Measurement\nof Covalent Bond Formation in Light-Curing\nHydrogels Predicts Physical Stability under Flow

Photo-crosslinking hydrogels are promising for tissue\nengineering\nand regenerative medicine, but challenges in reaction monitoring often\nleave their optimization subject to trial and error. The stability\nof crosslinked gels under fluid flow, as in the case of a microfluidic\ndevice, is particularly challenging to predict, both because of obstacles\ninherent to solid-state macromolecular analysis that prevent accurate\nchemical monitoring and because stability is dependent on size of\nthe patterned features. To solve both problems, we obtained <sup>1</sup>H NMR spectra of cured hydrogels which were enzymatically degraded.\nThis allowed us to take advantage of the high-resolution that solution\nNMR provides. This unique approach enabled the measurement of degree\nof cross-linking (DoC) and prediction of material stability under\nphysiological fluid flow. We showed that NMR spectra of enzyme-digested\ngels successfully reported on DoC as a function of light exposure\nand wavelength within two classes of photo-cross-linkable hydrogels:\nmethacryloyl-modified gelatin and a composite of thiol-modified gelatin\nand norbornene-terminated polyethylene glycol. This approach revealed\nthat a threshold DoC was required for patterned features in each material\nto become stable and that smaller features required a higher DoC for\nstability. Finally, we demonstrated that DoC was predictive of the\nstability of architecturally complex features when photopatterning,\nunderscoring the value of monitoring DoC when using light-reactive\ngels. We anticipate that the ability to quantify chemical cross-links\nwill accelerate the design of advanced hydrogel materials for structurally\ndemanding applications such as photopatterning and bioprinting.