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.

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.

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.

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.

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.

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.

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.

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.

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.

Are Endodontic Solvents Cytotoxic? An In Vitro Study on Human Periodontal Ligament Stem Cells.

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.

Binucleated human bone marrow-derived mesenchymal cells can be formed during neural-like differentiation with independence of any cell fusion events.

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.

Enforced mesenchymal stem cell tissue colonization counteracts immunopathology.

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.

Dexamethasone and Doxycycline Doped Nanoparticles Increase the Differentiation Potential of Human Bone Marrow Stem Cells.

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.

Immune recognition of syngeneic, allogeneic and xenogeneic stromal cell transplants in healthy retinas.

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.

Intravitreal and subretinal syngeneic bone marrow mononuclear stem cell transplantation improves photoreceptor survival but does not ameliorate retinal function in two rat models of retinal degeneration.

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.

In vitro biocompatibility testing of 3D printing and conventional resins for occlusal devices.

OBJECTIVE: To assess and compare the in vitro biocompatibility of new resins (Keysplint Soft (Keystone Industries), NextDent Ortho Rigid (3D System), and Freeprint Splint (Detax)) and traditional resins (Orthocryl (Dentaurum)) used for dental splints. METHODS: Standardized discs (n = 40) and 1:1, 1:2, and 1:4 extracts of the tested materials were prepared. Human gingival fibroblasts (hGFs) were isolated from gingival tissues. Different biological tests were carried out, including MTT assays to assess cell metabolic activity, cell migration assays, cell cytoskeleton staining, cell apoptosis, generation of intracellular reactive oxygen species (ROS), and scanning electron microscopy (SEM). Statistical analyses were performed using one-way ANOVA and Tukey's post hoc test (p<0.05). RESULTS: MTT experiments showed that Freeprint Splint significantly reduces the hGF metabolic activity (***p<0.001), whereas SEM analysis showed almost no cells adhered on its surface. Cell migration was significantly lower after exposure to undiluted extracts of Freeprint Splint at 48 and 72 h (***p<0.001). Cell cytoskeleton staining assays showed fewer attached cells in 1:1 and 1:2 dilutions of Freeprint Splint. Annexin-V and 7-AAD staining assays showed that only cells exposed to Keysplint Soft extracts displayed similar cell viability to the control group. Finally, ROS levels detected in undiluted extracts of all resins were significantly enhanced compared to the control group (***p<0.001). CONCLUSIONS: The 3D-printed resins and the conventional dental resin showed a similar biocompatibility, except for Freeprint Splint, which was the most cytotoxic on hGFs. CLINICAL SIGNIFICANCE: 3D printing has been on the rise in recent years and its use in daily clinical practice is expanding over time. Two of the three 3D-printed resins tested in this study performed as well in the cytotoxicity tests as the conventional one, supporting their use, but caution and further testing are required.

Chaperone-Mediated Autophagy Ablation in Pericytes Reveals New Glioblastoma Prognostic Markers and Efficient Treatment Against Tumor Progression.

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.

The Cytocompatibility of Silver Diamine Fluoride on Mesenchymal Stromal Cells from Human Exfoliated Deciduous Teeth: An In Vitro Study.

Silver diamine fluoride (SDF) has been used for many years for the treatment of caries, and minimally invasive dentistry concepts have made it popular again. The fact that its application does not require the administration of anesthesia makes its use in children more desirable. The aim of this study was to determine the cytotoxicity of two new commercial SDF products: Riva Star (SDI Dental Limited) and e-SDF (Kids-e-Dental) on mesenchymal stromal cells from human exfoliated deciduous teeth (SHEDs). SHEDs were exposed to SDF products at different concentrations (0.1%, 0.01% and 0.005%). Then different assays were performed to evaluate their cytocompatibility on SHEDs: IC50, MTT, cell migration (wound healing), cell cytoskeleton staining, cell apoptosis, generation of intracellular reactive oxygen species (ROS), and ion chromatography. Statistical analyses were performed using one-way ANOVA and Tukey's post hoc test (p < 0.05). Riva Star Step 2 showed the same cell metabolic activity when compared to the control condition at any time and concentration. Meanwhile, e-SDF displayed high cytotoxicity at any time and any concentration (*** p < 0.001), whereas Riva Star Step 1 displayed high cytotoxicity at any time at 0.1% and 0.01% (*** p < 0.001). Only e-SDF showed a statistically significant decreased cell migration rate (*** p < 0.001) at all times and in all concentrations. At 0.1%, e-SDF and Riva Star Step 1 only showed 4.37% and 4.47% of viable cells, respectively. These results suggest that Riva Star has better in vitro cytocompatibility on SHEDs than does e-SDF. Riva Star Step 1 was found to be as cytotoxic as e-SDF, but it had better biological properties when mixed with Riva Star Step 2. Our findings suggest that Riva Star is more suitable when used in deciduous teeth due to its lower cytotoxicity compared to e-SDF.

In Vitro Biocompatibility of Several Children's Toothpastes on Human Gingival Fibroblasts.

The European Academy of Paediatric Dentistry has recommended fluoride toothpastes from the eruption of the first teeth in children. Toothpastes stay in the mouth in contact with human gingival fibroblasts (hGFs) for a long time. Thus, the objective of this study was to compare the cytotoxicity of five different commonly used children's toothpastes: Oral B Kids +3 (Procter & Gamble, Alicante, Spain), Fluor Kin Calcium (Kin, Madrid, Spain), PHB Junior (PHB, Barcelona, Spain), Colgate +3 (Colgate Palmolive, Madrid, Spain) and Vitis Kids (Dentaid, Valencia, Spain) on hGFs. The children's toothpastes were exposed to hGFs at different concentrations (1:1, 1:2, 1:4). Afterwards, several tests were performed: MTT assays, cell cycle analyses, cell cytoskeleton staining assays, apoptosis/necrosis assays, and ICP-MS and ion chromatography. Oral B displayed the lowest cytotoxicity and was the toothpaste with the highest fluoride ion release; meanwhile, the other toothpastes were cytotoxic (*** p < 0.0001); Fluor Kin being the one with the lowest fluoride ion release. Among all the toothpastes analyzed, Oral B exhibited the best results in vitro in terms of biocompatibility. Future evaluations, both in vitro and in vivo, are required to confirm the biocompatibility of sodium lauryl sarcosinate and sodium lauryl sulfate containing toothpastes.

Bone marrow-derived mononuclear stem cells in the treatment of retinal degenerations.

Retinal degenerative diseases affecting the outer retina in its many forms (inherited, acquired or induced) are characterized by photoreceptor loss, and represent currently a leading cause of irreversible vision loss in the world. At present, there are very few treatments capable of preventing, recovering or reversing photoreceptor degeneration or the secondary retinal remodeling, which follows photoreceptor loss and can also cause the death of other retinal cells. Thus, these diseases are nowadays one of the greatest challenges in the field of ophthalmological research. Bone marrow derived-mononuclear stem cell transplantation has shown promising results for the treatment of photoreceptor degenerations. These cells may have the potential to slow down photoreceptor loss, and therefore should be applied in the early stages of photoreceptor degenerations. Furthermore, because of their possible paracrine effects, they may have a wide range of clinical applications, since they can potentially impact on several retinal cell types at once and photoreceptor degenerations can involve different cells and/or begin in one cell type and then affect adjacent cells. The intraocular injection of bone marrow derived-mononuclear stem cells also enhances the outcomes of other treatments aimed to protect photoreceptors. Therefore, it is likely that future investigations may combine bone marrow derived-mononuclear stem cell therapy with other systemic or intraocular treatments to obtain greater therapeutic effects in degenerative retinal diseases.