Thermosensitive Hydrogel Derived from a Human Amniotic Membrane Promotes Diabetic Wound Healing

Extracellular matrix (ECM) hydrogels are recognized as promising biomaterials for regenerative medicine owing to their ability to recapitulate the native tissue microenvironment. The human amniotic membrane (AM), readily available and posing little to no ethical concerns, is rich in ECM components with inherent wound-healing potential. This study aimed to develop and characterize thermosensitive hydrogels derived from a decellularized AM and assess their therapeutic potential for diabetic wound healing. The native AM was subjected to detergent-enzymatic decellularization to remove the cellular content while preserving the essential ECM. The resulting acellular AM was lyophilized, cryomilled, and digested with pepsin under acidic conditions at three different concentrations. The pregel solutions were neutralized and thermally induced to form AM ECM hydrogels at 37 °C. The physicochemical properties, including gelation kinetics, swelling, porosity, mechanical stiffness, and biodegradation, were evaluated. The biological evaluation was assessed using fibroblasts, keratinocytes, and endothelial cells through live/dead staining, the MTS assay, and analyses of ROS production, apoptosis, cytoskeletal organization, and cell migration. Proteomic profiling was conducted to identify the retained matrisome proteins. The in vivo performance was tested in a diabetic murine full-thickness wound model. AM ECM hydrogels exhibited temperature-dependent gelation (<i>t</i>_1/2: ∼12.75-27 min), high water content (>97%), and >60% porosity. All formulations supported >70% cell viability at 24 h and >300% proliferation at 72 h, with negligible ROS production, minimal apoptosis, and preserved cytoskeletal integrity. The proteomic analysis confirmed the maintenance of matrisome proteins related to epithelial differentiation, angiogenesis, and tissue repair. The in vivo study demonstrated that the AM ECM hydrogel accelerated wound healing, evidenced by early wound closure, along with vascular stabilization, regulated inflammatory response, and ECM stabilization compared to those of the control group. These findings collectively demonstrate that AM ECM hydrogel treatment in diabetic mice ameliorates wound pathology, as evidenced by reduced severity, a modulated inflammatory response, and decreased fibrotic burden.