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4.1 INTRODUCTION
Wound healing is a dynamic, interactive process involving soluble
mediators, blood cells, extracellular matrix and parenchymal cells (Singer and
Clark 1999). Acute and chronic wounds are at opposite ends of a spectrum of
wound healing types that progress towards healing at different rates. In acute
wounds, there is a precise balance between production and degradation of
matrix proteins such as collagen; in chronic wounds this balance is lost and
degradation plays too large a role.
A burn is an injury that occurs as a result of exposure of the tissues to
thermal, chemical, or electrical insults. Burns are classified based both on
their depth and the surface area of the skin that is involved. First-degree burns
that involve only the epidermal layer result in pain and erythema and usually
heal within a few days without any scarring. Second-degree burns involve the
entire epidermis and part of the underlying dermis. They are further classified
as superficial partial-thickness or deep partial-thickness burns based on the
depth of injury to the dermis. This distinction is important because many deep
partial-thickness burns heal with significant scarring. Superficial partial-
thickness burns are characterized by erythema, blister formation, and
weeping. They are very painful, and the skin remains sensitive to touch and
blanches when pressure is applied, indicating preservation of the dermal
circulation. These superficial partial-thickness burns generally heal within 2
weeks with minimal scarring. In contrast, deep partial-thickness wounds
involve the reticular as well as papillary layers of the dermis and are
characterized by the presence of a nonelastic, red or white layer on top of the
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burn that often does not blanch with pressure. Because many of the epithelial
appendages that give rise to restoration of the epidermis are destroyed, these
burns require up to 3 weeks healing and may be associated with significant
scarring. Full-thickness or third-degree burns involve the entire epidermis and
dermis and may appear as white, thick brown or tan and have a leathery
texture. The extent of the burn is expressed as the percentage of the total body
surface area (TBSA) that is involved and can be calculated using specialized
age-specific body charts, such as the Lund Browder chart (Lund and Browder
1944).
Significant advances have been made in understanding the cascade of
events (inflammation, tissue formation and remodeling) in normal healing
process. But, these phases are altered from their normal sequence in case of
infection. Especially, Pseudomonas aeruginosa, a gram-negative
opportunistic pathogen causes serious infection in burn wounds leading to
septicemia and if untreated results in mortality (Richard et al 1994).
Pathogenesis of Pseudomonas aeruginosa in burn wound is due to
various extracellular virulence factors such as elastase, exotoxins and
exozymes (Stieritz and Holder 1975), which are shown to influence directly
or indirectly the healing process. Since thermal injury induces an immuno
compromised state, infection due to Pseudomonas aeruginosa further
exacerbates the normally occurring array of events after burn injury.
Primarily, virulence factors may trigger serious weight loss (Steinstraesser et
al 2005). They contribute to delayed re-epithelialization, early dehiscence and
alter content of collagen (Smith and Enquist 1967, Robson 1997). Persistent
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infection after thermal injury impedes epidermal migration and maturation
under provisional matrix, resulting in increased scarring (Singer and McLain
2002). A burn wound infection is also known to spread systemically and
develop into sepsis with associated production of inflammatory cytokines,
mainly Intereukin-1β (IL-1β), Tumor Necrosis Factor-α (TNF- α) and
Intereukin -6 (IL-6). Bacterial endotoxins are the causative factors that induce
the production of these pro-inflammatory cytokines (Freudenberg et al 1993).
There exists a balance of inflammatory cytokines related to severity and
mortality (Walley et al 1996); hence control is of paramount importance.
Especially IL-1β and TNF- α are commonly reported, exhibiting synergism
and hence the net effect should be considered when correlating cytokines
levels and severity of disease (Dinarello 2000). These cytokines also play a
major role in regulation of immune response, hematopoiesis and inflammation
(Reddy et al 2001). Amongst these cytokines IL-6 has short peak time in
normal healing process, while IL-1β and TNF- α are shown to persist for
longer time (Moulin 1995, Neely et al 1996, Grellner 2002). Major cellular
infiltrates that secrete these cytokines are neutrophils and macrophages and
their localization during granulation is detrimental to healing process
(Appleton et al 1993). Impeded epidermal migration mentioned above is due
to altered expression pattern of major matrix remodeling component, Matrix
metalloproteinases (MMPs) (Armstrong and Jude 2002). These MMPs
constitute large family of Zn2+ and Ca2+ dependent endopeptidases, implicated
in tissue remodeling and chronic inflammation. They possess broad and
overlapping specificities and collectively have the capacity to degrade all
components of extracellular matrix (ECM) (Werb 1997, Shapiro 1998).
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Despite the systemic antibiotic therapy, prevention of infection at the
wound site greatly influences the inflammatory and remodeling events. Hence
the use of topical antibiotics are shown to be more effective in reducing the
incidence of wound infection (Halasz 1977, Scher and Peoples 1991). Topical
treatment offers the advantage of immediate effect, lower systemic levels and
higher local tissue bioavailability.
The present in vivo experiment is aimed at investigating qualitatively
the difference between Doxycycline loaded microspheres treated and control
rats, with deep second degree burn wounds challenged with Pseudomonas
aeruginosa, through histological, immunohistochemical localization of
proinflammatory cytokines. In addition quantitative assessment of collagen
turn over, tissue level expression of MMP-1, MMP-2 and MMP-9 to assess
the efficacy of Doxycycline loaded microspheres treated rats to combat
bacterial challenge and subsequently to exhibit faster healing in comparison to
controls. The influence of early infection on various cascading phases during
healing process has been analyzed with special reference to efficient
granulation tissue formation and effective remodeling.
4.2 MATERIALS AND METHODS
4.2.1 Materials
Reagents for biochemical estimation of Collagen, Hexosamine and
zymogram analysis of MMPs, Hydroxyproline, Glucosamine HCl, Para
Dimethyl Amino Benzaldehyde (PDAB), acetyl acetone, chloramine-T,
Bicinchoninic acid (BCA), Bovine Serum Albumin (BSA) standard, MMP-2
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(EC 3.4.24.24) and MMP-9 (EC 3.4.24.35) from human fibroblasts were from
SIGMA, USA. Antibodies used for immunohistochemical identification of
proinflammatory cytokines IL-1β, IL-6 and TNF- α and the basement
membrane marker, collagen IV, were from Santa Cruz Biotechnology, INC,
CA, USA. For immunohistochemical localization polyclonal rabbit anti IL-
1β, IL-6 and TNF- α were used. Respective secondary antibodies to probe the
proinflammatory cytokines were alkaline phosphatase tagged monoclonal
goat anti rabbit IgG. Alkaline phosphatase chromogen–Nitro Blue
Tetrazolium (NBT), 5-bromo, 4-chloro, 3-indolylphosphate (BCIP) and fast
red substrate was procured from SIGMA. All microbiological chemicals such
as Mueller-Hinton Broth (MHB) and Mueller-Hinton Agar (MHA) were from
Hi-media, Mumbai, India and Pseudomonas aeruginosa (ATCC 25619)
culture was procured from IMTECH, Chandigarh, India. All other chemicals
used for experimental purpose were of analytical grade.
4.2.2 Methods
4.2.2.1 Animals and study design
Female white Wistar rats, weighing 180-220 grams were used in this
experiment. All the animals were procured from Venkateswara enterprises,
Bangalore. The rats were acclimatized to laboratory conditions and fed with
standard rat chow and tap water ad libitum.
CLRI Institutional animal ethical committee approved all the animal
experimental protocols. Animal maintenance and care were according to the
Committee for the Purpose of Control and Supervision of Experiments on
Animals (CPCSEA) guidelines, India.
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4.2.2.2 Instrument design for thermal source
The instrument for inflicting burn injury in animal models was
designed by us using a commercially available solder rod with wooden
handle. The soldering edge was cut, tapered and replaced by a circular iron
disc of thickness 1.25 cm and diameter of 1.5 cm. In the non-contacting
surface of the disc a thermal sensor was soldered, with the other end
connected to a digital temperature read-out. The total weight of the modified
solder rod was 500 grams. The desired temperature was attained by heating
the solder rod electrically and controlled using a temperature controller.
Schematic diagram of the thermal source is given in Figure 4.1.
4.2.2.3 Creation of second degree burn wound in rats
The circular disc was heated to 820C-850C and allowed to stabilize
for 20 seconds, after which the external contacting surface of circular iron
disc was placed over the shaved dorsal side of rats, for 20 seconds, without
exerting any external pressure. Before creation of the burn wound all the rats
were anaesthetized using Thiopental sodium (Thiopental*), 50 mg/kg body
weight. Figure 4.2 shows the burn wound creation procedure and
Figure 4.3 shows the measure of wound formed. The depth of the wound was
assessed through histological section of the skin biopsy (5-7μm thick) with
Hematoxylin and Eosin (H&E). To confirm the depth, skin section was
analyzed for collagen IV immunoreactivity and detailed protocol for
immunohistochemical staining is given in Section 4.2.2.11.b (Ho-Asjoe et al
1999).
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Figure 4.1 Diagramatic Representation of the Burn Inducing Instrument
Figure 4.2 Infliction of Burn Wound with Modified Soldering Unit
Electrically Heated through a Temperature Controller
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Figure 4.3 Photograph Showing a Standard 1.5 x 1.5 cm Deep Second
Degree Wound Created on the Dorsum of Rat
4.2.2.4 Induction of infection in rats
Infection was induced using standard pathogenic strain of
Pseudomonas aeruginosa. Initial inoculums of this strain were prepared by
inoculating five colonies from fresh cultures (overnight culture) in MHB at
350C until logarithmic growth phase. From which 100 μL of the sample was
transferred to 10 mL of fresh MHB and incubated to attain exponential phase
(≡ 0.5 McFarland) (NCCLS 2000). Appropriate 10-fold dilution was made to
prepare bacterial challenge inoculum (107 cfu / mL). For inducing infection
1 mL of the above inoculum was centrifuged and re-suspended in 100 mL of
sterile saline and 100 μL of the suspension was injected carefully between the
subcutaneous skin and paraspinus muscular layer (Grzybowski et al 1999).
The infection was allowed to set overnight (12 hours) and treatment protocol
was carried out there after. Wounds were considered infected by the presence
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of subcutaneous deep dermal neutrophils showing positive bacterial
colonization. To further confirm quantitatively, skin biopsies (1 cm2) were
taken from 4 rats, extracted with sterile saline (1 mL) and centrifuged. Cell
pellet was re-suspended in 1 mL of saline, serially diluted and number of
colonies was found by spread plate method overnight in MHA. Colonies
greater than 106cfu were considered to be challenging incoulum size for
inducing infection.
4.2.2.5 Treatment protocol
The infected rats were divided into two groups. The treated groups
(n = 30) were covered with the developed dressing, which was then fixed with
adhesive bandage. Dressings were sterilized by ethylene oxide sterilization
before being cut into the size of the wound and soaked in sterile saline for
2 min before application. Similarly the control groups (n = 30) were covered
with sterile gauze immersed in sterile saline and fixed with the adhesive
bandage till the next dressing change.
4.2.2.6 Body weight and burn size determination
All the rats were monitored throughout the study period and body
weight was taken at the same time every day and expressed as mean ±
standard deviation (SD). The burn wound size was determined by tracing the
margin of wound area on to a transparent graph sheet and expressed as
percentage surface area reduction at each time interval.
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4.2.2.7 Tissue sample collection
Granulation tissue from the burn area was collected carefully after
euthanication of the rats at 3 days time interval till complete remodeling
(except for day 3 of control group rats, where granulation was not observed
due to severe infection). Tissue samples were stored at -200C and
appropriately processed for various experimental protocols.
4.2.2.8 Quantitative assessment of microbial infection
The severity of infection in both groups was assessed in tissue
biopsies. After euthanication the biopsy sample (1 cm2) was extracted
2-3 times with 5 mL of sterile saline, and number of cfu was assessed by
plating method. The counts were represented as cfu/cm2 against time interval.
4.2.2.9 Determination of collagen and total hexosamine content
A known amount of granulation tissue collected at regular time
intervals (as mentioned in 4.2.2.7) was dried to constant weight by
lyophilization and equal amount of dried tissue (5 mg) was subjected to acid
hydrolysis using 6 N HCl for 22 hours. The digested samples were made up to
standard volume and hydroxyproline content was estimated using the method
of Woessner 1961. The collagen content was calculated by multiplying
hydroxyproline content by the factor 7.46 and expressed as collagen content
in μg/mg of granulation tissue (dry weight). The glycosaminoglycan (GAG)
content in granulation tissue was determined, at various time intervals, by
processing the granulation tissue as done for hydroxyproline determination,
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except that the acid hydrolysis was carried out using 2 N HCl for 6 hours. The
GAG was measured as total hexosamine content using the method of Elson
and Morgan 1933 and expressed as total hexosamine content in μg/mg of
granulation tissue (dry weight).
4.2.2.10 Matrix metalloproteinases expression
Expression of active MMPs in granulation tissue of burn wounds in
both groups at various time intervals was determined by gelatin zymography.
4.2.2.10.a Sample preparation
Granulation tissue obtained was thoroughly rinsed with deionized
water to remove adhering blood components and homogenized using HEPES
Buffer (20 mM, pH 7.2) under cold conditions (40C). The homogenate was
centrifuged (Sigma 3K3 high speed refrigerated centrifuge, USA) at
18,000 rpm for 20 minutes and supernatant collected was stored in aliquots of
0.5 mL at -800C for further analysis. The protein concentration in the tissue
lysate was determined by bicinchoninic acid protein assay (Smith and Enquist
1985).
4.2.2.10.b Gelatin zymography
In the present investigation, the expression of MMP-2 and 9 at
various time intervals in both groups were analyzed. Granulation tissue lysate
(containing 20 μg) protein was mixed with non-reducing Laemmli’s buffer
(0.125 M Tris, pH 6.8, SDS-4%, glycerol 20% and and 0.02% w/v of
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bromophenol blue) and electrophoresed on a 10% polyacrylamide gel
copolymerized with gelatin (1 mg / mL) (containing 0.167% w/v of
N, N′-methylene bis acrylamide, 0.1% w/v of SDS dissolved in 0.375 M
Tris-HCl, pH 8.8 and polymerized using 10% w/v of APS and 0.02%
TEMED) with anodic and cathodic buffer of Tris-glycine (Tris-7 g, glycine-
14 g and SDS-1g per liter at pH 8.3). After electrophoresis, protein bands
were visualized by staining with 0.1% w/v of Coomassie Brilliant Blue R-250
(dissolved in a mixture containing 25 mL methanol, 10 mL acetic acid and
65 mL of water) (Madlener et al 1998). Standard MMP-2 and 9 were used as
markers. After electrophoresis, the gel was washed with 2.5% Triton X-100
for 1 hour and 30 minutes and then incubated with enzyme buffer (50 mM of
Tris-HCl, 150 mM NaCl, 5 mM CaCl2 and 0.05% sodium azide) at 370C for
20 hours to allow reactivation of MMPs. Gel was then stained with 0.5%
Coomassie Brilliant Blue R-250, and destained with10%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.
4.2.2.11 Histological observation
4.2.2.11.a H & Eosin staining pattern of granulation tissue
The granulation tissue biopsies collected at various time intervals
were fixed in buffered formalin (Formalin, 10%, 100 mL, anhydrous sodium
phosphate dibasic 6.5 g, sodium phosphate monobasic 4.0 g, distilled water
900 mL), paraffin embedded and 4-5 μm thick sections were cut using a
microtome and mounted on glass slides. Histological sections were then
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de-paraffinzed and stained with H&E to detect the dermal and connective
tissue changes. The staining was performed according to the standard protocol
(Culling 1974).
4.2.2.11.b Immunohistochemical analysis
The early and late course of inflammatory response during healing
was observed by the proinflammatory cytokines IL-1β, IL-6 and TNF-α
expression. Immunohistochemistry was performed according to previously
mentioned procedure (Moulin 1995). The sections were de-paraffinized using
xylene, air-dried and serially hydrated using aqueous ethanol (gradient of
70%) and finally hydrated completely. The hydrated sections were then
incubated for 1 hour with 2 N HCl at 370C (for antigen exposure) and kept
immersed in PBS (containing 0.5% Tween 20) overnight. The sections of
different post burn days (till day 12) were incubated for 1-2 hours at 370C,
individually with polyclonal rabbit antibody for IL-1β, IL-6 and TNF-α to
detect the inflammatory status, while that of control rats taken on day 1 and
day 9 were probed with collagen IV to observe the burn depth (as mentioned
in 4.2.2.3). All the primary antibodies were used at a dilution of 1:100 in 2%
BSA. Then the sections were incubated with alkaline phosphatase tagged anti
rabbit IgG (secondary antibody, diluted to 1:200 dilutions) for 45 mins–1 hr at
370C. Sections were then detected with fast red substrate (prepared with
buffer provided in the kit by manufacturers), washed overnight in water (in
dark), counterstained with Mayer’s Haematoxylin for 15 minutes and finally
mounted using crystal mountant.
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4.2.2.12 Statistical analysis
All graphical illustrations in this study are represented as mean ± S.D
and analyzed with Man Whitney test using SPSS software. Test for
significance was performed with confidence limit of 95%, i.e., p<0.05 was
considered statistically significant.
4.3 RESULTS
The present investigation deals with several responses in magnitude
and temporal pattern of wound repair in Pseudomonas aeruginosa (ATCC
25619) challenged standard deep second-degree rat burn model. It was
observed that there exists a strong relationship between severity of early
infection and subsequent healing events.
4.3.1 Burn Depth Observation
The depth of burn wound created using the thermal device designed
by us resulted in deep second-degree burn wounds. The tissue section excised
along with surrounding healthy skin stained with H&E shows (Figure 4.4) the
complete loss of epidermis, destruction of dermis spreading just above the
muscle layer (thin line of adnexial dermis can be visualized) with complete
loss of hair follicles and vessel walls. To show the comparison of the depth, a
section of unwounded tissue with healthy dermis, intact suprabasal layer with
partial dissociation of the epidermis due to spreading of sub-epidermal
blistering is shown in inset (marked as b). The depth was confirmed by the
presence of Type IV collagen in hair follicle by immuno staining. Tissue
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biopsy on day 1 (Figure 4.4) showed scattered spots of collagen IV in deep
dermis. To reaffirm the observations, section on day 9, when immunostained,
shows (Figure 4.5) presence of positive Type IV collagen just above the
subcutaneous-paraspinal region, over which thin layer of granulation can be
observed.
Figure 4.4 Determination of Wound Depth by Hematoxylin and Eosin Staining (i) Day 1 tissue section (inset) shows the wound depth along
with the adnexial intact epidermis (ep). Serrated arrow shows sub dermal blistering (b). Curved line shows the borderline of wound surface along the dermal-cutaneous layer (c).
(ii) Partially adherent dermis (d) above the muscle layer (m)
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Figure 4.5(i) Immunohistochemical Localization of Collagen IV on Day 1
Day 1 section showing Collagen IV spotted along borderline of adherent tissue layer (arrow), above the muscle layer (m).
Figure 4.5(ii) Immunohistochemical Localization of Collagen IV on Day 9 Immunoreaction of Collagen IV, on day 9, between the granulation (gt)-cutaneous layer junction (arrow heads)
4.3.2 Body Weight Assessment
The average body weight of the rats (n=60) was 184 grams. The
control group showed a constant decrease (5%) from their initial weight till
day 15, after which only a marginal increase is seen but they did not attain the
initial weight until complete healing. In treated group there was marginal
weight loss (1%) till day 9 but they regained their initial weight and showed
an increase of 3% from their initial body weight as shown in Figure 4.6.
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Figure 4.6 Determination of Body Weight at Various Time Intervals
4.3.3 Wound Closure Examination
The control group showed a significant increase in wound size
till day 15 (20% from initial size), followed by a positive healing, which was
slow when compared to the treated groups. The treated group showed an
increase of wound size till day 9, after which it exhibited positive healing
response achieving complete healing by day 24, which was significant when
compared with the control rats (complete healing observed only by 37 days).
From this investigation it is noteworthy that, control group rats exhibited
significantly larger wound size against treated groups at all time points
(p<0.05) (Figures 4.7 and 4.8).
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Figure 4.7 Efficiency of Doxy-MS-CS on Burn wounds in comparison
to control group
Figure 4.8 Percentage Wound Closure
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4.3.4 Influence of Infection on Healing
The control and treated rats infected with Pseudomonas aeruginosa
(107 cfu) exhibited differential response with regard to their microbial load
over the course of healing. As it can be observed from Figure 4.9, that on day
3 the number of cfu significantly increased by 100 fold in control group
(2x105 cfu –107 cfu, microbial count determined in the slough of the wound
since granulation was not obtained), while the treated rats did not show a
significant increase or decrease (2 fold increase, from 2x105 cfu – 4x105cfu).
However on day 6, in treated group a significant decrease (3 fold) in
microbial load was observed (4x105 cfu to 2x102 cfu), which constantly
reduced on post burn days. Treated rats showed 99.9% decrease by day 9
whilst, in control group, the number of cfu did not decrease significantly till
day 15. Almost 75% of control group showed mild to severe purulent
discharge, another sign indicating the onset of positive infection. The severity
of infection was obvious in control group, leading to mortality of 3 animals
indicating local infection has detrimental effect on healing.
Figure 4.9 Quantitative Determination of Microbial Burden in Control and Treated Rats
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4.3.5 Collagen Turn Over
A common pattern of change was observed in both the groups that a
steep increase in collagen content was observed followed by equally a steep
decrease of the collagen content from day 9 for treated and day 15 for control
group. In control group, collagen content (63.8 mg / mg) attained the peak on
day 15, whereas peak levels are found to be on day 9 for treated group. The
treated group exhibited a significant difference in collagen content till day 12
when compared with control (Figure 4.10).
Figure 4.10 Collagen Content in Granulation Tissue
4.3.6 Total Hexosamine Content
Proteoglycan deposition mainly hyaluronic acid, heparan sulphate,
chondroitin sulphate and dermatan sulphate in granulation tissue facilitates an
environment for cell movement and collagen deposition. Hexosamine (marker
for proteoglycan) expression levels are significant in both groups
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(Figure 4.11) (p<0.05). Peak concentration levels were observed on day 12
for control group, while treated group exhibited peak level on day 6.
Figure 4.11 Total Hexosamine Content in Granulation Tissue
4.3.7 Differential Expression of MMP
4.3.7.1 Detection of MMPs by gelatin zymography
Changes in the expression of pro and active form of MMPs during
post burn days in granulation tissue extracts collected at different days were
analyzed by gelatin zymography. Due to severe infection, granulation did not
occur on day 3. Figure 4.12 shows the relative changes in MMPs over time.
As can be observed, overall expression of MMP-9 was higher.
The overall expression of both MMP-2 and 9 were significantly
lower, thus hindering active remodeling in infected groups. It is noteworthy
that expression of both MMP-9 and 2, though less, can be observed for longer
duration especially till day 18 in case of control group.
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MMP 8
Whereas treated group showed appreciable levels of expression of
MMPs till day 6, after which MMP-2 levels declined, while MMP-9
expression could be still observed. After day 12, expression of both MMPs
diminished, which can be accounted for the control of microbial burden from
day 9. The expression of MMP-9 even on day 15 in control group indicates
slower onset of remodeling, whereas in treated group the MMP expression
subsided completely by day 12, hence faster remodeling was observed.
Figure 4.12 Gelatin Zymogram of Granulation Tissue Extract on
Various Post Burn Days: Differential Expression of MMP-2
and MMP-9
4.3.8 Histological Evaluation of Healing Process
4.3.8.1 H & E Staining of Granulation Tissue
Figures 4.13 and 4.14 show tissue sections stained with H & E on
various days during the course of healing. Both the groups showed destruction
of deep dermal region, after debridement of dead tissue (day1 – Figure 4.3).
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Day 3 histological sections exhibit complete loss of epidermis and spongy
layer of dermis, and the adnexial dermis is also completely devascularized.
Examination of sections of day 6 shows large amount of inflammatory cell
infiltration, which even spreads deep inside the muscle layer in both groups.
The control rats showed significant infiltration of eosinophilic stained layer
spreading all over the dermal portion showing prolific infiltrations of
polymorphonuclear leukocytes (PMNLs) even deep in the skeletal muscle
layers. The level of inflammatory cell infiltration was much lesser in case of
treated group rats. Sections of control group rats show necrotic slough with
clumps of bacteria covering the wound surface, whereas treated group rats
exhibited lesser infiltration of bacterial cells, during initial days, which was
not present in later days and from day 9 the infiltration of inflammatory cells
subsided drastically with less necrosis. On progressive post burn days the
treated group showed well-defined granulation by day 12, with proliferating
fibroblasts. Control group rats did not show any sign of defined matrix till day
15, after which there was loose matrix formation showing prominent
inflammatory cell infiltration as shown in day 18. The day 18 sections of
treated group show well-defined matrix with appreciable infiltration of
fibroblasts. Treated group exhibits well-defined dermis with thin epithelial
layer along the borderline whereas control group rats did not show any
epidermal demarcation. Complete remodeling was observed on day 21 in
treated group and day 37 for control group with well defined epidermal-
dermal margins, invaginating papillary dermis along with hair follicles.
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Figure 4.13 H & E Stained sections of Granulation Tissue at Various
Days in Control Group
Figure 4.14 H & E Stained sections of Granulation Tissue at Various
Days in Treated Group
4.3.8.2 Influx of proinflammatory cytokines
Proinflammatory cytokines IL-6, IL1-β and TNF-α play important
functions in early course of trauma and healing (Saren et al 1996). Especially
the proinflammatory cytokines influence the subsequent remodeling phase as
MMPs get exacerbated by proinflammatory cytokines particularly by IL-1β
and TNF- α. In the present study, expression of these cytokines
(IL-6 - Figure 4.15, IL1- β - Figure 4.16 and TNF-α - Figure 4.17) showed
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variation in treated and control groups. Increased expression of IL1-β was
obtained in both the groups when compared to the expression of other
cytokines. In control group IL1-β expression was persistent in both dermal
and subcutaneous regions till day 9. (Figure 4.16, inset of day 9). Treated rats
exhibited spreading out of IL1-β over the dermal margin, which significantly
decreased by day 6.
IL-6 generally showed a steep increase in levels during the
inflammatory phase and decrease after achieving the peak levels. Persistent
IL-6 was followed to ascertain the severity of trauma as well as infection. On
day 3, IL-6 expression in treated group was seen around the same margin of
the disrupted dermis. While on day 6 treated group did not show IL-6
expression, indicating its transient expression. The control group exhibited
widespread expression over the disrupted dermal layer even on day 6 due to
severe infection.
Another important proinflammatory cytokine TNF-α is a key
mediator of late inflammatory phase response due to trauma and infection. It
is expressed in the chronic granulation tissue, with marked persistence over
time. Though its level of expression was lower in comparison to IL-6 and
IL1-β, its tangible expression for 9 days post burn in control group shows
severity of the trauma. Initial days of expression of control group (on day 3)
were comparable with treated group showing slightly lesser expression. While
on day 6 significant differences in its level were observed. In control group
TNF-α level was more prominent and spread out in the granulation and
subcutaneous region due to severe infection even on day 9. In treated group,
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decrease in TNF-α level was seen on day 3 and completely subsided by day 9,
indicating the ability of the developed dressing to mitigate infection.
Control Treated
Figure 4.15 Immunohistochemical Localization of IL-6 Expression
Control Treated
Figure 4.16 Immunohistochemical Localization of IL-1β Expression
Control Treated
Figure 4.17 Immunohistochemical Localization of TNF-α Expression
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4.4 DISCUSSION
Despite major advances in burn wound management and other
supportive care regimen, infection remains the leading cause of morbidity and
mortality in burn wounds, as it exacerbates the impairment. Burn trauma and
resulting extensive tissue damage depletes the intrinsic cellular immune
components and makes the wound susceptible to infection. To create
reproducible burn wounds in animal models, the instrument designed by us
was used. In the standard deep second degree burn created, tissue damage
progressed for 9 days.
The burn depth was assessed by histological examination since it is
undeniably the most accurate method established so far (Ho-Asjoe et al
1999). Immunostain of collagen IV along with regular Haematoxylin and
Eosin (H&E) gave a clear picture of burn depth. Collagen IV in the hair
follicle has been used in this investigation to assess burn depth for two main
reasons – a. The immunostain is known to reach deep dermal regions and b.
It’s staining correlates with actual tissue damage rather than changes in
staining intensity.
It can be observed (Figure 4.4) that there was a complete loss of
collagen IV immunostain, with few scattered appearance on the deep dermal
layer due to disrupted basement beneath the hair follicle. This was supported
by the H&E stained sections and was in good accordance with the earlier
studies, which provided evidence (Papp et al 2004) for loss of immunostain
for collagen IV along the hair follicle when the time and depth of injury
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increased. Due to the trauma caused, the burn wounds are vulnerable to
infection. In humans infection is spontaneous, whereas in rat models the
infection has to be induced. The magnitude of pathogenic assault in the
present study (Subcutaneous injection of 107 cfu/mL of Pseudomonas
aeruginosa) caused severe infection. The influence of inoculum size
mentioned above is crucial, and its importance was shown by Raju et al
(1977). Their investigation established that only with 106 cfu infection could
be sustained. However wounds infected with 107 cfu of Pseudomonas
aeruginosa was found to exhibit noticeable and severe infection. Enlargement
of wound size was observed in both control and treated groups.
Severe infection causes hypermetabolic response, (Barrow et al 2001)
which affects the nutritional balance and directly influences the body weight
during post wound days. Treated rats did not show any significant weight loss
on post burn days indicating control of microbial load in improving the
nutritional status. They started to regain their body weight once the microbial
load receded completely (after 9 days) exhibiting up to 3% increase in body
weight at the end of healing process.
A noteworthy observation is that, the Doxy-MS-CS exerted a static
antimicrobial effect for the initial days due to the nature of the system to
deliver doxycycline in a controlled manner. After which there was a sharp
reduction in number of cfu from (107 to 102) by day 9, indicating the
accomplishment of the equilibrium drug release state.
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Thermal burns exhibit slow re-epithelialization in both early and later
time points due to loss/damage of follicles residing in deep dermal regions.
Bacterial load aggravates the damage by gaining entry through the follicular
path, reside deep in the follicular-basement juncture and further infiltrates into
deep dermal and paraspinal muscular layers. This is of paramount importance
since keratinocytes use the same portal for re-epithelialization (Schaffer et al
1997). Though the normal wounds and infected wounds share similar process
of granulation characteristics, existence of large bacterial load in infected rats
exerted a detrimental effect on rate of granulation and re-epithelialization.
Bacterial infiltration was found in both groups on day 3, but it persisted deep
in dermis in control group rats even on day 9. Due to persistent infection
epidermal maturation was also affected. Treated group rats exhibited
significant proliferation and migration of epidermal cells over fibrin matrix,
resulting in faster granulation covering the entire wound surface. Another
striking feature is the appearance of spongious collagen layer as early as in
day 21 in case of treated rats. A probable reason for faster remodeling in
treated group may be due to application of Doxy-MS-CS, which induces
active cellular infiltration.
Consistent with the histological appearance, quantitative assessment
of collagen content provided insight into the matrix deposition during healing.
Granulation tissue is a combination of cellular elements, fibroblasts and
inflammatory cells along with new capillaries embedded in provisional matrix
of collagen and GAG (Young and Grinnell 1994). The collagen deposition
due to Doxy-MS-CS treatment was much faster than control groups. Due to
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faster cellular infiltration and fibroplasia GAG content also reached the
maximum at a faster rate. Early onset of inflammatory response due to
expression of IL-6, IL-1β and TNF-α demonstrated the wound status after
infection. Especially IL-1β and TNF-α regarded as early phase inflammatory
cytokines, provide ample evidence for severity of infection in triggering
subsequent inflammatory reaction. In normal healing process an appropriate
balance exists in host inflammatory response. IL-6 expression reaches the
peak level rapidly and declines, while expression of IL-1β and TNF-α can be
observed for longer time. The immunohistochemical analysis shows control
group exhibiting prolonged expression of IL-6 (72 hours) along with IL-1β
and TNF-α, indicating the severity of inflammation, whereas, treated group
showed the normal expression pattern of proinflammatory cytokines.
Heavy microbial burden seems to affect the subsequent healing
phase, mainly the remodeling phase. Delay in epidermal regeneration is one
of the important observations in control group. Prolonged and/or higher
expression levels of MMPs may cause destruction of the early provisional
matrix, thus hindering healing, whereas treated group hastened the healing in
comparison to control. Active MMPs observed during initial days are required
for debridement, paving way for neo-dermis deposition. Early detection of
MMPs in treated group indicates the active clearance of dead tissue. Another
important observation is the delayed onset and prolonged expression of MMP
1, 2 and 9 in control group. One of the major factors that regulate MMP
activity is the bacterial exotoxins (Miyajima et al 2001) along with excessive
inflammatory cell infiltration at the wound site. In addition regulation of
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MMP-TIMP (tissue inhibitor of metalloproteinases) association during
healing process plays a crucial role in matrix remodeling (Armstong and Jude
2002). The Doxy-MS-CS reduces the bacterial load and is found to lower the
proinflammatory cytokines thus positively modulating MMP activity. Rats
treated with Doxy-MS-CS were able to hasten the healing process at a much
faster rate (35%) than the infected control rats.
Collectively, this investigation reveals that control of inflammation
and MMP regulation depends on the therapeutic efficiency of the initial
chemoprophylaxis applied to control infection. In addition, it will be a
constructive proposition if the dressing product could assist in controlling the
proteolytic wound environment to actuate faster and healthier remodeling.
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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. Although major advances have been
achieved in burn wound management and supportive care, infection remains
the leading cause of morbidity in burn patients. The studies on the
microorganisms that infiltrate wounds reveal that, Staphylococcus aureus and
Pseudomonas aeruginosa are predominant species responsible for wound
sepsis. The heavy infiltration of inflammatory cells further causes prolonged
or increased level of endopeptidases, mainly the matrix metalloproteinases
(MMPs), resulting in delayed healing. In general, chronicity of a wound
appears to be due to bacterial toxins, which in turn induce elevated MMP
expression and imbalance between activation and inhibition. Thus therapeutic
intervention to control infection and to positively regulate MMP balance is
considered vital in achieving faster remodeling of wounds.
Based on the wound type and status, dressing materials should be
selected. 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. 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 as scaffold. Bovine and porcine type I collagen provide a
readily available source of scaffold materials for various biomedical
applications. These sources have some potential risk of infectious diseases
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such as bovine spongiform encephalopathy (BSE) or transmissible
spongiform encephalopathy (TSE). In order to attenuate the risk to
manufacture biomaterials an alternate safe collagen must be considered. Type
I collagen from aquatic animals may provide an alternative collagen source
and shark skin (Rhizoprionodon acutus) collagen in particular is potentially
important.
Acid soluble and pepsin soluble collagen was isolated from the shark
skin and the chain composition was assessed by SDS – PAGE. Identical
bands were obtained for both ASC and PSC in SDS PAGE, showed
characteristic α1[I] and α2[I] chains of Type I collagen and type III collagen
is absent in fish skin. Based on the hydroxyproline index, the percentage
extractability of PSC showed higher value than the ASC, which is about 54%
and 32% respectively. The PSC also benefits from the removal of the
antigenic P-determinants located in the non helical ends which otherwise
provoke milder immune response. Hence PSC was used for further scaffold
development and characterization.
The incorporation of chitosan into the 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. Chitosan as
well as Aloe extract, a herbal source having the major constituent as
acetylated mannan have been utilized to enhance the physicochemical
property and therapeutic potency of reconstituted collagen scaffolds as they
have been already demonstrated to be potential wound healing agents.
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Chitosan (1% in 0.05 M acetic acid) and aloe extract (with respect to
sugar content) was incorporated during fibrillation at various ratios of 10:0.5,
10:1 and 10:2 w/w of collagen and chitosan /aloe extract respectively. The
bulk surface morphology of the collagen scaffold was assessed by SEM, fibril
band pattern and fiber width were directly observed through AFM. Heat
denaturation property of the collagen scaffold was analyzed using DSC and
the scaffolds were subjected to load-deformation analysis using Instron
automated material testing system 1.04 in uniaxial tension mode. The bulk
surface property of the developed scaffold observed through SEM image
showed densely packed porous structure adding evidence to the fact that the
fibrils formed network due to close association in both lateral and transverse
manner. FT-IR spectrum of the developed shark skin collagen scaffolds
displayed the characteristic absorption bands at 1660 and 1550 cm-1, which
represents the characteristic amide I and amide II absorption bands
respectively. The study well correlated with the thermodynamic properties of
the developed scaffolds that the ratio 10:1 displayed the maximum
mechanical strength and aloe incorporated scaffolds showed higher tensile
strength than the chitosan and pepsin soluble collagen scaffolds. Thus the
collagen scaffolds incorporated with aloe extract at the ratio of 10:1
demonstrated superior physicochemical and biocompatible properties required
for further applications.
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
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for many MMP associated disorders like chronic wounds, periodontal disease,
rheumatoid arthritis, corneal erosion and skeletal muscle reperfusion injuries.
Doxycycline possesses the ability to bind with divalent metal ions to cross the
tissue barrier. This phenomenon finds application to inhibit MMPs by
sequestering Zn2+. This is an important reason to select doxycycline as an
antibiotic especially in cases of chronic wounds. Chitosan was selected as the
carrier material to deliver doxycycline, since it is a unique polycationic
polymer with excellent biocompatible and biodegradable characteristics.
Chitosan microspheres were prepared by ionic gelation through KOH as
crosslinking agent. The spheres were prepared by w/o emulsion technique and
were further utilized to entrap doxycycline by equilibrium swelling method.
The drug entrapped in the spheres was further impregnated into the fish skin
collagen scaffold.
In this novel process, chitosan was ionically crosslinked using KOH,
(dissolved in n-octanol by sonication), which enables the ions to reach the
core of the micelles, without affecting their shape. The particle size
distribution curve showed sharp distribution range of microspheres, with 90%
spheres of 208 μm size and only 10% were undersized (< 150 μm). The
process variables like speed of emulsification, crosslinking rate and % w/v of
chitosan used are key variables in obtaining spheres of desired range.
Maximum percentage entrapment of doxycycline was observed at
1:10% w/w Doxycycline: CSM and it was found to be 8.4% w/w. The
morphological features of CSM and Doxy-CSM were assessed by both light
microscopic and scanning electron microscopic (SEM) techniques. The
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biocompatibility of the doxycycline and Doxy-CSM was demonstrated by
performing MTT assay. The drug-loaded microspheres did not show any
toxicity even at 400 µg. 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).
Gelatin zymography was carried out with extracts of human post-burn
granulation tissue, as a source of MMPs. There was a concentration
dependent inhibition of MMPs by doxycycline and complete inhibition was
achieved at 95 μg concentration of doxycycline.
Doxy – CSM was impregnated in the PSC scaffold and its surface
morphology was studied by LM and SEM. Spheres were entangled in the
fiber network and the interconnecting pores between fiber attachments to the
microspheres. Release of doxycycline would purely depend on swelling of
collagen and diffusion controlled release through chitosan microspheres. The
initial burst release (38%) exerts immediate chemo prophylaxis and
subsequent equilibrium concentration maintenance in a controlled manner for
antibacterial susceptibility. Another important aspect in controlling the release
of the drug is to reduce the host cell toxicity. It is evident from MTT assay
that controlled release of doxycycline induces less percentage of toxicity.
Moreover fluorescent assay shows CSM to act as template in inducing cell
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migration and proliferation. Doxy-CSM may provide moist environment for
healing as well as deliver the drug at required concentration to control
infection and further release of doxycycline may be reduced or arrested when
exudation decreases. The MMP inhibition study further confirms that
doxycycline concentration required to kill pathogens would be sufficient to
control MMPs. This would enable the system to prevent matrix degradation
and induce positive tissue healing.
Burn trauma and resulting extensive tissue damage depletes the
intrinsic cellular immune components and makes the wound susceptible to
infection. To create reproducible burn wounds in animal models, the
instrument designed by us was used. The burn depth was assessed by
histological examination since it is undeniably the most accurate method
established so far. Immunostain of collagen IV along with regular
Haematoxylin and Eosin (H&E) gave a clear picture of burn depth. The
magnitude of pathogenic assault in the present study (Subcutaneous injection
of 107 cfu/mL of Pseudomonas aeruginosa) caused severe infection. However
wounds infected with 107 cfu of Pseudomonas aeruginosa was found to
exhibit noticeable and severe infection. Enlargement of wound size was
observed in both control and treated groups. Treated rats did not show any
significant weight loss on post burn days indicating control of microbial load
in improving the nutritional status. They started to regain their body weight
once the microbial load receded completely (after 9 days) exhibiting up to 3%
increase in body weight at the end of healing process. Though the normal
wounds and infected wounds share similar process of granulation
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characteristics, existence of large bacterial load in infected rats exerted a
detrimental effect on rate of granulation and re-epithelialization. Treated
group rats exhibited significant proliferation and migration of epidermal cells
over fibrin matrix, resulting in faster granulation covering the entire wound
surface. The collagen deposition due to Doxy-MS-CS treatment was much
faster than control groups. Due to faster cellular infiltration and fibroplasia,
GAG content also reached the maximum at a faster rate. The
immunohistochemical analysis showed control group exhibiting prolonged
expression of IL-6 (72 hours) along with IL-1β and TNF-α, indicating the
severity of inflammation, whereas, treated group showed the normal
expression pattern of proinflammatory cytokines. Delay in epidermal
regeneration is one of the important observations in control group. Prolonged
and/or higher expression levels of MMPs may cause destruction of the early
provisional matrix, thus hindering healing, whereas treated group hastened the
healing in comparison to control. The Doxy-MS-CS reduced the bacterial
load and was found to lower the proinflammatory cytokines thus positively
modulating MMP activity. Rats treated with Doxy-MS-CS were able to
hasten the healing process at a much faster rate (35%) than the infected
control rats.
To conclude, the present work provides evidence to the fact that basic
drug delivery system designed by us in combination with novel therapeutic
agents and collagen based biomaterials enabled control of infection and
inflammation and accelerated healing and has good scope in clinical
applications.
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FUTURE PROSPECTS
• The scaffold developed with shark skin collagen can be
evaluated as a substrate in tissue engineering.
• The efficacy of the developed Doxycycline loaded chitosan
microspheres impregnated with shark skin type I collagen with
aloe needs to be ascertained in patients with chronic wounds.