García Bernal, David

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García Bernal, David

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García Bernal, David

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Universidad de Murcia. Departamento de Bioquímica y Biología Molecular"B" e Inmunología

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Open Access
Biocompatibility of three new calcium silicate-based endodontic sealers on human periodontal ligament stem cells
(Willey, 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.
Open Access
Chaperone-mediated autophagy sustains pericyte stemness necessary for brain tissue homeostasis
(Elsevier, 2025-04-24) Martínez, Carlos M.; Martínez-Morga, Marta; Rodríguez-Madoz, Juan R.; Prósper, Felipe; Zapata, Agustín G.; Moraleda, José María; Martínez, Salvador; García Bernal, David; Roca Soler, Francisco José; Salinas Hidalgo, María Dolores; Valdor Alonso, Rut; Bioquímica y Biología Molecular B e Inmunología
Introduction: Pericytes (PCs) are mural cells exhibiting some mesenchymal stem cell (MSC) properties and contribute to tissue regeneration after injury. We have previously shown that glioblastoma cancer cells induce in PCs, a pathogenic upregulation of chaperone-mediated autophagy (CMA) which modulates immune functions and MSC-like properties to support tumor growth. Objectives: The aim of the study was to interrogate the role of CMA-regulated MSC properties in PCs in the context of tissue repair during inflammation triggered by a demyelinating injury. Methods: Studies of RNA-seq were done PCs with (WT) and without (LAMP-2A KO) CMA. Cell characterization related to stemness, lineage and morphology was done in WT and KO PCs. Secretome analysis and cell differentiation assay using the supernatants from CMA-efficient and deficient PCs cultures was done in mesenchymal cells. Inflammatory response of brain cells was assessed with WT and KO PCs secretome. To corroborate in vitro results, CMA modulation in response to inflammation in PCs and tissue repair markers were measured in the lesion areas of a demyelination mouse model and correlated with the tissue reparation after intravenous PC administration. An inflammatory mediator was used to study effects on PC-CMA activity. Results We found that inflammatory mediators such as IFNγ downregulate CMA in PCs, suppressing PC stemness and promoting a pro-inflammatory secretome. Restoration of PC CMA activity during inflammation maintains PC MSC properties and induces an MSC-like proteome which decreases inflammation and promotes tissue repair. We identified secreted proteins involved in regenerative and protective processes, and therefore, necessary to restore brain tissue homeostasis after inflammation induced by a demyelinating injury. Conclusion: we show that manipulation of CMA activity in host PCs could be a useful therapeutical approach in the context of brain inflammation, which might be extended to other diseases where the pericyte has a key role in response to inflammation.
Embargo
GuttaFlow Bioseal promotes spontaneous differentiation of human periodontal ligament stem cells into cementoblast-like cells.
Rodríguez Lozano, Francisco Javier; Tomás-Catalá, C.J.; López, S.; Moraleda, J.M.; Murcia, L.; Collado-González, Mar; García Bernal, David; 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.
Open Access
Comparative analysis of the biological effects of the endodontic bioactive cements MTA Angelus, MTA Repair HP, and NeoMTA Plus on human dental pulp stem cells
(Willey, 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.
Open Access
Endothelial cell activation mediated by cold ischemia-released mitochondria is partially inhibited by defibrotide and impacts on early allograft function following liver transplantation
(Elsevier, 2023-09-18) Villalba-Lopez, Francisco; Mateo, Sandra V.; Vidal-Correoso, Daniel; Jover-Aguilar, Marta; Martínez-Alarcón, Laura; López-López, Víctor; Pons Miñano, José Antonio; Baroja-Mazo, Alberto; García Bernal, David; Pelegrín Vivancos, Pablo; Alconchel Gago, Felipe; Cascales Campos, Pedro Antonio; Ramírez, Pablo (Ramírez Romero); Rios Zambudio, Antonio; Medicina
DAMPs (danger-associated molecular patterns) are self-molecules of the organism that appear after damage. The endothelium plays several roles in organ rejection, such as presenting alloantigens to T cells and contributing to the development of inflammation and thrombosis. This study aimed to assess whether DAMPs present in the organ preservation solution (OPS) after cold ischemic storage (CIS) contribute to exacerbating the endothelial response to an inflammatory challenge and whether defibrotide treatment could counteract this effect. The activation of cultured human umbilical vein endothelial cells (HUVECs) was analyzed after challenging with endischemic OPS (eiOPS) obtained after CIS. Additionally, transwell assays were performed to study the ability of eiOPS to attract lymphocytes across the endothelium. The study revealed that eiOPS upregulated the expression of MCP-1 and IL-6 in HUVECs. Moreover, eiOPS increased the membrane expression of ICAM-1and HLA-DR, which facilitated leukocyte migration toward a chemokine gradient. Furthermore, eiOPS demonstrated its chemoattractant ability. This activation was mediated by free mitochondria. Defibrotide was found to partially inhibit the eiOPS-mediated activation. Moreover, the eiOPS-mediated activation of endothelial cells (ECs) correlated with early allograft dysfunction in liver transplant patients. Our finding provide support for the hypothesis that mitochondria released during cold ischemia could trigger EC activation, leading to complications in graft outcomes. Therefore, the analysis and quantification of free mitochondria in the eiOPS samples obtained after CIS could provide a predictive value for monitoring the progression of transplantation. Moreover, defibrotide emerges as a promising therapeutic agent to mitigate the damage induced by ischemia in donated organs.
Open Access
Are denture adhesives safe for oral cells?
(Wiley, 2020-07-12) López García, Sergio; Pecci Lloret, María Pilar; Guerrero Gironés, Julia; Pecci Lloret, Miguel Ramón; Rodríguez Lozano, Francisco Javier; García Bernal, David; Dermatología, Estomatología, Radiología y Medicina Física
Purpose: To compare the cytotoxicity of six commercially available denture adhesives on human gingival cells: Poligrip Flavour Free Fixative Cream, Fixodent Pro Duo Protection, Novafix cream, FittyDent, Polident Total Action, and Fixodent ProPlus Duo Protection. Material and Methods: Eluates of denture adhesives were brought into contact with human gingival cells and compared to untreated cells (w/o any dental adhesive elute). Cell toxicity was assessed by measuring cell viability (3-(4,5-imethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assays), cell morphology (immunofluorescenceassays), induction of apoptosis/necrosis and production of reactive oxygen species (ROS) (flow cytometry assays). In addition, the pH of each sample was determined. Data were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons test. Results: All denture adhesives tested led to a reduction in pH, especially Fixodent Pro Duo Protection and Fixodent Pro Plus Duo Protection. The cell viability assays showed that Fixodent Pro Duo Protection (1:1 72 hours, p = 3.04 × 10−6; 1:2 72 hours, p = 2.07 × 10−6; 1:4 72 hours, p = 2.04 × 10−6) and Fixodent Pro Plus Duo Protection (1:1 72 hours, p = 2.01 × 10−6; 1:2 72 hours, p = 3.03 × 10−6; 1:4 72 hours, p = 2.02 × 10−6) significantly decreased cell viability at all dilutions. Compared to the control group and the rest of the adhesives, Poligrip Flavour Free Fixative Cream (PFF 1:1 72 hours, p = 2.24 × 10−6; 1:2 72 hours, p = 2.44 × 10−6; 1:4 72 hours, p = 2.04 × 10−6) showed a significantly higher cell viability score at all dilutions. Fixodent Pro Duo Protection and Fixodent Pro Plus Duo Protection, both adhesives containing zinc salts in their composition, were responsible for necrosis, and the number of cells was much reduced, with aberrant morphology and pyknotic nucleus. Finally, Fixodent (1:2, p = 2.04 × 10−6, 1:4, p = 0.00036; 1:2, p = 8.82 × 10−6, 1:4, p = 2.30 × 10−6) products significantly promoted ROS production in gingival cells. Conclusions: The results suggest that denture adhesives containing zinc in their composition could be responsible of the decrease of cell viability, ROS production, aberrant cell morphology, and induction of apoptosis and cell death. However, other possible additional cytotoxic factors must be considered. Thus, more studies are necessary to confirm this hypothesis.
Open Access
Pericytes for use as a medicament
(2023-06-28) Valdor Alonso, Rut; García Bernal, David; Moraleda Jiménez, José María; Martínez Pérez, Salvador; Bioquímica y Biología Molecular B e Inmunología
The present invention refers to a substantially pure population of pericytes, or cell suspension comprising pericytes, wherein the pericytes are at least 80% of the total cell population or suspension, characterized in that CMA is inhibited, for example by inhibiting the expression or deleting the gene LAMP2A. In a preferred embodiment, said population or suspension of pericytes is used as a medicament, for instance in the treatment of cancer, preferably glioblastoma
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.
Open Access
Chaperone-mediated autophagy ablation in pericytes reveals new glioblastoma prognostic markers and efficient treatment against tumor progression
(2022-03-18) Molina Gallego, María Luisa; Aparicio, Pedro; Moraleda, José M.; Martínez, Salvador; García Bernal, David; Rubio Pedraza, Gonzalo; Salinas Hidalgo, María Dolores; Valdor Alonso, Rut; Bioquímica y Biología Molecular B e Inmunología
Background: The lack of knowledge of the progression mechanisms of glioblastoma (GB), the most aggressive brain tumor, contributes to the absence of successful therapeutic strategies. Our team has recently demonstrated a crucial new role for chaperone-mediated autophagy (CMA) in pericytes (PC)-acquired immunosuppressive function, which prevents anti-tumor immune responses and facilitates GB progression. The possible impact that GB-induced CMA in PC has on other functions that might be useful for future GB prognosis/treatment, has not been explored yet. Thus, we proposed to analyze the contribution of CMA to other GB-induced changes in PC biology and determine if CMA ablation in PC is a key target mechanism for GB treatment. Methods: Studies of RNA-seq and secretome analysis were done in GB-conditioned PC with and without CMA (from knockout mice for LAMP-2A) and compared to control PC. Different therapeutic strategies in a GB mouse model were compared. Results: We found several gene expression pathways enriched in LAMP2A-KO PC and affected by GB-induced CMA in PC that correlate with our previous findings. Phagosome formation, cellular senescence, focal adhesion and the effector function to promote anti-tumor immune responses were the most affected pathways, revealing a transcriptomic profiling of specific target functions useful for future therapies. In addition, several molecules associated with tumor mechanisms and related to tumor immune responses such as gelsolin, periostin, osteopontin, lumican and vitamin D, were identified in the PC secretome dependent on GB-induced CMA. The CMA ablation in PC with GB cells showed an expected immunogenic phenotype able to phagocyte GB cells and a key strategy to develop future therapeutic strategies against GB tumor progression. A novel intravenous therapy using exofucosylated CMA-deficient PC was efficient to make PC reach the tumor niche and facilitate tumor elimination. Conclusion: Our results corroborate previous findings on the impaired immunogenic function of PC with GB-induced CMA, driving to other altered PC functions and the identifications of new target markers related to the tumor immune responses and useful for GB prognosis/therapy. Our work demonstrates CMA ablation in PC as a key target mechanism to develop a successful therapy against GB progression.
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.