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Wound healing evaluation of benzalkonium chloride-loaded hydrocolloid in

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Journal of Pharmaceutical Investigation (2012) 42:327–333
DOI 10.1007/s40005-012-0043-2
RESEARCH ARTICLE
Wound healing evaluation of benzalkonium chloride-loaded
hydrocolloid in the wound infection model
Jeong Hwan Kim • Sung Giu Jin • Sae Kwang Ku • Doo Hyun Nam •
Young Taek Sohn • Dong Sung Ryu • Eui-Seon Do • Sun Woo Jang •
Mi Woon Son • Chul Soon Yong • Han-Gon Choi • Jong Oh Kim
Received: 10 August 2012 / Accepted: 7 October 2012 / Published online: 28 October 2012
Ó The Korean Society of Pharmaceutical Sciences and Technology 2012
Abstract An adequate environment for wound healing
improves cell proliferation and cell activity, rearranges
fibroblasts, and suppresses the multiplication of bacteria.
The aim of this study was to evaluate the wound-healing
effects of a semi-occlusive hydrocolloid wound dressing
(HWD) containing a topical antiseptic drug, benzalkonium
chloride (BKC) in an infectious wound model. In vivo
wound-healing profiles in rats exhibited that BKC-loaded
HWD successfully promoted epithelialization rate, com-
pared with gauze control or HWD without BKC. Further-
more, the significant decrease in desquamated epithelial
Jeong Hwan Kim and Sung Giu Jin contributed equally in this
manuscript.
J. H. Kim  D. H. Nam  C. S. Yong (&)  J. O. Kim (&)
College of Pharmacy, Yeungnam University, 214-1, Dae-Dong,
Gyeongsan 712-749, South Korea
e-mail: cysong@yu.ac.kr
J. O. Kim
e-mail: jongohkim@yu.ac.kr
S. G. Jin  D. S. Ryu  E.-S. Do  S. W. Jang  M. W. Son
Pharmaceutical Product Research Laboratories,
Dong-A Pharmaceutical Co. Ltd., Kyunggi-Do,
Yongin-Si 449-905, South Korea
S. K. Ku
College of Oriental Medicine, Daegu Haany University,
Gyeongsan 712-715, South Korea
Y. T. Sohn
College of Pharmacy, Duksung Women’s University,
Seoul 132-714, South Korea
H.-G. Choi (&)
College of Pharmacy, Hanyang University, 1271,
Sa-3-Dong, Ansan 426-791, South Korea
e-mail: hangon@hanyang.ac.kr
regions, numbers of microvessels and inflammatory cells
infiltrated in granulation tissues and granulation tissue
areas were observed in the histopathological and histo-
morphometrical profiles of BKC-loaded HWD. In conclu-
sion, these results suggest that BKC-loaded HWD
facilitates wound healing and re-epithelization, and has
promising effects for the treatment of infected wounds, as
compared to HWD without BKC.
Keywords Benzalkonium chloride  Hydrocolloid 
Wound dressing  Wound healing
Introduction
Wound healing is a fundamental response to tissue injury.
The healing process can be related to inflammation leading
to epithelisation, formation of granulation tissue and tissue
remodeling (Evans 1980). Wound healing involves over-
lapping steps composed of inflammation, cell migration,
and proliferation, neovascularization, extracellular matrix
production and remodeling, and multiplication of collagen
which is a major component of the extracellular matrix
(Froget et al. 2003). Inflammation followed by tissue repair
is also a complex physiological process aimed at restora-
tion of normal function after injury (Singer and Clark
1999). In addition, angiogenesis is also treated as an
important criterion in wound healing especially during the
later stages of tissue repair as a process essential to the
restoration of tissue damaged by injury and inflammation
(Halper et al. 2003). Thus, wound healing is a complex
pathophysiological process involving the interplay of sev-
eral cell biochemical processes (Kapoor et al. 2004).
Currently, different types of wound dressings have been
developed in the market and they are targeted to produce
123
328
beneficial conditions (Abdelrahman and Newton 2011;
Kim et al. 2008). Among the modern wound dressings, the
dressings which can maintain a moist state have been
widely preferred for wound care, because the healing is
promoted by the dressings which inhibit aridness of
wounds. Specifically, hydrocolloid dressings have the
ability to allow excess fluid to escape without permitting
wound desiccation. In addition, hydrocolloid dressing can
form a gel upon contact with wound exudates and high
absorption occurs via strong hydrogel formation (Lanel
et al. 1997; Thu et al. 2012).
The process of dermal wound healing can be accelerated
by the application of active ingredients, such as ben-
zalkonium chloride (BKC). BKC is a widely used disin-
fectant as well as a bactericidal to prevent infection in
wounds. Its antimicrobial activity has been proven in many
previous studies (Conroy et al. 1999; Tarbox et al. 1998).
In particular, BKC is effective as an irrigating agent for
eradicating Staphylococcus aureus, which is currently the
most common cause of infections from a contaminated
orthopedic wound (Tarbox et al. 1998; Archer 1998).
Hence, a new hydrocolloid formulation with a BKC
content of 0.5 % was developed, and its wound-healing
efficacy was investigated, in comparison to a gauze control
and hydrocolloid without BKC in an infected animal
model. A histological analysis was further performed to
determine whether BKC-loaded hydrocolloid wound
dressing (HWD) could enhance wound healing rate
accompanied by an eradication of micro-organisms.
Materials and methods
Materials
Hydrocolloid wound dressings (HWD) and HWDs con-
taining 0.5 % benzalkonium chloride (BKC-loaded HWD)
were obtained from Dong-A Pharmaceutical Co. Ltd.,
(Yongin, South Korea). Zoletil 50Ò (tiletamine/zolazepam)
and RompunÒ (xylazine hydrochloride) were purchased
from Virbac S.A. and Bayer, respectively. All of the other
chemicals were of reagent grade and were used without
further purification.
In vitro release
One side of the BKC-loaded HWD was attached to a
Teflon frame instrument that was immersed into 50 ml pH
7.4 phosphate buffer solution (PBS) as a dissolution med-
ium at 37 °C. It was stirred at the paddle speed of 50 rpm
for 48 h. At predetermined time intervals, 5 ml of the
medium was withdrawn, diluted with the dissolution
medium and filtered using a 0.45 lm syringe filter. The
123
J. H. Kim et al.
concentration of the drug was determined at 208 nm using
a Waters 2795 HPLC system consisted of Waters 2795
Separation module and Waters 2996 Photodiode Array
detector. Column used was Spherisorb CN-RP (4.6 mm
I.D 9 250 mm, 5 lm) (Waters Inc.). Mobile phase con-
sisted of acetonitrile and 0.1 % Na2HPO4 buffer (50:50
v/v) adjusted to pH 5.2 with 10 % phosphoric acid. Mobile
phase was filtered through 0.45 lm membrane filter and
eluted at a flow rate of 1.5 ml/min. The effluent was
monitored at a UV absorption wavelength of 208 nm. All
standard curves showed excellent linearity with
R2 = 0.999 and relative standard deviation at different
concentrations and time was less than 3 %.
In vivo wound healing
Sprague–Dawley rats weighing 250–300 g (Orient, Seoul,
South Korea) were used to evaluate the efficacy of HWD
and BKC-loaded HWD on wound healing. Briefly, the rats
were anesthetized with a mixture of ZoletilÒ and RompunÒ
(5:2), and two full thickness wounds (1.5 9 1.5 cm) were
created on back skin. Then, 50 ll of S. aureus solution
(KCCM 40050, 3.2 9 108/ml) was dropped on every site
to develop infectious wounds, and each wound was treated
with a sterile gauze (control group), a product without BKC
(HWD group) or a wound dressing containing BKC (BKC-
loaded HWD), respectively (Lim et al. 2010). All materials
were fixed with elastic adhesive tapes, MicroporeÒ 3 M
and replaced with new ones at the proper time. Every rat
was cared for in detached cages, and digital images of the
sites were taken every 3 days by using a digital camera.
These macroscopic data were used to measure the epi-
thelializing rate by using Adobe Acrobat 9 ProfessionalÒ.
The epithelializing rate was calculated as follows:
Et
Epithelializing rate ð%Þ ¼  100
W t þ Et
where Et and Wt are the epithelialized area and the wound
area at time t, respectively.
Multiple comparisons test was performed to statistically
clarify the differences between the groups. The acquired
data were analyzed by one way ANOVA test followed by
the least significant difference (LSD) method. Handling of
all animals and the experimental procedures were approved
by the Yeungnam University Animal Care and Use
Committee.
Histopathological study
Infected wound areas of skin were sampled containing both
the dermis and hypodermis, and one part of each sample
based on the wounds was crossly trimmed, maintaining the
central regions. All trimmed skins were fixed in 10 %
Evaluation of benzalkonium chloride-loaded hydrocolloid
neutral buffered formalin. After paraffin embedding,
3–4 lm sections were prepared. Representative sections
were stained with hematoxylin and eosin (H&E) for light
microscopic examination or Masson’s trichrome for col-
lagen fibers (Sung et al. 2010). Subsequently, the histo-
logical profiles of individual skins were observed under a
light microscope (Nikon; E400, Japan).
To observe more detail of the histopathological changes,
desquamated epithelium regions (mm), the number of
microvessels in granulation tissues (vessels/mm2 of field),
number of infiltrated inflammatory cells in granulation
tissues (cells/mm2 of field), percentages of collagen occu-
pied region in granulation tissues (%/mm2 of field) and
granulation tissue areas (mm2/crossly trimmed central
regions of wounds) were measured on the prepared crossly
trimmed individual histological skin samples using a dig-
ital image analyzer (DMI-300, DMI, Korea) according to
the previous methods (Lee et al. 2010; Hwang et al. 2010).
The histopathologist was blinds to group distribution when
this analysis was made. In addition, re-epithelization (%)
was also calculated according to some modification from
the known method as follows (Hirose et al. 2007).
Totallengthofwound  Desquamatedepitheliumregion ðmmÞ
Re-epithelization ð%Þ ¼
Total length of wound
Statistical analysis
Multiple comparisons tests for different those groups were
conducted. Variance homogeneity was examined using the
Levene test (Levene 1981). If the Levene test indicated no
significant deviations from variance homogeneity, the
obtained data were analyzed by one-way ANOVA test
followed by LSD multi-comparison test to determine
which pairs of group comparison were significantly dif-
ferent. When significant deviations from variance homo-
geneity were observed with the Levene test, a non-
parametric comparison test, the Kruskal–Wallis H test was
conducted. When a significant difference was observed by
the Kruskal–Wallis H test, the Mann–Whitney U (MW)
test was conducted to determine which the specific pairs of
group comparisons were significantly different. Statistical
analysis was conducted using SPSS for Windows (Release
14.0 K, SPSS Inc., USA) (Ludbrook 1997). Moreover, to
evaluate the comparable efficacy of gauze control and test
materials, their changes were calculated as follows:
329
Percentage changes compared with gauze control ð%Þ
¼ ðAT  AC Þ=AC  100
where AT and AC are data of tested groups and gauze
control, respectively.
Results and discussion
In vivo wound healing
The wound healing ability of the BKC-loaded HWD was
compared with HWD without BKC in the infected rat
model. After contamination with S. aureus, a sterile gauze
as control, HWD and BKC-loaded HWD were applied to
wound spots in the rat dorsum. The representative macro-
scopic images of the wounds treated with a sterile gauze,
HWD and BKC-loaded HWD at various days of post-
operation are illustrated in Fig. 1. During this experiment,
all rats survived until the sacrifice, and there was no evi-
dence of necrosis of the epidermal tissue. Throughout the
experiment, a little contraction, inflammation, and bleeding
were detected. On the third postoperative day, inflamma-
 100
tion was detected in all rats. However, severe symptoms,
including inflammation and hemorrhage were observed in
wounds treated with sterile gauzes only. Furthermore, a
moist surface was discovered in HWD-applied wounds,
whereas wounds in the control group showed a dry surface
and a scab. On the sixth postoperative day, the group
treated with only gauze had scabs on the surface, while no
blood clot was detected in groups treated with HWD and
BKC-loaded HWD. After 12 postoperative days, exposure
of the wounded area was still detected in the control group.
On the other hand, HWD and BKC-loaded HWD applied
groups were mostly healed and almost sealed. In addition,
HWD and BKC-loaded HWD accelerated the recovery of
the epidermis and reduced the size of the wounds without
the second damage on the surface faced with materials. In
particular, BKC-loaded HWD applied wounds showed
accelerated epithelialization and remarkable expansion of
keratinocytes compared with HWD-applied wounds.
As a result, HWD and BKC-loaded HWD-applied
groups definitely exhibited higher epithelializing rates than
123
330
Day 0 Day 3 Day 6
Fig. 1 Representative photographs of the macroscopic appearance of wounds treated with a gauze control, b HWD without BKC, and c BKC-
loaded HWD
Fig. 2 Epithelialization rates (%) of wounds treated with a gauze
control (filled circle), HWD without BKC (open square), and BKC-
loaded HWD (filled square). *p \ 0.05
the control group (Fig. 2). As shown in Fig. 2, HWD and
BKC-loaded HWD applied groups exhibited similar ten-
dencies. However, the rate of wound recovery after BKC-
loaded HWD treatment showed a more favorable result
(p \ 0.05). This may be because the BKC was rapidly
released from the HWD (Fig. 3), which helps the acceler-
ation of the wound healing process by deactivating bacte-
rial in the infected wounds. Obviously, BKC-loaded HWD
exhibited a two-stage drug release profiles of the faster
release at the initial period, accompanied by a decreased
123
J. H. Kim et al.
Day 9 Day 12 Day 15
Fig. 3 Drug release profile from the BKC-loaded hydrocolloid
wound dressing (filled square)
release rate with time. Furthermore, more than 60 % of the
BKC was released within 6 h. The initial fast release
probably results from the rapid swelling of HWD, which
increased the surface area of HWD for drug release. The
faster release of BKC would produce an antibacterial
action to protect against secondary infections. Thus, our
results indicated that the addition of BKC to HWD pro-
duced more significant improvements in wound healing
and higher re-epithelisation rates than the addition of the
gauze and HWD without BKC in the infected wound
healing process.
Evaluation of benzalkonium chloride-loaded hydrocolloid
Fig. 4 The representative histopathological profiles of infected skin
wounds (granulation tissues) of a–c a gauze control, d–f HWD without
BKC, and g–i BKC-loaded HWD applied rats. DE dermis; EP
regenerated epithelium; LP lamina propria; Squares means the enlarged
areas in right columns; a, b, d, e, g, and h: H&E stain; c, f, and i:
Masson’s trichrome stain, scale bars = 160 lm. Note that re-epitheli-
zation and reductions in desquamated epithelial regions were more
obvious in HWD and BKC-loaded HWD applied wounds as compared
Histopathological analysis
The histomorphometrical changes in the full thickness
wounds of rat dorsal back skins—desquamated epithelium
regions, re-epithelization, numbers of microvessels (neo-
vascularization), infiltrated inflammatory cells, and per-
centages of collagen-occupied regions in granulation
tissues, and granulation tissue areas are listed in Fig. 4, and
the representative histological profiles of study groups are
shown in Fig. 5.
Analysis of the histopathological and histomorphomet-
rical profiles revealed significant reductions in desqua-
mated epithelial regions in HWD and BKC-loaded HWD
331
with gauze control wounds. The inflammatory cell infiltrations and
neovascularization (arrows) in granulation tissues (b, e, h) in HWD and
BKC-loaded HWD treated groups were also less than that of gauze
control, and more numerous collagen proliferations (c, f, i; green colors)
were detected in both HWD and BKC-loaded HWD applied groups as
compared with gauze controls, respectively. These wound-healing signs
were more obvious in BKC-loaded HWD applied infected wounds than
in those of HWD without BKC. (Color figure online)
applied groups (p \ 0.01), in comparison with gauze con-
trols. Consequently, the re-epithelization rates were marked
and significantly increased in both HWD (47.86 ±
7.01 %) and BKC-loaded HWD applied wounds (63.74 ±
6.58 %, p \ 0.01). Furthermore, in both HWD and BKC-
loaded HWD-applied groups, the number of microvessels
(means neovascularization) and inflammatory cells infil-
trated in granulation tissues, and granulation tissue area
itself were also significantly reduced in comparison with
gauze controls (p \ 0.01), and significant increases in col-
lagen fibers were observed (Fig. 4). In the present study,
applications of HWD and BKC-loaded HWD on the infec-
ted full-thickness wounds facilitated the re-epithelization
123
332 J. H. Kim et al.

Fig. 5 Histomorphometrical
values: a desquamated
epithelium regions; b re-
epithelization rates; c number of
microvessels in granulation
tissues; d number of infiltrated
inflammatory cells in
granulation tissues;
e percentages of collagen
occupied regions in granulation
tissues; f granulation tissue
areas. Values are expressed as
mean ± SD of five wounds.
a
p \ 0.01 as compared with a
gauze control using the LSD
test. bp \ 0.01 as compared
with a gauze control using the
MW test. cp \ 0.01 and
d
p \ 0.05 as compared with
HWD using the LSD test.
e
p \ 0.01 as compared with
HWD using the MW test

and reconstruction of skin tissues as compared with gauze
controls.
In particular, histopathological observations showed that
significantly (p \ 0.01 or p \ 0.05) more rapid recon-
structions of granulation tissues, lower inflammatory cell
infiltrations, lower neovascularization, and more favorable
collagen fiber regenerations were demonstrated in BKC-
loaded HWD treated wounds in comparison to HWD
without BKC treated wounds. Obviously, these results
provide direct evidences that HWD with BKC has more
potential for the wound healing than HWD without BKC.
These results are consistent with those of previous wound
healing studies. Thus, these findings clearly suggested that
the newly developed BKC-loaded HWD could favorably
facilitate the re-epithelization and disappearance of gran-
ulation tissues or exchanges to normal skin tissues and
could be used as an effective therapeutic agent for treat-
ment of the infected wounds.

123
Evaluation of benzalkonium chloride-loaded hydrocolloid
Conclusion
Elastic and flexible HWDs with BKC significantly
enhanced wound recovery after infection by S. aureus.
Moreover, this type of dressing can effectively absorb the
exudate that comes from the wounded site and endure
mechanical stresses caused by swelling. Histopathological
studies reveal the beneficial effects of BKC-loaded HWD
on wound healing and the normalization of infected
wounds. Accordingly, the newly developed BKC-loaded
HWD is anticipated to have beneficial roles in the treat-
ment of infected wounds.
Acknowledgments This research was supported by the Basic Sci-
ence Research Program through the National Research Foundation of
Korea (NRF), funded by the Ministry of Education, Science and
Technology (No. 2010-0024185), and by a grant from the Korean
Health Technology R&D Project, Ministry for Health, Welfare and
Family Affairs, Republic of Korea (A092018).
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