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We show how to control the formation and alignment of gel ‘noodles’. Nanostructure alignment can be achieved reproducibly by extensional deformation as the filaments form. Using a spinning technique, very long and highly aligned filaments can be made. The Young's moduli of the gel noodles are similar to that of a bulk gel. By using two syringe pumps in a concentric flow setup, we show that a filament-in-filament morphology can be created., Extensional deformations induce nanostructure alignment in low molecular weight gel noodles during injection.

Supplemental Digital Content is available in the text., Rationale: Despite increasing understanding of the prognostic importance of vascular stiffening linked to perivascular fibrosis in hypertension, the molecular and cellular regulation of this process is poorly understood. Objectives: To study the functional role of microRNA-214 (miR-214) in the induction of perivascular fibrosis and endothelial dysfunction driving vascular stiffening. Methods and Results: Out of 381 miRs screened in the perivascular tissues in response to Ang II (angiotensin II)-mediated hypertension, miR-214 showed the highest induction (8-fold, P=0.0001). MiR-214 induction was pronounced in perivascular and circulating T cells, but not in perivascular adipose tissue adipocytes. Global deletion of miR-214−/− prevented Ang II-induced periaortic fibrosis, Col1a1, Col3a1, Col5a1, and Tgfb1 expression, hydroxyproline accumulation, and vascular stiffening, without difference in blood pressure. Mechanistic studies revealed that miR-214−/− mice were protected against endothelial dysfunction, oxidative stress, and increased Nox2, all of which were induced by Ang II in WT mice. Ang II-induced recruitment of T cells into perivascular adipose tissue was abolished in miR-214−/− mice. Adoptive transfer of miR-214−/− T cells into RAG1−/− mice resulted in reduced perivascular fibrosis compared with the effect of WT T cells. Ang II induced hypertension caused significant change in the expression of 1380 T cell genes in WT, but only 51 in miR-214−/−. T cell activation, proliferation and chemotaxis pathways were differentially affected. MiR-214−/− prevented Ang II-induction of profibrotic T cell cytokines (IL-17, TNF-α, IL-9, and IFN-γ) and chemokine receptors (CCR1, CCR2, CCR4, CCR5, CCR6, and CXCR3). This manifested in reduced in vitro and in vivo T cell chemotaxis resulting in attenuation of profibrotic perivascular inflammation. Translationally, we show that miR-214 is increased in plasma of patients with hypertension and is directly correlated to pulse wave velocity as a measure of vascular stiffness. Conclusions: T-cell–derived miR-214 controls pathological perivascular fibrosis in hypertension mediated by T cell recruitment and local profibrotic cytokine release.

Life is a dynamic event, and cells represent its smallest structural and functional units that can exist on their own. Cells and the surrounding extra cellular matrix are in a constant flux of change imposed by each other. The aim of this chapter is to look into the complex interplay of physical and chemical factors imposed on cells, in particular mesenchymal stem cells (MSCs), via their microenvironment and how such factors influence MSCs response. Some of the defined and independently varied factors such as surface chemistry, modulus and topography are reviewed in this chapter, with a focus on cell physicochemical memory formation. Acknowledging the importance of mechanical and chemical stimuli experienced by cells and their polarity, while investigating the interdependent relationship between them, will not only help develop more fitting culture systems, for instance to maintain stem cell multipotency, but advance the understanding of how such extracellular stimuli are understood and acted upon by cells during their lifetime.

The extracellular matrix is a highly complex microenvironment, whose various components converge to regulate cell fate. Hydrogels, as water-swollen polymer networks composed by synthetic or natural materials, are ideal candidates to create biologically active substrates that mimic these matrices and target cell behaviour for a desired tissue engineering application. Indeed, the ability to tune their mechanical, structural, and biochemical properties provides a framework to recapitulate native tissues. This review explores how hydrogels have been engineered to harness the chondrogenic response of stem cells for the repair of damaged cartilage tissue. The signalling processes involved in hydrogel-driven chondrogenesis are also discussed, identifying critical pathways that should be taken into account during hydrogel design.

Extracellular matrix (ECM) proteins are key mediators of cell/material interactions. The surface density and conformation of these proteins adsorbed on the material surface influence cell adhesion and the cellular response. We have previously shown that subtle variations in surface chemistry lead to drastic changes in the conformation of adsorbed fibronectin (FN). On poly(ethyl acrylate) (PEA), FN unfolds and displays domains for cell adhesion and FN-FN interaction, whereas on poly(methyl acrylate) (PMA) – with only one methyl group less – FN remains globular as it is in solution. The effect of the strength of the protein/material interaction in cell response, and its relation to protein density and conformation, has received limited attention so far. In this work, we used FN-functionalized AFM cantilevers to evaluate, via force spectroscopy, the strength of interaction between fibronectin and the underlying polymer which controls FN conformation (PEA and PMA). We found that the strength of FN/PEA interaction is significantly higher than FN/PMA, which limits the mobility of FN layer on PEA, reduces the ability of cells to mechanically reorganize FN and then leads to enhanced proteolysis and degradation of the surrounding matrix with compromised cell viability. By contrast, both PEA and PMA support cell adhesion when FN density is increased and also in the presence of serum or other serum proteins, including vitronectin (VN) and bovine serum albumin (BSA), which provide a higher degree of mobility to the matrix. Statement of Significance The identification of parameters influencing cell response is of paramount importance for the design of biomaterials that will act as synthetic scaffolds for cells to anchor, grow and, eventually, become specialised tissues. Cells interact with materials through an intermediate layer of proteins adsorbed on the material surface. It is known that the density and conformation of these proteins determine cell behaviour. Here we show that the strength of protein/material interactions, which has received very limited attention so far, is key to understand the cellular response to biomaterials. Very strong protein/material interactions reduce the ability of cells to mechanically reorganize proteins at the material interface which results in enhanced matrix degradation, leading ultimately to compromised cell viability.

Significance Tissues are viscoelastic in nature and their physical properties play a fundamental role in development, tumorigenesis, and wound healing. Cell response to matrix elasticity is well understood through a “molecular clutch” which engages when stiffness is sufficiently high to expose binding sites in mechanosensitive proteins. Here we show that cell response to pure viscous surfaces (i.e., with no elastic component) can be explained through the same molecular clutch. Mechanisms used by cells to sense rigidity are more universal and can be used to unveil cell interaction with complex viscoelastic environments. The research presents a tool to understand cells within tissues and in turn opens new avenues to incorporate viscosity into the design of synthetic cellular microenvironments., Cell response to matrix rigidity has been explained by the mechanical properties of the actin-talin-integrin-fibronectin clutch. Here the molecular clutch model is extended to account for cell interactions with purely viscous surfaces (i.e., without an elastic component). Supported lipid bilayers present an idealized and controllable system through which to study this concept. Using lipids of different diffusion coefficients, the mobility (i.e., surface viscosity) of the presented ligands (in this case RGD) was altered by an order of magnitude. Cell size and cytoskeletal organization were proportional to viscosity. Furthermore, there was a higher number of focal adhesions and a higher phosphorylation of FAK on less-mobile (more-viscous) surfaces. Actin retrograde flow, an indicator of the force exerted on surfaces, was also seen to be faster on more mobile surfaces. This has consequential effects on downstream molecules; the mechanosensitive YAP protein localized to the nucleus more on less-mobile (more-viscous) surfaces and differentiation of myoblast cells was enhanced on higher viscosity. This behavior was explained within the framework of the molecular clutch model, with lower viscosity leading to a low force loading rate, preventing the exposure of mechanosensitive proteins, and with a higher viscosity causing a higher force loading rate exposing these sites, activating downstream pathways. Consequently, the understanding of how viscosity (regardless of matrix stiffness) influences cell response adds a further tool to engineer materials that control cell behavior.

Cell migration is a fundamental process involved in a wide range of biological phenomena. However, how the underlying mechanisms that control migration are orchestrated is not fully understood. In this work, we explore the migratory characteristics of human fibroblasts using different organisations of fibronectin (FN) triggered by two chemically similar surfaces, poly(ethyl acrylate) (PEA) and poly(methyl acrylate) (PMA); cell migration is mediated via an intermediate layer of fibronectin (FN). FN is organised into nanonetworks upon simple adsorption on PEA whereas a globular conformation is observed on PMA. We studied cell speed over the course of 24 h and the morphology of focal adhesions in terms of area and length. Additionally, we analysed the amount of cell-secreted FN as well as FN remodelling. Velocity of human fibroblasts was found to exhibit a biphasic behaviour on PEA, whereas it remained fairly constant on PMA. FA analysis revealed more mature focal adhesions on PEA over time contrary to smaller FAs found on PMA. Finally, human fibroblasts seemed to remodel adsorbed FN more on PMA than on PEA. Overall, these results indicate that the cell–protein–material interface affects cell migratory behaviour. Analysis of FAs together with FN secretion and remodelling were associated with differences in cell velocity providing insights into the factors that can modulate cell motility.

Pancreatic ductal adenocarcinoma is particularly metastatic, with dismal survival rates and few treatment options. Stiff fibrotic stroma is a hallmark of pancreatic tumours, but how stromal mechanosensing affects metastasis is still unclear. Here, we show that mechanical changes in the pancreatic cancer cell environment affect not only adhesion and migration, but also ATP/ADP and ATP/AMP ratios. Unbiased metabolomic analysis reveals that the creatine-phosphagen ATP-recycling system is a major mechanosensitive target. This system depends on arginine flux through the urea cycle, which is reflected by the increased incorporation of carbon and nitrogen from L-arginine into creatine and phosphocreatine on stiff matrix. We identify that CKB is a mechanosensitive transcriptional target of YAP, and thus it increases phosphocreatine production. We further demonstrate that the creatine-phosphagen system has a role in invasive migration, chemotaxis and liver metastasis of cancer cells.

Bone marrow and adipose tissue human mesenchymal stem cells were seeded in highly performing 3D gelatin–chitosan hybrid hydrogels of varying chitosan content in the presence of human platelet lysate and evaluated for their proliferation and osteogenic differentiation. Both bone marrow and adipose tissue human mesenchymal stem cells in gelatin–chitosan hybrid hydrogel 1 (chitosan content 8.1%) or gelatin–chitosan hybrid hydrogel 2 (chitosan 14.9%) showed high levels of viability (80%–90%), and their proliferation and osteogenic differentiation was significantly higher with human platelet lysate compared to fetal bovine serum, particularly in gelatin–chitosan hybrid hydrogel 1. Mineralization was detected early, after 21 days of culture, when human platelet lysate was used in the presence of osteogenic stimuli. Proteomic characterization of human platelet lysate highlighted 59 proteins mainly involved in functions related to cell adhesion, cellular repairing mechanisms, and regulation of cell differentiation. In conclusion, the combination of our gelatin–chitosan hybrid hydrogels with hPL represents a promising strategy for bone regenerative medicine using human mesenchymal stem cells.

[EN] While much work has been done in the design of biomaterials to control integrin-mediated adhesion, less emphasis has been put on functionalization of materials with cadherin ligands. Yet, cell-cell contacts in combination with cell-matrix interactions are key in driving embryonic development, collective cell migration, epithelial to mesenchymal transition, and cancer metastatic processes, among others. This review focuses on the incorporation of both cadherin and integrin ligands in biomaterial design, to promote what is called the "adhesive crosstalk." First, the structure and function of cadherins and their role in eliciting mechanotransductive processes, by themselves or in combination with integrin mechanosensing, are introduced. Then, biomaterials that mimic cell-cell interactions, and recent applications to get insights in fundamental biology and tissue engineering, are critically discussed., This work was supported by an EPSRC Programme Grant (No. EP/P001114/1). The authors acknowledge support from a grant from the UK Regenerative Medicine Platform "Acellular/Smart Materials - 3D Architecture" (Grant No. MR/R015651/1) and MRC funding (Grant No. MR/S005412/1).

Here we report on the development of a new type of hybrid fibrinogen-polylactic acid (FBG-PLA) nanofibres (NFs) with improved stiffness, combining the good mechanical properties of PLA with the excellent cell recognition properties of native FBG. We were particularly interested in the dorsal and ventral cell response to the nanofibres' organization (random or aligned), using human umbilical endothelial cells (HUVECs) as a model system. Upon ventral contact with random NFs, the cells developed a stellate-like morphology with multiple projections. The well-developed focal adhesion complexes suggested a successful cellular interaction. However, time-lapse analysis shows significantly lowered cell movements, resulting in the cells traversing a relatively short distance in multiple directions. Conversely, an elongated cell shape and significantly increased cell mobility were observed in aligned NFs. To follow the dorsal cell response, artificial wounds were created on confluent cell layers previously grown on glass slides and covered with either random or aligned NFs. Time-lapse analysis showed significantly faster wound coverage (within 12 h) of HUVECs on aligned samples vs. almost absent directional migration on random ones. However, nitric oxide (NO) release shows that endothelial cells possess lowered functionality on aligned NFs compared to random ones, where significantly higher NO production was found. Collectively, our studies show that randomly organized NFs could support the endothelization of implants while aligned NFs would rather direct cell locomotion for guided neovascularization. Copyright © 2016 John Wiley & Sons, Ltd.

Basement membranes (BMs) are specialised extracellular matrices that provide structural support to tissues as well as influence cell behaviour and signalling. Mutations in COL4A1/COL4A2, a major BM component, cause a familial form of eye, kidney and cerebrovascular disease, including stroke, while common variants in these genes are a risk factor for intracerebral haemorrhage in the general population. These phenotypes are associated with matrix defects, due to mutant protein incorporation in the BM and/or its absence by endoplasmic reticulum (ER) retention. However, the effects of these mutations on matrix stiffness, the contribution of the matrix to the disease mechanism(s) and its effects on the biology of cells harbouring a collagen IV mutation remain poorly understood. To shed light on this, we employed synthetic polymer biointerfaces, poly(ethyl acrylate) (PEA) and poly(methyl acrylate) (PMA) coated with ECM proteins laminin or fibronectin (FN), to generate controlled microenvironments and investigate their effects on the cellular phenotype of primary fibroblasts harbouring a COL4A2(+/G702D) mutation. FN nanonetworks assembled on PEA induced increased deposition and assembly of collagen IV in COL4A2(+/G702D) cells, which was associated with reduced ER size and enhanced levels of protein chaperones such as BIP, suggesting increased protein folding capacity of the cell. FN nanonetworks on PEA also partially rescued the reduced stiffness of the deposited matrix and cells, and enhanced cell adhesion through increased actin-myosin contractility, effectively rescuing some of the cellular phenotypes associated with COL4A1/4A2 mutations. The mechanism by which FN nanonetworks enhanced the cell phenotype involved integrin β(1)-mediated signalling. Collectively, these results suggest that biomaterials and enhanced integrin signalling via assembled FN are able to shape the matrix and cellular phenotype of the COL4A2(+/G702D) mutation in patient-derived cells.

Surface functionalization strategies of synthetic materials for regenerative medicine applications comprise the development of microenvironments that recapitulate the physical and biochemical cues of physiological extracellular matrices. In this context, material-driven fibronectin (FN) nanonetworks obtained from the adsorption of the protein on poly(ethyl acrylate) provide a robust system to control cell behavior, particularly to enhance differentiation. This study aims at augmenting the complexity of these fibrillar matrices by introducing vitronectin, a lower-molecular-weight multifunctional glycoprotein and main adhesive component of serum. A cooperative effect during co-adsorption of the proteins is observed, as the addition of vitronectin leads to increased fibronectin adsorption, improved fibril formation, and enhanced vitronectin exposure. The mobility of the protein at the material interface increases, and this, in turn, facilitates the reorganization of the adsorbed FN by cells. Furthermore, the interplay between interface mobility and engagement of vitronectin receptors controls the level of cell fusion and the degree of cell differentiation. Ultimately, this work reveals that substrate-induced protein interfaces resulting from the cooperative adsorption of fibronectin and vitronectin fine-tune cell behavior, as vitronectin micromanages the local properties of the microenvironment and consequently short-term cell response to the protein interface and higher order cellular functions such as differentiation.

Engineering biomaterials for regenerative medicine involves a myriad of aspects to be considered for the successful design, interaction with cells and integration with living tissues (i.e. pore size, mechanical properties, degradation rate, biological activity). Among different technologies used to functionalise synthetic biomaterials and promote cell adhesion, cell growth and cell differentiation, this review focuses on strategies to organise extracellular matrix (ECM) proteins in a biomimetic way, as cells do in natural tissues in vivo (the ECM is a mesh of proteins that surrounds cells, and therefore, constitutes the scaffolding of a tissue), but using functional materials instead of living cells. The authors critically review material-based strategies to organise fibronectin (FN), a core component in the ECM of many tissues, and engineer microenvironments that recapitulate the structure and properties of the ECM. Material-driven organisation of FN in analogy with their natural cell-mediated assembly i...

We present a detailed characterization of fibronectin (FN) adsorption and cell adhesion on poly(ethyl acrylate) (PEA) and poly(methyl acrylate) (PMA), two polymers with very similar physicochemical properties and chemical structure, which differ in one single methyl group in the lateral chain of the polymer. The globular solution conformation of FN was retained following adsorption onto PMA, whereas spontaneous organization of FN into protein (nano) networks occurred on PEA. This distinct distribution of FN at the material interface promoted a different availability, measured via monoclonal antibody binding, of two domains that facilitated integrin binding to FN: FNIII10 (RGD sequence) and FNIII9 (PHSRN synergy sequence). The enhanced exposure of the synergy domain on PEA compared to PMA triggered different focal adhesion assemblies: L929 fibroblasts showed a higher fraction of smaller focal plaques on PMA (40%) than on PEA (20%). Blocking experiments with monoclonal antibodies against FNIII10 (HFN7.1) and FNIII9 (mAb1937) confirmed the ability of these polymeric substrates to modulate FN conformation. Overall, we propose a simple and versatile material platform that can be used to tune the presentation of a main extracellular matrix protein (FN) to cells, for applications than span from tissue engineering to disease biology.

[EN] Poly-l-lactic acid (PLLA) has been used as a biodegradable polymer for many years; the key characteristics of this polymer make it a versatile and useful resource for regenerative medicine. However, it is not inherently bioactive. Thus, here, a novel process is presented to functionalize PLLA surfaces with poly(ethyl acrylate) (PEA) brushes to provide biological functionality through PEA's ability to induce spontaneous organization of the extracellular matrix component fibronectin (FN) into physiological-like nanofibrils. This process allows control of surface biofunctionality while maintaining PLLA bulk properties (i.e., degradation profile, mechanical strength). The new approach is based on surface-initiated atomic transfer radical polymerization, which achieves a molecularly thin coating of PEA on top of the underlying PLLA. Beside surface characterization via atomic force microscopy, X-ray photoelectron spectroscopy and water contact angle to measure PEA grafting, the biological activity of this surface modification is investigated. PEA brushes trigger FN organization into nanofibrils, which retain their ability to enhance adhesion and differentiation of C2C12 cells. The results demonstrate the potential of this technology to engineer controlled microenvironments to tune cell fate via biologically active surface modification of an otherwise bioinert biodegradable polymer, gaining wide use in tissue engineering applications., The authors acknowledge the EPSRC (EP/P001114/1) and MRC (MR/S005412/1) funding. The authors also acknowledge the EPSRC funding as part of the Doctoral Training Centre EP/F500424/1. This work was also funded by a grant from the UK Regenerative Medicine Platform. X-ray photoelectron spectroscopy was conducted by the National EPSRC XPS Users' Service (NEXUS), Newcastle.

In article number 1800361, Matthew J. Dalby, Manuel Salmeron‐Sanchez, and co‐workers coat structural material, e.g., particles or scaffolds, with a bioactive synthetic material that organizes fibronectin to present BMP‐2 with high efficiency. This is used as a novel regenerative approach to heal critical‐sized bone defects. Simultaneous integrin and growth factor receptor synergistic signaling drives stem cell differentiation leading to bone regeneration in the first veterinary patient.

Cells assemble fibronectin (FN) into fibrils in a process mediated by integrins. For this process to occur, it is known that the presence of other serum proteins is necessary. However, the individual effect of these proteins on FN fibrillogenesis has not been addressed so far. In this study, the effect of vitronectin (VN), an ECM adhesion protein, on material-driven FN fibrillogenesis and cell-mediated FN reorganization is investigated. Poly(ethyl acrylate), PEA, which has previously shown the ability to induce the organization of FN into well-developed physiological-like networks upon adsorption, was employed as a material substrate. FN adsorption, cell adhesion and cellular FN reorganization in the presence or absence of VN were studied. Both FN surface density, quantified via western blot, and its distribution on PEA surfaces, determined via atomic force microscopy, were altered when FN was adsorbed competitively with VN at certain compositions. Moreover, the presence of VN on the material surfaces enhanced cell-mediated FN reorganization and secretion, in comparison with the process which took place in the presence of serum proteins., Support from the European Research Council thorough the ERC-StG_20111012 is acknowledged.

Surface nanotopography is widely employed to control cell behavior and in particular controlled disorder has been shown to be important in cell differentiation/maturation. However, extracellular matrix proteins, such as fibronectin (FN), initially adsorbed on a biomaterial surface are known to mediate the interaction of synthetic materials with cells. In this work, we examine the effect of nanotopography on cell behavior through this adsorbed layer of adhesive proteins using a nanostructured polycarbonate surface comprising 150 nm-diameter pits originally defined using electron beam lithography. We address the effect of this nanopitted surface on FN adsorption and subsequently on cell morphology and behavior using C2C12 myoblasts. Wettability measurements and atomic force microscopy imaging showed that protein is adsorbed both within the interpits spaces and inside the nanopits. Cells responded to this coated nanotopography with the formation of fewer but larger focal adhesions and by mimicking the pit patterns within their cytoskeleton, nanoimprinting, ultimately achieving higher levels of myogenic differentiation compared to a flat control. Both focal adhesion assembly and nanoimprinting were found to be dependent on cell contractility and are adversely affected by the use of blebbistatin. Our results demonstrate the central role of the nanoscale protein interface in mediating cell-nanotopographical interactions and implicate this interface as helping control the mechanotransductive cascade.

Researchers develop a simple technique to enhance the activity of growth factors during tissue healing., Growth factors (GFs) are powerful signaling molecules with the potential to drive regenerative strategies, including bone repair and vascularization. However, GFs are typically delivered in soluble format at supraphysiological doses because of rapid clearance and limited therapeutic impact. These high doses have serious side effects and are expensive. Although it is well established that GF interactions with extracellular matrix proteins such as fibronectin control GF presentation and activity, a translation-ready approach to unlocking GF potential has not been realized. We demonstrate a simple, robust, and controlled material-based approach to enhance the activity of GFs during tissue healing. The underlying mechanism is based on spontaneous fibrillar organization of fibronectin driven by adsorption onto the polymer poly(ethyl acrylate). Fibrillar fibronectin on this polymer, but not a globular conformation obtained on control polymers, promotes synergistic presentation of integrin-binding sites and bound bone morphogenetic protein 2 (BMP-2), which enhances mesenchymal stem cell osteogenesis in vitro and drives full regeneration of a nonhealing bone defect in vivo at low GF concentrations. This simple and translatable technology could unlock the full regenerative potential of GF therapies while improving safety and cost-effectiveness.

Computational fluid dynamic (CFD) techniques were used to optimize the microenvironment inside scaffolds for hematopoietic stem cell (HSC) culture in a perfusion bioreactor. These matrices are meant to be seeded with adherent bone marrow stromal cells and then co-cultivated with HSCs; the scaffold micro-architecture and the fluid-dynamic conditions have to be optimized to avoid non-adherent stem cells being dragged away while ensuring adequate nutrient supply. The insertion of longitudinal microchannels was tested as a tool to improve perfusion in a homogeneous porous scaffold. Models of microchannel-provided scaffolds, characterized by different values of geometric parameters concerning pores and channels, were built, and numerical fluid-dynamic and oxygen-transfer analyses were carried out. The results of the computations indicated that the microchannels created preferential paths for culture medium flow, causing low shear stresses and drag forces within the pores; meanwhile, they improved oxygen delivery by forcing its penetration into the scaffold bulk. In particular, an 85% porous, 3-mm-thick scaffold with 175-microm-diameter pores was considered; at a constant average drag force guaranteeing stem cell suspension inside this porous bulk, the addition of approximately 260-microm-diameter, 700-microm-spaced channels resulted in 34% higher oxygen partial pressure at the exit (approximately 135 vs 101 mmHg), maintaining a wall shear stress median value of approximately 0.14 mPa. The present work demonstrates the capacity of microchannel-provided scaffolds to ensure suitable conditions for HSC culture and shows that CFD methods are a valuable tool to retrieve significant clues for the design of the culture environment.

Based on the hypothesis that material substrates are able to trigger the regeneration of a cell population by making use of tissue engineering techniques. Within the context of the cell-protein-material interaction paradigm, this chapter presents a comprehensive description of the interface between material surfaces and living cells, including protein adsorption, cell adhesion, and matrix remodeling phenomena at the cell-material interface. Cell adhesion involves different physicochemical phenomena, in which several biological molecules participate: extracellular matrix (ECM) proteins, cell membrane proteins, and cytoskeleton proteins. The initial cell–material interaction usually involves the adsorption of proteins such as fibronectin (FN), vitronectin (VN), fibrinogen (FG), representing the so-called soluble matrix proteins in the biological fluids. The adsorption of proteins onto the surface of a biomaterial from the surrounding fluid phase is a complex, dynamic, energy-driven process, controlled by protein properties, material surface properties, and solution conditions.

Perfusion culture systems are widely used in tissue engineering applications for enhancing cell culture viability in the core of three-dimensional scaffolds. In this work, we present a multichamber confined-flow perfusion system, designed to provide a straightforward platform for three-dimensional dynamic cell cultures. The device comprises 6 culture chambers allowing independent and simultaneous experiments in controlled conditions. Each chamber consists of three parts: a housing, a deformable scaffold-holder cartridge, and a 7 mL reservoir, which couples water-tightly with the housing compressing the cartridge. Short-term dynamic cell seeding experiments were carried out with MC3T3-E1 cells seeded into polycaprolactone porous scaffolds. Preliminary results revealed that the application of flow perfusion through the scaffold favored the penetration of the cells to its interior, producing a more homogeneous distribution of cells with respect to dropwise or injection seeding methods. The culture chamber layout was conceived with the aim of simplifying the user operations under laminar flow hood and minimizing the risks for contamination during handling and operation. Furthermore, a compact size, a small number of components, and the use of bayonet couplings ensured a simple, fast, and sterility-promoting assembling. Finally, preliminaryin vitrotests proved the efficacy of the system in enhancing cell seeding efficiency, opening the way for further studies addressing long-term scaffold colonization.

[EN] In this study, we propose a methodology to obtain a family of biomimetic substrates with a hierarchical rough topography at the micro and nanoscale that span the entire range of wettability, from the superhydrophobic to the superhydrophilic regime, through an Ar-plasma treatment at increasing durations. Moreover, we employ the same approach to produce a superhydrophobic-to- superhydrophilic surface gradient along centimetre-length scale distances within the same sample. We characterize the biological activity of these surfaces in terms of protein adsorption and cell response, using fibronectin, a major component of the extracellular matrix, and C2C12 cells, a myoblast cell line. Fibronectin conformation, assessed via binding of the monoclonal antibody HFN7.1, exhibits a non-monotonic dependence on surface wettability, with higher activity on hydrophilic substrates (WCA = 38.6 ± 8.1°). On the other hand, the exposition of cell-binding epitopes is diminished on the surfaces with extreme wetting properties, the conformation being particularly altered on the superhydrophobic substrate. The assessment of cell response via the myogenic differentiation process reveals that a gradient surface promotes a different response with respect to cells cultured on discrete uniform samples: even though in both cases the same non-monotonic differentiation pattern is found, the differential response to the various wettabilities is enhanced along the gradient while the overall levels of differentiation are diminished. On a gradient surface cells are in fact exposed to a range of continuously changing stimuli that foster cell migration and detain the differentiation process. © 2013 The Royal Society of Chemistry., The support of the Spanish Ministry of Science and Innovation through project MAT2009-14440-C02-01 is acknowledged. CIBER-BBN is an initiative funded by the VI National R&D&I Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund.

Material-driven fibronectin fibrillogenesis is a novel route to engineer the network structure and biological activity of fibronectin fibrillar matrices in analogy with their physiological cell-mediated assembly. We identify specific surface chemistries that promote the cell-free formation of physiological-like fibronectin fibrils in a time- and concentration-dependent process. Our most recent and relevant results, reviewed in the chapter, demonstrate the role of this material-induced fibrillogenesis in cell adhesion, extracellular matrix organization and degradation, and cell differentiation.

[EN] Fibronectin (FN) assembles into fibrillar networks by cells through an integrin-dependent mechanism. We have recently shown that simple FN adsorption onto poly(ethyl acrylate) surfaces (PEA), but not control polymer (poly(methyl acrylate), PMA), also triggered FN organization into a physiological fibrillar network. FN fibrils exhibited enhanced biological activities in terms of myogenic differentiation compared to individual FN molecules. In the present study, we investigate the influence of topological cues on the material-driven FN assembly and the myogenic differentiation process. Aligned and random electrospun fibers were prepared. While FN fibrils assembled on the PEA fibers as they do on the smooth surface, the characteristic distribution of globular FN molecules observed on flat PMA transformed into non-connected FN fibrils on electrospun PMA, which significantly enhanced cell differentiation. The direct relationship between the fibrillar organization of FN at the material interface and the myogenic process was further assessed by preparing FN gradients on smooth PEA and PMA films. Isolated FN molecules observed at one edge of the substrate gradually interconnected with each other, eventually forming a fully developed network of FN fibrils on PEA. In contrast, FN adopted a globular-like conformation along the entire length of the PMA surface, and the FN gradient consisted only of increased density of adsorbed FN. Correspondingly, the percentage of differentiated cells increased monotonically along the FN gradient on PEA but not on PMA. This work demonstrates an interplay between material chemistry and topology in modulating material-driven FN fibrillogenesis and cell differentiation. © 2011 Springer Science+Business Media, LLC., The support of the Spanish Ministry of Science and Innovation through project MAT2009-14440-C02-01 is acknowledged. CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008–2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. This work was supported by funds for research in the field of Regenerative Medicine through the collaboration agreement from the Conselleria de Sanidad (Generalitat Valenciana), and the Instituto de Salud Carlos III.

Haematopoietic stem cell (HSC) transplantation has been widely used to treat patients that have undergone high-dose chemotherapy or radiotherapy for haematological or non-haematological malignancies, and is currently investigated for the treatment of several other pathologic conditions. Nevertheless, present and expected clinical applications are hindered by the shortage of cells available for transplantation. Hence, many researchers have attempted to achieve an in vitro expansion of HSCs, using different experimental set-ups and approaches, which range from traditional static monolayer cultures to three-dimensional (3D) dynamic systems. Specifically, several bioreactor systems have been proposed, including perfusion chambers, stirred, rotating, hollow fiber, and packed bed reactors [1]. Taken together, literature studies suggest that a dynamic 3D culture system may provide superior expansion of HSCs.Copyright © 2009 by ASME

Plasma polymerization was used to produce novel nanometric coatings able to direct fibronectin adsorption and cell response. Using ethyl acrylate as a monomer, we obtain coatings whose chemical composition maintains some of the characteristic functionalities of the photo-initiated polymer, while the water contact angle increases monotonically with the duration of the plasma discharge. Enhanced surface mobility of the polymer chains due to a decrease of the thickness of the coating justifies this increase in wettability at lower treatment times. The coatings with higher surface mobility are shown to promote a more active conformation of the adsorbed protein, as proved by binding of the monoclonal antibodies HFN7.1 and mAb1937. Culture of MC3T3-E1 osteoblast-like cells onto the fibronectin-coated substrates further proves that the more mobile surfaces support better initial cell adhesion, even at low fibronectin surface density, as well as stronger cell-mediated fibronectin reorganization., The authors would like to acknowledge the financial support of the Spanish Ministry of Science and Innovation through the project MAT2009-14440-C02-01.

Porous polymeric materials play a key role in regenerative medicine, serving as three-dimensional scaffolds for cell culture. Hence, the definition of their micro-architecture should be regarded as a pivotal design issue, that has to be wittingly addressed while engineering a cell culture system. Computational fluid dynamics techniques (CFD) appear to be very valuable in this respect, since they have been appreciably applied in recent literature as a means to analyze fluid dynamics and mass transport inside scaffold or bioreactor models [1]; moreover, leading researchers in tissue engineering have acknowledged the role of numerical methodology in the issue of defining optimal flow conditions for three-dimensional dynamic culture systems.Copyright © 2008 by ASME