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.
Keywords: Human amniotic fluid stem cells; cartilage regeneration; injectable biomaterials; mesenchymal stromal cell spheroids; thermosensitive hydrogel; tissue engineering.