Cytotoxic, migration, and angiogenic effects of intracanal irrigants on cells involved in revascularization of immature teeth.

OBJECTIVE: To evaluate protocols of root canal irrigation and dentin pretreatment in a cell culture model simulating immature teeth. Cytotoxic, migration, and angiogenic effects of Sodium hypochlorite associated with EDTA (NaOCl/EDTA), NaOCl associated with Smear Clear (NaOCl/SC), and QMix were compared. DESIGN: Three roots of mandibular first premolars had their length and root canal diameter standardized. Root canals were irrigated, and the resulting solutions were diluted in culture medium. Sulforhodamine B (SRB) assay was performed with apical papilla cells and with endothelial cells (HUVECs) to assess cytotoxicity. Polarity index and migration assays of apical papilla cells and sprouting of HUVECs were evaluated. Data were analyzed by ANOVA and Tukey post-hoc tests (p < .05). RESULTS: In apical papilla cells, NaOCl/SC and QMix promoted higher cytotoxicity, decreased fraction of elongated cells, and had lower migration speed and shorter migration distance of cells compared to NaOCl/EDTA. Also, HUVECs treated with NaOCl/SC and QMix showed decreased tubule formation in comparison with NaOCl/EDTA. CONCLUSIONS: NaOCl/SC and QMix showed unfavorable biological responses of cells involved in revascularization in comparison to NaOCl/EDTA. Further studies with other intracanal irrigants should be performed to improve the balance of root canal disinfection with biological responses.

Effects of serum-free culture media on human apical papilla cells properties.

OBJECTIVE: Aiming at more effective and safer cell therapies, the objective of this study was to evaluate the biological properties of human apical papilla cells cultured in the absence of serum supplementation in comparison to cells cultured with fetal bovine serum (FBS). DESIGN: Two apical papilla cell populations were isolated from third molars with incomplete rhizogenesis, and cultured in four different media: minimum essential Eagle medium - alpha modification (alpha-MEM); alpha-MEM supplemented with FBS (alpha-MEM + FBS); Dulbecco's modified Eagle medium/nutrient mixture F-12 (DMEM/F12); and DMEM/F12 supplemented with FBS (DMEM/F12 + FBS). We evaluated their proliferation, clonogenicity, and in vitro osteogenic and chondrogenic differentiation potential. RESULTS: Apical papilla cells cultured in DMEM/F12 + FBS and alpha-MEM + FBS were more proliferative than those grown in serum-free media, and also exhibited greater efficiency in colony cell formation. Despite this, all study groups showed immunostaining for the marker of mitosis anti-PHH3. Also, alpha-MEM + FBS, alpha-MEM, and DMEM/F12 + FBS exhibited higher amount of mineralized deposits in vitro than DMEM/F12, while only cells cultured with FBS were able to form spheres in chondrogenic differentiation assay. CONCLUSIONS: Our results showed that, although the cultivation of apical papilla cells in a serum-free medium has reduced the properties of cell proliferation and differentiation, these cells are still capable of maintaining their desirable characteristics.

Expression of Normal or Mutated X-Linked BCOR Transcripts in OFCD iPSCs.

Reprogramming diseased cells with mutated genes into induced pluripotent stem cells (iPSCs) can allow studies of disease mechanism and correct the mutation. Oculofaciocardiodental (OFCD) syndrome is a developmental disorder caused by heterozygous mutations in the X-linked BCL-6 corepressor (BCOR) gene. In this present study, we aimed to reprogram stem cells from a tooth apical papilla (SCAP) of a patient with OFCD, termed SCAP-O, into iPSCs. The SCAP-O carry a copy of the BCOR gene having 1 nucleotide deletion in 1 of the alleles, therefore harboring a mixture of cells expressing either normal (SCAP-OBCOR-WT) or mutated (SCAP-OBCOR-mut) BCOR transcripts. We subcloned SCAP-O and separated SCAP-OBCOR-WT and SCAP-OBCOR-mut as verified by sequencing. The selected subclone SCAP-OBCOR-mut expressed only the mutated BCOR transcripts and remained in such condition after multiple passages. We reprogrammed SCAP-O and subclone SCAP-OBCOR-mut into transgene-free iPSCs using an excisable lentiviral vector system (hSTEMCCA-loxP) carrying 4 reprogramming factors in a single cassette, followed by removal of transgenes via Cre-mediated excision. We found that after reprogramming SCAP-O or subclone SCAP-OBCOR-mut into iPSCs, some of the iPSC clones expressed either solely the normal BCOR-WT or BCOR-mut transcripts, while other clones expressed both BCOR-WT and BCOR-mut transcripts. This is our first step toward establishing OFCD study models by generating isogenic control BCOR-WT iPSCs versus BCOR-mut iPSCs.

Dental Mesenchymal Stem Cells: Dental Pulp Stem Cells, Periodontal Ligament Stem Cells, Apical Papilla Stem Cells, and Primary Teeth Stem Cells-Isolation, Characterization, and Expansion for Tissue Engineering.

Dental stem cells (DSCs) have been shown to possess great potential for multiple biomedical applications, especially for dental tissue regeneration. They are a special type of subpopulation of mesenchymal stem/stromal cells (MSCs) and present subtle differences from other types of MSCs. Therefore, it requires a specialized expertise to isolate, culture, and characterize these cells in vitro and in vivo. The purpose of this chapter is to share our experience in studying these cells. We will describe in detail laboratory protocols outlining how the cells are isolated, cultured, expanded, and characterized using various in vitro cellular and biochemical analyses, as well as an in vivo study model using immunocompromised mice to observe tissue regeneration after transplantation of these DSCs.

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.

Cyclic Adenosine Monophosphate Promotes Odonto/Osteogenic Differentiation of Stem Cells from the Apical Papilla via Suppression of Transforming Growth Factor Beta 1 Signaling.

INTRODUCTION: Stem cells from the apical papilla (SCAPs) possess strong odonto/osteogenic differentiation potential. This study investigated the effect of cyclic adenosine monophosphate (cAMP) on odonto/osteogenic differentiation of SCAPs and the underlining interplay between cAMP and transforming growth factor beta 1 (TGF-ß1). METHODS: SCAPs were stimulated with an activator of cAMP (forskolin) in the presence of either TGF-ß1 or a TGF-ß1 inhibitor. The amounts of calcium mineral deposition and alkaline phosphatase activity were determined. Quantitative real-time polymerase chain reaction was performed to elucidate cAMP on the TGF-ß1-mediated odonto/osteogenic differentiation of SCAPs. The effect of cAMP on the phosphorylation of Smad2/Smad3 and extracellular-regulated kinase (ERK)/P38 induced by TGF-ß1 was analyzed by Western blotting. RESULTS: Cotreatment with forskolin and a TGF-ß1 inhibitor enhanced alkaline phosphatase activity and deposition of calcium minerals in SCAPs. Moreover, the TGF-ß1 inhibitor synergized the effect of forskolin on the expression of type I collagen and runt-related transcription factor 2. The results of Western blotting revealed that forskolin attenuated the unregulated expression of the phosphorylation of Smad3 and ERK induced by TGF-ß1, and a cAMP inhibitor (H89) antagonized this effect. CONCLUSIONS: This study showed that cAMP signaling exerts its up-regulating effects on the odonto/osteogenic differentiation of SCAPs by interfering with TGF-ß1 signaling via inhibiting Smad3 and ERK phosphorylation.

Direct and Indirect Effect of Chlorhexidine on Survival of Stem Cells from the Apical Papilla and Its Neutralization.

INTRODUCTION: Several irrigants have been used for disinfection in regenerative endodontic procedures including chlorhexidine (CHX). In this context, the antibacterial properties of disinfectants are mainly in focus of research even though they may have an undesirable impact on the fate of stem cells. In this study, we hypothesized that CHX has both a direct effect when applied to stem cells of the apical papilla (SCAPs) and an indirect effect when SCAPs are exposed to dentin previously conditioned with CHX. METHODS: Cell toxicity was evaluated in vitro using the CellTox green fluorescence assay (Promega, Madison, WI) and CellTiter-Glo (Promega) after SCAPs were exposed directly to a dynamic concentration range of CHX; apical papilla explant cultures were stained with ApopTag (Merck Millipore, Billerica, MA) after culture with CHX. Furthermore, standardized slabs from human dentin were treated with CHX and consecutively rinsed in EDTA, L-α-lecithin (Sigma-Aldrich, St Louis, MO), or L-α-lecithin followed by EDTA. After that, SCAPs were cultured on the slabs for 5 days, and cellular viability was determined (indirect effect). Data were treated nonparametrically and analyzed using the Krukal-Wallis test (P ≤ .05). RESULTS: Direct exposure of SCAPs to CHX highly affected cell viability at concentrations above 10-3%, whereas lower concentrations had no adverse effect. During the initial 60 minutes, concentrations of 10-2% CHX or higher resulted in early pronounced toxicity with a maximum effect within 15 minutes after exposure. Likewise, CHX-conditioned dentin slabs were detrimental to SCAP survival; however, the deleterious effects were completely reversed by neutralization with L-α-lecithin. CONCLUSIONS: Chlorhexidine is toxic to SCAPs when applied directly or indirectly via conditioned dentin. If applied for a short time and neutralized by L-α-lecithin, it can be a gentle and cell-preserving disinfectant before endodontic regeneration.

The Effects of Mitogen-activated Protein Kinase Signaling Pathways on Lipopolysaccharide-mediated Osteo/Odontogenic Differentiation of Stem Cells from the Apical Papilla.

INTRODUCTION: Odontogenic differentiation of human stem cells from the apical papilla (SCAPs) is a prerequisite step in the root development of immature permanent teeth. However, little is known about the effects of an inflammatory environment on osteo/odontogenic differentiation of SCAPs. The purpose of this study was to investigate the effects of lipopolysaccharide (LPS) on the proliferation and osteo/odontogenic differentiation of SCAPs and the role of mitogen-activated protein kinase (MAPK) signaling pathways in LPS-mediated osteo/odontogenic differentiation of SCAPs. METHODS: SCAPs of human third permanent molars were cultured. Cell viability was analyzed. Alkaline phosphatase activity and mineralization ability were investigated. Gene expression of osteo/odontogenic differentiation and MAPK signaling pathways was evaluated during osteo/odontogenic differentiation of SCAPs. RESULTS: In the 0.1 µg/mL LPS-treated group, cell proliferation, alkaline phosphatase activity, and mineralization of SCAPs were up-regulated. Real-time quantitative polymerase chain reaction revealed that dentin sialophosphoprotein, runt-related transcription factor 2, and bone sialoprotein were increased. However, we did not detect any change of osteocalcin expression. In addition, the expression of p-ERK and p-p38 in SCAPs was enhanced by LPS treatment, whereas the inhibition of ERK and p38 MAPK pathways markedly suppressed the differentiation of LPS-treated SCAPs. CONCLUSIONS: Our findings showed that LPS at the appropriate concentration promoted the proliferation and osteo/odontogenic differentiation of SCAPs. ERK and p38 MAPK signaling pathways are involved in LPS-mediated osteo/odontogenic differentiation of SCAPs.

Interaction of intracanal medicaments with apical papilla stem cells: quantitative cytotoxicity assessment by methyl thiazolyl tetrazolium, trypan blue and lactate dehydrogenase.

BACKGROUND: Chemical residues often have cytotoxic effects on the stem cells. This study aimed to assess the cytotoxic effects of intracanal medicaments on stem cells of the apical papilla (SCAPs) using methyl thiazolyl tetrazolium (MTT), trypan blue exclusion (TBE) and lactate dehydrogenase release (LDHR) assays. METHODS: SCAPs were cultured and exposed to 0.125, 0.25, 1, 5 and 10 mg/mL concentrations of modified triple antibiotic paste (mTAP)/distilled water (DW), mTAP/chlorhexidine (CHX), calcium hydroxide(Ca(OH)2)/CHX and Ca(OH)2/DW. Cell viability was quantitatively analyzed using the MTT, LDHR and TBE assays. Data were analyzed using one-way ANOVA and Tukey's test. RESULTS: All three assessment methods yielded the same results. Ca(OH)2/ DW resulted in the highest and mTAP/CHX resulted in the lowest cell viability. In contrast to Ca(OH)2, mTAP decreased cell viability in a dose-dependent manner. Also, addition of CHX to mTAP and Ca(OH)2 increased their cytotoxicity. CONCLUSIONS: In contrast to the proliferative effects of Ca(OH)2, even low concentrations of mTAP have cytotoxic effects on SCAPs.

The periodontal stem/progenitor cell inflammatory-regenerative cross talk: A new perspective.

Adult multipotent stem/progenitor cells, with remarkable regenerative potential, have been isolated from various components of the human periodontium. These multipotent stem/progenitor cells include the periodontal ligament stem/progenitor cells (PDLSCs), stem cells from the apical papilla (SCAP), the gingival mesenchymal stem/progenitor cells (G-MSCs), and the alveolar bone proper stem/progenitor cells (AB-MSCs). Whereas inflammation is regarded as the reason for tissue damage, it also remains a fundamental step of any early healing process. In performing their periodontal tissue regenerative/reparative activity, periodontal stem/progenitor cells interact with their surrounding inflammatory micro-environmental, through their expressed receptors, which could influence their fate and the outcome of any periodontal stem/progenitor cell-mediated reparative/regenerative activity. The present review discusses the current understanding about the interaction of periodontal stem/progenitor cells with their surrounding inflammatory micro-environment, elaborates on the inflammatory factors influencing their stemness, proliferation, migration/homing, differentiation, and immunomodulatory attributes, the possible underlying intracellular mechanisms, as well as their proposed relationship to the canonical and noncanonical Wnt pathways.

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.

Apical Papilla Cells Are Capable of Forming a Pulplike Tissue with Odontoblastlike Cells without the Use of Exogenous Growth Factors.

INTRODUCTION: Dental pulp is a complex tissue with highly differentiated cells, which makes its reconstruction a challenging task. The apical papilla is an undifferentiated tissue considered as the remnant of the dental papilla that forms the dentin-pulp complex. Aiming to analyze morphologic features of the tissue formed in an in vivo pulp model, we used human apical papilla as a cell source without the use of exogenous growth factors. METHODS: A construct was built using newborn mice molar crowns treated with TrypLE (Fisher Scientific, Loughborough, UK) and EDTA. The crowns were filled with PuraMatrix (Corning Inc, Corning, NY) and a pool population of human apical papilla cells. As a control, we used crowns filled only with PuraMatrix and empty crowns. The constructs were transplanted under severe combined immunodeficient mice kidney capsules. Immunohistochemistry for lamin A, dentin sialophosphoprotein, and dentin matrix protein 1 was performed. RESULTS: Morphologic analysis of all transplanted crowns showed the formation of a loose connective tissue of variable cellularity with the presence of well-formed functional vessels. In the study group, lamin A-positive cells represented the majority of cells within the pulp chamber and a few cells in the vessel lining. We also found positivity for dentin sialophosphoprotein and dentin matrix protein 1, an indicator of odontoblast differentiation. CONCLUSIONS: In our study model, human transplanted apical papilla cells mixed with the host cells and formed a vascularized viable tissue, and these cells were able to differentiate into odontoblastlike cells without the use of exogenous growth factors.

Secreted products of oral bacteria and biofilms impede mineralization of apical papilla stem cells in TLR-, species-, and culture-dependent fashion.

Regenerative endodontics exploits the mineralization potential of stem cells from the apical papilla (SCAPs) in order to promote root maturation of permanent immature teeth. SCAPs may encounter post-disinfection residual bacteria either in planktonic or in biofilm growth mode. Bacterial components bind to Toll-like receptors (TLRs) and trigger pro-inflammatory responses. We hypothesized that biofilm-triggered TLR activation affects the mineralization potential of human SCAPs. SCAPs were challenged with conditioned media derived from standardized dual-species biofilms and planktonic bacterial cultures and their inflammatory status and mineralization capacity were studied. Bacterial products from both growth modes (planktonic vs. biofilm) compromised cell viability, proliferation and mineralization capacity of SCAPs, but in a species- and growth mode-dependent fashion. While TLR4 expression remained unaffected, TLR2 expression was upregulated coinciding with a pro-inflammatory activation of SCAPs. Moreover, TLR and its downstream TGF-ß-associated kinase (TAK1) appeared to be blocking mineralization, as inhibition of these factors restored it. In conclusion, bacterial products promoted the pro-inflammatory status and inhibited mineralization of human SCAPs in a TLR-, species-, and culture-dependent fashion. TLR2 emerged as the pivotal mediator of these responses and further research is warranted towards the judicious manipulation of SCAPs in order to modify the untoward events of TLR-priming and signaling.

[Effect of exogenous stem cells from apical papillae in the pulp revascularization treatment for the immature permanent tooth with periapical periodontitis].

Objective: To evaluate the effect of exogenous stem cells from apical papillae (SCAP) in the pulp revascularization treatment for the immature permanent tooth with periapical periodontitis in animal model. Methods: After the SCAP were isolated and cultured from the Beagle dogs, stem cell properties of these cells were characterized by analyzing their colony-forming ability, the expression of mesenchymal stem cell markers and the multidifferentiation characteristics including osteogenic, adipogenic, and chondrogenic potentials. Models of young permanent tooth with periapical periodontitis were established in dogs and the infection in each of the model tooth was eliminated by root canal irrigation and intracanal medication. After that, all of the model teeth were randomly divided into 4 groups: Group 1: normal developing teeth with no treatment applied;Group 2: teeth that periapical tissues were irritated to induce blood flowing into the root canals;Group 3: teeth that peripheral blood was delivered into the root canals;Group 4: teeth that SCAP were resuspended in peripheral blood and delivered into the root canals. In Group 2-4, firm coronal seal was performed after revascularization procedure and radiographs were taken periodically in order to observe the development of roots. After a 12-week-period, alveolar samples were collected and observed histologically. Results: The isolated SCAP showed clonogenic ability and multilineage differentiation ability including osteogenic, adipogenic and chondrogenic potentials. These cells also expressed the mesenchymal stem cell markers such as STRO-1 and CD146, while no cytokeratin was detected. The thickening of canal wall was observed radiographically 12 weeks after procedures of infection control and revascularization. Histologically, the newly formed tissues on the inner canal wall were found bone lacuna like structure in Group 2 and 3, and the new tissue formed in the Group 3 seemed easy to separate from the canal wall. The newly formed tissues in Group 4 were much thicker compare to those in the Group 2 and 3, and the dentine tubule like structure instead of bone lacuna was noticed although the orientation of these tubules were various. Conclusions: SCAP seem to play an important role in the tissue regeneration procedure when infection is well controlled in young permanent teeth with periapical periodontitis. It is difficult to achieve real tissue regeneration due to the lack of endogenous SCAP in apical area, therefore delivering adequate exogenous SCAP isolated and cultured in vitro could be a promising approach to overcome the challenge.

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.

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.

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.

Cytokine Expression of Stem Cells Originating from the Apical Complex and Coronal Pulp of Immature Teeth.

INTRODUCTION: The aim of this study was to measure and compare the expression levels of cytokines from developing apical complex cells (DACCs) and dental pulp stem cells (DPSCs) of the immature tooth. METHODS: DPSC-conditioned medium (CM) and DACCs-CM were obtained from human young teeth, and 174 cytokines secreted from each CM were identified and compared. A cytokine membrane array and enzyme-linked immunosorbent assay were used to measure and compare the expression levels of the cytokines. Immunocytochemistry targeting insulin-like growth factor-1 and neurotrophin-3 was additionally performed. RESULTS: There were statistically significant differences in the expression levels of 25 cytokines: 22 and 3 were expressed more strongly in DPSCs-CM and DACCs-CM, respectively. Odontoblast differentiation-related cytokines were more strongly expressed in DPSCs-CM, while cell-proliferation-related cytokines were more strongly expressed in DACCs-CM. Proinflammatory and anti-inflammatory cytokines were predominantly expressed in DPSCs-CM and DACCs-CM, respectively. CONCLUSIONS: DPSCs may exert a stronger paracrine effect than DACCs on regeneration of the dentin-pulp complex, in terms of odontoblast differentiation.

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.