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OBJECTIVES: To assess the biocompatibility, bioactivity, and immunomodulatory properties of three new calcium silicate cement-based sealers: Ceraseal (CS), Totalfill BC Sealer (TFbc) and WellRoot ST (WR-ST) on human periodontal ligament stem cells (hPDLSCs). MATERIALS AND METHODS: HPDLSCs were isolated from extracted third molars from healthy patients. Eluates (1:1, 1:2, and 1:4 ratio) and sample discs of CS, TFbc and WR-ST after setting were prepared. A series of assays were performed: cell characterization, cell metabolic activity (MTT assay) cell attachment and morphology (SEM assay), cell migration (wound-healing assay), cytoskeleton organization (phaloidin-based assay); IL-6 and IL-8 release (ELISA); differentiation marker expression (RT-qPCR assay), and cell mineralization (Alizarin Red S staining). HPDLSCs cultured in unconditioned (negative control) or osteogenic (positive control) culture media were used as a comparison. Statistical significance was established at p < 0.05. RESULTS: All the tested sealers exhibited similar results in the cytocompatibility assays (cell metabolic activity, migration, attachment, morphology, and cytoskeleton organization) compared with a negative control group. CS and TFbc exhibited an upregulation of at least one osteo/cementogenic marker compared to the negative and positive control groups. CS and TFbc also showed a significantly higher calcified nodule formation than the negative and positive control groups. Both the marker expression and calcified nodule formation were significantly higher in CS-treated cells than TFbc treated cells. WR-ST exhibited similar results to the control group. CS and TFbc-treated cells exhibited a significant downregulation of IL-6 after 72 h of culture compared to the negative control group (p < 0.05). CONCLUSION: All the tested sealers exhibited an adequate cytocompatibility. CS significantly enhances cell differentiation by upregulating the expression of key genes associated with bone and cementum formation. Additionally, CS was observed to facilitate the mineralization of the extracellular matrix effectively. In contrast, the effects of TFbc and WR-ST on these processes were less pronounced compared to CS. Furthermore, both CS and TFbc exhibited an anti-inflammatory potential, contributing to their potential therapeutic benefits in regenerative endodontics. CLINICAL RELEVANCE: This is the first study to compare the biological properties and immunomodulatory potential of Ceraseal, Totalfill BC Sealer, and WellRoot ST. The results act as supporting evidence for their use in root canal treatment.
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Materiales Biocompatibles , Compuestos de Calcio , Ensayo de Materiales , Ligamento Periodontal , Silicatos , Compuestos de Calcio/farmacología , Silicatos/farmacología , Humanos , Ligamento Periodontal/citología , Ligamento Periodontal/efectos de los fármacos , Materiales Biocompatibles/farmacología , Técnicas In Vitro , Células Cultivadas , Células Madre/efectos de los fármacos , Materiales de Obturación del Conducto Radicular/farmacología , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Adhesión Celular/efectos de los fármacos , Tercer MolarRESUMEN
Mesenchymal stem/stromal cells (MSCs) are one of the most widely used cell types in advanced therapies due to their therapeutic potential in the regulation of tissue repair and homeostasis, and immune modulation. However, their use in cancer therapy is controversial: they can inhibit cancer cell proliferation, but also potentially promote tumour growth by supporting angiogenesis, modulation of the immune milieu and increasing cancer stem cell invasiveness. This opposite behaviour highlights the need for careful and nuanced use of MSCs in cancer treatment. To optimize their anti-cancer effects, diverse strategies have bioengineered MSCs to enhance their tumour targeting and therapeutic properties or to deliver anti-cancer drugs. In this review, we highlight the advanced uses of MSCs in cancer therapy, particularly as carriers of targeted treatments due to their natural tumour-homing capabilities. We also discuss the potential of MSC-derived extracellular vesicles to improve the efficiency of drug or molecule delivery to cancer cells. Ongoing clinical trials are evaluating the therapeutic potential of these cells and setting the stage for future advances in MSC-based cancer treatment. It is critical to identify the broad and potent applications of bioengineered MSCs in solid tumour targeting and anti-cancer agent delivery to position them as effective therapeutics in the evolving field of cancer therapy.
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Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas , Neoplasias , Humanos , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/citología , Neoplasias/terapia , Neoplasias/patología , Neoplasias/tratamiento farmacológico , Trasplante de Células Madre Mesenquimatosas/métodos , Animales , Bioingeniería/métodos , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Vesículas Extracelulares/metabolismo , Sistemas de Liberación de MedicamentosRESUMEN
OBJECTIVE: Drug-loaded non-resorbable polymeric nanoparticles (NPs) are proposed as an adjunctive treatment for pulp regenerative strategies. The present in vitro investigation aimed to evaluate the effectiveness of tideglusib-doped nanoparticles (TDg-NPs) in mitigating the adverse effects of bacterial lipopolysaccharide endotoxin (LPS) on the viability, morphology, migration, differentiation and mineralization potential of human dental pulp stem cells (hDPSCs). METHODS: Cell viability, proliferation, and differentiation were assessed using a MTT assay, cell migration evaluation, cell cytoskeleton staining analysis, Alizarin Red S staining and expression of the odontogenic related genes by a real-time quantitative polymerase chain reaction (RT-qPCR) were also performed. Cells were tested both with and without stimulation with LPS at various time points. One-way ANOVA and Tukey's test were employed for statistical analysis (p < 0.05). RESULTS: Adequate cell viability was encountered in all groups and at every tested time point (24, 48, 72 and 168 h), without differences among the groups (p > 0.05). The analysis of cell cytoskeleton showed nuclear alteration in cultures with undoped NPs after LPS stimulation. These cells exhibited an in blue diffuse and multifocal appearance. Some nuclei looked fragmented and condensed. hDPSCs after LPS stimulation but in the presence of TDg-NPs exhibited less nuclei changes. LPS induced down-regulation of Alkaline phosphatase, Osteonectin and Collagen1 gene markers, after 21d. LPS half-reduced the cells production of calcium deposits in all groups (p < 0.05), except in the group with TDg-NPs (decrease about 10 %). SIGNIFICANCE: LPS induced lower mineral deposition and cytoskeletal disorganization in hDPSCs. These effects were counteracted by TDg-NPs, enhancing osteogenic differentiation and mineralization.
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INTRODUCTION: The aim of this study was to compare the biological effects of four chemical caries removal materials and to assess their cytotoxicity using human dental pulp stem cells (hDPSCs). METHODS: The products evaluated are: 1 - papain-based product (BRIX 3000®); 2 - papain/chloramine based products (NATURAL-CARE and Papacárie Duo®); and 3 - chloramine based product (Cariesolut). The following in vitro experiments were carried out: IC50 measurement, cell metabolic activity (MTT) assay, cell migration, immunofluorescence experiment, cell apoptosis analysis, and reactive oxygen species (ROS) production analysis. Statistical analyses were performed using one-way ANOVA followed by Tukey's post hoc test (p < 0.05). RESULTS: The IC50 values were: Brix 3000: 0.596%; Papacárie Duo: 0.052%; NATURAL CARE: 1.034%; and Cariesolut: 0.020%. The MTT assays showed non-adequate cell viability of all chemical-mechanical caries removal tested at 2% at 24, 48, and 72 h (p < 0.001). The same behaviour was observed at 0.1% in the Papacárie Duo and Cariesolut groups. In contrast, 0.1% of Brix 3000 at all times and NATURAL CARE at 24 h treated cells showed cell viability rates similar to the control group. At 0.01% only Brix 3000 did not show statistically significant differences at any time. Delayed cell migration was observed in all hDPSCs treated with Papacárie Duo and Cariesolut (p < 0.01 and p < 0.001). Phalloidin staining images showed a high confluence of cells in the presence of NATURAL CARE, similar to the control group. On the contrary, no cells were observed in Brix 3000 and Cariesolut at 2% and 0.1% concentrations. Papacárie Duo showed cells at all concentrations, but hDPSCs treated at 0.01% concentration exhibited better proliferation and spreading than those in the control group. Apoptosis essay showed that Brix 3000 at both 0.1% and 0.01% had a percentage of live cells higher than 99%, with 68.4% live cells at 2%, 3.69% early apoptotic cells, and 27.9% late apoptotic cells. Conversely, the rest of the materials showed an abundance of apoptotic cells, even at low concentrations. 0.1% and 0.01% of BRIX 3000 did not affect the ROS production levels, while 2% of BRIX 3000 counterpart very significantly increased the percentage of CM-H2DCFDA positive cells. Again, all concentrations of Cariesolut showed significantly higher levels of ROS production than those observed in control cells. CONCLUSION: Our results suggest that Brix 3000 would be the most suitable material for chemical caries removal, with Papacárie Duo and NATURAL CARE also being good options, and discourage the use of Cariesolut due to its low cytocompatibility on dental pulp stem cells.
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INTRODUCTION: BioRoot Flow (BRF) is a novel premixed bioceramic sealer indicated for endodontic treatments, but the biological and immunomodulatory effects of this endodontic sealer on human periodontal ligament stem cells (hPDLSCs) have not been elucidated. METHODS: To ascertain the biological impact of BRF, TotalFill BC Sealer (TFbc), and AH Plus (AHP) on human Periodontal Ligament Stem Cells (hPDLSCs), assessments were conducted to evaluate the cytocompatibility, cellular proliferation, migratory capacity, osteo/cementogenic differentiation potential, the ability to form mineralized nodules, and the immunomodulatory characteristics of hPDLSCs following treatment with these endodontic sealers. RESULTS: Biological assays showed adequate cell metabolic activity and cell migration in BRF, while SEM assay evidenced that TFbc and BRF groups demonstrated a superior cell adhesion process, including substrate adhesion, cytoskeleton development, and spreading on the niche-like structures of the cement as compared to the AHP group. TFbc and BRF-treated groups exhibited a significantly lower IL6 and IL8 production than AHP (*â¯pâ¯<.05). The bioceramic sealers stimulated heightened expression of BSP, CEMP-1, and CAP genes within a 7-14â¯day period. Notably, BRF and TFbc demonstrated a significant enhancement in the mineralization of hPDLSCs when compared to the negative control. Among these, cells treated with BRF showed a more substantial accumulation of calcium (***â¯pâ¯<â¯.001). CONCLUSIONS: Taken together, these findings indicate that BRF can potentially enhance cell differentiation by promoting the expression of essential genes related to bone and cement formation. In addition, BRF and TFbc displayed anti-inflammatory effects.
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Antiinflamatorios , Diferenciación Celular , Humanos , Diferenciación Celular/efectos de los fármacos , Antiinflamatorios/farmacología , Materiales de Obturación del Conducto Radicular/farmacología , Ligamento Periodontal/citología , Ligamento Periodontal/efectos de los fármacos , Ligamento Periodontal/metabolismo , Células Madre/efectos de los fármacos , Células Madre/metabolismo , Proliferación Celular/efectos de los fármacos , Cerámica/farmacología , Materiales Biocompatibles/farmacología , Materiales Biocompatibles/química , Movimiento Celular/efectos de los fármacos , Adhesión Celular/efectos de los fármacosRESUMEN
The aim of this study was to assess the influence of eucalyptol and menthol on the cell viability, migration, and reactive oxygen species production of human gingival fibroblasts (GFs) in vitro. Three different concentrations of eucalyptol and menthol were prepared following ISO 10993-5 guidelines (1, 5, and 10 mM). GFs were isolated from extracted teeth from healthy donors. The following parameters were assessed: cell viability via MTT, Annexin-V-FITC and 7-AAD staining, and IC50 assays; cell migration via horizontal scratch wound assay; and cell oxidative stress via reactive oxygen species assay. Data were analyzed using one-way ANOVA and Tukey's post hoc test. Statistical significance was established at p < 0.05. Eucalyptol and Menthol exhibited high cytotoxicity on gingival fibroblasts, as evidenced by cytotoxicity assays. Eucalyptol showed lower levels of cytotoxicity than menthol, compared to the control group. The cytotoxicity of the tested substances increased in a concentration-dependent manner. The same occurred in a time-dependent manner, although even 10 min of exposure to the tested substances showed a high cytotoxicity to the GFs. Commercially available products for oral application with these substances in their composition should be tested for cytotoxicity before their use.
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Mesenchymal stem/stromal cells (MSCs) are being increasingly used in cell-based therapies due to their broad anti-inflammatory and immunomodulatory properties. Intravascularly-administered MSCs do not efficiently migrate to sites of inflammation/immunopathology, but this shortfall has been overcome by cell surface enzymatic fucosylation to engender expression of the potent E-selectin ligand HCELL. In applications of cell-based therapies, cryopreservation enables stability in both storage and transport of the produced cells from the manufacturing facility to the point of care. However, it has been reported that cryopreservation and thawing dampens their immunomodulatory/anti-inflammatory activity even after a reactivation/reconditioning step. To address this issue, we employed a variety of methods to cryopreserve and thaw fucosylated human MSCs derived from either bone marrow or adipose tissue sources. We then evaluated their immunosuppressive properties, cell viability, morphology, proliferation kinetics, immunophenotype, senescence, and osteogenic and adipogenic differentiation. Our studies provide new insights into the immunobiology of cryopreserved and thawed MSCs and offer a readily applicable approach to optimize the use of fucosylated human allogeneic MSCs as immunomodulatory/anti-inflammatory therapeutics.
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Inmunomodulación , Células Madre Mesenquimatosas , Humanos , Glicosilación , Células Madre Mesenquimatosas/metabolismo , Criopreservación/métodos , Antiinflamatorios/metabolismoRESUMEN
To evaluate the effects of premixed calcium silicate based ceramic sealers on the viability and osteogenic/cementogenic differentiation of human periodontal ligament stem cells (hPDLSCs). The materials evaluated were TotalFill BC Sealer (TFbc), AH Plus Bioceramic Sealer (AHPbc), and Neosealer Flo (Neo). Standardized discs and 1:1, 1:2, and 1:4 eluates of the tested materials were prepared. The following in vitro experiments were carried out: ion release, cell metabolic activity 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, cell migration, immunofluorescence experiment, cell attachment, gene expression, and mineralization assay. Statistical analyses were performed using one-way ANOVA followed by Tukey's post hoc test (p < .05). Increased Ca2+ release was detected in TFbc compared to AHPbc and Neo (*p < .05). Biological assays showed a discrete cell metabolic activity and cell migration in Neo-treated cell, whereas scanning electronic microscopy assay exhibited that TFbc group had a better cell adhesion process of substrate attachment, spreading, and cytoskeleton development on the niche-like structures of the cement than AHPbc and Neo. The sealers tested were able to induce overexpression of the CEMP-1, ALP, and COL1A1 genes in the first days of exposure, particularly in the case of TFbc (***p < .001). All materials tested significantly increased the mineralization of hPDLSCs when compared to the negative control, although more pronounced calcium deposition was observed in the TFbc-treated cells (***p < .001). Our results suggested that TFbc promotes cell differentiation, both by increasing the expression of key osteo/odontogenic genes and by promoting mineralization of the extracellular matrix, whereas this phenomenon was less evident in Neo and AHPbc. RESEARCH HIGHLIGHTS: TFbc group had a better cell adhesion process of substrate attachment, spreading, and cytoskeleton development on the niche-like structures of the cement than AHPbc and Neo. The sealers tested were able to induce overexpression of the CEMP-1, ALP, and COL1A1 genes in the first days of exposure, particularly in the case of TFbc. All materials tested significantly increased the mineralization of hPDLSCs when compared to the negative control, although more pronounced calcium deposition was observed in the TFbc-treated cells.
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Compuestos de Calcio , Diferenciación Celular , Cerámica , Osteogénesis , Ligamento Periodontal , Silicatos , Células Madre , Humanos , Ligamento Periodontal/citología , Ligamento Periodontal/efectos de los fármacos , Compuestos de Calcio/farmacología , Compuestos de Calcio/química , Silicatos/farmacología , Silicatos/química , Diferenciación Celular/efectos de los fármacos , Cerámica/química , Células Madre/efectos de los fármacos , Células Madre/citología , Osteogénesis/efectos de los fármacos , Células Cultivadas , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cementogénesis/efectos de los fármacos , Microscopía Electrónica de RastreoRESUMEN
OBJECTIVES: To evaluate the cytocompatibility, bioactivity, and anti-inflammatory potential of the new pre-mixed calcium silicate cement-based sealers Bioroot Flow (BrF) and AH Plus Bioceramic Sealer (AHPbcs) on human periodontal ligament stem cells (hPDLSCs) compared to the epoxy resin-based sealer AH Plus (AHP). MATERIALS AND METHODS: Standardized discs and 1:1, 1:2, and 1:4 eluates of BrF, AHPbcs and AHP after setting were prepared. The following assays were performed: cell attachment and morphology via SEM, cell viability via a MTT assay, cell migration/proliferation via a wound-healing assay, cytoskeleton organization via immunofluorescence staining; cytokine release via ELISA; osteo/cemento/odontogenic marker expression via RT-qPCR, and cell mineralized nodule formation via Alizarin Red S staining. HPDLSCs were isolated from extracted third molars from healthy patients. Comparisons were made with hPDLSCs cultured in unconditioned (negative control) or osteogenic (positive control) culture media. Statistical significance was established at p < 0.05. RESULTS: Both BrF and AHPbcs showed significantly positive results in the cytocompatibility assays (cell metabolic activity, migration, attachment, morphology, and cytoskeleton organization) compared with a negative control group, while AHP showed significant negative results. BrF exhibited an upregulation of at least one osteo/cementogenic marker compared to the negative and positive control groups. BrF showed a significantly higher calcified nodule formation than AHPbcs, the negative and positive control groups, while AHPbcs was higher than the negative control group. Both were also significantly higher than AHP group. CONCLUSION: BrF and AHPbcs exhibit adequate and comparable cytocompatibility on hPDLSCs. BrF also promoted the osteo/cementogenic differentiation of hPDLSCs. Both calcium silicate-based sealers favored the downregulation of the inflammatory cytokine IL-6 and the calcified nodule formation from hPDLSCs. BrF exerted a significantly higher influence on cell mineralization than AHPbcs. CLINICAL RELEVANCE: This is the first study to elucidate the biological properties and immunomodulatory potential of Bioroot Flow and AH Plus Bioceramic Sealer. The results act as supporting evidence for their use in root canal treatment.
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Alopecia/congénito , Compuestos de Calcio , Resinas Epoxi , Ligamento Periodontal , Materiales de Obturación del Conducto Radicular , Silicatos , Humanos , CitocinasRESUMEN
Background: Neurological dysfunction and glial activation are common in severe infections such as sepsis. There is a sexual dimorphism in the response to systemic inflammation in both patients and animal models, but there are few comparative studies. Here, we investigate the effect of systemic inflammation induced by intraperitoneal administration of lipopolysaccharide (LPS) on the retina of male and female mice and determine whether antagonism of the NLRP3 inflammasome and the extrinsic pathway of apoptosis have protective effects on the retina. Methods: A single intraperitoneal injection of LPS (5 mg/kg) was administered to two months old C57BL/6J male and female mice. Retinas were examined longitudinally in vivo using electroretinography and spectral domain optical coherence tomography. Retinal ganglion cell (RGC) survival and microglial activation were analysed in flat-mounts. Retinal extracts were used for flow cytometric analysis of CD45 and CD11b positive cells. Matched plasma and retinal levels of proinflammatory cytokines were measured by ELISA. Retinal function and RGC survival were assessed in animals treated with P2X7R and TNFR1 antagonists alone or in combination. Results: In LPS-treated animals of both sexes, there was transient retinal dysfunction, loss of vision-forming but not non-vision forming RGCs, retinal swelling, microglial activation, cell infiltration, and increases in TNF and IL-1ß. Compared to females, males showed higher vision-forming RGC death, slower functional recovery, and overexpression of lymphotoxin alpha in their retinas. P2X7R and TNFR1 antagonism, alone or in combination, rescued vision-forming RGCs. P2X7R antagonism also rescued retinal function. Response to treatment was better in females than in males. Conclusions: Systemic LPS has neuronal and sex-specific adverse effects in the mouse retina, which are counteracted by targeting the NLRP3 inflammasome and the extrinsic pathway of apoptosis. Our results highlight the need to analyse males and females in preclinical studies of inflammatory diseases affecting the central nervous system.
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Inflamasomas , Proteína con Dominio Pirina 3 de la Familia NLR , Humanos , Ratones , Masculino , Femenino , Animales , Lactante , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Inflamasomas/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Lipopolisacáridos/farmacología , Ratones Endogámicos C57BL , Retina , Células Ganglionares de la Retina/metabolismo , Inflamación/metabolismoRESUMEN
BACKGROUND: The neuronal transdifferentiation of adult bone marrow cells (BMCs) is still considered an artifact based on an alternative explanation of experimental results supporting this phenomenon obtained over decades. However, recent studies have shown that following neural induction, BMCs enter an intermediate cellular state before adopting neural-like morphologies by active neurite extension and that binucleated BMCs can be formed independent of any cell fusion events. These findings provide evidence to reject the idea that BMC neural transdifferentiation is merely an experimental artifact. Therefore, understanding the intermediate states that cells pass through during transdifferentiation is crucial given their potential application in regenerative medicine and disease modelling. METHODS: In this study, we examined the functional significance of the variety of morphologies and positioning that cell nuclei of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) can adopt during neural-like differentiation using live-cell nuclear fluorescence labelling, time-lapse microscopy, and confocal microscopy analysis. RESULTS: Here, we showed that after neural induction, hBM-MSCs enter an intermediate cellular state in which the nuclei are able to move within the cells, switching shapes and positioning and even generating cellular protrusions as they attempt to contact the cells around them. These findings suggest that changes in nuclear positioning occur because human cell nuclei somehow sense their environment. In addition, we showed the process of direct interactions between cell nuclei, which opens the possibility of a new level of intercellular interaction. CONCLUSIONS: The present study advances the understanding of the intermediate stage through which hBM-MSCs pass during neural transdifferentiation, which may be crucial to understanding the mechanisms of these cell conversion processes and eventually harness them for use in regenerative medicine. Importantly, our study provides for the first time evidence that the nuclei of hBM-MSC-derived intermediate cells somehow sense their environment, generating cellular protrusions to contact other cells. In summary, human mesenchymal stromal cells could not only help to increase our understanding of the mechanisms underlying cellular plasticity but also facilitate the exact significance of nuclear positioning in cellular function and in tissue physiology.
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Células Madre Adultas , Neuronas , Adulto , Humanos , Diferenciación Celular/fisiología , Extensiones de la Superficie Celular , Núcleo Celular , Células de la Médula Ósea , Células CultivadasRESUMEN
OBJECTIVES: Silver fluoride (SF) is a preventive and therapeutic option for dental pathological processes involving structural alterations of the hard tissues, either during their formation or those caused by caries or other pathological reasons. This study aimed to compare the biological properties of two commercial SF products, one of them with ammonium (Riva Star; SDF) and the other ammonium-free (Riva Star Aqua; AgF), both with or without potassium iodide (KI), by the assessment of the cytotoxicity of the materials' eluates. METHODS: Human dental pulp stem cells (hDPSCs) were obtained from healthy 18-23-year-old donors. Three dilutions were prepared for the tested materials (0.005%, 0.0005%, and 0.0001%). The following groups were assessed: (AgF, AgF+KI, SDF, SDF+KI, KI, negative control). A series of cytocompatibility assays were performed: MTT assay, IC50 assay, wound healing (migration) assay, cell cytoskeleton staining, analysis of cell apoptosis and necrosis, and reactive oxygen species production. The normality in the distribution of the data was previously confirmed via a Q-Q plot. Statistical significance was tested using one way ANOVA and Tukey's post hoc test. RESULTS: The incorporation of KI improved the cytocompatibility of both SF products in terms of viability, migration, morphology, apoptosis, and reactive oxygen species production. This difference was higher in the AgF group. The lowest dilutions of SF+KI and AgF+KI showed a similar cytocompatibility to that of the control group (MTT assay (p > 0.05 after 24, 48, and 72 h of culture); migration assay (p > 0.05 after 24, 48, and 72 h of culture); reactive oxygen species production (p > 0.05 after 72 h of culture). SIGNIFICANCE: Riva Star Aqua shows lower cytotoxicity than Riva Star on hDPSCs. It can be considered as a good alternative in the conservative treatment of dental caries and in the preservation and remineralisation of viable dentine tissue.
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Caries Dental , Fluoruros , Compuestos de Plata , Humanos , Adolescente , Adulto Joven , Adulto , Caries Dental/tratamiento farmacológico , Pulpa Dental , Especies Reactivas de Oxígeno , Dentina , Yoduro de Potasio/uso terapéutico , Células MadreRESUMEN
OBJECTIVES: The current in vitro study aims to evaluate silk fibroin with and without the addition of graphene as a potential scaffold material for regenerative endodontics. MATERIAL AND METHODS: Silk fibroin (SF), Silk fibroin/graphene oxide (SF/GO) and silk fibroin coated with reduced graphene oxide (SF/rGO) scaffolds were prepared (n = 30). The microarchitectures and mechanical properties of scaffolds were evaluated using field emission scanning electron microscopy (FESEM), pore size and water uptake, attenuated total reflectance fourier transformed infrared spectroscopy (ATR-FTIR), Raman spectroscopy and mechanical compression tests. Next, the study analyzed the influence of these scaffolds on human dental pulp stem cell (hDPSC) viability, apoptosis or necrosis, cell adhesion, odontogenic differentiation marker expression and mineralized matrix deposition. The data were analyzed with ANOVA complemented with the Tukey post-hoc test (p < 0.005). RESULTS: SEM analysis revealed abundant pores with a size greater than 50 nm on the surface of tested scaffolds, primarily between 50 nm and 600 µm. The average value of water uptake obtained in pure fibroin scaffolds was statistically higher than that of those containing GO or rGO (p < 0.05). ATR-FTIR evidenced that the secondary structures did not present differences between pure fibroin and fibroin coated with graphene oxide, with a similar infrared spectrum in all tested scaffolds. Raman spectroscopy showed a greater number of defects in the links in SF/rGO scaffolds due to the reduction of graphene. In addition, adequate mechanical properties were exhibited by the tested scaffolds. Regarding biological properties, hDPSCs attached to scaffolds were capable of proliferating at a rate similar to the control, without affecting their viability over time. A significant upregulation of ALP, ON and DSPP markers was observed with SF/rGO and SF/GO groups. Finally, SF/GO and SF/rGO promoted a significantly higher mineralization than the control at 21 days. SIGNIFICANCE: Data obtained suggested that SF/GO and SF/rGO scaffolds promote hDPSC differentiation at a genetic level, increasing the expression of key osteo/odontogenic markers, and supports the mineralization of the extracellular matrix. However, results from this study are to be interpreted with caution, requiring further in vivo studies to confirm the potential of these scaffolds.
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Fibroínas , Grafito , Humanos , Fibroínas/química , Andamios del Tejido/química , Ingeniería de Tejidos/métodos , Grafito/química , Pulpa Dental , Diferenciación Celular , Agua , Proliferación Celular , Células MadreRESUMEN
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 end-ischemic 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.
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The aim of this study was to assess the influence of eucalyptol, chloroform, and Endosolv on the proliferative capability, cell viability, and migration rates of human periodontal ligament stem cells (hPDLSCs) in vitro. Solvent eluates were formulated following ISO 10993-5 guidelines, and 1%, 0.25%, and 0.1% dilutions were prepared. The HPDLSCs were isolated from the extracted third molars of healthy donors. The following parameters were assessed: cell viability via trypan blue and IC50 assays, cell migration via horizontal wound healing assay, cell morphology via cell cytoskeleton staining (phalloidin labeling), and cell oxidative stress via reactive oxygen species assay. The data were analyzed using one-way ANOVA and Tukey's posthoc tests, and their significance was established at p < 0.05. Chloroform and eucalyptol exhibited significantly higher cytotoxicity on the hPDLSCs in vitro compared to the control group, as shown by the cell viability, migration, morphology, and reactive oxygen species release assays. Alternatively, Endosolv showed adequate cytotoxicity levels comparable to those of the control group. The cytotoxicity of the tested endodontic solvents increased in a dose-dependent manner. The results from the present study highlight the cytotoxicity of chloroform and eucalyptol. Thus, their limited and cautious use is recommended, avoiding solvent extrusion.
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Although it has been reported that bone marrow-derived cells (BMDCs) can transdifferentiate into neural cells, the findings are considered unlikely. It has been argued that the rapid neural transdifferentiation of BMDCs reported in culture studies is actually due to cytotoxic changes induced by the media. While transplantation studies indicated that BMDCs can form new neurons, it remains unclear whether the underlying mechanism is transdifferentiation or BMDCs-derived cell fusion with the existing neuronal cells. Cell fusion has been put forward to explain the presence of gene-marked binucleated neurons after gene-marked BMDCs transplantation. In the present study, we demostrated that human BMDCs can rapidly adopt a neural-like morphology through active neurite extension and binucleated human BMDCs can form with independence of any cell fusion events. We also showed that BMDCs neural-like differentiation involves the formation of intermediate cells which can then redifferentiate into neural-like cells, redifferentiate back to the mesenchymal fate or even repeatedly switch lineages without cell division. Furthermore, we have discovered that nuclei from intermediate cells rapidly move within the cell, adopting different morphologies and even forming binucleated cells. Therefore, our results provide a stronger basis for rejecting the idea that BMDCs neural transdifferentiation is merely an artefact.
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Médula Ósea , Comunicación Celular , Humanos , Fusión Celular , Diferenciación Celular , NeuronasRESUMEN
Mesenchymal stem/stromal cells (MSCs) are distributed within all tissues of the body. Though best known for generating connective tissue and bone, these cells also display immunoregulatory properties. A greater understanding of MSC cell biology is urgently needed because culture-expanded MSCs are increasingly being used in treatment of inflammatory conditions, especially life-threatening immune diseases. While studies in vitro provide abundant evidence of their immunomodulatory capacity, it is unknown whether tissue colonization of MSCs is critical to their ability to dampen/counteract evolving immunopathology in vivo. To address this question, we employed a murine model of fulminant immune-mediated inflammation, acute graft-versus-host disease (aGvHD), provoked by donor splenocyte-enriched full MHC-mismatched hematopoietic stem cell transplant. aGvHD induced the expression of E-selectin within lesional endothelial beds, and tissue-specific recruitment of systemically administered host-derived MSCs was achieved by enforced expression of HCELL, a CD44 glycoform that is a potent E-selectin ligand. Compared to mice receiving HCELL- MSCs, recipients of HCELL+ MSCs had increased MSC intercalation within aGvHD-affected site(s), decreased leukocyte infiltrates, lower systemic inflammatory cytokine levels, superior tissue preservation, and markedly improved survival. Mechanistic studies reveal that ligation of HCELL/CD44 on the MSC surface markedly potentiates MSC immunomodulatory activity by inducing MSC secretion of a variety of potent immunoregulatory molecules, including IL-10. These findings indicate that MSCs counteract immunopathology in situ, and highlight a role for CD44 engagement in unleashing MSC immunobiologic properties that maintain/establish tissue immunohomeostasis.
RESUMEN
Non-resorbable polymeric nanoparticles (NPs) are proposed as an adjunctive treatment for bone regenerative strategies. The present in vitro investigation aimed to evaluate the effect of the different prototypes of bioactive NPs loaded with zinc (Zn-NPs), doxycycline (Dox-NPs) or dexamethasone (Dex-NPs) on the viability, morphology, migration, adhesion, osteoblastic differentiation, and mineralization potential of human bone marrow stem cells (hBMMSCs). Cell viability, proliferation, and differentiation were assessed using a resaruzin-based assay, cell cycle analysis, cell migration evaluation, cell cytoskeleton staining analysis, Alizarin Red S staining, and expression of the osteogenic-related genes by a real-time quantitative polymerase chain reaction (RT-qPCR). One-Way ANOVA and Tukey's test were employed. The resazurin assay showed adequate cell viability considering all concentrations and types of NPs at 24, 48, and 72 h of culture. The cell cycle analysis revealed a regular cell cycle profile at 0.1, 1, and 10 µg/mL, whereas 100 µg/mL produced an arrest of cells in the S phase. Cells cultured with 0.1 and 1 µg/mL NP concentrations showed a similar migration capacity to the untreated group. After 21 days, mineralization was increased by all the NPs prototypes. Dox-NPs and Dex-NPs produced a generalized up-regulation of the osteogenic-related genes. Dex-NPs and Dox-NPs exhibited excellent osteogenic potential and promoted hBMMSC differentiation. Future investigations, both in vitro and in vivo, are required to confirm the suitability of these NPs for their clinical application.
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BACKGROUND: Advanced therapies using adult mesenchymal stromal cells (MSCs) for neurodegenerative diseases are not effectively translated into the clinic. The cross talk between the transplanted cells and the host tissue is something that, despite its importance, is not being systematically investigated. METHODS: We have compared the response of the mouse healthy retina to the intravitreal transplantation of MSCs derived from the bone marrow in four modalities: syngeneic, allogeneic, xenogeneic and allogeneic with immunosuppression using functional analysis in vivo and histology, cytometry and protein measurement post-mortem. Data were considered significant (p < 0.05) after nonparametric suitable statistical tests. RESULTS: Transplanted cells remain in the vitreous and are cleared by microglial cells a process that is quicker in allotransplants regardless of immunosuppression. All transplants cause anatomical remodelling which is more severe after xenotransplants. Xeno- and allotransplants with or without immunosuppression cause macro- and microglial activation and retinal functional impairment, being xenotransplants the most detrimental and the only ones that recruit CD45+Iba1-cells. The profile of proinflammatory cytokines changes in all transplantation settings. However, none of these changes affect the retinal ganglion cell population. CONCLUSIONS: We show here a specific functional and anatomical retinal response depending on the MSC transplantation modality, an aspect that should be taken into consideration when conducting preclinical studies if we intend a more realistic translation into clinical practice.
Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas , Animales , Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/metabolismo , Ratones , Retina/patología , Células Ganglionares de la Retina/patologíaRESUMEN
PURPOSE: To study and compare effects of syngeneic bone marrow mononuclear stem cells (BM-MNCs) transplants on inherited retinal degeneration in two animal models with different etiologies: the RCS and the P23H-1 rats. To compare the safety and efficacy of two methods of intraocular delivery: subretinal and/or intravitreal. METHODS: A suspension of BM-MNCs was injected subretinally or intravitreally in the left eyes of P23H-1 and RCS rats at post-natal day (P) 21. At different survival intervals after the injection: 7, 15, 30 or 60 days, the retinas were cross-sectioned, and photoreceptor survival and glial cell responses were investigated using immunodetection of cones (anti-cone arrestin), synaptic connections (anti-bassoon), microglia (anti-Iba-1), astrocytes and Müller cells (anti-GFAP). Electroretinographic function was also assessed longitudinally. RESULTS: Intravitreal injections (IVIs) or subretinal injections (SRIs) of BM-MNCs did not produce adverse effects. The transplanted cells survived for up to 15 days but did not penetrate the retina. Both IVIs and SRIs increased photoreceptor survival, decreased synaptic degeneration and glial fibrillary acidic protein (GFAP) expression in Müller cells but did not modify microglial cell activation and migration or the electroretinographic responses. CONCLUSIONS: Intravitreal and subretinal syngeneic BM-MNCs transplantation decreases photoreceptor degeneration and shows anti-gliotic effects on Müller cells but does not ameliorate retinal function. Moreover, syngeneic BM-MNCs transplants are more effective than the xenotransplants of these cells. BM-MNC transplantation has potential therapeutic effects that merit further investigation.