(462b) Measurements of Cell-Mediated Degradation of Poly(ethylene glycol)-Norbornene Hydrogels with Non-Linear Chemical Gradients of Cytokines Using Microrheology

Implantable, degradable hydrogels are being designed as vehicles to deliver human mesenchymal stem cells (hMSCs) to treat wounds. A current challenge is ensuring cells migrate from the hydrogel to the wound site where they can begin the healing process. Cell migration could potentially be increased or directed by presenting physical or chemical cues in the scaffold. In the human body, wounds direct stem cell migration out of their niches by releasing a cytokine concentration gradient. We take inspiration from this process and create hydrogels with a tethered concentration gradient of cytokines to direct cell migration out of the gel to the wound. We use a combination of multiple particle tracking microrheology (MPT) and live cell imaging to measure pericellular cell-mediated degradation, cell persistence and changes in cell morphology. The goal of the work is to inform and improve the design of new scaffolds for cell delivery that use a the microenvironment to instruct basic cellular functions. The scaffold is composed of 4-arm poly (ethylene glycol)-norbornene (PEG-N), a matrix metalloproteinase (MMP)-degradable peptide cross-linker, a photoinitiator and a cellular adhesion ligand. The scaffold is photopolymerized using thiol:ene chemistry. Prior to scaffold formation, we use Traut’s reagent to attach a thiol group to each cytokine, which enables the cytokine to react with the PEG-N and be tethered into the network. Using a custom-built microfluidic device, we create hydrogels with a tethered gradient of either a pro-inflammatory cytokine, tumor necrosis factor-α (TNF- α), or an anti-inflammatory cytokine, transforming growth factor-β (TGF-β). The microfluidic device allows a source of thiolated cytokines to diffuse through a polymerized gel, after the gradient has formed, we again expose the hydrogel to UV light to tether the cytokine gradient in place. A modified indirect ELISA is used to confirm the cytokine is immobilized and present in the scaffold. The concentration of the cytokine gradient within the hydrogel is chosen to mimic the concentration in the native wound environment and is tethered to enable individual encapsulated cells to interact with multiple cytokines in their microenvironment. 3D encapsulated cells secrete MMPs, cleaving our peptide cross-linker inducing a material phase transition from gel to sol. MPT is used to measure this phase transition around individual hMSCs by measuring embedded fluorescent probe particles within the hydrogel and calculating their mean-squared displacements. We measure an increase in encapsulated cell motility, elongation and cell-mediated degradation when cytokines are uniformly tethered into the scaffold. Building on this experimental data, when cytokines are presented in a gradient, we can guide cells out of a hydrogel. We hypothesize this system will be effective in controlling cell motility within a scaffold for cell delivery and offer a solution to hindered cell migration out of a hydrogel.