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International Wound Journal ISSN 1742-4801

ORIGINAL ARTICLE

Comparison of the effects of topical fusidic acid and rifamycin

on wound healing in rats
Mehmet S Gurel1 , Sillan Naycı2 , Aslı V Turgut1 & Erol R Bozkurt3
1 Dermatology Department, Istanbul Educational and Research Hospital, Istanbul, Turkey
2 Dermatology Department, Istanbul Leprosy Dermatology and Venereology Hospital, Istanbul, Turkey
3 Pathology Department, Istanbul Educational and Research Hospital, Istanbul, Turkey

Key words Gurel MS, Naycı S, Turgut AV, Bozkurt ER. Comparison of the effects of topical
Rifamycin; Topical fusidic acid; Wound fusidic acid and rifamycin on wound healing in rats. Int Wound J 2015; 12:106–110
healing
Abstract
Correspondence to
S Naycı, MD Wound healing is an active and dynamic process that begins from the moment of
Istanbul Leprosy Dermatology and injury. Any delay in the initiation of the response to injury can prolong the healing
Venereology Hospital process. The aim of this study was to investigate the effects of topically applied
Bakırkoy 34147 fusidic acid and rifamycin on wound healing in a full-thickness wound model. Ten
Istanbul, Turkey female Sprague-Dawley rats, aged 4 months and weighing 200–250 g, were used. Four
E-mail: silankartal@gmail.com
rifamycin (R), four fusidic acid (F) and four control (K) areas were generated on their
backs by using a 5-mm punch biopsy pen. On the 4th, 7th, 14th and 21st days, biopsies
doi: 10.1111/iwj.12060
were taken from each wound area of all the rats. Fusidic acid group demonstrated a
statistically significant increase of collagen and intensity of fibroblast proliferation on
the 21st day of wound healing, whereas in the rifamycin group, healing time was, as
expected, similar to physiological wound-healing phases. Despite the limited number
of subjects, topical fusidic acid was found to delay wound healing by prolonging
fibroblast proliferation.

now resistant to methicillin (6). Apart from its antibacterio-

Introduction
logical effects, limited information is available regarding the
The most important role of the skin is to protect the body by effectiveness of fusidic acid as a topical treatment to promote
forming a barrier against external factors. Besides traumatic wound healing (7).
skin injuries, controlled skin losses are used for diagnosis or In this study, we aim to investigate the effects of topically
treatment purposes in many fields, particularly in the field of applied fusidic acid and rifamycin on wound healing in a
dermatological surgery (1). full-thickness wound model.
Wound healing is an active and dynamic process that begins
from the moment of injury; various mechanisms are operative
at different times in the process. Any delay in the initiation
of the response to injury can prolong the healing process. The Key Messages
purpose of topical and systemic agents is to prevent this delay
• in this research, the effects of topically applied fusidic
and provide an ideal setting for wound healing and scar forma-
acid and rifamycin on wound healing in a full-thickness
tion by regulating the factors that affect wound healing (2,3).
wound model were evaluated and compared using
Rifamycin is a widely used drug with proven effectiveness
histopathological examination
as an antituberculosis agent. It is derived from Streptomyces
• the evaluation results indicated that wound healing was
mediterranei and is a semisynthetic antibiotic. It has bac-
realised more rapidly in the areas on which rifamycin
tericidal effects against gram-negative and gram-positive
was applied in comparison to the control areas, whereas
microorganisms, particularly Staphylococcus aureus (4).
topical fusidic acid could delay wound healing by
The existing literature provides little information regard-
prolonging fibroblast proliferation
ing the use of rifamycin for wound care (5). Similarly,
• in conclusion, rifamycin and fusidic acid, both of which
fusidic acid, a narrow-spectrum antibiotic obtained from the
have antibacterial effects, may be used successfully to
Fusidium coccineum fungus, is especially effective against
heal infected skin lesions
S. aureus and Streptococcus epidermidis strains, which are
© 2013 The Authors
106 International Wound Journal © 2013 Medicalhelplines.com Inc and John Wiley & Sons Ltd

M. S. Gurel et al.
Materials and methods
Experimental animals
Ten female Sprague-Dawley rats, aged 4 months and weighing
200–250 g, were used in this study. Control and experimental
treatments were performed on the back of each rat. Rats were
fed with standard water and pellets and kept in a ventilated
room with two rats per cage at 21◦ C in a 12-hour day/night
cycle. Approval was obtained from the Ethical Committee
of the Experimental Medicine Research Center of İstanbul
University prior to conducting the study.
Formation of skin defects and subsequent wound care
The experimental animals were anaesthetised with intraperi-
toneal 70 mg/kg ketamine and 3 mg/kg chlorpromazine. Post
anaesthesia, an area 8 × 10 cm2 on the back of each rat was
shaved using an electric shaver. The area was cleansed with
0·9% NaCl and dried. Thereafter 12 areas were marked: 4 each
for rifamycin (R), fusidic acid (F) and control (K) groups. A
template was used to maintain a distance of 2 cm between
the areas. Photographs of the subjects were taken before the
procedure (Figure 1).
A full-thickness defect was created in each marked area
using a sterile 5-mm punch biopsy pen and a no. 11 surgical
knife; pressure was immediately applied firmly to the ulcer
areas to control bleeding. After controlling the bleeding,
wound-care phase was performed separately on previously
marked areas; in binary groups, 0·1 cc of rifamycin (R) and
normal saline solution (K) and half a fingertip unit (0·25 g) of
fusidic acid (F) were applied directly on the marked areas for
all ten rats. Following this, the wound areas were left open,
and photographs of the subjects were again taken (Figure 2).
The same wound care procedure was repeated separately
on each wound area once a day for a period of 7 days
approximately at the same time as the first application. No
anaesthetics were used during this phase, and following the
applications rats were kept immobile for 10 minutes and
marking was repeated on alternate days.
Taking biopsies in the wound areas
On the 4th, 7th, 14th and 21st days of wound healing, biopsies
were taken from R, F and K wound areas from each rat under
anaesthesia. A 5-mm punch biopsy pen was used and half
Figure 1 Preparation of the subjects before the procedure.
© 2013 The Authors
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Effects of topical fusidic acid and rifamycin on wound healing
Figure 2 Photographs of the subjects after forming full-thickness defect
and following wound care.
the wound tissue was taken for histopathological examination.
Totally, 120 biopsy samples were taken and kept in bottles
containing 10% formalin. All biopsy samples were delivered
to a pathologist who was blinded to the subject matter except
about the day of biopsy.
Histopathological examination
All 120 punch biopsy specimens of 5 mm were placed in
paraffin blocks. Prepared sections from every block were
stained with haematoxylin–eosin.
In all biopsy samples, scabbing, fibrin clot and oedema were
evaluated in the form of ‘yes’ or ‘no’ at the 4th, 7th, 14th
and 21st days of wound healing. Neutrophil, lymphocyte and
macrophage infiltration, proliferation of vascular, epithelial
and fibroblast tissue, intensity of collagen fibre and granulation
tissue were evaluated separately according to severity of den-
sity in the form of ‘1: mild, 2: moderate and 3: intense’ at the
same biopsy days. Previously prepared histopathologic exam-
ination form (Table 1) was filled out during the evaluation.
Statistical analysis
Data were analysed using SPSS, Chicago, IL for Windows 100
software. Kruskal–Wallis and Mann–Whitney U -tests were
used for comparison of the wound groups in terms of features
such as scabbing, fibrin clot, oedema, cell infiltration density,
vascular proliferation, epithelial and fibroblast proliferation,
intensity of collagen fibres and texture of granulation. P values
<0·05 were considered statistically significant.
Findings
No obvious macroscopic differences were observed between
the groups in terms of wound healing. Biopsy materials taken
on days 4, 7, 14 and 21 were stained with haematoxylin–eosin
and evaluated by a blinded pathologist.
For all biopsy samples taken, the R, F and K areas
showed no statistically significant difference when they were
compared in terms of scabbing and intensity of fibrin and
oedema. The intensity of neutrophil infiltration and vascular
proliferation was significantly higher in the control group on
day 21 (Figures 3 and 4), but no differences were found in
terms of macrophage and lymphocyte infiltration.
107
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Effects of topical fusidic acid and rifamycin on wound healing M. S. Gurel et al.

Table 1 Histopathologic examination form*

Neutrophil Lymphocyte Macrophage Vascular Epithel Fibroblast Granulation Collagen
Scabbing Fibrin clot Oedema infilt. infilt. infilt. prolif. prolif. prolif. tissue fibre
Rat No. (yes/no) (yes/no) (yes/no) (0/1/2/3) (0/1/2/3) (0/1/2/3) (0/1/2/3) (0/1/2/3) (0/1/2/3) (0/1/2/3) (0/1/2/3)
R1
F1
K1
R2
F2
K2
R3
F3
K3
R4
F4
K4
R5
F5
K5
R6
F6
K6
R7
F7
K7
R8
F8
K8
R9
F9
K9
R10
F10
K10
*Biopsy days: 4th/7th/14th/21st.

When the groups were compared in terms of epithelial
proliferation, no statistically significant differences were noted
on days 4, 7 and 14. The intensity of epithelial proliferation in
the control areas was statistically higher on day 21 (Figure 5).
When we investigated the intensity of fibroblast accumu-
lation, a significantly higher level was found in the F and K
areas on day 21 (Figure 6). A significant increase in collagen
fibres was found in the R and F areas on day 14 and in the
control area on day 21.
Discussion
The most important role of the skin is to protect the body
by forming a barrier against external factors. Wounds are
formed on the skin in both dermatological and surgical clinics
for diagnosis and treatment purposes. The purpose after skin
losses, either as a result of a trauma or under controlled
conditions, is to ensure that healing is rapid and cosmetically
acceptable. In addition to a suitable surgical technique, locally
and systemically suitable conditions must be provided in
order to ensure proper wound healing. Factors such as severe
anaemia, diabetes, immunosuppression, nutrition disorders,
smoking and infections play important roles in delaying
wound healing (8).
Numerous topical agents, such as bacitracin, polymycin B
sulphate, neomycin, neosporin, povidone-iodine, silver sul-
phadiazine, mafenide acetate, nystatin, nitrofurazone, gen-
tamycin, benzoyl peroxide, acetic acid, sodium hypochlorite,
108
silver nitrate and chlorhexidine gluconate, have been used
for wound care (9). Studies have shown that many antisep-
tic agents used in wound care can prolong the inflammatory
process and delay epithelialisation and collagen synthesis by
exerting a toxic effect on keratinocytes and fibroblasts (10).
Although topical antibiotics are often used in practice
when clinically infected wounds are treated with systemic
antibiotics, no general consensus has been reached regarding
the validity of this treatment. The use of topical antibiotics is
generally not proposed for the treatment of uninfected wounds.
However, its use is proposed for the treatment of ischaemic
injuries such as diabetic feet, pressure sores and burns where
vascularisation is not sufficient and systemic antibiotics would
be expected to have less impact on the wounded area (10,11).
Although rifamycin and fusidic acid are typically used for
wound care, sufficient data are not available regarding their
effect on wound healing. Nitrofurazone has been shown to
delay wound healing when used alone, but no negative effect
has been seen on full-layer wound healing when used in
combination with rifamycin (12).
One of the greatest advantages of using animal models
in wound healing is that it offers the possibility of follow-
ing up the wound-healing process histologically in addition
to performing macroscopic, biochemical and biomechanical
measurements (13). In the studies reported in the literature,
parameters such as intensity of scabbing, oedema, fibrin clots,
infiltration of neutrophils, white blood cells and inflammatory
© 2013 The Authors
International Wound Journal © 2013 Medicalhelplines.com Inc and John Wiley & Sons Ltd
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M. S. Gurel et al. Effects of topical fusidic acid and rifamycin on wound healing

Figure 3 Comparison of the neutrophil infil-

tration in R, F and K groups on days 4, 7, 14
and 21.

Figure 4 Comparison of the vascular pro-

liferation in R, F and K groups on days 4, 7,
14 and 21.

Figure 5 Comparison of the epithelial pro-

liferation in R, F and K groups on days 4, 7,
14 and 21.

Figure 6 Comparison of the fibroblast

intensity in R, F and K groups on days 4, 7,
14 and 21.

© 2013 The Authors

International Wound Journal © 2013 Medicalhelplines.com Inc and John Wiley & Sons Ltd 109

Effects of topical fusidic acid and rifamycin on wound healing
cells such as macrophages, intensity of fibroblast intensity,
angiogenesis, texture of granulation, reepithelialisation and
collagen intensity have been examined for histopathological
evaluation of wound healing (14,15). In the present case,
biopsies were taken on the days in which cell parameters
showed variability. Indeed, we were careful to perform biop-
sies on the days that were compatible with changes in these
parameters.
While no significant differences were observed between
the wound areas in terms of lymphocyte and macrophage
intensities, neutrophil infiltration was found to be higher in
the control group in the later days. Reactive oxygen species
and enzymes are released from excessively accumulated
neutrophils during the wound-healing process, and this is an
indication that the inflammatory phase has been prolonged,
and consequently, the wound-healing process delayed (16).
We separately evaluated the indicators of the proliferative
phase of wound healing, namely reepithelialisation, angio-
genesis, texture of granulation and also intensity of fibroblast
accumulation, which is the indicator of the fibroblast period.
While there was no significant difference between the treat-
ment groups in terms of epithelial and vascular proliferation
according to the biopsy samples taken on days 4, 7 and 14,
reepithelialisation and angiogenesis continued to day 21 in
the control group.
On the other hand, proliferation of fibroblasts was more
intense on day 21 in both fusidic acid and control areas
in comparison to the rifamycin areas. We noticed that the
proliferative phase, which typically begins on day 4 and is
expected to be completed by day 21, became prolonged in the
control and fusidic acid-treated areas, whereas it was similar
to physiological wound healing in the rifamycin group.
When the groups were evaluated in terms of intensity of
collagen fibre formation, we observed that collagen synthesis
began on day 7 in the rifamycin areas but there was no
statistically significant difference during that time. On day
21, the intensity of collagen fibres in the control and fusidic
acid areas was statistically more significant. The net amount
of collagen production shows a continuous increase until
day 21, on average, subsequent to infliction of the wound.
A decrease in collagen synthesis begins after that phase. The
higher intensity of collagen fibre in the control and fusidic
acid areas on day 21 is believed to be due to the longer
proliferative phase in these areas.
This study was carried out with a small number of
subjects and the wound-healing process was evaluated using
histopathological data alone. Despite these limitations, wound
healing in the areas on which rifamycin was applied was
more rapid compared with that of the control areas. However,
when we examined the physiological process, we observed
that wound healing with rifamycin was realised within the
expected time period, and consequently, it could not be
determined whether rifamycin had a positive or negative effect
on wound healing. On the other hand, fibroblast proliferation
was prolonged and the intensity of collagen increased in the
areas where fusidic acid was applied. Fibroblast proliferation,
which was expected to decrease in 2 weeks on average, and
still continued until day 21, was accepted as an indicator of
the prolongation of the proliferative wound-healing phase.
110
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M. S. Gurel et al.
Conclusion
In this research, the effects of topically applied fusidic acid
and rifamycin on wound healing in a full-thickness wound
model were evaluated and compared using histopathological
examination.
The evaluation results indicated that wound healing was
realised more rapidly in the areas on which rifamycin was
applied in comparison to the control areas and in agreement
with natural physiological processes. No negative effects from
rifamycin were observed on wound healing. On the other
hand, topical fusidic acid could delay wound healing by
prolonging fibroblast proliferation.
It can be concluded that rifamycin and fusidic acid, both
of which have antibacterial effects, may be used successfully
to heal infected skin lesions. However, future experiments
employing more subjects are needed to validate their effects
on wound healing.
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