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Aim Methodology Results Conclusions To assess Spanish dentists' management preferences for deep caries removal and exposed pulps in permanent teeth.A web‐based open and anonymous survey was distributed by social media and a specific website for this project amongst dentists practicing in Spain. The questionnaire comprised 40 questions, divided into five sections: (1) demographic data and professional activity; (2) carious tissue removal; (3) decision‐making regarding pulp exposure; (4) direct pulp capping and (5) pulpotomy procedures in permanent teeth. Results were descriptively analysed. Logistic regression (95% CI) analyses and X2 tests were carried out.A total of 538 responses were received. Half the respondents (53.7%) preferred to perform complete caries excavation for shallow and moderate carious dentin lesions, and selective excavation to firm dentin for deep lesions (57.8%). Selective removal to soft dentin and stepwise removal were much less indicated (15.4% and 10.9%, respectively). Exposed pulps in asymptomatic teeth were treated by direct pulp capping (over 80%), decreasing in the presence of reversible pulpitis symptoms (57.1%). If irreversible pulpitis was diagnosed, a pulpectomy would be performed by 53.5% and 89.9% of the respondents in, respectively, immature and mature teeth. Pulpotomy was performed routinely only by 26.4% of the clinicians. Patients' attitudes and priorities were the most relevant criteria when performing direct pulp capping and pulpotomy, together with the history of pain and the presence of bleeding. Regarding the clinical procedure, dry cotton was preferred to obtain haemostasis and Biodentine was the material of election.Caries removal preferences and management of pulp exposure by dentists practicing in Spain deviated from vital pulp treatment guidelines, mainly regarding indications and case selection. Pulp exposure was managed by direct pulp capping in asymptomatic cases, whilst immature permanent molars favoured the indication of pulpotomy when pulpitis was diagnosed. Most clinicians used hydraulic calcium silicate cement, specifically Biodentine, to perform vital pulp treatments. Postgraduate formation and continuing education in caries lesions management and vital pulp treatments were consistently related to more conservative and updated decisions. [ABSTRACT FROM AUTHOR]

Aim Methodology Results Conclusions To investigate the level and distribution of apoptosis, pyroptosis, necroptosis, and NETosis in pulpitis with or without necrosis on a basis of histological classification. Additionally, to examine the effect of pulpitis with necrosis (PWN) on the number and activation of peripheral and bone marrow (BM) neutrophils, as well as spleen lymphocytes, in a mouse model of pulpitis.The material comprised 20 permanent teeth, with or without caries, which were classified into three histological categories based on the distribution of inflammatory cells and the presence or absence of necrosis: (i) healthy pulp (HP), (ii) pulpitis without necrosis (PWON), and (iii) PWN. The levels of the four regulated cell death (RCD) pathways were detected by immunohistochemical and immunofluorescent staining with specific markers: apoptosis (caspase‐8, cleaved caspase‐3), pyroptosis (cleaved caspase‐1, membrane‐binding gasdermin D), necroptosis (receptor‐interacting kinase 3, phosphorylated MLKL), and NETosis (myeloperoxidase, citrullinated histone H3). Acute pulpitis was induced in C57BL/6J mice via pulp exposure, and the mice were divided into four groups: (i) control (no tooth preparation, n = 6), (ii) Day 1 (sacrificed at 1 day after pulp exposure, n = 3), (iii) Day 3 (n = 3), and (iv) Day 5 (n = 7). The control and Day 5 groups were used for further immunofluorescent analysis to assess the levels of RCD and flow cytometry to monitor the changes in peripheral and BM neutrophils, as well as spleen lymphocytes. Human dental pulp stem cells (hDPSCs) were isolated and cultured from extracted healthy third molars. Apoptosis and necroptosis in hDPSCs were induced by staurosporine, whilst pyroptosis was induced by lipopolysaccharide and nigericin. One‐way analysis of variance (ANOVA) with Tukey's test, Welch's ANOVA with Tamhane's test, and Student's t‐tests were used to compare immunohistochemical labelling and flow cytometric data amongst groups (p < .05).The pulpal tissue of PWN can be divided into the abscess core (PWN‐AC) and fibrous tissue (PWN‐FT). The ratio of total necrotic cells (TUNEL‐positive) in PWN‐AC was significantly higher than in PWN‐FT and PWON (both p < .01). Compared with HP, the expression levels of markers for apoptosis and pyroptosis were increased in PWON, whilst the expression levels of markers for apoptosis, pyroptosis, and NETosis were elevated in PWN, primarily detected in PWN‐AC. Interestingly, myeloperoxidase (MPO) was exclusively observed in PWN‐AC, with minimal detection in PWN‐FT and PWON. Additionally, the frequency of MPO+ cells was significantly higher than that of MB‐GSDMD+ cells and Cl‐cas3+ cells in PWN‐AC (both p < .01). Histological observation and TUNEL staining showed abundant necrotic cells in mouse pulpal tissue after pulp exposure, indicating a simulation of human PWN. In mouse pulpitis tissue, markers of apoptosis, pyroptosis, and NETosis were detected. In vitro, various cell deaths including apoptosis, pyroptosis, and necroptosis were also triggered in hDPSCs under various cell death treatments. Furthermore, in terms of systemic changes, pulp exposure‐induced pulpitis could increase the number (p < .05) and cellular activity (p < .01) of neutrophils from BM in a mouse model. No significant changes in peripheral blood neutrophils, spleen T cells, B cells, or the CD4/CD8 ratio were detected between the control and pulpitis mice.Our findings uncover distinct patterns of mixed cell death at different histological stages of human pulpitis and the impact of pulpitis on the number and activity of BM neutrophils. Notably, NETosis occurs specifically and predominates in the abscess area of pulpitis, suggesting a potential effect of neutrophil extracellular traps (NETs) on pulpitis progression and NETs‐targeted diagnostic strategy may play a role in decision making for vital pulp therapy. [ABSTRACT FROM AUTHOR]

Aim: To evaluate the role of biomimetic pulp scaffolds derived from the extracellular matrix derived of stem cells from human exfoliated deciduous teeth (SHED‐ECM‐PS) in promoting pulp–dentine complex regeneration. Methodology: SHED‐ECM‐PS was prepared through cell aggregation and decellularization techniques. Histological and immunofluorescence analyses, scanning electron microscopy, and DNA quantification assays were used to characterize the SHED‐ECM‐PS. Additionally, a tooth slice implantation model was established to evaluate the effects of SHED‐ECM‐PS on regeneration of the pulp–dentine complex in vivo. Extraction medium for SHED‐ECM‐PS was prepared, and its effect on bone marrow mesenchymal stem cells (BMMSCs) was assessed in vitro. Cell counting kit‐8 and Ki‐67 staining assays were performed to determine cell proliferation. The rate of apoptosis was evaluated by flow cytometry. Wound healing and transwell assays were conducted to evaluate cell migration. Alizarin red S staining was performed to examine mineralized nodule formation. Western blotting was used to detect the expression of osteogenic and odontogenic markers. The results were analysed using an independent two‐tailed Student's t‐test. p <.05 was considered statistically significant. Results: SHED‐ECM‐PS was successfully constructed, exhibiting a striped dental pulp‐like shape devoid of nuclear structures or DNA components, and rich in fibronectin, collagen I, DMP1 and DSPP. Notably, SHED‐ECM‐PS showed no impact on the proliferation or apoptosis of BMMSCs. Histological analysis revealed that dental pulp fibroblasts formed an interwoven mesh in the root canal, and angiogenesis was observed in the SHED‐ECM‐PS group. Moreover, a continuous, newly formed tubular dentine layer with polarized odontoblast‐like cells was observed along the inner wall of the root canal. SHED‐ECM‐PS promoted the migration, polar alignment and mineralized nodule formation of BMMSCs and specifically elevated the expression levels of odontogenic markers, but not osteogenic markers, compared with the control group in vitro. Conclusion: SHED‐ECM‐PS exhibited no cytotoxicity and promoted pulp‐dentine complex regeneration in vivo as well as cell migration and odontogenic differentiation of BMMSCs in vitro. These findings provide evidence that SHED‐ECM‐PS, as a novel biological scaffold, has the potential to improve the outcomes of REPs. [ABSTRACT FROM AUTHOR]

Aim Methodology Results Conclusion The aim of this study is to investigate the expression of inflammatory biomarkers (TNF‐α, IL‐10, IL‐1β) and the pulpitis‐associated miRNA (miR‐30a‐5p and miR‐128‐3p) in pulp tissue samples from unrestored teeth with a vital normal pulp (NP), teeth with symptomatic irreversible pulpitis (IP) and in unrestored teeth with periodontal disease, unresponsive to periodontal therapy, and a vital pulp (EP).Thirty patients were included in this observational study (10 teeth with NP, 10 teeth with IP, 10 teeth with EP). Dental pulp tissues samples were collected from patients during root canal treatment (RCT). RNA was extracted and qRT‐PCR of target genes (tumour necrosis factor [TNF]‐α, interleukin [IL]‐1β, IL‐10) and miRNAs (has‐miR‐30a‐5p, has‐miR‐128‐3p) performed to assess the expression profile. Fold‐change in expression was calculated using the formula 2−(ΔCt(Exp)−ΔCt(Ctrl)). One‐way anova with post‐hoc Tukey's was used to determine significant differences between groups. The significance level was set at 5% (p < .05). All teeth were also followed up clinically for 1 year and evaluated for a range of endodontic and periodontal‐related outcomes.All investigated genes significantly increased in expression and miRNAs significantly decreased in expression in the IP and EP groups compared with the NP group (p < .05). With regards to TNF‐α and IL‐1β there were no significant differences in expression between the IP and EP groups (p > .05), whereas IL‐10 expression levels were significantly reduced in the EP compared with the IP group (p < .05). Both miR‐30a‐5p and miR‐128‐3p showed significantly reduced expression in both IP and EP lesions, compared with NP (p < .05); however, no significant differences in miRNA expression were observed between IP and EP groups (p > .05). One year after root canal treatment and periodontal maintenance, tooth mobility and probing depth were significantly reduced in the EP group (p < .05).Pulp tissues from teeth with IP and EP presented similar levels of altered inflammatory markers compared with NP. TNF‐α, IL‐10, IL‐1β cytokines and miRNAs (miR‐30a‐5p and miR‐128‐3p) are potential objective biomarkers to indicate pulpal inflammatory status, aiding diagnosis and directing clinical decision‐making. RCT may be beneficial to improve stage III periodontitis unresponsive to non‐surgical periodontal treatment, but further research is required. [ABSTRACT FROM AUTHOR]

Aim Methodology Results Conclusions Angiogenesis is a key event in the successful healing of pulp injuries, and hypoxia is the main stimulator of pulpal angiogenesis. In this study, we investigated the effect of hypoxia on the proangiogenic potential of human dental pulp stem cells (hDPSCs) and the role of miR‐143‐5p in the process.Human dental pulp stem cells were isolated, cultured and characterized in vitro. Cobalt chloride (CoCl2) was used to induce hypoxia in hDPSCs. CCK‐8 and Transwell assays were used to determine the effect of hypoxia on hDPSCs proliferation and migration. Quantitative real‐time polymerase chain reaction (qRT‐PCR), Western blotting (WB) and ELISA were performed to assess the mRNA and protein levels of HIF‐1α and angiogenic cytokines in hDPSCs. The effect of hypoxia on hDPSCs proangiogenic potential was measured in vitro using Matrigel tube formation and chick chorioallantoic membrane (CAM) assays. Recombinant lentiviral vectors were constructed to stably overexpress or inhibit miR‐143‐5p in hDPSCs, and the proangiogenic effects were assessed using qRT‐PCR, WB, and tube formation assays. miR‐143‐5p target genes were identified and verified using bioinformatics prediction tools, dual‐luciferase reporter assays and RNA pull‐down experiments. Finally, a subcutaneous transplantation model in nude mice was used to determine the effects of hypoxia treatment and miR‐143‐5p overexpression/inhibition in hDPSCs in dental pulp regeneration.Hypoxia promotes hDPSCs proliferation, migration and proangiogenic potential. The in vivo experiments showed that hypoxia treatment (50 and 100 μM CoCl2) promoted pulp angiogenesis and dentine formation. In contrast to the levels of proangiogenic factors, miR‐143‐5p levels decreased with increasing CoCl2 concentration. miR‐143‐5p inhibition significantly promoted proangiogenic potential of hDPSCs, whereas miR‐143‐5p overexpression inhibited angiogenesis in vitro. Dual‐luciferase reporter assay identified retinoic acid receptor‐related orphan receptor alpha (RORA) as an miR‐143‐5p target gene in hDPSCs. RNA pull‐down experiments demonstrated that HIF‐1α and RORA were pulled down by biotin‐labelled miR‐143‐5p, and the levels of HIF‐1α and RORA bound to miR‐143‐5p in the hypoxia group were lower than those in the normoxia group. Inhibition of miR‐143‐5p expression in hDPSCs promoted ectopic dental pulp tissue regeneration.CoCl2‐induced hypoxia promotes hDPSCs‐driven paracrine angiogenesis and pulp regeneration. The inhibition of miR‐143‐5p upregulates the proangiogenic potential of hDPSCs under hypoxic conditions by directly targeting HIF‐1α and RORA. [ABSTRACT FROM AUTHOR]

Aims: Dental pulp stem cells (DPSCs) contain a population of stem cells with a broad range of differentiation potentials, as well as more lineage‐committed progenitors. Such heterogeneity is a significant obstacle to experimental and clinical applications. The aim of this study is to isolate and characterize a homogenous neuronal progenitor cell population from human DPSCs. Methodology: Polysialylated‐neural cell adhesion molecule (PSA‐NCAM+) neural progenitors were isolated from the dental pulp of three independent donors using magnetic‐activated cell sorting (MACS) technology. Immunofluorescent staining with a panel of neural and non‐neural markers was used to characterize the magnetically isolated PSA‐NCAM+ fraction. PSA‐NCAM+ cells were then cultured in Neurobasal A supplemented with neurotrophic factors: dibutyryl cyclic‐AMP, neurotrophin‐3, B27 and N2 supplements to induce neuronal differentiation. Both PSA‐NCAM+ and differentiated PSA‐NCAM+ cells were used in Ca2+ imaging studies to assess the functionality of P2X3 receptors as well as membrane depolarization. Results: PSA‐NCAM+ neural progenitors were isolated from a heterogeneous population of hDPSCs using magnetic‐activated cell sorting and anti‐PSA‐NCAM MicroBeads. Flow cytometry analysis demonstrated that immunomagnetic sorting significantly increased the purity of PSA‐NCAM+ cells. Immunofluorescent staining revealed expression of pan‐neuronal and mature neuronal markers, PGP9.5 and MAP2, respectively, as well as weak expression of the mature sensory markers, peripherin and islet1. ATP‐induced response was mediated predominately by P2X3 receptors in both undifferentiated and differentiated cells, with a greater magnitude observed in the latter. In addition, membrane depolarizations were also detected in cells before and after differentiation when loaded with fast‐voltage‐responding fluorescent molecule, FluoVolt™ in response to potassium chloride. Interestingly, only differentiated PSA‐NCAM+ cells were capable of spontaneous membrane oscillations. Conclusions: In summary, DPSCs contain a population of neuronal progenitors with enhanced neural differentiation and functional neural‐like properties that can be effectively isolated with magnetic‐activated cell sorting (MACS). [ABSTRACT FROM AUTHOR]

Background: The European Society of Endodontology published in 2023, the S3‐level clinical practice guidelines, which supersede the Quality Guidelines for Endodontic Treatment published in 2006. Objectives: This review aims to summarize and compare the above guidelines to support their dissemination. Method: A narrative synthesis of the main differences alongside tabulation according to the main themes. Results: Three tables were prepared according to the following themes: diagnosis of pulpal and apical condition; treatment of pulpitis; and treatment of nonvital pulp and apical periodontitis. Conclusions: A compared and simplified message regarding the most recent clinical practice guidelines has been prepared. Registration: Not applicable as a narrative review. [ABSTRACT FROM AUTHOR]

Aim Methodology Results Conclusions The purpose of this study was to investigate the role of calcium‐sensing receptor (CaSR) in the angiogenic differentiation of lipopolysaccharide (LPS)‐treated human dental pulp cells (hDPCs).The LPS‐induced hDPCs were cultured in the medium with different combinations of CaSR agonist R568 and antagonist Calhex231. The cell proliferation, migration, and angiogenic capacity were measured by Cell Counting Kit‐8 (CCK‐8), scratch wound healing, and tube formation assays, respectively. Enzyme‐linked immunosorbent assay (ELISA), quantitative real‐time polymerase chain reaction (qRT‐PCR), and western blot were conducted to determine the gene/protein expression of CaSR, inflammatory mediators, and angiogenic‐associated markers. The activation of phosphoinositide 3‐kinase (PI3K) and protein kinase B (Akt) was assessed by western blot analysis.The cell proliferation was elevated in response to R568 or Calhex231 exposure, but an enhanced cell migration was only found in cultures supplemented with Calhex231. Furthermore, R568 was found to potentiate the formation of vessel‐like structure, up‐regulated the protein expression of tumour necrosis factor (TNF)‐α, vascular endothelial growth factor (VEGF), and stromal cell‐derived factor (SDF)‐1; comparable influences were also observed in R568‐stimulated cells in the presence of PI3K inhibitor LY294002. In contrast, Calhex231 obviously inhibited the tube formation and VEGF protein level, whereas promoted the production of IL‐6, TNF‐α, and eNOS; however, in the presence of LY294002, Calhex231 showed a significant promotion on the protein expression of CaSR, VEGF, and SDF‐1. In addition, R568 exhibited a promotive action on the Akt phosphorylation, which can be reversed by LY294002.Our results demonstrated that CaSR can regulate the angiogenic differentiation of LPS‐treated hDPCs with an involvement of the PI3K/Akt signalling pathway. [ABSTRACT FROM AUTHOR]

Aim Methodology Results Conclusion Lack of adequate mechanical strength and progressive shrinkage over time remain challenges in scaffold‐free microtissue‐based dental pulp regeneration. Surface collagen cross‐linking holds the promise to enhance the mechanical stability of microtissue constructs and trigger biological regulations. In this study, we proposed a novel strategy for surface preconditioning microtissues using a natural collagen cross‐linker, proanthocyanidin (PA). We evaluated its effects on cell viability, tissue integrity, and biomineralization of dental pulp stem cell (DPSCs)‐derived 3D cell spheroids.Microtissue and macrotissue spheroids were fabricated from DPSCs and incubated with PA solution for surface collagen cross‐linking. Microtissue viability was examined by live/dead staining and 3‐(4,5‐dimethylthiazol‐2‐yl)−2,5‐diphenyltetrazolium bromide (MTT) assay, with transverse dimension change monitored. Microtissue surface stiffness was measured by an atomic force microscope (AFM). PA‐preconditioned microtissues and macrotissues were cultured under basal or osteogenic conditions. Immunofluorescence staining of PA‐preconditioned microtissues was performed to detect dentin sialophosphoprotein (DSPP) and F‐actin expressions. PA‐preconditioned macrotissues were subjected to histological analysis, including haematoxylin–eosin (HE), alizarin red, and Masson trichrome staining. Immunohistochemistry staining was used to detect alkaline phosphatase (ALP) and dentin matrix acidic phosphoprotein 1 (DMP‐1) expressions.PA preconditioning had no adverse effects on microtissue spheroid viability and increased surface stiffness. It reduced dimensional shrinkage for over 7 days in microtissues and induced a larger transverse‐section area in the macrotissue. PA preconditioning enhanced collagen formation, mineralized nodule formation, and elevated ALP and DMP‐1 expressions in macrotissues. Additionally, PA preconditioning induced higher F‐actin and DSPP expression in microtissues, while inhibition of F‐actin activity by cytochalasin B attenuated PA‐induced dimensional change and DSPP upregulation.PA surface preconditioning of DPSCs spheroids demonstrates excellent biocompatibility while effectively enhancing tissue structure stability and promoting biomineralization. This strategy strengthens tissue integrity in DPSC‐derived spheroids and amplifies osteogenic differentiation potential, advancing scaffold‐free tissue engineering applications in regenerative dentistry. [ABSTRACT FROM AUTHOR]

Aim Methodology Results Conclusions The regenerative capacity of dental pulp relies on the odonto/osteogenic differentiation of dental pulp cells (DPCs), but dynamic microenvironmental changes hinder the process. Bone morphogenetic protein 9 (BMP9) promotes differentiation of DPCs towards an odonto/osteogenic lineage, forming dentinal‐like tissue. However, the molecular mechanism underlying its action remains unclear. This study investigates the role of DLX6 antisense RNA 1 (DLX6‐AS1) in odonto/osteogenic differentiation induced by BMP9.Custom RT2 profiler PCR array, quantitative Real‐Time PCR (qRT‐PCR) and western blots were used to investigate the expression pattern of DLX6‐AS1 and its potential signal axis. Osteogenic ability was evaluated using alkaline phosphatase and alizarin red S staining. Interactions between lncRNA and miRNA, as well as miRNA and mRNA, were predicted through bioinformatic assays, which were subsequently validated via RNA immunoprecipitation and dual luciferase reporter assays. Student's t‐test or one‐way ANOVA with post hoc Tukey HSD tests were employed for data analysis, with a p‐value of less than .05 considered statistically significant.DLX6‐AS1 was upregulated upon BMP9 overexpression in DPCs, thereby promoting odonto/osteogenic differentiation. Additionally, miR‐128‐3p participated in BMP9‐induced odonto/osteogenic differentiation by interacting with the downstream signal MAPK14. Modifying the expression of miR‐128‐3p and transfecting pcMAPK14/siMAPK14 had a rescue impact on odonto/osteogenic differentiation downstream of DLX6‐AS1. Lastly, miR‐128‐3p directly interacted with both MAPK14 and DLX6‐AS1.DLX6‐AS1 could regulate the odonto/osteogenic differentiation of DPCs under the control of BMP9 through the miR‐128‐3p/MAPK14 axis. [ABSTRACT FROM AUTHOR]

Aim: To analyse the effect of ultrasonic irrigant activation (UIA) and the GentleWave (GW) multisonic irrigation (GW) with minimal instrumentation on the root canal microbial diversity in an ex vivo model that used extracted molars with a history of pulp necrosis. Methodology: Twenty‐three mandibular molars were prepared ex vivo for collection of superficial (surface control), pre‐treatment and post‐treatment samples 24 h after extraction. Samples were divided into two groups: UIA using 6% NaOCl (n = 11) and GW group (n = 12). All samples were processed using quantitative real‐time polymerase chain reaction (qPCR) and 16S rRNA next‐generation sequencing to measure microbial diversity before and after the antimicrobial treatment. For qPCR, a t‐test (α =.05) was used to compare the log10 reduction. The Chao1 and Shannon indices evaluated alpha diversity. Differences in community composition (beta diversity) were evaluated by analysis of similarity (ANOSIM). Kruskal–Wallis test with Bonferroni corrections was performed to evaluate the differences in abundances genera in the samples. Results: Quantitative real‐time polymerase chain reaction revealed an estimated 1.6 and 2.6 log10 reduction for UIA and GW groups respectively (p =.048). An average of 5 ± 4 and 3 ± 5 operational taxonomic units (OTUs) were found in surface's samples in the UIA and GW group respectively. These values were significantly lower (p <.001) compared to the number of preoperative OTUs in those groups (155 ± 79 and 187 ± 121). In assessing beta diversity, there were no significant differences found in pre‐treatment samples (R =.090, p =.070 ANOSIM with Bonferroni corrections). Also, no significant differences in community composition were observed in post‐treatment samples (R = −.05, p =.829). After treatment, there was a significant reduction of Eubacterium using conventional treatment with UIA and a significant reduction of Prevotella using minimal instrumentation with GW irrigation (p =.007 and p =.002 respectively). Conclusion: Quantitative PCR analysis revealed a significant reduction in microbial load for GW group. Overall, diversity changes were similar between UIA and GW irrigation in this ex vivo model that used extracted teeth with a history of pulp necrosis. OTUs obtained from the surface sample were negligible and did not affect the statistical outcome of the study. [ABSTRACT FROM AUTHOR]

Aim Methodology Results Conclusions This study investigated the effects of the inflammatory microenvironment of moderate pulpitis on biological properties of human dental pulp stem cells (DPSCs) and further explored the mechanism involved in osteo‐/odontogenic induction of the inflammatory microenvironment.Healthy DPSCs (hDPSCs) and inflammatory DPSCs (iDPSCs) were isolated from human‐impacted third molars free of caries and clinically diagnosed with moderate pulpitis, respectively. Healthy DPSCs were treated with lipopolysaccharides (LPS) to mimic iDPSCs in vitro. The surface markers expressed on hDPSCs and iDPSCs were detected by flow cytometry. A CCK‐8 assay was performed to determine cell proliferation. Flow cytometric analysis was used to evaluate cell apoptosis. The osteo‐/odontogenic differentiation of DPSCs was evaluated by western blot, alkaline phosphatase staining, and Alizarin Red S staining. The functions of the genes of differentially expressed mRNAs of hDPSCs and iDPSCs were analysed using gene set enrichment analysis. Transmission electron microscopy and western blot were used to evaluate the autophagy changes of LPS‐treated DPSCs.Compared with hDPSCs, iDPSCs showed no significant difference in proliferative capacity but had stronger osteo‐/odontogenic potential. In addition, the mRNAs differentially expressed between iDPSCs and hDPSCs were considerably enriched in autophagosome formation and assembly‐related molecules. In vitro mechanism studies further found that low concentrations of LPS could upregulate DPSC autophagy‐related protein expression and autophagosome formation and promote its odontogenic/osteogenic differentiation, whereas the inhibition of DPSC autophagy led to the weakening of the odontogenic/osteogenic differentiation induced by LPS.This explorative study showed that DPSCs isolated from teeth with moderate pulpitis possessed higher osteo‐/odontogenic differentiation capacity, and the mechanism involved was related to the inflammatory microenvironment‐mediated autophagy of DPSCs. This helps to better understand the repair potential of inflamed dental pulp and provides the biological basis for pulp preservation and hard tissue formation in minimally invasive endodontics. [ABSTRACT FROM AUTHOR]

Aim Methodology Results Conclusions The European Society of Endodontology outlines best practices for the management of deep caries and the pulp. Despite evidence supporting vital pulp treatments (VPTs) as predictable alternatives to conventional endodontic treatment, studies have shown they are not widely adopted in the UK. This study aimed to explore the barriers to implementation of VPTs by primary care general dental practitioners (GDPs).Qualitative one‐to‐one semi‐structured online interviews were conducted with purposively sampled UK GDPs. Interview transcripts were analysed using reflexive thematic analysis. Recurring themes were iteratively refined as additional transcripts were reviewed.Eleven participants were interviewed. A range of barriers to the provision of VPTs were identified, which aligned with two core themes: ‘Motivational barriers to service provision’ and ‘Educational access & opportunities’. Sub‐themes included lack of access to materials and equipment, deficiencies in knowledge of treatment (including protocols, outcomes and prognosis), lack of confidence (in treatment efficacy and clinical ability), time constraints and public dental service funding and remuneration.This study identifies barriers to the widespread adoption of VPTs among primary care GDPs in public and private settings. Economic constraints, practitioner confidence, time limitations and educational gaps are key challenges. Addressing these may require systemic changes such as policy interventions, education and improved resource allocation. [ABSTRACT FROM AUTHOR]

Background: Although several studies indicate the harmful effects of bleaching on pulp tissue, the demand for this procedure using high concentrations of hydrogen peroxide (HP) is high. Objectives: To investigate the influence of bleaching on the pulp tissue. Methods: Electronic searches were conducted (PubMed/MEDLINE, Scopus, Cochrane Library and grey literature) until February 2021. Only in vivo studies that evaluated the effects of HP and/or carbamide peroxide (CP) bleaching gels on the inflammatory response in the pulp tissue compared with a non‐bleached group were included. Risk of bias was performed according to a modified Methodological Index for Non‐Randomized Studies scale for human studies and the Systematic Review Centre for Laboratory Animal Experimentation's RoB tool for animal studies. Meta‐analysis was unfeasible. Results: Of the 1311 studies, 30 were eligible. Of these, 18 studies evaluated the inflammatory response in animal models. All these studies reported a moderate‐to‐strong inflammatory response in the superficial regions of pulp, characterized by cell disorganization and necrotic areas, particularly during the initial periods following exposure to 35%–38% HP, for 30–40 min. In the evaluation of human teeth across 11 studies, seven investigated inflammatory responses, with five observing significant inflammation in the pulp of bleached teeth. In terms of tertiary dentine deposition, 11 out of 12 studies noted its occurrence after bleaching with 35%–38% HP in long‐term assessments. Additionally, three studies reported significant levels of osteocalcin/osteopontin at 2 or 10 days post‐treatment. Other studies indicated an increase in pro‐inflammatory cytokines ranging from immediately up to 10 days after bleaching. Studies using humans' teeth had a low risk of bias, whereas animal studies had a high risk of bias. Discussion: Despite the heterogeneity in bleaching protocols among studies, High‐concentrations of HP shows the potential to induce significant pulp damage. Conclusions: High‐concentrations of bleaching gel increases inflammatory response and necrosis in the pulp tissue at short periods after bleaching, mainly in rat molars and in human incisors, in addition to greater hard tissue deposition over time. However, further well‐described histological studies with long‐term follow‐up are encouraged due to the methodological limitations of these studies. Registration: PROSPERO (CRD42021230937). [ABSTRACT FROM AUTHOR]

Aim: Loss‐of‐function mutations in FAM20A result in amelogenesis imperfecta IG (AI1G) or enamel‐renal syndrome, characterized by hypoplastic enamel, ectopic calcification, and gingival hyperplasia, with some cases reporting spontaneous tooth infection. Despite previous reports on the consequence of FAM20A reduction in gingival fibroblasts and transcriptome analyses of AI1G pulp tissues, suggesting its involvement in mineralization and infection, its role in deciduous dental pulp cells (DDP) remains unreported. The aim of this study was to evaluate the properties of DDP obtained from an AI1G patient, providing additional insights into the effects of FAM20A on the mineralization of DDP. Methodology: DDP were obtained from a FAM20A‐AI1G patient (mutant cells) and three healthy individuals. Cellular behaviours were examined using flow cytometry, MTT, attachment and spreading, colony formation, and wound healing assays. Osteogenic induction was applied to DDP, followed by alizarin red S staining to assess their osteogenic differentiation. The expression of FAM20A‐related genes, osteogenic genes, and inflammatory genes was analysed using real‐time PCR, Western blot, and/or immunolocalization. Additionally, STRING analysis was performed to predict potential protein–protein interaction networks. Results: The mutant cells exhibited a significant reduction in FAM20A mRNA and protein levels, as well as proliferation, migration, attachment, and colony formation. However, normal FAM20A subcellular localization was maintained. Additionally, osteogenic/odontogenic genes, OSX, OPN, RUNX2, BSP, and DSPP, were downregulated, along with upregulated ALP. STRING analysis suggested a potential correlation between FAM20A and these osteogenic genes. After osteogenic induction, the mutant cells demonstrated reduced mineral deposition and dysregulated expression of osteogenic genes. Remarkably, FAM20A, FAM20C, RUNX2, OPN, and OSX were significantly upregulated in the mutant cells, whilst ALP, and OCN was downregulated. Furthermore, the mutant cells exhibited a significant increase in inflammatory gene expression, that is, IL‐1β and TGF‐β1, whereas IL‐6 and NFκB1 expression was significantly reduced. Conclusion: The reduction of FAM20A in mutant DDP is associated with various cellular deficiencies, including delayed proliferation, attachment, spreading, and migration as well as altered osteogenic and inflammatory responses. These findings provide novel insights into the biology of FAM20A in dental pulp cells and shed light on the molecular mechanisms underlying AI1G pathology. [ABSTRACT FROM AUTHOR]

Aims: This study aimed to investigate the anti‐inflammatory and odontoblastic effects of cerium‐containing mesoporous bioactive glass nanoparticles (Ce‐MBGNs) on dental pulp cells as novel pulp‐capping agents. Methodology: Ce‐MBGNs were synthesized using a post‐impregnation strategy based on the antioxidant properties of Ce ions and proposed the first use of Ce‐MBGNs for pulp‐capping application. The biocompatibility of Ce‐MBGNs was analysed using the CCK‐8 assay and apoptosis detection. Additionally, the reactive oxygen species (ROS) scavenging ability of Ce‐MBGNs was measured using the 2,7‐Dichlorofuorescin Diacetate (DCFH‐DA) probe. The anti‐inflammatory effect of Ce‐MBGNs on THP‐1 cells was further investigated using flow cytometry and quantitative real‐time polymerase chain reaction (RT‐qPCR). Moreover, the effect of Ce‐MBGNs on the odontoblastic differentiation of the dental pulp cells (DPCs) was assessed by combined scratch assays, RT‐qPCR, western blotting, immunocytochemistry, Alizarin Red S staining and tissue‐nonspecific alkaline phosphatase staining. Analytically, the secretions of tumour necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β) were detected with enzyme‐linked immunosorbent assay (ELISA). Results: Ce‐MBGNs were confirmed to effectively scavenge ROS in THP‐1‐derived macrophages and DPCs. Flow cytometry and RT‐qPCR assays revealed that Ce‐MBGNs significantly inhibited the M1 polarization of macrophages (Mφ). Furthermore, the protein levels of TNF‐α and IL‐1β were downregulated in THP‐1‐derived macrophages after stimulation with Ce‐MBGNs. With a step‐forward virtue of promoting the odontoblastic differentiation of DPCs, we further confirmed that Ce‐MBGNs could regulate the formation of a conductive immune microenvironment with respect to tissue repair in DPCs, which was mediated by macrophages. Conclusions: Ce‐MBGNs protected cells from self‐produced oxidative damage and exhibited excellent immunomodulatory and odontoblastic differentiation effects on DPCs. As a pulp‐capping agent, this novel biomaterial can exert anti‐inflammatory effects and promote restorative dentine regeneration in clinical treatment. We believe that this study will stimulate further correlative research on the development of advanced pulp‐capping agents. [ABSTRACT FROM AUTHOR]

Aim: To explore the influence of PDGF‐AA on cell communication between human dental pulp stem cells (DPSCs) by characterizing gap junction intercellular communication (GJIC) and its potential biomechanical mechanism. Methodology: Quantitative real‐time PCR was used to measure connexin family member expression in DPSCs. Cell migration and CCK‐8 assays were utilized to examine the influence of PDGF‐AA on DPSC migration and proliferation. A scrape loading/dye transfer assay was applied to evaluate GJIC triggered by PDGF‐AA, a PI3K/Akt signalling pathway blocker (LY294002) and a PDGFR‐α blocker (AG1296). Western blotting and immunofluorescence were used to test the expression and distribution of the Cx43 and p‐Akt proteins in DPSCs. Scanning electron microscopy (SEM) and immunofluorescence were used to observe the morphology of GJIC in DPSCs. Results: PDGF‐AA promoted gap junction formation and intercellular communication between human dental pulp stem cells. PDGF‐AA upregulates the expression of Cx43 to enhance gap junction formation and intercellular communication. PDGF‐AA binds to PDGFR‐α and activates PI3K/Akt signalling to regulate cell communication. Conclusions: This research demonstrated that PDGF‐AA can enhance Cx43‐mediated GJIC in DPSCs via the PDGFR‐α/PI3K/Akt axis, which provides new cues for dental pulp regeneration from the perspective of intercellular communication. [ABSTRACT FROM AUTHOR]

Aim: To investigate novel diagnostic markers for pulpitis and validate by clinical samples from normal and inflamed pulp. To explore the relationship between diagnostic markers and immune cells or their phenotypes during pulp inflammation. Methodology: Two microarray datasets, GSE77459 and GSE92681, and identified differential expression genes were integrated. To understand immune features, gene functions, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Disease Ontology (DO) and ImmuneSigDB Gene Set Enrichment Analysis (GSEA) were analysed. For predictive purposes, machine learning techniques were applied to detect diagnostic markers. Immune infiltration in inflamed pulp was studied using CIBERSORT. The relationship between diagnostic markers and immune cells was investigated and validated their gene expression in clinical samples from the normal or inflamed pulp by qRT‐PCR. Finally, the correlation between one marker, secreted phosphoprotein 1 (SPP1), encoding osteopontin (OPN), and dendritic cells (DCs)/macrophages was identified via HE staining and multiplex immunohistochemistry. An in vitro inflammatory dental pulp microenvironment model of THP‐1 macrophages cocultured with dental pulp cells derived conditioned media (DPCs‐CM) to investigate OPN production and macrophage phenotypes was established. Results: Analysis revealed unique immunologic features in inflamed pulp. Three diagnostic markers for pulpitis: endothelin‐1 (EDN1), SPP1, and purine nucleoside phosphorylase (PNP), and validated them using qRT‐PCR were predicted. Multiplex immunohistochemistry demonstrated OPN co‐localized with activated DCs and M2 macrophages during pulp inflammation. In vitro experiments showed that THP‐1 macrophages produced the highest levels of OPN when stimulated with DPCs‐CM derived from the 20 μg/mL LPS pre‐conditioned group, suggesting an M2b‐like phenotype by increasing surface marker CD86 and expression of IL6, TNFα, IL10, and CCL1 but not CCL17 and MerTK. Levels of CCL1 and IL10 elevated significantly in the macrophages' supernatant from the 20 μg/mL LPS pre‐conditioned CM group. OPN was proven co‐localizing with CD86 in the inflamed pulp by immunofluorescence. Conclusions: The current findings suggest that OPN can serve as a promising biomarker for pulpitis, correlated with DCs and macrophages. OPN+ macrophages in the inflamed pulp are associated with M2b‐like phenotypes. These insights offer the potential for improved diagnosis and targeted therapy. [ABSTRACT FROM AUTHOR]

Aim: This study evaluated the transdentinal cytotoxic effects of enzymatic agents (EA) for chemomechanical carious tissue removal on human dental pulp cells. Methodology: The groups were based on the performed dentine treatments (n = 8): G1: Positive Control (PC ‐ no treatment); G2: Negative Control (NC ‐ 35% H2O2 for 2 min); G3: Brix 3000™ (BX) for 30 s; G4: BX for 2 min; G5: Papacarie Duo™ (PD) for 30 s; G6: PD for 2 min. The cells were evaluated for viability (VB; MTT assay) and production of reactive oxygen species (ROS; DCFH‐DA assay) and nitric oxide (NO; Griess reagent). A scanning electron microscope provided morphological chemical analyses and energy‐dispersive X‐ray spectroscopy. The data were submitted to the one‐way anova statistical test complemented by Tukey (p <.05). Results: Cell viability decreased by 21.1% and 58.4% in G5 and G6, respectively. ROS production in G3 and G4 maintained basal levels but increased by 171.2% and 75.1% in G5 and G6, respectively. Conclusions: The Brix3000™ enzymatic agent did not cause indirect cytotoxic effects on pulp cells, regardless of the application time. Conversely, Papacarie Duo™ reduced viability and increased ROS production by pulp cells. [ABSTRACT FROM AUTHOR]

Aim: Guanylate‐binding protein 5 (GBP5) is an interferon (IFN)‐inducible GTPase that plays a crucial role in the cell‐autonomous immune response against microbial infections. In this study, we investigated the immunoregulatory role of GBP5 in the pathogenesis of dental pulpitis. Methodology: Gene‐set enrichment analysis (GSEA) was utilized to evaluate the IFN‐γ signalling pathway, and the differential expression of GBP mRNA in normal versus inflamed dental pulp tissues was screened, based on Gene Expression Omnibus (GEO) datasets associated with pulpitis. Both normal pulp tissues and inflamed pulp tissues were used for experiments. The expression of IFNs and GBPs was determined by qRT–PCR. Immunoblotting and double immunofluorescence were performed to examine the cellular localization of GBP5 in dental pulp tissues. For the functional studies, IFN‐γ priming or lentivirus vector‐delivered shRNA was used to, respectively, overexpress or knock down endogenous GBP5 expression in human dental pulp stem cells (HDPSCs). Subsequently, LPS was used to stimulate HDPSCs (overexpressing or with knocked‐down GBP5) to establish an in vitro model of inflammation. qRT–PCR and ELISA were employed to examine the expression of proinflammatory cytokines (IL‐6, IL‐8 and IL‐1β) and cyclooxygenase 2 (COX2). Every experiment has three times of biological replicates and three technical replicates, respectively. Statistical analysis was performed using the Student's t‐test and one‐way ANOVA, and a p‐value of <.05 was considered statistically significant. Results: GSEA analysis based on the GEO dataset revealed a significant activation of the IFN‐γ signalling pathway in the human pulpitis group. Among the human GBPs evaluated, GBP5 was selectively upregulated in inflamed dental pulp tissues and predominantly expressed in dental pulp cells. In vitro experiments demonstrated that IFN‐γ robustly induced the expression of GBP5 in HDPSCs. Knockdown of GBP5 expression in HDPSCs significantly amplified the LPS‐induced upregulation of inflammatory mediators (IL‐6, IL‐8, IL‐1β and COX2) both with and without IFN‐γ priming. Conclusion: Our findings demonstrated that GBP5 partook in the pathogenesis of dental pulpitis. The involvement of GBP5 in pulpitis appeared to coordinate the regulation of inflammatory cytokines. Knockdown of GBP5 contributed to the exacerbation of LPS‐mediated inflammation. [ABSTRACT FROM AUTHOR]

Aim: To develop a new coculture system that allows exposure of dental pulp cells (DPCs) to Streptococcus mutans and dentine matrix proteins (eDMP) to study cellular interactions in dentine caries. Methodology: Dental pulp cells and S. mutans were cocultured with or without eDMP for 72 h. Cell proliferation and viability were assessed by cell counting and MTT assays, while bacterial growth and viability were determined by CFU and LIVE/DEAD staining. Glucose catabolism and lactate excretion were measured photometrically as metabolic indicators. To evaluate the inflammatory response, the release of cytokines and growth factors (IL‐6, IL‐8, TGF‐β1, VEGF) was determined by ELISA. Non‐parametric statistical analyses were performed to compare all groups and time points (Mann–Whitney U test or Kruskal–Wallis test; α =.05). Results: While eDMP and especially S. mutans reduced the number and viability of DPCs (p ≤.0462), neither DPCs nor eDMP affected the growth and viability of S. mutans during coculture (p >.0546). The growth of S. mutans followed a common curve, but the death phase was not reached within 72 h. S. mutans consumed medium glucose in only 30 h, whereas in the absence of S. mutans, cells were able to catabolize glucose throughout 72 h, resulting in the corresponding amount of l‐lactate. No change in medium pH was observed. S. mutans induced IL‐6 production in DPCs (p ≤.0011), whereas eDMP had no discernible effect (p >.7509). No significant changes in IL‐8 were observed (p >.198). TGF‐β1, available from eDMP supplementation, was reduced by DPCs over time. VEGF, on the other hand, was increased in all groups during coculture. Conclusions: The results show that the coculture of DPCs and S. mutans is possible without functional impairment. The bacterially induced stimulation of proinflammatory and regenerative cytokines provides a basis for future investigations and the elucidation of molecular biological relationships in pulp defence against caries. [ABSTRACT FROM AUTHOR]

Aim: To investigate the effect of IWP‐2, Wnt inhibitor, on human dental pulp stem cells (hDPSCs) responses. Methodology: hDPSCs were isolated from human dental pulp tissues. Cells were treated with 25 μM IWP‐2 for 24 h, and subsequently, the gene expression profile was examined using high‐throughput RNA sequencing. The mRNA expression was analysed using qPCR. The effect of IWP‐2 was investigated in both normal and LPS‐induced hDPSCs (inflamed hDPSCs). CD4+ T cells and CD14+ monocyte‐derived macrophages were cultured with conditioned media of IWP‐2 treated hDPSCs to observe the immunosuppressive property. Results: RNA sequencing indicated that IWP‐2 significantly downregulated several KEGG pathways, including cytokine‐cytokine receptor interaction, IL‐17 signalling pathway, and TNF signalling pathway. In both normal and inflamed conditions, IWP‐2 markedly upregulated TGFB1 mRNA expression while the mRNA expression of pro‐inflammatory cytokines, TNFA, IL1B, IFNG, and IL6, was inhibited. In the inhibition experiment, the pretreatment with p38, MAPK, or PI3K inhibitors abolished the effects of IWP‐2 in LPS‐induced inflammation. In terms of immune cells, IWP‐2‐treated‐inflamed hDPSCs conditioned media attenuated T cell proliferation and regulated regulatory T cell differentiation. In addition, the migratory property of macrophage was decreased after being exposed to IWP‐2‐treated inflamed hDPSCs conditioned media. Conclusion: IWP‐2 suppressed inflammatory cytokine expression in both normal and inflamed hDPSCs. Moreover, hDPSCs exerted the immunosuppressive property after IWP‐2 treatment. These results suggest the role of Wnt in inflammatory responses and immunomodulation in dental pulp tissues. [ABSTRACT FROM AUTHOR]

Aim: Dental pulp is richly innervated by nerve fibres, which are mainly involved in the sensation of pain. Aside from pain sensation, little is known regarding the role of dental innervation in reparative dentine formation. We herein generated a mouse model of experimental dentine injury to examine nerve sprouting within the odontoblast and subodontoblastic layers and investigated the potential effects of this innervation in reparative dentinogenesis. Methodology: Mouse tooth cavity model (bur preparation + etching) was established, and then nerve sprouting, angiogenesis and reparative dentinogenesis were determined by histological and immunofluorescent staining at 1, 3, 7, 14 and 28 days postoperatively. We also established the mouse‐denervated molar models to determine the role of sensory and sympathetic nerves in reparative dentinogenesis, respectively. Finally, we applied calcitonin gene‐related peptide (CGRP) receptor antagonist to analyse the changes in angiogenesis and reparative dentinogenesis. Results: Sequential histological results from dentine‐exposed teeth revealed a significant increase in innervation directly beneath the injured area on the first day after dentine exposure, followed by vascularisation and reparative dentine production at 3 and 7 days, respectively. Intriguingly, abundant type H vessels (CD31+Endomucin+) were present in the innervated area, and their formation precedes the onset of reparative dentine formation. Additionally, we found that sensory denervation led to blunted angiogenesis and impaired dentinogenesis, while sympathetic denervation did not affect dentinogenesis. Moreover, a marked increase in the density of CGRP+ nerve fibres was seen on day 3, which was reduced but remained elevated over the baseline level on day 14, whereas the density of substance P‐positive nerve fibres did not change significantly. CGRP receptor antagonist‐treated mice showed similar results as those with sensory denervation, including impairments in type H angiogenesis, which confirms the importance of CGRP in the formation of type H vessels. Conclusions: Dental pulp sensory nerves act as an essential upstream mediator to promote angiogenesis, including the formation of type H vessels, and reparative dentinogenesis. CGRP signalling governs the nerve‐vessel‐reparative dentine network, which is mostly produced by newly dense sensory nerve fibres within the dental pulp. [ABSTRACT FROM AUTHOR]

Aim: This study aimed to investigate the upstream regulators and specific mechanisms of metastasis‐associated lung adenocarcinoma transcript 1 (MALAT1) in the odontoblastic differentiation of human dental pulp stem cells (hDPSCs). Methodology: Human dental pulp stem cells were isolated and cultured, followed by conducting loss‐ or gain‐of‐function experiments on ATF4 and loss experiments on MALAT1 to elucidate their respective biological functions in odontoblastic differentiation. Chromatin immunoprecipitation assays and RNA immunoprecipitation were performed to uncover the interaction between ATF4‐MALAT1 and MALAT1‐JMJD3, respectively. The odontoblastic differentiation was estimated by the mRNA and protein of DSPP and DMP1, as well as alkaline phosphatase staining. Results: Expression of MALAT1 was upregulated in the hDPSCs cultured in an odontoblastic medium, and MALAT1 downregulation suppressed the odontoblastic differentiation of the hDPSCs. Subsequent experiments confirmed that ATF4 promoted odontoblastic differentiation and induced MALAT1 expression by binding to the MALAT1 promoter region. Further experiments revealed that nuclear MALAT1 interacted with JMJD3. MALAT1 knockdown decreased the JMJD3 protein level and demethylase activity, and it enhanced H3K27me3 occupancy of the promoter region of DSPP and DMP1, resulting in the inhibition of DSPP and DMP1 transcription. Importantly, JMJD3 overexpression significantly attenuated the inhibition of odontoblastic differentiation induced by MALAT1 knockdown. Conclusions: ATF4‐regulated MALAT1 plays a positive regulatory role in odontoblastic differentiation of hDPSCs through JMJD3‐mediated H3K27me3 modifications of the DSPP and DMP1 promoters. [ABSTRACT FROM AUTHOR]

Aim: This study aimed to compare the outcome of SCR and Pulpotomy in teeth with deep caries extending at least 75% into dentine. Methodology: This two‐armed, parallel‐group, randomized, superiority trial included vital mature permanent teeth with deep primary or secondary caries diagnosed radiographically as being at least 75% into the thickness of dentine, without clinical signs of symptomatic irreversible pulpitis or radiographic evidence of a periapical lesion. Carious teeth were blindly allocated to receive either SCR or Pulpotomy using computer‐generated randomized patient codes concealed in opaque envelopes. All teeth were reviewed clinically and radiographically at 6 months and 1 year post‐treatment. Using a significance level of p <.05, the log rank test and Cox proportional hazards regression were used to compare the outcome of SCR and Pulpotomy and to identify potential prognostic factors, respectively. Results: In all, 58 teeth in the SCR group and 55 teeth in the pulpotomy group completed treatment, after excluding 6 teeth because they did not complete the allocated treatment and another due to severe periodontal disease. At one year, 57/58 (98.3%) teeth from the SCR group and 48/55 (87.3%) teeth from the Pulpotomy group were available for analysis. One tooth in the Pulpotomy group (2.1%) and eight teeth in the SCR group (14.0%) required the further intervention of root canal treatment (p <.05). There were no other significant prognostic factors for survival. Overall, 91.4% of teeth treated with either SCR or Pulpotomy survived without requiring further intervention over a period of one year. No other adverse events occurred over the review period. Conclusion: Within the limitations of this study, Pulpotomy fares better than SCR in preserving the remaining pulp and periapical health. As a treatment modality, Pulpotomy carries greater cost outlay to patient and takes a longer time to complete treatment than SCR. Long‐term follow‐up is needed to study the pulpal and restorative outcomes of Pulpotomy and SCR. [ABSTRACT FROM AUTHOR]

Aim: The primary goal of this study was to investigate the potential effects of A5G81 in inducing reparative dentine (RD) formation both in vitro and in vivo. Methodology: Cell adhesion was observed by crystal violet staining and quantified by Sodium Dodecyl Sulphate (SDS) extraction. Cell proliferation was investigated using Cell Counting Kit‐8 (CCK‐8) assay. Spreading of cytoskeleton was visualized using immunofluorescence staining. Protein expression level of Akt signalling pathway was compared in a human Akt pathway phosphorylation array. Genes that were up or downregulated by A5G81 were identified by RNA sequencing. The mRNA expression of odontoblastic markers was detected by quantitative real‐time polymerase chain reaction (qPCR). Moreover, mineralization of human dental pulp cells (hDPCs) was visualized by alizarin red staining and quantified using cetylpyridinium chloride (CPC). A direct pulp‐capping model was established in SD rats and the RD formation at 2 weeks after operation was observed using HE staining. Results: A5G81 (optimal coating concentration: 0.5 mg/mL) promoted hDPCs adhesion and proliferation to a level that was similar to Type I collagen (COL‐1). Meanwhile, A5G81 activated Akt signalling pathway, albeit to a lesser extent than COL‐1. An inhibition test indicated that A5G81 induced hDPCs adhesion by activating PI3K pathway. A5G81 induced the expression of ECM remodelling genes and odontoblastic genes, which were demonstrated by RNA‐seq and qPCR, respectively. In addition, A5G81 efficiently accelerated the mineralization of hDPCs in both immobilized and soluble forms, a property that makes it more applicable in dental clinic. Finally, the pulp‐capping study in rats suggested that use of A5G81 could successfully induce the formation of RD within 2 weeks. Conclusion: Coating of A5G81 to non‐tissue culture–treated polystyrene facilitates spreading, proliferation and differentiation of hDPCs, resulting in rapid RD formation in artificially exposed pulp. [ABSTRACT FROM AUTHOR]

Aim: This ex vivo study aimed to compare protein expression of advanced glycation end‐products (AGE) and receptor (RAGE), and the levels of selected genes associated with inflammation and collagen within dental pulp tissue from patients with type 2 (T2D) diabetes and non‐T2D. Methodology: Noncarious extracted permanent molar teeth from patients with well‐controlled T2D (n = 19) and non‐T2D (controls) (n = 19) were collected and compared. The coronal pulp was examined using immunohistochemistry (IHC) (n = 10 per group) for anti‐AGE and anti‐RAGE. Quantitative PCR (n = 9 per group) was used to analyse the gene expression levels of NFKB, S100A12 and COLIA1. Data analyses were performed between the groups using GraphPad Prism using Pearson correlation, Shapiro–Wilk and Mann–Whitney U‐tests, and multiple regression using SPSS. Results: AGEs were distributed diffusely throughout the pulp extracellular matrix associated with collagen fibres and were present on several cell types. RAGE was expressed at the pulp–dentine interface and was observed on odontoblasts, immune cells, endothelial cells and fibroblasts. Semi‐quantitative analysis of IHC samples showed significantly increased expression of AGE (p <.0001) and RAGE (p =.02) in T2D samples compared with controls. The expression of NFKB (p <.0001), S100A12 (p <.0001) and COLIA1 (p =.01) genes were significantly higher in the T2D pulp, and multivariate logistic regression analysis showed that these findings were not affected by age. Conclusion: T2D may exert a similar glycation response in the dental pulp to other body sites. This could occur through activation of NF‐κB pathways with a concomitant increase in genes associated with inflammation and collagen. [ABSTRACT FROM AUTHOR]

Aim: The objective of this study was to assess and compare the cytocompatibility of decellularized porcine small intestine submucosal dural graft from Biodesign (BD) and polyester urethane‐based Neuro‐Patch (NP) dural substitute with the mineral trioxide aggregate (MTA) and the cyanoacrylate‐based Histoacryl surgical adhesive. Furthermore, the study evaluated the inflammatory response and osteogenic differentiation of human dental pulp stem cells (hDPSCs) when cultured in direct contact with the dural substitutes in comparison with MTA. Methodology: The viability of hDPSCs in direct contact with the tested materials was investigated in vitro by a CCK‐8 assay. Additionally, the effects of dural substitutes and MTA on the expression of the inflammatory mediator interleukin‐6 (IL‐6) was investigated via enzyme‐linked immunosorbent assay (ELISA), whilst effects on the differentiation were evaluated using alizarin red staining, alkaline phosphatase staining, ELISA and energy‐dispersive X‐ray elemental mapping. Results: The dural substitutes were cytocompatible and promoted cellular adhesion. The Histoacryl and MTA demonstrated cytotoxicity in fresh preparations but showed a more favourable cellular reaction when set. Investigations of biological activity indicated that dural substitute membranes did not induce an inflammatory response or osteogenic differentiation of hDPSCs. In contrast, MTA induced the expression of IL‐6 and alkaline phosphatase activity contributing to enhanced differentiation and mineralization. Conclusions: The dural substitute membranes showed cytocompatibility, did not provoke an inflammatory response and maintained the stemness of hDPSCs better than MTA. Additionally, the set Histoacryl surgical adhesive demonstrated good biocompatibility. Taken together, these results highlight the potential use of dural substitutes in regenerative endodontic procedures as coronal barriers alternative to MTA to reduce the incidence of intracanal calcifications. [ABSTRACT FROM AUTHOR]

Aim: The regulation of human dental pulp inflammation is not fully understood. This study aims to investigate the effect of miR‐4691‐3p on the cGAS–STING signalling cascade and its downstream cytokines production in human dental pulp cells (HDPCs). Methodology: Normal dental pulp tissue and pulp tissue with irreversible pulpitis from third molars were collected. HDPCs were isolated from pulp tissue. The expression of STING mRNA and miR‐4691‐3p was measured by quantitative real‐time PCR. Bioinformatic computation via TargetScanHuman 8.0 and a luciferase reporter assay was used to identify the targets of miR‐4691‐3p. A miR‐4691‐3p mimic and inhibitor were used to upregulate or downregulate miR‐4691‐3p expression in HDPCs. HDPCs were transfected with c‐di‐AMP, c‐di‐GMP, cGAMP, interferon stimulatory DNA (ISD) and bacterial genomic DNA. Immunoblot was performed to detect the phosphorylation of TBK1, p65 and IRF3. Enzyme‐linked immunoassay was performed to detect the cytokines including IFN‐β, TNF or IL‐6 downstream of cGAS–STING. Results: MiR‐4691‐3p expression was increased in human dental pulp tissue with irreversible pulpitis. Treatment of HDPCs using recombinant human IFN‐β, TNF or IL‐6 also upregulated miR‐4691‐3p. The bioinformatic prediction and luciferase reporter assay confirmed that STING was a direct target of miR‐4691‐3p. The miR‐4691‐3p mimic suppressed STING expression, the phosphorylation of TBK1, p65 and IRF3, and the IFN‐β, TNF or IL‐6 production. In contrast, the miR‐4691‐3p inhibitor enhanced the STING expression, the phosphorylation of TBK1, p65 and IRF3 and the IFN‐β, TNF or IL‐6 production. Conclusions: MiR‐4691‐3p negatively regulates the cGAS–STING pathway by directly targeting STING. This provides insight to utilize miRNA‐dependent regulatory effect to treat endodontic disease as well as STING‐dependent systemic inflammatory disease. [ABSTRACT FROM AUTHOR]

Background: Preoperative pulpal status may influence the outcomes of root canal treatment (RCTx) according to various measures used. Objectives: To compare the effectiveness of RCTx of teeth with a vital pulp versus a necrotic pulp, using a range of clinical and patient‐related outcomes proposed for the development of S3‐level clinical practice guidelines. Methods: A search was conducted in the PubMed‐MEDLINE, Scopus, EMBASE, Google scholar databases and available repositories, followed by hand searches, until 29 March 2022. Clinical studies published in the English language comparing the stipulated outcomes of RCTx of teeth with vital versus necrotic pulp were included. The Newcastle–Ottawa Scale was adapted to assess study quality. Effects of pulpal status were estimated and expressed as risk ratio (RR) using fixed‐ and random‐effect meta‐analyses. The quality of evidence was assessed through the Grading of Recommendations Assessment, Development and Evaluation tool. Results: Twenty‐eight studies published between 1961 and 2021 were included. Five studies have investigated the "tooth survival" outcome, four reported pulpal status was not a significant predictor, consistent with meta‐analysis findings (RR: 1.00; 95% CI: 1.00, 1.00; n = 3). Seven studies reported pulpal status had no significant influence on postoperative pain, regardless of duration after treatment. Sixteen studies have analysed "periapical health," and 11 revealed pulpal status had no significant influence. Meta‐analyses revealed the influence was not significant if preoperative periapical radiolucency was absent (RR: 0.95; 95% CI: 0.90, 1.00; n = 9) but significant if it was present (RR: 1.12; 95% CI: 1.05, 1.19; n = 11). Most studies were classified as "some concerns" (n = 17) to "low" (n = 9) risk of bias RoB. Discussion: Evidence is limited and only available for three outcomes when comparing the effectiveness of RCTx in permanent teeth with vital pulp versus pulp necrosis. Nevertheless, the quality of available evidence was moderate to high. The "periapical health" data heterogeneity could be explained by preoperative radiolucency, thus RCTx was found more effective for prevention than the resolution of apical periodontitis. Conclusions: There was no significant difference in the "tooth survival," "postoperative pain" and "evidence of apical radiolucency" outcomes of RCTx in teeth with vital or necrotic pulps. Registration: PROSPERO database (CRD42021260280). [ABSTRACT FROM AUTHOR]

Background: Regenerative endodontics has evolved in recent years with tissue engineering concepts in particular appearing promising. Endodontic tissue engineering (ETE) describes the various approaches based on the orthograde introduction of scaffolds or biomaterials (with or without cells) into the root canal to achieve pulp tissue regeneration. There are currently no systematic reviews investigating whether ETE is a suitable method for the treatment of endodontic disease in both mature and immature permanent teeth. Objectives: The purpose of this systematic review was to determine the effectiveness of ETE in permanent teeth with pulp necrosis in comparison with conventional endodontic treatment. Methods: We searched MEDLINE, Embase and the Cochrane Library for published reports as well as Google Scholar for grey literature up to November 2021. Included were studies of patients with permanent immature or mature teeth and pulp necrosis with or without signs of apical periodontitis (P) comparing ETE (I) with calcium hydroxide apexification, apical plug and root canal treatment (C) in terms of tooth survival, pain, tenderness, swelling, need for medication (analgesics and antibiotics), radiographic evidence of reduction in apical lesion size, radiographic evidence of normal periodontal ligament space, function (fracture and restoration longevity), the need for further intervention, adverse effects (including exacerbation, restoration integrity, allergy and discolouration), oral health‐related quality of life (OHRQoL), presence of sinus tract and response to sensibility testing (O). An observation period of at least 12 months was mandatory (T) and the number of patients in human experimental studies or longitudinal observational studies had to be at least 20 (10 in each arm) at the end (S). Risk of bias was appraised using the Cochrane risk‐of‐bias (RoB 2) tool. Two authors independently screened the records, assessed full texts for eligibility and evaluated risk of bias. Heterogeneity of outcomes and limited body of evidence did not allow for meta‐analysis. Results: Two randomized clinical trials investigating cell transplantation approaches with a total of 76 participants (40 treated immature teeth and 36 treated mature teeth) were included for qualitative analysis. Both studies had moderate concerns in terms of risk of bias. Due to the lack of homogeneity a meta‐analysis was not possible. Tooth survival for ETE, root canal treatment and apexification was 100% after 12 months. Teeth treated with ETE showed a higher number of cases with positive pulpal responses to sensitivity tests and with blood perfusion compared with root canal treatment or apexification. Discussion: This systematic review highlights that there is limited evidence for ETE approaches. Even though the results of this review suggest a high survival with ETE in mature and immature teeth, there is a moderate risk of bias due to methodological limitations in the included studies, so the overall results should be interpreted with caution. Lack of a robust control group was a common problem during literature screening, and outcomes besides dental survival were reported inconsistently. Future clinical trials need to address methodical as well as assessment concerns and report long‐term results. Conclusion: The benefits and high survival rates reported for ETE techniques suggest that this procedure might be an alternative to conventional procedures for permanent teeth with pulpal necrosis. However, more appropriate studies are needed to derive clinical recommendations. Registration: PROSPERO (CRD42021266350). [ABSTRACT FROM AUTHOR]

Background: Revitalization procedures primarily aim to eliminate clinical symptoms and heal periapical lesions. Objectives: The objective of the study was to elucidate the effectiveness of revitalization in treating apical periodontitis in necrotic mature and immature permanent teeth based on the following PICO question: In patients with permanent immature or mature teeth and pulp necrosis with or without signs of apical periodontitis (P) what is the effectiveness of revitalization (I) in comparison with calcium hydroxide apexification, apical plug and root canal treatment (C) in terms of tooth survival, pain, tenderness, swelling, need for medication (analgesics and antibiotics), radiographic evidence of reduction of apical lesion size, radiographic evidence of normal periodontal ligament space, radiographic evidence of increased root thickness and length (not for mature teeth), tooth function (fracture and restoration longevity), need for further intervention, adverse effects (including exacerbation, restoration integrity, allergy and discolouration), oral health‐related quality of life (OHRQoL), presence of sinus tract and response to sensibility testing (O). (T) = Defined as a minimum of 1 year and maximum of as long as possible for all outcome measures, except 'pain, tenderness, swelling, need for medication (analgesics)', which is a minimum of 7 days and maximum of 3 months and OHRQoL which is minimum of 6 months and a maximum of as long as possible. Methods: Three databases (PubMed, Embase and Cochrane Library) were searched for human, experimental and observational studies in English, complemented with hand search, until 31/10/2021. Studies recruiting teeth with pulp necrosis (with/without apical periodontitis), with minimum 10 teeth/arm at the end of the study and with a follow‐up of at least 1 year, were included. Records without an abstract and a full text were excluded. The qualitative analysis of the included (non‐) randomized controlled clinical trials was performed with the Revised Cochrane risk‐of‐bias tools (RoB 2 and ROBINS‐I). Meta‐analysis for survival and success (including a subgroup analysis for mature/immature permanent teeth) was performed using the Mantel–Haenszel method. The certainty of evidence was assessed using GRADE (Grades of Recommendation, Assessment, Development and Evaluation). Results: From the 365 identified records, five met the inclusion criteria. The 12 months survival rate was 100% for all (im)mature permanent teeth in all groups (3 studies). The success rate at 12 months was 100% for immature permanent teeth for I and C (1 study), however, reduced to 92% and 80% for mature teeth in I and C respectively (1 study, p >.05). The risk of bias for the most critical outcome (survival) was high for two studies and low for one. For the critical outcome success, all assessed studies were highly biased. Meta‐analyses provided pooled relative risk with no statistically significant difference between I and C for both survival (RR = 1.00, 95%CI = 0.96–1.04, p = 1.00) and success (RR = 1.06; 95%CI = 0.83–1.35, p =.66). The evidence level for survival was kept 'low' and for success was downgraded to 'very low' due to inconsistency and imprecision. Discussion: The survival and success rates were favourable in all included studies and for all groups; however, these outcomes are not reliable due to the low certainty level. Clinically, the most reported adverse event was tooth discolouration, hence the application of bismuth oxide containing calcium silicate cements should be avoided in revitalization. Radiographically, caution is needed when assessing periapical bone healing and further root development with periapical radiographs, due to multifactorial inaccuracies of this imaging technique. Methodological and assessment concerns need to be addressed in future clinical trials. Long‐term results are necessary for studies reporting revitalization of mature permanent teeth, as they seem to be experimental so far. Conclusions: No robust evidence was discovered to support that revitalization is effective to treat apical periodontitis in (im)mature permanent teeth. The success and survival rates of revitalized and fully pulpectomized (im)mature permanent teeth did not differ significantly. Registration: Prospero: CRD42021262466. [ABSTRACT FROM AUTHOR]

Background: Inflammatory biomarkers are potentially useful targets for pulpal diagnostic tests that can identify pulp status and predict vital pulp treatment (VPT) outcome, however, their accuracy is unknown. Objectives: (1) Calculate sensitivity, specificity and diagnostic odds ratio (DOR) of previously investigated pulpitic biomarkers; (2) Determine if biomarker levels discriminate between clinical diagnoses of pulpitis based on the presence or absence of spontaneous pain (3) Evaluate if biomarker level can predict VPT outcome. Methods: Searches: PubMed/MEDLINE, Ovid SP, Cochrane Central Register of Controlled Trials (CENTRAL), International Clinical Trials Registry Platform (ICTRP), ClinicalTrials.gov, Embase, Web of Science and Scopus in May 2023. Inclusion: prospective and retrospective observational studies and randomized trials. Participants were humans with vital permanent teeth and a well‐defined pulpal diagnosis. Exclusion: deciduous teeth, in vitro and animal studies. Risk of bias was assessed with modified‐Downs and Black quality assessment checklist. Meta‐analysis was performed using bivariate random effect model in Meta‐DiSc 2.0 and RevMan and the quality of the evidence was assessed using Grading of Recommendations Assessment, Development and Evaluation. Results: Fifty‐six studies were selected, reporting >70 individual biomolecules investigating pulpal health and disease at the gene and protein level. Most studies were of low and fair quality. Among the biomolecules investigated, IL‐8 and IL‐6 demonstrated a level of diagnostic accuracy with high sensitivity, specificity and DOR to discriminate between healthy pulps and those exhibiting spontaneous pain suggestive of IRP (low‐certainty evidence). However, none was shown to have high DOR and the ability to discriminate between pulpitic states (very low certainty evidence). Limited data suggests high levels of matrix metalloproteinase 9 correlate with poorer outcomes of full pulpotomy. Discussion: The inability of identified molecular inflammatory markers to discriminate between dental pulps with spontaneous and non‐spontaneous pain should shift the focus to improved study quality or the pursuit of other molecules potentially associated with healing and repair. Conclusions: Low‐quality evidence suggests IL‐8 and IL‐6 demonstrated level of diagnostic accuracy to discriminate between healthy pulps and those exhibiting spontaneous pain. There is a need for standardized biomarker diagnostic and prognostic studies focusing on solutions that can accurately determine the degree of pulp inflammation. Registration: PROSPERO CRD42021259305. [ABSTRACT FROM AUTHOR]

Aim: Previous studies have evaluated the pulpal responses to calcium silicate cements (CSCs) on normal dental pulp, but investigations on the effects of CSCs on inflamed pulp are limited. This study aimed to test the inflammatory response and odontogenic differentiation of inflamed rat dental pulp after direct pulp capping with CSCs. Methodology: Wistar rat molars pulps were exposed for 48 h to induce inflammation and then capped with ProRoot MTA (Dentsply), Biodentine (Septodont), RetroMTA (Bio MTA) and Dycal (Dentsply Caulk). The degree of pulpal inflammation and hard tissue formation was evaluated by histological analysis. Immunofluorescence staining for interleukin (IL)‐6, osteocalcin (OCN) and runt‐related transcription factor 2 (RUNX2) was also performed. Results: After 4 weeks, complete recovery from inflammation was evident in 22%, 37.5%, 10% and none of the ProRoot MTA, Biodentine, RetroMTA and Dycal samples, respectively. Heavy hard tissue deposition as a continuous hard tissue bridge was observed in 77.8%, 75%, 70% and 60% of the ProRoot MTA, Biodentine, RetroMTA and Dycal samples, respectively. IL‐6, OCN and RUNX2 were detected in all materials, mainly adjacent to areas of inflammation and reparative dentine formation. At one, two and 4 weeks, significant differences were not observed between the inflammation and hard tissue formation scores of the four material groups (p >.05). Conclusions: In this study, pulpal inflammation was still present in most specimens at 4 weeks after pulp capping and a significant number of samples showed incomplete and discontinuous dentine bridge formation. The results of this study suggest that initial inflammatory conditions of the pulp may risk the prognosis of teeth treated with CSCs. [ABSTRACT FROM AUTHOR]

Aim: Prevascularization is vital to accelerate functional blood circulation establishment in transplanted engineered tissue constructs. Mesenchymal stem cells (MSCs) or mural cells could promote the survival of implanted endothelial cells (ECs) and enhance the stabilization of newly formed blood vessels. However, the dynamic cell–cell interactions between MSCs, mural cells and ECs in the angiogenic processes remain unclear. This study aimed to explore the interactions of human umbilical vein ECs (HUVECs) and dental pulp stem cells (DPSCs) in an in vitro cell coculture model. Methodology: Human umbilical vascular ECs and DPSCs were directly cocultured or indirectly cocultured with transwell inserts in endothelial basal media‐2 (EBM‐2) supplemented with 5% FBS for 6 days. Expression of SMC‐specific markers in DPSCs monoculture and HUVEC+DPSC cocultures was assessed by western blot and immunofluorescence. Activin A and transforming growth factor‐beta 1 (TGF‐β1) in conditioned media (CM) of HUVECs monoculture (E‐CM), DPSCs monoculture (D‐CM) and HUVEC+DPSC cocultures (E+D‐CM) were analysed by enzyme‐linked immunosorbent assay. TGF‐β RI kinase inhibitor VI, SB431542, was used to block TGF‐β1/ALK5 signalling in DPSCs. Results: The expression of SMC‐specific markers, α‐SMA, SM22α and Calponin, were markedly increased in HUVEC+DPSC direct cocultures compared to that in DPSCs monoculture, while no differences were demonstrated between HUVEC+DPSC indirect cocultures and DPSCs monoculture. E+D‐CM significantly upregulated the expression of SMC‐specific markers in DPSCs compared to E‐CM and D‐CM. Activin A and TGF‐β1 were considerably higher in E+D‐CM than that in D‐CM, with upregulated Smad2 phosphorylation in HUVEC+DPSC cocultures. Treatment with activin A did not change the expression of SMC‐specific markers in DPSCs, while treatment with TGF‐β1 significantly enhanced these markers' expression in DPSCs. In addition, blocking TGF‐β1/ALK5 signalling inhibited the expression of α‐SMA, SM22α and Calponin in DPSCs. Conclusions: TGF‐β1 was responsible for DPSC differentiation into SMCs in HUVEC+DPSC cocultures, and TGF‐β1/ALK5 signalling pathway played a vital role in this process. [ABSTRACT FROM AUTHOR]

Aim: Inducing odontogenic differentiation and tubular dentine formation is extremely important in dentine repair and tooth regeneration. Bone morphogenic proteins (BMPs) signalling plays a critical role in dentine development and tertiary dentine formation, whilst how BMPR1A‐mediated signalling affects odontoblastic differentiation of Axin2‐expressing (Axin2+) odontogenic cells and tubular dentine formation remains largely unknown. This study aims to reveal the cellular and molecular mechanisms involved in the formation of secondary dentine. Methodology: Axin2lacZ/+ mice harvested at post‐natal 21 (P21) were used to map Axin2+ mesenchymal cells. Axin2CreERT2/+; R26RtdTomato/+ mice and Axin2CreERT2/+; R26RDTA/+; R26RtdTomato/+ mice were generated to observe the tempo‐spatial distribution pattern of Axin2‐lineage cells and the effect of ablation of Axin2+ cells on dentinogenesis, respectively. A loss‐of‐function model was established with Axin2CreERT2/+; Bmpr1afl/fl; R26RtdTomato/+ (cKO) mice to study the role of BMP signalling in regulating Axin2+ cells. Micro‐computed tomography, histologic and immunostainings, and other approaches were used to examine biological functions, including dentine formation, mineralization and cell differentiation in cKO mice. Results: The results showed rich expression of Axin2 in odontoblasts at P21. Lineage tracing assay confirmed the wide distribution of Axin2 lineage cells in odontoblast layer and dental pulp during secondary dentine formation (P23 to P56), suggesting that Axin2+ cells are important cell source of primary odontoblasts. Ablation of Axin2+ cells (DTA mice) significantly impaired secondary dentine formation characterized with notably reduced dentine thickness (Mean of control: 54.11 μm, Mean of DTA: 27.79 μm, p =.0101). Furthermore, malformed osteo‐dentine replaced the tubular secondary dentine in the absence of Bmpr1a with irregular cell morphology, abnormal cellular process formation and lack of cell–cell tight conjunction. Remarkably increased expression of osteogenic markers like Runx2 and DMP1 was detected, whilst DSP expression was observed in a dispersed manner, indicating an impaired odontogenic cell fate and failure in producing tubular dentine in cKO mice. Conclusions: Axin2+ cells are a critical population of primary odontoblasts which contribute to tubular secondary dentine formation, and BMP signalling pathway plays a vital role in maintaining the odontogenic fate of Axin2+ cells. [ABSTRACT FROM AUTHOR]

Aim: Biallelic loss‐of‐function FAM20A mutations cause amelogenesis imperfecta (AI) type IG, better known as enamel renal syndrome (ERS), characterized by severe enamel hypoplasia, delayed/failed tooth eruption, intrapulpal calcifications, gingival hyperplasia and nephrocalcinosis. FAM20A binds to FAM20C, the Golgi casein kinase (GCK) and potentiates its function to phosphorylate secreted proteins critical for biomineralization. While many FAM20A pathogenic mutations have been reported, the pathogeneses of orodental anomalies in ERS remain to be elucidated. This study aimed to identify disease‐causing mutations for patients with ERS phenotypes and to discern the molecular mechanism underlying ERS intrapulpal calcifications. Methodology: Phenotypic characterization and whole exome analyses were conducted for 8 families and 2 sporadic cases with hypoplastic AI. A minigene assay was performed to investigate the molecular consequences of a FAM20A splice‐site variant. RNA sequencing followed by transcription profiling and gene ontology (GO) analyses were carried out for dental pulp tissues of ERS and the control. Results: Biallelic FAM20A mutations were demonstrated for each affected individual, including 7 novel pathogenic variants: c.590‐5T>A, c.625T>A (p.Cys209Ser), c.771del (p.Gln258Argfs*28), c.832_835delinsTGTCCGACGGTGTCCGACGGTGTC CA (p.Val278Cysfs*29), c.1232G>A (p.Arg411Gln), c.1297A>G (p.Arg433Gly) and c.1351del (p.Gln451Serfs*4). The c.590‐5T>A splice‐site mutation caused Exon 3 skipping, which resulted in an in‐frame deletion of a unique region of the FAM20A protein, p.(Asp197_Ile214delinsVal). Analyses of differentially expressed genes in ERS pulp tissues demonstrated that genes involved in biomineralization, particularly dentinogenesis, were significantly upregulated, such as DSPP, MMP9, MMP20 and WNT10A. Enrichment analyses indicated overrepresentation of gene sets associated with BMP and SMAD signalling pathways. In contrast, GO terms related to inflammation and axon development were underrepresented. Among BMP signalling genes, BMP agonists GDF7, GDF15, BMP3, BMP8A, BMP8B, BMP4 and BMP6 were upregulated, while BMP antagonists GREM1, BMPER and VWC2 showed decreased expression in ERS dental pulp tissues. Conclusions: Upregulation of BMP signalling underlies intrapulpal calcifications in ERS. FAM20A plays an essential role in pulp tissue homeostasis and prevention of ectopic mineralization in soft tissues. This critical function probably depends upon MGP (matrix Gla protein), a potent mineralization inhibitor that must be properly phosphorylated by FAM20A‐FAM20C kinase complex. [ABSTRACT FROM AUTHOR]

Aim: Pyroptosis is a type of inflammatory cell death and is related to pulpitis and apical periodontitis. In this study, the aim was to investigate how periodontal ligament fibroblasts (PDLFs) and dental pulp cells (DPCs) respond to pyroptotic stimuli and explore whether dimethyl fumarate (DMF) could block pyroptosis in PDLFs and DPCs. Methodology: Three methods (stimulation with lipopolysaccharide [LPS] plus nigericin, poly(dA:dT) transfection and LPS transfection) were used to induce pyroptosis in PDLFs and DPCs, two types of fibroblasts related to pulpitis and apical periodontitis. THP‐1 cell was used as a positive control. Afterwards, PDLFs and DPCs were treated with or without DMF before inducing pyroptosis to examine the inhibitory effect of DMF. Pyroptotic cell death was measured by lactic dehydrogenase (LDH) release assays, cell viability assays, propidium iodide (PI) staining and flow cytometry. The expression levels of cleaved gasdermin D N‐terminal (GSDMD NT), caspase‐1 p20, caspase‐4 p31 and cleaved PARP were examined by immunoblotting. Immunofluorescence analysis was used to detect the cellular distribution of GSDMD NT. Results: Periodontal ligament fibroblasts and DPCs were more sensitive to cytoplasmic LPS‐induced noncanonical pyroptosis than to canonical pyroptosis induced by stimulation with LPS priming plus nigericin or by poly(dA:dT) transfection. In addition, treatment with DMF attenuated cytoplasmic LPS‐induced pyroptotic cell death in PDLFs and DPCs. Mechanistically, it was shown that the expression and plasma membrane translocation of GSDMD NT were inhibited in DMF‐treated PDLFs and DPCs. Conclusions: This study indicates that PDLFs and DPCs are more sensitive to cytoplasmic LPS‐induced noncanonical pyroptosis and that DMF treatment blocks pyroptosis in LPS‐transfected PDLFs and DPCs by targeting GSDMD, suggesting DMF might be a promising drug for the management of pulpitis and apical periodontitis. [ABSTRACT FROM AUTHOR]

Aim: This clinical study was undertaken to assess the effect of treatment with regenerative endodontic procedures (REPs) on 20 teeth with pulp necrosis, apical periodontitis, and external root resorption (ERR). Methodology: Teeth were treated with REPS utilizing the American Association of Endodontists (AAE) protocol. Quantitative assessment of changes in radiographic root area (RRA) were statistically analysed to assess changes in root dimensions after an average follow‐up period of 3 years. Results: All 20 teeth survived, 14 teeth (70%) were classified successful, and only 1 tooth (5%) failed throughout the study period. Based on the radiographic examination, all 20 teeth showed complete repair of the periapical lesions and arrested ERR. However, 5 teeth (25%) subsequently developed replacement resorption. The RRA between baseline and 3‐year follow‐up showed a significant difference for the total 20 teeth (p =.009). An analysis according to the trauma type and the extra‐oral time showed the RRA increase was significantly different in the non‐avulsion group (p =.015) and for the avulsion group with an extra‐oral time less than 60 min (p =.029). The RRA increase was not statistically significant in the avulsion group of extra‐oral time more than 60 min (p =.405). Nine teeth (45%) and 10 teeth (50%) responded to cold and electric pulp testing, respectively. Conclusions: Within the limitations of this study, the favourable outcomes of REPs were further confirmed for traumatized permanent necrotic teeth with ERR in terms of periapical lesion healed and a significant increase in RRA. The study contributes further evidence of the role of REPs in arresting ERR. [ABSTRACT FROM AUTHOR]

Aim: To evaluate the efficacy of a novel ultrasonic irrigation device, remotely‐generated irrigation with a non‐invasive sound field enhancement (RINSE) system, in removing biofilm‐mimicking hydrogel from a simulated isthmus model and compare it with sonically‐ and ultrasonically‐activated irrigation systems. Methodology: A polycarbonate root canal model containing two standardized root canals (apical diameter of 0.20 mm, 4% taper, 18 mm long with a coronal reservoir) connected by three isthmuses (0.40 mm deep, 2 mm high, 4 mm long) was used as the test model. The isthmuses were filled with a hydroxyapatite powder‐containing hydrogel. The canals were filled with irrigant, and the models were randomly assigned to the following activation groups (n = 15): EndoActivator (EA), ultrasonically activated irrigation (UAI), and RINSE system (RS). Syringe irrigation (SI) with a 30G needle served as the control. Standardized images of the isthmuses were taken before and after irrigation, and the amount of hydrogel removed was determined using image analysis software and compared across groups using anova (p <.05). Results: Hydrogel removal was significantly higher with the RS (83.7%) than with UAI, EA, or SI (p ≤.01). UAI (69.2%) removed significantly more hydrogel than SI and EA (p <.05), while there was no significant difference between SI (24.3%) and EA (25.7%) (p =.978). Conclusions: RINSE system resulted in the most hydrogel removal, performing better than UAI or EA. The effect of RS was also not reliant on the insert or tip entering the pulp chamber or root canal, making it particularly useful in conservative endodontics. [ABSTRACT FROM AUTHOR]

Aim: To compare the odontogenic differentiation potential of a composite scaffold (CSHA) comprising of nano‐hydroxyapatite (nHAp) and carboxymethyl chitosan (CMC) with Biodentine on human dental pulp stem cells (hDPSCs). Methodology: A CSHA scaffold was prepared through an ultrasonication route by adding nHAp and CMC (1:5 w/w) in water medium followed by freeze‐drying. Physicochemical characterization was achieved using scanning electron microscopy with energy‐dispersive X‐ray spectroscopy, X‐ray diffraction and Fourier transform infrared spectroscopy. In‐vitro bioactivity and pH assessments were done by soaking in simulated body fluid (SBF) for 28 days. The angiogenic and odontogenic differentiation abilities were assessed by expression of vascular endothelial growth factor (VEGF) and Dentine sialophosphoprotein (DSPP) markers on cultured hDPSCs by flow cytometry and RT‐qPCR at 7, 14 and 21 days. Cell viability/proliferation and biomineralization abilities of CSHA were compared with Biodentine by MTT assay, alkaline phosphatase (ALP) activity, Alizarin Red Staining (ARS) and osteopontin (OPN) expression on hDPSCs following 7 and 14 days. Data were statistically analysed with Kruskal Wallis and Friedman tests as well as one way anova followed by appropriate post hoc tests (p <.05). Results: Characterization experiments revealed a porous microstructure of CSHA with pore diameter ranging between 60 and 200 μm and 1.67 Ca/P molar ratio along with the characteristic functional groups of both HAp and CMC. CSHA displayed bioactivity in SBF by forming apatite‐like crystals and maintained a consistent pH value of 7.70 during 28 days' in vitro studies. CSHA significantly upregulated VEGF and DSPP levels on hDPSCs on day 21 compared with day 7 (p <.05). Further, CSHA supported cell viability/proliferation over 14 days like Biodentine with no statistical differences (p >.05). However, CSHA exhibited increased ALP and ARS activity with an intense OPN staining compared with Biodentine after 14 days (p <.05). Conclusion: The results highlighted the odontogenic differentiation and biomineralization abilities of CSHA on hDPSCs with significant VEGF and DSPP gene upregulations. Further, CSHA exhibited enhanced mineralization activity than Biodentine, as evidenced by increased ALP, ARS and OPN activity on day 14. The nHAp‐CMC scaffold has the potential to act as an effective pulp capping agent; however, this needs to be further validated through in‐vivo animal studies. [ABSTRACT FROM AUTHOR]

Aim: To explore the effects of phase‐transited lysozyme (PTL) coated dentine slices on cell adhesion, migration and odontogenic differentiation of human dental pulp cells (HDPCs). Methodology: Cell growth and cell cycle analysis were conducted to verify the biocompatibility of PTL for HDPCs. Cell adhesion, cell morphology and proliferation were explored by DiI staining, Scanning electron microscopy and MTT assay. Cell migration was investigated by Transwell assay. The effects of PTL on odontogenesis and mineralization of HDPCs were assessed by real‐time quantitative polymerase chain reaction and Western blot. The mineralization of HDPCs was evaluated by Alizarin red staining. HDPCs were isolated from extracted third molars. The level of statistically significant difference was accepted at p <.05. Results: PTL showed no negative effect on cell cycle of HDPCs and compared with the blank group, HDPCs labelled with DiI staining showed significantly more adhered cells at 48 h (p <.05), extending cell processes and more finger‐like or reticular pseudopodia on PTL‐coated dentine slices. The results of MTT and Transwell assay showed that PTL promoted the proliferation (p <.05) and migration (p <.01) of HDPCs, respectively. Compared with the blank group, the gene expression of dentine sialophosphoprotein (DSPP), osteopontin and bone sialoprotein in HDPCs cultured on PTL was significantly upregulated on day 3 and 7 (p <.05), while the protein expression of DSPP showed no significant change on both day 7 and day 14. Alizarin red staining showed that PTL promoted more mineralization nodules formation of HDPCs (p <.05). Conclusions: PTL promoted the adhesion, proliferation and migration of HDPCs on dentine slices, and positively affected odontogenic differentiation and mineralization of HDPCs. [ABSTRACT FROM AUTHOR]

Aim: Tideglusib is a small molecule agonist of the canonical Wnt pathway. The present study investigated the influence of Tideglusib on human dental pulp stem cell (hDPSC) proliferation, apoptosis, migration and odonto/osteogenic differentiation. Methodology: hDPSCs were treated with 50, 100 nM or 200 nM Tideglusib. β‐catenin accumulation was detected by immunofluorescence staining. Colony‐forming unit ability was assessed by staining with Coomassie blue. Cell cycle progression and cell apoptosis were investigated using flow cytometry. Cell migration was examined using an in vitro wound‐healing assay. Osteogenic differentiation was examined using alkaline phosphatase (ALP) staining, alizarin red S staining and osteogenic‐related gene expression. The gene expression profile was examined using a high‐throughput RNA sequencing technique. All experiments were repeated using cells derived from at least four different donors (n = 4). The Mann–Whitney U‐test was used to identify significant differences between two independent group comparisons. For three or more group comparisons, statistical differences were assessed using the Kruskal–Wallis test followed by pairwise comparison. The significance level was set at 5% (p <.05). Results: Tideglusib activated the Wnt signalling pathway in hDPSCs as demonstrated by an increase in cytoplasmic β‐catenin accumulation and nuclear translocation. Tideglusib did not affect hDPSC proliferation, cell cycle progression, cell apoptosis or cell migration. In contrast, 50 and 100 nM Tideglusib significantly enhanced mineralization and osteogenic marker gene expression (RUNX2, ALP, BMP2 and DSPP; p <.05). Conclusions: Tideglusib enhanced the odonto/osteogenic differentiation of hDPSCs. Therefore, incorporating this bioactive molecule in a pulp‐capping material could be a promising strategy to promote dentine repair. [ABSTRACT FROM AUTHOR]

Aim: This randomized clinical trial aimed to evaluate and compare the effect of mineral trioxide aggregate (MTA)‐based bioceramic‐ and resin‐based sealers on the incidence and intensity of post‐obturation pain and treatment outcome in asymptomatic vital molar teeth in single‐visit root canal treatment. Methodology: One hundred asymptomatic mandibular molar teeth with vital pulp were randomly assigned into two groups according to the sealer used: MTA Fillapex or AH Plus sealer. WaveOne Gold instruments were used for root canal preparations. Root canals were obturated with WaveOne Gold gutta‐percha cones corresponding in size and taper to the last instrument used. MTA Fillapex was used in the first group and AH Plus was used in the second group as the root canal sealer. The effects of age, gender, tooth types and the level of root canal filling on the treatment outcome were evaluated after 1 and 2 years. Postoperative pain at 6, 12, 24, 48, 72 h, 4, 5, 6, 7 and 30 days after root canal treatment was measured with the Numerical Rating Scale. Chi‐square test was used for the treatment outcome and Mann–Whitney U, chi‐Square and Spearman correlation analyses were used for postoperative pain. α =.05 was the level of significance. Results: Twelve patients did not complete the 2‐year follow‐up and the study finished with 88 patients. In the MTA Fillapex group 41 correct, one short, two extruded, and in the AH Plus group 40 correct, one short and three extruded root canal fillings were detected. No significant differences were observed between the first‐ and second‐year treatment outcomes between the groups (p >.05).There was no association between age, gender, tooth number, root canal filling length and the treatment success in all evaluated teeth (p >.05). The differences between the groups in terms of postoperative pain incidence and intensity at all time‐points were not significant (p >.05). There was no correlation between the age and postoperative pain (p >.05), but pain was statistically higher in women (p <.05). Conclusions: Mineral trioxide aggregate Fillapex showed similar success rate and post‐obturation pain incidence and intensity as AH Plus sealer and can be used as a root canal sealer in asymptomatic mandibular molars with irreversible pulpitis. [ABSTRACT FROM AUTHOR]

Aim: To evaluate the expression and function of the nod‐like receptor pyrin domain containing 3 (NLRP3) inflammasome in caries induced pulpitis. Methodology: NLRP3 expression was determined with immunohistochemistry in the dental pulp and qPCR in dental pulp cells (DPCs). THP‐1 macrophages expressing the apoptosis‐related speck‐like protein (ASC) and green fluorescent protein (GFP) fusion protein were used to assess NLRP3 inflammasome activation by live cell imaging, following treatment with lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Caspase I inhibitor was used to confirm inflammasome activation. An ex‐vivo pulpitis model in which the DPCs were co‐cultured with THP‐1 macrophages was used to study the effect of the NLRP3 inflammasome inhibitor (MCC950), and cytokines were measured using ELISA and multiplex array. Data were analysed using the t‐test or anova followed by a Bonferroni post hoc test with the level of significance set at p ≤.05. Results: NLRP3 inflammasome was differentially expressed in dental pulp of sound and carious teeth. Treatment of DPCs with LTA significantly upregulates NLRP3 and IL‐1 β‐expression (p <.05) and in induces more ASC specks formation compared to LPS. IL‐β release in response to LTA treatment is significantly reduced with Caspase I inhibitor suggesting inflammasome dependent mechanism (p <.01). NLRP3‐specific inhibitor, MCC950, significantly reduced IL‐1β and IL‐6 in an ex‐vivo pulpitis model (p <.01) but had no effect on IL‐8 or matrix metalloproteinase‐9 (MMP‐9). Conclusions: Expression and upregulation of NLRP3 inflammasome with caries and LTA treatment suggest a role in caries‐induced pulpitis. NLRP3 inhibitor attenuated the release of selective inflammatory cytokines and could be a potential treatment target that merit further investigation. [ABSTRACT FROM AUTHOR]

Aim: To determine the effects of lipopolysaccharides (LPS), hydrogen peroxide (H2O2), and both combined on cell proliferation/differentiation, inflammation, mitochondrial dynamics as indicated by mitochondrial fission/fusion, antioxidants as indicated by superoxide dismutase 2 (SOD2), and apoptosis of human dental pulpal cells (HDPCs). Methodology: Pulpal tissues from eight healthy subjects (n = 8) were collected from Faculty of Dentistry, Chiang Mai University. Isolated HDPCs from healthy donors were divided into four experimental groups: vehicle, 20 μg/ml LPS, 400 μM H2O2, and the two combined. All experimental groups were investigated to assess cell proliferation, mineralization, differentiation, inflammation, mitochondrial dynamics, antioxidants, and apoptosis. Results: H2O2 and combined agents decreased cell proliferation of HDPCs equally. LPS, H2O2, and both combined decreased mineralization and differentiation with an increase in tumour necrosis factor‐alpha (TNF‐α) levels. Surprisingly, LPS and combined agents increased SOD2 expression and caused an imbalance in mitochondrial dynamics. A significant increase in apoptosis was observed in the case of H2O2 and combined agents. Conclusions: These findings suggest that LPS induced inflammation, imbalanced mitochondrial dynamics, and reduced cell differentiation without altering apoptosis and cell proliferation. However, H2O2 decreased cell proliferation, and differentiation, and increased inflammation, and apoptosis without interfering with mitochondrial dynamics. Based on our findings, combining LPS and H2O2 could be potentially used as the inducers in in vitro study to mimic the clinical pulpitis. [ABSTRACT FROM AUTHOR]

Aim: To investigate the role of RAD54B in the proliferation of inflamed human dental pulp cells (hDPCs) induced by lipopolysaccharide (LPS). Methodology: Normal, carious and pulpitic human dental pulp tissues were collected. Total RNA was subjected to RNA‐sequencing (seq) and gene expression profiles were studied by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Differentially expressed genes (DEGs) in homologous recombination repair (HRR) were validated with qRT‐PCR. The expression of RAD54B and TNF‐α in human dental pulp tissues was detected using immunohistochemistry. HDPCs were cultured and RAD54B level in hDPCs was detected after LPS stimulation using western blot. CCK‐8 was used to investigate the proliferation of hDPCs transfected with negative control (Nc) small interfering RNA (siRNA), RAD54B siRNA, P53 siRNA or both siRNAs with or without LPS stimulation. Flow cytometry was used to detect the cell cycle distribution, and western blot and immunofluorescence were used to analyse the expression of RAD54B, P53 and P21 under the above treatments. One‐way and two‐way anova followed by least significant difference posttest were used for statistical analysis. Results: RNA‐seq results identified DEGs amongst the three groups. KEGG pathway analysis revealed enrichment of DEGs in the replication and repair pathway. HRR and non‐homologous end joining (NHEJ) components were further verified and qRT‐PCR results were basically consistent with the sequencing data. RAD54B, an HRR accessory factor highly expressed in carious and pulpitic tissues as compared to that in normal pulps, was chosen as our gene of interest. High RAD54B expression was confirmed in inflamed human dental pulp tissues and LPS‐stimulated hDPCs. Upon RAD54B knockdown, P53 and P21 expressions in hDPCs were upregulated whereas the proliferation was significantly downregulated, accompanied by increased G2/M phase arrest. After inhibiting P53 expression in RAD54B‐knockdown hDPCs, P21 expression and cell proliferation were reversed. Conclusions: Gene expression profiles of normal, carious and pulpitic human dental pulp tissues were revealed. HRR components were elucidated to function in dental pulp inflammation. Amongst the DEGs in HRR, RAD54B regulated the proliferation of inflamed hDPCs via P53/P21 signalling. This research deepens our understanding of dental pulp inflammation and provides new insight to clarify the underlying mechanisms. [ABSTRACT FROM AUTHOR]

Aim: To analyse the influence of ethylenediaminetetraacetic acid (EDTA) on the repair process in immature rat molars after a regenerative endodontic procedure (REP). Methodology: The lower first molars of 12 4‐week‐old Wistar rats underwent pulpectomy in the mesial root and were divided into the following groups: sodium hypochlorite (NaOCl; n = 6) – the mesial canals were irrigated with 2.5% NaOCl for 5 min, and NaOCl‐EDTA (n = 6) – the canals were irrigated with 2.5% NaOCl, followed by 17% EDTA for 5 min each. After evoking bleeding using a size 10 K‐file, the cavities were sealed. Three molars on the untreated side were randomly used as control (control‐15 d; n = 3), and three molars from the other three rats untreated were used as immediate control (n = 3). After 15 days (NaOCl, NaOCl‐EDTA and control‐15 d groups) or immediately (control‐immediate), the animals were euthanized, and the teeth were subjected to histologic evaluation of tissue regeneration and presence of collagen fibres. Mann–Whitney U‐test was used (p <.05). Results: The experimental groups had newly formed cementum‐like tissue and increased root length and thickness. Half of the specimens in NaOCl‐EDTA group showed apical foramen closure, whilst the NaOCl group had partial apical closure. The experimental groups showed inflammatory infiltrate extending mainly to the medium third of the root canal. These parameters were similar between experimental groups (p >.05). Newly formed connective tissue in the pulp space was significantly higher in the NaOCl‐EDTA group than in NaOCl group (p <.05). Regarding the collagen fibres, the NaOCl‐EDTA group had more collagen fibres in the root tip, but there was no significant difference compared to NaOCl group, and both groups showed greater amount of immature fibres in this area; in the centre of the apical third of root canal, there was equivalence between mature and immature fibres from both groups (p >.05). Conclusions: Ethylenediaminetetraacetic acid irrigation improved newly formed intracanal connective tissue after REP in immature molars of rats; however, EDTA did not influence cementum‐like tissue formation, apical closure, inflammatory infiltrate and maturation of collagen fibres. [ABSTRACT FROM AUTHOR]

Aims: Lipopolysaccharides (LPS)‐activated human dental pulp stem cells (hDPSCs) and macrophage co‐cultures showed downregulated TNF‐α secretion that is modulated by hDPSCs through IDO axis, whereas the secretory levels of IL‐1β remained unchanged. Therefore, sustained production of IL‐1β could contribute to progressive dental pulp inflammation. However, the role of interleukin‐1 receptor antagonist (IL‐1RA) in downregulating the secretion of IL‐1β and TNF‐α in LPS‐activated M0/M1/M2 macrophage and hDPSCs co‐culture has not been studied yet. Therefore, the aim of the present study was to determine the immunomodulatory role of blocking IL‐1 receptors in DPSCs macrophage co‐culture activated with LPS. Methodology: Human monocytic cell line THP‐1 was polarized to M0, M1 and M2 macrophages and co‐cultured with hDPSCs. The viability of the co‐cultured cells was assessed by apoptosis assay. Co‐cultures were activated with LPS followed by the assessment of gene expression and protein levels of IL‐1β and TNF‐α with and without IL‐1RA blocking via qRT‐PCR and cytokine flex assay by flow cytometry. Data from three separate experiments were analysed using one‐way anova followed by Tukey's post hoc test and a p‐value of <.05 was considered statistically significant. Results: THP‐1‐derived M0, M1 and M2 macrophages co‐cultured with hDPSCs showed spindle and round‐shaped cells, with >90% viability when assessed by apoptosis assay. Inflammatory TNF‐α and IL‐1β profiles in stimulated co‐cultures showed upregulated IL‐1β, whereas TNF‐α was downregulated (p <.05). Anti‐inflammatory gene expression levels of IL‐10 and TGF‐β were downregulated (p <.05). Blocking with IL‐1RA resulted in a remarkable decrease in IL‐1β at the gene expression and protein production levels whilst TNF‐α levels remained low (p <.05). Levels of anti‐inflammatory cytokine IL‐10 showed no significant difference. Conclusion: Blocking the IL‐1 receptor in hDPSCs and macrophage (M0, M1, M2) co‐cultures activated with LPS resulted in downregulation of inflammatory cytokines IL‐1β and TNF‐α. These findings highlight the immunomodulatory effect of IL‐1RA in inflammatory conditions of dental pulp infections. [ABSTRACT FROM AUTHOR]

Background: Pulp–dentine complex regeneration via tissue engineering is a developing treatment modality that aims to replace necrotic pulps with newly formed healthy tissue inside the root canal. Designing and fabricating an appropriate scaffold is a crucial step in such a treatment. Objectives: The present study aimed to review recent advances in the design and fabrication of scaffolds for de novo regeneration of pulp–dentine complexes via tissue engineering approaches. Methods: A literature search was conducted using PubMed, Europe PMC, Scopus and Google Scholar databases. To highlight bioengineering techniques for de novo regeneration of pulp–dentine complexes, both in vitro and in vivo studies were included, and clinical studies were excluded. Results: In the present review, four main classes of scaffolds used to engineer pulp–dentine complexes, including bioceramic‐based scaffolds, synthetic polymer‐based scaffolds, natural polymer‐based scaffolds and composite scaffolds, are covered. Additionally, recent advances in the design, fabrication and application of such scaffolds are analysed along with their advantages and limitations. Finally, the importance of vascular network establishment in the success of pulp–dentine complex regeneration and strategies used to create scaffolds to address this challenge are discussed. Discussion: In the tissue engineering platform, scaffolds provide structural support for cells to adhere and proliferate and also regulate cell differentiation and metabolism. Up to now, considerable progress has been achieved in the field of pulp–dentine complex tissue engineering, and a spectrum of scaffolds ranging from bioceramic‐based to naturally derived scaffolds has been fabricated. However, in designing a suitable scaffold for engineering pulp–dentine complexes, a variety of characteristic parameters related to biological, structural, physical and chemical features should be considered. Conclusion: The variety of biomaterials and fabrication techniques provides a great opportunity to address some of the requirements for scaffolds in regenerative endodontics. However, more studies are required to develop an ideal scaffold for use in a clinical setting. [ABSTRACT FROM AUTHOR]