Comparative bioactivity and immunomodulatory potential of the new Bioroot Flow and AH Plus Bioceramic sealer: An in vitro study on hPDLSCs.

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.

Glioblastoma ablates pericytes antitumor immune function through aberrant up-regulation of chaperone-mediated autophagy.

The contractile perivascular cells, pericytes (PC), are hijacked by glioblastoma (GB) to facilitate tumor progression. PC's protumorigenic function requires direct interaction with tumor cells and contributes to the establishment of immunotolerance to tumor growth. Cancer cells up-regulate their own chaperone-mediated autophagy (CMA), a process that delivers selective cytosolic proteins to lysosomes for degradation, with pro-oncogenic effects. However, the possible impact that cancer cells may have on CMA of surrounding host cells has not been explored. We analyzed the contribution of CMA to the GB-induced changes in PC biology. We have found that CMA is markedly up-regulated in PC in response to the oxidative burst that follows PC-GB cell interaction. Genetic manipulations to block the GB-induced up-regulation of CMA in PC allows them to maintain their proinflammatory function and to support the induction of effective antitumor T cell responses required for GB clearance. GB-induced up-regulation of CMA activity in PC is essential for their effective interaction with GB cells that help tumor growth. We show that CMA inhibition in PC promotes GB cell death and the release of high immunogenic levels of granulocyte-macrophage colony stimulating factor (GM-CSF), through deregulation of the expression of cell-to-cell interaction proteins and protein secretion. A GB mouse model grafted in vivo with CMA-defective PC shows reduced GB proliferation and effective immune response compared to mice grafted with control PC. Our findings identify abnormal up-regulation of CMA as a mechanism by which GB cells elicit the immunosuppressive function of PC and stabilize GB-PC interactions necessary for tumor cell survival.

Are Denture Adhesives Safe for Oral Cells?

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 Pro Plus 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-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assays), cell morphology (immunofluorescence assays), 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.

Defibrotide inhibits donor leucocyte-endothelial interactions and protects against acute graft-versus-host disease.

Allogeneic hematopoietic stem cell transplantation (allo-HCT) is an effective therapy for the treatment of high-risk haematological malignant disorders and other life-threatening haematological and genetic diseases. Acute graft-versus-host disease (aGvHD) remains the most frequent cause of non-relapse mortality following allo-HCT and limits its extensive clinical application. Current pharmacologic agents used for prophylaxis and treatment of aGvHD are not uniformly successful and have serious secondary side effects. Therefore, more effective and safe prophylaxis and therapy for aGvHD are an unmet clinical need. Defibrotide is a multi-target drug successfully employed for prophylaxis and treatment of veno-occlusive disease/sinusoidal obstruction syndrome. Recent preliminary clinical data have suggested some efficacy of defibrotide in the prevention of aGvHD after allo-HCT. Using a fully MHC-mismatched murine model of allo-HCT, we report here that defibrotide, either in prophylaxis or treatment, is effective in preventing T cell and neutrophil infiltration and aGvHD-associated tissue injury, thus reducing aGvHD incidence and severity, with significantly improved survival after allo-HCT. Moreover, we performed in vitro mechanistic studies using human cells revealing that defibrotide inhibits leucocyte-endothelial interactions by down-regulating expression of key endothelial adhesion molecules involved in leucocyte trafficking. Together, these findings provide evidence that defibrotide may represent an effective and safe clinical alternative for both prophylaxis and treatment of aGvHD after allo-HCT, paving the way for new therapeutic approaches.

Bone Marrow-Derived Mononuclear Cell Transplants Decrease Retinal Gliosis in Two Animal Models of Inherited Photoreceptor Degeneration.

Inherited photoreceptor degenerations are not treatable diseases and a frequent cause of blindness in working ages. In this study we investigate the safety, integration and possible rescue effects of intravitreal and subretinal transplantation of adult human bone-marrow-derived mononuclear stem cells (hBM-MSCs) in two animal models of inherited photoreceptor degeneration, the P23H-1 and the Royal College of Surgeons (RCS) rat. Immunosuppression was started one day before the injection and continued through the study. The hBM-MSCs were injected in the left eyes and the animals were processed 7, 15, 30 or 60 days later. The retinas were cross-sectioned, and L- and S- cones, microglia, astrocytes and Müller cells were immunodetected. Transplantations had no local adverse effects and the CD45+ cells remained for up to 15 days forming clusters in the vitreous and/or a 2-3-cells-thick layer in the subretinal space after intravitreal or subretinal injections, respectively. We did not observe increased photoreceptor survival nor decreased microglial cell numbers in the injected left eyes. However, the injected eyes showed decreased GFAP immunoreactivity. We conclude that intravitreal or subretinal injection of hBM-MSCs in dystrophic P23H-1 and RCS rats causes a decrease in retinal gliosis but does not have photoreceptor neuroprotective effects, at least in the short term. However, this treatment may have a potential therapeutic effect that merits further investigation.

Biological effects of acid-eroded MTA Repair HP and ProRoot MTA on human periodontal ligament stem cells.

OBJECTIVE: The aim of this study was to analyze the biological effects of MTA Repair HP and ProRoot MTA on human periodontal ligament stem cells (hPDLSCs) after exposure to acidic and neutral environments. MATERIALS AND METHODS: Discs of each material (n = 30) were exposed to phosphate buffered saline (pH = 7.4) or butyric acid (pH = 5.2) for 7 days, and biological testing was carried out in vitro on hPDLSCs. Cell viability and apoptosis assays were performed using eluates of each root-end filling material. To evaluate cell attachment to the different materials, hPDLSCs were directly seeded onto the material surfaces and analyzed by scanning electron microscopy. The chemical composition of the root-end filling materials was determined by energy-dispersive x-ray and eluates were analyzed by inductively coupled plasma-mass spectrometry. Statistical differences were assessed by ANOVA and Tukey test (p < 0.05). RESULTS: Under an acidic environment, both materials displayed similar ion release abilities, with the increased release of Si and Ca ions. Substantial changes in microstructure were observed for both materials after exposure to acidic pH. In addition, material exposure to an acidic environment showed a similar degree of cell adherence, and, surprisingly, MTA Repair HP exhibited higher cell viability rates at pH 5.2 than ProRoot MTA. CONCLUSIONS: Exposure to an acidic environment promoted Si and Ca ion release from ProRoot MTA and MTA Repair HP. Moreover, we observed optimal biological properties of ProRoot MTA and MTA Repair HP in terms of cell viability, cell death, and cell attachment in both environments. CLINICAL RELEVANCE: These results may suggest that MTA Repair HP and ProRoot exhibited optimal biological properties in terms of cell viability, cell death and cell attachment in acidic environment, being considered as materials for root-end filling and perforations.

Production via good manufacturing practice of exofucosylated human mesenchymal stromal cells for clinical applications.

BACKGROUND: The regenerative and immunomodulatory properties of human mesenchymal stromal cells (hMSCs) have raised great hope for their use in cell therapy. However, when intravenously infused, hMSCs fail to reach sites of tissue injury. Fucose addition in α(1,3)-linkage to terminal sialyllactosamines on CD44 creates the molecule known as hematopoietic cell E-/L-selectin ligand (HCELL), programming hMSC binding to E-selectin that is expressed on microvascular endothelial cells of bone marrow (BM), skin and at all sites of inflammation. Here we describe how this modification on BM-derived hMSCs (BM-hMSCs) can be adapted to good manufacturing practice (GMP) standards. METHODS: BM-hMSCs were expanded using xenogenic-free media and exofucosylated using α(1,3)-fucosyltransferases VI (FTVI) or VII (FTVII). Enforced fucosylation converted CD44 into HCELL, and HCELL formation was assessed using Western blot, flow cytometry and cell-binding assays. Untreated (unfucosylated), buffer-treated and exofucosylated BM-hMSCs were each analyzed for cell viability, immunophenotype and differentiation potential, and E-selectin binding stability was assessed at room temperature, at 4°C, and after cryopreservation. Cell product safety was evaluated using microbiological testing, karyotype analysis, and c-Myc messenger RNA (mRNA) expression, and potential effects on genetic reprogramming and in cell signaling were analyzed using gene expression microarrays and receptor tyrosine kinase (RTK) phosphorylation arrays. RESULTS: Our protocol efficiently generates HCELL on clinical-scale batches of BM-hMSCs. Exofucosylation yields stable HCELL expression for 48 h at 4°C, with retained expression after cell cryopreservation. Cell viability and identity are unaffected by exofucosylation, without changes in gene expression or RTK phosphorylation. DISCUSSION: The described exofucosylation protocol using xenogenic-free reagents enforces HCELL expression on hMSCs endowing potent E-selectin binding without affecting cell viability or native phenotype. This described protocol is readily scalable for GMP-compliant clinical production.

Chemokine-induced Zap70 kinase-mediated dissociation of the Vav1-talin complex activates alpha4beta1 integrin for T cell adhesion.

Lymphocyte integrins mediate cell arrest on endothelium during immune surveillance after activation by chemokine-stimulated inside-out signals. Here we show that a Vav1-talin complex in T cells is a key target for chemokine-triggered inside-out signaling leading to integrin alpha4beta1 activation. Thus, Vav1 dissociation from talin was required to generate high-affinity alpha4beta1 conformations. Assembly of the Vav1-talin complex required PtdIns(4,5)P(2), which was provided by talin-bound phosphatidylinositol phosphate kinase Igamma. Chemokine-promoted Vav1 dissociation from talin followed an initial increase in talin binding to alpha4beta1. This process was dependent on ZAP-70, which binds to and phosphorylates Vav1 in the complex, leading to further alpha4beta1 activation and cell adhesion strengthening. Moreover, Vav1-talin dissociation was needed for Rac1 activation, thus indicating that alpha4beta1 and Rac1 activation can be coupled by chemokine-stimulated ZAP-70 function. Our data suggest that Vav1 might function as a repressive adaptor of talin that must dissociate from alpha4beta1-talin complexes for efficient integrin activation.

Analysis of the Adherence of Dental Pulp Stem Cells on Two-Dimensional and Three-Dimensional Silk Fibroin-Based Biomaterials.

Among various biomaterials used as scaffolds in tissue engineering, silk fibroin is a highly attractive material. A scaffold should be biocompatible and nontoxic, with optimal physical features and mechanical properties. For this reason, tissue-engineering approaches in regenerative medicine have focused on investigating the biocompatibility of possible biomaterials by analyzing cell-scaffold interaction properties. The aim of the present study was to examine the biocompatibility of silk fibroin as a film (two-dimensional [2D]) and a scaffold (three-dimensional [3D]) after being cellularized with human dental pulp stem cells (hDPSCs). Human dental pulp stem cells were isolated from healthy patients aged between 18 and 31 years. Further, silk fibroin-based 2D films and 3D scaffolds were prepared. Human dental pulp stem cells were directly seeded onto the biomaterial surfaces and their proliferation, adherence, and cell morphology were analyzed after 24, 120, and 168 hours. Additionally, the characteristics of the silk fibroin 2D films and 3D scaffolds before and after cell seeding were analyzed by scanning electron microscopy. After the initial 24 hours, silk fibroin-based 3D scaffolds displayed more adhered cells with a suitable fibroblastic morphology than those displayed on the 2D films. After longer culture times, hDPSCs proliferated sufficiently to cover the entire surface of the 3D silk fibroin scaffold, whereas the 2D films were only partially covered. Our results indicate the good in vitro biocompatibility of silk fibroin-based biomaterials, especially when 3D scaffolds rather than 2D films are used.

Sphingosine-1-phosphate activates chemokine-promoted myeloma cell adhesion and migration involving α4ß1 integrin function.

Myeloma cell adhesion dependent on α4ß1 integrin is crucial for the progression of multiple myeloma (MM). The α4ß1-dependent myeloma cell adhesion is up-regulated by the chemokine CXCL12, and pharmacological blockade of the CXCL12 receptor CXCR4 leads to defective myeloma cell homing to bone marrow (BM). Sphingosine-1-phosphate (S1P) regulates immune cell trafficking upon binding to G-protein-coupled receptors. Here we show that myeloma cells express S1P1, a receptor for S1P. We found that S1P up-regulated the α4ß1-mediated myeloma cell adhesion and transendothelial migration stimulated by CXCL12. S1P promoted generation of high-affinity α4ß1 that efficiently bound the α4ß1 ligand VCAM-1, a finding that was associated with S1P-triggered increase in talin-ß1 integrin association. Furthermore, S1P cooperated with CXCL12 for enhancement of α4ß1-dependent adhesion strengthening and spreading. CXCL12 and S1P activated the DOCK2-Rac1 pathway, which was required for stimulation of myeloma cell adhesion involving α4ß1. Moreover, in vivo analyses indicated that S1P contributes to optimizing the interactions of MM cells with the BM microvasculture and for their lodging inside the bone marrow. The regulation of α4ß1-dependent adhesion and migration of myeloma cells by CXCL12-S1P combined activities might have important consequences for myeloma disease progression.

The nuclei of human adult stem cells can move within the cell and generate cellular protrusions to contact other cells.

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.

Assessment of the anti-inflammatory and biological properties of Bioroot Flow: A novel bioceramic sealer.

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 (* p <.05). The bioceramic sealers stimulated heightened expression of BSP, CEMP-1, and CAP genes within a 7-14 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 (*** p < .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.

In vitro biocompatibility of ammonia-free silver fluoride products on human dental pulp stem cells.

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.

Premixed calcium silicate-based ceramic sealers promote osteogenic/cementogenic differentiation of human periodontal ligament stem cells: A microscopy study.

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.

Optimizing cryopreservation conditions for use of fucosylated human mesenchymal stromal cells in anti-inflammatory/immunomodulatory therapeutics.

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.

Retinal response to systemic inflammation differs between sexes and neurons.

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.

Comparative Cytotoxicity of Menthol and Eucalyptol: An In Vitro Study on Human Gingival Fibroblasts.

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.

3D Graphene/silk fibroin scaffolds enhance dental pulp stem cell osteo/odontogenic differentiation.

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.

RGS10 restricts upregulation by chemokines of T cell adhesion mediated by α4ß1 and αLß2 integrins.

Chemokines rapidly and transiently upregulate α4ß1 and αLß2 integrin-mediated adhesion during T lymphocyte extravasation by activating Gα-dependent inside-out signaling. To limit and terminate Gα-mediated signaling, cells can use several mechanisms, including the action of regulator of G protein signaling (RGS) proteins, which accelerate the GTPase activity of Gα subunits. Using human T cells silenced for or overexpressing RGS10, we show in this article that RGS10 functions as an inhibitor of Gα(i)-dependent, chemokine-upregulated T cell adhesion mediated by α4ß1 and αLß2. Shear stress-dependent detachment and cell spreading analyses revealed that RGS10 action mainly targets the adhesion strengthening and spreading phases of α4ß1-mediated cell attachment. Associated with these observations, chemokine-stimulated Vav1-Rac1 activation was longer sustained and of higher intensity in RGS10-silenced T cells, or inhibited in cells overexpressing RGS10. Of importance, expression of constitutively activated Rac1 forms in cells overexpressing RGS10 led to the rescue of CXCL12-stimulated adhesion to VCAM-1 to levels similar to those in control transfectants. Instead, adhesion under flow conditions, soluble binding experiment, flow cytometry, and biochemical analyses revealed that the earlier chemokine-triggered integrin activation step was mostly independent of RGS10 actions. The data strongly suggest that RGS10 opposes activation by chemokines of the Vav1-Rac1 pathway in T cells, leading to repression of adhesion strengthening mediated by α4ß1. In addition to control chemokine-upregulated T cell attachment, RGS10 also limited adhesion-independent cell chemotaxis and activation of cdc42. These results identify RGS10 as a key molecule that contributes to the termination of Gα-dependent signaling during chemokine-activated α4ß1- and αLß2-dependent T cell adhesion.

Endothelial cell activation mediated by cold ischemia-released mitochondria is partially inhibited by defibrotide and impacts on early allograft function following liver transplantation.

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.