Dynamic Compression of Spheroid-Laden Alginate Granular Composites Induces Hypertrophic Chondrocyte Phenotype

This preprint describes a granular hydrogel strategy for engineering hypertrophic cartilage as a template for endochondral bone formation. The authors combined photocrosslinked alginate microgels, mesenchymal stromal cell spheroids, and cell-secreted decellularized extracellular matrix to create composite scaffolds, then applied dynamic compression to promote hypertrophic maturation. Decellularized ECM enhanced chondrogenic priming of spheroids, increased glycosaminoglycan production, elevated alkaline phosphatase activity, and supported hypertrophic progression with increased VEGFA expression and hydroxyapatite deposition. In granular scaffolds, low-RGD formulations combined with dECM-loaded spheroids produced stronger osteogenic and hypertrophic responses, including increased SPP1, COL1A1, ALP activity, and mineralization. Dynamic compression further promoted spheroid sprouting, cell proliferation, matrix remodeling, and mineral deposition, while YAP1 inhibition reduced hypertrophic marker expression, implicating Hippo-pathway mechanotransduction in compression-driven maturation. Overall, the study shows that dynamic compression, granular scaffold architecture, and dECM-loaded spheroids act together to generate mineralizable hypertrophic cartilage for bone repair applications.