Download 1. INTRODUCTION The skin is the largest organ in the human body

1.
INTRODUCTION
The skin is the largest organ in the human body comprising 12-15% of
body weight, with a surface area of 1-2 meters. Skin is continuous with, but
structurally distinct from mucous membranes that line the mouth, anus,
urethra, and vagina. Two distinct layers occur in the skin, namely the dermis
and epidermis. The basal cells lie directly on the basal membrane that forms a
definite border between the dermis and epidermis. The basal cells acting as
mother cells, by cell division, provide for the continuous regeneration of the
skin. The basal cell layer is comprised mostly of keratinocytes which are
either dividing or non-dividing. In the basal layer are also found the
melanocytes, which are the pigment producing cells. The skin functions in
thermoregulation, protection, metabolic functions and sensation. The dermis
is attached to an underlying hypodermis, also called subcutaneous connective
tissue, which stores adipose tissue. The skin is made up of cells like
fibroblasts, macrophages and adipocytes embedded in an extra cellular matrix
[ECM] consisting of ground substance and fibers formed by collagen and
elastin
along
with
glycosaminoglycans
[GAG],
proteoglycans
and
glycoproteins.
1.1
WOUNDS
Wound can be defined as a disruption of the normal anatomical
relationship of tissues as a result of injury. Wounds can be of open and closed
type. Open wounds have an opening or break in the skin, whereas in closed
wounds, skin is not broken, but the impact from the damaging object would
1
have injured or crushed tissues lying below the tissue point. These wounds are
commonly known as bruises and it will be visible due to discoloration
resulting from the rupturing of blood capillaries in the injured area. Different
types of open wounds are based on the manner in which the skin or tissue is
broken and there are six general kinds of wounds: abrasions, incisions,
lacerations, punctures, avulsions, and amputations. Many wounds, of course,
are combinations of two or more of these basic types (Linares 1996, Calvin
1998).
Figure 1.1 Anatomy of skin
1.1.1 Ulcer
An ulcer is an open sore of the skin, eyes or mucous membrane, often
caused, but not exclusively, by an initial abrasion and generally maintained by
an inflammation, infection, and/or medical conditions which impede healing.
2
In other words, it is a macroscopic discontinuity of the normal epithelium.
Other causes of skin ulcerations include pressure from various sources and
venous insufficiency, which is seen very often in diabetes milletus, resulting
in serious pathological conditions.
1.1.2 Burns
Burns form the most traumatic of all skin injuries, which may be due to
thermal, chemical or electrical means. In burn injuries the trauma and
subsequent repair relies greatly on the extension and the depth of the wound.
First-degree burns affect the outer layer of the skin, causing pain, redness, and
swelling. Second-degree burns affect both the outer and underlying layers of
the skin, causing pain, redness, swelling, and blistering. Third-degree burns
extend into deeper tissues, causing brown or blackened skin that may be
numb (Ho-Asjoe et al 1999).
1.2
WOUND HEALING
The process of wound healing involves interaction of a variety of
different cell types and matrix components. Various types of cells respond to
environmental signals in a specific manner in order to carry out their genetic
programme of proliferation, differentiation and function. Cells synthesize
different proteins for their proliferation and migration, all of which are
controlled in a phased manner. The healing process starts from clotting of
blood at the site of wound and gets completed with remodeling of the tissue.
However the restoration of the disrupted cellular and anatomic continuity of
organisms or its parts by way of healing to ensure wound closure and
3
functional restoration of the damaged tissue is a marvelous process that is not
yet fully and clearly understood (Oliver et al 1992).
1.3
PHYSIOLOGICAL BASIS OF WOUND HEALING
Wound healing is a fundamental biological process that allows the
individual to maintain structural and functional integrity in response to
trauma. Healing process is a well-orchestrated cascade of overlapping events
with haemostasis, inflammation, proliferation and remodeling phases. After
injury the cells release soluble products termed cytokines. Cytokines are
polypeptides, which allow communication between cells, causing major
changes in cell behavior. These cellular factors initiate events that lead to the
subsequent phases of wound healing (Diegelmann and Evans 2004).
1.3.1 Coagulation
Platelets agglutinate at the site of injury and a fibrin clot forms by the
activation
of
the
coagulation
cascade.
Thrombin
induces
platelet
degranulation, leading to the release of growth factors viz., platelet derived
growth factor (PDGF), transforming growth factor alpha (TGF-α),
transforming growth factor beta (TGF-β), epidermal growth factor (EGF) as
well as adhesive glycoprotein such as fibronectin. In addition to providing
haemostasis, the fibrin clot acts as a matrix for colonization of inflammatory
cells, which are attached to the wound site via chemotaxis by PDGF and
TGF-α.
4
1.3.2 Inflammation
Inflammation, the second phase of wound healing, occurs 1 – 5 days
after injury. Migrating inflammatory cells accumulate in the healing wound
with neutrophils predominating in the early hours of inflammation.
Neutrophils help to decontaminate the wound through phagocytosis of
bacteria
and
foreign
bodies,
after
which
macrophages
outnumber
lymphocytes. Macrophages release chemotactic factors (eg. Fibronectin) to
attract fibroblasts to the wound area. New blood vessels growth follows a
gradient of angiogenic factors produced by hypoxic macrophages because
macrophages do not produce these angiogenic factors when either fully
oxygenated or anoxic. Macrophages can be considered as factories for growth
factor production, including PDGF, fibroblast growth factor, vascular
endothelial growth factor, TGF-β, and TGF-α (Falanga 1993). These
cytokines are important in inducing cell migration as well as proliferation and
matrix production. Macrophages thus appear to play a pivotal role in the
transition between inflammation and repair.
1.3.3 Proliferation
The third phase of wound healing is tissue formation, which occurs
between 3-12 days. Re-epithelialization begins at the wound edges as early as
24 hours post-injury and granulation of the wound starts around post-injury
day 5 for the re-establishment of the integrity of the epidermis and dermis at
the wound site. Granulation tissue is formed as macrophages, fibroblasts and
endothelial cells move into the wound space. Fibroplasia begins with
5
fibroblast proliferation and collagen deposition stimulated by TGF-β. Wound
contraction occurs in full thickness injuries and reaches a peak between 5 and
15 days after injury. Contraction of the wound is mediated by myofibroblasts
and this continues through this phase leading to remodeling (Moulin 1995).
1.3.4 Remodeling
Tissue starts to reorganize itself as early as day 3 and continues until it
returns to a normal tissue structure. During which the accumulation of
collagen ceases and net loss of collagen begins by day 21. Although new
collagen continues to be deposited, net resorption occurs due to increased
degradation of formed collagen by collagenase. As macrophages begin to
disappear, angiogenesis and fibroblast proliferation also decrease. (Singer and
Clark 1999, Diegelmann and Evans 2004).
1.4
INFECTION – MAJOR CONTRIBUTOR FOR CHRONICITY
Among various types of skin wounds, injury due to burns account for
more than 60% of morbidity and mortality. Burn wounds represent a
breakdown of the skin’s haemostatic function, resulting in fluid and
electrolyte loss and infection, which may be life threatening alone or in
combination (Tsipouras et al 1995). Although major advances have been
achieved in burn wound management and supportive care, infection remains
the leading cause of morbidity in burn patients (Greenfield and McManus
1997). A comprehensive review by Bowler (1998), regarding the
microorganisms that infiltrate wounds reveal that, aerobic microorganisms,
Staphylococcus aureus and Pseudomonas aeruginosa are predominant species
6
responsible for wound sepsis. In addition Escherichia coli, Klebsiella
pneumoniae and Streptococcus sp. are other aerobes that contribute to
infection in chronic and acute wounds. The progressive bacterial colonization
of burns, probably within matter of hours has remained an inseparable phase
of healing process. Many times bacterial colonization causes acute wounds to
transform into a chronic state, most frequently associated with a predisposing
condition that ultimately compromises the dermal and epidermal tissues.
During these process bacteria attracts neutrophils through chemo attractants,
which in turn engulfs them. The phagocytosed products are secreted out along
with a representative tri-peptide called f-met-leu-phe, which in turn gives
signals for further recruitment of neutrophils (Tschaikowsky et al 1993).
Neutrophils will engorge themselves until they are filled with bacteria and
constitute what is called as ‘laudable pus’ in the wound. (Thurston 2000) The
heavy infiltration of inflammatory cells further causes prolonged or increased
level of endopeptidases, mainly the matrix metalloproteinases (MMPs),
resulting in delayed healing.
1.5
ROLE OF MMP IN HEALING PROCESS
Matrix metalloproteinases (MMPs) play a major role in healing
process, as they are responsible for transformation, organization and
maintenance
of
extracellular
matrix.
MMPs
are
zinc-dependent
endopeptidases that share common structural and functional elements and are
able to cleave one or several extracellular matrix constituents as well as nonmatrix proteins. Ample evidence exists on the role of MMPs in normal and
pathological
processes
including
embryogenesis,
wound
healing,
7
inflammation, and cancer. Endopeptidases modulate the matrix morphology
through a highly controlled intracellular and extracellular process. This
modulation of ECM is significantly different at various time points during the
course of healing for different wound types. Thus it facilitates cellular
migration, adhesion, wound contraction and re-epithelialization (Armstrong
and Jude 2002). There are various types of MMPs identified which have
specific matrix substrates (McCawley and Matrisian 2001) and their activity
appears to be controlled at three basic levels,
A.
Genetic
:
Controlled through transcriptional factors
B.
Molecular
:
Controlled by factors responsible for conversion
of proform to active form
C.
TIMPS
:
Tissue inhibitors of matrix metalloproteinases
(TIMPS) regulate MMP level through feed back
inhibition.
MMPs are secreted as proenzymes, which become active by removal
of propeptide domain. All these MMPs are either bound to cell membrane or
secreted into extracellular space. To be secreted they are induced by several
factors including PDGF, IL-1, and TNF-α (Mauviel 1993; Kerr et al 1990).
Natural inhibitors, TIMPS, growth factor like TGF-β and synthetic inhibitors
bring about inhibition of their activity. The activation of the MMPs follows
the opening up of catalytic zinc domain. These zinc domains are activated
through a “cysteine-zinc” switch mechanism (Figure 1.2). The propeptide
8
forms a cap over the active MMP site with a critical cysteine residue
providing the fourth co-ordination ligand to a catalytic zinc ion. Dissociation
occurs only at the cysteine–Zn2+ linkage, whereas the Zn2+ complexation with
the three histidines is not affected. As a consequence, the MMPs are activated
and are now able to degrade appropriate matrix substrate (Kroncke 2001).
Figure 1.2 Activation of MMP through Cysteine – Zinc Switch
(Kroncke 2001)
In general, chronicity of a wound appears to be due to bacterial toxins,
which in turn induce elevated MMP expression (Miyajima et al 2001) and
imbalance between activation and inhibition (Trengove et al 1999). Thus
therapeutic intervention to control infection and to positively regulate MMP
balance is considered vital in achieving faster healing of wounds.
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1.5.1 MMP inhibitors
The MMP activity is closely regulated by endogenous inhibitors. The
best characterised of these are the tissue inhibitors of metalloproteinases
(TIMPs), which comprise a family of four protease inhibitors: TIMP-1,
TIMP-2, TIMP-3 and TIMP-4. Their inhibitory activity results from the
formation of a non covalent complex of the TIMPs to the C –terminal domain
of the proMMPs. Over the past 20 years, impressive efforts have been
dedicated to the development of synthetic compounds interacting potently and
selectively with the active site of MMPs. Despite these efforts, inhibitors able
to differentiate fully one MMP from the others have not been identified so far
(Cuniasse et al 2005). The catalytically indispensable zinc ion at the active
site has been exploited to design synthetic inhibitors to this enzyme family. In
addition, MMPs contain a second zinc atom and atleast two calcium atoms
that are not part of the catalytic center but seem to play roles in stabilising
tertiary structure (Willenbrock et al 1995). Thus the development of inhibitors
for zinc Metalloproteinases has relied on the use of a peptide sequence,
recognized by the targeted protease, to which have been grafted different
chemical functionalities able to interact potently with the zinc ion of the
active-site. This strategy has allowed the identification of several potent
pseudo-peptide inhibitors of MMPs. Based on the chemical structure of the
zinc-binding group, four different classes of zinc metalloprotease inhibitors
have been developed, those incorporating a hydroxamate (CONH–O–), a
carboxylate (COO–), a thiolate (S–) or a phosphinyl (PO2–) groups.
Hydroxamate exhibited ideal bidentate bonding with the catalytic zinc atom
10
for stable divalent metal coordination within the active site (Whittaker et al
1999).
Doxycycline is the only clinically approved drug used to inhibit MMP
activity mainly by disrupting the active zinc domains (Nordstrom et al 1998)
and is marketed under the name Periostat (Collagenex) for use in the
treatment of periodontitis (Peterson 2004). Doxycycline is a member of the
tetracycline family of antibiotics and is known to exert biological effects that
are independent of its antimicrobial activity. Based on this MMP inhibition
activity, doxycycline has been used for many MMP associated disorders like
chronic wounds (Chin et al 2003), periodontal disease (Gapski et al 2004),
rheumatoid arthritis (Nordstrom et al 1998), corneal erosion (Dursun et al
2001) and skeletal muscle reperfusion injuries (Roach et al 2002).
1.6
WOUND DRESSING
Based on the wound type and status, dressing materials should be
selected. In general a wound dressing may be of any type, but should possess
following basic characteristics (Thomas 1990, Winter 1962).
•
Maintenance of a moist wound environment
•
Wound protection from secondary infections by acting as
bacterial barrier
•
Provision of adequate gaseous exchange
•
Provision of thermal insulation free from particulate or toxin
contaminants
•
Elastic and non-antigenic
11
Availability of wide range of wound dressings till date is probably
matched by the diversity in wound types. In addition a wide variety of wound
dressings with targeted therapy in mind to address specific problems is
emerging in recent days. Food and Drug Administration (FDA 1999) has
categorized the type of dressings broadly as –
1.
Non-resorbable gauze/sponge dressing for external use,
2.
Hydrophilic wound dressing,
3.
Occlusive wound dressing,
4.
Hydrogel wound and burn dressing and
5.
Interactive wound and burn dressings.
The choice of dressing is dictated by the type and condition of the
wound, is enumerated in Table 1.1
Table 1.1 Types of wound dressings (Young 1997)
Wound type
Clean, Medium-to-high exudates
(epithelialising)
Clean, dry, low exudates
(epithelialising)
Dressing type
•
•
Paraffin gauze
Knitted varicose primary dressing
•
Absorbent perforated plastic filmfaced dressing
Vapor-permeable adhesive film
dressings
•
Clean, exudating (granulating)
•
•
•
Hydrocolloids
Foams
Alginates
Slough-covered
•
•
Hydrocolloids
Hydrogels
Dry, necrotic
•
•
Hydrocolloids
Hydrogels
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The design of a wound dressing depends largely on appropriate
selection of material. A number of materials have been experimentally and /or
clinically studied. Irrespective of the type of material used, it must possess the
following characteristics, (Hutmacher 2001)
•
Three-dimensional and highly porous with an interconnected
porous network for cell / tissue growth and flow transport of
nutrients and metabolic waste.
•
Biodegradable or bioresorbable with controlled degradation and
resorption rate to match cell / tissue growth in vitro and / or in
vivo.
•
Suitable surface chemistry for cell attachment, proliferation and
differentiation
•
Mechanical properties to match the tissues at the site of
implantation and
•
Easily processed into a variety of shapes and sizes.
The application of inert or bioactive synthetic dressing materials
remain largely limited to the healing process and minimization of scarring.
Similarly moist wound dressings, which possess clinical benefits, pose
problems by providing a favorable environment for microbial proliferation
(Eaglstein 1993). Among recently available dressings, collagen-based
dressings have shown to actively influence the healing process by intervening
with various tissue components. Moreover they possess all the characteristics
of ideal material for scaffolding as mentioned previously.
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1.7
BIOMEDICAL POTENTIALS OF COLLAGEN
The collagen family has been well described for many years by various
authors (Harkness 1966, Prockop and Kivirikko 1995). In humans 25 distinct
types of collagen have been identified to date on the basis of protein and / or
DNA sequence information (Gelse et al 2003). Collagen is distinct from other
proteins as it is composed of three polypeptide chains forming an unique
triple helical structure. Organization of individual helices into fibrils and
further into fibrillar bundles, results in stable macromolecular structure.
Though collagen has phylogenetically well conserved primary sequence and
triple helical structure (Tanzer and Kimura 1988), it exhibits structural
variations from tissue to tissue with reference to alignment of fibrils, diameter
and density of packing. This fibrillar diversity leads to specific mechanical
and structural properties, characteristic of each tissue. For example collagen
preparation from bovine skin contains 80 - 85% of type I collagen and 1520% of type III collagen and in cartilages and vasculatures type II is present
(Kivirikko 1998). As a consequence of this structural and functional
significance, collagen can be fabricated into various physical forms (Table
1.2). Thus collagen based wound dressings offer a high degree of plasticity
enabling application in wound treatment (Pachence 1996).
14
Table 1.2 Commercially available collagen based dressings
(Pachence 1996)
Materials Type
Partially purified skin
Company (Brand name)
LifeCell
Integra Life Sciences Corp. (Helistat)
Collagen sponges
Johnson & Johnson (Instat) MedChem
(Acti-Foam) BioCore (SkinTemp)
Collagen fibers or fleeces
Collagen Powder
Collagen composite
dressing
Hydrolyzed Collagen
1.8
Integra
LifeSciences
Corp.
(Helitene)
MediChem (Avitene)
Medifil (BioCore)
Fibracol (Johnson & Johnson) Biobrane
(Mylin)
Chronicure (Derma Sciences)
SHARK SKIN COLLAGEN
The molecular structure and biological functions of collagen type I
from land animals have been extensively investigated. Bovine and porcine
type I collagen provide a readily available source of scaffold materials for
various biomedical applications. However these sources have some potential
risk of infectious diseases such as bovine spongiform encephalopathy (BSE)
or transmissible spongiform encephalopathy (TSE) (Nomura et al 2000). In
order to attenuate the risk to manufacture biomaterials an alternate safe
collagen must be considered.
15
Type I collagen from aquatic animals may provide an alternative
collagen source and shark skin collagen in particular is potentially important.
Shark skin, which constitutes about 11% of the body weight, is mainly
discarded as industrial waste, except for its use as leather. The main protein in
shark skin is type I collagen, the structure basically resembles that from
domestic animals and also has the ability of self assembly to form fibrils as
that of mammalian type I collagen (Kimura et al 1981). The content of imino
acids and the type of cross-linkages vary from mammalian collagen, which
reflects the low denaturation temperature of shark skin collagen (Nomura et al
2000). However, the low denaturation temperature can be improved by
exogenous crosslinking for biomedical applications. Easy extractability and
better yield of collagen are advantageous for applications. Hence, shark
collagen is a promising source of type I collagen for various biomedical
applications.
The milk shark, Rhizoprionodon acutus, is a requiem shark such as the
tiger shark, blue shark, bull shark, etc. of the family Carcharhinidae, found in
the tropical waters of the Indo-West Pacific and eastern Atlantic oceans
between latitudes 35° N and 30° S, from the surface to 200 m. Its length is
upto about 1.75 m for a specimen of unknown gender and 80 cm for females.
The milk shark is a small shark with a long, narrow, snout, big eyes without
notches, long labial furrows, and oblique-cusped teeth, which may be smoothedged or weakly, serrated. The second dorsal fin is small, low and behind the
larger anal fin, with no interdorsal ridge. It is found on continental shelves,
often on sandy beaches and rarely in estuaries, also reported to enter
16
freshwater and recorded several times from Cambodia as far upstream as the
Great Lake. Occurs near the surface in shallow waters, and feeds mainly on
small pelagic and benthic bony fishes, also cephalopods and other
invertebrates. It is viviparous and the meat is utilized fresh and dried salted
for human consumption and for fishmeal.
Figure 1.3 Milk shark (Rhizoprionodon acutus)
1.9
CROSSLINKING
1.9.1 Natural crosslinking
The systematic packaging of the triple-helices lends strength and
resilience to the collagen fibers. Additional mechanical and chemical stability
derives from intra- and intermolecular crosslinks. Initially, the formation of
crosslinks is mediated by lysyl oxidase during fibril formation (Yamauchi and
Mechanic 1988). The enzymatic activity is limited to the non-helical
telopeptide regions and leads to the conversion of selective lysyl and
hydroxylysyl residues to the corresponding aldehydes allysine and
hydroxyallysine. While the fibrils associate, the aldehydes can spontaneously
react. Intramolecular crosslinks form between two α-chains in the non-helical
17
section of the same molecule by aldol condensation of two aldehydes
(Yamauchi and Mechanic 1988, Reiser et al 1992). Intermolecular crosslinks
occur between the telopeptide region of one collagen molecule and the helical
region of a quarterly staggered, adjacent molecule. These bridges between
two different tropocollagen molecules result from aldimine formation
between aldehyde residues and ε-amino groups presented by lysine and
hydroxylysine (Reiser et al 1992). The interchain bifunctional crosslinks are
still reactive and continue to form polyfunctional crosslinks through multiple
condensations with histidine, lysine, or hydroxylysine residues (Graham and
Gallop 1994). Hence, through specific self-aggregation and crosslinking,
collagen can form fibers of unusual strength and stability, the primary reason
for the usefulness of collagen in medical devices.
1.9.2 Exogenous crosslinking
Natural crosslinking gives high tensile strength and proteolytic
resistance to collagen. Due to dissociation of crosslinks in the course of the
above described isolation processes, reconstituted forms of collagen such as
films, fibers, or sponges can lack sufficient strength and may disintegrate
upon handling or collapse under the pressure from surrounding tissue in vivo.
Furthermore, the rate of biodegradation has to be customized based on the
specific application. For example as a hemostat, collagen has accomplished its
mission once the blood clot has formed, whereas for tissue augmentation an
implant has to maintain its scaffolding properties while it is gradually
replaced by host collagen (Van et al 1994). Thus, it is often necessary to
confer mechanical firmness and collagenase resistance by introduction of
18
exogenous crosslinking into the molecular structure. Several physical and
chemical crosslinking methods have been reported. Physical methods include
dehydrothermal treatment and UV irradiation (Welz and Ofner 1992).
However, these methods cannot control the degree of crosslinking. Chemical
methods include formaldehyde, glutaraldehyde, hexamethylenediisocyanate,
carbodiimide, polyepoxy compounds and acyl azide. A major handicap of
chemical crosslinking agents is the potential toxic effect of residual molecules
and / or compounds formed during in vivo degradation (Friess 1998).
Glycosaminoglycans (GAGs) are long, unbranched polysaccharides
that are a significant component of proteoglycans, do not elicit an immune
response on their own, and have been used extensively for tissue engineering
applications. Copolymerization of collagen with GAGs has been observed to
increase the stiffness and toughness and decrease the degradation rate of
collagen (Yannas et al 1975). While the precise mechanism leading to these
effects have yet to be elucidated, collagen copolymerization with GAG is
used as an alternative to heavy crosslinking of collagen which can often
render the material brittle. Scaffolds fabricated from type I collagen and a
glycosaminoglycan have been used to study cell migration and contraction in
vitro (Ueno et al 2001, Freyman et al 2001, Sethi et al 2002) as well as induce
regeneration of the skin, conjunctiva, and peripheral nerves in vivo ( Yannas
1990).
Chitosan is a natural polysaccharide, which is structurally similar to
GAGs. It consists of β (1-4) linked D-glucosamine residues, and has been
reported to be nontoxic, bioabsorbable and to promote wound healing
19
(Francis and Howard 2000). Furthermore, the incorporation of chitosan into a
collagen scaffold is known to increase its mechanical strength, as it forms an
ionic complex between the positively charged chitosan and the negatively
charged collagen (Taravel and Domard 1996). Chitosan has also been
reported to stimulate the activity of growth factors (Ueno et al 2001) and to
contribute to the maintenance of the chondrogenic phenotype, especially in
terms of its morphology (Sechriest et al 2000). One of the interesting features
of chitosan is its cationic nature occurring from primary amine groups, thus
providing a high charge density in an acidic solution. The cationic charges
allow chitosan to form water-insoluble ionic complexes with a variety of
polyanionic substances such as sulfate, citrate, and tripolyphosphate (TPP) for
drug delivery applications (Shu and Zhu 2002). Furthermore, this
characteristic can be used for the delivery of biologically active polyanions
such as DNA (Lee et al 1998, Roy et al 1999).
1.10
COLLAGEN BASED DRUG DELIVERY SYSTEMS
Major advantages using collagen as a biomaterial for development of
delivery systems are – available in abundance and easily purified, nonantigenic, biodegradable and bioresorbable, non-toxic and biocompatible,
synergic with bioactive components, high tensile strength, easily modifiable
as desired by utilizing functional groups comparable with synthetic polymers
(Werkmeister and Ramshaw 1992). The use of collagen as a drug delivery
system is very comprehensive and diverse (Friess 1998). Table 1.3
enumerates the types of collagen drug delivery applications. Any preparation
that uses collagen as a matrix may act as (a) Reservoir – increase contact time
20
with the host, (b) Reversible – bind drug such that they are released in a
delayed mode, (c) Reduce the likelihood of systemic toxicity. Most of the
delivery devices mentioned have been designed to sustain the release of
therapeutics. Controlled delivery is essential for drugs, which have narrow
line of demarcation between therapeutic and toxic concentration. An
extensive compilation on different types of collagen based delivery systems
for varied applications, including antibacterial, recombinant proteins, growth
factors and gene therapy has been comprehensively described (Ruszczak and
Friess 2003).
1.11
LOCAL
DELIVERY
THERAPEUTICS
FOR
WOUND
HEALING
The importance of drug delivery is not only pertinent to physiological
response but also to the mechanism of drug release, especially as a carrier for
on-site delivery of antibacterials. The scaffolds used for this purpose are thin
films, gels, sponges and bilayered dressings with or without chemical
modifications (Ruszczak and Friess 2003). Fibrillar collagen matrices alone
are capable of only moderating the release of drugs hence a significant nonfibrillar collagen is necessary to modulate the diffusive parameters. Further
chemical modifications like succinylation, electrostatic charge interactions are
some of the strategies adopted to improve the release pattern of drugs. In this
category several wound healing materials like composite collagen –
chondrotin 4 and 6 sulphate (Berthod et al 1994), polyurethane coated
atelocollagen – rifampicin composite (Suh et al 1994) materials, bilayered
dressing with carboxymethyl – chitin hydrogel materials and chitosan acetate
21
thin films were able to sustain the antibiotic delivery. Emerging systems
deliver growth factors either through implants or through bioactive thin films
(Royce et al 1995, Mao et al 2005). Though collagen alone can be tailored
into various forms to deliver therapeutics, many of these systems are unable
to control the release pattern in a steady state for prolonged time (Friess
1998).
Table 1.3 Applications of collagen based drug delivery system
(Ruszczak 2003)
Application mode
Drug
Indications
erythromycin, Infection, glaucoma
Pilocarpine,
gentamycin,
Gentamycin,
vancomycin,
tobramycin,
Polymyxin
B, Infection, glaucoma
trimethoprim, amphotericin B, 5Shields
FU,
pilocarpine,
steroids, Glaucoma, Inflammation
flurbiprofene,
Particles
Cyclosporine
Allograft implication
Gels
Keterolac
Inflammation
111
Vinblastine, cisplatine, 5-FU, In,
90
Y labeled monoclonal antibodies, Cancer Treatment
Aqueous injection
TGF-β, FGF, Insulin, Growth Wound Repair
Hormone
Gentamycin, cefotaxim, fusidic
acid,
clindamycin,
all-trans Infection, cervical dysplasia
Sponges
retinoic acid, Growth factors, bone Tissue regeneration
morphogenic proteins
Medroxyprogestrone
acetate,
human
growth
hormone, Tissue
repair
and
Films
immunostimulants,
tetracycline, regeneration
growth factors
Retinol,
tretinoin,
tetracain, Local anaesthetic, dermal
Microparticles
lidocaine, ethracridine lactate
Minocycline,
lysozyme, Infection
and
Wound
Monolithic devices
intereukin-2, interferon
Repair
Inserts
22
To accomplish controlled release, drugs can be entrapped in micro-carriers
like micro/nano spheres, liposomes or as microencapsules. The following
section explains in detail the advantages of microspheres in controlling the
release pattern and the polymers utilized to develop microcarriers.
1.12
IMPACT
OF
CONTROLLED
DELIVERY
DEVICES
IN
WOUND HEALING
The term ‘Controlled Release’ has become associated with those
systems from which therapeutic agents may be delivered at a predetermined
rate over long time. These include situations requiring the slow release of
water-soluble drugs, drug delivery to specific sites and release of two or more
agents with same formulations. Among the recent advancements in wound
care management, slow release of drugs exhibited by hydrogels provide scope
for developing novel wound dressings (Kim et al 1992, Peppas et al 2000,
Lay-Flurrie 2004). The ideal drug delivery system should be inert,
biocompatible, mechanically strong, and comfortable for the patients, capable
of achieving optimum drug loading, simple to administer and remove, safe
from accidental release, and easy to formulate and sterilize. Figure 1.4
represents a comparison between release profiles obtained with traditional
dosage form and controlled release dosage system. In traditional drug
administration the blood level of the agent should remain between a
maximum value, which may represent a toxic level, and a minimum value,
below which the drug is no longer effective. In controlled drug delivery
systems designed for long-term administration, the drug level in the blood
remains constant, between the desired maximum and minimum, for an
extended period of time (Allen et al 2005).
23
Figure 1.4
Comparison between traditional dosage form and controlled
drug release system (Allen et al 2005)
1.13
CONTROLLED RELEASE MECHANISM
There are three primary mechanisms by which active agents can be
released from a delivery system: diffusion, degradation, and swelling
followed by diffusion. Any or all of these mechanisms may occur in a given
release system. Diffusion occurs when a drug or other active agent passes
through the polymer that forms the controlled-release device. Figure 1.5.1
reference shows the general mode of drug release through diffusion, either on
24
a macroscopic scale, as through pores in the polymer matrix on a molecular
level, by passing between polymer chains. Figure 1.5.2 shows the type of
system (reservoir system) most widely used for oral delivery of drugs.
Figure 1.5.3 shows a typical, dermal and/or the trans-dermal system used in
delivering drug to the wound site. In this type of system a reservoir of solid
drug, or dilute solution, or highly concentrated drug solution within a polymer
matrix is surrounded by a film or membrane of a rate controlling material by
which a fairly sustained rate of drug release can be maintained. In order to
achieve controlled release of drug in a desired rate, the most widely used
systems are swelling-controlled release devices. These systems are initially
dry and, when placed in the body will absorb water or other body fluids and
swell. The swelling increases the aqueous solvent content within the
formulation as well as the polymer mesh size, enabling the drug to diffuse
through the swollen network into the external environment. This may be
achieved through two phenomena – by diffusion through the polymer matrix
(Figure 1.5.4) and matrix swelling-controlled release systems (Figure 1.5.5).
Other systems used for controlled delivery of drugs include biodegradable
systems in which polymer degrades in the body, environmentally responsive
systems, and externally triggered controlled delivery systems (Robinson and
Lee 1987, Peppas et al 1997).
1.14
BIOMATERIALS
FOR
DEVELOPING
CONTROLLED
RELEASE SYSTEMS
Wide ranges of materials (both synthetic and natural) are available for
developing controlled release systems. Natural polymers have gained special
25
interest over synthetic polymers mainly because of better biocompatibility,
degradation products being biocompatible (in contrast to some of the
synthetic monomer), cost effectiveness, and bulk availability. The role of
natural polymers in biomedical application is pivotal, especially hydrogels,
carrier systems, wound dressings, oral dosage forms, and implants.
Biopolymers from marine sources have been studied and utilized in
pharmaceutical and biotechnological product development for a number of
years (Peppas et al 1997), particularly chitosan as biodegradable biopolymers
in drug delivery systems (Skaugrud et al 1999).
Figure 1.5
Schematic
representation
of
controlled
drug
release
mechanism through polymer matrix (Peppas et al 1997)
26
1.15
CHITOSAN
–
BIOMEDICAL
AND
PHARMACEUTICAL
APPLICATIONS
This class of marine polysaccharides possesses wide range of
biomedical and pharmaceutical applications owing to their excellent
biocompatibility and biodegradability (Muzzarelli et al 1988). Chitosan is a
linear, randomly distributed, binary heteropolysaccharide consisting of (1–4)linked 2-acetamido-2-deoxy-β-D-glucose and 2-amino-2-deoxy-β-D-glucan
units (Figure 1.6). It is derived by alkaline N-deacetylation of chitin, obtained
from marine crustaceans. Based on degree of N-deacetylation and molecular
weight, it’s physicochemical (e.g. solubility, viscosity), biological (e.g.
bioadhesion, absorption enhancement) and formulation properties can be
tailored accordingly. Networks formed by ionic crosslinking (Figure 1.7) of
chitosan are mainly used for drug delivery. They generally exhibit
pH-sensitive swelling and drug release by diffusion through their porous
structure (Ruel-Gariepy et al 2000, Remunan-Lopez and Bodmeier 1997).
Hence the micro and nano carriers of chitosan are widely used for controlled
delivery of wide range of therapeutics. The carriers can be developed by
various techniques like emulsion cross-linking, coacervation/precipitation,
spray drying, ionic gelation and sieving method. Among these, ionic gelation
is preferred for delivering therapeutics required to be delivered for a short
time period in a controlled fashion. Especially, in cases where drug
concentration has to be achieved in a short duration (burst release) and further
to be maintained in a steady state. This strategy is most suitable for materials
intended for wound healing application. Apart from that, it is a
Glycosaminoglycan (GAG) having natural ability to interact with host cells,
27
and also similar to the integral component of ECM (Agnihotri et al 2004,
Berger et al 2004).
The advancement in wound dressing strategies, specificities and
availability of wide variety of biomaterials provide impetus in developing
wound dressings for our desired need. Implication of controlled drug delivery
phenomenon along with the understanding of wound healing phases make it
possible to design scaffolds to tackle wounds of varied degree of pathology.
Figure 1.6 Structural unit of chitosan (Agnihotri et al 2004)
Figure 1.7 Schematic representation of ionically crosslinked chitosan
(Berger et al 2004).
28
AIM OF THE WORK
To develop collagen based controlled delivery system for wound
healing, with the main emphasis on mitigating infection and to hasten healing
with balanced remodeling.
Collagen, especially in the form of membranes/ films has gained more
importance in recent past, particularly for its healing properties. Biomedical
use of bovine and porcine skin type I collagen have been well documented.
But due to potential risk of Bovine spongiform encephalopathy (BSE) and
Transmissible spongiform encephalopathy (TSE) there is an increasing
demand for an alternate source especially collagen from aquatic animals like
squids and fish. Squids are more glycosylated and the collagen yield is low.
The collagen yield from fish skin is high and rich in type I collagen but the
disadvantage is the presence of scales. Whereas shark could be potential
alternative because shark skin is treated as waste and has very less and tiny
scales
and
the
collagen
extractability
is
high.
Collagen
and
Glycosaminoglycan (GAG), identical constituents of native tissues, are
widely utilized to fabricate scaffolds serving as an active analog of
extracellular matrix (ECM). The combination of collagen and GAG is unique
in skin regeneration since collagen or GAG alone cannot heal full thickness
wounds. Chitosan and Aloe vera which are GAG analogs have numerous
biomedical applications. Therefore, a reconstituted shark skin type I collagen
composite scaffold with chitosan or Aloe vera impregnated with doxycycline
loaded chitosan microspheres was developed, characterized and evaluated
in vivo.
29
The present work encompasses the following goals:
•
Isolation and characterization of collagen from the skin of milk
shark (Rhizoprionodon acutus).
•
Development of reconstituted shark skin collagen scaffolds
incorporated with chitosan and parenchymal Aloe extract and
their characterization, including assessment of physicochemical, morphological and biocompatible properties.
•
Development
of
doxycycline
hyclate
loaded
chitosan
microspheres, and drug loaded microsphere incorporated
collagen
scaffold,
and
assessment
of
morphological,
antibacterial and in vitro release studies.
•
Evaluation of the efficiency of doxycycline hyclate loaded
chitosan microspheres impregnated collagen scaffold against
infection challenged burn wound in rat model. Assessment of its
efficiency through healing rate, decrease in microbial load,
collagen and Glycosaminoglycan (GAG) turn over, expression
of proinflammatory cytokines (IL-6, IL-1β and TNF-α) and
Matrix Metalloproteinases (MMPs) expression, and efficient
remodeling.
30