Engineering of a CuO-Releasing 3D-Printed Bilayer Skin Substitute with Improved Angiogenesis: In Vitro and In Vivo Evaluations

The amniotic membrane (AM) is used in repairing skin wounds, but its poor strength and quick degradation are major issues. Additionally, a key problem in skin wound healing is the lack of blood vessel formation, known as angiogenesis. Copper ions can help promote this process and are affordable; however, their cytotoxicity is dose-dependent. This study created a bioink from chitosan (CTS) and alginate (Alg) loaded with varying amounts of copper oxide (CuO at 0. 4, 0. 8, and 1.6% w/w). The bioink was directly printed onto decellularized AM to form a bilayer scaffold that had better mechanical properties and could release copper nanoparticles to support angiogenesis. In vitro tests evaluated the appearance, copper release, strength, and biological characteristics of the scaffolds loaded with CuO. The optimized scaffold (AM-CTS/Alg + Cu) was tested on full-thickness wounds in animals, and its healing and angiogenic effects were compared with those treated with decellularized AM, bilayer scaffolds without CuO (AM-CTS/Alg), and untreated wounds (control). Histological analysis confirmed effective decellularization of the tissue and showed that collagen fibers were preserved. The 3D printing on the AM improved the scaffold’s mechanical properties and water absorption. Only AM-CTS/Alg + 1.6Cu showed cytotoxicity for fibroblast cells during 1, 3, and 7 days of cell culture follow-up. The AM-CTS/Alg + 0.8Cu demonstrated the optimized blood vessel formation and encouraged significant upregulation of gene expression (VEGFa, bFGF, Col I, and Col III) in vivo. Animal tests showed this optimized scaffold accelerated healing and provided a safe environment for cell growth and blood vessel development.