Tuning the crosslinking and degradation of hyaluronic acid/gelatin hydrogels using hydrogen peroxide for muscle cell sheet fabrication

Abstract

Cell sheets have immense potential for medical and pharmaceutical applications including tissue regeneration, drug testing, and disease modelling. In this study, composite hydrogels were prepared from a mixture of phenolated hyaluronic acid (HA-Ph) and gelatin (Gelatin-Ph), with a controlled degree of polymer crosslinking and degradation, to fabricate muscle cell sheets from myoblasts. These hydrogels were obtained via hydrogen peroxide (H₂O₂)-mediated crosslinking catalysed by horseradish peroxidase (HRP) and peroxide-mediated cleavage of the polymer chains. The degrees of crosslinking and degradation were modulated by altering the exposure time to air containing H₂O₂. The results showed that exposing a solution of 2% w/v HA-Ph, 0.75% w/v Gelatin-Ph, and 1 unit mL⁻¹ HRP to air with 16 ppm H₂O₂ for 60 min yielded a stiffer hydrogel (7.16 kPa Young's modulus) than exposure times of 15 min (0.46 kPa) and 120 min (3.98 kPa). Moreover, mouse myoblast C2C12 cells cultured on a stiff hydrogel and induced to undergo myogenic differentiation formed longer and higher-density myotubes than those on softer hydrogels. The cell sheets were readily detached within 5 min by immersing the HA-Ph/Gelatin-Ph hydrogels covered with a monolayer of cells in a medium containing hyaluronidase. Our findings demonstrate that composite hydrogels with properties tuned by controlling the exposure time to H₂O₂, show great promise as platforms for muscle cell sheet fabrication.