Injectable thermosensitive methylcellulose–carboxymethyl chitosan hydrogel for the delivery of human amniotic fluid mesenchymal stromal cell spheroids in cartilage regeneration

Cartilage injuries caused by trauma or degenerative conditions such as osteoarthritis have limited intrinsic healing capacity and often require invasive interventions. This study proposes a minimally invasive therapeutic strategy using spheroids of human amniotic fluid-derived mesenchymal stromal cells (AF-MSCs) embedded in a thermosensitive hydrogel composed of 10% (w/v) methylcellulose and 1% (w/v) carboxymethyl chitosan (M10C1). AF-MSCs spheroids, generated in 3D culture under TGF-β3 stimulation, exhibited high viability and robust extracellular matrix production, including type II collagen and aggrecan. Rheological characterisation confirmed that M10C1 displays shear-thinning behaviour and gelation near physiological temperatures (∼30 °C), making it suitable for injection and 3D bioprinting. Importantly, confocal microscopy and fusion assays demonstrated that the hydrogel preserved spheroid bioassembly and viability. Co-culture with human cartilage explants further showed that M10C1 promoted spheroid adhesion without hindering integration into native tissue. These findings highlight the potential of this AF-MSCs spheroid-hydrogel system as an injectable, biocompatible platform for cartilage repair, with promising applications in regenerative medicine and bioengineered tissue therapies.