Generation of small diameter, branched vascular systems by a combination of inkjet printing and multiphoton polymerization

Introduction: To date only single in vitro engineered tissues are transferred to clinical approaches due to todays inability to fabricate suitable, artifical vascular systems. Combining inkjet printing with high-resolution multiphoton polymerization (MPP) enables us to generate branched, tubular systems with diameters << 1 mm. New synthetic polymers were tailored to match the needs of the technical building process and the elastic properties of blood vessels. The polymers were biofunctionalized to achieve a close coating with endothelial cells (ECs). Experimental Methods: Based on numerical simulations, branched tubular scaffolds were fabricated by combining inkjet printing and MPP. Precursor polymers, cross linking agent, photo initiators and solvent additives were optimized to yield photo reactive inks with customizd E-moduli. Crosslinked polymers were modified with derivatized heparin and RGD and analyzed by XPS and colorimetric methods. Viability, proliferation, functionality of primary human microvascular ECs on the substrates was determined, using several assays and immunocytological stainings. Results: A set-up for integrating inkjet printing and MPP has been designed with which branched vessel scaffolds have been fabricated. The diameter of the tubes can range between 20 µm and several millimeters. Material compositions have been developed to achieve E-Moduli of 2-2000 MPa after crosslinking, the lower are similar to natural blood vessels. Suitable aftertreatment ensured biocompatibility of the processed polymers, thereafter thio-heparin and RGD have been covalently bound on the surface. On these biofunctionalized substrates an increased adhesion, viability and proliferation of ECs has been determined in comparison with unmodified substrates. EC-typical antigene expression has been observed by immunocytological stainings on all substrates. Conclusion: The presented combination of rapid prototyping techniques makes it possible to generate small diameter vessel-like systems that can be applied for supplying in vitro engineered tissues in a larger scale.