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Contains fulltext : 287291.pdf (Publisher’s version ) (Open Access) 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.

ObjectiveThe 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.MethodsOne 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.ResultsAll 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.SignificanceThe 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.

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.

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.

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.

The aim of this chapter is to expound on the methods for the development of a synthetic analog of the extracellular matrix (ECM) that, because of a careful choice of its characteristics, can regulate cell behavior and tissue progression for applications in neural regenerative medicine. Neural cells—neurons—are the smallest building blocks of the central and peripheral nervous systems. The function of neurons is to elaborate the information that a man receives from the environment, share it with other neurons, and use it to activate complex functions such as language, behavior and surviving, reasoning, and self-correction. In the body, neurons are linked to other neurons and are supported by the ECM. The scaffolds are an artificial analog of the ECM: they are designed and fabricated using a combination of chemical, physical, and engineering techniques. Thus the scope of tissue engineering and neural regenerative medicine is to optimize the characteristics of the scaffold to assure the best performance of the neurons cultivated in them. Performance is another word for efficiency: depending on the variables that one want to maximize, one can have different definitions of efficiency. Thus as for some examples, scaffolds can be designed to optimize cell adhesion, growth, proliferation, clustering, or activity. In this chapter, we will explain how one can use micro- and nanofabrication techniques to produce scaffolds with a tight control over its characteristics, including the physical, chemical, geometrical, and mechanical characteristics. Then, we will see how a combination of characteristics can influence cell behavior, and to what extent.

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.

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.

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 4 mm 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 22 MPa and 19 MPa 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 11.5 MPa and 7.5 MPa were recorded in the dentin and restoration, respectively, whereas stresses of 17 MPa and 9 MPa 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 4 mm deep class I cavities in lower molars versus “single-layer” bulk fill composite technique.

In many cell types, shape and function are intertwined. In vivo, vascular endothelial cells (ECs) are typically elongated and aligned in the direction of blood flow; however, near branches and bifurcations where atherosclerosis develops, ECs are often cuboidal and have no preferred orientation. Thus, understanding the factors that regulate EC shape and alignment is important. In vitro , EC morphology and orientation are exquisitely sensitive to the composition and topography of the substrate on which the cells are cultured; however, the underlying mechanisms remain poorly understood. Different strategies of substrate patterning for regulating EC shape and orientation have been reported including adhesive motifs on planar surfaces and micro- or nano-scale gratings that provide substrate topography. Here, we explore how ECs perceive planar bio-adhesive versus microgrooved topographic surfaces having identical feature dimensions. We show that while the two types of patterned surfaces are equally effective in guiding and directing EC orientation, the cells are considerably more elongated on the planar patterned surfaces than on the microgrooved surfaces. We also demonstrate that the key factor that regulates cellular morphology is focal adhesion clustering which subsequently drives cytoskeletal organization. The present results promise to inform design strategies of novel surfaces for the improved performance of implantable cardiovascular devices.

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.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.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.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 wrapping of water silhouettes creates 3D polymeric membranes in a single step., 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.

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 are 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) is 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, i.e. soft aligned matrices ameliorate the spontaneous adipogenic differentiation, whereas stiff aligned matrices reduce cross-differentiation. We infer that mechanical signalling 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 This article is protected by copyright. All rights reserved.

In this work, Vapor Annealing Sintering (VAS) process was introduced for low-cost pressureless producing dense Zinc Oxide (ZnO) thin films deposited from nanoparticles at near-room temperature (50 °C). Spontaneous densification evolution from nanoparticulate to a dense film via a dissolution-diffusion-reprecipitation mechanism was observed exposing ZnO layers to the vapor of an acetic acid aqueous solution at isothermal condition. The influence of the annealing on the optical properties of the treated films was investigated in order to study the structural changes. The proposed method can allow new opportunities for simple and low-cost ceramics thin film manufacturing also involving pressure and temperature-sensitive materials.

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.

Cell response to exogenous cues is the result of a complex integration of multiple biochemical/biophysical signals, which might occur simultaneously and might be characterized by specific spatial and temporal patterns. Among these signals, surface topography plays an important role in affecting cell functions and fate. However, the current understanding of the interplay between cells and topography relies on static environment. Here the intrinsic light-responsive properties of azopolymers and the versatility of laser-based confocal microscope technique is exploited, aiming to induce spatio-temporal dynamic topographic changes in situ during cell culture. Diverse patterns can be designed on cell-populated azopolymer films with high control on time, space, and on-off signal modification. The technique proposed in this study enables the development of synthetic platforms that finely control cell orientation and migration both in time and space. The results may pave the way to unravel complex processes involved in cell-topography interactions, thus allowing to define the spatio-temporal features that most effectively influence cell functions.

The enormous variety and specificity of cell functions observed in vivo are the result of multifaceted interplays occurring between adjacent cells or between cells and the extracellular environment. Arrays of signals whose presentation is tightly controlled in time and space regulate different aspects of cell behavior including differentiation, quiescence, proliferation and biosynthesis [1]. The integration of such a complex set of information within synthetic platforms has acquired large interest from the scientific community in order to fabricate biomaterials and devices that precisely control cell functions and fate in vitro. The field of functional biointerfaces is widely interdisciplinary, spanning from biochemistry, molecular and cell biology, microand nanotechnologies, material synthesis and functionalization. The successful translation of the scientific findings into the engineering of devices for practical biotechnological applications relies on integrating those disciplines. In this context, diverse but complementary contributions need to be integrated into new biomaterials, that is, multisignal patterning methodologies, multiscale modeling, advanced characterization and processing technologies. These should be optimized and match the desired biomedical or biotechnological applications. Thus, the concept of functional biointerfaces is rapidly evolving and constantly gaining new valences [2,3]. When a cell lands on a material surface, an integrin-mediated material recognition occurs and clusters of integrins assemble in the form of punctuate-wide nascent adhesions (

Stem cells (SCs) can self-renew or differentiate into different cell types, which makes them an ideal cell source for therapies based on tissue engineering. Despite these characteristics, the employment of SCs in clinics has seen alternating fortunes because of our limited understanding of the signals governing SC functions and fate, which impairs our ability to engineer systems to deliver SCs in vivo and to guide their correct biological processes. However, experimental evidence demonstrated that SCs are able to recognize biochemical and biophysical signals displayed by material surfaces; most importantly, cells integrate these signals to elaborate fate decisions. Although the mechanisms underlying signal recognition and response have not been thoroughly characterized, there is a general consensus that the cell adhesion process plays a central role. Adhesion represents a communication gate between exogenous signals and intracellular signaling cascades involving the cytoskeleton and the nucleus. In this work we present recent findings on materials engineered to control cell functions through adhesion processes. In particular, we emphasize the role of material signals on SC behavior. Finally, we discuss a few sensing and transductive molecular mechanisms in an effort to draw out unifying elements concerning cell recognition of and reaction to biophysical/biochemical material signals aimed at controlling cell fate through cell adhesion. Microneedles and nanomedicine are two out of several active innovative approaches used to enhance the transdermal drug and vaccine delivery. Their application individually or in combination have shown to be very promising and attracted considerable interest by researchers from both industry and academia over the last 2 decades. Combining the two technologies has growing interest and has shown to be promising approach not only in enhancing the transdermal drug and vaccine permeation for even difficult drug molecules, such as hydrophilic and macromolecules, but also impart protection and controlling the drug release rates. In Subchapter 2.2, we aim to highlight the advances, which has been made in using these two technologies as an individual or in combination with drug and vaccine delivery. Metal implants, in the form of screws, plates, or pins, are extensively used in the treatment of fractures and nonunions or as replacements for malfunctioned joints. These bones implants face many challenges for acceptance and survival upon insertion in the human body. These challenges include severe inflammation, bacterial invasion, and poor biointegration with the traumatized tissue. Titania (TiO 2 ) nanotubes (TNTs) arrays engineered on the surface of Ti implants by simple and scalable electrochemical anodization process, which have been widely explored as a new nanoengineering approach to improve the process of osseointegration and at the same time to be used as depots for loading drugs and their controllable release for localized delivery, and therapeutic purpose. Several advanced functions can be introduced into these multifunctional implants, including biopolymers, nanoparticles or external stimulation (e.g., electrical, electromagnetic and ultrasound) to release the loaded therapeutic agents in a desired manner when required. Subchapter 2.3 highlights the developed concepts of drug releasing implants based on TNTs for enhancing osteogenesis at the bone-implant interface, as an alternative approach to systemic delivery of therapeutic agents. The reconstruction of skeletal defects is a continuing challenge. Bone and dentin are mineralized hard tissues. The primary inorganic component of these two tissues is crystalline hydroxyapatite and the primary organic component is type I collagen, and these two components are cell-manufactured materials. Tissue-engineering strategies using novel biomaterials have emerged as a promising potential for the treatment of mineralization defects. Some of the recent bioinspired biomaterials utilize proteins and peptides as nanoscale building blocks. A key functional property is that biomaterials need to be cell-compatible and mimic the dynamic nature of the extracellular matrix. ECM is a complex environment comprising a plethora of macromolecules. Another class of novel biomaterials that are currently being developed are peptide-based hydrogels. Peptide-based engineered scaffolds present several advantages over traditional protein scaffolds as the control over hydrogel properties and can be easily tailored to the requirement of the tissue. Biomaterials generated by the self-assembly process have varied applications as it mimics nature’s method of material synthesis. Therefore, concepts of protein-based self-assembly can be utilized for constructing useful biomaterials.

Research on biomaterials and related subjects has been active in Italy. Starting from the very first examples of biomaterials and biomedical devices, Italian researchers have always provided valuable scientific contributions. This trend has steadily increased. To provide a rough estimate of this, it is sufficient to search PubMed, a free search engine accessing primarily the MEDLINE database of references and abstracts on life sciences and biomedical topics, with the keywords "biomaterials" or "tissue engineering" and sort the results by affiliation. Again, even though this is a crude estimate, the results speak for themselves, as Italy is the third European country, in terms of publications, with an astonishing 3,700 products in the last decade.

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.

In "situ" tissue engineering is a promising approach in regenerative medicine, envisaging to potentiate the physiological tissue repair processes by recruiting the host's own cellular progenitors at the lesion site by means of bioactive materials. Despite numerous works focused the attention in characterizing novel chemoattractant molecules, only few studied the optimal way to present signal in the microenvironment, in order to recruit cells more effectively. In this work, we have analyzed the effects of gradients of stromal derived factor-1 (SDF-1) on the migratory behavior of human mesenchymal stem cells (MSCs). We have characterized the expression of the chemokine-associated receptor, CXCR4, using cytofluorimetric and real-time PCR analyses. Gradients of SDF-1 were created in 3D collagen gels in a chemotaxis chamber. Migration parameters were evaluated using different chemoattractant concentrations. Our results show that cell motion is strongly affected by the spatio-temporal features of SDF-1 gradients. In particular, we demonstrated that the presence of SDF-1 not only influences cell motility but alters the cell state in terms of SDF-1 receptor expression and productions, thus modifying the way cells perceive the signal itself. Our observations highlight the importance of a correct stimulation of MSCs by means of SDF-1 in order to implement on effective cell recruitment. Our results could be useful for the creation of a "cell instructive material" that is capable to communicate with the cells and control and direct tissue regeneration. Biotechnol. Bioeng. 2014;111: 2303-2316. © 2014 Wiley Periodicals, Inc.

Material features proved to exert a potent influence on cell behaviour in terms of adhesion, migration and differentiation. In particular, biophysical and biochemical signals on material surfaces are able to affect focal adhesion distribution and cytoskeletal assemblies, which are known to regulate signalling pathways that ultimately influence cell fate and functions. However, a general, unifying model that correlates cytoskeletal-generated forces with genetic events has yet to be developed. Therefore, it is crucial to gain a better insight into the material-cytoskeleton crosstalk in order to design and fabricate biomaterials able to govern cell fate more accurately. In this work, we demonstrate that confining focal adhesion distribution and growth dramatically alters the cytoskeleton's structures and dynamics, which in turn dictate cellular and nuclear shape and polarization. MC3T3 preosteoblasts were cultivated on nanograted polydimethylsiloxane substrates and a thorough quantification – in static and dynamic modes – of the morphological and structural features of focal adhesions and cytoskeleton was performed. Nanoengineered surfaces provided well-defined zones for focal adhesions to form and grow. Unique cytoskeletal structures spontaneously assembled when focal adhesions were confined and, in fact, they proved to be very effective in deforming the nuclei. The results here presented provide elements to engineer surfaces apt to guide and control cell behaviour through the material-cytoskeleton-nucleus axis.

The Y-box binding protein 1 (YB-1) belongs to the cold-shock domain protein superfamily, one of the most evolutionarily conserved nucleic acid-binding proteins currently known. YB-1 performs a wide variety of cellular functions, including transcriptional and translational regulation, DNA repair, drug resistance, and stress responses to extracellular signals. Inasmuch as the level of YB-1 drastically increases in tumor cells, this protein is considered to be one of the most indicative markers of malignant tumors. Here, we present evidence that ΔNp63α, the predominant p63 protein isoform in squamous epithelia and YB-1, can physically interact. Into the nucleus, ΔNp63α and YB-1 cooperate in PI3KCA gene promoter activation. Moreover, ΔNp63α promotes YB-1 nuclear accumulation thereby reducing the amount of YB-1 bound to its target transcripts such as that encoding the SNAIL1 protein. Accordingly, ΔNp63α enforced expression was associated with a reduction of the level of SNAIL1, a potent inducer of epithelial to mesenchymal transition. Furthermore, ΔNp63α depletion causes morphological change and enhanced formation of actin stress fibers in squamous cancer cells. Mechanistic studies indicate that ΔNp63α affects cell movement and can reverse the increase of cell motility induced by YB-1 overexpression. These data thus suggest that ΔNp63α provides inhibitory signals for cell motility. Deficiency of ΔNp63α gene expression promotes cell mobilization, at least partially, through a YB-1-dependent mechanism.

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.

Material signals in the form of surface topographies, proved to be potent regulators of cell functions and fate, through mechanotransduction pathways. While a wealth of data is related to regular topographic patterns, i.e., lines, pit, or protrusions, there are comparatively few studies addressing the effects of circular, concentric patterns. Yet, curvatures affecting cell shape dramatically alter cell contractility and behavior. Additionally, the vast majority of patterned surfaces are static in nature and this prevents to understand how cells perceive and respond to the topographic patterns. Here, a technique is exploited for dynamically embossing micrometerscale circular pattern on azopolymeric substrates using a confocal laser microscope and it is analyzed how NIH‐3T3 reacts to the underlying topography in terms of changes in shape and mechanical properties. A characteristic pattern arrangement is found which most effectively alters cell morphology and orientation. Cells perceive the concentric pattern and reconfigure as fast as 2 h after pattern inscription. The changes in morphology also reflect dramatic changes in cell mechanics and cytoskeletal arrangements. The reported method is useful to manipulate cell shape and mechanics in a facile and cost‐effective manner and most importantly enables investigate mechanotransduction events dynamically.

Dermis is a heterogeneous tissue in which extracellular matrix components change in relative amount and spatial assembly across the tissue thickness. The effect of the microstructural and compositional heterogeneities on the overall mechanical response of dermis is, however, largely ignored. In this work, we aimed at gaining a better insight into the effect of extracellular matrix microstructure and composition on the mechanical behaviour of different dermal strata by combining mechanical analysis and selective enzymatic digestion of extracellular matrix components. The dynamical-mechanical tests we performed on bovine dermal splits show that the upper dermal stratum, which is richer in papillary dermis, is characterized by higher mechanical properties than the lower one, which is almost composed of reticular dermis. Moreover, the depletion of interfibrillar proteins, proteoglycans and glycosamminoglycans decreases the dynamic moduli of dermis, especially at small frequencies. Of the two dermal layers tested, the upper dermal layer is more sensitive to the enzymatic treatment than the lower layer. Interestingly, the disruption of the elastic network greatly influenced the viscoelastic properties of upper dermis, inducing a dramatic decrease of both storage (G') and loss (G '') moduli, suggesting that the spatial assembly of the elastin and collagen networks as well as their mutual interactions dominate the dynamical mechanical response of the tissue.

Physical and chemical characteristics of materials are important regulators of cell behavior. In particular, cell elasticity is a fundamental parameter that reflects the state of a cell. Surface topography finely modulates cell fate and function via adhesion mediated signaling and cytoskeleton generated forces. However, how topographies alter cell mechanics is still unclear. In this work we have analyzed the mechanical properties of peripheral and nuclear regions of NIH-3T3 cells on azopolymer substrates with different topographic patterns. Micrometer scale patterns in the form of parallel ridges or square lattices of surface elevations were encoded on light responsive azopolymer films by means of contactless optical methods. Cell mechanics was investigated by atomic force microscopy (AFM). Cells and consequently the cell cytoskeleton were oriented along the linear patterns affecting cytoskeletal structures, e.g., formation of actin stress fibers. Our data demonstrate that topographic substrate patterns are recognized by cells and mechanical information is transferred by the cytoskeleton. Furthermore, cytoskeleton generated forces deform the nucleus, changing its morphology that appears to be related to different mechanical properties in the nuclear region.

The deflection of magnetic beads in a microfluidic channel through magnetophoresis can be improved if the particles are somehow focused along the same streamline in the device. We design and fabricate a microfluidic device made of two modules, each one performing a unit operation. A suspension of magnetic beads in a viscoelastic medium is fed to the first module, which is a straight rectangular-shaped channel. Here, the magnetic particles are focused by exploiting fluid viscoelasticity. Such a channel is one inlet of the second module, which is a H-shaped channel, where a buffer stream is injected in the second inlet. A permanent magnet is used to displace the magnetic beads from the original to the buffer stream. Experiments with a Newtonian suspending fluid, where no focusing occurs, are carried out for comparison. When viscoelastic focusing and magnetophoresis are combined, magnetic particles can be deterministically separated from the original streamflow to the buffer, thus leading to a high deflection efficiency (up to ~96%) in a wide range of flow rates. The effect of the focusing length on the deflection of particles is also investigated. Finally, the proposed modular device is tested to separate magnetic and non-magnetic beads.

The initial conditions for morphogenesis trigger a cascade of events that ultimately dictate structure and functions of tissues and organs. Here we report that surface nanopatterning can control the initial assembly of focal adhesions, hence guiding human mesenchymal stem cells (hMSCs) through the process of self-organization and differentiation. This process self-sustains, leading to the development of macroscopic tissues with molecular profiles and microarchitecture reminiscent of embryonic tendons. Therefore, material surfaces can be in principle engineered to set off the hMSC program toward tissuegenesis in a deterministic manner by providing adequate sets of initial environmental conditions.

Topography of material surfaces is known to influence cell behavior at different levels: from adhesion up to differentiation. Different micro- and nano-patterning techniques have been employed in order to create patterned surfaces to investigate various aspects of cell behavior, most notably cellular mechanotransduction. Nevertheless, conventional techniques, once implemented on a specific substrate, fail in allowing dynamic changes of the topographic features. Here we investigated NIH-3T3 cell response to reversible topographic signals encoded on light responsive azopolymer films. Switchable patterns were fabricated by means of a well established holographic set-up. Surface relief gratings (SRGs) were realized with Lloyd's mirror system and erased with circular polarized or incoherent light. Cell cytoskeleton organization and focal adhesions assembly proved to be very sensitive to the underlying topographic signal. Thereafter, pattern reversibility was tested in air and wet environment by using temperature or light as triggers. Additionally, pattern modification was dynamically performed on substrates with living cells. This study paves the way towards an in-situ and real-time investigation of the material-cytoskeleton crosstalk owing to the intrinsic proprieties of azopolymers.

Topographic patterns are known to affect cellular processes such as adhesion, migration and differentiation. However, the optimal way to deliver topographic signals to provide cells with precise instructions has not been defined yet. In this work, we hypothesize that topographic patterns may be able to control the sensing and adhesion machinery of cells when their interval features are tuned on the characteristic lengths of filopodial probing and focal adhesions (FAs). Features separated by distance beyond the length of filopodia cannot be readily perceived; therefore, the formation of new adhesions is discouraged. If, however, topographic features are separated by a distance within the reach of filopodia extension, cells can establish contact between adjacent topographic islands. In the latter case, cell adhesion and polarization rely upon the growth of FAs occurring on a specific length scale that depends on the chemical properties of the surface. Topographic patterns and chemical properties may interfere with the growth of FAs, thus making adhesions unstable. To test this hypothesis, we fabricated different micropatterned surfaces displaying feature dimensions and adhesive properties able to interfere with the filopodial sensing and the adhesion maturation, selectively. Our data demonstrate that it is possible to exert a potent control on cell adhesion, elongation and migration by tuning topographic features’ dimensions and surface chemistry.

Contaminants in water streams, in the form of oily/organic phases or nanoparticles, affect a large variety of activities, from laboratory practice up to environmental systems. To date, a large number of materials for the absorption and retention of these pollutants have been developed. Such materials, however, rarely conjugate high oil uptake, nanoparticle sequestration, biodegradability, and cost effectiveness simultaneously. In this work, we exploited the structural properties of dermal collagen networks and simple chemical manipulations to fabricate an original material that proved effective in separating water from organic and nanoparticulated contaminants. Our material is reusable, biodegradable, safe, and cost-effective and shows a high absorptive capacity of a large variety of organic compounds; it is also able to capture metallic nanoparticles. These features allow our material to effectively separate water from oily/nanoparticulate phases, thus making it an appropriate absorber for chemical processes and environmental protection.