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.

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Hydrogel biomaterials show promise as implantable cell delivery vehicles that enhance tissue regeneration and the natural healing process. The design of these materials requires that they mimic the natural environment to retain native cell function. Biological tissues often have spatially varying stiffness, allowing them to have a variety of functions within the body. However, this makes them challenging to mimic mechanically with a synthetic scaffold. To enable these complex designs, characterization techniques that measure nonuniform mechanical properties are required, but these methods are limited. Bulk rheological measurements average the stiffness of the sample, microrheological methods cannot characterize high moduli materials (despite being able to resolve spatial variability), and atomic force microscopy measurements can be a function of the selected tip geometry and measurement procedure. We present a new method for determining the stiffness of nonuniform hydrogels. Our technique measures the hydrogel’s autofluorescent brightness, which is related to its degree of cross-linking, and relates this brightness to elastic modulus. We use a well-established 3D cell encapsulation platform. This photopolymerized polymer–peptide hydrogel is composed of poly­(ethylene glycol)–norbornene and a matrix metalloproteinase (MMP)-degradable peptide. We first develop a relationship between hydrogel elastic modulus and brightness, which are systematically varied by controlling UV light exposure during photopolymerization. We then relate elastic modulus and autofluorescent brightness at each exposure time. This relationship enables images of hydrogels that measure brightness to be converted into stiffnesses. To demonstrate the technique, we fabricate hydrogels with nonuniform stiffness profiles: (1) step changes and (2) smooth gradients in elastic moduli. These are made by controlling UV light exposure spatially with a photomask. We then characterize these gels with the new technique. This work provides an alternative characterization method for hydrogels with spatially nonuniform stiffnesses. To effectively design materials for cell encapsulation, they must be characterized so that their properties are finely tuned to match native tissue. This will improve the effectiveness of these scaffolds as cell delivery vehicles and in promoting tissue regeneration.

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...

Implantable hydrogels are designed to treat wounds by providing structure and delivering additional cells to damaged tissue. These materials must consider how aspects of the native wound, including environmental chemical cues, affect and instruct delivered cells. One cell type researchers are interested in delivering are human mesenchymal stem cells (hMSCs) due to their importance in healing. Wound healing involves recruiting and coordinating a variety of cells to resolve a wound. hMSCs coordinate the cellular response and are signaled to the wound by cytokines, including transforming growth factor-β (TGF-β) and tumor necrosis factor-α (TNF-α), present in vivo. These cytokines change hMSC secretions, regulating material remodeling. TGF-β, present from inflammation through remodeling, directs hMSCs to reorganize collagen, increasing extracellular matrix (ECM) structure. TNF-α, present primarily during inflammation, cues hMSCs to clear debris and degrade ECM. Because cytokines change how hMSCs degrade their microenvironment and are naturally present in the wound, they also affect how hMSCs migrate out of the scaffold to conduct healing. Therefore, the effects of cytokines on hMSC remodeling are important when designing materials for cell delivery. In this work, we encapsulate hMSCs in a polymer–peptide hydrogel and incubate the scaffolds in media with TGF-β or TNF-α at concentrations similar to those in wounds. Multiple particle tracking microrheology (MPT) measures hMSC-mediated scaffold degradation in response to these cytokines, which mimics aspects of the in vivo microenvironment post-implantation. MPT uses video microscopy to measure Brownian motion of particles in a material, quantifying structure and rheology. Using MPT, we measure increased hMSC-mediated remodeling when cells are exposed to TNF-α and decreased remodeling after exposure to TGF-β when compared to untreated hMSCs. This agrees with previous studies that measure: (1) TNF-α encourages matrix reorganization and (2) TGF-β signals the formation of new matrix. These results enable material design that anticipates changes in remodeling after implantation, improving control over hMSC delivery and healing.