[Effects and mechanism of thermo-sensitive hydrogel on the wound healing of full-thickness skin defects in diabetic mice].

To explore the effects and mechanism of (. ) thermo-sensitive hydrogel on the wound healing of full-thickness skin defects in diabetic mice. (1) According to the volume ratio of bacteria to medium of 1∶100, about 5×10(8) colony forming units/mL (the same concentration below) . was cultured in M17GS liquid medium. The growth conditions were observed at 0 (immediately), 2, 4, 6, 8, 10, and 12 h of culture with a microplate reader. In addition, another colony of the bacteria was taken and cultured under the same condition mentioned above. The culture medium was collected at the same time points as mentioned above, and the supernatant of bacterial culture was isolated. With the supernatant, the pH value was measured with a desktop pH meter, and the concentration of L-lactic acid at 0 (immediately), 2, 4, 8, and 12 h of culture was determined by the L-lactic acid detection and analysis kit (=3). (2) To prepare a simple thermo-sensitive hydrogel, the poloxamer thermo-sensitive polymer and M17GS liquid medium were mixed thoroughly according to the mass-volume ratio of 0.2 g∶1 mL. . was added to the simple thermo-sensitive hydrogel according to the volume ratio of bacteria to hydrogel of 1∶100, and the . thermo-sensitive hydrogel was prepared after thorough mixing. Afterwards, the morphology of . thermo-sensitive hydrogel was observed after 4 ℃, 37 ℃ incubation and again at 4 ℃ incubation after gelation. The storage modulus and loss modulus of the . thermo-sensitive hydrogel at 10-40 ℃ were measured by rheometer, and the gel forming temperature was observed. After freeze-drying the . thermo-sensitive hydrogel, the surface and the morphological structure of . in the hydrogel were observed by scanning electron microscope. (3) Mouse macrophages Raw264.7 cells were M1-type polarization stimulated by culturing with lipopolysaccharide and interferon γ in the final mass concentration of 100 and 10 ng/mL respectively for 24 h. The cells were divided into blank control group (without other treatment), . thermo-sensitive hydrogel group, and lactic acid group. . thermo-sensitive hydrogel in the volume of 1 mL was added to the cells of . thermo-sensitive hydrogel group, while lactic acid with the final molarity of 30 mmol/L was added to the cells in lactic acid group. After being cultured at 37 ℃ for 24 h, mRNA expressions of the markers arginase 1 and CD206 of M2-type macrophages were detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-PCR) (=3), and the immunofluorescence method was used to detect the protein localization and expression of arginase 1 and CD206. (4) Fifteen female BALB/c mice aged 8-10 weeks were induced into diabetic mouse models by the method of streptozotocin combined with high-sugar and high-fat diet, and a full-thickness wound with the diameter of 6 mm was made on the back of each mouse. The mice were divided into blank control group (without other treatment), thermo-sensitive hydrogel alone group, and . thermo-sensitive hydrogel group according to the random number table, with 5 mice in each group. The mice in the hydrogel treatment two groups were dripped with 200 μL corresponding hydrogel to the wound surface immediately after injury, and the hydrogel was replaced every day. After treatment for 0 (immediately), 3, 6, 9, and 12 days in the hydrogel treatment two groups, wound healing was observed, and wound area was measured. After 12 days of treatment, the wound tissue was taken to observe the thickness of granulation tissue by hematoxylin-eosin staining and CD206 and the marker of M1-type macrophages of inducible nitric oxide synthase (iNOS) positive cells by immunofluorescence method. The mice in blank control group were observed at the same time points as mentioned above. (5) Nine female BALB/c mice aged 8-10 weeks were induced into diabetic mouse models by the same method of experiment (4). Then, they were divided into normal skin group (without other treatment), wound alone group, and . thermo-sensitive hydrogel group according to the random number table, with 3 mice in each group. Mice in wound alone group and . thermo-sensitive hydrogel group were prepared with full-thickness skin defect wounds according to the method of experiment (4). Mice in the former group was left untreated after injury, and in the latter group, 200 μL . thermo-sensitive hydrogel was dripped onto the wound surface immediately after injury. After treatment for 1 day in hydrogel treatment group, the wound tissue of mice was taken, and the mRNA expressions of interleukin 1β (IL-1β), tumor necrosis factor α (TNF-α), and nuclear factor κB were detected by real-time fluorescence quantitative RT-PCR; after the eyeball blood was collected, the leukocyte count, lymphocyte count, and monocyte count in peripheral blood were measured by an automatic blood cell analyzer, and the serum L-lactic acid concentration was measured by the L-lactic acid detection and analysis kit. At the same time point mentioned above, normal skin tissue was taken from the corresponding parts of mice in normal skin group, wound tissue was taken from mice in wound alone group, and blood was taken from mice of the two groups for corresponding detection. Data were statistically analyzed with one-way analysis of variance, analysis of variance for repeated measurement, Tukey, and Dunnett test. (1) The growth of . reached the plateau in about 6 h of culture. In the culture supernatant of . the pH value gradually decreased, reaching the nadir about 4.9 after 8 h of culture, and the L-lactic acid concentration gradually increased, which peaked about 70 mmol/L after 8 h of culture. (2) The . thermo-sensitive hydrogel was a liquid at 4 ℃, and a solid gel at 37 ℃. After gelation, it became a liquid again after incubating at 4 ℃. The gel forming temperature was about 25 ℃. The storage modulus was about 3 000 Pa, and the loss modulus was about 1 000 Pa after gelation. Under the scanning electron microscope, the . thermo-sensitive hydrogel showed a loose three-dimensional porous structure, and the . had an ellipsoidal shape being wrapped inside the hydrogel. (3) After 24 h of culture, compared with those in blank control group, the expression of arginase 1 increased significantly (=11.620, 15.250, <0.01), the expression of CD206 mRNA increased significantly (=16.770, 19.030, <0.01), and the expression of CD206 protein located in the cell membrane and arginase 1 protein located in the cytoplasm increased significantly in the macrophages of . thermo-sensitive hydrogel group and lactic acid group. The expressions of arginase 1 and CD206 mRNA in the macrophages between lactic acid group and . thermo-sensitive hydrogel group were similar (=3.629, 2.259, >0.05). (4) After 3-12 days of treatment, compared with those in blank control group and thermo-sensitive hydrogel alone group, the wound of mice in . thermo-sensitive hydrogel group healed faster, the wound area was significantly reduced, and the inflammation of the wound edge tissue was reduced. After treatment of 3, 6, 9, 12 days, the wound areas of mice in . thermo-sensitive hydrogel group were (25.8±5.9), (21.2±4.6), (16.0±2.4), (8.4±2.4) mm(2) respectively, which were significantly smaller than (31.8±5.3), (28.0±3.4), (22.6±3.7), (17.0±1.0) mm(2) in blank control group (=3.506, 3.973, 3.856, 5.025, <0.05 or <0.01). After treatment of 3 and 6 days, the wound areas of mice in . thermo-sensitive hydrogel group were significantly smaller than those in thermo-sensitive hydrogel alone group (=3.739, 3.739, <0.05). After 12 days of treatment, compared with those in blank control group and thermo-sensitive hydrogel alone group, the wound granulation tissue of mice in . thermo-sensitive hydrogel group was thicker, with significantly reduced iNOS positive cells and increased CD206 positive cells in wound tissue. (5) After 1 day of treatment, the mRNA expressions of IL-1β, TNF-α, and nuclear factor κB in the wound tissue of mice in wound alone group were significantly higher than those of normal skin tissue of mice in normal skin group (=9.253, 4.819, 6.020, <0.01) but similar to those in . thermo-sensitive hydrogel group (=2.850, 2.735, 2.556, >0.05). The peripheral blood leukocyte count, lymphocyte count, and monocyte count of mice in wound alone group were significantly higher than those in normal skin group (=3.523, 5.373, 5.279, <0.05 or <0.01) but similar to those in . thermo-sensitive hydrogel group (=0.621, 1.240, 1.293, >0.05). The serum L-lactic acid concentration of mice in the three groups remained within the normal range and the overall comparison among them was not statistically significant (=4.095, >0.05). The . thermo-sensitive hydrogel was safe to use locally on the wounds of diabetic mice with full-thickness skin defects. It can produce and deliver lactic acid in situ, promote the polarization of macrophages from M1 to M2, reshape the wound healing microenvironment, and promote efficient wound healing.