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Dental pulp stem cells (DPSC) are pluripotent stem cells with high differentiation potential isolated from dental pulp. Using DPSC for vascular regeneration may be a good option. Hypoxia inducible factor-1α (HIF-1α) is an upstream gene of vascular endothelial growth factor (VEGF), and the small ubiquitin like protease 1 (SENP1) can reverse the small ubiquitin like (SUMO) modification of HIF-1α. Through the regulation of SENP1/HIF-1α, good vascular regeneration characteristics have been demonstrated in many in vitro and in vivo experiments. The SENP1/HIF-1α signaling axis has varying degrees of promoting and inhibiting effects on many solid tumors. Although there is relatively little literature on the role of the SENP1/HIF-1α signaling axis in dental pulp stem cells, it can be determined that SENP1/HIF-1α plays an important role in the angiogenesis of dental pulp stem cells. This article will elucidate the SENP1/HIF-1α signaling pathway and its mechanism of promoting vascular differentiation of DPSC.
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Abstract This study evaluated the influence of hydrofluoric acid (HF) concentration and thermal cycling on the microshear bond strength (µSBS) of a resin luting agent to IPS e.max® CAD and Rosetta® SM. Ceramic specimens (12.0 x 14.0 x 1.5mm) were randomized into 8 groups (n=10) according to HF concentration, commercial brand, and aging. Immediately after polishing, and etching, all specimens were silanized and a layer of adhesive was applied. A PVS mold of 3 mm thickness and 10mm diameter with (four) 1.0mm holes was fabricated, placed on each specimen, and then filled with a resin luting agent. Half of the specimens were subjected to the µSBS test using an Instron at a speed of 1.0 mm/min, following a 24-hour storage in deionized water at 37ºC. The remaining specimens were subjected to thermal cycling (5ºC-55ºC, 30 seconds per bath) and µSBS. The data were evaluated utilizing a three-way ANOVA and Tukey's post-hoc test (α=0.05). Significant differences were found for HF concentration and aging (p<0.0001). No significant difference in µSBS was found for commercial brands (p=0.085). The interaction between brand and HF concentration (p=0.358), brand and aging (p=0.135), and HF concentration and aging (p=0.138) were not statistically significant. The triple interaction among these factors was not statistically significant (p=0.610). In conclusion, the bond strength is affected by the HF concentration. No statistical difference was observed between the two ceramics. Thermal cycling significantly reduced µSBS.
Resumo Este estudo avaliou a influência da concentração do ácido fluorídrico (AF) e da ciclagem térmica na resistência de união ao microcisalhamento (RUµC) de um cimento resinoso para IPS e.max® CAD e Rosetta® SM. Espécimes cerâmicos (12,0 x 14,0 x 1,5mm) foram divididos em 8 grupos (n=10) de acordo com concentração do HF, marca comercial e envelhecimento. Imediatamente após o polimento e condicionamento ácido, todos os espécimes foram silanizados e uma camada de adesivo foi aplicada. Um molde PVS de 3 mm de espessura e 10 mm de diâmetro com (quatro) orifícios de 1,0 mm foi confeccionado, colocado em cada espécime e preenchido com o cimento resinoso. Metade dos espécimes foi submetida ao teste RUµC na Instron a velocidade de 1,0 mm/min, após 24 horas de armazenamento em água deionizada a 37ºC. Os espécimes restantes foram submetidos a ciclagem térmica (5ºC-55ºC, 30 segundos por banho) e a RUµC. Os dados foram avaliados por ANOVA de três fatores e ao teste post-hoc de Tukey (α=0,05). Diferenças significativas foram encontradas para concentração de HF e envelhecimento (p<0,0001). Nenhuma diferença significativa na RUµC foi encontrada para cada marca comercial (p=0,085). A interação entre marca comercial e a concentração do HF (p=0,358), marca comercial e envelhecimento (p=0,135) e concentração do HF e envelhecimento (p=0,138) não foram estatisticamente significativas. A tripla interação entre esses fatores não foi estatisticamente significativa (p=0,610). Concluindo, a resistência de união é afetada pela concentração de HF. Não foi observada diferença estatística entre as duas cerâmicas. A ciclagem térmica reduziu significativamente a resistência de união ao microcisalhamento.
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Abstract Studies regarding cytotoxic effects attributed to the use of adhesive bonding agents on pulp tissue are not conclusive. To point out whether these materials are safe for clinical use, in vivo exposure of dental pulp to adhesive bonding agents was simulated using an experimental setup in which Human Dental Pulp Stem Cells (hDPSC) are exposed to the action of two kinds of adhesives: self-etching adhesives and two-step bonding agents through a dentine barrier. Cytotoxic effects on these cells were evaluated by MTT assay protocol and fluorescence microscopy, and their results were contrasted to those obtained through Raman spectra taken on single hDPSCs. Overall, no significant cytotoxic effects were observed by combining all the techniques, and cell viability close to 90% was achieved for a dentine barrier of at least 1 mm thick. Moreover, Raman spectroscopy was able to detect structural DNA damage in some dental pulp cells when exposed to two-step bonding agents, suggesting that this technique could be considered a complementary tool with the potential to evaluate cell toxicity beyond cell viability.
Resumo Os estudos sobre os efeitos citotóxicos atribuídos ao uso de agentes de união adesivo no tecido pulpar não são conclusivos. Para determinar se esses materiais são seguros para uso clínico, a exposição in vivo da polpa dentária a agentes de união adesiva foi simulada por meio de uma configuração experimental na qual as células-tronco da polpa dentária humana (hDPSC) são expostas à ação de dois tipos de adesivos: adesivos autocondicionantes e agentes de união de duas etapas por meio de uma barreira de dentina. Os efeitos citotóxicos nessas células foram avaliados pelo protocolo de ensaio MTT e microscopia de fluorescência, e seus resultados foram contrastados com os obtidos por meio de espectros Raman obtidos em hDPSCs individuais. De modo geral, não foram observados efeitos citotóxicos significativos com a combinação de todas as técnicas, e a viabilidade celular próxima a 90% foi obtida para uma barreira de dentina de pelo menos 1 mm de espessura. Além disso, a espectroscopia Raman foi capaz de detectar danos estruturais ao DNA em algumas células da polpa dentária quando expostas a agentes de colagem de duas etapas, sugerindo que essa técnica poderia ser considerada uma ferramenta complementar com potencial para avaliar a toxicidade celular além da viabilidade celular.
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Abstract Objective This study aims to explore the regulatory mechanism of long noncoding RNA X inactive specific transcript (lncRNA XIST) in the odontogenic differentiation of human dental pulp stem cells (hDPSCs). hDPSCs were obtained from freshly extracted third molars and identified by flow cytometry. Methodology Odontogenic differentiation was induced in mineralized culture medium, and hDPSCs were infected with shRNA lentivirus targeting XIST or fused in sarcoma (FUS), followed by detection of alkaline phoshpatase (ALP) activity, alizarin red staining of mineralized nodules, Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) quantification of XIST expression, and Western blot analysis of FUS, ZBTB16, and odontogenic differentiation markers (DSPP and DMP1). IF-FISH was performed to detect the cellular localization of XIST and FUS. RIP assay validated the XIST and FUS binding. ZBTB16 mRNA stability was tested after actinomycin D treatment. hDPSCs were infected with oe-ZBTB16 lentivirus and further treated with sh-XIST for a combined experiment. Results LncRNA XIST was highly expressed in hDPSCs with odontogenic differentiation. Downregulation of XIST or FUS weakened the ALP activity of hDPSCs, reduced mineralized nodules, diminished DSPP and DMP1 expressions. XIST binds to FUS to stabilize ZBTB16 mRNA and promote ZBTB16 expression. ZBTB16 overexpression partially reversed the inhibitory effect of XIST silencing on odontogenic differentiation of hDPSCs. Conclusion In conclusion, XIST stabilizes ZBTB16 mRNA and promotes ZBTB16 expression by binding to FUS, thereby facilitating the odontogenic differentiation of hDPSCs.
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Objective @# To investigate the effects of angiopoietin 4 (ANGPT4) on the odontogenic differentiation of human dental pulp stem cells. @* Methods @#This study has been reviewed and approved by the Ethics Committee, and informed consent has been obtained from patients. Human premolars were fixed, decalcified, dehydrated, embedded, and sectioned. Immunofluorescence staining was used to observe the expression and localization of ANGPT4. Human dental pulp stem cells (hDPSCs) were isolated and cultured in vitro. The growth state and morphology of hDPSCs were observed under an inverted phase contrast microscope. The expression of cell surface-related molecular markers was detected by flow cytometry. Alkaline phosphatase and alizarin red S staining were used to detect the odontogenic differentiation potential of hDPSCs. Oil-red O staining was used to detect the adipogenic differentiation potential of hDPSCs. RNA was extracted from hDPSCs at different time points after odontogenic induction, and RT-qPCR was used to analyze the mRNA expression of ANGPT4 and odontogenic-related genes during the odontogenic differentiation of hDPSCs in vitro. siRNA gene silencing technology was used to silence the expression of ANGPT4 in hDPSCs, and the silencing efficiency was detected by RT-qPCR and Western Blot. After silencing ANGPT4 in hDPSCs for 24 h, odontogenic induction was performed. Alkaline phosphatase and alizarin red S staining were performed on the 7th and 14th of induction to detect the odontogenic differentiation ability of hDPSCs after silencing ANGPT4@*Results @# Immunofluorescence staining of human premolars showed that ANGPT4 was expressed in odontoblasts and sub-odontoblastic cell-rich zone. hDPSCs were in good condition after 14 days of isolation and culture. Under the microscope, multiple cell colonies were observed, and the cell morphology was uniform and long spindle-shaped. The results of flow cytometry showed that hDPSCs expressed mesenchymal stem cell markers CD105 (90.42%) and CD90 (97.15%), but did not express hematopoietic cell markers CD45 (0.01%) and CD34 (0.08%). After odontogenic and adipogenic induction of hDPSCs, alkaline phosphatase staining, alizarin red S staining and oil red O staining were positive. The results of RT-qPCR after the odontogenic induction of hDPSCs showed that ANGPT4 was highly expressed on the 5th, 7th, 11th and 14th days of differentiation of hDPSCs (P<0.05), with the highest expression level on the 5th day. After hDPSCs were transfected with si-ANGPT4, the expression of ANGPT4 mRNA and protein was significantly down-regulated (P<0.05). The results of alkaline phosphatase staining showed that ALP staining intensity and area in the si-ANGPT4 group were significantly lower than those in the negative control. Alizarin red S staining showed that the formation of calcium nodules in the si-ANGPT4 group was significantly lower than that in the negative control.@* Conclusion@#ANGPT4 was expressed in odontoblasts and sub-odontoblastic cell-rich zone of human premolars. ANGPT4 may be a factor to promote the odontogenic differentiation of hDPSCs.
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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 study the effect of stem cell factor (SCF) on the angiogenic ability of cocultured dental pulp stem cells (DPSCs) and human umbilical vein endothelial cells (HUVECs).@*Methods @#This study has been reviewed and approved by the Ethics Committee. The experiment was split into the HUVECs, SCF+HUVECs, DPSCs+HUVECs, and SCF+DPSCs+HUVECs groups. A mixture of SCF and culture medium was used to prepare a mixed culture medium with an SCF concentration of 100 ng/mL. In vitro coculture of DPSCs and HUVECs was performed at a 1∶5 ratio. CCK-8 proliferation assay was used to observe the proliferative capacity of cells in each group on days 1, 3, 5, and 7. Wound healing and Transwell migration assays were used to detect the effect of SCF on cell migration under either direct or indirect coculture conditions, respectively. In vitro angiogenesis experiments were performed to detect the angiogenic capacity of the cells in each group. The vascular endothelial growth factor A (VEGFA) concentration in the cell culture supernatant was detected using ELISAs, and the protein expression levels of CD31, CD34, and VEGFA were detected using Western blot analysis. @*Results @# Wound healing and Transwell migration experiments showed that SCF significantly promoted the migration of cocultured DPSCs and HUVECs (P<0.05). The in vitro angiogenesis experiment showed that the number of branches and the total length of branches of tubular structures in the SCF+DPSCs+HUVECs group were significantly greater than those of the other groups (P<0.05), and the expression levels of the vascular-related proteins CD31, CD34, and VEGFA in this group were greater (P<0.01). @*Conclusion @# SCF can enhance the migration and in vitro angiogenesis of cocultured DPSCs and HUVECs.
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@#Cell therapy based on mesenchymal stem cells (MSCs) has been a hot research topic in recent years, including the traditional cell therapy strategy based on living cells and the new cell-free therapy strategy based on soluble proteins or bioactive molecules such as extracellular vesicles (EVs). At present, MSC-induced cells have mature functions and specific structures, and insitu transplantation combined with biomaterials or organic technology has greatly improved the settlement rate and function. On the other hand, as the large-scale culture technique and EVs separation technique evolve, it is possible to obtain a large number of pure EVs, and EVs are gradually becoming a hot spot of current research. An increasing number of studies have shown that the therapeutic effect of MSCs not only occurs by implantation and differentiation but also manifests as the paracrine effect of MSCs. In this review, we discuss the emerging outcomes of cell therapies and acellular therapies to alleviate these pathological conditions.
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Objective To investigate the impacts of tanshinone ⅡA(Tan ⅡA)on lipopolysaccharide(LPS)induced proliferation and apoptosis of dental pulp stem cells by regulating the fatty acid synthase(Fas)/fatty acid synthase ligand(FasL)signaling pathway.Methods Identification of human pulp stem cells(hDPSCs)isolated from the third molar of 18~20 years old patients requiring orthodontics.Lps-induced hDPSCs were treated with low,medium and high doses of Tan ⅡA,and then human recombinant FasL protein(rh FasL)was used to intervene the LPS-induced hDPSCs after high dose Tan ⅡA.Proliferation and apoptosis of hDPSCs,levels of tumor necrosis factor-α(TNF-α)and interleukin(IL)-6 in hDPSCs supernatant,proliferating cell nuclear antigen(PCNA),Cleaved aspartate-specific cysteine proteinase-3(Cleaved Caspase-3),Fas,FasL protein expression were detected.Results hDPSCs were successfully isolated.In a dose-dependent manner,Tan ⅡA promoted LPS-induced proliferation,inhibited apoptosis,up-regulated PCNA protein expression,and inhibited TNF-α,IL-6 level,Cleaved Caspase-3,Fas,and FasL protein expression.The effect of rh FasL on LPS-induced dental pulp stem cells was opposite to the above indexes(P<0.05).rh FasL attenuates the effect of high-dose Tan ⅡA on pro-liferation and apoptosis of LPS-induced dental pulp stem cells.Conclusion Tan ⅡA may promote LPS induced hDPSCs proliferation and inhibit apoptosis by inhibiting the Fas/FasL signaling pathway.
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Objective @#To investigate the effects of Lin28 overexpression on the proliferation and osteogenic differentiation of human dental pulp stem cells (hDPSCs) through mTOR signaling pathway.@*Methods @#After transfecting lentiviral vectors of Lin28 gene in hDPSCs , the relative expression of Lin28 was detected by Real⁃time PCR. CCK⁃8 assay was applied to detect the effect on cell proliferation. qRT⁃PCR was used to research the expression levels of alkaline phosphatase (ALP) , osteopontin (OPN) and osteocalcin (OCN) . Western blot assay was processed to investigate the effects on the relative expression levels of ALP and OPN proteins. Alizarin red staining was utilized to detect the mineralized nodules. @*Results @#Compared with the control group , the cell proliferation of transfection group was promoted (P < 0. 05) ; The mRNA and protein expression levels of ALP , OPN and OCN in transfection group were significantly lower than those in control group (P < 0. 05) , the expression level of ALP apparently decreased after the addition of mTOR inhibitor rapamycin (P < 0. 05) ; Alizarin red staining showed that the size and number of mineralized nodules formed in transfection group were markedly declined compared with empty carrier group (P < 0. 05) .@*Conclusion @#Overexpression of Lin28 can inhibit the osteogenic differentiation of hDP⁃ SCs through suppress mTOR signaling pathway.
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Introduction: Mesenchymal stem cells (MSCs) are the adult stem cells with potential to differentiate into various tissues. Like in other tissues, MSCs also reside in dental pulp after toot development and help in repair and regeneration by differentiating into odontoblasts. Dental Pulp Stem Cells (DPSCs) and Stem cells from Apical Papilla (SCAP) are the type of MSCs from dental papilla and apical papilla respectively. Aim: The aim of this paper is to highlight the characteristics of DPSCs and SCAP. Method: Information was obtained and compiled from published literature and electronic database search engine from PubMed and Google Scholar. Results: In spite of both DPSCs and SCAP having similar cell population origin they possess some different characteristics. Conclusion: The Dental stem cells with different characteristics of similar origin can be utilized in the stem cell based tissue engineering.
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Abstract An endodontic material must be minimally harmful to stem cells since they are essential, thanks to their capacity for cell proliferation, self-renewal, and differentiation. For this reason, in this in vitro study, the cell viability and the expression of genes involved in cell plasticity and differentiation were investigated in stem cells recovered from human dental pulp (hDPSCs) that were in contact with four endodontic materials (Endofill, MTA, Pulp Canal Sealer, and Sealer 26). The viability of HDPSCs was assessed by MTT and trypan blue exclusion assays. PCR evaluated cellular plasticity by determining the CD34, CD45, Nestin, CD105, Nanog, and OCT4 expressions. The effect on cell differentiation was determined by RT-PCR expression of the RUNX2, ALP, OC/BGLAP, and DMP1 genes. The data were analyzed using ANOVA with Bonferroni correction (p <0.05). Pulp Canal Sealer and Endofill decreased cell viability after 48 hours (p <0.001). MTA and Sealer 26 did not disrupt cell viability (p> 0.05). When cultivated in the presence of MTA and Sealer 26, hDPSCs expressed Nestin, CD105, NANOG, and OCT-4 and did not express CD34 and CD45. MTA and Sealer 26 interfered with DMP1, OC/BGLAP and RUNX2 expressions (p <0.05) but did not change ALP gene expression (p> 0.05). MTA and Sealer 26 showed biological compatibility in the presence of hDPSCs.
Resumo Um material endodôntico deve ser minimamente prejudicial às células-tronco, uma vez que essas células são extremamente importantes, devido à sua capacidade de proliferação, autorrenovação e diferenciação celular. Por esse motivo, a viabilidade celular e a expressão de genes envolvidos na plasticidade e diferenciação celular foram investigadas em células-tronco recuperadas de polpa dentária humana (HDPSCs) que estiveram em contato com quatro materiais endodônticos (Endofill, MTA, Pulp Canal Sealer e Sealer 26). A viabilidade das HDPSCs foi avaliada pelos ensaios MTT e de exclusão de azul de tripano. A plasticidade celular foi avaliada pela determinação das expressões dos genes CD34, CD45, Nestin, CD105, Nanog e OCT4 por PCR. O efeito na diferenciação celular foi determinado pela expressão dos genes RUNX2, ALP, OC/BGLAP e DMP1 por RT-PCR. Os dados foram analisados por ANOVA com correção de Bonferroni (p <0,05). Em comparação com o controle, Pulp Canal Sealer e Endofill diminuíram a viabilidade celular após 48 horas (p <0,001). MTA e Sealer 26 não interromperam a viabilidade celular (p> 0,05). Quando cultivado na presença de MTA e Sealer 26, as HDPSCs expressaram Nestin, CD105, NANOG e OCT-4 e não expressaram CD34 e CD45. MTA e Sealer 26 interferiram nas expressões de DMP1, OC / BGLAP e RUNX2 (p <0,05), mas não alteraram a expressão do gene ALP (p> 0,05). Sendo assim, MTA e Sealer 26 demonstraram compatibilidade biológica na presença de HDPSCs.
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Objective@#To investigate the effects of apoptotic bodies (ABs) derived from dental pulp stem cells (DPSCs) on macrophage polarization and inflammation response in vivo. @*Methods @#Human DPSCs were extracted, cultured and identified. Staurosporine was used to apoptosis induction and differential methods were performed for ABs identification. The in vitro cultured macrophages were divided into 3 groups: solvent control, lipopolysaccharide (LPS), and the LPS+ABs. The macrophages were stimulated with LPS to induce inflammation followed by ABs treatment. In the untreated group, macrophages were added with an equal amount of solvent. The specific uptake of ABs by macrophages, the expression level of CD206 and the levels of inflammatory cytokines were analyzed. The mouse models of cutaneous wounds and dextran sulfate sodium (DSS)-induced colitis were established, and the mice were randomly divided into 3 groups: the PBS-treated group, the DPSCs-treated group, and the ABs-treated group. The mice were injected with the same volume of PBS, DPSCs and ABs, respectively. The body weight, histological pathology, the expression levels of CD206 and cytokines, and the extent of tissue regeneration were measured.@* Results @#DPSCs and ABs derived from DPSCs were successfully isolated and characterized. ABs could be taken up by macrophage. While lipopolysaccharide(LPS) induced production of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), ABs significantly reduced the levels of these pro-inflammatory cytokines and increased the expression of transforming growth factor-β (TGF-β) and CD206 (P < 0.01). In the cutaneous inflammatory wound model, the wound closure rate in mice intravenously injected with ABs was significantly accelerated (P < 0.05). The administration of ABs markedly reduced the pro-inflammatory factors levels and increased the CD206+ cell number. In the colitis model, treatment with ABs markedly reduced the loss in bodyweight (P < 0.05), recovered the colon length (P < 0.01), and significantly increased the CD206+ cell number.@* Conclusion@# DPSCs-derived ABs could enhance macrophage M2 polarization and attenuate inflammation. Therefore, ABs could be used as a promising cell replacement for inflammatory regulation and tissue regeneration.
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Objective @#To study the regulatory effect of coiled-coil domain containing 134 (CCDC134) on the osteogenic differentiation of human dental pulp stem cells (hDPSCs).@*Methods @# HDPSCs were isolated and cultured from dental pulp tissue and transfected with NC-CCDC134, shCCDC134 and CCDC134 lentiviruses. They were divided into the control group, negative control group, CCDC134 downregulation (shCCDC134) group and CCDC134 overexpression (CCDC134) group. Surface markers of hDPSCs (Stro-1, CD105, CD34, CD45) were detected by flow cytometry; colony formation was analyzed by toluidine blue staining; ALP expression was estimated by ALP staining; mineralized nodule formation was evaluated by alizarin red staining; lipid droplet formation was examined by oil red staining; and gene and protein expression of CCDC134, Runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), bone morphogenetic protein-2 (BMP-2), and mothers against decapentaplegic homolog 1 (SMAD1) was detected by qPCR and western blot, respectively. Further, a BMP-2 activator (BMP-2) and inhibitor (Dorsomorphin) were used to down-regulate and up-regulate CCDC134, respectively (shCCDC134, shCCDC134+BMP-2, CCDC134, CCDC134+Dorsomorphin), in hDPSCs. The hDPSC aggregates were subcutaneously transplanted into nude mice for 2 months, and new bone formation was detected by H&E staining. The BMP-2/SMAD1 signaling in each group was detected by qPCR.@*Results@#hDPSCs showed high expression of mesenchymal markers and low expression of hematopoietic markers. Compared with the control group, the expression of CCDC134 was increased in the osteogenic-induced hDPSCs (P < 0.05). Compared with the negative control group, the expression of CCDC134 was decreased in the shCCDC134 group, whereas it was increased in the CCDC134 group (P < 0.05). The mineralized nodules, osteogenic genes and proteins in the shCCDC134 group were decreased (P < 0.05), while they were increased in the CCDC134 group (P < 0.05). The expression of BMP-2/SMAD1 signaling decreased in the shCCDC134 group, while it increased in the CCDC134 group (P < 0.05). Compared to the shCCDC134 group, osteogenic genes and proteins increased in the shCCDC134+BMP-2 group, and subcutaneous new bone formation increased in nude mice (P < 0.05). The indexes of the CCDC134+Dorsomorphin group decreased compared with the CCDC134 group (P < 0.05).@*Conclusion@#CCDC134 promotes the osteogenic differentiation of hDPSCs by regulating the BMP-2/SMAD1 signaling pathway.
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Objective @#To investigate the effects of N-cadherin silencing on the proliferation and migration of human dental pulp stem cells (DPSCs) and to provide experimental evidence for DPSCs-based dental pulp regeneration.@* Methods@# DPSCs were transfected with N-cadherin shRNA lentivirus, and the knockdown efficiency of N-cadherin at both the mRNA and protein levels was confirmed by qRT-PCR and Western blot. The experiment included a negative control group (shRNA -NC) and an N-cadherin shRNA silencing group. Cell proliferation was detected by the CCK-8 method. Cell cycle and apoptosis were assessed by flow cytometry, and cell migration was detected using the Transwell method.@*Results@#N-cadherin shRNA significantly reduced the expression levels of N-cadherin mRNA and protein in DPSCs (P<0.001). The proliferation activity of the N-cadherin shRNA group was significantly greater than that of the shRNA-NC group on the 3rd and 4th days after cell inoculation and lower than that of the shRNA-NC group from the 6th to 8th days (P<0.05). On the 3rd day after cell inoculation, the proportion of cells in S phase and G2 phase in the N-cadherin shRNA group was greater than that in the shRNA-NC group (P<0.05). On the 6th day after cell inoculation, the proportion of cells in S phase and G2 phase in the N-cadherin shRNA group was lower than that in the shRNA-NC group (P<0.05), and the proportion of apoptotic cells in the N-cadherin shRNA group was greater than that in the shRNA-NC group (P<0.01). Low densities cells and high densities cells were inoculated into Transwell upper chamber for 20 h, the number of cells passing through the membrane pores of upper chamber in the N-cadherin shRNA group was greater than that in the shRNA-NC group (P<0.001).@*Conclusion@#Silencing N-cadherin expression can promote the early proliferation and migration of DPSCs.
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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 investigate the effect of tumor necrosis factor-α(TNF-α) on biological properties of dental pulp stem cells (DPSCs) in low-oxidation stress. Methods The experimental groups were as follows: normox, normox control plasmid, normox-TNF-α, 3% O
RÉSUMÉ
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)
Sujet(s)
Cellules souches , Collagène de type I , Sous-unité alpha 1 du facteur CBFRÉSUMÉ
Objective: 1) To critically review the published literature on applications of dental stem cells in the regeneration of intraoral tissues. 2) To provide an evidence-based level on research regarding application of dental stem cells in intraoral tissues regeneration. Methodology: This systematic review is conducted as per the JBI guidelines and reported as per the PRISMA. An initial literature search of papers published between 2004 and 2018 yielded 421 manuscripts. Nineteen studies satisfied the inclusion / exclusion criteria and were included for qualitative synthesis. Studies were categorized as animal (11) and human (8) trials. Five independent reviewers critically assessed the included studies. Risk of bias was assessed using SYstematic Review Centre for Laboratory animal Experimentation (SYRCLE) bias risk tool, robins-I tool for non-randomised clinical trial and Cochrane Collaboration's Tool for randomised clinical trial. Evidence levels were assessed based on JBI Criteria. Results: Animal trials mainly focused on periodontal regeneration. A high or unclear Risk of bias was more commonly found amongst animal studies. Laboratory, clinical and radiographic evaluation were used to assess the outcome. A total of Eight Human studies were conducted on a total samples size of 153 upon a wide age ranging from seven years to 60 years. Nearly 70% of the human studies used DPSC for regenerating alveolar bone defects. Conclusion: Appropriate well designed double-blind randomized clinical trials of longer duration are yet to be performed. Evidence for the included studies were 1C and 1D as per the JBI Criteria. Stem cell therapy demonstrated promising results in Periodontal tissue and alveolar bone regeneration. However, the number of studies to claim such a benefit are very limited (AU)
Objetivo: 1) Revisar criticamente a literatura publicada sobre aplicações de células-tronco dentárias na regeneração de tecidos intraorais. 2) Fornecer um nível baseado em evidências sobre pesquisas relacionadas à aplicação de células-tronco dentárias na regeneração de tecidos intraorais. Metodologia: Esta revisão sistemática é conduzida de acordo com as diretrizes do JBI e relatada de acordo com o PRISMA. Uma pesquisa bibliográfica inicial de artigos publicados entre 2004 e 2018 resultou em 421 manuscritos. Dezenove estudos satisfizeram os critérios de inclusão / exclusão e foram incluídos para síntese qualitativa. Os estudos foram categorizados como ensaios em animais (11) e humanos (8). Cinco revisores independentes avaliaram criticamente os estudos incluídos. O risco de viés foi avaliado usando a ferramenta de risco de viés do Centro de Revisão Sistemática para Experimentação com Animais de Laboratório (SYRCLE), a ferramenta robins-I para ensaios clínicos não randomizados e a Ferramenta da Colaboração Cochrane para ensaios clínicos randomizados. Os níveis de evidência foram avaliados com base nos critérios JBI. Resultados: Os ensaios em animais focaram principalmente na regeneração periodontal. Um risco alto ou pouco claro de viés foi mais comumente encontrado entre os estudos com animais. Avaliações laboratorial, clínica e radiográfica foram utilizadas para avaliar o resultado. Um total de oito estudos em humanos foram conduzidos em um tamanho total de amostras de 153 com ampla faixa etária, variando de sete a 60 anos. Quase 70% dos estudos em humanos usaram DPSC para regeneração de defeitos ósseos alveolares. Conclusão: Ensaios clínicos randomizados duplo-cegos apropriados e bem elaborados de maior duração ainda precisam ser realizados. As evidências para os estudos incluídos foram 1C e 1D de acordo com os critérios JBI. A terapia com células-tronco demonstrou resultados promissores na regeneração do tecido periodontal e do osso alveolar. No entanto, o número de estudos para reivindicar tal benefício é muito limitado (AU)
Sujet(s)
Humains , Animaux , Cellules souches , Dent de lait , Régénération tissulaire guidée parodontale , Pulpe dentaireRÉSUMÉ
BACKGROUND: Functional bioengineered tooth regeneration using autologous or allogeneic alternative differentiated cells sources are thought to have a great potential in replacing conventional dentures. This study investigated the potential of dental pulp stem cells (DPSCs) conditioned medium for odontoblastic differentiation of Wharton's jelly mesenchymal stem cells (WJMSCs). The DPSCs derived from healthy adult permanent first molars were cultured at high confluence prior to conditioned medium collection. The WJMSCs were cultured in six different treatments, with varying ratios of culture media to DPSCs-conditioned medium. MTT assay was used to measure the rate of proliferation of WJMSCs, while immunocytochemistry staining was utilised to detect the expression of dental matrix protein 1 (DMP-1). The deposited calcium was detected and analysed via Alizarin-Red Staining (ARS). RESULTS: It was found that the proliferation of WJMSCs cultured under the mixture of complete medium and DPSCs conditioned medium showed significantly lower than the control; presumably the cells started to exit proliferative state prior differentiation. In 14 days of induction, the cells in all treatments showed osteoblastic-like morphology, calcium compound deposits were observed at day 7, 10 and 14 of differentiation suggested that DPSCs conditioned medium could lead to osteoblastic/odontoblastic differentiation. However, the DMP-1 protein can be seen only expressed minimally at day 14 of conditioned medium induction. CONCLUSIONS: In conclusion, DPSCs conditioned medium appeared as a potential odontoblastic induction approach for WJMSCs. To further investigate the stimulatory effects by DPSCs conditioned medium, specific signalling pathway need to be elucidated to enhance the differentiation efficiency.