Heterochromatin-Driven Nuclear Softening Protects the Genome against Mechanical Stress-Induced Damage.

Tissue homeostasis requires maintenance of functional integrity under stress. A central source of stress is mechanical force that acts on cells, their nuclei, and chromatin, but how the genome is protected against mechanical stress is unclear. We show that mechanical stretch deforms the nucleus, which cells initially counteract via a calcium-dependent nuclear softening driven by loss of H3K9me3-marked heterochromatin. The resulting changes in chromatin rheology and architecture are required to insulate genetic material from mechanical force. Failure to mount this nuclear mechanoresponse results in DNA damage. Persistent, high-amplitude stretch induces supracellular alignment of tissue to redistribute mechanical energy before it reaches the nucleus. This tissue-scale mechanoadaptation functions through a separate pathway mediated by cell-cell contacts and allows cells/tissues to switch off nuclear mechanotransduction to restore initial chromatin state. Our work identifies an unconventional role of chromatin in altering its own mechanical state to maintain genome integrity in response to deformation.

ATP allosterically stabilizes integrin-linked kinase for efficient force generation.

SignificanceThe pseudokinase integrin-linked kinase (ILK) is a central component of focal adhesions, cytoplasmic multiprotein complexes that integrate and transduce biochemical and mechanical signals from the extracellular environment into the cell and vice versa. However, the precise molecular functions, particularly the mechanosensory properties of ILK and the significance of retained adenosine triphosphate (ATP) binding, are still unclear. Combining molecular-dynamics simulations with cell biology, we establish a role for ATP binding to pseudokinases. We find that ATP promotes the structural stability of ILK, allosterically influences the interaction between ILK and its binding partner parvin at adhesions, and enhances the mechanoresistance of this complex. On the cellular level, ATP binding facilitates efficient traction force buildup, focal adhesion stabilization, and efficient cell migration.

Hydrostatic pressure prevents chondrocyte differentiation through heterochromatin remodeling.

Articular cartilage protects and lubricates joints for smooth motion and transmission of loads. Owing to its high water content, chondrocytes within the cartilage are exposed to high levels of hydrostatic pressure, which has been shown to promote chondrocyte identity through unknown mechanisms. Here, we investigate the effects of hydrostatic pressure on chondrocyte state and behavior, and discover that application of hydrostatic pressure promotes chondrocyte quiescence and prevents maturation towards the hypertrophic state. Mechanistically, hydrostatic pressure reduces the amount of trimethylated H3K9 (K3K9me3)-marked constitutive heterochromatin and concomitantly increases H3K27me3-marked facultative heterochromatin. Reduced levels of H3K9me3 attenuates expression of pre-hypertrophic genes, replication and transcription, thereby reducing replicative stress. Conversely, promoting replicative stress by inhibition of topoisomerase II decreases Sox9 expression, suggesting that it enhances chondrocyte maturation. Our results reveal how hydrostatic pressure triggers chromatin remodeling to impact cell fate and function.This article has an associated First Person interview with the first author of the paper.

Ablation of integrin-mediated cell-collagen communication alleviates fibrosis.

OBJECTIVES: Activation of fibroblasts is a hallmark of fibrotic processes. Besides cytokines and growth factors, fibroblasts are regulated by the extracellular matrix environment through receptors such as integrins, which transduce biochemical and mechanical signals enabling cells to mount appropriate responses according to biological demands. The aim of this work was to investigate the in vivo role of collagen-fibroblast interactions for regulating fibroblast functions and fibrosis. METHODS: Triple knockout (tKO) mice with a combined ablation of integrins α1ß1, α2ß1 and α11ß1 were created to address the significance of integrin-mediated cell-collagen communication. Properties of primary dermal fibroblasts lacking collagen-binding integrins were delineated in vitro. Response of the tKO mice skin to bleomycin induced fibrotic challenge was assessed. RESULTS: Triple integrin-deficient mice develop normally, are transiently smaller and reveal mild alterations in mechanoresilience of the skin. Fibroblasts from these mice in culture show defects in cytoskeletal architecture, traction stress generation, matrix production and organisation. Ablation of the three integrins leads to increased levels of discoidin domain receptor 2, an alternative receptor recognising collagens in vivo and in vitro. However, this overexpression fails to compensate adhesion and spreading defects on collagen substrates in vitro. Mice lacking collagen-binding integrins show a severely attenuated fibrotic response with impaired mechanotransduction, reduced collagen production and matrix organisation. CONCLUSIONS: The data provide evidence for a crucial role of collagen-binding integrins in fibroblast force generation and differentiation in vitro and for matrix deposition and tissue remodelling in vivo. Targeting fibroblast-collagen interactions might represent a promising therapeutic approach to regulate connective tissue deposition in fibrotic diseases.

Emerging roles of mechanical forces in chromatin regulation.

Cells are constantly subjected to a spectrum of mechanical cues, such as shear stress, compression, differential tissue rigidity and strain, to which they adapt by engaging mechanisms of mechanotransduction. While the central role of cell adhesion receptors in this process is established, it has only recently been appreciated that mechanical cues reach far beyond the plasma membrane and the cytoskeleton, and are directly transmitted to the nucleus. Furthermore, changes in the mechanical properties of the perinuclear cytoskeleton, nuclear lamina and chromatin are critical for cellular responses and adaptation to external mechanical cues. In that respect, dynamic changes in the nuclear lamina and the surrounding cytoskeleton modify mechanical properties of the nucleus, thereby protecting genetic material from damage. The importance of this mechanism is highlighted by debilitating genetic diseases, termed laminopathies, that result from impaired mechanoresistance of the nuclear lamina. What has been less evident, and represents one of the exciting emerging concepts, is that chromatin itself is an active rheological element of the nucleus, which undergoes dynamic changes upon application of force, thereby facilitating cellular adaption to differential force environments. This Review aims to highlight these emerging concepts by discussing the latest literature in this area and by proposing an integrative model of cytoskeletal and chromatin-mediated responses to mechanical stress.

Monitoring the mass, eigenfrequency, and quality factor of mammalian cells.

The regulation of mass is essential for the development and homeostasis of cells and multicellular organisms. However, cell mass is also tightly linked to cell mechanical properties, which depend on the time scales at which they are measured and change drastically at the cellular eigenfrequency. So far, it has not been possible to determine cell mass and eigenfrequency together. Here, we introduce microcantilevers oscillating in the Ångström range to monitor both fundamental physical properties of the cell. If the oscillation frequency is far below the cellular eigenfrequency, all cell compartments follow the cantilever motion, and the cell mass measurements are accurate. Yet, if the oscillating frequency approaches or lies above the cellular eigenfrequency, the mechanical response of the cell changes, and not all cellular components can follow the cantilever motions in phase. This energy loss caused by mechanical damping within the cell is described by the quality factor. We use these observations to examine living cells across externally applied mechanical frequency ranges and to measure their total mass, eigenfrequency, and quality factor. The three parameters open the door to better understand the mechanobiology of the cell and stimulate biotechnological and medical innovations.

Mechanical stimulation and electrophysiological monitoring at subcellular resolution reveals differential mechanosensation of neurons within networks.

A growing consensus that the brain is a mechanosensitive organ is driving the need for tools that mechanically stimulate and simultaneously record the electrophysiological response of neurons within neuronal networks. Here we introduce a synchronized combination of atomic force microscopy, high-density microelectrode array and fluorescence microscopy to monitor neuronal networks and to mechanically characterize and stimulate individual neurons at piconewton force sensitivity and nanometre precision while monitoring their electrophysiological activity at subcellular spatial and millisecond temporal resolution. No correlation is found between mechanical stiffness and electrophysiological activity of neuronal compartments. Furthermore, spontaneously active neurons show exceptional functional resilience to static mechanical compression of their soma. However, application of fast transient (∼500 ms) mechanical stimuli to the neuronal soma can evoke action potentials, which depend on the anchoring of neuronal membrane and actin cytoskeleton. Neurons show higher responsivity, including bursts of action potentials, to slower transient mechanical stimuli (∼60 s). Moreover, transient and repetitive application of the same compression modulates the neuronal firing rate. Seemingly, neuronal networks can differentiate and respond to specific characteristics of mechanical stimulation. Ultimately, the developed multiparametric tool opens the door to explore manifold nanomechanobiological responses of neuronal systems and new ways of mechanical control.

Controlling self-renewal and differentiation of stem cells via mechanical cues.

The control of stem cell response in vitro, including self-renewal and lineage commitment, has been proved to be directed by mechanical cues, even in the absence of biochemical stimuli. Through integrin-mediated focal adhesions, cells are able to anchor onto the underlying substrate, sense the surrounding microenvironment, and react to its properties. Substrate-cell and cell-cell interactions activate specific mechanotransduction pathways that regulate stem cell fate. Mechanical factors, including substrate stiffness, surface nanotopography, microgeometry, and extracellular forces can all have significant influence on regulating stem cell activities. In this paper, we review all the most recent literature on the effect of purely mechanical cues on stem cell response, and we introduce the concept of "force isotropy" relevant to cytoskeletal forces and relevant to extracellular loads acting on cells, to provide an interpretation of how the effects of insoluble biophysical signals can be used to direct stem cells fate in vitro.

Mechanochemical control of epidermal stem cell divisions by B-plexins.

The precise spatiotemporal control of cell proliferation is key to the morphogenesis of epithelial tissues. Epithelial cell divisions lead to tissue crowding and local changes in force distribution, which in turn suppress the rate of cell divisions. However, the molecular mechanisms underlying this mechanical feedback are largely unclear. Here, we identify a critical requirement of B-plexin transmembrane receptors in the response to crowding-induced mechanical forces during embryonic skin development. Epidermal stem cells lacking B-plexins fail to sense mechanical compression, resulting in disinhibition of the transcriptional coactivator YAP, hyperproliferation, and tissue overgrowth. Mechanistically, we show that B-plexins mediate mechanoresponses to crowding through stabilization of adhesive cell junctions and lowering of cortical stiffness. Finally, we provide evidence that the B-plexin-dependent mechanochemical feedback is also pathophysiologically relevant to limit tumor growth in basal cell carcinoma, the most common type of skin cancer. Our data define a central role of B-plexins in mechanosensation to couple cell density and cell division in development and disease.

Development and biological validation of a cyclic stretch culture system for the ex vivo engineering of tendons.

INTRODUCTION: An innovative approach to the treatment of tendon injury or degeneration is given by engineered grafts, made available through the development of bioreactors that generate tendon tissue in vitro, by replicating in vivo conditions. This work aims at the design of a bioreactor capable of applying a stimulation of cyclic strain on cell constructs to promote the production of bioartificial tissue with mechanical and biochemical properties resembling those of the native tissue. METHODS: The system was actuated by an electromagnet and design specifications were imposed as follows. The stimulation protocol provides to scaffolds a 3% preload, a 10% deformation, and a stimulation frequency rate set at 0.5, 1, and 2 Hz, which alternates stimulation/resting phases. Porcine tenocytes were seeded on collagen scaffolds and cultured in static or dynamic conditions for 7 and 14 days. RESULTS: The culture medium temperature did not exceed 37°C during prolonged culture experiments. The applied force oscillates between 1.5 and 4.5 N. The cyclic stimulation of the engineered constructs let both the cells and the scaffold fibers align along the strain direction in response to the mechanical stimulus. CONCLUSION: We designed a pulsatile strain bioreactor for tendon tissue engineering. The in vitro characterization shows a preferential cell alignment at short time points. Prolonged culture time, however, seems to influence negatively on the survival of the cells indicating the need of further optimization concerning the culture conditions and the mechanical stimulation.

3D Stem Cell Niche Engineering via Two-Photon Laser Polymerization.

A strategy to modulate the behavior of stem cells in culture is to mimic structural aspects of the native cell-extracellular matrix (ECM) interaction. An important example of such artificial microenvironments for stem cell culture is the so-called "synthetic niche." Synthetic niches can be defined as polymeric culture systems mimicking at least one aspect of the interactions between stem cells and the extracellular surroundings, including biochemical factors (e.g., the delivery of soluble factors) and/or biophysical factors (e.g., the microarchitecture of the ECM). Most of the currently available approaches for scaffold fabrication, based on self-assembly methods, do not allow for a submicrometer control of the geometrical structure of the substrate, which might play a crucial role in stem cell fate determination. A novel technology that overcomes these limitations is laser two-photon polymerization (2PP). Femtosecond laser 2PP is a mask-less direct laser writing technique that allows manufacturing three dimensional arbitrary microarchitectures using photosensitive materials. Here, we report on the development of an innovative culture substrate, called the "nichoid," microfabricated in a hybrid organic-inorganic photoresist called SZ2080, to study mesenchymal stem cell mechanobiology.

Scaling-Up Techniques for the Nanofabrication of Cell Culture Substrates via Two-Photon Polymerization for Industrial-Scale Expansion of Stem Cells.

Stem-cell-based therapies require a high number (106-108) of cells, therefore in vitro expansion is needed because of the initially low amount of stem cells obtainable from human tissues. Standard protocols for stem cell expansion are currently based on chemically-defined culture media and animal-derived feeder-cell layers, which expose cells to additives and to xenogeneic compounds, resulting in potential issues when used in clinics. The two-photon laser polymerization technique enables three-dimensional micro-structures to be fabricated, which we named synthetic nichoids. Here we review our activity on the technological improvements in manufacturing biomimetic synthetic nichoids and, in particular on the optimization of the laser-material interaction to increase the patterned area and the percentage of cell culture surface covered by such synthetic nichoids, from a low initial value of 10% up to 88% with an optimized micromachining time. These results establish two-photon laser polymerization as a promising tool to fabricate substrates for stem cell expansion, without any chemical supplement and in feeder-free conditions for potential therapeutic uses.

An experimental-numerical investigation on the effects of macroporous scaffold geometry on cell culture parameters.

INTRODUCTION: Perfused bioreactors have been demonstrated to be effective in the delivery of nutrients and in the removal of waste products to and from the interior of cell-populated three-dimensional scaffolds. In this paper, a perfused bioreactor hosting a macroporous scaffold provided with a channel is used to investigate transport phenomena and culture parameters on cell growth. METHODS: MG63 human osteosarcoma cells were seeded on macroporous poly​(ε-caprolactone) scaffolds provided with a channel. The scaffolds were cultured in a perfused bioreactor and in static conditions for 5 days. Cell viability and growth were assessed while the concentration of oxygen, glucose and lactate were measured. An in silico, multiphysics, numerical model was set up to study the fluid dynamics and the mass transport of the nutrients in the perfused bioreactor hosting different scaffold geometries. RESULTS: The experimental and numerical results indicated that the specific cell metabolic activity in scaffolds cultured under perfusion was 30% greater than scaffolds cultured under static conditions. In addition, the scaffold provided with a channel enabled the shear stress to be controlled, the initial seeding density to be retained, and adequate mass transport and waste removal. CONCLUSIONS: We show that the macroporous scaffold provided with a channel cultured in a macroscale bioreactor can be a robust reference experimental model system to systematically investigate and assess crucial culture parameters. We also show that such an experimental model system can be employed as a simplified "representative unit" to improve the performance of both perfused culture systems and hollow, fiber-integrated scaffolds for large-scale tissue engineering.

Synthetic niche substrates engineered via two-photon laser polymerization for the expansion of human mesenchymal stromal cells.

The present study reports on the development of an innovative culture substrate, micro-fabricated by two-photon laser polymerization (2PP) in a hybrid organic-inorganic photoresin. It was previously demonstrated that this substrate is able to guide spontaneous homing and colonization of mesenchymal stromal cells by the presence of synthetic microniches. Here, the number of niches covering the culture substrate was increased up to 10% of the total surface. Human bone marrow-derived mesenchymal stromal cells were expanded for 3 weeks and then their proliferation, clonogenic capacity and bilineage differentiation potential towards the osteogenic and adipogenic lineage were evaluated, both by colorimetric assays and by real-time polymerase chain reaction. Compared with cells cultured on glass substrates, cells expanded on 2PP substrates showed a greater colony diameter, which is an index of clonogenic potential. Following medium conditioning on 2PP-cultured cells, the expression of RUNX2 and BSP genes, as well as PPAR-gamma, was significantly greater than that measured on glass controls. Thus, human cells expanded on the synthetic niche substrate maintained their proliferative potential, clonogenic capacity and bilineage differentiation potential more effectively than cells expanded on glass substrates and in some aspects were comparable to non-expanded cells. © 2016 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd.

The effect of scaffold pore size in cartilage tissue engineering.

INTRODUCTION: The effect of scaffold pore size and interconnectivity is undoubtedly a crucial factor for most tissue engineering applications. The aim of this study was to examine the effect of pore size and porosity on cartilage construct development in different scaffolds seeded with articular chondrocytes. METHODS: We fabricated poly-L-lactide-co-trimethylene carbonate scaffolds with different pore sizes, using a solvent-casting/particulate-leaching technique. We seeded primary bovine articular chondrocytes on these scaffolds, cultured the constructs for 2 weeks and examined cell proliferation, viability and cell-specific production of cartilaginous extracellular matrix proteins, including GAG and collagen. RESULTS: Cell density significantly increased up to 50% with scaffold pore size and porosity, likely facilitated by cell spreading on the internal surface of bigger pores, and by increased mass transport of gases and nutrients to cells, and catabolite removal from cells, allowed by lower diffusion barriers in scaffolds with a higher porosity. However, both the cell metabolic activity and the synthesis of cartilaginous matrix proteins significantly decreased by up to 40% with pore size. We propose that the association of smaller pore diameters, causing 3-dimensional cell aggregation, to a lower oxygenation caused by a lower porosity, could have been the condition that increased the cell-specific synthesis of cartilaginous matrix proteins in the scaffold with the smallest pores and the lowest porosity among those tested. CONCLUSIONS: In the initial steps of in vitro cartilage engineering, the combination of small scaffold pores and low porosity is an effective strategy with regard to the promotion of chondrogenesis.

Computational prediction of strain-dependent diffusion of transcription factors through the cell nucleus.

Nuclear spreading plays a crucial role in stem cell fate determination. In previous works, we reported evidence of multipotency maintenance for mesenchymal stromal cells cultured on three-dimensional engineered niche substrates, fabricated via two-photon laser polymerization. We correlated maintenance of multipotency to a more roundish morphology of these cells with respect to those cultured on conventional flat substrates. To interpret these findings, here we present a multiphysics model coupling nuclear strains induced by cell adhesion to passive diffusion across the cell nucleus. Fully three-dimensional reconstructions of cultured cells were developed on the basis of confocal images: in particular, the level of nuclear spreading resulted significantly dependent on the cell localization within the niche architecture. We assumed that the cell diffusivity varies as a function of the local volumetric strain. The model predictions indicate that the higher the level of spreading of the cell, the higher the flux across the nucleus of small solutes such as transcription factors. Our results point toward nuclear spreading as a primary mechanism by which the stem cell translates its shape into a fate decision, i.e., by amplifying the diffusive flow of transcriptional activators into the nucleus.

Hollow fiber bioreactor technology for tissue engineering applications.

Hollow fiber bioreactors are the focus of scientific research aiming to mimic physiological vascular networks and engineer organs and tissues in vitro. The reason for this lies in the interesting features of this bioreactor type, including excellent mass transport properties. Indeed, hollow fiber bioreactors allow limitations to be overcome in nutrient transport by diffusion, which is often an obstacle to engineer sizable constructs in vitro. This work reviews the existing literature relevant to hollow fiber bioreactors in organ and tissue engineering applications. To this purpose, we first classify the hollow fiber bioreactors into 2 categories: cylindrical and rectangular. For each category, we summarize their main applications both at the tissue and at the organ level, focusing on experimental models and computational studies as predictive tools for designing innovative, dynamic culture systems. Finally, we discuss future perspectives on hollow fiber bioreactors as in vitro models for tissue and organ engineering applications.

Direct photo-patterning of hyaluronic acid baits onto a fouling-release perfluoropolyether surface for selective cancer cell capture and immobilization.

A simple photolithographic process for directly patterning glycidyl methacrylate modified hyaluronic acid features onto UV curable perfluoropolyether-based surfaces is presented. Due to the versatility of the developed method, HA spotted areas with different geometrical features could be rapidly and inexpensively designed. In addition, the excellent antifouling and fouling-release properties of the substrates enabled direct HA baits photo-grafting onto PFPEs without further surface passivation or chemical modification to avoid not specific adsorption. The aim of the study was to locally switch the surface properties of the PFPEs from cells and protein repulsive to adherent. Particularly, we exploited HA well-known preferential interactions with CD44 transmembrane receptors to selectively immobilize cancer cells. Living cell arrays offer a higher-resolution visualization of HA-CD44 interactions and may provide a deep insight into understanding molecular mechanisms needed to develop selective therapies and diagnosis against tumor growth.

Two-photon polymerized "nichoid" substrates maintain function of pluripotent stem cells when expanded under feeder-free conditions.

BACKGROUND: The use of pluripotent cells in stem cell therapy has major limitations, mainly related to the high costs and risks of exogenous conditioning and the use of feeder layers during cell expansion passages. METHODS: We developed an innovative three-dimensional culture substrate made of "nichoid" microstructures, nanoengineered via two-photon laser polymerization. The nichoids limit the dimension of the adhering embryoid bodies during expansion, by counteracting cell migration between adjacent units of the substrate by its microarchitecture. We expanded mouse embryonic stem cells on the nichoid for 2 weeks. We compared the expression of pluripotency and differentiation markers induced in cells with that induced by flat substrates and by a culture layer made of kidney-derived extracellular matrix. RESULTS: The nichoid was found to be the only substrate, among those tested, that maintained the expression of the OCT4 pluripotency marker switched on and, simultaneously, the expression of the differentiation markers GATA4 and α-SMA switched off. The nichoid promotes pluripotency maintenance of embryonic stem cells during expansion, in the absence of a feeder layer and exogenous conditioning factors, such as the leukocyte inhibitory factor. CONCLUSIONS: We hypothesized that the nichoid microstructures induce a genetic reprogramming of cells by controlling their cytoskeletal tension. Further studies are necessary to understand the exact mechanism by which the physical constraint provided by the nichoid architecture is responsible for cell reprogramming. The nichoid may help elucidate mechanisms of pluripotency maintenance, while potentially cutting the costs and risks of both feed-conditioning and exogenous conditioning for industrial-scale expansion of stem cells.

A strain-dependent diffusivity model to study the nuclear import of mechanobiological transcription factors.

Nuclear spreading plays a crucial role in stem cell fate determination. In previous works, we reported evidence of multipotency maintenance of mesenchymal stromal cells cultured on three-dimensional engineered niche substrates fabricated via two-photon laser polymerization (2PP). We correlated multipotency maintenance to a more roundish nuclear morphology of cells cultured in the 2PP-fabricated niches, with respect to those on flat substrates. To interpret these findings, here we present a multiphysics model coupling nuclear strains induced by cell adhesion to diffusive transport across the cell nucleus. We reconstructed the cell nuclear geometry from confocal Z-stack images of 2PP-cultured cells, and we estimated the volume, surface and shape factors. The levels of nuclear spreading significantly varied depending on the cell localization within the niche architecture. We assumed the cell diffusivity as a function of the local volumetric strain. The computational model also indicate that the larger the nuclear deformation (e.g. in spread nuclei), the higher the nuclear flux of small solutes such as transcription factors through the nuclear membrane. Our results point towards nuclear deformation as a primary mechanism by which the stem cell translates its shape into a fate decision, i.e. through a strain-dependent amplification of the diffusive flow of signaling molecules into the nucleus.