Periodic Reporting for period 4 - ENABLE (Advancing cell based therapies by supporting implant survival)
Periodo di rendicontazione: 2022-07-01 al 2023-06-30
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 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 maximize the performance of our self-oxygenating tissues, we minimized hydrogen peroxide toxicity by designed incorporation of catalase. Surprisingly, we demonstrated that self-oxygenation of tissues is not only important to maintaining tissues survival, but can also guide cell fate by for example being osteo-inductive. 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 oxygen generating micromaterials with a new material that we have developed. Specifically, 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 based on the photo-annealing of micromaterials to create perfusable vascular beds. Uniquely, this allowed for the creation of high density capillary beds within engineered tissues in a scalable manner. We also invented a novel embedded bioprinting approach based on leveraging aqueous two phase systems, which enabled fast yet stable printing of low viscous materials, which was previously not possible. This allowed for the vast expansion of printable materials and made the print resolution independent of the print nozzle. We combined this print technology with our photoannealable micromaterials to create engineered tissues containing vascular trees composed of large vascular channels that branch into a 3D high density capillary network, and vice versa. We also demonstrated that our mechanism of crosslinking, which is based on oxidative crosslinking of phenolic compounds, not only is effective in creating these clinically-sized and highly perfused tissues, but also orchestrates mechanotransduction to encapsulated cells in a previously unknown manner. This mechanism relies on the fact that instead of placing cells in a material and then form reversible interactions, we crosslinking the material directly to cells and their microenvironment in a semi-permanent manner. Together, ENABLE has provided numerous innovations that represent a highly useful and versatile 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) A method to minimize the potential cytotoxicity of self-oxygenation of tissue by converting hydrogen peroxide using catalase.
4) We have been the first to demonstrate that – surprisingly – implant vascularization can be improved by increasing the implant’s oxygen tension.
5) We have been the first to demonstrate that – surprisingly – self-oxygenation can steer cell fate e.g. by rendering GelMA hydrogels osteo-inductive
6) We have developed the first fabrication strategy for ultra-high throughput of oxygen generating micromaterials.
7) We have developed the first 3D designed microfluidic droplet generator by exploring the use of 3D printing of microfluidic devices.
8) We have the developed a novel class of spatiotemporally controlled biomaterials based on supramolecular complexation and displacement.
9) We have developed a novel method based on photo-annealing of microgels to create living implants with high density microvascular networks in a scalable manner.
10) We have developed a novel method to 3D bioprint low viscous liquids by pioneering aqueous two phase stabilization of liquid prints.
11) We have enabled the fabrication of large living tissues containing hierarchical blood vessel networks by combining low viscous bioprinting and photo-annealing of microgels.
12) We have pioneered a novel method for the microfluidic mass production of distinct types of living microgels using in-air microfluidics, which allows for scalable production of living microbuilding blocks and organoids.
12) We have developed a novel method of mechanotransduction, which is based on the discrete on-cell crosslinking of polymers using enzymatic oxidative phenolic crosslinking.
13) We have created engineered clinically sized organs that remained viable for at least three weeks in the absence of host nutrients to enable the in growth of the host vasculature.
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) A method to minimize the potential cytotoxicity of self-oxygenation of tissue by converting hydrogen peroxide using catalase.
4) We have been the first to demonstrate that – surprisingly – implant vascularization can be improved by increasing the implant’s oxygen tension.
5) We have been the first to demonstrate that – surprisingly – self-oxygenation can steer cell fate e.g. by rendering GelMA hydrogels osteo-inductive
6) We have developed the first fabrication strategy for ultra-high throughput of oxygen generating micromaterials.
7) We have developed the first 3D designed microfluidic droplet generator by exploring the use of 3D printing of microfluidic devices.
8) We have the developed a novel class of spatiotemporally controlled biomaterials based on supramolecular complexation and displacement.
9) We have developed a novel method based on photo-annealing of microgels to create living implants with high density microvascular networks in a scalable manner.
10) We have developed a novel method to 3D bioprint low viscous liquids by pioneering aqueous two phase stabilization of liquid prints.
11) We have enabled the fabrication of large living tissues containing hierarchical blood vessel networks by combining low viscous bioprinting and photo-annealing of microgels.
12) We have pioneered a novel method for the microfluidic mass production of distinct types of living microgels using in-air microfluidics, which allows for scalable production of living microbuilding blocks and organoids.
12) We have developed a novel method of mechanotransduction, which is based on the discrete on-cell crosslinking of polymers using enzymatic oxidative phenolic crosslinking.
13) We have created engineered clinically sized organs that remained viable for at least three weeks in the absence of host nutrients to enable the in growth of the host vasculature.