(L-447) GMHA-Collagen Constructs as Biomimetic Scaffolds for an Engineered Cortical Tissue Model

Hydrogels are great material platforms in tissue engineering, as injectable vehicles as well as native-like microenvironment where cells can effectively function. In recent years, researchers have designed and supplemented hydrogels to mimic the brain’s native extracellular matrix (ECM) and support cell growth and function. One strategy involves incorporating hyaluronic acid (HA), a primary component of the brain’s ECM and its most abundant glycosaminoglycan. HA not only provides structural support within the brain, but also mediates cell signaling and adhesion. However, without tunable crosslinking motifs, HA swells in water and rapidly degrades in biological environments. Therefore, to enable tunable HA-based hydrogels, we have incorporated glycidyl methacrylate (GM), a UV photo crosslinking chemical that enables control over gel stiffness. We hypothesize that this glycidyl methacrylate-hyaluronic acid (GMHA) hydrogels, when mixed with type-I collagen, will recapitulate the mechanical and morphological properties of the brain's native ECM. Furthermore, these brain ECM-mimetic hydrogels will promote embryonic cortical neuron’s prolonged viability and neurite extension versus collagen-only hydrogels. By modeling both the mechanical, morphological, and protein-composition of the brain ECM within a tunable and customizable hydrogel platform could provide significant benefits in more accurately modeling brain tissue degeneration.

GMHA was synthesized by first dissolving hyaluronic acid powder in a mixture of acetone and water. Glycidyl methacrylate and triethylamine were then added to the dissolved HA. This reaction mixture was precipitated in acetone twice, dialyzed, frozen, and lyophilized for 7 days to obtain a purified GMHA powder. To fabricate the GMHA hydrogel constructs, GMHA powder was dissolved in a mixture of menthol, DPBS and photoinitiator, Igracure 2959, then the solution was combined with collagen which was dissolved in DMEM. Combining this solution with a collagen-DMEM solution. The resulting mixture was added to 24-well plates and thermally crosslinked in a 37 °C incubator for 90 min. Then it was photocrosslinked under UV light (365 nm) for 10 min. Collagen gels were used as control and prepared in a similar manner. We then removed the hydrogel samples from the 24 well plates and placed them on a metal mesh sheet with nylon meshes on both sides to contain the gels. Weights were placed on top of both the GMHA-collagen and pure collagen hydrogel samples for 5 mins to release excess water prior to testing. The impact of GMHA in hydrogels were compared with control, pure collagen hydrogels and the mechanical and physical properties of the gels were assessed via compression tests, swelling, and in vitro water loss studies. The stress-strain response was evaluated to determine the mechanical properties of the material.

The methacrylation of HA was successfully performed and the degree of methacrylation was assessed via ¹H-NMR and found as 16%. GMHA was combined with collagen and the blend hydrogels were formed through a two-step crosslinking process involving thermal and UV exposure. Water loss testing revealed that GMHA-collagen hydrogels were able to retain more water after applied compression compared to pure collagen hydrogel controls. This indicates that the addition of GMHA enhanced the water retention capacity of the hydrogels, as expected. However, swelling ratio assays over a 7-day degradation study showed that GMHA-collagen hydrogels had lower swelling ratios that progressively declined over time, whereas pure collagen gels displayed higher initial swelling that was maintained for the duration. This shows that most of the unbonded HA degrades within the first week of swelling. Scanning electron microscopy (SEM) characterization of hydrogel microstructure revealed key differences between GMHA-collagen and pure collagen networks. 
    In conclusion, we have fabricated tunable GMHA-collagen hydrogels that mimic the brain’s native extracellular matrix. The inclusion of glycidyl methacrylate-hyaluronic acid allowed customization of the gel’s mechanics by increasing interfibrillar space. Future studies will focus on the encapsulation of embryonic cortical mouse dorsal root ganglion’s and quantifying cell viability over time.
    This study demonstrated the potential of GMHA-collagen hydrogels to serve as tunable, biomimetic scaffolds to model the brain's ECM and support neural cell growth. However, further research is required to optimize these constructs. Future goals include focusing on quantifying neurite outgrowth and neuron viability within the gels. The final goal is to quantify long-term cellular viability and network stability by culturing encapsulated neural cells within the optimized GMHA-collagen hydrogels for over 4 weeks in vitro. This will assess the potential of these scaffolds to support the development of mature, functional neural networks. The biological characteristics of the gels will be determined by encapsulating E18 embryonic mouse cortical neuron cells and Live/Dead analysis will be performed each week for 4 weeks.