Stem Cells Of The Apical Papilla Research Articles

Chitosan-based endodontic irrigation solutions and TGF-β1 treatment: Creating the most favourable environment for the survival and proliferation of stem cells of the apical papilla in vitro.

The dental pulp's environment is essential for the regulation of mesenchymal stem cells' homeostasis and thus, it is of great importance to evaluate the materials used in regenerative procedures. To assess invitro (i) the effect of chitosan nanoparticles, 0.2% chitosan irrigation solution, Dual Rinse®, 17% EDTA, 10% citric acid and 2.5% NaOCl on DSCS viability; (ii) the effect of different concentrations of TGF-β1 on DCSC proliferation; and (iii) whether treatment with TGF-β1 following exposure to the different irrigation solutions could compensate for their negative effects. (i) DSCS were treated with three dilutions (1:10, 1:100 and 1:1000) of the six irrigation solutions prepared in DMEM for 10 and 60 min to assess the effect on viability. (ii) The effect of different concentrations (0, 1, 5 and 10 ng/mL) of TGF-β1 on DCSC proliferation was assessed at 1, 3 and 7 days. (iii) The proliferative effect of TGF-β1 following 10-min exposure to 1:10 dilution of each irrigation solution was also tested. We used MTT assay to assess viability and proliferation. We performed statistical analysis using Prism software. (i) The different endodontic irrigation solutions tested showed a significant effect on cell viability (p ≤ .0001). Significant interactions between the endodontic irrigation solutions and their dilutions were also found for all parameters (p ≤ .0001). Chitosan nanoparticles and 0.2% chitosan irrigation solution were the least cytotoxic to DSCS whilst 2.5% NaOCl was the most cytotoxic followed by 17% EDTA. (ii) TGF-β1 at concentrations of 1 and 5 ng/mL resulted in significantly higher proliferation compared to the control group. (iii) Exposure to 17% EDTA or 2.5% NaOCl for 10 min was sufficient to make DSCS cells refractory to the proliferative effects of TGF-β1. DSCS groups treated with TGF-β1 following exposure to chitosan nanoparticles, 0.2% chitosan irrigation solution, Dual Rinse® and 10% CA demonstrated significantly higher proliferation compared to non-TGF-β1-treated groups (p ≤ .0001, p ≤ .0001, p ≤ .0001 and p = .01 respectively). The current study offers data that can be implemented to improve the outcome of regenerative endodontic procedures by using less toxic irrigation solutions and adding TGF-β1 to the treatment protocol.

Effect of magnesium oxide nanoparticles and LED irradiation on the viability and differentiation of human stem cells of the apical papilla.

Currently, regenerative endodontic treatments are gaining more and more attention, and stem cells play a significant role in these treatments. In order to enhance stem cell proliferation and differentiation, a variety of methods and materials have been used. The purpose of this study was to determine the effects of magnesium oxide nanoparticles and LED irradiation on the survival and differentiation of human stem cells from apical papilla. The MTT test was used to measure the cell survival of SCAPs that had been exposed to different concentrations of magnesium oxide nanoparticles after 24 and 48h, and the concentration with the highest cell survival rate was picked for further studies. The cells were classified into four distinct groups based on their treatment: (1) control, which received no exposure, (2) exposure to magnesium oxide nanoparticles, (3) exposure to light emitting diode (LED) irradiation (635nm, 200mW/cm2) for 30s, (4) exposure simultaneously with magnesium oxide nanoparticles and LED irradiation. A green approach was employed to synthesize magnesium oxide nanoparticles. Quantitative real time PCR was used to measure the gene expression of osteo/odontogenic markers such as BSP, DSPP, ALP and DMP1 in all four groups after treatment, and Alizarin red S staining (ARS) was used to determine the osteogenic differentiation of SCAPs by demonstrating the Matrix mineralization. The highest viability of SCAPs was observed after 24h in concentration 1 and 10µg/mL and after 48h in concentration 1µg/mL, which were not significantly different from the control group. In both times, the survival of SCAPs decreased with increasing concentration of magnesium oxide nanoparticles (MgONPs). According to the results of Real-time PCR, after 24 and 48h, the highest differentiation of BSP, DMP1, ALP and DSPP genes was observed in the LED + MgONPs group, followed by MgONPs and then LED, and in all 3 experimental groups, it was significantly higher than control group (P < 0.05). Also, after 24 and 48h, the density of ARS increased in all groups compared to the control group, and the highest density was observed in the MgONPs + LED and MgONPs groups. This research concluded that exposure to SCAPs, MgONPs, and LED irradiation has a significant effect on enhancing gene expression of odontogenic/osteogenic markers and increasing matrix mineralization.

Polymeric nanoparticles delivery circumvents bacterial resistance to ciprofloxacin

Polymeric nanoparticles delivery circumvents bacterial resistance to ciprofloxacin

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.

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.

Tissue Stabilization, Bacterial Adhesion, and Stem Cell Viability in Trans-cinnamaldehyde-conditioned Dentin

IntroductionThis laboratory study aimed to evaluate the effect of trans-cinnamaldehyde (TC) conditioning on dentin tissue stabilization, bacterial adhesion, and stem cell toxicity. MethodsDentin beams (n = 204) from extracted human molars were demineralized in phosphoric acid and treated with TC (2.5, 5, and 7.5%), 50% ethanol-water mixture (vehicle control) or 2.5% glutaraldehyde (GA) (positive control) for 30 minutes. Demineralized but untreated specimens served as the negative control. After treatment, collagen crosslinking was characterized by measuring the elastic modulus (Er) and hardness (n = 5). Biodegradation resistance was examined by determining the loss of dry mass (n = 8), hydroxyproline release (n = 4) and scanning electron microscopy (n = 2), after exposure to bacterial collagenase. Inhibition of bacterial adhesion was investigated by colony counting assay (n = 12) and scanning electron microscopy (n = 2). Viability of stem cells of the apical papilla on TC-conditioned dentin was determined using the Cell Counting Kit-8 assay (n = 8). Data were statistically analyzed using one-way analysis of variance (ANOVA) test followed by Dunnett's multiple comparisons at a significance level of 5%. ResultsTC-conditioned dentin showed a concentration-dependent increase in Er and hardness. The Er and hardness of 5% and 7.5% TC-conditioned dentin were significantly greater than that of the negative control and vehicle control groups (P < .05). There was no significant difference in the biodegradation resistance between GA and 5% TC-conditioned dentin (P > .05). TC-conditioned dentin showed a well-preserved collagen fibril network with clear cross-banding, comparable to GA-conditioned dentin. All concentrations of TC inhibited bacterial adhesion on dentin, significantly greater than the negative control (P < .05). There was no reduction in viability of stem cells of the apical papilla viability on TC-conditioned dentin compared to the negative control (P > .05). ConclusionsTC conditioning stabilized the dentin and protected it from enzymatic degradation. TC prevented bacterial adhesion on the dentin but maintained stem cell viability.

Growth Factors Released from Advanced Platelet-Rich Fibrin in the Presence of Calcium-Based Silicate Materials and Their Impact on the Viability and Migration of Stem Cells of Apical Papilla.

Advanced platelet-rich fibrin (A-PRF) provides the scaffold and growth factors necessary for stem cells to proliferate and differentiate in successful regenerative endodontic procedures. This study investigates the release of transforming growth factor-β1 (TGF-β1), platelet-derived growth factor (PDGF), and vascular endothelial growth factor (VEGF) from A-PRF in cell culture media in the presence and absence of mineral trioxide aggregate (MTA) or Biodentine. Additionally, this research assesses the viability and migration of stem cells of the apical papilla (SCAP) in previously conditioned media. A-PRF obtained from 14 participants were incubated for 7 days in cell culture media alone or via layering with MTA or Biodentine discs and the release of selected growth factors in the media was evaluated using ELISA. The viability of SCAP grown in conditioned media was measured using the CCK8 assay, while SCAP migration was assessed via a transwell assay by counting migrated cells. The release of TGF-β1, PDGF, and VEGF was significantly higher in media with A-PRF alone than in the presence of either calcium-based silicate material (p < 0.05), which showed no difference from the no-A-PRF control (p < 0.05). None of the tested growth factors released in the A-PRF-conditioned media correlated with clot weight. A-PRF-conditioned media, both with and without calcium-based silicate materials, did not impact SCAP viability and migration (p > 0.05). This study shows that SCAP behavior is not impacted by the decrease in growth factor released in the presence of calcium-based silicate materials and that their role in REPs warrants further investigation.

Exosomes from hypoxia-conditioned apical papilla stem cells accelerate angiogenesis in vitro through Notch/JAG1/VEGF signaling

Dental pulp angiogenesis is a committed step in pulp regeneration therapy, and exosomes provide a new cell-free choice for tissue regeneration. This study revealed the underlying regulatory mechanism of exosomes from stem cells of the apical papilla (SCAPs) under hypoxic state on angiogenesis of human umbilical vein endothelial cells (HUVECs) in vitro. Exosomes extracted from normoxia or hypoxia-pretreated SCAPs were co-cultured with HUVECs, and hypoxia pretreatment increased the release of exosomes and the internalization of exosomes by HUVECs. Compared to normoxic SCAPs-derived exosomes, exosomes from hypoxic SCAPs were found to promote cell proliferation and migration in HUVECs, as it was respectively determined by Cell Counting Kit-8, RT-qPCR and Transwell assay. Besides, hypoxia-educated SCAPs-exosomes especially enhanced the angiogenesis abilities of HUVECs in vitro, which were confirmed by tube formation assay and RT-qPCR detection of angiogenesis-related molecular markers. Interestingly, we found that the hypoxia inducible factor-1α (HIF-1α)/Notch1 signaling pathway was activated in hypoxic SCAPs, and protein jagged-1 (JAG1) was delivered by hypoxic SCAPs-derived exosomes to increase vascular endothelial growth factor (VEGF) production in HUVECs. Moreover, exogenous interference of JAG1 expression in HUVECs partially neutralized the activities of hypoxic SCAPs-exosomes in promoting cell proliferation, migration and tube formation of HUVECs. In summary, this study elucidates that exosomes from hypoxic SCAPs shows high potential to promote angiogenesis in vitro through the HIF-1α/JAG1/VEGF signaling cascade, which may provide a new perspective for the development of vascular reconstruction measures during dental regeneration engineering.

METTL3 Promotes Osteo/Odontogenic Differentiation of Stem Cells by Inhibiting miR-196b-5p Maturation.

Mesenchymal stem cells (MSCs) have been considered a potential method for the regeneration of tooth and maxillofacial bone defects based on the multidirectional differentiation characteristics of MSCs. miRNAs have been found to play a key role in the differentiation of MSCs. However, its effectiveness still needs to be improved, and its internal mechanism is still unclear. In the present study, our data discovered that the knockdown of miR-196b-5p promoted alkaline phosphatase (ALP) activity assay, mineralization in vitro, and expressions of osteo/odontogenic differentiation markers DSPP and OCN and enhanced in vivo osteo/odontogenic differentiation of stem cells of the apical papilla (SCAPs). Mechanistically, the results indicated that METTL3-dependent N6-methyladenosine (m6A) methylation inhibited miR-196b-5p maturation by the microprocessor protein DGCR8. Moreover, miR-196b-5p indirectly negatively regulates METTL3 in SCAPs. Then, METTL3 was found to strengthen the ALP activity assay, mineralization, and expressions of osteo/dentinogenic differentiation markers. Taken together, our findings highlight the critical roles of the METTL3-miR-196b-5p signaling axis in an m6A-dependent manner in osteo/odontogenic differentiation of SCAPs, identifying some potential targets for tooth and maxillofacial bone defects.

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

ObjectiveTo 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. MethodsSCAPs 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. ResultsAll 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 ConclusionP11–4 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 significanceBased 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.

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

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. 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. 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. 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.

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. 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. 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. 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.

PEG–PLGA nanoparticles for encapsulating ciprofloxacin

Antibiotic medications have been found to hinder the success of regenerative endodontic treatment due to the rapid degradation of the drug, and the acidic nature of ciprofloxacin (CIP) can be harmful to stem cells of the apical papilla (SCAPs), the cells responsible for regeneration. In this study, a nanocarrier system was used for controlled drug release for longer drug activity and less cytotoxicity to the cells. CIP was loaded in poly (ethylene glycol) methyl ether-block-poly (lactide-co-glycolide) (PEG–PLGA) nanoparticles (NPs) with an ion-pairing agent. The NPs demonstrated a monodispersed spherical morphology with a mean diameter of 120.7 ± 0.43 nm. The encapsulation efficiency of the CIP-loaded PEG–PLGA NPs was 63.26 ± 9.24%, and the loading content was 7.75 ± 1.13%. Sustained CIP release was achieved over 168 h and confirmed with theoretical kinetic models. Enhanced NP bactericidal activity was observed against Enterococcus faecalis. Additionally, CIP-loaded PEG–PLGA NPs had a low cytotoxic effect on SCAPs. These results suggest the use of a nanocarrier system to prolong the antibiotic activity, provide a sterile environment, and prevent reinfection by the bacteria remaining in the root canal during regenerative endodontic treatment.

Endocannabinoids Modulate Production of Osteoclastogenic Factors by Stem Cells of the Apical Papilla In Vitro

IntroductionMany mediators are produced during pulp inflammation and necrosis, including endocannabinoids (ECbs), which might affect the function of stem cells of the apical papilla (SCAP), cells of paramount importance for root formation, and regenerative endodontic treatment. The aim of this study was to evaluate the production of osteoclastogenesis-related mediators by SCAP modulated by ECbs and lipopolysaccharide (LPS) in vitro. MethodsSCAP were cultured and treated with ECb anandamide (AEA), 2-arachidonoylglycerol, or N-arachidonoylaminophenol. All groups were incubated in the presence of a vehicle or LPS and the antagonist of transient receptor potential cation channel subfamily V member 1, capsazepine. After 24 hours, the culture medium supernatants were collected for further quantification of tumor necrosis factor alpha, CCL2, macrophage colony-stimulating factor, osteoprotegerin, and receptor activator of nuclear factor kappa B ligand. ResultsSmall amounts of tumor necrosis factor alpha and receptor activator of nuclear factor kappa B ligand were detected in SCAP supernatants, and none of the experimental conditions altered their production. A down-regulation in constitutive CCL2 production was observed in the AEA group compared with that in the LPS group. The production of macrophage colony-stimulating factor was significantly increased in all groups treated with AEA compared with the control and LPS-treated groups. Osteoprotegerin was significantly increased by AEA alone and by 2-arachidonoylglycerol and N-arachidonoylaminophenol in the presence of LPS and capsazepine. ConclusionsAEA modulates some of the osteoclastogenic factors produced by SCAP in a bone resorption protective fashion.

N-Acetylcysteine Protects the Stem Cells of the Apical Papilla

ObjectivePrevious research has demonstrated that stem cells of the apical papilla (SCAP) have a lower differentiation potential and are less resistant to cell death as compared to other stem cells. N-acetyl cysteine (NAC) prevents apoptosis of the dental pulp stem cells (DPSCs) by inducing differentiation of these cells. The use of NAC with SCAP could possibly, enhance their differentiation and resistance to cytotoxicity. Hence, the aim of this study was to determine if NAC could prevent apoptosis of SCAP by promoting proliferation and differentiation of these cells thereby contributing to the success of Regenerative endodontic procedures (REPs).MethodsHuman SCAP were cultured with and without 2-hydroxyethyl methacrylate (HEMA), 20 mM NAC and Dexamethasone (Dex). Proliferation rates were analyzed at days 4 and 7. Flow cytometric analysis was used to analyze the levels of cell death. Differentiation of the cells was analyzed using Real-time PCR and an ALP assay. Data were analyzed using ANOVA with a post-hoc Tukey test.ResultsThe NAC-treated cells had similar cell viability compared with the controls. The cells treated with NAC + HEMA had significantly higher rates of proliferation as compared to the HEMA only treated groups and displayed more cell viability when these groups were compared with flow cytometric analysis. Real-time PCR and the ALP assay demonstrated that the NAC group upregulated ALP, RUNX-2, and DSPP genes.ConclusionThe data demonstrated that NAC protects the SCAP from apoptosis and enhances the proliferation and differentiation potential of these cells suggesting that NAC could be used effectively during REPs.

The human dental apical papilla promotes spinal cord repair through a paracrine mechanism.

Traumatic spinal cord injury is an overwhelming condition that strongly and suddenly impacts the patient's life and her/his entourage. There are currently no predictable treatments to repair the spinal cord, while many strategies are proposed and evaluated by researchers throughout the world. One of the most promising avenues is the transplantation of stem cells, although its therapeutic efficiency is limited by several factors, among which cell survival at the lesion site. In our previous study, we showed that the implantation of a human dental apical papilla, residence of stem cells of the apical papilla (SCAP), supported functional recovery in a rat model of spinal cord hemisection. In this study, we employed protein multiplex, immunohistochemistry, cytokine arrays, RT- qPCR, and RNAseq technology to decipher the mechanism by which the dental papilla promotes repair of the injured spinal cord. We found that the apical papilla reduced inflammation at the lesion site, had a neuroprotective effect on motoneurons, and increased the apoptosis of activated macrophages/ microglia. This therapeutic effect is likely driven by the secretome of the implanted papilla since it is known to secrete an entourage of immunomodulatory or pro-angiogenic factors. Therefore, we hypothesize that the secreted molecules were mainly produced by SCAP, and that by anchoring and protecting them, the human papilla provides a protective niche ensuring that SCAP could exert their therapeutic actions. Therapeutic abilities of the papilla were demonstrated in the scope of spinal cord injury but could very well be beneficial to other types of tissue.

Mode of cell death of co-cultured dental mesenchymal stem cells following exposure to endodontic irrigating solutions

Aims: This in vitro study aimed to determine the mode of cell death of co-cultured dental mesenchymal stem cells following exposure to different irrigating solutions.Methods and Material: Mesenchymal stem cells of the apical papilla (SCAPs) and periodontal ligament stem cells (PDLSCs) were isolated from the periapical region of a third molar of an 18-year-old patient and cultured. Adequate number of cells in each well, 6-well plate inserts were placed over the wells. The cells were then randomly exposed to 17% ethylenediaminetetraacetic acid (EDTA), 1.5% sodium hypochlorite (NaOCL), 2% chlorhexidine (CHX) and saline. After 1, 5, and 10 minutes cells were trypsinized and underwent flow cytometry. Apoptotic and necrotic cells were quantified. Data were analyzed using the One-way ANOVA and Tukey’s post-hoc test. Results: With increasing exposure time, cell viability is decrease and the mode of cells death was necrosis in CHX and NaOCL groups and necrosis plus late apoptosis in 1 and 10 minutes in EDTA group.Conclusion: Maximum cell death occurred following exposure to EDTA while minimum cell death occurred following exposure to CHX. Necrosis was the dominant mode of cell death in all groups.

Characterization of human stem cells of the apical papilla

Characterization of human stem cells of the apical papilla

Endocannabinoids Regulate Stem Cells of the Apical Papilla via a Cannabinoid Receptor and TRPV1-Independent Mechanism

IntroductionEndogenous cannabinoids (endocannabinoids [eCBs]) have been shown to have a multitude of functions including neurotransmission and immune modulatory effects. This study aimed to evaluate if stem cells of the apical papilla (SCAP) express the receptors and enzymes of the endocannabinoid system (ECS) and whether eCBs regulate their proliferation and mineralization potential. MethodsGene expression of the main components of the ECS and transient receptor potential vanilloid 1 (TRPV1) was evaluated in SCAP cultures. SCAP were treated with 2 concentrations of eCBs and/or capsazepine, a TRPV1 antagonist. SCAP viability was evaluated after 1, 4, and 7 days. Osteogenic differentiation was assessed after 14 days, and the gene expression of mineralization markers was assessed after 7 days. ResultsThe enzymes of ECS and TRPV1 but not the cannabinoid receptors (cannabinoid receptors 1 and 2) were expressed in SCAP. Anandamide, 2-arachidonoylglycerol, and N-arachidonoylphenolamine (AM-404) reduced SCAP viability in all experimental periods at the highest concentration compared with the group with no treatment. Anandamide and AM-404 did not inhibit SCAP differentiation potential, but 2-arachidonoylglycerol at the highest concentration did. SCAP treated with AM-404 presented a down-regulation in gene expression of alkaline phosphatase (ALP), dentin matrix protein 1 (DMP-1), and dentin sialophosphoprotein (DSPP) compared with the proliferation medium group but not with control group. ConclusionsSCAP expressed the genes of the main components of ECS and TRPV1, and eCBs can affect SCAP viability, mineralization, and gene expression.