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To evaluate the osteogenic potential of platelet-rich fibrin (PRF) and low-level laser therapy (LLLT) on human stem cells from the apical papilla (SCAP) we isolated, characterized, and then cultured in an osteogenic medium cells with PRF and/or LLLT (660 nm, 6 J/m2-irradiation). Osteogenic differentiation was assessed by bone nodule formation and expression of bone morphogenetic proteins (BMP-2 and BMP-4), whereas the molecular mechanisms were achieved by qRT-PCR and RNA-seq analysis. Statistical analysis was performed by ANOVA and Tukey's post hoc tests (p < 0.05* and p < 0.01**). Although PRF and LLLT increased bone nodule formation after 7 days and peaked at 21 days, the combination of PRF + LLLT led to the uppermost nodule formation. This was supported by increased levels of BMP-2 and -4 osteogenic proteins (p < 0.005). Furthermore, the PRF + LLLT relative expression of specific genes involved in osteogenesis, such as osteocalcin, was 2.4- (p = 0.03) and 28.3- (p = 0.001) fold higher compared to the PRF and LLLT groups, and osteopontin was 22.9- and 1.23-fold higher, respectively (p < 0.05), after 7 days of interaction. The transcriptomic profile revealed that the combination of PRF + LLLT induces MSX1, TGFB1, and SMAD1 expression, after 21 days of osteogenic differentiation conditions exposition. More studies are required to understand the complete cellular and molecular mechanisms of PRF plus LLLT on stem cells. Overall, we demonstrated for the first time that the combination of PRF and LLLT would be an excellent therapeutic tool that can be employed for dental, oral, and craniofacial repair and other tissue engineering applications.
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Osteogênese , Fibrina Rica em Plaquetas , Humanos , Fibrina Rica em Plaquetas/metabolismo , Proliferação de Células , Células Cultivadas , Células-Tronco , Diferenciação Celular , LasersRESUMO
Los procedimientos endodónticos regenerativos (REPs) representan una evolución significativa en el campo de la endodoncia, buscando no sólo tratar la infección o lesión presente en el diente, sino tam-bién promover la regeneración de los tejidos denta-rios afectados. El presente caso clínico muestra un incisivo lateral superior izquierdo con apexogénesis incompleta y diagnóstico de absceso alveolar crónico reagudizado en una paciente de 22 años, en el que se aplicó un procedimiento de endodoncia regenerativa (REPs). La estrategia terapéutica elegida se basó en los principios de ingeniería tisular, incorporando la novedosa aplicación de la membrana amniótica hu-mana liofilizada esterilizada como andamio bioactivo intraconducto. Las evaluaciones clínicas, radiográ-ficas y tomográficas a corto, mediano y largo plazo revelaron el éxito de la terapia. La resolución exitosa mostró en los controles a la pieza dentaria asintomá-tica, con una notable remisión de la patología apical, aumento de la longitud radicular y disminución del calibre apical. Se ha podido destacar la eficacia de los REPs, con una exitosa aplicabilidad de la membra-na amniótica como andamio innovador (AU)
Regenerative endodontic procedures (REPs) represent a significant evolution in the field of endodontics, aiming not only to address the infection or injury within the tooth, but also to promote the regeneration of the affected dental tissues. In this clinical case, an upper left lateral incisor with incomplete apexogenesis and diagnosis of acute exacerbation of a chronic periapical lesion in a 22-year-old patient is presented. A regenerative endodontic procedure (REPs) was applied. The chosen therapeutic strategy was based on tissue engineering principles, incorporating the innovative use of sterilized lyophilized human amniotic membrane as an intraconduct bioactive scaffold. Clinical, radiographic, and tomographic assessments at short, medium, and long-term follow-up revealed the success of the therapy. Successful resolution demonstrated an asymptomatic tooth in the follow-up, with a notable resolution of apical pathology, increased root length, and decreased apical caliber. The effectiveness of REPs has been highlighted, demonstrating the successful applicability of amniotic membrane as an innovative scaffold (AU)
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Humanos , Feminino , Adulto , Células-Tronco/fisiologia , Alicerces Teciduais , Argentina , Faculdades de Odontologia , Papila Dentária , Liofilização/métodosRESUMO
OBJECTIVE: To analyze the effect of P11-4 self-assembly peptide on cell viability and osteogenic capacity of SCAPs through mineral deposition and gene expression of osteogenic markers. METHODS: SCAPs were seeded in contact with P11-4 (10 µg/ml, 100 µg/ml and 1 mg/ml) solution. Cell viability was evaluated using a colorimetric assay MTT: 3-(4,5-dimethyl-thiazolyl-2)-2,5- diphenyltetrazolium bromide) in an experimental time of 24, 48 and 72 h (n = 7). Mineral deposition and quantification provided by the cells was tested using the Alizarin Red staining and Cetylpyridinium Chloride (CPC), respectively, after 30 days (n = 4). Gene expression of Runt-related transcription factor 2 (RUNX2), Alkaline phosphatase (ALP) and Osteocalcin (OCN) was quantified using quantitative polymerase chain reaction (RT-qPCR), at 3 and 7 days with Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the housekeeping gene, and relative gene expression was measured using the ΔΔCq method. Data were analyzed using Kruskall-Wallis followed by multiple comparisons, and T-test for gene expression with α=0.05. RESULTS: All tested concentrations (10 µg/ml, 100 µg/ml and 1 mg/ml) were not cytotoxic at time 24 and 48 h. After 72 h, a slight decrease in cell viability was observed for the lowest concentration (10 µg/ml). The concentration of 100 µg/ml P11-4 showed the highest mineral deposition. However, qPCR analysis of P11-4 (10 µg/ml) showed upregulation of RUNX2 and OCN at 3 days, with downregulation of ALP at 3 and 7d CONCLUSION: P11-4 did not affect cell viability, induced mineral deposition in SCAPs, and upregulated the expression of RUNX2 and OCN genes at 3 days, while downregulating ALP expression at 3 and 7 days. CLINICAL SIGNIFICANCE: Based on the results obtained in this study it can be stated that self-assembling peptide P11-4 is a potential candidate to induce mineralization on dental stem cells for regenerative purposes and also for a clinical use as a capping agent without compromising the cells health.
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Subunidade alfa 1 de Fator de Ligação ao Core , Osteogênese , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Osteogênese/genética , Papila Dentária/metabolismo , Diferenciação Celular/genética , Células-Tronco/metabolismo , Proliferação de Células , Células CultivadasRESUMO
Dental pulp under physiological conditions has a defense function, repair capacity, and important mechanisms in pathological processes. In addition, the dental papilla is involved in important defense processes and an essential function in the pulp revascularization process. It is known that dental pulp and apical papilla undergo a natural aging process, in addition to stressful situations such as bruxism, inflammation, and infections. Both aging and stressful situations can lead to cellular senescence. Some evidence indicates that the changes resulting from this cellular state can directly affect the efficiency of cells in these tissues and affect conservative and regenerative clinical treatments. Thus, it is necessary to understand the causes and consequences of cellular senescence in addition to the development of methods for senescence prevention. This review aims to provide an overview of possible causes and consequences of senescence in dental pulp and stem cells from apical papilla and discusses possible methods to prevent this cellular state.
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Senescência Celular , Polpa Dentária , Humanos , Células-Tronco/fisiologia , Envelhecimento , Inflamação , Diferenciação CelularRESUMO
This study aimed to evaluate the expression of several differentiation markers in the apical papilla (AP) and dental pulp (DP) of human permanent teeth. Twenty young human teeth were extracted and classified according to three Moorrees tooth development stages: initial root formation (Ri), root length ½ (R1/2), and root length complete (Rc). Immunohistochemical assays were performed using STRO-1, VEGF Receptor-2, Neurofilament heavy (NFH), and Nestin antibodies and analyzed under light microscopy. Decalcified, formalin fixed paraffin embedded tooth sections stained with hematoxylin and eosin showed an apical cell rich zone between the DP and AP. The AP revealed fewer vascular and cellular components than the DP. STRO-1 was expressed on vascular and neuronal elements beneath the odontoblast (OB) and in the sub-odontoblastic (SOB) zone, and VEGFR-2 positive cells were observed in the endothelium, arterioles, and blood vessels. Neuroepithelial stem cell protein (Nestin) was highly expressed in differentiated odontoblasts in the predentin odontotoblast and odontoblast cell processes. Neurofilament heavy (NFH) was expressed in mature axons throughout the DP. STRO-1 and VEGFR-2 microvascular expression was higher at the stages Ri and R1/2 while STRO-1 and NFH expression showed strong spatial distribution of Rc neuronal elements as compared to Ri and R1/2. Differentiated OB and SOB cells showed Nestin expression, indicating a reservoir of newly differentiated odontoblast-like cells.
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Polpa Dentária , Receptor 2 de Fatores de Crescimento do Endotélio Vascular , Humanos , Nestina , Antígenos de Diferenciação , Células-Tronco , BiomarcadoresRESUMO
Abstract Fetal bovine serum (FBS) is the most used supplement in culture media; however, it may interfere with in vitro assays via effects on cell proliferation and cytokine production. The ideal FBS concentration for assays using apical papilla cells (APCs) remains unknown. Therefore, this study aimed to evaluate the effects of FBS on APC activation, cell viability/proliferation, and cytokine production. Methodology Human APCs were cultured, plated, and maintained in media containing increasing concentrations of FBS for 24 h, 48 h, 72 h, 7 days, and 14 days in the presence of Lipopolysaccharide (LPS - 1 µg/mL). At each time point, the cells were subjected to the MTT assay. The cytokines transforming growth factor (TGF)-β1, osteoprotegerin (OPG), and interleukin (IL)-6, along with the chemokine CCL2, were quantified using the enzyme-linked immunosorbent assay at the 24-h time-point. Statistical analysis was performed using two-way analysis of variance (ANOVA) followed by Tukey's post-hoc test (p<0.05). Results In general, APCs exhibited increasing metabolic activity in an FBS concentration-dependent fashion, regardless of the presence of LPS. In contrast, FBS interfered with the production of all the cytokines evaluated in this study, affecting the response induced by the presence of LPS. Conclusion FBS increased APC metabolism in a concentration-dependent manner and differentially affected the production of TGF-β1, OPG, IL-6, and CCL2 by APCs in vitro.
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INTRODUCTION: The research for alternative irrigating solutions is ongoing, since no "ideal" solution has yet been found. Octenidine dihydrochloride (OCT) has been indicated as an endodontic irrigant because it has adequate antimicrobial and biological properties. The present study aimed to assess the effects of OCT on proliferation, migration, and induction of the osteogenic phenotype of stem cells from human dental pulp and apical papilla. METHODS: Cells were collected from human third molars and exposed to different doses of OCT, chlorhexidine (CHX), sodium hypochlorite (NaOCl), and ethylenediaminetetraacetic acid (EDTA) to determine cell viability by alamarBlue assay; proliferation by bromodeoxyuridine incorporation; migration by the Transwell assay; alkaline phosphatase activity by thymolphthalein release; and production of mineralized nodules by alizarin red staining. The results were analyzed by 1- or 2-way analysis of variance and Tukey (α = .05). RESULTS: CHX promoted lower cell viability, followed by OCT, NaOCl, and EDTA, especially at intermediate doses (P < .05). Cells exposed to CHX had less proliferation than the other groups (P < .05). The Transwell assay revealed no differences among OCT, EDTA, and culture medium (control group) (P > .05). OCT and EDTA induced greater migration than CHX and NaOCl (P < .05). OCT and EDTA induced higher alkaline phosphatase activity than NaOCl and CHX (P < .05). No difference was detected among the groups using alizarin red staining (P > .05). CONCLUSIONS: OCT induced high migration, proliferation, and alkaline phosphatase activity of stem cells from human dental pulp and apical papilla, which could be advantageous for regenerative endodontic procedures.
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Osteogênese , Irrigantes do Canal Radicular , Humanos , Irrigantes do Canal Radicular/farmacologia , Ácido Edético/farmacologia , Fosfatase Alcalina , Polpa Dentária , Hipoclorito de Sódio/farmacologia , Clorexidina/farmacologia , Células-Tronco , Proliferação de Células , Papila DentáriaRESUMO
INTRODUCTION: Many mediators are produced during pulp inflammation and necrosis, including endocannabinoids (ECbs), which might affect the function of stem cells of the apical papilla (SCAP), cells of paramount importance for root formation, and regenerative endodontic treatment. The aim of this study was to evaluate the production of osteoclastogenesis-related mediators by SCAP modulated by ECbs and lipopolysaccharide (LPS) in vitro. METHODS: SCAP were cultured and treated with ECb anandamide (AEA), 2-arachidonoylglycerol, or N-arachidonoylaminophenol. All groups were incubated in the presence of a vehicle or LPS and the antagonist of transient receptor potential cation channel subfamily V member 1, capsazepine. After 24 hours, the culture medium supernatants were collected for further quantification of tumor necrosis factor alpha, CCL2, macrophage colony-stimulating factor, osteoprotegerin, and receptor activator of nuclear factor kappa B ligand. RESULTS: Small amounts of tumor necrosis factor alpha and receptor activator of nuclear factor kappa B ligand were detected in SCAP supernatants, and none of the experimental conditions altered their production. A down-regulation in constitutive CCL2 production was observed in the AEA group compared with that in the LPS group. The production of macrophage colony-stimulating factor was significantly increased in all groups treated with AEA compared with the control and LPS-treated groups. Osteoprotegerin was significantly increased by AEA alone and by 2-arachidonoylglycerol and N-arachidonoylaminophenol in the presence of LPS and capsazepine. CONCLUSIONS: AEA modulates some of the osteoclastogenic factors produced by SCAP in a bone resorption protective fashion.
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Osteogênese , Osteoprotegerina , Fator Estimulador de Colônias de Macrófagos/farmacologia , Ligante RANK , Endocanabinoides/farmacologia , Lipopolissacarídeos/farmacologia , Fator de Necrose Tumoral alfa , Células-Tronco , Células Cultivadas , OsteoclastosRESUMO
INTRODUCTION: Endogenous cannabinoids (endocannabinoids [eCBs]) have been shown to have a multitude of functions including neurotransmission and immune modulatory effects. This study aimed to evaluate if stem cells of the apical papilla (SCAP) express the receptors and enzymes of the endocannabinoid system (ECS) and whether eCBs regulate their proliferation and mineralization potential. METHODS: Gene expression of the main components of the ECS and transient receptor potential vanilloid 1 (TRPV1) was evaluated in SCAP cultures. SCAP were treated with 2 concentrations of eCBs and/or capsazepine, a TRPV1 antagonist. SCAP viability was evaluated after 1, 4, and 7 days. Osteogenic differentiation was assessed after 14 days, and the gene expression of mineralization markers was assessed after 7 days. RESULTS: The enzymes of ECS and TRPV1 but not the cannabinoid receptors (cannabinoid receptors 1 and 2) were expressed in SCAP. Anandamide, 2-arachidonoylglycerol, and N-arachidonoylphenolamine (AM-404) reduced SCAP viability in all experimental periods at the highest concentration compared with the group with no treatment. Anandamide and AM-404 did not inhibit SCAP differentiation potential, but 2-arachidonoylglycerol at the highest concentration did. SCAP treated with AM-404 presented a down-regulation in gene expression of alkaline phosphatase (ALP), dentin matrix protein 1 (DMP-1), and dentin sialophosphoprotein (DSPP) compared with the proliferation medium group but not with control group. CONCLUSIONS: SCAP expressed the genes of the main components of ECS and TRPV1, and eCBs can affect SCAP viability, mineralization, and gene expression.
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Papila Dentária , Osteogênese , Diferenciação Celular , Endocanabinoides , Receptores de Canabinoides , Células-Tronco , Canais de Cátion TRPV/genéticaRESUMO
BACKGROUND: This study aimed to evaluate the biological response of human apical papilla cells to different calcium hydroxide formulations and three tricalcium silicate-based materials. METHODS: Primary cells were obtained from explants of young immature premolars. 20,000 cells adhered for 24 h over discs of Biodentine™, ProRoot®MTA, BioRoot®RCS and calcium hydroxide mixed either with sodium chloride 0.9%w/v or polyethylene glycol and UltraCal® were used to evaluate cell adhesion by scanning electron microscopy and cell viability by MTT assay. RESULTS: Cells adhered to ProRoot®MTA showed an increase of F-actin like protrusions, suggesting bioactivity. Cells adhered to UltraCal® show protrusion such as filopodia. On the contrary, cells adhered to BioRoot®RCS showed no signs of any cellular protrusion. Regarding viability between the materials, we found a higher percentage of viability in cells cultured over discs of Biodentine™ and ProRoot®MTA. CONCLUSION: ProRoot®MTA and Biodentine™ exhibit a better cellular response of human apical papilla cells in vitro conditions compared to BioRoot® and calcium hydroxide diluted in sodium chloride.
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Hidróxido de Cálcio , Materiais Restauradores do Canal Radicular , Compostos de Alumínio/farmacologia , Compostos de Cálcio/farmacologia , Hidróxido de Cálcio/farmacologia , Combinação de Medicamentos , Humanos , Teste de Materiais , Microscopia Eletrônica de Varredura , Óxidos , Silicatos/farmacologiaRESUMO
This report describes the unexpected formation of root-like structures following the avulsion of immature permanent teeth without replantation. A 6-year-old female patient had avulsed the four permanent mandibular incisors and the two deciduous mandibular canines. The patient was seen in an emergency healthcare unit but did not receive specialized treatment for tooth replantation. As follow-up treatment, she received a removable prosthesis. After 4 years of follow-up, an image obtained by panoramic radiography showed formations similar to four root structures in the alveolus of the previously avulsed permanent teeth. This finding was confirmed by periapical radiography and computed tomography. This case report demonstrates that in teeth with incomplete root development, even after avulsion without replantation, cells from the pulp stump may have the capacity to form mineralized structures that appear radiographically comparable to root dentin.
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Avulsão Dentária , Criança , Necrose da Polpa Dentária , Dentição Permanente , Feminino , Humanos , Incisivo/diagnóstico por imagem , Avulsão Dentária/diagnóstico por imagem , Reimplante DentárioRESUMO
INTRODUCTION: This study evaluated photodynamic therapy (PDT) and photobiomodulation therapy (PBM) as adjuncts to pulp revascularization using cultures of apical papilla cells (APCs) and endothelial cells (HUVECs). METHODS: The root canal and apical foramen of 2 mandibular first premolars were enlarged to simulate immature teeth. The canal of 1 tooth was filled with 1 mL 0.005% methylene blue (MB). After that, the canals of both teeth were irrigated with 20 mL 1.5% sodium hypochlorite (NaOCl) and 20 mL 17% EDTA. The resulting solutions were diluted in cell culture media at a concentration of 0.5% (0.5% MB + NaOCl + EDTA and 0.5% NaOCl + EDTA). After PDT (0.5% MB + NaOCl + EDTA + PDT) and PBM (0.5% NaOCl + EDTA + PBM) applications, the effects were evaluated to determine cytotoxicity, polarity index, APC migration, and HUVEC sprouting, and results were compared with those of their controls (solutions without laser application). Cell culture media (CT) was also used as a control. Data were analyzed using 1-way analysis of variance and the Tukey post hoc test (P ≤ .05). RESULTS: PDT and PBM promoted greater APC viability than their controls, and PDT had greater cell viability than CT (P < .05). All protocols reduced APC migration when compared with CT (P < .05). HUVEC sprouts grown out of spheroids in PBM had a greater ratio area than their control (P ≤ .01), and the PDT ratio of the spheroid area was similar to that of its control (P > .05). CONCLUSIONS: PBM and PDT seem to be potentially effective adjuncts to revascularization in nonvital immature teeth.
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Fotoquimioterapia , Cavidade Pulpar , Ácido Edético , Células Endoteliais , Fármacos Fotossensibilizantes/uso terapêutico , Irrigantes do Canal Radicular/uso terapêutico , Preparo de Canal Radicular , Hipoclorito de Sódio/farmacologiaRESUMO
Introduction: Low-level laser therapy (LLLT) has been reported to improve cell proliferation and differentiation. The stem cells derived from dental apical papilla (SCAPs) are a promising therapy because they are easily obtained from immature human teeth. The effect of LLLT over SCAPs is still unknown. This study aimed to evaluate the proliferation and osteogenic potential of the SCAPs stimulated with LLLT. Methods: SCAPs were isolated from the third molars of a healthy donor and characterized according to the minimum established criteria. SCAPs were cultured for 24 hours before being exposed to LLLT. Cells were exposed to different doses, energy, and wavelengths for selecting the irradiation parameters. SCAPs proliferation was evaluated with the MTT assay at 24 hours and 7-day post-laser exposure. VEGF and TGFß2 expression were assessed with a specific enzyme-linked immunosorbent assay (ELISA). The osteogenic differentiation potential was analyzed with alizarin red staining, and the nodule quantification was performed by the relative optical density (ROD) analysis using ImageJ software. Results: The cells isolated from the apical papilla showed phenotype and stem cell properties. SCAPs irradiated with one dose at 6 J/m2 and 650 nm exhibited significantly higher proliferation (P>0.05) than the controls nonirradiated. LLLT stimulated SCAPs' expression of factors VEGF and TGFß2. Also, SCAPs irradiated showed higher osteogenic activity (P<0.05). Conclusion: LLLT promotes proliferation, osteogenic differentiation, and VEGF and TGFß2 expression on SCAPs. LLLT is a practical approach for the preconditioning of SCAPs in vitro for future regenerative therapies. More studies are needed to determine the underlying molecular processes that determine the mechanism of the LLLT.
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Fibronectin (FN)-loaded nanofiber scaffolds were developed and assessed concerning their bioactive potential on human apical papilla cells (hAPCs). First, random (NR) and aligned (NA) nanofiber scaffolds of polycaprolactone (PCL) were obtained by electrospinning technique and their biological properties were evaluated. The best formulations of NR and NA were loaded with 0, 5, or 10 µg/ml of FN and their bioactivity was assessed. Finally, FN-loaded NR and NA tubular scaffolds were prepared and their chemotactic potential was analyzed using an in vitro model to mimic the pulp regeneration of teeth with incomplete root formation. All scaffolds tested were cytocompatible. However, NR and NA based on 10% PCL promoted the highest hAPCs proliferation, adhesion and spreading. Polygonal and elongated cells were observed on NR and NA, respectively. The higher the concentration of FN added to the scaffolds, greater cell migration, viability, proliferation, adhesion and spreading, as well as collagen synthesis and gene expression (ITGA5, ITGAV, COL1A1, COL3A1). In addition, tubular scaffolds with NA loaded with FN (10 µg/ml) showed the highest chemotactic potential on hAPCs. It was concluded that FN-loaded NA scaffolds may be an interesting biomaterial to promote hAPCs-mediated pulp regeneration of endodontically compromised teeth with incomplete root formation.
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Materiais Biocompatíveis/química , Fibronectinas/química , Nanofibras/química , Poliésteres/química , Alicerces Teciduais/química , Adolescente , Adesão Celular , Proliferação de Células , Colágeno/química , Cadeia alfa 1 do Colágeno Tipo I/genética , Cadeia alfa 1 do Colágeno Tipo I/metabolismo , Colágeno Tipo III/genética , Colágeno Tipo III/metabolismo , Polpa Dentária , Feminino , Regeneração Tecidual Guiada , Humanos , Integrinas/genética , Integrinas/metabolismo , Masculino , Regeneração , Engenharia TecidualRESUMO
The regenerative effects of stem cells derived from dental tissues have been previously investigated. This study assessed the potential of human tooth stem cells from apical papilla (SCAP) on nerve regeneration. The SCAP collected from nine individuals were characterized and polarized by exposure to interferon-γ (IFN-γ). IFN-γ increased kynurenine and interleukin-6 (IL-6) production by SCAP, without affecting the cell viability. IFN-γ-primed SCAP exhibited a decrease of brain-derived neurotrophic factor (BDNF) mRNA levels, followed by an upregulation of glial cell-derived neurotrophic factor mRNA. Ex vivo, the co-culture of SCAP with neurons isolated from the rat dorsal root ganglion induced neurite outgrowth, accompanied by increased BDNF secretion, irrespective of IFN-γ priming. In vivo, the local application of SCAP reduced the mechanical and thermal hypersensitivity in Wistar rats that had been submitted to sciatic chronic constriction injury. The SCAP also reduced the pain scores, according to the evaluation of the Grimace scale, partially restoring the myelin damage and BDNF immunopositivity secondary to nerve lesion. Altogether, our results provide novel evidence about the regenerative effects of human SCAP, indicating their potential to handle nerve injury-related complications.
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Papila Dentária/citologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Regeneração Nervosa/fisiologia , Adolescente , Animais , Diferenciação Celular , Polaridade Celular/efeitos dos fármacos , Quimiocinas/metabolismo , Doença Crônica , Constrição Patológica , Modelos Animais de Doenças , Gânglios Espinais/metabolismo , Humanos , Inflamação/patologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Interferon gama/farmacologia , Masculino , Neurônios/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Wistar , Receptor 3 Toll-Like/agonistas , Receptor 3 Toll-Like/metabolismo , Receptor 4 Toll-Like/agonistas , Receptor 4 Toll-Like/metabolismo , Adulto JovemRESUMO
INTRODUCTION: Stem cells of apical papilla (SCAP) may be affected by inflammatory mediators released by activation with lipopolysaccharide (LPS) from infected pulpal cavities of necrotic immature teeth. Therefore, this study aimed to investigate the presence of a local renin-angiotensin system (RAS) and the role of angiotensin II (Ang II) on the modulation of SCAP in vitro. METHODS: Primary cultures of SCAP were incubated with LPS (0.1-10 µg/mL) for cell viability and quantification of the chemokine CCL2. Components of RAS were searched by gene expression of angiotensinogen (AGTN), angiotensin converting enzyme (ACE), renin, angiotensin receptor 1 (AT1) and 2 (AT2), and Mas receptor. Ang II was investigated in SCAP supernatants. Immunofluorescence was used to detect AGTN and AT1. Next, cells were treated with Ang II for viability/proliferation assessment, quantification of CCL2 and interleukin 6, and mineralization assay. Data were evaluated by analysis of variance using Tukey post hoc comparisons or the Student t test. P values <.05 were considered to be significant. RESULTS: LPS increased CCL2 production at 1 and 10 µg/mL. The gene expression of AGTN, renin, ACE, and AT1 was detected, but only ACE was increased by LPS. Ang II peptide was found in SCAP supernatants but unaltered by LPS. Both AGTN and AT1 proteins were detected by immunostaining. Ang II significantly induced SCAP proliferation, increased CCL2 production, down-regulated IL-6 release, and reduced the SCAP mineralization rate. CONCLUSIONS: A local RAS was found at the apical papilla, and Ang II was able to modulate SCAP function in vitro.
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Angiotensina II , Sistema Renina-Angiotensina , Proliferação de Células , Humanos , Lipopolissacarídeos , Células-TroncoRESUMO
INTRODUCTION: Root canal treatment of immature necrotic teeth is a major challenge in current endodontics. The effect of inflammatory mediators, such as prostaglandin, on the modulation of stem cells of the apical papilla (SCAP) is not completely understood. The aim of this study was to investigate the role of prostaglandin E2 (PGE2) on SCAP activation by Escherichia coli lipopolysaccharide (LPS) in vitro. METHODS: SCAP cultures were established and characterized. Increasing concentrations of lipopolysaccharide (0.1-10 µg/mL) were used to investigate cyclooxygenase-2 (COX-2/PTGS2) and PGE2 receptors (EP1-4) gene expression. Then, SCAP were treated with a COX-2 inhibitor (indomethacin) before treatment with different concentrations of LPS. The levels of the chemokine CCL2/monocyte chemoattractant protein 1 and interleukin (IL)-6 were detected in cell supernatants (24 hours) by enzyme-linked immunosorbent assay. Data analysis was performed using analysis of variance followed by the Tukey post test. RESULTS: The expression of COX-2 was up-regulated in the group treated with LPS at 1µg/mL compared with that in the control group. EP1-4 were detected in all experimental conditions at similar levels. SCAP treated with indomethacin presented a down-regulation in the production of LPS-induced CCL2 and the secretion of IL-6. CONCLUSIONS: SCAP showed increased COX-2 (PTGS2) gene expression induced by LPS and a PGE2-dependent production of IL-6 and CCL2.
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Quimiocina CCL2 , Ciclo-Oxigenase 2 , Interleucina-6 , Receptores de Prostaglandina E , Ápice Dentário , Células Cultivadas , Quimiocina CCL2/metabolismo , Ciclo-Oxigenase 2/metabolismo , Humanos , Interleucina-6/fisiologia , Lipopolissacarídeos , Receptores de Prostaglandina E/fisiologia , Células-Tronco , Ápice Dentário/metabolismoRESUMO
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.
Assuntos
Diferenciação Celular , Papila Dentária/citologia , Papila Dentária/fisiologia , Polpa Dentária , Odontoblastos , Ápice Dentário/citologia , Ápice Dentário/fisiologia , Animais , Animais Recém-Nascidos , Diferenciação Celular/genética , Transplante de Células , Papila Dentária/transplante , Polpa Dentária/citologia , Polpa Dentária/fisiologia , Proteínas da Matriz Extracelular/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos Endogâmicos , Fosfoproteínas/metabolismo , Regeneração , Sialoglicoproteínas/metabolismo , Ápice Dentário/transplanteRESUMO
To analyze the expression of transforming growth factor-beta 1 inheterotopic grafts of adult dental apical papilla. Methodology: The apical papilla of adult Wistar rats was grafted in the ear of the same donor rats. 1, 3, 7 and14 days after grafting, rats were perfused and the tissue containing the graft was processed for histological conventional technique and for immunohistochemical detection of transforming growth factor-beta 1. Results: Heterotopically grafted apical papilla developed osteoid dentine. In an early post-grafting stage, odontoblast-like cells organized themselves in palisade and synthesized dentine. However, newly formed dentine possessed the structural appearance of reactive osteoid dentine, which was systematically destroyed by the activity of osteoclaste-like cells. Transforming Growth Factor-beta 1 was observed in mesenchymal cells, extracellular matrix of the graft and surrounding host tissue, while odontoblast-like cells were systematically devoid of immunoreactivity. Conclusion: The different expression of transforming growth factor-beta 1 between normal tissue and grafted tissue development suggests that in heterotopic graft conditions the inflammatory mediation of the transforming growth factor-beta 1 prevails against its morphogenetic role...
Analizar la expresión del factor transformador del crecimiento-beta1 en trasplantes heterotópicos de papila dental del incisivo de la rata adulta. Metodología: La papila apical del incisivo de 12 ratas Wistar adultas fue trasplantada en la oreja de las mismas ratas donantes, y perfundidas 1, 3, 7 y 14 días postrasplante. El tejido fue procesado para histología convencionaly para la detección inmunohistoquímica del factor transformador del crecimiento-beta1. Resultados: La papila apical trasplantada desarrolló osteodentina. En fases tempranas postrasplante se observaron células parecidas a los odontoblastos que se organizaron en empalizada y segregaron dentina que se depositó sobre su superficie apical o secretora. Esta dentina evolucionó a osteodentina caracterizada por perder su estructura tubular e incluir a las células odontoblásticas en lagunas de su matriz. Finalmente, la osteodentina presentó procesos líticos mediados por células de tipo osteoclasto. Durante todo el proceso la expresión del factor transformador del crecimiento-beta1 se restringió a las células mesenquimales, a la matriz del trasplante y a las zonas circundantes del huésped, estando ausente en los odontoblastos, a diferencia de lo que sucede durante la odontogénesis normal. Conclusión: La diferente localización de la expresión del Factor Transformador de crecimiento beta1 entre el tejido hospedero y el trasplantado sugieren que en condiciones de trasplante heterotópico de papila dental la mediación inflamatoria del Factor Transformador de crecimiento beta1 prevalece sobre su papel morfogenético...