Engineering of a <scp>3D</scp> Printed Bilayer Artificial Skin Bio‐Mimicking Epidermis and Dermis for Skin Tissue Engineering Applications

ABSTRACT The limited strength and quick breakdown of amniotic membrane (AM) have restricted its use in skin wound treatment. This research involved direct 3D printing of a bioink prepared from chitosan (CTS) and alginate (Alg) onto AM to produce a two‐layer membrane with enhanced biomechanical properties, allowing for the healing of full‐thickness skin wounds. The AM‐CTS/Alg bilayer scaffold was fully characterized in vitro and in vivo. The suture retention and tensile strength of AM‐CTS/Alg showed a significant increase compared to AM based on the results. This bilayer scaffold exhibited excellent properties of cytobiocompatibility and cell attachment for fibroblast cells. Additionally, when AM was layered with CTS/Alg 3D printed scaffold, the degradation rate of the bilayer notably decreased. In vivo studies revealed both AM and AM‐CTS/Alg supported excellent skin wound healing. Histological analysis of the treated skin wounds demonstrated complete skin re‐epithelialization, granulation, collagen synthesis, and increased expression of pro‐angiogenesis genes (VEGFa and bFGF) in the full‐thickness skin wounds at days 10 and 20 posttreatment with AM‐CTS/Alg. In contrast, the control wounds showed persistent ruptured and unhealed epithelium. The optimized AM‐CTS/Alg demonstrates itself as a promising bilayer skin substitute possessing exceptional biomechanical and biological characteristics for effective treatment of full‐thickness wounds.