Apical Papilla Research Articles

In vitro and in vivo evaluation of iRoot BP Plus as a coronal sealing material for regenerative endodontic procedures

ObjectivesTo investigate in vitro effects of a nanoparticle bioceramic material, iRoot BP Plus, on stem cells from apical papilla (SCAP) and in vivo capacity to induce pulp-dentin complex formation. Materials and methodsThe sealing ability of iRoot BP Plus was measured via scanning electron microscopy (SEM). SCAP were isolated and treated in vitro by iRoot BP Plus conditioned medium, with mineral trioxide aggregate (MTA) conditioned medium and regular medium used as controls, respectively. Cell proliferation was assessed by BrdU labeling and MTT assay and cell migration was evaluated with wound healing and transwell assays. Osteo/odontogenic potential was evaluated by Alizarin red S staining and qPCR. Pulp-dentin complex formation in vivo was assessed by a tooth slice subcutaneous implantation model.ResultsiRoot BP Plus was more tightly bonded with the dentin. There was no difference in SCAP proliferation between iRoot BP Plus and control groups (P > 0.05). iRoot BP Plus had a greater capacity to elevated cell migration (P < 0.05) and osteo/odontogenic marker expression and mineralization nodule formation of SCAP compared with MTA groups (P < 0.05). Furthermore, the new continuous dentine layer and pulp-like tissue was observed in the iRoot BP Plus group in vivo.ConclusionsiRoot BP Plus showed excellent sealing ability, promoted the migration and osteo/odontogenesis of SCAP and induced pulp-dentin complex formation without affecting the cell proliferation, which indicated iRoot BP Plus was a promising coronal sealing material in REPs.Clinical relevanceThe coronal sealing materials play crucial roles for the outcomes of REPs. This study showed that iRoot BP Plus has good coronal sealing and promote pulp-dentin complex formation compared with MTA, providing experimental evidences for the clinical application of iRoot BP Plus as a promising coronal seal material in REPs.

The effect of injectable platelet-rich fibrin and platelet-rich fibrin in regenerative endodontics: a comparative in vitro study.

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). 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. 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). 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.

Effect of circular RNA hsa_circ_0008016 on the Osteogenic and Odontogenic Ability of Stem Cells from Apical Papilla

Effect of circular RNA hsa_circ_0008016 on the Osteogenic and Odontogenic Ability of Stem Cells from Apical Papilla

The Differentiation Potential of Apical Papilla Cells in Relation to Tenascin-C and Syndecan-1 Expression and Their Potential Role in Regeneration.

Introduction: This study investigated the distribution pattern of tenascin-C and syndecan-1 in the dental mesenchyme during root development of immature swine teeth in order to define the differentiation dynamics of both pulp tissue progenitors and apical papilla cells, as well as to assess the adequacy criticize of the apical papilla to induce dentin-pulp regeneration. Methods: Three 7-month-old miniature swine were used in this study. A total of 12 teeth, including two immature permanent incisors and two premolar teeth of each case, were extracted and processed for histological and immunohistochemical analysis. Different populations of mesenchymal cells located at the root apex were morphologically evaluated in hematoxylin-eosin serial sections. Additionally, the distribution patterns of tenascin-C and syndecan-1 were assessed immunohistochemically. Results: Syndecan-1 was strongly expressed in the dental pulp, particularly along the odontoblasts of the root and the newly deposited predentin layer. Tenascin-C was intensely expressed in the dental pulp. The apical papilla and dental follicle showed no expression of either molecule. Conclusions: Cell differentiation potential in the developing swine apex is progressively restricted to the newly formed dental pulp, whereas phenotypic expression of apical papilla cells remains undetermined unless the new microenvironment triggers cell differentiation towards the odontoblastic lineage.

The potential use of ascorbic acid to recover the cellular senescence of lipopolysaccharide-induced human apical papilla cells: an in vitro study.

To examine the effect of lipopolysaccharide (LPS) on cellular senescence induction of human apical papilla cells (hAPCs) and evaluate the potential use of 50 μg/ml ascorbic acid to recover cellular senescence and regenerative functions. hAPCs were treated with LPS at 1 and 10 μg/ml either with or without 50 μg/ml ascorbic acid for 48 h. The cellular senescence biomarkers were analyzed by senescence-associated β-galactosidase (SA-β-gal) staining and senescence-related gene expression, p16 and p21. Cell migration, at 12 h and 24 h, was evaluated using a scratch wound assay. Mineralization potential was assessed at 21 days using Alizarin red S staining and dentine sialophosphoprotein (DSPP) and bone sialoprotein (BSP) gene expression. 1 μg/ml and 10 μg/ml LPS stimulation for 48 h induced cellular senescence, as shown by remarkable SA-β-gal staining and p16 and p21 gene expression. The percentage of wound closure and mineralized formation was reduced. The co-incubation with ascorbic acid significantly down-regulated the level of SA-β-gal staining. The reduction of senescence-associated gene expressions was observed. Ascorbic acid improved cell migration, mineralized nodule formation, and the expression of DSPP and BSP genes in LPS-treated hAPCs. LPS significantly promoted cellular senescence on hAPCs and diminished the cell function capacity. Co-presence of ascorbic acid could impede cellular senescence and possibly improve the regenerative capacity of LPS-induced senescent hAPCs in vitro. The data support the in vitro potential benefit of ascorbic acid on cellular senescence recovery of apical papilla cells.

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

ObjectiveThe 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. DesignSCAPs 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. ResultsLPS 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. ConclusionIn 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.

A comparative study of biological properties of three root canal sealers.

The aim of this study was to evaluate the effects of Hiflow with other two kinds of root canal sealers on the biological behavior of stem cells from the apical papilla (SCAP), the influence on inflammatory cytokines release and its antibacterial effects. Material extracts of Hiflow, iRoot SP, and AH Plus were prepared. Then, SCAP was incubated with extracts. The effects were evaluated by CCK-8, wound healing assay, ALP staining, alizarin red staining, and qRT-PCR. Meanwhile, polymorphonuclears (PMNs) and monocytes were isolated and treated with extracts for 4h and 24h respectively. Cell viability was analyzed by Annexin-V/PI double staining flow cytometry. The effects on the release of cytokines were observed by ELISA. The antibacterial effects of different sealers were tested against three kinds of bacteria found in chronic apical periodontitis. A series of results of SCAP showed that Hiflow and iRoot SP could promote cell proliferation, migration, and osteogenesis (p < 0.05). Although Hiflow was associated with greater cell apoptosis and necrosis when incubated with PMNs and monocytes (p < 0.05), it had an approximate release of anti-inflammatory cytokines with iRoot SP, which was higher than AH plus (p < 0.05). The co-culture showed that Hiflow and iRoot SP inhibited the colony formation of F. nucleatum (p < 0.05). However, both sealers had no obvious antibacterial effect on E. faecalis and P. gingivalis (p > 0.05). In summary, Hiflow and iRoot SP both had positive biological stimulus on SCAP. Meanwhile, Hiflow showed a better induction on anti-inflammatory cytokines over the others. All the properties mentioned above and its antibacterial effect of F. nucleatum promise Hiflow a bright application prospect in endodontic uses. References for clinical work to use BC Sealer Hiflow as a good biological root canal sealer.

Electrospun dimethyloxallylglycine sustained release scaffold for promoting the migration and multidirectional differentiation of stem cells from the apical papilla

Electrospun dimethyloxallylglycine sustained release scaffold for promoting the migration and multidirectional differentiation of stem cells from the apical papilla

Polymeric nanoparticles delivery circumvents bacterial resistance to ciprofloxacin

Polymeric nanoparticles delivery circumvents bacterial resistance to ciprofloxacin

Nanodispersion of TiO2 in hypochlorous acid and its antimicrobial effect against oral pathogens

The continuous and inappropriate use of traditionally used antimicrobial agents has caused the rise of multidrug-resistant (MDR) bacterial strains and the mutation of microorganisms in the field of dentistry. Therefore, various nanoparticles have been developed to combat resistant pathogens. Titanium dioxide (TiO2) nanoparticles have been attractive antimicrobial agents because of their chemical stability, non-toxicity, and inexpensive precursors. Therefore, TiO2-based nanodispersions were explored by preparing them with well-known antimicrobial agents, such as hypochlorous acid (HOCl), to enhance the antimicrobial effect. In this study, sol-gel-based TiO2 NPs-HOCl nanodispersions were synthesized and characterized. The antimicrobial effect was assessed by a microdilution assay using S. mutans, S. aureus, E. faecalis, and C. albicans strains by incubating different concentrations of the nanodispersions. To evaluate the cytotoxic effects, stem cells from the apical papilla (SCAPs) were inoculated and evaluated using the MTT assay. The nanodispersion exhibited an enhanced antimicrobial effect, with almost no cytotoxicity. The HOCl-based nanodispersion exhibited a greater antimicrobial effect and high stability. Thus, it can be used as a promising antimicrobial agent for the treatment of various dental pathogens.

Gelatin-Chitosan Hydrogel Biological, Antimicrobial and Mechanical Properties for Dental Applications.

To investigate the mechanical properties of gelatin hydrogels based on decaethylated chitosan and antimicrobial activity against Streptococcus mutans and their biological effects with stem cells from apical papilla (SCAPs). Gelatin-chitosan hydrogels were synthesized at concentrations of 0%, 0.2% and 0.5%. Enzymatic and hydrolytic degradation, along with swelling capacity, was assessed. Fourier transform infrared spectroscopy (FTIR) analysis was employed to characterize the hydrogels. The interaction between hydrogels and SCAPs was examined through initial adhesion and cell proliferation at 24 and 48 h, using the Thiazolyl Blue Tetrazolium Bromide (MTT assay). The antimicrobial effect was evaluated using agar diffusion and a microdilution test against S. mutans. Uniaxial tensile strength (UTS) was also measured to assess the mechanical properties of the hydrogels. The hydrogels underwent hydrolytic and enzymatic degradation at 30, 220, 300 min and 15, 25, 30 min, respectively. Significantly, (p < 0.01) swelling capacity occurred at 20, 40, 30 min, respectively. Gelatin-chitosan hydrogels' functional groups were confirmed using vibrational pattern analysis. SCAPs proliferation corresponded to 24 h = 73 ± 2%, 82 ± 2%, 61 ± 6% and 48 h = 83 ± 11%, 86 ± 2%, 44 ± 2%, respectively. The bacterial survival of hydrogel interaction was found to be 96 ± 1%, 17 ± 1.5% (p < 0.01) and 1 ± 0.5% (p < 0.01), respectively. UTS showed enhanced (p < 0.05) mechanical properties with chitosan presence. Gelatin-chitosan hydrogels displayed favorable degradation, swelling capacity, mild dose-dependent cytotoxicity, significant proliferation with stem cells from apical papilla (SCAPs), substantial antimicrobial effects against S. mutans and enhanced mechanical properties. These findings highlight their potential applications as postoperative care dressings.

Exploring the impact of oral bacteria remnants on stem cells from the Apical papilla: mineralization potential and inflammatory response.

Bacterial persistence is considered one of the main causal factors for regenerative endodontic treatment (RET) failure in immature permanent teeth. This interference is claimed to be caused by the interaction of bacteria that reside in the root canal with the stem cells that are one of the essentials for RET. The aim of the study was to investigate whether prolonged exposure of stem cells from the apical papilla (SCAP) to bacterial remnants of Fusobacterium nucleatum, Actinomyces gerensceriae, Slackia exigua, Enterococcus faecalis, Peptostreptococcaceae yurii, commonly found in infected traumatized root canals, and the probiotic bacteria Lactobacillus gasseri and Limosilactobacillus reuteri, can alter SCAP's inflammatory response and mineralization potential. To assess the effect of bacterial remnants on SCAP, we used UV-C-inactivated bacteria (as cell wall-associated virulence factors) and bacterial DNA. Histochemical staining using Osteoimage Mineralization Assay and Alizarin Red analysis was performed to study SCAP mineralization, while inflammatory and osteo/odontogenic-related responses of SCAPs were assessed with Multiplex ELISA. We showed that mineralization promotion was greater with UV C-inactivated bacteria compared to bacterial DNA. Immunofluorescence analysis detected that the early mineralization marker alkaline phosphatase (ALP) was increased by the level of E. coli lipopolysaccharide (LPS) positive control in the case of UV-C-inactivated bacteria; meanwhile, DNA treatment decreased the level of ALP compared to the positive control. SCAP's secretome assessed with Multiplex ELISA showed the upregulation of pro-inflammatory factors IL-6, IL-8, GM-CSF, IL-1b, neurotrophic factor BDNF, and angiogenic factor VEGF, induced by UV-C-killed bacteria. The results suggest that long term stimulation (for 21 days) of SCAP with UV-C-inactivated bacteria stimulate their mineralization and inflammatory response, while DNA influence has no such effect, which opens up new ideas about the nature of RET failure.

A Potential Intracanal Medicament, 2-Hydroxyisocaproic Acid (HICA): Cytotoxicity, Genotoxicity, and Its Effect on SCAP Differentiation.

Intracanal medicaments with maximal antimicrobial efficacy and minimal damage to resident stem cells are essential for successful regenerative endodontic procedures. 2-Hydroxyisocaproic acid (HICA) could have the attributes of a potential intracanal medicament. This study evaluates its cytotoxicity, genotoxicity, and effects on the odontogenic and osteogenic differentiation of the stem cells of the apical papilla (SCAP). Cytotoxicity and cell viability assays were performed on cells treated for 24, 48, and 72 h with varying concentrations of HICA and compared to the standard intracanal medicament, calcium hydroxide. The genotoxicity was assessed via immunofluorescence for two markers of DNA double-strand breaks: phosphorylated γH2AX and 53BP1. The SCAP differentiation was evaluated based on the alkaline phosphatase activity, Alizarin Red staining, and expression of odontogenic and osteogenic genes (DSPP1, BSP1, OCN, RUNX2) in the presence of selected HICA concentrations. HICA was not cytotoxic at concentrations up to 10 mg/mL, regardless of the exposure time, although it was cytostatic at all tested concentrations. HICA was not genotoxic at concentrations below 5 mg/mL. No difference in cytotoxicity or genotoxicity was found between HICA and calcium hydroxide at 1 mg/mL. HICA retained about 70% of the osteogenic differentiation potential at 1 mg/mL. Within the limitations of this in vitro study, we show that HICA at 1 mg/mL could be a potential intracanal medicament for REPs.

Dental stem cells and their applications in pediatric dentistry

Regenerative medicine has garnered significant attention due to its transformative impact on disease management and the restoration of optimal bodily functions. Stem cells, a cornerstone of regenerative medicine, are particularly renowned for their remarkable capacity to facilitate tissue regeneration and repair, revolutionizing modern healthcare by offering diverse treatment avenues for multiple conditions. Dental pulp stem cells (DPSCs), a subset of postnatal stem cells, are especially noteworthy for their extensive proliferation and ability to differentiate into various specialized cell types. In the realm of pediatric dentistry, there exists a pressing need for advancements in regenerative medicine. Thorough research in this domain has the potential to propel evidence-based pediatric dentistry, ensuring that children receive tailored, high-quality care. Stem cells hold immense promise in pediatric dentistry by offering less invasive and regenerative solutions for a range of childhood dental issues and congenital anomalies. This research, initiated on 04 October 2023, was instigated following an exhaustive review of existing literature utilizing databases like PubMed, Web of Science, and Cochrane. The literature search encompassed a wide array of medical terminologies. Dental stem cells are categorized into four major types: DPSCs, stem cells from human exfoliated deciduous teeth (SHED), stem cells of the apical papilla (SCAP), and periodontal ligament stem cells (PDLSCs). Pediatric dentistry, encompassing endodontics, orthodontics, periodontics, and the treatment of craniofacial defects, stands to benefit significantly from the potential of dental stem cells. The preservation of dental pulp stem cells extracted from deciduous and permanent teeth through tooth banking offers a source for future regenerative therapies. The preservation process involves multiple comprehensive steps. In summary, dental stem cells present a promising avenue within pediatric dentistry, offering multifaceted applications ranging from pulpal regeneration and dentin repair to orthodontic support and the treatment of craniofacial anomalies.

Differential expression of circular RNAs in interleukin 6-promoted osteogenic differentiation of human stem cells from apical papilla.

Studies have shown that interleukin 6 (IL-6) can regulate stem cell osteogenic differentiation; however, the exact mechanism is not clear. Circular RNAs (circRNAs) are closed circular non-coding RNAs that are involved in the process of stem cell osteogenic differentiation. Therefore, the purpose of this present study was to investigate the effect of IL-6 treatment on osteogenic differentiation of human apical tooth papillae stem cells (hSCAPs), and to detect the difference in circRNA expression using gene microarray technology. After extraction and identification of hSCAPs, alkaline phosphatase (ALP) activity, alizarin red staining, and calcium ion quantitative assay were used to determine the changes of ALP enzyme, mineralized nodules, and matrix calcium levels before and after IL-6 treatment of hSCAPs gene microarray technology was used to analyze the changes in circRNA expression levels before and after IL-6 induction of mineralization. The four selected circRNAs were validated by qRT-PCR. Moreover, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to predict the potential functions and biological signaling pathways of circRNAs. Finally, these data are integrated and analyzed to construct circRNA-microRNA-mRNA networks. Alp and Alizarin red staining confirmed that IL-6 promoted the osteogenic differentiation of hSCAPs. The gene microarray results identified 132 differentially expressed circRNAs, of which 117 were upregulated and 15 were downregulated. Bioinformatic analysis predicted that the circRNA-406620/miR-103a-3p/FAT atypical cadherin 4 (FAT4) pathway might be involved in regulating IL-6 to promote osteogenic differentiation of hSCAPs. Differentially expressed circRNAs might be closely involved in regulating IL-6 to promote osteogenic differentiation of hSCAPs.

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.

Osteogenic potential of apical papilla stem cells mediated by platelet-rich fibrin and low-level laser

To evaluate the osteogenic potential of platelet-rich fibrin (PRF) and low-level laser therapy (LLLT) on human stem cells from the apical papilla (SCAP) we isolated, characterized, and then cultured in an osteogenic medium cells with PRF and/or LLLT (660 nm, 6 J/m2-irradiation). Osteogenic differentiation was assessed by bone nodule formation and expression of bone morphogenetic proteins (BMP-2 and BMP-4), whereas the molecular mechanisms were achieved by qRT-PCR and RNA-seq analysis. Statistical analysis was performed by ANOVA and Tukey’s post hoc tests (p < 0.05* and p < 0.01**). Although PRF and LLLT increased bone nodule formation after 7 days and peaked at 21 days, the combination of PRF + LLLT led to the uppermost nodule formation. This was supported by increased levels of BMP-2 and -4 osteogenic proteins (p < 0.005). Furthermore, the PRF + LLLT relative expression of specific genes involved in osteogenesis, such as osteocalcin, was 2.4- (p = 0.03) and 28.3- (p = 0.001) fold higher compared to the PRF and LLLT groups, and osteopontin was 22.9- and 1.23-fold higher, respectively (p < 0.05), after 7 days of interaction. The transcriptomic profile revealed that the combination of PRF + LLLT induces MSX1, TGFB1, and SMAD1 expression, after 21 days of osteogenic differentiation conditions exposition. More studies are required to understand the complete cellular and molecular mechanisms of PRF plus LLLT on stem cells. Overall, we demonstrated for the first time that the combination of PRF and LLLT would be an excellent therapeutic tool that can be employed for dental, oral, and craniofacial repair and other tissue engineering applications.

Expression of Toll-like Receptors in Stem Cells of the Apical Papilla and Its Implication for Regenerative Endodontics.

Regenerative therapies to replace cells and tissues damaged due to trauma and dental infections require temporal and spatial controlled recruitment and the differentiation of progenitor/stem cells. However, increasing evidence shows microbial antigens can interfere with this process. Toll-like receptors (TLRs) are crucial in recognizing pathogen-associated molecular patterns. Stem cells of the apical papilla (SCAP) are required for normal dental development and are intimately involved in the reparative and regenerative capacity of developing teeth. We hypothesized that TLRs are expressed in SCAP and that the activation of TLR2/TLR4 or TLR3 by different ligands results in differential cellular fate, impacting their differentiation into a mineralizing phenotype. We found that most TLRs are expressed as detected by PCR except TLR7 and TLR8; exposure to heat-killed E. coli results in upregulating TLR2 and TLR4 and reducing mineralization capacity. In addition, bacterial exposure resulted in the upregulation of 11 genes, of which 9 were chemokines whose proteins were also upregulated and released, promoting in vitro macrophage migration. On the other hand, TLR3 activation resulted in increased proliferation and a dramatic inhibition of osteogenic and odontoblastic differentiation, which was reversed by inhibition or the knockdown of TLR3 expression. The profound effects of TLR activation resulting in different cell fates that are ligand and receptor-specific warrants further evaluation and represents an important therapeutic target to make regenerative approaches more predictable following dental infections.

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

IntroductionStem 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. MethodsSCAPs 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 β-tubulin III, alpha-smooth muscle actin, and CD31 were used to study neural gene expression, neurite formation, and neurovascular cell interactions. ResultsNeural induction medium with small molecules rapidly induced SCAP differentiation toward neural-like cells. Gene expression of Nestin, β-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. ConclusionsiSCAP-formed spheres interact with SCAPs and HUVECs, promoting vasculogenesis and neurogenesis.

MLL1 inhibits the neurogenic potential of SCAPs by interacting with WDR5 and repressing HES1

Mesenchymal stem cell (MSC)-based therapy has emerged as a promising treatment for spinal cord injury (SCI), but improving the neurogenic potential of MSCs remains a challenge. Mixed lineage leukemia 1 (MLL1), an H3K4me3 methyltransferases, plays a critical role in regulating lineage-specific gene expression and influences neurogenesis. In this study, we investigated the role and mechanism of MLL1 in the neurogenesis of stem cells from apical papilla (SCAPs). We examined the expression of neural markers, and the nerve repair and regeneration ability of SCAPs using dynamic changes in neuron-like cells, immunofluorescence staining, and a SCI model. We employed a coimmunoprecipitation (Co-IP) assay, real-time RT-PCR, microarray analysis, and chromatin immunoprecipitation (ChIP) assay to investigate the molecular mechanism. The results showed that MLL1 knock-down increased the expression of neural markers, including neurogenic differentiation factor (NeuroD), neural cell adhesion molecule (NCAM), tyrosine hydroxylase (TH), βIII-tubulin and Nestin, and promoted neuron-like cell formation in SCAPs. In vivo, a transplantation experiment showed that depletion of MLL 1 in SCAPs can restore motor function in a rat SCI model. MLL1 can combine with WD repeat domain 5 (WDR5) and WDR5 inhibit the expression of neural markers in SCAPs. MLL1 regulates Hairy and enhancer of split 1 (HES1) expression by directly binds to HES1 promoters via regulating H3K4me3 methylation by interacting with WDR5. Additionally, HES1 enhances the expression of neural markers in SCAPs. Our findings demonstrate that MLL1 inhibits the neurogenic potential of SCAPs by interacting with WDR5 and repressing HES1. These results provide a potential therapeutic target for promoting the recovery of motor function in SCI patients.