| 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.
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