Exploring Gelation and Physicochemical Behavior of in Situ Bioresponsive Silk Hydrogels for Disc Degeneration Therapy

Hydrogels have received considerable attention in the field of tissue engineering because of their unique structural and compositional resemblance to the highly hydrated human tissues. In addition, controlled fabrication processes benefit them with desirable physicochemical features for injectability in minimally invasive manner and cell survival within hydrogels. Formulation of biologically active hydrogels with desirable characteristics is one of the prerequisites for successful applications like nucleus pulposus (NP) tissue engineering to address disc degeneration. To achieve such a benchmark, in this study, two naturally derived silk fibroin proteins (Bombyx mori, BM SF; and Antheraea assamensis, AA SF) were blended together to allow self-assembly and transformation to hydrogels in absence of any cross-linker or external stimuli. A comprehensive study on sol-gel transition of fabricated hydrogels in physiological fluid microenvironment (pH, temperature, and ionic strength) was conducted using optical and fluorescence analysis. Tunable gelation time (∼8-40 min) was achieved depending on combinations. The developed hydrogels were validated by extensive physicochemical characterizations which include confirmation of secondary structure, surface morphology, swelling and degradation. Mechanical behavior of the hydrogels was further analyzed in various in vitro-physiological-like conditions with varying pH, ionic strength, diameter, storage time, and strain values to determine their suitability in native physiological environments. Rheological study, cytocompatibility using primary porcine NP cells and ex vivo biomechanics of hydrogels were explored to validate their in situ applicability in minimally invasive manner toward potential disc regeneration therapy.