The Porosity and Stiffness of Articular Cartilage Extracellular Matrix Derived Scaffolds Modulate Chondrogenesis of Mesenchymal Stromal Cells.

Articular cartilage (AC) regeneration remains a major challenge in musculoskeletal tissue engineering. Cartilage extracellular matrix (ECM)-derived scaffolds have shown promise as tissue-specific biomaterials, but the extent to which the fabrication process can be tuned to modulate scaffold pore architecture and mechanics and how this might influence the chondrogenic differentiation of mesenchymal stromal/stem cells (MSCs) remains incompletely defined. The aim of this study was to develop solubilized porcine AC-ECM scaffolds with controlled differences in pore size and matrix stiffness, and to evaluate how these factors regulate chondrogenesis of MSCs. Scaffolds with different pore sizes were fabricated by modifying the freezing cycle during lyophilization through the inclusion of an annealing step, while scaffold stiffness was varied by adjusting the AC-ECM concentration from 10 (1% ECM) to 50 mg/mL (5% ECM). Incorporation of an annealing step led to a ~2.5-fold increase in the mean scaffold pore size and was associated with improved construct elasticity and greater early cellular penetration. Increasing the AC-ECM concentration increased scaffold stiffness, with the ramp modulus increasing from approximately 5 kPa in the 1% scaffolds to approximately 60 kPa in the 5% scaffolds. All scaffold groups supported cartilage-matrix deposition during chondrogenic culture. Higher ECM concentrations were associated with greater total collagen accumulation, while the stiffest 5% scaffolds supported increased Type X collagen staining relative to the softer scaffolds. The expression of key chondrogenic genes SOX9, ACAN, and COL2A1 was higher in the 2% scaffolds compared to 5% scaffolds, while the expression of the hypertrophic marker COL10A1 was higher in the 5% scaffolds. In summary, scaffold processing provided a practical means to tune pore architecture and bulk mechanical behavior in solubilized AC-ECM scaffolds, and these differences were associated with changes in cell distribution and phenotype. These findings can potentially be leveraged to engineer scaffolds supportive of zonal cartilage tissue engineering or for osteochondral defect repair.