Heterogeneity of T cells in periapical lesions and in vitro validation of the proangiogenic effect of GZMA on HUVECs.

AIM: T cells are key immunomodulatory cells in periapical lesions. This study aimed to explore the roles of T cells in chronic apical periodontitis (CAP) using single-cell RNA sequencing and to further investigate Granzyme A (GZMA) in angiogenesis regulation. METHODOLOGY: A total of five CAP samples were collected for single-cell RNA sequencing. We performed subcluster and lineage-tracing analyses for T cells. According to differential gene expression, distinct biological functions enriched in T cells of CAP were presented by gene set enrichment analysis (GSEA) and compared with healthy gingiva (data obtained from the GEO database). CellChat was used to explore potential ligand-receptor interactions between T cells and endothelial cells in CAP. The coculture of primary human umbilical vein endothelial cells (HUVECs) and Jurkat T cells, as well as the addition of GZMA recombinant protein, was used to validate the predicted pair of GZMA and coagulation factor II thrombin receptor (F2R) by RT-PCR, angiogenesis and migration assays. RESULTS: A transcriptomic atlas of 44 746 individual cells was constructed from the periapical lesions of five patients with CAP by single-cell RNA-seq, and eight cell types were identified. We identified nine subsets of T cells and deciphered the cellular heterogeneity of T cells in CAP at the functional level by subclustering and GSEA. Lineage tracing revealed a distinct lineage of T cells in CAP and predicted the transition of the T cellular state upon CAP. GSEA revealed multiple biological processes and relevant angiogenesis genes upregulated in CAP T cells. GZMA-F2R pairs were predicted by cell-cell interactions in CAP. High expression of GZMA and F2R was observed in the coculture of HUVECs and Jurkat T cells, and the proangiogenic capacity of the GZMA recombinant protein was emphasized by in vitro experiments. CONCLUSIONS: Our study provides novel insights into the heterogeneity of T cells in periapical lesions and reveals the potential role of GZMA in T cells in regulating angiogenesis in HUVECs.

Effect of different endodontic access preparations on the biomechanical behavior of lithium disilicate and resin nanoceramic onlay restorations: An in vitro and 3D finite element analysis study.

STATEMENT OF PROBLEM: The effect of different sizes of endodontic access preparations on the performance of lithium disilicate glass-ceramic and resin nanoceramic onlay restorations is unclear. PURPOSE: The purpose of this in vitro and 3D finite element analysis study was to assess the effect of a conservative endodontic access cavity and a traditional endodontic access cavity on the fracture resistance and stress distribution of lithium disilicate glass-ceramic and resin nanoceramic onlays. MATERIAL AND METHODS: Sixty caries-free human mandibular molars were anatomically prepared for onlays and divided into 6 groups. After restoration with a lithium disilicate glass-ceramic (N=30) or resin nanoceramic (N=30), each material was further divided into traditional or conservative endodontic access cavity or intact tooth groups. After endodontic therapy and thermocycling, all specimens were submitted to a cycle fatigue test and then loaded until fracture. Failure type and location after debonding or fracture were classified and recorded. Furthermore, stress distribution in the 6 models was analyzed by using a finite element analysis software program. The data were compared by using a 2-way ANOVA test and the Tukey post hoc test (α=.05). The Weibull modulus and Weibull failure probabilities were also estimated for each group. RESULTS: The lithium disilicate glass-ceramic onlays had lower fracture resistance values than the resin nanoceramic onlays in both the traditional and conservative endodontic access cavity groups (P<.05). The fracture resistance of the 2 materials for onlays with endodontic access was significantly lower than that for the intact restorations (P<.05). No significant difference was found between the fracture resistance of Lava Ultimate restorations with traditional endodontic access and conservative endodontic access, while the fracture resistance of EMAX restorations with traditional endodontic access was significantly lower than that of restorations with conservative endodontic access (P<.05). A higher percentage of irreparable fractures was found in the 3 resin nanoceramic restoration groups. The von Mises stresses were higher in the lithium disilicate glass-ceramic restorations than in the resin nanoceramic restorations with the same access cavities. The von Mises stresses in the tooth structure were higher with the resin nanoceramic restorations than with the lithium disilicate glass-ceramic restorations with the same access cavities. CONCLUSIONS: An endodontic access cavity had more influence on the lithium disilicate glass-ceramic onlays than on the resin nanoceramic onlays, and a traditional endodontic access cavity significantly decreased the fracture resistance of lithium disilicate glass-ceramic onlays.

An in vitro study on the effects of serum proteins on Enterococcus faecalis adhesion to three types of root sealers and gutta-percha.

BACKGROUND: The extrusion of overfilled materials that extend beyond the apical foramina into the periradicular tissue may serve as a reservoir for bacterial adhesion and further affect recovery from periapical diseases. The aim of this study was to evaluate the effects of serum proteins on Enterococcus faecalis adhesion and survival on the surface of a calcium hydroxide-based root canal sealer (Apexit Plus), an epoxy resin sealer (AH-Plus) and a bioceramic sealer (iRoot SP). METHODS: Apexit Plus, AH-Plus and iRoot SP were evenly coated on gutta-percha, using gutta-percha alone as the control. After root canal sealer setting, the number of E. faecalis adhering to the root canal sealers and gutta-percha was counted in fetal bovine serum (FBS) or tryptic soy broth supplemented with 1% glucose (TSBG) by viable cell plate counts. The morphology of 7-day-old E. faecalis biofilms in FSB and TSBG was observed by scanning electron microscopy (SEM). Furthermore, E. faecalis biofilms on the three root canal sealers were labeled with a LIVE/DEAD BacLight™ Bacterial Viability Kit, and the ratios of viable to dead cells were analyzed using laser scanning microscopy operative software (Zen software). RESULTS: In the assays, after 1 and 7 days, the number of E. faecalis adhering to the root canal sealers or gutta-percha in FBS were significantly lower than those in TSBG (P < 0.05). In FBS, E. faecalis adhesion to iRoot SP and gutta-percha was reduced to a greater extent than that adhered to Apexit Plus and AH-Plus. Few E. faecalis accumulated on iRoot SP in FBS, whereas many bacteria assembled on iRoot SP and formed biofilms in TSBG. The ratio of viable cells in the E. faecalis biofilm on iRoot SP was the lowest. CONCLUSIONS: Calcium hydroxide-based root canal sealers, epoxy resin sealers and bioceramic sealers may provide a substrate for E. faecalis adhesion, and the bioceramic sealer in this study showed the least E. faecalis adhesion in the presence of serum proteins compared to the other two sealers.

Sodium fluoride induces apoptosis through reactive oxygen species-mediated endoplasmic reticulum stress pathway in Sertoli cells.

Excessive fluoride exposure is known to contribute to reproductive system dysfunction, ultimately leading to pathological damage and apoptosis in cells. Although both oxidative and endoplasmic reticulum (ER) stresses have been implicated in fluorosis, the signaling pathways and their roles in sodium fluoride (NaF)-induced apoptosis of Sertoli cells have been sparsely described. In this study, oxidative damage, ER stress, and apoptosis were analyzed after Sertoli cells were treated with varying doses of NaF for 24hr. Moreover, the antioxidant N-acetylcysteine (NAC) and pro-apoptotic transcription factor CHOP knockdown were used to clarify the precise interplay between reactive oxygen species (ROS), ER stress and their roles in NaF-induced apoptosis in Sertoli cells. The present study indicated that NaF significantly decreased cell viability and induced apoptosis in Sertoli cells. In addition, NaF exposure facilitated the accumulation of ROS and increased nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) in Sertoli cells. Treatment with NAC caused remarkable recovery from these NaF-induced responses. Meanwhile, excessive NaF triggered ER stress as evidenced by up-regulated glucose-regulated protein 78 kDa (GRP78), PKR-like ER kinase (PERK), phosphorylation of eukaryotic translation initiation factor 2α (p-eIF2α) and CCAAT/enhancer-binding protein-homologous protein (CHOP), without affecting total eukaryotic translation initiation factor 2α (eIF2α). NAC effectively blocked the activation of ER stress, suggesting that NaF-induced ROS is an early event that triggers ER stress. Taken together, the results demonstrate that the ROS-mediated ER stress pathway is the crucial mechanistic event involved in NaF-induced apoptosis of Sertoli cells.

Hypoxia Alters the Proteome Profile and Enhances the Angiogenic Potential of Dental Pulp Stem Cell-Derived Exosomes.

Dental pulp stem cells (DPSCs) and their exosomes (Exos) are effective treatments for regenerative medicine. Hypoxia was confirmed to improve the angiogenic potential of stem cells. However, the angiogenic effect and mechanism of hypoxia-preconditioned DPSC-Exos are poorly understood. We isolated exosomes from DPSCs under normoxia (Nor-Exos) and hypoxia (Hypo-Exos) and added them to human umbilical vein endothelial cells (HUVECs). HUVEC proliferation, migration and angiogenic capacity were assessed by CCK-8, transwell, tube formation assays, qRT-PCR and Western blot. iTRAQ-based proteomics and bioinformatic analysis were performed to investigate proteome profile differences between Nor-Exos and Hypo-Exos. Western blot, immunofluorescence and immunohistochemistry were used to detect the expression of lysyl oxidase-like 2 (LOXL2) in vitro and in vivo. Finally, we silenced LOXL2 in HUVECs and rescued tube formation with Hypo-Exos. Hypo-Exos enhanced HUVEC proliferation, migration and tube formation in vitro superior to Nor-Exos. The proteomics analysis identified 79 proteins with significantly different expression in Hypo-Exos, among which LOXL2 was verified as being upregulated in hypoxia-preconditioned DPSCs, Hypo-Exos, and inflamed dental pulp. Hypo-Exos partially rescued the inhibitory influence of LOXL2 silence on HUVEC tube formation. In conclusion, hypoxia enhanced the angiogenic potential of DPSCs-Exos and partially altered their proteome profile. LOXL2 is likely involved in Hypo-Exos mediated angiogenesis.

Cyclic fatigue resistance for six types of nickel titanium instruments at artificial canals with different angles and radii of curvature.

This study evaluated the cyclic fatigue resistance for six types of 25# NiTi instruments. A traditional manufacturing instrument, an M wire instrument, a gold treatment instrument, a controlled memory (CM) wire instrument, a CM wire instrument with electrical discharge machining (EDM), and an R-phase heat treatment instrument, were operated in the different curved artificial canals. The fracture time (FT) and number of cycles to fracture (NCF) of the NiTi instruments were higher at 45° angles and double-curvature canals than at 60° angles. Except for the instruments with gold technology and EDM technology, others showed the longest FT and the highest NCF at an 8 mm radius of curvature. Morphological characteristics of cyclic fatigue were exhibited on the cross-section and lateral view of fracture fragments. The use of M-wire, R-phase wire, CM-wire, gold technology, EDM technology, and reciprocating movement were beneficial to enhance the cyclic fatigue resistance of NiTi files.