Click-crosslinked nanogels integrated into 3D stem cell spheroids enhance regenerative function for swallowing muscle repair

Muscle injury or degeneration in the head and neck region can impair daily swallowing function. Mesenchymal stem cell (MSC) therapy has shown potential for muscle regeneration but faces challenges like poor cell survival and limited engraftment. Click-crosslinked nanogel-based hydrogels have emerged as promising cell delivery systems. This study introduces nanogel-microfiber fragments (NG-MF) as structural spacers within spheroids to enhance cell viability and function. By modifying cholesterol-bearing pullulan with acryloyl groups (CHPA) nanogels, we synthesized NG-MF using freeze-thaw cycles and sonication. NG-MF were then combined with adipose-derived MSCs (ADSCs) to fabricate hybrid spheroids. The NG-MF to ADSC ratio was optimized in hybrid spheroids, resulting in approximately a 5.6-fold increase in cell viability relative to cell-only spheroids. Hybrid spheroids also showed elevated secretion of key repair-associated factors, including interleukin (IL)-6, IL-10, and hepatocyte growth factor. This enhanced secretory function was maintained after the spheroids were refined to a smaller, injection-compatible size of approximately 100 μm for in vivo delivery. In rats with injured swallowing muscles, hybrid spheroid injection enhanced cell engraftment, reduced fibrosis, accelerated myogenin stabilization, and improved swallowing muscle function compared to cell-only spheroids. Cell engraftment rose by 20.8% from in vivo biodistribution analysis, and swallowing amplitude improved by 9.0% from electromyographic data three weeks post-treatment. The addition of NG-MF spacers in spheroids facilitates in situ injection-based delivery of ADSCs, thereby potentially enhancing in vivo swallowing muscle regeneration.