MicroRNA expression in JAG1/Notch-activated periodontal ligament stem cells.

OBJECTIVES: The study explored the expression profile of miRNAs in Notch-activated periodontal ligament stem cells (PDLSCs) and examined their potential cellular targets. METHODS: PDLSCs were cultured and treated with indirect immobilized Jagged1. The miRNA expression profile was examined using NanoString analysis. Bioinformatic analysis was performed together with enrichment, and miRNA expression was evaluated and validated using a quantitative polymerase chain reaction (qPCR). RESULTS: A total of 26 miRNAs were differentially expressed in Jagged1 treated PDLSCs compared with the controls. Pathway analysis revealed that altered miRNAs were significantly associated with the transforming growth factor ß (TGF-ß) signaling pathway. Target prediction analysis demonstrated that 11,170 genes as predictable targets of these altered miRNAs. Enrichment of predicted target genes revealed that they were related to ErbB, Ras and MAPK signaling pathways and small GTPase transduction. CONCLUSIONS: The research concludes that several miRNAs are differentially expressed in jagged-1 treated PDLSCs. In translational terms the differential functionality of these miRNAs offer promise for the development of targeted regenerative materials that are necessary for managing lost tissue replacement in periodontal diseases.

Toll-like receptor and C-type lectin receptor agonists attenuate osteogenic differentiation in human dental pulp stem cells.

BACKGROUND: This study aimed to investigate the effects of various toll-like receptor (TLR) and C-type lectin receptor (CLR) ligands on osteogenic differentiation in human dental pulp stem cells (hDPSCs). METHODS: hDPSCs were cultured and treated with various concentrations (0.01, 0.1, 1.0, and 10 µg/mL) of TLR or CLR agonists (PG-LPS, E.coli LPS, poly(I:C), Pam3CSK4, Furfurman, and Zymosan). Cell viability was determined by MTT assay. The effects of TLR and CLR agonists on osteogenic differentiation of hDPSCs were measured by alkaline phosphatase (ALP) activity, Alizarin Red S staining, and Von Kossa staining. In addition, the mRNA expression of osteogenesis-related genes (ALP, COL1A1, RUNX2, OSX, OCN and DMP1) was examined by RT-qPCR. A non-parametric analysis was employed for the statistical analyses. The statistically significant difference was considered when p < 0.05. RESULTS: Treatment with TLR and CLR agonists was associated with an increase in hDPSCs' colony-forming unit ability. Compared with the control group, TLR and CLR agonists significantly inhibited the osteogenic differentiation of hDPSCs by decreasing the ALP activity, mineralised nodule formation, and mRNA expression levels of osteogenesis-related genes (ALP, COL1A1, RUNX2, OSX, OCN and DMP1). The inhibition of TRIF but not Akt signalling rescued the effects of TLR and CLR agonist attenuating hDPSCs' mineralisation. CONCLUSIONS: The activation of TLRs or CLRs exhibited an inhibitory effect on osteogenic differentiation of hDPSCs via the TRIF-dependent signalling pathway.

Periostin-integrin interaction regulates force-induced TGF-ß1 and α-SMA expression by hPDLSCs.

OBJECTIVE: Periostin (PN), a major matricellular periodontal ligament (PDL) protein, modulates the remodeling of the PDL and bone, especially under mechanical stress. This study investigated the requirement of PN-integrin signaling in force-induced expression of transforming growth factor-beta 1 (TGF-ß1) and alpha-smooth muscle actin (α-SMA) in human PDL stem cells (hPDLSCs). METHODS: Cells were stimulated with intermittent compressive force (ICF) using computerized controlled apparatus. Cell migration was examined using in vitro scratch assay. The mRNA expression was examined using real-time polymerase chain reaction. The protein expression was determined using immunofluorescent staining and western blot analysis. RESULTS: Stimulation with ICF for 24 h increased the expression of PN, TGF-ß1, and α-SMA, along with increased SMAD2/3 phosphorylation. Knockdown of POSTN (PN gene) decreased the protein levels of TGF-ß1 and pSMAD2/3 upon force stimulation. POSTN knockdown of hPDLSCs resulted in delayed cell migration, as determined by a scratch assay. However, migration improved after seeding these knockdown cells on pre-PN-coated surfaces. Further, the knockdown of αVß5 significantly attenuated the force-induced TGF-ß1 expression. CONCLUSION: Our findings indicate the importance of PN-αVß5 interactions in ICF-induced TGF-ß1 signaling and the expression of α-SMA. Findings support the critical role of PN in maintaining the PDL's tissue integrity and homeostasis.

Functional consequences of C-terminal mutations in RUNX2.

Cleidocranial dysplasia (CCD) is a genetic disorder caused by mutations in the RUNX2 gene, affecting bone and teeth development. Previous studies focused on mutations in the RUNX2 RHD domain, with limited investigation of mutations in the C-terminal domain. This study aimed to investigate the functional consequences of C-terminal mutations in RUNX2. Eight mutations were analyzed, and their effects on transactivation activity, protein expression, subcellular localization, and osteogenic potential were studied. Truncating mutations in the PST region and a missense mutation in the NMTS region resulted in increased transactivation activity, while missense mutations in the PST showed activity comparable to the control. Truncating mutations produced truncated proteins, while missense mutations produced normal-sized proteins. Mutant proteins were mislocalized, with six mutant proteins detected in both the nucleus and cytoplasm. CCD patient bone cells exhibited mislocalization of RUNX2, similar to the generated mutant. Mislocalization of RUNX2 and reduced expression of downstream genes were observed in MSCs from a CCD patient with the p.Ser247Valfs*3 mutation, leading to compromised osteogenic potential. This study provides insight into the functional consequences of C-terminal mutations in RUNX2, including reduced expression, mislocalization, and aberrant transactivation of downstream genes, contributing to the compromised osteogenic potential observed in CCD.

Alteration of extracellular matrix proteins in atrophic periodontal ligament of hypofunctional rat molars.

OBJECTIVES: The aim of this study was to investigate the effect of mechanical force on possible dynamic changes of the matrix proteins deposition in the PDL upon in vitro mechanical and in vivo occlusal forces in a rat model with hypofunctional conditions. MATERIALS AND METHODS: Intermittent compressive force (ICF) and shear force (SF) were applied to human periodontal ligament stem cells (PDLSCs). Protein expression of collagen I and POSTN was analyzed by western blot technique. To establish an in vivo model, rat maxillary molars were extracted to facilitate hypofunction of the periodontal ligament (PDL) tissue of the opposing mandibular molar. The mandibles were collected after 4-, 8-, and 12-weeks post-extraction and used for micro-CT and immunohistochemical analysis. RESULTS: ICF and SF increased the synthesis of POSTN by human PDLSCs. Histological changes in the hypofunctional teeth revealed a narrowing of the PDL space, along with a decreased amount of collagen I, POSTN, and laminin in perivascular structures compared to the functional contralateral molars. CONCLUSION: Our results revealed that loss of occlusal force disrupts deposition of some major matrix proteins in the PDL, underscoring the relevance of mechanical forces in maintaining periodontal tissue homeostasis by modulating ECM composition.

Intermittent compressive force regulates dentin matrix protein 1 expression in human periodontal ligament stem cells.

Background/purpose: Mechanical force differentially regulates periodontal ligament functions depending on types, magnitudes, and duration of stimulation. Intermittent compressive force (ICF) promotes an in vitro mineralization in human periodontal ligament cells. The present study investigated the effect of ICF on dentin matrix protein-1 (DMP1) expression in human periodontal ligament stem cells (hPDLSCs). Materials and methods: Cells were treated with ICF in a serum-free culture medium for 24 h The mRNA and protein expression were examined using real-time polymerase chain reaction, immunofluorescence staining and Western blot analysis, respectively. Results: The exposure to ICF in a serum-free condition significantly induced DMP1 expression in both mRNA and protein levels. The effect of ICF-induced DMP1 expression was inhibited by pretreatment with cycloheximide, indicating the requirement of the intermediated molecule(s). Pretreatment with transforming growth factor ß (TGF-ß) receptor inhibitor (SB431542) or neutralized antibody against TGF-ß1 prior to ICF application abolished the effect of ICF-induced DMP1 expression. Further, recombinant TGF-ß1 treatment stimulated DMP1 expression. Conclusion: The present study illustrated that ICF induces DMP1 expression in hPDLSCs via the regulation of TGF-ß signaling pathway.

Factors affecting the reduction rate of odontogenic cysts after decompression based on 3-dimensional volumetric analysis.

Purpose: This study aimed to investigate the potential factors that could affect the reduction rate of odontogenic cysts following decompression using cone-beam computed tomography (CBCT) for 3-dimensional volumetric analysis. Materials and Methods: The study sample consisted of CBCT images of 41 individuals who underwent decompression of odontogenic cysts at the Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Chulalongkorn University, between 2010 and 2022. Preoperative and postoperative CBCT results were collected, and a volumetric analysis was conducted to evaluate the differences in the reduction rate and the percentage of volume reduction of cystic lesions based on different parameters. Correlations between these parameters were analyzed to determine associations. Results: In this study, the average time of decompression for odontogenic cysts was 316 days. Males demonstrated a higher reduction rate than females (P<0.05). The reduction rate was directly proportional to initial cyst volume, with higher reduction rates for cysts with large initial volume than those with small initial volume (P<0.05). Spearman's rank correlation coefficient indicated a weak positive correlation between the initial cyst volume and the duration of decompression. Additionally, a strong positive correlation was observed between the initial volume and the reduction rate. Conclusion: Knowledge of the reduction rate of odontogenic cysts is vital for surgeons to evaluate the duration of decompression before enucleation and to determine a definitive treatment plan. Sex and initial lesion volume had significant effects on the reduction rate.

Utilization of rapid antigen tests for screening SARS-CoV-2 prior to dental treatment.

Potential aerosols containing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral particles can be generated during dental treatment. Hence, patient triage is essential to prevent the spread of SARS-CoV-2 in dental clinical settings. The present study described the use of rapid antigen tests for SARS-CoV-2 screening prior to dental treatment in an academic dental clinical setting in Thailand during the pandemic. The opinions of dental personnel toward the use of rapid antigen test screening prior to dental treatment were also assessed. From August 25 to October 3, 2021, dental patients who were expected to receive aerosols generating dental procedures were requested to screen for SARS-CoV-2 using a rapid antigen test before their treatment. A total of 7,618 cases completed the screening process. The average was 212 cases per day. Only five patients (0.07%) were positive for SARS-CoV-2 in the rapid antigen screening tests. All positive cases exhibited mild symptoms. For the questionnaire study, experienced dental personnel frequently and consistently agreed with the use of the rapid antigen test for SARS-CoV-2 screening, which made them feel safer during their patient treatment. However, implementing rapid antigen tests for SARS-CoV-2 may increase the total time spent on a dental appointment. In conclusion, a rapid antigen test could detect the infected individual prior to dental treatment. However, the specificity of rapid antigen tests for SARS-CoV-2 must be taken into account for consideration as a screening process before dental treatment. The enhanced infection control protocols in dental treatment must be consistently implemented.

Effects of decellularized extracellular matrix derived from Jagged1-treated human dental pulp stem cells on biological responses of stem cells isolated from apical papilla.

Objective: Indirect Jagged1 immobilization efficiently activates canonical Notch signaling in human dental pulp stem cells (hDPSCs). This study aimed to investigate the characteristics of the Jagged1-treated hDPSC-derived decellularized extracellular matrix (dECM) and its biological activity on odonto/osteogenic differentiation of stem cells isolated from apical papilla (SCAPs). Methods: Bioinformatic database of Jagged1-treated hDPSCs was analyzed using NetworkAnalyst. hDPSCs seeded on the Jagged1 immobilized surface were maintained with normal or osteogenic induction medium (OM) followed by decellularization procedure, dECM-N, or dECM-OM, respectively. SCAPs were reseeded on each dECM with either the normal medium or the OM. Cell viability was determined by MTT assay. Characteristics of dECMs and SCAPs were evaluated by SEM, EDX, immunofluorescent staining, and alcian blue staining. Mineralization was assessed by alizarin red S, Von Kossa, and alkaline phosphatase staining. Statistical significance was considered at p < 0.05. Results: RNA-seq database revealed upregulation of several genes involved in ECM organization, ECM-receptor interaction, and focal adhesion in Jagged1-treated hDPSCs. Immobilized Jagged1 increased the osteogenesis of the hDPSC culture with OM. dECMs showed fibrillar-like network structure and maintained major ECM proteins, fibronectin, type I-collagen, and glycosaminoglycans. A decrease in calcium and phosphate components was observed in dECMs after the decellularized process. Cell viability on dECMs did not alter by 7 days. Cell attachment and f-actin cytoskeletal organization of SCAPs proliferated on Jagged1-treated dECMs were comparable to those of the control dECMs. SCAPs exhibited significantly higher mineralization on dECM-N in OM and markedly enhanced on dECM-OM with normal medium or OM conditions. Conclusion: Jagged1-treated hDPSC-derived dECMs are biocompatible and increase odonto/osteogenic differentiation of SCAPs. The results suggested the potential of Jagged1 dECMs, which could be further developed into ECM scaffolds for application in regenerative medicine.

The association between thread pitch and cortical bone thickness influences the primary stability of orthodontic miniscrew implants: a study in human cadaver palates.

OBJECTIVE: The purpose of this study was to mathematically evaluate the influence of variations in thread pitch and cortical bone thickness on the maximum insertion torque (MIT) and implant stability (IS) of miniscrew implants (MIs). METHODS: Sixty custom made MIs with a 0.4-, 0.6-, 0.8-, 1.0-, or 1.2-mm thread pitch,12 for each pitch, were randomly placed into the palates of 10 embalmed human maxillae. The MIT was measured with a hand-operated digital torque reader screwdriver with a holding guide, and the IS test was performed using Anycheck. Conebeam computerized tomography was used to measure the cortical bone thickness(CBT) at each MI site. One-way ANOVA, Tukey post hoc test, Pearson's correlation,and multiple linear regression models were performed using the SPSS program. RESULTS: The MIT and IS tests demonstrated a pitch-dependent decrease. The pitch had a strong negative correlation with MIT and IS, while the CBT had a strong positive correlation with those outcomes. The association between pitch and CBT significantly influenced MI primary stability. Moreover, a strong correlation was found between MIT and IS. CONCLUSIONS: The MI primary stability, MIT, and IS are strongly influenced by theassociation between MI thread pitch and CBT.

Reduced ELANE and SLPI expression compromises dental pulp cell activity.

BACKGROUND: Patients with ELANE variants and severe congenital neutropenia (SCN) commonly develop oral complications. Whether they are caused only by low neutrophil count or the combination of neutropenia and aberrant dental cells is unknown. METHODS: Genetic variant was identified with exome sequencing. Dental pulp cells isolated from the SCN patient with an ELANE mutation were investigated for gene expression, enzyme activity, proliferation, colony formation, wound healing, apoptosis, ROS, attachment, spreading and response to lipopolysaccharide. RESULTS: ELANE cells had diminished expression of ELANE and SLPI and reduced neutrophil elastase activity. Moreover, ELANE cells exhibited impaired proliferation, colony forming, migration, attachment and spreading; and significantly increased ROS formation and apoptosis, corresponding with increased Cyclin D1 and MMP2 levels. The intrinsic levels of TGF-ß1 and TNF-α were significantly increased; however, IL-6, IL-8 and NF-kB1 were significantly decreased in ELANE cells compared with those in controls. After exposure to lipopolysaccharide, ELANE cells grew larger, progressed to more advanced cell spreading stages and showed significantly increased SLPI, TNF-α and NF-kB1 and tremendously increased IL-6 and IL-8 expression, compared with controls. CONCLUSION: This study, for the first time, suggests that in addition to neutropenia, the aberrant levels and functions of ELANE, SLPI and their downstream molecules in pulp cells play an important role in oral complications in SCN patients. In addition, pulp cells with diminished neutrophil elastase and SLPI are highly responsive to inflammation.