RESUMO
Fibroblast growth factor 2 (FGF2) is a crucial factor in odontoblast differentiation and dentin matrix deposition, which facilitates pulpodentin repair and regeneration. Nevertheless, the specific biological function of FGF2 in odontoblastic differentiation remains unclear because it is controlled by complex signalling pathways. This study aimed to investigate the mechanism underlying the effect of FGF2 on osteo/odontogenic differentiation of stem cells from the apical papilla (SCAP). SCAP were pretreated with conditioned media containing FGF2 for 1 week, followed by culturing in induced differentiation medium for another week. RNA sequencing (RNA-seq) combined with quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to evaluate the pathways affected by FGF2 in SCAP. Osteo/odontogenic differentiation of SCAP was determined using Alizarin red S staining, alkaline phosphatase staining, RT-qPCR, and western blotting. Pretreatment with FGF2 for 1 week increased the osteo/odontogenic differentiation ability of SCAP. RNA-seq and Kyoto Encyclopedia of Genes and Genomes pathway analyses revealed that phosphatidylinositol 3-kinase (PI3K)/AKT signalling is involved in the osteogenic function of FGF2. RT-qPCR results indicated that SCAP expressed FGF receptors, and western blotting showed that p-AKT was reduced in FGF2-pretreated SCAP. The activation of the PI3K/AKT pathway partially reversed the stimulatory effect of FGF2 on osteo/odontogenic differentiation of SCAP. Our findings suggest that pretreatment with FGF2 enhances the osteo/odontogenic differentiation ability of SCAP by inhibiting the PI3K/AKT pathway.
Assuntos
Diferenciação Celular , Papila Dentária , Fator 2 de Crescimento de Fibroblastos , Odontogênese , Osteogênese , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Células-Tronco , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Diferenciação Celular/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Papila Dentária/citologia , Papila Dentária/metabolismo , Humanos , Odontogênese/efeitos dos fármacos , Células-Tronco/metabolismo , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Células CultivadasRESUMO
Stem cells are crucial in tissue engineering, and their microenvironment greatly influences their behavior. Among the various dental stem cell types, stem cells from the apical papilla (SCAPs) have shown great potential for regenerating the pulp-dentin complex. Microenvironmental cues that affect SCAPs include physical and biochemical factors. To research optimal pulp-dentin complex regeneration, researchers have developed several models of controlled biomimetic microenvironments, ranging from in vivo animal models to in vitro models, including two-dimensional cultures and three-dimensional devices. Among these models, the most powerful tool is a microfluidic microdevice, a tooth-on-a-chip with high spatial resolution of microstructures and precise microenvironment control. In this review, we start with the SCAP microenvironment in the regeneration of pulp-dentin complexes and discuss research models and studies related to the biological process.
Assuntos
Papila Dentária , Dispositivos Lab-On-A-Chip , Células-Tronco , Humanos , Células-Tronco/citologia , Papila Dentária/citologia , Animais , Microambiente Celular , Polpa Dentária/citologia , Engenharia Tecidual/instrumentação , Nicho de Células-Tronco , Dentina/citologiaRESUMO
OBJECTIVE: The molecular regulation of odontoblasts in dentin formation remains largely uncharacterized. Using neohesperidin (NEO), a well-documented osteoblast regulator, we investigated whether and how NEO participates in odontoblast regulation through longitudinal treatments using various doses of NEO. DESIGN: Mouse dental papilla cell-23 (MDPC-23) served as a model for odontoblasts. MDPC-23 were treated with various doses of NEO (0, 1, 5, 10, 15, 20 µmol/L). Proliferation was assessed using the Cell counting kit-8 assay. Survival/apoptosis was assayed by live/dead ratio. Migration capability was assessed using scratch healing and Transwell migration assays. Mineralization was assessed using alkaline phosphatase staining and alizarin red staining. The expression levels of four key genes (Runx2, osteocalcin [OCN], ß-catenin, and bone morphogenetic protein [BMP]-2) representing NEO-induced differentiation of MDPC-23 were measured by quantitative reverse transcription polymerase chain reaction. RESULTS: The proliferation trajectories of MDPC-23 treated with the five doses of NEO demonstrated similar curves, with a rapid increase in the 10 µmol/L NEO condition after 48 h of treatment. Similar dose-dependent trajectories were observed for survival/apoptosis. All four key genes representing odontogenic differentiation were upregulated in MDPC-23 induced by NEO treatments at two optimal doses (5 µmol/L and 10 µmol/L). Optimal migration and mobility trajectories were observed in MDPC-23 treated with 10 µmol/L NEO. Optimal mineralization was observed in MDPC-23 treated with 5 µmol/L NEO. CONCLUSION: NEO can subtly regulate odontoblast proliferation, differentiation, migration, and mineralization in vitro. NEO at 5-10 µmol/L offers a safe and effective perspective for clinical promotion of dentin bridge formation in teenagers.
Assuntos
Apoptose , Diferenciação Celular , Movimento Celular , Proliferação de Células , Papila Dentária , Hesperidina , Odontoblastos , Animais , Camundongos , Proliferação de Células/efeitos dos fármacos , Hesperidina/farmacologia , Hesperidina/análogos & derivados , Diferenciação Celular/efeitos dos fármacos , Odontoblastos/efeitos dos fármacos , Papila Dentária/citologia , Papila Dentária/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Osteocalcina/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Técnicas In Vitro , Proteína Morfogenética Óssea 2/farmacologia , Sobrevivência Celular/efeitos dos fármacos , beta Catenina/metabolismo , Fosfatase Alcalina/metabolismo , Células Cultivadas , Reação em Cadeia da Polimerase em Tempo RealRESUMO
BACKGROUND: Circular RNA (circRNA) is a key player in regulating the multidirectional differentiation of stem cells. Previous research by our group found that the blue light-emitting diode (LED) had a promoting effect on the osteogenic/odontogenic differentiation of human stem cells from apical papilla (SCAPs). This research aimed to investigate the differential expression of circRNAs during the osteogenic/odontogenic differentiation of SCAPs regulated by blue LED. MATERIALS AND METHODS: SCAPs were divided into the irradiation group (4 J/cm2) and the control group (0 J/cm2), and cultivated in an osteogenic/odontogenic environment. The differentially expressed circRNAs during osteogenic/odontogenic differentiation of SCAPs promoted by blue LED were detected by high-throughput sequencing, and preliminarily verified by qRT-PCR. Functional prediction of these circRNAs was performed using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) and the circRNA-miRNA-mRNA networks were also constructed. RESULTS: It showed 301 circRNAs were differentially expressed. GO and KEGG analyses suggested that these circRNAs were associated with some signaling pathways related to osteogenic/odontogenic differentiation. And the circRNA-miRNA-mRNA networks were also successfully constructed. CONCLUSION: CircRNAs were involved in the osteogenic/odontogenic differentiation of SCAPs promoted by blue LED. In this biological process, circRNA-miRNA-mRNA networks served an important purpose, and circRNAs regulated this process through certain signaling pathways.
Assuntos
Diferenciação Celular , Papila Dentária , Luz , Odontogênese , Osteogênese , RNA Circular , Células-Tronco , RNA Circular/genética , RNA Circular/metabolismo , Humanos , Osteogênese/genética , Diferenciação Celular/genética , Células-Tronco/metabolismo , Células-Tronco/citologia , Odontogênese/genética , Papila Dentária/citologia , Papila Dentária/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Ontologia Genética , Células Cultivadas , Perfilação da Expressão Gênica/métodos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Redes Reguladoras de Genes , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Regulação da Expressão Gênica/efeitos da radiação , Luz AzulRESUMO
OBJECTIVE: To explore the feasibility of injectable platelet-rich fibrin (i-PRF) in regenerative endodontics by comparing the effect of i-PRF and platelet-rich fibrin (PRF) on the biological behavior and angiogenesis of human stem cells from the apical papilla (SCAPs). METHODOLOGY: i-PRF and PRF were obtained from venous blood by two different centrifugation methods, followed by hematoxylin-eosin (HE) staining and scanning electron microscopy (SEM). Enzyme-linked immunosorbent assay (ELISA) was conducted to quantify the growth factors. SCAPs were cultured with different concentrations of i-PRF extract (i-PRFe) and PRF extract (PRFe), and the optimal concentrations were selected using the Cell Counting Kit-8 (CCK-8) assay. The cell proliferation and migration potentials of SCAPs were then observed using the CCK-8 and Transwell assays. Mineralization ability was detected by alizarin red staining (ARS), and angiogenesis ability was detected by tube formation assay. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed to evaluate the expression of genes related to mineralization and angiogenesis. The data were subjected to statistical analysis. RESULTS: i-PRF and PRF showed a similar three-dimensional fibrin structure, while i-PRF released a higher concentration of growth factors than PRF ( P <.05). 1/4× i-PRFe and 1/4× PRFe were selected as the optimal concentrations. The cell proliferation rate of the i-PRFe group was higher than that of the PRFe group ( P <.05), while no statistical difference was observed between them in terms of cell mitigation ( P >.05). More importantly, our results showed that i-PRFe had a stronger effect on SCAPs than PRFe in facilitating mineralization and angiogenesis, with the consistent result of RT-qPCR ( P <.05). CONCLUSION: This study revealed that i-PRF released a higher concentration of growth factors and was superior to PRF in promoting proliferation, mineralization and angiogenesis of SCAPs, which indicates that i-PRF could be a promising biological scaffold for application in pulp regeneration.
Assuntos
Proliferação de Células , Ensaio de Imunoadsorção Enzimática , Peptídeos e Proteínas de Sinalização Intercelular , Microscopia Eletrônica de Varredura , Neovascularização Fisiológica , Fibrina Rica em Plaquetas , Reação em Cadeia da Polimerase em Tempo Real , Endodontia Regenerativa , Humanos , Proliferação de Células/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Endodontia Regenerativa/métodos , Células Cultivadas , Reprodutibilidade dos Testes , Movimento Celular/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Fatores de Tempo , Estudos de Viabilidade , Análise de Variância , Papila Dentária/efeitos dos fármacos , Papila Dentária/citologia , Valores de ReferênciaRESUMO
OBJECTIVE: Non-surgical therapeutics to reconstruct lost interdental papilla are evolving; these include hyaluronic acid injection. The aim of this systematic review is to evaluate the efficacy, safety, and long-term outcomes of hyaluronic acid injection in the treatment of black triangles and reconstruction of lost interdental papilla in anterior teeth. MATERIALS AND METHODS: The protocol was registered in PROSPERO (CRD42023446875) and in accordance with the Cochrane Handbook of Systematic Reviews of Interventions and the Preferred Reporting Items for Systematic Reviews and Meta-Analysis 'PRISMA'. The search involved four databases, PubMed/MEDLINE, The Cochrane Library, Google Scholar, and ProQuest for ''grey literature' with additional manual search for studies published up to May 2024. Human clinical studies of a prospective nature (randomised clinical trials and prospective cohort studies) were included. Exclusion criteria were case reports, case series, review articles, letter to editor, personal opinion, and animal studies. Furthermore, studies which utilised hyaluronic acid injection in conjunction with other therapeutic material, tissue graft, or any surgical procedure were also excluded. The data were extracted independently by the two authors and incorporated after consensus. The risk of bias was assessed using the RoB2: the revised Cochrane risk of bias tool for randomised clinical trials and the Newcastle Ottawa scale for prospective cohort studies. RESULTS: 24 studies, 15 prospective clinical studies and nine randomised clinical trials, were included with a total of 898 interdental papillae injected with hyaluronic acid. The studies showed promising outcomes in the reconstruction of lost interdental papilla with minimal adverse reactions. Risk of bias assessment among prospective clinical studies revealed 13 good quality studies with only two poor studies while the randomised clinical trials consisted of three with low, one with some concern, and five studies with high risk of bias. However, due to the high heterogeneity, a meta-analysis was not feasible. Conclusion: Hyaluronic acid injection is an effective minimally invasive approach in treating black triangles and reconstructing lost interdental papilla in the anterior teeth. Further long-term well-designed randomised clinical trials employing standardised procedures are essential to validate this treatment and provide better quality of evidence.
Assuntos
Ácido Hialurônico , Ácido Hialurônico/administração & dosagem , Humanos , Papila DentáriaRESUMO
Methyltransferase-like 3 (METTL3) is a crucial element of N6-methyladenosine (m6A) modifications and has been extensively studied for its involvement in diverse biological and pathological processes. In this study, we explored how METTL3 affects the differentiation of stem cells from the apical papilla (SCAPs) into odonto/osteoblastic lineages through gain- and loss-of-function experiments. The m6A modification levels were assessed using m6A dot blot and activity quantification experiments. In addition, we employed Me-RIP microarray experiments to identify specific targets modified by METTL3. Furthermore, we elucidated the molecular mechanism underlying METTL3 function through dual-luciferase reporter gene experiments and rescue experiments. Our findings indicated that METTL3+/- mice exhibited significant root dysplasia and increased bone loss. The m6A level and odonto/osteoblastic differentiation capacity were affected by the overexpression or inhibition of METTL3. This effect was attributed to the acceleration of pre-miR-665 degradation by METTL3-mediated m6A methylation in cooperation with the "reader" protein YTHDF2. Additionally, the targeting of distal-less homeobox 3 (DLX3) by miR-665 and the potential direct regulation of DLX3 expression by METTL3, mediated by the "reader" protein YTHDF1, were demonstrated. Overall, the METTL3/pre-miR-665/DLX3 pathway might provide a new target for SCAP-based tooth root/maxillofacial bone tissue regeneration.
Assuntos
Diferenciação Celular , Proteínas de Homeodomínio , Metiltransferases , MicroRNAs , Células-Tronco , Fatores de Transcrição , Animais , Camundongos , Adenosina/análogos & derivados , Adenosina/metabolismo , Diferenciação Celular/genética , Papila Dentária/citologia , Papila Dentária/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/genética , Metilação , Metiltransferases/metabolismo , Metiltransferases/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Células-Tronco/metabolismo , Células-Tronco/citologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genéticaRESUMO
OBJECTIVES: This study aimed to investigate the regulatory mechanisms governing dental mesenchymal cell commitment during tooth development, focusing on odontoblast differentiation and the role of epigenetic regulation in this process. METHODS: We performed single-cell RNA sequencing (scRNA-seq) of dental cells from embryonic day 14.5 (E14.5) mice to understand the heterogeneity of developing tooth germ cells. Computational analyses including gene regulatory network (GRN) assessment were conducted. We validated our findings using immunohistochemistry (IHC) and in vitro loss-of-function analyses using the DNA methyltransferase 1 (DNMT1) inhibitor Gsk-3484862 in primary dental mesenchymal cells (DMCs) isolated from E14.5 mouse tooth germs. Bulk RNA-seq of Gsk-3484862-treated DMCs was performed to identify potential downstream targets of DNMT1. RESULTS: scRNA-seq analysis revealed diverse cell populations within the tooth germs, including epithelial, mesenchymal, immune, and muscle cells. Using single-cell regulatory network inference and clustering (SCENIC), we identified Dnmt1 as a key regulator of early odontoblast development. IHC analysis showed the ubiquitous expression of DNMT1 in the dental papilla and epithelium. Bulk RNA-seq of cultured DMCs showed that Gsk-3484862 treatment upregulated odontoblast-related genes, whereas genes associated with cell division and the cell cycle were downregulated. Integrated analysis of bulk RNA-seq data with scRNA-seq SCENIC profiles was used to identify the potential Dnmt1 target genes. CONCLUSIONS: Dnmt1 may negatively affect odontoblast commitment and differentiation during tooth development. These findings contribute to a better understanding of the molecular mechanisms underlying tooth development and future development of hard-tissue regenerative therapies.
Assuntos
Diferenciação Celular , DNA (Citosina-5-)-Metiltransferase 1 , Papila Dentária , Odontoblastos , Análise de Célula Única , Germe de Dente , Animais , Camundongos , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Germe de Dente/metabolismo , Germe de Dente/citologia , Germe de Dente/embriologia , Papila Dentária/citologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Odontoblastos/citologia , Odontoblastos/metabolismo , Odontoblastos/efeitos dos fármacos , Análise de Sequência de RNA/métodos , Odontogênese/genética , Odontogênese/efeitos dos fármacos , Transcriptoma , Imuno-Histoquímica , Redes Reguladoras de Genes/efeitos dos fármacosRESUMO
Odontoblast differentiation is a key process in dentin formation. Mouse dental papilla cells (mDPCs) are pivotal in dentinogenesis through their differentiation into odontoblasts. Odontoblast differentiation is intricately controlled by transcription factors (TFs) in a spatiotemporal manner. Previous research explored the role of RUNX2 and KLF4 in odontoblast lineage commitment, respectively. Building on bioinformatics analysis of our previous ATAC-seq profiling, we hypothesized that KLF4 potentially collaborates with RUNX2 to exert its biological role. To investigate the synergistic effect of multiple TFs in odontoblastic differentiation, we first examined the spatiotemporal expression patterns of RUNX2 and KLF4 in dental papilla at the bell stage using immunostaining techniques. Notably, RUNX2 and KLF4 demonstrated colocalization in preodontoblast. Further, immunoprecipitation and proximity ligation assays verified the interaction between RUNX2 and KLF4 in vitro. Specifically, the C-terminus of RUNX2 was identified as the interacting domain with KLF4. Functional implications of this interaction were investigated using small hairpin RNA-mediated knockdown of Runx2, Klf4, or both. Western blot analysis revealed a marked decrease in DSPP expression, an odontoblast differentiation marker, particularly in the double knockdown condition. Additionally, alizarin red S staining indicated significantly reduced mineralized nodule formation in this group. Collectively, our findings highlight the synergistic interaction between RUNX2 and KLF4 in promoting odontoblast differentiation from mDPCs. This study contributes to a more comprehensive understanding of the regulatory network of TFs governing odontoblast differentiation.
Assuntos
Diferenciação Celular , Subunidade alfa 1 de Fator de Ligação ao Core , Papila Dentária , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like , Odontoblastos , Fator 4 Semelhante a Kruppel/metabolismo , Odontoblastos/metabolismo , Odontoblastos/citologia , Animais , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Papila Dentária/citologia , Papila Dentária/metabolismoRESUMO
AIM: Three-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. METHODOLOGY: A 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. RESULTS: The 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. CONCLUSION: In 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 SIGNIFICANCE: This 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.
Assuntos
Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Papila Dentária , Dentina , Gelatina , Dispositivos Lab-On-A-Chip , Engenharia Tecidual , Engenharia Tecidual/métodos , Humanos , Papila Dentária/citologia , Células-Tronco/citologia , Odontogênese , Osteogênese/fisiologia , Metacrilatos , Técnicas de Cultura de Células , Microfluídica/métodos , Microfluídica/instrumentação , Técnicas de Cultura de Células em Três Dimensões/métodos , Técnicas de Cultura de Células em Três Dimensões/instrumentação , Células CultivadasRESUMO
The coordination between odontoblastic differentiation and directed cell migration of mesenchymal progenitors is necessary for regular dentin formation. The synthesis and degradation of hyaluronan (HA) in the extracellular matrix create a permissive niche that directly regulates cell behaviors. However, the role and mechanisms of HA degradation in dentin formation remain unknown. In this work, we present that HA digestion promotes odontoblastic differentiation and cell migration of mouse dental papilla cells (mDPCs). Hyaluronidase 2 (HYAL2) is responsible for promoting odontoblastic differentiation through degrading HA, while hyaluronidase 1 (HYAL1) exhibits negligible effect. Silencing Hyal2 generates an extracellular environment rich in HA, which attenuates F-actin and filopodium formation and in turn inhibits cell migration of mDPCs. In addition, activating PI3K/Akt signaling significantly rescues the effects of HA accumulation on cytodifferentiation. Taken together, the results confirm the contribution of HYAL2 to HA degradation in dentinogenesis and uncover the mechanism of the HYAL2-mediated HA degradation in regulating the odontoblastic differentiation and migration of mDPCs.
Assuntos
Diferenciação Celular , Movimento Celular , Papila Dentária , Ácido Hialurônico , Hialuronoglucosaminidase , Odontoblastos , Animais , Hialuronoglucosaminidase/metabolismo , Hialuronoglucosaminidase/genética , Camundongos , Ácido Hialurônico/metabolismo , Odontoblastos/metabolismo , Odontoblastos/citologia , Papila Dentária/citologia , Papila Dentária/metabolismo , Transdução de Sinais , Proteínas Ligadas por GPI/metabolismo , Proteínas Ligadas por GPI/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Células Cultivadas , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol 3-Quinases/genéticaRESUMO
AIM: 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. METHODOLOGY: 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 in vitro. Fluorescence in situ 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. RESULTS: 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, in vivo experiments showed that SCAPs overexpressing circ-ZNF236 promoted bone formation in a rat skull defect model. CONCLUSIONS: 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.
Assuntos
MicroRNAs , Osteogênese , Humanos , Animais , Ratos , Osteogênese/genética , RNA Circular/genética , RNA Circular/metabolismo , RNA Circular/farmacologia , Hibridização in Situ Fluorescente , Papila Dentária , Diferenciação Celular , Células-Tronco , Proliferação de Células , Células Cultivadas , MicroRNAs/genética , MicroRNAs/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMO
OBJECTIVE: The aim of this study was to analyze the effect of lipopolysaccharides (LPS) on the biological properties of stem cells from the apical papilla (SCAPs), such as viability, adhesion to dentin, odontoblast-like differentiation, mineralization, and release of immunomodulatory cytokines. DESIGN: SCAPs were isolated from immature teeth of three donors (10 to 15 years old) and cultured in mineralizing media with or without 1 µg/mL lipopolysaccharide (LPS). Cells were seeded and cultured under standardized conditions; viability was assessed by MTT assay on days 1, 3, 5, and 7; adhesion to dentin was analyzed using an environmental scanning electron microscope after 2 days; the expression of odontogenic and mineralization genes (DSPP, DMP-1, OCN, Col1A1) was evaluated through qPCR after 14 days, mineralization was evaluated with alizarin red staining after 21 days; and the release of immunomodulatory cytokines (IL-6 and IL-10) was measured by ELISA after 1 and 7 days. The Kruskal-Wallis test was performed to detect the effect of LPS on SCAPs, followed by the Dunn-Sidak test. RESULTS: LPS presence in the culture media affected SCAPs viability on day 5 and increased IL-6 secretion by day 7, however, SCAPs retained the adhesion to dentin and mineralization capacities, as well as the differentiation capacity into a mineralizing phenotype. CONCLUSION: In conclusion, within the limitations of this in vitro study, and under the inflammatory microenvironment simulated in this study, stem cells from the apical papilla were found with retained adhesion capacity to dentin, differentiation into a mineralizing phenotype, mineralization, and release of IL-10.
Assuntos
Interleucina-10 , Lipopolissacarídeos , Lipopolissacarídeos/farmacologia , Lipopolissacarídeos/metabolismo , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Células Cultivadas , Papila Dentária , Osteogênese , Células-Tronco , Diferenciação Celular , Proliferação de CélulasRESUMO
PURPOSE: The important role of non-coding RNAs in odontoblastic differentiation of dental tissue-derived stem cells has been widely demonstrated; however, whether piRNA (a subclass of non-coding RNA) involved in the course of odontoblastic differentiation is not yet available. This study aimed to investigate the expression profile of piRNA during odontogenic differentiation of mDPCs and the potential molecular mechanism in vitro. MATERIALS AND METHODS: The primary mouse dental papilla cells (mDPCs) were isolated from the first molars of 1-day postnatal Kunming mice. Then, they were cultured in odontogenic medium for 9 days. The expression profile of piRNA was detected by Small RNA sequencing. RT-qPCR was used to verify the elevation of piR-368. The mRNA and protein levels of mineralization markers were examined by qRT-PCR and Western blot analysis. Alkaline phosphatase (ALP) activity and alizarin red S staining were conducted to assess the odontoblastic differentiation ability. RESULTS: We validated piR-368 was significantly upregulated and interference with piR-368 markedly inhibited the odontogenic differentiation of mDPCs. In addition, the relationship between Smad1/5 signaling pathway and piR-368-induced odontoblastic differentiation has been discovered. Finally, we demonstrated Smurf1 as a target gene of piR-368 using dual-luciferase assays. CONCLUSION: This study was the first to illustrate the participation of piRNA in odontoblastic differentiation. We proved that piR-368 promoted odontoblastic differentiation of mouse dental papilla cells via the Smad1/5 signaling pathway by targeting Smurf1.
Assuntos
Proteínas da Matriz Extracelular , RNA de Interação com Piwi , Animais , Camundongos , Diferenciação Celular/genética , Células Cultivadas , Papila Dentária/química , Papila Dentária/metabolismo , Polpa Dentária/química , Proteínas da Matriz Extracelular/metabolismo , Odontoblastos , Transdução de Sinais , Proteína Smad1/metabolismoRESUMO
INTRODUCTION: Stem cell-based dental pulp regeneration has been extensively studied, mainly focusing on exploiting dental stem cells' osteogenic and angiogenic potentials. Dental stem cells' neurogenic role is often overlooked. Stem cells from apical papilla (SCAPs), originating from the neural crest and capable of sphere formation, display potent neurogenic capacity. This study aimed to investigate the interactions of neuronally induced stem cells from apical papilla (iSCAP) spheres, SCAPs, and human umbilical vascular endothelial cells (HUVECs) on vasculogenesis and neurogenesis. METHODS: SCAPs were isolated and characterized using flow cytometry and multilineage differentiation assays. SCAP monolayer culture and spheres were neuronally induced by a small molecule neural induction medium, and the neural gene expression and neurite formation at days 0, 3, and 7 were evaluated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and using phase-contrast light and fluorescence microscopy. Direct coculture or pulp-on-chip was used to investigate iSCAP sphere interaction with SCAPs and HUVECs. RT-qPCR, fluorescence microscopy, and immunostaining with ß-tubulin III, alpha-smooth muscle actin, and CD31 were used to study neural gene expression, neurite formation, and neurovascular cell interactions. RESULTS: Neural induction medium with small molecules rapidly induced SCAP differentiation toward neural-like cells. Gene expression of Nestin, ß-tubulin III, microtubule-associated protein 2, neuron-specific enolase, and NeuN was higher in iSCAP spheres than in iSCAPs. iSCAP spheres formed more and longer neurites compared with iSCAPs. iSCAP sphere, HUVEC, and SCAP direct coculture significantly enhanced vessel formation along with up-regulated VEGF (P < .001) and multiple neural markers, such as Nestin (P < .01), microtubule-associated protein 2 (P < .001), S100 (P < .001), and NG2 (P < .001). iSCAP spheres, SCAPs, and HUVECs cultured in a pulp-on-chip system promoted endothelial and neural cell migration toward each other and alpha-smooth muscle actin-positive and CD31-positive cells assembling for the vascular constitution. CONCLUSIONS: iSCAP-formed spheres interact with SCAPs and HUVECs, promoting vasculogenesis and neurogenesis.
Assuntos
Polpa Dentária , Células Endoteliais , Humanos , Nestina/metabolismo , Papila Dentária , Tubulina (Proteína)/metabolismo , Actinas/metabolismo , Regeneração , Células-Tronco/fisiologia , Diferenciação Celular , Neurogênese , Células Cultivadas , Proteínas Associadas aos Microtúbulos/metabolismo , OsteogêneseRESUMO
Los procedimientos endodónticos regenerativos (REPs) representan una evolución significativa en el campo de la endodoncia, buscando no sólo tratar la infección o lesión presente en el diente, sino tam-bién promover la regeneración de los tejidos denta-rios afectados. El presente caso clínico muestra un incisivo lateral superior izquierdo con apexogénesis incompleta y diagnóstico de absceso alveolar crónico reagudizado en una paciente de 22 años, en el que se aplicó un procedimiento de endodoncia regenerativa (REPs). La estrategia terapéutica elegida se basó en los principios de ingeniería tisular, incorporando la novedosa aplicación de la membrana amniótica hu-mana liofilizada esterilizada como andamio bioactivo intraconducto. Las evaluaciones clínicas, radiográ-ficas y tomográficas a corto, mediano y largo plazo revelaron el éxito de la terapia. La resolución exitosa mostró en los controles a la pieza dentaria asintomá-tica, con una notable remisión de la patología apical, aumento de la longitud radicular y disminución del calibre apical. Se ha podido destacar la eficacia de los REPs, con una exitosa aplicabilidad de la membra-na amniótica como andamio innovador (AU)
Regenerative endodontic procedures (REPs) represent a significant evolution in the field of endodontics, aiming not only to address the infection or injury within the tooth, but also to promote the regeneration of the affected dental tissues. In this clinical case, an upper left lateral incisor with incomplete apexogenesis and diagnosis of acute exacerbation of a chronic periapical lesion in a 22-year-old patient is presented. A regenerative endodontic procedure (REPs) was applied. The chosen therapeutic strategy was based on tissue engineering principles, incorporating the innovative use of sterilized lyophilized human amniotic membrane as an intraconduct bioactive scaffold. Clinical, radiographic, and tomographic assessments at short, medium, and long-term follow-up revealed the success of the therapy. Successful resolution demonstrated an asymptomatic tooth in the follow-up, with a notable resolution of apical pathology, increased root length, and decreased apical caliber. The effectiveness of REPs has been highlighted, demonstrating the successful applicability of amniotic membrane as an innovative scaffold (AU)
Assuntos
Humanos , Feminino , Adulto , Células-Tronco/fisiologia , Alicerces Teciduais , Argentina , Faculdades de Odontologia , Papila Dentária , Liofilização/métodosRESUMO
Background: Dental papilla cells (DPCs) are one of the key stem cells for tooth development, eventually forming dentin and pulp. Previous studies have reported that PER2 is expressed in a 24-hour oscillatory pattern in DPCs in vitro. In vivo, PER2 is highly expressed in odontoblasts (which are differentiated from DPCs). However, whether PER2 modulates the odontogenic differentiation of DPCs is uncertain. This research was to identify the function of PER2 in the odontogenic differentiation of DPCs and preliminarily explore its mechanisms. Methods: We monitored the expression of PER2 in DPCs differentiated in vivo. We used PER2 overexpression and knockdown studies to assess the role of PER2 in DPC differentiation and performed intracellular ATP content and reactive oxygen species (ROS) assays to further investigate the mechanism. Results: PER2 expression was considerably elevated throughout the odontoblastic differentiation of DPCs in vivo. Overexpressing Per2 boosted levels of odontogenic differentiation markers, such as dentin sialophosphoprotein (Dspp), dentin matrix protein 1 (Dmp1), and alkaline phosphatase (Alp), and enhanced mineralized nodule formation in DPCs. Conversely, the downregulation of Per2 inhibited the differentiation of DPCs. Additionally, downregulating Per2 further affected intracellular ATP content and ROS levels during DPC differentiation. Conclusion: Overall, we demonstrated that PER2 positively regulates the odontogenic differentiation of DPCs, and the mechanism may be related to mitochondrial function as shown by intracellular ATP content and ROS levels.
Assuntos
Papila Dentária , Odontoblastos , Espécies Reativas de Oxigênio , Diferenciação Celular/genética , Trifosfato de AdenosinaRESUMO
Supernumerary teeth are advantaged sources for high-quality stem cell preparation from both apical papilla (SCAP-Ss) and dental pulp (DPSCs). However, the deficiency of the systematic and detailed comparison of the biological and transcriptomic characteristics of the aforementioned stem cells largely hinders their application in regenerative medicine. Herein, we collected supernumerary teeth for SCAP-S and DPSC isolation and identification by utilizing multiple biological tests (e.g., growth curve, cell cycle and apoptosis, adipogenic and osteogenic differentiation, and quantitative real-time polymerase chain reaction). Furthermore, we took advantage of transcriptome sequencing and multifaceted bioinformatic analyses to dissect the similarities and diversities between them. In this study, we found that SCAP-Ss and DPSCs showed indistinctive signatures in morphology and immunophenotypes, whereas with diversity in cell vitality and multi-lineage differentiation as well as gene expression profiling and differentially expressed genes-associated gene ontology and signaling pathways. Collectively, our data indicated the diversity of the multifaceted signatures of human supernumerary teeth-derived stem cells both at the cellular and molecular levels, which also supplied new references for SCAP-Ss serving as splendid alternative stem cell sources for regenerative medicine purposes.
Assuntos
Dente Supranumerário , Transcriptoma , Humanos , Osteogênese/genética , Dente Supranumerário/genética , Polpa Dentária , Células-Tronco , Diferenciação Celular , Perfilação da Expressão Gênica , Proliferação de Células , Células Cultivadas , Papila DentáriaRESUMO
Stem cells from the apical papilla (SCAPs) are used to regulate the microenvironment of nerve defects. KDM6B, which functions as an H3K27me3 demethylase, is known to play a crucial role in neurogenesis. However, the mechanism by which KDM6B influences the neurogenesis potential of SCAPs remains unclear. We evaluated the expression of neural markers in SCAPs by using real-time RT-PCR and immunofluorescence staining. To assess the effectiveness of SCAP transplantation in the SCI model, we used the BBB scale to evaluate motor function. Additionally, toluidine blue staining and Immunofluorescence staining of NCAM, NEFM, ß-III-tubulin, and Nestin were used to assess nerve tissue remodeling. Further analysis was conducted through Microarray analysis and ChIP assay to study the molecular mechanisms. Our results show that KDM6B inhibits the expression of NeuroD, TH, ß-III tubulin, and Nestin. In vivo studies indicate that the SCAP-KDM6Bsh group is highly effective in restoring spinal cord structure and motor function in rats suffering from SCI. Our findings suggest that KDM6B directly binds to the HES1 promoter via regulating H3K27me3 and HES1 expression. In conclusion, our study can help understand the regulatory role of KDM6B in neurogenesis and provide more effective treatments for nerve injury.
Assuntos
Histonas , Tubulina (Proteína) , Ratos , Animais , Histonas/metabolismo , Nestina/genética , Nestina/metabolismo , Diferenciação Celular , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Células-Tronco/metabolismo , Neurogênese , Papila Dentária/metabolismo , Células Cultivadas , OsteogêneseRESUMO
OBJECTIVE: To analyze the effect of P11-4 self-assembly peptide on cell viability and osteogenic capacity of SCAPs through mineral deposition and gene expression of osteogenic markers. METHODS: SCAPs were seeded in contact with P11-4 (10 µg/ml, 100 µg/ml and 1 mg/ml) solution. Cell viability was evaluated using a colorimetric assay MTT: 3-(4,5-dimethyl-thiazolyl-2)-2,5- diphenyltetrazolium bromide) in an experimental time of 24, 48 and 72 h (n = 7). Mineral deposition and quantification provided by the cells was tested using the Alizarin Red staining and Cetylpyridinium Chloride (CPC), respectively, after 30 days (n = 4). Gene expression of Runt-related transcription factor 2 (RUNX2), Alkaline phosphatase (ALP) and Osteocalcin (OCN) was quantified using quantitative polymerase chain reaction (RT-qPCR), at 3 and 7 days with Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the housekeeping gene, and relative gene expression was measured using the ΔΔCq method. Data were analyzed using Kruskall-Wallis followed by multiple comparisons, and T-test for gene expression with α=0.05. RESULTS: All tested concentrations (10 µg/ml, 100 µg/ml and 1 mg/ml) were not cytotoxic at time 24 and 48 h. After 72 h, a slight decrease in cell viability was observed for the lowest concentration (10 µg/ml). The concentration of 100 µg/ml P11-4 showed the highest mineral deposition. However, qPCR analysis of P11-4 (10 µg/ml) showed upregulation of RUNX2 and OCN at 3 days, with downregulation of ALP at 3 and 7d CONCLUSION: P11-4 did not affect cell viability, induced mineral deposition in SCAPs, and upregulated the expression of RUNX2 and OCN genes at 3 days, while downregulating ALP expression at 3 and 7 days. CLINICAL SIGNIFICANCE: Based on the results obtained in this study it can be stated that self-assembling peptide P11-4 is a potential candidate to induce mineralization on dental stem cells for regenerative purposes and also for a clinical use as a capping agent without compromising the cells health.