Human Dental Pulp Stem Cells Research Articles

Antimicrobial Peptide- and Dentin Matrix-Functionalized Hydrogel for Vital Pulp Therapy via Synergistic Bacteriostasis, Immunomodulation, and Dentinogenesis.

The preservation of vital pulps is crucial for maintaining the physiological functions of teeth; however, vital pulp therapy (VPT) of pulpitis teeth remains a substantial challenge due to uncontrolled infection, excessive inflammation, and limited regenerative potential. Current pulp capping agents have restricted effects in the infectious and inflammatory microenvironment. To address this, a multifunctional hydrogel (TGH/DM) with antibacterial, immunomodulatory, and mineralization-promoting effects is designed. The antimicrobial peptide (AMP) and demineralized dentin matrix are incorporated into the hydrogel, achieving sustainable delivery of AMP and a cocktail of growth factors. In vitro results show that TGH/DM could kill endodontic microbiota, ameliorate inflammatory responses of human dental pulp stem cells (hDPSCs), and prompt odontogenic differentiation of inflammatory hDPSCs via activation of peroxisome proliferator-activated receptor gamma. In vivo results suggest that TGH/DM is capable of inducing M2 phenotype transformation of macrophages in mice and fostering the regeneration of the dentin-pulp complex in inflamed pulps of beagle dogs. Overall, this study first proposes the synergistic regulation of AMP and tissue-specific extracellular matrix for the treatment of pulpitis, and the advanced hydrogel provides a facile and effective way for VPT.

Peptide KN-17-Loaded Supramolecular Hydrogel Induces the Regeneration of the Pulp-Dentin Complex.

Regenerating the pulp-dentin complex remains a decisive factor during apexification for immature permanent teeth. Peptide KN-17, which was modified based on the structure of cecropin B, could effectively interfere with bacterial growth and induce the migration of human bone marrow stromal cells (hBMSCs). This study aimed to investigate the effect of KN-17 on the tissue regeneration. To our surprise, KN-17 can significantly stimulate angiogenesis in vitro and in vivo, which may provide a guarantee for apical closure. Herein, a novel peptide/KN-17 coassembled hydrogel is developed via a heating-cooling process. Npx-FFEY/KN-17 supramolecular hydrogel can induce vessel development, stimulate odontogenic differentiation of human dental pulp stem cells (hDPSCs), and exert an antibacterial effect on Enterococcus faecalis (E. faecalis). Furthermore, coronal pulp excised rat molars are supplied with KN-17 or KN-17-loaded hydrogel and transplanted subcutaneously in BALB/c-nu mice. After 4 weeks, the hydrogel Npx-FFEY/KN-17 stimulates the formation of multiple odontoblast-like cells and dentin-like structures. Our findings demonstrate that the KN-17-loaded hydrogel can promote the regeneration of the pulp-dentin complex for continued root development.

Osteoblastic differentiation and changes in the redox state in pulp stem cells by laser treatment

The aim of this study was to determine the effect of low-level laser therapy (LLLT) on cell proliferation, mitochondrial membrane potential changes (∆Ψm), reactive oxygen species (ROS), and osteoblast differentiation of human dental pulp stem cells (hDPSCs). These cells were irradiated with 660- and 940-nm lasers for 5 s, 50 s, and 180 s. Cell proliferation was assessed using the resazurin assay, cell differentiation by RUNX2 and BMP2 expression, and the presence of calcification nodules using alizarin-red S staining. ROS was determined by the dichlorofluorescein-diacetate technique and changes in ∆Ψm by the tetramethylrhodamine-ester assay. Data were analyzed by a Student’s t-test and Mann–Whitney U test. The 940-nm wavelength for 5 and 50 s increased proliferation at 4 days postirradiation. After 8 days, a significant decrease in proliferation was observed in all groups. Calcification nodules were evident in all groups, with a greater staining intensity in cells treated with a 940-nm laser for 50 s, an effect that correlated with increased RUNX2 and BMP2 expression. ROS production and Δψm increased independently of irradiation time. In conclusion, photobiomodulation (PBM) with LLLT induced morphological changes and reduced cell proliferation rate, which was associated with osteoblastic differentiation and increased ROS and Δψm, independent of wavelength and time.

Effects of Semiconductor Laser Irradiation on Differentiation of Human Dental Pulp Stem Cells in Co-Culture with Dentin.

This study aimed to determine the effect of photobiomodulation therapy induced by semiconductor laser irradiation on human dental pulp stem cell (hDPSC) proliferation and their differentiation into odontoblast-like cells (OLCs). The effects of various semiconductor laser irradiation conditions on hDPSCs were examined. Three groups were evaluated: a single laser irradiation at 6 h post-seeding, multiple laser irradiations up to four times every 4 days after the first dose, and a control with no laser irradiation. The cells were irradiated at 10, 30, and 150 mW using a semiconductor laser. The effect of laser irradiation on hDPSC differentiation into OLCs was also determined. Four groups were evaluated, including co-culture using basic medium and dentin discs, simple culture using OLC differentiation-inducing medium, co-culture using OLC differentiation-inducing medium and dentin discs, and control culture with basic medium. The expression of the nestin, ALP, DSPP, and DMP-1 genes was measured using real-time PCR. The multiple irradiation group irradiated at 30 mW exhibited significantly more cell proliferation than the control. The expression of nestin associated with differentiation into OLCs during each culture period tended to be lower, whereas DSPP and ALP expression was higher compared with that of the control. Multiple laser irradiations at a low power of 30 mW induced significant hDPSC proliferation and might induce differentiation into OLCs.

Lin28 affects the proliferation and osteogenic differentiation of human dental pulp stem cells by directly inhibiting let-7b maturation

ObjectiveActivation of Lin28 gene under certain conditions promotes tissue damage repair. However, it remains unknown whether conditional expression of Lin28 facilitates the recovery of damaged pulp tissue. In the study, we focus on exploring the effects and possible regulatory mechanisms of Lin28 on the proliferation and differentiation of human dental pulp stem cells (hDPSCs).Materials and methodsWe adopted techniques such as the ethynyl-2ʹ-deoxyuridine (EdU) incorporation assay, RNA-protein immunoprecipitation (RIP) analysis, and luciferase assays to study the regulation of hDPSCs by Lin28. Furthermore, gain-of-function and loss-of-function analyses were also used in explored factors regulating hDPSCs activation.ResultsThe results show that Lin28 inhibited osteogenic differentiation by directly targets pre-let-7b. Through bioinformatics sequencing and dual luciferase experiments we learned that let-7b directly targets the IGF2BP2 3’UTR. Silencing of IGF2BP2 showed a similar biological effect as overexpression of let-7b. Overexpression of IGF2BP2 counteracted the differentiation-promoting effects produced by let-7b overexpression.Discussion/conclusionsIn conclusion, the RNA-binding protein Lin28 regulates osteogenic differentiation of hDPSCs by inhibiting let-7 miRNA maturation. And mature let-7b directly regulated the expression of IGF2BP2 by targeting the 3’UTR region of IGF2BP2 mRNA thus further inhibiting the differentiation of hDPSCs.

Bio-functionalized carbon dots for signaling immuno-reaction of carcinoembryonic antigen in an electrochemical biosensor for cancer biomarker detection

Early diagnosis of cancer demands sensitive and accurate detection of cancer biomarkers in blood. Carbon dots (CDs) bio-functionalization with antibodies, peptides or aptamers have played significant role in cancer diagnosis and targeted cancer therapy. Herein, a biosensor for detection of cancer biomarker carcinoembryonic antigen (CEA) in blood serum has been designed using CDs bio-functionalized with HRP-conjugated CEA antibody (CUCDs@CEAAb2) as detection probe. CDs were synthesized by upscaling of cow urine, a nitrogen rich biomass waste, by hydrothermal method. Detection probe based on CDs resulted in 3.5 times higher sensitivity as compared to conventional electrochemical sandwich immunoassay. To further improve the sensor performance, hyper-branched polyethylenimine grafted poly amino aniline (PEI-g-PAANI) was used as the sensing interface, which enabled immobilization of higher amount of capture antibody. Detection of CEA in human blood serum coupled with wide linear range (0.5–50 ng/ml), good specificity, stability, reproducibility and low detection limit (10 pg/ml) signified the excellence of CUCDs based CEA immunosensor. CUCDs exhibited excitation wavelength dependent fluorescence property and showed strong blue emission under UV irradiation. MTT assay indicated that the material is not toxic towards human dental pulp stem cells (hDPSCs) and MG63 osteosarcoma cells (cell viability > 90%). The present study demonstrates a methodology for valorization of animal waste to a cost-effective carbon based functional nanomaterial for clinical detection of cancer biomarkers.

Hsa_circ_0036872 has an important promotional effect in enhancing osteogenesis of dental pulp stem cells by regulating the miR-143-3p/IGF2 axis

BackgroundCircular RNAs (circRNAs), an extremely stable group of RNAs, possess a covalent closed-loop configuration. Numerous studies have highlighted the involvement of circRNAs in physiological processes and the development of various diseases. The present study aimed to investigate how circRNA regulates the osteogenic differentiation of human dental pulp stem cells (hDPSCs). MethodsWe isolated hDPSCs from dental pulp and used next-generation sequencing analysis to determine the differentially-expressed circRNAs during osteogenic differentiation. Bioinformatics and dual-luciferase reporter assays identified the downstream targets. The role of circRNAs in osteogenic differentiation was further confirmed through the use of heterotopic bone models. ResultsWe found that hsa_circ_0036872 expression was increased during osteogenic differentiation of hDPSCs, and downregulation of hsa_circ_0036872 inhibited their osteogenic differentiation. Dual-luciferase reporter assays showed that both miR-143-3p and IGF2 were downstream targets of hsa_circ_0036872. Overexpression of IGF2 or inhibition of miR-143-3p restored the osteogenic differentiation ability of hDPSCs after silencing hsa_circ_0036872. Overexpression of IGF2 reversed the inhibitory effect of miR-143-3p on osteogenic differentiation. ConclusionTaken together, our results show that hsa_circ_0036872 exerts an important promotional effect in enhancing the osteogenesis of dental pulp stem cells by regulating the miR-143-3p/IGF2 axis. These data suggest a novel therapeutic strategy for osteoporosis treatment and periodontal tissue regeneration.

PDGF-AA guides cell crosstalk between human dental pulp stem cells invitro via the PDGFR-α/PI3K/Akt axis.

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. 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. 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. 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.

Enhancing the inhibition of dental erosion and abrasion with quercetin-encapsulated hollow mesoporous silica nanocomposites.

Introduction: Dental erosion and abrasion pose significant clinical challenges, often leading to exposed dentinal tubules and dentine demineralization. The aim of this study was to analyse the efficacy of quercetin-encapsulated hollow mesoporous silica nanocomposites (Q@HMSNs) on the prevention of dentine erosion and abrasion. Method: Q@HMSNs were synthesized, characterized, and evaluated for their biocompatibility. A total of 130 dentine specimens (2mm × 2mm × 2mm) were prepared and randomly distributed into 5 treatment groups (n = 26): DW (deionized water, negative control), NaF (12.3mg/mL sodium fluoride, positive control), Q (300μg/mL quercetin), HMSN (5.0mg/mL HMSNs), and Q@HMSN (5.0mg/mL Q@HMSNs). All groups were submitted to in vitro erosive (4 cycles/d) and abrasive (2 cycles/d) challenges for 7days. The specimens in the DW, NaF, and Q groups were immersed in the respective solutions for 2min, while treatment was performed for 30s in the HMSN and Q@HMSN groups. Subsequently, the specimens were subjected to additional daily erosion/abrasion cycles for another 7days. The effects of the materials on dentinal tubule occlusion and demineralized organic matrix (DOM) preservation were examined by scanning electron microscopy (SEM). The penetration depth of rhodamine B fluorescein into the etched dentine was assessed using confocal laser scanning microscopy (CLSM). The erosive dentine loss (EDL) and release of type I collagen telopeptide (ICTP) were measured. The data were analysed by one-way analysis of variance (ANOVA) with post hoc Tukey's test (α = 0.05). Results: Q@HMSNs were successfully synthesized and showed minimal toxicity to human dental pulp stem cells (HDPSCs) and gingival fibroblasts (HGFs). Q@HMSNs effectively occluded the dentinal tubules, resulting in a thicker DOM in the Q@HMSN group. The CLSM images showed more superficial penetration in the HMSN and Q@HMSN groups than in the quercetin, NaF, and DW groups. The Q@HMSN group exhibited a significantly lower EDL and reduced ICTP levels compared to the other groups (p < 0.05). Conclusion: Q@HMSNs hold promise for inhibiting dentine erosion and abrasion by promoting tubule occlusion and DOM preservation.

Role of Physico-Chemical and Cellular Conditions on the Bone Repair Potential of Plastically Compressed Collagen Hydrogels.

Since their first description nearly 20 years ago, dense collagen hydrogels obtained by plastic compression have become popular scaffolds in tissue engineering. In particular, when seeded with dental pulp stem cells, they have demonstrated a great in vivo potential in cranial bone repair. Here, we investigated how physico-chemical and cell-seeding conditions could influence the formation and in vitro mineralization of these cellularized scaffolds. A qualitative assessment demonstrated that the gel stability before and after compression was highly sensitive to the conditions of fibrillogenesis, especially initial acid acetic and buffer concentrations. Gels with similar rheological properties but different fibrillar structures that exhibited different stabilities when used for the 3D culture of Stem cells from Human Exfoliated Deciduous teeth (SHEDs) could be prepared. Finally, in our optimal physico-chemical conditions, mineralization could be achieved only using human dental pulp stem cells (hDPSCs) at a high cell density. These results highlight the key role of fibrillogenic conditions and cell type/density on the bone repair potential of cell-laden plastically compressed collagen hydrogels.

Effect of Octamer-Binding Transcription Factor 4 Overexpression on the Neural Induction of Human Dental Pulp Stem Cells

Stem cell-based therapy is a potential alternative strategy for brain repair, with neural stem cells (NSC) presenting as the most promising candidates. Obtaining sufficient quantities of NSC for clinical applications is challenging, therefore alternative cell types, such as neural crest-derived dental pulp stem cells (DPSC), may be considered. Human DPSC possess neurogenic potential, exerting positive effects in the damaged brain through paracrine effects. However, a method for conversion of DPSC into NSC has yet to be developed. Here, overexpression of octamer-binding transcription factor 4 (OCT4) in combination with neural inductive conditions was used to reprogram human DPSC along the neural lineage. The reprogrammed DPSC demonstrated a neuronal-like phenotype, with increased expression levels of neural markers, limited capacity for sphere formation, and enhanced neuronal but not glial differentiation. Transcriptomic analysis further highlighted the expression of genes associated with neural and neuronal functions. In vivo analysis using a developmental avian model showed that implanted DPSC survived in the developing central nervous system and respond to endogenous signals, displaying neuronal phenotypes. Therefore, OCT4 enhances the neural potential of DPSC, which exhibited characteristics aligning with neuronal progenitors. This method can be used to standardise DPSC neural induction and provide an alternative source of neural cell types.Graphical

Effect of polydatin on the viability and odontogenic differentiation of human dental pulp stem cells: An in-vitro study

Background/purposeVarious materials have been used to promote human dental pulp stem cells (hDPSCs) differentiation to produce dentin bridge formation with less-than-optimal results. Polydatin (PD), a naturally present material with osteogenic properties can be a promising material in the pulp regeneration/repair process. The aim of this study was to evaluate the effect of (PD) on the viability and differentiation of human dental pulp stem cells. Materials and methodsPD effect on hDPSCs in terms of cellular viability, alkaline phosphatase (ALP) production, and messenger RNAs (mRNA) of odontogenic markers production using quantitative reverse transcription polymerase chain reaction (RT-qPCR) were evaluated. In addition, mineral deposits were detected with Alizarin red stain. ResultsThe viable hDPSCs in the presence of 0.01 μM and 0.1 μM PD were significantly higher than the control on days 3 and 7, respectively. In addition, ALP activity of hDPSCs was significantly increased with 0.01, 0.1, and 1 μM of PD. In addition, increased expression mRNAs of ALP, osteocalcin (OC), osteonectin (ON), osteopontin (OP), Runt-related transcription factor-2 (RUNX-2), dentin sialophosphoprotein (DSPP), and dentin matrix protein-1 (DMP-1) was observed after PD treatment, however, the difference was not statistically significant. Furthermore, increased size of mineral deposits was observed with PD. ConclusionPD promoted the expression of markers associated with odontogenic differentiation and mineralized tissue deposition in hDPSCs.

Enhanced Osteogenic Differentiation Based on Combining Pulp Stem Cells with Ultralong Hydroxyapatite Nanowires and Cellulose Fibers

Previous studies have confirmed the excellent biocompatibility, osteogenic properties, and angiogenic ability of hydroxyapatite (HAP), as well as the good osteoblast differentiation ability of dental pulp stem cells. We hypothesized that combining dental pulp stem cells with ultralong hydroxyapatite nanowires and cellulose fibers could more effectively promote osteoblast differentiation, making it a potential biomaterial for enhancing bone wound healing. Therefore, based on the optimal ratio of ultralong hydroxyapatite nanowires and cellulose fibers (HAPNW/CF) determined in previous studies, we added human dental pulp stem cells (hDPSCs) to investigate whether this combination can accelerate cell osteogenic differentiation. hDPSCs were introduced into HAPNW/CF scaffolds, and in vitro experiments revealed that: (1) HAPNW/CF scaffolds exhibited no cytotoxicity toward hDPSCs; (2) HAPNW/CF scaffolds enhanced alkaline phosphatase staining activity, an early marker of osteogenic differentiation, and significantly upregulated the expression level of osteogenic-related proteins; (3) co-culturing with hDPSCs in HAPNW/CF scaffolds significantly increased the expression of angiogenesis-related factors compared to hDPSCs alone when tested using human umbilical vein endothelial cells (hUVECs). Our study demonstrates that combining hDPSCs with HAPNW/CF can enhance osteogenic differentiation more effectively, potentially through increased secretion of angiogenesis-related factors promoting osteoblast differentiation.

Novel combination of nanostructured calcium hydroxide and natural materials: Formulation and characterization.

Calcium hydroxide is used as a reparative dentin inducer. The combination of cocoa pod husk and calcium hydroxide has also been studied as a reparative dentin inducer, whereas anchovy was identified as a good calcium source. The aim of the study was to investigate the optimal ratio of a nano-combination of calcium hydroxide, cocoa pod husk extract and anchovy powder as a reparative dentin inducer by analyzing their material, antioxidant and cytotoxic properties. The ratios of the 3 elements were established and their performance was compared in terms of viscosity, setting time, pH, and solubility rate. The optimal ratio was then examined for its antioxidant capacity using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. Additionally, its cytotoxicity against human dental pulp stem cells (hDPSCs) was evaluated through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. A ratio of 1:1:1 of calcium hydroxide, cocoa pod husk extract and anchovy powder was identified as the optimal ratio. It exhibited the greatest flowability, the shortest final setting time, had a pH range of 8.5-10.5, and the second highest solubility rate in comparison with other ratios. Moreover, it demonstrated active radical scavenging activity and stimulated the proliferation of hDPSCs. Based on the material, antioxidant and cytotoxic properties, a ratio of 1:1:1 of nano-calcium hydroxide, cocoa pod husk extract and anchovy powder was determined to be optimal for use as a reparative dentin inducer.

Preparation of PDA-GO/CS composite scaffold and its effects on the biological properties of human dental pulp stem cells

Reduced graphene oxide (rGO) is an graphene oxide (GO) derivative of graphene, which has a large specific surface area and exhibited satisfactory physicochemical characteristics. In this experiment, GO was reduced by PDA to generate PDA-GO complex, and then PDA-GO was combined with Chitosan (CS) to synthesize PDA-GO/CS composite scaffold. PDA-GO was added to CS to improve the degradation rate of CS, and it was hoped that PDA-GO/CS composite scaffolds could be used in bone tissue engineering. Physicochemical and antimicrobial properties of the different composite scaffolds were examined to find the optimal mass fraction. Besides, we examined the scaffold’s biocompatibility by Phalloidin staining and Live and Dead fluorescent staining.Finally, we applied ALP staining, RT-qPCR, and Alizarin red S staining to detect the effect of PDA-GO/CS on the osteogenic differentiation of human dental pulp stem cells (hDPSCs). The results showed that PDA-GO composite was successfully prepared and PDA-GO/CS composite scaffold was synthesized by combining PDA-GO with CS. Among them, 0.3%PDA-GO/CS scaffolds improves the antibacterial activity and hydrophilicity of CS, while reducing the degradation rate. In vitro, PDA-GO/CS has superior biocompatibility and enhances the early proliferation, migration and osteogenic differentiation of hDPSCs. In conclusion, PDA-GO/CS is a new scaffold materialsuitable for cell culture and has promising application prospect as scaffold for bone tissue engineering.

Gallic acid–coated nanolayer on mineral trioxide aggregate for regulating the inflammatory and differentiation cellular response profile of human dental pulp stem cells

Mineral trioxide aggregate (MTA) is a biomaterial used in endodontics due to its high bioactivity, antibacterial behaviors, and good biocompatibility. However, it has several disadvantages related to certain physical properties, including a long setting time and poor mechanical properties. Therefore, we hypothesized that coating MTA with gallic acid (GA) would regulate physical properties and lead to enhanced adhesion, proliferation, and secretion of angiogenic- and odontogenic-related proteins. This study examined whether MTA can be modified with GA to form GAMTA cement and evaluated its physicochemical and biological behaviors and immune suppression, angiogenesis, and odontogenesis capabilities. We soaked MTA in Tris buffer containing different concentrations of GA to coat the MTA powders. X-ray diffraction (XRD) and diametral tensile strength analyses were conducted to assess the physical characteristics of GAMTA. Then, we immersed GAMTA cement in simulated body fluid to evaluate its hydroxyapatite precipitation capabilities and silicon ion release profile. We used human dental pulp stem cells to assess the cell responses and the immunosuppressive, angiogenic, and odontogenic capabilities of GAMTA. The XRD results showed that GA was successfully coated onto MTA without the disruption or loss of MTA’s advantageous original structural properties. GAMTA demonstrated excellent physicochemical and biological properties, improving hydroxyapatite formation and angiogenic, odontogenic, and anti-inflammatory properties compared with MTA alone. Our results indicate that the GAMTA cement is a useful dental material for future tissue engineering and clinical applications.

C5L2 CRISPR KO enhances dental pulp stem cell-mediated dentinogenesis via TrkB under TNFα-induced inflammation.

Background and Objectives: Dental caries is one of the most common human pathological conditions resulting from the invasion of bacteria into the dentin. Current treatment options are limited. In many cases, endodontic therapy leads to permanent pulp tissue loss. Dentin-pulp complex regeneration involves dental pulp stem cells (DPSCs) that differentiate into odontoblast-like cells under an inflammatory context. However, limited information is available on how DPSC differentiation processes are affected under inflammatory environments. We identified the crucial role of complement C5a and its receptor C5aR in the inflammation-induced odontoblastic DPSC differentiation. Methodology: Here, we further investigated the role of a second and controversial C5a receptor, C5L2, in this process and explored the underlying mechanism. Human DPSCs were examined during 7-, 10-, and 14-day odontogenic differentiation treated with TNFα, C5L2 CRISPR, and tyrosine receptor kinase B (TrkB) antagonist [cyclotraxin-B (CTX-B)]. Results: Our data demonstrate that C5L2 CRISPR knockout (KO) enhances mineralization in TNFα-stimulated differentiating DPSCs. We further confirmed that C5L2 CRISPR KO significantly enhances dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP-1) expression after 14-day odontoblastic DPSC differentiation, and treatment with CTX-B abolished the TNFα/C5L2 CRISPR KO-induced DSPP and DMP-1 increase, suggesting TrkB's critical role in this process. Conclusion and Key applications: Our data suggest a regulatory role of C5L2 and TrkB in the TNFα-induced odontogenic DPSC differentiation. This study may provide a useful tool to understand the mechanisms of the role of inflammation in dentinogenesis that is required for successful DPSC engineering strategies.

Evaluation of the genotoxicity, cytotoxicity, and bioactivity of calcium silicate-based cements

BackgroundAs calcium silicate-based cements (CSCs) have found success in various vital pulp therapy applications, several new CSC products have emerged. This study aimed to assess the genotoxicity, cytotoxicity, and bioactivity of four CSCs by comparing the newly introduced materials Bio MTA+ and MTA Cem with previously studied materials, Biodentine and NeoMTA.MethodsGenotoxicity was evaluated using the micronucleus (MN) assay in human peripheral blood lymphocyte cells, measuring MN frequency and nuclear division index (NDI). Cytotoxicity was assessed in human dental pulp stem cells through the Water-Soluble Tetrazolium Salt-1 (WST-1) colorimetric assay. Bioactivity was determined by ELISA, measuring the levels of angiogenic and odontogenic markers (BMP-2, FGF-2, VEGF, and ALP). Statistical analyses included ANOVA, Dunnet and Sidak tests, and Wald chi-square test. (p < .05).ResultsThe MN frequency in the groups was significantly lower than that in the positive control group (tetraconazole) (p < .05). NDI values decreased with increasing concentration (p < .05). Bio MTA+ and NeoMTA showed decreased cell viability at all concentrations in 7-day cultures (p < .01). All materials increased BMP-2, FGF-2, and VEGF levels, with Biodentine and NeoMTA showing the highest levels of BMP-2 and FGF-2 on day 7. Biodentine displayed the highest VEGF levels on day 7. Biodentine and NeoMTA groups exhibited significantly higher ALP activity than the Bio MTA+ and MTA Cem groups by day 7.ConclusionBio MTA+ and MTA Cem demonstrated no genotoxic or cytotoxic effects. Moreover, this study revealed bioactive potentials of Bio MTA+ and MTA Cem by enhancing the expression of angiogenic and osteogenic growth factors.

Notch signaling regulates mineralization via microRNA modulation in dental pulp stem cells.

This study investigated the miRNA expression profile in Notch-activated human dental stem pulp stem cells (DPSCs) and validated the functions of miRNAs in modulating the odonto/osteogenic properties of DPSCs. DPSCs were treated with indirect immobilized Jagged1. The miRNA expression profile was examined using NanoString analysis. Bioinformatic analysis was performed, and miRNA expression was validated. Odonto/osteogenic differentiation was examined using alkaline phosphatase staining, Alizarin Red S staining, as well as odonto/osteogenic-related gene and protein expression. Fourteen miRNAs were differentially expressed in Jagged1-treated DPSCs. Pathway analysis revealed that altered miRNAs were associated with TGF-β, Hippo, ErbB signalling pathways, FoxO and Ras signalling. Target prediction analysis demonstrated that 7604 genes were predicted to be targets for these altered miRNAs. Enrichment analysis revealed relationships to various DNA bindings. Among differentially expressed miRNA, miR-296-3p and miR-450b-5p were upregulated under Jagged1-treated conditions. Overexpression of miR-296-3p and miR-450b-5p enhanced mineralization and upregulation of odonto/osteogenic-related genes, whereas inhibition of these miRNAs revealed opposing results. The miR-296-3p and miR-450b-5p inhibitors attenuated the effects of Jagged1-induced mineralization in DPSCs. Jagged-1 promotes mineralization in DPSCs that are partially regulated by miRNA. The novel understanding of these miRNAs could lead to innovative controlled mechanisms that can be applied to modulate biology-targeted dental materials.

Investigation of the Potential Targets and Mechanism Actions of Berberine in the Treatment of Pulpitis Based on Bioinformatics Analysis

Berberine, an active compound extracted from the Chinese herb, was reported to have an antibacterial effect and an anti-inflammatory effect and promote osteogenic differentiation of human dental pulp stem cells. However, the underlying therapeutic mechanism of berberine in pulpitis is still unknown. Here, bioinformatics analysis was performed to investigate the potential mechanism of berberine against pulpitis. First, we identified the collective targets of berberine and pulpitis from several databases using a Venny online tool. The pattern of interaction between berberine and the targeted protein was visualized by molecular docking. Moreover, we performed GSEA, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses to obtain potential pathways. Cell experiments were performed to validate the bioinformatics analysis results. Prostaglandin-endoperoxide synthase 2 (PTGS2) was identified as the crucial antipulpitis target of berberine via the CytoHubba plugin. Docking analysis indicated that berberine could fit in the binding pocket of the PTGS2 protein. Additionally, berberine exerted its therapeutic effects against pulpitis via multiple pathways. Overall, berberine possessed therapeutic effects against pulpitis through the inactivation of toll-like receptor and NOD-like receptor signaling pathways. The present research proposes a novel approach to explore the therapeutic mechanism of natural products.