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MECHANISMS INVOLVED IN THE EFFECT OF CHITOSAN
IN ENHANCING HEALING

presented by

Asmaa Mohamed Saeed Elassad

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MECHANISMS INVOLVED IN THE EFFECT OF CHITOSAN IN ENHANCIING
HEALING

By

Asmaa Mohamed Saeed Elassad

A THESIS
Submitted to
Michigan State University
In partial fulfillment of the requirements
For the degree of

MASTER OF SCIENCE
Department of Pathobiology and Diagnostic Investigation

2006

ABSTRACT

MECHANISMS INVOLVED IN THE EFFECT OF CHITOSAN IN ENHANCIING
HEALING

By

Asmaa Mohamed Saeed Elassad

Chitosan is a natural biopolymer that consists of a poly [3 (1-4)
glucosamine. It has been described as being a wound healing accelerator. In this
study, two approaches were taken to investigate the biological basis of wound
healing enhancement by chitosan. Firstly, the antibacterial effect of chitosan was
tested in-vitro, focusing on two bacteria strains, Eco/i and S.aureus using the
microbroth dilution assay and the gel diffusion test. The microbroth assay
showed that chitosan and the acetic acid solvent had the same minimum
inhibitory concentration (MIC) ranges. Likewise, no zone of growth inhibition
was detected by gel diffusion test. In the second experimental approach, the
cellular response to the intra-pen'toneal implantation of chitosan pads in August
rats was defined over time and compared to Gelfoam®. ln-vivo, as early as 4
hours post-implantation, the cellular response induced by chitosan was
significantly more than that to the Gellfoam®. However, the reaction declined
with time with chitosan, but continued to increase to the Gellfoam ® pads. After
two weeks, there was minimum collagen deposition, i.e. minimal scarring,
associated with chitosan pads compared to that of the Gelfoam pads. The
cellular response to chitosan was seen to include cells of the immune system.

These results support the concept that chitosan enhances wound healing.

To my Mother, Aziza
&
To my brothers and sisters

Without their love and sustained support,
This could not be achieved

iii

ACKNOWLEDGEMENT
I must first sincerely thank my mentor Dr. Charles Mackenzie, to whom
I am grateful for his endless support, guidance and patience throughout my
study program, and whose help made this dissertation possible. Furthermore,
I would like to thanks my committee members Dr. Linda Mansfield, Dr. Rob.
Eversole and Dr. Matti Kiupel for their advice and invaluable input during the
meetings.

My special thanks to Dr. Linda Mansfield for her help and
encouragement and her permission to carry out the antimicrobial work in her
laboratory Thanks to all her laboratory members especially Dr. Julia Bell for
her continuous help, and Dr. Geetha Parthasarathy, and Dave Wilson for their
help in the experimental work.

My sincere thanks to Dr. Rob Eversole at Western Michigan University,
Kalamazoo, for his permission to carry out the rat experiment in his laboratory,
and also my thanks to Jeff Muston for his help in the rat expen'ments.

My thanks go to the members of Histology laboratory, Department of
Physiology, Amy Porter, Kathy Campbell and Rick Rosebury for their help in the
histology and immunohistochemistry part of the study. Special thanks to Denise
Harrison and Sherry Lennman at Pathobiology and Diagnostic Investigation
Department for their help in the administrative departmental issues.

My greatest thanks to my family and friends in US and back home, for

their lasting support and encouragement.

iv

TABLE OF CONTENTS

LIST OF TABLES .......................................................................... viii
LIST OF FIGURES ........................................................................ ix
CHAPTER 1. LITERATURE REVIEW ................................................ 1
Chitin ........................................................................................ 2
Chitosan .............................................................................. 5
Uses of chitosan ..................................................................... 8
Medical application ................................................................. 9
Antimicrobial activity ............................................................... 9

Wound healing ...................................................................... 10

Anticoagulant properties .......................................................... 1 3

Delivery of drugs and vaccines ................................................. 14

Immune response ................................................................... 15

Degradation of chitin and chitosan ............................................. 18

Hypothesis and objectives ........................................................ 19

CHAPTER 2. MATERIALS AND METHODS ....................................... 20

1. Microbial experiments ................................................................. 21

1.1. Measurement of the antibacterial activity of chitosan by

microbroth di|ution assay ..................................................... 21

Acid solvent effect ..................................................... 22

PH effect ................................................................. 22

Mode of sterilization effect .......................................... 22

Preparation of media ................................................. 22

Growth of bacteria .................................................... 23

Controls .................................................................. 23

Preparation of stock antibiotics .................................... 24

Microbroth dilution assay ............................................ 24

Microbroth procedure ................................................. 25

Microbroth data analysis ............................................ 26

1.2. Agar diffusion assay .......................................................... 26

Preparation of chitosan solution ................................... 26

Strains of bacteria .................................................... 26

Controls .................................................................. 27

Agar diffusion procedure ............................................. 27

Agar diffusion analysis ............................................... 27

2. Cellular response to intra-peritoneal implantation of chitosan
Animals .................................................................................. 28

Study plan ............................................................................ 29

Surgery ................................................................................ 29

Histotechnology ..................................................................... 29
Sectioning ............................................................... 29
Staining .................................................................. 30
Microscopy .............................................................. 31
Assessment ............................................................. 31
Scoring of the cellular response in the reactive zone ......... 31
Masson’s Trichome Procedure .................................... 33
Data analysis ........................................................... 33
3. Immunohistochemistry ............................................................... 33
Controls ............................................................................... 36
Grading system ...................................................................... 36
CHAPTER 3. RESULTS ................................................................. 38
1. Microbiological results ................................................................. 39
1.1 Microbroth dilution assay results ........................................... 39
Acid solvent effect ..................................................... 39
PH effect ................................................................. 43
Mode of sterilization effect .......................................... 44
1.2 Agar disk diffusion assay ..................................................... 45
2. Cellular response to intra-peritoneal implantation of
chitosan ...................................................................................... 47
2.1 Effect of chitosan implantation on the mononuclear and
polymorphonuclear cells ...................................................... 47
2.2 Effect of chitosan on the multinuclear cells .............................. 54
2.3 Effect of chitosan on the formation of blood vessels .................. 56
2.4 Effect of chitosan on the lymphocytes cells ........................ 57
2.5 Effect of chitosan on Collagen deposition .............................. 58
3. Immunohistochemistry findings ..................................................... 60
Characterization of T cells and B cells using CD3 and CDZO
Antibodies ............................................................................. 60
CHAPTER 4. DISSCUSSION .......................................................... 65
The antimicrobial activity of chitosan 66
The Cellular response to intra-peritoneal implantation of
chitosan ................................................................................ 70
Conclusion ............................................................................ 75
APPENDICES
Appendix A ........................................................................... 77
Appendix B ........................................................................... 78

vi

Appendix C ........................................................................... 80
REFERENCES ........................................................................... 81

vii

LIST OF TABLES
Table 1. Chitin content and types from different sources .......................... 3
Table 2. Chemical characteristics of chitosan from different sources .......... 8

Table 3. Characteristics of the microbroth dilution assay

samples .............................................................................. 21
Table 4. Reaction scheme in the Automiser staining system ..................... 35
Table 5. The MIC values of chitosan forms at different acid solvents ........... 39
Table 6. The MIC values of chitosan forms and controls at various pH ........ 44

Table 7. Effect of mode of sterilization on the chitosan antibacterial
Activity ................................................................................ 44

Table 8. The antibacterial activity of chitosan and controls against gram-
negative and gram -positive bacteria by agar disk diffusion
assay ................................................................................ 46

Table 9. The cell types in the reactive zone after implantation of chitosan
and Gelfoam® pads at various time points .................................... 48

Table 10. ANOVA result of the effect of the different treatment on the
magnitude of the reaction and the cell types at various

time points ............................................................................

54
Table 11. The cell types and the blood vessels in the rat tissues
After implantation of chitosan and Gelfoam® pads at various
time points ....................................................................... 55
Table 12. Collagen deposition in chitosan and Gelfoam® groups at various
time points ......................................................................... 59
Table 13 CDB and CD20 cells immunophenotyping results ........................ 61

viii

LISTS OF FIGURES

Figure 1. The structure of chitin ..........................................................
Figure 2. The structure of chitosan ......................................................
Figure 3. Production of chitin and chitosan ...........................................
Figure 4. Microbroth dilution assay plate design .....................................

Figure 5.1. Bactericidal activity of cellulose sample MSU033 against Eco/i
dissolved in acetic acid ...................................................

Figure 5.2. Bactericidal activity of chitosan sample MSU034 against Eco/i
dissolved in acetic acid .......................................................

Figure 5.3. Bactericidal activity of chitosan sample MSU035 against E.coli
dissolved in acetic acid .......................................................

Figure 6.1. Bactericidal activity of cellulose sample MSU030 against Eco/i
dissolved in Formic acid ....................................................

Figure 6.2. Bactericidal activity of cellulose sample MSU033 against E. coli
dissolved in Formic acid .................................................

Figure 6.3. Bactericidal activity of chitosan sample MSU034 against Eco/i
dissolved in Formic acid .................................................

Figure 6.4. Bactericidal activity of chitosan sample MSU035 against Eco/i
dissolved in Formic acid ..................................................

Figure 7.The antibacterial activity of chitosan and controls against Eco/i by
agar diffusion test ..............................................................

Figure 8. The antibacterial activity of chitosan and controls against
S.aureus by agar diffusion test .............................................
Figure 9. The reactive zone size of chitosan and Gelfoam® at various
time point ........................................................................

Figure 10.1 .The cellular reaction surrounding the chitosan pad at 4 hours
post-implantation ...............................................................

Figure 10.2. The cellular reaction surrounding Gelfoam® pad at 4 hours
post-implantation ...............................................................

ix

4
5
6

25

4O

40

41

41

42

42

43

45

46

49

50

50

Figure 11.1. The cellular reaction surrounding the chitosan pad at 4 days
post-implantation ............................................................... 51

Figure 11.2. The cellular reaction surrounding Gelfoam® pad at 4 days
post-implantation ............................................................... 51

Figure 12.1. The cellular reaction surrounding the chitosan pad at 7 days
post-implantation ............................................................... 52

Figure 12.2. The cellular reaction surrounding Gelfoam® pad at 7 days
post-implantation ............................................................... 52

Figure 13.1. The cellular reaction surrounding the chitosan pad at 15 days
post-implantation ............................................................... 53

Figure 13.2. The cellular reaction surrounding Gelfoam® pad at 15 days
post-implantation ............................................................... 53

Figure 14. Giant cells in the reactive zone in a sample from the Gelfoam®
group ............................................................................ 56

Figure 15. The blood vessels in the reactive zone of chitosan and
Gelfoam® group at various time points. ............................... 57

Figure 16. Lymphocytes in the reactive zone in a sample from the chitosan
group ............................................................................... 58

Figure 17.1. Reaction around chitosan after 4 days of implantation. Minimal
collagen deposition with Masson’s Trichome
stain ............................................................................. 59

Figure 17.2. Reaction around chitosan after 15 days of implantation. High
collagen deposition with Masson’s Trichome
stain ......................................................................... 60

Figure 18.1. Immunohistochemistry: Example of level 1 (Low)
CD3 positivity in chitosan group .................................... 61

Figure 18.2. Immunohistochemistry: Example of level 2 (Intermediate)
CD3 positivity in chitosan group ......................................... 62

Figure 18.3. Immunohistochemistry : Example of level 3 (High) CDS 62
positivity in chitosan group ................................................

Figure 19.1. Immunohistochemistry: Example of level 1 (Low) C020
positivity in chitosan group ................................................ 63

Figure 19.2. Immunohistochemistry : Example of level 2 (Intermediate)
CDZO positivity in chitosan group ....................................... 63

Figure 19.3. Immunohistochemistry : Example of level 3 (High) CD20

positivity in chitosan group .............................................. 64

“Images in this thesis/dissertation are presented in color”

xi

CHAPTER 1 .

LITERATURE REVIEW AND OBJECTIVES

LITERATURE REVIEW

Chitin was first described by the French scientist, Braconnot (1811), when he
isolated it from mushrooms using diluted alkali; he called it “fungine”. Later, Odier
(1823) isolated the same substance from insects and called it chitin, using the
Greek word for “tunic envelope”. Rouget (1859) isolated chitosan by boiling
chitin in a very concentrated solution of potassium hydroxide, the product being
soluble in organic acids (Muzzarelli 1977). The natural deacetylated form of
chitosan was identified in 1954 from the cell walls of the fungus Phycomyces
blackesleeanus (Kreger 1954). It was also found in the walls of Zygomycete soil

fungi and in green algae such as Chlorella ellipsoidea (Mihara, 1961 ).

Chitin:

Chitin is widely abundant in nature and is second only to cellulose in
availability. It is mainly found as a component of the exoskeletons of crustaceans
and insects, as well as in the cell walls of some bacteria, fungi, algae, hydrozoa,
annelida, molluscans and nematode worms (Muzzarelli 1977). The proportion of
chitin present is different for the various species (Table 1). The traditional and
commercial sources of chitin are the shells of crab, shrimp and krill, all of which
are wastes from marine food processing. The worldwide annual production of
crustacean shells has estimated to be 1.2 x 106 tons and the production of chitin
and protein from this waste can be considered as a major additional source of

commercial income (Knorr 1991 ).

Table 1. Chitin content and types from different sources.

(Modified from Rhazi et al. 2000 and Rudrapatnam et al. 2003)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Chitin source Chitin content Chitin source Chitin content

(%) (%)

Crustacea: Insects:

Marbled crab 10 Blatella(cockroach) 18.4

Red crab 10 Colcoptera (beetle) 27-35

Spider crab 16 Bombyx (silk worm) 44.2

Cancer crab 72.1 Calleria (wax worm) 33.7

Carcinus crab 64.2 Molluscan orLans:

Lobster 17 Clam shell 6.1

Locust lobster 25 Oyster shell 3.6

Spiny lobster 32 Sguid, skeletal pen 41

Homarus lobster 60-70 Krill 40

Crayfish 36 Fungi:

Shrimp 22 Aspergillus niger 42

Squilla 24 Pencillium notatum 18.5

Cephalopda: Sacharomyces 2 9

cerevrseae
Cuttlefish 20 Mucor rouxii 44.5
Squid 40 Mushroom 19

 

 

 

 

 

Chitin is a white nitrogenous linear polysaccharide polymer, consists of
poly I3.- (1-4) linked N-acetyI-D-glucosamine with a molecular weight reaching
millions of Daltons (Figure 1). It is tough, relatively inert, and insoluble in water
and in organic acids. However, most of the different forms of chitin are soluble in
various concentrations of chloro-alcohols conjugated with mineral acids or

organic acid solutions (Austin 1975).

Figure 1.The structure of chitin (after Mochizuki et al.1989)

cups-I

H2014
‘ o

        
     

monsoon, NHCOCI-I3 NHCOCHg

Chitin is present in three different crystallographic forms,oc, B, and 7,
which differ in the arrangement of the polysaccharide molecular chains within the
crystal cells (Rudall, 1963). The chains of a—chitin are arranged in anti-parallel
manner with strong intermolecular hydrogen bonding (Minke et al. 1978). The 8 -
chitin is characterized by presence of parallel chains with relatively weak
intermolecular forces (Gardner and Blackwell 1975). The 7 - chitin has one chain
directed down after every two chains directed up. The most abundant and stable

form is the a — chitin (Minke et al. 1978) which is commonly found in crab and
shrimp shells. Most of crustaceans have a— chitin while cephalopods contain 8-

chitin form (Rhazi et al. 2000).

Currently, the chemical method used for commercial production of chitin is
a thenno-chemical process based on demineralization and de-proteination of
shellfish waste (Roberts 1997). Due to various drawbacks of the ‘chemical

method, an alternative biological method has recently been utilized by some

processors, and is found to be efficient and readily applicable (Jung et al. 2006).
This is based on the fermentation of shell wastes with proteolytic microorganisms

e.g. Lactobacillus paracasei subsp, tolerans KCTC-3074, and Serratia

marcescens FS-3.

Chitosan:

Chitosan is semi crystalline poly IS - (1-4) — gluco‘samine with molecular
weight varies from about 10,000 to 2 million Daltons (Figure 2). It is produced
from chitin by deacetylation (Figure 3). Chitosan preparations can vary

considerably in the degree of acetylation, solubility and the molecular weight.

Figure 2. The sthcture of Chitosan (after Mochizuki et al. 1989)

c1120"

Cl-IZOH cnzon

       

Figure 3. Production of chitin and chitosan (after Zikakis 1984)

Crustacean shell
Size reII uction
Protein separation «— NaOH
WasIhing
Demineralization <— HCI

I
Washing and dewatering

I
Chitin
I
Deaceytylation +— NaOH

I
Washing and dewatering

I
Chitosan

Deacetylation of chitin to produce chitosan is performed either by using
strong alkai (Roberts 1997) or chitin deacetylase enzyme, e.g. fungal chitin
deacetylases (Tsigos et al. 2000). The chemical method has disadvantages of
both consuming considerable amount of energy and using a large amount of
concentrated alkali that can lead to environmental pollution and production of
impure heterogeneous compounds. On the other hand, the deacetylase method
results in the production of novel, chemically and physically, well-defined
chitosan oligomers and polymers which attain the prerequisite quality required for

this compound’s many medical uses.

Chitosan and chitin can be fabricated in many forms such as powder, gels,
flakes, membranes and sponges. A major physical difference between chitin and
chitosan lies in their solublization, which is controlled by the degree of
acetylation (DA). The DA is defined as the molar fraction of N-acetyl-glucosamine

residues with respect to total units. In general, when the DA is <60% the polymer

is termed chitosan and when it is >60% the polymer is called chitin (Sorlier et al.

2003).

Chitosan is highly basic polysaccharide that is insoluble in neutral and
alkaline pH, but forms soluble salts with inorganic and organic acids such as
glutamic, hydrochloric, lactic and acetic acids. Different degrees of
deacetylation or depolymerization produce different physiochemical properties,
and by changing the degree of deacetylation the solubility of chitosan is also
modified. Another characteristic of chitosan is its high viscosity in an acidic
environment. The level of viscosity of chitosan is influenced by the molecular
weight, concentration, degree of deacetylation, ionic strength, pH and the
temperature (Wang and Xu 1994) (Table 2). Moreover, both chitin and chitosan
have high nitrogen content (6.89%), which allows them to act as chelating agents

(Rabea et al. 2003).

Table 2. Chemical characteristics of chitosan from different sources.

(Modified from Rhazi et al. 2000)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Chitin source Yield of Intrinsic Average Degree of
chitosan viscosity molecular Acetylation
from chitin (ml 9") weights Mv (%)
wt (%) (g mol'1) (NMR)
Barnacle 78 357 66 000 13
Marbled crab 65 313 55 000 4
Red crab 76 315 56 000 9
Spider crab 75 266 45 000 3
Lobster 77 384 72 000 10
Locust lobster 77 485 98 000 16
Spiny lobster 78 422 82 000 12
Crayfish 79 426 83 000 8
Shrimp 78 582 125 000 10
Squilla 67 429 84 000 4
Cuttlefish 64 74 8300 3
Squid 70 128 17 000 0.5

 

 

Uses of chitosan:

By changing the degree of acetylation of chitin and the level of viscosity,
3 wide range of chitosan forms can be produced. These different forms display
diverse potential uses in the industrial and medical fields. Commercially, due to
its cationic nature, chitosan is used to purify water from heavy metals and
pesticides (Onsoyen and Skaugrud 1990). In agriculture it has been used to coat
seeds of the plants as well as directly as a fertilizer that results in an increase of
crop yields (Hadwiger et al. 1984). Also, it used as an animal feed additive due to
its high dietary fiber content and its hypocholesterolemic effect (Rudrapantam et

al. 2003). In addition, due to its high viscosity, film-forming and the moisture

retention properties, chitosan has been used in cosmetic industry products such

as hair shampoo and conditioner (Chen & Heh 2000).

Medical applications:

Chitosan is a physically and biologically functional compound
characterized by lack of toxicity and allergenicity. In addition, it is biocompatible
and biodegradable and can be modified chemically and enzymatically. All these
features provide a stimulus for investigators to devise methods of utilizing
chitosan for various medical and pharmaceutical applications. For example, it is
used as a wound-healing agent, an antimicrobial, a haemostatic agent, and as a
medium for drug and vaccine delivery. (Loke et al. 2000, Mackenzie et al 2006,

Ueno et 2001, lllum et al 2000).

1. Antimicrobial activity:

Most of the evidence of the antimicrobial activity of chitosan is reported with
food and water borne microorganisms and is associated with the use of chitosan
as plant protectants and food preservatives (Taha et al. 2002). The antibacterial
effect was found against several strains of bacteria, including Escherichia coli,
Bacillus cereus, Staphylococcus aureus, and Salmonella typhimurium
(Sudarshan et al 1992, Rabea et al. 2003) Generally, chitosan is thought to show
a more powerful bactericidal effect against gram-positive bacteria than gram—

negative bacteria (Senel & McClure 2004). Chitosan also has antifungal effect,

 

and is found to be effective against Candida albicans and F. oxysporium (Tsai
2002)

Chitosan is also used as a wound dressing and has shown to control the
bacterial multiplication and invasion in Wound injuries (Loke et al. 2000, Mi et al.
2001 ). Loke et al. (2000) formulated a chitosan dressing consisting of two
layers; chitosan hydrogel in the upper layer and an anti-microbial impregnated
biomaterial in the lower layer. An inhibition zone around discs of chitosan
acetate incorporated with chlorehexidine gluconate was observed and the
antimicrobial capability of the dressing was found to be more effective against
gram-positive bacteria than the gram- negative ones. Similarly, Mi et al. (2001),
carried out an in vivo study using chitosan incorporated with silver sulfadiazine in
rats injected with Pseudomonas aeruginosa and Staphlycoccus aureus strains. A
significant decrease in the number of colony-forrning units was observed in the
tissues surrounding the infected wound. Thus chitosan membranes have the
capability of preventing the penetration of the bacteria into wounds and acts as a

prophylaxis against wound sepsis (Mi et al. 2001).

2. Wound healing:

The use of chitosan and chitin in wound healing goes back to ancient
times. Koreans used the pen of the octopus as a source of chitin for the
treatment of abrasions, and Mexicans used mushroom to accelerate the healing
of wounds (Allan et al.1984). In current times, the use of chitin as a wound-

healing accelerator begins with the studies of Prudden et al. (1957, 1970) who

10

noticed that shark cartilage promote wound healing and stated that the N-acetyl
glucosamine units (GlcNac) play the major role in healing.

Chitosan has many physiochemical properties that make it a good
candidate for wound healing. When chitin and chitosan incorporated with other
materials, they can form tough, water absorbent biocompatible and interactive
films that are capable of absorbing the exudates generated from wounds (Seo et
al. 2000). Also, they are characterized by high water vapor transmission, which
prevents the accumulation of fluids in wounds. When chitosan membranes are
applied to human skin, they adhere uniformly to the wound surface, and prevent
formation of air pockets and the accumulation of exudate fluid (Azad et al. 2003).
Chitosan has high oxygen permeability (7x10 '"cm2/s), which reduces oxygen
deprivation of the tissues, and it is slowly degraded by Iysozymes that are
transported to the wound area by various inflammatory cells (Allan, at al 1984).

Chitosan induces an inflammatory response and enhance leukocyte
migration and accumulation of neutrophils and lymphocytes (Hidaka et al 1999).
It also stimulates the production of interiukin 8 (IL-8) which chemoattracts and
recruits neutrophils to wounds (Mori et al. 1997). Chitosan scaffold implanted
intra-peritoneally and subcutaneously in mice did not induce any pathological
inflammatory tissue response to chitosan such as erythema and edema, but
there was a chemotactic effect on neutrophils (VandeVord et al. 2001). Also,
Chellat et al. (2000) demonstrated that after the implantation no signs of cell
damage such as swollen mitochondria and rough endoplasmic reticulum , are

seen.

11

Additionally, chitosan stimulates macrophages to produce more essential
cytokines which may speed up the wound healing (Mori et al. 1997, Nishimura et
al.1986). Ueno et al (1999) in their experiments with beagle dogs, found
infiltration of PMN and migration of macrophages and giant cells at an early
stage, as well as an increase in the phagocytic activity and the oxidative burst of
macrophages and PMN. The activation of peritoneal macrophages by chitosan
was indicated by the presence of activation markers including MHC I and MHC
II, Fc and mannose receptors.

The second phase of natural wound healing is characterized by the
presence of granulation tissue followed by the proliferation of fibroblasts and
production of ground substances and extracellular matrix (ECM) filling of the
dead spaces. Chitosan accelerates the reformation of connective tissues and
angiogenesis of the healing wound (Muzzarelli et al. 1988). Open wounds heal
by the initiation of an acute inflammatory phase and cellular response, which
then leads to granulation of tissue formation, re-epithelization and wound
contraction (Adam & Clark 1999). Both chitin and chitosan enhance the
proliferation of fibroblasts and keratinocytes (Howeling et al. 2001). Likewise,
Azad et al. (2003) observed abundant keratinocytes in the areas dressed with
chitosan membrane with a resultant faster re-epithelization of wounds.
Histologically, most of the tissue found after 7 days of implantation of chitin and
chitosan is thought to be granulation tissue (Kojima et al. 2004).

Synthesis of collagen, and the formation and remodeling of the

extracellular matrix, characterize the third phase of wound healing. Chitosan

12

promotes the tensile strength of tissue by speeding up the formation of
fibroblasts and the synthesis of collagen with more thickly and consolidated
collagen fibers (Azad et al. 2003). Lysozymes slowly hydrolyze chitosan
membranes and produce chito-oligmers that stimulate deposition, and the
assembly of collagen fibrils in the extracellular matrix components (Muzzarelli et
al. 1999). An increase in collagen synthesis associated with chitin and chitosan
application has been measured using prolyl-hydroxylase enzyme (PHL) activity

(Kojima et al. 2004).

3- Anticoagulant properties:

Both chitin and chitosan show a haemostatic activity when applied in
bleeding wounds and their muco-adhesive nature make them ideal haemostatic
agents (Evans et al. 1962). The contact of GlcNAc derivatives with platelets
leads to their activation, aggregation and a change of morphology that suggested
the irreversible activation of platelets (Thatte et al, 2004). Chou et al. (2003)
demonstrated that the chitosan enhancement of platelet adhesion and
aggregation varies with both time and dose. This aggregation is accompanied by
an increase of calcium level and expression of glycoprotein lllb/llla complexes
on the platelet membranes and leads to interaction of platelets with damaged
tissues and the promotion of wound healing.

A freeze-dried chitosan-based hemostatic dressing can control severe
parenchymal and large venous hemorrhage in a swine model with liver injury

(Pusateri et al. 2003). Recently, chitosan-based haemostatic dressings were

13

used to reduce hemonhage in military forces in Iraq and Afghanistan (Wedmore
et al. 2006). The dressings were applied to wounds in extremities, chest and
groin. In 97% of the cases the bleeding was stopped or greatly improved.
Various hypothesis were put fonivard to explain why chitosan enhances clotting,
Chou et al. (2003) suggested that chitosan dressing enhances platelet function,
while Klokkevold et al. (1991) suggested the incorporation of red blood cells into
the clot was the key. However, Wedmore et al. (2006) reported that the main

factor by which chitosan enhances clotting, is its muco-adhesive property.

4- Deliveronf drugs and vaccines:

Chitosan microparticles have great potential in mucosal vaccine delivery
systems and potentially have adjuvant activities. Suspensions of microparticles
have shown an immuno-stimulatory activity in macrophage proliferation, in tumor
growth suppression, in cytokine inducement and antibody production. They have
the ability to deliver drugs across the mucosal lining, and with their cationic
muco-adhesive property, they interacts with negatively charged cell surfaces and
cause redistribution of protein zones enhancing the permeability of epithelia to
the uptake of peptides and proteins, and thus increasing the contact of such
antigens with the immune system (lllum et al. 2000; Hwang et al. 2000).

Chitosan has been used as adjuvant to enhance the immune response
elicited to a number of human and veterinary vaccine antigens. Chitosan
stimulates non-specific resistance against Sendi virus, and E. coli when given

prior to challenge infection of mice. The intranasal and intravenous

14

administration of chitosan as an adjuvant was seen to be more effective than the
subcutaneous or intra-peritoneal administration. It appears to act early on the
non-specific elements of the immune response such as the accumulation and
activation of macrophages, the activation of natural killer cells and the induction
of IFN- y (lida et al. 1987). The 70% deacetylated chitin stimulates the
production of monokines such as CSF, IL-1 and TNF-a, and this adjuvant activity
may account for the induction of delayed hypersensitivity, circulating antibody
and cytotoxic T-Iymphocytes (Nishimura et al. 1986).

In a study carried out on BALB/c mice injected with recombinant protein
beta-human chorionic gonadotropin (rBhCG) combined with two different
formulations of chitosan, more than a 100- fold increase in antibodies titers was
achieved; the chitosan also primed the animals for a specific DTH response
(Seferian & Martinez 2001). Strong augmentation of both systemic and local
immune responses, with significant enhancement of IgG production, was also
seen when mice were vaccinated orally and nasally with Diptheria toxoid (DT)
incorporated with chitosan (Inez et al. 2003). Chitosan fed to rats elicits the
release of IL-10 and the expression of lL-4 and TGF—B mRNA in mucosal tissues
and Peyer’s patches, as well as with the activation of CD3“ T cells in the spleen

(Porporatto et al. 2005).
Immune response:

Chitin with its rigid crystalline structure has the potential to stimulate the

immune system when they carry certain chemical groups. For example, the

15

residual amino group, as well as the imino derivatives of chitosan, may stimulate
the immune system despite the low pK of the amino group of chitosan, which
ranges between 6.364 (Tokura et al.1999). Normally the implantation of the
polymer leads to inflammation; however a moderate inflammation may be useful.
During the inflammatory reaction the production of specific enzymes and NO by
macrophage and other cells augment polymer degradation and allow normal
growth of the tissues (Peluso et al. 1994).

Various research groups have reported properties of chitin and chitosan to
stimulate immune responses. The degree of deacetylation affects the stimulatory
activity of chitin and chitosan (Hidaka et al.1999 ) . Nishimura et al. (1984)
showed that mice peritoneal macrophages were activated when chitin of different
degrees of deacetylation and substituting groups were injected intra-peritoneally.
Macrophage activation indicators such as the percentage of lysis, stasis and the
release of H202 were found to be highest with 70 % DDA chitin. The activation
induced by carbomethyl-chitin is same as the 70% DDA chitin, whilst
phosphorlyated and sulphonated-chitin did not activate macrophages, and this
suggested that the free amino group likely plays the major role in activating the
peritoneal macrophages.

The activation of macrophages is important in host immune responses,
but their uncontrolled activation may lead to septic shock and death (Car et al.
1994). To control the unlimited inflammation and activation of macrophages,

chitosan induces Pas-mediated apoptosis of the macrophages of BALB/c mice

16

through the mannose receptor, which may be a mechanism by which it enhances
wound healing (Mori et al. 2005).

Chitosan also has an effect on the synthesis of nitric oxide (NO). When
macrophages stimulated with combination of chitosan and IFN-ry, their synthesis
of NO, cytotoxicity to tumor cells and their TNF-or secretion are increased (Seo et
al. 2000). Macrophages cultured in medium containing chitosan produce an
increase amount of nitrate (Pelsuo et al. 1994). The production of NO is mainly
attributed to the N-acetylglucosamine (NAGA) unit rather than the glucosamine
residues. 7

Chitosan can enhance the production of the main components of adaptive
immunity, as well as cell-mediated and antibody-mediated responses. It induces
production of circulating antibodies for the bacterial a-amylase and dinitrophenyl-
ovalbumin. 30% DDA chitin and S-chitin were the best among water-soluble
derivatives for production of circulating antibodies when used as an adjuvant
(Nishimura et al. 1985). Also, chitosan can promote the stimulation of delayed-
typed hypersensitivity to (ABA-N-acetyl tyrosine) as measured by skin reaction
test; the highest immunogencity was found associated with 70% DAA chitin
(Nishimura et al. 1985).

Chitosan affects the production of cytokines. It induces monocytes,
through binding to CD14, to produce TNF-ot; this effect depends on the
molecular weight and the neutral solubility (Otterlei et al. 1994). A high A
concentration of the poly-ionic chitosan-xanthan (CH-X) complex stimulates

macrophages to produce TNF-a and lL-18 that are important mediators of

17

inflammatory response involved in cell proliferation, differentiation and apoptosis

(Chellat et al. 2000).

Degradation of Chitin and chitosan:

One of the main characteristics of chitin and chitosan that favors their use
in medical areas is their biodegradability (Tomihata & lkada 1997). Chitinase is
an enzyme, which hydrolyzes chitin to free N-acetylaglucosamine (GlcNAc); it
consists of two hydrolases; chitin glycanohydrolase, and chitobiase. These are
found in the microorganisms as well as in various plants and animal species
(Muzzarelli 1977). Chitinases break down the chitin polymer into intermediate
sized chitin oligosaccharides, which then are hydrolyzed to chitobiose with
production of small amount of GlcNAc. Finally, chitobiase splits chitobiose into
molecules of GlcNAc. Chitosan can be degraded by Iysozymes, and related
enzymes such as N-acetyl-B-D-glucaminidases, which occur in the human body

(Bourbouze et al. 1991). The mannose receptor is the major macrophage
receptor for polysaccharides (Marodi et al. 1993). After binding to mannose
receptors, chitin and chitosan are internalized by the cell and then degraded by
lysozyme to N-acetyl D-glucosamine (Pangbum et al. 1982, Tomihata& lkada
1997). The rate of chitosan degradation is inversely related to its degree of

crystallinity, and the degree of acetylation (Hirano et al. 1989).

18

Hypothesis and objectives:
The present study addresses two main hypotheses associated with the
role and effect of chitosan in wound healing;
Hypothesis 1: Chitosan has antibacterial activity which will enhance wound
heafing.
Hypothesis 2: Chitosan improves the healing process and reduces the amount

of resultant scar tissue.

Two approaches were adopted to test these hypotheses. Firstly, to
measure the antibacterial effect of chitosan in vitro against two common bacterial
strains associated with wounds, Escherichia coli and Staphylococcus aureus.
The second approach was to define the cellular response to intra-peritoneal
implantation of chitosan and compared it with the regularly used material,
Gelfoam® (a collagen based material used to prevent intra-peritoneal bleeding

and promote healing).

19

CHAPTER 2.

MATERIALS AND METHODS

20

MATERIALS AND METHODS

1. Microbiological experiments:

1. 1: Measurement of the antibacterial activity of chitosan by microbroth

dilution assay:

Four different compounds were used, MSU 030, MSU 033, (controls), and
MSU 034 and MSU 035 (Chitosan ) (Table 3) (Vanson Halosource Company).
The bactericidal activity of chitosan was examined under varying environmental

factors. Factors tested were acid solvent, pH and the mode of sterilization:

Table 3. Characteristics of the microbroth dilution assay samples.

 

Test sample

MW

Type DDA (Daltons)

Source

 

MSU030

2% aqueous gel of
hydroxyl propyl NA ND
methyl cellulose

Crab

 

MSU 033

Hydroxy propyl
methyl cellulose NA ND
powder

Crab

 

MSU034

6.6% aqueous
solution of ethylene <7% 97,000
glycol chitosan

Crab

 

 

MSU035

 

Ethylene glycol
chitosan lyophilized <7% 97,000
powder

 

 

 

Crab

 

NA: Not applicable , ND: data not available

21

 

Acid solvents effect:

To assess the effect of acid solvent, two organic acids were used; acetic
acid and formic acid. In the first set of experiments, two concentrations of
chitosan were used, 1% and 2%, and they dissolved in either 1% or 2% of acetic

acid. In the second set, 2500 ppm chitosan was dissolved in 0.2M formic acid.

pH effect:

The effect of pH was assessed in the experiments where acetic acid was
used as the solvent. The experiments were carried out by adjusting the pH to
2.6, 4.2, 5.0, and 6.0. While, in the experiments where formic acid used as

solvent, pH 5.0 was used throughout all experiments.

Mode of sterilization effect:
In a set of experiments, chitosan solution was sterilized by autoclaving at
121 ° C for 15 minutes. As a control, a second set of experiments without

sterilization was performed.

Preparation of media:

Muellor Hinton lI Agar media (MHA): 38/.k grams of the MHA powder (B 11438,
Becton Dickinson, MD) was suspended in 1 liter of distilled water, boiled for 1
minute and autoclaved at 121° C for 15 minutes. The mixture was poured into
petri dishes to obtain a 3-4 mm deep agar, dried and stored in sealed plastic

bags at 4° C.

22

Muellor Hinton broth (MHB): 22 grams of the MHB powder (211443 Becton
Dickinson, MD) were dissolved in 1 liter water heated with frequent agitation and
then boiled for 7 min. The melted broth was dispensed into sterile capped bottles,

autoclaved for 20 min at 121° C and stored at room temperature.

Growth of bacteria:

A Gram-negative Escherichia coli ATCC 25922 strain was grown on MHA
media for 20-24 hours at 37 ° C. The inoculum of the bacteria was prepared by
swabbing off the bacteria from the plate and suspending the cells in MHB. The
spectrophotometeric absorbance was adjusted to 0.1 0D. at 560 nm which
contains approximately 5x108 CFU/ml. The suspension was further diluted 500
times with MBH before it was used in the assay, at concentration of 1x106

CFU/ml bacteria for use in each plate.

Controls:

Two forms of cellulose were used as controls. In addition, in each assay,
two positive controls were used; Kanamycin monosulfate antibiotic (K-4378,
Sigma, MO), and a “column” in the plate containing only the bacteria and the
broth. Similarly two negative controls were used; 1% acetic acid, and a “column”

in the plate containing only the broth.

23

Preparation of stock antibiotics:
The following equation was applied to determine the weight of Kanamycin
antibiotics to be used (NCCLS, 1997)
Weight (mg) = Desired concentration (ugjml) X mine (ml)
Potency of the antibiotic (pg/ml)
The potency of Kanamycin is 781 pg/ml (Sigma, MO). The powder was dissolved
in sterile water, and further diluted to make up the required volume. Stock

antibiotics were aliquoted and stored at —80 ° C for no more than 6 months.

Microbroth dilution assay:

96 round-bottom well plates (3799, Coming incorporated Costar, NY) were
used to obtain both a qualitative and a quantitative measures of any antibacterial
activity of chitosan. Plates were evaluated for growth as indicated by the
presence of either turbidity or pellets of the U shaped bottom well after 24 hours.
The antibiotics were diluted to twice the required starting concentration with

MBH.

24

Figure 4. Microbroth dilution assay plate design.

DILUTIONS -> _,,e We

 

 

 

CH OOOOOOOOOOQO‘
00000 0000000 2
CO 0000 0000003

“GO @000 000000 4

AC OOOOOOOOOOOOs

__OO 0000 000000 a

 

 

 

 

 

1 2 3 4 5 8 7 8 9 10 11 12

CH: Chitosan AC: Acetic acid AB: Antibiotics

Microbroth procedure:

100 pl of 1% chitosan were placed in the first 4 rows of the first column in
the plate, followed by 100 pl of 1% acetic acid in rows 5 and 6. In the last two
rows, 7 and 8, 100 pl of antibiotics were placed. 50pl of MHB was placed in all of
the remaining wells of the plate. Then, 50pl of 1% chitosan were transferred
from column 1 to column 2 mixed and so on transferred by serial dilution until
reaching column 10 ( 0.0019% concentration) . Similariy, Soul of 1% acetic acid
and Soul of antibiotics were transferred by serial dilution until column 10. Finally,
50pl of the bacterial suspension was added to column 12 and then from column 2
till the 10th column. Column 12 is the positive control; it contains the bacterial

suspension only. Column 11 is the negative control containing only the broth.

25

The plate was incubated at 37°C, in 5% C02, till growth was seen in column 12,

usually after 20-24 hours.

Microbroth analysis:

The lowest concentration of chitosan or the controls where no growth was
seen in the well was taken as the minimum inhibitory concentration (MIC). The
MIC tested for the antibiotics is within the range of 0.5 pg /ml to 128 pg /m
(NCCLS 1997). Images of the plates were taken using a camera with an

Alphaimager software program (Version 3.3).

1.2: Agar diffusion assay:
Preparation of chitosan solution:

Amphoteric form of chitosan, chitosan succinate, was dissolved in sterile
deionized water, and used at concentrations of 1%, 0.5% and 0.25% in the
assay. To desalt the chitosan, the solution was passed through filter paper No.1

and then sterilized by passage through 45 pm filter membrane.

Strains of bacteria:

The gram-negative Escherichia coli ATCC 25922 , and the gram-positive
Staphylococcus aureus ATCC 29213 strains were used in this assay. They were
grown on MHA media for 20-24 hours, and then the bacteria suspended in MHB

at a concentration of 1x108 CFU/ml throughout the experiments,

26

Controls:
Kanamycin antibiotic was used as a positive control at a concentration of 30 pg.

1% sodium succinate (82378, Sigma, and MO) was used as a negative control.

Agar diffusion procedure:

A bacterial suspension at a concentration of 1x108 CFU/m was streaked
out on a 100mm x 15mm MHA plate. Susceptibility discs (1599-33 Difco
laboratories, Detroit) were used and placed in the plate. The optimal
concentration of the chitosan solution needed to saturate the disc was found to
be 20 pl. In each assay 20 pl of 1%, 0.5% and 0.25% chitosan were used. In
addition, 20 pl of Kanamycin antibiotics and 20 pl of 1% sodium succinate

were used as controls in each plate.

Agar diffusion analysis:

The zones of growth inhibition surrounding each disc were measured in
each plate after incubation for 18 hours at 37° C. The susceptibility of E. coli and
S. aureus to chitosan was determined to be either resistant (- ve) or sensitive
(+ve). Images of the plates were taken using a camera with an Alphaimager

software program (Version 3.3)

27

2. Cellular response to intra-peritoneal implantation of chitosan:

Animals:

The animal models used in this investigation were of August rat inbred
strain (Originating at the MRC, London, UK). August rats were used in this
study for the following reasons:

1- They are inbred

2- The peritoneal cell population is extremely stable after they reach 1809m

body weight, and remains so until at least two years of age. Also, there

are no differences in cell populations that exist between males and
females (Mackenzie et al.1981).

3- Pilot studies with chitosan indicated that responses in the peritoneal cavity

of these rats strains paralleled that seen in the skin.

The approval for animal use was provided by the Institutional Animal Care
and Use Committee of Western Michigan University, Kalamazoo (Approval No.
LACUC protocol N0.03-06-01, and LACUC protocol NO. 06-06-01 ). The rats
were kept at the laboratory animal house, Western Michigan University, on 12-
hour light/dark cycle and given water and Purina Rodent Chow food ad libitum.
Both males and females were included in the study. The age of the rats at the
beginning of the study ranged from 5-14 months. A total of 32 rats were included
in the study. All the rats included in the study appeared to be in good health and

their average weight was 200 g.

28

Study plan:

The study period was approximately 2 weeks divided to four time points:
4 hours, 4 days, 7 days, and 15 days. 85% deacetylated chitosan (MW, 600.000
Daltons) sponge-like 1 cm x 1 cm x 0.4 cm pads, (Vanson Halosource
Company) was used throughout the study. Gelfoam® (Pfizer Corporation) pads
of a similar size were used as the control material for the study. Four replicates

for each time point were used for both chitosan and control groups.

Surgery:

Rats were anesthetized with Halothane® (approximately 2.5-3.0%),
abdominal hair shaved and the skin sterilized, and incisions made at about 1 cm
from the ventral midline. Chitosan pads were placed through the incision site
onto the omentum adjacent to spleen, and the abdominal incisions were closed
using standard suture material. The rats were sacrificed at 4 hours, 4 days, 7
days, and 15 days. The pads and the attached mesentery and peritoneal tissues
were taken and fixed in 10% phosphate-buffered formalin. In addition, the spleen,

mesenteric lymph node, thymus and the pancreas were taken in many animals.

Histotechnology:

Sectioning:

Tissues were fixed in 10% phosphate-buffered formalin, then processed

on a vacuum infiltration tissue processor (Thenno Electron Excellsior), passed

29

through a graded series of ethanol; 75% for 5 hours , 90% for 30 minutes , 95%
for 30 minutes, 100% for 30 minutes and another two times in 100% for one
hour each. Then they were passed two times through xylene for 30 minutes
each and one time for 1 hour successively. Following clearing by xylene, the
tissues were infiltrated with paraffin three times; twice for 30 minutes each and
one time for 1 hour. Afterward, the tissues were embedded in paraffin blocks on
a Tissue Tek ll (Cryoconsole scientific model 4585, Sakura USA), and then
sectioned on a rotary microtome at 5 microns (Reichert Jung, Model 2030). The
sections were floated on 37°C - 39°C waterbath (Tissue floating bath Model 135)

and dried in a warm oven at 56°C - 59°C for 1-24 hours.

Staining:

Hematoxylin and Eosin was used to stain the majority of sections
(Appendix A) . The sections were placed two times in Xylene for 5 minutes each
followed by two times in 100% ethanol and twice in 95% ethanol. Then, they
were rinsed in distilled water and placed in Lerner 2 hematoxylin for one and a
half minutes. After that, the sections were dipped 2-3 times in 1% glacial acetic
water and then placed into running tap water for 2-3 minutes followed by rinsing
in 95% ethanol. Then, they were placed in Eosin-Phloxine for 2 minutes and
rinsed in 95% ethanol. Subsequently, the sections were rinsed four times in
100% ethanol, 3 times in Xylene and coverslipped using synthetic mounting

media.

30

Microscopy:
The slides were scanned under 20X power magnification , and analyzed
under 40X magnification so as to aid identification of the cells involved in the

reaction associated with the chitosan and Gelfoam® pads.

Assessment:
For each section, the pad region and the surrounding host tissue were divided
into three zones:
A) The pad zone represented by the region in the center which contains the
remaining chitosan pad.
B) The reactive zone is the region of tissues surrounding the pad zone where
the cellular reaction to the pad was occurred.
C) The omental zone is the omentum outer layer which surrounds the pad

and the reactive zone.

Scoring of the cellular response in the reactive zone:

The diameter of the reactive zone was assessed as a measure of the
magnitude of the host reactions towards the chitosan and the Gelfoam® pads.
12 replicate measurements separated by about 20 mm each were counted for
each section. These 12 readings were used to calculate and compare the size of
the reactive zones. Additionally, from these 12 replicated readings across the

reactive zone the following parameters were also measured;

31

1-

The number and the types of the cells present in the reactive zone. Cells
were distinguished as either granulocytes, macrophages/ monocytes,
giant cells, mast cells, lymphocytes or plasma cells. The average

percentage of each cell types was calculated at each time point.

2- Cells were divided to the following groups (according to their morphology);

a) Mononuclear cell type which includes macrophages, lymphocytes and
plasma cells.

b) Multinuclear cell type which is mainly consisted of giant cells.

c) Polymorphonuclear cell type consisting of neutrophils , eosinophils
and basophils.

d) The fibrocytic cell type composed of fibrocytes and fibroblasts.

The changes in cell number and in the number of the vessels in the
reactive zone were estimated for each time point.

The amount of fibrosis was defined by the amount of collagen present (as
defined by the histochemical staining procedure of Masson’s Trichome).
The area of collagen positivity was measured using Metamorph® Image
Analysis program. The area of the pad was subtracted from the area of
the collagen histochemical positivity to provide the area of collagen

deposition surrounding the pad.

32

Masson’s Trichome Procedure :

The sections were deparaffinzed and hydrated before the Masson’s stain
was applied (Appendix B). Sections were rinsed in Bouin’s solution for one hour
at 56° C or overnight at room temperature (RT). Next, they were washed in tap
water and rinsed in distilled water, followed by staining in Weigert’s hematoxylin
for 10 minutes. Then rinsed in running tap water for 10 minutes and again in
distilled water. AftenNards, these were stained in Biebrich Scarlet- acid Fuchsin
solution for 1 minute and then rinsed. The sections were then placed in a
phosphmolybdic-phosphotungstic acid solution for 15 minutes, stained with
aniline blue for 3 minutes and rinsed in distilled water. Finally, they were placed
in 1% glacial acetic water for 3 minutes , rinsed, dehydrated through graded
series of alcohol, cleared in several changes of xylene, mounted , and

coverslipped.

Data Analysis:

ANOVA statistical test was used to compare differences of the
magnitude of reactive zones, as well as the differences in the number of
various cell types between chitosan and Gelfoam ® at different time points. The

level used to define a significance difference was a p-value of 0.05.

3- Immunohistochemistry:
The specific immune cells infiltrated in the tissues surrounding the

chitosan and the Gelfoam® pads were immuophenotyped by using CD3 and

33

CD20 monoclonal antibodies. The immunhistochemical assay was carried out
on forrnalin-fixed, paraffin embedded tissues. The sections were first
deparaffinized in two changes of xylene for 10 minutes each at RT, then rinsed
in three changes of 100% ethanol, two changes of 95%, one change of,
80% , 70%, 50% ethanol, and then rinsed several times in distilled water, to be
finally placed in Tris buffered saline (TBS) for a minimum of 5 minutes. For
antigen retrieval, the slides were treated with heat induced epitope retrieval
(HIER) solution (Antigen retrieval ClTRA HK086-9K Biogenex, CA). The HIER
was dissolved in TRIS/EDTA buffer in coplin jar and preheated to 95%-100%
(appendix C), then the sections were placed in the jar, incubated in a rice
steamer for 30 minutes and then rinsed in several changes of distilled water.
To block the endogenous peroxidase, the sections were placed in 6% hydrogen
peroxidase/methanol bath for 30 minutes at RT, rinsed in running tap water for 5
minutes followed by rinsing in several changes of distilled water. The slides were
placed in TBS+ Tween 20 (TBT999 ScyTek, Utah) for 5 minutes at RT. The
subsequent steps were carried out in Dako automiser universal staining system

(software version V3.1 .1) with the following scheme .

34

Table 4 Reaction scheme in the Automiser staining system.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Template step Staining information

Protein block Normal horse in NAD 1:28 —30 minutes

Buffer- blow Blow

Auxiliary reagents Avidin-15 minutes

Buffer rinse TBS-T20

Auxiliary reafints Biotin-15 minutes

Primary antibody CD3 1:50 in NAD for 60 minutes and
for control Normal Horse serum 1:28

Buffer rinse TBS-T20

Secondary antibody Horse anti-mouse (rat-absorbed) at
1:100 for 30 minutes

Buffer rinse TBS-T20

Tertiary reagent R.T.U. ABC Elite Peroxidase reagent
for 30 minutes

Buffer rinse TBS-T20

Substrate batch Nova Red for 15 minutes

Distilled water rinse Distilled water

 

 

 

The sections were incubated in normal horse serum (S-2000 Vector, CA)
at 1/28 concentration for 30 minutes, and then rinsed in tap water. 0.01% of
Avidin D (A-2000 Vector, CA) solution was applied in the sections for 15 minutes
at RT , and rinsed in three changes of TBS+T20. Afterwards, 0.03% of Biotin
(B4501 Sigma MO) solution was applied for 15 minutes at RT, and then the
sections rinsed with three changes of TBS+T20 . The primary antibody with
specificity for CD3 (Pc3/188A, Sc-20047 Santa-Cruz Biotechnology ) was
diluted in normal antibody diluent (ADT 999, ScyTek, Utah) and used at 1/50
concentration. The same dilution procedure was followed when primary
antibody CDZO (M-20, Sc - 77735 Santa-Cruz Biotechnology) was used. The
primary antibody was added to the sections for one hour at RT, then sections

were rinsed with three changes of TBS+T20 and stayed in the last change for 2

35

minutes. The secondary antibody, biotinlyated anti-mouse (rat absorbed) ( BA-
2001, Vector CA) IgG H+ L (made in the horse) was diluted to 1:100 in the
normal antibody diluent and applied for 30 minutes at RT, and the sections
rinsed in three changes of TBS+T20 . The tertiary reagent, R.T.U. Elite
peroxidase ( PK-7100, Vector CA) was added and the slides were incubated for
30 minutes at RT, and rinsed with three changes of TBS+T20 and stayed in the
last change for 2 minutes . To detect the positive cells, Nova RED (SK-4800,
Vector, CA) substrate was applied for 15 min at RT, and then the slides were
rinsed in distilled water. After the slides were taken from the automiser system,
they were counterstained by Lerner’s Haematoxylin for 1% minute, dipped 2-3
times in 1% aqueous glacial acetic acid , and placed under running tap water for
2 minutes. Finally, they were dehydrated through 2 changes of 95% ethanol, 3

changes of absolute alcohol , cleared in three changes of xylene and mounted.

Controls:
For the negative control, the primary antibody was replaced by the normal
horse sera in the assay . For the positive controls; thymic tissue was used as a

control for CD3 , and splenic tissue for CD20 antibodies .

Grading system:
The lymphocytes were enumerated and categorized either as positive or
negative based upon the binding of the CD3 and CDZO antibody. The result

assessed on a grade scale ; level 1 (+) where <20 cells were positively

36

stained, level 2 (++) where moderate (50-75) cells were stained, and level 3

(+++) was interpreted as high positivity where >100 cells were positive,

37

CHAPTER 3.

RESULTS

38

RESULTS
1. Microbiological results:
1.1 Microbroth dilution assay results:
Acid solvent effect:

Two organic acids, acetic and formic acid were used as solvents for
the test samples. The bactericidal activity of two different forms of chitosan and
control substances against Eco/i ATCC 25922 strain was measured at pH 5.0.
No samples inhibit the growth of bacteria with either acid. At 1% acetic
concentration, the E. coli strain was two- to four -fold more sensitive to MSU 033
(62.5 pg/ml) than to other forms . (Table 5). The MIC of both MSU 035 form and
the acetic acid is (250 pg/ml) (Figure 5.1, 5.2 & 5.3). The MIC of Kanamcyin
ranged between 02-04 pg/ml. However, at 0.2M formic acid neither the
chitosan forms at any concentration, nor the formic acid inhibit the growth of
E.coli. (Figure 6.1, 6.2, 6.3,& 6.4).

Table 5. The MIC values of chitosan forms at different acid solvents .

 

 

 

 

 

 

 

 

 

Test samples Minimum inhibitory concentration (MIC)
lug/ml)

Acetic acid Formic acid
MSU 033 62.5 0
MSU 034 125 0
MSU 035 250 0
Acetic acid 250 NA
Formic acid NA 0
Kanamycin 0.2-0.4 0.2-0.4

 

 

 

 

NA: not applicable

39

Figure 5.1. Bactericidal activity of cellulose sample MSU033 against Eco/i
Strain dissolved in acetic acid

CH

AC

 

 

DIlution ——-—'——§
AB= Antibiotic, AC= Acid, CI-I= Chitosan

Figure 5.2. Bactericidal activity of chitosan sample MSU034 against Eco/i strain
dissolved in acetic acid

 

 

Dilution >

 

40

Figure 5 .3. Bactericidal activity of chitosan sample MSU035 against E.coli strain
dissolved in acetic acid .

 

 

 

CH
AC
AB ‘ . , a a
-- iieisisfs‘i
Dilution é/

 

AB= Antibiotic, AC= Acid, CI-I= Chitosan

Figure 6 .1. Bactericidal activity of cellulose sample MSU030 against E.coli
strain dissolved in Formic acid

. isisiegeisjcja
e, e ..

 

AC

 

AB

 

 

 

 

 

Dilution >

41

Figure 6.2. Bactericidal activity of cellulose sample MSU033 against Eco/i strain
dissolved in Formic acid .

 

    

CH

 

AC

AB

 

 

Dilution ——j>

AB= Antibiotic, AC= Acid, CH= Chitosan

Figure 6 .3. Bactericidal activity of chitosan sample MSU034 against Eco/i
strain dissolved in Formic acid at.

 

 

42

Figure 6.4. Bactericidal activity of chitosan sample MSU035 against Eco/i strain
dissolved in Formic acid .

 

CH

 

AC

 

AB

 

 

 

 

 

Dilution >

pH effect:

The pH effect was examined in two concentrations of acetic acid, 1% and
2%, and the pH was adjusted to 2.6, 4.2, 5.0, and 6.0. All forms of chitosan at
1% or 2% acetic acid did not show antibacterial activity. No difference was

found between the different pH (Table 6)

Table 6. The MIC values of chitosan forms and controls at various pH.

 

 

 

 

 

 

 

 

Test Sample , MIC (pg/ml)
1% acetic acid 2% acetic acid

pH 2.6 pH 5.0 pH 4.2 pH 6.0
MSU 033 125 62.5 0 0
MSU 034 250 125 0 0
MSU 35 ND 250 0 0
Acetic acid 62.5 250 125 125
Kanamycin 0.2-0.4 0.2-0.4 0.2-0.4 0.2-0.4

 

 

 

 

 

 

 

ND= Data not available,

Mode of sterilization effect:
No difference was observed In the MIC values between the autoclaved
and non-autoclaved test samples solutions at pH 2.6 (Table 7) . The E. coli strain

was more sensitive to acetic acid than to chitosan .

Table 7. Effect of mode of sterilization on the chitosan antibacterial activity.

 

 

 

 

 

 

Test sample MIC (pg/ml)
Autoclaving No autoclave
MSU 033 125 125
MSU 034 250 250
Acetic acid 62.5 62.5
Kanamycin 0.2-0.4 0.2-0.4

 

 

 

 

 

44

1.2 Agar disk diffusion assay:

An amphoteric form of chitosan was used in these specific experiments.
Paper disks were saturated with different concentrations of chitosan, and placed
in agar plates, and the susceptibility of the bacteria to chitosan was determined
as either resistant (- ve) or sensitive (+ve). The three different concentrations of
chitosan did not inhibit the growth of the two strains of bacteria as no inhibition
zone was observed (Figure 7 &8). However, the two strains were sensitive to
Kanamycin antibiotics, and the diameter of the inhibition of the growth zone for

S.aureus was 18mm and for Eco/i was 20mm (Table 8)

Figure 7. The antibacterial activity of amphoteric chitosan and controls against
E. coli by agar diffusion test.

 

A= Kanamycin, B: 0.5% chitosan, C: 0.25 chitosan. D= 1% chitosan, E= Sodium succinate.

45

Figure 8. The antibacterial activity of amphoteric chitosan and controls against
S.aureus by agar diffusion test.

 

A= Kanamycin, B: 0.5% chitosan, C: 0.25 chitosan, D: 1% chitosan, E= Sodium succinate.

Table 8. The antibacterial activity of chitosan and ”controls against gram- negative
and gram -positive bacteria by agar disk diffusion assay.

 

Test samples S. aureus zone of E. coli zone of

inhibition (mean inhibition

diameter in mm)
*

 

1% chitosan

 

0.5 % chitosan

 

0.25 % chitosan

 

Sodium succinate

 

Kanamycin + 0(18) "' (20)

 

 

 

 

 

0 Negative no zone of inhibition, 0 positive with zone of inhibition.

46

2. Cellular response to intra-peritoneal implantation of chitosan:

2.1 Effect of chitosan implantation on the mononuclear and

polymorphonuclear cells:

The size of the reactive zone, the area of cellular reaction surrounding the
pads, was increased with time reaching its peak at 7 days post-implantation
(16.9 mm), and then declining after 15 days in the chitosan group of rats (9.2
mm) (Table 9). In the Gelfoam® group, on the other hand, it continued to

increase until day 15 (36.9 mm) (Table 9 & Figure 9).

47

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Figure 9. The reactive zone size of chitosan and Gelfoam® at various time point.

80
70
60
50

 

I Chitosan

40 III Gelfoam®

 

 

 

30

Zone size (mm)

20
10

 

o
4 4 7 7
"’0‘”, 0% Days 50%

Time

The most common cell type found after 4 hours of implantation were mast
cells and macrophages. In both the chitosan and Gelfoam® group, there was a
diffuse infiltration of inflammatory cells, macrophages and polymorphonuclear
cells (PMN), in the reactive area surrounding the pad that started as early as 4
hours post- implantation

(Figure 10.1 & 10.2).

49

Figure 10.1. The cellular reaction surrounding the chitosan pad at 4 hours post-
implantation.

 

CH= Chitosan, M= Mast cells, PMN: Polymorphonuclear cells

Figure 10.2. The cellular reaction surrounding the Gelfoam® pad at 4 hours post-
implantation.

 

CF= Gelfoam®, M= Mast cells, PMN: Polymorphonuclear cells

50

After 4 days of implantation the number of mononuclear cells and
fibrocytic cells continued to increase, while the PMN cells were decreased for

both chitosan and Gelfoam ® groups (6.2% and 1% respectively) (Table 9 and
Figure 11.1 & 11.2).

Figure 11.1 The cellular reaction surrounding the chitosan pad at 4 days post-
implantation.

 

CH=chitosan, O= Omentum

Figure 11.2 The cellular reaction surrounding the Gelfoam® pad at 4 days post-
implantation.

     

.m. s. 4‘

G: Giant cells, GF= Gelfoam®, M= Mast cells,
MQ= Macrophages, PMN: Polymorphonuclear cells

51

Large numbers of mononuclear cells and low numbers of mast cells and
PMN cells were observed at day 7 post- implantation in both chitosan and

Gelfoam® groups (Figure 12.1 &12.2).

Figure. 12.1 The cellular reaction surrounding the chitosan pad at 7 days post-
implantation

 

Figure 12.2. The cellular reaction surrounding the Gelfoam® pad at 7 days post-
implantation.

 

F= Fibrocytic cells, 6: Giant cells, GF= Gelfoam®, L= Lymphocytes

52

At 15 days post-implantation there was a decrease in the zone size and
number of mononuclear cells samples from the chitosan group (9.2 mm and
74.6% respectively) compared to the Gelfoam® group (36.9 mm and 82.2%

respectively) (Figure 13.1 & 13.2).

Figure. 13.1 The cellular reaction surrounding the chitosan pad at 15 days post-
implantation.

 

BV= blood vessel, CH= chitosan, MQ= Macrophages, L= Lymphocytes

Figure 13.2. The cellular reaction surrounding the Gelfoam® pad at 15 days post-
Implantation.

 

GF= Gelfoam®, MO: Macrophages, L= Lymphocytes

53

ANOVA tests showed that the size of the reactive zone of the chitosan
group was statistically significant from the zone of the Gelfoam® group (p=<0.05)
(Table 10). No significant difference was seen between chitosan and Gelfoam®
groups in terms of the inflammatory cells, mononuclear and PMN, (p>0.05)
(Table 10). However, the number of PMN cells significantly decreased with time

in each group (p=<0.005)

Table 10. ANOVA result of the effect of different treatment on the magnitude of
the reaction and the cell types at various time points.

 

 

 

 

 

 

 

 

 

Parameters Time Treatment Time &Treatment
Zone size
0011* 0.304 0.021*
Mononuclear cell
0.146 0.823 0.136
Multinuclear
0023* 0.001* 0.045*
Polymorphonuclear
cells 0.001 * 0.359 0.697
Lymphocytic cells 0.157 0001* 0005*
Blood vessels
0.002* 0.213 0.364

 

 

 

 

Time: 4hrours & 4,7 15 days ;Treatment: Chitosan or Gelfoam; * p-value is significant at p<0.05

2.2. Effect of chitosan on the multinuclear cells:

The number of multinuclear cells (giant cells) present in the reactive zone
increased with time in both the chitosan and control groups (Table 11). However,
they were significantly increased in the Gelfoam group ® compared to the

chitosan group (p<0.05) (Table 10, Figure 14).

54

 

 

 

 

 

 

 

 

 

 

 

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55

Figure 14. Giant cells in the reactive zone in a sample from the Gelfoam®
group.

 

2.3. Effect of chitosan on the formation of blood vessels:

It appears from the observations of the reactive zone surrounded the pads
that chitosan has greater angiogenic activity than Gelfoam ® (T able 11). The
number of blood vessels adjacent to pads was greater specifically at day 4 and
day 7 post-implantation with chitosan than with Gelfoam ® (Table 10 & 11).
However, after 15 days of implantation, the blood vessels numbers decreased in
the chitosan group compared to the Gelfoam® group (107/mm and 123/mm
respectively). Overall, neovascularization was more prominent in the chitosan

group compared to the Gelfoam® group (Figure 15).

56

Figure 15. The blood vessels in the reactive zone of chitosan and Gelfoam®
group at various time points.

160
140 /
120
100

80
I Chitosan

60 Gelfoam®

Blood vessels count

40

20

 

m

0

?

Time

2.4 Effect of chitosan on the lymphocyte cells:

At all points in the time course that were studied, the lymphocytic cells, i.e.
lymphocytes and plasma cells, were significantly greater in the tissues with
chitosan than with Gelfoam® (Table 10). Starting as early as 4 days post-
implantation lymphocytes were present, and they increased with time in the
chitosan group and reached a peak at day 15 post-implantation. In contrast,

these cells in the Gelfoam® group decreased with time. (Figure 16).

57

Figure 16. Lymphocytes in the reactive zone in a sample from the chitosan
group.

 

BV= blood vessel, L= Lymphocytes

2.5. Effect of chitosan on Collagen deposition:

Collagen presence was used as index of the amount of scar tissues that
develops around the pads. Both chitosan and Gelfoam® groups showed
increased amounts of collagen until day 7 post-implantation (Figure 17.1 & 17.2).
However, at 15 days post-implantation, the two groups showed different patterns
of collagen deposition. The amount of collagen was significantly less in the
chitosan group (2.51 umz), compared to the Gelfoam® group where it increased

significantly (p<0001) (Table 12).

58

Table 12. Collagen deposition in the chitosan and Gelfoam® groups at various
time points.

 

Figure 17.1. Reaction around chitosan after 4 days of implantation. Minimal
collagen deposition with Masson’s Trichome stain.

 

C= Collagen, CH= Chitosan

59

Figure 17.2. Reaction around chitosan after 15 days of implantation. High
collagen deposition with Masson’s Trichome stain

 

O= Omentum, C = Collagen

 

3. ' L' ‘ L ' ‘ y findings:
Characterization of T cells and B cells using anti-CD3 and anti- C020
antibodies:

The chitosan group of rats, showed high number of CD3 positive cells at
day 4 and 7 post-implantation, and this declined by day 15. On the other hand,
the Gelfoam® group showed a moderate number of 003 positive cells at day 4
and 7, which then increased by day 15 post-implantation (Table 13, Figure 18.1,
18.2 & 18.3). Also, the pattem of reaction of chitosan and Gelfoam® groups was
different when CD20 antibodies were used. The tissues in the chitosan group
had high counts of positive cells (level 3, +++) at day 7, which then slightly

decreased to level 2 (++) at day 15 (Figure 19.1, 19.2, & 19.3). However, low

60

numbers of CD20 positive cells were found in the Gelfoam® group at day 4 and 7

level 1(+), and these moderately increased by day 15 (Table 13)

Table 13. CD3 and 0020 immunophenotyping results.

Chitosan

 

Gelfoam

Figure 18.1. Immunohistochemistry : Example of level 1 (Low) CDS positivity in
chitosan group.

 

P= positive

61

Figure 18.2 Immunohistochemistry : Example of level 2 (lnterrnediate) cos
positivity in chitosan group

 

P= positive

Figure 18.3 Immunohistochemistry : Example of level 3 (High) cos positivity in
chitosan group

 

P= positive

62

Figure 19.1 Immunohistochemistry: Example of level 1 (Low) CD20 positivity in
chitosan group.

 

P= positive

Figure 19.2. Immunohistochemistry: Example of level 2 (lnterrnediate) CD20
positivity in chitosan group

 

63

Figure 19.3 Immunohistochemistry: Example of level 3(High) CDZO positivity in
chitosan group

 

P= positive

CHAPTER 4.

DISCUSSION

65

DISCUSSION

The antibacterial activity of chitosan:

The number of practical applications of chitin and chitosan in the medical
field has grown rapidly in recent years. Chitosan, a biodegradable, nontoxic and
biocompatible polymer, has been used as an antimicrobial compound in
agriculture, and as an agglutinating and flocculating agent in wastewater
treatment, as well as a pharmaceutical agent in biomedicine. In this present
study, two forms of chitosan were evaluated for their role in wound healing. Two
aspects were studied in exploring the compounds role in wound healing. First we
explored chitosan’s capability to inhibit the growth and penetration of bacteria in
vitro, because the presence of micro-organisms greatly inhibits wound healing.

' Second, the role of chitosan in stimulating the cellular components of healing,
particularly those associated with immunological responses, were also studied in

an intra-peritoneal rat model.

Through testing using both agar diffusion tests and microbroth dilution
assays, Two forms of chitosan were found to lack any bactericidal effect against
two strains of bacteria; E. coli AT0025922 and S.aureus ATCC 29213 . In
contrast, many reports have indicated that chitosan has antimicrobial activity
against several microorganisms including fungi, algae and different strains of

bacteria (Rabea et al. 2003, Taha et al 2002, and Tsai et al 2002). In the first

66

phase of study a microbroth dilution assay was used and the MIC of each
chitosan form was calculated against an Eco/i bacterial strain. At pH 5.0, the
MIC values for MSU034 chitosan was 125 ug/ml, and for MSU035 chitosan was
250 ug/ml. However, the MIC values for the controls, cellulose and the acetic
acid, were within the same range as the chitosan samples. The Mle for
cellulose samples were 62.5 and 125 ug/ml, and for the acetic acid they ranged
from 62.5 to 250 ug/ml. These results are in concordance with a study carried out
by Je et al. (2006), where they found that the MIC value of aminoethyl-chitin
(AEC) is 62.5 ug/ml, 125 u/ml for AEC90, and 250 pg/ml for
dimethylaminoethyl-chitin (DMAEC 90) when tested against the same bacteria.
It could be interpreted that as chitosan produced an MIC value similar to that of
acetic acid, the bactericidal effect of chitosan previousely observed may have
been entirely due to the presence of the acetic acid alone. The effect of acetic
acid was demonstrated by Jia et al (2001) who reported that the antibacterial
activity of quaternary ammonium chitosan in acetic acid medium is stronger than
this same compound in water, and also that the effects increased as the

concentration of the acetic acid increased .

Previous reports showed that the form of acid used as the solvent has an
effect on bactericidal activity of chitosan. This study showed that in experiments
where formic acid was used as the solvent at pH 5.0, there was no bactericidal
effect. This is contrary to the results reported by Chung et al. (2003) who

concluded that formic acid without chitosan had best antibacterial effect when

67

compared to other organic and inorganic acids. Nevertheless, in these
experiments the addition of chitosan to the acid greatly enhanced the bactericidal
activity. In general, organic acids solvents with lower carbon contents had higher

antibacterial effects (Chung et al. 2003).

The amphoteric form of chitosan also did not inhibit the growth of E.coli
and S.aureus bacterial strains when it was used in the agar gel diffusion assay.
Loke et al (2000) had a similar experience and did not observe growth inhibition
zones against Pseudomonas aeroginosa and S.aureus strains when chitosan
acetate dressing was used. However, zones of growth inhibition were detected
when the chitosan acetate dressing was impregnated with Chlorhexidine
gluconate compound. Probably, either the form of chitosan used in our
experiments contained some acetate salt, which may explain the lack of
bactericidal activity, or that chitosan may need to be incorporated with additional

antimicrobial substances to improve its antibacterial activity.

The physiochemical, and the consequent biological characteristics of
chitosan, vary with the degree of acetylation, the source of chitin, and the overall
molecular weight (Rhazi et al 2000). The chitosan forms used in this study have
different degrees of acetylation and molecular weight; this fact may explain their
weak bactericidal effects. Furthermore, the antimicrobial action is affected by the
degree of polymerization, the host, the natural nutrient consistency, the chemical
or nutrient constituents of substrates, the environmental conditions, the pH of the
growth medium, and the presence or absence of interfering substances such as

lipids or proteins (Rabea et al, 2003). Sudarshan et al (1992) showed that

68

chitosan is no more actively bactericidal at pH 7 due to presence of more
uncharged amino groups and poor solubility. Chitosan with MW of 10,000-
100,000 has a greater bactericidal action than chitosan of low MW (Rabea et al
2003), though, the chitosan form used in the microbroth assay in our studies has

a MW of 97,000 Daltons.

In addition, the antibacterial activity increases with the ionic strength, but
is inversely proportional to the pH and metal ion concentration (Chung et al.
2003). It is possible that chitosan compounds used in the present study contains
EDTA; Chung et al. (2003) has suggested that the antibacterial activity of
chitosan against S. aureus decreases with the addition of EDTA and metal ions .
It is possible that the media used in this study, Muellor Hinton broth, contains

some metal ions or other inhibitory ingredients.

In all likelihood, there are a number of different mechanisms by which
chitosan kills microorganisms and inhibits infections. One of these mechanisms,
involves the fact that chitosan is a positively charged molecule; this allows the
compound to interact with the negatively charged macromolecules on the surface
of the microorganisms (Liu et al 2004). These interactions of the different
charged molecules leads to the increase in the permeability of the outer and
inner membranes of bacteria, thus resulting in the disruption of membrane
integrity and the release of the contents of the cells (Hui et al 2004) . Moreover,
chitosan may also bind the DNA in the nuclei and consequently inhibit RNA

synthesis (Hadwiger et al 1986). Lastly, as discussed above, chitosan can act as

69

a chelating agent and will binds trace metals like Mg, that are essential to the

growth of bacteria (Cuero et al. 1991).
The cellular response to intra-peritoneal implantation to chitosan:

In the second phase of the study, the cellular responses towards the intra-
peritoneal chitosan implants, including those reflecting activation of an immune
response, were correlated with the rate of the wound healing. Wound healing is a
dynamic, complex process requiring the integration of many different cell types
controlled by a variety of cell growth factors and soluble mediators (Singer and
Clark 1999). Healing consists of three main overlapping phases; inflammation,
tissue formation and tissue remodeling. Chitin and chitosan are both thought to
have stimulatory effects on the tissue reactions involved in these three phases of

healing.

Several mechanisms may be in play when chitosan and chitin accelerate
the healing of wounds. In the present study, the zone containing reactive cells
surrounding the chitosan pad was found to be statistically significantly different
from that surrounding the Gelfoam® pads in a number of ways. Cells were seen
in this location as early as 4 hours post-implantation of the chitosan with the cell
number in this zone decreased with time. In contrast, the number of cells in the
zone area surrounding the Gelfoam® pads continued to increase until day 15
post—implantation. A significant decrease in the capsule thickness surrounding
chitosan was seen with time in our study as was seen in earlier studies
(VandeVord et al. 2001). The most common cells accumulate within the reactive

zone of chitosan and Gelfoam® groups were polymorphonuclear (PMN),

7O

macrophage and mast cells. After 4 hours of implantation, the percentage of
PMN was estimated to be 28.4% for the chitosan group and 18.5% for the
Gelfoam® group. However, mononuclear cells, consisting mainly of
macrophages were the most dominant cell type, the percentages being 48.7% for
the chitosan group and 63.5% for the Gelfoam® group. This finding was similar
to Ueno et al (1999) who observed higher counts of PMN and macrophages in
chitosan treated beagles 3 days post-wounding compared to non-treated
controls. They also observed that at day 6 the inflammation become milder in
chitosan treated animals. Macrophage activation was also reported by Pelsuo et
al (1994) who stated that chitosan activates macrophages to produce more
nitrate, and that it has chemotactic properties to the macrophages. Macrophage
activation is essential for wound healing, and results in increases in the metabolic
activity, and the secretion of transforming growth factors (TGF-B), platelet derived
growth factor (PDGF), and other cytokines (Mori et al 1997, Nishimura 1986).
The inflammatory reaction induced by chitosan varies with the degree of
deacetylation (DDA). The DDA of chitosan used in this study was 85%. Hidaka
et al. (1999) recounted that chitosan with a DDA ranging from 65% to 80%
provokes swelling, severe inflammation and marked neutrophils infiltration which
diminished in 2 weeks; whilst the reaction to the chitosan with 94% DDA was
only mild and was characterized by fibrosis. These inflammatory cells which are
stimulated by chitosan application are critical in wound healing since they are

capable of production and secretion of a large range of proinflammatory

71

cytokines, such as tumor necrosis factor (TNF-a), lL-1, lL-8 and other growth
factors, at early stages of healing (Ueno et al 2001).

Various theories has been postulated for the mechanisms involved in
the stimulation of inflammation , the enhancement of leukocyte migration, and
the accumulation of neutrophils and lymphocytes caused by chitosan. The
triggering of inflammation may be due to the activation of Hageman factor and
other plasma proteins by the negative charge of the chitosan acetyl group, which
elicits blood coagulation, kinin and plasmin production (Hidaka et al. 1999). In
the same way, Suzuki et al. (2000) found that chitosan activates complement and
induces the release of arachidonic acid products and increase the plasma C3
concentration. The migration of neutrophils may be due to the interaction of
chitosan with neutrophil receptors such as selectins (VandeVord et al. 2001).
Also, Mon’ et al (1997) reported that chitosan elicited release of lL-8 from dermal
fibroblasts, a factor that is a potent chemokine activator for neutrophils.

Another feature observed in this study was the augmentation of
neovascularization by chitosan. More angiogenic activity was associated with
chitosan compared to Gelfoam®, specifically at day 7 post-implantation where
about 145 blood vessels/mm were counted. Overall, more blood vessels and new
vessels were found in reactive zone surrounding the chitosan pads. This result is
in concordance with the results reported by Ueno et al. (1999) and VandeVord et
al. (2001). A remarkable neovascuolarization was observed in upper layer of
wound at day 6 by Ueno et al. (1999). VandeVord et al. (2001) observed an

increased angiogenic action associated with the chitosan scaffold that reached

72

about 4.3 capillaries /mm2 in week 8. Azad et al 2003 stated that the inducement
of angiogenesis by chitosan is attributed to the fact that chitosan speeds up the
formation of fibroblasts at early stages of healing. Neovascularization supplies
the wound with oxygen, and white blood cells, all of which accelerate wound
healing, and defend wounds from infection (Kjolseth et al.1994).

In the present study, the number of multinuclear cells (giant cells),
present in the reactive zones surrounding the chitosan pad was found to be
significantly less than in the zones surrounding the Gelfoam® pad (p<0.05).
This may enhance the potential of chitosan in wound healing, as excessive
granulation would cause delay in epithelization and result in delay of wound
healing (Johnson 1990)

Another important aspect this study demonstrates was the significant
difference in the collagen deposition between samples of chitosan and the
control group. The amount of collagen was increased with time in both treated rat
groups that reached its peak (5.77 pmz) at day 7 in the chitosan group. It then
declined by 15 post-implantation in the chitosan group, though, it continued to
increase in the Gelfoam® group (7.2 umz). A similar result was observed in a
parallel study by our lab in the punch biopsy wounds in rat’s dorsal skin
(Mackenzie et al 2006). By using Immunohistochemical staining, Ueno et al
(1999) also noticed high amounts of collagen and more granulation tissues at
day 9 post-wounding in the chitosan group. However, in their study the thickness
of the granulation tissue of the chitosan group was continued to increase more

than in the control group until day 15. Our results are in agreement with Azad et

73

 

al (2003) who observed a higher degree of epithelization and a lack of scarring
when chitosan mesh was applied on patients wounds compared to Bactigras
bandages. Our result was at odds with Kojima (2004) who found collagen

synthesis increased up to day 14.

The decrease in the collagen deposition associated with chitosan could be
explained by the action of chitosan in inhibiting fibroblast-mediated contraction of
collagen lattices and so reduce contraction of the wound and scarring (Howling et
al. 2002). Similarly, Taravel and Domard (1995) observed that when chitosan is

in excess it can denature collagen.

Lastly, the immunological nature of the cellular response was
investigated by determining the presence of particular lymphocyte cell types
involved in the immune complex — T and B cells using two antibodies, CD3 and
C020 respectively. The CD3 immunostain revealed that more T-cells were
associated with chitosan pads (>100) at day 4 and 7 post-implantation compared
to the Gelfoam® pad. However, at day 15 CD3 positive cells declined in chitosan
group and increased in the control group. On the hand, B- cells detected by
CD20 immunostaining were about (50-75 positive cells) in samples from both
chitosan and Gelfoam® groups atday 15 post-implantation. However, in this
study when the plasma cells and lymphocytes were counted in the H+E sections,
the chitosan group samples showed an increase of these cell types with time,
and in the Gelfoam® group they decreased. Chitosan was reported to have
immunomodulatory action and effects as an immune adjuvant (Nishimura 1985,

Seferian and Martinez 2001). Obminska-Mrukowicz et al (2006) reported that

74

 

administration of chitosan adipate increases the CD4+ and CD19+ cells but not
the CD3+ in the mesenteric lymph nodes. Contrary to our results VandeVord et
al. (2001) detected low to negative cellular or antibody response in their study to
assess the biocompatibility of chitosan. They interpreted their findings to show
that chitosan had chemotactic effect on the immune cells but did not lead to a

specific humoral immune response (VandeVord et al.2001).

Conclusion:

This study supported the concept that chitosan contributes positively to
the healing of wounds. So far, the two forms of chitosan used in this study did
not exhibit bactericidal activity against E. coli and S. aureus strains. Their MIC
values were similar to controls, cellulose and acetic acid. Chitosan implanted in
the peritoneal cavity induced a significant cellular response with recruitment of
inflammatory cells as well as other immune cells to the wound at an early stage
post-wounding. However, throughout the 15 day period of the study, the cellular
response decreased and became milder in comparison to the Gelfoam®.
Another important feature associated with chitosan treatment, is the minimal
deposition of collagen and subsequent reduced scarring. Lastly, the use of CD3
and CD20 antibody markers indicated that chitosan stimulates the rapid

+accumulation of T cells and B cells in the responding tissue reaction.

75

 

APPENDICES

76

 

Appendix A
HEMATOXYLIN 8. Eosin

Lerner 2 hematoxylin

Eosin Phyloxine:
95% Ethyl alcohol
1% Eosin Y (aqueous)
Phloxine b (1% aqueous)
Glacial acetic acid

1% glacial acetic water:

glacial acetic acid (concentrated)
Distilled water

77

800 ml
100 ml
10 ml
4 ml

9 ml
99.1 ml

Appendix B

MASSON’S TRICHOME CHEMICALS

Bouin’s solution:
Picric acid
37%-40% Forrnalin
Glacial acetic acid

Weigert’s Hematoxylin stock -solution A
Hematoxylin
95% Ethyl alcohol

29% Ferric Chloride:
Ferric chloride lumps
Distilled water

Weigert’s Hematoxylin stock —solution B
29% Ferric Chloride
Distilled water
HCL , concentrated

Weigert’s Hematoxylin working solution :
Weigert’s Hematoxylin stock -solution A
Weigert’s Hematoxylin stock —solution B
Filter before use

1% Aqueous Biebrich Scarlet stock solution:
Biebrich Scarlet
Distilled water

1% Aqueous acid Fuchsin stock solution:
Acid Fuchsin
Distilled water

Biebrich Scarlet- acid Fuchsin working solution:

1% Aqueous Biebrich Scarlet stock solution
1% Aqueous acid Fuchsin stock solution

78

750.0 ml
250 ML
50 ml

19m
100 ml

29 gm
100 ml

4 ml
95 ml
1 ml

20 ml
20 ml

19m
100 ml

1 ml
100 ml

45 ml
5 ml

Glacial acetic acid, concentrated

Phosphmolybdic-phosphotungstic acid working solution:

Phosphmolybdic acid
Phosphotungstic acid
Distilled water

Aniline blue:
Aniline blue
Distilled water
Glacial acetic acid, concentrated

79

1ml

5 gm
5 gm
200 ml

2.5 gm
100 ml
2 ml

Appendix C
Immunohistochemistry reagents

Tris HCL 0.1 M, PH 7.6:
Tris HCL (Sigma No. T4253) 7.45
Distilled water 1L
Adjust PH to 7.6

Hydrogen Peroxide/methanol

30% hydrogen peroxide 4 ml
distilled water 16 ml
diethyl alcohol 80 ml

HIER (working) CITRA method:

CITRA retrieval concentrates 1 part
Distilled water 9 parts

80

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