Project summary Interactions between human mesenchymal stem cells (hMSCs) and their environment are a main factor in the function of these cells. Although the importance of cell-material interactions is well established, it has been diffi- cult to characterize this complex interplay, especially in vivo. During in vivo experiments, a stem cell treatment's efficacy can be assessed, but the underlying cellular processes and change in the surrounding microenvironment that leads to these outcomes remain a black box. Positive outcomes may result from these experiments even if there is loss of function or integrity in the tissue due to failed cell-material interactions, making aspects of the stem cell treatment ineffective and potentially unnecessary. Since real-time measurement of these interactions has not been realized in vivo, in vitro models provide an alternative approach to measuring cell-material inter- actions in both 2D and 3D culture. The use of scaffolds to mimic aspects of native tissue provide controlled environments where cell-material interactions can be quantitatively characterized. These scaffolds are used for 3D cell encapsulation and are designed to be remodeled by cells. This creates a feedback loop where the cell remodels the pericellular region and responds to the dynamically changing cues in the environment. Real-time characterization of these dynamic cell-material interactions continues to be a challenge. We propose to char- acterize dynamic cell-material interactions by measuring real-time hMSC-mediated scaffold remodeling using microrheological characterization and the resulting cellular processes using cell staining and inhibition. We will use an hMSC-laden synthetic hydrogel scaffold that mimics aspects of native microenvironments to present cues to cells. To characterize hMSC function, we will use techniques including cell staining and pharmacological inhibi- tion of molecules for cellular contractility and matrix adhesion. Our unique approach will characterize the scaffold microenvironment in real-time during cell-material interactions. We will use multiple particle tracking microrhe- ology (MPT) to measure hMSC-mediated scaffold remodeling and degradation. This technique quantifies the spatio-temporal evolution of the rheology in the pericellular region, which is part of the feedback loop that defines cell-material interactions. Together, these measurements will provide a relationship between cellular function and cell-engineered pericellular rheology as the complexity of the scaffold microenvironment is increased. The pro- posed research program will focus on characterizing cell-material interactions during specific critical processes that are not fully understood. The processes we will study are (1) cellular adhesion, (2) hMSC motility in re- sponse to scaffold viscoelasticity and (3) hMSC-material interactions when signaling molecules are presented in the environment. The proposed work will support the overarching goal of u...