www.elsevier.com/locate/burns

Burns 32 (2006) 319–327

An investigation on burn wound healing in rats with chitosan gel

formulation containing epidermal growth factor

Ceren Alemdaroglu a, Zelihagul Degim a,*, Nevin Celebi a, Fatih Zor b,Serdar Ozturk b, Deniz Erdogan c

a Department of Pharmaceutical Technology, Gazi University, Faculty of Pharmacy, 06330 Etiler, Ankara, Turkeyb Department of Plastic and Reconstructive Surgery, Gulhane Military Medical Academy, 06010 Etlik, Ankara, Turkey

c Department of Histology, Gazi University, Faculty of Medicine, 06300 Besevler, Ankara, Turkey

Abstract

Various studies have shown that chitosan is effective in promoting wound healing. In this study, we aimed to develop an effective chitosan

gel formulation containing epidermal growth factor (EGF), and to determine the effect on healing of second-degree burn wounds in rats. Ten

micrograms per millilitre EGF in 2% chitosan gel was prepared. In an in vitro study to investigate release of EGF from the formulations, the

release rate was 97.3% after 24 h. In in vivo studies, animals were divided into six groups as follows: silver sulfadiazine [Silverdin1 cream

(SIL)], chitosan gel with and without EGF (EJ, J), EGF solution (ES) and untreated control groups [unburned (S) and untreated (Y) rats]

applied groups, respectively. A uniform deep second-degree burn of the backskin was performed with water heated to 94 � 1 8C during a 15-s

exposure. The EGF formulations were repeatedly applied on the burned areas with a dose of 0.160 mg/cm2 for 14 days (one application per

day). Healing of the wounds was evaluated immunohistochemically, histochemically and histologically on the tissue samples. When the

results were evaluated immunohistochemically, there were significant increases in cell proliferation observed in the EGF containing gel

applied group ( p < 0.001). The histochemical results showed that the epithelization rate in the EJ group was the highest compared to the ES

group results ( p < 0.001). The histological results indicated and supported these findings. It can be concluded that a better and faster

epithelization was observed in the EJ group compared to the other groups.

# 2005 Elsevier Ltd and ISBI. All rights reserved.

Keywords: Epidermal growth factor; Chitosan; Burn; Wound healing

1. Introduction

Disruption of the skin generally leads to increased fluid

loss, infection, hypothermia, scarring, compromised immu-

nity and change in body image [1–3]. All these factors

together are very important; furthermore, large skin damage

can cause mortality. The mortality rate from burns has

declined in the past decade; however, it is still high if more

than 70% of the body surface is injured or burned [4].

Burns are classified according to the depth of the injury.

In superficial second-degree burns, the epidermis and the

superficial dermis are mainly affected. These kinds of burns

are very painful. The main causes of a superficial second-

degree burn are hot liquids [5].

* Corresponding author. Tel.: +90 312 212 21 07; fax: +90 312 212 79 58.

E-mail addresses: zdegim@gazi.edu.tr, tunc@tr.net (Z. Degim).

0305-4179/$30.00 # 2005 Elsevier Ltd and ISBI. All rights reserved.

doi:10.1016/j.burns.2005.10.015

Wound healing is a complex process involving various

mechanisms, such as coagulation, inflammation, matrix

synthesis and deposition, angiogenesis, fibroplasia, epithe-

lization, contraction and remodeling. Growth factors are

polypeptides that control the growth, differentiation and

metabolism of cells and regulate the process of tissue repair

[6–9]. The fluids at wound sites may be an important reservoir

of growth factors that promote the wound healing process

[10]. Growth factors bind to specific high-affinity receptors

on the cell-surface to stimulate cell growth. Although they are

present in small amounts, they exert a powerful influence on

the process of wound repair [9]. There are some studies

reported dealing with growth factors in burn wound healing,

in which it is has been suggested that they may play an

important role in the healing process [11–13].

Several defined peptide growth factors, including

epidermal growth factor (EGF), platelet derived growth

C. Alemdaroglu et al. / Burns 32 (2006) 319–327320

factor (PDGF), fibroblast growth factor (FGF) and

transforming growth factor-beta (TGF-b), have been shown

to stimulate cellular proliferation and synthesis of the

extracellular matrix [6,14,15].

Chitosan is a polysaccharide comprising copolymers of

glucosamine and N-acetylglucosamine. It is derived by

partial deacetylation of chitin from crustacean shells [16].

Due to its high molecular weight of 50–2000 kDa, chitosan

exhibits a positive charge and film-forming and gelation

characteristics [17]. Previously published papers indicate

that chitosan enhances the functions of inflammatory cells

such as polymorphonuclear leukocytes, macrophages and

fibroblasts; thus, it promotes granulation and organization.

Therefore, chitosan can be used for large open wounds

[17,18].

In experimental animal models, chitosan was shown to

influence all stages of wound repair [19]. The hemostatic

activity of chitosan can be seen in the inflammatory phase. It

also interacts with and regulates the migration of neutrophils

and macrophages acting on repairing processes such as

fibroplasia and reepithelization [19,20]. During the inflam-

matory stage, chitosan accelerates the infiltration of

inflammatory cells such as neutrophils; therefore, the wound

area is cleaned from foreign agents. At the new tissue

formation period, formation of granulation tissue takes place

within the wound space. Simultaneously, fibroplasia begins.

Generally wide-open wounds become a hypertrophic scar,

due to the imbalance of type I and type III collagens.

However, as an advantage, open wounds in dogs and cats

which were treated with chitosan did not leave a wide scar

[7].

Chitosan gel also acts as an ideal wound dressing. It is

biocompatible, biodegradable, hemostatic, anti-infective

and, more importantly, it accelerates wound healing [20].

Chitosan gel has a strong tissue-adhesive property. A

previous study showed that chitosan-treated wounds were

epithelized when compared with wounds of the control

group after the treatment [20].

EGF is a small polypeptide of 53 amino acid residues and

has a molecular weight of 6216 Da [10]. EGF has been

reported to accelerate cellular proliferation and synthesis of

the extracellular matrix in numerous papers [9,10,21,22].

EGF acts by binding to the EGF receptor – tyrosine kinase –

thereby initiating a series of events which regulate cell

proliferation [23–25]. Recently, several formulations of EGF

have been studied regarding their ability to accelerate wound

healing. The most commonly used form is solution; there are

only a few studies reported dealing with bioadhesive gel,

microemulsion and liposome [9,10,21,22]. The commonly

used form is ointment formulations of EGF in burn wound

healing [12]. These results suggest that EGF formulations

may play an important role in wound healing after burns.

In vivo, several studies have proven that EGF is effective

for the acceleration of epithelization in human and animal

wounds [9,10,21,26]. EGF stimulates the proliferation of

keratinocytes in culture, and topical administration of EGF

accelerates dermal regeneration of partial thickness burns or

split-thickness incisions in vivo [27], but no study has been

done for the treatment of second-degree burn wounds with

EGF containing chitosan.

In light of the above, we aimed to develop a chitosan gel

formulation of EGF for the treatment of second-degree burn

wounds in rats. The results of the in vivo experiments were

evaluated immunohistochemically, histochemically and

histologically.

2. Materials and methods

2.1. Materials

Human epidermal growth factor (hEGF) was purchased

from Sigma, USA. Chitosan-H was kindly provided by

Dainichiseika Color & Chemicals Mfg. Co. Ltd., Japan.

Bromodeoxyuridine (BrdU) and anti-BrdU were purchased

from Sigma. Glacial acetic acid was supplied from Merck,

Germany. All other chemicals and solvents were of

analytical grade.

2.2. Methods

2.2.1. In vitro studies

2.2.1.1. Preparation of the chitosan gel. Glacial acetic

acid (0.5%) was added into half of the required water. The

weighed amount of chitosan was added and stirred slowly.

After the swelling, the remaining amount of water was

added and mixed. Gel was kept at room temperature

overnight before the application in order to remove the air

bubbles. The pH of the gel was measured as 5.32. After the

preparation of the chitosan gel, the required amount of EGF

solution was added and the final concentration was 10 mg/

mL. The molecular range of chitosan is 650,000 and

viscosity value of 2% chitosan solution is 7903 mPa at

25 8C.

2.2.1.2. In vitro release studies of EGF from the chitosan

gel formulation. An in vitro release study of EGF from the

chitosan gel formulation was also performed. The release

properties of EGF from the formulation were studied

according to the previously reported procedures [22].

Briefly, 1 mL of chitosan gel–EGF formulation with a

concentration of 2 mg/mL was placed in a dialysis sac

having a pore size of 12,000 Da, and the sac was immersed

in a constantly stirred receiver vessel containing a 15-fold

higher volume of the drug free phosphate buffer (pH 5.8) at

32 � 0.5 8C. At the designated periods, the sample (3 mL)

was removed from the receiver vessel and replenished with

fresh buffer. The samples were then analyzed using a

Shimadzu RF-1501 spectrofluorometer [28], and release

profile was observed. The excitation wavelengths were

342 nm with an emission wavelength of 260 run.

C. Alemdaroglu et al. / Burns 32 (2006) 319–327 321

Table 1

Design of experimental animal groups

Group codes Treatment

S Unburned

Y Burned but untreated

J Burned and treated by chitosan gel without EGF

EJ Burned and treated by chitosan gel with EGF

SIL Burned and treated by Silverdin1

ES Burned and treated by EGF solution

Fig. 1. In vitro release profile of EGF from chitosan–gel at pH 5.8

phosphate buffer at 32 � 0.5 8C (n = 6).

2.2.2. In vivo studies

2.2.2.1. Design of animal experiments. All animal experi-

ments were conducted under the protocols approved by the

Animal Care and Use Committee of Gulhane Military

Medical Academy. For the in vivo experiments, female

Sprague–Dawley rats weighing 250 � 10 g were used.

Rats were housed in individual cages with unrestricted

food and water access. The animals were divided into six

groups. The unburned group consisted of 4 rats and there

were 12 animals in each group. The design of the animal

groups is shown in Table 1.

2.2.2.2. Formation of the burn wounds. The trauma was

performed by exposing the shaved backskin of anesthetized

animals to hot water. For this procedure, a cylindrical shaped

bar with the radius of 1 cm was placed on the backs of rats

and then hot water (94 � 1 8C) was poured into this bar and

held for 15 s [29]. After the formation of standard, second-

degree burns wounds, the formulations were repeatedly

applied (one application every day) to the burned areas for

14 days. Full thickness skin biopsies were collected at the

3rd, 7th and 14th days after wound formation, and the degree

of healing was evaluated both immunohistochemically and

histochemically.

2.2.2.3. Immunohistochemical studies. For the evaluation

of the healing, bromodeoxyuridine technique was used.

BrdU is a pyrimidine analogue that is incorporated into

DNA-synthesizing nuclei. In immunohistochemical studies,

the BrdU incorporated into DNA has been detected using

antibodies against BrdU [30].

One hour before animals were sacrificed, BrdU (100 mg/

kg body weight) dissolved in saline was injected i.p. The

animals were then sacrificed and full thickness skin biopsies

were collected.

After all the steps of immunohistochemical staining had

been performed, the samples were assessed using Ks400

Vision Imaging Analysis Program under Zeiss Axioskop

light microscope. The values were represented as BrdU per

10 high power field (HPF).

2.2.2.4. Histochemical studies. The skin biopsies taken

from all groups were embedded in paraffin and the sections

were stained using hematoxylin and eosine and trichrome

staining techniques.

2.2.2.5. Measurement of epidermis thicknesses. Increment

in the epidermis thickness is one of the important indicators

of wound healing. Measurements were carried out using

Ks400 Vision Screening Analysis Program.

2.2.2.6. Measurement of fibroblast nucleus area. The area

of the fibroblast nuclei was measured using Ks400 Vision

Screening Analysis Program under Zeiss Axioskop light

microscope after trichrome staining. The increment in the

fibroblast nucleus sizes indicates a faster healing process.

Fifteen different areas from each preparation were examined.

2.2.2.7. Histological investigation. In order to compare the

effects on EJ, ES, SIL and J groups and untreated control

groups [unburned (S) and untreated (Y) rats] histologically,

structural changes in the skin layers were examined using

transmission electron microscope (TEM 911 Carl Zeiss).

Tissues were fixed in phosphate-buffered solution contain-

ing 2.5% glutaraldehyde for 2 h, then they were post-fixed in

1% osmium tetroxide (OsO4) and dehydrated in a series of

graded alcohols. After passing through propylene oxide, the

specimens were embedded in Araldyt CY212, 2-dodecen-1-

yl succinic anhydride (DDSA) and benzyldimethyl amine

(BDMA). Ultra-thin sections were stained with uranyl

acetate and lead citrate and examined with the electron

microscope.

2.2.2.8. Statistical analysis. All data are expressed as

means � S.D. Statistical analysis of data was performed

using one-way ANOVA.

3. Results

3.1. In vitro studies

3.1.1. In vitro release of EGF from the gel formulation

The release of EGF from the chitosan gel was found to be

97.3% to the pH 5.8 phosphate buffer during 24 h. The in

vitro release of EGF is shown in Fig. 1. The release kinetics

C. Alemdaroglu et al. / Burns 32 (2006) 319–327322

Table 2

The average stained cell number labeled with BrdU at various days of

treatment for the different groups

Groups The average stained cell number

labeled with BrdU (%) � S.D.

3rd day 7th day 14th day

S 2.11 � 0.12 2.10 � 0.10 2.13 � 0.15

Y –a 1.27 � 0.15 2.33 � 0.38

J 1.20 � 0.17 2.43 � 0.61 3.10 � 0.68

EJ 1.63 � 0.35 4.20 � 0.18 4.95 � 0.39

ES 1.43 � 0.28 4.25 � 0.13 3.90 � 0.64

SIL 1.48 � 0.29 3.27 � 0.15 4.53 � 0.53

a No healing.

Fig. 2. The average cell number labeled with BrdU at the: (a) 3rd day, (b)

7th day and (c) 14th day of the treatment for the different groups (%) (n = 6)

( p < 0.001).

Fig. 3. Stained proliferated cell (shown with arrows).

from the gel formulation were found to be the first order.

This result indicated that the release rate of EGF from the gel

varies with time.

3.2. In vivo studies

3.2.1. Evaluation of the immunohistochemical studies

Full thickness skin biopsies from the 3rd, 7th and 14th

days of therapy from all animal groups were examined

immunohistochemically using BrdU technique.

The average numbers of proliferating cells (%) labeled

with BrdU are shown in Table 2 and Fig. 2a–c at the 3rd, 7th

and 14th days, respectively. When the results were evaluated

immunohistochemically, the rate of healing in the EJ group

was found to be increased ( p < 0.001).

A sample image of the proliferated cell stained with BrdU

technique is shown in Fig. 3.

3.2.2. Evaluation of the histochemical studies

The full thickness skin samples of the untreated group,

the gel without EGF applied group, the EGF–gel formula-

tion applied group and the Silverdin1 (commercial silver

sulfadiazine cream) applied group were examined at the

14th day of the treatment after trichrome staining.

The epidermis thickness (Table 3; Fig. 4a–e) and

fibroblast nucleus areas (Fig. 5a–c) were also examined.

The histochemical findings of EJ and ES groups were found

to be similar.

Fibroblast nucleus areas in J, EJ, ES and control groups

were comparable at the 3rd day. The largest nucleus areas

were observed in EJ, ES and SIL groups at the 7th day. At the

14th day, the areas of the nucleus in the EJ group were

observed to have reached normal size (10.3 mm2 considering

S group) (Fig. 5a–c) ( p > 0.05). The results of the areas of

fibroblast nucleus are shown in Fig. 5a–c.

3.3. Histological studies

The wound tissues were investigated by ultra-thin

sectional preparation under a transmission electron micro-

scope at the 14th day of treatment. The results are

summarized as follows:

C. Alemdaroglu et al. / Burns 32 (2006) 319–327 323

Table 3

The 14th day epidermis thickness results according to group

Groups Average epidermis thickness (mm) � S.D.

S 6.03 � 0.09

Y –a

J –a

EJ 10.2 � 0.0

SIL 9.02 � 0.05

ES 11.5 � 0.4

a Ulcered tissue.

Fig. 4. The epidermis thickness of skin samples from different experimental groups: (a

(c) gel formulation without EGF applied group (J), (d) EGF–gel formulation applied

applied group (SIL), at the 14th day of treatment after trichrome staining (�5).

Healthy experimental group—S: Skin layers were clearly

identified, epidermis and dermis were observed to be

normal. A significant number of fibrils were also

observed (Fig. 6a).

Burn wound made but no treatment received group—Y:

Inflamed cells observed around the burn wound. Burned

cells were observed but detailed structure could not be

identified. Cell response was observed not to be proper at

deeper layer and epithelial tissues and collagen fibers

were irregular. Neutrophilic infiltrations were present at

) untreated group (Y), (b) EGF solution formulation applied group (ES),

group (EJ) and (e) Silverdin1 (commercial silver sulfadiazine cream)

C. Alemdaroglu et al. / Burns 32 (2006) 319–327324

Fig. 5. The areas of the fibroblast nucleus at the: (a) 3rd day (b) 7th day and

(c) 14th day of the treatment according to group ( p > 0.05).

Fig. 6. The microscopic image of the epidermis and dermis from the different experime

the treatment. E: epidermis; D: dermis (�3000).

the dermal surface and early development of scar tissue

was observed. The healing process was found to be

started after 14th day (Fig. 6b).

Burn wound made and a gel formulation without EGF

applied group—J: continuation of fibroblastic activity

was observed. Although no differentiation of epithelial

basal cell was observed, some degeneration of mitochon-

dria and loss of crista were noted. There was some wound

healing, indicated by active fibroblasts observed at

dermis (Fig. 7a).

Burn wound made and EGF containing gel applied

group—EJ: Emphasized epidermis layers and swollen

cells were observed under the wound scar. Some

vacuolization of the dermal cells and healing were

observed. An infiltration of inflamed cells was noticed.

The structures of surface epithelial cells were normal, but

some active fibroblasts and inflamed cells were present at

connective tissue layer. The microscopic image of the

EGF–gel formulation applied group at the 14th day of the

treatment is shown in Fig. 7b.

Burn wound made and EGF solution applied group—ES:

Although the epidermis could not be observed under the

wound scab, some inflammatory cells were found.

Infiltrations were observed; irregular myofibroblast

distributions, collagen fibers and many fibroblasts were

present. The continuation of fibroblastic activity, wound

scab and invasive niflammatory cell infiltrations under-

neath were also noticed. A degeneration of cells at

epithelial surface was seen, but some myofibroblasts,

accepted as one of the indications of wound healing, were

observed at dermis. The image of the EGF solution

applied group at the 14th day of the treatment is shown in

Fig. 7c.

ntal groups: (a) healthy group and (b) untreated group, at the 14th day of

C. Alemdaroglu et al. / Burns 32 (2006) 319–327 325

Fig. 7. The microscopic image of the epidermis and dermis from the different experimental groups: (a) gel formulation without EGF applied group, (b) EGF–

gel formulation applied group, (c) EGF solution applied group and (d) Silverdin1 (commercial silver sulfadiazine cream) applied group, at the 14th day of the

treatment. E: epidermis; Mf: myofibroblast; F: fibroblast (�3000).

Burn wound made and commercial Silverdin1 cream

applied group—SIL: Inflammatory cell infiltrations were

observed. At intercellular space of enlarged epidermis

cells and irregular formations at dermis were noticed.

Random distributions of myofibroblasts were present.

There were also some vacuoles; the healing process of the

wound was not completed. Epithelization was found to be

higher than in J and Y groups, but wound scab was still

present. The image of Silverdin1 (commercial silver

sulfadiazine cream) applied group at the 14th day of the

treatment is shown in Fig. 7d.

4. Discussion

Healing of skin wounds is quite a complicated process

involving epidermal regeneration, fibroblast proliferation,

neovascularization and synthesis. Although there have been

some advances, the best treatment remains undecided. Many

investigators have studied acceleration and whether the

duration of wound healing could be shortened. Some studies

have shown that exogenous application of growth factors

may decrease the healing period in burns [13,31].

Brown and colleagues [9] found that application of

epidermal growth factor accelerated healing in burns and

shortened the time and improved the quality of healing.

However, efficient delivery of the growth factor must be

considered. Trials have included liquids, gels and collagen

sponges as delivery vehicles for the growth factor. Chitosan

was proposed and used in burn wounds as a polymer for

delivery in this study. Chitosan is a biodegradable polymer

and it accelerates wound healing [32,33]. It has been

reported that chitosan permits regeneration of tissue

elements in skin wounds and has positive application

effects on wound healing [34]. Also, chitosan exhibits many

advantages for topical application, including good flow

properties, non-irritancy, some antibacterial effect and a

potential for a suitable release rate from the dosage form

[34]. It was shown in this study that treatment with EGF–

chitosan gel formulation decreased the wound healing

period, accelerated epidermal regeneration and stimulated

granulation; tissue formation could be obtained.

C. Alemdaroglu et al. / Burns 32 (2006) 319–327326

Monteglione et al. determined the tertiary structure of

murine EGF at pH 3.1 and a temperature of 28 8C using

NMR analysis and distance geometry calculations and

restrained energy minimization [35]. The molecular

architecture of murine EGF was found to be as same as

that described previously. Kohda et al. have done similar

experiments and the tertiary structure of mouse epidermal

growth factor in solution (28 8C, at pH 2.0) was studied by

two-dimensional NMR spectroscopy. The chain folds in the

two structural domains of mouse EGF and again the

structure was found to be very similar to those previously

reported ones [35]. The tertiary structure of the EGF was

found to be similar to other reported structures in acidic

medium [36]. The isoelectric point of the EGF was 4.5 [37].

According to the report of Medved et al., the stability of the

EGF was found to be decreased if the pH of the solution is

below than 3.8 [38]. Both epidermal growth factor and

transforming growth factor bind to EGF receptors and TGF

has been reported to be more potent than EGF as far as many

biological effects are concerned. One possible reason for this

is thought to be the difference in their dissociation from the

receptors in intracellular acidic compartments, which may

affect the final pathway (lysosomal degradation or recycling

to cell-surface) of endocytosed ligands [38]. According to

the report of Meada et al., there are some experiments that

have been performed to clarify the relationship between

intracellular dissociation from the receptors and the fate of

the endocytosed ligands. In these experiments, the

magnitude of the dissociation rate constants were deter-

mined for each ligand at pH 6.0, which is reported to be

similar to that inside early endosomes. pH 6.0 was reported

as a suitable pH for experiments with EGF considering the

stability in the solution, intracellular ligand/receptor

interaction and a dissociation reported as a minimal effect

on the affinity to the receptors on the cell-surface [39]. After

consideration of all these, pH 5.8 was chosen as a suitable

medium for in vitro EGF release experiments.

According to the in vitro release studies, the release of

EGF from the chitosan gel was found to be 97.3% after 24 h.

The release kinetics from the gel formulation were found to

be of the first order, and the release rate of EGF from the gel

varied with time. The burst effect or faster release was

observed from the gel at the initial period, after which EGF

was released for a longer time period at a lower rate; the

occlusive effect was also observed.

After the formation of second-degree burn wounds, the

EGF formulations were repeatedly applied on the burned

areas at a dose of 0.160 mg/cm2 for 14 days (one application

per day). Skin biopsies were collected at the 3rd, 7th and

14th days after the wound formation and the degree of

healing was evaluated histologically, immunohistochemi-

cally and histochemically.

When the results were evaluated immunohistochemically

at the 7th day of the therapy, the maximum cell proliferation

was found in the EJ group. The healing in the EJ and ES

groups was also found to be rather good, and it was seen that

the healing ratios were very similar ( p > 0.05). It was also

observed that the cell proliferation in J and other control

groups (S and Y groups) was extremely low. There were

significant increases in cell proliferation observed in the EGF

containing gel applied group ( p < 0.001). At the 14th day of

the therapy, the healing in the EJ and ES groups was noted as

being faster than that of other groups. When EJ, ES and SIL

groups were compared, the healing and the rate of healing in

the EJ group were found to be increased ( p < 0.001).

The histochemical results showed that the increase in the

epidermis thickness in the EJ group was the highest (Fig. 4a–

e). This observation indicates that the maximum healing

effect was in the EJ group. An increment in the diameter of

the fibroblasts is one of the indications of accelerated wound

healing. The areas of the fibroblast cell nucleus were also

measured in this study (Fig. 5a–c). The area of the cell

nucleus is related to the diameter of the fibroblast cells.

Therefore, the area of the cell nucleus was used for

comparison and conclusion. When the fibroblast nuclei areas

were evaluated at the 7th day, the healing rates in the ES and

EJ groups were similar, and the healing rate in the EJ group

was found to be the fastest. At the 14th day, the healing in the

EJ group was found to be the fastest compared with the ES

and SIL groups results.

Although an effect on the fibroblastic cells was observed

with the use of commercially available Silverdin1 ointment,

it can be used for a small burn wound [40]. Therefore, silver

sulfadiazine containing preparation may be useful for small

burn wounds.

The histological results indicated and supported that the

healing in the EJ group was better and more rapid when

compared with the other groups.

In conclusion, when the results were evaluated, it was

determined that EGF-containing formulations are effective

in the wound healing process. Growth factor formulations,

which play an important role in burn wound treatment, are

found to be promising for use in humans.

Acknowledgements

This study was supported by a research grant from Gazi

University (SBE-11/2002-14). The authors are grateful to

Assoc. Prof. Dr. Mustafa Deveci and Prof. Dr. Mustafa

Sengezer for providing the facilities for the in vivo

experiments; to Dr. Ahmet Nacar and Prof. Dr. Candan

Ozogul for their kind help in histological analysis; to Dr.

Melih Alomeroglu for analyzing the samples for the

immunohistochemical and histochemical studies.

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