Second harmonic generation microscopy of collagen organization in tunable, environmentally responsive alginate...

We fabricated photocrosslinked, environmentally responsive alginate hydrogels for tissue engineering applications. Methacrylated alginate (ALGMA) hydrogels were prepared across a variety and combination of ionic and covalent (chain growth, step growth, and mixed mode) crosslinking strategies to obtain a range of compressive moduli from 9.3 ± 0.2 kPa for the softest ionically crosslinked hydrogels to 22.6 ± 0.3 kPa for the dually crosslinked ionic mixed mode gels. The swelling behavior of the alginate hydrogels was significantly higher under basic pH conditions. Stiffer gels consistently swelled to a lesser degree compared to softer gels for all conditions. These hydrogels were stable - retaining >80% of their original mass for three weeks - when incubated in a basic solution of diluted sodium hydroxide, which mimicked accelerated degradation conditions. Encapsulated NIH/3T3 fibroblasts remained viable and proliferated significantly more in stiffer hydrogel substrates compared to softer gels. Additionally, the collagen secreted by encapsulated fibroblasts was quantifiably compared using second harmonic generation (SHG) imaging. Fibroblasts encapsulated in the softer hydrogels secreted significantly less collagen than the stiffer gels. The collagen in these softer gels was also more aligned than the stiffer gels. The ability to tune collagen organization using hydrogels has potential applications ranging from corneal wound healing where organized collagen is desired to epithelial wound scaffolds where a random organization is preferable.