John A. McGlynn’s research while affiliated with Lehigh University and other places

Human mesenchymal stem cells (hMSCs) are instrumental in the wound healing process. They migrate to wounds from their native niche in response to chemical signals released during the inflammatory phase of healing. At the wound, hMSCs downregulate inflammation and regulate tissue regeneration. Delivering additional hMSCs to wounds using cell-laden implantable hydrogels has the potential to improve healing outcomes and restart healing in chronic wounds. For these materials to be effective, cells must migrate from the scaffold into the native tissue. This requires cells to traverse a step-change in material properties at the implant-tissue interface. Migration of cells in material with highly varying properties is not well characterized. We measure 3D encapsulated hMSC migration and remodeling in a well-characterized hydrogel with a step-change in stiffness. This cell-degradable hydrogel is composed of 4-arm poly(ethylene glycol)-norbornene cross-linked with an enzymatically-degradable peptide. The scaffold is made with two halves of different stiffnesses separated by an interface where stiffness changes rapidly. We characterize changes in structure and rheology of the pericellular region using multiple particle tracking microrheology (MPT). MPT measures Brownian motion of embedded particles and relates it to material rheology. We measure more remodeling in the soft region of the hydrogel than the stiff region on day 1 post-encapsulation and similar remodeling everywhere on day 6. In the interface region, we measure hMSC-mediated remodeling along the interface and migration towards the stiff side of the scaffold. These results can improve materials designed for cell delivery from implants to a wound to enhance healing.

Biological materials have length scale dependent structure enabling complex cell-material interactions and driving cellular processes. Synthetic biomaterials are designed to mimic aspects of these biological materials for applications including enhancing cell delivery during wound healing. To mimic native microenvironments, we must understand how cells manipulate their surroundings over several length scales. Our work characterizes length scale dependent rheology in a well-established 3D cell culture platform for human mesenchymal stem cells (hMSCs). hMSCs re-engineer their microenvironment through matrix metalloproteinase (MMP) secretions and cytoskeletal tension. Remodeling occurs across length scales: MMPs degrade cross-links on nanometer scales resulting in micrometer-sized paths that hMSCs migrate through, eventually resulting in bulk scaffold degradation. We use multiple particle tracking microrheology (MPT) and bi-disperse MPT to characterize hMSC-mediated length scale dependent pericellular remodeling. MPT measures particle Brownian motion to calculate rheological properties. We use MPT to measure larger length scales with 4.5 µm particles. Bi-disperse MPT simultaneously measures two different length scales (0.5 and 2.0 µm). We measure that hMSCs preferentially remodel larger length scales measured as a higher mobility of larger particles. We inhibit cytoskeletal tension by inhibiting myosin-II and no longer measure this difference in particle mobility. This indicates that cytoskeletal tension is the source of cell-mediated length scale dependent rheological changes. Particle mobility correlates with cell speed across length scales, relating material rheology to cell behavior. These results quantify length scale dependent pericellular remodeling and provide insight into how these microenvironments can be designed into materials to direct cell behavior.

Multiple particle tracking microrheology (MPT) is a passive microrheological technique that measures the Brownian motion of probe particles embedded in a sample to characterize material rheological properties. MPT is a powerful tool that quantifies material rheology in the low moduli range while requiring only small sample volumes and relatively simple data acquisition using video microscopy. MPT quantitatively characterizes spatiotemporal rheological properties and is particularly well suited for the investigation of evolving materials with complex microenvironments. MPT has expanded the study of a variety of materials including biofilms, colloidal gels, hydrogels, stimuli-responsive materials, and cell-laden biomaterials. The aim of this Tutorial is to summarize the fundamentals, illustrate the versatility, and highlight recent advances in MPT. In each application, we will highlight how MPT is uniquely positioned to gather rheological properties, which would be difficult, if not impossible, to attain with other rheological characterization techniques and highlight how MPT can be used to supplement other measurement techniques. This Tutorial should provide researchers with the fundamental basis and skills needed to use MPT and develop new MPT techniques to characterize materials for their unique applications.