Dentin Sialophosphoprotein Research Articles

Bioactive glass 45S5 promotes odontogenic differentiation of apical papilla cells through autophagy.

The mechanism of the odontogenic differentiation of apical papillary cells (APCs) stimulated by bioactive glass 45S5 is still unclear. This study aims to investigate the effect of autophagy on the odontogenic differentiation of APCs stimulated by bioactive glass 45S5. APCs were isolated and cultured in vitro, and the cell origin was identified by flow cytometry. The culture medium was prepared with 1 mg/mL 45S5, and its pH and ion concentration were determined. The experiments were divided into control, 45S5, and 3-methyladenine (3-MA) 45S5 groups. In the 45S5 group, APCs were induced to culture with 1 mg/mL 45S5. In the 3-MA 45S5 group, the autophagy inhibitor 3-MA was added to 1 mg/mL 45S5. Protein immunoblotting assay (Western blot) was used to detect the expression of autophagy-associated proteins of microtubule-associated protein 1 light-chain 3β (LC3B) and P62 after 24 h of induction culture in each group. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression of bone sialoprotein (BSP), Runt-related transcription factor 2 (Runx2), dentin sialophosphoprotein (DSPP), and dentin matrix protein-1 (DMP-1) after 7 d of induction culture. Cellular alkaline phosphatase (ALP) staining analyzed cellular ALP activity at 7 d of induction, and alizarin red staining evaluated the formation of mineralized nodules at 21 d of induction. The pH of the 45S5 extract culture medium was 8.65±0.01, which was not significantly different from that of the control group (P>0.05). The silicon ion concentration of the 45S5 induction culture medium was (1.56±0.07) mmol/L, which was higher than that of the control group (0.08±0.01) mmol/L (P<0.05). The calcium ion concentration of the 45S5 induction culture was (1.57±0.15) mmol/L, which was not significantly different from that of the control group (P>0.05). Western blot results showed that LC3B-Ⅱ/Ⅰ ratio increased and P62 expression decreased in the 45S5 group compared with those in the control group (P<0.05). By contrast, the ratio decreased and the expression increased in the 3-MA 45S5 group compared with those in the 45S5 group (P<0.05). RT-qPCR results showed that the expression of BSP, Runx2, DMP-1, and DSPP enhanced in the 45S5 group compared with that in the control group (P<0.05), but the expression decreased in the 3-MA 45S5 group compared with that in the 45S5 group (P<0.05). Semi-quantitative analysis of ALP staining and alizarin red staining showed that the ALP activity was enhanced, and the formation mineralized nodule increased in the 45S5 group compared with those in the control group. The ALP activity weakened, and the formation mineralized nodules were reduced in the 3-MA 45S5 group compared with that those in the 45S5 group. Cell autophagy participates in the odontogenic differentiation of APCs induced by 1 mg/mL 45S5 in vitro.

The effect of concentrated growth factor on the biological performance of human dental pulp stem cells under oxidative stress status

Objective: To investigate the effect of concentrated growth factor (CGF) on the biological performance of human dental pulp stem cells (hDPSCs) under oxidative stress status induced by hydrogen peroxide (H2O2). Methods: The hDPSCs were isolated by using tissue block separation method from healthy permanent teeth extracted for orthodontic reason. hDPSCs surface markers CD34, CD45, CD90 and CD105 were detected by flow cytometry. Alkaline phosphatase (ALP), alizarin red S (ARS), oil red O staining and colony formation assay were used to identify hDPSCs. After the cell counting kit-8 (CCK-8) detection, the optimal H2O2 concentration was used to construct the hDPSCs oxidative stress model. CGF conditioned medium was prepared by repeated freeze-thaw methods. After CCK-8 detection, the optimum CGF concentration was chosen for the subsequent experiments. The hDPSCs were divided into control group, H2O2 (only H2O2 processing), H2O2+CGF group (H2O2 processing in combination with the CGF) and CGF group (only CGF processing). Subsequent experiments were performed according to these groups. The oxidative stress model was verified by reactive oxygen species, β-galactosidase staining and Western blotting. The effects of CGF on the proliferation and migration of hDPSCs under oxidative stress status were detected by CCK-8 and cell scratch assay, respectively. ALP activity and ARS staining were used to detect the effect of CGF on the osteogenic differentiation of hDPSCs under oxidative stress status. The mRNA expression levels of odontogenesis related genes were detected by real-time fluorescence quantitative PCR (RT-qPCR), and the expression levels of odontogenesis and osteogenesis related proteins were detected by Western blotting. Results: Isolated hDPSCs showed positive expression of mesenchymal stem cells surface markers of CD90, CD105, and negative expression of hematopoietic stem cells surface markers CD34, CD45. The hDPSCs were proved to have the capacity of osteogenic, adipogenic differentiation and clone formation. The optimal concentration to construct the oxidative stress model was 200 μmol/L H2O2. Twenty percent CGF was the optimal concentration for subsequent experiments. Compared with the control group, the expression of aging protein p53 was significantly up-regulated from (0.82±0.12) to (1.19±0.14) in H2O2 group (P<0.05), with deepened β-galactosidase staining and increased fluorescence intensity of reactive oxygen species. The proliferative capacity of cells in H2O2+CGF group on day 1, 3, 5 and 7 (0.23±0.01, 0.50±0.02, 1.60±0.07, 1.80±0.21) were all higher than in H2O2 group (0.15±0.01, 0.14±0.02, 0.50±0.03, 0.90±0.09) (P<0.01). Cell healing capacity of cells in H2O2+CGF group at 12 h and 24 h (0.47±0.07, 0.58±0.44) also increased compared with the H2O2 group (0.36±0.02, 0.44±0.02) (P<0.05), and similar results in the activity of ALP and the formation of mineralized nodules. On day 28, the mRNA expressions of dentin sialophosphoprotein (0.52±0.16) and dental matrix protein 1(DMP-1) (0.39±0.13) in H2O2 group were all significantly lower than those in H2O2+CGF group (0.96±0.24, 0.83±0.30, respectively) and CGF group (1.12±0.18, 1.23±0.19, respectively) (P<0.05). On day 28, the expressions of odontogenesis related protein DMP-1 (0.27±0.04) and osteogenesis related protein Runt-related transcription factor-2 (0.42±0.15) in H2O2 group were all significantly lower than those in H2O2+CGF group (0.66±0.18, 0.68±0.04) and CGF group (1.15±0.13, 1.06±0.19, respectively) (P<0.05). Conclusions: H2O2 can induce oxidative stress in hDPSCs, while CGF can promote proliferation, migration, odontogenic and osteogenic differentiation of hDPSCs under oxidative stress status.

The proneural transcription factor Atoh1 promotes odontogenic differentiation in human dental pulp stem cells (DPSCs)

BackgroundBioengineering of human teeth for replacement is an appealing regenerative approach in the era of gene therapy. Developmentally regulated transcription factors hold promise in the quest because these transcriptional regulators constitute the gene regulatory networks driving cell fate determination. Atonal homolog 1 (Atoh1) is a transcription factor of the basic helix-loop-helix (bHLH) family essential for neurogenesis in the cerebellum, auditory hair cell differentiation, and intestinal stem cell specification. The functional versatility of Atoh1 prompted us to test the possibility that Atoh1 may intersect the dental pulp stem cell (DPSC) gene regulatory network governing odontogenic differentiation.MethodsWe isolated DPSCs from human dental pulps and treated the cells with a replication-deficient adenoviral vector to achieve robust ectopic expression of Atoh1, following which the growth and odontogenic differentiation profiles of DPSCs were characterized.ResultsDPSCs harboring the Atoh1 expression vector exhibited an approximately 3,000-fold increase in the expression of Atoh1 compared to the negative control, leading to increased DPSC proliferation in the growth medium (P < 0.05). In the odontogenic medium, Atoh1 caused an early induction of BMP2 (P < 0.001) followed by a late induction of BMP7 (P < 0.01) and increased Wnt signaling (P < 0.01). The increased BMP/Wnt signaling led to up to 8-fold increased expression of the master osteogenic transcription factor Osterix (P < 0.005) while exhibiting no significant effect on Runx2 or Dlx5, which are abundantly expressed in DPSCs. Atoh1 stimulated expression of type I collagen (P < 0.005) and small integrin-binding ligand, N-linked glycoproteins (SIBLINGs) such as bone sialoprotein (P < 0.001), dentin matrix protein 1 (P < 0.05), dentin sialophosphoprotein (P < 0.005), and osteopontin (P < 0.001), resulting in increased dentin matrix mineralization (P < 0.05). The odontogenic phenotype is associated with metabolic remodeling marked by enhanced glycolytic flux and attenuated mitochondrial metabolic enzyme activities.ConclusionsAtoh1, despite being a proneural transcription factor in development, possesses a novel odontogenic function upon ectopic expression in DPSCs. This in vitro study demonstrates a novel odontogenic mechanism mediated by ectopic expression of the transcription factor Atoh1 in human DPSCs. The finding may offer an innovative strategy for gene-based regeneration of the pulp-dentin complex.

Mild heat stress promotes the differentiation of odontoblast-like MDPC-23 cells via yes-associated protein

Purpose Dentin hypersensitivity (DH) is a prevalent condition, but long-term effective treatments are scarce. Differentiation of odontoblast-like cells is promising for inducing tertiary dentinogenesis and ensuring sustained therapeutic efficacy against DH. This study examined the effects and mechanism of action of mild heat stress (MHS) on the differentiation of odontoblast-like MDPC-23 cells. Methods We used a heating device to accurately control the temperature and duration, mimicking the thermal microenvironment of odontoblast-like cells. Using this device, the effects of MHS on cell viability and differentiation were examined. Cell viability was assessed using the MTT assay. The expression and nucleoplasmic ratio of the yes-associated protein (YAP) were examined by western blotting and immunofluorescence. The gene expression levels of heat shock proteins (HSPs) and dentin matrix protein-1 (DMP1) were measured using qPCR. Dentin sialophosphoprotein (DSPP) expression was evaluated using immunofluorescence and immunoblotting. Verteporfin was used to inhibit YAP activity. Results Mild heat stress (MHS) enhanced the odontoblast differentiation of MDPC-23 cells while maintaining cell viability. MHS also increased YAP activity, as well as the levels of HSP25 mRNA, HSP70 mRNA, HSP90α mRNA, DMP1 mRNA, and DSPP protein. However, after YAP inhibition, both cell viability and the levels of HSP90α mRNA, DMP1 mRNA, and DSPP protein were reduced. Conclusion YAP plays a crucial role in maintaining cell viability and promoting odontoblast differentiation of MDPC-23 cells under MHS. Consequently, MHS is a potential therapeutic strategy for DH, and boosting YAP activity could be beneficial for maintaining cell viability and promoting odontoblast differentiation.

Effects of icariin on dental pulp stem cells and its potential applications in dentin repair

ObjectivesAs dental pulp therapy evolves towards regenerative approaches, biomolecules such as icariin, derived from Epimedium flowers, are being evaluated for their therapeutic potential. This study investigates icariin's effectiveness in promoting odontogenic differentiation in human dental pulp stem cells (hDPSCs) in vitro and as a pulp-capping agent in vivo. DesignThe study explored the effects of icariin on hDPSCs at concentrations of 10, 20, and 40 µM. Cell viability and migration assays were conducted to evaluate cytotoxicity and chemotaxis. Odontogenic differentiation was assessed using alkaline phosphatase staining and alizarin red S (ARS) staining, complemented by real-time PCR and Western blot analyses of key markers such as RUNX family transcription factor 2 (RUNX2), collagen type I alpha 1 chain (COL1A1), alkaline phosphatase (ALPL), and dentin sialophosphoprotein (DSPP). Additionally, the in vivo effects of icariin were tested in a rat maxillary molar model, where icariin-treated collagen sponges were used for direct pulp capping to evaluate its potential to induce reparative dentin formation. ResultsIcariin showed no cytotoxic effects on hDPSCs at any tested concentration, enhanced migratory activity in a dose-dependent manner, and significantly increased alkaline phosphatase activity and calcium deposition. Gene and protein expression analyses revealed a dose-dependent increase in odontogenic differentiation markers in icariin-treated hDPSCs. In vivo, icariin effectively promoted reparative dentin formation in exposed rat pulp. ConclusionsIcariin enhances odontogenic differentiation of hDPSCs and has promising potential as a pulp-capping agent for vital pulp therapy.

CREB3L1 deficiency impairs odontoblastic differentiation and molar dentin deposition partially through the TMEM30B

Odontoblasts are primarily responsible for synthesizing and secreting extracellular matrix proteins, which are crucial for dentinogenesis. Our previous single-cell profile and RNAscope for odontoblast lineage revealed that cyclic adenosine monophosphate responsive element-binding protein 3 like 1 (Creb3l1) was specifically enriched in the terminal differentiated odontoblasts. In this study, deletion of Creb3l1 in the Wnt1+ lineage led to insufficient root elongation and dentin deposition. Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and RNA sequencing were performed to revealed that in CREB3L1-deficient mouse dental papilla cells (mDPCs), the genes near the closed chromatin regions were mainly associated with mesenchymal development and the downregulated genes were primarily related to biological processes including cell differentiation, protein biosynthesis and transport, all of which were evidenced by a diminished ability of odontoblastic differentiation, a significant reduction in intracellular proteins, and an even greater decline in extracellular supernatant proteins. Dentin matrix protein 1 (Dmp1), dentin sialophosphoprotein (Dspp), and transmembrane protein 30B (Tmem30b) were identified as direct transcriptional regulatory targets. TMEM30B was intensively expressed in the differentiated odontoblasts, and exhibited a significant decline in both CREB3L1-deficient odontoblasts in vivo and in vitro. Deletion of Tmem30b impaired the ability of odontoblastic differentiation, protein synthesis, and protein secretion in mDPCs. Moreover, overexpressing TMEM30B in CREB3L1-deficient mDPCs partially rescued the extracellular proteins secretion. Collectively, our findings suggest that CREB3L1 participates in dentinogenesis and facilitates odontoblastic differentiation by directly enhancing the transcription of Dmp1, Dspp, and other differentiation-related genes and indirectly promoting protein secretion partially via TMEM30B.

Preventive and reparative effects of low-level laser therapy on orthodontically induced inflammatory root resorption-An animal study.

Orthodontically induced inflammatory root resorption (OIIRR) is one of the most important side effects of orthodontic treatment. Low-level laser therapy (LLLT) is a useful way to reduce the orthodontic treatment duration and may have some effect on preventing and repairing OIIRR. However, the specific effects of LLLT on OIIRR remain unknown. Our research aimed to evaluate the Dentin Sialophosphoprotein (DSPP) expression level and root resorption volume during treatment and retention to explore the role of LLLT in preventing and repairing OIIRR. Thirty-seven 6-week-old male Sprague-Dawley rats were selected to establish an OIIRR model; the rats were divided into Group B (blank), Group F (force), Group F(LLLT) (force and LLLT), Group F+R (force and retention) and Group F+R(LLLT) (force, retention and LLLT). The root resorption volume of the distal buccal root and mesial root in the maxillary left first molar was calculated by micro-CT, and the DSPP expression level on the compression side of the periodontal ligament was analysed by immunohistochemical staining. The resorption volume in Group F was greater than that in Group F(LLLT). For the mesial root, the volume in Group F was greater than that in Groups F+R and F+R(LLLT). For the distal buccal root, the volume in Groups F and F+R was greater than that in Group F+R(LLLT). The DSPP level in Group F(LLLT) was greater than that in Group F and there was no difference between Groups F+R and F+R(LLLT). LLLT has a certain preventive effect and a limited reparative effect on OIIRR in rats.

Dysregulation of lncRNA TFAP2A-AS1 is involved in the pathogenesis of pulpitis by the regulation of microRNA-32-5p.

This study was designed to evaluate TFAP2A-AS1 expression in the dental pulp of teeth with or without pulpitis and to determine the function of TFAP2A-AS1 in pulp cells. GSE92681 was analyzed to filter out differentially expressed lncRNAs. Pulp samples from teeth with pulpitis and healthy teeth (control) were examined using real-time (RT) quantitative polymerase chain reaction (qPCR). Human dental pulp stem cells (hDPSCs) were cultured in a specific medium for osteogenic induction, or treated with lipopolysaccharide (LPS) to simulate inflammation. The viability and apoptosis of human DPSCs (hDPSCs) were determined by XTT assay and apoptosis detection kit. Inflammation was induced by LPS and assessed by measuring the expression and release of inflammatory cytokines after TFAP2A-AS1 knockdown. Osteogenic differentiation of hDPSCs was investigated by determining expression levels of osteogenic markers and alkaline phosphatase (ALP) activity after TFAP2A-AS1 overexpression. The downstream microRNA (miRNA) was predicted. Dual-luciferase reporter was used to confirm the binding between miR-32-5p and TFAP2A-AS1. The expression of TFAP2A-AS1 was evaluated in inflamed pulp using RT-qPCR. TFAP2A-AS1 had a discriminatory ability for healthy individuals and patients with pulpitis. The expression of TFAP2A-AS1 decreased upon the osteogenic differentiation of hDPSCs, and increased upon the LPS induction. TFAP2A-AS1 can reverse the osteogenic differentiation of hDPSCs, as evidenced by decreased levels of dentine sialophosphoprotein, dentin matrix protein-1, and ALP activity. TFAP2A-AS1 knockdown can promote cell proliferation of hDPSCs and relieve LPS-induced inflammation, as evidenced by decreased levels of TNF-α, IL-1β, and IL-6. miR-32-5p was identified as a downstream miRNA of TFAP2A-AS1. This study demonstrated the expression and potential function of TFAP2A-AS1 in the human dental pulp. TFAP2A-AS1 can inhibit odontogenic differentiation but promote inflammation in pulp cells.

Role and Molecular Mechanism of miR-586 in the Differentiation of Dental Pulp Stem Cells into Odontoblast-like Cells.

Dental pulp stem cells (DPSCs) are a class of cells with the potential of self-replication and multi-directional differentiation, which are widely considered to have great application value. It was to investigate miR-586 in DPSCs differentiated into odontoblast-like cells. In this article, human dental pulp stem cells (hDPSCs) were used as samples, and hDPSCs were co-cultured with endothelial progenitor cells (EPCs). Furthermore, a lentiviral expression vector for the miR-586 inhibitor was established. The effect of miR-586 inhibitor expression vector on the activity of hDPSCs was detected by Cell Counting Kit-8 (CCK-8). The differentiation of hDPSCs was tested by mineralized nodule staining. The expression of miR-586 and a gene related to dental cell differentiation in the pulp was subjected to detection by real-time quantitative PCR (qRT-PCR). As against the normal hDPSCs and the empty vector, the miR-586 lentivirus expression inhibition vector could visibly raise the expression of dentin sialophosphoprotein (DSPP) in hDPSCs; and the cell proliferation activity was visibly enhanced; In addition, the mRNA expressions of dentin-matrix acidic phosphoprotein 1 (DMP-1) and alkaline phosphatase (ALP) were visibly raised in the miR-586 lentivirus expression inhibition vector (all P < 0.05). Additionally, ALP activity was significantly enhanced (P < 0.05). The number of mineralized nodules was significantly increased (P < 0.05). MiR-586 plays a key regulatory function in DPSCs differentiated into odontoblast-like cells and is associated with specific molecular mechanisms.

Proanthocyanidin surface preconditioning of dental pulp stem cell spheroids enhances dimensional stability and biomineralization invitro.

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.

In vitro Analysis of DSPP and BSP Expression: Comparing the Odontogenic Influence of Bio-C Repair and Biodentine in hDPSCs.

This study compared the ability of BIO-C Repair (BC) and Biodentine (BD) in relation to odontogenic differentiation by evaluating the dentin sialophosphoprotein (DSPP) and bone sialoprotein (BSP) expression and mineral deposition of human dental pulp stem cells (hDPSCs). BC and BD were pulverized and sterilized (ISO 10993-5:2009). The hDPSCs were the result of primary cultures that were 80% confluent (having gone through the stem cell marker tests CD90 98%, CD105 99.7%, CD73 94%, and LinNeg 0.5%) and reached P2-3 by means of serum starvation for 24 hours. This study involved seven groups, in which the hDPSCs were cultured on osteogenic media with the addition of either BD (Septodont, United States) at concentrations of 1:1, 1:2, or 1:5; BC (Angelus, Brazil) at concentrations of 1:1, 1:2, or 1:5; or the negative control (Dulbecco's modified eagle medium + osteogenic media). The hDPSC differentiation was determined via enzyme-linked immunosorbent assays of DSPP and BSP expression performed on days 7 and 14 and alizarin red staining performed on day 21. The data were analyzed using a one-way analysis of variance, followed by Tamhane's post hoc test, to compare the differences between groups. The t-test dependent was also used to identify differences between groups. BC and BD at 1:1 concentration, there was a statistically significant difference in DSPP and BSP expression. However, at concentrations of 1:2 and 1:5, there was no significant difference observed in either duration of observation (p > 0.05). The highest DSPP and BSP concentrations after 7 and 14 days of observation were observed with BD and BC at 1:5 concentration (6.6-6.71 and 13.20-13.47 ng/mL). The study shows that BC is as effective as BD in enhancing DSPP and BSP expression and mineral deposition in hDPSCs. The 1:5 concentration of BC showed the highest levels of DSPP and BSP expression and mineral deposition.

Enhancing the Physical, Antimicrobial, and Osteo/Odontogenic Properties of a Sol-Gel-Derived Tricalcium Silicate by Graphene Oxide for Vital Pulp Therapies.

This study developed a sol-gel tricalcium silicate/graphene oxide (TCS-GO) composite and examined its physicochemical properties, antimicrobial activity, and osteo/odontogenic effect on dental pulp stem cells. Tricalcium silicate was synthesized and combined with graphene oxide at three different concentrations, namely 0.02%, 0.04%, and 0.08% w/w, while tricalcium silicate and mineral trioxide aggregate served as controls. The setting time, compressive strength, pH, and calcium ion release of the composites were evaluated, as well as antimicrobial properties against Streptococcus mutans and Lactobacillus acidophilus. Additionally, the viability of dental pulp stem cells; apatite forming ability; and the gene expression of Alkaline phosphatase, Dentin sialophosphoprotein, and Runt-related transcription factor 2 were assessed. TCS-GO (0.08%) showed a significantly shorter setting time and higher compressive strength when compared to MTA (p < 0.05). Additionally, tricalcium silicate and TCS-GO groups showed a higher release of Ca ions than MTA, with no significant difference in pH values among the different groups. TCS-GO (0.08%) also demonstrated a significantly stronger antimicrobial effect against Lactobacillus acidophilus compared to MTA (p < 0.05). ALP expression was higher in TCS-GO (0.08%) than MTA on days 3 and 7, while DSPP expression was higher in TCS-GO (0.08%) than MTA on day 3 but reversed on day 7. There was no significant difference in RUNX2 expression between TCS-GO (0.08%) and MTA on days 3 and 7. The TCS-GO (0.08%) composite demonstrated superior physicochemical characteristics and antimicrobial properties compared to MTA. Moreover, the early upregulation of ALP and DSPP markers in TCS-GO (0.08%) indicates that it has the potential to promote and enhance the osteo/odontogenic differentiation of DPSCs.

Chrysin-Mediated Promotion of Odontoblastic Differentia- tion: A Pathway Towards Dental Pulp Regeneration

Dental pulp, a critical component in tooth development and maintenance, plays a vital role in dentin formation, nutrient supply, and signal transmission. When damaged, pulp can become infected and necrotic, leading to pain and a decline in the quality of life. Current clinical treatments, such as root canal therapy, though effective, result in the loss of pulp vitality and associated sensory and defensive functions. Recognizing the potential of regenerative approaches, our study explored the odontogenic differentiation effects of Chrysin, a natural flavonoid, on human dental pulp cells (hDPCs). Our investigations revealed that Chrysin, particularly at a concentration of 10 μM, exhibited no cytotoxic effects on hDPCs over a 5-day period. Notably, this concentration demonstrated optimal effects on alkaline phosphatase (ALP) activity and mineralization capacity, indicating its potential as an agent for odontogenic differentiation. Further analyses at the molecular level confirmed increased expression of odontogenic markers, including dentin matrix protein 1 (DMP-1), dentin sialophosphoprotein (DSPP), and ALP, supporting the pro-odontogenic effects of chrysin. In conclusion, our study underscores the positive effects of Chrysin on odontogenic differentiation in hDPCs, offering a promising avenue for further exploration in dental regenerative medicine. Future research should delve into in vivo applications and clinical assessments to validate the translational impact of Chrysin in preserving the vital pulp and advancing dental tissue regeneration.

Odontogenic and anti-inflammatory effects of magnesium-doped bioactive glass in vital pulp therapy

This study aimed to investigate the effects of magnesium-doped bioactive glass (Mg-BG) on the mineralization, odontogenesis, and anti-inflammatory abilities of human dental pulp stem cells (hDPSCs). Mg-BG powders with different Mg concentrations were successfully synthesized via the sol-gel method and evaluated using x-ray diffraction, Fourier-transform infrared spectroscopy, scanning electron microscopy, and transmission electron microscopy. Apatite formation was observed on the surfaces of the materials after soaking in simulated body fluid. hDPSCs were cultured with Mg-BG powder extracts in vitro, and no evident cytotoxicity was observed. Mg-BG induced alkaline phosphatase (ALP) expression and mineralization of hDPSCs and upregulated the expression of odontogenic genes, including those encoding dentin sialophosphoprotein, dentin matrix protein 1, ALP, osteocalcin, and runt-related transcription factor 2. Moreover, Mg-BG substantially suppressed the secretion of inflammatory cytokines (interleukin [IL]-4, IL-6, IL-8, and tumor necrosis factor-alpha). Collectively, the results of this study suggest that Mg-BG has excellent in vitro bioactivity and is a potential material for vital pulp therapy of inflamed pulps.

Functional extracellular vesicles from SHEDs combined with gelatin methacryloyl promote the odontogenic differentiation of DPSCs for pulp regeneration

BackgroundPulp regeneration is a novel approach for the treatment of immature permanent teeth with pulp necrosis. This technique includes the combination of stem cells, scaffolds, and growth factors. Recently, stem cell-derived extracellular vesicles (EVs) have emerged as a new methodology for pulp regeneration. Emerging evidence has proven that preconditioning is an effective scheme to modify EVs for better therapeutic potency. Meanwhile, proper scaffolding is of great significance to protect EVs from rapid clearance and destruction. This investigation aims to fabricate an injectable hydrogel loaded with EVs from pre-differentiated stem cells from human exfoliated deciduous teeth (SHEDs) and examine their effects on pulp regeneration.ResultsWe successfully employed the odontogenic induction medium (OM) of SHEDs to generate functional EV (OM-EV). The OM-EV at a concentration of 20 µg/mL was demonstrated to promote the proliferation and migration of dental pulp stem cells (DPSCs). The results revealed that OM-EV has a better potential to promote odontogenic differentiation of DPSCs than common EVs (CM-EV) in vitro through Alizarin red phalloidin, alkaline phosphatase staining, and assessment of the expression of odontogenic-related markers. High-throughput sequencing suggests that the superior effects of OM-EV may be attributed to activation of the AMPK/mTOR pathway. Simultaneously, we prepared a photocrosslinkable gelatin methacryloyl (GelMA) to construct an OM-EV-encapsulated hydrogel. The hydrogel exhibited sustained release of OM-EV and good biocompatibility for DPSCs. The released OM-EV from the hydrogel could be internalized by DPSCs, thereby enhancing their survival and migration. In tooth root slices that were subcutaneously transplanted in nude mice, the OM-EV-encapsulated hydrogel was found to facilitate dentinogenesis. After 8 weeks, there was more formation of mineralized tissue, as well as higher levels of dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP-1).ConclusionsThe effects of EV can be substantially enhanced by preconditioning of SHEDs. The functional EVs from SHEDs combined with GelMA are capable of effectively promoting dentinogenesis through upregulating the odontogenic differentiation of DPSCs, which provides a promising therapeutic approach for pulp regeneration.

Evaluation of dental pulp stem cells response to flowable nano-hybrid dental composites: A comparative analysis.

Flowable resin composites (FRC) are tooth-colored restorative materials that contain a lower filler particle content, and lower viscosity than their bulk counterparts, making them useful for specific clinical applications. Yet, their chemical makeup may impact the cellular population of the tooth pulp. This in-vitro study assessed the cytocompatibility and odontogenic differentiation capacity of dental pulp stem cells (DPSCs) in response to two recent FRC material extracts. Extracts of the FRC Aura easyflow (AEF) and Polofil NHT Flow (PNF) were applied to DPSCs isolated from extracted human teeth. Cell viability of DPSCs was assessed using MTT assay on days 1, 3 and 7. Cell migration was assessed using the wound healing assay. DPSCs' capacity for osteo/odontogenic differentiation was assessed by measuring the degree of mineralization by Alizarin Red S staining, alkaline phosphatase enzyme (ALP) activity, and monitoring the expression of osteoprotegerin (OPG), RUNX Family Transcription Factor 2 (RUNX2), and the odontogenic marker dentin sialophosphoprotein (DSPP) by RT-PCR. Monomer release from the FRC was also assessed by High-performance liquid chromatography analysis (HPLC). DPSCs exposed to PNF extracts showed significantly higher cell viability, faster wound closure, and superior odontogenic differentiation. This was apparent through Alizarin Red staining of calcified nodules, elevated alkaline phosphatase activity, and increased expression of osteo/odontogenic markers. Moreover, HPLC analysis revealed a higher release of TEDGMA, UDMA, and BISGMA from AEF. PNF showed better cytocompatibility and enhancement of odontogenic differentiation than AEF.

Histone Deacetylase Inhibitors Restore the Odontogenic Differentiation Potential of Dental Pulp Stem Cells under Hyperglycemic Conditions.

Complications arising from diabetes can result in stem cell dysfunction, impairing their ability to undergo differentiation into various cellular lineages. The present study evaluated the effect of histone deacetylase inhibitors, Valproic acid and Trichostatin A, on the odontogenic differentiation potential of dental pulp stem cells under hyperglycemic conditions. Streptozotocin (STZ) induced diabetes mellitus in 12 male Wistar rats. Dental parameters were examined using micro-computed tomography. The odontogenic potential of human pulp stem cells exposed to 30 mM glucose was assessed through alkaline phosphatase assays, examination of gene expression for dentin matrix protein 1 and dentin sialoprotein using real-time PCR, and alizarin red staining for calcium deposition. Along with reduced dentin thickness and root length in diabetic rats, the results revealed a significant increase in histone deacetylase 3 and 2 gene expressions in isolated diabetic pulp tissues compared to the control groups. The gene expression of odontogenic-related markers and alkaline phosphatase activity in human cultured pulp stem cells under hyperglycemic conditions significantly decreased. Adding Valproic acid and Trichostatin A restored the odontogenic differentiation markers, including calcium deposition, gene expression of dentin sialophosphoprotein, dentin matrix protein 1, and alkaline phosphatase activity. The data suggests that hyperglycemic conditions negatively impact the odontogenic potential of pulp mesenchymal stem cells. However, histone deacetylase inhibitors improve the impaired odontogenic differentiation capacity. This study implies that histone deacetylases may represent a potential therapeutic target for enhancing the regenerative mineralization of pulp cells in diabetic patients.

Effects of Tenascin-C on Dental Pulp Tissue in Mice In Vivo And on The Proliferation and Differentiation of Dental Pulp Stem Cells into Odontoblasts and Calcification In Vitro

This study aimed to investigate the effects of tenascin-C (TN-C) on dental pulp tissue and on dental pulp stem cells (DPSCs). In in vivo studies, A collagen sponge with phosphate-buffered saline (PBS) for the control group or with TN-C for the experimental group was placed over the dental pulp of mice. The root pulp was excised at 7 and 21 days postoperatively and was observed microscopically using HE staining and immunohistochemistry. Inflammatory cells were found in the entire pulp tissue in the control group but no inflammatory cells were identified in the pulp tissue in the TN-C-treated experimental group. Further, nesting-positive cells at 7 days and dentin sialo phosphoprotein (DSPP)-positive cells at 21 days were seen in the experimental group. In in vitro studies, DPSCs were cultured in a medium with or without TN-C, after which the proliferation rate of DPSCs was measured mRNA expression levels were examined using quantitative reverse transcription polymerase chain reaction (qRT-PCR), and the formation of calcified nodules was investigated using alizarin red staining. There was no significant difference in the cell proliferation rate of DPSCs between the experimental and control groups during day 3 or 5, using the proliferation rate on day 1 as the baseline value. The expression of nesting mRNA on day 7 was significantly higher in the experimental group than in the control group (P&lt;0.05), but the expression of osteocalcin (OCN) mRNA was significantly higher in the control group than in the experimental group (P&lt;0.05). More calcified nodules formed and the area of calcified nodules in the control group was significantly greater than in the TN-C-treated experimental group on day 14 and day 21 (P &lt; 0.05). These results suggest that TN-C regulates inflammation during the healing process in the dental pulp and induces the differentiation of dental pulp into odontoblast-like cells. Further, TN-C promotes the early differentiation of DPSCs into odontoblast-like cells, which suggests that TN-C may further contribute to the inhibition of excessive dentin formation. From the above, TN-C is involved in differentiating dental pulp into mineralized tissue-forming cells and may have a beneficial effect in treatments that utilize this function

Effect of Low-Level Diode Laser and Red Light-Emitting Diode on Survival and Osteogenic/Odontogenic Differentiation of Human Dental Pulp Stem Cells.

Background: This investigation set out to compare the impacts of low-level diode laser (LLDL) and red light-emitting diode (LED) on the survival of human dental pulp stem cells (hDPSCs) and osteogenic/odontogenic differentiation. Methods and materials: In this ex vivo experimental study, the experimental groups underwent the irradiation of LLDL (4 J/cm2 energy density) and red LED in the osteogenic medium. Survival of hDPSCs was assessed after 24 and 48 h (n = 9) using the methyl thiazolyl tetrazolium (MTT) assay. The assessment of osteogenic/odontogenic differentiation was conducted using alizarin red staining (ARS; three repetitions). The investigation of osteogenic and odontogenic gene expression was performed at two time points, specifically 24 and 48 h (n = 12). This analysis was performed utilizing real-time reverse-transcription polymerase chain reaction (RT-PCR). The groups were compared at each time point using SPSS version 24. To analyze the data, the Mann-Whitney U test, analysis of variance, Tukey's test, and t-test were utilized. Results: The MTT assay showed that LLDL significantly decreased the survival of hDPSCs after 48 h, compared with other groups (p < 0.05). The qualitative results of ARS revealed that LLDL and red LED increased the osteogenic differentiation of hDPSCs. LLDL and red LED both upregulated the expression of osteogenic/odontogenic genes, including bone sialoprotein (BSP), alkaline phosphatase (ALP), dentin matrix protein 1 (DMP1), and dentin sialophosphoprotein (DSPP), in hDPSCs. The LLDL group exhibited a higher level of gene upregulation (p < 0.0001). Conclusions: The cell survival of hDPSCs was reduced, despite an increase in osteogenic/odontogenic activity. Clinical relevance: Introduction of noninvasive methods in regenerative endodontic treatments.

Effect of Andrographis paniculata Herbal Extract on Cytotoxicity, Proliferation and Osteogenic/Odontogenic Differentiation of Human Dental Pulp Stem Cells: An In Vitro Study.

Research has been conducted to assess the regenerative potential of dental pulp stem cells (DPSCs) following pretreatment of stem cells with certain molecules, bioactive compounds, plant extract and physical stimulation. Andrographis paniculata (AP) herbal extract with important medicinal properties is proven to have a preosteogenic effect on osteoblasts. This study aimed to determine the effect of various concentrations of AP extract on the cytotoxicity and osteogenic and odontogenic potential of DPSCs. Dental pulp stem cells were subjected to treatment with various concentrations of AP herbal extract (7 ug/ml, 5.2 ug/ml, 3.5 ug/ml, 1.7 ug/ml and 0.8 ug/ml), following which the cells were subjected to tests-3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) analysis for cytotoxicity and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) test for expression of genes (bone morphogenetic protein (BMP), osteocalcin (OCN), dentin sialophosphoprotein (DSPP) and alkaline phosphatase (ALP)). AP extract at concentration of 0.8 ug/ml-5.2 ug/ml had no cytotoxicity supporting cell growth. 3.5 ug/ml showed significant upregulation of genes on the third day. AP, a commonly occurring medicinal plant through its effect on DPSCs, could serve as an effective pretreatment modality for cell-based regenerative therapy and vital pulp therapy.