Engineering of synthetic extracellular matrix analogues supporting and guiding neurons

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Author
Date
2018Type
- Doctoral Thesis
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Abstract
This thesis aimed to develop engineered matrices supporting high survival of encapsulated neurons and fast neurite outgrowth, as well as the tools enabling control of the growth pattern in 3D. To this purpose, we designed a number of new hydrogels and two-photon patterning protocols.
We found that a dynamic phase separation triggered by cross-linking of synthetic polyethylene glycol (PEG) hydrogels in the presence of some polysaccharides enabled to create interconnected porous structures, with tunable size ranging from submicron to dozens of microns. The resulting gels were simultaneously more permissive to invasion through the interconnected porous network, and more stable. The new protocols were shown to support an array of 3D neuronal cultures in vitro, as well as in vivo regeneration in a sciatic nerve injury model. This new method to control the macroporosity, which has the outstanding advantages of being compatible with injection and cell encapsulation, could be useful for a wide array of applications in hydrogel-based tissue engineering.
We then developed more biomimetic hydrogels based on hyaluronan as a polymer and using enzymatic reactions for cross-linking. In particular, the human coagulation factor XIII could be used to cross-link gels with good kinetics and biocompatibility, supporting neural network formation from encapsulated neurons. Later on, the bacterial ligase sortase A was shown to provide improved up-scalability and reagent stability, as well as even better kinetics, with the possibility to reach nearly instantaneous gelation, without sacrificing on cytocompatibility and biocompatibility. These enzymatic hydrogels could also have a wide scope of application, and we already demonstrated their use for cartilage tissue engineering. In particular, hydrogels encapsulating human chondroprogenitor cells, initially soft and brittle, reached a stiffness of the same order of magnitude as native hyaline cartilage after 3 weeks of culture.
Several of the defined gels developed or of the natural matrices classically used for neurons were also used in a systematic screening to identify a defined matrix supporting the growth of epithelial organoids (small intestine, liver and pancreas), traditionally grown in basal membrane extract from murine sarcoma. A fibrin/laminin minimal hydrogel was identified. These gels pave the way to a wide range of applications in high content screening and regenerative medicine.
Finally, we established a method making use of the enzymatic systems used/introduced earlier, to enable two-photon patterning of sensitive growth factors. Thanks to the improved orthogonality provided by the sortase A, the method works not only in inert gels such as alginate, but also in the common mammalian matrices collagen, hyaluronan, fibrin and laminin-rich basal membrane extract. This enabled us to demonstrate the guidance of encapsulated sensory neurons in 3D with patterns of nerve growth factor.
Overall, the new tools introduced give exceptional control over hydrogel properties, including their porosity, gelation kinetics, spatial patterning, as well as their cyto/biocompatibility and bio-orthogonality. These tools have already advanced the possibilities in neural tissue engineering, as well as cartilage tissue engineering and organoid cultures, and we hope that they will find widespread further usage for neural applications as well as in applications to other tissues. Show more
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https://doi.org/10.3929/ethz-b-000307984Publication status
publishedExternal links
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Publisher
ETH ZurichSubject
Tissue Engineering; Neural tissue engineering; Neurobiology; Cartilage engineering; Hydrogels; ENZYMATIC SYNTHESIS + ENZYMATIC REACTIONS (ORGANIC CHEMISTRY); Two-photon patterning; BiofabricationOrganisational unit
03949 - Zenobi-Wong, Marcy / Zenobi-Wong, Marcy
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