Research and development of microenvironment's influence on stem cells from the apical papilla - construction of novel research microdevices: tooth-on-a-chip.

Stem cells are crucial in tissue engineering, and their microenvironment greatly influences their behavior. Among the various dental stem cell types, stem cells from the apical papilla (SCAPs) have shown great potential for regenerating the pulp-dentin complex. Microenvironmental cues that affect SCAPs include physical and biochemical factors. To research optimal pulp-dentin complex regeneration, researchers have developed several models of controlled biomimetic microenvironments, ranging from in vivo animal models to in vitro models, including two-dimensional cultures and three-dimensional devices. Among these models, the most powerful tool is a microfluidic microdevice, a tooth-on-a-chip with high spatial resolution of microstructures and precise microenvironment control. In this review, we start with the SCAP microenvironment in the regeneration of pulp-dentin complexes and discuss research models and studies related to the biological process.

Expert consensus on endodontic therapy for patients with systemic conditions.

The overall health condition of patients significantly affects the diagnosis, treatment, and prognosis of endodontic diseases. A systemic consideration of the patient's overall health along with oral conditions holds the utmost importance in determining the necessity and feasibility of endodontic therapy, as well as selecting appropriate therapeutic approaches. This expert consensus is a collaborative effort by specialists from endodontics and clinical physicians across the nation based on the current clinical evidence, aiming to provide general guidance on clinical procedures, improve patient safety and enhance clinical outcomes of endodontic therapy in patients with compromised overall health.

Construction of dentin-on-a-chip based on microfluidic technology and tissue engineering.

AIM: Three-dimensional (3D) cell culture systems perform better in resembling tissue or organism structures compared with traditional 2D models. Organs-on-chips (OoCs) are becoming more efficient 3D models. This study aimed to create a novel simplified dentin-on-a-chip using microfluidic chip technology and tissue engineering for screening dental materials. METHODOLOGY: A microfluidic device with three channels was designed for creating 3D dental tissue constructs using stem cells from the apical papilla (SCAP) and gelatin methacrylate (GelMA). The study investigated the effect of varying cell densities and GelMA concentrations on the layer features formed within the microfluidic chip. Cell viability and distribution were evaluated through live/dead staining and nuclei/F-actin staining. The osteo/odontogenic potential was assessed through ALP staining and Alizarin red staining. The impact of GelMA concentrations (5 %, 10 %) on the osteo/odontogenic differentiation trajectory of SCAP was also studied. RESULTS: The 3D tissue constructs maintained high viability and favorable spreading within the microfluidic chip for 3-7 days. A cell seeding density of 2 × 104 cells/µL was found to be the most optimal choice, ensuring favorable cell proliferation and even distribution. GelMA concentrations of 5 % and 10 % proved to be most effective for promoting cell growth and uniform distribution. Within the 5 % GelMA group, SCAP demonstrated higher osteo/odontogenic differentiation than that in the 10 % GelMA group. CONCLUSION: In 3D culture, GelMA concentration was found to regulate the osteo/odontogenic differentiation of SCAP. The study recommends a seeding density of 2 × 104 cells/µL of SCAP within 5 % GelMA for constructing simplified dentin-on-a-chip. CLINICAL SIGNIFICANCE: This study built up the 3D culture protocol, and induced odontogenic differentiation of SCAP, thus forming the simplified dentin-on-a-chip and paving the way to be used as a well-defined biological model for regenerative endodontics. It may serve as a potential testing platform for cell differentiation.

Expert consensus on difficulty assessment of endodontic therapy.

Endodontic diseases are a kind of chronic infectious oral disease. Common endodontic treatment concepts are based on the removal of inflamed or necrotic pulp tissue and the replacement by gutta-percha. However, it is very essential for endodontic treatment to debride the root canal system and prevent the root canal system from bacterial reinfection after root canal therapy (RCT). Recent research, encompassing bacterial etiology and advanced imaging techniques, contributes to our understanding of the root canal system's anatomy intricacies and the technique sensitivity of RCT. Success in RCT hinges on factors like patients, infection severity, root canal anatomy, and treatment techniques. Therefore, improving disease management is a key issue to combat endodontic diseases and cure periapical lesions. The clinical difficulty assessment system of RCT is established based on patient conditions, tooth conditions, root canal configuration, and root canal needing retreatment, and emphasizes pre-treatment risk assessment for optimal outcomes. The findings suggest that the presence of risk factors may correlate with the challenge of achieving the high standard required for RCT. These insights contribute not only to improve education but also aid practitioners in treatment planning and referral decision-making within the field of endodontics.

Expert consensus on irrigation and intracanal medication in root canal therapy.

Chemical cleaning and disinfection are crucial steps for eliminating infection in root canal treatment. However, irrigant selection or irrigation procedures are far from clear. The vapor lock effect in the apical region has yet to be solved, impeding irrigation efficacy and resulting in residual infections and compromised treatment outcomes. Additionally, ambiguous clinical indications for root canal medication and non-standardized dressing protocols must be clarified. Inappropriate intracanal medication may present side effects and jeopardize the therapeutic outcomes. Indeed, clinicians have been aware of these concerns for years. Based on the current evidence of studies, this article reviews the properties of various irrigants and intracanal medicaments and elucidates their effectiveness and interactions. The evolution of different kinetic irrigation methods, their effects, limitations, the paradigm shift, current indications, and effective operational procedures regarding intracanal medication are also discussed. This expert consensus aims to establish the clinical operation guidelines for root canal irrigation and a position statement on intracanal medication, thus facilitating a better understanding of infection control, standardizing clinical practice, and ultimately improving the success of endodontic therapy.

Influence of operator's experience on complications of root canal treatment using contemporary techniques: a retrospective study.

BACKGROUND: Endodontic treatment has benefited from the development of new techniques and equipment. Few clinical studies have been published on the complications associated with root canal preparations performed by doctors with different working experiences using contemporary techniques. This study aimed to analyze the complications of endodontic treatment performed by residents and endodontic specialists in a teaching stomatology hospital using contemporary techniques. METHODS: Cases of root canal treatment (RCT) and non-surgical root canal retreatment (ReRCT) performed by residents with 1-3 years of experience and endodontic specialists with 5-7 years of experience were collected from the electronic medical system of the Department of Endodontics, Beijing Stomatology Hospital, from September 1, 2020 to August 31, 2021. The cases were examined in terms of patient age, sex, type of tooth, diagnosis, treatment modality (RCT or ReRCT), number of appointments, whether an operating microscope was used, presence of ledges, canal transportation, perforations, missed canals, separated instruments, flare-ups and clinical incidence of second mesiobuccal (MB2) root canal in the maxillary molars. RESULTS: In total, 859 teeth from 820 patients were included in the analysis. The overall incidence of complications in the resident group was significantly higher than that in the specialist group. More ledges and flare-ups were observed in the resident group (p < 0.05). The clinical incidence of MB2 was significantly higher in the specialist group (p < 0.05). There were no significant differences in root canal transportation, perforation, or instrument separation between the two groups (p < 0.05). Multivariate analysis showed that the incidence of root canal preparation complications was related to operator experience, tooth type and treatment modality. CONCLUSIONS: Technical advancements could reduce the effect of working experience on RCT complications between residents and endodontic specialists in a teaching stomatology hospital.

Expert consensus on digital guided therapy for endodontic diseases.

Digital guided therapy (DGT) has been advocated as a contemporary computer-aided technique for treating endodontic diseases in recent decades. The concept of DGT for endodontic diseases is categorized into static guided endodontics (SGE), necessitating a meticulously designed template, and dynamic guided endodontics (DGE), which utilizes an optical triangulation tracking system. Based on cone-beam computed tomography (CBCT) images superimposed with or without oral scan (OS) data, a virtual template is crafted through software and subsequently translated into a 3-dimensional (3D) printing for SGE, while the system guides the drilling path with a real-time navigation in DGE. DGT was reported to resolve a series of challenging endodontic cases, including teeth with pulp obliteration, teeth with anatomical abnormalities, teeth requiring retreatment, posterior teeth needing endodontic microsurgery, and tooth autotransplantation. Case reports and basic researches all demonstrate that DGT stand as a precise, time-saving, and minimally invasive approach in contrast to conventional freehand method. This expert consensus mainly introduces the case selection, general workflow, evaluation, and impact factor of DGT, which could provide an alternative working strategy in endodontic treatment.

Experts consensus on the procedure of dental operative microscope in endodontics and operative dentistry.

The dental operative microscope has been widely employed in the field of dentistry, particularly in endodontics and operative dentistry, resulting in significant advancements in the effectiveness of root canal therapy, endodontic surgery, and dental restoration. However, the improper use of this microscope continues to be common in clinical settings, primarily due to operators' insufficient understanding and proficiency in both the features and established operating procedures of this equipment. In October 2019, Professor Jingping Liang, Vice Chairman of the Society of Cariology and Endodontology, Chinese Stomatological Association, organized a consensus meeting with Chinese experts in endodontics and operative dentistry. The objective of this meeting was to establish a standard operation procedure for the dental operative microscope. Subsequently, a consensus was reached and officially issued. Over the span of about four years, the content of this consensus has been further developed and improved through practical experience.

Bonding effect of a Zr/Si coating prepared on zirconia using a sol-gel method.

STATEMENT OF PROBLEM: Zirconia has been widely used as a dental prosthetic material. However, bonding to zirconia is challenging, and whether a Zr/Si coating would improve bonding is unclear. PURPOSE: The purpose of this in vitro study was to prepare a Zr/Si coating on zirconia ceramics using a sol-gel method and to determine whether the bonding to resin is improved. MATERIAL AND METHODS: Presintered zirconia specimens were prepared and divided into 5 groups: 4 experimental groups with ratios of the binary sol-gel precursor (zirconium oxychloride/tetraethoxysilane) set as 2:1 (Z2), 1:1 (Z1), 0.5:1 (Z0.5), and 0.25:1 (Z0.25) and Group C as the control group. In addition to surface roughness measurements, scanning electron microscopy (SEM), energy dispersion spectroscopy (EDS), and X-ray diffraction (XRD) were carried out to characterize the surface. Each group was divided into 2 subgroups according to whether a silane coupling agent was applied. Half of the bond specimens were stored in deionized water for 24 hours; the remaining half were aged using 5000 thermocycles. The shear bond strength (SBS) of resin bonded to specimens was tested for the initial and durable bond strength, and the bonding interface was also observed by SEM after debonding. Data were subjected to 1-way ANOVA and the post hoc Tukey honestly significant difference test (α=.05). RESULTS: The Zr/Si coating formed on zirconia ceramics. Z0.5 had the greatest mean ±standard deviation roughness (2.13 ±0.15 µm) and had the highest silicon content (21.7 ±0.21%). t-ZrO2, m-ZrO2, c-SiO2, and ZrSiO4 were detected by XRD in Z1. The SBS values were decreased by aging but were significantly increased by Zr/Si coating, especially for Z0.5, with the application of silane (initial: 22.92 ±2.79 MPa; aged: 9.91 ±0.92 MPa). CONCLUSIONS: The Zr/Si coating significantly improved the initial and aged bond strength, and the optimal Zr/Si ratio of the sol-gel appeared to be 0.5:1.

[Study of cross-sectional morphology of root canals in Tibetan mandibular incisors by micro-computed tomography].

Objective: To study the cross-sectional morphology of root canal system of Tibetan mandibular incisors by micro-CT. Methods: From October 2019 to October 2020, one hundred and thirty-six mandibular incisors were collected from Tibetan patients [(51.9±16.4) years old, range from 25 to 80 years] who underwent teeth extraction due to severe periodontitis at the Department of Stomatology, Tibetan Hospital of Traditional Tibetan Medicine, including 84 mandibular central incisors and 52 mandibular lateral incisors. These teeth were scanned at 23 µm voxel size resolution. Root lengths from cemento-enamel junction (CEJ) to apex of mandibular incisors were measured. According to the length, the root was divided as cervical 1/3, middle 1/3 and apical 1/3, and the numbers of root canals were recorded simultaneously. The major diameter, minor diameter, and dimension were measured per millimeter in cross section for mandibular incisor with single root canal, and the ratio of major diameter to minor diameter (Dmax/Dmin) as well as roundness were calculated for morphological analysis. The diversions and conversions from CEJ to apex in cross section were recorded for mandibular incisor with multiple root canals. Results: For mandibular central incisors with single root canal, the Dmax/Dmin was highest in middle 1/3 of the root [1.99 (1.31, 2.79)], which was significantly higher than cervical 1/3 and apical 1/3 (P=0.010, P=0.003). The roundness was least in middle 1/3 [0.47 (0.31, 0.66)], which was significantly lower than cervical 1/3 and apical 1/3 (P=0.010, P=0.001). For mandibular central incisor with multiple root canals, the highest incidence of multiple root canals was 40.5% (34/84), and mainly detected in middle 1/3 of the root [32.1% (27/84)]. For mandibular lateral incisor with single root canal, the roundness was greatest in apical 1/3 of the root [0.61 (0.49, 0.71)], which was significantly higher than cervical 1/3 (P=0.001) and middle 1/3 (P=0.001). The highest incidence of multiple root canals was 34.6% (18/52), all of which were detected in apical 1/3. Conclusions: In Tibetan mandibular central incisors, the cross-sectional morphology of root canals was long and narrow in middle 1/3, and multiple root canals were more likely to be found here. In Tibetan mandibular lateral incisors, the cross-sectional anatomy of root canal was relatively close to circle in apical 1/3, but the shape was still so irregular that one root canal may divide into two here.

Evaluation of Silk Fibroin-RGD-Stem Cell Factor Scaffold Effect on Adhesion, Migration, and Proliferation of Stem Cells of Apical Papilla.

This study explored the effects of a silk fibroin-RGD-stem cell factor (SF-RGD-SCF) scaffold on the migration, proliferation, and attachment of stem cells of apical papilla (SCAPs). SF, SF-RGD, SF-SCF, and SF-RGD-SCF scaffolds were prepared, and laser confocal microscopy was used to observe the adhesion and growth status of SCAPs on the scaffolds. Furthermore, the numbers of SCAPs on the scaffolds were counted by a digestion counting method to evaluate their proliferation. Cells on the SF-RGD-SCF scaffold proliferated more than those on the other scaffolds and showed a more obvious tendency to migrate to the scaffold's deep porous structure after 7 d seeding. Live/dead cell staining results showed that almost all the adhered cells were alive after 7 d. Furthermore, cell counting showed that the number of cells on the SF-RGD-SCF scaffold was highest after both 1 and 7 d (P < 0.05). Thus, the SF-RGD-SCF composite is biocompatible and promotes the migration, adhesion, and proliferation of SCAPs, making it of potential use as a scaffold for cell-homing pulp regeneration.

Randomized controlled clinical trial of a highly filled flowable composite in non-carious cervical lesions: 3-year results.

OBJECTIVES: This prospective, randomized, split-mouth clinical trial assessed the 3-year clinical performance of a highly filled flowable composite and a conventional paste-type composite in non-carious cervical lesions (NCCLs). MATERIALS AND METHODS: A total of 84 NCCLs in 27 subjects were included in this split-mouth design study and randomly divided into two groups: a highly filled flowable composite Clearfil Majesty ES Flow group (ES, n = 42) and a conventional paste-type composite Majesty group (MJ, n = 42). Clearfil SE Bond was used following the manufacturer's instructions. The restorations were evaluated at baseline (BL) and 1, 2, and 3 years using FDI (World Dental Federation) criteria. Data were analysed by a paired chi-squared test for intergroup comparisons and the Friedman test for intragroup comparisons (α = 0.05). RESULTS: Both groups had a 97.3% retention rate at the 3-year evaluation. The acceptable scores (FDI scores 1-3) for each criterion exhibited no significant difference between the MJ and ES groups at any time point (p = 1.00). The marginal adaptation performance of ES was significantly better than that of MJ at every evaluation point (p < 0.05). CONCLUSIONS: The 3-year clinical performance of ES in NCCLs was similar to that of MJ. When the restorations were clinically acceptable, ES showed better marginal adaptation than MJ. CLINICAL RELEVANCE: Compared with conventional paste-type composites, highly filled flowable composites showed similar clinical performance and better marginal adaptation for restoring NCCLs after 3 years. TRIAL REGISTRATION: TRN: ChiCTR1900028484 . Date of registration: December 22, 2019, retrospectively registered.

Microbial Communities in the Extraradicular and Intraradicular Infections Associated With Persistent Apical Periodontitis.

Microorganisms in the complex root canal system and the extraradicular regions, including the periapical lesions and extraradicular biofilm may cause root canal treatment failures. However, few studies described the difference between the intraradicular and extraradicular infections from the same tooth associated with persistent apical periodontitis. This study aimed to characterize the microbiome present in the root canal, extraradicular biofilm, and periapical lesions associated with persistent apical periodontitis. The microbial communities in the root canal, extraradicular biofilm, and periapical lesions were investigated by Illumina high-throughput sequencing using Illumina Hiseq 2500 platform. The dominant phyla in the extraradicular and intraradicular infections associated with persistent apical periodontitis were Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria, and the genera Fusobacterium, Morganella, Porphyromonas, Streptococcus, and Bifidobacterium dominated across all samples. Although extraradicular infection sites showed higher OTU richness and ß-diversity compared to intraradicular samples, the occurrence of sinus tract rather than the sampling sites demarcated the microbial communities in the infections associated with persistent apical periodontitis. PERMANOVA analysis confirmed that the samples with or without sinus tracts contained significantly different microbial communities. Porphyromonas, Eubacterium, Treponema, and Phocaeicola were found in significantly higher levels with sinus tracts, whilst Microbacterium and Enterococcus were more abundant in samples without sinus tracts. In conclusion, diverse bacteria were detected in both intraradicular and extraradicular infections associated with persistent apical periodontitis, which might be influenced by the occurrence of the sinus tract. The results may provide new insight into the pathogenesis of persistent apical periodontitis.

Characteristics of the Irrigant Flow in a Simulated Lateral Canal Under Two Typical Laser-Activated Irrigation Regimens.

BACKGROUND AND OBJECTIVES: Photon-induced photoacoustic streaming (PIPS) and shockwave-enhanced emission photoacoustic streaming (SWEEPS) are two promising laser-activated irrigation (LAI) methods for root canal irrigation. Their performance in driving irrigant flush in a complex root canal system will be evaluated by microscale particle image velocimetry (µPIV) measurement and will be compared with that of ultrasonic-activated irrigation (UAI). STUDY DESIGN/MATERIALS AND METHODS: A µPIV system with 7 µm fluorescent tracer particles was adopted to measure two-dimensional (2D) velocity fields around the junction region, with a size of 1.8 mm × 1.5 mm, between one main canal and one lateral canal in an endodontic training block, which was driven by SWEEPS (Er:YAG laser) operating at 15 Hz and 20 mJ. The flow field driven by PIPS (Er:YAG laser) at the same frequency and energy, as well as by UAI (with non-cutting insert) operating at 40% unit power, was also measured for a direct comparison. RESULTS: It was found that both SWEEPS and PIPS can activate a so-called "breath mode" during the irrigation. Namely, the induced irrigant flush presented a back-and-forth oscillation along both the main canal and the lateral one. The maximum flow speed in the lateral canal was observed to be up to 10 m/s in the SWEEPS modality, while reduced to around 7 m/s in the PIPS modality. The penetration length in the lateral canal in both modalities was estimated to be larger than 1 mm. In comparison, the flow field induced by UAI was characterized as irregular vortical structures, the maximum flow speed in the lateral canal was 0.15 m/s and significantly lower than LAI (P < 0.01), and the penetration length was less than 300 µm and lower than LAI (P < 0.05). CONCLUSION: Compared to UAI, PIPS, and SWEEPS are more capable of delivering the irrigant deeper into the lateral canal. Furthermore, the back-and-forth flush in the breath mode is ideal for removing debris during irrigation. Lasers Surg. Med. © 2020 Wiley Periodicals LLC.

Identification of bacteria associated with periapical abscesses of primary teeth by sequence analysis of 16S rDNA clone libraries.

OBJECTIVE: This study aims to detect the predominant bacteria in acute periapical abscesses of primary teeth using culture-independent molecular methods based on 16S ribosomal DNA cloning. METHODS: Purulent material was collected from nine children diagnosed with abscesses of endodontic origin. DNA was extracted and the 16S rRNA gene amplified with universal primer pairs 27F and 1492R. Amplified genes were cloned, sequenced by Applied Biosystems, and identified by comparison with known 16S rRNA gene sequences. RESULTS: Bacterial DNA was present in all nine purulence samples. A total of 681 clones were classified into 8 phyla, 78 genera, and 125 species/phylotypes. The phyla were Firmicutes, Proteobacteria, Fusobacteria, Bacteroidetes, Actinobacteria, Tenericutes, Deinococcus-Thermus, and Spirochaetes. The most dominant genera were Streptococcus (13.3%), Fusobacterium (11.8%), Parvimonas (7.8%), Prevotella (6.7%), Sphingomonas (5.8%), and Hafnia (5.2%). Fusobacterium nucleatum (11.5%), Parvimonas micra (7.8%), Streptococcus intermedius (6.6%), Sphingomonas echinoides (5.3%), Hafnia alvei (5.2%), and Citrobacter freundii (4.9%) were the most common species/phylotypes. Among these species/phylotypes, F.nucleatum was the most prevalent (7/9). C. freundii, Carnobacterium maltaromaticum, and H. alvei were seldom detected species in acute periapical abscesses but had relatively high abundance and prevalence. CONCLUSION: Acute periapical abscesses are polymicrobial with certain prevalent bacteria, especially anaerobic bacterium. The most predominant and prevalent bacteria of acute periapical abscesses in children was F. nucleatum.

Depletion of PRDM9 enhances proliferation, migration and chemotaxis potentials in human periodontal ligament stem cells.

PURPOSE: Periodontal ligament mesenchymal stem cells (PDLSCs) are important for periodontal tissue regeneration, but how these cells are regulated remains unclear. PRDM (PRDI-BF1 and RIZ homology domain containing) genes play key roles in cell proliferation and differentiation. The present study aimed to investigate the role of one PRDM gene, PRDM9, in the proliferation, migration and chemotaxis potential of PDLSCs. MATERIALS AND METHODS: Cell proliferation was examined on the basis of the cell doubling time, cell counting kit-8 (CCK8) assays, and flow cytometry analysis of the cell cycle. Gene expression was detected by Western blotting and real-time RT-PCR. Scratch migration and Transwell chemotaxis assays were used to analyse cell migration and chemotaxis abilities. Microarray analysis and ChIP assays were used to examine the downstream genes of PRDM9 and the corresponding mechanism. RESULTS: The results showed that knock-down of PRDM9 enhanced cell proliferation by promoting cell cycle progression and rapid transition from the G1 to S phase via downregulation of p21 and p27 and upregulation of cyclin E. Additionally, depletion of PRDM9 increased the migration and chemotaxis potential of PDLSCs. Microarray results showed that 13 genes, including IGFBP5, IFI44L, and POSTN, were upregulated and 34 genes, including PIP, were downregulated after the depletion of PRDM9. Furthermore, we observed that the depletion of PRDM9 promoted the transcription of IGFBP5 by increasing H3K4me3 methylation in the IGFBP5 promoter. CONCLUSION: These discoveries indicated that depletion of PRDM9 increased the cell proliferation, migration and chemotaxis potential of PDLSCs and revealed important downstream genes.

Effects of Ultrasonic Removal of Fractured Files from the Middle Third of Root Canals on the Resistance to Vertical Root Fracture.

INTRODUCTION: This study aimed to analyze the effects of ultrasonic removal of fractured files from the middle third of root canals on the vertical root fracture resistance. METHODS: This study was an extension of a previous study assessing the effects of fractured file removal on dentinal microcracks. It included 18 bilaterally matched pairs of human mandibular incisors prepared and analyzed in the previous study. Briefly, 1 member of each pair was assigned to an ultrasonic or a control group. In the ultrasonic group, K-files were fractured in the middle third of canals followed by ultrasonic file removal and canal preparation. In the control group, the root canals were simply prepared. Micro-computed tomographic imaging was performed before and after treatment, and the cross-sectional root images were screened for microcracks. For the present study, 3-dimensional reconstruction was performed for volumetric assessments. The fracture resistance was measured using a universal testing machine. All data were statistically analyzed. RESULTS: Fracture loads were significantly smaller in the ultrasonic group (P < .05). The percentage increase in the canal volume significantly influenced the root fracture resistance (P < .05), whereas microcracks had no significant effect (P > .05). CONCLUSIONS: Ultrasonic removal of fractured instruments from the middle third of root canals lowers the vertical root fracture resistance, with increased dentin removal being the primary cause.

Analysis of the characteristics and expression profiles of coding and noncoding RNAs of human dental pulp stem cells in hypoxic conditions.

BACKGROUND: Human dental pulp stem cell (DPSC)-mediated regenerative endodontics is a promising therapy for damaged teeth; however, the hypoxic environment in root canals can affect tissue regeneration. In this study, we investigate the characteristics and possible regulatory mechanisms of DPSC function under hypoxic conditions. METHODS: Human DPSCs were cultured under normoxia (20% O2) and hypoxia (3% O2). DPSC proliferation and osteo/odontogenic differentiation potential were assessed by Cell Counting Kit-8 (CCK8) assay, carboxyfluorescein succinimidyl ester (CFSE) assay, alkaline phosphatase (ALP) activity, Alizarin red staining, real-time RT-PCR assays, and western blot analysis. Microarray and bioinformatic analyses were performed to investigate the differences in the mRNA, lncRNA, and miRNA expression profiles of DPSCs. RESULTS: DPSCs exhibited a more powerful proliferation ability and lower osteo/odontogenic differentiation potential in hypoxic conditions. A total of 60 mRNAs (25 upregulated and 35 downregulated), 47 lncRNAs (20 upregulated and 27 downregulated), and 14 miRNAs (7 upregulated and 7 downregulated) in DPSCs were differentially expressed in the hypoxia group compared with the normoxia group. Bioinformatic analysis identified that 7 mRNAs (GRPR, ERO1L, ANPEP, EPHX1, PGD, ANGPT1, and NQO1) and 5 lncRNAs (AF085958, AX750575, uc002czn.2, RP3-413H6.2, and six-twelve leukemia (STL)) may be associated with DPSCs during hypoxia according to CNC network analysis, while 28 mRNAs (including GYS1, PRKACB, and NQO1) and 13 miRNAs (including hsa-miR-3916 and hsa-miR-192-5p) may be involved according to miRNA target gene network analysis. The depletion of one candidate lncRNA, STL, inhibited the osteo/odontogenic differentiation potentials of DPSCs. CONCLUSIONS: Our results revealed that hypoxia could enhance the proliferation ability and impair the osteo/odontogenic differentiation potential of DPSCs in vitro. Furthermore, our results identified candidate coding and noncoding RNAs that could be potential targets for improving DPSC function in regenerative endodontics and lead to a better understanding of the mechanisms of hypoxia's effects on DPSCs.

Quorum sensing LuxS/autoinducer-2 inhibits Enterococcus faecalis biofilm formation ability.

OBJECTIVE: To investigate the relation between biofilm formation ability and quorum sensing gene LuxS/AI-2. MATERIALS AND METHODS: Enterococcus faecalis (E. faecalis) standard strain ATCC 29212 was used in the study. Long flanking homology polymerase chain reaction method was used to build the LuxS gene knockout strain. Sequential culture turbidity measurement and CFU counting were used to assess the proliferation ability of E. faecalis after the depletion of LuxS. 96-well plate assay was used to quantify the biofilm formation ability; CLSM was used to observe the attached bacteria areas, while scanning electron microscopy (SEM) was performed to observe biofilm microstructure conditions. RESULTS: LuxS gene knockout strains were successfully constructed and identified. The results showed that proliferation ability of E. faecalis was not affected by the depletion of the luxS gene, and the biofilm formation ability of ΔLuxS 29212 significantly decreased (P<0.05). CONCLUSIONS: Collectively, our studies provide the LuxS gene's key role in controlling biofilm formation of E. faecalis, which presented a negative regulation, and furthermore, providing us a possible way to conquer the persistent apical periodontitis.