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.

2016-09-26, Collado-González, Mar, García Bernal, David, Oñate Sánchez, Ricardo Elías, Ortolani-Seltenerich, P.S., Lozano, Adrián, Forner, Leopoldo, Llena, Carmen, Rodríguez Lozano, Francisco Javier, Dermatología, Estomatología, Radiología y Medicina Física

Aim To evaluate the biocompatibility of three calcium silicate-based endodontic sealers, Bioroot BC Sealer (Septodont, Saint-Maur-des-Fosses, France), Endoseal MTA (EndoSeal, Maruchi, Seoul, Korea) and Nano-ceramic Sealer (B&L Biotech, Fairfax, VA, USA) (NCS), on human periodontal ligament stem cells (hPDLSCs). Methodology human periodontal ligament stem cells were cultured in the presence of various endodontic sealer eluates for 24 h. Cell viability was determined using the MTT assay. Cell death and changes in phenotype induced by the set endodontic sealer eluates were evaluated through flow cytometry. Also, an in vitro scratch wound-healing model was used to determine their effects in cell migration. Finally, to assess cell morphology and attachment to the different sealers, hPDLSCs were directly seeded onto the material surfaces and analysed by scanning electron microscopy (SEM). One-way analysis of variance (ANOVA) followed by a Bonferroni post-test was performed (P < 0.05). Results At 24 h, cell spreading was evident in the presence of Bioroot BC Sealer (BR) and Nano-ceramic Sealer (NCS), but not Endoseal MTA (ES). At 72 h, BR and NCS exhibited high and moderate cell proliferation, respectively, whereas ES revealed low rates of cell proliferation (P < 0.05). Similar results were obtained in a cell death assay. In addition, hPDLSCs maintained their mesenchymal phenotype in all conditions although their capacity to migrate was higher in the presence of BR. Finally, SEM studies revealed a good degree of proliferation, cell spreading and attachment, especially when using BR and NCS discs. Conclusions BR and NCS were associated with better cytocompatibility than ES. Further in vitro and in vivo investigations are required to confirm the suitability of these calcium silicate-based endodontic sealers for clinical application.

2016-11-09, Collado-González, Mar, Guardiola Abellán, Francisco Antonio, Cuartero, María, Díaz Baños, F. Guillermo, Cuesta Peñafiel, Alberto, Moriñigo, Miguel Angel, Esteban Abad, María de los Ángeles, Biología Celular e Histología

Recently, interest in mucosal surfaces, more specifically fish skin and its secreted mucus, has greatly increased among immunologists. The abundance of terminal carbohydrates, several enzymes (proteases, lysozyme, peroxidase, alkaline phosphatase, esterases and ceruloplasmin), bactericidal activity against fish pathogenic and non-pathogenic bacteria and several physico-chemical parameters (protein concentration, pH, conductivity, redox potential, osmolarity, density and viscosity) in the skin mucus of Senegalese sole (Solea senegalensis, Kaup) have been evaluated. Present results evidence the abundance of N-acetylneuraminic acid, mannose, glucose and N-acetyl-galactosamine in skin mucus. The levels of lysozyme, proteases, esterases and alkaline phosphatase were very similar (from 20 to 30 Units mg−1 protein). However, 93 Units mg−1 protein were detected of ceruloplasmin and only 4′88 Units mg−1 protein of peroxidase. Skin mucus of S. senegalensis showed high bactericidal activity against the tested pathogen bacteria but weak activity against non-pathogenic bacteria. Finally, a clear relationship between mucus density and temperature was detected, while viscosity showed a direct shear- and temperature-dependent behaviour. These results could be useful for better understanding the role of the skin mucus as a key component of the innate immune system, as well as, for elucidating possible relationships between biological and physico-chemical parameters and disease susceptibility.

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.

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.

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.

2022-08-26, Collado-González, Mar, Esteban Abad, María de los Ángeles, Biología Celular e Histología

Nanoparticles-based treatments is of utmost importance for aquaculture. In this scenario, chitosan-based nanoparticles have been proposed due to the properties of chitosan, which include mucoadhesiveness. Nevertheless, pivotal parameters of chitosan, such as degree of acetylation and molecular weight, are commonly underestimated in the available literature despite the influence they seem to have on the properties of chitosan-based nanoparticles. In this systematic review, the immunomodulator capacity of chitosan nanoparticles used as mucosal vaccines on teleost fish has been evaluated paying special attention to the chitosan properties. Four databases were used for literature search, yielding 486 documents, from which 14 meet the inclusion criteria. Only 21% of the available studies reported properly chitosan properties, which should be improved in future works to generate reproducible data as well as valuable information. To the best of our knowledge, this work objectively compares for the first time, by quantifying the mg of chitosan/g of fish applied in each study, the chitosan nanoparticle preparation and doses applied to fish, as well as the effects of the treatments applied on fish immune status.

2020-01-15, Collado-González, Mar, Freitas, Alessandra R., Santos, Ana Claudia, Ferreira, Nuno F., Ferreri, Maria Cristina, Sequeira, Joana A.D., Díaz Baños, F. Guillermo, Víllora Cano, Gloria, Veiga, Francisco, Ribeiro, Antonio, Carissimi, Guzmán, Química Física

Polyelectrolyte nanocomposites rarely reach a stable state and aggregation often occurs. Here, we report the synthesis of nanocomposites for the oral delivery of insulin composed of alginate, dextran sulfate, poly-(ethyleneglycol) 4000, Poloxamer 188®, chitosan, and BSA. Chitosan seems to be essential for the final size of the nanocomposites. When the chitosan concentration used in the synthesis is 10-4 gcm-3, nanocomposites, microcomposites and milimetric composites are obtained. Reducing the chitosan concentration to 10-5 or 10-7 gcm-3 yields nanocomposites of 400-600 nm or 800-900 nm hydrodynamic diameter, respectively, measured by DLS. According to our results, the molecular weight of chitosan has little influence on the size of the composites. The enhanced stability of the newest nanocomposites synthesized was assessed with LUMiSizer®. The rounded shapes of the nanocomposites were confirmed by scanning electron microscopy. Analysis by HPLC revealed that, after loaded with insulin, about 80% of the drug was released from our nanocomposites.

2023-03-14, Pessoa, Bruno , Sandri, Giuseppina , Ribeiro, António , Collado-González, Mar, Biología Celular e Histología

The design of nanoparticle formulations composed of biopolymers, that govern the physicochemical properties of orally delivered insulin, relies on improving insulin Stability and absorption through the intestinal mucosa while protecting it from harsh conditions in the gastrointestinal (GI) tract. Chitosan/polyethylene glycol (PEG) and albumin coating of alginate/dextran sulfate hydrogel cores are presented as a ultilayer complex protecting insulin within the nanoparticle. This study aims to optimize a anoparticle formulation by assessing the relationship between design parameters and experimental data using response surface methodology through a 3-factor 3-level optimization Box–Behnken design. While the selected independent variables were the concentrations of PEG, chitosan and albumin, the dependent variables were particle Size, polydispersity index (PDI), zeta potential, and insulin release. Experimental results showed a nanoparticle size ranging from 313 to 585 nm, with PDI from 0.17 to 0.39 and zeta potential ranging from 􀀀29 to 􀀀44 mV. Insulin bioactivity was maintained in simulated GI media with over 45% cumulative release after 180 min in a simulated intestinal medium. Based on the experimental responses and according to the criteria of desirability on the experimental region’s constraints, solutions of 0.03% PEG, 0.047% chitosan and 1.20% albumin provide an optimum nanoparticle formulation for insulin oral delivery.

2017-09-11, Tomás Catalá, Christopher J., Oñate-Sánchez, Ricardo E., Forner, Leopoldo , Llena, Carmen, Lozano, Adrián , Castelo-Baz, P., Moraleda, José M., Rodríguez-Lozano, Francisco J., Collado-González, Mar, García Bernal, David, Biología Celular e Histología

Aim To evaluate the biological effects in vitro of MTAAngelus (MTA-Ang; Angelus, Londrina, PR, Brazil), MTA Repair HP (MTA-HP; Angelus) and NeoMTA Plus (NeoMTA-P; Avalon Biomed Inc, Bradenton, FL, USA) on human dental pulp stem cells (hDPSCs). Methodology Cell viability and cell migration assays were performed using eluates of each material. To evaluate cell morphology and cell attachment to the different materials, hDPSCs were directly seeded onto the material surfaces and analysed by immunocytofluorescence and scanning electron microscopy, respectively. The chemical composition of the materials was determined by energy-dispersive X-ray (EDX), and eluates were analysed by inductively coupled plasma–mass spectrometry (ICP-MS). Statistical analysis was performed with the analysis of variance and Bonferroni or Tukey post-test (a < 0.05). Results Undiluted MTA-Ang, MTA-HP and NeoMTA-P displayed a significant increase in cell viability greater than that obtained using complete medium alone (control) (*P < 0.05; **P < 0.01; ***P < 0.001). Moreover, a cell migration assay revealed cell migration rates after incubation with extracts of MTA-Ang, MTA-HP and NeoMTA-P that were similar to levels obtained in the control group. In addition, stretched cytoskeletal F-actin fibres were detected in the cells treated with the three material extracts. SEM studies revealed a high degree of cell proliferation and attachment on all three materials. EDX analysis demonstrated similar weight percentages of C, O and Ca in all three materials, whilst other elements such as Al, Si and S were also found. Conclusions MTA-Ang, MTA-HP and NeoMTA-P were associated with biological effects on hDPSCs in terms of cell proliferation, morphology, migration and attachment.