
bones, ligaments and internal organs. It is a dynamic organ in a constant state
of change where cells of the outer layers are continuously shed and replaced
by the inner cells moving up to the surface (Bensouilah et al., 2007). The
skin is a complex metabolically active organ which interfaces with the
environment and performs many important physiological functions such as
protecting the body against excessive water loss (Carola et al., 1990) and
pathogens (Bensouilah et al., 2007). Thermoregulation, sensation
,insulation, synthesis of vitamin D and the protection of vitamin B folates are
also skin functions.
Skin wounds are types of injuries in which the skin may be compromised
with exposing the underlying tissues (Open Wounds) or may not be torn with
formation of trauma to the underlying structures (Closed Wounds). The
wounds may be acute which normally proceed through an orderly and timely
reparative process through four highly programmed phases: hemostasis,
inflammation, proliferation and remodeling, occurring in the proper time
frame and sequence resulting in sustained restoration of the anatomic and
functional integrity through healing (Cohen et al., 1999), or chronic that fail
to proceed with the previously ordered sequence where many factors can
interfere with one or more of these phases causing improper or impaired
wound healing (Lazarus et al., 1994).
There are many types of wound dressings such as films, non-adherent,
hydrogels, hydrocolloids, hydrofibres, foam dressings and topical
chemotherapies for wounds of different types. Each dressing type has certain
properties and a mechanism of action.

2
Dressings made of the biomaterials, chitosan and/or alginate have got
attention due to their peculiar properties, hemostatic, biodegradability,
bioactivity and remodeling properties (Otterlei et al., 1991; Azad et al.,
2004; Lin et al., 2006), so many types of dressings of each one alone, a
combination of them or with other materials as well have been synthesized
and their efficacies have been proved.
Bioengineering is considered one of the most innovative approaches
tackling many diseases and body parts that need to be replaced. This term
applies to the efforts that span interdisciplinary boundaries and connects the
engineering and physical sciences to the biological sciences and medicine in
a multidisciplinary setting to develop or apply new treatment technologies as
well as performing specific biochemical functions with a major dependence
on cells within artificially-created support system, called scaffold (Zhao et
al., report) whose properties depend primarily on the nature and properties
of the used materials. Novel free form fabrication methods for engineering
polymeric scaffolds have gained interests due to their repeatability and
capability of usage with high accuracy in the fabrication resolution at the
macro and micro scales. For example, ionically cross-linked alginates have
great potential as scaffolds where they can form highly hydrated hydrogels
representing hospitable environment for the transplanted cells and cellular
infiltration. An ideal wound dressing should control evaporative water loss,
prevent dehydration, protect the wound from bacterial infection, allow
diffusion of oxygen and carbon dioxide, absorb wound exudate and enhance
its healing (Kirker et al., 2002).
Wound assessment is essential for effective wound management and for
investigating the effect of certain dressing on the healing cascade (NHS,
2008) with monitoring the wound closure rate and any changes to it.

3
Histological examinations for wounds beds are also essential for
assessing the skin maturity and testing the influence of the dressing in the
histo-architectural organization of the wound area. Angiogenesis and
neovascularization are critical determinants of wound healing outcomes
where the newly formed blood vessels participate in the healing process with
providing nutrition and oxygen to the growing tissues. Accordingly; to better
determine the functionality of the developing vasculature, the angiogenic
response is studied by the quantitative measurement of expression of the
angiogenesis-controlling genes using the molecular biology technique,
Polymerase Chain Reaction (PCR). Nowadays, Molecular biology plays
important roles in understanding structures, actions and regulations of
various cellular compartments and can be used efficiently for targeting
new drugs, diagnosis of diseases and studying the physiology of cells.
Aim of Work
This study aims at: (1) preparing a hydrogel made of a new extracted
chitosan and chemically modified alginates with irradiation and oxidation in
the form of alginate-chitosan coacervates under controlled conditions for
casting into homogeneous films utilizing a new method.
(2)The designing of a general scheme for choosing the best suitable hydrogel
that can act as a scaffold for engineering dermal and epidermal tissues and as
a controlled release system for drugs to the skin aiming to accelerating the
wound healing.
(3) Its biological effects for treatment of rat skin wound models will be
investigated using histological and molecular biological methods with
measuring the expression of certain angiogenic genes (VEGF and vWF) for
assessing the potential effect of the chosen hydrogel on the skin wound and
its promotion for the corresponding angiogenic responses.

Review of Literature
4
2. REVIEW OF LITERATURE
(1): Skin and Wound Healing
2.1.1 Skin Structure:
The skin is a physiologically and anatomically specialized boundary lamina
essential to life and has several functions such as forming a physical barrier to
environment to allow and limit the inward and outward passage of water,
electrolytes and various substances with providing protection against toxic agents,
microorganisms, Ultra-Violet radiation (U.V) and mechanical insults. It occupies
almost 1.8 m2
of the surface area in average adults, accounting for 16% of the body
mass making it its largest organ (Bensouilah et al., 2007).
Skin can be classified according to its thickness that varies with age of the
individual and the anatomical part of the body where it is found. It may be thin,
hairy (hirsute), constituting the majority of the body‘s surface (e.g., Skin on the
eyelids is less than 0.5 mm thick), or may be thick, hairless (glabrous) skin such as
skin covering the palms, soles and flexor surfaces of the digits and skin on the
middle of the upper back which is more than 5mm thick (Gray, 1987; Carola et
al., 1990).
2.1.2. Skin Anatomy:
Skin is a structurally complex and highly specialized organ, consisting of two
intimately associated main layers called: (1) The epidermis, the outermost layer of
skin, and (2) The dermis (corium), a thicker layer beneath the epidermis. Certain
appendages such as hair follicles and sweat glands span both the 2 layers and
penetrate into the subcutaneous adipose tissue beneath the dermis (Alberts et al.,
2002; Carola et al., 1990). Fig. (1) illustrates the general architecture of the skin
and the epidermal layers (Studyblue site).

5
(I) Epidermis:
It is composed of keratinized stratified squamous epithelium with no blood vessels,
so rupture of its old cells usually occurs without bleeding (Carola et al., 1990).
The main component cells are keratinocytes, in addition to other cell types such as
Langerhans cells and melanocytes (Alberts et al., 2002). Certain skin appendages
(e.g., Nails, hair and its follicles) are formed by the in-growth or other
modifications in this layer (Gray, 1987).
Figure (1): (A) Cross-section of skin showing its different layers.
(B) Cross-section in the Epidermis layer.
Epidermis is divided into a number of strata representing different stages in
keratinocytes maturation in a constant state of transition from the deep to
superficial layers (Carola et al., 1990) (fig. (1B)) as follows:
(1) Stratum Basale (Stratum Germinativum):
The innermost layer of epidermis that lays adjacent to the dermis. It includes a
single layer of columnar cells which undergo cell division to produce new cells
due to its content of stem and progenitor cells, so can replace those being sheared
off in the exposed corneal layer (Carola et al., 1990). The proportion of basal cell
population is Langerhans cells and melanocytes stretching between relatively large
numbers of neighboring keratinocytes.

6
Melanin pigment from melanocytes provides protection against (U.V) radiation.
Merkel cells are closely associated with cutaneous nerves and found with large
numbers in touch-sensitive sites (e.g., Finger tips and lips).
(2)Stratum Spinosum (Prickle Cell Layer):
As basal cells reproduce and mature, they move towards the outer skin layer
forming initially the (Stratum Spinosum) that composes of several layers of mature
keratinocytes (polyhedral cells with delicate intercellular bridges of desmosomes,
(prickles) to give support to this binding layer) (Carola et al., 1990). Langerhans
cells are dendritic, immunologically active cells, derived from bone marrow and
found on all epidermal surfaces, but mainly located in the middle of this layer for
their antigen-presenting functions.
(3) Stratum Granulosum:
This layer lies just above the (Spinosum layer) with (2-4) cell thickness resulting
from maturation of lower layer cells and continue to flatten during their continuous
transition to the surface with loss of nuclei and the cytoplasm appears granular at
this level. The cells contain keratohyaline crystals, the precursor of soft keratin for
initiating the keratinisation process, associated with the process of cell death
(Carola et al., 1990).
(4) Stratum Corneum:
This is the flat outermost epidermal layer with relative thickness. It consists of
corneocytes (non-viable cornified cells of hexagonal shape arranged in parallel
rows) with the final outcome of keratinocytes maturation; each cell is surrounded
by a protein envelope of (fillagrin) and filled with water-retaining keratin whose
orientations with cells shape strengthen this layer. Stacked lipid bilayers surround
the cells in the extracellular space to give a structure that provides the natural
physical and water-retaining barrier functions of skin where the corneocyte can
absorb water, 3 times its weight.

7
Based on the location of skin, this layer varies from only a few cells thick (e.g., in
the scalp) to more than 50 cells thick with the palms and soles having the most.
The layer cells are constantly shed through normal abrasion and are replaced by
new cells formed by cell division and pushed up from the germinative layers below
during the epidermal transit time to take on the function of the cells they replace
(5) Stratum Lucidum:
A subdivision of the (Stratum Corneum) that only appears in glabrous skin where
it acts as a protective shield against the (U.V) rays of the sun, thus prevents
sunburn to these areas (Carola et al., 1990). It consists of translucent, flat layers of
dead cells containing the protein eleidin, a transitional substance between the
precursor of soft keratin in the stratum granulosum and the soft keratin of the
corneum layer.
(II) Basement Membrane (Dermo-Epidermal Junction):
A specialized sheet-like Extra-Cellular Matrix (ECM) with complex structure that
allows the epidermis to obtain nutrients and dispose wastes via diffusion through
dermal papillae from the papillary dermis projecting perpendicular to the skin
surface (Gray et al., 1987). It is responsible for the epidermal mechanical
stabilization (Carola et al., 1990) and any abnormalities within the structure and
functions of the membrane result in the expression of rare skin diseases as well as
flattening during ageing accounting in part for some of its visual signs
(Bensouilah, 2007). It is composed of the following two layers:
Reticular Lamina (Lamina Densa): A deeper lamina on the dermal side that
grades into its connective tissue. Its structure includes networks of type IV
collagen molecules, fibronectin, epidermolysis bullosa acquisita antigen
glycoprotein (Type VII Collagen) and various proteoglycans.

8
It limits the passage of macromolecules from the dermis to epidermis, suppresses
differentiation of keratinocytes in the (Stratum Basale) and regulates other cellular
activities in the epidermis (Bensouilah, 2007).
(2)Basal Lamina (Lamina Lucida): It is a strong adhesive layer to the overlying
cells of the (Stratum Basale) with a thickness (about 80 nm). It is occupied by
various macromolecules including, laminin, heparan sulfate proteoglycan and
bullous pemphigoid antigen skin protein which give the layer a finely granular or
filamentous appearance (Carola et al., 1990).
(III) Dermis:
The dermis lies beneath the epidermis and Basement membrane constituting the
majority of skin. It varies in thickness, ranging from 0.3 mm on the eyelids to 3mm
on the back, palms and soles. It is composed of a tough, supportive cell matrix
including endothelial cells, smooth muscle cells, fibroblasts, macrophages and
immuno-competent mast cells (Supp and Boyce, 2005). Bulk of the dermis is
made of (ECM) of irregular, moderately dense, soft connective tissue consisting of
interwoven collagenous meshwork, mainly of type I collagen with various amounts
of elastin fibers, structural proteoglycans and fibronectin (Gray et al. 1987;
Carola et al., 1990). Collagen fibers make up 70% of the layer giving it strength
and toughness. Elastin maintains normal elasticity with flexibility and the
proteoglycans provide viscosity and hydration. Dermis is highly flexible and
reliant, but on stretching beyond its limits, collagenous and elastic fibers can be
torn resulting in (stretch marks) from the repaired scar tissue (Carola et al., 1990).
Embedded within its fibrous tissue are the dermal vasculature, lymphatics, sweat
glands, hair roots, small quantities of striated muscles, nerve cells and fibers. Two
well-defined layers compromise the dermis as follows:

9
(1) Reticular Layer:
A netlike inner dermal layer, made up of dense connective tissue with coarse
collagenous fibers and fiber bundles that criss-cross in random organization to
form strong and elastic network with different directional patterns in each area of
the body. The deepest region contains smooth muscle fibres, especially in the
genital and nipple areas and at the base of hair follicles (Carola et al., 1990).
(2)Papillary Layer:
This is a sub-epithelial layer that lies below the epidermis and connects with it. It
consists of fairly loose, packed connective tissue with thin bundles of collagenous
fiber housing rich networks of sensory nerve endings, blood vessels and tiny
papillae that join it to the epidermis through the Dermo-epidermal junctions at their
interfaces (Gray et al., 1987; Carola et al., 1990). Most of these papillae contain
capillary loops that nourish the epidermis while others have special nerve endings
called corpuscles of touch (Meissner`s corpuscles) serving as sensitive touch
receptors. In glabrous skin, double rows of papillae produce ridges to provide
mechanical anchorage, metabolic support and trophic maintenance to the overlying
epidermal tissue by keeping the skin from tearing and improving the grip on
surfaces. The overlying epidermis follows the corrugated contours of the
underlying dermis, and therefore, these papillae produce distinct fingerprint
patterns on the finger pads (Carola, 1990).
(3) Subcutis Layer (Hypodermis):
This is a dermal layer of skin within certain positions in the body and can be up to
3 cm thick on the abdomen (Gray et al. 1987). It consists of loose connective
tissue with fat.

10
2.1.3. Functions of Skin:
1- Prevents Loss of Moisture:
The layered sheets of epithelial tissue and a nearly waterproof layer of soft keratin
in the (Stratum Corneum) are responsible for the moisturizing effect of skin (Gray
et al., 1987; Carola et al., 1990). As the degenerating cells move towards the
outer layer, enzymes break down the keratin-fillagrin complex in the granules of
the (granular layer). When moisture content of the skin reduces, fillagrin is further
broken down in the (Stratum Corneum) under the action of specific proteolytic
enzymes into free amino acids which along with other components known as
Natural Moisturizing Factors (NMF: e.g., Lactic acid, urea and salts) are
responsible for keeping the skin moist and pliable due to their ability to attract and
hold water (Presland et al., 2009).
2-Thermo-regulation & Excretion:
The skin can act as a sheet of insulation to retain body heat and assist in its
cooling. Dense beds of blood vessels in the dermis dilate to allow heat loss through
evaporation of sweat from the surface and increased radiation of heat from the
blood. To assist in heat retention, the vessels constrict to reduce the radiation
(Gray et al., 1987; Carola et al., 1990). Perspiration also allows the excretion of
small amounts of waste products such as urea; up to 1 gram of waste nitrogen is
excreted every hour (Carola et al., 1990).
3-Acts as a Sensory Organ:
Sensation is a critical function of the skin (Clark et al., 2007). It contains sensory
receptors for heat, pain, cold, touch, pressure and allows us to make adjustments
for maintaining homeostasis. Merkel cells at the base of epidermis play a role in
sensory transduction. Keratinocytes are involved in the detection of physical and
chemical stimuli. Hair cells are also involved in cutaneous sense (Lumpkin and
Caterina, 2007).

11
4-Plays Roles in Immunological Surveillance:
The skin is very important as a passive barrier with immunological roles where it
defends the body against diseases and entry of harmful microorganisms. The skin
immune components are summarized in the report of (Bensouilah and Buck,
2007). It normally contains all the elements of cellular immunity including T-
lymphocytes, Langerhans cells, mast cells, keratinocytes, cytokines, Major
Histocompatibility Complexes (MHC), and complement cascade components with
the exception of B-cells.
5-Reduces the Harmful Effects of UV Radiation:
Melanocytes, located in the deepest part of the (Stratum Basale), have rounded cell
bodies and produce the dark pigment (melanin), packaged into melanosomes and
delivered to keratinocytes of the different layers to form a protective shield over
their nuclei and the genetic material to screen the harmful UV rays. If too much
UV light penetrates the skin (e.g., In sunburn): due to inadequate protection, the
radiation may cause damage of enzymes, cell membranes, interfere with its
metabolism and may cause epidermal cell death as well (Carola et al., 1990).
Epidermal neoplasms may occur after chronic exposure because of damage to the
basal cell's DNA resulting in squamous cell carcinoma. If tissue destruction is
extensive, toxic waste products and other resulting debris can enter the blood
stream and produce fever, associated with sun stroke.
6-Synthesis of Vitamin D3 (Cholecalciferol):
Although most of the UV rays are screened out by the skin, it permits the entry of
small amount to be consumed in converting (7-dehydrocholesterol) in the skin to
vitamin D3 (Cholecalciferol) in the two innermost strata, the stratum basale and
stratum spinosum. Vitamin D is essential for proper growth of bones and teeth and
its leakage impairs the calcium absorption from the intestine into the blood stream

12
7-It provides a protective barrier against mechanical, thermal, physical injury and
noxious agents.
8-Skin has also importance in the cosmetic, social and sexual associations.
2.1.4. Skin Wounds
2.1.4.1. Definition:
When the integrity of any tissue is compromised (e.g., Skin breaks, muscle tears,
burns, or bone fractures), a wound occurs. Skin wounds may be result of a fall,
surgical procedures; an infectious disease or by an underlying condition.
2.1.4.2. Description:
Types and causes of skin wounds are wide ranging with different ways of
classification. They may be acute wounds which normally proceed through an
orderly and timely reparative process resulting in sustained restoration of anatomic
and functional integrity through healing (Cohen et al., 1999). The other type is the
chronic wound that has failed to proceed through an orderly and timely process to
produce the required integrity due to compromised wound physiology (Lazarus et
al., 1994); examples include skin ulcers caused by diabetes, venous stasis or
prolonged local pressure.
2.1.4.3. Classification of Wounds:
(1) Open Wounds: Wounds in which the skin has been compromised and the
underlying tissues were exposed. The acute open wounds can be categorized
according to the relevant mechanism of injury into:
I-Abrasions (Scrapes): Superficial wounds in which the topmost layer of skin is
scraped off and rubbed away by friction against a rough surface.
II-Avulsions: Occur when an entire structure or part of it is forcibly pulled away
(e.g., Loss of a permanent tooth or an ear lobe, also with animal bites).

13
III- Fish-hooks: Injury caused by fishhook becoming embedded in soft tissue IV-
Crush Wounds: Occur when a heavy object falls onto a person, splitting the skin
and shattering or tearing underlying structures.
V-Cuts: Slicing wounds made with a sharp instrument leaving even edges. They
may be as minimal as paper cut or as significant as surgical incision.
VI-Incised Wounds: Any sharp cut in which the tissues are not severed; a clean
cut caused by a keen cutting instrument.
VII-Lacerations (Tears): Irregular tear-like wounds that produce ragged edges
resulting from a tremendous force against the body, either from an internal source
as in childbirth, or from an external source like a punch.
(2) Closed Wounds: Wounds in which the skin has not been compromised, but
trauma to the underlying structures has occurred and include:
I-Contusions (Bruises): They result from a forceful trauma that injures an internal
structure without breaking the skin. Blows to the chest, abdomen or head with a
blunt instrument (e.g., a football or a fist) can cause contusions.
II-Hematomas (Blood tumors): They are caused by damage to a blood vessel.
This in turn causes blood to collect under the skin.
III-Crushing Injuries: They are caused by an extreme amount of force applied
over a long period of time.
2.1.5. Skin Ulcers:
The ulcer can be defined as a gradual disturbance of tissues by underlying, and
thus internal etiology/pathology, but the wound results from acute disturbance of
tissues by an external force. The observed differences in demographics,
appearance, anatomical locations, pathology and physiology as well as the required
medical interventions, possible medical options and outcomes have become great
deal (Armstrong et al., 1998).

14
2.1.6. Wound Healing:
Wound healing (Cicatrisation) is a complex and dynamic process that results in the
restoration of anatomical continuity and function (Lazarus et al., 1994) through a
predictable chain of complex biochemical and molecular events taking place in a
closely orchestrated cascade involving complex interaction among (ECM)
molecules, soluble mediators, resident and infiltrating inflammatory cells which
either restore or at least secure the damaged tissue. These events are classically
divided into 4 main distinct but overlapping phases in time and duration:
Hemostasis, Inflammation, Proliferation and Tissue Remodeling (Maturation) as
summarized in (fig.(2))
Briefly, within minutes post-injury, platelets aggregate at the injury site to form a
fibrin clot which acts to control active bleeding (Hemostasis). The speed of wound
healing can be impacted by many factors including the bloodstream levels of
hormones (Poquérusse, 2012). In the inflammatory phase, bacteria and debris are
phagocytosed and removed. Certain growth factors and cytokines are released to
activate further migration and division of cells involved in the proliferation. During
the proliferative phase, new blood vessels are sprouting from existing blood
vessels in the skin by vascular ECs through the angiogenic cascades (Chang et al.,
2004).
During fibroplasia and granulation tissue formation, fibroblasts grow and form a
new, provisional (ECM) by secreting collagen and fibronectin (Midwood et al.,
2004). Concurrently, re-epithelialization of the epidermis occurs during the
proliferation and 'crawling' of epithelial cells atop the wound bed provides a cover
for the new tissue (Garg, 2000). The wound is made smaller by the action of
myofibroblasts which establish a grip on the wound edges and contract themselves.
When the cells' roles are close to complete, unneeded cells undergo apoptosis
(Midwood et al., 2004).

15
During the maturation phase, collagen is remodeled and realigned along tension
lines and the cells that are no longer needed are removed by apoptosis. Wound
healing is time dependent as illustrated in (fig. (3)).
Figure (2): Summary for phases of wound healing (Babensee et al., 1998;
Singer and Clark, 1999; MacNeil, 2007).
Figure (3): Summary for the overlapping periods of healing stages MacNeil,
2007).
Recently, a complementary model has been described (Nguyen et al., 2009) such
that the many elements of wound healing are more-clearly delineated where the
wound healing process is divided into (2) major phases: (1) The Early Phase:
begins immediately following skin injury and involves cascading molecular and
cellular events which lead to hemostasis with formation of an early makeshift
(ECM) that provides structural support for cellular attachment and subsequent
cellular proliferation.

16
(2) The Cellular Phase: follows the previous phase and involves several types of
cells working together to mount inflammatory response, synthesize granulation
tissue and restore the epithelial layer. Subdivisions of this phase are: (1)
Macrophages and Inflammatory components (within 1–2 days). (2) Epithelial-
mesenchymal interactions: re-epithelialization with change in the phenotype within
hours; migration begins on day 1 or 2. (3) Fibroblasts and Myofibroblasts:
progressive alignment, collagen production and matrix contraction (Days: 4-14).
(4) Endothelial cells and angiogenesis (begin on Day 4). (5) Dermal matrix:
elements of fabrication (begins on Day 4& lasts for 2 weeks) and alteration/
remodeling (begins after 2 weeks and lasts for weeks to months based on wound
size (Nguyen and Murphy, 2009). The importance of this new model became
more apparent through its utility in the fields of regenerative medicine and tissue
engineering.
Winter's study, in 60's, showed that occluded wounds in domestic pigs healed
much faster than dry ones and moist healing environment optimize the healing
rates (Winter, 1962). (Hinman and Maibach,1963) reported; later, similar
findings in human beings. An open wound which is directly exposed to the
atmosphere will dehydrate and a scab (eschar) containing a superficial part of the
dermis will be formed. This incorporation of dermis increases with the increase in
drying conditions to form a mechanical barrier to the migrating epidermal cells and
act as an inhibitor to natural wound healing through reaction with the wound area
(Winter and Scales, 1963). Moist healing prevents the formation of these crusts
and the epidermal cells will migrate over the dermal surface with a rate double
than their migration through the fibrous tissues. Designing many modern wound
care products provides these warm and moist conditions and the dressing will
maintain beneficial electrical gradients between the wounded and normal skin
(Eaglstein et al., 1988).

17
2.1.7. Classification of Wound Dressing Products:
2.1.7.1. Major Classifications of Wound Dressing Products:
Today, there is a wide variety of products to choose from that can lead to
confusion and, sometimes, choosing the wrong type for treating a particular
wound. Knowing the available types of dressings, their uses and the limits of usage
for certain wounds may be a difficult decision in the management of wound care.
Although there are hundreds of them to choose from, the dressings fall into the
following few categories from a clinical point of view.
1-Film Dressings:
These can be used as primary or secondary dressings acting as barriers to protect
an area of the body that might be experiencing friction or shear forces. The
transparent film allows oxygen to penetrate through to the wound while
simultaneously allows the release of moisture vapor with keeping the wound bed
dry. It can stay in place for up to one week, may stick to some wounds, promote
peri-wound maceration due to its occlusive nature and may not be suitable for
heavily draining wound. It aids in autolytic debridement, prevents friction against
the wound bed and does not need to be removed to visualize it.
Examples of these dressings include: [Mepore Film®
(Mölnlycke) & Askina
Derm®
(B Braun) & Bioclusive™
(Systagenix)].
2-Non-Adherent Dressings:
Removal of an adherent dressing during the frequent changes can tear away any
new granulation or epithelialising tissue within the wound bed resulting in
bleeding and distressing for patients. The dressing is designed not to stick to the
wound secretions, thereby causes less pain and trauma on removal. Its primary
function is to keep the wound dry by allowing evaporation of wound exudates and
preventing the entry of harmful bacteria. Examples: [Urgotul ®
(Urgo Medical) &
Mepitel®
(Mölnlycke) & Adaptic ™
(Systagenix)].

18
Paraffin gauze dressings and synthetic bandages belong to this category, but they
are no longer recommended for use on open wounds (NICE, 2008), though they
are readily available and cheaper than others.
3-Simple Island Dressings:
Examples include dressings with central pad of cellulose material to be used over a
suture line of wounds closed by primary intention to absorb any oozing during the
first post-surgery 24 hours. Other examples include [Alldress®
(Mölnlycke) &
Primapore®
(Smith and Nephew) & Medipore™
Pad (3M™
)].
4-Moist Dressings:
These types of dressings function by either actively donating moisture to the area
or preventing the skin surrounding the wound from losing moisture. The moist
dressing accentuates the body’s process of ridding itself of dead tissue through the
autolytic debridement process. It can be divided into 2 groups as follows:
A- Hydrogel Dressings:
These are moist dressings which contain water with different percentages
(generally between 60–70%) with combining the features of moist healing, good
fluid absorbance and transparency to allow wounds monitoring. They are applied
to wounds with necrotic or dead tissues which become hard and desiccated due to
the loss of blood supply, so can donate water to rehydrate and soften the wound
bed and aid the body’s process of autolytic debridement with loss of the dead
tissues. Some of them require a secondary one, either film or a hydrocolloid
dressing to hold it close against the wound bed. Some of them require changing
every 2–3 days with taking care not to macerate the surrounding skin with
excessive amounts of hydrogel. Examples for hydrogels and hydrogel sheets
include: [Intrasite Gel®
(Smith& Nephew)& Nu-Gel™
(Systagenix)& ActiformCool
Gel ™
(Activa Healthcare)]

19
B- Hydrocolloid Dressings:
A very absorbent type of dressings with strong adhesive packing and may be left in
place for several days. The dressing contains colloidal particles (e.g.,
Methylcellulose, gelatin or pectin) that swell into a gel-like mass on coming in
contact with exudates and form a ‘seal’ at the wound surface to prevent the normal
daily evaporation of moisture from the skin. They can be used to accelerate healing
of wounds due to burns, pressure and venous ulcers but cannot be used to prevent
infection. Examples include [Duoderm Signal®
(ConvaTec)& Tegasorb™
(3M™
)&
Nu-Derm™
(Systagenix)].
5-Absorbent Dressings:
Most difficult tasks in wound management are the containment of exudates that
may cause skin maceration if they were not contained within a suitable dressing so
there are vast numbers of different absorbent dressings. Wounds may be flat or
present as cavities that need to be lightly filled with dry absorbent primary dressing
and covered with a further absorbent2ry
one. Leaking and wet dressings and
clothing cause distress to patients and must be avoided. Examples:
A- Hydrofiber Dressings:
White fibrous dressing such as (100% Hydrofiber®
sodium carboxymethyl-
cellulose) is applied in dry form and transformed into a gel-like sheet on absorbing
of exudates. They are used for moderate to heavily exuding wounds and then
changed on saturation with exudate. Examples: [Aquacel AG®
(ConvaTec) &
ActivHeal AquaFiber®
(Advanced Medical Solutions)].
B- Foam Dressings:
Film coated highly absorbent gels for exudates which either lock fluid within the
core of the dressing or transform into gelling foam. They are non-occlusive
dressings and indicate when they need to be changed through the spreading of
discoloration on the dressing according to the amount of wound exudates.

20
If not changed often enough, this may promote peri-wound maceration. Some
foam may not be suitable for certain wounds, such as those that are infected or
tunneling. Examples include: [Allevyn AG®
(Smith and Nephew) &Mepilex
Border®
(Mölnlycke)].
C-Alginate Dressings:
They absorb exudates to form gel-like covering over the wound and the way of
absorption is dependent on the alginate makeup. They have many different
available non-adherent types which encourage the autolytic debridement. Some
alginate dressings retain their integrity and can be removed in one piece; others
disintegrate and need to be irrigated away from the wound bed. Alginate dressing
may be used for venous ulcers, infected wounds and those with tunneling or heavy
exudates. It can be used to lightly fill a cavity but needs to be covered by a
secondary one.
6-Composite Dressings (Composites):
This category involves a combination of types of dressings that may be used for a
variety of wounds either as primary or secondary dressings. These types are merely
of moisture retentive properties, in addition to using gauze dressing. Despite their
wide availability and usage simplicity, they may be more expensive and difficult to
store than other types with less choice/flexibility in use indications
Wound dressings may be also classified based on their nature of action as:
A-Passive Products: Include the traditional dressings which account for the
largest market product level (e.g., Gauze and tulle dressings) with a minimal role
in the healing process (Yannas and Burke, 1980).
B-Interactive Products: Include dressings in polymeric forms that are
recommended for low exuding wounds. These films are generally transparent,
permeable to water vapor and oxygen but not to bacteria.

21
C-Bioactive Products: They deliver active substances to wounds during healing,
may be bioactive compounds or the dressing itself is constructed from materials
having endogenous activities. These materials include proteoglycans, collagen,
non-collagenous proteins, alginates and chitosan. Properties and different types of
alginate as well as chitosan-based wound dressings are summarized in the review
of (Paul and Sharma, 2004).
2.1.7.2. Topical Chemotherapy for Wounds:
Several studies have been performed to identify fundamental substances of
angiogenic activities and direct action in promoting the repair process with
improving the survival of wounded patients. The following are examples:
1-Some enzyme-based ointments (e.g., DNAses and collagenases) act to promote
wound debridement and assist in the restoration of tissue (Hebda et al., 1990).
2-Some growth factors are among the substances, used in topical chemotherapies
where they demonstrate good abilities to accelerate tissue repair on topical
application to the wounds in experimental animals (Pierce and Tarpley, 1994)
(e.g., Recombinant human Platelet-Derived Growth Factor (PDGF)-based drugs
were found to directly interfere with the healing steps to favor the repair process
with showing good results in the healing of diabetic ulcers) (Steed, 1998). Some
angiogenic growth factors and inhibitors are listed in (Table: 1); they have begun
to receive U.S. Food and Drug Administration (FDA) approval by 2003.
3-Silver is reemerging as a viable treatment option for infections encountered in
burns, open wounds and chronic ulcers. It may be in the form of Silver salts (e.g.,
AgNO3), Silver compounds (e.g., Silver sulfadiazine (SSD)), Silver proteins,
electrically charged colloidal silver solutions and sustained silver releasing systems
such as Nano-crystalline silver (Carneiro et al., 2002; Carsin et al., 2004).

22
Table (1): Antiangiogenic agents, approved by FDA (Ribatti, 2009):
4-Activated carbon has large pore volume and surface area giving it a unique
adsorption capacity (Baker et al., 1992). On application onto a wound, activated
charcoal dressing adsorbs bacteria, wound degradation products and locally
released toxins, thereby promotes its healing (Kerihuel, 2009). The first available
charcoal-based dressing was (Actisorb Silver 220; Systagenix) composing of
added silver to charcoal cloth. This can help in killing adsorbed bacteria within the
carbon matrix. It is possible that this helps to promote healing in stagnating chronic
wounds which have a high bioburden (Singh and Barbul, 2008; Martin et al.,
2010).
5-The dressing (Vulnamin®
Professional Dietetics, Milano, Italy) contains (4)
essential amino acids (Gly, L-pro, L-lys and L-Leu) for the synthesis of collagen
and elastin.

23
It can modulate the inflammatory response with a reduction in the number of
inflammatory cells, an increase in fibroblast distribution density and it aids in the
synthesis of thin collagen fibers resulting in reduction in the healing time (Corsetti
et al., 2010).
6-The polysaccharides, chitosan and alginates in particular, are ideal materials for
the construction of dressings suitable for wound healing during its various phases
due to their specific biological properties including hemostasis, granulation and
epithelisation (Muzzarelli, 1993) as will be explained in section (5) of the review.
2.1.7.3. Bioengineering and Hydrogels in Wound Healing:
2.1.7.3.1. Bioengineering and Scaffolds System:
Bioengineering is defined as the science that puts efforts in designing and
manufacturing of spare parts for functional restoration of the impaired organs and
replacement of lost parts due to disease, trauma or tumors (Reddi, 1998), so it
rapidly became one of the most promising treatment options for patients suffering
from tissue failure. It is a multidisciplinary field incorporating the principles of
developmental biology, physiological modeling, chemistry, physics
,morphogenesis, kinetics, microfluidics and cell targeting gearing toward creating
biological substitutes of native tissues to replace, repair or augment diseased
tissues and it concerns itself more with the biological questions. Biomaterials,
Tissue engineering, Biomedical Engineering, Drug delivery and Biomechanics are
considered Bioengineering fields because of their strong dependence on the basic
science with more translational/medical applications. (Biomaterials) is a term used
for both: (1) The engineering of materials for and from biology; and (2) The study
of the interaction of materials with biology.
Tissue Engineering refers, generally, to the process of engineering or directing the
repair of tissues, but can also be applied to technologies outside of the body such
as to the building of tissues constructs for in vitro experimentation.

24
Regenerative medicine is often used synonymously with tissue engineering,
although those involved in regenerative medicine place more emphasis on the use
of stem cells to produce tissues. There are four fundamental technologies in
bioengineering: (1) The scaffolding for cell proliferation and differentiation, (2)
The isolation and culturing of cells, (3) The drug delivery system (DDS) of bio-
growth factor and (4) The maintenance of space to induce tissue regeneration.
The cells can be seeded on biodegradable polymer which serves several purposes:
It functions as a cell-delivery system that enables the transplantation of many cells
into an organism and creates a three-dimensional (3D) space for cells growth
serving as a template which can provide structural cues to direct tissue
development. The matrix temporarily provides the necessary biomechanical
support in the construct while the cells lay down their own ECM which ultimately
provides the structural integrity and biomechanical profile of the engineered tissue
(Terada et al., 2000). One of the essential properties of the used tissue guiding
scaffold is to be biodegradable while providing therapeutic functions on degrading
during replacement of the artificial matrix with a physiological one of the cellular
system. If the polymer is completely absorbed into the body, the long term foreign
body reaction can be eliminated with leaving only the natural regenerated matrix.
Nature of the material has been a subject of extensive studies including different
types of both natural and synthetic origins; the issue of optimal guidance for the
ECM is crucial one (Zhao et al., report).
For successful regeneration therapy of tissues and organs, it is important and
indispensable to develop the technology and methodology of tissue engineering
with molecular designing of a biomaterial acting as an intact scaffold for cells as
well as the DDS technologies of bio-signaling molecules for creating a local
environment which enhances the proliferation of cells and induces cell-based tissue
regeneration.

25
Growth factors are often required to promote tissue regeneration; they can induce
angiogenesis to promote sufficient supply of oxygen and nutrients for maintaining
the biological functions of cells transplanted for effective organ substitution.
2.1.7.3.2. Hydrogels as Wound Dressings:
Hydrogels are polymeric three-dimensional networks imbibing a large fraction of
aqueous medium and yet remain intact even given infinite time period without
dissolving. The hydrophilic polymer chains ensemble in the hydrogel, representing
the skeleton of gel, is somehow interacting with each other either by virtue of
covalent bonds or by interacting physically in cross-linking points as a network or
single mass (Kim et al., 1992) so as to keep the individual chains from diffusing
away into the aqueous milieu. The liquid in gel prevents its network from
collapsing into a compact mass and the network prevents its flowing away
(Tanaka et al., 1981). The network strands can be surrounded with the solvent
molecules, thereby push neighbor chains away and swell with occupying larger
volume. Thus, the hydrogel can be considered as intermediate matter state between
solid and liquid with maintaining its shape under the stress of its own weight.
Many extracellular structures which embed cells in the body can be considered as
(Hydrogels). The (ECM) of soft tissues and cartilage, for example, exists as a
network of glycoproteins and proteoglycans that both interact with each other
biophysically. Hydrogels of both natural and synthetic origin have been proposed
also as ECM analogues (Fonseca et al., 2011) due to their structural similarities to
the body macro molecular-based components so they met numerous applications.
Examples include: drugs delivery, medical prosthetic materials, antistatic coatings,
encapsulation materials for immunoisolation-based cell therapeutics, wound
dressings (Stile et al., 1999 ;Lee et al., 2001), as well as in soft contact lenses, gel
electrophoresis, anti-adhesion materials, environmental and chemical detectors
(Silva et al., 2006).

26
These hydrogels are also used as tissue engineering scaffolds, structures for filling
the irregularly shaped defects. In addition, they are used in general
macromolecular research with easy means of delivery for the bioactive molecules
into the body in a minimally invasive manner (Lee et al., 2001).They can be
designed to provide instructive environments for the 3D assembly of vascular
networks.
Hydrogels made from natural polymers such as alginate, chitosan, collagen,
hyaluronate (Denuzière et al., 2000; Chen and Cheng, 2009) or dextran (Kikuchi
et al., 1997) are frequently used as scaffolding materials in tissue regeneration
strategies as they are either components of or have similar macromolecular
structure to constituents of the natural tissues (ECM). Many studies of hydrogel-
based scaffolds have focused on their applications in the healing of wounds
(Balakrishnan et al., 2005b; Boucard et al., 2007; Kim et al., 2009; Shepherd
et al., 2011). They can also deliver growth factors (Kiyozumi et al., 2006), cells
(Liu et al., 2009) and antibiotics (Shepherd et al., 2011) to allow complete skin
regeneration.

27
(2): Review on Alginates
2.2.1. Chemical Structure of Alginate:
Based on description of the British chemist E. E. C. Stanford in 1881, alginate is a
random unbranched heteropolysaccharide with repeated two kinds of (1→4)
covalently linked monomers [ß-D-mannuronate (M) and its C5 epimer α-L-
guluronate (G)] in different sequences of varying proportions. They appear in
homopolymeric blocks fashion of consecutive G-residues (Polyguluronates; GGG-
blocks), consecutive M-residues (Poly mannuronates; MMM-blocks) and
heteropolymeric blocks of alternating randomly organized uronates (MGM-
blocks) (Sutherland et al., 1991).
-As shown in (fig. (4)), the monomers in the polymer chain have a tendency to
stay in their most energetically favorable structure. For M-M, this is the 4
C1 chair
form, linked by β-(1, 4) glycosidic bond, but it is the 1
C4 chair form for G-G,
linked by α-(1, 4) glycosidic bond (Yang et al., 2006). (G) and (M) residues adopt
axial and equatorial configurations, respectively; the M blocks have extended
ribbon form, G blocks are rigid and buckled and the MG-regions are of
intermediate rigidity (Grant et al., 1973).
Figure (4): Schematically drawn alginate block structure with a segment
showing structure of the molecules (Smidsrød et al., 1995).

28
Alginate solubility is affected by primary structure of the polymer, ionic strength
and pH (d’Ayala et al., 2008). Due to its functional groups (-COO-
and OH-
),
alginate can react readily with amino and amino derivative groups of other
polymers via electro-static interactions or with formation of Schiff bases or
amides.
2.2.2. Sources of Alginates:
Polysaccharides of algal origins are gaining particular attention due to their
peculiar chemical composition, renewability and abundance. For example, agar
and carrageenan that are extracted from red seaweeds (Hopkins et al., 2009) and
alginate from the brown seaweeds. Alginates are mainly alkaline extracted from
brown algae (phaeophyta, classe des Phaeophyceae), including the giant kelp
Macrocystis pyrifera, Ascophyllum nodosum and various species of Laminaria
with alginate contents (20-40 % of the dry weight) (Black, 1950). Amount and
properties of alginate vary based on the organism species, its reproductive cycle,
growing conditions and the tissue it is isolated from (Haug, 1964; Moe et al.,
1995). Alginate is located in the intercellular matrix and cell wall in a gel form
containing Ca+2
, Mg+2
and other multivalent cations (Haug and Smidsrød, 1967)
with mainly skeletal functions by conferring both mechanical strength and
flexibility to the algal tissue for growth so plants growing in rough waters provide
alginate richer in (G-residues) compared to plants of the same species from calmer
waters (Ertesvåg et al., 1996).
Alginate-like polymers are synthesized by number of bacterial strains as
exocellular secretions. The gram-negative bacterium, Pseudomonas aeruginosa,
and the soil bacterium, Azotobacter vinelandii that can fix nitrogen under aerobic
growth conditions are examples for these genera (Johnson et al., 1997).

29
2.2.3. Properties of Alginate:
1-Alginates, unlike other natural polysaccharides, look very promising due to their
unique biocompatibility with both host and enclosed cells, low mitogenic activity
and toxicity (Wang et al., 2011), abundance and renewability (Matsumoto et al.,
2003). They are amenable to sterilization and storage with ease of chemical
modification through simple chemistries (Briand and Tang, 2007). Under normal
physiological conditions, alginate is bioerodible with non-inflammatory
degradation products and has easy solubility without any harsh reaction
conditions.
2-Alginates could be candidates in many biomedical applications for preparing
many artificial matrices aiming to the regeneration of damaged tissues including
cartilage (Bouhadir et al., 2001), bone (Alsberg et al., 2001), liver (Chung et al.,
2002), cardiac tissue remodeling (Dar et al., 2002), dermatology and regeneration
of skin (Hashimoto et al., 2004).
3-Because they can mild gelate over wide range of temperatures with the ability to
retain water (d’Ayala et al., 2008), alginates have been successfully used as
matrices for the entrapment and/or delivery of biological agents (e.g., Drugs and
growth factors) without loss of the biological activity of these mitogenic molecules
and also as artificial matrices with scaffolding action for cells (Chinen et al.,
2003).
4- Alginate has a recognized GRAS status (Generally Recognized As Safe) with
constantly ensured quality (Ghidoni et al., 2008), so it has been widely used over
the last few years in food industries (e.g., Juices, stabilizer in ice cream) and many
other industrial interests (e.g., Salad dressings, cosmetics, slimming aids, paper
and textile scaffold manufacturing, waterproofing and fireproofing fabrics
(Bartels et al., 2011).

30
5-With its different biomedical and pharmaceutical applications, alginate can be
used alone, in composites with other materials as well as in blends with certain
modifications, especially due to its limited interaction with the majority of
mammalian cells due to its hydrophilic character (Wang et al., 1995) that
promotes limited protein adsorption (Lee and Mooney, 2001). Examples of these
reacting positively charged materials include: Ethyl cellulose (Bodmeier and
Wang, 1993), Eudragit (Gürsoy et al., 1998), Pectin (Liu and Krishnan, 1999)
and Chitosan (Sezer and Akbuga, 1999). This improves the deficiencies within
the alginate structure, helps solve the problems with drug leaching during
preparation and imports it innovative properties (d’Ayala et al., 2008). Thus,
alginate can compete with the synthetic biodegradable excipients available in the
market with opening more and more new perspectives and potential applications in
the future.
2.2.4. Alginate Gelation:
In several applications of alginate, strong thermo-stable gels can be prepared prior
to use or spontaneously formed in situ in physiological fluids. Alginate gelation
can be achieved by one of the following methods:
1-Photo-Polymerization of alginate monomers allows creating a hydrogel
independent of the divalent cation levels to control the gelation timing and kinetics
(Jeon et al., 2009; Rouillard et al., 2011).
2-Enzymatic Cross-linking: (Martinsen et al., 1991).
3-Chemical gelation: It can be achieved by one of the 2 following methods:
A-Lowering the pH of Alginate Solution: Induces the formation of acid gel
(Alginic acid) by physical hydrogen bonding.
B-Chemical Cross-linking: This method involves the covalent and ionic cross-
linking via crosslinker ions. The covalent cross-linked alginate gels show higher
stability than those cross-linked ionically (Eiselt et al., 1999).

31
I-Covalent Cross-linking:
Carried out via cross-linking agents (e.g., Carbodiimide (Rees and Welsh,
1977), glutaraldehyde or adipic dihydrazide (Maiti et al., 2009) where the (-
COO-
) groups on the alginate chains are left unperturbed.
II-Ionic Cross-linking via metal ions (Gelling Salt) [Egg-Box Model]:
As a hydrophilic polyelectrolyte, alginate can be cross-linked with exchange of
monovalent ions from guluronates with multivalent counter ions at certain
stoichiometric ratios (Martinsen et al., 1989). The diaxially-linked G-residues
spontaneously form electronegative cavities functioning as binding sites for some
di and polyvalent cations (e.g., Ca+2
, Sr+2
, Ba+2
, Fe+3
, Al+3
) (Patil et al., 2010)
when the polyguluronate segment exceeds the critical length (Stokke et al., 1991)
with small distances between the junctions and of the same order of magnitude as
the Kuhn statistical segment length (Smidsrød et al., 1974) for cancelling the
negative charges by these ions. Alternatively, other multivalent cations (e.g.,
Mg+2
) form soluble polymers on binding to the G-residues (Smidsrød et al.,
1970).
(Calcium alginate gels) are produced in calcium setting bath by (2) cooperative
inter-chains binding mechanisms responsible for the formation of the junction
zones (Smidsrød et al., 1972):
1-Calcium ions in the solution make ionic bridges for two carboxyl group moieties
on the adjacent polymer chains (Coviello et al., 2007).
2-The other energetically favorable mechanism is the crosslinking via (-COO-
groups) by (primary valences) and via the electronegative oxygen atoms of the
[OH-
groups: O (5) and O (4) in one unit and O (2) and O (3) in the preceding unit]
by (secondary valences) (Smidsrød et al., 1972; Angyal et al., 1973) making an
insoluble polymeric network described as the so called” Egg-Box model”,
illustrated in (fig.(5)) (Grant et al., 1973).

32
Coordinate bonds extend to two nearby (OH-
groups) of a third unit that may be in
the same chain to retain the macromolecule’s coiled shape or in another chain
resulting in the formation of a huge molecule with a (3D) net-like structure
(Whittington, 1971). (Donati et al., 2005) have found out that GGG and MGM
blocks can form mixed junctions, but no such effects were observed with MMM
blocks. The main function of MG blocks was suggested to be for binding to water
than forming junctions (Smidsrød et al., 1972).
Stiffness of the cross-linked alginates and the relative extension in aqueous (0.1 M
NaCl) and in the unperturbed state as well increases: (MG<MM < GG blocks)
(Smidsrød et al., 1973). Elasticity (flexibility) increases in the backward direction
(Draget et al., 2001) Therefore, the M/G ratio, length of polymeric chains and the
ratio of homologous to heterologous chains must be carefully tuned to optimize the
resulting gels and microcapsules.
On cross-linking of sufficient blocks containing L-guluronate, stable junctions
seem to be introduced which hinder the MMM-blocks aggregation and function as
single chain segments between the gel junctions. These segments, in between, are
very restricted in their movement so the applied energy for compressing the gel
can be transferred through the stiff network structure to cause partial rupture of the
junctions (Smidsrød et al., 1972).
Figure (5): The binding of a divalent cation to contiguous dimers of
guluronate residues (Smidsrød et al., 1995).

33
The majorities of cross-links in the alginate gels are not permanent but move or
break when they are sheared (Mancini et al., 1999). While calcium levels are a
convenient means for controlling the properties of these gels (Brandl et al., 2007),
their physiological roles are important in many systems (Allgrove et al., 2009).
They can be either administered separately or added as part of the formulation
within the pharmaceutical preparation.
The solution viscosity, overall molecular weights, the block-wise structure of
alginate (Morris et al., 1980), Ca+2
ions concentration during gelation (Dumitriu,
1988), degree of cross-linking (Mitchell, 1980), method of gelation (Nunamaker
et al., 2007), number of monomers in a strand (N), the fraction of overall
guluronate residues in the polymer (FG value), number-average of guluronate units
in G-blocks (NG) (de Gennes, 1979), the sequential order of these residues
(Dumitriu, 1998), functionality of the cross-linking point (number of strands
connected to one crosslink, F), the average weight of strands between two
neighboring crosslinks (Mc), and sometimes, the presence of excipients in the
gelation bath (e.g., Na-hexametaphosphate (Van Wazer, 1958) and Glucono-δ-
lactone (Nussinovitch et al., 1990)) are all fundamentals to determine the
physicochemical properties of alginate, physiological and gelling properties,
mechanical strength, porosity, swelling, biocompatibility (Thu et al.,1996),
effectiveness in a given application and uniformity of the resultant gels (Klock et
al., 1994). Rate of diffusion of the reactants is considered the rate limiting step in
the gelation process (Martinsen et al., 1989).
In addition, (Amsden et al., 1999) reported that the greater the (G-content) of gel,
the higher affinity for cross-linkers and the greater is the restriction to solute
transport. Accordingly, alginates of high (G-content) can create transparent fibers
of more porous cross-linked gels with good stability towards competing Na+
ions.

34
It will have also maintained mechanical integrity and rigidity for long periods
(Martinsen et al., 1989) and low degree of swelling. Conversely, alginate rich in
mannuronates can develop extra turbid elastic softer aggregates (Smidsrød et al.,
1972) with a high degree of swelling on calcium cross-linking (Grant et al., 1973)
and less proneness to syneresis (Nussinovitch, 1997). Alginates with high content
of the alternating sequence are characterized by low modulus, high volume and
flexibility.
The equilibrium of a freely swelling gel is determined by the interactions between
its network and the solvent. Generally in vitro, as Ca+2
ions are removed by
outward fluxing into the surrounding medium, the crosslinking in gel decreases
and becomes destabilized with loss of the mechanical stiffness over time (LeRoux
et al., 1999) due to the increased electrostatic repulsion between the (-COO-
anions) of alginate with increased swelling/ erosion (Kikuchi et al.,1997). The gel
is dissolved into dissociated individual chains with leakage of any entrapped
materials (Shoichet et al., 1996). These interactions are highly sensitive to
external conditions such as temperature, pH, presence of ions and external fields
(e.g., Magnetic, electric or pressure fields) (Vervoort, 2006).
Similar mechanism takes place in vivo where no hydrolytic or enzymatic chain
breakages occur within the alginate chains, but only softening of gel takes place
under physiological conditions forming absorbable alginate. This causes limited
quantities be safely left in situ accompanied with gradual disappearance of the
hydrogel and evacuation of the dissociated chains to be excreted by the kidneys,
especially on using alginates of modified molecular weights (Alshamkhani and
Duncan, 1995).

35
2.2.5. Modification of Alginate:
It is widely assumed that the critical parameter in the different approaches of
Bioengineering and designing drug delivery vehicles is the ability of the used
material to degrade over time in body in concert with new tissue formation to
provide new space for matrix deposition and allow formation of the desired tissue
around each cell or coalescence of cell clusters into one interconnected tissue
structure with increased mechanical functions (Nerem and Sambanis, 1996).
This is why the polymer that biodegrades too rapidly may not serve as a space-
filling scaffold for supporting the development of new tissue.
Controlling of both the degradation and adhesion characteristics of the prepared
scaffold (Example: Ca-Alginate gels) is considered a powerful tool in regulating
the regeneration processes of a broad range of tissues. Unfortunately, these gels;
on reaching maximum swelling, begin to dissolve in an uncontrollable manner
with releasing high molecular weight strands which may have difficulty to be
cleared from the body where clearing occurs slowly under physiological
conditions (Shoichet et al., 1996), in addition to the absence of hydrolytic and
enzymatic chain breakages within the alginate chains (Alshamkhani and
Duncan, 1995).
Mechanical stiffness of the ionically cross-linked alginate hydrogel and its
degradation can be controlled by adjusting the M/G ratio (Stokke et al., 1991;
Wang et al., 2003), alginate molecular weight (King, 1994) and/or concentrations
of the binding cations (Mancini et al., 1999). It is believed; however, that
controlling alginate concentration and the Molecular Weight Distribution (MWD)
of the properly tailored polymer chains are the most straightforward effective
factors irrespective of the method of cross-linking (Kong et al., 2002; Kong et
al., 2004).

36
Increasing concentration of the High Molecular Weight-Alginate (HMW-Alg)
typically used to form hydrogel increases viscosity of the pre-gelled solution
greatly resulting in non-uniform mixing with calcium slurry to make a gel with a
slow degradation rate and this may significantly limit this approach (Alsberg et
al., 2003). Alternatively, preparing a hydrogel with high Low Molecular Weight-
Alginate (LMW-Alg) concentration may limit this increase in viscosity while
enhancing stiffness of the hydrogel due to increased solids concentration. This
approach may not be ideal due to the potential brittleness of the resulting gel and
the high strains imposed on the material in the body which predicts its failure in
many applications. Additionally, the resulting device will biodegrade rapidly and
may not be able to serve as a space-filling scaffold capable of supporting new
tissue development (IAEA, 2009). At very low intrinsic viscosity, it is impossible
to make gels with low alginate concentrations (Martinsen et al., 1989).
Alginate properties can be regulated in a refined manner utilizing a bimodal
MWD system including a mixture of (HMW-polymer) and a polymer tailored to
have a lower MW but still able to participate in gel formation, so can decouple the
dependence of properties of the two fractions from the overall concentrations
(Kong et al., 2002) and alter the degradation rate of gels over a broad range
(Kong et al., 2004). Flexible (HMW-Alg) chains are more liable to form
intramolecular cross-links along a single molecule; the fraction of these cross-
links can be reduced with the incorporation of stiffer (LMW-Alg) chains of more
stretched conformation with improving the formation of intermolecular cross-
links between (HMW, LMW-alginates chains and the cross-linking ions). This
improves the capability of gel to transfer the deformation energy throughout its
entire (Kong et al., 2002). Several techniques have been reported to promote the
reaction rate of depolymerization process and reduce the MW of alginate
including:

37
1-Treatment with enzymes from some microorganisms:(Shimokawa et al.,1996).
2- Acid hydrolysis: Using HCl (Bouhadir et al., 2000), H2SO4 (Muramatsu et
al., 1993), formic acid (Sherbrock et al., 1984) or oxalic acid. Although the
chemical procedures are convenient, their common disadvantage is the low
recovery of oligosaccharides.
3- Heating (Thermal degradation or homolysis): (Ren, 2008).
4- Irradiation: (Kume et al., 1983; Nagasawa et al., 2000).
5- Oxidation: (Bouhadir et al., 2001).
Several reports indicated that certain radiation intensities and degrees of
oxidation do not damage the gel-forming ability of alginates while decrease
length of the polymer chains, so with partial oxidation or degradation of alginate
and using combination of polymers with distinct MWDs to form gels,
controllable degradation kinetics within a desirable time-frame for tissue repair
can be provided (Kong et al., 2004) with allowing to control the release kinetics
of the incorporating factors (Hao et al., 2007).
2.2.5.1. Irradiation of Alginate:
Radiation induced degradation technology is a new and promising application of
ionizing radiation to develop pulp, viscose, paper, natural bioactive agents,
pharmaceutical products and food preservatives. Polysaccharides and their
derivatives, exposed to the ionizing radiation have been recognized as degradable
polymers based on the reduction of their M.Ws (Potthast et al., 2006; El-Sawy
et al., 2010; Hassan et al., 2011). In spite of its disastrous effect on both
solutions and dry powder of alginate, gamma (γ)-irradiation is widely utilized in
multiple studies due to several reasons, for instances:
* The degradation process can be performed at room temperature.
*The degraded polysaccharides can be used without further purification.
*The simplicity to control the whole process.

38
*Economic competitiveness to the other alternative chain scissoring methods as
it offers a clean one step method for the formation of low molecular weight
polysaccharides in both the solid state and aqueous solutions even at high
concentrations.
Irradiating alginate up to a dose (50 KGy) does not affect the length of the GGG-
blocks or MMM-blocks (Kong et al., 2002) where chain scission, up to this
dose, occurs mainly in the bonds between (M& G) residues with preservation of
both overall G-content and G-block length that maintain the gel-forming ability
of the polymer. In contrast, the irradiated alginates at higher doses demonstrate a
decrease in the G-block length, along with the decreased molecular weight and
form extremely soft, weak gels.
For preparation of oligosaccharides with different molecular weights suitable for
using in the Bioengineering fields, higher degrading irradiation doses are
required when the polymer exists in solid form; however, such technology is not
economic. It was found out that the molecular weight of (Na-Alg) decreases with
using (γ-radiation) or oxidizing agent (initiator) alone (Li et al., 2010).
Meanwhile, combining both agents can accelerate the degradation rate and
decrease its (M.W) dramatically (Abdel-Rehim et al., 2011) and this is
considered a more economical way to produce alginate oligosaccharide units.
There are many types of initiators that can be combined with radiation, such as
ammonium persulphate (APS) and hydrogen peroxide (H2O2), but (H2O2) is
preferred in our study because of the following properties:
1-It is an effective and environmentally friendly oxidant that has been used to
oxidize many chain-scissoring polysaccharides (e.g., Starch (Poutanen et al.,
1995) & Cellulose (Zeronian and Inglesby, 1995) & Dextran (Ahrgren and de
Belder, 1975) and Chitosan (Kabal’nova et al., 2001; Qin et al., 2002).

39
2-The decay rate of the radicals in the presence of (H2O2) is much lower than the
decay for samples irradiated in presence of (APS).
3-The oxidation method do not only depolymerize the polysaccharide, but also
can change the structure of the main chain after irradiation.
4-It does do not require further treatment or purification steps, unlike irradiation
with (APS) which requires further fractionation steps.
Splitting of the polymeric macromolecules to form free radicals is employed for
synthesizing modified polymers. The mechanism is based on breakdown of the
ordered system of inter and intramolecular hydrogen bonds within the irradiated
chains. This influences the chains rigidity with a decrease in degree of
crystallinity of the material (von Sonntage and Schuchmann, 2001). There are
2 proposed mechanisms for the degrading effect of ionizing radiation:
(I) The Direct Reaction of Alginate with Irradiation:
Localization of the energy initiates dehydrogenation and degradation reactions
after irradiation (Ershov, 1998).
Figure (6): Proposed mechanism for degradation of alginate in the solid
state (Abdel-Rehim et al. 2011).

40
Alginate undergoes ionization on exposing to high-energy radiation in dry state,
then most of the kicked out electrons are thermalized and eventually recombined
with their parent ions to produce excited fragments of the polymer.
These fragments decompose with cleavage of the chemical links, mostly splitting
of carbon-bonded hydrogen leading to the formation of free radicals on polymer
chains, especially the substituted side chains and hydrogen atoms. A proposed
mechanism is illustrated in (fig. (6)).
(II) Irradiating Alginate in Solutions or with Oxidizing Agents :
The degradation follows indirect way where the interaction of radiation with
water causes ionization and excitation effects to produce water radiolysis
products including fast electrons and short-lived H2O+
radical-cations with
electronically-excited water molecules (H2O*
). These molecules are unstable and
decompose within 10-13
s to form OH•
and H•
radicals (IAEA, 2010) which can
create alginate macro radicals by abstracting Hydrogen atoms from the polymer
chain. Hence, (humidity) enhances the yield of the degraded alginates.
Irradiation (IR) + H2OH2O+
+ H2O*
H2O+
+H2O H3O+
+OH•
H2O*
H•
+OH•
2.2.5.2. Oxidation of Alginate:
Diols commonly found in carbohydrate groups may be oxidized by the natural
ageing in the presence of oxygen and light, enzymatically (Kristiansen, 2009),
or with chemical processing deliberately or un-deliberately (e.g., With periodate
or (2,2,6,6-tetramethylpiperidine-1-Oxy radical (TEMPO)) (Saito, 2006).
Periodate oxidation is commonly utilized as a ‘tool’ to control gel strength due to
the following reasons:
1-Although TEMPO oxidation introduces one carbonyl group at the C6 position
in the monosaccharide unit (Potthast et al., 2006), the product activity is lower
than that of periodate oxidation product.

41
2-Periodate oxidation has been a useful tool in glycochemistry for a long time
where it is known to act randomly upon alginate (Painter and Larsen, 1970)
giving wide range of molecular weights in relatively short period of time
(Alsberg et al., 2001).
The mechanism of periodate action is based on reducing the stiffness of alginate
chains briefly as follows:
I. The α-glycol groups are split under mild oxidation conditions with cleavage of
the C2-C3 bond carrying the 2-cis vicinal diols making two aldehyde groups in
the monosaccharide unit (Malaprade et al., 1928).
II. Open-chain adduct is formed within the alginate polysaccharide chain as
conversion of the relative rigid pyranoid ring to oxidized fraction alters its
conformational structure by the spontaneous formation of six-membered
hemiacetal rings between (-CHO groups) of the oxidized hexa-uronic-acid
residues with the closest (OH) groups on two adjacent non-oxidized uronates
(Balakrishnan et al., 2005a; Gao et al., 2009).
Figure (7): Suggested reaction scheme describing periodate oxidation of a
mannuronan residue within the alginate chain [modified from (Perlin,
2006)] (M+
:The metal ion bound to alginate anion (Na+
, K+
,...).

42
The formed adduct reduces the steric hindrance of the main chain and allows free
rotation of the β-glycosidic linkages to make it behave like an acetal group with
reduced stability to hydrolysis (Bruneel and Schacht, 1993) (Fig.(7)).
III. The resulting chains have the inability to form ionic bridges with the ionic
cross-linkers at the adduct sites where their formation requires an average of 20
adjacent guluronate groups and the breakage of one unit is expected to weaken
these ionic junctions (Bouhadir et al., 2001).
Although the periodate oxidation offers an interesting way for changing the
chemical structure of alginate and makes it more reactive, it leads to some
depolymerisation even when carried out in dark (Laurienzo et al., 2005) due to
the following reasons:
A-The involved degradation is presumably through a free radical mediated
mechanism, may be due to the oxidation of impurities present. The degradation
seems to be unavoidable even in the presence of free radicals scavengers
(Balakrishnan et al., 2005a).
B-The total MW of alginates decreases in proportion to the molar ratio of the
added NaIO4 reagent to the reaction (Kong et al., 2004).
C-Chemically, the oxidized residues can be degraded hydrolytically much faster
than the glycosidic linkages between the intact G and M residues. This can offer
a way to control degradation under mild acidic conditions to make the resulting
oxidized polymer suitable for various drug delivery and tissue engineering
approaches (Kristiansen, 2009).
Periodate oxidation is considered a selected approach to activate polysaccharides
where the new added (-CHO) groups are more reactive than the (OH) and(-COO-
) groups initially within the alginate structure (d’Ayala et al., 2008) and they
offer new sites for binding new materials and drugs for introducing to the body.

43
2.2.6. Purification of Alginate
A major hurdle to the successful medical applications of any biomaterial is its
immunogenicity and the lack of reproducible biocompatibility (Orive et al.,
2004). Alginate, as a natural polymer is limited by its tendency to contain various
fractions of impurities which exhibit mitogenic activity in in vitro tests and could
favor the overgrowth of macrophages and fibroblasts in experimental small and
large animals causing graft failure (De Vos et al., 2002; Van Hoogmoed et al.,
2003). Cellular reactions surrounding the implanted biomaterial could also lead
to the production of toxic cytokines (Cole et al., 1992) or depletion of oxygen
and nutrients (Colton, 1995).
Alginate immunogenicity is affected by number of variables as follows:
1-The Starting Material: The availability of freshly harvested algae for alginate
extraction was depicted to increase its quality (Jork et al., 2000).
2-The Industrial Extraction Process of alginate perhaps introduces additional
contaminants into the extracted raw material (Qi et al., 2009).
3-The Guluronic/Mannuronic Acid Ratio: The chosen alginate content in this
study is (61% M and 39 % G). High M % alginate has been reported to be less
biocompatible than a high G %-alginate due to the mitogenic properties of
mannuronic acid component (Otterlei et al., 1991). In spite of that, certain
protocols proved that the both alginates have the same biocompatibility (Klöck
et al., 1994; Duvivier-Kali et al., 2001) under the same main control of
purification degree.
4-The Molecular Weight: (King et al., 2000).
5-The Nature and Quantity of Residual Contaminants, introduced during
the extraction steps: There are three common contaminant types detected in
alginates, and used also as contamination indicators:

44
A-Proteins: These are the main contaminants in the alginate extract from algae
representing about 40% of the macro-components distribution of the different
seaweeds (Surialink et al., 2001). (Kanagaraja et al., 1999; Godek et al.,
2004) have found out that these impurities are responsible for provoking the host
immune reactions, so their removal is of paramount importance for enhancing the
biocompatibility of the used biomaterial, alginate. Proteins removal is more
difficult than that of the other 2 main contaminant types, polyphenols and
endotoxins.
B-Polyphenols and Polyphenol-like Compounds (PC): These are aromatic
compounds responsible for the chemical defense against herbivores in the brown
seaweeds (Pereira et al., 1999), so they are normally extracted with alginate.
These impurities are biorecalcitrants and can possibly accumulate in the body
(Skjak-Braek et al., 1989; W.H.O, 1994), so can be dangerous for humans. It
was proved that they can be mostly removed by simple chemical treatment steps.
C-Endotoxins: These are chemical compounds belonging to the pyrogen family
(Dusseault et al., 2006) and comprise the integral part of the outer cell
membrane of Gram-Negative Bacteria (Raetz, 1990) with organization and
stability responsibility (Vaara and Nikaido, 1984). In spite of that, they are
continuously liberated into the surrounding media during the cells growth,
division and after death, so found everywhere and their high concentrations are
found where bacteria accumulate specially during the bioprocesssing. These
molecules are very stable and their biologically active part survives extremes of
temperature and pH (Sharma et al., 1986), so their removal from alginates
requires routine temperatures within the range (180-250o
C) with acids or alkalis
of at least 0.1M.

45
6-The Method of Purification: Achieving a suitable biocompatibility level
requires highly purified alginates (Orive et al., 2002) and since the reporting of
immunogenicity of alginate in the early 90`s, several research protocols have
been described and many in-house methods have been developed including the
following methods:
1-Free Flow electrophoresis (FFE) method: (Zimmermann et al.,1992).
2-Klock method (Klock procedures and Saline Dialysis (K+SD), Pur. K):
Briefly, it involves (3) chloroform extraction repeats for alginate which then
treated with acid-washed as well as neutral activated charcoal. BaCl2 is a
jellifying reagent; the prepared beads are immersed then in acetic acid ,sodium
citrate and ethanol to remove the impurities (Klock et al., 1994).
3-Prokop-Wang method (Pur. P): It has the same procedures of (pur. K
method) without the chemical extractions on the alginate beads (Prokop and
Wang, 1997).
4-De Vos method (Pur. D): It uses Sodium Ethylene GlycolTetraAcetic Acid
(EGTA) solution of alginate with adjusted pH and involves continuous washing
with (HCl+ NaCl) solution, followed with several repeats of extraction with
Sevag Reagent, filtrations with several washings, and then ethanol precipitation
(De Vos et al., 1997).
5-Vidal-Serp D.S method (Pur. V): It depends mainly on acetone as a
purification reagent with several filtration and continuous washing steps with
(Vidal-Serp and Wandery, 2005).
6-Purification Preparative method: It uses the same technique of Size
Exclusion Chromatography (SEC); the eluent is KCl to reduce electrostatic
interactions among the proteins and alginate molecules (Ménard et al., 2010). It
is expensive method, needs special columns for carbohydrates purification and
better standardization for pure alginate preparation for clinical applications, so
considered a restricted method to laboratories.

46
(3): Review on Chitosans
2.3.1. Chemical Structure of Chitosan:
History of chitosan dates back to the 19th
century with the study of Rouget for the
deacetylated forms of its parent polymer, chitin (Dodane and Vilivalam, 1998).
During the past 20 years, a substantial amount of work has been reported on
chitosan and its various potential biomedical applications.
-The major chemical structure of chitin is composed of the monomers (Vinyl
Glucosamine and D-Glucosamine). Chitin becomes chitosan when the C-2s of its
monomers substitute total or partial Vinyl amines with amine groups (Knill et al.,
2004b) to give the unbranched cationic copolymer chitosan with a structure
consisting of 2 main repeated units linked by β (1→4) glycosidic bonds; these are:
(2-amino-2-deoxy-β-glucopyranose or D-Glucosamine) and (2-acetamido-2-
deoxy-β-D-glucopyranose or N-acetyl Glucosamine) with the energetically
favorable ( 4
C1 chair) form (Roberts, 1992) available in different grades depending
upon the degree of acetylated moieties (Hoppe-Seiler, 1994) (Fig. (8)). In addition
to the M.W of chains; these units provide specific structural properties for several
chitosans giving them different chemical and biological properties (Knill et al.,
2004b).
Figure(8):The chemical structures of Chitin and Chitosan (Collins,1998)
(GLcN refers to glucosamine& GlcNAc refers to N-acetylglucosamine).

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