RESUMEN
At least one-third of patients with epithelial ovarian cancer (OC) present ascites at diagnosis and almost all have ascites at recurrence especially because of the propensity of the OC cells to spread in the abdominal cavity leading to peritoneal metastasis. The influence of ascites on the development of pre-metastatic niches, and on the biological mechanisms leading to cancer cell colonization of the mesothelium, remains poorly understood. Here, we show that ascites weakens the mesothelium by affecting the morphology of mesothelial cells and by destabilizing their distribution in the cell cycle. Ascites also causes destabilization of the integrity of mesothelium by modifying the organization of cell junctions, but it does not affect the synthesis of N-cadherin and ZO-1 by mesothelial cells. Moreover, ascites induces disorganization of focal contacts and causes actin cytoskeletal reorganization potentially dependent on the activity of Rac1. Ascites allows the densification and reorganization of ECM proteins of the mesothelium, especially fibrinogen/fibrin, and indicates that it is a source of the fibrinogen and fibrin surrounding OC spheroids. The fibrin in ascites leads to the adhesion of OC spheroids to the mesothelium, and ascites promotes their disaggregation followed by the clearance of mesothelial cells. Both αV and α5ß1 integrins are involved. In conclusion ascites and its fibrinogen/fibrin composition affects the integrity of the mesothelium and promotes the integrin-dependent implantation of OC spheroids in the mesothelium.
Asunto(s)
Ascitis , Fibrina , Fibrinógeno , Integrina alfa5beta1 , Neoplasias Ováricas , Esferoides Celulares , Microambiente Tumoral , Humanos , Femenino , Neoplasias Ováricas/patología , Neoplasias Ováricas/metabolismo , Ascitis/patología , Ascitis/metabolismo , Integrina alfa5beta1/metabolismo , Esferoides Celulares/metabolismo , Esferoides Celulares/patología , Fibrinógeno/metabolismo , Fibrina/metabolismo , Neoplasias Peritoneales/secundario , Neoplasias Peritoneales/metabolismo , Neoplasias Peritoneales/patología , Carcinoma Epitelial de Ovario/metabolismo , Carcinoma Epitelial de Ovario/patología , Línea Celular Tumoral , Receptores de Vitronectina/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Adhesión Celular , Peritoneo/patología , Peritoneo/metabolismo , Epitelio/metabolismo , Epitelio/patología , Cadherinas/metabolismo , Células Tumorales CultivadasRESUMEN
Over the past decades, many cell-penetrating peptides (CPP) have been studied for their capacity to cross cellular membranes, mostly in order to improve cellular uptake of therapeutic agents. Even though hydrophobic and anionic CPPs have been described, many of them are polycationic, due to the presence of several arginine (Arg) residues. Noteworthy, however, the presence of aromatic amino acids such as tryptophan (Trp) within CPPs seems to play an important role to reach high membranotropic activity. RW9 (RRWWRRWRR) is a designed CPP derived from the polyarginine R9 presenting both features. In general, when interacting with membranes, CPPs adopt an optimal conformation for membrane interactions - an amphipathic helical secondary structure in the case of RW9. Herein, we assumed that the incorporation of a locally constrained amino acid in the peptide sequence could improve the membranotropic activity of RW9, by facilitating its structuration upon contact with a membrane, while leaving a certain plasticity. Therefore, two cyclized Trp derivatives (Tcc and Aia) were synthesized to be incorporated in RW9 as surrogates of Trp residues. Thus, a series of peptides containing these building blocks has been synthesized by varying the type, position, and number of modifications. The membranotropic activity of the RW9 analogs was studied by spectrofluorescence titration of the peptides in presence of liposomes (DMPG), allowing to calculate partition coefficients (Kp). Our results indicate that the partitioning of the modified peptides depends on the type, the number and the position of the modification, with the best sequence being [Aia4]RW9. Interestingly, both NMR analysis and molecular dynamic (MD) simulations indicate that this analog presents an extended conformation similar to the native RW9, but with a much-reduced structural flexibility. Finally, cell internalization properties were also confirmed by confocal microscopy.
Asunto(s)
Péptidos de Penetración Celular , Péptidos de Penetración Celular/farmacología , Péptidos de Penetración Celular/química , Membrana Celular/metabolismo , Secuencia de Aminoácidos , Liposomas/química , Simulación de Dinámica MolecularRESUMEN
The complex impregnation of a multifilament yarn by a cementitious matrix leads to a difficult prediction of the mechanical behaviour of textile reinforced concrete and its less spread use than steel-reinforced concrete. To solve this problem, several models were established but they are not based on direct observations of the embedded yarn or quantification of its impregnation. In order to improve those models, a double resin impregnation process followed by confocal microscopy was set up, after pullout test was performed on each sample. Several parameters were then computed from the obtained images, which enables to quantify the impregnation of the yarn for each sample and to compare it with the pullout mechanical results. The number of fully impregnated filaments is found to be the crucial point to explain the pullout maximum load. The type of failure is also defined using those same parameters, computed along the embedded length, and it was found that the shape of the extracted volume of yarn is cylindrical and so the failure of the filaments is not telescopic. A pre-existing model was then improved, considering all those conclusions obtained by microscopic observations, and a good match between the numerical and the experimental results was found. Multifilament yarns are a continuous and flexible textile structure composed of hundreds of filaments that are maintain together using a chemical product called sizing. Those multifilament yarns can be used to reinforce mortar or concrete, like steel in steel-reinforced concrete. The performances of those types of composites depend strongly on the strength of the reinforcement/mortar bond. In case of textile /mortar composite, this bond depends on the level of penetration of the mortar into the yarn, so the impregnation of the yarn by the mortar. Since they are both very heterogeneous material, this impregnation is random and incomplete. Consequently, it is very difficult to predict the strength of this bond and so, the strength of the yarn/mortar composite. As a result, this type of composite cannot be widely used. Some models were established to predict the strength of those composite; however, they are not based on direct observation of the textile/mortar bond. To improve those models, a new visualisation method of the impregnation of the yarn by the mortar was set up. A double moulding of the impregnated yarn by the mortar was manufactured, using resins and two different fluorescent dyes, after underwent a mechanical test of pulling out the yarn from the mortar (pullout test). First, a resin with a red fluorescent dye was used for the moulding of the yarn shortly after the test, and, second, after dissolution of the mortar around the yarn in acid, a resin with a green fluorescent dye. A number of cross sections of the yarn is observed using a microscope that detects those two dyes and the obtained images are analysed allowing a clear differentiation, location and counting of fully impregnated filaments of the yarn by the mortar and those partially or not impregnated. The results are compared to the mechanical parameters of the pullout test. The number of fully impregnated filaments is found to be crucial to explain the strength of the composite. The behaviour during the pullout test is also explained using parameters computed along the length of the yarn in the mortar. It is found that filaments extracted by pulling-out take a cylindrical shape and so the pullout behaviour is not conical shape as it was suggested in a number of models. Considering all those conclusions obtained by observations, a pre-existing model is improved, and a good match between the numerical and experimental results is found.
RESUMEN
Fibronectin (Fn) is an extracellular matrix (ECM) multifunctional glycoprotein essential for regulating cells behaviors. Within ECM, Fn is found as polymerized fibrils. Apart from fibrils, Fn could also form other kind of supramolecular assemblies such as aggregates. To gain insight into the impact of Fn aggregates on cell behavior, we generated several Fn oligomeric assemblies. These assemblies displayed various amyloid-like properties but were not cytotoxic. In presence of the more amyloid-like structured assemblies of Fn, the cell-ECM networks were altered and the cell shapes shifted toward extended mesenchymal morphologies. Additionnaly, the Fn amyloid-like aggregates promoted a single-cell and sparsed migration of SKOV3 cancer cells, which was associated with a relocalization of αv integrins from plasma membrane to perinuclear vesicles. These data pointed out that the features of supramolecular Fn assemblies could represent a higher level of fine-tuning cell phenotype, and especially migration of cancer cells.
Asunto(s)
Proteínas Amiloidogénicas/metabolismo , Células Epiteliales/metabolismo , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Agregado de Proteínas , Proteínas Amiloidogénicas/química , Animales , Células CHO , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Forma de la Célula , Cricetulus , Células Epiteliales/química , Células Epiteliales/ultraestructura , Matriz Extracelular/química , Matriz Extracelular/ultraestructura , Fibronectinas/química , Cadenas alfa de Integrinas/química , Cadenas alfa de Integrinas/metabolismo , Análisis de la Célula IndividualRESUMEN
Fibronectin, a ≈ 450-kDa protein with 4-9% (w/w) glycosylation, is a key component of extracellular matrices and has a high conformational lability regarding its functions. However, the accessibility and the role of glycosylated moieties associated with the conformational changes of fibronectin are poorly understood. Using lectins as probes, we developed an approach comprising dynamic light scattering, turbidimetry measurements, and isothermal titration calorimetry to assess the accessibility of glycosylated moieties of fibronectin undergoing thermal-induced conformational changes. Among a set of 14 lectins, fibronectin mainly reacted with mannose-binding lectins, specifically concanavalin A. When temperature was raised from 25 to 50 °C, fibronectin underwent progressive unfolding, but the conformation of concanavalin A was unaffected. Dynamic light scattering, turbidimetry measurements, and isothermal titration calorimetry showed increased concanavalin A binding to fibronectin during progressive thermal-induced unfolding of the protein core. Such data suggest that mannosylated residues are progressively exposed as fibronectin unfolds. Because oligosaccharide moieties can be differently exposed to cells, and the cell's responses could be modified physiologically or pathologically, modulation of fibronectin sugar chains could be relevant to its biological functions. Thus, lectins might be useful tools to probe the glycosylation accessibility accompanying changes in protein core folding, for which a better understanding would be of value for biological and biomedical research.
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Fibronectinas/metabolismo , Lectinas de Unión a Manosa/metabolismo , Calorimetría , Dispersión Dinámica de Luz , Glicosilación , Humanos , Nefelometría y Turbidimetría , Unión Proteica , Conformación Proteica , Pliegue de ProteínaRESUMEN
BACKGROUND: Intra-abdominal ascites is a complication of ovarian cancers and constitutes a permissive microenvironment for metastasis. Since fibronectin and vitronectin are key actors in ovarian cancer progression, we investigated their occurrence and molecular characteristics in various ascites fluids and the influence of these ascites-derived proteins on cell behavior. METHODS: Fibronectin and vitronectin were investigated by immunoblotting within various ascites fluids. A combined affinity-based protocol was developed to purify both proteins from the same sample. Each purified protein was characterized with regard to its molecular features (molecular mass of isoforms, tryptophan intramolecular environment, hydrodynamic radii), and its influence on cell adhesion. RESULTS: Fibronectin and vitronectin were found in all tested ascites. Several milligrams of purified proteins were obtained from ascites of varying initial volumes. Molecular mass isoforms and conformational lability of proteins differed according to the ascites of origin. When incorporated into the cancer cell environment, ascites-derived fibronectin and vitronectin supported cell adhesion and migration with various degrees of efficiency, and induced the recruitment of integrins into focal contacts. CONCLUSIONS: To our knowledge, this is the first combined purification of two extracellular matrix proteins from a single pathological sample containing a great variety of bioactive molecules. This study highlights that ascites-derived fibronectin and vitronectin exhibit different properties depending on the ascites. GENERAL SIGNIFICANCE: Investigating the relationships between the molecular properties of ascites components and ovarian cancer cell phenotype according to the ascites may be critical for a better understanding of the recurrence of this lethal disease and for further biomarker identification.
Asunto(s)
Ascitis/metabolismo , Fibronectinas/metabolismo , Neoplasias Ováricas/metabolismo , Vitronectina/metabolismo , Femenino , Fibronectinas/química , Humanos , Neoplasias Ováricas/patología , Conformación Proteica , Vitronectina/químicaRESUMEN
Commercially available bioactive glasses (BAGs) are exclusively used in powder form, due to their tendency to crystallize. Silicate BAG 1393 was developed to allow fiber drawing and scaffold sintering, but its slow degradation limits its potential. To enable scaffold manufacturing while maintaining glass dissolution rate close to that of commercially available BAGs, the borosilicate glass 1393B20 was developed. This study investigates the potential of 1393B20 scaffolds to support bone regeneration and mineralization in vitro and in vivo, in comparison to silicate 1393. Both scaffolds supported human adipose stem cells proliferation, either in direct contact for the 1393, or mainly around for the 1393B20. Similarly, both BAGs induced osteogenesis and angiogenesis in vitro, with a better pro-angiogenic influence of the 1393B20. In addition, these scaffolds supported bone regeneration and osteoclast/osteoblast activity in vivo in critical-sized rat calvarial defect. Nevertheless, mineralization and collagen formation were significantly enhanced for the 1393B20, at 3-months post-implantation, assigned to faster and more complete dissolution of the scaffolds. Thus, 1393B20 demonstrates greater promise for bone tissue engineering certainly due to its time-controlled release of boron and silicon. STATEMENT OF SIGNIFICANCE: Bioactive glasses (BAGs) show great promise in bone tissue engineering as they effectively bond with bone tissue, fostering integration and regeneration. Silicate BAG 1393 was developed to allow fiber drawing and scaffold sintering, but its slow degradation limits its potential. To enable scaffold manufacturing while maintaining glass dissolution rate close to that of commercially available BAGs, the borosilicate glass 1393B20 was developed. Both BAGs induced osteogenesis and angiogenesis in vitro, with a better pro-angiogenic influence of the 1393B20. The presence of boron in the 1393B20 enhanced mineralization and collagen formation in vivo compared to 1393, probably due to its faster dissolution rate. Here, 1393B20 demonstrated greater promise for bone tissue engineering compared to the well-known 1393 BAG.
RESUMEN
In vivo, cells reside in a 3D porous and dynamic microenvironment. It provides biochemical and biophysical cues that regulate cell behavior in physiological and pathological processes. In the context of fundamental cell biology research, tissue engineering, and cell-based drug screening systems, a challenge is to develop relevant in vitro models that could integrate the dynamic properties of the cell microenvironment. Taking advantage of the promising high internal phase emulsion templating, we here designed a polyHIPE scaffold with a wide interconnected porosity and functionalized its internal 3D surface with a thin layer of electroactive conducting polymer poly(3,4-ethylenedioxythiophene) (PEDOT) to turn it into a 4D electroresponsive scaffold. The resulting scaffold was cytocompatible with fibroblasts, supported cellular infiltration, and hosted cells, which display a 3D spreading morphology. It demonstrated robust actuation in ion- and protein-rich complex culture media, and its electroresponsiveness was not altered by fibroblast colonization. Thanks to customized electrochemical stimulation setups, the electromechanical response of the polyHIPE/PEDOT scaffolds was characterized in situ under a confocal microscope and showed 10% reversible volume variations. Finally, the setups were used to monitor in real time and in situ fibroblasts cultured into the polyHIPE/PEDOT scaffold during several cycles of electromechanical stimuli. Thus, we demonstrated the proof of concept of this tunable scaffold as a tool for future 4D cell culture and mechanobiology studies.
Asunto(s)
Polímeros , Estirenos , Andamios del Tejido , Andamios del Tejido/química , Porosidad , Polímeros/farmacología , Polímeros/química , Técnicas de Cultivo de Célula , Ingeniería de Tejidos/métodosRESUMEN
Touch DNA, which can be found at crime scenes, consists of invisible biological traces deposited through a person's skin's contact with an object or another person. Many factors influence touch DNA transfer, including the "destination" substrate's surface. The latter's physicochemical characteristics (wettability, roughness, surface energy, etc.) will impact touch DNA deposition and persistence on a substrate. We selected a representative panel of substrates from objects found at crime scenes (glass, polystyrene, tiles, raw wood, etc.) to investigate the impact of these characteristics on touch DNA deposition and detection. These were shown to impact cell deposition, morphology, retention, and subsequent touch DNA genetic analysis. Interestingly, cell-derived fragments found within keratinocyte cells and fingermarks using in vitro touch DNA models could be successfully detected whichever the substrates' physicochemistry by targeting cellular proteins and carbohydrates for two months, indoors and outdoors. However, swabbing and genetic analyses of such mock traces from different substrates produced informative profiles mainly for substrates with the highest surface free energy and therefore the most hydrophilic. The substrates' intrinsic characteristics need to be considered to better understand both the transfer and persistence of biological traces, as well as their detection and collection, which require an appropriate methodology and sampling device to get informative genetic profiles.
Asunto(s)
ADN , Tacto , Humanos , ADN/química , Propiedades de Superficie , Piel/metabolismo , Piel/química , Queratinocitos/metabolismo , Dermatoglifia del ADN/métodosRESUMEN
At a crime scene, investigators are faced with a multitude of traces. Among them, biological traces are of primary interest for the rapid genetic-based identification of individuals. "Touch DNA" consists of invisible biological traces left by the simple contact of a person's skin with objects. To date, these traces remain undetectable with the current methods available in the field. This study proposes a proof-of-concept for the original detection of touch DNA by targeting cell-derived fragments in addition to DNA. More specifically, adhesive-structure proteins (laminin, keratin) as well as carbohydrate patterns (mannose, galactose) have been detected with keratinocyte cells derived from a skin and fingermark touch-DNA model over two months in outdoor conditions. Better still, this combinatory detection strategy is compatible with DNA profiling. This proof-of-concept work paves the way for the optimization of tools that can detect touch DNA, which remains a real challenge in helping investigators and the delivery of justice.
Asunto(s)
Criminales , Humanos , Piel , Dermatoglifia del ADN , Tacto , ADN/genéticaRESUMEN
Tissue engineering is a promising alternative to current full thickness circumferential esophageal replacement methods. The aim of our study was to develop a clinical grade Decellularized Human Esophagus (DHE) for future clinical applications. After decontamination, human esophagi from deceased donors were placed in a bioreactor and decellularized with sodium dodecyl sulfate (SDS) and ethylendiaminetetraacetic acid (EDTA) for 3 days. The esophagi were then rinsed in sterile water and SDS was eliminated by filtration on an activated charcoal cartridge for 3 days. DNA was removed by a 3-hour incubation with DNase. A cryopreservation protocol was evaluated at the end of the process to create a DHE cryobank. The decellularization was efficient as no cells and nuclei were observed in the DHE. Sterility of the esophagi was obtained at the end of the process. The general structure of the DHE was preserved according to immunohistochemical and scanning electron microscopy images. SDS was efficiently removed, confirmed by a colorimetric dosage, lack of cytotoxicity on Balb/3T3 cells and mesenchymal stromal cell long term culture. Furthermore, DHE did not induce lymphocyte proliferation in-vitro. The cryopreservation protocol was safe and did not affect the tissue, preserving the biomechanical properties of the DHE. Our decellularization protocol allowed to develop the first clinical grade human decellularized and cryopreserved esophagus.
Asunto(s)
Matriz Extracelular , Andamios del Tejido , Ratones , Animales , Humanos , Andamios del Tejido/química , Ingeniería de Tejidos/métodos , Criopreservación , Dodecil Sulfato de Sodio/química , EsófagoRESUMEN
The success of stable and long-term implant integration implies the promotion, control, and respect of the cell microenvironment at the site of implantation. The key is to enhance the implant-host tissue cross talk by developing interfacial strategies that guarantee an optimal and stable seal of soft tissue onto the implant, while preventing potential early and late infection. Indeed, implant rejection is often jeopardized by lack of stable tissue surrounding the biomaterial combined with infections which reduce the lifespan and increase the failure rate of implants and morbidity and account for high medical costs. Thin films formed by the layer-by-layer (LbL) assembly of oppositely charged polyelectrolytes are particularly versatile and attractive for applications involving cell-material contact. With the combination of the extracellular matrix protein fibronectin (Fn, purified from human plasma) and poly-L-lysine (PLL, exhibiting specific chain lengths), we proposed proactive and biomimetic coatings able to guarantee enhanced cell attachment and exhibiting antimicrobial properties. Fn, able to create a biomimetic interface that could enhance cell attachment and promote extracellular cell matrix remodeling, is incorporated as the anionic polymer during film construction by the LbL technic whereas PLL is used as the cationic polymer for its capacity to confer remarkable antibacterial properties.
RESUMEN
Hybrid scaffolds based on bioactive glass (BAG) particles (<38 µm), covalently linked to gelatin (G*) using 3-glycidoxypropyltrimethoxysilane (GPTMS), have been studied for bone bioengineering. In this study, two glass compositions (13-93 and 13-93B20 (where 20% of the SiO2 was replaced with B2O3)) were introduced in the gelatin matrix. The Cfactor (gelatin/GPTMS molar ratio) was kept constant at 500. The hybrids obtained were found to be stable at 37 °C in solution, the condition in which pure gelatin is liquid. All hybrids were characterized by in vitro dissolution in Tris(hydroxymethyl)aminomethane (TRIS) solution (for up to 4 weeks) and Simulated Body Fluid (SBF) (for up to 2 weeks). Samples processed with 13-93B20 exhibited faster initial dissolution and significantly faster precipitation of a hydroxyapatite (HA) layer. The faster ion release and HA precipitation recorded from the G*/13-93B20 samples are attributable to the higher reactivity of borosilicate compared to silicate glass. The MC3T3-E1 cell behavior in direct contact with the hybrids was investigated, showing that the cells were able to proliferate and spread on the developed biomaterials. Tailoring the glass composition allows us to better control the material's dissolution, biodegradability, and bioactivity. Bioactive (especially with 13-93B20 BAG) and biocompatible, the hybrids are promising for bone application.
Asunto(s)
Materiales Biocompatibles/química , Regeneración Ósea/fisiología , Huesos/fisiología , Gelatina/química , Vidrio/química , Silanos/química , Animales , Materiales Biocompatibles/síntesis química , Materiales Biocompatibles/farmacología , Regeneración Ósea/efectos de los fármacos , Huesos/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Durapatita/química , Ratones , Osteoblastos/citología , Dióxido de Silicio/química , Ingeniería de Tejidos/métodos , Andamios del Tejido/químicaRESUMEN
Additive manufacturing is a rising field in bone tissue engineering. Additive fabrication offers reproducibility, high precision and rapid manufacture of custom patient-specific scaffolds. The development of appropriate composite materials for biomedical applications is critical to reach clinical application of these novel biomaterials. In this work, medical grade poly(lactic-co-glycolic) acid (PLGA) was mixed with hydroxyapatite nanoparticles (nHA) to fabricate 3D porous scaffolds by Fused Deposition Modeling. We have first confirmed that the composite material could be printed in a reproductive manner. Physical characterization demonstrated a low degradation of the material during manufacturing steps and an expected loading and homogeneous distribution of nHA. In vitro biodegradation of the scaffolds showed modifications of morphological and physicochemical properties over time. The composite scaffolds were biocompatible and high cell viability was observed in vitro, as well as a maintain of cell proliferation. As expected, the addition of nHA displayed a positive impact on osteodifferentiation in vitro. Furthermore, a limited inflammatory reaction was observed after subcutaneous implantation of the materials in the rat. Overall, this study suggests that this composite material is suitable for bone tissue engineering applications.
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Ingeniería de Tejidos , Andamios del Tejido , Animales , Materiales Biocompatibles , Huesos , Durapatita , Humanos , Impresión Tridimensional , Ratas , Reproducibilidad de los ResultadosRESUMEN
Fibrin gels are of interest as biomaterials for regenerative medicine but present poor mechanical properties, undergo fast degradation and strongly contract in presence of cells. To face these drawbacks, a fibrin network can be associated with another polymer network, in an Interpenetrating Polymer Network (IPN) architecture. In this study, we report the properties of an IPN comprising a fibrin (Fb) network and a silk fibroin (SF) network. This IPN is synthesized through the action of 2 enzymes, each one being specific of one protein gelation, i.e. thrombin (Tb) for Fb gelation, and horseradish peroxidase (HRP) for SF gelation. The effective formation of both Fb and SF networks in an IPN architecture was first verified at qualitative and quantitative levels. The resulting IPN was easily manipulable, displayed high viscoelastic properties and showed homogeneous macro- and micro-structure. Then the degradability of the IPN by two proteases, thermolysin (TL) and trypsin (TRY), obeying different mechanisms was presented. Finally, two-dimensional culture of human fibroblasts on the IPN surface induced little material contraction, while fibroblasts showed healthy morphology, displayed high viability and produced mature extracellular matrix (ECM) proteins. Taken together, the results suggest that this new IPN have a strong potential for tissue engineering and regenerative medicine.
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Fibrina/farmacología , Fibroínas/farmacología , Peroxidasa de Rábano Silvestre/metabolismo , Polímeros/farmacología , Trombina/metabolismo , Proliferación Celular , Forma de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Colágeno Tipo I/biosíntesis , Fibronectinas/biosíntesis , Humanos , Recién Nacido , MasculinoRESUMEN
Fibronectin (FN) is a multifunctional glycoprotein of the extracellular matrix (ECM) playing critical roles in physiological and pathological cell processes like adhesion, migration, growth, and differentiation. These various functions of FN are modulated by its supramolecular state. Indeed, FN can polymerize into different types of assemblies like fibrils and aggregates. However, the mechanism of polymerization and the effects of such assemblies on cell behaviors still remain to be elucidated. Here we show that upon irreversible thermal denaturation, human blood plasma fibronectin forms high molecular weight aggregates. These compact and globular aggregates show amyloid features: they are stabilized by intermolecular b-sheets, they bind Thioflavin T and they are resistant to reducing and denaturing agents. Their characterization by electrospray ionization charge detection mass spectrometry shows that two populations can be distinguished according to the mass and charge density. Despite their amyloid features and the presence of hydrophobic patches on their surface, these aggregates are not toxic for cells. However, their binding abilities to gelatin and RGD are drastically decreased compare to native FN, suggesting possible effects on ECM-cell interactions.
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Amiloide/química , Fibronectinas/sangre , Fibronectinas/química , Multimerización de Proteína , Línea Celular Tumoral , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Humanos , Estructura Secundaria de Proteína , Desplegamiento Proteico , TemperaturaRESUMEN
Design of new osteoinductive biomaterials to reproduce an optimized physiological environment capable of recruiting stem cells and instructing their fate towards the osteoblastic lineage has become a priority in orthopaedic surgery. This work aims at evaluating the bioactivity of BMP combined with human plasma fibronectin (FN/BMP) delivered in solution or coated onto titanium-hydroxyapatite (TiHA) surfaces. Herein, we focus on the comparison of in vitro osteogenic efficacy in mouse C2C12 pre-osteoblasts of three BMP members, namely: BMP-2, BMP-6 and BMP-7. In parallel, we evaluated the molecular binding strength between each BMP with FN using the Surface Plasmon Resonance (SPR) technology. The affinity of BMPs for FN was found totally different and dependent on BMP type. Indeed, the combination of FN with BMP-2 on TiHA surfaces potentiates the burst of gene-mediated osteogenic induction, while it prolongs the osteogenic activity of BMP-6 and surprisingly annihilates the BMP-7 one. These results correlate with FN/BMP affinity for TiHA, since BMP-6>BMP-2>BMP-7. In addition, by analyzing the osteogenic activity in the peri-implant environment, we showed that osteoinductive paracrine effects were significantly decreased upon (FN/BMP-6), as opposed to (FN/BMP-2) coatings. Altogether, our results support the use of FN/BMP-6 to develop a biomimetic microenvironment capable to induce osteogenic activity under physiological conditions, with minimum paracrine signalization. STATEMENT OF SIGNIFICANCE: The originality of our paper relies on the first direct comparison of the in vitro osteogenic potential of three osteogenic BMPs (BMP-2, -6 and -7) combined with native human plasma fibronectin delivered in solution or coated by laser transfer onto titanium hydroxyapatite surfaces. We confirm that BMP association with fibronectin enhances the osteogenic activity of BMP-2, -6 and -7, but with essential discrepancies, depending on the BMP member, and in agreement with the affinity of BMPs for fibronectin. Moreover, we bring elements to explain the origin of the BMP-2 medical life-threatening side-effects by analyzing in vitro paracrine effects. Finally, this work supports the alternative use of FN/BMP-6 to induce osteogenic activity under physiological conditions, with minimum side effects.
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Materiales Biomiméticos , Proteína Morfogenética Ósea 2 , Proteína Morfogenética Ósea 6 , Proteína Morfogenética Ósea 7 , Materiales Biocompatibles Revestidos , Durapatita , Fibronectinas , Osteoblastos/metabolismo , Titanio , Animales , Materiales Biomiméticos/química , Materiales Biomiméticos/farmacología , Proteína Morfogenética Ósea 2/química , Proteína Morfogenética Ósea 2/farmacología , Proteína Morfogenética Ósea 6/química , Proteína Morfogenética Ósea 6/farmacología , Proteína Morfogenética Ósea 7/química , Proteína Morfogenética Ósea 7/farmacología , Línea Celular , Materiales Biocompatibles Revestidos/química , Materiales Biocompatibles Revestidos/farmacología , Durapatita/química , Durapatita/farmacología , Fibronectinas/química , Fibronectinas/farmacología , Humanos , Ratones , Osteoblastos/citología , Titanio/química , Titanio/farmacologíaRESUMEN
Cancer staging is a way to classify cancer according to the extent of the disease in the body. The stage is usually determined by several factors such as the location of the primary tumor, the tumor size, the degree of spread in the surrounding tissues, etc. The study of E-cadherin (EC) expression on cancerous cells of patients has revealed variations in the molecular expression patterns of primary tumors and metastatic tumors. The detection of these cells requires a long procedure involving conventional techniques, thus, the requirement for development of new rapid devices that permit direct and highly sensitive detection stimulates the sensing field progress. Here, we explore if E-cadherin could be used as a biomarker to bind and detect epithelial cancer cells. Hence, the sensitive and specific detection of E-cadherin expressed on epithelial cells is approached by immobilizing anti-E-cadherin antibody (AEC) onto aminosilanized indium-tin oxide (ITO) surface. The immunosensing surfaces have been characterized by electrochemical measurements, wettability and confocal microscopy and their performance has been assessed in the presence of cancer cell lines. Under optimal conditions, the resulting immunosensor displayed a selective detection of E-cadherin expressing cells, which could be detected either by fluorescence or electrochemical techniques. The developed immunosensing surface could provide a simple tool that can be applied to cancer staging.
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Biomarcadores/análisis , Cadherinas/análisis , Anticuerpos/análisis , Biomarcadores/química , Cadherinas/química , Técnicas Electroquímicas/métodos , Humanos , Microscopía Confocal , Compuestos de Estaño/química , HumectabilidadRESUMEN
Fibronectin (Fn) is widely reported to promote cell adhesion and spreading, and recent reports attest to the synergistic effect of coadsorbed albumin (unexpected due to the passivating character of the latter protein). In this study, the sequential adsorption of fibronectin and albumin, and the morphology of cultured MC3T3-E1 preosteoblastic cells are investigated on three important biomaterial surfaces: silicon oxide, poly(styrene) (PS), and hydroxyapatite (HA). Using quartz crystal microgravimetry with dissipation analysis, the adsorbed protein composition and mechanics are determined. Interestingly, cell morphological changes correlate neither with the amount of Fn nor the rigidity of the protein layer. On the PS surface, Alb is seen to significantly diminish cell spreading, possibly due to Alb aggregation with a partially denatured initially placed Fn layer. HA appears to be a particularly favorable substrate for osteoblast adhesion, despite having low Fn adsorption and protein layer rigidity.
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Materiales Biocompatibles/química , Fibronectinas/química , Albúmina Sérica/química , Adsorción , Animales , Materiales Biocompatibles/farmacología , Adhesión Celular/efectos de los fármacos , Línea Celular , Durapatita/química , Humanos , Ratones , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Poliestirenos/química , Tecnicas de Microbalanza del Cristal de Cuarzo , Dióxido de Silicio/química , Propiedades de SuperficieRESUMEN
The release of molecules entrapped within biogels is dictated by diffusion laws. Innovative biogel architectures are conceived and tested to control small molecule delivery from gelatin gels. The ionic interactions modulate the release of small molecules. Alginate is then added to gelatin gels and further hydrolyzed; the influence of viscosity is discussed. Next, various mixed gels are compared, such as a gelatin-alginate IPN and the original architecture of an alginate gel entrapped in a gelatin gel with or without a polysaccharidase. The relative influence of ionic interactions and diffusional constraints on the delivery of small charged molecules is explored, and a solution for controlling diffusion is proposed for any situation.