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Epidermal Growth Factorcontaining Wound Closure Enhances Wound Healing İn Diabetic And Non-Diabetic Rats
MAKALE #4390 © Yazan Dr.Semra DOĞAN | Yayın Şubat 2010 | 3,019 Okuyucu
ORIGINAL ARTICLE


Epidermal growth factorcontaining

wound closure
enhances wound healing in
non-diabetic and diabetic
rats

S Dogan, S Demirer, I Kepenekci, B Erkek, A Kiziltay, N Hasirci,

S Mu¨ ftu¨ ogˇ lu, A Nazikogˇ lu, N Renda, UD Dincer, A Elhan, E Kuterdem

Dogan S, Demirer S, Kepenekci I, Erkek B, Kiziltay A, Hasirci N, Mu¨ ftu¨ ogˇ lu S, Nazikogˇ lu A, Renda N, Dincer UD,

Elhan A, Kuterdem E. Epidermal growth factor-containing wound closure enhances wound healing in non-diabetic
and diabetic rats. Int Wound J 2009; 6:107–115

ABSTRACT


Background:
This study was designed to elucidate the in vivo efficacy of epidermal growth factor (EGF) on

wound healing in non diabetic and diabetic rats.

Methods:
Ninety-six male Wistar-Albino rats were randomly divided into six groups. Saline-moistened gauze,

pure gelatin or EGF in gelatin-microsphere dressings were used in a dermal excision model in both normal and
streptomycin-induced diabetic rats. Wound healing was evaluated on day 7 and 14. Reduction in wound area,
hydroxypyroline content and tensile strength of the wound were evaluated in each rat. Tissue samples taken from
the wounds were examined histopathologically for reepithelialisation, cellular infiltration, number of fibroblasts,
granulation and neovascularisation.

Results:
On day 7, the use of EGF-containing dressing was observed to reduce the wound area better when

compared with the other dressings tested. This effect was significant in normal rats rather than diabetic rats. The
difference in reduction of wound area did not persist on day 14. No significant effect on hydroxyproline content
of the wound was found with EGF-containing dressing in either normal or diabetic rats. There was a statistically
significant increase in tensile strength values of EGF-applied non diabetic rats over the 14 day period. An increase
in tensile strength was prominent in also EGF-applied diabetic rats on day 14. Histological examination revealed
higher histopathologic scores in EGF-applied diabetic and non diabetic rats.

Conclusion:
These findings implicate that use of EGF in gelatin-microsphere dressings improves wound healing

both in normal and diabetic rats.

Key words:
Diabetes mellitus • Epidermal growth factor • Hydroxyproline content • Tensile strength • Wound healing


Authors:
S Dogan, Ankara University School of Medicine, Department of General Surgery, Ankara, Turkey; S Demirer, Ankara University

School of Medicine, Department of General Surgery, Ankara, Turkey; I Kepenekci, Ankara University School of Medicine, Department
of General Surgery, Ankara, Turkey; B Erkek, Ankara University School of Medicine, Department of General Surgery, Ankara, Turkey;
A Kiziltay, Middle East Technical University, Department of Chemistry, Ankara, Turkey; N Hasirci, Middle East Technical University,
Department of Chemistry, Ankara, Turkey; S Mu¨ ftu¨ odgˇ u, Hacettepe University School of Medicine, Department of Histology–Embryology,
Ankara, Turkey; A Nazikogˇ lu, Hacettepe University School of Medicine, Department of Histology–Embryology, Ankara, Turkey; N Renda,
Hacettepe University School of Medicine, Department of Biochemistry, Ankara, Turkey; UD Dincer, Ankara University School of Pharmacy,
Department of Pharmacy, Ankara, Turkey; A Elhan, Ankara University School of Medicine, Department of Biostatistics, Ankara, Turkey;
E Kuterdem, Ankara University School of Medicine, Department of General Surgery, Ankara, Turkey

Address for correspondence:
I Kepenekci, A.U.T.F. Ibni Sina Hastanesi, Genel Cerrahi AD. 2. Kat A Blok, 06100

Sıhhiye-Ankara, Turkey

E-mail:
ilknurkpn@yahoo.com


©
2009 The Authors. Journal Compilation © 2009 Blackwell Publishing Ltd and Medicalhelplines.com Inc • International Wound Journal • Vol 6 No 2 107


EGF-containing wound closure enhances wound healing


INTRODUCTION


Tissue repair and wound healing is a complex,

dynamic process, involving overlapping
steps of haemostasis and inflammation,migration
and proliferation of certain cells, angiogenesis,
extracellular matrix deposition, maturation
and remodelling (1). Normal wound

Key Points



normal wound healing requires

the interactions of various
types of cells, including inflammatory
cells, fibroblasts, keratinocytes
and endothelial
cells, as well as the involvement
of growth factors and
enzymes


numerous growth factors are

involved in the wound healing
process and act by stimulating
chemotaxis, cellular proliferation,
extracellular matrix
formation and angiogenesis


diabetes mellitus is perhaps

the most common and most
studied disease leading to
impaired wound healing


it is also well known that,

diabetics are more susceptible
to wound infection because
of impaired neutrophil chemotaxis
and phagocytosis


new and advanced topical

dressings are designed to
modulate levels of biological
molecules, such as growth
factors, that may promote
wound healing


early experimental studies have

shown the potential of EGF in
promoting wound healing but
there are only a few clinical
trials which have documented
the efficacy of EGF in chronic
wounds, and more evidence is
needed to clarify the clinical
recommendations related to
the use of EGF in wound
management


we designed this study to

elucidate the in vivo efficacy of
EGF on epidermal regeneration
and wound healing in non
diabetic and diabetic rats

healing requires the interactions of various

types of cells, including inflammatory cells,
fibroblasts, keratinocytes and endothelial cells,
as well as the involvement of growth factors
and enzymes. Numerous growth factors are
involved in wound healing process and act by
stimulating chemotaxis, cellular proliferation,
extracellular matrix formation and angiogenesis
(1,2).
Local and systemic factors can disrupt the
complex process of wound repair and alter the
healing trajectory (3). Diabetes mellitus perhaps
is the most common and most studied
disease leading to impairedwound healing (4).
The compromised wound healing in diabetics
is due to immune system and inflammatory
defects inherent to diabetes mellitus (5). It was
reported that a number of growth factors were
markedly reduced in wound fluid from diabetic
wounds (6,7). In diabetic patients, the
inflammatory process is abnormal and the proliferation
of fibroblasts and endothelial cells
is impaired (8–10). High serum glucose levels
lead to a decrease in collagen deposition
and impaired wound remodelling. Abnormal
wound contraction and a reduction in breaking
strength is observed in diabetes (11,12). It
is also well known that, diabetics are more
susceptible to wound infection because of
impaired neutrophil chemotaxis and phagocytosis
(13,14).
There are many sophisticated dressings in
the market to aid in wound management, and
several ongoing studies focus on the agents
modulating the aspects ofwoundbiology.New
and advanced topical dressings are designed to
modulate levels of biological molecules, such
as growth factors, that may promote wound
healing. Growth factor treatments and their
potential use in healing chronic wounds have
recently been described and both in vivo and
in vitro data have demonstrated the efficacy
of growth factors in enhancing wound healing
(15,16). Platelet derivated growth factor
(PDGF), fibroblast growth factor (FGF), transforming
growth factor and epidermal growth
factor (EGF) are the most studied growth factors
(17–31). Early experimental studies have
shown the potential of EGF in promoting
wound healing but there are only a fewclinical
trials which have documented the efficacy of
EGF in chronic wounds, and more evidence
is needed to clarify the clinical recommendations
related to the use of EGF in wound
management.
We designed this study to elucidate the in
vivo efficacy of EGF on epidermal regeneration
andwound healing in non diabetic anddiabetic
rats.

METHODS


Our study protocol was reviewed and

approved by Ankara University ethical committee.
All animals received humane care in
accordance with the Guide for the Care and
Use of Laboratory Animals prepared by the
National Academy of Sciences and published
by the National Institutes of Health (NIH publication
No. 85–23, revised 1985).

Preparation of EGF-containing


microspheres, gelatin sponge


and gelatin sponge with EGF-loaded


microspheres


Gelatin microspheres were prepared by a

modified coacervation technique reported by
Nastruzzi’s (32). In order to prepare the EGFcontaining
microspheres, EGF (human recombinant

EGF, Sigma
®, USA) (50 or 750 μg in


1 ml phosphate buffer, pH
= 7??4) and heparin


(50
μl) were added into the aqueous 10%


gelatin (Difco
®, USA) solution, then the solution


was added dropwise into paraffin oil
while the mixture was mechanically stirred
at 1000 rpm to form a water–oil emulsion.
The solution was rapidly cooled by immersing
in ice-water medium. The formed EGFcontaining
gelatin microspheres were filtered,
washed with acetone and dried at room
temperature.
In order to prepare gelatin sponges (GSs),
aqueous gelatin solutions stirred at about
2000 rpm for 30 minutes at room temperature
and glutaraldehyde solutions were added to
form cross-linkings. Then the solutions were
poured into molds, frozen in liquid nitrogen
and freeze-dried for 24 hours. GSs with EGFcontaining
microspheres were prepared by
adding the microspheres containing EGF

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© 2009 The Authors. Journal Compilation © 2009 Blackwell Publishing Ltd and Medicalhelplines.com Inc


EGF-containing wound closure enhances wound healing


just before pouring it into molds. Then the

general procedure of sponge formation was
followed (33). GSs were sterilised by UV
exposure and 1 cm

3 of the sponges were


applied to the full thickness skin wounds.

Formation of non diabetic and diabetic


rat groups


Adult male Wistar–Albino rats weighing

between 250 and 300 g were used in this study.
All rats were housed in temperature- and
humidity-controlled rooms and were allowed
free access to standard rat chow and tap water
ad lib for 1 week before the start of the
experiment.
Diabetes was induced by a single dose of
45 mg/kg streptozocin (Zanosar

®, Pharmacia


& Up John, USA) through tail vein of the
rats. Diabetes was defined as a blood glucose
level of greater than 200 mg/dl as measured
by glucometer (Glucotrend
®, Isse, Turkey).


The study was initiated four weeks after the
diabetic and non diabetic rat groups were
firmly established and blood glucose readings
of the rats were taken routinely during the
study period.
Ninety six rats were randomly divided into
six (three non diabetic and three diabetic)
groups. Moistened gauze (MS), GS and GS
with EGF-loaded microspheres (GS-EGF) were
applied to non diabetic and diabetic rat groups.
Rats in each group were further divided into
two groups in order to evaluate the wound
healing on 7th and 14th days.

Surgical procedure


Under ketamine anesthesia (80 mg/kg), asepsis

was provided by 10% povidine iodine
(Betadine

®) and the back of rats were shaved.


A circular, full thickness, standard wound was
created on the back of each rat by using a
0
??5-cm sterile punch (18 mm2). Three different


techniques of wound dressing were applied to
non diabetic and diabetic groups of rats. MG
was applied to group 1 and 4, GS was applied
to group 2 and 5, and GS-EGF was applied to
group 3 and 6, and then covered with polythene
sheet (Op-Site
®, Smith&Nephew,UK).


The wounds were evaluated for the evidence
of infection and the decrease in wound size
every 3 days. Wound size was measured by
using a grid scale.
Wound healing was assessed on 7th and
14th days. A rectangular shaped 10-mm wide
skin samples, each having the original wound
at its center, were excised. Mechanical test
was performed on samples immediately, and
tensile strength of the wound tissue was
calculated. After mechanical measurements,
the rectangular samples were divided into
two equal parts and one-half was preserved
in 10% formalin solution for histopathological
examination. A tissue sample containing
wound and normal skin excised from otherhalf
was preserved in liquid nitrogen tanks
for determination of hydroxyproline levels.
Finally, rats were euthanised by administering
an overdose of sodium pentothal.

Mechanical tests


Lloyd LRX 5 K
® mechanical test device was

used for the assessment of tensile strength
(Lloyd Instruments Limited, Hampshire, UK).
Tensile forcewas appliedto the long edge of the
rectangular skin samples. The pull interval and
pull rate were designated as 30

??2 mm and


30 mm/minute, and the testwas terminated as
the skin samplewas completely torn apart (34).
The formula
σ = Fmax/ A was utilised in


calculation of tensile force. In this formula,
σ,


Fmax and A, respectively denote tensile force
(Mega Pascal), maximum force encountered
during pull test (Newton) and section surface
area of skin sample before the pull test (mm
2).


Section surface area was calculated by multiplying
the short edge of rectangle (10 mm)
with skin thickness (2 mm on average).

Determination of hydroxyproline levels


Hydroxyproline content of the wounds were

determined by using the Bergmanmethod (35).
Absorbance was assessed by ‘Shimatzu

®


spectrophotometer UV-120-02
device.


Histological examination


For histological examination, tissue samples
were fixed in 10% phosphate-buffered formalin,
dehydrated and embedded in paraffin.
Sections were cut at 4–5
μm and stained with


hematoxylin–eosin and Masson’s trichrome
reagents. Photomicrographs were obtained
under light microscope. At least 10 sections
from each wound were examined. Histological
evaluations were performed by modified

©
2009 The Authors. Journal Compilation © 2009 Blackwell Publishing Ltd and Medicalhelplines.com Inc 109


EGF-containing wound closure enhances wound healing


method of Tsuboi and Rifkin (36). The parameters

measured were degree of reepithelialisation,
granulation tissue thickness, number
of infiltrated cells, and neovascularisation for
the entire wound area. Each of the parameters
was graded numerically to permit average
scores to be complied. Degree of reepithelialisation
was rated on a scale of 0–4 (

0 = no


closure;
1 =< 30%; 2 => 31,< 60%; 3 => 61%,


<
99%; and 4 =complete reepithelialisation by


keratinocytes). Granulation tissue formation
was rated on a scale of 0–3 (
0 = not found;


1
= thin granulation layer; 2 = moderate granulation;


and
3 = thick granulation layer). As


an index of degree of cellular infiltration,
the number of fibroblasts and macrophages
were estimated. Polymorphonuclear cells and
lymphocytes were not counted. Cellular infiltration
was rated on a scale of 0–3 (
1 = few


cells,
2 = moderate number of cells, 3 = many


cells). Neovasculature were scored by counting
the number of capillary lumens in the complete
wound section at
??100 magnification.


Only mature vessels that contained erythrocytes
were counted and given a value of 0–3.
(
0 = 0–4 capillaries per wound, 1 = 5–14 capillaries


per wound,
2 = 15–24 capillaries per


wound,
3 => 24 capillaries per wound. Five


micron-thick sections were stained with hematoxylin–
eosin and Mallory Azan. Afterwards,
they were examined and photographed under
Olympus-BH2
® light microscope (Olympus,


Tokyo, Japan).

Statistical analysis


Differences among groups for wound area,

hydroxyproline level and histopathological
scores were evaluated by Kruskal–Wallis
variance analysis. When the

P-value from the


Kruskal–Wallis test statistics is statistically
significant, multiple comparison test was
used to know which groups differ from
which others (37). Student’s t-test was used
for evaluation of mechanical test results.
Comparison between day 7 and day 14
was assessed by Mann–Whitney U-test. The
Bonferroni correction was applied for all
possible multiple comparisons. Because of
the number of tests undertaken, the level of
significance is set at 0.008 for Mann–Whitney
U-test. SPSS 11.5 (SPSS Inc., Chicago, IL, USA)
was used for statistical analysis. A
P-value


<
0??05 was considered significant.


RESULTS


Macroscopic observations


One rat in N MG day 14, two rats in N GSEGF
day 14 and two rats in D GS-EGF day
7 and another two rats in D GS-EGF day 14
groups died during the course of the study.
Death rats were not replaced and the study
was completed with the remaining ones.
On 7th day, skin defects were observed in
all lesions and all the wounds were covered
with hemorrhagic crutes.Wound infectionwas
observed in one rat from D GS-EGF group. On
14th day, size of the lesion was significantly
reduced but there were no complete healing in
non diabetic and diabetic rats treated withMG
and GS. In N GS-EGF group, size of the lesion
was reduced in all rats except the two with
wound infection. Furthermore, no skin defect
was visible in three rats from this group. In D
GS-EGF group, size of the lesion was reduced
in all rats except the one with wound infection.
Skin defects were also not visible in the three
rats from this group.

Reduction in wound area


On 7th day, for non diabetic rats, the reduction

in the mean surface area of wounds in GSEGF
group was significantly higher than
that in MG (

P = 0??008) and GS (P = 0??001)


groups. For diabetic rats, decrease in wound
size on 7th day was more prominent in
GS-EGF group, but this difference did not
reach statistical significance. On 14th day, no
statistically significant difference was found
between any of the groups by means of wound
size (
P > 0??05) (Figure 1).


Tensile strength


The maximum pull force of normal skin samplewas
found to be 102
??82 Newton (kgm/sn2).


Maximumpull force of skin samples of non diabetic
and diabetic rats are shown in Figure 2.
Tensile strength was calculated relying on
pull force and area of skin slice. In non diabetic
rats, 7th and 14th day assessments revealed
that tensile strength of wounds treated with
GS-EGF was higher than those treated either
with MG or GS (
P < 0??05), but there were


no difference among groups treated with
MG or GS (
P > 0??05) (Figure 3). In diabetic


rats, no difference was observed between the
tensile strengths of skin samples between
MG-, GS- and GS-EGF-applied groups on 7th

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© 2009 The Authors. Journal Compilation © 2009 Blackwell Publishing Ltd and Medicalhelplines.com Inc


EGF-containing wound closure enhances wound healing


day (
P > 0??05). However, tensile strength of

samples fromGS-EGF-applied ratswere found
to be higher than those treated with MG or GS
on 14th day (

P < 0??05) (Figure 3).


Hydroxyproline content of wound


For both non diabetic and diabetic rats,
hydroxyproline contents of wounds treated
with the three different modalities were
similar on 7th day (
P > 0??05). For the 14th


day assessment, we noted that type of the
wound dressing in non diabetic rats had no
significant effect on hydroxyproline contents of
wounds (
P > 0??05). However in diabetic rats,


hydroxyproline contents of wounds in GSapplied
group were statistically significantly
higher than both MG (
P = 0??004) and GS-EGFapplied


groups (
P = 0??011) (Figure 4).


0
2
4
6
8
10
12
14
MG GS GS-EGF MG GS GS-EGF
Wound Area (mm
2)


Day 7
Day 14
Non-diabetic rats Diabetic rats

*





Figure 1.
Wound areas of non diabetic and diabetic rats on day 7 and 14. (∗P = 0??008; †P = 0??001).


Strength (N)


102,82

50,72
47,83
53,05
62,83
48,25 50,72
86,90
53,92
56,24
48,26 52,15 66,08

0

10
20
30
40
50
60
70
80
90
100
110
120
Normal Skin
Sample
MG GS GS-EGF MG GS GS-EGF

day 7

day 14

Non-diabetic rats Diabetic rats


Figure 2.
Maximum pull force of normal skin sample and skin samples from non diabetic and diabetic rats.


Tensile Strength (MPa)

0
1
2
3
4
5
6
Normal Skin Sample
MG
GS
GS-EGF
MG
GS
GS-EGF
Day 7
Day 14
Non-diabetic rats Diabetic rats
* †

$ ¶
#

Figure 3.
Tensile strenght values of the normal skin sample and samples from non diabetic and diabetic rats on day 7 and 14.

(

∗P = 0??002; †P = 0??021; ‡P = 0??00002; §P = 0??00003; ¶P = 0??00081; #P = 0??00078).


©
2009 The Authors. Journal Compilation © 2009 Blackwell Publishing Ltd and Medicalhelplines.com Inc 111


EGF-containing wound closure enhances wound healing

Histological findings


There were significant differences in comparisons

of normal and diabetic rats, particularly
in terms of reepithelialisation, granulation
tissue formation and neovascularisation
(Figures 5 and 6). On day 7, scores for
reepithelialisation, granulation tissue formation
and neovascularisation were significantly
higher in diabetic rats treated with GS-EGF
than diabetic rats treated with GS (

P = 0??002,


P
<
0??001 and P < 0??001, respectively). In non


diabetic rats scores for reepithelialisation were

0

5
10
15
20
25
MG GS GS-EGF MG GS GS-EGF
Hydroxyproline content
(

μg/mg d)


day 7
day 14
Non-diabetic rats Diabetic rats

*



Figure 4.
Hydroxyproline content of wounds on day 7 and 14. (∗P = 0??004; †P = 0??011).


Figure 5.
(A) N-MG on day 7. Section from healing region covered by thick crust and a vascular fibrin remnants. A few number

of cells can be seen (haematoxylin–eosin,

??10). (B) N-SF on day 14. Completed epithelialisation and granulation tissue with


vascularisation and inflammatory cell is observed. Also a dermal papilla can be noticed (haematoxylin–eosin,
??4). (C) N-GS-EGF on


day 14. Epithelialisation is completed. Multiple dermal papilla are present. Connective tissue appearance of dermis hair follicules and
sebaceous glands of the surrounding peripheral normal skin can also be noticed (Masson’s trichrome,
??4).


Figure 6.
(A) D-MG on day 7. Wound area is filled with necrotic cells and covered by crust. (haematoxylin–Eosin, ??20). (B) D-MG


on day 14. Epithelialisation is completed, granulation tissue rich in vascularisation and inflammatory cell infiltration can be seen
(haematoxylin–eosin,
??4). (C) D-GS-EGF on day 7. Completed epithelialisation was seperated from granulation artificially during


sectioning. Crust covers epidermis but is surrounded and limited by the corneum layer. A piece of gelatin and infiltrative cells can
be noticed in highly vascularised granulation tissue (haematoxylin–eosin,
??4). (D) D-GS-EGF on day 14. Completed epithelialisation


and formation of multiple dermal papilla can be seen, collagen rich connective tissue in dermis is prominent. (Masson’s trichrome,

??
4).


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© 2009 The Authors. Journal Compilation © 2009 Blackwell Publishing Ltd and Medicalhelplines.com Inc


EGF-containing wound closure enhances wound healing


not different in GS-EGF group than the other

groups, but granulation tissue formation (

P =


0
??01) and neovascularisation (P < 0??001) were


enhanced. On day 14, scores for reepithelialisation
(
P = 0??008) and granulation tissue formation


(
P = 0??011) were higher in diabetic rats


treated with GS-EGF than the rats treated with
other dressings. In non diabetic groups also
higher scores for reepithelialisation (
P = 0??036)


and granulation tissue formation (
P = 0??011)


were observed.

DISCUSSION


Wound healing is a complex cellular and biochemical

process. Cytokines and growth factors
are soluble factors that regulate wound
healing. Application of exogenous growth factors
has been shown to positively influence
wound healing in animal models and also
in clinical trials (17–31). Acute wound therapy
with exogenous growth factors accelerates
the appearance of fibroblasts and collagen into
the wound, shortening the natural inflammatory
phase for gain in injured tissue tensile
strength (38).
Over the past two decades several recombinant
growth factors have been tested for
their ability to accelerate wound healing. EGF,
the first-discovered growth factor, is known to
stimulate fibroblast replication, collagen formation
and reepithelialisation. Laato et al. (22)
confirmed that EGF is a potent dose-dependent
mitogen for the granulation fibroblast. Previous
studies have demonstrated that a number
of growth factors were markedly reduced
in wound fluid from chronic wounds compared
with acute wounds (28). Bennett and
Schultz (15) postulated increased destruction
or inhibition of growth factors by elevated
levels of proinflammatory cytokines and metallomatrix
protein following repeated trauma
and infection. Early experimental studies have
shown the potential of EGF in promoting
wound healing (23,24).However, there are also
some studies in the literature which failed
to show any benefit of EGF in wound healing
(39–41). In these studies EGF was applied
with a simple vehicle that does not provide
sustained release. Buckley et al. (22) andCohen
and Carpenter (42), based on in vitro results,
concluded that these failuresmay be because of
the experimental conditions that did not provide
sufficient continuous exposure of residual
epithelial cells to EGF. It is known that
mitogenic effect of EGF requires continuous
exposure of target cells to EGF for a minimum
6–12 hours. The half life of EGF in the
body is too short when applied via injection
or in free form. Many enzymes are increased
within the early wound because of cytokines
and growth factors. It is known that topical
applications of growth factors are generally
ineffective, because they are rapidly degraded
by local enzymatic activity (38). Sufficient continuous
exposure of epithelial cells to EGF is
required for effect. Therefore, incorporation or
encapsulation of EGF into a polymer matrix
and its sustained release from this matrix may
enhance its in vivo efficiency. It has been shown
that sustained release of growth factors leads to
superior outcome (25–27, 43–44), as a matter of
fact that, the vehicle that will be used to deliver
the growth factor requires attention when
constructing a study design. Strength of the
current report is the use of a novel drug delivery
preparation – gelatin microspheres – with
which to introduce the growth factor into the
wound. GS was used as the wound dressing.
Gelatin is a non toxic, non immunogenic
and biodegradable material. Prepared sponges
have a very soft, porous and highly elastic
structure (33). A porous and biodegradable
matrix would serve as the host for the proliferating
cells and would degrade spontaneously
without creating any adverse effects while the
tissue regenerates. EGF was added into these
sponges in gelatinmicrospheres thatwould act
as sustained release vehicle.

Key Points



application of exogenous

growth factors has been shown
to positively influence wound
healing in animal models and
also in clinical trials


acute wound therapy with

exogenous growth factors
accelerates the appearance of
fibroblasts and collagen into
the wound, shortening the natural
inflammatory phase for
gain in injured tissue tensile
strength


strength of the current report

is the use of a novel drug
delivery preparation – gelatin
microspheres – with which to
introduce the growth factor
into the wound


EGF was added into these

sponges in gelatin microspheres
that would act as sustained
release vehicle


the present study was aimed

to evaluate the effectiveness of
EGF on wound healing


we used GS with EGF-loaded

microsphere for wound closure
in a dermal excision model in
both normal and streptozocin
induced diabetic rats


on the 7th day the use of GS

with EGF-loaded microsphere
was observed to reduce the
wound area better when compared
with the other dressings
tested

The present study was aimed to evaluate

the effectiveness of EGF on wound healing.
We used GS with EGF-loaded microsphere for
wound closure in a dermal excision model
in both normal and streptozocin-induced diabetic
rats. We evaluated the reduction in
wound area, hydroxypyroline content and
tensile strength of the wound. The tissue samples
taken from the wounds were examined
histopathologically for inflammatory cell infiltration,
fibroblast activity, granulation, vascularisation
and reepithelialisation. These assessments
were performed on 7th day and again
on the 14th day after wounding.
On 7th day the use of GS with EGFloaded
microsphere was observed to reduce
the wound area better when compared with
the other dressings tested. This effect was
significant in normal rats rather than diabetic
rats. The difference in reduction of wound

©
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EGF-containing wound closure enhances wound healing


area did not persist on day 14. Although, no

significant effect on hydroxyproline content
of the wound was found with GS with
EGF-loaded microsphere in either normal or
diabetic rats; there was a statistically significant
increase in tensile strength values of EGFapplied
non diabetic rats over the 14 day
period. An increase in tensile strength was
prominent in also EGF-applied diabetic rats
on day 14. This result is consistent with the
previous studies (29,45). The balance between
synthesis and breakdown and so deposition
of collagen is important in wound healing
and development of wound strength. Previous
studies have reported that, EGF application
increased hydroxyproline and collagen levels
in wounds (17,46). In our study, we observed
no benefit of EGF on hydroxyproline content
of the wounds. It is well known that healthy
dermis mostly consists of type I collagen
however, in granulation tissue there are 30%
type III collagen. Studies designed to assess
the specific collagen level of wounds may be
needed to clarify this issue.

Key Points



in conclusion, results of the

current study provide evidence
to suggest that EGF in gelatinmicrosphere
dressings may be
used in wound management to
enhance the healing process


although these results are

promising, there is a need for
much more research in this
area for integration of this
experience in clinical practice


growth factors may be used

in combination with alternative
wound dressings or activators
of other signal transduction
pathways such as hyperbaric
oxygen to potentiate their
effects. Clearly, this would be
54 another interesting area of
future research

EGF is known as a potent mitogen for epidermal

cells in normal skin, but there is less
knowledge about its effect on poor granulation
tissue and remodelling in diabetics (29). In the
present study, histopathological examination
and scoring revealed that there was a significant
difference by means of wound healing
scores in EGF-applied normal and diabetic rats
on day 7 and also 14. GS with EGF-loaded
microsphere seems to decrease the maturation
time of granulation tissue and wound
contractionwhich means that it enhances reepithelialisation,
but no significant effect was
detected in inflammatory infiltration and number
of fibroblasts in time-dependent activity.
In conclusion, results of the current study
provide evidence to suggest that EGF in
gelatin-microsphere dressings may be used in
wound management to enhance the healing
process. Although these results are promising,
there is a need for much more research in
this area for integration of this experience in
clinical practice. There are many sophisticated
dressings in the market to aid in wound
management. Growth factors may be used in
combination with alternative wound dressings
or activators of other signal transduction
pathways such as hyperbaric oxygen to
potentiate their effects. Clearly, this would be
another interesting area of future research.

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