Medical Honey and Silver Dressings - Key Attributes
Medical Honey and Silver Dressings - Key Attributes
In order to ascertain whether the addition of silver-containing dressings interferes with the primary sugars or osmotic strength of medical honey, 5 silver containing dressings (1 soluble glass powder [Arglaes Powder], 1 hydrogel [SilvaSorb Gel], 1 collagen [Puracol Plus Ag+], 1 foam [Optifoam Ag+ Non-Adhesive], and 1 alginate [Maxorb Extra Ag+]) were combined with a 100% medical honey gel dressing (TheraHoney Gel, Manuka Honey, no additives) and compared to the dressings or medical honey alone (control). All products were manufactured my Medline Industries, Inc, Mundelein, IL.
For these experiments, the honey was diluted (1/10 fold) to ensure reproducible amounts of honey were introduced to the test products (n = 6); to enable consistent mixing, thus ensuring maximum exposure of honey's sugars to the potential interacting substances; and for compatibility with the assays and equipment used. Test products were also incubated in phosphate buffered saline (PBS) (n = 5) to control for changes due to the PBS diluent.
Fructose and glucose were measured with a well-established mix-and-read assay (FA-20, Sigma Aldrich, St. Louis, MO). The assay has 2 components. The first is a dye that changes color in the presence of glucose; the second is an enzyme that turns fructose into glucose. Two separate assays were performed for each sample, 1 to measure glucose only, the other treated by the enzyme and then used to measure the glucose. The first value represents the total glucose, the second represents total glucose and fructose, and by subtracting the 2 values, the individual concentrations of fructose and glucose can be determined. Any chemical modifications to the fructose or glucose would be expected to preclude the ability to be quantified by this assay.
The authors used vapor pressure osmometry to determine any changes in gross osmolarity. A vapor pressure osmometer (OSMOMAT 070, Gonotec, Berlin, Germany) was calibrated with normal saline (308 mOsmol/kg). The osmometer was then used to measure the diluted honey stocks and the honey post-incubation with the silver-containing products.
The agar plate zone of inhibition assay was performed using standard techniques adapted from clinical microbiology testing for antibiotic sensitivity of clinical isolates. Medical-grade honey gel and 5 silver dressings were applied alone and combined to planktonic cultures of Pseudomonas aeruginosa (PA, strain PA01) or methicillin-resistant Staphylococcus aureus (MRSA, strain SA35556) grown as confluent lawns of planktonic bacterial on agar plates. Plates using cotton gauze dressing without honey or silver were tested as a negative control. Briefly, 300 mL of 1.5% tryptic soy agar (TSA) was autoclaved and allowed to cool to 45ºC-50ºC, then inoculated with 1 mL of a suspension culture of PA01 or SA35556 containing a total of 10 colony forming units (CFUs). The inoculated TSA agar was poured into 24 cm x 24 cm rectangular petri plates and allowed to gel at room temperature. The honey gel was applied as a thin continuous layer to 8 mm disks of oxidized regenerated cellulose/collagen dressing and the disks were placed onto the inoculated TSA agar plates with the honey gel in direct contact with the agar surface. Then, 8 mm disks or regions of application of the 5 silver dressings alone were also directly applied to surface of the inoculated TSA agar plates. To test the combination of honey gel and silver dressings, a thin, continuous layer of the honey gel was applied to 1 side of 8 mm disks of the silver dressings, or applied to the surface of the powder and gel dressings, then the silver disks were placed onto the surface of the inoculated TSA agar plates with the honey gel side in direct contact with agar. Fifty μL of sterile water was then applied to the top surface of each of the dressings and the TSA agar plates were placed in humidified incubators at 37ºC. After 24 hours of exposure, diameters of the zones of killing on the agar plates were measured visually using a millimeter ruler to the nearest 1 mm diameter distance. Triplicate replicates were performed for each test condition.
The authors also tested the combinations in a pig skin explant model, using minor modifications of the previously described procedure, to test the antimicrobial efficacy of these devices in a model more closely mimicking a true wound. Briefly, large sheets of fresh pig skin approximately 20 cm by 30 cm were obtained from a commercial meat processing company and thoroughly cleaned with hair closely trimmed. The subcutaneous fat layer was trimmed to leave approximately 1–2 mm thickness of subcutaneous fat. A large, partial-thickness wound approximately 0.508 mm deep was mechanically created using an electric Paget's dermatome. Individual explants 12 mm in diameter were punched from the wound area using 12 mm skin biopsy punch. The pig skin explants were sterilized using chlorine gas for 45 minutes then washed 3 times in sterile saline. Seven explants were transferred to 90 mm diameter petri dishes containing 0.5% soft TSA supplemented with antibiotics (50 μg gentamicin per ml for P. aeruginosa or 20 μg doxycycline per mL for S. aureus) to limit overgrowth of bacteria to the bottom of the explants. Sterile explants were inoculated with 50 μL of 107 CFUs of PA01or SA35556, and the explants were incubated for 12 hours at 37ºC. Explants were then transferred to fresh 90 mm petri dishes with 0.5% soft TSA plus antibiotics, again, to limit overgrowth of bacteria to the bottom of the explants. A thin, continuous layer of medical honey gel ~1 mm thick was then applied to the top surface of pig skin explants alone and covered with gauze or with 1 of the 5 silver dressings; 1.5 mL of sterile water was added to the surface of the top dressing. A sterile glass slide (25 mm x 75 mm x 1 mm) was placed on top of the gauze or silver dressings to reduce drying of the dressings and to enhance contact of the honey gel with the surface of the pig skin explants. After 24 hours of incubation, the honey gel and silver dressings were removed, explants were placed in 24 well culture plates, then washed 3 times for 5 minutes with sterile distilled water. Explants were transferred into 5 mL of PBS containing 5 ppm Tween 20, sonicated 5 times for 90 seconds with 1 minute between sonications. Suspensions were vortexed for 10 seconds, triplicate serial dilutions were plated, and CFUs were measured after 24 hours of incubation at 37ºC. Three to five replicate explants were performed for each test condition and the mean was calculated to represent the average CFUs remaining after each treatment condition.
No animals or humans were subjects to the experiments described herein. All of the work described was done in vitro.
Materials and Methods
In order to ascertain whether the addition of silver-containing dressings interferes with the primary sugars or osmotic strength of medical honey, 5 silver containing dressings (1 soluble glass powder [Arglaes Powder], 1 hydrogel [SilvaSorb Gel], 1 collagen [Puracol Plus Ag+], 1 foam [Optifoam Ag+ Non-Adhesive], and 1 alginate [Maxorb Extra Ag+]) were combined with a 100% medical honey gel dressing (TheraHoney Gel, Manuka Honey, no additives) and compared to the dressings or medical honey alone (control). All products were manufactured my Medline Industries, Inc, Mundelein, IL.
Biochemical Assessment of Honey Sugars
For these experiments, the honey was diluted (1/10 fold) to ensure reproducible amounts of honey were introduced to the test products (n = 6); to enable consistent mixing, thus ensuring maximum exposure of honey's sugars to the potential interacting substances; and for compatibility with the assays and equipment used. Test products were also incubated in phosphate buffered saline (PBS) (n = 5) to control for changes due to the PBS diluent.
Fructose and glucose were measured with a well-established mix-and-read assay (FA-20, Sigma Aldrich, St. Louis, MO). The assay has 2 components. The first is a dye that changes color in the presence of glucose; the second is an enzyme that turns fructose into glucose. Two separate assays were performed for each sample, 1 to measure glucose only, the other treated by the enzyme and then used to measure the glucose. The first value represents the total glucose, the second represents total glucose and fructose, and by subtracting the 2 values, the individual concentrations of fructose and glucose can be determined. Any chemical modifications to the fructose or glucose would be expected to preclude the ability to be quantified by this assay.
Assessment of Osmotic Strength
The authors used vapor pressure osmometry to determine any changes in gross osmolarity. A vapor pressure osmometer (OSMOMAT 070, Gonotec, Berlin, Germany) was calibrated with normal saline (308 mOsmol/kg). The osmometer was then used to measure the diluted honey stocks and the honey post-incubation with the silver-containing products.
Agar Plate Zone of Inhibition Assay
The agar plate zone of inhibition assay was performed using standard techniques adapted from clinical microbiology testing for antibiotic sensitivity of clinical isolates. Medical-grade honey gel and 5 silver dressings were applied alone and combined to planktonic cultures of Pseudomonas aeruginosa (PA, strain PA01) or methicillin-resistant Staphylococcus aureus (MRSA, strain SA35556) grown as confluent lawns of planktonic bacterial on agar plates. Plates using cotton gauze dressing without honey or silver were tested as a negative control. Briefly, 300 mL of 1.5% tryptic soy agar (TSA) was autoclaved and allowed to cool to 45ºC-50ºC, then inoculated with 1 mL of a suspension culture of PA01 or SA35556 containing a total of 10 colony forming units (CFUs). The inoculated TSA agar was poured into 24 cm x 24 cm rectangular petri plates and allowed to gel at room temperature. The honey gel was applied as a thin continuous layer to 8 mm disks of oxidized regenerated cellulose/collagen dressing and the disks were placed onto the inoculated TSA agar plates with the honey gel in direct contact with the agar surface. Then, 8 mm disks or regions of application of the 5 silver dressings alone were also directly applied to surface of the inoculated TSA agar plates. To test the combination of honey gel and silver dressings, a thin, continuous layer of the honey gel was applied to 1 side of 8 mm disks of the silver dressings, or applied to the surface of the powder and gel dressings, then the silver disks were placed onto the surface of the inoculated TSA agar plates with the honey gel side in direct contact with agar. Fifty μL of sterile water was then applied to the top surface of each of the dressings and the TSA agar plates were placed in humidified incubators at 37ºC. After 24 hours of exposure, diameters of the zones of killing on the agar plates were measured visually using a millimeter ruler to the nearest 1 mm diameter distance. Triplicate replicates were performed for each test condition.
Ex Vivo Pig Skin Explant Model
The authors also tested the combinations in a pig skin explant model, using minor modifications of the previously described procedure, to test the antimicrobial efficacy of these devices in a model more closely mimicking a true wound. Briefly, large sheets of fresh pig skin approximately 20 cm by 30 cm were obtained from a commercial meat processing company and thoroughly cleaned with hair closely trimmed. The subcutaneous fat layer was trimmed to leave approximately 1–2 mm thickness of subcutaneous fat. A large, partial-thickness wound approximately 0.508 mm deep was mechanically created using an electric Paget's dermatome. Individual explants 12 mm in diameter were punched from the wound area using 12 mm skin biopsy punch. The pig skin explants were sterilized using chlorine gas for 45 minutes then washed 3 times in sterile saline. Seven explants were transferred to 90 mm diameter petri dishes containing 0.5% soft TSA supplemented with antibiotics (50 μg gentamicin per ml for P. aeruginosa or 20 μg doxycycline per mL for S. aureus) to limit overgrowth of bacteria to the bottom of the explants. Sterile explants were inoculated with 50 μL of 107 CFUs of PA01or SA35556, and the explants were incubated for 12 hours at 37ºC. Explants were then transferred to fresh 90 mm petri dishes with 0.5% soft TSA plus antibiotics, again, to limit overgrowth of bacteria to the bottom of the explants. A thin, continuous layer of medical honey gel ~1 mm thick was then applied to the top surface of pig skin explants alone and covered with gauze or with 1 of the 5 silver dressings; 1.5 mL of sterile water was added to the surface of the top dressing. A sterile glass slide (25 mm x 75 mm x 1 mm) was placed on top of the gauze or silver dressings to reduce drying of the dressings and to enhance contact of the honey gel with the surface of the pig skin explants. After 24 hours of incubation, the honey gel and silver dressings were removed, explants were placed in 24 well culture plates, then washed 3 times for 5 minutes with sterile distilled water. Explants were transferred into 5 mL of PBS containing 5 ppm Tween 20, sonicated 5 times for 90 seconds with 1 minute between sonications. Suspensions were vortexed for 10 seconds, triplicate serial dilutions were plated, and CFUs were measured after 24 hours of incubation at 37ºC. Three to five replicate explants were performed for each test condition and the mean was calculated to represent the average CFUs remaining after each treatment condition.
Ethical Considerations
No animals or humans were subjects to the experiments described herein. All of the work described was done in vitro.
