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Nanomaterials 2020, 10, 390 4 of 16 and alginate-gelatin with AgNPs were evaluated. Briefly, L929 cells were seeded in 96-well culture plates (2 × 104 cells/well) and cultured in Dulbecco’s Modified Eagle Medium (DMEM) medium containing NaHCO3 (1.2 g/L,), ampicillin (0.025 g/L), streptomycin (0.1 g/L), supplemented with 10% fetal bovine serum (FBS). The negative control group was treated with the vehicle used to dilute the drug (DMSO 5%). For positive control, doxorubicin solution (100 μg/mL) was used [29]. Cell viability was assessed by the colorimetric method using Methyl-thiazolyl-tetrazolium (MTT). All reagents used in cell culture have been supplied by Sigma Chemical Co. (St. Louis, MO, USA). MTT solution (0.05%) was placed in contact with the cells, which were incubated at 37 ◦C for 3 h. After that, MTT was removed and added dimethyl sulfoxide (DMSO) for 10 min for solubilization of the tetrazolium salt crystals, and then the optical density (OD) reading was performed on an automated plate reader (ELISA) at a wavelength of 570 nm. The tests were conducted in quadruplicate and then normalized. The percentage of cell viability was calculated using the following equation, in which Abs stands for the absorbance of each respective solution: %Cell viability = Abs (treated cells) − Abs (blank) × 100 (1) Abs (negative control) − Abs (blank) 2.7.2. Minimum Inhibitory Concentration (MIC) Staphylococcus aureus (ATCC 25923) and Pseudomonas aeruginosa (ATCC 27853) strains were used for MIC assay. Colonies were harvested and resuspended to 1.5 × 108 CFU/mL (turbidity equivalent to 0.5 McFarland standard scale). The bacterial solution was diluted to 1 × 105 CFU/mL in Mueller Hinton Broth with 100 μL of gelatin, sodium alginate, AgNO3, and hydrogel (1, 2, and 4 mM). The negative control was 0.1 mL of Mueller Hinton Broth, and the positive control was 0.05 mL Mueller Hinton Broth and 0.05 mL bacterial solution at 1 × 105 CFU/mL dilution. The microdilution method has been used [30]. Plates were incubated at 37 ◦C for 20 h. 2.7.3. Wound Healing Test Adult Females Wistar rats (250 ± 50 g) were used. The Animal Research Ethics Committee of the Tiradentes University approved the in vivo procedures set in the Protocol No. 011116R, in compliance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. The animals were divided into three groups (n = 9, total 27 animals) and housed under conditions of controlled temperature (22 ± 1 ◦C) with a light/dark cycle of 12/12 h with free access to food and water. It was anesthetized with intraperitoneal injection composed of 1 mL ketamine (50 mg/kg) and 1 mL xylazine (20 mg/kg). The dorsal region was trichotomized, sterilized with a solution of polyvinylpyrrolidone-iodine, and the wound was done with a punch with 8 mm of width. In the immediate postoperative period, the animals received 10 mg/kg of ketoprofen intramuscularly for three days as a prophylactic dose of postoperative symptomatology, and the formulation was applied in the wound. Groups and each group identified all animals according to the number of days for analysis of the cicatricial process (3rd, 7th, and 14th day) until euthanasia in a CO2 chamber. The groups were divided into Control Group (GCTR), Group hydrogel sodium alginate/gelatin (80:20) (GH), and Group hydrogel with AgNP 4 mM AgNO3 (GHP) [31]. In the postoperative period, the lesions were controlled with photographic images (Sony brand camera, 10.1 megapixels) and measured with a digital caliper at the inner edges of the diameter at zero, three, seven, and fourteen postoperative days. The images were processed with the ImageJ® software. After the animals were sacrificed, the equivalent specimens were removed from the scar area with margins of 3.0 mm for histological characterization. 2.7.4. Histomorphology Analysis Removal of the specimens was equivalent to a scar area with a 0.5 cm margin of whole skin around the lesion, with depth up to the first muscle layer. The removed samples were fixed in 10% buffered formalin solution (pH 7.4) for 48 h. Subsequently, they were dehydrated in ethanol solutions at 70, 95,PDF Image | Wound Healing Silver Nanoparticles-Composing Hydrogel
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