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Objective @#To study the effect of light-emitting diode (LED) red light on the osteogenic/odontogenic differentiation of human dental pulp stem cells (hDPSCs) and its mechanism were discussed. @*Methods@#This study has been reviewed and approved by the Ethics Committee. hDPSCs were cultured by tissue block enzyme digestion. The proliferative capacity of hDPSCs was detected by the CCK-8 at days 1, 3, 5 and 7 under stimulation with 0, 1, 5 and 10 μg/mL lipopolysaccharide (LPS), and the LPS stimulatory concentration was screened. The CG group (mineralization induction), LPS+CG group, and LPS+CG+ (2, 4, 6, 8, and 10 J/cm2) LED red light groups were set. On day 7, alkaline phosphatase (ALP) staining and ALP activity were determined. Relative expression levels of the ALP, osterix (OSX), dentin matrix protein-1 (DMP-1) and dentin sialophosphoprotein (DSPP) genes were measured by qRT-PCR. On day 21, alizarin red staining and calcium nodule quantitative determination were performed to screen the best light energy. The LPS+CG group and LPS+CG+LED group (optimal energy) were set up, and the secretion and expression levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were detected by ELISAs on days 1, 3, 5 and 7. The relative expression levels of the extracellular regulated protein kinases 1/2 (ERK1/2), p38, c-Jun N-terminal kinase (JNK), and extracellular regulated protein kinases 5 (ERK5) proteins and their phosphorylated proteins in the MAPK signaling pathway were detected by Western blots. After the pathway was blocked, the relative expression levels of the ALP, OSX, DMP-1, and DSPP proteins after LED red light irradiation on day 7 were detected by Western blots.@*Results@# CCK-8 assays showed that the proliferation of hDPSCs induced by 10 μg/mL LPS was lower than that of the 0, 1, and 5 μg/mL groups on the 5th and 7th days (P<0.05), and 10 μg/mL was selected as the LPS stimulatory concentration in the follow-up experiment. ALP staining, ALP activity, gene expression levels of ALP, OSX, DMP-1 and DSPP and calcium nodule quantification in the LPS+CG+4 J/cm2 group were higher than those in the other treatment groups (P<0.05). 4 J/cm2 LED red light had the strongest ability to promote osteogenic/odontogenic differentiation and was used as the LED light energy density in subsequent experiments. ELISA showed that the secretion and expression levels of TNF-α and IL-1β in the LPS+CG+LED group were lower than those in the LPS+CG group on the 5th and 7th days (P<0.05). Western blot analysis showed that 4 J/cm2 LED red light promoted the expression levels of the p-ERK1/2, p-p38, p-JNK and p-ERK5 proteins. After the MAPK pathway was blocked, the expression levels of the ALP, OSX, DMP-1, and DSPP proteins in the LPS+CG+LED+U0126 (ERK1/2 inhibitor), SP600125 (JNK inhibitor), and BIX02189 (ERK5 inhibitor) groups were lower than those in the LPS+CG+LED group (P<0.001). The protein expression levels of ALP, OSX and DMP-1 in the LPS+CG+LED+SB203580 (p38 inhibitor) group were not significantly different from those in the LPS+CG+LED group (P>0.05).@*Conclusion@#In inflammatory conditions, LED red light promotes osteogenic/odontogenic differentiation of hDPSCs. This effect may be attributed to enhancement of the ERK1/2, JNK, and ERK5 signaling pathways, which reduces the production of the inflammatory cytokines TNF-α and IL-1β.
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Objective@#To explore the effects of long noncoding-RNA (lncRNA) taurine upregulated gene 1 (TUG1) on the proliferation and osteogenic/odontoblast differentiation of human dental pulp stem cells (hDPSCs). @*Methods @# hDPSCs were isolated and cultured. The surface antigens CD44, CD45, CD73, CD90, CD133 and STRO-1 were detected by flow cytometry. Alkaline phosphatase (ALP) staining and alizarin red staining were used to identify the ability of cells to differentiate. RNA was collected on Days 0, 7 and 14 of the osteogenic induction of hDPSCs, and qRT-PCR was used to detect the relative expression of TUG1. The hDPSCs were stably transfected with a lentiviral vector containing the TUG1-silenced pSLenti-U6-shRNA(TUG1)-CMV-EGFP-F2A-Puro-WPRE to silence TUG1. The ability of hDPSCs to proliferate was assessed with the CCK-8 method. ALP and alizarin red staining and quantitative detection were used to detect the ALP activity and formation of mineralized nodules of hDPSCs. The expression levels of dentin sialophosphoprotein (DSPP), dentin matrix protein-1 (DMP-1), Runt-associated transcription factor 2 (Runx2), osteocalcin (OCN) and osteopontin (OPN) genes and proteins were measured by qRT-PCR and Western blot.@*Results @#The hDPSCs were successfully isolated and cultured, and TUG1 expression was significantly increased during osteogenic differentiation (P<0.05). The hDPSCs proliferation was suppressed after silencing TUG1(P<0.05). After osteogenic induction, ALP and alizarin red staining showed that ALP activity and mineralized nodules were suppressed by silencing TUG1. The expression levels of the odontogenic differentiation gene DSPP and DMP-1 and the osteogenic differentiation gene Runx2, OCN and OPN were also significantly decreased (P<0.05).@*Conclusion @# Knocking down TUG1 can inhibit the proliferation and osteogenic/odontogenic differentiation of hDPSCs.
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Objective: To assess the effect of application of Biodentine (BD), Photobiomodulation (PBM) using 810 nm diode laser and both on the proliferation and odontogenic differentiation of human dental pulp stem cells (HDPSCs). Material and Methods: HDPSCs were collected, isolated, and characterized and then divided into six groups: groups 1, control; groups 2, biodentine (BD); group 3, irradiation at 1 J/cm 2 of 810-nm diode laser; group 4, irradiation at 1 J/cm 2 and culture with BD; group 5, irradiation at 2 J/cm 2, and group 6, irradiation at 2 J/cm 2 and culture with BD. Viability assay was measured through MTT assay and Alkaline phosphatase (ALP) enzyme activity and mRNA levels of RUNX2, collagen 1 (Col-1) and BMP2 were also assessed. Results: Photobiomodulation at 1 and 2 J/cm 2 combined with biodentine significantly promoted HDPSCs proliferation (in MTT assay results) and odontogenic differentiation (through the gene expression of RUNX2, Col-1 and BMP2 levels (p < 0.05). Conclusion: Photobiomodulation at 2 J/cm 2 combined with biodentine enhanced proliferation and odontogenic differentiation of cultured HDPSCs and thus could further be beneficial for dentin regeneration (AU)
Objetivo: Avaliar o efeito da aplicação de Biodentina (BD), Fotobiomodulação (PBM) usando diodo de laser de 810 nm e ambos na proliferação e diferenciação odontogênica de células tronco cultivadas da polpa dental (HDPSCs). Material e Métodos: HDPSCs foram coletadas, isoladas, caracterizadas e então divididas em seis grupos: grupo 1, controle; grupo 2, biodentina (BD); grupo 3, irradiação com diodo de laser a 1 J/cm2 de 810- nm; grupo 4, irradiação a 1 J/cm 2 e cultivo com BD; grupo 5, irradiação a 2 J/cm2, e grupo 6, irradiação a 2 J/cm2 e cultivo com BD. A viabilidade foi mensurada através do teste MTT e a atividade da enzima Fosfatase alcalina (ALP), e níveis de RNAm de RUNX2, de colágeno 1 (Col-1) e de BMP2 foram também mensurados. Resultados: Fotobiomodulação a 1 e 2 J/cm 2 combinada com biodentina promoveu significativa proliferação de HDPSCs (nos resultados do teste MTT) e diferenciação odontogênica (através da expressão genética dos níveis de RUNX2, Col-1 e BMP2 (p < 0.05)). Conclusão: Fotobiomodulação a 2 J/cm2 combinada com biodentina aumentou a proliferação e diferenciação odontogênica de HDPSCs cultivadas e dessa forma poderia ser benéfica para a regeneração dentinária. (AU)
Subject(s)
Stem Cells , Collagen Type I , Core Binding Factor Alpha 1 SubunitABSTRACT
BACKGROUND: Human dental pulp stem cells are important oral mesenchymal stem cells with strongproliferation and multidirectional differentiation functions. In-depth studies on the Human Genome Project make people gradually reali ze that functional non-coding RNAs play an extraordinary role in regulating gene expression. OBJECTIVE: To discuss the function and application of non-coding RNAs in human dental pulp stem cells. METHODS: Using “ncRNAs, human dental pulp stem cells, regenerative medicine” as keywords in English and Chinese, the first author searched PubMed, Medline, CNKI, and WanFang for relevant articles published from 2005 to 2019. Literatures unrelated to the purpose of the study and repetitive literatures were excluded, and 71 articles that meet the criteria were included for review. RESULTS AND CONCLUSION: It is now generally believed that non-coding RNAs can be used as a signal of specific cell state, providing prognostic value and even providing treatment options for patients. With the continuous development of regenerative medicine applications, human dental pulp stem cells are arousing increasing attentions. Exploration on the relationship between non-coding RNAs and human dental pulp stem cells provides a new approach for the clinical application of human dental pulp stem cells.
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BACKGROUND: Prostaglandin E1 and basic fibroblast growth factor can promote the proliferation of human dental pulp stem cells, but the effects of their combinations on the proliferation of human dental pulp stem cells and angiogenesis are unknown. OBJECTIVE: To investigate the effects of prostaglandin E1 combined with basic fibroblast growth factor on the proliferation and angiogenesis of human dental pulp stem cells. METHODS: (1) Human dental pulp stem cells were isolated and cultured in vitro. After detection and identification of surface markers, prostaglandin E1 and basic fibroblast growth factor at concentrations of 5, 10, 20, 50 and 100 μg/L were used to treat human dental pulp stem cells in vitro. The untreated cells served as control group. The cell proliferation was detected by cell counting kit-8 assay, and the optimum drug concentration and time of drug action were screened. (2) The in vitro cultured human dental pulp stem cells were divided into four groups: blank control group, prostaglandin E1 group, basic fibroblast growth factor group and combination group. The cell proliferation was detected by cell counting kit-8 assay. Human dental pulp stem cell conditioned medium was extracted. The levels of vascular endothelial growth factor and endostatin in the culture medium were detected by ELISA. The in vitro tubular formation ability of human umbilical vein endothelial cells after culture in conditioned medium was tested by tubule formation experiment. RESULTS AND CONCLUSION: The optimum concentration of prostaglandin E1 and basic fibroblast growth factor was 20 μg/L, and the optimum time of action was 2 days. Compared with the blank control group, the relative proliferation rate, level of vascular endothelial growth factor and the angiogenesis ability of human umbilical vein endothelial cell in vitro in the prostaglandin E1, basic fibroblast growth factor and combination groups were significantly increased (P < 0.05), while the level of endostatin was significantly decreased (P < 0.05). All above index levels in the combination group were significantly superior to those in the prostaglandin E1 and basic fibroblast growth factor groups (P < 0.05). In summary, prostaglandin E1 combined with basic fibroblast growth factor can promote the proliferation of human dental pulp stem cells and enhance the tubular formation ability of human umbilical vein endothelial cells in vitro.
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BACKGROUND: The major challenge of tissue engineering is to develop constructions with suitable properties which would mimic the natural extracellular matrix to induce the proliferation and differentiation of cells. Poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone) (PCL-PEG-PCL, PCEC), chitosan (CS), nano-silica (n-SiO₂) and nano-hydroxyapatite (n-HA) are biomaterials successfully applied for the preparation of 3D structures appropriate for tissue engineering. METHODS: We evaluated the effect of n-HA and n-SiO₂ incorporated PCEC-CS nanofibers on physical properties and osteogenic differentiation of human dental pulp stem cells (hDPSCs). Fourier transform infrared spectroscopy, field emission scanning electron microscope, transmission electron microscope, thermogravimetric analysis, contact angle and mechanical test were applied to evaluate the physicochemical properties of nanofibers. Cell adhesion and proliferation of hDPSCs and their osteoblastic differentiation on nanofibers were assessed using MTT assay, DAPI staining, alizarin red S staining, and QRT-PCR assay. RESULTS: All the samples demonstrated bead-less morphologies with an average diameter in the range of 190–260 nm. The mechanical test studies showed that scaffolds incorporated with n-HA had a higher tensile strength than ones incorporated with n-SiO₂. While the hydrophilicity of n-SiO₂ incorporated PCEC-CS nanofibers was higher than that of samples enriched with n-HA. Cell adhesion and proliferation studies showed that n-HA incorporated nanofibers were slightly superior to n-SiO₂ incorporated ones. Alizarin red S staining and QRT-PCR analysis confirmed the osteogenic differentiation of hDPSCs on PCEC-CS nanofibers incorporated with n-HA and n-SiO₂. CONCLUSION: Compared to other groups, PCEC-CS nanofibers incorporated with 15 wt% n-HA were able to support more cell adhesion and differentiation, thus are better candidates for bone tissue engineering applications.
Subject(s)
Humans , Biocompatible Materials , Bone and Bones , Cell Adhesion , Chitosan , Dental Pulp , Durapatite , Extracellular Matrix , Hydrophobic and Hydrophilic Interactions , Nanofibers , Nanoparticles , Osteoblasts , Silicon Dioxide , Spectroscopy, Fourier Transform Infrared , Stem Cells , Tensile Strength , Tissue EngineeringABSTRACT
Objective:To investigate the effects and its mechanism of lipopolysaccharide on the proliferation,differentiation and apoptosis of human dental pulp stem cells by Notch signaling pathway. Methods:Human dental pulp stem cells( hDPSCs) was isolated from dental pulp tissue;cell proliferation was detected after 0,0. 1,1,10 μg/ml treated cells for 1,3,5,7 days by CCK8 test;related mRNA expression ALP,DSPP,DMP1 gene was detected after 1 μg/ml lipopolysaccharide treated hDPSCs for 0,3,7,14,21 day by RT-PCR;cell apoptosis was detected after 1 μg/ml lipopolysaccharide treated hDPSCs for 0,7,14,21 day by flow cytometry. Cleaved caspase3,Notch1,Hes1 protein expression was detected by Western blot. Results:Cell proliferation after different concentrations lipopo-lysaccharide stimulated hDPSCs for 1, 3, 5 days had no significant difference, significantly lower at 7 day than 0 μg/ml lipopolysaccharide group(P<0. 01). ALP,DSPP,DMP1 mRNA expression lipopolysaccharide treated hDPSCs at 3 day compared with the control group had no statistical significance(P>0. 05),significantly higher at 7,14,21 day than control group(P<0. 01);cells apoptosis rate and Cleaved caspase3,Notch1,Hes1 protein expression lipopolysaccharide treatment hDPSCs at 7,14 and 21day was sig-nificantly higher than the control group(P<0. 01);ALP,DSPP,DMP1 mRNA expression and apoptosis rate and Cleaved caspase3, Notch1,Hes1 protein expression at 21 day was a downward trend. Conclusion:Lipopolysaccharide can decrease the proliferation of hDPSCs and promote its mineralization and apoptosis,which may be related to the activation of Notch signaling pathway.
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Objective:To investigate the effects and its mechanism of lipopolysaccharide on the proliferation,differentiation and apoptosis of human dental pulp stem cells by Notch signaling pathway. Methods:Human dental pulp stem cells( hDPSCs) was isolated from dental pulp tissue;cell proliferation was detected after 0,0. 1,1,10 μg/ml treated cells for 1,3,5,7 days by CCK8 test;related mRNA expression ALP,DSPP,DMP1 gene was detected after 1 μg/ml lipopolysaccharide treated hDPSCs for 0,3,7,14,21 day by RT-PCR;cell apoptosis was detected after 1 μg/ml lipopolysaccharide treated hDPSCs for 0,7,14,21 day by flow cytometry. Cleaved caspase3,Notch1,Hes1 protein expression was detected by Western blot. Results:Cell proliferation after different concentrations lipopo-lysaccharide stimulated hDPSCs for 1, 3, 5 days had no significant difference, significantly lower at 7 day than 0 μg/ml lipopolysaccharide group(P<0. 01). ALP,DSPP,DMP1 mRNA expression lipopolysaccharide treated hDPSCs at 3 day compared with the control group had no statistical significance(P>0. 05),significantly higher at 7,14,21 day than control group(P<0. 01);cells apoptosis rate and Cleaved caspase3,Notch1,Hes1 protein expression lipopolysaccharide treatment hDPSCs at 7,14 and 21day was sig-nificantly higher than the control group(P<0. 01);ALP,DSPP,DMP1 mRNA expression and apoptosis rate and Cleaved caspase3, Notch1,Hes1 protein expression at 21 day was a downward trend. Conclusion:Lipopolysaccharide can decrease the proliferation of hDPSCs and promote its mineralization and apoptosis,which may be related to the activation of Notch signaling pathway.
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The corneal reconstruction is tosurgial recover the structure integrity and corneal function after suffered from various trauma, inflammation and degenerative diseases.The corneal diseases caused millions of people worldwide suffering from eyesight damages and even blindness.At present, the corneal transplant is the main therapy for corneal blindness.However, the shortage in donor corneal issue is a worldwide problem and the failure due to the immunologic rejection of host is common.Nowadays, with the development of tissue culture and bioengineering technology, the application prospect of autologous stem cell transplantation is becoming more and more popular which might replace the allogeneic transplantation, becoming an important clinical treatment of regenerative medicine.Human dental pulp stem cells (hDPSCs) is a class of adult stem cell divided from the third molar teeth.Both hDPSCs and corneal cell are from the cranial nerve in embryonic ectoderm.Extensive researches show that the hDPSCs have the potentialities in corneal cell differentiation without causing immunologic rejection of the recipient.These findings manifested the potentials of hDPSCs in the clinical applications related to ocular surface reconstruction.In this paper, the features and current investigation status of hDPSCs in ocular surface reconstruction are reviewed.
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Objective:To evaluate the effects of human dental pulp stem cells-conditioned medium(HDPSCs-CM)on the osteogenic differentiation of human dental follicle stem cells(HDFSCs) in vitro.Methods:HDFSCs were in vitro cultured,purified and identified.CCK-8 assay was applied to evaluate the HDFSCs viability after 7 days cultured by HDPSCs-CM;the morphological changes of HDFSCs were observed;the osteogenic differentiation was studied by alkaline phosphatase(ALP) staining and alizarin red staining.The mRNA expression of POSTN,Col-Ⅰ,ALP,BSP and OPN was detected by Real-Time PCR.Results:Induced by HDPSCs-CM,HDFSCs exhibited several characteristics of cementoblast or osteoblast lineages.In the induction group the viability of HDFSCs was inhibited(P<0.05 or P<0.01),ALP staining was stronger and there were more mineralized nodules,the expression levels of POSTN,Col-Ⅰ,ALP,BSP and OPN were upregulated(P<0.05 or P<0.01).Conclusion:HDPSCs-CM can promote the osteogenic differentiation of HDFSCs.
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We investigated the potential of human dental pulp stem cells (hDPSCs) to differentiate into dopaminergic neurons in vitro as an autologous stem cell source for Parkinson's disease treatment. The hDPSCs were expanded in knockout-embryonic stem cell (KO-ES) medium containing leukemia inhibitory factor (LIF) on gelatin-coated plates for 3-4 days. Then, the medium was replaced with KO-ES medium without LIF to allow the formation of the neurosphere for 4 days. The neurosphere was transferred into ITS medium, containing ITS (human insulin-transferrin-sodium) and fibronectin, to select for Nestin-positive cells for 6-8 days. The cells were then cultured in N-2 medium containing basic fibroblast growth factor (FGF), FGF-8b, sonic hedgehog-N, and ascorbic acid on poly-l-ornithine/fibronectin-coated plates to expand the Nestin-positive cells for up to 2 weeks. Finally, the cells were transferred into N-2/ascorbic acid medium to allow for their differentiation into dopaminergic neurons for 10-15 days. The differentiation stages were confirmed by morphological, immunocytochemical, flow cytometric, real-time PCR, and ELISA analyses. The expressions of mesenchymal stem cell markers were observed at the early stages. The expressions of early neuronal markers were maintained throughout the differentiation stages. The mature neural markers showed increased expression from stage 3 onwards. The percentage of cells positive for tyrosine hydroxylase was 14.49%, and the amount was 0.526 ± 0.033 ng/mL at the last stage. hDPSCs can differentiate into dopaminergic neural cells under experimental cell differentiation conditions, showing potential as an autologous cell source for the treatment of Parkinson's disease.
Subject(s)
Animals , Humans , Mice , Brain/pathology , Cell Differentiation/drug effects , Cells, Cultured , Culture Media/chemistry , Dental Pulp/cytology , Dopaminergic Neurons/cytology , Enzyme-Linked Immunosorbent Assay , Glial Fibrillary Acidic Protein/genetics , Mice, Inbred ICR , Myelin Basic Protein/genetics , Real-Time Polymerase Chain Reaction , Stage-Specific Embryonic Antigens/genetics , Stem Cells/cytology , Tubulin/genetics , Tyrosine 3-Monooxygenase/analysisABSTRACT
We investigated the potential of human dental pulp stem cells (hDPSCs) to differentiate into dopaminergic neurons in vitro as an autologous stem cell source for Parkinson's disease treatment. The hDPSCs were expanded in knockout-embryonic stem cell (KO-ES) medium containing leukemia inhibitory factor (LIF) on gelatin-coated plates for 3-4 days. Then, the medium was replaced with KO-ES medium without LIF to allow the formation of the neurosphere for 4 days. The neurosphere was transferred into ITS medium, containing ITS (human insulin-transferrin-sodium) and fibronectin, to select for Nestin-positive cells for 6-8 days. The cells were then cultured in N-2 medium containing basic fibroblast growth factor (FGF), FGF-8b, sonic hedgehog-N, and ascorbic acid on poly-l-ornithine/fibronectin-coated plates to expand the Nestin-positive cells for up to 2 weeks. Finally, the cells were transferred into N-2/ascorbic acid medium to allow for their differentiation into dopaminergic neurons for 10-15 days. The differentiation stages were confirmed by morphological, immunocytochemical, flow cytometric, real-time PCR, and ELISA analyses. The expressions of mesenchymal stem cell markers were observed at the early stages. The expressions of early neuronal markers were maintained throughout the differentiation stages. The mature neural markers showed increased expression from stage 3 onwards. The percentage of cells positive for tyrosine hydroxylase was 14.49%, and the amount was 0.526 ± 0.033 ng/mL at the last stage. hDPSCs can differentiate into dopaminergic neural cells under experimental cell differentiation conditions, showing potential as an autologous cell source for the treatment of Parkinson's disease.
Subject(s)
Animals , Humans , Mice , Brain/pathology , Cell Differentiation/drug effects , Cells, Cultured , Culture Media/chemistry , Dental Pulp/cytology , Dopaminergic Neurons/cytology , Enzyme-Linked Immunosorbent Assay , Glial Fibrillary Acidic Protein/genetics , Mice, Inbred ICR , Myelin Basic Protein/genetics , Real-Time Polymerase Chain Reaction , Stage-Specific Embryonic Antigens/genetics , Stem Cells/cytology , Tubulin/genetics , Tyrosine 3-Monooxygenase/analysisABSTRACT
O hidróxido de cálcio (HCa), o agregado de trióxido mineral (MTA) e o óleo-resina de copaíba (COP) isoladamente apresentam características biológicas do material de capeamento pulpar direto mais apropriado. Com o pressuposto que associados poderiam originar materiais mais apropriados para serem aplicados em capeamento pulpar direto, este estudo objetivou analisar in vitro proliferação, diferenciação e migração de células-tronco de polpa de dente decíduo humano esfoliado (SHEDs) em resposta a substâncias liberadas pelo COP isolado ou associado ao HCa ou ao MTA. Proliferação, diferenciação e migração de SHEDs (Linhagem PDH3) foram analisadas através do ensaio de redução do MTT; da atividade de fosfatase alcalina (ALP), formação de nódulos mineralizados pelo ensaio de Vermelho de Alizarina e expressão dos genes (BGLAP, DSPP, DMP1 e HSP-27) pelo qRT-PCR; e, do ensaio do Scratch, respectivamente. As células foram submetidas à ação de meios condicionados pelos biomateriais, de acordo com os seguintes grupos experimentais: COP isolado (COP); HCa isolado (HCa); HCa associado ao COP (HCa+COP); MTA isolado (MTA) e MTA associado ao COP (MTA+COP). Células crescidas em meio de cultura fresco serviram de controle. Os dados foram comparados utilizando ANOVA complementado pelo teste de Tukey (p 0,05). O grupo HCa apresentou número de células viáveis significativamente menor que o dos demais grupos, inclusive o do grupo HCa+COP (p<0,01) em todos os tempos experimentais...
The calcium hydroxide (CaH), the mineral trioxide aggregate (MTA) and the oil-resin copaiba (COP) by themselves have biological characteristics of the ideal direct pulp capping material. With the assumption that when associated they could originate materials more appropriated for direct pulp capping, this study aimed to analyze the in vitro proliferation, differentiation and migration of stem cells from human exfoliated deciduous teeth (SHEDs) in response to substances leached from COP alone or associated with CaH or MTA. Proliferation, differentiation and migration of SHEDs (PDH3 lineage) were analyzed through the MTT reduction assay; alkaline phosphatase (ALP) activity, mineralized nodules formation using the Alizarin Red assay and gene expression (BGLAP, DSPP, DMP1 e HSP-27) using qRT-PCR; and the Scratch assay, respectively. The cells were submitted to the culture medium conditioned by the biomaterials according to the following experimental groups: COP alone (COP); CaH alone (CaH); CaH associated to COP (CaH+COP); MTA alone (MTA) and MTA associated to COP (MTA+COP). Cells grown in fresh culture medium served as control. Data were compared by ANOVA complemented by the Tukey´s test (p 0.05). The CaH group presented number of viable cells significantly smaller (p<0.01) than those of all other experimental groups, including the CaH+COP, during whole experimental time. The ALP activity in 14 days was similar in all experimental groups. In 21 days, the COP group presented the amount of mineralization higher (p<0.01) than those of all other groups. The gene DMP1 was not expressed by the cells in all experimental groups. The COP group presented the smallest expression (p<0.01) of BGLAP, DSPP and HSP-27 genes. The SHEDs of the MTA+COP group presented superexpression of the BGLAP, DSPP and HSP-27 genes, and in the CaH+COP group the cells superexpressed the BGLAP and HSP-27 genes...
Subject(s)
Biocompatible Materials , Stem Cells/metabolism , Dental Pulp CappingABSTRACT
This study investigated the genes involved in the differentiation of odontoblasts derived from human dental pulp stem cells (hDPSCs). hDPSCs isolated from human tooth pulp were validated by fluorescence activated cell sorting (FACS). After odontogenic induction, hDPSCs were analyzed investigated by Alizaline red-S staining, ALP assay, ALP staining and RT-PCR. Differential display-polymerase chain reaction (DD-PCR) was performed to screen differentially expressed genes involved in the differentiation of hDPSCs. By FACS analysis, the stem cell markers CD24 and CD44 were found to be highly expressed in hDPSCs. When hDPSCs were treated with agents such as beta-glycerophosphate (beta-GP) and ascorbic acid (AA), nodule formation was exhibited within six weeks. The ALP activity of hDPSCs was found to elevate over time, with a detectable up-regulation at 14 days after odontogenic induction. RT-PCR analysis revealed that dentin sialophosphoprotein (DSPP) and osteocalcin (OC) expression had increased in a time-dependent manner in the induction culture. Through the use of DD-PCR, several genes were differentially detected following the odontogenic induction. These results suggest that these genes may possibly be linked to a variety of cellular process during odontogenesis. Furthermore, the characterization of these regulated genes during odontogenic induction will likely provide valuable new insights into the functions of odontoblasts.
Subject(s)
Humans , Ascorbic Acid , Dental Pulp , Dentin , Extracellular Matrix Proteins , Flow Cytometry , Glycerophosphates , Mass Screening , Odontoblasts , Odontogenesis , Osteocalcin , Phosphoproteins , Sialoglycoproteins , Stem Cells , Tooth , Up-RegulationABSTRACT
Dlx3 and Dlx5 are homeobox domain proteins and are well-known regulators of osteoblastic differentiation. Since possible reciprocal relationships between osteogenic and adipogenic differentiation in mesenchymal stem cells exist, we examined the regulatory role of Dlx3 and Dlx5 on adipogenic differentiation using human dental pulp stem cells. Over-expression of Dlx3 and Dlx5 stimulated osteogenic differentiation but inhibited adipogenic differentiation of human dental pulp stem cells. Dlx3 and Dlx5 suppressed the expression of adipogenic marker genes such as C/EBPalpha, PPARgamma, aP2 and lipoprotein lipase. Adipogenic stimuli suppressed the mRNA levels of Dlx3 and Dlx5, whereas osteogenic stimuli enhanced the expression of Dlx3 and Dlx5 in 3T3-L1 preadipocytes. These results suggest that Dlx3 and Dlx5 exert a stimulatory effect on osteogenic differentiation of stem cells through the inhibition of adipogenic differentiation as well as direct stimulation.