Layer-by-layer tissue microfabrication supports cell proliferation in vitro and in vivo

Layer-by-layer biofabrication represents a novel strategy to create three-dimensional living structures with a controlled internal architecture, using cell micromanipulation technologies. Laser assisted bioprinting (LAB) is an effective printing method for patterning cells, biomolecules, and biomaterials in two dimensions. "Biopapers," made of thin polymer scaffolds, may be appropriate to achieve three-dimensional constructs and to reinforce mechanical properties of printed materials. The aim of this work was to evaluate the effect of the tridimensional organization of cells and biomaterials on cell proliferation in vitro and in vivo. The experimental LAB setup was comprised of an infrared laser, focused onto a glass ribbon coated with an absorbing layer of gold. The cell bioink was made of MG63 cells (50 millions cells/mL in culture medium and 1% alginate), transduced with Luciferase gene for tracking and quantification. The printing substrate was a 100-μm-thick polycaprolacton (PCL) electrospun scaffold. The building sequence comprised sequential layers of cells and PCL scaffolds stacked using two different tridimensional arrangements, which were compared in this study (layer-by-layer vs. seeding on a single locus of the scaffolds). Then the cell-seeded materials were cultured in vitro or implanted in vivo in NOD-SCID mice. The qualitative follow-up involved scanning electron microscopy (SEM) observations, live-dead assays, and histology. The cell amount was quantified by photon imager during 21 days in vitro and 2 months in vivo. Live- dead assay and SEM revealed that the cells survived after printing and spread onto PCL membranes. Circle-shaped patterns were maintained in vitro during the first week but they were no longer observable after 2 weeks, due to cell proliferation. Luciferase tracking displayed that the cell amount was increased in vitro and in vivo when the materials and the cells where stacked layer by layer. Histological sections of the in vivo samples revealed a thicker fibrous tissue in the layer-by-layer samples. We have demonstrated in this study that PCL electrospun biopapers can act as a shock-absorbing mattress for cell printing and could further support cell proliferation. The layer-by-layer printing provided an appropriate 3D environment for cell survival and enhanced cell proliferation in vitro and in vivo.