Spatial patterning of PCL µ-scaffolds directs 3D vascularized bio-constructs morphogenesis in vitro

Modular tissue engineering (mTE) strategies aim to build three-dimensional tissue analogues in vitro by the sapient combination of cells, micro-scaffolds (μ-scaffs) and bioreactors. The translation of these newly engineered tissues into current clinical approaches is, among other things, dependent on implant-to-host microvasculature integration, a critical issue for cells and tissue survival in vivo. In this work we reported, for the first time, a computer-aided modular approach suitable to build fully vascularized hybrid (biological/synthetic) constructs (bio-constructs) with micro-metric size scale control of blood vessels growth and orientation. The approach consists of four main steps, starting with the fabrication of polycaprolactone μ-scaffs by fluidic emulsion technique, which exhibit biomimetic porosity features. In the second step, layers of μ-scaffs following two different patterns, namely ordered and disordered, were obtained by a soft lithography-based process. Then, the as obtained μ-scaff patterns were used as template for human dermal fibroblasts and human umbilical vein endothelial cells co-culture, aiming to promote and guide the biosynthesis of collagenous extracellular matrix and the growth of new blood vessels within the mono-layered bio-constructs. Finally, bi-layered bio-constructs were built by the alignment, stacking and fusion of two vascularized mono-layered samples featuring ordered patterns. Our results demonstrated that, if compared to the disordered pattern, the ordered one provided better control over bio-constructs shape and vasculature architecture, while minor effect was observed with respect to cell colonization and new tissue growth. Furthermore, by assembling two mono-layered bio-constructs it was possible to build 1 mm thick fully vascularized viable bio-constructs and to study tissue morphogenesis during 1 week of in vitro culture. In conclusion, our results highlighted the synergic role of μ-scaff architectural features and spatial patterning on cells colonization and biosynthesis, and pave the way for the possibility to create in silico designed vasculatures within modularly engineered bio-constructs.