Hypoxia preconditioned DPSC-derived exosomes regulate angiogenesis via transferring LOXL2.

Hypoxia was proved to enhance the angiogenesis of stem cells. However, the mechanism of the angiogenic potential in hypoxia-pretreated dental pulp stem cells (DPSCs) is poorly understood. We previously confirmed that hypoxia enhances the angiogenic potential of DPSC-derived exosomes with upregulation of lysyl oxidase-like 2 (LOXL2). Therefore, our study aimed to illuminate whether these exosomes promote angiogenesis via transfer of LOXL2. Exosomes were generated from hypoxia-pretreated DPSCs (Hypo-Exos) stably silencing LOXL2 after lentiviral transfection and characterized with transmission electron microscopy, nanosight and Western blot. The efficiency of silencing was verified using quantitative real-time PCR (qRT-PCR) and Western blot. CCK-8, scratch and transwell assays were conducted to explore the effects of LOXL2 silencing on DPSCs proliferation and migration. Human umbilical vein endothelial cells (HUVECs) were co-incubated with exosomes to assess the migration and angiogenic capacity through transwell and matrigel tube formation assays. The relative expression of angiogenesis-associated genes was characterized by qRT-PCR and Western blot. LOXL2 was successfully silenced in DPSCs and inhibited DPSC proliferation and migration. LOXL2 silencing in Hypo-Exos partially reduced promotion of HUVEC migration and tube formation and inhibited the expression of angiogenesis-associated genes. Thus, LOXL2 is one of various factors mediating the angiogenic effects of Hypo-Exos.

Integration of single-cell RNA sequencing of endothelial cells and proteomics to unravel the role of ICAM1-PTGS2 communication in apical periodontitis: A laboratory investigation.

AIM: Endothelial cells (EDs) play a key role in angiogenesis and are associated with granulomatous lesions in patients with chronic apical periodontitis (CAP). This study aimed to investigate the diversity of EDs using single-cell ribonucleic acid sequencing (scRNA-seq) and to evaluate the regulation of intercellular adhesion molecule 1 (ICAM1) on the ferroptosis-related protein, prostaglandin-endoperoxide synthase 2 (PTGS2), in CAP. METHODOLOGY: EDs from the uploaded scRNA-seq data of five CAP samples (GSE181688 and GSE197680) were categorized using distinct marker genes. The interactions between vein EDs (veinEndo) and other cell types were analysed using CellPhoneDB. Differentially expressed proteins in the proteomics of human umbilical vein EDs (HUVECs) and THP-1-derived macrophages infected with Porphyromonas gingivalis were compared with the differentially expressed genes (DEGs) of VeinEndo in scRNA-seq of CAP versus healthy control periodontal tissues. The protein-protein interaction of ICAM1-PTGS2 in macrophages and HUVECs was validated by adding recombinant ICAM1, ICAM1 inhibitor and PTGS2 inhibitor using real-time polymerase chain reaction (PCR), western blotting, and immunofluorescence staining. RESULTS: EDs in patients with CAP were divided into eight subclusters: five vein ED, capillaries, arterials and EC (PLA). There were 29 mutually upregulated DEGs and two mutually downregulated DEGs in vein cells in the scRNA-seq data, as well as differentially expressed proteins in the proteomics of HUVECs. Real-time PCR and immunofluorescence staining showed that ICAM1 and PTGS2 were highly expressed in CAP, infected HUVECs, and macrophages. Recombinant protein ICAM1 may improve PTGS2 expression, reactive oxygen species (ROS), and Fe2+ levels and decrease glutathione peroxidase 4 (GPX4) and SLC7A11 protein levels. ICAM1 inhibitor may inverse the above changes. CONCLUSIONS: scRNA-seq revealed the diversity of EDs in CAP and identified the possible regulation of ICAM1 by the ferroptosis-related protein, PTGS2, in infected HUVECs and macrophages, thus providing a basis for therapeutic approaches that target the inflammatory microenvironment of CAP.

Single-cell RNA analysis of chemokine expression in heterogeneous CD14+ monocytes with lipopolysaccharide-induced bone resorption.

Lipopolysaccharide (LPS)-induced bone resorption has normally been found in inflammatory bone diseases, but the underlying mechanism is currently unclear. Since LPS binds to CD14 and activates Toll-like receptor 4 (TLR4) in monocytes, the present study focused on CD14+ monocytes and observed their responses after LPS treatment during the progression of local bone destruction. CD14+ monocytes were obtained from human peripheral blood mononuclear cells (PBMCs) by magnetic cell separation (MACS), and their classification was confirmed by fluorescence-activated cell sorting (FACS). Single-cell RNA sequencing (scRNA-seq) was further utilized to analyze their subpopulations, and the results showed that physiological CD14+ monocytes were heterogeneous and divided into 6 subsets, that could be easily agitated. After priming with a suitable concentration of LPS, heterogeneous CD14+ monocytes became pathological and expressed a large number of chemokines as a "cascade effect". Some of these chemokines have been validated in an animal model of mouse calvarial bone invasion. Taken together, our research has linked enhanced chemokine expression with stimulation of heterogeneous CD14+ monocytes, and indicated that inflammatory responses caused by microbiome infection are responsible for the recruitment and mobilization of CD14+ monocytes into bone resorption sites, which may explain the pathogenesis of LPS-associated bone diseases.

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.

The synergetic effect of pulp chamber extension depth and occlusal thickness on stress distribution of molar endocrowns: a 3-dimensional finite element analysis.

The aim of this study was to evaluate the effects of butt margin, occlusal thickness and pulp chamber extension depth on stress distributions on mandibular molar endodontically treated teeth (ETT) with EMAX endocrown restoration using 3-dimensional finite element analysis (FEA). The FEA models of endocrown with flat surface or curve surface of butt margin were firstly evaluated stress distributions, and then 9 FEA models of endocrown with 1-, 2- or 3-mm pulp chamber extension depth and 1-, 2- or 3-mm occlusal thickness were generated using curve surface of butt margin. In all of FEA models, a 200 N of vertical load or horizontal load was applied, and the von Mises stress (VMS) were evaluated. The results showed that curve surface of butt margin offered more adhesive area of enamel, though VMS on the prepared teeth was similar in flat surface and curve surface models. In 9 endocrown models, 2-mm occlusal thickness showed the lowest VMS on restorations, teeth tissue and root furcations, and 2-mm extension depth displayed the lowest VMS on root furcations under vertical load. Also, 2-mm extension depth exhibited the lowest VMS on restorations and teeth tissue under horizontal load. Within the limitations of this FEA study, the results of this study could be used as an aid for dentists to better devise endocrown restorations. Graphical abstract.

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.

Resistance fracture of minimally prepared endocrowns made by three types of restorative materials: a 3D finite element analysis.

A thin endocrown restoration was often applied in endodontically treated teeth with vertical bite height loss or inadequate clinical crown length. A model of mandibular molars made by endocrown restoration with 1 mm thickness and 2 mm depth of pulp chamber was constructed and imported into FEA ANSYS v18.0 software. The three CAD/CAM materials, feldspathic (Mark2), lithium disilicate (EMAX), and lava ultimate (LU), were assigned, and the five load indenters were loaded on the full occlusal (FO), occlusal center (OC), central fossa (CF), buccal groove (BG), and mesiobuccal cusp (MC) of restoration in the model. The MinPS and MaxPS of the thin endocrown were significantly higher than those of tooth tissue in five types of loads except for the LU endocrown loaded in the FO group. The smaller the contact surface of the load was, the higher MaxPS and MinPS were. MaxPS and MinPS of the MC were the highest, followed by the BG and CF in the restoration. In the stress distribution of tooth tissue, MaxPS in the LU endocrown accumulated at the external edge of enamel and was significantly higher than MaxPS in Mark2 and EMAX endocrown concentrated on the chamber wall of dentin under OC, CF and BG loads. Within the limitations of this FEA study, the LU endocrown transferred more stress to tooth tissue than Mark2 and EMAX, and the maximum principal stress on endocrown restoration and tooth tissue at the mesiobuccal cusp load was higher than that at the central fossa and buccal groove load.

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.

Parenchymal and stromal tissue regeneration of tooth organ by pivotal signals reinstated in decellularized matrix.

Cells are transplanted to regenerate an organs' parenchyma, but how transplanted parenchymal cells induce stromal regeneration is elusive. Despite the common use of a decellularized matrix, little is known as to the pivotal signals that must be restored for tissue or organ regeneration. We report that Alx3, a developmentally important gene, orchestrated adult parenchymal and stromal regeneration by directly transactivating Wnt3a and vascular endothelial growth factor. In contrast to the modest parenchyma formed by native adult progenitors, Alx3-restored cells in decellularized scaffolds not only produced vascularized stroma that involved vascular endothelial growth factor signalling, but also parenchymal dentin via the Wnt/ß-catenin pathway. In an orthotopic large-animal model following parenchyma and stroma ablation, Wnt3a-recruited endogenous cells regenerated neurovascular stroma and differentiated into parenchymal odontoblast-like cells that extended the processes into newly formed dentin with a structure-mechanical equivalency to native dentin. Thus, the Alx3-Wnt3a axis enables postnatal progenitors with a modest innate regenerative capacity to regenerate adult tissues. Depleted signals in the decellularized matrix may be reinstated by a developmentally pivotal gene or corresponding protein.

Expression of high mobility group box 1 in inflamed dental pulp and its chemotactic effect on dental pulp cells.

High mobility group box 1 protein (HMGB1) is a chromatin protein which can be released extracellularly, eliciting a pro-inflammatory response and promoting tissue repair process. This study aimed to examine the expression and distribution of HMGB1 and its receptor RAGE in inflamed dental pulp tissues, and to assess its effects on proliferation, migration and cytoskeleton of cultured human dental pulp cells (DPCs). Our data demonstrated that cytoplasmic expression of HMGB1 was observed in inflamed pulp tissues, while HMGB1 expression was confined in the nuclei in healthy dental pulp. The mRNA expression of HMGB1 and RAGE were significantly increased in inflamed pulps. In in vitro cultured DPCs, expression of HMGB1 in both protein and mRNA level was up-regulated after treated with lipopolysaccharide (LPS). Exogenous HMGB1 enhanced DPCs migration in a dose-dependent manner and induced the reorganization of f-actin in DPCs. Our results suggests that HMGB1 are not only involved in the process of dental pulp inflammation, but also play an important role in the recruitment of dental pulp stem cells, promoting pulp repair and regeneration.

Efficacy and safety of probiotics in the treatment of Candida-associated stomatitis.

This study aimed at evaluating the short-term efficacy and safety of probiotics as an aid in the treatment of Candida-associated stomatitis in a randomised controlled trial. A total of 65 patients were randomly assigned to receive oral local antifungal agents alone (gargle 2% sodium bicarbonate solution for 30 s, wait 10 min and then apply 2% nystatin paste) or these agents plus local probiotics (the mixture of Bifidobacterium longum, Lactobacillus bulgaricus and Streptococcus thermophilus) three times per day for 4 weeks. Parameters related to hyperaemia, visual analogue scale scores, culture of resting saliva and a lingual dorsum swab and adverse reactions were assessed or recorded in the beginning, middle and end of treatment. Although the baseline characteristics of the participants were similar, both groups showed a significant reduction in pain level and hyperaemia on the tongue mucosa (P = 0.000) after 4-week application. However, despite the reduction in hyperaemia in the probiotic group, these improvements did not display statistically significant differences. The detection rate of Candida spp. was 100% before treatment and 8.21% in the experimental group and 34.6% in the control group after treatment. The detection rate of Candida spp. decreased (P = 0.000) in both groups and was significantly lower in the probiotic group than the control group (P = 0.038). Other analysed micro-organisms, including the decreased detection rate for Lactobacillus spp. (P = 0.049) and the increased detection rate for Staphylococcus epidermidis (P = 0.019), did not display consistent change trends in the probiotics group. Compared with conventional antifungal therapies for oral candidiasis, the inclusion of locally administered probiotics helped improve certain clinical conditions and reduced the prevalence of Candida spp., although the impact of probiotics on oral bacterial species remains to be further studied.

Single-cell RNA sequencing combined with proteomics of infected macrophages reveals prothymosin-α as a target for treatment of apical periodontitis.

INTRODUCTION: Chronic apical periodontitis (CAP) is a common infectious disease of the oral cavity. Immune responses and osteoclastogenesis of monocytes/macrophages play a crucial role in CAP progression, and this study want to clarify role of monocytes/macrophages in CAP, which will contribute to treatment of CAP. OBJECTIVES: We aim to explore the heterogeneity of monocyte populations in periapical lesion of CAP tissues and healthy control (HC) periodontal tissues by single-cell RNA sequencing (scRNA-seq), search novel targets for alleviating CAP, and further validate it by proteomics and in vitro and in vivo evaluations. METHODS: ScRNA-seq was used to analyze the heterogeneity of monocyte populations in CAP, and proteomics of THP-1-derived macrophages with porphyromonas gingivalis infection were intersected with the differentially expressed genes (DEGs) of macrophages between CAP and HC tissues. The upregulated PTMA (prothymosin-α) were validated by immunofluorescence staining and quantitative real time polymerase chain reaction. We evaluated the effect of thymosin α1 (an amino-terminal proteolytic cleavage product of PTMA protein) on inflammatory factors and osteoclast differentiation of macrophages infected by P. gingivalis. Furthermore, we constructed mouse and rat mandibular bone lesions caused by apical periodontitis, and estimated treatment of systemic and topical administration of PTMA for CAP. Statistical analyses were performed using GraphPad Prism software (v9.2) RESULTS: Monocytes were divided into seven sub-clusters comprising monocyte-macrophage-osteoclast (MMO) differentiation in CAP. 14 up-regulated and 21 down-regulated genes and proteins were intersected between the DEGs of scRNA-seq data and proteomics, including the high expression of PTMA. Thymosin α1 may decrease several inflammatory cytokine expressions and osteoclastogenesis of THP-1-derived macrophages. Both systemic administration in mice and topical administration in the pulp chamber of rats alleviated periapical lesions. CONCLUSIONS: PTMA upregulation in CAP moderates the inflammatory response and prevents the osteoclastogenesis of macrophages, which provides a basis for targeted therapeutic strategies for CAP.

A ROS-responsive hydrogel incorporated with dental follicle stem cell-derived small extracellular vesicles promotes dental pulp repair by ameliorating oxidative stress.

Pulpitis, an inflammatory disease of dental pulp tissues, ultimately results in the loss of pulp defense properties. Existing clinical modalities cannot effectively promote inflamed pulp repair. Oxidative stress is a major obstacle inhibiting pulp repair. Due to their powerful antioxidative capacity, mesenchymal stem cell-derived small extracellular vesicles (MSC-sEVs) exhibit potential for treating oxidative stress-related disorders. However, whether MSC-sEVs shield dental pulp tissues from oxidative damage is largely unknown. Here, we showed that dental follicle stem cell-derived sEVs (DFSC-sEVs) have antioxidative and prohealing effects on a rat LPS-induced pulpitis model by enhancing the survival, proliferation and odontogenesis of H2O2-injured dental pulp stem cells (DPSCs). Additionally, DFSC-sEVs restored the oxidative/antioxidative balance in DPSC mitochondria and had comparable effects on ameliorating mitochondrial dysfunction with the mitochondrion-targeted antioxidant Mito-Tempo. To improve the efficacy of DFSC-sEVs, we fabricated an intelligent and injectable hydrogel to release DFSC-sEVs by combining sodium alginate (SA) and the ROS sensor RhB-AC. The newly formed SA-RhB hydrogel efficiently encapsulates DFSC-sEVs and exhibits controlled release of DFSC-sEVs in a HClO/ClO- concentration-dependent manner, providing a synergistic antioxidant effect with DFSC-sEVs. These results suggest that DFSC-sEVs-loaded SA-RhB is a promising minimally invasive treatment for pulpitis by enhancing tissue repair in the pulp wound microenvironment.

Small extracellular vesicles derived from hypoxic preconditioned dental pulp stem cells ameliorate inflammatory osteolysis by modulating macrophage polarization and osteoclastogenesis.

Extensive macrophage inflammatory responses and osteoclast formation are predominant during inflammatory or infective osteolysis. Mesenchymal stem cell (MSC)-derived small extracellular vesicles (MSC-sEV) have been shown to exert therapeutic effects on bone defects. However, cultured MSCs are typically exposed to normoxia (21% O2) in vitro, which differs largely from the oxygen concentration in vivo under hypoxic conditions. It is largely unknown whether sEV derived from dental pulp stem cells (DPSCs) cultured under hypoxic conditions (Hypo-sEV) exert better therapeutic effects on lipopolysaccharide (LPS)-induced inflammatory osteolysis than those cultured under normoxic conditions (Nor-sEV) by simultaneously inhibiting the macrophage inflammatory response and osteoclastogenesis. In this study, we show that hypoxia significantly induces the release of sEV from DPSCs. Moreover, Hypo-sEV exhibit significantly improved efficacy in promoting M2 macrophage polarization and suppressing osteoclast formation to alleviate LPS-induced inflammatory calvarial bone loss compared with Nor-sEV. Mechanistically, hypoxia preconditioning markedly alters the miRNA profiles of DPSC-sEV. MiR-210-3p is enriched in Hypo-sEV, and can simultaneously induce M2 macrophage generation and inhibit osteoclastogenesis by targeting NF-κB1 p105, which attenuates osteolysis. Our study suggests a promising potential for hypoxia-induced DPSC-sEV to treat inflammatory or infective osteolysis and identifies a novel role of miR-210-3p in concurrently hindering osteoclastogenesis and macrophage inflammatory response by inhibiting NF-kB1 expression.

Hypoxia­induced mitophagy regulates proliferation, migration and odontoblastic differentiation of human dental pulp cells through FUN14 domain­containing 1.

FUN14 domain­containing 1 (FUNDC1) is a receptor that has been previously reported to activate hypoxia­induced mitophagy. However, the potential role of FUNDC1 in the pathophysiology of dental pulp diseases remains unknown. Therefore, present study first collected tissue specimens from patients with pulpitis and from healthy individuals. The results of reverse transcription­quantitative PCR and immunohistochemical staining revealed markedly increased FUNDC1 and hypoxia­inducible factor­1α expression in pulpitis tissue specimens compared with those from healthy individuals. To provide a theoretical basis for the study of the occurrence, development and reparative mechanisms in the dental pulp after tissue injury, the present study then investigated the role of hypoxia­induced mitophagy in the regulation of proliferation, migration and odontoblastic differentiation in human dental pulp cells (HDPCs), in addition, to the possible involvement of FUNDC1. The surface markers and multipotent differentiation capabilities of HDPCs were performed by flow cytometry (surface markers), alizarin red (osteogenic capabilities), alcian blue (chondrogenic capabilities) and oil red O (adipogenic capabilities). Following culture under hypoxia conditions (1% O2) for varying time periods, the proliferation, migration and odontoblastic differentiation of HDPCs were measured using Cell Counting Kit­8, wound healing and Transwell migration assays, alkaline phosphatase staining and activity tests and western blotting (runt­related transcription factor 2, collagen I, osterix and osteopontin), respectively. Immunofluorescence and western blotting were performed to measure the expression levels of hypoxia­inducible factor­1α, pro­fission dynamin­related protein 1, mitochondria­related proteins translocase of inner mitochondrial membrane 23 and translocase of outer mitochondrial membrane 20, in addition to those of autophagy markers (p62, LC3II, Beclin­1 and autophagy­related 5). Transmission electron microscopy was also used to image the autophagosomes and mitochondrial morphology. In addition, to study the functional role of FUNDC1, its expression was silenced by liposome­mediated transfection with small interfering RNA into HDPCs. Compared with those in HDPCs cultured under normoxic conditions (21% O2), the ability of autophagy in HDPCs cultured under hypoxic conditions for 18 h was markedly increased, whilst the proliferation, migration and odontoblastic differentiation were also enhanced. Increased numbers of autophagosomes could also be observed in the hypoxic group. However, FUNDC1 knockdown in HDPCs reversed the aforementioned effects. Overall, data from the present study suggest that hypoxia can promote the proliferation, migration and odontoblastic differentiation of HDPCs, where the underlying mechanism may be associated with the activation of mitophagy downstream of FUNDC1.

Caspase-1 Inhibition Reduces Occurrence of PANoptosis in Macrophages Infected by E. faecalis OG1RF.

To investigate the effect of caspase-1 inhibition on PANoptosis in macrophages infected with Enterococcus faecalis OG1RF. RAW264.7 cells with and without pretreatment by caspase-1 inhibitor were infected with E. faecalis OG1RF at multiplicities of infection (MOIs). A live cell imaging analysis system and Western blot were applied to evaluate the dynamic curve of cell death and the expression of executor proteins of PANoptosis. The mRNA expression of IL-1ß and IL-18 was quantified by RT-qPCR. Morphological changes were observed under scanning electron microscopy. We found that PI-positive cells emerged earlier and peaked at a faster rate in E. faecalis-infected macrophages (Ef-MPs) at higher MOIs. The expression of the N-terminal domain of the effector protein gasdermin D (GSDMD-N), cleaved caspase-3 and pMLKL were significantly upregulated at MOIs of 10:1 at 6 h and at MOI of 1:1 at 12 h postinfection. In Ef-MPs pretreated with caspase-1 inhibitor, the number of PI-positive cells was significantly reduced, and the expression of IL-1ß and IL-18 genes and cleaved caspase-1/-3 and GSDMD-N proteins was significantly downregulated (p < 0.05), while pMLKL was still markedly increased (p < 0.05). Ef-MPs remained relatively intact with caspase-1 inhibitor. In conclusion, E. faecalis induced cell death in macrophages in an MOI-dependent manner. Caspase-1 inhibitor simultaneously inhibited pyroptosis and apoptosis in Ef-MPs, but necroptosis still occurred.

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.

Micro-CT Evaluation of Different Root Canal Irrigation Protocols on the Removal of Accumulated Hard Tissue Debris: A Systematic Review and Meta-Analysis.

Accumulated hard tissue debris (AHTD) is an inevitable by-product during endodontic treatment and is difficult to remove completely using traditional syringe and needle irrigation (SNI). Adjunctive irrigation is proposed to assist the clean-up of AHTD. This systematic review and meta-analysis aimed to evaluate the AHTD removal efficacy of different root canal irrigation devices using micro-computed tomography (Micro-CT). A literature search was carried out within the main scientific databases until 20 June 2022. All results were screened with detailed eligibility criteria. Eleven studies were included for analysis. SNI, passive ultrasonic irrigation (PUI), negative pressure systems, sonically activated irrigation (SAI), mechanical-activated system and laser-activated irrigation (LAI) were assessed. PUI is superior to SNI for debris removal and LAI has better AHTD removal performance than PUI. The negative pressure system and mechanical-activated system were proved to be less effective. Registration: PROSPERO (CRD42021273892).

The practicability of different preparation of mandibular molar restored by modified endocrown with intracanal extension: Computational analysis using finite element models.

BACKGROUND AND OBJECTIVE: Post-core-crown (PCC) and endocrown are two common restorative methods for severely damaged molars, but exhibit disadvantages. This study aimed to explore the practicability of modified endocrown with a 2 mm intracanal extension (MED) to restore defective teeth using finite element analysis (FEA). METHODS: Five groups of numerical models of mandibular molars restored by three MEDs, a PCC, and a routine endocrown after root canal treatment were devised by FEA software. We constructed 4 mm, 3 mm, and 2 mm thickness of MED restorations to restore mandibular molars that were prepared to 1 mm, 2 mm, and 3 mm from the cemento-enamel junction (CEJ). Furthermore, PCC and routine endocrown were used to compare the stress distribution with MED. Lithium disilicate glass-ceramics (EMAX) and resin nanoceramics (LU) were considered restorative materials, and a vertical load of 600 N and an oblique load of 200 N were applied to the restorations. RESULTS: In three MEDs by LU, 2 mm thickness of restoration generated the highest stress on prepared teeth, while the thickness of EMAX did not significantly influence the stress value. MED by LU generated higher stress around the CEJ, and reduced the stress on the middle and lower root compared to MEDs by EMAX, PCC by EMAX, and PCC by LU. MED by EMAX caused lower stress around the CEJ, and generated higher stress in the chamber walls after extended root canals compared with MED by LU, endocrowns by LU, and endocrowns by EMAX. There was an evident stress concentration at the last but one layer, which was a thin area of the tooth root in all restorative models. CONCLUSIONS: The use of modified endocrown may be considered an effective restorative method to restore defective mandibular molar, but suitable restorative material must be selected based on the tooth preparation method and deficiencies in the tooth structure.

Clinical outcome of bioceramic sealer iRoot SP extrusion in root canal treatment: a retrospective analysis.

BACKGROUND: During the obturation procedure, sealer extrusion occurs in some cases. iRoot SP is a kind of bioceramic sealer with superior physicochemical and biological properties. This article reports the outcome of iRoot SP extrusion in root canal treatment and the potential factors associated with the outcome. METHODS: Ninety-nine patients and one hundred and eighty-five teeth treated between 2014 and 2020 were included in this retrospective study. All of the cases were filled with a single-cone technique and the iRoot SP sealer. The minimum follow-up visit period was 1 year. The outcome was evaluated by clinical examination and radiographic examination at recall and was classified as healed, healing (success), or not healed (failure). RESULTS: The overall success rate of all teeth was 96.8%. The success rate of adequately filled teeth was 97.3%, while that of iRoot SP extrusion was 95.8%; the difference was not statistically significant. Factors such as gender, age, tooth position, follow-up visit period, size of periapical lesion, treatment type and extruding sealer amount had no influence on the outcome of iRoot SP extruded teeth. CONCLUSIONS: The results suggested that iRoot SP extrusion has no adverse effect on the outcome of root canal treatment, which may contribute to the endodontic treatment.