INJECTABLE SCAFFOLDED SPHEROIDS FOR INTERVERTEBRAL DISC TISSUE ENGINEERING

Intervertebral disc (IVD) degeneration is influenced by excessive mechanical stress, trauma, nutrient deficiencies, and genetic predispositions. While many studies have explored cell-based and scaffold-based therapies to address this issue, their effectiveness is often limited by the high mechanical load and challenging microenvironment. To overcome these challenges, we propose to use injectable scaffolded spheroids (S-SPH) [1] with optimal size, architecture, mechanical properties, and a nucleus pulposus (NP)-like phenotype to enhance IVD regeneration. The PCL-based resin (DEGRAD INX, BIO INX, Ghent, Belgium), was utilized as a to produce highly porous scaffolds (Diameter - 200 μm) using a custom-built two-photon polymerization (2PP) system based on a resonant scanner [2]. The optimal cell seeding density for the scaffolds was evaluated using human bone marrow-derived stromal cells (hBMSC). Further, Growth and Differentiation Factor -5 (GDF-5) and Transforming Growth Factor β1 (TGFβ1) supplemented medium was used for the differentiation of the hBMSC spheroids (SPH) towards NP-like lineage with or without the scaffolds (2000 cells/spheroid). The biochemical and biomechanical properties were assessed, while the gene expression of NP-specific genes (ACAN, COL2a1, KRT18, SOX9, COL10a1) was studied. Lastly, the injectability of the SPH with or without the scaffolds were evaluated. The 2PP processing parameters were optimised to produce a stable scaffold with an elastic modulus of 160 ± 31 kPa. 2000 cells/SPH was identified as an optimal cell seeding population for the scaffolds resulting in S-SPH with a maximum ferret diameter of 213 ± 37 μm. S-SPH showed higher metabolic activity (100 ± 26 %, SPH - 85 ± 12 %) during the injectability test and lower expression of stress-associated genes (IL-6 and MMP13) than SPH. GDF-5 promoted the differentiation of S-SPH into an NP-like lineage, as indicated by elevated glycosaminoglycan content, upregulation of NP-specific genes such as ACAN/COL2a1 (∼135-fold) and KRT18 (∼2-fold), and the downregulation of COL10a1 (hypertrophic marker) than the TGFβ1 group. Under GDF-5 stimulation, the S-SPH (91 ± 9 kPa) exhibited a higher elastic modulus compared to the SPH (18 ± 14 kPa). S-SPH present a promising strategy for creating NP-like microtissue units. The next research work is to validate the regenerative potential of the scaffolded spheroids in an ex vivo setup. This work demonstrated the potential of S-SPHs for IVD regeneration and applications in regenerative medicine.