Materials
The four test gums were purchased from Merck Pvt. Ltd., Mumbai, India. Roswell Park Memorial Institute Medium (RPMI)—1640 medium, Histopaque®-1077 and Thiazolyl-blue-tetrazolium-bromide (MTT), 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) were obtained from Sigma-Aldrich, St. Louis, USA. Surgispon®, used as comparative positive control dressing, was purchased from local market. All other chemicals were of analytical grade.
Ethics statement
All animal procedures and care protocols were followed as per the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) regulations and the experimental protocol was approved by the Institutional Animal Ethics Committee vide approval no. INM/IAEC/2018/23.
Experimental animals
Sprague Dawley (SD) male rats weighing 200-250g were obtained from Experimental Animal Facility of the institute. All animals were housed in standard environmental conditions of temperature (21-24 °C), humidity (45-50%), and a 12 h light and 12 h dark cycle in polypropylene cages and were provided with standard feed (Golden Feed Ltd., Delhi, India) and water ad libitum.
In vitro studies
Determination of whole blood clotting time
In vitro blood clotting time was measured using the Lee and White method [10]. One milliliter of anticoagulated fresh whole blood was collected in glass tubes from the retro-orbital plexus of healthy Sprague Dawley rats and treated with varying concentrations of each of the four test gums (range, 7-35 mg/ml for GT; 1-4 mg/ml for XG; 1-8 mg/ml for GG; and 1-20 mg/ml for GA) in combination with a fixed amount of optimized calcium gluconate (10mg/ml) for determining concentration of gum required for clotting of blood in least time. Tube contained blood without any anticoagulant taken as control. The tubes were tilted at 45° angle every 30 s to check for blood clot formation and the time taken for clotting was noted. The experiment was repeated five times for each of the test material.
Erythrocyte hemagglutination assay
Erythrocyte hemagglutination assay was carried out by the method reported by Pogorielov et al. [11]. Five milliliters of blood from the retro-orbital plexus of healthy Sprague Dawley rats was pooled in 10% potassium ethylene diamine tertaacetic acid (EDTA)-coated tubes. The tubes were centrifuged to separate erythrocytes. Separated erythrocytes were washed 3 times with phosphate-buffered saline (PBS, pH 7.4) to remove any leucocytes, plasma, platelets, and other cell debris. One milliliter of erythrocytes mass was then suspended in 19 ml PBS to make 5% erythrocyte dilution. Fifty microliters of this dilution was transferred to “U” bottom 96-well microplates and treated with sample gum solutions in a ratio of 1:1. The sample solutions contained varying concentration of each of the four test gum; viz., 0.5, 0.1, 0.01, and 0.001%, while control wells contained only PBS. The microplates were incubated at 37 °C for 1 h and visual and photographic evaluation was done. The experiments were performed in triplicates.
In vitro cytotoxicity study
Any potential cytotoxicity of the four biopolymeric gums was evaluated in isolated human lymphocytes using the standard 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay [12]. Lymphocytes were isolated from whole blood by density gradient centrifugation method using Histopaque®-1077 and cultured in RPMI 1640 media containing 10% fetal bovine serum, 100 unit/ml penicillin, 100 μg/ml streptomycin and buffered with HEPES. The cells were seeded into 96-well plates at a density of 1×104 per well and incubated for 24 h at 37 °C in a humidified atmosphere containing 5% carbon dioxide. After incubation, the cells were treated with autoclaved solution of the four test gums separately in varying concentrations (0.5, 0.1, 0.01, and 0.001%) for different time intervals, i.e., 1 h, 4 h, 16 h, and 24 h. The samples were treated with 10 μl of 5% MTT solution in PBS at the respective time intervals and incubated for 4 h at 37 °C for the formazan crystals to be formed. After incubation, the precipitated formazan crystals were dissolved in dimethyl sulfoxide (DMSO) and again incubated for 20 min for the colored product to be formed. The readings were recorded using enzyme-linked immunesorbent assay (ELISA) plate reader at a wavelength of 570 nm and percent cell viability was calculated for all the samples.
In vivo experiment: evaluation of hemostatic potential in Sprague Dawley rats
In vivo hemostatic potential of the four gums, GT, GG, GA, and XG was evaluated in Sprague Dawley (SD) rats using tail bleeding model as reported earlier by Sogut et al. [13]. Sixty healthy SD rats (200-250g) were randomly selected by using simple randomization method and assigned in ten groups of 6 rats each as mentioned below. The assessment was carried out in a non-blinded manner.
Group I: Treated with plain sponge without any coating; negative control
Group II: Treated with Surgispon®, a marketed hemostatic sponge; positive control
Group III: Treated with gum tragacanth (420 mg)-CG coated sponge
Group IV: Treated with gum tragacanth (420 mg)-CG in powder form
Group V: Treated with xanthan gum (36mg)-CG coated sponge
Group VI: Treated with xanthan gum (36mg)-CG in powder form
Group VII: Treated with guar gum (96 mg)-CG coated sponge
Group VIII: Treated with guar gum (96 mg)-CG in powder form
Group IX: Treated with gum acacia (60 mg)-CG coated sponge
Group X: Treated with gum acacia (60 mg)-CG in powder form
For preparing gum-coated sponge dressings, commercially available sponge material was taken and cut in circular shapes of 4 cm diameter. Each test gum in powder form was individually mixed with 120 mg of calcium gluconate (CG) using a mortar and pestle. The optimized amount of each gum and CG was calculated from the in vitro blood clotting experiment results described in earlier section. Water (q.s.) was added to these mixtures to make them into a thick paste. Using dip coating method, each test gum was individually coated onto different circular sponges. After coating, all sponge dressings were dried in a tray dryer at 37 °C for 2 h. Finally, gum-coated sponge dressings as well as each gum in simple powder form were evaluated for its hemostatic potential in subsequent rat tail bleeding experiments.
Animals were anesthetized by intraperitoneal injection of ketamine (80 mg/kg) and xylazine (10 mg/kg) [14]. Tails of all rats were disinfected using 98% alcohol. Four centimeters length of tail from the tail tip was amputated longitudinally with the help of a surgical blade. The bleeding tails were immediately covered with respective gum-coated sponge dressing/gum powder. The test material was initially applied along with direct pressure for 30 s. Pressure was then withdrawn and the wound was observed for any reduction in bleeding. Following parameters were studied for the evaluation of hemostatic potential as per previously published literature [15]:
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a)
Adherence strength of the dressing(s)
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b)
Time for complete hemostasis
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c)
Amount of blood absorbed by the dressing(s)
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d)
Incidence of re-bleeding, if any
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e)
Survival of animal
At the end of the experiment, animal wounds were cleaned with normal saline and povidone iodine was applied. Antibiotic (Amikacin sulfate @ 80 mg/kg body weight given through intraperitoneal injection) and analgesic (Diclofenac sodium @ 40 mg/kg body weight given through subcutaneous injection) treatments were given to the animals for 5 consecutive days followed by dressing with povidone iodine to prevent the occurrence of any infection and pain. Animals were kept under observation for 1 week. Thereafter, animals were rehabilitated at the Experimental Animal Facility of the institute for their use in future experiments.
Statistical analysis
The results were expressed as mean ± S.D. The data were analyzed statistically using GraphPad Prism 5.00. Statistical analysis was performed by one way ANOVA followed by Tukey’s post hoc test. For in vitro cytotoxicity study, the results were analyzed by two way ANOVA followed by Bonferroni test. A value of p ˂0.01 was considered significant in all cases.
