61 3.1 INTRODUCTION Despite a control over bacterial colonization due to improved materials, implants, clean room techniques as well as systemic chemo prophylaxis, infection still remains a complication during injuries to skin. Conventional dressings form a natural barrier to the migrating epidermal cells, forcing them to move beneath the drying eschar, thus prolonging healing time and loss of healthy tissue. Further, use of these types of dressings, in cases like partial thickness burns, can result in progressive dehydration followed by devitalization and necrosis. The coagulum which forms after applying such type of dressings eventually dries up to form a difficult eschar, which is hard to remove and causes further trauma when removed (Thomas 1990). An ideal wound dressing is one, which induces host cells to regenerate, prevents infection, acts like an osmotic regulator providing optimal environment for healing to take place quickly (Varghese et al 1986, Ho et al 2001). The modern dressing materials available, such as films, hydrogels and other bioactive materials have their own merits and demerits. For instance hydrogels exhibit tendency to adhere due to simple intrinsic viscosity and stickiness of serum itself. When such a type of dressing is removed, the band between the eschar and the underlying tissue will fail first, resulting in considerable damage to newly formed epithelia. On the other hand, film dressings, by virtue of their semi permeable nature, are claimed to provide a barrier to bacteria as well complying with most of the ideal characteristics needed for an effective wound dressing (Drake 1984).
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3.1 INTRODUCTION
Despite a control over bacterial colonization due to improved
materials, implants, clean room techniques as well as systemic chemo
prophylaxis, infection still remains a complication during injuries to skin.
Conventional dressings form a natural barrier to the migrating epidermal
cells, forcing them to move beneath the drying eschar, thus prolonging
healing time and loss of healthy tissue. Further, use of these types of
dressings, in cases like partial thickness burns, can result in progressive
dehydration followed by devitalization and necrosis. The coagulum which
forms after applying such type of dressings eventually dries up to form a
difficult eschar, which is hard to remove and causes further trauma when
removed (Thomas 1990). An ideal wound dressing is one, which induces host
cells to regenerate, prevents infection, acts like an osmotic regulator providing
optimal environment for healing to take place quickly (Varghese et al 1986,
Ho et al 2001). The modern dressing materials available, such as films,
hydrogels and other bioactive materials have their own merits and demerits.
For instance hydrogels exhibit tendency to adhere due to simple intrinsic
viscosity and stickiness of serum itself. When such a type of dressing is
removed, the band between the eschar and the underlying tissue will fail first,
resulting in considerable damage to newly formed epithelia. On the other
hand, film dressings, by virtue of their semi permeable nature, are claimed to
provide a barrier to bacteria as well complying with most of the ideal
characteristics needed for an effective wound dressing (Drake 1984).
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Infection in wounds greatly impairs the wound healing process
leading to chronicity of the wounds. A technical approach to control infection
can be through the delivery of antibacterial/ antibiotics at the site of wound.
There is an enormous advancement in developing wound care products
capable of mitigating infection. Amongst which, collagen is of special interest
due to its versatile structure and ability to interact with cells and proteins
during wound repair. Collagen films, by virtue of their capability to regulate
release profiles of incorporated drugs, have been used widely as carrier
constructs (Rubin et al 1973, Sato et al 1996, Thacharodi and Rao 1996).
Thus it has added new dimension to the design of biomaterial-based delivery
systems. Currently only a few collagens based dressings incorporated with
active therapeutic agents capable of controlling infection during healing
process are developed (Shanmugasundaram et al 2006). Considering the
ability of such types of dressings to control the release profiles of drugs, as in
excessive burns, plastic surgeons prescribe such appropriate temporary
dressings to the wound site in order to reduce bacterial infection and sepsis.
Products are available commercially, which utilize collagen as carrier
material for local antibacterial/ antibiotic delivery. In these systems
therapeutic agents are either delivered by spreading the drug in the form of
powder (Ruszczak and Friess 2003, Rushton 1997), by direct conjugation of
drug to amino or carboxyl terminal of the collagen membrane or through
microcarriers (Rao 1995, Raghunath et al 1985, Steffan et al 1985, Rossler
et al 1995, Maffia 1994). Collagen is also used for designing protein delivery
and gene delivery systems (Fujioka et al 1995, Honma et al 2004, Zhang et al
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2006). The systems mentioned above are intended to deliver therapeutic
moieties to modulate particular phase of wound healing like infection,
inflammation, re-epithelialization or remodeling. The remodeling event,
which is mainly carried out by MMPs are seldom targeted. Especially MMP-2
and MMP-9 are important as they are expressed in both early and late phases
of remodeling. The importance of MMPs during healing and their modulation
in response to inflammatory and infection status were investigated (Beckett et
al 1996). Hence the development of a delivery system, which can positively
modulate MMP activity, is of great significance.
In general MMP inhibitors (MMPI) developed so far are towards
cancer therapy or for control of arthritis (Brown 1998, Drummond et al 1999).
Whereas very few MMPIs that control the enzyme levels in tissue injury are
reported. Doxycycline, a tetracycline antibiotic, is one of the well-reported
MMPIs. Its sub antimicrobial dose has been reported to inhibit MMPs in
chronic periodontitis (Choi et al 2004), but its application targeting MMPs in
skin injury is not reported.
Figure 3.1 Structure of doxycycline hyclate
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In order to develop a controlled release system, a wide variety of
polymers are available. Among them biopolymers like chitosan, alginate, and
gelatin are preferred. Chitosan (poly (β-(1, 4)-2- amino-2-deoxy-D-
glucopyranose), a cationic polysaccharide, derived by deacetylation of chitin
is of special interest. The polyaminosaccharides in contrast to other polymers
carry a positive charge and are mucoadhesive in nature (Hirano et al 1988,
Issa et al 2005). The excellent biocompatibility and wide availability render
them ideal for drug delivery systems. Chitosan spheres prepared using a
covalent cross linker like glutaraldehyde is so far available (Thanoo et al
1992, Jameela et al 1996, Genta et al 1997, Blanco et al 2000). Due to
reported toxicity of glutaraldehyde, alternative methods of microsphere
preparation like spray drying (Lorenzo et al 1998, Mi et al 1999) and ionic
gelation using Na2SO4 or tripolyphosphate are carried out (Berthold et al
1996, Calvo et al 1997). In the present study, chitosan microspheres are
prepared by ionic gelation through potassium hydroxide (KOH) as
crosslinking agent. The spheres are prepared by a w/o emulsion technique and
are further utilized to entrap doxycycline hyclate by equilibrium swelling
method. The drug entrapped in the spheres is further impregnated into the
shark skin collagen scaffold.
The design and development of wound dressing, which could combat
both infection and elevated MMP levels through unit dosage application, is
indeed a novel approach to promote accelerated healing. In cases of severe
infection the local concentration of the administered antibiotic through
systemic route may not be sufficient to control the infection. Therefore,
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topical antimicrobial therapy remains one of the most important methods of
skin wound care. The local application of antibiotics can provide high drug
concentrations at the site of infection and would avoid systemic effects (Park
et al 2004). The design encompasses development of shark skin collagen Aloe
scaffold impregnated with doxycycline loaded chitosan microspheres. The
morphological characteristics of scaffolds are analyzed through light
microscopy and scanning electron microscopy. The current investigation also
provides design validation for percentage doxycycline entrapment, its in vitro
release profile from the scaffold and distribution homogeneity of impregnated
spheres. Hence this work describes the development and characterization of
reconstituted shark skin collagen scaffold impregnated with doxycycline-
loaded chitosan microspheres, capable of delivering the drug in a controlled
manner.
3.2 MATERIALS
Doxycycline hyclate, Soya oil and low viscosity grade Chitosan
(75-85% deacetylation, Mr: 90000-100000) were obtained from Sigma,
USA.Span-80 was obtained from Fluka, Switzerland; n-octanol was procured
from Merck, Germany. Trypsicase Soy Broth (TSB) Trypsicase Soy Agar
(TSA), Mueller Hinton Broth (MHB) and Mueller Hinton Agar (MHA) were
obtained from HI-MEDIA, India Ltd., Mumbai. All other chemicals used in
this work are of analytical grade. Microbial cultures - Pseudomonas
ml) was added to each well and incubated for 4 h at 370C (Mosmann 1983).
After which medium containing microspheres was removed gently and 500 μl
of dimethylsulfoxide (DMSO) was added to solubilize the formazan complex
and read in a micro plate reader (BIORAD, Model 680, USA) at 570 nm.
The % cell viability relative to control was calculated by [A] test / [A]
control x 100. Control was maintained same as above but omitting
doxycycline and Doxy-CSM. Values reported are the mean of 3
determinations.
CSM was further evaluated for its ability to act as a scaffold for
fibroblasts by allowing the cells to migrate into the microspheres and viewing
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the cells by calcein AM fluorescent dye. CSM was added in duplicates to a 24
well plate containing fibroblasts (2 x 104 cells / well) with 1ml of culture
medium and incubated for 96 hours in 5 % CO2, 95 % air atmosphere. After
which, to each well, 0.2 μM of calcein AM was added and incubated for
20 min. CSM were transferred into eppondorf tube and excess of calcein AM
was removed by washing with 500 μl of PBS. Few drops of CSM suspension
in PBS was dropped in cavity slide and viewed under phase contrast
fluorescent microscope (Leica DMIRB, USA). Photomicrographs were taken
to visualize the metabolically active cells inside the CSM using blue filter.
3.3.6 In Vitro Antibacterial activity of Doxy-CSM
Antibacterial activity of the drug-loaded spheres was examined
against Pseudomonas aeruginosa (ATCC 25619), Staphylococcus aureus
(ATCC 9144), Klebsiella pneumoniae (ATCC 15380), and E. coli (ATCC
25922). Initial inoculums of these strains were prepared by inoculating five
colonies from fresh cultures (overnight cultured) in TSB for S. aureus,
K. pneumoniae, and E. coli, and in MHB for Pseudomonas aeruginosa,
incubated at 35°C till logarithmic growth phase. From this culture, 100 µl of
the sample was transferred to 10 ml of respective media and incubated at
35°C to attain exponential logarithmic phase (≡ 0.5 McFarland). The cultures
were appropriately diluted to get 5x107 cfu / ml and used as primary inoculum
(NCCLS 2000). Different amounts of Doxy-CSM containing drug
concentration equivalent to 10-100 µg per ml was introduced into the flasks
containing various cultures and incubated at 35°C for 72 hours. Samples were
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withdrawn at different time intervals to determine the minimal bactericidal
concentration (MBC) (99.9% kill from initial inoculums) and mean number of
survivors at MBC.
3.3.7 Evaluation of doxycycline as MMP inhibitor by gelatin
zymography
Post burn granulation tissue (Day 7) was collected from human burn
patients after prior consent, complying with the ethical guidance of Central
Leather Research Institute (CLRI) and Kilpauk Medical College and Hospital
(KMCH), Chennai. Tissue was washed 4 times with water and then with
20 mM HEPES buffer (pH 7.5) to remove adhering blood clot and other
debris. The tissue was then homogenized for 2 min in buffer solution (50 mM
Tris-HCl, pH 7.5) on ice bath, centrifuged (SIGMA 3K30, USA) at
10,000 rpm, 40C for 15 min and the supernatant was collected, aliquoted and
stored at – 700C.
Granulation tissue lysate (containing 20 mg of protein) was incubated
alone (control) as well as with various concentrations of doxycycline for 15 h
at 37oC. The incubated samples were mixed with non-reducing laemmli’s
sample buffer and electrophoresed using a 7.5% PAGE containing 0.1%
gelatin substrate. Standard MMP 2 and MMP 9 activated by
4-aminophenylmercuric acetate (APMA) buffer (0.5 mM APMA, 50 mM Tris
HCl, pH 7.5, 50 mM NaCl, 0.005% Triton X-100), were simultaneously
electrophoresed. After electrophoresis, the gel was washed with 2.5% Triton
X-100 for 1 h and then incubated with enzyme buffer (50 mM of Tris-HCl,
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150 mM NaCl, 5mM CaCl2 and 0.05% sodium azide) at 370C for 20 h to
allow reactivation of MMPs. Gels were then stained with 0.5% Coomassie
Brilliant Blue R-250 (Fredericks and Mook 2004) and destained with 10% v/v
of acetic acid containing 30 % v/v of methanol. The MMPs were visualized as
clarified bands corresponding to zones of digestion of substrate gelatin.
3.3.8 Preparation of doxycycline-loaded microspheres impregnated
shark skin collagen – Aloe scaffold
For scaffold development, a known amount (300mg) of shark skin
collagen (PSC) was dissolved in 25 ml of 0.05 M acetic acid and 30mg
equivalent weight of Aloe extract was added as described in chapter 2. Fibril
formation was initiated by adding appropriate amount of 0.2 M phosphate
buffer (2ml), 2M NaCl and adjusting the pH using 1M sodium hydroxide to
6.9 – 7.2, until turbidity appeared. To the collagen solution a known amount
(0.2 grams) of drug loaded microspheres was added and gently stirred to
distribute the spheres homogeneously throughout the solution. After fibril
initiation the viscous solution was uniformly cast over horizontally placed
polypropylene platforms of 10x10cm dimension. The scaffolds were then
allowed to dry at a constant temperature of 300C until a thin scaffold was
obtained. The scaffold was stored in a light proof desiccator for further
experimental purpose.
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3.3.9 Determination of distribution homogeneity of microspheres in the
scaffold
To determine whether the drug-loaded microspheres are distributed in
a homogenous fashion, equal contours of 2x2 cm were cut randomly from
different regions of drug impregnated scaffolds and were subjected to drug
analysis by UV-Spectrophotometer and the amount of drug incorporated in
each contour was determined.
3.3.10 Morphological assessment of microspheres impregnated collagen
scaffolds
The surface and cross sectional features of doxycycline loaded
microspheres impregnated collagen scaffold (Doxy-MS-CS) were assessed
through both light microscope and SEM (with the same instruments as used to
analyze microspheres). Light microscopic images were taken to examine the
bulk property of the spheres in the scaffold, while SEM images were taken to
obtain information regarding the features of each microsphere and their mode
of entanglement with the collagen fibrils.
3.3.11 In Vitro release studies
The in vitro release study was carried out using the Franz diffusion
model finite dosage apparatus as schematically illustrated in Figure 3.2 (Franz
1978). A 12.5 cm2 of microspheres-impregnated scaffold was placed over the
aperture of Franz apparatus (Shanmugasundaram et al 2006) and subjected to
release rate experiment under constant stirring, using a magnetic stirrer, at
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370C. The reservoir solution of the Franz apparatus contained physiological
synthetic serum electrolyte solution (SSES, composed of 0.601g of NaCl,
0.235g of NaHCO3, 0.0283g of Na2HPO4, and 0.0284g of Na2SO4 / 100mL)
(Tsipouras et al 1995). To determine the release characteristics, aliquots of
samples were withdrawn at regular time intervals (3, 6, 12, 24, 48 and
72 hours) and analyzed for the amount of doxycycline released using
UV – spectrophotometer at 267nm. The experiments were carried out in
triplicate and the amount of drug released was expressed as % drug released
with respect to time.
Figure 3.2 Diagrammatic representation of Franz diffusion apparatus
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3.4 RESULTS
3.4.1 Preparation of chitosan microspheres
Chitosan microspheres were prepared by w/o emulsification process
with simultaneous ionic coacervation technique. In this process a mixture of
oil phase (soya oil and n-octanol) was used since it provided continuous phase
of optimal Relative Hydrophilic Lipophilic Balance (RHLB) to form uniform
aqueous phase chitosan micelles, which eventually transform into rigid
spheres, without any agglomeration. The oil mixtures of various ratios were
used for different batches of microsphere preparation. Oil mixture ratio of 2:1,
1:1, (soya oil : n-octanol) caused spheres of irregular morphology. In these
ratios, chitosan spheres obtained were discrete, but the high viscosity gradient
of soya oil had certain impact on surface morphology. The spheres appeared
sickle due to leaching of soya oil through the pores during recovery. However
oil mixture ratio of 1:2 (soya oil : n-octanol) resulted in formation of porous
microspheres without affecting the morphology of microspheres. Further
enhancement of n-octanol, resulted in the aggregation of spheres.
Due to low RHLB of oil phase mixture, Span 80 with low HLB (4.7)
was used. A 5% v/v of span 80 was found to be optimal to form microspheres
of uniform surface morphology with the size range of 200 - 225 μm, below
which aggregation of spheres occurred. Span 80 at 6 and 7% v/v causes
discrete spherical microspheres with the size range of 150-200 μm and
75-180 μm respectively. The most critical step in preparation of microsphere
is percentage w/v of chitosan used for preparation of spheres. Earlier studies
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(Janes et al., 2001) showed that concentration of chitosan below 3% w/v
results in coacervates of weak structures, which are susceptible to breakage
during the process of agitation. Hence, 3% w/v of chitosan prepared in 0.5 M
acetic acid was used in various concentrations. The 6% v/v of chitosan
solution was found to yield spheres of desirable size (200-225 μm) and
morphology. When volume of chitosan was increased to 7% v/v, the spheres
aggregated and strongly bound to each other, with the size range of
200-350 μm. Below 6% v/v discrete spheres were obtained, but they were
found to be fragile and some of the spheres disintegrated into smaller
particles. Moreover, with lower concentrations of chitosan the size of the
spheres varied widely. To obtain microspheres of uniform size and spherecity,
different batches of spheres were prepared at various speeds. The spheres
prepared with 1600 rpm was >95% uniformly sized and discrete.
Microspheres prepared with 1800 rpm resulted in formation of discrete
spheres with >50% size variation. Batch experiments carried out with speed
>2000 rpm resulted in heavily undersized (<100 μm), while those <1600 rpm
yielded agglomerated and oversized spheres (>300 μm). Hence the speed
range 1600-1700 rpm was used for further optimization and microsphere
preparation.
Crosslinking of microspheres was carried out using potassium
hydroxide. In order to improve the crosslinking it is essential that the anions
move uniformly from the core of the spheres to surface of matrix. In most of
the cases aqueous anionic solutions are used, but this resulted in crosslinking
of surface at faster rate than the core accounting for uneven crosslinking and
77
formation of fragile structures. Hence in this process KOH was dissolved in
n-octanol at various concentrations by sonication and batch experiments were
carried out individually. With 3% w/v of KOH, spheres adhered to the vessel
walls and more than 25% polymer settled down as jelly matrix. Batches
prepared with 2% w/v of KOH resulted in much better yield of spheres (>85%
recovery), but surface aggregation was inevitable. Hence the process was
validated with 1% w/v of KOH, which resulted in matrix congealing in a
uniform manner with >95% recovery of spheres in 200-225 μm size. The rate
and volume of addition of KOH was also considered equally important. Slow
crosslinking by drop wise addition at the rate of 1.5 ml/ min of 1% KOH at
every 15 min time interval was found to be optimal. Total time consumed to
obtain rigidized spheres was 4 h and final process of collection involves slow
and complete recovery of microspheres from oil phase using acetone. The
spheres with above conditions were found to be ideal and the spheres obtained
were of uniform size with spherical morphology and were completely
rigidized.
3.4.2 Particle size distribution and swelling ratio
The particle size distribution curve (Figure 3.3) shows sharp
distribution range of microspheres, with 90% spheres of 208 μm size and only
10% were undersized (<150 μm). To assess the ability of chitosan
microspheres to swell, 25 mg was placed in deionized water and increase in
size was assessed through particle size analysis at regular time intervals. A
plot of the swelling ratio with respect to time (Figure 3.4) shows that the
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Figure 3.3 Particle size distributions of chitosan microspheres
1
1.2
1.4
1.6
1.8
2
0 0 .25 0 .5 0 .75 1 2 3 4 5 6 12 24 48
Time (h)
Swel
ling
Rat
io
Figure 3.4 Swelling ratio of chitosan microspheres determined by
Malvern particle size analyzer
79
microspheres were able to swell 1.59 fold than the original size in 1 h and
remained in equilibrium for 6 h. After which, only a slight increase (≈ 2%) in
size occurred for 48 h. The spheres retained their morphology throughout the
study, without any disruptions.
3.4.3 Doxycycline entrapment
Maximum percentage entrapment of doxycycline was observed at
1:10 % w/w Doxycycline: CSM and found to be 8.4 % w/w. At higher
concentration (as detailed in methods) there was no significant increase in
percentage entrapment. Higher amount of CSM with respect to drug did not
show any significant improvement in entrapment efficiency.
3.4.4 Morphological features of CSM and Doxy-CSM
The chitosan microspheres exhibited typical characteristic features of
an ionically crosslinked coacervated matrix. Figure 3.6.a shows the light
microscopic images, of spheres with uniform size and shape. The SEM
images (Figure 3.6.b and Figure 3.6.c) further confirmed the uniformity and
also showed the porous nature of the microspheres due to interconnecting
network of chitosan. Light microscopic image of Doxy-CSM (Figure 3.6.d)
indicated that the spheres did not loose their morphology after entrapment.
SEM images confirmed further that the porous nature is retained after drug
loading (Figures 3.6.e and 3.6.f).
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3.4.5 Biocompatibility
The biocompatibility of the doxycycline and Doxy-CSM was
demonstrated by performing MTT assay. The free doxycycline showed dose
dependant inhibition of fibroblast growth and the drug concentration beyond
200 μg, was found to be toxic to the cells (Figure 3.5). The drug-loaded
microspheres did not show any toxicity even at 400 µg.
0
20
40
60
80
100
120
Control Doxycycline Doxy-CSM
% C
ell v
iabi
lity
100 200 300 400 500
Figure 3.5 MTT assay of human dermal fibroblast, treated for 48 h
with free doxycycline and doxycycline loaded chitosan
microspheres
81
(b)
(c)
(e)
(d) (a)
(f)
Figure 3.6 Morphological features of chitosan microspheres and doxycycline loaded chitosan microspheres. Photomicrograph (a) (20x) shows uniformly sized spheres with smooth surface. The SEM image (b) and (c) show the porous nature of the chitosan microspheres. Photomicrograph (d) is light micrographic image of Doxy-CSM taken at 20x. SEM images of Doxy-CSM (e) and (f) show that the morphological features of the microspheres are maintained after entrapment
82
a
* *
*
b
The fluoroprobe Calcein AM was used to indicate the ability of CSM
to act as a scaffold for fibroblasts. Calcein AM is a membrane permeant,
non-fluorescent molecule, once inside the cells it is hydrolyzed by
endogenous esterases into green fluorescent calcein and retained in the
cytoplasm of the live cells (Figure 3.7.a). During the study the cells were
observed to migrate towards the microspheres and penetrated through the
pores of the microspheres rather than spreading over the entire culture
surface. The fluorescent photomicrographs (Figure 3.7.b) of CSM exposed to
the fibroblasts for 96 h, showed deeply embedded metabolically active cells in
CSM. The study reveals that the developed CSM is a good substratum and
can be utilized as a scaffold for various biomedical applications.
Figure 3.7 Fluorescent photomicrograph of fibroblast cultured over cover slip as well as with chitosan microspheres. a) Shows the green fluorescence of the cytoplasm of the spindle shaped viable cells after treatment with calcein AM (2μM) for 20 min. b) Photomicrograph showing deeply embedded fibroblasts as fluorescence in chitosan microspheres (indicated by asterisk), focused beyond the boundaries of the microspheres). The inset of the figure (20x) is the image at normal focus
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3.4.6 Antibacterial activity
Antibacterial efficiency of the Doxy-CSM was assessed by
determining the MBC by standard tube dilution method against four standard
pathogenic strains (ATCC) in mid-logarithmic phase cultures. The MBC was
found to be nearly equal for Klebsiella pneumoniae (16.5 µg/ ml) and E. coli
(17.4 µg/ ml). Pseudomonas aeruginosa required higher drug concentrations
(98.3 µg/ ml), while Staphylococcus aureus exhibited susceptibility (MBC)
with lesser concentration levels (11.2 µg/ ml). Though the number of
survivors observed varied with different time points, they were found to be
well within the susceptibility limits (20-300 cfu).
3.4.7 MMP inhibition activity of doxycycline
Gelatin zymography was carried out with extracts of human post-burn
granulation tissue, as a source of MMPs. Lane 1 in Figure 3.8 is the standard
enzymes of human MMPs 2 & 9 activated by APMA. Lane 2 shows the
MMPs in the granulation tissue extracts, in which active form of MMP 2 and
MMP 9 are major enzymes expressed. It can be clearly seen that there is a
concentration dependent inhibition of MMPs (lane 3-6) by doxycycline and
complete inhibition (lane 5) was achieved at 95 μg concentration of
doxycycline.
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3.4.8 Determination of distribution homogeneity of microspheres in the
scaffold
In order to find whether impregnated microspheres in collagen
scaffold are evenly distributed, randomly cut, equally sized (2 x 2 cm)
contours of specific area of microsphere-impregnated scaffolds were
subjected to drug analysis. The drug content was found to be 196 ± 9 μg and
there was no significant difference observed among the samples. Hence they
show great deal of homogeneous distribution pattern.
Figure 3.8 Gelatin zymogram showing MMP inhibitory effect of doxycycline with extracts of human post-burn granulation tissue. Lane 1 is the standard enzyme, MMP 2 and MMP 9. Lane 2 shows the MMPs expressed in the granulation tissue extracts in which active form of MMP 2 and MMP 9 are major enzymes expressed. Lane 3, 4, 5 and 6 show the inhibition of MMPs with increasing concentrations of
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