CRAFTING THE MICROVASCULAR CANVAS: BIOENGINEERED MATERIALS OR NATIVE EXTRACELLULAR MATRIX FOR THE CENTRAL NERVOUS SYSTEM

De Luca C¹, Virtuoso A¹, Cirillo G¹, De Angelis F¹, Guedán A², Maduna T³, Morin A⁴, Sepulveda Diaz J⁴, Vlachos A^3,5,6, Panetsos F⁷, Pérez-Rigueiro J⁸ and Papa M^1,9 | ¹Laboratory of Morphology of Neuronal Networks and Systems Biology, Department of Mental and Physical Health and Preventive Medicine, University of Campania Luigi Vanvitelli, Naples, Italy; ²Bioactive Surfaces S.L., Madrid, Spain; ³Department of Neuroanatomy, Institute of Anatomy and Cell Biology, Faculty of Medicine, University of Freiburg, Freiburg, Germany; ⁴Microfluidics Innovation Center, Paris, France; ⁵Center for Basics in Neuromodulation (NeuroModulBasics), Faculty of Medicine. University of Freiburg, Freiburg, Germany ⁶Center BrainLinks-BrainTools, University of Freiburg, Freiburg, Germany; ⁷Universidad Complutense de Madrid, Madrid, Spain ⁸Universidad Politécnica de Madrid, Spain; ⁹SYSBIO Centre of Systems Biology ISBE-IT, Milan, Italy

The THOR project (European Innovation Council-Pathfinder Programme, Grant Agreement number 101099719) addresses the fundamental challenge of developing a durable, working microvasculature that accurately reflects the crucial neurovascular unit (NVU) for CNS vitality and regenerative therapies. This work aims to reconstruct the complex natural process of vessel invasion into the CNS extracellular matrix (ECM) seen during embryogenesis. We utilized an innovative technique to spin and precisely deposit individual, 10-20 μm high-performance functionalized silk fibers in tailored configurations, serving as biomimetic scaffolds. We report on experiments where C57BL/6 Mouse Embryonic Brain Endothelial Cells were successfully seeded on silk scaffolds and tubes modified with either vascular endothelial growth factor (VEGF) or the IKVAV peptide from the laminin alpha-1 chain. We observed successful vascular invasion of both scaffold types and the formation of tube-like structures expressing markers for the blood-brain barrier (claudin−5) and associated pericytes (CD13, PDGFRβ). Critically, we confronted the results obtained on the functionalized silk scaffolds with those obtained using decellularized ECM derived from adult brain slices. This comparison validated the effectiveness of the engineered silk in guiding cell organization. The resulting endothelium secreted basal lamina proteins (collagen-IV, laminin) with alignments that were directed by the silk fibers (parallel, perpendicular, or anchored). The resulting biohybrid endothelium has been cultivated within a sophisticated microfluidic system featuring a pressurized lid, parallel channels, and integrated flow sensors to maintain precise culturing conditions. Our findings indicate that diverse peptides linked to the silk and the advanced microfluidics can effectively direct embryonic cell self-organization within this engineered framework to support brain tissue, acting as an engineered substitute for the complex cues of the native embryonic ECM. This work represents a significant advance toward constructing intricate microvascular networks and progressing regenerative therapies for the CNS.