A bioactive three-layered skin substitute based on ECM components effectively promotes skin wound healing and regeneration

To overcome the limitations of conventional skin tissue engineering (TE), 3D biofabrication approaches are being developed. However, tissue mimicry should be further improved in skin models. Here, we developed and characterized biomimetic hydrogels to obtain a biofabricated three-layered (BT) skin substitute based on the main components found in the epidermal, dermal, and hypodermal skin layers. Hydrogels for dermal and hypodermal skin layers were based on a mix of agarose and type I collagen, supplemented with skin-related extracellular matrix (ECM) components (dermatan sulfate, hyaluronic acid, and elastin) and loaded with human dermal fibroblasts (hDFs) or human mesenchymal stem/stromal cells (hMSCs), respectively. The epidermal hydrogel was formulated using type I collagen supplemented with keratin and sphingolipids, and seeded with human epidermal keratinocytes (hEKs). Physicochemical results revealed adequate viscosity, gelling times, and pH for each hydrogel solution. The BT Skin also showed good swelling and degradation kinetics, and mechanical properties in a similar range of human skin. The hydrogels and BT Skin demonstrated stable cell viability and metabolic activity, as well as intercellular communication through the release of growth factors. Moreover, the BT Skin demonstrated controlled inflammation <i>in vivo</i>, and produced results comparable to autografting in a mouse skin wound model. This bioactive and biomimetic three-layered BT Skin has a composition that attempts to mimic the natural ECM of the skin, formulated with the characteristic cells and biomolecules present in each skin layer, and offers promising properties for its clinical application in the treatment of patients with skin injuries.