Biofabrication of Heterogeneous, Multi-Layered, and Human-Scale Tissue Transplants Using Eluting Mold Casting

The creation of multi-tissue auricular transplants for the treatment of microtia is a challenge due to the complex and layered structure of this anatomical tissue. A novel casting technique for the 3D biofabrication of heterogeneous, multi-layered, and human-scale tissue transplants using eluting agarose molds is presented. The molds are generated by casting agarose into custom 3D-printed containers, termed metamolds, optimized to facilitate the hydrogel casting process based on geometric and topological constraints. Casting yields high resolution (50 mu m) and allows for subsequent casting of further hydrogel layers on the transplant. Multi-layered auricular constructs are fabricated on a cartilage core consisting of a hyaluronic acid-alginate double network and an adjacent gelatin-based dermal layer. Bonding between adjacent layers is achieved by orthogonal physical and enzymatic crosslinking of residual functional groups between each layer. Material composition and culture duration are optimized for each layer allowing for maturation into cartilaginous and pre-vascularized dermal tissues. To demonstrate the scalability of this technique for the biofabrication of human-sized transplants, bi-layered human-sized ears are cast. Overall, this novel casting technique offers a promising approach for the fabrication of complex tissue grafts, overcoming the limitations of other traditional biofabrication methods. A novel casting technique to create multi-layered, high-resolution, and human-sized tissue transplants for craniofacial deformations is developed using crosslinker-eluting agarose molds. Bi-layered human-sized ears made of cartilage and dermis are successfully biofabricated, cultured, and analyzed for tissue maturation, demonstrating the scalability of the technique.image