Hydrogels are commonly used in tissue engineering due to their biocompatibility, high water content, and similar mechanical properties to natural tissues. Traditional bulk hydrogels are hindered by nanoporosity which limits cell migration and interaction. While porosity can be introduced with strategies such as porogen leaching or freeze-drying, these steps are often incompatible with cells and limit the injectability of the hydrogel. Granular, microgel annealed scaffolds are an emerging platform which addresses these issues due to their modularity and inherent microporous void space. The ability to tune individual microgels allows for complex scaffold designs well suited to heterogeneous tissue found throughout the body. This work establishes a rapid annealing method for fabricating microgel scaffolds and investigates how microgel size, stiffness, and biochemical cues can be tuned to influence cell spreading and phenotype.

First, we developed a light-based technique for rapidly annealing microgels across a range of diameters. Utilizing 8-arm PEG-vinyl sulfone, we stoichiometrically controlled the number of arms available for crosslinking, functionalization, and annealing. We fabricated small and large microgels to explore how microgel diameter impacts void space and the role of porosity on cell spreading, cell aggregation, and macrophage polarization. Mesenchymal stromal cells (MSCs) spread rapidly in both formulations, yet the smaller microgels supported a higher cell density. When seeded with macrophages, the smaller microgels promoted an M1 phenotype, while larger microgels promoted a more regenerative M2 phenotype. As another application, we leveraged the inherent porosity of annealed microgels to induce cell aggregation. Finally, we implanted our microgels to examine how different size microgels influence endogenous cell invasion and macrophage polarization. The use of ultraviolet light allows for microgels to be noninvasively injected into a desired mold or wound defect before annealing, and microgels of different properties combined to create a heterogeneous scaffold.

We next use our established microgel platform to develop microgels for the repair of bone and cartilage using instructive peptides and changes in stiffness to create osteogenic and chondrogenic microgels, respectively. The microgels outperformed bulk hydrogel controls evidenced by significant upregulation of osteogenic and chondrogenic markers by MSCs. We leveraged this microgel platform to create a bilayer scaffold and assess the ability of microgels to spatially control the differentiation of MSCs. Osteochondral bilayer scaffolds exhibited distinct regions of osteogenic and chondrogenic differentiation as a function of microgel population. Spatial transcriptomics confirmed osteogenic and chondrogenic genes were upregulated in their respective microgel regions. These studies highlight the modularity of microgels and the importance of microporous void space.

Finally, we combined microgels with poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) to explore the interplay of void volume and conductivity to synergistically promote myogenic differentiation. PEDOT:PSS increased the conductivity of microgels over 2-fold while maintaining stiffness, annealing strength, and viability of associated myoblastic cells. Murine C2C12 myoblasts exhibited an upregulation of the late-stage differentiation marker myosin heavy chain as a function of both porosity and conductivity. The earlier stage marker, myogenin, was influenced only by porosity. Human skeletal muscle derived cells upregulated Myod1, IGF-1, and IGFBP-2, at earlier timepoints on conductive microgel scaffolds compared to non-conductive scaffolds. These data further demonstrate how the inherent porosity in microgel scaffolds is beneficial to cell differentiation.

Collectively, this dissertation demonstrates the versatility of microgels to serve as an instructive niche to regulate cell function. The engineering of microgel scaffolds could direct cell spreading, macrophage phenotype, MSC differentiation, and myoblast differentiation. Microgel size, stiffness, and biochemical cues are all factors that can be tuned to match specific tissue properties.