Differentiation Of Stem Cells From The Apical Papilla Research Articles

Construction of dentin-on-a-chip based on microfluidic technology and tissue engineering

AimThree-dimensional (3D) cell culture systems perform better in resembling tissue or organism structures compared with traditional 2D models. Organs-on-chips (OoCs) are becoming more efficient 3D models. This study aimed to create a novel simplified dentin-on-a-chip using microfluidic chip technology and tissue engineering for screening dental materials. MethodologyA microfluidic device with three channels was designed for creating 3D dental tissue constructs using stem cells from the apical papilla (SCAP) and gelatin methacrylate (GelMA). The study investigated the effect of varying cell densities and GelMA concentrations on the layer features formed within the microfluidic chip. Cell viability and distribution were evaluated through live/dead staining and nuclei/F-actin staining. The osteo/odontogenic potential was assessed through ALP staining and Alizarin red staining. The impact of GelMA concentrations (5 %, 10 %) on the osteo/odontogenic differentiation trajectory of SCAP was also studied. ResultsThe 3D tissue constructs maintained high viability and favorable spreading within the microfluidic chip for 3–7 days. A cell seeding density of 2 × 104 cells/μL was found to be the most optimal choice, ensuring favorable cell proliferation and even distribution. GelMA concentrations of 5 % and 10 % proved to be most effective for promoting cell growth and uniform distribution. Within the 5 % GelMA group, SCAP demonstrated higher osteo/odontogenic differentiation than that in the 10 % GelMA group. ConclusionIn 3D culture, GelMA concentration was found to regulate the osteo/odontogenic differentiation of SCAP. The study recommends a seeding density of 2 × 104 cells/μL of SCAP within 5 % GelMA for constructing simplified dentin-on-a-chip. Clinical SignificanceThis study built up the 3D culture protocol, and induced odontogenic differentiation of SCAP, thus forming the simplified dentin-on-a-chip and paving the way to be used as a well-defined biological model for regenerative endodontics. It may serve as a potential testing platform for cell differentiation.

Circ-ZNF236 mediates stem cells from apical papilla differentiation by regulating LGR4-induced autophagy.

Human stem cells from the apical papilla (SCAPs) are an appealing stem cell source for tissue regeneration engineering. Circular RNAs (circRNAs) are known to exert pivotal regulatory functions in various cell differentiation processes, including osteogenesis of mesenchymal stem cells. However, few studies have shown the potential mechanism of circRNAs in the odonto/osteogenic differentiation of SCAPs. Herein, we identified a novel circRNA, circ-ZNF236 (hsa_circ_0000857) and found that it was remarkably upregulated during the SCAPs committed differentiation. Thus, in this study, we showed the significance of circ-ZNF236 in the odonto/osteogenic differentiation of SCAPs and its underlying regulatory mechanisms. The circular structure of circ-ZNF236 was identified via Sanger sequencing, amplification of convergent and divergent primers. The proliferation of SCAPs was detected by CCK-8, flow cytometry analysis and EdU incorporation assay. Western blotting, qRT-PCR, Alkaline phosphatase (ALP) and Alizarin red staining (ARS) were performed to explore the regulatory effect of circ-ZNF236/miR-218-5p/LGR4 axis in the odonto/osteogenic differentiation of SCAPs invitro. Fluorescence insitu hybridization, as well as dual-luciferase reporting assays, revealed that circ-ZNF236 binds to miR-218-5p. Transmission electron microscopy (TEM) and mRFP-GFP-LC3 lentivirus were performed to detect the activation of autophagy. Circ-ZNF236 was identified as a highly stable circRNA with a covalent closed loop structure. Circ-ZNF236 had no detectable influence on cell proliferation but positively regulated SCAPs odonto/osteogenic differentiation. Furthermore, circ-ZNF236 was confirmed as a sponge of miR-218-5p in SCAPs, while miR-218-5p targets LGR4 mRNA at its 3'-UTR. Subsequent rescue experiments revealed that circ-ZNF236 regulates odonto/osteogenic differentiation by miR-218-5p/LGR4 in SCAPs. Importantly, circ-ZNF236 activated autophagy, and the activation of autophagy strengthened the committed differentiation capability of SCAPs. Subsequently, invivo experiments showed that SCAPs overexpressing circ-ZNF236 promoted bone formation in a rat skull defect model. Circ-ZNF236 could activate autophagy through increasing LGR4 expression, thus positively regulating SCAPs odonto/osteogenic differentiation. Our findings suggested that circ-ZNF236 might represent a novel therapeutic target to prompt the odonto/osteogenic differentiation of SCAPs.

The development of a dental light curable PRFe-loaded hydrogel as a potential scaffold for pulp-dentine complex regeneration: An in vitro study.

The study aimed to develop a bicomponent bioactive hydrogel formed in situ and enriched with an extract of platelet-rich fibrin (PRFe) and to assess its potential for use in pulp-dentine complex tissue engineering via cell homing. A bicomponent hydrogel based on photo-activated naturally derived polymers, methacrylated chitosan (ChitMA) and methacrylated collagen (ColMA), plus PRFe was fabricated. The optimized formulation of PRFe-loaded bicomponent hydrogel was determined by analysing the mechanical strength, swelling ratio and cell viability simultaneously. The physical, mechanical, rheological and morphological properties of the optimal hydrogel with and without PRFe were determined. Additionally, MTT, phalloidin/DAPI and live/dead assays were carried out to compare the viability, cytoskeletal morphology and migration ability of stem cells from the apical papilla (SCAP) within the developed hydrogels with and without PRFe, respectively. To further investigate the effect of PRFe on the differentiation of encapsulated SCAP, alizarin red S staining, RT-PCR analysis and immunohistochemical detection were performed. Statistical significance was established at p < .05. The optimized formulation of PRFe-loaded bicomponent hydrogel can be rapidly photocrosslinked using available dental light curing units. Compared to bicomponent hydrogels without PRFe, the PRFe-loaded hydrogel exhibited greater viscoelasticity and higher cytocompatibility to SCAP. Moreover, it promoted cell proliferation and migration in vitro. It also supported the odontogenic differentiation of SCAP as evidenced by its promotion of biomineralization and upregulating the gene expression for ALP, COL I, DSPP and DMP1 as well as facilitated angiogenesis by enhancing VEGFA gene expression. The new PRFe-loaded ChitMA/ColMA hydrogel developed within this study fulfils the criteria of injectability, cytocompatibility, chemoattractivity and bioactivity to promote odontogenic differentiation, which are fundamental requirements for scaffolds used in pulp-dentine complex regeneration via cell-homing approaches.

Effects of CEM cement and emdogain on proliferation and differentiation of human stem cells from the apical papilla: a comparative in vitro study.

This study compared the effects of calcium-enriched mixture (CEM) cement, Emdogain (EMD), and their combination (CEM/Emdogain) on the differentiation and proliferation of stem cells from the apical papilla (SCAPs). In this in vitro, experimental study, SCAPs were isolated from two sound immature impacted third molars and cultured. After ensuring their stemness by detecting cell surface markers they were exposed to CEM cement, Emdogain, and CEM cement coated with Emdogain for 24 and 72h. The control cells did not undergo any intervention. Cell viability [by methyl thiazolyl tetrazolium (MTT) assay], expression of odontogenic differentiation genes [by quantitative reverse-transcription polymerase chain reaction (qRT-PCR)], and alkaline phosphatase (ALP) activity (by ALP staining kit) were evaluated. Data were analyzed by one-way ANOVA, t-test, and Mann-Whitney test (α = 0.05). Cell viability in the CEM cement and CEM/Emdogain groups decreased compared with the control group at 72h (P < 0.05). Expression of dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP1), bone sialoprotein (BSP) genes, and ALP activity significantly increased in all three experimental groups compared with the control group at both 24 and 72h. This increase was substantially more significant in CEM/Emdogain group (P > 0.05). The number of mineralized nodules significantly increased in all groups at 72h, with a higher rate in the CEM/Emdogain group. All biomaterials increased the differentiation of SCAPs, expression of odontogenic differentiation genes, and ALP activity, but CEM/Emdogain was considerably more effective for this purpose.

Effect of diode low level laser and red light emitting diode irradiation on cell proliferation and osteogenic/odontogenic differentiation of stem cells from the apical papilla

BackgroundThis experimental study aimed to assess the effect of irradiation of red light-emitting diode (LED) and Diode low-level laser (LLL) on osteogenic/odontogenic differentiation of stem cells from the apical papilla (SCAPs).Materials and methodsSCAPs were isolated from the human tooth root. The experimental groups were subjected to 4 J/cm2 diode low level laser and red LED irradiation in osteogenic medium. The control group did not receive any irradiation. Cell viability/proliferation of SCAPs was assessed by the methyl thiazolyl tetrazolium (MTT) assay on days 1 and 2 (n = 9). Osteogenic differentiation was evaluated by alizarin red staining (ARS) (n = 3), and expression of osteogenic genes by real-time polymerase chain reaction (RT-PCR) (n = 12) on days 1 and 2. SPSS version 18 was used for data evaluation. The Kruskal-Wallis and Mann-Whitney tests were used to compare the groups at each time point.ResultsThe MTT assay showed no significant difference in cell viability/proliferation of SCAPs in the low level laser, red LED, and control groups at 24 or 48 h (P < 0.001). The ARS assessment showed that low level laser and red LED irradiation enhanced osteogenic differentiation of SCAPs. low level laser and red LED irradiation both induced over-expression of osteogenic/dentinogenic genes including alkaline phosphatase (ALP), dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP-1), and bone sialoprotein (BSP) in SCAPs. Up-regulation of genes was significantly greater in low level laser irradiation group than red LED group (P < 0.001).ConclusionDiode low level laser irradiation with 4 J/cm2 energy density and red LED irradiation enhanced osteogenic differentiation of SCAPs without adversely affecting cell viability.

Assessment of Cytotoxicity and Odontogenic/Osteogenic Differentiation Potential of Nano-Dentine Cement Against Stem Cells from Apical Papilla.

Assessment of the cytotoxicity of novel calcium silicate-based cement is imperative in endodontics. This experimental study aimed to assess the cytotoxicity and odontogenic/osteogenic differentiation potential of a new calcium silicate/pectin cement called Nano-dentine against stem cells from the apical papilla (SCAPs). In this experimental study, the cement powder was synthesized by the sol-gel technique. Zirconium oxide was added as opacifier and Pectin, a plant-based polymer, and calcium chloride as the liquid to prepare the nano-based dental cement. Thirty-six root canal dentin blocks of human extracted single-canal premolars with 2 mm height, flared with #1, 2 and 3 Gates-Glidden drills were used to prepare the cement specimens. The cement, namely mineral trioxide aggregate (MTA), Biodentine, and the Nano-dentine were mixed according to the manufacturers' instructions and applied to the roots of canal dentin blocks. The cytotoxicity and odontogenic/osteogenic potential of the cement were evaluated by using SCAPs. SCAPs were characterized by the expression of routine mesenchymal cell markers and differentiation potential to adipocytes, osteoblasts, and chondrocytes. Cement displayed no significant differences in cytotoxicity or calcified nodules formation. Gene expression analysis showed that all three types of cement induced significant down- regulation of COLA1; however, the new cement induced significant up-regulation of RUNX2 and SPP1 compared to the control group and MTA. The new cement also induced significant up-regulation of TGFB1 and inducible nitric oxide synthase (iNOS) compared with Biodentine and MTA. The new Nano-dentin cement has higher odontogenic/osteogenic potential compared to Biodentine and MTA for differentiation of SCAPs to adipocytes, osteoblasts, and chondrocytes.

Effect of biodentine coated with emdogain on proliferation and differentiation of human stem cells from the apical papilla.

This study assessed the effect of Biodentine coated with Emdogain (Biodentine/Emdogain) on proliferation and differentiation of human stem cells from the apical papilla (SCAPs). METHODS ANDRESULTS: In this in vitro, experimental study, SCAPs were isolated from two immature impacted third molars and cultured. After ensuring the stemness of the cells by assessing the cell surface markers, they were exposed to Biodentine, Emdogain, and Biodentine/Emdogain for 24 and 72 h. The control cells did not receive any intervention. Cell viability was evaluated by the methyl thiazolyl tetrazolium assay. Expression of odontogenic differentiation genes was analyzed by the quantitative reverse transcription polymerase chain reaction. Alkaline phosphatase (ALP) activity was quantified by the respective kit. Data were analyzed by one-way ANOVA, t-test, and Mann-Whitney test (α = 0.05). Cell viability did not change after 24 h of exposure to biomaterials. At 72 h, the viability of the cells exposed to Biodentine and Biodentine/Emdogain decreased compared with the control group. The expression of dentin sialophosphoprotein, dentin matrix protein 1, and bone sialoprotein genes, and ALP activity significantly increased in all three experimental groups, compared with the control group at both 24 and 72 h; this increase was significantly greater in Biodentine/Emdogain group. The number of mineralized nodules significantly increased in all groups after 72 h with a greater rate in Biodentine/Emdogain group. All biomaterials increased the differentiation of SCAPs, expression of odontogenic genes, and ALP activity, but Biodentine/Emdogain was significantly more effective for this purpose.

The Antimicrobial Peptide LL-37 Promotes Migration and Odonto/Osteogenic Differentiation of Stem Cells from the Apical Papilla through the Akt/Wnt/β-catenin Signaling Pathway

IntroductionThe antimicrobial peptide LL-37, in addition to its broad spectrum of antibacterial function, can promote odontogenesis and osteogenesis. Stem cells from the apical papilla (SCAPs) are essential for the formation of dentin/bonelike tissues. However, little information on these cells is available in regenerative endodontics. This study aimed to evaluate the effects of LL-37 on the proliferation, migration, and differentiation of SCAPs. MethodsSCAPs were isolated, cultured, and characterized. Cell viability was analyzed by Cell Counting Kit-8 assays (Dojindo, Kumamoto, Japan). Cell migration was investigated by transwell assays. Dentin sialophosphoprotein, dentin matrix protein 1, runt-related transcription factor 2, and osterix were assessed by quantitative polymerase chain reaction and Western blots. Alkaline phosphatase (ALP) activity and ALP staining were assessed to determine the in vitro potential for osteogenic differentiation. The involvement of the Akt/Wnt/β-catenin signaling pathway was also studied. ResultsIn the 2.5-μg/mL LL-37 -treated group, cell proliferation and migration were up-regulated. Quantitative polymerase chain reaction and Western blot assays both revealed that LL-37 at 2.5 μg/mL up-regulated odonto/osteogenic markers (dentin sialophosphoprotein, dentin matrix protein 1, runt-related transcription factor 2, and osterix). LL-37 at 2.5 μg/mL significantly promoted ALP activity and increased the staining in SCAPs. In addition, the p-Akt and p-glycogen synthase kinase-3β levels were increased in LL-37–treated SCAPs. The migratory and odonto/osteogenic differentiation capacities of SCAPs were inhibited after treatment with inhibitors LY294002 and XAV-939. ConclusionsOur study showed that LL-37 at 2.5 μg/mL promoted the migration and odonto/osteogenic differentiation of SCAPs by activating the Akt/Wnt/β-catenin signaling pathway.

Platelet-rich Fibrin Improves the Osteo-/Odontogenic Differentiation of Stem Cells from Apical Papilla via the Extracellular Signal–regulated Protein Kinase Signaling Pathway

IntroductionThe aim of this study was to investigate the cytobiological effects of platelet-rich fibrin (PRF) on stem cells from the apical papilla (SCAPs) in vitro and to further explore the underlying molecular mechanisms. MethodsSCAPs were isolated from immature third molars. Different concentrations of PRF conditioned medium (one eighth, one quarter, and one half PRF) were prepared. After pretreatment with PRF, the proliferation rate and migration capacity of SCAPs were examined by the Cell Counting Kit-8 assay and wound healing assay, respectively. Alizarin red S staining was performed to examine mineralized nodule formation. Western blot analysis was used to detect the expression of osteo-/odontogenic markers and the extracellular signal–regulated kinase (ERK) pathway in SCAPs. Data were analyzed by one-way analysis of variance. P values <.05 were considered statistically significant. ResultsThe Cell Counting Kit-8 assay showed that one eighth PRF improved the proliferation rate and the migration capacity of SCAPs (P < .005), whereas one quarter PRF and one half PRF showed no significant difference compared with the control group. The expression of osteo-/odontogenic markers and the capacity to form mineralized nodules of SCAPs were promoted by one eighth PRF and one quarter PRF. In addition, PRF activated ERK signaling, and the ERK inhibitor attenuated PRF-induced osteo-/odontogenesis of SCAPs. ConclusionsPRF improved the osteo-/odontogenic differentiation of SCAPs by activating the ERK pathway; meanwhile, PRF improved the proliferation and migration of SCAPs, and one eighth PRF achieved the most obvious promotion effect. The favorable cytobiological effects of PRF on SCAPs might serve as basis for PRF applications in regenerative endodontic treatment.

Irradiation with blue light-emitting diode enhances osteogenic differentiation of stem cells from the apical papilla.

This study aimed to evaluate the effects of low-energy blue LED irradiation on the osteogenic differentiation of stem cells from the apical papilla (SCAPs). SCAPs were derived from human tooth root tips and were irradiated with 0 (control group), 1J/cm2, 2J/cm2, 3J/cm2, or 4J/cm2 blue light in osteogenic induction medium. Cell proliferation was analyzed using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. Osteogenic differentiation activity was evaluated by monitoring alkaline phosphatase (ALP), alizarin red staining, and real-time polymerase chain reaction (RT-PCR). The results of the MTT assay indicated that SCAPs in the LED groups exhibited a lower proliferation rate than those in the control group, and there were statistically differences between the 2J/cm2, 3J/cm2, and 4J/cm2 groups and the control group (P < 0.05). The results of the ALP and alizarin red analyses showed that blue LED promoted osteogenic differentiation of the SCAPs. And 4J/cm2 blue light upregulates the expression levels of the osteogenic/dentinogenic genes ALP, dentin sialophosphoprotein (DSPP), dentin matrix protein-1 (DMP-1), and osteocalcin (OCN) in SCAPs. Our results confirmed that low-energy blue LED at 1J/cm2, 2J/cm2, 3J/cm2, and 4J/cm2 could inhibit the proliferation of SCAPs and promotes osteogenic differentiation of SCAPs. Further in vitro studies are required to explore the mechanisms of the effects by low-energy blue LED.

Intermittent Administration of Parathyroid Hormone Enhances Odonto/Osteogenic Differentiation of Stem Cells from the Apical Papilla via JNK and P38 MAPK Pathways.

Objective Parathyroid hormone (PTH) is considered to be essential during the tooth development. Stem cells from the apical papilla (SCAPs) are responsible for dentine formation. However, the interaction between PTH and SCAPs remains unclear. This study was aimed at investigating the effects of PTH on odonto/osteogenic differentiation capacity of SCAPs and elucidating the underlying molecular mechanisms. Materials and Methods. Here, SCAPs were isolated and identified in vitro. Effects of PTH on the proliferation of SCAPs were determined by Cell Counting Kit-8 (CCK-8), flow cytometry (FCM), and EdU. Alkaline phosphatase (ALP) activity, alizarin red staining, Western blot, and RT-PCR were carried out to detect the odonto/osteogenic differentiation of PTH-treated SCAPs as well as the participation of the MAPK signaling pathway. Results An ALP activity assay determined that 10−8 mol/L PTH was the optimal concentration for the induction of SCAPs with no significant influence on the proliferation of SCAPs as indicated by CCK-8, FCM, and EdU. The expression of odonto/osteogenic markers was significantly upregulated in mRNA levels and protein levels. Moreover, intermittent treatment of PTH also increased phosphorylation of JNK and P38, and the differentiation was suppressed following the inhibition of JNK and P38 MAPK pathways. Conclusion PTH can regulate the odonto/osteogenic differentiation of SCAPs via JNK and P38 MAPK pathways.

Micropatterned hydrogels and cell alignment enhance the odontogenic potential of stem cells from apical papilla in-vitro

IntroductionAn understanding of the extracellular matrix characteristics which stimulate and guide stem cell differentiation in the dental pulp is fundamental for the development of enhanced dental regenerative therapies. Our objectives, in this study, were to determine whether stem cells from the apical papilla (SCAP) responded to substrate stiffness, whether hydrogels providing micropatterned topographical cues stimulate SCAP self-alignment, and whether the resulting alignment could influence their differentiation towards an odontogenic lineage in-vitro. MethodsExperiments utilized gelatin methacryloyl (GelMA) hydrogels of increasing concentrations (5, 10 and 15%). We determined their compressive modulus via unconfined compression and analyzed cell spreading via F-actin/DAPI immunostaining. GelMA hydrogels were micropatterned using photolithography, in order to generate microgrooves and ridges of 60 and 120μm, onto which SCAP were seeded and analyzed for self-alignment via fluorescence microscopy. Lastly, we analyzed the odontogenic differentiation of SCAP using alkaline phosphatase protein expression (ANOVA/Tukey α=0.05). ResultsSCAP appeared to proliferate better on stiffer hydrogels. Both 60 and 120μm micropatterned hydrogels guided the self-alignment of SCAP with no significant difference between them. Similarly, both 60 and 120μm micropattern aligned cells promoted higher odontogenic differentiation than non-patterned controls. SignificanceIn summary, both substrate mechanics and geometry have a statistically significant influence on SCAP response, and may assist in the odontogenic differentiation of dental stem cells. These results may point toward the fabrication of cell-guiding scaffolds for regenerative endodontics, and may provide cues regarding the development of the pulp-dentin interface during tooth formation.

Potential immunomodulatory effects of stem cells from the apical papilla on Treg conversion in tissue regeneration for regenerative endodontic treatment

To evaluate the expression of Foxp3-positive lymphocytes around newly formed tissue after regenerative endodontic treatment (RET) in vivo and investigate the effects of stem cells from theapical papilla (SCAP) on the conversion of CD4+ CD25- T cells to CD4+ CD25+ Foxp3+ regulatory T cells (Tregs) in vitro. Three 6-month-old beagles with nine doubled-rooted premolars in each dog were randomly assigned to the RET group and the control group. RET was performed after apical periodontitis had been induced in the experimental immature teeth. Three months later, the expression of Foxp3 was detected in the histological sections by immunofluorescent staining. Human SCAP and CD4+ CD25- T cells from mice spleens (1:1 and 1:5) were co-cultured in cell-cell contact or in Transwells, respectively, for 24 and 72h in vitro. The percentage of Tregs was evaluated by flow cytometry. The results were analysed using theFisher's exact test and analysis of variance. P<0.05 was regarded as statistically significant. Inflammatory cells were present with tissue regeneration in the RET group, and Foxp3-positive T cells were enriched around the newly formed tissues. SCAP promoted Treg conversion after 72h in vitro. Cell-cell contact played an important role after the 24 h co-culture, whilst soluble factors were also involved after 72h (P<0.05). SCAP promoted the conversion of pro-inflammatory T cells to Tregs in vitro. Tregs were enriched around the regenerating tissues in the root canals after RET, which may create a suitable immune microenvironment for the differentiation of SCAP. This study provides an underlying mechanism for tissue regeneration during RET.

Stromal-derived Factor-1α signaling is involved in bone morphogenetic protein-2-induced odontogenic differentiation of stem cells from apical papilla via the Smad and Erk signaling pathways

Stromal-derived factor-1α (SDF-1α) is a chemokine signaling molecule that binds to the transmembrane receptor CXC chemokine receptor-4 (CXCR4) and carries out important functions in development tissue homeostasis. SDF-1α signaling via CXCR4 regulates the recruitment of stem and precursor cells to support tissue-specific repair or regeneration. In this study, we examined the contribution of SDF-1α signaling to the odontogenic differentiation of stem cells from the apical papilla (SCAP) induced by bone morphogenic protein 2 (BMP-2). CXCR4 expression was detected in cultured SCAP and SDF-1α promoted the migration of SCAP in Transwell assays. Blocking SDF-1α signaling by treatment with siRNA significantly affected BMP-2-induced mineralized nodule formation and alkaline phosphatase (ALP) activity. Moreover, blocking SDF-1α signaling inhibited the BMP-2-induced early expression of runt-related factor-2 (Runx-2) and strongly suppressed the induction of dentin matrix protein 1 (DMP-1) and dentin sialophosphoprotein (DSPP) expression by BMP-2. Furthermore, the interaction between SDF-1α and BMP-2 signaling was mediated via intracellular Smads and Erk activation. In conclusion, our results demonstrated that SDF-1α can significantly promote the migration of SCAP. Moreover, we revealed corequirement of the SDF-1α/CXCR4 signaling pathways in the BMP-2-induced odontogenic differentiation of SCAP, and these findings may be applied in new strategies for dental pulp regeneration.

The Effects of Mitogen-activated Protein Kinase Signaling Pathways on Lipopolysaccharide-mediated Osteo/Odontogenic Differentiation of Stem Cells from the Apical Papilla

IntroductionOdontogenic differentiation of human stem cells from the apical papilla (SCAPs) is a prerequisite step in the root development of immature permanent teeth. However, little is known about the effects of an inflammatory environment on osteo/odontogenic differentiation of SCAPs. The purpose of this study was to investigate the effects of lipopolysaccharide (LPS) on the proliferation and osteo/odontogenic differentiation of SCAPs and the role of mitogen-activated protein kinase (MAPK) signaling pathways in LPS-mediated osteo/odontogenic differentiation of SCAPs. MethodsSCAPs of human third permanent molars were cultured. Cell viability was analyzed. Alkaline phosphatase activity and mineralization ability were investigated. Gene expression of osteo/odontogenic differentiation and MAPK signaling pathways was evaluated during osteo/odontogenic differentiation of SCAPs. ResultsIn the 0.1 μg/mL LPS–treated group, cell proliferation, alkaline phosphatase activity, and mineralization of SCAPs were up-regulated. Real-time quantitative polymerase chain reaction revealed that dentin sialophosphoprotein, runt-related transcription factor 2, and bone sialoprotein were increased. However, we did not detect any change of osteocalcin expression. In addition, the expression of p-ERK and p-p38 in SCAPs was enhanced by LPS treatment, whereas the inhibition of ERK and p38 MAPK pathways markedly suppressed the differentiation of LPS-treated SCAPs. ConclusionsOur findings showed that LPS at the appropriate concentration promoted the proliferation and osteo/odontogenic differentiation of SCAPs. ERK and p38 MAPK signaling pathways are involved in LPS-mediated osteo/odontogenic differentiation of SCAPs.

Inhibition of autophagy suppresses osteogenic differentiation of stem cells from apical papilla

To investigate the effects of autophagy on osteogenic differentiation of stem cells from the apical papilla (SCAPs) in the presence of tumor necrosis factor-α (TNF-α) stimulation in vitro. SCAPs treated with TNF-α (0, 5, and 10 ng/mL) with or without 5 mmol/L 3-MA were examined for the expression of autophagy marker LC3-Ⅱ using Western blotting. The cells were transfected with GFP-LC3 plasmid and fluorescence microscopy was used for quantitative analysis of intracellular GFP-LC3; AO staining was used to detect the acidic vesicles in the cells. The cell viability was assessed with CCK-8 assays and the cell apoptosis rate was analyzed using flow cytometry. The cells treated with TNF-α or with TNF-α and 3-MA were cultured in osteogenic differentiation medium for 3 to 14 days, and real- time PCR was used to detect the mRNA expressions of osteogenesis-related genes (ALP, BSP, and OCN) for evaluating the cell differentiation. TNF-α induced activation of autophagy in cultured SCAPs. Pharmacological inhibition of TNF-α-induced autophagy by 3-MA significantly decreased the cell viability and increased the apoptosis rate of SCAPs (P &lt; 0.05). Compared with the cells treated with TNF-α alone, the cells treated with both TNF-α and 3-MA exhibited decreased expressions of the ALP and BSP mRNA on days 3, 7 and 14 during osteogenic induction (P &lt; 0.05) and decreased expression of OCN mRNA on days 3 and 7 during the induction (P &lt; 0.05). Autophagy may play an important role during the osteogenic differentiation of SCAPs in the presence of TNF-α stimulation.

The potential application of concentrated growth factor in regenerative endodontics.

To investigate the potential application of concentrated growth factor (CGF) to promote pulp regeneration within immature teeth. Concentrated growth factor clots produced from peripheral blood samples were investigated histologically by haematoxylin-eosin (HE) staining and evaluated morphologically by scanning electron microscope (SEM). The cytokines were extracted from the CGF, and representative cytokines were quantified by enzyme-linked immunosorbent assay (ELISA). The biological effects of the CGF on human stem cells from the apical papilla (SCAPs) were then investigated and quantified, including cell proliferation, cell migration, mineralized nodule formation, and the gene expression of alkaline phosphatase (ALP), dentine sialophosphoprotein (DSPP) and dentine matrix protein (DMP)-1. The results were analysed statistically using one-way analysis of variance (anova). Concentrated growth factor had a complex three-dimensional structure with a high density of platelets and nucleated cells. Representative growth factors including PDGF-BB, IGF-1, TGF-β1, bFGF and VEGF were detected. The growth rate and migratory cell numbers of the CGF groups were significantly greater than those in the control groups (P<0.05). The mineralization areas in the CGF groups were significantly larger than those in the control groups (P<0.05). The expression levels of ALP, DSPP and DMP-1 were significantly up-regulated after induction by CGF (P<0.05). Concentrated growth factor promoted the proliferation, migration and differentiation of SCAPs and could be a promising biomaterial applied in regenerative endodontics.

Guanine and nucleotide binding protein 3 promotes odonto/osteogenic differentiation of apical papilla stem cells via JNK and ERK signaling pathways.

Odonto/osteogenic differentiation of stem cells from the apical papilla (SCAPs) is a key process in tooth root formation and development. However, the molecular mechanisms underlying this process remain largely unknown. In the present study, it was identified that guanine and nucleotide binding protein 3 (GNAI3) was at least in part responsible for the odonto/osteogenic differentiation of SCAPs. GNAI3 was markedly induced in mouse tooth root development in vivo and in human SCAPs mineralization in vitro. Notably, knockdown of GNAI3 by lentiviral vectors expressing short-hairpin RNAs against GNAI3 significantly inhibited the proliferation, cell cycle progression and migration of SCAPs, as well as odonto/osteogenic differentiation of SCAPs in vitro, suggesting that GNAI3 may play an essential role in tooth root development. The promotive role of GNAI3 in odonto/osteogenic differentiation was further confirmed by downregulation of odonto/osteogenic makers in GNAI3-deficient SCAPs. In addition, knockdown of GNAI3 effectively suppressed activity of c-Jun N-terminal kinase (JNK) and extracellular-signal regulated kinase (ERK) signaling pathways that was induced during SCAPs differentiation, suggesting that GNAI3 promotes SCAPs mineralization at least partially via JNK/ERK signaling. Taken together, the present results implicate GNAI3 as a critical regulator of odonto/osteogenic differentiation of SCAPs in tooth root development, and suggest a possible role of GNAI3 in regeneration processes in dentin or other tissues.

SFRP2 enhances the osteogenic differentiation of apical papilla stem cells by antagonizing the canonical WNT pathway

BackgroundExploring the molecular mechanisms underlying directed differentiation is helpful in the development of clinical applications of mesenchymal stem cells (MSCs). Our previous study on dental tissue-derived MSCs demonstrated that secreted frizzled-related protein 2 (SFRP2), a Wnt inhibitor, could enhance osteogenic differentiation in stem cells from the apical papilla (SCAPs). However, how SFRP2 promotes osteogenic differentiation of dental tissue-derived MSCs remains unclear. In this study, we used SCAPs to investigate the underlying mechanisms.MethodsSCAPs were isolated from the apical papilla of immature third molars. Western blot and real-time RT-PCR were applied to detect the expression of β-catenin and Wnt target genes. Alizarin Red staining, quantitative calcium analysis, transwell cultures and in vivo transplantation experiments were used to study the osteogenic differentiation potential of SCAPs.ResultsSFRP2 inhibited canonical Wnt signaling by enhancing phosphorylation and decreasing the expression of nuclear β-catenin in vitro and in vivo. In addition, the target genes of the Wnt signaling pathway, AXIN2 (axin-related protein 2) and MMP7 (matrix metalloproteinase-7), were downregulated by SFRP2. WNT1 inhibited the osteogenic differentiation potential of SCAPs. SFRP2 could rescue this WNT1-impaired osteogenic differentiation potential.ConclusionsThe results suggest that SFRP2 could bind to locally present Wnt ligands and alter the balance of intracellular Wnt signaling to antagonize the canonical Wnt pathway in SCAPs. This elucidates the molecular mechanism underlying the SFRP2-mediated directed differentiation of SCAPs and indicates potential target genes for improving dental tissue regeneration.

The transcription factor cyclic adenosine 3',5'-monophosphate response element-binding protein enhances the odonto/osteogenic differentiation of stem cells from the apical papilla.

To investigate the role of cAMP response element-binding protein (CREB) in the regulation of odonto/osteogenic differentiation of stem cells from the apical papilla (SCAPs). Stem cells from the apical papilla were obtained from human impacted third molars (n=15). Isolated SCAPs were transfected with CREB overexpressing/silenced lentivirus. Transfected cells were stained with alizarin red to investigate mineralized nodule formation. The expression of the mineralization-related genes, alkaline phosphatase (ALP), collagen type I (Col I), runt-related transcription factor 2 (RUNX2), osterix (OSX) and osteocalcin (OCN), was determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Protein expression of the odontogenic-related marker dentine sialoprotein (DSP) and the osteogenic-related marker RUNX2 was measured by Western blotting analysis. One-way analysis of variance (anova) and Student's t-test were used for statistical analysis (a=0.05). The overexpression of CREB enhanced mineralized nodule formation and up-regulated (P<0.05) the mRNA levels of odonto/osteogenic-related markers, including ALP, Col I, RUNX2, OSX and OCN, and also increased (P<0.05) the protein expression of DSP and RUNX2. In contrast, the silencing of CREB inhibited (P<0.05) the mineralization capacity of the SCAPs and decreased (P<0.05) the expression of odonto/osteogenic-related markers. Up-regulation of CREB expression promoted odonto/osteogenic differentiation of SCAPs and provided a potential method for the regeneration of the dentine-pulp complex.