The general model of most tissue-engineering strategies rests on the use of exogenous biocompatible scaffolds in which cells can be seeded and matured in vitro or in vivo, to grow the tissue of interest. Scaffolds have been subject to prolific research and development over the last thirty years and, in general, offer the advantage of good biocompatibility, cell attachment and proliferation, while providing the biological, chemical, and mechanical clues to guide the eventual cell differentiation and assembly into a three-dimensional tissue construct. Scaffold-based tissue engineering has led to significant results in the reconstruction of various tissues and organs and, in some cases, has been translated to clinical practice.
Biomaterials-based exogenous scaffolds, though promising, still face general as well as specific challenges. Scaffolds may elicit adverse host responses and interfere with direct cell-cell interaction, as well as assembly and alignment of cell-produced extracellular matrix. Thus, fabrication techniques for production of scaffold-free engineered tissue constructs have recently emerged. Here we describe a novel fully biological self-assembly approach, which we implement through a rapid prototyping bioprinting method. The approach utilizes bio-ink particles, convenient multicellular units, either spheroids or cylinders of controlled diameter (300 to 500 μm), that are deposited with specifically designed bio-printers. The cellular composition of the bio-ink and the deposition scheme are respectively consistent with the cellular composition and topology of the desired biological construct. We use the method for scaffold-free small diameter vascular reconstruction and nerve regeneration. Various vascular cell types, including smooth muscle cells and fibroblasts, are aggregated into the bio-ink units. These are printed layer-by-layer concomitant with agarose rods, used here as a molding template. The post-printing fusion of the discrete units results in single- and double-layered small diameter vascular tubes. A unique aspect of the method is the ability to engineer vessels of distinct shapes, complex internal geometry, in case of nerve grafts, and hierarchical trees that combine tubes of distinct diameters, in the case of vascular grafts. The technique is quick and easily scalable.