Wound healing evaluation of benzalkonium chloride-loaded hydrocolloid in the wound infection model

Journal of Pharmaceutical Investigation (2012) 42:327–333 DOI 10.1007/s40005-012-0043-2 RESEARCH ARTICLE Wound healing evaluation of benzalkonium ch...
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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, compared with gauze control or HWD without BKC. Furthermore, the significant decrease in desquamated epithelial

regions, numbers of microvessels and inflammatory cells infiltrated in granulation tissues and granulation tissue areas were observed in the histopathological and histomorphometrical profiles of BKC-loaded HWD. In conclusion, 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.

Jeong Hwan Kim and Sung Giu Jin contributed equally in this manuscript.

Introduction

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: [email protected]

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 overlapping 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 restoration 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 several 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

J. O. Kim e-mail: [email protected] 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: [email protected]

Keywords Benzalkonium chloride  Hydrocolloid  Wound dressing  Wound healing

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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 benzalkonium chloride (BKC). BKC is a widely used disinfectant 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 containing 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 medium 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

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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 consisted 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 (BKCloaded 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 epithelializing rate by using Adobe Acrobat 9 ProfessionalÒ. The epithelializing rate was calculated as follows: Epithelializing rate ð%Þ ¼

Et  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 collagen fibers (Sung et al. 2010). Subsequently, the histological 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 occupied 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 digital 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).

Re-epithelization ð%Þ ¼

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 macroscopic images of the wounds treated with a sterile gauze, HWD and BKC-loaded HWD at various days of postoperation are illustrated in Fig. 1. During this experiment, all rats survived until the sacrifice, and there was no evidence of necrosis of the epidermal tissue. Throughout the experiment, a little contraction, inflammation, and bleeding were detected. On the third postoperative day, inflamma-

Totallengthofwound  Desquamatedepitheliumregion ðmmÞ  100 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 different. When significant deviations from variance homogeneity were observed with the Levene test, a nonparametric 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:

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

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Day 0

Day 3

Day 6

Day 9

Day 12

Day 15

A

B

C

Fig. 1 Representative photographs of the macroscopic appearance of wounds treated with a gauze control, b HWD without BKC, and c BKCloaded HWD

Fig. 2 Epithelialization rates (%) of wounds treated with a gauze control (filled circle), HWD without BKC (open square), and BKCloaded 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 tendencies. However, the rate of wound recovery after BKCloaded 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 acceleration of the wound healing process by deactivating bacterial 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

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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 produced 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.

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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-epithelization and reductions in desquamated epithelial regions were more obvious in HWD and BKC-loaded HWD applied wounds as compared

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)

Histopathological analysis

applied groups (p \ 0.01), in comparison with gauze controls. 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 BKCloaded HWD-applied groups, the number of microvessels (means neovascularization) and inflammatory cells infiltrated in granulation tissues, and granulation tissue area itself were also significantly reduced in comparison with gauze controls (p \ 0.01), and significant increases in collagen fibers were observed (Fig. 4). In the present study, applications of HWD and BKC-loaded HWD on the infected full-thickness wounds facilitated the re-epithelization

The histomorphometrical changes in the full thickness wounds of rat dorsal back skins—desquamated epithelium regions, re-epithelization, numbers of microvessels (neovascularization), infiltrated inflammatory cells, and percentages 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 histomorphometrical profiles revealed significant reductions in desquamated epithelial regions in HWD and BKC-loaded HWD

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Fig. 5 Histomorphometrical values: a desquamated epithelium regions; b reepithelization 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 reconstructions of granulation tissues, lower inflammatory cell infiltrations, lower neovascularization, and more favorable collagen fiber regenerations were demonstrated in BKCloaded HWD treated wounds in comparison to HWD without BKC treated wounds. Obviously, these results

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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 granulation tissues or exchanges to normal skin tissues and could be used as an effective therapeutic agent for treatment of the infected wounds.

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 treatment of infected wounds. Acknowledgments This research was supported by the Basic Science 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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