RESUMEN
BACKGROUND AND AIM: Regenerative endodontic procedures (REPs) are oriented by the principles of tissue engineering, incorporating dental pulp stem cells (DPSC), crucial growth factors like Transforming growth factor-ß (TGF-ß1), and scaffolds to facilitate the regeneration of dental pulp tissues. The present study aimed to investigate the effect of photobiomodulation (PBM) therapy, using an 808 nm diode laser on cellular modulation mechanisms in REPs. METHOD AND MATERIAL: A total of 108 human dentin discs obtained from intact single root teeth were randomly assigned into six groups (n = 8): 1. Positive control (EDTA), 2. PBM-1 (3 J/cm2), 3. PBM-2 (5 J/cm2), 4. EDTA+PBM-1, 5. EDTA+PBM-2, and 6. Negative control (NaOCl). Then, an extract solution was prepared from each disc and the concentration of released TGF-ß1 from the discs was measured using enzyme-linked immunosorbent assay (ELISA). Moreover, the extract solution was added to DPSC culture medium to evaluate cell viability and migration through MTT assay and scratch test, respectively. RESULT: The group exposed to PBM-1 showed the highest cell viability, while treatment with EDTA and EDTA+PBM-2 decreased cellular viability. Also, the PBM-treated groups showed significantly higher release of TGF-ß1 compared to the negative control. EDTA and EDTA+PBM-1 showed the highest release among all the groups. No significant difference was found between EDTA and EDTA+PBM-1, as well as between PBM-1 and PBM-2. Moreover, the PBM-1 group exhibited the highest migration after 24 h, which was significantly greater than other groups, except for the PBM-2 group. CONCLUSION: According to the obtained data, 808 nm mediated-PBM (3 J/cm2), both independently and in conjunction with EDTA, enhanced the release of TGF-ß1 from dentin and improved cell viability and migration of DPSCs. It seems that, PBM under the specific parameters employed in this study, could be an effective adjunctive therapy in REPs.
Asunto(s)
Terapia por Luz de Baja Intensidad , Endodoncia Regenerativa , Humanos , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Ácido Edético/farmacología , Dentina/metabolismo , Pulpa Dental/metabolismo , Células Madre/metabolismoRESUMEN
Pain within the trigeminal system, particularly dental pain, is poorly understood. This study aimed to determine whether single or multiple dental pulp injuries induce persistent pain, its association with trigeminal central nociceptive pathways and whether electroacupuncture (EA) provides prolonged analgesic and neuroprotective effects in a persistent dental pain model. Models of single dental pulp injury (SDPI) and multiple dental pulp injuries (MDPI) were used to induce trigeminal neuropathic pain. The signs of dental pain-related behavior were assessed using the mechanical head withdrawal threshold (HWT). Immunofluorescence and western blot protocols were used to monitor astrocyte activation, changes in apoptosis-related proteins, and GABAergic interneuron plasticity. SDPI mice exhibited an initial marked decrease in HWT from days one to 14, followed by progressive recovery from days 21 to 42. From days 49 to 70, the HWT increased and returned to the control values. In contrast, MDPI mice showed a persistent decrease in HWT from days one to 70. MDPI increased glial fibrillary acidic protein (GFAP) and decreased glutamine synthetase (GS) and glutamate transporter-1 (GLT1) expression in the Vi/Vc transition zone of the brainstem on day 70, whereas no changes in astrocytic markers were observed on day 70 after SDPI. Increased expression of cleaved cysteine-aspartic protease-3 (cleaved caspase-3) and Bcl-2-associated X protein (Bax), along with decreased B-cell lymphoma/leukemia 2 (Bcl-2), were observed at day 70 after MDPI but not after SDPI. The downregulation of glutamic acid decarboxylase (GAD65) expression was observed on day 70 only after MDPI. The effects of MDPI-induced lower HWT from days one to 70 were attenuated by 12 sessions of EA treatment (days one to 21 after MDPI). Changes in astrocytic GFAP, GS, and GLT-1, along with cleaved caspase-3, Bax, Bcl-2, and GAD65 expression observed 70 days after MDPI, were reversed by EA treatment. The results suggest that persistent dental pain in mice was induced by MDPI but not by SDPI. This effect was associated with trigeminal GABAergic interneuron plasticity along with morphological and functional changes in astrocytes. EA exerts prolonged analgesic and neuroprotective effects that might be associated with the modulation of neuron-glia crosstalk mechanisms.
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Electroacupuntura , Neuralgia , Fármacos Neuroprotectores , Ratones , Animales , Astrocitos/metabolismo , Fármacos Neuroprotectores/metabolismo , Caspasa 3/metabolismo , Proteína X Asociada a bcl-2 , Electroacupuntura/métodos , Pulpa Dental/metabolismo , Neuralgia/metabolismo , Analgésicos/metabolismo , Interneuronas/metabolismoRESUMEN
High doses of tumor necrosis factor-α (TNF-α) suppress osteogenic differentiation of human dental pulp stem cells (hDPSCs). In the present study, we aimed to explore the role and potential regulatory mechanism of microRNA-138 (miR-138) in the osteogenic differentiation of hDPSCs after treatment with a high dose of TNF-α. The hDPSCs were cultured in osteogenic medium with or without 50 ng/ml TNF-α. The miR-138 levels were upregulated during osteogenic differentiation of the hDPSCs following TNF-α treatment. The miR-138 overexpression accelerated but miR-138 knockdown alleviated the TNF-α-induced suppression of the alkaline phosphatase activity, calcium deposition, and protein abundance of dentin sialophosphoprotein, dentin matrix protein 1, bone sialoprotein, and osteopontin during osteogenic differentiation induction of hDPSCs. Additionally, miR-138 overexpression accelerated but miR-138 knockdown alleviated the suppression of the focal adhesion kinase- (FAK-) extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway during osteogenic differentiation induction of hDPSCs under TNF-α treatment. In conclusion, miR-138 accelerates TNF-α-induced suppression of osteogenic differentiation of hDPSCs. Inactivation of the FAK-ERK1/2 signaling pathway may be one of the mechanisms underlying the effect of miR-138. Inhibition of miR-138 expression may be a strategy to weaken the inhibitory effect of high-dose TNF-α on the osteogenic differentiation of hDPSCs.
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MicroARNs , Osteogénesis , Diferenciación Celular/genética , Células Cultivadas , Pulpa Dental/metabolismo , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Osteogénesis/genética , Células Madre/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
BMP-7 has shown inductive potential for in vitro osteogenic differentiation of mesenchymal stem cells, which are an ideal resource for regenerative medicine. Externally applied, recombinant BMP-7 was able to induce the osteogenic differentiation of DPSCs but based on our previous results with BMP-2, we aimed to study the effect of the tetracyclin-inducible BMP-7 expression on these cells. DPSC, mock, and DPSC-BMP-7 cell lines were cultured in the presence or absence of doxycycline, then alkaline phosphatase (ALP) activity, mineralization, and mRNA levels of different osteogenic marker genes were measured. In the DPSC-BMP-7 cell line, the level of BMP-7 mRNA significantly increased in the media supplemented with doxycycline, however, the expression of Runx2 and noggin genes was upregulated only after 21 days of incubation in the osteogenic medium with doxycycline. Moreover, while the examination of ALP activity showed reduced activity in the control medium containing doxycycline, the accumulation of minerals remained unchanged in the cultures. We have found that the induced BMP-7 expression failed to induce osteogenic differentiation of DPSCs. We propose three different mechanisms that may worth investigating for the engineering of expression systems that can be used for the induction of differentiation of mesenchymal stem cells.
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Proteína Morfogenética Ósea 7/metabolismo , Diferenciación Celular , Pulpa Dental/citología , Doxiciclina/farmacología , Osteogénesis , Células Madre/citología , Fosfatasa Alcalina/metabolismo , Antibacterianos/farmacología , Proliferación Celular , Células Cultivadas , Pulpa Dental/efectos de los fármacos , Pulpa Dental/metabolismo , Humanos , Células Madre/efectos de los fármacos , Células Madre/metabolismoRESUMEN
Nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy has been implicated in the ferroptosis in cancer cells and hematopoiesis in the bone marrow. However, the role of iron metabolism, especially NCOA4-mediated degradation of ferritin, has not been explored in the proliferation of mesenchymal stem cells. The present study was designed to explore the role of NCOA4-mediated ferritinophagy in hypoxia-treated dental pulp stem cells (DPSCs). Hypoxia treatment increased ROS generation, boosted cytosolic labile iron pool, increased expression of transferrin receptor 1 and NCOA4. Moreover, colocalization of LC3B with NCOA4 and ferritin was observed in hypoxia-treated DPSCs, indicating the development of ferritinophagy. Hypoxia promoted the proliferation of DPSCs, but not ferroptosis, under normal serum supplement and serum deprivation. NCOA4 knock-down reduced ferritin degradation and inhibited proliferation of DPSCs under hypoxia. Furthermore, the activation of hypoxia inducible factor 1α and p38 mitogen-activated protein kinase signaling pathway was involved in the upregulation of NCOA4 in hypoxia. Therefore, our present study suggested that NCOA4-mediated ferritinophagy promoted the level of labile iron pool, leading to enhanced iron availability and elevated cell proliferation of DPSCs. Our present study uncovered a physiological role of ferritinophagy in the proliferation and growth of mesenchymal stem cells under hypoxia.
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Hipoxia de la Célula/fisiología , Pulpa Dental/patología , Ferritinas/metabolismo , Hierro/metabolismo , Coactivadores de Receptor Nuclear/metabolismo , Células Madre/patología , Autofagia , Proliferación Celular/fisiología , Pulpa Dental/metabolismo , Humanos , Células Madre/metabolismoRESUMEN
Dental pulp is exposed to resin monomers leaching from capping materials. Toxic doses of the monomer, triethyleneglycol dimethacrylate (TEGDMA), impact cell growth, enhance inflammatory and oxidative stress responses, and lead to tissue necrosis. A therapeutic agent is required to rescue growth-arrested tissues by continuing their development and modulating the exacerbated responses. The functionality of N-Acetyl Cysteine (NAC) as a treatment was assessed by employing a 3D dental pulp microtissue platform. Immortalized and primary microtissues developed and matured in the extracellular matrix (ECM). TEGDMA was introduced at various concentrations. NAC was administered simultaneously with TEGDMA, before or after monomer addition during the development and after the maturation stages of the microtissue. Spatial growth was validated by confocal microscopy and image processing. Levels of inflammatory (COX2, NLRP3, IL-8) and oxidative stress (GSH, Nrf2) markers were quantified by immunoassays. NAC treatments, in parallel with TEGDMA challenge or post-challenge, resumed the growth of the underdeveloped microtissues and protected mature microtissues from deterioration. Growth recovery correlated with the alleviation of both responses by decreasing significantly the intracellular and extracellular levels of the markers. Our 3D/ECM-based dental pulp platform is an efficient tool for drug rescue screening. NAC supports compromised microtissues development, and immunomodulates and maintains the oxidative balance.
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Acetilcisteína/farmacología , Pulpa Dental/efectos de los fármacos , Matriz Extracelular/efectos de los fármacos , Inflamación/tratamiento farmacológico , Proliferación Celular/efectos de los fármacos , Resinas Compuestas/farmacología , Pulpa Dental/crecimiento & desarrollo , Pulpa Dental/metabolismo , Evaluación Preclínica de Medicamentos , Matriz Extracelular/genética , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Humanos , Inflamación/inducido químicamente , Inflamación/genética , Inflamación/patología , Interleucina-8/genética , Factor 2 Relacionado con NF-E2/genética , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Estrés Oxidativo/efectos de los fármacos , Polietilenglicoles/farmacología , Ácidos Polimetacrílicos/farmacologíaRESUMEN
Irreversible pulpitis is an extremely painful condition and its consequence in the central nervous system (CNS) remains unclear. A mouse model of dental pulp injury (DPI) resembles the irreversible pulpitis profile in humans. This study sought to determine whether pain induced by DPI activates microglia and astrocytes in the trigeminal subnucleus caudalis (Vc), as well as increases levels of proinflammatory cytokines, and whether electroacupuncture (EA) can be a potential analgesic and neuroprotective therapy following DPI. Pain behavior was measured via head-withdrawal threshold (HWT) and burrowing behavior at days 1, 3, 7, 14 and 21 after DPI. A marked decrease in HWT and burrowing activity was observed from day 1 to 14 after DPI and no changes were seen on day 21. Microglial and astrocytes activation; along with high cytokine (TNFα, IL-1ß, and IL-6) levels, were observed in the Vc at 21 days after DPI. These effects were attenuated by verum (local and distal) EA, as well as oral ibuprofen administration. The results suggest that DPI-induced pain and glial activations in the Vc and EA exert analgesic efficacy at both local and distal acupoints. Furthermore, verum (local and distal) EA might be associated with the modulations of microglial and astrocytes activation.
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Analgésicos/farmacología , Pulpa Dental/efectos de los fármacos , Pulpa Dental/lesiones , Electroacupuntura , Fármacos Neuroprotectores/farmacología , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Conducta Animal , Citocinas/genética , Citocinas/metabolismo , Pulpa Dental/metabolismo , Pulpa Dental/patología , Modelos Animales de Enfermedad , Electroacupuntura/métodos , Expresión Génica , Histocitoquímica , Mediadores de Inflamación/metabolismo , Microglía/efectos de los fármacos , Microglía/metabolismo , Pulpitis/tratamiento farmacológico , Pulpitis/etiología , Pulpitis/metabolismo , Pulpitis/patología , Ratas , Núcleos del Trigémino/citología , Núcleos del Trigémino/efectos de los fármacos , Núcleos del Trigémino/metabolismoRESUMEN
Orofacial clefts (OFCs) are among the most common congenital craniofacial malformations, including cleft lip with or without cleft palate as the core symptoms. Developmental or functional defects in neural crest cells (NCCs) that contribute to craniofacial morphogenesis are involved in OFC development. Previous studies have suggested that oxidative stress in NCCs is involved in the development of OFCs, suggesting that the anti-oxidative activity of folic acid (FA) could have protective effects. However, studies of human-derived NCCs are limited, as these cells are predominantly active during the embryonic stage. In this study, the effects of oxidative stress and FA were evaluated in human OFCs. In particular, NCC-derived stem cells from human exfoliated deciduous teeth (SHEDs) were obtained from 3 children with non-syndromic cleft lip with cleft palate (CLPs) and from 3 healthy children (CTRLs). Mitochondrial reactive oxygen species (ROS) levels were significantly higher in CLPs than in CTRLs and were associated with lower mRNA expression levels of superoxide dismutase 1 (SOD1) and decreased cell mobility. In addition, significantly greater vulnerability to pyocyanin-induced ROS, mimicking exogenous ROS, was observed in CLPs than in CTRLs. These vulnerabilities to endogenous and exogenous ROS in CLPs were significantly improved by FA. These results indicated that the transcriptional dysregulation of SOD1 in NCCs is an oxidative stress-related pathological factor in OFCs, providing novel evidence for the benefits of perinatal anti-oxidant supplementation, including FA, for the management of these common deformities.
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Antioxidantes/uso terapéutico , Labio Leporino/tratamiento farmacológico , Fisura del Paladar/tratamiento farmacológico , Ácido Fólico/uso terapéutico , Estrés Oxidativo/efectos de los fármacos , Diente Primario/efectos de los fármacos , Células Cultivadas , Niño , Labio Leporino/metabolismo , Fisura del Paladar/metabolismo , Pulpa Dental/citología , Pulpa Dental/efectos de los fármacos , Pulpa Dental/metabolismo , Humanos , Células Madre/citología , Células Madre/efectos de los fármacos , Células Madre/metabolismo , Diente Primario/citología , Diente Primario/metabolismo , Complejo Vitamínico B/uso terapéuticoRESUMEN
Vital pulp therapy (VPT) is to preserve the nerve and maintain healthy dental pulp tissue. Laser irradiation (LI) is beneficial for VPT. Understanding how LI affects dental pulp cells and tissues is necessary to elucidate the mechanism of reparative dentin and dentin regeneration. Here, we show how Er:YAG-LI and diode-LI modulated cell proliferation, apoptosis, gene expression, protease activation, and mineralization induction in dental pulp cells and tissues using cell culture, immunohistochemical, genetic, and protein analysis techniques. Both LIs promoted proliferation in porcine dental pulp-derived cell lines (PPU-7), although the cell growth rate between the LIs was different. In addition to proliferation, both LIs also caused apoptosis; however, the apoptotic index for Er:YAG-LI was higher than that for diode-LI. The mRNA level of odontoblastic gene markers-two dentin sialophosphoprotein splicing variants and matrix metalloprotease (MMP)20 were enhanced by diode-LI, whereas MMP2 was increased by Er:YAG-LI. Both LIs enhanced alkaline phosphatase activity, suggesting that they may help induce PPU-7 differentiation into odontoblast-like cells. In terms of mineralization induction, the LIs were not significantly different, although their cell reactivity was likely different. Both LIs activated four MMPs in porcine dental pulp tissues. We helped elucidate how reparative dentin is formed during laser treatments.
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Apoptosis/efectos de la radiación , Proliferación Celular/efectos de la radiación , Pulpa Dental/efectos de la radiación , Animales , Diferenciación Celular/efectos de la radiación , Línea Celular , Pulpa Dental/citología , Pulpa Dental/metabolismo , Proteínas de la Matriz Extracelular/análisis , Proteínas de la Matriz Extracelular/genética , Regulación de la Expresión Génica/efectos de la radiación , Láseres de Semiconductores , Terapia por Luz de Baja Intensidad , Metaloproteinasa 20 de la Matriz/análisis , Metaloproteinasa 20 de la Matriz/genética , Odontoblastos/citología , Odontoblastos/metabolismo , Odontoblastos/efectos de la radiación , Fosfoproteínas/análisis , Fosfoproteínas/genética , Sialoglicoproteínas/análisis , Sialoglicoproteínas/genética , PorcinosRESUMEN
Stem cells derived from human exfoliated deciduous teeth (SHED) represent a promising cell source for bone tissue regeneration. This study evaluated the effects of interleukin-17A (IL-17A) on the osteogenic differentiation of SHED. SHED were cultured in complete alpha minimum essential medium supplemented with osteoinducing reagents and treated with recombinant IL-17A. The cells were quantitatively analysed for proliferative activity by MTS assay, cell markers expression, and apoptotic activity by flow cytometry. For osteogenic differentiation, alkaline phosphatase (ALP) activity was quantified; mineralization assays were carried out using von Kossa and Alizarin red, and expression of osteogenic markers were analysed by real-time polymerase chain reaction and Western blot. The results showed that treatment with IL-17A increased proliferative activity in a dose-dependent manner, but reduced the expression of stem cell markers (c-Myc and Nanog) as the days progressed. IL-17A induced osteogenic differentiation in SHED as evidenced by high ALP activity, increased matrix mineralization, and upregulation of the mRNA expression of the osteogenic markers ALP, alpha 1 type 1 collagen (Col1A1), runt-related transcription factor 2 (RUNX2), osteopontin (OPN), osteocalcin (OCN), and osteoprotegerin (OPG) but downregulation of receptor activator of nuclear factor κB ligand (RANKL) as well as altering the OPG/RANKL ratio. Findings from our study indicate that IL-17A enhances proliferation and osteogenic differentiation of SHED by regulating OPG/RANKL mechanism thus suggests therapeutic potential of IL-17A in bone regeneration.
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Diferenciación Celular/efectos de los fármacos , Pulpa Dental/metabolismo , Interleucina-17/farmacología , Osteogénesis/efectos de los fármacos , Osteoprotegerina/biosíntesis , Ligando RANK/biosíntesis , Células Madre/metabolismo , Diente Primario/metabolismo , Pulpa Dental/citología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Células Madre/citología , Diente Primario/citologíaRESUMEN
INTRODUCTION: Compelling evidence pinpoints that pulp tissue engineering after the transplantation of stem cells is possible. Although intriguing, severe problems regarding clinical feasibility remain. Cell homing has been proposed as a viable alternative in which dentin-derived growth factors in a conducive scaffold may attract resident cells to form pulplike tissue. In this study, an ectopic animal model for in situ dental pulp tissue engineering was developed to evaluate whether pulplike tissue formation in empty root canals after the attraction of stem cells was possible and whether this could be enhanced by dentin-derived growth factors. METHODS: Three types of fibrin (custom-made fibrin, fibrin sealant, and plasma rich in growth factors [PRGF]) as well as a self-assembling peptide were evaluated in vivo in a modified tooth root model using human teeth. Root canal dentin was conditioned with EDTA, tooth roots were filled with growth factor-laden scaffolds, and dental pulp stem cells in collagen were placed at the root tip. Constructs were implanted into immunocompromised mice for 4 weeks and subsequently analyzed histologically. Differential interference contrast and second harmonic generation imaging were performed for selected sections. RESULTS: For custom-made fibrin and fibrin sealant with dentin matrix proteins, migration into the roots and the formation of a pulplike tissue were observed, whereas the peptide-based scaffold appeared less suitable. PRGF supported tissue formation regardless of the addition of dentin matrix proteins. In the test groups with dentin matrix proteins and EDTA conditioning, differentiated odontoblastlike cells extended cellular processes into the dentinal tubules, which coincided with the deposition of the newly formed collagenous dentin matrix. CONCLUSIONS: This new cell homing model provides evidence that fibrin derivatives make applicable scaffolds and that dentin-derived proteins induce chemotaxis and pulplike tissue formation.
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Pulpa Dental/fisiología , Dentina/metabolismo , Proteínas de la Matriz Extracelular/uso terapéutico , Endodoncia Regenerativa/métodos , Ingeniería de Tejidos/métodos , Adulto , Animales , Movimiento Celular , Pulpa Dental/metabolismo , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos , Andamios del Tejido , Raíz del Diente/fisiologíaRESUMEN
OBJECTIVE: To explore suitable concentration of recombinant human transforming growth factor ß1 (rhTGF-ß1) usage and study the effect of rhTGF-ß1 on differentiation of dental pulp stem cells (DPSCs). METHODS: DPSCs were isolated from the undiseased third molars of people aged 18-25 years and cultured according to instructions in vitro. Different concentrations (1 , 6 , 10 µg/L) of rhTGF-ß1 were added to the culture medium to examine DPSCs proliferation by CCK-8 (cell counting kit-8) assay.The suitable concentration was then selected. For differentiation, the DPSCs were incubated for 7 or 14 days with rhTGF-ß1 supplemented with osteo/odontoblastic induction medium containing 10 nmol/L dexamethasone, 10 mmol/L b-glycerophosphate, 50 g/L ascorbate phosphate, 10 nmol/L 1,25-dihydroxyvitamin D3 and 10% fetal bovine serum. The cells were then washed 3 times with phosphate-buffered saline and sonicated with 1%Triton X-100 for 30 minutes on ice. Cellular alkaline phosphatase (ALP) activity was assayed with p-nitrophenyl phosphate as the substrate. The enzyme activity was expressed as p-nitrophenyl produced per milligram of protein [bicinchoninic acid (BCA) protein assay kit]. To examine mineral nodule formation, the cultured cells were fixed in 4% paraformaldehyde and washed in water, and the mineralization of the extracellular matrix was assayed by 1% alizarin red S staining and elution of staining was examined as optical density (D) under microplate reader. The mean difference was considered significant at 0.05 and 95% confidence interval. RESULTS: The DPSCs had typical fibroblast morphology and could form mineral nodules after being cultured with osteo/odontoblstic induction medium for 14 days. 6 µg/L rhTGF-ß1 significantly promoted the DPSCs proliferation on the 3rd and 5th days. After the incubation of osteo/odontoblastic induction medium, the DPSCs with the 6 µg/L rhTGF-ß1 increased ALP activities compared with the control; D values in the 6 µg/L rhTGF-ß1 group was 0.31±0.03, while the control group was 0.02±0.01 (P<0.05). The total protein content in the 6 µg/L rhTGF-ß1 group was (2 775.46±83.54) mg/L, and the control group was (1 432.20±110.83) mg/L (P<0.05). To eliminate the cells proliferation influence, relative ALP activities, which was defined as the total ALP divided by the total protein content, the 6µg/L rhTGF-ß1 group was 6 times higher than the control group. Alizarin red S staining showed increased mineral nodule formation in the rhTGF-ß1 group. The elution of staining under microplate reader also showed more optical density in the 6 µg/L rhTGF-ß1-treated cells (0.83±0.02) than that in the control groups (0.55±0.05, P<0.05). CONCLUSION: 6 µg/L rhTGF-ß1 could significantly promote DPSCs proliferation and odontoblastic differentiation in vitro.
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Proliferación Celular , Pulpa Dental , Proteínas Recombinantes , Células Madre , Factor de Crecimiento Transformador beta1 , Adolescente , Adulto , Fosfatasa Alcalina , Diferenciación Celular , Células Cultivadas , Pulpa Dental/citología , Pulpa Dental/efectos de los fármacos , Pulpa Dental/metabolismo , Humanos , Proteínas Recombinantes/farmacología , Células Madre/efectos de los fármacos , Factor de Crecimiento Transformador beta1/farmacología , Adulto JovenRESUMEN
OBJECTIVES: Vital pulp therapy aims to treat reversible pulpal injuries via protective dentinogenesis and to preserve more tooth structure. Mineral trioxide aggregate (MTA)-based capping materials demonstrate prolonged setting time increases the risk of pulpal infection during multi-visit treatment. Their non-degradable property occupies pulp space and limits dentin-pulp regeneration. This study reports an inorganic degradable biomaterial that presents a short initial setting time and acts as a growth factor reservoir to promote reparative dentinogenesis. METHODS: We synthesize nanocrystalline calcium sulfate hemihydrate (nCS), hydroxyapatite (HAp) and calcium sulfate hemihydrate (CS) as a reservoir to which transforming growth factor-beta 1 (TGF-ß1) and vascular endothelial growth factor (VEGF) are added (denoted as nCS/HAp/CS/TGF-ß1/VEGF). In vitro biocompatibility and mineralization (the activity and expression of alkaline phosphatase, ALP) were evaluated. Rat animal model was created to test in vivo efficacy. RESULTS: Cultured human dental pulp cells (HDPCs) showed that nCS/HAp/CS/TGF-ß1/VEGF cement has excellent biocompatibility and the potential to elevate the activity and expression of ALP. The in vivo efficacy (rat animal model) indicates protective dentin by micro-computed tomography (µ-CT) measurements and histological analyses. The 3D µ-CT non-destructive analysis also determines volume changes during pulpotomy, suggesting that the degraded space of the nCS/HAp/CS/TGF-ß1/VEGF cement is repaired by the formation of dentin-pulp tissue. SIGNIFICANCE: These findings demonstrate that nCS/HAp/CS cement acts as a potent reservoir for the sustained release of growth factors, and that nCS/HAp/CS/TGF-ß1/VEGF cement has a high potential to form the reparative dentinogenesis in vivo.
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Pulpa Dental/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Sulfato de Calcio/farmacología , Durapatita/farmacología , Humanos , Ratas , Microtomografía por Rayos XRESUMEN
Here we present a new model for alveolar jaw bone regeneration, which uses human dental pulp cells (hDPCs) combined with tyrosine-derived polycarbonate polymer scaffolds [E1001(1k)] containing beta-tricalcium phosphate (ß-TCP) [E1001(1k)/ß-TCP]. E1001(1k)/ß-TCP scaffolds (5 mm diameter × 1 mm thickness) were fabricated to fit a 5 mm rat mandibular ramus critical bone defect. Five experimental groups were examined in this study: (1) E1001(1k)/ß-TCP scaffolds seeded with a high density of hDPCs, 5.0 × 10(5) hDPCs/scaffold (CH); (2) E1001(1k)/ß-TCP scaffolds seeded with a lower density of hDPCs, 2.5 × 10(5) hDPCs/scaffold (CL); (3) acellular E1001(1k)/ß-TCP scaffolds (SA); (4) acellular E1001(1k)/ß-TCP scaffolds supplemented with 4 µg recombinant human bone morphogenetic protein-2 (BMP); and (5) empty defects (EDs). Replicate hDPC-seeded and acellular E1001(1k)/ß-TCP scaffolds were cultured in vitro in osteogenic media for 1 week before implantation for 3 and 6 weeks. Live microcomputed tomography (µCT) imaging at 3 and 6 weeks postimplantation revealed robust bone regeneration in the BMP implant group. CH and CL groups exhibited similar uniformly distributed mineralized tissue coverage throughout the defects, but less than the BMP implants. In contrast, SA-treated defects exhibited sparse areas of mineralized tissue regeneration. The ED group exhibited slightly reduced defect size. Histological analyses revealed no indication of an immune response. In addition, robust expression of dentin and bone differentiation marker expression was observed in hDPC-seeded scaffolds, whereas, in contrast, BMP and SA implants exhibited only bone and not dentin differentiation marker expression. hDPCs were detected in 3-week but not in 6-week hDPC-seeded scaffold groups, indicating their survival for at least 3 weeks. Together, these results show that hDPC-seeded E1001(1k)/ß-TCP scaffolds support the rapid regeneration of osteo-dentin-like mineralized jaw tissue, suggesting a promising new therapy for alveolar jaw bone repair and regeneration.
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Regeneración Ósea , Pulpa Dental/metabolismo , Mandíbula/metabolismo , Traumatismos Mandibulares/terapia , Cemento de Policarboxilato/química , Trasplante de Células Madre , Células Madre/metabolismo , Andamios del Tejido/química , Animales , Femenino , Humanos , Mandíbula/patología , Traumatismos Mandibulares/metabolismo , Traumatismos Mandibulares/patología , Osteogénesis , Ratas , Ratas Desnudas , Tirosina/químicaRESUMEN
OBJECTIVES: The aim of this study was to evaluate the level of odontogenic differentiation of dental pulp stem cells (DPSCs) on hydrogel scaffolds derived from bone extracellular matrix (bECM) in comparison to those seeded on collagen I (Col-I), one of the main components of dental pulp ECM. METHODS: DPSCs isolated from human third molars were characterized for surface marker expression and odontogenic potential prior to seeding into bECM or Col-I hydrogel scaffolds. The cells were then seeded onto bECM and Col-I hydrogel scaffolds and cultured under basal conditions or with odontogenic and growth factor (GF) supplements. DPSCs cultivated on tissue culture polystyrene (TCPS) with and without supplements were used as controls. Gene expression of dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP-1) and matrix extracellular phosphoglycoprotein (MEPE) was evaluated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and mineral deposition was observed by Von Kossa staining. RESULTS: When DPSCs were cultured on bECM hydrogels, the mRNA expression levels of DSPP, DMP-1 and MEPE genes were significantly upregulated with respect to those cultured on Col-I scaffolds or TCPS in the absence of extra odontogenic inducers. In addition, more mineral deposition was observed on bECM hydrogel scaffolds as demonstrated by Von Kossa staining. Moreover, DSPP, DMP-1 and MEPE mRNA expressions of DPSCs cultured on bECM hydrogels were further upregulated by the addition of GFs or osteo/odontogenic medium compared to Col-I treated cells in the same culture conditions. SIGNIFICANCE: These results demonstrate the potential of the bECM hydrogel scaffolds to stimulate odontogenic differentiation of DPSCs.
Asunto(s)
Matriz Ósea/química , Diferenciación Celular/efectos de los fármacos , Pulpa Dental/efectos de los fármacos , Hidrogeles/química , Células Madre/efectos de los fármacos , Andamios del Tejido , Biomarcadores/metabolismo , Adhesión Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Colágeno Tipo I/química , Pulpa Dental/citología , Pulpa Dental/metabolismo , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Expresión Génica/efectos de los fármacos , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Hidrogeles/farmacología , Péptidos y Proteínas de Señalización Intercelular/farmacología , Diente Molar , Odontogénesis/efectos de los fármacos , Odontogénesis/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Poliestirenos/química , Cultivo Primario de Células , ARN Mensajero/genética , ARN Mensajero/metabolismo , Sialoglicoproteínas/genética , Sialoglicoproteínas/metabolismo , Células Madre/citología , Células Madre/metabolismoRESUMEN
Glycans of cell surface glycoproteins are involved in the regulation of cell migration, growth, and differentiation. N-acetyl-glucosaminyltransferase V (GnT-V) transfers N-acetyl-d-glucosamine to form ß1,6-branched N-glycans, thus playing a crucial role in the biosynthesis of glycoproteins. This study reveals the distinct expression of GnT-V in STRO-1 and CD-146 double-positive dental pulp stem cells (DPSCs). Furthermore, we investigated three types of hexosamines and their N-acetyl derivatives for possible effects on the osteogenic differentiation potential of DPSCs. Our results showed that exogenous d-glucosamine (GlcN), N-acetyl-d-glucosamine (GlcNAc), d-mannosamine (ManN), and acetyl-d-mannosamine (ManNAc) promoted DPSCs' early osteogenic differentiation in the absence of osteogenic supplements, but d-galactosamine (GalN) or N-acetyl-galactosamine (GalNAc) did not. Effects include the increased level of TGF-ß receptor type I, activation of TGF-ß signaling, and increased mRNA expression of osteogenic differentiation marker genes. The hexosamine-treated DPSCs showed an increased mineralized matrix deposition in the presence of osteogenic supplements. Moreover, the level of TGF-ß receptor type I and early osteogenic differentiation were abolished in the DPSCs transfected with siRNA for GnT-V knockdown. These results suggest that GnT-V plays a critical role in the hexosamine-induced activation of TGF-ß signaling and subsequent osteogenic differentiation of DPSCs.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , N-Acetilglucosaminiltransferasas/genética , Proteínas Serina-Treonina Quinasas/biosíntesis , Receptores de Factores de Crecimiento Transformadores beta/biosíntesis , Células Madre/metabolismo , Factor de Crecimiento Transformador beta/genética , Acetilglucosamina/administración & dosificación , Proliferación Celular/efectos de los fármacos , Pulpa Dental/citología , Pulpa Dental/efectos de los fármacos , Pulpa Dental/metabolismo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Glucosamina/administración & dosificación , Glucosamina/análogos & derivados , Hexosaminas/administración & dosificación , Humanos , N-Acetilglucosaminiltransferasas/antagonistas & inhibidores , N-Acetilglucosaminiltransferasas/metabolismo , Osteogénesis/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/biosíntesis , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/genética , Transducción de Señal/efectos de los fármacos , Células Madre/citología , Células Madre/efectos de los fármacos , Factor de Crecimiento Transformador beta/biosíntesis , Adulto JovenRESUMEN
Mesenchymal stem cell (MSC) osteogenic differentiation potential varies according to factors such as tissue source and cell population heterogeneity. Pre-selection of cell subpopulations harboring higher osteopotential is a promising strategy to achieve a thorough translation of MSC-based therapies to the clinic. Here, we searched for novel molecular markers predictive of osteopotential by comparing MSC populations from two sources harboring different osteogenic potentials. We show that MSCs from human deciduous teeth (SHED) have an intrinsically higher osteogenic potential when compared with MSCs from human adipose tissue (hASCs) under the same in vitro controlled induction system. Transcriptome profiling revealed IGF2 to be one of the top upregulated transcripts before and during early in vitro osteogenic differentiation. Further, exogenous IGF2 supplementation enhanced alkaline phosphatase activity and matrix mineralization, and inhibition of IGF2 lessened these parameters in SHED and hASCs, validating IGF2 as an osteogenic factor in these MSCs. Further, we found IGF2 to be biallelically expressed in SHED, but not in hASCs. We observed a 4 % methylation increase in the imprinting control region within the IGF2-H19 locus in SHED, and this is mainly due to 2 specific CpG sites. Thus, we suggest that IGF2 upregulation in SHED is due to loss of imprinting. This study unravels osteogenic properties in SHED, implying IGF2 as a potential biomarker of MSCs with higher osteopotential, and unveils IGF2 loss-of-imprinting in SHED.
Asunto(s)
Tejido Adiposo/metabolismo , Diferenciación Celular/genética , Pulpa Dental/metabolismo , Factor II del Crecimiento Similar a la Insulina/genética , Osteogénesis/genética , Tejido Adiposo/citología , Adulto , Anciano , Secuencia de Bases , Benzopiranos/farmacología , Western Blotting , Niño , Análisis por Conglomerados , Pulpa Dental/citología , Perfilación de la Expresión Génica/métodos , Humanos , Factor II del Crecimiento Similar a la Insulina/antagonistas & inhibidores , Factor II del Crecimiento Similar a la Insulina/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Activación Transcripcional/efectos de los fármacosRESUMEN
Hyperpolarization-activated cyclic nucleotide-gated channel 1 (HCN1) and 2 (HCN2) are abundantly expressed in primary sensory neurons and contribute to neuronal excitability and pathological pain. We studied the expression of HCN1 and HCN2 in the rat trigeminal ganglion (TG) neurons and axons in the dental pulp, and the changes in their expression following inflammation, using light- and electron-microscopic immunocytochemistry and quantitative analysis. HCN1 and HCN2 were expressed predominantly in large-sized, neurofilament 200-immunopositive (+) or parvalbumin+ soma in the TG whereas they were expressed mostly in unmyelinated and small myelinated axons in the sensory root. The expression was particularly strong along the plasma membrane in the soma. In the dental pulp, majority of HCN1+ and HCN2+ axons coexpressed calcitonin gene-related peptide. They were expressed mainly in the peripheral pulp and pulp horn where the axons branch extensively in the dental pulp. The expression of HCN1 and HCN2 in TG neurons increased significantly in rats with experimentally induced inflammation of the dental pulp. Our findings support the notion that HCN1 and HCN2 are expressed mainly by both the soma of mechanosensitive neurons in the TG and peripheral axons of nociceptive neurons in the sensory root, and may play a role in the mechanisms of inflammatory pain from the dental pulp.
Asunto(s)
Pulpa Dental/metabolismo , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Canales de Potasio/metabolismo , Células Receptoras Sensoriales/metabolismo , Ganglio del Trigémino/metabolismo , Animales , Axones/metabolismo , Axones/ultraestructura , Péptido Relacionado con Gen de Calcitonina/metabolismo , Pulpa Dental/ultraestructura , Enfermedades de la Pulpa Dental/metabolismo , Enfermedades de la Pulpa Dental/patología , Modelos Animales de Enfermedad , Adyuvante de Freund , Inflamación/metabolismo , Inflamación/patología , Masculino , Fibras Nerviosas Mielínicas/metabolismo , Fibras Nerviosas Mielínicas/ultraestructura , Proteínas de Neurofilamentos/metabolismo , Parvalbúminas/metabolismo , Ratas Sprague-Dawley , Células Receptoras Sensoriales/ultraestructura , Ganglio del Trigémino/ultraestructuraRESUMEN
BACKGROUND: There is increasing evidence that peripheral glutamate signaling mechanism is involved in the nociceptive transmission during pathological conditions. However, little is known about the glutamate signaling mechanism and related specific type of vesicular glutamate transporter (VGLUT) in the dental pulp following inflammation. To address this issue, we investigated expression and protein levels of VGLUT1 and VGLUT2 in the dental pulp and trigeminal ganglion (TG) following complete Freund's adjuvant (CFA) application to the rat dental pulp by light microscopic immunohistochemistry and Western blot analysis. RESULTS: The density of VGLUT2- immunopositive (+) axons in the dental pulp and the number of VGLUT2+ soma in the TG increased significantly in the CFA-treated group, compared to control group. The protein levels of VGLUT2 in the dental pulp and TG were also significantly higher in the CFA-treated group than control group by Western blot analysis. The density of VGLUT1+ axons in the dental pulp and soma in the TG remained unchanged in the CFA-treated group. CONCLUSIONS: These findings suggest that glutamate signaling that is mediated by VGLUT2 in the pulpal axons may be enhanced in the inflamed dental pulp, which may contribute to pulpal axon sensitization leading to hyperalgesia following inflammation.
Asunto(s)
Axones/efectos de los fármacos , Ácido Glutámico/metabolismo , Hiperalgesia/genética , Inflamación/genética , Proteína 1 de Transporte Vesicular de Glutamato/genética , Proteína 2 de Transporte Vesicular de Glutamato/genética , Animales , Axones/metabolismo , Axones/patología , Pulpa Dental/efectos de los fármacos , Pulpa Dental/metabolismo , Pulpa Dental/fisiopatología , Adyuvante de Freund/administración & dosificación , Regulación de la Expresión Génica , Hiperalgesia/etiología , Hiperalgesia/fisiopatología , Inflamación/inducido químicamente , Inflamación/complicaciones , Inflamación/fisiopatología , Masculino , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Ganglio del Trigémino/efectos de los fármacos , Ganglio del Trigémino/metabolismo , Ganglio del Trigémino/fisiopatología , Proteína 1 de Transporte Vesicular de Glutamato/metabolismo , Proteína 2 de Transporte Vesicular de Glutamato/metabolismoRESUMEN
BACKGROUND: Intracanal medicaments are used to disinfect the root canal system, reduce interappointment pain and inflammation, and prevent resorption. Bacterial components such as lipopolysaccharide (LPS) are implicated in the development of pulpal and periapical inflammation and inducing osteoclastogenesis. Propolis is a natural, non-toxic substance collected from bee's wax that has been used for many years in folk medicine. Propolis has been demonstrated to have antibacterial and anti-inflammatory properties. Our previous studies have shown that propolis inhibits osteoclast maturation. However, the effect of propolis on the inflammatory response of pulp cells and osteoclasts has not been explored. AIM: The purpose of this study was to evaluate whether propolis alters the inflammatory response of three endodontically relevant cell lines: mouse odontoblast-like cells (MDPC-23), macrophages (RAW264.7), and osteoclasts. MATERIAL AND METHODS: Cells were exposed to 0-20 ug ml(-1) LPS to induce an inflammatory response, in the presence of propolis or vehicle control. Culture supernatants were collected after 6 and 24 h, and expression of multiple soluble mediators was determined using Luminex(®) multiplex technology. RESULTS: Propolis was effective in reducing secretion of the LPS-induced inflammatory cyto/chemokines: IL-1α, IL-6, IL-12(p70), IL-15, G-CSF, TNF-α, MIP-1α, MCP-1, and IP-10. CONCLUSION: Our results demonstrate that propolis suppresses the LPS-induced inflammatory response of key cells within the root canal system.