Effect of 1,25-Dihydroxyvitamin D3 on Stem Cells from Human Apical Papilla: Adhesion, Spreading, Proliferation, and Osteogenic Differentiation.

Currently, it still remains a difficult problem to treat apical insufficiency of young permanent teeth resulted from pulp necrosis or periapical periodontitis. Previous studies have demonstrated that the treatment of revascularization using stem cells from apical papilla (SCAPs) results in increased root length and thickness of traumatized immature teeth and necrotic pulp. In this study, we investigated the role of 1,25-dihydroxyvitamin D3 in regulating the adhesion, spreading, proliferation, and osteogenic differentiation of SCAP, laying the foundation for subsequent clinical drug development. The immature tooth samples were collected in clinical treatment. SCAPs with stable passage ability were isolated and cultured. The multidifferentiation potential was determined by directed induction culture, while the stem cell characteristics were identified by flow cytometry. There were three groups: group A-SCAPs general culture group; group B-SCAPs osteogenesis induction culture group; and group C-SCAPs osteogenesis induction culture+1,25-dihydroxyvitamin D3 group, and the groups were compared statistically. The proliferation of SCAPs in each groups was detected through CCK-8 assay. RT-qPCR was used to detect the transcription levels of Runx2, ALP, Col I, and OCN of SCAPs in each groups. Results exhibited that the isolated SCAPs had multidifferentiation potential and stem cell characteristics. After 24 h culturing, cells in group C spread better than those in groups A and B. The proliferation activity of SCAPs factored by CCK-8 ranked as group C > group B > group A, while the transcription levels of Runx2, ALP, Col I, and OCN leveled as group C > group B > group A. These results suggested that 1,25-dihydroxyvitamin D3 can significantly promote the adhesion, spreading, and proliferation of SACPs and improve the osteogenic differentiation of SCAPs by means of regulating upward the transcription level of osteogenic differentiation marker.

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

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

Effect of angiotensin II and angiotensin-(1-7) on proliferation of stem cells from human dental apical papilla.

The effects of the renin-angiotensin system (RAS) on stem cells isolated from human dental apical papilla (SCAPs) are completely unknown. Therefore, the aim of this study was to identify RAS components expressed in SCAPs and the effects of angiotensin (Ang) II and Ang-(1-7) on cell proliferation. SCAPs were collected from third molar teeth of adolescents and maintained in cell culture. Messenger RNA expression and protein levels of angiotensin-converting enzyme (ACE), ACE2, and Mas, Ang II type I (AT1) and type II (AT2) receptors were detected in SCAPs. Treatment with either Ang II or Ang-(1-7) increased the proliferation of SCAPs. These effects were inhibited by PD123319, an AT2 antagonist. While Ang II augmented mTOR phosphorylation, Ang-(1-7) induced ERK1/2 phosphorylation. In conclusion, SCAPs produce the main RAS components and both Ang II and Ang-(1-7) treatments induced cell proliferation mediated by AT2 activation through different intracellular mechanisms.

Layer-by-layer self-assembly polyelectrolytes loaded with cyclic adenosine monophosphate enhances the osteo/odontogenic differentiation of stem cells from apical papilla.

Cyclic adenosine monophosphate (cAMP) is a second messenger involved in the dental regeneration. However, efficient long-lasting delivery of cAMP that is sufficient to mimic the in vivo microenvironment remains a major challenge. Here, cAMP was loaded in stem cells from apical papilla (SCAPs) using layer-by-layer self-assembly with gelatin and alginate polyelectrolytes (LBL-cAMP-SCAPs). LBL-cAMP-SCAPs expressed cAMP and increased the phosphorylation level of cAMP-response element-binding protein (CREB) which were evaluated by immunofluorescence and western blotting (WB). Enzyme-linked immunosorbent assay (ELISA) demonstrated that a sustained release of cAMP and vascular endothelial growth factor (VEGF) were present up to 14 days. Scanning electron microscopy (SEM) found LBL-coated SCAPs exhibited a spheroid-like morphology. CCK8 and live/dead staining showed that LBL treatment had no significant effect on cell proliferation and viability. LBL-cAMP-SCAPs enhanced mineralized nodule formation and up-regulated the mRNA levels of the osteogenesis-related genes, as well as related transcription factor-2 protein level which were revealed by Alizarin red staining, RT-PCR and WB, respectively. In conclusion, LBL self-assembly loaded with cAMP promoted the osteo/odontogenic differentiation of SCAPs, thereby providing a potential strategy for bioactive molecular delivery in dental regeneration.

Melatonin regulates mitochondrial function and biogenesis during rat dental papilla cell differentiation.

OBJECTIVE: The aim of this study was to investigate the effect of melatonin on mitochondria of dental papilla cells (DPCs) during the odontogenic differentiation process. MATERIALS AND METHODS: Primary DPCs were obtained from the first molar dental papilla of neonatal rats and cultured in osteogenic (OS) or basal medium supplemented with melatonin at different concentrations (0, 1 pM, 0.1 nM, 10 nM, and 1 µM) for differentiation in vitro. Effects of melatonin on differentiation, mitochondrial respiratory function, and mitochondrial biogenesis of DPCs were analyzed. RESULTS: Upon odontogenic induction, Alkaline phosphatase (ALP) activity, dentin sialophosphoprotein (DSPP), and dentin matrix protein (DMP1) expression were significantly enhanced, with a peaked expression at 10 nM of melatonin treatment. During DPCs differentiation, 10 nM melatonin could significantly induce the increase of intracellular Adenosine triphosphate (ATP), the decrease of the oxidized form of nicotinamide adenine dinucleotide (NAD+)/NADH ratio and reactive oxygen species (ROS). The mRNA and protein levels of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), nuclear respiratory factor 1 (NRF-1), and mitochondrial transcription factor A (TFAM) were significantly increased, and the peak level of expression was found in cells treated with 10 nM of melatonin. Furthermore, the mitochondria DNA (mtDNA) copy number was significantly decreased during DPCs differentiation. CONCLUSIONS: These findings suggest that melatonin can promote the differentiation of rat DPCs and regulate mitochondrial energy metabolism, ROS scavenging, and mitochondrial biogenesis.

High Glucose Enhances the Odonto/Osteogenic Differentiation of Stem Cells from Apical Papilla via NF-KappaB Signaling Pathway.

OBJECTIVE: The transport and metabolism of glucose are important during mammalian development. High glucose can mediate the biological characteristics of mesenchymal stem cells (MSCs). However, the role of high glucose in the odonto/osteogenic differentiation of stem cells from apical papilla (SCAPs) is unclear. MATERIALS AND METHODS: SCAPs were isolated and identified in vitro. Then, SCAPs were cultured in normal α-MEM and high glucose α-MEM separately. MTT assay was applied to observe the proliferation of SCAPs. ALP activity, alizarin red staining, real-time RT-PCR, and western blot were used to detect the odonto/osteogenic capacity of SCAPs as well as the participation of NF-κB pathway. RESULTS: SCAPs in 25mmol/L glucose group expressed the maximum proteins of RUNX2 and ALP as compared with those in 5, 10, and 15 mmol/L groups. MTT assay showed that 25 mmol/L glucose suppressed the proliferation of SCAPs. ALP assay, alizarin red staining, real-time RT-PCR, and western blot showed 25 mmol/L high glucose can obviously enhance the odonto/osteogenic capacity of SCAPs. Moreover, the NF-κB pathway was activated in 25mmol/L glucose-treated SCAPs and the odonto/osteogenic differentiation was inhibited following the inhibition of NF-κB signaling pathway. CONCLUSIONS: High glucose can enhance the odonto/osteogenic capacity of SCAPs via NF-κB pathway.

Root canal dressings for revascularization influence in vitro mineralization of apical papilla cells.

Endodontic revascularization is based on cell recruitment into the necrotic root canal of immature teeth after chemical disinfection. The clinical outcome depends on the ability of surviving cells from the apical tissue to differentiate and promote hard tissue deposition inside the dentinal walls. OBJECTIVE: To investigate the effect of calcium hydroxide (CH) and modified triple antibiotic paste (mTAP - ciprofloxacin, metronidazole and cefaclor) on the viability and mineralization potential of apical papilla cells (APC) in vitro . MATERIAL AND METHODS: APC cultures were kept in contact with CH or mTAP (250-1000 µg/mL) for 5 days, after which cell viability was assessed using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Next, APCs were subjected to CH or mTAP at 250 µg/mL for 5 days before inducing the differentiation assay. After 14 and 21 days, calcium deposition was assessed by the Alizarin Red S staining method, followed by elution and quantification using spectrophotometry. Data were analyzed using ANOVA followed by Tukey post hoc test. RESULTS: CH induced cell proliferation, whereas mTAP showed significant cytotoxicity at all concentrations tested. APC treated with CH demonstrated improved mineralization capacity at 14 days, while, for mTAP, significant reduction on the mineralization rate was observed for both experimental periods (14 and 21 days). CONCLUSION: Our findings showed that CH induces cell proliferation and improves early mineralization, whereas mTAP was found cytotoxic and reduced the mineralization potential in vitro of APCs.

Cytotoxicity of intracanal dressings on apical papilla cells differ upon activation with E. faecalis LTA.

OBJECTIVE: The aim of this study was to investigate the cytotoxic effects of modified triple antibiotic paste and an experimental composition using calcium hydroxide on lipoteichoic acid (LTA)-primed apical papilla cells (APC). MATERIAL AND METHODS: Human APC were tested for in vitro cytotoxicity of modified Triple Antibiotic Paste (mTAP - Ciprofloxacin, Metronidazole and Cefaclor at 1:1:1) and of a paste of Ciprofloxacin, Metronidazole and Calcium hydroxide (CMC - 1:1:2) and modified CMC (mCMC - 2:2:1) by using MTT assay. The substances were reconstituted in DMEM at 1,000 µg/mL and » serially diluted before being kept in contact with cells for 1, 3, 5 and 7 days. Further, cells were primed with 1 µg/mL of Enterococcus faecalis LTA for 7 days prior to the viability test with 1,000 µg/mL of each substance. Statistical analysis was performed using one-way analysis of variance (ANOVA) and two-way ANOVA respectively followed by Tukey's post-test. Significance levels were set at p<0.05. RESULTS: In the first assay, the higher cytotoxic rates were reached by mTAP for all experimental periods. CMC was found toxic for APC at 5 and 7 days, whereas mCMC did not affect the cell viability. Only CMC and mCMC were able to induce some cellular proliferation. In the second assay, when considering the condition with medium only, LTA-primed cells significantly proliferated in comparison to LTA-untreated ones. At this context, mTAP and CMC showed similar cytotoxicity than the observed for LTA-untreated cells, while mCMC was shown cytotoxic at 7 days only for LTA-primed APC. Comparing the medications, mTAP was more cytotoxic than CMC and mCMC. CONCLUSION: mTAP showed higher cytotoxicity than CMC and mCMC and the effect of topic antimicrobials might differ when tested against apical papilla cells under physiological or activated conditions.

Root canal dressings for revascularization influence in vitro mineralization of apical papilla cells

Abstract Endodontic revascularization is based on cell recruitment into the necrotic root canal of immature teeth after chemical disinfection. The clinical outcome depends on the ability of surviving cells from the apical tissue to differentiate and promote hard tissue deposition inside the dentinal walls. Objective To investigate the effect of calcium hydroxide (CH) and modified triple antibiotic paste (mTAP - ciprofloxacin, metronidazole and cefaclor) on the viability and mineralization potential of apical papilla cells (APC) in vitro . Material and Methods APC cultures were kept in contact with CH or mTAP (250-1000 µg/mL) for 5 days, after which cell viability was assessed using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Next, APCs were subjected to CH or mTAP at 250 µg/mL for 5 days before inducing the differentiation assay. After 14 and 21 days, calcium deposition was assessed by the Alizarin Red S staining method, followed by elution and quantification using spectrophotometry. Data were analyzed using ANOVA followed by Tukey post hoc test. Results CH induced cell proliferation, whereas mTAP showed significant cytotoxicity at all concentrations tested. APC treated with CH demonstrated improved mineralization capacity at 14 days, while, for mTAP, significant reduction on the mineralization rate was observed for both experimental periods (14 and 21 days). Conclusion Our findings showed that CH induces cell proliferation and improves early mineralization, whereas mTAP was found cytotoxic and reduced the mineralization potential in vitro of APCs.

Cytotoxicity of intracanal dressings on apical papilla cells differ upon activation with E. faecalis LTA

Abstract Objective The aim of this study was to investigate the cytotoxic effects of modified triple antibiotic paste and an experimental composition using calcium hydroxide on lipoteichoic acid (LTA)-primed apical papilla cells (APC). Material and Methods Human APC were tested for in vitro cytotoxicity of modified Triple Antibiotic Paste (mTAP - Ciprofloxacin, Metronidazole and Cefaclor at 1:1:1) and of a paste of Ciprofloxacin, Metronidazole and Calcium hydroxide (CMC - 1:1:2) and modified CMC (mCMC - 2:2:1) by using MTT assay. The substances were reconstituted in DMEM at 1,000 µg/mL and » serially diluted before being kept in contact with cells for 1, 3, 5 and 7 days. Further, cells were primed with 1 µg/mL of Enterococcus faecalis LTA for 7 days prior to the viability test with 1,000 µg/mL of each substance. Statistical analysis was performed using one-way analysis of variance (ANOVA) and two-way ANOVA respectively followed by Tukey's post-test. Significance levels were set at p<0.05. Results In the first assay, the higher cytotoxic rates were reached by mTAP for all experimental periods. CMC was found toxic for APC at 5 and 7 days, whereas mCMC did not affect the cell viability. Only CMC and mCMC were able to induce some cellular proliferation. In the second assay, when considering the condition with medium only, LTA-primed cells significantly proliferated in comparison to LTA-untreated ones. At this context, mTAP and CMC showed similar cytotoxicity than the observed for LTA-untreated cells, while mCMC was shown cytotoxic at 7 days only for LTA-primed APC. Comparing the medications, mTAP was more cytotoxic than CMC and mCMC. Conclusion mTAP showed higher cytotoxicity than CMC and mCMC and the effect of topic antimicrobials might differ when tested against apical papilla cells under physiological or activated conditions.

Effect of Bioceramic Materials on Proliferation and Odontoblast Differentiation of Human Stem Cells from the Apical Papilla.

INTRODUCTION: In regenerative endodontic treatment (RET), practitioners favor the placement of bioceramics as sealing materials over blood clots. It is important to understand the interaction between sealing material and cells in the root canal. The purpose of this study was to compare the effectiveness of various bioceramic materials (ProRoot MTA [Dentsply, Tulsa, OK], Biodentine [Septodont, Saint-Maur-des-Fossés, France], and RetroMTA [BioMTA, Seoul, Korea]) as sealing materials in RET for the proliferation and differentiation of stem cells from the apical papilla (SCAPs). METHODS: SCAPs were seeded at 20,000 cells/well and cultured with soluble agents of testing materials through a transwell culture plate. The proliferation of SCAPs was investigated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay on days 1, 3, 7, and 14 of testing. Alizarin red staining and quantitative real-time polymerase chain reaction were used for SCAP differentiation at different time points (1, 7, 14, and 21 days). The odontoblast genes expressed are dentin matrix acidic phosphoprotein 1, dentin sialophosphoprotein, osteocalcin, and matrix extracellular phosphoglycoprotein, which were used in this study. The SCAPs were cultured in odonto/osteogenic induction medium and also contacted soluble agents from the testing materials. RESULTS: All 3 tested biomaterials induced SCAP proliferation. The Biodentine, ProRootMTA, and RetroMTA groups showed significant SCAP proliferation on days 7 and 14 compared with the control. In regard to odontoblastic differentiation, only Biodentine showed positive alizarin red staining. The highest expressions of dentin matrix acidic phosphoprotein 1, dentin sialophosphoprotein, and matrix extracellular phosphoglycoprotein were found on day 21 in the Biodentine group. The expression of osteocalcin was found to be significant on day 7. CONCLUSIONS: Biodentine, ProRootMTA, and RetroMTA can induce SCAP proliferation. Biodentine induced significant SCAP differentiation among the 3 materials.

Release of TGF-ß1 into root canals with various final irrigants in regenerative endodontics: an in vitro analysis.

AIM: To investigate the release of growth factors into the root canal space after various final irrigants during regenerative endodontic procedures. The residual cytotoxic effect of final irrigants on stem cells from the apical papilla (SCAP) was also examined. METHODOLOGY: To measure the release of TGF-ß1, root segments (8 mm long) were irrigated with 1.5% NaOCl followed by 20 mL of final irrigants; Saline, 17% EDTA, 10% citric acid, 10% or 37% phosphoric acid. Specimens were then immersed into culture medium for 24 h and the supernatants were collected to measure TGF-ß1 by ELISA. For the cytotoxicity of residual final irrigants, dentine chips (5 × 5 × 1 mm) treated with irrigants as above were placed in the upper chamber of transwell system. Stem cells from the apical papilla were incubated indirectly in the lower chamber for 24 h and MTS assay was performed after 24 h. The surfaces of irrigated root canals were examined for smear layer with a scanning electron microscope (SEM). Log transformation was performed for ELISA data to compare different groups (one-way ANOVA, α = 0.05). RESULTS: Ten percent citric acid released the greatest amount of TGF-ß1 amongst all groups, which was significantly different to 17% EDTA (P < 0.01). All dentine chips irrigated with the irrigants showed no significant difference of cytotoxicity on SCAP compared to nonirrigated dentine (P > 0.05). SEM revealed completely open dentinal tubules in 10% citric acid, whereas 17% EDTA was associated with partially open dentinal tubules. CONCLUSIONS: Ten percent citric acid was effective as a final irrigant for releasing TGF-ß1 with good biocompatibility in regenerative endodontics.

Extracellular calcium increases fibroblast growth factor 2 gene expression via extracellular signal-regulated kinase 1/2 and protein kinase A signaling in mouse dental papilla cells.

We previously reported that elevated extracellular calcium (Ca2+) levels increase bone morphogenetic protein 2 expression in human dental pulp (hDP) cells. However, it is unknown whether extracellular Ca2+ affects the expression of other growth factors such as fibroblast growth factor 2 (FGF2). The present study aimed to examine the effect of extracellular Ca2+ on FGF2 gene expression in hDP and immortalized mouse dental papilla (mDP) cells. Cells were stimulated with 10 mM CaCl2 in the presence or absence of cell signaling inhibitors. FGF2 gene expression was assessed using real-time polymerase chain reaction. The phosphorylation status of signaling molecules was examined by Western blotting. Extracellular Ca2+ increased FGF2 gene expression in mDP and hDP cells. Gene expression of the calcium-sensing receptor and G protein-coupled receptor family C group 6 member A, both of which are extracellular Ca2+ sensors, was not detected. Ca2+-mediated Fgf2 expression was reduced by pretreatment with the protein kinase A (PKA) inhibitor H-89 or extracellular signal-regulated kinase (ERK) 1/2 inhibitor PD98059 but not by pretreatment with the protein kinase C inhibitor GF-109203X or p38 inhibitor SB203580. Extracellular Ca2+ increased PKA activity and ERK1/2 phosphorylation. Ca2+-induced PKA activity decreased by pretreatment with PD98059. These findings indicate that elevated extracellular Ca2+ levels led to increased Fgf2 expression through ERK1/2 and PKA in mDP cells and that this mechanism may be useful for designing regenerative therapies for dentin.

A Comparative Evaluation of Concentrated Growth Factor and Platelet-rich Fibrin on the Proliferation, Migration, and Differentiation of Human Stem Cells of the Apical Papilla.

INTRODUCTION: Concentrated growth factor (CGF) is considered to be a natural biomaterial that is better than platelet-rich fibrin (PRF) in bone regeneration, but there is little information acquired in regenerative endodontics. Therefore, the purpose of this study was to evaluate their effects on the proliferation, migration, and differentiation of human stem cells of the apical papilla (SCAPs). METHODS: CGF- and PRF-conditioned medium were prepared using the freeze-dried method. SCAPs were isolated and identified. The proliferative potential of SCAPs was investigated using the Cell Counting Kit-8 (KeyGen Biotech, Nanjing, China). The migration capacity was analyzed using transwell assays, and the mineralization ability was determined by alizarin red S staining. The expression levels of alkaline phosphatase, bone sialoprotein, dentin matrix protein 1, and dentin sialophosphoprotein were determined by quantitative polymerase chain reaction. RESULTS: The cultured cells exhibited mesenchymal stem cell characteristics. The growth rate and migratory cell numbers of the CGF and PRF groups were significantly greater than those of the control group. The mineralized areas in the CGF and PRF groups were significantly larger than those in the control group after incubation for 7 days and 14 days. The expression levels of osteogenic/odontoblast-related genes were reduced on day 7, but they were dramatically enhanced on day 14, and the related gene expression levels in the PRF group were higher than those in the CGF group. CONCLUSIONS: Both CGF and PRF can promote the proliferation, migration, and differentiation of SCAPs. CGF may be a promising alternative in regenerative endodontics.

Effect of 3 Bioceramic Materials on Stem Cells of the Apical Papilla Proliferation and Differentiation Using a Dentin Disk Model.

INTRODUCTION: There is a complex interaction between biomaterials placed as a coronal barrier with stem cells and dentin in regenerative procedures. In this study, the effect of Biodentine (BD; Septodont, Saint-Maurdes-Fossés, France), Endosequence BC Root Repair Material-Putty (ES; Brasseler, Savannah, GA), Endosequence BC Root Repair Material-Putty Fast set (ES-fast, Brasseler), and ProRoot (Dentsply Tulsa Dental Specialties, Johnson City, TN) mineral trioxide aggregate (MTA) on the viability and differentiation of stem cells of the apical papilla (SCAP) was evaluated using an ex vivo dentin disk model. METHODS: Standardized human dentin disks were treated using an established protocol. Disk lumens were filled with BD, ES, ES-fast, or MTA, and SCAP were cultured directly onto the samples. Cell viability was measured at 7 days, whereas differentiation into a mineralizing phenotype was evaluated by real-time reverse-transcription polymerase chain reaction and alizarin red staining at 21 days in culture. Results were analyzed using 1-way analysis of variance with the Bonferroni post hoc test or the Mann-Whitney U test (P ≤ .05). RESULTS: All materials promoted SCAP viability and proliferation with a greater response in the BD and ES groups. Also, a greater expression of alkaline phosphatase messenger RNA and dentin sialophosphoprotein was noted in the BD and ES groups, whereas MTA promoted a greater expression of the osteoblastic marker IBSP. Interestingly, no difference in alizarin red staining was observed with MTA, BD, or ES, which were significantly greater than ES-fast. CONCLUSIONS: These data suggest that BD and ES promoted greater survival and differentiation of SCAP and the increase of the odontoblastic marker DSPP, whereas MTA appeared to promote greater osteoblastic differentiation. Thus, BD and ES can be considered for regenerative and vital pulp therapies.

Odontoclastogenesis of mouse papilla-derived MDPC-23 cells induced by lipopolysaccharide.

AIM: To investigate the role of Lipopolysaccharide (LPS) in the odontoclast differentiation of MDPC-23 cells. It was hypothesized that MDPC-23 odontoblast-like cells may function as odontoclasts under the influence of LPS. METHODOLOGY: MDPC-23 cells were cultured in the presence of 0.1 or 1 µg mL-1 LPS for 6 days. Cell viability was determined using the CCK8 assay. TRAP staining, dentine resorption assay and ROS detection by confocal laser scanning microscope were used to test the odontoclast-like function of the induced cells. In additional, the related protein expression was confirmed by Western blotting and ELISA. An unpaired Student's t-test and one-way anova were used in statistical analysis. RESULTS: TRAP-positive cells, which are multinucleated, on the dentine slice were significantly increased in 1 µg mL-1 LPS-induced cells (P < 0.05). Osteoclast-specific proteins such as TRAP cathepsin K and Rac1 were upregulated in the 1 µg mL-1 LPS-treated cells (P < 0.05), whilst the expression of marker proteins of the RANKL-RANK signalling pathway (RANKL, RANK and TRAF6) in the induced cells was not significantly changed (P > 0.05). ROS production was observed in the 1 µg mL-1 LPS treatment group (P < 0.05), but no significant differences were observed in the level of RANKL in the cell supernatant between the LPS-treated group and the control group (P > 0.05). CONCLUSIONS: A known value of 1 µg mL-1 LPS might induce odontoblast-like MDPC-23 cells to generate odontoclast-like cells or to function as odontoclasts. The data might provide a new explanation for the precursors of odontoclasts and root resorption.

EDTA Treatment for Sodium Hypochlorite-treated Dentin Recovers Disturbed Attachment and Induces Differentiation of Mouse Dental Papilla Cells.

INTRODUCTION: The disturbance of cellular attachment to dentin by sodium hypochlorite (NaOCl) may hamper pulp tissue regeneration. The aims of this study were to examine the recovering effect of EDTA on the attachment/differentiation of stemlike cells and to address the mechanisms of EDTA-induced recovery under the hypothesis that attachment to the exposed dentin matrix and the subsequent activation of integrin/phosphatidylinositol 3-kinase (PI3K) signaling play a crucial role. METHODS: Mouse dental papilla (MDP) cells were cultured on bovine dentin disks treated with NaOCl (0%, 1.5%, or 6%) followed by EDTA (0%, 3%, or 17%). Cell attachment was evaluated by cell density, viability, and scanning and transmission electron microscopy. Odonto-/osteoblastic gene expression in attached MDP cells was analyzed with or without a pan-PI3K inhibitor (LY294002) using real-time polymerase chain reaction. RESULTS: NaOCl treatment (1.5%, 10 minutes) significantly diminished attached MDP cells (P < .00001), but EDTA treatment (3% and 17%, ≥10 minutes) of NaOCl-pretreated dentin induced a significant increase in attached cells (P < .05). Ultrastructurally, MDP cells on EDTA-treated dentin showed attachment to exposed collagen fibers. MDP cells cultured on EDTA-treated disks (with or without 1.5% NaOCl pretreatment) showed significant up-regulation of alkaline phosphatase, dentin matrix protein 1, and dentin sialophosphoprotein messenger RNAs (P < .05). Alkaline phosphatase expression was down-regulated by LY294002 (P < .05). CONCLUSIONS: Under the present experimental conditions, 10 minutes of EDTA treatment was sufficient to recover attachment/differentiation of MDP cells on 1.5% NaOCl-pretreated dentin. EDTA-induced exposure of collagen fibers and subsequent activation of integrin/PI3K signaling may contribute, at least partly, to the recovery.

The Effects of Irrigants on the Survival of Human Stem Cells of the Apical Papilla, Including Endocyn.

INTRODUCTION: Endocyn, a pH-neutral solution of hypochlorous acid and hypochlorite has been developed for use as an endodontic irrigant. The purpose of this study was to evaluate the effect of Endocyn on human periodontal ligament (PDL) fibroblasts, rat osteosarcoma cells (UMR-106), and stem cells of the apical papilla (SCAP) compared with other commonly used endodontic irrigants. METHODS: To determine cytotoxicity, cells were exposed to various concentrations of Endocyn, 6% sodium hypochlorite (NaOCl), 17% EDTA, and 2% chlorhexidine for 10 minutes, 1 hour, or 24 hours. Cell survival was measured fluorescently using calcein AM. Endocyn also was tested for its ability to inhibit SCAP proliferation and alkaline phosphatase activity. Finally, SCAP transcript expression was examined via reverse-transcriptase polymerase chain reaction. RESULTS: Endocyn was no more toxic to PDL and UMR cells than water for up to 24 hours. Endocyn concentrations of 50% were toxic to SCAP after 1 hour of exposure. Endocyn concentrations of >20% inhibited SCAP proliferation, whereas concentrations of ≥10% inhibited alkaline phosphatase activity. Exposure of SCAP to 10% Endocyn for 3 days did not alter most transcript expression, but did significantly reduce the expression of alkaline phosphatase, fibromodulin, and osteomodulin. CONCLUSION: Endocyn was significantly less cytotoxic to PDL, UMR-106, and SCAP cells compared with other commonly used endodontic irrigants. High concentrations of Endocyn did inhibit some transcript expression and alkaline phosphatase activity, indicating a potential reduction in the osteogenic potential of stems cells exposed to Endocyn.

Extracellular calcium increases fibroblast growth factor 2 gene expression via extracellular signal-regulated kinase 1/2 and protein kinase A signaling in mouse dental papilla cells

Abstract We previously reported that elevated extracellular calcium (Ca2+) levels increase bone morphogenetic protein 2 expression in human dental pulp (hDP) cells. However, it is unknown whether extracellular Ca2+ affects the expression of other growth factors such as fibroblast growth factor 2 (FGF2). Objective: The present study aimed to examine the effect of extracellular Ca2+ on FGF2 gene expression in hDP and immortalized mouse dental papilla (mDP) cells. Materials and Methods: Cells were stimulated with 10 mM CaCl2 in the presence or absence of cell signaling inhibitors. FGF2 gene expression was assessed using real-time polymerase chain reaction. The phosphorylation status of signaling molecules was examined by Western blotting. Results: Extracellular Ca2+ increased FGF2 gene expression in mDP and hDP cells. Gene expression of the calcium-sensing receptor and G protein-coupled receptor family C group 6 member A, both of which are extracellular Ca2+ sensors, was not detected. Ca2+-mediated Fgf2 expression was reduced by pretreatment with the protein kinase A (PKA) inhibitor H-89 or extracellular signal-regulated kinase (ERK) 1/2 inhibitor PD98059 but not by pretreatment with the protein kinase C inhibitor GF-109203X or p38 inhibitor SB203580. Extracellular Ca2+ increased PKA activity and ERK1/2 phosphorylation. Ca2+-induced PKA activity decreased by pretreatment with PD98059. Conclusions: These findings indicate that elevated extracellular Ca2+ levels led to increased Fgf2 expression through ERK1/2 and PKA in mDP cells and that this mechanism may be useful for designing regenerative therapies for dentin.

Effects of Lipopolysaccharide on the Proliferation and Osteogenic Differentiation of Stem Cells from the Apical Papilla.

INTRODUCTION: Stem cells from the apical papilla (SCAPs) were suggested as the stem cell source in regenerative endodontic procedures. However, bone and/or cementum-like structure were observed in root canals. Lipopolysaccharide (LPS) in infected root canals might alter SCAPs' osteogenic differentiation pattern. The objectives of this study were to investigate the effects of LPS on SCAPs' proliferation and osteogenic differentiation. METHODS: The mesenchymal stem cell characteristics of SCAPs were confirmed. Cell viability was tested with Porphyromonas gingivalis LPS at concentration between 0.001 and 5 µg/mL. SCAPs were pretreated with those concentrations for 168 hours. Then SCAPs were further investigated for cell proliferation by resazurin-based assay. Mineralization capacity was determined by alizarin red S staining. Odontoblast marker was determined by DSPP gene expression. General bone and cementum markers, BSP and OPN, were also determined. Determination of the expression levels of these genes was performed by polymerase chain reaction. RESULTS: SCAPs demonstrated the mesenchymal stem cell characteristics. All LPS concentrations did not affect cell viability. Pretreatment with LPS also did not affect cell proliferation and mineralization in every concentration. There was no significant difference between DSPP and OPN gene expression levels at all concentrations. However, LPS at 5 µg/mL significantly increased BSP gene expression. CONCLUSIONS: Under the limitations of this in vitro study, LPS did not affect SCAP proliferation and mineralization. However, LPS at high concentration, 5 µg/mL, increased BSP gene expression.