Design Rules to Control Mechanical Properties in 3D Hydrogels for Optimal Neuron Growth


Neurons are sensitive to the mechanical properties of their environment and show better growth, survival and differentiation when they are cultured in soft environments with mechanical properties similar to those of the brain compared to other tissues. Within the central nervous system (CNS), there is also a range of mechanical properties that vary with age, presence/absence of pathology and anatomic location. However, it is difficult to recapitulate the subtle differences of mechanical niches within the CNS with currently available 3D culture materials such as collagen and MatriGel. Moreover, these materials only allow the manipulation of a single mechanical variable - stiffness (G’) - and even then cannot do so while keeping other biologically important variables such as matrix morphology and biodegradation rate constant. This work also investigated the use of the 3D hydrogel for neuronal culture that addresses these fundamental deficiencies in that it enables the orthogonal control of different mechanical variables while preserving the peptide content and supramolecular morphology of the hydrogel. To solve this problem of confounding variables, a class of peptide amphiphiles (PAs) that contain a hydrophobic alkyl tail, a β-sheet-forming peptide sequence and a solubilizing, charged amino acid sequence was used. Under physiological conditions, these PA molecules self-assemble into 1D nanofibers that form 3D gels when electrostatically screened with multivalent cations, which provides two orthogonal handles for controlling the gelation process and the resulting mechanical properties of the hydrogel: the nanofiber and the gelator. Polycations such as oligo-L-lysines gel negatively charged PA nanofibers by decreasing electrostatic repulsion between nanofibers so that entropic exclusion of water drives the formation of supramolecular nanofiber bundles. The nanofiber bundling process is proposed here as the mechanism of gelation, and thus the storage modulus (G’) of bulk gels was investigated as a function of molecular weight of the oligo-l-lysine added to nanofiber solutions. Using oligo-l-lysines of different molecular lengths, incremental control over G’ was achieved: for each additional lysine monomer in the oligo-l-lysine chain, G′ was found to increase by 10.5 Pa without changing the peptide content of the gel or nanofiber morphology. Furthermore, this platform was used to demonstrate that even small reductions in G’ on the order of 70 Pa improve survival, neurite growth, and the tyrosine hydroxylase-positive population in induced pluripotent stem cell (iPSC)-derived dopaminergic (DA) neurons. Next, the contribution of PA nanofiber structure was examined by increasing the amount of hydrogen bonding within self-assembled PA nanofibers. This was accomplished by substituting glycine for an aza-glycine (azaG) within the β sheet-forming region of the PA molecule, thereby increasing the number of hydrogen bonds each molecule could make with its neighbors. Increasing the number of PA molecules that contain azaG from 0 – 5% increases the persistence length of a nanofiber fivefold. Then, when these more rigid nanofibers are used to make 3D hydrogels, the presence of azaG increases neurite outgrowth while simultaneously doubling tyrosine hydroxylase expression independently of the G’ as controlled by the gelator. The results described here provide insight into how nanoscale interactions and self-assembly translate to bulk material properties of hydrogels. Electrostatic interactions between gelators are responsible for gelation and G’ while hydrogen bonding networks control the persistence length of individual nanofibers. Moreover, these two interactions may be varied orthogonally to one another to achieve a mechanically optimized scaffold. The strategy developed here enables mechanical customization of hydrogels for applications in 3D cell culture. This technology may be useful for improving yields of iPSC-derived DA in vitro for use in drug screening applications, developmental studies and for eventual in vivo use as palliation for Parkinson’s Disease.

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