Biological properties of stem cells from the apical papilla exposed to lipopolysaccharides: An in vitro study.

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.

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

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.

piR-368 promotes odontoblastic differentiation of dental papilla cells via the Smad1/5 signaling pathway by targeting Smurf1.

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.

Interactions of Neuronally Induced Stem Cells from Apical Papilla Spheres, Stems Cells from Apical Papilla, and Human Umbilical Vascular Endothelial Cells on Vasculogenesis and Neurogenesis.

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.

PER2 regulates odontoblastic differentiation of dental papilla cells in vitro via intracellular ATP content and reactive oxygen species levels.

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.

Decoding the multifaceted signatures and transcriptomic characteristics of stem cells derived from apical papilla and dental pulp of human supernumerary teeth.

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.

KDM6B Negatively Regulates the Neurogenesis Potential of Apical Papilla Stem Cells via HES1.

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.

The Self-assembling peptide P11-4 influences viability and osteogenic differentiation of stem cells of the apical papilla (SCAP).

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.

Effects of EDTA and saline as the final irrigation in regenerative endodontic procedures on the migration, proliferation, and differentiation of human stem cells from the apical papilla.

OBJECTIVES: To evaluate the effect of EDTA and saline as the final irrigation in regenerative endodontic procedures (REPS) on the attachment, proliferation, migration, and differentiation of stem cells from the apical papilla (SCAPs). MATERIALS AND METHODS: Dentin specimens from 140 human third molars were irrigated with various protocols-group 1: normal sterile saline (NSS), group 2: EDTA, group 3: EDTA then 5 mL NSS, or group 4: EDTA then 20 mL NSS. The specimens were used in cell assays. For cell proliferation, SCAPs were seeded on dentin, and the cell viability on days 1, 3, and 7 was determined using an MTT assay. At day 3, the attached cells' morphology was observed using SEM, and cell migration was investigated using a transwell migration assay. The ALP activity and odonto/osteogenic differentiation gene expression were evaluated at days 7, 14, and 21 using an ALP activity assay and RT-qPCR. RESULTS: On days 3 and 7, group 4 demonstrated more viable cells than group 1 (p < 0.01). The amount of migrated cells in groups 2, 3, and 4 was greater compared with group 1 (p < 0.05). Moreover, SCAP differentiation was similar between groups. CONCLUSIONS: Irrigating dentin with EDTA alone or with EDTA then NSS promoted SCAP migration. However, a final irrigation with 20 mL NSS after EDTA promoted SCAP proliferation without affecting their differentiation. CLINICAL RELEVANCE: When using a blood clot as a scaffold, a final flushing with 20 mL NSS after EDTA could be beneficial for clinical REP protocols.

Reactive Oxygen Species-Induced Inhibition of Odontoblastic Differentiation of Mouse Dental Papilla Cells Is Mediated by Downregulation of Importin 7.

Tooth dentin is a crucial tooth structure. The biological process of odontoblast differentiation is essential for formation of normal dentin. Accumulation of reactive oxygen species (ROS) leads to oxidative stress, which can influence the differentiation of several cells. As a member of the importin-ß superfamily, importin 7 (IPO7) is essential for nucleocytoplasmic transport and plays an important role in the processes of odontoblast differentiation and oxidative stress. Nevertheless, the association between ROS, IPO7, and odontoblast differentiation in mouse dental papilla cells (mDPCs) and the underlying mechanisms remain to be elucidated. In this study, we confirmed that ROS suppressed odontoblastic differentiation of mDPCs as well as the expression and nucleocytoplasmic shuttle of IPO7 in cells, while overexpression of IPO7 can rescue these effects. ROS resulted in increased phosphorylation of p38 and cytoplasmic aggregation of phosphorylated p38 (p-p38), which was able to be reversed by overexpression of IPO7. p-p38 interacted with IPO7 in mDPCs without hydrogen peroxide (H2O2) treatment, but in the presence of H2O2, the interaction between p-p38 and IPO7 was significantly decreased. Inhibition of IPO7 increased the expression level and nuclear translocation of p53, which are mediated by cytoplasmic aggregation of p-p38. In conclusion, ROS inhibited odontoblastic differentiation of mDPCs, which is mediated by downregulation and damaged nucleocytoplasmic shuttle of IPO7.

Effects of L-Chg10-Teixobactin on Viability, Proliferation, and Osteo/Odontogenic Differentiation of Stem Cells from Apical Papilla.

INTRODUCTION: Intracanal medicament is one of the essential steps for ensuring success in regenerative endodontic procedures. L-Chg10-teixobactin is a novel antimicrobial agent that exhibited potent antibacterial and antibiofilm effects against Enterococcusfaecalis at low concentrations compared with ampicillin. At the same time, its cytotoxicity on dental stem cells has not been studied. This study aimed to investigate the effects of L-Chg10-teixobactin on the viability, proliferation, migration, and osteo/odontogenic differentiation of stem cells from apical papilla (SCAPs). MATERIALS AND METHODS: SCAPs isolated from immature human third molars were treated with various concentrations of L-Chg10-teixobactin, calcium hydroxide, and dimethyl sulfoxide. The viability and proliferation of SCAPs were assessed using the LIVE/DEAD Viability/Cytotoxicity Kit and Cell Counting Kit-8. A scratch wound healing test was used to evaluate the lateral migration capacity of SCAPs. Alkaline phosphatase (ALP) activity, calcium mineralization ability tests -ie, ALP staining and alizarin red S staining, and quantitative real-time polymerase chain reaction were performed to assess the osteo /odontogenic differentiation of SCAPs. RESULTS: The tested concentrations of L-Chg10-teixobactin (0.01, 0.02, and 0.03 mg/mL), 1 mg/mL calcium hydroxide, and 0.03% dimethyl sulfoxide had no significant cytotoxic effect on SCAPs at any time point (P > .05). Besides, there were no significant differences between the control and experimental groups in SCAPs' viability, proliferation, and migration. L-Chg10-teixobactin upregulated the gene expression of osteo/odontogenic markers in SCAPs, while no significant difference was found in the ALP activity and alizarin red S staining. CONCLUSIONS: L-Chg10-teixobactin demonstrated excellent biocompatibility on SCAPs at concentrations from 0.01 to 0.03 mg/mL and potentially enhance the osteo/odontogenic differentiation of SCAPs; suggesting its promising role as root canal medicament for regenerative endodontic procedures.

KDM4D enhances osteo/dentinogenic differentiation and migration of SCAPs via binding to RPS5.

OBJECTIVES: Stem cells of the apical papilla (SCAPs) provide promising candidates for dental pulp regeneration. Despite great advances in the transcriptional controls of the SCAPs fate, little is known about the regulation of SCAP differentiation. MATERIALS AND METHODS: Short hairpin RNAs and full-length RNA were used to deplete or overexpress lysine demethylase 4D (KDM4D) gene expression. Western blotting, real-time RT-PCR, alizarin red staining, and scratch migration assays were used to study the role of KDM4D and the ribosomal protein encoded by RPS5 in SCAPs. RNA microarray, chromatin Immunoprecipitation (ChIP), and co-immunoprecipitation (Co-IP) assays were performed to explore the underlying molecular mechanisms. RESULTS: KDM4D enhanced the osteo/dentinogenic differentiation, migration, and chemotaxis of SCAPs. The microarray results revealed that 88 mRNAs were differentially expressed in KDM4D-overexpressed SCAPs. ChIP results showed knock-down of KDM4D increased the level of H3K9me2 and H3K9me3 in CNR1 promoter region. There were 37 possible binding partners of KDM4D. KDM4D was found to combine with RPS5, which also promoted the osteo/dentinogenic differentiation, migration, and chemotaxis of SCAPs. CONCLUSIONS: KDM4D promoted the osteo/dentinogenic differentiation and migration potential of SCAPs in combination with RPS5, which provides a therapeutic clue for improving SCAPs-based dental tissue regeneration.

Dental Stem Cells SV40, a new cell line developed in vitro from human stem cells of the apical papilla.

AIM: To establish and fully characterize a new cell line from human stem cells of the apical papilla (SCAPs) through immortalization with an SV40 large T antigen. METHODOLOGY: Human SCAPs were isolated and transfected with an SV40 large T antigen and treated with puromycin to select the infected population. Expression of human mesenchymal surface markers CD73, CD90 and CD105 was assessed in the new cell line named Dental Stem Cells SV40 (DSCS) by flow cytometry at early and late passages. Cell contact inhibition and proliferation were also analysed. To evaluate trilineage differentiation, quantitative polymerase chain reaction and histological staining were performed. RESULTS: DSCS cell flow cytometry confirmed the expression of mesenchymal surface markers even in late passages [100% positive for CD73 and CD90 and 98.9% for CD105 at passage (P) 25]. Fewer than 0.5% were positive for haematopoietic cell markers (CD45 and CD34). DSCS cells also showed increased proliferation when compared to the primary culture after 48 h, with a doubling time of 23.46 h for DSCS cells and 40.31 h for SCAPs, and retained the capacity to grow for >45 passages (150 population doubling) and their spindle-shaped morphology. Trilineage differentiation potential was confirmed through histochemical staining and gene expression of the chondrogenic markers SOX9 and COL2A1, adipogenic markers CEBPA and LPL, and osteogenic markers COL1A1 and ALPL. CONCLUSIONS: The new cell line derived from human SCAPs has multipotency, retains its morphology and expression of mesenchymal surface markers and shows higher proliferative capacity even at late passages (P45). DSCS cells can be used for in vitro study of root development and to achieve a better understanding of the regenerative mechanisms.

Apical Papilla Regulates Dental Follicle Fate via the OGN-Hh Pathway.

Root apical complex, including Hertwig's epithelial root sheath, apical papilla, and dental follicle (DF), is the germinal center of root development, wherein the DF constantly develops into periodontal tissue. However, whether DF development is regulated by the adjacent apical papilla remains largely unknown. In this study, we employed a transwell coculture system and found that stem cells from the apical papilla (SCAPs) inhibit the differentiation and maintain the stemness of dental follicle stem cells (DFSCs). Meanwhile, partial SCAP differentiation markers were upregulated after DFSC coculture. High-throughput RNA sequencing revealed that the Hedgehog (Hh) pathway was significantly downregulated in DFSCs cocultured with SCAPs. Upregulation or downregulation of the Hh pathway can respectively activate or inhibit the multidirectional differentiation of DFSCs. Osteoglycin (OGN) (previously known as mimecan) is highly expressed in the dental papilla, similarly to Hh pathway factors. By secreting OGN, SCAP regulated the stemness and multidirectional differentiation of DFSCs via the OGN-Hh pathway. Finally, Ogn-/- mice were established using the CRISPR/Cas9 system. We found that the root length growth rate was accelerated during root development from PN0 to PN30 in Ogn-/- mice. Moreover, the hard tissues (including dentin and cementum) of the root in Ogn-/- mice were thicker than those in wild-type mice. These phenotypes were likely due to Hh pathway activation and the increased cell proliferation and differentiation in both the apical papilla and DF. The current work elucidates the molecular regulation of early periodontal tissue development, providing a theoretical basis for future research on tooth root biology and periodontal tissue regeneration.

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.

BACKGROUND: This 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 METHODS: SCAPs 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. RESULTS: The 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). CONCLUSION: Diode low level laser irradiation with 4 J/cm2 energy density and red LED irradiation enhanced osteogenic differentiation of SCAPs without adversely affecting cell viability.

The Effect of Octenidine on Proliferation, Migration, and Osteogenic Differentiation of Human Dental Pulp and Apical Papilla Stem Cells.

INTRODUCTION: The research for alternative irrigating solutions is ongoing, since no "ideal" solution has yet been found. Octenidine dihydrochloride (OCT) has been indicated as an endodontic irrigant because it has adequate antimicrobial and biological properties. The present study aimed to assess the effects of OCT on proliferation, migration, and induction of the osteogenic phenotype of stem cells from human dental pulp and apical papilla. METHODS: Cells were collected from human third molars and exposed to different doses of OCT, chlorhexidine (CHX), sodium hypochlorite (NaOCl), and ethylenediaminetetraacetic acid (EDTA) to determine cell viability by alamarBlue assay; proliferation by bromodeoxyuridine incorporation; migration by the Transwell assay; alkaline phosphatase activity by thymolphthalein release; and production of mineralized nodules by alizarin red staining. The results were analyzed by 1- or 2-way analysis of variance and Tukey (α = .05). RESULTS: CHX promoted lower cell viability, followed by OCT, NaOCl, and EDTA, especially at intermediate doses (P < .05). Cells exposed to CHX had less proliferation than the other groups (P < .05). The Transwell assay revealed no differences among OCT, EDTA, and culture medium (control group) (P > .05). OCT and EDTA induced greater migration than CHX and NaOCl (P < .05). OCT and EDTA induced higher alkaline phosphatase activity than NaOCl and CHX (P < .05). No difference was detected among the groups using alizarin red staining (P > .05). CONCLUSIONS: OCT induced high migration, proliferation, and alkaline phosphatase activity of stem cells from human dental pulp and apical papilla, which could be advantageous for regenerative endodontic procedures.

The glycoprotein Wnt6 regulates human dental papilla cells differentiation by canonical Wnt signaling pathway.

OBJECTIVE: The aim of this study was to test the hypothesis in vitro and in vivo, that the glycoprotein Wnt6 can regulate human dental papilla cell differentiation by ß-catenin signaling. DESIGN: The expression of Wnt6 was detected by quantitative polymerase chain reaction (qPCR). Wnt6 stealth RNA was used to knockdown the expression of Wnt6. The Wnt canonical signaling was detected by immunofluorescence staining, qPCR, and TOPflash/FOPflash dual-luciferase reporter assay. The differentiation was investigated by alkaline phosphatase staining or Alizarin Red staining after osteo/odontogenic medium culture and by Masson trichrome staining after subcutaneous transplantation. There are at least three samples in one group for each experiment. RESULTS: Wnt6 protein and mRNA were high expressed in dental mesenchyme tissue and cells. In human dental papilla cells, Wnt6 over-expression could activate ß-catenin dependent pathway, including ß-catenin accumulation in cell nuclei, lymphoid enhancer factor 1 mRNA up-regulation, and enhanced ß-catenin transcriptional activity. Wnt6 activated ß-catenin pathway in a similar way to Wnt3a but at a lower level. Wnt6 inhibited human dental papilla cells differentiation as alkaline phosphatase activity in vitro, and promoted differentiation as mineralization after subcutaneous transplantation in vivo, as same trend as Wnt3a but at a lower level. The Wnt/ß-catenin inhibitor XAV939 treatment attenuated Wnt6- or Wnt3a-induced human dental papilla cells mineralization. CONCLUSIONS: Wnt6 activated ß-catenin dependent pathway and regulated human dental papilla cells differentiation. Potential mechanism of Wnt6-regulated cell differentiation is the activation of Wnt/ß-catenin signaling pathway.

Efficacy of platelet-rich fibrin and connective tissue graft in papilla reconstruction.

PURPOSE: To evaluate efficacy of platelet-rich fibrin (PRF) or connective tissue graft (CTG) in papilla reconstruction (PR) with the semilunar incision (SI) technique. MATERIALS AND METHODS: The analysis consisted of 55 sites (27 CTG and 28 PRF) from 20 patients who underwent PR with either PRF or CTG placed in the maxillary anterior region with SI technique. Baseline (BL) and follow-up (T1 , first month, T3 , third month, T6 , sixth month) clinical data including periodontal evaluations (gingival index (GI), plaque index (PI), pocket depth (PD), keratinized tissue width (KTW), gingival recession), papilla-associated recordings (alveolar crest-interdental contact point [AC-IC], alveolar crest-papilla tip [AC-PT], papilla tip-interdental contact point [PT-IC], papilla height loss [PHL], interdental tissue stroke [ITS] and papilla presence index [PPI]) and patient satisfaction were analyzed. RESULTS: CTG provided better PR outcomes. GI, PI, and PD showed a slight increase at T1 and then, turned to their BL levels. The other periodontal parameters showed significant improvement after both treatment modalities. No inter-group difference was found except for KTW, which was in favor of CTG. CONCLUSION: Based on the results, CTG is recommended over PRF in PR treatment due to its superior outcomes with less recurrence risk. CLINICAL SIGNIFICANCE: Connective tissue graft provides superior results than platelet-rich fibrin in papilla reconstruction with the semilunar incision technique.

PRMT6/LMNA/CXCL12 signaling pathway regulated the osteo/odontogenic differentiation ability in dental stem cells isolated from apical papilla.

Tooth loss and maxillofacial bone defect are common diseases, which seriously affect people's health. Effective tooth and maxillofacial bone tissue regeneration is a key problem that need to be solved. In the present study, we investigate the role of PRMT6 in osteo/odontogenic differentiation and migration capacity by using SCAPs. Our results showed that knockdown of PRMT6 promoted the osteo/odontogenic differentiation compared with the control group, as detected by alkaline phosphatase activity, alizarin red staining, and the indicators of osteo/odontogenic differentiation measured by Western blot. In addition, overexpression of PRMT6 inhibited the osteo/odontogenic differentiation potentials of SCAPs. Then, knockdown of PRMT6 promoted the migration ability and overexpression of PRMT6 inhibited the migration ability in SCAPs. Mechanically, we discovered that the depletion of PRMT6 promoted the expression of CXCL12 by decreasing H3R2 methylation in the promoter region of CXCL12. In addition, PRMT6 formed a protein complex with LMNA, a nuclear structural protein. Depletion of LMNA inhibited the osteo/odontogenic differentiation and CXCL12 expression and increased the intranucleus PRMT6 in SCAPs. To sum up, PRMT6 might inhibit the osteo/odontogenic differentiation and migration ability of SCAPs via inhibiting CXCL12. And LMNA might be a negative regulator of PRMT6. It is suggested that PRMT6 may be a key target for SCAP-mediated bone and tooth tissue regeneration.

Comparative Effects of Concentrated Growth Factors on the Biological Characteristics of Periodontal Ligament Cells and Stem Cells from Apical Papilla.

INTRODUCTION: During cell-free regenerative endodontic therapy, both stem cells from apical papilla (SCAPs) and periodontal ligament cells (PDLCs) are possible cell sources because of their proximity. Nonetheless, the regenerative ability of PDLCs and SCAPs under the induction of concentrated growth factors (CGFs) remains unclear. METHODS: PDLCs and SCAPs were treated with various concentrations of CGF-conditioned medium (CCM). The effects of CCM with or without Porphyromonas gingivalis lipopolysaccharide (LPS) on cell migration, odonto/osteogenic differentiation, and the expression of inflammatory cytokines were assessed. Dentin matrix transplants composed of PDLCs or SCAPs cell sheets coupled with CGF were put subcutaneously in immunocompromised mice for 8 weeks to explore their regenerative characteristics in vivo. RESULTS: CCM dose dependently enhanced the migration, proliferation, and odonto/osteogenic differentiation of PDLCs and SCAPs. CCM alleviated LPS-inhibited odonto/osteogenic differentiation of PDLCs and SCAPs as well as the LPS-induced up-regulation of inflammatory cytokines. In vivo, the newly regenerated tissue and microvessels formed by PDLCs and SCAPs were significantly increased under the induction of CGF. SCAPs mainly regenerated pulp/dentinlike tissues and a large number of microvessels, whereas PDLCs mainly formed bone/cementumlike structures. CONCLUSIONS: Overall, PDLCs excelled in cell proliferation, migration, and osteogenic differentiation, whereas SCAPs outperformed PDLCs in terms of angiogenic and odontogenic differentiation. The biological differences between PDLCs and SCAPs provided a possible theoretical basis for the formation of bone/cementum/periodontal ligament-like tissues after cell-free regenerative endodontic therapy.