(258g) Measuring the Effect of TGF-? and TNF-? on Human Mesenchymal Stem Cell Remodeling of Synthetic Polymer-Peptide Hydrogels Using Multiple Particle Tracking Microrheology

The resolution of a wound involves the cooperation of a variety of cell types with different functions, guided by human mesenchymal stem cells (hMSCs). These cells are instructed in part by cytokines, such as transforming growth factor-β (TGF-β) and tumor necrosis factor-α (TNF-α), which instruct cell function through each wound stage. hMSCs are present at each stage of the wound healing process, signaling macrophages to clear debris and kill bacteria at the early stages and encouraging collagen deposition and extracellular matrix (ECM) development at later stages. Because of their importance in wound healing, implantable materials are being designed to deliver additional hMSCs to a wound to improve outcomes and accelerate healing. A unique challenge in designing these materials is understanding how hMSCs remodel these materials to enable migration through the scaffold and deliver of these cells to the wounded tissue. In addition to signals presented in the synthetic scaffold, signals in the wound environment, including cytokines, will change hMSC-mediated scaffold degradation and motility. Our work aims to understand how hMSC remodeling occurs in the presence of cytokines. This will allow us to assess how these materials will perform in the wound environment. In this work, we encapsulate hMSCs in a well-defined poly(ethylene glycol)-based hydrogel scaffold which is cross-linked with a peptide that is cleaved by hMSC-secreted enzymes called matrix metalloproteinases (MMPs) enabling remodeling. We measure cellular remodeling around hMSCs treated with the cytokines, either TGF-β or TNF-α, and compare this to remodeling around untreated hMSCs. Remodeling is quantified using multiple particle tracking microrheology (MPT). MPT measures the Brownian motion of embedded probe particles in a material to quantify spatial and temporal changes in viscoelastic properties and material structure. Using MPT, we measure that cells treated with TNF-α remodel their microenvironment significantly more than cells in the other treatment conditions, degrading their surroundings to a viscoelastic fluid state on later days. Additionally, cells in all treatment conditions which have remodeled to a viscoelastic fluid are more likely to migrate in a persistent manner. We attribute these differences between treatment conditions to the effects of each cytokine on cell behavior. TNF-α is prominent early in the wound healing process and encourages the production of MMPs which break down damaged ECM in preparation for new matrix to be formed. It also decreases the production of tissue inhibitors of metalloproteinase (TIMPs) enabling more MMPs to be active. The increase in MMP and decrease in TIMP secretion results in more significant remodeling of the scaffold by hMSCs treated with TNF-α. Conversely, TGF-β, which is more prominent at later stages of wound healing discourages the production of MMPs and increases the production of TIMPs. This allows the wound to maintain structure as collagen is reorganized and the tissue is strengthened. These factors cause cells treated with TGF-β to have pericellular regions which remain in the gel state. These results have important implications for the design of new implantable materials for hMSC delivery as they characterize hMSC remodeling in the presence of TGF-β and TNF-α, cytokines which will be present in a wound when these materials are implanted as cell delivery vehicles.