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Advancing cell based therapies by supporting implant survival

Periodic Reporting for period 2 - ENABLE (Advancing cell based therapies by supporting implant survival)

Reporting period: 2019-07-01 to 2020-12-31

Bioengineering of tissues and organs has the potential to generate functional replacement organs. ENABLE is inspired by the fact that we have become progressively more adapt at creating small tissues to treat small animals e.g. mice and rats, but have relentlessly struggled to create large viable tissues to treat humans, which should be the ultimate end game. Although small tissues can rely on diffusion of oxygen and glucose from the hosts, large tissues suffer from nutrient diffusion limitations, which results in starvation and death of the implant. Overcoming this key limitation is required for the creation of engineered replacement organs. In ENABLE, we aim to overcome this grand challenge by endowing engineered tissues with a continuous supply of nutrients will enable the survival and function of large solid tissues in vivo. I will accomplish this through the marriage of three fields: i) developmental bioengineering, which has taught us the basics of tissue formation, ii) microfluidics, which has provided powerful enabling platforms for micromaterial fabrication, and iii) 3D bioprinting, which has enabled the engineering of prevascularized tissues. Combining these techniques is expected to enabling the survival and function of clinically sized engineered implants, which is required to unleash the potential of man-made organs for organ replacement therapies.
We engineered self-oxygenating tissues, which was achieved via the incorporation of hydrophobic oxygen-generating micromaterials (HOGs) into engineered tissues. Self-oxygenation of tissues offered temporary metabolic protection from the formation of anoxic microenvironments by in situ elevating the oxygen tension within living tissues in a homogenous and tissue size-independent manner. This capability was leveraged to transform anoxic stress into hypoxic stimulation, which resulted in the sustained production of high quantities of vascular endothelial growth factor. Self-oxygenation of tissues was therefore proved as a novel and effective strategy to protect and vascularize living implants for organ transplantation and regenerative medicine applications. To aid clinical and commercial translation of these micromaterials, we build a custom designed in-air microfluidic set-up that allowed for ultra-high throughput fabrication of micromaterials. We have combined these HOGs with a new material that we have developed. Specifically, an hydrogels were orthogonally post-functionalized with desthiobiotinylated moieties using multivalent neutravidin. In situ exchange of desthiobiotin by biotin enables spatiotemporal material functionalization as demonstrated by the formation of long-range, conformal, and contradirectional biochemical gradients within complex-shaped 3D hydrogels. Temporal control over engineered tissue biochemistry is further demonstrated by timed presentation and sequestration of growth factors using desthiobiotinylated antibodies. The method’s universality is confirmed by modifying hydrogels with biotinylated fluorophores, peptides, nanoparticles, enzymes, and antibodies. Overall, this work provides a facile, cytocompatible, and universal strategy to spatiotemporally functionalize materials, which is of great value as the desired osteogenic bio-ink as well as the vascular nanocoating, which is used for our efforts on bioprinting capillary blood vessel networks. We have created a method to bioprint fractal patterns to create seamlessly integrated vascular trees and vascular beds. Together, this provides an innovative and highly useful toolbox to allow the survival and function of clinically sized implant.
We have delivered on the following breakthroughs that we consider to be beyond state-of-the art:
1) A novel and highly controllable oxygen generating micromaterial that allows for long term oxygen release at physiologically relevant concentrations.
2) Creation of self-oxygenating tissues that were proven to survive and function following implantation.
3) We have been the first to demonstrate that – surprisingly – implant vascularization can be improved by increasing the implant’s oxygen tension.
4) We have developed the first fabrication strategy for ultra-high throughput of oxygen generating micromaterials.
5) We have developed the first 3D designed microfluidic droplet generator by exploring the use of 3D printing of microfluidic devices.
6) We have the developed a novel class of spatiotemporally controlled biomaterials based on macromolecular complex formation and displacement.
7) We developed a novel method to create and guide vascularization and angiogenesis by controlling the living implant’s microporosity with an unprecedented capillary density.

We expect that we will deliver the following results before the finalization of the project:
1) Enabling the fabrication of hierarchical blood vessel networks: smoothly going from a single large artery into a high density microcapillary bed that will converge back into a single vein.
2) Gaining fundamental insights into what drives cell death and implant failure following implantation and how this could be rescued to enable survival and function of implanted engineered tissues.
3) Creation of engineered clinically sized organs that will remain viable for at least three weeks in the absence of host nutrients to enable the in growth of the host vasculature.
Summarizing overview of ERC StG ENABLE