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

This study addresses key barriers limiting minimally invasive cell-based therapies for intervertebral disc degeneration (IVDD), namely injection-induced shear damage and poor cell survival and phenotype maintenance in the nucleus pulposus (NP) microenvironment. The authors developed injectable “scaffolded spheroids” (S-SPH) by integrating human bone marrow–derived mesenchymal stromal cell (hBMSC) spheroids into high-resolution 3D-printed polycaprolactone-based microscaffolds (MS; ~200 μm diameter) fabricated by two-photon polymerization. The work systematically optimized spheroid seeding density (~2000 cells/spheroid) and MS printing parameters, then evaluated discogenic/NP-like differentiation using growth differentiation factor 5 (GDF5) (with comparisons to TGFβ1 and combinatory cues) under standard culture and under NP-mimetic “healthy in vivo-like” conditions (hypoxia at 2% O2 and low glucose). Across viability, morphology, biochemical assays (GAG/DNA), histology/immunostaining (e.g., aggrecan and collagen II), and RT-qPCR, GDF5 emerged as the most effective cue to drive an NP-like phenotype characterized by elevated ACAN, increased ACAN/COL2A1 ratios, and suppression of hypertrophic COL10A1. Importantly, S-SPH showed enhanced biomechanical performance relative to scaffold-free spheroids and better retention of structure during cyclic compression. Under hypoxic/low-glucose priming, S-SPH further increased NP-marker expression (e.g., KRT18, HIF1α) and ECM accumulation, producing compressive properties approaching reported native human NP mechanical ranges. Finally, S-SPH demonstrated improved injectability through a 26G needle, maintaining higher post-injection viability and exhibiting reduced inflammatory/catabolic stress responses (lower IL6 and MMP13) compared with scaffold-free spheroids. Collectively, the study proposes S-SPH as a mechanically protective, injectable microtissue building block for NP regeneration that warrants further ex vivo and preclinical evaluation for IVDD therapy.