PROJECT SUMMARY/ABSTRACT Three-dimensional (3D) bioprinting has been making a revolutionary impact on building living tissues and organs. Despite several attempts, vascularization is still an unmet problem for 3D bioprinting of scalable tissues and organs. In spite of significant efforts reported to generate vascular networks in 3D printed tissues, the gold standard is still the use of sacrificial inks, which has several shortcomings such as the need for extensive post- processing efforts to remove sacrificial inks, inability to remove these inks from low aspect-ratio features (i.e., highly thin, long vascular networks) and the residuals remaining from inks that usually interfere with biological function of cells inhibiting their adhesion and spread. In this project, we propose a highly novel technology through harnessing the power of compressibility of air in yield-stress gels and unveil 3D printing of air (3DAirP), which is an intangible ink. The process will induce open-channel network generation in yield-stress gels in a single step, which will overcome the outstanding limitations with the use of sacrificial inks. Moreover, the proposed 3DAirP technology will facilitate the generation of vascular channels (up to the minimum diameter of ~125 µm) at an unprecedented printing speed (i.e., >10-folds faster than 3D printing of sacrificial inks). In Specific Aim 1, we propose to develop 3DAirP technology, which has the capability of rapidly generating open stable channels in yield-stress gels. We will investigate the interplay among the viscoelastic properties of yield-stress gels and governing physical forces during 3DAirP, and how such interplay will enable us to dissect the knowledge for directly laying down the air channels to produce stable vascular networks in engineered tissues. To exemplify the technology, we will demonstrate two unique applications including (i) a scalable vascularized bone tissue and (ii) an atherosclerosis model on a chip, both in vitro. In Specific Aim 2, we will reconfigure the technology for 3DAirP intraoperatively that will facilitate air channel generation under surgical settings. To exemplify its utility, we will demonstrate two unique applications including (i) intraoperative 3DAirP within bioprinted bone constructs in critical-sized rat calvarial defects and (ii) coupling intraoperative 3DAirP with a new microsurgery technique to rapidly induce angiogenesis and orient vascular ingrowth in rat hindlimbs. In this regard, we have formed a complementary collaboration that merges essential domain knowledge in bioprinting, 3D printing process and instrument development, biomaterials, vascularization, microsurgery, craniofacial surgery, biomechanics and simulation, and bone and vascular tissue engineering with the depth necessary to propel the proposed work towards meaningful advances that would otherwise not be possible. Successful completion of the proposed work is anticipated to give rise to an advanced 3D printing technology for rapid generation of vascular networks towards fabrication of scalable tissues and organs.

PROJECT NARRATIVE The proposed work will demonstrate a new three dimensional (3D) printing technique for generation of vascular channels in hydrogel matrices via the use of air as a new ink and explore the use of such a technique for fabrication of vascularized tissues. Successful completion of the proposed work will have a great potential to pioneer the development of a disruptive 3D printing technology and advance its utilization in fabrication of vascularized scalable tissues.