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.

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.

Characterization of the Cellular Responses of Dental Mesenchymal Stem Cells to the Immune System.

INTRODUCTION: Dental stem cells have gained importance recently and are being used for various purposes in regenerative medicine and dentistry. Although much research has been done to show the various properties of these dental stem cells, the immunomodulatory properties of some of these stem cells are still unknown. This is important considering these cells are being used routinely. Therefore, the aim of this study was to investigate the interactions between the activated immune cells and 3 types of dental-derived mesenchymal stem cells: dental pulp stem cells, stem cells from human exfoliated deciduous teeth, and stem cells of the apical papilla (SCAP). METHODS: SCAP, dental pulp stem cells, stem cells from human exfoliated deciduous teeth, and periodontal ligament fibroblasts were cultured, and various assays were performed including a proliferation assay, flow cytometric analysis, lactate dehydrogenase and chromium-51 cytotoxicity assays, and an enzyme-linked immunosorbent assay to evaluate the interactions of these dental stem cells when cocultured with either peripheral blood mononuclear cells or natural killer cells. RESULTS: SCAP were less resistant to immune cell-mediated cytotoxicity as seen from the results obtained from the LDH and chromium-51 cytotoxicity assays. The flow cytometric analysis showed a lower resilience of SCAP to cytotoxic compounds. The enzyme-linked immunosorbent assay results demonstrated that the SCAP induced high levels of proinflammatory cytokine secretion compared with the other dental stem cells. CONCLUSIONS: SCAP did not perform as well as the other dental stem cells. This could in turn affect their survival and differentiation abilities as well as their functionality. This may be an important aspect to consider when selecting dental stem cells for various regenerative procedures.

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.

Obese Subjects With Specific Gustatory Papillae Microbiota and Salivary Cues Display an Impairment to Sense Lipids.

Some obese subjects overeat lipid-rich foods. The origin of this eating behavior is unknown. We have here tested the hypothesis that these subjects could be characterized by an impaired fatty taste sensitivity linked to a change in the gustatory papillae microbial and salivary environment. The composition of microbiota and saliva surrounding the circumvallate papillae was analyzed in combination with the orosensory lipid detection threshold in normal weight (NW) and obese (O) adults. Microbial architecture was similar to what was known in feces, but with an increased frequency of Proteobacteria. No difference in the orosensory sensitivity to lipids and composition of oral microbiota and saliva was observed between NW and O subjects. By contrast, specific bacterial and salivary signatures were found in lipid non-tasters, irrespectively of BMI. A multivariate approach highlighted that the salivary flow, lysozyme activity, total antioxidant capacity and TM7 bacterial family discriminated between tasters and non-tasters. Subgroup analysis of obese tasters (OT) versus obese non-tasters (ONT) identified specific bacterial metabolic pathways (i.e. phosphotransferase and simple sugar transport systems) as being higher in ONT. Altogether with the identification of a set of significant salivary variables, our study suggests that an "obese tongue" phenotype is associated with decreased orosensory sensitivity to lipids in some obese subjects.

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.

Evaluation of the papilla level adjacent to implants placed in fresh, healing or healed sites: A systematic review.

A better understanding of factors that can lead to papilla formation or recession, such as the type of site where the implant was placed, is of fundamental importance to the aesthetic success of the rehabilitation. The aim of this study was to perform a systematic review of the literature regarding the formation or recession of papilla adjacent to implants placed in fresh, healing or healed sites. The protocol for this study was registered in the PROSPERO database (registration number CRD 42016033784). An electronic search was performed by two independent reviewers who applied the inclusion and exclusion criteria on the PubMed/MEDLINE, Scopus, and Embase databases from January 2005 up to February 2016. The initial screening yielded 1,065 articles, from which 15 were selected for a systematic review after applying the inclusion and exclusion criteria. Nine studies compared fresh and healed sites, four studies compared healing and healed sites, one study compared fresh and healing sites, and one study analysed all three sites. The majority of studies identified by this systematic review showed no difference between groups after the longer follow-up period. The sites where the implants were placed did not have a long-term influence on papilla formation or recession.

Rac1 Regulates the Proliferation, Adhesion, Migration, and Differentiation of MDPC-23 Cells.

INTRODUCTION: Stem cells are responsible for replacing damaged pulp tissue; therefore, promoting their survival and inducing their adhesion to dentin are vital. As a member of the Rho family of guanosine triphosphatases, Rac1 is an important regulator of osteoblast functions. However, little is known about its role in regenerative endodontic procedures. The current study examined the role of Rac1 in the proliferation, migration, and odontoblastic differentiation of MDPC-23 cells. METHODS: MDPC-23 cells were transfected with small interfering RNA to knock down Rac1 expression, and then their proliferation, migration, adhesion, and odontoblastic differentiation were examined in vitro. RESULTS: MDPC-23 cells transfected with si-Rac1 exhibited the increased expression of several key odontogenic protein markers, including Dmp1, Dspp, Runx2, and alkaline phosphatase, as well as decreased proliferation and migration in vitro. The results suggest that Rac1 might regulate nuclear factor kappa B signaling in MDPC-23 cells. CONCLUSION: Rac1 may have vital roles in the proliferation, migration, adhesion, and odontoblastic differentiation of MDPC-23 cells.

Evaluation of a Commercially Available Hyaluronic Acid Hydrogel (Restylane) as Injectable Scaffold for Dental Pulp Regeneration: An In Vitro Evaluation.

INTRODUCTION: Regenerative endodontic procedures (REPs) are viable alternatives for treating immature teeth, yet these procedures do not predictably lead to pulp-dentin regeneration. A true bioengineering approach for dental pulp regeneration requires the incorporation of a scaffold conducive with the regeneration of the pulp-dentin complex. Several materials have been proposed as scaffolds for REPs; nonetheless, the majority are not eligible for immediate clinical chairside use. Thus, the aim of this study was to evaluate Restylane, a Food and Drug Administration-approved hyaluronic acid-based gel, as possible scaffold for REPs. METHODS: Stem cells of the apical papilla (SCAP) were cultured either alone or in mixtures with either Restylane or Matrigel scaffolds. Groups were cultured in basal culture medium for 6, 24, and 72 hours, and cell viability was assessed. For the mineralizing differentiation experiments, groups were cultured in differentiation medium either for 7 days and processed for alkaline phosphatase activity or for 14 days and processed for gene expression by using quantitative reverse-transcription polymerase chain reaction. SCAP in basal medium served as control. RESULTS: Cell encapsulation in either Restylane or Matrigel demonstrated reduced cell viability compared with control. Nonetheless, cell viability significantly increased in the Restylane group in the course of 3 days, whereas it decreased significantly in the Matrigel group. Restylane promoted significantly greater alkaline phosphatase activity and upregulation of dentin sialophosphoprotein, dentin matrix acidic phosphoprotein-1, and matrix extracellular phosphoglycoprotein, compared with control. CONCLUSIONS: A Food and Drug Administration-approved hyaluronic acid-based injectable gel promoted SCAP survival, mineralization, and differentiation into an odontoblastic phenotype and may be a promising scaffold material for REPs.

Characterization of a Vascular Endothelial Growth Factor-loaded Bioresorbable Delivery System for Pulp Regeneration.

INTRODUCTION: Vascular endothelial growth factor (VEGF) is a signal protein that stimulates angiogenesis and vasculogenesis and has been used in tissue regeneration and pulp regeneration experimental models. The purpose of this study was to develop a delivery system composed of a biodegradable fiber and controlled release of VEGF to promote cell viability and secure an adequate blood supply for the survival of human stem cells of the apical papilla (SCAP) favoring endodontic regenerative procedures. METHODS: We developed a polydioxanone fiber, 50 µm in diameter, loaded with VEGF at a linear concentration of 12.2 ng/cm. Cytotoxic effects of the VEGF-loaded fiber (VF) on SCAP and mouse fibroblasts were assessed by using a multiparametric assay kit (XTT-NR-CVDE [Xenometrix, Allschwil, Switzerland]). We evaluated VF-induced mRNA expression of downstream growth factors by using a human growth factor Taqman array in real-time polymerase chain reaction. We also assessed the in vivo subcutaneous reaction of C57BL/6 mice to implants of VF alone and human root fragments (10 mm in length) filled with VF after 10, 20, and 45 days. Statistical analyses were performed by using analysis of variance and Student t tests or non-parametric alternatives. RESULTS: Enzyme-linked immunosorbent assay verified detectable concentrations of released VEGF in solution for 25 days. No cytotoxicity was observed on SCAP and mouse fibroblasts treated with VEGF. In addition, VEGF treatment also induced the expression of additional growth factors with roles in tissue and blood vessel formation and neuroprotective function. Implantation of VF and root fragments filled with VF showed biocompatibility in vivo, promoting new blood vessels and connective tissue formation into the root canal space with negligible inflammation. CONCLUSIONS: Our results show that the VF used in this study is biocompatible and may be a promising scaffold for additional optimization and use in endodontic regenerative procedures.

Effects of a Bioactive Scaffold Containing a Sustained Transforming Growth Factor-ß1-releasing Nanoparticle System on the Migration and Differentiation of Stem Cells from the Apical Papilla.

INTRODUCTION: This 2-part study hypothesized that a bioactive scaffold containing a sustained transforming growth factor (TGF)-ß1-releasing nanoparticle system will promote migration and enhance differentiation of stem cells from the apical papilla (SCAP). The study aimed to develop and characterize a novel modified chitosan-based scaffold containing TGF-ß1-releasing chitosan nanoparticles (TGF-ß1-CSnp) to enhance migration and differentiation of SCAP. METHODS: Part I concerns the synthesis and characterization of a carboxymethyl chitosan-based scaffold and TGF-ß1-CSnp. Part II examines the effect of sustained TGF-ß1 release from scaffold containing TGF-ß1-CSnp on odontogenic differentiation of SCAP. RESULTS: The scaffold demonstrated properties conducive to cellular activities. The incorporation of TGF-ß1 in CSnp allowed sustained release of TGF-ß1, facilitating delivery of a critical concentration of TGF-ß1 at the opportune time. TGF-ß1 bioactivity was maintained for up to 4 weeks. SCAP showed greater viability, migration, and biomineralization in the presence of TGF-ß1-CSnp than in the presence of free TGF-ß1. SCAP cultured in TGF-ß1-CSnp + scaffold showed significantly higher dentin matrix protein-1 and dentin sialophosphoprotein signals compared with free TGF-ß1 + scaffold or CSnp + scaffold. CONCLUSIONS: These experiments highlighted the potential of a carboxymethyl chitosan-based scaffold with growth factor releasing nanoparticles to promote migration and differentiation of SCAP. The results of this study may have direct application to improve current endodontic regenerative protocols.

EphrinB2 Stabilizes Vascularlike Structures Generated by Endothelial Cells and Stem Cells from Apical Papilla.

INTRODUCTION: This study aimed to investigate the roles of ephrinB2 in stabilizing vascularlike structures generated by stem cells from apical papilla (SCAPs) and human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs were seeded alone or with SCAPs concurrently or 12 hours later. Angiogenesis and ephrinB2 phosphorylation were assayed at different time points. Additionally, ephrinB2 expression in SCAPs and HUVECs was silenced with small interfering RNA, and vascularlike structure formation within coculture was assessed; 1 × 10(5) HUVECs were seeded in transwell inserts, and 6 × 10(5) SCAPs were plated in lower wells with or without ephrinB2-Fc. Migratory cells were stained and counted. Delayed addition of ephrinB2-Fc to the coculture of HUVECs and SCAPs was performed to evaluate the role of ephrinB2 on the stabilization of vascularlike structures. RESULTS: Concurrent coculture of SCAPs and HUVECs yielded significantly longer tubule lengths at 4, 8, and 12 hours (P < .05). Delayed addition of SCAPs to coculture with HUVECs resulted in vascularlike structures persisting longer than the HUVEC monoculture. Western blot confirmed that ephrinB2 phosphorylation was initiated at 0.5 hours of coculture and peaked at 1 hour. Silencing ephrinB2 expression in SCAPs and HUVECs resulted in the absence of vascularlike structures. Enhanced migration of HUVECs by SCAPs could be inhibited by ephrinB2-Fc. When ephrinB2-Fc was added at 3 hours of coculture, the vascularlike structures were stabilized for more than 12 hours as compared with 9 hours in the control group. CONCLUSIONS: EphrinB2 plays an important role in the stabilization of vascularlike structures generated by HUVECs and SCAPs.

Composition of Mineral Produced by Dental Mesenchymal Stem Cells.

Mesenchymal stem cells isolated from different dental tissues have been described to have osteogenic/odontogenic-like differentiation capacity, but little attention has been paid to the biochemical composition of the material that each produces. Here, we used Raman spectroscopy to analyze the mineralized materials produced in vitro by different dental cell populations, and we compared them with the biochemical composition of native dental tissues. We show that different dental stem cell populations produce materials that differ in their mineral and matrix composition and that these differ from those of native dental tissues. In vitro, BCMP (bone chip mass population), SCAP (stem cells from apical papilla), and SHED (stem cells from human-exfoliated deciduous teeth) cells produce a more highly mineralized matrix when compared with that produced by PDL (periodontal ligament), DPA (dental pulp adult), and GF (gingival fibroblast) cells. Principal component analyses of Raman spectra further demonstrated that the crystallinity and carbonate substitution environments in the material produced by each cell type varied, with DPA cells, for example, producing a more carbonate-substituted mineral and with SCAP, SHED, and GF cells creating a less crystalline material when compared with other dental stem cells and native tissues. These variations in mineral composition reveal intrinsic differences in the various cell populations, which may in turn affect their specific clinical applications.

Role of ALK5/Smad2/3 and MEK1/ERK Signaling in Transforming Growth Factor Beta 1-modulated Growth, Collagen Turnover, and Differentiation of Stem Cells from Apical Papilla of Human Tooth.

INTRODUCTION: Transforming growth factor ß1 (TGF-ß1) plays an important role in cell proliferation, matrix formation, and odontogenesis. This study investigated the effects of TGF-ß1 on stem cells from apical papilla (SCAPs) and its signaling by MEK/ERK and Smad2. METHODS: SCAPs were exposed to TGF-ß1 with/without pretreatment and coincubation by SB431542 (an ALK5/Smad 2/3 inhibitor) or U0126 (a MEK/ERK inhibitor). Cell growth was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide assay or direct counting of viable cells. Collagen content was determined by using the Sircol collagen assay (Biocolor Ltd, Newtownabbey, Northern Ireland). Cell differentiation was evaluated by measuring alkaline phosphatase (ALP) activity. Smad2 and ERK1/2 phosphorylation was analyzed by Western blotting or PathScan phospho-enzyme-linked immunosorbent assay (Cell Signaling Technology Inc, Danvers, MA). RESULTS: TGF-ß1 stimulated the growth and collagen content of cultured SCAPs. TGF-ß1 stimulated ERK1/2 and Smad2 phosphorylation within 60 minutes of exposure. Pretreatment by U0126 and SB431542 effectively prevented the TGF-ß1-induced cell growth and collagen content in SCAPs. TGF-ß1 stimulated ALP activity at lower concentrations (0.1-1 ng/mL) but down-regulated ALP at higher concentrations (>5 ng/mL). U0126 prevented 0.5 ng/mL TGF-ß1-induced ALP activity but showed little effect on 10 ng/mL TGF-ß1-induced decline of ALP in SCAPs. Interestingly, SB431542 attenuated both the stimulatory and inhibitory effects on ALP by TGF-ß1. CONCLUSIONS: TGF-ß1 may affect the proliferation, collagen turnover, and differentiation of SCAPs via differential activation of ALK5/Smad2 and MEK/ERK signaling. These results highlight the future use of TGF-ß1 and SCAP for engineering of pulpal regeneration and apexogenesis.

Papilla regeneration by injectable stem cell therapy with regenerative medicine: long-term clinical prognosis.

Black triangle (BT), an open interproximal space between teeth, can cause aesthetic concerns, food impaction, phonetic difficulties and periodontitis. The aim of this study was to determine the possibility and long-term prognosis of novel papilla regeneration with regenerative medicine, i.e. tissue-engineered papilla (TEP), and to investigate the potential of a tissue-engineering method for soft-tissue augmentation, especially aesthetic improvement of BT, with mesenchymal stem cells (MSCs) as the isolated cells, platelet-rich plasma (PRP) as the growth factor and hyaluronic acid (HA) as the scaffold. The parameters were assessed from a clinical point of view by measuring the distance from the tip of the interproximal papilla to the base of the contact area in each study region. The mean volumes, operation times and follow-up periods of TEP were 1.32 ± 0.25 ml, 2.2 ± 1.62 times and 55.3 ± 17.7 months; the mean improved BT values were 2.55 ± 0.89 mm. An aesthetic improvement was achieved. TEP was able to provide aesthetic improvement of black triangle and predictable results, and could emerge as another novel option for periodontal regenerative therapy in periodontal diseases.

Influência de medicações intracanal utilizadas em procedimentos endodônticos regenerativos na sobrevivência de células da papila apical in vitro / Influence of intracanal medications used in regenerative endodontic procedures on survival of apical papilla cells in vitro

Procedimentos endodônticos regenerativos proporcionaram mudanças no tratamento de pacientes com dentes imaturos e periodontite apical possibilitando desenvolvimento radicular completo e menor incidência de fratura dental. Medicações intracanal são utilizadas para a realização da desinfecção; entretanto, o efeito delas sobre às células da papila apical é pouco elucidado. Adicionalmente, pouco se conhece a respeito do efeito destas substâncias sobre células previamente submetidas à condição pró- inflamatória. O objetivo deste estudo foi avaliar a citotoxicidade de medicações intracanal empregadas em procedimentos regenerativos em Endodontia sobre células de papila humana em cultura em condição fisiológica e ativada. Cultura de células foi estabelecida a partir da papila apical removida de um terceiro molar imaturo extraído. As substâncias estudadas foram a pasta tripla antibiótica modificada: ciprofloxacina, metronidazol e cefalosporina (1:1:1); CFC: ciprofloxacina, metronidazol e hidróxido de cálcio (1:1:2) e CFC modificado: ciprofloxacina, metronidazol e hidróxido de cálcio (2:2:1). Parte das células foram estimuladas previamente por ácido lipoteicóico (LTA) de Enterococcus faecalis por 7 dias. Após plaqueamento, células foram expostas a concentrações crescentes das medicações por 1, 3, 5 e 7 dias. Foram avaliados viabilidade celular por meio de brometo de 3-(4,5-dimetiliazol-2-il)-2,5-difeniltetrazólio (MTT) e liberação de óxido nítrico (NO) pelo método de Griess

A análise estatística foi realizada por meio de análise de variância a 1 critério (ANOVA) seguida de pós teste de Tukey, com nível de significância de 5%. O CFC modificado foi a medicação que demonstrou menor efeito citotóxico sobre a viabilidade celular nos tempos experimentais estudados, o CFC promoveu queda da viabilidade celular especialmente após 7 dias de contato. A pasta tripla antibiótica modificada resultou em comprometimento importante da viabilidade podendo ser considerada a mais citotóxica. A ativação celular por LTA resultou em níveis aumentados de atividade mitocondrial para todas as medicações sendo mais evidente nos períodos experimentais mais longos. A ativação celular também contribuiu para níveis maiores de óxido nítrico. Conclui-se que o efeito citotóxico das medicações testadas é dependente de sua concentração, tempo de contato e condição celular, sendo a pasta tripla antibiótica modificada a mais citotóxica em concentrações elevadas podendo implicar clinicamente na diminuição da viabilidade das células da papila apical podendo diminuir o sucesso dos procedimentos regenerativos.

Regenerative Endodontic procedures have provided changes in treatment of patients with immature teeth and apical periodontitis enabling full root development and lower incidence of dental fracture. Intracanal dressings are used for disinfection; however, their effect on apical papilla cells is poorly elucidated. Additionally, the effect of these substances on cells previously subjected to proinflammatory condition is still unknown. The aim of this study was to evaluate the cytotoxicity of intracanal dressings used in Endodontics regenerative procedures on cultured human apical papilla cells at physiologic and activated condition. Cell culture was established from the apical papilla removed from an extracted immature third molar. The substances studied were triple antibiotic modified paste: ciprofloxacin, metronidazole and cephalosporin (1:1:1); CFC: ciprofloxacin, metronidazole and calcium hydroxide (1:1:2) and modified CFC: ciprofloxacin , metronidazole and calcium hydroxide (2:2:1). Part of the cells was stimulated previously with lipotheichoic acid (LTA) of Enterococcus faecalis por 7 days. Once plated, cells were exposed to increasing concentration of the medications for 1, 3, 5 and 7 days. Cell viability was evaluated by means of 3-bromide (4.5-dimetiliazol-2-yl) -2.5-difeniltetraze (MTT) and Nitric Oxide (NO) release was assessed by the Griess method.

The statistical analysis was done through analysis of variance with 1 criteria (ANOVA) followed by Tukey test with 5% of significance level. Modified CFC was the medication that demonstrated the less cytotoxic effect on cell viabilityat the experimental periods studied while CFC promoted significant decrease on cell viability specially after 7 days of contact. The modified triple antibiotic paste resulted in important alteration of cell viability being considered the most citotoxic. Cellular activation by LTA resulted in increased levels of mitochondrial activity for all medications being more evident at the longer experimental periods. Cellular activation also contributed to higher levels of nitric oxide release. In conclusion, the cytotoxic effect of the tested medications is dependent on concentration, time of contact and cellular condition, being the triple antibiotic modified paste the most cytotoxic in high concentrations leading clinically in the decreased of the cells viability of the apical papilla, decreasing the success of regenerative procedures.

A comparison of bovine bone and hydroxyapatite scaffolds during initial bone regeneration: an in vitro evaluation.

OBJECTIVES: To evaluate the different behavior of 3-dimensional biomaterial scaffolds-Bovine Bone (BB; Bio-Oss) and Hydroxyapatite (HA; ENGIpore)-during initial bone healing and development. MATERIALS AND METHODS: Human dental papilla stem cells (hDPaSCs) were selected with FACsorter cytofluorimetric analysis, cultured with osteogenic medium, and analyzed with Alizarin red stained after differentiation. The obtained osteoblast-like cells (OCs) were cultured with BB and HA. alkaline phosphatase (ALP), OC, MEPE, and runt-related transcription factor 2 (RUNX2) expression markers were investigated performing Western blot and reverse transcription-polymerase chain reaction (RT-PCR) analysis. After 40 days, samples were analyzed by light and electron microscopy. RESULTS: All the samples showed high in vitro biocompatibility and qualitative differences of OCs adhesion. RT-PCR and Western blot data exhibited similar marker rate, but ALP, OC, MEPE, and RUNX2expression, during initial healing and bone regeneration phase, was higher and faster in human dental papilla onto BB than in HA scaffolds. In biomaterials growth, RUNX2 seems to play an important role as a key regulator in human OCs from dental papilla bone development. CONCLUSION: Different surface BB scaffold characteristics seem to play a critical role in OCs differentiation showing different time of bone regeneration morphological characteristics as well as higher and faster levels of all observed markers.

Epiregulin can promote proliferation of stem cells from the dental apical papilla via MEK/Erk and JNK signalling pathways.

OBJECTIVES: Mesenchymal stem cells (MSCs) are a reliable resource for tissue regeneration, but their molecular mechanisms of differentiation and proliferation remain unclear; this situation has restricted use of MSCs to a limited number of applications. A previous study of ours found a member of the epidermal growth factor family, epiregulin (EREG), to be involved in regulation of MSC differentiation. In the present study, we have used human dental stem cells from the apical papilla (SCAPs) to investigate the role of EREG on proliferation of MSCs. MATERIALS AND METHODS: SCAPs were isolated from apical papillae of immature third molars. Retroviral short hairpin RNA (shRNA) was used to silence EREG gene expression, and human recombinant EREG protein was used to stimulate SCAPs. SCAP proliferation was examined using tetrazolium dye colorimetric assay/cell growth curve. Western blotting was performed to detect expressions of extracellular signal-regulated protein kinases 1 and 2 (Erk1/2), mitogen-activated protein kinases 1 and 2 (MEK1/2), protein kinase B (Akt), p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun N-terminal kinase (JNK). RESULTS: Depletion of EREG with shRNA inhibited SCAP proliferation and repressed phosphorylation of Erk1/2 and JNK. Human recombinant EREG protein promoted cell proliferation and enhanced Erk1/2, MEK and JNK phosphorylation in SCAPs. Furthermore, blocking MEK/Erk signalling with specific Erk1/2 inhibitor PD98059, or JNK signalling with specific inhibitor SP600125, abolished effects of EREG on cell proliferation. CONCLUSION: These findings indicate that EREG could enhance cell proliferation in dental tissue-derived MSCs by activating MEK/Erk and JNK signalling pathways.

Transgene-free induced pluripotent dental stem cells for neurogenic differentiation.

A stem-cell-based therapy could be the ultimate strategy for the regeneration of degenerated nervous tissues. While neural progenitor cells are limited, the generation of functional nervous tissue cells from non-neural somatic cells (for example, dental stem cells) is highly desired. The recent publication in Stem Cell Research and Therapy by Huang and colleagues is an interesting contribution to this topic. The present commentary puts this paper in context with contemporary reports about (transgene-free) induced pluripotent stem cells and neurogenic differentiation.