Cone-beam computed tomography accuracy in pulp chamber size evaluation: An ex vivo study

This study aimed to assess ex vivo the accuracy of cone-beam computed tomography (CBCT), as compared to operative microscope, for evaluating pulp chamber size. A total of forty teeth were extracted for periodontal reasons and a horizontal section was done at the most apical level of the cement-enamel junction. The pulp chamber was photographed using a digital camera connected to an optical microscope. Then, the tooth was scanned with CBCT and the horizontal slide matching the anatomical section of pulp chamber was digitally stored. The pulp chamber section area was measured through image analysis software. The two methods provided similar results, either for monoradicular (P = 0.14) or multiradicular teeth (P = 0.93). Correlation was statistically significant (P < 0.0001), being the coefficient r = 0.89 and 0.94 for monoradicular and multiradicular teeth, respectively. Conclusively, CBCT is suitable for pulp chamber morphology evaluation. However, it has limitations in detecting the anatomical variability of small branches in root canal system

Adhesive restoration of endodontically treated premolars: influence of posts on cuspal deflection

To determine, by means of a non-destructive experimental procedure, the effectiveness of adhesive restorations in reducing the cuspal deflection of endodontically treated premolars, with or without root canal fiber posts.To determine, by means of a non-destructive experimental procedure, the effectiveness of adhesive restorations in reducing the cuspal deflection of endodontically treated premolars, with or without root canal fiber posts. MATERIALS AND METHODS: The cuspal deflection of ten sound, intact maxillary premolars was evaluated. A loading device induced deformation by axial force (ranging from 98 to 294 N) applied on the occlusal surface of teeth while laser sensors registered the amount of deflection. Once tested, teeth were endodontically treated and the marginal ridges were removed. The teeth were randomly divided into two groups and restored with: group 1) dual curing adhesive, flowable composite, and microhybrid composite; group 2) the same materials associated with root canal glass fiber post and composite cement. The cuspal deflection test was repeated with the same protocol after restorative procedures, allowing a direct comparison of the same samples. Statistical analysis was performed using ANOVA at a significance level of 0.05. RESULTS: Different average cuspal deflection was detected in the two groups: composite resin with post insertion resulted in lower deformation compared with composite alone. Mean deflection ranged from 3.43 to 12.17 μm in intact teeth, from 14.42 to 26.93 μm in group 1, and from 15.35 to 20.39 μm in group 2. ANOVA found significant differences (p = 0.02). CONCLUSION: Bonded composite restorations with fiber posts may be more effective than composite alone in reducing the cuspal deflection in endodontically treated premolars in which the marginal ridges have been lost

Strumenti rotanti in lega nichel-titanio per il ritrattamento: un'analisi pre-clinica

RiassuntoIntroduzioneL'impiego di strumenti in lega nichel-titanio (NiTi) per la rimozione dei materiali da otturazione è stato di recente prospettato con soddisfacenti risultati. Obiettivo di questo lavoro è valutare due sistemi di strumenti meccanici rotanti in lega NiTi per il ritrattamento.Materiali e metodiSono stati selezionati 60 elementi dentali monoradicolati (premolari superiori e inferiori) privi di carie e fratture, estratti per motivi parodontali. Una volta sagomati, detersi e otturati mediante condensazione verticale della guttaperca, gli elementi sono stati suddivisi in due gruppi omogenei denominati rispettivamente R-Endo e D-Endo. Gli strumenti impiegati per eseguire i ritrattamenti nei due gruppi sono stati, rispettivamente, i sistemi R-Endo e D-Endo, impiegati secondo le norme descritte dai produttori; al fine di rendere omogenee le zone apicali di strumentazione in entrambi i gruppi, sono state rifinite, rispettivamente, nel gruppo R-Endo con il file Rs e nel gruppo D-Endo con un Protaper F3 (entrambi ISO 30 in punta). Sono stati valutati i tempi di raggiungimento e di rifinitura del limite apicale di strumentazione, l'estrusione di detriti apicali alla fine della strumentazione per il ritrattamento, il diametro apicale medio al termine della fase di ritrattamento, le fratture di strumenti, i blocchi all'avanzamento degli strumenti in direzione apicale e infine i residui radiograficamente apprezzabili all'interno dei canali radicolari al termine della procedura di ritrattamento. I dati sono stati analizzati statisticamente mediante analisi della varianza (ANOVA) con una significatività posta a p <0,05; i dati qualitativi sono stati analizzati mediante test U di Mann-Whitney con una significatività posta a p <0,05.RisultatiIl tempo medio di sagomatura per la tecnica R-Endo è stato di 6,2 minuti, mentre quello per la metodica D-Endo è stato di 5,1. Tale differenza è risultata significativa (p=0,0003). La quantità di detriti espulsi apicalmente è stata pari a 0,024g nel gruppo R-Endo e di 0,031g nel gruppo D-Endo. Il diametro medio apicale nel gruppo R-Endo è stato di 26,4 ISO, quello nel gruppo D-Endo di 28,1. Questa differenza non è risultata statisticamente significativa. Il numero di fratture di strumenti è stato pari a due nel gruppo D-Endo e a uno nel gruppo R-Endo, mentre abbiamo verificato un'errata progressione nel limite apicale (trasporto o falsa strada) in tre casi nel gruppo R-Endo e in due casi nel gruppo D-Endo. La differenza non è risultata statisticamente significativa. Tutti i campioni hanno dimostrato la presenza di residui di guttaperca all'interno dei canali apprezzabili radiograficamente.ConclusioniI sistemi per ritrattamento presi in esame, pur non essendo, in termini assoluti, altamente efficaci, hanno dimostrato di poter raggiungere il limite apicale di strumentazione in tempi ragionevolmente brevi, di avere pochi effetti collaterali e di produrre una sagomatura del limite apicale rispettosa dell'anatomia. Entrambe le metodiche hanno tuttavia dimostrato, in senso assoluto, un'incompleta rimozione del contenuto intracanalare del materiale da otturazione.SummaryIntroductionEndodontic files made in nickel-titanium (NiTi) alloys have been used not only for shaping procedures, but also for retreatment shaping purposes with satisfactory clinical results. The aim of this paper was to evaluate the efficacy of two NiTi file systems in retreatment.Material and methodsSixty monorooted teeth – upper and lower premolars – caries-free and without any fracture, extracted for periodontal reasons, were carefully selected. After a normal root canal treatment finished by vertical condensation of warm gutta-percha, these teeth were randomly divided into two homogeneous groups, namely R-Endo and D-Endo. The NiTi files for retratment were respectively R-Endo files and D-Endo; the whole shaping procedure during the retreatment phase was accomplished according to the manufacturer's instructions, except at the end of the retreatment. At that point, to make tha final shaping comparable the R-Endo group had an apical finishing with the Rs instrument, while the D-Endo group had the same final instrumentation by Protaper F3 (both ISO 30 at the tip). In the study, the time needed to reach the apical terminus, the weight of debris extruded from the apical foramen, the mean apical size in the two groups, instruments blocks and fractures and finally the presence of residual gutta-percha into the root canals after the whole retreatment procedure were addressed. Data were analyzed by analysis of variance (ANOVA) with a significance level p<0.05 and non-parametric Mann-Whitney U test with the same level of significance.ResultsThe mean shaping time was respectively 6.2minutes in the R-Endo group and 5.1minutes in the D-Endo group (p=0.0003). The amount of debris extruded beyond the apical terminus was 0.024g in the R-Endo group and 0.031g in the D-Endo group; no statistically significance was reported. The mean apical size was 26.4 ISO in the R-Endo group and 28.1 in the D-Endo group; again, no significant difference was shown. In the D-Endo group two instruments fractured, whereas in the R-Endo group only one instrument fractured and three cases were dropped for canal blockage. In these cases as well no statistically significant difference was reported. All the samples in both groups showed a remarkable presence of gutta-percha into the root canals at the end of the retreatment procedure.ConclusionsBoth retreatment system file groups were effective, reaching the apical part of the root canal in relatively short times with a low percentage of instrument breakage or intracanal blocks; the amount of debris was similar and the anatomy of the apical part was carefully preserved. However, the incomplete removal of filling debris was observed in all the samples

Xeno-free cultured mesenchymal stromal cells release extracellular vesicles with a "therapeutic" miRNA cargo ameliorating cartilage inflammation in vitro

Rationale: Mesenchymal stromal cells (MSCs)-derived extracellular vesicles (EVs) emerged as an innovative strategy for the treatment of chronic disorders such as osteoarthritis (OA). Biological activity of EVs is generally driven by their cargo, which might be influenced by microenvironment. Therefore, pre-conditioning strategies, including modifications in culture conditions or oxygen tension could directly impact on MSCs paracrine activity. In this study we selected an appropriate preconditioning system to induce cells to perform the most suitable therapeutic response by EV-encapsulated bioactive factors. Methods: A xeno-free supplement (XFS) was used for isolation and expansion of MSCs and compared to conventional fetal bovine serum (FBS) culture. Bone Marrow-derived MSCs (BMSCs) were pre-conditioned under normoxia (20% O2) or under hypoxia (1% O2) and EVs production was evaluated. Anti-OA activity was evaluated by using an in vitro inflammatory model. miRNA content was also explored, to select putative miRNA that could be involved in a biological function. Results: Modulation of IL-6, IL-8, COX-2 and PGE2 was evaluated on hACs simultaneously treated with IL-1a and BMSC-derived EVs. FBS-sEVs exerted a blunt inhibitory effect, while a strong anti-inflammatory outcome was achieved by XFS-sEVs. Interestingly, in both cases hypoxia pre-conditioning allowed to increase EVs effectiveness. Analysis of miRNA content showed the upregulation in XFS-hBMSC-derived EVs of miRNA known to have a chondroprotective role, such as let-7b-5p, miR-17, miR-145, miR-21-5p, miR-214-3p, miR-30b-5p, miR-30c-5p. Activated pathways and target genes were investigated in silico and upregulated miRNAs functionally validated in target cells. MiR-145 and miR-214 were found to protect chondrocytes from IL-1a-induced inflammation and to reduce production of pro-inflammatory cytokines. Conclusions: XFS medium was found to be suitable for isolation and expansion of MSCs, secreting EVs with a therapeutic cargo. The application of cells cultured exclusively in XFS overcomes issues of safety associated with serum-containing media and makes ready-to-use clinical therapies more accessible

Ultrastructural examination of lung "cryobiopsies" from a series of fatal COVID-19 cases hardly revealed infected cells

Ultrastructural analysis of autopsy samples from COVID-19 patients usually suffers from significant structural impairment possibly caused by the rather long latency between death of the patient and an appropriate sample fixation. To improve structural preservation of the tissue, we obtained samples from ventilated patients using a trans-bronchial "cryobiopsy" within 30&nbsp;min after their death and fixed them immediately for electron microscopy. Samples of six COVID-19 patients with a documented histopathology were systematically investigated by thin section electron microscopy. The different samples and areas inspected revealed the ultrastructural correlates of the different phases of diffuse alveolar damage, including detachment of the alveolar epithelium, hyperplasia of type 2 cells, exudates, and accumulation of extracellular material, such as the hyaline membranes and fibrin. Macrophages and neutrophilic granulocytes were regularly detected. Structural integrity of endothelium was intact in regions where the alveolar epithelium was already detached. Aggregates of erythrocytes, leukocytes with fibrin, and thrombocytes were not observed. Coronavirus particles were only found in and around very few cells in one of the six patient samples. The type and origin of these cells could not be assessed although the overall structural preservation of the samples allowed the identification of pulmonary cell types. Hence, the observed alveolar damage is not associated with virus presence or structural impairment due to ongoing replication at later stages of the disease in fatal cases, which implies that the lung damage in these patients is at least propagated by alternative mechanisms, perhaps, an inappropriate immune or stress response

Pathogenic variants of Valosin-containing protein induce lysosomal damage and transcriptional activation of autophagy regulators in neuronal cells

Aim: Mutations in the valosin-containing protein (VCP) gene cause various lethal proteinopathies that mainly include inclusion body myopathy with Paget's disease of bone and frontotemporal dementia (IBMPFD) and amyotrophic lateral sclerosis (ALS). Different pathological mechanisms have been proposed. Here, we define the impact of VCP mutants on lysosomes and how cellular homeostasis is restored by inducing autophagy in the presence of lysosomal damage. Methods: By electron microscopy, we studied lysosomal morphology in VCP animal and motoneuronal models. With the use of western blotting, real-time quantitative polymerase chain reaction (RT-qPCR), immunofluorescence and filter trap assay, we evaluated the effect of selected VCP mutants in neuronal cells on lysosome size and activity, lysosomal membrane permeabilization and their impact on autophagy. Results: We found that VCP mutants induce the formation of aberrant multilamellar organelles in VCP animal and cell models similar to those found in patients with VCP mutations or with lysosomal storage disorders. In neuronal cells, we found altered lysosomal activity characterised by membrane permeabilization with galectin-3 redistribution and activation of PPP3CB. This selectively activated the autophagy/lysosomal transcriptional regulator TFE3, but not TFEB, and enhanced both SQSTM1/p62 and lipidated MAP1LC3B levels inducing autophagy. Moreover, we found that wild type VCP, but not the mutants, counteracted lysosomal damage induced either by trehalose or by a mutant form of SOD1 (G93A), also blocking the formation of its insoluble intracellular aggregates. Thus, chronic activation of autophagy might fuel the formation of multilamellar bodies. Conclusion: Together, our findings provide insights into the pathogenesis of VCP-related diseases, by proposing a novel mechanism of multilamellar body formation induced by VCP mutants that involves lysosomal damage and induction of lysophagy

Rapamycin Combined with Anti-CD45RB mAb and IL-10 or with G-CSF Induces Tolerance in a Stringent Mouse Model of Islet Transplantation

Background: A large pool of preexisting alloreactive effector T cells can cause allogeneic graft rejection following transplantation. However, it is possible to induce transplant tolerance by altering the balance between effector and regulatory T (Treg) cells. Among the various Treg-cell types, Foxp3 +Treg and IL-10-producing T regulatory type 1 (Tr1) cells have frequently been associated with tolerance following transplantation in both mice and humans. Previously, we demonstrated that rapamycin+IL-10 promotes Tr1-cell-associated tolerance in Balb/c mice transplanted with C57BL/6 pancreatic islets. However, this same treatment was unsuccessful in C57BL/6 mice transplanted with Balb/c islets (classified as a stringent transplant model). We accordingly designed a protocol that would be effective in the latter transplant model by simultaneously depleting effector T cells and fostering production of Treg cells. We additionally developed and tested a clinically translatable protocol that used no depleting agent. Methodology/Principal Findings: Diabetic C57BL/6 mice were transplanted with Balb/c pancreatic islets. Recipient mice transiently treated with anti-CD45RB mAb+rapamycin+IL-10 developed antigen-specific tolerance. During treatment, Foxp3 +Treg cells were momentarily enriched in the blood, followed by accumulation in the graft and draining lymph node, whereas CD4 +IL-10 +IL-4 - T (i.e., Tr1) cells localized in the spleen. In long-term tolerant mice, only CD4 +IL-10 +IL-4 - T cells remained enriched in the spleen and IL-10 was key in the maintenance of tolerance. Alternatively, recipient mice were treated with two compounds routinely used in the clinic (namely, rapamycin and G-CSF); this drug combination promoted tolerance associated with CD4 +IL-10 +IL-4 - T cells. Conclusions/Significance: The anti-CD45RB mAb+rapamycin+IL-10 combined protocol promotes a state of tolerance that is IL-10 dependent. Moreover, the combination of rapamycin+G-CSF induces tolerance and such treatment could be readily translatable into the clinic. © 2011 Gagliani et al

Molecular and functional heterogeneity of IL-10-producing CD4 + T cells

IL-10 is a prototypical&nbsp;anti-inflammatory cytokine, which is fundamental to the maintenance of immune homeostasis, especially in the intestine. There is an assumption that cells producing IL-10 have an immunoregulatory function. However, here we report that IL-10-producing CD4 + T cells are phenotypically and functionally heterogeneous. By combining single cell transcriptome and functional analyses, we identified a subpopulation of IL-10-producing Foxp3 neg CD4 + T cells that displays regulatory activity unlike other IL-10-producing CD4 + T cells, which are unexpectedly pro-inflammatory. The combinatorial expression of co-inhibitory receptors is sufficient to discriminate IL-10-producing CD4 + T cells with regulatory function from others and to identify them across different tissues and disease models in mice and humans. These regulatory IL-10-producing Foxp3 neg CD4 + T cells have a unique transcriptional program, which goes beyond the regulation of IL-10 expression. Finally, we found that patients with Inflammatory Bowel Disease demonstrate a deficiency in this specific regulatory T-cell subpopulation

PIM-induced phosphorylation of Notch3 promotes breast cancer tumorigenicity in a CSL-independent fashion

Dysregulation of the developmentally important Notch signaling pathway is implicated in several types of cancer, including breast cancer. However, the specific roles and regulation of the four different Notch receptors have remained elusive. We have previously reported that the oncogenic PIM kinases phosphorylate Notch1 and Notch3. Phosphorylation of Notch1 within the second nuclear localization sequence of its intracellular domain (ICD) enhances its transcriptional activity and tumorigenicity. In this study, we analyzed Notch3 phosphorylation and its functional impact. Unexpectedly, we observed that the PIM target sites are not conserved between Notch1 and Notch3. Notch3 ICD (N3ICD) is phosphorylated within a domain, which is essential for formation of a transcriptionally active complex with the DNA-binding protein CSL. Through molecular modeling, X-ray crystallography, and isothermal titration calorimetry, we demonstrate that phosphorylation of N3ICD sterically hinders its interaction with CSL and thereby inhibits its CSL-dependent transcriptional activity. Surprisingly however, phosphorylated N3ICD still maintains tumorigenic potential in breast cancer cells under estrogenic conditions, which support PIM expression. Taken together, our data indicate that PIM kinases modulate the signaling output of different Notch paralogs by targeting distinct protein domains and thereby promote breast cancer tumorigenesis via both CSL-dependent and CSL-independent mechanisms.</p