Functionalized Annealed Microgels for Spatial Control of Osteogenic and Chondrogenic Differentiation

The biophysical heterogeneity of the bone-cartilage interface requires complex materials to mimic differences in bone density, extracellular matrix composition, and mineralization. Biomaterial approaches to repair osteochondral tissue typically use multilayer scaffolds, which require multi-step fabrication and may undergo delamination at the construct interface. This work describes the development of functionalized microgels for the repair of osteochondral tissues using an N-cadherin peptide, BMP-2 peptide, and changes in stiffness to create pro-osteogenic and pro-chondrogenic microgels. Microgels, when annealed into a scaffold, outperformed bulk hydrogel controls evidenced by upregulation of osteogenic and chondrogenic markers in mesenchymal stromal cells (MSCs). The macroporous void space present in microgel annealed scaffolds enabled robust cell proliferation and ECM deposition throughout the entire scaffold. We then created a bilayer functionalized annealed microgel scaffold and assessed the ability to spatially control the differentiation of MSCs. Osteochondral bilayer scaffolds exhibited distinct regions of osteogenic and chondrogenic protein expression as a function of microgel population upon immunostaining for osteocalcin and aggrecan, respectively. Spatial transcriptomics confirmed osteogenic and chondrogenic genes were upregulated in their respective microgel regions. These studies highlight the tunable and functionalizable nature of microgels and the importance of macroporous void space.