Molecular Biological Studies to Evaluate the Treatment Role of Irradiated Scaffolds in Ulcers and...

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)

DEDICATION

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

List of Abbreviations 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

http://www.pda.org/Publications_1/PDA-Publications/Technical-Reports.aspx


x

Abbreviation Name

R.T Room Temperature (25oC)

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.

http://en.wikipedia.org/wiki/Integumentary_system

http://www.google.com.eg/search?tbo=p&tbm=bks&q=inauthor:%22Philippa+Buck%22


























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.

http://en.wikipedia.org/wiki/Cell_(biology)

http://en.wikipedia.org/wiki/Drug

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).

http://www.google.com.eg/search?tbo=p&tbm=bks&q=inauthor:%22Janetta+Bensouilah%22


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).

http://en.wikipedia.org/wiki/Fibrin

http://en.wikipedia.org/wiki/Bleeding

http://en.wikipedia.org/wiki/Phagocytosed

http://en.wikipedia.org/wiki/Fibroblast

http://en.wikipedia.org/wiki/Wound_healing#cite_note-Midwood-5


http://en.wikipedia.org/wiki/Epithelial_cell

http://en.wikipedia.org/wiki/Myofibroblast



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.

http://en.wikipedia.org/wiki/Apoptosis


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).

http://en.wikipedia.org/wiki/Macrophage

http://en.wikipedia.org/wiki/Epithelial

http://en.wikipedia.org/wiki/Mesenchymal

http://en.wikipedia.org/wiki/Phenotype

http://en.wikipedia.org/wiki/Fibroblasts

http://en.wikipedia.org/wiki/Myofibroblasts

http://en.wikipedia.org/wiki/Endothelial_cell

http://en.wikipedia.org/wiki/Regenerative_medicine

http://en.wikipedia.org/wiki/Tissue_engineering

http://en.wikipedia.org/wiki/Tissue_engineering


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)].

http://www.woundsource.com/product-category/dressings/transparent-films


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.

http://www.woundsource.com/blog/burns-and-wound-care-symptoms-causes-and-treatments


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 4C1 chair

form, linked by β-(1, 4) glycosidic bond, but it is the 1C4 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).

http://pubs.acs.org/action/doSearch?action=search&author=Eiselt%2C+Petra&qsSearchArea=author


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:

http://www.ncbi.nlm.nih.gov/pubmed?term=Alsberg%20E%5BAuthor%5D&cauthor=true&cauthor_uid=14578503


http://www.ncbi.nlm.nih.gov/pubmed?term=Martinsen%20A%5BAuthor%5D&cauthor=true&cauthor_uid=18587846


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) + H2OH2O+ + 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:

http://www.ncbi.nlm.nih.gov/pubmed?term=Kl%C3%B6ck%20G%5BAuthor%5D&cauthor=true&cauthor_uid=7764423

http://www.ncbi.nlm.nih.gov/pubmed?term=Kl%C3%B6ck%20G%5BAuthor%5D&cauthor=true&cauthor_uid=7764423

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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 (180-250oC) 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 ( 4C1 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).


47

2.3.1.1. Degree of Deacetylation (DD):

It refers to the percentage of average number of D-Glucosamine units present in

the polymer chains when the distribution of the 2 constitutive residues is random.

The term (Chitin) usually refers to a copolymer with (DD less than 70 %), while

(Chitosan) represents a series of copolymers of (DD <70 %) (Domard and

Domard, 2002) with lactic-average M.W in the range (10-103 KDa) (Roberts,

1992). Chitin is generally insoluble in standard polar and non-polar solvents, but

chitosan dissolves in diluted acidic solutions (Hayes et al., 1977) below pH 6.0,

suitable for quaternisation of the amine groups with macro intrinsic pKa value of

6.3 (Meng et al., 2010) giving it a high positive charge density to make water-

soluble cationic polyelectrolyte (New et al., 2008) able to form salts with inorganic

and organic acids (e.g., Glutamic and acetic acids) (Illum, 1998). Different

deacetylation conditions can be influenced by changing the inter and intra

molecular repulsion forces (Sashiwa et al., 1991).

Biodegradability in living organisms is (DD)-dependent where it increases as (DD)

decreases (Kurita et al., 2000; Yang et al., 2007), so lower DD chitosans induce

acute inflammatory responses, but those with high DDs produce a minimal

response due to their lower degradation rate. Chitosan is known to degrade

predominantly in vertebrates by lysozyme and by certain bacterial enzymes in the

colon (Kean and Thanou, 2009). (8) Human chitinases in the glycoside hydrolase

family have been identified and (3) of them have shown enzymatic activity

(Funkhouser and Aronson, 2007).

2.3.2. Sources of Chitosans:

Chitin is found primarily as supporting material in many marine organisms (e.g.,

Shrimp shells, lobster, krill, crab, cuttlefish and bone plates of squid).


48

As the second most abundant natural polymer after cellulose (Rinaudo, 2006),

chitin is a principal component in the exoskeletons of silkworms, many insects

(e.g., Drosophila melanogaster, Extatosoma tiaratum, Sipyloidea sipylus,

mosquitoes, cockroaches and honey bees) (Jeuniaux, 1996), some terrestrial

crustaceans (e.g., Porcellio scaber and Armadillidium vulgare), nematodes, some

algae (Nwe et al., 2010 and its cited references: Anantaraman and

Ravindranath, 1976; Florida and Carlberg, 1982; Veronico et al., 2001;

Pochanavanich and Suntornsuk, 2002; Nemtsev et al., 2004; Moussian et

al.,2005; Tauber, 2005; Paulino et al., 2006; Mau, 2007; Hild et al., 2008;

Mario et al., 2008), but it is fully absent in mammals. Chitosan (Ch) is also a

structural component in certain fungi such as (Mucor rouxii, yeasts, mushrooms

(e.g., Auricularia auriculajudae, Pleurotus sp., Agaricus bisporus, Lentinula

edodes, Trametes versicolor and Armillaria mellea) (Roberts, 1992). Production

of chitosan from fungi using fermentation methods is gaining much interest in

recent years (Nwe et al., 2002).

From economic and technological points of view, these byproducts represent

abundant, cheap, ecologically-friendly, renewable resources for the extraction of

chitin and its derivatives where several million tons of chitin are harvested

annually in the world (Roberts, 1992). For example, the crude shrimp head and

skin have low economical values and are treated as biowaste or sold to animal feed

manufacturers (Suchiva et al., 2002); these biowaste in the topical region contain

[(10-20 %) Calcium + (30-65%) Protein content + (8-10%) Chitin) on a dry basis

(Rao et al., 1996), so they are used as raw materials for the isolation of chitin

through inexpensive simple chemistries.

Chitosan; as a semi-synthetic polysaccharide, can be prepared by the controlled

alkaline deacetylation of chitin (Furda, 1983) with different physicochemical

characteristics, viscosities and (DD) (Rao et al., 1996).


49

There are three forms of chitin known as (α, β and γ) based on the origin of

polymer and its treatment during the extraction process (Wuolijoki et al., 1999).

The α-chitosan is the most commonly obtained one from the crustacean chitin of

crab and shrimp shell wastes (Roberts, 1992) accounting for approximately 70%

of the organic part.

2.3.3. Properties of Chitosan:

1-Chitosan is a pseudonatural, renewable cationic polysaccharide of superior

biodegradability (Hirano et al., 1990), ease of availability, biocompatibility

(Thanou et al., 2001), non-antigenicity (Berger et al., 2004a), low-toxicity (Kean

and Thanou, 2009) with bioabsorbable and easy chelating power. It is an

interesting natural glycosaminoglycan possessing rare bioactivity (Domard and

Domard, 2002) and the 4th

edition of the European Pharmacopoeia (2002)

included a monograph relating to (Chitosan hydrochloride). Chitosan has a gel-

forming capability either of the polymer itself (Ladet et al., 2008) or with other

compounds (Il’ina and Varlamov, 2005). It has the ability to be injected into the

body as a liquid below the Lower Critical Solution Temperature (LCST) and then

forms a gel in situ at body temperature (above the LCST) (Jeong et al., 2002).

2-Due to chitin’s poor solubility in aqueous solutions and organic solvents, it does

not find practical applications; whereas its artificial variants; chitosan oligomers,

with their interesting multidimensional properties have been examined and found

to have a wide range of applications.

Chitosans` applications are biomedical (Paul, 2000) as well as applications in

biotechnology, aquaculture, environmental engineering and several industries such

as in agrochemicals, cosmetics, pharmaceutics and food industries. In addition,

they are pervasively used as drug delivery carriers of the polymer alone (Lubben

et al., 2001).


50

3-Combination of chitosan with other polymers can provide the required

physicochemical properties for designing of new drug delivery systems (Berger et

al., 2004b), synthesis of surgical threads (Lou, 2008), repairing of hyaline

cartilage (Montembault et al., 2006), preparing media for pancreatic islets

culturing (Cui et al., 2001), manufacturing of bone healing materials in different

forms such as sponges (Lee et al., 2000), synthesis of new preparations of

antitumor activities (Suzuki et al., 1986) and for using in gene transferring studies

(Leong et al., 1998).

4-Many synthetic chemicals such as the phenolic compounds are found to be

strong radical scavengers; however, the use of synthetic antioxidants is under strict

regulation due to their potential health hazards (Je et al., 2004). Therefore, the

usage of natural antioxidants as alternatives is of great importance. Recently, the

antioxidant activity of chitosan and its derivatives has attracted an increased

attention (Chiang et al., 2000).

5-The unique properties of chitosan are on account of its amino groups that carry

positive charges at pH values below 6 making cationic polyelectrolyte structures

(Sandford, 1989) that have the activity to bind to negatively charged materials

(e.g., Nucleic acids and enzymes) and interact with the negatively charged

glycosaminoglycans of the ECM and cells surfaces (e.g., Cells of skin) (Sandford,

1992).

6-As a cationic polymer, chitosan was studied as a partner of several Inter-

Macromolecular Complexes (IMC) with natural or synthetic anionic

polyelectrolytes, such as Chondroitin sulfate (Denuziere et al., 1998), Poly

(acrylic acid) (Wang et al., 1997), Poly (galacturonic acid) (Martins, 2007)

,Alginate (Knill et al., 2004a), K-carageenan (Zhang and Zhang, 2012), Xanthan

(Chellat et al.,2000), Carboxymethyldextran (Chen , 2003) and

Carboxymethylcellulose (Fukuda, 1980; Roscaa et al., 2005).

http://www.ncbi.nlm.nih.gov/pubmed?term=Zhang%20Y%5BAuthor%5D&cauthor=true&cauthor_uid=22667145

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51

7-Chitosan is the most useful derivative of chitin (Meyers et al., 1989). The

available primary (-NH2 and OH- groups) on the molecule are active sites that can

be used to chemically alter its properties through the formation of number of

linkages including amide and ester bonds as well as Schiff base formation

employing mild reaction conditions (Bergeret al., 2004; Hoare and Kohane,

2008). For example: Chitosan characteristics such as solubility, hemostatic and

cationic properties can be reversed by sulfating the amine groups to make anionic

and water-soluble molecules with new introduced anticoagulant properties (Suh

and Matthew, 2000). This facile derivatization makes chitosan an ideal candidate

for bio-fabrication with ample biomedical applications (Denuzière et al., 2000; Yi

et al., 2005).

8-Due to its cationic nature, chitosan has been claimed to entrap lipids in intestine

so utilized in treatment of some hyperlipidemia cases (Kanauchi et al., 1995;

Wuolijoki et al., 1999). It can enhance the paracellular route of absorption through

interaction with cell membrane resulting in structural reorganization of tight

junction-associated proteins. This mechanism is important in the transporting of

hydrophilic compounds (e.g., Therapeutic peptides and antisense oligonucleotides)

across the membrane.

9-Due to the several biological activities and excellent bioadhesive properties of

chitosan oligomers, they have become important biomaterials for wound

management (Azad et al., 2004; Lin et al., 2006). They were found to have anti-

acid and anti-ulcer activities that prevent or weaken drug irritation in the stomach

(Onal and Zihnioglu, 2002). The antimicrobial activities of chitosan and its

derivatives were reported where its (HMW- molecules) were suggested to stack on

the surface of bacterial cell wall to prevent the supply of nutrients (Tokura et al.,

1997).

http://www.ncbi.nlm.nih.gov/pubmed?term=Denuzi%C3%A8re%20A%5BAuthor%5D&cauthor=true&cauthor_uid=10669813


52

2.3.4. Production of Chitosan from Chitin of Shrimp Shells:

Shrimp industries generate large quantities of shrimp biowaste during processing;

approximately 45-55% of the weight of raw shrimp which can be used to produce

value-added products because of its richness in protein, carotenoids and chitin.

Many methods have been adopted for the extraction of chitin derivatives from

shells waste, maybe with the aid of some enzymes (Win et al., 2000; Win and

Stevens, 2001), by fermentation with certain microorganisms (Rao et al., 2000) or

with direct isolation using refluxing method (Sehol et al., 2002). The chemical

method of extraction is widely used to obtain chitin with a high quality by

removing protein, pigments, lipids and inorganic materials (mainly CaCO3). In

many chitin producing industries, the deproteination step is carried out prior to the

demineralization (Decalcification) step (Roberts, 1992) where deproteination is

achieved by treatment with NaOH and HCl is used in the demineralization step.


53

(4): Review on PolyElectrolyete Complexes (PECs)

Polyelectrolyte complexes (PECs) represent a special class of polymeric

compounds (Michaels, 1965) consisting of oppositely charged polyions which

interact together spontaneously to yield a complex in different forms that

essentially contains stoichiometric equivalents of the component polyions. These

complexes have been studied since the early 60’s and the nature and extent of such

reactions were proven to be sensitive to the molecular properties of the reacting

polymers including molecular weights and structures, as well as to the

complexation conditions including temperature, period, pH, moisture content,

mixing ratio, ionic strength of the system, cross-linking agents and density of the

formed complex (Dumitriu et al., 1994; Dumitriu and Chornet, 1997).

2.4.1. Properties of the Alginate/Chitosan PECs:

With preparing a hydrogel matrix of (alginate-chitosan) in the form of PECs, the

resulting structure will have new unique composite properties over the properties

of each used one alone, but with retaining some of their properties. These overall

properties will suit various pharmaceutical and biomedical interests (Ulbricht,

2006) and can be summarized in the following points:

A-For ideal absorptive properties of the hydrogel, it must be rich moisture

resistant. For hydrogel films of single polymer, hydration is problematic due to

limited inter-chain interactions as water-polymer interactions compete effectively

with those between the polymer chains. Based on the forces driving intermolecular

interactions, polymer-polymer interactions can be strengthened by combining

polymers of different structures and predominantly introducing charge interactions

rather than (H-bonding) (Hoare and Kohane, 2008).

B-No auxiliary molecules (e.g., Catalysts or initiators) are needed for the

complexation reactions which are performed generally in aqueous solution; this

represents the main advantage over covalently crosslinked networks.


54

This favors biocompatibility and avoids purification before administration. This is

very important for the drugs to be entrapped (Lee et al., 1999) and more effective

in limiting the release of encapsulated materials compared to either polymer alone

(Filipović-Grčić, 1995; Alexakis et al., 1995).

C-Both alginate and its membranes are non-adhesive so their structure must be

modified for enhancing their biocompatibility and bioadhesion properties on using

as dressing materials. There are several methods for mitigating that including the

usage of Schiff reaction for coupling (-COO- groups) with (-NH2) groups,

carbodiimide chemistry for binding sequence of amino acids (e.g., The crosslinker

1-ethyl-3 (-3-dimethylaminopropyl) carbodiimide. HCl (EDC) is commonly

employed with alginate as a tissue fixative where it binds to a certain sequence of

amino acids) (Hunt et al., 2009), or through the coupling of alginate chains with

positively charged polymers (Eiselt et al., 1999; Meng et al., 2010). D-It was

discovered that hydrogels of high alginate composition have high water uptake

attributed to its hydrophilicity that may cause the release of HMW chains on

degradation of the prepared scaffold (Tolba et al., 2010). Accordingly, the

possibility to change the cross-linking density within alginate hydrogels with

adjustment of their swellability through binding with other polymers may make

them potential for wound dressing applications.

E-Chitosan has low flexibility so it is majorly used with modification through

several methods such as cross-linking, grafting and blending (da Silva et al., 2008;

Hong et al., 2009). Blending is an effective and convenient method to improve the

performance of the polymers.

F-Hydrogels composed of chitosan alone are limited by the lack of an efficient

control for drug delivery, their poor Tensile Strength (TS) and elasticity (Berger et

al., 2004a; Berger et al.,2004b), so the addition of other polymers is necessary to

form PEC films with improved mechanical strength and elasticity while

maintaining the properties of the interacting polymers.

http://www.ncbi.nlm.nih.gov/pubmed?term=Meng%20X%5BAuthor%5D&cauthor=true&cauthor_uid=20101440

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55

Several research papers concerning the preparation of Inter Penetrating Networks

(IPNs)-based on chitosan and its derivatives with other polyanionic

macromolecules as well as physical gels have been published (Al-Kahtani et al.,

2009; Zhou et al., 2009).

In view of the limitations encountered in pure alginate, chitosan beads and

hydrogel systems, the concept of Alginate/Chitosan PECs (Alg/Ch-PECs) gained

acceptance (Murata et al., 1993; Huguet and Dellacherie, 1996) as the formed

complex has new properties involving those of the individual polymers with

circumventing their problems issues. For example, (Alg/Ch-PECs) were found to

still biocompatible and mechanically stronger at low pH values at which chitosan

dissolves (Hein et al., 2008), but also can degrade partially through means of

hydrolysis that can be regulated by change of the components ratios, so (PECs)

have high potential in bioengineering as scaffolds and supporting materials (Li et

al., 2009). The coacervation rate between chitosan and high-G alginates was found

to be higher than that with the high M-alginates (Gaserod et al., 1998).

2.4.2. Principle of Formation of Alginate/ Chitosan PECs:

The general PEC formation conception and its main driving force is the gain in

entropy caused by the release of low-molecular-weight counter-ions (fig.(9)). The

Hydrogen bonding and hydrophobic interactions can contribute to the process of

complexation. The structure formation is mainly determined by the fast kinetics of

the process which depend on the electrolytes with low molecular mass and the

polyelectrolytes concentrations, followed by a slower stage in which the chains

redistribute to a PEC conformation closer to equilibrium (Bakeev et al. 1992).

Other factors affecting the structure formation are the mixing procedures, medium

conditions and the macromolecular characteristics of the reacting polyelectrolytes

such as molecular weight, chain flexibility and linear charge density (LCD).


56

Figure (9): Schematic diagram of the release of counter-ions upon PEC formation.

There are three main categories of the PEC structures including soluble, colloidally

stable and coacervate complexes. Water-soluble PECs are formed by mixing

polyions with significantly different molecular weights and weak ionic groups in a

mixture of non-stoichiometric proportions under certain salt conditions; the longer

chains host several small guest chains (Kabanov 2003). PEC formation between

strong polyelectrolytes results in highly aggregated or macroscopic flocculated

systems of Colloidally stable PECs (Thünemann et al. 2004). In extremely dilute

solutions, the aggregation can be stopped at a colloidal level (with diameters of

10–100 nm) and a polydisperse system of nearly spherical particles is usually

achieved. A coacervate is formed when the mutual binding of opposite

polyelectrolytes is of moderate strength as a result of low charge density. The

coacervate is a liquid-like, mobile and reversible structure (Biesheuvel and

Stuart, 2004).

The electrostatic interactions between chitosan and other polyelectrolytes such as

alginate are stronger than other secondary binding interactions such as (H-bonding)

due to their polyelectrolyte nature. The complexation is generally reversible and

straightforward (Samoilova et al.,2010). Considerable researches have previously

discussed different methods for designing different PECs forms of alginate and

chitosan to suit special biomedical as well as industrial applications. Examples:

PEC hydrocolloids for delivery of protein drugs (George and Abraham, 2006).


57

(Alg/Ch) Beads for the biological molecules encapsulation (e,g., Hemoglobin

(Hucuet et al., 1994) and DNA microcapsules (Alexakis et al., 1995)), drugs

delivery as in the report of (Liu et al., 1997) and in the form of dry-coated tablets

(Takeuchi et al., 2000).

Based on the principles of gel network engineering, polymer chemistry and

physics; a ‘permanent interpenetrating polyion-complex hydrogel’ can be prepared

from these poly-pyranoid saccharides composing of a primary entangled network

of Ca+2

-crosslinked alginate chains (the chemical gel part) that connects and

encases physical hydrogel regions in which blends of alginate and chitosan

networks interact with each other. These interactions are initially via electrostatic

mechanisms neutralizing their charges (e.g.,ionic binding among Chitosan (NH3+)

and alginate (-COO-) groups at certain pH values. Hydrogen bonding between

chains, Coulomb forces, Vander Waals forces and transfer forces share in the

complexation process (Takahashi et al., 1990; Hoffman, 2002) as well with the

association of hydrophobic contributions of the polymers chains (Abe et al., 1978).

These complexes are supposed to be as in (fig. (10)).

Figure (10): Schematic interpretation of the (Alginate-Chitosan physical complex and

Semi-IPN complex) (with modification from (Knill et al., 2004a).

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58

(5): The Biological Effects of Alginates and Chitosans -based

Dressings on Wound Healing

2.5.1. Alginates as Wound Dressings:

Surgical applications for alginates were suggested many years ago with the first

published report on its hemostatic activity dating from 1947 (Blaine, 1947), then

met many development stages that were slow at first, but the manufacturing of

alginate fibers and quality of the resulting textiles were then improved with the

development of wet spinning and new textile technologies (Groves and

Lawrence, 1986). Among the various fibrous and hydrogel products, alginate-

based products are currently one of the most popular types used in wound

management. They have different physical and chemical properties based on the

proportion and arrangement of mannuronate and guluronate residues and their

(Ca+2

& Na+ ions-content) and they offer many advantages over conventional

dressings such as cotton gauze (Qin and Gilding, 1996) as follows:

1-Alginates are gel-forming materials with hemostatic properties (Blaine, 1947)

and enough strength for usage as an aid to retract and grip the tissues well.

2-In contact with body fluids, alginates are dissolved and absorbed where the

wound exudate converts the calcium to sodium salt facilitating removal of the

dressing by dissolution. Any residual fibers remaining within the wound are

degradable based on its composition (Burrow and Welch,1983).

3-Although a greater inflammatory response in the form of an increase in the

number of leukocytes was seen in wounds treated with the biodegradable (Ca-

Alginate) compared to the control wounds, this does not detract from its efficacy

(Barnett and Varley, 1987).


59

4-If it is the main polymer in the medical product dressing; on absorption of wound

exudate, alginate can be rinsed away with saline irrigation, so the removal of

dressing does not disturb the healing tissues (Choi et al., 1999).

5-The value of treating wounds with alginates is based on their effects in

maintaining a physiologically optimal moist microenvironment that promotes

healing with formation of granulation tissue (Motta, 1989).

6-(Ca-Alginate) has been shown to be (4) times as absorbent per unit weight as

gauze. This reduces the number of swabs used; the time spent in mopping up blood

or other wound fluids, avoids excessive use of diathermy (Blair et al., 1990) and

makes the dressing usage be very useful for moderately-to-heavily exuding

wounds.

7-Topical alginate treatment exerts its bioactivity by augmenting the natural wound

healing with concomitant induction of cytokines production by human monocytes

(Otterlei et al., 1991) and inhibition of the cytokines associated with fibrosis

resulting in increased epithelial proliferation and decreased wound size (Lee et al.,

2009).

Many previous studies described alginate based wound-dressings as follows:

I. (Ca-Alginate), on contact with blood, releases Ca+2

ions in exchange for Na+

ions and activates coagulation by stimulating platelets and the clotting factors VII,

IX, X and VIII-vWF (Born and Cross, 1964) based on its Ca+2

-content (Jarvis et

al., 1987). Calcium alginate (BritCair Ltd., Aldershot, UK) was first introduced as

a wound dressing in the study of (Blair, 1990) and evaluated as a possible surgical

hemostat; especially in major surgery, particularly where the excessive blood loss

leads to the need for transfusion.

II. (Thomas and Tucker, 1989) prepared an alginate-based dressing and proved

its efficacy in the treatment of richly exuding wounds (e.g., Chronic leg ulcers and

pressure sores).


60

In laboratory tests, alginate dressings have been found out to have an increased

capacity in absorbing blood compared to cotton gauze with promoting hemostasis

(Groves and Lawrence, 1986; Blair, 1990).

III. Through the active portion of alginate, the mannuronate residues have been

shown to stimulate human monocytes to produce elevated levels of IL-1b, IL-6 and

TNF-α (Gensheimer, 1993). This provides a convenient marker for macrophages

activation in the wound environment (Chensue et al.,1990) with exerting a

transient pro-inflammatory effect to recruit fresh leukocytes from blood (Groves et

al., 1995) and initiate the cascade of healing events.

IV. Alginate dressing was found to suppress the expression of fibronectin, TGF-β1

and VEGF effectively and blocks the accumulation of angiogenesis and ECM in

the wound but activates collagen-1 expression (Lee et al.,2009).

V. A hydrogel dressing made of cross-linked oxidized alginate with gelatin in the

presence of borax was prepared. This biodegradable, non-toxic composite matrix

has the hemostatic effects of gelatin, the wound healing-promoting features of

alginate and the antiseptic property of borax to make it a potential wound dressing

material (Balakrishnan et al., 2005b).

VI. The ability of alginate dressings to accelerate epithelialization has been

demonstrated in many experimental and clinical investigations (Agren, 1996).

Kaltostat and Sorbsan alginate dressings have been reported recently to exert

cytotoxic effects on fibroblasts. Numerous patents detailed the production of

alginate fibers and dressings (Cole and Nelson, 1993; Thompson, 1996; Griffiths

and Mahoney, 1997; Fenton et al., 1998; Mahoney et al., 1999; Qin and

Gilding, 2000; Horsler, 2000). Commercial alginate-based dressings are

summarized in (Table: 2).


61

Table (2): Summary for commercial Alginate and Chitosan Dressings

Drug name Composition Company Action

Algisite®M Non-woven fibers of

(Calcium alginate)

Smith and

Nephew

-Induce inflammation by

the soluble endotoxins

-The Activity is

associated with the

insoluble fibers and

macrophages activation.

Algosteril® Ca-Alginate Beiersdorf

Kaltocarb® Ca-Alginate fiber ConvaTec

Kaltogel® Ca/Na-Alginate gelling fiber ConvaTec

Kaltostat®

hemostat

Guluronic-rich Ca-Alginate

fibers in non-woven pads,

ConvaTec

Melgisorb® Ca/Na-Alginate gelling fibre Molnlycke -Significant haemostatic

properties on utilizing to

control the blood loss

from skin graft donor

sites (BMA, 2001;

Kennedy et al., 2001).

Seasorb ® Ca/Na-Alginate gelling fibre Coloplast

Sorbalgon® Ca-Alginate Hartman

Sorbsan ® Ca-Alginate fibers in non-

woven pads (Groves and

Lawrence, 1986).

Maersk

Chitopack S® Chitin in sponge form

Eisai Co

Chitopack P® Non-woven dressing made

of chitin-modified PET

Chitopack C® Non-woven cotton-chitosan

Non-woven dressing of chitosan fibers

(Muzzarelli, 2003)

Concern

Nikita Co

Tegasorb® Chitosan gel American

firm 3M

Designed for the healing

of wide internal wounds. Tegaderm® Chitosan hydrocolloid form

HemCon

Patch PRO

Chitosan-based hemostatic

dressings in the sponge form.

HemCon

Medical

Syvek Patch -Chitosan-modified cellulose Marine

pol. Tech.

(Fischer et al., 2004;

Alam et al., 2005) -Chitin sponge RDH


62

2.5.2. Chitosans as Wound Dressings:

The enhancement in chitosan purification and textile processing of fibers enhanced

the quality and yield of chitosan-based products in powder, film, bead, fiber and

fabric forms. In recent years, it has become important biomaterial for wound

management and the synthesis of dressings as one of its main uses (Azad et al.,

2004; Lin et al., 2006) where:

1-Chitosan was observed to have hemostatic properties independent of the normal

clotting cascade (Howling et al., 2001) and allow the promotion of normal

histoarchitectural organization (William and Herbert, 1985).

2-It can accelerate tensile strength of wounds as well as the infiltration of

inflammatory cells (Huang et al, link; Hiroshi et al., 1999) with activating the

migration and proliferation of fibroblasts (Hiroshi and Haruo, 1999; Wang et al.,

2003) as well as keratinocytes (Chatelet et al., 2001) in the wound area through

the induction of interleukin release.

3-Different chitosans-based dressings with bacteriostatic and fungistatic activities

were prepared (Tomihata and Ikada, 1997) due to the antimicrobial activities of

chitosan (Tokura et al., 1997).

4-Many Antibiotics can be included with many chitosan dressings (Mi et al.,

2002).

5-The presence of chitin/chitosan in a dressing was proven to speed the fibroblastic

synthesis of collagen in the first few days of healing (Chung et al., 1994) and

affect macrophages function to help in faster healing (Mattioli-Belmonte et al.,

1997; Muzzarelli et al., 1999).

6-A sponge constituted of (bovine type I collagen /chondroitin-4,6-sulfate/

chitosan) for skin reconstruction was developed and achieved a permanent skin

tissue regeneration with good functional and aesthetic characteristics (Li et

al.,1997). Some Chitosan-based dressings are summarized in (Table: 2).


63

2.5.3. Alginate/ Chitosan-Based Wound Dressings:

One of the most common uses for composites made of (alginate/ chitosan) is the

synthesis of wound dressings that may be in form of (Alginate /Chitosan

/Polylactic Acid/ Cotton /Tencel composite) wound dressings (Huang et al., link),

Ca-Alginate filaments coated with Chitosan (Tamura et al., 2002), membrane

sheets (Wang et al., 2002) or sponge dressings (Lai, 2003).

Flexible transparent membranes of (Chitosan/Alginate (Ch/Alg) -PECs), cast from

aqueous suspensions of (Ch-Alg coacervates) with CaCl2 were evaluated as

potential wound-dressing materials that can accelerate healing of incision wounds

in rat models, comparing to conventional gauze dressing. They can facilitate

remodeling of scar tissue by increasing the rate of collagen synthesis, compaction

of its organized fibers into thicker bundles with alignment into parallel bundles,

epidermal architecture maturation with keratinized surfaces of normal thickness

and a subsided inflammation in the dermis with fibroblasts maturation (Wang et

al., 2002).

2.5.4. Angiogenesis and Angiogenesis-Controlling Genes:

Diverse processes such as the embryonic development, tumor growth and tissue

repair are linked by the absolute requirements for a vascular bed to deliver oxygen

and nutrients (e.g., Glucose and amino acids) to the metabolically active cells and

help the removal of debris (Colton, 1995). Under normal conditions, a tissue or

tumor cannot grow beyond 1 to 2 mm in diameter without neovascularization; this

distance is defined by the limits in the diffusion of oxygen and metabolites

(Folkman, 1971). A healthy human /animal being is bleeding permanently in

small arterioles and veins without even getting aware of, so wound healing is a

vitally important adjustment of nature to prevent continuous bleeding into the

tissue after damage of blood vessels.


64

Achieving instantaneous repair and closure in wounds require sealing of numerous

blood clotting factors to vessel lesions for stopping the excessive blood loss,

provision of adequate oxygen, protein delivery and other nutritional factors to the

tissue, maintaining moist environment, appropriate inflammatory milieu,

debridement, appropriate management of infection and correction of the

contributing medical diagnoses.

Angiogenesis, the formation of new blood vessels from pre-existing vessels, is a

crucial process in wound tissue repairing in which various growth factors,

cytokines, and adhesion molecules are involved (Battegay, 1995; Martin, 1997).

Most blood vessels are formed during fetal development, but adult tissues can

induce angiogenesis in response to injury. This capability is governed by both

angiogenic agonists and antagonists that have been identified throughout the body

at various times during the repair (DiPietro et al., 1996) suggesting changes for

the net angiogenic stimuli as the balance of these factors alternatively favors either

vessel growth or regression (Martin, 1997).

2.5.4.1. Vascular Endothelial Growth Factor (VEGF):

2.5.4.1.1. Functions of VEGF:

VEGF was initially recognized as the best-characterized endothelial-specific

growth factor (Nissen et al., 1998), though other growth factors serve redundant

and overlapping functions(e.g., Angiogenin, angiotropin, angiopoetin-1, Epidermal

Growth Factor (EGF), Transforming Growth Factor -β (TGF- β) and Fibroblast

Growth Factors (FGFs)) (Folkman and D'Amore, 1996). Its biology is gaining in

complexity due to its different regulatory biological functions:

1- VEGF plays critical roles in the formation and development of blood vessels

networks, modulating thrombogenicity (Miyagami and Katayama, 2005) and

enhancing the vascular permeability with increasing the hydraulic conductivity

(Bates and Curry, 1997) and fenestration (Esser et al., 1998).


65

It has three main mechanisms of action: (1) It may increase the vessel

permeability to water, small solutes and macromolecules (Nagy and Benjamin,

2008), (2) It can increase the blood flow to tissues by acting as a potent vasodilator

(Bates and Harper, 2002), and (3) It may reduce the distance of tissue cells from

the nearest blood vessel by stimulating angiogenesis.

2-VEGF has been implicated in the wound healing process (Hayashi et al., 2004).

Its administration improves healing in nondiabetic ischemic wounds (Corral et al.,

1999). VEGF Blocking with neutralizing antibodies impedes tissue repair

(Howdieshell et al., 2001). An increase in the total levels of VEGF in fetal skin

correlates with the transition from scarless to fibrotic healing and exposure of

wounds that normally heal in a scarless manner to exogenous VEGF can lead to

scar formation (Wilgus et al., 2008).

3-VEGF can interact with many body cells, some of them are as follows:

*It could act as Endothelial cells (ECs)-specific mitogen (De Vries et al., 1992).

This potently induces their migration and proliferation in vitro and angiogenesis in

vivo (Ferrara et al., 1992; Dvorak et al., 1995).

*It plays a role in the chemotaxis of monocytes/macrophages that are crucial in the

inflammatory reactions and in wound-repair processes by inducing monocytes

activation and migration (Clauss et al., 1990) via the (VEGF receptor-1; VEGFR-

1) (Barleon et al., 1996).

*There is a possibility that VEGF could indirectly modulate the contraction and

growth of smooth muscle cells (SMCs) (Doi et al., 1996).

4- It may play a role in tumor growth (Senger et al., 1993), age-related macular

degeneration, numerous pathological situations, rheumatoid arthritis, proliferative

retinopathy and collateral formation in the ischemic tissues (Dvorak et al., 1995;

Ferrara and Bunting, 1996).


66

5-(Lerman et al., 2003) showed that targeted VEGF supplementation may be

useful as a potential new therapeutic agent for occlusive vascular diseases. Intra-

arterial administration of recombinant human VEGF165 markedly increases the

development of collateral vessels and associated perfusion in a rabbit model with

chronic hindlimb ischemia (Takeshita et al., 1994).

2.5.4.1.2. VEGF Gene:

The human VEGF gene has been mapped to chromosome 6 (location: 6p12) (Wei

et al., 1996) with cytogenic band (6p21.1) (fig. (11)) (genecards.org). It is made

up of eight exons: Exons 1–5 and 8 are always present in VEGF mRNA, whereas

the expression of exons 6 and 7 is regulated by alternative splicing. The VEGF

gene shows the same exonic structure in rodents and humans (Shima et al., 1996).

Murine VEGFA gene location is (9q12).

Figure (11):Genomic location of human VEGFA Gene on chromosome (6)

2.5.4.1.3. Structure of VEGF Protein and VEGF Receptors:

VEGF is a disulfide linked dimeric glycoprotein of 34 to 40 KDa. Several VEGF

homologues: VEGF-A,-B,-C,-D,-E and Placental Growth Factor (PlGF) have been

cloned (Werner and Grose, 2003). Most of the biologically important known

functions of VEGF are ascribed to (VEGF-A), synthesized from internal

rearrangements "alternative splicing" of mRNA, encoding (7) isoforms with 121 to

206 amino acids (Bates and Harper, 2002; Ferrara et al., 2003) of similar

biological activities but different binding affinities to heparin and the ECM (Roth

and Piekarek, 2006). Among these, VEGF121, VEGF145, VEGF165, VEGF189 and

VEGF206 are the predominant isoforms in human (Kessler et al., 2007).


67

Murine VEGF-As are denoted VEGF-A120, VEGFA144, VEGF-A164, VEGF-A188

and A205 (Tamayose et al., 1996) and each one is shorter than the corresponding

human isoform by 1 amino acid with a 95% protein homology (fig. (12))

(Burchardt et al., 1999). The small isoforms (121 to 165 amino acids) are

secreted in soluble form, while larger ones have trans-membrane domains, being

initially associated with cells where they are released and activated by proteolysis.

The isoforms VEGF121 and VEGF165 appear to be mainly involved in the

angiogenesis process (Watkins et al., 1999) and they are characteristic products of

the ovary (Olson et al., 1994). The VEGF121 is an acidic protein, while the others

have a basic isoelectric point. VEGF165 has heparin-binding properties and most of

it is freely secreted. VEGF189, after secretion, bind avidly to cell by heparin and the

ECM, although it may be released as a soluble form by heparin, heparinase or

plasmin. VEGF110 was discovered by the screening and sequencing of VEGF

cDNA clones from rat’s penis (Burchardt et al., 1999).

Figure (12): Exon structure & function of rat VEGFA. The nucleotide length

of the exon boxes and corresponding amino acid number are indicated below

the VEGF 205 & above the VEGF 110 forms (The alternate splicing results in

generation of several VEGF-A isoforms that differ in expression of the heparin-

binding domain; alternate expression of this domain results in different isoform

functions (Burchardt et al., 1999; Eming and Krieg, 2006).


68

The VEGF family members exert their biological functions by differential

interactions with three receptor protein tyrosine kinases (RTKs): VEGF receptor-1

(VEGFR-1) [similar to Fms tyrosine kinase (Flt-1)], VEGFR-2: [fetal liver kinase

(Flk-1)] and VEGFR-3 (Flt-4). Every VEGF isoform has a different affinity for

each receptor as in (fig. (13)). The VEGF tyrosine kinase receptor possesses an

extracellular domain containing 7 immunoglobulin-like loops, a single

hydrophobic membrane-spanning domain and a large cytoplasmic domain

comprising a single catalytic domain containing all the conserved motifs found in

other RTKs.

The receptors VEGFR-1 and VEGFR-2 are restricted to the vascular endothelium,

while VEGFR-3, together with its preferred ligands, VEGF-C and VEGF-D seem

to be involved in the growth of lymphatic endothelium (Barrientos and

Stojadinovic, 2008). These receptors are responsible for the VEGF-mediated

angiogenesis, blood vessels growth, regulation and then the regression during the

maturation stage of wound healing cascades. Neuropilins and heparan sulfate

proteoglycans are co-receptors lacking enzymatic activity, but each modulate the

signal output by VEGF receptors.

Figure (13): Different VEGF Receptors and their corresponding binding

cytokines.


69

2.5.4.1.4. Sources of VEGF:

Vascular endothelial growth factor (VEGF) (or vascular permeability factor

(VPF)) was first described in 1983 (Senger et al., 1983) and proved to be involved

in angiogenesis after its isolation for the first time from bovine pituitary follicular

cells in 1989 (Ferrara and Henzel, 1989) where it was identified as an

endothelial-specific growth factor. VEGF was proven to be produced by malignant

tumor cells of several types as well as macrophages (Berse et al., 1992), cultured

Smooth Muscle cells (SMCs) (Williams et al., 1995), ECs (Namiki et al., 1995)

and platelets (Bao et al., 2009). Fibroblasts (Ben-Av et al., 1995; Ferrara et al.,

2003), skin epithelial cells (Takamiya et al., 2002) as well as glioma cells,

hypertrophic chondrocytes (Miyagami and Katayama, 2005) and osteoblasts

(Gerber et al., 1999) demonstrate VEGF immuno-reactivity also. (Brown et al,

1992) identified macrophage-like cells as sources of VEGF in mouse skin wounds.

The study of (Nicholas et al.,1998) demonstrated that the fibroblasts and

macrophages are primary sources of surgical wound VEGF. Up-regulation of

VEGF production was found in keratinocytes in wounds of rats and guinea pigs as

well (Brown et al., 1992).

2.5.4.1.5. VEGF and Wound Healing:

I-VEGF directly recruits bone marrow-derived progenitor cells through a

chemotactic gradient via VEGF receptors expressed in these cells. It may signals

the Endothelial Progenitor Cells (EPCs) releasing from the marrow via its soluble

circulating factors and may act as a local signal mediating the recruitment to injury

sites (via its heparin-binding non-soluble isoforms) (Moore, 2002). This

recruitment may be also by altering the expression of other stem cell trafficking

molecules in the dermis and blood vessels during the cutaneous repair (Fedyk et

al., 2001). Examples include Chemokine Receptor type 4 (CXCR-4) and the

Stromal Cell-Derived Factor-1 (SDF-1) (Jo et al., 2000; Shen et al., 2001; Petit et

al., 2002).


70

II-In wound sites, Reactive Oxygen Species are generated by a NADPH oxidase-

dependent respiratory burst mechanism in both phagocytic (Babior et al., 1973)

and non-phagocytic cells (Suh et al., 1999). They may promote wound

angiogenesis by inducing VEGF expression in wound-related cells such as

keratinocytes and macrophages (Khanna et al., 2001; Sen et al., 2002).

III-VEGF is most likely to act through receptors in the endothelium to increase the

production of nitric oxide (NO) and prostacyclin (PGI2) and augment intracellular

endothelial cell survival signaling.

IV-VEGF-A controls wound repair by inducing vascular permeability; may be by

inducing vascular leakage (Senger et al., 1983; Dvorak et al., 1995).

V-There is an increasing evidence that VEGF contributes to inflammation

(Detmar et al.,1998) as the endothelial activation is important for (VEGF-induced

inflammation) (Yano et al., 2006). VEGFR-2 plays a main role in this induction

through both phospholipase C (PLC-γ)-dependent (in large part) and PKA-

dependent (to a lesser extent) signaling pathways (Xiong et al., 2009). VEGR-1

plays a role in inflammation by promoting the migration and secretion of

monocytes/ macrophages in which only VEGFR-1 is expressed (Pickkers et al.,

2005).

VI-Effect of Hypoxia in VEGF Secretion:

While hypoxia can initiate neovascularization by inducing angiogenic factors

expression, it cannot sustain it as a threshold level of oxygenation is required to

support the metabolic needs of tissue remodeling. Chronic hypoxia impairs wound

angiogenesis with causing dysfunction and death of tissue (Allen et al., 1997), but

acute hypoxia facilitates the angiogenic process (Semenza, 2000). On one hand,

hypoxia is a potent trigger of the inducible expression of VEGF as well as its

receptors (Berra et al., 2000).


71

VEGF production and VEGF-mediated angiogenic activity would rise in the early

hypoxic wounds and then fall when the neovascularization is completed under the

action of anti-angiogenic mediators with restoring wound perfusion and capillary

regression (Shweiki et al., 1992; Detmar et al., 1997). On the other hand,

hyperoxia was proven to induce VEGF secretion (Maniscalco et al., 1995;

Darrington et al., 1997; Sheikh et al., 2000).

2.5.4.1.6. Expression of VEGF Gene:

1-The neutrophiles, through their characteristic “respiratory burst” activity,

produce O2-., a very well-known critical reactive oxygen species for defense

against bacteria and other pathogens (Babior et al., 1973). It may be rapidly

converted to a variety of species such as H2O2, .OH or ONOO

- depending on the

local conditions. It has been previously reported that H2O2 induces the VEGF

expression in human keratinocytes and the .OH radical is a stronger oxidizing

agent than H2O2 (Khanna et al., 2001).

2-The granulation tissues formation with their content of fibroblasts, macrophages

and ECs are all essential to maintain the VEGF secretion.

3-The VEGF gene expression is up-regulated by hypoxia as well as by TGF- β,

angiotensin II, (FGF-2) and Interleukin-1 (Il-1) (Williams et al., 1995; Ferrara

and Bunting, 1996; Hayashi et al., 2004; Nagata et al., 2005; Jin et al., 2006).

(Nogami et al., 2007) reported that the VEGF mRNA levels significantly increase

24 h after the incisional wounding of skin compared with the 3rd

and 7th

days, but

fibroblasts, ECs and CD68-positive mononuclear cells sustain VEGF expression 7

days after the wounding.

4-(Nogami et al., 2007) reported also that the presence of VEGF in the early

stages of wound healing indicates the possibility of its involvement in the

vascularization during healing. (Hayashi et al., 2004) observed VEGF expression

in a relatively late stage of skin injury.


72

In murine cutaneous wounds, VEGF expression was detected in the epidermal cells

after 24 h of wounding and lasted for 144 h (Takamiya et al., 2002).

5-VEGF protein was suppressed with wounds treated with alginate than those

treated with Vaseline and normal healed wounds (Lee et al., 2009).

6-Although VEGF is considered a primary angiogenic mediator in surgical wounds

for several days postoperatively, it does not appear to participate in the initiation

phase of angiogenesis where FGF-2 and VEGF act in concert to provide a

sustained angiogenic stimulus in surgical wounds. The angiogenic stimulus

initiated by FGF-2 is subsequently maintained by VEGF where the early

endothelial proliferation is supported by FGF-2 and the later capillary growth and

differentiation are directed by VEGF (Asahara et al., 1995; Koolwijk et al.,

1996). The preformed FGF-2, sequestered in uninjured tissue is likely released at

the time of surgery either by cell damage or by enzymes such as thrombin

(Cordon-Cordo et al., 1990; Ben-Ezra et al., 1993; Vallaschi and Nicosia,

1993) and was proven to up-regulate VEGF production (Stavri et al., 1995).

2.5.4.2. von Willebrand Factor (vWF):

(vWF) is frequently used as a biochemical marker for endothelial cells. Despite

this, little is known about the relative expression and regulation levels of this

hemostatic factor in ECs in different vascular beds in vivo.

2.5.4.2.1. Functions of vWF:

1-vWF behaves as an acute phase protein, because its plasma concentration may

increase several fold in response to variety of physiological and patho-

physiological conditions (Suffredini et al., 1989; Borchiellini et al., 1996).

2-vWF initiates the hemostatic process by mediating platelets adhesion to the

subendothelium which becomes exposed with disruption of the continuous

endothelial layer (Tschopp et al., 1974; Meyer and Baumgartner, 1983).


73

3-With the help of collagen, vWF plays a key role in promoting platelet adhesion

to the damaged vessel walls, subendothelial matrix and endothelial surfaces

(Sakariassen et al., 1979; Girma et al., 1980; Baumgartner et al., 1980; Huang

et al., 2009) and in platelets activation (Farndale, 2006).

4-vWF acts as a genuine matrix protein that promotes the organization of

microfilaments and adhesion plaques via a trans-membrane mechanism with

keeping the anatomical integrity of the internal vessel lining (Dejana et al., 1989).

In addition, it promotes EC adhesion and supports their cytoskeletal organization

(Cheresh, 1987; Charo et al., 1987).

5-(factor VIII-related antigen): It acts as a stabilizer for the coagulation factor VIII

against proteolytic degradation inside the blood stream (Wagner, 1990) and its

carrier to the destination point in the circulation (Huang et al., 2009).

6-vWF plays essential roles in hemostasis: its deficiency or dysfunction results in

increased angiogenesis (Starke et al., 2011) with causing von Willebrand Diseases

(VWD); the most common congenital bleeding disorder in humans (Sadler, 2005).

Its increased levels are clinical markers of risk associated with atherosclerosis and

coronary thrombosis (Spiel et al., 2008).

7-Endothelial vWF is also involved in the regulation of inflammation by

modulating leukocytes adhesion through direct and indirect mechanisms (Denis et

al., 2001; Pendu et al., 2006).

2.5.4.2.2. vWF Gene:

-Human vWF gene is located on the short arm of chromosome 12 (Location:

12p13) with the cytogenetic band (12p13.31) (fig. (14)) (NCBI)) and consists of

178.000 bps. Murine vWF gene location is (4q42) (http://rgd).

Figure (14): Genomic location of human vWF gene on chromosome (12).

http://www.ensembl.org/Homo_sapiens/geneview?gene=ENSG00000110799

http://rgd/


74

2.5.4.2.3. Structure and Biosynthesis of vWF Protein and its Receptors:

The human vWF gene is transcribed into an mRNA copy containing 52 exons

(Mancuso et al., 1989). In the ribosomes, the first translation product,

prepropolypeptide (prepro vWF); consisting of 2813 amino acids, undergoes a

complex series of post-translational modifications (Jaffe et al., 1973).

In the endoplasmic reticulum, biosynthesis of vWF protein includes the formation

of dimers through disulfide bond linkages at the carboxy-terminal ends of the

molecule (”Tail-to-tail” dimerization) (Wagner and Marder, 1984; Marti et al.,

1987) involving only amino acids within the last and cysteine rich 150 residues

(Sadler, 1998).

These vWF dimers subsequently multimerize in the acidic environment of the

Golgi apparatus and post-Golgi compartments through disulfide bond linkages in

the amino termini forming (pro-vWF) (Fretto et al., 1986; Marti et al., 1987)

which becomes mature with the cleavage of 741 amino acids (95 KDa) (Mayadas

and Wagner, 1989) (fig. (15)). Thus mature polypeptide subunit consists of (2050

amino acids) is formed and modified then by 12 N-linked and 10 O-linked

carbohydrate side chains (Titani et al., 1986) with the sulfation of N-linked

carbohydrates (Carew et al., 1990). Finally, vWF is formed as a large multimeric

plasma glycoprotein (GP) with estimated molecular mass ranging from 500 KD to

over 10,000 KD (fig. (15)) (Ruggeri and Zimmerman, 1981).

Figure (15): Structure of vWF Protein: The amino acids are numbered in ascending

order beginning at the amino end of the protein. Capital letters represent associations of

amino acids into functional domains. The prepro VWF molecule contains a signaling

peptide (residues 1 to 22), a large propeptide (23-763) and the mature subunit (764-2813)

residues. (Sadler, 1998).


75

vWF may be stored in special compartments, called Weibel-Palade Bodies

(WPBs), or released into the blood stream (Wagner and Marder, 1984) under the

induction of a variety of chemical stimuli (e.g., VEGF stimulators) that may be

induced by injuries of vessels walls or increased levels of adrenaline in the

surrounding blood (Sporn et al.,1986; Wagner et al., 1991). The deposited vWF

in the endothelial ECM consists of higher molecular weight multimers than

plasmatic vWF (Tannenbaum et al., 1989; Baruch et al., 1991) with increased

capacity to bind to the platelets (Moake et al., 1986). The largest vWF multimers

were found to bind preferentially to a fibroblast ECM (Sporn et al., 1987).

The two human platelet vWF glycoprotein receptors (GpIb and GpIIb-IIIa) belong

to the superfamily of cell adhesion receptors "integrins" (Hynes, 1987). This

complex has several structural and functional homologies and recognizes numbers

of ECM components via a common Arg-Gly-Asp (RGD) sequence (Ruoslahti and

Pierschbacher,1986) (Fig. (15)). They are structurally unrelated and recognize

different specific binding domains on the vWF molecule (Girma et al., 1987;

Nurden,1987) resulting in the rolling of platelets over the endothelium with their

firm adhesion and activation (Ruggeri and Mendolicchio, 2007).

2.5.4.2.4. Sources of vWF:

Besides being a plasma protein, vWF is also a constitutive component of the

subendothelial matrix (Giddings, 1986) where megakaryocytes and (ECs) are its

only synthesizing cells (Jaffe et al., 1973; Wagner, 1990; Zanetta et al., 2000).

They can release it in the blood stream with organization in close association with

other matrix proteins within the subendothelial basement lamina (Giddings, 1986).

The synthesized vWF by ECs can be stored within the WPBs with other angiogenic

regulators (e.g., Angiopoietin (Ang-2) (Starke et al., 2011)) that is associated with

hemostasis and vascular tone (Valentijn et al., 2011; Metcalf et al.,2008) and

with unidentified proteins.


76

The normal biology of the WPBs is disrupted with deletion of vWF gene (Denis et

al., 2001). vWF and other molecules are released simultaneously and a temporal

association of vWF and Ang-2 release has been observed (Fiedler et al., 2004;

Starke et al., 2011). Rat endothelial cells store vWF in the endoplasmic reticulum

(Reidy et al., 1989). The synthesized protein may be secreted from the bovine ECs

as that observed in the work of (Lynch et al, 1983) without any significant net

intracellular accumulation of the precursor product. (Hormia et al., 1984; Warhol

and Sweet, 1984; Cramer et al., 1985) have visualized vWF in the Golgi

apparatus. The α-granules are the most abundant granules in platelets and contain a

multifunctional array of proteins (e.g., vWF, thrombospondin, P-selectin and

fibrinogen) (Sabrkhany et al., 2011). The physical structure of these organelles

with the observed longitudinal tubules seem to be due to the multimeric vWf

molecule itself indicating its capability for directing the formation of its own

storage granules (Wagner et al., 1991; Wagner, 1993; Voorberg et al., 1993).

2.5.4.2.5. vWF and Wound Healing:

I-vWF and Hemostasis:

Both the circulating and secreted vWF from the WPBs contribute to wound healing

and blood clotting. Under normal healing conditions, a stable blood clot seals an

injured blood vessel in a complex process involving more than a dozen of blood

coagulation factors, accompanied by alteration of local streaming conditions,

exposure of various connective tissues and the contraction of the injured blood

vessels to limit bleeding which affect strongly the streaming properties of the

flowing blood (Girma et al., 1987). The vWF network represents the basis for

wound healing and the aggregation of platelets to this network is the starting point

for the plug growing which finally seals the vessel damage. The primary

hemostasis process is the initial step to stop blood loss by the formation of (vWF-

platelet plugs) and is based on conformational changes within the protein from an

inactive globular into an elongated activated state.


77

Plasma vWF does not bind to the circulating platelets, but on immobilizing on a

collagen surface, high shear stress with binding of vWF to the subendothelium

induces changes in the vWF conformation to allow platelets adhesion

(Sakariassen et al., 1984). At the vascular damage sites, activated vWF comprises

specific receptors in its A1 and A3 domain to the counterpart in the collagen

matrix.

On binding to the surface, vWF molecules interconnect to form macroscopic

networks on the exposed layers of collagenous components of the subendothelium

and on additional non-collagenous site(s) for vWF (Wagner and Marder, 1984;

De Groot et al., 1988). Platelets are enriched near the vessel wall with drastic

increase on their binding probability (Fåhræus–Lindqvist effect) (Perkkioe et al.,

1987), mediated by two distinct platelet receptors, GPIb and GP IIb/IIIa (integrin

αIIbβ3) (Girma et al., 1987).

A weak bond with a high dissociation constant is formed between VWF’s A1

domain and the platelet receptor GPIbα, followed with a stronger and irreversible

bond involving VWF’s C1 domain and the platelet integrin αIIbβ3 with a low

dissociation rate enough to form (fig. (15)) (Ruggeri, 1997). These interactions

lead to subsequent platelets spreading and aggregates formation on their membrane

comprising even more binding sites for VWF while reducing the opposing drag

forces at the same time (Weiss et al., 1986; Sakariassen et al., 1986; Fressinaud

et al., 1988). As a result, vWF can function as a bridge between platelets and

collagen (Fredrickson et al., 1998) and serve as glue between multiple platelets

layers. This platelet plaque grows till further loss of blood is prevented (Steppich

et al., 2009), especially if the rent in the vessel wall is small.

Under some circumstances, such as after thrombin activation, vWF also binds to

the platelet membrane glycoprotein complex (GpIIb-IIIa).


78

This promotes the platelets aggregation (Fujimoto et al., 1982; Ruggeri et al.,

1982; Ruggeri et al., 1983). During secondary hemostasis: Larger damage

requires further stabilization of the platelets aggregates so water-soluble fibrinogen

is transformed to water insoluble fibrin forming a mesh-work for embedding both

erythrocytes and platelets (Guyton and Hall, 2000). The production of fibrin

involves a cascade of multiple blood clotting factors activating each other and the

dysfunction or deficiency of one of these factors can decelerate or even stop blood

clotting (Steppich et al., 2009).

II-vWF and Inflammation:

vWF is stored with P-selectin in the endothelial WPBs and secreted in response to

the same signals (e.g., Vascular injury or inflammatory mediators) which

externalize P-selectin to the plasma membrane and this suggests the possibility of

overlapping functions between the two proteins. The inflammatory role of vWF

could be largely secondary, with the adherent activated platelets providing a

surface for leukocyte recruitment so its deficiency in mice was found to lessen the

inflammatory response in wound healing (Methia et al., 2001) due largely to the

mispackaging and defective secretion of P-selectin associated with VWF

deficiency (Lo´pez, 2006). vWF could recruit leukocytes when conditions favor

the persistence of its hyper-adhesive forms on the endothelial surfaces as occurs

under the influence of inflammatory cytokines which stimulate VWF secretion and

inhibit its processing by ADAMTS13 (Bernardo et al., 2004). Its levels are

elevated in both chronic and acute inflammation (Vischer, 2006). WPBs were

reported also to play roles in inflammation through the stored IL-8 in the

microvascular ECs (Utgaard et al., 1988; Wolff et al., 1998).


79

III-vWF and Angiogenesis (Relation between vWF and VEGF):

vWF is required to control the formation of the vascular network (Starke et al.,

2011). Its leakage results in increased vascularization, suggesting that VEGF-

dependent angiogenesis is enhanced in VWF-deficient cases. Increased VEGF

expression and signaling have been linked to vascular malformations (Thurston et

al.,1999; Ozawa et al.,2004). vWF has an inhibitory activity in the constitutive

VEGFR-2–dependent pathway(s) that promote EC migration; however, there is

partial effect for the extracellular VWF on this process (Starke et al., 2011).

vWF regulates Ang-2 release from ECs. Decreased/ dysfunctional vWF can cause

decrease or loss in production of WPB and cause defective packaging of these

proteins as a consequence resulting in constitutive release of its components (e.g.,

Ang-2) that destabilizes the blood vessels and acts synergistically with VEGF-A to

promote angiogenesis (Yu and Stamenkovic, 2001; Lobov, 2002; Hashizume et

al., 2010). The over-expression of Ang-2 in in vivo studies results in the formation

of unstable, dysplastic vessels (Feng et al., 2007). An increase in plasma levels of

(Ang-2) in patients with VWD might contribute to promoting blood vessel

plasticity and thus exacerbate angiodysplasia.

2.5.4.2.6. Expression of vWF Gene:

Variety of perturbations (e.g., Renal failure (Warrell et al., 1979), irradiation

(Sporn et al., 1984) and cerebral thrombosis (Zheng,1983) cause increase in the

circulating levels of vWF that is thought to be an indicative of endothelial injury.

vWF is heterogeneously distributed through the vascular tree and is associated

with regional variations in the mRNA levels (Rand et al., 1987; Wu et al., 1987;

Bahnak et al., 1989; Coffin et al., 1991; Page et al., 1992; Aird et al., 1995;

Smith et al., 1996). Low levels of vWF are present within the sinusoidal ECs of

liver and spleen. In en face preparations of the rat aorta, expression of vWF

appeared to vary from one EC to another (Senis et al., 1996).

Materials and Methods

80

3. MATERIALS and METHODS

3.1. Materials:

*Sodium alginate (Na-Alginate) (A2033, medium viscosity, M/G ratio (1.56)) and

Potassium Bromide: Sigma-Aldrich, USA.

*Coomassie Brilliant Blue G-250 & Ethylene glycol (ANALAR) & Diethyl ether

and Fehling solutions: Fisher scientific, USA.

*Bovine Serum Albumin (BSA) (Biotechnology Grade): AMResco, Canada.

*Haematoxylin, Eosin and MT: Surgipath Europe Ltd, UK.

*Sodium chloride: Riedel-de Haën, Germany.

*Acetic acid: FEINchemie Schwebda, Germany.

*Streptozotocin (STZ): Merck Chemical, Germany.

*Sodium (meta) periodate: LOBA-Chemie, India.

*Calcium chloride, Ferrous chloride, Ammonium thiocyanate, Charcoal, Sodium

hydroxide (Analytical grade), Hydroxylamine hydrochloride, absolute Ethanol,

Butanol, Methanol, Sulphoric, Hydrochloric, Nitric, Phosphoric acids (Analytical

grades), Formaldehyde and Chloroform: Elnasr Pharmaceutical Company

(ADWIC, Egypt).

*Hydrogen peroxide (Analytical grade): Piochem, Egypt.

*α-tocopherol: CID pharmaceutical company, Egypt.

3.2. Preparation of reagents:

3.2.1.Hydroxylamine.HCl (NH2OH.HCl) reagent (0.25 M)

According to (Huiru and Heindel, 1991), 17 gm dried (NH2OH.HCl) powder

were dissolved in 150 ml distilled water with heating at 65oC till complete

dissolving. The solution was cooled; then 6 ml (Methyl Orange reagent, 0.05 %)

were added and completed to 1 L with adjusted pH at 4.

3.2.2. Sevag Reagent:

Chloroform and n-butanol solutions were mixed together at a ratio (4:1).


81

3.2.3. Phosphate buffered saline (PBS):

I.Potassium dihydrogen phosphate (0.1 M): (6.81 gm KH2PO4 (from (ADWIC-

Egypt)) were dissolved in distilled water to 500 ml.

II.Disodium hydrogen phosphate (0.1 M): (8.91 gm Na2HPO4.2H2O (from

(ADWIC-Egypt)) were dissolved in distilled water to 500 ml.

III.Preparation of Phosphate Buffered Saline (PBS, 0.1 M, pH 7.4): 80.8 ml

(Na2HPO4, 0.1M) were mixed with 19.2 ml of (KH2PO4, 0.1 M).

3.2.4. Sodium Citrate buffer (0.1 M, pH 4.5):

44.5 ml (Citric acid. H2O, 0.1M) were mixed with 55.5ml (Trisodium citrate.

2H2O, 0.1M).

3.2.5. Reagents for the RNA Extraction based on (Fermentus, Thermo Fisher

Scientific Inc, UK) extraction kit:

*The working Lysis Buffer: 24 µl β-mercaptoethanol were added to each 1 ml of

the kit Lysis Buffer to prepare the required working buffer.

*TE-Buffer: Consisted of (10 mM Tris-Cl (pH 7.5), 1 mM EDTA (pH 8)).

*Proteinase-K: Diluted enzyme was prepared by adding 10 µl Proteinase K to 590

µl of the prepared TE-Buffer.

*Preparation of Wash Buffer (1): 10 ml of absolute ethanol were added to 40 ml

concentrated wash buffer (1) to reach a total volume (50 ml).

*Preparation of Wash Buffer (2): 39 ml of absolute ethanol were added to 23 ml

concentrated wash buffer (2) to reach a total volume (62 ml).

3.3. Modification of Alginate:

3.3.1. Irradiation of Sodium Alginate:

Degraded Na-Alginates were prepared by the following methods:

1-(LMW-Alginate) was obtained through γ-irradiation of sodium alginate from

(Cobalt-60 (Gamma-Cell 220), Atomic energy of Canada Limited, installed at

Radioisotope Department of the Egyptian AEA, Cairo-Egypt) at the 2 doses (50

and 100 KGy) at R.T on air with dose rate (5.6 KGy/ hour).


82

2-Sharp decreasing for the molecular weight was obtained by mixing Na-alginate

(powder) with 10 (wt. %) oxidizing agent (H2O2) for its obtaining in the paste form

that was then packaged tightly in polyethylene bags and subjected to γ-irradiation

at the doses (50 and 100 KGy) under the same previous conditions. The resulting

alginate was denoted (VLMW: Very Low Molecular Weight–Alg).

3.3.2. Oxidation of Sodium Alginate:

This was carried out according to the modified protocol of (Bouhadir et al., 2001).

8 Erlenmeyer flasks; each one contained 1 % homogeneous alginate solution in

100 ml NaCl solution (10 mM), were wrapped with aluminum foil, and divided

into (2) groups; each one of 4 flasks and denoted as follows:

-Group I (Multi Aldehyde Sodium Alginate-I (MASA-I)): each flask was charged

with 1.0 gm un-irradiated sodium alginate (HMW-Alg). -Group II

(Multi Aldehyde Sodium Alginate-II (MASA-II): each flask was charged with 1.0

gm irradiated sodium alginate (LMW-Alg).

4 different concentrations of Na-periodate solutions (0.25, 0.5, 0.75, and 1.0 M)

were prepared and 20 ml from each one were added to one flask of each group

with stirring the mixtures for 24 h at R.T. Ethylene glycol (0.5 ml) was added to

the mixture of each flask to quench the reactions by reducing any unreacted

periodate (Lee et al., 2002) and then stirred for additional 0.5 h at R.T. 2.5 gm

NaCl were then added to the solution of each flask, followed by precipitation with

excess of absolute ethanol (200 ml for each solution). The precipitates, collected

by centrifuging the different alginate products were redissolved in distilled water

and re-precipitated with ethanol. After their concentrating, the precipitates were

dried to yield a white product (about 0.7-0.8 gm).


83

3.3.3. Characterization of the different modified alginates:

3.3.3.1. Test for Presence of Aldehyde Groups:

Different aqueous treated alginates solutions were heated with Fehling solution and

tested for the formation of red precipitates of cuprous oxide.

3.3.3.2. Fourier Transform-Infra Red Spectroscopic Characterization:

Different modified alginate samples (LMW and MASA) were prepared in the

forms of (KBr disks) by mixing 2 mg of each sample powder with 200 mg of

anhydrous KBr, triturated with agate mortar, then pressed under vacuum and the

Infra-Red spectra were recorded using (JASCO FT/IR-6100 type A

spectrophotometer, Japan) with a resolution of 4 cm-1

at ambient temperature over

the region (400- 4000 cm-1

) with repetition of 64 scans. For the oxidized alginates,

the analysis was concentrated in the range (1000- 2500 cm-1

). All spectra were

compared with that of the unmodified alginates.

3.3.3.3. Determination of Degree of Aldehyde Substitution:

The degree of aldehyde substitution was measured according to the steps adopted

by Maiti et al (Maiti et al., 2009). 10 ml of 2.5 % aqueous solutions of the

different oxidized alginates (MASA) were prepared and the pH was adjusted to 4

with dil. HCl. An excess of NH2OH.HCl solution (about 50 ml) was added drop

wise to each stirred solution, mixed together and allowed to stand for 2 hours till

no further reduction in solution pH was detected. pH of mixture was then

maintained at 4 via titration with standardized NaOH solution (0.08 M) and the

reaction was monitored by the following methods:

A-Potentiometric Titration Monitoring:

The end point of the titrated mixture was monitored potentiometrically to an

apparent (pH 4) with a calomel electrode and pH meter. The strength of HCl

generated from the reaction between NH2OH.HCl and the oxidized alginate was

calculated from the titrant volume that maintained the pH.


84

The degree of aldehyde substitution was calculated based on the report of Zhao

and Heindel (Zhao and Heindel, 1991) from the following equation:

B-The titration continued until the red (of the acidic liberated HCl-rich solution) -

to yellow (The neutral solution) end point was achieved by matching the color of

the samples solutions with that of a blank one.

3.3.3.4. Determination of the Average Molecular Weights:

3.3.3.4.1. The Viscometric Method:

Dynamic viscosities for the raw and modified alginates solutions (in 10 mM NaCl

solvent) were measured using (Synchro-Lectric rotational Viscometer: Model DV-

IV+ Pro, Brookfield Engineering Labs Inc., Middleboro, MA, USA), operated at

25°C using the spindle (No. CPE 41) rotating at 25 RPM. The spindle was

immersed at first on the solvent, then on each alginate type in ascending order of

concentrations. For the raw alginate, measurements were performed with the

concentrations (C: 0.1, 0.2, 0.4, 0.6, 0.8, and 1 %), but they were (0.5, 1, 2, 3, 4,

and 5%) for the degraded forms. The viscosity measurements of each sample were

performed in triplicates to obtain accurate readings for calculating the molecular

weights as follows:

(I) The obtained measurements are the coefficients of viscosity as follows:

-solvent): denotes the solvent NaCl (10 mM) viscosity.

- (sol`n): denotes the prepared alginate solutions viscosities.

(II) To a very good approximation, the Relative Viscosity (rel) is calculated from

the ratio between (stated sol`n and solvent) at the same temperature.

(III) The solution Specific Viscosity: (solvent

solventnsolsp

' =rel-1)

(IV) Reduced Viscosity (dl/gm): (redsp/ C).

(V) Inherent Viscosity (dl/gm): (inh=lnrel/ C).


85

With plotting inh and red versus the concentration and extrapolating to C=0, the

intrinsic viscosity (int) was determined by averaging the values of the intercept

(Russo et al., 1986). The average Molecular Weight was calculated on the basis of

the following Mark–Houwink–Sakurada equation (MHS) (Tanford, 1961):

Where: *int: the intrinsic viscosity.

*M or (Mv): viscosity average Molecular Weight of the polymer.

*The equation constants: (K= 4.85×10-3

ml/gm and a= 0.97) at which Mw ≈

Mv for alginate (Stokke et al., 2000).

3.3.3.4.2. Gel Permeation Chromatography (GPC) method:

The number-average Molecular Weight (Mn) and weight-average Molecular

Weight (Mw) for the unirradiated, irradiated and oxidized samples were measured

by ((GPC) 1100 Agilant instrument) equipped with organic and aqueous GPC-SEC

start up kits with a flow rate of 2 ml/min, maximum pressure 150 bar, minimum

pressure 5 bar, injection volume 50 µl and column temperature thermostat 25°C).

The eluent was monitored by a refractive index detector of optical unit temperature

25°C and peak width 0.1 min. The polymer concentration was 0.1 (wt. %) and the

M.Ws were determined from a calibration curve using polyethylene oxide

standards for aqueous systems. The used method for determining (Mn) and (Mw)

followed (Liu et al., 2006; Li and Liang 2007).


86

3.4. Purification Protocol of Sodium Alginate:

3.4.1. Acid-washing of Activated Charcoal:

This was accomplished based on (Asada et al., 2006) method with 3 trial

batches. Each 1 gm of pre-dried charcoal in each patch was completed to 100 ml

with 1 M H2SO4, HNO3 or HCl, then mixed together at R.T for 5 hours, filtrated

and washed several times with distilled water. The resulting charcoals were then

dried at 100oC overnight and tested as well as the raw type in the pellet form with

(JASCO FT/IR-6100-type A spectrophotometer) according to the procedures,

stated in section (3.3.3.2). The spectral range of analysis was (400-4000 cm-1

)

with 100 scans and a resolution of 4 cm-1

. The same washing steps were repeated

then in patches system with the chosen washing acid after the FT-IR testing.

3.4.2. Method of Alginate Purification:

At first, the alginates to be purified were separated into batches. All steps of

preparation and testing were carried out using double distilled water, tested for its

pH (7 ± 0.15) and calcium concentration (less than 3 ppm). All the used glass

flaks and containers were sterilized with saturated water steam before the

purification steps which were performed according to the protocol of (Qi et al.,

2009) with some modifications as follows:

A-Precipitation of Proteins from Alginate Solutions:

For all steps, the alginate concentration was expressed according to the weight

percent (wt. %). For each patch, (1% homogeneous solution, pH 7) was prepared

in 10 mM NaCl solution at R.T with continuous shaking. For each chilled 100 ml

solution, 25 ml Sevag reagent were added (Staub et al., 1965), shaken vigorously

for 30 min and centrifuged at 3000 RPM leaving the solution divided into 3 layers:

top organic layer, a middle layer containing the precipitated proteins; these two

layers were aspirated and only the bottom aqueous one containing the alginate

polymer remained. This step was repeated for each patch.

http://www.researchgate.net/researcher/39258946_Takashi_Asada/


87

B-Precipitation of the (PC) from Alginate Solutions:

Equivalent alginate weight of acid-washed activated charcoal was added to each

solution, mixed well, then filtrated to remove the adsorbent charcoal with several

washings with dist. water till its complete removal.

C-Precipitation of Alginate:

200 ml ethanol were added to each 100 ml of alginate solution, mixed

continuously, allowed to stand for 30 min and filtered for separating the alginate

that was redissolved in water and re-precipitated then with ethanol. After

concentrating, the precipitates were frozen at -70oC over-night, lyophilized for 2

days and then the following tests were applied for testing the effects of

purification on the treated alginates.

3.4.3. Testing the Effects of Purification in Alginate:

3.4.3.1. Viscosity Measurements for the Raw and Purified Alginates:

Dynamic viscosities of the different alginate solutions (0.5 % w/v) before and

after purification were checked using a rotational Viscometer (Synchro-Lectric

Brookfield rotational Viscometer) operated at 20oC and the spindle (No. CPE 41)

was selected by rotating at 25 RPM.

3.4.3.2. Quantification of Impurities Concentrations:

3.4.3.2.1. Proteins Content Quantification:

I-Preparation of Reagents:

*Dye Stock: According to (Bradford, 1976), 100 mg Coomassie Brilliant Blue

G-250 were dissolved in 50 ml methanol (95%), 100 ml phosphoric acid (85%)

were then added. After complete dissolving of the dye, the solution was diluted

to 200 ml with dist. water to give a dark red solution with pH 0.08.

Concentrations of the final reagent components were (0.5 mg /ml Coomassie

Blue G, 25% methanol and 42.5% phosphoric acid).

http://academic.research.microsoft.com/Author/10699737/marion-m-bradford


88

*Assay Reagent: Prepared by diluting 1 vol. of the dye stock with 4 volumes of

dist. water to give brown colored solution, stable in dark at 4oC.

*Blank Solution: 0.15M NaCl.

*Protein Standards: Series of Bovine Serum Albumin (BSA) standards were

utilized for building a convenient standard curve and prepared by dilution with

(150 mM NaCl) with the following concentrations:

-0, 250, 500, 750, 1000, 1500 and 2000 µg/ml: for the standard assay.

-0, 1.5, 7.5, 10, 30, 40 and 50 µg/ml: for the micro-assay.

*Alginates Samples: For increasing the accuracy of measurements, unpurified

and purified (HMW and irradiated alginates) were prepared in different

concentrations using the same solvent (150 mM NaCl).

II-Method of Quantification (The Microassay):

1ml of the prepared assay reagent was added to 100 µl of each concentration

from both the standards and alginate samples and incubated at R.T for 10 min.

The absorbance of each resulted solution as well as the dye only (zero protein)

were measured using blanked spectrophotometer at 590 nm. Plastic cuvettes were

used and all measurements were in ascending order. For presentation of the

results, the concentrations were converted to the equivalent mg of proteins per

gm of dry alginate.

3.4.3.2.2. Polyphenolic-like Compounds (PC) Quantification:

The fluorescence spectra of unirradiated and irradiated alginate solutions (1%

w/v in dil. NaCl) were obtained using a spectrofluorimeter (JASCO FP-6500,

Japan, source: Xenon arc lamp 150 Watt, both the slit band width of excitation

monochromator and emission monochromator were 5 nm and PMT gain was

medium). An excitation wavelength of 366 nm and emission wavelength of 445

nm were applied.


89

The relative quantities of the Polyphenol-like compounds were detected by the

appearance of a characteristic absorbance peak at 445 nm (Skjak-Braek et al.,

1989) and were measured and expressed in terms of Arbitrary Fluorescence Units

(AFU) with correlation to Raman bands.

3.4.3.3. Characterization of the Purified Alginates with (FT-IR): Fourier

Transform-Infra Red (FT-IR) spectroscopic analysis was utilized to investigate

the effect of purification on the structural properties of the alginate chains and the

binding behavior of their functional groups (C-OH and COONa). Structures of

the purified products were confirmed through (FTIR) applied to (KBr-Alginate

pellets), prepared as previously described in section (3.3.3.2). The bands

corresponding to the functional groups for both the raw and purified alginates

within I.R charts were compared.


90

3.5. Preparation of Chitosan:

3.5.1. The Extraction and Deacetylation Steps:

These were carried out according to (Gopalakannan et al., 2000) with some

modifications. Frozen dried shrimp shells waste was cut into pieces, washed with

tap water and deproteinized by boiling in 1 N NaOH (1:5 w/v) for 30 min. The

alkali was drained, replaced with another amount and the process was repeated for

the removal of residual proteins. Colors of the solutions were noticed till becoming

clear and the pieces were washed with tap water. The shells were demineralised by

soaking in 1 N HCl (1:5 w/v) at 40°C for 30 min. The acid was then drained off

and the pieces were washed thoroughly with tap water till reaching the neutrality

followed with dist. water washing. The chitin product was dried at R.T and stored

under dry conditions till the further deacetylation procedures.

The deacetylation steps were carried out according to (Roberts, 1992); the chitin

was soaked in 50 % aqueous NaOH (1:20 w/v) and divided into two different

patches which were boiled at 100°C for 2 hours divided into 4 equal times with the

changing of NaOH. The first patch was then washed with water and nominated as

(Ch-1). The second patch, (Ch-2) was stored till cooled, then reboiled in 50 %

aqueous NaOH for additional 2 hours, re-cooled and reboiled for additional 1 hour.

After deacetylation of the (2) chitosan patches with draining off the alkali and

washing by tap water, they were washed by dist. water and dried at R.T for several

days.

3.5.2. Characterization of the Prepared Chitosan products:

3.5.2.1. Solubility Test:

Solubility of each patch product as well as the parent compound, chitin was tested

by formation of soluble polymer on preparation of (0.5 % solution in 1 % Acetic

acid solution (HAc)) with continuous heating and shaking.


91

3.5.2.2. (FT-IR) Spectroscopic Characterization:

Chitosans samples were prepared in the form of KBr pellets based on the method

of (Sabnis and Block, 1997) where the pellets (powdered chitosan: KBr=1: 3)

were dried at 80°C in hot air oven for 24 hours before analysis, then characterized

by infrared spectrophotometer in the range of (800-4000 cm-1

). The % N-

deacetylation values were determined using the equation proposed by (Terayama,

1952; Domszy and Roberts, 1985) but with using the baseline, proposed by

(Baxter et al., 1992) for comparing the intensity of the (amide I) band in chitosan

with that of the (-OH groups). The following equation was applied:

%DD=100- [(A1655/A3450) *100/1.33]

Where: A1655: The absorbance at 1655 cm-1

for the amide (N-H) band as a measure

of the N-acetyl group content.

A3450: Absorbance at 3450 cm-1

for the (OH) band as (internal standard) to correct

for film thickness or differences in chitosan powder concentration. 1.33:

Correction factor in case of (A1655/A3450) for fully N-acetylated chitosan (it reaches

zero for the fully deacetylated chitosans).

3.5.2.3. Measurement of the Protein Content:

After complete drying, the protein content % of chitin as well as each patch

product was measured according to the standard method, mentioned in section

(3.4.3.2.1) in triplicate measurements for each patch. The standard curve was built

using high BSA concentrations (1, 2, 4, 6, 8 and 10 mg/ml)

3.5.2.4. Measurement of the Ash Content (Purity):

For each chitosan patch, ash content was measured after heating the samples (in

triplicates for each patch) at 100°C for 24 h.


92

3.5.2.5. Determination of the Total Antioxidant Activity of Chitosans:

Each sample (1.3 mg) of the different polymer powder was dissolved to 10 ml with

50 mM phosphate buffer (pH 7) and added to 10 ml absolute ethanol containing

130µl linoleic acid. The total volume was then adjusted to 25 ml with distilled

water. After incubation of the closed flasks at 37°C in dark for 6 days, the degree

of oxidation was evaluated by measuring the level of ferric thiocyanate according

to (Mitsuda et al., 1996) as follows:

100 µl of the resulting oxidized linoleic acid solution were mixed with [4.7 ml

(75% ethanol), 100 µl (30% ammonium thiocyanate) and 100 µl (0.02 M FeCl2

solution, prepared in 3.5 % HCl)], stirred for 3 min and the absorbance was

measured at 500 nm. Taking dist. water as a control and α-tocopherol as a standard

substance, the capacity of each polymer to inhibit the peroxide formation in the

linoleic acid system was calculated as follows:

(%) Inhibition (Anti-oxidative capacity) = [1 - (A sample/A control)] *100

Where: Asample and Acontrol are the absorbance of samples and control, respectively.

3.5.2.6. Determination of Molecular Weights of the Chitosans:

Chitin M.W was not easily determined due to its low solubility. M.Ws of chitosans

from the two patches were determined by the following methods:

3.5.2.6.1. The viscometric Method:

(Synchro-Lectric rotational Viscometer) was used for measuring viscosities for the

solvent (1% acetic acid) at first and then for the 2 chitosan solutions of the

concentrations (0.1, 0.2, 0.4, 0.6, 0.8 and 1 %). The (Mv) value for each chitosan

type was calculated on the basis of the (MHS) equation:

Where: *ƞint: the intrinsic viscosity

*M (Mv): the polymer molecular weight

*The values of the equation constants for chitosan are (K=0.076 ml/gm and

a=0.76) (Rinaudo et al., 1993).


93

3.5.2.6.2. Gel Permeation Chromatography (GPC) Method:

The Mn and Mw values of both chitosans were measured by GPC instrument

(1100 Agilant) equipped with GPC-SEC start up kits with a flow rate of 2 ml/min,

maximum pressure 150 bar, minimum pressure 5 bar, injection volume 50µL and

column temperature thermostat 25°C. The eluent was monitored by a refractive

index detector of optical unit temperature 25°C and peak width 0.1 min. The

molecular weights were determined from calibration curve with polyethylene

oxide standards and the method used for determination followed (Liu et al., 2006;

Li and Liang, 2007).

3.6. Method of Preparing the (Alginate-Chitosan PECs Hydrogels):

For future references, Alginate will be referred to as (Alg) and Chitosan as (Ch).

Homogeneous chitosan solution (Ch-1, 0.4 %, pH 5) was prepared in 0.5 % acetic

acid. Alginate solutions (of different ratios of alginates, 0.4 %) were prepared in 10

mM NaCl with adjusted pH at 6.84.

For preparation of PECs Films: All polymers solutions were fresh to avoid any

intrinsic changes in them with time and they were autoclaved before using. The

used volume of alginate solution was 25 ml and chitosan solution was added

dropwise with continuous gentle shaking to the resulting mixture till ending the

reaction of coacervation with formation of PECs. For preparation of the (semi-

IPNs), CaCl2 solution (700 µl) was added dropwise with continuous shaking. After

ending the reaction, selected viscous suspensions were kept aside for some time till

the removal of air bubbles then casted into glass Petri dishes and allowed to dry at

40oC for 48 hours. The dried gels were then stored in polyethylene bags under

nitrogen atmosphere at R.T and sterilized by (γ-radiation) from (Co60

irradiation

source) at (25 KGy) with a dose rate (5.2 KGy/hour).


94

3.7. Major Steps for Choosing the Best Type of Hydrogels:

3.7.1. In Vitro Swelling of Hydrogels in Simulated Wound Fluids:

The kinetics of swelling for the different formulations was characterized in the

terms of % weight gain/loss at different time intervals using conventional

gravimetric procedures (Berens and Hopfenberg, 1978; Park et al., 2001). After

drying to a constant weight in a desiccator, each gel formulation was divided into

weighed pieces (1cm×1cm); all weights were within the range (0.06-0.08 gm) and

each one was placed into a well of a multi-wells plate containing (3 ml of the

prepared PBS, pH 7.4) representing a simulated wound fluid, covered to minimize

water loss by evaporation and incubated at 37oC. After 30 min, they were

withdrawn, soaked with tissue paper, kept in desiccator till getting a constant

weight and each one was weighed separately. The same steps were repeated with

periodic monitoring of pieces weights at the time intervals (1,2 ,4, 8, 12 and 24 hrs)

with the continuous medium changing. Three individual experiments were

performed at each time point with each gel with gaining the average values. The

fold swelling of the different formulations was calculated from the following

equation according to (Yeole et al., 2006):

Degree of Swelling % = [(Ms/Md)-1] x 100

Where: Ms = Mass of swollen film at time t.

Md = Mass of dried film at (t=0).

3.7.2. Stability Characterization Studies:

As a rapid test for strength of the prepared gels, a part of each prepared

formulation gel was tested for its physical stability by weak shaking of a small

piece (2 cm ×2 cm) from each for 60 seconds with visual establishing of the gels

integrity.

3.7.3. Blood Compatibility Tests:

The chosen hydrogels were tested for their hemocompatibility as follows:


95

3.7.3.1. Thrombus Formation Test:

This was judged by the blood clot formation test (Mishra and Chaudhar, 2010).

Each dried hydrogel film (2 gm) was equilibrated with 2 ml saline water and

incubated in a water bath (37oC) for 24 hrs. To each swollen hydrogel, 500 µl

Human Citrated Blood (HCB) were added, followed by 30 µl CaCl2 solution (4 M)

to start the thrombus formation with incubation at 37oC for 5 min. The reaction

was terminated by adding 4 ml dist. water to each sample and soaking at R.T for

10 min. The clots were fixed with 2 ml formaldehyde solution (37 %) for another

10 min., placed then in water for more 10 min and the weights corresponding to

the different gels and glass surface were recorded after drying.

3.7.3.2. Hemolysis Assay:

According to (Mishra and Chaudhar, 2010), 2 ml (0.9 % w/v) saline water were

added for each dry hydrogel film (2 gm) and incubated in water bath (37oC) for 24

hrs. To each swollen hydrogel, 250 µl (HCB) were added, mixed well and

incubated at 37oC for 20 min. 2 ml saline were added to each film for reaction

termination with incubation at 37oC for 60 min. Positive and negative controls

were obtained by adding 250 µl of (HCB) and saline solution, respectively to 2 ml

distilled water. The incubated samples were centrifuged for 45 min and absorbance

of the supernatants was recorded at 545 nm using a spectrophotometer. Each

absorbance data point was obtained by measuring three samples with determining

the deviations of the three tests. The percentage of hemolysis was calculated from

the following equation (Thomas, 1996):

Hemolysis (%) = [(Atest sample – A (-) control)/ (A (+) control – A (-) control)] x 100 where:

A is the absorbance.

3.7.4. Rate of Evaporation of Water from Gel:

The chosen dried hydrogels were kept at 37oC and 35 % relative humidity with

measuring their weights at regular intervals of time (Balakrishnan et al., 2005b).


96

Three specimens were used at each time point for each gel film. The following

equation was used to find out the weight remaining (%):

Hydrogel weight remaining (%) = (Mt/Mo) × 100

Where: Mo and Mt are initial mass and mass after time‘t’, respectively.

3.7.5. In vitro Degradation of the Prepared Hydrogel Films:

The chosen dried hydrogel samples were weighed and incubated under sterilized

conditions in 50 ml PBS at 37oC with changing the medium each 2 days. Gel

degradation behavior was monitored by measuring the dry weight loss of the gels

at predetermined time points after keeping in a desiccator for 1 day (Hian, 2009).

Three Specimens were used with each weight at each time point for each film to

obtain the degradation curve. The mass loss % was calculated from the following

equation:

Mass loss %= (Mi -Md) / Mi ×100

Where:(Mi& Md) are the initial mass and mass of degraded gel, respectively.

3.7.6. Primary Skin Irritation Test for the Hydrogels:

This is a characterizing test for properties of the prepared hydrogel and its irritation

effects were compared with gels before irradiation for only confirming the test

results. According to the (NIH Publication No. 85-23, 1996), six male rats

weighing (180-200 gm) were lightly anesthetized with diethyl ether; the dorsal

surface skin was completely shaved and cleaned with alcohol (70 %). The rats

were then returned to their cages; each one in individual cage for 24 hours to allow

any edema caused by shaving to recede. Application fields were outlined on the

right dorsal side of each rat with marking pen and single full thickness wound of

area (1 cm2) was created by circular excising in skin of the dorsum with forceps

and scissors. The wounds in 3 of the rats were covered with cut pieces of the

irradiated hydrogel membrane but the other 3 with unirradiated pieces and fixed

with plasters.


97

The shaved skin on the left side remained intact but also covered with the

corresponding type of gel on each rat. Rats were returned to their cages and the

plaster covers with the hydrogel remnants were removed from all wounds after 4

hours and examined for the irritation signs, edema and swelling after 1, 24, 48 and

72 hours. Effects of the tested hydrogels were scored according to the attached

grading system as reported in (Procter and Gamble, 1977) for calculating the

Primary Dermal Irritation Index (PDII) corresponding to each hydrogel from the

following equation:

PDII= (sum. of averages (1, 24, 48, 72 hrs))

(Number of observation periods times (4))

3.7.7. Testing the Optimum Composition of Hydrogel:

The following tests were carried out to reach the optimum composition of the

prepared hydrogel and its optimum preparation conditions:

3.7.7.1 Detection of the best concn. for the used cross-linker (CaCl2):

This test was applied to the two formulae (F-18, 20) using the same swelling

procedures, stated in section (3.7.1). Three different concentrations of CaCl2 (1%,

2%, and 2.5%) were used with each one and the swelling degree was calculated

after 4 hours of immersing in PBS (pH 7.4) at 37oC from the following equation

taking (n=3) for each concentration of each film.

Q %=(Ms/Md) ×100.

Where: Ms = Mass of swollen film at time t.

Md = Mass of dried film at (t=0).

3.7.7.2. Characterizing the Effects of (γ-irradiation) on Hydrogel (F-20):

3.7.7.2.1. Testing the Bacterial Growth with the Irradiated Hydrogels:

3 gel pieces (1cm×1cm) were taken from unirradiated gel samples and randomly

chosen irradiated hydrogels, placed aseptically in 24 multi-well plates; each in a

well with adding sufficient FBS nutrient medium amount. The plate was shaken to

achieve complete immersion of the pieces and better contact with the medium.


98

All wells were covered with para -film and the plate was covered, incubated at

37oC for 2 weeks and observed then with a light microscope. Interpretation of the

results was done then by turbidimetric analysis for the immersed gels; the cloudy

medium indicated the proliferation of bacteria and the clear one indicated the

growth absence.

3.7.7.2.2. Hemocompatibility Assays:

Both the anti-thrombogenic potentials of the hydrogel surfaces and hemolysis %

were measured for the irradiated and un-irradiated hydrogels of (F-20 and 5) as

previously described in the section (3.7.3).

3.7.7.2.3. Swelling Study for the Irradiated and Unirradiated gels:

The irradiated formulae (F-20 and 5) were tested using the same previous swelling

steps as in section (3.7.1). Their swelling behaviors were compared with the

corresponding unirradiated gels (F-20(un)& 5(un)). The CaCl2 concentration was

(2 %) and the (D.S) was recorded after immersing in PBS (pH 7.4) at 37oC for 24

hours (n=3 for each time point of the gel). (F-5) was used to confirm results of the

test.

3.7.7.2.4. (FT-IR) Spectroscopic Characterization:

Experimental spectra for the prepared irradiated and unirradiated (F-20) films were

obtained by mixing the dried film with KBr. Pellets of the alginates powder as well

as physical mixture of chitosan and the used alginates (1:1) were also obtained on

mixing 2 mg with 98 mg KBr, pressed with hydraulic pressure at 13 tons and the

disks were examined with (JASCO FT/IR 6100 type A spectrophotometer). The

spectra were recorded as (% T) at 32 scans and resolution of 4 cm-1

in the range

(500-4000 cm-1

).


99

3.7.7.2.5. Scanning Electron Microscopy (SEM):

Representative air dried samples from unirradiated and irradiated PEC sheets were

taken and analyzed by (SEM). The films were sectioned with a blade, mounted on

an aluminum sample support by means of a conductive and double-sided adhesive

tape before sputter-coating with an ultra thin gold layer (300 Ao) under vacuum in

a Polaron E-coating apparatus. The Morphologies of films' surfaces were then

analyzed using a (JEOL-JSM-5400 Scanning Electron Microscope, Japan) at

accelerating voltage (30 KV).

3.7.7.3. Choosing the Best Working Film Structure:

3 different forms of the formula (F-20) were prepared with changing the ratio

between (A& B and D chains) according to the following composition:

(F-20/I): [A+B+D (2:1:1) + (CaCl2)] + (Ch).

(F-20/II): [A+B+D (1:2:1) + (CaCl2)] + (Ch).

(F-20/III): [A+B+D (1:1:2) + (CaCl2)] + (Ch).

Their properties were compared to reach the structure of the best working film by

application of the following tests:

3.7.7.3.1. Swelling Studies:

With the same previous swelling steps in section (3.7.1) using CaCl2 (2%) as a

cross-linker, the swelling degrees for these forms were recorded after immersing in

PBS (pH 7.4) at 37oC for 24 hours (n=3).

3.7.7.3.2. Rate of Evaporation of Water from Gel:

The chosen hydrogels (F-20/I & F-20) were kept at 37oC and 35% relative

humidity. Their weights were measured at regular intervals of time and the weight

remaining % was calculated from the equation stated in section (3.7.4) (n=3).


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3.7.7.3.3. In vitro Degradation of the Prepared Hydrogel Films:

The dried hydrogel samples were weighed and incubated under sterilized

conditions in 50 ml PBS at 37oC with changing the medium each 2 days. The gel

degradation behavior was calculated by the equation stated in section (3.7.5) (n=3).

3.7.7.3.4. Capability of drugs loading to the hydrogel forms (F-20, 20/I):

Two Hydrogel films of each form (F-20 and F-20/I) were prepared. One of each

contained a model protein, (BSA) representing a high molecular weight drug and

the other 2 preparations contained (Curcumin) representing a low molecular weight

drug:

3.7.7.3.4.1. Encapsulation of Albumin into the Hydrogel Network:

10 ml BSA (0.5 %) were mixed with alginate solution and the same steps of

preparing the PEC hydrogels were performed as detailed in section (3.6).

3.7.7.3.4.2. Encapsulation of Curcumin into the Hydrogel Network:

10 ml Curcumin (0.5%) were mixed with chitosan solution with repeating the same

steps of PEC hydrogels preparation in section (3.6).

3.7.7.3.4.3. Scanning electron microscopy for the prepared loaded films

To indemnify the ability of drug loading, representative air dried samples from

irradiated and un-irradiated PEC sheets loaded with Curcumin or BSA were

investigated through analyzing morphologies of the films surfaces with (SEM)

according to the procedures stated in section (3.7.7.2.5).

3.8. Statistical Analyses:

In all assays concerning the quantification of impurities, modification of alginate,

characterization of the prepared chitosans and the steps of characterizing the

hydrogels properties, triplicate treatments were tested individually with each.


101

The SPSS Computer program (Version: 14) aided in statistical analysis of the

results. Data were analyzed using one way Analysis Of Variance (ANOVA)

followed by post Hoc LSD test. The data were expressed as (Mean ± Standard

Error). Differences were considered Statistically Significant at (p≤ 0.05) and

Highly Significant at (p≤ 0.01).


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3.9. Wounding and Wound Healing Assessment:

3.9.1. Animals:

The care and use of laboratory animals were cooperated in accordance with the

Ethics Committee of the National Research Center, conformed to the “Guide for

the Care and Use of Laboratory Animals” published by the U.S National Institute

of Health (NIH Publication No. 85-23, 1996). (160) adult male albino rats (Rattus

norvigicus); purchased from the animal farm of the Egyptian Holding Company

for Biological Products and Vaccines (VACSERA), Giza, Egypt, were used in the

present investigation. Their body weights ranged from (120-150 gm) and were

acclimatized to laboratory conditions for one week under well-ventilation

conditions in the animal house building of the National Center for Radiation

Research and Technology (NCRRT) with a 12:12-hour light dark cycle and had

free access to food and water prior to initiation of the study.

3.9.2. Wounding Procedures:

Before any step, approximate fasting glucose for blood samples from tail veins of

all rats was determined with (glucometer& reagent strip, BIONINE GM-100,

China) to make sure all rats; under the study, are not diabetic. An excision wound

model was created according to (Morton and Malone, 1972) method. At first, the

rats were lightly anesthetized with diethyl-ether; the dorsal surface skin was then

digitally shaved completely and cleaned with alcohol (70 %). The rats were

returned to their cages; each one in individual cage for 24 hours to allow any

edema, caused by shaving to recede. Application fields were outlined then with a

marking pen and single right full thickness circular wound of area (1 cm2) was

created by circular excising in the dorsal skin and the underlying (panniculus

carnosus) using toothed forceps and scissors. The day of skin excision was defined

as (D 0) and the animals were randomly assigned into the following three groups:


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Group (1: Dressing/ Dre-group): Composed of 75 rats. Immediately on excision,

the wound was covered with the chosen hydrogel cut pieces. The PEC film

adhered well to the moist wound, but additional adhesive material was used to

prevent its removal by the rat itself. The wounds were monitored daily with

removal of the old hydrogel residues and redressing it approximately each 2 days.

Group (2:Fusidin/Fus-group):Composed of 10 rats. The wound was treated with

topical application of a commercial cream; fusidin, 2 times per day.

Group (3: Control/ Con-group): Composed of 75 rats serving as untreated

controls. The wounds were only disinfected with ethanol and kept opened.

3.9.3. Wound and Skin Assessment:

The involved methods for assessment of the potential wound healing activity of the

chosen hydrogel and in the other groups were as follows:

3.9.3.1. Monitoring the Visible Changes in Wounds during Healing:

Daily, the wounds of all groups were grossly monitored for infection and rats with

its signs were excluded. Photographing of the wounds was performed during the

post wounding days from a standard height to monitor any changes during healing

using a digital camera (Samsung, South Korea).

3.9.3.2. Measurement of Residual Wound Area:

By using a previously standardized tracing paper and thin marker method for

tracing wound healing (Thomas and Wysocki, 1990), all wounds margins were

drawn daily beginning with (D 0), then the paper pieces were weighed and surface

areas of the wounds at their continuous healing days were calculated. The %

reduction in area with respect to that of (D 0) and the original wound dimensions

was calculated according to the equation:

% Reduction= (SAi-SAc) ×100/ SAi Where: SAi: The wound Surface Area at (zero day) (D 0).

SAc: The wound Surface Area, currently.


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3.9.3.3. Histological Studies:

I-Histological Stains:

The most commonly used stains are (Haematoxylin (H) and Eosin (E)).

Haematoxylin stains cell nuclei and other negatively charged structures(e.g.,

Alginate) blue-black while Eosin stains cytoplasm and the connective tissue fibers

in varying shades of pink, orange and red (Bancroft and Gamble, 2007).

Generally the (H& E staining) was used to assess the skin maturity by

demonstrating the inflammatory response at the wound area, dermal closure,

remodeling and general tissues architectures while advanced granulation tissues

formation as well as tissues remodeling were assessed via Masson`s-Trichrome

(MT) staining. Collagens are the most abundant component of ECM in most

tissues including the dermal layer and they are strongly stained with the acid dyes

due to the affinity of cationic groups of these proteins for the anionic reactive

groups of these dyes. Collagen fibers of different thicknesses are colored

differently.

II- Samples Preparations:

The wound tissues including the scab and complete epithelial margin for the

control (G:3) as well as the dressed wounds with hydrogel remnants (G:1) were

rapidly extirpated during different post-wounding days with surrounding skin

tissue at the edges and a supporting layer of body wall musculature as a mass. 10%

(v/v) formal-saline was prepared by dissolving 10 ml formaldehyde (37 %) in 90

ml saline solution (0.9%) and used to fix 3D samples for 24h at R.T. Acetone was

used then to fix cell monolayers for 30 min at R.T and 3D samples were

transferred to Cell Safe biopsy inserts (CellPath Ltd, UK) that were placed in

cassettes, then processed by progressive dehydration in graded ethanol and

immersed in xylene using a Citadel 1000 Tissue Processor.

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The samples were then transferred to liquid paraffin (Tissue-Tek III Embedding

Wax, Sakura, UK) and embedded in paraffin using Tissue-Tek TEC Tissue

Embedding Console System. They were heated to 60°C (4.5 min), then immersed

in xylene (1 min), IMS 99 (40 sec), 95% IMS 99 (40sec), rinsed in tap water (20

sec), immersed in Haematoxylin (2.5min), rinsed in water (20 sec), immersed in

0.3% w/v acetic acid in dist. water (20 sec), 0.3% HCl in 70% IMS 99, rinsed in

water (20 sec), immersed in tap water substitute (40 sec), washed in water (20 sec),

immersed in Eosin diluted 1:1 with 70% IMS 99 (20 sec), rinsed in water (20 sec),

immersed in IMS 99 (1 min) and xylene (50 sec). Following this, the samples were

held in xylene for up to 4h. Coverslips were then mounted on the slides and viewed

with a light microscope. As part of the histological evaluation, all slides were

examined by a pathologist without knowledge of the group with treatment.

3.9.3.4. Quantification of RNA Corresponding to (VEGF and vWF):

In rats of the 1st and 3

rd groups, the expression of VEGF and vWF genes has been

quantified by the real-time PCR method (Isaksson and Nilsson, 2006, Benoy et

al., 2006; Graham et al., 2006). Specificity of the amplicons was verified by

building the melting curves for the PCR products. The extraction kit (Fermentus,

Thermo Fisher Scientific Inc, UK) was used to extract RNA from tissue and blood

samples according to the manufacturer’s protocol based on the (Chomczynski and

Sacchi, 1987; Boom et al., 1990) methods.

I. RNA Extraction from Wound Tissues:

Step (A): Cell Lysate Preparation

1-At the post-wounding days 1, 2, 3, 5, 7, 11, 15, 21 and 28, about 30 mg of fresh

tissue were excised from the subcutaneous area of both control and dressed

wounds. The harvested intact pieces were disrupted by grinding in liquid nitrogen

thoroughly with mortar and pestle till becoming powder.


106

2-The tissue powder was transferred immediately into a microcentrifuge tube (1.5

ml) containing 300 µl of the working buffer, and then mixed thoroughly with

vortexing for 10 sec.

3-600 µl diluted Proteinase K were added, vortexed to mix thoroughly and

incubated at 15-25°C for 10 min, centrifuged for 5 min. at 12000 x g and the

supernatant was transferred into new RNase-free microcentrifuge tube.

4-450 µl of absolute ethanol were finally added and mixed by pipetting.

Step (B): Binding RNA to Column:

5-Up to 700 µl of lysate were transferred to the RNA Purification Column inserted

in a 2 ml collection tube and centrifuged for 1 min at ≥ 12000 x g.

6-The flow-through was discarded and the purification column was placed back

into the collection tube with repeating step (5) till transferring all the lysate into the

Column. The collection tube was discarded with the flow-through solution and the

Column was placed into a new collection tube.

Step (C): Column Washing:

7-700 µl of (Wash Buffer 1) were added to the RNA Purification Column and

centrifuged for 1 min at ≥ 12000 x g with discarding the flow-through and the

Column was placed back into the collection tube.

8-600 µl of (Wash Buffer 2) were added to the RNA Purification Column and

centrifuged for 1 min at ≥ 12000 x g with discarding the flow-through and the

Column was placed back into the collection tube.

9-Washing with the same buffer (250 µl) was repeated and the Purification

Column was transferred to a sterile 1.5 ml RNase-free microcentrifuge tube after

discarding the collection tube containing the flow-through solution.


107

Step (D): RNA Elution:

10- 40 µl of nuclease-free water were added to the center of the RNA Purification

Column membrane and centrifuged for 1 min at ≥ 12000 x g to elute the RNA. The

purification column was discarded then and the purified RNA was used for the

synthesis of (cDNA).

II. RNA Extraction from Blood:

1-Blood cells were collected by centrifugation of 0.5 ml of fresh whole blood

(containing EDTA), collected from the studied rats of groups (1 and 3) at 400 x g

for 5 min at 4ºC to generate a pellet of about 60-70% of the total sample volume

after removal of the clear supernatant (plasma) with a pipette. The pellet was

resuspended in 600 µl of the working Lysis Buffer and pipetted to mix thoroughly.

2-450 µl of absolute ethanol were added and mixed by pipetting. The same RNA-

extraction steps from tissue were repeated then beginning with the step of binding

RNA to the column till getting RNA ready for cDNA synthesis.

III. Synthesis of (cDNA) via Reverse Transcription:

cDNA was synthesized from RNA extracted from both tissue and blood samples

after quantification of their concentrations with nano-dropper according to the

instructions of cDNA synthesis kit (Fermentus, Thermo Fisher Scientific Inc, UK)

(Wiame et al., 2000) as follows:

Step (A): The following components were mixed together in a chilled thin-walled

reaction tube which then incubated at 70oC for 5 min:

Sterile H2O…………………………………………….............................(6 µl).

Random Hexamer (400 ng/ µl)…………………………..........................(1 µl).

RNA (50 ng/µl).…........................................................………………..(5 µl).

Drop of oil………………………………………………………………(40 µl).


108

Step (B): The following components were also mixed in thin-walled reaction tube,

placed on ice and incubated at (37oC for 5 min) then at (25°C for 5 min):

Reaction Buffer X……………………………………………………….(4 µl).

dNTPs mix ……………………………………………….........................(2 µl).

RNAse inhibitors………………………………………….. ……………(1 µl).

Step (C):

1 µl of the Reverse Transcriptase enzyme (RT) was added to each mixture and

incubated at (42oC for 1 hour) then at (70

oC for 10 min). Negative RT reaction was

conducted according to the previous steps without using the (RT enzyme) to make

sure that there are no DNA-contaminants with the extracted RNA.

IV. The real time Polymerase Chain Reaction (r.t. PCR):

PCR amplifications for VEGF and vWF cDNAs were accomplished in (Rotor -

Gene 2000 real-time fluorescence thermal cycler (Corbett Ltd., Australia)) with a

heated lid (105oC) based on the programs shown in (table: 3). Each cDNA

fragment was amplified in duplicate for each gene as well as β-actin. All primers

were purchased from (Jena Bioscience, Germany). VEGF primers were designed

according to (Gomez et al., 2002) to amplify a region common to all VEGF

isoforms in the tissues cDNAs while the intron spanning primers for vWF of blood

cDNAs were according to(Bu et al.,2010

PCR Mastermix per sample was prepared as follows:

1-DNase-free water……………………………………….……………(7.5 µl).

2-Primer (F) and Primer (R) (0.25 Pmol/µl)……...…(1 µl from each primer).

4-cDNA Sample………………………………………………………..(3 µl).

5-SyberGreen mix………………………………………….……….(12.5 µl).

The (β-actin) expression as a housekeeping gene was normalized between samples

in each set, so that the relative expression of genes of interest could be established.


109

(Table: 3) summarizes the oligonucleotide sequences of VEGF, vWF and β-actin

primers.

Table (3): Primers sequences, expected product length and PCR programs for

amplification of (β-actin, VEGF and vWF genes):

Each gene was amplified in a separate well. Fluorescence measurement at the end

of elongation step of every PCR cycle was performed to monitor the increasing

amount of amplified DNA. The relative expression of the real-time PCR products

and the fold change in the targets genes were determined by the (ΔΔCt method)

(Livak and Schmittgen, 2001; Zhang et al., 2013). The second derivative

maximum (Ct) for VEGF, vWF and β-actin RNAs was determined from the

amplification curves and the fold change in VEGF and vWF genes was expressed

as delta Ct (ΔCt) with respect to (β-actin RNA). (ΔΔCt) was calculated from the

difference between (ΔCt) of each wound (undressed and treated with the dressing)

during the different healing days and the corresponding (ΔCt) of the first healing

day in each group. This expresses the change of VEGF and vWF mRNAs in the

different healing days comparing with the mRNAs during the first healing day

(Fleige et al., 2006). For verifying the amplification specificity and distinguishing

any artifacts from the specific amplicons, melting curves were generated by

denaturing the PCR products by slowly increasing the temperature from (65-99°C)

with the rate (0.1oC/sec).


110

Optical data were collected throughout the duration of temperature drop with

dramatic increase in fluorescence seen when the denatured strands on melting

reannealed. For the statistical analysis of results, every test was replicated three

times for the quantification of each gene corresponding to each time point of group

and the exponential Ct values were averaged before calculating the amplification

and the results were statistically analyzed as mentioned in section (3.8). The results

of each day were compared with the previous one in the same group as well as the

corresponding day in the other group.

3.9.3.5. Screening of kidney Functions:

Blood samples from both (Dre (1) and Con (2)-groups) of wounded rats were

collected in EDTA during the different post wounding-days, centrifuged

immediately and the plasma was stored at -20°C till further analysis. Later, the

samples were analyzed for renal function tests. A triplet sample of each group per

day was analyzed for optimal screening and the results were statistically analyzed

as mentioned in section (3.8).

3.9.3.5.1. Quantitative Estimation of (Blood Urea Nitrogen) in Plasma:

The enzymatic colorimetric method of (Tabacco et al., 1979) was the base for the

quantitative estimation of (Blood Urea Nitrogen (BUN)) in plasma using a

diagnostic kit supplied by (Diamond company-Egypt).

Principle:

The reaction involved in the assay system was as follows:

1-Sample urea is hydrolyzed enzymatically with (Urease) into (NH4+

+ CO2).

2-The formed (NH4+) ions react with salicylate and hypochlorite (NaClO) in the

presence of (catalyst-nitroprusside) to form green indophenol whose intensity is

proportional to the urea concentration in the sample.

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111

Reagents Composition:

Reagent (1): R1 (Urea standard)……………………………….. (50 mg/dl).

Reagent (2): R2 (Urease)…………………………………………(30000 U/l).

Reagent (3): R3 (Buffer): Phosphate (pH 6.7, 50 mmol/l) with NaOH (150 mmol/l)

& EDTA (2 mmol/l) & Sodium Salicylate (400 mmol/l) and Sodium Nitroprusside

(10 mmol/l).

Reagent (4): R4 (NaClO)………………………………………(140 mmol/l).

Procedures: The following components were pipetted in tubes and mixed together:

After incubation for 5 minutes at R.T, the following reagent was added:

R4 (NaClO) (µl) 200 200 200

These components were mixed together and incubated at R.T for 10 minutes and

Absorbance of the specimens (Asm) and standards (Ast) were measured against

reagent blank within 60 min after incubation.

Calculations:

Blood Urea Nitrogen (mg/dl) = (Asm/Ast) × 50/2.14

Where: The standard urea Concentration was (50 mg/dl).

3.9.3.5.2. Quantitative Estimation of (Creatinine) in Plasma:

A-Principle:

Creatinine was quantitatively estimated in plasma according to (Young and

Friedman, 2001) based on Jaffe colorimetric-kinetic method using a diagnostic kit

supplied by (Diamond company-Egypt).

Reagents Blank Standard Sample

R3 (Buffer) (ml) 1 1 1

R2 (Urease) 1 drop 1 drop 1 drop

R1 (Standard) (µl) - 10 -

Sample (µl) - - 10


112

Creatinine reacts with (alkaline sodium picrate) forming a red complex whose

intensity is proportional to the creatinine concentration in the sample.

B-Reagents Composition:

Reagent (1): R1 (Creatinine Standard)…………………………..(2 mg/dl).

Reagent (2): R2 (Picric acid)………………………………… (17.5 mmol/l).

Reagent (3): R3 (Alkaline reagent, NaOH)………………. …..(0.29 mmol/l).

C-Procedures:

1-The working reagent was prepared by mixing equal volume of (R2 and R3)

2-The following components were pipetted into cuvettes then mixed:

Reagents Blank Standard Sample

Working Reagent (ml) 1 1 1

Standard (µl) - 100 -

Sample (µl) - - 100

The absorbance (A1) after (30 sec) and (A2) after (120 sec) of sample addition was

taken with a spectrophotometer adjusted to zero with the Blank at (λ=492 nm) and

R.T.

Calculations:

ΔA=A2-A1

Creatinine concentration (mg/dl) = (ΔAsample - ΔABlank)× 2

(ΔAstandard – ΔA Blank)

Where: The standard creatinine concentration was (2 mg/dl).

Results

113

4. RESULTS

4.1. Characterization of the Modified Alginates:

4.1.1. Test for Aldehyde Groups:

The oxidized MASA samples formed red precipitates on reacting with the

Fehling reagent but no precipitates were formed with the raw alginate.

4.1.2. Fourier Transform-Infra Red Spectroscopic Characterization:

The FTIR charts for both the irradiated (VLMW-50 KGy) and the oxidized

alginate (MASA-I A) are only shown in (fig. (16)) due to their importance in

preparation of the gel membranes. Spectrum of the irradiated alginate shows

conservation for most bands of its native alginate with little differences. The

oxidized alginate spectrum shows some differences from the native form due

to the introduction of new functional groups including the aldehyde groups.

4.1.3. Degree of Aldehyde Substitution:

On expression of the results, the oxidized alginates (MASA-I, and MASA-II)

were denoted according to the concentrations of Na-periodate (0.25, 0.5, 0.75

and 1 M) to be (A, B, C and D), respectively. (Table: 4) displays the aldehyde

analyses for the different oxidized as well as non oxidized alginates. There

was no significant difference in the degree of aldehyde substitution of

(MASA-I A (p=0.434) and MASA-II A (p=0.175)) versus the raw alginate but

differed significantly versus the other oxidized alginates (p≤0.05).

4.1.4. Average Molecular Weights Measurements:

The values of (Mv) according to the calculations of (MHS-equation) for the

raw, irradiated and oxidized alginates as well as the values of Mn and Mw

according to the (GPC-method) are summarized in (table: 5).

Results

114

Figure (16): FT-IR spectra for the Irradiated and Oxidized Sodium Alginates:

(A: 3444 cm-1

, B: 1298 cm-1,

C: 1090 cm-1

, D: 3450 cm-1

, E: 1715 cm-1

, F: 790 cm-1

,

G: 2931 cm-1

, H: 1623 cm-1

, I: 1419 cm-1

).

Table (4):Aldehyde analyses Table (5): Average Molecular Weights

for the different alginates for the different alginates:

(Formyls/ Mol. of alginate) (n=3):

(HMW-Alg= A & LMW-Alg (50 KGy) =A` & LMW-Alg (100 KGy) =A`` &

VLMW (50 KGy) =B & VLMW (100 KGy) =C & MASA-I A=D & MASA-II A

=E) (The letters: A`, A``, B, C, D, E denote the symbols of the different alginates

for their subsequent usage in preparation of the hydrogels)

Results

115

4.2. Alginate purification:

4.2.1. Testing the Purification of Activated Charcoal:

Fig. (17) shows the I.R chart for the raw activated charcoal and different acid

treated charcoals and (Appendix (A)) shows the corresponding peaks tracking.

The HCl treated charcoal was the only one who conserved most bands of the

native type.

Figure (17): FT-IR Spectra showing the changes within the activated charcoal

after washing with different acids.

4.2.2. Characterization of the Purified Alginates:

4.2.2.1. Dynamic Viscosity Measurements:

Dynamic viscosity values for the raw and purified (HMW-Alginates) were (62

± 1 and 68 ± 2 cPs, respectively). The viscosity values for the raw and purified

(VLMW-Alginates) were (25 ± 1 and 28 ± 1 cPs, respectively).

4.2.2.2. Quantification of the Impurities Concentrations:

The values of proteins and polyphenols impurities for the raw HMW and

VLMW-Alginates as well as their purified forms only are illustrated in

(figures 18 A, B and C) due to their importance in the following steps:

4.2.2.2.1. Protein Content Quantification:

Fig. (18 A) shows a significant difference in protein concentrations in each

purified alginate versus its raw type.

Results

116

4.2.2.2.2. Polyphenol-like Compounds Content Quantification:

Fig. (18 B) displays the efficiency of polyphenols purification for the

different alginates as a function of the corresponding (AFU). Significant

difference was observed in each purified form versus the raw type. Fig. (18

C) shows a decrease in the AFUnits corresponding to the peak at 445 nm

with repeating the purification step with Sevag reagent. Neither the (HMW

(p-2) nor (VLMW (p-2)) reached the value of (AFU) for blank solvent (3.9).

Results

117

Figure (18): Quantitative evaluation of the major alginate contaminants

before/ after purification: (HMW (Raw): Unpurified High Molecular Weight

Alginate & VLMW (Raw): Unpurified Very Low Molecular Weight Alginate &

(p-1): Product after one purification step & (p-2): Product after two purification

steps. (A): Protein content measured by the Bradford assay; the results are

represented in (mg proteins/ gm Alginate) (n=3). Bars show means and SEM.

(B): Polyphenols content represented in arbitrary fluorescence units (AFU)

(n=2) detected by the appearance of a characteristic absorbance peak at 445 nm

(C). (*: p≤0.001 versus HMW (Raw), Φ: p≤0.001 versus VLMW (Raw), Ж:

p≤0.05 versus the Blank solvent).

Figure (19): FT-IR spectra of (Na-Alginate) before and after purification:

(A: 3444 cm-1

, B: 2931 cm-1

, C: 1623 cm-1

, D: 1419 cm-1

, E:1298 cm-1

, F:1095 cm-1

,G: 947 cm-1

).

Results

118

4.2.2.3. Characterization of the raw and purified alginates with(FT-IR):

Fig. (19) and (Appendix (B)) show the I.R charts for the raw and purified

alginate and the corresponding peaks tracking, respectively. Most raw alginate

characteristic bands were conserved after purification.

4.3. Characterization of the Prepared Chitosans:

4.3.1. Solubility Test:

Chitin is insoluble in most solvents. Difference in solubility between the (2)

chitosans was significant after fast shaking (p≤‎0.001)as displayed in (table:6

4.3.2. FTIR-Analysis:

Fig. (20 A) and Appendix (C) illustrates the I.R chart for (Ch-1 and Ch-2).

There was significant difference in the degrees of deacetylation between the 2

types where the calculated (DD %) for (Ch-1) was (90 % ± 0.577), but for

(Ch-2) was about (98 % ± 0.265) as shown in (figures (20 C and D)).

4.3.3. Protein Content:

The results are displayed in (Table: 6) where (Ch-2) showed significant less

protein content than (Ch-1) (p≤ 0.001).

Table(6): Properties of the prepared chitosans (DD: Degree of deacetylation):

Parameter Chitosan-1 (Ch-1) Chitosan-1 (Ch-1)

Mv (Da) 2.15 *104 2.14* 10

4

Mn (Da) 4.3*103

3.8*103

Mw (Da) 2.2*104 2.19*10

4

Solubility % 92 ± 0.577 98.5 ± 0.29

Ash content % ± SEM 1.84 ± 0.012 0.78 ± 0.012

Protein content % ± SEM 0.875 ± 0.009 0.589 ± 0.006

DD % ± SEM 90 ± 0.577 98 ± 0.265

Inhibition % ± SEM 51.5 ± 0.577 78.5 ± 1.155

Absorbance 0.613 0.272

Results

119

Figure (20): (FT-IR) spectra for the Chitosans (1, 2). (A) I.R spectra within

range (500-4000 cm-1

). (B) I.R spectra within range (1200-1800 cm-1

). (C) and (D)

show the base lines for calculating the DD for (Chitosan-2) and (Chitosan-1),

respectively and values of the corresponding (A1655 and A3450):

(A: 3450 cm-1

, B: 2900 cm-1

, C: 1620 cm-1

, D: 1545 cm-1

.

Results

120

4.3.4. Ash Content:

(Ch-2) had significant less ash content than (Ch-1) (p≤ 0.001) (Table: 6).

4.3.5. Total Antioxidant Activity of the Different Chitosan Products:

The absorbance (A) and % inhibition of the prepared chitosan samples are

summarized in (Table: 6). Absorbance for control (Acontrol) was 1.263 and for

(Ast) was 0.555 with inhibition % (56.1±1.155). Versus (α-tocopherol), there

was a significant difference in the % inhibition for both Ch-1(p=0.017) and

Ch-2 (p≥ 0.001).

4.3.6. Average Molecular Weights Measurement:

The values of Viscosity, Number and Weight Average Molecular Weights for

(Ch-1 and Ch-2) are summarized in (Table: 6).

4.4. Major Steps for Choosing the Best Type of Hydrogels:

4.4.1. In vitro Swelling of the Hydrogels& Stability characterization:

Although for some samples, Qm>1 (swelling) and for others, Qm<1 (syneresis),

all points belonging to the same gel type seemed to collapse into a single

curve. The results representing change in the degrees of swelling as a function

of time are shown in (fig. (21)); divided into 3 groups for facilitating the

comparison. Composition of the different formulae and a general overview for

the swelling and stability tests results are given in (Table: 7). The water

sorption curves for most formulae are non-linear and can be divided into two

regimes: the first one includes an exponential increase in water uptake during

the first hours of immersing in medium followed by a second one with low

amount of water absorbed to make its maximum absorption at the end of the

first regime to somewhat constant. Some curves displayed degree of syneresis

with the immersion in medium.

Results

121

Figure (21): Swelling kinetics for different formulae immersed in PBS medium

(pH 7.4) for 24 hours. (A): Curves for the formulae (F 23, 16, 21, 10, 9, 15, 2, 25,

1, 12) with 2 alginate fractions containing (A-chains). (B): Curves for formulae

(F-11, 14, 26, 4, 7, 22, 18, 8) with 2 alginate fractions containing no (A-chains).

(C): Curves for the formulae (F-13, 6, 19, 17, 32, 31, 34, 33, 20, 5, 24, 3) with 3

alginate fractions containing (A-chains).

Results

122

Table (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. (α denotes the unstable gel β, weakly stable gel, κ,

intact gel):

Alginate fraction for

each formula

Properties Alginate

fraction

Properties

1-[A+B + (CaCl2)] κ & Very low (D.S) 12-[A+B] κ & High (D.S), but

increases weakly

15-[A+C+ (CaCl2)] β & Lower (D.S) 9-[A+C] κ & High (D.S)

10-[A+D+ (CaCl2)] β & Lower (D.S) 21-[A+D] κ & Low (D.S)

16-[A+E+ (CaCl2)] κ &Lower (D.S) 23-[A+E] κ & Low (D.S)

20-[A+B+D+ (CaCl2)] κ & High (D.S) 5-[A+B+D] β & High (D.S)

19-[A+B+E+ (CaCl2)] κ & Lower (D.S) 17-[A+B+E] κ & Low (D.S)

2-[A+ (CaCl2)] κ & Very low (D.S) 25-[A] κ & Low (D.S)

13-[A+C+D+ (CaCl2)] α & Low (D.S) 6-[A+C+D] β & High (D.S)

24-[A+C+E+ (CaCl2)] κ & Lower (D.S) 3-[A+C+E] κ & Low (D.S)

7-[B+E+ (CaCl2)] κ & Lower (D.S) 22-[B+E] κ & Low (D.S)

18-[B+D+ (CaCl2)] κ & High (D.S),but

increases weakly

8-[B+D] β& High (D.S) then

Syneresis after 2hrs

14-[C+D+ (CaCl2)] κ & Lower (D.S) 11-[C+D] κ & Low (D.S)

26-[C+E+ (CaCl2)] κ & Lower (D.S) 4-[C+E] κ & High (D.S)

32-[A+B+G+ (CaCl2)] β & Low (D.S) 31-[A+B+G] β & Low (D.S)

34-[A+B+F+ (CaCl2)] κ & Very low (D.S) 33-[A+B+F] β & Degrades in the

medium

(20/1) [A+B+D+ (100

mM CaCl2)]

Β & High (D.S)

then syneresis

(20/2.5) [A+B+

D+(250 mM

CaCl2)]

κ & Less (D.S)

(18/1)[B+D+ (100 mM

CaCl2)]

β & High (D.S) (18/2.5)[B+D+

(250 mM CaCl2)

κ & Less (D.S)

20 (Irr) -[A+B+D+

(CaCl2)]

κ & Higher (D.S) 20 (un)-[A+B +

D+(CaCl2)]

κ & Lower (D.S)

5 (Irr) -[A+B+D] β & High (D.S) 5(un)-[A+B+D] β & Lower (D.S)

Results

123

Alginate fraction for

each formula

Properties Alginate fraction Properties

(20/I ) (Irr) -[A+B

+D (2:1:1) +

(CaCl2)]

κ & high (D.S) then

syneresis after 4hrs

(20/I) (un)-[A+B

+ D (2:1:1) +

(CaCl2)]

κ& Less (D.S) then

weak syneresis after

10 hrs

(20/II) (Irr)-[A+B +

D (1:2:1)+ (CaCl2)]

κ & Low (D.S) (20/III)(Irr)-[A+B

+D(1:1:2)+

(CaCl2)]

β& Low (D.S)

4.4.2. Blood Compatibility Assays for the Hydrogels:

The results obtained for the test of hemolysis of human blood with different

formulae compared with glass surface and the corresponding formed clot

weights are displayed in (Table: 8). The positive reference (100% lysis) was

blood/water mixture and the negative reference (0% lysis) was blood/saline

mixture. The OD values of positive control (A (+)control) and (A (-)control) were

1.46 and 0.008, respectively. The values of hemolysis % differed significantly

versus controls (p≥ 0.05). There were no significant differences in the

thrombogenic potentials between (F-4, 6 and 9) (p<0.05), but differed

significantly versus the other formulae (p ‏≤‏ 0.001) which also showed no

significant differences versus each other except between the 2 formulae (F-5)

and its Ca-containing formula (F- 20) (p≥ 0.05).

4.4.3. Rate of Evaporation of Water from Gel:

All hydrogels showed sharp linear decrease in water content during the first

day to reach 20 % of water remained with little differences in the following

days (fig. (22)).

4.4.4. In vitro Degradation of the Prepared Hydrogel Films:

Results for the degradation behaviors are summarized in (fig.(23)) where the

formulae (F-5 and 18) showed a faster solubilization rate than (F-20).

Results

124

Table (8): Blood compatibility parameters for different hydrogels (Results are

expressed as (Mean ± SEM):

Sample Weight of clot,

formed (gm)

Haemolysis %

(A) (%)

4-([C+E] +Chitosan)

5-([A+B+D] +Chitosan)

6-([A+C+D] +Chitosan)

9-([A+C] +Chitosan)

18-([B+D+ (CaCl2)] +Chitosan)

20-([A+B+D+ (CaCl2)] +Chitosan)

(F-20 (un))([A+B+D+(CaCl2)]+Ch)

(F-20 (Irr))([A+B+D+(CaCl2)]+Ch)

(F-5(un))([A+B+D]+Ch)

(F-5 (Irr))([A+B+D]+Ch)

Glass

0.037± 0.001

0.060 ± 0.002

0.044 ± 0.001

0.040 ± 0.002

0.070 ± 0.003

0.075 ± 0.003

0.060 ± 0.033

0.075 ± 0.003

0.020±0.0017

0.06 ± 0.002

0.08

0.127

0.07

0.091

0.01

0.066

0.046

0.081

0.046

0.1

0.07

8.2 ± 0.115

4.3 ± 0.075

5.7 ± 0.03

0.14 ± 0.092

4 ± 0.085

2.6 ± 0.003

5 ± 0.577

2.6 ± 0.003

6.34 ± 0.03

4.3 ± 0.075

100

Figure (22): Time dependence of water loss from the 3 different hydrogel

formulae (F-20, 18 and 5).

Results

125

Figure (23): Rate of degradation for the formulae (F-20, 18 and 5).

Table (9): PDI test results for the Non-irradiated Hydrogel

PDII= (5.6/4) = 1.4

Table (10): PDI test results for the irradiated Hydrogel

PDII= (1.5/4) =0. 375

Results

126

4.4.5. Primary Skin Irritation Test for the Hydrogels:

(Tables: 9 and 10) display the PDII results for the prepared irradiated

hydrogel and compared with the results of the un-irradiated hydrogel. The

PDII values for non-irradiated and irradiated dressings were calculated to be

1.4 and 0.375, respectively


4.4.6.1 Detection of the best concentration for the cross-linker (CaCl2):

The swelling degrees for the 2 gels of 3 different (CaCl2) concentrations are

represented in fig. (24) and a summary for their swelling behavior and stability

study is shown in (Table: 7). (D.S) of the 2 hydrogels falls with increasing the

used CaCl2 concn. but with no significant difference in (D.S) between the two

(F-20) forms with the CaCl2 concentrations (1 and 2 %).

4.4.6.2. Characterization of the effects of irradiation on gel (F-20):

4.4.6.2.1. Testing the bacterial growth with the irradiated Hydrogels:

The terminal applied dose (25 KGy) to the hydrogels appeared to be effective

in killing off any microorganisms within the prepared films where the

turbidity appeared with the monitored tested non irradiated gel pieces

disappeared on examining the irradiated gel pieces.

4.4.6.2.2. Hemocompatibility Assays:

The results of blood compatibility tests for the irradiated and unirradiated (F-

20 and 5) are shown in (Table: 8) with significant difference in blood

hemolysis % and clot weight between the corresponding formulae (P≥‏ 0.05).

4.4.6.2.3. Swelling Study for the Irradiated and Unirradiated Gels:

The modes of swelling for (F-20) forms as models for the (semi-IPNs) and (F-

5) forms as physical hydrogels are illustrated in (fig. (25)). A summary for the

swelling and stability studies is shown in (Table: 7). The unirradiated forms

showed less swelling rates than the corresponding irradiated ones.

Results

127

Figure (26): The influence of cross-linking agent (CaCl2) concentration on the

swelling degree for the formulae (F-18 and 20) after 4 hours of immersing in

PBS (pH 7.4) at 37oC. Bars show means and SEM.

Figure (25): Comparison of the swelling kinetics for the unirradiated and

irradiated formulae (F-20 and 5).

Results

128

4.4.6.2.4. (FT-IR) Spectroscopic Characterization:

Fig. (26) displays the FTIR-spectra for the physical mixture of alginates,

mixture of (Alg/Ch), unirradiated and irradiated PECs from (F-20) formula.

The later 2 charts showed different characteristic bands than the others.

4.4.6.2.5. Scanning Electron Microscopy (SEM):

The (SEM) images for unirradiated and irradiated PECs of both Ca-containing

formula (F-20) and Ca-free one (F-5) are shown in (fig. (27)).

4.4.5.3. Choosing the Best Working Film Structure:

4.4.5.3.1. Swelling Studies:

The 2 forms (F-20/II, 20/III) showed weak swelling unlike (F-20/I) that

showed higher (D.S) at the beginning of immersion in medium; but (Q %)

decreased after the 1st hour leaving more than 2-fold increase in mass despite

showing syneresis with time (fig. (28)).

Figure (26): FTIR spectra for the physical mixture of alginates, alginate with

chitosan, the unirradiated and irradiated PECs (F-20): (A: 2931 cm-1

, B: 1415

cm-1

, C: 1262 cm-1

, D: 1623 cm-1

, E: 1632 cm-1

, F: 1084 cm-1

, G: 1432 cm-1

, H:

3444 cm-1

, I: 2925 cm- 1

).

Results

129

Figure (27): Scanning Electron Micrographs for the surfaces of different

formulae based on (Alginate/Chitosan PECs). (A) Unirradiated (F-20, mag.

1500X), (B) Irradiated (F-20, 1500X), (C) Irradiated (F-20, 1500X), (D)

Irradiated (F-20, 750X), (E)Unirradiated (F-5, 1500X), (F) Irradiated (F-5,

1500X), (G) Irradiated Albumin-loaded (F-20/I, 2000X), (H) Irradiated

Albumin-loaded (F-20, 2000X), (I) Irradiated Curcumin-loaded (F-20/I, 2000 X),

(J) Irradiated Curcumin-loaded (F-20, 500X).

Results

130

4.4.5.3.2. Rate of Evaporation of Water from Gel:

Fig. (29) illustrates that both the 2 hydrogel forms (F-20 and 20/I) showed a

sharp linear decrease in water content after the 1st day. About 80 and 70 %

water loss was observed for F-20 and F-20/I, respectively during the 1st day,

then decreased slowly in the following days to retain about (15 and 20 %,

respectively) of water content after 7 days.

4.4.5.3.3. In vitro Degradation of the Prepared Hydrogel Films:

Fig. (30) displays the results of degradation for both the unirradiated and

irradiated forms (F-20 and 20/I). The irradiated forms showed higher

degradation rates than the corresponding un-irradiated forms and both the

unirradiated and irradiated (F-20/I) forms showed faster degradation than the

corresponding (F-20) forms

4.4.5.3.4. Scanning Electron Microscopy for the 2 Drug-loaded forms:

The (SEM) images (28 G and 28 H) represent the irradiated albumin-loaded

membrane of the forms (F-20/I & F-20, respectively) and (28 I & 28 J) are for

the irradiated curcumin-loaded membrane of the corresponding forms.

Figure (28): Comparison of the swelling kinetics for the different forms of the

formula (F-20): (20/I: [A+B+D (2:1:1) + (CaCl2)] + (Ch) & F-20/II: (F-[A+B+ D

(1:2:1) +(CaCl2)]+(Ch) & (F-20/III:[A+B+D (1:1:2)+ (CaCl2)] +(Ch)).

Results

131

Figure (29): Time dependence of water loss from the 2 hydrogel forms (F-20, and

F-20/I).

Figure (30): Rate of degradation for the unirradiated and irradiated forms (F-20

and F-20/I).

Results

132

4.5. Wounding and Wound Healing

Most rats showed normal (FBG) levels within the range (50-135 mg/dl=13.9

mmol/dl). Few of them had higher levels and were excluded from the study.

4.5.1. Gross Examination of Wounds:

Closure rate and the appearance of full thickness skin excisional wounds,

treated with the tested PEC gel films were compared with the fusidin-treated

and un-treated wounds. Fig. (31) displays that wound healing was grossly

normal with complete closure for rats of the (Dre-group) on the 11th

post-

healing day. Indeed, the growth of hair started on day 3 and its density was

significantly heavier in the center of hydrogel-treated wounds than in the

surrounding non-wounded skin area at the end of healing days with continuous

treatment. Wounds of the (Fus-group); in contrast, showed weak hair growth

for the wounded and non-wounded areas with complete closure on the

postoperative day 13, but without the signs of complete skin repair. No repair

occurred completely with the untreated wounds even after 12 post-wounding

days. Some rats of this group showed signs of infection with pus secretion and

the skin was hemorrhagic with a few of them.

Figure (31): Representative digital photographs assessment of healing

progression during the first 2 post-operative weeks.

Abbreviations: Con: Untreated wounds & Dre: Wounds treated with the

Dressing & Fus: Wounds treated with fusidin.

Results

133

4.5.2. Residual Wound Area Measurements:

The healing curves for wounds of large rats treated with the new hydrogel or

fusidin cream as well as the control wounds can fit into number of curves

representing a change in wound closure as a function of the post-wounding

days. No difference was observed in all wounds sizes during the first 2 post-

wounding days, but the significant reduction % for wounds treated with the

(Dre) was about 50 % in day 3 to reach 90 % after 6 days and about 95 % after

10 days (fig.(32)). The % reduction was only 30 % in case of wounds treated

with (Fus) and (Con) groups with no significant difference between them till

day 3 to reach about 55% after 6 days and 95 % after 13 days for the treated

wounds with significant difference than the Control wounds that showed weak

reduction in size to reach its maximum (95 %) after 2 weeks.

Fig.(33) displays 2 model curves for the reduction in area of wounds of small

rats treated with the (Dre) and a model curve for normal healing of wound.

Wounds treated with the (Dre) showed no significant change in size during the

first 2 days but rapid significant contraction was observed then till the 4th

day,

followed by a slight decrease with slight differences in wound area in the

following days to reach max wound size reduction in the 11th

or 13th

day. The

normal wound closure took place slowly with, to somewhat, equal rate from

each day to the following one reaching maximum level after 16 days.

4.5.3. Histological Examinations for Wounds Tissues:

Fig. (34) shows the histological images for the control and dressed wounds

tissues during the post wounding days (1 and 3) and stained with (H, E and

MT stains). The changes occurred during the post-wounding day (7) and days

(11& 15) are illustrated in (figures (35& 36)), respectively. Fig. (37) displays

the last healing day for the stained dressed wound (Dre-group) with (H, E and

MT).

Results

134

Figure (32): Rate of closure of open wounds in large rats, treated with the

prepared dressing, fusidin cream and the untreated wounds. (ᶑ: p≤0.05 for the

dressed wound versus control, ᵳ: p≤0.05 for the fusidin-treated wound versus

control).

Figure (33): Comparison of the healing models between dressed and undressed

wounds in small rats. (ᶑ: p≤0.05 for the dressed wound versus control).

Results

135

Fig (34): Histology of wounds sections stained with Hematoxylin (H), Eosin (E)

and Masson`s Trichome (MT) under polarized light after 1 and 3 days of

wounding. (a) Day(1): Dre (H,E, mag. 100X)& (b) Day(1): Con (H,E, 100X)& (c)

Day(1): Dre (H,E, 200X)& (d) Day(3): Con (MT, 200X).

Abbreviation: (EGT): Early Granulation Tissue.

Figure (35): Representative images of (H, E and MT) histologically stained

wounds sections. (a) Day(7): Dre (H,E, mag. 200X)& (b) Day(7): Con (H,E,

200X)& (c)Day(7): Dre (MT, 200X). Abbreviations: (SSCs): Stratum Spinosum

Cells, (SGCs): Stratum Granulosum Cells, (SCCs): Stratum Corneum Cells.

Results

136

Figure (36): Representative images of (H, E and MT) stained wounds sections.

(a): Day(11): Con (H,E, mag. 100X)& (b): Day(15): Con (H,E, 200X)&

(c): Day(15): Con (MT, 200X) & (d): Day(11): Dre (H,E, mag. 100X)&

(e): Day(15): Dre (H,E, 100X)& (f): Day(15): Dre (H,E, 200X)&

(g): Day(11): Dre (MT, 200X)& (h): Day(15): Dre (MT, 200X).

Abbreviation: (A.T): Adipose tissues.

Results

137

Figure (37): Representative images of (H, E and MT) stained wounds sections.

(a): Day (16): Dre (H, E, mag. 100X)& (b): Day(16): Dre (MT, 200X).

Abbreviations: (HF): Hair Follicle, (SGl): Sebaceous Glands, (BCs): Basal Cells,

(SwG): Sweet Gland, (DSwG): Duct of Sweet Gland.

4.5.4. Quantification of mRNAs by real-time PCR:

There was no statistical significant difference in VEGF mRNA of dressed

excision wound between 2nd

and 3rd

days (P0.05>‏), but the following healing

days showed significant increase in the mRNA (P>0.001); each one versus the

previous day till the 7th

day where they began to decrease significantly (P>

0.05) (fig.(38)). For the non-dressed wounds, VEGF mRNA significantly

decreased during the first 3 post wounding days; each one versus the previous

day (P> 0.001), then no significant difference was observed till the 7th

day (P

<0.05) where it began then to increase significantly reaching its maximum

level during the 11th

day (P> 0.001) followed by a sharp decrease during the

following days. Tissues from the dressed wounds showed significant higher

levels of VEGF mRNA versus their corresponding non-dressed wounds (P>

0.05) at different time points except for the last healing days (28 C and 28 D)

where the difference wasn`t significant (P‏ < 0.05). The amplification curves

for VEGF and β-actin cDNAs of both treated and untreated wounds (Days: 5th

,

11th

and 21th

) are displayed in (fig. (39)). Single sharp melting curves for the

PCR products are illustrated in (fig. (40)) indicating the homogeneity of these

products.

Results

138

Figure (38): VEGF mRNA quantification by rt-PCR during the healing days of

both Dressed (D) and non-treated Control wounds (C). Bars show means and

SEM. (Ψ: P≤ 0.05 for each dressed wound versus the undressed one at the same

day & Ω: P≤ 0.05 for each dressed wound versus the previous day& Ϫ: P≤ 0.05

for each undressed wound versus the previous day).

Figure (39): Amplification curves for the Quantitative real time PCR of VEGF

and β-actin cDNAs from both the 2 wounding groups (Dressed wounds group (D)

and control untreated wounds group (C)).

Results

139

Figure (40):Melting curves for the PCR products of VEGF cDNA amplification

from wounds of both (Dressing group (D) and control untreated wounds group

(C)), built by their denaturing from 65 to 99°C.

The levels of mRNA corresponding to the (vWf gene) of blood decreased for

each type of wounded groups during the first five post-wounding days without

any significant difference between days (P< 0.05) (fig. (41)). The rate of

decrease with mRNAs of the healing dressed wounded group was less than the

rate with the un-treated group and there was also no significant difference in

the mRNA levels between each 2 corresponding healing days (P<0.05).The

vWF mRNA levels began to increase by the 7th

and 11th

healing day of the un-

treated and treated wounds, respectively with significant difference between

each day and the previous one (P> 0.05). In addition, the rate of increase in the

mRNA was higher with the undressed group than the corresponding dressed

group with significant difference between each 2 corresponding days (P>

0.05). The amplification curves for vWF and β-actin cDNAs of blood in both

treated and untreated wounds groups (Days: 5th

, 11th

and 21th

) are displayed in

(fig. (42)) and the melting curves for the PCR products as in (fig. (43)) show

narrow peaks indicating their purity.

Results

140

Figure (41): vWF mRNA quantification by rt-PCR during the healing days of

both Dressed (D) and non-treated Control wounds (C). Bars show means and

SEM. (Ψ: P≤ 0.05 for each dressed wound versus the undressed one at the same

day & Ω: P≤ 0.05 for each dressed wound versus the previous day& Ϫ: P≤ 0.05

for each undressed wound versus the previous day).

Figure (42): Amplification curves for the Quantitative real time PCR of vWF

and β-actin cDNAs from both the 2 wounding groups (Dressed wounds group

(D) and control untreated wounds group (C)).

Results

141

Figure (43): Melting curves of the PCR products of vWF cDNA amplification

from wounds of both (Dressing group (D) and control untreated wounds group

(C)), built by their denaturing from 65°C to 99°C.

4.5.5. Screening of kidney Functions:

(Table: 11) displays the mean values for BUN and Creatinine in the blood of

both Control and Dressed wounds groups. Values of both are in the normal

range (for Creatinine= 0.3-0.6 mg/dl & for BUN=12-20mg/dl (Johnson-

Delaney, 1996; Quesenberry and Carpenter, 2004) with no significant

differences neither among their values with the different days within the same

experimental group nor versus the corresponding days of the other group.

Table (11): Levels of (BUN (mg/dl) and Creatinine (mg/dl) ± SEM) in plasma:

Discussion

142

5. DISCUSSION 5.1.Characterization of the prepared Oxidized and Irradiated Alginates:

The Fehling reagent gave red cuprous oxide precipitates with the oxidized

products indicating the presence of aldehyde groups within their structures and

its importance as a rapid test for the oxidation of organic compounds as stated in

the report of (Wang et al., 2011).

Spectrum of the irradiated Na-Alg: showed conservation for most bands of its

native alginate with little differences. The band at (3444 cm–1

) for the (O-H

groups) stretching mode and (1090 cm–1

) for (C–O stretching) became broader

(fig. (16)); broadening increased with the increase of radiation dose (not

shown). The peak of skeletal vibrations appeared weaker at(1298 cm–1

).

Spectrum of the oxidized alginate: showed some differences from its native

structure. Some new bands appeared proving its oxidation including:(1) The

characteristic peak of (C=O) stretching for the aldehyde groups at (1715 cm–1

)

in addition to the characteristic vibrational peak of (COO−) of Na-alginate at

(1623 cm–1

) proving alginate oxidation although it is weak. (2) The FT-IR band

of mannuronate residues at (~ 790 cm–1

) with absence of guluronates peaks;

may be due to oxidation of most of its units with ring opening as concluded by

(García-Ríos et al., 2012) (fig. (16)).

Sodium alginate was oxidized with periodate; increasing its concentration lead

to the formation of alginates with higher aldehyde contents. Four different

concentrations were chosen and the results of gelation tests proved higher

concentrations made oxidized forms with weak ability to gelate. The results

revealed also that the forms (MASA-I C, D and MASA-II B, C, D) had weak

gelation properties. (Maiti et al., 2009) found out that higher aldehyde content

than 1 mol. % makes oxidized alginates with no ability to make gels.

Discussion

143

Both irradiation and oxidation caused long chains breakages with decrease in

the M.W and its severity increased with the increase in dose (Table: 5).

Oxidation, in addition, introduced new functional groups to the chains. Only the

molecular weight of (MASA-I A-oxidized alginate) is displayed in (Table :5)

because it, unlike other oxidized forms, was found to have strong effects on

characteristics of the prepared membranes with good gelation properties.

5.2. Alginate Purification:

The used method for purification of alginate in this study utilized simple steps

but were proven to be effective according to (Qi et al., 2009) but with some

modifications for better biocompatibility and simplicity. The used Sevag

reagent was proven to be efficient in protein purification from chilled alginate

solutions. The activated charcoal is carbonaceous non-toxic adsorbent of high

efficiency, porosity and convenience with a large internal area that is effective

in adsorption of contaminants and removal of the organic compounds such as

polyphenols from aqueous solution. It was essential to characterize the used

distilled water to deviate any side reactions during the preparation and

purification steps where Na-alginate precipitates at pH>3.5 and may degrade

(Jain et al., 2011). The high concentrations of polyvalent ions, especially

calcium ions lead to gelation of the alginate solution, so they must be at the

suitable lowest ppm.

5.2.1. Characterization of the Washed Activated Charcoal:

The ash content of activated carbon is a measure of the mineral content left in

during the manufacturing and activation processes, so it must be cleaned at first

with acid followed with water to remove any contaminants (Strand, 2001) for

usage in the alginate purification. The FT-IR spectra were studied in many

previous assignments as a rapid and accurate method to check the structure and

any changes with the charcoal after the different treatments.

Discussion

144

The references in (Table: 1, cited in article of (Terzyk et al., 2001)) are

examples of the papers which involve bands covering the same ranges of wave-

numbers as observed with our investigated carbons and were chosen with the

assignment to certain functional groups with referring after each wave-number.

Interpretation of the I.R charts (fig. (17) and Appendix (A)) could prove that

spectrum of the washed Charcoal with (HCl); unlike the Spectrum of Charcoal,

washed with (HNO3) and that washed with (H2SO4), showed similar bands to

those of the initial carbon, so (HCl) was chosen as the washing acid for charcoal

where the other acids introduced new groups to the charcoal making it

unsuitable for the purification purposes.

5.2.2. Characterization of the Purified Alginate:

The rise in alginate viscosity after purification may be due to the following

reasons: (1) The used activated charcoal and Sevag reagent helped in removal of

the contaminants interfering with the intermolecular interactions among the

alginate chains in addition to proteins that interfered with its hydrophilicity

(Ménard et al.,2010). (2) Low molecular weight oligomers were absorbed and

removed during the filtration steps and this essentially increased the average

MW as well as the solution viscosity (Dusseault et al., 2006). (3) The applied

method was absolutely physical with slight chemical effects in the alginate

chains so no noticeable degradation could be observed

One purification step was not sufficient to deproteinize the different alginates,

so repeating the extraction with Sevag reagent was essential to reach the

lowest protein levels that guaranteed their biocompatibility (fig. (18A)). No

purification steps were carried out on the oxidized alginates. Alternatively, its

(HMW-Alg precursor) was only purified to deviate any side reactions between

the purification reagents and the oxidized fractions.

Discussion

145

Purification of the viscous (HMW-Alg) took place slowly due to the strong

electrostatic interactions among its chains and the contaminants molecules,

but was faster with the less viscous (VLMW-Alg) giving higher yields of the

purified alginate. Both the (HMW and VLMW-Alginates) required 2 repeats

of (PC) extraction for efficient purification. Due to its high viscosity, removal

of (PC) from (HMW-Alg) was slow and the minimum (AFU) for the finally

purified HMW was (9). In contrast, removal of (PC) from the (VLMW-Alg)

was faster due to its lower viscosity to reach a value of (7) for the final

purified polymer, despite having initial AFU of (93.8).

The Purified Alginate Spectrum: exhibited slight differences from most of

the native alginate characteristic peaks (fig. (19) and appendix (B)). The peak

at (1623 cm–1

) was taken as the reference peak for comparing the native

polymer with the purified type due to the fact that carboxyl groups do not

change after degradation. The peak corresponding to the free (O-H groups)

appeared stronger with the purified alginate than the peak of the raw polymer.

The peak for (C-C stretching) appeared with broadening at (1095 cm-1

) with

disappearance of the other characteristic peaks. The peak at (947 cm–1

) is

assumed to be due to (C-O) stretching of the uronate residues that may have

been formed due to slight degradation of the polymer during purification

(Matsuhiro et al, 2008; Xiaoxia et al., 2010).

5.3. Preparation of Chitosan:

On beginning with the deproteination step during the extraction, a higher

reduction in the ash content is achieved than on beginning with the

demineralization as the high levels of retained proteins increase the amount of

minerals that remain bound to the chitin solid fraction (Stevens, 2001). The 1st

step deproteination followed with 2nd

step demineralization have hydrolyzing

effects on the resulting polymer (Stevens, 2001).

Discussion

146

In spite of that, such degrading effects can be controlled with controlling the

demineralization period to get the required chitosan molecular weight for the

subsequent applications. NaOH concentration, reaction temperature, period and

the interaction between them play dominant roles in influencing the DD

(Lertsutthiwong et al., 2002, Sehol et al., 2002; Toan, 2009).

The schematic interpretation of chitin backbone structure is illustrated in (fig.

(44)). It shows a sandwich of thin layers of chitin between two thicker layers of

proteins, called carrier proteins (CP), surrounded by mineralized proteinous

matrix (MM) which is embedded in layers of CaCO3. The linkages between

chitin part and protein moieties are through amide formation between the free (-

NH2) groups in chitin and the (-COOH) groups in the side chains of proteins

(Roberts, 1992). NaOH, with heating have hydrolyzing effects on these bonds

(Lertsutthiwong et al.,2002) to overcome the resistance of such groups

imposed by the trans-arrangement of the C2-C3 substituents on the sugar ring

(Horton and Lineback, 1965) for removal of acetyl groups in the form of (Na-

acetate) through a mechanism illustrated in (fig. (45)).

(Roberts, 1997; Chinadit et al., 1998) reported that a multi-step process is

required to obtain high degrees of deacetylation at low temperatures. However,

high DD at high temperature can be reached in one step as well. The DD is an

important characteristic influencing the performance of polymer in many of its

applications. The interactions between chitosan and the cells increase as it

increases due to the increase in the free amino groups-content. Other biological

properties (e.g., Analgesic, antitumor, hypocholesterolemic, hemostatic,

antimicrobial and antioxidant properties) are also affected by the chemical and

physical properties of chitosan (Koide, 1998; Kumar, 2000; Kumar et al.,

2004).

Discussion

147

Figure (44): Schematic interpretation of the chitin backbone structure (Poulicek

et al., 1986).

-OH

O-Na

+

O-Na

+

NH C CH3

NH C CH3

:O:

H

CH3 C ONa

O

HO

O

H

H

H

CH2OH

H

H

O_

Na+OH

-

O-Na

+

:O:

+NH2 C CH3

NH2

O

NH C CH3+

::

:

+

is_

Figure (45): Deacetylation mechanism for chitin into chitosan (Sehol et al., 2002).

5.3.1. Characterization of the Prepared Chitosan:

Solubility is a rapid test to distinguish between the prepared chitin and its

deacetylated form, chitosan (Ch). Chitin is insoluble in most solvents; but on

heating with (NaOH), converts into (Ch) that is soluble in diluted organic acids

so the solubility refers to formation of chitosan and the efficiency of

deacetylation. This relates to the DD; as its raise makes the solubility easier.

Discussion

148

Many methods were proposed to measure the D.D (e.g., Dye adsorption

measurement, residual salicylaldehyde determination and hydrobromide salt

titration (Baxter et al., 1992), but the I.R-spectroscopy provides a rapid,

accurate technique with a high level of precision to detect the different DDs.

(Fig. (20 A) and Appendix (C)). Due to the overlapping between the bands in

the spectral region (1100-1000 cm–1

) for both chitosans, it was difficult to

identify the peaks corresponding to the stretching of (O-H) groups of carbon

atoms (C3 and C6) as well as the C-O stretching vibration and bridge oxygen

stretching bands.

There were significant differences in protein and ash contents between the 2

prepared chitosans (Table: 6). This refers that the prolonged deacetylation steps

on heating with (NaOH) share in more deproteination as well as more removal

for the retained CaCO3 within the chitosan products. These results were also

observed by (Lertsutthiwong et al., 2002). The increases in DD as well lead to

increase in the inhibition ability and antioxidant activity of chitosan, so (Ch-2)

as well as (α-tocopherol) had significant higher antioxidant activities than (Ch-

1) (Table: 6). In addition, the results of (Park et al., 2004) showed that the

chitosans with high DDs exhibit also higher scavenging activity than those with

lower DDs.

The current study shows the ability to prepare 2 chitosan polymers of different

DDs with different periods of heating with NaOH, but they had similar M.Ws,

lower than those of many commercial chitosans (Table: 6). This is very

important result as preparing chitosans of high deacetylation degrees is known

to associate with their degradation and decrease in bulk density (Sehol et al.,

2002; Trung et al., 2006). The low M.W-chitosan is preferred to allow chitosan

penetration into the alginate chains to reinforce preparation of the desired Poly

Electrolyte complex (PEC).

Discussion

149

This would enhance tensile properties of the produced sheet structure (Knill et

al., 2004a). The very high M.W-chitosan; even at low concentrations,

precipitates in the presence of Ca+2

ions resulting in very low levels of chitosan

incorporated into the resulting PEC (Tamura et al., 2002).

5.4. Preparation and Choosing the Best Hydrogel Structure:

An aim of this study focused on preparing different (Alg/Ch) hydrogels and

evaluating their properties. For these purposes, different alginate and chitosan

components derivatives with different ratios as well as the optimal crosslinking

conditions were investigated aiming to producing biocompatible and highly

hydrophilic hydrogels with suitable structure/properties supposed to accelerate

the wound healing with preventing its dehydration and scab formation.

Accordingly, the designed preparation method was as follows:

A-The Preparation Method:

I. (Chitosan-1, DD=90 %) was chosen to prepare the desired PEC hydrogel with

alginate rather than (Chitosan-2, DD=98%) for the following reasons:

(1)With certain levels of (H3C-CO-NH-), the complete complexation between (-

COO-) of alginate and (-NH3

+) of chitosan can be deviated, so can control the

dissolution and degradation rates of the resulting (PEC) for its supposed

scaffolding action for cells. The amino groups in (Ch-2) chains would bind

stronger than (Ch-1) to the alginate chains forming more stable hydrogel.

(2) Many drugs can be loaded on the PEC hydrogel through binding with the (-

COO-) groups, so very high levels of (-NH3

+) groups were not needed.

(3) The suitability of (-NH3+ groups) to be modified for drugs loading is less

than that of (H3C-CO-NH-) groups, so certain levels of (H3C-CO-NH-) groups

were needed for further applications of the prepared PEC gels.

(4) Preparation of (Ch-2) as well as the conversion of (Ch-1) to (Ch-2) for

enhancing the DD requires more time, energy and effort.

Discussion

150

II. Concentrations of the Polymers:

Polyelectrolyte titration method was used to prepare the PEC from (alginate/

chitosan). Concentration of the polyelectrolyte in the beaker; alginate, into

which the chitosan is titrated, changes during titration. First, the solvent of the

titrant dilutes the solution and, second, the alginate in the beaker is consumed

by the ongoing complexation process. To counteract this dilution, the first

polyelectrolyte in beaker sometimes has a 2–10-times higher concentration than

the other one (Gärdlund et al. 2003). To circumvent that during the study, the

reaction was stopped directly after the observation of coacervates formation, so

the used volumes of chitosan and alginate were not equal. Special reactants

concentrations were used to prevent the precipitation of either one out of the

formed complex. Under certain dilution conditions, the prepared complex

disperses homogeneously as small particles in solution which finally grow to

fine fibrous structures on allowing standing for few days at certain temperatures

range (Tsuchida et al., 1980).

III. pH of the Polymers Solutions:

pH is the most pivotal factor affecting the strength and directing the formation

of (alginate/chitosan physical hydrogels). As a conclusion from our

experimental trials, mixing alginate solution (pH 6.84) and chitosan (pH 5) at

equimolar compositions may lead to reaching a suspending solution (pH 5.28)

at which: the chitosan protonated amino groups spontaneously associate with

the negatively charged (-COO-) groups of alginate to form the PEC in solution

which then mutually precipitates from it.

It was essential to control the pH of reactants for promoting the ionic

interactions between these two counter-charged biopolymers at which:

(1) The stoichiometric composition of strong charged polyelectrolyte (i.e.,

Strong Polyacid-Polybase complex) is retained (Tsuchida et al., 1980).

Discussion

151

(2) There is charge balance thus limiting repulsion.

(3)The interactions of polymers with water are greatly minimized with

maximization for the yielding dense of the (polymer-polymer interactions).

(4) The net charge does not reach zero to allow surplus electrical charging of the

resulting coacervates which is necessary for the solubilization of particles

(Gilsenan et al., 2003).

(5) For Alginate: Generally, at pH<3.5, the carboxylate groups become less

ionized with protonation to form alginic acid physical gel clusters through

(inter-chain interactions). The increase in pH makes free (-COO-) on the

polyanion chains. At pH>8, the high concentration of Na+

ions restrains the

extension of the tangled molecular chain on the hydrogel, therefore its swelling

may be inhibited (Liu et al., 2002).

(6) Chitosan precipitates above its pKa value of 6.3. Charge density of the

chitosan molecules is reduced with increasing the pH of solution and the

polymer chains become less extended (Ikeda et al., 1995). As pH increases

from 4.5 to 6.2, the ionization degree of chitosan decreases from 1.0 to 0.5

(Ikeda et al., 1995), the amino groups become less charged with reduction in

the charge density by almost 50% resulting in expected fewer ionic linkages

between any negatively charged polymer and chitosan.

In contrast, the chitosan chains become less extended with these changes at pH

6.2 with smaller radius of gyration and enhanced diffusion coefficient into the

(Alg/ Ch network) causing stronger combination with (Alg-chains) during a

specified reaction time than the linkages of the extended chains at pH 4.5.

Accordingly, PECs formed of chitosan at pH 6.2 have lower D.S than the

complex formed at 4.5. Raising the pH of chitosan solution from 4.5 to 5.5 with

a corresponding decrease in its total charge was observed to have no significant

effects on D.S of the complex gel (Argin-Soysal et al., 2009).

Discussion

152

IV. The Coacervation Reaction:

(1) Rapid coacervation for the reacting polymers at definite pH values induces

the formation of dense interphasic PEC membranes that separate the polymers

solutions and prevent further reaction and this is the common employed

technology to prepare microspheres of chitosan–alginate PECs (Tay et al.,

1993; Alexakis et al., 1995; Liu et al., 1997; Silva et al., 2006). This

technology was not used in the present study as it was unsuitable to produce

coacervates ready for casting into films. The extensive development of these

intermolecular interactions during hydrogel films preparation would generate

viscosities too high to distribute or matrices too solidified to flow. To prepare

coacervates for casting into homogeneous films, the rate of reaction between the

two polymers must be sufficiently slow to prevent the formation of such

membranes, so that the reaction can complete.

(2) Based on previous reports, (Wang et al., 2002; Ca´rdenas et al., 2003;

Meng et al., 2010), addition of water miscible organic solvent (e.g., Acetone ,

ethanol or PEG200) into the chitosan solution was proven effective to slow the

rate of coacervation. They have lower polarity so the chitosan chains will

assume less extended conformation in and this will restrict the fast reaction

between the two polyelectrolytes and so perfect complexation can occur.

(3) An aim of the current study was using the least number of chemicals, which

may cause skin irritation, inflammation or ulceration on application of the

prepared hydrogel film to the wound with a main dependence on the physical

activity for mixing the 2 polymers to produce a high quality of PECs through the

coacervation process. Thus, controlling the reactions speed through controlling

the rate of mixing of chitosan and alginate solutions together is undoubtedly the

pivotal point for success.

http://www.ncbi.nlm.nih.gov/pubmed?term=Alexakis%20T%5BAuthor%5D&cauthor=true&cauthor_uid=7702366

Discussion

153

(4) The high shaking rates with the titrant polymer solution addition promote

foam formation with opaque sheets having impaired barrier properties, more

permeability to gases, in addition to forming some suspensions of soluble

complexes with no ability to coacervate. The weak speeds lead to fast

coacervation with no ability to form gels. Accordingly, the moderate shaking

and mixing speeds (about 50 RPM) with controlled addition rate (about 300 µl/

min) were perfect to prepare ideal PECs suspensions of (alginate/ chitosan)

despite taking some time. The resulting hydrogel films were transparent that

allowed effective observation of changes in the wounds during skin repair.

(5) Multifaceted structure may have been formed when a chitosan drop of

certain concentration fell into alginate solution at the desired pH values.

Complexation may have occurred between the two oppositely charged

polyelectrolytes through the electrostatic interactions. By this way,

complexation between the common free (-COO-) groups of alginate chains and

the chitosan protonated amino groups (NH3+) occurred and chitosan may have

acted as the insolubilising cation for production of (alginate-chitosan Physical

complex) (Cole and Nelson, 1993; Pandit, 1998). With adding (CaCl2) into the

mixture during complexation, it may have diffused into the alginate core more

rapidly than chitosan because of its lower molecular weight to form a gel core.

Calcium cross-linking for alginate helped in its stabilization with forming (a

Semi Interpenetrating Network (Semi-IPN))of pH sensitivity and reversible

properties that were proved later through the characterization tests.

V. Preparation of the film dressings:

The stand-alone films from macromolecules are generally produced by

extrusion, deposition, or casting/solvent evaporation (Farris et al., 2009). For

(PEC-film) formation, all the involved physical changes must occur in situ

during the preparation in order to ensure formation of the desired complex

structure in place within the final film.

Discussion

154

Generating films by casting melted plastics or preparing bio-hydrogels involves

spreading of the film-forming solution onto non-adhering, flat and smooth

substrates, followed by removal of the solvent usually through a hot air flux.

This is the mostly used method in research as it is simple, practical, does not

require expensive equipment and has no adverse effects on properties of the

prepared films from bio-macromolecules on keeping under control. During the

step of membranes drying, the hot air evaporates water from the outermost

region of the nascent membrane to induce phase to coalesce and ultimately fuse

to form dense skin, then the underlying phase separation region tends to

coalesce with time and fuse with the dense skin to increase its thickness (Meng

et al., 2010). The unreacted Alg and Ch molecules have supporting effect to

help in forming the PECs coacervates that give the coherent flexible thin

membranes (Meng et al., 2010). The upper surface of the membrane is

responsible for controlling the loss of water through evaporation, inhibition of

body fluid loss, and protection from any external contamination.

B-The Dressing Form:

(Alginate/ chitosan PEC films of hydrogel nature) were selected as a test model

of wound dressings. The films were thin and transparent to help in easy

monitoring of any changes during healing. The hydrogel nature of the dressing

was desired due to the following distinct useful features that may make them are

highly accepted by the patient:

1- Mechanical flexibility.

2- Free permeability to small molecules such as dissolved gases and low

molecular weight nutrients and wastes.

3- Controllable permeability to larger molecules such as proteins.

http://www.amazon.com/s/ref=ntt_athr_dp_sr_1?_encoding=UTF8&field-author=Peter%20Zilla&search-alias=books&sort=relevancerank



Discussion

155

4- Due to their similar physical properties to the natural tissues (Jeon et al.,

2009), hydrogels are generally found to be easily tailored devices in vitro to suit

many functions of prosthetics on coming in contact with blood or tissues and

can be very well tolerated when implanted in vivo.

5- The wound dressing hydrogels are now considered good alternatives to more

conventional dosage forms (e.g., Creams, ointments and patches). The low

interfacial tension of hydrogels gives them humectants properties, allows good

surface swelling, skin moisturization (Yan et al., 2000), provides a better feel

on application to the skin and prevents any scabbing or drying out so that the

wound will be allowed to heal normally from the inside out (Winter and

Scales, 1963; Pinkus, 1970).

6-Because of the volume changing behavior of hydrogel with absorption of

fluid or medium, this is one of its important characteristics for the cells

migration in three dimensions (Hubbell, 1999).

7-Hydrogels; with their high effective surface area, can be used to store large

quantities of biologically active molecules that can be released in a triggerable

fashion to the ECM or wound bed.

8-The absorption of secretions from wound causes an expansion of the cross -

links between the polymer chains making rooms for the inclusion of foreign

bodies such as bacteria detritus and odorous molecules which are irreversibly

taken up along the liquid (Pal et al., 2006).

9- Since an ideal dressing should temporarily perform protective functions of

the epidermis while tissue repairing and granulation are going on with

replacement of the old layers of epidermis, the use of hydrogel as a wound

covering material was considered.

10- The thata solvent for the gel precursors is water; so there is a possibility to

apply dissolved hydrogel precursors in physiological saline to a tissue site for in

situ formation of the hydrogel (Balakrishnan, 2005b).

Discussion

156

C-Choosing the Type of Hydrogel for Usage as Wound Dressing:

5.4.1. In vitro swelling of the hydrogels and stability characterization:

Determination of the fluid absorbing capacity of hydrogel is an important

criterion for its biomedical applications. It was studied concerning the usage of

hydrogel as a wound dressing with probable scaffolding properties for the

following reasons:

1-Application of a hydrogel with good swelling properties maintains a moist

environment over the wound bed and provides a cooling barrier that allows

water to evaporate from the surface to produce cooling effects which help in

reducing the microcapillary circulation in the surface of skin and encourage a

reduction in erythema, building up of edema and reduced swelling to allow a

more even cicatrization to develop at the wound site (Smith et al., 1994).

2-Frequent changing of post-operative dressings is sometimes painful and

increases risks of trauma to the wound, thereby hinders wound healing and may

not be economically feasible. Patients need to move freely and take showers so

the spent nursing time needs to be greatly reduced (Hulten, 1994). Dressings

with large absorptive capacity reduce the problems concerned with the pain

related to maceration of the surrounding tissues and pressure caused by the

excessive exudates (Ferrell et al., 2001).

3-It is essential to understand the diffusion of medium into the scaffold hydrogel

and to detect its internal characteristics to determine viability of the

incorporated cells as their highly swollen state facilitates the transport of

nutrients into and cellular waste out (Drury and Mooney, 2003). In addition,

the water sorption isotherms are important for understanding what concerns the

interaction mechanisms between water and film components.

4-The drug release from dressing films is initially affected by D.S that can be

considered as its release rate-limiting step (Prajapati and Sawant, 2009).

Discussion

157

5-The D.S of PEC hydrogels and microcapsules has been studied in many

previous researches as indication of their cross-linking density. The increase in

cross-linking density reduces the pH-sensitivity by improving the stability of gel

network and restricts the high swelling due to decreased chain mobility with

less porous structures (Argin-Soysal et al., 2009).

As pointed out by (Velings and Mestdagh, 1995), changes in volume (V) of a

gel can be correlated to changes in its mass (M) due to mere polymer movement

as a linear correlation exists between the (V& M) changes with a slope (1.08

Kg/l), close to the water density value at 25oC. Characteristics of the swelling

and release profiles for (Alg/Ch complex) relate to its structure, crosslinking

density, pH and medium composition (Murata et al., 1993).

The swelling takes place in a medium based on the following mechanisms:

I. Calcium-mediated alginate cross-linking is disrupted due to the trapped Ca+2

ion leakage by monovalent ions of medium (example: Na+ ions).

II. The general mechanism of swelling for the prepared PECs is based on the pH

7.4 at which the electrostatic interactions between the oppositely charged chains

become weak (Liu et al., 2004; Bajpai et al., 2006). The -OH groups attract the

protons of the charged amino groups on chitosan surface to make them less

charged with more free (-COO- groups) on the alginates surface. (Argin-Soysal

et al., 2009)

III.There are (2) main sites for water holding capacity in chitosan within the

prepared hydrogels: (-OH and NH2 groups) (Cervera et al., 2004), but (3) sites

with alginate including (-COO- and OH groups), in addition to (-CHO) groups

of the oxidized alginates. Swelling occurs in aqueous medium by attracting its

molecules by these free surface polar hydrophilic groups which become

hydrated with the(1ry

Bound Water) and the network then exposes the

hydrophobic groups to interact with water molecules (2ry

Bound Water).


Discussion

158

IV. The network then imbibes additional water based on the osmotic driving

force of the network chains towards infinite dilution due to the difference in

concentrations of the ionic solutes in gel and the surrounding solution with

flexibility of the polymeric chains (Berger et al., 2004a; Argin-Soysal et al.,

2009).The additional swelling is assumed to fill the space among the network

chains and/or the centers of macropores and voids till reaching equilibrium

swelling level on equilibration of the osmotic pressure in the gel and medium

with a balance between repulsion and contractile forces within the network.

Greater flexibility of the polymer chains would allow larger expansion of free

spaces, thereby increasing the capacity to retain the fluid (Hian, 2009).

V. This behavior is opposed by the covalent or physical crosslinks leading to

retraction forces within the elastic network. The maximum fluid uptake capacity

(equilibrium swelling level) would be reached on balancing between the

swelling pressure and elastic resistance of the polymer network (Remuñán-

López and Bodmeier, 1997). On reaching a critical value of the ion-exchange

with the cross-linked degradable hydrogel, the whole crosslinked network will

be disjointed and the gel will begin to disintegrate and dissolve at a rate

depending on its composition (Hoffman, 2002).

The Formula (F-25) ([A] +Chitosan): includes components with the highest

M.Ws (Ch+HMW-Alg). Considering the reaction stoichiometry, the largest

amounts of coacervate complexes may have been generated (Li and Pelton,

1992) with the same previous swelling mechanism.

The Formula (F-2) ([A+ (CaCl2)]+Ch): Swelling occurred due to the same

reasons as (F-25) but the affinity of gel to swell H2O molecules is less. Though

its affinity to retain water is expected to be higher than (F-25) by the formed

net-like structure with the cross-linking ions, the firm coacervation between (F-

25) polymers chains helps in the stabilization of this network.




Discussion

159

D.S of this formula is the least among the corresponding (Ca-containing

hydrogels) of different constituents (fig. (21A)) due to the stronger cross-linking

of Ca+2

with the repeated longest (G-blocks).

The Formulae (F-12) ([A+B] +Ch) & (F-9) ([A+C] +Ch): The total bound

H2O is the first reason as in the case of (F-25), but short chains of the irradiated

alginates (B and C) are movable that may have increased the tendency of the

free (COO- groups) to imbibe more H2O molecules to make them have a higher

swelling degrees than (F-25).

The Formulae (F-1)([A+B+(CaCl2)]+Ch)& (F-15) ([A+C+(CaCl2)] +Ch):

They had less D.S than (F-12 and F-9), respectively where the (A-chains) seem

to be the controllable factor of swelling in these 4 formulae as in case of (F-25

and 2). The difference in D.S between (F-12 and 1) was not great, unlike that

between (F-9 and 15).

The Formulae (F-21) ([A+D] +Ch) & (F-23) ([A+E] +Ch): They had chains

of low M.Ws due to the hydrolysis on oxidation to make them more movable

with higher affinity to H2O than (F-25) but with less affinity than those of (F-

12, 9) due to the less polar aldehyde groups than (COO- groups).

The Formulae (F-10) ([A+D+ (CaCl2)] +Ch) & (F-16) ([A+E+ (CaCl2)]

+Ch: The affinity of (F-10) to (H2O) molecules was slightly less than (F-21)

with weak effects of the formed network by Ca+2

ions due to the presence of

oxidized alginate fractions. The (A-chains) maintained the gel properties. No

difference was observed in D.S between (F-16 and 23).

The Formula (F-5) ([A+B+D] +Ch): The polar groups are distributed on (A, B

and D chains). The increase in the number of small chains (B and D) than in (F-

25) may have been the reason for the increased affinity to (H2O).

Discussion

160

The free and total bound water are responsible for this increased affinity.

Though they displayed higher diffusion ability to coacervate with chitosan

chains, the resulting hydrogel had slight dispersion which may relate to the

decreased ratio of the long (A-chains).

The Formula (F-20) ([A+B+D+ (CaCl2)] +Ch): Generally, addition of (Ca+2

)

ions during the hydrogel preparation enhances its mechanical strength due to the

strengthened forces by which these ions bind (Alg-chains) and the cross-linking

between (G-units) and these ions generally requires a critical length of (G-

blocks). Accordingly, hydrolysis of the small chains takes place on immersing

in the aqueous medium at first due to their weaker cross-linkages which cause

increasing in the hydrogel affinity to bind H2O molecules causing its swelling.

The D.S was higher than that of (F-5); they may have the same swelling

mechanism but the cross-linking ions help to stabilize the gel and the formed

net store the medium molecules with a higher degree than (F-5) causing the

retaining of medium molecules with increasing the binding of the hydrogel

chains to water.

The Formulae (F-17) ([A+B+E] +Ch) & (F-3) ([A+C+E] +Ch): They showed

similar swellability with initial fast imbibing of water, but lower than (F-5)

which may relate to faster release of the small chains to medium; especially the

higher oxidized alginate chains. In spite of that, the swelling curves of these 2

formulae reach early plateau phases on equilibrium between the swellability of

the remaining chains and their hydrolysis.

The formulae (F-19) ([A+B+E+(CaCl2)]+Ch)& (F-24)([A+C+E+(CaCl2)]

+Ch) : have lower affinity to (H2O molecules) than (F-20); may relate to fast

solubilization of the small chains accompanied with swelling and weak cross -

linking (Yan et al., 2000) making weak nets to store medium molecules.

Discussion

161

The formulae (F-6) ([A+C+D] +Ch) & (F-13) ([A+C+D+ (CaCl2)]+Ch):

Alginate long chains are the main factors responsible for stability of the formed

PEC with chitosan. The new added short irradiated and oxidized chains with the

decrease in (A-chains %) may lead to increasing the binding affinity between

the 2 polymers and the formed gel becomes dispersed. The weak networks,

formed by Ca+2

ions with (F-13) store small amounts of medium molecules

accompanied with diffusion of the small chains.

The formulae (F-31) ([A+B+G] +Ch) & (F-33) ([A+B+F] +Ch) & (F-32)

([A+B+G+ (CaCl2)] +Ch) & (F-34) ([A+B+F+ (CaCl2)]+Ch): Increasing the

ratio of alginate short chains lead to disturbing stability of the formed PECs.

Both the oxidized alginates (F&G) have lower M.Ws than (D&E) with more

aldehyde groups to generate unstable (PECs) of faster diffusion for the weakly

bound chains to medium. The short (G-blocks) weaken the stabilizing action of

Ca+2

ions and facilitate the short chains diffusion.

The formulae (F-8) ([B+D] +Ch & (F-18) [B+D+ (CaCl2)] +Ch:

The short alginate chains seem to have higher affinity to (H2O molecules) than

the HMW-Alginate (A-Alg) with higher swellability. Syneresis of (F-8) in

medium began after one hour of strong swelling (fig. (21C)). This may relate to

the rapid diffusion of the short chains to medium with beginning of film matrix

solubilization with weight loss (Yan et al., 2000). This may be a good

indication of the importance of alginate long chains in stabilization of the

hydrogel structure. The cross-linking of the chains with Ca+2

ions helps to

stabilize the formula (F-18) hydrogel than (F-8). The formed network

maintained the storage of medium molecules but the swelling curve appeared

weak after 1 hour of immersing in medium due to the fast movement of the

short chains to medium against the action of the cross-linking Ca+2

ions.

Discussion

162

The formulae (F-22) ([B+E] +Ch) &(F-7) ([B+E+(CaCl2)]+Ch)& (F-11)

([C+D]+Ch) & (F-14) ([C+D+(CaCl2)]+Ch): With similar composition as (F-

8, 18) but less in swellability. The D.S was higher than (F-25), but slowly

increased with time to make them unsuitable for use as intended dressings.

The formulae (F-4) ([C+E] +Ch) & (F-26) ([C+E+(CaCl2)]+Ch): They

displayed normal increasing swelling curves where the swelling was initially

rapid, slowed down to reach a plateau after 6 hours in case of (F-4) and after 8

hours for (F-26 ). The behavior of (F-4) in medium is questionable where it

differed from (F-22 and 11) as good D.S was observed though these small

chains were expected to diffuse into the medium or to express small level of

swelling. The behavior of (F-26) was expected due to the stabilizing action of

Ca+2

ions for the hydrogel structure.

The formulae (F-27)(D+Ch)& (F-28)(E+Ch)&(F-29) (B+Ch)& (F-30)(C+

Ch): According to (Vanerek and van deVen, 2006), chitosan chains with high

M.W act as host molecules for the other much smaller alginate chains

(Irradiated alginates (B& C) and oxidized alginates (D& E)). Only soluble PECs

may have been formed without forming any colloidal or coacervate complexes,

so no gels were formed on casting.

(According to these results, the formulae (F-4, 5, 6, 9, 18 and 20) were

considered as the best prepared hydrogels in their stability and water uptake

abilities where the gels do not exude the fluids and retain the absorbed amounts

present. In clinical practice, these findings would translate to a greater wound

fluid absorption and longer wear time.)

5.4.2. Hemocompatibility Assays of Hydrogels:

Biocompatibility, especially blood compatibility, is the most important property

with regard to the biomedical materials.

Discussion

163

When the polymeric device comes in contact with blood, it must not induce

thrombosis, severe antigenic responses, thromboembolisms or destruction of

blood constituents or plasma proteins (Sevastianov, 1991). Hence, the

hemocompatibility tests have been conducted with respect to the different

formulae. When the RBCs come into contact with water, blood is hemolyzed.

(Dhandayuthapani et al., 2012) had reported that a value of up to (5%)

hemolysis is permissible for the biomaterial and the device (dressing) will be

incompatible with human blood on having hemolytic tendencies (Pharm Labs,

2008). Accordingly, hemolysis % for the formulae (F-5, 9, 18 and 20) located

in this permissible limit with considerable biocompatibility. The fast release of

small chains from the other 2 formulae in spite of their high D.S may be

responsible for their high hemolytic tendencies.

The different clot weights relate to the different abilities of hydrogels to activate

thrombus formation. The anti-thrombogenic potential of surfaces for the

different formulae films was judged by the blood clot formation test where there

were similar weak clot formation abilities for the formulae (F-4, 6 and 9)

without any significant differences between them (p< 0.05). These values

differed significantly versus each of the (Ca-containing formulae) (p>0.001,

Table: 8). The abilities of (F-5, 18 and 20) to activate clotting were similar

with no significant differences between the (Ca-containing formulae), but the

difference was between (F-5) and its (Ca-containing formula; F-20) (p>0.05)

confirming the supposed action of (Ca+2

) within the structure of (F-18 and 20).

The release of (Ca+2

ions) on swelling during the ion exchange with the soluble

monovalent ions of the used test mediums helps to activate the clotting reactions

in the in-vitro tests. This is similar to the hydrogel behavior on application to

the wound where the blood and wound fluid enable the film to rehydrate, swell

and turn gel.

Discussion

164

This helps in the moisturization of wound bed and platelets activation, in

addition to exchange of the Ca+2

ions of gel with the monovalent ions of these

fluids to activate their actions on skin injury. In addition, Calcium ion is the

(clotting factor: IV) responsible for promoting homeostasis. (Vernenkar, 2009)

The formula (F-9) displayed good hemolytic activity, but weak clotting ability

as well.

(According to these results, the formulae (F-5, 18 and 20) may be the best

candidates for using as dressing agents due to their better blood clotting and

homeostatic abilities, in addition to their good hydration/swelling properties)

5.4.3. The Extent of Evaporation/ Loss of water from the hydrogel: was

evaluated to examine its behavior on using as a dressing over a dry wound. The

physical interaction between the gel network and incorporated water makes

jelly-like structure containing firmly bound water. This property is essential for

materials to be used in wound covering, so it was essential to determine the

water loss from the prepared gels at R.T. When it covers a wound, the water

loss enables the gel to take up exudate and edema fluid from the wound by an

active upward-directed process and this will avoid dehydration (fig. (22). The

three prepared hydrogels (F-5, 18 and 20) have, to somewhat, equal water

evaporation rates. Accordingly, they will be more beneficial to wounds with

moderate exudates than dry ones.

5.4.4. The In vitro Degradation of the Hydrogel Films test: measured the

solubilization rate of the hydrogel chains without further studying of their in

vivo biochemical degradation sequence. It was determined as the mass loss (%)

over time. Fig. (23) shows that the degradation rates for (F-18 & F-5) were

faster than that of (F-20) despite of its higher (D.S). Diffusion of the (F-18)

small chains to the test medium had a faster rate than that of (F-20). containing

the long (A-chains) to initiate faster solubilization from the gel.

Discussion

165

This causes an increase in the degradation of gel-network. The greater extent of

degradation would reduce the ability of gel matrix to retain the absorbed fluid.

The scaffold degradation must be under moderate control to make rooms for the

growth of new cells and then substituted by new skin tissue. Unlike (F-18),

application of the hydrogel (F-20) is not supposed to require continuous

changing. Drug release from gel would be faster to the wound bed with

uncontrolled diffusion on its encapsulation within (F-18).

Different extents of degradation with medium fluid uptake are attributed to the

rigidity and cross-linking effects of Ca+2

in the hydrogel. Generally, a high

extent of cross-linking and uniform distribution of cross-linkages across the

matrix would favor a lower degree of degradation. On the contrary, the

progression of degradation would result in the release of cross-linked cations

from the network junctions. Unlike the other formulae, (F-5) lacks the

stabilizers Ca+2

ions, so expressed a faster rate of solubilization than (F-20) but

less than (F-18) where the long (A-chains) are shown to play important role in

the stabilization of the coacervate structure in gel. This behavior, in addition to

the weaker blood-clotting properties than (F-20 & 18) makes the usage of (F-5)

as a wound dressing material impractical with a final conclusion that (F-20)

may be the best one as a dressing formula with probable scaffolding properties

for healing, better swelling and hemocompatibility properties with more

practical usage.

5.4.5. The results of Primary Skin Irritation Test for the Hydrogels, displayed in

(Tables: 9 and 10) showed that the irritation which accompanied the application

of the un-irradiated hydrogel to the wound was minimized with its terminal

irradiation which has effects in killing any microorganisms within the hydrogel.

In spite of that, the sterilization effect of radiation should be studied with

comparing its effect with the effect of other methods.

Discussion

166


The formula (F-18) was used to confirm the results of this study due to its good

swelling properties. The fall in (D.S) of the 2 hydrogels with increasing the used

CaCl2 concentration is related to the formation of gels with higher network

crosslinking density and enhanced mechanical strength. This reduces the ability

of hydrogel to uptake water with significant reduction in the D.S. On the other

hand, the low concentration is linked to higher swelling due to its weaker

crosslinks which cause also poor mechanical strength, limited stability of the

hydrogel with continuous immersing in medium and weak hemostatic effects.

These results are in accordance with the results of (Draget et al., 1990). Based

on the presented results, the (2%) concentration of (CaCl2) was preferred in

both types of hydrogels. Intact gel was formed with this concentration having

good swelling properties and better hemostatic properties.

Characterizing the Effects of Irradiation on the Hydrogel (F-20):

During the pharmaceutical products manufacturing, sterilization is essential for

destroying all the microbial life including microorganisms and bacterial

endopores. It includes certain physical or chemical procedures, applied either

before mixing the filling components (i.e., Alginate, Chitosan, CaCl2 and their

solvents) (Aseptic manufacturing process) or with the (Terminal sterilization

of the final product in the final container) for delivery to the end user

(Yaman, 2001) and that was the applied method in the present study. The major

applied methods for sterilizing the medical devices include:

1-Gaseous Sterilization: A Chemical method that utilizes a sterilizing gas (e.g.,

Ethylene oxide, formaldehyde, glutaraldehyde, or propylene oxide) under

special conditions of pressure, temperature, application time and humidity.

Discussion

167

One of its disadvantages is the presence of toxic residues in the product

hazardous to the patient and can lead to adverse biological reactions such as

anaphylaxis and immune responses (Silver, 1994) which limits its usage as a

mean for sterilization (Rao and Sharma, 1995).

2-Saturated Water Steam Sterilization: The bacterial Death by moist heat

relates to the denaturation and coagulation of essential protein molecules

(enzymes) and cell constituents. With the saturated water steam, the chitosan -

based membranes become water insoluble and lose about 80% of their original

tensile strength with retaining only 28% of the initial strain at break, so it was

not applied in the present study (Boucard et al., 2007).

3-Dry Heat Sterilization: The killing of microorganisms by heat is a function

of the time-temperature combination used. With increasing temperature, the

time required for killing all the bacteria will then decrease (Sultana, 2007). The

Surgical dressings cannot be sterilized by dry heat as they will get charred at the

high temperatures (4my4141, 2013).

4-Microwave Sterilization: This method is based on heating the product by

microwave radiation energy. It is more preferred during the aseptic

manufacturing of the products (Yaman, 2001).

5-Pulse Light Sterilization: It utilizes radiation in the U.V-region, pulsed

through the product with producing an internal minimal temperature increase

(Furukawa et al., 1999). The effective range of wavelengths to kill

microorganisms is narrow (220 to 280 nm), especially those close to 253.7 nm

at which maximum biological efficiency exists. This is also the absorption peak

of isolated DNA, the target for UV-induced lethal events (4my4141, 2013) with

the formation of a dimer of (2) bonded adjacent pyrimidines.

Discussion

168

The photo-reactivation for some microorganisms can be actually caused by a

light activated enzyme that recognizes thiamine dimers in the DNA and cleaves

them so that its normal structure can be restored (Cook and Mcgrath, 1967).

The bactericidal UV-light may cause eye problems and skin erythema on

exposing for a prolonged period of time. Its usage is not practical for the

photodegradable products and its sterilization efficacy is less than the following

two methods (Fleck and Nielsen, 2004).

6-Electron Beam Sterilization: It is based on particles radiation resulting from

the artificially accelerated electrons to high energies to improve their ability to

penetrate a target to a good depth. It has similar sterilization conditions to (γ-

irradiation) (Komender et al., 1981; Li et al., 1997), but cannot be used for

sterilizing the prepared gel films in the dishes because the size of sterilization

chamber is limited and subsequently the number of pallets that can be processed

per the sterilization load is limited as well.

7-Gamma Irradiation: The 2 radionuclide isotopes (Cobalt(Co60

)& Cesium

(Cs137

)) are typically used as γ-rays sources without application of humidity,

heat, pressure, or vacuum with 2 possible mechanisms for their sterilizing

effects: (I)The γ-rays attack the microorganism and can cause irreversible

damage to its critical biomolecules, specially the DNA. (II) They cause the

ionization of water molecules within its body to produce free radicals that

oxidize the (OH.) free radicals and reduce the H

. radicals (Boyd et al., 1981)

The following points summarize the advantages of using γ-irradiation for

sterilization of objects according to (Mukherjee, IAEA):

(a) After pre-packing of the object in hermetically sealed packages impermeable

to microorganisms, the γ-rays can reach easily to all of its parts due to their high

penetrating ability without retardation by most materials making the sterile

shelf-life of the supplies practically indefinite.

Discussion

169

(b) At the applied sterilization dose, radiation causes no significant rise in

temperature. Being a 'cold' process, it permits the sterilization of heat-sensitive

materials. Radiation is certainly the best and often the only method for

sterilizing the biological tissues and preparations of biological origin.

(c) γ-irradiation leaves no residual radioactivity, the possible disadvantages of

its chemical reactivity are relatively low compared with the often highly

sterilizing reactive gases and doesn`t require quarantine for the out-gassing.

(d) The sterilizing effect of irradiation is instantaneous with permitting the

stopping of its effect at the desired moment.

(e) Radiation sterilization is suitable for a continuous, fully automated process

with a single controllable parameter, the time of exposure. Steam and chemical

sterilization are batch processes requiring more controls.

(f) This method can be used to sterilize biomolecules (polymers, proteins and

peptides) and the sterilized product may be parametrically released without

performing sterility testing (Yaman et al., 2001).

(g) As a constant and predictable sterilization method, gamma irradiation

provides benefits in safety, time and cost.

Over time and dependent on the dose, the absorption of radiation is cumulative

and some polymers that may be qualified for use in gamma-irradiated single-use

bioprocess systems may not be capable of withstanding more than 50 KGy

(AAMI TIR17, 1997) where γ-rays may cause main chain scissions with

changes to the physical or chemical properties of many polymers (Lim et al.,

1998; Hemmerich, 2000). The doses of γ-irradiation (≥8 KGy) are generally

adequate to eliminate low bioburden levels (Table 9 in ANSI, 2006). In the

cases with elevated bioburden levels (>1,000 colony forming units (cfu/unit)) as

may occur with very large single-use systems, higher doses may be required to

achieve sterility.

Discussion

170

The accepted overkill dose established by the Association for the Advancement

of Medical Instrumentation (AAMI) is 25 KGy with a sterility assurance level

(SAL) of 10–6

(PDA, 1988). Even with elevated bioburden levels, its reduction

can be achieved with lower probabilities of sterility (e.g., SAL of 10–5

or 10–4

).

The Products irradiated to such SALs are still sterile but have higher

probabilities of non-sterility and may not meet the standards for validated sterile

claims as specified in the industry standards for sterilization of health care

products. (Rosiak et al., 1992) had proved that the biocompatibility of chitosan

is not affected by a sterilizing dose of (25 KGy) and (Lim et al., 1999)

concluded that γ-irradiation was the best sterilization mean for the chitosan-

based products at this dose. Gamma irradiation at 25 KGy under anoxic

conditions was believed to be suitable for sterilization of chitin and chitosan

products (Rosiak et al., 1992; Khor and Lim, 2003; Lim et al., 2010). This

dose was also used for sterilization of the alginate-based as well as the alginate/

chitosan based wound dressings (Wang et al., 2002; Kucharska et al., 2008).

The breakage of chemical bonds within the microorganisms (e.g., Bacterium)

has the same mechanism by which the irradiation attacks the bioactive

macromolecule, so its effect on properties of the prepared films was studied.

The irradiation of hydrogel lead to decreasing in its hemolytic effect and

increasing the blood clotting abilities as illustrated in (Table:8). The change in

integrity of gel with calcium content after irradiation and immersion in the used

medium or in contact with the wound fluid facilitates a faster release of (Ca+2

)

ions to cause faster clotting of blood than the Ca-free gel.

The sorption capacity increased substantially after sterilization with γ-

irradiation that probably disturbed the integrity of the formed complex between

the polymers chains.

Discussion

171

The applied irradiation dose may have caused partial breakages for chitosan

chains leading to making more cavities or pores within the hydrogel network to

cause more hydration with 2-fold increase in water absorption for (F-20(Irr))

than(F-20(Un)) after 30 min of swelling and this fold remained to somewhat

constant over time (fig. (25)). For the (Ca-free formulae): Although the D.S was

similar in both after 30 min. of swelling, its folding increased for the irradiated

formula after 1 hour of immersion. The unirradiated one showed a small

increase with time.

The FTIR spectroscopy has been used to test and explain the interactions

between functional groups of the oppositely charged polyions. (Stuart, 2004)

demonstrated that when two immiscible polymers are brought together, the

resulting I.R spectrum will be expected to be the sum of spectra for the

individual compounds as they will have the same environment of their pure

states. When they are; by contrary, miscible, intermolecular interactions may

occur among their chains and this will be reflected in changes with the I.R

spectra of the mixture such as wave-number shifts, broadening of bands, the

disappearance or appearance of absorption bands.

The absorption bands for (N-H) stretching of amide I, margined at(1326 cm–1

)

and amide III at (1367 cm–1

) in chitosan disappeared with complexation. Some

characteristic peaks of the free (Na-Alg) appeared with both (PECs) charts (e.g.,

the asymmetric stretching mode of (CH2) group at (2931cm–1

) (Zhu et al.,

2012)). Shifting occurred for the band (1432 cm-1

) with the physical mixture of

polymers to (1415 cm-1

) with the charts of both PECs indicating the binding of

alginate with NH3 +

groups of chitosan via hydrogen bonding (Meng et al.,

2010) (fig. (26)). In addition, irradiation may have activated the formation of

free radicals which induced the binding of (-OH groups) together and with the

(NH3+

groups) of chitosan.

Discussion

172

This caused shifting for the stretching peak of (O-H groups) to appear at (3444

cm–1

). The 2 bands (1415 & 1262 cm–1

) with the Unirradiated PECs chart

indicate that most of alginate was deprotonated (Lawrie et al., 2007; Li et al.,

2009), but the band (1262 cm–1

) was absent with chart of the irradiated PEC.

The peaks for C–O stretching vibrations of (Irrad. PEC) appeared at (1084 and

1039 cm–1

) and at (1084 and 1047 cm–1

) for the (unirrad. PEC). These results

show that only slight breakages may have occurred for the bonds between the 2

polymers chains after irradiation.

Scanning electron microscopy was followed to evaluate the internal and

external structures for the different prepared films before/ after irradiation and

spot the changes in their morphological appearance. Fig. (27A) illustrates the

micrographs of the formula (F-20 ([A+B+D + (CaCl2)] +Ch)) prior to

irradiation. The shot magnification (1500) didn’t show detailed structure for the

film and no textured surface was observed which may imply that complete

complexation didn’t actually take place. On the other hand, (fig.(27B)) shows

the irradiated sample of the former membrane; changes in the surface were

captured, may be due to the existence of more favorable complex formation

conditions under the irradiation with γ-rays (mag. 1500).

The dose of 25 KGy was applied beneficially for presence of the natural

chitosan in the structure. It might degrade under high doses with breakages

formation for some of its chains that may induce modifying their interaction

behaviors with alginate chains in addition to the cross-linking with Ca+2

ions

and rough porous web structures formation (fig.(27B)) which may indicate

compatibility of the resulting membrane and prove it can withstand the

mechanical action for dressing purposes. The scanned surface with the taken

shot (mag. 1500, fig (27 C)) may be due to rupture of the strong bonds among

the polymers chains during preparing the films for the SEM analysis.

Discussion

173

Additionally, Fig (27 D, mag. 750) for the irradiated sample displays big to

medium clusters with porous layer form. The unirradiated physical gels as

Alg/Ch membranes (F-5 ([A+B+D] +Ch)) were monitored to claim the

difference between complexes with and without CaCl2 additives. A paste like

structure with no homogenous form is displayed in the shot (fig (27 E), shot

mag. 1500). This indicates the importance of cross-linking for smoothing the

surfaces and organizing the interactions between polymers chains.

Fig (27 F, mag. 1500) illustrates post irradiated (F-5) film and shows small

clusters in semi uniform distribution implying some interactions and slight

homogeneity upon irradiation. The boundary surfaces on the polymers chains

bind favorably with each other on applying γ-rays to form less rough surfaces

than the un-irradiated films, however increasing of the surface roughness over

that of the (Ca-containing formula) was detected. This dense form did not

produce a uniform permeable network with strong structure.

In general, as can be seen even after irradiation, morphological differences

could be spotted easily between the 2 irradiated (Alg/Ch/CaCl2 and Alg/Ch

/CaCl2-free membranes) (Figures 27 B, D, F, respectively). (Alg/Ch/CaCl2

film) showed more uniform network structure while the (Alg/Ch/CaCl2-free

film) indicated less homogeneity and porosity which were proved through the

hygroscopic characterization and physical tests mentioned in this study.

Lower stability and permeability of gel was indicated prior to irradiation and

this will affect negatively the main function of the membrane as a wound

dressing. Indeed, permeability is needed in the wound healing process with

accelerating the fibrous tissue formation. These findings were studied then with

a wound model in rats, accompanied with examining biopsy samples that were

eliminated after treatment as mentioned in the current study.

Discussion

174

The used ratio between the different incorporated alginates was (1:1:1), but

different ratios of alginates were supposed to have different effects on

properties of the previously chosen hydrogel, (F-20), so new three forms of the

same formula with the same components were prepared as follows: (F-20/I):

[A+B+D (2:1:1) + (CaCl2)] + (Ch)]& (F-20/II): [A+B+D (1:2:1) + (CaCl2)] +

(Ch) and (F-20/III): [A+B+D (1:1:2) + (CaCl2)] + (Ch). Their properties were

compared to reach the structure of the best working film with assessing their

different properties.

The alginate chains of low M.Ws seemed to interfere with the coacervation of

chitosan with the (HMW-Alg (Alg-A)) due to their easier diffusion to the

chitosan chains for a high degree of complexation. During swelling, diffusion of

the small chains to medium is limited due to the cross-linking with cations. As

can be concluded generally for (F-20) from (fig. (28)), maintaining constant

equal ratio between (A: B: D) gave the resulted gel high swellability for

medium molecules. The increase in ratio of (A-fraction) seemed to enhance the

stability of the resulted gel with stronger crosslinking.

With the increased initial imbibing ability for medium by the weak cross-linked

small and long chains, cleavage of these weak cross-linkages would be

accelerated. On swelling, the small chains begin to move faster at first to the

medium causing theoretical decrease in swelling, but the gel mass and

swellability were maintained by the coacervated alginate long chains. This is

shown with the swelling curve of the form (F-20/I) which after the early fast

swelling, the D.S began to decrease, but still higher than the other forms. The

irradiated (F-20/I) was compared with the corresponding unirradiated form. The

(F-20/I/Un) showed good D.S, but weaker than the irradiated one. These results

confirm the effects of irradiation on the complexation state within the gel

structure.

Discussion

175

Further studying for properties of the forms (F-20/II and F-20/III) was

cancelled due to their weak swelling abilities that may relate to the fast release

of the weakly cross-linked small chains to medium.

The extent of water loss from the hydrogels on exposure to air was evaluated to

examine their behavior on using as dressings over dry wounds. This water loss

can enable the gels (F-20 and 20/I) to take up the exudates from the wound by

an active upward-directed process to help in its moisturization for further

accelerating of healing (fig. (29)).

The irradiated (F-20/I) gel expressed higher rate of degradation in vitro than (F-

20) after (3) days, may be due to the initiation of gel syneresis after short

swelling period on immersing in medium during the first 24 hours, and it

continued in the following days. This degradation behaviour was compared with

that of the corresponding unirradiated formulae which displayed slower rates of

degradation (fig. (30)). The change in integrity of the gels after irradiation

accompanied with the faster swelling may have initiated this faster degradation.

(According to the previous results, the PEC hydrogel of the form (F-20) was

used as the model of dressings and its efficacy was tested in vivo with the

healing of wounds models).

For comparing the efficacy of drugs loading onto PEC hydrogel from the

formula (F-20), Albumin and Curcumin-loaded samples on the forms (F-20

&F-20/I) were prepared under optimum conditions and tested as drug loaded-

models. This highlights the ability of the prepared membranes to act as wound

dressing as well as drugs and antibiotics carriers for further in vitro and in vivo

release systems studies.

Discussion

176

During the preparation of loaded membranes, the used protein model was water

soluble and Curcumin was soluble in organic solvents. Unlike the SEM images

of their virgin membranes, the (irradiated Albumin loaded samples) showed

smooth yet cracked surface layer with more uniform distributed lamination over

the matrix of the prepared membrane in case of the film (F-20/I, fig. (27G))

than that of the loaded film (F-20, fig. (27 H)).

Curcumin loaded samples formed big non-uniform clusters with fusion form

and observed crystallization of the drug in case of (F-20-loaded film, fig. (27

J)) and (F-20/I-loaded film, fig. (27 I)) as well.

These results might indicate a homogeneity and well distribution of drugs in

case of (Albumin) over the prepared membranes in case of (Curcumin) samples

and this of course will be reflected in the texture, success percent, hygroscopic

characterization and performance of the complex during the healing process

cascades. Further studies for the mechanism of entrapment of each drug, the

changes within the hydrogel structure after entrapment, optimizing the

conditions of preparing these new devices and the release rate of the two drugs

from both (F-20 forms) are required to be studied as well.

Discussion

177

3.6. Wounding and Wound Healing:

Rodents are the indispensible tool of choice for most researchers as models for

human diseases where they require little space, easy to handle, inexpensive and

have accelerated healing compared with humans, thereby yield faster results

(Scherer et al., 2008). Murine male skin is 40% stronger than female skin due

to a much thicker dermis, while female skin exhibits a thicker epidermis and

hypodermis. (Azzi et al., 2005). Accordingly, using male rats in the study

would provide easier method for comparing effects of the hydrogel between the

treated and un-treated wounds.

Numerous wound healing components including hemostasis, inflammation,

angiogenesis and matrix deposition are impaired in wide variety of tissues

healing including myocardium, skeletal muscles, and nerves, so it was essential

to measure blood glucose level for the studied rats and exclude the diabetic

members.

Both the wound models, excisions and incisions, are valid in studying the

healing with well recognized advantages and limitations for both. These two

models have met some criticism because the main mechanism of healing in

rodents is contraction due to the presence of the panniculus carnosus muscle in

the subcutaneous tissue but human wounds; in contrast heal more through re-

epithelialization (Falanga, 2005). In spite of that along with observing the

healing of large open acute wounds in rodents, the human display a significant

amount of contraction with the excisional models but this is of less

consideration with the incisional ones that more closely represent the clinical

situation of acute surgical wounds. All excisional healing models are based on

the same principles (Scherer et al., 2008): (1) Robust

inflammation/angiogenesis are hallmarks of this model. (2) Granulation and

epithelialization can be examined with more accuracy (Wong et al., 2011).

Discussion

178

(3) This model supports the evaluation of new topical pharmacological

interventions as medications that can be applied directly to the wound bed

(Tsuboi and Rifkin, 1990; Toker et al., 2009). (4) The excision may be a

better model to evaluate cosmesis, despite the smaller scale of the injury

response (Wilgus and DiPietro, 2012).

A single standard outcome for wound healing does not exist, but some objective

and subjective measures are widely used by the researchers. Within health care

organizations, the % reduction in surface area is often used as an indicator for

the wound response to the treatment. No wound repair occurred completely for

the non-dressed wounds during the study period (Fig (31)) where the formation

of a scab loosely united the wound edges and contributed to a dry environment

that may also have retarded the healing (Hinman and Maibach, 1963). The

faster contraction in size of wounds treated with the hydrogel (Dre) than the

other groups may be due to the physical forces, initiated by the adsorption of

various protein molecules from the wound surface into the dressing

components, especially the chitosan fraction (figures (32, 33)).

3.6.1. The histological assessment of the healing progression:

A quantitative assessment strategy for wound healing cascades was established

based on histological criteria. They identified the different healing phases

including inflammatory response, re-epithelialization, cells migration, regulated

proliferation as well as the epidermal differentiation. Dermal closure, matrix

distribution and skin remodeling along with observing dissolution of the

hydrogel components during the timeline of healing progression occur in a

parallel and sequential manner and they were also identified with the

histological characterization as follows:

Discussion

179

During the 1st

and 3rd

observation days: Marked necrosis associated with

inflammatory cells infiltration and marked edema were observed in the post-

wounding days (1C, 1D, 3C, and 3D) of both the dressed and control wounds

within the superficial layers with mild hemorrhage, so it was difficult to

differentiate between the two groups (fig. (34 a), H, E). Inflammation is

prerequisite to healing and can be initiated by various causes (e.g., Injury or

may be due to the effect of the added dressing materials), so the main cause

couldn`t be judged. The dressed wound showed congested blood vessels with

few early granulation tissues (EGT) which have close linkage to the extensive

proliferation of fibroblasts (Fig. (34 c), H, E).

Abundant growth of new fibroblasts was observed subsequent to attenuation of

the initial inflammatory response (Liorabraiman-Wikeman, 2007) and they

have close association with the formation of new capillaries (Fig (34 c)), wound

contraction and movement of the entire dermis with the first manifestation of

the early matrix. (Ueno et al., 2001) have suggested that chitosan can accelerate

the granulation tissue formation. With (MT-staining, no collagen fibers were

observed except on the edges of the (V-shaped clefts). These clefts extended

down into the subcutaneous tissue after the 1st

day of treatment (data not shown)

and after 3 days (for control) (fig. (34d)).

During the 7th

observation day: The 2 groups could be distinguished. Figures

(35 a (7D) and b (7C), H, E) displayed degree of necrosis with inflammation

which were weaker with the dressed wounds sections. Focal regenerative

epidermal cell layer was observed with the dressed wound to derive the

reepithelialization process (fig.(35 a), H, E). Collagen formation is initiating

step of the healing process (Ruszczak, 2003 Asadi et al., 2010) and the tensile

strength of wound tissue depends on the amounts of its fibers, their orientation,

maturation and formation of crosslinks between them.

Discussion

180

The MT-stain showed massive collagen fibers deposition within the skin

epidermis for (7 D-stained sections (fig. (35 c)) and in the following days.

The stratum germinativum layer with prekeratin filaments, Cells of Stratum

Spinosum (SSCs) with interconnecting desmosomes for promoting their

adhesion, the Stratum Granulosum Cells (SGCs) with keratohyaline granules

and keratin squames of the Stratum Corneum Cells (SCCs) with intervening

spaces representing shrinkage artifacts have been observed with sections from

only treated wounds. The observation of these newly growing cells was in the

same period of observing a dressing remnants in the form of fragmented gel

structures in pink color as illustrated in (figures (35 a (H,E) and c (MT)). This

may suggest the breaking of the cross-linkages and other bonds within the PEC

structures with releasing free polymers chains at the same time with increased

proliferation of the new epidermal layer cells. (Kiyozumi et al., 2006) have

suggested that the infiltrating neutrophils to wound bed can promote chitosan

hydrogel degradation as well.

During the 11th

observation day: Granulation tissues were detected with the

new skin in sections of (11C& 11 D (figures (36 a and d),H, E). Moderate

collagen deposition was observed in the stained (11 D) sections (fig. (36 g),

MT), but deposition of massive collagen fibers was observed in the case of

stained control healed tissue (not shown) with healing patterns in the epidermal

and dermal layers, but with penetration of hemorrhage to the collagen fibers of

these stained sections.

For the 15th

day of observation: Sections of (15 C) showed regenerated

epidermal and dermal layers with inflammatory cells infiltration, granulation

and necrotic tissues formation (fig. (36 b), H, E). Adipose tissues (A.T) with

sections from both wounds tissues types were observed.

Discussion

181

The defect area became small with the treated wounds with the observation of

regenerating proliferated epidermal cells resting on granulation tissue (fig. (36

e), H, E) and absence of inflammatory cells. Marked collagen fibers deposition

was observed with the (15 C) sections of new skin within the hypodermis layer

(fig. (36 c), MT), but few collagen fibers deposition was observed with the

(15D) sections (fig. (36 h), MT). These observations may meet an aim of the

study for preparing a dressing that allows synthesis of thin collagen fibers with

little scar tissue that can result in a reduction in healing time. Although the

collagen deposition with using the hydrogel dressing was massive at the early

healing days (fig. (35 c, MT), its degree decreased with time in accordance with

the complete skin regeneration (figures (36 g, h and 37 b), MT)).

The (MT-staining) displayed also (melanin) in the (stratum germinativum) layer

with representation of melanocytes and keratinocytes (indicated by arrows) (fig.

(36 h), MT). In light skin, the melanin is broken down before it leaves the upper

part of the (stratum spinosum) so it was observed mainly in the deep epidermal

layers. It is illustrated also that the treatment of wounds with the gel dressing

allowed reconstruction of both the dermis and dermal–epidermal junctions with

re-epithelialization within the full thickness skin defects. Epidermal rete ridge-

like structures were observed with the (15 D) sections (figures (36 e, f, and h),

H,E) with much depth and projection into the dermis. Though the normal skin

of murines lacks these protrusions, they may become apparent during the rodent

wound healing and often described as pseudocarcinomatous hyperplasia

(Sundberg, 2004). Normally, they create a more extensive interface between

the dermis and epidermis and help the skin resist shearing forces. Generally,

they are increased at the sites with increased mechanical stress on the skin.

After about 1 day (Day: 16), most skin layers components were observed within

the stained sections of dressed wounds.

Discussion

182

The observed thickness of epidermis was large due to the thickness of the

(stratum corneum) that was more lightly stained than the deeper epidermal

portions (fig. (37 a), H, E). Sweat Glands (SwG) for secretion of watery sweat

all over the skin were also observed in the upper part of the hypodermis with

several Sweat Ducts (DSwG) passing through the epidermis (fig.(37 b), MT).

The skin seems to be regenerated with the development of Sebaceous glands

(SGl) from epithelial cells of the (hair follicles (HF)) and discharge their

secretions into the follicles from where they reach the skin surface to moisturize

and protect it. These sebaceous secretions include the entire cell which secretes

through the Holocene mode, in which the cells burst and therefore they need to

be replaced constantly in the functional gland. Cells at the periphery of these

glands are basal cells (BCs) which divide and replace those that are lost with

the secretions (fig. (37 a)).

Although gross examination of the healing wounds illustrated early hair growth

on margins of the Hydrogel-treated wounds, observing of complete hair

structure through the histological examination was after 2 weeks. These results

show that the prepared hydrogel promoted significant skin maturation with

mature epithelial morphology and hair follicles after two weeks. This may

suggest that it facilitates epithelial cell migration or homing to the wound area

and supports epithelial and other skin cells differentiation.

3.6.2. Quantification of mRNAs by real-time PCR:

The healing of a wound requires well-orchestrated integration of complex

biological and molecular events of cells migration, proliferation and ECM

deposition (Baum and Arpey, 2005). Each event is modulated by a vast array

of cytokines forming an elaborate communication network which coordinate the

healing process as a whole.

Discussion

183

New understandings in its complexities; particularly roles of the cytokines,

enable clinicians to manage superficial wounds (e.g., Skin flaps and even the

most difficult-to-heal types more effectively) (Brem and Tomic-Canic,2007).

To confirm the production and roles of VEGF during wound healing, we

quantified its gene expression by the sensitive and reliable real-time

fluorescence PCR technique. In the first phase of wound healing, VEGF

stimulates the coagulation factors in the ECs; therefore, the thrombocytes

accumulation and adhesion occur (Banks et al., 1998; Nogami et al., 2007). It

has been previously reported by neutralization experiments that Basic Fibroblast

Growth Factor (bFGF) accounts for a large part of the immediate angiogenic

stimulus in the wound area (Nissen et al., 1996; Takamiya et al., 2002)

without any participation of VEGF in the initiation of angiogenesis. The

neutrophils; recruited to wound area directly after wounding as a part of the

inflammatory response, have been described as (VEGF-negative secretory cells)

(Hayashi et al., 2004). These previous observations may explain why the

VEGF gene expression in our experiments was weaker at the first wounding day

than the 2nd

one (figures (38 and 39)).

For blood vessels formation, immuno-localization of VEGF increases in the

inflammatory cells during the inflammation phase as well as in ECs, fibroblasts

and macrophages in the following phases (Banks et al., 1998; Miyagami and

Katayama, 2005; Nogami et al., 2007). This recruiting along with activation of

the respiratory burst (Babior et al., 1973; Khanna et al., 2001) may explain the

significant rise in VEGF mRNA at day 2 of normal healing to attract

metabolites and oxygen to the healing region (Hayashi et al., 2004) with

maintaining these levels in the following days. Our results showed increased

VEGF gene expression levels after the 1st week of normal healing.Maintaining

high certain levels of VEGF expression and its mRNA is essential to activate

the required angiogenic steps for accelerating the healing.

Discussion

184

CD68-positive mononuclear cells, ECs and fibroblasts can sustain the VEGF

expression 7 days after the wounding indicating the possibility of its

involvement in the chemotaxis (Nissen et al., 1998) and vascularization

(Hayashi et al., 2004). Previous studies by (Nissen et al., 1998, Altavilla et al.,

2001; Nogami et al., 2007) have observed that VEGF mRNA has significant

high levels after one day of normal healing compared with the 3rd

and 7th

days.

There is an early appearance of fibroblasts in wounds treated with the dressing

along with the EGT (fig. (34 c)). These observations along with the reports of

(Okamoto et al., 1995; Ueno et al., 1999; Ueno et al., 2001; Alemdaroglu et

al., 2006) who found out faster recruiting of leukocytes and macrophages to the

wound area treated with chitosan may explain the early high levels of VEGF

during healing of the treated wounds with our hydrogel than the untreated ones

(fig. (38)). In addition, (Ueno et al., 2001; Inan and Saraydın, 2013) have

suggested that chitosan might stimulate recruiting and proliferation of

inflammatory cells during the early inflammation phase.

The growth factors including VEGF along with other fibroblasts cytokines in

the wound granulation tissue during the early healing phases might also increase

and last for only a few days (Alemdaroglu et al., 2006). Results of (Ying,

2011) as well found out that the effects of alginate on wound healing may

involve important roles associated with the expression of the cytokines VEGF

and TGF-β1. In summary, these reports showed increased expression of VEGF

during the healing days, especially the 7th

day of wounds treated with alginate

dressing and over that of the untreated wound. These are the same results

obtained using our newly prepared hydrogel although it consists of a complex of

alginate with chitosan. This refers that both the two polyelectrolytes within the

complex maintained their effects on VEGF gene expression with more

enhancement after complexation.

Discussion

185

(Lee et al., 2009) have observed suppression for VEGF protein with wounds

treated with alginate than those treated with Vaseline and normal healed

wounds. The discrepancy between our results and these observations may relate

to the prepared complex structure which may overcome the individual alginate

effects making the behavior of the complex membrane differ from some of its

individual component's behaviors.

(Blann and Taberner, 1995; Constans and Conri,2006) have observed that

the elevation in vWF levels is perhaps the most known marker of endothelial

injury. The decrease in level of blood vWF mRNA may relate to the direction of

its secretory cells towards the wound bed for the initiation of blood clotting.

During hemostasis, two steps in platelets adherence have been identified that

have a requirement for divalent cations. One step is the platelets spreading

reaction on subendothelium which has a need for factor VIII-von Willebrand

factor (FVIII-vWF) and Ca2+

or Mg2+

(Sakariassen et al., 1979; Bolhuis et al.,

1981). Some (vWF-related proteins) are already present in the subendothelium

(Rand et al., 1980), but the (FVIII-vWF) of plasma is required for optimal

adherence where it binds to the subendothelium after deendothelialization and

prior to the platelets adherence (Weiss et al.,1978; Sakariassen et al.,1979;

Bolhuis et al., 1981).

The second step is the facilitation of platelets adherence by (FVIII-vWF) that

has a requirement for Ca2+

(Sakariassen et al.,1984). Sufficient Ca2+

may be

associated with (FVIII-vWF) to change the microenvironment at the

subendothelium surface. This causes a small increase in platelets adherence

(Mikaelsson et al., 1983). Solubilization of the used dressing with releasing of

Ca2+

ions may be a reason for more activation of (FVIII-vWF) in wound bed

causing faster clotting.

Discussion

186

This may explain the reason for the decrease in blood levels of vWF mRNA

during the first post-wounding week comparable to the undressed wound though

the differences were not significant. The higher blood vWF in the un-treated

rats may also reflect higher release of vWF antigen from ECs and/or platelets

than in the treated rats.

vWF is a major part in the WPBs where it is involved in their biogenesis and

used as a marker of them (Rondaij et al., 2006; Sadler et al., 1991) and the

decrease in its expression causes the release of WBPs components including

(Ang-2) which may act synergistically with VEGF to promote angiogenesis.

This WPB exocytosis gives rise to a rapid release of vWF and other mediators

(e.g., interleukin-8 (IL-8)) (Babich et al., 2008) with translocation of P-selectin

from within granules to the endothelial surfaces for triggering leukocytes rolling

are all critical early events in the endothelial activation and vascular

inflammation (Wagner, 1993).

It has been reported that VEGF regulates the vWF/WPB release(Matsushita et

al., 2005) where it rapidly induces a time and dose-dependent vWF release from

human venous endothelial cells. Accordingly; as concluded from our results, the

raise in levels of VEGF from day 2 to reach its greatest levels at day 7 may

explain the corresponding decrease in vWF mRNA in the blood secretory cells.

Alternatively, the decrease in VEGF levels after the 7th

day with the untreated

wounds may explain the high corresponding increase in the mRNA of blood

vWF. For the dressed wound, the gradual decrease in VEGF mRNA is also

responsible for the gradual increase in vWF mRNA levels (fig. (41)).

Due to economic reasons, the expression of vWF gene has been quantified in

blood cells but further experiments are suggested to measure its levels in the

wound bed tissue with concentrating the studies in the blood vessels tree for

better understanding of the effects of the degraded gel in the wound bed.

Discussion

187

The overall result from these tests with using the prepared hydrogel as a

dressing without the addition of growth factors or cytokines seems to be the

enhancement of angiogenesis through accelerating the recruitment of ECs to the

wound area. This neovascularization facilitates cells and nutrients transportation

as well as oxygen and wastes exchange and these are all critical conditions for

the optimum wound healing with accelerating ECM production for perfect skin

regeneration.

Biomaterials are defined as the materials that can be interfaced with biological

systems in order to evaluate, treat, augment, or replace any tissue, organ or

function of the body. Any material to be used as a part of a biomaterial device

has to be biocompatible and its clinical applications should not cause any

adverse reactions in the organism nor endanger the life of patients. BUN and

creatinine levels were in the range of normal values with no significant

difference between the untreated rats and those treated with the hydrogel

(Table: 9). This refers that solubilization of its polymers chains does not affect

the physiological functions in the body.

Recommendations

188

Recommendations for Future Work Preparation of New Drugs-Loaded Wound Dressings:

Loading of 2 drug models was tested at the end of the study with (SEM), but more tests are

required to get an overview of the mechanism of binding of each one to the hydrogels as well

as the release mechanisms and rates in vitro and in vivo. Loading of new drugs is also

essential for enhancing the accelerating effects of the virgin hydrogel dressings on wound

healing, especially for the treatment of ulcers. Examples of these drugs may involve loading

of silver, charcoal, antibiotics, enzymes or nano-materials whose efficacies can be tested

through both in vitro and in vivo studies. These studies may require modifications for the

structures of the composing polymers, blending of new materials, as well as preparing new

hydrogels forms; maybe in the hydrocolloid, sponge, multi-membrane forms, and so on. This

requires more understanding of the chemical and physical properties of these materials and

may open the way to synthesize new materials to produce new wound care products.

Further studies for the healing wounds: More examinations are required for the treated healing wounds for understanding and giving

more proofs of the supposed scaffolding action of the hydrogels. The angiogenic response in

wounds beds was only studied with histological examinations and measuring the expression

of 2 angiogenic genes, but more experiments are required to get a complete overview on the

mechanism of angiogenesis and understanding the effects of the hydrogel on the normal

angiogenic cascades. Immuno-histochemical analysis for the infiltrating cells, measuring the

expression of more angiogenic genes and Doppler imaging of the wounds beds may be

suitable methods. This study focused on the angiogenic effects of the treating hydrogels and

skin maturation, but more studies are required to assess the complete mechanism of wound

healing as well as the scarring response after treatment at the molecular and cellular levels

with including the different cells involved in the healing stages. The different involved

molecular and cellular processes such as signal transduction, proliferation, migration and

growth factors production after the treatment should also be studied. More wounds models

can also be created (e.g., Ischemic flaps, mechanical load model, parabiosis model and

pressure ulcer model) in the future studies as well to overcome the differences in wound

healing mechanisms between rats and human.

Summary

189

Summary

Skin is the first line of defense from external factors all over the place. It holds

all organs together, protects our bodies, helps keep them at just the right

temperature and allows us to have the sense of touch. Skin wounds often occur

as a result of accident or injury. Surgical incisions, sutures and stitches also

cause wounds so the wound management is essential to promote healing of the

underlying soft tissue and treat or reduce the risk of infection.

We first prepared a Polyelectrolyte complex through the binding of alginate

with chitosan incorporating certain functional groups and proved its

biocompatibility and integration with the host tissue. A new type of wound

dressings in hydrogel film form based on this complex was prepared where the

2 polymers were mixed together with a new preparation method. At first, the

alginate molecular weight and chemical structure were tailored with (γ-

irradiation and oxidation), followed by characterization of their properties

through measuring their M.Ws and the degrees of oxidation for the modified

oxidized structures as well as assessing the resulting structures with (FT-IR).

The alginates were then purified and the purification degree was confirmed.

Different chitosans were extracted from shrimp shells and were characterized

by (FT-IR). Their degrees of purity, M.Ws and antioxidant properties were

investigated as well.

A scheme for choosing the best structure from different prepared hydrogels for

using as a dressing was suggested involving different physical, chemical,

spectroscopic and biological approaches. The chosen hydrogel was then

applied to excisional wound model in rats to improve the characterization of

its biological effects through monitoring of its effects on healing with

measuring the wounds closure rates.

http://www.nlm.nih.gov/medlineplus/injuries.html

Summary

190

The histological examinations for skin and wounds beds and tests for detecting

the expression of the angiogenic genes (VEGF& vWF) with the routine

measuring of kidney functions for the wounded rats were performed also to

test the effects of solubilization and degradation of these biomaterials

The chosen formula was (F-20) with the structure consisting of (chitosan and

different alginates of the types (High M.W+ Very low M.W+ Multi aldehyde

Sodium Alginate (Type I-A)). The in vivo study of its effects on wound

healing included (3) groups of wounded rats; single right wound was created

on the dorsal skin of each rat:

Group (1) (G. (1)): The created wounds were treated with the dressing.

Group (2) (G. (2)): The created wounds were treated with Fusidin cream.

Group (3) (Control group (G. (3)): The wounds were left untreated.

With continuous monitoring for wounds of these groups during the following

days, analysis of the results revealed that:(1) The wound closure rate % was

about 98 % after 11 days of healing for the hydrogel-treated wounds (G.(1)),

but after 2 weeks for the fusidin-treated wounds (G. (2)) and only 95 % was

achieved for the non-treated wounds (G. (3)) during the same period.

(2) The histological examination for skin and wounds beds of both (G. (1 and

3)) proved the efficiency of the used hydrogel to promote healing with

accelerating the remodeling of wounds tissues and its regeneration as well as

promoting the angiogenic response compared to the non-treated wounds.

(3) The hydrogel dressing (G. (1)) maintained certain levels of VEGF mRNA

in most post-wounding days and after the 7th

day with vWF which guaranteed

the acceleration of suitable blood supply to the injured tissues in addition to

performing their other functions. These genes expression levels were

significantly lower with the non-treated wounds (G. (3)).

References

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Appendices

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APPENDICES Appendix (A): The different groups frequency wave-numbers (cm

-1) for

the Raw charcoal and different washed charcoals:

Functional Groups Assignments Absorption Position (cm-1)

References

Raw Charcoal *isolated O–H groups 3765-3755 -Puziy et al., 2003;

Budinova et al.,2006

*O–H stretching mode of (-OH)

groups and adsorbed water 3440

-Puziy et al., 2005;

Özgül et al., 2007

*Aliphatic, asymmetric C-H

stretching vibration of (-CH3 groups) 2974

*Aliphatic, asymmetric C-H

stretching vibration of (-CH2 groups) 2921

*Symmetric C-H stretching of (-CH2) 2856 *Absorbed water molecules on KBr 1627 -Nakamoto, 1986;

Shaffer et al.,1998

*Ring vibrations in large aromatic

skeleton in carbonaceous materials 1590–1500 -Sun et al., 2001

*C-O stretching vibrations

in alcohols and ethers 1200-800 -Puziy et al.,2005; Guo

and Rockstraw, 2007 *(-CH2) bending vibrations 1454 -Painter and Starsinic,

1985; Ros et al., 2002 *Aromatic C-H out-of-plane

bending vibration 873 Painter and Starsinic,

1985 *Aromatic CH deformation 900-700 Guo and Bustin, 1998 *Out-of-plane (O-H) bending mode 706

Spectrum of Charcoal, washed with (HNO3) *Asymmetric stretching vibrations

of C=O 1644 Painter and Starsinic,

1985 *Increase in the already available

Oxygen functionalities indicating

stronger oxidation

1384 Naasser and

Hendawy, 2003

*(N–O) asymmetric stretching 1570-1500

*(N–O) symmetric stretching 1395 -1355

Spectrum of Charcoal, washed with (H2SO4) *Asymmetric stretching vibrations

of C=O 1644 -Painter and Starsinic,

1985

Spectrum of Charcoal, washed with (HCl) * sp

3 CH3 bending modes 1375

*alkane (C-C) stretching modes 822



Appendices

236

Appendix (B):The groups frequency wave-numbers for sodium Alginate


References

* free (O-H) groups stretching 3444 *Aliphatic, asymmetric C-H

stretching vibration of (-CH2 groups) 2931 -Zhu et al., 2012

*Asymmetric stretching vibrations

of C=O in (-COO-) groups

1623 -Soares et al., 2004;

Kalyani et al., 2008

*Symmetric stretching vibrations

of C=O in (-COO-) groups

1419

*Skeletal vibrations of the alginate

polysaccharide (C–C–H and

O–C–H stretching)

1298 -Lawrie et al., 2007

*(C-O stretching) vibrations 1090 & 1041

*(C–C stretching) vibrations 1050-900 -Sartori et al., 1997;

Xiaoxia et al., 2010

Appendices

237

Appendix (C): The different groups frequency wave-numbers (cm-1

) for

the two prepared chitosans (Ch-1 and Ch-2):


References

*O–H stretching mode of (-OH)

groups 3450

*Asymmetric C-H stretching vibration

of (-CH2 groups) and pyranose ring 2900

*Asymmetric stretching vibrations

of the superimposed (C=O groups)

of the (amide bond I), linked to (-OH)

groups by H-bonding

1620 -Limam et al.,

2011

*In-plane bending vibrations of (NH2

groups) in non-acetylated 2-amino-

glucose 1ry

amine

1605-1590 -Pearson et al.,

1960; Pawlak and

Mucha, 2003 * N–H deformation band of (amide I)

Or N-H stretching of (amide II) 1545 -Ravindra et al.,

1998; Duarte et

al., 2001 *(N-H) stretching of (amide I) 1430-1425 -Sankalia et al.,

2007; Silva et al.,

2008 * stretching vibration of (amide III) 1365-1360 -Zhang et al., 2011 *(CH2) bonds wagging 1330-1325 Brandenburg and

Seydel, 1996; Hein

et al., 2003 *(C-OH) stretching vibration 1250-1200 Coates, 2000;

Stuart, 2004 * (C-O-H) out of plane bending

and (CH2) twisting 950-750

الملخص العربى


يعد الجلد أكبر أعضاء الجسم ويتكون من عدة طبقات التى تحمى العضالت والعظام واألعضاء الداخلية ولها عدة وظائف حسية وفسيولوجية مثل الحماية من فقد الماء المتزايد

قد الجلدية الجروح. بالجسم باإلضافة إلى التنظيم الحرارى للجسم والحماية من الطفيليات

أو ( الجروح المفتوحة)وتعريض الطبقات الداخلية للهواء الجلد طبقات من أى إزالة من تنتجالتئام الجروح يتم من خالل مجموعة (. الجروح المغلقة)نتيجة لصدمات داخلية بدون جرح

التجلط ومنع النزف، االلتهاب، تنشيط انقسام )من المراحل المتتابعة والمتداخلة وتشمل (.وأخيرا إعادة بناء التركيب العام للجلد المنزوع الخاليا،

تهدف هذه الدراسة إلى وضع طريقة جديدة لتحضير أفالم رقيقة من هالم مائى

الكيتوزان المستخلص من قشر ) صنوع من متضاعف اليكتروليتى مكون منم( هيدروجيل)واقتراح خطة من مجموعة من االختبارات (الجمبرى واأللجينات المحور اشعاعيا وبالتأكسد

التى تهدف الختيار أفضل هالم يمكن استخدامه فيما بعد كضمادة للجروح فى صورة دعامة للخاليا الجديدة إلعادة هيكلة كل من طبقة البشرة و األدمة فى الجلد المتمزق وتكون قادرة

الجى للهالم المختار على الجروح ثم يتم دراسة التأثير الع. على مدها بالتركيبات العالجيةالمفتوحة فى جلد الفئران من خالل مجموعة من الدراسات النسيجية والبيولوجية الجزيئية

عامل نمو )عبر قياس تعبير بعض الجينات المسئولة عن تكوين األوعية الدموية وتشمل (.بطانة األوعية الدموية وعامل فون ويلبراند

لجينات أوال من الشوائب وتعديل تركيبه الكيميائى باستخدام اإلشعاع وقد تم تنقية األ

الجامى واألكسدة المتعمدة باالضافة إلى استخالص الكيتوزان من قشر الجمبرى وتم الدمج بينهما بعدة صور بطريقة جديدة لتحضير عدة تركيبات مشععة تم المفاضلة بينها من خالل

اختبارات االنتفاخ، التشتت، التوافق مع الدم، معدل )مجموعة من االختبارات التى تشمل وكذلك تم دراسة تأثير االشعاع على مواصفات التركيبات المختارة ( تبخير الماء والتحلل

تم اختبارها على الجروح المفتوحة فى ثمواختيار أفضل تركيبة وبنسب معينة بين مكوناتها .جلد الفئران

المكونة من F) -02)المختلفة السابقة تم اختيارالهالم اعتمادا على االختبارات

ذات الوزن الجزيئى )المستخلص ومخلوط األلجينات ( Ch-1)الكيتوزان VLMW-50)ذات الوزن الجزيئى المنخفض جدا بواسطة االشعاع +(HMW)العالى

KGy)+ االلجينات المؤكسد(MASA-I A) (1:1:1)بالنسب).

هالم المختار على نوع من الجروح الحادة فى الفئران والتى تم من تم أوال تجربة ال :مجموعات( 3)خاللها تقسيم الفئران المجروحة إلى

تم معالجة الجرح الظهرى فى جلد كل فأر من هذه المجموعة بتركيبة : المجموعة االولى- .الهالم المختارة


فأر بكريم الفيوسيدين تم معالجة الجرح الظهرى فى جلد كل: المجموعة الثانية-لم يتم معالجة الجرح الظهرى (: Control-1)( )1)المجموعة الضابطة )المجموعة الثالثة -

.ألى من هذه الفئران وتركت لتلتئم بدون أى معالجات

ها بالمجموعتين األخرتين على جلد الفئران ومقارنت تأثير الهالميمكن تلخيص نتائج و :يلى كما

الثالثة حيث تم معدل إغالق الجرح فى المجموعة األولى أسرع من الثانية أسرع من ( 1)فى المجموعة بعد الجرحشر عحادى فى اليوم ال %( 89)بصورة شبة تامة اغالق الجرح

،أما فى المجموعة الثالثة فقد ( 13)األولى ،وفى المجموعة الثانية تم أقصى إغالق فى اليوم .%( 89)للجرح بعد أسبوعين من الجرح بمعدل وصل أقصى إغالق

تم مقارنة الجروح فى المجموعتين االولى والثالثة حيث : على المستوى النسيجى( 0)

أظهرت الدراسة بداية مرحلة االلتهاب فى كال المجموعتين مصحوبة بتكوين خاليا ليفية فى ة لطبقات الجلد المختلفة مع أنسجة الجروح المعالجة بالهالم فقط مع ظهور الخاليا المكون

البقايا المتفتتة من الهالم فى اليوم السابع فى حالة المجموعة األولى واكتمال تكوين طبقات أما فى حالة المجموعة . الجلد المختلفة بكامل مكوناتها وتنظيمها بعد أسبوعين من الجرح

.(19)الثالثة لم يحدث اكتمال للتكوين حتى اليوم ال عامل نمو بطانة )بمقارنة التعبير الجينى لكل من : بالنسبة لقياسات البيولوجيا الجزيئية(3)

فى المجموعتين األولى والثالثة أظهرت المجموعة ( األوعية الدموية وعامل فون ويلبراندعامل نمو بطانة األوعية )األولى تحسنا ملموسا أثناء حدوث االلتئام فى أغلب األيام فى حالة

مقارنة بالمجموعة الثالثة ولكن بدا التحسن الملموس من اليوم السابع فى حالة ( دمويةال .(عامل فون ويلبراند) بعد قياس وظائف الكلى للفئران من المجموعتين األولى والثالثة فى أيام االلتئام المختلفة (4)

جموعتين مما يدل لم يتم التوصل الى اختالف معنوى فى نسب الكرياتنين والبولينا بين المعلى ان تحلل مكونات الهالم فى جروح فئران المجموعة األولى لم تؤثر على النشاط

.الطبيعى الكلى وان التحلل ال يؤثر على الوظائف العامة للجسم

المستخلص العربى

المستخلص العربى

م مغلق، حاد أم ان الجرح، نوعه ماإذا كان مفتوح أنواع الجروح حسب المسبب، مكتختلف أ

مزمن، وكذلك فسيولوجية االلتئام وعليه يلزم اختيار نوع معين من الضمادات من بين عشرات

هذه الدراسة إلى تصميم نوع جديد من ضمادات هدفت. المتوفرة لكل نوع من الجروحاالنواع

وعمل االختبارات الالزمة على ( الكيتوزان و األلجينات)الجروح من نوعين من البوليمرات هما

المتراكب المتكون للتاكد من مواصفاته الفيزيائية والتركيبية والتحللية المفترضة ليقوم بتأثيره

اسة تأثيراته البيولوجية على الجروح الحادة فى الفئران من المساعد إلعادة بناء منطقة الجرح ثم در

النسيجى للجلد المنزوع ،وكذلك خالل قياس تأثيره على معدل اغالق الجروح ،وإعادة التركيب

قياس التعبير الجينى لبعض الجينات المسئولة عن تكوين األوعية الدموية خصوصا فى منطقة

.الجرح

الطيفية والفيزيائية والكيميائية تم اختيار نوع معين من المتراكبات والتى وبعد اجراء التحاليل

:أثبت التحليل االحصائى لتاثيره على الجرح الحاد النتائج التالية

سرع للجروح المعالجة بالهالم المختار عنه فى حالة الجروح المعالجة بالكريم عن معدل التئام أ-1

.الجروح الغير معالجة

تحسنا واضحا فى (المجموعة المعالجة بالهالم()1)أظهرت المجموعة : المستوى النسيجىعلى -2

(.الغير معالجة)االلتئام مقارنة بالمجموعة الضابطة

تحسنا ملموسا ( 1)اظهرت المجموعة : بالنسبة لقياسات البيولوجيا الجزيئية للجينات المختبرة-3

.الضابطة أثناء االلتئام فى اغلب األيام عن المجموعة

1)أثبتت قياسات وظائف الكلى تشابه النشاط الفسيولوجى وحيوية الكلى فى كال المجموعتين -4

(.والضابطة

دراسبت بيولوجية جزيئية لتقيين الدور العالجى للدعبهبت الوشععة على التقرحبت والجروح فى جلد الفئراى

رسبلة هقدهة هي الدارس

عبده أمير محمد محمد على بكبلوريوس علوم

(9002قسن الكيويبء والكيويبء الحيوية )

كمتطلب جزئى للحصول على

درجة الماجستير فى العلوم )تخصص كيمياء حيوية(

تحت إشراف

الدغيدى المنعم عبد اجالل/ د.أ ىمهد السيد محمد االسيد/ د.أ

المتفرغ الحيوية الكيمياء أستاذ الحيوية الكيمياء أستاذ االشعاعية البيولوجيا قسم حلوان جامعة-العلوم كلية عميدو

االشعاع وتكنولوجيا لبحوث القومى المركز

المصرية الذرية الطاقة هيئة

المزين محمد المنعم عبد حاتم/دأ.م. المغربى هللا عبد خالد طارق/ د.أ

الكيمياء الحيوية مساعدأستاذ أستاذ البيولوجيا الجزيئية

جامعة حلوان-كلية العلوم المركز القومى لبحوث وتكنولوجيا االشعاع

المصرية هيئة الطاقة الذرية

قسم الكيمياء

جامعة حلوان –كلية العلوم

2012

دراسبت بيولوجية جزيئية لتقيين الدور العالجى

الجروح للدعبهبت الوشععة على التقرحبت و

فى جلد الفئراى

رسبلة هقدهة هي الدارس

عبده أمير محمد محمد على

بكبلوريوس علوم

(9002قسن الكيويبء والكيويبء الحيوية )

جبهعة الونصورة

للحصول علىجزء متطلب ك

)تخصص كيمياء حيوية(درجة الماجستير فى العلوم

لىإ

جبهعة حلواى-كلية العلوم

قسن الكيويبء

9002

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