Exploring Injectable Scaffolded Spheroids for Nucleus Pulposus Therapy in Degenerated Intervertebral Discs

Cell-based therapies for intervertebral disc degeneration (IVDD) treatment face significant challenges, including cell damage from injection-induced shear stress and poor survival in the harsh, nutrient-depleted microenvironment of the intervertebral disc. To overcome these challenges, we developed scaffolded spheroids (S-SPH) by integrating human bone marrow-derived mesenchymal stem cell (hBMSC) spheroids (SPH) into microscaffolds (MS) produced via high-resolution 3D printing, thereby forming injectable tissue-building blocks. We optimized cell seeding density (∼2000 cells/spheroid) and MS fabrication parameters and induced nucleus pulposus (NP)-like differentiation using growth differentiation factor-5 (GDF5) under both normoxic and hypoxic, low-glucose conditions mimicking a healthy <i>in vivo</i>-like environment. S-SPH maintained high cell viability and produced abundant extracellular matrix under both culture conditions. They also upregulated key NP markers, including aggrecan (ACAN), keratin-18 (KRT18), and hypoxia-inducible factor-1α (HIF1α), which indicated successful NP-like differentiation. They also exhibited improved compressive properties approaching those of native human IVD and retained structural integrity and cell viability following injection through a 26G needle. When differentiated into an NP-like phenotype, S-SPH fused and retained a high viability upon injection <i>in vitro</i>. These results demonstrate that S-SPH provide a promising <i>in vitro</i> strategy for NP regeneration, warranting further preclinical evaluation for IVDD therapy.