Collado-González,  Mar

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Collado-González, Mar

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Collado-González, Mar

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Universidad de Murcia. Departamento de Biología Celular e Histología

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Open Access
Citotoxicity of Guttaflow bioseal, Guttaflow2, MTA Fillapex, and AH Plus on human periodontal ligament stem cells.
(Elsevier Science, 2017-03-23) Collado-González, Mar; Tomás Catalá, Christopher Joseph; Oñate Sánchez, Ricardo Elías; Moraleda Jiménez, José María; Rodríguez Lozano, Francisco Javier; Dermatología, Estomatología, Radiología y Medicina Física
Introduction: The aim of the present study was to evaluate the in vitro cytotoxicity of endodontic sealers (GuttaFlow Bioseal, GuttaFlow2, and MTA Fillapex) on human periodontal ligament stem cells (hPDLSCs). As a reference, AH Plus was compared with the more recent endodontic sealers regarding cell viability and cell attachment. Methods: Biological testing was carried out in vitro on hPDLSCs. Cell viability assay was performed by using eluates from each endodontic sealer. To assess cell orphology and attachment to the different sealers, the hPDLSCs were directly seeded onto the material surfaces and analyzed by scanning electron microscopy. Chemical composition of the sealers was determined by energy-dispersive x-ray, and eluates were analyzed by inductively coupled plasma mass spectrometry. Statistical differences were assessed by analysis of variance and Tukey test (P < .05). Results: Cell viability was evident after 24 hours in the presence of GuttaFlow Bioseal and GuttaFlow 2 but not in the case of AH Plus or MTA Fillapex. At 168 hours, GuttaFlow Bioseal and GuttaFlow 2 exhibited high and moderate cell viability, respectively, whereas AH Plus and MTA Fillapex revealed low rates of cell cell viability (P < .001). Finally, scanning electron microscopy studies revealed a high degree of proliferation, cell spreading, and attachment, especially when using GuttaFlow Bioseal disks. Conclusions: GuttaFlow Bioseal and GuttaFlow2 showed lower cytotoxicity than MTA Fillapex and AH plus. Further in vitro and in vivo investigations are required to confirm the suitability of GuttaFlow Bioseal for clinical application.
Open Access
Description and comparative study of physico-chemical parameters of the teleost fish skin mucus.
(SAGE Publications, 2015-07-01) Guardiola Abellán, Francisco Antonio; Cuartero, María; Collado-González, Mar; Arizcún, Marta; Díaz Baños, F. Guillermo; Meseguer, José; Esteban Abad, María de los Ángeles; Biología Celular e Histología
The study of mucosal surfaces, and in particular the fish skin and its secreted mucus, has been of great interest recently among immunologists. Measurement of the viscosity and other physico-chemical parameters (protein concentration, pH, conductivity, redox potential, osmolality and density) of the skin mucus can help to understand its biological functions. We have used five marine species of teleost: gilthead seabream (Sparus aurata L.), European sea bass (Dicentrarchus labrax L.), shi drum (Umbrina cirrosa L.), common dentex (Dentex dentex L.) and dusky grouper (Epinephelus marginatus L.), all of them with commercial interest in the aquaculture of the Mediterranean area. Mucus showed a direct shear- and temperature-dependent viscosity, with a non-Newtonian behavior, which differed however between two groups: one with higher viscosity (D. labrax, U. cirrosa, D. dentex) and the other with lower viscosity (S. aurata, E. marginatus). In addition, there was a clear interrelation between density and osmolality, as well as between density and temperature. Taking into account that high values of viscosity should improve the barrier effect against pathogens but low values of viscosity are needed for good locomotion characteristics, our results may help elucidate the relationship between physico-chemical and biological parameters of skin mucus, and disease susceptibility.
Open Access
Silk fibroin and graphene oxide composites promote human periodontal ligament stem cell spontaneous differentiation into osteo/cementoblast-like cells
(Mary Ann Liebert, Inc, 2016-11-15) Sánchez Vera, María del Mar; Aznar-Cervantes, Salvador; Jover, Eva; García Bernal, David; Oñate Sánchez, Ricardo Elías; Hernández Romero, Diana; Moraleda Jiménez, José María; Collado-González, Mar; Rodríguez Lozano, Francisco Javier; Cenís, José L.; Dermatología, Estomatología, Radiología y Medicina Física
Graphene represents one of the most interesting additions to the tissue engineering toolbox. Novel graphenebased composites are required to improve the beneficial graphene properties in terms of tridimensional polymeric structure, conferring a higher mechanical strength and favoring the differentiation of human mesenchymal stem cells. Here, we have demonstrated in a wide range of composite combinations, the successful use of graphene and silk-fibroin constructs for future bioengineering applications in the field of clinical regenerative dentistry using human periodontal ligament stem cells. Our results provide exciting new data for the development of suitable scaffolds that allow good cell engrafting, preservation of cell viability and proliferation, promotion of spontaneous osteoblastic differentiation, and importantly, stimulation of a higher cementum physiological synthesis than using other different available biomaterials.
Open Access
Resveratrol lacks antifungal activity against Candida albicans.
(Springer, 2012-03-30 ) Collado-González, Mar; Guirao-Abad, José Pedro; Sánchez-Fresneda Pinto, Ruth; Belchí Navarro, Sarai; Argüelles Ordóñez, Juan Carlos; Genética y Microbiología
The putative candicidal activity of resveratrol is currently a matter of controversy. Here, the antifungal activity as well as the antioxidant response of resveratrol against Candida albicans, have been tested in a set of strains with a well-established genetic background At the doses usually employed in antifungal tests (10–40 lg/ml), resveratrol has no effect on the exponential growth of the C. albicans CAI.4 strain, a tenfold increase (400 lg/ml) was required in order to record a certain degree of cell killing, which was negligible in comparison with the strong antifungal effect caused by the addition of amphotericin B (5 lg/ml). An identical pattern was recorded in the prototrophic strains of C. albicans SC5314 and RM-100, whereas the oxidative sensitive trehalose-deficient mutant (tps1/tps1 strain) was totally refractory to the presence of resveratrol. In turn, the serum-induced yeast-to-hypha transition remained unaffected upon addition of different concentrations of resveratrol. Determination of endogenous trehalose and catalase activity, two antioxidant markers in C. albicans; revealed no significant changes in their basal contents induced by resveratrol. Collectively, our results seem to dismiss a main antifungal role as well as the therapeutic application of resveratrol against the infections caused by C. albicans.
Open Access
Chitosan as stabilizing agent for negatively charged nanoparticles
(Elsevier, 2016-12-24) Collado-González, Mar; García Montalbán, Mercedes; Peña-García, Jorge; Pérez-Sánchez, Horacio; Víllora Cano, Gloria; Díaz Baños, F. Guillermo; Biología Celular e Histología
Chitosan is a biocompatible polysaccharide with positive Z potential which can stabilize negative charged nanoparticles. Silk fibroin nanoparticles and citrate gold nanoparticles, both with negative Z potential, but they form aggregates at physiological ionic strength. In this work, we study the behavior of chitosan in solution when the ionic strength of the medium is increased and how the concentration of chitosan and the proportion of the two components (chitosan and AuNP or SFN) significantly affect the stability and size of the nanocomposites formed. In addition to experimental measurements, molecular modeling were used to gain insight into how chitosan interacts with silk fibroin monomers, and to identify the main energetic interactions involved in the process. The optimum values for obtaining the smallest and most homogeneous stable nanocomposites were obtained and two different ways of organization through which chitosan may exert its stabilizing effect were suggested.
Embargo
Thermo-setting glass ionomer cements promote variable biological responses of human dental pulp stem cells
(2018) Collado-González, Mar; Pecci Lloret, Miguel Ramón; Tomás Catalá, Christopher Joseph; García Bernal, David; Oñate Sánchez, Ricardo Elías; Llena, Carmen; Forner, Leopoldo; Rosa, Vinicius; Rodríguez Lozano, Francisco Javier; Dermatología, Estomatología, Radiología y Medicina Física
Objective: To evaluate the in vitro cytotoxicity of Equia Forte (GC, Tokyo, Japan) and Ionostar Molar (Voco, Cuxhaven, Germany) on human dental pulp stem cells (hDPSCs). Methods: hDPSCs isolated from third molars were exposed to several dilutions of Equia Forte and Ionostar Molar eluates (1/1, 1/2 and 1/4). These eluates were obtained by storing material samples in respective cell culture medium for 24 h (n = 40). hDPSCs in basal growth culture medium were the control. Cell viability and cell migration assays were performed using the MTT and wound-healing assays, respectively. Also, induction of apoptosis and changes in cell phenotype were evaluated by flow cytometry. Changes in cell morphology were analysed by immunocytofluorescence staining. To evaluate cell attachment to the different materials, hDPSCs were directly seeded onto the material surfaces and analyzed by scanning electron microscopy (SEM). The chemical composition of the materials was determined by energy dispersive X-ray (EDX) and eluates were analyzed by inductively coupled plasma-mass spectrometry (ICP-MS). Statistical analysis was performed with analysis of variance (ANOVA) and Student's t-test (α < 0.05). Results: Undiluted Equia Forte extracts led to a similar cell proliferation rates than the control group from 72 h onwards. There were no significance differences between Equia Forte and Ionostar Molar in terms of cell apoptosis and phenotype. However, in presence of Equia extracts the migration capacity of hDPSCs was higher than in presence of Ionostar Molar (p < 0.05). Also, SEM studies showed a higher degree of cell attachment when Equia Forte extracts were used. Finally, EDX analysis pointed to different weight percentages of C, O and Ca ions in glass ionomer cements, while other elements such as La, Al, Si, W, Mo and F were also detected. Significance: In summary, Equia Forte promoted better biological responses in hDPSCs than Ionostar Molar.
Open Access
Caracterización de nanoestructuras de quitosano, fibroína de seda y oro como liberadores de fármacos
(Universidad de Murcia, 2018-02-28) Collado-González, Mar; Víllora Cano, Gloria; Díaz Baños, F. Guillermo; Facultad de Química
Uno de los principales inconvenientes del uso de nanodispositivos para la liberación de fármacos es su tendencia a agregar al ser expuestos a condiciones fisiológicas. Se ha propuesto el uso de polímeros como agentes estabilizadores. Entre estos, el quitosano (CS) es un polielectrolito policatiónico en disolución que ha mostrado potenciales aptitudes en la estabilización de diferentes nanoestructuras cargadas negativamente. Los objetivos de este trabajo fueron sintetizar y caracterizar nanodispositivos de liberación de fármacos, en los que el CS de peso molecular medio (MMW) actuara como agente estabilizador de diferentes nanoestructuras cargadas negativamente. De forma concreta, nanodispositivos compuestos con CS MMW y nanopartículas de oro recubiertas de citrato (AuNP) o CS MMW y nanopartículas de fibroína de la seda (SFN). Estudiar la citotoxicidad del CS MMW, de las AuNP, de las SFN y de las nanoestructuras desarrolladas, CS MMW:AuNP y CS MMW:SFN sobre cultivos celulares usando para ello células HeLa. Por último, caracterizar nanodispositivos complejos formados a partir de CS MMW y otros biopolímeros complejos como el alginato, el sulfato dextrano y el polietilenglicol 4000 entre otros. Y evaluar estas nanoestructuras como dispositivos de liberación de fármacos usando insulina como molécula modelo. La caracterización de las estructuras se llevó a cabo mediante el estudio del diámetro hidrodinámico medido mediante dispersión dinámica de luz (DLS), del potencial Z, del espectro de absorción UV-Vis mediante espectrofotometría y análisis mediante fotocentrifugación. Se recurrió a la microscopía electrónica de transmisión (TEM) y de barrido (SEM) para la visualización de las nanoestructuras. Se determinó la citotoxicidad de los nanodispositivos mediante la reducción de una sal de tetrazolio (MTT) llevada a cabo por células HeLa. Para cuantificar la liberación de insulina por nanodispositivos polisacarídicos complejos, se aplicó cromatrografía líquida de alta resolución (HPLC). Los resultados de este trabajo permiten concluir que: 1. CS MMW en disolución a 10-5 g/mL y pH inferior a 6.5 presenta principalmente nanoestructuras que experimentan una reducción de su diámetro hidrodinámico en condiciones de elevada fuerza iónica como resultado del apantallamiento de cargas de la superficie de las cadenas. 2. El CS MMW en disolución acídica es un polímero policatiónico capaz de estabilizar nanoestructuras polianiónicas. Derivados del polímero como el gicol CS mantienen las propiedades estabilizadoras del polisacárido. 3. Las propiedades de las nanoestructuras obtenidas como resultado de la interacción del biopolímero y nanopartículas cargadas negativamente dependen de las concentraciones de los polielectrolitos y de las proporciones entre el biopolímero y las nanopartículas polianiónicas. 4. El CS MMW actúa como un andamiaje donde se adhieren las AuNP y quedan protegidas de la agregación en condiciones de bajo pH o elevada fuerza iónica. 5. El CS MMW se dispone en la superficie de la SFN donde la protege de la agregación en condiciones de elevada fuerza iónica. La SFN se postula como un material prometedor en nanotecnología debido a puede ser estabilizada mediante el uso de CS MMW y el gicol CS. 6. El CS MMW, las AuNP, la SFN y las nanoestructuras formuladas compuestas de AuNP:CS y CS:SFN, muestran biocompatibilidad sobre cultivos de células HeLa. 7. El CS MMW permite modular el tamaño y la agregación de nanocomposites complejos formulados con otros polisacáridos. One of the main problems when using drug delivery systems is their tendency to aggregate under physiological conditions. Polymers have been proposed as stabilizing agents for avoiding this issue. Among them, chitosan (CS) is a polycationic polyelectrolyte in solution, which has shown good abilities in the stabilization of several negatively charged nanostructures. The objectives of this work were sinthesizing and characterizing nanodispositives for drug delivery in which medium molecular weight (MMW) CS acts as stabilizing agent for different negatively charged nanostructures. Concretely, nanodispositives composed of MMW CS and gold nanopartícles coated with gold (AuNP) and MMW CS and silk fibroin nanoparticles (SFN). Evaluating the citotoxicity of MMW CS, AuNP, SFN, and the nanostructures developed, MMW CS:AuNP and MMW CS:SFN, in HeLa cell cultures. Finally, characterize complex nanodispositives composed of MMW CS and other complex biopolymers as alginate, dextran sulfate and polyethylene glycol 4000, among others. And, evaluate them as drug delivery systems using insulin as a model. In order to characterize nanostructures, hydrodynamic diameter by dynamic light scattering (DLS), Z potential, UV-Vis spectrophotometric analysis, and photocentrifugation analysis were developed. Trasmission electron microscopy and scanning electron microscopy were used to visualize nanostructures. Citotoxicity of the nanodispositives was evaluated by tetrazolium salt reduction (MTT) by HeLa cell cultures. High performance liquid chromatography (HPLC) was used to evaluate the insulin released by polysacharide complex nanodevices. The results obtained in this work let conclude 1. CS MMW in solution at 10-5 g/mL and pH lower than 6.5 shows mainly nanometric size. When increased ionic strength, this nanostructures diminished their size as a result of the screening effect. 2. CS MMW in acidic solution is a polycationic polymer capable of stabilizing polyanionic nanostructures. Its derivatives as glycol CS keep the stabilizing properties of the polyelectrolyte. 3. Properties of the nanostructures obtained as a result of the interaction of MMW CS and negatively charged nanoparticles depended on the polyelectrolyte concentrations and the proportions among the biopolymer and polyanionic nanoparticles. 4. MMW CS acts as scaffold in which AuNP got immobilized and protected from aggregation at low pH of high ionic strength conditions. 5. MMW CS is placed on the surface of SFN avoiding their aggregation at high ionic strength. SFN can be stabilized at high ionic strength by using MMW CS or glycol CS. Thus, SFN is a promising biomaterial for nanotechnology. 6. MMW CS modulated the size and the aggregation of the complex polyssacharyde nanodevices. 7. MMW CS, AuNP, SFN and nanodevices composed of MMW CS:AuNP and MMW CS:SFN showed biocompatibility in HeLa cell cultures.
Open Access
Size-exclusion chromatography of macromolecules: a brief tutorial overview on fundamentals with computational tools for data analysis and determination of structural information
(MDPI, 2025-02-22) Henández-Cifre, José Ginés; Collado-González, Mar; Díaz Baños, F. Guillermo; García de la Torre, José; Química Física; Ingeniería Química; Biología Celular e Histología
Size-exclusion chromatography (SEC) is presently a widely used and very informative technique for the characterization of macromolecules in solution. Beyond the first implementations of SEC—which required cumbersome column calibrations and were mainly intended for the determination of molecular weights—the modern SEC approach involving multiple detectors (md-SEC) is based on solution properties such as intrinsic viscosity and light scattering. Thus, md-SEC enables the direct and more efficient determination of molecular weights, as well as the determination of relationships between property and molecular weight, which can be quite useful in structural studies. Here, we first present a review of the fundamental aspects of the dilute-solution properties of macromolecules—particularly the differential refractive index, intrinsic viscosity, and scattering-related properties—on which the various detectors involved in md-SEC are based. Then, we developed SECtools, a suite of public-domain, open-source computer programs, which allow for the full analysis of md-SEC chromatograms. These analyses range from just the recorded raw signals (mV) of the detectors to a full determination of molecular weight averages and distributions. The use of these programs is illustrated through experimental studies using various samples.
Open Access
Cytotoxicity and bioactivity of various pulpotomy materials on stem cells from human exfoliated primary teeth
(Willey, 2017-02-07) Collado-González, Mar; García Bernal, David; Oñate Sánchez, Ricardo Elías; Ortolani-Seltenerich, P.S.; Álvarez-Muro, T.; Lozano, A.; Forner, L.; Llena, C.; Moraleda Jiménez, José María; Rodríguez Lozano, Francisco Javier; Dermatología, Estomatología, Radiología y Medicina Física
Aims To investigate the cytotoxicity and bioactivity of several pulpotomy materials: Biodentine (Septodont, Saint-Maur-des-Fosses, France) MTA (Angelus, Londrina, PR, Brazil), Theracal LC (Bisco Inc., Schamburg, IL, USA) and IRM (Dentsply DeTrey GmbH, Konstanz, Germany), after contact with stem cells isolated from human exfoliated primary teeth (SHEDs). Methodology SHEDs were cultured in the presence of the eluates of various pulpotomy materials for 24, 48 and 72 h. Cell viability was determined by mitochondrial dehydrogenase enzymatic (MTT) assay. Apoptosis and changes in cell phenotype were evaluated by flow cytometry. Also, an in vitro scratch wound-healing assay was used to determine their effects on cell migration. To assess cell morphology and attachment to the different pulpotomy materials, SHEDs were directly seeded onto the material surfaces and analysed by scanning electron microscopy (SEM). Finally, the deposition of a calcified matrix in presence of these materials was verified by Alizarin Red staining. Statistical analysis was performed with analysis of variance and Bonferroni or Tukey post-test (a = 0.05). Results Cell viability in the presence of Biodentine eluates was significantly higher to that obtained using complete medium alone (control; P < 0.01) and was also significantly higher than using MTA Angelus from 48 h of incubation (P < 0.01). However, Theracal LC and IRM were associated with low rates of cell viability (P < 0.001). Similar results were obtained in an apoptosis assay. In addition, SHEDs maintained their mesenchymal phenotype in all conditions although their capacity to migrate was higher in the presence of Biodentine. SEM studies revealed a suitable proliferation rate, cell spreading and attachment, especially when using Biodentine and MTA Angelus discs. Finally, Biodentine eluates significantly induced calcified matrix deposition from 7 days of culture (P < 0.01). Conclusions Biodentine exhibited better cytocompatibility and bioactivity than MTA Angelus, Theracal LC and IRM.
Embargo
GuttaFlow Bioseal promotes spontaneous differentiation of human periodontal ligament stem cells into cementoblast-like cells.
Rodríguez Lozano, Francisco Javier; Tomás-Catalá, C.J.; García-Bernal, D.; López, S.; Moraleda, J.M.; Murcia, L.; Collado-González, Mar; Dermatología, Estomatología, Radiología y Medicina Física
Objectives: To evaluate in vitro the cementogenic potential and the biological effects of GuttaFlow Bioseal, GuttaFlow 2, MTA Fillapex and AH Plus on human periodontal ligament stem cells (hPDLSCs). Methods: Cell viability, cell migration and cell morphology assays were performed using eluates of each material. To evaluate cell attachment, hPDLSCs were directly seeded onto the material surfaces and analyzed by scanning electron microscopy (SEM). The effects of endodontic sealers on cementum protein 1 (CEMP1), cementum-derived attachment protein (CAP), bone sialoprotein (BSP), ameloblastin (AMBN), amelogenin (AMELX) and alkaline phosphatase (ALP) gene expression on hPDLSCs were investigated by qPCR and immunofluorescence (IF). Statistical analysis was performed with analysis of variance and Bonferroni or Tukey post-test (α < 0.05). Results: More than 90% of viable cells were obtained using extracts of GuttaFlow Bioseal and GuttaFlow2 after 72 h of culture. By contrast, AH Plus and MTA Fillapex induced significantly lower levels of cell viability. GuttaFlow2 and GuttaFlow Bioseal promoted wound closure in a concentration-dependent manner, comparable to that observed with control extracts (*p < 0.05). However, with AH Plus and MTA Fillapex, cell migration was significantly lower than in the control (***p < 0.0001). SEM analysis pointed to an organized stress fiber assembly and high degree of cell adhesion on GuttaFlow Bioseal disks but low rates on GuttaFlow2, MTA Fillapex and AH Plus. When hPDLSCs were cultured with GuttaFlow Bioseal-conditioned media, qPCR assays and IF showed a higher level of AMELX, AMBN, CEMP1 and CAP expression than the control (*p < 0.05)), whereas no such expression was observed in the other sealers. Significance: Our results showed that GuttaFlow sealers were more cytocompatible than AH Plus and MTA Fillapex, while GuttaFlow Bioseal favored cementoblast differentiation of hPDLSCs in the absence of any growth factors.