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Controlling the assembly of high-order structures is central to soft-matter and biomaterial engineering. Angle-resolved linear dichroism can probe the ordering of chiral collagen molecules in the dense state. Collagen triple helices were aligned by solvent evaporation. Their ordering gives a strong linear dichroism (LD) that changes sign and intensity with varying sample orientations with respect to the beam linear polarization. Being complementary to circular dichroism, which probes the structure of chiral (bio)molecules, LD can shift from the molecular to the supramolecular scale and from the investigation of the conformation to interactions. Supported by multiphoton microscopy and X-ray scattering, we show that LD provides a straightforward route to probe collagen alignment, determine the packing density, and monitor denaturation. This approach could be adapted to any assembly of chiral (bio)macromolecules, with key advantages in detecting large-scale assemblies with high specificity to aligned and chiral molecules and improved sensitivity compared to conventional techniques.
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Ovarian cancer is the most lethal gynecologic cancer in developed countries. In the tumor microenvironment, the extracellular matrix (ECM) and flow shear stress are key players in directing ovarian cancer cells invasion. Artificial ECM models based only on ECM proteins are used to build an ovarian tumor-on-chip to decipher the crosstalk between ECM and shear stress on the migratory behavior and cellular heterogeneity of ovarian tumor cells. This work shows that in the shear stress regime of the peritoneal cavity, the ECM plays a major role in driving individual or collective ovarian tumor cells migration. In the presence of basement membrane proteins, migration is more collective than on type I collagen regardless of shear stress. With increasing shear stress, individual cell migration is enhanced; while, no significant impact on collective migration is measured. This highlights the central position that ECM and flow shear stress should hold in in vitro ovarian cancer models to deepen understanding of cellular responses and improve development of ovarian cancer therapeutic platforms. In this frame, adding flow provides significant improvement in biological relevance over the authors' previous work. Further steps for enhanced clinical relevance require not only multiple cell lines but also patient-derived cells and sera.
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Ovarian cancer (OC) is a disease of major concern with a survival rate of about 40% at five years. This is attributed to the lack of visible and reliable symptoms during the onset of the disease, which leads over 80% of patients to be diagnosed at advanced stages. This implies that metastatic activity has advanced to the peritoneal cavity. It is associated with both genetic and phenotypic heterogeneity, which considerably increase the risks of relapse and reduce the survival rate. To understand ovarian cancer pathophysiology and strengthen the ability for drug screening, further development of relevant in vitro models that recapitulate the complexity of OC microenvironment and dynamics of OC cell population is required. In this line, the recent advances of tridimensional (3D) cell culture and microfluidics have allowed the development of highly innovative models that could bridge the gap between pathophysiology and mechanistic models for clinical research. This review first describes the pathophysiology of OC before detailing the engineering strategies developed to recapitulate those main biological features.
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Neoplasias Ováricas , Humanos , Femenino , Neoplasias Ováricas/metabolismo , Técnicas de Cultivo de Célula , Microambiente TumoralRESUMEN
Cellular heterogeneity is associated with many physiological processes, including pathological ones, such as morphogenesis and tumorigenesis. The extracellular matrix (ECM) is a key player in the generation of cellular heterogeneity. Advances in our understanding rely on our ability to provide relevant in vitro models. This requires obtainment of the characteristics of the tissues that are essential for controlling cell fate. To do this, we must consider the diversity of tissues, the diversity of physiological contexts, and the constant remodeling of the ECM along these processes. To this aim, we have fabricated a library of ECM models for reproducing the scaffold of connective tissues and the basement membrane by using different biofabrication routes based on the electrospinning and drop casting of biopolymers from the ECM. Using a combination of electron microscopy, multiphoton imaging, and AFM nanoindentation, we show that we can vary independently protein composition, topology, and stiffness of ECM models. This in turns allows one to generate the in vivo complexity of the phenotypic landscape of ovarian cancer cells. We show that, while this phenotypic shift cannot be directly correlated with a unique ECM feature, the three-dimensional collagen fibril topology patterns cell shape, beyond protein composition and stiffness of the ECM. On this line, this work is a further step toward the development of ECM models recapitulating the constantly remodeled environment that cells face and thus provides new insights for cancer model engineering and drug testing.
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Colágeno , Matriz Extracelular , Colágeno/metabolismo , Matriz Extracelular/metabolismoRESUMEN
In the original article, there was a mistake published in Figure 2 [...].
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Major progress in the field of regenerative medicine is expected from the design of artificial scaffolds that mimic both the structural and functional properties of the ECM. The bionanocomposites approach is particularly well fitted to meet this challenge as it can combine ECM-based matrices and colloidal carriers of biological cues that regulate cell behavior. Here we have prepared bionanocomposites under high magnetic field from tilapia fish scale collagen and multifunctional silica nanoparticles (SiNPs). We show that scaffolding cues (collagen), multiple display of signaling peptides (SiNPs) and control over the global structuration (magnetic field) can be combined into a unique bionanocomposite for the engineering of biomaterials with improved cell performances.
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Colágeno/química , Dióxido de Silicio/química , Tilapia/metabolismo , Andamios del Tejido/química , Células 3T3 , Animales , Adhesión Celular , Campos Magnéticos , Ratones , Nanocompuestos/química , Medicina RegenerativaRESUMEN
Because the positioning and clustering of biomolecules within the extracellular matrix dictates cell behaviors, the engineering of biomaterials incorporating bioactive epitopes with spatial organization tunable at the nanoscale is of primary importance. Here we used a highly modular composite approach combining peptide amphiphile (PA) nanofibers and silica nanoparticles, which are both easily functionalized with one or several bioactive signals. We show that the surface of silica nanoparticles allows the clustering of RGDS bioactive signals leading to improved adhesion and spreading of fibroblast cells on composite hydrogels at an epitope concentration much lower than in PA-only based matrices. Most importantly, by combining the two integrin-binding sequences RGDS and PHSRN on nanoparticle surfaces, we improved cell adhesion on the PA nanofiber/particle composite hydrogels, which is attributed to synergistic interactions known to be effective only for peptide intermolecular distance of ca. 5 nm. Such composites with soft and hard nanostructures offer a strategy for the design of advanced scaffolds to display multiple signals and control cell behavior.
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Nanofibras , Nanopartículas , Análisis por Conglomerados , Matriz Extracelular , LigandosRESUMEN
Cells respond to biophysical and biochemical signals. We developed a composite filament from collagen and silica particles modified to interact with collagen and/or present a laminin epitope (IKVAV) crucial for cell-matrix adhesion and signal transduction. This combines scaffolding and signaling and shows that local tuning of collagen organization enhances cell differentiation.
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Materiales Biocompatibles/farmacología , Colágeno/farmacología , Células-Madre Neurales/efectos de los fármacos , Dióxido de Silicio/farmacología , Materiales Biocompatibles/química , Diferenciación Celular/efectos de los fármacos , Colágeno/química , Humanos , Dióxido de Silicio/químicaRESUMEN
Rebuilding biological environments is crucial when facing the challenges of fundamental and biomedical research. Thus, preserving the native state of biomolecules is essential. We use electrospinning (ES), which is an extremely promising method for the preparation of fibrillar membranes to mimic the ECM of native tissues. Here, we report for the first time (1) the ES of pure and native collagen into a self-supported membrane in absence of cross-linker and polymer support, (2) the preservation of the membrane integrity in hydrated media in absence of cross-linker, and (3) the preservation of the native molecular structure and recovery of the hierarchical assembly of collagen. We use a multiscale approach to characterize collagen native structure at the molecular level using circular dichroism, and to investigate collagen hierarchical organization within the self-supported membrane using a combination of multiphoton and electron microscopies. Finally, we show that the membranes are perfectly suited for cell adhesion and spreading, making them very promising candidates for the development of biomaterials and finding applications in biomedical research.
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Second harmonic generation (SHG) enables in situ imaging of fibrillar collagen architecture in connective tissues. Recently, Circular Dichroism SHG (CD-SHG) microscopy has been implemented to take advantage of collagen chirality to improve 3D visualization. It measures the normalized difference in the SHG signal obtained upon excitation by left versus right circular polarizations. However, CD-SHG signal is not well characterized yet, and quite different CD-SHG values are reported in the literature. Here, we identify two major artifacts that may occur in CD-SHG experiments and we demonstrate that thorough optimization and calibration of the experimental setup are required for CD-SHG imaging. Notably it requires a careful calibration of the incident circular polarizations and a perfect mechanical stabilization of the microscope stage. Finally, we successfully record CD-SHG images in human cornea sections and confirm that this technique efficiently reveals collagen fibrils oriented out of the focal plane.
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Artefactos , Dicroismo Circular , Colágeno/química , Imagenología Tridimensional , Animales , Córnea/anatomía & histología , Humanos , Movimiento , Ratas , Imagen de Lapso de TiempoRESUMEN
Silica-coated gold-silver alloy nanoshells were obtained via a bioinspired approach using gelatin and poly-l-lysine (PLL) as biotemplates for the interfacial condensation of sodium silicate solutions. X-ray photoelectron spectroscopy was used as an efficient tool for the in-depth and complete characterization of the chemical features of nanoparticles during the whole synthetic process. Cytotoxicity assays using HaCaT cells evidenced the detrimental effect of the gelatin nanocoating and significant induction of late apoptosis after silicification. In contrast, PLL-modified nanoparticles had less biological impact that was further improved by the silica layer, and uptake rates of up to 50% of those of the initial particles could be achieved. These results are discussed considering the effect of nanosurface confinement of the biopolymers on their chemical and biological reactivity.
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Increasing bacterial resistance calls for the simultaneous delivery of multiple antibiotics. One strategy is to design a unique pharmaceutical carrier that is able to incorporate several drugs with different physico-chemical properties. This is highly challenging as it may require the development of compartmentalization approaches. Here we have prepared core-shell silica particles allowing for the dual delivery of gentamicin and rifamycin. The effect of silica particle surface functionalization on antibiotic sorption was first studied, enlightening the role of electrostatic and hydrophobic interactions. This in turn dictates the chemical conditions for shell deposition and further sorption of these antibiotics. In particular, the silica shell deposition was favored by the positively charged layer of gentamicin coating on the core particle surface. Shell modification by thiol groups finally allowed for rifamycin sorption. The antibacterial activity of the core-shell particles against Staphylococcus aureus and Pseudomonas aeruginosa demonstrated the dual release and action of the two antibiotics.
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The quantification of collagen fibril size is a major issue for the investigation of pathological disorders associated with structural defects of the extracellular matrix. Second-harmonic generation microscopy is a powerful technique to characterize the macromolecular organization of collagen in unstained biological tissues. Nevertheless, due to the complex coherent building of this nonlinear optical signal, it has never been used to measure fibril diameter so far. Here we report absolute measurements of second-harmonic signals from isolated fibrils down to 30 nm diameter, via implementation of correlative second-harmonic-electron microscopy. Moreover, using analytical and numerical calculations, we demonstrate that the high sensitivity of this technique originates from the parallel alignment of collagen triple helices within fibrils and the subsequent constructive interferences of second-harmonic radiations. Finally, we use these absolute measurements as a calibration for ex vivo quantification of fibril diameter in the Descemet's membrane of a diabetic rat cornea.
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Colágeno Tipo I/ultraestructura , Lámina Limitante Posterior/ultraestructura , Matriz Extracelular/ultraestructura , Procesamiento de Imagen Asistido por Computador/estadística & datos numéricos , Microscopía Electrónica/instrumentación , Animales , Colágeno Tipo I/aislamiento & purificación , Lámina Limitante Posterior/patología , Diabetes Mellitus Experimental/patología , Matriz Extracelular/química , Microscopía Electrónica/métodos , Ratas , Cola (estructura animal)/químicaRESUMEN
The assembly of proteins into fibrillar structures is an important process that concerns different biological contexts, including molecular medicine and functional biomaterials. Engineering of hybrid biomaterials can advantageously provide synergetic interactions of the biopolymers with an inorganic component to ensure specific supramolecular organization and dynamics. To this aim, we designed hybrid systems associating collagen and surface-functionalized silica particles and we built a new strategy to investigate fibrillogenesis processes in such multicomponents systems, working at the crossroads of chemistry, physics and mathematics. The self-assembly process was investigated by bimodal multiphoton imaging coupling second harmonic generation (SHG) and 2 photon excited fluorescence (2PEF). The in-depth spatial characterization of the system was further achieved using the three-dimensional analysis of the SHG/2PEF data via mathematical morphology processing. Quantitation of collagen distribution around particles offers strong evidence that the chemically induced confinement of the protein on the silica nanosurfaces has a key influence on the spatial extension of fibrillogenesis. This new approach is unique in the information it can provide on 3D dynamic hybrid systems and may be extended to other associations of fibrillar molecules with optically responsive nano-objects.
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Colágeno/química , Nanopartículas/química , Adsorción , Animales , Fibrina/química , Concentración de Iones de Hidrógeno , Imagenología Tridimensional , Ensayo de Materiales , Microscopía Electrónica de Transmisión , Nanocompuestos/química , Nanoestructuras/química , Nanotecnología/métodos , Fotones , Polímeros/química , Conformación Proteica , Ratas , Dióxido de Silicio/química , Agua/químicaRESUMEN
During evolution, living organisms have learned to design biomolecules exhibiting self-assembly properties to build-up materials with complex organizations. This is particularly evidenced by the delicate siliceous structures of diatoms and sponges. These structures have been considered as inspiration sources for the preparation of nanoscale and nanostructured silica-based materials templated by the self-assembled natural or biomimetic molecules. These templates range from short peptides to large viruses, leading to biohybrid objects with a wide variety of dimensions, shapes and organization. A more recent strategy based on the integration of biological self-assembly as the driving force of silica nanoparticles organization offers new perspectives to elaborate highly-tunable, biofunctional nanocomposites.
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The bio-responsive reversible assembly of silica nanoparticles grafted with complementary DNA strands containing an ATP-sensitive aptamer sequence was studied. The optimal conditions for ATP-induced specific disassembly of the bionanocomposite network were identified, highlighting the existing competition between ATP/aptamer interactions and DNA duplex stability. A new, dialysis-based method for ATP removal was proposed, allowing partial re-formation of the initial DNA duplexes. Further disassembly was re-achieved by subsequent addition of ATP. These data constitute promising preliminary steps towards DNA-based fully reversible responsive bionanocomposite devices.
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Bio-hybrid networks are designed based on the self-assembly of surface-engineered collagen-silica nanoparticles. Collagen triple helices can be confined on the surface of sulfonate-modified silica particles in a controlled manner. This gives rise to hybrid building blocks with well-defined diameters and surface potentials. Taking advantage of the self-assembling properties of collagen, collagen-silica networks are further built-up in solution. The structural and specific recognition properties of the collagen fibrils are well-preserved within the hybrid assembly. A combination of calorimetry, dynamic light scattering, zetametry and microscopy studies indicates that network formation occurs via a surface-mediated mechanism where pre-organization of the protein chains on the particle surface favors the fibrillogenesis process. These results enlighten the importance of the nano-bio interface on the formation and properties of self-assembled bionanocomposites.
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Colágeno/química , Nanopartículas/química , Dióxido de Silicio/química , Calorimetría , Concentración de Iones de Hidrógeno , Luz , Dispersión de Radiación , Ácidos Sulfónicos/química , Propiedades de SuperficieRESUMEN
DNA is used to rationally build up networks of silica nanoparticles (SiNPs) based on the molecular recognition properties of complementary sequences. Network self-assembly is controlled from DNA covalently grafted at the surface of chemically modified SiNPs. Two strategies are compared, where grafted DNA sequences are designed in a three-strand system using noncomplementary sequences and an extra DNA linker, or in a two-strand approach for direct hybridization. In this paper, both systems are compared in terms of DNA hybridization stability, network size, and three-dimensional organization using a combination of dynamic light scattering and electron microscopy. The observed differences are discussed in terms of hybridization interactions between DNA sequences in particle-free systems through fluorescence, circular dichroism, and UV spectroscopy techniques.
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ADN/química , Modelos Moleculares , Nanopartículas/química , Tamaño de la Partícula , Dióxido de Silicio/química , Secuencia de Bases , ADN/genética , Electroforesis , Microscopía Electrónica , Hibridación de Ácido Nucleico , Análisis EspectralRESUMEN
Anionic quantum dots (QDs) are spontaneously enfolded by coordination networks self-assembled from nucleotide monophosphates and lanthanide ions in water; luminescent core-shell nanoparticles are specifically obtained for anionic QDs, which allows their separation from amine-modified QDs.