The Role of Methylacrylylated Hyaluronic Acid Hydrogels in Promoting Skin Wound Healing

Abstract

Background: Hyaluronic acid, as a natural extracellular matrix component, possesses excellent biocompatibility and moisturizing properties, and demonstrates unique advantages in wound repair. Objectives: To explore the role of hyaluronic acid methacryloyl (HAMA) hydrogels in skin wound healing and analyze the characteristics of the local microenvironment of wounds. Methods: Mice divided into a control group (n = 6) and a HAMA group (n = 6) at random were used to create a model of total cortical resection. 100μL of HAMA hydrogel was applied to the wound surface in the HAMA group, while 100 μL of lithium phenyl-2,4,6-trimethylbenzoyl hypophosphonate (LAP) was applied to the wound surface in the control group. Both were irradiated with an ultraviolet lamp for 20 seconds, and on days 0, 3, 7, 10, and 14, the residual wounds were measured. The effect of the HAMA hydrogel on wound healing was analyzed by measuring the remaining wound area and performing hematoxylin-eosin (H&E) staining. The cellular characteristic spectrum of the local skin of the wound on the 14th day was analyzed via single-cell sequencing technology, and the degree of type I and type III collagen expression, F4/80, CD206 and CD86 in the local wound were detected via immunohistofluorescence technology. The mRNA expression levels of Arg1, Nos2, Itgam and Itgb2 in the RAW264.7 mouse macrophage line coincubated with the HAMA hydrogel for 24 hours were detected by RT-qPCR. Cluster analysis of fibroblasts and macrophages in the local skin of the wounds on the 14th day in mice was conducted via the Seurat software package, and the communication status between fibroblasts and macrophages was analyzed via the CellChat software package. Results: The HAMA group's skin wounds healed considerably more quickly than the control group's did. While the wounds in the control group had not yet fully healed, those in the HAMA group had by the fourteenth day. Single-cell sequencing analysis revealed that the proportion of fibroblast subsets with high expression of Col3a1 in the HAMA group (90.2%) was greater than that in the control group (79.8%), whereas the proportion of fibroblast subsets with high expression of Colla1 (5.7%) was lower than that in the control group (15.9%). The results of the immunofluorescence analysis confirmed that the local type III collagen level in the wounds of the HAMA group was greater than that in the wounds of the control group (P = 0.035), whereas the type I collagen level was lower than that in the wounds of the control group (P = 0.044). There was no significant difference in the proportion of local macrophages on the wound surface between the HAMA group mice and the control group mice. However, both the single-cell sequencing analysis results and the in vitro treatment of Raw264.7 macrophages with the HAMA hydrogel revealed increased expression of Arg1 (P < 0.001) and decreased expression of Nos2 (P < 0.001). Moreover, in the HAMA group, macrophages at the wound site expressed higher levels of CD206 (P = 0.042) and lower levels of CD86 (P = 0.011). Conclusions: Treatment of the microenvironment with the HAMA hydrogel is conducive to the healing of skin wounds, and more anti-inflammatory macrophages and fibroblasts that secrete type III collagen accumulate locally in the wound healing tissue.