ORIGINAL ARTICLE
Efficacy of Silver-Loaded Nanofiber Dressings in Candida albicans–Contaminated Full-Skin Thickness Rat Burn Wounds N. Sinem Ciloglu, MD,* A. Irem Mert, MD,* Zarife Doğan,† Ali Demir,† Simin Cevan,* Sebahat Aksaray, MD,*Mustafa Tercan, MD*
In this experimental study, the effects of nanofiber dressings containing different forms of silver on full-thickness rat burn contaminated with Candida albicans was analyzed. A full-thickness skin burn was formed on a total of 32 Sprague-Dawley rats. After the burn wound was seeded with a 108 colony-forming units/ml standard strain of Candida albicans ATCC90028, the animals were divided into four groups. The effects of topical silver sulfadiazine and two recently designed nanofiber dressings containing nanosilver and silversulfadiazine as active materials were compared with the control group. There was a significant difference in the Candida growth on the burn eschar tissue among the groups. The difference for Candida growth in the burn eschar between the control group and the 1% silver sulfadiazine–containing nanofiber dressing group was statistically significant (P< 0.01). Silver sulfadiazine–containing nanofiber dressing was the most effective agent in the treatment of Candida albicans–contaminated burn wounds. Because of their regenerative potential, silver-loaded nanofiber dressings could be a good alternative for infected burn wounds. (J Burn Care Res 2013;35:e317–e320)
Burns are one of the most serious forms of injury that can be further complicated by infection, with increase in mortality and morbidity.1,2 Infection-control procedures such as broad-spectrum topical and systemic antimicrobial agents along with patient isolation showed successful benefits in the management of bacterial infections. In addition, the incidence of invasive fungal burn wound infections has increased among burn patients, and fungal infections is one of the most common causes of increased mortality and morbidity.3 Candida is the most frequent cause of burn wound colonization.4 It causes persistent infections, delays wound healing, and may be a cause of increased mortality in case of systemic infection.4,5 The antimicrobial effect of silver was first reported by Moyer and colleagues.6 Thereafter, various forms
From the *Haydarpasa Numune Training and Research Hospital, Istanbul; and †Istanbul Technical University, Istanbul. Address correspondence to N. Sinem Ciloglu, Haydarpasa Numune Training and Research Hospital, Tıbbiye Cad. 34688, Uskudar Istanbul, Turkey. Copyright © 2014 by the American Burn Association 1559-047X/2014 DOI: 10.1097/BCR.0b013e3182aa7143
of silver were launched in the market. Silver displays no antimicrobial activity in its metallic form but in solution it ionizes and becomes active. Silver acts on the cell wall, causing protein alteration and inhibits transcription via binding DNA.7 Silver promotes powerful antibacterial and antifungal activity.8,9 The nanoparticle structure of silver provides expansive surface area for moisture contact thus allowing silver to solubilize and increase in antimicrobial properties.7 Nanofibers are used in the medical field as wound dressings and artificial vessels because of their high porosity, specific surface area, ability to mimic the extracellular matrix, effective use as a drug carrier, and ability to provide a convenient environment for cell proliferation and replication.10 Nanofibers also have positive impact on wound healing. They supply enough oxygen, enable water vapor permeation, and protect the wound from infection and dehydration. Also, the rate of epithelization is increased and dermis is organized with nanofibers. Therefore nanofibers provide good support for wound healing. Because of these properties, two different wound dressings have been designed. The wound dressings are nanofiber in structure, one contains nanosilver particles and the other, silver sulfadiazine as active material. e317
Journal of Burn Care & Research September/October 2014
e318 Ciloglu et al
Nanotechnology and the ability to deliver silver from a nanocrystalline structure markedly improve the biologic value of silver. There is a marked increase in surface area for water to react with silver in nanocrystalline form. When moistened with water, the nanocrystalline silver continues to release silver ions into the wound surface.11 It reveals potent antibacterial, antiviral and antifungal activity. The nanosilver exerts antifungal activity through disrupting the structure of the cell membrane and inhibiting the normal budding process as a result of destruction of membrane integrity.12 Nanofibers have high porosity and specific surface areas. They have the ability to mimic the extracellular matrix. If a convenient drug is integrated with nanofibers, it can be released into the healing wound in a homogenous and controlled manner.10 The nanofiber wound dressings that have been developed and used in this study are three layered (Figure 1). They have both an upper and lower policaprolactone protective layer and in between a polyethylene oxide layer containing the active material. Electrospinning has been used for the production of the nanofibers. The active material consists of 1% nanosilver in the dressing used in the third group and 1% silver sulfadiazine in the dressing used in the fourth group. Policaprolactone and polyethylene oxide were used as raw materials because of their biocompatibility and biodegradability. A study was designed to compare the antimicrobial efficiency of topical silver sulfadiazine 1%, and the two newly formed nanofiber wound dressings, containing nanosilver and silversulfadiazine as active materials, against Candida albicans–contaminated full-thickness burns in rats.
METHODS The study protocol was approved by Marmara University Animal Studies Ethical Committee before the study began. Thirty-two male Sprague-Dawley rats weighing between 200 to 230 g were used. A fullthickness burn protocol was administered.13
The animals were anesthetized through inhalation of ether. The weights of the animals were measured followed by the shaving of their backs. The burn was induced to more than 15% of the rats’ body surface via submersion in water at 100°C for 8 sec. Fullthickness burn was confirmed by histologic findings. The animals were resuscitated with subcutaneous injections of lactated Ringer’s solution (2 ml) and paracetamol was added to their water at a dose of 200 mg/kg per day for analgesia. Ten minutes after burn injury, the burned area in each rat was swabbed with 0.5-ml broth containing 108 colony-forming units (CFUs) of Candida albicans (ATCC90028). The rats were individually housed at ambient room temperature and provided adequate water and laboratory chow throughout the study. After 24 hr, the rats were randomly divided into four groups with each containing eight rats. Group 1 was the control group and no topical agent was applied. Group 2 was the topical silver sulfadiazine 1%–treated group, which was treated daily by using a sterile tongue blade. Group 3 was the 1% nanosilvercontaining nanofiber dressing treated group. Group 4 was the 1% silver sulfadiazine–containing nanofiber dressing treated group. The dressings in groups 3 and 4, which were applied every 2 days, were wetted with sterile serum physiologic and stabilized with sterile gauze and skin staples. On the 7th day, all animals were killed with a high dose of intraperitoneal ketamine injection. Their weights were recorded. A weight loss of more than 7.5% of their body weight was considered indicative of systemic illness. Biopsies were obtained from the center of the burn eschar and the parvertebral muscles beneath the burn eschar. Thoracotomies were performed, blood was drawn from the left ventricle, and lung biopsies were taken. Tissue weighing 0.25 g, 0.15 g, and 0.10 g was obtained from the lung, eschar, and the muscle, respectively. All the materials were performed by using a sterile technique and were prepared for culture in the clinical microbiology
Figure 1. The structure of the newly developed nanofiber wound dressing.
Journal of Burn Care & Research Volume 35, Number 5
Ciloglu et al e319
Table 1. The number of rats with Candida albicans colonization in each group n(%); the results of minimum, maximum, and median values of culture results in Cfu/g of tissue Control (n=8) Eschar
Muscle
Lung Blood
n (%) Min–Max Median n (%) Min–Max Median n (%) n (%)
8 (%100) 1 × 104 −1 × 106 1 × 104 4 (%50.0) 0 − 2 × 103 500 0 (%0) 0 (%0)
%1 Silver Sulfadiazine (n=8) 8 (%100) 1 × 103 − 1 × 107 5.5 × 105 3 (%37.5) 0−2 × 105 0 0 (%0) 0 (%0)
%1 Nanosilver Nanofiber (n=8)
(n%1 Silver Sulfadiazine Nanofiber =8)
8 (%100) 1 × 103 − 1 × 107 1 × 105 2 (%25.0) 0 −2 × 106 0 0 (%0) 0 (%0)
7 (%87.5) 0 −1 × 106 1 × 103 2 (%25.0) 0 −1 × 103 0 0 (%0) 0 (%0)
P (Kruskal–Wallis Test) 0.045*
0.599 — —
*P < 0.05.
laboratory. The tissue samples were weighed in a balance inside sterile petri dishes on a balance and placed in 2 ml of brain–heart infusion broth. The samples were diluted in saline and 0.1-ml portions were plated in Sabouraud dextrose agar and 5% sheep blood agar with a calibrated loop. A smear was prepared for each sample for Gram stain. The plates were incubated for 24 and 48 hr at 35°C, and colonies were counted. All colonies isolated were compared with Gram staining. The quantitative counts for each colony type were determined by using Miles and Misra formula. Pediatric culture bottles were used for blood cultures, which were incubated and monitored by BacT/ Alert 3D (BioMerieux, France) automated system. All bottles flagged positive by the instrument were removed and subcultered with 5% sheep blood agar and saboraud–dextrose agar and were incubated for 24 to 48 hr at 35°C in aerobic conditions. Candida albicans was diagnosed with a germ tube test. The results were compared statistically among groups. For the statistical evaluation NCSS (Number Cruncher Statistical System) 2007&PASS (Power Analysis and Sample Size) 2008 Statistical software programme (UT) was used. Besides the descriptive statistical methods (median minimum, maximum, frequency, and rate) Kruskal–Wallis test has been used for the comparison of the parameters that are not showing normal distribution among groups. Mann–Whitney U test has been used for the comparison of the group showing difference. The significance level was set at P
Efficacy of Silver-Loaded Nanofiber Dressings in Candida albicans–Contaminated Full-Skin Thickness Rat Burn Wounds N. Sinem Ciloglu, MD,* A. Irem Mert, MD,* Zarife Doğan,† Ali Demir,† Simin Cevan,* Sebahat Aksaray, MD,*Mustafa Tercan, MD*
In this experimental study, the effects of nanofiber dressings containing different forms of silver on full-thickness rat burn contaminated with Candida albicans was analyzed. A full-thickness skin burn was formed on a total of 32 Sprague-Dawley rats. After the burn wound was seeded with a 108 colony-forming units/ml standard strain of Candida albicans ATCC90028, the animals were divided into four groups. The effects of topical silver sulfadiazine and two recently designed nanofiber dressings containing nanosilver and silversulfadiazine as active materials were compared with the control group. There was a significant difference in the Candida growth on the burn eschar tissue among the groups. The difference for Candida growth in the burn eschar between the control group and the 1% silver sulfadiazine–containing nanofiber dressing group was statistically significant (P< 0.01). Silver sulfadiazine–containing nanofiber dressing was the most effective agent in the treatment of Candida albicans–contaminated burn wounds. Because of their regenerative potential, silver-loaded nanofiber dressings could be a good alternative for infected burn wounds. (J Burn Care Res 2013;35:e317–e320)
Burns are one of the most serious forms of injury that can be further complicated by infection, with increase in mortality and morbidity.1,2 Infection-control procedures such as broad-spectrum topical and systemic antimicrobial agents along with patient isolation showed successful benefits in the management of bacterial infections. In addition, the incidence of invasive fungal burn wound infections has increased among burn patients, and fungal infections is one of the most common causes of increased mortality and morbidity.3 Candida is the most frequent cause of burn wound colonization.4 It causes persistent infections, delays wound healing, and may be a cause of increased mortality in case of systemic infection.4,5 The antimicrobial effect of silver was first reported by Moyer and colleagues.6 Thereafter, various forms
From the *Haydarpasa Numune Training and Research Hospital, Istanbul; and †Istanbul Technical University, Istanbul. Address correspondence to N. Sinem Ciloglu, Haydarpasa Numune Training and Research Hospital, Tıbbiye Cad. 34688, Uskudar Istanbul, Turkey. Copyright © 2014 by the American Burn Association 1559-047X/2014 DOI: 10.1097/BCR.0b013e3182aa7143
of silver were launched in the market. Silver displays no antimicrobial activity in its metallic form but in solution it ionizes and becomes active. Silver acts on the cell wall, causing protein alteration and inhibits transcription via binding DNA.7 Silver promotes powerful antibacterial and antifungal activity.8,9 The nanoparticle structure of silver provides expansive surface area for moisture contact thus allowing silver to solubilize and increase in antimicrobial properties.7 Nanofibers are used in the medical field as wound dressings and artificial vessels because of their high porosity, specific surface area, ability to mimic the extracellular matrix, effective use as a drug carrier, and ability to provide a convenient environment for cell proliferation and replication.10 Nanofibers also have positive impact on wound healing. They supply enough oxygen, enable water vapor permeation, and protect the wound from infection and dehydration. Also, the rate of epithelization is increased and dermis is organized with nanofibers. Therefore nanofibers provide good support for wound healing. Because of these properties, two different wound dressings have been designed. The wound dressings are nanofiber in structure, one contains nanosilver particles and the other, silver sulfadiazine as active material. e317
Journal of Burn Care & Research September/October 2014
e318 Ciloglu et al
Nanotechnology and the ability to deliver silver from a nanocrystalline structure markedly improve the biologic value of silver. There is a marked increase in surface area for water to react with silver in nanocrystalline form. When moistened with water, the nanocrystalline silver continues to release silver ions into the wound surface.11 It reveals potent antibacterial, antiviral and antifungal activity. The nanosilver exerts antifungal activity through disrupting the structure of the cell membrane and inhibiting the normal budding process as a result of destruction of membrane integrity.12 Nanofibers have high porosity and specific surface areas. They have the ability to mimic the extracellular matrix. If a convenient drug is integrated with nanofibers, it can be released into the healing wound in a homogenous and controlled manner.10 The nanofiber wound dressings that have been developed and used in this study are three layered (Figure 1). They have both an upper and lower policaprolactone protective layer and in between a polyethylene oxide layer containing the active material. Electrospinning has been used for the production of the nanofibers. The active material consists of 1% nanosilver in the dressing used in the third group and 1% silver sulfadiazine in the dressing used in the fourth group. Policaprolactone and polyethylene oxide were used as raw materials because of their biocompatibility and biodegradability. A study was designed to compare the antimicrobial efficiency of topical silver sulfadiazine 1%, and the two newly formed nanofiber wound dressings, containing nanosilver and silversulfadiazine as active materials, against Candida albicans–contaminated full-thickness burns in rats.
METHODS The study protocol was approved by Marmara University Animal Studies Ethical Committee before the study began. Thirty-two male Sprague-Dawley rats weighing between 200 to 230 g were used. A fullthickness burn protocol was administered.13
The animals were anesthetized through inhalation of ether. The weights of the animals were measured followed by the shaving of their backs. The burn was induced to more than 15% of the rats’ body surface via submersion in water at 100°C for 8 sec. Fullthickness burn was confirmed by histologic findings. The animals were resuscitated with subcutaneous injections of lactated Ringer’s solution (2 ml) and paracetamol was added to their water at a dose of 200 mg/kg per day for analgesia. Ten minutes after burn injury, the burned area in each rat was swabbed with 0.5-ml broth containing 108 colony-forming units (CFUs) of Candida albicans (ATCC90028). The rats were individually housed at ambient room temperature and provided adequate water and laboratory chow throughout the study. After 24 hr, the rats were randomly divided into four groups with each containing eight rats. Group 1 was the control group and no topical agent was applied. Group 2 was the topical silver sulfadiazine 1%–treated group, which was treated daily by using a sterile tongue blade. Group 3 was the 1% nanosilvercontaining nanofiber dressing treated group. Group 4 was the 1% silver sulfadiazine–containing nanofiber dressing treated group. The dressings in groups 3 and 4, which were applied every 2 days, were wetted with sterile serum physiologic and stabilized with sterile gauze and skin staples. On the 7th day, all animals were killed with a high dose of intraperitoneal ketamine injection. Their weights were recorded. A weight loss of more than 7.5% of their body weight was considered indicative of systemic illness. Biopsies were obtained from the center of the burn eschar and the parvertebral muscles beneath the burn eschar. Thoracotomies were performed, blood was drawn from the left ventricle, and lung biopsies were taken. Tissue weighing 0.25 g, 0.15 g, and 0.10 g was obtained from the lung, eschar, and the muscle, respectively. All the materials were performed by using a sterile technique and were prepared for culture in the clinical microbiology
Figure 1. The structure of the newly developed nanofiber wound dressing.
Journal of Burn Care & Research Volume 35, Number 5
Ciloglu et al e319
Table 1. The number of rats with Candida albicans colonization in each group n(%); the results of minimum, maximum, and median values of culture results in Cfu/g of tissue Control (n=8) Eschar
Muscle
Lung Blood
n (%) Min–Max Median n (%) Min–Max Median n (%) n (%)
8 (%100) 1 × 104 −1 × 106 1 × 104 4 (%50.0) 0 − 2 × 103 500 0 (%0) 0 (%0)
%1 Silver Sulfadiazine (n=8) 8 (%100) 1 × 103 − 1 × 107 5.5 × 105 3 (%37.5) 0−2 × 105 0 0 (%0) 0 (%0)
%1 Nanosilver Nanofiber (n=8)
(n%1 Silver Sulfadiazine Nanofiber =8)
8 (%100) 1 × 103 − 1 × 107 1 × 105 2 (%25.0) 0 −2 × 106 0 0 (%0) 0 (%0)
7 (%87.5) 0 −1 × 106 1 × 103 2 (%25.0) 0 −1 × 103 0 0 (%0) 0 (%0)
P (Kruskal–Wallis Test) 0.045*
0.599 — —
*P < 0.05.
laboratory. The tissue samples were weighed in a balance inside sterile petri dishes on a balance and placed in 2 ml of brain–heart infusion broth. The samples were diluted in saline and 0.1-ml portions were plated in Sabouraud dextrose agar and 5% sheep blood agar with a calibrated loop. A smear was prepared for each sample for Gram stain. The plates were incubated for 24 and 48 hr at 35°C, and colonies were counted. All colonies isolated were compared with Gram staining. The quantitative counts for each colony type were determined by using Miles and Misra formula. Pediatric culture bottles were used for blood cultures, which were incubated and monitored by BacT/ Alert 3D (BioMerieux, France) automated system. All bottles flagged positive by the instrument were removed and subcultered with 5% sheep blood agar and saboraud–dextrose agar and were incubated for 24 to 48 hr at 35°C in aerobic conditions. Candida albicans was diagnosed with a germ tube test. The results were compared statistically among groups. For the statistical evaluation NCSS (Number Cruncher Statistical System) 2007&PASS (Power Analysis and Sample Size) 2008 Statistical software programme (UT) was used. Besides the descriptive statistical methods (median minimum, maximum, frequency, and rate) Kruskal–Wallis test has been used for the comparison of the parameters that are not showing normal distribution among groups. Mann–Whitney U test has been used for the comparison of the group showing difference. The significance level was set at P
