关键词: 壳聚糖, 精氨酸, 水凝胶, 壳聚糖水凝胶, 巨噬细胞, 免疫, 创面修复

Abstract: BACKGROUND: Clinical skin wound healing continues to be a significant concern, and tissue repair research has moved to the forefront with the development of biomaterials with immunomodulatory properties. Therefore, it is crucial to research wound dressings that have immunomodulatory properties.
OBJECTIVE: To prepare chitosan hydrogels that have been modified by arginine with different configurations and assess their capacity to speed up wound healing in a rat animal model.
METHODS: (1) In vitro trial: Chitosan modified by pure L-arginine, pure D-arginine, and L-arginine and D-arginine was synthesized by EDC/NHS system, which was then crosslinked with aldehyde-modified four-arm polyethylene glycol. Different chitosan-based hydrogels (CS-L, CS-D, and CS-DL) were finally formed via the Schiff base reaction. Three kinds of hydrogel extracts were co-cultured with fibroblasts respectively. Hydrogel cytocompatibility was assessed using the CCK-8 assay and live/dead staining. The effect of hydrogel on the migration capacity of fibroblasts was assessed by using a scratch test. Three kinds of hydrogels were incubated with rat erythrocyte suspension respectively to evaluate the hemocompatibility of the hydrogels. The hydrogel extract was co-cultured with RAW264.7 macrophages to test the hydrogels’ capacity to enhance macrophage NO generation and polarize macrophage phenotype. (2) In vivo experiment: A total of 36 adult SD rats were divided into 4 groups with 9 rats in each group by the random number table method. Two full-layer skin defect wounds of 2 cm×2 cm were made on the back of each rat. Normal saline was added to the wounds of the control group, and corresponding hydrogel was added to the wounds of the CS-L, CS-D, and CS-DL groups, respectively, and then bandaged and fixed. The wound healing was observed regularly after operation. Hematoxylin-eosin staining was performed at 3, 10, and 21 days after operation. The samples were collected 10 days after operation and M2 macrophage immunofluorescence staining was performed.
RESULTS AND CONCLUSION: (1) In vitro experiments: Under scanning electron microscopy, the three kinds of hydrogels exhibited obvious interpenetrating network structures with pore sizes ranging from 70-200 µm. The three kinds of hydrogels have good swelling performance, degradation performance, self-healing performance, and suitable mechanical strength. The three kinds of hydrogels had good cytocompatibility and hemocompatibility and could promote the migration of fibroblasts. All three kinds of hydrogels had the ability to promote the polarization of macrophages, and CS-D hydrogels had the strongest ability to promote the polarization of macrophages. CS-L hydrogel could significantly promote the production of NO in macrophages. (2) In vivo experiment: 3 and 10 days after operation, the wound healing rate in the CS-L and CS-D groups was higher than that in the control group (P < 0.05). After 21 days, the wound healing rate of the three hydrogel groups was higher than that of the control group. Hematoxylin-eosin staining displayed that a large number of inflammatory cells were infiltrated in the wound tissue of rats in all groups, accompanied by neovessels and fibroblasts 3 days after operation. 10 days after operation, there was still more inflammatory cell infiltration in the wound of the control group, and the inflammation of the other three groups was improved, especially the decrease of inflammatory cells in the CS-D group was more obvious. 21 days after operation, the wound epithelium of each group was well repaired, and there was basically no inflammatory cell infiltration in the CS-L and CS-D groups, while there was still a small amount of inflammatory cell infiltration in the control group. Immunofluorescence staining revealed that the number of M2-type macrophages in the CS-D group was higher than that in the other three groups (P < 0.05). (3) The results conclude that chitosan hydrogels modified by different configurations of arginine can promote wound healing through different mechanisms. 

Key words: chitosan, arginine, hydrogel, chitosan hydrogel, macrophage, immunity, wound repair