Multiscale Hybrid Fabrication: Volumetric Printing Meets Two-Photon Ablation

The vascular tree spans length scales from centimeter to micrometer. Engineering multiscale vasculature, in particular from millimeter vessels to micrometer-size capillaries, represents an unmet challenge and may require the convergence of two or more printing modalities. Leveraging the great advances in light-based biofabrication, we herein introduce a hybrid strategy to tackle this challenge. By combining volumetric printing (VP) and high-resolution two-photon ablation (2PA), we demonstrate the possibility to create complex multiscale organotypic perfusable models with features ranging from mesoscale (VP) to microscale (2PA). To successfully combine these two methods, we first eliminated micrometer-size defects generated during VP process. Due to optical modulation instability of the laser source and selffocusing phenomenon that occurs when the light triggers the photoresin crosslinking, VP printed constructs feature micrometer-size filaments and channels. By optical tuning the refractive index of the photoresin, we demonstrate defect-free VP that can then be combined with 2PA. To facilitate the 2PA process and meet VP requirements, we introduce a purely protein-based photoclick photoresin combining gelatin-norbornene and gelatin-thiol. By optimizing defect-free VP and 2PA processes, we finally demonstrate the possibility to generate complex 3D vasculature-like constructs with features ranging from ~400 m of VP to ~2 m of 2PA. This hybrid strategy opens new possibilities to better recapitulate microtissues vasculature and complex architectures, with particular potential for microfluidics and organ/tissue-on-a-chip technologies.