Microrheology encompasses a range of methods to measure the mechanical properties of soft materials. By characterizing the motion of embedded microscopic particles, microrheology extends the probing length scale and frequency range of conventional bulk rheology. Microrheology can be characterized into either passive or active methods based on the driving force exerted on probe particles. Tracer particles are driven by thermal energy in passive methods, applying minimal deformation to the assessed medium. In active techniques, particles are manipulated by an external force, most commonly produced through optical and magnetic fields. Small-scale rheology holds significant advantages over conventional bulk rheology, such as eliminating the need for large sample sizes, the ability to probe fragile materials non-destructively, and a wider probing frequency range. More importantly, some microrheological techniques can obtain spatiotemporal information of local microenvironments and accurately describe the heterogeneity of structurally complex fluids. Recently, there has been significant growth in using these minimally invasive techniques to investigate a wide range of biomedical systems both in vitro and in vivo. Here, we review the latest applications and advancements of microrheology in mammalian cells, tissues, and biofluids and discuss the current challenges and potential future advances on the horizon.

Biomechanical cues such as shear stress, stretching, compression, and matrix elasticity are vital in the establishment of next generation physiological in vitro tissue models. Matrix elasticity, for instance, is known to guide stem cell differentiation, influence healing processes and modulate extracellular matrix (ECM) deposition needed for tissue development and maintenance. To better understand the biomechanical effect of matrix elasticity on the formation of articular cartilage analogs in vitro, this study aims at assessing the redifferentiation capacity of primary human chondrocytes in three different hydrogel matrices of predefined matrix elasticities. The hydrogel elasticities were chosen to represent a broad spectrum of tissue stiffness ranging from very soft tissues with a Young's modulus of 1 kPa up to elasticities of 30 kPa, representative of the perichondral-space. In addition, the interplay of matrix elasticity and transforming growth factor beta-3 (TGF-β3) on the redifferentiation of primary human articular chondrocytes was studied by analyzing both qualitative (viability, morphology, histology) and quantitative (RT-qPCR, sGAG, DNA) parameters, crucial to the chondrotypic phenotype. Results show that fibrin hydrogels of 30 kPa Young's modulus best guide chondrocyte redifferentiation resulting in a native-like morphology as well as induces the synthesis of physiologic ECM constituents such as glycosaminoglycans (sGAG) and collagen type II. This comprehensive study sheds light onto the mechanobiological impact of matrix elasticity on formation and maintenance of articular cartilage and thus represents a major step toward meeting the need for advanced in vitro tissue models to study both re- and degeneration of articular cartilage.

Many antibiotics and antimicrobial agents have the bacterial cell envelope as their primary target, interfering with functions such as synthesis of peptidoglycan, membrane stability and permeability, and attachment of surface components. The cell envelope is the outermost barrier of the bacterial cell, conferring protection against environmental stresses, and maintaining structural integrity and stability of the growing cell, while still allowing for required metabolism. In this work, inhibitory concentrations of several different cell envelope targeting antibiotics and antimicrobial agents were used to select for derivatives of lactic acid bacteria (LAB) with improved properties for dairy applications. Interestingly, we observed that for several LAB species a fraction of the isolates had improved milk texturizing capabilities. To further improve our understanding of the mechanisms underlying the improved rheology and to validate the efficacy of this method for strain improvement, genetic and physiological characterization of several improved derivatives was performed. The results showed that the identified genetic changes are diverse and affect also other cellular functions than the targeted cell surface. In short, this study describes a new versatile and powerful toolbox based on targeting of the cell envelope to select for LAB derivatives with improved phenotypic traits for dairy applications.

The high viscosities/yield stresses of lignocellulose slurries makes their industrial processing a significant challenge. However, little is known regarding the degree to which liquefaction and its enzymatic requirements are specific to a substrate's physicochemical and rheological properties. In the work reported here, the substrate- and rheological regime-specificities of liquefaction of various substrates were assessed using real-time in-rheometer viscometry and offline oscillatory rheometry when hydrolyzed by combinations of cellobiohydrolase (Trichoderma reesei Cel7A), endoglucanase (Humicola insolens Cel45A), glycoside hydrolase (GH) family 10 xylanase, and GH family 11 xylanase. In contrast to previous work that has suggested that endoglucanase activity dominates enzymatic liquefaction, all of the enzymes were shown to have at least some liquefaction capacity depending on the substrate and reaction conditions. The contribution of individual enzymes was found to be influenced by the rheological regime; in the concentrated regime, the cellobiohydrolase outperformed the endoglucanase, achieving 2.4-fold higher yield stress reduction over the same timeframe, whereas the endoglucanase performed best in the semi-dilute regime. It was apparent that the significant differences in rheology and liquefaction mechanisms made it difficult to predict the liquefaction capacity of an enzyme or enzyme cocktail at different substrate concentrations.

Introduction: In the view of 3D-bioprinting with cell models representative of neural cells, we produced inks to mimic the basic viscoelastic properties of brain tissue. Moving from the concept that rheology provides useful information to predict ink printability, this study improves and expands the potential of the previously published 3D-reactive printing approach by introducing pH as a key parameter to be controlled, together with printing time. Methods: The viscoelastic properties, printability, and microstructure of pectin gels crosslinked with CaCO3 were investigated and their composition was optimized (i.e., by including cell culture medium, HEPES buffer, and collagen). Different cell models representative of the major brain cell populations (i.e., neurons, astrocytes, microglial cells, and oligodendrocytes) were considered. Results and Discussion: The outcomes of this study propose a highly controllable method to optimize the printability of internally crosslinked polysaccharides, without the need for additives or post-printing treatments. By introducing pH as a further parameter to be controlled, it is possible to have multiple (pH-dependent) crosslinking kinetics, without varying hydrogel composition. In addition, the results indicate that not only cells survive and proliferate following 3D-bioprinting, but they can also interact and reorganize hydrogel microstructure. Taken together, the results suggest that pectin-based hydrogels could be successfully applied for neural cell culture.

Intracerebral hemorrhage (ICH) is a devastating subtype of stroke with high morbidity and mortality. However, there is no effective therapy method to improve its clinical outcomes to date. Here we report an injectable gelatin hydrogel that is capable of suppressing inflammation and enhancing functional recovery in a mouse model of ICH. Thiolated gelatin was synthesized by EDC chemistry and then the hydrogel was formed through Michael addition reaction between the thiolated gelatin and polyethylene glycol diacrylate. The hydrogel was characterized by scanning electron microscopy, porosity, rheology, and cytotoxicity before evaluating in a mouse model of ICH. The in vivo study showed that the hydrogel injection into the ICH lesion reduced the neuron loss, attenuated the neurological deficit post-operation, and decreased the activation of the microglia/macrophages and astrocytes. More importantly, the pro-inflammatory M1 microglia/macrophages polarization was suppressed while the anti-inflammatory M2 phenotype was promoted after the hydrogel injection. Besides, the hydrogel injection reduced the release of inflammatory cytokines (IL-1β and TNF-α). Moreover, integrin β1 was confirmed up-regulated around the lesion that is positively correlated with the M2 microglia/macrophages. The related mechanism was proposed and discussed. Taken together, the injectable gelatin hydrogel suppressed the inflammation which might contribute to enhance the functional recovery of the ICH mouse, making it a promising application in the clinic.