RESUMEN
Metastatic cancers can be highly heterogeneous, show large patient variability and are typically hard to treat due to chemoresistance. Personalized therapies are therefore needed to suppress tumor growth and enhance patient's quality of life. Identifying appropriate patient-specific therapies remains a challenge though, due mainly to non-physiological in vitro culture systems. Therefore, more complex and physiological in vitro human cancer microenvironment tools could drastically aid in development of new therapies. We developed a plasma-modified, electro-spun 3D scaffold (PP-3D-S) that can mimic the human cancer microenvironment for customized-cancer therapeutic screening. The PP-3D-S was characterized for optimal plasma-modifying treatment and scaffolds morphology including fiber diameter and pore size. PP-3D-S was then seeded with human fibroblasts to mimic a stromal tissue layer; cell adhesion on plasma-modified poly (lactic acid), PLA, electrospun mats vastly exceeded that on untreated controls. The cell-seeded scaffolds were then overlaid with alginate/gelatin-based hydrogel embedded with MDA-MB231 human breast cancer cells, representing a tumor-tissue interface. Among three different plasma treatments, we found that NH3 plasma promoted the most tumor cell migration to the scaffold surfaces after 7 days of culture. For all treated and non-treated mats, we observed a significant difference in tumor cell migration between small-sized and either medium- or large-sized scaffolds. In addition, we found that the PP-3D-S was highly comparable to the standard Matrigel® migration assays in two different sets of doxorubicin screening experiments, where 75% reduction in migration was achieved with 0.5 µM doxorubicin for both systems. Taken together, our data indicate that PP-3D-S is an effective, low-cost, and easy-to-use alternate 3D tumor migration model which may be suitable as a physiological drug screening tool for personalized medicine against metastatic cancers.
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Calidad de Vida , Andamios del Tejido , Técnicas de Cocultivo , Doxorrubicina/farmacología , Humanos , Hidrogeles/farmacologíaRESUMEN
Bone is a frequent site of tumor metastasis. The bone-tumor microenvironment is heterogeneous and complex in nature. Such complexity is compounded by relations between metastatic and bone cells influencing their sensitivity/resistance to chemotherapeutics. Standard chemotherapeutics may not show efficacy for every patient, and new therapeutics are slow to emerge, owing to the limitations of existing 2D/3D models. We previously developed a 3D interface model for personalized therapeutic screening, consisting of an electrospun poly lactic acid mesh activated with plasma species and seeded with stromal cells. Tumor cells embedded in an alginate-gelatin hydrogel are overlaid to create a physiologic 3D interface. Here, we applied our 3D model as a migration assay tool to verify the migratory behavior of different patient-derived bone metastasized cells. We assessed the impact of two different chemotherapeutics, Doxorubicin and Cisplatin, on migration of patient cells and their immortalized cell line counterparts. We observed different migratory behaviors and cellular metabolic activities blocked with both Doxorubicin and Cisplatin treatment; however, higher efficiency or lower IC50 was observed with Doxorubicin. Gene expression analysis of MDA-MB231 that migrated through our 3D hybrid model verified epithelial-mesenchymal transition through increased expression of mesenchymal markers involved in the metastasis process. Our findings indicate that we can model tumor migration in vivo, in line with different cell characteristics and it may be a suitable drug screening tool for personalized medicine approaches in metastatic cancer treatment.
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Neoplasias Óseas , Cisplatino , Humanos , Microambiente Tumoral , Neoplasias Óseas/metabolismo , Transición Epitelial-Mesenquimal , Doxorrubicina/farmacologíaRESUMEN
Since the earliest days of this field there has been an interest in correlating the structure of plasma polymer (PP) coatings with deposition parameters, most particularly with energy input per monomer molecule, Em. Both of our laboratories have developed methods for measuring Em (or somewhat equivalent, the apparent activation energy, Ea) in low- (LP) and atmospheric-pressure (AP) electrical discharge plasmas. We recently proposed a new parameter, energy conversion efficiency (ECE), which for the first time permits direct comparison of LP and AP experiments. Here, we report the case of small hydrocarbons, namely acetylene, ethylene and methane. "Critical" Em (or Ea) values that demarcate ECE regimes separating different reaction mechanisms are found to agree remarkably well, and to correlate with specific reaction mechanisms, including dissociation, recombination, gas-phase oligomerization, and surface processes.
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Biomaterials are frequently evaluated for pro-coagulant activity but usually in the presence of microparticles (MPs), cell-derived vesicles in blood plasma whose phospholipid surfaces allow coagulation factors to set up as functional assemblies. We tested the hypothesis that synthetic anionic surfaces can catalyze burst thrombin activation in human blood plasma in the absence of MPs. In a thromboelastography (TEG) assay with plastic sample cups and pins, recalcified human citrated platelet-poor plasma spontaneously burst-coagulated but with an unpredictable clotting time whereas plasma depleted of MPs by ultracentrifugation failed to coagulate. Coagulation of MP-depleted plasma was restored in a dose-dependent manner by glass microbeads, hydroxyapatite nanoparticles (HA NPs), and carboxylic acid-containing anionic nanocoatings of TEG cups and pins (coated by glow-discharge plasma-polymerized ethylene containing oxygen, L-PPE:O with 4.4 and 6.8 atomic % [COOH]). Glass beads lost their pro-coagulant activity in MP-depleted plasma after their surfaces were nanocoated with hydrophobic plasma-polymerized hexamethyl disiloxane (PP-HMDSO). In FXII-depleted MP-depleted plasma, glass microbeads failed to induce coagulation, however, FXIa was sufficient to induce coagulation in a dose-dependent manner, with no effect of glass beads. These data suggest that anionic surfaces of crystalline, organic, and amorphous solid synthetic materials catalyze explosive thrombin generation in MP-depleted plasma by activating the FXII-dependent intrinsic contact pathway. The data also show that microparticles are pro-coagulant surfaces whose activity has been largely overlooked in many coagulation studies to-date. These results suggest a possible mechanism by which anionic biomaterial surfaces induce bone healing by contact osteogenesis, through fibrin clot formation in the absence of platelet activation.
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Materiales Biocompatibles/farmacología , Coagulación Sanguínea/efectos de los fármacos , Micropartículas Derivadas de Células/química , Durapatita/farmacología , Nanopartículas/química , Siloxanos/farmacología , Aniones , Materiales Biocompatibles/química , Plaquetas/citología , Plaquetas/efectos de los fármacos , Durapatita/química , Factor XII/metabolismo , Vidrio/química , Humanos , Membranas Artificiales , Siloxanos/química , Propiedades de Superficie , Tromboelastografía , Trombina/metabolismoRESUMEN
Photoinitiated chemical vapor deposition (PICVD) has become attractive for selective and specific surface functionalization, because it relies on a single energy source, the photons, to carry out (photo-) chemistry. In the present wavelength (λ)-dependent study, thiol (SH)-terminated thin film deposits have been prepared from gas mixtures of acetylene (C2H2) and hydrogen sulfide (H2S) via PICVD using four different vacuum-ultraviolet (VUV) sources, namely, KrL (λpeak = 123.6 nm), XeL (λpeak = 147.0 nm), XeE (λpeak = 172.0 nm), and Hg (λ = 184.9 nm) lamps. Different λ influence the deposition kinetics and film composition, reflecting that photolytic reactions are governed by the gases' absorption coefficients, k(λ). Thiol concentrations, [SH], up to â¼7.7%, were obtained with the XeL source, the highest reported in the literature so far. Furthermore, all films showed islandlike surface morphology, regardless of λ.
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The patency of small-diameter (<6 mm) synthetic vascular grafts (VGs) is still limited by the absence of a confluent, blood flow-resistant monolayer of endothelial cells (ECs) on the lumen and of vascular smooth muscle cell (VSMC) growth into the media layer. In this research, electrospinning has been combined with bioactive coatings based on chondroitin sulfate (CS) to create scaffolds that possess optimal morphological and bioactive properties for subsequent cell seeding. We fabricated random and aligned electrospun poly(ethylene terephthalate), ePET, mats with small pores (3.2 ± 0.5 or 3.9 ± 0.3 µm) and then investigated the effects of topography and bioactive coatings on EC adhesion, growth, and resistance to shear stress. Bioactive coatings were found to dominate the cell behavior, which enabled creation of a near-confluent EC monolayer that resisted physiological shear-flow conditions. CS is particularly interesting since it prevents platelet adhesion, a key issue to avoid blood clot formation in case of an incomplete EC monolayer or partial cell detachment. Regarding the media layer, circumferentially oriented nanofibers with larger pores (6.3 ± 0.5 µm) allowed growth, survival, and inward penetration of VSMCs, especially when the CS was further coated with tethered, oriented epithelial growth factor (EGF). In summary, the techniques developed here can lead to adequate scaffolds for the luminal and media layers of small-diameter synthetic VGs.
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Prótesis Vascular , Sulfatos de Condroitina/química , Electroquímica , Células Endoteliales de la Vena Umbilical Humana/citología , Músculo Liso Vascular/citología , Nanofibras/química , Ingeniería de Tejidos/métodos , Animales , Aorta Torácica/citología , Adhesión Celular , Células Cultivadas , Humanos , Tereftalatos Polietilenos/química , Ratas , Estrés Mecánico , Andamios del TejidoRESUMEN
PURPOSE: To evaluate the potential of a bioactive coating based on chondroitin sulfate (CS) and tethered epidermal growth factor (EGF) for improvement of healing around stent grafts (SGs). MATERIALS AND METHODS: The impact of the bioactive coating on cell survival was tested in vitro on human vascular cells using polyethylene terephthalate films (PET) as a substrate. After being transferred onto a more "realistic" material (expanded polytetrafluoroethylene [ePTFE]), the durability and mechanical behavior of the coating and cell survival were studied. Preliminary in vivo testing was performed in a canine iliac aneurysm model reproducing type I endoleaks (three animals with one control and one bioactive SG for each). RESULTS: CS and EGF coatings significantly increased survival of human smooth muscle cells and fibroblasts compared with bare PET or ePTFE (P < .05). The coating also displayed good durability over 30 days according to enzyme-linked immunosorbent assay and cell survival tests. The coating did not affect mechanical properties of ePTFE and was successfully transferred onto commercial SGs for in vivo testing. No difference was observed on computed tomography and macroscopic examinations in endoleak persistence at 3 months, but the bioactive coating deposited on the abluminal surface of the SG (exposed to the vessel wall) increased the percentage of healed tissue in the aneurysm. No adverse effect, such as neointima formation or thrombosis, was observed. CONCLUSIONS: The bioactive coating promoted in vitro cell survival, displayed good durability, and was successfully transferred onto a commercial SG. Preliminary in vivo results suggest improved healing around bioactive SGs.
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Implantación de Prótesis Vascular/instrumentación , Prótesis Vascular , Sulfatos de Condroitina/administración & dosificación , Materiales Biocompatibles Revestidos , Factor de Crecimiento Epidérmico/administración & dosificación , Aneurisma Ilíaco/cirugía , Arteria Ilíaca/cirugía , Stents , Animales , Implantación de Prótesis Vascular/efectos adversos , Adhesión Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Angiografía por Tomografía Computarizada , Modelos Animales de Enfermedad , Perros , Endofuga/etiología , Endofuga/prevención & control , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Humanos , Aneurisma Ilíaco/diagnóstico por imagen , Aneurisma Ilíaco/patología , Arteria Ilíaca/diagnóstico por imagen , Arteria Ilíaca/patología , Ensayo de Materiales , Microscopía Confocal , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/patología , Proyectos Piloto , Tereftalatos Polietilenos , Politetrafluoroetileno , Diseño de Prótesis , Factores de Tiempo , Cicatrización de Heridas/efectos de los fármacosRESUMEN
In the context of porous bone void filler for oral bone reconstruction, peptides that suppress microbial growth and promote osteoblast function could be used to enhance the performance of a porous bone void filler. We tested the hypothesis that P15-CSP, a novel fusion peptide containing collagen-mimetic osteogenic peptide P15, and competence-stimulating peptide (CSP), a cationic antimicrobial peptide, has emerging properties not shared by P15 or CSP alone. Peptide-coated surfaces were tested for antimicrobial activity toward Streptoccocus mutans, and their ability to promote human mesenchymal stem cell (MSC) attachment, spreading, metabolism, and osteogenesis. In the osteogenesis assay, peptides were coated on tissue culture plastic and on thin films generated by plasma-enhanced chemical vapor deposition to have hydrophilic or hydrophobic character (water contact angles 63°, 42°, and 92°, respectively). S. mutans planktonic growth was specifically inhibited by CSP, whereas biofilm formation was inhibited by P15-CSP. MSC adhesion and actin stress fiber formation was strongly enhanced by CSP, P15-CSP, and fibronectin coatings and modestly enhanced by P15 versus uncoated surfaces. Metabolic assays revealed that CSP was slightly cytotoxic to MSCs. MSCs developed alkaline phosphatase activity on all surfaces, with or without peptide coatings, and consistently deposited the most biomineralized matrix on hydrophilic surfaces coated with P15-CSP. Hydrophobic thin films completely suppressed MSC biomineralization, consistent with previous findings of suppressed osteogenesis on hydrophobic bioplastics. Collective data in this study provide new evidence that P15-CSP has unique dual capacity to suppress biofilm formation, and to enhance osteogenic activity as a coating on hydrophilic surfaces.
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Péptidos Catiónicos Antimicrobianos/farmacología , Biopelículas/efectos de los fármacos , Materiales Biocompatibles Revestidos/química , Colágeno/farmacología , Osteogénesis/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Streptococcus/efectos de los fármacos , Secuencia de Aminoácidos , Péptidos Catiónicos Antimicrobianos/administración & dosificación , Péptidos Catiónicos Antimicrobianos/química , Proteínas Bacterianas/administración & dosificación , Proteínas Bacterianas/química , Proteínas Bacterianas/farmacología , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Colágeno/administración & dosificación , Colágeno/química , Proteínas de Unión al ADN/administración & dosificación , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/farmacología , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Datos de Secuencia Molecular , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/química , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/farmacología , Infecciones Estreptocócicas/prevención & control , Streptococcus/fisiología , HumectabilidadRESUMEN
In the quest to reduce risk of thrombosis in vascular grafts, it is essential to provide a surface with morphological and mechanical properties close to those of the extracellular matrix beneath the luminal endothelium, and to favor the growth of a confluent, stable monolayer of endothelial cells. This is accomplished here by combining electrospun poly(ethylene terephthalate) (PET) mats with an amine-rich thin plasma-polymerized coating, designated "L-PPE:N." Its deposition does not modify the open, highly porous mats and leads only to small changes in mechanical properties. L-PPE:N significantly improves the adhesion and growth of human umbilical vein endothelial cells (HUVEC) and their resistance to flow-induced shear stress. These properties favor the formation of desired confluent HUVEC monolayers on the topmost surface, unlike conventional vascular grafts (ePTFE or woven PET), where cells migrate inside the material. This combination is therefore highly advantageous for the pre-endothelialization of the luminal side of small-diameter vascular prostheses.
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Prótesis Vascular , Células Endoteliales/citología , Nanofibras/química , Plasma/química , Trombosis/prevención & control , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Análisis de Varianza , Fenómenos Biomecánicos , Técnica del Anticuerpo Fluorescente , Células Endoteliales de la Vena Umbilical Humana , Humanos , Microscopía Electrónica de Rastreo , Espectroscopía de Fotoelectrones , PolimerizacionRESUMEN
The mechanical properties of mammalian cells are largely determined by their cytoskeletons (CSKs), which comprise several distinct but interacting cytoplasmic molecular networks. To examine the influence of the CSK on cell mechanical properties, we deformed several mammalian cell-types (L929, CHO, HEK293, and U937) in suspension using time-varying non-uniform electric fields. Confocal fluorescent microscopy was also used to visualize and semi-quantitatively analyze CSK dimensions. We found mechanical properties of individually deformed cells to depend on cortical actin (CA) thickness. U937 and HEK293 cells with thin CA were more easily deformed than CHO and L929 cells, which bore thicker CA. In additional experiments, we treated U937 cells with latrunculin-A (Lat-A) and acrylamide (ACR), drugs that disrupt microfilaments (MF) and intermediate-filaments (IF), respectively, in order to assess their effects on the CSK and on the cell mechanical properties. We fit strain data using either a power-law or a viscoelasticity model of compliance. Our results demonstrated that maximal strain values observed under identical loading conditions were determined by the structural integrity and thickness of CA in suspended cells. Young's modulus values of individually deformed cells that were estimated using a power-law model showed a linear dependence on cortical actin thickness.
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Citoesqueleto de Actina/fisiología , Animales , Células CHO , Cricetinae , Cricetulus , Elasticidad , Estimulación Eléctrica , Células HEK293 , Humanos , Ratones , Estrés Mecánico , Suspensiones , Células U937 , ViscosidadRESUMEN
Bioactive coatings constitute an interesting approach to enhance healing around implants, such as stent-grafts used in endovascular aneurysm repair. Three different plasma techniques, namely NH3 plasma functionalization and atmospheric- or low-pressure plasma polymerization, are compared to create amino groups and covalently bind CS and EGF bioactive molecules on PET. The latter presents the greatest potential. CS + EGF coating is shown to strongly decrease cell apoptosis and cell depletion in serum-free medium, while increasing cell growth compared to unmodified PET. This versatile biomimetic coating holds promise in promoting vascular repair around stent-grafts, where resistance to apoptosis is a key issue.
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Apoptosis/efectos de los fármacos , Sulfatos de Condroitina , Materiales Biocompatibles Revestidos , Factor de Crecimiento Epidérmico , Proteínas Inmovilizadas , Stents , Aneurisma/terapia , Células Cultivadas , Sulfatos de Condroitina/química , Sulfatos de Condroitina/farmacología , Materiales Biocompatibles Revestidos/química , Materiales Biocompatibles Revestidos/farmacología , Factor de Crecimiento Epidérmico/química , Factor de Crecimiento Epidérmico/farmacología , Humanos , Proteínas Inmovilizadas/química , Proteínas Inmovilizadas/farmacologíaRESUMEN
Low seeding efficiency and poor cell retention under flow-induced shear stress limit the effectiveness of in vitro endothelialization strategies for small-diameter vascular grafts. Primary-amine-rich plasma-polymerized coatings (PPE:N) deposited using low- and atmospheric-pressure plasma discharges on PET and PTFE are evaluated for their ability to improve endothelial cells' kinetics and strength of attachment. PPE:N coatings increase cell adhesion and adhesion rate, spreading, focal adhesion, and resistance to flow-induced shear compared with bare and gelatin-coated PET and PTFE. In particular, about 90% of the cells remain on coated surfaces after 1 h exposure to shear. These coatings, therefore, appear as a promising versatile approach to improve cell seeding strategies for vascular grafts.
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Materiales Biocompatibles Revestidos , Endotelio Vascular/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Polietilenglicoles/química , Politetrafluoroetileno/química , Ingeniería de Tejidos/métodos , Aminas/química , Prótesis Vascular , Adhesión Celular , Materiales Biocompatibles Revestidos/síntesis química , Materiales Biocompatibles Revestidos/metabolismo , Endotelio Vascular/citología , Células Endoteliales de la Vena Umbilical Humana/citología , Humanos , Nitrógeno/química , Gases em Plasma/química , Polietilenglicoles/metabolismo , Tereftalatos Polietilenos , Politetrafluoroetileno/metabolismo , Presión , Reología , Estrés Mecánico , Propiedades de SuperficieRESUMEN
Mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into chondrocytes, osteoblasts, myocytes, adipocytes, and a variety of other cell types. Several studies have been directed toward using MSCs from patients with osteoarthritis (OA) for cartilage repair, not only because these are the ones that will require a source of autologous stem cells if biological repair of cartilage lesions is to be a therapeutic option, but also to further an understanding of stem cell differentiation. Previous studies have shown that a major drawback of current cartilage and intervertebral disc tissue repair is that human MSCs from OA patients express type X collagen (COL X). COL X, a marker of late-stage chondrocyte hypertrophy, is implicated in endochondral ossification. However, those studies also revealed that a novel plasma-polymerized thin film material, named nitrogen-rich plasma-polymerized ethylene (PPE:N), was able to inhibit COL X expression in committed MSCs. The specific aim of this present study was to determine if the suppression of COL X by PPE:N is maintained when MSCs are transferred to pellet cultures in serum-free media. Our results confirmed the potential of two different types of PPE:N surfaces (low-pressure-PPE:N [L-PPE:N] and high-pressure-PPE:N [H-PPE:N]) in suppressing COL X expression, more so on the latter. Interestingly, when MSCs were transferred to pellet cultures, the expression level of COL X was further decreased by preincubation on H-PPE:N, suggesting that these kinds of coatings show promise for tissue engineering of cartilage and disc tissues. Further studies are needed to assess the relative importance of surface-chemistry versus surface-morphology in the mechanism of COL X suppression.
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Colágeno Tipo X/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Nitrógeno/farmacología , Plasma/metabolismo , Polimerizacion/efectos de los fármacos , Anciano , Anciano de 80 o más Años , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Medio de Cultivo Libre de Suero , Etilenos/farmacología , Femenino , Humanos , Masculino , Células Madre Mesenquimatosas/efectos de los fármacos , Microscopía Electrónica de Rastreo , Persona de Mediana Edad , Osteoartritis/patología , Poliestirenos/farmacología , Propiedades de Superficie/efectos de los fármacosRESUMEN
BACKGROUND: Recent evidence indicates that osteoarthritis (OA) may be a systemic disease since mesenchymal stem cells (MSCs) from OA patients express type X collagen, a marker of late stage chondrocyte hypertrophy (associated with endochondral ossification). We recently showed that the expression of type X collagen was suppressed when MSCs from OA patients were cultured on nitrogen (N)-rich plasma polymer layers, which we call "PPE:N" (N-doped plasma-polymerized ethylene, containing up to 36 atomic percentage (at.% ) of N. METHODS: In the present study, we examined the expression of type X collagen in fetal bovine growth plate chondrocytes (containing hypertrophic chondrocytes) cultured on PPE:N. We also studied the effect of PPE:N on the expression of matrix molecules such as type II collagen and aggrecan, as well as on proteases (matrix metalloproteinase-13 (MMP-13) and molecules implicated in cell division (cyclin B2). Two other culture surfaces, "hydrophilic" polystyrene (PS, regular culture dishes) and nitrogen-containing cation polystyrene (Primaria®), were also investigated for comparison. RESULTS: Results showed that type X collagen mRNA levels were suppressed when cultured for 4 days on PPE:N, suggesting that type X collagen is regulated similarly in hypertrophic chondrocytes and in human MSCs from OA patients. However, the levels of type X collagen mRNA almost returned to control value after 20 days in culture on these surfaces. Culture on the various surfaces had no significant effects on type II collagen, aggrecan, MMP-13, and cyclin B2 mRNA levels. CONCLUSION: Hypertrophy is diminished by culturing growth plate chondrocytes on nitrogen-rich surfaces, a mechanism that is beneficial for MSC chondrogenesis. Furthermore, one major advantage of such "intelligent surfaces" over recombinant growth factors for tissue engineering and cartilage repair is potentially large cost-saving.
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Condrocitos/metabolismo , Colágeno Tipo X/metabolismo , Placa de Crecimiento/citología , Nitrógeno/metabolismo , Agrecanos/metabolismo , Animales , Bovinos , Técnicas de Cultivo de Célula , División Celular , Células Cultivadas , Condrocitos/citología , Colágeno Tipo II/metabolismo , Colágeno Tipo X/genética , Medios de Cultivo Condicionados , Ciclina B2/metabolismo , Regulación hacia Abajo , Placa de Crecimiento/metabolismo , Hipertrofia/metabolismo , Metaloproteinasa 13 de la Matriz/metabolismo , ARN Mensajero , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The development of new strategies for protein immobilization to control cell adhesion, growth and differentiation is of prime interest in the field of tissue engineering. Here we propose a versatile approach based on the interaction between two de novo designed peptides, Ecoil and Kcoil, for oriented immobilization of epidermal growth factor (EGF) on polyethylene terephthalate (PET) films. After amination of PET surfaces by ammonia plasma treatment, Kcoil peptides were covalently grafted in an oriented fashion using succinimidyl 6-[30-(2-pyridyldithio)-propionamido] hexanoate (LC-SPDP) linker, and the Kcoil-functionalized films were characterized by X-ray photoelectron spectroscopy (XPS). Bioactivity of Ecoil-EGF captured on Kcoil-functionalized PET via coiled-coil interactions was confirmed by EGF receptor phosphorylation analysis following A-431 cell attachment. We also demonstrated cell biological effects where tethered EGF enhanced adhesion, spreading and proliferation of human corneal epithelial cells compared to EGF that was either physically adsorbed or present in solution. Tethered EGF effects were most likely linked to the prolonged activation of both mitogen-activated protein kinase and phosphoinositidine 3-kinase pathways. Taken together, our results indicate that coiled-coil-based oriented immobilization is a powerful method to specifically tailor biomaterial surfaces for tissue engineering applications.
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Córnea/citología , Factor de Crecimiento Epidérmico/farmacología , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Factor de Crecimiento Epidérmico/química , Células Epiteliales/metabolismo , Receptores ErbB/metabolismo , Humanos , Inmunohistoquímica , Microscopía Confocal , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Espectroscopía de Fotoelectrones , Transducción de Señal/efectos de los fármacosRESUMEN
Recent evidence indicates that a major drawback of current cartilage- and intervertebral disc (IVD) tissue engineering is that human mesenchymal stem cells (MSCs) from patients with osteoarthritis rapidly express type X collagen (COL10A1), a marker of late stage chondrocyte hypertrophy associated with endochondral ossification. We recently demonstrated that COL10A1 expression was inhibited in MSCs from patients with osteoarthritis cultured on nitrogen-rich plasma polymerized (PPE:N) coatings. Here, we sought to understand the mechanisms of action of this effect by culturing MSCs on PPE:N surfaces in the presence of different inhibitors of kinases and cyclooxygenases. The effect of PPE:N surfaces on COL10A1 expression was found to be mimicked by the cyclooxygenase inhibitor NPPB, but not by daphnetin (an inhibitor of protein kinases) nor by genistein (an inhibitor of tyrosine kinases). COL10A1 expression was also suppressed by the specific cyclooxygenase-1 (COX-1: SC-560) and 5-lipoxygenase (5-LOX: MK-866) inhibitors, but not by COX-2 (COX-2 inhibitor 2) and 12-LOX (baicalein) inhibitors. Finally, the incubation of MSCs on PPE:N surfaces inhibited the expression of COX-1 while 5-LOX was not expressed in these cells. Taken together, these results indicate that PPE:N surfaces inhibit COL10A1 expression via the suppression of COX-1.
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Colágeno Tipo X/metabolismo , Ciclooxigenasa 1/metabolismo , Células Madre Mesenquimatosas/fisiología , Nitrógeno/química , Osteoartritis/metabolismo , Polímeros , Anciano , Anciano de 80 o más Años , Araquidonato 5-Lipooxigenasa/metabolismo , Materiales Biocompatibles/química , Materiales Biocompatibles/metabolismo , Células Cultivadas , Materiales Biocompatibles Revestidos , Inhibidores Enzimáticos/metabolismo , Humanos , Ensayo de Materiales , Células Madre Mesenquimatosas/citología , Persona de Mediana Edad , Nitrógeno/metabolismo , Osteoartritis/patología , Polímeros/química , Polímeros/metabolismo , ARN/metabolismo , Ingeniería de Tejidos/métodosRESUMEN
In many industrial applications, inadequate cell attachment can be a limitation, especially when serum-free media are used. Nitrogen-rich plasma-polymerised ethylene (PPE:N) exhibits high concentrations of polar groups that can help to promote the attachment of weakly adherent cell types. Tissue plasminogen activator-producing Chinese hamster ovary (CHO) cells, adapted to suspension, were grown in the presence PPE:N flakes and were found to adhere to them. The growth rate was reduced, but cell viability was enhanced and their metabolism was more efficient, with generally higher recombinant protein productivity. Finally, cell adhesion on PPE:N surfaces was found to be independent of integrins, and was probably mediated by certain non-specific interactions with the PPE:N surface.
Asunto(s)
Células CHO/metabolismo , Adhesión Celular/fisiología , Etilenos , Nitrógeno/química , Polímeros , Proteínas Recombinantes/biosíntesis , Animales , Materiales Biocompatibles Revestidos , Cricetinae , Cricetulus , Etilenos/química , Etilenos/metabolismo , Glucosa/metabolismo , Glutamina/metabolismo , Integrina beta1/metabolismo , Ácido Láctico/metabolismo , Ensayo de Materiales , Polímeros/química , Polímeros/metabolismo , Compuestos de Amonio Cuaternario/metabolismo , Proteínas Recombinantes/genética , Propiedades de Superficie , Activador de Tejido Plasminógeno/biosíntesisRESUMEN
We present a two-fold study designed to elucidate the adhesion mechanism of human U937 monocytes on novel N-rich thin films deposited by plasma- and VUV photo-polymerisation, so-called "PVP:N" materials. It is shown that there exist sharply-defined ("critical") surface-chemical conditions that are necessary to induce cell adhesion. By comparing the film chemistries at the "critical" conditions, we demonstrate the dominant role of primary amines in the cell adhesion mechanism. Quantitative real-time RT-PCR experiments using U937 cells that had adhered to PVP:N materials for up to 24 h are presented. The adhesion induces a transient expression of cytokines, markers of macrophage activation, as well as a more sustained expression of PPAR gamma and ICAM-I.
Asunto(s)
Adhesión Celular/fisiología , Monocitos/fisiología , Nitrógeno/química , Animales , Línea Celular , Citocinas/genética , Citocinas/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Técnicas Electroquímicas/instrumentación , Técnicas Electroquímicas/métodos , Humanos , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Monocitos/citología , PPAR gamma/genética , PPAR gamma/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
We report the use of dielectrophoresis (DEP) to position U-937 monocytes within a non-uniform electric field, prior to electroporation (EP) for gene delivery. DEP positioning and EP pulsing were both accomplished using a common set of inert planar electrodes, micro-fabricated on a glass substrate. A single-shell model of the cell's dielectric properties and finite-element modeling of the electric field distribution permitted us to predict the major features of cell positioning. The extent to which electric pulses increased the permeability of the cell membranes to fluorescent molecules and to pEGFPLuc DNA plasmids were found to depend on prior positioning. For a given set of pulse parameters, EP was either irreversible (resulting in cytolysis), reversible (leading to gene delivery), or not detectable, depending on where cells were positioned. Our results clearly demonstrate that position-dependent EP of cells in a non-uniform electric field can be controlled by DEP.