Use of Mouse Footpad Model to Test Effectiveness of Wound Dressings

Introduction Acemannan is a complex carbohydrate isolated from the clear gel in the center of the aloe vera leaf, which consists of polymerized beta-(1,4)-linked acetylmannose. Molecular weight after purification varies from 10-1000kDa with an average of 200kDa. The precise size depends on the degree of degradation during the manufacturing process. Acemannan does not appear to be mitogenic for monocytes, macrophages, or fibroblasts and is noncytotoxic even at high concentrations.[1–3] Many wound dressings, such as alginates and hydrocolloids, contain various forms of carbohydrates.[4] Differences in carbohydrate chemical structure can lend different properties and functions to wound dressings, such as improvement of absorption, increased binding, or improved retention of moisture. At a cellular level, some carbohydrates appear to stimulate cell functions of proliferation, migration, and cytokine production.[5–10] Early reports in the literature indicate carbohydrates can enhance wound repair by promoting early mobilization of macrophages to a wounded area.[11] Periwound injections of macrophages activated by the carbohydrate glucan caused an increase in fibroblast proliferation, fibrogenesis, collagen synthesis, epithelialization, and an increase in the tensile strength of the wound.[11] This was presumed to be due to the effect of glucan-induced interleukin-1 (IL-1), and tumor necrosis factor alpha (TNF-apha) on the wound fibroblasts.[11,12] Topical application of glucans to wounds seems to result in more rapid angiogenesis and reepithelialization compared to controls.[12] Other carbohydrate extracts have been shown to accelerate healing of open wounds and burns.[13,14] These extracts stimulate oxygen consumption, increase angiogenesis, and increase collagen synthesis in the wounds.[13] Because aloe vera sap and acemannan gel have been reported to enhance wound healing, the following experiments were designed to determine if acemannan could enhance healing and to investigate possible mechanisms of the activity of acemannan. The wound model used in this study was adopted and modified from the methods developed previously by others.[11,15] The method developed by these authors has the twin merits of simplicity and speed. The reader chooses which wound of a pair has healed (if any) as well as gives each wound a subjective score. When read blind, the presence of a control on the opposite foot provides an additional measure of wound healing by providing a basis for comparison as well as a control for any bias in the reader’s scoring. This model is simple, yet quick and reliable. The reproducibility and reliability come from the use of blinds and multiple controls. The basic experimental unit consisted of two groups, each of 30 mice. One group of mice in each experiment had controls in both feet, while the other group received control in one foot and treatment in the opposite foot. All experiments were performed blind. Materials and Methods Mice. Male Swiss-Webster mice weighing approximately 20g were obtained from Harlan Sprague Dawley, Inc. (Indianapolis, Indiana). Groups of 30 mice were wounded on the posterior tarsus of both feet after being anesthetized with 0.6mL of a mixture of sodium pentothal, propylene glycol, 90-percent ethanol, and sterile distilled water administered intraperitoneally. A 5mm incision was made through the thickness of the skin between the Achilles tendon and the external vestigial finger parallel to the posterior tarsal vein. Immediately after making the incision, one wound was treated with one drop of test material. A 2mm x 4mm piece of sterile parafilm (Parafilm™, American National Can, Chicago, Illinois) was inserted into the wounds to ensure even distribution of the solutions and to prevent rapid adhesion of the wound edges. The parafilm fell out within 24 hours without further intervention. All experiments consisted of one treatment group and one control group of 30 mice. The wounds were examined daily and measured by a subjective scoring system on a scale of 5 to 0. The basis of each score is as follows: 5 is open and totally unhealed; 4 is a small slit; 3 is a closed wound with a scab; 2 is a closed wound with scab shed; 1 is a healed wound with a visible scar; 0 is a healed wound and no visible scar. Wounds with represented scores are shown in Figure 1. Comparison of both feet of each animal was made at the same time. All wounds were made by the same individual and read by a different investigator. The wounds were read blind; therefore, the reader was unaware of treatment applied, which wound was the control, or if both wounded feet were treated with control material. Treatments. See Table 1 for a complete summary of test articles and concentrations used in the experiments. Solutions. The control solution was sterile 0.9-percent saline. The test solution was made from freeze-dried acemannan from lot numbers PD1COO2 and PD1COO3 (Carrington Laboratories Inc., Irving, Texas). Acemannan was provided as a freeze-dried pellet that was reconstituted with sterile saline to 1µg/mL. All acemannan dilutions were made from this stock solution. Sodium hydroxide was used to hydrolyze and deactivate acemannan. The acemannan was hydrolyzed and column purified by chemists. No visible toxic effects from the hydrolyzed acemannan were seen in cellular bioassays indicating little if any sodium hydroxide residue was present in the preparation. Hydrolyzed acemannan was reconstituted to 200 and 20µg/mL using sterile saline. Mannose, 99-percent pure (Aldrich, Milwaukee, Wisconsin), was diluted to 200 and 20µg/mL using sterile saline. Gels. A gel containing 0.1-percent acemannan in polyacrylic acid (Carbopol, Merck, West Point, Pennsylvania) was used. Polyacrylic acid gel without acemannan was used as a control and referred to as excipient. Injectables. One milliliter of saline or acemannan solution was injected into the peritoneal cavity of mice immediately after tarsal wounding. All injectable solutions were made from a single freshly constituted stock solution of acemannan Statistical analysis. All scores from these experiments had the variance and standard deviation calculated. Analysis of variance (ANOVA) was used to determine if the scores of treated and control wounds were significantly different. Statistical analysis was performed using Analyse-It for Microsoft Excel, version 1.62 (Analyse-It Software, Ltd., Leeds, England, UK). Cytokine assays. Harvesting wound macrophages can be difficult. Since peritoneal macrophages behave similarly to macrophages found in wounds, these cells were used to determine if specific carbohydrates could induce proinflammatory cytokine production. Naïve mice were killed and 3mL of RPMI 1640 supplemented with 50U/mL penicillin and 50µg/mL streptomycin (P/S) was injected into the peritoneal cavity and the abdominal region was massaged. The solution was removed and the peritoneal cavity was washed twice more with 3mL of RPMI. These washes were pooled with the original wash. The cells were centrifuged at 1000rpm for 20 minutes and the pellet was resuspended in 2mL of RPMI 1640 supplemented with 10-percent FBS and P/S. The cells were counted using a hemocytometer. The cell suspension was diluted in RPMI 1640 supplemented with 10-percent FBS and P/S to give a final concentration of 5 x 106 macrophages mL-1. A 12-well tissue culture plate was used for each cell concentration, and the cells were incubated at 37 degrees C in 5-percent CO2 for four hours. At the end of that time, the media was removed and replaced by RPMI 1640 without FBS containing 0.5, 10, 50, 100, or 200µg/mL acemannan or 0.5, 10, 20, 50, 100, or 200µg/mL hydrolyzed (deactivated) acemannan or 200µg/mL mannose. The cells were incubated for 24 hours; the media from each well was harvested and centrifuged at 2000rpm for 20 minutes; and the supernatants were frozen at –70 degrees C. The supernatant fluid was analyzed for the presence of TNF-alpha, IL-1beta, and IL-6 by ELISA assays (Genzyme, Cambridge, Massachusetts). Each fluid sample was assayed in duplicate. All test kits were used following the manufacturer’s recommendations with appropriate controls. Histology. Samples for histological examination were taken on alternate days after wounding. Two animals from each group were used resulting in eight specimens. Tissue was fixed within five minutes of death in 10-percent buffered neutral formalin (pH7). The tissue was decalcified with equal portions of A) 50g sodium citrate in 250mL distilled water and B) 125mL of 90-percent formic acid and 125mL distilled water for two to three weeks. Serial paraffin sections were stained with hematoxylin and eosin (H&E) for general differences and with Masson’s trichrome connective tissue stain to distinguish collagen and muscle tissue.[16] Twenty-five slides per specimen per day post-wounding were evaluated. Slides were randomized and read by a blinded evaluator. The evaluator used a descriptive analysis for each slide. In addition to general comments, the descriptive analysis utilized a 0 to 3 plus scale to consistently describe the epithelium migration and thickness, granulation tissue composition, and collagen deposition and thickness in order to objectively determine what, if any, descriptive differences were recorded as opposed to determining statistical differences. Results Control wounds. Throughout the study, the control group receiving controls in both feet consistently had the right foot score slightly better (1 score better) than the left foot. Because this occurred in every control group, it is assumed, therefore, the handedness bias of the same surgeon occurred in the treated groups of mice and the score difference was adjusted accordingly (no less than 0.83 and no more than 0.93 to each left foot score depending on control group differences) per discussions with a statistician. Response of wounds to aqueous acemannan. Three different doses of aqueous acemannan (5, 20, or 100µg/mL) or sterile saline were applied once to the wound at the time of injury. Wounds were read and scored daily for seven days. In all mice that received saline in one wound and acemannan solution in the other, the saline control wounds consistently healed more slowly. Wounds treated with 20µg/mL acemannan solution had better healing scores indicating the wounds healed rapidly compared to controls (Figure 2). The wound scores for the 20µg/mL solution were statistically significant for all seven days (p Response of wounds to acemannan gel. To determine if acemannan delivered in a gel enhanced wound healing compared to excipient control gel, groups of 30 mice were wounded as described above, and control wounds were given excipient gel. Thus, one group of 30 mice received excipient gel in both wounds, while the other group received excipient gel in one foot and 0.1-percent acemannan gel in the other. Wounds were read and scored daily for eight days. Wounds treated with acemannan gel consistently had lower scores than excipient gel wounds throughout the experiment (Figure 4). The wounds in the acemannan gel-treated group had statistically significantly better scores on days two through seven (p = 0.0024, p = 0.0012, p = 0.0119, p = 0.0003, p = 0.0003, p = 0.0151, respectively). By day eight, both types of wounds were essentially completely healed, and differences between them were insignificant. Systemic effect. To determine if acemannan exerted a systemic effect on the rate of tarsal wound healing, a slightly different form of experiment was devised. Wounded mice were injected intraperitoneally with 1mL of saline or with one of three doses of acemannan (25, 50, or 100µg/mL) immediately after wounding. Wounds were made on both feet and scored daily as described above. No statistical differences in the scores of wounds of saline controls or acemannan-treated mice were seen irrespective of the dose of acemannan administered. Other carbohydrate solutions. To determine if other carbohydrates could have an effect on healing, 20µg/mL solutions of mannose and hydrolyzed (deactivated) acemannan were used in the animal model. Neither 20µg/mL solution of mannose (Figure 5) or hydrolyzed acemannan (Figure 6) had an effect on wound healing scores. Both figures illustrate the reproducibility of the wound model and the scoring system. Cytokine release assays. Mouse peritoneal macrophages showed a dose-dependent response to acemannan in vitro (Figure 7). TNFa was released in greatest quantity with 1900pg/mL released following exposure to 200µg/mL acemannan. IL-1 was also released in a dose-dependent fashion with 569pg/mL being released after being exposed to 200µg/mL acemannan. IL-6 was released in an inverse response with 420pg/mL released after being exposed to 100µg/mL and 220pg/mL. released after being exposed to 200µg/mL acemannan. Macrophages exposed to 200µg/mL hydrolyzed acemannan did not release a detectable amount of TNF-alpha. Macrophages exposed to 200µg/mL mannose released 276pg/mL TNF-alpha. Discussion The results obtained from these experiments suggest that acemannan applied topically as a solution or as a gel enhanced wound healing in linear incisions in the tarsi of mice as depicted in the low healing scores. All acemannan-treated groups had lower scores, which represent faster wound closure than saline controls. Changing the chemical structure of acemannan rendered the wound healing effects of the molecule ineffective. Histological examinations correlated well with the healing scores. Histological samples indicated the acemannan-treated wounds appeared to reepithelialize and deposit collagen approximately two days sooner than saline control wounds. Wounds treated with acemannan had thicker collagen fibrils than saline control wounds, and it could be speculated that collagen I may be replacing collagen III earlier in these wounds. Experiments are under way to determine if this is the case. The collagen deposition in acemannan-treated wounds appeared to be more evenly distributed than the saline control wounds. Complex carbohydrates are believed to cause macrophage activation because they act as foreign bodies within the cell.[17] Activated macrophages release cytokines that may promote wound repair. Glucan-treated mouse serum has been shown to contain elevated colony-stimulating activity and macrophage colony-stimulating activity levels.[18] In addition, supernatant fluids from glucan, activated macrophage cultures, or topical glucan appears to increase the breaking strength of mouse wounds.[12,19] Acemannan is rapidly phagocytosed by mouse macrophages.[1,20] Although acemannan appears to be soluble and forms a clear solution when reconstituted, it is probable that small particles remain in suspension and may be readily ingested by macrophages. For example, acemannan labeled with 14C and injected intravenously or intraperitoneally was deposited in the liver and spleen of dogs within 48 hours.[21] Thus, the distribution of acemannan was consistent with removal of foreign particles by the mononuclear phagocytic system, since these organs are known to play major roles in host defenses by removing particulate antigen from the blood. The presence of acemannan may activate the macrophage foreign body response and, in turn, enhance wound healing. Alternatively, molecular aspects of acemannan may possibly bind to mannose receptors on the macrophage cell surface and subsequently trigger a signal that activates the macrophage similar to Pneumocystis cranii uptake.[22] The presence of the activated macrophages and the cytokines released may enhance the wound healing. In aged animals, macro-phage function is known to be a limiting factor in wound healing and the replacement of impaired macrophages improved wound healing.[23] Injection of acemannan into periwound tissue has been shown to reduce time to healing in aged animals.[24] These findings support the theory that acemannan interacts with macrophages within the wound, but these findings do not elucidate the mechanism of the interaction. Acemannan enhances both macrophage phagocytic activity and nonspecific cytotoxicity as well as the expression of MHC Class II antigens.[1] It is possible that this increased expression of MHC Class II molecules may enhance antigen presentation in acemannan-treated animals.[25] From the results shown here, it is now apparent that acemannan, once within macrophages, causes their activation and promotes the synthesis of TNF-alpha, IL-1, and IL-6.[1,9,10,26,27] All three of these cytokines have been shown to be involved in the wound healing process.[28–30] Indeed, creams that contain IL-1 have been shown to accelerate wound healing.[30] Systemic acemannan in the concentrations tested did not show an affect on healing, but acemannan applied topically does. The authors conclude acemannan must have been acting locally in the wound. Because acemannan and other carbohydrates can stimulate cytokine production by the macrophage, the authors hypothesize the increased synthesis of TNF-alpha, IL-1, and IL-6 in acemannan-treated wounds resulted in lower healing scores. Carbohydrates, like mannose and hydrolyzed acemannan, when tested at similar concentrations, did not stimulate cytokine production or have an effect on healing scores as acemannan did. This would indicate acemannan does not bind to the mannose receptor on the macrophage causing activation as previously thought. Further studies are underway to elucidate the mechanism of action for acemannan. Acknowledgments The authors would like to thank Bryan Maxwell and Yawei Ni for technical support. The gel containing 0.1-percent acemannan in polyacrylic acid was provided by Carrington Laboratories, Inc., Irving, Texas.