(221r) Rheological Characterization of Dynamic Remodeling of the Pericellular Region By Human Mesenchymal Stem Cell-Secreted Enzymes in Well-Defined Synthetic Hydrogel

Human mesenchymal stem cells (hMSCs) actively reengineer and degrade the native extracellular matrix (ECM) during differentiation and migration. Migration of hMSCs is important since these cells migrate from their native niche to injury sites and participate in the healing process by regulating inflammation. hMSCs secrete enzymes, matrix metalloproteinases (MMPs), which degrade the ECM. The ECM is complex and presents different physical and chemical cues to the cells. To simplify this complexity, synthetic hydrogels are widely used to mimic aspects of the ECM and control the physical and chemical cues presented to the cell. Here, we encapsulate hMSCs in 3D in a well-defined synthetic poly(ethylene glycol (PEG)-peptide hydrogel scaffold to characterize rheological changes due to cell-mediated degradation and remodeling in the pericellular region. Our hydrogel is composed of a 4-arm star PEG backbone end-functionalized with norbornene that is cross-linked with a MMP-degradable peptide sequence. We use multiple particle tracking (MPT) to measure the dynamic cellular reengineering and degradation of the pericellular region. MPT measures the Brownian motion of 1 micron fluorescently labeled probe particles embedded in the material. MPT measurements of the pericellular region around encapsulated hMSCs measure a degradation profile where the material remains in a gel phase around the cell and greatest degradation is measured 160 μm from the cell center. This is a reverse-reaction diffusion degradation profile where cross-link density decreases as distance from the cell increases. We hypothesized that this degradation is due to cell-secreted tissue inhibitor of metalloproteinases (TIMPs) which bind to MMPs and create MMP-TIMP complexes which inhibit MMPs from degrading the scaffold. Inhibition of MMPs by TIMPs maintains microenvironmental stiffness around the cells, which enables cell spreading and attachment to the network prior migration. This MMP-TIMP complex diffuses away from the cell and unbinds enabling MMP scaffold degradation. Four TIMPs have been identified (TIMP 1 – 4) and hMSCs derived from bone marrow secrete TIMP-1 and -2. In order to investigate the role of TIMPs in material degradation and remodeling we neutralize TIMPs and measure the degradation profile around joint and single TIMP inhibited hMSCs. Joint TIMP inhibition changes the degradation profile to a reaction-diffusion profile where the greatest degradation happens immediately around the cell. We measure both degradation profiles around the cells when a single TIMP is inhibited. We also measure significant increase in cell speed when joint or single TIMPs are inhibited. After TIMP inhibition, MMPs are actively degrading the scaffold and due to durotaxis, cell migration towards stiffer regions, or a decrease in the physical barrier around the cell, cells migrate faster toward the interfaces with higher stiffness. This simple chemical treatment can increase cell delivery to the wounded areas if these scaffolds are implanted at the wound site.