Odontogenic Differentiation Of Stem Cells Research Articles

Heparin-Functionalized Bioactive Glass to Harvest Endogenous Growth Factors for Pulp Regeneration.

Pulp and periapical diseases can lead to the cessation of tooth development, resulting in compromised tooth structure and functions. Despite numerous efforts to induce pulp regeneration, effective strategies are still lacking. Growth factors (GFs) hold considerable promise in pulp regeneration due to their diverse cellular regulatory properties. However, the limited half-lives and susceptibility to degradation of exogenous GFs necessitate the administration of supra-physiological doses, leading to undesirable side effects. In this research, a heparin-functionalized bioactive glass (CaO-P2O5-SiO2-Heparin, abbreviated as PSC-Heparin) with strong bioactivity and a stable neutral pH is developed as a promising candidate to addressing challenges in pulp regeneration. Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and thermogravimetric analysis reveal the successful synthesis of PSC-Heparin. Scanning electron microscopy and X-ray diffraction show the hydroxyapatite formation can be observed on the surface of PSC-Heparin after soaking in simulated body fluid for 12 h. PSC-Heparin is capable of harvesting various endogenous GFs and sustainably releasing them over an extended duration by the enzyme-linked immunosorbent assay. Cytological experiments show that developed PSC-Heparin can facilitate the adhesion, migration, proliferation, and odontogenic differentiation of stem cells from apical papillae. Notably, the histological analysis of subcutaneous implantation in nude mice demonstrates PSC-Heparin is capable of promoting the odontoblast-like layers and pulp-dentin complex formation without the addition of exogenous GFs, which is vital for clinical applications. This work highlights an effective strategy of harvesting endogenous GFs and avoiding the involvement of exogenous GFs to achieve pulp-dentin complex regeneration, which may open a new horizon for regenerative endodontic therapy.

Heat stress promotes osteogenic and odontogenic differentiation of stem cells from apical papilla via glucose-regulated protein 78-mediated autophagy

Background/purposeHeat stress is essential for improving the efficacy of mesenchymal stem cell (MSC)-based regeneration medicine. However, it is still unclear whether and how heat stress influences the differentiation of stem cells from apical papilla (SCAPs). This research aimed to explore the potential mechanism of glucose-regulated protein 78 (GRP78) in regulating differentiation under heat stress in SCAPs. Materials and methodsThe proliferation ability was assessed using the 5-Ethynyl-2’- deoxyuridine (EdU) assay, cell counting kit assay (CCK-8), and flow cytometry (FCM). The osteogenic and odontogenic differentiation capacities were investigated through Western blot, quantitative reverse transcription polymerase chain reaction (qRT-PCR), alkaline phosphatase (ALP) staining and activity assay, alizarin red S (ARS) staining, as well as immunofluorescence staining. Western blot and transmission electron microscopy (TEM) were used to detect autophagy. ResultsHeat stress enhanced the osteogenic and odontogenic differentiation of SCAPs, but it did not significantly affect proliferation. Besides, GRP78 has been confirmed to modulate the differentiation induced by heat stress. Autophagy triggered by GRP78 enhanced osteogenic and odontogenic differentiation of SCAPs, while the knockdown of GRP78 or the inhibitor of autophagy suppressed the differentiation. ConclusionHeat stress induces osteogenic and odontogenic differentiation of SCAPs through GRP78-mediated autophagy.

Short-term exposure to TNF-α promotes odontogenesis of stem cells of apical papilla on nanofibrous poly(l-lactic acid) scaffolds

The aim of this study was to investigate the effect of short-term (3 days) preconditioning with pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-α) on proliferation and odontogenic differentiation of stem cells of apical papilla (SCAP) seeded on a 3D scaffold. Our results showed that while TNF-α preconditioning at various concentrations did not affect SCAP proliferation, it enhanced alkaline phosphatase (ALP) activity and mineralized matrix deposition in culture. Preconditioning SCAP using TNF-α at a concentration of about 1 ng/ml optimally enhanced odontogenic gene expression of SCAP in culture. Highly porous nanofibrous (NF) poly(l-lactic acid) (PLLA) scaffolds, with nanofiber features mimicking the environment of the extracellular matrix, were fabricated using a thermally induced phase separation method. TNF-α preconditioning of SCAP at 1 ng/ml promoted ALP activity, calcium deposition and mineralized tissue formation of SCAP cultured on 3D NF-PLLA scaffolds in vitro. Moreover, TNF-α preconditioning at a concentration of 1 ng/ml promoted the formation of more mineralized tissue as well as stronger DSPP expression of SCAP seeded on NF-PLLA scaffolds in vivo. Collectively, this study suggests that short term TNF-α preconditioning, mimicking the transiently low concentration TNF-α present during inflammation, may serve as a feasible approach to enhance the odontogenic differentiation of stem/progenitor cells on a biomimetic scaffold for 3D dentin regeneration. These results present a first step in the development of a dental tissue engineering strategy where odontogenesis is enhanced by modulating inflammation.

Hsa-miR-27b-5p suppresses the osteogenic and odontogenic differentiation of stem cells from human exfoliated deciduous teeth via targeting BMPR1A: An ex vivo study.

Recently, miR-27b-5p was shown to be abundantly expressed in extracellular vehicles (EVs) from the inflammatory microenvironment. This study determined the role of miR-27b-5p in regulating osteogenic and odontogenic differentiation of stem cells from human exfoliated deciduous teeth (SHEDs) and further examined the regulatory mechanism of bone morphogenetic protein receptor type-1A (BMPR1A). Characteristics of SHEDs and SHEDs-EVs derived from SHEDs were evaluated respectively. The expression of miR-27b-5p in SHEDs and EVs was detected during osteo-induction. Mechanically, SHEDs were treated with miR-27b-5p mimics or an inhibitor, and the osteogenic/odontogenic differentiation and proliferation were assessed. Bioinformatic analysis and luciferase reporter were utilized for target gene prediction and verification. Finally, BMPR1A-overexpressed plasmids were transfected into SHEDs to investigate the participation of the BMPR1A/SMAD4 pathway. Data were analyzed by Student's t- test, One-way analysis of variance and Chi-square test. MiR-27b-5p was expressed in both SHEDs and EVs and was significantly increased at the initial stage of differentiation and then decreased in a time-dependent manner (p < .01). Upregulation of miR-27b-5p significantly suppressed osteogenic/odontogenic differentiation of SHEDs and inhibited proliferation (p < .05). Whereas inhibition of miR-27b-5p enhanced the differentiation (p < .05). Dual luciferase reporter assay and pull-down assay confirmed the binding site between miR-27b-5p and BMPR1A (p < .05). The overexpression of BMPR1A rescued the effect of miR-27b-5p, while contributed to the decrease of pluripotency (p < .05). Additionally, miR-27b-5p maintained pluripotency in BMPR1A-overexpressed SHEDs (p < .05). MiR-27b-5p in SHEDs/EVs was inversely associated with differentiation and suppressed the osteogenic and odontogenic differentiation of SHEDs and maintained the pluripotency of SHEDs partly by shuttering BMPR1A-targeting BMP signaling. Theoretically, inhibition of miR-27b-5p represents a potential strategy to promote osteanagenesis and dentinogenesis. However, miR-27b-5p capsuled EVs might maintain cell pluripotency and self-renewal for non-cell-targeted therapy.

Long non-coding RNA IGFBP7-AS1 accelerates the odontogenic differentiation of stem cells from human exfoliated deciduous teeth by regulating IGFBP7 expression

Stem cells from human exfoliated deciduous teeth (SHED) are attractive seed cells for dental tissue engineering. We identified the effect of the long noncoding RNA insulin-like growth factor-binding protein 7 antisense RNA 1 (lncRNA IGFBP7-AS1) in vivo and its underlying mechanism during SHED odontogenic differentiation. IGFBP7-AS1 and insulin-like growth factor-binding protein 7 (IGFBP7) were overexpressed using lentiviruses. IGFBP7 expression was knocked down with small interfering RNA. The effect of IGFBP7-AS1 in vivo was confirmed by animal experiments. The effect of IGFBP7 on SHED odontogenic differentiation was assessed with alkaline phosphatase staining, alizarin red S staining, quantitative reverse transcription-PCR, and western blotting. The relationship between IGFBP7-AS1 and IGFBP7 was confirmed by quantitative reverse transcription–PCR and western blotting. IGFBP7-AS1 promoted SHED odontogenesis in vivo, and regulated the expression of the coding gene IGFBP7 positively. Inhibiting IGFBP7 led to suppress SHED odontogenic differentiation while IGFBP7 overexpression had the opposite effect. IGFBP7-AS1 enhanced the stability of IGFBP7. IGFBP7-AS1 promoted SHED odontogenic differentiation in vivo. The underlying mechanism may involve the enhancement of IGFBP7 stability. This may provide novel potential targets for dental tissue engineering.

Long non-coding RNA IGFBP7-AS1 promotes odontogenic differentiation of stem cells from human exfoliated deciduous teeth through autophagy: An in vitro study

ObjectiveIn the present study, we aimed to investigate whether long non-coding RNA (lncRNA) insulin-like growth factor binding protein 7-antisense 1 (IGFBP7-AS1) regulates the odonto-differentiation of stem cells from human exfoliated deciduous teeth (SHED) and its underlying mechanism. DesignReal-time polymerase chain reaction (PCR) and correlation analysis were used to determine the expression of IGFBP7-AS1 during odontogenesis. Alkaline phosphate staining, alizarin red S staining, and real-time PCR in vitro were performed to investigate the effects of IGFBP7-AS1 during odontogenesis. Western blot and immunostaining (with or without chloroquine treatment) were applied to detect the expression of the autophagy-related markers, microtubule-associated proteins 1A/1B light chain 3B (LC3B) and p62. The autophagy inhibitor 3-methyladenine was used to further clarify the effect of autophagy in odonto-differentiation as promoted by IGFBP7-AS1. ResultsThe expression of lncRNA IGFBP7-AS1 is significantly upregulated during odonto-differentiation of SHED and promotes odontogenesis of SHED in vitro. IGFBP7-AS1 promotes autophagy during odontogenesis. ConclusionsIGFBP7-AS1 elicits odontogenic differentiation of SHED through autophagy. Furthermore, IGFBP7-AS1 shows promise as a gene target in the regeneration of dental hard tissue and dental–pulp complex.

The functional extracellular matrix on the regulation of odontogenic differentiation of stem cells

AbstractDental defects and loss are common oral diseases that seriously affect the chewing efficiency of patients and thereby affect their health. With the development of tissue engineering technology, dental tissue regeneration has emerged as a promising technique. Cell type, extracellular matrix (ECM) environment, cytokines and other factors which affect odontogenic differentiation and dental tissue regeneration have aroused significant interest. The functional ECM consists of extracellular components that control the initiation and growth of crystal deposition and regulate the differentiation and regeneration of endogenous or exogenous stem cells in the defect site. At present, the function and regulatory mechanism of the functional ECM in this process remain poorly understood. Therefore, elucidating the effect of the functional ECM on odontogenic differentiation and its mechanism has great scientific significance and potential clinical application. This review explored functional ECM components involved in odontogenic differentiation and dental tissue regeneration.

PD-1 Suppresses the Osteogenic and Odontogenic Differentiation of Stem Cells from Dental Apical Papilla via Targeting SHP2/NF-κB Axis.

Stem cells from the apical papilla (SCAPs) are important for tooth root development and regeneration of root dentin. Here, we examined the expression of programmed cell death protein-1 (PD-1) in SCAPs and investigated the effects of PD-1 on odontogenic and osteogenic differentiation, as well as the relationship between PD-1 and SHP2/NF-κB signals. SCAPs were obtained and cultured in the related medium. The proliferation ability was evaluated by the cell counting kit 8 (CCK-8) and the 5-ethynyl-20-deoxyuridine (EdU) assay. Alkaline phosphatase (ALP) activity assay, ALP staining, Western blot, real-time quantitative reverse-transcription polymerase chain reaction (RT-qPCR), Alizarin Red S (ARS) staining, and immunofluorescence (IF) staining were performed to explore the osteo/odontogenic potential and the involvement of SHP2/NF-κB pathways. Besides, we transplanted SCAPs components into mouse calvaria defects to evaluate osteogenesis in vivo. We found that human SCAPs expressed PD-1 for the first time. PD-1 knockdown enhanced the osteo/odontogenic differentiation of SCAPs by suppressing the SHP2 pathway and activating the NF-κB pathway. Overexpression of PD-1 inhibited the osteogenesis and odontogenesis of SCAPs via activation of SHP2 signal and inhibition of the NF-κB pathway. PD-1 activated SHP2 signal to block NF-κB signal and then played a vital role in osteo/odontogenic differentiation of SCAPs.

Odontogenic Differentiation Induced by TGF-β1 Binding Peptide–Modified Bioglass

Emerging evidence suggests that growth factors are crucial in regenerative endodontic therapy. To achieve the desired effects, the systematic administration of supraphysiologic concentrations of exogenous growth factors is commonly performed, but this is usually associated with high costs, technique, and safety issues. Here, we describe a novel biomaterial that can manipulate endogenous growth factors without the need for adding exogenous growth factors. Transforming growth factor β1 binding peptide (TGFp) was grafted onto the surface of a neutral pH phytic acid–derived bioactive glass (PSC) to synthesize modified bioactive glass (PSC-TGFp). Fourier transform infrared spectroscopy and thermogravimetric analysis results demonstrated that the TGFp was successfully grafted to the surface of the PSC. Scanning electron microscopy and x-ray diffraction showed that PSC-TGFp possessed good in vitro bioactivity. After soaking in simulated body fluid for 24 h, hydroxyapatite formed on the surface of PSC-TGFp. Enzyme-linked immunosorbent assay showed that PSC-TGFp could capture endogenous transforming growth factor β1 from dentin matrix–extracted proteins (DMEP) and release it slowly over 21 d. Cytologic experiments revealed that PSC-TGFp after adsorbing DMEP could enhance the adhesion, migration, viability, and odontogenic differentiation of stem cells from apical papilla. The results highlight that PSC-TGFp may be a promising biomaterial to manipulate endogenous growth factors for regenerative endodontic therapy in the future.

Cytokine co-stimulation effect on odontogenic differentiation of stem cells

The study aims to evaluate the effect of bone morphogenetic protein-2 (BMP-2) and transforming growth factor-beta 1 (TGF-β1) co-stimulation on odontogenic differentiation of human dental pulp stem cells (hDPSCs). The viability/proliferation of hDPSCs treated with BMP-2 (group B), TGF-β1 (group T), or BMP-2/TGF-β1 (group BT) were evaluated. The experiments on odontogenic differentiation were done for 14days. The following subgroups were added to investigate the effect of co-stimulation with different timing: subgroup B1, TGF-β1 co-stimulation in the first week; subgroup B2, TGF-β1 co-stimulation in the second week; subgroup T1, BMP-2 co-stimulation in the first week; and subgroup T2, BMP-2 co-stimulation in the second week. The mineralization was assessed using alizarin red staining. The expression of following genes was assessed using quantitative real-time polymerase chain reaction: dentin sialophosphoprotein (DSPP), dentin matrix protein-1 (DMP1), osteopontin (OPN), and alkaline phosphatase. All groups showed viability similar to the control group (P > .05). The greater mineralization was detected in B groups on day 14. The expressions of DSPP, DMP-1, and OPN increased on day 14 (P < .05). In the combination groups, the higher expressions of DSPP and DMP-1 were observed in subgroups B1 and B2 than groups B and T (P < .05). BMP-2 was the key in odontogenic differentiation of hDPSCs, which was further enhanced by co-stimulation with TGF-β1. Continuous stimulation with TGFβ-1 did not improve the differentiation of hDPSCs. Combined use of the BMP-2 and TGFβ-1 at the specific sequence can provide a tissue engineering approach for the future guided dentin regeneration.

Effect of two different concentrations of 1α,25-dihydroxyvitamin D3 on odontogenic differentiation of stem cells from human exfoliated deciduous teeth

Background:Stem cells from human exfoliated deciduous teeth (SHEDs) can transform into odontoblasts in vitro and in vivo. The role of 1α, 25-dihydroxyvitamin D3 (1α,25 vitD3) has been reported in the mineralization of hard tissues and teeth, as well as osteoblastic differentiation. This study aimed to assess the effect of different concentrations of 1α,25 vitD3 on odontogenic differentiation of SHEDs.Materials and Methods:In this experimental study, second-passage SHEDs were exposed to odontogenic medium along with 0, 10, 50, 100, and 150 nmol concentrations of in 1α, 25 vitD3 to determine its optimal concentration for odontogenic differentiation. The methyl thiazolyl tetrazolium (MTT) assay was performed. Odontogenic differentiation was evaluated by QRT- polymerase chain reaction for dentin matrix protein (DMP1) and dentin sialophosphoprotein (DSPP) genes. Morphology of differentiated cells was studied by Scanning Electron Microscopy. Data were analyzed using the Kruskal–Wallis, Mann–Whitney, Friedman, and Chi-square test. P < 0.05 is considered statistically significant.Results:MTT test result showed the two groups of odontogenic medium + 10 nm 1α,25 vitD3 and odontogenic medium + 150 nm 1α,25 vitD3 provided the most suitable conditions for cell viability at 72 h. Expression of both genes significantly increased in the presence of 1α,25 vitD3 (P < 0.001). Expression of both genes was significantly higher at 14 days compared with 7 days (P < 0.01). At both time points, expression of both genes was significantly higher in the presence of 150 nm 1α,25 vitD3 compared with 10 nm (P < 0.01). The accumulation of cells with odontoblastic morphology, cell interactions, and calcifications were evident.Conclusion:1α,25 vitD3 upregulates DMP1 and DSPP and results in odontogenic differentiation of SHEDs in odontogenic medium. This upregulation increases with time and by an increase in concentration of 1α,25 vitD3.

Epigallocatechin-3-Gallate Promotes Osteo-/Odontogenic Differentiation of Stem Cells from the Apical Papilla through Activating the BMP-Smad Signaling Pathway.

Stem cells from apical papilla (SCAPs) are desirable sources of dentin regeneration. Epigallocatechin-3-gallate (EGCG), a natural component of green tea, shows potential in promoting the osteogenic differentiation of bone mesenchymal stem cells. However, whether EGCG regulates the odontogenic differentiation of SCAPs and how this occurs remain unknown. SCAPs from immature human third molars (16–20 years, n = 5) were treated with a medium containing different concentrations of EGCG or bone morphogenic protein 2 (BMP2), with or without LDN193189 (an inhibitor of the canonical BMP pathway). Cell proliferation and migration were analyzed using a CCK-8 assay and wound-healing assay, respectively. Osteo-/odontogenic differentiation was evaluated via alkaline phosphatase staining, alizarin red S staining, and the expression of osteo-/odontogenic markers using qPCR and Western blotting. We found that EGCG (1 or 10 μM) promoted the proliferation of SCAPs, increased alkaline phosphatase activity and mineral deposition, and upregulated the expression of osteo-/odontogenic markers including dentin sialophosphoprotein (Dspp), dentin matrix protein-1 (Dmp-1), bone sialoprotein (Bsp), and Type I collagen (Col1), along with the elevated expression of BMP2 and phosphorylation level of Smad1/5/9 (p < 0.01). EGCG at concentrations below 10 μM had no significant influence on cell migration. Moreover, EGCG-induced osteo-/odontogenic differentiation was significantly attenuated via LDN193189 treatment (p < 0.01). Furthermore, EGCG showed the ability to promote mineralization comparable with that of recombinant BMP2. Our study demonstrated that EGCG promotes the osteo-/odontogenic differentiation of SCAPs through the BMP–Smad signaling pathway.

Mesoporous Bioactive Glass Nanoparticles Promote Odontogenesis and Neutralize Pathophysiological Acidic pH

Pathophysiological acidic-pH hinders the dental biomaterial-based pulp-dentin regeneration. Bioactive glass (BG) synthesized by sol-gel methods had shown odontogenic and pulp regeneration potential. However, the pathophysiological acidic-pH neutralizing potential of BG has not been tested yet. In this study, we aimed to design mesoporous BG-nanoparticles by a well-established sol-gel method and test its odontogenic and acidic-pH neutralizing potential. BG-nanoparticles were synthesized and further characterized by SEM, EDS, XRD, and FTIR. Mono-dispersed and spherical mesoporous BG-nanoparticles with size 300-500 nm were successfully fabricated. Effect of BG ionic extraction in DMEM with 0.1-2.5 g/L BG concentrations on proliferation and odontogenic differentiation of stem cells from human exfoliated deciduous teeth (SHED) was analyzed. All the BG ionic extractions did not affect SHEDs proliferation at early time points (day 1 and 3). BG ionic extraction 0.5 g/L robustly enhanced odontogenic differentiation of SHEDs, as shown by the expression pattern of ALP, Col1, DSPP, and matrix mineralization results. The chemical composition of the BG ionic extraction-induced mineralized matrix resembled natural dentin. BG-nanoparticles/alginate paste neutralized the butyric acid solution (pH 5.4) and buffered within pH 8.3 for a month. Our findings showed the odontogenic and pathophysiological acidic-pH neutralizing potential of BG-nanoparticles, indicating its possible application in pulp-dentin regeneration under pathophysiologically challenged acidic environment.

Functionalization of Graphene Oxide Nanosheets Can Reduce Their Cytotoxicity to Dental Pulp Stem Cells

Background. The dental pulp is a heterogeneous soft tissue that supplies nutrients and acts as a biosensor to identify pathogenic stimuli. Regeneration of the dental pulp is one of the desirable topics for researchers. Graphene oxide nanosheets (nGOs) help overexpression of the genes related to odontogenic differentiation of stem cells from dental pulps and increases attachment and proliferation of dental pulp stem cells. Despite its benefits, nGO may be considered as a threat to the environment and human health. Therefore, the purpose of this study was to evaluate the biocompatibility potential of graphene oxide (nGO), chitosan functionalized graphene oxide (nGO-CS), and carboxylated graphene (nGO-COOH) when exposed to human dental pulp stem cells (hDPSCs). Material and Methods. Some different aspects of biocompatibility of nGO, nGO-CS, and nGO-COOH were synthesized, and several intracellular effects induced by different concentrations of graphene-based nanosheets, including cell viability, intracellular oxidative damages, and various factors such as LDH, GSH, SOD, MDA, and MMP, were studied on hDPSCs. Results. According to results, IC50 was determined as 232.01, 467.81, and ≥1000 μg/mL for nGO, nGO-CS, and nGO-COOH, respectively. These results demonstrated the lower toxicity and higher cytocompatibility of nGO-CS and nGO-COOH compared to nGO. nGO-COOH not only has any adverse effect on the cell membrane and mitochondrial activity but also shows slight antioxidant activity at some concentrations. Conclusion. The findings help design safe and cytocompatible nGO derivatives for biomedical applications in dental fields.

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.

Lipopolysaccharide Downregulates the Osteo-/Odontogenic Differentiation of Stem Cells From Apical Papilla by Inducing Autophagy

IntroductionThe dentinogenesis potential of stem cells during dentin-pulp tissue regeneration may be compromised by microorganism components. Here we aimed to investigate the cell viability and osteo-/odontogenic differentiation of stem cells from apical papilla (SCAP) exposed to bacterial lipopolysaccharide (LPS) and to evaluate the molecular mechanism in vitro. MethodsCCK8 assay was used to assess the SCAP proliferation rate on exposure to different concentrations of LPS in medium. Dentin matrix protein-1 (DMP-1), runt-related transcription factor-2 (Runx-2), and alkaline phosphatase (ALP) expression and mineralized nodule formation were detected by Western blotting and alizarin red S staining to evaluate SCAP osteo-/odontogenic differentiation. Autophagosomes in SCAP and the autophagy-related markers Beclin 1, autophagy-related gene 5 (Atg5), and microtubule-associated proteins light chain 3 (LC3) were detected by transmission electron microscopy and Western blotting, respectively. Effects of the autophagy inhibitor 3-methyladenine on LPS-treated SCAP osteo-/odontogenic differentiation were also examined. ResultsSCAP cell viability was decreased by 5 μg/mL LPS treatment on day 3. LPS (5 μg/mL) inhibited SCAP osteo-/odontogenic differentiation and decreased DMP-1, Runx-2, and ALP expression. Moreover, LC3, Atg5, and Beclin 1 expression and autophagosome number were elevated. Conversely, autophagy inhibition rescued the number of mineralized nodules and DMP-1, Runx-2, and ALP expression in the LPS-treated SCAP. ConclusionsOur findings indicated that autophagy was involved in the suppression of SCAP osteo-/odontogenic differentiation in an LPS-induced inflammatory environment. This study discloses autophagy modulation as a potential new strategy to improve SCAP osteo-/odontogenic differentiation in an inflammatory environment.

Lineage-specific exosomes promote the odontogenic differentiation of human dental pulp stem cells (DPSCs) through TGF\u03b21/smads signaling pathway via transfer of microRNAs

BackgroundExosomes derived from dental pulp stem cells (DPSCs) can be used as biomimetic tools to induce odontogenic differentiation of stem cells, but the regulatory mechanisms and functions of exosome-encapsulated microRNAs are still unknown. The present study aimed to clarify the role of microRNAs contained in the exosomes derived from human DPSCs and their potential signaling cascade in odontogenic differentiation.MethodsExosomes were isolated from human DPSCs cultured undergrowth and odontogenic differentiation conditions, named UN-Exo and OD-Exo, respectively. The microRNA sequencing was performed to explore the microRNA profile contained in UN-Exo and OD-Exo. Pathway analysis was taken to detect enriched pathways associated with the predicted target genes of microRNAs. The regulatory roles of a highly expressed microRNA in OD-Exo were investigated through its inhibition or overexpression (miRNA inhibitors and miRNA mimics). Automated western blot was used to identify the function of exosomal microRNA and the roles of TGFβ1/smads pathway in odontogenic differentiation of DPSCs. A luciferase reporter gene assay was used to verify the direct target gene of exosomal miR-27a-5p.ResultsEndocytosis of OD-Exo triggered odontogenic differentiation of DPSCs by upregulating DSP, DMP-1, ALP, and RUNX2 proteins. MicroRNA sequencing showed that 28 microRNAs significantly changed in OD-Exo, of which 7 increased and 21 decreased. Pathway analysis showed genes targeted by differentially expressed microRNAs were involved in multiple signal transductions, including TGFβ pathway. 16 genes targeted by 15 differentially expressed microRNAs were involved in TGFβ signaling. Consistently, automated western blot found that OD-Exo activated TGFβ1 pathway by upregulating TGFβ1, TGFR1, p-Smad2/3, and Smad4 in DPSCs. Accordingly, once the TGFβ1 signaling pathway was inhibited by SB525334, protein levels of p-Smad2/3, DSP, and DMP-1 were significantly decreased in DPSCs treated with OD-Exo. MiR-27a-5p was expressed 11 times higher in OD-Exo, while miR-27a-5p promoted odontogenic differentiation of DPSCs and significantly upregulated TGFβ1, TGFR1, p-Smad2/3, and Smad4 by downregulating the inhibitory molecule LTBP1.ConclusionsThe microRNA expression profiles of exosomes derived from DPSCs were identified. OD-Exo isolated under odontogenic conditions were better inducers of DPSC differentiation. Exosomal microRNAs promoted odontogenic differentiation via TGFβ1/smads signaling pathway by downregulating LTBP1.

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.

Acetylsalicylic Acid Treatment and Suppressive Regulation of AKT Accelerate Odontogenic Differentiation of Stem Cells from the Apical Papilla

IntroductionStem cells isolated from the root apical papilla of human teeth (stem cells from the apical papilla [SCAPs]) are capable of forming tooth root dentin and are a feasible source for bioengineered tooth root regeneration. In this study, we examined the effect of acetylsalicylic acid (ASA) on odontogenic differentiation of SCAPs in vitro and in vivo. MethodsSCAPs were cultured under odontogenic conditions supplemented with or without ASA. ASA-treated SCAPs were also subcutaneously transplanted into immunocompromised mice. ResultsASA accelerates in vitro and in vivo odontogenic differentiation of SCAPs associated with down-regulation of runt-related nuclear factor 2 and up-regulation of specificity protein 7, nuclear factor I C, and dentin phosphoprotein. ASA up-regulated the phosphorylation of AKT in the odontogenic SCAPs. Of interest, pretreatments with phosphoinositide 3-kinase inhibitor LY294402 and small interfering RNA for AKT promoted ASA-induced in vitro and in vivo odontogenic differentiation of SCAPs. LY294402 and small interfering RNA for AKT also suppressed the ASA-induced expression of runt-related nuclear factor 2 and enhanced ASA-induced expression of specificity protein 7, nuclear factor I C, and dentin phosphoprotein in SCAPs. ConclusionsThese findings suggest that a combination of ASA treatment and suppressive regulation of the phosphoinositide 3-kinase–AKT signaling pathway is a novel approach for SCAP-based tooth root regeneration.

A dentin-derived hydrogel bioink for 3D bioprinting of cell laden scaffolds for regenerative dentistry

Recent studies in tissue engineering have adopted extracellular matrix (ECM) derived scaffolds as natural and cytocompatible microenvironments for tissue regeneration. The dentin matrix, specifically, has been shown to be associated with a host of soluble and insoluble signaling molecules that can promote odontogenesis. Here, we have developed a novel bioink, blending printable alginate (3% w/v) hydrogels with the soluble and insoluble fractions of the dentin matrix. We have optimized the printing parameters and the concentrations of the individual components of the bioink for print accuracy, cell viability and odontogenic potential. We find that, while viscosity, and hence printability of the bioinks, was greater in the formulations containing higher concentrations of alginate, a higher proportion of insoluble dentin matrix proteins significantly improved cell viability; where a 1:1 ratio of alginate and dentin (1:1 Alg-Dent) was most suitable. We further demonstrate high retention of the soluble dentin molecules within the 1:1 Alg-Dent hydrogel blends, evidencing renewed interactions between these molecules and the dentin matrix post crosslinking. Moreover, at concentrations of 100 μg ml−1, these soluble dentin molecules significantly enhanced odontogenic differentiation of stem cells from the apical papilla encapsulated in bioprinted hydrogels. In summary, the proposed novel bioinks have demonstrable cytocompatibility and natural odontogenic capacity, which can be a used to reproducibly fabricate scaffolds with complex three-dimensional microarchitectures for regenerative dentistry in the future.