<p>With the rising incidence of severe foot ulcers in diabetes, pressure sores in the elderly and critically ill, as well as deep burns, there is an increasing demand for therapies that significantly improve the performance of current wound healing approaches. This study aimed to investigate the mechanisms underlying skin regeneration facilitated by stem cells incorporated into natural material-based wound healing agents, which are designed for convenient application and effective treatment of wounds. In this study, we demonstrated on stem cells (Bone marrow derived mesenchymal stem cells, MSCs) incorporation into an injectable wound healing agent with sodium alginate, chitosan, tempo-oxidized cellulose nanofiber (TOCN), and natural porcine skin dSECM (A₁C_0.1T₁E₁₀ + MSCs) on the regeneration of skin defects. The characterization results indicated that the hydrogel possesses self-healing, biodegradable, and antibacterial properties. Biocompatible hydrogels loaded with MSCs actively interact with the biological environment, thereby improving the microenvironment for tissue repair. Results from in vitro and in vivo analysis demonstrated that the hydrogel with stem cells promoted cell proliferation, ECM deposition, neovascularization, modulation of inflammatory responses, re-epithelialization, and the enhancement of hair follicle and gland formation. The synergistic effect of stem cells with the hydrogel accelerated wound healing by activating the Wnt signaling pathway. Based on our research, it is suggested that the A₁C_0.1T₁E₁₀ + MSCs composite hydrogel holds potential for applications in a range of in vivo therapeutic fields.</p>