Actuation of Soft Thermoresponsive Hydrogels Mechanically Stimulates Osteogenesis in Human Mesenchymal Stem Cells without Biochemical Factors.

Mesenchymal stem cells (MSCs) have the potential to differentiate into multiple lineages and can be harvested relatively easily from adults, making them a promising cell source for regenerative therapies. While it is well-known how to consistently differentiate MSCs into adipose, chondrogenic, and osteogenic lineages by treatment with biochemical factors, the number of studies exploring how to achieve this with mechanical signals is limited. A relatively unexplored area is the effect of cyclic forces on the MSC differentiation. Recently, our group developed a thermoresponsive N-ethyl acrylamide/N-isopropylacrylamide (NIPAM/NEAM) hydrogel supplemented with gold nanorods that are able to convert near-infrared light into heat. Using light pulses allows for local hydrogel collapse and swelling with physiologically relevant force and frequency. In this study, MSCs are cultured on this hydrogel system with a patterned surface and exposed to intermittent or continuous actuation of the hydrogel for 3 days to study the effect of actuation on MSC differentiation. First, cells are harvested from the bone marrow of three donors and tested for their MSC phenotype, meeting the following criteria: the harvested cells are adherent and demonstrate a fibroblast-like bipolar morphology. They lack the expression of CD34 and CD45 but do express CD73, CD90, and CD105. Additionally, their differentiation potential into adipogenic, chondrogenic, and osteogenic lineages is validated by the addition of standardized differentiation media. Next, MSCs are exposed to intermittent or continuous actuation, which leads to a significantly enhanced cell spreading compared to nonactuated cells. Moreover, actuation results in nuclear translocation of Runt-related transcription factor 2 and the Yes-associated protein. Together, these results indicate that cyclic mechanical stimulation on a soft, ridged substrate modulates the MSC fate commitment in the direction of osteogenesis.

A keratin code defines the textile nature of epithelial tissue architecture.

We suggest that the human body can be viewed as of textile nature whose fabric consists of interconnected fiber systems. These fiber systems form highly dynamic scaffolds, which respond to environmental changes at different temporal and spatial scales. This is especially relevant at sites where epithelia border on connective tissue regions that are exposed to dynamic microenvironments. We propose that the enormous heterogeneity and adaptability of epithelia are based on a "keratin code", which results from the cell-specific expression and posttranslational modification of keratin isotypes. It thereby defines unique cytoskeletal intermediate filament networks that are coupled across cells and to the correspondingly heterogeneous fibers of the underlying extracellular matrix. The resulting fabric confers unique local properties.

Mechanobiological implications of age-related remodelling in the outer retina.

The outer retina consists of the light-sensitive photoreceptors, the pigmented epithelium, and the choroid, which interact in a complex manner to sustain homeostasis. The organisation and function of these cellular layers are mediated by the extracellular matrix compartment named Bruch's membrane, situated between the retinal epithelium and the choroid. Like many tissues, the retina experiences age-related structural and metabolic changes, which are relevant for understanding major blinding diseases of the elderly, such as age-related macular degeneration. Compared with other tissues, the retina mainly comprises postmitotic cells, making it less able to maintain its mechanical homeostasis over the years functionally. Aspects of retinal ageing, like the structural and morphometric changes of the pigment epithelium and the heterogenous remodelling of the Bruch's membrane, imply changes in tissue mechanics and may affect functional integrity. In recent years, findings in the field of mechanobiology and bioengineering highlighted the importance of mechanical changes in tissues for understanding physiological and pathological processes. Here, we review the current knowledge of age-related changes in the outer retina from a mechanobiological perspective, aiming to generate food for thought for future mechanobiology studies in the outer retina.

Combining Image Restoration and Traction Force Microscopy to Study Extracellular Matrix-Dependent Keratin Filament Network Plasticity.

Keratin intermediate filaments are dynamic cytoskeletal components that are responsible for tuning the mechanical properties of epithelial tissues. Although it is known that keratin filaments (KFs) are able to sense and respond to changes in the physicochemical properties of the local niche, a direct correlation of the dynamic three-dimensional network structure at the single filament level with the microenvironment has not been possible. Using conventional approaches, we find that keratin flow rates are dependent on extracellular matrix (ECM) composition but are unable to resolve KF network organization at the single filament level in relation to force patterns. We therefore developed a novel method that combines a machine learning-based image restoration technique and traction force microscopy to decipher the fine details of KF network properties in living cells grown on defined ECM patterns. Our approach utilizes Content-Aware Image Restoration (CARE) to enhance the temporal resolution of confocal fluorescence microscopy by at least five fold while preserving the spatial resolution required for accurate extraction of KF network structure at the single KF/KF bundle level. The restored images are used to segment the KF network, allowing numerical analyses of its local properties. We show that these tools can be used to study the impact of ECM composition and local mechanical perturbations on KF network properties and corresponding traction force patterns in size-controlled keratinocyte assemblies. We were thus able to detect increased curvature but not length of KFs on laminin-322 versus fibronectin. Photoablation of single cells in microprinted circular quadruplets revealed surprisingly little but still significant changes in KF segment length and curvature that were paralleled by an overall reduction in traction forces without affecting global network orientation in the modified cell groups irrespective of the ECM coating. Single cell analyses furthermore revealed differential responses to the photoablation that were less pronounced on laminin-332 than on fibronectin. The obtained results illustrate the feasibility of combining multiple techniques for multimodal monitoring and thereby provide, for the first time, a direct comparison between the changes in KF network organization at the single filament level and local force distribution in defined paradigms.

Quantitative mapping of keratin networks in 3D.

Mechanobiology requires precise quantitative information on processes taking place in specific 3D microenvironments. Connecting the abundance of microscopical, molecular, biochemical, and cell mechanical data with defined topologies has turned out to be extremely difficult. Establishing such structural and functional 3D maps needed for biophysical modeling is a particular challenge for the cytoskeleton, which consists of long and interwoven filamentous polymers coordinating subcellular processes and interactions of cells with their environment. To date, useful tools are available for the segmentation and modeling of actin filaments and microtubules but comprehensive tools for the mapping of intermediate filament organization are still lacking. In this work, we describe a workflow to model and examine the complete 3D arrangement of the keratin intermediate filament cytoskeleton in canine, murine, and human epithelial cells both, in vitro and in vivo. Numerical models are derived from confocal airyscan high-resolution 3D imaging of fluorescence-tagged keratin filaments. They are interrogated and annotated at different length scales using different modes of visualization including immersive virtual reality. In this way, information is provided on network organization at the subcellular level including mesh arrangement, density and isotropic configuration as well as details on filament morphology such as bundling, curvature, and orientation. We show that the comparison of these parameters helps to identify, in quantitative terms, similarities and differences of keratin network organization in epithelial cell types defining subcellular domains, notably basal, apical, lateral, and perinuclear systems. The described approach and the presented data are pivotal for generating mechanobiological models that can be experimentally tested.

Integrin α5ß1 nano-presentation regulates collective keratinocyte migration independent of substrate rigidity.

Nanometer-scale properties of the extracellular matrix influence many biological processes, including cell motility. While much information is available for single-cell migration, to date, no knowledge exists on how the nanoscale presentation of extracellular matrix receptors influences collective cell migration. In wound healing, basal keratinocytes collectively migrate on a fibronectin-rich provisional basement membrane to re-epithelialize the injured skin. Among other receptors, the fibronectin receptor integrin α5ß1 plays a pivotal role in this process. Using a highly specific integrin α5ß1 peptidomimetic combined with nanopatterned hydrogels, we show that keratinocyte sheets regulate their migration ability at an optimal integrin α5ß1 nanospacing. This efficiency relies on the effective propagation of stresses within the cell monolayer independent of substrate stiffness. For the first time, this work highlights the importance of extracellular matrix receptor nanoscale organization required for efficient tissue regeneration.

Mechanobiology of Epithelia From the Perspective of Extracellular Matrix Heterogeneity.

Understanding the complexity of the extracellular matrix (ECM) and its variability is a necessary step on the way to engineering functional (bio)materials that serve their respective purposes while relying on cell adhesion. Upon adhesion, cells receive messages which contain both biochemical and mechanical information. The main focus of mechanobiology lies in investigating the role of this mechanical coordination in regulating cellular behavior. In recent years, this focus has been additionally shifted toward cell collectives and the understanding of their behavior as a whole mechanical continuum. Collective cell phenomena very much apply to epithelia which are either simple cell-sheets or more complex three-dimensional structures. Researchers have been mostly using the organization of monolayers to observe their collective behavior in well-defined experimental setups in vitro. Nevertheless, recent studies have also reported the impact of ECM remodeling on epithelial morphogenesis in vivo. These new concepts, combined with the knowledge of ECM biochemical complexity are of key importance for engineering new interactive materials to support both epithelial remodeling and homeostasis. In this review, we summarize the structure and heterogeneity of the ECM before discussing its impact on the epithelial mechanobiology.

The role of basement membrane laminins in vascular function.

The extracellular matrix is an integral component of the vasculature, contributing to both developmental processes and structural and functional homeostasis. We describe here the types of extracellular matrices that occur in different blood vessel types, ranging from capillaries to veins, venules and arteries, and focus on the endothelial basement membranes and the laminin family of proteins. We summarize data on the molecular composition of endothelial basement membranes, the structure and in vivo expression patterns of the main endothelial laminin isoforms (laminins 411 and 511) and their, to date, deciphered functions in the vasculature. A significant portion of the review focuses on postcapillary venules and leukocyte extravasation and how the endothelial laminins affect adhesion and migration of different leukocyte types, but also how laminins affect endothelial barrier function by modulating expression and localization of endothelial cell-cell junction molecules, and how these effects differ in CNS versus non-CNS tissues. Comparisons are made to small artery dilation in response to shear flow, which has been shown to be dependent on endothelial laminins and junctional complexes. The data discussed support a central role for basement membrane laminins in different aspects of micro- and macro-vessel endothelial function, but also reveal that many open questions remain, including the contribution of perivascular cells which are either embedded or in direct contact with the endothelial cell basement membrane laminins.

Surface Immobilized E-Cadherin Mimetic Peptide Regulates the Adhesion and Clustering of Epithelial Cells.

Cadherin mimetic peptides are widely used in synthetic biomaterials to mimic cell-cell adhesion in cell microniches. This mimicry regulates various cell behaviors. Although the interaction between immobilized cadherin and cells is investigated in numerous studies, the exact manner of functioning of cadherin mimetic peptides is yet to be fully understood. Cadherin mimetic peptides mimic only the critical amino acid sequence of cadherin and are not equal to these proteins in function. Compared to the cadherin proteins, mimetic peptides are more stable, easier to fabricate, and exhibit a precise chemical composition. In this study the E-cadherin mimetic peptide His-Ala-Val (HAV) on material surfaces is immobilized and epithelial cell adhesion and clustering are studied. The results suggest that immobilized HAV peptides specifically interact with E-cadherin on the cell membrane, resulting in an increased expression of E-cadherin and its downstream signaling protein ß-catenin. This interaction relocates E-cadherin-based adhesion from the cell-cell interface to the cell-materials interface, which promotes cell adhesion via mechanosensing and initiates a transition in the cell cluster from a solid-like to a fluid-like state. The study presents an overview of the interactions between E-cadherin mimetic peptide and epithelial cells to aid in the design of novel biomaterials.

NTA-Co3+-His6 versus NTA-Ni2+-His6 mediated E-Cadherin surface immobilization enhances cellular traction.

Understanding the biological impact of strategies for protein immobilization onto bioactive surfaces is crucial for the design of biomimetic materials. A common strategy used to immobilize or label recombinant proteins is to exploit the Ni2+-mediated interaction of nitrilotriacetic acid (NTA) with the hexahistidine tag (His6-tag) present on recombinant proteins. While this method ensures a controlled orientation and functionality of the protein, the kinetically labile nature of the bond ensures only its weak immobilization onto the surface. Recently, it has been shown that the oxidation of Co2+ to Co3+ greatly stabilizes the bond between NTA and the His6-tagged proteins, making it inert to ligand exchange and resistant to chelators. This approach not only has the potential to improve the quality of biomimetic material functionalization and molecule labeling but could also affect cellular mechanical responses for which the mechanical strength of the protein-surface bond is crucial. Here, we compared gold (Au) nanopatterned polyacrylamide (PAA) hydrogels functionalized with E-cadherin via Co3+ with those functionalized via Ni2+ for studying adhesion-mediated responses in keratinocytes. We show that keratinocytes develop higher and a broader range of adhesion forces, leading to extended cell spreading and colony organization on Co3+ vs. Ni2+. This work uniquely shows that stabilizing the NTA/His6-tag bond via Co3+ for protein immobilization significantly impacts cellular phenotype on biomimetic materials by impacting cell signaling.

Mechanical interactions among followers determine the emergence of leaders in migrating epithelial cell collectives.

Regulating the emergence of leaders is a central aspect of collective cell migration, but the underlying mechanisms remain ambiguous. Here we show that the selective emergence of leader cells at the epithelial wound-margin depends on the dynamics of the follower cells and is spatially limited by the length-scale of collective force transduction. Owing to the dynamic heterogeneity of the monolayer, cells behind the prospective leaders manifest locally increased traction and monolayer stresses much before these leaders display any phenotypic traits. Followers, in turn, pull on the future leaders to elect them to their fate. Once formed, the territory of a leader can extend only to the length up-to which forces are correlated, which is similar to the length up-to which leader cells can transmit forces. These findings provide mechanobiological insight into the hierarchy in cell collectives during epithelial wound healing.

Cell-matrix signals specify bone endothelial cells during developmental osteogenesis.

Blood vessels in the mammalian skeletal system control bone formation and support haematopoiesis by generating local niche environments. While a specialized capillary subtype, termed type H, has been recently shown to couple angiogenesis and osteogenesis in adolescent, adult and ageing mice, little is known about the formation of specific endothelial cell populations during early developmental endochondral bone formation. Here, we report that embryonic and early postnatal long bone contains a specialized endothelial cell subtype, termed type E, which strongly supports osteoblast lineage cells and later gives rise to other endothelial cell subpopulations. The differentiation and functional properties of bone endothelial cells require cell-matrix signalling interactions. Loss of endothelial integrin ß1 leads to endothelial cell differentiation defects and impaired postnatal bone growth, which is, in part, phenocopied by endothelial cell-specific laminin α5 mutants. Our work outlines fundamental principles of vessel formation and endothelial cell differentiation in the developing skeletal system.

Endothelial Basement Membrane Laminin 511 Contributes to Endothelial Junctional Tightness and Thereby Inhibits Leukocyte Transmigration.

Endothelial basement membranes constitute barriers to extravasating leukocytes during inflammation, a process where laminin isoforms define sites of leukocyte exit; however, how this occurs is poorly understood. In addition to a direct effect on leukocyte transmigration, we show that laminin 511 affects endothelial barrier function by stabilizing VE-cadherin at junctions and downregulating expression of CD99L2, correlating with reduced neutrophil extravasation. Binding of endothelial cells to laminin 511, but not laminin 411 or non-endothelial laminin 111, enhanced transendothelial cell electrical resistance (TEER) and inhibited neutrophil transmigration. Data suggest that endothelial adhesion to laminin 511 via ß1 and ß3 integrins mediates RhoA-induced VE-cadherin localization to cell-cell borders, and while CD99L2 downregulation requires integrin ß1, it is RhoA-independent. Our data demonstrate that molecular information provided by basement membrane laminin 511 affects leukocyte extravasation both directly and indirectly by modulating endothelial barrier properties.

Endothelial basement membrane laminin 511 is essential for shear stress response.

Shear detection and mechanotransduction by arterial endothelium requires junctional complexes containing PECAM-1 and VE-cadherin, as well as firm anchorage to the underlying basement membrane. While considerable information is available for junctional complexes in these processes, gained largely from in vitro studies, little is known about the contribution of the endothelial basement membrane. Using resistance artery explants, we show that the integral endothelial basement membrane component, laminin 511 (laminin α5), is central to shear detection and mechanotransduction and its elimination at this site results in ablation of dilation in response to increased shear stress. Loss of endothelial laminin 511 correlates with reduced cortical stiffness of arterial endothelium in vivo, smaller integrin ß1-positive/vinculin-positive focal adhesions, and reduced junctional association of actin-myosin II In vitro assays reveal that ß1 integrin-mediated interaction with laminin 511 results in high strengths of adhesion, which promotes p120 catenin association with VE-cadherin, stabilizing it at cell junctions and increasing cell-cell adhesion strength. This highlights the importance of endothelial laminin 511 in shear response in the physiologically relevant context of resistance arteries.

The regulation of immune cell trafficking by the extracellular matrix.

The extracellular matrix (ECM) comes in different structural forms and biochemical compositions, which determine both its biophysical properties and its ability to convey specific signals to immune cells encountering or navigating through it. Traditionally, the role of the individual ECM molecules on cell migration has been investigated independent of considerations such as the tension/mechanical strength constituted by the ECM. However, more recently, this aspect has attracted considerable attention and data suggest that rigidity and molecular signals derived from the ECM define the mode of cell migration. We here review the different types of ECM encountered by migrating immune cells in vivo, as well as current information on how both molecular components of the ECM and their supramolecular structure can impact on modes of immune cell migration.

Hippocampal long term memory: effect of the cholinergic system on local protein synthesis.

The present study was aimed at establishing a link between the cholinergic system and the pathway of mTOR and its downstream effector p70S6K, likely actors in long term memory encoding. We performed in vivo behavioral experiments using the step down inhibitory avoidance test (IA) in adult Wistar rats to evaluate memory formation under different conditions, and immunohistochemistry on hippocampal slices to evaluate the level and the time-course of mTOR and p70S6K activation. We also examined the effect of RAPA, inhibitor of mTORC1 formation, and of the acetylcholine (ACh) muscarinic receptor antagonist scopolamine (SCOP) or ACh nicotinic receptor antagonist mecamylamine (MECA) on short and long term memory formation and on the functionality of the mTOR pathway. Acquisition test was performed 30 min after i.c.v. injection of RAPA, a time sufficient for the drug to diffuse to CA1 pyramidal neurons, as demonstrated by MALDI-TOF-TOF imaging. Recall test was performed 1 h, 4 h or 24 h after acquisition. To confirm our results we performed in vitro experiments on live hippocampal slices: we evaluated whether stimulation of the cholinergic system with the cholinergic receptor agonist carbachol (CCh) activated the mTOR pathway and whether the administration of the above-mentioned antagonists together with CCh could revert this activation. We found that (1) mTOR and p70S6K activation in the hippocampus were involved in long term memory formation; (2) RAPA administration caused inhibition of mTOR activation at 1 h and 4 h and of p70S6K activation at 4 h, and long term memory impairment at 24 h after acquisition; (3) scopolamine treatment caused short but not long term memory impairment with an early increase of mTOR/p70S6K activation at 1 h followed by stabilization at longer times; (4) mecamylamine plus scopolamine treatment caused short term memory impairment at 1 h and 4 h and reduced the scopolamine-induced increase of mTOR/p70S6K activation at 1 h and 4 h; (5) mecamylamine plus scopolamine treatment did not impair long term memory formation; (6) in vitro treatment with carbachol activated mTOR and p70S6K and this effect was blocked by scopolamine and mecamylamine. Taken together our data reinforce the idea that distinct molecular mechanisms are at the basis of the two different forms of memory and are in accordance with data presented by other groups that there exist molecular mechanisms that underlie short term memory, others that underlie long term memories, but some mechanisms are involved in both.