Volumetric Evaluation of Voids and Gaps of Different Calcium-Silicate Based Materials Used in Furcal Perforations: A Micro-CT Study.

This study aimed at evaluating volumetrically gaps and voids of calcium-silicate based materials of different generations and handling properties (BC­Endosequence BC RRM-Fast Set Condensable Putty, MTA­ProRoot MTA, and BIO­Biodentine) in simulated furcal perforations in an ex vivo setup by microcomputed tomography (Micro-CT) analysis. Thirty-six extracted human mandibular molars with sound furcation areas were selected. Standardized perforations were created in the furcation area of the pulp chamber using #4 diamond burs. The specimens were randomly assigned to three groups (BC, MTA and BIO; n = 12). Samples were then scanned (SkyScan 1172; Bruker-microCT, Kontich, Belgium), and three-dimensional (3D) images reconstructed. The relative volume of gaps (VG%) and voids (VV%) present on each material was calculated. Data were analyzed using one-way analysis of variance (ANOVA) and Tukey's HSD test (p < 0.05). Mean VG% for BC, MTA, and BIO groups were, respectively, 0.513%, 1.128%, 1.460%, with BC presenting statistically (p < 0.05) fewer gaps formation than the other groups. Mean VV% were, respectively, 0.018%, 0.037%, and 0.065%. The was no statistical difference regarding VV%. There were no gap-free and void-free samples. BC group had the lowest VG% among the groups with a significant statistical difference (p < 0.05).

SCF/C-Kit Signaling Induces Self-Renewal of Dental Pulp Stem Cells.

INTRODUCTION: The maintenance of a stem cell pool is imperative to enable healing processes in the dental pulp tissue throughout life. As such, knowing mechanisms underlying stem cell self-renewal is critical to understand pulp pathophysiology and pulp regeneration. The purpose of this study was to evaluate the impact of stem cell factor (SCF) signaling through its receptor tyrosine kinase (c-Kit) on the self-renewal of human dental pulp stem cells (hDPSCs). METHODS: The hDPSCs were stably transduced with lentiviral vectors expressing shRNA-c-Kit or vector control. The impact of the SCF/c-Kit axis on hDPSC self-renewal was evaluated by using a pulpsphere assay in low attachment conditions and by evaluating the expression of polycomb complex protein Bmi-1 (master regulator of self-renewal) by Western blot and flow cytometry. RESULTS: The c-Kit-silenced hDPSCs formed fewer pulpspheres when compared with hDPSCs transduced with control vector (P < .05). Evaluation of pulpsphere morphology revealed the presence of 3 distinct sphere types, ie, holospheres, merospheres, and paraspheres. Although c-Kit silencing decreased the number of holospheres compared with control cells (P < .05), it had no effect on the number of merospheres and paraspheres. Recombinant human stem cell factor (rhSCF) increased the number of holospheres (P < .05) and induced dose-dependent Bmi-1 expression in hDPSCs. As expected, the inductive capacity of rhSCF on Bmi-1 expression and fraction of Bmi-1-positive cells was inhibited when we silenced c-Kit in hDPSCs. CONCLUSIONS: These results unveiled the role of SCF/c-Kit signaling on the self-renewal of hDPSCs and suggested that this pathway enables long-term maintenance of stem cell pools in human dental pulps.

Filling ability of three bioceramic root-end filling materials: A micro-computed tomography analysis.

This study aimed at evaluating the volume of gaps and voids, and the total porosity percentage of three calcium-silicate-based materials in mandibular molars apicoectomy by Micro-CT analysis. Thirty-three mesial roots of extracted human mandibular molars were instrumented and obturated. The apical 3mm of each root was resected and prepared. Root-end cavities were filled with EndoSequence BC Putty (BC); ProRoot MTA (MTA) and Biodentine (BIO). Samples were scanned using a Micro-CT scanner and the tridimensional images reconstructed. Percentage of gaps (VG%) and of voids (VV%) were obtained. Porosity percentage (Po%) was also assessed. Data were analysed using Student's t-test (P < 0.05). All materials presented gaps and voids. VG% was 2.006 (BC), 1.882 (MTA) and 1.450 (BIO), and VV% was 0.039 (BC), 0.021 (MTA) and 0.024 (BIO) with no statistical difference. Po% were 56.73 (BC), 51.94 (MTA) and 50.45 (BIO), with BC being statistically (P > 0.05) more porous.

Incorporation of AuNP-PLL nanocomplexes in DPSC: a new tool for 3D analysis in pulp regeneration.

OBJECTIVES: To assess the viability of dental pulp stem cells loaded with gold nanoparticles complexed with poly (L-lysine) (AuNP-PLL) and to track the cellular behavior in a 3D analysis by micro-CT. MATERIALS AND METHODS: DPSC (dental pulp stem cells) were cultured and incorporated with AuNP-PLL (0.2 mg/ml) and assessed for cell viability (24 h, 48 h, and 72 h) using MTS assay. Apoptosis/cell death index and cell cycle were analyzed by propidium iodide. AuNP-PLL-RITC were used for observation in confocal microscopy and quantification of the incorporation rates. Cells were also suspended in agarose and analyzed three-dimensionally in µCT, assessing their radiopacity. Quantitative data (cell viability and apoptosis) were analyzed by t test (p < 0.05). RESULTS: AuNP-PLL labeling did not affect cellular viability in any of the periods analyzed nor interfered with the apoptosis index of DPSC. AuNP-PLL nanocomplexes were identified in the cytoplasm of cells by immunofluorescence, mainly in the perinuclear region. The observed incorporation rate was 98%. Micro-CT analysis has shown that incorporated cells are now visible using x-ray, with a clear increase in radiopacity when compared to the control group (non-incorporated cells). CONCLUSION: These results indicate that it is possible to incorporate AuNP-PLL complex into DPSC and track the cells by using µCT; furthermore, this incorporation of 0.2 mg/ml of AuNP-PLL does not interfere in the DPSC basic behavior. CLINICAL RELEVANCE: This methodology can be a useful tool for cellular labeling to observe cell behavior and their interaction with scaffolds in a 3D manner, opening an array of new approaches in regenerative endodontics.

Lipoprotein Receptor-related Protein 6 Signaling is Necessary for Vasculogenic Differentiation of Human Dental Pulp Stem Cells.

The aim of this study was to evaluate the effects of Wnt signaling through lipoprotein receptor-related protein 6 (LRP6) and Frizzled6 on the endothelial differentiation of dental pulp stem cells (DPSCs). DPSCs were stably transduced with enhanced green fluorescent protein (EGFP)-tagged lentiviral vectors (short hairpin RNA-LRP6, short hairpin RNA-Frizzled6, or empty vector controls). We evaluated the effects of LRP6 and Frizzled6 on expression of endothelial markers and on capillary tube formation mediated by DPSCs induced with recombinant human Wnt1 (rhWnt1) and/or recombinant human vascular endothelial growth factor165 (rhVEGF165). In vivo, tooth slices/scaffolds were seeded with LRP6-silenced, Frizzled6-silenced, or vector control DPSC cells and transplanted into immunodeficient mice. The density of blood vessels generated by DPSCs differentiated into vascular endothelial cells was analyzed by immunohistochemistry for EGFP. The rhWnt1 and rhVEGF165 induced expression of active ß-catenin in control DPSCs and in Frizzled6-silenced DPSCs, but not in LRP6-silenced DPSCs. Furthermore, VEGF and interleukin-8 were downregulated in LRP6-silenced DPSCs, but not in control DPSCs or in Frizzled6-silenced DPSCs (P < .05). Likewise, rhWnt1 and rhVEGF165 induced expression of the endothelial marker VEGF receptor-2 in control DPSCs and in Frizzled6-silenced DPSCs, but not in LRP6-silenced DPSCs. These data correlated with a trend for lower density of capillary sprouts generated by LRP6-silenced DPSCs when compared with control DPSCs in Matrigel. In vivo, tooth slice/scaffolds seeded with DPSC-short hairpinRNA-LRP6 cells showed lower density of human blood vessels (ie, EGFP-positive blood vessels), when compared with tooth slice/scaffolds seeded with vector control cells (P < .05). Collectively, these data demonstrated that LRP6 signaling is necessary for the vasculogenic differentiation of human DPSCs.

Wnt/ß-Catenin Signaling Determines the Vasculogenic Fate of Postnatal Mesenchymal Stem Cells.

Vasculogenesis is the process of de novo blood vessel formation observed primarily during embryonic development. Emerging evidence suggest that postnatal mesenchymal stem cells are capable of recapitulating vasculogenesis when these cells are engaged in tissue regeneration. However, the mechanisms underlining the vasculogenic differentiation of mesenchymal stem cells remain unclear. Here, we used stem cells from human permanent teeth (dental pulp stem cells [DPSC]) or deciduous teeth (stem cells from human exfoliated deciduous teeth [SHED]) as models of postnatal primary human mesenchymal stem cells to understand mechanisms regulating their vasculogenic fate. GFP-tagged mesenchymal stem cells seeded in human tooth slice/scaffolds and transplanted into immunodeficient mice differentiate into human blood vessels that anastomize with the mouse vasculature. In vitro, vascular endothelial growth factor (VEGF) induced the vasculogenic differentiation of DPSC and SHED via potent activation of Wnt/ß-catenin signaling. Further, activation of Wnt signaling is sufficient to induce the vasculogenic differentiation of postnatal mesenchymal stem cells, while Wnt inhibition blocked this process. Notably, ß-catenin-silenced DPSC no longer differentiate into endothelial cells in vitro, and showed impaired vasculogenesis in vivo. Collectively, these data demonstrate that VEGF signaling through the canonical Wnt/ß-catenin pathway defines the vasculogenic fate of postnatal mesenchymal stem cells. Stem Cells 2016;34:1576-1587.

Accuracy of cone-beam computed tomography and periapical radiography in apical periodontitis diagnosis.

INTRODUCTION: This study aimed to investigate the correlation and the agreement between periapical radiography (PR) and cone-beam computed tomography (CBCT) correlating to histologic findings in the diagnosis of apical periodontitis (AP). METHODS: One hundred thirty-four premolar root canals from 10 dogs were treated after AP induction. Four months later, the animals were killed, and standard digital PRs were obtained. The area of AP was measured by using ImageJ software. CBCT (i-CAT) images from each arch were obtained, and AP area and volume were measured by using Osiri-X software. The apical inflammatory infiltrate was evaluated under light microscopy. The correlation between imaging methods was evaluated by using the Pearson coefficient. The Bland-Altman method was used to assess the agreement between PR and CBCT data. The Spearman coefficient was used to correlate the imaging data and histologic findings. RESULTS: Despite a strong correlation between PR and CBCT areas, the agreement limits were very broad (95% limits of agreement, 0.19-1.08). PR only measured, on average, 63% of CBCT values. Although there was a strong correlation between PR area and CBCT volume, the Bland-Altman method suggests that the larger the CBCT volume, the more underestimated the PR value. When APs had a volume smaller than 6 mm(3), the PR estimation of CBCT data was unpredictable. A positive correlation was found for PR area, CBCT area, CBCT volume, and histology data. CONCLUSIONS: The diagnosis of AP based on PR data is clinically limited, and it should not be used for scientific investigations.