Mechanobiology of myeloid cells.

Tissue-resident myeloid cells sense and transduce mechanical signals such as stiffness, stretch and compression. In the past two years, our understanding of the mechanosensitive signalling pathways in myeloid cells has significantly expanded. Moreover, it is increasingly clear which mechanical signals induce myeloid cells towards a pro- or anti-inflammatory phenotype. This is especially relevant in the context of altered matrix mechanics in immune-related pathologies or in the response to implanted biomaterials. A detailed understanding of myeloid cell mechanosensing may eventually lead to more effective cell-based immunotherapies for cancer, the development of mechanically inspired therapies to target fibrosis, and the engineering of safer implants. This review covers these recent advances in the emerging field of mechanoimmunology of myeloid cells.

CRIPTO-based micro-heterogeneity of mouse muscle satellite cells enables adaptive response to regenerative microenvironment.

Skeletal muscle repair relies on heterogeneous populations of satellite cells (SCs). The mechanisms that regulate SC homeostasis and state transition during activation are currently unknown. Here, we investigated the emerging role of non-genetic micro-heterogeneity, i.e., intrinsic cell-to-cell variability of a population, in this process. We demonstrate that micro-heterogeneity of the membrane protein CRIPTO in mouse-activated SCs (ASCs) identifies metastable cell states that allow a rapid response of the population to environmental changes. Mechanistically, CRIPTO micro-heterogeneity is generated and maintained through a process of intracellular trafficking coupled with active shedding of CRIPTO from the plasma membrane. Irreversible perturbation of CRIPTO micro-heterogeneity affects the balance of proliferation, self-renewal, and myogenic commitment in ASCs, resulting in increased self-renewal in vivo. Our findings demonstrate that CRIPTO micro-heterogeneity regulates the adaptative response of ASCs to microenvironmental changes, providing insights into the role of intrinsic heterogeneity in preserving stem cell population diversity during tissue repair.

Collagen-Based Biomimetic Systems to Study the Biophysical Tumour Microenvironment.

The extracellular matrix (ECM) is a pericellular network of proteins and other molecules that provides mechanical support to organs and tissues. ECM biophysical properties such as topography, elasticity and porosity strongly influence cell proliferation, differentiation and migration. The cell's perception of the biophysical microenvironment (mechanosensing) leads to altered gene expression or contractility status (mechanotransduction). Mechanosensing and mechanotransduction have profound implications in both tissue homeostasis and cancer. Many solid tumours are surrounded by a dense and aberrant ECM that disturbs normal cell functions and makes certain areas of the tumour inaccessible to therapeutic drugs. Understanding the cell-ECM interplay may therefore lead to novel and more effective therapies. Controllable and reproducible cell culturing systems mimicking the ECM enable detailed investigation of mechanosensing and mechanotransduction pathways. Here, we discuss ECM biomimetic systems. Mainly focusing on collagen, we compare and contrast structural and molecular complexity as well as biophysical properties of simple 2D substrates, 3D fibrillar collagen gels, cell-derived matrices and complex decellularized organs. Finally, we emphasize how the integration of advanced methodologies and computational methods with collagen-based biomimetics will improve the design of novel therapies aimed at targeting the biophysical and mechanical features of the tumour ECM to increase therapy efficacy.

A switchable light-responsive azopolymer conjugating protein micropatterns with topography for mechanobiological studies.

Stem cell shape and mechanical properties in vitro can be directed by geometrically defined micropatterned adhesion substrates. However, conventional methods are limited by the fixed micropattern design, which cannot recapitulate the dynamic changes of the natural cell microenvironment. Current methods to fabricate dynamic platforms usually rely on complex chemical strategies or require specialized apparatuses. Also, with these methods, the integration of dynamic signals acting on different length scales is not straightforward, whereas, in some applications, it might be beneficial to act on both a microscale level, that is, cell shape, and a nanoscale level, that is, cell adhesions. Here, we exploited a confocal laser-based technique on a light-responsive azopolymer displaying micropatterns of adhesive islands. The laser light promotes a directed mass migration and the formation of submicrometric topographic relieves. Also, by changing the surface chemistry, the surfacing topography affects cell spreading and shape. This method enabled us to monitor in a non-invasive manner the dynamic changes in focal adhesions, cytoskeleton structures, and nucleus conformation that followed the changes in the adhesive characteristic of the substrate. Focal adhesions reconfigured after the surfacing of the topography, and the actin filaments reoriented to coalign with the newly formed adhesive island. Changes in cell morphology also affected nucleus shape, chromatin conformation, and cell mechanics with different timescales. The reported strategy can be used to investigate mechanotransduction-related events dynamically by controlling cell adhesion at cell shape and focal adhesion levels. The integrated technique enables achieving a submicrometric resolution in a facile and cost-effective manner.

A role for nuclear stretching and NPCs changes in the cytoplasmic-nuclear trafficking of YAP: An experimental and numerical modelling approach.

Mechanical forces, acting on eukaryotic cells, are responsible for cell shape, cell proliferation, cell polarity, and cell differentiation thanks to two cells abilities known as mechanosensing and mechanotransduction. Mechanosensing consists of the ability of a cell to sense mechanical cues, while mechanotransduction is the capacity of a cell to respond to these signals by translating mechanical stimuli into biochemical ones. These signals propagate from the extracellular matrix to the nucleus with different well known physical connections, but how the mechanical signals are transduced into biochemical ones remains an open challenge. Recent findings showed that the cell-generated forces affect the translocation of transcription factors (TFs) from the cytoplasm to the nucleus. This mechanism is affected by the features of nuclear pore complexes. Owing to the complex patterns of strains and stresses of the nuclear envelope caused by cytoskeletal forces, it is likely that the morphology of NPC changes as cytoskeleton assemblies' change. This may ultimately affect molecular transport through the nucleus, hence altering cell functions. Among the various TFs, Yes-associated protein (YAP), which is typically involved in cell proliferation, survival, and differentiation, is able to activate specific pathways when entrapped into the cell nucleus. Here, starting from experimental results, we develop a multiscale finite element (FE) model aimed to simulate the macroscopic cell spreading and consequent changes in the cell mechanical behaviour to be related to the NPCs changes and YAP nuclear transport.

Simple yet effective methods to probe hydrogel stiffness for mechanobiology.

In spite of tremendous advances made in the comprehension of mechanotransduction, implementation of mechanobiology assays remains challenging for the broad community of cell biologists. Hydrogel substrates with tunable stiffness are essential tool in mechanobiology, allowing to investigate the effects of mechanical signals on cell behavior. A bottleneck that slows down the popularization of hydrogel formulations for mechanobiology is the assessment of their stiffness, typically requiring expensive and sophisticated methodologies in the domain of material science. Here we overcome such barriers offering the reader protocols to set-up and interpret two straightforward, low cost and high-throughput tools to measure hydrogel stiffness: static macroindentation and micropipette aspiration. We advanced on how to build up these tools and on the underlying theoretical modeling. Specifically, we validated our tools by comparing them with leading techniques used for measuring hydrogel stiffness (atomic force microscopy, uniaxial compression and rheometric analysis) with consistent results on PAA hydrogels or their modification. In so doing, we also took advantage of YAP/TAZ nuclear localization as biologically validated and sensitive readers of mechanosensing, all in all presenting a suite of biologically and theoretically proven protocols to be implemented in most biological laboratories to approach mechanobiology.

The role of cortical zone level and prosthetic platform angle in dental implant mechanical response: A 3D finite element analysis.

OBJECTIVE: The aim of this study was to evaluate the influence of three different dental implant neck geometries, under a combined compressive/shear load using finite element analysis (FEA). The implant neck was positioned in D2 quality bone at the crestal level or 2 mm below. METHODS: One dental implant (4.2 × 9 mm) was digitized by reverse engineering techniques using micro CT and imported into Computer Aided Design (CAD) software. Non-uniform rational B-spline surfaces were reconstructed, generating a 3D volumetric model similar to the digitized implant. Three different models were generated with different implant neck configurations, namely 0°, 10° and 20°. D2 quality bone, composed of cortical and trabecular structure, was modeled using data from CT scans. The implants were included in the bone model using a Boolean operation. Two different fixture insertion depths were simulated for each implant: 2 mm below the crestal bone and exactly at the level of the crestal bone. The obtained models were imported to FEA software in STEP format. Von Mises equivalent strains were analyzed for the peri-implant D2 bone type, considering the magnitude and volume of the affected surrounding cortical and trabecular bone. The highest strain values in both cortical and trabecular tissue at the peri-implant bone interface were extracted and compared. RESULTS: All implant models were able to distribute the load at the bone-implant contact (BIC) with a similar strain pattern between the models. At the cervical region, however, differences were observed: the models with 10° and 20° implant neck configurations (Model B and C), showed a lower strain magnitude when compared to the straight neck (Model A). These values were significantly lower when the implants were situated at crestal bone levels. In the apical area, no differences in strain values were observed. SIGNIFICANCE: The implant neck configuration influenced the strain distribution and magnitude in the cortical bone and cancellous bone tissues. To reduce the strain values and improve the load dissipation in the bone tissue, implants with 10° and 20 neck configuration should be preferred instead of straight implant platforms.

Effects of surface nanopatterning on internalization and amyloid aggregation of the fragment 264-277 of Nucleophosmin 1.

The mechanical interpretation of the plethora of factors that governs cellular localization of amyloid aggregates is crucial for planning novel therapeutical interventions in neurodegenerative diseases since these aggregates exert a primary role in the proteostasis machinery. The uptake of Cell Penetrating Peptides (CPPs) conjugated with different amyloid polypeptides occurs via different endocytic processes regulated by cytoskeleton organization and cell morphology. Herein, we deepened the internalization of an amyloid system in cells cultured on nanopatterned surfaces that represent a powerful tool to shape cell and regulate its contractility. We analyzed the behavior of an amyloid model system, employing NPM1264-277 sequence, covalently conjugated to Tat fragment 48-60 as CPP. To investigate its internalization mechanism, we followed the formation of aggregates on two kinds of substrates: a flat and a nanopatterned surface. Herein, investigations during time were carried out by employing both confocal and second harmonic generation (SHG) microscopies. We showed that modifications of cellular environment affect peptide localization, its cytoplasmic translocation and the size of amyloid aggregates.

Decellularized matrices for tumor cell modeling.

Collagen is the main component of the extracellular matrix and it plays a key role in tumor progression. Commercial collagen solutions are derived from animals, such as rat-tail and bovine or porcine skin. Their cost is quite high and the product is stable only at low temperature, with the disadvantage of a short expiring date. Most importantly, lot-to-lot variability can occur and the reconstituted collagen gels differ significantly from native tissues in terms of both structure and stiffness. In this chapter, we describe a straightforward method to use native, collagen rich skin samples derived from by-products of the tanning industry. The protocol proposed preserves the microstructure of the ovine skin collagen network, offering structurally competent and more relevant model to investigate cell behavior in vitro. Other advantages of the proposed procedure consist in the cost-effectiveness of the process and an increased level of reproducibility. The decellularized ovine skin samples support the adhesion and growth of different cancer cell lines (pancreatic, breast and melanoma cells). The proposed decellularized skin scaffolds are meant as future low-cost competitors for conventional porous scaffold derived by biomaterials, since they offer a biomimetic environment for the cells.

Topographic Cues Impact on Embryonic Stem Cell Zscan4-Metastate.

The extracellular microenvironment proved to exert a potent regulatory effect over different aspects of Embryonic Stem Cells (ESCs) behavior. In particular, the employment of engineered culture surfaces aimed at modulating ESC self-organization resulted effective in directing ESCs toward specific fate decision. ESCs fluctuate among different levels of functional potency and in this context the Zscan4 gene marks the so-called "metastate," a cellular state in which ESCs retain both self-renewal and pluripotency capabilities. Here we investigated the impact of topographic cues on ESCs pluripotency, differentiation and organization capabilities. To this aim, we engineered culturing platforms of nanograted surfaces with different features size and we investigated their impact on ESCs multicellular organization and Zscan4 gene expression. We showed that the morphology of ESC-derived aggregates and Zscan4 expression are strictly intertwined. Our data suggest that ESC Zscan4 metastate can be promoted if the adhesive surface conditions guide cellular self-aggregation into 3D dome-like structure, in which both cell-material interactions and cell-cell contact are supportive for Zscan4 expression.

Adhesive class I restorations in sound molar teeth incorporating combined resin-composite and glass ionomer materials: CAD-FE modeling and analysis.

OBJECTIVES: To investigate the influence of different resin composite and glass ionomer cement material combinations in a "bi-layer" versus a "single-layer" adhesive technique for class I cavity restorations in molars using numerical finite element analysis (FEA). MATERIALS AND METHODS: Three virtual restored lower molar models with class I cavities 4mm deep were created from a sound molar CAD model. A combination of an adhesive and flowable composite with bulk fill composite (model A), of a glass ionomer cement with bulk fill composite (model B) and of an adhesive with bulk fill composite (model C), were considered. Starting from CAD models, 3D-finite element (FE) models were created and analyzed. Solid food was modeled on the occlusal surface and slide-type contact elements were used between tooth surface and food. Polymerization shrinkage was simulated for the composite materials. Physiological masticatory loads were applied to these systems combined with shrinkage. Static linear analyses were carried out. The maximum normal stress criterion was adopted as a measure of potential damage. RESULTS: All models exhibited high stresses principally located along the tooth tissues-restoration interfaces. All models showed a similar stress trend along enamel-restoration interface, where stresses up to 22MPa and 19MPa was recorded in the enamel and restoration, respectively. A and C models showed a similar stress trend along the dentin-restoration interface with a lower stress level in model A, where stresses up to 11.5MPa and 7.5MPa were recorded in the dentin and restoration, respectively, whereas stresses of 17MPa and 9MPa were detected for model C. In contrast to A and C models, the model B showed a reduced stress level in dentin, in the lower restoration layer and no stress on the cavity floor. SIGNIFICANCE: FE analysis supported the positive effect of a "bi-layer" restorative technique in a 4mm deep class I cavities in lower molars versus "single-layer" bulk fill composite technique.

Aligned fibrous decellularized cell derived matrices for mesenchymal stem cell amplification.

Biochemical and biophysical stimuli of stem cell niches finely regulate the self-renewal/differentiation equilibrium. Replicating this in vitro is technically challenging, making the control of stem cell functions difficult. Cell derived matrices capture certain aspect of niches that influence fate decisions. Here, aligned fibrous matrices synthesized by MC3T3 cells were produced and the role of matrix orientation and stiffness on the maintenance of stem cell characteristics and adipo- or osteo-genic differentiation of murine mesenchymal stem cells (mMSCs) was investigated. Decellularized matrices promoted mMSC proliferation. Fibrillar alignment and matrix stiffness work in concert in defining cell fate. Soft matrices preserve stemness, whereas stiff ones, in presence of biochemical supplements, promptly induce differentiation. Matrix alignment impacts the homogeneity of the cell population, that is, soft aligned matrices ameliorate the spontaneous adipogenic differentiation, whereas stiff aligned matrices reduce cross-differentiation. We infer that mechanical signaling is a dominant factor in mMSC fate decision and the matrix alignment contributes to produce a more homogeneous environment, which results in a uniform response of cells to biophysical environment. Matrix thus produced can be obtained in vitro in a facile and consistent manner and can be used for homogeneous stem cell amplification or for mechanotransduction-related studies.

Mechanical behavior of Class I cavities restored by different material combinations under loading and polymerization shrinkage stress. A 3D-FEA study.

PURPOSE: To examine the influence of different bulk and block composite and flowable and glass-ionomer material combinations in a multi-layer technique and in a unique technique, in deep Class I dental restorations. METHODS: 3D CAD of the sound tooth were built-up from a CT scan dataset using reverse engineering techniques. Four restored tooth models with Class I cavity were virtually created from a CAD model of a sound tooth. 3D-finite element (FE) models were created and analyzed starting from CAD models. Model A with flowable resin composite restoring the lower layer and bulk-fill resin composite restoring the upper layer, model B with glass-ionomer cement (GIC) restoring the lower layer and bulk-fill resin composite restoring the upper layer, model C with block composite as the only restoring material and model D with bulk-fill resin composite as the only restoring material. Polymerization shrinkage was simulated with the thermal expansion approach. Physiologic masticatory loads were applied in combination with shrinkage effect. Nodal displacements on the lower surfaces of FE models were constrained in all directions. Static linear analyses were carried out. The maximum normal stress criterion was used to assess the influence of each factor. RESULTS: Considering direct restoring techniques, models A, B and D exhibited a high stress gradient at the tooth/restorative material interface. Models A and D showed a similar stress trend along the cavity wall where a similar stress trend was recorded in the dentin and enamel. Model B showed a similar stress trend along enamel/restoration interface but a very low stress gradient along the dentin/restoration interface. Model C with a restoring block composite material showed a better response, with the lowest stress gradient at the dentin, filling block composite and enamel sides. CLINICAL SIGNIFICANCE: Bulk resin-based composite materials applied in a multilayer technique to deep and large Class I cavities produced adverse stress distributions versus block resin composite. Polymerization shrinkage and loading determined high stress levels in deep Class I cavities with bulk multi-layer restorations, while its impact on adhesion in block composite restorations was insignificant.

Quick liquid packaging: Encasing water silhouettes by three-dimensional polymer membranes.

One of the most important substances on Earth is water. It is an essential medium for living microorganisms and for many technological and industrial processes. Confining water in an enclosed compartment without manipulating it or by using rigid containers can be very attractive, even more if the container is biocompatible and biodegradable. Here, we propose a water-based bottom-up approach for facile encasing of short-lived water silhouettes by a custom-made adaptive suit. A biocompatible polymer self-assembling with unprecedented degree of freedom over the water surface directly produces a thin membrane. The polymer film could be the external container of a liquid core or a free-standing layer with personalized design. The membranes produced have been characterized in terms of physical properties, morphology and proposed for various applications from nano- to macroscale. The process appears not to harm cells and microorganisms, opening the way to a breakthrough approach for organ-on-chip and lab-in-a-drop experiments.

Spatio-Temporal Control of Cell Adhesion: Toward Programmable Platforms to Manipulate Cell Functions and Fate.

Biophysical and biochemical signals of material surfaces potently regulate cell functions and fate. In particular, micro- and nano-scale patterns of adhesion signals can finely elicit and affect a plethora of signaling pathways ultimately affecting gene expression, in a process known as mechanotransduction. Our fundamental understanding of cell-material signals interaction and reaction is based on static culturing platforms, i.e., substrates exhibiting signals whose configuration is time-invariant. However, cells in-vivo are exposed to arrays of biophysical and biochemical signals that change in time and space and the way cells integrate these might eventually dictate their behavior. Advancements in fabrication technologies and materials engineering, have recently enabled the development of culturing platforms able to display patterns of biochemical and biophysical signals whose features change in time and space in response to external stimuli and according to selected programmes. These dynamic devices proved to be particularly helpful in shedding light on how cells adapt to a dynamic microenvironment or integrate spatio-temporal variations of signals. In this work, we present the most relevant findings in the context of dynamic platforms for controlling cell functions and fate in vitro. We place emphasis on the technological aspects concerning the fabrication of platforms displaying micro- and nano-scale dynamic signals and on the physical-chemical stimuli necessary to actuate the spatio-temporal changes of the signal patterns. In particular, we illustrate strategies to encode material surfaces with dynamic ligands and patterns thereof, topographic relieves and mechanical properties. Additionally, we present the most effective, yet cytocompatible methods to actuate the spatio-temporal changes of the signals. We focus on cell reaction and response to dynamic changes of signal presentation. Finally, potential applications of this new generation of culturing systems for in vitro and in vivo applications, including regenerative medicine and cell conditioning are presented.

On the influence of surface patterning on tissue self-assembly and mechanics.

Extracellular matrix assembly and composition influence the biological and mechanical functions of tissues. Developing strategies to control the spatial arrangement of cells and matrix is of central importance for tissue engineering-related approaches relying on self-assembling and scaffoldless processes. Literature reports demonstrated that signals patterned on material surfaces are able to control cell positioning and matrix orientation. However, the mechanisms underlying the interactions between material signals and the structure of the de novo synthesized matrix are far from being thoroughly understood. In this work, we investigated the ordering effect provided by nanoscale topographic patterns on the assembly of tissue sheets grown in vitro. We stimulated MC3T3-E1 preosteoblasts to produce and assemble a collagen-rich matrix on substrates displaying patterns with long- or short-range order. Then, we investigated microstructural features and mechanical properties of the tissue in uniaxial tension. Our results demonstrate that patterned material surfaces are able to control the initial organization of cells in close contact to the surface; then cell-generated contractile forces profoundly remodel tissue structure towards mechanically stable spatial patterns. Such a remodelling effect acts both locally, as it affects cell and nuclear shape and globally, by affecting the gross mechanical response of the tissue. Such an aspect of dynamic interplay between cells and the surrounding matrix must be taken into account when designing material platform for the in vitro generation of tissue with specific microstructural assemblies.

A straightforward method to produce decellularized dermis-based matrices for tumour cell cultures.

Decellularized matrices are steadily gaining popularity to study the biology of cells and tissues, as they represent a biomimetic environment in which cells can recapitulate certain behaviours that share similarities with those observed in vivo. Basically, biochemistry, microstructure and mechanics of the decellularized matrices are the most valuable properties that differentiate these culturing systems from conventional bidimensional models. Several procedures to decellularize tissues have been proposed so far, with the common aim to preserve the tissue chemical/physical properties of the original tissue. However, these processes are complex, time-consuming and expensive. In this work, we propose a cost-effective, easy-to-produce decellularized dermal matrix, derived from animal skin. The chemical/physical processes to obtain the matrices proved to not alter matrix structure and did not induce cytotoxicity issues. To test the validity of the decellularized matrices as a model to study the behaviour of tumour cells in vitro, we performed microstructural and mechanical investigations as well as cell proliferation assays. In particular, three different tumour cell lines were used, which proliferated and invaded the matrix with no additional treatments. Decellularized skin scaffold, presented in this work, could be a strong competitor for conventional 3D systems like synthetic porous scaffolds or hydrogels. Copyright © 2016 John Wiley & Sons, Ltd.

Engineering Cell Instructive Materials To Control Cell Fate and Functions through Material Cues and Surface Patterning.

Mastering the interaction between cells and extracellular environment is a fundamental prerequisite in order to engineer functional biomaterial interfaces able to instruct cells with specific commands. Such advanced biomaterials might find relevant application in prosthesis design, tissue engineering, diagnostics and stem cell biology. Because of the highly complex, dynamic, and multifaceted context, a thorough understanding of the cell-material crosstalk has not been achieved yet; however, a variety of material features including biological cues, topography, and mechanical properties have been proved to impact the strength and the nature of the cell-material interaction, eventually affecting cell fate and functions. Although the nature of these three signals may appear very different, they are equated by their participation in the same material-cytoskeleton crosstalk pathway as they regulate cell adhesion events. In this work we present recent and relevant findings on the material-induced cell responses, with a particular emphasis on how the presentation of biochemical/biophysical signals modulates cell behavior. Finally, we summarize and discuss the literature data to draw out unifying elements concerning cell recognition of and reaction to signals displayed by material surfaces.

Controlling Cell Functions and Fate with Surfaces and Hydrogels: The Role of Material Features in Cell Adhesion and Signal Transduction.

In their natural environment, cells are constantly exposed to a cohort of biochemical and biophysical signals that govern their functions and fate. Therefore, materials for biomedical applications, either in vivo or in vitro, should provide a replica of the complex patterns of biological signals. Thus, the development of a novel class of biomaterials requires, on the one side, the understanding of the dynamic interactions occurring at the interface of cells and materials; on the other, it requires the development of technologies able to integrate multiple signals precisely organized in time and space. A large body of studies aimed at investigating the mechanisms underpinning cell-material interactions is mostly based on 2D systems. While these have been instrumental in shaping our understanding of the recognition of and reaction to material stimuli, they lack the ability to capture central features of the natural cellular environment, such as dimensionality, remodelling and degradability. In this work, we review the fundamental traits of material signal sensing and cell response. We then present relevant technologies and materials that enable fabricating systems able to control various aspects of cell behavior, and we highlight potential differences that arise from 2D and 3D settings.