Unveiling the Neurodegenerative Alterations through Oral Stem Cells.

Parkinson's disease (PD) is a neurodegenerative condition characterized by the progressive and selective loss of dopaminergic (DAergic) neurons in the midbrain. The replacement of neuromelanin (NM)-containing DAergic neurons in the substantia nigra and the enhancement of NM concentration could offer a promising and safe approach to treating PD symptoms. The objective of this study was to investigate and compare the potential of human periapical-cysts mesenchymal stem cells (hPCy-MSCs) and dental pulp stem cells (DPSCs) to differentiate into DAergic NM-producing neurons and to generate functional 3-dimensional (3D) midbrain-like organoids in vitro. We assessed the changes in morphology and behavior of neuron-like cells (NLCs) as well as the expression of molecular markers characterizing the DAergic neurons. Furthermore, we observed electrically active and functionally mature DAergic neurons by means of electrophysiological assays, NM dosage assays, and the quantification of dopamine release by high-performance liquid chromatography. Our results demonstrate for the first time that both hPCy-MSCs and DPSCs are capable of differentiating into NLCs, further confirmed by the increase in lactate levels in the medium of cells exposed to neurogenic conditions. Importantly, we have induced such NLCs to further differentiate into functional DAergic NM-producing neurons. Finally, 3D midbrain-like organoids have been produced from oral stem cells: they appear as neurosphere-like structures diffusely expressing the neural marker ß-III tubulin and containing NM-like granules. Our findings open up a novel and fascinating opportunity to rethink oral stem cells, and the derived 3D disease models, as a strategic and reliable tool for unveiling the neurodegenerative alterations.

MicroRNA-27a-5p Downregulates Expression of Proinflammatory Cytokines in Lipopolysaccharide-Stimulated Human Dental Pulp Cells via the NF-κB Signaling Pathway.

MicroRNA-27a-5p (miR-27a-5p) was significantly upregulated in dental pulp inflammation, yet its underlying mechanisms remain unclear. This study investigated the effect of miR-27a-5p on the expression of proinflammatory cytokines in human dental pulp cells (hDPCs) stimulated by lipopolysaccharide (LPS). LPS-stimulated hDPCs showed concurrent increases in the expression of miR-27a-5p and proinflammatory cytokines (IL-6, IL-8, and MCP1), and the increased expression was suppressed by NF-κB inhibitor BAY 11-0785. Transfection of the miR-27a-5p mimic downregulated the expression of proinflammatory cytokines, NF-κB activity, and the expression of NF-κB signaling activators (TAB1, IRAK4, RELA, and FSTL1) in LPS-stimulated hDPCs. Luciferase reporter assays revealed that miR-27a-5p bound directly to the 3'-UTR of TAB1. siTAB1 downregulated NF-κB activity and proinflammatory cytokine expression. Downregulation of proinflammatory cytokine expression, NF-κB activity, and NF-κB signaling activator expression (TAB1, IRAK4, and RELA) was also found in LPS-stimulated rat incisor pulp tissue explants following transfection with the miR-27a-5p mimic ex vivo. MiR-27a-5p, whose expression was induced by NF-κB signaling, negatively regulated the synthesis of proinflammatory cytokines via targeting NF-κB signaling. In particular, TAB1, a potent NF-κB activator, was targeted by miR-27a-5p. These results provide insights into the negative regulatory effects of miR-27a-5p, particularly those targeting the TAB1-NF-κB signaling pathway, on pulp inflammation.

Assessment of the onset of analgesia and length of analgesia following the use of PBM with different wavelengths: a clinical study.

This clinical study assessed photobiomodulation (PBM) induced analgesic effects of diode lasers and an LED light source on the dental pulp. Baseline responses to electric pulp testing (EPT) were recorded in 93 healthy unrestored premolar teeth in 26 adults (age range 22-63 years) attending a private dental practice. The teeth were irradiated on buccal and lingual aspects of the crown, by placing the tips on the middle third of the crown of the teeth, on separate days for each of 4 different light sources (660, 808, or 904 nm diode lasers, or a novel multi-wavelength LED light source (700-1100 nm)) using comparable parameters (100 mW, 30 s, 6 J). EPT scores were measured after a further 1-, 2-, 5- and 20-min. Discomfort caused by PBM therapy was recorded using the Wong-Baker scale. EPT changes were tracked over time using repeated measures analysis of variance. Baseline EPT scores were very consistent between different days (linear regression r2 0.9422-0.9648). All PBM devices caused a significant elevation in EPT at 5 min, with an earlier onset at 2 mins for 904 nm and LED. The LED was the only light source that elevated scores at 20 min. Across 2-20 min, when ranked by effectiveness, the greatest EPT elevations were seen for LED, followed by 904 nm, then 660 nm and finally 808 nm. Discomfort during PBM was most common with 904 nm, followed by 808 nm. No discomfort occurred from the LED. Among the light sources utilized, the LED multi-wavelength system demonstrated the largest increase in EPT readings, suggesting its potential as a non-pharmacological alternative for achieving dental analgesia compared to diode lasers.

Examining the level of inflammatory cytokines TNF-α and IL-8 produced by osteoblasts differentiated from dental pulp stem cells.

BACKGROUND: The use of dental pulp stem cells (DPSCs) in clinical applications instead of bone marrow stem cells is a very promising method capable of significantly changing the future of medical treatment. If further studies prove that DPSCs and the cells differentiated from them do not stimulate the immune system, these cells can be used more reliably in treatment of autoimmune diseases. METHODS: In this research, we examined the isolated DPSCs and differentiated osteoblasts from them in medium without inflammatory stimulants in terms of TLR3 and TLR4 gene expression and inflammatory cytokines, including TNF-α and IL-8 using qRT-PCR, and measured the concentration of inflammatory cytokines IL-8 and TNF-α produced by these two types of cells through ELISA. RESULTS: The obtained results showed that the expression level of inflammatory cytokines IL-8 and TNF-α in differentiated osteoblasts is significantly different as compared with DPSCs. However, no significant difference was observed in TLR-4 expression between two groups. An increase in TNF-α expression level was found to directly correlate with an increase in the expression of IL-8. The concentration of cytokine TNF-α in osteoblasts was significantly higher than that of IL-8 in DPSCs. CONCLUSION: In comparison to DPSCs, osteoblast cells first lead to inflammatory responses. These responses reduce overtime. However, DPSCs retain their immunomodulatory properties and do not show inflammatory responses.

Effect of four direct pulp capping agents on human dental pulp tissue.

Direct pulp capping (DPC) is a vital pulp therapy, wherein accidental or carious pulp exposures can be capped with various materials to induce reparative dentine formation. One of the major factors that determine the success of direct pulp capping procedures is the material used. Therefore, it is of interest to evaluate and compare the efficacy of a new material, Tristrontium aluminate in comparison with the widely used materials such as ProRoot MTA, Biodentine & TheraCal LC. Hence, 40 premolars scheduled for extraction for orthodontic treatment, were equally divided into 4 groups and iatrogenic DPC was done using one of the study materials. The premolars were then extracted after 90 days & assessed for the formation of Dentine Bridge and pulpal response elicited by the materials using CBCT and histopathological analysis. All the materials tested in the study were successful in inducing dentine bridge formation & maintaining pulp vitality. Thus, the novelty of Tristrontium aluminate for potential applications in future is eminent.

Research progress of dental pulp regeneration treatment. / å†ç”Ÿæ€§ç‰™é«“æ²»ç–—çš„ç ”ç©¶è¿›å±•.

The dental pulp is the only soft tissue structure within the tooth, serving functions such as sensation and nutrition. However, the dental pulp is highly susceptible to necrosis due to external factors. Currently, root canal therapy is the most commonly used treatment for pulp necrosis. Nevertheless, teeth treated with root canal therapy are prone to secondary infections and adverse outcomes like vertical root fractures. Regenerative endodontic therapy has emerged as a solution, aiming to replace damaged tooth structures, including dentin, root structure, and the pulp-dentin complex cells. This approach demonstrates significant advantages in addressing clinical symptoms and achieving regeneration of the root and even the pulp. Since the discovery of dental pulp stem cells, regenerative endodontic therapy has gained new momentum. Advances in cell transplantation and cell homing techniques have rapidly developed, showing promising potential for clinical applications.

Post-mortem Interval estimate based on dental pulp: A histomorphology approach.

Estimating the post-mortem interval (PMI) of human remains based on the histomorphology of dental pulp parameters is promising, but available evidence is scarce and sometimes contradictory without a scientific model. The aim of the study is to characterise the histomorphological changes of dental pulp associated with the decomposition of human remains by a qualitative and quantitative approach. The main aim is to establish a correlation based on post-mortem (PM) dental pulp histomorphology and the PMI, and whether pulp degradation could be an available medico-legal tool for PMI estimation beyond the first week after death (late PMI). The eligible sample consisted of 27 sound teeth from 16 healthy patients aged 16 to 72 years due to orthodontic or oral surgery treatment, to create PMI's simulating the death of the subject as the time elapsed from tooth avulsion. Data collected from patients (sex, date of birth, tooth position, date and hour of the avulsion, date and hour of pulp extraction) were anonymised in accordance with the requirements of Faculty of Dental Medicine of the University of Lisbon. The sample was divided into 9 groups of 3 teeth according to different PMI sets from T0 (baseline) up to 2 weeks (T0, 7, 12, 24, 36, 48, and 72 hours, 1 and 2 weeks). All the dental samples were stored at room temperature up to the time of pulp extraction and then prepared with haematoxylin and eosin stain. High-resolution microscopy was performed to obtain histological images. An operator performed the qualitative evaluation of blood vessels, collagen fibres, and the extra-cellular matrix (ECM) in PM pulps and measured the variation in cells/nuclei density by counting 6 different ROIs (Regions of Interest) for each pulp manually and automatically (quantitative analysis). Qualitative results showed that the degeneration of dental pulp appears 7 hours after death but histological changes in vessels, fibres, and ECM in PM dental pulp are characterised by high variability, consequently it is not possible to generalise the results for early PMIs. Quantitative measurements proved that cell count cannot be standardised due to the presence of superimposed layers of cells and nuclei fragmentation. Odontoblasts did not demonstrate evidence of cellular or nuclear lysis up to 14 PM suggesting their applicability in late PMIs. Future research will focus on late PMIs and different techniques of tooth preparation.

Chemical preconditioning escalates chondrogenic activity in explant cultured human dental pulp stem cell study model for future temporomandibular joint regeneration.

Context: Human dental pulp stem cells (hDPSC) derived from dental pulp in conducive environment activated by chemicals can enhance chondrogenic cells for future animal model temporomandibular joint model. Aim: The study aims at evaluating the chemicals preconditioning (curcumin and rapamycin) efficacy toward chondrogenic proliferation of human dental pulp stem cells. Settings and Design: The in vitro study model with 10 premolar teeth extirpated pulp was processed under sterile chemical conditions. The cells viability was checked with calorimetric assay for adipogenic and chondrogenic, osteogenic lineages. The viability of the cells and the concentration of curcumin (CU) and rapamycin (RP) required for cell differentiation toward chondrogenic lineage were assessed. Material and Methods: The hDPSC was evaluated after explant long-term cultivation with characterization and chemical conditioning with dimethyl sulfoxide (DMSO) as control. MTT assay was used for cytotoxicity evaluation, cell viability, and proliferation. The dose optimization was observed with RP and CU. Chondrogenic proliferation was assessed with standard staining method of 0.1% Safranin O and 0.1% Alcian blue. Statistical Design: The flow cytometry analysis revealed good results for CD 90 compared to others. The intergroup analysis was done by ANOVA, and intragroup analysis was done by Post hoc Tukey's test. The intragroup analysis showed P value < 0.05 for RP in comparison between the various preconditioning agents CU and RP. The dosage of 10 µg/ml RP was considered statistically significant. Results: The flow cytometer analysis revealed good results for CD 90 compared to other surface markers. The dosage of 10 µg/ml RP was having good chondrogenic cell proliferation. The intragroup analysis showed P value < 0.05 for RP in comparison between the various preconditioning agents CU and RP. The calorimetric assay (MTT) quantitative analysis of the chondrogenic cells with Safranin O stain the standard deviation (SD = 0.017 for rapamycin), Alcian blue (SD = 0.49 for RP) in comparison to DMSO (control) and CU. Conclusion: RP activates mTOR pathway and hence stabilizes the stem cell maintenance of human dental pulp stem cell and the dose quantified can be used for future animal temporomandibular joint animal model.

Efficacy of topical application of corticosteroids in the remineralization of dental pulp tissue. A systematic review of the literature.

OBJECTIVES: The aim of this systematic review was to demonstrate the efficacy of topical application of corticosteroids in remineralization of dental pulp tissues to preserve their vitality and function. DATA, SOURCES AND STUDY SELECTION: An electronic search was performed using MEDLINE by PubMed, EMBASE, Web of Science (WOS), and Scopus databases. The inclusion criteria were in vitro studies that employed dental pulp tissue obtained from extracted healthy permanent human teeth and were subjected to topical administration of corticosteroids and evaluated tissue remineralization by performing any mineralization assay. A total of 11 studies were selected for inclusion. PRISMA guidelines were followed, and the methodological quality and risk of bias of the included studies were evaluated using the RoBDEMAT guidelines. Also, tables were designed for data extraction, including tissue mineralization and osteogenic differentiation as primary and secondary outcomes, respectively. CONCLUSIONS: Alizarin Red S (ARS) has been able to demonstrate a possible mineralizing power of corticosteroids, applied at an adequate dose. The up-regulation of Alkaline phosphatase (ALP), osteocalcin (OCN), osteopontin (OSP), sialophosphoprotein (DSPP), runt-related transcription factor 2 (RUNX2), collagen type 1 alpha 1(COL1α1) and dentin matrix protein 1 (DMP-1) induced the osteogenic/odontogenic differentiation of dental pulp stem cells (DPSCs). CLINICAL SIGNIFICANCE: Deep carious lesions treatment is still challenging in restorative dentistry. Some treatments have been focused on dental pulp tissue remineralization to maintain the function and vitality. After corticosteroids topical application, mineral deposition and osteogenic differentiation have been detected.

Hybrid scaffolds for bone tissue engineering: Integration of composites and bioactive hydrogels loaded with hDPSCs.

Bone tissue regeneration remains a significant challenge in clinical settings due to the complexity of replicating the mechanical and biological properties of bone environment. This study addresses this challenge by proposing a hybrid scaffold designed to enhance both bioactivity and physical stability for bone tissue regeneration. This research is the fisrt to develop a rigid 3D structure composed of polycaprolactone (PCL) and hydroxyapatite nanoparticles (nHA) integrated with a bioink containing human dental pulp stem/stromal cells (hDPSCs), alginate, nHA and collagen (Col). The biofabricated constructs were extensively characterized through cytocompatibility tests, osteogenic differentiation assessment, and biocompatibility evaluation in a rat model. In vitro results demontrated that the hybrid scaffolds presented significantly higher cell viability after 168 h compared to the control group. Furthermore, the hybrid scaffolds showed increased osteogenic differentiation relative to other groups. In vivo evaluation indicated good biocompatibility, characterized by minimal inflammatory response and successful tissue integration. These findings highlight the scaffold's potential to support bone tissue regeneration by combining the mechanical strength of PCL and nHA with the biological activity of the alginate-nHA-Col and hDPSCs bioink. The current study provides a promising foundation for the development of biomaterials aimed at improving clinical outcomes in bone repair and regeneration, particulary for the treatment of critical-size bone defects, targeted drug administration, and three-dimensional models for bone tissue engineering.

Pulp Sensitivity Testing in Multiple Sclerosis: Disease Duration and Sensory/Motor Associations-A Cross-Sectional Study.

Introduction: This study explores a relatively unexplored aspect of multiple sclerosis (MS) by examining the sensitivity threshold of dental pulp as a potential indicator of neuropathy in MS patients. Building upon earlier research that focused on assessing the response to electrical pulp testing in MS patients who did not have a history of trigeminal neuralgia, this survey is aimed at delving into the relationship between MS duration and the threshold for stimulation in response to pulp sensitivity tests. Materials and Methods: This study encompassed a total of 124 maxillary central incisors from patients diagnosed with relapsing-remitting multiple sclerosis (RRMS). The participants were uniform in terms of age, falling within the 18-50 years range, and all had RRMS with no history of trigeminal neuralgia. The electric pulp sensitivity test was conducted on all samples, and the results of the electric pulp testing (EPT) were recorded according to the grade of the pulp tester that elicited a response. The threshold was considered reached when the patient first experienced a burning sensation after EPT application and the use of 1,1,1,2-tetrafluoroethane spray. Data analysis employed paired t-tests, Fisher's exact test, and Spearman correlation, with a significance level set at p < 0.05. Results: Based on the study's findings, the average response value to EPT was 2.69 ± 1.17, while the response time to the cold test was 2.61 ± 1.03 s. There was no statistically significant difference in the response to the cold test based on age (p = 0.45). However, it was observed that the mean response time to the cold test was significantly longer among male participants (p = 0.001). No significant differences were identified in the pulpal response to EPT or the cold test between patients with and without sensory-motor involvement (p > 0.05). Furthermore, Spearman's analysis revealed a noteworthy positive correlation between the electrical pulp threshold and the time taken to respond to the cold test (p = 0.025, r = 0.2). Conclusions: The utilization of the pulpal sensitivity test in MS patients holds promise for practical clinical use. Notably, individuals with a more extended duration of the disease exhibited a notably elevated threshold for both the EPT and the cold test conducted on their maxillary central incisors.

Inconsistent decision making in dental caries diagnosis and treatment: A case-based questionnaire survey.

Background and Aims: Delayed implementation of new knowledge into clinical practice poses patient safety risks. This study investigates agreement on use of the dental caries interventions, sealing, and stepwise excavation. Methods: A cross-sectional questionnaire survey, based on 11 constructed cases with descriptions of patient symptoms, radiographic, and clinical findings. Interrater agreement on dental caries- and pulp diagnoses and interventions were measured with Cohen's and Light's κ. The data collection period was September 28 to November 5, 2021. To explore variations in use and knowledge factors, we examined Danish dentists' attitudes toward continuing education. Results: Based on 243 responses, moderate interrater agreement for dental caries and pulp diagnoses and weak agreement on interventions were seen. The agreement with the gold standard for caries was moderate. No agreement was found for dental pulp diagnosis, and for interventions the agreement was weak. No pattern in agreement with the gold standard was seen in relation to case difficulty level. The majority reported knowing of and using stepwise excavation, in conflict with findings that less than half chose stepwise excavation in cases, where considered appropriate. One in four (25%) reported to be unfamiliar with sealing, and half (50%) use sealing regularly. Better access to continuing education and for universities to offer continuing education as alternatives to one-sided private market were requested. Conclusion: Some patients may receive too radical treatment despite available less invasive evidence-based effective treatments. Dentists acknowledge the importance of continuing education. Easier access and perhaps more incentives for seeking out high-quality continuing education from trustworthy sources are needed.

Endodontic characteristics of mandibular premolar with dens evaginatus: a retrospective study.

Objectives: This study aimed to investigate the endodontic characteristics of mandibular premolars with dens evaginatus (DE) that require endodontic treatment. Materials and Methods: Patients who underwent endodontic treatment were enrolled. The inclusion criteria were patients who underwent root canal treatment in the lower permanent teeth with DE and were followed up for at least 1 year. Preoperative clinical and radiographic variables were obtained. The frequency distribution of the preoperative variables was compared using the χ2 or Fisher's exact tests. The significance of the change in periapical health index (PAI) and root development stages before and after treatment was examined using the Wilcoxon signed-rank test. Results: A total of 150 teeth of 134 patients with an average age of 15.3 years were included. The percentage distribution comparison of the preoperative variables and obturation techniques revealed significant differences in pulpal and periapical diagnosis, and percussion, and especially regarding age, root development stage, and PAI. Age was the only statistically significant preoperative variable associated with root growth (p < 0.05). Conclusions: Approximately, 60% of DEs requiring endodontic treatment had immature roots. Age being the most significant predisposing factor, early treatment provides the greatest opportunity for full root development.

Sclerostin Antibody-Loaded Dense Collagen Hydrogels Promote Critical-Size Bone Defect Repair.

The management of extensive bone loss remains a clinical challenge. Numerous studies are underway to develop a combination of biomaterials, biomolecules, and stem cells to address this challenge. In particular, the systemic administration of antibodies against sclerostin, a regulator of bone formation, was recently shown to enhance the bone repair efficiency of dense collagen hydrogels (DCHs) hosting murine dental pulp stem cells (mDPSCs). The aim of the present study was to assess whether these antibodies, encapsulated and released from DCHs, could promote craniofacial bone repair by the local inhibition of sclerostin. In vitro studies showed that antibody loading modified neither the hydrogel structure nor the viability of seeded mDPSCs. When implanted in a mouse calvaria critical-size bone defect, antibody-loaded DCHs showed repair capabilities similar to those of acellular unloaded DCHs combined with antibody injections. Importantly, the addition of mDPSCs provided no further benefit. Altogether, the local delivery of antisclerostin antibodies from acellular dense collagen scaffolds is highly effective for bone repair. The drastic reduction in the required amount of antibody compared to systemic injection should reduce the cost of the procedure, making the strategy proposed here a promising therapeutic approach for large bone defect repair.

Root canal configuration of human permanent anterior teeth: A cross-sectional study using cone-beam computed tomography in the Pakistani population.

Objectives: To analyse and document internal root morphological variations in permanent anterior teeth using conebeam computed tomography. METHODS: The cross-sectional, retrospective study was conducted from January 21 to June 19, 2021, at Jinnah Magnetic Resonance Imaging and Body Scan, and comprised data from July to December 2020 related to all the patients who had been referred to the centre for dental cone-beam computed tomography scan. The scans were evaluated at the Radiology Department of the School of Dentistry at Shaheed Zulfiqar Ali Bhutto Medical University, Islamabad, Pakistan. The morphology of the roots and root canals in all permanent anterior teeth was examined. Root canal configuration was categorised using Vertucci's classification. Data was analysed using SPSS 20. RESULTS: Of the 211 patients, 116(55%) were females and 95(45%) were males. The overall mean age was 39.9±1.02 years (range: 15-65 years). All the permanent anterior teeth exhibited a single root, with the exception 2(0.9%) mandibular right and 4(1.9%) left canines which had double roots. Type I root canals were observed in all 211 (100%) maxillary right central incisors, maxillary left central incisors and maxillary left canines. Among maxillary right canines, 208(98.6%) had type-I root canals, and the corresponding value for maxillary right lateral incisors was 209(99.1%), and for maxillary left lateral incisors, the value was 210(99.5%). Root canal polymorphism had no significant association with gender (p>0.05). CONCLUSIONS: The presence of type-I root canal was common in permanent anterior teeth, and there was significant association of root canal polymorphism with gender.

Biocompatibility and antimicrobial effect of demineralised dentin matrix hydrogel for dental pulp preservation.

Regeneration of dentin and preserving pulp vitality are essential targets for vital pulp therapy. Our study aimed to evaluate a novel biomimetic pulp capping agent with increased dentin regenerative activities. To produce demineralised dentin matrix (DDM) particles, human extracted teeth were ground and treated with ethylene diamine tetra-acetic acid solution. DDM particles were added to sodium alginate and this combination was dripped into a 5% calcium chloride to obtain DDM hydrogel (DDMH). The eluants of both DDMH and mineral trioxide aggregate (MTA) were tested using an MTT assay to detect their cytotoxic effect on dental pulp stem cells (DPSC). Collagen-I (COL-I) gene expression was analysed on DPSC exposed to different dilutions of pulp capping material eluants by real-time quantitative polymerase chain reaction. Acridine orange staining was used to monitor the cell growth over the tested materials. Agar diffusion assay was utilised to test the antibacterial effect of DDMH and MTA compared to controls. MTT assay revealed that neat eluates of DDMH promoted DPSC viability. However, neat eluates of MTA were cytotoxic on DPSC after 72 h of culture. Moreover, DPSC were capable of growth and attached to the surface of DDMH, while they showed a marked reduction in their number when cultured on the MTA surface for one week, as shown by the acridine orange stain. In DPSC cultured with DDMH eluates, the COL-I gene was overexpressed compared to those cultured with MTA eluants. DDMH had significant antimicrobial activity in comparison to MTA after 24 h incubation. This in vitro study showed that DDMH could be an alternative pulp capping agent for regenerative endodontics.

Apoptosis Regulation in Dental Pulp Cells and PD-1/PD-L1 Expression Dynamics Under Ozone Exposure - A Pilot Approach.

This study aimed to determine the effect of ozone on the expression of Bax and Bcl-2 genes in dental pulp cells. Additionally, the programmed cell death protein 1, programmed death-ligand 1, and CD200 antigens were determined in lymphocytes to assess their surface expression. Dental pulp cells were cultured from extracted healthy third molars and characterized as dental pulp stromal cells. Gene expression of Bcl-2 and Bax was analyzed at 0 s, 6 s, and 12 s of ozone exposure using real-time PCR. Lymphocytes from dental pulp were subjected to ozone exposure for 12 s and PD-1, PD-L1, and CD200/CD200R expression was analyzed by flow cytometry. Upon exposure to ozone for 6 s, the Bcl-2 expression decreased significantly to -0.09, and at 12 s, it increased significantly to 0.3. Bax gene expression level increased significantly to 0.188 after 6 s exposure, and at 12 s, to 0.16. Lymphocytes exposed to ozone for 12 s showed minimal changes in PD-1, PD-L1, and CD200/CD200R expression levels, indicating that oxidative stress does not impact the signaling pathways regulating these molecules. The significant upregulation of Bcl-2 at 12 s highlights the cells' effort to protect themselves from prolonged oxidative stress, possibly tipping the balance toward cell survival and tissue repair. However, the absence of changes in PD-1 and PD-L1 expression on lymphocytes under oxidative stress suggests that these molecules are not sensitive to oxidative stress in this context.

Evaluation of the cytotoxicity of new formulations of cariostatic agents containing nano silver fluoride: an in vitro study.

The objective of the study was to assess the indirect cytotoxicity of 600 ppm and 1500 ppm nano silver fluoride (NSF) compared to other commercial cariostatic agents. 56 dentin discs with 0.4 mm in thickness were obtained from intact human molars and adapted to artificial pulp chambers (APCs). The discs were divided into seven groups according to treatment (n = 8): no treatment (positive control-PC), 29% hydrogen peroxide (negative control-NC), 30% Cariestop (CS30), 38% Riva Star (RS38), 38% Advantage Arrest (AA38), 600 ppm NSF (NSF600), and 1500 ppm NSF (NSF1500). The cariostatic agents were applied on the occlusal surface of the dentin discs (facing upward), and the pulp surface (facing downward) remained in contact with the culture medium. Immediately after the treatments, the extracts (DMEM + cariostatic agent components diffused through the discs) were collected and applied to MDPC-23 cells, which were assessed for viability (CV-alamarBlue, live/dead), adhesion/spreading (F-actin), alkaline phosphatase (ALP) activity, and mineralization nodule (MN) formation. The data were statistically analyzed by ANOVA/Games-Howell (p = 0.05). CV and ALP activity in CS30, RS38, AA38, and NSF600 were similar to PC (p > 0.05). MN formation significantly decreased only in NC, CS30, RS38, and AA38 compared to PC (p < 0.001). Only NSF600 and NSF1500 did not differ from PC (p > 0.05) with mineralization nodules, and this specific cell activity significantly decreased in all other groups (p < 0.05). NSF solutions (600 ppm and 1500 ppm) did not cause transdentinal toxicity on MDPC-23 cells.

GREM1 Negatively Regulates Osteo-/Dentinogenic Differentiation of Dental Pulp Stem Cells via Association with YWHAH.

OBJECTIVE: To investigate the biological regulatory function of Gremlin1 (GREM1) and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein eta (YWHAH) in dental pulp stem cells (DPSCs), and determine the underlying molecular mechanism involved. METHODS: Alkaline phosphatase (ALP) activity, alizarin red staining, scratch migration assays and in vitro and in vivo osteo-/dentinogenic marker detection of bone-like tissue generation in nude mice were used to assess osteo-/dentinogenic differentiation. Coimmunoprecipitation and polypeptide microarray assays were employed to detect the molecular mechanisms involved. RESULTS: The data revealed that knockdown of GREM1 promoted ALP activity, mineralisation in vitro and the expression of osteo-/dentinogenic differentiation markers and enhanced osteo-/ dentinogenesis of DPSCs in vivo. GREM1 bound to YWHAH in DPSCs, and the binding site was also identified. Knockdown of YWHAH suppressed the osteo-/dentinogenesis of DPSCs in vitro, and overexpression of YWHAH promoted the osteo-/dentinogenesis of DPSCs in vitro and in vivo. CONCLUSION: Taken together, the findings highlight the critical roles of GREM1-YWHAH in the osteo-/dentinogenesis of DPSCs.

Osteogenic potency of dental stem cell-composite scaffolds in an animal cleft palate model.

Objective: To evaluate the osteogenic potency of stem cells isolated from human exfoliated deciduous teeth (SHED) in polycaprolactone with gelatin surface modification (PCL-GE) and poly (lactic-co-glycolic acid)-bioactive glass composite (PLGA-bioactive glass (BG)) scaffolds after implantation in a rat cleft model. Methods: Cleft palate-like lesions were induced in Sprague-Dawley rats by extracting the right maxillary first molars and drilling the intact alveolar bone. Rats were then divided into five groups: Control, PCL-GE, PCL-GE-SHED, PLGA-BG, and PLGA-BG-SHED, and received corresponding composite scaffolds with/without SHED at the extraction site. Tissue samples were collected at 2, 3, and 6 months post-implantation (4 rats per group). Gross and histological analyses were conducted to assess osteoid or bone formation. Immunohistochemistry for osteocalcin and human mitochondria was performed to evaluate bone components and human stem cell viability in the tissue. Results: Bone tissue formation was observed in the PCL-GE and PLGA-BG groups compared to the control, where no bone formation occurred. PLGA-BG scaffolds demonstrated greater bone regeneration potential than PCL-GE over 2-6 months. Additionally, scaffolds with SHED accelerated bone formation compared to scaffolds alone. Osteocalcin expression was detected in all rats, and positive immunoreactivity for human mitochondria was observed in the regenerated bone tissue with PCL-GE-SHED and PLGA-BG-SHED. Conclusion: PCL-GE and PLGA-BG composite scaffolds effectively repaired and regenerated bone tissue in rat cleft palate defects. Moreover, scaffolds supplemented with SHED exhibited enhanced osteogenic potency. Clinical significance: PCL-GE and PLGA-BG scaffolds, augmented with SHED, emerge as promising biomaterial candidates for addressing cleft repair and advancing bone tissue engineering endeavors.