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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

116

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.