Substrate viscoelasticity affects human macrophage morphology and phagocytosis.

Viscoelasticity is an inherent characteristic of many living tissues and, in an attempt to better recapitulate this aspect in cell culture, hydrogel biomaterials have been engineered to exhibit time-dependent energy-dissipative mechanical behavior. Viscoelastic hydrogel culture platforms have been instrumental in understanding the biological effects of viscoelasticity. Although viscoelasticity has been shown to regulate fundamental cell processes such as spreading and differentiation in adherent cells, the influence of viscoelasticity on macrophage behavior has not been explored. Here, we use a tunable viscoelastic polyacrylamide hydrogel culture system to demonstrate that viscoelasticity is an important biophysical regulator of macrophage function. After biologically validating our system with HS-5 fibroblasts to show behavior consistent with existing reports, we seed human THP-1 monocytes on these viscoelastic substrates and differentiate them into macrophages. THP-1 macrophages become smaller and rounder, and less efficient at phagocytosis on more viscous polyacrylamide hydrogel substrates. Since macrophages play key roles in mounting responses such as inflammation and fibrosis, these results indicate that viscoelasticity is an important parameter in the design of immunomodulatory biomaterials.

Hydrophilic Mechano-Bactericidal Nanopillars Require External Forces to Rapidly Kill Bacteria.

Nanopillars have been shown to mechanically damage bacteria, suggesting a promising strategy for future antibacterial surfaces. However, the mechanisms underlying this phenomena remain unclear, which ultimately limits translational potential toward real-world applications. Using real-time and end-point analysis techniques, we demonstrate that in contrast to initial expectations, bacteria on multiple hydrophilic "mechano-bactericidal" surfaces remained viable unless exposed to a moving air-liquid interface, which caused considerable cell death. Reasoning that normal forces arising from surface tension may underlie this mechano-bactericidal activity, we developed computational and experimental models to estimate, manipulate, and recreate the impact of these forces. Our experiments together demonstrate that a critical level of external force acting on cells attached to nanopillar surfaces can rapidly deform and rupture bacteria. These studies provide fundamental physical insight into how nanopillar surfaces can serve as effective antibacterial materials and suggest use-conditions under which such nanotechnology approaches may provide practical value.

Microfluidic Shear Assay to Distinguish between Bacterial Adhesion and Attachment Strength on Stiffness-Tunable Silicone Substrates.

Tuning surface composition and stiffness is now an established strategy to improve the integration of medical implants. Recent evidence suggests that matrix stiffness affects bacterial adhesion, but contradictory findings have been reported in the literature. Distinguishing between the effects of bacterial adhesion and attachment strength on these surfaces may help interpret these findings. Here, we develop a precision microfluidic shear assay to quantify bacterial adhesion strength on stiffness-tunable and biomolecule-coated silicone materials. We demonstrate that bacteria are more strongly attached to soft silicones, compared to stiff silicones; as determined by retention against increasing shear flows. Interestingly, this effect is reduced when the surface is coated with matrix biomolecules. These results demonstrate that bacteria do sense and respond to stiffness of the surrounding environment and that precisely defined assays are needed to understand the interplay among surface mechanics, composition, and bacterial binding.

Fracture-based fabrication of normally closed, adjustable, and fully reversible microscale fluidic channels.

Adjustable fluidic structures play an important role in microfluidic systems. Fracture of multilayered materials under applied tension has been previously demonstrated as a convenient, simple, and inexpensive approach to fabricate nanoscale adjustable structures; here, it is demonstrated how to extend this concept to the microscale. This is achieved by a novel pairing of materials that leverages fracture mechanics to limit crack formation to a specified region, allowing to create size-controllable and adjustable microfluidic structures. This technique can be used to fabricate "normally closed" microfluidic channels that are completely reversible, a feature that is challenging to achieve in conventional systems without careful engineering controls. The adjustable microfluidic channels are then applied to mechanically lyse single cells, and subsequently manipulate the released nuclear chromatin, creating new possibilities for epigenetic analysis of single cells. This simple, versatile, and robust technology provides an easily accessible pathway to construct adjustable microfluidic structures, which will be useful in developing complex assays and experiments even in resource-limited settings.

Engineering placental trophoblast fusion: A potential role for biomechanics in syncytialization.

The process by which placental trophoblasts fuse to form the syncytiotrophoblast around the chorionic villi is not fully understood. Mechanical features of the in vivo and in vitro culture environments have recently emerged as having the potential to influence fusion efficiency, and considering these mechanical cues may ultimately allow predictive control of trophoblast syncytialization. Here, we review recent studies that suggest that biomechanical factors such as shear stress, tissue stiffness, and dimensionally-related stresses affect villous trophoblast fusion efficiency. We then discuss how these stimuli might arise in vivo and how they can be incorporated in cultures to study and enhance villous trophoblast fusion. We believe that this mechanical paradigm will provide novel insight into manipulating the syncytialization process to better engineer improved models, understand disease progression, and ultimately develop novel therapeutic strategies.

Effects of Surface Topography and Cellular Biomechanics on Nanopillar-Induced Bactericidal Activity.

Bacteria are mechanically resistant biological structures that can sustain physical stress. Experimental data, however, have shown that high-aspect-ratio nanopillars deform bacterial cells upon contact. If the deformation is sufficiently large, it lyses the bacterial cell wall, ultimately leading to cell death. This has prompted a novel strategy, known as mechano-bactericide technology, to fabricate antibacterial surfaces. Although adhesion forces were originally proposed as the driving force for mechano-bactericidal action, it has been recently shown that external forces, such as capillary forces arising from an air-water interface at bacterial surfaces, produce sufficient loads to rapidly kill bacteria on nanopillars. This discovery highlights the need to theoretically examine how bacteria respond to external loads and to ascertain the key factors. In this study, we developed a finite element model approximating bacteria as elastic shells filled with cytoplasmic fluid brought into contact with an individual nanopillar or nanopillar array. This model elucidates that bacterial killing caused by external forces on nanopillars is influenced by surface topography and cell biomechanical variables, including the density and arrangement of nanopillars, in addition to the cell wall thickness and elastic modulus. Considering that surface topography is an important design parameter, we performed experiments using nanopillar arrays with precisely controlled nanopillar diameters and spacing. Consistent with model predictions, these demonstrate that nanopillars with a larger spacing increase bacterial susceptibility to mechanical puncture. The results provide salient insights into mechano-bactericidal activity and identify key design parameters for implementing this technology.

3D Bioprinting of Food Grade Hydrogel Infused with Living Pleurotus ostreatus Mycelium in Non-sterile Conditions.

Mycelium is the root-like network of fungi. Mycelium biocomposites prepared by template replication (molding) can function as environmentally friendly alternatives to conventional polystyrene foams, which are energy- and carbon-intensive to manufacture. Recently, several studies have shown that 3D bioprinting technologies can be used to produce high value functional mycelium products with intricate geometries that are otherwise difficult or impossible to achieve via template replication. A diverse range of nutrients, thickeners, and gelling agents can be combined to produce hydrogels suitable for 3D bioprinting. 3D bioprinting with hydrogel formulations infused with living fungi produces engineered living materials that continue to grow after bioprinting is complete. However, a hydrogel formulation optimized for intricate 3D bioprinting of Pleurotus ostreatus mycelium, which is among the strains most commonly used in mycelium biocomposite fabrication, has yet to be described. Here, we design and evaluate a versatile hydrogel formulation consisting of malt extract (nutrient), carboxymethylcellulose and cornstarch (thickeners), and agar (gelling agent), all of which are easily sourced food grade reagents. We also outline a reproducible workflow to infuse this hydrogel with P. ostreatus liquid culture for 3D bioprinting of intricate structures comprised of living P. ostreatus mycelium and characterize the changes in height and mass as well as hardness of the prints during mycelium growth. Finally, we demonstrate that the workflow does not require a sterile bioprinting environment to achieve successful prints and that the same mycelium-infused hydrogel can be supplemented with additives such as sawdust to produce mycelium biocomposite objects. These findings demonstrate that 3D bioprinting using mycelium-based feedstocks could be a promising biofabrication technique to produce engineered living materials for applications such as mushroom cultivation, food preparation, or construction of the built environment.

Directed biomechanical compressive forces enhance fusion efficiency in model placental trophoblast cultures.

The syncytiotrophoblast is a multinucleated structure that arises from fusion of mononucleated cytotrophoblasts, to sheath the placental villi and regulate transport across the maternal-fetal interface. Here, we ask whether the dynamic mechanical forces that must arise during villous development might influence fusion, and explore this question using in vitro choriocarcinoma trophoblast models. We demonstrate that mechanical stress patterns arise around sites of localized fusion in cell monolayers, in patterns that match computational predictions of villous morphogenesis. We then externally apply these mechanical stress patterns to cell monolayers and demonstrate that equibiaxial compressive stresses (but not uniaxial or equibiaxial tensile stresses) enhance expression of the syndecan-1 and loss of E-cadherin as markers of fusion. These findings suggest that the mechanical stresses that contribute towards sculpting the placental villi may also impact fusion in the developing tissue. We then extend this concept towards 3D cultures and demonstrate that fusion can be enhanced by applying low isometric compressive stresses to spheroid models, even in the absence of an inducing agent. These results indicate that mechanical stimulation is a potent activator of cellular fusion, suggesting novel avenues to improve experimental reproductive modelling, placental tissue engineering, and understanding disorders of pregnancy development.

In Situ Measurement of Intra-tumoral Tissue Rigidity.

Local tissue scale mechanical properties are essential for understanding cell fate and function; however, few methods to measure stiffness at this length scale exist, and applications in 3D tissues can present further challenges. To address this need, microgel-based sensors fabricated out of the thermally responsive hydrogel poly(N-isopropylacrylamide) were developed allowing internal architectures of tissues to be mapped by optically measuring microgel response when actuated in a matrix. These robust probes are widely applicable for in vitro and in vivo studies of tissue mechanics providing tissues can be fluorescently imaged. Here we describe the fabrication of these thermally responsive hydrogel sensors, calibration of the microgels using phantom tissues, and image processing techniques used to make the measurements.

pH-Responsive Reversible Granular Hydrogels Based on Metal-Binding Mussel-Inspired Peptides.

Taking advantage of their thixotropic behavior, microporosity, and modular properties, granular hydrogels formed from jammed hydrogel microparticles have emerged as an exciting class of soft, injectable materials useful for numerous applications, ranging from the production of biomedical scaffolds for tissue repair to the therapeutic delivery of drugs and cells. Recently, the annealing of hydrogel microparticles in situ to yield a porous bulk scaffold has shown numerous benefits in regenerative medicine, including tissue-repair applications. Current annealing techniques, however, mainly rely either on covalent connections, which produce static scaffolds, or transient supramolecular interactions, which produce dynamic but mechanically weak hydrogels. To address these limitations, we developed microgels functionalized with peptides inspired by the histidine-rich cross-linking domains of marine mussel byssus proteins. Functionalized microgels can reversibly aggregate in situ via metal coordination cross-linking to form microporous, self-healing, and resilient scaffolds at physiological conditions by inclusion of minimal amounts of zinc ions at basic pH. Aggregated granular hydrogels can subsequently be dissociated in the presence of a metal chelator or under acidic conditions. Based on the demonstrated cytocompatibility of these annealed granular hydrogel scaffolds, we believe that these materials could be developed toward applications in regenerative medicine and tissue engineering.

Ultrasoft edge-labelled hydrogel sensors reveal internal tissue stress patterns in invasive engineered tumors.

Measuring internal mechanical stresses within 3D tissues can provide important insights into drivers of morphogenesis and disease progression. Cell-sized hydrogel microspheres have recently emerged as a powerful technique to probe tissue mechanobiology, as they can be sufficiently soft as to deform within remodelling tissues, and optically imaged to measure internal stresses. However, measuring stresses at resolutions of ∼10 Pa requires ultrasoft, low-polymer content hydrogel formulations that are challenging to label with sufficiently fluorescent materials to support repeated measurements, particularly in optically dense tissues over 100 µm thick, as required in cancer tumor models. Here, we leverage thermodynamic partitioning of hydrogel components to create "edge-labelled" ultrasoft hydrogel microdroplets, in a single polymerization step. Bright and stable fluorescent nanoparticles preferentially polymerize at the hydrogel droplet interface, and can be used to repeatedly track sensor surfaces over long-term experiments, even when embedded deep in light-scattering tissues. We utilize these edge-labelled microspherical stress gauges (eMSGs) in inducible breast cancer tumor models of invasion, and demonstrate distinctive internal stress patterns that arise from cell-matrix interactions at different stages of breast cancer progression. Our studies demonstrate a long-term macroscale compaction of the tumor during matrix encapsulation, but only a short-term increase in local stress as non-invasive tumors rapidly make small internal reorganizations that reduce the mechanical stress to baseline levels. In contrast, once invasion programs are initiated, internal stress throughout the tumor is negligible. These findings suggest that internal tumor stresses may initially prime the cells to invade, but are lost once invasion occurs. Together, this work demonstrates that mapping internal mechanical stress in tumors may have utility in advancing cancer prognostic strategies, and that eMSGs can have broad utility in understanding dynamic mechanical processes of disease and development.

Compressive molding of engineered tissues via thermoresponsive hydrogel devices.

Biofabrication of tissues requires sourcing appropriate combinations of cells, and then arranging those cells into a functionally-useful construct. Recently, organoids with diverse cell populations have shown great promise as building blocks from which to assemble more complex structures. However, organoids typically adopt spherical or uncontrolled morphologies, which intrinsically limit the tissue structures that can be produced using this bioassembly technique. Here, we develop microfabricated smart hydrogel platforms in thermoresponsive poly(N-isopropylacrylamide) to compressively mold microtissues such as spheroids or organoids into customized forms, on demand. These Compressive Hydrogel Molders (CHyMs) compact at cell culture temperatures to force loaded tissues into a new shape, and then expand to release the tissues for downstream applications. As a first demonstration, breast cancer spheroids were biaxially compacted in cylindrical cavities, and uniaxially compacted in rectangular ones. Spheroid shape changes persisted after the tissues were released from the CHyMs. We then demonstrate long-term molding of spherical brain organoids in ring-shaped CHyMs over one week. Fused bridges formed only when brain organoids were encased in Matrigel, and the resulting ring-shaped organoids expressed tissue markers that correspond with expected differentiation profiles. These results demonstrate that tissues differentiate appropriately even during long-term molding in a CHyM. This platform hence provides a new tool to shape pre-made tissues as desired, via temporary compression and release, allowing an exploration of alternative organoid geometries as building blocks for bioassembly applications.

Engineering physical microenvironments to study innate immune cell biophysics.

Innate immunity forms the core of the human body's defense system against infection, injury, and foreign objects. It aims to maintain homeostasis by promoting inflammation and then initiating tissue repair, but it can also lead to disease when dysregulated. Although innate immune cells respond to their physical microenvironment and carry out intrinsically mechanical actions such as migration and phagocytosis, we still do not have a complete biophysical description of innate immunity. Here, we review how engineering tools can be used to study innate immune cell biophysics. We first provide an overview of innate immunity from a biophysical perspective, review the biophysical factors that affect the innate immune system, and then explore innate immune cell biophysics in the context of migration, phagocytosis, and phenotype polarization. Throughout the review, we highlight how physical microenvironments can be designed to probe the innate immune system, discuss how biophysical insight gained from these studies can be used to generate a more comprehensive description of innate immunity, and briefly comment on how this insight could be used to develop mechanical immune biomarkers and immunomodulatory therapies.

The case for cancer-associated fibroblasts: essential elements in cancer drug discovery?

Although cancer-associated fibroblasts (CAFs) have gained increased attention for supporting cancer progression, current CAF-targeted therapeutic options are limited and failing in clinical trials. As the largest component of the tumor microenvironment (TME), CAFs alter the biochemical and physical structure of the TME, modulating cancer progression. Here, we review the role of CAFs in altering drug response, modifying the TME mechanics and the current models for studying CAFs. To provide new perspectives, we highlight key considerations of CAF activity and discuss emerging technologies that can better address CAFs; and therefore, increase the likelihood of therapeutic efficacy. We argue that CAFs are crucial components of the cancer drug discovery pipeline and incorporating these cells will improve drug discovery success rates.

Recent advances in cancer research have improved our understanding of disease progression; however, the number of drugs failing in clinical trials remains high and therefore, present a critical challenge for cancer drug discovery. Although the interactions of the tissue surrounding the tumor, the tumor microenvironment, are now considered key targets for new interventions in cancer, the role of microenvironment is largely absent in drug discovery pipelines. Here we explore the role of the most prominent cell type in the tumor microenvironment, cancer-associated fibroblasts (CAFs), in altering cancer therapy response and ultimately patient outcome. To provide new perspectives for future studies, we draw attention to key complications of CAF biology and highlight emerging technologies that could be used to address this. We believe including CAFs in drug discovery, whether for targeting cancer cells or the microenvironment, will allow for a better understanding of therapeutic efficacy and ultimately improve clinical outcome.

Integrating mechanical sensor readouts into organ-on-a-chip platforms.

Organs-on-a-chip have emerged as next-generation tissue engineered models to accurately capture realistic human tissue behaviour, thereby addressing many of the challenges associated with using animal models in research. Mechanical features of the culture environment have emerged as being critically important in designing organs-on-a-chip, as they play important roles in both stimulating realistic tissue formation and function, as well as capturing integrative elements of homeostasis, tissue function, and tissue degeneration in response to external insult and injury. Despite the demonstrated impact of incorporating mechanical cues in these models, strategies to measure these mechanical tissue features in microfluidically-compatible formats directly on-chip are relatively limited. In this review, we first describe general microfluidically-compatible Organs-on-a-chip sensing strategies, and categorize these advances based on the specific advantages of incorporating them on-chip. We then consider foundational and recent advances in mechanical analysis techniques spanning cellular to tissue length scales; and discuss their integration into Organs-on-a-chips for more effective drug screening, disease modeling, and characterization of biological dynamics.

Microfluidic Study of Bacterial Attachment on and Detachment from Zinc Oxide Nanopillars.

Nanopillars can influence how bacterial cells attach to a surface. Herein, we investigated whether self-assembled zinc oxide (ZnO) nanopillars synthesized on glass substrates via the conventional hydrothermal route possess anti-biofouling properties either by reducing the amount of initially attached cells or promoting the detachment of cells from the surface or both. To avoid complications associated with manual intervention methods of assessing bacterial attachment on nanopillar surfaces, we implemented a microfluidic approach. In our study, we synthesized two nanopillar topographies: a low surface density of ZnO nanopillars and a high surface density of ZnO nanopillars. Next, we mounted microfluidic channels to each of these substrates. This microfluidic approach allowed us to gently flow Pseudomonas aeruginosa, Staphylococcus aureus, or Bacillus subtilis cells onto the nanopillars for initial attachment before systematically increasing the flowrate to attempt to detach remaining attached cells without introducing air-liquid interface artefacts during the assay. Generally, initial bacterial attachment was similar across all substrates. However, cells consistently detached more readily from high-surface-density nanopillars compared to low-surface-density nanopillars. Electron microscopy revealed that cells that attached to high-surface-density nanopillars rested atop the nanopillars, fully exposed to microfluidic shear, whereas many cells became trapped in the void space between neighboring low-surface-density nanopillars, shielding these cells from detachment. Our findings indicate that self-assembled ZnO nanopillars can provide anti-biofouling properties under submerged flow but only if synthesized at high surface density.

Surface Wettability Is a Key Feature in the Mechano-Bactericidal Activity of Nanopillars.

Nanopillar-textured surfaces are of growing interest because of their ability to kill bacteria through physical damage without relying on antimicrobial chemicals. Although research on antibacterial nanopillars has progressed significantly in recent years, the effect of nanopillar hydrophobicity on bactericidal activity remains elusive. In this study, we investigated the mechano-bactericidal efficacy of etched silicon nanopillars against Pseudomonas aeruginosa at nanopillar hydrophobicities from superhydrophilic to superhydrophobic. Assessing cell viability and bacterial morphology in immersed wet conditions, we observed negligible bactericidal activity; however, air/liquid interface displacement during water evaporation established a bactericidal effect that strongly depends on substrate hydrophobicity. Specifically, bactericidal activity was highest on superhydrophilic surfaces but abated with increasing hydrophobicity, diminishing at substrate contact angles larger than 90°. Calculation of the surface tension and Laplace pressure forces during water evaporation for each substrate subsequently highlighted that the total capillary force, as an external driving force responsible for bacterial deformation, is significantly weaker on hydrophobic substrates. These findings suggest that superhydrophilic nanopillared surfaces are a superior choice for mechano-bactericidal activity, whereas superhydrophobic surfaces, although not bactericidal, may have antibiofouling properties through their self-cleaning effect. These findings provide new insights into the design and application of nanopillared surfaces as functional antibacterial materials.

Decellularized extracellular matrix: New promising and challenging biomaterials for regenerative medicine.

Extracellular matrix is rich in biomolecules including structural proteins, glycosaminoglycans, and small molecules that are important for the maintenance and repair of tissue. Decellularized extracellular matrix (dECM) is expected to retain these key biomolecules and makes it a promising biomaterial candidate for regenerative medicine applications. To date, dECM-particle based biomaterials have been developed to engineer over 15 tissue types or organs, with the ultimate goal of mimicking specific biological and physical properties of the native tissue. The most common scaffold types are injectable hydrogels, electrospun scaffolds and bioprinted scaffolds. The purpose of this review paper is to highlight key challenges, fabrication methods and progress made for each tissue type, along with the discussion of other elements that are integral to push dECM biomaterials towards effective and specialized tissue repair.

Engineered models for placental toxicology: Emerging approaches based on tissue decellularization.

Recent increases in prescriptions and illegal drug use as well as exposure to environmental contaminants during pregnancy have highlighted the critical importance of placental toxicology in understanding and identifying risks to both mother and fetus. Although advantageous for basic science, current in vitro models often fail to capture the complexity of placental response, likely due to their inability to recreate and monitor aspects of the microenvironment including physical properties, mechanical forces and stiffness, protein composition, cell-cell interactions, soluble and physicochemical factors, and other exogenous cues. Tissue engineering holds great promise in addressing these challenges and provides an avenue to better understand basic biology, effects of toxic compounds and potential therapeutics. The key to success lies in effectively recreating the microenvironment. One strategy to do this would be to recreate individual components and then combine them. However, this becomes challenging due to variables present according to conditions such as tissue location, age, health status and lifestyle. The extracellular matrix (ECM) is known to influence cellular fate by working as a storage of factors. Decellularized ECM (dECM) is a recent tool that allows usage of the original ECM in a refurbished form, providing a relatively reliable representation of the microenvironment. This review focuses on using dECM in modified forms such as whole organs, scaffold sheets, electrospun nanofibers, hydrogels, 3D printing, and combinations as building blocks to recreate aspects of the microenvironment to address general tissue engineering and toxicology challenges, thus illustrating their potential as tools for future placental toxicology studies.

Hydrogel Mechanics Influence the Growth and Development of Embedded Brain Organoids.

Brain organoids are three-dimensional, tissue-engineered neural models derived from induced pluripotent stem cells that enable studies of neurodevelopmental and disease processes. Mechanical properties of the microenvironment are known to be critical parameters in tissue engineering, but the mechanical consequences of the encapsulating matrix on brain organoid growth and development remain undefined. Here, Matrigel was modified with an interpenetrating network (IPN) of alginate, to tune the mechanical properties of the encapsulating matrix. Brain organoids grown in IPNs were viable, with the characteristic formation of neuroepithelial buds. However, organoid growth was significantly restricted in the stiffest matrix tested. Moreover, stiffer matrixes skewed cell populations toward mature neuronal phenotypes, with fewer and smaller neural rosettes. These findings demonstrate that the mechanics of the culture environment are important parameters in brain organoid development and show that the self-organizing capacity and subsequent architecture of brain organoids can be modulated by forces arising from growth-induced compression of the surrounding matrix. This study therefore suggests that carefully designing the mechanical properties of organoid encapsulation materials is a potential strategy to direct organoid growth and maturation toward desired structures.